Alpha-polyglutamine oxidized pemetrexed and its use

Abstract

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

Description

Background Art

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

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

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

[0004] Pemetrexed is a multi-target folate antagonist that exerts its effects by disrupting folate-dependent metabolic processes essential for cellular homeostasis and replication. Pemetrexed inhibits three enzymes necessary for purine and pyrimidine biosynthesis: thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of these enzymes suppresses new nucleotide biosynthesis, leading to disruption of cellular homeostasis and an imbalance of purine and pyrimidine precursors, which in turn prevents accurate DNA replication in cells and ultimately leads to cell death.

[0005] Pemetrexed is transported into cells by the reduced folate carrier (RFC) system and folate receptors (FR) α and β, as well as by the proton-coupled folate transporter (PCFT), which is most active in sub-normal pH environments. RFC is the primary transporter for pemetrexed at physiological pH and is widely expressed in normal and diseased cells. Consequently, pemetrexed is often subject to dose-limiting toxicity, a major obstacle in cancer chemotherapy. Once inside the cell, pemetrexed is polyglutamine-oxidized by FPGAS, which can add up to six L-glutamyl groups to the L-gammacarboxyl group binding to pemetrexed. L-gamma polyglutamine oxidation of pemetrexed by FPGAs serves at least two primary therapeutic purposes: (1) it greatly enhances the affinity and inhibitory activity of pemetrexed against several folate-dependent enzymes, including thymidylate synthase and GARFTase; and (2) it facilitates the accumulation of polyglutamate-oxidized pemetrexed, which, unlike pemetrexed (monoglutamate), is not readily transported out of cells by the cell efflux pump.

[0006] Pemetrexed acts during DNA and RNA synthesis, resulting in significant toxic effects against rapidly dividing cells such as malignant and myeloid cells. Myelosuppression is typically a dose-limiting toxicity of pemetrexed therapy and limits its clinical application. To mitigate the most common side effects associated with pemetrexed therapy, including myelosuppression, fatigue, and rash, pretreatment with folic acid and vitamin B is now used.

[0007] Resistance to pemetrexed therapy is typically associated with one or more of the following: (a) increased cell efflux pump activity, (b) increased thymidylate synthetase activity, (c) decreased folyl polygamma-glutamate synthase (FPGS) activity, and (d) increased gamma-glutamyl hydrolase (GGH) activity, which cleaves gamma-polyglutamate chains bound to folate and folate antimetabolites.

[0008] The problem with the long-term (>30-year) observation that higher levels of polyglutamates of various folate antimetabolites are far more potent than lower levels of glutamates has been that the scientific community has relied on intracellular FPGS-mediated mechanisms to convert low levels of glutamates to their higher-level forms. This invention provides a means for directly delivering higher-level polyglutamate forms of folate antimetabolites into cells, without relying on cellular mechanisms to achieve this goal.

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

[0010] This disclosure generally relates to novel alpha-polyglutamine oxidized pemetrexed (PMX) compositions, and to methods for manufacturing and using these compositions to treat diseases including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

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

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

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

[10] , wherein the polyglutamate is linear;

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

[10] , wherein the polyglutamate is a branched chain;

[13] Liposome composition (Lp-αPPMX) containing alpha-polyglutamine oxidized pemetrexed as described in any one of items [1] to

[12] ;

[14] A LαPP composition according to item

[13] , wherein alpha-polyglutamine oxidized pemetrexed comprises an L-type glutamyl group having an alpha-carboxyl group bond;

[15] A composition of Lp-αPPMX as described in item

[13] or

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

[16] Lp-αPPMX compositions according to item

[13] or

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

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

[13] to

[16] , wherein the liposomes contain alpha-polyglutamine oxidized pemetrexed having 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups;

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

[13] to

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

[19] A composition according to any one of items

[13] to

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

[20] A composition according to any one of items

[13] to

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

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

[13] to

[19] , wherein the liposomes contain alpha-tetraglutamine oxidized pemetrexed, alpha-pentaglutamine oxidized pemetrexed, or alpha-hexaglutamine oxidized pemetrexed, or alpha-polyglutamine oxidized pemetrexed;

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

[13] to

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

[23] A composition (PαLp-αPPMX) according to any one of items

[13] to

[22] , wherein the liposomes are pegylated;

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

[28] A composition of Lp-αPPMX as described in item

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

[29] Lp-αPPMX compositions according to item

[27] or

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

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

[27] to

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

[31] A composition according to any one of items

[27] to

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

[32] A composition of Lp-αPPMX as described in item

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

[33] A composition of Lp-αPPMX as described in item

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

[33] , wherein the liposomes are cationic;

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

[13] to

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

[40] Lp-αPPMX compositions as described in item

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

[41] A composition of Lp-αPPMX as described in item

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

[42] A composition of Lp-αPPMX as described in item

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

[43] A composition of Lp-αPPMX as described in any one of items

[39] to

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

[44] A composition according to any one of items

[39] to

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

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

[39] to

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

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

[39] to

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

[47] A composition according to any one of items

[13] to

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

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

[13] to

[47] , wherein the liposomes contain fewer than 500,000 or fewer than 200,000 alpha-polyglutamine oxidized pemetrexed molecules;

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

[13] to

[48] , wherein the liposomes contain 10 to 100,000 or any range in between alpha-polyglutamine oxidized pemetrexed molecules;

[50] A composition according to any one of items

[13] to

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

[51] Lp-αPPMX compositions as described in item

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

[52] A composition relating to the Lp-αPPMX composition described in item

[50] or

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

[53] A composition according to any one of items

[50] to

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

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

[50] to

[53] , wherein the targeting portion is measured by BIACORE® analysis to be 0.5 x 10 -10 ~10x10 -6 A composition that binds to a surface antigen with an equilibrium dissociation constant (Kd) within the range of [value];

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

[50] to

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

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

[50] to

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

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

[50] to

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

[58] A composition of Lp-αPPMX as described in item

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

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

[39] to

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

[60] Lp-αPPMX composition as described in item

[58] or

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

[61] A composition of Lp-αPPMX described in any one of items

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] Lp-αPPMX compositions as described in item

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

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

[13] to

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

[65] Targeted compositions comprising any one of items [1] to

[64] ;

[66] Non-targeting compositions comprising any one of items [1] to

[49] ;

[67] A composition according to any one of items

[13] to

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

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

[13] to

[67] ;

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

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

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

[71] Use of any one of the compositions described in 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 of which treatment or prevention is in need, comprising the step of administering a composition of any one of items [1] to

[70] to the subject;

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

[13] to

[69] to the subject;

[74] A method for killing overgrown cells, comprising the step of bringing the overgrown cells into contact with a composition described in any one of items [1] to

[69] ;

[75] A method for killing hyperproliferating cells, comprising the step of contacting the hyperproliferating cells with a liposome alpha-polyglutamine oxidized pemetrexed composition described in any one of items

[13] to

[69] ;

[76] Methods relating to item

[74] or

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

[77] A method for treating cancer, comprising the step of administering an effective amount of 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 a liposomal alpha-polyglutamine oxidized pemetrexed composition described in any one of items

[13] to

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

[79] Methods according to item

[77] or

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

[80] Methods relating 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] Methods relating to item

[77] or

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

[82] Methods relating to item

[77] or

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

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

[50] to

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

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

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

[85] Maintenance therapy comprising the step of administering an effective amount of the liposomal alpha-polyglutamine oxidized pemetrexed composition described in any one of items

[13] to

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

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

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

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

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

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

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

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

[13] to

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

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

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

[91] A method for preparing an alpha-polyglutamine oxidized pemetrexed composition comprising the liposome alpha-polyglutamine oxidized pemetrexed composition described in any one of items

[13] to

[69] , comprising the steps of: forming a mixture containing liposome components and alpha-polyglutamine oxidized pemetrexed in solution; homogenizing the mixture in solution to form liposomes; and processing the mixture to form liposomes containing alpha-polyglutamine oxidized pemetrexed;

[92] A method for preparing an alpha-polyglutamine oxidized pemetrexed composition comprising the liposome alpha-polyglutamine oxidized pemetrexed composition described in any one of items

[13] to

[69] , comprising the steps of: forming a mixture containing liposome components and alpha-polyglutamine oxidized pemetrexed in solution; and processing the mixture to form liposomes containing alpha-polyglutamine oxidized pemetrexed;

[93] A method according to item

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

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

[13] to

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

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

[50] to

[69] , comprising the steps of: forming a mixture in solution comprising a liposome component and alpha-polyglutamine oxidized pemetrexed; processing the mixture to form liposomes that encapsulate and / or contain alpha-polyglutamine oxidized pemetrexed; and imparting a targeting portion to the surface of the liposomes, wherein the targeting portion has specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ);

[96] A method relating to item

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

[97] A method according to any one of items

[94] to

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

[98] A method according to any one of items

[94] to

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

[99] A method according to any one of items

[91] to

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

[0012] In some embodiments, the disclosure provides alpha-polyglutamine oxidized pemetrexed (αPPMX) compositions in which at least one glutamyl residue of alpha-polyglutamine oxidized pemetrexed is linked by an alpha-carboxyl group. In some embodiments, αPPMX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl groups of pemetrexed). In some embodiments, αPPMX contains two or more L-type glutamyl groups. In other embodiments, αPPMX contains D-type glutamyl groups. In further embodiments, αPPMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, αPPMX contains two or more glutamyl groups having gamma bonds. In some embodiments, at least one glutamyl group has both alpha and gamma bonds.

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

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

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

[0016] In further embodiments, the disclosure provides compositions comprising delivery carriers such as liposomes filled (i.e., encapsulated) and / or otherwise bound to alpha-polyglutamate-oxidized pemetrexed, and methods for preparing αPPMX-filled / bound delivery carrier compositions and methods for using them to deliver alpha-polyglutamate-oxidized pemetrexed to diseased (e.g., cancerous) cells and / or target cells. These compositions have applications including, but are not limited to, the treatment of diseases including, for example, hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria. The αPPMX-filled / bound delivery carrier compositions result in improved efficacy and safety of pemetrexed delivery to cancer cells by providing selective delivery of a higher cytotoxic payload (e.g., polyglutamate-oxidized pemetrexed) compared to the cytotoxicity of pemetrexed (PMX) administered in monoglutamate form.

[0017] In further embodiments, the disclosure provides compositions (Lp-αPPMX) comprising liposomes encapsulated (filled) with alpha-polyglutamine oxidized pemetrexed. In some embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 20 glutamyl groups (including the glutamyl groups of pemetrexed). In some embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX contains two or more L-type glutamyl groups. In other embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX contains D-type glutamyl groups. In further embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX contains D-type glutamyl groups and two or more L-type glutamyl groups. In further embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX contains two or more glutamyl groups having gamma bonds. In further embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX contains one or more glutamyl groups having both alpha and gamma bonds. In some embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX contains 2 to 10 or any range in between, glutamyl groups having both alpha and gamma bonds. In some embodiments, the polyglutamate chain of the alpha-polyglutamine oxidized pemetrexed is linear. In some embodiments, the polyglutamate chain of the alpha-polyglutamine oxidized pemetrexed is branched.

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

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

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

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

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

[0023] In further embodiments, the liposomal alpha-polyglutamine oxidized pemetrexed composition is pegylated (PLp-αPPMX).

[0024] In some embodiments, the liposomal alpha-polyglutamine oxidized pemetrexed composition is untargeted (NTLp-αPPMX). That is, the NTLp-αPPMX composition does not have specific affinity for epitopes expressed on the surface of target cells of interest (e.g., epitopes on surface antigens). In further embodiments, the untargeted liposomal alpha-polyglutamine oxidized pemetrexed composition is pegylated (NTPLp-αPPMX).

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

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

[0027] In certain embodiments, the targeting moiety comprises a polypeptide that specifically binds to a folate receptor. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the folate receptor bound by the targeting moiety specifically binds to one or more folate receptors selected from the group consisting of folate receptor alpha (FR-α, FOLR1), folate receptor beta (FR-β, FOLR2), and folate receptor delta (FR-δ, FOLR4). In some embodiments, the folate receptor bound by the targeting moiety is folate receptor alpha (FR-α). In some embodiments, the folate receptor bound by the targeting moiety is folate receptor beta (FR-β). In some embodiments, the targeting moiety specifically binds to FR-α and FR-β.

[0028] In further embodiments, the αPPMX composition comprises one or more immunostimulants, detectable markers, and maleimides, disposed on at least one of the PEG or outer surfaces of the liposome. In some embodiments, the liposome αPPMX composition (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX) is cationic. In other embodiments, the liposome αPPMX composition (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX) is anionic or neutral. In further embodiments, the liposomes of the liposome-αPPMX composition (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX) have a diameter in the range of 20 nm to 500 nm, or any range in between. In further embodiments, the liposomes of the liposome-αPPMX composition have a diameter in the range of 80 nm to 120 nm, or any range in between. In some embodiments, the liposome-αPPMX composition is pegylated (e.g., PLp-αPPMX, NTPLp-αPPMX, or TPLp-αPPMX). In some embodiments, the liposome-αPPMX composition is targeted (e.g., TLp-αPPMX or TPLp-αPPMX). In further embodiments, the liposome-αPPMX composition is both pegylated and targeted (e.g., TPLp-αPPMX). In some embodiments, the liposomal αPPMX composition comprises alpha-polyglutamine oxidized pemetrexed containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomal αPPMX composition comprises alpha-tetraglutamine oxidized pemetrexed. In some embodiments, the liposomal αPPMX composition comprises alpha-pentaglutamine oxidized pemetrexed. In other embodiments, the liposomal αPPMX composition comprises alpha-hexaglutamine oxidized pemetrexed.

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

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

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

[0032] Liposome compositions comprising αPPMX-encapsulated liposomes are also provided. In some embodiments, the liposome composition comprises a pegylated αPPMX composition. In some embodiments, the liposome composition comprises an αPPMX composition bound to or otherwise incorporated therein a targeting moiety. In further embodiments, the liposome composition comprises a pegylated αPPMX composition bound to or otherwise incorporated therein a targeting moiety. In some embodiments, the liposome composition comprises αPPMX comprising 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome composition comprises alpha-tetraglutamine oxidized pemetrexed. In some embodiments, the liposome composition comprises alpha-pentaglutamine oxidized pemetrexed. In other embodiments, the liposome composition comprises alpha-hexaglutamine oxidized pemetrexed.

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

[0034] Pharmaceutical compositions are also provided that include alpha-polyglutamine oxidized pemetrexed (αPPMX) containing a delivery carrier such as liposome αPPMX. In some embodiments, the pharmaceutical composition includes a pegylated αPPMX composition. In some embodiments, the pharmaceutical composition includes an αPPMX composition bonded to or otherwise incorporated therein a targeting moiety. In further embodiments, the pharmaceutical composition includes a pegylated αPPMX composition bonded to or otherwise incorporated therein a targeting moiety. In some embodiments, the pharmaceutical composition includes αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition includes alpha-tetraglutamine oxidized pemetrexed. In some embodiments, the pharmaceutical composition includes alpha-pentaglutamine oxidized pemetrexed. In other embodiments, the pharmaceutical composition includes alpha-hexaglutamine oxidized pemetrexed.

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

[0036] In further embodiments, the disclosure provides a method for killing cells, the method comprising the step of contacting cells with a composition comprising an alpha-polyglutamine oxidized pemetrexed (αPPMX) composition. In some embodiments, the cells to be contacted are mammalian cells. In further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are overgrowth cells. In further embodiments, the overgrowth cells are cancer cells. In further embodiments, the cancer cells to be contacted are primary cells, or cells derived from cell lines obtained from / derived from cancers selected from the group consisting of, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasmacytic dysplasia or cachexia. In some embodiments, the method is carried out in vivo. In other embodiments, the method is carried out in vitro. In some embodiments, αPPMX contains 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the αPPMX composition contains alpha-tetraglutamine oxidized pemetrexed. In some embodiments, the αPPMX composition comprises alpha-pentaglutamine oxidized pemetrexed. In other embodiments, the αPPMX composition comprises alpha-hexaglutamine oxidized pemetrexed.

[0037] In further embodiments, the disclosure provides a method for killing cells, the method comprising the step of contacting cells with liposomes containing alpha-polyglutamine oxidized pemetrexed (i.e., Lp-αPPMX such as PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX). In some embodiments, the cells to be contacted are mammalian cells. In further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are overgrowth cells. In further embodiments, the overgrowth cells to be contacted are cancer cells. In further embodiments, cancer cells are primary cells, or cells derived from cell lines obtained from / derived from cancers selected from the group consisting of, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasmacytic dysplasia or cachexia. In some embodiments, the method is carried out in vivo. In other embodiments, the method is carried out in vitro. In some embodiments, the liposomes contain αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes contain alpha-tetraglutamine oxidized pemetrexed. In some embodiments, the liposomes contain alpha-pentaglutamine oxidized pemetrexed. In other embodiments, the liposomes contain alpha-hexaglutamine oxidized pemetrexed.

[0038] In further embodiments, the disclosure provides a method for treating cancer, comprising the step of administering an effective amount of a delivery carrier (e.g., an immune complex or liposome) containing alpha-polyglutamine oxidized pemetrexed to a subject having or at risk of having cancer. In some embodiments, the delivery carrier is an antibody-containing immune complex (e.g., including a full-length IgG antibody, a bispecific antibody, or scFv). In some embodiments, the delivery carrier is a liposome (e.g., Lp-αPPMX such as PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX). In some embodiments, the delivery carrier to be administered is pegylated. In some embodiments, the delivery carrier to be administered is not pegylated. In further embodiments, the delivery carrier to be administered includes a targeting moiety having specific affinity for an antigen epitope on the surface of cancer cells.In further embodiments, the delivery carrier includes a targeting moiety that specifically binds to a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β, or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, fibronectin extradomain 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, CD10 5, 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, integrins (e.g., integrins αvβ3, αvβ5, or αvβ6), C 242 antigens, 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 includes a targeting moiety that specifically binds to a cell surface antigen identified as originating from or expressed on a specific target cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety specifically binds to a cell surface antigen identified as originating from or expressed on a specific target tumor, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the delivered delivery carrier includes αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the delivered delivery carrier includes alpha-tetraglutamine oxidized pemetrexed. In some embodiments, the delivered delivery carrier includes alpha-pentaglutamine oxidized pemetrexed. In other embodiments, the delivered delivery carrier includes alpha-hexaglutamine oxidized pemetrexed. In some embodiments, the delivered delivery carrier includes L-alpha-polyglutamine oxidized pemetrexed. In some embodiments, the delivered delivery carrier includes D-alpha-polyglutamine oxidized pemetrexed. In some embodiments, the delivery carrier administered comprises L and D alpha-polyglutamine oxidized pemetrexed. In some embodiments, the cancer is selected from the group consisting of non-hematological malignancies, including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies, including, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasia or cachexia. 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 cell 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.

[0039] In further embodiments, the disclosure provides a method for treating cancer, the method comprising the step of administering an effective amount of liposomes containing alpha-polyglutamine oxidized pemetrexed (e.g., Lp-αPPMX such as PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX) to a subject having or at risk of having cancer. In some embodiments, the liposomes are pegylated. In some embodiments, the liposomes are not pegylated. In further embodiments, the liposomes include a targeting moiety having specific affinity for an antigen epitope on the surface of cancer cells.In further embodiments, the liposome includes a targeting moiety that specifically binds to a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β, or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, fibronectin extradomain 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, CD10 5, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrins αvβ3, αvβ5, or αvβ6), C 242 antigens, 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, CD44, CD138, and CD15. In some embodiments, the liposomes include a targeting moiety that specifically binds to a cell surface antigen that is determined to originate from or be expressed on a specific target tumor, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the liposomes include αPPMX containing 4, 5, 6, 2–10, 4–6, or more than 5 glutamyl groups. In some embodiments, the liposomes include alpha-tetraglutamine oxidized pemetrexed. In some embodiments, the liposomes include alpha-pentaglutamine oxidized pemetrexed. In other embodiments, the liposomes include alpha-hexaglutamine oxidized pemetrexed. In some embodiments, the liposomes include L-alpha-polyglutamine oxidized pemetrexed. In some embodiments, the liposomes include D-alpha-polyglutamine oxidized pemetrexed. In some embodiments, the liposomes contain L and D alpha-polyglutamine oxidized pemetrexed. In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, and hematological malignancies (e.g., leukemia or lymphoma). In some embodiments, the cancer is lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer is breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer cells are ovarian cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is osteosarcoma.

[0040] In further embodiments, the present disclosure provides a method for treating cancer, comprising the step of administering an effective amount of a liposome composition comprising liposomes having a targeting moiety having specific affinity for alpha-polyglutamine oxidized pemetrexed and an epitope of an antigen on the surface of a cancer to a subject having or at risk of having cancer.In some embodiments, the liposomes include a targeting moiety that specifically binds to a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β, or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P Cadherin, fibronectin extradomain 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, CD10 5, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrins αvβ3, αvβ5, or αvβ6), C 242 antigens, Apo2, PSGR, NGEP, PSCA, TMEFF2, Endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the administered liposomes include a targeting moiety that specifically binds to cell surface antigens determined to originate from or be expressed on a specific target tumor, such as a neoantigen. In some embodiments, the administered liposome composition includes pegylated liposomes (e.g., TPLp-αPPMX). In some embodiments, the administered liposome composition includes non-pegylated liposomes. In some embodiments, the liposomes of the administered liposome composition include αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition include alpha-tetraglutamine oxidized pemetrexed. In some embodiments, the liposomes of the administered liposome composition include alpha-pentaglutamine oxidized pemetrexed. In other embodiments, the liposomes of the administered liposome composition include alpha-hexaglutamine oxidized pemetrexed. In some embodiments, the liposome composition is administered to treat cancers selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma and other plasma cell dysplasia or cachexia, as well as leukemia, lymphoma and other B-cell 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 liposomal composition is administered to treat pancreatic cancer. In some embodiments, the liposomal composition is administered to treat liver cancer. In some embodiments, the liposomal composition is administered to treat head and neck cancer.In some embodiments, liposomal compositions are administered to treat osteosarcoma.

[0041] In further embodiments, the Disclosure provides a method for treating cancer, comprising the step of administering an effective amount of a liposome composition to a subject having or at risk of having cancer expressing folate receptors on its cell surface, wherein the liposome composition comprises liposomes comprising (a) alpha-polyglutamine oxidized pemetrexed (αPPMX) and (b) a targeting moiety having a specific binding affinity to folate receptors. In some embodiments, the targeting moiety has a specific binding affinity to folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has a specific binding affinity to folate receptor alpha (FR-α) and folate receptor beta (FR-β). In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., TPLp-αPPMX). In some embodiments, the administered liposome composition comprises non-pegylated liposomes. In some embodiments, the liposomes of the administered liposome composition contain αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition contain alpha-tetraglutamine oxidized pemetrexed. In some embodiments, the liposomes of the administered liposome composition contain alpha-pentaglutamine oxidized pemetrexed. In other embodiments, the liposomes of the administered liposome composition contain alpha-hexaglutamine oxidized pemetrexed. In some embodiments, the liposome composition is administered to treat non-hematological malignancies, including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and cancers selected from the group consisting of, for example, hematological malignancies, including leukemia, lymphoma and other B-cell malignancies, myeloma and other plasmacytic dysplasia or cachexia. In some embodiments, the liposome composition is administered to treat lung cancer (e.g., NSCLC or mesothelioma).In some embodiments, the liposomal composition is administered to treat breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the liposomal composition is administered to treat colorectal cancer. In some embodiments, the liposomal composition is administered to treat ovarian cancer. In some embodiments, the liposomal composition is administered to treat endometrial cancer. In some embodiments, the liposomal composition is administered to treat pancreatic cancer. In some embodiments, the liposomal composition is administered to treat liver cancer. In some embodiments, the liposomal composition is administered to treat head and neck cancer. In some embodiments, the liposomal composition is administered to treat osteosarcoma.

[0042] In further embodiments, the Disclosure provides a method for maintenance therapy of cancer, the method comprising the step of administering an effective amount of a liposomal composition comprising liposomes containing alpha-polyglutamine oxidized pemetrexed (Lp-αPPMX) to a subject receiving or formerly receiving cancer therapy. In some embodiments, the administered liposomal composition is PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX. In some embodiments, the administered liposomal composition comprises pegylated liposomes (e.g., PLp-αPPMX, NTPLp-αPPMX, or TPLp-αPPMX). In some embodiments, the administered liposomal composition comprises targeted liposomes (e.g., TLp-αPPMX or TPLp-αPPMX). In some embodiments, the administered liposomal composition comprises pegylated and targeted liposomes (e.g., TPLp-αPPMX). In some embodiments, the liposomes of the administered liposome composition contain alpha-polyglutamine oxidized pemetrexed, which contains 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition contain alpha-tetraglutamine oxidized pemetrexed. In some embodiments, the liposomes of the administered liposome composition contain alpha-pentaglutamine oxidized pemetrexed. In other embodiments, the liposomes of the administered liposome composition contain alpha-hexaglutamine oxidized pemetrexed.

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

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

[0045] In further embodiments, the Disclosure provides a method for preparing a liposome composition comprising a liposome alpha-polyglutamine oxidized pemetrexed (αPPMX) composition, the method comprising the steps of: forming a mixture comprising liposome components and α-polyglutamine oxidized pemetrexed in solution; homogenizing the mixture in solution to form liposomes; and processing the mixture to form liposomes comprising polyglutamine oxidized pemetrexed. In some embodiments, alpha-polyglutamine oxidized pemetrexed comprises 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the polyglutamine oxidized pemetrexed composition comprises alpha-tetraglutamine oxidized pemetrexed. In some embodiments, the polyglutamine oxidized pemetrexed composition comprises alpha-pentaglutamine oxidized pemetrexed. In other embodiments, the polyglutamine oxidized pemetrexed composition comprises alpha-hexaglutamine oxidized pemetrexed.

[0046] In one embodiment, the Disclosure provides a kit comprising an alpha-polyglutamine oxidized pemetrexed composition and / or an αPPMX delivery carrier such as a liposome containing the αPPMX and αPPMX immune complex (e.g., ADC) described herein. [Brief explanation of the drawing]

[0047] [Figure 1]Figures 1A to 1L show the chemical formulas of pemetrexed (Figure 1A), representative alpha-pemetrexed alpha-polyglutamate, pemetrexed diglutamate (Figure 1B), pemetrexed triglutamate (Figures 1C and 1D), pemetrexed tetraglutamate (Figures 1E and 1F), pemetrexed pentaglutamate (Figures 1G and 1H), pemetrexed hexaglutamate (Figures 1I and 1J), pemetrexed heptaglutamate (Figures 1K and 1L), pemetrexed octaglutamate (Figures 1M and 1N), representative alpha-pemetrexed polyglutamate (Figure 1O), and representative pemetrexed analogs (Figures 1P and 1Q). Figures 1R to 1U show the structures of representative branched pemetrexed polyglutamates, including branched polyglutamates having a gamma-glutamyl skeleton and an alpha-glutamyl branch (Figure 1S) and branched polyglutamates having an alpha-glutamyl skeleton and a gamma-glutamyl branch (Figure 1T). [Figure 2] This study demonstrates the relative potency of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6) and its enantiomer, liposomal alpha-D hexaglutamate (liposomal aDG6), compared to pemetrexed, after 48 hours of exposure to cancer cell lines SW620 (CRC), HT-29 (colon cancer), H1806 (triple-negative breast cancer), OAW28 (ovarian cancer), H292 (NSCLC, adenocarcinoma subtype), and H2342 (NSCLC, adenocarcinoma subtype). [Figure 3] This example shows the dose-response relationship, expressed as the percentage of surviving cells after 48 hours of treatment, for free pemetrexed L-gammahexaglutamate (gG6), liposomal pemetrexed L-gammahexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha-targeted antibody (FR1Ab) liposomal pemetrexed L-gammahexaglutamate (liposomal gG6-FR1Ab) in NCI H2342 non-small cell lung cancer (NSCLC), adenocarcinoma subtype. Folate receptor alpha-targeted liposomes containing alpha-polyglutamate pemetrexed are predicted to successfully target NCI H2342 non-small cell lung cancer cells and reduce the survival rate of these cancer cells. [Figure 4] This example shows the 48-hour dose-response relationship of free pemetrexed L-gammahexaglutamate (gG6), liposomal pemetrexed L-gammahexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha-targeted antibody (FR1Ab) liposomal pemetrexed L-gammahexaglutamate (liposomal gG6-FR1Ab) in HT-29 (colon cancer) cells. Folate receptor alpha-targeted liposomes containing alpha-polyglutamic pemetrexed are also predicted to successfully target HT-29 (colon cancer) cells and reduce their survival rate. [Figure 5] This study demonstrates the therapeutic efficacy of liposomal pemetrexed alpha-L hexaglutamate (Lps Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate (Lps Hexa aDG6), and pemetrexed against HCC1806 triple-negative breast cancer after 48 hours of exposure. [Figure 6] This study demonstrates the therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (LPS Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate (LPS Hexa aDG6), and pemetrexed on OAW28 ovarian cancer cells after 48 hours of exposure. [Figure 7] The therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (Lps Hexa aG6) and liposomal pemetrexed alpha-D hexaglutamate (Lps Hexa aDG6) on H292 non-small cell lung cancer cells after 48 hours of exposure are shown compared to pemetrexed alone. [Figure 8]This study demonstrates the therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed on H292 non-small cell lung cancer cells after 48 hours of exposure at various dose levels ranging from 16 to 128 nM. In each tested dose range, the liposomal pemetrexed aG6 formulation showed superior suppression of H292 non-small cell lung cancer cells compared to pemetrexed. [Figure 9] This study demonstrates the therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed on HCC1806 triple-negative breast cancer cells after 48-hour exposure at various dose levels ranging from 16 to 128 nM. In each tested dose range, the liposomal pemetrexed aG6 formulation was superior to pemetrexed in suppressing HCC1806 triple-negative breast cancer cells. [Figure 10] This study demonstrates the therapeutic effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed on OAW28 ovarian cancer cells after 48-hour exposure at a range of concentrations. At a dose of 128 nM, pemetrexed appears to be more effective than the liposomal pemetrexed aG6 formulation, but at doses of 32 nM and 64 nM, the liposomal formulations exhibit superior therapeutic effects compared to pemetrexed, and at 16 nM, the therapeutic effect of liposomal pemetrexed aG6 is similar to that of pemetrexed. [Figure 11] The toxicity of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed to differentiated human neutrophils at 64 nM, 128 nM, and 264 nM levels. The figure shows that liposomal pemetrexed aG6 is significantly less toxic to differentiated human neutrophils than pemetrexed. [Figure 12] This study demonstrates the effects on neutrophils (differentiated from CD34+ cells) after 48 hours of exposure to various dose levels ranging from 16 to 128 nM of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal alpha-D hexaglutamate (liposomal aDG6), and the corresponding pemetrexed agents. [Figure 13] This study demonstrates the effects on AML12 hepatocytes after 48-hour exposure to liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and the corresponding 16 nM, 32 nM, 64 nM, and 128 nM doses of pemetrexed. Notably, none of the tested dose levels of the liposomal agents appeared to be toxic to AML12 hepatocytes after treatment with liposomal pemetrexed aG6. In contrast, pemetrexed treatment resulted in approximately a 40% reduction in AML12 hepatocyte counts at all doses investigated. [Figure 14] This study demonstrates the effects of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and the effects of pemetrexed at 16 nM, 32 nM, 64 nM, and 128 nM concentrations on CCD841 colon epithelial cells after 48 hours of exposure. At all concentrations tested, pemetrexed resulted in a reduction of approximately 50% or more in CCD841 colon epithelial cell counts, compared to a reduction of approximately 20% or less after treatment with each of the liposomal compositions tested. [Figure 15] The structures of the polyglutamate folate antagonist, cisplatin (CDDP), and two possible aG6-cisplatin complexes are shown. The pH-dependent formation of interchain and / or intrachain coordination between the carboxyl group of the polyglutamate folate antagonist and cisplatin may lead to degradation into separate molecules of aG6 and cisplatin upon encountering acidic lysosomes (pH 4-5) and in the presence of intracellular chloride ions. [Figure 16]Hematological parameters: The effects of liposomal aG6 treatment in mice with weekly administration of 40 mg / kg and 80 mg / kg for 4 weeks are shown on white blood cell (WBC) count, neutrophil count, and platelet count. No significant decrease in mean neutrophils, mean white blood cell count, or mean platelet count was observed. [Figure 17] This study demonstrates the effects of liposomal aG6 therapy on hemoglobin and reticulocyte count index in mice, administered weekly at doses of 40 mg / kg and 80 mg / kg for 4 weeks. Higher dose levels show a minimal decrease in mean hemoglobin concentration, while simultaneously exhibiting a slight increase in mean reticulocyte count index. [Figure 18] This study demonstrates the effects of liposomal aG6 therapy in mice with weekly doses of 40 mg / kg and 80 mg / kg for 4 weeks on liver markers including serum albumin, serum aspartate aminotransferase (AST), and serum alanine aminotransferase (ALT). No significant increase was observed in mean AST or mean ALT levels, and no change in mean albumin levels was observed. [Figure 19] This shows the relative tumor volume of immunodeficient female Nu / J mice (6-8 weeks old) inoculated with NCI-H292 (non-small cell lung cancer) cells and administered intravenously once every 3 weeks at doses of control, pemetrexed, and liposomal aG6 at 167 mg / kg. As these preliminary data show, liposomal aG6 results in reduced tumor control compared to pemetrexed. [Figure 20] The results of a survival study using liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6) in a xenograft model of NSCLC (H292) are shown. The survival curve for mice (10) administered 90 mg / kg of liposomal aG6 intravenously once a week for 4 weeks (90 mg / kg administered subcutaneously weekly for 6 weeks) is shown as a solid circle. The survival curve for mice (10) administered pemetrexed (167 mg / kg intravenously every 3 weeks for 6 weeks) is shown as a solid triangle. Administration of 167 mg / kg of pemetrexed to mice is equivalent to administration of 500 mg / m2 in humans. The survival curve for control mice (10) is shown as a hollow diamond. [Figure 21A-F] Liposomal pemetrexed alpha-L triglutamate was administered over 48 hours to H2342 (NSCLC, adenocarcinoma subtype) (Figure 21A), H292 (NSCLC, adenocarcinoma subtype) (Figure 21B), HT-29 (colon cancer) (Figure 21C), HCC1806 (triple-negative breast cancer) (Figure 21D), MCF7 (ER+ breast cancer) (Figure 21E), and OAW28 (ovarian cancer) (Figure 21F). The dose-response relationships for liposome aG3), liposome pemetrexed alpha-L pentaglutamate (liposome aG5), liposome pemetrexed alpha-L octaglutamate (liposome aG7), and the combination of liposome pemetrexed alpha-L hexaglutamate (aG6) and alpha-L dodecaglutamate (aG12) (liposome aG6 and aG12) are shown. Cell viability was measured by the CellTiter-Glo® (CTG) luminescent cell viability assay, basically as described in Example 1. As shown for all cell lines, the potency of each polyglutamate-oxidized pemetrexed liposome composition well exceeded the potency of the liposome carrier and the empty liposome control. [Modes for carrying out the invention]

[0048] Generally, this disclosure relates to a novel alpha-polyglutamine oxidized pemetrexed composition. The composition represents an advance to the treatment of existing hyperproliferative diseases such as cancer. Methods for manufacturing, delivering, and using the alpha-polyglutamine oxidized pemetrexed composition are also provided. The alpha-polyglutamine oxidized composition has applications including, but is not limited to, the treatment or prevention of hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0049] I. Definition Unless otherwise specified, all technical and scientific terms used herein shall have the same meaning as those generally interpreted by those skilled in the art to which this disclosure belongs.

[0050] Whenever an embodiment is described herein with the term “comprising,” other similar embodiments described with the terms “containing,” “consisting of,” and / or “consisting essentially of,” are also provided. However, when used as transitional clauses in the claims, each should be interpreted separately and in the appropriate legal and factual context (for example, in the claims, the transitional clause “comprising” is considered a more open phrase, “consisting of” is considered more exclusive, and “consisting essentially of” is considered intermediate between these).

[0051] As used herein, the singular forms "a," "an," and "the" refer to multiple references unless otherwise specified or unless the context clearly indicates that multiple references are not intended.

[0052] In this specification, the term "and / or" as used 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" as used in expressions such as "A, B and / or C" each encompasses the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

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

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

[0055] The terms "polyglutamate," "polyglutamine oxidation," or their variations refer to a composition comprising at least one chain of two or more bonded glutamyl groups. Polyglutamate chains can be linear or branched. Linear polyglutamate chains may, for example, contain glutamyl groups with alpha-carboxyl or gamma-carboxyl group bonds. Branched polyglutamate chains may contain, for example, one or more glutamyl groups with both alpha-carboxyl and gamma-carboxyl group bonds to other glutamyl groups, thereby providing branching points in the polyglutamate. Representative branched polyglutamates are shown in Figures 1R-1U. Polyglutamate chains contain an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of a polyglutamate chain does not bond to another glutamyl group via its amino group, but rather to one or more glutamyl groups via its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of polyglutamine oxidized pemetrexed is the glutamyl group of pemetrexed. The C-terminal glutamyl groups (one or more) of the polyglutamate chain bond to another glutamyl group via their amino groups, but not via their carboxylic acid groups.

[0056] The terms “polyglutamine oxidized pemetrexed,” “polyglutamine oxidized PMX,” “PMX-PG,” “PPMX,” and their repeats are used herein to mean the same thing, and refer to a pemetrexed composition (i.e., PMX-PG) that contains at least one glutamyl group in addition to the glutamyl group in pemetrexed. n This means n≧1). References to the number of glutamyl groups in αPPMX (PMX-PG) herein take into account the glutamyl groups in pemetrexed. For example, a PMX-PG composition containing five glutamyl residues in addition to the glutamyl groups of PMX is herein referred to as hexaglutamic oxidized pemetrexed or pemetrexed hexaglutamate.

[0057] The terms “alpha-glutamyl group,” “alpha-glutamate,” and “alpha bond” refer to a glutamyl group containing an alpha-carboxyl bond when they relate to the bond of a glutamyl group. In some embodiments, the alpha bond is an amide bond between an alpha-carboxyl group of one glutamyl group and a second glutamyl group. The alpha bond may be a bond between a glutamyl group and a glutamyl group in pemetrexed, or between a glutamyl group and a second glutamyl group, such as a glutamyl group in a polyglutamate chain that is not present in pemetrexed but is bonded to pemetrexed.

[0058] The terms “gamma-glutamyl group,” “gamma-glutamate,” and “gamma bond” refer to a glutamyl group containing a gamma-carboxyl bond when these relate to the bonding of a glutamyl group. As discussed herein, when pemetrexed enters a cell, it is polyglutamic oxidized by the enzyme folyl polygamma-glutamate synthase (FPGS), which sequentially adds L-glutamyl groups to the gamma-carboxyl groups of glutamates in pemetrexed. Thus, alpha-polyglutamic pemetrexed compositions are not formed in cells during pemetrexed therapy. In some embodiments, the gamma bond is an amide bond between the gamma-carboxyl group of one glutamyl group and a second glutamyl group. A gamma bond can be a bond between a glutamyl group and a glutamyl group in pemetrexed, or between a glutamyl group and a second glutamyl group, such as a glutamyl group in a polyglutamate chain bonded to pemetrexed that is not present in pemetrexed. In some embodiments, a gamma bond refers to an amide bond of the glutamyl group in pemetrexed. Unless otherwise specified or clearly indicated by the context, references to gamma bonds include gamma bonds of the glutamyl group in pemetrexed.

[0059] Unless otherwise specified, the terms “alpha-polyglutamine oxidized pemetrexed,” “αPPMX,” “alpha-PMX-PG,” and their repetitions are used herein to mean the same thing and refer to a polyglutamine oxidized pemetrexed composition comprising at least one glutamyl group containing an alpha bond. For example, a pentaglutamine oxidized PMX composition in which the second glutamyl group has an alpha bond, but each of the other glutamyl groups has a gamma bond, is considered alpha-PMX-PG in this disclosure. In some embodiments, each glutamyl group of PMX-PG other than the glutamyl group of PMX has an alpha bond (for example, PMX-PG in n=5 where G1, G2, G3, G4, and G5 each have an alpha bond). n ). In some embodiments, the C-terminal glutamyl group(s) or each glutamyl group of PMX-PG other than the glutamyl group of PMX has an alpha linkage (for example, PMX-PG when n=5 and each of G1, G2, G3, and G4 has an alpha linkage). n ). In some embodiments, each glutamyl group of PMX-PG other than the C-terminal glutamyl group(s) has an alpha linkage (for example, PMX-PG when n=5 and each of the glutamyl groups of PMX and G1, G2, G3, and G4 has an alpha linkage). n ).

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

[0061] As used herein, the term “targeting portion” means a molecule that provides enhanced affinity to a selective target, such as a cell, cell type, tissue, organ, region of the body, or compartment, such as a cell, tissue, or compartment of an organ. Targeting portions can include a wide variety of substances. Targeting portions may include native molecules, or recombinant or synthetic molecules. In some embodiments, the targeting portion is an antibody-antigen-binding antibody fragment, a bispecific antibody, or other antibody-based molecule or compound. In some embodiments, the targeting portion is an aptamer, avimer, receptor-binding ligand, nucleic acid, biotin-avidin bond pair, peptide, protein, carbohydrate, lipid, vitamin, toxin, microbial component, hormone, receptor ligand, or any derivative thereof. Other targeting portions are known in the Art and are included herein.

[0062] The term “specific affinity” or “specifically binding” means that a targeting moiety, such as an antibody or antigen-binding antibody fragment, reacts to or binds to an epitope, protein, or target molecule more frequently, more rapidly, for longer periods, with greater affinity, or in some combination of these, than it would to another substance containing a protein unrelated to the target epitope. Due to sequence identity between homologous proteins in different species, specific affinity, in some embodiments, may include a binding agent that recognizes two or more proteins or targets in different species. Similarly, due to homology within specific regions of polypeptide sequences of different proteins, the term “specific affinity” or “specific binding” may include a binding agent that recognizes two or more proteins or targets. In certain embodiments, 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 (though may include) exclusive binding, e.g., binding to only one target. Therefore, a targeting moiety may, in certain embodiments, specifically bind to two or more targets. In certain embodiments, multiple targets may be combined by the same targeting portion.

[0063] The term "epitope" refers to a portion of an antigen that can be recognized and specifically bound to a targeting portion (i.e., a binding site) such as an antibody. When the antigen is a polypeptide, the epitope can be formed from both continuous and discontinuous amino acids juxtaposed by the protein's tertiary folding. Epitopes formed from continuous amino acids are usually retained during protein denaturation, while epitopes formed by tertiary folding are usually lost during protein denaturation. Epitopes typically contain at least three amino acids, more commonly at least five or eight to ten amino acids, within a distinctive spatial higher-order structure.

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

[0065] The term “delivery carrier” typically refers to any composition that assists, promotes, or facilitates the entry of alpha-polyglutamine oxidized pemetrexed into cells. Such delivery carriers are known in the art and are not limited to, but include liposomes, lipospheres, polymers (e.g., polymer complexes), peptides, proteins such as antibodies (e.g., immune complexes such as antibody-drug conjugates (ADCs) and antigen-binding antibody fragments and their derivatives), cellular components, cyclic oligosaccharides (e.g., cyclodextrins), micelles, microparticles (e.g., microspheres), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), hydrogels, lipoprotein particles, viral sequences, viral substances, or lipid or liposome formulations, and combinations thereof. The delivery carrier can be directly or indirectly bound to the targeting moiety. In some examples, the targeting moiety is selected from polymers, proteins, peptides, monoclonal antibodies, or fatty acid lipids.

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

[0067] As used herein, “effective dose” means a dose of the drug sufficient to produce the medically desired result. The effective dose may vary depending on the desired outcome, the specific condition being treated or prevented, the age and health status of the person being treated, the severity of the condition, the duration of treatment, the nature of any concurrent or adjunctive therapies, the specific route of administration, and similar factors within the scope of the knowledge and professional opinion of a healthcare practitioner. The “effective dose” can be determined experimentally and routinely in relation to the stated purpose. In the case of cancer, an effective dose of the drug may reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow to some extent, preferably stop) the invasion of cancer cells into surrounding organs; inhibit (i.e., slow to some extent, preferably stop) the metastasis of the tumor; inhibit (i.e., slow to some extent, preferably stop) the growth of the tumor; and / or alleviate to some extent one or more of the symptoms associated with the disorder. Depending on the extent to which the drug can prevent and / or kill existing cancer cells, the drug may be inhibitory and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by evaluating survival time, progression-free survival (PFS) time, response rate (RR), response duration, and / or quality of life.

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

[0069] The terms “cancer,” “tumor,” or “malignant tumor” are used synonymously to mean any of many diseases characterized by uncontrolled, abnormal proliferation of cells, localized or metastatic spread of infected cells to other parts of the body via the bloodstream and lymphatic system, and numerous distinctive structural and / or molecular features. As used herein, “tumor” means all neoplastic cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. “Malignant tumor,” or “malignant cell,” is understood to be a cell that has specific structural characteristics, lacks differentiation, and is capable of invasion and metastasis. Cancers that can be treated with the αPPMX compositions provided herein include, but are not limited to, non-hematological malignancies, such as lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies, such as leukemia, lymphoma and other B-cell malignancies, myeloma and other plasmacytic dysplasia or cachexia. Other types of cancers and tumors that can be treated with the αPPMX compositions are described herein or known in the art. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive when used herein.

[0070] Terms such as “to treat,” “to cure,” or “to treat” mean both (a) therapeutic means that cure, slow, reduce, and / or halt the progression of the symptoms of a diagnosed condition or disorder, and (b) preventive or protective means that prevent and / or delay the onset of a targeted disease or condition. Accordingly, subjects requiring treatment include subjects who already have the cancer, disorder or disease, subjects at risk of developing the cancer or condition, and subjects for whom infection or a condition should be prevented. Subjects are identified using well-known medical and diagnostic techniques as “at risk of having” cancer, infectious disease, immune system disorder, hyperproliferative disorder, or another disease or disorder as referred herein. In certain embodiments, if a subject exhibits overall, partial, or temporary remission or elimination of symptoms associated with a disease or condition (e.g., cancer, rheumatoid arthritis), the subject is “treated” by the methods provided herein. In certain embodiments, the terms “treating,” “treatment,” or “treat” mean improvement of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not be identifiable by the patient. In other embodiments, the terms “treating,” “treatment,” or “treat” mean suppression of the progression of a proliferative disorder, for example, physically by stabilizing identifiable symptoms, or physiologically, for example, by stabilizing physical parameters, or both. In other embodiments, the terms “treating,” “treatment,” or “treat” mean reduction or stabilization of size, tumor cell proliferation or survival, or cancer cell number. For treatment, the α-PPMX composition may be used alone or in combination with additional therapeutic agents.

[0071] The terms “subject,” “patient,” and “animal” are used synonymously and mean human patients and mammals such as non-human primates, as well as laboratory animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, mammals and non-mammals such as chickens, amphibians, and reptiles. As used herein, “mammal” means any member of the class Mammalia, including, but not limited to, humans and non-human primates, such as chimpanzees and other apes and monkey species; domestic animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals such as rodents such as mice, rats, and guinea pigs, and other members of the class Mammalia known in the art. In certain embodiments, the patient is human.

[0072] As used herein, “treatment of proliferative disorders” includes maintaining or reducing the size of the tumor of the subject of the proliferative disorder, inducing tumor reduction (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, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma. In one embodiment, the proliferative disorder is a hematological malignancy. Such hematological malignancies include, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasmacytotic dysplasia or cachexia.

[0073] As used herein, the term “autoimmune disease” is defined as a disorder resulting from an autoimmune reaction. Autoimmune diseases are the result of an inappropriate and excessive reaction to autoantigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes mellitus (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthritis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.

[0074] As used herein, the term “therapeutic agent” means a drug or derivative thereof that interacts with overgrowth cells, such as cancer cells or immune cells, thereby reducing the proliferative state of the cells and / or killing them. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, cytotoxic agents, platinum-based drugs (e.g., cisplatin, carboplatin, oxaliplatin), taxanes (e.g., Taxol®), etoposide, alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate (MTX)), 5-fluorouracil, gemcitabine, or derivatives thereof), antitumor antibiotics (e.g., mitomycin, doxorubicin), and plant-derived antitumor agents (e.g., vincristine, vindesine, Taxol®). Such agents include, but are not limited to, the anticancer agents trimethrexate, temozolomide, larcitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benziguanidine (6-BG), bis-chloronitrosourea (BCNU), and camptothecin, or any therapeutic derivative thereof. Further examples of therapeutic agents that may be suitable for use by the methods of this disclosure include, but are not limited to, anti-restenotic agents, proliferative or antiproliferative agents, anti-inflammatory agents, antineoplastic agents, antimitotic agents, antiplatelet agents, anticoagulants, antifibrin agents, antithrombin agents, cell proliferation inhibitors, antibiotics and other anti-infective agents, anti-enzyme agents, antimetabolite agents, angiogenic agents, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, and / or cardioprotective agents. "Therapeutic agent" also means the salts, acids, and free base forms of the above agents.

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

[0076] As used herein, the term “antometabolic antagonist” means a therapeutic agent that inhibits the utilization of a metabolite or its prodrug. Examples of antimetabolic antagonists include methotrexate, pemetrexed, 5-fluorouracil, 5-fluorouracil prodrugs such as capecitabine, 5-fluorodeoxyuridine monophosphate, cytarabine, nerarabine and other cytarabine prodrugs, 5-azacitidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine. Nucleoside analogs, including purines or pyrimidine analogs, are useful antimetabolic antagonists for carrying out the methods disclosed herein. In some embodiments, alpha-polyglutamine oxidized pemetrexed compositions are used in combination with an antimetabolite selected from the group consisting of fluoropyrimidine, 5-fluorouracil, 5-fluoro-2'-deoxycytidine, cytarabine, gemcitabine, troxacitabine, decitabine, azacitidine, pseudoisocytidine, zebralin, ancitabine, fazarabine, 6-azacitidine, capecitabine, N4-octadecylcytarabine, elaidic acid cytarabine, fludarabine, cladribine, clopharabine, nerarabine, folodesine, and pentostatin, or derivatives thereof. In one example, the nucleoside analog is a substrate of a nucleoside deaminase, which is adenosine deaminase or cytidine deaminase. In some examples, the nucleoside analog is selected from fludarabine, cytarabine, gemcitabine, decitabine, and azacitidine or their derivatives. In certain embodiments, the antimetabolite is 5-fluorouracil.

[0077] As used herein, “taxane” is an anticancer agent that interferes with or disrupts microtubule stability, formation, and / or function. Taxanes include paclitaxel and docetaxel and their derivatives, the derivatives of which function on microtubules in the same mode of action 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-conjugated paclitaxel (nab-paclitaxel; Abraxane®), DHA-paclitaxel, or PG-paclitaxel.

[0078] The terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable carrier" refer to components in a pharmaceutical preparation other than the active ingredient that are non-toxic to the target. pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents, or preservatives. Examples of pharmaceutically acceptable carriers include one or more compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans or other subjects.

[0079] This disclosure generally relates to novel alpha-polyglutamine oxidized pemetrexed (PMX) compositions, and to methods for manufacturing and using these compositions to treat diseases including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0080] In some embodiments, this disclosure provides the following: [1] A composition comprising alpha-polyglutamine oxidized pemetrexed, wherein at least one glutamyl group has an alpha-carboxyl group bond; [2] A composition according to item [1], wherein alpha-polyglutamine oxidized pemetrexed comprises 1 to 10 glutamyl groups having alpha-carboxyl group bonds; [3] A composition according to item [1] or [2], wherein alpha-polyglutamine oxidized pemetrexed comprises 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups; [4] A composition according to any one of items [1] to [3], comprising alpha-tetraglutamine oxidized pemetrexed; [5] A composition according to any one of items [1] to [3], comprising alphapentaglutamine oxidized pemetrexed; [6] A composition according to any one of items [1] to [3], comprising alpha-hexaglutamine oxidized pemetrexed; [7] A composition described in any one of items [1] to [6], wherein the composition is as follows: (a) Two or more glutamyl groups having alpha-carboxyl group bonds, (b) Each glutamyl group other than the glutamyl group of pemetrexed has an alpha-carboxyl group bond, (c) A composition having two or more glutamyl groups bonded to a gammacarboxyl group; [8] A composition described in any of items [1] to [6], which is as follows: (a) Each glutamyl group other than the C-terminal glutamyl group and the glutamyl group of pemetrexed has an alpha-carboxyl group bond; or (b) A composition in which each glutamyl group other than the C-terminal glutamyl group(s) has an alpha-carboxyl group bond; [9] A composition according to any one of items [1] to [8], wherein at least one glutamyl group has both an alpha-carboxyl group bond and a gamma-carboxyl group bond;

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

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

[10] , wherein the polyglutamate is linear;

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

[10] , wherein the polyglutamate is a branched chain;

[13] Liposome composition (Lp-αPPMX) containing alpha-polyglutamine oxidized pemetrexed as described in any one of items [1] to

[12] ;

[14] A LαPP composition according to item

[13] , wherein alpha-polyglutamine oxidized pemetrexed comprises an L-type glutamyl group having an alpha-carboxyl group bond;

[15] A composition of Lp-αPPMX as described in item

[13] or

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

[16] Lp-αPPMX compositions according to item

[13] or

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

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

[13] to

[16] , wherein the liposomes contain alpha-polyglutamine oxidized pemetrexed having 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups;

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

[13] to

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

[19] A composition according to any one of items

[13] to

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

[20] A composition according to any one of items

[13] to

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

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

[13] to

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

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

[13] to

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

[23] A composition (PαLp-αPPMX) according to any one of items

[13] to

[22] , wherein the liposomes are pegylated;

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

[28] A composition of Lp-αPPMX as described in item

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

[29] Lp-αPPMX compositions according to item

[27] or

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

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

[27] to

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

[31] A composition according to any one of items

[27] to

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

[32] A composition of Lp-αPPMX as described in item

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

[33] A composition of Lp-αPPMX as described in item

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

[33] , wherein the liposomes are cationic;

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

[13] to

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

[40] Lp-αPPMX compositions as described in item

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

[41] A composition of Lp-αPPMX as described in item

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

[42] A composition of Lp-αPPMX as described in item

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

[43] A composition of Lp-αPPMX as described in any one of items

[39] to

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

[44] A composition according to any one of items

[39] to

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

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

[39] to

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

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

[39] to

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

[47] A composition according to any one of items

[13] to

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

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

[13] to

[47] , wherein the liposomes contain fewer than 500,000 or fewer than 200,000 alpha-polyglutamine oxidized pemetrexed molecules;

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

[13] to

[48] , wherein the liposomes contain 10 to 100,000 or any range in between alpha-polyglutamine oxidized pemetrexed molecules;

[50] A composition according to any one of items

[13] to

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

[51] Lp-αPPMX compositions as described in item

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

[52] A composition relating to the Lp-αPPMX composition described in item

[50] or

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

[53] A composition according to any one of items

[50] to

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

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

[50] to

[53] , wherein the targeting portion is measured by BIACORE® analysis to be 0.5 x 10 -10 ~10x10 -6 A composition that binds to a surface antigen with an equilibrium dissociation constant (Kd) within the range of [value];

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

[50] to

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

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

[50] to

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

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

[50] to

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

[58] A composition according to item

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

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

[39] to

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

[60] Lp-αPPMX composition as described in item

[58] or

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

[61] A composition of Lp-αPPMX described in any one of items

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] Lp-αPPMX compositions as described in item

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

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

[13] to

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

[65] Targeted compositions comprising any one of items [1] to

[64] ;

[66] Non-targeting compositions comprising any one of items [1] to

[49] ;

[67] A composition according to any one of items

[13] to

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

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

[13] to

[67] ;

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

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

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

[71] Use of any one of the compositions described in 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 of which treatment or prevention is in need, comprising the step of administering a composition of any one of items [1] to

[70] to the subject;

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

[13] to

[69] to the subject;

[74] A method for killing overgrown cells, comprising the step of bringing the overgrown cells into contact with a composition described in any one of items [1] to

[69] ;

[75] A method for killing hyperproliferating cells, comprising the step of contacting the hyperproliferating cells with a liposome alpha-polyglutamine oxidized pemetrexed composition described in any one of items

[13] to

[69] ;

[76] Methods relating to item

[74] or

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

[77] A method for treating cancer, comprising the step of administering an effective amount of 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 a liposomal alpha-polyglutamine oxidized pemetrexed composition described in any one of items

[13] to

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

[79] Methods according to item

[77] or

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

[80] Methods relating 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] Methods relating to item

[77] or

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

[82] Methods relating to item

[77] or

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

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

[50] to

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

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

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

[85] Maintenance therapy comprising the step of administering an effective amount of the liposomal alpha-polyglutamine oxidized pemetrexed composition described in any one of items

[13] to

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

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

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

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

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

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

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

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

[13] to

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

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

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

[91] A method for preparing an alpha-polyglutamine oxidized pemetrexed composition comprising the liposome alpha-polyglutamine oxidized pemetrexed composition described in any one of items

[13] to

[69] , comprising the steps of: forming a mixture containing liposome components and alpha-polyglutamine oxidized pemetrexed in solution; homogenizing the mixture in solution to form liposomes; and processing the mixture to form liposomes containing alpha-polyglutamine oxidized pemetrexed;

[92] A method for preparing an alpha-polyglutamine oxidized pemetrexed composition comprising the liposome alpha-polyglutamine oxidized pemetrexed composition described in any one of items

[13] to

[69] , comprising the steps of: forming a mixture containing liposome components and alpha-polyglutamine oxidized pemetrexed in solution; and processing the mixture to form liposomes containing alpha-polyglutamine oxidized pemetrexed;

[93] A method according to item

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

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

[13] to

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

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

[50] to

[69] , comprising the steps of: forming a mixture in solution comprising a liposome component and alpha-polyglutamine oxidized pemetrexed; processing the mixture to form liposomes that encapsulate and / or contain alpha-polyglutamine oxidized pemetrexed; and imparting a targeting portion to the surface of the liposomes, wherein the targeting portion has specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ);

[96] A method relating to item

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

[97] A method according to any one of items

[94] to

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

[98] A method according to any one of items

[94] to

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

[99] A method according to any one of items

[91] to

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

[0081] II. Alpha-polyglutamine oxidized pemetrexed (αPPMX) Generally, this disclosure relates to alpha-polyglutamine-oxidized pemetrexed (αPPMX) compositions. αPPMX compositions contain at least one glutamyl group having an alpha bond. These compositions are structurally different from L-gamma-polyglutamine-oxidized pemetrexed (LαPPMX), which is produced in cells by the enzyme folyl polygamma-glutamate synthase (FPGS) during pemetrexed therapy.

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

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

[0084] In some embodiments, alpha-polyglutamine-oxidized pemetrexed is triglutamine-oxidized. That is, alpha-polyglutamine-oxidized pemetrexed contains two additional glutamyl groups in addition to the glutamyl group of pemetrexed (αPMX-PG2). In some embodiments, each of the two additional glutamyl groups has an alpha bond. In other embodiments, one of the two additional glutamyl groups has an alpha bond and the other glutamyl group has a gamma bond. In some embodiments, one of the two additional glutamyl groups has an alpha bond. In some embodiments, one of the two additional glutamyl groups has a gamma bond. In some embodiments, two of the three glutamyl groups have alpha bonds. In other embodiments, one of the three glutamyl groups has an alpha bond and the other glutamyl group has a gamma bond. In some embodiments, one glutamyl group has both an alpha bond and a gamma bond. In some embodiments, each glutamyl group in alpha-triglutamine oxidized pemetrexed is L-type. In other embodiments, alpha-triglutamine oxidized PMX contains D-type glutamyl groups. In further embodiments, each glutamyl group in alpha-triglutamine oxidized pemetrexed, other than the glutamyl group in pemetrexed, is D-type. In further embodiments, triglutamine oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0085] In some embodiments, alpha-polyglutamine-oxidized pemetrexed is tetraglutamine-oxidized and therefore contains three additional glutamyl groups in addition to the glutamyl groups in pemetrexed (αPMX-PG3). In some embodiments, each of the three glutamyl groups has an alpha bond. In other embodiments, one or two of the three additional glutamyl groups have an alpha bond, and the remaining two or one glutamyl group each have a gamma bond. In some embodiments, two of the three additional glutamyl groups have an alpha bond. In other embodiments, one of the three glutamyl groups has an alpha bond, and another additional glutamyl group has a gamma bond. In other embodiments, one of the three additional glutamyl groups has both an alpha and a gamma bond. In other embodiments, three of the four glutamyl groups have an alpha bond. In some embodiments, at least one glutamyl group has both an alpha and a gamma bond. In some embodiments, alpha-tetraglutamine oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-tetraglutamine oxidized pemetrexed is L-type. In other embodiments, alpha-tetraglutamine oxidized PMX contains D-type glutamyl groups. In further embodiments, each glutamyl group in alpha-tetraglutamine oxidized pemetrexed, other than the glutamyl group in pemetrexed, is D-type. In further embodiments, tetraglutamine oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0086] In some embodiments, alpha-polyglutamine-oxidized pemetrexed is pentaglutamine-oxidized (αPMX-PG4) and contains a chain of four additional glutamyl groups bonded to the glutamyl group of pemetrexed. In some embodiments, each of the four additional glutamyl groups in the chain has an alpha bond. In some embodiments, each of the four additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In other embodiments, one, two, or three of the four additional glutamyl groups have an alpha bond, and the remaining three, two, or one glutamyl group are each bonded to the molecule's glutamyl group via gamma bonds. In other embodiments, one or two of the four additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl group is bonded to the molecule's glutamyl group via gamma bonds. In some embodiments, at least one additional glutamyl group has both an alpha and a gamma bond. In some embodiments, at least one of the five glutamyl groups has both an alpha and a gamma bond. In some embodiments, each of the five glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, alpha-pentaglutamine oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-pentaglutamine oxidized pemetrexed is L-type. In other embodiments, alpha-pentaglutamine oxidized PMX contains D-type glutamyl groups. In further embodiments, each glutamyl group in alpha-pentaglutamine oxidized pemetrexed, other than the glutamyl groups in pemetrexed, is D-type. In further embodiments, pentaglutamine oxidized PMX contains D-type glutamyl groups and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0087] In some embodiments, alpha-polyglutamine-oxidized pemetrexed is hexaglutamine-oxidized (αPMX-PG5) and contains a chain of five additional glutamyl groups bonded to the glutamyl group of pemetrexed. In some embodiments, each of the five additional glutamyl groups in the chain has an alpha bond. In some embodiments, each of the five additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, four of the five additional glutamyl groups in the chain have alpha bonds. In other embodiments, one, two, three, or four of the five additional glutamyl groups are bonded to the glutamyl group of the molecule via alpha bonds, and the remaining four, three, two, or one glutamyl group are each bonded to the glutamyl group of the molecule via gamma bonds. In other embodiments, one, two, three, or four of the five additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the six glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the six glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, five of the six glutamyl groups have alpha bonds. In some embodiments, alpha-hexaglutamine oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-hexaglutamine oxidized pemetrexed is L-type. In other embodiments, alpha-hexaglutamine oxidized PMX contains D-type glutamyl groups. In further embodiments, each glutamyl group in alpha-hexaglutamic pemetrexed, other than the glutamyl group in pemetrexed, is D-type. In further embodiments, hexaglutamic pemetrexed comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0088] In some embodiments, alpha-polyglutamine-oxidized pemetrexed is heptaglutamine-oxidized (αPMX-PG6) and therefore contains a chain of six additional glutamyl groups bonded to the glutamyl group of pemetrexed. In some embodiments, each of the six additional glutamyl groups has an alpha bond. In some embodiments, each of the six additional glutamyl groups in the chain, excluding the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, five of the six additional glutamyl groups in the chain have alpha bonds. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have alpha bonds, and the remaining five, four, three, two, or one glutamyl group each have a gamma bond. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the glutamyl group of the molecule via gamma bonds. In some embodiments, at least one additional glutamyl group has both an alpha and a gamma bond. In some embodiments, at least one of the seven glutamyl groups has both an alpha and a gamma bond. In some embodiments, each of the seven glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, six of the seven glutamyl groups have an alpha bond. In some embodiments, alpha-heptaglutamine oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-heptaglutamine oxidized pemetrexed is L-type. In other embodiments, alpha-heptaglutamine oxidized PMX contains D-type glutamyl groups. In further embodiments, each glutamyl group in alpha-heptaglutamine oxidized pemetrexed, other than the glutamyl group in pemetrexed, is D-type. In further embodiments, heptaglutamine-oxidized PMX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0089] In some embodiments, alpha-polyglutamine-oxidized pemetrexed is octaglutamine-oxidized (αPMX-PG7) and therefore contains a chain of seven additional glutamyl groups bonded to the glutamyl group of pemetrexed. In some embodiments, each of the seven additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, six of the seven additional glutamyl groups in the chain have an alpha bond. In some embodiments, each of the seven additional glutamyl groups has an alpha bond. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have an alpha bond, and the remaining six, five, four, three, two, or one glutamyl group each have a gamma bond. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl group is bonded to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha and a gamma bond. In some embodiments, at least one of the eight glutamyl groups has both an alpha and a gamma bond. In some embodiments, each of the eight glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, seven of the eight glutamyl groups have an alpha bond. In some embodiments, alpha-octaglutamine oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-octaglutamine oxidized pemetrexed is L-type. In other embodiments, alpha-octaglutamine oxidized PMX contains D-type glutamyl groups. In further embodiments, each glutamyl group in alpha-octaglutamine oxidized pemetrexed, other than the glutamyl group in pemetrexed, is D-type. In further embodiments, octaglutamine oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

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

[0091] In some embodiments, alpha-polyglutamine-oxidized pemetrexed is deca-glutamine-oxidized (αPMX-PG9) (i.e., it contains a chain of nine additional glutamyl groups bonded to the glutamyl group of pemetrexed). In some embodiments, each of the nine additional glutamyl groups has an alpha bond. In some embodiments, each of the nine additional glutamyl groups in the chain, excluding the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, eight of the nine additional glutamyl groups in the chain have alpha bonds. In other embodiments, one, two, three, four, five, six, seven, or eight of the nine additional glutamyl groups have alpha bonds, and the remaining eight, seven, six, five, four, three, two, or one glutamyl group each have a gamma bond. In other embodiments, one, two, three, four, five, six, seven, or eight of the nine additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the ten glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the ten glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, nine of the ten glutamyl groups have alpha bonds. In some embodiments, alpha-decaglutamine oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-decaglutamine oxidized pemetrexed is L-type. In other embodiments, alpha-decaglutamine oxidized PMX contains D-type glutamyl groups. In further embodiments, each glutamyl group in alphadecaglutamine-oxidized pemetrexed, other than the glutamyl group in pemetrexed, is D-type. In further embodiments, decaglutamine-oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

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

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

[0094] In some embodiments, alpha-polyglutamine-oxidized pemetrexed is triskaide-de-glutamine-oxidized (αPMX-PG 12In some embodiments, each of the 12 additional glutamyl groups has an alpha bond. In some embodiments, each of the 12 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 11 of the 12 additional glutamyl groups in the chain have alpha bonds. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have alpha bonds, and the remaining 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both an alpha and a gamma bond. In some embodiments, at least one of the 13 glutamyl groups has both an alpha and a gamma bond. In some embodiments, each of the 13 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 12 of the 13 glutamyl groups have an alpha bond. In some embodiments, alpha-triskaidecaglutamine oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-triskaidecaglutamine oxidized pemetrexed is L-type. In other embodiments, alpha-triskaidecaglutamine oxidized PMX contains D-type glutamyl groups. In further embodiments, each glutamyl group in alpha-triskaidecaglutamine oxidized pemetrexed, other than the glutamyl groups in pemetrexed, is D-type. In further embodiments, triskaidecaglutamine oxidized PMX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

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

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

[0097] In some embodiments, alpha-polyglutamine-oxidized pemetrexed is hexadeca-glutamine-oxidized (αPMX-PG 15In some embodiments, each of the 15 additional glutamyl groups has an alpha bond. In some embodiments, each of the 15 additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, 14 of the 15 additional glutamyl groups in the chain have alpha bonds. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have alpha bonds, and the remaining 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each have a gamma bond. In other embodiments, one, two, three, fourteen, five, sixteen, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen of the fifteen additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the sixteen glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the sixteen glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, fifteen of the sixteen glutamyl groups have alpha bonds. In some embodiments, alpha-hexadeca-glutamine oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-hexadeca-glutamine oxidized pemetrexed is L-type. In other embodiments, alpha-hexadeca-glutamine oxidized PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of alpha-hexadeca-glutamine oxidized pemetrexed, other than the glutamyl group of pemetrexed, is D-type. In further embodiments, hexadeca-glutamine oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0098] In other embodiments, alpha-polyglutamine-oxidized pemetrexed is heptadeca-glutamine-oxidized (αPMX-PG 16In some embodiments, each of the 16 additional glutamyl groups has an alpha bond. In some embodiments, each of the 16 additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, 15 of the 16 additional glutamyl groups in the chain have alpha bonds. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have alpha bonds, and the remaining 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each have a gamma bond. In other embodiments, one, two, three, four, five, sixteen, seventeen, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen of the sixteen additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the seventeen glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the seventeen glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, sixteen of the seventeen glutamyl groups have alpha bonds. In some embodiments, alpha-heptadeca-glutamine oxidized PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-heptadeca-glutamine oxidized pemetrexed is L-type. In other embodiments, alphaheptadecaglutamine oxidized PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group in alphaheptadecaglutamine oxidized pemetrexed, other than the glutamyl group in pemetrexed, is D-type. In further embodiments, heptadecaglutamine oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

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

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

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

[0102] In some embodiments, alpha-polyglutamine-oxidized pemetrexed is icosica henaglutamine-oxidized (αPMX-PG 20In some embodiments, each of the 20 additional glutamyl groups has an alpha bond. In some embodiments, each of the 20 additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, 19 of the 20 additional glutamyl groups in the chain have alpha bonds. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have alpha bonds, and the remaining 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the 21 glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the 21 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 20 of the 21 glutamyl groups have alpha bonds. In some embodiments, Alpha Kosika Hena Glutamate Oxide PMX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in Alpha Kosika Henaglutamine Oxide Pemetrexed is L-type. In other embodiments, Alpha Kosika Henaglutamine Oxide PMX contains D-type glutamyl groups. In further embodiments, each glutamyl group in Alpha Kosika Henaglutamine Oxide Pemetrexed, other than the glutamyl group in Pemetrexed, is D-type. In further embodiments, Ikosika Henaglutamine Oxide PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear.In other embodiments, the polyglutamate chain is a branched chain.

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

[0104] In one embodiment, alpha-polyglutamine-oxidized pemetrexed is tetraglutamine-oxidized, and each of the three glutamyl groups in the polyglutamate chain bonded to pemetrexed contains an alpha linkage. In another embodiment, alpha-polyglutamine-oxidized pemetrexed is tetraglutamine-oxidized, and each of the three glutamyl groups in the polyglutamate chain bonded to pemetrexed, except for the C-terminal glutamyl group(s), contains an alpha linkage. In some embodiments, each of the four glutamyl groups is L-type. In some embodiments, each of the glutamyl groups in alpha-tetraglutamine-oxidized pemetrexed, other than the glutamyl group of pemetrexed, is D-type. In other embodiments, at least two of the glutamyl groups in alpha-tetraglutamate-pemetrexed are L-type, and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0105] In one embodiment, alpha-polyglutamine-oxidized pemetrexed is pentaglutamine-oxidized, and each of the four glutamyl groups in the polyglutamate chain bonded to pemetrexed contains an alpha linkage. In another embodiment, alpha-polyglutamine-oxidized pemetrexed is pentaglutamine-oxidized, and each of the four glutamyl groups in the polyglutamate chain bonded to pemetrexed, except for the C-terminal glutamyl group(s), contains an alpha linkage. In some embodiments, each of the four glutamyl groups is L-type. In some embodiments, each of the glutamyl groups in alpha-pentaglutamine-oxidized pemetrexed, other than the glutamyl group of pemetrexed, is D-type. In other embodiments, at least two of the glutamyl groups in alpha-pentaglutamine-oxidized pemetrexed are L-type, and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0106] In one embodiment, alpha-polyglutamine-oxidized pemetrexed is hexaglutamine-oxidized, and each of the five glutamyl groups in the polyglutamate chain bonded to pemetrexed contains an alpha linkage. In one embodiment, alpha-polyglutamine-oxidized pemetrexed is hexaglutamine-oxidized, and each of the five glutamyl groups in the polyglutamate chain bonded to pemetrexed, except for the C-terminal glutamyl group(s), contains an alpha linkage. In some embodiments, each of the five glutamyl groups is L-type. In some embodiments, each of the glutamyl groups in alpha-hexaglutamine-oxidized pemetrexed, other than the glutamyl group of pemetrexed, is D-type. In other embodiments, at least two of the glutamyl groups in alpha-hexaglutamine-oxidized pemetrexed are L-type, and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is a branched chain.

[0107] In another embodiment, alpha-polyglutamine-oxidized pemetrexed is heptaglutamine-oxidized, and each of the six glutamyl groups in the polyglutamate chain bonded to pemetrexed contains an alpha linkage. In yet another embodiment, alpha-polyglutamine-oxidized pemetrexed is heptaglutamine-oxidized, and each of the six glutamyl groups in the polyglutamate chain bonded to pemetrexed, except for the C-terminal glutamyl group(s), contains an alpha linkage. In some embodiments, each of the six glutamyl groups is L-type. In some embodiments, each of the glutamyl groups in alpha-heptaglutamine-oxidized pemetrexed, other than the glutamyl group of pemetrexed, is D-type. In other embodiments, at least two of the glutamyl groups in alpha-heptaglutamine-oxidized pemetrexed are L-type, and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is a branched chain.

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

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

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

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

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

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

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

[0115] In some embodiments, the alpha-polyglutamine oxidized pemetrexed compositions provided herein can accept one or more additional glutamyl groups, i.e., the compositions can serve as substrates for FPGAs (folyl polyglutamate synthetase). Reagents, assays, and reagents for measuring the ability of alpha-polyglutamine oxidized pemetrexed compositions to act as substrates for FPGAs (e.g., human FPGAs or rat liver FPGAs) are readily available and can be performed as part of routine procedures.

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

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

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

[0119] In some embodiments, the alpha-polyglutamine-oxidized pemetrexed compositions provided herein have lower toxic side effects than pemetrexed. In some embodiments, the alpha-polyglutamine-oxidized pemetrexed compositions provided herein result in lower frequency or less severe toxic side effects than pemetrexed in in vivo assays. In some embodiments, the in vivo assay is performed in an in vivo mouse model. In some embodiments, the alpha-polyglutamine-oxidized pemetrexed compositions provided herein result in lower frequency or less severe hematological or hepatotoxic side effects than pemetrexed. In some embodiments, hematological side effects are assessed by mean neutrophils, mean leukocytes, or mean platelet counts. In some embodiments, hepatotoxic side effects are assessed by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the in vivo assay involves administering a 40 mg / kg or 80 mg / kg alpha-polyglutamine oxidized pemetrexed composition once a week for four weeks. In some embodiments, alpha-polyglutamine oxidized pemetrexed is hexaglutamine oxidized pemetrexed.

[0120] In some embodiments, treatment with the alpha-polyglutamine oxidized pemetrexed compositions provided herein does not induce significant hematological or hepatic toxic side effects in in vivo mouse models. In some embodiments, hematological side effects are assessed by mean neutrophil, mean leukocyte, or mean platelet count. In some embodiments, hepatic toxic side effects are assessed by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the alpha-polyglutamine oxidized pemetrexed compositions provided herein do not significantly decrease mean neutrophil, mean leukocyte, or mean platelet count. In some embodiments, the alpha-polyglutamine oxidized pemetrexed compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the alpha-polyglutamine oxidized pemetrexed compositions provided herein do not significantly decrease serum albumin levels. In some embodiments, the in vivo assay involves administering a 40 mg / kg or 80 mg / kg alpha-polyglutamine oxidized pemetrexed composition once a week for four weeks. In some embodiments, alpha-polyglutamine oxidized pemetrexed is hexaglutamine oxidized pemetrexed.

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

[0122] Alpha-polyglutamine oxidized pemetrexed (αPPMX) compositions and their uses are further described in U.S. Patent Applications No. 62 / 374,458, No. 15 / 675,695, No. 15 / 675,701, and No. 62 / 583,432, as well as in International Patent Applications PCT / US2017 / 046666 and PCT / US2017 / 046667, respectively. The contents of each of these are incorporated herein by reference in their entirety.

[0123] A. Polyglutamine oxidized pemetrexed analogs and derivatives This disclosure also encompasses alpha-polyglutamine-oxidized pemetrexed derivatives and analogs. The compositions and methods disclosed herein are intended to be applied to any and all known polyglutamine-oxidized pemetrexed derivatives or analogs. In some embodiments, polyglutamine-oxidized pemetrexed analog or derivative compositions prepared and used according to the disclosed compositions and methods are shown in Figures 1I and 1J. In some embodiments, the analog corresponds to a modified form of pemetrexed, in which case the glutamyl group of pemetrexed is not bound to the remainder of the pemetrexed molecule via a gamma-peptide bond. In some embodiments, the analog is a variant of pemetrexed, in which case the glutamyl group in pemetrexed is of the D type. In some embodiments, the polyglutamine-oxidized form of pemetrexed, or the polyglutamine-oxidized pemetrexed analog or derivative, is not fluorinated.

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

[0125] B.PMX-PG synthesis The pemetrexed polyglutamate compositions provided herein are obtained by the following known synthetic methods in the art: the procedure for synthesizing pemetrexed (including different pharmaceutically acceptable salts or acids (e.g., pemetrexed disodium) and crystalline and amorphous forms) and intermediates for synthesizing pemetrexed, but is not limited to U.S. Patents No. 8,507,508, 8,362,245, 7,138,521, 6,262,262, 6,066,732, and 5,416,2 Patent No. 11, No. 5,344,932; U.S. Patent Application Publication No. 2013 / 0165654A1; European Patent Nos. 0905128, 2882753B1, 0905128 and 2409978B1; International Publication Nos. 2014 / 024164A1, 2012 / 056285A1, 2008 / 021410A1 and 2001 / 14379A2, and Barnett Examples include those described in et al., Org. Proc Res & Develop. 3:184-188 (1999); Taylor et al., J. Org. Chem. 68:9938-9947 (2003); Taylor et al., Tetrahedron Lett. 40:4023 (1999); Barnett et al., Org. Proc. Res. & Develop. 3:184-188 (1999); and Kjell et al., Org. Proc. Res. Dev. 9:738 (2005).

[0126] The addition of glutamyl residues to the glutamyl residues of pemetrexed can be carried out using synthetic methods known in the art. In some embodiments, glutamyl residues are sequentially added to the glutamyl residues of pemetrexed. In further embodiments, polyglutamates are added to the glutamyl residues of pemetrexed using "click chemistry" or other bioconjugate chemistry methods known to those skilled in the art. Alternatively, a peptide of glutamyl residues of desired length can be generated and added to a precursor of pemetrexed that does not contain glutamyl residues. The peptide can be prepared using methods known in the art. In some embodiments, the initial glutamyl residues are bound to a wangle resin, and additional glutamyl residues are sequentially added by solid-phase peptide synthesis using F-moc chemistry. After the addition of the final glutamyl residue, the pemetrexed precursor is bound to the peptide, and the molecule is cleaved from the resin.

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

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

[0129] In some embodiments, the disclosure provides compositions comprising a complex of αPPMX with a therapeutic agent or a salt or acid thereof. In further embodiments, the αPPMX / therapeutic agent complex comprises one or more αPPMX containing 2-150, 2-100, 2-75, 2-50, 2-24, 2-30, 2-20, 2-19, 2-15, 2-10, or 2-5 glutamyl groups. In some embodiments, the αPPMX / therapeutic agent complex comprises one or more αPPMX containing 3-10, 3-9, 3-8, or 3-7 glutamyl groups, or any range in between. In other embodiments, the αPPMX / therapeutic agent complex comprises one or more αPPMX containing 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 glutamyl groups, or any range in between. In a particular embodiment, the complex comprises one or more αPPMX containing 3-10 glutamyl groups. In further embodiments, the αPPMX / therapeutic complex comprises one or more αPPMX compounds containing 3 to 7 glutamyl groups. In another embodiment, the αPPMX / therapeutic complex comprises one or more αPPMX compounds containing 5 glutamyl groups. In yet another embodiment, the αPPMX / therapeutic complex comprises one or more αPPMX compounds containing 6 glutamyl groups. In some embodiments, the therapeutic agent is a cytotoxic compound or its salt or acid. In further embodiments, the therapeutic agent is a chemotherapeutic agent or its salt or acid. In another embodiment, the chemotherapeutic agent is a platinum-based drug. In yet another embodiment, the chemotherapeutic agent is a taxane-based drug. In further embodiments, the molar ratio of αPPMX / therapeutic agent in the complex is in the range of 1 to 10:1. In some embodiments, the molar ratio of αPPMX / therapeutic agent in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50.In some embodiments, the molar ratio of αPPMX / therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In some embodiments, the αPPMX / therapeutic agent complex is encapsulated in liposomes (e.g., as described herein or by other methods known in the art).

[0130] In alternative embodiments, the αPPMX complex comprises αPPMX and cyclodextrin. In some embodiments, the molar ratio of αPPMX (e.g., αPPMX salt) / cyclodextrin in the complex is in the range of 1 to 20:1, or any range in between. In some embodiments, the molar ratio of αPPMX / cyclodextrin in the complex is in the range of 1 to 10:1, or any range in between. In further embodiments, the molar ratio of αPPMX / cyclodextrin in the complex is in the range of 2 to 8:1, or any range in between. In some embodiments, the molar ratio of αPPMX / cyclodextrin in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of αPPMX / cyclodextrin in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range in between. In some embodiments, the molar ratio of αPPMX / cyclodextrin in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In some embodiments, the αPPMX / cyclodextrin complex is encapsulated in liposomes (e.g., as described herein or by other methods known in the art).

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

[0132] In further embodiments, the αPPMX / platinum-based chemotherapeutic agent conjugate comprises cisplatin, carboplatin, oxaliplatin analogs, or their salts or acids. In some embodiments, the molar ratio of αPPMX / platinum-based analogs in the conjugate is in the range of 1 to 20:1, or any range in between. In some embodiments, the molar ratio of αPPMX / platinum-based analogs in the conjugate is in the range of 1 to 10:1, or any range in between. In further embodiments, the molar ratio of αPPMX / platinum-based agent in the conjugate is in the range of 2 to 8:1, or any range in between. In some embodiments, the molar ratio of αPPMX / platinum analogs in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In some embodiments, the molar ratio of αPPMX / platinum-based drug in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPPMX / platinum-based analog complex is encapsulated in liposomes (e.g., as described herein or by other methods known in the art).

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

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

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

[0136] In further embodiments, the Disclosure provides a conjugate comprising αPPMX and a platinum-based chemotherapeutic agent (platinum) selected from the group consisting of nedaplatin, heptaplatin, lovaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine, satoraplatin, enloplatin, JM216, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or salts or acids thereof. In other embodiments, the αPPMX / platinum-based chemotherapeutic agent conjugate includes nedaplatin, heptaplatin, lovaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine, satoraplatin, enloplatin, JM216, NK121, CI973, DWA2114R, NDDP, or analogues of nedaplatin, or salts or acids thereof. In some embodiments, the molar ratio of αPPMX / platinum (or platinum salt or acid) in the conjugate is in the range of 1 to 20:1, or any range in between. In further embodiments, the molar ratio of αPPMX / platinum (or platinum salt or acid) in the conjugate is in the range of 1 to 10:1, or any range in between. In further embodiments, the molar ratio of αPPMX / platinum (or a platinum salt or acid) in the complex is in the range of 2 to 8:1, or any range in between. In some embodiments, the molar ratio of αPPMX / platinum (or a 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 some embodiments, the molar ratio of αPPMX / platinum (or a salt or acid of platinum) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPPMX / platinum (or a salt or acid or analog thereof) complex is encapsulated in liposomes (e.g., as described herein or by other methods known in the art).

[0137] In some embodiments, the Disclosure provides compositions comprising an αPPMX / taxane chemotherapeutic agent (taxane) conjugate. In some embodiments, the taxane 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 the αPPMX / taxane agent in the conjugate is in the range of 1 to 20:1, or any range in between. In further embodiments, the molar ratio of the αPPMX / taxane (or a salt or acid of a taxane) in the conjugate is in the range of 1 to 10:1, or any range in between. In further embodiments, the molar ratio of the αPPMX / taxane (or a salt or acid of a taxane) in the conjugate is in the range of 2 to 8:1, or any range in between. In some embodiments, the molar ratio of αPPMX / taxane (or salt or acid of taxane) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In some embodiments, the molar ratio of αPPMX / taxane (or salt or acid of taxane) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPPMX / taxane-based drug complex is encapsulated in liposomes (e.g., as described herein or by other methods known in the art).

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

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

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

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

[0142] In further embodiments, the Disclosure provides a complex comprising αPPMX and another antimetabolite, or a salt or acid thereof. An antimetabolite is a chemical substance that is similar in structure to metabolites required for normal biochemical reactions but is sufficiently different to interfere with the normal function of one or more cells, such as cell division. In some embodiments, the Disclosure provides a complex comprising αPPMX and pemetrexed (PMX), or a salt or acid thereof. In some embodiments, the Disclosure provides a complex comprising αPPMX and an antimetabolite selected from the group consisting of gemcitabine, fluorouracil, capecitabine, antiphorates (e.g., methotrexate, larcitrexed), tegafur, cytosine arabinoside, thioguanine, 5-azacitidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as any pharmaceutically acceptable salt or acid (one or more) or derivative thereof. In some embodiments, the molar ratio of αPPMX / antimetabolite (or salt or acid of the antimetabolite) in the complex is in the range of 1 to 20:1, or any range in between. In further embodiments, the molar ratio of αPPMX / antimetabolite (or salt or acid of the antimetabolite) in the complex is in the range of 1 to 10:1, or any range in between. In further embodiments, the molar ratio of αPPMX / antimetabolite (or salt or acid of the antimetabolite) in the complex is in the range of 2 to 8:1, or any range in between. In some embodiments, the molar ratio of αPPMX / antimetabolite (or salt or acid of the antimetabolite) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In some embodiments, the molar ratio of αPPMX / antimetabolite (or salt or acid of the antimetabolite) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50.In further embodiments, the αPPMX / antimetabolite (or salt or acid of the antimetabolite) complex is encapsulated in a liposome (for example, as described herein or by other methods known in the art).

[0143] In further embodiments, the disclosure provides complexes of αPPMX (e.g., αPPMX as disclosed herein) and cyclodextrins. Cyclodextrins (CDs) are a group of cyclic oligosaccharides that have been shown to improve the physicochemical properties of many drugs through the formation of complexes. CDs are cyclic oligosaccharides composed of several D-glucose units linked by α-(1,4) bonds. This cyclic structure provides a hydrophobic internal cavity, giving CDs a shortened conical shape. Many hydroxyl groups are located on the ends of the ring, which makes CDs both lipophilic and water-soluble. As a result, CDs can form complexes with a wide variety of hydrophobic drugs, thereby altering the physicochemical properties of these complexed drugs.

[0144] The term "cyclodextrin" or "CD" usually refers to a parent or derivatized cyclic oligosaccharide capable of forming a complex with pemetrexed-PG, containing a variable number of (α-1,4)-linked D-glucopyranoside units, unless otherwise specified. Each cyclodextrin glucopyranoside subunit has a secondary hydroxyl group at positions 2 and 3 and a primary hydroxyl group at position 6. The terms "parent," "non-derivativeized," or "inactive" cyclodextrins contain a D-glucopyranoside unit and have the basic formula C6H 12This refers to cyclodextrins that have an O6 and glucose structure and no additional chemical substituents (e.g., α-cyclodextrin consisting of 6 D-glucopyranoside units, β-cyclodextrin consisting of 7 D-glucopyranoside units, and γ-cyclodextrin consisting of 8 D-glucopyranoside units). The physical and chemical properties of parent cyclodextrins can be modified by derivatizing the hydroxyl group with other functional groups. Any substance located in the cyclodextrin internal phase is said to be "complexed" with the cyclodextrin, or to form a complex (inclusion complex) with the cyclodextrin.

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

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

[0147] In some embodiments, the "ionizable" or "charged" portion of the CD derivative is weakly ionizable. The weakly ionizable portion is either a weakly basic or weakly acidic portion. The weakly basic functional group (W) has a pKa of CH3-W in the range of approximately 6.0–9.0, 6.5–8.5, 7.0–8.0, 7.5–8.0, and any range in between (including endpoints). Similarly, the weakly acidic functional group (X) has a logged dissociation constant (pKa) of CH3-X in the range of approximately 3.0–7.0, 4.0–6.5, 4.5–6.5, 5.0–6.0, 5.0–5.5, and any range in between (including endpoints). Typical anionic moieties include, but are not limited to, carboxylates, carboxymethyls, succinyls, sulfonyls, phosphates, sulfoalkyl ethers, sulfate carbonates, thiocarbonates, dithiocarbonates, phosphates, phosphonates, sulfonates, nitrates, and borate groups. Typical cationic moieties include, but are not limited to, aminos, guanidines, and quaternary ammonium groups.

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

[0149] In another embodiment, the derivatized cyclodextrin is a “chargeable amphiphilic substance.” “Chargeable” means that the amphiphilic substance has a pK in the range of pH 4 to pH 8 or 8.5. The charged amphiphilic substance can therefore be a weak acid or a base. As used herein, “amphoteric” means a derivatized cyclodextrin having ionizable groups with both anionic and cationic properties, wherein (a) at least one of the optionally cationic and anionic amphiphilic substances has at least one charge group having a pK between 4 and 8 to 8.5, (b) cationic charge is dominant at pH 4, and (c) anionic charge is dominant at pH 8 to 8.5.

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

[0151] Any one, some, or all of the hydroxyl groups of the α-D-glucopyranoside units of any one, some, or all of the cyclodextrins can be modified into ionizable chemical groups as described herein. Since each cyclodextrin hydroxyl group has a different chemical reactivity, the reaction with the modified portion can produce an amorphous mixture of positional and optical isomers. Alternatively, specific chemistry can be used to react the groups to form a homogeneous product of the pre-modified α-D-glucopyranoside units.

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

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

[0154] Additional cyclodextrin derivatives that can be complexed with therapeutic agents in the disclosed liposome composition include sugammadex or Org-25969, in which the 6-hydroxyl group on γ-CD is substituted with a carboxythioacetate ether bond and hydroxybutenyl-β-CD. Alternative forms of cyclodextrin include 2,6-di-O-methyl-β-CD (DIMEB), 2-hydroxylpropyl-3-cyclodextrin (HP-β-CD), random-methylated-β-cyclodextrin (RAMEB), and sulfobutyl ether Examples include β-cyclodextrin (SBE-β-CD), sulfobutyl ether-γ-cyclodextrin (SBEγCD), sulfobutylated-beta-cyclodextrin sodium salt, (2-hydroxypropyl)-alpha-cyclodextrin, (2-hydroxypropyl)-beta-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, 2,6-di-O-methyl)-beta-cyclodextrin (DIMEB-50 heptakis), 2,3,6-tri-O-methyl)-beta-cyclodextrin (TRIMEB heptakis), methyl-beta-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-gamma-cyclodextrin.

[0155] In some embodiments, cyclodextrin has high solubility in water to facilitate the encapsulation of larger amounts of cyclodextrin in the liposome's internal phase. In some embodiments, the solubility in water of 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 higher. In some embodiments, the water solubility of cyclodextrin is within the ranges of 10–150 mg / mL, 20–100 mg / mL, 20–75 mg / mL, and any range in between (including both ends).

[0156] In some embodiments, a large binding constant between cyclodextrin and αPPMX and / or other therapeutic agents complexed with cyclodextrin is preferred, which can be achieved 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)). If the binding constant is pH-dependent, the cyclodextrin can be selected such that the binding constant is large at the pH of the liposome intraphase. As a result, the solubility (nominal solubility) of the therapeutic agent in the presence of cyclodextrin can be further improved. In some embodiments, the binding constant between cyclodextrin and 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 in between.

[0157] In some embodiments, the cyclodextrin in the αPPMX / cyclodextrin complex and / or cyclodextrin / therapeutic complex is non-derivativeized.

[0158] In some embodiments, the cyclodextrin in the αPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic complex is non-derivativeized. In further embodiments, the cyclodextrin derivative of the complex is of formula I: [ka] It has the structure, where n is 4, 5, or 6; R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently -H, a linear or branched C1-C8 alkylene group, or an optionally substituted linear or branched C1-C6 group, and at least one of R1, R2, R3, R4, R5, R6, R7, R8, and R9 is a linear or branched C1-C8 alkylene (e.g., C1-C8-(alkylene)-SO3) - It is the basis.

[0159] In some embodiments, the cyclodextrin derivative of the αPPMX / cyclodextrin complex and / or cyclodextrin / therapeutic complex is of formula II: [ka] It has the structure, where n is 4, 5, or 6; R1, R2, R3, R4, R5, R6, R7, R8, and R9 are each independently -O- or -O-(C2-C6 alkylene)-SO3 - The group is; at least one of R1 and R2 is independently -O-(C2-C6 alkylene)-SO3 - The group is; S1, S2, S3, S4, S5, S6, S7, S8, and S9 are each independently pharmaceutically acceptable cations. In further embodiments, the pharmaceutically acceptable cations are Li + kaNa + , or K + Alkali metals such as Ca 2+ , or Mg 2+ The ammonium ions and amine cations are selected from alkaline earth metals such as (C1-C6)-alkylamines, piperidines, pyrazines, (C1-C6)-alkanolamines, and (C4-C8)-cycloalkanolamines. In some embodiments, at least one of R1 and R2 is independently -O-(CH2) mThe SO3- group is an -O-(C2-C6 alkylene)-SO3- group, where m is 2 to 6, preferably 2 to 4 (e.g., -O-CH2CH2CH2SO3- or -O-CH2CH2CH2CH2SO3-); and S1, S2, S3, S4, S5, S6, S7, S8, and S9 are each independently H or a pharmaceutically acceptable cation, which includes, for example, alkali metals (e.g., Li + kaNa + , K + ), alkaline earth metals (e.g., Ca 2+ Mg 2+ Examples include ammonium ions and amine cations such as (C1-C6)-alkylamines, piperidines, pyrazines, (C1-C6)-alkanolamines, and (C4-C8)-cycloalkanolamines.

[0160] In some embodiments, the cyclodextrin derivatives of the αPPMX / cyclodextrin complex and / or cyclodextrin / therapeutic complex are cyclodextrins disclosed in U.S. Patents 6,133,248, 5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, and 6,869,939; and International Publication 02005 / 117911. The contents of each of these patent documents are incorporated herein by reference in a preferential manner.

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

[0162] In some embodiments, the cyclodextrin derivative of the αPPMX / cyclodextrin complex and / or cyclodextrin / therapeutic complex is formula III: [ka] It is a compound of the form, where R is, (a)(H) 21-X Or (-(CH2)4-SO3Na) X , and x = 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0; (b)(H) 21-X Or (-(CH2CH(OH)CH3) X , and x = 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0; (c)(H) 21-X or (sulfoalkyl ether) X , and x = 1.0~10.0, 1.0~5.0, 6.0~7.0, or 8.0~10.0; or (d)(H) 21-X Or (-(CH2)4-SO3Na) X , and x = 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0.

[0163] In further embodiments, the αPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic complex are encapsulated in liposomes (for example, as described herein or by other methods known in the art).

[0164] III. αPPMX Delivery Carrier In alternative embodiments, the disclosure provides αPPMX delivery systems and their use for delivering αPPMX payloads to cells(s) in vitro or in vivo. In some embodiments, αPPMX is complexed with or incorporated into a delivery carrier. Such delivery carriers are known in the Art and not limited to, but include liposomes, lipospheres, polymers, peptides, proteins, antibodies (e.g., ADCs such as antibody-αPPMX complexes), cellular 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 specific embodiments, the delivery carrier is an antibody or antigen-binding antibody fragment.

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

[0166] In some embodiments, the provided liposomes further include an immunostimulant, a detectable marker, or both, disposed on the outer surface of the liposome. The immunostimulant or detectable marker can be ionic or covalently bonded to the outer surface of the liposome, and in this case, optionally, this may include binding to, for example, a steric-stabilizing component of the liposome.

[0167] The term "immunostimulatory agent," also known as "immunostimulant" or "immunostimulator," refers to a substance that stimulates immunity (including pre-existing immune responses) by inducing the activation or increased activity of any component of the immune system. These immunostimulators include one or more of the following: haptens, adjuvants, protein immunostimulants, nucleic acid immunostimulants, and chemoimmunostimulants. Many adjuvants contain substances designed to stimulate the immune response, such as lipid A, Bordetella pertussis, or Mycobacterium tuberculosis proteins. Specific adjuvants include, for example, Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Mich.); Merck adjuvant 65 (Merck and Company, Inc., Rahway, NJ); 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 sugars; polysaccharides derivatized with cationic or anionic agents; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; IFN gamma, IFN alpha, FLT3 ligand; and immunostimulant antibodies (e.g., commercially available as anti-CTLA-4, anti-CD28, anti-CD3; 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 may be at least one selected from the group consisting of fluorescein, DNP, beta-glucan, beta-1,3-glucan, and beta-1,6-glucan. In a further preferred embodiment, the immunostimulant is a Toll-like receptor (TLR) modulator. In a further embodiment, the Toll-like receptor (TLR) modulator is one or more of oxidized low-density lipoproteins (e.g., OXPAC, PGPC), erythril lipids (e.g., E5564), and resolvins.In some embodiments, the liposome contains fluorescein isothiocyanate (FITC), which, based on our experiments, surprisingly functions as both an immunostimulant and a detectable marker.

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

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

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

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

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

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

[0174] In some embodiments, the liposomes in the provided liposome composition include agents such as membrane permeabilizing agents that facilitate the delivery of liposomes across the cell membrane and confer upon liposomes the ability to bypass the endocytosis pathway and the harsh environment of lysosomes. Membrane permeabilizing agents are known in the art and can be routinely used and applied to the manufacture and use of the provided liposome composition. In some embodiments, the membrane permeabilizing 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 include a membrane permeabilizing agent selected from the following group: RKKRRQRRR (SEQ ID NO: 7), GRKKRRQRRRTPQ (SEQ ID NO: 8), YGRKKRRQRRR (SEQ ID NO: 9), AAVALLPAVLLALLA (SEQ ID NO: 10), MGLGLHLLVLAAALQ (SEQ ID NO: 11), GALFLGFLGAAGSTM (SEQ ID NO: 12), AGYLLGKINLKALAALAKKIL (SEQ ID NO: 13), RVIRVWFQNKRCKDKK (SEQ ID NO: 14). ,RQIKIWFQNRRMKWKK(SEQ ID NO: 15),GLFEAIAGFIENGWEGMIDG(SEQ ID NO: 16),GWTLNSAGYLLGKIN(SEQ ID NO: 17),RSQSRSRYYRQRQRS(SEQ ID NO: 18),LAIPEQEY(SEQ ID NO: 19),LGIAEQEY(SEQ ID NO: 20),LGIPAQEY(SEQ ID NO: 21),LGIPEAEY(SEQ ID NO: 22),LGIPEQAY(SEQ ID NO: 23),LGIAEAEY(SEQ ID NO: 24),LGIPEAAY(SEQ ID NO: 25),LGIAEQAY(SEQ ID NO: 15) 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), G WTLNSAGYLLGKINLKALAALAKKIL (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), RRRRRRRRRRRR (SEQ ID NO: 43), and YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG(R)n (wherein n=2~15R in L- and / or D-type) (SEQ ID NO: 44), or these cell-permeable fragments.

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

[0176] In some embodiments, the liposome composition comprises pegylated liposomes (PLp-αPPMX). In some embodiments, the pegylated liposomes in the liposome composition comprise αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX comprises two or more L-type glutamyl groups. In other embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX comprises D-type glutamyl groups. In further embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX comprises two or more glutamyl groups having gamma bonds. In some embodiments, at least one glutamyl group has both alpha and gamma bonds. In some embodiments, the liposome composition comprises pegylated liposomes containing α-pentaglutamine oxidized PMX. In further embodiments, the liposomes comprise L-α-pentaglutamine oxidized PMX, D-α-pentaglutamine oxidized PMX, or L- and D-α-pentaglutamine oxidized PMX. In some embodiments, the liposome composition comprises pegylated liposomes containing α-hexaglutamine oxidized PMX. In further embodiments, the liposomes comprise L-α-hexaglutamine oxidized PMX, D-α-hexaglutamine oxidized PMX, or L- and D-α-hexaglutamine oxidized PMX. In some embodiments, the liposome composition comprises pegylated liposomes that are anionic or neutral. In some embodiments, the liposome composition comprises pegylated liposomes that are cationic. In some embodiments, the PLp-αPPMX composition is untargeted (NTPLp-αPPMX). In other embodiments, the PLp-αPPMX composition is targeted (TPLp-αPPMX). In further embodiments, the liposome composition comprises pegylated liposomes containing 30-70%, 30-60%, or 30-50%, or any range in between, liposome-encapsulated alpha-polyglutamine oxidized pemetrexed.In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% (w / w) of alpha-polyglutamine oxidized pemetrexed is encapsulated in PLp-αPPMX. In some embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 20 nm to 500 nm. In some embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 20 nm to 200 nm. In further embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 80 nm to 120 nm.

[0177] In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamic oxidized pemetrexed in the composition has 4 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamic oxidized pemetrexed in the provided liposome composition is tetraglutamine-oxidized. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamic oxidized pemetrexed in the provided liposome composition is pentaglutamine-oxidized. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the polyglutamic oxidized pemetrexed in the provided liposome composition is hexaglutamine-oxidized.

[0178] In some embodiments, the alpha-polyglutamine oxidized pemetrexed composition (e.g., a delivery carrier such as polyglutamates and polyglutamates-containing liposomes) is in an aqueous solution. In some embodiments, the αPPMX composition is used as a liposome composition, with a volume of 1 square meter (m²). 2In a further embodiment, the αPPMX composition is administered as a liposome composition at a dose of approximately 0.005 to approximately 5000 mg of αPPMX per square meter of body surface area, or in any range between those doses.

[0179] (1) Liposome composition The liposome lipids and other components contained in a liposome composition can be any lipid, combination and ratio of lipids, or combination of lipids and other liposome components and their respective ratios known in the art. However, it will be understood by those skilled in the art that the liposome encapsulation of any particular drug, such as alpha-polyglutamine oxidized PMX, which is considered herein, may involve substantially standardized experiments to obtain useful and functional liposome formulations. Generally, the liposomes provided may have any liposome structure, e.g., a structure having an inner space isolated from an outer medium by one or more lipid bilayers, or any microcapsule structure having a semipermeable membrane with a lipophilic central portion whose membrane isolates the interior. The lipid bilayer can be an amphiphilic molecule of any structure characterized by a hydrophilic portion and a hydrophobic portion. Typically, the amphiphilic molecules in the bilayer are arranged on a two-dimensional sheet, where the hydrophobic portion faces inward on the sheet and the hydrophilic portion faces outward. The amphiphilic molecules that form the provided liposomes may be any known or hereafter discovered amphiphilic molecules (e.g., synthetic or naturally occurring lipids or biocompatible lipids). Liposomes can be formed from amphiphilic polymers and surfactants, e.g., polymerosomes and niosomes. In this disclosure, but not limited to these, these liposome-forming materials are also referred to as “lipids.”

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

[0181] In some embodiments, liposomes contain steric stabilizers that extend their lifespan in circulation. One or more steric stabilizers, such as hydrophilic polymers (polyethylene glycol (PEG)), glycolipids (monosialoganglioside (GM1)), or others, occupy spaces directly adjacent to the liposome surface, excluding other polymers from this space. As a result, access to and binding of plasma opsonins to the liposome surface is hindered, and therefore, macrophage interaction with such liposomes, or any other removal mechanism, is suppressed, extending the lifespan of circulating liposomes. In some embodiments, the steric stabilizer or group of steric stabilizers is PEG or a combination including PEG. In further embodiments, the steric stabilizer is PEG or a combination including PEG having a number-average molecular weight (Mn) of 200 to 5000 Daltons. These PEGs can be in any structure, such as linear, branched, star-shaped, or comb-shaped, and are commercially available.

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

[0183] The properties of liposomes are influenced by the properties of the lipids used to construct them. A wide variety of lipids have been used to construct liposomes. These include cationic, anionic, and neutral lipids. In some embodiments, liposomes containing alpha-polyglutamine oxidized pemetrexed are anionic or neutral. In other embodiments, the provided liposomes are cationic. The determination of charge (e.g., anionic, neutral, or cationic) can be determined by a standard procedure by measuring the zeta potential of the liposome. The zeta potential of a liposome can be positive, zero, or negative. In some embodiments, the zeta potential of a liposome is zero or less. In some embodiments, the zeta potential of a liposome is in the range of 0 to -150 mV. In another embodiment, the zeta potential of a liposome is in the range of -30 to -50 mV.

[0184] In some embodiments, cationic liposomes, commonly used as gene transfer agents, are constructed using cationic lipids. The positive charge on the cationic liposome enables interaction with the negative charge on the cell surface. After binding of the cationic liposome to the cell, the liposome is transported into the cell interior by endocytosis.

[0185] In some preferred embodiments, neutral to anionic liposomes are used. In preferred embodiments, anionic liposomes are used. For example, anionic liposomes are formed by using a mixture of neutral lipids such as HSPC and anionic lipids such as PEG-DSPE, which are less likely to bind nonspecifically to normal cells. Specific binding to tumor cells can be achieved using tumor-targeting antibodies, such as folate receptor antibodies including folate receptor alpha antibodies, folate receptor beta antibodies and / or folate receptor delta antibodies.

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

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

[0188] Lipids containing liposomes provided herein may 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 selective pH. At physiological pH, such lipids include, for example, dioleoyl phosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, kephalins, cholesterol, cerebrosides, and diacylglycerols. Examples of zwitterionic lipids include, but are not limited to, dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), and dioleoyl phosphatidylserine (DOPS). Anionic lipids are negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and anionic modifying groups attached to neutral lipids.

[0189] In this specification, anionic and neutral lipids are collectively referred to as noncationic lipids. Such lipids may contain phosphorus, but are not limited to it. Examples of noncationic lipids include lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), and distearoylphosphatidyl 1-Ethanolamine (DSPE), Palmitoyloleoylphosphatidylethanolamine (POPE), Palmitoyloleoylphosphatidylcholine (POPC), Egg phosphatidylcholine (EPC), Distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), Dioleoylphosphatidylglycerin (DOPG), Dipalmitoylphosphatidylglycerin (DPPG), Palmitoyloleyolphosphatidylglycerin (POPG), 16-0-MonomethylPE, 16-0-DimethylPE, 18-1-Trans Examples include PE, palmitoyloleoylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleoylphosphatidiethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, and cholesterol.

[0190] Liposomes can be constructed using any liposome assembly method with liposomal components (also called liposome components) known in the art. Liposome components include, for example, lipids such as DSPE, HSPC, and cholesterol, and derivatives of these components. Other suitable lipids are commercially available, for example, from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). A list of some of the available negatively or neutrally charged lipids suitable for the production of anionic liposomes may 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, Tetramyristoylcardiolipin·(Na)2, DSPE-mPEG-2000·Na, DSPE-mPEG-5000·Na, and DSPE-MaleimidePEG-2000·Na.

[0191] In some embodiments, the αPPMX compositions provided herein are incorporated into liposomes containing cationic lipids. In one embodiment, but not limited to, the cationic lipids are as described in International Publication Nos. 2012 / 040184, 2011 / 153120, 2011 / 149733, 2011 / 090965, 2011 / 043913, 2011 / 022460, 2012 / 061259, 2012 / 054365, and 2012 The cationic lipids are selected from those described in Patent Nos. / 044638, 2010 / 080724, 2010 / 21865, and 2008 / 103276, U.S. Patent Nos. 7,893,302, 7,404,969, and 8,283,333, U.S. Patent Application Publication No. 20100036115, and U.S. Patent Application Publication No. 20120202871. Each of these patent documents is incorporated herein by reference in its entirety. In another embodiment, the cationic lipid may be selected from formula A as described in International Publication Nos. 2012 / 040184, 2011 / 153120, 2011 / 1149733, 2011 / 090965, 2011 / 043913, 2011 / 022460, 2012 / 061259, 2012 / 054365, and 2012 / 044638, but not limited to these. Each of these patent documents is incorporated herein by reference in its entirety. In yet another embodiment, the cationic lipid may be selected from, but is not limited to, formulas CLI-CLXXIX of International Publication No. 2008103276, formulas CLI-CLXXIX of U.S. Patent No. 7,893,302, formulas CLI-CLXXXXII of U.S. Patent No. 7,404,969, and formulas I-VI of U.S. Patent Application Publication No. 20100036115. Each of these patent documents is incorporated herein by reference in its entirety. As a non-limiting example, cationic lipids may be selected from the following: (20Z,23Z)-N,N-dimethylnonacosa-20,23-diene-10-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-diene-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-diene-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-diene-5-amine, (12Z,(15Z)-N,N-dimethylhenicosa-12,15-diene-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-diene-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-diene-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-diene-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-diene-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-diene-4-amine, (19Z,22Z)-N,N- Dimeihyroctacosa-19,22-diene-9-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-diene-8-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-diene-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-diene-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-diene-10-amine, (21Z,24Z)-N,N-dimethyl-triaconta-21,24-diene-9-amine, (18Z)-N,N-dimethylhe Ptacosa-18-en-10-amine, (17Z)-N,N-dimethylhexacosa-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-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-nonylicosa-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacosa-20-en-10-amine, (15Z)-N,N-dimethylhepta Cosa-15-en-10-amine, (14Z)-N,N-dimethylnonacosa-14-en-10-amine, (17Z)-N,N-dimethylnonacosa-17-en-10-amine, (24Z)-N,N-dimethyltriaconta-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-nonylhenicosa-12,15-Diene-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecane-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]henicosane-10-amine,N, N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan--10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1R,2S)-2-undecyl-cyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimeth Luoctadecane-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-diene-1-yloxy]-3-(octyloxy)propane-n-2-amine, S--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-3-(octyloxy)propane-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-3-[(5Z-)-octa-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,(12Z)-Octadeca-9,12-diene-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-Octadeca-9,12-diene-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-Octadeca-9,12-diene-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)propane-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-diene-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propane-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-diene-1-yloxy]-N,N-dimethylpropane-2-amine, 1-[(11Z,14Z)-icosa-11,14-diene-1-yloxy [Xy]-N,N-dimethyl-1-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-diene-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-diene-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(1 3Z)-docosa-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadeca-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-Dien-1-yloxylpropan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,23-triene-10-amine or a pharmaceutically acceptable salt or acid or stereoisomer thereof.,

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

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

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

[0195] In some embodiments, the αPPMX composition is incorporated into 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 20120178702, which is incorporated herein by reference in its entirety. Non-limiting examples include, but are not limited to, cationic peptides or polypeptides such as polylysine, polyornithine, and / or polyarginine, and cationic peptides described in International Publication 2012 / 013326. In another embodiment, which is incorporated herein by reference in its entirety, αPPMX is incorporated into a lipid-polycation complex, which further includes, but is not limited to, a neutral lipid such as cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0196] The components of the liposome may include any molecule (i.e., chemicals / reagents / proteins) that binds to it, and in some embodiments, the components of the liposome provided include at least a member selected from the group DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide. In some embodiments, the components of the liposome provided 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.

[0197] In further embodiments, the liposomes of the liposome compositions provided herein contain oxidized phospholipids. In some embodiments, the liposomes contain oxidized phospholipids that are members selected from the group consisting of phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, and 1-palmitoyl-2-arachidonoyl-sn-glycero-2-phosphate. In some embodiments, the phospholipids have unsaturated bonds. In some embodiments, the phospholipids are arachidonic acid containing phospholipids. In further embodiments, the phospholipids are sn-2-oxygenated. In further embodiments, the phospholipids are not fragmented.

[0198] In some embodiments, the liposomes of the disclosed liposome composition contain oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-holylcholine (OxPAPC). As used herein, the term “oxPAPC” means the lipid produced by the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-holylcholine (PAPC), thereby producing a mixture of oxidized phospholipids containing fragmented or fully oxygenated sn-2 residues. Characteristic oxidative fragmented species contain 5-carbon sn-2 residues having an omegaaldehyde or omegacarboxyl group. Oxidation of arachidonic acid residues also produces phospholipids containing esterified isoprostanes. Among the many oxidized products present in oxPAPC, oxPAPC includes, 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-folylcholine (PECPC), and / or 1-palmitoyl-2-(epoxyisoprostane E2)-sn-glycero-4-phosphocholine (PEIPC). In some embodiments, the phospholipid has an unsaturated bond. In some embodiments, the phospholipid is arachidonic acid containing the phospholipid. In further embodiments, the phospholipid is sn-2-oxygenated. In further embodiments, the phospholipid is not fragmented.

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

[0200] In some embodiments, the liposomes of the disclosed liposome composition contain lipids selected from: 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC); 1-palmitoyl-2-(9′oxo-nonanoyl)-sn-glycero-3-phosphocholine; 1-palmitoyl-2-araquinodoyl-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-acetoyl-sn-glycero-3-phosphocholine. In further embodiments, the liposomes contain PGPC.

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

[0202] In some embodiments, at least one component of the liposomal lipid bilayer is functionalized (or reactive). As used herein, the functionalized component is a component containing a reactive group that can be used to crosslink reagents and portions to lipids. When a lipid is functionalized, any liposomes it forms are functionalized. In some embodiments, the reactive group reacts with a crosslinking agent (or other portion) to form a crosslink. The reactive group in the liposomal lipid bilayer is located somewhere in the lipid that comes into contact with the crosslinking agent and enables crosslinking with another portion (e.g., a steric stabilizer or targeting portion). In some embodiments, the reactive group is located in the tip group of a 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 dithiothreitol (DTT), but are not limited to these.

[0203] It should be understood that the use of other functionalized lipids, other reactive groups, and other crosslinking agents beyond those mentioned above is further intended. In addition to maleimide groups, other examples of intended reactive groups 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, imide ester groups, N-hydroxysuccinimide esters, sulfhydryl groups, and pyridyl disulfide groups.

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

[0205] (2) Liposome internal space In further non-limiting embodiments, the liposomes provided include an internal space. In some embodiments, the internal space includes, but is not limited to, an aqueous solution. In some embodiments, the internal space includes alpha-polyglutamine oxidized pemetrexed, provided herein. In further embodiments, the internal space of the liposome includes an isotonic agent. 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 in between. In some embodiments, the internal space of the liposome includes 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 in between. In some embodiments, the pH of the internal space of the liposome is pH 2 to 8, or any range in between. In some embodiments, the pH of the solution containing the liposome composition is pH 5 to 8, or any range in between. In some embodiments, the pH of the solution containing the liposome composition is pH 6 to 7, or any range in between. In some embodiments, the pH of the solution containing the liposome composition is 6 to 7.5, 6.5 to 7.5, 6.7 to 7.5, or 6.3 to 7.0, or any range in between. In some embodiments, the internal space contains a buffer. In further embodiments, the buffer is selected from HEPES, citrate, or sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer has a concentration of 15 to 200 mM, or any range in between.In further embodiments, the buffer solution is concentrated in concentrations 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 in between. In some embodiments, the buffer solution is HEPES concentrated in concentrations of 15–200 mM, or any range in between. In some embodiments, the buffer solution is citrate concentrated in concentrations of 15–200 mM, or any range in between. In some embodiments, the buffer solution is sodium phosphate concentrated in concentrations of 15–200 mM, or any range in between. In some embodiments, the internal space of the liposome contains a total concentration of sodium acetate and calcium acetate of 5 mM–500 mM, or 50 mM–500 mM, or any range in between.

[0206] 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%, 5–20%, or 1–50%, or any range in between. In yet another embodiment, the concentration (wt%) of trehalose is 1–15%, or any range in between. In an additional embodiment, the trehalose is present in an amount of about 5–20% (wt%) of trehalose, or any combination of one or more lyophilized protective agents or cryoprotective substances in 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 in between. In some embodiments, the internal space contains a buffer. In some embodiments, the buffer is selected from HEPES, citrate, or sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer is concentrated in a range of 15–200 mM or any range in between. In further embodiments, the HBS citrate buffer is concentrated in a range 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 in between. In some embodiments, the buffer is HEPES at a concentration of 15–200 mM or any range in between. In some embodiments, the buffer is citrate at a concentration of 15–200 mM or any range in between. In some embodiments, the buffer is sodium phosphate at a concentration of 15–200 mM or any range in between. 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 ranging from 5 mM to 500 mM, or 50 mM to 500 mM, or any range in between.

[0207] In some embodiments, the internal space of the liposome contains dextrose. In further embodiments, the concentration (wt%) of dextrose is 0.1–20%, 1–20%, 0.5–15%, 1–15%, 5–20%, or 1–50%, or any range in between. In yet another embodiment, the concentration (wt%) of dextrose is 1–15%, or any range in between. In additional embodiments, dextrose is present at a dextrose concentration of about 5–20% (wt%), or any combination of one or more lyophilized protective agents 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 in between. In some embodiments, the internal space contains a buffer. In some embodiments, the buffer is selected from HEPES, citrate, or sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer is concentrated in a range of 15–200 mM or any range in between. In further embodiments, the buffer is concentrated in a range 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 in between. In some embodiments, the buffer is HEPES at a concentration of 15–200 mM or any range in between. In some embodiments, the buffer is citrate at a concentration of 15–200 mM or any range in between. In some embodiments, the buffer is sodium phosphate at a concentration of 15–200 mM or any range in between. 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 ranging from 5 mM to 500 mM, or 50 mM to 500 mM, or any range in between.

[0208] In further embodiments, the disclosure provides liposome compositions comprising liposomes encapsulated (i.e., filled) with alpha-polyglutamine oxidized pemetrexed (e.g., αPPMX as disclosed herein). In some embodiments, the liposomes in the liposome composition comprise αPPMX comprising 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups (including the glutamyl groups of pemetrexed). In some embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX comprises two or more L-type glutamyl groups. In other embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX comprises D-type glutamyl groups. In further embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX contains two or more glutamyl groups having gamma-carboxyl bonds. In some embodiments, the liposome composition includes liposomes containing α-pentaglutamine oxidized PMX. In further embodiments, the liposomes include L-α-pentaglutamine oxidized PMX, D-α-pentaglutamine oxidized PMX, or L- and D-α-pentaglutamine oxidized PMX. In some embodiments, the liposome composition includes liposomes containing α-hexaglutamine oxidized PMX (Lp-αPPMX). In further embodiments, the liposomes include L-α-hexaglutamine oxidized PMX, D-α-hexaglutamine oxidized PMX, or L- and D-α-hexaglutamine oxidized PMX.

[0209] In some embodiments, targeted pegylated liposome alpha-polyglutamine-oxidized (e.g., pentaglutamine-oxidized or hexaglutamine-oxidized) pemetrexed comprises a medium comprising a liposome having an internal space; aqueous alpha-polyglutamine-oxidized pemetrexed disposed within the internal space; and a targeting moiety containing a protein having specific affinity for at least one folate receptor, the targeting moiety being located 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 above-mentioned freeze-drying protective agents or cryoprotective substances. In some embodiments, the cryoprotective substance is mannitol, trehalose, sorbitol, or sucrose.

[0210] In some embodiments, liposomes encapsulating alpha-polyglutamine oxidized pemetrexed (i.e., Lp-αPPMX including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) have an internal space containing fewer than 500,000 or fewer than 200,000 alpha-polyglutamine oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains 10 to 100,000 or any range in between alpha-polyglutamine oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains 10,000 to 100,000 or any range in between alpha-polyglutamine oxidized pemetrexed molecules. In some embodiments, the liposome is not pegylated and has an internal space containing fewer than 500,000 or fewer than 200,000 alpha-polyglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are not pegylated, and the internal space of the liposomes contains 10 to 100,000 or any range in between alpha-polyglutamine oxidized pemetrexed molecules. In further embodiments, the liposomes are not pegylated, and the internal space of the liposomes contains 10,000 to 100,000 or any range in between alpha-polyglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are targeted and also not pegylated (TLp-αPPMX), and have an internal space containing fewer than 500,000 or fewer than 200,000 alpha-polyglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are targeted and also not pegylated, and the internal space of the liposomes contains 10 to 100,000 or any range in between alpha-polyglutamine oxidized pemetrexed molecules. In a further embodiment, the liposomes are targeted and not pegylated, and the internal space of the liposomes contains 10,000 to 100,000 or any range in between alpha-polyglutamine oxidized pemetrexed molecules.In some embodiments, the liposomes are untargeted and unpegylated (NTLp-αPPMX) and have an internal space containing fewer than 500,000 or fewer than 200,000 alpha-polyglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are untargeted and unpegylated, and the internal space of the liposomes contains 10 to 100,000 or any range in between alpha-polyglutamine oxidized pemetrexed molecules. In further embodiments, the liposomes are untargeted and unpegylated, and the internal space of the liposomes contains 10,000 to 100,000 or any range in between alpha-polyglutamine oxidized pemetrexed molecules.

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

[0212] In some embodiments, the liposome encapsulates alpha-polyglutamine oxidized pemetrexed (i.e., Lp-αPPMX including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) and has an internal space containing fewer than 500,000 or fewer than 200,000 alpha-tetraglutamine oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains 10 to 100,000 or any range in between alpha-tetraglutamine oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains 10,000 to 100,000 or any range in between alpha-tetraglutamine oxidized pemetrexed molecules. In some embodiments, the liposome is not pegylated and has an internal space containing fewer than 500,000 or fewer than 200,000 alpha-tetraglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are not pegylated, and the internal space of the liposomes contains 10 to 100,000 or any range in that range of alpha-tetraglutamine oxidized pemetrexed molecules. In further embodiments, the liposomes are not pegylated, and the internal space of the liposomes contains 10,000 to 100,000 or any range in that range of alpha-tetraglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are targeted and not pegylated (TLp-αPPMX), and have an internal space containing fewer than 500,000 or fewer than 200,000 alpha-tetraglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are targeted and not pegylated, and the internal space of the liposomes contains 10 to 100,000 or any range in that range of alpha-tetraglutamine oxidized pemetrexed molecules. In a further embodiment, the liposomes are targeted and not pegylated, and the internal space of the liposomes contains 10,000 to 100,000 or any range in between alpha-tetraglutamine oxidized pemetrexed molecules.In some embodiments, the liposomes are untargeted and unpegylated (NTLp-αPPMX) and have an internal space containing fewer than 500,000 or fewer than 200,000 alpha-tetraglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are untargeted and unpegylated, and the internal space of the liposomes contains 10 to 100,000 or any range in between alpha-tetraglutamine oxidized pemetrexed molecules. In further embodiments, the liposomes are untargeted and unpegylated, and the internal space of the liposomes contains 10,000 to 100,000 or any range in between alpha-tetraglutamine oxidized pemetrexed molecules.

[0213] In some embodiments, the liposomes encapsulate alpha-pentaglutamine oxidized pemetrexed (i.e., Lp-αPPMX including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) and have an internal space containing fewer than 500,000 or fewer than 200,000 alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains 10 to 100,000 or any range in between alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains 10,000 to 100,000 or any range in between alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are not pegylated and have an internal space containing fewer than 500,000 or fewer than 200,000 alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are not pegylated, and the internal space of the liposome contains 10 to 100,000 or any range in that range of alpha-pentaglutamine oxidized pemetrexed molecules. In further embodiments, the liposomes are not pegylated, and the internal space of the liposome contains 10,000 to 100,000 or any range in that range of alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are targeted and not pegylated (TLp-αPPMX), and have an internal space containing fewer than 500,000 or fewer than 200,000 alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are targeted and not pegylated, and the internal space of the liposome contains 10 to 100,000 or any range in that range of alpha-pentaglutamine oxidized pemetrexed molecules. In a further embodiment, the liposomes are targeted and not pegylated, and the internal space of the liposomes contains 10,000 to 100,000 or any range in between alpha-pentaglutamine oxidized pemetrexed molecules.In some embodiments, the liposomes are untargeted and unpegylated (NTLp-αPPMX) and have an internal space containing fewer than 500,000 or fewer than 200,000 alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are untargeted and unpegylated, and the internal space of the liposomes contains 10 to 100,000 or any range in between alpha-pentaglutamine oxidized pemetrexed molecules. In further embodiments, the liposomes are untargeted and unpegylated, and the internal space of the liposomes contains 10,000 to 100,000 or any range in between alpha-pentaglutamine oxidized pemetrexed molecules.

[0214] In some embodiments, the liposome encapsulates alpha-hexaglutamine oxidized pemetrexed (i.e., Lp-αPPMX including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) and has an internal space containing fewer than 500,000 or fewer than 200,000 alpha-hexaglutamine oxidized pemetrexed molecules. In some embodiments, the internal space of the liposome contains 10 to 100,000 or any range in between alpha-hexaglutamine oxidized pemetrexed molecules. In further embodiments, the internal space of the liposome contains 10,000 to 100,000 or any range in between alpha-hexaglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are not pegylated and have an internal space containing fewer than 500,000 or fewer than 200,000 alpha-hexaglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are not pegylated and the internal space of the liposome contains 10 to 100,000 or any range in between alpha-hexaglutamine oxidized pemetrexed molecules. In further embodiments, the liposomes are not pegylated and the internal space of the liposome contains 10,000 to 100,000 or any range in between alpha-hexaglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are targeted and not pegylated (TLp-αPPMX) and have an internal space containing fewer than 500,000 or fewer than 200,000 alpha-hexaglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are targeted and not pegylated, and the internal space of the liposomes contains 10 to 100,000 or any range in between alpha-hexaglutamine oxidized pemetrexed molecules. In further embodiments, the liposomes are targeted and not pegylated, and the internal space of the liposomes contains 10,000 to 100,000 or any range in between alpha-hexaglutamine oxidized pemetrexed molecules.In some embodiments, the liposomes are untargeted and unpegylated (NTLp-αPPMX) and have an internal space containing fewer than 500,000 or fewer than 200,000 alpha-hexaglutamine oxidized pemetrexed molecules. In some embodiments, the liposomes are untargeted and unpegylated, and the internal space of the liposomes contains 10 to 100,000 or any range in between alpha-hexaglutamine oxidized pemetrexed molecules. In further embodiments, the liposomes are untargeted and unpegylated, and the internal space of the liposomes contains 10,000 to 100,000 or any range in between alpha-hexaglutamine oxidized pemetrexed molecules.

[0215] In some embodiments, the Disclosure provides liposomal alpha-polyglutamine oxidized pemetrexed compositions, wherein the liposomes encapsulate alpha-polyglutamine 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 1% to 50% (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 therein. In some embodiments, the internal ...

Claims

[Claim 1] A liposome composition comprising liposomes encapsulating alpha-polyglutamine-oxidized pemetrexed and one or more polyglutamine-oxidizable folate antimetabolites or polyglutamine-oxidizable folate antimetabolites, The alpha-polyglutamine oxidized pemetrexed comprises 2 to 15 glutamyl groups having alpha-carboxyl group bonds; (a) At least two of the glutamyl groups of the alpha-polyglutamine oxidized pemetrexed are L-type, (b) Whether each glutamyl group of the alpha-polyglutamine oxidized pemetrexed is L-type, (c) At least one of the glutamyl groups of the alpha-polyglutamine oxidized pemetrexed is of type D, (d) Each of the glutamyl groups of the alpha-polyglutamine oxidized pemetrexed, other than the glutamyl group of pemetrexed, is of type D, or (e) At least two of the glutamyl groups of the alpha-polyglutamine oxidized pemetrexed are L-type and at least one of the glutamyl groups is D-type; The liposomes have a diameter of 50 nm to 150 nm. Liposome composition.

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