WO2019157120A1 - Alpha polyglutamated tetrahydrofolates and uses thereof - Google Patents

Alpha polyglutamated tetrahydrofolates and uses thereof Download PDF

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Publication number
WO2019157120A1
WO2019157120A1 PCT/US2019/016955 US2019016955W WO2019157120A1 WO 2019157120 A1 WO2019157120 A1 WO 2019157120A1 US 2019016955 W US2019016955 W US 2019016955W WO 2019157120 A1 WO2019157120 A1 WO 2019157120A1
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WIPO (PCT)
Prior art keywords
alpha
thf
polyglutamated
tetrahydrofolate
apthf
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PCT/US2019/016955
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English (en)
French (fr)
Inventor
Clet Niyikiza
Victor Mandla MOYO
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LEAF Holdings Group LLC
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LEAF Holdings Group LLC
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Publication date
Priority to EP19751561.2A priority Critical patent/EP3749319A4/en
Priority to CN201980024661.9A priority patent/CN111936146A/zh
Priority to CA3090506A priority patent/CA3090506A1/en
Priority to US16/967,621 priority patent/US12336994B2/en
Priority to JP2020542422A priority patent/JP7514533B2/ja
Application filed by LEAF Holdings Group LLC filed Critical LEAF Holdings Group LLC
Publication of WO2019157120A1 publication Critical patent/WO2019157120A1/en
Anticipated expiration legal-status Critical
Priority to JP2024101225A priority patent/JP7674775B2/ja
Priority to US19/022,038 priority patent/US20250319188A1/en
Priority to JP2025069103A priority patent/JP2025108646A/ja
Priority to US19/208,668 priority patent/US20260048054A1/en
Ceased legal-status Critical Current

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    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D475/00—Heterocyclic compounds containing pteridine ring systems
    • C07D475/02—Heterocyclic compounds containing pteridine ring systems with an oxygen atom directly attached in position 4
    • C07D475/04—Heterocyclic compounds containing pteridine ring systems with an oxygen atom directly attached in position 4 with a nitrogen atom directly attached in position 2
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
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    • C07D475/08—Heterocyclic compounds containing pteridine ring systems with a nitrogen atom directly attached in position 4 with a nitrogen atom directly attached in position 2
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    • C07D—HETEROCYCLIC COMPOUNDS
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    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
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    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • This disclosure generally relates to polyglutamated alpha tetrahydrofolate compositions, including delivery vehicles such as liposomes containing the polyglutamated alpha tetrahydrofolate compositions, and methods of making and using the compositions to treat diseases including hyperproliferative diseases such as cancer, disorders of the immune system such as rheumatoid arthritis, infectious diseases such as HIV and malaria.
  • delivery vehicles such as liposomes containing the polyglutamated alpha tetrahydrofolate compositions
  • diseases including hyperproliferative diseases such as cancer, disorders of the immune system such as rheumatoid arthritis, infectious diseases such as HIV and malaria.
  • the polyglutamated alpha tetrahydrofolate compositions also have uses in combination therapy with one or more therapeutic agents such as a chemotherapeutic drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a “chemoprotectant” (e.g., in combination with antifolates such as methotrexate) to reduce toxic side effects associated with the therapeutic agent(s).
  • a chemotherapeutic drug e.g ., 5-fluorouracil
  • chemoprotectant e.g., in combination with antifolates such as methotrexate
  • Folate is an essential cofactor that mediates the transfer of one-carbon units involved in nucleotide biosynthesis and DNA repair, the remethylation of homocysteine (Hey), and the methylation of DNA, proteins, and lipids.
  • the only circulating forms of folates in the blood are monoglutamates and folate monoglutamates are the only form of folate that is transported across the cell membrane - likewise, the monoglutamate form of tetrahydrofolate, are transported across the cell membrane.
  • intracellular tetrahydrofolate is polyglutamated by the enzyme folylpoly-gamma- glutamate synthetase (FPGS).
  • the polyglutamation of tetrahydrofolate by FPGS serves at least 2 main therapeutic purposes: (1) it greatly enhances tetrahydrofolate affinity for DHFR; and (2) it facilitates the accumulation of polyglutamated tetrahydrofolate, which unlike tetrahydrofolate (monoglutamate), is not easily transported out of cells by cell efflux pumps.
  • the provided polyglutamated alpha tetrahydrofolate compositions deliver a strategy for improving the therapeutic efficacy of tetrahydrofolate.
  • This disclosure generally relates polyglutamated alpha tetrahydrofolate (THF) compositions and methods of making and using the compositions to treat diseases including hyperproliferative diseases such as cancer, disorders of the immune system such as inflammation and rheumatoid arthritis, and infectious disease such as HIV and malaria.
  • the polyglutamated alpha tetrahydrofolate compositions also have uses in combination therapy with one or more therapeutic agents such as a chemotherapeutic drug (e.g ., 5- fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a “chemoprotectant” (e.g., in combination with antifolates such as methotrexate) to reduce toxic side effects associated with the therapeutic agent(s).
  • chemotherapeutic drug e.g ., 5- fluorouracil
  • chemoprotectant e.g., in combination with antifolates such as methotrexate
  • the disclosure provides:
  • composition comprising a polyglutamated alpha tetrahydrofolate.
  • polyglutamated 5-formyl-THF e.g, polyglutamated [6S]-5-formyl-THF
  • polyglutamated lO-formyl-THF e.g, polyglutamated [6R]-l 0-formyl - THF
  • polyglutamated 5,l0-methenyl-THF e.g, polyglutamated [6R]-5,lO- methenyl-THF
  • polyglutamated 5-methyl-THF e.g, polyglutamated [6S]-5-methyl-THF
  • polyglutamated tetrahydrofolate e.g, polyglutamated [6S]- Tetrahydrofolate THF
  • polyglutamated 5,l0-methylene-THF e.g, polyglutamated [6R]-5,lO- methylene-THF
  • polyglutamated 5-formimino-THF e.g, polyglutamated [6S]-5-formimino- THF.
  • pentaglutamated alpha tetrahydrofolate e.g., [6R]-5,lO- methenyl-THF, [6S]-5-formyl-THF and/or [6R]-lO-formyl-THF.
  • [6] the composition according to any of [l]-[3], wherein the polyglutamated alpha tetrahydrofolate is hexaglutamated alpha tetrahydrofolate (e.g, [6R]-5,lO- methenyl-THF, [6S]-5-formyl-THF and/or [6R]-lO-formyl-THF).
  • polyglutamated alpha tetrahydrofolate is hexaglutamated alpha tetrahydrofolate (e.g, [6R]-5,lO- methenyl-THF, [6S]-5-formyl-THF and/or [6R]-lO-formyl-THF).
  • tetrahydrofolate has an alpha carboxyl group linkage
  • each of the glutamyl groups of the polyglutamated alpha tetrahydrofolate is in the L-form
  • each of the glutamyl groups of the polyglutamated alpha tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form, or
  • tetrahydrofolate are in the L-form and at least 1 of the glutamyl groups is in the D-form.
  • a liposomal composition comprising the polyglutamated alpha tetrahydrofolate according to any of [l]-[l 1] (Lp-aPTHF);
  • glutamyl groups of the polyglutamated alpha tetrahydrofolate is in the L-form
  • the Lp-aPTHF composition according to any of [12]-[15], wherein the liposome comprises a polyglutamated alpha tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5, glutamyl groups;
  • the Lp-aPTHF composition according to any of [12]-[19], wherein the liposome comprises a polyglutamated alpha tetrahydrofolate containing tetraglutamated alpha tetrahydrofolate, pentaglutamated alpha tetrahydrofolate, or
  • the Lp-aPTHF composition according to any of [12]-[19], wherein the liposome comprises a polyglutamated alpha tetrahydrofolate containing tetraglutamated alpha tetrahydrofolate, pentaglutamated alpha tetrahydrofolate, or
  • poly glutamate is linear or branched
  • liposome is pegylated (PaLp-aPTHF);
  • liposomes comprise at least 1% weight by weight (w/w) of the polyglutamated alpha tetrahydrofolate or wherein during the process of preparing the Lp- aPTHF, at least 1% of the starting material of polyglutamated alpha THF is encapsulated (entrapped) in the aPTHF;
  • the Lp-aPTHF composition according to any of [l2]-[24], wherein the liposome has a diameter in the range of 20 nm to 500 nm or 20 nm to 200 nm;
  • components comprise at least one of an anionic lipid and a neutral lipid
  • the Lp-aPTHF composition according to [27] or [28], wherein the liposomal components comprise at least one selected from the group consisting of: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol;
  • cholesterol-PEG and cholesterol-maleimide
  • liposomal components comprise at least one selected from the group consisting of: DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC;
  • methacrylamide amphiphilic poly-N-vinylpyrrolidones; L-amino-acid-based polymer; oligoglycerol, copolymer containing polyethylene glycol and polypropylene oxide, Poloxamer 188, and polyvinyl alcohol;
  • liposome is anionic or neutral
  • liposome has a zeta potential that is less than or equal to zero;
  • liposome has a zeta potential that is between 0 to -150 mV;
  • liposome has a zeta potential that is between -30 to -50 mV;
  • liposome is cationic
  • liposome has an interior space comprising the polyglutamated alpha tetrahydrofolate and an aqueous pharmaceutically acceptable carrier;
  • a tonicity agent such as dextrose, mannitol, glycerine, potassium chloride, sodium chloride, at a concentration of greater than 1%;
  • pharmaceutically acceptable carrier comprises 1% to 15 weight of dextrose
  • the Lp-aPTHF composition according to any of [39] -[43], wherein the interior space of the liposome comprises 5% dextrose suspended in an HEPES buffered solution;
  • pharmaceutically acceptable carrier comprises a buffer such as HEPES Buffered Saline (HBS) or similar, at a concentration of between 1 to 200 mM and a pH of between 2 to 8;
  • HBS HEPES Buffered Saline
  • pharmaceutically acceptable carrier comprises a total concentration of sodium acetate and calcium acetate of between 50 mM to 500 mM;
  • liposome comprises less than 500,000 or less than 200,000 molecules of the polyglutamated alpha tetrahydrofolate;
  • liposome comprises between 10 to 100,000 molecules of the polyglutamated alpha tetrahydrofolate, or any range therein between;
  • the targeting moiety comprises a targeting moiety and wherein the targeting moiety has a specific affinity for a surface antigen on a target cell of interest;
  • targeting moiety is an antibody or an antigen binding fragment of an antibody
  • targeting moiety binds the surface antigen with an equilibrium dissociation constant (Kd) in a range of 0.5 x 10 10 to 10 x 10 6 as determined using BIACORE® analysis;
  • targeting moiety specifically binds one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-a), folate receptor beta (FR- b), and folate receptor delta (FR-d);
  • targeting moiety comprises one or more selected from the group consisting of: an antibody, a humanized antibody, an antigen binding fragment of an antibody, a single chain antibody, a single-domain antibody, a bi-specific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody;
  • pegylated liposome comprises from 1 to 1000 or 30-200 targeting moieties
  • the Lp-aPTHF composition according to any of [39]-[57], further comprising one or more of an immunostimulatory agent, a detectable marker and a maleimide, wherein the immunostimulatory agent, the detectable marker or the maleimide is attached to said PEG or the exterior of the liposome;
  • immunostimulating agent is at least one selected from the group consisting of: a protein immunostimulating agent; a nucleic acid immunostimulating agent; a chemical immunostimulating agent; a hapten; and an adjuvant;
  • a fluorescein e.g., a fluorescein isothiocyanate (FITC)
  • a DNP e.g., a beta glucan
  • immunostimulatory agent and the detectable marker is the same;
  • cryoprotectant selected from the group consisting of mannitol; trehalose; sorbitol; and sucrose;
  • a targeted composition comprising the composition according to any of [l]-[64];
  • a pharmaceutical composition comprising the liposomal polyglutamated alpha tetrahydrofolate composition according to any of [l2]-[67];
  • composition of any of [l]-[69], for use in the treatment of disease [71] use of the composition of any of [l]-[70], in the manufacture of a medicament for the treatment of disease and/or for use in combination therapy with one or more therapeutic agents such as a chemotherapeutic drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a chemotherapeutic drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a chemotherapeutic drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a chemotherapeutic drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a chemotherapeutic drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a chemotherapeutic drug (e.g ., 5-
  • chemoprotectant e.g., in combination with an antifolate such as
  • methotrexate to reduce a toxic side effect associated with the therapeutic agent(s);
  • [72] a method for treating or preventing disease in a subject needing such treatment or prevention, the method comprising administering the composition of any of [l]-[70] to the subject;
  • polyglutamated alpha tetrahydrofolate composition of any of [l2]-[69] to the subject;
  • the hyperproliferative cell is a cancer cell, a mammalian cell, and/or a human cell;
  • [77] a method for treating cancer that comprises administering an effective amount of the composition of any of [l]-[69] to a subject having or at risk of having cancer;
  • [78] a method for treating cancer that comprises administering an effective amount of the liposomal polyglutamated alpha tetrahydrofolate composition of any of [l2]-[68] to a subject having or at risk of having cancer;
  • a non-hematologic malignancy including such as for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma ( e.g osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and a hematologic malignancy such as for example, a leukemia, a lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias;
  • a hematologic malignancy such as for example, a leukemia, a lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias
  • colorectal cancer lung cancer, breast cancer, head and neck cancer, and pancreatic cancer;
  • [83] a method for treating cancer that comprises administering an effective amount of the Lp-aPTHF composition of any of [50]-[66] to a subject having or at risk of having a cancer cell that expresses on its surface a folate receptor bound by the targeting moiety;
  • a maintenance therapy for subjects that are undergoing or have undergone cancer therapy that comprise administering an effective amount of the composition of any of [l]-[69] to a subject that is undergoing or has undergone cancer therapy;
  • a maintenance therapy for subjects that are undergoing or have undergone cancer therapy that comprise administering an effective amount of the liposomal polyglutamated alpha tetrahydrofolate composition of any of [l2]-[69] to a subject that is undergoing or has undergone cancer therapy;
  • the disorder of the immune system is selected from: inflammation (e.g ., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn disease, dermatomyositis/polymyositis, systemic lupus erythematosus, and Takayasu, and psoriasis;
  • the liposomal polyglutamated alpha tetrahydrofolate composition of any of [8]-[69] to a subject having or at risk of having a disorder of the immune system, optionally wherein the disorder of the immune system is selected from: inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn disease, dermatomyositis/polymyositis, systemic lupus erythematosus, and Takayasu, and psoriasis;
  • inflammation e.g., acute and chronic
  • systemic inflammation e.g., rheumatoid arthritis
  • IBD inflammatory bowel disease
  • Crohn disease e.g., dermatomyositis/polymyositis
  • systemic lupus erythematosus e.g., rheumatoid arthritis
  • Takayasu p
  • composition according to any of [l]-[69] to a subject having or at risk of having leukopenia;
  • cardiovascular disease or metabolic disease that comprises administering an effective amount of the composition according to any of [l]-[69] to a subject having or at risk of having an infectious disease, cardiovascular disease, or another disease, wherein the disease is a member selected from:
  • CVD cardiovascular disease
  • coronary artery disease myocardial infarction
  • stroke stroke
  • metabolic syndrome a gestational trophoblastic disease
  • ectopic pregnancy cardiovascular disease
  • an autoimmune disease that comprises administering an effective amount of the composition according to any of [l]-[69] to a subject having or at risk of having an autoimmune disease;
  • rheumatoid arthritis that comprises administering an effective amount of the composition according to any of [l]-[69] to a subject having or at risk of having rheumatoid arthritis;
  • an inflammatory condition that comprises administering an effective amount of the composition according to any of [l]-[69] to a subject having or at risk of having inflammation, optionally wherein the inflammation is acute, chronic, and/or systemic inflammation; or
  • a skin condition that comprises administering an effective amount of the composition according to any of [l]-[69] to a subject having or at risk of having a skin condition, optionally wherein the skin condition is psoriasis;
  • liposomal polyglutamated alpha tetrahydrofolate composition of any of [l2]-[69] to a subject having or at risk of having an infectious disease
  • a method of preparing a polyglutamated alpha tetrahydrofolate composition comprising the liposomal polyglutamated alpha tetrahydrofolate composition of any of [12] -[69], the method comprising: forming a mixture comprising: liposomal components and polyglutamated alpha antifolate in solution;
  • [92] a method of preparing the composition of any of [l2]-[69] comprising the steps of: forming a mixture comprising: liposomal components and polyglutamated alpha tetrahydrofolate in a solution; homogenizing the mixture to form liposomes in the solution; processing the mixture to form liposomes entrapping and/or encapsulating polyglutamated alpha tetrahydrofolate; and providing a targeting moiety on a surface of the liposomes, the targeting moiety having specific affinity for at least one of folate receptor alpha (FR-a), folate receptor beta (FR-b) and folate receptor delta (FR-d);
  • FR-a folate receptor alpha
  • FR-b folate receptor beta
  • FR-d folate receptor delta
  • the processing step includes one or more steps of: thin film hydration, extrusion, in-line mixing, ethanol injection technique, freezing-and-thawing technique, reverse-phase evaporation, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze- dried double emulsion, 3D printing, membrane contactor method, and stirring; and/or
  • processing step includes one or more steps of modifying the size of the liposomes by one or more of steps of extrusion, high-pressure microfluidization, and/or sonication.
  • the disclosure provides a polyglutamated alpha tetrahydrofolate (aPTHF) composition wherein at least 2 of the glutamyl residues of the polyglutamated alpha tetrahydrofolate have a alpha carboxyl group linkage.
  • the aPTHF contains 2-20, 2-15, 2-10, 2-5, or more than 5, glutamyl groups (including the glutamyl group in tetrahydrofolate).
  • the aPTHF comprises two or more glutamyl groups in the L-form.
  • the aPTHF comprises a glutamyl group in the D-form.
  • the aPTHF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In additional embodiments, the aPTHF comprises two or more glutamyl groups that have an alpha linkage. In some embodiments, at least one glutamyl group has both an alpha linkage and a gamma linkage.
  • the aPTHF composition contains a chain of 3 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., a tetraglutamated tetrahydrofolate).
  • the tetraglutamated THF comprises two or more glutamyl groups in the L-form.
  • the tetraglutamated THF comprises a glutamyl group in the D-form.
  • the tetraglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L- form.
  • the tetraglutamated THF comprises two or more glutamyl groups that have a gamma linkage.
  • the aPTHF composition contains a chain of 4 glutamyl groups attached to the gamma glutamyl group of tetrahydrofolate (e.g ., a-pentaglutamated tetrahydrofolate).
  • the pentaglutamated alpha THF comprises two or more glutamyl groups in the L-form.
  • the pentaglutamated alpha THF comprises a glutamyl group in the D-form.
  • the pentaglutamated alpha THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the pentaglutamated THF comprises two or more glutamyl groups that have a gamma linkage.
  • the aPTHF composition contains a chain of 5 glutamyl groups attached to the gamma glutamyl group of tetrahydrofolate (e.g., a-hexaglutamated tetrahydrofolate).
  • the hexaglutamated alpha THF comprises two or more glutamyl groups in the L-form.
  • the hexaglutamated alpha THF comprises a glutamyl group in the D-form.
  • the hexaglutamated alpha THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the hexaglutamated THF comprises two or more glutamyl groups that have a gamma linkage.
  • compositions containing delivery vehicles such as liposomes filled with (e.g, encapsulating) and/or otherwise associated with polyglutamated alpha tetrahydrofolate, and methods of making and using the aPTHF filled/associated delivery vehicle compositions (DV-aPTHF) to deliver polyglutamated alpha tetrahydrofolate to diseased (e.g, cancerous) and/or targeted cells.
  • delivery vehicles such as liposomes filled with (e.g, encapsulating) and/or otherwise associated with polyglutamated alpha tetrahydrofolate
  • DV-aPTHF aPTHF filled/associated delivery vehicle compositions
  • polyglutamated alpha tetrahydrofolate in the DV-aPTHF contains 2-20, 2-15, 2-10, 2-5, more than 5, or more than 20, glutamyl groups (including the glutamyl group in tetrahydrofolate).
  • the DV- aPTHF filled/associated delivery vehicle compositions provide improvements to the efficacy and safety of delivering tetrahydrofolate to cancer cells by providing the preferential delivery of a more cytotoxic payload (e.g, polyglutamated tetrahydrofolate) compared to the cytotoxicity of tetrahydrofolate administered in its monoglutamate state (THF).
  • a more cytotoxic payload e.g, polyglutamated tetrahydrofolate
  • compositions containing delivery vehicles such as liposomes filled with (e.g, encapsulating) and/or otherwise associated with polyglutamated alpha tetrahydrofolate in combination therapy with one or more therapeutic agents such as a chemotherapeutic drug (e.g, 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a “chemoprotectant” (e.g, in combination with antifolates such as methotrexate) to reduce toxic side effects associated with the therapeutic agent(s).
  • chemotherapeutic drug e.g, 5-fluorouracil
  • chemoprotectant e.g, in combination with antifolates such as methotrexate
  • polyglutamated alpha tetrahydrofolate in the DV-aPTHF contains 2-20, 2-15, 2-10, 2-5, more than 5, or more than 20, glutamyl groups (including the glutamyl group in tetrahydrofolate).
  • the DV- aPTHF filled/associated delivery vehicle compositions provide improvements to the efficacy and safety of delivering tetrahydrofolate to cancer cells by providing the preferential delivery of a more cytotoxic payload (e.g, polyglutamated tetrahydrofolate) compared to the cytotoxicity of tetrahydrofolate administered in its monoglutamate state (THF).
  • a more cytotoxic payload e.g, polyglutamated tetrahydrofolate
  • the disclosure provides a composition comprising a polyglutamated alpha tetrahydrofolate (aPTHF).
  • aPTHF polyglutamated alpha tetrahydrofolate
  • the disclosure provides a composition comprising a polyglutamated alpha 5 -formyl -THF.
  • the polyglutamated alpha 5-formyl-THF polyglutamated alpha [6S]-5-formyl-THF.
  • the composition comprises polyglutamated alpha [6R,S]-5-formyl-THF.
  • the composition comprises polyglutamated alpha [6R]-5-formyl-THF.
  • the composition contains polyglutamated alpha 5-formyl-THF that has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including the glutamyl group in 5-formyl-THF).
  • the polyglutamated alpha 5-formyl-THF comprises 1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage.
  • the composition contains polyglutamated alpha 5-formyl-THF that has two or more glutamyl groups in the L-form.
  • the composition contains polyglutamated alpha 5-formyl-THF that has a glutamyl group in the D-form.
  • the composition contains polyglutamated alpha 5-formyl-THF that has a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamated alpha tetrahydrofolate in the Lp-aPTHF comprises two or more glutamyl groups that have an alpha linkage.
  • the polyglutamated alpha tetrahydrofolate in the Lp-aPTHF comprises one or more glutamyl groups that have both an alpha linkage and a gamma linkage.
  • the polyglutamated alpha tetrahydrofolate in the Lp-aPTHF comprises 2- 10 glutamyl groups that have both an alpha linkage and a gamma linkage, or any range therein between.
  • the polyglutamate chain of the polyglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamate chain of the polyglutamated alpha 5-formyl-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5-formyl-THF that contains a chain of 3 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., tetraglutamated 5-formyl-THF).
  • the composition comprises tetraglutamated alpha [6S]-5-formyl-THF.
  • the composition comprises tetraglutamated alpha [6R,S]-5-formyl- THF.
  • the composition comprises tetraglutamated alpha [6R]-5- formyl-THF.
  • the tetraglutamated alpha 5-formyl-THF comprises 1, 2, or 3, glutamyl groups that have an alpha linkage. In some embodiments, the tetraglutamated alpha 5-formyl-THF comprises two or more glutamyl groups in the I form. In other embodiments, the tetraglutamated alpha 5-formyl-THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamated alpha 5- formyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the tetraglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alpha 5-formyl-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5-formyl-THF that contains a chain of 4 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., pentaglutamated 5-formyl-THF).
  • the composition comprises pentaglutamated alpha [6S]-5-formyl-THF.
  • the composition comprises pentaglutamated alpha [6R,S]-5-formyl- THF.
  • the composition comprises pentaglutamated alpha [6R]-5- formyl-THF.
  • the pentaglutamated alpha 5-formyl-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alpha linkage. In some embodiments, the pentaglutamated 5-formyl-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the pentaglutamated 5-formyl-THF comprises a glutamyl group in the D-form. In further embodiments, the pentaglutamated 5-formyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5- formyl-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5-formyl-THF that contains a chain of 5 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated 5-formyl-THF).
  • the composition comprises hexaglutamated alpha [6S]-5-formyl-THF.
  • the composition comprises hexaglutamated alpha [6R,S]-5-formyl- THF.
  • the composition comprises hexaglutamated alpha [6R]-5- formyl-THF.
  • the hexaglutamated alpha 5-formyl-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have an alpha linkage. In some embodiments, the hexaglutamated alpha 5-formyl-THF comprises two or more glutamyl groups in the L- form. In other embodiments, the hexaglutamated THF comprises a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5-formyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the hexaglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamate chain of the hexaglutamated alpha 5- formyl-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5,l0-methenyl-THF.
  • the composition comprises polyglutamated alpha [6R]-5,lO-methenyl-THF.
  • the composition comprises polyglutamated alpha [6R,S]-5,lO-methenyl-THF.
  • the composition comprises polyglutamated alpha [6S]-5,lO-methenyl- THF.
  • the composition contains polyglutamated alpha 5,10- methenyl-THF that has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including the glutamyl group in 5,l0-methenyl-THF).
  • the polyglutamated alpha 5,l0-methenyl-THF comprises 1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage.
  • the composition contains polyglutamated alpha 5-formyl-THF that has two or more glutamyl groups in the L-form.
  • the composition contains polyglutamated alpha 5,l0-methenyl- THF that has a glutamyl group in the D-form.
  • the composition contains polyglutamated alpha 5,l0-methenyl-THF that has a glutamyl group in the D- form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the polyglutamated alpha 5,l0-methenyl-THF is linear. In some embodiments, the polyglutamate chain of the polyglutamated alpha 5,l0-methenyl-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5,l0-methenyl-THF that contains a chain of 3 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., tetraglutamated 5,l0-methenyl- THF).
  • the composition comprises tetraglutamated alpha [6R]- 5,l0-methenyl-THF.
  • the composition comprises tetraglutamated alpha [6R,S]-5,lO-methenyl-THF.
  • the composition comprises tetraglutamated alpha [6S]-5,lO-methenyl-THF.
  • the tetraglutamated alpha 5,l0-methenyl-THF comprises 1, 2, or 3, glutamyl groups that have an alpha linkage. In some embodiments, the tetraglutamated alpha 5,l0-methenyl- THF comprises two or more glutamyl groups in the L-form. In other embodiments, the tetraglutamated alpha 5,l0-methenyl-THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamated alpha 5,l0-methenyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the 5,l0-methenyl-THF tetrahydrofolate is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alpha 5,10- methenyl-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5,l0-methenyl-THF that contains a chain of 4 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., pentaglutamated 5,l0-methenyl- THF).
  • the composition comprises pentaglutamated alpha [6R]- 5,l0-methenyl-THF.
  • the composition comprises pentaglutamated alpha [6R,S]-5,lO-methenyl-THF.
  • the composition comprises pentaglutamated alpha [6S]-5,lO-methenyl-THF.
  • the pentaglutamated alpha 5,l0-methenyl-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alpha linkage.
  • the pentaglutamated alpha 5,l0-methenyl- THF comprises two or more glutamyl groups in the L-form.
  • the pentaglutamated alpha 5,l0-methenyl-THF comprises a glutamyl group in the D-form.
  • the pentaglutamated alpha 5,l0-methenyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the pentaglutamated alpha 5,l0-methenyl-THF is linear. In some embodiments, the polyglutamate chain of the pentaglutamated alpha
  • the disclosure provides a composition comprising a polyglutamated alpha 5,l0-methenyl-THF that contains a chain of 5 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated 5,l0-methenyl- THF).
  • the composition comprises hexaglutamated alpha [6R]-
  • the composition comprises hexaglutamated alpha [6R,S]-5,lO-methenyl-THF. In some embodiments, the composition comprises hexaglutamated alpha [6S]-5,lO-methenyl-THF. In some embodiments, the hexaglutamated alpha 5,l0-methenyl-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have an alpha linkage. In some embodiments, the hexaglutamated alpha 5,10- methenyl-THF comprises two or more glutamyl groups in the L-form.
  • the hexaglutamated alpha 5,l0-methenyl-THF comprises a glutamyl group in the D-form.
  • the hexaglutamated alpha 5,l0-methenyl- THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • 5.10-methenyl-THF is linear.
  • the polyglutamate chain of the hexaglutamated alpha 5,l0-methenyl-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5-methyl-THF.
  • the composition comprises polyglutamated alpha [6S]-5-methyl-THF.
  • the composition comprises polyglutamated alpha [6R,S]-5-methyl-THF.
  • the composition comprises polyglutamated alpha [6R]-5-methyl-THF.
  • the composition contains polyglutamated alpha 5-methyl-THF that has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including the glutamyl group in 5-methyl-THF).
  • the polyglutamated alpha 5-methyl-THF comprises 1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage.
  • the composition contains polyglutamated alpha 5-methyl-THF that has two or more glutamyl groups in the L-form.
  • the composition contains polyglutamated alpha 5-methyl-THF that has a glutamyl group in the D-form.
  • the composition contains polyglutamated alpha 5-methyl-THF that has a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the polyglutamated alpha 5-methyl- THF is linear.
  • the polyglutamate chain of the polyglutamated alpha 5-methyl-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5-methyl-THF that contains a chain of 3 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., tetraglutamated 5-methyl-THF).
  • the composition comprises tetraglutamated alpha [6S]-5-methyl-THF.
  • the composition comprises tetraglutamated alpha [6R,S]-5-methyl- THF.
  • the composition comprises tetraglutamated alpha [6R]-5- methyl-THF.
  • the tetraglutamated alpha 5-methyl-THF comprises 1, 2, or 3, glutamyl groups that have a gamma linkage In some embodiments, the tetraglutamated alpha 5-methyl-THF comprises two or more glutamyl groups in the L- form. In other embodiments, the tetraglutamated alpha 5-methyl-THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamated alpha 5- methyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the tetraglutamated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alpha 5-methyl-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5-methyl-THF that contains a chain of 4 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., pentaglutamated 5-methyl-THF).
  • the composition comprises pentaglutamated alpha [6S]-5-methyl-THF.
  • the composition comprises pentaglutamated alpha [6R,S]-5-methyl- THF.
  • the composition comprises pentaglutamated alpha [6R]-5- methyl-THF.
  • the pentaglutamated alpha 5-methyl-THF comprises 1, 2, 3, or 4, glutamyl groups that have a gamma linkage. In some embodiments, the pentaglutamated alpha 5-methyl-THF comprises two or more glutamyl groups in the L- form. In other embodiments, the pentaglutamated alpha 5-methyl-THF comprises a glutamyl group in the D-form. In further embodiments, the pentaglutamated alpha 5- methyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5-methyl-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5-methyl-THF that that contains a chain of 5 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated 5-methyl-THF).
  • the composition comprises hexaglutamated alpha [6 S] -5-methyl- THF.
  • the composition comprises hexaglutamated alpha [6R,S]-5- methyl-THF.
  • the composition comprises hexaglutamated alpha [6R]- 5-methyl-THF.
  • the hexaglutamated alpha 5-methyl-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have an alpha linkage. In some embodiments, the hexaglutamated alpha 5-methyl-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamated alpha 5-methyl-THF comprises a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5-methyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the hexaglutamated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamate chain of the hexaglutamated alpha 5-methyl-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha Tetrahydrofolate THF.
  • the composition comprises polyglutamated alpha [6S]- Tetrahydrofolate THF.
  • the composition comprises polyglutamated alpha [6R,S]- Tetrahydrofolate THF.
  • the composition comprises polyglutamated alpha [6R]- Tetrahydrofolate THF.
  • the composition contains polyglutamated alpha Tetrahydrofolate THF that has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including the glutamyl group in Tetrahydrofolate THF).
  • the alpha polyglutamated Tetrahydrofolate-THF comprises 1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage.
  • the composition contains polyglutamated alpha Tetrahydrofolate THF that has two or more glutamyl groups in the L-form.
  • the composition contains polyglutamated alpha Tetrahydrofolate THF that has a glutamyl group in the D-form.
  • the composition contains polyglutamated alpha Tetrahydrofolate THF that has a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the poly glutamate chain of the polyglutamated alpha Tetrahydrofolate THF is linear.
  • the polyglutamate chain of the polyglutamated alpha Tetrahydrofolate THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha Tetrahydrofolate THF that contains a chain of 3 glutamyl groups attached to the glutamyl group of Tetrahydrofolate THF (i.e tetraglutamated Tetrahydrofolate THF).
  • the composition comprises tetraglutamated alpha [6S] Tetrahydrofolate THF.
  • the composition comprises tetraglutamated alpha [6R,S]- Tetrahydrofolate THF.
  • the composition comprises tetraglutamated alpha [6R]-Tetrahydrofolate THF.
  • the tetraglutamated alpha Tetrahydrofolate-THF comprises 1, 2, or 3, glutamyl groups that have an alpha linkage. In some embodiments, the tetraglutamated alpha Tetrahydrofolate THF comprises two or more glutamyl groups in the L-form. In other embodiments, the tetraglutamated alpha Tetrahydrofolate THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamated alpha Tetrahydrofolate THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the tetraglutamated alpha Tetrahydrofolate THF is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alpha Tetrahydrofolate THF is branched. [0027] In some embodiments, the disclosure provides a composition comprising a polyglutamated alpha Tetrahydrofolate THF that contains a chain of 4 glutamyl groups attached to the glutamyl group of THF ( i.e ., pentaglutamated Tetrahydrofolate THF). In some embodiments, the composition comprises pentaglutamated alpha [6S] Tetrahydrofolate THF.
  • the composition comprises pentaglutamated alpha [6R,S]- Tetrahydrofolate THF. In some embodiments, the composition comprises pentaglutamated alpha [6R]- Tetrahydrofolate THF. In some embodiments, the pentaglutamated alpha Tetrahydrofolate-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alpha linkage. In some embodiments, the pentaglutamated alpha THF comprises two or more glutamyl groups in the L-form. In other embodiments, the pentaglutamated alpha Tetrahydrofolate THF comprises a glutamyl group in the D- form.
  • the pentaglutamated alpha Tetrahydrofolate THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L- form.
  • the polyglutamate chain of the pentaglutamated alpha Tetrahydrofolate THF is linear. In some embodiments, the polyglutamate chain of the pentaglutamated alpha THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha Tetrahydrofolate THF that contains a chain of 5 glutamyl groups attached to the glutamyl group of Tetrahydrofolate THF ⁇ i.e., hexaglutamated Tetrahydrofolate THF).
  • the composition comprises hexaglutamated alpha [6S] Tetrahydrofolate THF.
  • the composition comprises hexaglutamated alpha [6R,S]- Tetrahydrofolate THF.
  • the composition comprises hexaglutamated alpha [6R]- Tetrahydrofolate THF.
  • the hexaglutamated alpha Tetrahydrofolate-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have an alpha linkage.
  • the hexaglutamated alpha Tetrahydrofolate Tetrahydrofolate THF comprises two or more glutamyl groups in the L-form.
  • the hexaglutamated Tetrahydrofolate THF comprises a glutamyl group in the D-form.
  • the hexaglutamated alpha Tetrahydrofolate THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the hexaglutamated alpha Tetrahydrofolate THF is linear. In some embodiments, the polyglutamate chain of the hexaglutamated alpha Tetrahydrofolate THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5,l0-methylene-THF.
  • the composition comprises polyglutamated alpha [6R]-5,lO-methylene-THF.
  • the composition comprises polyglutamated alpha [6R,S]-5,lO-methylene-THF.
  • the composition comprises polyglutamated alpha [6S]-5,lO-methylene- THF.
  • the composition contains polyglutamated alpha 5,10- methylene-THF that has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including the glutamyl group in 5,l0-methylene-THF).
  • the polyglutamated alpha 5,l0-methylene-THF comprises 1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage.
  • the composition contains polyglutamated alpha 5,l0-methylene-THF that has two or more glutamyl groups in the L-form.
  • the composition contains polyglutamated alpha 5,10- methylene-THF that has a glutamyl group in the D-form.
  • the composition contains polyglutamated alpha 5,l0-methylene-THF that has a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the polyglutamated alpha 5,l0-methylene-THF is linear. In some embodiments, the polyglutamate chain of the polyglutamated alpha
  • the disclosure provides a composition comprising a polyglutamated alpha 5,l0-methylene-THF that contains a chain of 3 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., tetraglutamated 5,l0-methylene- THF).
  • the composition comprises tetraglutamated alpha [6R]-
  • the composition comprises tetraglutamated alpha [6R,S]-5,lO-methylene-THF. In some embodiments, the composition comprises tetraglutamated alpha [6S]-5,lO-methylene-THF. In some embodiments, the tetraglutamated alpha 5,lO-methylene-THF comprises 1, 2, or 3 glutamyl groups that have an alpha linkage. In some embodiments, the tetraglutamated alpha 5,l0-methylene- THF comprises two or more glutamyl groups in the L-form. In other embodiments, the tetraglutamated alpha 5,l0-methylene-THF comprises a glutamyl group in the D-form.
  • the tetraglutamated alpha 5,l0-methylene-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the tetraglutamated alpha 5,l0-methylene-THF is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alpha
  • the disclosure provides a composition comprising a polyglutamated alpha 5,l0-methylene-THF that contains a chain of 4 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., pentaglutamated lO-methylene- THF).
  • the composition comprises pentaglutamated alpha [6R]
  • the composition comprises pentaglutamated alpha [6R,S]-5,lO-methylene-THF. In some embodiments, the composition comprises pentaglutamated alpha [6S]-5,lO-methylene-THF. In some embodiments, the pentaglutamated alpha 5,l0-methylene-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alpha linkage. In some embodiments, the pentaglutamated alpha 5,l0-methylene-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the pentaglutamated alpha 5,l0-methylene-THF comprises a glutamyl group in the D-form.
  • the pentaglutamated alpha 5,10- methylene-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the pentaglutamated alpha 5,l0-methylene-THF is linear. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5,l0-methylene-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5,l0-methylene-THF that contains a chain of 5 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated 5,l0-methylene- THF).
  • the composition comprises hexaglutamated alpha [6R] 5,l0-methylene-THF.
  • the composition comprises hexaglutamated alpha [6R] 5,l0-methylene-THF.
  • the composition comprises hexaglutamated alpha [6R,S]-5,lO-methylene-THF.
  • the composition comprises hexaglutamated alpha [6S]-5,lO-methylene-THF.
  • the alpha hexa glutamated 5,l0-methylene-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have an alpha linkage.
  • the hexaglutamated alpha 5,l0-methylene-THF comprises two or more glutamyl groups in the L-form.
  • the hexaglutamated alpha 5,l0-methylene-THF comprises a glutamyl group in the D-form.
  • the hexaglutamated alpha 5,10- methylene-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the hexaglutamated alpha 5,l0-methylene-THF is linear. In some embodiments, the polyglutamate chain of the hexaglutamated alpha 5,l0-methylene-THF is branched.
  • the disclosure provides a composition comprising a polyglutamated alpha 5-formimino-THF.
  • the composition comprises polyglutamated alpha [6S]-5-formimino-THF.
  • the composition comprises polyglutamated alpha [6R,S]-5-formimino-THF.
  • the composition comprises polyglutamated alpha [6R]-5-formimino-THF.
  • the composition contains polyglutamated alpha 5-formimino- THF that has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including the glutamyl group in 5-formimino-THF).
  • the polyglutamated alpha 5-formimino-THF comprises 1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage.
  • the composition contains polyglutamated alpha 5- formimino-THF that has two or more glutamyl groups in the L-form.
  • the composition contains polyglutamated alpha 5-formimino-THF that has a glutamyl group in the D-form.
  • the composition contains polyglutamated alpha 5-formimino-THF that has a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the polyglutamated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamate chain of the polyglutamated alpha 5-formimino-THF is branched.
  • the composition comprises a polyglutamated alpha 5-formimino-THF that contains a chain of 3 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., tetraglutamated 5-formimino-THF).
  • the composition comprises tetraglutamated alpha [6S]-5-formimino-THF.
  • the composition comprises tetraglutamated alpha [6R,S]-5- formimino-THF.
  • the composition comprises tetraglutamated alpha [6R] 5-formimino-THF.
  • the tetraglutamated alpha 5- formimino-THF comprises 1, 2, or 3, glutamyl groups that have an alpha linkage. In some embodiments, the tetraglutamated alpha 5-formimino-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the tetraglutamated alpha 5- formimino-THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamated alpha 5-formimino-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the tetraglutamated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alpha 5-formimino-THF is branched.
  • the composition comprises a polyglutamated alpha
  • the composition comprises pentaglutamated alpha [6S]-5-formimino- THF.
  • the composition comprises pentaglutamated alpha [6R,S]- 5-formimino-THF.
  • the composition comprises pentaglutamated alpha [6R] 5-formimino-THF.
  • the pentaglutamated alpha 5- formimino-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alpha linkage.
  • the pentaglutamated alpha 5-formimino-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the pentaglutamated alpha 5- formimino-THF comprises a glutamyl group in the D-form. In further embodiments, the pentaglutamated alpha 5-formimino-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5-formimino-THF is branched.
  • the composition comprises a polyglutamated alpha 5- formimino-THF that contains a chain of 5 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated 5-formimino-THF).
  • the composition comprises hexaglutamated alpha [6S]-5-formimino-THF.
  • the composition comprises hexaglutamated alpha [6R,S]-5-formimino- THF.
  • the composition comprises hexaglutamated alpha [6R] 5-formimino-THF.
  • the hexaglutamated alpha 5-formimino-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have an alpha linkage. In some embodiments, the hexaglutamated alpha 5-formimino-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamated alpha 5- formimino-THF comprises a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5-formimino-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the hexaglutamated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamate chain of the hexaglutamated alpha 5-formimino-THF is branched
  • the disclosure provides a composition comprising a liposome encapsulating (filled with) polyglutamated alpha tetrahydrofolate (Lp-aPTHF).
  • Lp-aPTHF polyglutamated alpha tetrahydrofolate
  • the disclosure provides a composition comprising a liposome encapsulating (filled with) polyglutamated alpha 5-formyl-THF.
  • the liposome comprises polyglutamated alpha [6S]-5-formyl-THF.
  • the liposome comprises polyglutamated alpha [6R,S]-5-formyl-THF.
  • the liposome comprises polyglutamated alpha [6R]-5-formyl-THF.
  • the liposome contains polyglutamated alpha 5-formyl-THF that has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including the glutamyl group in 5-formyl-THF).
  • the polyglutamated alpha 5-formyl-THF comprises 1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage.
  • the liposome contains polyglutamated alpha 5-formyl-THF that has two or more glutamyl groups in the L-form.
  • the liposome contains polyglutamated alpha 5-formyl-THF that has a glutamyl group in the D-form.
  • the liposome contains polyglutamated alpha 5-formyl-THF that has a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the polyglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamate chain of the polyglutamated alpha 5- formyl-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5-formyl-THF that contains a chain of 3 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., tetraglutamated 5-formyl-THF).
  • the liposome comprises tetraglutamated alpha [6S]-5-formyl-THF.
  • the liposome comprises tetraglutamated alpha [6R,S]-5-formyl-THF.
  • the liposome comprises tetraglutamated alpha [6R]-5-formyl-THF.
  • the tetraglutamated alpha 5-formyl-THF comprises 1, 2, or 3, glutamyl groups that have a gamma linkage. In some embodiments, the tetraglutamated alpha 5-formyl-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the tetraglutamated alpha 5-formyl-THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamated alpha 5-formyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the tetraglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alpha 5- formyl-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5-formyl-THF that contains a chain of 4 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., pentaglutamated 5-formyl-THF).
  • the liposome comprises pentaglutamated alpha [6S]-5-formyl-THF.
  • the liposome comprises pentaglutamated alpha [6R,S]-5-formyl-THF.
  • the liposome comprises pentaglutamated alpha [6R]-5-formyl-THF.
  • the pentaglutamated alpha 5-formyl-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alpha linkage. In some embodiments, the pentaglutamated 5-formyl-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the pentaglutamated 5-formyl-THF comprises a glutamyl group in the D- form. In further embodiments, the pentaglutamated 5-formyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5- formyl-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5-formyl-THF that contains a chain of 5 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated 5-formyl-THF).
  • the liposome comprises hexaglutamated alpha [6S]-5-formyl-THF.
  • the liposome comprises hexaglutamated alpha [6R,S]-5-formyl-THF.
  • the liposome comprises hexaglutamated alpha [6R]-5-formyl-THF.
  • the hexaglutamated alpha 5-formyl-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have an alpha linkage. In some embodiments, the hexaglutamated alpha 5-formyl-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamated THF comprises a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5-formyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the hexaglutamated alpha 5-formyl-THF is linear. In some embodiments, the polyglutamate chain of the hexaglutamated alpha 5- formyl-THF is branched.
  • the disclosure provides a composition comprising a liposome encapsulating (filled with) polyglutamated alpha 5,l0-methenyl-THF.
  • the liposome comprises polyglutamated alpha [6R]-5,lO-methenyl-THF.
  • the liposome comprises polyglutamated alpha [6R,S]-5,lO- methenyl-THF.
  • the liposome comprises polyglutamated alpha [6S]-5,lO-methenyl-THF.
  • the liposome contains polyglutamated alpha 5,l0-methenyl-THF that has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including the glutamyl group in 5,l0-methenyl-THF).
  • the polyglutamated alpha 5,l0-methenyl-THF comprises 1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage.
  • the liposome contains polyglutamated alpha 5-formyl-THF that has two or more glutamyl groups in the L-form.
  • the liposome contains polyglutamated alpha 5,l0-methenyl-THF that has a glutamyl group in the D-form.
  • the liposome contains polyglutamated alpha 5,l0-methenyl-THF that has a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the polyglutamated alpha 5,l0-methenyl-THF is linear. In some embodiments, the polyglutamate chain of the polyglutamated alpha 5-formyl-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5,l0-methenyl-THF that contains a chain of 3 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., tetraglutamated 5,l0-methenyl-THF).
  • the liposome comprises tetraglutamated alpha [6R]-5,lO-methenyl-THF.
  • the liposome comprises tetraglutamated alpha [6R,S]-5,lO- methenyl-THF.
  • the liposome comprises tetraglutamated alpha [6S]-5,lO-methenyl-THF.
  • the tetraglutamated alpha 5,10- methenyl-THF comprises 1, 2, or 3, glutamyl groups that have an alpha linkage.
  • the tetraglutamated alpha 5,l0-methenyl-THF comprises two or more glutamyl groups in the L-form.
  • the tetraglutamated alpha 5,10- methenyl-THF comprises a glutamyl group in the D-form.
  • the tetraglutamated alpha 5,l0-methenyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the 5,l0-methenyl-THF tetrahydrofolate is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alpha 5,l0-methenyl-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5,l0-methenyl-THF that contains a chain of 4 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., pentaglutamated 5,l0-methenyl-THF).
  • the liposome comprises pentaglutamated alpha [6R]-5,lO-methenyl-THF.
  • the liposome comprises pentaglutamated alpha [6R,S]-5,lO- methenyl-THF.
  • the liposome comprises pentaglutamated alpha [6S]-5,lO-methenyl-THF.
  • the pentaglutamated alpha 5,10- methenyl-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alpha linkage.
  • the pentaglutamated alpha 5,l0-methenyl-THF comprises two or more glutamyl groups in the L-form.
  • the pentaglutamated alpha 5,l0-methenyl-THF comprises a glutamyl group in the D-form.
  • the pentaglutamated alpha 5,l0-methenyl-THF comprises a glutamyl group in the D- form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the pentaglutamated alpha 5,l0-methenyl-THF is linear. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5,l0-methenyl-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5,l0-methenyl-THF that contains a chain of 5 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated 5,l0-methenyl-THF).
  • the liposome comprises hexaglutamated alpha [6R]-5,lO-methenyl-THF.
  • the liposome comprises hexaglutamated alpha [6R,S]-5,lO- methenyl-THF.
  • the liposome comprises hexaglutamated alpha [6S]-5,lO-methenyl-THF.
  • the hexaglutamated alpha 5,10- methenyl-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have an alpha linkage.
  • the hexaglutamated alpha 5,l0-methenyl-THF comprises two or more glutamyl groups in the L-form.
  • the hexaglutamated alpha 5,l0-methenyl-THF comprises a glutamyl group in the D-form.
  • the hexaglutamated alpha 5, lO-methenyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the hexaglutamated alpha 5, lO-methenyl-THF is linear.
  • the polyglutamate chain of the hexaglutamated alpha 5, lO-methenyl-THF is branched.
  • the disclosure provides a composition comprising a liposome encapsulating (filled with) polyglutamated alpha 5-methyl-THF.
  • the liposome comprises polyglutamated alpha [6S]-5-methyl-THF. In some embodiments, the liposome comprises polyglutamated alpha [6R,S]-5-methyl- THF. In some embodiments, the liposome comprises polyglutamated alpha [6R]-5- methyl-THF. In some embodiments, the liposome contains polyglutamated alpha 5- methyl-THF that has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including the glutamyl group in 5-methyl-THF). In some embodiments, the polyglutamated alpha 5-methyl-THF comprises 1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage.
  • the liposome contains polyglutamated alpha 5-methyl- THF that has two or more glutamyl groups in the L-form. In other embodiments, the liposome contains polyglutamated alpha 5-methyl-THF that has a glutamyl group in the D-form. In further embodiments, the liposome contains polyglutamated alpha 5-methyl- THF that has a glutamyl group in the D-form and two or more glutamyl groups in the L- form. In some embodiments, the polyglutamate chain of the polyglutamated alpha 5- methyl-THF is linear. In some embodiments, the polyglutamate chain of the polyglutamated alpha 5-methyl-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5-methyl-THF that contains a chain of 3 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., tetraglutamated 5-methyl-THF).
  • the liposome comprises tetraglutamated alpha [6S]-5-methyl-THF.
  • the liposome comprises tetraglutamated alpha [6R,S]-5-methyl-THF.
  • the liposome comprises tetraglutamated alpha [6R]-5-methyl-THF.
  • the tetraglutamated alpha 5-methyl-THF comprises 1, 2, or 3, glutamyl groups that have an alpha linkage. In some embodiments, the tetraglutamated alpha 5-methyl-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the tetraglutamated alpha 5-methyl-THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamated alpha 5-methyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the tetraglutamated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alpha 5- methyl-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5-methyl-THF that contains a chain of 4 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., pentaglutamated 5-methyl-THF).
  • the liposome comprises pentaglutamated alpha [6S]-5-methyl-THF.
  • the liposome comprises pentaglutamated alpha [6R,S]-5-methyl-THF.
  • the liposome comprises pentaglutamated alpha [6R]-5-methyl-THF.
  • the pentaglutamated alpha 5-methyl-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alpha linkage. In some embodiments, the pentaglutamated alpha 5-methyl-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the pentaglutamated alpha 5-methyl-THF comprises a glutamyl group in the D-form. In further embodiments, the pentaglutamated alpha 5-methyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5- methyl-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5-methyl-THF that that contains a chain of 5 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated 5-methyl-THF).
  • the liposome comprises hexaglutamated alpha [6S]-5-methyl-THF.
  • the liposome comprises hexaglutamated alpha [6R,S]-5-methyl- THF.
  • the liposome comprises hexaglutamated alpha [6R]-5- methyl-THF.
  • the hexaglutamated alpha 5-methyl-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have an alpha linkage. In some embodiments, the hexaglutamated alpha 5-methyl-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamated alpha 5-methyl-THF comprises a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5-methyl-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the hexaglutamated alpha 5-methyl-THF is linear. In some embodiments, the polyglutamate chain of the hexaglutamated alpha 5-methyl-THF is branched.
  • the disclosure provides a composition comprising a liposome encapsulating (filled with) polyglutamated alpha Tetrahydrofolate THF.
  • the liposome comprises polyglutamated alpha [6S]-Tetrahydrofolate THF.
  • the liposome comprises polyglutamated alpha [6R,S]- Tetrahydrofolate THF.
  • the liposome comprises polyglutamated alpha [6R]-Tetrahydrofolate THF.
  • the liposome contains polyglutamated alpha Tetrahydrofolate THF that has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including the glutamyl group in Tetrahydrofolate THF).
  • the polyglutamated alpha Tetrahydrofolate THF comprises 1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage.
  • the liposome contains polyglutamated alpha Tetrahydrofolate THF that has two or more glutamyl groups in the L-form.
  • the liposome contains polyglutamated alpha Tetrahydrofolate THF that has a glutamyl group in the D-form.
  • the liposome contains polyglutamated alpha Tetrahydrofolate THF that has a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the polyglutamated alpha Tetrahydrofolate THF is linear. In some embodiments, the polyglutamate chain of the polyglutamated alpha Tetrahydrofolate THF is branched.
  • the Lp-aPTetrahydrofolate THF composition comprises a polyglutamated alpha tetrahydrofolate that contains a chain of 3 glutamyl groups attached to the glutamyl group of Tetrahydrofolate THF (i.e tetraglutamated tetrahydrofolate).
  • the liposome comprises tetraglutamated alpha [6S] tetrahydrofolate.
  • the liposome comprises tetraglutamated alpha [6R,S]-Tetrahydrofolate THF.
  • the liposome comprises tetraglutamated alpha [6R]-Tetrahydrofolate THF.
  • the tetraglutamated alpha Tetrahydrofolate THF comprises 1, 2, or 3 glutamyl groups that have an alpha linkage. In some embodiments, the tetraglutamated alpha Tetrahydrofolate THF comprises two or more glutamyl groups in the L-form. In other embodiments, the tetraglutamated alpha Tetrahydrofolate THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamated alpha Tetrahydrofolate THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the tetraglutamated alpha tetrahydrofolate is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alpha tetrahydrofolate is branched.
  • the Lp-aPTetrahydrofolate THF composition comprises a polyglutamated alpha tetrahydrofolate that contains a chain of 4 glutamyl groups attached to the glutamyl group of Tetrahydrofolate THF (i.e pentaglutamated tetrahydrofolate).
  • the liposome comprises pentaglutamated alpha [6S] tetrahydrofolate.
  • the liposome comprises pentaglutamated alpha [6R,S]-Tetrahydrofolate THF.
  • the liposome comprises pentaglutamated alpha [6R]-Tetrahydrofolate THF.
  • the pentaglutamated alpha Tetrahydrofolate THF comprises 1, 2, 3, or 4, glutamyl groups that have an alpha linkage. In some embodiments, the pentaglutamated alpha Tetrahydrofolate THF comprises two or more glutamyl groups in the L-form. In other embodiments, the pentaglutamated alpha Tetrahydrofolate THF comprises a glutamyl group in the D-form. In further embodiments, the pentaglutamated alpha Tetrahydrofolate THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the pentaglutamated alpha Tetrahydrofolate THF is linear. In some embodiments, the polyglutamate chain of the pentaglutamated alpha Tetrahydrofolate THF is branched.
  • the Lp-aPTetrahydrofolate THF composition comprises a polyglutamated alpha tetrahydrofolate that contains a chain of 5 glutamyl groups attached to the glutamyl group of tetrahydrofolate ⁇ i.e., hexaglutamated tetrahydrofolate).
  • the liposome comprises hexaglutamated alpha [6S] tetrahydrofolate.
  • the liposome comprises hexaglutamated alpha [6R,S]- Tetrahydrofolate THF.
  • the liposome comprises hexaglutamated alpha [6R]-Tetrahydrofolate THF.
  • the hexaglutamated alpha Tetrahydrofolate THF comprises 1, 2, 3, 4, or 5 glutamyl groups that have an alpha linkage.
  • the hexaglutamated alpha Tetrahydrofolate THF comprises two or more glutamyl groups in the L-form.
  • the hexaglutamated Tetrahydrofolate THF comprises a glutamyl group in the D-form.
  • the hexaglutamated alpha Tetrahydrofolate THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the hexaglutamated alpha tetrahydrofolate is linear. In some embodiments, the polyglutamate chain of the hexaglutamated alpha tetrahydrofolate is branched.
  • the disclosure provides a composition comprising a liposome encapsulating (filled with) polyglutamated alpha 5,l0-methylene-THF.
  • the liposome comprises polyglutamated alpha [6R]-5,lO-methylene- THF.
  • the liposome comprises polyglutamated alpha [6R,S]-5,lO- methylene-THF.
  • the liposome comprises polyglutamated alpha [6S]-5,lO-methylene-THF.
  • the liposome contains polyglutamated alpha 5,l0-methylene-THF that has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including the glutamyl group in 5,l0-methylene-THF).
  • the polyglutamated alpha 5,l0-methylene-THF comprises 1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage.
  • the liposome contains polyglutamated alpha 5,l0-methylene-THF that has two or more glutamyl groups in the L-form.
  • the liposome contains polyglutamated alpha 5,10- methylene-THF that has a glutamyl group in the D-form.
  • the liposome contains polyglutamated alpha 5,l0-methylene-THF that has a glutamyl group in the D-form and two or more glutamyl.
  • the polyglutamate chain of the polyglutamated alpha 5,l0-methylene-THF is linear. In some embodiments, the polyglutamate chain of the polyglutamated alpha 5,l0-methylene-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5,l0-methylene-THF that contains a chain of 3 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., tetraglutamated 5,l0-methylene-THF).
  • the liposome comprises tetraglutamated alpha [6R]-5,lO-methylene-THF.
  • the liposome comprises tetraglutamated alpha [6R,S]-5,lO- methylene-THF.
  • the liposome comprises tetraglutamated alpha [6S]-5,lO-methylene-THF.
  • the tetraglutamated alpha 5,10- methylene-THF comprises 1, 2, or 3, glutamyl groups that have an alpha linkage. In some embodiments, the tetraglutamated alpha 5,l0-methylene-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the tetraglutamated alpha 5,10- methylene-THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamated alpha 5,l0-methylene-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the tetraglutamated alpha 5,l0-methylene-THF is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alpha 5,l0-methylene-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5,l0-methylene-THF that contains a chain of 4 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., pentaglutamated lO-methylene-THF).
  • the liposome comprises pentaglutamated alpha [6R] 5, lO-methylene-THF.
  • the liposome comprises pentaglutamated alpha [6R,S]-5,lO- methylene-THF.
  • the liposome comprises pentaglutamated alpha [6S]-5, lO-methylene-THF.
  • the pentaglutamated alpha 5,10- methylene-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alpha linkage.
  • the pentaglutamated alpha 5, lO-methylene-THF comprises two or more glutamyl groups in the L-form.
  • the pentaglutamated alpha 5, lO-methylene-THF comprises a glutamyl group in the D-form.
  • the pentaglutamated alpha 5, lO-methylene-THF comprises a glutamyl group in the D- form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the pentaglutamated alpha 5, lO-methylene-THF is linear. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5,l0-methylene- THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5,l0-methylene-THF that contains a chain of 5 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated 5,l0-methylene-THF).
  • the liposome comprises hexaglutamated alpha [6R] 5,l0-methylene-THF.
  • the liposome comprises hexaglutamated alpha [6R] 5,10- methylene-THF.
  • the liposome comprises hexaglutamated alpha [6R,S]-5,lO-methylene-THF.
  • the liposome comprises hexaglutamated alpha [6S]-5,lO-methylene-THF.
  • the hexaglutamated alpha 5,l0-methylene-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have an alpha linkage.
  • the hexaglutamated alpha 5,10- methylene-THF comprises two or more glutamyl groups in the L-form.
  • the hexaglutamated alpha 5,l0-methylene-THF comprises a glutamyl group in the D-form.
  • the hexaglutamated alpha 5,l0-methylene- THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the hexaglutamated alpha 5,l0-methylene-THF is linear. In some embodiments, the polyglutamate chain of the hexaglutamated alpha 5,l0-methylene-THF is branched.
  • the disclosure provides a composition comprising a liposome encapsulating (filled with) polyglutamated alpha 5-formimino-THF.
  • the liposome comprises polyglutamated alpha [6S]-5-formimino-THF.
  • the liposome comprises polyglutamated alpha [6R,S]-5-formimino- THF.
  • the liposome comprises polyglutamated alpha [6R]-5- formimino-THF.
  • the liposome contains polyglutamated alpha 5- formimino-THF that has 2-20, 2-15, 2-10, 2-5, or more than 20, glutamyl groups (including the glutamyl group in 5-formimino-THF).
  • the polyglutamated alpha 5-formimino-THF comprises 1, 2, 3, or more than 3, glutamyl groups that have an alpha linkage.
  • the liposome contains polyglutamated alpha 5-formimino-THF that has two or more glutamyl groups in the L- form.
  • the liposome contains polyglutamated alpha 5-formimino- THF that has a glutamyl group in the D-form.
  • the liposome contains polyglutamated alpha 5-formimino-THF that has a glutamyl group in the D- form and two or more glutamyl groups in the L-form.
  • the poly glutamate chain of the polyglutamated alpha 5-formimino-THF is linear.
  • the polyglutamate chain of the polyglutamated alpha 5-formimino-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5-formimino-THF that contains a chain of 3 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., tetraglutamated 5-formimino-THF).
  • the liposome comprises tetraglutamated alpha [6S]-5-formimino-THF.
  • the liposome comprises tetraglutamated alpha [6R,S]-5-formimino- THF.
  • the liposome comprises tetraglutamated alpha [6R] 5- formimino-THF.
  • the tetraglutamated alpha 5-formimino-THF comprises 1, 2, or 3, glutamyl groups that have an alpha linkage. In some embodiments, the tetraglutamated alpha 5-formimino-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the tetraglutamated alpha 5-formimino-THF comprises a glutamyl group in the D-form. In further embodiments, the tetraglutamated alpha 5-formimino-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the tetraglutamated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamate chain of the tetraglutamated alpha 5-formimino-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha 5-formimino-THF that contains a chain of 4 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., pentaglutamated 5-formimino-THF).
  • the liposome comprises pentaglutamated alpha [6S]-5-formimino-THF.
  • the liposome comprises pentaglutamated alpha [6R,S]-5-formimino- THF.
  • the liposome comprises pentaglutamated alpha [6R] 5- formimino-THF.
  • the pentaglutamated alpha 5-formimino-THF comprises 1, 2, 3, or 4, glutamyl groups that have an alpha linkage. In some embodiments, the pentaglutamated alpha 5-formimino-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the pentaglutamated alpha 5-formimino-THF comprises a glutamyl group in the D-form. In further embodiments, the pentaglutamated alpha 5-formimino-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamate chain of the pentaglutamated alpha 5-formimino-THF is branched.
  • the Lp-aPTHF composition comprises a polyglutamated alpha -5-formimino-THF that contains a chain of 5 glutamyl groups attached to the glutamyl group of tetrahydrofolate (i.e., hexaglutamated 5-formimino-THF).
  • the liposome comprises hexaglutamated alpha [6S]-5-formimino-THF.
  • the liposome comprises hexaglutamated alpha [6R,S]-5-formimino- THF.
  • the liposome comprises hexaglutamated alpha [6R] 5- formimino-THF.
  • the hexaglutamated alpha 5-formimino-THF comprises 1, 2, 3, 4, or 5, glutamyl groups that have an alpha linkage. In some embodiments, the hexaglutamated alpha 5-formimino-THF comprises two or more glutamyl groups in the L-form. In other embodiments, the hexaglutamated alpha 5- formimino-THF comprises a glutamyl group in the D-form. In further embodiments, the hexaglutamated alpha 5-formimino-THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain of the hexaglutamated alpha 5-formimino-THF is linear. In some embodiments, the polyglutamate chain of the hexaglutamated alpha 5-formimino-THF is branched
  • the Lp-aPTHF composition is cationic.
  • the Lp-aPTHF liposome is cationic and has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therein between.
  • the Lp-aPTHF liposome is cationic and the composition has a diameter in the range of 80 nm to 120 nm, or any range therein between.
  • the cationic Lp-aPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the polyglutamated alpha THF.
  • at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, of the starting material of polyglutamated alpha THF is encapsulated (entrapped) in the cationic Lp-aPTHF.
  • the polyglutamated alpha tetrahydrofolate encapsulated by the liposome is in a HEPES buffered solution within the liposome.
  • Lp-aPTHF composition is anionic or neutral.
  • the Lp-aPTHF liposome is anionic or neutral and has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therein between.
  • the Lp-aPTHF liposome is anionic or neutral and the composition has a diameter in the range of 80 nm to 120 nm, or any range therein between.
  • the Lp-aPTHF liposome is anionic and has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therein between. In further embodiments, the Lp-aPTHF liposome is anionic and the composition has a diameter in the range of 80 nm to 120 nm, or any range therein between.
  • the Lp-aPTHF liposome is neutral and has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therein between.
  • the anionic or neutral Lp- aPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the polyglutamated alpha THF.
  • the anionic or neutral Lp-aPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the tetraglutamated alpha THF.
  • the anionic or neutral Lp-aPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the pentaglutamated alpha THF. In some embodiments, the anionic or neutral Lp-aPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the hexaglutamated alpha THF. In additional embodiments, the polyglutamated alpha tetrahydrofolate encapsulated by the liposome is in a HEPES buffered solution within the liposome.
  • the liposomal polyglutamated alpha tetrahydrofolate composition is pegylated (PLp-aPTHF).
  • the liposomal polyglutamated alpha tetrahydrofolate composition is non-targeted (NTLp-aPTHF). That is, the NTLp-aPTHF composition does not have specific affinity towards an epitope (e.g ., an epitope of a surface antigen) expressed on the surface of a target cell of interest.
  • the non- targeted liposomal polyglutamated alpha tetrahydrofolate composition is pegylated (NTPLp-aPTHF).
  • the liposomal polyglutamated alpha tetrahydrofolate composition is targeted (TLp-aPTHF). That is, the TLp-aPTHF composition contains a targeting moiety that has specific affinity for an epitope (surface antigen) on a target cell of interest. In some embodiments, the targeting moiety of the TLp-aPTHF or TPLp- aPTHF is not attached to the liposome through a covalent bond. In other embodiments, the targeting moiety of the TLp-aPTHF or TPLp-aPTHF is attached to one or both of a PEG and the exterior of the liposome.
  • Targeted liposomal polyglutamated alpha tetrahydrofolate compositions provide further improvements over the efficacy and safety profile of tetrahydrofolate, by specifically delivering polyglutamated alpha (e.g., a-pentaglutamated and/or a-hexaglutamated) tetrahydrofolate to target cells such as cancer cells.
  • polyglutamated alpha e.g., a-pentaglutamated and/or a-hexaglutamated
  • the targeted liposomal polyglutamated alpha tetrahydrofolate composition is pegylated (TPLp- aPTHF).
  • the targeting moiety of the TLp-aPTHF or TPLp-aPTHF is attached to one or both of a PEG and the exterior of the liposome. In some embodiments, the targeting moiety of the TLp-aPTHF or TPLp-aPTHF is attached to the liposome through a covalent bond.
  • Function of the targeting moiety of the TLp-aPTHF and/or TPLp-aPTHF compositions include but are not limited to, targeting the liposome to the target cell of interest in vivo or in vitro; interacting with the surface antigen for which the targeting moiety has specific affinity, and delivering the liposome payload (aPTHF) into the cell.
  • Suitable targeting moieties are known in the art and include, but are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides, and peptides.
  • the targeting moiety is a polypeptide.
  • the targeting moiety is a polypeptide that comprises at least 3, 5, 10, 15, 20, 30, 40, 50, or 100, amino acid residues.
  • the targeting moiety of the TLp-aPTHF or TPLp-aPTHF is an antibody or an antigen-binding antibody fragment.
  • the targeting moiety comprises one or more of an antibody, a humanized antibody, an antigen binding fragment of an antibody, a single chain antibody, a single-domain antibody, a bi- specific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody.
  • the targeting moiety of the TLp-aPTHF or TPLp-aPTHF has specific affinity for an epitope that is preferentially expressed on a target cell such as a tumor cell, compared to normal or non-tumor cells.
  • the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on a tumor cell but absent or inaccessible on a non-tumor cell. In some embodiments, the targeting moiety binds an epitope of interest with an equilibrium dissociation constant (Kd) in a range of 0.5 x 10 10 to 10 x 10 6 as determined using BIACORE® analysis.
  • Kd equilibrium dissociation constant
  • the TLp-aPTHF or TPLp-aPTHF targeting moiety comprises a polypeptide that specifically binds a folate receptor.
  • the targeting moiety is an antibody or an antigen-binding antibody fragment.
  • the folate receptor bound by the targeting moiety is one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-a, FOLR1), folate receptor beta (FR-b, FOLR2), and folate receptor delta (FR-d, FOLR4).
  • the folate receptor bound by the targeting moiety is folate receptor alpha (FR-a).
  • the folate receptor bound by the targeting moiety is folate receptor beta (FR-b).
  • the targeting moiety specifically binds FR-a and FR-b.
  • the Lp-aPTHF composition comprises one or more of an immunostimulatory agent, a detectable marker, and a maleimide, disposed on at least one of the PEG and the exterior of the liposome.
  • the liposome aPTHF composition e.g, Lp-aPTHF, PLp-aPTHF, NTLp-aPTHF, NTPLp-aPTHF, TLp-aPTHF, or TPLp-aPTHF
  • Lp-aPTHF, PLp-aPTHF, NTLp-aPTHF, NTPLp-aPTHF, TLp-aPTHF, or TPLp-aPTHF is cationic.
  • the liposome aPTHF composition e.g, Lp-aPTHF, PLp-aPTHF, NTLp-aPTHF, NTPLp-aPTHF, TLp-aPTHF or TPLp-aPTHF
  • Lp-aPTHF, PLp-aPTHF, NTLp-aPTHF, NTPLp-aPTHF, TLp-aPTHF or TPLp-aPTHF is anionic or neutral.
  • the liposome of the liposome aPTHF composition (e.g, Lp-aPTHF, PLp-aPTHF, NTLp-aPTHF, NTPLp- aPTHF, TLp-aPTHF or TPLp-aPTHF) has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, 50 nm to 150 nm, or any range therein between. In some embodiments, the liposome of the liposome-aPTHF composition has a diameter in the range of 30 nm to 175 nm or 50 nm to 150 nm, or any range therein between.
  • the liposome of the liposome aPTHF composition has a diameter in the range of 80 nm to 120 nm, or any range therein between.
  • the liposome aPTHF composition is pegylated (e.g, PLp-aPTHF, NTPLp-aPTHF, or TPLp- aPTHF).
  • the liposome aPTHF composition comprises a targeting moiety (e.g, TLp-aPTHF or TPLp-aPTHF).
  • the liposome aPTHF composition is pegylated and targeted (e.g, TPLp-aPTHF).
  • the liposome aPTHF composition comprises polyglutamated alpha tetrahydrofolate that contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, the liposome aPTHF composition comprises tetraglutamated alpha tetrahydrofolate. In some embodiments, the liposome aPTHF composition comprises pentaglutamated alpha tetrahydrofolate. In other embodiments, the liposome aPTHF composition comprises hexaglutamated alpha tetrahydrofolate.
  • the liposome compositions comprise a polyglutamated alpha tetrahydrofolate that contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the polyglutamated alpha THF.
  • the Lp-aPTHF composition comprises polyglutamated alpha tetrahydrofolate that contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups and l%- 98.5% w/w of the polyglutamated alpha THF.
  • the liposomes comprise polyglutamated alpha tetrahydrofolate that contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups and wherein during the process of preparing the Lp-aPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of polyglutamated alpha THF is encapsulated (entrapped) in the Lp-aPTHF.
  • the liposome compositions comprise a tetraglutamated alpha tetrahydrofolate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the tetraglutamated alpha THF.
  • the Lp-aPTHF composition comprises tetraglutamated alpha tetrahydrofolate and l%-98.5% w/w of the tetraglutamated alpha THF.
  • the liposomes comprise tetraglutamated alpha tetrahydrofolate and wherein during the process of preparing the Lp-aPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of tetraglutamated alpha THF is encapsulated (entrapped) in the Lp-aPTHF.
  • the liposome compositions comprise a pentaglutamated alpha tetrahydrofolate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the pentaglutamated alpha THF.
  • the Lp-aPTHF composition comprises pentaglutamated alpha tetrahydrofolate and l%-98.5% w/w of the pentaglutamated alpha THF.
  • the liposomes comprise pentaglutamated alpha tetrahydrofolate and wherein during the process of preparing the Lp-aPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of pentaglutamated alpha THF is encapsulated (entrapped) in the Lp- aPTHF.
  • the liposome compositions comprise a hexaglutamated alpha tetrahydrofolate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the hexaglutamated alpha THF.
  • the Lp-aPTHF composition comprises hexaglutamated alpha tetrahydrofolate and l%-98.5% w/w of the hexaglutamated alpha THF.
  • the liposomes comprise hexaglutamated alpha tetrahydrofolate and wherein during the process of preparing the Lp-aPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of the starting material of pentaglutamated alpha THF is encapsulated (entrapped) in the Lp- aPTHF.
  • Liposomal compositions comprising liposomes encapsulating aPTHF are also provided.
  • the liposomal composition comprises a pegylated aPTHF composition.
  • the liposomal composition comprises a aPTHF composition that is linked to or otherwise associated with a targeting moiety.
  • the liposomal composition comprises a aPTHF composition that is pegylated and linked to or otherwise associated with a targeting moiety.
  • the liposomal composition comprises aPTHF that contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • the liposomal composition comprises tetraglutamated alpha tetrahydrofolate.
  • the liposomal composition comprises pentaglutamated alpha tetrahydrofolate.
  • the liposomal composition comprises hexaglutamated alpha tetrahydrofolate.
  • the liposomal composition comprises a liposome aPTHF
  • the liposome aPTHF is pegylated (e.g, NTPLp-aPTHF, and TPLp-aPTHF).
  • the liposome aPTHF comprises a targeting moiety that has a specific affinity for an epitope of an antigen on the surface of a target cell of interest such as a cancer cell (e.g, TLp-aPTHF or TPLp-aPTHF)).
  • the liposomal composition comprises a liposome aPTHF that is pegylated and further comprises a targeting moiety that has a specific affinity for an epitope of an antigen on the surface of a target cell of interest such as a cancer cell (e.g ., TPLp-aPTHF).
  • a target cell of interest such as a cancer cell
  • the liposomal composition comprises a liposome aPTHF that is cationic.
  • the liposomal composition comprises a liposome aPTHF that is anionic or neutral.
  • the liposomal composition comprises a liposome aPTHF that has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, or any range therein between. In further embodiments, the liposome aPTHF has a diameter in the range of 80 nm to 120 nm, or any range therein between.
  • compositions comprising polyglutamated alpha tetrahydrofolate
  • the pharmaceutical composition comprises a pegylated aPTHF composition.
  • the pharmaceutical composition comprise a aPTHF composition that is linked to or otherwise associated with a targeting moiety.
  • the pharmaceutical composition comprise a aPTHF composition that is pegylated and linked to or otherwise associated with a targeting moiety.
  • the pharmaceutical composition comprises aPTHF that contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • the pharmaceutical composition comprises tetraglutamated alpha tetrahydrofolate.
  • the pharmaceutical composition comprises pentaglutamated alpha tetrahydrofolate.
  • the pharmaceutical composition comprises hexaglutamated alpha tetrahydrofolate.
  • the pharmaceutical compositions comprise a liposome aPTHF (e.g., Lp-aPTHF, PLp-aPTHF, NTLp-aPTHF, NTPLp-aPTHF, TLp-aPTHF, and TPLp-aPTHF).
  • a liposome aPTHF e.g., Lp-aPTHF, PLp-aPTHF, NTLp-aPTHF, NTPLp-aPTHF, TLp-aPTHF, and TPLp-aPTHF.
  • the liposome aPTHF composition is pegylated (e.g, NTPLp-aPTHF, and TPLp-aPTHF).
  • the liposome aPTHF comprises a targeting moiety that has a specific affinity for an epitope of an antigen on the surface of a target cell of interest such as a cancer cell (e.g, TLp-aPTHF or TPLp- aPTHF)).
  • the pharmaceutical composition comprises a liposome aPTHF composition that is pegylated and further comprises a targeting moiety that has a specific affinity for an epitope of an antigen on the surface of a target cell of interest such as a cancer cell ( e.g TPLp-aPTHF).
  • the pharmaceutical composition comprises a liposome aPTHF that is cationic.
  • the pharmaceutical composition comprises a liposome aPTHF that is anionic or neutral.
  • the pharmaceutical composition comprises a liposome aPTHF that has a diameter in the range of 20 nm to 500 nm or 20 nm to 500 nm, or any range therein between.
  • the liposome aPTHF composition has a diameter in the range of 80 nm to 120 nm, or any range therein between.
  • the disclosure provides a method of modulating the activation, chemokine production, or metabolic activity of a cell that comprises contacting the cell with a composition comprising a polyglutamated alpha tetrahydrofolate (aPTHF) composition.
  • aPTHF polyglutamated alpha tetrahydrofolate
  • the contacted cell is a mammalian cell.
  • the contacted cell is a human cell.
  • the contacted cell is a hyperproliferative cell.
  • the cell is an immune cell.
  • the method is performed in vivo. In other embodiments, the method is performed in vitro.
  • the aPTHF contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • the aPTHF composition comprises tetraglutamated alpha tetrahydrofolate. In some embodiments, the aPTHF composition comprises pentaglutamated alpha tetrahydrofolate. In other embodiments, the aPTHF composition comprises hexaglutamated alpha tetrahydrofolate.
  • the disclosure provides a method of modulating the activation, chemokine production, or metabolic activity of a cell that comprises contacting the cell with a liposome comprising a polyglutamated alpha tetrahydrofolate (aPTHF) composition.
  • aPTHF polyglutamated alpha tetrahydrofolate
  • the contacted cell is a mammalian cell.
  • the contacted cell is a human cell.
  • the contacted cell is a hyperproliferative cell.
  • the cell is an immune cell.
  • the method is performed in vivo. In other embodiments, the method is performed in vitro.
  • the aPTHF contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • the aPTHF composition comprises tetraglutamated alpha tetrahydrofolate. In some embodiments, the aPTHF composition comprises pentaglutamated alpha tetrahydrofolate. In other embodiments, the aPTHF composition comprises hexaglutamated alpha tetrahydrofolate.
  • the disclosure provides a method of killing a cell that comprises contacting the cell with a composition comprising a polyglutamated alpha tetrahydrofolate (aPTHF) composition (e.g, a aPTHF herein).
  • aPTHF polyglutamated alpha tetrahydrofolate
  • the contacted cell is a mammalian cell.
  • the contacted cell is a human cell.
  • the contacted cell is a hyperproliferative cell.
  • the hyperproliferative cell is a cancer cell.
  • the contacted cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from the group consisting of: a non-hematologic malignancy including such as for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g, osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and a hematologic malignancy such as for example, a leukemia, a lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias.
  • a non-hematologic malignancy including such as for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric
  • the contacted cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, stomach cancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, and chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma (desmoid tumors, aggressive fibromatosis, bladder cancer, and central Nervous System (CNS) lymphoma.
  • a cancer selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, stomach cancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, and chor
  • the cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from colorectal cancer, breast cancer, gastric cancer (e.g, stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g, non small cell lung cancer and/or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell cancer.
  • the cancer cell is a primary cell or a cell from a cell line obtained/derived from colorectal cancer.
  • the aPTHF contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • the aPTHF contains 4 glutamyl groups.
  • the aPTHF contains 5 glutamyl groups.
  • the aPTHF contains 6 glutamyl groups.
  • the method is performed in vivo. In other embodiments, the method is performed in vitro.
  • the disclosure provides a method of killing a cell that comprises contacting the cell with a liposome containing polyglutamated alpha tetrahydrofolate (e.g ., an Lp-aPTHF such as, PLp-aPTHF, NTLp-aPTHF, NTPLp- aPTHF, TLp-aPTHF or TPLp-aPTHF).
  • a liposome containing polyglutamated alpha tetrahydrofolate e.g ., an Lp-aPTHF such as, PLp-aPTHF, NTLp-aPTHF, NTPLp- aPTHF, TLp-aPTHF or TPLp-aPTHF.
  • the contacted hyperproliferative cell is a cancer cell.
  • the cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from the group consisting of: a non-hematologic malignancy including such as for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and a hematologic malignancy such as for example, a leukemia, a lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias.
  • a non-hematologic malignancy including such as for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer
  • the contacted cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, stomach cancer, osteosarcoma, Non- Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, and chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma (desmoid tumors, aggressive fibromatosis, bladder cancer, and central Nervous System (CNS) lymphoma.
  • a cancer selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, stomach cancer, osteosarcoma, Non- Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, and chor
  • the cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from colorectal cancer, breast cancer, gastric cancer (e.g, stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g, non-small cell lung cancer and/or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell cancer.
  • the cancer cell is a primary cell or a cell from a cell line obtained/derived from colorectal cancer.
  • the method is performed in vivo. In other embodiments, the method is performed in vitro.
  • the liposome contains a aPTHF containing 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, the liposome comprises a aPTHF containing 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, the liposome comprises a aPTHF containing 4 glutamyl groups. In some embodiments, the liposome comprises a aPTHF containing 5 glutamyl groups. In some embodiments, the liposome comprises a aPTHF containing 6 glutamyl groups. In some embodiments, the aPTHF contains 1, 2, 3, or more than 3, glutamyl groups having a gamma linkage.
  • the disclosure provides a method for treating cancer that comprises administering an effective amount of a delivery vehicle (e.g ., an immunoconjugate or liposome) comprising polyglutamated alpha tetrahydrofolate to a subject having or at risk of having cancer.
  • a delivery vehicle e.g ., an immunoconjugate or liposome
  • the delivery vehicle is an antibody-containing immunoconjugate (comprising e.g., a full-length IgG antibody, abispecific antibody, or a scFv).
  • the delivery vehicle is a liposome (e.g, an Lp-aPTHF such as, PLp-aPTHF, NTLp-aPTHF, NTPLp-aPTHF, TLp-aPTHF, or TPLp-aPTHF).
  • the administered delivery vehicle is pegylated. In some embodiments, the administered delivery vehicle is not pegylated. In additional embodiments, the administered delivery vehicle comprises a targeting moiety that has a specific affinity for an epitope of an antigen on the surface of a cancer cell.
  • the delivery vehicle comprises a targeting moiety that has specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g, folate receptor-a, folate receptor-b or folate receptor-d), Mucin 1 (METC-l), 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-l (ZIP6), CGEN-15027, P cadherin, fibronectin extra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, Errofi
  • the delivery vehicle comprises a targeting moiety that specifically binds a cell surface antigen(s) derived from, or determined to be expressed on, a specific subject’s cancer (tumor) such as a neoantigen.
  • the targeting moiety has specific affinity for an epitope of a cell surface antigen(s) derived from or determined to be expressed on a specific subject’s tumor such as a neoantigen.
  • the targeting moiety is an antibody or an antigen binding antibody fragment.
  • the administered delivery vehicle comprises aPTHF containing 4, 5, 2-10, 4-6, or more than 5, -glutamyl groups.
  • the administered delivery vehicle comprises aPTHF containing 4 glutamyl groups. In some embodiments, the administered delivery vehicle comprises aPTHF containing 5 glutamyl groups. In some embodiments, the administered delivery vehicle comprises aPTHF containing 6 glutamyl groups.
  • the aPTHF is a member selected from: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF); (b) polyglutamated lO-formyl-THF (e.g, polyglutamated [6R]-lO-formyl-THF); (c) polyglutamated 5, l0-methenyl-THF (e.g, polyglutamated [6R]-5,lO-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g, polyglutamated [6S]-5-methyl-THF); (e) polyglutamated Tetrahydrofolate THF (e.g, polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated 5,l0-methylene-THF (e.g, polyglutamated [6R]-5,lO- methylene-THF); and (g) polyglutamated 5,
  • the aPTHF is polyglutamated 5,10- methylene-THF. In further embodiments, the aPTHF is polyglutamated [6R]-5,lO- methylene-THF. In other embodiments, the aPTHF is polyglutamated [6R,S]-5,lO- methylene-THF. In some embodiments, the aPTHF is polyglutamated 5-methyl-THF. In further embodiments, the aPTHF is [6S]-5-methyl-THF. In other embodiments, the aPTHF is [6R,S]-5-methyl-THF. In some embodiments, the aPTHF is polyglutamated 5-formyl-THF.
  • the aPTHF is polyglutamated [6S]-5-formyl- THF. In other embodiments, the aPTHF is polyglutamated [6R,S]-5-formyl-THF.
  • the cancer is selected from the group consisting of: a non- hematologic malignancy including such as for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma, brain cancer, central nervous system cancer, and melanoma; and a hematologic malignancy such as for example, a leukemia, a lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias.
  • a non- hematologic malignancy including such as for example, lung cancer
  • the cancer is selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, stomach cancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, and chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma (desmoid tumors, aggressive fibromatosis, bladder cancer, and central Nervous System (CNS) lymphoma.
  • NHL Non-Hodgkin's lymphoma
  • ALL acute lymphoblastic leukemia
  • mycosis fungoides cutaneous T-cell lymphoma
  • choriocarcinoma choriocarcinoma
  • chorioadenoma nonleukemic meningeal cancer
  • soft tissue sarcoma desmoid tumors, aggressive fibromatosis, bladder cancer
  • CNS central Nervous
  • the cancer is selected from the group consisting of: colorectal cancer, breast cancer, gastric cancer (e.g ., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and/or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell cancer.
  • the cancer is colorectal cancer.
  • the disclosure provides a method for treating cancer that comprises administering an effective amount of a liposome comprising polyglutamated alpha tetrahydrofolate (e.g, an Lp-aPTHF such as, PLp-aPTHF, NTLp- aPTHF, NTPLp-aPTHF, TLp-aPTHF, or TPLp-aPTHF) to a subject having or at risk of having cancer.
  • a liposome is pegylated.
  • the liposome is not pegylated.
  • the liposome comprises a targeting moiety that has a specific affinity for an epitope of an antigen on the surface of a cancer cell.
  • the liposome comprises a targeting moiety that has specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g., folate receptor-a, folate receptor-b or folate receptor-d), Mucin 1 (METC-l), 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-l (ZIP6), CGEN- 15027, P cadherin, fibronectin extra-domain B (ED- B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER
  • the liposome comprises a targeting moiety that has specific affinity for an epitope of a cell surface antigen(s) derived from or determined to be expressed on a specific subject’s tumor such as a neoantigen.
  • the targeting moiety is an antibody or an antigen binding antibody fragment.
  • the liposome comprises aPTHF containing 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • the administered liposome comprises a aPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF); (b) polyglutamated lO-formyl-THF (e.g., polyglutamated [6R]-lO-formyl-THF); (c) polyglutamated 5, l0-methenyl-THF (e.g, polyglutamated [6R]-5,lO-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g, polyglutamated [6S]-5-methyl-THF); (e) polyglutamated Tetrahydrofolate THF (e.g, polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated 5,l0-methylene-THF (e.g., polyglutamated [6R]-5,lO- methylene-THF);
  • the administered liposome comprises polyglutamated 5, l0-methylene-THF. In further embodiments, the administered liposome comprises polyglutamated [6R]-5, lO-methylene-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5, lO-methylene-THF. In some embodiments, the administered liposome comprises polyglutamated 5-methyl- THF. In further embodiments, the administered liposome comprises [6S]-5-methyl-THF. In other embodiments, the administered liposome comprises [6R,S]-5-methyl-THF. In some embodiments, the administered liposome comprises polyglutamated 5-formyl- THF.
  • the administered liposome comprises polyglutamated [6S]-5-formyl-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5-formyl-THF. In some embodiments the administered liposomal composition comprises tetraglutamated aPTHF. In some embodiments the administered liposomal composition comprises pentaglutamated aPTHF. In some embodiments the administered liposomal composition comprises hexaglutamated aPTHF. In some embodiments, a liposome of the administered liposomal composition comprises aPTHF containing 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • a liposome of the administered liposomal composition comprises a aPTHF containing 1, 2, 3, or more than 3, glutamyl groups having a gamma linkage.
  • the cancer is selected from the group consisting of: lung (e.g, non-small 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, biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma ( e.g ., osteosarcoma), brain cancer, central nervous system cancer, melanoma, and a hematologic malignancy (e.g., a leukemia or lymphoma).
  • lung e.g, non-small lung cancer
  • pancreatic cancer breast cancer, ovarian cancer, prostate cancer, head and neck cancer
  • gastric cancer gastrointestinal cancer, colorectal cancer, esophageal cancer
  • the cancer cell is a primary cell or a cell from a cell line obtained/derived from a cancer selected from colorectal cancer, breast cancer, gastric cancer (e.g, stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g, non-small cell lung cancer and/or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell cancer.
  • the cancer cell is a primary cell or a cell from a cell line obtained/derived from colorectal cancer.
  • the disclosure provides a method for treating cancer that comprises administering to a subject having or at risk of having cancer, an effective amount of a liposomal composition comprising a liposome that comprises polyglutamated alpha tetrahydrofolate and a targeting moiety that has a specific affinity for an epitope of an antigen on the surface of the cancer.
  • the liposome comprises a targeting moiety that has specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, a folate receptor (e.g, folate receptor-a, folate receptor-b or folate receptor-d), Mucin 1 (METC-l), 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-l (ZIP6), CGEN-15027, P cadherin, fibronectin extra-domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3,
  • the liposome comprises a targeting moiety that a targeting moiety that has specific affinity for an epitope of a cell surface antigen(s) derived from, or determined to be expressed on, a specific subject’s cancer (tumor) such as a neoantigen.
  • the targeting moiety is an antibody or an antigen binding antibody fragment.
  • the liposome comprises aPTHF containing 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • the administered liposome comprises aPTHF containing 4 glutamyl groups.
  • the administered liposome comprises aPTHF containing 5 glutamyl groups.
  • the administered liposome comprises aPTHF containing 6 glutamyl groups.
  • the administered liposome comprises a aPTHF selected from: (a) polyglutamated 5-formyl- THF (e.g, polyglutamated [6S]-5-formyl-THF); (b) polyglutamated lO-formyl-THF ( e.g.
  • polyglutamated [6R]- lO-formyl-THF polyglutamated [6R]- lO-formyl-THF); (c) polyglutamated 5,l0-methenyl-THF (e.g, polyglutamated [6R]-5, lO-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g, polyglutamated [6S]-5-methyl-THF); (e) polyglutamated Tetrahydrofolate THF (e.g, polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated 5, l0-methylene- THF (e.g, polyglutamated [6R]-5, lO-methylene-THF); and (g) polyglutamated 5- formimino-THF (e.g, polyglutamated [6S]-5-formimino-THF).
  • the administered liposome comprises polyglutamated 5, l0-methylene-THF. In further embodiments, the administered liposome comprises polyglutamated [6R]-5,lO- methylene-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5, lO-methylene-THF. In some embodiments, the administered liposome comprises polyglutamated 5-methyl-THF. In further embodiments, the administered liposome comprises [6S]-5-methyl-THF. In other embodiments, the administered liposome comprises [6R,S]-5-methyl-THF. In some embodiments, the administered liposome comprises polyglutamated 5-formyl-THF.
  • the administered liposome comprises polyglutamated [6S]-5-formyl-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5- formyl-THF. In some embodiments, the liposome comprises a aPTHF containing 1, 2, 3, or more than 3, glutamyl groups having a gamma linkage.
  • the administered liposomal composition comprises pegylated liposomes (e.g ., TPLp-aPTHF). In some embodiments, the administered liposomal composition comprises liposomes that are not pegylated. In some embodiments, liposomes of the administered liposomal composition comprise a aPTHF containing 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, the administered liposome comprises aPTHF containing 4 glutamyl groups. In some embodiments, the administered liposome comprises aPTHF containing 5 glutamyl groups. In some embodiments, the administered liposome comprises aPTHF containing 6 glutamyl groups.
  • pegylated liposomes e.g ., TPLp-aPTHF
  • the administered liposomal composition comprises liposomes that are not pegylated.
  • liposomes of the administered liposomal composition comprise a aPTHF containing 4, 5, 2-10, 4-6, or
  • the administered liposome comprises a aPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl- THF); (b) polyglutamated lO-formyl-THF (e.g, polyglutamated [6R]-lO-formyl-THF); (c) polyglutamated 5,l0-methenyl-THF (e.g, polyglutamated [6R]-5,lO-methenyl- THF); (d) polyglutamated 5-methyl-THF (e.g, polyglutamated [6S]-5-methyl-THF); (e) polyglutamated Tetrahydrofolate THF (e.g, polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated 5,l0-methylene-THF (e.g, polyglutamated [6R]-5,lO- methylene-THF); and (g) polyglutama
  • the administered liposome comprises polyglutamated 5,l0-methylene-THF. In further embodiments, the administered liposome comprises polyglutamated [6R]-5,lO-methylene-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5,lO-methylene-THF. In some embodiments, the administered liposome comprises polyglutamated 5-methyl- THF. In further embodiments, the administered liposome comprises [6S]-5-methyl-THF. In other embodiments, the administered liposome comprises [6R,S]-5-methyl-THF. In some embodiments, the administered liposome comprises polyglutamated 5-formyl- THF.
  • the administered liposome comprises polyglutamated [6S]-5-formyl-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5-formyl-THF. In some embodiments, a liposome of the administered liposomal composition comprises a aPTHF containing 1, 2, 3, or more than 3, glutamyl groups having a gamma linkage.
  • the liposomal composition is administered to treat a cancer selected from the group consisting of: lung cancer (e.g ., non-small cell), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma, a leukemia and a lymphoma.
  • lung cancer e.g ., non-small cell
  • pancreatic cancer breast cancer, ovarian cancer, prostate cancer, head and neck cancer
  • gastric cancer gastrointestinal cancer
  • colorectal cancer esophageal cancer
  • cervical cancer cervical cancer
  • liver cancer liver cancer
  • kidney cancer biliary duct cancer
  • gallbladder cancer gallbladder cancer
  • bladder cancer s
  • the liposomal composition is administered to treat a cancer selected from the group consisting of: colorectal cancer, breast cancer, gastric cancer (e.g, stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g, non-small cell lung cancer and/or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell cancer.
  • a cancer selected from the group consisting of: colorectal cancer, breast cancer, gastric cancer (e.g, stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g, non-small cell lung cancer and/or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell cancer.
  • the liposomal composition is administered to treat colorectal cancer.
  • the disclosure provides a method for treating cancer that comprises administering an effective amount of a liposomal composition to a subject having or at risk of having a cancer that expresses folate receptor on its cell surface, wherein the liposomal composition comprises liposomes that comprise (a) polyglutamated alpha tetrahydrofolate (aPTHF) and (b) a targeting moiety that has specific binding affinity for a folate receptor.
  • the targeting moiety has specific binding affinity for folate receptor alpha (FR-a), folate receptor beta (FR-b), and/or folate receptor delta (FR-d).
  • the targeting moiety has a specific binding affinity for folate receptor alpha (FR-a), folate receptor beta (FR-b), and/or folate receptor delta (FR-d). In some embodiments, the targeting moiety has a specific binding affinity for folate receptor alpha (FR-a) and folate receptor beta (FR-b).
  • the administered liposomal composition comprises pegylated liposomes (e.g, TPLp-aPTHF). In some embodiments, the administered liposomal composition comprises liposomes that are not pegylated. In some embodiments, liposomes of the administered liposomal composition comprises a aPTHF containing 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • the administered liposome comprises aPTHF containing 4 glutamyl groups. In some embodiments, the administered liposome comprises aPTHF containing 5 glutamyl groups. In some embodiments, the administered liposome comprises aPTHF containing 6 glutamyl groups.
  • the administered liposome comprises a aPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g., polyglutamated [6S]-5-formyl-THF); (b) polyglutamated lO-formyl-THF (e.g., polyglutamated [6R]-lO-formyl-THF); (c) polyglutamated 5, l0-methenyl-THF (e.g, polyglutamated [6R]-5,lO-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g, polyglutamated [6S]-5-methyl-THF); (e) polyglutamated Tetrahydrofolate THF (e.g, polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated 5,l0-methylene-THF (e.g., polyglutamated [6R]-5,lO- methylene-THF);
  • the administered liposome comprises polyglutamated 5, l0-methylene-THF. In further embodiments, the administered liposome comprises polyglutamated [6R]-5, lO-methylene-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5, lO-methylene-THF. In some embodiments, the administered liposome comprises polyglutamated 5-methyl- THF. In further embodiments, the administered liposome comprises [6S]-5-methyl-THF. In other embodiments, the administered liposome comprises [6R,S]-5-methyl-THF. In some embodiments, the administered liposome comprises polyglutamated 5-formyl- THF.
  • the administered liposome comprises polyglutamated [6S]-5-formyl-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5-formyl-THF. In some embodiments, a liposome of the administered liposomal composition comprises 1, 2, 3, or more than 3 glutamyl groups containing a gamma linkage.
  • the liposomal composition is administered to treat a cancer selected from the group consisting of: a non-hematologic malignancy including such as for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma ( e.g ., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and a hematologic malignancy such as for example, a leukemia, a lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias.
  • a cancer selected from the group consisting of: a non-hematologic malignancy including such as for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gas
  • the liposomal composition is administered to treat a cancer is selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, stomach cancer, osteosarcoma, Non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, and chorioadenoma, nonleukemic meningeal cancer, soft tissue sarcoma (desmoid tumors, aggressive fibromatosis, bladder cancer, and central Nervous System (CNS) lymphoma.
  • NHL Non-Hodgkin's lymphoma
  • ALL acute lymphoblastic leukemia
  • mycosis fungoides cutaneous T-cell lymphoma
  • choriocarcinoma choriocarcinoma
  • chorioadenoma nonleukemic meningeal cancer
  • soft tissue sarcoma desmoid tumors, aggressive fibromatos
  • the liposomal composition is administered to treat a cancer selected from the group consisting of: colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g, non-small cell lung cancer and/or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell cancer.
  • a cancer selected from the group consisting of: colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g, non-small cell lung cancer and/or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell cancer.
  • the disclosure provides a method for cancer maintenance therapy that comprises administering an effective amount of a liposomal composition comprising liposomes that contain polyglutamated alpha tetrahydrofolate (Lp-aPTHF) to a subject that is undergoing or has undergone cancer therapy.
  • the administered liposomal composition is a PLp-aPTHF, NTLp-aPTHF, NTPLp-aPTHF, TLp-aPTHF or TPLp-aPTHF.
  • the administered liposomal composition comprises pegylated liposomes (e.g, PLp-aPTHF, NTPLp- aPTHF, or TPLp-aPTHF).
  • the administered liposomal composition comprises targeted liposomes (e.g, TLp-aPTHF or TPLp-aPTHF).
  • the administered liposomal composition comprises liposomes that are pegylated and comprise a targeting moiety (e.g, TPLp-aPTHF).
  • a liposome of the administered liposomal composition comprises polyglutamated alpha tetrahydrofolate that contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • the administered liposome comprises aPTHF containing 4 glutamyl groups.
  • the administered liposome comprises aPTHF containing 5 glutamyl groups.
  • the administered liposome comprises aPTHF containing 6 glutamyl groups.
  • the administered liposome comprises a aPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g, polyglutamated [6S]-5-formyl-THF); (b) polyglutamated lO-formyl-THF (e.g., polyglutamated [6R]- lO-formyl-THF); (c) polyglutamated 5,l0-methenyl-THF (e.g, polyglutamated [6R]-5,lO-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g, polyglutamated [6S]-5-methyl-THF); (e) polyglutamated Tetrahydrofolate THF (e.g, polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated 5,l0-methylene-THF (e.
  • the administered liposome comprises polyglutamated 5,l0-methylene-THF. In further embodiments, the administered liposome comprises polyglutamated [6R]-5,lO- methylene-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5,lO-methylene-THF. In some embodiments, the administered liposome comprises polyglutamated 5-methyl-THF. In further embodiments, the administered liposome comprises [6S]-5-methyl-THF. In other embodiments, the administered liposome comprises [6R,S]-5-methyl-THF. In some embodiments, the administered liposome comprises polyglutamated 5-formyl-THF.
  • the administered liposome comprises polyglutamated [6S]-5-formyl-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5- formyl-THF. In some embodiments, a liposome of the administered liposomal composition comprises 1, 2, 3, or more than 3 glutamyl groups containing a gamma linkage.
  • the disclosure provides a method for treating a disorder of the immune system that comprises administering an effective amount of a liposomal composition comprising liposomes that contain polyglutamated alpha tetrahydrofolate (e.g, Lp-aPTHF, PLp-aPTHF, NTLp-aPTHF, NTPLp-aPTHF, TLp- aPTHF or TPLp-aPTHF) to a subject having or at risk of having a disorder of the immune system.
  • the liposomal composition is administered to treat an autoimmune disease.
  • the liposomal composition is administered to treat rheumatoid arthritis.
  • the liposomal composition is administered to treat inflammation.
  • the administered liposomal composition comprises pegylated liposomes (e.g ., PLp-aPTHF, NTPLp-aPTHF, or TPLp-aPTHF).
  • the administered liposomal composition comprises targeted liposomes (e.g., TLp-aPTHF or TPLp-aPTHF) that contain a targeting moiety having a specific affinity for a surface antigen on a target cell of interest (e.g, an immune cell).
  • the administered liposomal composition comprises liposomes that are pegylated and comprise a targeting moiety (e.g, TPLp-aPTHF)).
  • a liposome of the administered liposomal composition comprises pentaglutamated alpha tetrahydrofolate that contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • the administered liposome comprises aPTHF containing 4 glutamyl groups.
  • the administered liposome comprises aPTHF containing 5 glutamyl groups.
  • the administered liposome comprises aPTHF containing 6 glutamyl groups.
  • the administered liposome comprises a aPTHF selected from: (a) polyglutamated 5-formyl- THF (e.g, polyglutamated [6S]-5-formyl-THF); (b) polyglutamated lO-formyl-THF (e.g, polyglutamated [6R]- lO-formyl-THF); (c) polyglutamated 5,l0-methenyl-THF (e.g, polyglutamated [6R]-5, lO-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g, polyglutamated [6S]-5-methyl-THF); (e) polyglutamated Tetrahydrofolate THF (e.g, polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated 5,l0-methylene-THF (e.g, polyglutamated [6R]-5, lO-methylene-THF); and (
  • the administered liposome comprises polyglutamated 5, l0-methylene-THF. In further embodiments, the administered liposome comprises polyglutamated [6R]-5,lO- methylene-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5,lO-methylene-THF. In some embodiments, the administered liposome comprises polyglutamated 5-methyl-THF. In further embodiments, the administered liposome comprises [6S]-5-methyl-THF. In other embodiments, the administered liposome comprises [6R,S]-5-methyl-THF. In some embodiments, the administered liposome comprises polyglutamated 5-formyl-THF.
  • the administered liposome comprises polyglutamated [6S]-5-formyl-THF. In other embodiments, the administered liposome comprises polyglutamated [6R,S]-5- formyl-THF. In some embodiments, a liposome of the administered liposomal composition comprises 1, 2, 3, or more than 3 glutamyl groups containing a gamma linkage.
  • the disclosure also provides a method of delivering polyglutamated alpha tetrahydrofolate to a tumor and/or cancer cell that comprises: administering to a subject having the tumor, a composition comprising polyglutamated alpha tetrahydrofolate (L- aPTHF) and a targeting moiety that has a specific binding affinity for an epitope of a surface antigen on the tumor cell or cancer cell.
  • the administered targeting moiety is associated with a delivery vehicle.
  • the delivery vehicle is an antibody or an antigen binding fragment of an antibody.
  • the delivery vehicle is a liposome.
  • the antibody, antigen-binding antibody fragment, or liposome is pegylated.
  • the administered composition comprises polyglutamated alpha tetrahydrofolate that contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups. In some embodiments, the administered composition comprises tetraglutamated alpha tetrahydrofolate. In some embodiments, the administered composition comprises pentaglutamated alpha tetrahydrofolate. In other embodiments, the administered composition comprises hexaglutamated alpha tetrahydrofolate.
  • the administered composition comprises a aPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g, polyglutamated [6S]-5- formyl-THF); (b) polyglutamated lO-formyl-THF (e.g., polyglutamated [6R]-lO-formyl- THF); (c) polyglutamated 5,l0-methenyl-THF (e.g, polyglutamated [6R]-5,lO-methenyl- THF); (d) polyglutamated 5-methyl-THF (e.g, polyglutamated [6S]-5-methyl-THF); (e) polyglutamated Tetrahydrofolate THF (e.g, polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated 5,l0-methylene-THF (e.g., polyglutamated [6R]-5,lO-methylene- THF); and (g) polyglutamated 5,
  • the administered composition comprises polyglutamated 5,l0-methylene-THF. In further embodiments, the administered composition comprises polyglutamated [6R]-5,lO-methylene-THF. In other embodiments, the administered composition comprises polyglutamated [6R,S]-5,lO- methylene-THF. In some embodiments, the administered composition comprises polyglutamated 5 -methyl -THF. In further embodiments, the administered composition comprises [6S]-5-methyl-THF. In other embodiments, the administered composition comprises [6R,S]-5-methyl-THF. In some embodiments, the administered composition comprises polyglutamated 5-formyl-THF. In further embodiments, the administered composition comprises polyglutamated [6S]-5-formyl-THF. In other embodiments, the administered composition comprises polyglutamated [6R,S]-5-formyl-THF.
  • the disclosure provides a method of preparing a liposomal composition that comprises a liposomal polyglutamated alpha tetrahydrofolate (aPTHF) composition, the method comprising: forming a mixture comprising: liposomal components and a polyglutamated tetrahydrofolate in solution; homogenizing the mixture to form liposomes in the solution; and processing the mixture to form liposomes containing polyglutamated tetrahydrofolate.
  • the polyglutamated alpha tetrahydrofolate contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • the aPTHF composition comprises pentaglutamated alpha tetrahydrofolate. In some embodiments, the aPTHF composition comprises tetraglutamated alpha tetrahydrofolate. In other embodiments, the aPTHF composition comprises hexaglutamated alpha tetrahydrofolate. In some embodiments, the polyglutamated alpha tetrahydrofolate contains 1, 2, 3, or more than 3, glutamyl groups containing a gamma linkage. In some embodiments, the aPTHF composition contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, glutamyl groups in the D-form.
  • the aPTHF composition contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10, glutamyl groups in the L- form. In some embodiments, the aPTHF composition contains 2, 3, 4, 5, or more than 5, glutamyl groups in the L-form, and 1, 2, 3, 4, 5 or more than 5, glutamyl groups in the D- form.
  • composition comprises a aPTHF selected from: (a) polyglutamated 5-formyl-THF (e.g, polyglutamated [6S]-5-formyl-THF); (b) polyglutamated lO-formyl-THF (e.g., polyglutamated [6R]-lO-formyl-THF); (c) polyglutamated 5,l0-methenyl-THF (e.g, polyglutamated [6R]-5,lO-methenyl-THF); (d) polyglutamated 5-methyl-THF (e.g, polyglutamated [6S]-5-methyl-THF); (e) polyglutamated Tetrahydrofolate THF (e.g, polyglutamated [6S]-Tetrahydrofolate THF); (f) polyglutamated 5,l0-methylene-THF (e.g, polyglutamated [6R]-5,lO-methylene- THF); and (g) polyglutamated
  • the composition comprises polyglutamated 5,l0-methylene-THF. In further embodiments, the composition comprises polyglutamated [6R]-5,lO-methylene-THF. In other embodiments, the composition comprises polyglutamated [6R,S]-5,lO-methylene-THF. In some embodiments, the composition comprises polyglutamated 5-methyl-THF. In further embodiments, the composition comprises [6S]-5-methyl-THF. In other embodiments, the composition comprises [6R,S]-5-methyl-THF. In some embodiments, the composition comprises polyglutamated 5-formyl-THF. In further embodiments, the composition comprises polyglutamated [6S]-5-formyl-THF. In other embodiments, the administered composition comprises polyglutamated [6R,S]-5-formyl-THF.
  • the disclosure provides a kit comprising a polyglutamated alpha tetrahydrofolate composition and/or a aPTHF delivery vehicle such as a liposome containing aPTHF or an aPTHF immunoconjugate (e.g, an ADC) described herein.
  • a aPTHF delivery vehicle such as a liposome containing aPTHF or an aPTHF immunoconjugate (e.g, an ADC) described herein.
  • FIGS. 1A-1N show exemplary chemical formulas of alpha tetrahydrofolates: alpha 5-10 methylene THF (FIG. 1A), alpha 5-10 methylene THF diglutamate (FIG. IB), alpha 5- 10 methylene THF triglutamate (FIGS. 1C and ID), alpha 5-10 methylene THF tetraglutamates (FIGS. IE and IF), alpha 5-10 methylene THF pentaglutamates (FIGS. 1G and 1H), alpha 5-10 methylene THF hexaglutamates (FIGS. II and 1J), alpha 5-10 methylene THF heptaglutamates (FIGS.
  • FIGS. 1R-1U present depictions of exemplary branched 5-10 methylene THF polyglutamate structures, including a branched polyglutamate having an alpha glutamyl backbone and gamma glutamyl branches (FIG.
  • FIG. IT a branched polyglutamate having a gamma glutamyl backbone and alpha glutamyl branches
  • FIG. 1U a branched polyglutamate having an alpha glutamyl backbone and both gamma glutamyl branches and alpha glutamyl branches
  • FIG. 2 presents the relative potency of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6) and its mirror image, liposomal alpha-D hexaglutamate (liposomal aDG6) relative to pemetrexed following exposure of the 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), over 48 hours.
  • CRC cancer cell lines
  • HT-29 colon cancer
  • H1806 triple negative breast cancer
  • OAW28 ovarian cancer
  • H292 NSCLC, adenocarcinoma subtype
  • H2342 NSCLC, adenocarcinoma subtype
  • FIG. 3 presents an example dose response relationship of free pemetrexed L- gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha targeting antibody (FRlAb) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FRlAb) in the NCI H2342 non small cell lung cancer (NSCLC), adenocarcinoma subtype depicted as the percentage of viable cells after 48 hours of treatment.
  • NCI H2342 non small cell lung cancer NCI H2342 non small cell lung cancer
  • Folate receptor alpha targeted liposomes containing alpha polyglutamated pemetrexed are expected to also be successful in targeting and reducing the viability of NCI H2342 non-small cell lung cancer cells.
  • FIG. 4 presents an example dose response relationship of free pemetrexed L- gamma hexaglutamate (gG6), liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha targeting antibody (FRlAb) liposomal pemetrexed L-gamma hexaglutamate (liposomal gG6-FRlAb) in the HT-29 (colon cancer) at 48 hours.
  • Folate receptor alpha targeted liposomes containing alpha polyglutamated pemetrexed are expected to also be successful in targeting and reducing the viability of HT-29 (colon cancer) cells.
  • FIG. 5 presents the treatment effect on HCC1806 triple negative breast cancer cells following exposure of liposomal pemetrexed alpha-L hexaglutamate (Lps Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate (Lps Hexa aDG6), and to pemetrexed over 48 hours.
  • Lps Hexa aG6 liposomal pemetrexed alpha-L hexaglutamate
  • Lps Hexa aDG6 liposomal pemetrexed alpha-D hexaglutamate
  • FIG. 6 presents the treatment effect on OAW28 ovarian cancer cells following exposure of liposomal pemetrexed alpha-L hexaglutamate (Lps Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate (Lps Hexa aDG6), and to pemetrexed over 48 hours.
  • Lps Hexa aG6 liposomal pemetrexed alpha-L hexaglutamate
  • Lps Hexa aDG6 liposomal pemetrexed alpha-D hexaglutamate
  • FIG. 7 presents the treatment effect on H292 non-small cell lung cancer cells following exposure of liposomal pemetrexed alpha-L hexaglutamate (Lps Hexa aG6), liposomal pemetrexed alpha-D hexaglutamate (Lps Hexa aDG6), as compared to pemetrexed over 48 hours.
  • Lps Hexa aG6 liposomal pemetrexed alpha-L hexaglutamate
  • Lps Hexa aDG6 liposomal pemetrexed alpha-D hexaglutamate
  • FIG. 8 presents the treatment effect on H292 non-small cell lung cancer cells following exposure of various dose levels ranging from 16 to 128 nM of liposomal pemetrexed alpha-L hexaglutamate (Liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (Liposomal aDG6), and pemetrexed over 48 hours.
  • the liposomal pemetrexed aG6 formulation is superior to inhibiting H292 non-small cell lung cancer cells compared to pemetrexed.
  • FIG. 9 presents the treatment effect on HCC1806 triple negative breast cancer cells following exposure of various dose levels ranging from 16 to 128 nM of liposomal pemetrexed alpha-L hexaglutamate (Liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (Liposomal aDG6), and pemetrexed over 48 hours.
  • the liposomal pemetrexed aG6 formulation is superior to pemetrexed in inhibiting HCC1806 triple negative breast cancer cells.
  • FIG. 10 presents the treatment effect on OAW28 ovarian cancer cells of liposomal pemetrexed alpha-L hexaglutamate (Liposomal aG6), liposomal alpha-D hexaglutamate (Liposomal aDG6), and pemetrexed following exposure over 48 hours following exposure over a range of concentrations.
  • pemetrexed appears to more effective than the Liposomal pemetrexed aG6 liposomal formulation, whereas the liposomal formulation at the dose of 32 nM and 64 nM has a better treatment effect than pemetrexed; at 16 nM the Liposomal pemetrexed aG6 treatment effect is similar in to pemetrexed.
  • FIG. 11 shows the toxicity of liposomal pemetrexed alpha-L hexaglutamate (Liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (Liposomal aDG6), and pemetrexed on differentiating human neutrophils at 64 nM, 128 nM, and 264 nM.
  • the figure demonstrates that liposomal pemetrexed aG6 is significantly less toxic to differentiating human neutrophils than pemetrexed.
  • FIG. 12 shows the effect of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal alpha-D hexaglutamate (liposomal aDG6), and pemetrexed on neutrophils (differentiated from CD34+ cells) following exposure of various dose levels ranging from 16 to 128 nM of the corresponding agent over 48 hours.
  • FIG. 13 shows the effect of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed on AML12 liver cells following exposure over 48 hours at 16 nM, 32 nM, and 64 nM, and 128 nM of the corresponding agent. Strikingly, there does not appear to be any toxicity to the AML12 liver cells following treatment with a liposomal pemetrexed aG6 at any of the liposomal agents at the dose levels tested. In contrast, pemetrexed treatment results in a reduction in the AML 12 liver cell counts of approximately 40% at all doses studied.
  • FIG. 14 shows the effect of liposomal pemetrexed alpha-L hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D hexaglutamate (liposomal aDG6), and pemetrexed on CCD841 colon epithelium cells following exposure over 48 hours at 16 nM, 32 nM, and 64 nM, and 128 nM, of the corresponding agent. At all of the concentrations tested, pemetrexed leads to approximately a >50% decrease in the number of CCD841 colon epithelium cells compared to approximately a 20% or less decrease in cell number after treatment with each of the liposome compositions tested. [00106] FIG.
  • FIG. 16 presents the effects of liposomal aG6 treatment of mice with 40 mg/kg and 80 mg/kg given once weekly for 4 weeks upon the hematologic parameters: white blood cell (WBC) counts, neutrophil counts and as platelet counts. No appreciable decrease in mean neutrophil, mean white blood cell or mean platelet counts was observed.
  • WBC white blood cell
  • FIG. 17 presents the effects of liposomal aG6 treatment of mice with 40 mg/kg and 80 mg/kg given once weekly for 4 weeks upon hemoglobin and reticulocyte indices. There is a minimal decrease in mean hemoglobin concentrations at the higher dose level. In parallel there is a slight increase in mean reticulocytosis indices
  • FIG. 18 presents the effects of liposomal aG6 treatment of mice with 40 mg/kg and 80 mg/kg given once weekly for 4 weeks upon hepatic markers including serum aspartate transaminase (AST) and serum alanine transaminase (ALT) along with serum albumin.
  • AST serum aspartate transaminase
  • ALT serum alanine transaminase
  • FIG. 19 presents 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 control, pemetrexed, and Liposomal aG6 intravenously at 167 mg/kg once every three weeks.
  • liposomal aG6 provides reduced tumor control compared to pemetrexed.
  • FIGS. 20A-F present the dose response relationship of liposomal pemetrexed alpha-L tri glutamate (Liposomal aG3), liposomal pemetrexed alpha-L pentaglutamate (Liposomal aG5), liposomal pemetrexed alpha-L octaglutamate (Liposomal aG7), and a combination of liposomal pemetrexed alpha-L hexaglutamate (aG6) and alpha-L dodecaglutamate (aGl2) (Liposomal aG6 and aGl2), over 48 hours on H2342 (NSCLC, adenocarcinoma subtype)(FIG.
  • NSCLC adenocarcinoma subtype
  • H292 NSCLC, adenocarcinoma subtype
  • HT-29 colon cancer
  • HCC1806 triple negative breast cancer
  • MCF7 ER+ breast cancer
  • OAW28 ovarian cancer
  • the disclosure generally relates to polyglutamated alpha tetrahydrofolate compositions.
  • the compositions provide advances over prior treatments of hyperproliferative diseases such as cancer.
  • Methods of making, delivering and using the polyglutamated alpha tetrahydrofolate compositions are also provided.
  • the polyglutamated alpha compositions have uses that include but are not limited to treating (e.g, treating and/or preventing) hyperproliferative diseases such as cancer, disorders of the immune system such as inflammation and rheumatoid arthritis, and infectious disease such as HIV and malaria.
  • the polyglutamated alpha compositions also have uses in combination therapy with one or more therapeutic agents such as a chemotherapy drug (e.g, 5-fluorouracil) to enhance the effectiveness of the therapeutic agent or as a “chemoprotectant” (e.g, in combination with an antifolate such as methotrexate) to reduce toxic side effects associated with the therapeutic agent(s).
  • a chemotherapy drug e.g, 5-fluorouracil
  • chemoprotectant e.g, in combination with an antifolate such as methotrexate
  • transitional phrase“comprising” is considered more of an open-ended phrase while the transitional phrases“consisting of’ is more exclusive and“consisting essentially of’ achieves a middle ground).
  • the term“and/or” as used in a phrase such as“A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term“and/or” as used in a phrase such as“A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • Headings and subheadings are used for convenience and/or formal compliance only, do not limit the subject technology, and are not referred to in connection with the interpretation of the description of the subject technology.
  • Features described under one heading or one subheading of the subject disclosure may be combined, in various embodiments, with features described under other headings or subheadings. Further it is not necessarily the case that all features under a single heading or a single subheading are used together in embodiments.
  • tetrahydrofolate and“THF” are used interchangeably to include a salt, acid and and/or free base form of a tetrahydrofolate (e.g., tetrahydrofolate disodium).
  • a tetrahydrofolate e.g., tetrahydrofolate disodium
  • “THF(s)” and“tetrahydrofolate(s)” include natural and unnatural THF forms, including one carbon-substituted THF derivatives.
  • THF(s) and“tetrahydrofolate(s)” include diasteromeric compositions having a [6R] configuration at the C-6 atom of the tetrahydropterin component of the THF, diasteromeric compositions having a [6S] configuration at the C-6 atom, and/or mixtures of [6,R,S] diastereomers (e.g, 1 : 1).
  • “THF(s)” and“tetrahydrofolate(s)” include: (a) 5-formyl-THF (e.g., [6S], [6R,S], or [6R] -5- formyl-THF); (b) 5-formyl-THF (e.g., [6S], [6R,S], or [6R] -5-formyl-THF); (c) 5,10- methenyl-THF (e.g., [6R], [6R,S], or [6S], -5,l0-methenyl-THF) ; (d) 5-methyl-THF (e.g., [6S], [6R,S], or [6R], -5-methyl-THF); (e) Tetrahydrofolate THF ((2S)-2- ⁇ [4-( ⁇ [2- amino-4-oxo-l,4,5,6,7,8-hexahydropteridin
  • the disclosure provides a composition containing a THF diastereomer selected from (a) [6S]-5-formyl-THF; (b) [6R]-5-formyl-THF; (c) [6R]-5,lO-methenyl- THF; (d) [6S]-5-methyl-THF; (e) [6S]-Tetrahydrofolate THF; (f) [6R]-5,lO-methylene- THF; and (g) [6S]-5-formimino-THF.
  • a THF diastereomer selected from (a) [6S]-5-formyl-THF; (b) [6R]-5-formyl-THF; (c) [6R]-5,lO-methenyl- THF; (d) [6S]-5-methyl-THF; (e) [6S]-Tetrahydrofolate THF; (f) [6R]-5,lO-methylene- THF;
  • the disclosure provides a composition containing a THF diastereomer mixture (e.g, a diastereoisomeric mixture [6R,S]-5-methyl-THF (1 : 1), and/or a diastereoisomeric mixture [6R,S]-5-CHO-THF (1 : 1
  • a THF diastereomer mixture e.g, a diastereoisomeric mixture [6R,S]-5-methyl-THF (1 : 1), and/or a diastereoisomeric mixture [6R,S]-5-CHO-THF (1 : 1
  • Compositions containing a THF salt may further contain any of a variety of cations, such as Na + , Mg 2+ , K + , NH 4 + , and/or Ca 2+ .
  • the salts are pharmaceutically acceptable salts.
  • the THF salt contains Na + .
  • Tetrahydrofolate contains one L-gamma glutamyl group, and is therefore
  • THF Tetrahydrofolate THF
  • A“Tetrahydrofolate THF” may also be referred to herein as a species of tetrahydrofolate (THF).
  • polyglutamate refers to a composition comprising at least one chain of 2 or more linked glutamyl groups.
  • Polyglutamate chains can be linear or branched.
  • Linear polyglutamate chains can contain for example, glutamyl groups containing either an alpha carboxyl group or a gamma carboxyl group linkage.
  • Branched polyglutamate chains can comprise for example, one or more glutamyl groups that contain both an alpha carboxyl group and a gamma carboxyl group linkage to other glutamyl groups, thereby providing a branch point of the polyglutamate.
  • Exemplary branched polyglutamates are depicted in FIGS. 1R-1U.
  • Polyglutamate chains comprise anN-terminal glutamyl group and one or more C-terminal glutamyl groups.
  • the N-terminal glutamyl group of a polyglutamate chain is not linked to another glutamyl group via its amine group, but is linked to one or more glutamyl group via its carboxylic acid group.
  • the N-terminal glutamyl group of a polyglutamated-tetrahydrofolate is the glutamyl group of tetrahydrofolate.
  • the C-terminal glutamyl group or groups of a polyglutamate chain are linked to another glutamyl group via their amine group, but are not linked to another glutamyl group via their carboxylic acid group.
  • polyglutamated-tetrahydrofolate refers to a tetrahydrofolate composition that comprises at least one glutamyl group in addition to the glutamyl group of tetrahydrofolate (i.e., THF-PGn, wherein n > 1).
  • THF-PGn the glutamyl group of tetrahydrofolate
  • Reference to the number of glutamyl groups in a aPTHF (aTHF-PG) herein takes into account the glutamyl group of tetrahydrofolate.
  • THF-PG composition containing 5 glutamyl residues in addition to the glutamyl group of THF is referred to herein as hexaglutamated tetrahydrofolate or tetrahydrofolate hexaglutamate.
  • polyglutamated-tetrahydrofolate is a member selected from: (a) polyglutamated 5- formyl-THF; (b) polyglutamated lO-formyl-THF; (c) polyglutamated 5,l0-methenyl- THF; (d) polyglutamated 5-methyl-THF; (e) polyglutamated tetrahydrofolate ((2S)-2- ⁇ [4- ( ⁇ [2-amino-4-oxo-l,4,5,6,7,8-hexahydropteridin-6-yl]methyl ⁇ amino)phenyl] formamido ⁇ pentanedioic acid) (( e.g [6S], [6R,S], and [6R,] -((2S)-2- ⁇ [4-( ⁇ [2-amino-4- oxo-l,4,5,6,7,8-hexahydropteridin-6-yl]methyl ⁇ amino) phenyl] formamido ⁇ pentane
  • the polyglutamated-tetrahydrofolate is a member selected from: (a) polyglutamated [6S]-5-formyl-THF; (b) polyglutamated [6R]- lO-formyl-THF; (c) polyglutamated[6R]-5,lO-methenyl-THF; (d) polyglutamated [6S]-5- methyl-THF; (e) polyglutamated [6S]-Tetrahydrofolate THF; (f) polyglutamated [6R]- 5,l0-methylene-THF; and (g) polyglutamate[6S]-5-formimino-THF.
  • the polyglutamated-tetrahydrofolate is [6R]-5,lO-methylene-THF. In some embodiments, the polyglutamated-tetrahydrofolate is [6S]-5-methyl-THF. In some embodiments, the polyglutamated-tetrahydrofolate is [6S]-5-formyl-THF.
  • the polyglutamated-tetrahydrofolate is a [6R,S]-5,lO-methylene-THF diastereomeric mixture, a [6R,S]-5-methyl-THF diastereomeric mixture, or a [6R,S]-5- formyl-THF diastereomeric mixture (e.g., 1 : 1 w/w).
  • alpha glutamyl group refers to a glutamyl group that contains an alpha carboxyl group linkage.
  • the alpha linkage is an amide bond between the alpha carboxyl group of one glutamyl group and a second glutamyl group.
  • the alpha linkage can be between a glutamyl group and the glutamyl group of tetrahydrofolate, or between the glutamyl group and a second glutamyl group that is not present in tetrahydrofolate, such as a glutamyl group within a polyglutamate chain attached to tetrahydrofolate.
  • an“alpha glutamyl group” of a provided polyglutamated alpha tetrahydrofolate has both an alpha carboxyl group linkage and a gamma carboxyl group linkage.
  • the alpha glutamyl group is in the L-form.
  • the alpha glutamyl group is in the D-form.
  • the glutamyl group is in the L-form. In some embodiments, one or more glutamyl groups in the polyglutamated alpha tetrahydrofolate are in the L form and one or more glutamyl groups in the polyglutamated alpha tetrahydrofolate are in the D form.
  • polyglutamated alpha tetrahydrofolate “a-polyglutamated tetrahydrofolate”,“aPTHF”,“polyglutamated alpha-tetrahydrofolate”,“polyglutamated alpha THF”,“aTHF-PG”, and iterations thereof, are used interchangeably herein to refer to a tetrahydrofolate composition that comprises at least one glutamyl group having an alpha carboxyl group linkage (e.g., THF-PGn, wherein n > 1 a glutamyl group).
  • THF-PGn alpha carboxyl group linkage
  • aTHF-PG a aTHF-PG composition containing 5 glutamyl groups in addition to the glutamyl group of THF, and wherein at least one of the glutamyl groups has a alpha carboxyl linkage, may be referred to herein as alpha hexaglutamated tetrahydrofolate, hexaglutamated alpha tetrahydrofolate or alpha tetrahydrofolate hexaglutamate.
  • the terms“gamma glutamyl group”,“gamma glutamate”, and“gamma linkage”, as they relate to the linkage of a glutamyl group, refers to a glutamyl group that contains a gamma carboxyl group linkage.
  • the gamma linkage is an amide bond between the gamma carboxyl group of one glutamyl group and a second glutamyl group.
  • the gamma linkage can be between a glutamyl group and the glutamyl group of tetrahydrofolate, or between the glutamyl group and a second glutamyl group that is not present in tetrahydrofolate, such as a glutamyl group within a polyglutamate chain attached to tetrahydrofolate.
  • one or more gamma linked glutamyl groups in the polyglutamated alpha tetrahydrofolate is in the L form.
  • one or more gamma linked glutamyl groups in the polyglutamated alpha tetrahydrofolate is in the D form.
  • one or more gamma linked glutamyl groups in the polyglutamated alpha tetrahydrofolate is in the L form and one or more gamma linked glutamyl groups in the polyglutamated alpha tetrahydrofolate is in the D form.
  • Isolated refers to a composition which is in a form not found in nature.
  • Isolated polyglutamated alpha compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature.
  • a polyglutamated alpha tetrahydrofolate which is isolated is substantially pure.
  • Isolated compositions will be free or substantially free of material with which they are naturally associated such as other cellular components such as proteins and nucleic acids with which they may potentially be found in nature, or the environment in which they are prepared ( e.g cell culture).
  • the polyglutamated alpha compositions may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example, the polyglutamated alpha compositions will normally be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.
  • the isolated polyglutamated alpha compositions e.g ., alpha polyglutamates and delivery vehicles such as liposomes containing the alpha polyglutamate contain less than 1% or less than 0.1% undesired DNA or protein content.
  • the alpha polyglutamate compositions e.g., alpha polyglutamate and delivery vehicles such as liposomes containing the alpha polyglutamate
  • targeting moiety is used herein to refer to a molecule that provides an enhanced affinity for a selected target, e.g, a cell, cell type, tissue, organ, region of the body, or a compartment, e.g, a cellular, tissue or organ compartment.
  • the targeting moiety can comprise a wide variety of entities. Targeting moieties can include naturally occurring molecules, or recombinant or synthetic molecules. In some embodiments, the targeting moiety is an antibody, antigen-binding antibody fragment, bispecific antibody or other antibody-based molecule or compound.
  • the targeting moiety is an aptamer, avimer, a receptor-binding ligand, a nucleic acid, a biotin-avidin binding pair, a peptide, protein, carbohydrate, lipid, vitamin, toxin, a component of a microorganism, a hormone, a receptor ligand or any derivative thereof.
  • Other targeting moieties are known in the art and are encompassed by the disclosure.
  • the terms“specific affinity”,“specifically binds”, and“enhanced affinity”, mean that a targeting moiety such as an antibody or antigen binding antibody fragment, reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including proteins unrelated to antigens containing the target epitope. Because of the sequence identity between homologous proteins in different species, specific affinity can, in several embodiments, include a binding agent that recognizes an epitope on a protein and/or target molecule in more than one species.
  • the term“specific affinity” or“specifically binds” can include a binding agent that recognizes an epitope that is present on more than one protein and/or target molecule. It is understood that, in certain embodiments, a targeting moiety that specifically binds a first target may or may not specifically bind a second target. As such,“specific affinity” does not necessarily require (although it can include) exclusive binding, e.g., binding to an epitope on a single target. Thus, a targeting moiety may, in certain embodiments, specifically bind an epitope that is present on more than one target. In certain embodiments, multiple targets may be bound by the same targeting moiety specifically binds an epitope that is present on multiple targets.
  • epitopope refers to that portion of an antigen capable of being
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • Expressions like“binding affinity for a target”,“binding to a target”,“enhanced affinity”, and analogous expressions known in the art refer to a property of a targeting moiety which may be directly measured through the determination of the affinity constants, e.g, the amount of targeting moiety that associates and dissociates at a given antigen concentration.
  • Different methods can be used to characterize the molecular interaction, such as, but not limited to, competition analysis, equilibrium analysis and microcalorimetric analysis, and real-time interaction analysis based on surface plasmon resonance interaction (for example using a Biacore® instrument).
  • delivery vehicle refers generally to any compositions that acts to assist, promote or facilitate entry of polyglutamated alpha tetrahydrofolate into a cell.
  • delivery vehicles include, but are not limited to, liposomes, lipospheres, polymers ( e.g ., polymer-conjugates), peptides, proteins such as antibodies (e.g, immunoconjugates, such as Antibody Drug Conjugates (ADCs) and antigen binding antibody fragments and derivatives thereof), 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 material, or lipid or liposome formulations, and combinations thereof.
  • the delivery vehicle can be linked directly or indirectly
  • A“subject” refers to a human or vertebrate mammal including but not limited to a dog, cat, horse, goat and primate, e.g, monkey.
  • the invention can also be used to treat diseases or conditions in non-human subjects. For instance, cancer is one of the leading causes of death in companion animals (e.g, cats and dogs).
  • companion animals e.g, cats and dogs.
  • the subject is a human.
  • the term “subject” and“patient” is used interchangeably and has the same meaning. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.
  • an“effective amount” refers to a dosage of an agent sufficient to provide a medically desirable result.
  • the effective amount will vary with the desired outcome, the particular condition being treated or prevented, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent or combination therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
  • An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
  • the effective amount of an agent may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.
  • the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • efficacy in vivo can, for example, be measured by assessing the duration of survival, duration of progression free survival (PFS), the response rates (RR), duration of response, and/or quality of life.
  • the terms “hyperproliferative disorder”, “proliferative disease”, and “proliferative disorder”, are used interchangeably herein to pertain to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
  • the proliferative disease is cancer or tumor disease (including benign or cancerous) and/or any metastases, wherever the cancer, tumor and/or the metastasis is located.
  • the proliferative disease is a benign or malignant tumor.
  • the proliferative disease is a non-cancerous disease.
  • the proliferative disease is a hyperproliferative condition such as hyperplasias, fibrosis (especially pulmonary, but also other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis and smooth muscle proliferation in the blood vessels, such as stenosis or restenosis following angioplasty.
  • hyperproliferative condition such as hyperplasias, fibrosis (especially pulmonary, but also other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis and smooth muscle proliferation in the blood vessels, such as stenosis or restenosis following angioplasty.
  • Cancer “tumor”, or“malignancy”, are used as synonymous terms and refer to any of a number of cell types or diseases that are characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (metastasize) and/or any of the characteristic structural and/or molecular features known to be associated with these cell types or diseases.
  • “Tumor”, as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • A“cancerous tumor”, or“malignant cell” is understood as a cell having specific structural properties, lacking differentiation and being capable of invasion and metastasis.
  • a cancer that can be treated using a aPTHF composition provided herein includes without limitation, a non-hematologic malignancy including such as for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma ( e.g ., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and a hematologic malignancy such as for example, a leukemia, a lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias.
  • a non-hematologic malignancy including such as for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal
  • the cancer is selected from the group consisting of: colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g, non-small cell lung cancer and/or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell cancer.
  • gastric cancer e.g., stomach cancer
  • pancreatic cancer liver cancer
  • lung cancer e.g, non-small cell lung cancer and/or adenocarcinoma
  • head and neck cancer ovarian cancer
  • gallbladder cancer gallbladder cancer
  • basal cell cancer basal cell cancer
  • Terms such as “treating”, “treatment”, or “to treat”, refer to both (a) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (b) prophylactic or preventative measures that prevent and/or slow the development of a targeted disease or condition.
  • subjects in need of treatment include those already with the cancer, disorder or disease; those at risk of having the cancer or condition; and those in whom the infection or condition is to be prevented.
  • Subjects are identified as“having or at risk of having” cancer, an infectious disease, a disorder of the immune system, a hyperproliferative disease, or another disease or disorder referred to herein using well-known medical and diagnostic techniques.
  • a subject is successfully “treated” according to the methods provided herein if the subject shows, e.g, total, partial, or transient amelioration or elimination of a symptom associated with the disease or condition (e.g, cancer, inflammation, and rheumatoid arthritis).
  • the terms treating”, or “treatment”, or “to treat” refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient.
  • the terms treating”, or “treatment”, or “to treat” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom,
  • the terms treating”, or “treatment”, or “to treat”, refer to the reduction or stabilization of tumor size, tumor cell proliferation or survival, or cancerous cell count. Treatment can be with a a-PTHF composition, alone or in combination with an additional therapeutic agent.
  • mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all
  • vertebrates e.g, mammals and non-mammals, such as chickens, amphibians, and reptiles.
  • “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and other members of the class Mammalia known in the art.
  • the subject is a human.
  • Treatment of a proliferative disorder is used herein to include maintaining or decreasing tumor size, inducing tumor regression (either partial or complete), inhibiting tumor growth, and/or increasing the life span of a subject having the proliferative disorder.
  • the proliferative disorder is a solid tumor.
  • 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, biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g, osteosarcoma), brain cancer, central nervous system cancer, and melanoma.
  • the proliferative disorder is a hematologic malignancy.
  • hematologic malignancies include for example, a leukemia, a lymphoma and other B cell malignancies, myeloma and other plasma cell dysplasias or dyscrasias.
  • autoimmune disease as used herein is defined as a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriate and excessive response to a self-antigen.
  • autoimmune diseases include but are not limited to, Addison’s disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, inflammation and rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis, among others.
  • therapeutic agent is used herein to refer to an agent or a derivative thereof that can interact with a hyperproliferative cell such as a cancer cell or an immune cell, thereby reducing the proliferative status of the cell and/or killing the cell.
  • therapeutic agents include, but are not limited to, chemotherapeutic agents, cytotoxic agents, platinum-based agents (e.g ., cisplatin, carboplatin, oxaliplatin), taxanes (e.g., TAXOL®), etoposide, alkylating agents (e.g, cyclophosphamide, ifosamide), metabolic antagonists (e.g, tetrahydrofolate (THF), 5- fluorouracil gemcitabine, or derivatives thereof), antitumor antibiotics (e.g, mitomycin, doxorubicin), plant-derived antitumor agents (e.g, vincristine, vindesine, TAXOL®).
  • chemotherapeutic agents e.g ., cytotoxic agents, platinum-based agents (e.g ., cisplatin, carboplatin, oxaliplatin), taxanes (e.g., TAXOL®), etoposide, alky
  • Such agents may further include, but are not limited to, the anticancer agents trimetrexate, temozolomide, S-(4-Nitrobenzyl)-6- thioinosine (NBMPR), 6-benzyguanidine (6-BG), bis-chloronitrosourea (BCNU) and camptothecin, or a therapeutic derivative of any thereof.
  • NBMPR S-(4-Nitrobenzyl)-6- thioinosine
  • 6-BG 6-benzyguanidine
  • BCNU bis-chloronitrosourea
  • camptothecin or a therapeutic derivative of any thereof.
  • therapeutic agents include, without limitation, anti-restenosis, pro- or anti-proliferative, anti-inflammatory, anti-neoplastic, antimitotic, anti -platelet, anticoagulant, antifibrin, antithrombin, cytostatic, antibiotic and other anti-infective agents, anti-enzymatic, anti-metabolic, angiogenic, cytoprotective, angiotensin converting enzyme (ACE) inhibiting, angiotensin II receptor antagonizing and/or cardioprotective agents.
  • “Therapeutic agents” also refer to salts, acids, and free based forms of the above agents.
  • the term“chemotherapeutic agent” when used in relation to cancer therapy refers to any agent that results in the death of cancer cells or inhibits the growth or spread of cancer cells.
  • chemotherapeutic agents include alkylating agents, antibiotics, antimetabolites, plant-derived agents, and hormones.
  • the disclosed polyglutamated alpha tetrahydrofolate compositions are used in combination with a chemotherapeutic agent.
  • the chemotherapeutic agent is 5-fluorouracil.
  • the chemotherapeutic agent is cisplatin.
  • the chemotherapeutic agent is carboplatin.
  • the chemotherapeutic agent is oxaliplatin. In other embodiments, the chemotherapeutic agent is gemcitabine. In other embodiments, the chemotherapeutic agent is doxorubicin. In particular embodiments, the chemotherapeutic agent is a pyrimidine analog (e.g ., a fluorpyrimidine such as 5-fluorouracil (5-FU)).
  • a fluorpyrimidine such as 5-fluorouracil (5-FU)
  • antimetabolite is used herein to refer to a therapeutic agent that inhibits the utilization of a metabolite or a prodrug thereof.
  • antimetabolites include 5-FU and 5-FU metabolites and/or prodrugs such as 5-FUMP, 5-FUDP, 5- FdUMP, capecitabine, tegafur 5-fluorodeoxyuridine monophosphate; and cytarabine and cytarabine prodrugs such as nelarabine, 5-azacytidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine.
  • Anti-metabolites useful for practicing the disclosed methods include nucleoside analogs, including a purine or pyrimidine analogs.
  • the polyglutamated alpha tetrahydrofolate compositions are used in combination with an antimetabolite selected from the group consisting of: a fluoropyrimidine, 5-fluorouracil, 5-fluoro-l-(oxolan-2-yl)pyrimidine-2,4-dione, 5-fluoro-2'-deoxycytidine, cytarabine, gemcitabine, troxacitabine, decitabine, Azacytidine, pseudoisocytidine, Zebularine, Ancitabine, Fazarabine, 6-azacytidine, capecitabine, N4-octadecyl-cytarabine, elaidic acid cytarabine, fludarabine, cladribine, clofarabine, nelarabine, forodesine, and pentostatin
  • the nucleoside analog is a substrate for a nucleoside deaminase that is adenosine deaminase or cytidine deaminase.
  • the nucleoside analog is selected from among fludarabine, cytarabine, gemcitabine, decitabine and azacytidine or derivatives thereof.
  • the nucleoside analog is selected from N3-alkylated analogues of 5-fluorouracil, 5- fluorouracil derivatives with l,4-oxaheteroepane moieties, 5-fluorouracil and nucleoside analogues, cis- and trans-5-fluoro-5,6-dihydro-6-alkoxyuracil, cyclopentane 5- fluorouracil analogues, A-OT-fluorouracil, N4-trimethoxybenzoyl-5'-deoxy-5-fluoro- cytidine and 5'-deoxy-5-fluorouridine, l-hexylcarbamoyl-5-fluorouracil, B-3839, uracil- l-(2-tetrahydrofuryl)-5-fluorouracil, l-(2'-deoxy-2'-fluoro-P-D-arabinofuranosyl)-5-fl-
  • a“taxane” is an anti-cancer agent that interferes with or disrupts microtubule stability, formation and/or function.
  • Taxane agents include paclitaxel and docetaxel as well as derivatives thereof, wherein the derivatives function against microtubules by the same mode of action as the taxane from which they are derived.
  • the taxane is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel.
  • the taxane is paclitaxel (TAXOL®), docetaxel (TAXOTERE®), albumin-bound paclitaxel (nab-paclitaxel; ABRAXANE®), DHA-paclitaxel, or PG-paclitaxel.
  • pharmaceutically-acceptable carrier refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, carrier, excipient, stabilizer, diluent, or preservative.
  • Pharmaceutically-acceptable carriers can include for example, one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other subject.
  • This disclosure generally relates to polyglutamated alpha tetrahydrofolate (aPTHF) compositions and methods of making and using the compositions to treat diseases including hyperproliferative diseases such as cancer, disorders of the immune system such as rheumatoid arthritis, and infectious disease such as HIV and malaria.
  • the gamma polyglutamated compositions also have uses in combination therapy with one or more therapeutic agents such as a chemotherapy drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a “chemoprotectant” (e.g., in combination with an antifolate such as methotrexate) to reduce toxic side effects associated with the therapeutic agent(s).
  • a chemotherapy drug e.g ., 5-fluorouracil
  • chemoprotectant e.g., in combination with an antifolate such as methotrexate
  • the disclosure provides:
  • composition comprising a polyglutamated alpha tetrahydrofolate.
  • polyglutamated 5-formyl-THF e.g, polyglutamated [6S]-5-formyl-THF
  • polyglutamated lO-formyl-THF e.g, polyglutamated [6R]-l 0-formyl - THF
  • polyglutamated 5,l0-methenyl-THF e.g, polyglutamated [6R]-5,lO- methenyl-THF
  • polyglutamated 5-methyl-THF e.g, polyglutamated [6S]-5-methyl-THF
  • polyglutamated tetrahydrofolate e.g, polyglutamated [6S]- Tetrahydrofolate THF
  • polyglutamated 5,l0-methylene-THF e.g, polyglutamated [6R]-5,lO- methylene-THF
  • polyglutamated 5-formimino-THF e.g, polyglutamated [6S]-5-formimino- THF.
  • composition of [1] or [2], wherein the polyglutamated alpha tetrahydrofolate contains 4, 5, 2-10, 4-6, or more than 5, glutamyl groups having alpha carboxyl group linkages.
  • polyglutamated alpha tetrahydrofolate is tetraglutamated alpha tetrahydrofolate (e.g [6R]-5,lO- methenyl-THF, [6S]-5-formyl-THF and/or [6R]-lO-formyl-THF).
  • pentaglutamated alpha tetrahydrofolate e.g., [6R]-5,lO- methenyl-THF, [6S]-5-formyl-THF and/or [6R]-lO-formyl-THF.
  • [6] the composition according to any of [l]-[3], wherein the polyglutamated alpha tetrahydrofolate is hexaglutamated alpha tetrahydrofolate (e.g, [6R]-5,lO- methenyl-THF, [6S]-5-formyl-THF and/or [6R]-lO-formyl-THF).
  • polyglutamated alpha tetrahydrofolate is hexaglutamated alpha tetrahydrofolate (e.g, [6R]-5,lO- methenyl-THF, [6S]-5-formyl-THF and/or [6R]-lO-formyl-THF).
  • tetrahydrofolate has an alpha carboxyl group linkage
  • each of the glutamyl groups other than the C-terminal glutamyl group or groups and the glutamyl group of tetrahydrofolate has an alpha carboxyl group linkage
  • each of the glutamyl groups other than the C-terminal glutamyl group or groups has an alpha carboxyl group linkage.
  • each of the glutamyl groups of the alpha polyglutamated tetrahydrofolate is in the L-form
  • each of the glutamyl groups of the alpha polyglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form, or
  • tetrahydrofolate are in the L-form and at least 1 of the glutamyl groups is in the D-form;
  • a liposomal composition comprising the alpha polyglutamated tetrahydrofolate according to any of [l]-[l2] (Lp-aPTHF);
  • glutamyl groups of the alpha polyglutamated tetrahydrofolate is in the L-form
  • the Lp-aPTHF composition according to any of [13]-[16], wherein the liposome comprises an alpha polyglutamated tetrahydrofolate containing 4, 5, 2-10, 4-6, or more than 5, glutamyl groups;
  • the Lp-aPTHF composition according to any of [l3]-[20], wherein the liposome comprises an alpha polyglutamated tetrahydrofolate containing alpha
  • polyglutamate is linear or branched
  • the Lp-aPTHF composition according to any of [l3]-[23], wherein the liposomes comprise at least 1% weight by weight (w/w) of the alpha polyglutamated tetrahydrofolate or wherein during the process of preparing the Lp- aPTHF, at least 1% of the starting material of alpha polyglutamated THF is encapsulated (entrapped) in the aPTHF;
  • the Lp-aPTHF composition according to any of [l3]-[24], wherein the liposome has a diameter in the range of 20 nm to 500 nm or 20 nm to 200 nm;
  • the Lp-aPTHF composition according to [27] or [28], wherein the liposomal components comprise at least one selected from the group consisting of: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol- PEG; and cholesterol-maleimide;
  • the Lp-aPTHF composition according to any of [27]-[29], wherein the liposomal components comprise at least one selected from the group consisting of: DSPE; DSPE-PEG; D SPE-PEG-FIT C ; DSPE-PEG-maleimide; cholesterol; and HSPC;
  • PEG polyethylene glycol
  • PLL poly- L-lysine
  • GM1 monosialoganglioside
  • PVP poly(vinyl pyrrolidone)
  • PAA poly(acrylamide)
  • PAA poly(2-methyl-2-oxazoline)
  • poly(2-ethyl-2-oxazoline) phosphatidyl polyglycerol
  • amphiphilic poly-N-vinylpyrrolidones L-amino-acid-based polymer
  • oligoglycerol copolymer containing polyethylene glycol and polypropylene oxide, Poloxamer 188, and polyvinyl alcohol;
  • a tonicity agent such as dextrose, mannitol, glycerine, potassium chloride, sodium chloride, at a concentration of greater than 1%;
  • acceptable carrier is trehalose
  • pharmaceutically acceptable carrier comprises 1% to 15 weight of dextrose
  • pharmaceutically acceptable carrier comprises a buffer such as HEPES Buffered Saline (HBS) or similar, at a concentration of between 1 to 200 mM and a pH of between 2 to 8;
  • HBS HEPES Buffered Saline
  • pharmaceutically acceptable carrier comprises a total concentration of sodium acetate and calcium acetate of between 50 mM to 500 mM;
  • the targeting moiety comprises a targeting moiety and wherein the targeting moiety has a specific affinity for a surface antigen on a target cell of interest;
  • the Lp-aPTHF composition according to any of [50]-[52], wherein the targeting moiety is an antibody or an antigen binding fragment of an antibody;
  • Kd equilibrium dissociation constant
  • the Lp-aPTHF composition according to any of [50]-[55], wherein the targeting moiety specifically binds one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-a), folate receptor beta (FR-b), and folate receptor delta (FR-d);
  • the targeting moiety specifically binds one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-a), folate receptor beta (FR-b), and folate receptor delta (FR-d);
  • the Lp-aPTHF composition according to any of [50]-[56], wherein the targeting moiety comprises one or more selected from the group consisting of: an antibody, a humanized antibody, an antigen binding fragment of an antibody, a single chain antibody, a single-domain antibody, a bi-specific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody;
  • pegylated liposome comprises from 1 to 1000 or 30-200 targeting moieties; [58] the Lp-aPTHF composition according to any of [39]-[57], further comprising one or more of an immunostimulatory agent, a detectable marker and a maleimide, wherein the immunostimulatory agent, the detectable marker or the maleimide is attached to said PEG or the exterior of the liposome;
  • agent is at least one selected from the group consisting of: a fluorescein; a fluorescein isothiocyanate (FITC); a DNP; a beta glucan; a beta-l,3-glucan; a beta-l,6-glucan; a resolvin (e.g, a Resolvin D such as D n -6DPA or D n -3DPA, a Resolvin E, or a T series resolvin); and a Toll-like receptor (TLR) modulating agent such as, an oxidized low-density lipoprotein (e.g, OXPAC, PGPC), and an eritoran lipid (e.g, E556);
  • a fluorescein fluorescein isothiocyanate
  • DNP a beta glucan
  • beta-l,3-glucan e.g, a Resolvin D such as D n -6DPA or D n -3DPA, a Resolv
  • immunostimulatory agent and the detectable marker is the same;
  • cryoprotectant selected from the group consisting of mannitol
  • trehalose trehalose
  • sorbitol and sucroseat least one cryoprotectant selected from the group consisting of mannitol; trehalose; sorbitol; and sucrose;
  • a targeted composition comprising the composition according to any of [l]-[64];
  • An non-targeted composition comprising the composition according to any of [l]-[49]; [67] the Lp-aPTHF composition according to any of [l3]-[66], which further comprises carboplatin and/or pembroluzumab;
  • a pharmaceutical composition comprising the liposomal alpha polyglutamated tetrahydrofolate composition according to any of [l3]-[67];
  • composition of any of [l]-[70] in the manufacture of a medicament for the treatment of disease and/or for use in combination therapy with one or more therapeutic agents such as a chemotherapeutic drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a chemotherapeutic drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a chemotherapeutic drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a chemotherapeutic drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a chemotherapeutic drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a chemotherapeutic drug (e.g ., 5-fluorouracil) to enhance the effectiveness of the therapeutic agent(s) or as a chemo
  • chemoprotectant e.g., in combination with an antifolate such as
  • methotrexate to reduce a toxic side effect associated with the therapeutic agent(s);
  • [73] a method for treating or preventing disease in a subject needing such treatment or prevention, the method comprising administering the composition of any of [l]-[69] to the subject;
  • polyglutamated alpha tetrahydrofolate composition of any of [l3]-[69] to the subject;
  • hyperproliferative cell with the liposomal polyglutamated alpha tetrahydrofolate composition of any of [l3]-[69]; [77] the method of [75] or [76], wherein the hyperproliferative cell is a cancer cell, a mammalian cell, and/or a human cell;
  • [78] a method for treating cancer that comprises administering an effective amount of the composition of any of [l]-[69] to a subject having or at risk of having cancer;
  • [79] a method for treating cancer that comprises administering an effective amount of the liposomal polyglutamated alpha tetrahydrofolate composition of any of [l3]-[68] to a subject having or at risk of having cancer;
  • a non-hematologic malignancy including such as for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, biliary duct cancer, gallbladder cancer, bladder cancer, sarcoma ( e.g osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and a hematologic malignancy such as for example, a leukemia, a lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias;
  • a hematologic malignancy such as for example, a leukemia, a lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias
  • colorectal cancer lung cancer, breast cancer, head and neck cancer, and pancreatic cancer;
  • [84] a method for treating cancer that comprises administering an effective amount of the Lp-aPTHF composition of any of [50]-[66] to a subject having or at risk of having a cancer cell that expresses on its surface a folate receptor bound by the targeting moiety;
  • cancer therapy that comprise administering an effective amount of the composition of any of [l]-[69] to a subject that is undergoing or has undergone cancer therapy;
  • cancer therapy that comprise administering an effective amount of the liposomal polyglutamated alpha tetrahydrofolate composition of any of [l3]-[69] to a subject that is undergoing or has undergone cancer therapy;
  • the disorder of the immune system is selected from: inflammation (e.g ., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn disease, dermatomyositis/polymyositis, systemic lupus erythematosus, and Takayasu, and psoriasis;
  • the liposomal polyglutamated alpha tetrahydrofolate composition of any of [l3]-[69] to a subject having or at risk of having a disorder of the immune system, optionally wherein the disorder of the immune system is selected from: inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn disease, dermatomyositis/polymyositis, systemic lupus erythematosus, and Takayasu, and psoriasis; [89] a method for treating:
  • inflammation e.g., acute and chronic
  • systemic inflammation e.g., rheumatoid arthritis
  • IBD inflammatory bowel disease
  • Crohn disease e.g., Crohn disease
  • dermatomyositis/polymyositis e.g., systemic erythematosus
  • Takayasu p
  • composition according to any of [l]-[69] to a subject having or at risk of having leukopenia;
  • cardiovascular disease or metabolic disease that comprises administering an effective amount of the composition according to any of [l]-[69] to a subject having or at risk of having an infectious disease, cardiovascular disease, or another disease, wherein the disease is a member selected from: atherosclerosis, cardiovascular disease (CVD), coronary artery disease, myocardial infarction, stroke, metabolic syndrome, a gestational trophoblastic disease, and ectopic pregnancy;
  • CVD cardiovascular disease
  • CVD cardiovascular disease
  • coronary artery disease myocardial infarction
  • stroke stroke
  • metabolic syndrome a gestational trophoblastic disease
  • ectopic pregnancy ectopic pregnancy
  • an autoimmune disease that comprises administering an effective amount of the composition according to any of [l]-[69] to a subject having or at risk of having an autoimmune disease;
  • rheumatoid arthritis that comprises administering an effective amount of the composition according to any of [l]-[69] to a subject having or at risk of having rheumatoid arthritis;
  • an inflammatory condition that comprises administering an effective amount of the composition according to any of [l]-[69] to a subject having or at risk of having inflammation, optionally wherein the inflammation is acute, chronic, and/or systemic inflammation; or
  • a skin condition that comprises administering an effective amount of the composition according to any of [l]-[69] to a subject having or at risk of having a skin condition, optionally wherein the skin condition is psoriasis;
  • a method for treating an infectious disease that comprises administering an effective amount of the liposomal alpha polyglutamated tetrahydrofolate composition of any of [l3]-[69] to a subject having or at risk of having an infectious disease;
  • [91] a method of delivering alpha polyglutamated tetrahydrofolate to a tumor expressing a folate receptor on its surface, the method comprising: administering the Lp-aPTHF composition of any of [l]-[69] to a subject having the tumor in an amount to deliver a therapeutically effective dose of the alpha polyglutamated tetrahydrofolate to the tumor;
  • the method comprising: forming a mixture comprising: liposomal components and alpha polyglutamated antifolate in solution; homogenizing the mixture to form liposomes in the solution; and processing the mixture to form liposomes containing alpha polyglutamated tetrahydrofolate;
  • the method comprising: forming a mixture comprising: liposomal components and alpha polyglutamated tetrahydrofolate in solution; and processing the mixture to form liposomes containing alpha polyglutamated tetrahydrofolate,
  • [95] a method of preparing the composition of any of [5l]-[70] comprising the steps of: forming a mixture comprising: liposomal components and alpha polyglutamated tetrahydrofolate in a solution; homogenizing the mixture to form liposomes in the solution; processing the mixture to form liposomes entrapping and/or encapsulating alpha polyglutamated tetrahydrofolate; and providing a targeting moiety on a surface of the liposomes, the targeting moiety having specific affinity for at least one of folate receptor alpha (FR-a), folate receptor beta (FR-b) and folate receptor delta (FR-d); [96] a method of preparing the composition of any of [5l]-[70], comprising the steps of: forming a mixture comprising: liposomal components and alpha polyglutamated tetrahydrofolate in a solution; processing the mixture to form liposomes entrapping and/or encapsulating alpha poly
  • the processing step includes one or more steps of: thin film hydration, extrusion, in-line mixing, ethanol injection technique, freezing-and-thawing technique, reverse-phase evaporation, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor method, and stirring; and/or
  • processing step includes one or more steps of modifying the size of the liposomes by one or more of steps of extrusion, high-pressure microfluidization, and/or sonication; and/or
  • the disclosure generally relates to polyglutamated alpha tetrahydrofolate (aPTHF) compositions.
  • the aPTHF compositions comprise at least one glutamyl group having an alpha carboxyl group linkage. These compositions are structurally distinct from the L-gamma polyglutamated forms of tetrahydrofolate (that are produced by the enzyme folylpoly-gamma-
  • the aPTHF composition contains 2-20, 2-15, 2- 10, 2-5, 2-6, or more than 5, glutamyl groups (including the glutamyl group in tetrahydrofolate).
  • each of the glutamyl groups in the aPTHF other than the glutamyl group of tetrahydrofolate have an alpha linkage. In some embodiments, each of the glutamyl groups in the aPTHF other than the C-terminal glutamyl group or groups and the glutamyl group of tetrahydrofolate, have an alpha linkage. In some embodiments, each of the glutamyl groups in the aPTHF other than the C-terminal glutamyl group or groups have an alpha linkage. In some embodiments, 2 or more of the glutamyl groups in the aPTHF have a gamma linkage.
  • At least one glutamyl group of the alpha polyglutamated tetrahydrofolate has both an alpha carboxyl group linkage and a gamma carboxyl group linkage.
  • each of the glutamyl groups in the aPTHF is in the L-form.
  • each of the glutamyl groups in the aPTHF other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the aPTHF comprises two or more glutamyl groups in the L-form and one or more glutamyl groups in the D-form.
  • the polyglutamate chain of the aPTHF is linear (not branched). In some embodiments, the polyglutamate chain of the aPTHF is branched.
  • the alpha polyglutamated tetrahydrofolate is diglutamated.
  • the alpha polyglutamated tetrahydrofolate contains 1 additional glutamyl group in addition to the glutamyl group of tetrahydrofolate (aTHF-PGi), and the additional glutamyl group is linked to the glutamyl group in tetrahydrofolate through an alpha linkage.
  • each of the glutamyl groups of the alpha diglutamated tetrahydrofolate is in the L-form.
  • the alpha diglutamated THF comprises a glutamyl group in the D-form.
  • the alpha polyglutamated tetrahydrofolate is triglutamated. That is, the alpha polyglutamated tetrahydrofolate contains 2 additional glutamyl groups in addition to the glutamyl group of tetrahydrofolate (aTHF-PG2). In some embodiments, each of the 2 additional glutamyl groups have an alpha linkage. In other embodiments, one of the 2 additional glutamyl groups have an alpha linkage and the other glutamyl group has a gamma linkage. In some embodiments, one of the 2 additional glutamyl groups has an alpha linkage. In some embodiments, one of the 2 additional glutamyl groups has a gamma linkage.
  • each of the glutamyl groups of the alpha triglutamated tetrahydrofolate is in the L-form.
  • the alpha triglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha triglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the triglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is tetraglutamated and thus contains 3 additional glutamyl groups in addition to the glutamyl group in tetrahydrofolate (aTHF-PG3).
  • each of the 3 additional glutamyl groups have an alpha linkage.
  • 1 or 2 of the 3 additional glutamyl groups have an alpha linkage and the remaining 2 or 1 glutamyl groups, respectively, have a gamma linkage.
  • 2 of the 3 additional glutamyl groups have an alpha linkage.
  • one of the 3 additional glutamyl groups has an alpha linkage and another additional glutamyl group has a gamma linkage. In other embodiments, one of the 3 additional glutamyl groups has an alpha linkage and a gamma linkage. In other embodiments, three of the four glutamyl groups have an alpha linkage. In some embodiments, at least one glutamyl group has both an alpha linkage and a gamma linkage. In some embodiments, the alpha tetraglutamated THF comprises two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alpha tetraglutamated tetrahydrofolate is in the L-form.
  • the alpha tetraglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha tetraglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the tetraglutamated THF comprises a glutamyl group in the D- form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is pentaglutamated (aTHF-PG 4 ) and contains a chain of 4 additional glutamyl groups attached to the glutamyl group of tetrahydrofolate.
  • each of the 4 additional glutamyl groups in the chain have an alpha linkage.
  • each of the 4 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 1, 2, or 3, of the 4 additional glutamyl groups have an alpha linkage and the remaining 3, 2, or 1, glutamyl groups, respectively, are linked to a glutamyl group of the molecule through a gamma linkage.
  • 1 or 2 of the 4 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • at least one of the 5 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 5 glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • the alpha pentaglutamated THF comprises two or more glutamyl groups in the L-form.
  • each of the glutamyl groups of the alpha pentaglutamated tetrahydrofolate is in the L-form.
  • the alpha pentaglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha pentaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the pentaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is hexaglutamated (a.THF-PGs) and contains a chain of 5 additional glutamyl groups attached to the glutamyl group of tetrahydrofolate.
  • each of the 5 additional glutamyl groups in the chain have an alpha linkage.
  • each of the 5 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 4 of the 5 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, or 4 of the 5 additional glutamyl groups are linked to a glutamyl group of the molecule through an alpha linkage and the remaining 4, 3, 2, or 1, glutamyl groups, respectively, are linked to a glutamyl group of the molecule through a gamma linkage.
  • 1, 2, 3, or 4 of the 5 additional glutamyl groups have an alpha linkage and the remaining non- C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • At least one of the 6 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 6 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 5 of the 6 glutamyl groups have an alpha linkage.
  • the alpha hexaglutamated THF comprises two or more glutamyl groups in the L-form.
  • each of the glutamyl groups of the alpha hexaglutamated tetrahydrofolate is in the L-form.
  • the alpha hexaglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha hexaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the hexaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is heptaglutamated (a.THF-PGr,) and thus contains a chain of 6 additional glutamyl groups attached to the glutamyl group of tetrahydrofolate.
  • each of the 6 additional glutamyl groups have an alpha linkage.
  • each of the 6 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 5 of the 6 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, 4, or 5, of the 6 additional glutamyl groups have an alpha linkage and the remaining 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, or 5 of the 6 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • at least one of the 7 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 7 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage. In some embodiments, 6 of the 7 glutamyl groups have an alpha linkage. In some embodiments, the alpha heptaglutamated THF comprises two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alpha heptaglutamated tetrahydrofolate is in the L-form. In other embodiments, the alpha heptaglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha heptaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the heptaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L- form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is octaglutamated (aTHF-PG7) and thus contains a chain of 7 additional glutamyl groups attached to the glutamyl group of tetrahydrofolate.
  • each of the 7 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 6 of the 7 additional glutamyl groups in the chain have an alpha linkage.
  • each of the 7 additional glutamyl groups have an alpha linkage.
  • 1, 2, 3, 4, 5, or 6, of the 7 additional glutamyl groups have an alpha linkage and the remaining 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, or 6 of the 7 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • at least one of the 8 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 8 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage. In some embodiments, 7 of the 8 glutamyl groups have an alpha linkage.
  • the alpha octaglutamated THF comprises two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alpha octaglutamated tetrahydrofolate is in the L-form. In other embodiments, the alpha octaglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha octaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the octaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L- form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is nonaglutamated (a.THF-PGx) and contains a chain of 8 additional glutamyl groups attached to the glutamyl group of tetrahydrofolate.
  • each of the 8 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 7 of the 8 additional glutamyl groups in the chain have an alpha linkage.
  • each of the 8 additional glutamyl groups have an alpha linkage.
  • 8 additional glutamyl groups have an alpha linkage and the remaining 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, or 7 of the 8 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • at least one of the 9 glutamyl groups has both an alpha linkage and a gamma linkage.
  • the alpha nonaglutamated THF comprises two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alpha nonaglutamated tetrahydrofolate is in the L-form. In other embodiments, the alpha nonaglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha nonaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the nonaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is decaglutamated (aTHF-PGi») (i.e., contains a chain of 9 additional glutamyl groups attached to the glutamyl group of tetrahydrofolate).
  • each of the 9 additional glutamyl groups have an alpha linkage.
  • each of the 9 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 8 of the 9 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, or 8, of the 9 additional glutamyl groups have an alpha linkage and the remaining 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • at least one of the 10 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 10 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage. In some embodiments, 9 of the 10 glutamyl groups have an alpha linkage.
  • the alpha decaglutamated THF comprises two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alpha decaglutamated tetrahydrofolate is in the L-form. In other embodiments, the alpha decaglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha decaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the decaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is undecaglutamated (aTHF-PGio).
  • each of the 10 additonal glutamyl groups have an alpha linkage.
  • each of the 10 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 9 of the 10 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, or 9, of the 10 additional glutamyl groups have an alpha linkage and the remaining 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • at least one of the 11 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 11 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 10 of the 11 glutamyl groups have an alpha linkage.
  • the alpha undecaglutamated THF comprises two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alpha undecaglutamated tetrahydrofolate is in the L-form. In other embodiments, the alpha undecaglutamated THF comprises a D glutamyl group. In further embodiments, each of the glutamyl groups of the alpha undecaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate, is in the D-form. In additional embodiments, the undecaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched. [00158] In some embodiments, the alpha polyglutamated tetrahydrofolate is dodecaglutamated (aTHF-PGn). In some embodiments, each of the 11 additonal glutamyl groups have an alpha linkage. In some embodiments, each of the 11 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage. In some embodiments, 10 of the 11 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11, additional glutamyl groups have an alpha linkage and the remaining 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • At least one of the 12 glutamyl groups has both an alpha linkage and a gamma linkage. In some embodiments, each of the 12 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage. In some embodiments, 11 of the 12 glutamyl groups have an alpha linkage.
  • the alpha dodecaglutamated THF comprises two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alpha dodecaglutamated tetrahydrofolate is in the L-form. In other embodiments, the alpha dodecaglutamated THF comprises a glutamyl group in the D- form.
  • each of the glutamyl groups of the alpha dodecaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the dodecaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is triskaidecaglutamated (aTHF-PGi2).
  • each of the 12 additional glutamyl groups have an alpha linkage.
  • each of the 12 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 11 of the 12 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, of the 12 additional glutamyl groups have an alpha linkage and the remaining 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • at least one of the 13 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 13 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 12 of the 13 glutamyl groups have an alpha linkage.
  • the alpha triskaidecaglutamated THF comprises two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alpha triskaidecaglutamated tetrahydrofolate is in the L-form. In other embodiments, the alpha triskaidecaglutamated THF comprises a glutamyl group in the D-form. In further embodiments, each of the glutamyl groups of the alpha triskaidecaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate, is in the D-form.
  • the triskaidecaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is tetradecaglutamated (a.THF-PGn).
  • each of the 13 additional glutamyl groups have an alpha linkage.
  • each of the 13 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 12 of the 13 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, of the 13 additional glutamyl groups have an alpha linkage and the remaining 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • At least one of the 14 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 14 glutamyl groups other than the C- terminal glutamyl group or groups have an alpha linkage.
  • 13 of the 14 glutamyl groups have an alpha linkage.
  • the alpha tetradecaglutamated THF comprises two or more glutamyl groups in the L-form.
  • each of the glutamyl groups of the alpha tetradecaglutamated tetrahydrofolate is in the L-form.
  • the alpha tetradecaglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha tetradecaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the tetradecaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is pentadecaglutamated (aTHF-PGi4).
  • each of the 14 additional glutamyl groups have an alpha linkage.
  • each of the 14 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 13 of the 14 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, of the 14 additional glutamyl groups have an alpha linkage and the remaining 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • At least one of the 15 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 15 glutamyl groups other than the C- terminal glutamyl group or groups have an alpha linkage.
  • 14 of the 15 glutamyl groups have an alpha linkage.
  • the alpha pentadecaglutamated THF comprises two or more glutamyl groups in the L-form.
  • each of the glutamyl groups of the alpha pentadecaglutamated tetrahydrofolate is in the L-form.
  • the alpha pentadecaglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha pentadecaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the pentadecaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is hexadecaglutamated (a.THF-PGis)
  • each of the 15 additional glutamyl groups have an alpha linkage.
  • each of the 15 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 14 of the 15 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, of the 15 additional glutamyl groups have an alpha linkage and the remaining 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • At least one of the 16 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 16 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 15 of the 16 glutamyl groups have an alpha linkage.
  • the alpha hexadecaglutamated THF comprises two or more glutamyl groups in the L-form.
  • each of the glutamyl groups of the alpha hexadecaglutamated tetrahydrofolate is in the L-form.
  • the alpha hexadecaglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha hexadecaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the hexadecaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is heptadecaglutamated (aTHF-PGi 6 ).
  • each of the 16 additional glutamyl groups have an alpha linkage.
  • each of the 16 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 15 of the 16 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, of the 16, additional glutamyl groups have an alpha linkage and the remaining 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • At least one of the 17 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 17 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 16 of the 17 glutamyl groups have an alpha linkage.
  • the alpha heptadecaglutamated THF comprises two or more glutamyl groups in the L-form.
  • each of the glutamyl groups of the alpha heptadecaglutamated tetrahydrofolate is in the L-form.
  • the alpha heptadecaglutamated THF comprises a D glutamyl group.
  • each of the glutamyl groups of the alpha heptadecaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the heptadecaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is octadecaglutamated (aTHF-PGn).
  • each of the 17 additional glutamyl groups have an alpha linkage.
  • each of the 17 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 16 of the 17 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, of the 17 additional glutamyl groups have an alpha linkage and the remaining 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have an alpha linkage and the remaining non-C- terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • At least one of the 18 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 18 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 17 of the 18 glutamyl groups have an alpha linkage.
  • the alpha octadecaglutamated THF comprises two or more glutamyl groups in the L-form.
  • each of the glutamyl groups of the alpha octadecaglutamated tetrahydrofolate is in the L-form.
  • the alpha octadecaglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha octadecaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the octadecaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is enneadecaglutamated (a.THF-PGix).
  • each of the 18 additional glutamyl groups have an alpha linkage.
  • each of the 18 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 17 of the 18 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, of the 18 additional glutamyl groups have an alpha linkage and the remaining 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alpha linkage and the remaining non- C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • At least one of the 19 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 19 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 18 of the 19 glutamyl groups have an alpha linkage.
  • the alpha enneadecaglutamated THF comprises two or more glutamyl groups in the L-form.
  • each of the glutamyl groups of the alpha enneadecaglutamated tetrahydrofolate is in the L-form.
  • the alpha enneadecaglutamated THF comprises a D glutamyl group.
  • each of the glutamyl groups of the alpha enneadecaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the enneadecaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is icosiglutamated (aTHF-PGi9).
  • each of the 19 additional glutamyl groups have an alpha linkage.
  • each of the 19 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 18 of the 19 additional glutamyl groups in the chain have an alpha linkage. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
  • glutamyl groups respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • at least one of the 20 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 20 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage. In some embodiments, 19 of the 20 glutamyl groups have an alpha linkage. In some embodiments, the alpha icosiglutamated THF comprises two or more glutamyl groups in the L-form. In further embodiments, each of the glutamyl groups of the alpha icosiglutamated tetrahydrofolate is in the L-form. In other embodiments, the alpha icosiglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha icosiglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the icosiglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is icosikaihenaglutamated (aTHF-PG2o).
  • each of the 20 additional glutamyl groups have an alpha linkage.
  • each of the 20 additional glutamyl groups in the chain other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 19 of the 20 additional glutamyl groups in the chain have an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, of the 20 additional glutamyl groups have an alpha linkage and the remaining 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, glutamyl groups, respectively, have a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have an alpha linkage and the remaining non-C-terminal glutamyl groups are linked to a glutamyl group of the molecule through a gamma linkage.
  • at least one additional glutamyl group has both an alpha linkage and a gamma linkage.
  • At least one of the 21 glutamyl groups has both an alpha linkage and a gamma linkage.
  • each of the 21 glutamyl groups other than the C-terminal glutamyl group or groups have an alpha linkage.
  • 20 of the 21 glutamyl groups have an alpha linkage.
  • the alpha icosikaihenaglutamated THF comprises two or more glutamyl groups in the L-form.
  • each of the glutamyl groups of the alpha icosikaihenaglutamated tetrahydrofolate is in the L- form.
  • the alpha icosikaihenaglutamated THF comprises a glutamyl group in the D-form.
  • each of the glutamyl groups of the alpha icosikaihenaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • the icosikaihenaglutamated THF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • each of the 4-7 attached glutamyl groups is in the L-form. In other embodiments, each of the 4-7 attached glutamyl groups is in the D-form. In other embodiments, the 4-7 attached glutamyl groups are in the L-form and the D-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched. [00169] In one embodiment, the alpha polyglutamated tetrahydrofolate is tetraglutamated and each of the 3 glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate contains an alpha linkage.
  • the alpha polyglutamated tetrahydrofolate is tetraglutamated and each of the 3 glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate other than the C-terminal glutamyl group or groups contains an alpha linkage.
  • each of the 4 glutamyl groups is in the L-form.
  • each of the glutamyl groups in the alpha tetraglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • At least two glutamyl groups in the alpha tetraglutamate tetrahydrofolate are in the L-form and at least one glutamyl group is in the D-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is pentaglutamated and each of the 4 glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate contains an alpha linkage.
  • the alpha polyglutamated tetrahydrofolate is pentaglutamated and each of the 4 glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate other than the C-terminal glutamyl group or groups contains an alpha linkage.
  • each of the 4 glutamyl groups is in the L-form.
  • each of the glutamyl groups in the alpha pentaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • at least two glutamyl groups in the alpha pentaglutamated tetrahydrofolate are in the L-form and at least one glutamyl group is in the D-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is hexaglutamated and each of the 5 glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate contains an alpha linkage.
  • the alpha polyglutamated tetrahydrofolate is hexaglutamated and each of the 5 glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate other than the C-terminal glutamyl group or groups contains an alpha linkage.
  • each of the 5 glutamyl groups is in the L-form.
  • each of the glutamyl groups in the alpha hexaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • at least two glutamyl groups in the alpha hexaglutamated tetrahydrofolate are in the L-form and at least one glutamyl group is in the D-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate is heptaglutamated and each of the 6 glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate contains an alpha linkage.
  • the alpha polyglutamated tetrahydrofolate is heptaglutamated and each of the 6 glutamyl groups in the polyglutamate chain attached to the tetrahydrofolate other than the C-terminal glutamyl group or groups contains an alpha linkage.
  • each of the 6 glutamyl groups is in the L-form.
  • each of the glutamyl groups in the alpha heptaglutamated tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • at least two glutamyl groups in the alpha heptaglutamated tetrahydrofolate are in the L-form and at least one glutamyl group is in the D-form.
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate contains a total of 1-15, 1-10, 2-15, 2-10, 3-15, 3-10, 3-6, 3-5, 4-10, 4-7, or 4-6, glutamyl groups including the glutamyl group in tetrahydrofolate, or any range therein between.
  • each of the glutamyl groups in the aPTHF other than the glutamyl group of tetrahydrofolate have an alpha linkage.
  • each of the glutamyl groups in the aPTHF other than the C-terminal glutamyl group or groups and the glutamyl group of tetrahydrofolate has an alpha linkage.
  • each of the glutamyl groups in the aPTHF other than the C-terminal glutamyl group or groups has an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, of the glutamyl groups in the aPTHF have an alpha linkage.
  • the aPTHF comprises glutamyl groups in the L-form and the D-form.
  • each of the glutamyl groups in the polyglutamate structure of the polyglutamated tetrahydrofolate is in the L-form.
  • each of the glutamyl groups in the aPTHF other than the glutamyl group of tetrahydrofolate is in the D-form. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.
  • the alpha polyglutamated tetrahydrofolate contains a total of 2-20, 2-15, 2-10, 2-5, glutamyl groups including the glutamyl group in tetrahydrofolate, or any range therein between.
  • each of the glutamyl groups in the aPTHF other than the glutamyl group of tetrahydrofolate have an alpha linkage.
  • each of the glutamyl groups in the aPTHF other than the C-terminal glutamyl group or groups and the glutamyl group of tetrahydrofolate has an alpha linkage.
  • each of the glutamyl groups in the aPTHF other than the C-terminal glutamyl group or groups has an alpha linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, of the glutamyl groups have an alpha linkage.
  • the aPTHF contains two or more glutamyl groups having a gamma linkage.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, of the glutamyl groups in the aPTHF other than the glutamyl group of tetrahydrofolate have an alpha linkage and 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or none, of the glutamyl groups, respectively, has a gamma linkage.
  • each of the glutamyl groups in the aPTHF is in the L-form.
  • each of the glutamyl groups in the aPTHF other than the glutamyl group of tetrahydrofolate is in the D-form.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, of the glutamyl groups in the aPTHF are in the L-form.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, glutamyl groups in the aPTHF is in the D- form.
  • the alpha polyglutamated tetrahydrofolate contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, glutamyl groups in addition to the glutamyl group in tetrahydrofolate).
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, of the additional glutamyl groups have an alpha linkage.
  • 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, of the glutamyl groups in the alpha polyglutamated tetrahydrofolate have a gamma linkage.
  • at least one glutamyl group has both an alpha linkage and a gamma linkage.
  • the glutamyl group in tetrahydrofolate has an alpha linkage. In some embodiments, the glutamyl group in tetrahydrofolate has both an alpha linkage and a gamma linkage.
  • a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, glutamyl groups in the polyglutamated alpha tetrahydrofolate are in the L-form, the D- form, or in the L-form and the D-form.
  • each of the glutamyl groups of the polyglutamated alpha tetrahydrofolate is in the L-form.
  • each of the glutamyl groups of the polyglutamated alpha tetrahydrofolate other than the glutamyl group of tetrahydrofolate is in the D-form.
  • At least two of the glutamyl groups in the polyglutamated alpha tetrahydrofolate are in the L-form and at least one of the glutamyl groups in the polyglutamated alpha tetrahydrofolate is in the D-form.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 glutamyl groups in the polyglutamated alpha tetrahydrofolate are in the L-form.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, glutamyl groups in the polyglutamated alpha tetrahydrofolate are in the D-form.
  • at least one glutamyl group has both an alpha linkage and a gamma linkage.
  • the polyglutamated alpha tetrahydrofolate contains 20-100, 20-75, 20-50, 20-40, 20-30, 20-25, or more than 100, alpha glutamyl groups, or any range therein between.
  • each of the glutamyl groups of the aPTHF is in the L-form.
  • each of the glutamyl groups of the aPTHF other than the glutamyl group of tetrahydrofolate is in the D-form.
  • at least two of the glutamyl groups in the aPTHF are in the L-form and at least one of the glutamyl groups in the aPTHF is in the D-form.
  • at least one glutamyl group has both an alpha linkage and a gamma linkage.
  • the provided compositions comprise a aPTHF that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20, glutamyl groups that have alpha linkages.
  • the aPTHF contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20, glutamyl groups in the L-form.
  • the aPTHF contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20, glutamyl groups in the D-form.
  • the aPTHF contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20, glutamyl groups in the L-form and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 or 1-20, glutamyl groups in the D- form.
  • the polyglutamated alpha tetrahydrofolate contains at least 1 glutamyl group that has both an alpha linkage and a gamma linkage. In some embodiments, the polyglutamated alpha tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10 glutamyl groups that have both an alpha linkage and a gamma linkage.
  • the alpha-polyglutamated tetrahydrofolate contains a least 1 glutamyl group having an alpha linkage and contains 2, 3, 4, 5, 6, 7, 8, 9, 1-10, 1-20, or more, glutamyl groups having a gamma linkage.
  • the polyglutamated alpha tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, L-alpha glutamyl group linkages and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, L-gamma glutamyl group linkages.
  • the polyglutamated alpha tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, L-alpha glutamyl group linkages and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, D-gamma glutamyl group linkages.
  • the polyglutamated alpha tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, D-alpha glutamyl group linkages and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, D-gamma glutamyl group linkages.
  • the polyglutamated alpha tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10, D- gamma glutamyl group linkages and further contains 1, 2, 3, 4, 5, 6, or 1-10, L-gamma glutamyl group linkages.
  • the polyglutamated alpha tetrahydrofolate contains at least 1 glutamyl group that has both an alpha linkage and a gamma linkage.
  • the polyglutamated alpha tetrahydrofolate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10, glutamyl groups that have both an alpha linkage and a gamma linkage.
  • the aPTHF composition provided herein is capable of adding one or more additional glutamyl groups that, is the composition is able to act as a substrate for by FPGS (folylpolyglutamate synthetase).
  • FPGS fluorescence-activated glutamate synthetase
  • Reagents and assays and reagents for determining the ability of a aPTHF composition to act as a substrate for FPGS e.g, human FPGS, or rat liver FPGS
  • the rate of uptake of naked alpha PTHF compositions disclosed herein are taken up by hepatic cells at a significantly reduced rate compared to the uptake rate of tetrahydrofolate under the same physiological conditions.
  • the rate of hepatic cell uptake of the naked alpha PTHF composition is less than 30%, 20%, 15%, or 10% compared to the rate of tetrahydrofolate.
  • the rate of the efflux (transport out) of alpha PTHF compositions disclosed herein from hepatic-cells occurs at a rate that is significantly reduced compared to tetrahydrofolate (e.g, less than 30%, 20%, 15%, or 10%) compared to the rate of tetrahydrofolate.
  • an alpha polyglutamated tetrahydrofolate composition provided herein is more cytotoxic to hyperproliferative cells than tetrahydrofolate.
  • the hyperproliferative cells are cancer cells.
  • the hyperproliferative cells a colorectal carcinoma cells, colon cancer cells, breast cancer cells, or ovarian cancer cells.
  • the cancer cells are mesothelioma cells or non-small cell lung carcinoma cells.
  • cytotoxicity is measured in an in vitro assay.
  • the alpha polyglutamated tetrahydrofolate is a hexaglutamated tetrahydrofolate.
  • an alpha polyglutamated tetrahydrofolate composition provided herein has lower toxic side effects than tetrahydrofolate. In some embodiments, the alpha polyglutamated tetrahydrofolate composition provided herein is less toxic to non-hyperproliferative cells than tetrahydrofolate. In some embodiments, the alpha polyglutamated tetrahydrofolate composition provided herein is less toxic to neutrophils, liver cells, or to colon epithelium cells than tetrahydrofolate. In some embodiments, the neutrophils human neutrophils, differentiating human neutrophils, or neutrophils differentiated from CD34+ cells. In some embodiments, the liver cells are AML12 liver cells.
  • the colon epithelium cells are CCD841 colon epithelium cells.
  • the toxicity is measured in an in vitro assay.
  • the alpha polyglutamated tetrahydrofolate is a hexaglutamated tetrahydrofolate.
  • an alpha polyglutamated tetrahydrofolate composition provided herein has lower toxic side effects than to tetrahydrofolate.
  • an alpha polyglutamated tetrahydrofolate composition provided herein causes fewer or less severe toxic side effects in an vivo assay than tetrahydrofolate.
  • the in vivo assay is an in vivo murine model.
  • an alpha polyglutamated tetrahydrofolate composition provided herein causes fewer or less severe hematological or hepatic toxic side effects than tetrahydrofolate.
  • hematological side effects are assessed by measuring mean neutrophil, mean white blood cell or mean platelet counts.
  • hepatic toxic side effects are assessed by measuring serum aspartate transaminase (AST), serum alanine transaminase (ALT), and/or serum albumin levels.
  • the in vivo assay comprises administering 40 mg/kg or 80 mg/kg of the alpha polyglutamated tetrahydrofolate composition once weekly for 4 weeks.
  • the alpha polyglutamated tetrahydrofolate is a hexaglutamated tetrahydrofolate.
  • treatment with an alpha polyglutamated tetrahydrofolate composition provided herein does not induce significant hematological or hepatic toxic side effects in an in vivo murine model.
  • hematological side effects are assessed by measuring mean neutrophil, mean white blood cell or mean platelet counts.
  • hepatic toxic side effects are assessed by measuring serum aspartate transaminase (AST), serum alanine transaminase (ALT), and/or serum albumin levels.
  • AST serum aspartate transaminase
  • ALT serum alanine transaminase
  • an alpha polyglutamated tetrahydrofolate composition provided herein does not significantly decrease mean neutrophil, mean white blood cell or mean platelet counts.
  • an alpha polyglutamated tetrahydrofolate composition provided herein does not significantly increase serum aspartate transaminase (AST) and serum alanine transaminase (ALT) levels. In some embodiments, an alpha polyglutamated tetrahydrofolate composition provided herein does not significantly decrease serum albumin levels. In some embodiments, the in vivo assay comprises administering 40 mg/kg or 80 mg/kg of the alpha polyglutamated tetrahydrofolate composition once weekly for 4 weeks. In some embodiments, the alpha polyglutamated tetrahydrofolate is a hexaglutamated tetrahydrofolate.
  • the aPTHF compositions do not contain a fluorine atom.
  • the aPTHF compositions do not contain a 4-fluoroglutamyl group.
  • aPTHF alpha tetrahydrofolate
  • the disclosure also encompasses aPTHF derivatives and analogs.
  • the compositions and methods disclosed herein are envisioned to apply to any and every known derivative or analog of tetrahydrofolate that is polyglutamated.
  • the polyglutamated tetrahydrofolate analog or derivative composition prepared and used according to the disclosed compositions and methods is depicted in FIGS. 1I-1J
  • the analog corresponds to a modified form of tetrahydrofolate wherein the glutamyl group of tetrahydrofolate is not linked to the remainder of tetrahydrofolate molecule through a gamma peptide linkage.
  • the analog is a variant form of tetrahydrofolate wherein the glutamyl group of tetrahydrofolate in in the D-form.
  • the polyglutamated form of tetrahydrofolate, or polyglutamated tetrahydrofolate analog or derivative is not fluorinated.
  • the polyglutamated alpha tetrahydrofolate derivative or analog has a variant polyglutamate chain.
  • the polyglutamate chain contains one or more natural or synthetic residues other than glutamate.
  • the polyglutamate chain contains one or more glutamyl groups that do not contain an amide linkage.
  • one or more of the glutamyl groups of the polyglutamate chain is derivatized.
  • the tetrahydrofolate polyglutamate compositions provided herein may be obtained by following synthetic procedures using available reagents and synthetic intermediates.
  • the addition of glutamyl residues to the glutamyl residues of tetrahydrofolate can be accomplished using synthetic procedures known in the art.
  • glutamyl residues are added serially to the glutamyl residue of tetrahydrofolate.
  • polyglutamates are added to the glutamyl reside of tetrahydrofolate using “click chemistry” methods or other bioconjugate chemistries known to those in the art.
  • a peptide of glutamyl residues can be generated of the desired length and added to a precursor of tetrahydrofolate which does not have a glutamyl residue.
  • the peptide can be produced using synthetic procedures known in the art.
  • an initial glutamyl residue is bonded to wang resin and additional glutamyl residues are added serially via solid phase peptide synthesis using F-moc chemistry. After the final glutamyl residue is added the tetrahydrofolate precursor is coupled to the peptide and the molecule is cleaved from the resin.
  • glutamyl residues to the glutamyl residues of tetrahydrofolate can be accomplished using synthetic procedures known in the art.
  • glutamyl residues are added serially to the glutamyl residue of tetrahydrofolate.
  • polyglutamates are added to the glutamyl reside of tetrahydrofolate using“click chemistry” methods or other bioconjugate chemistries known to those in the art.
  • a peptide of glutamyl residues can be generated of the desired length and added to a precursor of tetrahydrofolate which does not have a glutamyl residue. The peptide can be produced using synthetic procedures known in the art.
  • an initial glutamyl residue is bonded to wang resin and additional glutamyl residues are added serially via solid phase peptide synthesis using F- moc chemistry. After the final glutamyl residue is added the tetrahydrofolate precursor is coupled to the peptide and the molecule is cleaved from the resin.
  • aPTHF tetrahydrofolate
  • the disclosure provides a complex of a aPTHF (e.g, a aPTHF disclosed herein) and a therapeutic agent or a salt or acid thereof.
  • the aPTHF/complex comprise aPTHF and a therapeutic agent.
  • the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent.
  • the aPTHF/complex contains a platinum-based drug such as platinum-based chemotherapeutic agent (e.g, cisplatin, carboplatin and oxaliplatin).
  • the aPTHF/complex contains a taxane-based chemotherapeutic agent (e.g, paclitaxel and docetaxel).
  • the aPTHF/complex contains a cyclodextrin.
  • the aPTHF/complex is encapsulated in a liposome
  • the disclosure provides a composition comprising a complex of a aPTHF and a therapeutic agent or a salt or acid thereof.
  • the aPTHF/therapeutic agent complex comprises one or more aPTHF 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.
  • the aPTHF/therapeutic agent complex comprises one or more aPTHF containing 3-10, 3-9, 3-8, or 3-7, glutamyl groups, or any range therein between.
  • the aPTHF/therapeutic agent complex comprises one or more aPTHF containing 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5, glutamyl groups, or any range therein between.
  • the complex comprises one or more aPTHF containing 3-10 glutamyl groups.
  • the aPTHF/therapeutic agent complex comprises one or more aPTHF containing 3-7 glutamyl groups.
  • the aPTHF/therapeutic agent complex comprises one or more aPTHF containing 5 glutamyl groups.
  • the aPTHF/therapeutic agent complex comprises one or more aPTHF containing 6 glutamyl groups.
  • the therapeutic agent is a cytotoxic compound or a salt or acid thereof. In a further embodiment, the therapeutic agent is a chemotherapeutic agent or a salt or acid thereof. In another embodiment, the therapeutic agent is a platinum-based drug. In another embodiment, the therapeutic agent is a taxane-based drug. In additional embodiments, the molar ratio of aPTHF/therapeutic agent in the complex is in the range 1-10:1.
  • the molar ratio of aPTHF/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.
  • the molar ratio of aPTHF/therapeutic agent in the complex is in the range 1 : 1-20, 1 : 1-10, or 1 :2-8, or any range therein between.
  • the molar ratio of aPPTHF/therapeutic agent is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, l:(2l-50), or 1 :>50.
  • the aPTHF/therapeutic agent complex is encapsulated in a liposome ( e.g ., as described herein or otherwise known in the art).
  • the aPTHF complex comprises aPTHF and cyclodextrin.
  • the molar ratio of aPTHF (e.g., aPTHF salt)/cyclodextrin in the complex is in the range 1-20: 1, or any range therein between.
  • the molar ratio of aPTHF/cyclodextrin in the complex is in the range 1-10:1, or any range therein between.
  • the molar ratio of aPTHF/cyclodextrin in the complex is in the range 2-8:1, or any range therein between.
  • the molar ratio of aPTHF/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.
  • the molar ratio of aPTHF/cyclodextrin in the complex is in the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between.
  • the molar ratio of aPTHF/cyclodextrin is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, l:(2l-50), or 1 :>50.
  • the aPTHF/cyclodextrin complex is encapsulated in a liposome ( e.g ., as described herein or otherwise known in the art).
  • the disclosure provides a composition comprising a aPTHF/platinum-based chemotherapeutic agent complex.
  • the platinum-based chemotherapeutic agent is selected from the group consisting of: cisplatin, carboplatin, and oxaliplatin, or a salt or acid thereof.
  • the aPTHF/platinum-based chemotherapeutic agent complex comprises an analog of a cisplatin, carboplatin, oxaliplatin, or a salt or acid thereof.
  • the molar ratio of aPTHF/platinum-based agent in the complex is in the range 1-20: 1, or any range therein between.
  • the molar ratio of aPTHF/platinum-based agent in the complex is in the range 1-10:1, or any range therein between. In further embodiments, the molar ratio of aPTHF/platinum-based agent in the complex is in the range 2-8:1, or any range therein between. In some embodiments, the molar ratio of aPTHF/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, (2l-50):l, or >50:1.
  • the molar ratio of aPTHF/platinum-based agent in the complex is in the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between.
  • the molar ratio of aPTHF/platinum-based agent is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, l:(2l-50), or l:>50.
  • the aPTHF//platinum-based agent complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
  • the aPTHF/platinum-based chemotherapeutic agent complex comprises an analog of a cisplatin, carboplatin, oxaliplatin, or a salt or acid thereof.
  • the molar ratio of aPTHF/platinum-based analog in the complex is in the range 1-20:1, or any range therein between.
  • the molar ratio of aPTHF/platinum-based analog in the complex is in the range 1-10: 1, or any range therein between.
  • the molar ratio of aPTHF/platinum-based agent in the complex is in the range 2-8:1, or any range therein between.
  • the molar ratio of aPTHF/platinum-based analog in the complex is 1 : 1, 2: 1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50): 1, or >50:1.
  • the molar ratio of aPTHF/platinum- based analog in the complex is in the range 1 : 1-20, 1 : 1-10, or 1 :2-8, or any range therein between.
  • the molar ratio of aPTHF/platinum-based analog is: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, l:(2l-50), or l:>50.
  • the aPTHF//platinum-based analog complex is encapsulated in a liposome ( e.g as described herein or otherwise known in the art).
  • the disclosure provides a complex containing aPTHF and cisplatin or a salt or acid thereof.
  • the molar ratio of aPTHF/cisplatin (or cisplatin salt or acid) in the complex is in the range 1-20:1, or any range therein between.
  • the molar ratio of aPTHF/cisplatin (or cisplatin salt or acid) in the complex is in the range 1-10: 1, or any range therein between.
  • the molar ratio of aPTHF/cisplatin (or cisplatin salt or acid) in the complex is in the range 2-8:1, or any range therein between.
  • the molar ratio of aPTHF/cisplatin (or cisplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50): 1, or >50:1.
  • the molar ratio of aPTHF/cisplatin (or cisplatin salt or acid) in the complex is in the range 1 : 1-20, 1 : 1-10, or 1 :2-8, or any range therein between.
  • the molar ratio of aPTHF/cisplatin (or cisplatin salt or acid) 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, l:(2l-50), or 1 :>50.
  • the aPTHF//cisplatin (or cisplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
  • the disclosure provides a complex containing aPTHF and carboplatin or a salt or acid thereof.
  • the molar ratio of aPTHF/carboplatin (or carboplatin salt or acid) in the complex is in the range 1-20:1, or any range therein between.
  • the molar ratio of aPTHF/carboplatin (or carboplatin salt or acid) in the complex is in the range 1-10:1, or any range therein between.
  • the molar ratio of aPTHF/carboplatin (or carboplatin salt or acid) in the complex is in the range 2-8:1, or any range therein between.
  • the molar ratio of aPTHF/carboplatin (or carboplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 -50): 1 , or >50:1.
  • the molar ratio of aPTHF/carboplatin (or carboplatin salt or acid) in the complex is in the range 1 : 1-20, 1:1- 10, or 1:2-8, or any range therein between.
  • the molar ratio of aPTHF/carboplatin (or carboplatin salt or acid) 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, l:(2l-50), or l:>50.
  • the aPTHF/carboplatin (or carboplatin salt or acid) complex is encapsulated in a liposome (e.g ., as described herein or otherwise known in the art).
  • the disclosure provides a complex containing aPTHF and oxaliplatin, or a salt or acid thereof.
  • the molar ratio of aPTHF/oxaliplatin (or oxaliplatin salt or acid) in the complex is in the range 1-20:1, or any range therein between.
  • the molar ratio of aPTHF/oxaliplatin (or oxaliplatin salt or acid) in the complex is in the range 1-10:1, or any range therein between.
  • the molar ratio of aPTHF/oxaliplatin (or oxaliplatin salt or acid) in the complex is in the range 2-8:1, or any range therein between.
  • the molar ratio of aPTHF/oxaliplatin (or oxaliplatin salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50): 1, or >50:1.
  • the molar ratio of aPTHF/oxaliplatin (or oxaliplatin salt or acid) in the complex is in the range 1:1-20, 1:1-10, or 1:2-8, or any range therein between.
  • the molar ratio of aPTHF/oxaliplatin (or oxaliplatin salt or acid) 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, l:(2l-50), or l:>50.
  • the aPTHF/oxaliplatin (or oxaliplatin salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
  • the disclosure provides a complex comprising aPTHF and a platinum-based chemotherapeutic agent (platinum) selected from the group consisting of: nedaplatin, heptaplatin, lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum -triamine, traplatin, enloplatin, JM216, NK121, CI973, DWA 2114R, NDDP, and dedaplatin, or a salt or acid thereof.
  • platinum-based chemotherapeutic agent platinum-based chemotherapeutic agent selected from the group consisting of: nedaplatin, heptaplatin, lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin,
  • the aPTHF/platinum-based chemotherapeutic agent complex comprises an analog of nedaplatin, heptaplatin, lobaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iproplatin, ormaplatin, zeniplatin, platinum-triamine, traplatin, enloplatin, JM216, NK121, CI973, DWA 2114R, NDDP, or dedaplatin, or a salt or acid thereof.
  • the molar ratio of aPTHF/platinum (or platinum salt or acid) in the complex is in the range 1-20: 1, or any range therein between. In further embodiments, the molar ratio of aPTHF/platinum (or platinum salt or acid) in the complex is in the range 1-10: 1, or any range therein between. In further embodiments, the molar ratio of aPTHF/platinum (or platinum salt or acid) in the complex is in the range 2-8:1, or any range therein between.
  • the molar ratio of aPTHF/platinum (or platinum salt or acid) in the complex is 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7:1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14:1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20:1, (21-50): 1, or >50: 1.
  • the molar ratio of aPTHF/platinum (or platinum salt or acid) in the complex is in the range 1 : 1-20, 1: 1- 10, or 1 :2-8, or any range therein between.
  • the molar ratio of aPTHF/platinum (or platinum salt or acid) 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, l :(2l-50), or l :>50.
  • the aPTHF/platinum (or salt or acid or analog thereof) complex is encapsulated in a liposome (e.g as described herein or otherwise known in the art).
  • the disclosure provides a composition comprising a aPTHF/taxane-based chemotherapeutic agent (taxane) complex.
  • the taxane -based chemotherapeutic agent is selected from the group consisting of: paclitaxel (PTX), docetaxel (DTX), larotaxel (LTX), and cabazitaxel (CTX), or a salt or acid thereof.
  • the molar ratio of aPTHF/taxane-based agent in the complex is in the range 1-20:1, or any range therein between.
  • the molar ratio of aPTHF/taxane (or taxane salt or acid) in the complex is in the range 1- 10:1, or any range therein between. In further embodiments, the molar ratio of aPTHF/taxane (or taxane salt or acid) in the complex is in the range 2-8:1, or any range therein between. In some embodiments, the molar ratio of aPTHF/taxane (or taxane salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50): 1, or >50:1.
  • the molar ratio of aPTHF/taxane (or taxane salt or acid) in the complex is in the range 1:1- 20, 1 : 1-10, or 1 :2-8, or any range therein between.
  • the molar ratio of a aPTHF/taxane (or taxane salt or acid) 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, l:(2l-50), or l:>50.
  • the aPTHF/taxane-based agent complex is encapsulated in a liposome ( e.g as described herein or otherwise known in the art).
  • the disclosure provides a complex comprising aPTHF and paclitaxel (PTX), or a salt or acid thereof.
  • the aPTHF/taxane- based chemotherapeutic agent complex comprises an analog of paclitaxel (PTX), or a salt or acid thereof.
  • the molar ratio of aPTHF/paclitaxel (or paclitaxel salt or acid) in the complex is in the range 1-20: 1, or any range therein between.
  • the molar ratio of aPTHF/paclitaxel (or paclitaxel salt or acid) in the complex is in the range 1-10:1, or any range therein between.
  • the molar ratio of aPTHF/paclitaxel (or paclitaxel salt or acid) in the complex is in the range 2-8:1, or any range therein between. In some embodiments, the molar ratio of aPTHF/paclitaxel (or paclitaxel salt or acid) in the complex is 1 : 1, 2: 1, 3 : 1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50): 1, or >50:1.
  • the molar ratio of aPTHF/paclitaxel (or paclitaxel salt or acid) in the complex is in the range 1 : 1-20, 1 : 1-10, or 1 :2-8, or any range therein between.
  • the molar ratio of aPTHF/paclitaxel (or paclitaxel salt or acid) 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, l:(2l-50), or l:>50.
  • the aPTHF/paclitaxel (or paclitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
  • the disclosure provides a complex comprising aPTHF and docetaxel (DTX), or a salt or acid thereof.
  • the aPTHF/taxane- based chemotherapeutic agent complex comprises an analog of docetaxel (DTX), or a salt or acid thereof.
  • the molar ratio of aPTHF/docetaxel (or docetaxel salt or acid) in the complex is in the range 1-20: 1, or any range therein between.
  • the molar ratio of aPTHF/docetaxel (or docetaxel salt or acid) in the complex is in the range 1-10: 1, or any range therein between.
  • the molar ratio of aPTHF/docetaxel (or docetaxel salt or acid) in the complex is in the range 2-8:1, or any range therein between. In some embodiments, the molar ratio of aPTHF/docetaxel (or docetaxel 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, (2l-50):l, or >50:1.
  • the molar ratio of aPTHF/docetaxel (or docetaxel salt or acid) in the complex is in the range 1 : 1-20, 1 : 1-10, or 1 :2-8, or any range therein between.
  • the molar ratio of aPTHF/docetaxel (or docetaxel salt or acid) 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, l:(2l-50), or 1 :>50.
  • the aPTHF/docetaxel (or docetaxel salt or acid) complex is encapsulated in a liposome (e.g, as described herein or otherwise known in the art).
  • the disclosure provides a complex comprising aPTHF and larotaxel (LTX), or a salt or acid thereof.
  • the aPTHF/taxane- based chemotherapeutic agent complex comprises an analog of larotaxel (LTX), or a salt or acid thereof.
  • the molar ratio of aPTHF/larotaxel (or larotaxel salt or acid) in the complex is in the range 1-20: 1, or any range therein between.
  • the molar ratio of aPTHF/larotaxel (or larotaxel salt or acid) in the complex is in the range 1-10:1, or any range therein between.
  • the molar ratio of aPTHF/larotaxel (or larotaxel salt or acid) in the complex is in the range 2-8:1, or any range therein between. In some embodiments, the molar ratio of aPTHF/larotaxel (or larotaxel 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, (2l-50):l, or >50:1.
  • the molar ratio of aPTHF/larotaxel (or larotaxel salt or acid) in the complex is in the range 1 : 1-20, 1 : 1-10, or 1 :2-8, or any range therein between.
  • the molar ratio of aPTHF/larotaxel (or larotaxel salt or acid) 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, l:(2l-50), or 1 :>50.
  • the aPTHF/larotaxel (or larotaxel salt or acid) complex is encapsulated in a liposome ( e.g ., as described herein or otherwise known in the art).
  • the disclosure provides a complex comprising aPTHF and cabazitaxel (CTX), or a salt or acid thereof.
  • CTX cabazitaxel
  • the aPTHF/taxane-based chemotherapeutic agent complex comprises an analog of cabazitaxel (CTX), or a salt or acid thereof.
  • the molar ratio of aPTHF/cabazitaxel (or cabazitaxel salt or acid) in the complex is in the range 1-20:1, or any range therein between.
  • the molar ratio of aPTHF/cabazitaxel (or cabazitaxel salt or acid) in the complex is in the range 1-10:1, or any range therein between. In further embodiments, the molar ratio of aPTHF/cabazitaxel (or cabazitaxel salt or acid) in the complex is in the range 2-8:1, or any range therein between.
  • the molar ratio of aPTHF/cabazitaxel (or cabazitaxel salt or acid) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50): 1, or >50:1.
  • the molar ratio of aPTHF/cabazitaxel (or cabazitaxel salt or acid) in the complex is in the range 1 : 1-20, 1:1-10, or 1:2-8, or any range therein between.
  • the molar ratio of aPTHF/cabazitaxel (or cabazitaxel salt or acid) 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, l:(2l-50), or l:>50.
  • the aPTHF/cabazitaxel (or cabazitaxel salt or acid) complex is encapsulated in a liposome (e.g., as described herein or otherwise known in the art).
  • the disclosure provides a complex comprising aPTHF and another anti-metabolite, or a salt or acid thereof.
  • An anti-metabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with one or more normal functions of cells, such as cell division.
  • the disclosure provides a complex comprising aPTHF and tetrahydrofolate (THF), or a salt or acid thereof.
  • the disclosure provides a complex comprising aPTHF and an anti -metabolite selected from the group consisting of, gemcitabine, fluorouracil, capecitabine, an antifolate (e.g., tetrahydrofolate, tetrahydrofolate), tegafur, cytosine arabinoside, thioguanine, 5- azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salt or acids, acids, or derivatives of any of these.
  • an anti -metabolite selected from the group consisting of, gemcitabine, fluorouracil, capecitabine, an antifolate (e.g., tetrahydrofolate, tetrahydrofolate), tegafur, cytosine arabinoside, thioguanine, 5- azacytidine, 6-mer
  • the molar ratio of aPTHF/anti- metabolite (or anti-metabolite salt or acid) in the complex is in the range 1-20: 1, or any range therein between. In further embodiments, the molar ratio of aPTHF/anti-metabolite (or anti-metabolite salt or acid) in the complex is in the range 1-10: 1, or any range therein between. In further embodiments, the molar ratio of aPTHF/anti-metabolite (or anti metabolite salt or acid) in the complex is in the range 2-8: 1, or any range therein between.
  • the molar ratio of aPTHF/anti-metabolite (or anti-metabolite salt or acid) in the complex is 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14:1, 15: 1, 16: 1, 17: 1, 18:1, 19: 1, 20: 1, (21-50): 1, or >50: 1.
  • the molar ratio of aPTHF/anti-metabolite (or anti-metabolite salt or acid) in the complex is in the range 1 : 1-20, 1 : 1-10, or 1 :2-8, or any range therein between.
  • the molar ratio of aPTHF/anti-metabolite (or anti-metabolite salt or acid) 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.
  • the aPTHF/anti-metabolite (or anti metabolite salt or acid) complex is encapsulated in a liposome (e.g, as described herein or otherwise known in the art).
  • the disclosure provides a complex of aPTHF (e.g, a aPTHF disclosed herein) and a cyclodextrin.
  • Cyclodextrins are groups of cyclic oligosaccharides which have been shown to improve physicochemical properties of many drugs through formation of complexes.
  • CDs are cyclic oligosaccharides composed of several D-glucose units linked by a-(l,4) bonds. This cyclic configuration provides a hydrophobic internal cavity and gives the CDs a truncated cone shape. Many hydroxyl groups are situated on the edges of the ring which make the CDs both lipophilic and soluble in water. As a result, CDs are able to form complexes with a wide variety of hydrophobic agents, and thus change the physical-chemical properties of these complexed agents.
  • cyclodextrin refer generally to a parent or derivatized cyclic oligosaccharide containing a variable number of (a-l,4)-linked D-glucopyranoside units that is able to form a complex with a tetrahydrofolate-PG.
  • Each cyclodextrin glucopyranoside subunit has secondary hydroxyl groups at the 2 and 3 positions and a primary hydroxyl group at the 6-position.
  • cyclodextrin refer to a cyclodextrin containing D- glucopyranoside units having the basic formula C6H12O6 and a glucose structure without any additional chemical substitutions (e.g ., a-cyclodextrin consisting of 6 D- glucopyranoside units, a b-cyclodextrin consisting of 7 D-glucopyranoside units, and a a- cyclodextrin consisting of 8 D-glucopyranoside units).
  • the physical and chemical properties of a parent cyclodextrin can be modified by derivatizing the hydroxyl groups with other functional groups. Any substance located within the cyclodextrin internal phase is said to be "complexed" with the cyclodextrin, or to have formed a complex (inclusion complex) with the cyclodextrin.
  • the cyclodextrin component of the aPTHF/cyclodextrin complexes there are no particular limitations on the cyclodextrin component of the aPTHF/cyclodextrin complexes so long as the cyclodextrins can form complexes with the aPTHF.
  • the cyclodextrins have been derivatized to bear ionizable (e.g., weakly basic and/or weakly acidic) functional groups to facilitate complex formation with aPTHF and/or liposome encapsulation.
  • the cyclodextrin of the aPTHF/cyclodextrin complex has at least 2, 3, 4, 5, 6, 6, 7, 8, 9, or 10, hydroxyl group substituted with an ionizable chemical group.
  • charged cyclodextrin refers to a cyclodextrin having one or more of its hydroxyl groups substituted with a charged moiety. Such a moiety can itself be a charged group or it can comprise an organic moiety (e.g a C1-C6 alkyl or C1-C6 alkyl ether moiety) substituted with one or more charged moieties.
  • the "ionizable" or “charged” moieties of a CD derivative are weakly ionizable.
  • Weakly ionizable moieties are those that are either weakly basic or weakly acidic.
  • Weakly basic functional groups (W) have a pKa of between about 6.0-9.0, 6.5-8.5, 7.0-8.0, 7.5-8.0, and any range in between inclusive according to CH3-W.
  • weakly acidic functional groups (X) have a log dissociation constant (pKa) of between about 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 inclusive according to CH3-X.
  • Representative anionic moieties include, without limitation, carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkyl ether, sulphate carbonate, thiocarbonate, dithiocarbonate, phosphate, phosphonate, sulfonate, nitrate, and borate groups.
  • Representative cationic moieties include, without limitation, amino, guanidine, and quartemary ammonium groups.
  • the derivatized cyclodextrin is a "polyanion" or
  • poly cation A polyanion is a derivatized cyclodextrin having more than one negatively charged group resulting in net a negative ionic charge of more than two units.
  • a polycation is a derivatized cyclodextrin having more than one positively charged group resulting in net positive ionic charger of more than two units.
  • the derivatized cyclodextrin is a "chargeable amphiphile.”
  • chargeable is meant that the amphiphile has a pK in the range pH 4 to 8, or pH 4 to 8.5.
  • a chargeable amphiphile may therefore be a weak acid or base.
  • amphoteric herein is meant a derivatized cyclodextrin having a ionizable groups of both anionic and cationic character wherein: (a) at least one, and optionally both, of the cation and anionic amphiphiles is chargeable, having at least one charged group with a pK between 4 and 8 to 8.5, (b) the cationic charge prevails at pH 4, and (c) the anionic charge prevails at pH 8 to 8.5.
  • the "ionizable" or “charged” derivatized cyclodextrin as a whole, whether polyionic, amphiphilic, or otherwise, are weakly ionizable (e.g have a pKai of between 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 inclusive).
  • Any one, some, or all hydroxyl groups of any one, some or all a-D-glucopyranoside units of a cyclodextrin can be modified to an ionizable chemical group as described herein. Since each cyclodextrin hydroxyl group differs in chemical reactivity, reaction with a modifying moiety can produce an amorphous mixture of positional and optical isomers. Alternatively, certain chemistry can allow for pre-modified a-D-glucopyranoside units to be reacted to form uniform products.
  • the aggregate substitution that occurs for cyclodextrin derivatives in a mixture is described by a term referred to as the degree of substitution.
  • a 6- ethylenediamino- b-cyclodextrin with a degree of substitution of seven would be composed of a distribution of isomers of 6-ethyl enedi ami hob-cyclodextrin i n which the average number of ethyl enedi ami no groups per 6-ethyl enedi ami ho-b-cy cl odextri n molecule is seven.
  • the degree of substitution for a cyclodextrin derivative mixture can routinely be determined using mass spectrometry or nuclear magnetic resonance spectroscopy.
  • At least one hydroxyl moieties facing away from the cyclodextrin interior is substituted with an ionizable chemical group.
  • the C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and all three of C2-C3-C6 hydroxyls of at least one a-D- glucopyranoside unit are substituted with an ionizable chemical group.
  • Any such combination of hydroxyls can similarly be combined with at least two, three, four, five, six, seven, eight, nine, ten, eleven, up to all of the alpha-D-glucopyranoside units in the modified cyclodextrin as well as in combination with any degree of substitution described herein.
  • SAE-CD sulfoalkyl ether cyclodextrin
  • SBE-P-CD beta cyclodextrin
  • Additional cyclodextrin derivatives that may be complexed with therapeutic agents in the disclosed liposome compositions include sugammadex or Org-25969, in which the 6-hydroxy groups on g-CD have been replaced by carboxythio acetate ether linkages, and hydroxybutenyl-P-CD.
  • cyclodextrin examples include: 2,6-Di- O-methyl-P-CD (DIMEB), 2-hydroxylpropyl-3 -cyclodextrin (HP-P-CD), randomly methylated-P-cyclodextrin (RAMEB), sulfobutyl ether P-cyclodextrin (SBE-P-CD), and sul fobuty 1 ether-y-cy cl odextri n (SBEaCD), sulfobutylated beta-cyclodextrin sodium salt, (2-Hydroxypropyl)-alpha-cyclodextrin, (2-Hydroxypropyl)-beta-cyclodextrin, (2- Hydroxypropyl)-y-cyclodextrin, 2,6-di-0-methyl)-beta-cyclodextrin (DIMEB-50 Heptakis), 2,3,6-tri-0-methyl)-beta-cyclo
  • the cyclodextrin(s) has a high solubility in water in order to facilitate entrapment of a larger amount of the cyclodextrin in the liposome internal phase.
  • the water solubility of the cyclodextrin is at least 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL or higher.
  • the water solubility of the cyclodextrin(s) is within a range of 10-150 mg/mL, 20-100 mg/mL 20-75 mg/mL, and any range in between inclusive.
  • a large association constant between the cyclodextrin and the aPTHF and/or other therapeutic agent complexed with cyclodextrin is preferable and can be obtained by selecting the number of glucose units in the cyclodextrin based on the size of the therapeutic agent (see, for example, Albers et al ., Crit. Rev. Therap. Drug Carrier Syst. 12:311-337 (1995); Stella et al, Toxicol. Pathol. 36:30-42 (2008).
  • the association constant depends on pH
  • the cyclodextrin can be selected such that the association constant becomes large at the pH of the liposome internal phase.
  • the solubility (nominal solubility) of the therapeutic agent in the presence of cyclodextrin can be further improved.
  • the association constant of the cyclodextrin with the therapeutic agent is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or higher.
  • the association constant of the cyclodextrin with the therapeutic agent is in the range 100-1, 200, 200-1,000, 300-750, and any range therein between.
  • the cyclodextrin of the aPTHF/cyclodextrin complex and/or cyclodextrin/therapeutic agent complex is underivatized.
  • the cyclodextrin of the aPTHF/cyclodextrin complex and/or cyclodextrin/therapeutic agent complex is derivatized.
  • the cyclodextrin derivative of the complex has the structure of Formula I:
  • n 4, 5, or 6;
  • Ri, R2, R3, R4, Rs, R 6 , R7, Rx, and R9 are each, independently, -H, a straight chain or branched Ci-Cs- alkylene group, or an optionally substituted straight-chain or branched C1-C6 group, wherein at least one of Ri, R2, R3, R4, Rs, R 6 , R7, Rs and R9 is a straight-chain or branched Ci-Cs- alkylene (e.g., Ci-C8-(alkylene)-S0 3 group);
  • the cyclodextrin derivative of the aPTHF/cyclodextrin complex and/or cyclodextrin/therapeutic agent complex has the structure of formula II:
  • n 4, 5, or 6;
  • Ri, R 2 , R3, R4, Rs, R 6 , R7, Rs, and R9 are each, independently, -O- or a -0-(C2-C 6 alkylene)-S03- group; wherein at least one of Ri and R2 is independently a -0-(C2-C 6 alkylene)-S0 3 group; and Si, S2, S3, S 4 , Ss, S 6 , S7, Ss, and S9 are each, independently, a pharmaceutically acceptable cation.
  • the pharmaceutically acceptable cation is selected from: an alkali metal such as Li + , Na + , or K + ; an alkaline earth metal such as Ca +2 , or Mg +2 and ammonium ions and amine cations such as the cations of (Cl-C6)-alkylamines, piperidine, pyrazine, (Cl-C6)-alkanolamine and (C4- C8)-cycloalkanolamine.
  • At least one of Rl and R2 is independently a -0-(C2-C6 alkylene)-S03- group that is a -0-(CH2)mS03- group, wherein m is 2 to 6, preferably 2 to 4, ( e.g ., -O-CH2CH2CH2S03- or -O- CH2CH2CH2CH2S03 -); and Si, S2, S3, S 4 , S5, S6, S7, Ss, and S9 are each, independently, H or a pharmaceutically cation which includes for example, alkali metals (e.g., Li + , Na + , K + ) alkaline earth metals (e.g, Ca +2 , Mg +2 ), ammonium ions and amine cations such as the cations of (Cl-C6)-alkylamines, piperidine, pyrazine, (Ci-C 6 )-alkanol-amine and (C 4 -C8)
  • a cyclodextrin derivative of the aPTHF/cyclodextrin complex and/or cyclodextrin/therapeutic agent complex is a cyclodextrin disclosed in U. S. Pat. Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; and Intl. Appl. Publ. No. WO 02005/117911, the contents each of which is herein incorporated by reference in its priority.
  • the cyclodextrin derivative of the aPTHF/cyclodextrin complex and/or cyclodextrin/therapeutic agent complex is a sulfoalkyl ether cyclodextrin.
  • the cyclodextrin derivative of complex is a sulfobutyl ether-3- cyclodextrin such as CAPTISOL® (CyDex Pharma. Inc., Lenexa, Kansas. Methods for preparing sulfobutyl ether-3- cyclodextrin and other sulfoalkyl ether cyclodextrins are known in the art.
  • the cyclodextrin derivative in of the aPTHF/cyclodextrin complex and/or cyclodextrin/therapeutic agent complex is a compound of Formula III:
  • the aPTHF/cyclodextrin complex and/or cyclodextrin/therapeutic agent complex is encapsulated in a liposome (e.g, as described herein or otherwise known in the art).
  • the disclosure provides aPTHF delivery systems and their use to deliver a payload of aPTHF to a cell or cells in vitro or in vivo.
  • aPTHF is complexed with or incorporated into a delivery vehicle.
  • Such delivery vehicles include, but are not limited to, liposomes, lipospheres, polymers, peptides, proteins, antibodies (e.g ., ADCs such as Antibody- aPTHF conjugates), cellular components, cyclic oligosaccharides (e.g., cyclodextrins), nanoparticles (e.g, lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), lipoprotein particles, and combinations thereof.
  • the delivery vehicle is a liposome.
  • the delivery vehicle is an antibody or an antigen binding antibody fragment.
  • the disclosure provides liposomal compositions that comprise a liposome encapsulating (i.e., filled with) an aPTHF (e.g, a aPTHF disclosed herein).
  • a liposome in the liposomal composition comprises a aPTHF containing 4, 5, 2-10, 4-6, or more than 5, glutamyl groups (including the glutamyl group in tetrahydrofolate).
  • the aPTHF in the Lp-aPTHF comprises two or more glutamyl groups in the L-form.
  • the aPTHF in the Lp-aPTHF comprises a glutamyl group in the D-form.
  • the aPTHF in the Lp-aPTHF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form. In additional embodiments, the aPTHF in the Lp-aPTHF comprises two or more glutamyl groups that have a gamma carboxyl linkage. In some embodiments, the polyglutamated alpha tetrahydrofolate in the Lp-aPTHF comprises at least one glutamyl group that has both an alpha carboxyl linkage and a gamma carboxyl linkage. In some embodiments, the liposomal composition comprises a liposome comprising a tetraglutamated THF.
  • the liposomal composition comprises a liposome comprising a pentaglutamated THF. In some embodiments, the liposomal composition comprises a liposome comprising a hexaglutamated aPTHF (Lp- aPTHF). In some embodiments, the liposomal composition comprises a liposome that is anionic or neutral. In some embodiments, the liposomal composition comprises a liposome that is cationic. In some embodiments, the Lp-aPTHF composition is unpegylated. In some embodiments, the Lp-aPTHF composition is non-targeted (NTLp- aPTHF). In other embodiments, the Lp-aPTHF composition is targeted (TLp-aPTHF).
  • the liposomal composition comprises a liposome having a diameter in the range of 20 nm to 500 nm, or any range therein between. In some embodiments, the liposomal composition comprises a liposome having a diameter in the range of 20 nm to 400 nm, or any range therein between. In some embodiments, the liposomal composition comprises a liposome having a diameter in the range of 20 nm to 300 nm or any range therein between. In some embodiments, the liposomal composition comprises a liposome having a diameter in the range of 20 nm to 200 nm, or any range therein between.
  • the liposomal composition comprises a liposome having a diameter in the range of 20 nm to 150 nm, or any range therein between. In further embodiments, the liposomal composition comprises a liposome having a diameter in the range of 80 nm to 120 nm, or any range therein between. In additional embodiments, 30-70%, 30-60%, or 30-50% w/w aPTHF, or any range therein between, is encapsulated (entrapped) in the Lp-aPTHF during the process of preparing the liposomes.
  • the Lp-aPTHF composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the polyglutamated alpha THF. 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, aPTHF, is encapsulated in the Lp-aPTHF during the process of preparing the liposomes.
  • the provided liposomes further comprise an immunostimulatory agent, a detectable marker, or both disposed on its exterior.
  • the immunostimulatory agent or detectable marker can be ionically bonded or covalently bonded to an exterior of the liposome, including, for example, optionally to a steric stabilizer component of the liposome.
  • immunostimulatory agents also known as“immunostimulants”, and “immunostimulators”, refer to substances that stimulate an immune (including a preexisting immune response) by inducing activation or increasing activity of any of the components of the immune system.
  • immunostimulatory agents can include one or more of a hapten, an adjuvant, a protein immunostimulating agent, a nucleic acid immunostimulating agent, and a chemical immunostimulating agent.
  • Many adjuvants contain a substance designed to stimulate immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
  • adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, PA.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; IFN-alpha, IFN-alpha, FLT3-ligand; and immunostimulatory antibodies (e.g, anti-CTLA-4, anti-CD28, anti- CD3.
  • immunostimulatory antibodies e.g, anti-CTLA-4, anti-CD28, anti- CD3.
  • Cytokines such as GM-CSF, interleukin-2, -7, -12, and -15, and other like growth factors, can also be used as adjuvants.
  • the immunostimulant can be at least one selected from the group consisting of fluorescein, DNP, beta glucan, beta-l,3-glucan, beta-l,6-glucan.
  • the immunostimulant is a Toll-like receptor (TLR) modulating agent.
  • TLR Toll-like receptor
  • the Toll-like receptor (TLR) modulating agent is one or more of: an oxidized low-density lipoprotein (e.g .,.
  • the liposomes comprise fluorescein isothiocyanate (FITC) which, based on our experiments, surprisingly serves as both an immunostimulant and a detectable marker.
  • FITC fluorescein isothiocyanate
  • the liposomes comprise a detectable marker.
  • a detectable marker may, for example, include, at least, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator, an enzyme, a dye, an ink, a magnetic compound, a biocatalyst or a pigment that is detectable by any suitable means known in the art, e.g., magnetic resonance imaging (MRI), optical imaging, fluorescent/luminescent imaging, and/or nuclear imaging techniques.
  • MRI magnetic resonance imaging
  • optical imaging fluorescent/luminescent imaging
  • nuclear imaging techniques e.g., nuclear imaging techniques.
  • the immunostimulatory agent and/or detectable marker is attached to the exterior by co-incubating it with the liposome.
  • the immunostimulatory agent and/or detectable marker may be associated with the liposomal membrane by hydrophobic interactions or by an ionic bond such as an avi din/biotin bond or a metal chelation bond (e.g, Ni-NTA).
  • the immunostimulatory agent or detectable marker may be covalently bonded to the exterior of the liposome such as, for example, by being covalently bonded to a liposomal component or to the steric stabilizer which is the PEG.
  • the liposomes further comprise an agent that increases the uptake of liposomes into a cellular compartment of interest including the cytosol.
  • the liposomes comprise a mitochondrial-targeting agent.
  • the liposomes comprise triphenylphosphonium (TPP).
  • TPP triphenylphosphonium
  • Methods and mechanisms for surface functionalizing liposomes with TPP are known in the art (e.g ., attaching TPP to the lipid anchor via a peg spacer group and modifying TPP with a stearyl group (stearyl triphenylphosphonium (STPP)).
  • STPP stearyl triphenylphosphonium
  • the liposomes comprise high-density octa-arginine.
  • the liposomes comprise sphingomyelin and/or a sphingomyelin metabolite.
  • Sphingomyelin metabolite used to formulate the liposomes of the present invention can include, for example ceramide, sphingosine or sphingosine 1 -phosphate.
  • the liposomes comprise Rhodamine 123.
  • the liposomes comprise, a mitochondria penetrating peptide.
  • the liposomes comprise, a mitochondria penetrating agent selected from the group consisting of: a mitofusin peptide, a mitochondrial targeting signal peptide, and Antennapedia helix III homeodomain cell-penetrating peptide (ANT) (e.g., comprising RQIKIWF QNRRMKWKKRKKRRQRRR (SEQ ID NO: l), RKKRRXR RRGC where X is any natural or non-natural amino acid (SEQ ID NO:2), CCGCCAAGAAGCG (SEQ ID NO:3), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAG CCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGGGG GC GTCAT (SEQ ID NO:4),
  • ANT Antennapedia helix III homeodomain cell-penetrating peptide
  • liposomes in the provided liposome compositions comprise a mitochondria penetrating agent selected from the group: a guanidine-rich peptoid, tetraguanidinium, triguanidinium, diguanidinium, monoguanidinium, a guanidine-rich polycarbamate, a beta-oligoarginine, a proline-rich dendrimer, and a phosphonium salt (e.g., methyltriphenyl-phosphonium and/or tetraphenylphosphonium).
  • a mitochondria penetrating agent selected from the group: a guanidine-rich peptoid, tetraguanidinium, triguanidinium, diguanidinium, monoguanidinium, a guanidine-rich polycarbamate, a beta-oligoarginine, a proline-rich dendrimer, and a phosphonium salt (e.g., methyltriphenyl-phosphonium and
  • liposomes in the provided liposome compositions comprise sphingomyelin and/or stearyl-octa-arginine. In some embodiments, the liposomes comprise sphingomyelin and/or stearyl-octa-arginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyl-octa-arginine sphingomyelin and stearyl-octa-arginine.
  • the liposomes comprise DOPE, sphingomyelin, stearyl-octa-arginine sphingomyelin and stearyl-octa-arginine at a molar ratio of 9:2: 1.
  • the liposomes comprise the MITO-porter® system or a variant thereof.
  • liposomes in the provided liposome compositions comprise an agent such as a cell penetrating agent that that facilitates delivery of the liposome across a cell membrane and provides the liposome with the ability to bypass the endocytic pathway and the harsh environment of lysosomes.
  • Cell penetrating agents are known in the art and can routinely be used and adapted for manufacture and use of the provided liposome compositions.
  • the cell penetrating/lysosome bypassing agent is chloroquine.
  • the cell penetrating agent is a cell penetrating peptide.
  • liposomes in the provided liposome compositions comprise a cell penetrating agent selected from the group: RKKRRQRRR (SEQ ID NO: 7), GRKKRRQRRRTPQ (SEQ ID NO: 8), Y GRKKRRQRRR (SEQ ID NO: 9), A AVAL LPAVLLALLA (SEQ ID NO: 10), MGLGLHLLVLAAALQ (SEQ ID NO: 11), GALFL GFLGAAGSTM (SEQ ID NO: 12), AGYLLGKINLKALAALAALAKKIL (SEQ ID NO: 13), RVIRVWF QNKRCKDKK (SEQ ID NO: 14), RQIKIWF QNRRMKWKK (SEQ ID NO: 15),
  • GLFEAIAGFIENGWEGMIDG SEQ ID NO: 16
  • GWTLN S AGYLLGKIN
  • SEQ ID NO: 17 RS Q SRSR YYRQRQRS
  • LAIPEQEY SEQ ID NO: 19
  • LGIAEQEY SEQ ID NO:20
  • LGIPAQEY SEQ ID NO:2l
  • LGIPEAEY SEQ ID NO:22
  • LGIPEQAY SEQ ID NO:23
  • LGIAEAEY SEQ ID NO:24
  • LGIPEAAY SEQ ID NO:25
  • LGIAEQAY SEQ ID NO:26
  • LGIAEAAY SEQ ID NO:27
  • LLIILRRRIRKQAHAHSK SEQ ID NO:28
  • LKALAALAKKIL SEQ ID NO:29
  • KLALKLALKALKAALKLA SEQ ID NO: 30
  • KETWWETWWTEW SQPKKKRKV SEQ ID NO:3 l
  • the liposomes may comprise a steric stabilizer that can increase their longevity in circulation.
  • the steric stabilizer may be at least one member selected from the group consisting of polyethylene glycol (PEG), poly-L4ysine (PLL), monosialoganglioside (GM1), poly(vinyl pyrrolidone) (PVP), poly(acrylamide) (PAA), poly(2-methyl-2- oxazoline), poly(2-ethyl-2-oxazoline), phosphatidyl polyglycerol, poly[N-(2- hydroxypropyl) methacrylamide], amphiphilic poly-N-vinylpyrrolidones, L-amino-acid- based polymer, oligoglycerol, copolymer containing polyethylene glycol and polypropylene oxide, Poloxamer 188, and polyvinyl alcohol.
  • PEG polyethylene glycol
  • PLL poly-L4ysine
  • GM1 monosia
  • the steric stabilizer or the population of steric stabilizer is PEG.
  • the steric stabilizer is a PEG.
  • the PEG has a number average molecular weight (Mn) of 200 to 5000 daltons.
  • Mn number average molecular weight
  • the liposomal composition comprises a pegylated liposome (PLp-aPTHF).
  • a pegylated liposome in the liposomal composition comprises a aPTHF containing 4, 5, 2-10, 4-6, or more than 5, glutamyl groups.
  • the aPTHF in the Lp-aPTHF comprises two or more glutamyl groups in the L-form.
  • the aPTHF in the Lp-aPTHF comprises a glutamyl group in the D-form.
  • the aPTHF in the Lp-aPTHF comprises a glutamyl group in the D-form and two or more glutamyl groups in the L-form.
  • the polyglutamated alpha tetrahydrofolate in the Lp-aPTHF comprises two or more glutamyl groups that have an alpha linkage.
  • at least one glutamyl group has both an alpha linkage and a gamma linkage.
  • the liposomal composition comprises a pegylated liposome comprising a pentaglutamated THF.
  • the liposome comprises an L-pentaglutamated THF, a D-pentaglutamated THF, or an L- and D- pentaglutamated THF.
  • the liposomal composition comprises a pegylated liposome comprising a hexaglutamated THF.
  • the liposome comprises an L-hexaglutamated aPTHF, a D-hexaglutamated THF, or an L- and D- hexaglutamated THF.
  • the liposomal composition comprises a pegylated liposome that is anionic or neutral.
  • the liposomal composition comprises a pegylated liposome that is cationic.
  • the PLp-aPTHF composition is non-targeted (NTPLp-aPTHF).
  • the PLp-aPTHF composition is targeted (TPLp-aPTHF).
  • the liposomal composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the aPTHF.
  • the liposomal composition comprises a pegylated liposome comprising at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75%, w/w of the aPTHF is encapsulated (entrapped) in the PLp-aPTHF during the process of preparing the liposomes.
  • the liposomal composition comprises a pegylated liposome having a diameter in the range of 20 nm to 500 nm.
  • the liposomal composition comprises a pegylated liposome having a diameter in the range of 20 nm to 400 nm.
  • the liposomal composition comprises a pegylated liposome having a diameter in the range of 20 nm to 300 nm. In some embodiments, the liposomal composition comprises a pegylated liposome having a diameter in the range of 20 nm to 200 nm. In further embodiments, the liposomal composition comprises a pegylated liposome having a diameter in the range of 80 nm to 120 nm.
  • greater than 70%, 80% or 90% of the polyglutamated tetrahydrofolate in a provided liposomal composition is pentaglutamated. In some embodiments, greater than 70%, 80% or 90% of the polyglutamated tetrahydrofolate in a provided composition is hexaglutamated. In some embodiments, greater than 70%, 80% or 90% of the polyglutamated tetrahydrofolate in the composition has 4-10, 4-6, or more than 5, glutamyl groups.
  • the aPTHF compositions are in an aqueous solution.
  • the aPTHF composition is administered in a liposomal composition at a dose of between 0.005 and 5000 mg of aPTHF per square meter (m 2 ) of body surface area, or any range therein between.
  • the aPTHF composition is administered in a liposomal composition at a dose of between 0.1 and 1000 mg aPTHF per square meter of body surface area, or any range therein between.
  • the lipids and other components of the liposomes contained in the liposomal compositions can be any lipid, lipid combination and ratio, or combination of lipids and other liposome components and their respective ratios known in the art.
  • liposomal encapsulation of any particular drug such as, and without limitation, the polyglutamated alpha THF discussed herein, may involve substantial routine experimentation to achieve a useful and functional liposomal formulation.
  • the provided liposomes may have any liposome structure, e.g, structures having an inner space sequestered from the outer medium by one or more lipid bilayers, or any microcapsule that has a semi-permeable membrane with a lipophilic central part where the membrane sequesters an interior.
  • the lipid bilayer can be any arrangement of amphiphilic molecules characterized by a hydrophilic part (hydrophilic moiety) and a hydrophobic part (hydrophobic moiety).
  • amphiphilic molecules in a bilayer are arranged into two dimensional sheets in which hydrophobic moieties are oriented inward the sheet while hydrophilic moieties are oriented outward.
  • Amphiphilic molecules forming the provided liposomes can be any known or later discovered amphiphilic molecules, e.g, lipids of synthetic or natural origin or biocompatible lipids.
  • the liposomes can also be formed by amphiphilic polymers and surfactants, e.g, polymerosomes and niosomes.
  • these liposome-forming materials also are referred to as "lipids”.
  • the liposome composition formulations provided herein can be in liquid or dry form such as a dry powder or dry cake.
  • the dry powder or dry cake may have undergone primary drying under, for example, lyophilization conditions or optionally, the dry cake or dry powder may have undergone both primary drying only or both primary drying and secondary drying.
  • the powder or cake may, for example, have between 1% to 6% moisture, for example, such as between 2% to 5% moisture or between 2% to 4% moisture.
  • One example method of drying is lyophilization (also called freeze-drying, or cyrodessication).
  • Any of the compositions and methods of the disclosure may include liposomes, lyophilized liposomes or liposomes reconstituted from lyophilized liposomes.
  • the disclosed compositions and methods include one or more lyoprotectants or cryoprotectants.
  • These protectants are typically polyhydroxy compounds such as sugars (mono-, di-, and polysaccharides), polyalcohols, and their derivatives, glycerol, or poly ethyleneglycol, trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, or aminoglycosides.
  • the lyoprotectants or cryoprotectants comprise up to 10% or up to 20% of a solution outside the liposome, inside the liposome, or both outside and inside the liposome.
  • the liposomes include a steric stabilizer that increases their longevity in circulation.
  • a steric stabilizer such as a hydrophilic polymer (Polyethylene glycol (PEG)), a glycolipid (monosialoganglioside (GM1)) or others occupies the space immediately adjacent to the liposome surface and excludes other macromolecules from this space. Consequently, access and binding of blood plasma opsonins to the liposome surface are hindered, and thus interactions of macrophages with such liposomes, or any other clearing mechanism, are inhibited and longevity of the liposome in circulation is enhanced.
  • PEG Polyethylene glycol
  • GM1 glycolipid
  • the steric stabilizer or the population of steric stabilizers is a PEG or a combination comprising PEG.
  • the steric stabilizer is a PEG or a combination comprising PEG with a number average molecular weight (Mn) of 200 to 5000 daltons.
  • Mn number average molecular weight
  • These PEG(s) can be of any structure such as linear, branched, star or comb structure and are commercially available.
  • the diameter of the disclosed liposomes is not particularly limited. In some embodiments, the liposomes have a diameter in the range of for example, 30-150 nm (nanometer). In other embodiments, the liposomes have a diameter in the range of 40-70 nm.
  • the properties of liposomes are influenced by the nature of lipids used to make the liposomes.
  • lipids have been used to make liposomes. These include cationic, anionic and neutral lipids.
  • the liposomes comprising the aPTHF are anionic or neutral.
  • the provided liposomes are cationic.
  • the determination of the charge e.g anionic, neutral or cationic
  • the determination of the charge can routinely be determined by measuring the zeta potential of the liposome.
  • the zeta potential of the liposome can be positive, zero or negative.
  • the zeta potential of the liposome is less than or equal to zero.
  • the zeta potential of the liposome is in a range of 0 to -150 mV. In another embodiment, the zeta potential of the liposome is in the range of -30 to -50 mV.
  • cationic lipids are used to make cationic liposomes which are commonly used as gene transfection agents.
  • the positive charge on cationic liposomes enables interaction with the negative charge on cell surfaces. Following binding of the cationic liposomes to the cell, the liposome is transported inside the cell through endocytosis.
  • a neutral to anionic liposome is used.
  • an anionic liposome is used.
  • Using a mixture of, for example, neutral lipids such as HSPC and anionic lipids such as PEG-DSPE results in the formation of anionic liposomes which are less likely to non-specifically bind to normal cells.
  • Specific binding to tumor cells can be achieved by using a tumor targeting antibody such as, for example, a folate receptor antibody, including, for example, folate receptor alpha antibody, folate receptor beta antibody and/or folate receptor delta antibody.
  • At least one (or some) of the lipids is/are amphipathic lipids, defined as having a hydrophilic and a hydrophobic portions (typically a hydrophilic head and a hydrophobic tail).
  • the hydrophobic portion typically orients into a hydrophobic phase (e.g ., within the bilayer), while the hydrophilic portion typically orients toward the aqueous phase (e.g., outside the bilayer).
  • the hydrophilic portion can comprise polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups.
  • the hydrophobic portion can comprise apolar groups that include without limitation long chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted by one or more aromatic, cyclo-aliphatic or heterocyclic group(s).
  • amphipathic compounds include, but are not limited to, phospholipids, aminolipids and sphingolipids.
  • the lipids are phospholipids.
  • Phospholipids include without limitation phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and the like. It is to be understood that other lipid membrane components, such as cholesterol, sphingomyelin, and cardiolipin, can be used.
  • the lipids comprising the liposomes provided herein can be anionic and neutral (including zwitterionic and polar) lipids including anionic and neutral phospholipids.
  • Neutral lipids exist in an uncharged or neutral zwitterionic form at a selected pH.
  • such lipids include, for example, dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacylglycerols.
  • DOPG dioleoylphosphatidylglycerol
  • zwitterionic lipids include without limitation dioleoylphosphatidylcholine (DOPC), dimyristoylphos-phatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS).
  • DOPC dioleoylphosphatidylcholine
  • DMPC dimyristoylphos-phatidylcholine
  • DOPS dioleoylphosphatidylserine
  • Anionic lipids are negatively charged at physiological pH.
  • lipids include without limitation phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dode- canoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphos- phatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
  • POPG palmitoyloleyolphos- phatidylglycerol
  • non-cationic lipids Such lipids may contain phosphorus but they are not so limited.
  • non-cationic lipids include lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethan-olamine, dioleoylphosphati- dylethanolamine (DOPE), dipalmitoyl phosphatidyl ethanol-amine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidy l-ethan-olamine (DSPE), palmitoyloleoyl- phosphatidylethanolamine (POPE) palmitoyl-oleoyl-phosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidyl-choline (DSPC), dioleoylphosphatidylcholine
  • the liposomes may be assembled using any liposomal assembly method using liposomal components (also referred to as liposome components) known in the art.
  • Liposomal components include, for example, lipids such as DSPE, HSPC, cholesterol and derivatives of these components.
  • Other suitable lipids are commercially available for example, by Avanti Polar Lipids, Inc. (Alabaster, Alabama, ETSA).
  • a partial listing of available negatively or neutrally charged lipids suitable for making anionic liposomes can be, for example, at least one of the following: DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA'Na, DPPAvNa, DOPA'Na, DMPG'Na, DPPGvNa, DOPGvNa, DMPSvNa, DPPS*Na, DOPS*Na, DOPE-Glutaryl*(Na)2, Tetramyristoyl Cardiolipin *(Na)2, DSPE-mPEG-2000*Na, DSPE-mPEG-5000*Na, and DSPE-
  • the aPTHF compositions provided herein are formulated in a liposome comprising a cationic lipid.
  • the cationic lipid is selected from, but not limited to, a cationic lipid described in Intl. Appl. Publ. Nos.
  • the cationic lipid may be selected from, but not limited to, formula A described in Intl. Appl. Publ. Nos. WO2012/040184, WO2011/153120, WO201/1149733, WO2011/090965, WO2011/043913, WO2011/022460,
  • the cationic lipid may be selected from, but not limited to, formula CLI-CLXXIX of International Publication No. W02008103276, formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formula CLI-CLXXXXII of U.S. Pat. No. 7,404,969 and formula I- VI of US Patent Publication No. US20100036115; each of which is herein incorporated by reference in their entirety.
  • the cationic lipid may be selected from (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-l0-amine, (l7Z,20Z)-N,N-dimemyl- hexacosa-l7,20-dien-9-amine, (lZ,l9Z)-N5N-dimethylpentacosa-l6, l9-dien-8-amine, (l3Z,l6Z)-N,N-dimethyl-docosa-l3,l6-dien-5-amine, (12Z,15Z)-N,N- dimethylhenicosa-l2,l5-dien-4-amine, (l4Z,l7Z)-N,N-dimethyltricosa-l4,l7-dien-6- amine, (15Z,18Z)-N,N- dimethyltetracosa-l5,l8-dien-7-amine, (18Z,21Z)
  • the lipid may be a cleavable lipid such as those described in in Inti. Publ. No. WO2012/170889, which is herein incorporated by reference in its entirety
  • the cationic lipid can routinely be synthesized using methods known in the art and/or as described in Intl. Publ. Nos. WO2012/040184, WO2011/153120, WO2011/149733, WO2011/090965, W0201/1043913, WO2011/022460,
  • Lipid derivatives can include, for example, at least, the bonding (preferably covalent bonding) of one or more steric stabilizers and/or functional groups to the liposomal component after which the steric stabilizers and/or functional groups should be considered part of the liposomal components.
  • Functional groups comprises groups that can be used to attach a liposomal component to another moiety such as a protein. Such functional groups include, at least, maleimide.
  • steric stabilizers include at least one from the group consisting of: polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2 -hydroxy-propyl) methacrylamide]; amphiphilic poly-N-vinylpyrrolidones; L- amino-acid-based polymer; and polyvinyl alcohol.
  • PEG polyethylene glycol
  • PLL poly-L-lysine
  • GM1 monosialoganglioside
  • PVP poly(vinyl pyrrolidone)
  • PAA poly(acrylamide)
  • PAA poly(2-methyl-2-oxazoline)
  • the aPTHF compositions are formulated in a lipid- polycation complex.
  • the formation of the lipid-polycation complex may be accomplished using methods known in the art and/or as described in U.S. Pub. No. 20120178702, herein incorporated by reference in its entirety.
  • the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine and the cationic peptides described in International Pub. No. WO2012/013326; herein incorporated by reference in its entirety.
  • the aPTHF is formulated in a lipid-polycation complex which further includes a neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • the components of a liposome can include any molecule(s) (e.g chemical/reagent/protein) that is bound to it
  • the components of the provided liposomes 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.
  • the components of the provided liposomes include DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC- maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide.
  • the liposomal components that make up the liposome comprises DSPE; DSPE-FITC; DSPE-maleimide; cholesterol; and HSPC.
  • the liposomes of the liposome compositions provided herein comprise oxidized phospholipids.
  • the liposomes comprise an oxidize phospholipid of a member selected from the group consisting of phosphatidylserines, phosphatidylinositols, phosphatidylethanolamines, phosphatidyl cholines and l-palmytoyl-2-arachidonoyl-sn-glycero-2-phosphate.
  • the phospholipids have unsaturated bonds.
  • the phospholipids are arachidonic acid containing phospholipids.
  • the phospholipids are sn-2-oxygenated.
  • the phospholipids are not fragmented.
  • the liposomes of the disclosed liposome compositions comprise oxidized l-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC).
  • OxPAPC oxidized l-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine
  • PAPC l-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine
  • Oxidation of arachidonic acid residue also produces phospholipids containing esterified isoprostanes.
  • OxPAPC includes HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC species, among other oxidized products present in oxPAPC.
  • the oxPAPCs are epoxyisoprostane-containing phospholipids.
  • the oxPAPC is l-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), l-palmitoyl-2-(epoxy-cyclo-pentenone)-sn-glycero-3-phosphorylcholine (PECPC) and/or l-palmitoyl-2-(epoxy-isoprostane E2)-sn-glycero-4-phosphocholine (PEIPC).
  • the phospholipids have unsaturated bonds.
  • the phospholipids are arachidonic acid containing phospholipids.
  • the phospholipids are sn-2-oxygenated.
  • the phospholipids are not fragmented.
  • the liposomal aPTHF composition is pegylated (i.e., a pegylated liposomal polyglutamated alpha (e.g pentaglutamated or hexaglutamated) antifolate (PLp-aPTHF or TPLp-aPTHF)).
  • PLp-aPTHF or TPLp-aPTHF is water soluble. That is, the PLp-aPTHF or TPLp-aPTHF is in the form an aqueous solution.
  • the liposomes of the disclosed liposome compositions comprise a lipid selected from: l-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC); l-palmitoyl-2-(9'oxo-nonanoyl)-sn-glycero-3-phosphocholine; l-palmitoyl-2- arachinodoyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-myristoyl-sn-glycero-3- phosphocholine; 1 -palmitoyl-2-hexadecyl-sn-glycero-3 -phosphocholine; 1 -palmitoyl-2- azelaoyl-sn-glycero-3-phosphocholine; and l-palmitoyl-2-acetoyl-sn-glycero-3- phospho-choline.
  • PGPC l-pal
  • the pH of solutions comprising the liposome composition is from pH 5 to 8 or from pH 2 to 6. 2 to 8, or any range therein between. In some embodiments, the pH of solutions comprising the liposome composition is from pH 5 to 8, or any range therein between. In some embodiments, the pH of solutions comprising the liposome composition is from pH 6 to 7, or any range therein between. In some embodiments, the pH of solutions comprising the liposome composition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, or any range therein between.
  • At least one component of the liposome lipid bilayer is functionalized (or reactive).
  • a functionalized component is a component that comprises a reactive group that can be used to crosslink reagents and moieties to the lipid. If the lipid is functionalized, any liposome that it forms is also functionalized.
  • the reactive group is one that will react with a crosslinker (or other moiety) to form crosslinks.
  • the reactive group in the liposome lipid bilayer is located anywhere on the lipid that allows it to contact a crosslinker and be crosslinked to another moiety (e.g ., a steric stabilizer or targeting moiety).
  • the reactive group is in the head group of the lipid, including for example a phospholipid.
  • the reactive group is a maleimide group.
  • Maleimide groups can be crosslinked to each other in the presence of dithiol crosslinkers including but not limited to dithiolthrietol (DTT).
  • the provided liposomes enclose an interior space.
  • the interior space comprises, but is not limited to, an aqueous solution.
  • the interior space comprises a aPTHF as provided herein.
  • the interior space of the liposome comprises a tonicity agent.
  • the concentration (weight percent) of the tonicity agent is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 1-50%, or any range therein between.
  • the interior space of the liposome includes a sugar (e.g ., trehalose, maltose, sucrose, lactose, mannose, mannitol, glycerol, dextrose, fructose, etc.).
  • the concentration (weight percent) of the sugar is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 1-50%, or any range therein between.
  • the pH of the interior space of the liposome is from pH 2 to 8, or any range therein between.
  • the pH of solutions comprising the liposome composition is from pH 5 to 8, or any range therein between.
  • the pH of solutions comprising the liposome composition is from pH 6 to 7, or any range therein between. In some embodiments, the pH of solutions comprising the liposome composition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, or any range therein between.
  • the interior space comprises buffer.
  • the buffer a buffer selected from HEPES, citrate, or sodium phosphate (e.g., monobasic and/or dibasic sodium 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, monobasic and/or dibasic sodium phosphate).
  • the buffer is at a concentration of 15 to 200 mM, or any range therein between. In yet further embodiments, the buffer is at a concentration of between 5 to 200 mM, 15-200, between 5 to 100 mM, between 15 to 100 mM, between 5 to 50 mM, between 15 to 50 mM, between 5 to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or any range therein between. In some embodiments, the buffer is HEPES at a concentration of 15 to 200 mM, or any range therein between. In some embodiments, the buffer is citrate at a concentration of 15 to 200 mM, or any range therein between.
  • the buffer is sodium phosphate at a concentration of 15 to 200 mM, or any range therein between.
  • the interior space of the liposome comprises a total concentration of sodium acetate and calcium acetate of between 5 mM to 500 mM, or 50 mM to 500 mM, or any range therein between.
  • the interior space of the liposome includes trehalose.
  • the concentration weight percent of trehalose is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 5-20%, or any range therein between.
  • the concentration (weight percent) of trehalose is 1-15%, or any range therein between.
  • the trehalose is present at about 5% to 20% weight percent of trehalose or any combination of one or more lyoprotectants or cryoprotectants at a total concentration of 5% to 20%.
  • the pH of solutions comprising the liposome composition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, or any range therein between.
  • the interior space comprises buffer.
  • the buffer is selected from HEPES, citrate, or sodium phosphate ( e.g ., monobasic and/or dibasic sodium phosphate).
  • the buffer is HEPES.
  • the buffer is citrate.
  • the buffer is sodium phosphate (e.g., monobasic and/or dibasic sodium phosphate).
  • the buffer is at a concentration of 15 to 200 mM, or any range therein between.
  • the buffer is at a concentration of between 5 to 200 mM, 15-200, between 5 to 100 mM, between 15 to 100 mM, between 5 to 50 mM, between 15 to 50 mM, between 5 to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or any range therein between.
  • the buffer is HEPES at a concentration of 15 to 200 mM, or any range therein between.
  • the buffer is citrate at a concentration of 15 to 200 mM, or any range therein between.
  • the buffer is sodium phosphate at a concentration of 15 to 200 mM, or any range therein between.
  • the interior space of the liposome comprises sodium acetate and/or calcium acetate. In some embodiments, the interior space of the liposome comprises a total concentration of sodium acetate and calcium acetate of between 5 mM to 500 mM, or 50 mM to 500 mM, or any range therein between.
  • the interior space of the liposome includes dextrose.
  • the concentration weight percent of dextrose is 0.1-20%, 1-20%, 0.5-15%, 1-15%, or 5-20%, or any range therein between.
  • the concentration (weight percent) of dextrose is 1-15%, or any range therein between.
  • the dextrose is present at about 5% to 20% weight percent of dextrose or any combination of one or more lyoprotectants or cryoprotectants at a total concentration of 5% to 20%.
  • the pH of solutions comprising the liposome composition is from 6 to 7.5, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, or any range therein between.
  • the interior space comprises buffer.
  • the buffer is selected from HEPES, citrate, or sodium phosphate ( e.g ., monobasic and/or dibasic sodium phosphate).
  • the buffer is HEPES.
  • the buffer is citrate.
  • the buffer is sodium phosphate (e.g., monobasic and/or dibasic sodium phosphate).
  • the buffer is at a concentration of 15 to 200 mM, or any range therein between.
  • the buffer is at a concentration of between 5 to 200 mM, 15-200, between 5 to 100 mM, between 15 to 100 mM, between 5 to 50 mM, between 15 to 50 mM, between 5 to 25 mM, between 5 to 20 mM, between 5 to 15 mM, or any range therein between.
  • the buffer is HEPES at a concentration of 15 to 200 mM, or any range therein between.
  • the buffer is citrate at a concentration of 15 to 200 mM, or any range therein between.
  • the buffer is sodium phosphate at a concentration of 15 to 200 mM, or any range therein between
  • the interior space of the liposome comprises sodium acetate and/or calcium acetate.
  • the interior space of the liposome comprises a total concentration of sodium acetate and calcium acetate of between 5 mM to 500 mM, or 50 mM to 500 mM, or any range therein between.
  • the disclosure provides liposomal compositions that comprise a liposome encapsulating (filled with) a aPTHF (e.g., a aPTHF disclosed herein).
  • a liposome in the liposomal composition comprises a aPTHF containing 4, 5, 2-10, 4-6, or more than 5, glutamyl groups (including the glutamyl group in tetrahydrofolate).
  • the aPTHF in the Lp- aPTHF comprises two or more glutamyl groups that have a gamma carboxyl linkage.
  • the liposomal composition comprises a liposome comprising a tetraglutamated THF.
  • the liposomal composition comprises a liposome comprising a pentaglutamated THF.
  • the liposomal composition comprises a liposome comprising a hexaglutamated THF.
  • the medium is an aqueous solution.
  • the interior space, the exterior space (e.g, the medium), or both the interior space and the medium contains one or more lyoprotectants or cryoprotectants which are listed above.
  • the cryoprotectant is mannitol, trehalose, sorbitol, or sucrose.
  • the liposome encapsulating aPTHF (i.e., Lp-aPTHF, including PLp-aPTHF, TPLp-aPTHF, TLp-aPTHF, and NTLp-aPTHF) has an interior space that contains less than 500,000 or less than 200,000 molecules of aPTHF.
  • the liposome interior space contains between 10 to 100,000 molecules of aPTHF, or any range therein between.
  • the liposome interior space contains between 10,000 to 100,000 molecules of aPTHF, or any range therein between.
  • the liposome is unpegylated and has an interior space that contains less than 500,000 or less than 200,000 molecules of aPTHF.
  • the liposome is unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of aPTHF, or any range therein between. In further embodiments, the liposome is unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of aPTHF, or any range therein between. In some embodiments, the liposome is targeted and unpegylated (TLp-aPTHF) and has an interior space that contains less than 500,000 or less than 200,000 molecules of aPTHF. In some embodiments, the liposome is targeted and unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of aPTHF, or any range therein between.
  • TLp-aPTHF targeted and unpegylated
  • the liposome is targeted and unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of aPTHF, or any range therein between.
  • the liposome is non-targeted and unpegylated (NTLp-aPTHF) and has an interior space that contains less than 500,000 or less than 200,000 molecules of aPTHF.
  • NTLp-aPTHF non-targeted and unpegylated
  • the liposome is non-targeted and unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of aPTHF, or any range therein between.
  • the liposome is non-targeted and unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of aPTHF, or any range therein between.
  • the liposome encapsulates polyglutamated alpha containing 2-10 glutamyl groups (i.e., Lp-aPTHF, including PLp-aPTHF, TPLp-aPTHF, TLp-aPTHF, and NTLp-aPTHF) and has an interior space that contains less than 500,000 or 200,000 molecules of aPTHF containing 2-10 glutamyl groups.
  • the liposome interior space contains between 10 to 100,000 molecules of aPTHF containing 2-10 glutamyl groups, or any range therein between.
  • the liposome interior space contains between 10,000 to 100,000 molecules of aPTHF containing 2-10 glutamyl groups, or any range therein between.
  • the liposome is unpegylated and has an interior space that contains less than 500,000 or 200,000 molecules of aPTHF containing 2-10 glutamyl groups. In some embodiments, the liposome is unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of aPTHF containing 2-10 glutamyl groups, or any range therein between. In further embodiments, the liposome is unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of aPTHF containing 2-10 glutamyl groups, or any range therein between.
  • the liposome is targeted and unpegylated (TLp-aPTHF) and has an interior space that contains less than 500,000 or 200,000 molecules of aPTHF containing 2-10 glutamyl groups. In some embodiments, the liposome is targeted and unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules aPTHF containing 2-10 glutamyl groups, or any range therein between. In further embodiments, the liposome is targeted and unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules aPTHF containing 2-10 glutamyl groups, or any range therein between.
  • TLp-aPTHF targeted and unpegylated
  • the liposome is non-targeted and unpegylated (NTLp- aPTHF) and has an interior space that contains less than 500,000 or 200,000 molecules of aPTHF containing 2-10 glutamyl groups. In some embodiments, the liposome is non- targeted and unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of aPTHF containing 2-10 glutamyl groups, or any range therein between. In further embodiments, the liposome is non-targeted and unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of aPTHF containing 2-10 glutamyl groups, or any range therein between.
  • the liposome encapsulates tetraglutamated alpha tetrahydrofolate (i.e., Lp-aPTHF, including PLp-aPTHF, TPLp-aPTHF, TLp-aPTHF, and NTLp-aPTHF) and has an interior space that contains less than 500,000 or 200,000 molecules of tetraglutamated alpha tetrahydrofolate. In some embodiments, the liposome interior space contains between 10 to 100,000 molecules of tetraglutamated alpha tetrahydrofolate, or any range therein between.
  • Lp-aPTHF tetraglutamated alpha tetrahydrofolate
  • the liposome interior space contains between 10 to 100,000 molecules of tetraglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome interior space contains between 10,000 to 100,000 molecules of tetraglutamated alpha tetrahydrofolate, or any range therein between. In some embodiments, the liposome is unpegylated and has an interior space that contains less than 500,000 or 200,000 molecules of tetraglutamated alpha tetrahydrofolate. In some embodiments, the liposome is unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of tetraglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of tetraglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is targeted and unpegylated (TLp-aPTHF) and has an interior space that contains less than 500,000 or 200,000 molecules of tetraglutamated alpha tetrahydrofolate.
  • the liposome is targeted and unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of tetraglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is targeted and unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of tetraglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is non-targeted and unpegylated (NTLp-aPTHF) and has an interior space that contains less than 500,000 or 200,000 molecules of tetraglutamated alpha tetrahydrofolate.
  • the liposome is non-targeted and unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of tetraglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is non-targeted and unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of tetraglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome encapsulates pentaglutamated alpha tetrahydrofolate (i.e., Lp-aPTHF, including PLp-aPTHF, TPLp-aPTHF, TLp-aPTHF, and NTLp-aPTHF) and has an interior space that contains less than 500,000 or 200,000 molecules of pentaglutamated alpha tetrahydrofolate. In some embodiments, the liposome interior space contains between 10 to 100,000 molecules of pentaglutamated alpha tetrahydrofolate, or any range therein between.
  • pentaglutamated alpha tetrahydrofolate i.e., Lp-aPTHF, including PLp-aPTHF, TPLp-aPTHF, TLp-aPTHF, and NTLp-aPTHF
  • the liposome interior space contains between 10 to 100,000 molecules of pentaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome interior space contains between 10,000 to 100,000 molecules of pentaglutamated alpha tetrahydrofolate, or any range therein between. In some embodiments, the liposome is unpegylated and has an interior space that contains less than 500,000 or 200,000 molecules of pentaglutamated alpha tetrahydrofolate. In some embodiments, the liposome is unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of pentaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of pentaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is targeted and unpegylated (TLp-aPTHF) and has an interior space that contains less than 500,000 or 200,000 molecules of pentaglutamated alpha tetrahydrofolate.
  • the liposome is targeted and unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of pentaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is targeted and unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of pentaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is non-targeted and unpegylated (NTLp- aPTHF) and has an interior space that contains less than 500,000 or 200,000 molecules of pentaglutamated alpha tetrahydrofolate.
  • NTLp- aPTHF non-targeted and unpegylated
  • the interior space of the liposome contains between 10 to 100,000 molecules of pentaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is non-targeted and unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of pentaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome encapsulates hexaglutamated alpha tetrahydrofolate (i.e., Lp-aPTHF, including PLp-aPTHF, TPLp-aPTHF, TLp-aPTHF, and NTLp-aPTHF) and has an interior space that contains less than 500,000 or 200,000 molecules of hexaglutamated alpha tetrahydrofolate. In some embodiments, the liposome interior space contains between 10 to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, or any range therein between.
  • Lp-aPTHF hexaglutamated alpha tetrahydrofolate
  • the liposome interior space contains between 10 to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome interior space contains between 10,000 to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is unpegylated and has an interior space that contains less than 500,000 or 200,000 molecules of hexaglutamated alpha tetrahydrofolate.
  • the liposome is unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is targeted and unpegylated (TLp-aPTHF) and has an interior space that contains less than 500,000 or 200,000 molecules of hexaglutamated alpha tetrahydrofolate.
  • the liposome is targeted and unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is targeted and unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is non-targeted and unpegylated (NTLp-aPTHF) and has an interior space that contains less than 500,000 or 200,000 molecules of hexaglutamated alpha tetrahydrofolate.
  • the liposome is non-targeted and unpegylated and the interior space of the liposome contains between 10 to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, or any range therein between.
  • the liposome is non-targeted and unpegylated and the interior space of the liposome contains between 10,000 to 100,000 molecules of hexaglutamated alpha tetrahydrofolate, or any range therein between.
  • the disclosure provides a liposomal aPTHF composition wherein the liposome encapsulates aPTHF or a salt or acid thereof, and one or more aqueous pharmaceutically acceptable carriers.
  • the liposome interior space contains trehalose. In some embodiments, the liposome interior space contains 5% to 20% weight of trehalose. In some embodiments, the liposome interior space contains HBS at a concentration of between 1 to 200 mM and a pH of between 2 to 8. In some embodiments, liposome interior space has a pH 5-8, or any range therein between. In some embodiments, liposome interior space has a pH 6-7, or any range therein between. In some embodiments, the liposome interior space has a total concentration of sodium acetate and calcium acetate of between 50 mM to 500 mM, or any range therein between.
  • the liposome aPTHF (e.g, Lp-aPTHF, including PLp- aPTHF, TPLp-aPTHF, TLp-aPTHF, and NTLp-aPTHF) compositions comprise aPTHF e.g, a aPTHF disclosed herein) and one or more non-polyglutamated, polyglutamatable antifolate compositions.
  • the Lp-aPTHF (e.g, PLp-aPTHF, TPLp-aPTHF, TLp- aPTHF, and NTLp-aPTHF) comprises aPTHF (e.g, a aPTHF disclosed herein) and tetrahydrofolate (THF).
  • aPTHF e.g, a aPTHF disclosed herein
  • THF tetrahydrofolate
  • the Lp-aPTHF (i.e., liposome alpha polyglutamated tetrahydrofolate) comprises alpha polyglutamated tetrahydrofolate and a polyglutamatable antifolate selected from the group consisting of: tetrahydrofolate (THF), methotrexate (MTX), pemetrexed (PMX), lometrexol (LMX), raltitrexed (RTX), pralatrexate, AG2034, GW1843, aminopterin, and LY309887.
  • the Lp-aPTHF comprises alpha polyglutamated tetrahydrofolate and lometrexol.
  • the Lp-aPTHF comprises alpha polyglutamated tetrahydrofolate and pemetrexed. In some embodiments, the Lp-aPTHF comprises alpha polyglutamated tetrahydrofolate and leucovorin. In some embodiments, the Lp-aPTHF comprises alpha polyglutamated tetrahydrofolate and a triazine antifolate derivative (e.g., a sulphonyl fluoride triazine such as NSC 127755). In some embodiments, the Lp-aPTHF comprises alpha polyglutamated tetrahydrofolate and a serine hydroxymethyltransferase (SHMT2) inhibitor. In some embodiments, the SHMT2 inhibitor is an antifolate (e.g., a polyglutamatable or nonpolyglutamatable antifolate). In some embodiments, the SHMT2 inhibitor is an antifolate.
  • SHMT2 inhibitor is an antifolate (e.g., a

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EP19751561.2A EP3749319A4 (en) 2018-02-07 2019-02-07 TETRAHYDROFOLATES ALPHA POLYGLUTAMATE AND THEIR USES
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CA3090506A CA3090506A1 (en) 2018-02-07 2019-02-07 Alpha polyglutamated tetrahydrofolates and uses thereof
US16/967,621 US12336994B2 (en) 2018-02-07 2019-02-07 Alpha polyglutamated tetrahydrofolates and uses thereof
JP2020542422A JP7514533B2 (ja) 2018-02-07 2019-02-07 アルファポリグルタミン酸化テトラヒドロ葉酸およびその使用
JP2024101225A JP7674775B2 (ja) 2018-02-07 2024-06-24 アルファポリグルタミン酸化テトラヒドロ葉酸およびその使用
US19/022,038 US20250319188A1 (en) 2018-02-07 2025-01-15 Alpha polyglutamated antifolates and uses thereof
JP2025069103A JP2025108646A (ja) 2018-02-07 2025-04-18 アルファポリグルタミン酸化テトラヒドロ葉酸およびその使用
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