Ionic liquids for drug delivery

Abstract

Compositions comprising ionic liquids for the stabilization and delivery of active compounds are provided.SOLUTION: A composition is provided comprising at least one ionic liquid comprising an anion that is at least one of (a) a carboxylic acid that is not a fatty acid, (b) a carboxylic acid comprising an aliphatic chain of 4 carbons or less, (c) an aromatic anion, and / or (d) an anion having a LogP of less than 1.0, and a cation comprising a quaternary ammonium.SELECTED DRAWING: Figure 19D

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 939,088, filed November 22, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy, created on November 19, 2020, has the file name 002806-096230WOPT_SL.txt and is 25,930 bytes in size.

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

[0004] background The uptake of many active compounds, for example, pharmaceutically active compounds, can be improved by delivering the compound in a solvent.However, most of these solvents exhibit toxic side effects and / or act as irritants at the time of delivery, so this approach is often inappropriate for in vivo use.These toxic and irritating effects are serious enough to suppress any increase in the uptake or performance of active compounds. Summary of the Invention

[0005] overview As shown herein, the inventors have identified characteristics of ionic liquids that provide surprisingly superior active compound uptake kinetics for certain classes of active compounds. Accordingly, compositions and methods related to these unexpectedly highly effective ionic liquids (ILs) are described herein.

[0006] In one aspect of any embodiment, described herein is a composition comprising at least one ionic liquid comprising an anion that is at least one of the following: a) a carboxylic acid that is not a fatty acid; b) a carboxylic acid that includes an aliphatic chain of four carbons or less; c) an aromatic anion; and / or d) an anion that has a LogP of less than 1.0; and a cation that includes a quaternary ammonium.

[0007] In some embodiments of any aspect, the anion has a LogP of less than 1.0 and is a) a carboxylic acid that is not a fatty acid; b) a carboxylic acid containing an aliphatic chain of four carbons or less; or c) an aromatic anion. In some embodiments of any aspect, the fatty acid contains an aliphatic chain of three carbons or less. In some embodiments of any aspect, the anion contains only one carboxylic acid group (e.g., an R-COOH group). 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; isovaleric acid; hydrocinnaminic acid; 4-phenolsulfonic acid; phenylphosphoric acid; and biphenyl-3-carboxylic acid.

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

[0009] In some embodiments of any aspect, the ionic liquid comprises a ratio of cations to anions of about 2:1 to about 1:1. In some embodiments of any aspect, the ionic liquid comprises a ratio of cations to anions of about 2:1. In some embodiments of any aspect, the ionic liquid has a cation:anion ratio of less than 1:1. In some embodiments of any aspect, the ionic liquid has a cation:anion ratio with a cation excess.

[0010] In some embodiments of any aspect, the composition further comprises at least one active compound in combination with the at least one ionic liquid.

[0011] In some embodiments of any aspect, the active compound comprises a polypeptide. In some embodiments of any aspect, the polypeptide is an antibody or antibody reagent. In some embodiments of any aspect, the active compound has a molecular weight greater than 450. In some embodiments of any aspect, the active compound has a molecular weight greater than 500. In some embodiments of any aspect, the anion has a LogP 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 embodiments of any aspect, active compound comprises nucleic acid.In some embodiments of any aspect, nucleic acid is inhibitory nucleic acid.In some embodiments of any aspect, nucleic acid is siRNA.In some embodiments of any aspect, anion has LogP less than 1.0 and is a) non-fatty acid carboxylic acid; or b) carboxylic acid that comprises 4 carbons or less aliphatic chain; and / or c) aromatic anion.

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

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

[0015] In some embodiments of any aspect, the active compound is provided at a dose of 1 to 40 mg / kg. In some embodiments of any aspect, the composition further comprises at least one non-ionic surfactant. In some embodiments of any aspect, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments of any aspect, the composition is provided in a degradable capsule. In some embodiments of any aspect, the composition is an admixture. In some embodiments of any aspect, the composition is provided in one or more nanoparticles. In some embodiments of any aspect, the composition comprises one or more nanoparticles comprising the active compound, the nanoparticles being in solution or suspension in a composition comprising an ionic liquid.

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

[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 an anti-human TNF-α mouse IgG1 antibody (clone MAb11) isolated from CGLY variants at concentrations ranging from 20 to 90% by volume. Figure 1C shows the circular dichroism spectrum of anti-human TNF-α IgG isolated from CGLY variants. The IgG was dispersed in 50% by volume of the CGLY variants, stored at room temperature (25°C) for 1 hour, and then dialyzed for 48 hours. The beta-sheet secondary structure of the IgG was retained after exposure to the CGLY solution. Figure 1D shows an SDS-PAGE of anti-human TNF-α IgG isolated from CGLY variants. [Figure 1-2] See description of Figure 1-1. [Figure 2] Figures 2A-2B show in vitro studies of CGLY variants on Caco-2 cell viability and IgG transport. Figure 2A shows Caco-2 cell viability treated with CGLY variants. Data are presented as mean ± SE (n = 6). Figure 2B shows enhanced FITC-IgG transport across Caco-2 monolayers in the presence of 30 mM CGLY variants. Data are presented as mean ± SE (n = 5); (*p < 0.05; CGLY 1:1 and CGLY 2:1 treatment compared to CGLY 1:2). (##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 by CGLY2:1. In the presence of various CGLY2:1 concentrations, enhanced transport of FITC-IgG (Figure 3A) and Lucifer Yellow (Figure 3B) across Caco-2 monolayers was observed. Data are presented as mean ± SE (n = 5). Figure 3C shows the effect on tight junction integrity of Caco-2 cells after treatment with various concentrations of CGLY. Data are presented as mean ± SE (n = 5); (*p < 0.05; **p < 0.001; all CGLY2:1-treated compared to no CGLY2:1 treatment). Figure 3D shows FITC-IgG transport across Caco-2 monolayers after 24 h of incubation in the presence of 55 mM CGLY2:1 and with or without transcytosis inhibitors. Data are presented as mean ± SE (n = 5). [Figure 3-2] See description of 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, 12.5, 25, and 50 vol% CGLY2:1 in saline. CGLY2:1 treatment was added to the mucus, followed by gentle shaking and then measurement after 30 minutes of equilibration. Data are presented as the mean (n = 3). Black circles = 0 vol%, dark gray circles = 12.5 vol%, light gray circles = 25 vol%, and open circles = 50 vol%. Figure 4B shows the mean viscosity values ​​of porcine mucus in the presence of 0, 12.5, 25, and 50 vol% CGLY2:1 in saline at a shear rate of 49.87 1 / s. 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 triplicate, and representative images are shown. The scale bar represents 200 μm. Figure 5A shows an oral toxicity study of CGLY2:1. CGLY2:1 (50% by volume) or saline was administered by oral gavage at a dose of 1250 mg / kg (n = 2) for 15 days. Results were evaluated by body weight monitoring, blood chemistry, and H&E staining of the GI tract and major organs. Figure 5D shows fluorescence quantification of FITC-IgG per unit area of ​​the villi from Figures 5A-5C. Data are expressed as mean ± SE (n = 10). FIG. 5E shows in vivo plasma anti-human TNF-α IgG concentrations after intrajejunal injection of CGLY2:1 or IgG in saline, as quantified by ELISA. [Figure 5-2] See description of Figure 5-1. [Figure 6A] Figures 6A-6C show an in vivo toxicity study of CGLY2:1. Rats were orally administered CGLY2:1 or saline once daily for 7 consecutive days. Figure 6A shows rat weight records from days 0 to 7 during the study. Data are expressed as mean ± SE (n = 6). Figure 6B shows the results of rats sacrificed on day 7, and GI tract sections were processed with hematoxylin and eosin (H&E) for histological staining. The scale bar represents 100 μm. Figure 6C shows a comprehensive metabolic panel of 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 legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7] FIG. 7 shows a diagram of drug delivery. [Figure 8] Figure 8 shows functional antibody stability as measured by ELISA in the indicated ILs. There is a general trend for smaller anions to be more compatible with antibodies than larger anions. [Figure 9]Figure 9 shows functional antibody stability in the indicated ILs as measured by size exclusion chromatography. The antibody used was anti-human TNFα (mouse) (clone MAb11) that was dialyzed for 2 days. [Figure 10] Figure 10 shows functional antibody stability as measured by circular dichroism in the indicated ILs. The antibody used was anti-human TNFα (mouse) (clone MAb11) that was dialyzed for 2 days. [Figure 11] Figure 11 shows a graph of antibody concentrations in serum following intrajejunal administration in the indicated compositions. The dose was 200 μg / kg, n=3. [Figure 12] FIG. 12 shows the experimental design for in vivo mAb local delivery. [Figure 13] FIG. 13 shows the results of in vivo localized mAb delivery. [Figure 14] FIG. 14 shows compatibility testing of CGLY2:1 with other antibodies. [Figure 15] Figure 15 shows H&E staining of major organs in the toxicity study of 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 processed by H&E for histological staining. No differences were observed between the CGLY2:1 and saline control groups. The scale bar represents 100 μm. [Figure 16] Figure 16 shows H&E staining of the GI tract in the toxicity study of 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 processed with H&E for histological staining. No differences were observed between the CGLY2:1 and saline control groups. The scale bar represents 100 μm. [Figure 17] FIG. 17 shows the structures of the ILs tested for siRNA delivery capabilities. [Figure 18]Figure 18 shows representative confocal microscopy images of transwell membranes covered with a layer of Caco-2 cells and incubated for 5 hours with various concentrations of FITC-IgG dispersed in CGLY2:1. Images were taken at 40x magnification. Images show DAPI-labeled nuclei, FITC-IgG, and an overlay of DAPI staining and FITC-IgG. The scale bar represents 50 μm. [Figure 19A] Figures 19A-19E show the screening of cholinium-based bioactive IL-RNA complexes for enhanced epidermal accumulation. (Figure 19A) CD spectrum of siRNA in phosphate-buffered saline (PBS) after 30 min of incubation with IL (50% by volume) and 72 h of dialysis. (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 h of incubation in the presence of a 1:1 IL combination (CAGE+CAPA). From left to right: merge, Cy5, differential interference contrast (DIC). Scale bar, 50 μm. (Figures 19D and 19E) Transport of Cy5-labeled siRNA into different layers of the skin in the presence of individual ILs at a concentration of 50% by volume (Figure 19D) and IL combinations at a concentration of 50% by volume (Figure 19E) as determined by tape stripping (n=3). For Figures 19D-19E, data are means ± SEM and were determined to be nonparametric by normality tests and Kruskal-Wallis statistics. *P<0.05. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 19D] See legend to Figure 19A. [Figure 19E] See legend to Figure 19A. [Figure 20-1]Figures 20A-20F show MD simulations identifying the extent of IL-siRNA interactions for solvation and enhanced stability. (Figures 20A and 20B) Snapshots of the simulated unit cell of CAGE and siRNA (Figure 20A) and CAGE components seen within 10 Å of the siRNA (Figure 20B) under periodic boundary conditions for 500 ns. (Figures 20C and 20D) Snapshots of the simulated unit cell of an optimized IL combination (CAGE and CAPA, 1:1) and siRNA (Figure 20C) and IL species seen within 10 Å of the siRNA (Figure 20D) under similar conditions. (Figures 20E and 20F) Radius of gyration (RGYR) (Figure 20E) and root mean square deviation (RMSD) (Figure 20F) obtained over 500 ns for CAPA and the IL combination (CAGE and CAPA) relative to CAGE (control). [Figure 20-2] See description of Figure 20-1. [Figure 21-1] Figures 21A-21E show three MD simulations establishing the lipid bilayer interaction enhancement and transfer mechanism of IL combinations. (Figure 21A) Lipid bilayer simulation with aggregates of choline, geranic acid, and phenylpropanoic acid highlighted by circles. (Figure 21B) Zoomed-in view of ionic species from the circle showing their closed interactions with the phospholipid heads and tails. The aggregate contains all three ionic species contributing to the interaction with the lipid membrane. (Figure 21C) Representative snapshots viewed perpendicular to the membrane in the plane of the lipid bilayer. (Figures 21D and 21E) Average lipid membrane thickness (Figure 21D) and average area per lipid (Figure 21E) during simulations in the presence of CAPA and IL combinations (CAGE and CAPA) versus CAGE (control). For Figures 21D-21E, all data are mean ± SEM and were determined to be nonparametric by normality and Kruskal-Wallis statistics. ****P<0.0001. [Figure 21-2] See description of 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 of the topical application schedule. (Figure 22B) Representative histology [hematoxylin and eosin (H&E)] images 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 are means ± SEM and were determined to be nonparametric by normality tests and Kruskal-Wallis statistics. *P<0.05, ***P<0.001, and ****P<0.0001. (Figure 22E) GAPDH levels in skin samples were determined using a GAPDH enzyme-linked immunosorbent assay. Data are mean ± SEM, and statistics were calculated by one-way ANOVA with 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 description of Figure 22-1. [Figure 23-1]Figures 23A-23J show that local inhibition of NFKBIZ by topical IL-siRNA suppresses imiquimod-induced psoriasis-like skin inflammation and other key psoriasis-related genes. (Figure 23A) Schematic of the application schedule for disease induction and topical IL-siRNA administration. (Figure 23B) Psoriasis-induced mice were topically treated 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 blinded daily scoring using the human PASI scoring system on a scale of 0 (no change) to 4 (very clear change). (Figure 23G) Heat map of the expression levels of various psoriasis-related genes after treatment with IL-NFKBIZ siRNA compared to untreated (control) and IL-siCon-treated groups. (Figures 23H-23J) mRNA expression levels for NFKBIZ, TNF-α, and IL-17A, respectively, were measured by qPCR, and β-actin mRNA expression was used for normalization. Data are mean ± SEM, and statistics were calculated by one-way ANOVA with Tukey 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 description of Figure 23-1. [Figure 23-3] See description of Figure 23-1. [Figure 24-1]Figures 24A-24E show the design and synthesis of an in-house cholinium-based IL library for improved biocompatibility and interaction with RNA. (Figure 24A) A cholinium-based IL library containing various anions synthesized using CAGE as the reference IL. (Figure 24B) General synthetic scheme of salt metathesis used in the synthesis of ILs. (Figure 24C) Synthetic scheme of an optimized IL combination (CAGE+CAPA) for siRNA delivery. (Figure 24D) H-NMR spectra of the synthesized ILs, (a) CAGE, (b) CAVA, (c) CAPA, and (d) CADA, that remained viscous at RT. (Figure 24E) Relative density of siRNA bands after IL incubation measured with Image J software. [Figure 24-2] See description of Figure 24-1. [Figure 24-3] See description of Figure 24-1. [Figure 25-1] Figures 25A-25D show improved epidermal accumulation of Cy5-labeled siRNA in the presence of ILs. (Figure 25A) Schematic of the Franz diffusion cell (FDC) setup for ex vivo pig skin permeation studies. (Figure 25B) Representative confocal images of control, naked siRNA, and siRNA in the presence of CAGE. (Figure 25C) Epidermal accumulation of Cy5-siRNA in the presence of newly synthesized cholinium-based ILs and a 1:1 combination after 24 hours of incubation in pig skin. Left to right: merge, Cy5, differential interference contrast (DIC). Scale bar, 50 μm. (Figure 25D) Transport of Cy5-labeled siRNA into different layers of the skin as determined by tape stripping (n = 3). Data are means ± SEM and were determined to be nonparametric by normality tests and Kruskal-Wallis statistics. [Figure 25-2] See description of Figure 25-1. [Figure 26]Figures 26A-26B show the main contribution of IL species mobility in IL-lipid bilayer interaction and permeation. (Figure 26A) Lipid bilayer simulation in the presence of IL combinations (highlighted by circles). (Figure 26B) Trajectories of individual ionic species within IL combinations, CAGE+CAPA simulation using the python library MDAnalysis. [Figure 27] Figures 27A-27D show highly biocompatible IL formulations without toxicity or irritation after topical application. (Figure 27A) Application site of a healthy mouse topically treated with IL-GAPDH siRNA compared with water and IL-siCon groups. (Figure 27B) H&E staining of a skin section from a healthy mouse topically treated with IL-siCon for 4 consecutive days. Scale bar, 100 μm; magnification, 10×. (Figure 27C) Skin sections from a healthy mouse were analyzed for hyperproliferation by staining with the proliferation marker Ki67. Scale bar, 100 μm. Quantitative analysis for IHC was not performed because no areas of proliferation 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. Statistics were based on one-way ANOVA with 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 show the characterization of the effects of IL-siCon in mice with imiquimod-induced psoriasis. (Figure 28A) Psoriasis-induced mice were topically treated with IL-siCon for four consecutive days. (Figure 28B) H&E staining of skin sections from mice with imiquimod-induced psoriasis that were topically treated with IL-siCon. Scale bar, 50 μm; magnification, 10×. (Figure 28C) Skin sections from mice with psoriasis 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 Image J software. Data are mean ± SEM; statistics were based on one-way ANOVA with Tukey HSD post-hoc test. *P<0.05, ****P<0.0001. [Figure 29] Figures 29A-29C show the effect of IL-NFKBIZ siRNA on imiquimod-induced psoriasis-like skin inflammation in mice. Mice with imiquimod-induced psoriasis were monitored by double skinfold thickness (DSFT) and analyzed for cumulative score (Figure 29A), body weight (Figure 29B), and skin thickness (Figure 29C) during the 5-day induction / application period. 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, and IL-36G (Figures 30A-30F); chemokine CCL 20 (Figure 30G); S100 protein S100A9 (Figure 30H); antimicrobial protein lipocalin-2, LCN2, and β-defensin-2, DEFB4 (Figure 30J). β-actin mRNA expression was used for normalization. Data are mean ± SEM. Statistics were calculated by one-way ANOVA with 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 description of Figure 30-1. [Figure 30-3] See description of Figure 30-1. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description of the Invention The data provided herein demonstrate that the anion of an ionic liquid (IL) exerts a significant influence on whether a particular active agent is transported across a biological barrier (e.g., an epithelial layer such as the dermis). Anions with low hydrophobicity and / or aromatic groups offer improved drug delivery properties for antibody and siRNA cargo molecules over anions in previously described ILs, such as CAGE (choline and geranic acid). When selecting a cation to pair with an anion, the primary concern is that the cation not be too closely associated with the anion, as this would cause the anion to be retained on the first side of the biological barrier.

[0019] Thus, in one aspect of any embodiment, described herein is a compound comprising: 1) an anion that is at least one of the following: a) a carboxylic acid that is not a fatty acid; b) carboxylic acids containing aliphatic chains of four carbons or less; c) an aromatic anion; and / or d) anions with a LogP of less than 1.0; and 2) Cations containing quaternary ammonium The composition comprises at least one ionic liquid,

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

[0021] As used herein, the term "ionic liquid (IL)" refers to an organic salt or mixture of organic salts that are in a liquid state at room temperature. This class of solvents has been shown to be useful in a variety of fields, including industrial processing, catalysis, pharmaceuticals, and electrochemistry. Ionic liquids contain at least one anionic component and at least one cationic component. Ionic liquids can contain additional hydrogen bond donors (i.e., any molecule that can provide an -OH or -NH group); examples include, but are not limited to, alcohols, fatty acids, and amines. The at least one anionic component and the at least one cationic component can be present in any molar ratio. Exemplary molar ratios (cation:anion) include, but are not limited to, 1:1, 1:2, 2:1, 1:3, 3:1, 2:3, 3:2, and ranges between these ratios. For further discussion of ionic liquids, see, e.g., Hough, et al., "The third evolution of ionic liquids: active pharmaceutical ingredients," New Journal of Chemistry, 31: 1429 (2007) and Xu, et al., "Ionic Liquids: Ion Mobilities, Glass Temperatures, and Fragilities," Journal of Physical Chemistry B, 107(25): 6170-6178 (2003), each of which is incorporated by reference in its entirety. In some embodiments of any aspect, the ionic liquid or solvent exists as a liquid below 100°C. In some embodiments of any aspect, the ionic liquid or solvent exists as a liquid at room temperature.

[0022] As described herein, anions with 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, the improved drug delivery properties include reduced denaturation or degradation of the cargo molecule. In some embodiments, the improved drug delivery properties include an increased ability to cross biological barriers (e.g., increased permeability). In some embodiments of any aspect, anions with 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 with 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 the ILs described herein is hydrophobic.

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

[0025] Carboxylic acids are compounds having the structure of Formula I, where R can be any group. TIFF2026021383000002.tif32128

[0026] In general, the anion is RX - where X is CO2 - , SO3 - , OSO3 2- or OPO3 2- and R is an optionally substituted C1-C 10 Alkyl, optionally substituted C2-C 10 Alkenyl or optionally substituted C2-C 10 It is alkynyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0027] In some embodiments, R is an optionally substituted straight-chain or branched C1-C9 alkyl. For example, R is a C1-C9 alkyl optionally substituted with 1, 2, 3, 4, 5, or 6 substituents independently selected from the group consisting of C1-C3 alkyl, hydroxy (OH), halogen, oxo (=O), carboxy (CO2), cyano (CN), and aryl. In some embodiments, R is a C1-C6 alkyl optionally 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 alkyl optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of methyl, ethyl, hydroxyl, carboxy, and phenyl. Exemplary alkyls for R include, but are not limited to, methyl, carboxymethyl, hydroxymethyl, ethyl, 1-hydroxyethyl, 2-phenylethyl, propyl, prop-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 an optionally substituted straight-chain or branched C2-C8 alkenyl. For example, R is a C2-C9 alkenyl optionally 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 optionally 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 optionally 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 optionally substituted aryl or heteroaryl. For example, R is an aryl or heteroayl optionally 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 optionally 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 phenyl 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-biphen-4-yl.

[0030] In some embodiments, X is CO -and R is methyl, carboxymethyl, hydroxymethyl, ethyl, 1-hydroxyethyl, 2-phenylethyl, propyl, prop-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-biphen-4-yl. - and R is methyl, carboxymethyl, hydroxymethyl, ethyl, 1-hydroxyethyl, 2-phenylethyl, propyl, prop-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-biphen-4-yl. 2- or SO3 - and R is 2-hydroxyphenyl, 3-hydroxyphenyl, or 4-hydroxyphenyl.

[0031] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight-chain (i.e., unbranched) or branched carbon chain (or carbons), or combinations thereof, which may be fully saturated, monounsaturated, or polyunsaturated, having the specified number of carbon atoms (i.e., C1 to C6). 10(meaning 1 to 10 carbons) and can include mono-, di-, and polyvalent radicals. Alkyl is an acyclic chain. 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, (cyclohexyl)methyl, and homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, and n-octyl. "Alkenyl" is an unsaturated alkyl group, one having one or more double bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), and higher homologs and isomers.

[0032] The term "aryl," unless otherwise specified, refers to a polyunsaturated, aromatic, hydrocarbon substituent, which may be a single ring or multiple rings (preferably 1 to 3 rings) fused together (i.e., fused-ring aryl) or covalently linked together. Fused-ring aryl refers to multiple rings fused together, where 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 fused-ring heteroaryl groups (i.e., multiple rings fused together, where at least one of the fused rings is a heteroaromatic ring). 5,6-fused-ring heteroarylene refers to two rings fused together, where one ring has five members and the other has six members, and at least one ring is a heteroaryl ring. Similarly, a 6,6-fused ring heteroarylene refers to two rings fused together, one ring having 6 members and the other ring having 6 members, and at least one ring is a heteroaryl ring. Also, a 6,5-fused ring heteroarylene refers to two rings fused together, one ring having 6 members and the other ring having 5 members, and at least one ring is a heteroaryl ring. The heteroaryl group can be attached to the rest of the molecule through a carbon or heteroatom.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- These include, but are not limited to, thienyl, 3-thienyl, pyridine, 2-pyridyl, naphthyridinyl, 3-pyridyl, 4-pyridyl, benzophenone pyridyl, pyridazinyl, 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, and the like.

[0033] The term "optionally substituted" means that the specified group or moiety is unsubstituted or substituted with one or more (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" refers to a group "substituted" on the group substituted at any atom of the substituted group. Suitable substituents include, but are not limited to, halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido. In some cases, two substituents, together with the carbons to which they are attached, may form a ring.

[0034] As used herein, a "fatty acid" refers to a fatty acid having a structure in which R comprises a saturated or unsaturated aliphatic chain, e.g., R is of the formula C n H 2n+1 In some embodiments of any aspect, the fatty acid is a monocarboxylic acid. The fatty acid can be natural or synthetic. The aliphatic chain of the fatty acid can be saturated, unsaturated, branched, straight-chain, and / or cyclic. In some embodiments of any aspect, the aliphatic chain does not include an aromatic group. In some embodiments of any aspect, the aliphatic chain comprises, consists of, or consists essentially of an alkyl or alkene chain.

[0035] Exemplary carboxylic acids that are not fatty acids can include, but are not limited to, lactic acid; glycolic acid; malonic acid; maleic acid; glutaric acid; citric acid; gluconic acid; and adipic acid. TIFF2026021383000003.tif24060

[0036] In some embodiments, the non-fatty acid carboxylic acid contains five or fewer carbons in the R group, in either a linear or branched configuration. In some embodiments, the non-fatty acid carboxylic acid contains a hydroxy 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 contains 5 or fewer carbons in the R group, in either a linear or branched configuration, and contains a hydroxy group in the R group. In some embodiments, the non-fatty acid carboxylic acid contains 1-5 carbons in the R group, in either a linear or branched configuration, and contains a hydroxy group in the R group.

[0038] In some embodiments, the non-fatty acid carboxylic acids contain five or fewer carbons in the R group, in either a linear or branched configuration, and contain one or more carboxylic acid groups in the R group. In some embodiments, the non-fatty acid carboxylic acids contain one to five carbons in the R group, in either a linear or branched configuration, and contain one or more carboxylic acid groups in the R group.

[0039] In some embodiments, the non-fatty acid carboxylic acid contains 1-5 carbons in the R group, in either a linear or branched configuration, and contains one carboxylic acid group in the R group.

[0040] When the number of carbons in a chain is referred to herein, it is intended to refer to the total number of carbons 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 comprises one carboxylic acid group.

[0042] Exemplary carboxylic acids containing an aliphatic chain of four carbons or less can 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). TIFF2026021383000004.tif168128

[0043] Exemplary alternative anions contemplated herein include decanoic acid and ethylhexyl sulfate. TIFF2026021383000005.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 phenylphosphate. TIFF2026021383000006.tif21273

[0045] Hydrophobicity may be assessed by analysis of logP. "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 an immiscible solvent, as a neutral molecule. Partition coefficient, P = [organic] / [aqueous], where [ ] = concentration Log P=log 10 (Partition coefficient) = log 10 P In practice, LogP values ​​will vary depending on the measurement conditions and the choice of partitioning solvent. A LogP value of 1 means that the concentration of the compound is 10 times higher in the organic phase than in the aqueous phase. An increase in logP value of 1 indicates a 10-fold increase in the compound concentration in the organic phase compared to the aqueous phase.

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

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

[0048] In some embodiments of any aspect, the anion of an IL described herein has a pKa of less than 4.0. In some embodiments of any aspect, the anion of an IL 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 one of ordinary skill 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 catalog listings. The pKa and LogP values ​​of exemplary anions are provided in Table 1 herein.

[0050] Exemplary, non-limiting anions are provided in Table 1 below.

[0051] [Table 1]

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

[0053] In some embodiments of any aspect, the anion is an unsubstituted alkane. In some embodiments of any aspect, the anion is an unsubstituted alkene. In some embodiments of any aspect, the carbon chain backbone of the carboxylic acid comprises one or more substituents. In some embodiments of any aspect, the carbon chain of the carboxylic acid comprises one or more substituents, wherein each substituent comprises at least one carbon atom. In some embodiments of any aspect, the carbon chain of the carboxylic acid comprises one or more substituents, wherein each substituent is an alkyl, aryl, heteroalkyl, heteroaryl, alkane, or alkene. In some embodiments of any aspect, the carbon chain of the carboxylic acid comprises one or more substituents, wherein each substituent is an unsubstituted alkyl, unsubstituted aryl, unsubstituted heteroalkyl, unsubstituted heteroaryl, unsubstituted alkane, or unsubstituted alkene.

[0054] As described herein, when selecting a cation to pair with an anion, the primary concern is that the cation not be too closely associated with the anion, which would 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. Thus, the cations of the ILs described herein can be cations containing quaternary ammonium. Quaternary ammonions have the structure NR4 + where each R is independently an alkyl or aryl group.

[0055] The general term "quaternary ammonium" is NH4 + It refers to any compound that can be considered derived from ammonium hydroxide or an ammonium salt by replacing all four hydrogen atoms of the ion with organic groups. For example, quaternary ammonium is NR4 + wherein each R is hydroxyl, optionally substituted C1-C 10 Alkyl, optionally substituted C2-C 10 Alkenyl, optionally substituted C2-C 10is independently selected from alkynyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0056] In some embodiments of any aspect, the cation has a molar mass equal to or greater than choline, e.g., a molar mass equal to or greater than 104.1708 g / mol. In some embodiments of any aspect, the cation has a molar mass greater than choline, e.g., a molar mass equal to or greater than 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. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an alkyl, alkane, or alkene. In some embodiments of any aspect, each R group of the quaternary ammonium independently comprises an alkane or alkene. 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 comprises a carbon chain of 10 carbon atoms or less, e.g., 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 a carbon chain of 12 carbon atoms or less. 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 some embodiments of any aspect, each R group of the quaternary ammonium independently comprises a carbon chain of 20 carbon atoms or less.

[0059] In some embodiments of any aspect, each R group of the quaternary ammonium independently includes an alkyl group of 10 carbon atoms or less, e.g., 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 includes an alkyl group of 12 carbon atoms or less. In some embodiments of any aspect, each R group of the quaternary ammonium independently includes an alkyl group of 15 carbon atoms or less. In some embodiments of any aspect, each R group of the quaternary ammonium independently includes an alkyl group of 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 includes a hydroxy group. In some embodiments of any aspect, one R group of the quaternary ammonium includes a hydroxy group. In some embodiments of any aspect, only one R group of the quaternary ammonium includes a hydroxy group.

[0062] Exemplary non-limiting cations can include choline and any of the cations designated C1-C7 as defined by the following structure: TIFF2026021383000008.tif113128

[0063] Further non-limiting examples of cations include: 1-(hydroxymethyl)-1-methylpyrrolidin-1-ium 1-(2-hydroxyethyl)-1-methylpyrrolidin-1-ium 1-Ethyl-1-(3-hydroxypropyl)pyrrolidin-1-ium 1-(3-hydroxypropyl)-1-methylpyrrolidin-1-ium 1-(4-hydroxybutyl)-1-methylpyrrolidin-1-ium 1-Ethyl-1-(4-hydroxybutyl)pyrrolidin-1-ium 1-(4-hydroxybutyl)-1-propylpyrrolidin-1-ium 1-(5-hydroxypentyl)-1-propylpyrrolidin-1-ium 1-Ethyl-1-(5-hydroxypentyl)pyrrolidin-1-ium 1-(5-hydroxypentyl)-1-methylpyrrolidin-1-ium 1-(hydroxymethyl)-1-methylpiperidin-1-ium 1-(2-hydroxyethyl)-1-methylpiperidin-1-ium 1-Ethyl-1-(2-hydroxyethyl)piperidin-1-ium 1-Ethyl-1-(3-hydroxypropyl)piperidin-1-ium 1-(3-hydroxypropyl)-1-propylpiperidin-1-ium 1-(3-hydroxypropyl)-1-methylpiperidin-1-ium 1-(4-hydroxybutyl)-1-methylpiperidin-1-ium 1-Ethyl-1-(4-hydroxybutyl)piperidin-1-ium 1-(4-hydroxybutyl)-1-propylpiperidin-1-ium 1-Butyl-1-(5-hydroxypentyl)piperidin-1-ium 1-(5-hydroxypentyl)-1-propylpiperidin-1-ium 1-Ethyl-1-(5-hydroxypentyl)piperidin-1-ium 1-(5-hydroxypentyl)-1-methylpiperidin-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)pyridin-1-ium 1-(4-hydroxybutyl)pyridin-1-ium 1-(3-hydroxypropyl)pyridin-1-ium 1-(2-hydroxyethyl)pyridin-1-ium 1-(hydroxymethyl)pyridin-1-ium 1-Hydroxypyridin-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 cation is choline, C1, C6, and / or C7. In some embodiments of any aspect, the cation is C1, C6, and / or C7.

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

[0066] Non-limiting exemplary combinations of cations and anions are provided in Table 2 below.

[0067] [Table 2]

[0068] In some embodiments of any aspect, the ionic liquid is not CAGE (choline and geranate). In some embodiments of any aspect, the cation of the ionic liquid is not choline. In some embodiments of any aspect, the anion of the ionic liquid is not geranate or geranic acid. In some embodiments of any aspect comprising a plurality of ionic liquids, the first ionic liquid is not CAGE (choline and geranate). In some embodiments of any aspect comprising a plurality of ionic liquids, the cation of the first ionic liquid is not choline. In some embodiments of any aspect comprising a plurality of ionic liquids, the anion of the first ionic liquid is not geranate 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); phenylphosphoric 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); phenylphosphoric 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. Combinations of two, three, four, five, or more of any of the ionic liquids described herein are contemplated. As a non-limiting example, the following table includes exemplary pairwise combinations of ionic liquids contemplated herein: TIFF2026021383000010.tif119170

[0071] In some embodiments of any aspect in which the composition includes multiple ionic liquids, the first and second ionic liquids have the same cation, e.g., choline. In some embodiments of any aspect in which the composition includes multiple ionic liquids, the first and second ionic liquids have different anions. For example, the first ionic liquid and the second ionic liquid can 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); phenylphosphoric acid; and biphenyl-3-carboxylic acid. In some embodiments of any aspect in which the composition includes 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 where the composition includes a plurality of ionic liquids, the first ionic liquid is choline and geranic acid (CAGE). In some embodiments of any aspect where the composition includes a plurality of ionic liquids, the second ionic liquid is choline and dimethylacrylic acid (CADA), choline and isovaleric acid (CAVA), choline and phenylphosphoric 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 where the composition includes a plurality of 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 phenylphosphoric 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 where the composition includes a plurality of 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 where the composition includes a plurality of 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 embodiments of any aspect, the IL is at a concentration of at least 0.01% w / v. In some embodiments of any aspect, the IL is at a concentration of at least 0.05% w / v. In some embodiments of any aspect, the IL is at a concentration of at least 0.1% w / v. In some embodiments of any aspect, the IL is at a concentration of at least 0.2% w / v, at least 0.3% w / v, at least 0.4% w / v, at least 0.5% w / v, at least 1% w / v or more. In some embodiments of any aspect, the IL is at a concentration of about 0.01% w / v to about 1% w / v. In some embodiments of any aspect, the IL is at a concentration of 0.01% w / v to 1% w / v. In some embodiments of any aspect, the IL is at a concentration of about 0.05% w / v to about 0.5% w / v. In some embodiments of any aspect, the IL is at a concentration of 0.05% w / v to 0.5% w / v.

[0075] In some embodiments of any aspect, the IL is at a concentration of at least 25% w / w. In some embodiments of any aspect, the IL is at a concentration of at least 25% w / w in water. In some embodiments of any aspect, the IL is at a concentration of at least 25% w / w in saline or a physiologically compatible buffer.

[0076] In some embodiments of any aspect, the IL is at a concentration of about 5% w / w to about 75% w / w. In some embodiments of any aspect, the IL is at a concentration of 5% w / w to 75% w / w. In some embodiments of any aspect, the IL is at a concentration of about 5% w / w to about 75% w / w in water, saline, or a physiologically compatible buffer. In some embodiments of any aspect, the IL is at a concentration of 5% w / w to 75% w / w in water, saline, or a physiologically compatible buffer.

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

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

[0079] In some embodiments of any aspect, the IL is 100% w / w or 100% w / v.

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

[0081] In some embodiments of any aspect, the IL is at a concentration of at least 25% w / w and has a cation:anion ratio of at least 1:3. In some embodiments of any aspect, the IL is at a concentration of at least 25% w / w in water and has a cation:anion ratio of at least 1:3. In some embodiments of any aspect, the IL is at a concentration of at least 25% w / w and has a cation:anion ratio of 1:3 or 1:4. In some embodiments of any aspect, the IL is at a concentration of at least 25% w / w in water and has a cation:anion ratio of 1:3 or 1:4. In some embodiments of any aspect, the IL is a gel, or a shear-thinning Newtonian gel.

[0082] In some embodiments of any aspect, the IL has a cation:anion ratio of about 10:1 to about 1:10. In some embodiments of any aspect, the IL has a cation:anion ratio of 10:1 to 1:10. In some embodiments of any aspect, the IL has a cation:anion ratio of about 5:1 to about 1:5. In some embodiments of any aspect, the IL has a cation:anion ratio of 5:1 to 1:5. In some embodiments of any aspect, the IL has a cation:anion ratio of about 2:1 to about 1:4. In some embodiments of any aspect, the IL has a cation:anion ratio of 2:1 to 1:4. In some embodiments of any aspect, the IL has a cation:anion ratio of about 2:1 to about 1:10. In some embodiments of any aspect, the IL has a cation:anion ratio of about 2:1 to about 1:1. In some embodiments of any aspect, the IL has a cation:anion ratio of 2:1 to 1:10. In some embodiments of any aspect, the IL has a cation:anion ratio of 2:1 to 1:1. In some embodiments of any aspect, the IL has a cation:anion ratio such that there is a greater amount of anion, for example, a ratio of less than 1:1. In some embodiments of any aspect, the IL has a cation:anion ratio such that there is an excess of anion. In some embodiments of any aspect, the IL has a cation:anion ratio of about 1:1 to about 1:10. In some embodiments of any aspect, the IL has a cation:anion ratio of 1:1 to 1:10. In some embodiments of any aspect, the IL has a cation:anion ratio of about 1:1 to about 1:4. In some embodiments of any aspect, the IL has a cation:anion ratio of 1:1 to 1:4. In some embodiments of any aspect, the IL has a cation:anion ratio of about 1:1 to about 1:3. In some embodiments of any aspect, the IL has a cation:anion ratio of 1:1 to 1:3. In some embodiments of any aspect, the IL has a ratio of cations:anions of about 1:1 to about 1:2. In some embodiments of any aspect, the IL has a ratio of cations:anions of 1:1 to 1:2.In some embodiments of any aspect, the IL has a cation:anion ratio of about 1:1, 1:2, 1:3, or 1:4. In some embodiments of any aspect, the IL has a cation:anion ratio of 1:1, 1:2, 1:3, or 1:4. In some embodiments of any aspect, the IL has a cation:anion ratio of less than about 1:1. In some embodiments of any aspect, the IL has a cation:anion ratio of less than 1:1. Without wishing to be bound by theory, compositions having a higher amount of anions relative to cations exhibit greater hydrophobicity.

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

[0084] In some embodiments of any aspect, for example, when one or more nucleic acid molecules are provided in combination with an IL, the ratio of cations:anions is greater than 1:1, e.g., greater than 1:2, from about 1:2 to about 1:4, or from 1:2 to 1:4.

[0085] In some embodiments of any aspect, the IL is at a concentration of at least 20 mM. In some embodiments of any aspect, the IL is at a concentration of at least about 20 mM. In some embodiments of any aspect, the IL is at a concentration of at least 25 mM. In some embodiments of any aspect, the IL is at a concentration of at least about 25 mM. In some embodiments of any aspect, the IL is at a concentration of at least 50 mM. In some embodiments of any aspect, the IL is at a concentration of at least about 50 mM. In some embodiments of any aspect, the IL is at a concentration of at least 100 mM, 500 mM, 1 M, 2 M, 3 M or more. In some embodiments of any aspect, the IL is at a concentration of at least about 100 mM, 500 mM, 1 M, 2 M, 3 M or more.

[0086] In some embodiments of any aspect, the IL is at a concentration of about 50 mM to about 4 M. In some embodiments of any aspect, the IL is at a concentration of 50 mM to 4 M. In some embodiments of any aspect, the IL is at a concentration of about 500 mM to about 4 M. In some embodiments of any aspect, the IL is at a concentration of 500 mM to 4 M. In some embodiments of any aspect, the IL is at a concentration of about 1 M to about 4 M. In some embodiments of any aspect, the IL is at a concentration of 1 M to 4 M. In some embodiments of any aspect, the IL is at a concentration of about 2 M to about 4 M. In some embodiments of any aspect, the IL is at a concentration of 2 M to 4 M.

[0087] In some embodiments of any aspect, the IL concentration in the composition or formulation is about 0.1 mM to 20 mM. In some embodiments of any aspect, the IL concentration in the composition or formulation is about 0.5 mM to 20 mM, 0.5 mM to 18 mM, 0.5 mM to 16 mM, 0.5 mM to 14 mM, 0.5 mM to 12 mM, 0.5 mM to 10 mM, 0.5 mM to 8 mM, 1 mM to 20 mM, 1 mM to 18 mM, 1 mM to 16 mM, 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 M, 10 mM to 16 mM, 10 mM to 14 mM, 10 mM to 12 mM, 12 mM to 20 mM, 12 mM to 18 mM, 12 mM to 16 mM, 12 mM to 14 mM, 14 mM to 20 mM, 14 mM to 18 mM, 14 mM to 16 mM, 16 mM to 20 mM, 16 mM to 18 mM, or 18 mM to 20 mM. In some embodiments of any aspect, the IL concentration in the composition or formulation is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM.

[0088] It is specifically contemplated that the compositions or combinations described herein can include one, two, three, or more of any type of component described herein. For example, a composition can include a mixture, solution, combination, or emulsion of multiple different ionic liquids (e.g., different ionic liquids described herein), and / or a mixture, solution, combination, or emulsion of multiple different nonionic surfactants, and / or a mixture, solution, combination, or emulsion of multiple different active compounds.

[0089] In some embodiments of any aspect, one or more ILs can be combined with at least one compound. As used herein, "combined with" refers to two or more substances present in the same formulation, such as in an admixture, solution, mixture, suspension, colloid, or emulsion, in any molecular or physical arrangement. The formulation can be a homogeneous or heterogeneous mixture. In some embodiments of any aspect, the active compound can be contained in a solution, mixture, admixture, suspension, or the like with the IL by a superstructure, such as a nanoparticle, liposome, vector, cell, scaffold, or the like.

[0090] As used herein, an "active compound" or "active agent" is any agent that has an effect on a target cell or target organism. The terms "compound" and "agent" refer to any entity that is not normally present or present at levels administered and / or provided to a cell, tissue, or subject. The agent can be selected from a group including: chemicals; small organic or inorganic molecules; signaling molecules; nucleic acid sequences; nucleic acid analogs; proteins; peptides; enzymes; aptamers; peptidomimetics, peptide derivatives, peptide analogs, antibodies; intrabodies; biopolymers, extracts made from biological materials such as bacterial, plant, fungal, or animal cells or tissues; natural or synthetic compositions or functional fragments thereof. In some embodiments, the agent is any chemical, entity, or moiety, including, but not limited to, synthetic and natural non-proteinaceous entities. The agent can be known to have a desired activity and / or property, or can be selected from a library of diverse compounds. Non-limiting examples of active compounds contemplated for use in the methods described herein include small molecules, polypeptides, nucleic acids, chemotherapeutic compounds, antibodies, antibody reagents, vaccines, GLP-1 polypeptides or mimetics / analogs thereof, insulin, acarbose, or ruxolitinib.

[0091] As described herein, nucleic acid molecule can be vector, expression vector, inhibitory nucleic acid, aptamer, template molecule or cassette (for example, for gene editing), or targeting molecule (for example, for CRISPR-Cas technology), or any other nucleic acid molecule that is desired to be delivered to cell.Nucleic acid molecule can be RNA, DNA, or their synthetic or modified version.In some embodiments of any aspect, nucleic acid is inhibitory nucleic acid, for example siRNA.

[0092] In one aspect of any of the embodiments, described herein is a method of delivering a nucleic acid molecule to a cell, comprising contacting the cell with a nucleic acid molecule in combination with one or more ILs as described herein. In some embodiments of any of the aspects, the cell is a cell in a subject, and the contacting comprises administering the nucleic acid molecule in combination with one or more ILs to the subject. In some embodiments of any of the aspects, the cell is in vitro, in vivo, or ex vivo. In some embodiments of any of the aspects, the cell is a eukaryotic organism. In some embodiments of any of the aspects, the cell is a mammalian cell. In some embodiments of any of the aspects, the cell is an epithelial cell, e.g., an intestinal epithelial cell. In some embodiments of any of the aspects, the cell is an epithelial cell.

[0093] In some embodiments of any aspect where the active compound comprises a nucleic acid, 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 that contains an aliphatic chain of 4 carbons or less; or c) an aromatic anion. In some embodiments of any aspect where the active compound comprises a nucleic acid, the anion has a LogP of less than 1.0 and is an aromatic anion. In some embodiments of any aspect where the active compound comprises a nucleic acid, the anion is an aromatic anion.

[0094] As used herein, the term "small molecule" refers to a chemical agent that may include, but is not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds having a molecular weight of less than about 10,000 grams per mole (i.e., including heteroorganic and organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0095] In some embodiments of any aspect, the active compound can be a therapeutic compound or drug, for example, an agent or compound that is therapeutically effective for treating at least one condition in a subject. Therapeutic compounds are known in the art for various conditions, see, for example, the database available on the web at drugs.com or the catalog of FDA-approved compounds available on the web at catalog.data.gov / dataset / drugsfda-database; each of which is incorporated herein by reference in its entirety.

[0096] By way of non-limiting example, exemplary antibodies and / or antibody reagents suitable for use as active compounds / therapeutic compounds herein include: abciximab; 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 tiuxetan; idarucizumab; infliximab; infliximab-abda; infliximab-dyyb; ipilimumab; ixekizumab; mepolizumab; natalizumab; necitumumab; nivolumab; obiltoxin Mabs; obinutuzumab; ocrelizumab; ofatumumab; olaratumab; omalizumab; palivizumab; panitumumab; pembrolizumab; pertuzumab; ramucriumab; ranibizumab; raxibacumab; reslizumab; rituximab; secukinumab; siltuximab; tocilizumab; trastuzumab; ustekinumab; vedolizumab; sarilumab; guselkumab; inotuzumab ozogamicin; inotuzumab; adalimumab-adbm, gemtuzumab ozogamicin; gemtuzumab; bevacizumab-awwb; bevacizumab inralizumab; emicizumab; emicizumab-kxwh; trastuzumab-dkst; infliximab-qbtx; ibalizumab; ibalizumab-uiyk; tildrakizumab; tildrakizumab-asmn; burosumab; burosumab-twza; erenumab; erenumab-aooe; tositumomab; mogamulizumab; moxetumomab; moxetumomab-pasudotox; cemiplimab; polatuzumab; catumaxomab; polatuzumab vedotin; and combinations thereof, including bispecific antibodies made by combining portions of the foregoing.

[0097] By way of non-limiting example, exemplary inhibitory nucleic acids suitable for use as active / therapeutic compounds herein include: patisiran; and combinations thereof, including bispecific antibodies made by combining portions of the foregoing.

[0098] As used herein, the term "chemotherapeutic agent" refers to any chemical or biological agent that has therapeutic utility in treating diseases characterized by abnormal cell proliferation. Such diseases include tumors, neoplasms, and cancers, as well as diseases characterized by hyperplastic growth. These agents may function to inhibit cellular activities on which cancer cells depend for continued growth. In some aspects of all embodiments, the chemotherapeutic agent is a cell cycle inhibitor or cytostatic agent. Categories of chemotherapeutic agents useful in the methods of the present invention include alkylating / alkaloid agents, antimetabolites, hormones or hormone analogs, and a wide variety of antitumor drugs. Most of these agents are directly or indirectly toxic to cancer cells. In one embodiment, the chemotherapeutic agent is a radioactive molecule.

[0099] In some embodiments of either aspect, the active compound is a polypeptide. In some embodiments of either aspect, the active compound is an antibody or an antibody reagent. As used herein, the term "antibody reagent" refers to a polypeptide that comprises at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds 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 comprises 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 embodiments of any aspect wherein the active compound comprises a polypeptide (e.g., an antibody or antibody reagent), 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 that contains an aliphatic chain of four carbons or less; or c) an aromatic anion. In some embodiments of any aspect wherein the active compound comprises a polypeptide (e.g., an antibody or antibody reagent), 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 that contains an aliphatic chain of four carbons or less. In some embodiments of any aspect wherein the active compound comprises a polypeptide (e.g., an antibody or antibody reagent), the anion has a LogP of less than 1.0 and is ... is not a fatty acid; or b) a carboxylic acid that contains an aliphatic chain of four carbons or less. In some embodiments of any aspect wherein the active compound comprises a polypeptide (e.g., an antibody or antibody reagent), the anion has a LogP of less than 1.0.

[0101] In some embodiments of any aspect, the active compound has a molecular weight greater than about 450. In some embodiments of any aspect, the active compound has a molecular weight greater than about 500. In some embodiments of any aspect, the active compound has a molecular weight greater than 450, e.g., greater than 450, greater than 500, greater than 550, greater than 600, greater than 1000, or more. In some embodiments of any aspect, the active compound is polar.

[0102] In some embodiments of any aspect where the active agent is an inhibitory nucleic acid, the composition comprises a plurality of ionic liquids, and a first ionic liquid is choline and geranic acid (CAGE). In some embodiments of any aspect where the active agent is an inhibitory nucleic acid, the composition comprises a plurality of ionic liquids, and a second ionic liquid is choline and dimethylacrylic acid (CADA); choline and isovaleric acid (CAVA); choline and phenylphosphoric acid (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 where the active agent is an inhibitory nucleic acid, the composition comprises a plurality of ionic liquids, and 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 phenylphosphoric 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 where the active agent is an inhibitory nucleic acid, the composition comprises a plurality of ionic liquids, and the first ionic liquid is selected from the group consisting of choline and geranic acid (CAGE), choline and dimethylacrylic acid (CADA), 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 where the active agent is an inhibitory nucleic acid, the composition comprises a plurality of ionic liquids, wherein a first ionic liquid is choline and geranic acid (CAGE) and a second ionic liquid is 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, binds to NFKBIZ mRNA and inhibits the expression of NFKBIZ.As used herein, "NFKBIZ" or "NFKB inhibitor zeta" refers to the inhibitor of nuclear factor κB (IκB) protein IκBζ, which plays an important role in the regulation of 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 signal transduction, neutrophil chemotaxis and leukocyte activation.Therefore, provided herein is a method for treating psoriasis, for example, by administering a composition described herein, which comprises an active agent that is an NFKBIZ inhibitor, for example, an NFKBIZ inhibitory nucleic acid. The sequences of NFKBIZ from several species are known in the art, for example, the human NFKBIZ sequence is available in the NCBI database under 64332 Gene ID (e.g., mRNAs NM_001005474.3 (SEQ ID NO: 37) and NM_031419.4 (SEQ ID NO: 38)). One skilled in the art can easily design NFKBIZ inhibitory nucleic acids, for example, using the automated tools described hereinabove. 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, e.g., binds to TNF-α mRNA and inhibits expression of TNF-α. As used herein, "tumor necrosis factor α" or "TNF-α" refers to a pro-inflammatory cytokine implicated in autoimmune diseases, psoriasis, and other conditions. Accordingly, provided herein are methods of treating inflammatory conditions (e.g., psoriasis) and / or reducing or inhibiting inflammation, e.g., by administering a composition described herein that includes an active agent that is an inhibitor of TNF-α, 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)). One skilled in the art can readily design TNF-α inhibitory nucleic acids, for example, using the automated tools described herein above. 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, e.g., binds to IL-17 mRNA and inhibits expression of IL-17. As used herein, "interleukin-17" or "IL-17" refers to a proinflammatory cytokine produced by activating T cells that has been implicated in autoimmune diseases, psoriasis, rheumatoid arthritis, multiple sclerosis, and other conditions. Accordingly, provided herein are methods of treating inflammatory conditions (e.g., psoriasis) and / or reducing or inhibiting inflammation, e.g., by administering a composition described herein that includes an active agent that is an inhibitor of IL-17, e.g., an IL-17 inhibitory nucleic acid. IL-17 sequences 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)). One of skill in the art can readily design IL-17 inhibitory nucleic acids, e.g., using automated tools described herein above. 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 embodiment, provided herein is a method of treating an inflammatory condition and / or reducing inflammation in a subject in need thereof, comprising administering to the subject a composition described herein comprising at least one IL and at least one anti-inflammatory agent. In some embodiments of any aspect, the anti-inflammatory agent is an inhibitory nucleic acid that targets 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 harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by organisms to eliminate harmful stimuli and initiate tissue healing processes. Thus, the term "inflammation" includes any cellular process that results from the production of inflammatory cytokines, inflammatory mediators, and / or the action of the cytokines so produced, leading to associated downstream cellular events, such as fever, fluid accumulation, swelling, abscess formation, and cell death. 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 cell types involved. Acute inflammation often involves polymorphonuclear neutrophils; chronic inflammation is usually characterized by lymphohistiocytic and / or granulomatous responses.

[0109] Inflammatory conditions are any disease conditions characterized by inflammatory tissue (e.g., infiltrates of leukocytes such as lymphocytes, neutrophils, macrophages, eosinophils, mast cells, basophils, and dendritic cells) or inflammatory processes that induce or contribute to the abnormal clinical and histological characteristics of disease conditions.Inflammatory conditions include, but are not limited to, inflammatory conditions of the skin, inflammatory conditions of the lungs, inflammatory conditions of the joints, inflammatory conditions of the intestines, inflammatory conditions of the eye, inflammatory conditions of the endocrine system, inflammatory conditions of the cardiovascular system, inflammatory conditions of the kidneys, inflammatory conditions of the liver, inflammatory conditions of the central nervous system, or sepsis-related conditions.In some embodiments, the inflammatory conditions are associated with wound healing.In some embodiments, the inflammation treated according to the methods described herein can be skin inflammation; inflammation caused by substance abuse or drug dependence; inflammation associated with infection; inflammation of the cornea; inflammation of the retina; inflammation of the spinal cord; inflammation associated with organ regeneration; and pulmonary inflammation.

[0110] In some embodiments, the inflammatory condition is an inflammatory condition of the skin. In some embodiments of this aspect, the inflammatory condition is an autoimmune disease.

[0111] Non-limiting examples of inflammatory skin conditions include psoriasis, e.g., Sweet's syndrome, pyoderma gangrenosum, subcorneal pustular dermatitis, erythema elevatum, Behcet's disease or acute generalized exanthematous pustulosis, bullous disorders, psoriasis, conditions that produce pustular lesions, acne, acne vulgaris, dermatitis (e.g., contact dermatitis, atopic dermatitis, seborrheic dermatitis, eczematous dermatitis, fissure eczema), and the like. These may include eczema, ulcers and erosions due to 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 due to mechanical shearing of the skin, skin atrophy due to topical use of corticosteroids, and inflammation of the mucous membranes (e.g., cheilitis, chapped lips, nasal irritation, mucositis, and vulvovaginitis).

[0112] In some embodiments, the inflammatory condition can be an autoimmune disease. Non-limiting examples of autoimmune diseases can include type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, inflammatory bowel disease, Crohn's disease, and autoimmune thyroiditis.

[0113] By way of non-limiting example, the inflammatory condition may be a pulmonary inflammatory condition, such as asthma, bronchitis, chronic bronchitis, bronchiolitis, pneumonia, sinusitis, emphysema, adult respiratory distress syndrome, pulmonary inflammation, pulmonary fibrosis, and cystic fibrosis (which may additionally or alternatively involve the gastrointestinal tract or other tissues). By way of non-limiting example, the inflammatory condition may be a joint inflammatory condition, such as rheumatoid arthritis, rheumatoid spondylitis, juvenile rheumatoid arthritis, osteoarthritis, gouty arthritis, infectious arthritis, psoriatic arthritis, and other arthritic conditions. By way of non-limiting example, the inflammatory condition may be a gastrointestinal or intestinal inflammatory condition, such as inflammatory bowel disease, Crohn's disease, ulcerative colitis, and distal proctitis. By way of non-limiting example, the inflammatory condition may be an ocular inflammatory condition, such as dry eye syndrome, uveitis (including iritis), conjunctivitis, scleritis, and keratoconjunctivitis sicca. Non-limiting examples of inflammatory conditions include inflammatory conditions of the endocrine system, such as autoimmune thyroiditis (Hashimoto's disease), Graves' disease, type I diabetes, and acute and chronic inflammation of the adrenal cortex. Non-limiting examples of inflammatory conditions include inflammatory conditions of the cardiovascular system, such as coronary artery infarction injury, peripheral vascular disease, myocarditis, vasculitis, revascularization of stenosis, atherosclerosis, and vascular disease associated with type II diabetes. Non-limiting examples of inflammatory conditions include inflammatory conditions of the kidney, 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. Non-limiting examples of inflammatory conditions include inflammatory conditions of the liver, such as hepatitis (arising from viral infection, autoimmune response, drug treatment, toxins, environmental factors, or secondary to primary disorders), biliary atresia, primary biliary cirrhosis, and primary sclerosing cholangitis. By way of non-limiting example, the inflammatory condition may be an inflammatory condition of the central nervous system, for example, multiple sclerosis and neurodegenerative diseases such as Alzheimer's disease or dementia associated with HIV infection.By way of non-limiting example, the inflammatory condition may be an inflammatory condition of the central nervous system, such as MS; all types of encephalitis and meningitis; acute disseminated encephalomyelitis; acute transverse myelitis; neuromyelitis optica; focal demyelinating syndromes (e.g., Barrault concentric sclerosis and the Marburg variant of MS); progressive multifocal leukoencephalopathy; subacute sclerosing panencephalitis; acute hemorrhagic leukoencephalitis (Hurst disease); human T-lymphotropic 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 neuropathies; and myasthenia gravis. By way of non-limiting example, the inflammatory condition may be a sepsis-related condition, such as systemic inflammatory response syndrome (SIRS), septic shock, or multiple organ dysfunction syndrome (MODS).Further non-limiting examples of inflammatory conditions include endotoxic shock, periodontal disease, polychondritis; periarticular disorders; pancreatitis; 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's 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 thyroiditis), pemphigus, pernicious anemia, canine polyarthritis, 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; fibrosing alveolitis, hypersensitivity pneumonitis, allergic aspergillosis, idiopathic pulmonary eosinophilia, bronchiolitis obliterans matrix pneumonia; urticaria; lupoid hepatitis; familial common cold autoinflammatory syndrome, Muckle-Wells syndrome, neonatal-onset multisystem inflammatory disease, transplant rejection (including allograft rejection and graft-versus-host disease), otitis, chronic obstructive pulmonary disease, sinusitis, chronic prostatitis, reperfusion injury, silicosis, inflammatory myopathies, hypersensitivity reactions, and migraine. In some embodiments, the inflammatory condition is associated with an infection, e.g., a viral, bacterial, fungal, parasitic, or prion infection. In some embodiments, the inflammatory condition is associated with an allergic reaction. In some embodiments, the inflammatory condition is associated with pollutants (e.g., asbestosis, silicosis, or beryllium disease).

[0114] In some embodiments, the inflammatory condition can be a localized condition, such as a rash or an allergic reaction. In some embodiments, the inflammation is associated with a wound.

[0115] Anti-inflammatory agents are known in the art, and non-limiting examples may include nonsteroidal 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 drugs; cyclophosphamide; pro-resolving drugs; mycophenolate; or opiates (e.g., endorphins, enkephalins, and dynorphins), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and inhibitors of inflammatory gene products (e.g., the inhibitory nucleic acids described hereinabove). Inflammatory genes are known in the art and include, by way of non-limiting example, 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, unless otherwise specified, a "composition" refers to any IL, combination of ILs, or combination of one or more ILs with one or more active agents described herein.

[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 mucosal membranes, such as the nasal, oral, or vaginal membranes. In some embodiments of any aspect, the oral formulation can be a degradable capsule containing a composition comprising at least one IL and optionally an active compound.

[0118] In some embodiments of any aspect, described herein are compositions comprising at least one IL and at least one active compound described herein. In some embodiments of any aspect, described herein are compositions consisting essentially of at least one IL and at least one active compound described herein. In some embodiments of any aspect, described herein are compositions consisting of at least one IL and at least one active compound described herein. In some embodiments of any aspect, a composition comprising at least one IL and at least one active compound described herein is administered as monotherapy, e.g., no other treatment for the condition is administered to the subject.

[0119] In one aspect of any embodiment, described herein are pharmaceutical compositions 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 described herein and one or more active compounds. In some embodiments, the pharmaceutical composition consists essentially of at least one IL described herein and one or more active compounds. In some embodiments, the pharmaceutical composition consists of at least one IL described herein and one or more active compounds. In some embodiments, the pharmaceutical composition consists essentially of an aqueous solution of at least one IL described herein and one or more active compounds. In some embodiments, the pharmaceutical composition consists of an aqueous solution of at least one IL described herein and one or more active compounds.

[0120] The compositions, formulations, and combinations described herein can include at least one IL described herein, e.g., one IL, two ILs, three ILs, or more. In some embodiments of any aspect, the compositions, formulations, or combinations described herein can include at least one IL described herein and CAGE (choline and geranate).

[0121] In some embodiments of any aspect, the 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 appreciably separate in aqueous media. The properties of nonionic surfactants depend largely on the ratio of hydrophilic to hydrophobic groups in the molecule. Hydrophilic groups include oxyethylene groups (--OCH2CH2--) and hydroxy groups. By varying the number of these groups in hydrophobic molecules such as fatty acids, a range of substances can be obtained, from strongly hydrophobic and water-insoluble compounds such as glyceryl monostearate to strongly hydrophilic and water-soluble compounds such as macrogol. Between these two extremes are compounds with a more evenly balanced ratio of hydrophilic and hydrophobic groups, such as macrogol esters and ethers and sorbitan derivatives. Suitable non-ionic surfactants can be found in Martindale, The Extra Pharmacopoeia, 28th Edition, 1982, The Pharmaceutical Press, London, Great Britain, pp. 370 to 379.Non-limiting examples of nonionic surfactants include polysorbates, Tween™, block copolymers of ethylene oxide and propylene oxide, glycol and glyceryl esters of fatty acids and their derivatives, polyoxyethylene esters of fatty acids (macrogol esters), polyoxyethylene ethers of fatty acids and their derivatives (macrogol ethers), polyvinyl alcohol, and sorbitan esters, sorbitan monoesters, ethers formed from fatty alcohols and polyethylene glycols, polyoxyethylene-polypropylene glycols, alkyl polyglycosides, cetomacrogol 1000, cetostearyl alcohol, cetyl alcohol, cocamide DEA, cocamide MEA, decyl glucoside, decyl polyglucose, glycerol monostearate, IGEPAL CA-630, isoceteth-20, lauryl glucoside, maltoside, monolaurin, mycosubtilin, 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 tallow amine, polyglycerol polyricinoleate, sorbitan, sorbitan monolaurate, sorbitan monostearate, sorbitan tristearate, stearyl alcohol, surfactin, Triton X-100, etc. In some embodiments of any aspect, the 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 fatty acids. Common trade 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 embodiments of any aspect, the 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 embodiments of any aspect, the 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 embodiments of any aspect, the 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 embodiments of any aspect, the at least one nonionic surfactant (e.g., at least one polysorbate) is present at a concentration of 1% to 5% w / v. In some embodiments of any aspect, the 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 embodiments of any aspect, the at least one nonionic surfactant (e.g., at least one polysorbate) is present at a concentration of 3% to 10% w / v. In some embodiments of any aspect, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of less than about 5% w / v. In some embodiments of any aspect, the at least one non-ionic surfactant (e.g., at least one polysorbate) is present at a concentration of less than 5% w / v.

[0124] In some embodiments of any aspect, the combination of at least one active compound and at least one IL described herein is provided in one or more nanoparticles. In some embodiments of any aspect, the combination of at least one active compound and at least one IL described herein comprises nanoparticles comprising the active compound, wherein the nanoparticles are in solution or suspension in a composition comprising at least one IL described herein.

[0125] In some embodiments of any aspect, the compositions described herein, e.g., compositions comprising at least one IL and an active compound, may further comprise a pharmaceutically acceptable carrier. As used herein, the terms "pharmaceutically acceptable" and "physiologically tolerable," and their grammatical variations, are used interchangeably when referring to compositions, carriers, diluents, and reagents, and indicate that the material can be administered to or on a mammal without producing undesirable physiological effects, such as nausea, dizziness, or stomach upset. A pharmaceutically acceptable carrier does not promote an immune response to the agent with which it is admixed, unless so desired. The preparation of pharmacological compositions containing active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared for injection as either a liquid solution or suspension, but may also be prepared in solid forms suitable for dissolution or suspension in liquid prior to use. The preparation may also be emulsified or presented as a liposomal composition. The active ingredient can be mixed with pharmaceutically acceptable excipients compatible with the active ingredient in amounts appropriate for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient. The therapeutic compositions of the present disclosure may contain pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, tartaric acid, mandelic acid, and the like (formed with the free amino groups of the polypeptide). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Physiologically tolerable carriers are well known in the art.Exemplary liquid carriers are sterile aqueous solutions containing no materials in addition to the active ingredient and water, or buffers such as sodium phosphate, physiological saline, or both, e.g., phosphate-buffered saline, at physiological pH values. Additionally, aqueous carriers can contain multiple buffer salts and salts such as sodium and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent used in the methods described herein that will be effective in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field. For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of the active ingredient in 0.9% sodium chloride solution.

[0126] The term "carrier" in the context of a pharmaceutical carrier refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated as suppositories, using traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co., 1990). The formulation should suit the mode of administration.

[0127] Pharmaceutically acceptable carriers and diluents include physiological saline, aqueous buffer solutions, solvents and / or dispersion media.The use of such carriers and diluents is well known in the art.Some non-limiting examples of materials that can serve as pharmaceutically acceptable carriers include:(1) sugars such as lactose, glucose, and sucrose;(2) starches such as corn starch and potato starch;(3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate;(4) powdered tragacanth;(5) malt;(6) gelatin;(7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc;(8) excipients such as cocoa butter and suppository wax;(9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (24) C2-C3 ethanol; 12 Alcohol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient," "carrier," and "pharmaceutically acceptable carrier" are used interchangeably herein. In some embodiments, the carrier inhibits degradation of the active compound. The term "pharmaceutically acceptable carrier" excludes tissue culture medium.

[0128] In some embodiments of any aspect, the compositions described herein, e.g., 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 can be a degradable capsule containing a composition described herein, e.g., 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, the IL significantly enhances the permeability of the active compound through the skin compared to a control in the absence of the at least one IL.

[0130] In one aspect of any embodiment, described herein is a method of administering at least an active compound to a subject using a catheter coated with at least one IL described herein. In one aspect of any embodiment, described herein is a method of withdrawing bodily fluids by placing a catheter coated with at least one IL described herein within the body.

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

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

[0133] In some embodiments, the methods described herein relate to treating a subject who has or has been diagnosed with a condition with a composition described herein, for example, comprising at least one IL and an active compound. A subject with a condition, such as diabetes, can be identified by a physician using current 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. For example, tests that can be useful in diagnosing diabetes include, but are not limited to, blood tests (e.g., fasting glucose level). A family history of diabetes or exposure to risk factors for diabetes (e.g., being overweight) can also be useful in determining whether a subject is likely to have diabetes or in making a diagnosis of diabetes.

[0134] The compositions and methods described herein can be administered to a subject having or diagnosed with a condition described herein. In some embodiments, the methods described herein include administering to a subject an effective amount of a composition described herein, e.g., a composition comprising at least one IL and an active compound described herein, to alleviate the symptoms of a condition described herein. As used herein, "alleviating symptoms" refers to an improvement in any marker or symptom associated with the condition. Compared to an equivalent untreated control, such a 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, airway (aerosol), pulmonary, cutaneous, injection, or intratumoral administration. Administration can be local or systemic.

[0135] In some embodiments of any aspect, the administration is transdermal. In some embodiments of any aspect, the administration is transdermal, mucosal (e.g., nasal, oral, or vaginal) administration, oral, subcutaneous, intradermal, parenteral, intratumoral, or intravenous administration.

[0136] Oral administration may include providing a liquid such as, but not limited to, a tablet (including, but not limited to, scored or coated tablets), a pill, a caplet, a capsule, a chewable tablet, a powder packet, a cachet, a troche, a wafer, an aerosol spray, or a syrup, elixir, a solution, or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Oral formulations may include separate dosage forms such as, but not limited to, a liquid such as, but not limited to, a tablet (including, but not limited to, scored or coated tablets), a pill, a caplet, a capsule, a chewable tablet, a powder packet, a cachet, a troche, a wafer, an aerosol spray, or a syrup, elixir, a solution, or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of CAGE and at least one active compound and may be prepared by pharmaceutical methods well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia, PA. (2005).

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

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

[0139] Suitable vehicles that can be used to provide parenteral dosage forms of compositions containing at least one IL (e.g., CAGE) in combination with at least one active compound disclosed within the scope are well known to those skilled in the art. Examples include, but are not limited to: sterile water; USP Water for Injection; saline; glucose solution; aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of ingredients in the compositions disclosed herein can also be incorporated into the parenteral dosage forms of the present disclosure, including regular 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 variations in drug concentrations in a patient's blood and other tissues. These variations can affect several parameters, such as administration frequency, onset of action, duration of effectiveness, maintenance of therapeutic blood levels, toxicity, and side effects. As previously described 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 contemplated herein that the methods and compositions may be utilized in controlled-release formulations in some embodiments. For example, controlled-release formulations can be used to control the drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or sustained-release dosage forms or formulations can be used to ensure maximum drug efficacy while minimizing potential side effects and safety concerns that can arise from both underdosing (i.e., below the minimum therapeutic level) and exceeding toxic drug levels. In some embodiments, compositions comprising at least one IL in combination with at least one active compound can be administered in a sustained-release formulation.

[0141] Controlled-release pharmaceutical products share a common goal: improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of optimally designed controlled-release preparations in medical treatment is characterized by the use of a minimum amount of drug substance to cure or control a medical condition in a minimal amount of time. Advantages of controlled-release formulations include: 1) extended drug activity; 2) reduced dosing frequency; 3) improved patient compliance; 4) less total drug use; 5) reduced local or systemic side effects; 6) minimized drug accumulation; 7) reduced blood level fluctuations; 8) improved therapeutic efficacy; 9) reduced synergism or loss of drug activity; and 10) improved rate of disease or condition control. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0142] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that immediately produces the desired therapeutic effect, and then gradually and continuously release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. To maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that replaces the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions, including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0143] Various known controlled-release or sustained-release dosage forms, formulations and devices can be adapted for use with the salts and compositions of the present disclosure.Examples include but are not limited to those described in U.S. Patent No. 3,845,770; U.S. Patent No. 3,916,899; U.S. Patent No. 3,536,809; U.S. Patent No. 3,598,123; U.S. Patent No. 4,008,719; U.S. Patent No. 5,674,533; U.S. Patent No. 5,059,595; U.S. Patent No. 5,591,767; U.S. Patent No. 5,120,548; U.S. Patent No. 5,073,543; U.S. Patent No. 5,639,476; U.S. Patent No. 5,354,556; U.S. Patent No. 5,733,566; and U.S. Patent No. 6,365,185 B1, each of which is incorporated herein by reference. These dosage forms can be used to provide sustained or controlled release of one or more active ingredients using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or combinations thereof to provide desired release profiles at various rates.

[0144] The term "effective amount" as used herein refers to the amount of a composition required to alleviate at least one or more symptoms of a disease or disorder, and relates to a sufficient amount of a pharmacological composition to produce a desired effect. Thus, the term "therapeutically effective amount" refers to an amount of a composition sufficient to produce a specific effect when administered to a typical subject. As used herein, the term "effective amount" also includes, in various contexts, an amount sufficient to delay the onset of disease symptoms, alter the course of disease symptoms (for example, but not limited to, delaying the progression of disease symptoms), or reverse disease symptoms. Therefore, it is generally not feasible to specify an exact "effective amount." However, for any given case, one skilled in the art can determine an appropriate "effective amount" using only routine experimentation.

[0145] Effective doses, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Dosages can vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. The therapeutically effective dose can be initially estimated from cell culture assays. Alternatively, doses can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of active compound that achieves a half-maximal inhibition of symptoms) determined in cell culture or a suitable animal model. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dosage can be monitored by appropriate bioassays, such as blood glucose assays, among others. Dosages can be determined by a physician and can be adjusted, as necessary, to achieve the observed therapeutic effect.

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

[0147] Glucagon-like peptide-1 (GLP-1) is an incretin derived from the transcription product of the proglucagon gene, which is known to reduce food intake and hunger in humans and contributes to glucose homeostasis. GLP-1 mimetics are currently used to treat type 2 diabetes. Recent clinical trials have shown that these treatments not only improve glucose homeostasis but also successfully induce weight loss. As used herein, "GLP-1 polypeptide" refers to various pre- and pro-peptides and cleavage products of GLP-1, such as GLP-1(1-37) (SEQ ID NO: 2), GLP-1(7-36) (SEQ ID NO: 3), and GLP-1(7-37) (SEQ ID NO: 4) in humans. In some embodiments, the GLP-1 polypeptide can be GLP-1(7-36) and / or GLP-1(7-37) or related polypeptides derived from species other than humans. GLP-1 polypeptide sequences are known in the art for numerous species, such as human GLP-1 (NCBI Gene ID: 2641) polypeptide (e.g., NCBI Ref Seq: NP_002045.1; SEQ ID NO: 1) and SEQ ID NOs: 2-4. In some embodiments, pre- or pro-peptides of GLP-1, such as glucagon preproprotein (e.g., SEQ ID NO: 1), can be used in the methods or compositions described herein. Naturally occurring alleles or variants of any of the polypeptides described herein are also specifically contemplated for use in the methods and compositions described herein.

[0148] SEQ ID NO: 1 TIFF2026021383000011.tif13128SEQ ID NO: 2 TIFF2026021383000012.tif4128SEQ ID NO: 3 TIFF2026021383000013.tif4128SEQ ID NO: 4 TIFF2026021383000014.tif4128

[0149] A variety of GLP-1 mimetics are known in the art and are used for the treatment of diabetes.GLP-1 mimetics (or analogues) can include exendin-4 (Heloderma lizard polypeptide with human GLP-1 homology) and its derivatives, the GLP-1 analogues modified to be DPP-IV resistant, or the human GLP-1 polypeptides conjugated with various additional active substances, for example, to extend half-life.GLP-1 mimetics / analogues can include, for example, exenatide, lixisenatide, dulaglutide, semaglutide, albiglutide, LY2189265, liraglutide and taspoglutide. Examples of such molecules and further discussion of their production and activity can be found in the art, for example, in Gupta. Indian J. Endocrinol Metab 17:413-421 (2013); Garber. Diabetes Treatments 41:S279-S284 (2018); U.S. Patent Publication No. US 2009 / 0181912; and International Patent Publication No. WO 2011 / 080103, each of which is incorporated herein by reference in its entirety.

[0150] In some embodiments of either aspect, the active compound can be a chemotherapeutic agent or an agent effective in treating cancer. As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide uncontrollably and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymphatic system. There are several major types of cancer. Carcinomas are cancers that develop in the tissues that line or cover the skin or internal organs. Non-carcinomas are cancers that develop in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissues. Leukemia is cancer that develops in blood-forming tissues, such as the bone marrow, causing large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that develop in cells of the immune system. Central nervous system cancers are cancers that develop in the tissues of the brain and spinal cord.

[0151] In some embodiments of either aspect, the cancer is a primary cancer. In some embodiments of either aspect, the cancer is a malignant cancer. As used herein, the term "malignant" refers to a cancer in which tumor cells exhibit one or more of uncontrolled proliferation (i.e., division beyond normal limits), invasion (i.e., invading and destroying adjacent tissues), and metastasis (i.e., spreading to other parts of the body via the lymphatics or blood). As used herein, the term "metastasizing" refers to the spread of cancer from one part of the body to another. A tumor formed by spread cells is called a "metastatic tumor" or "metastatic cancer." A metastatic tumor contains cells similar to cells in the original (primary) tumor. As used herein, the terms "benign" or "non-malignant" refer to a tumor that may grow larger but does not spread to other parts of the body. Benign tumors are self-limited and usually do not invade or metastasize.

[0152] "Cancer cell" or "tumor cell" refers to an individual cell of a cancerous growth or tissue. A tumor generally refers to a swelling or lesion formed by abnormal cell proliferation and may be benign, premalignant, or malignant. Most cancer cells form tumors, but some, such as leukemia, do not necessarily form tumors. For tumor-forming cancer cells, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0153] As used herein, the term "neoplasm" refers to any new and abnormal growth of tissue, e.g., an abnormal tissue mass whose growth is in excess of and uncoordinated with that of normal tissue. Thus, a neoplasm can be a benign neoplasm, a premalignant neoplasm, or a malignant neoplasm.

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

[0155] Examples of cancer include carcinoma, lymphoma, blastoma, non-cancerous carcinoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; stomach cancer (including gastrointestinal cancer); glioblastoma (GBM); liver cancer; hepatocellular carcinoma; intraepithelial neoplasia; kidney or renal carcinoma; Laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; seminal follicular carcinoma; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; and other epithelial and non-epithelial malignancies; and B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; and bulky mass disease NHL) ; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phacomatosis, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0156] "Cancer cells" are cancerous, precancerous, or transformed cells, either in vivo, ex vivo, or in tissue culture, that undergo spontaneous or induced phenotypic changes that do not necessarily involve the incorporation of new genetic material. Transformation can result from infection with a transforming virus and the incorporation of new genomic nucleic acid, or from the incorporation of exogenous nucleic acid, but can also result from spontaneous or subsequent exposure to carcinogens, resulting in mutations of endogenous genes. Transformation / cancer is associated with morphological changes, cellular immortalization, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor-specific markers, invasiveness or metastasis, and tumor growth in a suitable animal host, such as nude mice.

[0157] In some embodiments of either aspect, a composition described herein, e.g., a composition comprising at least one IL described herein in combination with at least one active compound, is administered as a monotherapy, e.g., in the absence of another treatment for the condition.

[0158] In some embodiments of any aspect, the methods described herein may further include administering a second agent and / or treatment to the subject, either in a composition described herein, e.g., a composition comprising at least one IL described herein in combination with at least one active compound, or as a separate formulation, e.g., as part of a combination therapy. For example, non-limiting examples of second agents and / or treatments for cancer treatment include radiation therapy, surgery, gemcitabine, cisplatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metuledopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); ostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictine; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, fenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Enediyne antibiotics (e.g., antibiotics such as the calicheamicins, particularly calicheamicin gamma 1I and calicheamicin omega 1I (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, Detrubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin , lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, and doxif Pyrimidine analogues such as uridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziconazole;Elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including treatment regimens of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;Leucovorin (LV); oxaliplatin, including oxaliplatin treatment regimens (FOLFOX); lapatinib (Tykerb.RTM.); PKC-alpha inhibitors that reduce cell proliferation, Raf inhibitors, H-Ras inhibitors, EGFR inhibitors (e.g., erlotinib (Tarceva®)), and VEGF-A inhibitors, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above. In addition, the treatment method may further include the use of radiation or radiotherapy. Furthermore, the treatment method may further include the use of surgical treatment.

[0159] In certain embodiments, an effective dose of a composition described herein, e.g., 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 a composition described herein, e.g., a composition comprising at least one IL described herein in combination with at least one active compound, can be administered to a patient multiple times. For systemic administration, a subject can receive a therapeutic amount of a composition described herein, e.g., a composition comprising at least one IL described herein in combination with at least one active compound, e.g., 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, the at least one active compound is present in the combination at a dose of about 1.0 to 40.0 mg / kg. In some embodiments of any aspect, at least one active compound is present in the combination at a dose of 1.0 to 40.0 mg / kg. In some embodiments of any aspect, at least one active compound is present in the combination at a dose of about 1.0 to 20.0 mg / kg. In some embodiments of any aspect, 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 dosage can be about 1 U / kg to about 20 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be 1 U / kg to 20 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be less than 20 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be about 2 U / kg to about 10 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be 2 U / kg to 10 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be about 2 U / kg to about 5 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be 2 U / kg to 5 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be about 5 U / kg to about 10 U / kg. In some embodiments, the active compound is insulin, and the insulin concentration or dosage can be 5 U / kg to 10 U / kg. In some embodiments, the active compound is insulin, and the concentration or dosage of insulin can be 2 U / kg, 5 U / kg, or 10 U / kg.

[0161] In one aspect of any embodiment, described herein is a method of treating a disease in a subject in need thereof by administering to the subject an active compound in combination with at least one IL described herein via injection into the affected tissue. In some embodiments, the diseased tissue is a tissue containing diseased cells. In some embodiments, the diseased tissue is a tissue that exhibits symptoms of a disease. Non-limiting examples of suitable diseased tissues include tumor tissue, fat tissue, adipose tissue, etc. In some embodiments of any aspect, the disease is a disease caused by tissue proliferation, e.g., unwanted, abnormal, or pathological tissue proliferation. A disease caused by tissue proliferation can be any disease caused by or characterized by a rate of tissue proliferation, location of tissue proliferation, or pattern / structure of tissue proliferation that differs from that normal for that type of tissue in a healthy subject. Non-limiting examples of such diseases are tumors, cancer, adiposity / obesity, and / or hyperplasia. In some embodiments of any aspect, such a disease is a tumor, cancer, adiposity / obesity, and / or hyperplasia.

[0162] In some embodiments, after the initial treatment regimen, treatment can be administered less frequently. For example, after 3 months of treatment every other week, treatment can be repeated once a month for 6 months or a year or more. Treatment according to the methods described herein can reduce the level of markers or symptoms of a 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 can be adjusted accordingly depending on the observed therapeutic effect. Regarding the duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine when the treatment provides therapeutic benefit and determine whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment plan. The dosing schedule can vary from once a week to daily, depending on several clinical factors, such as the subject's sensitivity to the active compound. The desired dose or amount of the active substance can be administered once, or divided into subdoses, e.g., two to four subdoses, and administered over a period of time, e.g., at appropriate intervals throughout the day, or according to other suitable schedules. In some embodiments, administration can be chronic, e.g., with one or more doses and / or treatments daily for a period of several weeks or months. Exemplary dosing and / or treatment schedules are daily, twice daily, three times daily, or four or more times daily for a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more. The compositions described herein, e.g., compositions comprising at least one IL in combination with at least one active compound, can be administered over a period of time, e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes.

[0164] The dosage range of the compositions described herein for administration by the methods described herein depends, for example, on the form of active compound, its efficacy, and the degree of desired reduction of the symptoms, markers, or indicators of the conditions described herein, for example, the percentage of desired reduction of symptoms or markers.Dosage should not be so high as to cause adverse side effects.Generally, dosage varies according to the age, condition, and sex of patients, and can be determined by those skilled in the art.In the case of any complications, dosage can also be adjusted by individual physicians.

[0165] For example, the effectiveness of a composition described herein in treating a condition described herein or the effectiveness of a composition for inducing a response described herein can be determined by a skilled clinician. However, if one or more of the signs or symptoms of a condition described herein are modified in a beneficial manner, other clinically recognized symptoms are improved or even ameliorated, or a desired response is induced, for example, by at least 10% after treatment by a method described herein, as the term "effective treatment" is used herein, the treatment is considered "effective treatment." Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or incidence of the condition treated by a method described herein, or any other suitable measurable parameter. Efficacy can also be measured by the lack of deterioration of the individual, as assessed by hospitalization, or the lack of need for medical intervention (i.e., the progression of the disease has stopped). Methods for measuring these indicators are known to those skilled in the art and / or are described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or animals) and includes: (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of the disease, e.g., causing regression of symptoms. An effective amount for treating a disease means an amount that, when administered to a subject in need thereof, is sufficient to result in effective treatment, as the term effective treatment is defined herein, for that disease. The effectiveness of an agent can be determined by assessing physical indicators of the condition or desired response. It is well within the capabilities of one skilled in the art to monitor the effectiveness of administration and / or treatment by measuring any one or any combination of such parameters. Efficacy can be assessed in animal models of the conditions described herein, e.g., the treatment of diabetes or cancer. When using experimental animal models, the effectiveness of treatment is demonstrated when a statistically significant change in the marker is observed.

[0166] Provided herein are in vitro and animal model assays that allow for the evaluation of a given dose of the compositions described herein, e.g., compositions comprising at least one IL in combination with at least one active compound.

[0167] In some embodiments of any aspect, the subject to whom a composition comprising at least one IL described herein, for example, in combination with an active compound, is administered is a subject who has or has been diagnosed with obesity, excess weight, or who needs treatment for obesity, excess weight, or prevention of weight gain. In some embodiments, the subject is overweight. The methods described herein include methods for treating obesity, reducing weight gain, preventing weight gain, promoting weight loss, etc. Such methods can, for example, promote metabolic health and prepare patients for surgical interventions pursued for aesthetic reasons and / or contraindicated for patients with high BMI or weight. In some embodiments, for example, when a subject is overweight and / or obese, weight loss can be medically necessary and / or medically indicated. In some embodiments, for example, when a subject desires weight loss, regardless of whether weight loss is medically necessary and / or medically indicated, weight loss can be for cosmetic purposes.

[0168] The term "obesity" refers to excess body fat. Obesity can be determined by any measure recognized and utilized by those skilled in the art. Currently, the recognized measure of obesity is the body mass index (BMI), which is a measure of body weight in kilograms relative to the square of height in meters. Generally, for adults over the age of 20, a BMI between about 18.5 and 24.9 is considered normal, a BMI between about 25.0 and 29.9 is considered overweight, a BMI of about 30.0 or higher is considered obese, and a BMI of about 40 or higher is considered morbidly obese. (See, e.g., Gallagher et al. (2000) Am J Clin Nutr 72:694-701.) These BMI ranges are based on the impact of body weight on increased risk of disease. Some common conditions associated with high BMI and obesity include cardiovascular disease, high blood pressure (i.e., hypertension), osteoarthritis, cancer, and diabetes. BMI correlates with body fat, but the relationship between BMI and actual body fat varies by age and gender. For example, for the same BMI, women are more likely to have a higher body fat percentage than men.In addition, the BMI threshold for distinguishing between normal, overweight and obesity can vary depending on, for example, age, sex, ethnicity, health and body type, among other factors.In some embodiments, obese subjects have a body mass index (BMI) of at least about 25 kg / m before undergoing the treatment described herein. 2 In some embodiments, a subject with obesity has a body mass index of at least about 30 kg / m prior to treatment as described herein. 2 The subject may have a body mass index of

[0169] In some embodiments of any aspect, the subject to whom a composition comprising at least one IL described herein, for example, in combination with at least one active compound, is administered is a subject who has, has been diagnosed with, or needs treatment for metabolic disorder or metabolic syndrome. The term "metabolic disorder" refers to any disorder associated with or exacerbated by impaired or altered glucose regulation or glycemic control, such as insulin resistance. Such disorders include, but are not limited to, obesity; excess adipose tissue; diabetes; fatty liver disease; nonalcoholic fatty liver disease; metabolic syndrome; dyslipidemia; hypertension; hyperglycemia; and cardiovascular disease. "Metabolic syndrome" is distinct from metabolic disorder and 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. By way of example only, the WHO defines metabolic syndrome as the presence of any one of diabetes, impaired glucose tolerance, impaired fasting glucose or insulin resistance, and two of the following: blood pressure of 140 / 90 mmHg or higher, dyslipidemia, central obesity, and microalbuminuria. In some embodiments, the metabolic disorder can be selected from the group consisting of obesity; excess adipose tissue; diabetes; and cardiovascular disease.

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

[0171] For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise stated or implied from the context, the following terms and phrases have the meanings provided below. Since the scope of the present invention is limited only by the claims, the definitions are provided to aid in the description of particular embodiments and are not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0172] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

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

[0174] In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), and more preferably 20 or fewer carbon atoms. Likewise, preferred cycloalkyls have from 3 to 10 carbon atoms in their ring structure, and 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 referring to an alkyl moiety having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.

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

[0176] Substituents of substituted alkyls can include halogen, hydroxy, nitro, thiol, amino, azido, imino, amido, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfate, sulfonamide, sulfamoyl and sulfonate), and silyl groups, as well as ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylate, and ester), -CF3, -CN, and the like.

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

[0178] As used herein, the term "alkynyl" refers to an unsaturated hydrocarbon radical having at least one carbon-carbon triple bond. x Alkynyl and C x ~C y Alkynyl is typically used, where X and Y indicate the number of carbon atoms in the chain. For example, C2-C6 alkynyl includes alkynyls having a chain of 1 to 6 carbons and at least one triple bond, such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, isopentynyl, 1,3-hexadiyn-yl, n-hexynyl, 3-pentynyl, 1-hexen-3-ynyl, and the like. Alkynyl represented with another radical (e.g., as in arylalkynyl) refers to a straight-chain or branched, alkynyl divalent radical having the indicated number of atoms. The alkynyl backbone can optionally be inserted with one or more heteroatoms, such as N, O, or S.

[0179] As used herein, the term "halogen" or "halo" refers to an atom selected from fluorine, chlorine, bromine, and iodine. The term "halogen radioisotope" or "haloisotope" refers to a radionuclide of an atom selected from fluorine, chlorine, bromine, and iodine. A "halogen-substituted moiety" or "halo-substituted moiety," as an isolated group or part of a larger group, refers to an aliphatic, alicyclic, or aromatic moiety, as described herein, substituted with one or more "halo" atoms, as such terms are defined in this application. For example, halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl, and the like (e.g., halo-substituted (C1-C3) alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (-CF3), 2,2,2-trifluoroethyl, perfluoroethyl, 2,2,2-trifluoro-1,1-dichloroethyl, and the like).

[0180] The term "cyclyl" or "cycloalkyl" refers to saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and for example, 3 to 6 carbons.x Cyclyl and C x ~C y Cyclyl is typically used, where X and Y indicate the number of carbon atoms in the ring system. Cycloalkyl groups can be further optionally substituted, for example, with 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, adamantan-1-yl, decahydronaphthyl, oxocyclohexyl, dioxocyclohexyl, thiocyclohexyl, 2-oxobicyclo[2.2.1]hept-1-yl, and the like.

[0181] The term "heterocyclyl" refers to a non-aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having 1 to 3 heteroatoms if monocyclic, 1 to 6 heteroatoms if bicyclic, or 1 to 9 heteroatoms if tricyclic, selected from O, N, or S (e.g., carbon atoms and 1 to 3, 1 to 6, or 1 to 9 N, O, or S heteroatoms in the monocyclic, bicyclic, or tricyclic ring systems, respectively). x Heterocyclyl and C x ~C y Heterocyclyl is typically used, where X and Y indicate the number of carbon atoms in the ring system. In some embodiments, 1, 2, or 3 hydrogen atoms in each ring can be replaced by a substituent. Exemplary heterocyclyl groups include, but are not limited to, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolidinyl, 1,4-diazaperhydroepynyl, 1,3-dioxanyl, 1,4-dioxanyl, etc.

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

[0183] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 N, O, or S heteroatoms in the monocyclic, bicyclic, or tricyclic ring systems, respectively). x Heteroaryl and C x ~C yHeteroaryl is typically used, where X and Y indicate the number of carbon atoms in the ring system. Heteroaryl includes 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, indolizine, imidazo[l,2a]pyridine, quinoline, isoquinoline, phthalazine, quinoxaline, naphthyridine, quinolizine, indole, isoindole, indazole, indoline, benzoxazole, benzyl benzoate, ... 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, pyrrolo l,2-dihydropyrrolo[3,2,1-hi]indole, indolizine, pyrido[l,2-a]indole, 2(lH)-dihydropyrrolo[3,2,1-hi ... -pyridinone, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, Venn diagram thiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b] tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octamethylphenyl Hydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, Piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydrofuranyl Some exemplary heteroaryl groups include, but are not limited to, those derived from pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, pyridazinyl, pyrazinyl, quinolinyl, indolyl, thiazolyl, naphthyridinyl, 2-amino-4-oxo-3,Examples include, but are not limited to, 4-dihydropyridin-6-yl, tetrahydroisoquinolinyl, and the like. In some embodiments, 1, 2, 3, or 4 hydrogen atoms on each ring may be replaced by a substituent.

[0184] The term "substituted," as used herein, refers to the independent replacement of one or more hydrogen atoms on the substituted moiety with a substituent independently selected from, but not limited to, alkyl, alkenyl, heterocycloalkyl, alkoxy, aryloxy, hydroxy, amino, amido, 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 1, 2, 3, 4, or 5) hydrogen atoms on the substituted moiety with substituents independently selected from the group of substituents listed below or otherwise specified in the definition of "substituent." In general, a non-hydrogen substituent may be any substituent that may be bound to an atom of the given moiety that is specified to be substituted. Examples of substituents include acyl, acylamino, acyloxy, aldehyde, alicyclic, aliphatic, alkanesulfonamido, alkanesulfonyl, alkaryl, alkenyl, alkoxy, alkoxycarbonyl, alkyl, alkylamino, alkylcarbanoyl, alkylene, alkylidene, alkylthio, alkynyl, amide, amido, amino, amino, aminoalkyl, aralkyl, aralkylsulfonamido, arenesulfonamido, arenesulfonyl, aromatic, aryl, arylamino, arylcarbanoyl, aryloxy, azido, carbamoyl, carbonyl, carbonyls (including ketone, carboxy, carboxylate), CF3, cyano( CN), cycloalkyl, cycloalkylene, ester, ether, haloalkyl, halogen, heteroaryl, heterocyclyl, hydroxy, 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 carbons to which they are attached, can also form a ring.

[0186] The aryl and heteroaryl may be optionally substituted at one or more positions with one or more substituents, such as halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moiety, -CF3, -CN, and the like.

[0187] The term "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, n-propyloxy, isopropyloxy, n-butyloxy, isobutyloxy, and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Thus, the alkyl substituent that makes the alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl. An aroxy may be represented by -O-aryl or -O-heteroaryl, where aryl and heteroaryl are defined below. Alkoxy and aroxy groups may be substituted as previously described for alkyl.

[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] The term "alkylthio" as used herein refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio, ethylthio, and the like. 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-. It is noted that the sulfinyl radical may be further substituted with a variety of substituents to form different sulfinyl groups including sulfinic acids, sulfinamides, sulfinyl esters, sulfoxides, and the like.

[0191] The term "sulfonyl" refers to the radical -SO2-. It is noted that the sulfonyl radical can be further substituted with a variety of substituents to form different sulfonyl groups including sulfonic acid (-SO3H), sulfonamides, sulfonate esters, sulfones, and the like.

[0192] The term "thiocarbonyl" refers to the radical -C(S)-. It is noted that the thiocarbonyl radical may be further substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, thioketones, and the like.

[0193] As used herein, the term "amino" refers to -NH2. The term "alkylamino" refers to a nitrogen moiety having at least one straight-chain or branched unsaturated aliphatic, cyclyl, or heterocyclyl radical attached to the nitrogen. For example, representative amino groups include -NH2, -NHCH3, -N(CH3), -NH(C1-C2), ... 10 alkyl), -N(C1-C 10The term "alkylamino" includes "alkenylamino," "alkynylamino," "cyclylamino," and "heterocyclylamino." The term "arylimino" refers to a nitrogen moiety having at least one aryl radical attached to the nitrogen. For example, -NHaryl and -N(aryl)2. The term "heteroarylamino" refers to a nitrogen moiety having at least one heteroaryl radical attached to the nitrogen. For example, -NHheteroaryl and -N(heteroaryl)2. Optionally, the two substituents can also form a ring together with the nitrogen. Unless otherwise specified, compounds described herein containing an amino moiety can include protected derivatives thereof. Suitable protecting groups for amino moieties include acetyl, tertbutoxycarbonyl, 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 positioned between the carbon atoms of the alkyl, alkenyl, or alkynyl. For example, a (C2-C6)aminoalkyl refers to a chain comprising 2 to 6 carbon atoms and one or more nitrogen atoms positioned between the carbon atoms.

[0195] The term "alkoxyalkoxy" means -O-(alkyl)-O-(alkyl), such as -OCHCHOCH and the like. The term "alkoxycarbonyl" means -C(O)O-(alkyl), such as -C(=O)OCH, -C(=O)OCHCH and the like. The term "alkoxyalkyl" means -(alkyl)-O-(alkyl), such as -CHOCH, -CHOCHCH and the like. The term "aryloxy" means -O-(aryl), such as -O-phenyl, -O-pyridinyl and the like. The term "arylalkyl" means -(alkyl)-(aryl), such as benzyl (i.e., -CHphenyl), -CH-pyrindinyl and the like. The term "arylalkyloxy" means -O-(alkyl)-(aryl), such as -O-benzyl, -O-CH-pyridinyl and the like. The term "cycloalkyloxy" means -O-(cycloalkyl), such as -O-cyclohexyl and the like. The term "cycloalkylalkyloxy" means -O-(alkyl)-(cycloalkyl, e.g., -OCHcyclohexyl, etc. The term "aminoalkoxy" means -O-(alkyl)-NH, e.g., -OCHNH, -OCHCHNH, etc. The term "mono- or di-alkylamino" means -NH(alkyl) or -N(alkyl)(alkyl), respectively, e.g., -NHCH, -N(CH). The term "mono- or di-alkylaminoalkoxy" means -O-(alkyl)-NH(alkyl) or -O-(alkyl)-N(alkyl)(, respectively. The term "arylamino" means -NH(aryl), for example, -NH-phenyl, -NH-pyridinyl, and the like. The term "arylalkylamino" means -NH-(alkyl)-(aryl), for example, -NH-benzyl, -NHCH-pyridinyl, and the like. The term "alkylamino" means -NH(alkyl), for example, -NHCH, -NHCHCH, and the like. The term "cycloalkylamino" means -NH-(cycloalkyl), for example, -NH-cyclohexyl, and the like.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-ended in the sense that they may include additional substituents other than those specified. Thus, C1 alkyl indicates that there is one carbon atom, but does not indicate what the substituents on the carbon atom are. Thus, C1 alkyl includes methyl (i.e., -CH3) as well as CR a R b R c where R a , R b , and R c may each independently be hydrogen or any other substituent where the atom alpha to the carbon is a heteroatom or cyano. Thus, CF, CHOH, and CHCN are all C alkyls.

[0197] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms, for example, the replacement of a hydrogen atom by deuterium or tritium, or 13 C- or 14 Compounds having the present structure, except for the replacement of a carbon atom with a C-enriched carbon, are within the scope of this invention.

[0198] As used herein, the term "isomer" refers to compounds that have the same molecular formula but different structures. Isomers that differ only in configuration and / or conformation 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 are non-superimposable. Other terms used to designate or refer to enantiomers include "stereoisomers" (due to different configurations or stereochemistry around the chiral center; all enantiomers are stereoisomers, but not all stereoisomers are enantiomers) or "enantiomers" (due to the optical activity of pure enantiomers, which 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 points and boiling points, and also the same spectroscopic properties. Enantiomers can differ from each other with respect to their interaction with plane-polarized light and with respect to 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 would have 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] The terms "resolving" or "resolution" when used in reference to a racemic mixture refers to the separation of a racemate into its two mirror image forms (i.e., (+) and (-); or (R) and (S) forms). The term can also refer to the enantioselective conversion of a racemate into a single isomeric product.

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

[0203] Whether referred to as a "purified enantiomer" or "pure enantiomer" or "resolved enantiomer" or "enantiomeric excess of a compound," the term is intended to indicate the amount of one enantiomer in excess of the other. Thus, when referring to an enantiomeric preparation, the percent (e.g., mole or weight or volume) of the major enantiomer and / or the percent enantiomeric excess of the major enantiomer may be used to determine whether the preparation represents a purified enantiomeric preparation.

[0204] The terms "enantiomeric purity" or "enantiomeric purity" of an isomer refer to a qualitative or quantitative measure of a purified enantiomer; typically, the measurement is expressed in terms of ee or enantiomeric excess.

[0205] The terms "substantially purified enantiomer," "substantially resolved enantiomer," and "substantially purified enantiomeric preparation" are intended to refer to a preparation (e.g., derived from non-optically active starting materials, substrates, or intermediates) that is enriched in one enantiomer over the other, more preferably in which the other enantiomer represents less than 20%, more preferably less than 10%, more preferably less than 5%, and even more preferably less than 2% of the enantiomer or enantiomeric preparation.

[0206] The terms "purified enantiomer," "resolved enantiomer," and "purified enantiomer preparation" are intended to refer to a preparation (e.g., derived from non-optically active starting materials, substrates, or intermediates) that is enriched in one enantiomer (e.g., the R-enantiomer) over the other, and more preferably, the other enantiomer (e.g., the S-enantiomer) accounts for less than 30%, preferably less than 20%, more preferably less than 10% of the preparation (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%. A 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 "enantioselectivity," also known as the enantiomeric ratio, denoted by the symbol "E," refers to the selective ability of an enzyme to produce one enantiomer from a racemic substrate over the other enantiomer in a racemic product mixture; i.e., it is a measure of the enzyme's ability to distinguish between enantiomers. While a nonselective reaction has an E of 1, resolutions with an E of 20 or greater are generally considered useful in synthesis or resolution. Enantioselectivity resides in the difference in the rates of conversion between the enantiomers of interest. The reaction product is enriched in one of the enantiomers; conversely, the remaining substrate is enriched in the other enantiomer. For practical purposes, it is generally desirable to obtain a large excess of one enantiomer. This is achieved by stopping the conversion process at a certain degree of conversion.

[0208] CAGE (choline and geranate) is an ionic liquid comprising the cation choline (see, e.g., Structure I) and the anion geranate or geranic acid (see, e.g., Structures II and III). Preparation of CAGE can be as described, for example, in International Patent Publication WO 2015 / 066647, which is incorporated herein by reference in its entirety, or as described in the Examples herein. TIFF2026021383000015.tif167128

[0209] The terms "reduce," "reduced," "reduction," or "inhibit" are all used herein to mean a statistically significant reduction. In some embodiments, "reduce," "reduction," or "reduce" or "inhibit" generally refers to a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and may include, for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or greater reduction. As used herein, "reduction" or "inhibition" does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The decrease can preferably be down to a level that is accepted as within the normal range for individuals without the given disorder.

[0210] The terms "increased," "increase," "enhance," or "activate" are all used herein to mean a statistically significant increase. In some embodiments, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of less than or equal to 100%, or any increase between 10-100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

[0211] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate such as a primate, a rodent, a livestock animal, or a game animal. Primates include chimpanzees, macaques such as cynomolgus monkeys, spider monkeys, and rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, and wolves, bird species such as chickens, emus, and ostriches, and fish such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, e.g., a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0212] Preferably, the subject is a mammal.The mammal can be, but is not limited to, human, non-human primate, mouse, rat, dog, cat, horse, or cow.Non-human mammals can be advantageously used as subjects to represent the animal model of the pathology described herein.The subject can be male / male or female / female.

[0213] The subject may have been previously diagnosed with, identified as suffering from, or have a condition requiring treatment or one or more complications associated with such a condition, and may optionally have already been treated for the condition or one or more complications associated with the condition. Alternatively, the subject may not have been previously diagnosed with the condition or one or more complications associated with the condition. For example, the subject may exhibit one or more risk factors for the condition or one or more complications associated with the condition, or may not exhibit risk factors.

[0214] A subject "in need" of treatment for a particular condition can be a subject who has the condition, has been diagnosed with the condition, or is at risk of developing the condition.

[0215] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues connected to each other by peptide bonds between the alpha amino and carboxy groups of adjacent residues. The terms "protein" and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, the usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0216] In various embodiments described herein, it is further intended to encompass any variants (natural or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of the specific polypeptides described. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that modify a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," in which the modification results in the substitution of an amino acid with a chemically similar amino acid and the desired activity of the polypeptide is retained. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

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

[0218] Amino acids can be grouped by similarities in the properties of their side chains (A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on shared side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another. Particular conservative substitutions include, for example, Ala for Gly or Ser; Arg for Lys; Asn for Gln or His; Asp for Glu; Cys for Ser; Gln for Asn; Glu for Asp; Gly for Ala or Pro; His for Asn or Gln; Ile for Leu or Val; Leu for Ile or Val; Lys for Arg, Gln, or Glu; Met for Leu, Tyr, or Ile; Phe for Met, Leu, or Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp; and / or Phe for Val, Ile, or Leu.

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

[0220] In some embodiments, the polypeptides described herein may be variants of the sequences described herein. In some embodiments, the variants are conservatively modified variants. Conservative substitution variants can be obtained, for example, by mutation of a native nucleotide sequence. As referred to herein, a "variant" is a polypeptide that is substantially homologous to a native or reference polypeptide but has an amino acid sequence that differs from that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide encompasses sequences encoding a mutant protein or fragment thereof that contains one or more nucleotide additions, deletions, or substitutions compared to a native or reference DNA sequence but retains activity. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art.

[0221] The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to the native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined by comparing the two sequences, for example, using freely available computer programs widely used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0222] In some embodiments of either aspect, the variant can be a polypeptide that has at least 90%, at least 95%, at least 98% or more sequence identity to one of the reference sequences provided herein and retains the wild-type activity, e.g., incretin activity, of that reference sequence. In some embodiments of either aspect, the variant can be a polypeptide that has at least 90%, at least 95%, at least 98% or more sequence identity to one of the naturally occurring reference sequences provided herein and retains the wild-type activity, e.g., incretin activity, of that reference sequence. In some embodiments of either aspect, the variant can be a naturally occurring polypeptide that has at least 90%, at least 95%, at least 98% or more sequence identity to one of the reference sequences provided herein and retains the wild-type activity, e.g., incretin activity, of that reference sequence.

[0223] Alterations to the native amino acid sequence can be achieved by any of a number of techniques known to those of skill in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing the mutated sequence flanked by restriction sites that allow ligation to a fragment of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to provide modified nucleotide sequences with specific codons altered by the required substitution, deletion, or insertion. Techniques for making such modifications are very well established and include, for example, those disclosed in Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entireties. Any cysteine ​​residue not involved in maintaining the correct conformation of the polypeptide can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) can be added to a polypeptide to improve its stability or promote oligomerization.

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

[0225] As used herein, the term "antibody reagent" refers to a polypeptide that contains at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds to a given antigen. An antibody reagent may include an antibody or a polypeptide comprising the antigen-binding domain of an antibody. In some embodiments, an antibody reagent may include a monoclonal antibody or a polypeptide comprising the antigen-binding domain of a monoclonal antibody. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibody (dAb) fragments as well as complete antibodies.

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

[0227] As used herein, the term "nanobody" or single domain antibody (sdAb) refers to antibodies (VHHs) containing small single variable domains of antibodies obtained from camels and dromedaries. Antibody proteins obtained from members of the camel and dromedary families (Camelus baclrianus and Calelus dromaderius), including New World members such as llama species (alpacas, lamas, and vicuñas), have been characterized for size, structural complexity, and antigenicity to human subjects. Certain IgG antibodies from this family of mammals found in nature lack light chains and are therefore structurally distinct from the typical four-chain quaternary structure with two heavy chains and two light chains of antibodies from other animals. See PCT / EP93 / 02214 (WO 94 / 04678 published March 3, 1994; which is incorporated herein by reference in its entirety).

[0228] Regions of camelid antibodies, which are small single variable domains identified as VHHs, can be genetically engineered to generate small proteins with high affinity for targets, resulting in low molecular weight antibody-derived proteins known as "camelid nanobodies." See U.S. Patent No. 5,759,808, issued June 2, 1998; see also 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 which is incorporated by reference in its entirety. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, the amino acid sequences of camelid antibodies can be recombinantly modified to obtain sequences that more closely resemble human sequences, i.e., the nanobodies can be "humanized." Thus, the naturally low antigenicity of camelid antibodies to humans can be further reduced.

[0229] Camelid nanobodies have a molecular weight approximately one-tenth that of human IgG molecules, and the protein has a physical diameter of only a few nanometers. One significance of their small size is that they can bind to antigenic sites functionally invisible to larger antibody proteins, making them useful as reagents for detecting antigens obscured using classical immunological techniques and as potential therapeutic agents. Another significance of their small size is that camelid nanobodies can bind to and inhibit specific sites in grooves or narrow clefts of target proteins, thus acting in a capacity that more closely resembles the function of classical low-molecular-weight drugs than classical antibodies. The low molecular weight and small size also result in camelid nanobodies being highly heat-resistant, stable to extreme pH and proteolytic digestion, and having low antigenicity. See U.S. Patent Application No. 20040161738, published August 19, 2004; this is incorporated herein by reference in its entirety. These characteristics, combined with low antigenicity to humans, indicate great therapeutic potential.

[0230] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. Nucleic acids can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, a nucleic acid can be DNA. In another aspect, a nucleic acid can be RNA. Suitable DNA can include, for example, cDNA. Suitable RNA can include, for example, mRNA.

[0231] As used herein, "inhibitory nucleic acid" refers to a nucleic acid molecule that can inhibit the expression of a target, such as a double-stranded RNA (dsRNA), an inhibitory RNA (iRNA), etc. In some embodiments of any aspect, the inhibitory nucleic acid can be a silencing RNA (siRNA), a microRNA (miRNA), or a short hairpin RNA (shRNA). The inhibitory nucleic acid can also include a guide sequence molecule (e.g., a guide RNA), which, for example, functions in combination with an enzyme to induce insertion, deletion, indel, and / or mutation of the target, thereby inhibiting the expression of the target.

[0232] Double-stranded RNA molecules (dsRNA) have been shown to block gene expression through a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein can be up to 30 nucleotides in length, i.e., 15-30 nucleotides in length, typically 19-24 nucleotides in length, and contain an RNA strand (antisense strand) with a region substantially complementary to at least a portion of a targeted mRNA transcript. These iRNAs can be used to target and degrade mRNA transcripts, resulting in decreased expression and / or activity of the target.

[0233] As used herein, the term "iRNA" refers to an agent that includes RNA (or modified nucleic acids as described herein below) and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. In some embodiments of either aspect, the iRNA described herein results in inhibition of target expression and / or activity. In some embodiments of either aspect, contacting a cell with an inhibitor (e.g., iRNA) results in a reduction in target mRNA levels in the cell of at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or 100% of the target mRNA levels found in the cell in the absence of iRNA. In some embodiments of either aspect, administration of an inhibitor (e.g., an iRNA) to a subject results in a decrease in target mRNA levels in the subject of at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, 100%, or less, including the target mRNA levels found in the subject in the absence of the iRNA.

[0234] In some embodiments of either aspect, the iRNA can be a dsRNA. The dsRNA comprises two RNA strands sufficiently complementary to hybridize and form a duplex structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally completely complementary, to the target sequence. The target sequence can be derived from the sequence of an mRNA formed during expression of the target; for example, it can span one or more intron boundaries. The other strand (the sense strand) comprises a region complementary to the antisense strand such that, when combined under appropriate conditions, the two strands hybridize to form a duplex structure. Typically, the duplex structure is between 15 and 30 base pairs in length, including 15 and 30 base pairs, more typically between 18 and 25 base pairs in length, including 18 and 25 base pairs in length, even more typically between 19 and 24 base pairs in length, including 19 and 24 base pairs in length, and most typically between 19 and 21 base pairs in length, including 19 and 21 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30 base pairs in length, including 15 and 30 base pairs, more commonly between 18 and 25 base pairs in length, including 18 and 25 base pairs in length, even more commonly between 19 and 24 base pairs in length, including 19 and 24 base pairs in length, and most commonly between 19 and 21 base pairs in length, including 19 and 21 base pairs in length. In some embodiments of either aspect, the dsRNA is between 15 and 20 nucleotides in length, including 15 and 20 nucleotides in length; in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, including 25 and 30 nucleotides in length. As one of skill in the art will recognize, the targeted region of an RNA that is targeted for cleavage is most often a portion of a larger RNA molecule, often an mRNA molecule. Where relevant, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). dsRNA with duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. In most cases, the target is at least 15 nucleotides in length, preferably 15-30 nucleotides in length.

[0235] The exemplary embodiment of inhibitory nucleic acid type can include, for example, siRNA, shRNA, miRNA and / or amiRNA, which are well known in the art.Those skilled in the art can, for example, use commonly available design tools to design additional siRNA, shRNA or miRNA for targeting the nucleic acid sequence of target gene or gene product (for example, mRNA).SiRNA, shRNA or miRNA is generally produced by companies such as Dharmacon (Layfayette, CO) or Sigma Aldrich (St. Louis, MO).

[0236] In some embodiments of either aspect, the RNA of the iRNA, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids described herein may be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, inverted linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, inverted linkage, etc.), (b) base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that form base pairs with an expanded repertoire of partners, base removal (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., 2'- or 4'-position) or sugar substitutions, and (d) backbone modifications, including modification or substitution of phosphodiester bonds. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to, RNA that contains modified backbones or does not contain natural internucleoside linkages.RNA with modified backbones include, among others, those that do not have a phosphorus atom in their backbones.For the purpose of this specification and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbones can also be considered oligonucleosides.In some embodiments of either aspect, modified RNAs have a phosphorus atom in their internucleoside backbones.

[0237] Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with opposite polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and other mixed N, O, S, and CH moieties. These include oligonucleosides having heteroatom backbones, and in particular those having -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- (known as the methylene(methylimino) or MMI backbone), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- (where the natural phosphodiester backbone is represented as -OPO-CH2-).

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

[0239] The RNA of iRNA can also be modified to contain one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with modified ribose moieties, which contain an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. Adding locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce nonspecific effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0240] Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, described herein, may contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Exemplary suitable modifications are O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments of either aspect, the dsRNA comprises one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents with similar properties. In some embodiments of either aspect, the modification comprises 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the O(CH)ON(CH) group, also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, as described in the Examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH-O-CH-N(CH), also described in the Examples herein below.

[0241] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and the 5' position of the 5'-terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0242] Inhibitory nucleic acids may also include modifications or substitutions of nucleobases (often referred to in the art simply as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal (8-hydroxyl Other synthetic and natural nucleobases include other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-dazaadenine, and 3-deazaguanine and 3-deazaadenine. Some of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are an exemplary base substitution, even more particularly when combined with a 2'-O-methoxyethyl sugar modification.

[0243] The preparation of the above-described modified nucleic acids, backbones, and nucleobases is well known in the art.

[0244] Another modification of the inhibitory nucleic acids featured in the invention involves chemically linking to the inhibitory nucleic acid one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA. Such moieties include cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterols (Oberhauser et al., Nucl. Acids Res., 1992, 4: 1053-1060), and the like. 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al.Lipid moieties include, but are not limited to, octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937), or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0245] In some embodiments of various aspects described herein, the inhibitory nucleic acid is a guide nucleic acid (gNA). As used herein, the term "guide nucleic acid," "guide sequence," "crRNA," "guide RNA," "single guide RNA," "gRNA," or "CRISPR guide sequence" refers to a nucleic acid that contains a sequence that determines the specificity of an enzyme, such as a Cas DNA-binding protein of a CRISPR / Cas system, for a polynucleotide target. A gNA can include a polynucleotide sequence that has at least partial complementarity with a target nucleic acid sequence sufficient to hybridize with the target nucleic acid sequence and direct the sequence-specific binding of an enzyme, such as a nuclease, to the target nucleic acid sequence.

[0246] In some embodiments, the enzyme guided by gNA is a gene editing protein, for example, any nuclease that induces a nick or double-strand break at the desired recognition site. Such enzymes can be natural or modified. These breaks can then be repaired by cells in one of two ways: non-homologous end joining and homology-directed repair (homologous recombination). In non-homologous end joining (NHEJ), double-strand breaks are repaired by direct ligation of the cut ends. Therefore, new nucleic acid material is not inserted into the site, but some nucleic acid material may be lost, resulting in deletion. In homology-directed repair, a donor polynucleotide that has homology with the cut target DNA sequence can be used as a template for repairing the cut target DNA sequence, causing 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. Targeted DNA modification by NHEJ and / or homology-directed repair can be used for gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, and the like.

[0247] In one embodiment, the gene editing protein is a CRISPR-associated nuclease.Natural prokaryotic CRISPR-associated nuclease system comprises a short repeat sequence (i.e., a cluster of regularly spaced short inverted repeats) with a fixed length of intervening variable sequence and an array of CRISPR-associated ("Cas") nuclease proteins.The transcribed RNA of the CRISPR array is processed by a subset of Cas proteins into small guide RNA, which generally has two components as discussed below.There are at least three different systems: Type I, Type II, and Type III.The enzymes involved in processing RNA into mature crRNA are different in the three systems.In natural prokaryotic systems, guide RNA ("gRNA") comprises two short non-coding RNA species called CRISPR RNA ("crRNA") and trans-acting RNA ("tracrRNA").In an exemplary system, gRNA forms a complex with a nuclease, for example, a Cas nuclease. The gRNA:nuclease complex binds to a target polynucleotide sequence that has a protospacer adjacent motif ("PAM") and a protospacer, a sequence 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-associated nuclease can be used in the systems and methods of the present invention. CRISPR nuclease systems are known to those skilled in the art, such as Cas9, Cas12, and Cas12a, and are described in patents / 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 WO 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 which is incorporated by reference in its entirety. The nuclease can also be a phage Cas nuclease, such as CasΦ (e.g., Pausch et al. Science 369: 333-7 (2020); which is incorporated by reference in its entirety).

[0249] The full-length guide nucleic acid chain can be any length.For example, the guide nucleic acid chain can be about 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 nucleotides in length, or about 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 nucleotides in length.In some embodiments of various aspects described herein, the nucleic acid chain is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length. For example, the guide nucleic acid sequence is 10 to 30 nucleotides in length.

[0250] In addition to the sequence that is complementary to target nucleic acid, in some embodiments, gNA also comprises scaffold sequence.The expression of gNA that encodes both the sequence that is complementary to target nucleic acid and scaffold sequence has the dual function of binding (hybridizing) to target nucleic acid and recruiting endonuclease to target nucleic acid, which can result in site-specific CRISPR activity.In some embodiments, this chimeric gNA can be called single guide RNA (sgRNA).

[0251] In some embodiments of various aspects described herein, the guide nucleic acid is designed using a guide design tool (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™; which are available on the web).

[0252] The term "vector" as used herein refers to a nucleic acid construct designed for delivery into a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that can replicate when associated with the correct control elements and transfer gene sequences into a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, recombinant vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, virions, etc.

[0253] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides from sequences linked to transcriptional regulatory sequences on the vector. The expressed sequences are often, but not necessarily, heterologous to the cell. An expression vector may contain additional elements; for example, an expression vector may have two replication systems, thus allowing it to be maintained in two organisms, such as human cells for expression and prokaryotic hosts for cloning and amplification. The term "expression" refers to the cellular processes involved in the production of RNA and proteins, and, if applicable, their secretion, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, as applicable. "Expression product" includes RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence that is transcribed into RNA (DNA) in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5' UTR) or "leader" sequence and 3' UTR or "trailer" sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0254] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. A viral vector can contain a nucleic acid encoding a polypeptide as described herein in place of a non-essential viral gene. The vector and / or particle can be used to transfer any nucleic acid into cells either in vitro or in vivo. Many forms of viral vectors are known in the art.

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

[0256] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatments whose purpose is to reverse, alleviate, ameliorate, inhibit, slow, or halt the progression or severity of symptoms associated with a disease or disorder, such as a condition or disease described herein. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least slowing of the progression or worsening of symptoms compared to that expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, a decrease in the extent of the disease, stabilization of the disease state (i.e., not worsening), a delay or slowing of disease progression, an improvement or palliative of the disease state, remission (whether partial or complete), and / or reduced mortality. The term "treatment" of a disease also includes bringing about the alleviation of symptoms or side effects of the disease (including symptomatic treatment).

[0257] As used herein, the term "pharmaceutical composition" refers to an active agent combined with a pharmaceutically acceptable carrier, e.g., a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be a carrier other than water. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be an artificial or engineered carrier, e.g., a carrier in which the active ingredient is not found to occur in nature.

[0258] As used herein, the term "administration" refers to placing a compound as disclosed herein in a subject by a method or route that results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions containing the compounds disclosed herein can be administered by any suitable route that results in effective treatment in the subject.

[0259] As used herein, " contact " refers to 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 into cell culture medium, perfusion, injection, or other delivery methods 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 operating a delivery device or machine.

[0260] The term "effective amount" refers to an amount of a composition sufficient to result in at least some improvement in symptoms associated with a condition. In one embodiment, "effective amount" refers to an amount of a composition that reduces a marker or symptom of a condition in a subject with the condition.

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

[0262] Other than in the working examples, or unless otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±1%.

[0263] As used herein, the terms "comprising" or "comprises" are used in reference to methods and compositions essential to the invention and their respective components, although they may include elements not specified, whether essential or not. As used herein, the term "comprising" means that other elements may be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[0264] The term "consisting of" refers to the compositions, methods, and each component thereof described herein, and does not include any element not recited in the description of that embodiment.

[0265] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0266] The term "specific binding" as used herein refers to the 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 a first entity to a 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 assay being used.

[0267] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin "exempli gratia," and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0268] Groupings of alternative elements or aspects of the invention disclosed herein should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, and deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification herein is deemed to include the group as modified, fulfilling the written description of all Markush groups used in the appended claims.

[0269] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art to which this disclosure belongs. It should be understood that the present invention is not limited to the particular methodology, protocols, and reagents, etc., described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305);Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4. thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414);Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X);Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542);Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons Sons, Inc. 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are incorporated herein by reference in their entireties.

[0270] Those skilled in the art can readily identify chemotherapeutic agents to use (see, e.g., Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff's Clinical Oncology, 2013 Elsevier; and Fischer DS (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

[0271] Other terms are defined herein within the description of various aspects of the invention.

[0272] All patents and other publications, including references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications, which may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or indication as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0273] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes; however, those skilled in the relevant art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are shown in a given order, in alternative embodiments, the functions may be performed in a different order, or functions may be performed substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods, as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, if necessary, to employ compositions, functions, and concepts from the above references and applications to provide still further embodiments of the present disclosure. Furthermore, given consideration of biological functional equivalence, some changes can be made to protein structure without affecting biological or chemical activity in type or amount. These and other changes can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0274] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Furthermore, although advantages associated with particular embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not necessarily all embodiments may exhibit such advantages to be within the scope of the present disclosure.

[0275] The technology described herein is further illustrated by the following examples, which should in no way be construed as further limiting.

[0276] Some aspects of the technology described herein can be defined by any of the following numbered clauses: 1. (a) Carboxylic acids that are not fatty acids, (b) a carboxylic acid containing an aliphatic chain of four carbons or less; (c) an aromatic anion, and / or (d) Anions with a LogP of less than 1.0 and an anion that is at least one of Quaternary ammonium cations 1. A composition comprising at least one ionic liquid, comprising: 2. The anion is has a LogP less than 1.0, and a. non-fatty acid carboxylic acids, b. Carboxylic acids containing an aliphatic chain of four carbons or less, or c. Aromatic anions That is, 10. The composition of any of the preceding claims. 3. The composition of any of the preceding paragraphs, wherein the fatty acid comprises an aliphatic chain of 3 carbons or less. 4. The composition of any of the preceding paragraphs, wherein the anion contains only one carboxylic acid group (e.g., an R-COOH group). 5. The composition of any of the preceding paragraphs, 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; phenylphosphonic acid; and biphenyl-3-carboxylic acid. 6. The composition of any of the preceding paragraphs, wherein the cation has a molar mass equal to or greater than that of choline. 7. Quaternary ammonium is NR4 + and at least one R group comprises a hydroxy group. 8. Quaternary ammonium is NR4 + The composition of any preceding paragraph, having the structure: and only one R group contains a hydroxy group. 9. The composition of any of the preceding paragraphs, wherein the cation is C1, C6, or C7. 10. The composition of any of the preceding paragraphs, wherein the ionic liquid comprises a ratio of cations to anions of about 2:1 to about 1:1. 11. The composition of any of the preceding paragraphs, wherein the ionic liquid comprises a ratio of cations to anions of about 2:1. 12. The composition of any of the preceding paragraphs, wherein the ionic liquid has a cation:anion ratio of less than 1:1. 13. The composition of any preceding paragraph, wherein the ionic liquid has a cation:anion ratio in cation excess. 14. The composition of any preceding paragraph, further comprising at least one active compound in combination with at least one ionic liquid. 15. The composition of any of the preceding paragraphs, wherein the active compound comprises a polypeptide. 16. The composition of paragraph 15, wherein the polypeptide is an antibody or antibody reagent. 17. The composition of any of paragraphs 15 to 16, wherein the active compound has a molecular weight greater than 450. 18. The composition of any of paragraphs 15 to 16, wherein the active compound has a molecular weight greater than 500. 19. An anion is has a LogP less than 1.0, and a. a carboxylic acid that is not a fatty acid; or b. Carboxylic acids containing aliphatic chains of four carbons or less That is, Item 19. The composition according to any one of items 15 to 18. 20. The composition of any of the preceding paragraphs, wherein the active compound comprises a nucleic acid. 21. The composition of paragraph 20, wherein the nucleic acid is an inhibitory nucleic acid. 22. The composition of paragraph 21, wherein the nucleic acid is an siRNA. 23. An anion is has a LogP less than 1.0, and a. a carboxylic acid that is not a fatty acid; or b. Carboxylic acids containing aliphatic chains of four carbons or less; and / or c. Aromatic anions That is, 23. The composition according to any one of items 20 to 22. 24. The composition of any of the preceding paragraphs, wherein the ionic liquid is at a concentration of at least 0.1% w / v. 25. The composition of any of the preceding paragraphs, wherein the ionic liquid is at a concentration of about 10 to about 70% w / v. 26. The composition of any of the preceding paragraphs, wherein the ionic liquid is at a concentration of about 30 to about 50% w / v. 27. The composition of any of the preceding paragraphs, wherein the ionic liquid is at a concentration of about 30 to about 40% w / v. 28. The composition of any of the preceding paragraphs, formulated for transdermal, mucosal, oral, subcutaneous, intradermal, parenteral, intratumoral, or intravenous administration. 29. The composition of paragraph 28, formulated for transdermal administration. 30. The composition of paragraph 28, wherein the mucosa is nasal mucosa, oral mucosa, or vaginal mucosa. 31. The composition of any of the preceding paragraphs, providing the active compound at a dose of 1 to 40 mg / kg. 32. The composition of any preceding paragraph, further comprising at least one nonionic surfactant. 33. The composition of any of the preceding paragraphs, further comprising a pharmaceutically acceptable carrier. 34. The composition of any of the preceding paragraphs provided in a degradable capsule. 35. A composition of any preceding paragraph which is an admixture. 36. The composition of any of the preceding paragraphs provided in one or more nanoparticles. 37. The composition of any preceding paragraph, comprising one or more nanoparticles comprising an active compound, the nanoparticles being in solution or suspension in a composition comprising an ionic liquid. 38. A method of administering at least one active compound, comprising administering the composition of any of paragraphs 14 to 37. 39. The method of paragraph 38, wherein the composition is administered once. 40. The method of any of paragraphs 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 clauses: 1. (a) Carboxylic acids that are not fatty acids, (b) a carboxylic acid containing an aliphatic chain of four carbons or less; (c) an aromatic anion, and / or (d) Anions with a LogP of less than 1.0 and an anion that is at least one of Quaternary ammonium cations 1. A composition comprising at least one ionic liquid, comprising: 2. The anion is has a LogP less than 1.0, and a. non-fatty acid carboxylic acids, b. Carboxylic acids containing an aliphatic chain of four carbons or less, or c. Aromatic anions That is, 10. The composition of any of the preceding claims. 3. The composition of any of the preceding paragraphs, wherein the fatty acid comprises an aliphatic chain of 3 carbons or less. 4. The composition of any of the preceding paragraphs, wherein the anion contains only one carboxylic acid group (e.g., an R-COOH group). 5. The composition of any of the preceding paragraphs, 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); phenylphosphoric acid; and biphenyl-3-carboxylic acid. 6. The composition of any of the preceding paragraphs, 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); phenylphosphoric acid; and biphenyl-3-carboxylic acid. 7. The composition of any of the preceding paragraphs, wherein the cation has a molar mass equal to or greater than that of choline. 8. Quaternary ammonium is NR4 + and at least one R group comprises a hydroxy group. 9. Quaternary ammonium is NR4 + The composition of any preceding paragraph, having the structure: and only one R group contains a hydroxy group. 10. The composition of any of the preceding paragraphs, wherein the cation is choline, C1, C6, or C7. 11. The composition of any of the preceding paragraphs, wherein the cation is choline. 12. The composition of any of the preceding paragraphs, wherein the cation is C1, C6, or C7. 13. The composition of any preceding paragraph, wherein the ionic liquid comprises a ratio of cations to anions of about 2:1 to about 1:1. 14. The composition of any of the preceding paragraphs, wherein the ionic liquid comprises a ratio of cations to anions of about 2:1. 15. The composition of any of the preceding paragraphs, wherein the ionic liquid has a cation:anion ratio of less than 1:1. 16. The composition of any preceding paragraph, wherein the ionic liquid has a cation:anion ratio in cation excess. 17. The composition of any preceding paragraph, comprising a first ionic liquid and at least a second ionic liquid. 18. The composition of paragraph 17, wherein each ionic liquid has a choline cation. 19. The composition of any of items 17 to 18, wherein the first ionic liquid and the second ionic liquid each contain a different anion. 20. The composition of paragraph 19, wherein the first ionic liquid and the second ionic liquid each comprise a different anion 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); phenylphosphoric acid; and biphenyl-3-carboxylic acid. 21. The composition of 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. The composition of any of paragraphs 17 to 21, wherein the first ionic liquid is choline and geranic acid (CAGE). 23. The composition of any of paragraphs 17 to 22, wherein the second ionic liquid is choline and dimethylacrylic acid (CADA); choline and isovaleric acid (CAVA); choline and phenylphosphonic acid (CAPP); choline and biphenyl-3-carboxylic acid (CABA); choline and 4-phenolsulfonic acid (CASA); or choline and phenylpropanoic acid (CAPA). 24. The composition of any of paragraphs 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 phenylphosphoric acid (CAPP); choline and biphenyl-3-carboxylic acid (CABA); choline and 4-phenolsulfonic acid (CASA); or choline and phenylpropanoic acid (CAPA). 25. The composition of any of paragraphs 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. The composition of any of paragraphs 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. The composition of any preceding paragraph, further comprising at least one active compound in combination with at least one ionic liquid. 28. The composition of any of the preceding paragraphs, wherein the active compound comprises a polypeptide. 29. The composition of paragraph 28, wherein the polypeptide is an antibody or antibody reagent. 30. The composition of any of paragraphs 28 to 29, wherein the active compound has a molecular weight greater than 450. 31. The composition of any of paragraphs 28 to 30, wherein the active compound has a molecular weight greater than 500. 32. An anion is has a LogP less than 1.0, and a. a carboxylic acid that is not a fatty acid; or b. Carboxylic acids containing aliphatic chains of four carbons or less That is, Item 32. The composition according to any one of items 28 to 31. 33. The composition of any of the preceding paragraphs, wherein the active compound comprises a nucleic acid. 34. The composition of paragraph 33, wherein the nucleic acid is an inhibitory nucleic acid. 35. The composition of paragraph 34, wherein the nucleic acid is an siRNA. 36. The composition of any of paragraphs 34 to 35, wherein the inhibitory nucleic acid is an NFKBIZ, TNFα, and / or IL-17 inhibitory nucleic acid. 37. An anion is has a LogP less than 1.0, and a. a carboxylic acid that is not a fatty acid; or b. Carboxylic acids containing aliphatic chains of four carbons or less; and / or c. Aromatic anions That is, Item 37. The composition according to any one of items 33 to 36. 38. The composition of any of the preceding paragraphs, wherein the ionic liquid is at a concentration of at least 0.1% w / v. 39. The composition of any of the preceding paragraphs, wherein the ionic liquid is at a concentration of about 10 to about 70% w / v. 40. The composition of any of the preceding paragraphs, wherein the ionic liquid is at a concentration of about 30 to about 50% w / v. 41. The composition of any of the preceding paragraphs, wherein the ionic liquid is at a concentration of about 30 to about 40% w / v. 42. The composition of any of the preceding paragraphs, formulated for transdermal, mucosal, oral, subcutaneous, intradermal, parenteral, intratumoral, or intravenous administration. 43. The composition of paragraph 42, formulated for transdermal administration. 44. The composition of paragraph 42, wherein the mucosa is nasal mucosa, oral mucosa, or vaginal mucosa. 45. The composition of any of the preceding paragraphs, providing the active compound at a dose of 1 to 40 mg / kg. 46. ​​The composition of any preceding paragraph, further comprising at least one nonionic surfactant. 47. The composition of any of the preceding paragraphs, further comprising a pharmaceutically acceptable carrier. 48. The composition of any of the preceding paragraphs provided in a degradable capsule. 49. A composition of any preceding paragraph which is an admixture. 50. The composition of any of the preceding paragraphs provided in one or more nanoparticles. 51. The composition of any preceding paragraph, comprising one or more nanoparticles comprising an active compound, the nanoparticles being in solution or suspension in a composition comprising an ionic liquid. 52. A method for administering at least one active compound to a subject, comprising administering the composition of any one of paragraphs 27 to 51. 53. The method of paragraph 52, wherein the composition is administered once. 54. The method of any of paragraphs 52 to 53, wherein the composition is administered in multiple doses. 55. The method of any of paragraphs 52 to 54, wherein administration is transdermal, mucosal, oral, subcutaneous, intradermal, parenteral, intratumoral, or intravenous. 56. The method of any of paragraphs 52-55, wherein the composition comprises NFKBIZ, TNFα, and / or IL-17 inhibitory nucleic acid, and the subject is in need of treatment for an inflammatory condition. 57. A method for treating an inflammatory condition in a subject in need thereof, comprising administering to the subject the composition of any of paragraphs 36 to 51. 58. The method of any of paragraphs 56 to 57, wherein the administration is topical. 59. The method of any of paragraphs 56 to 58, wherein the inflammatory condition is psoriasis. 60. The composition of any of paragraphs 27 to 51 for use in a method of administering at least one active compound to a subject. 61. The composition of paragraph 60, which is administered once. 62. The composition of paragraph 60, which is administered in multiple doses. 63. The composition of any of paragraphs 60 to 62, wherein the administration is transdermal, mucosal, oral, subcutaneous, intradermal, parenteral, intratumoral, or intravenous. 64. The composition of any of paragraphs 60 to 63, comprising an NFKBIZ, TNFα, and / or IL-17 inhibitory nucleic acid, and the subject is in need of treatment for an inflammatory condition. 65. The composition of any of paragraphs 36 to 51 for use in a method of treating an inflammatory condition in a subject in need thereof. 66. The composition of any of paragraphs 64 to 65, wherein the administration is topical. 67. The composition of any of paragraphs 64 to 66, wherein the inflammatory condition is psoriasis. [Example]

[0278] Example 1: Ionic Liquids for Oral Monoclonal Antibody Delivery Monoclonal antibodies (mAbs) are currently used to treat many conditions, including cancer, psoriasis, arthritis, and atopic dermatitis, among others. All mAbs are currently administered either intravenously or subcutaneously. Described herein is the use of the ionic liquid choline and glycolate (CGLY) as a platform for oral administration 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 for antibody transport. In vivo results in rats demonstrate that CGLY effectively delivers TNFα antibodies into the intestinal mucosa and the systemic circulation. A 1-week repeated-dose study, followed by histological examination and serum biochemistry analysis, showed that CGLY was well tolerated by rats. Overall, this study demonstrates the benefits of using a choline-based ionic liquid as an oral delivery platform for local and systemic delivery of therapeutic antibodies.

[0279] Therapeutic monoclonal antibodies (mAbs) are a very large class of protein-based therapeutic agents [1、2] There are more than 50 mAb-based products approved for commercial use and over 500 mAb-based therapies in clinical development. [3] Antibodies are used to treat a variety of diseases, including cancer, infectious diseases, inflammation, and autoimmune diseases. [1、4] However, mAbs are delivered as intravenous infusions or subcutaneous injections, which are associated with adverse effects such as systemic inflammatory reactions, infusion reactions, and poor 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 acceptability, and low manufacturing costs. In addition to providing a possible means for non-invasive systemic administration, oral administration also provides a means for localized delivery of antibodies into the gastrointestinal tract for the treatment of localized 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 results 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 allows the antibody to exhibit improved tolerance to intestinal proteases and resist degradation. In addition, the engineered anti-TNF antibody fragment also exhibits improved permeability to affected tissues in the GI tract.

[14] .

[0280] Described herein is an investigation of the potential of choline-based ILs for oral IgG delivery. To accomplish 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 Initial studies were conducted to evaluate the role of CGLY ionic stoichiometry on 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 saline at a range of 20–90% by volume. The IgG antibody was completely dissolved in all CGLY variants and concentrations, and no precipitation was observed. After 1 h of incubation at room temperature and 48 h of dialysis, the antibody samples were evaluated based on their antigen-binding capacity using ELISA (Figure 1B). CGLY 2:1 and CGLY 1:1 The IgG-CGLY formulations showed negligible effects on the intrinsic binding capacity of TNF-α IgG1 at CGLY concentrations up to 60% and 70% by volume, respectively. 1:2 The IgG antibody sample isolated from the IgG antibody induced a decrease in binding efficiency in the concentration range of 20 to 90% by volume.

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

[32] In the absence of CGLY, the model IgG appears as a single band at approximately 150 kDa (Figure 1D). IgGs from all CGLY variants are identified at the same band position. No other bands were observed above or below the 150 kDa band, suggesting that there was no detectable antibody fragmentation or aggregation from the formulation with CGLY. [32、33] Taken together, the antibody characterization results from ELISA, CD spectroscopy, and SDS-PAGE indicate that CGLY 2:1 and CGLY 1:1 have shown to have minimal effects on structure or antibody aggregation.

[0283] Effect of CGLY on Caco-2 cell viability and IgG transport Caco-2 cells are CGLY 2:1 and CGLY 1:1 was well tolerated, and no adverse effects on cell growth were observed up to high concentrations of >100 mM, whereas CGLY 1:2 CGLY reduced cell viability at fairly low concentrations (Fig. 2A). 2:1 , C.G.L.Y. 1:1 and CGLY 1:2 The IC50 values ​​of the compounds 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 across Caco-2 monolayers. These tests were performed using 30 mM CGLY, which is much lower than the IC50 of all CGLY variants. During the 5-hour test, FITC-IgG transport increased progressively over time in all CGLY groups, whereas no FITC-IgG transport was detected in control transwells without CGLY (Figure 2B). Notably, throughout all time points, CGLY was significantly more potent than any other CGLY variant. 2:1 showed the highest significant IgG transport. 2:1 The average IgG transport in monolayers treated with 2 So, this is CGLY 1:1 Treated cells (0.83 μg / cm 2 ) is more than twice as high as CGLY 1:2 Treated cells (1.06 μg / cm 2 ) was 1.6 times higher.

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

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

[0287] Paracellular and transcellular pathways are the main pathways involved in the transport of peptides and proteins across the intestinal epithelium. 2:1 To investigate the mechanism of IgG transport, we investigated the roles of both paracellular and transcellular transport. First, the paracellular pathway was assessed by transport of the paracellular transport marker Lucifer Yellow.

[34] The amount of transported Lucifer Yellow was 30–80 mM CGLY. 2:1 The effect was dramatically improved across all time points in cells treated with 30 mM CGLY (Figure 3B). 2:1 At the lowest range of 80 mM CGLY, Lucifer Yellow transport was enhanced approximately 2-fold. 2:1 At this concentration, Lucifer Yellow transport was enhanced 4-6 fold 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 assess tight junction integrity of Caco-2 monolayers and CGLY. 2:1 Paracellular involvement in accessory transport was further confirmed. For untreated wells, TEER measurements showed a slight increase within the range of 15% by the end of the 24-hour study, which is consistent with previous literature. [23、35] 30mM CGLY 2:1 The addition of CGLY reduced the TEER value by 11% in 1 hour, and the reduction was limited to a range of 11-16% in the first 5 hours. 2:1 The reduction in TEER due to CGLY was apparently temporary, with cells recovering 96% of their tight junction integrity within 24 hours. 2:1 The degree of decrease in TEER further increased with increasing concentration. 2:1TEER decreased by approximately 34% at 80mM CGLY 2:1 A 45% decrease in CGLY was observed, indicating tight junction opening. 2:1 The induced decrease in TEER still exhibited a transient behavior, and at 24 h, cells were treated with 55 mM and 80 mM CGLY. 2:1 The cells recovered 94% and 82% of the initial TEER values ​​at 30–80 mM CGLY, respectively. 2:1 The decrease and recovery of TEER measurements when treated with CGLY 2:1 These results suggest that CGLY may transiently open intestinal tight junctions and facilitate IgG transport across the intestinal epithelial barrier. 2:1 In the presence of CGLY, the increase in Lucifer Yellow transport and the decrease in TEER values ​​were 2:1 Characteristic paracellular transport was confirmed.

[0288] The contribution of the transcytosis pathway to IgG transport was assessed by the use of transcytosis inhibitors, including monodansylcadaverine (MDC; an inhibitor of clathrin-mediated endocytosis), filipin (an inhibitor of caveolae-mediated endocytosis), and wortmannin (an inhibitor of phosphatidylinositol 3-kinase, involved in micropinocytosis).

[36] The cumulative transport of FITC-IgG after 24 h of incubation showed no significant difference between cells treated with any of the inhibitors compared to the control without inhibitor (Figure 3D). This finding is consistent with the results of CGLY. 2:1 These results suggest that the improved delivery of FITC-IgG through Caco2 transwells by FITC-IgG was not primarily assisted through transcellular transport.

[0289] CGLY for mucus viscosity 2:1 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 effect of CGLY 2:1 The rheology of PIM treated with CGLY was evaluated. Figure 4A shows the rheology of PIM treated with CGLY at 0 to 50 vol%. 2:1Figure 1 shows the shear thinning properties of PIM samples after incubation with CGLY compared to untreated PIM. 2:1 The viscosity of the treated mucus showed a significant decrease throughout the shear range measured. For example, at a shear rate of 49.87 1 / s, the average viscosity of untreated PIM was measured to be 576.8 cP, which is comparable to previously reported literature values. [37、38] (Figure 4B). 12.5, 25, and 50% by volume of CGLY 2:1 The addition of CGLY significantly reduced the mucus viscosity to 317.9, 398.0, and 429.6 cP, respectively. 2:1 This ability may facilitate antibody delivery to the small intestinal epithelium.

[0290] CGLY 2:1 In vivo local and systemic antibody delivery of IgG by CGLY 2:1 FITC-IgG formulated in 50% by volume of CGLY was injected intrajejunally into Wistar rats. 2:1 Control rats received an equivalent saline injection with or without FITC-IgG. Two hours later, jejunal tissue was harvested and cryosections were prepared for imaging (Figures 5A-5C), and the fluorescent signal of FITC-IgG per unit area on the intestinal villi was quantified (Figure 5D). There was a significant difference in the FITC-IgG signal in the intestinal mucosa between the treatment groups. 2:1 The jejunal tissues of the treated group showed a significant signal of FITC-IgG in the intestinal villi (Figure 5B), and the measured fluorescent signal was CGLY 2:1 The FITC signal on the villi was more than 4.5-fold higher than that of the control without FITC (Figure 5D). On the other hand, for the control group with FITC-IgG in saline, the FITC signal on the villi was not significant compared to the negative control. Rather, the signal from FITC-IgG was strictly localized outside the villi, i.e., in the mucus layer (Figure 5C), indicating that the transport of IgG alone is severely impaired by the mucus barrier. This finding is consistent with the findings of CGLY. 2:1 These results show that IgG effectively enhances IgG permeation through intestinal mucus and epithelial layers.

[0291] In parallel studies, CGLY for enhancing IgG absorption was performed by measuring plasma IgG levels. 2:1 The benefits of using CGLY were assessed (Figure 5E). For the study, anti-human TNF-α IgG monoclonal antibody was used as a model antibody, and intrajejunal injection of CGLY was performed. 2:1 IgG concentrations increased gradually over the first 2 hours after injection. Between 3 and 5 hours of testing, CGLY 2:1 A significant increase in IgG concentration was observed in the treated group. 2:1 IgG concentrations in the treatment group were 5-fold higher than in the control group by the end of the study. Taken together, the excellent local FITC-IgG and plasma IgG concentration results demonstrated that CGLY 2:1 This study demonstrates that the IgG transporter allows the permeation and transport of IgG from the villi into the bloodstream. More importantly, the transported IgG was functionally preserved, as plasma IgG concentrations were detected by ELISA. Given the long circulating half-life of antibodies, repeated oral administration of IgG may sustainably increase blood concentrations and achieve much higher concentrations.

[0292] CGLY 2:1 In vivo toxicity evaluation of CGLY 2:1 The toxicity of CGLY was evaluated in adult male Wistar rats. 2:1 was orally administered at a dose of 625 mg / kg once daily for 7 consecutive days. Rats administered with saline were used as a negative control. 2:1 Rats treated with CGLY maintained a similar body weight compared to those treated with saline, and all rats showed a steady increase in body weight (Figure 6A). Neither group exhibited physiological symptoms such as lethargy, diarrhea, hunched posture, or unkempt fur. On day 7, the rats were sacrificed, and blood samples were collected for metabolic panel analysis. Major organs and gastrointestinal (GI) tissues were collected from the rats and stained with hematoxylin and eosin (H&E). 2:1Histological sections of the stomach, small intestine (duodenum, jejunum, and ileum), and colon from the treatment groups showed unchanged gastric and intestinal mucosal epithelial structures, including the size and number of crypts and villi, and mucosal thickness, compared with the saline control group (Figure 6B). There was no infiltration of immune cells, such as neutrophils, lymphocytes, or macrophages, into the mucosa, indicating no signs of tissue inflammation. H&E staining of major organs showed no hemorrhage, and CGLY 2:1 No differences were detected between the treatment groups and the saline control group (Figure 21). Comprehensive blood chemistry panel analysis showed no significant differences between the two groups (Figure 6C). 2:1 showed that CGLY did not produce any observable adverse effects on liver or kidney function in rats. 2:1 In vivo toxicity studies of CGLY showed no effects on rat body weight, blood metabolic panel or histopathological changes. 2:1 demonstrated safety for oral administration in a rat model.

[0293] conclusion Choline glycolate ILs with various ionic stoichiometries were synthesized. Among the CGLY variants, CGLY with a choline to glycolic acid molar ratio of 2:1 was synthesized. 2:1 demonstrated excellent cytocompatibility, IgG integrity preservation, and demonstrated the best ability for IgG antibody transport in vitro. 2:1 Further investigation of CGLY 2:1 can transiently disrupt intestinal tight junction integrity and inhibit the translocation of CGLY across Caco-2 cells 2:1 Enhanced IgG transport was revealed to occur via the paracellular pathway. 2:1 It is also possible to reduce the viscosity of mucus. 2:1 Intrajejunal administration of IgG from CGLY significantly improved antibody absorption into the rat intestinal villi, increasing the concentration of a model monoclonal antibody by up to five-fold compared to the negative control. 2:1 Treatment had no adverse effects on rat body weight, histological changes in the GI tract, or comprehensive metabolic panel of blood. Overall, this report demonstrates the efficacy of CGLY 2:1The promise and strength of this compound, along with its excellent biocompatibility, indicates that it is an oral delivery vehicle that can effectively improve both the local and systemic bioavailability of IgG antibodies.

[0294] Experimental section Materials: Glycolic acid, choline bicarbonate, dimethyl sulfoxide (DMSO), FITC-labeled immunoglobulin G (FITC-IgG, 20 mg / mL) from human serum, and 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 coating 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 gels, Tris / glycine / SDS running 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 the American Type Culture Collection (Manassas, VA, USA). Dulbecco's modified Eagle's medium (DMEM) with or without phenol red, fetal bovine serum (FBS), penicillin / streptomycin (P / S) solution, Hank's balanced salt solution (HBSS), Dulbecco's phosphate-buffered saline (DPBS), and 0.25% trypsin solution were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Basal seeding medium (BSM), enterocyte differentiation medium (EDM), and intestinal epithelial growth medium containing MITO + serum extender were purchased from Corning (Corning, NY, USA).Millicell®-PCF cell culture inserts (3.0 μm pore size, 12 mm diameter) and the TEER measurement device, Millicell®-ERS, were obtained from Millipore Sigma (Burlington, MA, USA), while the TEER measurement 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). Pig 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). BD lithium heparin-coated tubing was 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 variants and antibody-CGLY formulations: CGLY variants were synthesized as previously reported.

[27] Briefly, glycolic acid dissolved in the minimum amount of ultrapure water required for dissolution was reacted with choline bicarbonate (80 wt% solution) at 2:1, 1:2, and 1:2 molar ratios (choline:glycolic acid) at 40 °C with constant stirring for 12 h until CO evolution ceased. Residual water was removed by rotary evaporation at 20 mbar and 60 °C for 2 h, followed by drying in a vacuum oven at 60 °C for 48 h. Each CGLY formulation was characterized by nuclear magnetic resonance (NMR) spectroscopy. TIFF2026021383000016.tif39160 IgG-CGLY formulations were 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 the antibody in CGLY variants by ELISA, circular dichroism, and SDS-PAGE: To evaluate the antibody stability in CGLY variants, antibody-CGLY samples at 0.1 mg / mL anti-human TNF-α IgG antibody concentration were added to CGLY. 2:1 , C.G.L.Y. 1:1 and CGLY 1:2 The samples were incubated with or without 20–90% Tween 20 (vol / vol) at room temperature (25°C) for 1 hour 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 TNF-α IgG antibody-CGLY samples was assayed 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, after which serially diluted dialyzed anti-human TNF-α IgG antibody samples were added as the primary antibody. After a 2-hour incubation, the wells were washed three times with PBS containing 0.05% Tween 20 (PBST). HRP-conjugated goat anti-mouse IgG (Biolegend) was then used as the secondary antibody. Plates were incubated for 1 hour and then washed five times with PBST. ELISA plates were developed with TMB substrate (Biolegend) and 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 at 0.5 mg / mL anti-human TNF-α IgG antibody concentration were diluted with 50% by volume of CGLY. 2:1 , C.G.L.Y. 1:1and CGLY 1:2 The antibody samples were incubated with or without CGLY at room temperature (25°C) for 1 hour and then dialyzed for 48 hours in 10 mM pH 7.4 sodium phosphate buffer (Boston BioProducts). Prior to CD measurements, the antibody concentration was adjusted to 0.2 mg / mL. 400 μL of antibody sample was loaded into a rectangular quartz cell (1 mm path length, Starna Cells, 1-Q-1), and CD spectra in the far-UV region (190–250 nm), which indicates protein secondary structure, were collected using a CD spectrophotometer (Jasco J-1500). SDS-PAGE assays were performed to evaluate antibody aggregation in antibody-CGLY samples. Specifically, all samples were adjusted to equal antibody concentrations in Laemmli protein sample buffer. The samples were then separated on 4–15% 12-well precast polyacrylamide gels in Tris / glycine / SDS running buffer using a Mini-PROTEAN™ Tetra Cell Electrophoresis System (BioRad). Protein bands were stained for visualization with Bio-Safe™ Coomassie stain (BioRad) according to the manufacturer's protocol.

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

[0299] Caco-2 cell viability assessment of CGLY: Caco-2 cells suspended in supplemented DMEM were seeded at a density of 150,000 cells / mL and dispensed (100 μL / well) into a 96-well plate. 2:1 , C.G.L.Y. 1:1 and CGLY 1:2) was diluted in supplemented DMEM to concentrations ranging from 1.875 to 480 mM. The medium was aspirated from each well, and each dilution was dispensed (100 μL / well) into six wells (six cell replicates). Control wells were filled with medium alone. Cells were incubated with different concentrations of CGLY variants at 37°C and 5% CO2 for 5 hours, after which the medium was replaced with fresh DMEM (100 μL / well). Cells were grown for an additional 19 hours (up to a total of 24 hours). Cell viability was assessed using the Cell Titer 96 AQueous™ One Solution Cell Proliferation Assay (Promega Corporation) based on the compound MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium). Briefly, 20 μL of MTS reagent was added to each well, gently mixed, and incubated at 37°C for 4 hours. The absorbance of the 96-well plate was then read at 490 nm using a Spectramax i3 plate reader. The conversion of MTS tetrazolium to a formazan product, as measured by absorbance at 490 nm, is directly proportional to the number of viable cells. As suggested by the manufacturer's protocol, the average absorbance of the cell-free control wells was subtracted from all other experimental wells, and the percentage of cell viability was calculated by assuming that the average absorbance from wells containing untreated cells represented 100%.

[0300] Caco-2 Monolayer Culture Transwell: For transport experiments in transwells, a 3-day rapid Caco-2 expansion system was used. Cells were seeded at a density of 400,000 cells / mL in Corning® Basal Seeding Medium (BSM) supplemented with MITO serum + extender 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 basolateral side. After 24 hours of incubation at 37°C and 5% CO2, the medium was replaced with the same volume of MITO serum + extender-supplemented enterocyte differentiation medium and cultured for an additional 2–4 days. TEER was measured periodically and maintained at 200 Ω.cm, indicating sufficient tight junction integrity between cells. 2 If it exceeded this limit, a transport test was conducted.

[0301] FITC-IgG and Lucifer Yellow transport across Caco-2 monolayer transwells: Prior to the experiment, Caco-2 transwells were washed twice with HBSS and then incubated with DMEM without phenol red, FBS, or P / S for 30 minutes on both the apical (400 μL) and basolateral (600 μL) sides. The apical media was then 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 DMEM without phenol red, FBS, or P / S. Immediately after adding FITC-IgG to the apical side, a 150 μL aliquot was removed from the basolateral side and replaced with an equal volume of fresh DMEM. This procedure was repeated at 1, 2, 3, 4, and 5 h. During the test, the transwell plate was placed in a 37°C, 5% CO2 incubator on a rotating shaker at 100 rpm, and removed only to collect aliquots at the aforementioned time points. At the end of the 5-hour test, the FITC-IgG and Lucifer Yellow concentrations in the aliquots 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 FITC-IgG and Lucifer Yellow concentrations at each time point were calculated from calibration solutions of each fluorescent molecule and then plotted as basolateral chamber concentrations versus time.

[0302] For quantitative analysis of FITC-IgG uptake by Caco-2 cells, transwells from the FITC-IgG transport study were washed twice with HBSS at the end of the study, followed by the addition of 500 μL of 4% paraformaldehyde and maintaining overnight at 4°C. The following day, 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 covered with a coverslip. Confocal imaging of the membranes (ZEISS, Laser Scanning Confocal Microscope LSM 700) was performed at 40x magnification.

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

[0304] FITC-IgG transport with transcytosis inhibitors: Before the experiment, Caco-2 transwells were washed twice with HBSS and then incubated on both the apical (400 μL) and basolateral (600 μL) sides with phenol red, FBS, and P / S-free DMEM for 30 min, followed by transcytosis inhibitors containing 50 μM monodansylcadaverine (MDC), 1 μg / mL filipin, and 0.5 μM wortmannin.

[36] 500 μg / mL FITC-IgG, 55 mM CGLY, with or without 2:1 The apical medium was replaced with 400 μL of phenol red-, FBS-, and P / S-free DMEM containing 150 μL of FITC-IgG. After 24 h of incubation, 150 μL aliquots were removed from the basolateral side, and the FITC-IgG concentration in the aliquots 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 control wells that did not contain any transcytosis inhibitors.

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

[39] Porcine mucus was pooled and immediately examined. 10 μL of CGLY at 0, 12.5, 25, and 50% by volume in 0.9% saline was added to 200 μL of porcine mucus aliquots, and measurements were performed at 25°C over a shear rate range of 1 to 100 1 / s using an AR-G2 rheometer (TA Instruments, New Castle, DE, USA) with a 40 mm diameter steel parallel plate geometry.

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

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

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

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

[0308] In vivo toxicity testing: CGLY in vivo 2:1 To evaluate the acute toxicity of CGLY, adult male Wistar rats (n=6, each weighing 275-300 g) were dosed with 50% by volume of CGLY. 2:1Rats were orally administered saline at a dose of 625 mg / kg once daily for 7 consecutive days using the procedure described above. Control rats received an equivalent volume of saline. Rat body weights were monitored daily throughout the experimental period. On day 7, rats were sacrificed, blood samples were collected 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% formalin (vol / vol) for 18 hours, dehydrated in 70% ethanol, and then embedded in paraffin. Tissue sections were cut at 5 μm thickness, 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 SDS PAGE studies, experiments were performed in triplicate and representative images are shown. For fluorescence and bright-field imaging, experiments were performed in triplicate and representative images are shown. All other experiments were performed at least in triplicate. To test for statistical significance, an unpaired two-tailed t-test was performed in GraphPad Prism 8™, and a confidence level of P = 0.05 was considered significant.

[0310] References TIFF2026021383000017.tif71154TIFF2026021383000018.tif232160TIFF2026021383000019.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 antibodies than larger anions. Performance was maintained with different individual antibodies (Figure 14).

[0312] Oral or intrajejunal antibody delivery was also tested (Figs. 11-13). ILs were found to be non-toxic when administered orally (Figs. 15-16).

[0313] IL for siRNA delivery was also contemplated and transdermal delivery of siRNA was tested (FIGS. 17-18).

[0314] Example 3 Systemic antibodies targeting tumor necrosis factor α (TNF-α) and interleukin-17A (IL-17A) are effective in treating plaque psoriasis. Despite their popularity, safety concerns pose challenges for systemic biologics. While anti-TNF-α and anti-IL-17A antibodies effectively inhibit their respective proteins, we hypothesized that an approach based on local silencing of upstream targets, such as NFKBIZ, would be advantageous for the treatment of 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 an enabling technology, we describe herein the effective delivery of NFKBIZ siRNA to the skin and its therapeutic efficacy in a psoriasis model. Treatment with IL-siRNA suppressed aberrant gene expression and downregulated 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 the United States an estimated $135 billion annually (1). Its etiology and underlying mechanisms are still not fully understood. Nuclear factor κB (NF-κB), a widely expressed transcription factor, is considered a master regulator of immune responses and has been implicated in several autoimmune and inflammatory diseases, including psoriasis (2). Although several therapies targeting the NF-κB signaling pathway are available in the clinic, lack of specificity and concerns about side effects pose challenges (3). This challenge is particularly challenging because pleiotropic proteins like NF-κB provide essential basal activity as survival factors, and their systemic inhibition can lead to severe side effects. Network-centric approaches, including pathway-specific inhibitors, have gained 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 US Food and Drug Administration and are claimed to mediate their therapeutic effects through modulation of NF-κB activity ( 5 ).

[0316] NFKBIZ, a gene encoding the atypical inhibitor of nuclear factor κB (IκB) protein IκBζ, has gained interest in therapeutic intervention due to its critical 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, which are involved in inflammatory signaling, neutrophil chemotaxis, and leukocyte activation (8-11). In addition, strong expression of NFKBIZ in psoriasis patients may correlate with elevated IL-36 and IL-17A responses (12). Local silencing of NFKBIZ may be advantageous because it could potentially broaden the patient population that could benefit from treatment compared to treatment with a single antibody.

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

[0318] Described herein is a modular IL-based siRNA delivery approach for silencing various genes of interest.Specifically, described herein is a combination of IL, which simultaneously stabilizes siRNA and enhances the penetration of siRNA into the skin after topical application.The effectiveness of this formulation is shown in silencing NFKBIZ in vivo in imiquimod-induced psoriasis mouse model.

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

[0320] Effect of ILs on siRNA stability The effect of ILs on siRNA stability was evaluated. Circular dichroism (CD) spectroscopy of siRNA incubated with 50% (v / v) aqueous solutions of individual ILs revealed significant changes in the α-helical backbone in the presence of CAGE, CADA, and CABA (as confirmed by negative bands at 210 nm). Meanwhile, CAVA and CAPA preserved the secondary structure of siRNA (Figure 19A). The bands obtained from native gel electrophoresis were complemented by CD results (Figure 19B). The improved siRNA stability in the presence of CAPA suggested a possible synergistic effect between ILs prepared from two structurally distinct anions. Therefore, to determine whether CAPA's compatibility with siRNA provides additional protection against the deleterious effects of CAGE and CABA on siRNA structure, the effect of IL mixtures on siRNA stability was evaluated. The combination of CAGE (25% v / v) with CAPA (25% v / v) resulted in a prominent band indicating the retention of siRNA structure (Figures 24A–24E).

[0321] Screening of optimal IL combinations for siRNA delivery Individual ILs and their combinations were then evaluated for epidermal penetration of Cy5-labeled siRNA into porcine skin in Franz diffusion cells (FDC) (Figure 19C). In controls, some epidermal uptake of naked siRNA was observed. CAGE demonstrated the highest delivery of all ILs tested (Figure 19D). Approximately 0.20 nmol / cm2 of siRNA was delivered to the epidermis in the presence of CAGE (50% by volume), compared with 0.07 nmol / cm2 for naked siRNA. Because 50% CAGE may have an effect on siRNA structure, the ability of IL combinations to deliver siRNA to the skin was also measured. A combination of CAPA and CAGE (25% by volume each) delivered approximately 0.4 nmol / cm2 of siRNA to the skin (Figure 19E). The CAGE+CAPA combination resulted in the highest epidermal delivery and high stability, and was therefore selected as the lead formulation for further testing (Figures 25A-25D).

[0322] IL-induced intercalation and solvation effects on RNA To explore the mechanism by which the IL combination (CAGE+CAPA) stabilizes RNA, we performed molecular dynamics (MD) simulations. Fr...

Claims

1. (a) a carboxylic acid that is not a fatty acid, (b) Carboxylic acids containing aliphatic chains of four carbons or less; (c) an aromatic anion, and / or (d) Anions with a LogP of less than 1.0 and an anion that is at least one of Quaternary ammonium-containing cations 1. A composition comprising at least one ionic liquid, comprising:

2. The anion is has a LogP less than 1.0, and a. non-fatty acid carboxylic acids, b. Carboxylic acids containing an aliphatic chain of four carbons or less, or c. Aromatic anions That is, A composition according to any one of the preceding claims.

3. 10. The composition of claim 1, wherein the fatty acid comprises an aliphatic chain of 3 carbons or less.

4. 2. The composition of claim 1, wherein the anion comprises only one carboxylic acid group (e.g., an R-COOH group).

5. The anions are geranic acid; glycolic acid; propanoic acid; isobutyric acid; butyric acid; gallic acid; lactic acid; malonic acid; Maleic acid; glutaric acid; citric acid; 3. The composition of any one of the preceding claims, wherein the carboxylic acid is selected from the group consisting of 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; hydrocinnaminic acid (phenylpropanoic acid); phenylphosphoric acid; and biphenyl-3-carboxylic acid.

6. 10. The composition of any one of the preceding claims, 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); phenylphosphoric acid; and biphenyl-3-carboxylic acid.

7. 10. The composition of claim 9, wherein the cation has a molar mass equal to or greater than that of choline.

8. Quaternary ammonium is NR 4 + and at least one R group comprises a hydroxy group.

9. Quaternary ammonium is NR 4 + and wherein only one R group contains a hydroxy group.

10. 10. The composition of claim 1, wherein the cation is choline, C1, C6, or C7.

11. 10. The composition of claim 1, wherein the cation is choline.

12. 10. The composition of claim 1, wherein the cation is C1, C6, or C7.

13. 10. The composition of any one of the preceding claims, wherein the ionic liquid comprises a ratio of cations to anions of about 2:1 to about 1:

1.

14. 10. The composition of claim 1, wherein the ionic liquid comprises a ratio of cations to anions of about 2:

1.

15. 10. The composition of claim 1, wherein the ionic liquid has a cation:anion ratio of less than 1:

1.

16. 10. The composition of claim 9, wherein the ionic liquid has a cation:anion ratio in cation excess.

17. 10. The composition of any one of the preceding claims, comprising a first ionic liquid and at least a second ionic liquid.

18. 18. The composition of claim 17, wherein each ionic liquid has a choline cation.

19. 19. The composition of any one of claims 17-18, wherein the first ionic liquid and the second ionic liquid each comprise a different anion.

20. The first ionic liquid and the second ionic liquid are, respectively, geranic acid; glycolic acid; propanoic acid; isobutyric acid; butyric acid; gallic acid; lactic acid; malonic acid; Maleic acid; glutaric acid; citric acid; 20. The composition of claim 19, comprising different anions selected from 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); phenylphosphoric acid; and biphenyl-3-carboxylic acid.

21. 21. The composition of any one of claims 17 to 20, wherein the first ionic liquid has a geranate anion and the second ionic liquid has a phenylpropanoate anion.

22. 22. The composition of any one of claims 17 to 21, wherein the first ionic liquid is choline and geranic acid (CAGE).

23. 23. The composition of any one of claims 17-22, wherein the second ionic liquid is choline and dimethylacrylic acid (CADA); choline and isovaleric acid (CAVA); choline and phenylphosphonic acid (CAPP); choline and biphenyl-3-carboxylic acid (CABA); choline and 4-phenolsulfonic acid (CASA); or choline and phenylpropanoic acid (CAPA).

24. 22. The composition of any one of claims 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 phenylphosphonic acid (CAPP); choline and biphenyl-3-carboxylic acid (CABA); choline and 4-phenolsulfonic acid (CASA); or choline and phenylpropanoic acid (CAPA).

25. 22. The composition of any one of claims 17-21, 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. 23. The composition of any one of claims 17-22, wherein the first ionic liquid is choline and geranic acid (CAGE) and the second ionic liquid is choline and phenylpropanoic acid (CAPA).

27. 10. The composition of any one of the preceding claims, further comprising at least one active compound in combination with at least one ionic liquid.

28. 10. The composition of any one of the preceding claims, wherein the active compound comprises a polypeptide.

29. 29. The composition of claim 28, wherein the polypeptide is an antibody or antibody reagent.

30. 30. The composition of any one of claims 28 to 29, wherein the active compound has a molecular weight greater than 450.

31. The composition of any one of claims 28 to 30, wherein the active compound has a molecular weight greater than 500.

32. The anion is has a LogP less than 1.0, and a. a carboxylic acid that is not a fatty acid; or b. Carboxylic acids containing aliphatic chains of four carbons or less That is, The composition of any one of claims 28 to 31.

33. 10. The composition of any one of the preceding claims, wherein the active compound comprises a nucleic acid.

34. 34. The composition of claim 33, wherein the nucleic acid is an inhibitory nucleic acid.

35. 35. The composition of claim 34, wherein the nucleic acid is an siRNA.

36. The composition of any one of claims 34 to 35, wherein the inhibitory nucleic acid is an NFKBIZ, TNFα, and / or IL-17 inhibitory nucleic acid.

37. The anion is has a LogP less than 1.0, and a. a carboxylic acid that is not a fatty acid; or b. Carboxylic acids containing aliphatic chains of four carbons or less; and / or c. Aromatic anions That is, The composition of any one of claims 33 to 36.

38. 10. The composition of any one of the preceding claims, wherein the ionic liquid is at a concentration of at least 0.1% w / v.

39. 10. The composition of any one of the preceding claims, wherein the ionic liquid is at a concentration of about 10 to about 70% w / v.

40. 10. The composition of any one of the preceding claims, wherein the ionic liquid is at a concentration of about 30 to about 50% w / v.

41. 10. The composition of any one of the preceding claims, wherein the ionic liquid is at a concentration of about 30 to about 40% w / v.

42. 10. The composition of any one of the preceding claims, formulated for transdermal, mucosal, oral, subcutaneous, intradermal, parenteral, intratumoral, or intravenous administration.

43. 43. The composition of claim 42, formulated for transdermal administration.

44. 43. The composition of claim 42, wherein the mucosa is a nasal mucosa, an oral mucosa, or a vaginal mucosa.

45. 10. The composition of any one of the preceding claims, wherein the active compound is provided in a dose of 1 to 40 mg / kg.

46. 10. The composition of any one of the preceding claims, further comprising at least one non-ionic surfactant.

47. 10. The composition of any one of the preceding claims, further comprising a pharmaceutically acceptable carrier.

48. 10. The composition of any one of the preceding claims, provided in a degradable capsule.

49. 10. The composition of any one of the preceding claims, which is an admixture.

50. 10. The composition of any one of the preceding claims, provided in one or more nanoparticles.

51. 10. The composition of claim 1, comprising one or more nanoparticles comprising an active compound, said nanoparticles being in solution or suspension in a composition comprising an ionic liquid.

52. 52. A method of administering at least one active compound to a subject, comprising administering the composition of any one of claims 27-51.

53. 53. The method of claim 52, wherein the composition is administered once.

54. 54. The method of any one of claims 52-53, wherein the composition is administered in multiple doses.

55. 55. The method of any one of claims 52-54, wherein the administration is transdermal, mucosal, oral, subcutaneous, intradermal, parenteral, intratumoral, or intravenous.

56. The method of any one of claims 52-55, wherein the composition comprises an NFKBIZ, TNFα, and / or IL-17 inhibitory nucleic acid and the subject is in need of treatment for an inflammatory condition.

57. 52. A method of treating an inflammatory condition in a subject in need thereof, said method comprising administering to the subject the composition of any one of claims 36-51.

58. 58. The method of any one of claims 56-57, wherein the administration is topical.

59. 59. The method of any one of claims 56-58, wherein the inflammatory condition is psoriasis.

60. 52. The composition of any one of claims 27 to 51 for use in a method of administering at least one active compound to a subject.

61. 61. The composition of claim 60, administered once.

62. 61. The composition of claim 60, administered in multiple doses.

63. 63. The composition of any one of claims 60-62, wherein the administration is transdermal, mucosal, oral, subcutaneous, intradermal, parenteral, intratumoral, or intravenous.

64. 64. The composition of any one of claims 60-63, comprising an NFKBIZ, TNFα, and / or IL-17 inhibitory nucleic acid, and wherein the subject is in need of treatment for an inflammatory condition.

65. 52. The composition of any one of claims 36 to 51 for use in a method of treating an inflammatory condition in a subject in need thereof.

66. 66. The composition of any one of claims 64-65, wherein the administration is topical.

67. 67. The composition of any one of claims 64-66, wherein the inflammatory condition is psoriasis.

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