Gamma polyglutamated pemetrexed and uses thereof

Abstract

To provide compositions to treat diseases including hyperproliferative diseases such as cancer, disorders of the immune system such as inflammation and rheumatoid arthritis, and infectious disease such as HIV and malaria.SOLUTION: Provided is a liposome composition comprising: liposome encapsulating gamma-polyglutamated pemetrexed; and one or more polyglutamylatable antifolates which is not polyglutamylated, or one or more non-polyglutamylatable antifolates, the gamma-polyglutamated pemetrexed containing 2 to 15 glutamyl groups with gamma or alpha carboxyl linkages.SELECTED DRAWING: None

Description

Background Art

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

[0002] Pemetrexed disodium is the active ingredient of an antineoplastic product sold under the trade name ALIMTA® (Eli Lilly and Company), and its chemical name is 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 5N5Na2O6·7H2O). Pemetrexed is 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 also demonstrated activity in clinical trials in various tumor types including lung cancer, breast cancer, colorectal cancer, mesothelioma, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, and cervical cancer.

[0003] Folic acid is an essential cofactor involved in biosynthesis and DNA repair, remethylation of homocysteine (Hcy), and transfer of one-carbon units involved in 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 transported across cell membranes. Similarly, monoglutamic acid-type polyglutamylatable folic acid antagonists such as pemetrexed are not 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-targeted folate antimetabolite that exerts its action by disrupting folate-dependent metabolic processes essential for cell homeostasis and replication. Pemetrexed inhibits at least three enzymes required for purine and pyrimidine biosynthesis - thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycineamide ribonucleotide formyltransferase (GARFTase). Inhibition of these enzymes suppresses de novo nucleotide biosynthesis, leading to disruption of cell homeostasis and imbalance of purine and pyrimidine precursors, which prevents cells from undergoing accurate DNA replication and ultimately results in cell death.

[0005] Pemetrexed is transported into cells by the reduced folate carrier (RFC) system and folate receptors (FR) γ and β, as well as by the proton-coupled folate transporter (PCFT), which is most active in a lower pH environment than normal. RFC is the major pemetrexed transporter at physiological pH and is widely expressed in both normal and diseased cells. As a result, pemetrexed treatment often suffers from dose-limiting toxicity, a major obstacle in cancer chemotherapy. Once inside the cell, pemetrexed is polyglutamylated by FPGS, which allows up to six L-glutamyl groups to be added to pemetrexed during L-gamma carboxyl group linkage. 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 promotes the accumulation of polyglutamylated pemetrexed, which, unlike pemetrexed (monoglutamate), is not freely transported out of the cell by cellular efflux pumps.

[0006] Pemetrexed acts specifically 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 restricts the clinical application of pemetrexed. Pretreatment with folic acid and vitamin B is currently used to ameliorate the most frequent side effects associated with pemetrexed treatment, including myelosuppression, fatigue, and skin rash.

[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, making them more sensitive to efflux transport out of the cell.

[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 them from the lower level 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 use of gamma-polyglutamylated pemetrexed compositions provides a strategy to overcome the pharmacological issues related to dose-limiting toxicity and treatment resistance associated with pemetrexed therapy. The methods provided deliver potent cytotoxic gamma-polyglutamylated pemetrexed to target cells while (a) minimizing / reducing exposure to normal tissue cells, (b) optimizing / improving the cytotoxic effect of pemetrexed-based agents on target cells such as cancer cells, and (c) minimizing / reducing the effects of efflux pumps, alterations in the activity of enzymes in the folate metabolic pathway, and other resistance mechanisms that limit the therapeutic efficacy of pemetrexed. Summary of the Invention

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

[0011] In some embodiments, the present disclosure provides the following. [1] A composition comprising gamma-polyglutamylated pemetrexed; [2] The composition according to item [1], wherein the gamma-polyglutamylated pemetrexed comprises 1 to 10 glutamyl groups having a gamma-carboxyl group bond; [3] The composition according to item [1] or [2], wherein the gamma-polyglutamylated pemetrexed comprises 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups having a gamma-carboxyl group bond; [4] The composition according to any one of items [1] to [3], wherein the gamma-polyglutamylated pemetrexed is gamma-tetraglutamylated pemetrexed; [5] The composition according to any one of items [1] to [3], wherein the gamma-polyglutamylated pemetrexed is gamma-pentaglutamylated pemetrexed; [6] The composition according to any one of items [1] to [3], wherein the gamma-polyglutamylated pemetrexed is gamma-hexaglutamylated pemetrexed; [7] The composition according to any one of items [1] to [6], which is the following composition: (a) The gamma-polyglutamylated pemetrexed contains two or more L-type glutamyl groups having a gamma-carboxyl group bond, (b) Each of the glutamyl groups of the gamma-polyglutamylated pemetrexed is of the L-type and has a gamma-carboxyl group bond, (c) At least one of the glutamyl groups of the gamma-polyglutamylated pemetrexed is of the D-type and has a gamma-carboxyl group bond, (d) Each of the glutamyl groups of the gamma-polyglutamylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type and has a gamma-carboxyl group bond, or (e) The gamma-polyglutamylated pemetrexed contains two or more L-type glutamyl groups and at least one D-type glutamyl group having a gamma-carboxyl group bond. [8] The composition according to item [4], wherein (a) Each of the glutamyl groups is of the L-type and has a gamma-carboxyl group bond, or (b) each of the glutamyl groups other than the glutamyl group of pemetrexed is of the D-type and each of the glutamyl groups has a gamma-carboxyl group bond. [9] The composition according to item [5], which is (a) each of the glutamyl groups is of the L-type and has a gamma-carboxyl group bond, or (b) each of the glutamyl groups other than the glutamyl group of pemetrexed is of the D-type and each of the glutamyl groups has a gamma-carboxyl group bond;

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

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

[10] , wherein gamma-polyglutamylated aminopterin is polyglutamylatable by FGPS under physiological conditions and / or the polyglutamylated PMX has a lower uptake rate (less than 30%) by hepatocytes than PMX;

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

[11] ;

[13] The Lp-γPPMX composition according to item

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

[14] The Lp-γPPMX composition according to item

[12] or

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

[15] The Lp-γPPMX composition according to item

[12] or

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

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

[12] to

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

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

[12] to

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

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

[12] to

[17] , wherein the liposome contains gamma-tetra-glutamylated pemetrexed;

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

[12] to

[17] , wherein the liposome contains gamma-penta-glutamylated pemetrexed;

[20] An Lp-γPPMX composition according to any one of items

[12] to

[17] , wherein the liposome contains gamma hexaglutamine oxidized pemetrexed;

[21] An Lp-γPPMX composition according to any one of items

[12] to

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

[22] An Lp-γPPMX composition according to any one of items

[12] to

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

[23] An Lp-γPPMX composition according to any one of items

[12] to

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

[24] An Lp-γPPMX composition according to any one of items

[12] to

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

[25] An Lp-γPPMX composition according to any one of items

[12] to

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

[26] An Lp-γPPMX composition according to any one of items

[12] to

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

[27] An Lp-γPPMX composition according to any one of items

[12] to

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

[28] The Lp-γPPMX composition according to item

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

[29] The Lp-γPPMX composition according to item

[27] or

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

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

[27] to

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

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

[27] to

[30] , wherein one or more liposome components further comprise a steric stabilizer; Lp-γPPMX composition;

[32] The Lp-γPPMX 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; Lp-γPPMX composition;

[33] The Lp-γPPMX 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; Lp-γPPMX composition;

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

[12] to

[33] , wherein the liposome is anionic or neutral; Lp-γPPMX composition;

[35] An Lp-γPPMX composition according to any one of items

[12] to

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

[36] An Lp-γPPMX composition according to any one of items

[12] to

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

[37] An Lp-γPPMX composition according to any one of items

[12] to

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

[38] An Lp-γPPMX composition according to any one of items

[12] to

[33] , wherein the liposome is cationic;

[39] An Lp-γPPMX composition according to any one of items

[12] to

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

[40] The Lp-γPPMX 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-γPPMX composition according to item

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

[42] The Lp-γPPMX composition according to item

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

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

[39] to

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

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

[39] to

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

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

[39] to

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

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

[39] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

[51] The Lp-γPPMX 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-γPPMX composition according to item

[50] or

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

[53] An Lp-γPPMX composition according to any one of

[50] to

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

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

[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 ;

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

[50] to

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

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

[50] to

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

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

[50] to

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

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

[39] to

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

[59] An Lp-γPPMX composition according to any one of

[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-γPPMX 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 (e.g., D n-6DPA or D n-3DPA such as resorcin D, resorcin E, or T-series resorcin), oxidized low-density lipoprotein (e.g., OXPAC, PGPC), and toll-like receptor (TLR) regulators such as erythran lipids (e.g., E5564), and is at least one selected from the group consisting of; Lp-γPPMX composition;

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

[58] to

[60] , wherein the immunostimulant and the detectable marker are the same; Lp-γPPMX composition;

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

[58] to

[61] , further comprising a hapten; Lp-γPPMX composition;

[63] The Lp-γPPMX composition according to item

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

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

[12] to

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

[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-γPPMX composition according to any one of items

[12] to

[66] , further comprising carboplatin and / or pembrolizumab; Lp-γPPMX composition;

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

[12] to

[67] ;

[69] A pharmaceutical composition comprising the gamma 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 of such treatment or prevention, the method comprising the step of administering the composition according to any one of items [1] to

[70] to the subject;

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

[12] to

[69] to the subject;

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

[69] ;

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

[12] to

[69] ;

[76] The method according to item

[74] or

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

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

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

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

[12] to

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

[79] The method according to item

[77] or

[78] , wherein the cancer is a non-hematological tumor including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and a 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-γPPMX composition according to any one of items

[50] to

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

[84] 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 gamma-polyglutamylated pemetrexed composition according to any one of items

[12] 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 gamma-polyglutamylated pemetrexed composition according to any one of items [8] 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 gamma polyglutamate oxidized pemetrexed composition according to any one of items

[12] to

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

[90] A method for delivering gamma polyglutamate oxidized pemetrexed to a tumor expressing a folate receptor on its surface, comprising administering the Lp-γPPMX 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 gamma polyglutamate oxidized pemetrexed to the tumor;

[91] A method for preparing a gamma polyglutamate oxidized pemetrexed composition comprising the liposomal gamma polyglutamate oxidized pemetrexed composition according to any one of items

[12] to

[69] , comprising forming a mixture comprising a liposomal component and a gamma polyglutamate oxidized folate antagonist in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes encapsulating gamma polyglutamate oxidized pemetrexed;

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

[12] to

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

[93] The method according to item

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

[94] The method according to item

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

[0012] In some embodiments, the present disclosure provides a gamma polyglutamine oxidized pemetrexed (γPPMX) composition, wherein at least two glutamyl residues of gamma polyglutamine oxidized pemetrexed have gamma carboxyl group bonds. In some embodiments, γPPMX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including glutamyl groups in pemetrexed). In some embodiments, γPPMX contains two or more L-type glutamyl groups. In other embodiments, γPPMX contains a D-type glutamyl group. In further embodiments, γPPMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0013] In one embodiment, the γPPMX composition contains a chain of three glutamyl groups bonded to the glutamyl group of pemetrexed (i.e., tetraglutamine oxidized pemetrexed). In some embodiments, tetraglutamine oxidized PMX contains two or more L-type glutamyl groups. In other embodiments, tetraglutamine oxidized PMX contains a D-type glutamyl group. In further embodiments, tetraglutamine oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0014] In one embodiment, the γPPMX composition comprises a chain of four γ-glutamyl groups attached to the glutamyl group of pemetrexed (e.g., γ-pentaglutamyl oxidized pemetrexed). In some embodiments, gamma pentaglutamyl oxidized PMX comprises two or more L-type glutamyl groups. In other embodiments, gamma pentaglutamyl oxidized PMX comprises a D-type glutamyl group. In further embodiments, gamma pentaglutamyl oxidized PMX comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0015] In one embodiment, the γPPMX composition comprises a chain of five γ-glutamyl groups attached to the glutamyl group of pemetrexed (e.g., γ-hexaglutamyl oxidized pemetrexed). In some embodiments, gamma hexaglutamyl oxidized PMX comprises two or more L-type glutamyl groups. In other embodiments, gamma hexaglutamyl oxidized PMX comprises a D-type glutamyl group. In further embodiments, gamma hexaglutamyl oxidized PMX comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0016] In further embodiments, the present disclosure provides compositions comprising delivery carriers such as liposomes loaded (i.e., encapsulated) with and / or otherwise conjugated to gamma-polyglutamylated pemetrexed, methods of making gamma-polyglutamylated pemetrexed-loaded / conjugated delivery carrier compositions (DV-γPPMX), and methods of using the same to deliver gamma-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 inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria. In some embodiments, the gamma-polyglutamylated pemetrexed in DV-γPPMX comprises 2 to 20, 2 to 15, 2 to 10, 2 to 5, more than 5, or more than 20 glutamyl groups (including the glutamyl groups in pemetrexed). The DV-γPPMX-loaded / conjugated delivery carrier composition provides for the selective delivery of a higher cytotoxic payload (e.g., polyglutamylated pemetrexed) compared to the cytotoxicity of pemetrexed (PMX) administered in the monoglutamate state, resulting in improved efficacy and safety of pemetrexed delivery to cancer cells.

[0017] In further embodiments, the present disclosure provides a composition (Lp-γPPMX) comprising liposomes encapsulated (loaded) with gamma-polyglutamylated pemetrexed. In some embodiments, the gamma-polyglutamylated pemetrexed in Lp-γPPMX comprises 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 20 glutamyl groups (including the glutamyl groups in pemetrexed). In some embodiments, the gamma-polyglutamylated pemetrexed in Lp-γPPMX comprises two or more L-type glutamyl groups. In other embodiments, the gamma-polyglutamylated pemetrexed in Lp-γPPMX comprises D-type glutamyl groups. In further embodiments, the gamma-polyglutamylated pemetrexed in Lp-γPPMX comprises D-type glutamyl groups and two or more L-type glutamyl groups.

[0018] In one embodiment, the Lp-γPPMX composition comprises gamma polyglutamylated PMX containing a chain of three glutamyl groups attached to the glutamyl group of pemetrexed (i.e., tetraglutamylated pemetrexed). In some embodiments, the tetraglutamylated PMX contains two or more L-type glutamyl groups. In other embodiments, the tetraglutamylated PMX contains a D-type glutamyl group. In further embodiments, the tetraglutamylated PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0019] In one embodiment, the Lp-γPPMX composition comprises gamma polyglutamylated PMX containing a chain of four gamma glutamyl groups attached to the glutamyl group of pemetrexed (e.g., gamma pentaglutamylated pemetrexed). In some embodiments, the gamma pentaglutamylated PMX contains two or more L-type glutamyl groups. In other embodiments, the gamma pentaglutamylated PMX contains a D-type glutamyl group. In further embodiments, the gamma pentaglutamylated PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0020] In one embodiment, the Lp-γPPMX composition comprises gamma polyglutamylated PMX containing a chain of five gamma glutamyl groups attached to the glutamyl group of pemetrexed (e.g., gamma hexaglutamylated pemetrexed). In some embodiments, the gamma hexaglutamylated PMX contains two or more L-type glutamyl groups. In other embodiments, the gamma hexaglutamylated PMX contains a D-type glutamyl group. In further embodiments, the gamma hexaglutamylated PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

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

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

[0023] In a further embodiment, the liposomal gamma polyglutamine oxidized pemetrexed composition is pegylated (PLp-γPPMX).

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

[0025] In other embodiments, the liposomal gamma-polyglutamylated pemetrexed composition is targeted (TLp-γPPMX). That is, the TLp-γPPMX composition includes a targeting moiety having a specific affinity for an epitope (surface antigen) on the target cell of interest. In some embodiments, the targeting moiety of TLp-γPPMX or TPLp-γPPMX is not covalently bound to the liposome. In other embodiments, the targeting moiety of TLp-γPPMX or TPLp-γPPMX is bound to one or both of the PEG and the outer surface of the liposome. The targeted liposomal gamma-polyglutamylated pemetrexed compositions (TLp-γPPMX and TPLp-γPPMX) provide further improvement over the efficacy and safety profiles of pemetrexed by specifically delivering gamma-polyglutamylated (e.g., γ-pentaglutamylated and / or γ-hexaglutamylated) pemetrexed to target cells such as cancer cells. In some embodiments, the targeted liposomal gamma-polyglutamylated pemetrexed composition is pegylated (TPLp-γPPMX). In some embodiments, the targeting moiety of TLp-γPPMX or TPLp-γPPMX is bound to one or both of the PEG and the outer surface of the liposome. In some embodiments, the targeting moiety of TLp-γPPMX or TPLp-γPPMX is covalently bound to the liposome. The functions of the targeting moiety of the TLp-γPPMX and / or TPLp-γPPMX 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 (γPPMX) to the cell. Suitable targeting moieties are known in the art and include, but are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides, and peptides. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is a polypeptide comprising at least 3, 5, 10, 15, 20, 30, 40, 50, or 100 amino acid residues.

[0026] In some embodiments, the targeting moiety of TLp-γPPMX or TPLp-γPPMX 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 of TLp-γPPMX or TPLp-γPPMX 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 has an equilibrium dissociation constant (Kd) in the range of 0.5x10 -10 ~10x10 -6 and binds to the target epitope.

[0027] In certain embodiments, the TLp-γPPMX or TPLp-γPPMX 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 is one or more folate receptors selected from the group consisting of folate receptor gamma (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 LP-γPPMX 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 γPPMX composition (e.g., Lp-γPPMX, PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX, or TPLp-γPPMX) is cationic. In other embodiments, the liposomal γPPMX composition (e.g., Lp-γPPMX, PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX or TPLp-γPPMX) is anionic or neutral. In further embodiments, the liposomes of the liposomal γPPMX composition (e.g., Lp-γPPMX, PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX or TPLp-γPPMX) have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, 50 nm to 150 nm, or any range therebetween. In some embodiments, the liposomes of the liposomal γPPMX composition have a diameter in the range of 30 nm to 175 nm or 50 nm to 150 nm, or any range therebetween. In further embodiments, the liposomes of the liposomal γPPMX composition have a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the liposomal γPPMX composition is pegylated (e.g., PLp-γPPMX, NTPLp-γPPMX, or TPLp-γPPMX). In some embodiments, the liposomal γPPMX composition comprises a targeting moiety (e.g., TLp-γPPMX or TPLp-γPPMX). In further embodiments, the liposomal γPPMX composition is pegylated and targeted (e.g., TPLp-γPPMX). In some embodiments, the liposomal γPPMX composition comprises gamma-polyglutamylated pemetrexed containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomal γPPMX composition comprises gamma-tetra-glutamylated pemetrexed.In some embodiments, the liposomal γPPMX composition comprises gamma pentaglutamine oxidized pemetrexed. In other embodiments, the liposomal γPPMX composition comprises gamma hexaglutamine oxidized pemetrexed.

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

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

[0031] In some embodiments, the liposome composition is composed of gamma-pentaglutamyl 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 gamma-pentaglutamyl oxidized PMX. In some embodiments, the Lp-γPPMX composition contains gamma-pentaglutamyl oxidized pemetrexed and 1% - 98.5% w / w of gamma-pentaglutamyl oxidized PMX. In some embodiments, the liposome contains gamma-pentaglutamyl oxidized pemetrexed, and during the preparation process of Lp-γPPMX, starting materials of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of gamma-pentaglutamyl oxidized PMX are encapsulated (enclosed) in Lp-γPPMX. In some embodiments, the liposome composition is composed of gamma-hexaglutamyl 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 gamma-hexaglutamyl oxidized PMX. In some embodiments, the Lp-γPPMX composition contains gamma-hexaglutamyl oxidized pemetrexed and 1% - 98.5% w / w of gamma-hexaglutamyl oxidized PMX. In some embodiments, the liposome contains gamma-hexaglutamyl oxidized pemetrexed, and during the preparation process of Lp-γPPMX, starting materials of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of gamma-pentaglutamyl oxidized PMX are encapsulated (enclosed) in Lp-γPPMX.

[0032] Liposome compositions comprising γPPMX-encapsulated liposomes are also provided. In some embodiments, the liposome composition comprises a pegylated γPPMX composition. In some embodiments, the liposome composition comprises a γPPMX composition linked or otherwise conjugated to a targeting moiety. In further embodiments, the liposome composition comprises a pegylated γPPMX composition linked or otherwise conjugated to a targeting moiety. In some embodiments, the liposome composition comprises γPPMX comprising 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome composition comprises gamma tetrapeptidoglutamyl oxidized pemetrexed. In some embodiments, the liposome composition comprises gamma pentapeptidoglutamyl oxidized pemetrexed. In other embodiments, the liposome composition comprises gamma hexapeptidoglutamyl oxidized pemetrexed.

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

[0034] There is also provided a pharmaceutical composition comprising gamma polyglutamine oxidized pemetrexed (γPPMX) containing a delivery carrier such as liposomal γPPMX. In some embodiments, the pharmaceutical composition comprises a pegylated γPPMX composition. In some embodiments, the pharmaceutical composition comprises a γPPMX composition linked or otherwise attached to a targeting moiety. In further embodiments, the pharmaceutical composition comprises a pegylated γPPMX composition linked or otherwise attached to a targeting moiety. In some embodiments, the pharmaceutical composition comprises γPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises gamma tetraglutamine oxidized pemetrexed. In some embodiments, the pharmaceutical composition comprises gamma pentaglutamine oxidized pemetrexed. In other embodiments, the pharmaceutical composition comprises gamma hexaglutamine oxidized pemetrexed.

[0035] In some embodiments, the pharmaceutical composition comprises liposomal γPPMX (e.g., Lp-γPPMX, PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX, and TPLp-γPPMX). In some embodiments, the liposomal γPPMX composition is pegylated (e.g., NTPLp-γPPMX, and TPLp-γPPMX). In some embodiments, liposomal γPPMX comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TLp-γPPMX or TPLp-γPPMX). In further embodiments, the pharmaceutical composition comprises pegylated liposomal γPPMX and further comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TPLp-γPPMX). In some embodiments, the pharmaceutical composition comprises liposomal γPPMX that is cationic. In other embodiments, the pharmaceutical composition comprises liposomal γPPMX that is anionic or neutral. In further embodiments, the pharmaceutical composition comprises liposomal γPPMX 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 γPPMX 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 a gamma-polyglutamylated pemetrexed (γPPMX) composition (e.g., γPPMX as disclosed herein). In some embodiments, the cells to be contacted are mammalian cells. In further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are hyperproliferative cells. In further embodiments, the hyperproliferative cells are cancer cells. In further embodiments, the cancer cells to be contacted are primary cells or cells derived from a cell line obtained from / derived from 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 cachexia. In some embodiments, the cancer cells are cells derived from a primary cell or a cell line obtained from / derived from lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer cells are cells derived from a primary cell or a cell line obtained from / derived from breast cancer (e.g., HER2+ or triple-negative breast cancer). In some embodiments, the cancer cells are cells derived from a primary cell or a cell line obtained from / derived from colorectal cancer. In some embodiments, the cancer cells are cells derived from a primary cell or a cell line obtained from / derived from ovarian cancer. In some embodiments, the cancer cells are cells derived from a primary cell or a cell line obtained from / derived from endometrial cancer. In some embodiments, the cancer cells are cells derived from a primary cell or a cell line obtained from / derived from pancreatic cancer. In some embodiments, the cancer cells are cells derived from a primary cell or a cell line obtained from / derived from liver cancer. In some embodiments, the cancer cells are cells derived from a primary cell or a cell line obtained from / derived from head and neck cancer. In some embodiments, the cancer cells are cells derived from a primary cell or a cell line obtained from / derived from osteosarcoma. In some embodiments, the method is performed in vivo.In other embodiments, the method is performed in vitro. In some embodiments, γPPMX contains 4, 5, 6, 2 to 10, 4 to 6, or more than 5 gamma-glutamyl groups. In some embodiments, γPPMX contains D-type gamma-glutamyl groups. In some embodiments, γPPMX contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type gamma-glutamyl groups. In some embodiments, γPPMX contains L-type gamma-glutamyl groups. In some embodiments, γPPMX contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type gamma-glutamyl groups. In some embodiments, γPPMX contains both L-type and D-type gamma-glutamyl groups. In some embodiments, γPPMX contains 2, 3, 4, 5, or more than 5 L-type gamma-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type gamma-glutamyl groups. In some embodiments, the γPPMX composition contains gamma-tetra-glutamine oxidized pemetrexed. In some embodiments, the γPPMX composition contains gamma-penta-glutamine oxidized pemetrexed. In other embodiments, the γPPMX composition contains gamma-hexa-glutamine oxidized pemetrexed.

[0037] In further embodiments, the present disclosure provides a method of killing cells, the method comprising contacting the cells with liposomes comprising gamma polyglutamylated pemetrexed (e.g., Lp-γPPMX such as PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX or TPLp-γPPMX). In some embodiments, the cells to be contacted are mammalian cells. In still further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are hyperproliferative cells. In further embodiments, the hyperproliferative cells to be contacted are cancer cells. In further embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from cancers selected from the group consisting of non-hematological tumors including, for example, lung cancer (e.g., NSCLC or mesothelioma), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors including, for example, leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. In some embodiments, the cells are primary cells or cells derived from cell lines obtained from lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from breast cancer (e.g., HER2+ or triple-negative breast cancer). In some embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from colorectal cancer. In some embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from ovarian cancer. In some embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from endometrial cancer. In some embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from pancreatic cancer. In some embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from liver cancer. In some embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from head and neck cancer. In some embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from osteosarcoma.In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposome comprises γPPMX comprising 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises gamma tetraglutamine oxidized pemetrexed. In some embodiments, the liposome comprises gamma pentaglutamine oxidized pemetrexed. In other embodiments, the liposome comprises gamma hexaglutamine oxidized pemetrexed.

[0038] In some embodiments, the liposome comprises γPPMX comprising 4, 5, 6, 2-10, 4-6, or more than 5 gamma glutamyl groups. In some embodiments, the liposome comprises γPPMX comprising D-type gamma glutamyl groups. In some embodiments, the liposome comprises γPPMX comprising D-type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma glutamyl groups. In some embodiments, the liposome comprises γPPMX comprising L-type gamma glutamyl groups. In some embodiments, the liposome comprises γPPMX comprising L-type 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma glutamyl groups. In some embodiments, the liposome comprises γPPMX comprising both L-type and D-type gamma glutamyl groups. In some embodiments, the liposome comprises γPPMX comprising L-type 2, 3, 4, 5, or more than 5 gamma glutamyl groups and D-type 1, 2, 3, 4, 5, or more than 5 gamma glutamyl groups. In some embodiments, the liposome comprises gamma pentaglutamine oxidized pemetrexed. In other embodiments, the liposome comprises gamma hexaglutamine oxidized pemetrexed.

[0039] 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 an effective amount of a delivery carrier (e.g., an immune complex or liposome) comprising gamma-polyglutamyl 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 a scFv). In some embodiments, the delivery carrier is a liposome (e.g., an Lp-γPPMX such as PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX, or TPLp-γPPMX). In some embodiments, the administered delivery carrier is pegylated. In some embodiments, the administered delivery carrier is not pegylated. In further embodiments, the administered delivery carrier comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells.In a further embodiment, the delivery carrier comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin α. v β3, α v β5, or α vβ6), 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(s) derived from a specific subject's cancer (tumor), such as a neoantigen, or determined to be expressed on the tumor. In some embodiments, the targeting moiety has specific affinity for a cell surface antigen(s) derived from a specific tumor of the subject, such as a neoantigen, or determined to be expressed on the tumor. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the administered delivery carrier comprises γPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups. In some embodiments, the delivery carrier comprises γPPMX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups of the D type. In some embodiments, the delivery carrier comprises γPPMX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 γ-glutamyl groups of the L type. In some embodiments, the delivery carrier comprises γPPMX containing 2, 3, 4, 5, or more than 5 γ-glutamyl groups of the L type and 1, 2, 3, 4, 5, or more than 5 γ-glutamyl groups of the D type. In some embodiments, the administered delivery carrier comprises gamma-tetra-glutamyl oxidized pemetrexed. In some embodiments, the administered delivery carrier comprises gamma-penta-glutamyl oxidized pemetrexed. In other embodiments, the administered delivery carrier comprises gamma-hexa-glutamyl oxidized pemetrexed. In some embodiments, the administered delivery carrier comprises L-gamma-poly-glutamyl oxidized pemetrexed. In some embodiments, the administered delivery carrier comprises D-gamma-poly-glutamyl oxidized pemetrexed. In further embodiments, the administered delivery carrier comprises L and D gamma-poly-glutamyl oxidized pemetrexed.In some embodiments, the cancer is selected from the group consisting of non - hematological tumors, such as, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma, 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 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 is 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 for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of liposomes comprising gamma-polyglutamyl oxidized pemetrexed (e.g., Lp-γPPMX such as PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX or TPLp-γPPMX). In some embodiments, the liposomes are pegylated. In some embodiments, the liposomes are not pegylated. In a further embodiment, the liposomes comprise a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells.In a further embodiment, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin α. v β3, α v β5, or α vβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK. This also includes the use of cancer stem cell targeting moieties such as those targeting CD34, CD133 and CD44, CD138, and CD15. In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen(s) derived from a particular subject's tumor, such as a neoantigen, or determined to be expressed on the tumor. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the liposome comprises γPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposome of the administered liposome composition comprises γPPMX containing L-type γ-glutamyl groups. In some embodiments, the liposome of the administered liposome composition comprises γPPMX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposome of the administered liposome composition comprises γPPMX containing both L-type and D-type γ-glutamyl groups. In some embodiments, the liposome of the administered liposome composition comprises γPPMX containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the administered liposome composition comprises tetraglutamine oxidized γPPMX. In some embodiments, the administered liposome composition comprises pentaglutamine oxidized γPPMX. In some embodiments, the administered liposome composition comprises hexaglutamine oxidized γPPMX.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.

[0041] In a further embodiment, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a liposomal composition comprising liposomes containing gamma-polyglutamyl oxidized 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 having specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P-cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen(s) derived from a particular subject's cancer (tumor), such as a neoantigen, or determined to be expressed on the cancer. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the liposome comprises γPPMX comprising 4, 5, 6, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the liposome comprises γPPMX comprising L-type γ-glutamyl groups. In some embodiments, the liposome comprises γPPMX comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposome comprises γPPMX comprising D-type γ-glutamyl groups. In some embodiments, the liposome comprises γPPMX comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the liposome comprises gamma tetrapeptide oxidized pemetrexed. In some embodiments, the liposome comprises gamma pentapeptide oxidized pemetrexed. In other embodiments, the liposome comprises gamma hexapeptide oxidized pemetrexed.

[0042] In some embodiments, the administered liposomal composition comprises pegylated liposomes (e.g., TPLp-γPPMX). In some embodiments, the administered liposomal composition comprises non-pegylated liposomes. In some embodiments, the liposomes of the administered liposomal composition comprise γPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPPMX containing D-type gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPPMX containing D-type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPPMX containing L-type gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPPMX containing L-type 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPPMX containing both L-type and D-type gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPPMX containing L-type 2, 3, 4, 5, or more than 5 gamma-glutamyl groups and D-type 1, 2, 3, 4, 5, or more than 5 gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise gamma-tetra-glutamine oxidized pemetrexed. In some embodiments, the administered liposomal composition comprises gamma-penta-glutamine oxidized pemetrexed. In some embodiments, the liposomes of the administered liposomal composition comprise gamma-hexa-glutamine oxidized pemetrexed. In some embodiments, the liposomal composition is administered for treating cancer selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma, leukemia and lymphoma.In some embodiments, the liposome composition is administered to treat lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the liposome composition is administered to treat breast cancer (e.g., HER2++ or triple negative breast cancer). In some embodiments, the liposome composition is administered to treat colorectal cancer. In some embodiments, the liposome composition is administered to treat ovarian cancer. In some embodiments, the liposome composition is administered to treat endometrial cancer. In some embodiments, the liposome composition is administered to treat pancreatic cancer. In some embodiments, the liposome composition is administered to treat liver cancer. In some embodiments, the liposome composition is administered to treat head and neck cancer. In some embodiments, the liposome composition is administered to treat osteosarcoma.

[0043] In further embodiments, the present disclosure provides a method for treating cancer, the method comprising administering an effective amount of a liposomal composition to a subject having or at risk of having cancer that expresses a folate receptor on its cell surface, the liposomal composition comprising liposomes comprising (a) gamma polyglutamylated pemetrexed (γPPMX) and (b) a targeting moiety having specific binding affinity for the folate receptor. In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α) and folate receptor beta (FR-β). In some embodiments, the liposomal composition to be administered comprises pegylated liposomes (e.g., TPLp-γPPMX). In some embodiments, the liposomal composition to be administered comprises non-pegylated liposomes. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPPMX comprising 4, 5, 6, 2-10, 4-6, or more than 5 gamma glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPPMX comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma glutamyl groups of the D form. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPPMX comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma glutamyl groups of the L form. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPPMX comprising 2, 3, 4, 5, or more than 5 gamma glutamyl groups of the L form and 1, 2, 3, 4, 5, or more than 5 gamma glutamyl groups of the D form. In some embodiments, the liposomes of the liposomal composition to be administered comprise gamma pentaglutamylated pemetrexed. In other embodiments, the liposomes of the liposomal composition to be administered comprise gamma hexaglutamylated pemetrexed.In some embodiments, the liposome composition is administered to treat cancer selected from the group consisting of non - hematological tumors including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors including, for example, leukemia, lymphoma and other B - cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. In some embodiments, the liposome composition is administered to treat lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the liposome composition is administered to treat breast cancer (e.g., HER2 ++ or triple - negative breast cancer). In some embodiments, the liposome composition is administered to treat colorectal cancer. In some embodiments, the liposome composition is administered to treat ovarian cancer. In some embodiments, the liposome composition is administered to treat endometrial cancer. In some embodiments, the liposome composition is administered to treat pancreatic cancer. In some embodiments, the liposome composition is administered to treat liver cancer. In some embodiments, the liposome composition is administered to treat head and neck cancer. In some embodiments, the liposome composition is administered to treat osteosarcoma.

[0044] In a further embodiment, 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 (Lp-γPPMX) comprising gamma polyglutamine oxidized pemetrexed. In some embodiments, the liposomal composition to be administered is PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX or TPLp-γPPMX. In some embodiments, the liposomes of the liposomal composition to be administered comprise pegylated liposomes (e.g., PLp-γPPMX, NTPLp-γPPMX, or TPLp-γPPMX). In some embodiments, the liposomal composition to be administered comprises targeted liposomes (e.g., TLp-γPPMX or TPLp-γPPMX). In some embodiments, the liposomal composition to be administered comprises liposomes that are pegylated and comprise a targeting moiety (e.g., TPLp-γPPMX). In some embodiments, the liposomes of the liposomal composition to be administered comprise gamma polyglutamine oxidized pemetrexed having 4, 5, 6, 2-10, 4-6, or more than 5 gamma glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPPMX having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma glutamyl groups of the D form. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPPMX having 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma glutamyl groups of the L form. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPPMX having 2, 3, 4, 5, or more than 5 gamma glutamyl groups of the L form and 1, 2, 3, 4, 5 or more than 5 gamma glutamyl groups of the D form. In some embodiments, the liposomes of the liposomal composition to be administered comprise gamma tetraglutamine oxidized pemetrexed. In some embodiments, the liposomes of the liposomal composition to be administered comprise gamma pentaglutamine oxidized pemetrexed. In other embodiments, the liposomes of the liposomal composition to be administered comprise gamma hexaglutamine oxidized pemetrexed.

[0045] In a further embodiment, the present disclosure provides a method for 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 gamma polyglutamine oxidized pemetrexed (e.g., Lp-γPPMX, PLp-γPPMX, NTLp-γPPMX, NTPLp-γPPMX, TLp-γPPMX or TPLp-γPPMX). In some embodiments, the liposomal composition is administered for treating an autoimmune disease. In a further embodiment, the liposomal composition is administered for treating rheumatoid arthritis. In another embodiment, the liposomal composition is administered for treating inflammation. In some embodiments, the liposomal composition administered comprises a pegylated liposome (e.g., PLp-γPPMX, NTPLp-γPPMX, or TPLp-γPPMX). In some embodiments, the liposomal composition administered comprises a targeted liposome (e.g., TLp-γPPMX or TPLp-γPPMX) comprising a targeting moiety having specific affinity for a surface antigen on a target cell of interest (e.g., an immune cell). In a further embodiment, the liposomal composition administered comprises a liposome that is pegylated and comprises a targeting moiety (e.g., TPLp-γPPMX). In some embodiments, the liposome of the liposomal composition administered comprises gamma pentaglutamine oxidized pemetrexed comprising 4, 5, 6, 2-10, 4-6, or more than 5 gamma glutamyl groups. In some embodiments, the liposome of the liposomal composition administered comprises γPPMX comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma glutamyl groups of the D-type. In some embodiments, the liposome of the liposomal composition administered comprises γPPMX comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma glutamyl groups of the L-type. In some embodiments, the liposome of the liposomal composition administered comprises γPPMX comprising 2, 3, 4, 5, or more than 5 gamma glutamyl groups of the L-type and 1, 2, 3, 4, 5, or more than 5 gamma glutamyl groups of the D-type. In some embodiments, the liposome of the liposomal composition administered comprises gamma tetraglutamine oxidized pemetrexed.In some embodiments, the liposomes of the administered liposomal composition comprise gamma pentaglutamine oxidized pemetrexed. In other embodiments, the liposomes of the administered liposomal composition comprise gamma hexaglutamine oxidized pemetrexed.

[0046] The present disclosure also provides a method of delivering gamma polyglutamine oxidized pemetrexed to a tumor and / or cancer cell, the method comprising administering to a subject having a tumor a composition comprising gamma polyglutamine oxidized pemetrexed (L-γPPMX) and a targeting moiety having specific binding affinity for an epitope on the surface antigen of a tumor cell or cancer cell. In some embodiments, the administered targeting moiety is bound to a delivery carrier. In some embodiments, the delivery carrier is an antibody or an antigen-binding fragment of an antibody. In further embodiments, the delivery carrier is a liposome. In further embodiments, the antibody, antigen-binding antibody fragment, or liposome is a pegylated liposome (e.g., TPLp-γPPMX). In some embodiments, the administered composition comprises gamma polyglutamine oxidized pemetrexed having 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered composition comprises gamma tetraglutamine oxidized pemetrexed. In some embodiments, the administered composition comprises gamma pentaglutamine oxidized pemetrexed. In other embodiments, the administered composition comprises gamma hexaglutamine oxidized pemetrexed.

[0047] In a further embodiment, the present disclosure provides a method for preparing a liposomal composition comprising a liposomal gamma polyglutamate oxidized pemetrexed (γPPMX) composition, the method comprising: forming a mixture comprising liposomal components and gamma polyglutamate oxidized pemetrexed in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising polyglutamate oxidized pemetrexed. In some embodiments, the gamma polyglutamate oxidized pemetrexed comprises 4, 5, 6, 2 to 10, 4 to 6, or more than 5 gamma glutamyl groups. In some embodiments, the γPPMX composition comprises γPPMX comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma glutamyl groups of the D form. In some embodiments, the γPPMX composition comprises γPPMX comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gamma glutamyl groups of the L form. In some embodiments, the γPPMX composition comprises 2, 3, 4, 5, or more than 5 gamma glutamyl groups of the L form and 1, 2, 3, 4, 5, or more than 5 gamma glutamyl groups of the D form. In some embodiments, the γPPMX composition comprises gamma pentaglutamate oxidized pemetrexed. In some embodiments, the γPPMX composition comprises gamma tetraglutamate oxidized pemetrexed. In other embodiments, the γPPMX composition comprises gamma hexaglutamate oxidized pemetrexed.

[0048] In one embodiment, the present disclosure provides a kit comprising a gamma polyglutamate oxidized pemetrexed composition described herein and / or a γPPMX delivery carrier such as liposomes comprising γPPMX and γPPMX immune complexes (e.g., ADC). BRIEF DESCRIPTION OF THE DRAWINGS

[0049]

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Mode for Carrying Out the Invention

[0050] In general, the present disclosure relates to gamma - polyglutamylated pemetrexed compositions. The compositions provide an advance over prior treatments for hyperproliferative diseases such as cancer. Methods of making, delivering, and using gamma - polyglutamylated pemetrexed compositions are also provided. Gamma - polyglutamylated compositions have uses including, but not limited to, the treatment or prevention of hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0051] I. Definitions Unless otherwise indicated, 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.

[0052] When an embodiment is described herein with the term "comprising", another similar 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 the appropriate legal and factual context (e.g., in a claim, the transitional phrase "comprising" is considered a more open-ended phrase, the transitional phrase "consisting of" is considered more exclusive, and "consisting essentially of" is considered intermediate between these).

[0053] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise or unless it is otherwise clear from the context that only a single referent is intended.

[0054] As used herein, the term "and / or" in expressions such as "A and / or B" is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" in expressions such as "A, B, and / or C" includes 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.

[0055] 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 subheading are necessarily used together in some embodiments.

[0056] 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 any of various cations, such as Na + , Mg 2+ , K + , NH 4+ , and / or Ca 2+ . In certain embodiments, the salt is a pharmaceutically acceptable salt. In further certain embodiments, the PMX salt contains Na + . Pemetrexed can also be referred to as ALIMTA® , LY231514, MTA by the chemical name N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-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 contains one L-gamma-glutamyl group and is thus considered to be monoglutaminated for the purposes of this disclosure.

[0057] The terms "polyglutamylated pemetrexed", "polyglutamylated PMX", "PMX-PG", "PPMX" and variations thereof are used interchangeably herein and refer to pemetrexed compositions that contain at least one glutamyl group in addition to the glutamyl group of pemetrexed (i.e., PMX-PGn, n≧1). References herein to the number of glutamyl groups in γPPMX (PMX-PG) include the glutamyl group of pemetrexed. For example, a PMX-PG composition that contains five glutamyl residues in addition to the glutamyl group of PMX is referred to herein as hexaglutamylated pemetrexed or pemetrexed hexaglutamate. The polyglutamate chain contains an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of the polyglutamate chain does not bind to another glutamyl group via its amino group, but binds to one or more glutamyl groups via its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of polyglutamylated 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.

[0058] The terms "gamma-glutamyl group", "gamma-glutamyl group", and "gamma bond", when they relate to the bonding of glutamyl groups, mean a glutamyl group that includes a gamma-carboxyl group bond. A gamma bond can be a bond between a glutamyl group and the glutamyl group of pemetrexed, or between a glutamyl group and a second glutamyl group that is not present in pemetrexed (e.g., a glutamyl group within a polyglutamate chain attached to pemetrexed). In some embodiments, a gamma bond is an amide bond between the gamma-carboxyl group of one glutamyl group and a second glutamyl group. In some embodiments, a gamma bond means an amide bond of a glutamyl group in pemetrexed. In some embodiments, a gamma bond is an amide bond between the gamma-carboxyl group of one glutamyl group and a second glutamyl group. A reference to a gamma bond includes a gamma bond of a glutamyl group in pemetrexed, unless otherwise specified or clearly apparent from the context that it is not intended. In some embodiments, the gamma-glutamyl group is of the L-type. In some embodiments, the gamma-glutamyl group is of the D-type. As discussed herein, during pemetrexed therapy, pemetrexed enters cells and is polyglutamylated by the enzyme folylpoly-gamma-glutamate synthetase (FPGS), which adds L-glutamyl groups in series to the gamma-carboxyl group of the glutamate within the pemetrexed L-glutamyl group of pemetrexed. As a result, D-gamma-polyglutamylated pemetrexed compositions are not formed intracellularly during pemetrexed therapy.

[0059] The terms "gamma-polyglutamylated pemetrexed", "γ-polyglutamylated pemetrexed", "γPPMX", "gamma-polyglutamylated pemetrexed", "polyglutamylated PMX", "γPMX-PG", and iterations thereof are used herein interchangeably and refer to a pemetrexed composition having at least one gamma-glutamyl group having a gamma-carboxyl group bond in addition to the gamma-glutamyl group of pemetrexed (e.g., a gamma-glutamyl group of PMX-PGn, n≧1). References herein to the number of glutamyl groups (γPMX-PG) in γPPMX include the gamma-glutamyl groups of pemetrexed. For example, a γPMX-PG composition containing 5 gamma-glutamyl groups in addition to the glutamyl groups of PMX may be referred to herein as gamma-hexaglutamylated pemetrexed or gamma-pemetrexed hexaglutamate.

[0060] The terms "alpha-glutamyl group", "α-glutamyl group", and "alpha bond", when referring to the bond of a glutamyl group, mean a glutamyl group containing an alpha-carboxyl group bond.

[0061] As used herein, the term "isolated" means a composition in a form not found in nature. Isolated gamma polyglutamine oxidase compositions include those that are purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, the isolated gamma polyglutamine oxidized pemetrexed is substantially pure. An isolated composition is free or substantially free of substances that are naturally incorporated, such as other cellular components such as proteins and nucleic acids that may potentially be found in nature or in the environment in which they are produced (e.g., cell culture). Gamma polyglutamine oxidase compositions can be formulated with diluents or adjuvants and further isolated for more practical purposes - for example, when used in a diagnostic or therapeutic agent, gamma polyglutamine oxidase compositions are typically mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, an isolated gamma polyglutamine oxidase composition (e.g., a delivery vehicle such as a gamma polyglutamate and a liposome containing gamma polyglutamate) contains less than 1% or less than 0.1% of unwanted DNA or protein content. In some embodiments, a gamma polyglutamate composition (e.g., a delivery vehicle such as a gamma polyglutamate and a liposome containing gamma polyglutamate) is "isolated".

[0062] 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, region of the body, or compartment, e.g., a compartment of a cell, tissue or organ. A targeting moiety can include a wide variety of entities. 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 affimer, 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.

[0063] The terms "specific affinity", "specifically binds", and "enhanced affinity" mean that a targeting moiety, such as an antibody or an antigen-binding antibody fragment, reacts or binds to an epitope, protein, or target molecule with a higher frequency, more rapidly, for a longer period of time, with a greater affinity, or in some combination of these, than it does to another substance containing a protein unrelated to the antigen containing the target epitope. Due to sequence identity between homologous proteins in different species, in some embodiments, a particular affinity involves a binding substance that recognizes an epitope on a protein and / or target molecule 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 an epitope present on two or more proteins and / or target molecules. In certain embodiments, it is understood that a targeting moiety that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific affinity" does not necessarily require exclusive binding, e.g., binding to only one epitope on one target (although it can include this). Thus, in certain embodiments, a targeting moiety can specifically bind to epitopes present on two or more targets. In certain embodiments, multiple targets can be bound by the same targeting moiety that specifically binds to epitopes present on the multiple targets.

[0064] The term "epitope" means a portion of an antigen that can be recognized and specifically bound by a targeting moiety (i.e., a binding portion), such as an antibody. When the antigen is a polypeptide, the epitope can be formed from both contiguous and non-contiguous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed from contiguous amino acids are usually retained upon protein denaturation, whereas epitopes formed by tertiary folding are usually lost upon protein denaturation. An epitope typically contains at least 3 amino acids, more generally at least 5 or 8 - 10 amino acids, in a particular spatial conformation.

[0065] Expressions such as "binding affinity for a target", "binding to a target", "enhanced affinity", and similar expressions that are known in the art refer to properties of a targeting moiety that can be directly measured by determining an affinity constant, e.g., the amount of targeting moiety that binds and dissociates at a given antigen concentration. Without limitation, other methods such as competitive analysis, equilibrium analysis and microcalorimetry, and real-time interaction analysis based on surface plasmon resonance interactions (e.g., using a BIACORE® instrument) can be used to characterize intermolecular interactions. These methods are well known to those skilled in the art and are described, for example, in Neri et al., Tibtech 14:465-470 (1996), and Jansson et al., J. Biol. Chem. 272:8189-8197 (1997).

[0066] The term "delivery vehicle" generally means any composition that acts to assist, facilitate or ease the entry of gamma-polyglutamyl oxidized pemetrexed into cells. Such delivery vehicles are known in the art and include, without limitation, liposomes, lipospheres, polymers (e.g., polymer conjugates), peptides, proteins such as antibodies (e.g., immunoconjugates such as antibody-drug conjugates (ADCs) and antigen-binding antibody fragments and derivatives thereof), cell components, cyclic oligosaccharides (e.g., cyclodextrins), micelles, microparticles (e.g., microspheres), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), hydrogels, lipoprotein particles, viral sequences, viral substances, or lipid or liposome formulations, and combinations thereof. The delivery vehicle can be bound directly or indirectly to the targeting moiety. In some embodiments, the targeting moiety is selected from the group consisting of a polymer, protein, peptide, monoclonal antibody or fatty acid lipid.

[0067] "Subject" means a human or, without limitation, a vertebrate mammal including a dog, cat, horse, goat and primate, such as a monkey. Thus, the present invention can also be used to treat diseases or conditions in non-human subjects. For example, cancer is one of the leading causes of death in companion animals (e.g., cats and dogs). In some embodiments of the present invention, the subject is a human. In the present disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. Generally, it is preferred that the maximum dose, i.e., the maximum safe dose according to sound medical judgment, is used.

[0068] As used herein, "effective amount" means an amount of a drug sufficient to produce a medically desired result. The effective amount can vary depending on the desired outcome, the particular condition to be treated or prevented, the age and health status of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent or concomitant therapies (if any), the specific route of administration and similar factors within the knowledge and professional opinion of the health care provider. The "effective amount" can be determined experimentally or routinely in relation to the indicated purpose. In the case of cancer, an effective amount of a drug reduces the number of cancer cells; reduces the size of the tumor; inhibits the invasion of cancer cells into surrounding organs (i.e., slows down and preferably stops to some extent); inhibits the metastasis of the tumor (i.e., slows down and preferably stops to some extent); inhibits the growth of the tumor to some extent; and / or alleviates one or more of the symptoms associated with the disorder to some extent. Depending on the extent to which the drug can prevent and / or kill the growth of existing cancer cells, the drug can be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by evaluating survival time, progression-free survival (PFS) period, response rate (RR), response duration, and / or quality of life.

[0069] The terms "hyperproliferative disorder", "proliferative disease", and "proliferative disorder" are used interchangeably herein and relate to unwanted or abnormal cell growth, such as neoplastic or hyperplastic growth, whether in vitro or in vivo, of unwanted or uncontrolled cells. 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.

[0070] "Cancer", "tumor", or "malignant tumor" are used as synonymous terms and refer to any of a number of cell types or 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 / or any characteristic structures and / or molecular features known to be associated with these cell types or diseases. As used herein, "tumor" means all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. "Cancerous tumor", or "malignant cells" are understood to be cells that have specific structural characteristics, lack differentiation, and are capable of invasion and metastasis. Cancers that can be treated with the γPPMX 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, myeloma and other plasma cell dyscrasias or cachexia. Other types of cancers and tumors that can be treated with the γPPMX 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.

[0071] The terms "treating," "treatment," or "for treatment" mean both (a) therapeutic measures that cure, slow down, reduce the symptoms of, and / or halt the progression of a diagnosed medical condition or disorder and (b) prophylactic or preventive measures that prevent and / or delay the occurrence of a targeted disease or condition. Thus, subjects in need of treatment include subjects already having the cancer, disorder or disease, subjects at risk of becoming a cancer or condition, and subjects in which an infection or condition is to be prevented. A subject is identified as "at risk of having" a cancer, infectious disease, immune system disorder, hyperproliferative disease, or another disease or disorder mentioned herein using well-known medical and diagnostic techniques. In certain embodiments, a subject is "being treated successfully" by the methods provided herein if the subject exhibits, for example, complete, partial, or temporary remission or elimination of symptoms associated with a disease or condition (e.g., cancer, inflammation, and rheumatoid arthritis). In certain embodiments, the term "treating" or "treatment" or "for treatment" means improvement of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, that 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 identifiable symptoms or physiologically, for example, 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 γPPMX composition can be used alone or in combination with additional therapeutic agents for treatment.

[0072] "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, such as mammals and non-mammals, for example, chickens, amphibians, and reptiles. As used herein, "mammal" includes, but is not limited to, humans and non-human primate animals, such as chimpanzees and other apes and monkey species; domestic animals such as cows, sheep, pigs, goats, and horses; pet mammals such as dogs and cats; laboratory animals such as rodents, mice, rats, guinea pigs, and other members of the mammalian class known in the art. In certain embodiments, the subject is human.

[0073] As used herein, "treatment of a proliferative disorder" includes maintaining or reducing the tumor size of a subject with a proliferative disorder, inducing tumor shrinkage (partial or complete), suppressing tumor growth, and / or extending lifespan. In one embodiment, the proliferative disorder is a solid tumor. Such tumors include, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma. In one embodiment, the proliferative disorder is a hematological tumor. Such hematological tumors include, for example, leukemia, lymphoma and other B cell malignancies, multiple myeloma and other plasma cell dyscrasias or cachexia.

[0074] 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 and inflammation, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis are specifically mentioned.

[0075] As used herein, the term "therapeutic agent" means an agent, or a derivative or prodrug thereof, that can interact with hyperproliferative cells such as cancer cells or immune cells and thereby reduce the proliferative state of the cells and / or kill 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 derivatives thereof), 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 trimetrexate, temozolomide, raltitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), bis-chloronitrosourea (BCNU) and camptothecin, or any therapeutic derivatives thereof. Further examples of therapeutic agents that may be suitable for use by the methods of the present disclosure include, but are not limited to, anti-restenosis agents, pro-proliferative or anti-proliferative agents, anti-inflammatory agents, anti-neoplastic agents, anti-mitotic agents, antiplatelet agents, anticoagulants, antifibrin agents, antithrombin agents, cytostatic agents, antibiotics and other anti-infective agents, anti-enzymatic agents, antimetabolites, angiogenesis agents, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists and / or cardioprotective agents. "Therapeutic agent" also means salts, acids, and free base forms of the above agents.

[0076] As used herein, the term "chemotherapeutic agent" means, when used in the context of cancer therapy, 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.

[0077] 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, including purine or pyrimidine analogs. In some embodiments, the gamma-polyglutamylated 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, foladesine, 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 the group consisting of fludarabine, cytarabine, gemcitabine, decitabine, and azacitidine, or derivatives thereof. In certain embodiments, the antimetabolite is 5-fluorouracil.

[0078] As used herein, "taxane" is an anti-cancer agent that interferes with or disrupts microtubule stability, formation, and / or function. Taxane agents include paclitaxel and docetaxel and their derivatives, which function in the same mode of action with respect to microtubules as the taxanes from which they are derived. In certain embodiments, the taxane is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the taxane is paclitaxel (Taxol®), docetaxel (Taxotere®), albumin-bound paclitaxel (nab-paclitaxel; Abraxane®), DHA-paclitaxel, or PG-paclitaxel.

[0079] The term "pharmaceutically acceptable carrier" means a non-toxic component other than the active ingredient in a pharmaceutical formulation for a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents, or preservatives. Pharmaceutically acceptable carriers include, for example, one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other subjects.

[0080] The present disclosure generally relates to compositions containing gamma-polyglutamyl oxidized pemetrexed (PMX), and methods of making and using the compositions for treating diseases including proliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0081] In some embodiments, the present disclosure provides the following. [1] A composition comprising gamma-polyglutamyl oxidized pemetrexed; [2] The composition of item [1], wherein the gamma-polyglutamyl oxidized pemetrexed comprises 1 to 10 glutamyl groups having gamma-carboxyl group linkages. [3] The composition according to item [1] or [2], wherein the gamma-polyglutamyl oxidized pemetrexed contains 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups having a gamma-carboxyl group bond; [4] The composition according to any one of items [1] to [3], wherein the gamma-polyglutamyl oxidized pemetrexed is gamma-tetra-glutamyl oxidized pemetrexed; [5] The composition according to any one of items [1] to [3], wherein the gamma-polyglutamyl oxidized pemetrexed is gamma-penta-glutamyl oxidized pemetrexed; [6] The composition according to any one of items [1] to [3], wherein the gamma-polyglutamyl oxidized pemetrexed is gamma-hexa-glutamyl oxidized pemetrexed; [7] The composition according to any one of items [1] to [6], which is the following composition: (a) The gamma-polyglutamyl oxidized pemetrexed contains 2 or more L-type glutamyl groups having a gamma-carboxyl group bond, (b) Each of the glutamyl groups of the gamma-polyglutamyl oxidized pemetrexed is of the L-type and has a gamma-carboxyl group bond, (c) At least one of the glutamyl groups of the gamma-polyglutamyl oxidized pemetrexed is of the D-type and has a gamma-carboxyl group bond, (d) Each of the glutamyl groups of the gamma-polyglutamyl oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type and has a gamma-carboxyl group bond, or (e) The gamma-polyglutamyl oxidized pemetrexed contains 2 or more L-type glutamyl groups and at least one D-type glutamyl group having a gamma-carboxyl group bond. [8] The composition according to item [4], (a) Each glutamyl group is of the L-type and has a gamma-carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of pemetrexed is of the D-type and each glutamyl group has a gamma-carboxyl group bond. [9] The composition according to item [5], wherein (a) each glutamyl group is of the L-type and has a gamma-carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of pemetrexed is of the D-type and each glutamyl group has a gamma-carboxyl group bond;

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

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

[10] , wherein gamma-polyglutamylated aminopterin is polyglutamylatable by FGPS under physiological conditions and / or polyglutamylated PMX has a lower uptake rate (less than 30%) by hepatocytes than PMX;

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

[11] ;

[13] The Lp-γPPMX composition according to item

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

[14] The Lp-γPPMX composition according to item

[12] or

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

[15] The Lp-γPPMX composition according to item

[12] or

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

[19] An Lp-γPPMX composition according to any one of items

[12] to

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

[20] An Lp-γPPMX composition according to any one of items

[12] to

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

[21] An Lp-γPPMX composition according to any one of items

[12] to

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

[22] An Lp-γPPMX composition according to any one of items

[12] to

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

[23] An Lp-γPPMX composition according to any one of items

[12] to

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

[24] An Lp-γPPMX composition according to any one of items

[12] to

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

[25] An Lp-γPPMX composition according to any one of items

[12] to

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

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

[12] to

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

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

[12] to

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

[28] The Lp-γPPMX composition according to item

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

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

[27] to

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

[32] The Lp-γPPMX 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 is at least one selected from the group consisting of; an Lp-γPPMX composition;

[33] The Lp-γPPMX 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; an Lp-γPPMX composition;

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

[12] to

[33] , wherein the liposome is anionic or neutral; an Lp-γPPMX composition;

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

[12] to

[33] , wherein the liposome has a zeta potential of zero or less; an Lp-γPPMX composition;

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

[12] to

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

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

[12] to

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

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

[12] to

[33] , wherein the liposome is cationic; an Lp-γPPMX composition;

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

[12] to

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

[40] The Lp-γPPMX 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-γPPMX composition according to item

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

[42] The Lp-γPPMX composition according to item

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

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

[39] to

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

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

[39] to

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

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

[39] to

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

[46] The Lp-γPPMX 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 - 500 mM;

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

[12] to

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

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

[12] to

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

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

[12] to

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

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

[12] to

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

[51] The Lp-γPPMX 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-γPPMX composition according to item

[50] or

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

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

[50] to

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

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

[50] to

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

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

[50] to

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

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

[39] to

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

[59] An Lp-γPPMX 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-γPPMX 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 (e.g., D n-6DPA or D n-3DPA such as resorcin D, resorcin E, or T-series resorcin), and toll-like receptor (TLR) regulators such as oxidized low-density lipoprotein (e.g., OXPAC, PGPC), and erythran lipid (e.g., E5564);

[61] An Lp-γPPMX composition according to any one of items

[58] to

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

[62] An Lp-γPPMX composition according to any one of items

[58] to

[61] , further comprising a hapten, the Lp-γPPMX composition;

[63] An Lp-γPPMX composition according to item

[62] , wherein the hapten comprises one or more of fluorescein or beta-1,6-glucan, the Lp-γPPMX composition:

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

[12] to

[63] , further comprising at least one cryoprotectant selected from the group consisting of mannitol, trehalose, sorbitol, and sucrose, the Lp-γPPMX composition;

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

[64] ;

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

[49] ;

[67] An Lp-γPPMX composition according to any one of items

[12] to

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

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

[12] to

[67] ;

[69] A pharmaceutical composition comprising the gamma 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 of such treatment or prevention, the method comprising the step of administering the composition according to any one of items [1] to

[70] to the subject;

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

[12] to

[69] to the subject;

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

[69] ;

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

[12] to

[69] ;

[76] The method according to item

[74] or

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

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

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

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

[12] to

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

[79] The method according to item

[77] or

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

[80] The method according to item

[77] or

[78] , wherein the cancer is a member selected from the group consisting of lung cancer, breast cancer, colorectal 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; A method for treating cancer, comprising administering an effective amount of the Lp-γPPMX 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 on the surface thereof, which is 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 undergoing or has undergone cancer therapy;

[85] Maintenance therapy, comprising administering an effective amount of the liposomal gamma-polyglutamylated pemetrexed composition according to any one of items

[12] 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 gamma-polyglutamylated pemetrexed composition according to any one of items [8] 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 gamma-polyglutamylated pemetrexed composition according to any one of items

[12] to

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

[90] A method for delivering gamma-polyglutamylated pemetrexed to a tumor expressing a folate receptor on its surface, comprising administering the Lp-γPPMX 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 gamma-polyglutamylated pemetrexed to the tumor;

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

[12] to

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

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

[12] to

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

[93] The method according to item

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

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

[50] to

[69] , the method comprising: forming a mixture comprising a liposomal component and alpha-polyglutamylated pemetrexed in a solution; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes that capture and / or encapsulate alpha-polyglutamylated pemetrexed; and providing 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-γ), folate receptor beta (FR-β), and folate receptor delta (FR-δ);

[95] A method for preparing a 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 that capture and / or encapsulate alpha polyglutamine oxidized pemetrexed; and providing 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-γ), folate receptor beta (FR-β), and folate receptor delta (FR-δ);

[96] The method according to item

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

[97] The method according to any one of item

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

[98] The method according to any one of items

[95] to

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

[99] The method according to any one of items

[91] to

[98] , wherein at least 1% of the starting material of alpha polyglutamine oxidized pemetrexed is encapsulated or enclosed in the liposomes.

[0082] II. Gamma polyglutamine oxidized pemetrexed (γPPMX) Generally, the present disclosure relates to gamma-polyglutamylated pemetrexed (γPPMX) compositions. The γPPMX compositions include at least one glutamyl group having a gamma-carboxyl group bond. These are structurally different from L-gamma-polyglutamylated pemetrexed (Lγ1PPMX) produced by the enzyme folylpolyglutamate synthase (FPGS) in cells during pemetrexed therapy.

[0083] In some embodiments, the γPPMX composition includes 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl groups in pemetrexed). In some embodiments, each glutamyl group in γPPMX other than the glutamyl groups of pemetrexed has a gamma bond. In some embodiments, two or more glutamyl groups in γPPMX have a gamma bond. In some embodiments, each glutamyl group in γPPMX is of the L-type. In some embodiments, each glutamyl group in γPPMX other than the glutamyl groups of pemetrexed is of the D-type. In some embodiments, γPPMX includes two or more L-type glutamyl groups and one or more D-type glutamyl groups.

[0084] In some embodiments, the gamma-polyglutamylated pemetrexed is diglutamylated. That is, the gamma-polyglutamylated pemetrexed includes, in addition to the glutamyl groups of pemetrexed, one additional glutamyl group (γPMX-PG1), and the additional glutamyl group is bonded to the glutamyl group in pemetrexed via a gamma bond. In some embodiments, each glutamyl group of the gamma-diglutamylated pemetrexed is of the L-type. In other embodiments, the gamma-diglutamylated PMX includes a D-type glutamyl group.

[0085] In some embodiments, gamma-polyglutamylated pemetrexed is triglutamylated. That is, gamma-polyglutamylated pemetrexed contains two additional glutamyl groups in addition to the glutamyl group of pemetrexed (γPMX-PG2). In some embodiments, each glutamyl group of gamma-triglutamylated pemetrexed is of the L-type. In other embodiments, gamma-triglutamylated PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma-triglutamylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, gamma-triglutamylated PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0086] In some embodiments, gamma-polyglutamylated pemetrexed is tetraglutamylated and thus contains three additional glutamyl groups in addition to the glutamyl group in pemetrexed (γPMX-PG3). In some embodiments, gamma-tetraglutamylated PMX contains two or more L-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group of gamma-tetraglutamylated pemetrexed is of the L-type. In other embodiments, gamma-tetraglutamylated PMX contains a D-type γ-glutamyl group. In some embodiments, gamma-tetraglutamylated PMX contains two D-type γ-glutamyl groups. In some embodiments, each glutamyl group of gamma-tetraglutamylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, tetraglutamylated PMX contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0087] In some embodiments, gamma polyglutamylated pemetrexed is pentaglutamylated (γPMX-PG4) and includes a chain of four additional glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma pentaglutamylated PMX includes two or more L-type γ-glutamyl groups. In further embodiments, each glutamyl group of gamma pentaglutamylated pemetrexed is of the L-type. In other embodiments, gamma pentaglutamylated PMX includes a D-type glutamyl group. In some embodiments, gamma tetraglutamylated PMX includes two or three D-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group of gamma pentaglutamylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, pentaglutamylated PMX includes a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0088] In some embodiments, gamma polyglutamylated pemetrexed is hexaglutamylated (γPMX-PG5) and includes a chain of five additional glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma hexaglutamylated PMX includes two or more L-type γ-glutamyl groups. In further embodiments, each glutamyl group of gamma hexaglutamylated pemetrexed is of the L-type. In other embodiments, gamma hexaglutamylated PMX includes a D-type γ-glutamyl group. In some embodiments, gamma tetraglutamylated PMX includes two, three, four, or five D-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group of gamma hexaglutamylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, hexaglutamylated PMX includes a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0089] In some embodiments, gamma polyglutamylated pemetrexed is heptaglutamylated (γPMX-PG6) and thus contains a chain of six additional glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma heptaglutamylated PMX contains two or more L-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group of gamma heptaglutamylated pemetrexed is of the L-type. In other embodiments, gamma heptaglutamylated PMX contains a D-type γ-glutamyl group. In some embodiments, gamma tetraglutamylated PMX contains two, three, four, five, or six D-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group of gamma heptaglutamylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, heptaglutamylated PMX contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0090] In some embodiments, gamma polyglutamylated pemetrexed is octaglutamylated (γPMX-PG7) and thus contains a chain of seven additional glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma octaglutamylated PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma octaglutamylated pemetrexed is of the L-type. In other embodiments, gamma octaglutamylated PMX contains a D-type glutamyl group. In some embodiments, gamma octaglutamylated PMX contains two, three, four, five, six, or seven D-type γ-glutamyl groups. In further embodiments, each glutamyl group of gamma octaglutamylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, octaglutamylated PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0091] In some embodiments, gamma polyglutamate oxidized pemetrexed is nonaglutamate oxidized (γPMX-PG8) and includes a chain of eight additional glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma nonaglutamate oxidized PMX includes two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma nonaglutamate oxidized pemetrexed is of the L-type. In other embodiments, gamma nonaglutamate oxidized PMX includes a D-type glutamyl group. In further embodiments, each glutamyl group of gamma nonaglutamate oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, nonaglutamate oxidized PMX includes a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0092] In some embodiments, gamma polyglutamate oxidized pemetrexed is decaglutamate oxidized (γPMX-PG9) and includes a chain of nine additional glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma decaglutamate oxidized PMX includes two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma decaglutamate oxidized pemetrexed is of the L-type. In other embodiments, gamma decaglutamate oxidized PMX includes a D-type glutamyl group. In further embodiments, each glutamyl group of gamma decaglutamate oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, decaglutamate oxidized PMX includes a D-type glutamyl group and two or more L-type glutamyl groups.

[0093] In some embodiments, gamma polyglutamate oxidized pemetrexed is undecaglutamate oxidized (γPMX-PG 10) It contains a chain of 10 γ-glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma-undecaglutaminyl oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma-undecaglutaminyl oxidized pemetrexed is of the L-type. In other embodiments, gamma-undecaglutaminyl oxidized PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma-undecaglutaminyl oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, undecaglutaminyl oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0094] In some embodiments, gamma-polyglutamyl oxidized pemetrexed is dodecaglutaminylated (γPMX-PG 11 ) It contains a chain of 11 γ-glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma-dodecaglutaminyl oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma-dodecaglutaminyl oxidized pemetrexed is of the L-type. In other embodiments, gamma-dodecaglutaminyl oxidized PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma-dodecaglutaminyl oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, dodecaglutaminyl oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0095] In some embodiments, gamma-polyglutamyl oxidized pemetrexed is tridecaglutaminylated (γPMX-PG 12) It contains a chain of 12 gamma-glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma-triskadecakaglutaminoxylated PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma-triskadecakaglutaminoxylated pemetrexed is of the L-type. In other embodiments, gamma-triskadecakaglutaminoxylated PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma-triskadecakaglutaminoxylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, triskadecakaglutaminoxylated PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0096] In some embodiments, gamma-polyglutaminoxylated pemetrexed is tetradecakaglutaminoxylated (γPMX-PG 13 ) It contains a chain of 13 gamma-glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma-tetradecakaglutaminoxylated PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma-tetradecakaglutaminoxylated pemetrexed is of the L-type. In other embodiments, gamma-tetradecakaglutaminoxylated PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma-tetradecakaglutaminoxylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, tetradecakaglutaminoxylated PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0097] In some embodiments, gamma-polyglutaminoxylated pemetrexed is pentadecakaglutaminoxylated (γPMX-PG 14) It contains a chain of 14 gamma-glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma-pentadeca-glutaminyl oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma-pentadeca-glutaminyl oxidized pemetrexed is of the L-type. In other embodiments, gamma-pentadeca-glutaminyl oxidized PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma-pentadeca-glutaminyl oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, pentadeca-glutaminyl oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0098] In some embodiments, gamma-polyglutaminyl oxidized pemetrexed is hexadeca-glutaminylated (γPMX-PG 15 ) It contains a chain of 15 gamma-glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma-hexadeca-glutaminyl oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma-hexadeca-glutaminyl oxidized pemetrexed is of the L-type. In other embodiments, gamma-hexadeca-glutaminyl oxidized PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma-hexadeca-glutaminyl oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, hexadeca-glutaminyl oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0099] In other embodiments, gamma-polyglutaminyl oxidized pemetrexed is heptadeca-glutaminylated (γPMX-PG 16) It contains a chain of 16 γ-glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma-heptadeca-glutaminoxylated PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma-heptadeca-glutaminoxylated pemetrexed is of the L-type. In other embodiments, gamma-heptadeca-glutaminoxylated PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma-heptadeca-glutaminoxylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, heptadeca-glutaminoxylated PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0100] In some embodiments, gamma-polyglutaminoxylated pemetrexed is octadeca-glutaminoxylated (γPMX-PG 17 ) It contains a chain of 17 γ-glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma-octadeca-glutaminoxylated PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma-octadeca-glutaminoxylated pemetrexed is of the L-type. In other embodiments, gamma-octadeca-glutaminoxylated PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma-octadeca-glutaminoxylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, octadeca-glutaminoxylated PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0101] In some embodiments, gamma-polyglutaminoxylated pemetrexed is ennea-deca-glutaminoxylated (γPMX-PG 18) It contains a chain of 18 γ-glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma eicosaglutaminylated PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma eicosaglutaminylated pemetrexed is of the L-type. In other embodiments, gamma eicosaglutaminylated PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma eicosaglutaminylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, eicosaglutaminylated PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0102] In some embodiments, gamma polyglutaminylated pemetrexed is eicosaglutaminylated (γPMX-PG 19 ) It contains a chain of 19 γ-glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gamma eicosaglutaminylated PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma eicosaglutaminylated pemetrexed is of the L-type. In other embodiments, gamma eicosaglutaminylated PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma eicosaglutaminylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, eicosaglutaminylated PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0103] In some embodiments, gamma polyglutaminylated pemetrexed is heneicosaglutaminylated (γPMX-PG 20) It contains a chain of 20 γ-glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, gammahenicosaglutaminyl oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gammahenicosaglutaminyl oxidized pemetrexed is of the L-type. In other embodiments, gammahenicosaglutaminyl oxidized PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gammahenicosaglutaminyl oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, henicosaglutaminyl oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0104] In some embodiments, gamma polyglutamyl oxidized pemetrexed contains a chain of 4 to 7 glutamyl groups attached to pemetrexed (i.e., γPMX-PGn, n = 4 to 7), and each of the 4 to 7 linked glutamyl groups has a gamma linkage. In some embodiments, each of the 4 to 7 linked glutamyl groups is of the L-type. In other embodiments, each of the 4 to 7 linked glutamyl groups is of the D-type. In other embodiments, the 4 to 7 linked glutamyl groups are of the L-type and the D-type.

[0105] In one embodiment, gamma polyglutamyl oxidized pemetrexed is tetraglutaminylated, and each of the three glutamyl groups in the polyglutamate chain attached to pemetrexed contains a gamma linkage. In some embodiments, each of the 4 glutamyl groups is of the L-type. In some embodiments, each glutamyl group in gamma tetraglutaminyl oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In other embodiments, at least two glutamyl groups in gamma tetraglutaminyl oxidized pemetrexed are of the L-type and at least one glutamyl group is of the D-type.

[0106] In one embodiment, gamma-polyglutamyl oxidized pemetrexed is pentaglutamylated, and each of the four glutamyl groups in the polyglutamate chain attached to pemetrexed contains a gamma bond. In some embodiments, each of the four glutamyl groups is of the L-type. In some embodiments, each of the glutamyl groups in gamma-pentaglutamyl oxidized pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In other embodiments, at least two of the glutamyl groups in gamma-pentaglutamyl oxidized pemetrexed are of the L-type and at least one of the glutamyl groups is of the D-type.

[0107] In one embodiment, gamma-polyglutamyl oxidized pemetrexed is hexaglutamylated. In some embodiments, each of the five glutamyl groups is of the L-type. In some embodiments, each of the glutamyl groups in gamma-hexaglutamyl oxidized pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In other embodiments, at least two of the glutamyl groups in gamma-hexaglutamyl oxidized pemetrexed are of the L-type and at least one of the glutamyl groups is of the D-type.

[0108] In another embodiment, gamma-polyglutamyl oxidized pemetrexed is heptaglutamylated. In some embodiments, each of the six glutamyl groups is of the L-type. In some embodiments, each of the glutamyl groups in gamma-heptaglutamyl oxidized pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In other embodiments, at least two of the glutamyl groups in gamma-heptaglutamyl oxidized pemetrexed are of the L-type and at least one of the glutamyl groups is of the D-type.

[0109] In some embodiments, gamma-polyglutamylated pemetrexed (γPPMX) 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 groups in pemetrexed, or any range therebetween. In some embodiments, each glutamyl group in γPPMX other than the glutamyl groups of pemetrexed has a gamma bond. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in γPPMX have a gamma bond. In some embodiments, γPPMX contains L-type and D-type γ-glutamyl groups. 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 γPPMX other than the glutamyl groups of pemetrexed is of the D-type. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the glutamyl groups in γPPMX 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 γPPMX are of the D-type.

[0110] In further embodiments, gamma-polyglutamylated pemetrexed contains 20 to 100, 20 to 75, 20 to 50, 20 to 40, 20 to 30, 20 to 25 gamma-glutamyl groups, or more than 100, or any range therebetween. In some embodiments, each glutamyl group of gamma-polyglutamylated pemetrexed is of the L-type. In other embodiments, each glutamyl group of gamma-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 gamma-polyglutamylated pemetrexed are of the L-type and at least one of the glutamyl groups in gamma-polyglutamylated pemetrexed is of the D-type.

[0111] In further embodiments, the provided composition comprises gamma-polyglutamylated pemetrexed having 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 glutamyl groups having gamma-linkages. In some embodiments, the gamma-polyglutamylated pemetrexed comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-type glutamyl groups. In some embodiments, the gamma-polyglutamylated pemetrexed comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 D-type glutamyl groups. In some embodiments, the gamma-polyglutamylated pemetrexed comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-type glutamyl groups and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10 or 1-20 D-type glutamyl groups.

[0112] In some embodiments, the gamma-polyglutamylated pemetrexed composition provided herein can accept one or more additional glutamyl groups, i.e., the composition can serve as a substrate for FPGS (folylpolyglutamate synthetase). Reagents and assays for measuring the ability of the 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.

[0113] In some embodiments, the naked gamma-PPMX composition disclosed herein (e.g., gamma-PPMX not bound to a delivery carrier) is taken up by liver cells at a rate that is significantly reduced compared to the uptake rate of pemetrexed under the same physiological conditions. In some embodiments, the liver cell uptake rate of the naked gamma-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 naked gamma-PPMX composition disclosed herein from liver cells occurs at a rate that is significantly slower (e.g., less than 30%, 20%, 15%, or 10%) compared to the rate of pemetrexed.

[0114] In some embodiments, the gamma-polyglutamylated pemetrexed compositions provided herein are more cytotoxic to 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 gamma-polyglutamylated pemetrexed is hexaglutamylated pemetrexed.

[0115] In some embodiments, the gamma-polyglutamylated pemetrexed compositions provided herein have lower toxic side effects than pemetrexed. In some embodiments, the gamma-polyglutamylated pemetrexed compositions provided herein are less toxic to non-proliferating cells than pemetrexed. In some embodiments, the gamma-polyglutamylated pemetrexed compositions provided herein are less toxic to neutrophils, hepatocytes, 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 hepatocytes are AML12 hepatocytes. In some embodiments, the colon epithelial cells are CCD841 colon epithelial cells. In some embodiments, the toxicity is measured in an in vitro assay. In some embodiments, the gamma-polyglutamylated pemetrexed is hexaglutamylated pemetrexed.

[0116] In some embodiments, the gamma-polyglutamylated pemetrexed compositions provided herein have lower toxic side effects than pemetrexed. In some embodiments, the gamma-polyglutamylated pemetrexed compositions provided herein result in fewer or less severe toxic side effects than pemetrexed in in vivo assays. In some embodiments, the in vivo assays are performed in an in vivo mouse model. In some embodiments, the gamma-polyglutamylated pemetrexed compositions provided herein result in fewer or less severe hematological or liver 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 liver toxic side effects are evaluated by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the in vivo assay comprises administering a gamma-polyglutamylated pemetrexed composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the gamma-polyglutamylated pemetrexed is hexaglutamylated pemetrexed.

[0117] In some embodiments, treatment with the gamma-polyglutamyl oxidized 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 gamma-polyglutamyl oxidized pemetrexed compositions provided herein do not significantly decrease mean neutrophils, mean white blood cells, or mean platelet counts. In some embodiments, the gamma-polyglutamyl oxidized pemetrexed compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the gamma-polyglutamyl oxidized pemetrexed compositions provided herein do not significantly reduce serum albumin levels. In some embodiments, the in vivo assay involves administering the gamma-polyglutamyl oxidized pemetrexed composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the gamma-polyglutamyl oxidized pemetrexed is hexaglutamyl oxidized pemetrexed.

[0118] In some embodiments, the gamma-polyglutamyl oxidized pemetrexed composition does not contain a fluorine atom. In some embodiments, the gamma-polyglutamyl oxidized pemetrexed composition does not contain a 4-fluoroglutamyl group.

[0119] Gamma-polyglutamylated pemetrexed (αPPMX) compositions and their uses are further disclosed in U.S. Application Nos. 62 / 374,458, 15 / 675,695, 15 / 675,701, and 62 / 583,432; International Application Nos. PCT / US2017 / 046666 and PCT / US2017 / 046667, the contents of each of which are hereby incorporated by reference in their entirety.

[0120] Polyglutamylated pemetrexed analogs and derivatives The present disclosure also encompasses gamma-polyglutamylated pemetrexed derivatives and analogs. The above-described compositions and methods disclosed herein are contemplated for application to any and all known derivatives or analogs of polyglutamylated pemetrexed. In some embodiments, compositions of polyglutamylated pemetrexed analogs or derivatives prepared and used according to the disclosed compositions and methods are shown in FIGS. 1I-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 of pemetrexed is of the D-type. In some embodiments, the polyglutamylated form of pemetrexed, or polyglutamylated pemetrexed analog or derivative, is not fluorinated.

[0121] In further embodiments, the gamma-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.

[0122] A. PMX-PG synthesis The pemetrexed polyglutamate compositions provided herein are obtained by the following synthetic methods known in the art. The 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. Application Publication No. 2013 / 0165654, European Patents EP 0905128, EP 2882753, EP 0905128 and EP 2409978; International Application Publications WO 2014 / 024164, WO 2012 / 056285, WO 2008 / 021410, and WO 2001 / 14379, 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).

[0123] The addition of glutamyl residues to the glutamyl residues of pemetrexed can be carried out using synthetic methods known in the art. In some embodiments, the glutamyl residues are sequentially added to the glutamyl residues of pemetrexed. In further embodiments, the polyglutamate is added to the glutamyl residues of pemetrexed using "click chemistry" methods or other bioconjugate chemistries known to those skilled in the art.

[0124] B. Pemetrexed-PG Complex The inventors have made the surprising discovery that polyglutamine oxidized pemetrexed (γPPMX) can form a complex with other compositions, including therapeutic agents containing cytotoxic compounds such as platinum-based compounds. Accordingly, in some embodiments, the present disclosure provides a complex of γPPMX (e.g., γPPMX disclosed herein) with a therapeutic agent or a salt or acid thereof.

[0125] In some embodiments, the γPPMX / complex comprises γPPMX and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In further embodiments, the γPPMX / complex comprises a platinum-based drug such as a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, and oxaliplatin). In other embodiments, the γPPMX / complex comprises a taxane-based chemotherapeutic agent (e.g., paclitaxel and docetaxel). In other embodiments, the γPPMX / complex comprises cyclodextrin. In further embodiments, the γPPMX / complex is encapsulated in liposomes.

[0126] In some embodiments, the present disclosure provides a composition comprising a complex of γPPMX and a therapeutic agent or a salt or acid thereof. In further embodiments, the γPPMX / therapeutic agent complex comprises one or more γPPMX comprising 2 to 150, 2 to 100, 2 to 75, 2 to 50, 2 to 24, 2 to 30, 2 to 20, 2 to 19, 2 to 15, 2 to 10 or 2 to 5 glutamyl groups. In some embodiments, the γPPMX / therapeutic agent complex comprises one or more γPPMX comprising 3 to 10, 3 to 9, 3 to 8, or 3 to 7 glutamyl groups, or any range therebetween. In other embodiments, the γPPMX / therapeutic agent complex comprises one or more γPPMX comprising 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 glutamyl groups, or any range therebetween. In a particular embodiment, the complex comprises one or more γPPMX comprising 3 to 10 glutamyl groups. In further embodiments, the γPPMX / therapeutic agent complex comprises one or more γPPMX comprising 3 to 7 glutamyl groups. In further embodiments, the γPPMX / therapeutic agent complex comprises one or more γPPMX comprising 5 glutamyl groups. In another embodiment, the γPPMX / therapeutic agent complex comprises one or more γPPMX comprising 6 glutamyl groups. In some embodiments, the therapeutic agent is a cytotoxic compound or a salt or acid thereof. In further embodiments, the therapeutic agent is a chemotherapeutic agent or a salt or acid thereof. In another embodiment, the chemotherapeutic agent is a platinum-based drug. In another embodiment, the chemotherapeutic agent is a taxane-based drug. In further embodiments, the molar ratio of γPPMX / therapeutic agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of γPPMX / therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / therapeutic agent in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween.In some embodiments, the molar ratio of γPPMX / to the therapeutic agent 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 γPPMX / therapeutic agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0127] In alternative embodiments, the γPPMX complex comprises γPPMX and cyclodextrin. In some embodiments, the molar ratio of γPPMX (e.g., γPPMX salt) / cyclodextrin in the complex ranges from 1 - 20:1, or any range therebetween. In some embodiments, the molar ratio of γPPMX / cyclodextrin in the complex ranges from 1 - 10:1, or any range therebetween. In further embodiments, the molar ratio of γPPMX / cyclodextrin in the complex ranges from 2 - 8:1, or any range therebetween. In some embodiments, the molar ratio of γPPMX / 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 γPPMX / cyclodextrin in the complex ranges from 1:1 - 20, 1:1 - 10, or 1:2 - 8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / cyclodextrin is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In some embodiments, the γPPMX / cyclodextrin complex is encapsulated in liposomes (e.g., as described herein or as known in the art).

[0128] In some embodiments, the present disclosure provides a composition comprising a γPPMX / 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 γPPMX / platinum-based chemotherapeutic agent complex comprises cisplatin, carboplatin, an analogue of oxaliplatin, or salts or acids thereof. In some embodiments, the molar ratio of γPPMX / platinum-based agent in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPPMX / platinum-based agent in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPPMX / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPPMX / platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / platinum-based agent in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / platinum-based agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In some embodiments, the γPPMX / platinum-based agent complex is encapsulated in liposomes (e.g., as described herein or as known in the art).

[0129] In a further embodiment, the γPPMX / platinum-based chemotherapeutic agent complex comprises cisplatin, carboplatin, an oxaliplatin analog, or a salt or acid thereof. In some embodiments, the molar ratio of γPPMX / platinum-based analog in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPPMX / platinum-based analog in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of γPPMX / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPPMX / platinum-based analog in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / platinum-based analog 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 γPPMX / platinum-based analog is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:?20, 1:(21 to 50), or 1:>50. In a further embodiment, the γPPMX / platinum-based analog complex is encapsulated in liposomes (e.g., as described herein or as known in the art).

[0130] In a further embodiment, the present disclosure provides a complex comprising γPPMX and cisplatin or a salt or acid thereof. In some embodiments, the molar ratio of γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / cisplatin (or a salt or acid of cisplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / cisplatin (or a salt or acid of cisplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In a further embodiment, the γPPMX / cisplatin (or a salt or acid of cisplatin) complex is encapsulated in liposomes (e.g., as described herein or as known in the art).

[0131] In another embodiment, the present disclosure provides a complex comprising γPPMX and carboplatin or a salt or acid thereof. In some embodiments, the molar ratio of γPPMX / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPPMX / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPPMX / 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 γPPMX / carboplatin (or a salt or acid of carboplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In some embodiments, the molar ratio of γPPMX / carboplatin in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / carboplatin (or a salt or acid of carboplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In further embodiments, the γPPMX / carboplatin (or a salt or acid of carboplatin) complex is encapsulated in liposomes (e.g., as described herein or as known in the art).

[0132] In another embodiment, the present disclosure provides a complex comprising γPPMX and oxaliplatin or a salt or acid thereof. In some embodiments, the molar ratio of γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / oxaliplatin (or a salt or acid of oxaliplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / oxaliplatin (or a salt or acid of oxaliplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In a further embodiment, the γPPMX / oxaliplatin (or a salt or acid of oxaliplatin) complex is encapsulated in liposomes (e.g., as described herein or as known in the art).

[0133] In a further embodiment, the present disclosure provides a complex comprising a platinum-based chemotherapeutic agent (a "platinum") selected from the group consisting of γPPMX and nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platino, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or salts or acids thereof. In other embodiments, the γPPMX / platinum-based chemotherapeutic agent complex comprises nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platino, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, or an analogue of nedaplatin, or salts or acids thereof. In some embodiments, the molar ratio of γPPMX / platinum (or platinum salt or acid) in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of γPPMX / platinum (or platinum salt or acid) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of γPPMX / platinum (or platinum salt or acid) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPPMX / platinum (or platinum salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / platinum (or platinum salt or acid) 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 γPPMX / platinum (or a salt or acid of platinum) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In further embodiments, the γPPMX / platinum (or its salt or acid or analog) complex is encapsulated in liposomes (e.g., as described herein or as known in the art).

[0134] In some embodiments, the present disclosure provides a composition comprising a γPPMX / 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 γPPMX / taxane agent in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of γPPMX / taxane (or a salt or acid of taxane) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of γPPMX / 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 γPPMX / 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 other embodiments, the molar ratio of γPPMX / taxane (or a salt or acid of taxane) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / taxane (or a salt or acid of taxane) 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 a further embodiment, the γPPMX / taxane agent complex is encapsulated in liposomes (e.g., as described herein or as known in the art).

[0135] In further embodiments, the present disclosure provides a complex comprising γPPMX and paclitaxel (PTX) or a salt or acid thereof. In other embodiments, the γPPMX / taxane-based chemotherapeutic agent complex comprises an analog of paclitaxel (PTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / 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 other embodiments, the molar ratio of γPPMX / paclitaxel (or a salt or acid of paclitaxel) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / paclitaxel (or a salt or acid of paclitaxel) 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 further embodiments, the γPPMX / paclitaxel (or a salt or acid of paclitaxel) complex is encapsulated in liposomes (e.g., as described herein or as known in the art).

[0136] In further embodiments, the present disclosure provides a complex comprising γPPMX and docetaxel (DTX) or a salt or acid thereof. In other embodiments, the γPPMX / taxane-based chemotherapeutic agent complex comprises an analogue of docetaxel (DTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / docetaxel (or a salt or acid of docetaxel) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / docetaxel (or a salt or acid of docetaxel) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / docetaxel (or a salt or acid of docetaxel) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In further embodiments, the γPPMX / docetaxel (or a salt or acid of docetaxel) complex is encapsulated in liposomes (e.g., as described herein or as known in the art).

[0137] In a further embodiment, the present disclosure provides a complex comprising γPPMX and larotaxel (LTX) or a salt or acid thereof. In other embodiments, the γPPMX / taxane-based chemotherapeutic agent complex comprises larotaxel (LTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPPMX / 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 γPPMX / 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 γPPMX / 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 γPPMX / 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 other embodiments, the molar ratio of γPPMX / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPPMX / larotaxel (or a salt or acid of larotaxel) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In a further embodiment, the γPPMX / larotaxel (or a salt or acid of larotaxel) complex is encapsulated in liposomes (e.g., as described herein or as known in the art).

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

[0139] In a further embodiment, the present disclosure provides a complex comprising γPPMX 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 γPPMX and pemetrexed (PMX), or a salt or acid thereof. In some embodiments, the present disclosure provides a complex comprising γPPMX and an antimetabolite selected from the group consisting of gemcitabine, fluorouracil, capecitabine, folic acid antimetabolites (e.g., methotrexate, raltitrexed), tegafur, cytosine arabinoside, thioguanine, 5-azacitidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, and one or more pharmaceutically acceptable salts or acids, or derivatives thereof. In some embodiments, the molar ratio of γPPMX / 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 γPPMX / antimetabolite (or salt or acid of the antimetabolite, or prodrug) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of γPPMX / 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 γPPMX / antimetabolite (or salt or acid of the antimetabolite) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPPMX / antimetabolite (or salt or acid of the antimetabolite) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween.In some embodiments, the molar ratio of γPPMX / antimetabolite (or a salt or acid of the antimetabolite) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In further embodiments, the γPPMX / antimetabolite (or a salt or acid of the antimetabolite) complex is encapsulated in liposomes (e.g., as described herein or as known in the art).

[0140] In further embodiments, the present disclosure provides a complex of γPPMX (e.g., γPPMX as disclosed herein) and cyclodextrin. Cyclodextrin (CD) is a group of cyclic oligosaccharides that have been shown to improve the physicochemical properties of many drugs through complex formation. CD is a cyclic oligosaccharide composed of several D-glucose units linked by α-(1,4) bonds. This cyclic structure provides a hydrophobic internal cavity and gives CD a truncated cone shape. Many hydroxyl groups are located on the ends of the ring, which makes CD both lipophilic and water-soluble. As a result, CD can form complexes with a wide variety of hydrophobic agents, thereby changing the physicochemical properties of these complexed agents.

[0141] 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 positions 2 and 3 and a primary hydroxyl group at position 6. The terms "parent", "undenivatized", or "inactive" cyclodextrin refer to the basic formula C6H containing D-glucopyranoside units. 12Cyclodextrin 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 in the inner phase of the cyclodextrin is said to be "complexed" with the cyclodextrin or to form a complex (inclusion complex) with the cyclodextrin.

[0142] As used herein, there are no special restrictions on the cyclodextrin component of the γPPMX / cyclodextrin complex as long as the cyclodextrin can form a complex with γPPMX. 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 γPPMX and / or liposome encapsulates.

[0143] Modification of the hydroxyl groups of cyclodextrin, such as hydroxyl groups directed from the inner phase of the cyclodextrin using ionizable chemical groups, is known to facilitate the addition of cyclodextrin and therapeutic agents complexed with cyclodextrin. In some embodiments, the cyclodextrin of the γPPMX / 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 a charged group itself or an organic moiety substituted with one or more charged moieties (e.g., a C1-C6 alkyl or C1-C6 alkyl ether moiety).

[0144] In some embodiments, the "ionizable" or "charged" portion of the CD derivative is weakly ionizable. The weakly ionizable portion is a weakly basic or weakly acidic portion. The weakly basic functional group (W) has a pKa by CH3-W in the range of about 6.0 to 9.0, 6.5 to 8.5, 7.0 to 8.0, 7.5 to 8.0, and any range therebetween (including the endpoints). Similarly, the weakly acidic functional group (X) has a logarithmically recorded dissociation constant (pKa) by CH3-X in the range of about 3.0 to 7.0, 4.0 to 6.5, 4.5 to 6.5, 5.0 to 6.0, 5.0 to 5.5, and any range therebetween (including the endpoints). Representative anionic portions include, but are not limited to, carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkyl ether, sulfate carbonate, thiocarbonate, dithiocarbonate, phosphate, phosphonate, sulfonate, nitrate, and borate groups. Representative cationic portions include, but are not limited to, amino, guanidine, and quaternary ammonium groups.

[0145] 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.

[0146] 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" herein means a derivatized cyclodextrin having ionizable groups with both anionic and cationic characteristics, (a) at least one, optionally both, of the cationic and anionic amphiphiles being chargeable and having at least one charge group with a pK between 4 and 8 to 8.5, (b) the cationic charge being dominant at pH 4, and (c) the anionic charge being dominant at pH 8 to 8.5.

[0147] In some embodiments, "ionizable" or "charged" derivatized cyclodextrins are generally polyionic, amphiphilic, or otherwise, weakly ionizable (e.g., having a pKai of about 4.0 - 8.5, 4.5 - 8.0, 5.0 - 7.5, 5.5 - 7.5, 6.0 - 6.5, and any range therebetween (including both ends)).

[0148] Any one, some, or all of the hydroxyl groups of any one, some, or all of the γ-D-glucopyranoside units of any cyclodextrin can be modified to an ionizable chemical group as described herein. Since each cyclodextrin hydroxyl group has different chemical reactivity, the reaction with the modifying moiety can produce a mixture of positional and optical isomers. Alternatively, with specific chemistry, the reaction can be made to form a homogeneous product of pre-modified α-D-glucopyranoside units.

[0149] Aggregate substitution occurring in cyclodextrin derivatives in a mixture is described by a term called degree of substitution. For example, 6-ethylenediamino-β-cyclodextrin having a degree of substitution of 7 would be composed of the distribution of isomers of 6-ethylenediamino-β-cyclodextrin where the number of ethylenediamino groups per 6-ethylenediamino-β-cyclodextrin molecule is 7. The degree of substitution of a cyclodextrin derivative mixture can be measured routinely using mass spectrometry or nuclear magnetic resonance spectroscopy.

[0150] In one embodiment, at least one hydroxyl moiety oriented in the opposite direction from within the cyclodextrin is replaced by an ionizable chemical group. For example, at least one of the γ-D-glucopyranoside units of C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and all three of C2-C3-C6 hydroxyls is replaced by an ionizable chemical group. Any such combination of hydroxyls can be combined with any of the degrees of substitution described herein, in addition to being able to similarly combine at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, up to all of the gamma D-glucopyranoside units in the modified cyclodextrin. One such derivative is sulfalkyl ether cyclodextrin (SAE-CD). The sulfobutyl ether derivative of beta cyclodextrin (SBE-β-CD) has been shown to have significantly improved water solubility compared to the parent cyclodextrin.

[0151] 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 was replaced by a carboxythioacetate ether bond and hydroxybutenyl-β-CD. Alternative cyclodextrins include 2,6-di-O-methyl-β-CD (DIMEB), 2-hydroxylpropyl-3-cyclodextrin (HP-β-CD), randomly methylated-β-cyclodextrin (RAMEB), sulfobutyl ether β-cyclodextrin (SBE-β-CD), and sulfobutyl ether-γ-cyclodextrin (SBEγCD), sulfobutylated-β-cyclodextrin sodium salt, (2-hydroxypropyl)-gamma cyclodextrin, (2-hydroxypropyl)-beta-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, 2,6-di-O-methyl)-β-cyclodextrin (DIMEB-50 heptakis), 2,3,6-tri-O-methyl)-β-cyclodextrin (TRIMEB heptakis), methyl-β-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-γ-cyclodextrin.

[0152] In some embodiments, the cyclodextrin(s) have high solubility in water to facilitate capture of a greater amount of cyclodextrin in the liposomal inner phase. In some embodiments, the aqueous solubility of the cyclodextrin is at least 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, or more. In some embodiments, the water solubility of the cyclodextrin is in the range of 10 - 150 mg / mL, 20 - 100 mg / mL, 20 - 75 mg / mL, and within any range (including both ends) therebetween.

[0153] In some embodiments, a large binding constant between cyclodextrin and γPPMX 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.

[0154] In some embodiments, the cyclodextrin of the γPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is not derivatized.

[0155] In some embodiments, the cyclodextrin of the γPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is derivatized. In further embodiments, the cyclodextrin derivative of the complex has the structure of Formula I:

Chemical Formula

[0156] In some embodiments, the cyclodextrin derivative of the γPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is of formula II:

Chemical formula

[0157] In some embodiments, the cyclodextrin derivatives of the γPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex are the cyclodextrins disclosed in U.S. Patent Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; and International Publication No. 02005 / 117911. The content of each of these patent documents is hereby incorporated by reference into this specification preferentially.

[0158] In some embodiments, the cyclodextrin derivatives of the γPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex are sulfalkyl 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). The preparation methods of sulfobutyl ether-3-cyclodextrin and other sulfalkyl ether cyclodextrins are known in the art.

[0159] In some embodiments, the cyclodextrin derivative of the γPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is of Formula III:

Chemical formula

[0160] In a further embodiment, the γPPMX / 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).

[0161] III. γPPMX Delivery Carrier In alternative embodiments, the present disclosure provides γPPMX delivery systems and their use for delivering the payload of γPPMX to cells (one or more) in vitro or in vivo. In some embodiments, γPPMX 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-γPPMX 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.

[0162] A. Liposomes In some embodiments, the present disclosure provides a liposomal composition comprising liposomes encapsulating (i.e., filled with) gamma polyglutamylated pemetrexed (e.g., γPPMX as disclosed herein). In some embodiments, the liposomes in the liposomal composition comprise γPPMX comprising 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups (including the glutamyl groups in pemetrexed). In some embodiments, the gamma polyglutamylated pemetrexed in Lp-γPPMX comprises two or more L-type glutamyl groups. In other embodiments, the gamma polyglutamylated pemetrexed in Lp-γPPMX comprises a D-type glutamyl group. In further embodiments, the gamma polyglutamylated pemetrexed in Lp-γPPMX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the gamma polyglutamylated pemetrexed in Lp-γPPMX comprises two or more glutamyl groups having gamma carboxyl bonds. In some embodiments, the liposomal composition comprises liposomes comprising γ-pentaglutamylated PMX. In further embodiments, the liposomes comprise L-γ-pentaglutamylated PMX, D-γ-pentaglutamylated PMX, or L- and D-γ-pentaglutamylated PMX. In some embodiments, the liposomal composition comprises liposomes (Lp-γPPMX) comprising γ-hexaglutamylated PMX. In further embodiments, the liposomes comprise L-γ-hexaglutamylated PMX, D-γ-hexaglutamylated PMX, or L- and D-γ-hexaglutamylated 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-γPPMX composition is not pegylated. In some embodiments, the Lp-γPPMX composition is not targeted (NTLp-γPPMX). In other embodiments, the Lp-γPPMX composition is targeted (TLp-γPPMX). In some embodiments, the liposomal composition comprises liposomes having a diameter in the range of 20 nm to 500 nm, or any range therebetween.In some embodiments, the liposome composition comprises liposomes having a diameter in the range of 20 nm to 400 nm, or any range therebetween. In some embodiments, the liposome composition comprises liposomes having a diameter in the range of 20 nm to 300 nm, or any range therebetween. In some embodiments, the liposome composition comprises liposomes having a diameter in the range of 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposome composition comprises liposomes having a diameter in the range of 20 nm to 150 nm, or any range therebetween. In further embodiments, the liposome composition comprises liposomes having a diameter in the range of 80 nm to 120 nm, or any range therebetween. In further embodiments, 30 to 70%, 30 to 60%, or 30 to 50% w / w, or any range therebetween of gamma-polyglutamylated pemetrexed is encapsulated (enclosed) in Lp-γPPMX during the liposome preparation process. In some embodiments, the Lp-γPPMX composition comprises 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 gamma-polyglutamylated PMX. 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 gamma-polyglutamylated pemetrexed is encapsulated in Lp-γPPMX during the liposome preparation process.

[0163] In some embodiments, the liposomes provided further comprise an immunostimulant, a detectable marker, or both, disposed on the outer surface of the liposome. The immunostimulant or detectable marker can be ionically or covalently bound to the outer surface of the liposome, optionally including binding to the steric stabilizer component of the liposome.

[0164] 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 are, for example, Freund's incomplete adjuvant and complete adjuvant (Difco Laboratories, Detroit, Mich.); Merck adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, PA.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron, or zinc; insoluble suspensions of acylated tyrosine; acylated saccharides; polysaccharides derivatized cationically or anionicly; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; IFN alpha, IFN gamma, FLT3 ligand; and immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3) and are commercially available. Cytokines such as GM-CSF, interleukin 2, 7, 12, and 15, and other similar growth factors can also be used as adjuvants. In a preferred embodiment, the immunostimulant can be at least one selected from the group consisting of fluorescein, DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan. In a further preferred embodiment, the immunostimulant is a toll-like receptor (TLR) regulator. In a further embodiment, the toll-like receptor (TLR) regulator is one or more of oxidized low density lipoprotein (e.g., OXPC, PGPC), eritoran lipid (e.g., E5564), and resolvin.In some embodiments, the liposome contains fluorescein isothiocyanate (FITC), which, based on the inventors' experiments, surprisingly functions as both an immunostimulant and a detectable marker.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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 (e.g., RQIKIWFQNRRMKWKKRKKRRQR RR (SEQ ID NO: 1), RKKRRXR RRGC, wherein X is any natural or unnatural amino acid (SEQ ID NO: 2), CCGCCAAGAAGCG (SEQ ID NO: 3), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO: 4), ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTC ATCCGTCAGCTC (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.

[0169] In some embodiments, the liposomes in the provided liposome composition comprise a mitochondrial permeabilizing agent selected from the group consisting of guanidine-rich peptides, tetraguanidium, triguanidium, diguanidium, monoguanidium, guanidine-rich polycarbamate, beta-oligoarginine, proline-rich dendrimers, and phosphonium salts (e.g., methyltriphenylphosphonium and / or tetraphenylphosphonium).

[0170] In some embodiments, the liposomes in the provided liposome composition comprise sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes comprise sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyl octaarginine sphingomyelin and stearyl octaarginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyl octaarginine sphingomyelin and stearyl octaarginine in a molar ratio of 9:2:1. In some embodiments, the liposomes comprise the MITO-Porter® system or a variant thereof.

[0171] In some embodiments, the liposomes in the provided liposome composition include agents such as membrane permeabilizing agents that facilitate the delivery of the liposomes across the cell membrane and confer the ability to bypass the harsh environment of the endocytic pathway and lysosomes to the liposomes. Membrane permeabilizing agents are known in the art and are routinely used and applicable to the manufacture and use of the provided liposome composition. In some embodiments, the membrane 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 group consisting of: RKKRRQRRR (SEQ ID NO: 7), GRKKRRQRRRTPQ (SEQ ID NO: 8), YGRKKRRQRRR (SEQ ID NO: 9), AAVALLPAVLLALLA (SEQ ID NO: 10), MGLGLHLLVLAAALQ (SEQ ID NO: 11), GALFLGFLGAAGSTM (SEQ ID NO: 12), AGYLLGKINLKALAALAKKIL (SEQ ID NO: 13), RVIRVWFQNKRCKDKK (SEQ ID NO: 14), RQIKIWFQNRRMKWKK (SEQ ID NO: 15), GLFEAIAGFIENGWEGMIDG (SEQ ID NO: 16), GWTLNSAGYLLGKIN (SEQ ID NO: 17), RSQSRSRYYRQRQRS (SEQ ID NO: 18), LAIPEQEY (SEQ ID NO: 19), LGIAEQEY (SEQ ID NO: 20), LGIPAQEY (SEQ ID NO: 21), LGIPEAEY (SEQ ID NO: 22), LGIPEQAY (SEQ ID NO: 23), LGIAEAEY (SEQ ID NO: 24), LGIPEAAY (SEQ ID NO: 25), LGIAEQAY (SEQ ID NO: 26), LGIAEAAY (SEQ ID NO: 27), LLIILRRRIRKQAHAHSK (SEQ ID NO: 28), LKALAALAKKIL (SEQ ID NO: 29), KLALKLALKALKAALKLA (SEQ ID NO: 30), KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 31), DHQLNPAF (SEQ ID NO: 32), DPKGDPKG (SEQ ID NO: 33), VTVTVTVTVTGKGDPKPD (SEQ ID NO: 34), RQIKIWFQNRRMKWKK (SEQ ID NO: 35), GRKKRRQRRRPPQ (SEQ ID NO: 36), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 37), GRKKRRQRRR (SEQ ID NO: 38), RRRRRRR (SEQ ID NO: 39), RRRRRRRR (SEQ ID NO: 40), RRRRRRRRR (SEQ ID NO: 41), RRRRRRRRRR (SEQ ID NO: 42), RRRRRRRRRRR (SEQ ID NO: 43), and YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG(R)n (wherein n = 2 to 15 R in L- and / or D-form) (SEQ ID NO: 44), or cell permeable fragments thereof.

[0172] As discussed above, liposomes can contain steric stabilizers that can extend their lifespan in circulation. For these embodiments incorporating steric stabilizers, the steric stabilizer can be at least one member selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymers; 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 have any structure such as linear, branched, star or comb structures and are commercially available.

[0173] In some embodiments, the liposomal composition comprises pegylated liposomes (PLp-γPPMX). In some embodiments, the pegylated liposomes in the liposomal composition comprise γPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the gamma-polyglutamylated pemetrexed in Lp-γPPMX contains 2 or more L-type glutamyl groups. In other embodiments, the gamma-polyglutamylated pemetrexed in Lp-γPPMX contains D-type glutamyl groups. In further embodiments, the gamma-polyglutamylated pemetrexed in Lp-γPPMX contains D-type glutamyl groups and 2 or more L-type glutamyl groups. In some embodiments, the liposomal composition comprises pegylated liposomes containing γ-pentaglutamylated PMX. In further embodiments, the liposomes comprise L-γ-pentaglutamylated PMX, D-γ-pentaglutamylated PMX, or L- and D-γ-pentaglutamylated PMX. In some embodiments, the liposomal composition comprises pegylated liposomes containing γ-hexaglutamylated PMX. In further embodiments, the liposomes comprise L-γ-hexaglutamylated PMX, D-γ-hexaglutamylated PMX, or L- and D-γ-hexaglutamylated PMX. In some embodiments, the liposomal composition comprises pegylated liposomes that are anionic or neutral. In some embodiments, the liposomal composition comprises pegylated liposomes that are cationic. In some embodiments, the PLp-γPPMX composition is not targeted (NTPLp-γPPMX). In other embodiments, the PLp-γPPMX composition is targeted (TPLp-γPPMX). In some embodiments, the liposomal composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% (w / w) of gamma-polyglutamylated pemetrexed.In some embodiments, the liposome composition comprises pegylated liposomes containing at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% (w / w) of gamma-polyglutamylated pemetrexed, which is encapsulated (enclosed) in PLp-γPPMX during the liposome preparation process. 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 400 nm. In some embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 20 nm to 300 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.

[0174] In some embodiments, more than 70%, 80% or 90% of the polyglutamylated pemetrexed in the provided liposome composition is pentaglutamylated. In some embodiments, more than 70%, 80% or 90% of the polyglutamylated pemetrexed in the provided composition is hexaglutamylated. In some embodiments, more than 70%, 80% or 90% of the polyglutamylated pemetrexed in the composition has 4 to 10, 4 to 6, or more than 5 gamma-glutamyl groups.

[0175] In some embodiments, the gamma-polyglutamylated pemetrexed composition (e.g., delivery carriers such as gamma-polyglutamate and liposomes containing polyglutamate) is in an aqueous solution. In some embodiments, the γPPMK composition is in the form of a liposome composition, per square meter of body surface area (m 2)It is administered at a dose of 0.005 to 5000 mg of γPPMX per unit area, or any range therebetween. In a further embodiment, the γPPMX composition is administered as a liposomal composition at a dose of 0.1 to 1000 mg of γPPMX per square meter of body surface area, or any range therebetween.

[0176] (1) Liposomal composition The lipids and other components of the liposomes contained in the liposomal composition can be any lipid, combination and ratio of lipids, or combination of lipids and other liposomal components and their respective ratios known in the art. However, without limitation, it will be understood by those skilled in the art that the liposomal encapsulation of any particular drug, such as gamma polyglutamyl 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 separated from the outer medium by one or more lipid bilayers, or any microcapsule structure having a semipermeable membrane with a lipophilic central portion that the membrane isolates the interior. The lipid bilayer can be any amphiphilic molecule having a hydrophilic moiety (hydrophilic moiety hydrophilic moiety) and a hydrophobic moiety (hydrophobic moiety). Usually, the amphiphilic molecules in the bilayer are arranged in a two-dimensional sheet, where the hydrophobic moieties face the inside of the sheet, while the hydrophilic moieties face the outside. The amphiphilic molecules forming the liposomes provided can be any known or hereafter discovered amphiphilic molecules (e.g., synthetic or naturally derived 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".

[0177] The liposome composition formulations provided herein can be in liquid or dry forms such as dry powder or dry cake. The dry powder or dry cake can be subjected to primary drying, for example, under lyophilization conditions, or can be subjected to only primary drying or both primary and secondary drying. In the dry form, the powder or cake can have, for example, 1% to 6% moisture, such as 2% to 5% moisture or 2% to 4% moisture. An example of the drying method is lyophilization (also called freeze-drying or cyrodessication). Any of the compositions and methods of the present disclosure can include liposomes, lyophilized liposomes, or liposomes reconstituted from lyophilized liposomes. In some embodiments, the disclosed compositions and methods include one or more lyoprotectants or cryoprotective substances. These protectants are typically saccharides (monosaccharides, disaccharides, and polysaccharides), polyhydric alcohols, and derivatives thereof, polyhydroxy compounds such as 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 the liposome, inside the liposome, or both outside and inside the liposome.

[0178] 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)), and others, occupy the space directly adjacent to the liposome surface and exclude other polymers from this space. As a result, access and binding of plasma opsonins to the liposome surface are hindered, thus suppressing the interaction of such liposomes with macrophages or any other removal mechanism, and extending the lifespan of the liposomes in circulation. In some embodiments, the steric stabilizer or group of steric stabilizers is PEG or a combination containing PEG. In further embodiments, the steric stabilizer is PEG or a combination containing PEG with a number average molecular weight (Mn) in the range of 200 to 5000 daltons. These PEGs can have any structure, such as linear, branched, star-shaped, or comb-shaped structures, and are commercially available.

[0179] 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.

[0180] 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, liposomes containing pemetrexed gamma polyglutamate oxidation 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.

[0181] In some embodiments, cationic lipids are used to create cationic liposomes, which are commonly used as gene delivery agents. The positive charges on the cationic liposomes enable interaction with the negative charges on the cell surface. After binding of the cationic liposomes to the cells, the liposomes are transported into the interior of the cells by endocytosis.

[0182] In some preferred embodiments, neutral to anionic liposomes are used. In a preferred embodiment, anionic liposomes are used. For example, by using a mixture of neutral lipids such as HSPC and anionic lipids such as PEG-DSPE, anionic liposomes are formed, which have a low likelihood of non-specifically 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 gamma antibody, folate receptor beta antibody, and / or folate receptor delta antibody.

[0183] As an example, at least one (or several) lipids are amphiphilic lipids defined as having hydrophilic and hydrophobic moieties (usually a hydrophilic head and a hydrophobic tail). The hydrophobic moiety usually faces the hydrophobic phase (e.g., within the bilayer), while the hydrophilic moiety usually faces the aqueous phase (e.g., outside the bilayer). The hydrophilic moiety can include polar or charged groups such as carbohydrates, phosphates, carboxylic acids, sulfates, aminos, sulfhydryls, nitros, hydroxyls, and other similar groups. The hydrophobic moiety can include nonpolar groups including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted by one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids, and sphingolipids.

[0184] 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.

[0185] 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 a selected pH. At physiological pH, such lipids include, for example, dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol. Examples of zwitterionic lipids include, but are not limited to, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS). Anionic lipids are negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and anionic modifying groups attached to neutral lipids.

[0186] 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), palmitoyloleyolphosphatidylglycerol (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.

[0187] Liposomes can be constructed using liposomal components (also referred to as liposome components) known in the art, using any liposome assembly method. Liposome 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.

[0188] In some embodiments, the γPPMX compositions provided herein are formulated in liposomes comprising a cationic lipid. In one embodiment, without limitation, the cationic lipid is selected from the group consisting of 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. 2010 / 0036115, and 2012 / 0202871. Each of these patent documents is hereby incorporated by reference in its entirety. In another embodiment, the cationic lipid can be selected from, without limitation, 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, without limitation, Formulas CLI - CLXXIX of International Publication No. WO 2008 / 103276, Formulas CLI - CLXXIX of U.S. Pat. No. 7,893,302, Formulas CLI - CLXXXXII of U.S. Pat. No. 7,404,969, and Formulas I - VI of U.S. Patent Application Publication No. 2010 / 0036115. Each of these respective patent documents is hereby incorporated by reference in its entirety. By way of non-limiting example, the cationic lipid can be selected from the following: (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethyl-hexacosa-17,20-dien-9-amine, (1Z,19Z)-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-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicos-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacosa-20-en-10-amine, (15Z)-N,N-dimethyleptacosa-15-en-10-amine, (14Z)-N,N-dimethylnonacosa-14-en-10-amine, (17Z)-N,N-dimethylnonacosa-17-en-10-amine, (24Z)-N,N-dimethyltritriaconta-24-en-10-amine, (20Z)-N,N-dimethylnonacosa-20-en-10-amine, (22Z)-N,N-dimethylhentriaconta-22-en-10-amine, (16Z)-N,N-dimethylpentacosa-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-[(R1S,2R)-2-octylcyclopropyl]heneicosan-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecane-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecane-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecane-8-amine, R--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, S--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-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]propan-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-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-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.,

[0189] In one embodiment, the lipid can be a cleavable lipid such as those described in International Publication No. WO 2012 / 170889, which is hereby incorporated by reference in its entirety.,

[0190] The cationic lipids can be synthesized routinely using methods known in the art and / or as described in International Publication Nos. WO 2012 / 040184, WO 2011 / 153120, WO 2011 / 149733, WO 2011 / 090965, WO 2011 / 1043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, WO 2012 / 044638, WO 2010 / 080724 and WO 2010 / 21865, which are hereby incorporated by reference in their entirety.,

[0191] The lipid derivative can include, for example, at least one or more steric stabilizers and / or the binding (preferably covalent binding) of a functional group to the liposome component, after which the steric stabilizer and / or the functional group should be regarded as part of the liposome component. The functional group includes groups that can be used to bind the liposome component to another moiety such as a protein. Such functional groups include at least maleimide. These steric stabilizers include polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymers; and at least one selected from the group consisting of polyvinyl alcohol.

[0192] In some embodiments, the γPPMX 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, γPPMX is formulated in a lipid-polycation complex, which further includes neutral lipids such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0193] The components of the liposome can include any molecule that binds thereto (e.g., a chemical / drug / reagent / protein), and in some embodiments, the components of the provided liposome include at least members selected from the group consisting of DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide. In some embodiments, the components of the provided liposome include DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide. In preferred embodiments, the liposome components constituting the liposome include DSPE; DSPE-FITC; DSPE-maleimide; cholesterol; and HSPC.

[0194] In further embodiments, the liposomes of the liposome compositions provided herein include oxidized phospholipids. In some embodiments, the liposome includes an oxidized phospholipid that is a member selected from the group consisting of phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, and 1-palmitoyl-2-arachidonoyl-sn-glycero-2-phosphate. In some embodiments, the phospholipid has an unsaturated bond. In some embodiments, the phospholipid is arachidonic acid containing the phospholipid. In further embodiments, the phospholipid is sn-2-oxygenated. In further embodiments, the phospholipid is not fragmented.

[0195] In some embodiments, the liposomes of the disclosed liposome compositions comprise oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC). As used herein, the term "oxPAPC" means a lipid produced by the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), thereby resulting in a mixture of oxidized phospholipids containing fragmented or full-length oxygenated sn-2 residues. Characteristic oxidized fragmentation species contain 5-carbon sn-2 residues with omega aldehyde or omega carboxyl groups. Oxidation of the arachidonic acid residue also produces phospholipids containing esterified isoprostanes. OxPAPC includes, among many oxidized products present in oxPAPC, in particular, the HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC species. In further embodiments, oxPAPC is an epoxyisoprostane-containing phospholipid. In further embodiments, oxPAPC is 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2-(epoxycyclopentenone)-sn-glycero-3-phosphocholine (PECPC) and / or 1-palmitoyl-2-(epoxy-isoprostane E2)-sn-glycero-4-phosphocholine (PEIPC). In some embodiments, the phospholipid has an unsaturated bond. In some embodiments, the phospholipid is arachidonic acid containing the phospholipid. In further embodiments, the phospholipid is sn-2-oxygenated. In further embodiments, the phospholipid is not fragmented.

[0196] In some embodiments, the liposomal gamma-polyglutamyl oxidized pemetrexed composition is pegylated (i.e., pegylated liposomal gamma-polyglutamyl oxidized (e.g., pentaglutamyl oxidized or hexaglutamyl oxidized) folate antagonist (PLp-γPPMX or TPLp-γPPMX)). In some embodiments, PLp-γPPMX or TPLp-γPPMX is water-soluble. That is, PLp-γPPMX or TPLp-γPPMX is in the form of an aqueous solution.

[0197] In some embodiments, the liposomes of the disclosed liposome compositions comprise a lipid selected from the group consisting of: 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.

[0198] In some embodiments, the pH of the solution comprising the liposome composition is pH 5-8, or pH 2-6, 2-8, or any range therebetween. In some embodiments, the pH of the solution comprising the liposome composition is pH 5-8, or any range therebetween. In some embodiments, the pH of the solution comprising the liposome composition is pH 6-7, or any range therebetween. In some embodiments, the pH of the solution comprising the liposome composition is 6-7.5, 6.5-7.5, 6.7-7.5, or 6.3-7.0, or any range therebetween.

[0199] In some embodiments, at least one component of the liposomal lipid bilayer is functionalized (or reactive). As used herein, a functionalized component is a component that includes a reactive group that can be used to crosslink reagents and moieties to the lipid. When a lipid is functionalized, any liposomes it forms are also functionalized. In some embodiments, the reactive group is one that reacts with a crosslinking agent (or other moiety) to form a crosslink. The reactive groups in the liposomal lipid bilayer are positioned somewhere on the lipid such that upon contact with a crosslinking agent, crosslinking with another moiety (e.g., a steric stabilizer or targeting moiety) is enabled. In some embodiments, the reactive group is in the head group of the lipid, such as a phospholipid. In some embodiments, the reactive group is a maleimide group. Maleimide groups can crosslink with each other in the presence of a dithiol crosslinking agent such as, but not limited to, dithiothreitol (DTT).

[0200] It should be understood that the use of other functionalized lipids, other reactive groups, and other crosslinking agents beyond those described above is further contemplated. In addition to maleimide groups, other examples of reactive groups contemplated include, but are not limited to, other thiol-reactive groups, amino groups such as primary or secondary amines, carboxyl groups, hydroxyl groups, aldehyde groups, alkyne groups, azide groups, carbonyl groups, haloacetyl (e.g., iodoacetyl) groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydryl groups, and pyridyl disulfide groups.

[0201] Functionalized and non-functionalized lipids are available from many commercial sources such as Avanti Polar Lipids (Alabaster, AL) and Lipoid LLC (Newark, NJ).

[0202] (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 gamma-polyglutamyl oxidized pemetrexed provided herein. In further embodiments, the internal space of the liposome contains an isotonic agent. In some embodiments. In some embodiments, the concentration (wt%) of the isotonic agent is 0.1 - 20%, 1 - 20%, 0.5 - 15%, 1 - 15% or 1 - 50%, or any range therebetween. In some embodiments, the internal space of the liposome contains a sugar (e.g., trehalose, maltose, sucrose, lactose, mannose, mannitol, glycerol, dextrose, fructose, etc.). In further embodiments, the concentration (wt%) of the sugar is 0.1 - 20%, 1 - 20%, 0.5 - 15%, 1 - 15% or 1 - 50%, or any range therebetween. In some embodiments, the pH of the internal space of the liposome is pH 2 - 8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 5 - 8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 6 - 7, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is 6 - 7.5, 6.5 - 7.5, 6.7 - 7.5, or 6.3 - 7.0, or any range therebetween. In some embodiments, the internal space contains a buffer. In further embodiments, the buffer is a buffer selected from the group consisting of 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 a further embodiment, the buffer has a concentration of 5 to 200 mM, 15 to 200 mM, 5 to 100 mM, 15 to 100 mM, 5 to 50 mM, 15 to 50 mM, 5 to 25 mM, 5 to 20 mM, 5 to 15 mM, or any range therebetween. In some embodiments, the buffer is HEPES at a concentration of 15 to 200 mM, or any range therebetween. In some embodiments, the buffer is citrate at a concentration of 15 to 200 mM, or any range therebetween. In some embodiments, the buffer is sodium phosphate at a concentration of 15 to 200 mM, or any range therebetween. In some embodiments, the internal space of the liposome contains a total concentration of sodium acetate and calcium acetate of 5 mM to 500 mM, or 50 mM to 500 mM, or any range therebetween.

[0203] In some embodiments, the internal space of the liposome contains trehalose. In further embodiments, the concentration (wt%) of trehalose is 0.1 - 20%, 1 - 20%, 0.5 - 15%, 1 - 15%, or 5 - 20%, or any range therebetween. In further embodiments, the concentration (wt%) of trehalose is 1 - 15%, or any range therebetween. In additional embodiments, trehalose is about 5% - 20% (wt%) trehalose, or any combination of one or more lyoprotectants or cryoprotective substances is present at a total concentration of 5% - 20%. In some embodiments, the pH of the solution containing the liposome composition is 6 - 7.5, 6.5 - 7.5, 6.7 - 7.5, or 6.3 - 7.0, or any range therebetween. In some embodiments, the internal space contains a buffer solution. In some embodiments, the buffer solution is selected from the group consisting of HEPES, citrate, or sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer solution is HEPES. In some embodiments, the buffer solution is citrate. In some embodiments, the buffer solution is sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer solution is at a concentration of 15 - 200 mM, or any range therebetween. In further embodiments, the buffer solution is at a concentration of 5 - 200 mM, 15 - 200 mM, 5 - 100 mM, 15 - 100 mM, 5 - 50 mM, 15 - 50 mM, 5 - 25 mM, 5 - 20 mM, 5 - 15 mM, or any range therebetween. In some embodiments, the buffer solution is HEPES at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the buffer solution is citrate at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the buffer solution is sodium phosphate at a concentration of 15 - 200 mM, or any range therebetween. In further embodiments, the internal space of the liposome contains sodium acetate and / or calcium acetate.In some embodiments, the internal space of the liposome contains a total concentration of sodium acetate and calcium acetate in the range of 5 mM to 500 mM, or 50 mM to 500 mM, or any range therebetween.

[0204] In some embodiments, the internal space of the liposome contains dextrose. In further embodiments, the concentration (weight %) of dextrose is 0.1 - 20%, 1 - 20%, 0.5 - 15%, 1% - 15%, or 5 - 20%, or any range therebetween. In still further embodiments, the concentration (weight %) of dextrose is 1 - 15%, or any range therebetween. In additional embodiments, dextrose is present at a dextrose concentration of about 5% - 20% (weight %), or any combination of one or more 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 the group consisting of 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.

[0205] In further embodiments, the present disclosure provides a liposome composition comprising liposomes encapsulating (filling) gamma polyglutamylated pemetrexed (e.g., γPPMX disclosed herein). In some embodiments, the liposomes in the liposome composition contain γPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups (including the glutamyl groups of pemetrexed). In some embodiments, the gamma polyglutamylated pemetrexed in Lp-γPPMX contains two or more L-type glutamyl groups. In other embodiments, the gamma polyglutamylated pemetrexed in Lp-γPPMX contains a D-type glutamyl group. In further embodiments, the gamma polyglutamylated pemetrexed in Lp-γPPMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the gamma polyglutamylated pemetrexed in Lp-γPPMX contains two or more glutamyl groups having a gamma carboxyl bond. In some embodiments, the liposome composition comprises liposomes containing γ-pentaglutamylated PMX. In further embodiments, the liposome contains L-γ-pentaglutamylated PMX, D-γ-pentaglutamylated PMX, or L- and D-γ-pentaglutamylated PMX. In some embodiments, the liposome composition comprises liposomes containing γ-hexaglutamylated PMX (Lp-γPPMX). In further embodiments, the liposome contains L-γ-hexaglutamylated PMX, D-γ-hexaglutamylated PMX, or L- and D-γ-hexaglutamylated PMX.

[0206] In some embodiments, the targeted pegylated liposome gamma-polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) pemetrexed comprises a medium comprising liposomes that contain an internal space; aqueous gamma-polyglutamylated 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 contain one or more of the lyoprotectants or cryoprotective substances listed above. In some embodiments, the cryoprotective substance is mannitol, trehalose, sorbitol, or sucrose.

[0207] In some embodiments, gamma-polyglutamyl oxidized pemetrexed encapsulated liposomes (i.e., Lp-γPPMX including PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) have an internal space containing less than 500,000 or less than 200,000 gamma-polyglutamyl oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains from 10 to 100,000 or any range therebetween of gamma-polyglutamyl oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains from 10,000 to 100,000 or any range therebetween of gamma-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 gamma-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 gamma-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 gamma-polyglutamyl oxidized pemetrexed molecules. In some embodiments, the liposome is targeted and not pegylated (TLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma-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 gamma-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 gamma-polyglutamyl oxidized pemetrexed molecules. In some embodiments, the liposome is not targeted and not pegylated (NTLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma-polyglutamyl 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 gamma-polyglutamylated 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 gamma-polyglutamylated pemetrexed molecules.

[0208] In some embodiments, the liposome encapsulates gamma-polyglutamylated pemetrexed containing 2 to 10 glutamyl groups (i.e., Lp-γPPMX including PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX), and has an internal space containing less than 500,000 or less than 200,000 gamma-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the internal space of the liposome contains 10 to 100,000 or any range therebetween of gamma-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups. In further embodiments, the internal space of the liposome contains 10,000 to 100,000 or any range therebetween of gamma-polyglutamylated 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 gamma-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome is not pegylated, and the internal space of the liposome contains 10 to 100,000 or any range therebetween of gamma-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups. In further embodiments, the liposome is not pegylated, and the internal space of the liposome contains 10,000 to 100,000 or any range therebetween of gamma-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome is targeted and not pegylated (TLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma-polyglutamylated 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 10 to 100,000 or any range therebetween of gamma-polyglutamylated 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 between 10,000 and 100,000 or any range therebetween of gamma-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome is not targeted and not pegylated (NTLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome is not targeted and not pegylated, and the internal space of the liposome contains between 10 and 100,000 or any range therebetween of gamma-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups. In a further embodiment, the liposome is not targeted and not pegylated, and the internal space of the liposome contains between 10,000 and 100,000 or any range therebetween of gamma-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups.

[0209] In some embodiments, the liposome encapsulates gamma-tetrapeptide glutamine oxidized pemetrexed (i.e., Lp-γPPMX including PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma-pentapeptide glutamine oxidized pemetrexed molecules. In some embodiments, the internal space of the liposome contains from 10 to 100,000 or any range therebetween of gamma-tetrapeptide glutamine oxidized pemetrexed molecules. In some embodiments, the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of gamma-tetrapeptide glutamine 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 gamma-tetrapeptide glutamine 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 gamma-tetrapeptide glutamine oxidized 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 gamma-tetrapeptide glutamine oxidized pemetrexed molecules. In some embodiments, the liposome is targeted and not pegylated (TLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma-tetrapeptide glutamine 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 gamma-tetrapeptide glutamine oxidized 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 gamma-tetrapeptide glutamine oxidized pemetrexed molecules. In some embodiments, the liposome is not targeted and not pegylated (NTLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma-tetrapeptide glutamine oxidized pemetrexed molecules.In some embodiments, the liposomes are not targeted, not pegylated, and the internal space of the liposomes contains from 10 to 100,000 or any range therebetween of gamma tetrapeptide oxidized pemetrexed molecules. In further embodiments, the liposomes are not targeted, not pegylated, and the internal space of the liposomes contains from 10,000 to 100,000 or any range therebetween of gamma tetrapeptide oxidized pemetrexed molecules.

[0210] In some embodiments, the liposome encapsulates gamma pentaglutamine oxidized pemetrexed (i.e., Lp-γPPMX including PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains from 10 to 100,000 or any range therebetween of gamma pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains from 10,000 to 100,000 or any range therebetween of gamma 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 gamma 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 therebetween of gamma 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 therebetween of gamma pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposome is targeted and not pegylated (TLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma 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 therebetween of gamma 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 therebetween of gamma pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposome is not targeted and not pegylated (NTLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma pentaglutamine 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 gamma pentaglutamylated 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 gamma pentaglutamylated pemetrexed molecules.

[0211] In some embodiments, the liposome encapsulates gamma-hexaglutaminyl oxidized pemetrexed (i.e., Lp-γPPMX including PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma-hexaglutaminyl oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains 10 to 100,000 or any range therebetween of gamma-hexaglutaminyl oxidized pemetrexed molecules. In further embodiments, the liposome internal space contains 10,000 to 100,000 or any range therebetween of gamma-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 gamma-hexaglutaminyl oxidized pemetrexed molecules. In some embodiments, the liposome is not pegylated and the internal space of the liposome contains 10 to 100,000 or any range therebetween of gamma-hexaglutaminyl oxidized pemetrexed molecules. In further embodiments, the liposome is not pegylated and the internal space of the liposome contains 10,000 to 100,000 or any range therebetween of gamma-hexaglutaminyl oxidized pemetrexed molecules. In some embodiments, the liposome is targeted and not pegylated (TLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma-hexaglutaminyl oxidized pemetrexed molecules. In some embodiments, the liposome is targeted and not pegylated and the internal space of the liposome contains 10 to 100,000 or any range therebetween of gamma-hexaglutaminyl oxidized pemetrexed molecules. In further embodiments, the liposome is targeted and not pegylated and the internal space of the liposome contains 10,000 to 100,000 or any range therebetween of gamma-hexaglutaminyl oxidized pemetrexed molecules. In some embodiments, the liposome is not targeted and not pegylated (NTLp-γPPMX) and has an internal space containing less than 500,000 or less than 200,000 gamma-hexaglutaminyl oxidized pemetrexed molecules.In some embodiments, the liposomes are not targeted, not pegylated, and the internal space of the liposomes contains from 10 to 100,000 or any range therebetween of gamma-hexaglutaminyl oxidized pemetrexed molecules. In further embodiments, the liposomes are not targeted, not pegylated, and the internal space of the liposomes contains from 10,000 to 100,000 or any range therebetween of gamma-hexaglutaminyl oxidized pemetrexed molecules.

[0212] In some embodiments, the present disclosure provides a liposomal gamma-polyglytamyl oxidized pemetrexed composition, wherein the liposomes encapsulate gamma-polyglytamyl oxidized pemetrexed or a salt or acid thereof, and one or more aqueous pharmaceutically acceptable carriers. In some embodiments, the internal space of the liposomes contains trehalose. In some embodiments, the internal space of the liposomes contains from 5 wt% to 20 wt% trehalose. In some embodiments, the internal space of the liposomes contains HBS at a concentration of 1 to 200 mM and a pH of 2 to 8. In some embodiments, the internal space of the liposomes has a pH of 5 to 8, or any range therebetween. In some embodiments, the internal space of the liposomes has a pH of 6 to 7, or any range therebetween. In some embodiments, the internal space of the liposomes contains a total concentration of sodium acetate and calcium acetate in the range of 50 mM to 500 mM, or any range therebetween.

[0213] A non-polyglytamylated polyglutaminylatable folate antagonist In some embodiments, a liposomal gamma-polyglytamyl oxidized pemetrexed (e.g., Lp-γPPMX including PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) composition comprises gamma-polyglytamyl oxidized pemetrexed (e.g., γPPMX disclosed herein) and a composition of one or more non-polyglytamylated polyglutaminylatable folate antagonists.

[0214] In some embodiments, Lp-γPPMX (e.g., PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) comprises gamma-polyglutamylated pemetrexed (e.g., γPPMX as disclosed herein) and pemetrexed (PMX). In some embodiments, Lp-γPPMX (i.e., liposomal gamma-polyglutamylated pemetrexed) comprises a gamma-polyglutamylated pemetrexed and a polyglutamylatable folate antagonist selected from the group consisting of pemetrexed (PMX), methotrexate (MTX), raltitrexed (RTX), pralatrexate, AG2034, GW1843, aminopterin, and LY309887. In some embodiments, Lp-γPPMX comprises gamma-polyglutamylated pemetrexed and raltitrexed. In some embodiments, Lp-γPPMX comprises gamma-polyglutamylated pemetrexed and pemetrexed. In some embodiments, Lp-γPPMX comprises gamma-polyglutamylated pemetrexed and leucovorin. In some embodiments, Lp-γPPMX comprises gamma-polyglutamylated pemetrexed and a triazine folate antagonist derivative (e.g., a sulfonylfluoride triazine such as NSC127755). In some embodiments, Lp-γPPMX comprises gamma-polyglutamylated pemetrexed and a serine hydroxymethyltransferase (SHMT2) inhibitor. In some embodiments, the SHMT2 inhibitor is a folate antagonist (e.g., a polyglutamylatable or non-polyglutamylatable folate antagonist). In some embodiments, the SHMT2 inhibitor is a folate antagonist.

[0215] B Non-polyglutamylatable folate antagonist In some embodiments, Lp-γPPMX (e.g., PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX) comprises gamma-polyglutamylated pemetrexed (e.g., γPPMX as disclosed herein) and so-called “non-polyglutamylatable” folate antagonists. In some embodiments, the liposome comprises gamma-polyglutamylated pemetrexed and a non-polyglutamylatable folate antagonist that inhibits one or more enzymes in the folate cycle metabolic pathway. In further embodiments, the non-polyglutamylatable folate antagonist inhibits one or more enzymes selected from the group consisting of thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycineamide ribonucleotide (GAR) transformylase, and aminoimidazole carboxamide ribonucleotide (AICAR) transformylase. In some embodiments, the liposome comprises gamma-polyglutamylated pemetrexed and a non-polyglutamylatable folate antagonist that inhibits DHFR. In some embodiments, the liposome comprises gamma-polyglutamylated pemetrexed and a non-polyglutamylatable folate antagonist that inhibits TS. In some embodiments, the liposome comprises gamma-polyglutamylated pemetrexed and a non-polyglutamylatable folate antagonist that inhibits GAR or AICAR transformylase. In further embodiments, the non-polyglutamylatable folate antagonist is selected from the group consisting of trimethoprim (TMQ), pyrithioxime (BW301U), and talotrexed (PT523). In further embodiments, the non-polyglutamylatable folate antagonist is selected from the group consisting of nolatrexed (AG337), premetrexed (ZD9331, BGC9331), and BGC945 (ONX0801).

[0216] C Platinum In some embodiments, the liposome comprises gamma-polyglutamyl oxidized pemetrexed (e.g., Lp-γPPMX such as PLp-γPPMX, TPLp-γPPMX, TLp-γPPMX, and NTLp-γPPMX), and comprises gamma-polyglutamyl oxidized pemetrexed (e.g., γPPMX disclosed herein) and a platinum-based chemotherapeutic agent or a salt or acid thereof. In some embodiments, the liposome comprises a gamma-polyglutamyl oxidized pemetrexed / platinum-based drug complex (e.g., as described in Section IIB).

[0217] In some embodiments, Lp-γPPMX comprises a platinum-based chemotherapeutic agent selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or a salt or acid thereof. In some embodiments, Lp-γPPMX comprises an analog of a platinum-based chemotherapeutic agent selected from the group consisting of cisplatin, carboplatin, or oxaliplatin, or a salt or acid thereof.

[0218] In some embodiments, Lp-γPPMX comprises gamma-polyglutamyl oxidized pemetrexed and cisplatin or a salt or acid thereof. In some embodiments, Lp-γPPMX comprises gamma-polyglutamyl oxidized pemetrexed and a cisplatin analog or a salt or acid thereof.

[0219] In some embodiments, Lp-γPPMX comprises gamma-polyglutamyl oxidized pemetrexed and carboplatin or a salt or acid thereof. In some embodiments, the liposome comprises gamma-polyglutamyl oxidized pemetrexed and a carboplatin analog or a salt or acid thereof.

[0220] In some embodiments, Lp-γPPMX comprises gamma-polyglutamyl oxidized pemetrexed and oxaliplatin or a salt or acid thereof. In some embodiments, the liposome comprises gamma-polyglutamyl oxidized pemetrexed and an oxaliplatin analog or a salt or acid thereof.

[0221] In some embodiments, the liposome comprises a platinum-based chemotherapeutic agent selected from the group consisting of gamma polyglutamine oxidized pemetrexed (e.g., γPPMX disclosed herein) and nedaplatin, heptaplatin, and lobaplatin, nedaplatin, heptaplatin and lobaplatin or salts or acids thereof. In some embodiments, Lp-γPPMX comprises an analog of a platinum-based chemotherapeutic agent selected from the group consisting of gamma polyglutamine oxidized pemetrexed and nedaplatin, heptaplatin, and lobaplatin, or salts or acids thereof.

[0222] In some embodiments, Lp-γPPMX comprises a platinum-based chemotherapeutic agent selected from the group consisting of gamma polyglutamine oxidized pemetrexed and satraplatin, paraplatin, platinol, cisplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, 254-S, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or salts or acids thereof. In some embodiments, Lp-γPPMX comprises a platinum-based chemotherapeutic agent selected from the group consisting of gamma polyglutamine oxidized pemetrexed and satraplatin, paraplatin, platinol, cisplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, 254-S, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or salts or acids thereof.

[0223] In some embodiments, the liposome composition comprises liposomes further comprising one or more of an immunostimulant, a detectable marker, and maleimide disposed on at least one of the PEG or outer surface of the liposome.

[0224] D Cyclodextrin In further embodiments, the γPPMX liposomes comprise γPPMX (e.g., γPPMX disclosed herein) and cyclodextrin (e.g., the cyclodextrin of Section IIB herein).

[0225] In some embodiments, the γPPMX liposomes comprise a complex formed by cyclodextrin and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound or a salt or acid thereof. In further embodiments, the therapeutic agent is a chemotherapeutic agent or a salt or acid thereof. In another embodiment, the chemotherapeutic agent is a platinum-based drug. In another embodiment, the chemotherapeutic agent is a taxane-based drug. In further embodiments, the therapeutic agent of the cyclodextrin / therapeutic agent complex is a member selected from the group consisting of gemcitabine, gemcitabine-based therapeutic agents, doxorubicin, folic acid antagonists, folic acid antagonist-based chemotherapeutic agents, or salts or acids thereof, in acid form or free base form. In further embodiments, the molar ratio of cyclodextrin / therapeutic agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / therapeutic agent in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / therapeutic agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50.

[0226] In some embodiments, the γPPMX liposomes contain γPPMX and a cyclodextrin / platinum-based chemotherapeutic agent complex. In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the cyclodextrin / platinum-based chemotherapeutic agent complex contains cisplatin, carboplatin, an analog of oxaliplatin, or salts or acids thereof. In some embodiments, the molar ratio of cyclodextrin / platinum-based drug in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / platinum-based drug in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / platinum-based drug in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / therapeutic agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50.

[0227] In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the cyclodextrin / platinum-based chemotherapeutic agent complex contains cisplatin, carboplatin, an analogue of oxaliplatin, or salts or acids thereof. In some embodiments, the molar ratio of cyclodextrin / platinum-based agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, ⑨:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / platinum-based agent in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / platinum-based agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In further embodiments, the cyclodextrin / platinum-based agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0228] In a further embodiment, the present disclosure provides a complex comprising cyclodextrin and cisplatin or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / cisplatin (or a salt or acid of cisplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / cisplatin (or a salt or acid of cisplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In a further embodiment, the cyclodextrin / cisplatin (or a salt or acid of cisplatin) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0229] In another embodiment, the present disclosure provides a complex comprising cyclodextrin and carboplatin or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / carboplatin (or a salt or acid of carboplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / carboplatin (or a salt or acid of carboplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In a further embodiment, the cyclodextrin / carboplatin (or a salt or acid of carboplatin) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0230] In another embodiment, the present disclosure provides a complex comprising cyclodextrin and oxaliplatin or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / oxaliplatin (or a salt or acid of oxaliplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / oxaliplatin (or a salt or acid of oxaliplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In a further embodiment, the cyclodextrin / oxaliplatin (or a salt or acid of oxaliplatin) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0231] In a further embodiment, the present disclosure provides a complex comprising a cyclodextrin and a platinum-based chemotherapeutic agent selected from the group consisting of nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or a salt or acid thereof. In other embodiments, the cyclodextrin / platinum-based chemotherapeutic agent complex comprises nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, or an analogue of nedaplatin, or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / platinum-based chemotherapeutic agent (or a salt or acid or analogue thereof) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / platinum-based chemotherapeutic agent (or a salt or acid or analogue thereof) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween.In some embodiments, the molar ratio of cyclodextrin / platinum-based chemotherapeutic agent (or its salt or acid or analog) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In further embodiments, the cyclodextrin / platinum-based chemotherapeutic agent (or its salt or acid or analog) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0232] In some embodiments, the present disclosure provides a composition comprising a cyclodextrin / taxane-based chemotherapeutic agent complex. In some embodiments, the taxane-based chemotherapeutic agent is selected from the group consisting of paclitaxel (PTX), docetaxel (DTX), larotaxel (LTX), and cabazitaxel (CTX), or their salts or acids. In some embodiments, the molar ratio of cyclodextrin / taxane-based agent in the complex ranges from 1 - 10:1. In some embodiments, the molar ratio of cyclodextrin / taxane-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 cyclodextrin / taxane-based agent in the complex ranges from 1:1 - 20, 1:1 - 10, or 1:2 - 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / taxane-based agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In further embodiments, the cyclodextrin / taxane-based agent complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

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

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

[0235] In a further embodiment, the present disclosure provides a complex comprising cyclodextrin and larotaxel (LTX) or a salt or acid thereof. In other embodiments, the cyclodextrin / taxane-based chemotherapeutic agent complex comprises an analogue of larotaxel (LTX), or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / 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 other embodiments, the molar ratio of cyclodextrin / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / larotaxel (or a salt or acid of larotaxel) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:1...

Claims

【Claim 1】 Liposomes encapsulating gamma-polyglutamyl oxidized pemetrexed, and one or more non-gamma-polyglutamylated gamma-polyglutamylatable folate antagonists, or one or more non-gamma-polyglutamylatable folate antagonists, A liposome composition comprising: The gamma-polyglutamyl oxidized pemetrexed contains 2 to 15 glutamyl groups having gamma or alpha carboxyl group bonds, where (a) At least two of the glutamyl groups of the gamma-polyglutamyl oxidized pemetrexed are of the L-form, (b) Each glutamyl group of the gamma-polyglutamyl oxidized pemetrexed is of the L-form, (c) At least one of the glutamyl groups of the gamma-polyglutamyl oxidized pemetrexed is of the D-form, (d) Each of the glutamyl groups of the gamma-polyglutamyl oxidized pemetrexed, except for the pemetrexed of the glutamyl group, is of the D-form, or (e) At least two of the glutamyl groups of the gamma-polyglutamyl oxidized pemetrexed are of the L-form, and at least one of the glutamyl groups is of the D-form, The liposomes have a diameter in the range of 50 to 150 nm, Liposome composition.

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