The present invention also contemplates the use of rapamycin analogs and derivatives. Exemplary rapamycin analogs and derivatives that may be used in the present invention include, but are not limited to, those described in WO 2001/034816; WO

2013/093493; WO 2015/004455; U.S. Patent Nos. 7,795,252; 7,678,901, 6,015,815,

6,015,809; 6,004,973; 5,985,890; 5,955,457; 5,922,730; 5,912,253; 5,780,462; 5,665,772;

5,637,590; 5,567,709; 5,563,145; 5,559,122; 5,559,120; 5,559,119; 5,559,112; 5,550,133; 5,541, 192; 5,541, 191; 5,532,355; 5,530,121; 5,530,007; 5,525,610; 5,521,194; 5,519,031;

5,516,780; 5,508,399; 5,508,290; 5,508,286; 5,508,285; 5,504,291; 5,504,204; 5,491,231;

5,489,680; 5,489,595; 5,488,054; 5,486,524; 5,486,523; 5,486,522; 5,484,791; 5,484,790;

5,480,989; 5,480,988; 5,463,048; 5,446,048; 5,434,260; 5,411,967; 5,391,730; 5,389,639;

5,385,910; 5,385,909; 5,385,908; 5,378,836; 5,378,696; 5,373,014; 5,362,718; 5,358,944; 5,346,893; 5,344,833; 5,302,584; 5,262,424; 5,262,423; 5,260,300; 5,260,299; 5,233,036;

5,221,740; 5,221,670; 5,202,332; 5, 194,447; 5, 177,203; 5, 169,851; 5, 164,399; 5, 162,333;

5,151,413; 5,138,051; 5, 130,307; 5, 120,842; 5, 120,727; 5, 120,726; 5, 120,725; 5, 118,678;

5,118,677; 5,100,883; 5,023,264; 5,023,263; 5,023,262; all of which are hereby incorporated by reference. As used herein, the term rapamycin and rapamycin derivatives includes rapamycin, as well as pharmaceutically acceptable salts, esters, carbamates, and prodrugs thereof, as well as rapamycin derivatives having from one to ten modifications with respect to rapamycin (e.g., from one to five or from one to four alternative and/or additional substituents). Substituents may be independently selected from hydroxy, alkyl, alkenyl, carboxyl, alkyloxy, amino, amido, aryl, or halogen. In some embodiments, the derivative is an enantiomer of rapamycin.

The pharmaceutical composition of the invention comprises a nanoparticle or microparticle carrier to deliver the rapamycin or an analogue or derivative thereof to the liver. As used herein, the term "nanoparticle," refers to a particle having at least one dimension in the range of about 1 nm to about 1000 nm. The term "microparticle" includes particles having at least one dimension in the range of at least about one micrometer (μιη). The term "particle" includes nanoparticles and microparticles. The size of the particle carrier can impact the pharmacodynamics of the composition, including tissue distribution, cell internalization, and size of the payload, for example. In various embodiments, the particle may have a size (e.g., average diameter) in the range of about 25 nm to about 5 μιη. In various embodiments, the particle carrier may have a size in the range of about 25 nm to about 500 nm, or in the range of about 50 nm to about 300 nm, or in the range of about 50 nm to about 250 nm, or in the range of about 50 to 150 nm.

In some embodiments, the nanoparticle or microparticle is polymeric. For example, the particle carrier may comprise a material having one or more degradable linkages, such as an ester linkage, a disulfide linkage, an amide linkage, an anhydride linkage, and a linkage susceptible to enzymatic degradation. For example, the nanoparticle or microparticle may comprise polymers or copolymers selected from cyclodextrin, poly(D,L-lactic acid-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-Lactide) (PLLA), PLGA-b-poly(ethylene glycol)-PLGA (PLGA-bPEG-PLGA), PLLA-bPEG-PLLA, PLGA-PEG, poly(D,L-lactide-co-caprolactone), poly(D,L-Lactide- co-caprolactone-co-glycolide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (UPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes, polyalkylene oxides (PEO), polyalkylene terephthalates, polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), derivatized celluloses such as alkyl cellulose, hydroxy alkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as polymethylmethacrylate) (PMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly (isobutyl (meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), polyiisobutyl acrylate), poly(octadecyl acrylate) (poly acrylic acids), polydioxanone, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, polyvinylpyrrolidone, polyorthoesters, polyphosphazenes, and polyphosphoesters. In one embodiment, the nanoparticle or microparticle may comprise PLGA and/or PLGA-PEG polymers or PLA and/or PLA-PEG polymers. In alternative embodiments, the nanoparticle or microparticle may be a micellar assembly comprising surfactants or liposome. Various nanoparticle or microparticle carrier systems have been described, and find use with the invention, including those described in US 8,206,747, US 2014/0112881, US 2015/0202163, US 2015/0209447, and WO/2015/105549, which are hereby incorporated by reference in their entireties.

The nanoparticle or microparticle may be designed to provide desired pharmacodynamic advantages, including circulating properties, biodistribution, and degradation kinetics. Such parameters include size, surface charge, polymer composition, targeting ligand conjugation chemistry, among others. For example, in some embodiments, the particles have a PLGA polymer core, and a hydrophilic shell formed by the PEG portion of PLGA-PEG co-polymers. The hydrophilic shell may further comprise ester-endcapped PLGA-PEG polymers that are inert with respect to functional groups, such as PLGA-PEG-MeOH polymers. The nanoparticles can be tuned for a specific biodegradation rate in vivo by adjusting the LA:GA ratio and/or molecular weight of the PLGA polymer. In some embodiments, the PLGA is based on a LA:GA ratio of from 20: 1 to 1 :20, including compositions of L/G of: 5/95, 10/90, 15/85, 20/80, 25/75, 30/70, 35/65, 40/60, 45/55, 50/50, 55/45, 60/40, 65/35, 70/30, 75/25, 80/20, 85/15, 90/10, or 95/5. PLGA degrades by hydrolysis of its ester linkages. The time required for degradation of PLGA is related to the ratio of monomers: the higher the content of glycolide units, the lower the time required for degradation as compared to predominantly lactide units. In addition, polymers that are end-capped with esters (as opposed to the free carboxylic acid) have longer degradation half-lives. The molecular weights of the PLGA and PEG co-polymers allows for tunable particle size. For example, PLGA co-polymers may have a molecular weight within about 10K to about 100K, and PEG co-polymers may have a molecular weight within about 2K to about 20K.

The rapamycin or an analogue or derivative thereof may be non-covalently incorporated into the particle carrier. For example, the rapamycin or an analogue or derivative thereof may be non-covalently incorporated into a crosslinked or non- crosslinked network of polymer molecules, which are part of the polymeric carrier. In other embodiments, the rapamycin or an analogue or derivative thereof is covalently linked to the nanoparticle or microparticle carrier, and released upon degradation of the carrier. In some embodiments, the nanoparticle or microparticle carrier is formed by self- assembly in an aqueous environment. For example, the particles may be formed by self- crosslinking reactions with self-crosslinking polymer as described in US 2014/0112881, which is hereby incorporated by reference.

In particular embodiments, the carrier comprises an oligoethylene glycol (OEG) hydrophilic shell and a lipophilic interior comprising disulfide-crosslinked branch groups, allowing the carrier to degrade in the presence of intracellular concentrations of biochemical reductant, such as glutathione (GSH). In these embodiments, the particles may be formed from amphiphilic polymers comprising the hydrophilic OEG branch groups and the lipophilic branch groups. The oligoethylene glycol (OEG) groups include

, wherein p is an integer from about 5 to about 200 (e.g., from about 5 to about 150, from about 5 to about 100, from about 5 to about 50, from about 10 to about 200, from about 20 to about 200, from about 50 to about 200, from about 100 to about 200, from about 10 to about 30, from about 10 to about 50). In some embodiments, the OEG branch groups have from 5 to 50 ethylene glycol units. OEG units may introduce a charge-neutral hydrophilic functional group, which provides biocompatibility.

Lipophilic branch groups comprise a lipophilic moiety to drive particle assembly and allow crosslinking of the interior. For example, the lipophilic branch groups may comprise pyridyl disulfide (PDS) moieties. The lipophilic functionality provides a supramolecular amphiphilic nano-assembly in the aqueous phase, which helps avoid the use of any additional surfactant molecules to generate the nanogel. The amphiphilic nature of the nanoparticle or microparticle carrier (e.g. nanogel) and lipophilic environment provides the opportunity for lipophilic guest molecules or active agents to be sequestered within these nano-assemblies prior to crosslinking. The PDS functionality is reactive, but specific, to thiols and provides a mild method for disulfide crosslinking to form the nanogel. Furthermore, since the nanoparticle or microparticle carriers may be based on disulfide crosslinkers that can be cleaved by thiol-disulfide exchange reactions, these nanogels also have a pathway to release the stably encapsulated guest molecules. Further, because the nanoparticle or microparticle formation can be conducted with thiol-disulfide exchange or thiol reshuffling reactions, the use of organic solvents and metal containing catalysts or additional reagents can be avoided. In some embodiments, the disulfide exchange reaction may shuffle sulfhydryl groups of dithiothreitol (DTT) into the disulfides of disulfide-linked lipophilic branch groups. The OEG branch groups and the lipophilic branch groups may be present at a ratio of from 1 :4 to 4: 1. In one embodiment, the OEG branch groups and the lipophilic branch groups may be present at a ratio of about 1 :4, 1 : 3, 1 :2, 1 : 1, 2: 1, 3 : 1 or 4: 1. The amphiphilic co-polymer may be prepared by reversible addition fragmentation chain transfer (RAFT) polymerization of pyridyl disulfide ethyl methacrylate (PDSEMA) and oligoethylene glycol monomethyl ether methacrylate. The resulting polymer may be purified with precipitation methods. See, for example, US 2014/0112881, which is hereby incorporated by reference.

In some embodiments, the crosslinked network of the nanoparticle or microparticle may have a crosslinking density in the range of from 2% to 80%, relative to the total number of structural units in the polymer. For example, the crosslinked network of may have a crosslinking density from about 2% to about 70%, from about 2% to about 60%>, from about 2% to about 50%, from about 2% to about 40%, from about 2% to about 30%, from about 2% to about 20%, from about 2% to about 10%, from about 5% to about 80%, from about 10% to about 80%, from about 20% to about 80%, from about 30% to about 80%), from about 40% to about 80%, relative to the total number of structural units in the polymer. Other variations for formulation of particle carriers in accordance with this disclosure may be used, including those described in one or more of US 2014/0112881, US 2015/0202163, US 2015/0209447, and WO/2015/105549, which are hereby incorporated by reference in their entireties.

In another aspect, the invention relates to a method for making the pharmaceutical composition described herein. The method comprises incorporating the rapamycin or an analogue or derivative thereof into a nanoparticle or microparticle carrier, including by cross-linking of lipophilic branch groups as described above, or by nanoprecipitation using PLGA-PEG polymers or similar polymer constructs.

The rapamycin or an analogue or derivative thereof is released upon partial or complete degradation or de-crosslinking of polymer molecules at or near the biological site. For example, after transport of the nanoparticle or microparticle carrier to the liver, the carrier may be degraded or de-crosslinked, thereby releasing the active agent. In one embodiment, the degradation is triggered by an endosomal or intracellular environment upon cell internalization. For example, the degradation may be caused by breaking the disulfide bonds in the nanoparticle or microparticle carrier in a reducing environment. Alternatively, degradation of the nanoparticle or microparticle carrier may be triggered by low pH. In some embodiments, the active agent (i.e., rapamycin or an analogue or derivative thereof) is not substantially released at concentrations of reducing agent characteristic of blood plasma, so that active agent is only released after cell internalization.

In one aspect, the pharmaceutical composition of the present invention may comprise a targeting agent to direct the nanoparticle or microparticle to the liver. Such targeting may improve the efficiency and effectiveness of the active agent (i.e., rapamycin or an analogue or derivative thereof), as the local concentration of the active agent is elevated. In some embodiments, the targeting agent may be a liver selective targeting agent. In other embodiments, the target agent may be selective for one or more cells types found within the liver including but not limited to, hepatocytes, Kupffer cells, hepatic stellate cells, sinusoidal endothelial cells, bile duct epithelial cells, or hepatocellular carcinoma cells.

In some embodiments, the targeting moieties include, without limitation, an asialoorasomucoid (ASOR) polypeptide, a N-acetyl-galactosamine (NAG) sugar, an asialotrianntenary (A3) polypeptide, or a hyaluronan (HA) polypeptide. Without wishing to be bound by theory, it is believed that ASOR, NAG, A3, arabinogalactan, or any another synthetic or naturally occurring galactose-presenting molecules specifically target hepatocytes via the asialoglycoprotein receptors (ASGP-R), while HA, NAG or mannan specifically target the liver sinusoidal endothelial cells via the hyaluronan, NAG or mannose receptors, respectively. In some embodiments, the targeting agent is triantennary N-Acetylgalactosamine (GalNAc), dimeric GalNAc or monomeric GalNAc, which targets the particle carriers to hepatocytes.

In other embodiments, the targeting agent may be a statin, diethylenetriaminopentaacetic acid (DTP A), lactobionic acid, a liver-targeting peptide (e.g., conserved region 1 from circumsporozoite protein of Plasmodium sporozoite), any moieties with tri- and tetra-antennary N-linked sugar side chains with terminal galactose residues, a lysine based nitrile triacetic acid with saccharide group functionalities (e.g. O- GalNAc, O-Lactose), a targeting agent described in US 201 1/0077386, or any other targeting agents described in Mishra et al., (2013) BioMed Research International, 2013 : 1- 20, which is hereby incorporated by reference. Alternative targeting agents may be a cell- penetrating peptide (CPP).

In some embodiments, the targeting agent may be an antibody or antigen-binding fragment thereof. In other embodiments, the targeting agent may a peptide, aptamer, adnectin, polysaccharide, or biological ligand. The various formats for target binding include a single-domain antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin, a Tetranectin, an Affibody; a Transbody, an Anticalin, an AdNectin, an Affilin, a Microbody, a peptide aptamer, a phylomer, a stradobody, a maxibody, an evibody, a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody, a pepbody, a vaccibody, a UniBody, a DuoBody, a Fv, a Fab, a Fab', a F(ab')2, a peptide mimetic molecule, or a synthetic molecule, or as described in US Patent Nos. or Patent Publication Nos. US 7,417, 130, US 2004/132094, US 5,831,012, US 2004/023334, US 7,250,297, US 6,818,418, US 2004/209243, US 7,838,629, US 7, 186,524, US 6,004,746, US 5,475,096, US 2004/146938, US 2004/157209, US 6,994,982, US 6,794, 144, US 2010/239633, US 7,803,907, US 2010/1 19446, and/or US 7, 166,697, the contents of which are hereby incorporated by reference in their entireties. See also, Storz MAbs. 201 1 May- Jun; 3(3): 310-317. Exemplary targeting agents include antigen-binding antibody fragments, such as but not limited to F(ab')2 or Fab, a single chain antibody, a bi-specific antibody, or a single domain antibody. In an embodiment, the antibody or antibody fragment thereof may recognize and bind to an epitope on hepatic cells such as the asialoglycoprotein receptor (ASGP-R) which are exclusively found on hepatocytes. For example, the antibody or antibody fragment thereof may recognize and bind the HI and/or H2 subunit of the ASGP-R. In an embodiment, the antibody or antibody fragment thereof may comprise a domain antibody (dAb) that bind to ASGP-R as described in US 2013/0078216, the entire contents of which is hereby incorporated by reference.

The targeting agent can be chemically conjugated to the particles using any available process. Functional groups for conjugation include COOH, H₂, and SH. See, e.g., Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, New York, 1996. Activating functional groups include alkyl and acyl halides, amines, sulfhydryls, aldehydes, unsaturated bonds, hydrazides, isocyanates, isothiocyanates, ketones, and other groups known to activate for chemical bonding. Alternatively, the targeting agent can be conjugated through the use of a small molecule-coupling reagent. Non-limiting examples of coupling reagents include carbodiimides, maleimides, N-hydroxysuccinimide esters, bischloroethylamines, bifunctional aldehydes such as glutaraldehyde, anhydrides and the like.

In some embodiments, the targeting agent may be conjugated or attached to the surface of the nanoparticle or microparticle, e.g., through OEG or PEG terminus. In one embodiment, the targeting agent is an antibody or antibody fragment linked to the polymeric units on the surface of the nanoparticle or microparticle, either non-covalently or covalently.

In one aspect, the nanoparticle or microparticle may be directed by passive targeting, referring to the accumulation of the nanoparticle or microparticle into particular regions of the body due to the natural features and physiological role of the tissues and cells. In some embodiments, the nanoparticle or microparticle carrier may accumulate in the liver in the absence of a targeting agent. For example, the nanoparticle or microparticle carrier may accumulate in the liver, which is an organ of the reticulo-endothelial system (RES) that captures foreign substances and objects that reach the systemic circulation. The pharmaceutical composition may be formulated into liquid or solid dosage forms and administered systemically or locally. The pharmaceutical composition may be delivered, for example, in a timed- or sustained-low release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intra-sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections or other modes of delivery.

While the form and/or route of administration can vary, in some embodiments the pharmaceutical composition is administered parenterally (e.g., by subcutaneous, intravenous, or intramuscular administration). For injection, the agents of the disclosure may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.

In some embodiments, the route of administration is transdermal, such as by transdermal patch or gel.

In some embodiments employing oral administration or administration to the GI, the pharmaceutical composition may further comprise an enteric coating. The enteric coating controls the release of the nanoparticles to avoid harsh environments of the stomach for example, by employing a coating that is insoluble at low pH, but soluble at higher pH so as to release particle carriers in the small or large intestine.

In some embodiments, the pharmaceutical composition is administered by intravenous or intraarterial administration, oral administration, subcutaneous administration, transdermal administration, or direct administration to desired tissues. In some embodiments, the pharmaceutical composition is administered from once daily to about once monthly. In some embodiments, particles are formulated to have a longer half- life, and thereby allow for less frequent administration. In one aspect, the invention relates to using the pharmaceutical composition described herein to treat liver-related diseases and conditions. In some embodiments, the present invention relates to using the pharmaceutical compositions described herein to treat liver-related diseases and conditions caused by dysfunctional autophagy. Autophagy or "self eating" is an important physiological process that targets cytosolic components such as proteins, protein aggregates and organelles for degradation in lysosomes. The cell utilizes this process for antigen presentation, recycling of amino acids from damaged proteins, degradation of defunct organelles, and subsequent generation of metabolites for energetic requirements. Defective autophagy is associated with a number of diseases including diseases of the liver. For example, defective autophagy has been linked to liver diseases caused by alpha- 1 -antitrypsin deficiency as well as other genetic disease including, but not limited to, fibrinogen storage disease or a hepatic lipid storage disease, such as cholesterol ester storage disease. Defective autophagy has also been suggested to disrupt lipid metabolism and contribute to non-alcoholic fatty liver disease (NAFLD).

Accordingly, in various embodiments, the present invention provides pharmaceutical compositions that allow for the liver-specific delivery of rapamycin or an analogue or derivative thereof for induction of autophagy in hepatocytes. In various embodiments, methods of the invention are effective in inducing and/or restoring autophagy in patients with an impairment or loss of autophagy. Such patients may suffer from any of the liver diseases described herein including, but not limited to, NAFLD as well as liver diseases caused by alpha- 1 -antitrypsin deficiency as well as other genetic disease including, but not limited to, fibrinogen storage disease or a hepatic lipid storage disease, such as cholesterol ester storage disease.

In various embodiments, the present invention provides methods for treating or ameliorating a liver disease. In certain embodiments, the methods provided herein include treatment of acute and/or chronic liver disease. In an embodiment, the methods are for treatment of an acute liver disease. In another embodiment, the methods are for treatment of a chronic liver disease. In one embodiment, the methods are for reducing liver damage or livery injury associated with acute and/or chronic liver disease.

In some embodiments, the liver disease is a disorder that results from an injury to the liver. In an embodiment, the injury to the liver is caused by toxins, including alcohol, drugs, impurities in foods, and the abnormal build-up of normal substances in the blood. In another embodiment, the injury to the liver is caused by an infection or by an autoimmune disorder.

In some embodiments, the liver disease includes, but is not limited to non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis (e.g., primary biliary cirrhosis (PBC)), liver fibrosis, liver injury (e.g., due to alcohol or other chemical toxicity), hepatic ischemia-reperfusion injury, alpha- 1 -antitrypsin deficiency, fibrinogen storage disease, or a hepatic lipid storage disease (e.g., cholesterol ester storage disease), hepatitis (including viral and alcoholic hepatitis), and hepatocellular carcinoma. In some embodiments, the liver disease is associated with elevated liver enzymes (e.g., ALT and AST), which are pathological evidence of on-going liver damage as a result of steatosis (fatty liver), fibrosis, and/or cirrhosis.

In an embodiment, provided herein are methods for treatment of fatty liver (also called hepatic steatosis), including non-alcoholic fatty liver disease (NAFLD). Fatty liver is defined as an excessive accumulation of triglyceride inside the liver cells. The most common causes of non-alcoholic fatty liver are obesity, diabetes, and elevated serum triglyceride levels. Other causes include malnutrition, hereditary disorders of metabolism (such as the glycogen storage diseases, and drugs (such as corticosteroids, tetracycline and aspirin)). In an embodiment, the methods provided herein are useful in treating one or more of the symptoms of non-alcoholic fatty liver disease, such as jaundice, nausea, vomiting, pain, and abdominal tenderness.

In an embodiment, provided herein are methods for treatment of non-alcoholic steatohepatitis (NASH), which is fatty liver with liver inflammation not caused by alcohol. In certain embodiments, NASH can be caused by any of the causes mentioned above as possible causes of NAFLD. In an embodiment, the present invention provides methods for treatment of hepatitis or inflammation of the liver, including viral and alcoholic hepatitis. The viral hepatitis can be acute or chronic. In certain embodiments, the acute viral hepatitis is caused by hepatitis A, B, C, D or E virus. In other embodiments, the acute viral hepatitis is caused by hepatitis B or C virus. In certain embodiments, the methods provided are for treatment of chronic viral hepatitis. In an embodiment, the chronic viral hepatitis is caused by hepatitis B or C virus.

In an embodiment, provided are methods for treatment of alcoholic hepatitis. Alcoholic hepatitis (steatohepatitis) is a combination of fatty liver, diffuse liver inflammation, and liver necrosis, in certain embodiments, focal necrosis, all in various degrees of severity.

In an embodiment, the present invention provides methods for treating liver fibrosis, lobular hepatitis and/or periportal bridging necrosis in a patient. Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. In certain embodiments, advanced liver fibrosis results in cirrhosis and liver failure. In certain embodiments, the liver fibrosis is caused by hepatitis, chemical exposure, bile duct obstruction, autoimmune disease, obstruction of outflow of blood from the liver, heart and blood vessel disturbance, alpha- 1 -antitrypsin deficiency, high blood galactose level, high blood tyrosine level, glycogen storage disease, diabetes, malnutrition, Wilson Disease and/or hemochromatosis.

In an embodiment, provided herein is a method for treating or ameliorating cirrhosis. In some embodiments, cirrhosis may be associated with hepatitis C, the use of certain drugs, alcohol abuse, chemical exposure, bile duct obstruction, autoimmune diseases, obstruction of outflow of blood from the liver (i.e., Budd-Chiari syndrome), heart and blood vessel disturbances, alpha- 1 -antitrypsin deficiency, high blood galactose levels, high blood tyrosine levels, glycogen storage disease, diabetes, malnutrition, hereditary accumulation of too much copper (Wilson Disease) or iron (hemochromatosis).

In an embodiment, provided herein are methods of treating liver disease associated with a metabolic disorder. In an embodiment, provided herein are methods of treating liver disease in a patient at risk for or is suffering from a metabolic disorder. Exemplary metabolic disorders include, but are not limited to, metabolic syndrome, diabetes, obesity. In an embodiment, the metabolic disorder is metabolic syndrome. For example, the metabolic syndrome may be associated with elevated triglycerides, elevated low density lipoproteins, reduced high density lipoproteins, reduced lipoprotein index, elevated fasting glucose levels, elevated fasting insulin, reduced glucose clearance following feeding, insulin resistance, impaired glucose tolerance, obesity and combinations thereof. In another embodiment, the metabolic disorder is obesity. In such an embodiment, the patient may also be suffering from hyperlipidemia and hyperlipoproteinemia. In a further embodiment, the metabolic disease is diabetes (type 1 or type 2) or one or more of insulin resistance, prediabetes, impaired fasting glucose (IFG), and impaired glucose tolerance (IGT).

By stimulating autophagy, other treatment regimens may be made more effective, including treatments for liver disease and/or metabolic disease. In some embodiments, the present invention relates to treatment of a patient who is undergoing treatment with an anti-obesity agent. Illustrative agents include, but are not limited to, orlistat, lorcaserin, phentermine-topiramate, naltrexone-bupropion, sibutramine, rimonabant, exenatide, pramlintide, phentermine, benzphetamine, diethylpropion, phendimetrazine, bupropion, and metformin. In various embodiments, the additional agent is an agent that that interfere with the body's ability to absorb specific nutrients in food, such as orlistat, glucomannan, and guar gum. Agents that suppress appetite are also among the additional agents, e.g. catecholamines and their derivatives (such as phentermine and other amphetamine-based drugs), various anti-depressants and mood stabilizers (e.g. bupropion and topiramate), anorectics (e.g. dexedrine, digoxin). Agents that increase the body's metabolism are also among the additional agents. In some embodiments, additional agents may be selected from among appetite suppressants, neurotransmitter reuptake inhibitors, dopaminergic agonists, serotonergic agonists, modulators of GABAergic signaling, anticonvulsants, antidepressants, monoamine oxidase inhibitors, substance P ( K1) receptor antagonists, melanocortin receptor agonists and antagonists, lipase inhibitors, inhibitors of fat absorption, regulators of energy intake or metabolism, cannabinoid receptor modulators, agents for treating addiction, agents for treating metabolic syndrome, peroxisome proliferator-activated receptor (PPAR) modulators; and dipeptidyl peptidase 4 (DPP -4) antagonists. In some embodiments, additional agents may be selected from among amphetamines, benzodiazepines, sulfonyl ureas, meglitinides, thiazolidinediones, biguanides, beta-blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers, phenlermine, sibutramine, lorcaserin, cetilistat, rimonabant, taranabant, topiramate, gabapentin, valproate, vigabatrin, bupropion, tiagabine, sertraline, fluoxetine, trazodone, zonisamide, methylphenidate, varenicline, naltrexone, diethylpropion, phendimetrazine, repaglinide, nateglinide, glimepiride, pioglitazone, rosiglilazone, liraglutide, and sitagliptin.

In some embodiments, the present invention relates to treatment of a patient who is undergoing treatment with an agent for treating pre-diabetes, diabetes, type II diabetes, insulin resistance, glucose intolerance, or hyperglycemia. Examples of agents include, but are not limited to, alpha-glucosidase inhibitors, amylin analogs, dipeptidyl peptidase-4 inhibitors, GLP1 agonists, meglitinides, sulfonylureas, biguanides, thiazolidinediones (TZD), and insulin. Additional examples of such agents include bromocriptine and Welchol. Examples of alpha-glucosidase inhibitors include but are not limited to acarbose and miglitol. An example of an amylin analog is pramlintide. Examples of dipeptidyl peptidase-4 inhibitors include but are not limited to saxagliptin, sitagliptin, vildagliptin, linagliptin, and alogliptin. Examples of GLP1 agonist include but are not limited to liraglutide, exenatide, exenatide extended release. Examples of meglitinides include but are not limited to nateglinide, and repaglinide. Examples of sulfonylureas include but are not limited to chlorpropamide, glimepiride, glipizide, glyburide, tolazamide, and tolbutamide. Examples of biguanides include but are not limited to metformin, Riomet, Glucophage, Glucophage XR, Glumetza. Examples of thiazolidinedione include but are not limited to rosiglitazone and pioglitazone. Examples of insulin include but are not limited to Aspart, Detemir, Glargine, Glulisine, and Lispro. Examples of combination drugs include but are not limited to glipizide/metformin, glyburide/metformin, pioglitazone/glimepiride, pioglitazone/metformin, repaglinide/metformin, rosiglitazone/glimepiride, rosiglitazone/metformin, saxagliptin/metformin, sitagliptin/simvastatin, sitagliptin/metformin, linagliptin/metformin, alogliptin/metformin, and alogliptin/pioglitazone.

In some embodiments, the present invention relates to treatment of a patient who is undergoing treatment with an anti-viral agent that includes, but is not limited to, Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine, Famciclovir, and Foscarnet.

In some embodiments, the present invention relates to treatment of a patient who is undergoing treatment with an anti-inflammatory agent such as steroidal anti-inflammatory agents or non-steroidal anti-inflammatory agents (NSAIDS). Steroids, particularly the adrenal corticosteroids and their synthetic analogues, are well known in the art. Examples of corticosteroids useful in the present invention include, without limitation, hydroxyltriamcinolone, alpha-methyl dexamethasone, beta-methyl betamethasone, beclomethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, clobetasol valerate, desonide, desoxymethasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, clocortelone, clescinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate. (NSAIDS) that may be used in the present invention, include but are not limited to, salicylic acid, acetyl salicylic acid, methyl salicylate, glycol salicylate, salicylmides, benzyl-2,5-diacetoxybenzoic acid, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin. Additional anti-inflammatory agents are described, for example, in U.S. Patent No. 4,537,776, the entire contents of which are incorporated by reference herein.

In some embodiments, the present invention relates to treatment of a patient who is undergoing treatment with an agent used for treating alpha- 1 -antitrypsin deficiency such as carbarn azepine.

Claims

CLAIMS:

1. A pharmaceutical composition comprising rapamycin or an analogue thereof encapsulated in a pharmaceutically acceptable microparticle or nanoparticle carrier that selectively targets the liver.

2. The pharmaceutical composition, wherein the rapamycin or analogue thereof induces autophagy in hepatocytes.

3. The pharmaceutical composition of claim 1 or 2, wherein the rapamycin or analogue thereof is released upon degradation of the microparticle or nanoparticle.

4. The pharmaceutical composition of claim 3, wherein degradation is triggered by an endosomal environment.

5. The pharmaceutical composition of claim 3 or 4, wherein degradation is triggered by increased concentration of biochemical reductant or by acidic pH.

6. The pharmaceutical composition of claim 3, wherein degradation of the microparticle or nanoparticle occurs extracellularly.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the microparticle or nanoparticle has a size in the range of about 25 nm to about 500 nm, or in the range of about 50 nm to about 300 nm, or in the range of about 100 nm to about 250 nm.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the rapamycin or analogue is incorporated in the microparticle or nanoparticle non-covalently.

9. The pharmaceutical composition of any one of claims 1 to 7, wherein the rapamycin or analogue is incorporated in the microparticle or nanoparticle covalently.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the microparticle or nanoparticle is polymeric.

11. The pharmaceutical composition of claim 10, wherein the microparticle or nanoparticle comprises a crosslinked interior.

12. The pharmaceutical composition of claim 10, wherein the microparticle or nanoparticle comprises an oligoethylene glycol (OEG) or polyethylene glycol (PEG) hydrophilic shell.

13. The pharmaceutical composition of claim 12, wherein the microparticle or nanoparticle carrier degrades in the presence of intracellular concentrations of glutathione

(GSH), and the carrier comprises an oligoethylene glycol (OEG) hydrophilic shell and a lipophilic interior comprising disulfide-crosslinked branch groups.

14. The pharmaceutical composition of claim 13, wherein the carrier is formed by self- assembly in an aqueous environment.

15. The pharmaceutical composition of claim 13 or 14, wherein the microparticle or nanoparticle carrier is formed in the presence of the rapamycin or analogue and an amphiphilic copolymer, the amphiphilic copolymer comprising hydrophilic oligoethylene glycol branch groups and disulfide-linked lipophilic branch groups to drive micellar assembly and encapsulation, followed by cross-linking of lipophilic branch groups through disulfide exchange reactions.

16. The pharmaceutical composition of claim 15, wherein the rapamycin or analogue is not substantially released at concentrations of reducing agent characteristic of blood plasma.

17. The pharmaceutical composition of claim 15 or 16, wherein the OEG branch groups have from 5 to 50 ethylene glycol units.

18. The pharmaceutical composition of any one of claims 15 to 17, wherein the lipophilic branch groups comprise disulfide-linked aryl groups, and are optionally pyridyldisulfide (PDS).

19. The pharmaceutical composition of any one of claims 15 to 18, wherein the OEG branch groups and the lipophilic branch groups are present at a ratio of from about 1 :4 to about 4: 1.

20. The pharmaceutical composition of claim 19, wherein the amphiphilic co-polymer is prepared by RAFT polymerization of pyridyl disulfide ethyl methacrylate (PDSEMA) and oligoethylene glycol monomethyl ether methacrylate.

21. The pharmaceutical composition of claim 20, wherein the disulfide exchange reaction shuffles sulfhydryl groups of dithiothreitol (DTT) into the disulfides of disulfide- linked lipophilic branch groups.

22. The pharmaceutical composition of claim 21, wherein the crosslinking density of the nanoparticle or microparticle carrier is from 2% to 50%.

23. The pharmaceutical composition of any one of claims 1 to 22, wherein the particle delivery system is described in one or more of US 2014/0112881, US 2015/0202163, US

2015/0209447, and WO/2015/105549, which are hereby incorporated by reference in their entireties.

24. The pharmaceutical composition of claim 10, comprising one or more polymers or copolymers selected from cyclodextrin, poly(D,L-lactic acid-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), PLA-PEG, poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(L-lactic acid-co- glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-Lactide) (PLLA), PLGA-b- poly(ethylene glycol)-PLGA (PLGA-bPEG-PLGA), PLLA-bPEG-PLLA, PLGA-PEG, poly(D,L-lactide-co-caprolactone), poly(D,L-Lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L- lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes, polyalkylene oxides (PEO), polyalkylene terephthalates, polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), derivatized celluloses such as alkyl cellulose, hydroxy alkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as polymethylmethacrylate) (PMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly (isobutyl (meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), polyiisobutyl acrylate), poly(octadecyl acrylate) (poly acrylic acids), polydioxanone, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, polyvinylpyrrolidone, polyorthoesters, polyphosphazenes, and polyphosphoesters.

25. The pharmaceutical composition of claim 24, comprising PLGA and PLGA-PEG.

26. The pharmaceutical composition of claim 24 or 25, wherein the particle carrier is described in US 8,206,747, which is hereby incorporated by reference in its entirety.

27. The pharmaceutical composition of claim 8 or 9, wherein the particle is a micellar assembly comprising surfactants.

28. The pharmaceutical composition of any one of claims 1 to 27, wherein the particle carrier has a liver-targeting agent conjugated to the surface thereof.

29. The pharmaceutical composition of claim 28, wherein the targeting agent is triantennary N-Acetylgalactosamine (GalNAc), dimeric GalNAc or monomeric GalNAc.

30. The pharmaceutical composition of claim 28, wherein the targeting agent is a statin, diethylenetriaminopentaacetic acid (DTP A), lactobionic acid, liver-targeting peptide (conserved region 1 from circumsporozoite protein of Plasmodium sporozoite), lysine based nitrile triacetic acid with saccharide group functionalities (e.g. O-GalNAc, O- Lactose), or a targeting agent described in US 201 1/0077386, which is hereby incorporated by reference.

30. The pharmaceutical composition of claim 28, wherein the targeting agent is an antibody or antigen-binding fragment thereof.

31. The pharmaceutical composition of claim 28, wherein the targeting agent is a peptide, aptamer, adnectin, polysaccharide, or biological ligand.

32. The pharmaceutical composition of claim 30, wherein the antigen-binding fragment is F(ab')2 or Fab, a single chain antibody, a bi-specific antibody, or a single domain antibody.

33. The pharmaceutical composition of claim 28, wherein the targeting agent is a cell- penetrating peptide (CPP).

34. The pharmaceutical composition of claim 28, wherein the targeting agent is a single-domain antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin, a Tetranectin, an Affibody; a Transbody, an Anticalin, an AdNectin, an Affilin, a Microbody, a peptide aptamer, a phylomer, a stradobody, a maxibody, an evibody, a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody, a pepbody, a vaccibody, a UniBody, a DuoBody, a Fv, a Fab, a Fab', a F(ab')2, a peptide mimetic molecule, or a synthetic molecule, or as described in US Patent Nos. or Patent Publication Nos. US 7,417, 130, US 2004/132094, US 5,831,012, US 2004/023334, US 7,250,297, US 6,818,418, US 2004/209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US 2004/146938, US 2004/157209, US 6,994,982, US 6,794,144, US 2010/239633, US 7,803,907, US 2010/119446, and/or US 7,166,697, the contents of which are hereby incorporated by reference in their entireties. See also, Storz MAbs. 2011 May-Jun; 3(3): 310-317.

35. The pharmaceutical composition of claim 28, wherein the targeting agent induces receptor-dependent cellular uptake, or is internalization through an endocytic pathway.

36. The pharmaceutical composition of any one of claims 1 to 27, wherein the nanoparticle or microparticle carrier accumulates in the liver in the absence of a targeting agent.

37. The pharmaceutical composition of any one of claims 1 to 36, wherein the carrier encapsulates rapamycin.

38. The pharmaceutical composition of any one of claims 1 to 36, wherein the carrier encapsulates a rapamycin analogue selected from US 7,795,252, WO 2001/034816, WO 2013/093493, or WO 2015/004455, which are hereby incorporated by reference in their entireties.

39. The pharmaceutical composition of any one of claims 1 to 38, formulated for intravenous, intraarterial, subcutaneous, or oral administration.

40. A method for making the pharmaceutical composition of any one of claims 1 to 39, comprising: incorporating the rapamycin or analogue thereof into a nanoparticle or microparticle carrier that selectively targets the liver.

41. A method for treating a disease or condition of the liver, comprising administering an effective amount of the pharmaceutical composition of any one of claims 1 to 39 to a patient in need thereof.

42. The method of claim 41, wherein the pharmaceutical composition is administered by intravenous, intraarterial, subcutaneous, transdermal, or oral administration.

43. The method of claim 41 or 42, wherein the pharmaceutical composition is administered from once daily to about once monthly.

44. The method of any one of claims 42 to 44 wherein the patient has non-alcoholic fatty liver disease (NAFLD); liver fibrosis; liver injury due to alcohol; ischemia- reperfusion injury; alpha- 1 -antitrypsin deficiency; fibrinogen storage disease; or a hepatic lipid storage disease, which is optionally cholesterol ester storage disease.

45. The method of claim 41 or 42, wherein the patient has an impairment or loss of autophagy.

46. The method of claim 41 or 42, wherein the patient exhibits or is at risk of insulin resistance, metabolic disease, obesity, or type 2 diabetes.

47. The method of any one of claims 41 to 46, wherein the patient further received carbamazepine therapy.