Novel liver-targeting glucocorticoid prodrugs

Abstract

Glucocorticoids (GCs) are widely used in the treatment of inflammatory liver diseases and sepsis, but GC's various side effects on extrahepatic tissues limit their clinical benefits. Liver-targeting GC therapy may have multiple advantages over systemic GC therapy. Embodiments disclose herein are directed to novel liver-targeting GC prodrugs that provide improved methods for the treatment of inflammatory liver diseases and sepsis.

Description

NOVEL LIVER-TARGETING GLUCOCORTICOID PRODRUGSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 596,840, filed November 7, 2023. The content of this this earlier filed application is hereby incorporated by reference herein in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under grant numbers AA027349 and CA241781, awarded by the National Institute of Health. The government has certain rights in the invention.

[0004] BACKGROUND OF THE INVENTIONField of the Invention

[0005] Alcoholic liver disease (ALD) is a major cause of death worldwide.Patients with alcoholic hepatitis (AH) have severe inflammation and cholestatic liver injury, and severe AH (sAH) has a high mortality rate. Glucocorticoids (GCs) are currently the only available drug therapy for sAH, however, prednisolone, the current standard of care for sAH, only marginally reduces 28-day mortality without long-term improvement. Additionally, sepsis is a leading cause of death of sAH, and hepatic GR (glucocorticoid receptor) is essential to protect against liver failure and mortality in sepsis. GCs are also frequently used in the treatment of sepsis to control the hyperinflammation. GC treatment improves liver failure in patients with sepsis.However, there is no clear survival benefit for GC treatment of sepsis patients. Thus, there is an urgent unmet need to improve the current GC therapy of sAH and sepsis.

[0006] The markedly impaired urea synthesis in AH is associated with disease severity and hepatic encephalopathy. Glucocorticoid receptor (GR) controls hepatic urea cycle, and prednisolone restores urea synthesis in survivors of sAH.Additionally, cholestasis contributes to malnutrition and AH severity. Activation of GR in hepatocytes protects against apoptosis and inflammation. Activation of GR in hepatocytes induces the key bile acid (BA) transporters Na (+)-taurocholate transportprotein (NTCP) and bile salt export pump (BSEP), and GCs protect against cholestatic liver injury and steatohepatitis in patients and mice. These he pato protective and anti-inflammatory effects of GR activation on hepatocytes likely underly the benefits of current GC therapy in sAH and sepsis.

[0007] Long-term GC treatment can cause various adverse effects that limit AH therapy of sAH, the severity of which is proportional to the dose and duration of GC treatment. Activation of GR in extrahepatic tissues can promote alcohol consumption and psychiatric problems, adipose lipolysis, intestinal reabsorption of bile acid (BA) and cholestasis, gastrointestinal bleeding, and wasting of skeletal muscle. A recent study shows that activation of GR in intestinal epithelia cells aggravates alcoholic steatohepatitis and liver injury due to increased intestinal epithelial permeability and gut dysbiosis. Moreover, high doses of GC can aggravate alcoholic liver injury and impair liver regeneration by inhibiting macrophage-mediated phagocytic and hepatic regenerative functions. In contrast, GR deficiency in hepatocytes delays liver regeneration. Therefore, extrahepatic and hepatic non- parenchymal side effects of GCs are likely the major factors that limit the therapeutic efficacy of GC therapy during high-dose and long- term GC treatment of sAH.

[0008] Vamorolone, a dissociative GC that only maintains GC’s transrepression activity, was developed to ameliorate GCs side effects; however, it has weaker antiinflammatory effects and increases hepatic necrosis in mice with sick cell disease. In contrast to GR, activation of mineralocorticoid receptor (MR) by GCs promotes steatohepatitis and fibrosis, vascular damage, and acute kidney injury (AKI). Prednisolone, the current standard of care for sAH, strongly activates MR at 100 nM. Thus, sAH patients who are resistant to GR-mediated anti-inflammatory and cytoprotective effects will have elevated risk of the prednisolone-MR-mediated side effects such as AKI. In contrast, dexamethasone (DEX), a highly potent and selective GR agonist, prevents severe AKI in patients with kidney diseases. Budesonide is a 2nd generation GC with extensive hepatic first-pass metabolism and limited systemic exposure. In a small clinical study, budesonide has similar efficacy and diminished side effects in sAH patients compared to prednisolone. This provides a key proof of concept for our liver-targeting GC prodrug as improved sAH therapy. However, oral GCs, even for short-term treatment, increase the risk of Gl bleeding. Moreover, GCs promote intestinal BA reabsorption, and activation of GR inintestinal epithelia disrupts intestinal permeability and gut microbiome and worsens steatohepatitis in ALD.

[0009] Therefore, liver-specific delivery of GCs to activate hepatic GR improves GC therapy of sAH by ameliorating the adverse effects of GCs on extrahepatic tissues and immunosuppression. BA-drug conjugates with a hydrophilic linker for liver-specific uptake by the liver-specific BA transporter NTCP have been synthesized as oral prodrugs for liver-specific drug delivery. DEX is a lipophilic molecule, which compromises the liver-selectivity of prodrug. We further introduced ultra-hydrophilic zwitterionic moiety as a flexible linker to dramatically increase the water solubility of BA-drug conjugates to reduce the passive nonspecific diffusion into cells and maintain the high substrate specificity for NTCP.

[0010] As more fully discussed herein below, our novel liver-targeting GC prodrugs are characterized by comparing the differential effects of our liver-targeting GC prodrugs and the parent drug DEX on primary human hepatocytes (PHH), mouse macrophages, human whole blood, and mice with sepsis. Our results showed that compared to the parent drug DEX, our liver-targeting GC prodrugs demonstrated high water solubility, transporter-dependent cellular activity, less suppressive effects on immune cells, and better anti-inflammatory effects on the livers of septic mice.BRIEF SUMMARY OF THE INVENTION

[0011] Embodiments disclosed herein are directed to methods of treating inflammatory liver diseases in a subject in need thereof. The methods include administering a therapeutically effective amount of a prodrug composition having the following formula:wherein, TL is a targeting ligand, L is a multivalent linkage, D is a drug, Z is a zwitterionic group, and n, m, and p are integers from 1 to about 4.

[0012] Embodiments disclosed herein are directed to methods of treating cancer in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of at least one of the prodrug compositions disclosed herein. Additional embodiments are directed to methods of treating immune system diseases in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of at least one of the prodrug compositions herein.

[0013] Embodiments disclosed herein are directed to methods of treating inflammatory liver disease, wherein the inflammatory liver disease is alcoholic hepatitis (AH), severe alcoholic hepatitis (sAH), and / or autoimmune hepatitis. Further embodiments include methods of treating inflammatory liver disease, wherein the inflammatory liver disease further comprises sepsis.

[0014] Further embodiments disclosed herein are directed to novel prodrug compositions having targeting ligands (TL) that include bile acids (BA) and cholic acid (CA); multivalent linkages (L), which are used to prepare the prodrug compositions that include oligo amino acids and orthogonal and orthogonally protected tri-functional aromatic molecule; drugs (D), which are used to prepare the prodrug compositions that include glucocorticoid (GC), such as dexamethasone, prednisone, prednisolone, cortisone, hydrocortisone, and fludrocortisone; and zwitterionic groups (Z), which are used to prepare the prodrug compositions that include glycerophosphorylcholine (GPC), carboxylic betaine (CB), carnitine, choline phosphate (CP), sulfobetaines (SB), and trimethylamine N-oxide (TMAO). Other embodiments disclosed herein are directed to multivalent linkages (L) that include an oligolysine having from about 1 to about 16 lysine residues.

[0015] Further embodiments disclosed herein are directed to prodrug compositions wherein the prodrug compositions assemble into micelles having the targeting ligand and the zwitterionic groups the outside of the micelles and the therapeutic drug(s) resides within the inside of the micelles.

[0016] Further embodiments disclosed herein are directed to prodrug compositions that are comprised of nanoparticles having a diameter size from about 10 nm to about 20 nm.

[0017] Embodiments disclosed herein are directed to novel prodrug compositions, such as, CA-GPC-DEX, which has a MW of 1606.8, and the following structure:

[0018] CA-GPC-DEX can be identified as: (10S,17S,22S,27R)-17-carbamoyl-22- carboxy-10-(4-(4-(2-((8S,9R, 10S, 11 S, 13S, 14S, 16R, 17R)-9-fluoro-11 , 17-dihydroxy- 10,13,16-trimethyl-3-oxo-6,7,8,9,10,11 ,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)-4-oxobutanamido)butyl)-2-hydroxy- 5,8,11 ,19,24-pentaoxo-27-((3R,5S,7R,9S, 1 OS, 12S, 13R, 14S, 17R)-3,7, 12-trihydroxy- 10,13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)-4-oxa- 9,12,18,23-tetraazaoctacosyl (2-(trimethylammonio)ethyl) phosphate.

[0019] Embodiments disclosed herein are further directed to novel prodrug compositions, such as, CA-CB-DEX, which has a MW of 1438.78, and the following structure:

[0020] CA-CB-DEX can be identified as: (9S,16S,21S,26R)-16-carbamoyl-21- carboxy-9-(4-(4-(2-((8S,9R, 10S, 11 S, 13S, 14S, 16R, 17R)-9-fluoro-11 , 17-dihydroxy- 10, 13, 16-trimethyl-3-oxo-6,7,8,9, 10,11 ,12,13,14,15,16, 17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)-4-oxobutanamido)butyl)-3,3-dimethyl- 7,10,18,23-tetraoxo-26-((3R,5S,7R,9S, 1 OS, 12S, 13R, 14S, 17R)-3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl )-3 , 8 , 11 ,17,22- pentaazaheptacosan-3-iumoate.

[0021] The novel prodrug compositions incorporate ultra-hydrophilic zwitterionic groups into the design of the liver-targeting GC prodrugs, which markedly increases the water solubility and decrease the non-specific passive diffusion of GC prodrug across cell membranes, leading to markedly improved liver-specific delivery of GCs and the resultant enhanced anti-inflammatory effects on the liver and decreased immunosuppression on blood cells and side effects on extrahepatic tissues.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0022] FIG. 1 presents chemical structure of DEX prodrugs CA-DEX, CA-GPC- DEX, and CA-CB-DEX.

[0023] FIG. 2 presents the route of synthesis of DEX-COOH (A), CA-DEX (B), GPC-COOH (C), CA-GPC-DEX (D), and CA-CB-DEX (E).

[0024] FIG. 3 presents MALDI-TOF mass spectrum of CA-DEX (A), CA-GPC- DEX(B), CA-CB-DEX(C) and intermediates (D-J).

[0025] FIG. 4 presents the roles of transporters in mediating transactivation of BSEP promoter by bile acid- dexamethasone conjugates. HEK293 cells were cotransfected with BSEP promoter reporter vector, pRL-CMV reporter vector, and expression vectors for FXR, OATP1B1 / 1B3 (1B1 / 3), ASBT, and / or NTCP. Medium was replaced with chemical-containing medium 18 h after transfection and dualluciferase assay was conducted 6 h after chemical treatment. N = 4 per group, mean ± SE. * p < 0.05 versus control.

[0026] FIG. 5A presents the effects of dexamethasone (DEX) and DEX prodrugs on 111 b mRNA expression (qPCR) in mouse RAW264.7 cells stimulated with lipopolysaccharides (LPS) for 6 h; N = 3, mean ± SE. * p < 0.05 versus LPS. FIG. 5B - FIG. 5H present the effects of Prednisolone (Pred, 0.75 pM), DEX (0.1 pM), CA-GPC-DEX (GPC, 0.1 pM), and CA-CB-DEX (CB, 0.1 pM) on mRNA expression (qPCR) of TNF (FIG. 5B), IL6 (FIG. 5C), IL1B (FIG. 5D), IL1RN (FIG. 5E), IL10 (FIG. 5F), GILZ (FIG. 5G) and release of TNF (FIG. 5H) and IL6 (FIG. 5I) proteins in human whole blood 4 h (at 37 °C) after stimulation with LPS (2 ng / ml). N = 4 per group, mean ± SE. * p < 0.05 versus LPS; # p < 0.05 versus DEX.

[0027] FIG. 6A presents data mining of microarray analysis (GSE28619) of mRNAs in humans with severe AH (normalized to P-actin). Mean ± SE. N = 7-15 pergroup. FIG. 6B presents qPCR analysis of mRNAs in primary human hepatocytes (PHH) treated with 1 μM DEX, 1 μM CA-CB-DEX, or vehicle (0.1 % DMSO) for 6 h, normalized to GAPDH. N = 3 per group, mean ± SE. * p < 0.05 versus normal livers (FIG. 6A) or control PHH (FIG. 6B).

[0028] FIG. 7A presents the effects of CA-CB-DEX and DEX on weight loss, FIG. 7B presents blood glucose, FIG. 7C presents blood lymphocytes, FIG. 7D- FIG. 7F present qPCR quantification of hepatic mRNA expression (normalized to PGK1 ) 24 h after surgery in adult male sham and / or CLP mice ip injected CA-CB-DEX (0.25 pmole / kg), DEX (0.25 pmole / kg), or vehicle (VEH, 0.5 % DMSO 10 ml / kg) 3 h after surgery. N = 3 (sham groups) or 6 (CLP groups) per group, mean ± SE. * p < 0.05 versus sham control. # p < 0.05 versus VEH CLP group.

[0029] FIG. 8 is a Table listing the abbreviations used to describe the various embodiments presented herein.

[0030] FIG. S1 A presents a TLC analysis of the purity of CA-CB-DEX; FIG. S1 B presents a determination of the size of CA-CB-DEX nanoparticles by dynamic light scattering; and FIG. S1C presents a determination of the CMC of CA-CB-DEX nanoparticles by fluorescence of Nile Red.DEFINITIONS

[0031] As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0032] As used herein, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “a compound” include the use of one or more compound(s).

[0033] As used herein the terms “about,” “approximately,” and the like, when used in connection with a numerical variable, generally refers to the value of the variable and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval [Cl 95%] for the mean) or within ±10% of the indicated value, whichever is greater.

[0034] As used herein the term “excipient” or “adjuvant” refers to any inert substance.

[0035] As used herein the terms “drug product,” “pharmaceutical dosage form,” “dosage form,” “final dosage form” and the like, refer to a pharmaceutical compositionthat is administered to a subject in need of treatment and generally may be in the form of inhalers, tablets, capsules, sachets containing powder or granules, liquid solutions or suspensions, patches, and the like.

[0036] As used herein the term “pharmaceutically acceptable” substances refers to those substances, which are within the scope of sound medical judgment suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, and effective for their intended use.

[0037] As used herein the term “pharmaceutical composition” refers to the combination of one or more drug substances, one or more excipients, and one or more pharmaceutically acceptable vehicles with which the one or more drugs is administered to a subject.

[0038] As used herein, the term "amino acid" refers to a carboxylic acid bearing an amine functional group. Amino acids include the diamino carboxylic acids described above. Amino acids include naturally occurring .alpha. -amino acids, wherein the amine is bound to the carbon adjacent to the carbonyl carbon of the carboxylic acid. Examples of naturally occurring .alpha.-amino acids include, but are not limited to, L- aspartic acid, L-glutamic acid, L-histidine, L-lysine, and L-arginine. Amino acids may also include the D-enantiomers of naturally occurring .alpha.-amino acids, as well as .beta. -amino acids and other non-naturally occurring amino acids.

[0039] As used herein, the term "lipid" refers to lipid molecules that can include fats, waxes, steroids, cholesterol, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, phospholipids, sphingolipids, glycolipids, cationic or anionic lipids, derivatized lipids, and the like, as described in detail below. Lipids can form micelles, monolayers, and bilayer membranes.

[0040] As used herein, the term "hydrophobic group" refers to a chemical moiety that is water-insoluble or repelled by water. Examples of hydrophobic groups include, but are not limited to, long-chain alkanes and fatty acids, fluorocarbons, silicones, certain steroids such as cholesterol, and many polymers including, for example, polystyrene and polyisoprene.

[0041] As used herein, the term "hydrophilic group" refers to a chemical moiety that is water-soluble or attracted to water. Examples of hydrophilic groups include, but are not limited to, alcohols, short-chain carboxylic acids, quaternary amines, sulfonates, phosphates, sugars, and certain polymers such as polyethylene glycol) (PEG).

[0042] As used herein, the term "amphiphilic compound" refers to a compound having both hydrophobic portions and hydrophilic portions. For example, the amphiphilic compounds of the present invention can have one hydrophilic face of the compound and one hydrophobic face of the compound. Amphiphilic compounds useful in the present invention include, but are not limited to, cholic acid and cholic acid analogs and derivatives, and cholesterol formate.

[0043] As used herein, the term "cholic acid" refers to (R)-4-((3R, 5S, 7R, 8R, 9S, 10S, 12S, 13R, 14S, 17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1 H- cyclopenta[a]phenan- thren-17-yl)pentanoic acid. Cholic acid is also known as 3.alpha. ,7. alpha., 12. alpha. -trihydroxy-5. beta. -cholanoic acid; 3-.alpha.,7-.alpha.,12- .alpha.-Trihydroxy-5-.beta.-cholan-24-oic acid; 17-.beta.-(1-methyl-3- carboxypropyl)etiocholane-3. alpha. ,7. alpha., 12.alph- a.-triol; cholalic acid; and cholalin. Cholic acid derivatives and analogs, such as allocholic acid, pythocholic acid, avicholic acid, deoxycholic acid, and chenodeoxycholic acid are also useful in the present invention.

[0044] As used herein, the terms "drug" or "therapeutic agent" refers to an agent capable of treating and / or ameliorating a condition or disease. Some non-limiting examples of prednisone, prednisolone, dexamethasone. Hydrocortisone, cortisone, betamethasone, prednisone, prednisolone, methylprednisolone, triamcinolone, dexamethasone, and fludrocortisone. One of skill in the art will appreciate that other drugs are useful in the present invention.

[0045] As used herein, "forming a reaction mixture" refers to the process of bringing into contact at least two distinct species such that they mixed together and can react, either modifying one of the initial reactants or forming a third, distinct, species, a product. It should be appreciated, however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0046] As used herein, the term "solvent mixture" refers to a mixture of two or more solvents selected for suspension and / or dissolution of components in a reaction mixture. The solvents in the mixture and the volume ratio in which they are combined depend primarily on the polarity of the lipids and telodendrimers in the reaction mixture. Non-limiting examples of solvents for use in the solvent mixture includechloroform, dichloromethane, ethanol, methanol, acetone, hexanes, petroleum ether, diethyl ether, dioxane, tetrahydrofuran, and water.

[0047] As used herein, the term "buffer" refers to an aqueous solution capable of maintaining the pH of the solution at a nearly constant value. The buffer accomplishes this by including a weak acid and its conjugate base, such that the pH does not substantially change following addition of a small amount of acid or base. Representative buffering agents include citric acid, acetic acid, dipotassium phosphate (K2HPO4), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), and borate. Buffers commonly used include, but are not limited to, TAPS, bicine, tris, tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, SSC, MES and succinic acid.

[0048] As used herein, unless otherwise stated, the term "group” refers to a chemical entity that has one terminus that can be covalently bonded to other chemical species.

[0049] As used herein, unless otherwise stated, the term “moiety” refers to a chemical entity that has two or more termini that can be covalently bonded to other chemical species.

[0050] As used herein, the terms “nanocarrier” and “nanoparticle” refer to a micelle resulting from aggregation of dendrimer or telodendrimer conjugates of the present disclosure. The nanocarrier has a charged and / or hydrophobic core, which, optionally, comprises charged groups / moieties, and a hydrophilic exterior (e.g., a hydrophilic exterior with zwitterionic groups)

[0051] As used herein, the term “composition” refers to the specific arrangement, type, and ratio of atoms that make up a chemical substance, essentially describing the elements present in a compound and their relative proportions within it. A composition may be expressed using a chemical formula to represent the exact makeup of a molecule.

[0052] As used herein, the terms "monomer" and "monomer unit" refer to a diamino carboxylic acid, a dihydroxy carboxylic acid, or a hydroxyl amino carboxylic acid. Examples of diamino carboxylic acid groups of the present disclosure include, but are not limited to, 2,3-diamino propanoic acid, 2,4-diaminobutanoic acid, 2,5- diaminopentanoic acid (ornithine), 2,6-diaminohexanoic acid (lysine), (2-aminoethyl)- cysteine, 3-amino-2-aminomethyl propanoic acid, 3-amino-2~aminomethyl-2-methyl propanoic acid, 4-amino-2-(2-aminoethyl) butyric acid and 5-amino-2-(3- aminopropyl) pentanoic acid. Examples of dihydroxy carboxylic acid groups of thepresent disclosure include, but are not limited to, glyceric acid, 2,4-dihydroxybutyric acid, glyceric acid, 2,4-dihydroxybutyric acid, 2,2-bis(hydroxymethyi)propionic acid, and 2,2-bis(hydroxymethyl)butyric acid. Examples of hydroxyl amino carboxylic acids include, but are not limited to, serine and homoserine. One of skill in the art will appreciate that other monomer units can be used in the present disclosure.

[0053] As used herein, the term “linker” refers to a chemical moiety that links (e.g., via covalent bonds) one segment (e.g., a reactive portion of a segment, such as, for example, an amine) of a conjugate to another segment (e.g., a reactive portion of another segment, such as, for example, a carboxylic acid) of the conjugate. In various examples, a linker is a chemical moiety that links (e.g., via covalent bonds) a fragment of a functional group. The types of bonds used to link the linker to the segments functional group, but are not limited to, amides, amines, esters, carbamates, ureas, thioethers, thiocarbamates, thiocarbonate, and thioureas.

[0054] As used herein, the term “multivalent linkage” refers to a type of chemical bond where a single molecule or atom can form multiple bonds with other molecules or atoms simultaneously, essentially acting as a “multi-hook” that can connect to several partners at once; this is often seen in biological systems where molecules with multiple binding sites interact with other molecules with multiple complementary binding sites, creating a strong and specific interaction.

[0055] As used herein, the term “prodrug” is a therapeutic compound, i.e., drug, that undergoes metabolism and converts to its resultant active moiety for pharmacological action in a targeted tissue. The metabolic transformation necessary to convert the prodrug into the drug is catalyzed by specific enzymes and ideally this should selectively occur at the target tissue to prevent undesirable side effects.

[0056] As used herein, the term “zwitterionic group” refers to a functional group within a molecule that contains both a positively charged group (cation) and a negatively charged group (anion) within a proximity. The overall charge of a zwitterion is zero.

[0057] As used herein, the term “hydrophobic group” refers to a chemical moiety that is water-insoluble or repelled by water. Examples of hydrophobic groups include, but are not limited to, alkanes (e.g., long-chain alkanes) and faty acids, lipids, vitamins (e.g., vitamin E), natural compounds, herbal extracts, fluorocarbons, silicones, certain steroids such as cholesterol, bile acids, and certain polymers such as, for exampie, polystyrene and polyisoprene.

[0058] As used herein, the term “hydrophilic group” refers to a chemical moiety that is water-soluble or attracted to water. Examples of hydrophilic groups include, but are not limited to, alcohols, short-chain carboxylic acids, quaternary amines, sulfonates, phosphates, sugars, and certain polymers such as, for example, PEG, PVA, and zwitterionic polymers.

[0059] As used herein, the term “amphiphilic compound” refers to a compound having both hydrophobic portions and hydrophilic portions. For example, the amphiphilic compounds of the present disclosure can have one hydrophilic part of the compound and one hydrophobic part of the compound, for example, bile acids, cholic acids, riboflavin, chlorgenic acid, etc.

[0060] As used herein, the term “polar compound” refers to a compound having a non-zero vector sum of its bond dipoles.

[0061] As used herein, the term “RGD peptide” refers to a short peptide sequence composed of three amino acids: arginine (R), glycine (G), and aspartic acid (D), which acts as a key recognition site for integrin receptors on cell surfaces, playing a vital role in cell adhesion and signaling processes within the body; it is a small molecule that mimics a natural binding site for cells to attach to the extracellular matrix.

[0062] As used herein, the term “integrin targeting ligand” is a molecule that specifically binds to an integrin receptor on a cell surface, essentially acting as a key to unlock the integrin and trigger cellular responses like adhesion, migration, or signaling, by mimicking the natural proteins (like fibronectin or collagen) that integrins normally bind to in the extracellular matrix; often, these ligands are small peptides containing specific amino acid sequences like RGD (arginine-glycine- aspartic acid) that are recognized by certain integrins.

[0063] As used herein, the term “flexible linker” refers to spacer molecules with multiple rotatable single bonds.

[0064] As used herein, the term “Glu-urea-based PSMA ligand” is a small molecule that binds to prostate-specific membrane antigen (PSMA) to target prostate cancer and other cell types.

[0065] As used herein, the term “oligo amino acids” refers to a short chain of amino acids linked together by peptide bonds, i.e., a small peptide, where "oligo" indicates a small number of units. Typically, an oligopeptide consists of between 2 and 25 amino acids.

[0066] As used herein, the term “aromatic molecules” refer to cyclic compounds with a planar ring structure that exhibit special stability due to delocalized TT- electrons. As used herein, the term “Orthogonal Tri-functional Aromatic Molecules” refers to the arrangement of three functional groups in a way that allows them to react independently of each other. Orthogonal tri-functional aromatic molecules have applications, such as scaffolds for creating compounds with diverse properties.

[0067] As used herein, the terms “treat”, “treating” and “treatment” refer to any indicia of success in the treatment or amelioration of an injury, pathology, condition, or symptom (e.g., pain), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom or condition. The treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination.

[0041] As used herein, the term “subject” refers to animals such as mammals. Suitable examples of mammals include, but are not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like. In certain embodiments, the subject is a human.

[0042] As used herein, the terms “therapeutically effective amount or dose” or “therapeutically sufficient amount or dose” or “effective or sufficient amount or dose” refer to a dose that produces therapeutic effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, the therapeutically effective dose can often be lower than the conventional therapeutically effective dose for nonsensitized cells.

[0043] As used herein, the term “ultra-hydrophilic zwitterionic” refers to the charge pair are close enough to be 100% charged and fully hydrated with water molecules.

[0044] Before embodiments are further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, ofcourse, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0045] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0046] Unless defined otherwise, 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. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are entirely incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.DETAILED DESCRIPTION OF THE INVENTION

[0047] Glucocorticoids (GCs) are widely used in the treatment of inflammatory liver diseases and sepsis. However, the various side effects of GCs on extrahepatic tissues limit their clinical benefits. Liver-targeting GC therapy may have multiple advantages over systemic GC therapy. Embodiments disclosed herein are directed to novel liver-targeting GC prodrugs as improved treatment for inflammatory liver diseases and sepsis.

[0048] Certain embodiments disclosed herein are directed to novel prodrugs having the following formula:wherein the prodrug has three components, TL, D, and Z centralized about a center scaffold linkage. The three components, as more fully described herein, are bonded to multivalent multifunctional linkages that can be made up of oligo amino acids, orthogonal tri-functional aromatic molecules. In a two-component composition a linear ethyleneglycol based hydrophilic linkercan be used to reduce stereo hindrance.

[0049] The novel prodrugs may assemble into micelles having the hydrophobic drug within the micelles and the zwitterionic component and targeting ligand on the exterior or the outside of the micelles.

[0050] Further embodiments disclosed herein are directed to novel DEX-BA conjugates as liver-targeting GC prodrugs. In contrast to GR, activation of MR by GCs worsens non-alcoholic steatohepatitis (NASH) and liver fibrosis. Many natural GCs and synthetic corticosteroid hormones can activate both the GR and the MR, and the novel prodrugs disclosed herein avoid the deleterious side effects of activation of hepatic MR. Thus, the claimed BA conjugate prodrugs disclosed herein provide highly potent and selective agonist of GR over MR. BA conjugates have been successfully synthesized as oral prodrugs for liver-specific drug delivery. DEX is hydrophobic, so it is easily absorbed by essentially all types of cells. We decorate GC-BA with a hydrophilic flexible linkers or zwitterionic groups, namely carboxybetaine (CB) or glycerylphosphorylcholine (GPC), to generate CA-DEX, CA- CB-DEX, and CA-GPC-DEX, via solid-phase peptide chemistry. We purified these GC prodrugs with chromatography and confirmed the molecular weight and structure by mass spectrometry and nuclear magnetic resonance (NMR), respectively. We found that CA-DEX was slightly water-soluble, whereas the zwitterionic CA-CB-DEX and CA-GPC-DEX were highly water-soluble. The CA molecule allows the prodrug to be absorbed into hepatocytes by the liver-specific BA uptake transporter NTCP. This bridge of CA and DEX to generate CA-DEX, CA-CB-DEX and CA-GPC-DEX allows the prodrugs to be absorbed by the liver but minimizes the passive diffusion across the cell membrane.

[0051] Initially, we verified the transporter-dependent cellular activity of these prodrugs (FIG. 4). The liver-specific organic anion transporting polypeptide 1B1 (OATP1 B1 ) and OATP1 B3 are essential for hepatic uptake of unconjugated BAs,whereas NTCP and apical sodium-dependent BA transporter (ASBT) mediate hepatic and intestinal uptake of conjugated BAs. To verify the transporter-dependent cellular uptake, we co-transfected HEK293 cells with a luciferase reporter vector for bile salt export pump (BSEP), a target gene for the BA receptor farnesoid X receptor (FXR), and expression vectors for FXR, 0ATP1 B1, 0ATP1B3, ASBT, and / or NTCP. CA-DEX and CA-CB-DEX at 2 μM failed to activate BSEP promoter in the absence of transporters or the presence of 0ATP1 B1 / 0ATP1 B3 but caused moderate and strong transactivation of BSEP promoter in the presence of ASBT and NTCP, respectively. We found that the DEX prodrugs CA-DEX and CA-CB-DEX had very poor activities in the activation of reporter for GR in HEK293 cells, even in the presence of NTCP (data not shown), which is consistent with the dramatic decreased GR agonist activity when the 21 -hydroxyl of DEX is esterified. Thus, cellular uptake of CA-DEX and CA-CB-DEX are largely dependent on the liverspecific NTCP and intestine-predominant ASBT. CA-DEX and CA-CB-DEX will exhibit liver-selective uptake after iv / ip / sc injection and have the potential to be developed as oral drugs via the ASBT-NTCP pathway.

[0052] With respect to the differential effects of DEX prodrugs and DEX on LPS- stimulated macrophages, we note that macrophages have a high capacity of chemical uptake via endocytosis, which is independent of the BA transporters. To test whether the prodrugs have decreased uptake and activity in the macrophages, we conducted a comparative study using DEX and DEX-BAs (100 nM and 1 pM) to treat the macrophage RAW264.7 cells activated by LPS (200 ng / mL). Compared to vehicle (VEH), LPS treatment for 6 hr. caused a dramatic induction of 111b mRNA (Fig. 5A). DEX at 100 nM largely inhibited 111b induction by LPS. CA-DEX had weaker, and CA-CB-DEX had much weaker inhibition compared to DEX at 100 nM. Even at 1 μM of CA-CB-DEX, its inhibitor effect still tended to be weaker than the parent drug DEX at 100 nM (Fig. 5A).

[0053] With respect to the differential effects of DEX prodrugs and DEX on LPS- treated human whole blood, we note that in the blood circulation the ester bond of these GC prodrugs may be broken by esterases to release the parent drug DEX, and these prodrugs may be taken up by the blood cells via endocytosis and / or passive diffusion. We further compared the effects of our GC prodrugs with DEX and prednisolone, the current standard care for sAH, on the LPS- stimulated induction and release of cytokines in human whole blood. qPCR data showed that LPS (2ng / ml) tended to moderately induce TNF mRNA (FIG. 5B), and LPS strongly induced mRNA expression of proinflammatory IL6 (FIG. 5C) and IL1B (FIG. 5D) as well as the anti-inflammatory IL1 receptor antagonist (IL1RN, FIG. 5E) and IL10 (FIG. 5F). In contrast, LPS down-regulated the key GR-target anti-inflammatory gene glucocorticoid- induced leucine zipper (GILZ) (FIG. 5G). The equipotent dose of DEX (0.1 pM) and prednisolone (0.75 pM) caused similarly potent inhibition of the LPS- induction of IL6, IL1B, and IL1RN. Additionally, DEX and prednisolone further enhanced the LPS-induction of IL10 (FIG. 5F) and completely blocked the down regulation of GILZ by LPS (FIG. 5G). Compared to DEX group, the CA-GPC-DEX (GPC) group tended to have higher expression of TNF, IL6, and IL1B, whereas the CA-CB-DEX (CB) group had significantly higher expression of TNF (FIG. 5B), IL1 B (FIG. 5D), IL1RN (FIG. 5E) but lower IL10 (FIG. 5F). In contrast, all these GCs similarly reversed the LPS-down-regulation of GILZ, a major mediator of GC’s antiinflammatory effects (Fig. 5G).

[0054] Overexpression of GILZ in monocytes / macrophages inhibits the inflammatory response, enhances the phagocytosis of bacteria, and prolongs the survival of CLP mice. ELISA results showed that DEX and prednisolone similarly attenuated the LPS-induced release of TNF proteins (FIG. 5H), whereas only DEX attenuated the release of IL6 proteins in the whole blood (FIG. 5I). These results clearly demonstrate weaker immunosuppressive effects of GC prodrugs CA-CB-DEX and CA-GPC-DEX compared to DEX on the human blood cells. Because of the trend of lower GC activities of CA-CB-DEX compared to CA-GPC-DEX in nonhepatocytes, CA-CB-DEX was used in the further in vitro and in vivo studies.

[0055] Currently, alterations of hepatic GR signaling in human AH remain poorly understood. Thus, we have analyzed the microarray data of hepatic mRNA expression in patients with sAH (Maddrey’s discriminant function > 32) (GSE28619). Compared to normal liver, liver from sAH had moderate down-regulation of GR (NR3C1) and the GR-target bile acid uptake transporter NTCP (FIG. 6A), consistent with cholestasis in AH patients. Moreover, sAH liver had marked down-regulation of known GR-target genes hepatocyte nuclear factor 4A (HNF4A) ( |69 %), Kruppel-like factor 15 (KLF15,j,69 %), GILZ Q87 %), dual specificity protein phosphatase 1 (DUSP1, |92 %), growth arrest DNA damage-inducible gene 45|3 (GADD45B, |81 %), peroxisome proliferator activated receptor gamma coactivator 1a (PGC1a, j.65 %), glucose-6-phosphatase catalytic-subunit (G6PC, j82 %), pyruvatedehydrogenase kinase 4 (PDK4, |85%), glycine N-methyltransferase (GNMT, j.88 %), and metallothionein 1X (MT1XJ60 %) (FIG. 6A). Hepatic KLF15 enables rapid switch between lipogenesis and gluconeogenesis to ameliorate hypertriglyceridemia. DUSP1 is essential in protecting against TNF-induced inflammation in liver. GADD45p is a key hepatoprotective gene by inhibiting the JNK signaling. The PGC1a is a master regulator of mitochondria biogenesis. Hepatic deficiency of the key gluconeogenic enzyme G6PC aggravates HFD-induced steatosis and liver injury. Hepatic PDK4 is critical in FAO, and loss of PDK4 switches the hepatic NF-KB pathway from pro-survival to pro-apoptosis. GNMT maintains DNA methylation and protects against steatohepatitis and cholestatic liver injury. Metallothionein protects against non-alcoholic fatty liver disease and ALD by inhibiting oxidative stress. Thus, marked down-regulation of these known GR-target genes likely play important roles in steatohepatitis and cholestatic liver injury in sAH patients.

[0056] Our data mining found that sAH liver had marked dysregulation of a group of noncanonical GR-target genes (FIG. 6A), including the retinoic acid receptor- related orphan receptor alpha (RORA, |76 %), ERBB receptor feedback inhibitor 1 (ERRFI1 , |80 %), 6- phosphofructo-2-kinase / fructose-2,6-biphosphatase 3 (PFKFB3, j.74 %), hydroxy acid oxidase 2 (HAO2, |83 %), nicotinamide phosphoribosyl-transferase (NAMPT, j,93 %), and G0 / G1 switch gene 2 (G0S2, f 105 %). The orphan receptor RORa protects against NASH by inhibiting lipogenesis and inflammation. ERRFI1 , a negative EGFR regulator, protects against fatty liver and insulin resistance [99, 100]. Increased glycolysis can provide the energy and intermediate metabolites to permit the survival of hypoxic hepatocytes. The peroxisomal enzyme HAO2 promotes lipid catabolism to eliminate lipid accumulation. NAMPT is a rate-limiting enzyme for the biosynthesis of NAD+ that is depleted in AH. NAMPT is down-regulated in human AH and ethanol-fed mice. In contrast, NAMPT overexpression ameliorates alcoholic liver injury by restoring NAD + level and activity of Sirtuin 1 , a crucial mitochondrial biogenesis regulator that protects against AH. Conversely, G0S2 is a potent inhibitor of lipolysis and lipid droplet degradation. Thus, dysregulation of these noncanonical GR-target genes also plays an important role in steatohepatitis and cholestatic liver injury in AH patients.

[0057] Our analysis included the modulation of mRNA expression of GR-target genes in PHH by GC treatments. The PHH (Liver Tissue Cell Distribution System,Pittsburg) were cultured overnight in the serum-free medium for PHH without GC, and then treated with CA-CB-DEX and DEX at 1 μM for 6 h to determine the direct effect of GR activation on transcriptome by RNA-sequencing, followed by qPCR verification. DEX at 1 μM does not activate the human pregnane X receptor. We found that treatment of PHH with 1 μM DEX and CA-CB-DEX for 6 hr. caused similarly rapid and marked down-regulation of G0S2 and strong induction of all those known and noncanonical GR-target genes down-regulated in sAH (FIG. 6B). Taken together, hepatic GR signaling is markedly impaired in sAH patients, and GR in hepatocytes protects against AH via anti-inflammatory, anti-apoptotic, lipid-catabolic, and anti-cholestatic effects. Our novel highly hydrophilic CA-CB-DEX displayed good selectivity toward hepatocytes over immune cells which strongly supports the further development of CA-CB-DEX as liver-targeting GC prodrug to improve the GC therapy of sAH.

[0058] Systemic GC treatment may increase the risk of infection in AH patients. Currently, GC treatment is avoided in sAH patients with infections. In contrast, hepatic GR deficiency occurs in septic patients, and GR in hepatocytes is critical in protecting against liver failure and mortality in mice with sepsis induced by CLP. Thus, liver-selective activation of GR may improve the efficacy and decrease the side effects in AH patients with infections. We conducted a comparative study on the effects of CA-CB-DEX and DEX on CLP-induced sepsis in WT mice. CLP mice were ip injected DEX (0.25 pmole / kg = 0.1 mg / kg), CA-CB-DEX (0.25 pmole / kg), or vehicle 3 h after CLP surgery, and mice were sacrificed 24 hr. after surgery. CA-CB- DEX, but not DEX, attenuated weight loss in CLP mice (FIG. 7A). Both CA-CB-DEX and DEX attenuated hypoglycemia (FIG. 7B). White blood cells were markedly decreased in CLP mice (FIG. 7C); CA-CB-DEX group tended to have less lymphocytopenia than DEX group (p = 0.06). Hepatocytes are a major site of production of IL6 in mice which has a key role in hyperinflammation and weight loss in sepsis. CA-CB-DEX attenuated hepatic induction of IL6 (FIG. 7D) and IL1 b (FIG. 7E). Importantly, CA-CB-DEX had much weaker inhibitory effect than DEX on IL1 b induction in macrophages (FIG. 5A). Thus, the stronger inhibitory effects on hepatic IL1 b and IL6 by CA-CB-DEX than DEX in CLP mice are most likely due to pharmacokinetics, namely higher NTCP-mediated liver delivery of CA-CB-DEX (it is hard to determine DEX in liver 21 hr. after a low dose). Sepsis causes translational inhibition and induction of Eif4ebp3, a repressor of Eif4e assembly to the capcomplex. Both CA-CB-DEX and DEX treatment abolished hepatic induction of Eif4ebp3 in CLP mice (FIG. 7F), suggesting that GC may ameliorate sepsis-induced translational repression in the liver. Overall, these data strongly suggest that our novel liver-targeting GC prodrug will have better efficacy in sAH and may be safer in AH patients with infections, in view of the better anti-inflammatory effects of CA-CB- DEX in septic livers and the trend of less lymphocytopenia in CA-CB-DEX group than DEX group (FIG. 7C).

[0059] Embodiments disclosed herein are directed to the introduction of novel ultra-hydrophilic zwitterionic groups into the design of liver- targeting GC prodrug will markedly increase the water solubility and decrease the non- specific passive diffusion of GC prodrug across cell membranes, leading to markedly improved liverspecific delivery of GCs and the resultant enhanced anti-inflammatory effects on the liver and decreased immunosuppression on blood cells and side effects on extrahepatic tissues.

[0060] In embodiments disclosed herein, we developed the first-in-class livertargeting GC prodrugs and verified their transporter-dependent and liver-selective actions. CA-CB-DEX caused weaker immunosuppressive effects than DEX and prednisolone on human whole blood, whereas CA-CB-DEX exerted stronger antiinflammatory effects on livers in septic mice. Results from our work support the novel liver-targeting GC prodrug CA-CB-DEX as an improved therapy for sAH.

[0061] Hydrophilic linkers have been used to synthesize BA-drug conjugates to improve NTCP-mediated liver-selective drug delivery. We are the first to use a highly hydrophilic zwitterionic linker to synthesize novel second-generation BA-drug conjugates, CA-CB-DEX and CA-GPC-DEX, to further enhance liver-specific drug delivery via increasing the water solubility and decreasing the passive diffusion of BA-drug conjugate across the cell membrane. Compared to DEX and the first- generation, CA-DEX, CA-CB-DEX and CA-GPC-DEX exert much-enhanced water solubility and decreased immunosuppressive activities in mouse macrophages and / or human whole blood.

[0062] Importantly, our novel liver-targeting GC prodrugs comprise uniform nanoparticles (10 nm) by CA-CB-DEX, which likely contributes to its high-water solubility. Zwitterionic polymers are known to form self-assembled nanoparticles. The CA-CB-DEX nanoparticles are likely formed with a shell of zwitterionic CB and an inner core of CA and hydrophobic DEX. The CMC value of 91 μM for CA-CB-DEX suggests that the iv injected CA-CB- DEX will be in the form of nanoparticles in the blood initially. After dissociation from nanoparticle, the conjugated bile acids will mediate NTCP-dependent liver-specific uptake of the prodrug. The filtration clearance of nanoparticles by kidney has the effective size cutoff of 10 nm. The uptake of nanoparticles by macrophages decreases with decreasing particle size. Drugs delivered by nanoparticles have prolonged half-life due to the retention of drug nanoparticles in the circulation. Therefore, the formation of stable small (10-20 nm) nanoparticles is desirable for persistent liver-specific delivery of GC. In addition, the density of zwitterionic CB groups and the hydrophilic linkers can be adjusted to optimize the pharmacokinetics of enhanced liver-targeting DEX prodrugs. It can also be applied to other drugs to synergize liver disease treatments.

[0063] The ultra-hydrophilic zwitterionic linker can be used in the design of nextgeneration tissue / cell-specific prodrugs to markedly improve the water solubility and decrease the non-specific passive diffusion across cell membranes. Compared to DEX, CA-CB-DEX demonstrated good transporter-dependent hepatocyte-selectivity in vitro and better anti-inflammatory effects on livers of septic mice. Further test of the novel liver-targeting GC prodrug CA-CB-DEX as an improved GC therapy for inflammatory liver diseases and sepsis is warranted.EXAMPLES

[0064] In preparing our prodrugs we utilized a hydrophilic linker or an ultra- hydrophilic zwitterionic linker carboxylic betaine (CB) to bridge cholic acid (CA) and dexamethasone (DEX) to generate transporter-dependent liver-targeting GC prodrugs CA-DEX and the highly hydrophilic CA-CB-DEX. The efficacy of livertargeting DEX prodrugs and DEX were determined in primary human hepatocytes (PHH), macrophages, human whole blood, and / or mice with sepsis induced by cecal ligation and puncture.

[0065] As such, the following CA-DEX prodrugs were prepared as described herein below: Dexamethasone (DEX) was obtained from Sigma. Rink Amide Resin was purchased from Hecheng Company (Tianjin, China). (Fmoc)-Lys (Dde)-OH, Fmoc- Glu-OtBu, and N-hydroxy benzotriazole (HOBt) were purchased from AnaSpec Inc. Cholic acid (CA), D-o-tocopherol succinate, diisopropyl carbodiimide (DIC), dichloromethane (DCM), methanol (MeOH), N,N-Dimethylformamide (DMF),Triisopropyl silane (TIPS), trifluoroacetic acid (TFA) and other chemical reagents were acquired from Sig ma-Ald rich.

[0066] We synthesized three DEX prodrugs i.e. CA-DEX, CA-GPC-DEX, and CA- CB-DEX (FIG. 1), based on the rational design through a straightforward method. DEX-COOH was prepared by conjugation of succinic anhydride on DEX by ester bond (FIG. 2A), followed by the coupling of targeting ligand cholic acid and hydrophilic zwitterionic groups i.e. GPC and CB via solid phase peptide chemistry. The final products were purified by column chromatography and further confirmed by matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry (FIG. 3).

[0067] Dexamethasone prodrug CA-DEX was prepared by solid phase peptide synthesis as shown in FIG. 2B. Initially, dexamethasone, succinic anhydride and 4- dimethylaminopyridine (DMAP) were dissolved in DMF and reacted at 50 °C for 48 h to prepare DEX-COOH (Fig. 2A). Fmoc group of Rink Amide resin was deprotected via 20 % (v / v) 4-methylpiperidine in DMF and the NH2 group was conjugated with Fmoc-Lys (Dde)-OH (three equiv) through coupling reagents HOBt and DIC confirming by negative Kaiser test result. After deprotection of Fmoc group, Fmoc- Glu-OtBu and Cholic acid were coupled sequentially to achieve intermediate with cholic acid-targeting moiety. After removal of Dde group by 5 % (v / v) hydrazine in DMF, DEX-COOH was conjugated on NH2 group. The final product CA-DEX was acquired by deprotection of tert-butyl group and cleavage from rink resins by TFA: H2O: TIPs (18:1 :1 v / v / v) solution for 3 times with 1 h each time. The final product was purified by column chromatography through silica gel (40-60 pm, 60A, Acros Organics) with mobile phase of DCM and MeOH (4:1 v / v). The molecular weight of intermediates (FIG. 3D - FIG. 3G) and final product CA-DEX were detected by MALDI-TOF mass (Bruker Microflex) [M + Na]+, m / z 1162.400 (calculated), m / z 1162.804 (found) (FIG. 3A). The solubility test showed that CA-DEX was slightly water soluble (up to 0.1 mg / ml).

[0068] Dexamethasone prodrugs CA-GPC-DEX and CA-CB-DEX were prepared by solid phase peptide synthesis as shown in FIG. 2D and FIG. 2E. Fmoc group of Rink Amide resin was deprotected via 20 % (v / v) 4-methylpiperidine in DMF and the NH2 group was conjugated with Fmoc-Lys (Dde)-OH (three equiv) through coupling reagents HOBt and DIC confirming by negative Kaiser test result. After deprotection of Fmoc group, Fmoc- Glu-OtBu and Cholic acid were coupled sequentially toachieve intermediate with cholic acid-targeting moiety. After removal of Dde group by 5 % (v / v) hydrazine in DMF, Fmoc-Lys(Dde)-OH was coupled. Then glycerophosphorylcholine (GPC-COOH, FIG. 2C) or carboxylic betaine (CB) via both 4-(N,N-Dimethylamino)butanoic succinimide and tert- butyl bromoacetate at 60 °C and DEX-COOH were conjugated after deprotection of Fmoc and Dde groups, respectively. The final product CA-GPC-DEX and CA-CB-DEX were acquired by deprotection of tert-butyl group and cleavage from rink resins by TFA: H2O: TIPs (18:1:1 v / v / v) solution for 3 times with 1 h each time. The final products were purified by column chromatography through silica gel (40-60 pm, 60A, Acros Organics) with mobile phase of DCM and MeOH (2:1 v / v). The molecular weight of intermediates (FIG. 3D - FIG. 3 J) final product CA-GPC-DEX and CA-CB-DEX were detected by MALDI-TOF mass (Bruker Microflex) CA-GPC-DEX [M + H]+, m / z 1607.874 (calculated), m / z 1607.035 (found) (Fig. 3B); CA-CB-DEX [M + H]+, m / z 1438.838 (calculated), m / z 1438.834 (found), [M + Na]+, m / z 1460.820 (calculated), m / z 1460.801 (found) (FIG. 3C). The solubility test showed that CA-CB-DEX was highly water soluble (up to 5 mg / ml). Thin-layer chromatography (TLC) revealed a single spot of CA-CB-DEX as visualized under UV 254 nm, indicating excellent purity (FIG. S1A).

[0069] Characterization of CA-CB-DEX in solution: CA-CB-DEX can be dissolved in phosphate buffered saline (PBS, pH 7.4) at the concentration of 5 mg / mL (3.5 mM) and micelle nanoparticles was detected by dynamic light scattering particle sizer at 10 nm (FIG. S1B). The critical micelle concentration (CMC) of CA-CB-DEX was measured by fluorescence of Nile Red: 5 pL of 0.5 mg / mL methanol solution of Nile Red was added to the wells of 96-well plate. After methanol was evaporated under the vacuum, 100 pL of CA-CB-DEX solutions at different concentrations (1 to 1000 pg / mL) were added to each well and the 96-well plate was incubated with agitation overnight at room temperature covered with foil. Fluorescent intensity at 620 nm was measured using microplate reader (BioTek Synergy H1) with the excitation at 543 nm. CMC of CA-CB-DEX was determined as 131.18 pg / mL (91 pM) at the intersection of two fitting lines (Fig. S1C).

[0070] Cell culture and drug treatment with PHH with DEX and DEX prodrugs: PHH (Liver Tissue Cell Distribution System, Pittsburg) was replaced with RPCD1 medium (without DEX) in the afternoon and cultured overnight. DEX, the prodrug CA-CB-DEX (1 μM in DMSO), and vehicle (0.1 % DMSO) were added in the nextmorning (final DMSO concentration 0.1 %). PHH were collected 6 h after drug treatment for total RNA isolation and real-time PCR quantification of mRNAs, normalized to glyceraldehyde- 3-phosphate dehydrogenase (GAPDH).

[0071] Comparative study on effects of DEX and BA conjugates of DEX (DEX- BAs) on mouse macrophage activation by lipopolysaccharide (LPS, from escherichia coli O55:B5, L2880, Sigma-Aldrich): Mouse macrophage RAW 264.7 cells were seeded in 12-well plates (5 x 105 cells / well) and incubated at 37 °C for 18 hr. (overnight). Cells were co-treated with DEX or one of the three DEX-BA conjugates (100 nM and 1 uM) and LPS (200 ng / mL). After 6 h incubation periods, cells were collected for total RNA isolation and real-time PCR analysis of interleukin 1 beta (111b), normalized to beta-actin.

[0072] Comparative study on effects of DEX and DEX-BAs on LPS-stimulated expression and release of cytokines in human whole blood: Fresh human whole blood samples were obtained from a healthy male volunteer (IRB#754811— 13) in the morning via venipuncture into heparinized tube. Aliquots of 0.24 ml heparinized human whole blood were transferred to Eppendorf tubes that contain 0.24 ml of RPMI-1640 medium as well as test drugs and 2 ng / mL LPS. There were 6 treatment groups (N = 4 per group), namely DMSO control, LPS, LPS plus drugs (prednisolone 0.75 uM, DEX 0.1 uM, CA-CB-DEX 0.1 uM, and CA-GPC-DEX 0.1 uM). The final DMSO concentrations were 0.1 % in all the treatment groups. After 4 hr. incubation with LPS (at 37 °C x 250 rpm), samples were centrifuged at 1500 g for 10 min at 25 °C and supernatants stored at - 80 °C for analysis of tumor necrosis factor (TNF) and IL6 by ELISA. Total RNAs were prepared from the lower layer of blood cells using the RiboPure RNA Purification kit, blood (AM1928, Invitrogen) for qPCR determination of mRNAs, normalized to akirin 1 (AKIRIN1 ) which has been validated as the most stable housekeeping gene in human blood cells during inflammation and LPS treatment.

[0073] Animals and treatments The mouse model of polymicrobial sepsis induced by cecal ligation puncture (CLP) is a “gold standard” animal model of sepsis because it reproduces certain key features of secondary bacterial peritonitis in humans. Adult male C57 / BL6 mice underwent CLP- induced sepsis following our previous protocol. Briefly, after anesthesia with ketamine / xylazine and laparotomy, the cecum is ligated with a 5-0 silk suture at about 1 .3 cm position from distal pole to the base of cecum to avoid the bowel obstruction. The cecum is punctured twice with a 22G-gaugeneedle at top and bottom, respectively, and gently squeezed to extrude a 1-mm3 column of fecal material. The cecum is returned to the abdominal cavity. Control (sham) mice undergo laparotomy without CLP, and then the abdominal incision is closed with 5-0 silk suture. After CLP surgery, warm saline (1 ml sc) is given for resuscitation. Mice received 0.05 mg / kg buprenorphine every 12 h for pain control. CB-CA-DEX (0.25 pmole / kg), DEX (0.25 pmole / kg), or vehicle (0.5 % DMSO in saline 10 ml / kg) were ip administered at 3 h after CLP. Mice were sacrificed 24 h after CLP to collect blood for complete blood count using Auto Blood Analyzer and analyses of blood chemistry. Liver tissues were collected and snap-frozen in liquid nitrogen for storage at - 80C°. All procedures in animal experiments have been approved by Institutional Animal Care and Use Committee (IACUC) at SUNY Upstate Medical University (IACUC #459). RNA isolation and real-time PCR quantification of mRNA Total RNAs from liver tissues and / or cells were extracted using RNA STAT-60 (Tel-Test, Friendswood, TX, USA) according to the manufacturer’s instructions. Equal amount of total RNAs from each sample in the given group was mixed to prepare the pooled RNA samples. cDNAwas produced using a cDNA Synthesis kit (iScript™ cDNA Synthesis Kit, Bio-Rad, Hercules, CA, USA). The diluted cDNAwas used for real-time PCR quantification of mRNA using iTaq SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA) and CFX Real-Time PCR Detection System (Bio-Rad). A list of primers used for qPCR quantification of mouse and human genes is provided in Table 1 . The data were analyzed by CFX Maestro qPCR Analysis Software (Bio-Rad), and the amounts of mRNA were calculated using the CQ value, normalized to an endogenous reference gene.

[0074] Table 1. Primers for real-time PCR quantification of mRNAs:

[0075] 2.5. Statistical analysis Data are presented as mean ± standard error (SE).Statistical significance was set at * p <0.05. For comparison of two groups, Student’s t-test was used. For comparison of multiple groups, ANOVA tests and Dunnett T3 post hoc testing of selected pairings were utilized.

[0076] With respect to our results, we noted that the CA-DEX was moderately water soluble, whereas CA-CB-DEX was highly water soluble. CA-CB-DEX and CA- DEX displayed highly transporter-dependent activities in reporter assays. Data mining found marked dysregulation of many R-target genes (regulated by glucocorticoid receptor (GR)) important for lipid catabolism, cytoprotection, and inflammation in patients with severe alcoholic hepatitis. These key GR-target genes were similarly and rapidly (within 6 h) induced or down-regulated by CA-CB-DEX and DEX in PHH. CA-CB-DEX had much weaker inhibitory effects than DEX on endotoxin-induced cytokines in mouse macrophages and human whole blood. In contrast, CA-CB-DEX exerted more potent anti-inflammatory effects than DEX in livers of septic mice.

[0077] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.

Claims

WHAT IS CLAIMED IS:

1. A method of treating inflammatory liver disease in a subject in need thereof, said method comprising administering a therapeutically effective amount of a prodrug composition having the formula:wherein, TL is a targeting ligand, L is a multivalent linkage, D is a drug, Z is a zwitterionic group, and n, m, and p are integers from 1 to about 42. The method of treating inflammatory liver disease of claim 1 , wherein TL is at least one selected from the group consisting of bile acids (BA) and cholic acid (CA).

3. The method of treating inflammatory liver disease of claim 1 , wherein L is at least one selected from the group consisting of an oligo amino acid, an orthogonal and an orthogonally protected tri-functional aromatic molecule.

4. The method of treating inflammatory liver disease of claim 1 , wherein D is at least one glucocorticoid (GC) drug selected from the group consisting of dexamethasone (DEX), prednisone, prednisolone, cortisone, hydrocortisone, and fludrocortisone.

5. The method of treating inflammatory liver disease of claim 1 , wherein Z is at least one selected from the group consisting of glycerophosphorylcholine (GPC), carboxylic betaine (CB), carnitine, choline phosphate (CP), sulfobetaines (SB), and trimethylamine N-oxide (TMAO).

6. The method of treating inflammatory liver disease of claim 1 , wherein TL is CA.

7. The method of treating inflammatory liver disease of claim 1 , wherein L is an oligolysine having from about 1 to about 16 lysine residues.

8. The method of treating inflammatory liver disease of claim 1 , wherein Z is GPC.

9. The method of treating inflammatory liver disease of claim 1 , wherein Z is CB.

10. The method of treating inflammatory liver disease of claim 1 , wherein D is dexamethasone.

11. The method of treating inflammatory liver disease of claim 1 , wherein said prodrug composition assembles into micelles having the TL and Z reside on an outside of the micelles and the D resides within an inside of the micelles.

12. The method of treating inflammatory liver disease of claim 1 , wherein prodrug composition is at least one selected from the group consisting ofpharmaceutically acceptable salt thereof.

13. A prodrug composition having the formula:wherein, TL is a targeting ligand, L is multivalent linkage, D is a drug, Z is a zwitterionic group, and n, m, and p are integers from 1 to about 4.

14. The prodrug composition of claim 13, wherein TL is at least one selected from the group consisting of cholic acid (CA), folic acid (FA), mannose, RGD peptide, integrin targeting ligand, glu-urea-based PSMA ligand, phosphatidylserine, and maleic acid.

15. The prodrug composition of claim 13, wherein L is at least one selected from the group consisting of an oligo amino acid and an orthogonal tri-functional aromatic molecule.

16. The prodrug composition of claim 13, wherein D is at least one selected from the group consisting of dexamethasone, prednisone, prednisolone, cortisone, hydrocortisone, fludrocortisone, paclitaxel, docetaxel, carbazitaxel, SN-38, doxorubicin, daunorubicin, topotecan, camptothecin, epothilone, irinotecan, and etoposide.

17. The prodrug composition of claim 13, wherein Z is at least one selected from the group consisting of glycerophosphorylcholine (GPC), carboxylic betaine (CB), carnitine, choline phosphate (CP), sulfobetaines (SB), and trimethylamine N-oxide (TMAO).

18. The prodrug composition of claim 13, wherein L is an oligolysine having from about 1 to about 16 lysine residues.

19. The prodrug composition of claim 13, wherein Z is GPC.

20. The prodrug composition of claim 13, wherein Z is CB.

21. The prodrug composition of claim 13, wherein D is at least one selected from the group consisting of glucocorticoid (GC) drugs, chemotherapeutic drugs, and immunosuppressant drugs.

22. The prodrug composition of claim 13, wherein said prodrug assembles into micelles having the TL and Z reside on an outside of the micelles and the D resides within an inside of the micelles.

23. The prodrug composition of claim 13, wherein prodrug composition is at least one selected from the group consisting of, and or a pharmaceutically acceptable salt thereof.

24. A method of treating cancer in a subject in need thereof, said method comprising administering a therapeutically effective amount of a prodrug composition of claim 13.

25. A method of treating an immune system disease in a subject in need thereof, said method comprising administering a therapeutically effective amount of a prodrug composition of claim 13.

26. The method of treating inflammatory liver disease of claim 1 , wherein the inflammatory liver disease further comprises sepsis.

27. The method of treating inflammatory liver disease of claim 1 , wherein the inflammatory liver disease is alcoholic hepatitis (AH), severe alcoholic hepatitis (sAH), and / or autoimmune hepatitis.

28. The method of treating inflammatory liver disease of claim 1 , wherein the prodrug composition comprises nanoparticles having a diameter size that is from about 10nm to about 20nm.

29. The prodrug composition of claim 13, wherein the prodrug composition comprises nanoparticles having a diameter size that is from about 10nm to about 20nm.

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