Ionic liquids for intracorporeal delivery

Abstract

To provide a method for oral delivery of an active compound.SOLUTION: A method for the oral delivery of at least one active compound comprises orally administering the active compound in combination with a composition comprising the ionic liquid CholineAndGEranate (CAGE).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 588,008 filed November 17, 2017, U.S. Provisional Application No. 62 / 681,852 filed June 7, 2018, U.S. Provisional Application No. 62 / 681,856 filed June 7, 2018, U.S. Provisional Application No. 62 / 681,861 filed June 7, 2018, and U.S. Provisional Application No. 62 / 681,866 filed June 7, 2018, the contents of which are incorporated herein by reference in their entireties.

[0002] Technical Field The technology described herein relates to ionic liquids, such as CAGE, for the stabilization and delivery of active compounds. [Background technology]

[0003] background The uptake of many active compounds, for example, pharmaceutically active compounds, can be improved by delivering the compounds in a solvent.However, such approaches are often inappropriate for in vivo use, because most of these solvents exhibit toxic side effects and / or act as irritants at the time of delivery.These toxic and irritating effects are serious enough to suppress any increase in the uptake or performance of active compounds. Summary of the Invention

[0004] overview As demonstrated herein, the inventors have discovered that a particular ionic liquid, CAGE, surprisingly results in improved uptake kinetics of active compounds without causing adverse side effects. This surprising effect applies to multiple delivery routes that are typically susceptible to the toxicity and irritation caused by carrier solvents. This lack of toxic side effects is particularly surprising because CAGE is known to have antibacterial properties.

[0005] In one aspect of any embodiment, described herein is a method for oral delivery of at least one active compound, comprising orally administering the active compound in combination with a composition comprising the ionic liquid choline and geranate (CAGE). In one aspect of any embodiment, described herein is a method for delivery of at least one active compound, comprising subcutaneously, intradermally, or intravenously administering the active compound in combination with CAGE. In one aspect of any embodiment, described herein is a method for delivery of at least one active compound, comprising mucosally administering the active compound in combination with CAGE.

[0006] In one aspect of any of the embodiments, described herein is a method for parenteral delivery of at least one active compound, comprising parenterally administering an active compound in combination with CAGE. In some embodiments of any of the aspects, the administration comprises delivery to a tumor. In one aspect of any of the embodiments, described herein is a method for treating a disease in a subject in need thereof by administering to the subject an active compound in combination with CAGE by injection into the affected tissue. In some embodiments of any of the aspects, the disease is cancer, excess fat, adipose tissue, warts, hyperplasia, or any other disease resulting from unwanted tissue proliferation.

[0007] In one aspect of any of the embodiments, described herein is a composition comprising an active compound in combination with CAGE. In some embodiments of any of the aspects, the composition further comprises an additional pharmaceutically acceptable carrier.

[0008] In some embodiments of either aspect, the composition is formulated as an oral, subcutaneous, or parenteral formulation. In some embodiments of either aspect, the oral formulation is a degradable capsule containing the active compound and CAGE combination.

[0009] In some embodiments of either aspect, the CAGE is at a concentration of at least 0.1% w / v. In some embodiments of either aspect, the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. In some embodiments of either aspect, the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. In some embodiments of either aspect, the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:4. In some embodiments of either aspect, the anion of the ionic liquid comprises geranate and / or geranic acid.

[0010] In some embodiments of either aspect, the active compound in combination with CAGE is administered once. In some embodiments of either aspect, the active compound in combination with CAGE is administered in multiple doses.

[0011] In some embodiments of any aspect, the active compound comprises a nucleic acid molecule. In some embodiments of any aspect, the active compound comprises a small molecule. In some embodiments of any aspect, the active compound comprises a polypeptide. In some embodiments of any aspect, the active compound comprises a chemotherapeutic compound. In some embodiments of any aspect, the active compound comprises insulin. In some embodiments of any aspect, the active compound comprises an antibody or antibody reagent.

[0012] In some embodiments of either aspect, the biological activity of the active compound is improved or stabilized compared to the activity in the absence of CAGE.

[0013] In some embodiments of either aspect, the combination of the active compound and the CAGE is an admixture. In some embodiments of either aspect, the combination of the active compound and the CAGE includes nanoparticles comprising the active compound, wherein the nanoparticles are in solution or suspension in a composition comprising the CAGE.

[0014] As demonstrated herein, the inventors have found that a particular ionic liquid, CAGE, reduces the uptake of lipophilic molecules in the intestine, and thus CAGE can reduce fat absorption in a subject, providing a treatment for obesity or reducing body weight / weight gain.

[0015] In one aspect of any embodiment, provided herein is a method for treating obesity, preventing weight gain, or reducing the weight of a subject, comprising orally administering to the subject a composition comprising the ionic liquid choline and geranate (CAGE).

[0016] In some embodiments of either aspect, the CAGE is at a concentration of at least 0.1% w / v. In some embodiments of either aspect, the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. In some embodiments of either aspect, the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. In some embodiments of either aspect, the anion of the ionic liquid comprises geranate and / or geranic acid.

[0017] In some embodiments of either aspect, the composition further comprises an active compound. In some embodiments of either aspect, the active compound is therapeutically effective in treating obesity or an obesity-related disorder. In some embodiments of either aspect, the active compound is a small molecule, a polypeptide, or an antibody or antibody reagent.

[0018] As shown herein, salts (e.g., ionic liquids or CAGEs) exhibit a surprising increase in drug delivery efficacy (e.g., ability to cross cells and / or cell membranes) at concentrations of 0.05M or greater. Accordingly, described herein are methods for delivering at least one active compound, wherein the active compound is administered in combination with a salt-containing composition, wherein the salt is present at a concentration of at least 0.05M. Further described herein are methods for delivering CAGEs, wherein the CAGE is administered at a concentration of at least 0.05M. In one aspect of any embodiment, described herein is a composition comprising a salt and an active compound, wherein the salt is present at a concentration of at least 0.05M.

[0019] In some embodiments of either aspect, the salt is an ionic liquid. In some embodiments of either aspect, the ionic liquid is choline and geranate (CAGE). In some embodiments of either aspect, the cation is choline. In some embodiments of either aspect, the anion is geranate or geranic acid.

[0020] In some embodiments of either aspect, delivery is oral, subcutaneous, intradermal, intravenous, parenteral, or mucosal. In some embodiments of either aspect, delivery is oral.

[0021] In some embodiments of either aspect, the salt is present at a concentration of at least 0.05 M, 0.1 M, 0.5 M, 1 M, 1.5 M, 2 M, 2.5 M, 3 M, 3.5 M, 4 M, or higher. In some embodiments of either aspect, the salt is present at a concentration of about 0.05 M to about 4 M.

[0022] In some embodiments of either aspect, the salt dissolves after administration. In some embodiments of either aspect, the salt is a pure liquid or anhydrous liquid. In some embodiments of either aspect, the salt is an aqueous solution.

[0023] In some embodiments of either aspect, the administering is administering to a subject. In some embodiments of either aspect, the administering is contacting a cell and / or tissue.

[0024] In some embodiments of either aspect, the active compound comprises a nucleic acid molecule, a chemotherapeutic compound, a small molecule, a peptide, and / or an antibody or antibody reagent. In some embodiments of either aspect, the active compound is a component of a salt. In some embodiments of either aspect, the active compound comprises insulin.

[0025] In some embodiments of either aspect, the biological activity of the active compound is improved or stabilized compared to the activity in the absence of the salt. In some embodiments of either aspect, the combination of the active compound and the salt is a mixture. In some embodiments of either aspect, the combination of the active compound and the salt includes nanoparticles comprising the active compound, and the nanoparticles are in solution or suspension in the salt-containing composition.

[0026] As demonstrated herein, the ionic liquid choline and geranate (CAGE) has been demonstrated to have enzyme inhibitor activity, including inhibiting the degradation of insulin. Accordingly, provided herein are methods and compositions related to the use of ionic liquids, such as CAGE, to treat certain diseases. In one aspect of any embodiment, described herein is a method of administering enzyme inhibitor therapy to a subject in need thereof, comprising administering to the subject a composition comprising the ionic liquid choline and geranate (CAGE). In one aspect of any embodiment, described herein is a method of treating diabetes, ulcers, cancer, or fibrosis in a subject in need thereof, comprising administering to the subject a composition comprising the ionic liquid choline and geranate (CAGE).

[0027] In some embodiments of either aspect, the composition comprising CAGE does not include an additional therapeutically active agent. In some embodiments of either aspect, the subject is not administered an additional therapeutically active agent for the condition (e.g., diabetes, ulcer, cancer, or fibrosis) during the period while the subject is administered the composition comprising CAGE and / or during the period while the subject is undergoing a treatment regimen including the composition comprising CAGE.

[0028] In some embodiments of either aspect, administration is via injection or orally.

[0029] In some embodiments of either aspect, the CAGE is at a concentration of at least 0.1% w / v. In some embodiments of either aspect, the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. In some embodiments of either aspect, the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. In some embodiments of either aspect, the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:4. In some embodiments of either aspect, the anion of the ionic liquid comprises geranate and / or geranic acid. [The present invention 1001] A method for oral delivery of at least one active compound, comprising orally administering the active compound in combination with a composition comprising the ionic liquid choline and geranate (CAGE). [The present invention 1002] A method for delivering at least one active compound, comprising subcutaneously, intradermally, or intravenously administering the active compound in combination with a CAGE. [The present invention 1003] A method for delivering at least one active compound, comprising administering to a mucosal membrane an active compound in combination with a CAGE. [The present invention 1004] The method of claim 1003, wherein the mucosa is a nasal mucosa, an oral mucosa, or a vaginal mucosa. [The present invention 1005] A method for parenteral delivery of at least one active compound, comprising parenterally administering the active compound in combination with a CAGE. [The present invention 1006] The method of claim 1005, wherein administering comprises delivery to a tumor. [The present invention 1007] A method of treating a disease in a subject in need thereof by administering to the subject an active compound in combination with CAGE by injection into the affected tissue. [The present invention 1008] The method of claim 1007, wherein the disease is cancer, adipose, hyperplasia, or any other disease caused by tissue proliferation. [The present invention 1009] 1009. The method of any of claims 1001 to 1008, wherein the CAGE is at a concentration of at least 0.1% w / v. [The present invention 1010] 1009. The method of any of claims 1001-1009, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. [The present invention 1011] 1009. The method of any of claims 1001-1009, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. [The present invention 1012] 1009. The method of any of claims 1001-1009, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:4. [The present invention 1013] The method of any one of claims 1001 to 1012, wherein the anion of the ionic liquid comprises geranate and / or geranic acid. [The present invention 1014] The method of any of claims 1001 to 1013, wherein the active compound in combination with CAGE is administered once. [The present invention 1015] The method of any of claims 1001 to 1014, wherein the active compound in combination with CAGE is administered in multiple doses. [The present invention 1016] The method of any one of claims 1001 to 1015, wherein the active compound comprises a nucleic acid molecule. [The present invention 1017] The method of any one of claims 1001 to 1015, wherein the active compound comprises a small molecule. [The present invention 1018] The method of any one of claims 1001 to 1015, wherein the active compound comprises a polypeptide. [The present invention 1019] The method of any one of claims 1001 to 1015, wherein the active compound comprises an antibody or antibody reagent. [The present invention 1020] The method of any of claims 1001 to 1019, wherein the active compound comprises a chemotherapeutic compound. [The present invention 1021] The method of any one of claims 1001 to 1020, wherein the active compound comprises insulin. [The present invention 1022] The method of any of claims 1001 to 1020, wherein the active compound comprises a GLP-1 polypeptide or a mimetic or analog thereof. [The present invention 1023] The method of any of claims 1021 to 1022, wherein the active compound is provided at a dosage of 1 to 20 mg / kg. [The present invention 1024] A composition comprising an active compound in combination with a CAGE. [The present invention 1025] 1024. The composition of the present invention, wherein the CAGE is at a concentration of at least 0.1% w / v or 5% w / w. [The present invention 1026] 1026. The composition of any of claims 1024-1025, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. [The present invention 1027] 1027. The composition of any of claims 1024-1026, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. [The present invention 1028] 1028. The composition of any of claims 1024-1027, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:4. [The present invention 1029] 1029. The composition of any one of claims 1024 to 1028, wherein the active compound comprises a nucleic acid molecule. [The present invention 1030] The composition of any of claims 1024 to 1028, wherein the active compound comprises a small molecule. [The present invention 1031] 1029. The composition of any one of claims 1024 to 1028, wherein the active compound comprises a polypeptide. [The present invention 1032] 1022. The composition of any one of claims 1024 to 1031, wherein the active compound comprises an antibody or antibody reagent. [The present invention 1033] The composition of any of claims 1024 to 1032, wherein the active compound comprises a chemotherapeutic compound. [The present invention 1034] The composition of any one of claims 1024 to 1030, wherein the active compound comprises insulin. [This invention 1035] The composition of any of claims 1024 to 1031, wherein the active compound comprises a GLP-1 polypeptide or a mimetic or analog thereof. [The present invention 1036] 6. The composition of any of claims 1034 to 1035, wherein the active compound is provided in a dosage of 1 to 20 mg / kg. [This invention 1037] The composition of any one of claims 1024 to 1036, further comprising an additional pharmaceutically acceptable carrier. [The present invention 1038] Any of the compositions of 1024 to 1037 of the present invention formulated as an oral, subcutaneous, or parenteral formulation. [This invention 1039] Any of the compositions of claims 1024 to 1038 formulated for administration to a mucosa. [The present invention 1040] The composition of the present invention 1039, wherein the mucosa is nasal mucosa, oral mucosa, or vaginal mucosa. [The present invention 1041] The composition of claim 1038, wherein the oral formulation is a degradable capsule containing the active compound and a CAGE combination. [The present invention 1042] 1024-1041. The composition of any of claims 1024-1041, wherein the biological activity of the active compound is improved or stabilized compared to the activity in the absence of CAGE. [This invention 1043] The method or composition of any of claims 1001 to 1042, wherein the combination of the active compound and the CAGE is an admixture. [This invention 1044] The method or composition of any of claims 1001 to 1042, wherein the combination of active compound and CAGE comprises nanoparticles comprising the active compound, the nanoparticles being in solution or suspension in a composition comprising the CAGE. [This invention 1045] A method for delivering a nucleic acid molecule to a cell, comprising contacting the cell with the nucleic acid molecule in combination with a composition comprising the ionic liquid choline and geranate (CAGE). [The present invention 1046] The method of claim 1045, wherein the cell is a cell in a subject and the contacting step comprises administering to the subject the nucleic acid molecule in combination with a composition comprising the ionic liquid choline and geranate (CAGE). [This invention 1047] 1047. The method of any one of claims 1045 to 1046, wherein the nucleic acid molecule comprises a vector, an expression vector, or an inhibitory nucleic acid molecule. [This invention 1048] 8. The method of any of claims 1045 to 1047, wherein the CAGE is at a concentration of at least 0.1% w / v or 5% w / w. [This invention 1049] The method of any of claims 1045-1047, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. [The present invention 1050] The method of any of claims 1045-1047, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. [This invention 1051] The method of any of claims 1045 to 1047, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:4. [This invention 1052] The method of any one of claims 1045 to 1051, wherein the anion of the ionic liquid comprises geranate and / or geranic acid. [This invention 1053] The method of any one of claims 1045 to 1052, wherein the combination of the nucleic acid molecule and the CAGE is a mixture. [This invention 1054] The method of any of claims 1045 to 1053, wherein the combination of nucleic acid molecule and CAGE comprises nanoparticles comprising the nucleic acid molecule, the nanoparticles being in solution or suspension in a composition comprising the CAGE. [This invention 1055] At least one active compound in combination with a composition comprising the ionic liquid choline and geranate (CAGE) for oral delivery, mucosal delivery, parenteral delivery, or use in treating disease. [This invention 1056] The combination of claim 1055, wherein the mucosa is nasal mucosa, oral mucosa, or vaginal mucosa. [This invention 1057] The combination of 1055 of the present invention, wherein parenteral administration comprises delivery to a tumor. [This invention 1058] 1055. A combination of the present invention wherein the treatment comprises injection of said composition into the affected tissue. [This invention 1059] The combination of the present invention 1058, wherein the disease is cancer, adipose, hyperplasia, or any other disease caused by tissue proliferation. [The present invention 1060] Any of the combinations of 1055 to 1059, wherein the CAGE is at a concentration of at least 0.1% w / v or 5% w / w. [The present invention 1061] The combination of any of claims 1055 to 1059, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. [The present invention 1062] The combination of any of claims 1055 to 1061, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. [This invention 1063] The combination of any of claims 1055 to 1061, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:4. [The present invention 1064] Any of the combinations of 1055 to 1063, wherein the anion of the ionic liquid comprises geranate and / or geranic acid. [This invention 1065] Any of the combinations of 1055 to 1064, wherein the active compound in combination with CAGE is administered once. [The present invention 1066] Any of the combinations of 1055 to 1064, wherein the active compound in combination with CAGE is administered in multiple doses. [This invention 1067] 1067. The combination of any one of claims 1055 to 1066, wherein the active compound comprises a nucleic acid molecule. [The present invention 1068] Any of the combinations of 1055 to 1066, wherein the active compound comprises a small molecule. [The present invention 1069] 1067. The combination of any one of claims 1055 to 1066, wherein the active compound comprises a polypeptide. [The present invention 1070] The combination of any of claims 1055 to 1066, wherein the active compound comprises an antibody or antibody reagent. [This invention 1071] Any of the combinations of 1055 to 1066, wherein the active compound comprises a chemotherapeutic compound. [This invention 1072] Any of the combinations of claims 1055 to 1066, wherein the active compound comprises insulin. [This invention 1073] 1067. The combination of any of claims 1055 to 1066, wherein the active compound comprises a GLP-1 polypeptide or a mimetic or analog thereof. [This invention 1074] A combination of 1073 according to the invention, wherein the active compound is provided in a dosage of 1 to 20 mg / kg. [This invention 1075] A method for treating obesity, preventing weight gain, or reducing the weight of a subject, comprising orally administering to the subject a composition comprising the ionic liquid choline and geranate (CAGE). [This invention 1076] 1075. The method of claim 1075, wherein the CAGE is at a concentration of at least 0.1% w / v. [This invention 1077] The method of any of claims 1075-1076, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. [This invention 1078] The method of any of claims 1075-1077, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. [This invention 1079] The method of any one of claims 1075 to 1078, wherein the anion of the ionic liquid comprises geranate and / or geranic acid. [The present invention 1080] The method of any one of claims 1075 to 1079, wherein the composition further comprises an active compound. [This invention 1081] The method of claim 1080, wherein the active compound is therapeutically effective in treating obesity. [This invention 1082] The method of claim 1080, wherein the active compound is therapeutically effective in treating an obesity-related disorder. [This invention 1083] The method of any of claims 1080 to 1082, wherein the active compound comprises a small molecule. [This invention 1084] The method of any of claims 1080 to 1082, wherein the active compound comprises a polypeptide. [This invention 1085] The method of any of claims 1080 to 1082, wherein the active compound comprises an antibody or antibody reagent. [The present invention 1086] A composition comprising the ionic liquid choline and geranate (CAGE), for use in a method for treating obesity, preventing weight gain, or reducing weight in a subject, the composition being orally administered to a subject. [This invention 1087] 1086. The composition of claim 1086, wherein the CAGE is at a concentration of at least 0.1% w / v. [This invention 1088] 8. The composition of any of claims 1086-1087, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. [This invention 1089] 8. The composition of any of claims 1086-1087, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. [The present invention 1090] 1089. The composition of any one of claims 1086 to 1089, wherein the anion of the ionic liquid comprises geranate and / or geranic acid. [This invention 1091] The composition of any one of claims 1086 to 1090, further comprising an active compound. [This invention 1092] The composition of invention 1091, wherein the active compound is therapeutically effective in treating obesity. [This invention 1093] The composition of the present invention 1091, wherein the active compound has a therapeutic effect in treating an obesity-related disorder. [This invention 1094] The composition of any of claims 1091 to 1093, wherein the active compound comprises a small molecule. [This invention 1095] 14. The composition of any one of claims 1091 to 1093, wherein the active compound comprises a polypeptide. [This invention 1096] The composition of any of claims 1091 to 1093, wherein the active compound comprises an antibody or antibody reagent. [This invention 1097] A method of delivering at least one active compound comprising administering the active compound in combination with a salt-containing composition, wherein the salt is present at a concentration of at least 0.05M. [This invention 1098] 1097. The method of claim 10, wherein the salt is an ionic liquid. [This invention 1099] The method of claim 1098, wherein the ionic liquid is choline and geranate (CAGE). [The present invention 1100] The method of claim 1099, wherein the cation is choline. [The present invention 1101] 110. The method of any one of claims 1099 to 1100, wherein the anion is geranate or geranic acid. [The present invention 1102] The method of any of claims 1097 to 1101, wherein delivery is oral, subcutaneous, intradermal, intravenous, parenteral, or mucosal. [The present invention 1103] The method of any of claims 1097 to 1102, wherein the delivery is oral. [The present invention 1104] 1104. The method of any of claims 1097 to 1103, wherein the salt is present at a concentration of at least 0.05M, 0.1M, 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M, or higher. [This invention 1105] The method of any of claims 1097 to 1104, wherein the salt is present at a concentration of from about 0.05M to about 4M. [The present invention 1106] 16. The method of any one of claims 1097 to 1105, wherein the salt dissolves after administration. [This invention 1107] 1106. The method of any one of claims 1097 to 1105, wherein the salt is a pure liquid or an anhydrous liquid. [This invention 1108] 1106. The method of any one of claims 1097 to 1105, wherein the salt is in an aqueous solution. [This invention 1109] The method of any one of claims 1097 to 1108, wherein the administration is to a subject. [The present invention 1110] The method of any of claims 1097 to 1108, wherein the administering comprises contacting the cells and / or tissues. [The present invention 1111] The method of any of claims 1097-1110, wherein the active compound comprises a nucleic acid molecule, a chemotherapeutic compound, a small molecule, a peptide, and / or an antibody or antibody reagent. [The present invention 1112] The method of claim 1111, wherein the active compound is a component of a salt. [The present invention 1113] The method of claim 1111, wherein the active compound comprises insulin or a GLP-1 polypeptide or a mimetic or analog thereof. [This invention 1114] a. a salt present at a concentration of at least 0.05M; and b. Active compounds A composition comprising: [This invention 1115] 1114. The composition of claim 1114, wherein the salt is an ionic liquid. [The present invention 1116] The composition of claim 1115, wherein the ionic liquid is choline and geranate (CAGE). [This invention 1117] 1116. The composition of claim 1116, wherein the cation is choline. [This invention 1118] The composition of claim 1116 or 1117, wherein the anion is geranate or geranic acid. [This invention 1119] 1114. The composition of any of claims 1114-1118, wherein the salt is present in a concentration of at least 0.05M, 0.1M, 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M, or higher. [The present invention 1120] 1119. The composition of any of claims 1114 to 1119, wherein the salt is present in a concentration of from about 0.05M to about 4M. [This invention 1121] The composition of any one of claims 1114 to 1120, wherein the salt is a pure liquid or an anhydrous liquid. [This invention 1122] The composition of any one of claims 1114 to 1120, wherein the salt is in the form of an aqueous solution. [This invention 1123] The composition of any of claims 1114 to 1121, wherein the active compound comprises a nucleic acid molecule, a chemotherapeutic compound, a small molecule, a peptide, and / or an antibody or antibody reagent. [This invention 1124] 1123. The composition of the present invention, wherein the active compound is a component of a salt. [This invention 1125] The composition of the present invention 1123, wherein the active compound comprises an insulin or GLP-1 polypeptide or a mimetic or analog thereof. [Invention 1126] Any of the compositions of claims 1114 to 1125 for use in a delivery method or a treatment method. [This invention 1127] 17. The method or composition of any of claims 1097 to 1126, wherein the biological activity of the active compound is improved or stabilized compared to the activity in the absence of the salt. [This invention 1128] The method or composition of any of claims 1097 to 1127, wherein the combination of active compound and salt is an admixture. [This invention 1129] The method or composition of any of claims 1097 to 1128, wherein the combination of active compound and salt comprises nanoparticles comprising the active compound, the nanoparticles being in solution or suspension in a composition comprising the salt. [The present invention 1130] A method of treating diabetes, an ulcer, cancer, or fibrosis in a subject in need thereof, comprising administering to the subject a composition comprising the ionic liquid choline and geranate (CAGE) and no additional therapeutically active agent. [This invention 1131] The method of claim 1130, wherein the administration is via injection. [This invention 1132] The method of claim 1130, wherein the administration is oral. [This invention 1133] 1133. The method of any of claims 1130 to 1132, wherein the CAGE is at a concentration of at least 0.1% w / v. [This invention 1134] The method of any of claims 1130-1133, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. [This invention 1135] The method of any of claims 1130 to 1133, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. [This invention 1136] The method of any of claims 1130 to 1133, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:4. [This invention 1137] The method of any one of claims 1130 to 1136, wherein the anion of the ionic liquid comprises geranate and / or geranic acid. [This invention 1138] A composition comprising the ionic liquid choline and geranate (CAGE) and no further therapeutically active agent, for use in a method for treating diabetes, an ulcer, cancer, or fibrosis in a subject in need thereof. [This invention 1139] The composition of the present invention 1138, wherein administration is via injection. [The present invention 1140] The composition of the present invention 1138, wherein the administration is oral. [This invention 1141] 1138-1140. Any of the compositions of claims 1138-1140, wherein the CAGE is at a concentration of at least 0.1% w / v. [This invention 1142] 1138-1141. The composition of any of claims 1138-1141, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. [This invention 1143] 1138-1141. The composition of any of claims 1138-1141, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. [This invention 1144] 1138-1141. The composition of any of claims 1138-1141, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:4. [Invention 1145] The composition of any one of claims 1138 to 1144, wherein the anion of the ionic liquid comprises geranate and / or geranic acid. [Brief explanation of the drawings]

[0030] [Figure 1] Enhancement of FITC-insulin transport across Caco-2 monolayers in the presence of CAGE. Data presented as mean ± SE (n=6) (* p<0.01, ** p<0.001). [Figure 2] Figures 2A-2B show representative confocal micrograph images of transwell membranes covered with a monolayer of Caco-2 cells and incubated for 5 h with FITC-insulin dispersed in saline (Figure 2A) and FITC-insulin dispersed in 10 mM CAGE (Figure 2B). Images were taken at 60x magnification. The images show the overlap of DAPI-labeled nuclei (blue) and FITC-insulin (green). [Figure 3] Figure 1 shows the effect on tight junction integrity in Caco-2 cells upon treatment with CAGE. Data presented as mean ± SE (n=6); (* p<0.01, ** p<0.001). [Figure 4] Figure 1 shows the efficacy of insulin-CAGE in lowering blood glucose levels when administered intrajejunally in non-diabetic rats. Data are presented as mean ± SE (n=6). Significantly greater (p<0.05) efficacy was noted in 5 U / kg insulin-CAGE-treated rats compared to subcutaneous administration of 2 U / kg insulin at 2-3.5 and 5 hours (denoted by *). [Figure 5] Figure 1 shows the effectiveness of CAGE in enhancing the oral bioavailability of insulin. Data presented as mean ± SE (n=4). [Figure 6] Photograph of a CAGE placed inside an elongated size 9 capsule for oral administration is shown. [Figure 7] Figure 1 shows the in vivo efficacy of insulin-CAGE administered orally in capsules. Data are presented as mean ± SE (n=6). Significantly greater (p<0.05) efficacy of CAGE-insulin was observed at various time points (indicated by *) compared to subcutaneously administered insulin. [Figure 8] Figures 8A-8E show hematoxylin and eosin-stained micrographs of small intestinal tissue sections. (Figure 8A) Neat CAGE administered intrajejunally; (Figure 8B) Saline administered intrajejunally; (Figure 8C) Oral CAGE administration; (Figure 8D) Oral insulin-saline administration; (Figure 8E) Oral insulin-CAGE administration. Scale bar: 200 μm. Insets show the mucosal surface and 50 μm scale bar. [Figure 9] Circular dichroism spectra of insulin isolated from CAGE at different months. Insulin was dispersed in CAGE and stored at RT (25°C) or refrigerated at 4°C for up to 4 months. [Figure 10] Figure 1 shows the efficacy of insulin isolated from CAGE in reducing blood glucose levels at different time points in non-diabetic rats. At 1 and 2 hours after administration (indicated by *), a statistically significant difference (p<0.05) in bioactivity was noted between freshly prepared insulin and insulin-CAGE stored at RT (25°C) for 3 months. All data presented as mean ± SE (n=6). [Figure 11] Left: Effect of camptothecin (CPT) solubilized in CAGE on 4T1 cells; Right: Effect of paclitaxel solubilized in CAGE on 4T1 cells. Both graphs show that drugs solubilized in CAGE are as effective as those solubilized in DMSO. [Figure 12] Bioactivity of insulin (2 U / kg) delivered subcutaneously from CAGE (blue) and control (2 U / kg in saline, orange) and CAGE alone (green) is shown. Compared to standard insulin-saline injections, insulin in CAGE resulted in a substantially longer duration of hypoglycemia. [Figure 13]Figure 1 shows the bioactivity of insulin stored in CAGE at room temperature (RT) and 4° C. Bioactivity was maintained even after 2 months of storage at room temperature. [Figure 14] Figure 1 shows the effect of CAGE on dextran distribution in tissue. Compared to saline (leftmost panel), the addition of CAGE to the formulation enhances dextran distribution in the skin. The magnitude of enhancement is quantified in the table in Example 2. CAGE alone enhanced dextran distribution between 2.6 and 5.5 fold. [Figure 15] Circular dichroism spectra of insulin isolated from CAGE at different months are shown. Insulin was dispersed in CAGE and stored at room temperature (25°C) or refrigerated at 4°C for up to 4 months. The secondary conformation of the insulin alpha helix was maintained in CAGE for extended periods. [Figure 16] Figures 16A-16B show the efficacy of insulin-CAGE in lowering blood glucose levels when administered intrajejunally in nondiabetic rats. Figure 16A shows the efficacy of various formulations after normalization for blood glucose changes associated with the fasting effect. Animals injected with saline alone were considered the fasting group, and data were plotted after subtracting the blood glucose values ​​obtained from the saline group. Figure 16B shows blood glucose changes compared to initial levels without normalization to saline blood glucose levels. All data are expressed as mean ± standard error (SE) (n=6). Compared to subcutaneous administration of 2 U / kg insulin, significantly greater efficacy (p<0.05) was noted in 5 U / kg insulin-CAGE-treated rats at various time points of the study (denoted by *). [Figure 17] Figure 1 shows the effectiveness of CAGE in enhancing the oral bioavailability of insulin. Data presented as mean ± SE (n=4). [Figure 18]Figures 18A-18B show the in vivo efficacy of insulin-CAGE administered orally in capsules. Figure 18A shows the efficacy of various formulations after normalization for blood glucose changes associated with the fasting effect. Animals injected with empty capsules were considered the fasted group, and data were plotted after subtracting the blood glucose changes obtained from the empty capsule group. Figure 18B shows blood glucose changes compared to initial levels without normalization to blood glucose levels from empty capsules. All data are expressed as mean ± SE (n = 6). Significantly higher (p < 0.05) efficacy of CAGE-insulin was observed at various time points (denoted by *) compared to subcutaneously administered insulin. [Figure 19] Hematoxylin and eosin-stained micrographs of small intestinal tissue sections are shown. Sections were taken after intrajejunal administration of CAGE alone or saline; oral administration of CAGE alone, insulin-saline, or insulin-CAGE capsules; and repeated daily administration of insulin, CAGE, or insulin-CAGE capsules for 7 days. Scale bar: 200 μm. Inset shows the mucosal surface. Scale bar: 50 μm. [Figure 20] Figure 1 shows the blood glucose lowering efficacy of sequential administration of a CAGE-only capsule followed 0.5 hours later by a 10 U / kg insulin capsule. No significant difference in efficacy was observed between the 10 U / kg insulin solution and sequential capsule administration of CAGE and 10 U / kg insulin. Data presented as mean ± SE (n=6). [Figure 21] Figure 1 shows the reduction of mucus viscosity by CAGE. Average viscosity values ​​in cP at a shear rate of 50.12 1 / s are shown for simulated mucus without CAGE (0%), 1, and 5% w / v CAGE. Data presented as mean ± SE (n=3); (*p<0.001, CAGE treatment compared to no CAGE treatment). [Figure 22]Confocal images using GFP-plasmid are shown. P3000™ Reagent aids in efficient delivery of the delivery vehicle-conjugated plasmid to the nucleus, helping to improve transfection efficiency. P3000 is most likely a small molecule, not a liposome. Transfections were performed for 3 days before a complete medium change. [Figure 23] Concentration dependence is shown: CAGE (1:2) + P3000. % refers to CAGE concentration. The concentration of P3000 (enhancer reagent) was kept constant at 0.09% v / v. Transfection was performed for 3 days before a complete medium change. [Figure 24] The effect of CAGE composition is shown: 0.9% concentration. Maximal transfection occurred in the presence of excess geranic acid (1:2 and 1:4). P3000 enhancer concentration was kept constant at 0.09% v / v. Transfection was performed with IL+ plasmid for 1 day before a complete medium change. [Figure 25] Confocal images using GFP-plasmid are shown: Control. Transfection was carried out for 3 days before a complete medium change. [Figure 26] Wide-angle X-ray scattering patterns at room temperature for pure CAGE-1:1 (black solid line), CAGE-1:2 (gray solid line), CAGE-1:3 (black dashed line), and CAGE-1:4 (gray dashed line). The position of the pre-peak (low q peak) is affected by the molar ratio of geranic acid in CAGE, but the position of the adjacent peak (high q peak) is not. [Figure 27A]Figures 27A-27D show wide-angle X-ray scattering patterns showing hydration-induced nanostructural transitions at room temperature for (Figure 27A) CAGE-1:1, (Figure 27B) -1:2, (Figure 27C) -1:3, and (Figure 27D) -1:4. The position and intensity of the pre-peak (low q peak) and adjacent peaks are affected by the amount of water and the molar ratio of geranic acid in the CAGE. With higher molar ratios of geranic acid, such as -1:3 and -1:4, the nanostructure became more ordered, as reflected by the transition from a broad peak to a sharp peak. Black thick line: pure CAGE; gray solid line: 75% w / w CAGE; black thin line: 50% w / w CAGE; gray / thin dashed line: 25% w / w CAGE; and black / wide dashed line: 5% w / w CAGE. [Figure 27B] Figures 27A-27D show wide-angle X-ray scattering patterns showing hydration-induced nanostructural transitions at room temperature for (Figure 27A) CAGE-1:1, (Figure 27B) -1:2, (Figure 27C) -1:3, and (Figure 27D) -1:4. The position and intensity of the pre-peak (low q peak) and adjacent peaks are affected by the amount of water and the molar ratio of geranic acid in the CAGE. With higher molar ratios of geranic acid, such as -1:3 and -1:4, the nanostructure became more ordered, as reflected by the transition from a broad peak to a sharp peak. Black thick line: pure CAGE; gray solid line: 75% w / w CAGE; black thin line: 50% w / w CAGE; gray / thin dashed line: 25% w / w CAGE; and black / wide dashed line: 5% w / w CAGE. [Figure 27C]Figures 27A-27D show wide-angle X-ray scattering patterns showing hydration-induced nanostructural transitions at room temperature for (Figure 27A) CAGE-1:1, (Figure 27B) -1:2, (Figure 27C) -1:3, and (Figure 27D) -1:4. The position and intensity of the pre-peak (low q peak) and adjacent peaks are affected by the amount of water and the molar ratio of geranic acid in the CAGE. With higher molar ratios of geranic acid, such as -1:3 and -1:4, the nanostructure became more ordered, as reflected by the transition from a broad peak to a sharp peak. Black thick line: pure CAGE; gray solid line: 75% w / w CAGE; black thin line: 50% w / w CAGE; gray / thin dashed line: 25% w / w CAGE; and black / wide dashed line: 5% w / w CAGE. [Figure 27D] Figures 27A-27D show wide-angle X-ray scattering patterns showing hydration-induced nanostructural transitions at room temperature for (Figure 27A) CAGE-1:1, (Figure 27B) -1:2, (Figure 27C) -1:3, and (Figure 27D) -1:4. The position and intensity of the pre-peak (low q peak) and adjacent peaks are affected by the amount of water and the molar ratio of geranic acid in the CAGE. With higher molar ratios of geranic acid, such as -1:3 and -1:4, the nanostructure became more ordered, as reflected by the transition from a broad peak to a sharp peak. Black thick line: pure CAGE; gray solid line: 75% w / w CAGE; black thin line: 50% w / w CAGE; gray / thin dashed line: 25% w / w CAGE; and black / wide dashed line: 5% w / w CAGE. [Figure 28] Representative transitions in the rheological behavior of pure and 50% hydrated CAGE-1:2 and -1:4 at 25 °C and a shear rate of 1 s are shown. The viscosity of pure CAGE-1:2 decreased with the addition of 50% water, while the viscosity of pure -1:4 increased. Dark gray: pure; and light gray: hydrated. Inset: Photographs of less viscous -1:2 (50% HO, w / w) and gel-like -1:4 (50% HO, w / w). [Figure 29]Figure 29A shows a graph of the Newtonian properties of pure CAGE-1:4 (blue plot) and the non-Newtonian properties of 50% hydrated-1:4 (red plot). Figure 29B shows a graph of the shear thinning behavior of hydrated 50% hydrated-1:4. Dark grey: pure-1:4; light grey: 50% hydrated-1:4. Rheological experiments were performed at 25°C. [Figure 30] Figures 30A-30D show plots of the ratio of the first (I1) and third (I3) vibronic bands of pyrene emission versus concentration for (Figure 30A) CAGE-1:1, (Figure 30B) -1:2, (Figure 30C) -1:3, and (Figure 30D) -1:4, which correspond to steeper slopes. CAGE-1:3 and -1:4 exhibited two CMCs, likely due to their self-assembly into two distinct nanostructures. [Figure 31] Figures 31A-31F show cryo-TEM micrographs of DES nanostructures in water. Top panel: (Figure 31A) 5% w / w CAGE-1:2; (Figure 31B) 5% w / w CAGE-1:3; (Figure 31C) 5% w / w CAGE-1:4 oil-in-water microemulsions; (Figure 31D) 20% w / w CAGE-1:2 micelles; (Figure 31E) 20% CAGE-1:3 vesicles; and (Figure 31F) CAGE-1:4 vesicles. [Figure 32] Figure 1 shows graphs of PK and PD of semaglutide administered in CAGE. n=3, mean + / - SEM. The TO time point was positive for semaglutide via EIA kit and these TO values ​​have been subtracted in the dashed data set. [Figure 33] Enhancement of Lucifer Yellow transport across Caco-2 monolayers in the presence of different concentrations of CAGE. Data presented as mean ± SE (n=4) (* p<0.05, ** p<0.001, *** p<0.0001; all CAGE treatments compared to no CAGE treatment). [Figure 34] Figure 1 shows the effect of various concentrations of CAGE on the passive transcytosis of coumarin-6. Data are presented as mean ± SE (n = 4); (* p < 0.05; ** p < 0.0001; no CAGE treatment compared to 10 mM CAGE treatment group). [Figure 35] Figure 1 shows the viability of Caco-2 cells upon treatment with various concentrations of CAGE. After 5 hours, CAGE was removed from the culture medium and replaced with fresh DMEM. Cell viability was measured after an additional 19 hours (24 hours total) using the MTT assay. Data presented as mean ± SE (n=4). [Figure 36] Representative confocal micrograph images of transwell membranes covered with a layer of Caco-2 cells and incubated for 5 hours with various concentrations of FITC-insulin dispersed in CAGE or PPS are shown. Images were taken at 40x magnification. The images show DAPI-labeled nuclei (blue), FITC-insulin (green), and overlap of DAPI staining with FITC-insulin. [Figure 37] Figure 1 shows enhanced FITC-insulin transport across Caco-2 monolayers in the presence of CAGE. Data presented as mean ± SE (n=4) (* p<0.05, ** p<0.001; CAGE treatment compared to no CAGE treatment). [Figure 38] Enhancement of FITC-dextran transport across Caco-2 monolayers in the presence of different concentrations of CAGE. Data presented as mean ± SE (n=4) (*p<0.01, **p<0.0001; CAGE treatment compared to no CAGE treatment). [Figure 39] Figure 1 shows the effect of treatment with various concentrations of CAGE or 10 mM sodium caprate on tight junction integrity in Caco-2 cells. Data presented as mean ± SE (n=4); (* p<0.05; ** p<0.0001; all CAGE treatments compared to no CAGE treatment). [Figure 40] Figure 1 shows the enhanced stability of insulin against trypsin digestion by CAGE. Data presented as mean ± SE (n=3); (* p<0.01; ** p<0.001; insulin-PBS treatment compared to insulin-CAGE treatment). DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description As described herein, the inventors have demonstrated that the ionic liquid CAGE (choline and geranate or geranic acid) is not only safe for oral and / or parenteral use, but also significantly improves the delivery of active compounds contained in the CAGE solution without the negative side effects typically observed with most solvents. Indeed, because solvents as a class pose a common problem of toxicity, "solvent exposure" is a widely used medical term meant to encompass the risk of contact with one or more solvents. Solvent exposure in general, as well as exposure to select individual solvents, has been shown to contribute to the pathogenesis of numerous disease states. In light of this, the safety profile of CAGE demonstrated herein, particularly via oral and parenteral routes of administration, which bypass many of the body's natural defenses, is particularly surprising and unexpected.

[0032] Thus, in one aspect of any embodiment, described herein is a method for oral delivery of at least one active compound, comprising orally administering a composition comprising the active compound in combination with the ionic liquid choline and geranate (CAGE).

[0033] Oral administration may include providing a tablet (including, but not limited to, a scored or coated tablet), a pill, a caplet, a capsule, a chewable tablet, a powder packet, a cachet, a troche, a wafer, an aerosol spray, or a liquid such as, but not limited to, a syrup, an elixir, a solution or suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Oral formulations may include individual dosage forms such as, but not limited to, a tablet (including, but not limited to, a scored or coated tablet), a pill, a caplet, a capsule, a chewable tablet, a powder packet, a cachet, a troche, a wafer, an aerosol spray, or a liquid such as, but not limited to, a syrup, an elixir, a solution or suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of CAGE and at least one active compound and may be prepared by pharmaceutical methods well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia, PA. (2005).

[0034] In one aspect of any embodiment, described herein is a method for treating diabetes comprising orally administering an oral formulation of insulin in combination with CAGE. In one aspect of any embodiment, described herein is a method for treating diabetes comprising orally administering an oral formulation of a GLP-1 polypeptide or mimetic / analog thereof in combination with CAGE.

[0035] It is noted herein that oral and topical administration differ significantly, for example, in the characteristics of the cell layers that must be traversed to allow the active compound to penetrate the interior of the organism upon administration. In the skin, the first layer that an administered composition traverses is the epidermis, which consists of stratified squamous epithelium and then the basal layer. The epidermis has at least five layers, with the most commonly found cell types being Merkel cells, keratinocytes, malanocytes, and Langerhans cells. Notably, the outermost surface of the skin contains a layer of dead cells 25–30 cells deep. Beneath the epidermis is the dermis, which contains connective tissue, blood vessels, and various glands. In contrast, with oral administration, the composition immediately contacts the intestinal epithelium, which is a single layer of simple columnar epithelial cells with two thin underlying layers. Furthermore, while the intestinal epithelium forms tight junctions to provide impermeability, the skin contains keratin to form an external barrier to the organism. Thus, the problem of passing through each of these tissues without causing undue damage is significantly greater, with tissues encountered during oral administration clearly being much more susceptible to irritation and toxicity.

[0036] As described elsewhere herein, CAGEs can safely transport active compounds across sensitive membranes encountered during oral administration, as well as safely deliver active compounds across other sensitive internal tissues.

[0037] As described in the examples herein, CAGE provides both protease inhibitory activity and mucolytic activity.Therefore, it is particularly suitable as a delivery vehicle to / across mucosa.In one aspect of any embodiment, described herein is a method for delivering at least one active compound, comprising administering an active compound combined with CAGE to mucosa, for example, nasal mucosa, oral mucosa, or vaginal mucosa.

[0038] CAGE also solubilizes the drug, allowing for high-concentration injections. CAGE also diffuses into tissues at a faster rate than traditional solubilizing carriers such as EtOH. In some embodiments, without wishing to be bound by theory, CAGE can also diffuse into tissues, allowing for local precipitation of the drug. The precipitated drug can form a depot and exhibit sustained release.

[0039] In another embodiment, the drug may remain soluble in the CAGE even after local diffusion of the CAGE, leading to rapid and enhanced delivery to the circulation. Subcutaneous delivery often requires multiple doses or controlled-release formulations. Such approaches are not necessarily implemented due to any concerns about timing per se, but rather to ensure that the full dose is received before the active compound is degraded. Bolus administration is often ineffective because the active compound is degraded or metabolized before the full amount can be effective. Thanks to the ability of CAGE to stabilize the active compound, such approaches are unnecessary, i.e., the desired total amount can be delivered as a single dose without the need for a release control mechanism.

[0040] Thus, in one aspect of any embodiment, described herein is a method of delivering at least one active compound by subcutaneous, intradermal, or intravenous administration, comprising administering the active compound in combination with a CAGE. In some embodiments of any aspect, the subcutaneous, intradermal, or intravenous administration comprises administration via injection, catheter, port, etc.

[0041] The inventors further demonstrated that CAGE produces a cytotoxic effect at higher concentrations, killing more than 50% of cells exposed to CAGE. This is particularly useful for applications where the active compound itself is designed to be cytotoxic or cytostatic, or where a disease can be treated by inhibiting the growth of one or more cell types (e.g., cancer). The concentration of CAGE suitable for cytotoxicity may vary depending, for example, on the type of target tissue, the stoichiometry of the CAGE used, the amount of composition being administered, and / or the degree of the desired effect.

[0042] Thus, in one aspect of any embodiment, described herein is a method for parenteral delivery of at least one active compound, comprising parenterally administering the active compound in combination with CAGE. In some embodiments, the parenteral administration comprises delivery to a tumor, e.g., a cancer tumor.

[0043] In some embodiments, the composition comprising CAGE in combination with at least one active compound as described herein can be in a parenteral dosage form. Because administration of a parenteral dosage form typically bypasses the patient's natural defenses against contaminants, the parenteral dosage form is preferably sterile or can be sterilized before administration to the patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable injection vehicle, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration to the patient, including, but not limited to, DUROS®-type dosage forms and dose dumping.

[0044] Suitable vehicles that can be used to provide parenteral dosage forms of compositions comprising CAGE in combination with at least one active compound as disclosed herein are well known to those skilled in the art. Examples include, but are not limited to, sterile water; water for injection, USP; saline solution; glucose solution; aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of components in the compositions as disclosed herein, including conventional and controlled-release parenteral dosage forms, can also be incorporated into the parenteral dosage forms of the present disclosure.

[0045] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, the use of conventional dosage forms can lead to wide fluctuations in drug concentrations in a patient's blood and other tissues. These fluctuations can affect many parameters, such as dosing frequency, onset of action, duration of effectiveness, maintenance of therapeutic blood levels, toxicity, side effects, and the like. As noted hereinabove, compositions comprising CAGE in combination with at least one active compound can obviate certain reasons for using controlled-release formulations, but in some embodiments, it is contemplated herein that methods and compositions can be utilized in controlled-release formulations. For example, controlled-release formulations can be used to control the drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or sustained-release dosage forms or formulations can be used to ensure that maximum drug efficacy is achieved while minimizing potential adverse effects and safety concerns that may result from both underdosing (i.e., below the minimum therapeutic level) and exceeding toxic drug levels. In some embodiments, compositions comprising CAGE in combination with at least one active compound can be administered in a sustained release formulation.

[0046] Controlled-release pharmaceutical products share a common goal: improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of optimally designed controlled-release preparations in medical treatment is characterized by the use of a minimum amount of drug substance to cure or control a medical condition in a minimal amount of time. Advantages of controlled-release formulations include: 1) extended drug activity; 2) reduced dosing frequency; 3) improved patient compliance; 4) less total drug use; 5) reduced local or systemic side effects; 6) minimized drug accumulation; 7) reduced blood level fluctuations; 8) improved therapeutic efficacy; 9) reduced synergism or loss of drug activity; and 10) improved rate of disease or condition control. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0047] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that immediately produces the desired therapeutic effect, and then gradually and continuously release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. To maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that replaces the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions, including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0048] Various known controlled-release or sustained-release dosage forms, formulations and devices can be adapted for use with the salts and compositions of the present disclosure.Examples include but are not limited to those described in U.S. Patent No. 3,845,770; U.S. Patent No. 3,916,899; U.S. Patent No. 3,536,809; U.S. Patent No. 3,598,123; U.S. Patent No. 4,008,719; U.S. Patent No. 5,674,533; U.S. Patent No. 5,059,595; U.S. Patent No. 5,591,767; U.S. Patent No. 5,120,548; U.S. Patent No. 5,073,543; U.S. Patent No. 5,639,476; U.S. Patent No. 5,354,556; U.S. Patent No. 5,733,566; and U.S. Patent No. 6,365,185 B1, each of which is incorporated herein by reference. These dosage forms can be used to provide sustained or controlled release of one or more active ingredients using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or combinations thereof to provide desired release profiles at various rates.

[0049] In one aspect of any embodiment, described herein is a method of treating a disease in a subject in need thereof by administering to the subject an active compound in combination with CAGE by injection into the diseased tissue. In some embodiments, the diseased tissue is a tissue containing diseased cells. In some embodiments, the diseased tissue is a tissue that exhibits symptoms of a disease. Non-limiting examples of suitable diseased tissue include tumor tissue, fat tissue, warts, adipose tissue, etc.

[0050] In some embodiments of any aspect, the disease is caused by tissue proliferation, for example, unwanted, abnormal or pathological tissue proliferation.The disease caused by tissue proliferation can be any disease caused by or characterized by the rate of tissue proliferation, the location of tissue proliferation, or the pattern / structure of tissue proliferation that is different from the normal one for the type of tissue in healthy subjects.Non-limiting examples of such diseases are tumor, cancer, fat / obesity, wart, and / or hyperplasia.

[0051] As used herein, the term "ionic liquid (IL)" refers to an organic salt or mixture of organic salts that are in a liquid state at room temperature. This class of solvents has been shown to be useful in a variety of fields, including industrial processing, catalysis, medicine, and electrochemistry. Ionic liquids contain at least one anionic component and at least one cationic component. Ionic liquids can contain additional hydrogen bond donors (i.e., any molecule capable of providing an -OH or -NH group), examples of which include, but are not limited to, alcohols, fatty acids, and amines. The at least one anionic component and the at least one cationic component can be present in any molar ratio. Exemplary molar ratios (cation:anion) include, but are not limited to, 1:1, 1:2, 2:1, 1:3, 3:1, 2:3, 3:2, and ranges between these ratios. For further discussion of ionic liquids, see, e.g., Hough, et al., "The third evolution of ionic liquids: active pharmaceutical ingredients", New Journal of Chemistry, 31: 1429 (2007) and Xu, et al., "Ionic Liquids: Ion Mobilities, Glass Temperatures, and Fragilities", Journal of Physical Chemistry B, 107(25): 6170-6178 (2003), each of which is incorporated by reference in its entirety. In some embodiments of any aspect, the ionic liquid or solvent exists as a liquid below 100°C. In some embodiments of any aspect, the ionic liquid or solvent exists as a liquid at room temperature.

[0052] In some embodiments of any aspect, the ionic liquid described herein is CAGE (Choline and Geranate). CAGE is an ionic liquid containing the cation choline (see, e.g., Structure I) and the anion geranate or geranic acid (see, e.g., Structures II and III). Preparation of CAGE can be as described, for example, in International Patent Publication WO 2015 / 066647, which is incorporated herein by reference in its entirety, or as described in the Examples herein. TIFF2026012396000002.tif21379

[0053] In some embodiments of any aspect, the anion of the ionic liquid comprises geranate and / or geranic acid. In some embodiments of any aspect, the anion of the ionic liquid comprises geranate. In some embodiments of any aspect, the anion of the ionic liquid comprises geranic acid.

[0054] In some embodiments of either aspect, CAGE is at a concentration of at least 0.01% w / v. In some embodiments of either aspect, CAGE is at a concentration of at least 0.05% w / v. In some embodiments of either aspect, CAGE is at a concentration of at least 0.1% w / v. In some embodiments of either aspect, CAGE is at a concentration of at least 0.2% w / v, at least 0.3% w / v, at least 0.4% w / v, at least 0.5% w / v, at least 1% w / v, or higher.

[0055] In some embodiments of either aspect, the CAGE is at a concentration of about 0.01% w / v to about 1% w / v. In some embodiments of either aspect, the CAGE is at a concentration of 0.01% w / v to 1% w / v. In some embodiments of either aspect, the CAGE is at a concentration of about 0.05% w / v to about 0.5% w / v. In some embodiments of either aspect, the CAGE is at a concentration of 0.05% w / v to 0.5% w / v.

[0056] In some embodiments of either aspect, the CAGE is at a concentration of at least 25% w / w. In some embodiments of either aspect, the CAGE is at a concentration of at least 25% w / w in water. In some embodiments of either aspect, the CAGE is at a concentration of at least 25% w / w in saline or a physiologically compatible buffer.

[0057] In some embodiments of either aspect, the CAGE is at a concentration of about 5% w / w to about 75% w / w. In some embodiments of either aspect, the CAGE is at a concentration of 5% w / w to 75% w / w. In some embodiments of either aspect, the CAGE is at a concentration of about 5% w / w to about 75% w / w in water, saline, or a physiologically compatible buffer. In some embodiments of either aspect, the CAGE is at a concentration of 5% w / w to 75% w / w in water, saline, or a physiologically compatible buffer.

[0058] As described herein, different structures can be achieved by varying the concentration and ratio of CAGE. Increasing the degree of hydration disrupted the native molecular and ionic clusters, leading to nanoscale rearrangements that affected polar and nonpolar domains, atomic proximity, and intra- and intermolecular interactions. At higher hydration levels (>75% HO), DES nanostructures self-assembled into vesicles, spheroidal micelles, and oil-in-water microemulsions, depending on the precursor molar ratio.

[0059] In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of about 1:4 and provides a microemulsion at a concentration of about 5% w / w. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of about 1:3 to about 1:4 and provides a vesicle at a concentration of about 20% w / w. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of about 1:2 and provides a micelle at a concentration of about 20% w / w. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of about 1:4 and provides a sol-gel at a concentration of about 50% w / w.

[0060] In some embodiments of either aspect, the CAGE is a gel or a shear-thinning Newtonian gel.

[0061] In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of at least 1:3 at a concentration of at least 25% w / w. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of at least 1:3 at a concentration of at least 25% w / w in water. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of 1:3 or 1:4 at a concentration of at least 25% w / w. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of 1:3 or 1:4 at a concentration of at least 25% w / w in water. In some embodiments of either aspect, the CAGE is a gel or a shear-thinning Newtonian gel.

[0062] In some embodiments of either aspect, the CAGE is 100% w / w or w / v.

[0063] In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of about 10:1 to about 1:10. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of 10:1 to 1:10. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of about 5:1 to about 1:5. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of 5:1 to 1:5. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of about 2:1 to about 1:4. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of 2:1 to 1:4. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of about 1:1 to about 1:4. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of 1:1 to 1:4. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of about 1:1, 1:2, 1:3, or 1:4. In some embodiments of either aspect, the CAGE has a choline:geranic acid (or geranate) ratio of 1:1, 1:2, 1:3, or 1:4. The figures show data collected with a 1:2 ratio CAGE, except for FIG. 11, which utilized a 1:1 ratio CAGE. Without wishing to be bound by theory, compositions with higher ratios of geranic acid and / or geranate exhibit greater hydrophobicity and toxicity, while compositions with higher ratios of choline exhibit greater hydrophilicity and are more inactive. In some embodiments of either aspect, compositions with higher ratios of geranic acid and / or geranate exhibit greater hydrophobicity, while compositions with higher ratios of choline exhibit greater hydrophilicity.

[0064] In some embodiments of either aspect, for example when one or more nucleic acid molecules are provided in combination with CAGE, the ratio of choline:geranic acid (or geranate) is greater than 1:1, e.g., greater than 1:2, about 1:2 to about 1:4, or 1:2 to 1:4.

[0065] In some embodiments of either aspect, the concentration of ionic liquid (eg, CAGE) in the composition or formulation is between about 0.1 mM and 20 mM. In some embodiments of either aspect, the ionic liquid (e.g., CAGE) concentration in the composition or formulation is about 0.5 mM to 20 mM, 0.5 mM to 18 mM, 0.5 mM to 16 mM, 0.5 mM to 14 mM, 0.5 mM to 12 mM, 0.5 mM to 10 mM, 0.5 mM to 8 mM, 1 mM to 20 mM, 1 mM to 18 mM, 1 mM to 16 mM, 1 mM to 14 mM, 1 mM to 12 mM, 1 mM to 10 mM, 1 mM to 8 mM, 2 mM to 20 mM, 2 mM to 18 mM, 2 mM to 16 mM, 2 mM to 14 mM, 2 mM to 12 mM, 2 mM to 10 mM, 2 mM to 8 mM, 4 mM to 20 mM, 4 mM to 18 mM, 4 mM to 16 mM, 4 mM to 14 mM, 4 mM to 12 mM, 4 mM to 10 mM, 4 mM to 8 mM, 6 mM to 20 mM, 6 mM to 18 mM, 6 mM to 18 mM, 6 mM to 10 ... mm~14 mm, 6 mm~12 mm, 6 mm~10 mm, 6 mm~8 mm, 8 mm~20 mm, 8 mm~18 mm, 8 mm~16 mm, 8 mm~14 mm, 8 mm~12 mm, 8 mm~10 mm, 10 mm~20 mm, 10 mm~18 mm, 10 mm~16 mm, 10 mm~14 mm, 10 mm~12 mm, 12 mm~20 12 mM to 18 mM, 12 mM to 16 mM, 12 mM to 14 mM, 14 mM to 20 mM, 14 mM to 18 mM, 14 mM to 16 mM, 16 mM to 20 mM, 16 mM to 18 mM, or 18 mM to 20 mM. In some embodiments of either aspect, the concentration of ionic liquid (e.g., CAGE) in the composition or formulation is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM.

[0066] As used herein, "in combination with" refers to two or more substances present in any molecular or physical arrangement in the same formulation, e.g., in an admixture, solution, mixture, suspension, colloid, emulsion. The formulation may be a homogeneous or heterogeneous mixture. In some embodiments of either aspect, the active compound may be contained with the CAGE in a solution, mixture, admixture, suspension, etc., by a superstructure, e.g., a nanoparticle, liposome, vector, cell, scaffold, etc.

[0067] As used herein, an "active compound" or "active agent" is any agent that has an effect on a target cell or target organism. The terms "compound" and "agent" refer to any entity that is not normally present or present at levels administered and / or provided to a cell, tissue, or subject. The agent can be selected from a group including: chemicals; small organic or inorganic molecules; signaling molecules; nucleic acid sequences; nucleic acid analogs; proteins; peptides; enzymes; aptamers; peptidomimetics, peptide derivatives, peptide analogs, antibodies; intrabodies; biopolymers, extracts made from biological materials such as bacterial, plant, fungal, or animal cells or tissues; natural or synthetic compositions or functional fragments thereof. In some embodiments, the agent is any chemical, entity, or moiety, including, but not limited to, synthetic and natural non-proteinaceous entities. The agent can be known to have a desired activity and / or property, or can be selected from a library of diverse compounds. Non-limiting examples of active compounds contemplated for use in the methods described herein include small molecules, polypeptides, nucleic acids, chemotherapeutic compounds, antibodies, antibody reagents, vaccines, GLP-1 polypeptides or mimetics / analogs thereof, and insulin.

[0068] As described herein, nucleic acid molecule can be vector, expression vector, inhibitory nucleic acid, aptamer, template molecule or cassette (for example, for gene editing), or targeting molecule (for example, for CRISPR-Cas technology), or any other nucleic acid molecule that is desired to be delivered to cells.Nucleic acid molecule can be RNA, DNA, or their synthetic or modified versions.

[0069] In one aspect of any of the embodiments, described herein is a method for delivering a nucleic acid molecule to a cell, comprising contacting the cell with a nucleic acid molecule combined with a composition comprising the ionic liquid choline and geranate (CAGE). In some embodiments of any of the aspects, the cell is a cell in a subject, and the contacting step comprises administering to the subject the nucleic acid molecule combined with a composition comprising the ionic liquid choline and geranate (CAGE). In some embodiments of any of the aspects, the cell is in vitro, in vivo, or ex vivo. In some embodiments of any of the aspects, the cell is a eukaryotic organism. In some embodiments of any of the aspects, the cell is a mammalian cell. In some embodiments of any of the aspects, the cell is an epithelial cell, for example, an intestinal epithelial cell.

[0070] As used herein, the term "small molecule" refers to a chemical agent that may include, but is not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds having a molecular weight of less than about 10,000 grams per mole (i.e., including heteroorganic and organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0071] In some embodiments of either aspect, the active compound can be a therapeutic compound or drug, e.g., an agent or compound that is therapeutically effective in treating at least one medical condition in a subject. Therapeutic compounds for various medical conditions are known in the art; see, for example, the database available on the World Wide Web at drugs.com or the catalog of FDA-approved compounds available on the World Wide Web at catalog.data.gov / dataset / drugsfda-database, each of which is incorporated herein by reference in its entirety.

[0072] As used herein, the term "chemotherapeutic agent" refers to any chemical or biological agent that has therapeutic utility in treating diseases characterized by abnormal cell proliferation. Such diseases include tumors, neoplasms, and cancers, as well as diseases characterized by hyperplastic growth. These agents may function to inhibit cellular activities on which cancer cells depend for continued growth. In some aspects of all embodiments, the chemotherapeutic agent is a cell cycle inhibitor or cytostatic agent. Categories of chemotherapeutic agents useful in the methods of the present invention include alkylating / alkaloid agents, antimetabolites, hormones or hormone analogs, and a wide variety of antitumor drugs. Most of these agents are directly or indirectly toxic to cancer cells. In one embodiment, the chemotherapeutic agent is a radioactive molecule.

[0073] In some embodiments of either aspect, the active compound is a hydrophobic molecule, such as estradiol, testosterone, imiquimod, corticosterone, paclitaxel, doxorubicin, cisplatin, and / or camptothecin. In some embodiments of either aspect, the active compound is a hydrophobic molecule, such as estradiol, testosterone, paclitaxel, doxorubicin, cisplatin, and / or camptothecin.

[0074] In one aspect of any embodiment, described herein are compositions comprising at least one active compound in combination with CAGE. In some embodiments, the pharmaceutical composition comprises CAGE and one or more active compounds as described herein. In some embodiments, the pharmaceutical composition consists essentially of CAGE and one or more active compounds as described herein. In some embodiments, the pharmaceutical composition consists essentially of an aqueous solution of CAGE and one or more active compounds as described herein. In some embodiments, the pharmaceutical composition consists of an aqueous solution of CAGE and one or more active compounds as described herein.

[0075] In some embodiments of any aspect, the compositions described herein, e.g., compositions comprising a CAGE and an active compound, may further comprise a pharmaceutically acceptable carrier. As used herein, the terms "pharmaceutically acceptable" and "physiologically tolerable" and their grammatical variations are used interchangeably when referring to compositions, carriers, diluents, and reagents, and indicate that the material can be administered to or on a mammal without producing undesirable physiological effects, such as nausea, dizziness, or stomach upset. A pharmaceutically acceptable carrier does not promote an immune response to the agent with which it is admixed, unless so desired. The preparation of pharmacological compositions containing active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared for injection as either a liquid solution or suspension, but may also be prepared in solid forms suitable for dissolution or suspension in liquid prior to use. Preparations may also be emulsified or presented as liposomal compositions. The active ingredient can be mixed with pharmaceutically acceptable excipients compatible with the active ingredient in amounts appropriate for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient. The therapeutic compositions of the present disclosure may contain pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, tartaric acid, mandelic acid, and the like (formed with the free amino groups of the polypeptide). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Physiologically tolerable carriers are well known in the art.Exemplary liquid carriers are sterile aqueous solutions containing no materials in addition to the active ingredient and water, or buffers such as sodium phosphate, physiological saline, or both, e.g., phosphate-buffered saline, at physiological pH values. Additionally, aqueous carriers can contain multiple buffer salts and salts such as sodium and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent used in the methods described herein that will be effective in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field. For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of the active ingredient in 0.9% sodium chloride solution.

[0076] The term "carrier" in the context of a pharmaceutical carrier refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can be formulated as a suppository, using traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co., 1990). The formulation should be appropriate for the mode of administration.

[0077] Pharmaceutically acceptable carriers and diluents include physiological saline, aqueous buffer solutions, solvents and / or dispersion media.The use of such carriers and diluents is well known in the art.Some non-limiting examples of materials that can serve as pharmaceutically acceptable carriers include:(1) sugars such as lactose, glucose, and sucrose;(2) starches such as corn starch and potato starch;(3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate;(4) powdered tragacanth;(5) malt;(6) gelatin;(7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc;(8) excipients such as cocoa butter and suppository wax;(9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (24) C2-C3 ethanol; 12 Alcohol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient," "carrier," and "pharmaceutically acceptable carrier" are used interchangeably herein. In some embodiments, the carrier inhibits degradation of the active compound. The term "pharmaceutically acceptable carrier" excludes tissue culture medium.

[0078] In some embodiments of any aspect, a composition described herein, e.g., a composition comprising a CAGE and an active compound, can be formulated as an oral, subcutaneous, intravenous, intradermal, or parenteral formulation. In some embodiments of any aspect, the oral formulation can be a degradable capsule containing a composition described herein, e.g., a composition comprising a CAGE and an active compound.

[0079] In some embodiments of any aspect described herein, the biological activity of the active compound is improved or stabilized compared to the activity in the absence of CAGE. In some embodiments of any aspect described herein, the ionic liquid (e.g., CAGE) or solvent significantly enhances the permeation of insulin across the skin compared to control insulin in the absence of the ionic liquid or solvent.

[0080] In one aspect of any embodiment, described herein is a method of administering at least an active compound to a subject using a catheter coated with a CAGE. In one aspect of any embodiment, described herein is a method of collecting bodily fluids by placing a catheter coated with a CAGE within the body.

[0081] In some embodiments, the methods described herein relate to treating a subject with or diagnosed with a medical condition with a composition described herein, e.g., comprising a CAGE and an active compound. A subject with a medical condition, such as diabetes, can be identified by a physician using current methods for diagnosing diabetes. Symptoms and / or complications of diabetes that characterize these conditions and aid in diagnosis are well known in the art and include, but are not limited to, weight loss, delayed healing, polyuria, polydipsia, polyphagia headache, itchy skin, and fatigue. For example, tests that may aid in the diagnosis of diabetes include, but are not limited to, blood tests (e.g., for fasting glucose levels). A family history of diabetes or exposure to risk factors for diabetes (e.g., being overweight) can also help determine whether a subject is likely to have diabetes or make a diagnosis of diabetes.

[0082] The compositions and methods described herein can be administered to a subject who has or has been diagnosed with a condition described herein. In some embodiments, the methods described herein include administering to a subject an effective amount of a composition described herein, such as a composition comprising CAGE and an active compound, to alleviate the symptoms of a condition described herein. As used herein, "alleviating symptoms" refers to the improvement of any marker or symptom associated with the condition. Compared to an equivalent untreated control, such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or greater, as measured by any standard technique. Various means for administering the compositions described herein to a subject are known to those skilled in the art. Such methods may include, but are not limited to, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection, or intratumoral administration. Administration may be local or systemic.

[0083] The term "effective amount" as used herein refers to the amount of a composition required to alleviate at least one or more symptoms of a disease or disorder, and relates to a sufficient amount of a pharmacological composition to produce a desired effect. Thus, the term "therapeutically effective amount" refers to an amount of a composition sufficient to produce a specific effect when administered to a typical subject. As used herein, the term "effective amount" also includes, in various contexts, an amount sufficient to delay the onset of disease symptoms, alter the course of disease symptoms (for example, but not limited to, delaying the progression of disease symptoms), or reverse disease symptoms. Therefore, it is generally not feasible to specify an exact "effective amount." However, for any given case, one skilled in the art can determine an appropriate "effective amount" using only routine experimentation.

[0084] Effective doses, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Dosages can vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. The therapeutically effective dose can be initially estimated from cell culture assays. Alternatively, doses can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of active compound that achieves half-maximal inhibition of symptoms) determined in cell culture or a suitable animal model. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dosage can be monitored by appropriate bioassays, such as blood glucose assays, among others. Dosages can be determined by a physician and can be adjusted, as necessary, to achieve the observed therapeutic effect.

[0085] As used herein, "diabetes" refers to diabetes mellitus, a metabolic disease characterized by a deficiency or absence of insulin secretion by the pancreas. As used throughout this specification, unless otherwise specified herein, "diabetes" includes type 1, type 2, type 3, and type 4 diabetes. The onset of diabetes is usually due to a combination of genetic and environmental causes, resulting in abnormally high blood sugar levels (hyperglycemia). The two most common forms of diabetes are caused by either a decrease in insulin production (in type 1) or a decrease in the body's response to insulin (in type 2 and gestational diabetes). Both lead to hyperglycemia, which primarily causes the acute symptoms of diabetes: excessive urine production, resulting in compensatory thirst and increased fluid intake, blurred vision, unexplained weight loss, lethargy, and altered energy metabolism. Diabetes can lead to many complications. Acute complications (hypoglycemia, ketoacidosis, or nonketotic hyperosmolar coma) can occur if the disease is not properly controlled. Serious long-term complications (i.e., chronic side effects) include cardiovascular disease (doubled risk), chronic renal failure, retinal damage (which can lead to blindness), nerve damage (several types), and microvascular damage, which can cause impotence and poor wound healing. Poor wound healing, particularly of the feet, can lead to gangrene and possibly amputation. In some embodiments, the diabetes can be type 2 diabetes. Type 2 diabetes (non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes) is a metabolic disorder primarily characterized by insulin resistance (a reduced body response to insulin), relative insulin deficiency, and hyperglycemia. In some embodiments, the subject can have prediabetes, which can be characterized, for example, as having elevated fasting blood glucose or elevated postprandial blood glucose.

[0086] Glucagon-like peptide-1 (GLP-1) is an incretin derived from the transcription product of the proglucagon gene, which is known to reduce food intake and hunger in humans and contributes to glucose homeostasis. GLP-1 mimetics are currently used to treat type 2 diabetes. Recent clinical trials have shown that these treatments not only improve glucose homeostasis but also successfully induce weight loss. As used herein, "GLP-1 polypeptide" refers to various pre- and pro-peptides and cleavage products of GLP-1, such as GLP-1(1-37) (SEQ ID NO: 2), GLP-1(7-36) (SEQ ID NO: 3), and GLP-1(7-37) (SEQ ID NO: 4) in humans. In some embodiments, the GLP-1 polypeptide can be GLP-1(7-36) and / or GLP-1(7-37) or related polypeptides derived from species other than humans. GLP-1 polypeptide sequences are known in the art for numerous species, such as human GLP-1 (NCBI Gene ID: 2641) polypeptide (e.g., NCBI Ref Seq: NP_002045.1; SEQ ID NO: 1) and SEQ ID NOs: 2-4. In some embodiments, pre- or pro-peptides of GLP-1, such as glucagon preproprotein (e.g., SEQ ID NO: 1), can be used in the methods or compositions described herein. Naturally occurring alleles or variants of any of the polypeptides described herein are also specifically contemplated for use in the methods and compositions described herein. TIFF2026012396000003.tif54159

[0087] A variety of GLP-1 mimetics are known in the art and are used for the treatment of diabetes.GLP-1 mimetics (or analogues) can include exendin-4 (Heloderma lizard polypeptide with human GLP-1 homology) and its derivatives, the GLP-1 analogues modified to be DPP-IV resistant, or the human GLP-1 polypeptides conjugated with various additional active substances, for example, to extend half-life.GLP-1 mimetics / analogues can include, for example, exenatide, lixisenatide, dulaglutide, semaglutide, albiglutide, LY2189265, liraglutide and taspoglutide. Examples of such molecules and further discussion of their production and activity can be found in the art, for example, in Gupta. Indian J. Endocrinol Metab 17:413-421 (2013); Garber. Diabetes Treatments 41:S279-S284 (2018); U.S. Patent Publication No. US 2009 / 0181912; and International Patent Publication No. WO 2011 / 080103, each of which is incorporated herein by reference in its entirety.

[0088] As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide uncontrollably and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymphatic system. There are several main types of cancer. Carcinomas are cancers that begin in the tissues that line or cover the skin or internal organs. Non-carcinomas are cancers that begin in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissues. Leukemia is cancer that begins in blood-forming tissues, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.

[0089] In some embodiments of either aspect, the cancer is a primary cancer. In some embodiments of either aspect, the cancer is a malignant cancer. As used herein, the term "malignant" refers to a cancer in which tumor cells exhibit one or more of uncontrolled proliferation (i.e., division beyond normal limits), invasion (i.e., invading and destroying adjacent tissues), and metastasis (i.e., spreading to other parts of the body via the lymphatics or blood). As used herein, the term "metastasizing" refers to the spread of cancer from one part of the body to another. A tumor formed by spread cells is called a "metastatic tumor" or "metastatic cancer." A metastatic tumor contains cells similar to cells in the original (primary) tumor. As used herein, the terms "benign" or "non-malignant" refer to a tumor that may grow larger but does not spread to other parts of the body. Benign tumors are self-limited and usually do not invade or metastasize.

[0090] "Cancer cell" or "tumor cell" refers to an individual cell of a cancerous growth or tissue. A tumor generally refers to a swelling or lesion formed by abnormal cell proliferation and may be benign, premalignant, or malignant. Most cancer cells form tumors, but some, such as leukemia, do not necessarily form tumors. For tumor-forming cancer cells, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0091] As used herein, the term "neoplasm" refers to any new and abnormal growth of tissue, e.g., an abnormal tissue mass whose growth is in excess of and uncoordinated with that of normal tissue. Thus, a neoplasm can be a benign neoplasm, a premalignant neoplasm, or a malignant neoplasm.

[0092] A subject with cancer or tumor is a subject with objectively measurable cancer cells present in the subject's body.This definition includes malignant, actively growing cancers, as well as potentially dormant tumors or micrometastases.Cancer that migrates from its original location and disseminates to other vital organs can ultimately lead to the death of the subject through the functional deterioration of the affected organ.

[0093] Examples of cancer include carcinoma, lymphoma, blastoma, non-cancerous carcinoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; stomach cancer (including gastrointestinal cancer); glioblastoma (GBM); liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; kidney or renal carcinoma; Laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; seminal follicular carcinoma; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; and other epithelial and non-epithelial malignancies; and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; and bulky mass disease NHL) ; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phacomatosis, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0094] "Cancer cells" are cancerous, precancerous, or transformed cells, either in vivo, ex vivo, or in tissue culture, that undergo spontaneous or induced phenotypic changes that do not necessarily involve the incorporation of new genetic material. Transformation can result from infection with a transforming virus and the incorporation of new genomic nucleic acid, or from the incorporation of exogenous nucleic acid, but can also result from spontaneous or subsequent exposure to carcinogens, resulting in mutations of endogenous genes. Transformation / cancer is associated with morphological changes, cellular immortalization, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor-specific markers, invasiveness or metastasis, and tumor growth in a suitable animal host, such as nude mice.

[0095] In some embodiments of either aspect, a composition described herein, e.g., a composition comprising CAGE in combination with at least one active compound, is administered as a monotherapy, e.g., without another treatment for the condition.

[0096] In some embodiments of any aspect, the methods described herein may further include administering a second agent and / or treatment to the subject, either in a composition described herein, e.g., a composition comprising CAGE in combination with at least one active compound, or as a separate formulation, e.g., as part of a combination therapy. For example, non-limiting examples of second agents and / or treatments for cancer treatment include radiation therapy, surgery, gemcitabine, cisplatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metuledopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); ostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictine; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, fenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Enediyne antibiotics (e.g., antibiotics such as the calicheamicins, particularly calicheamicin gamma 1I and calicheamicin omega 1I (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, Detrubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin , lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, and doxif Pyrimidine analogues such as uridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziconazole;Elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;Leucovorin (LV); oxaliplatin, including oxaliplatin treatment regimens (FOLFOX); lapatinib (Tykerb.RTM.); PKC-alpha inhibitors that reduce cell proliferation, Raf inhibitors, H-Ras inhibitors, EGFR inhibitors (e.g., erlotinib (Tarceva®)), and VEGF-A inhibitors, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above. In addition, the treatment method may further include the use of radiation or radiotherapy. Furthermore, the treatment method may further include the use of surgical treatment.

[0097] In certain embodiments, an effective dose of the compositions described herein, for example, a composition comprising CAGE in combination with at least one active compound, can be administered to a patient once. In certain embodiments, an effective dose of the compositions described herein, for example, a composition comprising CAGE in combination with at least one active compound, can be administered to a patient repeatedly. For systemic administration, a therapeutic amount of the compositions described herein, for example, a composition comprising CAGE in combination with at least one active compound, can be administered to a subject at, for example, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more.

[0098] In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from about 1 U / kg to about 20 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from 1 U / kg to 20 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be less than 20 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from about 2 U / kg to about 10 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from 2 U / kg to 10 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from about 2 U / kg to about 5 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from 2 U / kg to 5 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from about 5 U / kg to about 10 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from 5 U / kg to 10 U / kg. In some embodiments, the active compound is insulin, and the concentration or dosage of insulin can be 2 U / kg, 5 U / kg, or 10 U / kg.

[0099] In some embodiments, after the initial treatment regimen, treatment can be performed less frequently.For example, after 3 months of treatment every other week, treatment can be repeated once a month for 6 months or a year or more.Treatment according to the methods described herein can reduce the level of markers or symptoms of disease state by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0100] The dosage of the compositions described herein can be determined by a physician and can be adjusted as necessary according to the observed therapeutic effect.Regarding the duration and frequency of treatment, a skilled clinician will usually monitor the subject to determine when treatment provides therapeutic benefit and determine whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment plan.The dosing schedule can vary from once a week to daily, depending on numerous clinical factors, such as the subject's sensitivity to the active compound.The desired dose or amount of the active substance can be administered once, or divided into smaller doses, such as two to four smaller subdoses, and administered over a period of time, for example, at suitable intervals throughout the day or according to other suitable schedules.In some embodiments, administration can be chronic, for example, with one or more doses and / or treatments daily for a period of several weeks or months. Exemplary dosing and / or treatment schedules are daily, twice daily, three times daily, or four or more times daily for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or longer. The compositions described herein, such as compositions comprising CAGE in combination with at least one active compound, can be administered over a period of time, for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes.

[0101] The dosage range for administering the compositions described herein by the methods described herein depends, for example, on the form of the active compound, its potency, and the degree of desired reduction of the symptoms, markers, or indicators of the disease conditions described herein, for example, the percentage of desired reduction of symptoms or markers.Dosage should not be so high as to cause adverse side effects.Generally, dosage varies according to the age, condition, and sex of the patient, and can be determined by those skilled in the art.In the event of any complications, dosage can also be adjusted by individual physicians.

[0102] For example, the effectiveness of a composition described herein in treating a condition described herein or the effectiveness of a composition for inducing a response described herein can be determined by a skilled clinician. However, if one or more of the signs or symptoms of a condition described herein are modified in a beneficial manner, other clinically recognized symptoms are improved or even ameliorated, or a desired response is induced, for example, by at least 10% after treatment with a method described herein, a treatment is considered "effective treatment," as that term is used herein. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or incidence of a condition treated by a method described herein, or any other suitable measurable parameter. Efficacy can also be measured by the absence of deterioration of an individual, as assessed by hospitalization, or the need for medical intervention (i.e., halting the progression of the disease). Methods for measuring these indicators are known to those skilled in the art and / or are described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or animals) and includes: (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of the disease, e.g., causing regression of symptoms. An effective amount for treating a disease means an amount sufficient, when administered to a subject in need thereof, to result in effective treatment for that disease, as that term is defined herein. The effectiveness of an agent can be determined by assessing physical indicators of the disease state or desired response. It is well within the capabilities of one of ordinary skill in the art to monitor the effectiveness of administration and / or treatment by measuring any one or any combination of such parameters. Efficacy can be assessed in animal models of the disease states described herein, e.g., for the treatment of diabetes or cancer. When using experimental animal models, the effectiveness of treatment is demonstrated when a statistically significant change in the marker is observed.

[0103] Provided herein are in vitro and animal model assays that allow the assessment of a given dose of the compositions described herein, such as compositions comprising CAGE in combination with at least one active compound. As a non-limiting example, the effect of a dose of a composition comprising CAGE in combination with insulin can be assessed by using the mouse model described in the Examples herein.

[0104] The incidence of obesity is increasing, and existing treatments, such as dietary therapy, have notoriously low long-term success rates. Additional treatments and strategies for reducing obesity or reducing the rate of weight gain are crucial to address both obesity itself and the number of pathologies caused or exacerbated by excess weight. As described herein, the inventors have demonstrated that the ionic liquid CAGE (choline and geranate or geranic acid) reduces the uptake of hydrophobic / lipophilic molecules in the intestine. Thus, provided herein are methods for treating obesity and / or reducing body weight / weight gain by administering CAGE to a subject in need thereof.

[0105] Additionally, CAGE can safely transport active compounds across sensitive membranes encountered during oral administration. Thus, in some embodiments of either aspect, a composition comprising CAGE further comprises an active compound / agent. Thus, in some embodiments of either aspect, a composition comprising CAGE in combination with an active compound / agent is administered to a subject.

[0106] In some embodiments of any aspect, the active compound is therapeutically effective in treating obesity. In some embodiments of any aspect, the active compound is therapeutically effective in treating a disease associated with obesity. In some embodiments of any aspect, the active compound is therapeutically effective in treating a disease caused by obesity. In some embodiments of any aspect, the active compound is therapeutically effective in treating a disease that causes obesity. In some embodiments of any aspect, the active compound is therapeutically effective in treating metabolic syndrome.

[0107] In some embodiments of either aspect, the active compound is a hydrophobic molecule, such as estradiol, testosterone, imiquimod, corticosterone, paclitaxel, doxorubicin, cisplatin, and / or camptothecin. In some embodiments of either aspect, the active compound is a hydrophobic molecule, such as estradiol, testosterone, corticosterone, paclitaxel, doxorubicin, cisplatin, and / or camptothecin.

[0108] In some embodiments of either aspect, the composition comprises CAGE. In some embodiments of either aspect, the composition consists essentially of CAGE. In some embodiments of either aspect, the composition consists of CAGE. In some embodiments of either aspect, the composition comprising CAGE is administered as a monotherapy, e.g., without another treatment for the condition.

[0109] In some embodiments of either aspect, the composition comprises CAGE and at least one active compound. In some embodiments of either aspect, the composition consists essentially of CAGE and at least one active compound. In some embodiments of either aspect, the composition consists of CAGE and at least one active compound. In some embodiments of either aspect, the composition comprising CAGE and at least one active compound is administered as a monotherapy, e.g., without another treatment for the condition.

[0110] In some embodiments of either aspect, the composition comprising CAGE and optionally an active compound can be formulated as an oral, subcutaneous, intravenous, intradermal, or parenteral formulation. In some embodiments of either aspect, the oral formulation can be a degradable capsule containing the composition comprising CAGE and optionally an active compound.

[0111] In some embodiments of either aspect, the subject to whom the composition comprising CAGE is administered has or has been diagnosed with obesity, excess weight, or is in need of treatment for obesity, excess weight, or prevention of weight gain. In some embodiments, the subject is overweight. The methods described herein include methods for treating obesity, reducing weight gain, preventing weight gain, promoting weight loss, etc. Such methods can, for example, promote metabolic health, be pursued for aesthetic reasons, and / or prepare patients for surgical interventions that are contraindicated for patients with high BMI or weight. In some embodiments, weight loss can be medically necessary and / or medically indicated, for example, when the subject is overweight and / or obese. In some embodiments, weight loss can be cosmetic, for example, when the subject desires to lose weight, regardless of whether weight loss is medically necessary and / or medically indicated.

[0112] The term "obesity" refers to excess body fat. Obesity can be determined by any measure recognized and utilized by those skilled in the art. Currently, the recognized measure of obesity is body mass index (BMI), which is a measure of body weight in kilograms compared to the square of height in meters. Generally, for adults over the age of 20, a BMI between about 18.5 and 24.9 is considered normal, a BMI between about 25.0 and 29.9 is considered overweight, a BMI of about 30.0 or higher is considered obese, and a BMI of about 40 or higher is considered morbidly obese. (See, e.g., Gallagher et al. (2000) Am J Clin Nutr 72:694-701.) These BMI ranges are based on the impact of body weight on increased risk of disease. Some common conditions associated with high BMI and obesity include cardiovascular disease, high blood pressure (i.e., hypertension), osteoarthritis, cancer, and diabetes. Although BMI correlates with body fat, the relationship between BMI and actual body fat varies with age and sex.For example, for the same BMI, women are more likely to have a higher body fat percentage than men.In addition, the BMI threshold for distinguishing between normal, overweight and obesity can vary with, among other factors, for example, age, sex, ethnicity, physical fitness and body type.In some embodiments, obese subjects are at least about 25 kg / m before undergoing the treatment described herein. 2 In some embodiments, a subject with obesity may have a body mass index of at least about 30 kg / m prior to treatment as described herein. 2 The subject may have a body mass index of

[0113] In some embodiments of any aspect, the subject to whom the composition comprising CAGE is administered has, has been diagnosed with, or is in need of treatment for a metabolic disorder or metabolic syndrome. The term "metabolic disorder" refers to any disorder associated with or exacerbated by impaired or altered glucose regulation or glycemic control, such as insulin resistance. Such disorders include, but are not limited to, obesity; excess adipose tissue; diabetes; fatty liver disease; nonalcoholic fatty liver disease; metabolic syndrome; dyslipidemia; hypertension; hyperglycemia; and cardiovascular disease. "Metabolic syndrome" is distinct from metabolic disorders and refers to a combination of medical disorders that, when occurring together, increase the risk of developing cardiovascular disease and diabetes. Numerous definitions of metabolic syndrome have been established, for example, by the American Heart Association and the International Diabetes Foundation. By way of example only, the WHO defines metabolic syndrome as the presence of any one of diabetes, impaired glucose tolerance, impaired fasting glucose, or impaired insulin resistance, and two of the following: blood pressure of 140 / 90 mmHg or greater, dyslipidemia, central obesity, and microalbuminuria. In some embodiments, the metabolic disorder may be selected from the group consisting of obesity, excess adipose tissue, diabetes, and cardiovascular disease.

[0114] In some embodiments, the methods described herein involve treating a subject with or diagnosed with a condition with a composition comprising a CAGE and an active compound. Subjects with a condition such as diabetes can be identified by a physician using current methods for diagnosing diabetes. Symptoms and / or complications of diabetes that characterize these conditions and aid in diagnosis are well known in the art and include, but are not limited to, weight loss, delayed healing, polyuria, polydipsia, polyphagia headache, itchy skin, and fatigue. For example, tests that may aid in the diagnosis of diabetes include, but are not limited to, blood tests (e.g., for fasting glucose levels). A family history of diabetes or exposure to risk factors for diabetes (e.g., overweight) can also help determine whether a subject is likely to have diabetes or make a diagnosis of diabetes.

[0115] In some embodiments of any aspect, the subject treated by the method is a subject who does not have or has not been diagnosed with diabetes. In some embodiments of any aspect, the subject treated by the method is a subject who is not receiving insulin. In some embodiments of any aspect, the composition comprising CAGE does not include insulin. In some embodiments of any aspect, the composition comprising CAGE does not include another active pharmaceutical ingredient and / or another agent that has a therapeutic effect in treating diabetes.

[0116] The uptake of many active compounds, for example, pharmaceutically active compounds, can be improved by delivering the compound in solvent.However, most of these solvents exhibit toxic side effects and / or act as irritants at the time of delivery, so this approach is often unsuitable for in vivo use.Described herein are methods and compositions that can provide improved delivery kinetics and low toxicity.

[0117] As described herein, the inventors have discovered that at 50 mM or greater, salts exhibit a surprising and significant increase in their ability to cross cells / cell membranes and to increase the ability of any associated molecules to do the same. Thus, described herein are methods of drug delivery involving the use of high molar concentrations of salt, e.g., greater than 0.05 M.

[0118] As used herein, "salt" refers to an ionic compound that contains at least one cation and at least one anion, such that the compound is electrically neutral. The salt may contain inorganic or organic, polyatomic or monoatomic ions. The salt may be an alkali salt. In some embodiments of either aspect, the salt is an ionic liquid.

[0119] In some embodiments of either aspect, the salt is at a concentration of at least 0.01% w / v. In some embodiments of either aspect, the salt is at a concentration of at least 0.05% w / v. In some embodiments of either aspect, the salt is at a concentration of at least 0.1% w / v. In some embodiments of either aspect, the salt is at a concentration of at least 0.2% w / v, at least 0.3% w / v, at least 0.4% w / v, at least 0.5% w / v, at least 1% w / v or more.

[0120] In some embodiments of either aspect, the salt is at a concentration of at least 20 mM. In some embodiments of either aspect, the salt is at a concentration of at least about 20 mM. In some embodiments of either aspect, the salt is at a concentration of at least 25 mM. In some embodiments of either aspect, the salt is at a concentration of at least about 25 mM. In some embodiments of either aspect, the salt is at a concentration of at least 50 mM. In some embodiments of either aspect, the salt is at a concentration of at least about 50 mM. In some embodiments of either aspect, the salt is at a concentration of at least 100 mM, 500 mM, 1 M, 2 M, 3 M, or more. In some embodiments of either aspect, the salt is at a concentration of at least about 100 mM, 500 mM, 1 M, 2 M, 3 M, or more.

[0121] In some embodiments of either aspect, the salt is at a concentration of about 50 mM to about 4 M. In some embodiments of either aspect, the salt is at a concentration of 50 mM to 4 M. In some embodiments of either aspect, the salt is at a concentration of about 500 mM to about 4 M. In some embodiments of either aspect, the salt is at a concentration of 500 mM to 4 M. In some embodiments of either aspect, the salt is at a concentration of about 1 M to about 4 M. In some embodiments of either aspect, the salt is at a concentration of 1 M to 4 M. In some embodiments of either aspect, the salt is at a concentration of about 2 M to about 4 M. In some embodiments of either aspect, the salt is at a concentration of 2 M to 4 M.

[0122] In some embodiments of either aspect, for example when one or more nucleic acid molecules are provided in combination with CAGE, the ratio of choline:geranic acid (or geranate) is greater than 1:1, e.g., greater than 1:2, about 1:2 to about 1:4, or 1:2 to 1:4.

[0123] In one aspect of any embodiment, described herein are compositions comprising at least one active compound in combination with a salt (e.g., at a concentration of 0.05 M or greater). In some embodiments, the pharmaceutical composition comprises a salt and one or more active compounds described herein. In some embodiments, the pharmaceutical composition consists essentially of a salt and one or more active compounds described herein. In some embodiments, the pharmaceutical composition consists essentially of an aqueous solution of a salt and one or more active compounds described herein. In some embodiments, the pharmaceutical composition consists essentially of an aqueous solution of a salt and one or more active compounds described herein. In some embodiments, the pharmaceutical composition consists of an aqueous solution of a salt and one or more active compounds described herein.

[0124] In some embodiments, the salt is an anhydrous salt, e.g., an ionic liquid that is not diluted or dissolved in water. In some embodiments, the salt is provided as an aqueous solution.

[0125] In some embodiments of either aspect, a composition comprising a salt (eg, at a concentration of 0.05 M or greater) and an active compound can further comprise a pharmaceutically acceptable carrier.

[0126] In some embodiments of either aspect, the composition comprising the salt (e.g., at a concentration of 0.05 M or greater) and the active compound can be formulated as an oral, subcutaneous, intravenous, intradermal, or parenteral formulation. In some embodiments of either aspect, the oral formulation can be a degradable capsule containing the composition comprising the salt and the active compound.

[0127] In another embodiment, the drug may remain soluble in a salt such as CAGE even after local diffusion of the CAGE, leading to rapid and enhanced delivery to the circulation. Subcutaneous delivery often requires multiple doses or controlled-release formulations. Such approaches are not necessarily implemented due to any concerns about timing per se, but rather to ensure that the full dose is received before the active compound is degraded. Bolus administration is often ineffective because the active compound is degraded or metabolized before the full amount can be effective. Thanks to the ability of CAGE to stabilize the active compound, such approaches are unnecessary, i.e., the desired total amount can be delivered as a single dose without the need for a release control mechanism.

[0128] In one aspect of any embodiment, described herein is a method of treating a disease in a subject in need thereof by administering to the subject an active compound in combination with a salt by injection into the diseased tissue. In some embodiments, the diseased tissue is a tissue containing diseased cells. In some embodiments, the diseased tissue is a tissue that exhibits symptoms of a disease. Non-limiting examples of suitable diseased tissue include tumor tissue, fat tissue, adipose tissue, warts, etc. In some embodiments of any aspect, suitable diseased tissue includes tumor tissue, fat tissue, adipose tissue, etc.

[0129] In some embodiments of any aspect, the disease is a disease caused by tissue proliferation, for example, unwanted, abnormal, or pathological tissue proliferation.Diseases caused by tissue proliferation can be any disease caused by or characterized by the rate of tissue proliferation, the location of tissue proliferation, or the pattern / structure of tissue proliferation that is different from that normal for the type of tissue in healthy subjects.Non-limiting examples of such diseases are tumors, cancer, fat / obesity, warts, and / or hyperplasia.In some embodiments of any aspect, such diseases are tumors, cancer, fat / obesity, and / or hyperplasia.

[0130] In some embodiments of any aspect delineated herein, the biological activity of the active compound is improved or stabilized compared to the activity in the absence of the salt at a concentration of greater than 0.05 M. In some embodiments of any aspect delineated herein, the salt (e.g., CAGE) or solvent significantly enhances the permeation of insulin across the skin compared to control insulin in the absence of the ionic liquid or solvent.

[0131] In one aspect of any embodiment, described herein is a method of administering at least an active compound to a subject using a catheter coated with a salt at a concentration greater than 0.05 M. In one aspect of any embodiment, described herein is a method of collecting bodily fluids by placing a catheter coated with a salt at a concentration greater than 0.05 M within the body.

[0132] In some embodiments, the methods described herein relate to treating a subject with or diagnosed as having a medical condition with a composition comprising a salt and an active compound at a concentration greater than 0.05 M. A subject with a medical condition such as diabetes can be identified by a physician using current methods for diagnosing diabetes. Symptoms and / or complications of diabetes that characterize these conditions and aid in diagnosis are well known in the art and include, but are not limited to, weight loss, delayed healing, polyuria, polydipsia, polyphagia headache, itchy skin, and fatigue. For example, tests that may aid in the diagnosis of diabetes include, but are not limited to, blood tests (e.g., for fasting glucose levels). A family history of diabetes or exposure to risk factors for diabetes (e.g., overweight) can also help determine whether a subject is likely to have diabetes or make a diagnosis of diabetes.

[0133] In some embodiments of either aspect, a composition comprising a salt at a concentration greater than 0.05 M is administered as a monotherapy, e.g., without another treatment for the condition, In such embodiments, the salt or a component thereof may have therapeutic efficacy for the disease being treated.

[0134] In some embodiments of either aspect, a composition comprising a salt at a concentration greater than 0.05 M in combination with at least one active compound described herein is administered as a monotherapy, e.g., without another treatment for the condition.

[0135] In some embodiments of any aspect, the methods described herein may further include administering to the subject a second agent and / or treatment, either in a composition comprising a salt at a concentration greater than 0.05 M in combination with at least one active compound, or as a separate formulation, e.g., as part of a combination therapy.

[0136] In certain embodiments, an effective dose of a composition comprising a salt at a concentration of 0.05 M or greater in combination with at least one active compound described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising a salt at a concentration of 0.05 M or greater in combination with at least one active compound can be administered to a patient repeatedly. For systemic administration, a therapeutic amount of a composition comprising a salt at a concentration of 0.05 M or greater in combination with at least one active compound can be administered to a subject, for example, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more.

[0137] In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from about 1 U / kg to about 20 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from 1 U / kg to 20 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be less than 20 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from about 2 U / kg to about 10 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from 2 U / kg to 10 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from about 2 U / kg to about 5 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from 2 U / kg to 5 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from about 5 U / kg to about 10 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be from 5 U / kg to 10 U / kg. In some embodiments, the active compound is insulin, and the concentration or dosage of insulin can be 2 U / kg, 5 U / kg, or 10 U / kg.

[0138] Enzyme inhibitors are a treatment option for many conditions, including diabetes, for which insulin-degrading enzyme inhibitors, ACE inhibitors, and alapha-glucosidase inhibitors have all been explored as therapeutic approaches. Therefore, safe and effective enzyme inhibitors are of interest in the treatment of many conditions. In one aspect of any embodiment, described herein is a method for treating diabetes, ulcers, cancer, or fibrosis in a subject in need thereof, comprising administering to the subject a composition comprising the ionic liquid choline and geranate (CAGE). In some embodiments, the composition comprising CAGE does not contain an additional therapeutically active agent.

[0139] The ionic liquid CAGE (choline and geranate or geranic acid) is safe for oral and / or parenteral use without the negative side effects typically observed with most solvents. Indeed, because solvents as a class commonly pose toxicity issues, "solvent exposure" is a widely used medical term meant to encompass the risks of contact with one or more solvents. Solvent exposure in general, as well as selective exposure to individual solvents, has been shown to contribute to the pathogenesis of numerous disease states. In light of this, the safety profile of CAGE, particularly via oral and parenteral routes of administration, which bypass many of the body's natural defenses, is particularly surprising and unexpected.

[0140] In one aspect of any embodiment, described herein is a composition comprising CAGE. In some embodiments, the composition is a pharmaceutical composition comprising CAGE. In some embodiments, the pharmaceutical composition consists essentially of CAGE. In some embodiments, the pharmaceutical composition consists of CAGE. In some embodiments, the pharmaceutical composition consists essentially of an aqueous solution of CAGE. In some embodiments, the pharmaceutical composition consists of an aqueous solution of CAGE.

[0141] In some embodiments of either aspect, the composition comprising CAGE can be formulated as an oral, subcutaneous, intravenous, intradermal, or parenteral formulation. In some embodiments of either aspect, the oral formulation can be a degradable capsule containing the composition comprising CAGE. In certain embodiments, an effective dose of the composition comprising CAGE can be administered to a patient once. In certain embodiments, an effective dose of the composition comprising CAGE can be administered to a patient repeatedly.

[0142] In one aspect of any of the embodiments, described herein are methods of treating a disease in a subject in need thereof by administering CAGE by injection into the diseased tissue. In some embodiments, the diseased tissue is a tissue containing diseased cells. In some embodiments, the diseased tissue is a tissue that exhibits symptoms of a disease. Non-limiting examples of suitable diseased tissue include tumor tissue, fat tissue, adipose tissue, warts, etc. In some embodiments of any of the aspects, the diseased tissue includes tumor tissue, fat tissue, adipose tissue, etc. The disease can be, for example, cancer, fibrosis, or an ulcer.

[0143] Fibrotic conditions benefit from the production and / or maintenance of extracellular matrix by reducing the accumulation of scar tissue in favor of extracellular matrix. As used herein, "fibrosis" refers to the formation of fibrous tissue as a repair or reactive process, rather than as a normal component of an organ or tissue. Fibrosis is characterized by the accumulation of fibroblasts and the deposition of collagen in excess of normal deposition in any particular tissue. Fibrosis can occur as a result of inflammation, irritation, or healing. A subject in need of treatment for a fibrotic condition is any subject who has, has been diagnosed with, or is at risk of having a fibrotic condition. Non-limiting examples of fibrotic conditions include pulmonary fibrosis; scarring; skin scarring; trauma; wounds; chronic wounds (e.g., in diabetic patients), corneal defects; corneal ulcers; corneal wounds; diabetic ulcers; ulcers; sepsis; arthritis; idiopathic pulmonary fibrosis; cystic fibrosis; liver cirrhosis; endomyocardial fibrosis; mediastinal fibrosis; myelofibrosis; retroperitoneal fibrosis; progressive massive fibrosis; nephrogenic systemic fibrosis; Crohn's disease; keloids; scleroderma; systemic sclerosis; arthrofibrosis; adhesive capsulitis; pulmonary fibrosis; and liver fibrosis. ; renal fibrosis; cardiac fibrosis; vascular fibrosis; skin fibrosis; ocular fibrosis; myelofibrosis; asthma; sarcoidosis; COPD; emphysema; schistosomiasis; cholangitis; diabetic nephropathy; lupus nephritis; aerial restenosis after angioplasty; atherosclerosis; burn scars; hypertrophic scars; nephrogenic fibrosing dermatosis; post-cataract surgery; proliferative vitreoretinopathy; Peyronie's disease; Dupuytren's contracture; dermatomyositis; and graft-versus-host disease.

[0144] As used herein, "ulcer" refers to the breakage or destruction of body membranes. In some embodiments, ulcers can be caused by inflammation and / or necrosis of affected tissue. Ulcers can be skin ulcers (e.g., pressure ulcers, diabetic ulcers, ulcerative dermatitis, etc.), corneal ulcers, oral ulcers, peptic ulcers, venous ulcers, stress ulcers, or ulcerative colitis.

[0145] In some embodiments of either aspect, the composition comprising CAGE is administered as a monotherapy, eg, without another treatment for the condition.

[0146] For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise specified or implied from the context, the following terms and phrases have the meanings provided below. Since the scope of the present invention is limited only by the claims, the definitions are provided to aid in the description of particular embodiments and are not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0147] For convenience, this specification collects here certain terms employed in the specification, examples, and appended claims.

[0148] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a statistically significant reduction. In some embodiments, "reduce," "reduction," or "reduce" or "inhibit" generally refers to a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and may include, for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or greater reduction. As used herein, "reduction" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease can preferably be down to a level that is accepted as within the normal range for individuals without the given disorder.

[0149] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean a statistically significant increase. In some embodiments, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of less than or equal to 100%, or any increase between 10-100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

[0150] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate such as a primate, a rodent, a livestock animal, or a game animal. Primates include chimpanzees, macaques such as cynomolgus monkeys, spider monkeys, and rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, and wolves, bird species such as chickens, emus, and ostriches, and fish such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, e.g., a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0151] Preferably, the subject is a mammal.The mammal can be, but is not limited to, human, non-human primate, mouse, rat, dog, cat, horse, or cow.Non-human mammals can be advantageously used as subjects to represent the animal model of the pathology described herein.The subject can be male / male or female / female.

[0152] The subject may have been previously diagnosed with, identified as suffering from, or have a condition requiring treatment or one or more complications associated with such a condition, and may optionally have already been treated for the condition or one or more complications associated with the condition. Alternatively, the subject may not have been previously diagnosed with the condition or one or more complications associated with the condition. For example, the subject may exhibit one or more risk factors for the condition or one or more complications associated with the condition, or may not exhibit risk factors.

[0153] A subject "in need" of treatment for a particular condition can be a subject who has the condition, has been diagnosed with the condition, or is at risk of developing the condition.

[0154] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues connected to each other by peptide bonds between the alpha amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, the usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0155] In various embodiments described herein, it is further intended to encompass any variants (natural or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of the specific polypeptides described. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that modify a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," in which the modification results in the substitution of an amino acid with a chemically similar amino acid and the desired activity of the polypeptide is retained. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0156] A given amino acid can be replaced with a residue having similar physiochemical characteristics, such as substituting one aliphatic residue for another (e.g., substituting Ile, Val, Leu, or Ala for each other), or substituting one polar residue for another (e.g., between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, such as the substitution of entire regions with similar hydrophobic characteristics, are well known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, e.g., the activity and specificity of the native or reference polypeptide, is retained.

[0157] Amino acids can be grouped by similarities in the properties of their side chains (A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on shared side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another. Particular conservative substitutions include, for example, Ala for Gly or Ser; Arg for Lys; Asn for Gln or His; Asp for Glu; Cys for Ser; Gln for Asn; Glu for Asp; Gly for Ala or Pro; His for Asn or Gln; Ile for Leu or Val; Leu for Ile or Val; Lys for Arg, Gln, or Glu; Met for Leu, Tyr, or Ile; Phe for Met, Leu, or Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp; and / or Phe for Val, Ile, or Leu.

[0158] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or section of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide as determined by the assays described herein below. Functional fragments may include conservative substitutions of the sequences disclosed herein.

[0159] In some embodiments, the polypeptides described herein may be variants of the sequences described herein. In some embodiments, the variants are conservatively modified variants. Conservative substitution variants can be obtained, for example, by mutation of a native nucleotide sequence. As referred to herein, a "variant" is a polypeptide that is substantially homologous to a native or reference polypeptide but has an amino acid sequence that differs from that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide encompasses sequences encoding a mutant protein or fragment thereof that contains one or more nucleotide additions, deletions, or substitutions compared to a native or reference DNA sequence but retains activity. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art.

[0160] The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to the native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined by comparing the two sequences, for example, using freely available computer programs widely used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0161] In some embodiments of either aspect, the variant can be a polypeptide that has at least 90%, at least 95%, at least 98% or more sequence identity to one of the reference sequences provided herein and retains the wild-type activity, e.g., incretin activity, of that reference sequence. In some embodiments of either aspect, the variant can be a polypeptide that has at least 90%, at least 95%, at least 98% or more sequence identity to one of the naturally occurring reference sequences provided herein and retains the wild-type activity, e.g., incretin activity, of that reference sequence. In some embodiments of either aspect, the variant can be a naturally occurring polypeptide that has at least 90%, at least 95%, at least 98% or more sequence identity to one of the reference sequences provided herein and retains the wild-type activity, e.g., incretin activity, of that reference sequence.

[0162] Alterations to the native amino acid sequence can be achieved by any of a number of techniques known to those of skill in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing the mutated sequence flanked by restriction sites that allow ligation to a fragment of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to provide modified nucleotide sequences with specific codons altered by the required substitution, deletion, or insertion. Techniques for making such modifications are very well established and include, for example, those disclosed in Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entireties. Any cysteine ​​residue not involved in maintaining the correct conformation of the polypeptide can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) can be added to a polypeptide to improve its stability or promote oligomerization.

[0163] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that immunospecifically binds to an antigen. The term also refers to various forms, including immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single-domain antibodies (dAbs), diabodies, multispecific antibodies, dual-specific antibodies, anti-idiotypic antibodies, bispecific antibodies, functionally active epitope-binding portions thereof, and / or bifunctional hybrid antibodies, antibodies composed of two immunoglobulin heavy chains and two immunoglobulin light chains, as well as full-length antibodies and antigen-binding portions thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a constant region of the light chain. The light chain constant region consists of a CL domain. The VH and VL regions may be further divided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with conserved regions called framework regions (FRs). Thus, each VH and VL region consists of three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.

[0164] As used herein, the term "antibody reagent" refers to a polypeptide that contains at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds to a given antigen. An antibody reagent may include an antibody or a polypeptide comprising the antigen-binding domain of an antibody. In some embodiments, an antibody reagent may include a monoclonal antibody or a polypeptide comprising the antigen-binding domain of a monoclonal antibody. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibody (dAb) fragments as well as complete antibodies.

[0165] Antibodies and / or antibody reagents may include immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies, diabodies, multispecific antibodies, dual specific antibodies, anti-idiotypic antibodies, bispecific antibodies, and functionally active epitope-binding portions thereof.

[0166] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acids can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, a nucleic acid can be DNA. In another aspect, a nucleic acid can be RNA. Suitable DNA can include, for example, cDNA. Suitable RNA can include, for example, mRNA.

[0167] As used herein, "inhibitory nucleic acid" refers to a nucleic acid molecule that can inhibit the expression of a target, eg, double-stranded RNA (dsRNA), inhibitory RNA (iRNA), etc.

[0168] Double-stranded RNA molecules (dsRNA) have been shown to block gene expression through a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein can be up to 30 nucleotides in length, i.e., 15-30 nucleotides in length, typically 19-24 nucleotides in length, and contain an RNA strand (antisense strand) with a region substantially complementary to at least a portion of a targeted mRNA transcript. These iRNAs can be used to target and degrade mRNA transcripts, resulting in decreased expression and / or activity of the target.

[0169] As used herein, the term "iRNA" refers to an agent that includes RNA (or modified nucleic acids as described herein below) and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. In some embodiments of either aspect, the iRNA described herein results in inhibition of target expression and / or activity. In some embodiments of either aspect, contacting a cell with an inhibitor (e.g., iRNA) results in a reduction in target mRNA levels in the cell of at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or 100% of the target mRNA levels found in the cell in the absence of iRNA. In some embodiments of either aspect, administration of an inhibitor (e.g., an iRNA) to a subject results in a decrease in target mRNA levels in the subject of at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, 100%, or less, including the target mRNA levels found in the subject in the absence of the iRNA.

[0170] In some embodiments of either aspect, the iRNA can be a dsRNA. The dsRNA comprises two RNA strands sufficiently complementary to hybridize and form a duplex structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally completely complementary, to the target sequence. The target sequence can be derived from the sequence of an mRNA formed during expression of the target; for example, it can span one or more intron boundaries. The other strand (the sense strand) comprises a region complementary to the antisense strand such that, when combined under appropriate conditions, the two strands hybridize to form a duplex structure. Typically, the duplex structure is between 15 and 30 base pairs in length, including 15 and 30 base pairs, more typically between 18 and 25 base pairs in length, including 18 and 25 base pairs in length, even more typically between 19 and 24 base pairs in length, including 19 and 24 base pairs in length, and most typically between 19 and 21 base pairs in length, including 19 and 21 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30 base pairs in length, including 15 and 30 base pairs, more commonly between 18 and 25 base pairs in length, including 18 and 25 base pairs in length, even more commonly between 19 and 24 base pairs in length, including 19 and 24 base pairs in length, and most commonly between 19 and 21 base pairs in length, including 19 and 21 base pairs in length. In some embodiments of either aspect, the dsRNA is between 15 and 20 nucleotides in length, including 15 and 20 nucleotides in length; in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, including 25 and 30 nucleotides in length. As one of skill in the art will recognize, the targeted region of an RNA that is targeted for cleavage is most often a portion of a larger RNA molecule, often an mRNA molecule. Where relevant, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). dsRNA with duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. In most cases, the target is at least 15 nucleotides in length, preferably 15-30 nucleotides in length.

[0171] Exemplary embodiments of types of inhibitory nucleic acids can include, for example, siRNA, shRNA, miRNA, and / or amiRNA, which are well known in the art.

[0172] In some embodiments of either aspect, the RNA of the iRNA, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids described herein may be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, inverted linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, inverted linkage, etc.), (b) base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that form base pairs with an expanded repertoire of partners, base removal (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., 2'- or 4'-position) or sugar substitutions, and (d) backbone modifications, including modification or substitution of phosphodiester bonds. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to, RNA that contains modified backbones or does not contain natural internucleoside linkages.RNA with modified backbones include, among others, those that do not have a phosphorus atom in their backbones.For the purpose of this specification and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbones can also be considered oligonucleosides.In some embodiments of either aspect, modified RNAs have a phosphorus atom in their internucleoside backbones.

[0173] Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with opposite polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and other mixed N, O, S, and CH moieties. These include oligonucleosides having heteroatom backbones, and in particular those having -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- (known as the methylene(methylimino) or MMI backbone), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- (where the natural phosphodiester backbone is represented as -OPO-CH2-).

[0174] In other RNA mimics suitable or intended for use in iRNA, both the sugar and internucleoside linkages, i.e., the backbone, of the nucleotide unit are replaced with novel groups. The base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone.

[0175] The RNA of iRNA can also be modified to contain one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with modified ribose moieties, which contain an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. Adding locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce nonspecific effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0176] Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, described herein, may contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Exemplary suitable modifications are O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments of either aspect, the dsRNA comprises one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents with similar properties. In some embodiments of either aspect, the modification comprises 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the O(CH)ON(CH) group, also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, as described in the Examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH-O-CH-N(CH), also described in the Examples herein below.

[0177] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and the 5' position of the 5'-terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0178] Inhibitory nucleic acids may also include modifications or substitutions of nucleobases (often referred to in the art simply as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal (8-hydroxyl Other synthetic and natural nucleobases include other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-dazaadenine, and 3-deazaguanine and 3-deazaadenine. Some of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are an exemplary base substitution, even more particularly when combined with a 2'-O-methoxyethyl sugar modification.

[0179] The preparation of the above-described modified nucleic acids, backbones, and nucleobases is well known in the art.

[0180] Another modification of the inhibitory nucleic acids featured in the invention involves chemically linking to the inhibitory nucleic acid one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA. Such moieties include cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterols (Oberhauser et al., Nucl. Acids Res., 1992, 4: 1053-1060), and the like. 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al.Lipid moieties include, but are not limited to, octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937), or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0181] The term "vector" as used herein refers to a nucleic acid construct designed for delivery into a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that can replicate when associated with the correct control elements and transfer gene sequences into a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, recombinant vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, etc.

[0182] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides from sequences linked to transcriptional regulatory sequences on the vector. The expressed sequences are often, but not necessarily, heterologous to the cell. An expression vector may contain additional elements; for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, such as human cells for expression and prokaryotic hosts for cloning and amplification. The term "expression" refers to the cellular processes involved in the production of RNA and proteins, and, if applicable, their secretion, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, as applicable. "Expression product" includes RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence that is transcribed into RNA (DNA) in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5' UTR) or "leader" sequence and 3' UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0183] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. A viral vector can contain a nucleic acid encoding a polypeptide as described herein in place of a non-essential viral gene. The vector and / or particle can be used to transfer any nucleic acid into cells either in vitro or in vivo. Many forms of viral vectors are known in the art.

[0184] By " recombinant vector " is meant a vector that contains a heterologous nucleic acid sequence or " transgene " that can be expressed in vivo. It should be understood that in some embodiments, the vectors described herein can be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means for maintaining the nucleotide of interest in high copy number outside of chromosomal DNA in a subject, thereby eliminating the potential effects of chromosomal integration.

[0185] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatments whose purpose is to reverse, alleviate, ameliorate, inhibit, slow, or halt the progression or severity of symptoms associated with a disease or disorder, such as a condition or disease described herein. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least slowing of the progression or worsening of symptoms compared to that expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, a decrease in the extent of the disease, stabilization of the disease state (i.e., not worsening), a delay or slowing of disease progression, an improvement or palliative of the disease state, remission (whether partial or complete), and / or reduced mortality. The term "treatment" of a disease also includes bringing about the alleviation of symptoms or side effects of the disease (including symptomatic treatment).

[0186] As used herein, the term "pharmaceutical composition" refers to an active agent combined with a pharmaceutically acceptable carrier, e.g., a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be a carrier other than water. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be an artificial or engineered carrier, e.g., a carrier in which the active ingredient is not found to occur in nature.

[0187] As used herein, the term "administering" refers to placing a compound as disclosed herein in a subject by a method or route that results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions containing the compounds disclosed herein can be administered by any suitable route that results in effective treatment in the subject.

[0188] The term "effective amount" refers to an amount of a composition sufficient to result in at least some improvement in symptoms associated with a condition. In one embodiment, an "effective amount" refers to an amount of a composition that reduces a marker or symptom of a condition in a subject with the condition.

[0189] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference greater than two standard deviations (2SD).

[0190] Other than in the working examples, or unless otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±1%.

[0191] As used herein, the terms "comprising" or "comprises" are used in reference to methods and compositions essential to the invention and their respective components, although they may include elements not specified, whether essential or not. As used herein, the term "comprising" means that other elements may be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[0192] The term "consisting of" refers to the compositions, methods, and each component thereof described herein, and does not include any element not recited in the description of that embodiment.

[0193] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0194] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin "exempli gratia," and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0195] Groupings of alternative elements or aspects of the invention disclosed herein should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, and deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification herein is deemed to include the group as modified, fulfilling the written description of all Markush groups used in the appended claims.

[0196] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art to which this disclosure belongs. It should be understood that the present invention is not limited to the particular methodology, protocols, and reagents, etc., described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305);Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4. thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414);Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X);Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542);Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons Sons, Inc. 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are incorporated herein by reference in their entireties.

[0197] Those skilled in the art can readily identify chemotherapeutic agents to use (see, e.g., Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff's Clinical Oncology, 2013 Elsevier; and Fischer DS (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

[0198] Other terms are defined herein within the description of various aspects of the invention.

[0199] All patents and other publications, including references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications, which may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or indication as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0200] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes; however, those skilled in the relevant art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are shown in a given order, in alternative embodiments, the functions may be performed in a different order, or functions may be performed substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods, as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, if necessary, to employ compositions, functions, and concepts from the above references and applications to provide still further embodiments of the present disclosure. Furthermore, given consideration of biological functional equivalence, some changes can be made to protein structure without affecting biological or chemical activity in type or amount. These and other changes can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0201] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Furthermore, although advantages associated with particular embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not necessarily all embodiments may exhibit such advantages to be within the scope of the present disclosure.

[0202] The technology described herein is further illustrated by the following examples, which should in no way be construed as further limiting.

[0203] Some aspects of the technology described herein can be defined by any of the following numbered clauses: 1. Ionic liquids or solvents, including a group of salts with organic cations and organic / inorganic anions. 2. The ionic liquid or solvent of item 1, wherein the organic cation and organic / inorganic anion are choline and geranate or geranic acid, respectively. 3. An ionic liquid or solvent according to item 1 or 2 that exists as a liquid below 100°C. 4. An ionic liquid or solvent of items 1, 2, or 3 that exists as a liquid at room temperature. 5. An ionic liquid or solvent of any of paragraphs 1-4 that significantly enhances the permeation of insulin across the skin compared to control insulin in the absence of the ionic liquid or solvent. 6. A composition comprising the ionic liquid according to any one of items 1 to 5 and insulin. 7. The composition of paragraph 6, further comprising a pharmaceutically acceptable carrier. 8. The composition of paragraph 6 or 7, formulated as an oral formulation. 9. A composition comprising an ionic liquid containing choline and geranate and insulin. 10. The composition of paragraph 9, further comprising a pharmaceutically acceptable carrier. 11. An oral formulation comprising an ionic liquid comprising choline and geranate, insulin, and a pharmaceutically acceptable carrier. 12. An insulin formulation for oral delivery, comprising the ionic liquid according to any one of items 1 to 5. 13. The insulin formulation for oral delivery of paragraph 12, further comprising a pharmaceutically acceptable carrier. 14. A method for orally delivering a therapeutic drug, comprising the steps of mixing the ionic liquid according to any one of items 1 to 5 with a therapeutic drug and orally administering the mixture to a subject. 15. The method of paragraph 14, wherein the admixture is an oral formulation. 16. A method for treating diabetes, comprising orally administering an oral formulation of insulin comprising choline and geranate and insulin, wherein the choline and geranate form an ionic liquid solvent. 17. The composition of any one of items 6 to 10, the formulation of any one of items 11 to 13, or the method of any one of items 14 to 16, wherein the concentration of the ionic liquid in the composition or formulation is about 0.1 mM to 20 mM. 18. The concentration of the ionic liquid in the composition or formulation is about 0.5 mM to 20 mM, 0.5 mM to 18 mM, 0.5 mM to 16 mM, 0.5 mM to 14 mM, 0.5 mM to 12 mM, 0.5 mM to 10 mM, 0.5 mM to 8 mM, 1 mM to 20 mM, 1 mM to 18 mM, 1 mM to 16 mM, 1 mM to 14 mM, 1 mM to 12 mM, 1 mM to 10 mM, 1 mM to 8 mM, 2 mM to 20 mM, 2 mM to 18 mM, 2 mM to 16 mM, 2 mM to 14 mM, 2 mM to 12 mM, 2 mM to 10 mM, 2 mM to 8 mM, 4 mM to 20 mM, 4 mM to 18 mM, 4 mM to 16 mM, 4 mM to 14 mM, 4 mM to 12 mM, 4 mM to 10 mM, 4 mM to 8 mM, 6 mM to 20 mM, 6 mM to 18 mM, 6 mM to 14 mM, 6 mM to 10 ... 16. The composition of any one of items 6 to 10, the formulation of any one of items 11 to 13, or the method of any one of items 14 to 16, wherein the saturation factor is 6 mM to 12 mM, 6 mM to 10 mM, 6 mM to 8 mM, 8 mM to 20 mM, 8 mM to 18 mM, 8 mM to 16 mM, 8 mM to 14 mM, 8 mM to 12 mM, 8 mM to 10 mM, 10 mM to 20 mM, 10 mM to 18 mM, 10 mM to 16 mM, 10 mM to 14 mM, 10 mM to 12 mM, 12 mM to 20 mM, 12 mM to 18 mM, 12 mM to 16 mM, 12 mM to 14 mM, 14 mM to 20 mM, 14 mM to 18 mM, 14 mM to 16 mM, 16 mM to 20 mM, 16 mM to 18 mM, or 18 mM to 20 mM. 19. The composition of paragraph 17, wherein the concentration of the ionic liquid in the composition is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM.

[0204] Some aspects of the technology described herein can be defined by any of the following numbered clauses: 1. A method for oral delivery of at least one active compound, comprising orally administering the active compound in combination with a composition comprising the ionic liquid choline and geranate (CAGE). 2. A method for delivering at least one active compound, comprising subcutaneously, intradermally, or intravenously administering the active compound in combination with CAGE. 3. A method for delivering at least one active compound, comprising administering to a mucosa the active compound in combination with a CAGE. 4. The method of item 3, wherein the mucosa is nasal mucosa, oral mucosa, or vaginal mucosa. 5. A method for parenteral delivery of at least one active compound, comprising parenterally administering the active compound in combination with CAGE. 6. The method of paragraph 5, wherein administering comprises delivery to a tumor. 7. A method of treating a disease in a subject in need thereof by administering to the subject an active compound in combination with CAGE by injection into the affected tissue. 8. The method of paragraph 7, wherein the disease is cancer, adipose tissue, warts, hyperplasia, or any other disease caused by tissue proliferation. 9. The method of paragraphs 1-8, wherein the CAGE is at a concentration of at least 0.1% w / v. 10. The method of paragraphs 1-9, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. 11. The method of paragraphs 1-9, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. 12. The method of paragraphs 1-9, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:4. 13. The method of paragraphs 1 to 12, wherein the anion of the ionic liquid comprises geranate and / or geranic acid. 14. The method of paragraphs 1 to 13, wherein the active compound in combination with CAGE is administered once. 15. The method of paragraphs 1-14, wherein the active compound in combination with CAGE is administered in multiple doses. 16. The method of paragraphs 1 to 15, wherein the active compound comprises a nucleic acid molecule. 17. The method of paragraphs 1 to 15, wherein the active compound comprises a small molecule. 18. The method of paragraphs 1 to 15, wherein the active compound comprises a polypeptide. 19. The method of paragraphs 1 to 15, wherein the active compound comprises an antibody or antibody reagent. 20. The method of paragraphs 1-19, wherein the active compound comprises a chemotherapeutic compound. 21. The method of paragraphs 1 to 20, wherein the active compound comprises insulin. 22. The method of paragraph 21, wherein the insulin is provided at a dosage of 1 to 20 mg / kg. 23. A composition comprising an active compound in combination with CAGE. 24. The composition of paragraph 23, wherein the CAGE is at a concentration of at least 0.1% w / v. 25. The composition of paragraphs 23-24, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. 26. The composition of paragraphs 23-24, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. 27. The composition of paragraphs 23-24, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:4. 28. The composition of paragraphs 23 to 27, wherein the active compound comprises a nucleic acid molecule. 29. The composition of paragraphs 23 to 27, wherein the active compound comprises a small molecule. 30. The composition of paragraphs 23 to 27, wherein the active compound comprises a polypeptide. 31. The composition of paragraphs 23 to 27, wherein the active compound comprises an antibody or antibody reagent. 32. The composition of paragraphs 23 to 31, wherein the active compound comprises a chemotherapeutic compound. 33. The composition of paragraphs 23 to 32, wherein the active compound comprises insulin. 34. The composition of paragraph 233, wherein the insulin is provided in a dosage of 1 to 20 mg / kg. 35. The composition of paragraphs 23 to 34, further comprising an additional pharmaceutically acceptable carrier. 36. The composition of any one of paragraphs 23 to 35, formulated as an oral, subcutaneous, or parenteral formulation. 37. The composition of paragraphs 23 to 36, formulated for administration to a mucosa. 38. The composition of paragraph 37, wherein the mucosa is nasal mucosa, oral mucosa, or vaginal mucosa. 39. The composition of paragraph 36, wherein the oral formulation is a degradable capsule containing the active compound and the CAGE combination. 40. The composition of paragraphs 23 to 39, wherein the biological activity of the active compound is improved or stabilized compared to the activity in the absence of CAGE. 41. The method or composition of paragraphs 1 to 40, wherein the combination of the active compound and the CAGE is an admixture. 42. The method or composition of paragraphs 1-40, wherein the combination of active compound and CAGE comprises nanoparticles containing the active compound, and the nanoparticles are in solution or suspension in a composition containing the CAGE. 43. A method for delivering a nucleic acid molecule to a cell, comprising contacting the cell with the nucleic acid molecule in combination with a composition comprising the ionic liquid choline and geranate (CAGE). 44. The method of paragraph 43, wherein the cell is a cell in a subject, and the contacting step includes administering to the subject the nucleic acid molecule in combination with a composition comprising the ionic liquid choline and geranate (CAGE). 45. The method of paragraphs 43-44, wherein the nucleic acid molecule comprises a vector, an expression vector, or an inhibitory nucleic acid molecule. 46. ​​The method of paragraphs 43-45, wherein the CAGE is at a concentration of at least 0.1% w / v. 47. The method of paragraphs 43-46, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. 48. The method of paragraphs 43-46, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. 49. The method of paragraphs 43-46, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:4. 50. The method of paragraphs 43 to 49, wherein the anion of the ionic liquid comprises geranate and / or geranic acid. 51. The method of paragraphs 43 to 50, wherein the combination of the nucleic acid molecule and CAGE is a mixture. 52. The method of paragraphs 43 to 50, wherein the combination of nucleic acid molecule and CAGE comprises nanoparticles containing the nucleic acid molecule, and the nanoparticles are in solution or suspension in a composition containing the CAGE. 53. At least one active compound in combination with a composition comprising the ionic liquid choline and geranate (CAGE) for oral delivery, mucosal delivery, parenteral delivery, or use in treating a disease. 54. The combination of paragraph 53, wherein the mucosa is nasal mucosa, oral mucosa, or vaginal mucosa. 55. The combination of paragraph 53, wherein parenteral administration includes delivery to a tumor. 56. The combination of paragraph 53, wherein treatment comprises injection of the composition into the affected tissue. 57. The combination of paragraph 56, wherein the disease is cancer, fat, wart, hyperplasia, or any other disease resulting from tissue proliferation. 58. A combination of paragraphs 53 to 57, wherein the CAGE is at a concentration of at least 0.1% w / v. 59. The combination of paragraphs 53 to 58, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:10. 60. The combination of paragraphs 53 to 59, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:4. 61. The combination of paragraphs 53 to 59, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:4. 62. The combination of items 53 to 61, wherein the anion of the ionic liquid includes geranate and / or geranic acid. 63. The combination of paragraphs 53 to 62, wherein the active compound in combination with CAGE is administered once. 64. The combination of paragraphs 53 to 62, wherein the active compound in combination with CAGE is administered in multiple doses. 65. The combination of paragraphs 53 to 64, wherein the active compound comprises a nucleic acid molecule. 66. The combination of paragraphs 53 to 65, wherein the active compound comprises a small molecule. 67. The combination of paragraphs 53 to 65, wherein the active compound comprises a polypeptide. 68. The combination of paragraphs 53 to 65, wherein the active compound comprises an antibody or antibody reagent. 69. The combination of paragraphs 53 to 65, wherein the active compound comprises a chemotherapeutic compound. 70. The combination of paragraphs 53 to 65, wherein the active compound comprises insulin. 71. A combination of paragraph 70, in which insulin is provided at a dose of 1 to 20 mg / kg. [Example]

[0205] Example 1: Choline-geranate as a highly effective solvent for oral delivery of insulin Abbreviation AUC Area under the concentration curve BSM Basal Seeding Medium CAGE Choline and Geranate CD circular dichroism CLSM Confocal Laser Scanning Microscopy DAPI 4',6-diamidino-2-phenylindole, dihydrochloride DMEM Dulbecco's Modified Eagle's Medium DMSO dimethyl sulfoxide F Bioavailability FBS Fetal Bovine Serum FITC Fluorescein isothiocyanate GIT gastrointestinal tract IC50 half-maximal inhibitory concentration IJ jejunum K el Disappearance rate constant MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide PBS Phosphate-buffered saline P / S Penicillin and Streptomycin RT room temperature SQ Subcutaneous t 1 / 2 Half-life TEER Transepithelial electrical resistance

[0206] With the rise in diabetes cases worldwide and lack of patient adherence to glycemic control using injectable insulin, the development of efficient oral insulin formulations is urgently needed. However, the gastrointestinal tract poses a formidable barrier to oral delivery of biologics. Here, we report the development of a highly effective oral insulin formulation using choline and geranate (CAGE) ionic liquids. In vivo, after jejunal administration in rats, insulin-CAGE demonstrated exceptional 51% pharmacokinetic bioavailability and 66% pharmacodynamic bioavailability. Low doses of insulin (3–10 U / kg) resulted in significant reductions in blood glucose levels that persisted for a longer period (up to 12 hours), unlike subcutaneously injected insulin. When 10 U / kg of insulin-CAGE was orally delivered in enteric-coated capsules via oral gavage, sustained reductions in blood glucose of up to 45% were observed. The formulation exhibited high biocompatibility and was stable for 2 months at room temperature and at least 4 months under refrigeration. Taken together, the results indicate that CAGE is a promising oral delivery vehicle that should be further explored for the oral delivery of insulin and other biologics currently marketed as injectable substances.

[0207] The oral route of drug administration is preferred over injections due to its ease of administration, high patient compliance, and low manufacturing costs. However, various gastrointestinal barriers to drug absorption make it unsuitable for the delivery of biologics. For example, insulin is an essential medication for the management of type 1 diabetes. It is currently administered as a subcutaneous injection, but its invasive nature has been associated with a lack of patient adherence. Orally delivered insulin can significantly enhance patient compliance and closely mimics the physiological pathway of pancreatic insulin. Oral / pancreatic insulin is transported to the liver via the portal vein, where 80% is retained, and the remainder reaches the systemic circulation, creating up to three-fold higher insulin concentrations in the portal vein compared with the systemic circulation. When insulin is injected subcutaneously, this portal-peripheral insulin gradient is disrupted due to higher systemic insulin concentrations compared with those in the portal vein (only approximately 20%), disrupting the liver's delicate balance between glycogen stores and glucose output. This often results in hyperglycemia, which, when treated with higher insulin doses, can lead to hypoglycemia.

[0208] The pursuit of oral insulin products began decades ago. While several strategies have been developed to overcome the gastrointestinal barriers to oral absorption of biologics, no formulations have successfully overcome all clinical hurdles, and therefore no oral insulin products are currently on the market. Products that have completed or are currently undergoing Phase II clinical trials include enteric-coated capsules containing additives to improve oral insulin uptake (Capsulin™ by Diabetology Ltd. and ORMD-0801 by Oramed Ltd.), hepatic-directed liposomal insulin (HDV-Insulin by Diasome Pharmaceuticals Inc.), insulin conjugated with polyethylene glycol (PEG) (IN-105 by Biocon Ltd.), insulin-proinsulin-c-peptide in the Oshadi carrier (Oshadli Icp by Oshadi Drug Administration Ltd.), and long-acting insulin analog tablets using gastrointestinal absorption enhancement technology (GIPET 1 by Novo Nordisk).

[0209] Furthermore, many products require multi-step formulation procedures, various additives, or chemical modifications of the protein, which have their own drawbacks.With the emerging global diabetes epidemic, there is an urgency to develop a safe, effective, and easily scalable oral insulin product.

[0210] We developed an ionic liquid (IL)-based oral formulation of insulin and determined its safety, efficacy, and long-term stability. Ionic liquids are composed of organic / inorganic salts with melting points below 100°C and are widely used in various novel chemical and pharmaceutical technologies. In this work, we utilized a room-temperature stable deep eutectic solvent of choline and geranate (CAGE), which previously showed remarkable efficacy in transdermal delivery of antibiotics and insulin. Insulin was dispersed in CAGE in a single-step process, and its safety and efficacy, as well as its storage stability, were assessed both in vitro and in vivo. This study demonstrates unprecedented improvements in the oral bioavailability of insulin with excellent oral efficacy, biocompatibility, and long-term stability.

[0211] Determination of FITC-insulin transport across intestinal monolayers We designed a 5-hour long transport experiment of FITC-insulin across Caco-2 monolayers using 10 mM CAGE or saline. In this study, insulin-CAGE-treated cells showed significantly higher transport from the start compared to the insulin-saline-treated group (Figure 1). For both groups, FITC-insulin transport steadily increased over time, and throughout the study, at least three-fold higher transport of insulin was observed in CAGE-treated monolayers compared to insulin-saline-treated cells across all time points. By the final 5-hour time point of the study, FITC-insulin transport in insulin-CAGE-treated cells reached 30%, compared to 10% in insulin-saline-treated cells.

[0212] These results were confirmed through confocal imaging of the transwell membranes at the end of the study, which clearly showed higher uptake of FITC-insulin by cells incubated with insulin-CAGE compared to insulin-saline (Figures 2A-2B).

[0213] TEER was measured to determine the integrity of tight junctions in Caco-2 cells upon treatment with CAGE. In insulin-saline-treated cells, TEER remained close to 100% of its original value over the first 3 h, decreasing by 2.5% to 98.5 ± 2.64% by the final 5-h time point of the study (Figure 3). However, in insulin-CAGE-treated cells, TEER significantly decreased to 88.35 ± 0.86 and 77.43 ± 3.48% at 2 and 3 h, respectively. Beyond 3 h, TEER began to rise, reaching 82.94 ± 2.64% of its initial value at 4 h and finally reaching 91.3 ± 1.73% at 5 h, which was not significantly different from insulin-saline-treated cells. These changes in TEER induced by CAGE suggest that CAGE transiently opens intestinal tight junctions to facilitate insulin transport across the cells. However, there may be multiple mechanisms besides the transient opening of tight junctions that come into play to facilitate the transport of insulin across these cells by CAGE that need to be elucidated.

[0214] In vivo efficacy of insulin-CAGE upon intrajejunal administration To measure the blood glucose lowering efficacy of insulin-CAGE, 3–5 U / kg insulin was dispersed in CAGE and administered intrajejunally to anesthetized rats, along with its control. Blood glucose was then monitored every 0.5 hours for a total of 5 hours (Figure 4). In rats treated with 3 U / kg insulin-CAGE, blood glucose began to steadily decline from 0.5 hours, reaching 55% of the initial value (55.37 ± 5.64%) by 2.5 hours. Beyond this point, blood glucose decline plateaued, reaching 53% of the initial value (53.12 ± 3.16%) by the final 5-hour time point of the study. The group treated with 5 U / kg insulin-CAGE showed a rapid decrease in blood glucose levels, reaching a drop of approximately 65% ​​within 1.5 and 2 hours (36.73 ± 4.46 and 35.37 ± 5.25%, respectively). As is typically observed in nondiabetic rats exposed to a rapid drop in blood glucose, blood glucose levels subsequently increased due to the body's glucose homeostasis mechanisms. At the end of the 5-hour period, blood glucose levels were approximately 74% (73.93 ± 5.72%) of the initial level. Rats receiving subcutaneous injections of 2 U / kg insulin also showed a similar pattern of blood glucose reduction compared with 5 U / kg intrajejunal administration of insulin-CAGE. However, the magnitude of blood glucose reduction was lower than that observed with 5 U / kg intrajejunally administered insulin-CAGE. A maximum reduction of 51% was observed at 1 hour (48.76 ± 7.55% of the initial level), which rapidly recovered to 100% (101.61 ± 6.69%) by the final 5-hour period of the study. At earlier time points, no significant difference in efficacy was noted between subcutaneously administered insulin and 5 U / kg insulin-CAGE. However, as clearly seen in Figure 6, CAGE significantly prolonged the insulin effect until the end of the study. Similar sustained insulin bioactivity was noted with intrajejunal administration of 3 U / kg insulin-CAGE. Other formulation controls, such as CAGE alone, saline, or 3 U / kg insulin-saline intrajejunal administration, did not produce the rapid drop in blood glucose levels observed in the aforementioned groups. In all these control groups, blood glucose decreased slowly (most likely due to continued fasting) to approximately 25% of initial levels in 5 hours.

[0215] The pharmacokinetics of insulin absorption and excretion were determined by measuring serum insulin levels at different time points (Figure 5). Insulin concentrations rapidly increased within 1 h after subcutaneous injection of 2 U / kg insulin and intrajejunal administration of 5 U / kg insulin-CAGE, followed by a subsequent decline, and excretion followed a similar pattern. In contrast, intrajejunally administered 3 U / kg insulin-saline showed little increase in serum insulin concentrations. Pharmacokinetic parameters calculated using serum insulin concentrations indicated that the elimination half-life of intrajejunally administered insulin-CAGE was approximately two-fold longer than that of SQ insulin (Table 1). The oral bioavailability of 5 U / kg IJ insulin-CAGE calculated in this way was found to be 50.6%.

[0216] Table 1. Pharmacokinetic parameters of intrajejunally administered insulin-CAGE and subcutaneously administered insulin solution TIFF2026012396000004.tif16169

[0217] Our studies have yielded a jejunal bioavailability of 51%, one of the highest observed among other oral insulin formulations. It should also be noted that the elimination half-life of insulin-CAGE is approximately two-fold longer than that of subcutaneously injected insulin, indicating a more sustained efficacy.

[0218] In vivo efficacy of insulin-CAGE delivered orally in capsules The considerable effectiveness of CAGE in enhancing the oral bioavailability of insulin when administered intrajejunally led us to investigate whether similar efficacy could be achieved using insulin-CAGE delivered orally in capsules. To this end, we placed 10 U / kg of insulin-CAGE and its control in elongated size 9 capsules as illustrated in Figure 6 and administered them via oral gavage to overnight-fasted rats.

[0219] The group treated with 10 U / kg insulin-CAGE showed a rapid drop in blood glucose levels 1 and 2 hours after the start of the study (68.58 ± 2.12 and 62.32 ± 1.58% of initial levels, respectively) (Figure 7). Beyond this time point, blood glucose slowly but steadily declined to approximately 56% (56.66 ± 2.31%) of initial levels at 12 hours. In contrast, subcutaneous administration of 2 U / kg insulin led to a rapid 50% drop in blood glucose levels (51.43 ± 5.25%) at 1 hour, followed by a steady increase to approximately 88% by 4 hours. Thereafter, blood glucose levels declined in a pattern similar to the CAGE-only, empty capsule, and insulin-saline formulation controls. When these controls were orally administered, a slow, steady decrease in blood glucose levels was observed over time (most likely due to continued fasting), culminating in a decrease of approximately 25% at 12 hours. It should be noted that, unlike subcutaneously administered insulin, and consistent with intrajejunal administration, CAGE led to a significantly sustained effect of insulin starting at 4 hours and continuing through the final 12 hour time point of the study.

[0220] Histological analysis of the intestine after intrajejunal and oral administration of CAGE Histological examination showed no significant differences in the morphology of small intestinal tissue between rats treated with CAGE or saline (Figures 8A-8E). Intestinal tissues were collected either 5 hours after intrajejunal administration or 12 hours after oral administration, and no significant structural damage was observed in the small intestinal tissue. Notably, finger-like villi were present in all tissues. The results clearly demonstrated the excellent biocompatibility of CAGE with the intestine, thereby validating its suitability for oral administration.

[0221] Secondary structure of insulin in CAGE Insulin possesses a unique alpha-helical conformation essential for its receptor binding and therefore biological activity. Previous studies have demonstrated that insulin retains its alpha-helical conformation in CAGE after 17 hours of storage at room temperature (RT, 25°C) [Banerjee et al., AHM, 2017]. However, it is unclear whether the conformation is preserved when insulin is dispersed in CAGE for longer periods. To this end, we stored insulin-CAGE at RT (avoiding direct sunlight) or under refrigeration at 4°C and assessed the secondary structure monthly over a 4-month period using CD. Results showed the presence of a double negative trough at approximately 207 and 222 nm, a typical representation of an alpha helix in a CD graph (Figure 9). No differences in the shape or degree of ellipticity were observed between fresh insulin and insulin stored at RT for up to 3 months or under refrigeration for up to 4 months. This result suggests that CAGE helps maintain insulin stability over long periods of time, which was subsequently confirmed through in vivo bioactivity assessment. Generally, lyophilized standard insulin is stable at room temperature for up to 3-4 weeks, while insulin solutions stored at 4°C are stable for only 2-7 days (see, for example, records available on the World Wide Web at prospecbio.com / Insulin_Human).

[0222] Confirmation of insulin stability during CAGE through in vivo bioactivity assessment Given the encouraging results obtained in the CD stability study, we sought to assess the biological activity of insulin in nondiabetic rats to confirm the stability results. To this end, overnight-fasted rats were subcutaneously injected with 1 U / kg insulin (isolated from CAGE and resuspended in sterile saline), and blood glucose was monitored over an 8-hour period (Figure 10). Freshly prepared insulin solution (insulin in saline) resulted in a ∼50% drop in blood glucose levels in 1 hour (50.73 ± 2.47%), which was maintained for the next hour (49.59 ± 4.4%). Blood glucose levels then rose to ∼92% of the original level (92.05 ± 4.8%) in 5 hours and then slowly dropped again to ∼30% of the initial value as a result of continued fasting. In comparison, insulin isolated from CAGE stored at various temperatures and for different time points followed a similar pattern, exhibiting similar blood glucose drop at 1 and 2 hours after injection, with no significant difference in the drop rate compared to fresh insulin. However, a significant decline in efficacy was observed for insulin stored at RT for 3 months. In this group, only a 38% drop in blood glucose levels was noted 1 and 2 hours after insulin injection (61.57 ± 2.38% and 61.98 ± 3.19%, respectively), indicating that insulin loses some of its bioactivity after 2 months of storage at RT. However, insulin stored at 4°C with CAGE did not show any loss of bioactivity even after 4 months of storage. This clearly demonstrates that CAGE is an excellent solvent for long-term storage of insulin. Further studies are needed to determine the maximum time of stability of insulin in CAGE at 4°C.

[0223] Consideration Over the past few decades, the prevalence of diabetes has grown so steadily that it is now being referred to as the "epidemic of the century" (1). Rising diabetes cases have been reported in all countries, with the fastest growth in low- and middle-income countries and the highest prevalence in the Middle East and North Africa region (1-3). A recent World Health Organization report indicated that diabetes was responsible for 1.5 million deaths worldwide in 2012, with an additional 3.2 million deaths due to hyperglycemia-related comorbidities (3). In the United States, 30.3 million people (approximately 9.4% of the population) were reported to be afflicted with the disease in 2015, with 1.5 million new cases diagnosed annually (4). Current suggestions for managing this disease include insulin therapy, either alone or in combination with oral hypoglycemic agents such as metformin (5). Individuals receiving insulin therapy alone often require the administration of either twice-daily intermediate-acting insulin or once-daily long-acting insulin (5). Insulin is not available as an oral pill in outpatient clinics and is administered exclusively as a subcutaneous injection. However, despite its effectiveness in managing hyperglycemia and mitigating the risk of neuropathy, nephropathy, and retinopathy, injectable insulin is associated with lower patient compliance due to pain, interference with daily activities, and embarrassment, resulting in willful neglect and poor long-term glycemic control in as many as 60% of patients (6, 7). This leads to elevated hemoglobin A1C levels and increased hospitalizations due to diabetes-related complications (6). To circumvent this problem, MannKind Corporation developed Afrezza®, an inhalable, fast-acting insulin formulation for postprandial glycemic control. However, associated pulmonary risks, such as higher incidence of lung cancer and diabetic ketoacidosis, decreased pulmonary function, and a higher risk of acute bronchospasm in patients with chronic lung disease, may deter patients from switching to inhaled insulin therapy (8). Given the exponential growth and scale of diabetes, it is essential to develop insulin therapies that are patient-appealing and avoid the adverse effects of formulation-based treatments.

[0224] Oral delivery enjoys high patient compliance, but it is not suitable for the delivery of macromolecules such as protein or peptide drugs. This is due to the fact that orally delivered drugs must traverse the acidic environment of the stomach, which can degrade protein / peptide drugs. This can be avoided by encapsulating them in enteric or other protective coating systems. However, upon release from their protective casing in the intestine, peptide / protein drugs are thrust into the intestinal proteolytic environment and easily cleaved into smaller amino acid units by resident enzymes. Even if a proportion of the drug escapes proteolysis, it is nearly impossible for it to be absorbed into the blood circulation as an intact molecule through the intestinal mucus layer and enterocytes. Oral insulin absorption is further hindered by erratic GI transit times and the lack of specific insulin uptake mechanisms in the intestine (9). Any disruption of insulin structure during GI transit may lead to significant denaturation and loss of biological activity. Therefore, not surprisingly, protein and peptide drugs have negligible oral bioavailability, less than 1%, a stark contrast to injectable formulations, in which 100% of the dose is available for pharmacological activity (10). Several researchers have attempted to solve the perennial problem of low oral insulin bioavailability by modifying the insulin molecule, encapsulating it in novel carriers, and using enteric coatings, absorption enhancers, or proteolytic inhibitors. Some examples include the use of PLGA (poly(lactic-co-glycolic acid))-based nanoparticles for oral insulin delivery. Pan et al. obtained a pharmacological bioavailability of 10.3% using 10 U / kg insulin loaded into PLGA nanoparticles, while Cui and colleagues obtained oral bioavailabilities of 3.68 and 6.27% using 20 U / kg insulin placed in PLGA and PLGA-55 nanoparticles, respectively (11, 12). Sarmento et al. encapsulated insulin using chitosan-dextran nanoparticles and observed pharmacological bioavailabilities of 5.6 and 3.4% after placing 50 and 100 U / kg insulin in the particles, respectively (13).Alginate-chitosan nanoparticles improved the oral bioavailability of insulin to 6.8% and 3.4% for insulin doses of 50 and 100 U / kg, respectively (14). Zhang and colleagues encapsulated 50 U / kg insulin in solid lipid nanoparticles (SLNs) and wheat germ agglutinin-modified SLNs, obtaining pharmacological bioavailabilities of 4.46% and 6.08%, respectively (15). Similarly, Ansari et al. reported a five-fold enhancement in the oral bioavailability of insulin using SLNs compared with orally administered insulin solution (8.26% vs. 1.7%) (16). High oral bioavailability of insulin (37.6%) was achieved by orally delivering 75 U / kg insulin in biodegradable polyisobutylcyanoacrylate nanospheres (17). Other strategies to improve oral protein delivery involve the use of protease inhibitors, such as sodium glycocholate, aprotinin, soybean trypsin inhibitor, bacitracin, and camostat mesilate, which have shown promise in improving the efficacy of orally delivered insulin (18). Chemical modifications of insulin, such as attaching targeting ligands like transferrin or cell-penetrating peptides like the TAT peptide, have been shown to aid insulin transcytosis across enterocytes (19, 20). The insulin analog IN-105, obtained through the conjugation of short chains of polyethylene glycol (PEG) to insulin, has been demonstrated to orally dose-dependently reduce blood glucose levels after meals in patients with T2DM through improved solubility, proteolytic stability, and intestinal absorption (21, 22). Absorption enhancers include bile salts, surfactants, fatty acids, calcium ion chelators, certain polymers such as chitosan / thiolated chitosan, and zona occludens toxins, which function either by modulating the cell membrane structure of the intestinal epithelium for transcellular uptake or by increasing tight junction permeability for paracellular transport (18).

[0225] ILs constitute a group of salts with organic cations and organic / inorganic anions, and are typically liquid below 100°C, although some exist as liquids at room temperature. (23) By pairing different ions, ILs can be tailored to possess distinct physicochemical properties, such as viscosity, hydrophobicity, solubility, and biodegradability, for a wide range of pharmaceutical applications, particularly in the fields of biocatalysis, enzymatic processes, protein stability, permeation enhancers, and solubilizers. (23-29) We recently designed a choline-geranate (CAGE)-based IL that significantly enhanced insulin permeation across the skin. (27)

[0226] The biocompatibility of orally delivered CAGE was first tested in vitro using the human intestinal Caco-2 cell line, which is widely used as a model of intestinal absorption and barrier function. (30, 31) IC values ​​were obtained for periods of 12–48 h of incubation. 50 The values ​​were ≧10 mM, suggesting that high concentrations of CAGE were not cytotoxic to intestinal cells, thus demonstrating the potential utility of CAGE as a safe oral drug delivery agent.

[0227] To determine the effectiveness of CAGE in transporting insulin across enterocytes, we dispersed insulin in 10 mM CAGE and incubated the formulation with Caco-2 cells for 5 hours. Compared to insulin in saline, CAGE significantly enhanced insulin transport by 3-4 fold throughout the study period. A transient but significant decrease in tight junction integrity in the cells was also noted, which is hypothesized to contribute to the enhanced insulin transport across the cells. However, the interplay of multiple other mechanisms may also have contributed to the enhanced insulin transcytosis and requires investigation. It is important to note that CAGE only caused a transient decrease in tight junction integrity, and the cells restored junction integrity to roughly basal levels within 5 hours. This clearly suggests that, unlike many other permeation enhancers, CAGE does not permanently or long-term damage to the intestinal epithelial structure, thereby mitigating any resulting toxicity.

[0228] material and method Preparation of CAGE CAGE solvent was synthesized according to our previous work [Zakrewsky PNAS 2014]. Briefly, 2 equivalents of geranic acid alone (20 g, 0.119 mol, Sigma-Aldrich, St. Louis, MO), which had been recrystallized at least five times in acetone at <-70 °C to remove impurities, were added to 1 equivalent of choline bicarbonate (80 wt% solution, 12.275 g, 0.059 mol, Sigma-Aldrich, St. Louis, MO) in a 500 mL round-bottom flask. The mixture was stirred at 40 °C until CO2 evolution ceased, and water was removed by rotary evaporation at 60 °C for 2 hours, followed by drying in a vacuum oven at 60 °C for 48 hours. Physical characterization at 25 °C showed good agreement with previous values. NMR spectra (collected using a 500-MHz Varian instrument, Palo Alto, CA) also showed good agreement with previous preparations: TIFF2026012396000005.tif27159

[0229] Caco-2 monolayer culture in 96-well plates and transwells Human epithelial colorectal adenocarcinoma cells (Caco-2, ATCC, Manassas, VA) were seeded at a density of 1,000 cells / well in 96-well plates and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S) (ThermoFisher Scientific, Waltham, MA) at 37°C and 5% CO for 21 days to fully differentiate and form a confluent monolayer. During this period, cell culture medium was changed every 2 days during the first week and every other day during the second and third weeks.

[0230] For transport experiments in transwells, a 3-day rapid Caco-2 proliferation system was used. Cells were plated at a density of 400,000 cells / ml in Corning® Basal Seeding Medium (BSM) supplemented with MITO Serum + Extender onto Millicell® PCF inserts placed inside 24-well plates. Following the manufacturer's recommendations, 500 μl of cells containing medium was placed apically, and 1000 μl of cell-free BSM was placed basolaterally. After 24 hours of incubation at 37°C and 5% CO2, the medium was replaced with an equal volume of enterocyte differentiation medium supplemented with MITO Serum + Extender for an additional 2–4 days. TEER was measured periodically and maintained at 200 ohms.cm, indicating sufficient tight junction integrity between cells. 2 Transport studies were carried out when it reached higher levels.

[0231] In vitro oral biocompatibility evaluation of CAGE This study utilized Caco-2 cells grown in 96-well plates. CAGE was diluted with DMEM to concentrations ranging from 25 to 3.125 mM. Three different sets of CAGE dilutions were prepared (three dilution replicates). The medium was aspirated from each well, and each dilution was distributed into six wells (100 μL per well) (six cell replicates). Control wells were filled with medium alone. Cells were incubated at 37°C and 5% CO2 for 12, 24, or 48 hours. At each time point, the CAGE-medium mixture was aspirated from the wells, and cell viability was assessed using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. MTT powder (ThermoFisher Scientific, Waltham, MA) was mixed with the medium to a concentration of 0.5 mg / mL and added to each well (100 μL) and incubated at 37°C and 5% CO2 for 4 hours. The MTT solution was removed, and 100 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO) was added to each well. The plates were wrapped in foil and shaken for 20 minutes, after which the absorbance was read at 570 nm using a microplate reader (M220 Infinite Pro, Tecan Group Ltd, Morrisville, NY). The viability values ​​of untreated cells were used to normalize the absorbance readings.

[0232] FITC-insulin transport assay Before starting the experiment, the existing medium in the transwells was replaced with DMEM without phenol red, FBS, and P / S on both the apical (200 μl) and basolateral (600 μl) sides, and the cells were incubated for 30 minutes. The apical medium was then replaced with 200 μl of 500 μg / ml FITC-insulin solubilized in DMEM without phenol red, FBS, and P / S, prepared with or without 10 mM CAGE. Immediately after adding FITC-insulin to the apical side, a 100 μl aliquot was removed from the basolateral side and replaced with an equal volume of fresh DMEM. This was repeated at 1, 2, 3, 4, and 5 hours. Throughout the study, the transwell plate was placed on a rotating shaker at 100 rpm inside a 37°C, 5% CO2 incubator, and removed and aliquots removed only during the aforementioned time periods. After the final 5-hour time point of the study, FITC-insulin concentrations in aliquots were measured using a plate reader (Tecan, Infinite M1000, Mannedorf, Switzerland) at excitation / emission wavelengths of 495 / 520 nm and plotted as % FITC-insulin transport versus time. Additionally, TEER was measured at all time points after removal of aliquots from the transwells and plotted as % change from initial value versus time.

[0233] For qualitative analysis of FITC-insulin uptake by Caco-2 cells, transwells from the FITC-insulin transport study were washed twice with PBS at the end of the study, followed by the addition of 100 μl of 4% paraformaldehyde and storage at 4°C overnight. The following day, the paraformaldehyde was aspirated from the wells, the membranes were washed twice with PBS, and the transwell membranes were excised. These membranes were then gently placed on glass slides, to which mounting medium containing 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Vectashield™ Hardset, Vector Laboratories, Burlingame, CA) was added, and the membranes were then covered with glass slides. Confocal imaging of the membranes was performed using an Olympus Fluoview 1000™ spectral confocal instrument at 60x magnification.

[0234] Assessment of efficacy and pharmacokinetic parameters of insulin-CAGE upon intrajejunal administration The efficacy of intrajejunal administration of insulin-CAGE was determined in nondiabetic adult male Wistar rats that were fasted overnight but allowed free access to water. Prior to the start of the study, rats were anesthetized, abdominal hair was clipped, and the area was surgically prepared using 70% ethanol and betadine. An abdominal incision was made to expose the intestine. The jejunum was located and injected with 100 μl of either 3 or 5 U / kg insulin dispersed in CAGE or 100 μl of control (3 U / kg insulin in saline, CAGE alone, and saline). Each formulation was tested in six rats, except for saline, which was tested in only three rats. The intestinal segment was then replaced into the abdomen, and the muscle and skin were sutured. Blood glucose was determined using a commercially available glucose meter at the beginning and every 0.5 hours until the final 5-hour time point of the study. Animals remained anesthetized throughout the entire study, at the end of which they were euthanized, and the intestinal segment around the injection site was removed for further histological examination to determine toxicity, if any. To compare efficacy, another group of 3 rats was injected subcutaneously with 2 U / kg insulin in saline. Results were plotted as % change in blood glucose relative to the initial reading at t=0 against time.

[0235] The pharmacokinetics of insulin delivered via CAGE were assessed by collecting approximately 250 μL of blood into BD Vacutainer® red-top tubes at 0, 1, 2, 3, and 5 h from rats injected intrajejunally with 5 U / kg insulin-CAGE, intrajejunally with 3 U / kg insulin-saline, and subcutaneously with 2 U / kg insulin-saline. Serum was separated from whole blood according to standard protocols. Briefly, blood samples were allowed to stand at room temperature for 15–30 min to allow clot formation, followed by centrifugation at 2,000 g for 10 min. The clear supernatant (serum) was then collected into clean tubes and stored on ice during the procedure, then at -20°C until further analysis of insulin content. To assess insulin concentrations in serum samples, a human insulin ELISA kit (Thermo Fisher Scientific, Waltham, MA) was used to determine insulin concentrations at each time point according to the manufacturer's protocol. The elimination rate constant (K ) was calculated from a plot of serum insulin concentration versus time. el ), elimination half-life (t 1 / 2 Pharmacokinetic parameters such as % oral bioavailability (F), area under the concentration curve (AUC), and % oral bioavailability (F) were calculated.

[0236] In vivo oral efficacy To determine the efficacy of insulin-CAGE administered via the oral route, elongated size 9 capsules (Torpac, Fairfield, NJ) capable of holding 80 μl of CAGE were used. These capsules were filled with either 80 μl of 10 U / kg insulin-CAGE, CAGE alone, or left empty. Following this, the capsules were enteric-coated three times with 12.5% ​​w / v Eudragit® L-100 dissolved in isopropanol to prevent capsule degradation in the acidic environment of the stomach and provide intestinal site-specific delivery of the encapsulated CAGE. For oral efficacy studies, nondiabetic adult male Wistar rats were fasted overnight but given free access to water. The next day, the capsules were administered to the rats via oral gavage, followed by subcutaneous administration of 5 mg / kg metoclopramide hydrochloride to promote gastric emptying. Blood glucose was then measured using a commercially available glucose meter, and subsequently measured hourly for up to 12 hours. The rats were fasted throughout the entire study period. A control of 10 U / kg insulin in saline was also tested by administering the formulation orally as a solution (not in a capsule). Additionally, the efficacy of 2 U / kg insulin-saline injected subcutaneously was also assessed. Groups of six rats per formulation were used for 10 U / kg insulin-CAGE and CAGE alone, while three rats per group were utilized to study the efficacy of empty capsules, 10 U / kg insulin solution, and 2 U / kg insulin-saline injected subcutaneously. Results were plotted as percent change in blood glucose levels over time.

[0237] Tissue histology Tissues were fixed in 10% buffered formalin, dehydrated in ethanol, and embedded in paraffin. Five-micron cross sections of intestinal tissue were deparaffinized, rehydrated, and stained with hematoxylin and eosin. Histological morphology was examined using a light microscope at 10x and 40x magnification (Olympus BX60™ upright compound microscope).

[0238] Assessment of insulin stability during CAGE Samples containing human insulin (100 U, 3.5 mg, Sigma-Aldrich, St. Louis, MO) were suspended in 1 mL of either CAGE or PBS in a 2 mL microcentrifuge tube and incubated at room temperature (25°C) or under refrigeration (4°C). After 1 month and approximately monthly thereafter for a total of 4 months, the samples were centrifuged at 10,000 × g for 10 minutes, the CAGE removed via pipette, and the soft insulin pellet washed with 1 mL of phosphate-buffered saline (PBS) and centrifuged again. The PBS-CAGE was removed, and the washing / centrifugation process was repeated until no insulin formed a pellet during centrifugation.

[0239] Circular dichroism (CD) studies: To collect spectra in the far-UV region (190–250 nm), which indicates the secondary structure of proteins, circular dichroism spectrophotometry (Jasco J-1500, Easton, MD) was performed using a rectangular quartz cell (1 mm path length, Starna Cells, 1-Qq, Atascadero, CA) loaded with 400 uL of sample.

[0240] In vivo biological efficacy assessment: The biological activity of insulin isolated from CAGE was assessed by subcutaneously injecting the resulting insulin into nondiabetic adult male Wistar rats at a dose of 1 U / kg. Blood glucose levels were monitored for 8 hours using a commercially available glucose meter and compared with freshly prepared insulin solutions. All animal experiments were performed in accordance with the animal care committee guidelines of the University of California, Santa Barbara, and the National Research Council's Guide for the Care and Use of Animals of the Institute of Laboratory Animal Resources. Rats were fasted overnight before insulin injection but had free access to water and remained fasted throughout the study to eliminate fluctuations in blood glucose due to food consumption. Results were plotted as the percentage change in blood glucose compared to initial levels over time.

[0241] Data analysis All data are presented as mean ± standard error (SE). Student's T-test was used for statistical analysis. Differences were considered significant at p<0.05. All experiments were performed at least in triplicate.

[0242] References TIFF2026012396000006.tif26153TIFF2026012396000007.tif238159TIFF2026012396000008.tif232159TIFF2026012396000009.tif19158

[0243] Example 2: Ionic Liquids for Delivery to Tissues Solubilization of hydrophobic drugs has long been a major hurdle in drug delivery. Many small molecules, including chemotherapy drugs, are highly hydrophobic in nature and require complex solubilization strategies. The present invention describes the use of ionic liquids for this purpose. Another object of this disclosure is to demonstrate the use of ionic liquids to enhance the dispersion of drugs from the injection site to tissues. Another object of this disclosure is to demonstrate the use of ionic liquids to enhance the bioavailability of drugs after delivery to the subcutaneous or intradermal space.

[0244] Deep eutectic solvents (DES) and ionic liquids (ILs) can enhance drug delivery and disrupt / neutralize biofilm-forming bacteria. Furthermore, compared to traditional chemical penetration enhancers such as ethanol, the compositions described herein exhibit lower toxicity to cells, thereby mitigating the tissue irritation problem characteristic of many chemical enhancers. DESs are mixtures of compounds that collectively have a lower freezing point than the individual components, while ILs are salts composed of organic cations and organic / inorganic anions that are stable and liquid at room temperature. Prior to the demonstrations provided herein, the ability of ionic liquids, including CAGEs, to solubilize hydrophobic drugs and biologicals for parenteral administration and their use to enhance drug dispersion and absorption had not been explored.

[0245] method Preparation of CAGE Choline geranate deep eutectic was synthesized. Briefly, 2 equivalents of geranic acid alone (50.0 g, 0.297 moles, Sigma Aldrich, St. Louis, MO) were recrystallized five times in acetone at -70°C in a 500 mL round-bottom flask and added to 1 equivalent of choline bicarbonate (80% wt solution, 30.7 g, 0.297 moles, Sigma Aldrich, St. Louis, MO). The mixture was stirred at room temperature until CO2 evolution ceased. Residual HO was removed by rotary evaporation at 60°C for 2 hours and drying in a vacuum oven at 60°C for 96 hours. Physical characterization at 25°C was in good agreement with published values ​​and is as follows: density, 0.989 ± 0.00 lg / mL; and conductivity, 0.0427 ± 0.0005 mS / cm. NMR spectrum is provided. NMR assignments are also in good agreement with published assignments and include the following: TIFF2026012396000010.tif26157

[0246] Physical characteristic evaluation NMR spectroscopy was performed to verify the identity of the final product. H and C NMR spectra were collected on a 600-MHz Varian instrument using a sample concentration of approximately 50 mM in DMSO-d6. H spectra were averaged over 128 scans with a 2-second relaxation delay between pulses. C spectra were averaged over 512 scans with a 2-second relaxation delay between pulses. Density was measured in triplicate using a 1-mL volumetric flask and an analytical balance. Conductivity was measured with a DS-71 conductivity meter (Horiba, Kyoto, Japan) equipped with a flow-type conductivity electrode (Horiba, Kyoto, Japan) calibrated with a KCl standard solution. Conductivity was measured in triplicate at 25°C for each sample using 0.25 mL of sample.

[0247] In vivo experiments The efficacy of insulin delivery from CAGE was evaluated in normal rats. Before the experimental day, animals were fasted overnight but allowed free access to water. Insulin was suspended in CAGE before administration. Animals were anesthetized and injected subcutaneously with insulin-CAGE. For control experiments, CAGE alone or insulin-saline was injected. Blood glucose levels were measured from the tail vein at different time intervals using a commercially available blood glucose meter.

[0248] In vitro cell culture experiments The in vitro anticancer efficacy of drug-loaded liposomes was determined using a calcein-AM cell viability assay (Life Technologies). 4T1 cells were seeded into 96-well cell culture plates at a density of 11,000 cells per well or 1,000 cells per well in a total volume of 100 μL of medium and allowed to adhere overnight. The medium was then replaced with fresh medium containing liposomes and incubated with 4T1 cells for 48 hours. After drug incubation, the medium was aspirated and replaced with 1 μM calcein-AM in PBS for 30 minutes at room temperature. The fluorescence intensity of intracellularly hydrolyzed calcein-AM was measured using excitation and emission wavelengths of 490 nm and 520 nm. Fractional cell inhibition was calculated by subtracting the fluorescence of live cells in the experimental wells from the fluorescence of untreated cells and normalizing to the fluorescence of untreated cells.

[0249] Effect of CAGE on molecular unfolding in tissues Experiments were performed to determine whether CAGE could be used to enhance the distribution of molecules from the injection point into tissue. These experiments were performed using skin as a model tissue. To this end, 1 mg / ml of labeled (FITC or Antonia Red) 150 kDa dextran was suspended in either 1:1 CAGE alone, 50% CAGE (diluted in saline), 10% CAGE (diluted in saline), or saline. 50 μl of each sample was injected intradermally into one side of a 5 cm × 5 cm square of pig skin. Two differently labeled samples with the same CAGE concentration were injected into the same square. Samples were incubated at RT for 2 hours (while we were undergoing training) and then imaged. Samples were incubated at 37°C for an additional 2 hours and imaged again. The 2-hour samples were imaged with different exposures (we were still learning the system). All 4-hour samples were imaged with the same exposure. For each sample and time point, the size of the fluorescent area was estimated by manually drawing an outline around the area using ImageJ™.

[0250] result Solubility: The solubility of two hydrophobic drugs, paclitaxel and camptothecin, in CAGE was determined. Both drugs are practically insoluble in water, requiring solubilization strategies or modifications to the drug chemistry. For example, the lack of solubility of paclitaxel in water led to the use of a solvent called Cremophor, currently used in outpatient clinics. However, Cremophor suffers from toxicity issues. Camptothecin, on the other hand, is not used clinically due to a lack of a solvent, and an alternative chemical form, irinotecan, is in clinical use. The ability of CAGE to solubilize both drugs offers a clear advantage over alternatives. Paclitaxel: 200 + / - 48 mg / ml Camptothecin: 54 mg / ml

[0251] This ability of CAGE to solubilize hydrophobic drugs can be used for a variety of drugs, including estradiol, testosterone, imiquimod, corticosterone, paclitaxel, doxorubicin, cisplatin, and camptothecin. These hydrophobic drugs currently require complex strategies involving solvents such as ethanol or DMSO, which preclude their use in many applications, especially injection-based delivery systems. Alternatively, strategies have been developed to solubilize drugs in micelles. While the use of micelles is useful for solubilization, it substantially changes the drug's morphology from a "small molecule" to a colloidal one. This transformation significantly alters therapeutic outcomes. Drug solubilization in CAGE directly addresses this limitation. CAGE can be used in several possible ways. In one approach, solubilized drugs can be formulated for topical application, such as oral or transdermal administration. The primary contribution of CAGE in this application is to retain the solubilized drug within the formulation to provide a high concentration gradient for drug diffusion across the barrier. In another application, the drug solution in the CAGE can be injected intradermally, subcutaneously, or intravenously. In such formulations, the CAGE serves to solubilize the drug, allowing for high-concentration injections. In one embodiment, upon injection, the CAGE can diffuse into tissues, allowing for local precipitation of the drug. The precipitated drug can form a depot and exhibit sustained release. In another embodiment, the drug may remain soluble in the CAGE even after local diffusion of the CAGE, leading to rapid and enhanced delivery to the circulation.

[0252] In vitro efficacy of chemotherapy drugs dissolved in CAGE: The effects of camptothecin and paclitaxel solubilized in CAGE on 4T1 cancer cells were tested. A positive control was used: the same drugs dissolved in DMSO. Note that DMSO is a solvent used in in vitro studies but is not a clinically acceptable alternative.

[0253] The embodiments described herein can be used in multiple ways. In one form, chemotherapy drugs are solubilized in CAGE and delivered directly into the tumor. The solubilized chemotherapy drugs may diffuse into the tumor, allowing for a therapeutic effect. It is noted that CAGE itself also has a cytotoxic effect at high concentrations. In one embodiment, CAGE can be injected directly into the tumor to achieve a localized cytotoxic effect. Such treatment can be used to treat solid tumors, such as tumors in the liver, pancreas, or breast. In one embodiment, CAGE can be injected directly into adipose tissue to promote lysis. CAGE can either directly lyse adipocytes by lysis or induce destruction sufficient to promote macrophage / immune cell-mediated clearance.

[0254] In vivo efficacy of insulin delivery from CAGE: The insulin delivered from CAGE was highly bioactive. A simple suspension of insulin in CAGE was injected subcutaneously. This formulation produced substantial hypoglycemia, superior to an insulin solution in saline (Figure 12).

[0255] The embodiment depicted in Figure 12 can be used in several ways. In one form, CAGE can be used to enhance the absorption of biologics into the circulation. Large biologics, such as antibodies, have limited absorption into the systemic circulation after injection into the subcutaneous space. The addition of CAGE can enhance their bioavailability. In another embodiment, CAGE can be used to alter the pharmacokinetics of injected drugs. For example, insulin, which is rapidly absorbed from a subcutaneous injection site, can be made to exhibit sustained absorption by the addition of CAGE (Figure 12).

[0256] Stabilization of biomolecules: The stability of insulin in CAGE was evaluated. Insulin was suspended in CAGE and left at room temperature or 4°C. After one or two months, insulin was injected subcutaneously, and its bioactivity was evaluated based on the blood glucose response (Figure 13). The bioactivity of insulin-CAGE stored at room temperature was comparable to that of native insulin after two months, demonstrating the effectiveness of CAGE in stabilizing biomolecules.

[0257] Stabilization of biomolecules by CAGE can be used to protect vaccines from degradation. Vaccines are typically formulated in aqueous formulations and require refrigeration to maintain activity. This cold chain requirement makes it difficult to deliver vaccines to remote areas of the world. CAGE can be used to protect vaccine activity at room temperature.

[0258] Increased organizational variance: All AntoniaRed-labeled dextran samples appeared to unfold moderately between the 2 and 4 hour time points. FTIC-labeled dextran samples did not appear to unfold between 2 and 4 hours, except for the CAGE-only sample, which showed an approximately 2-fold increase in area (see, e.g., Figure 14).

[0259] This embodiment can be used in several ways. In one form, CAGE can be used to enhance its own distribution into tissues after injection for the treatment of localized conditions such as tumors or warts. The effect of CAGE on local tissues can also be used to enhance the distribution of solubilized drugs such as chemotherapeutic agents.

[0260] As used herein, the term "ionic liquid" refers to an organic salt or mixture of organic salts that are in a liquid state at room temperature. This class of solvents has been shown to be useful in a variety of fields, including industrial processing, catalysis, medicine, and electrochemistry. Ionic liquids contain at least one anionic component and at least one cationic component. Optionally, the IL contains an additional hydrogen bond donor (i.e., any molecule that can provide an -OH or -NH group), examples of which include, but are not limited to, alcohols, fatty acids, and amines. In some embodiments, the cationic or anionic component is also a drug.

[0261] While the behavior of ILs is dominated by ionic interactions, deep eutectic solvents (DESs) exhibit strong contributions from hydrogen bonding. Classification of systems containing ionic and neutral species is complex, primarily due to limitations in current definitions. In this regard, the definitions and descriptions provided herein for compositions, ionic liquids, and deep eutectic solvents are intended to control terminology as they may be applied by others in the field.

[0262] The at least one anionic component and the at least one cationic component may be present in any molar ratio. Exemplary molar ratios (cation:anion) include, but are not limited to, 1:1, 1:2, 2:1, 1:3, 3:1, 2:3, 3:2, and ranges between these ratios. In some embodiments, the IL is a deep eutectic solvent (DES). DES is a type of ionic solvent with special properties: it is composed of a mixture that forms a eutectic with a melting point much lower than that of either of the individual components. Exemplary DES include, but are not limited to, choline oleate, choline hexanoate, choline geranate, choline malonate (disodium choline malonate), and urea-choline. In these formulations, the excess carboxylate prevents the ions from pairing 1:1, making the formulation a DES and not a true ionic liquid. The ions used in preparing the IL / DES may include molecules other than choline or generic acids. For example, a derivative or analog of choline can be used as the cation. At the same time, analogs, derivatives, or geranic acid can also be used as the anion.

[0263] The drugs to be delivered may include small molecules, peptides, proteins, nucleic acids, antibodies, or any other therapeutically active molecule.

[0264] Example 3: Ionic Liquids for Oral Insulin Delivery With the rise in diabetes cases worldwide and lack of patient adherence to glycemic control using injectable insulin, the development of efficient oral insulin formulations is urgently needed. However, the gastrointestinal tract poses a formidable barrier to oral delivery of biologics. Described herein is the development of a highly effective oral insulin formulation using choline and geranate (CAGE) ionic liquids. CAGE significantly enhanced paracellular transport of insulin, protecting it from enzymatic degradation and thinning it by interacting with the mucus layer. In vivo, insulin-CAGE demonstrated exceptional pharmacokinetic and pharmacodynamic results after jejunal administration in rats. Low doses of insulin (3–10 U / kg) resulted in significant reductions in blood glucose levels that lasted for a longer period (up to 12 hours), unlike subcutaneously injected insulin. When 10 U / kg of insulin-CAGE was orally delivered in enteric-coated capsules via oral gavage, a sustained reduction in blood glucose of up to 45% was observed. The formulation exhibited high biocompatibility and was stable for 2 months at room temperature and at least 4 months under refrigeration. Taken together, the results indicate that CAGE is a promising oral delivery vehicle for the oral delivery of insulin and other biologics currently marketed as injectable substances.

[0265] Although insulin is currently available as an injectable formulation, an oral product would enjoy higher patient compliance and significantly improve the quality of life for diabetic patients worldwide. However, oral delivery of proteins such as insulin is challenging due to various gastrointestinal barriers to oral absorption of macromolecules. Described herein is the development of a safe and highly effective ionic liquid-based oral insulin formulation that significantly enhances oral insulin absorption by efficiently bypassing gastrointestinal barriers. Moreover, the formulation exhibited good stability at room temperature and under refrigeration.

[0266] The oral route of drug administration is preferred over injections due to its ease of administration, high patient compliance, and low manufacturing costs. However, it is inappropriate for the delivery of biologics due to various gastrointestinal barriers to drug absorption. For example, insulin is an essential medication for the management of type 1 diabetes. Currently administered as a subcutaneous injection, it is associated with a lack of patient adherence due to injection-related pain and needle phobia (1). Orally delivered insulin can significantly enhance patient compliance. Furthermore, it closely mimics the physiological pathway of pancreatic insulin (2, 3). Oral / pancreatic insulin is transported to the liver via the portal vein, where 80% is retained, and the remainder reaches the systemic circulation, creating up to threefold higher insulin concentrations in the portal vein compared to the systemic circulation (3). When insulin is injected subcutaneously, this portal-peripheral insulin gradient is disrupted due to higher systemic insulin concentrations compared to those in the portal vein (only approximately 20%), disrupting the liver's delicate balance between glycogen stores and glucose output. This often results in hyperglycemia, which, if treated with higher insulin doses, can lead to hypoglycemia (3, 4).

[0267] The pursuit of oral insulin products began decades ago. Several strategies have been developed to overcome the gastrointestinal barriers to oral absorption of biologics. However, no formulation has successfully overcome all clinical hurdles, and therefore no oral insulin products are currently on the market. Products that have completed or are currently undergoing Phase II clinical trials include enteric-coated capsules containing additives to improve oral insulin uptake (Capsulin™ by Diabetology Ltd. and ORMD-0801 by Oramed Ltd.), hepatic-directed liposomal insulin (HDV-Insulin by Diasome Pharmaceuticals Inc.), insulin conjugated with polyethylene glycol (PEG) (IN-105 by Biocon Ltd.), insulin-proinsulin-c-peptide in the Oshadi carrier (Oshadli Icp by Oshadi Drug Administration Ltd.), and long-acting insulin analog tablets using gastrointestinal absorption enhancement technology (GIPET 1 by Novo Nordisk) (5-7). Furthermore, many products require multi-step formulation procedures, various additives, or chemical modifications of the protein, which have their own drawbacks.With the emerging global diabetes epidemic, there is an urgency to develop a safe, effective, and easily scalable oral insulin product.

[0268] Described herein is an ionic liquid (IL)-based oral formulation of insulin and its demonstration of safety, efficacy, and long-term stability. Ionic liquids are composed of organic / inorganic salts with melting points below 100°C (8-10). Herein, we utilized a deep eutectic solvent of choline and geranate (CAGE), which is stable at room temperature (11, 12). Insulin was dispersed in CAGE in a single-step process, and its safety and efficacy were assessed both in vitro and in vivo, as well as its storage stability. This study demonstrates unprecedented improvements in the oral bioavailability of insulin with excellent oral efficacy, biocompatibility, and long-term stability.

[0269] result Insulin was stable in CAGE for a long period of time Insulin possesses a unique alpha-helical conformation that is essential for its receptor interaction and, therefore, biological activity (13). Before performing functional studies of oral delivery with CAGE, we assessed whether insulin was stable in CAGE over long periods of time. Insulin-CAGE was stored at room temperature (avoiding direct sunlight) or refrigerated at 4°C, and the secondary structure of insulin isolated from CAGE was assessed monthly over a 4-month period using circular dichroism (CD). The results showed the presence of a double negative trough at approximately 207 and 222 nm, a typical representation of an alpha helix in a CD graph (Figure 15). No difference in the shape or degree of ellipticity was observed between freshly prepared insulin solutions and insulin stored in CAGE at RT or 4°C for up to 3 and 4 months, respectively. This result indicates that CAGE preserves insulin's secondary structure over long periods of time.

[0270] To validate the CD stability data, the bioactivity of insulin isolated from CAGE at different time points was assessed in nondiabetic rats. To this end, overnight-fasted rats were subcutaneously injected with insulin (isolated from CAGE and resuspended in sterile saline), and blood glucose was monitored over an 8-hour period. No significant differences in bioactivity were observed between fresh insulin and insulin-CAGE stored at RT for 1-2 months or at 4°C for 1-4 months (Figure 10). While a decline in potency was observed for insulin stored at RT for 3 months, insulin stored with CAGE at 4°C did not show any loss of bioactivity even after 4 months of storage. This clearly demonstrates that CAGE is an excellent solvent for long-term storage of insulin.

[0271] Upon intrajejunal administration, insulin-CAGE caused significant hypoglycemia and demonstrated exemplary pharmacokinetics. To measure the blood glucose-lowering efficacy of insulin-CAGE, 3–5 U / kg insulin was dispersed in CAGE and administered intrajejunally to anesthetized nondiabetic rats. Blood glucose was then monitored every 0.5 hours for a total of 5 hours (Figure 16). Rats treated with 3 U / kg insulin-CAGE showed a steady decline in blood glucose levels beginning at 0.5 hours, reaching a 45% drop from initial levels by 2.5 hours (55 ± 6%). Beyond this point, blood glucose levels plateaued, finishing at 47% by 5 hours. The group treated with 5 U / kg insulin-CAGE showed a rapid decrease in blood glucose levels, reaching a drop of approximately 65% ​​within 1.5 and 2 hours (37 ± 5% and 35 ± 5%, respectively). Blood glucose subsequently increased, a normal homeostatic response in nondiabetic rats exposed to a sudden and rapid drop in blood glucose levels. At the end of 5 hours, blood glucose levels were 74% of initial levels. Rats receiving subcutaneous injections of 2 U / kg insulin also showed a similar pattern of blood glucose decline, but to a lesser extent compared to 5 U / kg insulin-CAGE. A maximum decline of 51% was observed at 1 hour (49 ± 8% of initial levels), which rapidly recovered to 100% by the final 5 hours of the study (101 ± 7%). Controls, such as CAGE alone, saline, or intrajejunal administration of 3 U / kg insulin-saline, did not result in a significant decline in blood glucose. In all these control groups, blood glucose declined slowly (most likely due to continued fasting) to approximately 25% of initial levels by 5 hours. The pharmacokinetics of insulin absorption and excretion were determined by measuring serum insulin levels at different time points (Figure 17). Insulin concentrations rapidly increased and subsequently declined within 1 hour after SQ injection of 2 U / kg insulin and intrajejunal (IJ) administration of 5 U / kg insulin-CAGE, and excretion followed a similar pattern. Pharmacokinetic parameters calculated using serum insulin concentrations indicated that the elimination half-life of intrajejunally administered insulin-CAGE was approximately two-fold longer than that of SQ insulin (Table 2). The oral bioavailability of 5 U / kg IJ insulin-CAGE calculated in this way was found to be 51%.The pharmacodynamic bioavailability of the formulation calculated from the efficacy plot was 66%. Conversely, 5 U / kg insulin-saline administered IJ did not show any increase in insulin levels over time.

[0272] Table 2. Pharmacokinetic parameters of intrajejunally administered insulin-CAGE and subcutaneously administered insulin solution TIFF2026012396000011.tif29169 * Disappearance rate constant (K el ); Half-life (t 1 / 2 ); Area under the concentration curve (AUC); Bioavailability (F)

[0273] Orally administered insulin-CAGE capsules showed remarkable efficacy in lowering blood glucose levels The remarkable efficacy of CAGE in enhancing the oral bioavailability of insulin when administered intrajejunally led us to investigate its efficacy when delivered orally using capsules. To this end, 10 U / kg of insulin-CAGE or its control was placed in enteric-coated elongated size 9 capsules and administered to nondiabetic rats by oral gavage.

[0274] The group treated with 10 U / kg insulin-CAGE showed a rapid 38% drop in blood glucose levels within 2 hours after capsule administration (62 ± 2% of initial levels) (Figure 18). Beyond this time point, blood glucose slowly but steadily declined by approximately 45% over 10 hours (55 ± 3%). In contrast, subcutaneous administration of 2 U / kg insulin led to a rapid 49% drop in blood glucose levels at 1 hour (51 ± 5%), which steadily rose, subsequently reaching a peak of 88% of initial levels at 4 hours. Thereafter, blood glucose levels declined in a pattern similar to the CAGE-only, empty capsule, and insulin-saline formulation controls. As previously observed with IJ administration, insulin-CAGE in capsules, unlike SQ insulin, demonstrated significantly sustained efficacy through the end of the study.

[0275] To assess whether CAGE alone acts as a permeation enhancer, we administered CAGE alone in a capsule to rats, followed by 10 U / kg of insulin powder in a capsule after a 0.5-hour delay (Figure 20). While a significant difference in efficacy was observed between simultaneous and sequential administration of insulin-CAGE in the first few hours of the study (compare Figure 20 with Figure 18), no significant difference was observed between insulin solution (no CAGE) and sequential administration of insulin and CAGE throughout all time points (Figure 20). This study further demonstrates that administering insulin and CAGE together results in significant efficacy.

[0276] CAGE showed good oral biocompatibility in vivo Histological examination of small intestinal samples collected 5 hours after jejunal administration, 12 hours after oral capsule administration, or 7 days after repeated daily administration showed no significant differences in morphology between CAGE and saline- or insulin-treated animals (Figure 19). Furthermore, no significant structural damage was noted in the small intestinal tissue. Notably, finger-like villi were found in all tissues. The results clearly demonstrate the excellent biocompatibility of CAGE with intestinal tissue, thereby verifying the suitability of the formulation for oral administration.

[0277] Consideration Over the past few decades, the prevalence of diabetes has grown so steadily that it is now being referred to as the "epidemic of the century" (14). Rising diabetes cases have been reported in all countries, with the fastest growth in low- and middle-income countries and the highest prevalence in the Middle East and North Africa region (14-16). A recent World Health Organization report indicated that diabetes was responsible for 1.5 million deaths worldwide in 2012, with an additional 3.2 million deaths due to hyperglycemia-related comorbidities (16). In the United States, 30.3 million people (approximately 9.4% of the population) were reported to be afflicted with the disease in 2015, with 1.5 million new cases diagnosed annually (17). Current suggestions for managing this disease include insulin therapy, either alone or in combination with oral hypoglycemic agents such as metformin (18). Individuals receiving insulin therapy alone often require the administration of either twice-daily intermediate-acting insulin or once-daily long-acting insulin (18). Insulin is not available as an oral pill in outpatient clinics and is administered exclusively as a subcutaneous injection. However, despite its effectiveness in managing hyperglycemia and mitigating the risk of neuropathy, nephropathy, and retinopathy, injectable insulin leads to lower patient compliance due to pain, interference with daily activities, and embarrassment, resulting in willful neglect and poor long-term glycemic control in as many as 60% of patients (5, 19). This leads to elevated hemoglobin A1C levels and increased hospitalizations due to diabetes-related complications (19). To circumvent this problem, MannKind Corporation developed Afrezza®, an inhalable, fast-acting insulin formulation for postprandial glycemic control; however, it has been associated with pulmonary risks, including higher incidence of lung cancer and diabetic ketoacidosis, decreased lung function, and a higher risk of acute bronchospasm in patients with chronic lung disease (20). Given the rapid growth and scale of diabetes, it is essential to develop insulin therapies that are patient-friendly and avoid formulation-based adverse effects.

[0278] Oral delivery enjoys high patient compliance but is not suitable for the delivery of biologics. This is due to the fact that orally delivered drugs must traverse the acidic environment of the stomach, which can degrade protein / peptide drugs. This can be avoided by encapsulating them in enteric or other protective coating systems. However, upon release from their protective casing in the intestine, peptide / protein drugs are thrust into the intestinal proteolytic environment and easily cleaved into smaller amino acid units by resident enzymes. Even if a proportion of the drug escapes proteolysis, it is difficult for it to be absorbed as an intact molecule through the intestinal mucus layer and enterocytes into the systemic circulation. Oral insulin absorption is further hindered by erratic GI transit times and the lack of specific insulin uptake mechanisms in the intestine (21). Disruption of insulin structure during GI transit may lead to significant denaturation and loss of biological activity. Not surprisingly, therefore, protein and peptide drugs have negligible oral bioavailability, less than 1%, a stark contrast to injectable formulations, in which 100% of the dose is available for pharmacological activity (22). Several researchers have attempted to solve the perennial problem of low oral insulin bioavailability by modifying the insulin molecule, encapsulating it in novel carriers, or using enteric coatings, absorption enhancers, or proteolytic inhibitors. Some examples include the use of poly(lactic-co-glycolic acid) (PLGA) or chitosan-based nanoparticles for oral insulin delivery. Pan et al. obtained a pharmacological bioavailability of 10.3% using 10 U / kg insulin loaded in PLGA nanoparticles and 15.3% using 21 U / kg insulin in chitosan nanoparticles, whereas Cui and colleagues obtained oral bioavailabilities of 3.7 and 6.3%, respectively, using 20 U / kg insulin placed in PLGA and PLGA-55 nanoparticles. (23-25)Sarmento and colleagues encapsulated insulin using chitosan-dextran nanoparticles and observed 5.6 and 3.4% pharmacological bioavailability after placing 50 and 100 U / kg insulin in the particles, respectively. (26) Alginate-chitosan nanoparticles improved the oral bioavailability of insulin to 6.8 and 3.4%, respectively, for insulin doses of 50 and 100 U / kg. (27) Zhang and colleagues encapsulated 50 U / kg insulin in solid lipid nanoparticles (SLNs) and wheat germ agglutinin-modified SLNs, obtaining pharmacological bioavailability of 4.5 and 6.1%, respectively. (28) Similarly, Ansari et al. reported a fivefold enhancement in the oral bioavailability of insulin using SLNs compared with orally administered insulin solution (8.3% vs. 1.7%). (29) High oral bioavailability of insulin (37.6%) was achieved by orally delivering insulin in biodegradable polyisobutylcyanoacrylate nanospheres, but at a high dose of 75 U / kg (30). Other strategies to improve oral protein delivery involve the use of protease inhibitors such as sodium glycocholate, aprotinin, soybean trypsin inhibitor, bacitracin, and camostat mesilate (31). Chemical modifications of insulin, such as attaching targeting ligands like transferrin or cell-penetrating peptides like the TAT peptide, have been shown to aid insulin transcytosis across enterocytes (32, 33). The insulin analog IN-105, obtained through the conjugation of short chains of PEG to insulin, has been demonstrated to orally dose-dependently reduce postprandial blood glucose in patients with type 2 diabetes through improved solubility, proteolytic stability, and intestinal absorption (34, 35). Absorption enhancers include bile salts, surfactants, fatty acids, calcium ion chelators, certain polymers such as chitosan / thiolated chitosan, and zonula occludens toxins, which function either by modulating the cell membrane structure of the intestinal epithelium for transcellular uptake or by tight junction permeability for paracellular transport ( 31 ).

[0279] ILs comprise a group of salts with organic cations and organic / inorganic anions, and are typically liquid below 100°C (36). Choline and geranic acid are both recognized by the Food and Drug Administration (FDA) as GRAS (Generally Regarded as Safe) ingredients. Choline, an important component of lecithin, is present in both plants and animals, is necessary for various physiological functions, and has an oral lethal dose (LD50) of 3,400 mg / kg. 50 ), whereas geranic acid, which is widely used as a food flavoring, has an oral LD ​​of 3,700 mg / kg in rats. 50 (43, 44) Furthermore, in a comprehensive review of the toxicity of ionic liquids, choline was found to exhibit the lowest toxicity among various other cationic head groups (45). In this study, the oral CAGE dose after a single administration was 80 mg (80 μL), which is composed of approximately 27 mg of choline and 53 mg of geranic acid. Therefore, the oral LD ​​of each individual component was 50 Adequate intakes of choline are considered to be 550 mg per day for men and 425 mg per day for women (46). Gelatinous acid, on the other hand, is a common food additive found in cardamom, lemongrass, petitgrain, and other essential oils (47).

[0280] CAGE was found to significantly reduce the viscosity of mucin hydrogels at 1 and 5% w / v concentrations (Figure 21), indicating that CAGE aids in in vivo mucus penetration, a critical barrier for oral uptake of macromolecules.

[0281] In vivo, CAGE demonstrated remarkable efficacy in enhancing oral insulin uptake when administered either intrajejunally or in capsules. Intrajejunally injected 3 U / kg insulin-CAGE led to a 47% drop in blood glucose levels at 5 hours, comparable to the drop observed with subcutaneously injected 2 U / kg insulin. With 5 U / kg insulin-CAGE, an even more dramatic 65% drop in blood glucose levels was observed at 2 hours, and levels remained significantly lower compared with subcutaneously injected insulin for the remainder of the study. This is one of the lowest oral insulin doses found in the literature demonstrating such remarkable efficacy. Among other notable work, Pan and colleagues demonstrated a 52% drop in blood glucose levels at 4 hours when 10 U / kg insulin in PLGA particles was orally administered (24). Using vitamin B12-conjugated dextran nanoparticles loaded with 20 U / kg insulin, Chalasani et al. achieved a 70–75% reduction in blood glucose and an oral bioavailability of 29.4% (51). Intrajejunal administration of 10 U / kg insulin using bile salt mixed micelles in dogs resulted in an absolute bioavailability of 1.8% (52). In rats, placement of a rectangular mucoadhesive patch loaded with 50 U / kg insulin in the jejunum resulted in a relative bioavailability of 3.9%. In the presence of the permeation enhancer dimethyl palmitoyl ammoniopropanesulfonate (PPS), the relative bioavailability increased to 7.7% (53). Yin and colleagues demonstrated that intestinal injection of trimethylchitosan-cysteine ​​conjugate nanoparticles encapsulating 50 U / kg insulin reduced blood glucose levels by 70% in rats (54). Using Labrasol™ to improve oral absorption of insulin, Takada and colleagues observed bioavailabilities of 0.25 and 0.2% for intraileal and intracolonic administration, respectively (55).Intragastric delivery of a lecithin-based microemulsion containing 200 IU / kg resulted in bioavailabilities of 0.148 (without aprotinin) and 0.159 (with aprotinin) in normal rats ( 56 ).

[0282] Herein, high jejunal bioavailability of insulin was noted. The elimination half-life of insulin-CAGE was also noted to be approximately twofold higher than that of subcutaneously injected insulin, indicating a more sustained efficacy. To verify the significant efficacy previously observed, insulin-CAGE was further encapsulated in enteric-coated capsules and orally administered to rats. The enteric coating prevents capsule disintegration in the acidic environment of the stomach and releases the encapsulated material only in the more alkaline environment of the small intestine (57). Therefore, bypassing the stomach using enteric-coating technology prevents degradation of biologics by gastric acid. To account for dose dilution during insulin-CAGE release in the intestine, a higher insulin dose (10 U / kg) than that used for intravenous (IJ) delivery was used. Again, insulin-CAGE produced a similar degree of blood glucose reduction compared to subcutaneously injected insulin. However, unlike SQ insulin, the efficacy of insulin-CAGE persisted until the final 12-h time point of the study, demonstrating its suitability for long-acting oral insulin formulations. Administration of CAGE alone followed by insulin 0.5 hours later did not result in any significant blood glucose lowering efficacy (Figure 20), indicating that co-administration of insulin and CAGE is necessary to achieve significant in vivo efficacy.

[0283] In addition to high efficacy, CAGE exhibited biocompatibility and long-term stability for potential application in outpatient clinics (Figure 19). To determine its in vivo safety, small intestinal sections were isolated from rats treated with CAGE alone or insulin-CAGE after either a single dose or repeated daily doses for one week. No toxicity was found at the morphological level after intrajejunal or oral administration, demonstrating the excellent oral tolerability of CAGE in vivo.

[0284] As assessed through secondary structure and in vivo bioactivity assessments, insulin-CAGE was stable for 2 months at RT and at least 4 months at 4°C (Figures 15 and 10). Physicochemical degradation of insulin occurs primarily due to hydrolysis, aggregation, and intermolecular alteration reactions, leading to loss of potency (60). It has been reported that ionic liquids can prevent protein interactions with water molecules, while protic ionic liquids stabilize the native conformation of several amino acids and insulin, mitigating its tendency to self-aggregate (59, 61). Without wishing to be bound by theory, it is contemplated herein that storing insulin with CAGE alone prevents protein interactions with water, mitigates hydrolysis interactions, and stabilizes its alpha-helical secondary structure. Considering that the monomeric form of insulin is the bioactive form, the enhanced oral bioactivity of insulin in CAGE may also be partially attributed to the presentation of insulin molecules as monomers to enterocytes and the systemic circulation.

[0285] Overall, described herein is the development of a highly effective oral insulin formulation with tremendous potential for clinical use. Insulin-CAGE can be painlessly prepared in a single-step process, allowing for easy scaling for industrial production. This product does not require modification of the insulin structure or the development of complex nanostructures, preventing the generation of an immune response to the modified protein or loss of active ingredient during multi-step formulation development. Furthermore, this simple and robust formulation, consisting only of insulin and an ionic liquid made from GRAS components, eliminates the need for any additives to enhance efficacy. The formulation demonstrated significant efficacy in vivo at very low insulin doses. By delivering insulin-CAGE in an enteric-coated capsule, we successfully circumvented gastrointestinal degradation of insulin and enhanced its intestinal permeability, thereby overcoming barriers to oral delivery of biologics. Furthermore, this formulation is biocompatible and has good long-term stability. The results of this study may facilitate the realization of oral insulin delivery in outpatient clinics.

[0286] material and method material Geranic acid, choline bicarbonate, dimethyl sulfoxide (DMSO), FITC-insulin, FITC-dextran, sodium caprate (98% pure), mucin, and human insulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bovine trypsin was obtained from MP Biomedicals (Santa Ana, CA, USA). Caco-2 human colorectal adenocarcinoma cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Dulbecco's modified Eagle's medium (DMEM), Hank's balanced salt solution (HBSS), Dulbecco's phosphate-buffered saline (DPBS), and 0.25% trypsin solution were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Intestinal epithelial growth medium containing basal seeding medium (BSM), enterocyte differentiation medium (EDM), and MITO+ serum extender were purchased from Corning (Corning, NY, USA). Millicell®-PCF cell culture inserts (3.0 μm pore size, 12 mm diameter) and the TEER measurement device Millicell®-ERS were obtained from Millipore Sigma (Burlington, MA, USA). The TEER measurement electrode was obtained from World Precision Instruments, Inc. (Sarasota, FL, USA). Paraformaldehyde (16% w / v) and metoclopramide hydrochloride were purchased from Alfa Aesar (Ward Hill, MA, USA). Vectashield Hardset™ with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) was obtained from Vector Laboratories Inc. (Burlingame, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity kit and human insulin ELISA kit were obtained from ThermoFisher Scientific (Waltham, MA, USA).Male Wistar rats weighing between 200 and 300 g were purchased from Charles River Laboratories (Wilmington, MA, USA), and blood glucose measurement meters (Aimstrip plus) along with their strips were purchased from Fisher Scientific (Pittsburgh, PA, USA). Capsule oral gavage and size 9 elongated capsules were obtained from Torpac (Fairfield, MA, USA). Hematoxylin and eosin solution was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents used were of analytical grade.

[0287] Preparation of CAGE and insulin-CAGE CAGE was synthesized according to our previous work (38). Briefly, 2 equivalents of geranic acid alone (20 g, 0.119 mol), which had been recrystallized at least five times in acetone at <-70 °C to remove impurities, were added to 1 equivalent of choline bicarbonate (80 wt% solution, 12.275 g, 0.059 mol) in a 500 mL round-bottom flask. The mixture was stirred at 40 °C until CO2 evolution ceased, and water was removed by rotary evaporation at 60 °C for 2 h, followed by drying in a vacuum oven at 60 °C for 48 h. Physical characterization at 25 °C showed good agreement with previous values. NMR spectra (collected using a 500-MHz Varian instrument, Palo Alto, CA) also showed good agreement with previous preparations: TIFF2026012396000012.tif27160 Insulin-CAGE was prepared by adding a predetermined amount of insulin powder to a specific volume of CAGE, followed by vortexing for 5 minutes.

[0288] Assessment of insulin stability during CAGE Samples containing human insulin (100 U, 3.5 mg) were suspended in 1 mL of either CAGE or phosphate-buffered saline (PBS) in a 2 mL microcentrifuge tube and incubated at room temperature (25°C) or under refrigeration (4°C). After 1 month and approximately monthly thereafter for a total of 4 months, the samples were centrifuged at 10,000 × g for 10 minutes, the CAGE removed via pipette, and the soft insulin pellet washed with 1 mL of PBS and centrifuged again. The PBS-CAGE was removed, and the washing / centrifugation process was repeated until no insulin formed a pellet during centrifugation. The collected insulin was analyzed for its stability and in vivo bioactivity using CD (described in the SI section). To collect spectra in the far-UV region (190–250 nm), which indicates the secondary structure of proteins, CD spectrophotometry (Jasco J-1500, Easton, MD) was performed using a rectangular quartz cell (1 mm path length, Starna Cells, 1-Qq, Atascadero, CA) loaded with 400 μL of sample.

[0289] Determining the efficacy of insulin-CAGE upon intrajejunal administration and assessing pharmacokinetic parameters The efficacy of intrajejunally injected insulin-CAGE was determined in nondiabetic adult male Wistar rats that were fasted overnight but allowed free access to water. Fasting before antidiabetic efficacy studies is routinely performed (29, 62, 63). This helps to avoid fluctuations in blood glucose levels as a result of feeding, which can vary between animals based on the amount and time of food intake. All animal experiments were performed in accordance with the animal care committee guidelines of the University of California, Santa Barbara, and the National Research Council's Guide for the Care and Use of Animals of the Institute of Laboratory Animal Resources. Prior to the start of the study, rats were anesthetized, abdominal hair was clipped, and the surgical area was prepared using 70% ethanol and betadine. An abdominal incision was made to expose the intestine. The jejunum was located and injected with 100 μL of either 3 or 5 U / kg insulin-CAGE or 100 μL of control. After exposing the intestine, blood glucose was measured at time 0, i.e., immediately before injection. Each formulation was tested in six rats, except for saline, which was tested in only three rats. The intestinal segment was then returned to the abdomen, and the muscle and skin were sutured. Blood glucose was determined using a commercially available glucose meter at the beginning and every 0.5 hours until the final 5-hour time point of the study. Loss of body heat in the animals during anesthesia was prevented by placing them on a temperature-controlled heating pad before surgery, followed by an additional towel cover after surgery. The animals remained anesthetized throughout the entire study and were euthanized 5 hours later. At the final 5-hour time point of the study, a segment of intestine around the injection site was removed for further histological examination to determine toxicity, if any. To compare efficacy, another group of three rats was subcutaneously injected with 2 U / kg insulin in saline. Results were plotted as the percentage change in blood glucose relative to the initial reading over time.Additionally, the saline group was considered the fasting control, and graphs were plotted after subtracting the blood glucose levels of the saline group. The pharmacokinetics of insulin-CAGE was assessed by collecting approximately 250 μL of blood into BD Vacutainer® Red Top tubes (Becton, Dickinson and Company, Franklin Lanes, NJ, USA) at 0, 1, 2, 3, and 5 h from rats injected with 5 U / kg insulin-CAGE intrajejunally, 5 U / kg insulin-saline intrajejunally, and 2 U / kg insulin-saline subcutaneously. Serum was separated from whole blood according to standard protocols. Briefly, blood samples were allowed to clot at RT for 15–30 min, followed by centrifugation at 2,000 g for 10 min. The clear supernatant (serum) was then collected into clean tubes and stored on ice during the procedure and at -20°C until further analysis of insulin content. To assess insulin concentrations in serum samples, human insulin ELISA was used to determine insulin concentrations at each time point according to the manufacturer's protocol. The elimination rate constant (K) was calculated from a plot of serum insulin concentration versus time. el ), half-life (t 1 / 2 Pharmacokinetic parameters such as area under the concentration curve (AUC), and % bioavailability (F) were calculated. The AUC obtained from the efficacy plot was used to calculate the pharmacodynamic bioavailability.

[0290] In vivo oral efficacy To determine the efficacy of insulin-CAGE administered orally, elongated size 9 capsules were used. These capsules were filled with 80 μL of either 10 U / kg insulin-CAGE, CAGE alone, or left empty. Following this, the capsules were enteric-coated three times with 12.5% ​​w / v Eudragit® L-100 dissolved in isopropanol. For oral efficacy studies, nondiabetic adult male Wistar rats were fasted overnight but allowed free access to water. The next day, the capsules were administered to the rats via oral gavage, followed by subcutaneous administration of 5 mg / kg metoclopramide hydrochloride to promote gastric emptying. Blood glucose was then measured using a commercially available glucose meter, followed by hourly measurements for up to 12 hours. The rats were fasted throughout the entire study period. A control of 10 U / kg insulin in saline was also tested by orally administering the formulation as a solution (without capsules). Additionally, the efficacy of subcutaneously injected 2 U / kg insulin-saline was assessed. Groups of six rats per formulation were used for 10 U / kg insulin-CAGE and CAGE alone, while three rats per group were utilized to study the efficacy of empty capsules, 10 U / kg insulin solution, and subcutaneously injected 2 U / kg insulin-saline. Results were plotted as the percentage change in blood glucose levels over time. Additionally, the empty capsule group was considered the fasting control, and graphs were plotted after subtracting the blood glucose levels of the empty capsule group. Twelve hours after the study, the rats were euthanized, and small intestinal sections were collected for tissue histology.

[0291] Repeated dose biocompatibility The biocompatibility of CAGE during repeated dose administration was assessed in non-diabetic rats after repeated daily administration of CAGE-only capsules, 10 U / kg insulin-CAGE capsules, and 10 U / kg insulin in capsules over a 7-day period. All animals were euthanized on day 8, and their small intestinal tissue sections were collected for tissue histology.

[0292] Tissue histology Small intestinal tissue was fixed in 10% buffered formalin, dehydrated in ethanol, and embedded in paraffin. Five-micron cross sections of intestinal tissue were deparaffinized, rehydrated, and stained with hematoxylin and eosin. Histological morphology was examined using a light microscope at 10x and 40x magnification (Olympus BX60™ upright compound microscope).

[0293] Data analysis All data are presented as mean ± standard error (SE). Student's T-test was used for statistical analysis. Differences were considered significant at p<0.05. All experiments were performed at least in triplicate.

[0294] References TIFF2026012396000013.tif150160TIFF2026012396000014.tif231158TIFF20260123960 00015.tif223159TIFF2026012396000016.tif223160TIFF2026012396000017.tif187160

[0295] Example 4: Supplementary material to Example 3 result The biological activity of insulin isolated from CAGE was maintained even after long-term storage. To verify the long-term stability data of insulin-CAGE obtained using CD, the bioactivity of insulin isolated from CAGE at different months was assessed in nondiabetic rats (Figure 10). Freshly prepared insulin solution (insulin in saline) resulted in a drop in blood glucose levels of approximately 49% (51 ± 2%) in 1 hour, which persisted for the next hour (50 ± 4%). Blood glucose levels then rose to 92% (92 ± 5%) in 5 hours and then slowly dropped again to approximately 30% of the initial value as a result of continued fasting. In comparison, insulin isolated from CAGE stored at various temperatures and for different time points followed a similar pattern of blood glucose drop at 1 and 2 hours after injection, with no significant difference in the drop rate compared to fresh insulin. However, a significant reduction in efficacy was observed for insulin stored at room temperature (RT) for 3 months. In this group, a significantly lower 38% drop in blood glucose levels was noted 1 and 2 hours after insulin injection (62±2% and 62±3%, respectively), indicating that insulin loses some of its bioactivity after 2 months of storage at RT. However, insulin stored at 4°C with CAGE retained its bioactivity for at least 4 months of storage.

[0296] Co-administration of CAGE and insulin is necessary for significant therapeutic efficacy When CAGE alone and 10 U / kg insulin were administered separately after 0.5 hours, blood glucose decreased slowly and did not differ significantly at any time point compared to 10 U / kg insulin solution (Figure 20). At the 10-hour point in the study, blood glucose decreased by approximately 34% of the initial level for both groups (66.3±4.9 and 66.9±7.1% for sequential administration and insulin solution, respectively). This indicated that CAGE did not cause any long-term permeabilization of the intestinal membrane and significant blood glucose-lowering efficacy could only be achieved when insulin and CAGE were co-administered as an insulin-CAGE mixture.

[0297] Impact of CAGE on Caco-2 monolayer viability The effect of CAGE on Caco-2 intestinal cells was studied in vitro. Cell viability was first determined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay. Monolayers were exposed to various concentrations of CAGE for 5 hours, and cell viability values ​​were normalized to the control without CAGE treatment (Figure 21). No adverse effects on cell proliferation were observed with 10 mM CAGE, and a high cell viability of 86% was obtained using 25 mM CAGE. A slight decrease of 69% viability was obtained with 50 mM CAGE.

[0298] CAGE significantly enhanced insulin transport across the intestinal monolayer Five-hour long transport experiments of fluorescein isothiocyanate (FITC)-insulin across Caco-2 monolayers were developed using 0, 10, 25, and 50 mM CAGE. In both groups, FITC-insulin transport steadily increased with time throughout the study (data not shown).

[0299] These results were corroborated through confocal images of the transwell membranes at the final 5 h time point of the study, which clearly show a higher uptake of FITC-insulin by Caco-2 cells with increasing concentrations of CAGE compared to control cells (data not shown).

[0300] CAGE-mediated drug transport across enterocytes is primarily paracellular Transport of molecules across the intestinal epithelium can be either paracellular or transcellular. To assess the mechanism by which CAGE enhances drug transport across Caco-2 monolayers, both paracellular and passive transcellular transport were investigated using specific markers for these transports in the presence of various concentrations of CAGE. To this end, transport of 4 kDa FITC-dextran (a specific marker for paracellular transport) was assessed (1).

[0301] Transport of 4 kDa FITC-dextran, which has a size similar to that of insulin, was also significantly enhanced in the presence of 25 and 50 mM CAGE at various time points (data not shown). However, to confirm whether CAGE improved paracellular transport of molecules, we assessed the tight junction integrity of Caco-2 cells treated with various concentrations of CAGE by measuring transepithelial electrical resistance (TEER) and comparing it with that of sodium caprate, an established permeation enhancer (data not shown) (2). In control cells, TEER decreased slightly by approximately 10% over the first 3 hours, then recovered to its initial value by 5 hours and remained unchanged until the final 24-hour time point of the study. However, upon treatment with 10 mM CAGE, TEER decreased by 29% at 1 hour and was significantly different from the control at both the 1-hour and 5-hour time points. This decrease in TEER was found to be transient, and the cells fully restored tight junction integrity within 24 hours.

[0302] Although the drop in TEER caused by sodium caprate was similar to that of 50 mM CAGE, tight junction integrity in this group further decreased to 36% of initial levels by the final 24-hour time point of the study. The changes in TEER caused by 10–25 mM CAGE suggest that at these concentrations, CAGE transiently opens intestinal tight junctions, which aids in transcellular insulin transport. Other mechanisms of oral absorption enhancement mediated by CAGE, such as mucus penetration and stability against enzymatic degradation, were also subsequently assessed.

[0303] CAGE led to thinning of mucus Incubation of CAGE with simulated mucus (SM) resulted in a very significant drop in viscosity (Figure 21). SM exhibited a shear-thinning profile similar to that reported for gastric mucus (4), and the SM viscosity profile compared favorably with literature values ​​for healthy human duodenal gastric mucus (5). For example, at a shear rate of 46 l / s, the literature value was 12.3 cP, compared with the SM mean value of 11.3 cP measured at 50.12 l / s. Addition of 1 and 5% CAGE (23 and 115 mM, respectively) reduced viscosity throughout the entire shear range measured. The reduced viscosity indicates that CAGE aids in mucus penetration in vivo, thus facilitating insulin delivery to the intestinal epithelium while protecting it from proteolysis at luminal, mucosal, and epithelial sites.

[0304] method In vivo evaluation of long-term stability of insulin in CAGE The biological activity of insulin-CAGE stored at RT or 4°C was assessed at different months by subcutaneously injecting 1 U / kg of insulin isolated from CAGE into nondiabetic adult male Wistar rats. Blood glucose levels were monitored for 8 hours using a commercially available glucose meter and compared with a freshly prepared, subcutaneously injected 1 U / kg insulin solution. Rats were fasted overnight before insulin injection but had free access to water and continued fasting throughout the study to eliminate fluctuations in blood glucose due to food consumption. Results were plotted as the % change in blood glucose compared to initial levels over time.

[0305] Sequential administratio...

Claims

1. A method for oral delivery of at least one active compound, comprising orally administering the active compound in combination with a composition comprising the ionic liquid choline and geranate (CAGE).

2. A method for delivering at least one active compound, comprising subcutaneously, intradermally, or intravenously administering the active compound in combination with a CAGE.

3. A method for delivering at least one active compound, comprising administering to a mucosal membrane an active compound in combination with a CAGE.

4. 4. The method of claim 3, wherein the mucosa is a nasal mucosa, an oral mucosa, or a vaginal mucosa.

5. A method for parenteral delivery of at least one active compound, comprising parenterally administering the active compound in combination with a CAGE.

6. 6. The method of claim 5, wherein the administering comprises delivery to a tumor.

7. A method of treating a disease in a subject in need thereof by administering to the subject an active compound in combination with CAGE by injection into the affected tissue.

8. 8. The method of claim 7, wherein the disease is cancer, adipose, hyperplasia, or any other disease caused by tissue proliferation.

9. 9. The method of any one of claims 1 to 8, wherein the CAGE is at a concentration of at least 0.1% w / v.

10. 10. The method of any one of claims 1 to 9, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:

10.

11. 10. The method of any one of claims 1 to 9, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:

4.

12. 10. The method of any one of claims 1 to 9, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:

4.

13. 13. The method of any one of claims 1 to 12, wherein the anion of the ionic liquid comprises geranate and / or geranic acid.

14. 14. The method of any one of claims 1 to 13, wherein the active compound in combination with CAGE is administered once.

15. 15. The method of any one of claims 1 to 14, wherein the active compound in combination with CAGE is administered in multiple doses.

16. 16. The method of any one of claims 1 to 15, wherein the active compound comprises a nucleic acid molecule.

17. 16. The method of any one of claims 1 to 15, wherein the active compound comprises a small molecule.

18. 16. The method of any one of claims 1 to 15, wherein the active compound comprises a polypeptide.

19. 16. The method of any one of claims 1 to 15, wherein the active compound comprises an antibody or antibody reagent.

20. 20. The method of any one of claims 1 to 19, wherein the active compound comprises a chemotherapeutic compound.

21. 21. The method of any one of claims 1 to 20, wherein the active compound comprises insulin.

22. 21. The method of any one of claims 1 to 20, wherein the active compound comprises a GLP-1 polypeptide or a mimetic or analog thereof.

23. 23. The method of any one of claims 21-22, wherein the active compound is provided at a dosage of 1-20 mg / kg.

24. A composition comprising an active compound in combination with a CAGE.

25. 25. The composition of claim 24, wherein the CAGE is at a concentration of at least 0.1% w / v or 5% w / w.

26. 26. The composition of any one of claims 24-25, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:

10.

27. 27. The composition of any one of claims 24-26, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:

4.

28. 28. The composition of any one of claims 24-27, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:

4.

29. The composition of any one of claims 24 to 28, wherein the active compound comprises a nucleic acid molecule.

30. The composition of any one of claims 24 to 28, wherein the active compound comprises a small molecule.

31. The composition of any one of claims 24 to 28, wherein the active compound comprises a polypeptide.

32. The composition of any one of claims 24 to 31, wherein the active compound comprises an antibody or antibody reagent.

33. 33. The composition of any one of claims 24 to 32, wherein the active compound comprises a chemotherapeutic compound.

34. 31. The composition of any one of claims 24 to 30, wherein the active compound comprises insulin.

35. 32. The composition of any one of claims 24 to 31, wherein the active compound comprises a GLP-1 polypeptide or a mimetic or analog thereof.

36. 36. The composition of any one of claims 34-35, wherein the active compound is provided in a dosage of 1-20 mg / kg.

37. 37. The composition of any one of claims 24 to 36, further comprising an additional pharmaceutically acceptable carrier.

38. 38. The composition of any one of claims 24 to 37, formulated as an oral, subcutaneous, or parenteral formulation.

39. 39. The composition of any one of claims 24 to 38, formulated for administration to a mucosal membrane.

40. 40. The composition of claim 39, wherein the mucosa is a nasal mucosa, an oral mucosa, or a vaginal mucosa.

41. 40. The composition of claim 38, wherein the oral formulation is a degradable capsule containing the combination of the active compound and the CAGE.

42. 42. The composition of any one of claims 24 to 41, wherein the biological activity of the active compound is improved or stabilized compared to the activity in the absence of CAGE.

43. 43. The method or composition of any one of claims 1 to 42, wherein the combination of active compound and CAGE is an admixture.

44. 43. The method or composition of any one of claims 1 to 42, wherein the combination of active compound and CAGE comprises nanoparticles comprising the active compound, wherein the nanoparticles are in solution or suspension in a composition comprising the CAGE.

45. A method for delivering a nucleic acid molecule to a cell, comprising contacting the cell with the nucleic acid molecule in combination with a composition comprising the ionic liquid choline and geranate (CAGE).

46. 46. ​​The method of claim 45, wherein the cell is a cell in a subject, and the contacting step comprises administering to the subject the nucleic acid molecule in combination with a composition comprising the ionic liquid Choline And Geranate (CAGE).

47. 47. The method of any one of claims 45-46, wherein the nucleic acid molecule comprises a vector, an expression vector, or an inhibitory nucleic acid molecule.

48. 48. The method of any one of claims 45 to 47, wherein the CAGE is at a concentration of at least 0.1% w / v or 5% w / w.

49. 48. The method of any one of claims 45-47, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:

10.

50. 48. The method of any one of claims 45-47, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:

4.

51. 48. The method of any one of claims 45-47, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:

4.

52. 52. The method of any one of claims 45 to 51, wherein the anion of the ionic liquid comprises geranate and / or geranic acid.

53. 53. The method of any one of claims 45 to 52, wherein the combination of the nucleic acid molecule and the CAGE is an admixture.

54. 54. The method of any one of claims 45 to 53, wherein the combination of nucleic acid molecule and CAGE comprises nanoparticles comprising the nucleic acid molecule, the nanoparticles being in solution or suspension in a composition comprising the CAGE.

55. At least one active compound in combination with a composition comprising the ionic liquid Choline and Geranate (CAGE) for oral delivery, mucosal delivery, parenteral delivery, or use in treating disease.

56. 56. The combination of claim 55, wherein the mucosa is nasal, oral, or vaginal mucosa.

57. 56. The combination of claim 55, wherein the parenteral administration comprises delivery to a tumor.

58. 56. The combination of claim 55, wherein treatment comprises injection of said composition into the affected tissue.

59. 59. The combination of claim 58, wherein the disease is cancer, adipose, hyperplasia, or any other disease resulting from tissue proliferation.

60. 60. The combination of any one of claims 55 to 59, wherein the CAGE is at a concentration of at least 0.1% w / v or 5% w / w.

61. 60. The combination of any one of claims 55 to 59, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:

10.

62. 62. The combination of any one of claims 55 to 61, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:

4.

63. 62. The combination of any one of claims 55 to 61, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:

4.

64. 64. The combination of any one of claims 55 to 63, wherein the anion of the ionic liquid comprises geranate and / or geranic acid.

65. 65. The combination of any one of claims 55 to 64, wherein the active compound in combination with CAGE is administered once.

66. 65. The combination of any one of claims 55 to 64, wherein the active compound in combination with CAGE is administered in multiple doses.

67. 67. The combination of any one of claims 55 to 66, wherein the active compound comprises a nucleic acid molecule.

68. 67. The combination of any one of claims 55 to 66, wherein the active compound comprises a small molecule.

69. 67. The combination of any one of claims 55 to 66, wherein the active compound comprises a polypeptide.

70. 67. The combination of any one of claims 55 to 66, wherein the active compound comprises an antibody or antibody reagent.

71. 67. The combination of any one of claims 55 to 66, wherein the active compound comprises a chemotherapeutic compound.

72. 67. A combination according to any one of claims 55 to 66, wherein the active compound comprises insulin.

73. 67. A combination according to any one of claims 55 to 66, wherein the active compound comprises a GLP-1 polypeptide or a mimetic or analogue thereof.

74. 74. The combination of claim 73, wherein the active compound is provided in a dosage of 1 to 20 mg / kg.

75. A method for treating obesity, preventing weight gain, or reducing the weight of a subject, comprising orally administering to the subject a composition comprising the ionic liquid choline and geranate (CAGE).

76. 76. The method of claim 75, wherein the CAGE is at a concentration of at least 0.1% w / v.

77. 77. The method of any one of claims 75-76, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:

10.

78. 78. The method of any one of claims 75-77, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:

4.

79. 79. The method of any one of claims 75 to 78, wherein the anion of the ionic liquid comprises geranate and / or geranic acid.

80. 80. The method of any one of claims 75-79, wherein the composition further comprises an active compound.

81. 81. The method of claim 80, wherein the active compound is therapeutically effective in treating obesity.

82. 81. The method of claim 80, wherein the active compound is therapeutically effective in treating an obesity-related disorder.

83. 83. The method of any one of claims 80-82, wherein the active compound comprises a small molecule.

84. 83. The method of any one of claims 80-82, wherein the active compound comprises a polypeptide.

85. 83. The method of any one of claims 80-82, wherein the active compound comprises an antibody or antibody reagent.

86. A composition comprising the ionic liquid choline and geranate (CAGE), for use in a method for treating obesity, preventing weight gain, or reducing weight in a subject, the composition being orally administered to a subject.

87. 87. The composition of claim 86, wherein the CAGE is at a concentration of at least 0.1% w / v.

88. 88. The composition of any one of claims 86-87, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:

10.

89. 88. The composition of any one of claims 86-87, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:

4.

90. 90. The composition of any one of claims 86-89, wherein the anion of the ionic liquid comprises geranate and / or geranic acid.

91. 91. The composition of any one of claims 86-90, further comprising an active compound.

92. 92. The composition of claim 91, wherein the active compound is therapeutically effective in treating obesity.

93. 92. The composition of claim 91, wherein the active compound is therapeutically effective in treating an obesity-related disorder.

94. The composition of any one of claims 91-93, wherein the active compound comprises a small molecule.

95. 94. The composition of any one of claims 91-93, wherein the active compound comprises a polypeptide.

96. The composition of any one of claims 91-93, wherein the active compound comprises an antibody or antibody reagent.

97. A method of delivering at least one active compound comprising administering the active compound in combination with a salt-containing composition, wherein the salt is present at a concentration of at least 0.05M.

98. 98. The method of claim 97, wherein the salt is an ionic liquid.

99. 99. The method of claim 98, wherein the ionic liquid is choline and geranate (CAGE).

100. 100. The method of claim 99, wherein the cation is choline.

101. 101. The method of any one of claims 99-100, wherein the anion is geranate or geranic acid.

102. 102. The method of any one of claims 97-101, wherein delivery is oral, subcutaneous, intradermal, intravenous, parenteral, or mucosal.

103. The method of any one of claims 97-102, wherein the delivery is oral.

104. 104. The method of any one of claims 97-103, wherein the salt is present at a concentration of at least 0.05M, 0.1M, 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M, or higher.

105. 105. The method of any one of claims 97-104, wherein the salt is present at a concentration of from about 0.05M to about 4M.

106. 106. The method of any one of claims 97-105, wherein the salt dissolves after administration.

107. 106. The method of any one of claims 97-105, wherein the salt is a pure liquid or an anhydrous liquid.

108. 106. The method of any one of claims 97-105, wherein the salt is in an aqueous solution.

109. The method of any one of claims 97 to 108, wherein administering is to a subject.

110. The method of any one of claims 97 to 108, wherein administering is contacting a cell and / or tissue.

111. 111. The method of any one of claims 97-110, wherein the active compound comprises a nucleic acid molecule, a chemotherapeutic compound, a small molecule, a peptide, and / or an antibody or antibody reagent.

112. 112. The method of claim 111, wherein the active compound is a component of a salt.

113. 112. The method of claim 111, wherein the active compound comprises insulin or a GLP-1 polypeptide or a mimetic or analog thereof.

114. a. a salt present at a concentration of at least 0.05M; and b. Active compounds A composition comprising:

115. 115. The composition of claim 114, wherein the salt is an ionic liquid.

116. 116. The composition of claim 115, wherein the ionic liquid is choline and geranate (CAGE).

117. 117. The composition of claim 116, wherein the cation is choline.

118. 118. The composition of claim 116 or 117, wherein the anion is geranate or geranic acid.

119. 119. The composition of any one of claims 114-118, wherein the salt is present at a concentration of at least 0.05M, 0.1M, 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M, or greater.

120. 120. The composition of any one of claims 114-119, wherein the salt is present at a concentration of about 0.05M to about 4M.

121. 121. The composition of any one of claims 114-120, wherein the salt is a pure liquid or an anhydrous liquid.

122. 121. The composition of any one of claims 114-120, wherein the salt is in an aqueous solution.

123. The composition of any one of claims 114-121, wherein the active compound comprises a nucleic acid molecule, a chemotherapeutic compound, a small molecule, a peptide, and / or an antibody or antibody reagent.

124. 124. The composition of claim 123, wherein the active compound is a component of a salt.

125. 124. The composition of claim 123, wherein the active compound comprises insulin or a GLP-1 polypeptide or a mimetic or analog thereof.

126. 126. The composition of any one of claims 114 to 125 for use in a delivery method or a method of treatment.

127. 127. The method or composition of any one of claims 97-126, wherein the biological activity of the active compound is improved or stabilized compared to the activity in the absence of the salt.

128. 128. The method or composition of any one of claims 97-127, wherein the combination of active compound and salt is an admixture.

129. 129. The method or composition of any one of claims 97-128, wherein the combination of active compound and salt comprises nanoparticles comprising the active compound, wherein the nanoparticles are in solution or suspension in a composition comprising the salt.

130. A method of treating diabetes, an ulcer, cancer, or fibrosis in a subject in need thereof, comprising administering to the subject a composition comprising the ionic liquid choline and geranate (CAGE) and no additional therapeutically active agent.

131. 131. The method of claim 130, wherein the administration is via injection.

132. 131. The method of claim 130, wherein the administration is oral.

133. 133. The method of any one of claims 130-132, wherein the CAGE is at a concentration of at least 0.1% w / v.

134. 134. The method of any one of claims 130-133, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:

10.

135. 134. The method of any one of claims 130-133, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:

4.

136. 134. The method of any one of claims 130-133, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:

4.

137. 137. The method of any one of claims 130-136, wherein the anion of the ionic liquid comprises geranate and / or geranic acid.

138. A composition comprising the ionic liquid Choline and Geranate (CAGE) and no further therapeutically active agent, for use in a method for treating diabetes, an ulcer, cancer, or fibrosis in a subject in need thereof.

139. 139. The composition of claim 138, wherein administration is via injection.

140. 139. The composition of claim 138, wherein administration is oral.

141. The composition of any one of claims 138-140, wherein the CAGE is at a concentration of at least 0.1% w / v.

142. 142. The composition of any one of claims 138-141, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 2:1 to about 1:

10.

143. 142. The composition of any one of claims 138-141, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:1 to about 1:

4.

144. 142. The composition of any one of claims 138-141, wherein the CAGE comprises a ratio of choline:geranic acid or geranate of about 1:2 to about 1:

4.

145. 145. The composition of any one of claims 138-144, wherein the anion of the ionic liquid comprises geranate and / or geranic acid.

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