Ionic liquids for drug delivery

Abstract

The technology described herein relates to ionic liquids and methods of drug delivery. TIFF2023503899000027.tif86170

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits under Section 119(e) of U.S. Patent Provisional Application No. 62 / 939,088, filed on 22 November 2019, which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy, prepared on November 19, 2020, is filed 002806-096230WOPT_SL.txt and is 25,930 bytes in size.

[0003] Technical field The techniques described herein relate to ionic liquids for the stabilization and delivery of active compounds. [Background technology]

[0004] background The uptake of many active compounds, such as pharmaceutically active compounds, can be improved by delivering the compound in a solvent. However, most such solvents exhibit toxic side effects and / or act as irritants at the time of delivery, making such approaches often unsuitable for in vivo use. These toxic and irritant effects are often severe enough to suppress any increase in the uptake or performance of the active compound. [Overview of the project]

[0005] overview As described herein, the inventors have identified characteristics of ionic liquids that provide remarkably excellent uptake kinetics of certain types of active compounds. Accordingly, compositions and methods relating to these ionic liquids (ILs) that exhibit unexpectedly high efficacy are described herein.

[0006] In one aspect of any embodiment, the foregoing describes a composition comprising at least one ionic liquid comprising an anion which is at least one of the following: a) a non-fatty acid carboxylic acid; b) a carboxylic acid comprising an aliphatic chain of 4 or fewer carbon atoms; c) an aromatic anion; and / or d) an anion having a LogP of less than 1.0; and a cation which comprises a quaternary ammonium.

[0007] In some aspects of any given aspect, the anion has a LogP of less than 1.0 and is a) a non-fatty acid carboxylic acid; b) a carboxylic acid containing an aliphatic chain of 4 or fewer carbon atoms; or c) an aromatic anion. In some aspects of any given aspect, the fatty acid contains an aliphatic chain of 3 or fewer carbon atoms. In some aspects of any given aspect, the anion contains only one carboxylic acid group (e.g., an R-COOH group). In some aspects of any given aspect, the anion is selected from the group consisting of: glycolic acid; propanoic acid; isobutyric acid; butyric acid; gallic acid; lactic acid; malonic acid; maleic acid; glutaric acid; citric acid; 3,3-dimethylacrylic acid; dimethylacrylic acid; gluconic acid; adipic acid; sodium ethylhexyl sulfate; decanoic acid; hydroxybenzenesulfonic acid; 4-hydroxybenzenesulfonic acid; isovaleric acid; hydrocinnaminic acid; 4-phenolsulfonic acid; phenyl phosphoric acid; and biphenyl-3-carboxylic acid.

[0008] In some aspects of any configuration, the cation has a molar mass equal to or greater than that of choline. In some aspects of any configuration, the quaternary ammonium is NR4 + It has the structure and at least one R group contains a hydroxyl group. In some aspects of any configuration, the quaternary ammonium is NR4 + It has the structure and contains only one hydroxyl group. In some embodiments of any aspect, the cation is C1, C6, or C7.

[0009] In some aspects of any given plane, the ionic liquid contains a cation-to-anion ratio of about 2:1 to about 1:1. In some aspects of any given plane, the ionic liquid contains a cation-to-anion ratio of about 2:1. In some aspects of any given plane, the ionic liquid has a cation:anion ratio of less than 1:1. In some aspects of any given plane, the ionic liquid has a cation:anion ratio with excess cations.

[0010] In some aspects of any configuration, the composition further comprises at least one active compound combined with at least one ionic liquid.

[0011] In some aspects of any given aspect, the active compound comprises a polypeptide. In some aspects of any given aspect, the polypeptide is an antibody or antibody reagent. In some aspects of any given aspect, the active compound has a molecular weight greater than 450. In some aspects of any given aspect, the active compound has a molecular weight greater than 500. In some aspects of any given aspect, the anion has a LogP of less than 1.0 and is a) a carboxylic acid that is not a fatty acid; or b) a carboxylic acid containing an aliphatic chain of 4 carbons or less.

[0012] In some aspects of any given context, the active compound comprises a nucleic acid. In some aspects of any given context, the nucleic acid is an inhibitory nucleic acid. In some aspects of any given context, the nucleic acid is an siRNA. In some aspects of any given context, the anion has a LogP of less than 1.0 and is a) a non-fatty acid carboxylic acid; or b) a carboxylic acid containing an aliphatic chain of 4 or fewer carbon atoms; and / or c) an aromatic anion.

[0013] In some aspects of any given situation, the ionic liquid has a concentration of at least 0.1% w / v. In some aspects of any given situation, the ionic liquid has a concentration of about 10 to about 70% w / v. In some aspects of any given situation, the ionic liquid has a concentration of about 30 to about 50% w / v. In some aspects of any given situation, the ionic liquid has a concentration of about 30 to about 40% w / v.

[0014] In some aspects of any given context, the composition is formulated for transdermal administration, mucosal administration, oral administration, subcutaneous administration, intradermal administration, parenteral administration, intratumoral administration, or intravenous administration. In some aspects of any given context, the composition is formulated for transdermal administration. In some aspects of any given context, the mucosa is the nasal mucosa, oral mucosa, or vaginal mucosa.

[0015] In some aspects of any configuration, the active compound is provided in doses of 1 to 40 mg / kg. In some aspects of any configuration, the composition further comprises at least one nonionic surfactant. In some aspects of any configuration, the composition further comprises a pharmaceutically acceptable carrier. In some aspects of any configuration, the composition is provided in a biodegradable capsule. In some aspects of any configuration, the composition is a misform. In some aspects of any configuration, the composition is provided in one or more nanoparticles. In some aspects of any configuration, the composition comprises one or more nanoparticles containing the active compound, the nanoparticles being in a solution or suspension in a composition containing an ionic liquid.

[0016] In one aspect of any embodiment, the method described herein is a method for administering at least one active compound, comprising the step of administering the composition described herein. In some aspects of any embodiment, the composition is administered once. In some aspects of any embodiment, the composition is administered in multiple doses. [Brief explanation of the drawing]

[0017] [Figure 1-1]Figures 1A-1D. Figure 1A shows the chemical structures of choline and glycolic acid. CGLY variants (choline:glycolic acid molar ratios of 2:1, 1:1, and 1:2) were prepared by salt metathesis of choline bicarbonate and glycolic acid. Figure 1B shows the retained antigen-binding ability of anti-human TNF-α mouse IgG1 antibody (clone MAb11) isolated from CGLY variants in the concentration range of 20-90 vol%. Figure 1C shows the circular dichroism spectrum of anti-human TNF-αIgG isolated from CGLY variants. IgG was dispersed in 50 vol% CGLY variants, stored at RT (25°C) for 1 hour, and then dialyzed for 48 hours. The beta-sheet secondary structure of IgG was retained after exposure to CGLY solution. Figure 1D shows the SDS-PAGE of anti-human TNF-αIgG isolated from CGLY variants. [Figure 1-2] See the explanation in Figure 1-1. [Figure 2] Figures 2A and 2B show in vitro studies of CGLY variants on Caco-2 cell viability and IgG transport. Figure 2A shows the viability of Caco-2 cells treated with the CGLY variant. Data are expressed as mean ± SE (n=6). Figure 2B shows the enhancement of FITC-IgG transport across the Caco-2 monolayer in the presence of 30 mM CGLY variant. Data are expressed as mean ± SE (n=5); (*p<0.05; CGLY2:1 treatment compared to CGLY1:1 and CGLY1:2). (##p<0.01; all CGLY treatments compared to no CGLY treatment). [Figure 3-1]Figures 3A–3D show in vitro molecular transport across Caco-2 cell monolayers mediated by CGLY2:1. Enhancement of FITC-IgG (Figure 3A) and Lucifer Yellow (Figure 3B) transport across Caco-2 monolayers in the presence of various CGLY2:1 concentrations. Data are expressed as mean ± SE (n=5). Figure 3C shows the effect of various CGLY treatments on tight junction integrity in Caco-2 cells. Data are expressed as mean ± SE (n=5); (*p<0.05; **p<0.001; all CGLY2:1 treatments compared to no CGLY2:1 treatment). Figure 3D shows FITC-IgG transport across Caco-2 monolayers after 24-hour incubation in the presence of 55 mM CGLY2:1 and in the presence or absence of transcellular transport inhibitors. Data are expressed as mean ± SE (n=5). [Figure 3-2] See the explanation in Figure 3-1. [Figure 4] Figure 4A shows the viscosity of porcine small intestinal mucus plotted as a function of shear rate in the range of 10 to 80 1 / s in the presence of 0 vol%, 12.5 vol%, 25 vol% and 50 vol% CGLY2:1 in physiological saline. CGLY2:1 treatment was added to the mucus, followed by gentle shaking, and then measurement after equilibration for 30 minutes. Data are expressed as mean (n=3). Black circles = 0 vol%, dark gray circles = 12.5 vol%, light gray circles = 25 vol%, white circles = 50 vol%. Figure 4B shows the average viscosity of porcine mucus at a shear rate of 49.87 1 / s in the presence of 0 vol%, 12.5 vol%, 25 vol% and 50 vol% CGLY2:1 in physiological saline. Data are expressed as mean ± SE (n=3); (*p<0.05, **p<0.01, ***p<0.001; CGLY2:1 treatment compared to no CGLY treatment). [Figure 5-1]Figures 5A-5C show fluorescence microscopy images of intestinal villi after intrajejunal injection of FITC-IgG and CGLY2:1 (Figure 5B), FITC-IgG and saline (Figure 5C), and saline without FITC-IgG (Figure 5A). Fluorescence microscopy imaging was performed in triplicates, and representative images are shown. The scale bar represents 200 μm. Figure 5A shows the oral toxicity study of CGLY2:1. Participants were given oral nutrition of CGLY2:1 (50 vol%) or saline at a dose of 1250 mg / kg, n=2, for 15 days. Results were evaluated by weight monitoring, blood chemistry, GI tubes, and H&E staining of major organs. Figure 5D shows the fluorescence quantification of FITC-IgG per unit area of ​​villi from Figures 5A-5C. Data are expressed as mean ± SE (n=10). Figure 5E shows the in vivo plasma anti-human TNF-αIgG concentrations after jejunal injection of IgG in CGLY2:1 or physiological saline, as quantified by ELISA. [Figure 5-2] See the explanation in Figure 5-1. [Figure 6A] Figures 6A–6C show the in vivo toxicity study of CGLY2:1. Rats were orally administered either CGLY2:1 or saline once daily for seven consecutive days. Figure 6A shows the rat body weight records from day 0 to day 7 during the study. Data are expressed as mean ± SE (n=6). Figure 6B shows the results of treating GI tube sections with hematoxylin and eosin (H&E) for tissue staining after the rats were sacrificed on day 7. Scale bars represent 100 μm. Figure 6C shows the comprehensive metabolic panel of the rats (n=6). Blood tests performed on day 7 showed no significant changes between the two groups, indicating normal liver and kidney function after CGLY administration. All bars and markers represent mean ± SE. [Figure 6B] See the explanation in Figure 6A. [Figure 6C] See the explanation in Figure 6A. [Figure 7] Figure 7 shows a diagram of drug delivery. [Figure 8] Figure 8 shows the functional antibody stability of the ILs shown, as measured by ELISA. As a general trend, smaller anions have higher compatibility with the antibody than larger anions. [Figure 9]Figure 9 shows the functional antibody stability of the ILs shown, as measured by size exclusion chromatography. The antibody used was anti-human TNFα (mouse) (clone MAb11) that had been dialyzed for 2 days. [Figure 10] Figure 10 shows the functional antibody stability measured by circular dichroism in the indicated IL. The antibody used was anti-human TNFα (mouse) (clone MAb11) that had been dialyzed for 2 days. [Figure 11] Figure 11 shows a graph of antibody concentrations in serum after intrajejunal administration of the compositions shown. The dose was 200 μg / kg, and n=3. [Figure 12] Figure 12 shows the experimental design for in vivo mAb local delivery. [Figure 13] Figure 13 shows the results of in vivo mAb local delivery. [Figure 14] Figure 14 shows compatibility testing of CGLY2:1 with other antibodies. [Figure 15] Figure 15 shows H&E staining of major organs in the toxicity test shown in Figure 5A. Rats were orally administered CGLY2:1 or saline once daily for 7 consecutive days. On day 7, the rats were sacrificed, and major organs, including the heart, liver, spleen, lungs, and kidneys, were treated with H&E for histological staining. No differences were observed between the CGLY2:1 and saline control groups. Scale bars represent 100 μm. [Figure 16] Figure 16 shows H&E staining of GI tubes in the toxicity test shown in Figure 5A. Rats were orally administered CGLY2:1 or saline once daily for 7 consecutive days. On day 7, the rats were sacrificed, and major organs, including the heart, liver, spleen, lungs, and kidneys, were treated with H&E for histological staining. No difference was observed between the CGLY2:1 and saline control groups. The scale bar represents 100 μm. [Figure 17] Figure 17 shows the structure of the IL used for siRNA delivery performance testing. [Figure 18]Figure 18 shows representative confocal microscope images of Transwell membranes covered with layers of Caco-2 cells and incubated for 5 hours with FITC-IgG dispersed in various concentrations of CGLY2:1. Images were acquired at 40× magnification. The images show DAPI-labeled nuclei, FITC-IgG, and superimposed images of DAPI staining and FITC-IgG. The scale bar represents 50 μm. [Figure 19A] Figures 19A–19E show screening of Corinium-based bioactive IL-RNA complexes for enhanced epidermal accumulation. (Figure 19A) CD spectrum of siRNA in phosphate-buffered saline (PBS) after incubation with IL (50 vol%) for 30 minutes and dialysis for 72 hours. (Figure 19B) Representative native gel image of siRNA after IL incubation. bp, base pairs. (Figure 19C) Representative confocal images of siRNA (red) in different skin layers (a) stratum corneum (SC), (b) epidermis, and (c) dermis after 24-hour incubation in the presence of a 1:1 mixed IL combination (CAGE+CAPA). Left to right: Merge, Cy5, differential interference contrast (DIC). Scale bar, 50 μm. (Figures 19D and 19E) Transport of Cy5-labeled siRNA to different layers of skin in the presence of 50 vol% individual ILs (Figure 19D) and 50 vol% IL combinations (Figure 19E), as determined by tape stripping (n=3). For Figures 19D-19E, data are mean ± SEM and were determined to be nonparametric by normality tests and Kruskal-Wallis tests. *P<0.05. [Figure 19B] See the explanation in Figure 19A. [Figure 19C] See the explanation in Figure 19A. [Figure 19D] See the explanation in Figure 19A. [Figure 19E] See the explanation in Figure 19A. [Figure 20-1]Figures 20A–20F show MD simulations identifying the degree of IL-siRNA interaction for solvation and stability enhancement. (Figures 20A and 20B) Snapshots of simulation unit cells of CAGE and siRNA (Figure 20A) and CAGE components observed within 10 Å of siRNA (Figure 20B) under periodic boundary conditions at 500 ns. (Figures 20C and 20D) Snapshots of simulation unit cells of optimized IL combination (CAGE and CAPA, 1:1) and siRNA (Figure 20C) and IL species observed within 10 Å of siRNA (Figure 20D) under similar conditions. (Figures 20E and 20F) Radius of gyration (RGYR) (Figure 20E) and mean squared deviation (RMSD) (Figure 20F) obtained over 500 ns for CAPA and IL combination (CAGE and CAPA) compared to CAGE (control). [Figure 20-2] See the explanation in Figure 20-1. [Figure 21-1] Figures 21A–21E show three MD simulations establishing the enhancement and transition mechanism of lipid bilayer interactions in the IL combination. (Figure 21A) Lipid bilayer simulation with aggregates of choline, geranic acid, and phenylpropanoic acid, highlighted by circles. (Figure 21B) Magnified view of ion species from circles showing closed interactions of ion species with phospholipid heads and tails. The aggregates contain all three ion species contributing to the interaction with the lipid membrane. (Figure 21C) Representative snapshot of the lipid bilayer in a plane, viewed perpendicular to the membrane. (Figures 21D and 21E) Mean thickness of the lipid membrane (Figure 21D) and mean area per lipid (Figure 21E) during simulations in the presence of CAPA and the IL combination (CAGE and CAPA) compared to CAGE (control). For Figures 21D–21E, all data are mean ± SEM and were determined to be nonparametric by statistical tests for normality and Kruskal-Wallis test. ****P<0.0001. [Figure 21-2] See the explanation in Figure 21-1. [Figure 22-1]Figures 22A–22E show that IL-siRNA inhibits GAPDH expression in mice after topical application without toxicity. (Figure 22A) Schematic diagram of the topical application schedule. (Figure 22B) Representative histological images [hematoxylin and eosin (H&E)] of skin tissue 5 days after topical application of IL-siRNA. Scale bar, 100 μm; magnification, ×10. (Figure 22C) Confocal images of epidermal accumulation of Cy5-siRNA in mouse skin tissue in the presence and absence of IL. Scale bar, 50 μm. (Figure 22D) GAPDH mRNA expression was measured by qPCR. β-actin mRNA expression was used for normalization. Data were mean ± SEM and were determined to be nonparametric by normality tests and Kruskal-Wallis tests. *P<0.05, ***P<0.001, and ****P<0.0001. (Figure 22E) GAPDH levels in skin samples were determined using the GAPDH enzyme-linked immunosorbent assay. Data are mean ± SEM, and statistics were analyzed using one-way ANOVA and Tukey's HSD post-hoc test. ****P<0.0001 (control, n=5; naked siRNA, n=5; IL-siCon, n=4; IL-siRNA, n=8). [Figure 22-2] See the explanation in Figure 22-1. [Figure 23-1]Figures 23A–23J show that local inhibition of NFKBIZ by local IL-siRNA suppresses imiquimod-induced psoriasis-like skin inflammation and other major psoriasis-related genes. (Figure 23A) Schematic diagram of disease induction and application schedule of local IL-siRNA administration. (Figure 23B) Psoriasis-induced mice were treated locally with IL-NFKBIZ siRNA and compared with untreated and IL-treated groups. (Figure 23C) H&E staining of psoriasis-induced skin sections from treated or untreated mice. Scale bar, 50 μm; magnification, ×10. (Figure 23D) Skin sections from mice were analyzed by IHC for keratinocyte proliferation (proliferation marker, Ki67). Scale bar, 100 μm. (Figures 23E and 23F) Erythema and scaling scores obtained by daily blinded scoring using the human PASI scoring system on a scale of 0 (no change) to 4 (very clear change). (Figure 23G) Heatmaps of expression levels of various psoriasis-related genes after treatment with IL-NFKBIZ siRNA, compared to the untreated (control) and IL-siCon-treated groups. (Figures 23H-23J) mRNA expression levels for NFKBIZ, TNF-α, and IL-17A were measured by qPCR, and β-actin mRNA expression was used for normalization. Data are mean ± SEM, and statistics were performed using one-way ANOVA and Tukey's HSD post-hoc test. *P<0.05, **P<0.01, and ****P<0.0001 (control, n=4; IL, n=4; IL-siCon, n=4; IL-siRNA, n=8). [Figure 23-2] See the explanation in Figure 23-1. [Figure 23-3] See the explanation in Figure 23-1. [Figure 24-1]Figures 24A–24E show the design and synthesis of in-house Corinium-based IL libraries for improved biocompatibility and interaction with RNA. (Figure 24A) Corinium-based IL libraries containing various anions, synthesized with CAGE as the reference IL. (Figure 24B) General synthesis scheme of salt metathesis used in IL synthesis. (Figure 24C) Synthesis scheme of an optimized IL combination (CAGE+CAPA) for siRNA delivery. (Figure 24D) 1H-NMR spectra of synthetic IL as viscous at RT: (a) CAGE, (b) CAVA, (c) CAPA, and (d) CADA. (Figure 24E) Relative density of siRNA bands after IL incubation, measured with Image J software. [Figure 24-2] See the explanation in Figure 24-1. [Figure 24-3] See the explanation in Figure 24-1. [Figure 25-1] Figures 25A–25D show improved epidermal accumulation of Cy5-labeled siRNA in the presence of IL. (Figure 25A) Schematic diagram of the Franz diffusion cell (FDC) setup for the exvivovota skin penetration test. (Figure 25B) Representative confocal images of siRNA in the presence of control, bare siRNA, and CAGE. (Figure 25C) Epidermal accumulation of Cy5-siRNA in the presence of freshly synthesized Corinium-based IL and a 1:1 ratio combination after incubation of porcine skin for 24 hours. Left to right: Merge, Cy5, Differential Interference (DIC). Scale bar, 50 μm. (Figure 25D) Transport of Cy5-labeled siRNA to different layers of skin, determined by tape stripping (n=3). Data are mean ± SEM and were determined to be nonparametric by normality tests and Kruskal-Wallis tests. [Figure 25-2] See the explanation in Figure 25-1. [Figure 26]Figures 26A-26B show the main contributions of IL species mobility to IL-lipid bilayer interactions and permeation. (Figure 26A) Lipid bilayer simulation in the presence of IL combinations (highlighted with circles) (Figure 26B) Orbitals of individual ion species within IL combinations, CAGE+CAPA simulation using the Python library MDAnalysis. [Figure 27] Figures 27A-27D show highly biocompatible IL preparations with no toxicity or irritation after topical application. (Figure 27A) Application sites of healthy mice treated topically with IL-GAPDH siRNA were compared with water and the IL-siCon group. (Figure 27B) H&E staining of skin sections from healthy mice treated topically with IL-siCon for 4 consecutive days. Scale bar, 100 μm, magnification, 10×. (Figure 27C) Skin sections from healthy mice were analyzed for hyperproliferation by staining with the proliferation marker Ki67. Scale bar, 100 μm. Quantitative analysis of IHC was not performed because no proliferative areas were observed. (Figure 27D) TNF-α mRNA expression was measured by qPCR, and β-actin mRNA expression was used for normalization. Data are mean ± SEM, and statistics were performed using one-way ANOVA and Tukey's HSD post-hoc test. *P<0.05, **P<0.01, ****P<0.0001. (Control, n=5; Naked siRNA, n=5; IL-siCon, n=4; IL-siRNA, n=8). [Figure 28] Figures 28A-28D illustrate the characterization of the IL-siCon effect in imiquimod-induced psoriasis mice. (Figure 28A) Psoriasis-induced mice were topically treated with IL-siCon for 4 consecutive days. (Figure 28B) H&E staining of skin sections from imiquimod-induced psoriasis mice topically treated with IL-siCon. Scale bar, 50 μm, magnification, 10×. (Figure 28C) Skin sections from psoriasis mice were analyzed for hyperproliferation by staining with the proliferation marker Ki67. Scale bar, 100 μm. (Figure 28D) Epidermal thickness; mean epidermal thickness calculated based on 10-15 random site measurements using ImageJ software. Data are mean ± SEM, and statistics are performed using one-way ANOVA and Tukey's HSD post-hoc test. *P<0.05, ****P<0.0001. [Figure 29] Figures 29A–29C show the effects of IL-NFKBIZ siRNA on imiquimod-induced psoriasis-like skin inflammation in mice. Imiquimod-induced psoriasis mice were monitored by dual subcutaneous fat thickness (DSFT) during a 5-day induction / application period, and cumulative score (Figure 29A), body weight (Figure 29B), and skin thickness (Figure 29C) were analyzed. Data are mean ± SEM. (Control, n=4; IL, n=4; IL-siRNA, n=8). [Figure 30-1] Figures 30A–30J show the downstream effects of NFKBIZ silencing on psoriasis-related gene products. mRNA expression was measured by qPCR for cytokines IL-17C, IL-19, IL-22, IL-23A, IL-36A, IL-36G (Figures 30A–30F); chemokine CCL 20 (Figure 30G); S100 protein S100A9 (Figure 30H); antimicrobial proteins lipocalin-2, LCN2, and β-defensin-2, DEFB4 (Figure 30J), with β-actin mRNA expression used for normalization. Data are mean ± SEM, and statistics were performed using one-way ANOVA and Tukey's HSD post-hoc test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (Control, n=4; IL, n=4; IL-siCon, n=4; IL-siRNA, n=8). [Figure 30-2] See the explanation in Figure 30-1. [Figure 30-3] See the explanation in Figure 30-1. [Modes for carrying out the invention]

[0018] Detailed description of the invention The data provided herein demonstrate that anions in ionic liquids (ILs) significantly influence whether certain activators are transported across biological barriers (e.g., epithelial layers such as the dermis). Anions with low hydrophobicity and / or aromatic groups offer improved drug delivery properties for antibody and siRNA cargo molecules compared to previously described anions in ILs, such as CAGE (choline and geranic acid). A key concern in selecting cations to pair with anions is that the cations should not be too closely associated with the anion, as this close association causes the anion to be retained on the first side of the biological barrier.

[0019] Therefore, in any aspect of this specification, 1) anion which is at least one of the following: a) Carboxylic acids that are not fatty acids; b) Carboxylic acids containing aliphatic chains of 4 carbon atoms or less; c) Aromatic anions; and / or d) Anions having a LogP of less than 1.0; and 2) Cation containing quaternary ammonium The composition comprises at least one ionic liquid, including [the specified element].

[0020] In any aspect of this specification, the following is described herein: a composition comprising at least one ionic liquid comprising 1) an anion which is a carboxylic acid as described herein; and 2) a cation which comprises a quaternary ammonium.

[0021] As used herein, the term “ionic liquid (IL)” refers to an organic salt or mixture of organic salts that is in a liquid state at room temperature. Solvents of this class have been shown to be useful in a variety of fields, including industrial processing, catalysis, pharmaceuticals, and electrochemistry. Ionic liquids contain at least one anionic component and at least one cationic component. Ionic liquids may contain additional hydrogen bond donors (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. At least one anionic component and 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. For further consideration of ionic liquids, see, for example, 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 incorporated herein by reference. In some embodiments of any aspect, ionic liquids or solvents exist as liquids below 100°C. In some embodiments of any aspect, ionic liquids or solvents exist as liquids at room temperature.

[0022] As described herein, anions having low hydrophobicity, relatively short carbon chains, and / or aromatic groups provide improved drug delivery properties for large polypeptide (e.g., antibody) or nucleic acid cargo molecules. In some embodiments, improved drug delivery properties include reduced denaturation or degradation of the cargo molecule. In some embodiments, improved drug delivery properties include increased ability to cross a biological barrier (e.g., increased permeability). In some embodiments of any aspect, anions having low hydrophobicity and / or relatively short carbon chains provide improved drug delivery properties for large polypeptide (e.g., antibody) cargo molecules. In some embodiments of any aspect, anions having aromatic groups and / or relatively short carbon chains provide improved drug delivery properties for nucleic acid cargo molecules.

[0023] In some embodiments of any aspect, the anion of IL described herein is hydrophobic.

[0024] In some embodiments of any aspect, the anion of IL described herein comprises a carboxylic acid. In some embodiments of any aspect, the anion of IL described herein comprises a carboxylic acid that is not a fatty acid.

[0025] A carboxylic acid is a compound having the structure of Formula I, wherein R can be any group. TIFF0007917913000001.tif32128

[0026] Generally, the anion is R-X - , wherein X is CO2 - , SO3 - , OSO3 2- or OPO3 2- ; and R is optionally substituted C1 to C 10 alkyl, optionally substituted C2 to C 10 alkenyl, or optionally substituted C2 to C 10 alkynyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0027] In some embodiments, R is a linear or branched C1-C9 alkyl group which may be substituted. For example, R is a C1-C9 alkyl group which may be substituted with 1, 2, 3, 4, 5, or 6 substituents independently selected from the group consisting of C1-C3 alkyl groups, hydroxy(OH), halogens, oxo(=O), carboxy(CO2), cyano(CN), and aryl groups. In some embodiments, R is a C1-C6 alkyl group which may be substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of C1-C3 alkyl groups, hydroxy, carboxy, and phenyl groups. Preferably, R is a C1-C5 alkyl group which may be substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of methyl, ethyl, hydroxyl, carboxy, and phenyl groups. Exemplary alkyl groups for R include, but are not limited to, methyl, carboxymethyl, hydroxymethyl, ethyl, 1-hydroxyethyl, 2-phenylethyl, propyl, propa-2-yl, 1-methylpropyl, 2-methylpropyl, 3-carboxypropyl, 2,3-dicarboxymethyl-2-hydroxypropyl, butyl, pentyl, 1,2,3,4,5-pentahydroxypentyl, hexyl, 2-ethylhexyl, and nonyl.

[0028] In some embodiments, R is a linear or branched C2-C8 alkenyl which may be substituted. For example, R is a C2-C9 alkenyl which may be substituted with 1, 2, 3, 4, 5, or 6 substituents independently selected from the group consisting of C1-C3 alkyl, hydroxy, halogen, oxo, carboxy, cyano, and aryl. In some embodiments, R is a C2-C6 alkenyl which may be substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of C1-C3 alkyl, hydroxy, carboxy, and phenyl. Preferably, R is a C1-C5 alkenyl which may be substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of methyl, ethyl, hydroxyl, carboxy, and phenyl. Exemplary alkenyls for R include, but are not limited to, ethenyl, 2-carboxyethenyl, 1-methylpropenyl, and 2-methylpropenyl.

[0029] In some embodiments, R is an aryl or heteroaryl which may be substituted. For example, R is an aryl or heteroayl which may be substituted with 1, 2, 3, 4, 5, or 6 substituents independently selected from the group consisting of C1-C3 alkyl, hydroxy, halogen, oxo, carboxy, cyano, and aryl. In some embodiments, R is an aryl which may be substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of C1-C3 alkyl, hydroxy, carboxy, and phenyl. Preferably, R is a phenyl which is substituted with 1, 2, or 3 substituents independently selected from the group consisting of methyl, ethyl, hydroxyl, carboxy, and phenyl. Exemplary aryls for R include, but are not limited to, phenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, dihydroxyphenyl, trihydroxyphenyl, 3,4,5-trihydroxyphenyl, and 1,1-bifen-4-yl.

[0030] In some embodiments, X is CO2 -And R is methyl, carboxymethyl, hydroxymethyl, ethyl, 1-hydroxyethyl, 2-phenylethyl, propyl, propa-2-yl, 1-methylpropyl, 2-methylpropyl, 3-carboxypropyl, 2,3-dicarboxymethyl-2-hydroxypropyl, butyl, pentyl, 1,2,3,4,5-pentahydroxypentyl, hexyl, 2-ethylhexyl, nonyl, ethenyl, 2-carboxyethenyl, 1-methylpropenyl, 2-methylpropenyl, 3,4,5-trihydroxyphenyl, or 1,1-bifen-4-yl. In some other embodiments, X is OSO3 - And R is methyl, carboxymethyl, hydroxymethyl, ethyl, 1-hydroxyethyl, 2-phenylethyl, propyl, propa-2-yl, 1-methylpropyl, 2-methylpropyl, 3-carboxypropyl, 2,3-dicarboxymethyl-2-hydroxypropyl, butyl, pentyl, 1,2,3,4,5-pentahydroxypentyl, hexyl, 2-ethylhexyl, nonyl, ethenyl, 2-carboxyethenyl, 1-methylpropenyl, 2-methylpropenyl, 3,4,5-trihydroxyphenyl, or 1,1-bifen-4-yl. In some other embodiments, X is OPO3 2- or SO3 - And R is 2-hydroxyphenyl, 3-hydroxyphenyl, or 4-hydroxyphenyl.

[0031] The term "alkyl" means, by itself or as part of another substituent, unless otherwise specified, a straight (i.e., unbranched) or branched carbon chain (or carbon), or a combination thereof, which may be fully saturated, monounsaturated or polyunsaturated, and which have a specified number of carbon atoms (i.e., C1-C1). 10Alkyls can include monovalent, divalent, and polyvalent radicals (where 1 to 10 carbon atoms are involved). Alkyls are acyclic chains. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, and (cyclohexyl)methyl, as well as homologs and isomers such as n-pentyl, n-hexyl, n-heptyl, and n-octyl. "Alkenyl" is an unsaturated alkyl group having one or more double bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, clotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), as well as higher homologs and isomers.

[0032] The term "aryl," unless otherwise specified, means a polyunsaturated, aromatic, hydrocarbon substituent, which may be a single ring, a fused ring (i.e., a fused ring aryl), or a covalently bonded group of rings (preferably 1 to 3 rings). A fused ring aryl refers to a group of fused rings in which at least one of the fused rings is an aryl ring. The term "heteroaryl" refers to an aryl group (or ring) containing at least one heteroatom such as N, O, or S, where the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. Thus, the term "heteroaryl" includes a fused ring heteroaryl group (i.e., a group of fused rings in which at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to a group of two fused rings in which one ring has 5 members, the other ring has 6 members, and at least one of the rings is a heteroaryl ring. Similarly, a 6,6-fused heteroarylene refers to two fused rings, one having six members and the other having six members, with at least one ring being a heteroaryl ring. A 6,5-fused heteroarylene refers to two fused rings, one having six members and the other having five members, with at least one ring being a heteroaryl ring. The heteroaryl group may be bonded to the rest of the molecule via carbon or heteroatoms.Exemplary aryl and heteroaryl groups include phenyl, 4-nitrophenyl, 1-naphthyl, 2-naphthyl, biphenyl, 4-biphenyl, pyrrole, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, pyrazole, 3-pyrazolyl, imidazole, imidazolyl, 2-imidazolyl, 4-imidazolyl, benzimidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, thiazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2- This includes, but is not limited to, thienyl, 3-thienyl, pyridine, 2-pyridyl, naphthilidinyl, 3-pyridyl, 4-pyridyl, benzophenonepyridyl, pyridadinyl, pyrazinyl, 2-pyrimidyl, 4-pyrimidyl, pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, indolyl, 5-indolyl, quinoline, quinolinyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, 6-quinolyl, furan, furyl or furanyl, thiophene, thiophenyl or thienyl, diphenyl ether, diphenylamine, etc.

[0033] The term “may be substituted” means that the specified group or portion is either unsubstituted or substituted with one or more substituents (typically 1, 2, 3, 4, 5, or 6 substituents) independently selected from the group of substituents listed below or otherwise specified in the definition of “substituent.” The term “substituent” means a group that is “substituted” on a group that is substituted on any atom of the substituted group. Suitable substituents include, but are not limited to, halogens, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, cyclyl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, allensulfonyl, alkanesulfonamide, allensulfonamide, aralkylsulfonamide, alkylcarbonyl, acyloxy, cyano, or ureido. In some cases, two substituents, together with the carbon atom to which they are bonded, can form a ring.

[0034] As used herein, "fatty acid" refers to a fatty acid in which R is a saturated or unsaturated aliphatic chain, for example, R is of formula C n H 2n+1 This refers to a carboxylic acid having a specific characteristic. In some aspects of any aspect, a fatty acid is a monocarboxylic acid. Fatty acids can be natural or synthetic. The aliphatic chain of a fatty acid can be saturated, unsaturated, branched, linear, and / or cyclic. In some aspects of any aspect, the aliphatic chain does not contain an aromatic group. In some aspects of any aspect, the aliphatic chain contains, consists of, or is fundamentally composed of alkyl or alkene chains.

[0035] Exemplary carboxylic acids that are not fatty acids may include, but are not limited to, lactic acid; glycolic acid; malonic acid; maleic acid; glutaric acid; citric acid; gluconic acid; and adipic acid. TIFF0007917913000002.tif24060

[0036] In some embodiments, the non-fatty acid carboxylic acid has either a linear or branched configuration and contains five or fewer carbon atoms in the R group. In some embodiments, the non-fatty acid carboxylic acid contains a hydroxyl group in the R group. In some embodiments, the non-fatty acid carboxylic acid contains one or more carboxylic acids in the R group.

[0037] In some embodiments, the non-fatty acid carboxylic acid is either linear or branched, contains five or fewer carbon atoms in the R group, and contains a hydroxyl group in the R group. In some embodiments, the non-fatty acid carboxylic acid is either linear or branched, contains one to five carbon atoms in the R group, and contains a hydroxyl group in the R group.

[0038] In some embodiments, a non-fatty acid carboxylic acid is either linear or branched, contains five or fewer carbon atoms in the R group, and contains one or more carboxylic acid groups in the R group. In some embodiments, a non-fatty acid carboxylic acid is either linear or branched, contains one to five carbon atoms in the R group, and contains one or more carboxylic acid groups in the R group.

[0039] In some embodiments, non-fatty acid carboxylic acids are either linear or branched in configuration, containing 1 to 5 carbon atoms in the R group and containing 1 carboxylic acid group in the R group.

[0040] When the number of carbon atoms in a chain is referred to herein, it is intended to mean the total number of carbon atoms in the chain (including branches). In the case of a straight chain, this is the same as the carbon chain length. In the case of a branched chain, "chain length" refers to the longest carbon chain branch of the branched chain.

[0041] In some embodiments, the anion contains one carboxylic acid group.

[0042] Exemplary carboxylic acids containing aliphatic chains of four or fewer carbon atoms may include propanoic acid (fatty acid); isobutyric acid (fatty acid); butyric acid (fatty acid); 3,3-dimethylacrylic acid (fatty acid); dimethylacrylic acid (fatty acid); and isovaleric acid (fatty acid). TIFF0007917913000003.tif168128

[0043] Exemplary alternative anions intended herein include decanoic acid and ethylhexyl sulfate. TIFF0007917913000004.tif64128

[0044] Exemplary aromatic anions include, but are not limited to, gallic acid, hydrocinnamic acid, hydroxybenzenesulfonic acid, 4-hydroxybenzenesulfonic acid (4-phenolsulfonic acid), biphenyl-3-carboxylic acid, and phenyl phosphoric acid. TIFF0007917913000005.tif21273

[0045] Hydrophobicity can also be evaluated by logP analysis. "LogP" refers to the logarithm of P (partition coefficient). P is a measure of how well a substance partitions between lipids (oils) and water. P itself is a constant. It is defined as the ratio of the concentration of a compound in the aqueous phase to the concentration of the compound in the immiscible solvent, as a neutral molecule. The partition coefficient is P = [organic] / [aqueous], where [ ] = concentration. Log P = log 10 (Distribution coefficient) = log 10 P In reality, the LogP value will vary depending on the measurement conditions and the choice of partition solvent. A LogP value of 1 means that the concentration of the compound in the organic phase is 10 times higher than in the aqueous phase. An increase in the LogP value of 1 indicates a 10-fold increase in the concentration of the compound in the organic phase compared to the aqueous phase.

[0046] In some aspects of any given position, the anion has a LogP of less than 1.0. In some aspects of any given position, the anion has a LogP of less than 0.80. In some aspects of any given position, the anion has a LogP of less than 0.75. In some aspects of any given position, the anion has a LogP of less than 0.50. In some aspects of any given position, the anion has a LogP of less than 0.25. In some aspects of any given position, the anion has a LogP of less than 0.

[0047] In one aspect of any embodiment, the following is described herein: a composition comprising at least one ionic liquid comprising 1) an anion having a LogP of less than 1.0 and being a carboxylic acid that is not a fatty acid, and 2) a cation comprising a quaternary ammonium. In one aspect of any embodiment, the following is described herein: a composition comprising at least one ionic liquid comprising 1) an anion having a LogP of less than 1.0 and being a carboxylic acid containing an aliphatic chain of 4 carbons or less, and 2) a cation comprising a quaternary ammonium. In one aspect of any embodiment, the following is described herein: a composition comprising at least one ionic liquid comprising 1) an anion having a LogP of less than 1.0 and being aromatic, and 2) a cation comprising a quaternary ammonium.

[0048] In some aspects of any configuration, the IL anion described herein has a pKa of less than 4.0. In some aspects of any configuration, the IL anion described herein has a pKa of less than 4.0 and a LogP of less than 1.0.

[0049] The pKa and LogP values ​​of anions are known in the art and / or can be calculated by those skilled in the art. For example, PubChem and SpiderChem provide these values ​​for various anions, and chemical manufacturers typically provide them as part of their product catalogs. Exemplary pKa and LogP values ​​for anions are provided in Table 1 of this specification.

[0050] Examples of non-limiting anions are provided in Table 1 below.

[0051] [Table 1]

[0052] In some aspects of any configuration, the anion is an alkane. In some aspects of any configuration, the anion is an alkene. In some aspects of any configuration, the anion comprises a single carboxyl group. In some aspects of any configuration, the carbon chain of the carboxylic acid comprises one or more substituents. In some aspects of any configuration, the carbon chain skeleton of the carboxylic acid comprises one or more substituents, each substituent comprising at least one carbon atom. In some aspects of any configuration, the carbon chain skeleton of the carboxylic acid comprises one or more substituents, each substituent comprising at least one methyl group. In some aspects of any configuration, the carbon chain skeleton of the carboxylic acid comprises two substituents, each substituent comprising at least one carbon atom. In some aspects of any configuration, the carbon chain skeleton of the carboxylic acid comprises two substituents, each substituent comprising a methyl group. In some aspects of any configuration, the carbon chain skeleton of the carboxylic acid comprises two substituents, each substituent comprising a methyl group.

[0053] In some aspects of any configuration, the anion is an unsubstituted alkane. In some aspects of any configuration, the anion is an unsubstituted alkene. In some aspects of any configuration, the carbon chain skeleton of the carboxylic acid contains one or more substituents. In some aspects of any configuration, the carbon chain of the carboxylic acid contains one or more substituents, where each substituent contains at least one carbon atom. In some aspects of any configuration, the carbon chain of the carboxylic acid contains one or more substituents, where each substituent is an alkyl, aryl, heteroalkayle, heteroaryl, alkane, or alkene. In some aspects of any configuration, the carbon chain of the carboxylic acid contains one or more substituents, where each substituent is an unsubstituted alkyl, unsubstituted aryl, unsubstituted heteroalkayle, unsubstituted heteroaryl, unsubstituted alkane, or unsubstituted alkene.

[0054] As described herein, when selecting a cation to pair with an anion, the main concern is that the cation should not be too closely associated with the anion, as such close association can cause the anion to be retained on the first side of the biological barrier. Choline and its derivatives have been shown to be particularly suitable as IL cations for the types of anions described herein. Therefore, the IL cations described herein may include quaternary ammonium cations. Quaternary ammonium cations have the structure NR4. + It is a positively charged polyatomic ion, where each R is independently an alkyl group or an aryl group.

[0055] The common term "quaternary ammonium" refers to NH4 + This concerns any compound that can be considered to have been derived from ammonium hydroxide or an ammonium salt by replacing all four hydrogen atoms of the ion with an organic group. For example, quaternary ammonium is NR4 + It has a structure in which each R is hydroxyl, and C1-C may be substituted. 10 Alkyl, possibly substituted C2-C 10 Alkenyl, may be substituted C2-C 10The following are independently selected from alkynyls, optionally substituted aryls, or optionally substituted heteroaryls.

[0056] In some aspects of any given plane, the cation has a molar mass greater than or equal to choline, for example, a molar mass greater than 104.1708 g / mol. In some aspects of any given plane, the cation has a molar mass greater than choline, for example, a molar mass greater than equal to 104.1708 g / mol.

[0057] In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an alkyl, alkane, alkene, or aryl group. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an alkyl, alkane, or alkene group. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an alkane or alkene group. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises a carbon chain of 10 carbon atoms or less in length, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 carbon atoms or less in length. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises a carbon chain of 12 carbon atoms or less in length. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises a carbon chain of 15 carbon atoms or less in length. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises a carbon chain of 20 carbon atoms or less in length.

[0058] In some embodiments of any aspect, each R group of the quaternary ammonium independently contains a carbon chain of 10 carbon atoms or less, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 carbon atoms or less. In some embodiments of any aspect, each R group of the quaternary ammonium independently contains a carbon chain of 12 carbon atoms or less. In some embodiments of any aspect, each R group of the quaternary ammonium independently contains a carbon chain of 15 carbon atoms or less. In some embodiments of any aspect, each R group of the quaternary ammonium independently contains a carbon chain of 20 carbon atoms or less.

[0059] In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an alkyl group with 10 carbon atoms or less, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 carbon atoms or less. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an alkyl group with 12 carbon atoms or less. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an alkyl group with 15 carbon atoms or less. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an alkyl group with 20 carbon atoms or less.

[0060] In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an alkane, an alkene, an aryl, a heteroaryl, an alkyl, or a heteroalkyl. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an unsubstituted alkane, an unsubstituted alkene, an unsubstituted aryl, an unsubstituted heteroaryl, an unsubstituted alkyl, or an unsubstituted heteroalkyl. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an unsubstituted alkane. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an unsubstituted alkene. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises one or more substituents.

[0061] In some embodiments of any aspect, at least one R group of the quaternary ammonium contains a hydroxyl group. In some embodiments of any aspect, one R group of the quaternary ammonium contains a hydroxyl group. In some embodiments of any aspect, only one R group of the quaternary ammonium contains a hydroxyl group.

[0062] Exemplary non-limiting cations may include choline and any cations designated as C1-C7 as defined by the following structures. TIFF0007917913000007.tif113128

[0063] Further non-limiting examples of cations include the following: 1-(hydroxymethyl)-1-methylpyrrolidine-1-ium 1-(2-hydroxyethyl)-1-methylpyrrolidine-1-ium 1-Ethyl-1-(3-hydroxypropyl)pyrrolidine-1-ium 1-(3-hydroxypropyl)-1-methylpyrrolidine-1-ium 1-(4-hydroxybutyl)-1-methylpyrrolidine-1-ium 1-Ethyl-1-(4-hydroxybutyl)pyrrolidine-1-ium 1-(4-hydroxybutyl)-1-propylpyrrolidine-1-ium 1-(5-hydroxypentyl)-1-propylpyrrolidine-1-ium 1-Ethyl-1-(5-hydroxypentyl)pyrrolidine-1-ium 1-(5-hydroxypentyl)-1-methylpyrrolidine-1-ium 1-(hydroxymethyl)-1-methylpiperidine-1-ium 1-(2-hydroxyethyl)-1-methylpiperidine-1-ium 1-Ethyl-1-(2-hydroxyethyl)piperidine-1-ium 1-Ethyl-1-(3-hydroxypropyl)piperidine-1-ium 1-(3-hydroxypropyl)-1-propylpiperidine-1-ium 1-(3-hydroxypropyl)-1-methylpiperidine-1-ium 1-(4-hydroxybutyl)-1-methylpiperidine-1-ium 1-Ethyl-1-(4-hydroxybutyl)piperidine-1-ium 1-(4-hydroxybutyl)-1-propylpiperidine-1-ium 1-Butyl-1-(5-hydroxypentyl)piperidine-1-ium 1-(5-hydroxypentyl)-1-propylpiperidine-1-ium 1-Ethyl-1-(5-hydroxypentyl)piperidine-1-ium 1-(5-hydroxypentyl)-1-methylpiperidine-1-ium 3-ethyl-1-methyl-1H-imidazol-3-ium 1-Methyl-3-propyl-1H-imidazol-3-ium 3-Butyl-1-methyl-1H-imidazol-3-ium 1-Methyl-3-pentyl-1H-imidazol-3-ium 1,2-dimethyl-3-pentyl-1H-imidazol-3-ium 3-Butyl-1,2-dimethyl-1H-imidazol-3-ium 1,2-dimethyl-3-propyl-1H-imidazol-3-ium 3-(hydroxymethyl)-1,2-dimethyl-1H-imidazol-3-ium 3-(2-hydroxyethyl)-1,2-dimethyl-1H-imidazol-3-ium 3-(3-hydroxypropyl)-1,2-dimethyl-1H-imidazol-3-ium 3-(4-hydroxybutyl)-1,2-dimethyl-1H-imidazol-3-ium 3-(5-hydroxypentyl)-1,2-dimethyl-1H-imidazol-3-ium 3-(5-hydroxypentyl)-1-methyl-1H-imidazol-3-ium 3-(4-hydroxybutyl)-1-methyl-1H-imidazol-3-ium 3-(3-hydroxypropyl)-1-methyl-1H-imidazol-3-ium 3-(2-hydroxyethyl)-1-methyl-1H-imidazol-3-ium 3-(hydroxymethyl)-1,2,4,5-tetramethyl-1H-imidazol-3-ium 3-(2-hydroxyethyl)-1,2,4,5-tetramethyl-1H-imidazol-3-ium 3-(3-hydroxypropyl)-1,2,4,5-tetramethyl-1H-imidazol-3-ium 3-(4-hydroxybutyl)-1,2,4,5-tetramethyl-1H-imidazol-3-ium 3-(5-hydroxypentyl)-1,2,4,5-tetramethyl-1H-imidazol-3-ium 1-(5-hydroxypentyl)pyridine-1-ium 1-(4-hydroxybutyl)pyridine-1-ium 1-(3-hydroxypropyl)pyridine-1-ium 1-(2-hydroxyethyl)pyridine-1-ium 1-(hydroxymethyl)pyridine-1-ium 1-Hydroxypyridine-1-ium (Hydroxymethyl)trimethylphosphonium Triethyl(hydroxymethyl)phosphonium Triethyl(2-hydroxydiethyl)phosphonium (2-hydroxyethyl)tripropylphosphonium (3-hydroxypropyl)tripropylphosphonium Tributyl(3-hydroxypropyl)phosphonium (3-hydroxypropyl)tripentylphosphonium (4-hydroxybutyl)tripentylphosphonium (5-hydroxypentyl)tripentylphosphonium

[0064] In some embodiments of any aspect, the cations are choline, C1, C6, and / or C7.

[0065] In some embodiments of any given aspect, the cation is choline, C1, C6, and / or C7, and the anion is an anion selected from Table 1.

[0066] Table 2 below provides a non-limiting set of exemplary combinations of cations and anions.

[0067] [Table 2]

[0068] In some aspects of any aspect, the ionic liquid is not CAGE(choline and gelanate). In some aspects of any aspect, the cation of the ionic liquid is not choline. In some aspects of any aspect, the anion of the ionic liquid is not gelanate or geranic acid. In some aspects of any aspect, including multiple ionic liquids, the first ionic liquid is not CAGE(choline and gelanate). In some aspects of any aspect, including multiple ionic liquids, the cation of the first ionic liquid is not choline. In some aspects of any aspect, including multiple ionic liquids, the anion of the first ionic liquid is not gelanate or geranic acid.

[0069] In some embodiments of any aspect, the anion is selected from the group consisting of geranic acid; glycolic acid; propanoic acid; isobutyric acid; butyric acid; gallic acid; lactic acid; malonic acid; maleic acid; glutaric acid; citric acid; 3,3-dimethylacrylic acid; dimethylacrylic acid; gluconic acid; adipic acid; sodium ethylhexyl sulfate; decanoic acid; hydroxybenzenesulfonic acid; 4-hydroxybenzenesulfonic acid (4-phenolsulfonic acid); isovaleric acid; hydrocinnamic acid (phenylpropanoic acid); phenyl phosphoric acid; and biphenyl-3-carboxylic acid. In some embodiments of any aspect, the anion is selected from the group consisting of glycolic acid; propanoic acid; isobutyric acid; butyric acid; gallic acid; lactic acid; malonic acid; maleic acid; glutaric acid; citric acid; 3,3-dimethylacrylic acid; dimethylacrylic acid; gluconic acid; adipic acid; sodium ethylhexyl sulfate; decanoic acid; hydroxybenzenesulfonic acid; 4-hydroxybenzenesulfonic acid (4-phenolsulfonic acid); isovaleric acid; hydrocinnamic acid (phenylpropanoic acid); phenyl phosphoric acid; and biphenyl-3-carboxylic acid.

[0070] In some embodiments of any aspect, the composition comprises a first ionic liquid and at least a second ionic liquid. Two, three, four, five, or more combinations of any ionic liquids described herein are contemplated. As a non-limiting example, the following table includes exemplary pairs of ionic liquids contemplated herein. TIFF0007917913000009.tif119170

[0071] In some aspects of any configuration in which the composition comprises multiple ionic liquids, the first and second ionic liquids have the same cation, e.g., choline. In some aspects of any configuration in which the composition comprises multiple ionic liquids, the first and second ionic liquids have different anions. For example, the first and second ionic liquids may each contain different anions selected from: geranic acid; glycolic acid; propanoic acid; isobutyric acid; butyric acid; gallic acid; lactic acid; malonic acid; maleic acid; glutaric acid; citric acid; 3,3-dimethylacrylic acid; dimethylacrylic acid; gluconic acid; adipic acid; sodium ethylhexyl sulfate; decanoic acid; hydroxybenzenesulfonic acid; 4-hydroxybenzenesulfonic acid (4-phenolsulfonic acid); isovaleric acid; hydrocinnamic acid (phenylpropanoic acid); phenyl phosphoric acid; and biphenyl-3-carboxylic acid. In some aspects of any configuration in which the composition comprises multiple ionic liquids, the first ionic liquid has a geranic acid anion, and the second ionic liquid has a phenylpropanoic acid anion.

[0072] In some embodiments of any aspect in which the composition comprises multiple ionic liquids, the first ionic liquid is choline and geranic acid (CAGE). In some embodiments of any aspect in which the composition comprises multiple ionic liquids, the second ionic liquid is choline and dimethylacrylic acid (CADA); choline and isovaleric acid (CAVA); choline and phenyl phosphoric acid (CAPP); choline and biphenyl-3-carboxylic acid (CABA); choline and 4-phenolsulfonic acid (CASA); or choline and phenylpropanoic acid (CAPA).

[0073] In some embodiments of any aspect of the composition comprising multiple ionic liquids, the first and second ionic liquids are different ionic liquids selected from the group consisting of: choline and geranic acid (CAGE); choline and dimethylacrylic acid (CADA); choline and isovaleric acid (CAVA); choline and phenyl phosphoric acid (CAPP); choline and biphenyl-3-carboxylic acid (CABA); choline and 4-phenolsulfonic acid (CASA); or choline and phenylpropanoic acid (CAPA). In some embodiments of any aspect of the composition comprising multiple ionic liquids, the first ionic liquid is selected from the group consisting of: choline and geranic acid (CAGE); choline and dimethylacrylic acid (CADA); and choline and choline and biphenyl-3-carboxylic acid (CABA); and the second ionic liquid is selected from the group consisting of: isovaleric acid (CAVA); and choline and phenylpropanoic acid (CAPA). In some embodiments of any aspect of the composition comprising multiple ionic liquids, the first ionic liquid is choline and geranic acid (CAGE), and the second ionic liquid is choline and phenylpropanoic acid (CAPA).

[0074] In some aspects of any given situation, the IL concentration is at least 0.01% w / v. In some aspects of any given situation, the IL concentration is at least 0.05% w / v. In some aspects of any given situation, the IL concentration is at least 0.1% w / v. In some aspects of any given situation, the IL concentration is 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. In some aspects of any given situation, the IL concentration is from about 0.01% w / v to about 1% w / v. In some aspects of any given situation, the IL concentration is from 0.01% w / v to about 1% w / v. In some aspects of any given situation, the IL concentration is from about 0.05% w / v to about 0.5% w / v. In some aspects of any given situation, the IL concentration is from 0.05% w / v to 0.5% w / v.

[0075] In some aspects of any given situation, IL is at least 25% w / w in concentration. In some aspects of any given situation, IL is at least 25% w / w in concentration in water. In some aspects of any given situation, IL is at least 25% w / w in concentration in physiological saline or a physiologically compatible buffer.

[0076] In some aspects of any given situation, IL is concentrated at a concentration of approximately 5% w / w to approximately 75% w / w. In some aspects of any given situation, IL is concentrated at a concentration of approximately 5% w / w to approximately 75% w / w in water, saline, or a physiologically compatible buffer. In some aspects of any given situation, IL is concentrated at a concentration of 5% w / w to approximately 75% w / w in water, saline, or a physiologically compatible buffer.

[0077] In some aspects of any given situation, the IL concentration is at least about 0.1% w / w. In some aspects of any given situation, the IL concentration is at least 0.1% w / w. In some aspects of any given situation, the IL concentration is about 10% w / w to about 70% w / w. In some aspects of any given situation, the IL concentration is 10% w / w to 70% w / w. In some aspects of any given situation, the IL concentration is about 30% w / w to about 50% w / w. In some aspects of any given situation, the IL concentration is 30% w / w to 40% w / w. In some aspects of any given situation, the IL concentration is about 30% w / w to about 50% w / w. In some aspects of any given situation, the IL concentration is 30% w / w to 40% w / w.

[0078] In some aspects of any given situation, the %w / w concentration of IL is the %w / w concentration in water, saline, or a physiologically compatible buffer.

[0079] In some aspects of any given situation, IL is 100% w / w or 100% w / v.

[0080] In some embodiments, IL is an anhydrous salt, for example, an ionic liquid that is not diluted or dissolved in water. In some embodiments, IL is provided as an aqueous solution.

[0081] In some aspects of any given plane, IL has a concentration of at least 25% w / w and a cation:anion ratio of at least 1:3. In some aspects of any given plane, IL has a concentration of at least 25% w / w in water and a cation:anion ratio of at least 1:3. In some aspects of any given plane, IL has a concentration of at least 25% w / w and a cation:anion ratio of 1:3 or 1:4. In some aspects of any given plane, IL has a concentration of at least 25% w / w in water and a cation:anion ratio of 1:3 or 1:4. In some aspects of any given plane, IL is a gel, or a shear-thickened Newton gel.

[0082] In some aspects of any given phase, IL has a cation:anion ratio of about 10:1 to about 1:10. In some aspects of any given phase, IL has a cation:anion ratio of 10:1 to about 1:10. In some aspects of any given phase, IL has a cation:anion ratio of about 5:1 to about 1:5. In some aspects of any given phase, IL has a cation:anion ratio of 5:1 to about 1:5. In some aspects of any given phase, IL has a cation:anion ratio of about 2:1 to about 1:4. In some aspects of any given phase, IL has a cation:anion ratio of 2:1 to about 1:4. In some aspects of any given phase, IL has a cation:anion ratio of about 2:1 to about 1:10. In some aspects of any given phase, IL has a cation:anion ratio of about 2:1 to about 1:1. In some aspects of any given phase, IL has a cation:anion ratio of about 2:1 to about 1:10. In some aspects of any given phase, IL has a cation:anion ratio of 2:1 to 1:1. In some aspects of any given phase, IL has a cation:anion ratio such that there is a greater amount of anion, for example, a ratio less than 1:1. In some aspects of any given phase, IL has a cation:anion ratio such that there is an excess of anion. In some aspects of any given phase, IL has a cation:anion ratio of about 1:1 to about 1:10. In some aspects of any given phase, IL has a cation:anion ratio of 1:1 to 1:10. In some aspects of any given phase, IL has a cation:anion ratio of about 1:1 to about 1:4. In some aspects of any given phase, IL has a cation:anion ratio of 1:1 to 1:4. In some aspects of any given phase, IL has a cation:anion ratio of about 1:1 to about 1:3. In some aspects of any given phase, IL has a cation:anion ratio of 1:1 to 1:3. In some aspects of any given aspect, IL has a cation:anion ratio of approximately 1:1 to approximately 1:2.In some aspects of any given plane, IL has a cation:anion ratio of approximately 1:1, 1:2, 1:3, or 1:4. In some aspects of any given plane, IL has a cation:anion ratio of 1:1, 1:2, 1:3, or 1:4. In some aspects of any given plane, IL has a cation:anion ratio of less than approximately 1:1. In some aspects of any given plane, IL has a cation:anion ratio of less than 1:1. While we don't want to be bound by theory, compositions with a higher amount of anion relative to the cation exhibit greater hydrophobicity.

[0083] In some embodiments of any given aspect, IL has a cation:anion ratio in which cations are in excess.

[0084] In some embodiments of any aspect, for example, when providing one or more nucleic acid molecules in combination with IL, the cation:anion ratio is greater than 1:1, for example, greater than 1:2, about 1:2 to about 1:4, or 1:2 to 1:4.

[0085] In some aspects of any given situation, the IL concentration is at least 20 mM. In some aspects of any given situation, the IL concentration is at least about 20 mM. In some aspects of any given situation, the IL concentration is at least 25 mM. In some aspects of any given situation, the IL concentration is at least about 25 mM. In some aspects of any given situation, the IL concentration is at least 50 mM. In some aspects of any given situation, the IL concentration is at least about 50 mM. In some aspects of any given situation, the IL concentration is at least 100 mM, 500 mM, 1 M, 2 M, 3 M or higher. In some aspects of any given situation, the IL concentration is at least about 100 mM, 500 mM, 1 M, 2 M, 3 M or higher.

[0086] In some aspects of any given situation, the concentration of IL is approximately 50 mM to approximately 4 M. In some aspects of any given situation, the concentration of IL is 50 mM to approximately 4 M. In some aspects of any given situation, the concentration of IL is approximately 500 mM to approximately 4 M. In some aspects of any given situation, the concentration of IL is 500 mM to approximately 4 M. In some aspects of any given situation, the concentration of IL is approximately 1 M to approximately 4 M. In some aspects of any given situation, the concentration of IL is 1 M to approximately 4 M. In some aspects of any given situation, the concentration of IL is approximately 2 M to approximately 4 M. In some aspects of any given situation, the concentration of IL is 2 M to 4 M.

[0087] In some embodiments of any aspect, the IL concentration in the composition or formulation is approximately 0.1 mM to 20 mM. In some embodiments of any aspect, the IL concentration in the composition or formulation is approximately 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, 1mM~14mM, 1mM~12mM, 1mM~10mM, 1mM~8mM, 2mM~20mM, 2mM~18mM, 2mM~16mM, 2mM~14m M, 2mM~12mM, 2mM~10mM, 2mM~8mM, 4mM~20mM, 4mM~18mM, 4mM~16mM, 4mM~12mM, 4mM~1 0mM, 4mM~8mM, 6mM~20mM, 6mM~18mM, 6mM~14mM, 6mM~12mM, 6mM~10mM, 6mM~8mM, 8mM ~20mM, 8mM~18mM, 8mM~16mM, 8mM~14mM, 8mM~12mM, 8mM~10mM, 10mM~20mM, 10mM~18m The ranges are M, 10mM-16mM, 10mM-14mM, 10mM-12mM, 12mM-20mM, 12mM-18mM, 12mM-16mM, 12mM-14mM, 14mM-20mM, 14mM-18mM, 14mM-16mM, 16mM-20mM, 16mM-18mM, or 18mM-20mM. In some aspect of any given scenario, the IL concentration in the composition or formulation is approximately 1 mM, approximately 2 mM, approximately 3 mM, approximately 4 mM, approximately 5 mM, approximately 6 mM, approximately 7 mM, approximately 8 mM, approximately 9 mM, approximately 10 mM, approximately 11 mM, approximately 12 mM, approximately 13 mM, approximately 14 mM, approximately 15 mM, approximately 16 mM, approximately 17 mM, approximately 18 mM, approximately 19 mM, or approximately 20 mM.

[0088] The compositions or combinations described herein are specifically intended to include one, two, three, or more of any type of components described herein. For example, a composition may include a mixture, solution, combination, or emulsion of several different ionic liquids (e.g., the different ionic liquids described herein), and / or a mixture, solution, combination, or emulsion of several different nonionic surfactants, and / or a mixture, solution, combination, or emulsion of several different active compounds.

[0089] In some aspects of any aspect, one or more ILs can be combined with at least one compound. As used herein, “combined with” means two or more substances present in the same formulation, e.g., in a misc, solution, mixture, suspension, colloid, or emulsion, in any molecular or physical arrangement. The formulation may be a homogeneous or heterogeneous mixture. In some aspects of any aspect, the active compound may be contained together with the IL in a solution, mixture, misc, suspension, etc., by a superstructure, e.g., nanoparticles, liposomes, vectors, cells, scaffolds, etc.

[0090] As used herein, “active compound” or “active substance” is any substance that exerts an effect on a target cell or target organism. The terms “compound” and “substance” refer to any entity that does not normally exist or does not exist at a level that is administered and / or provided to a cell, tissue or subject. Substances can be selected from the group including: chemical substances; small organic or inorganic molecules; signaling molecules; nucleic acid sequences; nucleic acid analogs; proteins; peptides; enzymes; aptamers; peptide mimes, peptide derivatives, peptide analogs, antibodies; intracellular antibodies (intrabodies); biomacromolecules, extracts made from biological materials such as cells or tissues of bacteria, plants, fungi, or animals; natural or synthetic compositions or functional fragments thereof. In some embodiments, a substance is any chemical substance, entity, or part, including but not limited to synthetic and natural non-proteinogenic entities. Substances can be known to have the desired activity and / or properties, or can be selected from a diverse library of compounds. Non-limiting examples of active compounds intended for use in the manner described herein include small molecules, polypeptides, nucleic acids, chemotherapeutic / chemotherapy compounds, antibodies, antibody reagents, vaccines, GLP-1 polypeptides or their mimes / analogs, insulin, acarbose, or ruxolitinib.

[0091] As described herein, a nucleic acid molecule may be a vector, an expression vector, an inhibitory nucleic acid, an aptamer, a template molecule or cassette (e.g., for gene editing), or a targeting molecule (e.g., for CRISPR-Cas technology), or any other nucleic acid molecule to be delivered to a cell. The nucleic acid molecule may be RNA, DNA, or a synthetic or modified version thereof. In some embodiments of any aspect, the nucleic acid is an inhibitory nucleic acid, such as siRNA.

[0092] In one aspect of any aspect, the method described herein is a method for delivering a nucleic acid molecule to a cell, comprising the step of contacting the cell with a nucleic acid molecule combined with one or more ILs as described herein. In some aspects of any aspect, the cell is a cell in a subject, and the contact step comprises the step of administering the nucleic acid molecule combined with one or more ILs to the subject. In some aspects of any aspect, the cell is in vitro, in vivo, or ex vivo. In some aspects of any aspect, the cell is a eukaryote. In some aspects of any aspect, the cell is a mammalian cell. In some aspects of any aspect, the cell is an epithelial cell, such as an intestinal epithelial cell. In some aspects of any aspect, the cell is an epidermal cell.

[0093] In some aspects of any morphology in which the active compound contains nucleic acids, the anion has a LogP of less than 1.0 and is a) a non-fatty acid carboxylic acid; or b) a carboxylic acid containing an aliphatic chain of 4 or fewer carbon atoms; or c) an aromatic anion. In some aspects of any morphology in which the active compound contains nucleic acids, the anion has a LogP of less than 1.0 and is an aromatic anion. In some aspects of any morphology in which the active compound contains nucleic acids, the anion is an aromatic anion.

[0094] As used herein, the term “small molecule” refers to chemical substances that may include, but are not limited to, peptides, peptide mimes, 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., 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, as well as salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0095] In some aspects of any given situation, the active compound may be a therapeutic compound or drug, for example, a therapeutically effective agent or compound for treating at least one condition in a subject. Therapeutic compounds are known in the art for a variety of conditions; see, for example, the database available on drugs.com on the web or the catalog of FDA-approved compounds available on catalog.data.gov / dataset / drugsfda-database on the web; these are each incorporated herein by reference in their entirety.

[0096] As a non-limiting example, exemplary antibodies and / or antibody reagents suitable for use as active / therapeutic compounds in this specification include: absiximab; adalimumab; adlimumab-atto; ado-trastuzumab; ado-trastuzumab emtansine; alemtuzumab; alirocumab; atezolizumab; avelumab; basiliximab; belimumab; bevacizumab; bezlotoxumab; blinatumomab; brentuximab; brentuximab vedotin; brodalumab; canakinumab; capromab; Capromab pendetide; certolizumab; certolizumab pegol; cetuximab; daclizumab; daratumumab; denosumab; dinutuximab; dupilumab; durvalumab; eculizumab; elotuzumab; evolocumab; etanercept; etanercept-szzs; golimumab; ibritumomab; ibritumomab thiouxetan; idarucizumab; infliximab; infliximab-abda; infliximab-dyyb; ipilimumab; ixekizumab; mepolizumab; natalizumab; necitumumab; nivolumab; obiltoxaxi Mab; obinutuzumab; ocrelizumab; ofatumumab; oraratuzumab; omalizumab; palivizumab; panitumumab; pembrolizumab; pertuzumab; ramucriumab; ranibizumab; laxibakumab; reslizumab; rituximab; secukinumab; siltuximab; tocilizumab; trastuzumab; ustekinumab; vedolizumab; sarilumab; guselkumab; inotuzumab ozogamicin; inotuzumab; adalimumab-adbm, gemtuzumab ozogamicin; gemtuzumab; bevacizumab-awwb; be Combinations of bispecific antibodies, including those produced by combining inralizumab; emicizumab; emicizumab-kxwh; trastuzumab-dkst; infliximab-qbtx; ivalizumab; ivalizumab-uiyk; tildrakizumab; tildrakizumab-asmn; brosumab; brosumab-twza; erenumab; erenumab-aooe; tositumomab; mogamulizumab; moxetumomab; moxetumomab pasudotox; semiprimab; polatuzumab; catumakisomab; polatuzumab vedotin; and some of the aforementioned.

[0097] Examples of exemplary inhibitory nucleic acids suitable for use as active / therapeutic compounds in this specification include: patisirane; and combinations thereof, including bispecific antibodies prepared by combining some of the foregoing.

[0098] As used herein, the term “chemotherapeutic agent” refers to any chemical or biological agent that has therapeutic utility in the treatment of diseases characterized by abnormal cell proliferation. Such diseases include tumors, neoplasms, and cancers, as well as diseases characterized by hyperplastic proliferation. These agents may function to inhibit the cellular activity on which cancer cells depend for continued proliferation. In some aspects of all embodiments, chemotherapeutic agents are cell cycle inhibitors or cell division inhibitors. The category of chemotherapeutic agents useful in the methods of the present invention includes alkylating / alkaloids, antimetabolites, hormones or hormone analogs, and a wide range of antitumor agents. Most of these agents are directly or indirectly toxic to cancer cells. In one embodiment, the chemotherapeutic agent is a radioactive molecule.

[0099] In some aspects of any given context, the active compound is a polypeptide. In some aspects of any given context, the active compound is an antibody or an antibody reagent. As used herein, the term “antibody reagent” refers to a polypeptide comprising at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binding to a given antigen. An antibody reagent may comprise an antibody or a polypeptide comprising the antigen-binding domain of an antibody. In some aspects, an antibody reagent may comprise a monoclonal antibody or a polypeptide comprising the antigen-binding domain of a monoclonal antibody. For example, an antibody may comprise 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 may comprise 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).

[0100] In some aspects of any morphology involving the active compound a polypeptide (e.g., an antibody or antibody reagent), the anion has a LogP of less than 1.0 and is a) a non-fatty acid carboxylic acid; or b) a carboxylic acid containing an aliphatic chain of 4 carbons or less; or c) an aromatic anion. In some aspects of any morphology involving the active compound a polypeptide (e.g., an antibody or antibody reagent), the anion has a LogP of less than 1.0 and is a) a non-fatty acid carboxylic acid; or b) a carboxylic acid containing an aliphatic chain of 4 carbons or less. In some aspects of any morphology involving the active compound a polypeptide (e.g., an antibody or antibody reagent), the anion has a LogP of less than 1.0 and is a carboxylic acid containing an aliphatic chain of 4 carbons or less. In some aspects of any morphology involving the active compound a polypeptide (e.g., an antibody or antibody reagent), the anion is a) a non-fatty acid carboxylic acid; or b) a carboxylic acid containing an aliphatic chain of 4 carbons or less. In some aspects of any morphology involving the active compound a polypeptide (e.g., an antibody or antibody reagent), the anion has a LogP of less than 1.0.

[0101] In some aspects of any given plane, the active compound has a molecular weight greater than about 450. In some aspects of any given plane, the active compound has a molecular weight greater than about 500. In some aspects of any given plane, the active compound has a molecular weight greater than 450, for example, greater than 450, greater than 500, greater than 550, greater than 600, greater than 1000, or greater. In some aspects of any given plane, the active compound is polar.

[0102] In some embodiments of any aspect in which the active ingredient is an inhibitory nucleic acid, the composition comprises a plurality of ionic liquids, the first of which is choline and geranic acid (CAGE). In some embodiments of any aspect in which the active ingredient is an inhibitory nucleic acid, the composition comprises a plurality of ionic liquids, the second of which is choline and dimethylacrylic acid (CADA); choline and isovaleric acid (CAVA); choline and phenyl phosphate (CAPP); choline and biphenyl-3-carboxylic acid (CABA); choline and 4-phenolsulfonic acid (CASA); or choline and phenylpropanoic acid (CAPA).

[0103] In some embodiments of any aspect in which the active ingredient is an inhibitory nucleic acid, the composition comprises a plurality of ionic liquids, the first and second ionic liquids being different ionic liquids selected from the group consisting of: choline and geranic acid (CAGE); choline and dimethylacrylic acid (CADA); choline and isovaleric acid (CAVA); choline and phenyl phosphate (CAPP); choline and biphenyl-3-carboxylic acid (CABA); choline and 4-phenolsulfonic acid (CASA); or choline and phenylpropanoic acid (CAPA). In some embodiments of any aspect in which the active ingredient is an inhibitory nucleic acid, the composition comprises a plurality of ionic liquids, the first ionic liquid being choline and geranic acid (CAGE), and the second ionic liquid being choline and phenylpropanoic acid (CAPA). In some embodiments of any aspect, the composition is administered topically or formulated for topical administration.

[0104] In some embodiments, the inhibitory nucleic acid is an NFKBIZ inhibitory nucleic acid, for example, that binds to NFKBIZ mRNA and inhibits NFKBIZ expression. As used herein, “NFKBIZ” or “NFKB inhibitor zeta” refers to an inhibitor of the nuclear factor κB (IκB) protein IκBζ, which plays a crucial role in the regulation of the NF-κB complex. It is a direct transcriptional activator of TNF-α, IL-17A, and IL-36-induced psoriasis-related gene products, which are involved in inflammatory signaling, neutrophil chemotaxis, and leukocyte activation. Accordingly, provided herein is a method for treating psoriasis by administering a composition described herein, for example, an NFKBIZ inhibitor, such as an NFKBIZ inhibitory nucleic acid. Sequences of NFKBIZ from several species are known in the art; for example, human NFKBIZ sequences are available in the NCBI database under Gene ID 64332 (e.g., mRNAs NM_001005474.3 (SEQ ID NO: 37) and NM_031419.4 (SEQ ID NO: 38)). Those skilled in the art can easily design NFKBIZ inhibitory nucleic acids, for example, using the automated tools described herein. NFKBIZ inhibitory nucleic acids are also commercially available, for example, from Dharmacon (Lafayette, CO) under catalog number J-040680-06-0050.

[0105] In some embodiments, the inhibitory nucleic acid is a TNF-α inhibitory nucleic acid, for example, that binds to TNF-α mRNA and inhibits TNF-α expression. As used herein, “tumor necrosis factor α” or “TNF-α” refers to an inflammatory cytokine associated with autoimmune diseases, psoriasis, and other conditions. Accordingly, provided herein are methods for treating inflammatory conditions (e.g., psoriasis) and / or reducing or inhibiting inflammation by administering a composition described herein, for example, an active substance which is a TNF-α inhibitor, e.g., a TNF-α inhibitory nucleic acid. Sequences of TNF-α from several species are known in the art; for example, the human TNF-α sequence is available in the NCBI database under Gene ID 7124 (e.g., mRNA NM_000594.4 (SEQ ID NO: 39)). Those skilled in the art can easily design TNF-α inhibitory nucleic acids, for example, using the automated tools described herein. TNF-α inhibitory nucleic acids are also commercially available, for example, from Dharmacon (Lafayette, CO) under catalog numbers J-010546-09-0002, J-010546-10-0002, J-010546-11-0002, and J-010546-12-0002.

[0106] In some embodiments, the inhibitory nucleic acid is an IL-17 inhibitory nucleic acid, for example, one that binds to IL-17 mRNA and inhibits IL-17 expression. As used herein, “interleukin 17” or “IL-17” refers to an inflammatory cytokine produced by activating T cells, which is associated with autoimmune diseases, psoriasis, rheumatoid arthritis, multiple sclerosis, and other conditions. Accordingly, provided herein are methods for treating inflammatory conditions (e.g., psoriasis) and / or reducing or inhibiting inflammation by administering a composition described herein, for example, an IL-17 inhibitor, such as an IL-17 inhibitory nucleic acid. Sequences of IL-17 from several species are known in the art; for example, the human IL-17 sequence is available in the NCBI database under Gene ID 3605 (e.g., mRNA NM_002190.3 (SEQ ID NO: 40)). Those skilled in the art can easily design IL-17 inhibitory nucleic acids, for example, using the automated tools described herein. IL-17 inhibitory nucleic acids are also commercially available, for example, from Dharmacon (Lafayette, CO) under catalog numbers J-007937-05-0002, J-007937-06-0002, J-007937-07-0002, and J-007937-08-0002.

[0107] In one aspect of any aspect, the foregoing provides a method for treating an inflammatory condition and / or reducing inflammation in a subject in need thereof, the method comprising administering to the subject a composition according to the foregoing, comprising at least one IL and at least one anti-inflammatory agent. In some aspects of any aspect, the anti-inflammatory agent is an inhibitory nucleic acid targeting one or more inflammatory gene products, e.g., IL-17, TNF-α, and / or NFKBIZ.

[0108] As used herein, “inflammation” refers to a complex biological response to a harmful stimulus, such as a pathogen, damaged cells, or irritant. Inflammation is a biological protective attempt to eliminate the harmful stimulus and to initiate the tissue healing process. Therefore, the term “inflammation” includes any cellular process that causes associated downstream cellular events, such as fever, fluid accumulation, swelling, abscess formation, and cell death, resulting from the production of inflammatory cytokines, inflammatory mediators, and / or the action of cytokines thus produced. Inflammation can include both acute responses (i.e., responses in which the inflammatory process is active) and chronic responses (i.e., responses characterized by slow progression and the formation of new connective tissue). Acute and chronic inflammation may be distinguished by the types of cells involved. Acute inflammation often involves polymorphonuclear neutrophils; chronic inflammation is usually characterized by lymphohistiocytic and / or granulomatous responses.

[0109] An inflammatory state is any disease state characterized by inflammatory tissue (e.g., infiltrations of leukocytes such as lymphocytes, neutrophils, macrophages, eosinophils, mast cells, basophils, and dendritic cells) or inflammatory processes that induce or contribute to abnormal clinical and histological features of the disease state. Inflammatory states include, but are not limited to, inflammatory states of the skin, lungs, joints, intestines, eyes, endocrine systems, cardiovascular systems, kidneys, livers, central nervous systems, or sepsis-related states. In some aspects, inflammatory states are related to wound healing. In some aspects, inflammation treated according to the methods described herein may be cutaneous inflammation; inflammation caused by substance abuse or drug addiction; inflammation related to infection; corneal inflammation; retinal inflammation; spinal cord inflammation; inflammation related to organ regeneration; and pneumonia.

[0110] In some aspects, the inflammatory state is an inflammatory state of the skin. In some aspects of this context, the inflammatory state is an autoimmune disease.

[0111] Non-limited examples of inflammatory skin conditions include psoriasis, e.g., Sweet's syndrome, pyoderma gangrenosum, subcorneal pustular dermatitis, erythema elevata, Behçet's disease or acute systemic exanthematous pustulosis, bullous disorders, psoriasis, conditions producing pustular lesions, acne, acne vulgaris, dermatitis (e.g., contact dermatitis, atopic dermatitis, seborrheic dermatitis, eczematous dermatitis, fissured eczema) This may include craquelee, photoallergic dermatitis, phototoxic dermatitis, plant photodermatitis, radiation dermatitis, stasis dermatitis or allergic contact dermatitis, eczema, ulcers and erosions resulting from trauma, burns, ischemia of the skin or mucous membranes, some forms of ichthyosis, epidermolysis bullosa, hypertrophic scars, keloids, skin changes of natural aging, photoaging, friction blisters resulting from mechanical shearing of the skin, skin atrophy resulting from topical use of corticosteroids, and inflammation of mucous membranes (e.g., cheilitis, chapped lips, nasal irritation, mucositis and vulvovaginitis).

[0112] In some aspects, an inflammatory state can be an autoimmune disease. Non-exclusive examples of autoimmune diseases may include type 1 diabetes; systemic lupus erythematosus; rheumatoid arthritis; psoriasis; inflammatory bowel disease; Crohn's disease; and autoimmune thyroiditis.

[0113] In addition, inflammatory conditions may include inflammatory conditions of the lungs, e.g., asthma, bronchitis, chronic bronchitis, bronchiolitis, pneumonia, sinusitis, emphysema, adult respiratory distress syndrome, pneumonia, pulmonary fibrosis, and cystic fibrosis (which may additionally or alternatively involve the gastrointestinal tract or other tissues). In addition, inflammatory conditions may include inflammatory conditions of the joints, e.g., rheumatoid arthritis, rheumatoid spondylitis, juvenile rheumatoid arthritis, osteoarthritis, gouty arthritis, infectious arthritis, psoriatic arthritis, and other arthritis conditions. In addition, inflammatory conditions may include inflammatory conditions of the gastrointestinal tract or intestines, e.g., inflammatory bowel disease, Crohn's disease, ulcerative colitis, and distal proctitis. In addition, inflammatory conditions may include inflammatory conditions of the eyes, e.g., dry eye syndrome, uveitis (including iritis), conjunctivitis, scleritis, and keratoconjunctivitis sicca. In addition, inflammatory conditions may include endocrine inflammatory conditions such as autoimmune thyroiditis (Hashimoto's disease), Graves' disease, type 1 diabetes, and acute and chronic inflammation of the adrenal cortex. In addition, inflammatory conditions may include cardiovascular inflammatory conditions such as coronary artery infarction, peripheral vascular disease, myocarditis, vasculitis, vascular regeneration of stenosis, atherosclerosis, and vascular diseases associated with type 2 diabetes. In addition, inflammatory conditions may include renal inflammatory conditions such as glomerulonephritis, interstitial nephritis, lupus nephritis, and nephritis secondary to Wegener's disease, acute renal failure secondary to acute nephritis, post-obstructive syndrome, and tubular ischemia. In addition, inflammatory conditions may include hepatic inflammatory conditions such as hepatitis (resulting from viral infection, autoimmune response, drug treatment, toxins, environmental factors, or as a secondary consequence of primary injury), biliary atresia, primary biliary cirrhosis, and primary sclerosing cholangitis. As a non-limiting example, the inflammatory state may be an inflammatory state of the central nervous system, such as neurodegenerative diseases like multiple sclerosis and Alzheimer's disease or dementia associated with HIV infection.In addition, non-limiting inflammatory conditions may include inflammatory conditions of the central nervous system, e.g., MS; all types of encephalitis and meningitis; acute disseminated encephalomyelitis; acute transverse myelitis; neuromyelitis optica; focal demyelinating syndromes (e.g., Barlow concentric sclerosis and Marburg variant of MS); progressive multifocal leukoencephalopathy; subacute sclerosing panencephalitis; acute hemorrhagic leukoencephalitis (Hearst's disease); human T-lymphophilic virus type 1 associated myelopathy / tropical spastic paraparesis; Devic's disease; human immunodeficiency virus encephalopathy; human immunodeficiency virus vacuolar myelopathy; peripheral neuropathy; Guillain-Barré syndrome and other immune-mediated neuropathy; and myasthenia gravis. In addition, non-limiting inflammatory conditions may include sepsis-related conditions, e.g., systemic inflammatory response syndrome (SIRS), septic shock, or multiple organ dysfunction syndrome (MODS).Further non-limiting examples of inflammatory conditions include endotoxin shock, periodontal disease, polychondritis; periarthritis; pancreatitis; and systemic lupus erythematosus. Erythematosus; Sjögren's syndrome; vasculitis, sarcoidosis, amyloidosis; allergy; anaphylaxis; systemic mastocytosis; pelvic inflammatory disease; multiple sclerosis; multiple sclerosis (MS); celiac disease, Guillain-Barré syndrome, sclerosing cholangitis, autoimmune hepatitis, Raynaud's phenomenon, Goodpasture syndrome, Wegener's granulomatosis, polymyalgia rheumatica, temporal arteritis / giant cell arteritis, chronic fatigue syndrome (CFS), autoimmune Addison's disease, ankylosing spondylitis, acute disseminated encephalomyelitis, antiphospholipid syndrome, aplastic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis This includes thyroiditis, pemphigus, pernicious anemia, polyarthritis in dogs, Reiter's syndrome, Takayasu's arteritis, warm autoimmune hemolytic anemia, fibromyalgia (FM), autoinflammatory PAPA syndrome, familial Mediterranean fever, polymyalgia rheumatica, polyarteritis nodosa, Churg-Strauss syndrome; fibrous alveolitis, hypersensitivity pneumonitis, allergic aspergillosis, idiopathic pulmonary eosinophilia, bronchiolitis-associated pneumonia; urticaria; lupoid hepatitis; familial influenza autoinflammatory syndrome, Muckle-Wells syndrome, neonatal onset multi-organ inflammatory disease, graft rejection (including allograft rejection and graft-versus-host disease), otitis media, chronic obstructive pulmonary disease, sinusitis, chronic prostatitis, reperfusion injury, silicosis, inflammatory myopathy, hypersensitivity, and migraine. In some embodiments, the inflammatory state is associated with an infection, such as a viral, bacterial, fungal, parasitic, or prion infection. In some embodiments, the inflammatory state is associated with an allergic reaction. In some embodiments, the inflammatory state is associated with a contaminant (e.g., asbestos, silicosis, or beryllium disease).

[0114] In some embodiments, the inflammatory state may be a local condition, such as a rash or an allergic reaction. In some embodiments, inflammation is related to a wound.

[0115] Anti-inflammatory agents are known in the art and may include, in no particular way, non-steroidal anti-inflammatory drugs (NSAIDs - such as aspirin, ibuprofen, or naproxen); corticosteroids including glucocorticoids (e.g., cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclomethasone); methotrexate; sulfasalazine; leflunomide; anti-TNF agents; cyclophosphamide; pro-resolving drugs; mycophenolates; or opioids (e.g., endorphins, enkephalins, and dynorphins), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and inhibitors of inflammatory gene products (e.g., the inhibitory nucleic acids mentioned herein). Inflammatory genes are known in the art and include, but are not limited to, NKFBIZ, TNF-α, IL-17, IL-36 (IL-37α, IL-36β, and IL-36γ), IL-22, IL-17C, CXCL8, CCL20, IL23A, DEFB4, and LCN2.

[0116] As used herein, “composition” means any IL, any combination of ILs, or a combination of one or more ILs with one or more active substances described herein, unless otherwise specified.

[0117] In some embodiments of any aspect, the compositions or combinations described herein, comprising at least one IL and optionally an active compound, can be formulated as oral, subcutaneous, transdermal, intratumoral, intravenous, intradermal, or parenteral formulations. In some embodiments of any aspect, the compositions or combinations described herein can be formulated for delivery to mucous membranes, such as the membranes of the nose, mouth, or vagina. In some embodiments of any aspect, the oral formulation may be a degradable capsule comprising a composition comprising at least one IL and optionally an active compound.

[0118] In some aspects of any configuration, what is described herein is a composition comprising at least one IL and at least one active compound as described herein. In some aspects of any configuration, what is described herein is a composition essentially consisting of at least one IL and at least one active compound as described herein. In some aspects of any configuration, what is described herein is a composition consisting of at least one IL and at least one active compound as described herein. In some aspects of any configuration, the composition comprising at least one IL and at least one active compound as described herein is administered as monotherapy, for example, without administering any other treatment to the subject for the condition.

[0119] In any aspect of this specification, what is described herein is a pharmaceutical composition comprising at least one active compound in combination with at least one IL described herein. In some embodiments, the pharmaceutical composition comprises at least one IL and one or more active compounds described herein. In some embodiments, the pharmaceutical composition is essentially composed of at least one IL and one or more active compounds described herein. In some embodiments, the pharmaceutical composition consists of at least one IL and one or more active compounds described herein. In some embodiments, the pharmaceutical composition is essentially composed of an aqueous solution of at least one IL and one or more active compounds described herein. In some embodiments, the pharmaceutical composition consists of an aqueous solution of at least one IL and one or more active compounds described herein.

[0120] The compositions, formulations, and combinations described herein may comprise at least one IL described herein, for example, one IL, two ILs, three ILs, or more. In some embodiments of any aspect, the compositions, formulations, or combinations described herein may comprise at least one IL described herein and CAGE (choline and gelanate).

[0121] In some aspects of any given setting, at least one active compound and at least one ionic liquid are further combined with at least one nonionic surfactant. As used herein, “nonionic surfactant” refers to a surfactant that lacks a net ionic charge and does not visibly separate in an aqueous medium. The properties of a nonionic surfactant depend largely on the ratio of hydrophilic to hydrophobic groups in the molecule. Hydrophilic groups include oxyethylene groups (--OCH2 CH2--) and hydroxyl groups. By varying the number of these groups in hydrophobic molecules such as fatty acids, substances ranging from strongly hydrophobic and water-insoluble compounds such as glyceryl monostearate to strongly hydrophilic and water-soluble compounds such as macrogol can be obtained. Between these two extreme types are those with a more uniformly balanced ratio of hydrophilic and hydrophobic groups, such as macrogol esters and ethers, and sorbitan derivatives. Suitable nonionic surfactants can be found in Martindale, The Extra Pharmacopoeia, 28th Edition, 1982, The Pharmaceutical Press, London, Great Britain, pp. 370 to 379.Non-limited examples of nonionic surfactants include polysorbates, Tween®, block copolymers of ethylene oxide and propylene oxide, glycols and glyceryl esters of fatty acids and their derivatives, polyoxyethylene esters (macrogol esters) of fatty acids, polyoxyethylene ethers (macrogol ethers) of fatty acids and their derivatives, polyvinyl alcohol, and sorbitan esters, sorbitan monoesters, ethers derived from fatty alcohols and polyethylene glycol, polyoxyethylene-polypropylene glycol, alkyl polyglycosides, cetomacrogol 1000, cetostearyl alcohol, cetyl alcohol, cocamide DEA, cocamide MEA, decyl glucoside, decyl polyglucose, glycerol monostearate, and IGEPAL. This includes CA-630, Isoceteth-20, Lauryl Glucoside, Maltoside, Monolaurin, Mycosbutyrin, Nonidet P-40, Nonoxynol-9, Nonoxynol, NP-40, Octaethylene Glycol Monododecyl Ether, N-Octyl β-D-Thioglucopyranoside, Octyl Glucoside, Oleyl Alcohol, PEG-10 Sunflower Glyceride, Pentaethylene Glycol Monododecyl Ether, Polidocanol, Poloxamer, Poloxamer 407, Polyethoxylated Fatamine, Polyglycerol Polyricinoleate, Sorbitan, Sorbitan Monolaurate, Sorbitan Monostearate, Sorbitan Tristearate, Stearyl Alcohol, Surfactin, Triton X-100, and the like. In some aspects of any configuration, at least one nonionic surfactant has a neutral hydrophilic head group.

[0122] As used herein, “polysorbate” refers to surfactants derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with a fatty acid. Common trademark names for polysorbates include Scattics®, Alkest®, Canarcel®, and Tween®. Exemplary polysorbates include polysorbate 20 (polyoxyethylene(20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene(20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene(20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene(20) sorbitan monooleate).

[0123] In some aspects of any given situation, at least one nonionic surfactant (e.g., at least one polysorbate) is present at a concentration of about 0.1% to about 50% w / v. In some aspects of any given situation, at least one nonionic surfactant (e.g., at least one polysorbate) is present at a concentration of 0.1% to 50% w / v. In some aspects of any given situation, at least one nonionic surfactant (e.g., at least one polysorbate) is present at a concentration of about 1% to about 5% w / v. In some aspects of any given situation, at least one nonionic surfactant (e.g., at least one polysorbate) is present at a concentration of 1% to 5% w / v. In some aspects of any given situation, at least one nonionic surfactant (e.g., at least one polysorbate) is present at a concentration of about 3% to about 10% w / v. In some aspects of any given situation, at least one nonionic surfactant (e.g., at least one polysorbate) is present at a concentration of 3% to 10% w / v. In some aspects of any given situation, at least one nonionic surfactant (e.g., at least one polysorbate) is present at a concentration of less than about 5% w / v.

[0124] In some aspects of any configuration, the combination of at least one active compound and at least one IL described herein is provided in one or more nanoparticles. In some aspects of any configuration, the combination of at least one active compound and at least one IL described herein comprises nanoparticles containing the active compound, the nanoparticles being in the state of solution or suspension in a composition containing at least one IL described herein.

[0125] In some aspects of any of these, the compositions described herein, for example, compositions comprising at least one IL and an active compound, may further comprise a pharmaceutically acceptable carrier. Where used herein, the terms “pharmaceutically acceptable,” “physiologically tolerable,” and their grammatical variations are interchangeable when referring to compositions, carriers, diluents, and reagents, and mean 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 increased immune response to the active substance with which it is miscible, unless so desired. The preparation of pharmacological compositions containing active ingredients dissolved or dispersed therein is well understood in the art and does not need to be limited on a formulation basis. Such compositions are usually prepared for injection as either a liquid solution or a suspension, but may also be prepared in solid form suitable for dissolution in liquid or suspension before use. Preparations may also be emulsified or presented as liposome compositions. The active ingredient can be mixed with pharmaceutically acceptable and compatible excipients in an amount appropriate for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and combinations thereof. Furthermore, if desired, the composition may contain small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffers, etc., to enhance the efficacy of the active ingredient. The therapeutic compositions of the present disclosure may contain pharmaceutically acceptable salts of their components. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, tartaric acid, or mandelic acid (formed with the free amino group of the polypeptide). Salts formed with the free carboxyl group may 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, and procaine. Physiologically acceptable carriers are well known in the art.Exemplary liquid carriers are sterile aqueous solutions containing the active ingredient and water, either without any other materials, or containing a buffer such as sodium phosphate, physiological saline, or both, at a physiological pH value, e.g., phosphate-buffered saline. Furthermore, aqueous carriers may contain multiple buffer salts as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions may also contain liquid phases, both with and without water. Examples of such additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active ingredient effective in treating a particular disorder or condition, as used in the methods described herein, 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 art of the technique. For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of the active ingredient in a 0.9% sodium chloride solution.

[0126] In the context of pharmaceutical carriers, the term "carrier" refers to a diluent, adjuvant, excipient, or medium with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids such as water and oil, including those of petroleum, animal, plant, 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, as well as 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, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. If desired, the composition may also contain small amounts of wetting or emulsifying agents or pH buffers. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, or sustained-release formulations. The compositions can be formulated as suppositories using traditional binders and carriers such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. 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.

[0127] Pharmacopoeia-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, e.g., 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 waxes; (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) Water free of pyrogens; (17) Isotonic saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) pH buffer solution; (21) Polyesters, polycarbonates and / or polyanhydrides; (22) Expanders such as polypeptides and amino acids; (23) Serum components such as serum albumin, HDL, and LDL; (22) C2-C2 12 Alcohols; and (23) other non-toxic, compatible substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulations. The terms “excipient,” “carrier,” and “pharmaceutically acceptable carrier” are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active compound. The term “pharmaceutically acceptable carrier” is used with respect to tissue culture media.

[0128] In some embodiments of any aspect, the compositions described herein, for example, compositions comprising at least one IL and an active compound described herein, can be formulated as oral, subcutaneous, intravenous, intradermal, or parenteral formulations. In some embodiments of any aspect, the oral formulation may be a degradable capsule comprising the compositions described herein, for example, a composition comprising at least one IL and an active compound described herein.

[0129] 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 at least one IL. In some embodiments of any aspect described herein, IL greatly enhances the permeability of the active compound through the skin compared to a control in the absence of at least one IL.

[0130] In any aspect of this specification, a method for administering at least one active compound to a subject using a catheter coated with at least one IL as described herein. In any aspect of this specification, a method for collecting body fluids by placing a catheter coated with at least one IL as described herein inside the body.

[0131] In any aspect of this specification, the compositions or combinations described herein are for, for example, a method of administration or delivery of at least one active compound for the treatment of a disease. In any aspect of this specification, a method of administration of at least one active compound is described herein, comprising the step of administering the active compound in combination with at least one IL described herein. In any aspect of this specification, a method of treatment of a disease by administering at least one active compound is described herein, comprising the step of administering the active compound in combination with at least one IL described herein.

[0132] Diseases treated by the methods described herein may include, for example, cancer (breast cancer, leukemia, lymphoma, B-cell chronic lymphocytic leukemia, glioblastoma, carcinoma, urothelial carcinoma, lung cancer, colorectal cancer, lymphoblastic leukemia, lymphocytic leukemia, sarcoma, melanoma, prostate cancer, myeloma, multiple myeloma, non-Hodgkin lymphoma), neuroblastoma, diabetes, infections, inflammation, inflammatory diseases (e.g., rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, plaque psoriasis), autoimmune diseases, atopic dermatitis, gastrointestinal inflammation, inflammatory bowel disease (IBD), hypercholesterolemia, coronary artery disease, asthma, transplant / organ rejection, systemic lupus erythematosus, multiple sclerosis, osteoporosis, and others.

[0133] In some embodiments, the methods described herein relate to treating subjects having or diagnosed with a condition with a composition described herein, for example, comprising at least one IL and an active compound. Subjects having a condition, for example, diabetes mellitus, can be identified by a physician using existing diabetes diagnostic methods. 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, slow healing, polyuria, polydipsia, polyphagia, headache, itchy skin, and fatigue. Tests that may be helpful in diagnosing diabetes include, but are not limited to, blood tests (e.g., fasting glucose levels). A family history of diabetes or exposure to risk factors for diabetes (e.g., being overweight) may also be helpful in determining whether a subject may have diabetes or in making a diagnosis of diabetes.

[0134] The compositions and methods described herein can be administered to subjects having, or diagnosed with, the conditions described herein. In some embodiments, the methods described herein include the step of administering to a subject an effective amount of a composition described herein, for example, a composition comprising at least one IL and an active compound described herein, to alleviate the symptoms of the conditions described herein. As used herein, “alleviating symptoms” means improving 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 more, 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 include, but are not limited to, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), pulmonary, cutaneous, injection, or intratumoral administration. Administration may be local or systemic.

[0135] In some aspects of any given situation, administration is transdermal. In some aspects of any given situation, administration is transdermal, administered into a mucous membrane (e.g., nasal mucosa, oral mucosa, or vaginal mucosa), administered orally, subcutaneously, intradermally, parenterally, intratumorally, or intravenously.

[0136] Oral administration may include providing a liquid, such as tablets (including, but not limited to, scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, lozenges, wafers, aerosol sprays, or syrups, elixirs, liquids, or suspensions in aqueous liquids, non-aqueous liquids, oil-in-water emulsions, or water-in-oil emulsions. Oral formulations may include separated dosage forms, such as tablets (including, but not limited to, scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, lozenges, wafers, aerosol sprays, or syrups, elixirs, liquids, or suspensions in aqueous liquids, non-aqueous liquids, oil-in-water emulsions, or water-in-oil emulsions. Such compositions may contain a predetermined amount of CAGE and at least one active compound and may be prepared by compounding methods well known to those skilled in the art. See, in general, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

[0137] In one aspect of any aspect, the method described herein is a method for delivering at least one active compound by subcutaneous, intradermal, or intravenous administration, comprising the step of administering the active compound in combination with at least one IL described herein. In some aspects of any aspect, subcutaneous, intradermal, or intravenous administration includes administration via injection, catheter, port, etc.

[0138] In any aspect of this specification, a method for parenteral delivery of at least one active compound is described herein, comprising the step of parenterally administering the active compound in combination with at least one IL described herein. In some aspects, parenteral delivery includes delivery to a tumor, e.g., a cancerous tumor. In some aspects of this specification, the compositions or combinations described herein may be parenteral dosage forms. Since parenteral dosage form administration typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or sterilizable before administration to the patient. Examples of parenteral dosage forms include, but are not limited to, injectable liquids, dry formulations that can be dissolved or suspended in a pharmaceutically acceptable medium for injection, injectable suspensions, and emulsions. In addition, controlled-release parenteral dosage forms, including, but not limited to, DUROS® type dosage forms and dose-dumping, can be prepared for patient administration.

[0139] Suitable media that can be used to provide parenteral dosage forms of compositions comprising at least one IL (e.g., CAGE) in combination with at least one active compound disclosing within range are well known to those skilled in the art. Examples include: sterile water; United States Pharmacopeia water for injection; saline solution; glucose solution; sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's solution, among other aqueous media; water-miscible media, among other ethyl alcohol, polyethylene glycol, and propylene glycol, among other water-miscible media; and non-aqueous media, among other corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate, among other water-miscible media. Compounds that alter or modify the solubility of components in the compositions disclosed herein can also be incorporated into the parenteral dosage forms of this disclosure, including normal and controlled-release parenteral dosage forms.

[0140] 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 variability in drug concentrations in the patient's blood and other tissues. These variability can affect several parameters, including administration frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, and side effects. As previously stated herein, compositions comprising at least one IL in combination with at least one active compound can eliminate certain reasons for using controlled-release formulations, but it is intended herein that this method and composition may be utilized in controlled-release formulations in some embodiments. For example, controlled-release formulations can be used to control the onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels of a drug. In particular, controlled or sustained-release dosage forms or formulations can be used to ensure that the maximum efficacy of a drug is achieved while minimizing potential side effects and safety concerns that may arise from both drug overdose (i.e., below the minimum therapeutic level) and exceeding the drug's toxic level. In some embodiments, compositions comprising at least one IL in combination with at least one active compound can be administered in sustained-release formulations.

[0141] Controlled-release pharmaceutical products share the common goal of improving pharmacotherapy beyond what is achieved by their non-controlled-release counterparts. Ideally, the use of optimally designed controlled-release formulations in medical treatment is characterized by the minimum amount of drug substance used to cure or control a condition in the shortest amount of time. The 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 fluctuations in blood levels; 8) improved therapeutic efficacy; 9) reduced synergistic effects 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).

[0142] Most controlled-release formulations are designed to initially release an amount of the drug (active ingredient) that immediately produces the desired therapeutic effect, and then gradually and continuously release other amounts of the drug to maintain this level of therapeutic or preventive effect over a longer period. 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. The control of active ingredient release can be stimulated by a variety of conditions, including but not limited to pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0143] For use with the salts and compositions of this disclosure, a variety of known controlled-release or sustained-release dosage forms, formulations, and devices can be adapted. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 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, for example, by using hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (OROS® (Alza Corporation, Mountain View, Calif. USA, etc.)), or combinations thereof, to provide desired release profiles in various proportions.

[0144] As used herein, the term “effective dose” refers to the amount of a composition necessary 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 the desired effect. Thus, the term “therapeutic effective dose” refers to the amount of a composition sufficient to produce a particular effect when administered to a typical subject. As used herein, effective doses also include, in various contexts, amounts sufficient to delay the onset of disease symptoms, alter the course of a symptomatic disease (e.g., delay the progression of disease symptoms), or reverse the symptoms of a disease. Therefore, determining an exact “effective dose” is generally not feasible. However, for any given case, a person skilled in the art can determine a suitable “effective dose” using only routine experiments.

[0145] Effective dose, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals to determine, for example, the LD50 (lethal dose in 50% of the population) and ED50 (administered dose in 50% of the population). Dosage may vary depending on the dosage form used and the route of administration utilized. The dose-to-toxicity ratio is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compositions and methods exhibiting a large therapeutic index are preferred. The therapeutically effective dose can be initially estimated from cell culture assays. Dosage can also be formulated in animal models to achieve the circulating plasma concentration range, including the IC50 (i.e., the concentration of the active compound that achieves semi-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 dose can be monitored by appropriate bioassays, such as blood glucose assays. Dosage can be determined by a physician and may be adjusted as needed to match the observed therapeutic effect.

[0146] As used herein, “diabetes mellitus” refers to diabetes mellitus, a metabolic disorder characterized by a deficiency or absence of insulin secretion from the pancreas. As used throughout this specification, unless otherwise specified herein, “diabetes mellitus” includes type 1, type 2, type 3, and type 4 diabetes mellitus. The onset of diabetes mellitus is usually due to a combination of genetic and environmental causes, resulting in abnormally high blood glucose levels (hyperglycemia). The two most common forms of diabetes mellitus are caused by either decreased insulin production (in type 1) or decreased bodily response to insulin (in type 2 and pregnancy-related). Both lead to hyperglycemia and primarily cause acute signs of diabetes: excessive urine production, resulting in compensatory thirst and increased fluid intake, blurred vision, unexplained weight loss, lethargy, and altered energy metabolism. Diabetes mellitus can lead to many complications. If the disease is not adequately controlled, acute complications may occur (hypoglycemia, ketoacidosis, or nonketotic hyperosmolar coma). Severe long-term complications (i.e., chronic side effects) include cardiovascular disease (double the risk), chronic renal failure, retinal damage (which can lead to blindness), nerve damage (of several types), and microvascular damage, which can lead to impotence and poor wound healing. Poor wound healing, particularly in the feet, can lead to gangrene and possibly amputation. In some embodiments, diabetes may be type 2 diabetes. Type 2 diabetes (non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes mellitus) is a metabolic disorder characterized primarily by insulin resistance (a decreased response of the body to insulin), relative insulin deficiency, and hyperglycemia. In some embodiments, the subject may be prediabetic, which can be characterized, for example, by having elevated fasting blood glucose or elevated postprandial blood glucose.

[0147] Glucagon-like peptide-1 (GLP-1) is an incretin derived from the transcript of the proglucagon gene, which is known to reduce food intake and hunger in humans and contributes to glucose homeostasis. GLP-1 mimics 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 propeptides 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 may be an interrelated polypeptide derived from GLP-1(7-36) and / or GLP-1(7-37) or a non-human species. The sequences of GLP-1 polypeptides 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 NO: 2-4. In some embodiments, pre- or propeptides of GLP-1, such as glucagon preproprotein (e.g., SEQ ID NO: 1), can be used in the methods or compositions described herein. Natural alleles or variants of any polypeptide described herein are also specifically intended for use in the methods and compositions described herein.

[0148] SEQ ID NO: 1 TIFF0007917913000010.tif13128SEQ ID NO: 2 TIFF0007917913000011.tif4128SEQ ID NO: 3 TIFF0007917913000012.tif4128SEQ ID NO: 4 TIFF0007917913000013.tif4128

[0149] Various GLP-1 mimics are known in the art and are used in the treatment of diabetes. GLP-1 mimics (or analogs) may include exendin-4 (a Heloderma lizard polypeptide homologous to human GLP-1) and its derivatives, GLP-1 analogs modified to be DPP-IV resistant, or human GLP-1 polypeptides conjugated with various further active ingredients, for example, to extend half-life. GLP-1 mimics / analogs may include, for example, exenatide, lixisenatide, dulaglutide, semaglutide, albiglutide, LY2189265, liraglutide, and taspoglutide. Examples of such molecules, as well as further considerations of their manufacture and activity, can be found in the art, for example, Gupta. Indian J. Endocrinol Metab 17:413-421 (2013); Garber. Diabetes Treatments 41:S279-S284 (2018); U.S. Patent Publication US 2009 / 0181912; and International Patent Publication WO 2011 / 080103, each of which is incorporated herein by reference as a whole.

[0150] In some aspects of any aspect, the active compound may be a chemotherapeutic agent or a drug effective in treating cancer. As used herein, the term “cancer” generally refers to a category of diseases or conditions in which abnormal cells divide uncontrollably and invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymphatic systems. There are several main types of cancer. Epithelial malignancies are cancers that occur in the skin or tissues that line or cover internal organs. Non-epithelial malignancies are cancers that occur in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Leukemia is a cancer that occurs in hematopoietic tissue, such as bone marrow, causing the production and invasion of a large number of abnormal blood cells into the bloodstream. Lymphoma and multiple myeloma are cancers that occur in the cells of the immune system. Central nervous system cancers are cancers that occur in the tissues of the brain and spinal cord.

[0151] In some aspects of either aspect, cancer is primary cancer. In some aspects of either aspect, cancer is malignant cancer. As used herein, the term “malignant” refers to cancer in which a population of tumor cells exhibits one or more uncontrolled proliferation (i.e., division beyond the normal limit), invasion (i.e., invasion and destruction of adjacent tissues), and metastasis (i.e., spread to other parts of the body via the lymphatic system or blood). As used herein, the term “metastatic” refers to the spread of cancer from one part of the body to another. The tumor formed by the spread cells is called a “metastatic tumor” or “metastatic cancer.” A metastatic tumor contains cells similar to the cells in the original (primary) tumor. As used herein, the terms “benign” or “non-malignant” refer to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limiting and do not typically invade or metastasize.

[0152] "Cancer cells" or "tumor cells" refer to cancerous growth or individual cells in tissue. A tumor generally refers to a swelling or lesion formed by abnormal cell proliferation, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, such as leukemia, do not necessarily. The terms cancer(cells) and tumor(cells) are used interchangeably for cancer cells that form tumors.

[0153] As used herein, the term “neoplasm” refers to any new and abnormal growth of tissue, for example, an abnormal tissue mass in which the growth exceeds and is discoordinate with the growth of normal tissue. Thus, a neoplasm may be a benign neoplasm, a premalignant neoplasm, or a malignant neoplasm.

[0154] A subject with cancer or a tumor is a subject that has objectively measurable cancer cells present in its body. This definition includes malignant, actively growing cancers, as well as potentially dormant tumors or micrometastases. Cancer that has migrated from its original site and spread to other vital organs can ultimately lead to the subject's death through functional deterioration of the affected organs.

[0155] Examples of cancer include epithelial malignancies, lymphomas, blastomas, non-epithelial malignancies, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancers; endometrial cancer; esophageal cancer; eye cancer; head and neck cancers; stomach cancer (including gastrointestinal cancer); glioblastoma (GBM); liver cancer; hepatocellular carcinoma; carcinoma in situ; kidney cancer or renal cancer; 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 lymphoma and non-Hodgkin lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lips, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancers of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; sperm Focal cancer; thyroid cancer; uterine or endometrial cancer; urinary tract cancer; vulvar cancer; and other epithelial and non-epithelial malignancies; and B-cell lymphoma (low-grade / follicular non-Hodgkin 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-incisional nuclear cell NHL; giant tumor NHL) This includes, but is not limited to, mantle cell lymphoma; AIDS-associated lymphoma; and Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorders (PTLD), as well as nevus disorders, edema (such as that associated with brain tumors), and abnormal angiogenesis associated with Meigs syndrome.

[0156] "Cancer cells" are cancerous, precancerous, or transformed cells, either in vivo, ex vivo, or in tissue culture, that exhibit spontaneous or induced phenotypic changes without necessarily involving the uptake of new genetic material. Transformation can result from infection by a transforming virus and the incorporation of new genomic nucleic acids, or from the uptake of exogenous nucleic acids, but can also result from spontaneous or subsequent exposure to carcinogens, which leads to mutations in endogenous genes. Transformations / cancer are associated with morphological changes, cell immortalization, abnormal growth control, lesion formation, anchorage independence, malignancy, contact inhibition of growth and loss of density limiting, growth factor or serum independence, tumor-specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts, such as nude mice.

[0157] In some aspects of any of these, the compositions described herein, for example, compositions comprising at least one IL described herein in combination with at least one active compound, are administered as monotherapy, for example, not for the treatment of another medical condition.

[0158] In some embodiments of any aspect, the methods described herein may further include administering a second active substance and / or treatment to a subject, for example, as part of a combination therapy, in a composition described herein, for example, a composition comprising at least one IL described herein in combination with at least one active compound, or as another formulation. For example, non-limiting examples of secondary agents and / or treatments for cancer include radiotherapy, surgery, gemcitabine, cisplastin, 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 biposulfan; aziridines such as benzodopa, carbocon, metsuredopa, and uredopa; ethyleneimines and methylamelamines including altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethyloromelamamine; acetogenins (especially bratacin and bratacinone); camptothecin (including the synthetic analog topotecan); and buri Ostatin; calistatin; CC-1065 (including its synthetic analogues adzeresin, carzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleuterobin; pancratistatin; sarcodictin; spongistatin; chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembitine, fenestrine, prednimustine, trophosphamide, uracil mustard, and other nitrogen mustards; carmustine, chlorozotosine, fotemustine, lomustine, nimustine, and ranimnustine, and other nitrosureas;Endiin antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994))); dynemycin including dynemycin A; bisphosphonates such as clodronate; esperamycin; and neocardinostatin chromophores and related chromoprotein enediin antibiotic chromophores), acrasinomycin, actinomycin, autoramycin, azaserin, bleomycin, kakutinomycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, Detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin , rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamipurine, and thioguanine; ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxif Pyrimidine analogs such as rulizine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as phlophosphate; acegraton; aldofsphamide glycoside; aminolevulinic acid; enyluracil; amsacrine; bestrabusil; bisantren; edatraxate; defofamine; demecolsin; diazicon;Elformitin; eriptinium acetate; epotilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; ronidynin; meitansinoids such as meitansin and anthamitocin; mitogwazone; mitoxantrone; mopidammole; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Lazoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2”-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, Beraclin A, Loridine A, and Angidin); Urethane; Vindesine; Dacarbazine; Manomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., TAXOL® Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® Paclitaxel Cremophor-free, albumin-modified nanoparticle formulations (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® Doxetaxel (Rhone-Poulenc Rorer, Antony, France); Chlorambucil; GEMZAR® Gemcitabine; 6-Thiogunine; 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 with irinotecan, 5-FU, and leucovorin); Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids such as retinoic acid; Capecitabine; Combretastatin;The treatment method may include leucovorin (LV); oxaliplatin, including oxaliplatin treatment regimens (FOLFOX); lapatinib (Tykerb RTM); PKC-alpha inhibitors, Raf inhibitors, H-Ras inhibitors, EGFR inhibitors (e.g., erlotinib (Tarceva®)) and VEGF-A inhibitors that reduce cell proliferation, as well as any pharmaceutically acceptable salts, acids, or derivatives thereof. 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.

[0159] In certain embodiments, an effective dose of the composition described herein, for example, a composition comprising at least one IL described herein in combination with at least one active compound, can be administered to a patient once. In certain embodiments, an effective dose of the composition described herein, for example, a composition comprising at least one IL described herein in combination with at least one active compound, can be administered to a patient repeatedly. For systemic administration, a therapeutic dose of the composition described herein, for example, a composition comprising at least one IL described herein 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. In some embodiments of any aspect, at least one active compound is present in the combination at a dose of about 1.0 to 40.0 mg / kg. In some aspects of any given situation, at least one active compound is present in the combination at a dose of 1.0 to 40.0 mg / kg. In some aspects of any given situation, at least one active compound is present in the combination at a dose of approximately 1.0 to 20.0 mg / kg. In some aspects of any given situation, at least one active compound is present in the combination at a dose of 1.0 to 20.0 mg / kg.

[0160] In some embodiments, the active compound is insulin, and the insulin concentration or dose may be about 1 U / kg to about 20 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dose may be 1 U / kg to 20 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dose may be less than 20 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dose may be about 2 U / kg to about 10 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dose may be 2 U / kg to 10 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dose may be about 2 U / kg to about 5 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dose may be 2 U / kg to 5 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dose may be about 5 U / kg to about 10 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dose may be 5 U / kg to 10 U / kg. In some embodiments, the active compound is insulin, and the concentration or dose of insulin may be 2 U / kg, 5 U / kg, or 10 U / kg.

[0161] In one aspect of any aspect, what is described herein is a method for treating a disease in a subject requiring such treatment, which involves administering an active compound combined with at least one IL described herein into the affected tissue by injection. In some aspects, the affected tissue is tissue containing diseased cells. In some aspects, the affected tissue is tissue exhibiting symptoms of the disease. Non-limiting examples of suitable affected tissue include tumor tissue, fat tissue, adipose tissue, etc. In some aspects of any aspect, the disease is a disease resulting from tissue proliferation, e.g., unwanted, abnormal, or pathological tissue proliferation. A disease resulting from tissue proliferation may be any disease caused or characterized by a rate of tissue proliferation, location of tissue proliferation, or pattern / structure of tissue proliferation that differs from normal for the type of tissue in a healthy subject. Non-limiting examples of such diseases are tumors, cancers, adipose / obesity, and / or hyperplasia. In some aspects of any aspect, such diseases are tumors, cancers, adipose / obesity, and / or hyperplasia.

[0162] In some embodiments, treatment can be performed at a less frequent rate after the initial treatment plan. For example, after treatment every other week for three months, treatment can be repeated once a month for six months or more than one year. Treatment by the method described herein can reduce the level of markers or symptoms of the condition 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.

[0163] The dosage of the compositions described herein can be determined by a physician and may be adjusted as appropriate in accordance with the observed therapeutic effect. Regarding the duration and frequency of treatment, it is typical for an experienced clinician to monitor the subject to determine when the treatment provides therapeutic benefit and to decide whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue or resume the treatment, or make other changes to the treatment plan. The medication schedule may vary from once a week to daily, depending on several clinical factors, such as the subject's sensitivity to the active compound. The desired dose or amount of the active substance may be administered in a single dose, or in partial doses, e.g., divided into 2 to 4 partial doses and administered over a period of time, e.g., at appropriate intervals throughout the day, or on another appropriate schedule. In some embodiments, administration may be chronic, e.g., with one or more doses and / or treatments daily over a period of several weeks or months. Examples of medication and / or treatment schedules include daily, twice daily, three times daily, or four or more times daily administration over periods of one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, or six months, or longer. The compositions described herein, for example, compositions comprising at least one IL in combination with at least one active compound, may be administered over a period of time, for example, over periods of 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes.

[0164] The dosage range for administering the compositions described herein by the methods described herein depends, for example, the form of the active compound, its potency, and the degree to which the symptoms, markers, or indicators of the condition described herein are to be reduced, for example, the desired percentage reduction for the symptoms or markers. The dosage should not be so high as to cause adverse side effects. In general, the dosage will vary depending on the patient's age, condition, and sex and may be determined by those skilled in the art. In the event of any complications, the dosage may also be adjusted by the individual physician.

[0165] For example, the effectiveness of a composition described in this specification for the treatment of a condition described herein, or for inducing a response described herein, can be determined by a skilled clinician. However, a treatment is considered an “effective treatment,” where the term “effective treatment” is used herein, if one or more signs or symptoms of a condition described herein are modified in a beneficial manner, or other clinically recognized symptoms improve or even go into remission, or a desired response is induced in at least 10% of cases after treatment by the method described herein. Effectiveness can be assessed, for example, by measuring markers, indicators, symptoms, and / or incidences of a condition treated by the method described herein, or any other appropriate measurable parameter. Effectiveness can also be measured by the absence of deterioration of an individual as assessed by hospitalization, or the absence of the need for medical intervention (i.e., cessation of disease progression). Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of a disease in an individual or animal (in some non-limiting cases, 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 a regression of symptoms. An effective dose for treatment of a disease means an amount sufficient to produce an effective treatment for that disease, as defined herein, when administered to a subject in need of it. The effectiveness of an active substance can be determined by evaluating physical indicators of the condition or desired response. Monitoring the effectiveness of administration and / or treatment by measuring any one of such parameters or any combination of parameters is well within the capabilities of those skilled in the art. Effectiveness can be evaluated in animal models of the conditions described herein, e.g., diabetes or cancer. When using experimental animal models, the effectiveness of a treatment is demonstrated when a statistically significant change is observed in the markers.

[0166] This specification provides in vitro and animal model tests that enable the evaluation of a given dose of a composition described herein, for example, a composition comprising at least one IL in combination with at least one active compound.

[0167] In some embodiments of any aspect, a subject to which a composition comprising, for example, an active compound is administered is a subject who is obese, has, has been diagnosed with, or requires treatment for obesity, obesity, or prevention of weight gain. In some embodiments, the subject is overweight. Methods of the specification include methods for treating obesity, reducing weight gain, preventing weight gain, and promoting weight loss. Such methods can, for example, promote metabolic health and prepare a patient for surgical intervention pursued for aesthetic reasons and / or contraindicated for patients with a high BMI or weight. In some embodiments, for example, if the subject is overweight and / or obese, weight loss may be medically necessary and / or medically directed. In some embodiments, for example, if the subject desires weight loss, regardless of whether weight loss is medically necessary and / or medically directed, weight loss may be for cosmetic purposes.

[0168] The term "obesity" refers to excess body fat. Obesity can be determined by any scale recognized and used by those skilled in the art. Currently, the recognized measure of obesity is the Body Mass Index (BMI), which is a measure of weight in kilograms relative to the square of height in meters. Generally, for adults over 20 years of age, a BMI between approximately 18.5 and 24.9 is considered normal, a BMI between approximately 25.0 and 29.9 is considered overweight, a BMI of approximately 30.0 or higher is considered obese, and a BMI of approximately 40 or higher is considered morbidly obese. (See, for example, Gallagher et al. (2000) Am J Clin Nutr 72:694-701.) These BMI ranges are based on the impact of weight on the increased risk of disease. Some common conditions associated with high BMI and obesity include cardiovascular disease, hypertension (i.e., hypertension), osteoarthritis, cancer, and diabetes. BMI correlates with body fat, but 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. Furthermore, the BMI thresholds that distinguish between normal, overweight, and obese can vary depending on other factors, such as age, sex, ethnicity, health status, and body type. In some embodiments, subjects with obesity have a body fat percentage of at least about 25 kg / m² before undergoing the treatment described herein. 2 The subject may have an obesity index of at least 30 kg / m² before undergoing the treatment described herein. 2 This could be a subject with a certain obesity index.

[0169] In some aspects of any given situation, a subject to which a composition comprising at least one IL described herein, for example, in combination with at least one active compound, is administered is a subject having, diagnosed with, or requiring 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 blood glucose control, such as insulin resistance. Such disorders include, but are not limited to, obesity; excess adipose tissue; diabetes mellitus; fatty liver disease; non-alcoholic fatty liver disease; metabolic syndrome; dyslipidemia; hypertension; hyperglycemia; and cardiovascular disease. “Metabolic syndrome,” unlike metabolic disorders, refers to a combination of medical disorders that, when occurring together, increase the risk of developing cardiovascular disease and diabetes. Several definitions of metabolic syndrome have been established, for example, by the American Heart Association and the International Diabetes Foundation. As just one example, the WHO defines metabolic syndrome as the presence of one of the following: diabetes mellitus, impaired glucose tolerance, impaired fasting blood glucose, or insulin resistance, as well as two of the following: blood pressure ≥ 140 / 90 mmHg, dyslipidemia, central obesity, and microalbuminuria. In some aspects, metabolic disorders may be selected from the group consisting of: obesity; excess adipose tissue; diabetes mellitus; and cardiovascular disease.

[0170] The uptake of many active compounds, such as pharmaceutically active compounds, can be improved by delivering the compound in a solvent. However, most such solvents exhibit toxic side effects and / or act as irritants at the time of delivery, making such approaches often unsuitable for in vivo use. Described herein are methods and compositions that can provide low toxicity along with improved delivery kinetics.

[0171] For convenience, the meanings of several terms and phrases used herein, in the examples, and in the appended claims are provided below. Unless otherwise stated or implied by context, the following terms and phrases include the meanings provided herein. Since the scope of the present invention is limited only by the claims, these definitions are provided to facilitate 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 the present invention belongs. In the event of an apparent conflict between the usage of a term in the art and the definition provided herein, the definition provided herein shall prevail.

[0172] For convenience, certain terms used in the specification, the examples, and the appended claims are collected herein in this specification.

[0173] Carboxylic acid is a carbonyl-bearing functional group having the formula RCOOH, wherein R is aliphatic, heteroaliphatic, alkyl, or heteroalkyl.

[0174] In a preferred embodiment, a straight or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1 to C30 for straight chain, C3 to C30 for branched chain), more preferably 20 or fewer carbon atoms. Likewise, preferred cycloalkyls have 3 to 10 carbon atoms in their ring structure, more preferably 5, 6 or 7 carbons in the ring structure. As used throughout the specification, examples, and claims, the term "alkyl" (or "lower alkyl") is intended to include both "unsubstituted alkyl" and "substituted alkyl", the latter of which refers to an alkyl moiety having one or more substituents replacing hydrogen on one or more carbons of the hydrocarbon backbone.

[0175] Unless otherwise specified, the term "lower alkyl" as used herein means an alkyl group having 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, in its skeletal structure, as defined above. Similarly, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Throughout this application, preferred alkyl groups are lower alkyl groups. In preferred embodiments, substituents referred to as alkyl herein are lower alkyl groups.

[0176] Substituents in substituted alkyl groups may include halogens, hydroxyl, nitro, thiol, amino, azide, imino, amide, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfate, sulfonamide, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and the like.

[0177] As used herein, the term "alkenyl" refers to an unsaturated linear, branched, or cyclic hydrocarbon radical having at least one carbon-carbon double bond. x Alkenyl and C x ~C y Alkenyls are typically used, where X and Y indicate the number of carbon atoms in the chain. For example, C2-C6 alkenyls include alkenyls having a chain of 1-6 carbon atoms and at least one double bond (e.g., vinyl, allyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methylallyl, 1-hexenyl, 3-hexenyl, 3-hexenyl, etc.). Alkenyls expressed with another radical (e.g., aryl alkenyls) refer to a straight-chain or branched, divalent alkenyl radical having the indicated number of atoms. The alkenyl skeleton can optionally have one or more heteroatoms such as N, O, or S inserted.

[0178] As used herein, the term "alkynyl" refers to an unsaturated hydrocarbon radical having at least one carbon-carbon triple bond. x Alkinyl and C x ~C y Alkynnyls are typically used, where X and Y indicate the number of carbon atoms in the chain. For example, C2-C6 alkynyls include alkynyls having a chain of 1-6 carbon atoms and at least one triple bond, such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, isopentinyl, 1,3-hexa-diynyl, n-hexynyl, 3-pentynyl, and 1-hexen-3-ynyl. Alkynnyls expressed with another radical (e.g., arylalkynyls) refer to a linear or branched, divalent alkynyl radical having the indicated number of atoms. The alkynyl skeleton can optionally have one or more heteroatoms such as N, O, or S inserted.

[0179] As used herein, the terms “halogen” or “halo” refer to atoms selected from fluorine, chlorine, bromine, and iodine. The terms “halogen radioisotope” or “halo isotope” refer to radionuclides of atoms selected from fluorine, chlorine, bromine, and iodine. “Halogen-substituted moiety” or “halo-substituted moiety,” as an isolated group or as part of a larger group, means an aliphatic, alicyclic, or aromatic moiety as defined herein, substituted with one or more “halo” atoms. For example, halo-substituted alkyls include haloalkyls, dihaloalkyls, trihaloalkyls, perhaloalkyls, etc. (e.g., halo-substituted (C1-C3) alkyls include chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (-CF3), 2,2,2-trifluoroethyl, perfluoroethyl, 2,2,2-trifluoro-1,1-dichloroethyl, etc.).

[0180] The terms "cyclyl" or "cycloalkyl" refer to saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbon atoms, for example, 3 to 8 carbon atoms, and for example, 3 to 6 carbon atoms.x Cyclyl and C x ~C y Cyclyl groups are typically used, where X and Y indicate the number of carbon atoms in the ring system. Cycloalkyl groups can be further optionally substituted with, for example, 1, 2, 3, or 4 substituents. Examples of cyclyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, 2,5-cyclohexadienyl, cycloheptyl, cyclooctyl, bicyclo[2.2.2]octyl, adamantane-l-yl, decahydronaphthyl, oxocyclohexyl, dioxocyclohexyl, thiocyclohexyl, 2-oxobicyclo[2.2.1]hept-1-yl, and others.

[0181] The term "heterocyclyl" refers to a non-aromatic 5-8 member monocyclic, 8-12 member bicyclic, or 11-14 member tricyclic ring system having 1-3 heteroatoms in the case of a monocyclic, 1-6 heteroatoms in the case of a bicyclic, or 1-9 heteroatoms in the case of a tricyclic, where the heteroatoms are selected from O, N, or S (for example, monocyclic, bicyclic, or tricyclic ring systems have a carbon atom and 1-3, 1-6, or 1-9 N, O, or S heteroatoms, respectively). x Heterocyclyl and C x ~C y Heterocyclyls are typically used, where X and Y indicate the number of carbon atoms in the ring system. In some embodiments, one, two, or three hydrogen atoms in each ring may be substituted by substituents. Exemplary heterocyclyl groups include, but are not limited to, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolidinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl, and 1,4-dioxanyl.

[0182] The terms “bicyclic” and “tricyclic” refer to polycyclic ring assemblies linked by condensation, bridging, or single bonds. As used herein, the term “fused ring” refers to a ring bonded to another ring so as to form a compound having a bicyclic structure, where common ring atoms are directly bonded to each other in both rings. Non-exclusive examples of common fused rings include decalin, naphthalene, anthracene, phenanthrene, indole, furan, benzofuran, and quinoline. Compounds having fused ring systems can be saturated, partially saturated, cyclyl, heterocyclyl, aromatic, heteroaromatic, etc.

[0183] The term "heteroaryl" refers to aromatic 5-8 member monocyclic, 8-12 member fused bicyclic, or 11-14 member fused tricyclic ring systems, each having 1-3 heteroatoms in the case of a monocyclic, 1-6 heteroatoms in the case of a bicyclic, or 1-9 heteroatoms, selected from O, N, or S (for example, monocyclic, bicyclic, or tricyclic systems each have a carbon atom and 1-3, 1-6, or 1-9 N, O, or S heteroatoms, respectively). x Heteroaryl and C x ~C yHeteroaryl compounds are typically used, where X and Y represent the number of carbon atoms in the ring system. Heteroaryl compounds include benzo[b]furan, benzo[b]thiophene, benzimidazole, imidazo[4,5-c]pyridine, quinazoline, thieno[2,3-c]pyridine, thieno[3,2-b]pyridine, thieno[2,3-b]pyridine, indidine, imidazo[l,2a]pyridine, quinoline, isoquinoline, phthalazine, quinoxaline, naphthyridine, quinolidine, indole, isoindole, indazole, indoline, benzoxazole, and benz Zopyrazole, benzothiazole, imidazo[l,5-a]pyridine, pyrazolo[1,5-a]pyridine, imidazo[l,2-a]pyrimidine, imidazo[l,2-c]pyrimidine, imidazo[l,5-a]pyrimidine, imidazo[l,5-c]pyrimidine, pyrrolo[2,3-b]pyridine, pyrrolo[2,3cj]pyridine, pyrrolo[3,2-c]pyridine, pyrrolo[3,2-b]pyridine, pyrrolo[2,3-d]pyrimidine, pyrrolo[3,2-d]pyrimidine, pyrro [2,3-b]pyrazine, pyrazolo[1,5-a]pyridine, pyrrolo[l,2-b]pyridazine, pyrrolo[l,2-c]pyrimidine, pyrrolo[l,2-a]pyrimidine, pyrrolo[l,2-a]pyrazine, triazo[l,5-a]pyridine, pteridine, purine, carbazole, acridine, phenazine, phenothiazene, phenoxazine, l,2-dihydropyrrolo[3,2,1-hi]indole, indoridine, pyrido[l,2-a]indole, 2(lH) -Pyridinone, benzimidazolyl, benzofuranil, benzothiofuranil, benzothiophenyl, benzoxazolyl, benzoxazolinil, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinil, carbazolyl, 4aH-carbazolyl, carborinil, chromanil, clomenil, cinnolinil, decahydroquinolinil, 2H,6H-1,5,2-dithiadinyl, dihydrofloxacinyl[2,3b] Tetrahydrofuran, furanil, flazanil, imidazolidinil, imidazolinil, imidazolyl, 1H-indazolyl, indolenil, indolinil, indolidinil, indolyl, 3H-indolyl, isatinoyl, isobenzofuranil, isochromanil, isoindazolyl, isoindolinil, isoindolyl, isoquinolinil, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinil, naphthilidinil, octa Hydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxepanil, oxetanil, oxyindolyl, pyrimidinyl, phenanthrolinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, Piperidonil, 4-piperidonil, piperonil, pteridinil, prinil, pyranil, pyrazinil, pyrazolidinil, pyrazolinil, pyrazolyl, pyridadinil, pyridoxazole, pyridoimidazole, pyridothiazole, pyridinil, pyridyl, pyrimidinil, pyrrolidinil, pyrrolinil, 2H-pyrrolyl, pyrrrolyl, quinazolinil, quinolinil, 4H-quinolidinil, quinoxalinil, quinuclidinil, tetrahydrofuranil, tetrahydrofuranil Roisoquinolinyl, tetrahydropyranil, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiadinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl and xanthenyl are included but not limited to those derived from these groups. Some exemplary heteroalol groups include pyridyl, furyl or furanil, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, pyridadinyl, pyrazinyl, quinolinyl, indolyl, thiazolyl, naphthilidinyl, 2-amino-4-oxo-3,This includes, but is not limited to, 4-dihydropiteridine-6-yl and tetrahydroisoquinolinyl. In some embodiments, one, two, three, or four hydrogen atoms of each ring may be substituted by substituents.

[0184] As used herein, the term “substituted” refers to the independent substitution of one or more hydrogen atoms on the substituted moiety by substituents independently selected from, but not limited to, alkyl, alkenyl, heterocycloalkyl, alkoxy, aryloxy, hydroxy, amino, amide, alkylamino, arylamino, cyano, halo, mercapto, nitro, carbonyl, acyl, aryl, and heteroaryl groups.

[0185] As used herein, the term “substituted” refers to the independent replacement of one or more (typically one, two, three, four, or five) hydrogen atoms on a substituted moiety by substituents independently selected from the group of substituents listed below or otherwise specified in the definition of “substituents.” In general, non-hydrogen substituents can be any substituents that can bond to the atoms of a given moiety designated to be substituted. Examples of substituents include acyl, acylamino, acyloxy, aldehyde, alicyclic, aliphatic, alkanesulfonamide, alkanesulfonyl, alkalyl, alkenyl, alkoxy, alkoxycarbonyl, alkyl, alkylamino, alkylcarbanoyl, alkylene, alkylidene, alkylthio, alkynyl, amide, amino, aminoalkyl, aralkyl, aralkylsulfonamide, allensulfonamide, allensulfonyl, aromatic, aryl, arylamino, arylcarbanoyl, aryloxy, azide, carbamoyl, carbonyl, carbonyls (including ketones, carboxylates, CF3, cyano( This includes, but is not limited to, CN, cycloalkyl, cycloalkylene, ester, ether, haloalkyl, halogen, halogen, heteroaryl, heterocyclyl, hydroxy, hydroxyalkyl, imino, iminoketone, ketone, mercapto, nitro, oxaalkyl, oxo, oxoalkyl, phosphoryl (including phosphonate and phosphinate), silyl group, sulfonamide, sulfonyl (including sulfate, sulfamoyl and sulfonate), thiol, and ureido moieties, each of which may be substituted or unsubstituted. In some cases, two substituents, together with the carbon to which they are bonded, may form a ring.

[0186] The aryl and heteroaryl compounds may be substituted at one or more positions with one or more substituents, such as halogens, alkyls, aralkyls, alkenyls, alkynyls, cycloalkyls, hydroxyls, aminos, nitros, sulfhydryls, iminos, amides, phosphates, phosphonates, phosphinates, carbonyls, carboxyls, silyls, ethers, alkylthios, sulfonyls, ketones, aldehydes, esters, heterocyclyls, aromatic or heteroaromatic moieties, -CF3, -CN, etc.

[0187] As used herein, the terms “alkoxyl” or “alkoxy” refer to alkyl groups as defined above, having an oxygen radical bonded to them. Typical alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, n-propyloxy, isopropyloxy, n-butyloxy, and isobutyloxy. An “ether” is two hydrocarbons covalently bonded by oxygen. Therefore, alkyl substituents that make an alkyl group an ether are alkoxyl or similar, such as those represented by -O-alkyl, -O-alkenyl, and -O-alkynyl. Alloxy groups can be represented by -O-aryl or O-heteroaryl, where aryl and heteroaryl are defined below. Alkoxy and alloxy groups can be substituted as described above for alkyl groups.

[0188] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

[0189] As used herein, the term "alkylthio" refers to an alkyl group as defined above, having a sulfur radical bonded to it. In a preferred embodiment, the "alkylthio" portion is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl groups. Typical alkylthio groups include methylthio and ethylthio. The term "alkylthio" also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups. "Arylthio" refers to an aryl or heteroaryl group.

[0190] The term "sulfinyl" refers to the radical -SO-. Note that the sulfinyl radical can be further substituted with various substituents to form different sulfinyl groups, including sulfinic acids, sulfinamides, sulfinyl esters, and sulfoxides.

[0191] The term "sulfonyl" refers to the radical -SO2-. Note that sulfonyl radicals can be further substituted with various substituents to form different sulfonyl groups, including sulfonic acids (-SO3H), sulfonamides, sulfonate esters, and sulfones.

[0192] The term "thiocarbonyl" refers to the radical -C(S)-. Note that the thiocarbonyl radical can be further substituted with various substituents to form different thiocarbonyl groups, including thioacids, thioamides, thioesters, and thioketones.

[0193] As used herein, the term "amino" means -NH2. The term "alkylamino" means a nitrogen moiety having at least one linear or branched unsaturated aliphatic, cyclyl, or heterocyclyl radical bonded to nitrogen. For example, typical amino groups include -NH2, -NHCH3, -N(CH3)2, and -NH(C1~C 10 Alkyl), -N(C1~C 10alkyl)₂ and the like. The term “alkylamino” includes “alkenylamino”, “alkynylamino”, “cycloamino”, and “heterocyclylamino”. The term “arylamino” refers to a nitrogen moiety having at least one aryl radical bonded to nitrogen, for example, -NHaryl and -N(aryl)₂. The term “heteroarylamino” refers to a nitrogen moiety having at least one heteroaryl radical bonded to nitrogen, for example, -NHheteroaryl and -N(heteroaryl)₂. Optionally, two substituents may together form a ring with the nitrogen. Unless otherwise stated, compounds described herein containing an amino moiety can include protected derivatives thereof. Suitable protecting groups for an amino moiety include acetyl, tert-butoxycarbonyl, benzyloxycarbonyl and the like.

[0194] The term “aminoalkyl” refers to alkyl, alkenyl, and alkynyl as defined above, except that one or more substituted or unsubstituted nitrogen atoms (-N-) are located between carbon atoms of alkyl, alkenyl, or alkynyl. For example, (C₂~C₆)aminoalkyl refers to a chain containing 2 to 6 carbons and one or more nitrogen atoms located between carbon atoms.

[0195] The term "alkoxyalkoxy" means -O-(alkyl)-O-(alkyl), for example -OCH2CH2OCH3. The term "alkoxycarbonyl" means -C(O)O-(alkyl), for example -C(=O)OCH3, -C(=O)OCH2CH3, etc. The term "alkoxyalkyl" means -(alkyl)-O-(alkyl), for example --CH2OCH3, -CH2OCH2CH3, etc. The term "aryloxy" means -O-(aryl), for example -O-phenyl, -O-pyridinyl, etc. The term "arylalkyl" means -(alkyl)-(aryl), for example benzyl (i.e., -CH2phenyl), -CH2-pyridinyl, etc. The term "arylalkyloxy" means -O-(alkyl)-(aryl), for example -O-benzyl, -O-CH2-pyridinyl, etc. The term "cycloalkyloxy" means -O-(cycloalkyl), for example -O-cyclohexyl, etc. The term "cycloalkylalkyloxy" means -O-(alkyl)-(cycloalkyl, e.g., -OCH2 cyclohexyl). The term "aminoalkoxy" means -O-(alkyl)-NH2, e.g., -OCH2NH2, -OCH2CH2NH2. The terms "mono or dialkylamino" mean -NH(alkyl) or -N(alkyl)(alkyl), e.g., -NHCH3, -N(CH3)2. The terms "mono or dialkylaminoalkoxy" mean -O-(alkyl)-NH(alkyl) or -O-(alkyl)-N(alkyl)( The term "arylamino" means -NH (aryl), for example, -OCH2NHCH3, -OCH2CH2N(CH3)2, etc. The term "arylamino" means -NH (aryl), for example, -NH-phenyl, -NH-pyridinyl, etc. The term "arylalkylamino" means -NH-(alkyl)-(aryl), for example, -NH-benzyl, -NHCH2-pyridinyl, etc. The term "alkylamino" means -NH (alkyl), for example, -NHCH3, -NHCH2CH3, etc. The term "cycloalkylamino" means -NH-(cycloalkyl), for example, -NH-cyclohexyl, etc.The term "cycloalkylalkylamino" refers to -NH-(alkyl)-(cycloalkyl), such as -NHCH2-cyclohexyl.

[0196] It should be noted that with respect to all definitions provided herein, the definitions should be interpreted as open in the sense that they may include further substituents other than those specified. Therefore, C1 alkyl indicates the presence of one carbon atom, but does not indicate what substituents are on that carbon atom. Thus, C1 alkyl is methyl (i.e., -CH3) and CR a R b R c This includes R a , R b , and R c Each of these can independently be any other substituent where the alpha atom relative to hydrogen or carbon is a heteroatom or cyano. Therefore, CF3, CH2OH, and CH2CN are all C1 alkyl groups.

[0197] Unless otherwise specified, the structures described herein are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms, for example, the substitution of a hydrogen atom with deuterium or tritium, or 13 C- or 14 Except for the substitution of carbon atoms with 1C-enriched carbon, compounds having this structure are within the scope of the present invention.

[0198] As used herein, the term "isomer" refers to a compound that has the same molecular formula but differs in structure. Isomers that differ only in their configuration and / or stereostructure are called "stereoisomers." The term "isomer" is also used to refer to enantiomers.

[0199] The term "enantiomer" is used to describe one of a pair of molecular isomers that are mirror images of each other and cannot be superimposed. Other terms used to designate or refer to enantiomers include "stereoisomer" (due to different arrangements or stereochemistry around a chiral center; all enantiomers are stereoisomers, but not all stereoisomers are enantiomers) or "optical isomer" (due to the optical activity of pure enantiomers; this is the ability of different pure enantiomers to rotate plane-polarized light in different directions). Enantiomers generally have the same physical properties, such as melting and boiling points, and also the same spectroscopic properties. Enantiomers may differ from each other in terms of their interactions with plane-polarized light and in terms of biological activity.

[0200] The terms "racemic mixture," "racemic compound," or "racemate" refer to a mixture of two enantiomers of a compound. An ideal racemic mixture is one in which there is a 50:50 mixture of both enantiomers of a compound, such that the optical rotation of the (+) enantiomer cancels out the optical rotation of the (-) enantiomer.

[0201] When used in relation to racemic mixtures, the term "dividing" or "splitting" refers to the separation of a racemic mixture into its two enantiomer forms (i.e., (+) and (-); or (R) and (S) forms). The term can also refer to the enantiomer-selective transformation of a racemic mixture into a single isomer product.

[0202] The term "enantiomer excess" or "ee" refers to a reaction product in which one enantiomer is produced in excess of the other, in molar, weight, or volume ratio F. (+) and F (-) (F (+) and F (-) It is defined as a mixture of (+)- and (-)- enantiomers with a composition shown by the sum of (1). The enantiomer excess is *F (+) -F (-) * is defined as such, and the enantiomer excess percentage is 100 × *F (+) -F (-)*According to the rules. The "purity" of enantiomers is indicated by their ee or percentage ee value (%ee).

[0203] Whether expressed as “purified enantiomer,” “pure enantiomer,” “divided enantiomer,” or “enantiomer-excess compound,” the term is intended to indicate that the amount of one enantiomer exceeds the amount of the other. Therefore, when referring to an enantiomer formulation, both (or either) the percentage of the major enantiomer (e.g., moles, weight, or volume) and / or the percentage of enantiomer excess of the major enantiomer may be used to determine whether the formulation represents a purified enantiomer formulation.

[0204] The term "enantiomer purity" or "enantiomer purity" of an isomer refers to a qualitative or quantitative measure of purified enantiomers; typically, the measurement is expressed based on the enantiomer excess (ee).

[0205] The terms “substantially purified enantiomer,” “substantially divided enantiomer,” and “substantially purified enantiomer formulation” are intended to describe formulations (e.g., derived from non-optically active starting materials, substrates, or intermediates) in which one enantiomer is more concentrated than the other, and more preferably the other enantiomer is less than 20%, more preferably less than 10%, more preferably less than 5%, and even more preferably less than 2% of the enantiomer or enantiomer formulation.

[0206] The terms “purified enantiomer,” “divided enantiomer,” and “purified enantiomer formulation” are intended to describe a formulation (e.g., derived from a non-optically active starting material, substrate, or intermediate) in which one enantiomer (e.g., the R-enantiomer) is more concentrated than the other, and more preferably the other enantiomer (e.g., the S-enantiomer) is less than 30%, preferably less than 20%, more preferably less than 10% (e.g., in this particular case, the R-enantiomer is substantially free of the S-enantiomer), more preferably less than 5%, and even more preferably less than 2%. The purified enantiomer may be synthesized substantially free of the other enantiomer, or the purified enantiomer may be synthesized by a stereopreferential procedure followed by a separation step, or the purified enantiomer may be derived from a racemic mixture.

[0207] The term "enantiomerism," also known as the enantiomer ratio represented by the symbol "E," refers to the selective ability of an enzyme to produce one enantiomer from a racemic substrate relative to the other enantiomer in the racemic product mixture; that is, it is a measure of the enzyme's ability to distinguish between enantiomers. A non-selective reaction has an E of 1, while resolutions with an E of 20 or higher are generally considered useful in synthesis or resolution. Enantiomerism lies in the difference in conversion rates between the enantiomers in question. A reaction product enriched with one enantiomer is obtained; conversely, the remaining substrate is enriched with the other enantiomer. For practical purposes, it is generally desirable to obtain a large excess of one enantiomer. This is achieved by terminating the conversion step after a certain degree of conversion.

[0208] CAGE (choline and gelanate) is an ionic liquid comprising the cation choline (see, e.g., structure I) and the anion gelanate or geranic acid (see, e.g., structures II and III). Preparation of CAGE may be as described, for example, in International Patent Publication WO 2015 / 066647, which is incorporated herein by reference in whole, or as described in the examples herein. TIFF0007917913000014.tif167128

[0209] The terms “reduce,” “reduced,” “decrease,” or “inhibit” are all used herein to mean a reduction of a statistically significant amount. In some embodiments, “reduce,” “decrease,” or “decrease” or “inhibit” typically mean a reduction of at least 10% compared to a reference level (e.g., the absence of a given treatment or active ingredient), and may include reductions of, 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. As used herein, “decrease” or “inhibit” does not include complete inhibition or reduction compared to a reference level. “Complete inhibition” is 100% inhibition compared to a reference level. The reduction can preferably be lowered to a level that is considered within the normal range for a given non-disabled individual.

[0210] The terms “increased,” “enhance,” “enhance,” or “activate” are all used herein to mean an increase of a statically significant amount. In some embodiments, the terms “increased,” “enhance,” or “activate” may 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 including 100%, or any increase between 10% and 100% compared to a reference level, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 times and 10 times or more compared to a reference level. In the context of markers or symptoms, “increase” is a statistically significant increase at such a level.

[0211] As used herein, “subject” means human or animal. Typically, animals are vertebrates such as primates, rodents, domesticated animals, or game animals. Primates include macaques such as chimpanzees, crab-eating macaques, spider monkeys, and rhesus macaques. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domesticated 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, such as a human. The terms “individual,” “patient,” and “subject” are used interchangeably herein.

[0212] Preferably, the subject is a mammal. Mammals may be, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Non-human mammals can be advantageously used as subjects to represent animal models of the medical conditions described herein. The subject may be male or female.

[0213] Subjects may be those who have been previously diagnosed with, identified as suffering from, or have a medical condition requiring treatment or one or more complications related to such a condition, and may optionally have already received treatment for that condition or one or more complications related to it. Alternatively, subjects may not have been previously diagnosed with that condition or one or more complications related to it. For example, subjects may exhibit one or more risk factors for that condition or one or more complications related to it, or subjects may not exhibit any risk factors.

[0214] Those who "need treatment" for a particular medical condition may be individuals who have that condition, have been diagnosed with that condition, or are at risk of developing that condition.

[0215] As used herein, the terms “protein” and “polypeptide” are used interchangeably to refer to a set of amino acid residues linked to one another by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms “protein” and “polypeptide” refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. While “protein” and “polypeptide” are often used in reference to relatively large polypeptides, the term “peptide” is often used in reference to smaller polypeptides; however, the usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to gene products and their fragments. Thus, exemplary polypeptides or proteins include gene products, native proteins, homologs, orthologues, paralogs, fragments and other equivalents, variants, fragments, and analogs of those described above.

[0216] In the various embodiments described herein, it is further intended that any variants (natural or otherwise), alleles, homologs, conserved modified variants, and / or conserved substitution variants of any of the particular polypeptides described herein are included. With respect to amino acid sequences, those skilled in the art will recognize that any individual substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, or protein sequence that modifies a single amino acid or a small percentage of amino acids in the encoded sequence is a “conserved modified variant” in which the modification results in an amino acid substitution with a chemically similar amino acid and the desired activity of the polypeptide is preserved. Such conserved modified variants are added to, but not excluded from, the polymorphic variants, interspecific homologs, and alleles consistent with this disclosure.

[0217] A given amino acid can be replaced with a residue having similar physiological and chemical characteristics, such as by substituting one aliphatic residue with another (e.g., Ile, Val, Leu, or Ala with each other) or by substituting one polar residue with another (e.g., between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, such as substitutions of entire regions with similar hydrophobic characteristics, are well known. Polypeptides containing conservative amino acid substitutions can be tested in any of the assays described herein to confirm that the desired activity, e.g., the activity and specificity of the native or reference polypeptide, is preserved.

[0218] Amino acids can be grouped according to the similarities in the properties of their side chains (in AL 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) Non-charged: 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, natural residues can be classified into groups based on common 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 affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another. Certain conservative substitutions include, for example, Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0219] In some embodiments, the polypeptides described herein (or nucleic acids encoding such polypeptides) may be functional fragments of the amino acid sequences described herein. As used herein, “functional fragment” is a fragment or portion of a peptide that retains at least 50% of the activity of the wild-type reference polypeptide by the assays described herein below. Functional fragments may include conserved substitutions of the sequences disclosed herein.

[0220] In some embodiments, the polypeptides described herein may be variants of the sequences described herein. In some embodiments, the variants are conserved modified variants. Conserved substitution variants can be obtained, for example, by mutations in the native nucleotide sequence. The term “variant” as used herein is a polypeptide that is substantially homologous to the native or reference polypeptide but has a different amino acid sequence from that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding the variant polypeptide includes sequences encoding the variant protein or fragment thereof that, compared to the native or reference DNA sequence, contains one or more nucleotide additions, deletions, or substitutions 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.

[0221] Mutant amino acids or DNA sequences may be identical to the natural or reference sequence by 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. The degree of homology (identity rate) between the natural sequence and the mutant sequence can be determined by comparing the two sequences using a freely available computer program widely used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0222] In some aspects of any aspect, the variant may be a polypeptide having at least 90%, at least 95%, or at least 98% sequence homology to one of the reference sequences provided herein and retaining the wild-type activity of that reference sequence, such as incretin activity. In some aspects of any aspect, the variant may be a polypeptide having at least 90%, at least 95%, or at least 98% sequence homology to one of the natural reference sequences provided herein and retaining the wild-type activity of that reference sequence, such as incretin activity. In some aspects of any aspect, the variant may be a natural polypeptide having at least 90%, at least 95%, or at least 98% sequence homology to one of the reference sequences provided herein and retaining the wild-type activity of that reference sequence, such as incretin activity.

[0223] Modification of natural amino acid sequences can be achieved by any of the many techniques known to those skilled in the art. Mutations can be introduced to specific loci, for example, by synthesizing oligonucleotides containing mutant sequences located on the side of restriction sites that allow ligation to fragments of the natural sequence. After ligation, the resulting reconstructed sequence encodes analogs having the desired amino acid insertions, substitutions, or deletions. Alternatively, oligonucleotide site-specific mutagenesis procedures can be used to provide modified nucleotide sequences having specific codons modified by the required substitutions, deletions, or insertions. Techniques for making such modifications are very well established, including, 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. Patents 4,518,584 and 4,737,462, which are incorporated herein by reference as a whole. Any cysteine ​​residues that do not participate in maintaining the correct conformation of the polypeptide can also generally be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, cysteine ​​bonds can be added to polypeptides to improve their stability or promote oligomerization.

[0224] As used herein, the term “antibody” refers to an immunoglobulin molecule and a molecule containing the immunologically active portion of an immunoglobulin molecule, i.e., an antigen-binding site that binds immunospecifically to an antigen. The term also refers to various forms of antibodies, including, for example, immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-transplant antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, disulfide-bonded Fv, scFv, single-domain antibodies (dAb), diabodies, multispecific antibodies, dual-specific antibodies, anti-idiotype antibodies, bispecific antibodies, their functionally active epitope-binding sites, and / or bifunctional hybrid antibodies, as well as antibodies consisting of two immunoglobulin heavy chains and two immunoglobulin light chains, and full-length antibodies and their antigen-binding sites. Each heavy chain consists of a variable region of the heavy chain (abbreviated here as HCVR or VH) and a constant region of the heavy chain. The constant region of the heavy chain consists of three domains: CH1, CH2, and CH3. Each light chain consists of a variable region (hereinafter abbreviated as LCVR or VL) and a constant region. The constant region of the light chain consists of CL domains. The VH and VL regions may be further divided into hypervariable regions interwoven with a conserved region called the complementarity-determining region (CDR) and the framework region (FR). Thus, each VH and VL region consists of three CDRs and four FRs arranged from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.

[0225] As used herein, the term “antibody reagent” refers to a polypeptide comprising at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binding to a given antigen. An antibody reagent may comprise an antibody or a polypeptide comprising the antigen-binding domain of an antibody. In some embodiments, an antibody reagent may comprise a monoclonal antibody or a polypeptide comprising the antigen-binding domain of a monoclonal antibody. For example, an antibody may comprise 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 may comprise 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).

[0226] Antibodies and / or antibody reagents may include immunoglobulin molecules, monoclonal antibodies, chimeric antibodies, CDR-transplant antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab')2, Fv, disulfide-bonded Fv, scFv, single-domain antibodies, diabodies, multispecific antibodies, dual-specific antibodies, anti-idiotype antibodies, bispecific antibodies, and their functionally active epitope-binding moieties.

[0227] As used herein, the terms “nanobody” or “single-domain antibody” (sdAb) refer to antibodies (VHH) containing a small single variable domain, obtained from camels and dromedaries. Antibody proteins obtained from members of the Camelidae and Dromedariidae families (Camelus baclrianus and Calelus dromaderius), including New World members such as llama species (Lama paccos, lama glama, and lama vicugna), are characterized in terms of size, structural complexity, and antigenicity against human targets. Certain IgG antibodies from this family of naturally occurring mammals lack light chains and are therefore structurally distinct from the typical quaternary structure of antibodies from other animals, which have two heavy chains and two light chains. See PCT / EP93 / 02214 (WO 94 / 04678, published March 3, 1994; this is incorporated herein by reference in its entirety).

[0228] The camel antibody region, identified as a small, single variable domain (VHH), can be obtained through genetic engineering to generate small proteins with high affinity for their target, thereby yielding low molecular weight antibody-derived proteins known as "camel nanobodies." See U.S. Patent No. 5,759,808, issued 2 June 1998; Stijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J. 17: 3512-3520; each of these is incorporated herein by reference in whole. Modified libraries of camel antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. Similar to other antibodies of non-human origin, the amino acid sequences of camel antibodies can be modified by recombinant DNA to obtain sequences more closely resembling human sequences, i.e., the nanobodies can be "humanized." Therefore, the naturally low antigenicity of camel antibodies against humans can be further reduced.

[0229] Camel nanobodies have a molecular weight approximately one-tenth that of human IgG molecules, and the proteins have a physical diameter of only a few nanometers. One significance of their small size is that camel nanobodies can bind to antigen sites that are functionally invisible to larger antibody proteins; that is, camel nanobodies are useful as reagents for detecting antigens that are indistinct using classical immunological techniques, and as potential therapeutic agents. Thus, another significance of their small size is that camel nanobodies can inhibit target proteins as a result of binding to specific sites in grooves or narrow fissures, and therefore can act with a capability more closely resembling classical antibodies than classical low molecular weight drugs. Low molecular weight and small size further result in camel nanobodies that are highly heat-resistant, stable to extreme pH and proteolytic digestion, and have low antigenicity. See U.S. Patent Application No. 20040161738, published August 19, 2004; which is incorporated herein by reference in its entirety. These features, combined with low antigenicity to humans, indicate great therapeutic potential.

[0230] As used herein, the terms “nucleic acid” or “nucleic acid sequence” refer to any molecule, preferably a polymer molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acids can be single-stranded or double-stranded. A single-stranded nucleic acid may be one nucleic acid strand of denatured double-stranded DNA. Alternatively, it may be a single-stranded nucleic acid that does not originate from any double-stranded DNA. In one aspect, a nucleic acid may be DNA. In another aspect, a nucleic acid may be RNA. Suitable DNA may include, for example, cDNA. Suitable RNA may include, for example, mRNA.

[0231] As used herein, “inhibitory nucleic acid” refers to nucleic acid molecules capable of inhibiting the expression of a target, such as double-stranded RNA (dsRNA) or inhibitory RNA (iRNA). In some aspects of any given context, inhibitory nucleic acid may be silencing RNA (siRNA), microRNA (miRNA), or small hairpin RNA (shRNA). Inhibitory nucleic acid may also include guide sequence molecules (e.g., guide RNA) that function, for example, in combination with an enzyme, to induce insertion, deletion, insertion-deletion, and / or mutation of a target, thereby inhibiting the expression of the target.

[0232] Double-stranded RNA molecules (dsRNAs) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein may include RNA strands (antisense strands) that are 30 nucleotides or less in length, i.e., 15–30 nucleotides, and generally 19–24 nucleotides, and have a region substantially complementary to at least a portion of the targeted mRNA transcript. Using these iRNAs enables targeted degradation of the mRNA transcript, resulting in a reduction in the expression and / or activity of the target.

[0233] As used herein, the term “iRNA” refers to an agent comprising RNA (or modified nucleic acids as described herein below) that mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. In some aspects of any aspect, the iRNAs described herein result in inhibition of target expression and / or activity. In some aspects of any aspect, contact of cells with the inhibitor (e.g., iRNA) results in a decrease in target mRNA levels in the cells, including 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%, and 100% of the target mRNA levels found in the cells in the absence of the iRNA. In some aspects of any of these, administration of an inhibitor (e.g., iRNA) to a target results in a decrease in the target mRNA level in the target, including at least approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, and 100% of the target mRNA level found in the target in the absence of iRNA.

[0234] In some aspects of any given situation, iRNA can be dsRNA. A dsRNA contains two RNA strands that are sufficiently complementary to hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a complementarity region that is substantially, and generally fully complementary to, the target sequence. The target sequence can originate from the sequence of mRNA formed during the expression of the target, for example, it can extend to one or more intron boundaries. The other strand (the sense strand) contains a region complementary to the antisense strand such that, when combined under appropriate conditions, the two strands hybridize to form a double-stranded structure. Generally, double-stranded structures are between 15 and 30 base pairs in length, more generally between 18 and 25 base pairs in length, even more generally between 19 and 24 base pairs in length, and most generally between 19 and 21 base pairs in length. Similarly, complementary regions to target sequences are between 15 and 30 base pairs in length, more commonly between 18 and 25 base pairs in length, even more commonly between 19 and 24 base pairs in length, and most commonly between 19 and 21 base pairs in length, in nucleotide lengths, including 19 and 21 base pairs. In some embodiments of either aspect, the dsRNA is between 15 and 20 nucleotides in length, including 15 and 20 nucleotides, and in another embodiment, the dsRNA is between 25 and 30 nucleotides in length, including 25 and 30 nucleotides. As those skilled in the art will recognize, the targeted region of RNA that is targeted for cleavage is in most cases a portion of a larger RNA molecule, often an mRNA molecule. Where applicable, a “portion” of an mRNA target is a contiguous sequence of mRNA targets of sufficient length to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). A short double-stranded dsRNA of 9 base pairs can, under certain circumstances, mediate RNAi-designated RNA cleavage. In most cases, the target is at least 15 nucleotides long, preferably 15 to 30 nucleotides long.

[0235] Exemplary embodiments of inhibitory nucleic acid types may include, for example, siRNA, shRNA, miRNA, and / or amiRNA, which are well known in the art. Those skilled in the art can design further siRNA, shRNA, or miRNA to target the nucleic acid sequence of a target gene or gene product (e.g., mRNA), for example, using commonly available design tools. siRNA, shRNA, or miRNA are generally manufactured by companies such as Dharmacon (Layfayette, CO) or Sigma Aldrich (St. Louis, MO).

[0236] In some aspects of any of these, the RNA of 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, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Modifications include, for example, (a) terminal modifications, e.g., 5' terminal modifications (phosphorylation, conjugation, inverted linkage, etc.), 3' terminal modifications (conjugation, DNA nucleotide, inverted linkage, etc.), (b) base modifications, e.g., stabilizing bases, destabilizing bases, or substitutions of bases that form base pairs with the extended repertoire of a partner, base removal (debastic nucleotide), or conjugate bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) skeletal modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing a modified skeleton or RNAs that do not contain natural internucleoside bonds. RNAs containing a modified skeleton include, among other things, those that do not have a phosphorus atom in their skeleton. For the purposes of this specification and as is sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside skeleton may also be considered oligonucleosides. In some embodiments of any aspect, modified RNAs have a phosphorus atom in their internucleoside skeleton.

[0237] Modified RNA backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramides including 3'-aminophosphoramides and aminoalkyl phosphoramides, thionophosphoramides, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates having the usual 3'-5' linkage, their 2'-5' linked analogues, and those with reverse polarity where adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Modified RNA skeletons that do not contain phosphorus atoms have skeletons formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. These include morpholino bonds (partially formed from the sugar portion of nucleosides); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and other mixed N, O, S, and CH2 component parts. This includes those having a methyl group, oligonucleotides having a heteroatomic skeleton, and in particular those having -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as the methylene(methylimino) or MMI skeleton], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [where the natural phosphodiester skeleton is represented as -OPO-CH2-].

[0238] In other RNA mimics suitable or intended for use with iRNA, both the sugar and nucleoside bonds of the nucleotide unit, i.e., the backbone, are replaced by novel groups. The base unit is maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimic shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and are directly or indirectly bound to the aza nitrogen atom of the amide portion of the backbone.

[0239] The RNA in iRNA can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide that has a modified ribose moiety in which the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose during 3'-end 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).

[0240] Modified RNA may also contain one or more substituted sugar moieties. iRNAs described herein, e.g., dsRNAs, 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 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 1 to about 10. In some aspects of any aspect, dsRNA contains 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, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of iRNA, or group for improving the pharmacodynamic properties of iRNA, and other substituents having similar properties. In some aspects of any of these, the modification involves a 2'-methoxyethoxy (2'-O-CH2CH2OCH3, 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(CH2)2ON(CH3)2 group, also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, as described in the following examples herein, as well as 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2, as also described in the following examples herein.

[0241] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications may also be added to other positions on the iRNA RNA, particularly on the 3' terminal nucleotide or at the 3' position of the sugar in 2'-5' linked dsRNA and the 5' position of the 5' terminal nucleotide. The iRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars.

[0242] Inhibitory nucleic acids may also include modifications or substitutions of nucleic acid bases (often simply referred to in the art as “bases”). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases 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 native nucleic acid bases include other 8-substituted adenines and guanines, 5-halos, particularly 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-daazaadenine, and 3-deazaguanine and 3-deazaadenine. Some of these nucleic acid bases 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 the stability of nucleic acid double helix 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 even more exemplary when combined with 2'-O-methoxyethyl sugar modification.

[0243] The preparation of the modified nucleic acids, skeletons, and nucleic acid bases described above is well known in the art.

[0244] Another modification of the inhibitory nucleic acid characterized by the present invention involves chemically attaching one or more ligands, moieties, or conjugates to the inhibitory nucleic acid that enhance the activity, cell distribution, pharmacokinetic properties, or cellular uptake of the iRNA. Such parts include the cholesterol portion (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), and thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains, e.g., 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, e.g., 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 chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moiety (Mishra et al.This includes, but is not limited to, lipid moieties such as Biochim. Biophys. Acta, 1995, 1264:229-237, or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0245] In some embodiments of the various aspects described herein, the inhibitory nucleic acid is a guide nucleic acid (gNA). As used herein, the terms “guide nucleic acid,” “guide sequence,” “crRNA,” “guide RNA,” “single guide RNA,” “gRNA,” or “CRISPR guide sequence” refer to nucleic acids containing a sequence that determines the specificity of an enzyme, e.g., the Cas DNA-binding protein of the CRISPR / Cas system, to a polynucleotide target. A gNA may include a polynucleotide sequence that hybridizes with the target nucleic acid sequence and has at least partial complementarity with the target nucleic acid sequence sufficient to guide sequence-specific binding of an enzyme, e.g., a nuclease, to the target nucleic acid sequence.

[0246] In some embodiments, the enzyme guided by gNA is a gene editing protein, e.g., any nuclease that guides a nick or double-strand break to a desired recognition site. Such enzymes may be native or modified. These breaks can then be repaired by the cell in one of two ways: non-homologous end joining and homology-directed repair (homologous recombination). In non-homologous end joining (NHEJ), the double-strand break is repaired by direct ligation of the broken ends. Thus, no new nucleic acid material is inserted into the site, but some nucleic acid material may be lost, resulting in a deletion. In homology-directed repair, a donor polynucleotide homologous to the cleaved target DNA sequence can be used as a template for repairing the cleaved target DNA sequence, triggering the transfer of genetic information from the donor polynucleotide to the target DNA. Thus, new nucleic acid material can be inserted / copied into the site. Modification of target DNA by NHEJ and / or homology-directed repair can be used for gene correction, gene substitution, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, and more.

[0247] In one embodiment, gene-editing proteins are CRISPR-associated nucleases. The natural prokaryotic CRISPR-associated nuclease system comprises an array of CRISPR-associated ("Cas") nuclease proteins and short repetitive sequences (i.e., clusters of regularly spaced short reverse repeats) with intervening variable sequences of constant length. The RNA of the transcribed CRISPR array is processed into a small guide RNA by a subset of Cas proteins, which generally has two components, as will be discussed below. There are at least three distinct systems: Type I, Type II, and Type III. The enzymes involved in the processing of RNA to mature crRNA differ in the three systems. In the natural prokaryotic system, the guide RNA ("gRNA") includes two short non-coding RNA species called CRISPR RNA ("crRNA") and trans-acting RNA ("tracrRNA"). In the exemplary system, the gRNA forms a complex with a nuclease, e.g., a Cas nuclease. The gRNA:nuclease complex binds to a target polynucleotide sequence containing a protospacer-adjacent motif ("PAM") and a protospacer, which are sequences complementary to a portion of the gRNA. Recognition and binding of the target polynucleotide by the gRNA:nuclease complex induces cleavage of the target.

[0248] Any CRISPR-related nuclease can be used in the system and method of the present invention. CRISPR nuclease systems are known to those skilled in the art, such as Cas9, Cas12, Cas12a, etc., and are patented / applications 8,993,233, US 2015 / 0291965, US 2016 / 0175462, US 2015 / 0020223, US 2014 / 0179770, 8,697,359; 8,771,945; 8,795,965; WO 2015 / 191693; US 8,889,418; WO 2015 / 089351; WO 2015 / 089486; WO 2016 / 028682; WO 2016 / 049258; WO 2016 / 094867; WO See 2016 / 094872;WO 2016 / 094874;WO 2016 / 112242;US 2016 / 0153004;US 2015 / 0056705;US 2016 / 0090607;US 2016 / 0029604;8,865,406;8,871,445; each of these is incorporated herein by reference in its entirety. The nuclease may also be a phage Cas nuclease, e.g., CasΦ (e.g., Pausch et al. Science 369: 333-7 (2020); this is incorporated herein by reference in its entirety).

[0249] The full-length guide nucleic acid chain can be of any length. For example, the guide nucleic acid chain may have a nucleotide length of approximately 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more, or it may have a nucleotide length of approximately 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more. In some embodiments of the various aspects described herein, the nucleic acid chain has a nucleotide length of approximately 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less. For example, guide nucleic acid sequences are 10 to 30 nucleotides long.

[0250] In addition to a sequence complementary to the target nucleic acid, in some embodiments, gNAs also include a scaffold sequence. Expression of gNAs encoding both a sequence complementary to the target nucleic acid and a scaffold sequence has a dual function of both binding to the target nucleic acid (hybridization) and recruiting endonucleases to the target nucleic acid, which can result in site-specific CRISPR activity. In some embodiments, such chimeric gNAs may be called single guide RNAs (sgRNAs).

[0251] In some embodiments of the various aspects described herein, guide nucleic acids are designed using guide design tools (e.g., Benchling™; Broad Institute GPP™; CasOFFinder™; CHOPCHOP™; CRISPOR™; Deskgen™; E-CRISP™; Geneious™; GenHub™; GUIDES™ (e.g., for library design); Horizon Discovery™; IDT™; Off-Spotter™; and Synthego™; these are available on the web).

[0252] As used herein, the term “vector” refers to a nucleic acid construct designed for delivery to a host cell or for movement between different host cells. Where used herein, a vector may be viral or nonviral. The term “vector” encompasses any genetic element that, when associated with the correct regulatory elements, can replicate and move a gene sequence into a cell. Vectors may include, but are not limited to, cloning vectors, expression vectors, recombinant vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, and the like.

[0253] As used herein, the term “expression vector” refers to a vector that directs the expression of RNA or polypeptides from a sequence bound to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often, though not necessarily, heterogeneous to the cell. An expression vector may include additional elements; for example, an expression vector may have two replication systems, thus enabling it to be maintained in two organisms, e.g., in human cells for expression, and in a prokaryotic host for cloning and amplification. The term “expression” refers to cellular processes involved in the production of RNA and proteins, and, if applicable, the secretion of proteins, including, but not limited to, transcription, transcriptional processing, translation, and protein folding, modification, and processing. “Expression products” include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term “gene” means a nucleic acid sequence that is transcribed to RNA (DNA) in vitro or in vivo when functionally ligated to a suitable regulatory sequence. A gene may or may not include regions before and after the coding region, such as the 5' untranslated (5' UTR) or "leader" sequence and the 3' UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0254] As used herein, the term “viral vector” refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into a viral vector particle. Viral vectors may contain nucleic acids encoding polypeptides, such as those described herein, instead of non-essential viral genes. Vectors and / or particles may be used for the purpose of transferring any nucleic acid into cells, either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0255] The term "recombinant vector" refers to a vector containing a heterologous nucleic acid sequence or "transgene" that can be expressed in vivo. It should be understood that the vectors described herein, in some embodiments, can be combined with other suitable compositions and therapies. In some embodiments, the vectors are episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotides of interest outside of chromosomal DNA at a high copy number in a subject, thereby eliminating the potential effects of chromosomal integration.

[0256] As used herein, the terms “to treat,” “to treat,” “treating,” or “improving” refer to therapeutic actions aimed at reversing, mitigating, improving, inhibiting, delaying, or halting the progression or severity of a condition or disorder associated with a disease or disorder, such as, for example, a medical condition or disorder described herein. The term “treating” includes reducing or mitigating at least one adverse effect or symptom of a medical condition, disorder, or disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Or, treatment is “effective” if the progression of the disease is reduced or halted. That is, “treatment” includes not only improvement of symptoms or markers but also cessation or at least delay of the progression or worsening of symptoms compared to what would be expected without treatment. Beneficial or desirable clinical outcomes include, but are not limited to, relief of one or more symptoms, a decline in the severity of the disease, stabilization of the disease state (i.e., no worsening), delay or halting of disease progression, improvement or temporary relief of the disease state, remission (whether partial or complete), and / or a reduction in mortality, whether detectable or undetectable. The term "treatment" of a disease also includes reducing the symptoms or side effects of the disease (including symptomatic treatment).

[0257] As used herein, the term “pharmaceutical composition” refers to an active ingredient in combination with a pharmaceutically acceptable carrier, such as a carrier widely used in the pharmaceutical industry. The term “pharmaceutically acceptable” is used herein to mean a compound, material, composition, and / or dosage form suitable for use in contact with human and animal tissues, within the bounds of sound medical judgment, with a reasonable benefit / risk ratio, and without excessive toxicity, irritation, allergic reactions, or other problems or complications. In some aspects of any aspect, a pharmaceutically acceptable carrier may be a carrier other than water. In some aspects of any aspect, a pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticles, and / or ointment. In some aspects of any aspect, a pharmaceutically acceptable carrier may be an artificial or engineered carrier, such as a carrier in which the active ingredient is not found to occur naturally.

[0258] As used herein, the term “administer” means placing a compound as disclosed herein into a subject by a method or route that results in the delivery of at least a partial active substance 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.

[0259] As used herein, “contact” means any suitable means for delivering or exposing an active substance to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture media, perfusion, injection, or other delivery methods well known to those skilled in the art. In some embodiments, contact includes physical human activity, such as injection; dispensing, mixing, and / or decanting; and / or operation of a delivery device or machine.

[0260] The term “effective dose” means an amount of a composition sufficient to produce at least some improvement in the symptoms associated with a medical condition. In one embodiment, “effective dose” means an amount of a composition that reduces markers or symptoms of a medical condition in a subject having a medical condition.

[0261] The terms "statistically significant" or "significantly significant" refer to statistical significance, which generally means a difference greater than two standard deviations (2SD).

[0262] Except in the examples provided, or unless otherwise indicated, all numbers used herein to represent ingredient quantities or reaction conditions should be understood to be modified in all cases by the term "approximately." When used in relation to percentages, the term "approximately" may mean ±1%.

[0263] As used herein, the terms “contains” or “includes” 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 “contains” means that other elements may be present in addition to the elements defined herein. The use of “contains” indicates inclusion rather than limitation.

[0264] The term "consisting of" refers to the compositions, methods, and their respective components described herein, and does not include any elements not described in the description of their embodiments.

[0265] As used herein, the term “essentially derived from” refers to an element necessary for a given embodiment. This term allows for the presence of additional elements that do not substantially affect the basic and novel or functional features of that embodiment of the invention.

[0266] As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells, and / or particles in which a first entity binds to a second target entity with greater specificity and affinity than it binds to a third, non-target entity. In some embodiments, specific binding refers to the affinity of the first entity to the second target entity, which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or more than its affinity to a third, non-target entity. A reagent specific to a given target is one that exhibits specific binding to that target under the conditions of the test being performed.

[0267] The singular terms “a,” “an,” and “the” refer to multiple objects unless the context otherwise explicitly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context otherwise explicitly indicates otherwise. In the implementation or testing of this disclosure, similar or equivalent methods and materials may be used, but suitable methods and materials are described below. The abbreviation “e.g.” “exempli gratia” is derived from the Latin “exempli gratia” and is used herein to indicate non-limiting examples. Thus, the abbreviation “e.g.” “For example” is synonymous with the term “for example.”

[0268] The grouping of alternative elements or embodiments of the Invention disclosed herein should not be construed as limitation. Members of each group may be referred to and asserted 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 or removed from a group for convenience and / or patentability reasons. In the event of any such inclusion or removal, the specification shall be deemed to include the modified group and shall satisfy the written description of all Markush groups used in the appended claims.

[0269] Unless otherwise defined herein, scientific and technical terms used in connection with this application have meanings that are broadly understood by those skilled in the art to which this disclosure belongs. It should be understood that the present invention is not limited to, and therefore can be varied from, the specific methodologies, protocols, and reagents described herein. Technical terms used herein are for the sole purpose of describing specific aspects and are not intended to limit the scope of the present invention, which are defined solely by the claims.Definitions of common terms in immunology and molecular biology are 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. This can be found in 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 as a whole.

[0270] Those skilled in the art can easily identify the chemotherapeutic agents to be used (see, for example, 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).

[0271] Other terms are defined herein within the context of describing various aspects of the present invention.

[0272] All patents and other publications cited throughout this application, including references, issued patents, published patent applications, and concurrently pending patent applications, are expressly incorporated herein by reference for the purpose of explaining and disclosing methodologies described in such publications, which may, for example, be used in connection with the technology described herein. These publications are provided exclusively for their disclosures prior to the filing date of this application. In this regard, it should not be construed as an acknowledgment that the inventors are not granted any prior rights to such disclosures, either on the grounds of prior invention or for any other reason. All statements relating to dates or indications relating to the contents of these documents are based on information available to the applicants and do not constitute any acknowledgment of the accuracy of the dates or contents of these documents.

[0273] The description of the aspects of this disclosure is not intended to be comprehensive or to limit the disclosure to any specific form disclosed. Specific aspects and examples of this disclosure are described herein for illustrative purposes only, but various equivalent modifications are possible within the scope of this disclosure, as will be recognized by those skilled in the art. For example, while the steps or functions of a method are described in a given order, in alternative embodiments the functions may be performed in a different order, or the functions may be performed substantially simultaneously. The teachings of this disclosure provided herein can be applied to other procedures or methods as needed. Further embodiments can be provided by combining the various aspects described herein. Aspects of this disclosure can be modified to use the compositions, functions, and concepts of the above-mentioned references and applications, if necessary, to provide even further embodiments of this disclosure. Furthermore, given the equivalence of biological functions, several changes can be made to the protein structure without affecting the biological or chemical action in any way or amount. These and other changes can be made in this disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0274] Specific elements of any of the embodiments described above can be combined with or substituted for elements of other embodiments. Furthermore, while the advantages related to specific embodiments of this disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments are necessarily required to exhibit such advantages and be within the scope of this disclosure.

[0275] The technologies described herein are further illustrated by the following examples, but these examples should not be construed as further limitations.

[0276] Some aspects of the technology described herein can be defined by any of the following numbered sections: 1. (a) Carboxylic acids that are not fatty acids, (b) Carboxylic acids containing aliphatic chains of 4 carbon atoms or less, (c) Aromatic anions, and / or (d) Anions with a LogP of less than 1.0 An anion which is at least one of the following, and cations containing quaternary ammonium A composition comprising at least one ionic liquid, including the following. 2. Anion, Having a LogP of less than 1.0, a. Carboxylic acids that are not fatty acids, b. Carboxylic acids containing aliphatic chains of 4 carbon atoms or less, c. Aromatic anions That is, Any of the compositions described in the preceding paragraph. 3. Any composition according to the preceding item, wherein the fatty acid comprises an aliphatic chain of three carbon atoms or less. 4. Any composition according to the preceding item, wherein the anion comprises only one carboxylic acid group (e.g., an R-COOH group). 5. Any composition according to the preceding item, wherein the anion is selected from the group consisting of glycolic acid; propanoic acid; isobutyric acid; butyric acid; gallic acid; lactic acid; malonic acid; maleic acid; glutaric acid; citric acid; 3,3-dimethylacrylic acid; dimethylacrylic acid; gluconic acid; adipic acid; sodium ethylhexyl sulfate; decanoic acid; hydroxybenzenesulfonic acid; 4-hydroxybenzenesulfonic acid; isovaleric acid; hydrocinnamic acid; 4-phenolsulfonic acid; phenyl phosphoric acid; and biphenyl-3-carboxylic acid. 6. Any composition according to the preceding item, wherein the cation has a molar mass equal to or greater than that of choline. 7. Quaternary ammonium compounds are NR4 + A composition according to any of the above items, having the structure and comprising at least one R group comprising a hydroxyl group. 8. Quaternary ammonium is NR4 + A composition according to any of the preceding items, having the structure and containing only one hydroxyl group among the R groups. 9. Any composition according to the preceding item, wherein the cation is C1, C6, or C7. 10. Any composition according to the preceding item, wherein the ionic liquid contains a cation-to-anion ratio of about 2:1 to about 1:1. 11. Any composition according to the preceding item, wherein the ionic liquid contains a cation-to-anion ratio of about 2:1. 12. Any composition according to the preceding item, wherein the ionic liquid has a cation:anion ratio of less than 1:1. 13. Any composition according to the preceding item, wherein the ionic liquid has a cation:anion ratio in excess of cations. 14. Any composition according to the preceding item, further comprising at least one active compound in combination with at least one ionic liquid. 15. Any composition according to the preceding item, wherein the active compound comprises a polypeptide. 16. The composition of item 15, wherein the polypeptide is an antibody or antibody reagent. 17. A composition according to any of items 15 to 16, wherein the active compound has a molecular weight greater than 450. 18. A composition according to any of items 15 to 16, wherein the active compound has a molecular weight greater than 500. 19. Anion, Having a LogP of less than 1.0, a. Carboxylic acids that are not fatty acids; or b. Carboxylic acids containing aliphatic chains of 4 carbon atoms or less That is, A composition according to any of items 15 to 18. 20. Any composition according to the preceding item, wherein the active compound comprises a nucleic acid. 21. The composition of item 20, wherein the nucleic acid is an inhibitory nucleic acid. 22. The composition of item 21, wherein the nucleic acid is siRNA. 23. Anion, Having a LogP of less than 1.0, a. Carboxylic acid that is not a fatty acid; or b. Carboxylic acids containing aliphatic chains of 4 carbon atoms or less; and / or c. Aromatic anions That is, A composition according to any of items 20 to 22. 24. Any of the compositions described in the preceding paragraph, wherein the ionic liquid has a concentration of at least 0.1% w / v. 25. Any of the compositions described in the preceding paragraph, wherein the ionic liquid has a concentration of approximately 10 to approximately 70% w / v. 26. Any of the compositions described in the preceding paragraph, wherein the ionic liquid has a concentration of approximately 30 to approximately 50% w / v. 27. Any of the compositions described in the preceding paragraph, wherein the ionic liquid has a concentration of approximately 30 to approximately 40% w / v. 28. Any of the compositions described in the preceding paragraph, formulated for transdermal administration, mucosal administration, oral administration, subcutaneous administration, intradermal administration, parenteral administration, intratumoral administration, or intravenous administration. 29. The composition of item 28, formulated for transdermal administration. 30. The composition of item 28, wherein the mucous membrane is the nasal mucosa, oral mucosa, or vaginal mucosa. 31. Any composition according to the preceding item, which provides an active compound in a dose of 1 to 40 mg / kg. 32. Any composition according to the preceding item, further comprising at least one nonionic surfactant. 33. Any composition according to the preceding paragraph, further comprising a pharmaceutically acceptable carrier. 34. Any composition of the preceding item, provided in a biodegradable capsule. 35. A composition of any of the preceding items, which is a mixture. 36. Any composition according to the preceding item, provided in one or more nanoparticles. 37. Any composition according to the preceding item, comprising one or more nanoparticles containing an active compound, wherein the nanoparticles are in the state of solution or suspension in a composition containing an ionic liquid. 38. A method for administering at least one active compound, comprising the step of administering one of the compositions of items 14 to 37. 39. The method of item 38, wherein the composition is administered in a single dose. 40. Any method of items 38 to 39, wherein the composition is administered in multiple doses.

[0277] Some aspects of the technology described herein can be defined by any of the following numbered sections: 1. (a) Carboxylic acids that are not fatty acids, (b) Carboxylic acids containing aliphatic chains of 4 carbon atoms or less, (c) Aromatic anions, and / or (d) Anions with a LogP of less than 1.0 An anion which is at least one of the following, and cations containing quaternary ammonium A composition comprising at least one ionic liquid, including the following. 2. Anion, Having a LogP of less than 1.0, a. Carboxylic acids that are not fatty acids, b. Carboxylic acids containing aliphatic chains of 4 carbon atoms or less, c. Aromatic anions That is, Any of the compositions described in the preceding paragraph. 3. Any composition according to the preceding item, wherein the fatty acid comprises an aliphatic chain of three carbon atoms or less. 4. Any composition according to the preceding item, wherein the anion comprises only one carboxylic acid group (e.g., an R-COOH group). 5. Any composition according to the preceding item, wherein the anion is selected from the group consisting of geranic acid; glycolic acid; propanoic acid; isobutyric acid; butyric acid; gallic acid; lactic acid; malonic acid; maleic acid; glutaric acid; citric acid; 3,3-dimethylacrylic acid; dimethylacrylic acid; gluconic acid; adipic acid; sodium ethylhexyl sulfate; decanoic acid; hydroxybenzenesulfonic acid; 4-hydroxybenzenesulfonic acid (4-phenolsulfonic acid); isovaleric acid; hydrocinnamic acid (phenylpropanoic acid); phenyl phosphoric acid; and biphenyl-3-carboxylic acid. 6. Any composition according to the preceding item, wherein the anion is selected from the group consisting of glycolic acid; propanoic acid; isobutyric acid; butyric acid; gallic acid; lactic acid; malonic acid; maleic acid; glutaric acid; citric acid; 3,3-dimethylacrylic acid; dimethylacrylic acid; gluconic acid; adipic acid; sodium ethylhexyl sulfate; decanoic acid; hydroxybenzenesulfonic acid; 4-hydroxybenzenesulfonic acid (4-phenolsulfonic acid); isovaleric acid; hydrocinnamic acid (phenylpropanoic acid); phenyl phosphoric acid; and biphenyl-3-carboxylic acid. 7. Any composition according to the preceding item, wherein the cation has a molar mass equal to or greater than that of choline. 8. Quaternary ammonium is NR4 + A composition according to any of the above items, having the structure and comprising at least one R group comprising a hydroxyl group. 9. Quaternary ammonium is NR4 + A composition according to any of the preceding items, having the structure and containing only one hydroxyl group among the R groups. 10. Any composition according to the preceding item, wherein the cation is choline, C1, C6, or C7. 11. Any composition according to the preceding paragraph, wherein the cation is choline. 12. Any composition according to the preceding item, wherein the cation is C1, C6, or C7. 13. Any composition according to the preceding item, wherein the ionic liquid contains a cation-to-anion ratio of about 2:1 to about 1:1. 14. Any composition according to the preceding item, wherein the ionic liquid contains a cation-to-anion ratio of about 2:1. 15. Any composition according to the preceding item, wherein the ionic liquid has a cation:anion ratio of less than 1:1. 16. Any composition according to the preceding item, wherein the ionic liquid has a cation:anion ratio in excess of cations. 17. Any composition according to the preceding item, comprising a first ionic liquid and at least a second ionic liquid. 18. The composition of item 17, wherein each ionic liquid has a choline cation. 19. Any composition according to items 17 to 18, wherein the first ionic liquid and the second ionic liquid each contain different anions. 20. The composition of item 19, wherein the first ionic liquid and the second ionic liquid each contain different anions selected from geranic acid; glycolic acid; propanoic acid; isobutyric acid; butyric acid; gallic acid; lactic acid; malonic acid; maleic acid; glutaric acid; citric acid; 3,3-dimethylacrylic acid; dimethylacrylic acid; gluconic acid; adipic acid; sodium ethylhexyl sulfate; decanoic acid; hydroxybenzenesulfonic acid; 4-hydroxybenzenesulfonic acid (4-phenolsulfonic acid); isovaleric acid; hydrocinnamic acid (phenylpropanoic acid); phenyl phosphoric acid; and biphenyl-3-carboxylic acid. 21. A composition according to any one of items 17 to 20, wherein the first ionic liquid has a geranate anion and the second ionic liquid has a phenylpropanoate anion. 22. A composition according to any of items 17 to 21, wherein the first ionic liquid is choline and geranic acid (CAGE). 23. A composition according to any of items 17 to 22, wherein the second ionic liquid is choline and dimethylacrylic acid (CADA); choline and isovaleric acid (CAVA); choline and phenyl phosphoric acid (CAPP); choline and biphenyl-3-carboxylic acid (CABA); choline and 4-phenolsulfonic acid (CASA); or choline and phenylpropanoic acid (CAPA). 24. A composition according to any one of items 17 to 21, wherein the first and second ionic liquids are different ionic liquids selected from the group consisting of choline and geranic acid (CAGE); choline and dimethylacrylic acid (CADA); choline and isovaleric acid (CAVA); choline and phenyl phosphoric acid (CAPP); choline and biphenyl-3-carboxylic acid (CABA); choline and 4-phenolsulfonic acid (CASA); or choline and phenylpropanoic acid (CAPA). 25. A composition according to any one of items 17 to 21, wherein the first ionic liquid is selected from the group consisting of choline and geranic acid (CAGE); choline and dimethylacrylic acid (CADA); and choline and choline and biphenyl-3-carboxylic acid (CABA); and the second ionic liquid is selected from the group consisting of isovaleric acid (CAVA); and choline and phenylpropanoic acid (CAPA). 26. A composition according to any of items 17 to 22, wherein the first ionic liquid is choline and geranic acid (CAGE), and the second ionic liquid is choline and phenylpropanoic acid (CAPA). 27. Any composition according to the preceding item, further comprising at least one active compound in combination with at least one ionic liquid. 28. Any composition according to the preceding item, wherein the active compound comprises a polypeptide. 29. The composition of item 28, wherein the polypeptide is an antibody or antibody reagent. 30. A composition according to any of items 28 to 29, wherein the active compound has a molecular weight greater than 450. 31. A composition according to any of items 28 to 30, wherein the active compound has a molecular weight greater than 500. 32. Anion, Having a LogP of less than 1.0, a. Carboxylic acids that are not fatty acids; or b. Carboxylic acids containing aliphatic chains of 4 carbon atoms or less That is, A composition according to any of items 28 to 31. 33. Any composition according to the preceding item, wherein the active compound comprises a nucleic acid. 34. The composition of item 33, wherein the nucleic acid is an inhibitory nucleic acid. 35. The composition of item 34, wherein the nucleic acid is siRNA. 36. A composition according to any of items 34-35, wherein the inhibitory nucleic acid is NFKBIZ, TNFα, and / or IL-17 inhibitory nucleic acid. 37. Anion, Having a LogP of less than 1.0, a. Carboxylic acid that is not a fatty acid; or b. Carboxylic acids containing aliphatic chains of 4 carbon atoms or less; and / or c. Aromatic anions That is, A composition according to any of items 33 to 36. 38. Any composition according to the preceding item, wherein the ionic liquid has a concentration of at least 0.1% w / v. 39. Any of the compositions described in the preceding paragraph, wherein the ionic liquid has a concentration of approximately 10 to approximately 70% w / v. 40. Any of the compositions described in the preceding paragraph, wherein the ionic liquid has a concentration of approximately 30 to approximately 50% w / v. 41. Any of the compositions described in the preceding paragraph, wherein the ionic liquid has a concentration of approximately 30 to approximately 40% w / v. 42. Any composition according to the preceding paragraph, formulated for transdermal administration, mucosal administration, oral administration, subcutaneous administration, intradermal administration, parenteral administration, intratumoral administration, or intravenous administration. 43. The composition of item 42, formulated for transdermal administration. 44. The composition of item 42, wherein the mucous membrane is the nasal mucosa, oral mucosa, or vaginal mucosa. 45. Any composition according to the preceding item, which provides the active compound in a dose of 1 to 40 mg / kg. 46. ​​Any composition according to the preceding item, further comprising at least one nonionic surfactant. 47. Any composition according to the preceding item, further comprising a pharmaceutically acceptable carrier. 48. Any composition according to the preceding item, provided in a biodegradable capsule. 49. A composition of any of the preceding items, which is a mixture. 50. Any composition according to the preceding item, provided in one or more nanoparticles. 51. Any composition according to the preceding item, comprising one or more nanoparticles containing an active compound, wherein the nanoparticles are in the state of solution or suspension in a composition containing an ionic liquid. 52. A method for administering at least one active compound to a subject, comprising the step of administering one of the compositions of items 27 to 51. 53. The method of item 52, wherein the composition is administered in a single dose. 54. Any method of items 52 to 53, wherein the composition is administered in multiple doses. 55. Any method of administration described in paragraphs 52-54, wherein the administration is transdermal, mucosal, oral, subcutaneous, intradermal, parenteral, intratumoral, or intravenous. 56. Any method of paragraphs 52 to 55, wherein the composition comprises NFKBIZ, TNFα, and / or IL-17 inhibitory nucleic acids, and the subject requires treatment of an inflammatory condition. 57. A method for treating an inflammatory condition in a subject requiring the treatment thereof, comprising the step of administering any of the compositions of items 36 to 51 to the subject. 58. Any of the methods described in paragraphs 56-57, wherein the administration is local. 59. The inflammatory condition is psoriasis, by any of the methods described in paragraphs 56-58. 60. Any composition according to items 27 to 51 for use in a method of administering at least one active compound to a subject. 61. The composition of item 60, administered as a single dose. 62. The composition of item 60, administered in multiple doses. 63. A composition according to any of items 60 to 62, wherein the administration is transdermal, mucosal, oral, subcutaneous, intradermal, parenteral, intratumoral, or intravenous. 64. A composition according to any of items 60 to 63, comprising NFKBIZ, TNFα, and / or IL-17 inhibitory nucleic acids, wherein the subject requires treatment of an inflammatory condition. 65. Any composition of items 36 to 51 for use in a method of treating an inflammatory condition of an object that requires it. 66. Any composition of items 64 to 65, wherein the administration is local. 67. Any composition of items 64 to 66, wherein the inflammatory condition is psoriasis. [Examples]

[0278] Example 1: Ionic liquid for oral monoclonal antibody delivery Monoclonal antibodies (mAbs) are currently used to treat many conditions, particularly cancer, psoriasis, arthritis, and atopic dermatitis. All mAbs are currently administered either intravenously or subcutaneously. Described herein is the use of choline and glycolate (glycolic acid) (CGLY), an ionic liquid, as a platform for the oral delivery of therapeutic antibodies. CGLY maintained the stability and structure of TNFα antibodies. CGLY significantly enhanced paracellular transport of TNFα antibodies in vitro. CGLY also reduced the viscosity of intestinal mucus, another important barrier to antibody transport. In vivo results in rats indicate that CGLY effectively delivers TNFα antibodies to the intestinal mucosa and into systemic circulation. A 1-week repeated-dose study and subsequent histological and serum biochemical analyses showed that CGLY was well tolerated by rats. Overall, this study demonstrates the advantages of using choline-based ionic liquids as an oral delivery platform for topical and systemic delivery of therapeutic antibodies.

[0279] Therapeutic monoclonal antibodies (mAbs) constitute a very large class of protein-based therapeutic agents. [1、2] More than 50 mAb-based products have been approved, and over 500 mAb-based therapies are in clinical development. [3] Antibodies are used to treat a variety of diseases, including cancer, infections, inflammation, and autoimmune diseases. [1、4] However, mAbs are delivered as intravenous or subcutaneous injections, and these are associated with adverse effects such as systemic inflammatory responses, infusion reactions, and low patient compliance due to pain and injection phobia. [5-7] Oral administration of mAbs offers potential advantages over injection due to its simplicity of administration, high patient tolerance, and low manufacturing costs. In addition to providing a possible means for non-invasive systemic administration, oral administration also provides a means for local delivery of antibodies into the gastrointestinal tract for the treatment of local diseases such as inflammatory bowel disease. [8-10] Nevertheless, as with all oral delivery of proteins, many gastrointestinal barriers collectively limit protein-drug absorption. [11、12]This has motivated efforts to develop oral antibody formulations that can achieve therapeutic outcomes in a more effective manner. For example, recombinant antibodies against tumor necrosis factor (TNF) are under development to treat gastrointestinal infections and inflammatory bowel disease. [13-15] The recombinant portion resulted in improved tolerance of the antibody to intestinal proteases and resistance to degradation. In addition, the modified anti-TNF antibody fragment also showed improved permeability to affected tissue within GI tubules.

[14] .

[0280] This specification describes an investigation into the potential of choline-based ILs for oral IgG delivery. To achieve this, choline glycolate (CGLY) ionic liquids were prepared and evaluated for antibody stability, in vitro transport, and in vivo uptake.

[0281] result Physicochemical characterization of IgG-CGLY variant formulations The initial test was conducted to evaluate the role of ionic stoichiometry of CGLY in its compatibility with IgG antibodies. Three variants of CGLY were synthesized with choline:glycolic acid molar ratios of 2:1, 1:1, and 1:2 (Figure 1A). A model IgG antibody, anti-human TNF-α mouse IgG1 (clone MAb11), was dissolved at a concentration of 0.1 mg / mL in CGLY variants diluted in physiological saline in a range of 20–90 vol%. The IgG antibody dissolved completely in all CGLY variants and concentrations, and no precipitation was observed. After incubation at room temperature for 1 hour and dialysis for 48 hours, the antibody samples were evaluated based on their antigen-binding ability using ELISA (Figure 1B). 2:1 and CGLY 1:1 The IgG-CGLY formulations showed only a negligible effect on the intrinsic binding ability of TNF-αIgG1 at CGLY concentrations up to 60% and 70% by volume, respectively. On the other hand, CGLY 1:2 The IgG antibody samples isolated from the sample induced a decrease in binding efficiency in the concentration range of 20–90 volume percent.

[0282] To further elucidate the effects of CGLY on IgG antibodies, circular dichroism (CD) and SDS-PAGE analysis were performed. Because the presence of CGLY generates significant background CD noise, CD measurements were performed after dialysis of IgG-CGLY samples at room temperature for 48 hours. The far-UV wavelength spectra of anti-human TNF-αIgG showed no difference in shape or degree of ellipticity compared to initial IgG (Figure 1C). All CD spectra showed a minimum at 218 nm, which is a typical finding for β-sheets, the main secondary structure of IgG. [30、31] This result indicates that the structural conformation of IgG is retained even after exposure to the CGLY variant. Simultaneously, SDS-PAGE was also used to evaluate the effect of CGLY on anti-human TNF-αIgG, with particular focus on the possibility of IgG aggregation.

[32] In the absence of CGLY, model IgG appears as a single band at approximately 150 kDa (Figure 1D). IgG from all CGLY variants is identified at the same band position. No other bands are observed above or below the 150 kDa band, suggesting no detectable antibody fragmentation or aggregation from formulations with CGLY. [32、33] Combined, the antibody characterization results from ELISA, CD spectroscopy, and SDS-PAGE are CGLY 2:1 and CGLY 1:1 This demonstrated that it has minimal effect on structure or antibody aggregation.

[0283] Effects of CGLY on Caco-2 cell viability and IgG transport Caco-2 cells are CGLY 2:1 and CGLY 1:1 It was well tolerated, and no adverse effects on cell proliferation were observed up to high concentrations of >100 mM, but CGLY 1:2 It reduced cell viability at fairly low concentrations (Figure 2A). CGLY 2:1 CGLY 1:1 and CGLY 1:2 The IC50 values ​​were approximately 140.4 mM, 223.3 mM, and 40.78 mM, respectively.

[0284] The ability of CGLY to enhance transepithelial transport was tested using fluorescein isothiocyanate-labeled (FITC)-IgG passing through Caco-2 monolayers. These tests were performed using 30 mM CGLY, which is far lower than the IC50 of all CGLY variants. During the 5-hour test, FITC-IgG transport increased cumulatively over time in all CGLY groups, while no FITC-IgG transport was detected from control transwells without CGLY (Figure 2B). In particular, among all CGLY variants at all time points, CGLY 2:1 showed the highest and most significant IgG transport. At the end of the test, the average IgG transport in the monolayers treated with CGLY 2:1 was 1.70 μg / cm 2 , which is CGLY 1:1 more than twice that of treated cells (0.83 μg / cm 2 ), and 1.6 times higher than that of CGLY 1:2 treated cells (1.06 μg / cm 2 ).

[0285] Considering the results from IgG antibody-CGLY characterization and Caco-2 cell responses to CGLY, CGLY 2:1 was superior among the tested CGLY variants as the optimal ionic liquid for IgG antibody delivery. Therefore, CGLY 2:1 was selected for further in vitro and in vivo investigations thereafter.

[0286] CGLY transport across Caco-2 intestinal cells 2:1 Detailed analysis of mediated antibody transport CGLY 2:1 enhanced transepithelial transport of FITC-IgG across Caco-2 monolayers in a concentration-dependent manner (Figure 3A). As the CGLY 2:1 concentration increased from 30 mM to 80 mM, the transported amount increased from 1.70 μg / cm 2 to 9.32 μg / cm 2 . On the other hand, FITC-IgG transport is CGLY 2:1It is worth noting that detection was impossible in the absence of the substance. These results were consistent with transport assessed from confocal images of Caco-2 cells (Figure 20). Fluorescence images of cells at the end of the 5-hour experiment showed CGLY compared to the control well. 2:1 We clearly demonstrated that the uptake of FITC-IgG by Caco-2 cells increases with increasing concentration of FITC-IgG.

[0287] Paracellular and transcellular pathways are the main routes involved in the transport of peptides and proteins across the intestinal epithelium. CGLY across the Caco-2 monolayer. 2:1 To investigate the mechanisms of mediated IgG transport, we examined the roles of both paracellular and transcellular transport. First, the paracellular pathway was evaluated by the transport of the paracellular transport marker, Lucifer Yellow.

[34] The amount of Lucifer Yellow transported was 30-80 mM CGLY. 2:1 Cells treated with 30 mM CGLY showed dramatic improvement throughout all time points (Figure 3B). 2:1 At the lowest level, Lucifer Yellow transport was increased by approximately twofold. 80mM CGLY 2:1 In terms of concentration, Lucifer Yellow transport was enhanced 4-6 times at various time points. In parallel experiments, transepithelial electrical resistance (TEER) measurements were performed on Caco-2 transwells containing various concentrations of CGLY2:1 to evaluate the tight junction integrity of the Caco-2 monolayer, and CGLY2:1 2:1 We further confirmed the involvement of paracellular cells in auxiliary transport. In the untreated wells, TEER measurements showed a slight increase within 15% by the end of the 24-hour test, which is consistent with previous literature. [23、35] 30mM CGLY 2:1 The addition of [ingredient] reduced the TEER value by 11% in 1 hour, and the decrease remained within the range of 11-16% in the first 5 hours. However, CGLY 2:1 The decrease in TEER caused by CGLY was clearly temporary, and the cells recovered 96% of their tight junction integrity within 24 hours. 2:1 Increasing the concentration further increased the degree of TEER reduction. At 1–5 hours of the test, 55 mM CGLY 2:1Approximately 34% decrease in TEER, 80mM CGLY 2:1 A 45% decrease was observed, indicating the opening of a tight junction. Nevertheless, CGLY 2:1 Induced TEER reduction still exhibits a transient behavior, with cells showing 55 mM and 80 mM CGLY levels within 24 hours. 2:1 These restored 94% and 82% of the initial TEER values, respectively. Cells were treated with 30-80 mM CGLY. 2:1 The decrease and recovery of TEER measurements when processed with CGLY 2:1 This suggests that it can temporarily open intestinal tight junctions and promote IgG transport across the intestinal epithelial barrier. Combined, this leads to a gradual increase in CGLY 2:1 In the presence of CGLY, increased Lucifer Yellow transport and decreased TEER value are due to CGLY 2:1 Characteristic paracellular transport was observed.

[0288] The transcellular pathway's contribution to IgG transport is investigated using transcellular transport inhibitors, including monodansylcadaverine (MDC; an inhibitor of clathrin-mediated endocytosis), philipin (an inhibitor of caveolae-mediated endocytosis), and wartmanin (an inhibitor of phosphatidylinositol 3 kinase, which is involved in microphamiosis).

[36] The effect was evaluated by testing the cumulative transport of FITC-IgG after 24 hours of incubation. There was no significant difference between cells treated with any inhibitor compared to a control without the inhibitor (Figure 3D). This finding is relevant to CGLY. 2:1 The improved delivery of FITC-IgG via Caco2 Transwell was suggested not to be primarily aided by transcellular transport.

[0289] CGLY for the viscosity of mucus 2:1 The effect Intestinal mucus is one of the important components of the intestinal barrier.

[12] CGLY against porcine small intestinal mucus (PIM) 2:1 To investigate the effects of CGLY, 2:1 The rheology of PIM treated with was evaluated. Figure 4A shows CGLY of 0-50 volume%. 2:1This shows the shear viscosity reduction characteristics of the PIM sample after incubation with CGLY. Compared to untreated PIM, CGLY 2:1 The viscosity of the treated mucus showed a significant decrease throughout the entire measured shear range. For example, at a shear rate of 49.87 1 / s, the average viscosity of untreated PIM was measured at 576.8 cP, which was comparable to previously reported literature values. [37、38] (Figure 4B). CGLY at 12.5, 25, and 50 volume%. 2:1 The addition of significantly reduced the mucus viscosity to 317.9, 398.0, and 429.6 cP, respectively. CGLY reduces mucus viscosity. 2:1 This ability may facilitate antibody delivery to the small intestinal epithelium.

[0290] CGLY 2:1 In vivo topical and systemic antibody delivery of IgG CGLY 2:1 The formulated FITC-IgG was injected intrajejunally into Wistar rats (50 volume% CGLY). 2:1 (1 mg / mL of FITC-IgG). Control rats were injected with equivalent saline solutions, either with or without FITC-IgG. Two hours later, jejunal tissue was collected, frozen sections were prepared for imaging (Figures 5A-5C), and the fluorescence signal of FITC-IgG per unit area on the intestinal villi was quantified (Figure 5D). There was a significant difference in FITC-IgG signaling in the intestinal mucosa between the treatment groups. CGLY 2:1 The jejunal tissue from the treatment group showed a significant signal of FITC-IgG in the intestinal villi (Figure 5B), and the measured fluorescence signal was CGLY 2:1 The level was more than 4.5 times higher compared to the control group without FITC-IgG (Figure 5D). On the other hand, in the control group with FITC-IgG in physiological saline, the FITC signal on the villi was not significantly higher than in the negative control group. The signal from FITC-IgG was rather strictly localized to the outside of the villi, i.e., the mucus layer (Figure 5C), indicating that the transport of IgG alone is greatly impaired by the mucus barrier. This finding is relevant to CGLY. 2:1 This demonstrates that it effectively enhances IgG permeability through intestinal mucus and the epithelial layer.

[0291] In parallel studies, plasma IgG levels were measured to enhance IgG absorption using CGLY. 2:1 The benefits of using it were determined (Figure 5E). For the study, an anti-human TNF-αIgG monoclonal antibody was used as a model antibody and administered by intrajejunal injection to CGLY. 2:1 Administered at 200 μg / kg with or without [another substance]. IgG concentrations gradually increased during the first two hours after injection. CGLY increased during the 3-5 hour period of the study. 2:1 A significant increase in IgG concentration was observed in the treatment group, particularly in CGLY. 2:1 IgG concentrations in the treatment group were five times higher than in the control group by the end of the study. Combined, the superior local FITC-IgG and plasma IgG concentration results were found in CGLY. 2:1 This indicates that it enables the permeation and transport of IgG from the villi into the bloodstream. More importantly, since plasma IgG concentrations were detected by ELISA, the transported IgG was functionally conserved. Given the long circulating half-life of the antibody, repeated oral administration of IgG is intended to sustainably increase blood concentrations and potentially achieve much higher levels.

[0292] CGLY 2:1 In vivo toxicity assessment CGLY 2:1 The toxicity was evaluated in adult male Wistar rats. CGLY 2:1 The drug was administered orally once daily for seven consecutive days at a dose of 625 mg / kg. Rats administered with sodium chloride were used as a negative control group. During the study, CGLY 2:1 Rats administered with maintained nearly the same body weight as rats administered with saline, and all rats showed a steady increase in body weight (Figure 6A). Neither group exhibited any physiological symptoms such as lethargy, diarrhea, hunched posture, or coarse hair. On day 7, the rats were sacrificed, blood samples were taken for metabolic panel analysis, and major organ and gastrointestinal (GI) tissues were collected from the rats and stained with hematoxylin and eosin (H&E). CGLY 2:1Tissue sections of the stomach, small intestine (duodenum, jejunum, and ileum), and colon from the treatment group showed no changes compared to the salt control group, exhibiting gastric and intestinal mucosal epithelial structures, including crypt and villi size and number, as well as mucosal thickness (Figure 6B). There was no infiltration of immune cells such as neutrophils, lymphocytes, or macrophages into the mucosa, indicating no signs of tissue inflammation. No hemorrhage was observed in H&E staining of major organs, and CGLY was observed. 2:1 No difference was detected between the treatment group and the saline control group (Figure 21). Comprehensive blood chemistry panel analysis showed no significant difference between the two groups (Figure 6C), CGLY 2:1 It was shown that it did not cause any observable adverse effects on the liver and kidney function of rats. CGLY 2:1 In vivo toxicity studies showed no effect on rat body weight, blood metabolic panel, or histopathological changes, and CGLY 2:1 This study demonstrated that it is safe for oral administration in rat models.

[0293] conclusion We synthesized various ionic stoichiometric choline glycolates (IL). Among the CGLY varieties, we synthesized CGLY with a choline-to-glycolic acid molar ratio of 2:1. 2:1 It demonstrated excellent cytocompatibility and IgG integrity preservation, showing the best capability in in vitro IgG antibody transport. CGLY 2:1 Further investigation revealed that CGLY 2:1 This can temporarily disrupt the integrity of intestinal tight junctions and allow CGLY to pass through Caco-2 cells. 2:1 Enhanced IgG transport was revealed to occur via the paracellular pathway. (CGLY) 2:1 It can also reduce the viscosity of mucus. CGLY 2:1 Intrajejunal administration of IgG within the jejunum substantially improved antibody absorption into rat intestinal villi, increasing the concentration of the model monoclonal antibody up to five times compared to the negative control. In addition, CGLY 2:1 The treatment did not show adverse effects on rat body weight, histological changes in GI ducts, or a comprehensive metabolic panel of blood. Overall, this report is in accordance with CGLY. 2:1Its potential and strengths demonstrate that, along with excellent biocompatibility, it is an orally delivered medium that can effectively improve both the local and systemic bioavailability of IgG antibodies.

[0294] Experimental section Materials: Glycolic acid, choline bicarbonate, dimethyl sulfoxide (DMSO), human serum-derived FITC-labeled immunoglobulin G (FITC-IgG, 20 mg / mL), hematoxylin, and eosin solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). LEAF® purified anti-human TNF-α mouse IgG1 (clone Mab11), recombinant human TNF-α, ELISA-coated buffer, HRP-conjugated goat anti-mouse IgG (clone poly4053), and TMB substrate were purchased from Biolegend (San Diego, CA, USA). 10 mM sodium phosphate buffer, pH=7.4 was obtained from Boston BioProducts (Ashland, MA, USA), and 0.9% sterile saline solution was purchased from Teknova (Hollister, CA, USA). Laemmli protein sample buffer, 4-15% 12-well precast polyacrylamide gel, Tris / glycine / SDS electrophoresis buffer, Mini-PROTEAN® Tetra Cell Electrophoresis System, and Bio-Safe® Coomassie Stain were purchased from BioRad Laboratories (Hercules, CA, USA). Caco-2 human colorectal adenocarcinoma cells were purchased from American Type Culture Collection (Manassas, VA, USA), while Dulbecco's modified Eagle medium (DMEM) with or without phenol red, fetal bovine serum (FBS), penicillin / streptomycin (P / S) solution, Hanks equilibrium salt solution (HBSS), Dulbecco's phosphate-buffered saline (DPBS), and 0.25% trypsin solution were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Basic seeding medium (BSM), enteric cell differentiation medium (EDM), and enteric epithelial growth medium including MITO+ Seelam Extender were purchased from Corning (Corning, NY, USA).Millicell®-PCF cell culture inserts (pore size 3.0 μm, diameter 12 mm) and TEER analyzer, Millicell®-ERS, were obtained from Millipore Sigma (Burlington, MA, USA), while the TEER electrode was obtained from World Precision Instruments, Inc (Sarasota, FL, USA). Paraformaldehyde (16% w / v) was purchased from Alfa Aesar (Ward Hill, MA, USA). Vectashield Hardset® containing 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) was obtained from Vector Laboratories Inc. (Burlingame, CA, USA). Pork small intestine was obtained from CBSET Inc. (Lexington, MA, USA). Male Wistar rats weighing 275-300 g were purchased from Charles River Laboratories (Wilmington, MA, USA). The BD heparin lithium-coated tubes were purchased from Becton, Dickinson and Company (Franklin Lanes, NJ, USA). Lucifer Yellow was purchased from VWR (Radnor, PA, USA). All other reagents used were of analytical grade.

[0295] Preparation of CGLY variant and antibody-CGLY formulation: The CGLY variant was synthesized as previously reported.

[27] In short, glycolic acid dissolved in the minimum amount of ultrapure water required for dissolution was reacted with choline bicarbonate (80 wt% solution) in molar ratios of 2:1, 1:2, and 1:2 (choline:glycolic acid) at 40°C with constant stirring for 12 hours until CO2 generation ceased. Residual water was removed by rotary evaporation at 20 mbar and 60°C for 2 hours, followed by drying in a vacuum dryer at 60°C for 48 hours. Each CGLY formulation was characterized by nuclear magnetic resonance (NMR) spectroscopy. The IgG-CGLY formulation was prepared by adding a predetermined amount of antibody to a specific amount of CGLY, followed by gentle mixing for 1 minute.

[0296] Physicochemical evaluation of antibodies in CGLY variants by ELISA, circular dichroism, and SDS-PAGE: To evaluate antibody stability in CGLY variants, antibody-CGLY samples with an anti-human TNF-αIgG antibody concentration of 0.1 mg / mL were subjected to CGLY 2:1 CGLY 1:1 and CGLY 1:2 The samples were incubated at room temperature (25°C) for 1 hour with or without (20-90 vol%) of the TNFα-α-IgG antibody, and then dialyzed in 10 mM pH 7.4 sodium phosphate buffer (Boston BioProducts). After 48 hours, the antibody samples were collected and evaluated by enzyme-linked immunosorbent assay (ELISA). The TNFα-specific binding ability of the dialyzed anti-human TNFα-αIgG antibody-CGLY samples was tested by ELISA. A 96-well ELISA plate was first coated overnight with 2 μg / mL human TNFα using ELISA coating buffer (Polysciences, Inc.). The wells were then blocked with Superblock® Blocking Buffer (ThermoFisher Scientific) for 30 minutes, and then the serially diluted and dialyzed anti-human TNFα-αIgG antibody samples were added as the primary antibody. After 2 hours of incubation, the wells were washed three times with 0.05% Tween 20-containing PBS (PBST). Then, HRP-conjugated goat anti-mouse IgG (Biolegend) was used as the secondary antibody. After incubating the plates for 1 hour, they were washed five times with PBST. The ELISA plates were developed with TMB substrate (Biolegend), and the absorbance was measured at 450 nm using a Spectramax i3® plate reader.

[0297] To analyze antibody stability using circular dichroism (CD) and SDS-PAGE, antibody-CGLY samples with an anti-human TNF-αIgG antibody concentration of 0.5 mg / mL were mixed with 50 vol% CGLY. 2:1 CGLY 1:1and CGLY 1:2 The samples were incubated at room temperature (25°C) for 1 hour with or without the appropriate agent, and then dialyzed for 48 hours in 10 mM pH 7.4 sodium phosphate buffer (Boston BioProducts). Before CD measurement, the antibody concentration was adjusted to 0.2 mg / mL. 400 μL of antibody sample was packed into a rectangular quartz cell (1 mm path length, Starna Cells, 1-Q-1), and the CD spectrum in the far-UV region (190-250 nm) showing protein secondary structure was collected using CD spectrophotometry (Jasco J-1500). Antibody aggregation of antibody-CGLY samples was evaluated by SDS-PAGE assay. Specifically, all samples were adjusted to the same antibody concentration in Laemmli protein sample buffer. The samples were then separated on a 12-well precast polyacrylamide gel with 4-15% Tris / glycine / SDS electrophoresis buffer using a Mini-PROTEAN® Tetra Cell Electrophoresis System (BioRad). Protein bands were stained with Bio-Safe® Coomassie stain (BioRad) according to the manufacturer's protocol for observation.

[0298] Caco-2 cell culture: The Caco-2 cell line (human colorectal adenocarcinoma, ATCC HTB-37) was purchased from the American Type Culture Collection (ATCC) and maintained at 37°C in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U / mL penicillin and 100 μg / mL streptomycin) under a humidified atmosphere containing 5% CO2.

[0299] Evaluation of Caco-2 cell viability in CGLY: Supplemental Caco-2 cells suspended in DMEM were seeded at a density of 150,000 cells / mL and dispensed into 96-well plates (100 μL / well). Each CGLY variant (CGLY 2:1 CGLY 1:1 and CGLY 1:2The CGLY variant was diluted with supplemental DMEM to concentrations ranging from 1.875 to 480 mM. The culture medium was aspirated from each well and each dilution was dispensed into 6 wells (6 cell replications) (100 μL / well). The control well was filled with only culture medium. The cells were incubated with different concentrations of CGLY variant at 37°C and 5% CO2 for 5 hours, followed by replacement of the culture medium with fresh DMEM (100 μL / well). The cells were grown for a further 19 hours (up to a total of 24 hours). Cell viability was evaluated using the Cell Titer 96 AQueous® One Solution cell proliferation assay (Promega Corporation) based on the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) compound. Briefly, 20 μL of MTS reagent was added to each well, gently mixed, and incubated at 37°C for 4 hours. Subsequently, the absorbance of the 96-well plate was read at 490 nm using a Spectramax i3 plate reader. The conversion of MTS tetrazolium to formazan products, measured by absorbance at 490 nm, is directly proportional to the number of viable cells. As suggested in the manufacturer's protocol, the percentage of cell viability was calculated by subtracting the average absorbance of the cell-free control well from all other experimental wells and assuming that the average absorbance from the wells containing untreated cells represents 100%.

[0300] Caco-2 Monolayer Culture Transwell: For transport experiments in the Transwell, a 3-day rapid Caco-2 growth system was used. Cells were added to Corning® Basic Seeding Medium (BSM) supplemented with MITO Seelam + Extender and seeded at a density of 400,000 cells / mL onto Millicell® PCF inserts placed in 24-well plates. As recommended by the manufacturer, 500 μL of cell-containing medium was added to the apical side, while 1000 μL of cell-free BSM was added to the vasolateral side. After incubation at 37°C and 5% CO2 for 24 hours, the medium was replaced with the same volume of MITO Seelam + Extender-supplemented intestinal cell differentiation medium, and cultured for a further 2-4 days. TEER was measured periodically, and 200 Ω.cm was observed to indicate sufficient tight junction integrity between cells. 2 If it exceeded that, a transport test was conducted.

[0301] Transport of FITC-IgG and Lucifer Yellow through Caco-2 monolayer transwells: Prior to the experiment, Caco-2 transwells were washed twice with HBSS and then incubated for 30 minutes with phenol red, FBS, and P / S-free DMEM in both the apical (400 μL) and basolateral (600 μL) sides. Subsequently, the medium in the apical side was replaced with 400 μL of either 500 μg / mL FITC-IgG or Lucifer Yellow, prepared with 0, 30, 55, or 80 mM CGLY and solubilized in phenol red, FBS, and P / S-free DMEM. Immediately after adding FITC-IgG to the apical side, a fixed volume of 150 μL was taken from the basolateral side and replaced with an equal volume of fresh DMEM. This was repeated at 1, 2, 3, 4, and 5 hours. During the experiment, the Transwell plate was placed in a 37°C, 5% CO2 incubator on a shaker rotating at 100 rpm, and was removed only to collect a fixed amount during the aforementioned period. After the 5-hour experiment, the concentrations of FITC-IgG and Lucifer Yellow in a fixed amount were measured using a BioTek Synergy Neo2™ plate reader (Vermont, USA) at excitation / emission wavelengths of 485 / 520 nm and 485 / 530 nm, respectively. The concentrations of FITC-IgG and Lucifer Yellow at each time point were calculated from the calibration solution of each fluorescent molecule and then plotted as the batholateral chamber concentration against time.

[0302] For quantitative analysis of FITC-IgG uptake by Caco-2 cells, the transwells from the FITC-IgG transport test were washed twice with HBSS at the end of the test, followed by the addition of 500 μL of 4% paraformaldehyde, and maintained overnight at 4°C. The following day, the paraformaldehyde was aspirated from the wells, the membranes were washed twice with PBS, and the transwell membranes were cut and gently placed on glass slides. DAPI-containing mounting medium was added to the membranes, and they were covered with coverslips. Confocal imaging of the membranes (ZEISS, laser scanning confocal microscope LSM 700) was performed at 40× magnification.

[0303] CGLY 2:1TEER measurement of treated Caco-2 monolayer transwells: After washing the Caco-2 transwells, they were incubated with phenol red, FBS, and P / S-free DMEM for 30 minutes. TEER values ​​were recorded for each insert. The apical side was then incubated with 400 μL of 0, 30, 55, or 80 mM CGLY. 2:1 It was replaced with [the appropriate component]. During the test, the Transwell plate was placed in a 37°C, 5% CO2 incubator on a shaker rotating at 100 rpm, and was removed only to perform further TEER measurements at 1, 2, 3, 4, 5, and 24 hours to determine TEER recovery and tight junction reversibility. TEER was plotted as the percentage change from the initial value over time.

[0304] FITC-IgG transport with transcellular transport inhibitors: Before the experiment, Caco-2 transwells were washed twice with HBSS and then incubated for 30 minutes with phenol red, FBS, and P / S-free DMEM in both the apical (400 μL) and basolateral (600 μL) sides. Subsequently, transcellular transport inhibitors including 50 μM monodansyl cadaverine (MDC), 1 μg / mL Filipinopropylmethylamine, and 0.5 μM Wartmannin were incubated.

[36] With or without, 500 μg / mL of FITC-IgG, 55 mM CGLY 2:1 The apical culture medium was replaced with 400 μL of phenol red, FBS, and P / S-free DMEM containing [specific ingredient]. After 24 hours of incubation, a fixed 150 μL was taken from the basolateral side, and the FITC-IgG concentration in this fixed volume was measured at excitation / emission wavelengths of 485 / 520 nm using a BioTek, Synergy Neo2™ plate reader (Vermont, USA), and plotted as the percentage of FITC-IgG transport compared to a control well without any transcellular transport inhibitors.

[0305] Mucosal rheology test: Porcine small intestinal mucus was extracted from the porcine small intestine by gently scraping the washed mucosal surface with a small laboratory spatula, avoiding the removal of epithelial cells as much as possible.

[39] Pig mucus was pooled and immediately examined. 10 μL of CGLY at 0, 12.5, 25, and 50 vol% concentrations in 0.9% saline was added to a fixed amount of 200 μL of pig mucus, and measurements were taken at 25°C using a 40 mm diameter steel parallel plate AR-G2 rheometer (TA Instruments, New Castle, DE, USA) over a shear rate range of 1 to 100 1 / s.

[0306] In vivo local delivery of antibody-CGLY by intrajejunal administration: All experiments involving the use of animals were conducted according to protocols approved by the Institutional Animal Care and Use Committee of Harvard University. Prior to the experiment, adult male Wistar rats weighing 275-300g were fasted overnight, but were allowed free access to water. On the day of the experiment, the rats were anesthetized and administered 50 volume %CGLY. 2:1 Rats were injected with physiological saline or 200 μL of 1 mg / mL FITC-labeled IgG antibody (FITC-IgG) in physiological saline (n=3). Rats injected with equivalent physiological saline without FITC-IgG were used as a negative control group. After 2 hours, the rats were sacrificed, and jejunal tissue was collected and stored using the Swiss rolling technique.

[40] Next, the rolled tissue was fixed in 4% paraformaldehyde at 4°C for 12 hours, then transferred to 4.5% sucrose and treated at 4°C for 4 hours, and finally transferred to 20% sucrose and treated at 4°C for 12 hours.

[41] Next, the tissue was frozen at -80°C in the presence of an optimal cutting temperature (OCT) compound, and tissue sections were cut to a thickness of 25 μm. These sections were visualized using a slide scanner microscope (ZEISS Axio Scan.Z1), and the images were processed using Zen® (Blue edition) software.

[0307] In vivo systemic delivery of antibody-CGLY by jejunal administration: A study was conducted in adult male Wistar rats that were fasted overnight but allowed free access to water. Before the start of the study, the rats were anesthetized, the hair on their abdomens was shaved, and the surgical area was prepared using betadine and 70% ethanol. The abdomen was incised to expose the intestines, and the test formulation was injected into the jejunum. After intestinal exposure, i.e., immediately before injection, zero-time blood samples were collected. 50 volume %CGLY was administered to each group of six rats. 2:1 0.3 mg / mL of anti-human TNF-αIgG antibody (200 μg / kg) was injected into physiological saline or physiological saline alone. The intestinal portion was then returned to the abdomen, and the muscles and skin were sutured. Before surgery, the animals were placed on a temperature-controlled warming pad, and after surgery, an additional towel cover was used to prevent hypothermia in the animals during anesthesia. The animals were kept anesthetized throughout the experiment and euthanized after 5 hours. Anti-human TNF-αIgG concentrations in plasma were evaluated by taking approximately 250 μL of blood in heparin-coated tubes from the treated rats at 0, 0.5, 1, 1.5, 2, 3, and 5 hours. Plasma was separated from whole blood according to a standard protocol. Blood samples were centrifuged at 2,000 × g for 15 minutes. Plasma supernatant was immediately transferred to a clean tube and stored on ice during the procedure, and then stored at -20°C until further analysis of IgG content. The concentration of anti-human TNF-αIgG in plasma samples at each time point was evaluated using ELISA as previously described, and calculated from the calibration solution for anti-human TNF-αIgG.

[0308] In vivo toxicity testing: CGLY in vivo 2:1 To evaluate its acute toxicity, adult male Wistar rats (n=6, 275-300g each) were given 50% CGLY by volume. 2:1Physiological saline was administered orally once daily for 7 consecutive days at a dose of 625 mg / kg using the procedure described above. Control rats were administered the same amount of physiological saline. Rat body weight was monitored daily throughout the experiment. On day 7, the rats were sacrificed, blood samples were taken for comprehensive metabolic panel analysis, and major organs and gastrointestinal tissues were processed for histological examination. Heart, liver, spleen, lung, kidney, and gastrointestinal (stomach, small intestine, and colon) tissues were fixed in neutral buffered 10% by volume formalin for 18 hours, dehydrated in 70% ethanol, and then embedded in paraffin. Tissue sections were cut to a thickness of 5 μm, deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E). Histological morphology was visualized using a bright-field slide scanner microscope (ZEISS Axio Scan.Z1®), and images were processed using Zen® (Blue edition) software.

[0309] Statistical Analysis: All data are presented as mean ± SE. For the SDS-PAGE test, experiments were conducted in triplets, and representative images are shown. For fluorescence and bright-field imaging, experiments were conducted in triplets, and representative images are shown. All other experiments were conducted in at least triplets. To examine statistical significance, independent two-tailed t-tests were performed using GraphPad Prism 8 (trademark), with a confidence level of P=0.05 considered significant.

[0310] References TIFF0007917913000016.tif71154TIFF0007917913000017.tif232160TIFF0007917913000018.tif104159

[0311] Example 2 Multiple ILs were tested to determine how well they promoted functional antibody stability (Figures 8-10). As a general trend, smaller anions were more compatible with the antibody than larger anions. Performance was maintained across different individual antibodies (Figure 14).

[0312] Antibody delivery by oral or intrajejunal administration was also tested (Figures 11-13). IL was confirmed to be non-toxic when administered orally (Figures 15-16).

[0313] Il for siRNA delivery was also considered, and transdermal siRNA was tested (Figures 17-18).

[0314] Example 3 Systemic antibodies targeting tumor necrosis factor α (TNF-α) and interleukin-17A (IL-17A) are effective against psoriasis vulgaris. Despite their high reputation, safety concerns pose challenges for systemic biologics. While anti-TNF-α and anti-IL-17A antibodies effectively inhibit their respective proteins, the inventors hypothesized that an approach based on local silencing of upstream targets, such as NFKBIZ, would be advantageous for treating psoriasis. However, effective delivery of small interfering RNA (siRNA) to the skin presents significant hurdles due to the skin's barrier function and poor siRNA stability. Using ionic liquids as a realization technique, this specification describes the effective delivery of NFKBIZ siRNA to the skin and its therapeutic efficacy in a psoriasis model. Treatment with IL-siRNA suppressed abnormal gene expression and resulted in downregulation of psoriasis-related signals, including TNF-α and IL-17A. These results provide a framework for a local siRNA delivery platform.

[0315] Introduction Psoriasis is one of the most debilitating chronic skin diseases, affecting over 125 million people worldwide and costing an estimated $135 billion per year in the United States (1). Its etiology and underlying mechanisms are still not fully understood. Nuclear factor κB (NF-κB), a widely expressed transcription factor, is considered a major regulator of the immune response and is thought to be involved in several autoimmune inflammatory diseases, including psoriasis (2). Several therapies targeting the NF-κB signaling pathway are available in the clinic; however, a lack of specificity and concerns about side effects present challenges (3). This challenge is particularly difficult because plurifacetous proteins like NF-κB provide essential basal activity as survival factors, and their systemic inhibition can lead to serious side effects. Network-centered approaches, including pathway-specific inhibitors, have garnered considerable therapeutic interest (4). In this regard, infliximab and adalimumab [both anti-tumor necrosis factor α (TNF-α) monoclonal antibodies] and secukinumab [anti-interleukin 17A (IL-17A) antibody] have been approved by the U.S. Food and Drug Administration and are claimed to mediate their therapeutic effects by modulating NF-κB activity (5).

[0316] NFKBIZ, a gene encoding an atypical inhibitor of the nuclear factor-κB (IκB) protein IκBζ, has garnered interest in therapeutic intervention due to its crucial role in regulating the NF-κB complex (6, 7). It has been reported to be a direct transcriptional activator of TNF-α, IL-17A, and IL-36-induced psoriasis-related gene products involved in inflammatory signaling, neutrophil chemotaxis, and leukocyte activation (8-11). In addition, high expression of NFKBIZ in psoriasis patients may correlate with elevated IL-36 and IL-17A responses (12). Topical silencing of NFKBIZ may be advantageous because it could potentially broaden the patient population that could benefit from the treatment compared to treatment with a single antibody.

[0317] Silencing NFKBIZ by topical application of small interfering RNA (siRNA) provides a non-invasive, self-administered treatment option with minimal side effects (13). However, the biggest challenge in this pathway is that only a limited number of drugs with low molecular weight (up to several hundred daltons) and high octanol-water partition coefficients can be used for successful topical delivery (14). Transdermal and topical delivery of hydrophilic molecules, particularly macromolecules such as antibodies and nucleic acids, remains difficult due to their high molecular weight (15). Several reports have shown topical siRNA delivery using techniques such as globular nucleic acids (16) and self-assembling framework nucleic acids (17). Microneedles are also being explored for topical delivery of siRNA (18). Methods such as electroporation (19) and peptide carriers are also being explored (20-22). Strategies for delivering siRNA to treat skin wounds have also been developed (23, 24).

[0318] This specification describes a modular IL-based siRNA delivery approach for silencing various genes of interest. Specifically, this specification describes a combination of ILs that simultaneously stabilizes siRNA and enhances siRNA penetration into the skin after topical application. The efficacy of the formulation for in vivo silencing of NFKBIZ in an imiquimod-induced psoriasis mouse model is demonstrated.

[0319] result IL selection We designed and synthesized an IL library to evaluate siRNA delivery to the skin. For biocompatibility, cholinium was used as the cation in all ILs. Several different anions were used to synthesize the ILs (Figures 24A-24E). Geraneic acid was used as the reference anion in the IL library [i.e., choline and geraneic acid (CAGE) as the reference IL]. Other anions were selected for several reasons. First, to evaluate the effect of chain length on siRNA stability and delivery, we selected anions containing shorter linear carbon chains compared to geraneic acid. Anions with aromatic groups were selected because they can interact with stacked RNA base pairs via electrostatic, hydrophobic, and polar interactions. All ILs were prepared in a 1:2 (cation:anion) stoichiometric ratio and evaluated for stability and siRNA delivery. Of the synthesized interleukins (ILs), CAGE, choline and dimethylacrylic acid (CADA), choline and isovaleric acid (CAVA), and choline and phenylpropanoic acid (CAPA) remained viscous liquids at room temperature (RT), while choline and 4-phenolsulfonic acid (CASA), choline and phenyl phosphate (CAPP), and choline and biphenyl-3-carboxylic acid (CABA) solidified or formed gels (Figures 24A-24E). Representative 1H nuclear magnetic resonance (NMR) spectra can be found in Figures 24A-24E, confirming the good synthesis and purity of the ILs. In addition, since both interleukins and ILs are referred to as "IL," all interleukins are shown numerically throughout the manuscript for clarity.

[0320] Effect of IL on siRNA stability The effect of ILs on siRNA stability was evaluated. Circular dichroism (CD) spectroscopy of siRNA incubated with aqueous solutions of individual ILs at 50 vol% concentrations revealed significant changes in the α-helical skeleton in the presence of CAGE, CADA, and CABA (confirmed by a negative band at 210 nm). On the other hand, CAVA and CAPA preserved the secondary structure of siRNA (Figure 19A). Bands obtained from native gel electrophoresis complemented by CD results (Figure 19B). The improved stability of siRNA in the presence of CAPA suggested a possible synergistic effect between ILs prepared from two structurally different anions. Therefore, the effect of IL mixtures on siRNA stability was evaluated to determine whether the compatibility of CAPA with siRNA provides further protection against the adverse effects of CAGE and CABA on the siRNA structure. The combination of CAGE (25 vol%) and CAPA (25 vol%) yielded significant bands indicating preservation of the siRNA structure (Figures 24A-24E).

[0321] Screening for the optimal IL com...

Claims

1. Anions that are carboxylic acids containing aliphatic chains of 4 carbon atoms or less, and cations containing quaternary ammonium at least one ionic liquid containing; and Polypeptide-containing active compounds A composition comprising, wherein at least one ionic liquid is not choline and gelanate (CAGE).

2. The composition according to claim 1, wherein the anion is a carboxylic acid having a LogP of less than 1.0 and the formula R-COOH (wherein R is an unsubstituted alkyl or alkenyl with 4 or fewer carbon atoms).

3. The composition according to claim 1 or 2, wherein the carboxylic acid comprises an aliphatic chain of 3 carbon atoms or less.

4. The composition according to any one of claims 1 to 3, wherein the anion comprises only one carboxylic acid group.

5. The composition according to any one of claims 1 to 4, wherein the anion is selected from the group consisting of propanoic acid; isobutyric acid; butyric acid; 3,3-dimethylacrylic acid; dimethylacrylic acid; and isovaleric acid.

6. The composition according to any one of claims 1 to 5, wherein the cation has a molar mass equal to or greater than that of choline.

7. Quaternary ammonium is NR 4 + It has the structure and at least one R group contains a hydroxyl group, or Quaternary ammonium is NR 4 + The composition according to any one of claims 1 to 6, having the structure and comprising only one R group containing a hydroxyl group.

8. The cation is choline, or the structure shown below: A composition according to any one of claims 1 to 7, having the following characteristics.

9. The ionic liquid contains a cation-to-anion ratio of approximately 2:1 to approximately 1:1, 2:1, less than 1:1, or cation-excess, and / or The composition according to any one of claims 1 to 8, wherein the ionic liquid has a concentration of at least 0.1% w / v, 10 to 70% w / v, 30 to 50% w / v, or 30 to 40% w / v.

10. (a) Carboxylic acids that are not fatty acids, (b) Carboxylic acids containing aliphatic chains of 4 carbon atoms or less, (c) Aromatic anions, and / or (d) Anions with a LogP of less than 1.0 anion which is at least one of the following, Furthermore cations containing quaternary ammonium Further ionic liquids A composition according to any one of claims 1 to 9, further comprising:

11. Regarding each ionic liquid, (i) The cation has a molar mass equal to or greater than that of choline; (ii) Quaternary ammonium is NR 4 + Having the structure and containing at least one hydroxyl group; (iii) Quaternary ammonium is NR 4 + Having the structure and containing only one hydroxyl group in each R group; or (iv) The cation is choline, or the structure shown below: Do you have it? or Each ionic liquid has a choline cation, and / or Each ionic liquid contains a different anion; Further ionic liquids include geranic acid; glycolic acid; propanoic acid; isobutyric acid; butyric acid; gallic acid; lactic acid; malonic acid; Maleic acid; glutaric acid; citric acid; 3,3-Dimethylacrylic acid; Dimethylacrylic acid; Gluconic acid; Adipic acid; Sodium ethylhexyl sulfate; Decanoic acid; The anions include hydroxybenzenesulfonic acid; 4-hydroxybenzenesulfonic acid (4-phenolsulfonic acid); isovaleric acid; hydrocinnamic acid (phenylpropanoic acid); phenyl phosphoric acid; and biphenyl-3-carboxylic acid. The composition according to claim 10.

12. Each ionic liquid is a different ionic liquid selected from the group consisting of choline and geranic acid (CAGE); choline and dimethylacrylic acid (CADA); choline and isovaleric acid (CAVA); choline and phenyl phosphate (CAPP); choline and biphenyl-3-carboxylic acid (CABA); choline and 4-phenolsulfonic acid (CASA); or choline and phenylpropanoic acid (CAPA). The composition according to any one of claims 1 to 11.

13. The ionic liquid and / or further ionic liquid according to any one of claims 1 to 9 comprises a cation-to-anion ratio of about 2:1 to about 1:1, or a cation-to-anion ratio with a cation excess of 2:

1. or The ionic liquid and / or further ionic liquid according to any one of claims 1 to 9 includes a cation-to-anion ratio of less than 1:1, or a cation-to-anion ratio in excess of cations, and / or The ionic liquid and / or further ionic liquid according to any one of claims 1 to 9 is at a concentration of at least 0.1% w / v, at a concentration of 10 to 70% w / v, at a concentration of 30 to 50% w / v, or at a concentration of 30 to 40% w / v. The composition according to any one of claims 10 to 12.

14. (i) Is it a mixture? (ii) Provided in one or more nanoparticles, wherein the one or more nanoparticles are in the state of solution or suspension in a composition containing an ionic liquid; (iii) Provided in a biodegradable capsule; and / or (iv) Formulated for transdermal administration, mucosal administration, oral administration, subcutaneous administration, intradermal administration, parenteral administration, intratumoral administration, or intravenous administration, The composition according to any one of claims 1 to 13.

15. The polypeptide is an antibody or antibody reagent. The composition according to any one of claims 1 to 14.

16. The polypeptide is provided in doses of 1 to 40 mg / kg; or The polypeptide is an antibody or antibody reagent, and the polypeptide has a molecular weight greater than 450 or greater than 500. The composition according to any one of claims 1 to 15.

17. The composition according to any one of claims 1 to 16, further comprising at least one nonionic surfactant and / or a pharmaceutically acceptable carrier.

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