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• • A—HUMAN NECESSITIES
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  • A61K31/00—Medicinal preparations containing organic active ingredients
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  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/365—Lactones
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
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Definitions

• the present invention relates the use of inhibitors of autophagy for the treatment of fibrosis. More particularly, autophagy inhibitors and methods of their use are provided for the treatment of fibrosis of liver, lung, kidney, and other tissues, and for the inhibition of stellate cell activation.
• Fibrotic diseases affect hundreds of millions worldwide. For example, chronic liver disease and cirrhosis exact an unacceptable burden on US and global health, with a rising incidence of end-stage liver disease that is attributable to aging of the HCV-infected cohort, accelerating prevalence of obesity and increasing rates of alcohol abuse in some regions. Of those ⁇ 5 million patients with chronic HCV in the US, -25% are already cirrhotic, and by 2030, that proportion will rise to 45% (Davis, et al. 2010. Aging of hepatitis C virus (HCV)-infected persons in the United States: a multiple cohort model of HCV prevalence and disease progression. Gastroenterology. 138:513-21, 521 el-6).
• HCV hepatitis C virus
• Hepatic fibrosis or scarring of the liver, is a wound-healing response that engages a range of cell types and mediators to encapsulate injury. Sustained signals associated with chronic liver disease caused by infection, drugs, metabolic disorders, or immune attack, are required for significant fibrosis to accumulate, although even acute injury will activate mechanisms of fibrogenesis. Cirrhosis is the most advanced stage of fibrosis, and is associated with greater scarring than fibrosis alone, and also with distortion of the liver parenchyma associated with septae and nodule formation, altered blood flow, and risk of liver failure.
• HSC hepatic stellate cells
• ECM extracellular matrix
• cytokines produced by HSCs
• Activation refers to the transdifferentiation of quiescent, retinoid-laden non-proliferative cells into proliferative and contractile myofibroblasts that display well-characterized phenotypic features. These features include enhanced proliferation, contractility, fibrogenesis, and retinoid loss, along with enlarged ER and lysosomal compartments (Friedman, S.L. 2008. Hepatic Stellate Cells - Protean, Multifunctional, and Enigmatic Cells of the Liver. Physiological Reviews. 88: 125- 172).
• HSCs not only respond to inflammatory stimuli, but also orchestrate the immune response through enhanced Toll-like receptor signaling, antigen presentation and interactions with specific immune cell subsets (Gao, B., et al. 2008. Liver: An organ with predominant innate immunity. Hepatology. 47:729-36; Radaeva, et al. 2006. Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis- inducing ligand-dependent manners. Gastroenterology.
• liver diseases e.g., hepatitis B virus (HBV) and hepatitis C virus (HCV)
• HBV hepatitis B virus
• HCV hepatitis C virus
• the present invention is based on the discovery that stellate cell activation and fibrosis are linked to autophagy, and provides compositions and methods for treating fibrosis.
• the present invention advantageously provides the ability to generate new treatment approaches, which can reduce the morbidity, mortality and economic burden of fibrotic diseases, such as, but not limited to, end-stage liver disease.
• the invention thus provides a method for treating fibrosis comprising administering to a patient in need of such treatment an effective amount for treating fibrosis of at least one inhibitor of autophagy.
• the fibrosis is hepatic fibrosis, kidney fibrosis or lung fibrosis.
• the at least one inhibitor of autophagy is a nucleic acid molecule or small molecule that targets a signaling molecule in the autophagy signaling pathway.
• the nucleic acid molecule is an antisense oligonucleotide.
• the antisense oligonucleotide is an siRNA or shRNA molecule.
• the shRNA has a nucleic acid sequence selected from the group consisting of GTGAUCAACGAAAUGCAGA- UUCAAGAGA-UCUGCAUUUCGUUGAUCAC (SEQ ID NO: 8), CUUUCUUCAUAUUAGCAU-UUCAAGAGA-AAUGCUAAUAUGAAGAAAG (SEQ ID NO: 9), and GCAGCUCAUUGAUAACCAU-UUCAAGAGA-
• the inhibitor of autophagy is a member selected from the group consisting of 3-methyladenine (3 MA), Wortmannin, bafilomycin Al, and lysosomotropic alkalines.
• the lysosomotropic alkaline is chloroquine or 3 -OH chloroquine.
• an inhibitor of autophagy is administered to a patient as an expression vector capable of expressing the inhibitor of autophagy in vivo.
• the patient is a mammal.
• the mammal is a human.
• the invention provides a pharmaceutical formulation comprising at least one inhibitor of autophagy and a pharmaceutical carrier.
• the pharmaceutical formulation is useful for the treatment of hepatic fibrosis, kidney fibrosis and/or lung fibrosis.
• the invention provides a method for the treatment of fibrosis comprising administering to a patient in need of such treatment a therapeutically effective amount of a pharmaceutical formulation comprising at least one inhibitor of autophagy and a pharmaceutical carrier, wherein the fibrosis is afflicting an organ or tissue that is a member selected from the group consisting of liver, pancreas, lung, heart, nervous system, kidneys, bone marrow, lymph nodes, endomyocardium, and retroperitoneum.
• the invention provides a method for the treatment of a disease or condition which comprises administering to a patient in need of such treatment a therapeutically effective amount of the pharmaceutical formulation comprising at least one inhibitor of autophagy and a pharmaceutical carrier, wherein said disease or condition is a member selected from the group consisting of cirrhosis, diffuse parenchymal lung disease, post-vasectomy pain syndrome, tuberculosis, sickle-cell anemia, rheumatoid arthritis, progressive massive fibrosis, idiopathic pulmonary fibrosis, injection fibrosis, myelofibrosis, cardiac fibrosis, pancreatic fibrosis, skin fibrosis, scleroderma, intestinal fibrosis or strictures, vascular fibrosis, and mediastinal fibrosis.
• cirrhosis diffuse parenchymal lung disease, post-vasectomy pain syndrome, tuberculosis, sickle-cell anemia, rheumatoid arthritis, progressive massive fibrosis
• the vascular fibrosis is atherosclerosis or phlebosclerosis.
• the pharmaceutical formulation according to the present invention comprises an inhibitor of autophagy that is an siRNA or shRNA molecule.
• the shRNA molecule in the pharmaceutical formulation has a nucleic acid sequence selected from the group consisting of GTGAUCAACGAAAUGCAGA- UUCAAGAGA-UCUGCAUUUCGUUGAUCAC (SEQ ID NO: 8), CUUUCUUCAUAUUAGCAU-UUCAAGAGA-AAUGCUAAUAUGAAGAAAG (SEQ ID NO: 9), and GCAGCUCAUUGAUAACCAU-UUCAAGAGA-
• the invention provides a method for treating fibrosis comprising administering to a patient in need of such treatment an effective amount for treating fibrosis of at least one inhibitor of autophagy, wherein the inhibitor of autophagy inhibits the expression level or activity of an autophagy signaling molecule.
• the invention provides a method for treating fibrosis comprising administering to a patient in need of such treatment an effective amount for treating fibrosis of at least one inhibitor of autophagy, wherein the inhibitor of autophagy inhibits the expression level or activity of an autophagy signaling molecule, and wherein the autophagy signaling molecule is a member selected from the group consisting of VPS34, Beclin 1, Atg3, Atg4, Atg5, Atg7, Atg8, Atgl2, Atgl4 and Atgl6.
• Figure 1A illustrates immunoblot results showing decreases in collagen led and accumulation of LC3B-II after blocking autophagy with chloroquine compared to control at 12 and 24 hours and
• Figure IB is a graph of q-RT-PCR results in JS1 cells treated with chloroquine or control; fold-change in mRNA expression of collagen led (“Coll al(I)”), collagen 1 ⁇ 2 (“Coll a2(I)”), alpha SMA ("ASMA”), ⁇ -PDGF-R, TGFpi, and MMP-2 is shown; "*" indicates statistical significance with P ⁇ 0.01.
• Figure 2 is a immunoblot showing decreased conversion of LC3B-
• Figure 3 is a graph showing q-RT-PCR on JS1 cells treated either with 3MA or transduced with siAtg7; fold-change in mRNA expression of collagen led (“Coll al(I)”), collagen 1 ⁇ 2 (“Coll a2(I)”), ⁇ -PDGF-R, TGFpi, MMP-2 and adipose differentiation-related protein (“ADRP”) is shown; "*” means that P ⁇ 0.01 for collagen and ⁇ -PDGFR; and P ⁇ 0.05 for MMP2.
• Figure 4 is an immunoblot showing expression of collagen 1
• Col(I) Cold Cell(I)
• ADRP adipose differentiation-related protein
• GAP dehydrogenase GAP dehydrogenase
• Figure 5 shows electron micrographs showing increases in autophagic vacuoles and lipid droplets in autophagy deficient JS1 cells treated with siAtg7 or 3MA compared to controls. Lipid droplets are indicated by dashed arrows and autophagolysosomes are indicated by solid black arrows.
• FIG. 6A shows immunoblots for Atg7, collagen 1 ("COL 1”) and LC3B-I and LC3B-II at the indicated time points on lysed JS 1 cells transduced with either empty lentiviral vector (VEC) or vector expressing shRNA to the Atg7 ("siAtg7"). Protein ratios (normalized to GAPDH or Tubulin) were used to quantify fold change relative to control, and those values are shown below each blot. Data represent the mean value of at least 3 experiments; "*" indicates statistical significance with P ⁇ 0.05.
• FIG. 6B shows immunoblots for Atg5 and LC3B-I and II on lysed JS1 cells transduced with either empty lentiviral vector (VEC) or vector expressing shRNA to the Atg5 ("siAtg5"). Protein ratios (normalized to GAPDH) were used to quantify fold change relative to control, and are shown below each blot. Data represent the mean value of at least 3 experiments (*P ⁇ 0.05).
• Figure 6C is a graph quantifying the number of autophagic vacuoles (AV) per hundred cells; "**” indicates statistical significance with PO.001; error bars represent the standard error of the mean (“SEM”).
• Figures 6D-6G are graphs quantifying the fold change in mRNA expression of collagen led (“Col lal”), collagen 1 ⁇ 2 (“Col la2”), a-sma, Mmp2 and ⁇ - Pdgfr, in JS-1 stellate cells treated with 3MA, chloroquine (CQ), siAtg7, and siAtg5, respectively. Data represent the mean value of at least 3 experiments; "*" indicates statistical significance with P ⁇ 0.05; indicates statistical significance with PO.001.
• Figure 6H shows immunoblots for protein expression of collagen la(I) ("COL1"), ⁇ -PDGFR, a-SMA and MMP2 in JS-1 stellate cells treated with 3 MA, chloroquine (CQ), siAtg7, or siAtg5; "*" indicates that P ⁇ 0.05, ""**”” indicates statistical significance with P ⁇ 0.001; error bars give the SEM; protein ratios (normalized to GAPDH or Tubulin) were used to quantify fold change relative to control, and are shown below each blot. Data represent the mean value of at least 3 experiments.
• Figure 7 shows immunoblots for LC3B-I and II (left blot) and P62
• Figure 8 is a graph quantifying the average number per hundred cells of autophagic vacuoles ("AV") in stellate cells isolated from wild-type mice after acute liver injury induction with CC14;""**"" indicates statistical significance with PO.001.
• AV autophagic vacuoles
• Figure 9A is an immunoblot of LC3B-I and II and P62 in primary human stellate cells isolated from hepatitis B virus ("HBV”) infected liver or non-infected control. Protein ratios (normalized to GAPDH) were used to quantify fold change relative to control, and are shown below each blot.
• HBV hepatitis B virus
• Figure 9B is a graph quantifying the average number per hundred cells of autophagic vacuoles ("AV") in primary human stellate cells isolated from HBV infected liver or non-infected control indicates statistical significance with PO.001 ; error bars give the SEM. -
• Figure 10 is an immunoblot of LC3B-I and II, P62 and GAPDH
• NF non-fibrotic lung
• IPF idiopathic pulmonary fibrosis
• Figure 1 1A shows immunoblot results in human hepatic stellate cell line, LX-2, treated with or without 3MA and showing decreases in collagen led, PDGFR- ⁇ , and MMP9 after treatment with 3 MA
• Figure 1 IB shows q-RT-PCR results in LX-2 cells treated with or without 3MA and showing fold-change in mR A expression levels of ⁇ -PDGFR, TIMP1, TIMP2, MMP9 and collagen 1 ⁇ 2 ("Col Ia2”) normalized to GAPDH.
• Figure 12 shows immunoblots of LC3B-I and LC3B-II protein expression in control (“sham”) wild-type mice or wild-type mice that underwent bile duct ligation ("BDL").
• Figure 13 shows immunoblot results in primary HSCs isolated from either control or chloroquine ("CQ") treated mice showing protein expression levels of LC3B-I , LC3B-II, collagen 1 (“COLLAGEN”) and Tubulin (loading control).
• CQ chloroquine
• Figures 14A and 14B show immunoblots of stellate cell protein isolated from Atg7 F/F and Atg7 F/F -GFAP -ere mice demonstrating decreased expression of Atg7, LC3B-II and increased P62 (GAPDH shown as loading control) ( Figure 14A) and expression of CRE recombinase ("CRE") in stellate cells from Atg7 F/F -GFAP-cre mice compared to their Atg7 F/F littermates (calnexin shown as loading control) ( Figure 14B).
• CRE CRE recombinase
• Figure 15 is a graph quantifying the average number per hundred cells of autophagic vacuoles ("AV") as determined by electron microscopy in stellate cells isolated from Atg7 F/F and Atg7 F/F -GFAP -ere mice. indicates statistical significance with P ⁇ 0.001 and error bars represent the SEM.
• AV autophagic vacuoles
• Figure 16 shows an immunoblot of collagen type I ("COL1") and a-SMA protein expression in isolated stellate cells from Atg7 F/F and Atg7 F/F -GFAP-cre mice after chronic liver injury with CC14. Protein ratios (normalized to GAPDH or Tubulin) were used to quantify fold change relative to control, and those values are shown below each blot. Data represent the mean value of at least 3 experiment; "*" indicates statistical significance with PO.05.
• Figure 17 is a graph quantifying the liver to body weight ratio after CC14-induced chronic liver injury in Atg7 F/F and Atg7 F/F -GFAP -ere mice treated with CC14 for 6 weeks. Results are shown as mean ⁇ SEM; "*" indicates statistical significance with PO.05.
• Figures 18A and 18B are graphs quantifying the aspartate aminotransferase ("AST") ( Figure 18A) and alanine aminotransferase (“ALT”) ( Figure 18B) level in units/L (U/L) in Atg7 and Atg7 -GFAP-cre mice treated with CC14 for 6 weeks. Error bars represent the SEM; and "NS" indicates "not statistically significant.”
• Figures 19A and 19B are immunoblots of COL 1, -SMA and j3
• Figure 20 shows immunoblots of collagen l ed ("COL 1 "), alpha
• -SMA mouse embryonic fibroblasts
• WT Atg5 knockout mice
• protein ratios normalized to GAPDH or Tubulin
• Figures 21 and 22 are immunoblots of collagen 1 ("COL 1 "), ⁇ -
• Figures 23, 24A and 24B are graphs quantifying lipid content in
• Figures 25A and 25B are graphs quantifying triglycerides content
• Figures 26A and 26B are immunoblots showing protein expression of ADRP in 3MA or control treated ( Figure 26A) or siAtg7 or control vector ("VEC") ( Figure 26B) transduced JS l cells. Protein ratios (normalized to GAPDH) were used to quantify fold change relative to control, and those values are shown below each blot. Data represent the mean value of at least 3 experiments; "*" indicates statistical significance with PO.05.
• Figure 27 is an immunoblot showing protein expression of ADRP in stellate cells from Atg7 F/F and Atg7 F/F -GFAP-cre mice. GAPDH is shown as a loading control.
• Figure 28 is a graph quantifying ATP content (picomoles per 10 6 cells) in JS l stellate cells treated with 3MA or control, 12 h after treatment; "*" indicates statistical significance with P ⁇ 0.05; error bars represent SEM.
• Figure 29 shows immunoblots of collagen l ed ("COL 1"), alpha
• Figures 30 and 31 shows immunoblots of collagen led ("COL 1 "), j3 -PDGFR, alpha SMA (" -SMA”), and MMP2 protein expression in JS l stellate cells treated with 3MA or 3MA + oleic acid (“OA”) or JS l stellate cells transduced with siAtg7 and supplemented with oleic acid conjugated to BSA ("OA”) or with BSA alone (“control”) ( Figure 31). Protein ratios (normalized to GAPDH) were used to quantify fold change relative to control, and those values are shown below each blot. Data represent the mean value of at least 3 experiments; "*" indicates statistical significance with PO.05. DETAILED DESCRIPTION
• the present invention is based on the discovery that autophagy contributes to stellate cell activation in fibrosis. As demonstrated herein, expression levels of fibrogenic genes were reduced following inhibition of autophagy in fibrosis from diverse tissues, including hepatic fibrosis, kidney fibrosis and lung fibrosis, indicating that autophagy is a conserved process in fibrosis, and not limited to one particular cell type. It is also discovered that autophagy plays an essential role in providing energy to fuel fibrogenesis not only in hepatic stellate cells, but also in embryonic myofibroblasts and fibrogenic cells from kidney and lung. These discoveries yield collateral benefits to the study and treatment of fibrotic disorders of other organs, including heart and pancreas, among others. Further, the present methods encompass in part a technique for the treatment of fibrosis using inhibitors of autophagy.
• Autophagy refers to a mechanism for breaking down cellular components including organelles or long-lived proteins in a cell that is evolutionarily conserved between all eukaryotes.
• cytoplasmic components are engulfed by double-membrane-bound structures (autophagosomes) and delivered to lysosomes/vacuoles for degradation.
• Starvation is a classic stimulus of autophagy in vitro.
• the autophagy process is mediated by a variety of proteins, including, among others, "VPS34", “LC3B”, and "P62.”
• yeast In yeast, more than thirty genes are shown to be essential for autophagy (Tsukada M. and Ohsumi Y., FEBS Lett (1993);333, 169-74). Recently, the autophagy-related genes and the products of these genes were named ATG and Atg, respectively. These ATG (AuTophaGy-related) genes contribute either to the formation of the machinery for autophagy, or to its regulation in response to a variety of signals.
• Beclin 1 is the homologue of yeast Atg6/Vps30, and it is among the earliest characterized mammalian autophagy genes. Beclin 1 is required for phagophore formation.
• Class III PI3K a functional orthologue of yeast Vps34, is an activator of autophagy and plays a crucial role at an early step of phagophore formation in mammalian cells, and it is required for the nucleation of the sequestering membrane, through its fusion with Beclin 1.
• Non-limiting examples of critical genes involved in autophagy are VPS34, Beclin 1, ATG3, ATG4, ATG5, ATG7, ATG8, ATG12, ATG 14 and ATG16.
• the autophagy signaling pathway and the signaling molecules critically involve therein are described in detail in International Patent Application Publication No. WO/2010/030936 by Yue et al; Mizumisha, N. et al.
• Atg proteins are the discovery of two ubiquitin-like conjugation systems, Atgl2-Atg5 and Atg8- phosphatidylethanolamine (PE).
• Atgl2-Atg5 and Atg8- phosphatidylethanolamine PE
• half of the Atg genes essential for autophagy are involved in these conjugation systems, are well conserved among eukaryotes, and are required for the formation and elongation of the phagophore membrane.
• Atg5 and Atg7 are particularly preferred targets for the inhibition of autophagy, although other autophagy signaling molecules, such as those described above, are also contemplated as targets in the present invention.
• Atg7 is specific for autophagy, is essential not only for the conjugation of the Atgl2-Atg5 system but also for LC3B-II lipidation, Atg conjugation and autophagosome formation, and is required in multiple steps in the autophagic cascade, including LC3B lipidation and autophagosome elongation (Heath, R.J., and R.J. Xavier. "Autophagy, immunity and human disease.” (2009) Curr Opin Gastroenterol. 25:512-20).
• Atg7 is a homologue of the ubiquitin activating enzyme that activates both Atg8/LC3B and Atgl2 (Rubinsztein, D.C., et al. "Potential therapeutic applications of autophagy.” (2007) Nat Rev Drug Discov. 3:14-12).
• Atg7 tissue-specific knockout mice have dramatic phenotypes reflecting loss of autophagy, including liver- and CNS-specific knockouts (Iwata, J., et al. "Excess peroxisomes are degraded by autophagic machinery in mammals.” (2006) J Biol Chem. 281 :4035-41 ; Komatsu, et al.
• Atg5 knockouts are as equally informative as the Atg7 knockouts (Lee, H.K., et al. "Autophagy-dependent viral recognition by plasmacytoid dendritic cells.” (2007) Science. 315: 1398-401 ; Nakai, A., et al. "The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress.” (2007) Nat Med. 13 :619-24).
• LC3 or "LC3B” is the commonly used name for microtubule- associated protein 1 light chain 3, a ubiquitin-like molecule that is the mammalian homologue of the autophagy-related Atg8 encoded product in yeast.
• proLC3B proteolytically cleaved by Atg4 protease, resulting in the LC3B-I form with a carboxyterminal exposed glycine.
• LC3B-II Upon induction of autophagy, the exposed glycine of LC3B-I is conjugated by Atg7 (an El -like activity), Atg3 (an E2-like conjugating activity) and by Atgl2- Atg5-Atgl6L multimers (E3-like ligase activity) to the highly lipophilic phosphatidylethanolamine (PE) moiety to generate LC3B-IL.
• the PE group promotes integration of LC3B-II into lipid membranes at the phagophore and autophagosomes.
• LC3B-II is the only well-characterized protein that is specifically localized to autophagic structures throughout the process from phagophore to lysosomal degradation.
• LC3B In wild-type cells, LC3B is detected in 2 forms: LC3B-I (18 kDa) and LC3B-II (16 kDa) (see, Mizushima, N., and T. Yoshimori. "How to interpret LC3 immunoblotting” (2007) Autophagy. 3 :542-5).
• LC3B-I is located in the cytoplasm, while LC3B-II is a tightly membrane bound protein and is attached to PAS and autophagosomes.
• the relative amount of membrane-bound LC3B-II reflects the abundance of autophagosomes, so the induction and inhibition of autophagy can be monitored through measuring total and free LC3B-II levels by means of immunoassay.
• LC3B-I to LC3B-II is indicative of autophagic activity.
• Immunoblotting of LC3 usually reveals two bands: LC3B-I (18 kDa) and LC3B-II (16 kDa).
• the amount of LC3B-II correlates well with the number of autophagosomes. This characteristic conversion of LC3B can be used to monitor autophagic activity.
• the cytoplasmic form (LC3B- I) is processed and recruited to the autophagosomes, where LC3B-II is generated by site specific proteolysis and lipidation near to the C-terminus.
• autophagic activation is thus the formation of cellular autophagosome punctae containing LC3B-II, while autophagic activity is measured biochemically as the amount of LC3B-II that accumulates also in the absence of lysosomal activity.
• the adaptor molecule P62/SQSTM1 is also implicated in activating autophagy or in targeting cargo to the autophagosomes. Its expression levels can also be determined by immunoblot for measuring autophagic activity in a cell.
• P62/SQSTM1 is a selective autophagy adaptor molecule that binds polyubiquitinated proteins and aggregates by oligomerization and binds to LC3 on the auotphagosome membrane to target aggregates.
• P62/SQSTM1 interacts with polyubiquitinated protein aggregates through a ubiquitin-binding domain and with LC3B through its LC3B-binding domain, thereby targeting these aggregates for degradation at the autolysosome. Impairment of autophagy leads to the inability to eliminate P62 and therefore to its accumulation.
• autophagy inhibitors such as R Ai molecules targeted to signaling molecules involved in the autophagy pathway (e.g., Atg5 and Atg7), 3 MA and chloroquine, inhibit autophagy, as evidenced by decreased presence of autophagolysosomes. Further, inhibition of autophagy was discovered to lead to decreased HSC activation, as evidenced by decreased expression of a panel of fibrogenic genes, such as collagen led, collagen 1 ⁇ 2, Tgf l, ⁇ -Pdgfr, Type I TGFP-R, Timp 1 & 2, alpha SMA and MMP-2 (Friedman, S.L. "Mechanisms of hepatic fibrogenesis.” 2008; Gastroenterology.
• adipose differentiation-related protein (ADRP), which is a marker of quiescent HSCs (Lee, et al. (2010) "Downregulation of hepatic stellate cell activation by retinol and palmitate mediated by adipose differentiation-related protein (ADRP)." J Cell Physiol. 223 :648-57).
• the present Examples demonstrate, in a murine model of CCl 4 -induced hepatic fibrosis, that mice with acute liver injury have increased levels of autophagy, showing that autophagy is involved in liver injury and fibrosis, and represents a new therapeutic target for the treatment of fibrosis-related diseases and conditions.
• Murine models of hepatic fibrosis closely mimic characteristics of human hepatic fibrosis and provide a useful tool for understanding human hepatic fibrosis as well as fibroses of other organs.
• the present invention provides compositions and methods for treating fibrosis involving inhibition of autophagy in cells. These compositions and methods are useful for treating fibrosis of any organ, and in particular, for the treatment of hepatic fibrosis.
• the inhibitors of the invention decrease hepatic stellate cell activation.
• the term “inhibitor of autophagy” encompasses any and all agents which reduce or abrogate the bulk degradation of intracellular material engulfed in a double membrane in a cell by its fusion with the lysosome.
• the term “inhibit” is used to refer to any level of reduction in autophagy in a cell or in a population of cells. The level of reduction of autophagy may be assessed by different methods to obtain an accurate measurement; however, non-limiting examples include those presented herein, such as quantifying the number of autophagolysosomes in a cell with electron microcopy, LC3B-II conversion and/or P62 levels.
• an inhibitor of autophagy inhibits the
• Autophagy signaling molecules include without limitation, VPS34, Beclin-1, Atg3, Atg4, Atg5, Atg7, Atg8, Atgl2, Atgl4 and Atgl6.
• An autophagy inhibitor that inhibits the expression level of such autophagy signaling molecule can be, without limitation, an oligonucleotide, such as an siR A or shR A, or a small molecule.
• An autophagy inhibitor that inhibits the activity of such autophagy signaling molecules can be, for example and without limitation, a fragment of the autophagy signaling molecule (e.g., a dominant negative fragment), a small molecule, an aptamer, or an oligonucleotide. It is to be understood that these inhibitors are provided as non-limiting examples, and the skilled artisan can readily design those and other inhibitors to target the desired autophagy signaling molecule using methods known in the art.
• fibrosis regression means an improvement in any stage of the disease state encompassed by "fibrosis", “liver disease” or “cirrhosis”, including but not limited to decreased levels of collagen type I (lal and 1 ⁇ 2) or other extracellular matrix or scar molecules in the liver or other organ and/or decreased numbers of activated hepatic stellate cells, myofibroblasts, or other fibrosis-producing mesenchymal cells, whether derived from within the liver or extra-hepatic sites.
• an "antisense" nucleic acid molecule or oligonucleotide is a single stranded nucleic acid molecule, which may be DNA, RNA, a DNA-RNA chimera, or a derivative thereof, which, upon hybridizing under physiological conditions with complementary bases in an RNA or DNA molecule of interest, inhibits the expression of the corresponding gene or mRNA splice variant by inhibiting, e.g., mRNA transcription, mRNA splicing, mRNA transport, or mRNA translation or by decreasing mRNA stability.
• antisense broadly includes RNA-RNA interactions, RNA-DNA interactions, and RNaseH mediated arrest.
• Antisense nucleic acid molecules can be encoded by a recombinant gene for expression in a cell (see, e.g., U.S. Patents No. 5,814,500 and 5,81 1,234), or alternatively they can be prepared synthetically (see, e.g., U.S. Patent No. 5,780,607).
• Examples of synthetic antisense oligonucleotides envisioned for this invention include oligonucleotides that contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl, or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those with CH2-NH-0- CH2, CH2-N(CH3)-0-CH2, CH2-0-N(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and O- N(CH3)-CH2-CH2 backbones (where phosphodiester is 0-P02-0-CH2).
• 5,677,437 describes heteroaromatic oligonucleoside linkages. Nitrogen linkers or groups containing nitrogen can also be used to prepare oligonucleotide mimics (U.S. Patents Nos. 5,792,844 and 5,783,682). U.S. Patent No. 5,637,684 describes phosphoramidate and phosphorothioamidate oligomeric compounds. Also envisioned are oligonucleotides having morpholino backbone structures (U.S. Pat. No. 5,034,506).
• the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al, Science, 1991, 254: 1497).
• oligonucleotides may contain substituted sugar moieties comprising one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, 0(CH2) n NH2 or 0(CH2) n CH 3 where n is from 1 to about 10; CI to CIO lower alkyl, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3; OCF 3 ; 0-; S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH 3 ; S0 2 CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted sialyl; a fluorescein moiety; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligon
• Oligonucleotides may also have sugar mimetics such as cyclobutyls or other carbocyclics in place of the pentofuranosyl group.
• Nucleotide units having nucleosides other than adenosine, cytidine, guanosine, thymidine and uridine may be used, such as inosine.
• ribozyme is used to refer to a catalytic RNA molecule capable of cleaving RNA substrates. Ribozyme specificity is dependent on complementary RNA-RNA interactions (for a review, see Cech and Bass, Annu. Rev. Biochem. 1986; 55: 599-629). Two types of ribozymes, hammerhead and hairpin, have been described. Each has a structurally distinct catalytic center.
• the present invention contemplates the use of ribozymes designed on the basis of signaling molecules in the autophagy signaling pathway (e.g., Atg5 or Atg7) to induce catalytic cleavage of the corresponding mRNA of such signaling molecules, thereby inhibiting expression of these molecules.
• Ribozyme technology is described further in Intracellular Ribozyme Applications: Principals and Protocols, Rossi and Couture ed., Horizon Scientific Press, 1999.
• the term "RNA interference" or "R Ai” refers to the ability of double stranded RNA (dsRNA) to suppress the expression of a specific gene or mRNA isoform of interest in a homology-dependent manner.
• RNA interference acts post-transcriptionally by targeting mRNA molecules for degradation.
• RNA interference commonly involves the use of dsRNAs that are greater than 500 bp; however, it can also be mediated through small interfering RNAs (siRNAs) or small hairpin RNAs (shRNAs), which can be 10 or more nucleotides in length and are typically 18 or more nucleotides in length.
• siRNAs small interfering RNAs
• shRNAs small hairpin RNAs
• oligonucleotides with the following nucleotide sequences were used for the cloning of shRNA-encoding sequences into a lentiviral vector: ATG7: 5 '
• ATCCCCGCAGCTCATTGATAACCATTTCAAGAGAATGGTTATCAATGAGCTGCTT TTTC-3 ' (SEQ ID NO: 1); ATG5 no. 1 : 5 ' ATCCCCGTGATCAACGAAATGCAGATTC
• oligonucleotides were annealed and cloned into pSUPER (Ambion) at the Bglll-Xhol site.
• the invention provides Atg5 shRNAs encoded by the shRNA-encoding sequences: ATG5 no. 1 : 5 ' ATCCCCGTGATCAACGAAATGCAGATTCAAGAGATCTGCATTTCGTTGATCACTT TTTGGAAA-3 ' (SEQ ID NO: 2); and/or ATG5 no. 2: GATCCCCCTTTCTTCATATTAGCATTTCAAGAGAAATGCTAATATGAAGAAAGTT TTTGGAAA (SEQ ID NO: 3).
• the invention provides an Atg7 shRNA encoded by the shRNA-encoding sequence: 5 ' ATCCCCGCAGCTCATTGATAACCATTTCAAGAGAAT
• GGTTATCAATGAGCTGCTTTTTC-3' (SEQ ID NO: 1).
• an Atg5 shRNA of the invention has a nucleic acid sequence: GTGAUCAACGAAAUGCAGA-UUCAAGAGA- UCUGCAUUUCGUUGAUCAC (SEQ ID NO: 8) or CUUUCUUCAUAUUAGCAU- UUCAAGAGA-AAUGCUAAUAUGAAGAAAG (SEQ ID NO: 9).
• an Atg7 shRNA of the invention has a nucleic acid sequence: GCAGCUCAUUGAUAACCAU-UUCAAGAGA-AUGGUUAUCAAUGAGCUGC (SEQ ID NO: 10). The dashes in the shRNA sequences represent separation of sense, loop and antisense sequences, from left to right.
• RNAi and other oligonucleotide inhibitors are known in the art, and the skilled artisan can design such molecules to target signaling molecules in the autophagy signaling pathway, such as, but not limited to VPS34, Beclin-1, Atg3, Atg4, Atg5, Atg7, Atg8, Atgl2, Atgl4 and Atgl6.
• Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind almost any molecule.
• the systematic evolution of ligands by exponential enrichment (SELEX) process (Ellington et al, Nature 1990; 346:818-22; Tuerk et al, Science 1990; 249:505-10) can be used to design such aptamers.
• Such apatamers can be used to inhibit expression or function of a desired target molecule.
• an aptamer designed to target Atg5 or Atg7 can prevent these molecules from functioning in the autophagy signaling pathway, thereby inhibiting autophagy.
• oligonucleotide As used herein, the term "triplex-forming oligonucleotide" or
• TFO triple helix forming oligonucleotide
• TFOs bind to the purine- rich strand of the duplex through Hoogsteen or reverse Hoogsteen hydrogen bonding. They exist in two sequence motifs, either pyrimidine or purine. According to the present invention, TFOs can be employed as an alternative to antisense oligonucleotides. TFOs have also been shown to produce mutagenic events, even in the absence of tethered mutagens. TFOs can increase rates of recombination between homologous sequences in close proximity. TFOs of the present invention may be conjugated to active molecules.
• the above-defined antisense oligonucleotides, ribozymes, aptamers, RNAi molecules and TFOs of the present invention encompass molecules that are synthetically produced and delivered to cells directly (e.g., by liposomes), as well as molecules which are expressed within cells, e.g., using various vectors.
• vector refers to recombinant constructs including, e.g., plasmids, cosmids, phages, viruses, and the like, with which a nucleic acid molecule can be introduced into a host cell so as to, e.g., clone the vector or express the introduced nucleic acid molecule.
• Vectors may further comprise selectable markers.
• expression construct is meant a nucleic acid sequence comprising a target nucleic acid sequence or sequences whose expression is desired, operatively associated with expression control sequence elements which provide for the proper transcription and translation of the target nucleic acid sequence(s) within the chosen host cells.
• sequence elements may include a promoter and a polyadenylation signal.
• the "expression construct” may further comprise “vector sequences”.
• vector sequences any of several nucleic acid sequences established in the art which have utility in the recombinant DNA technologies of the invention to facilitate the cloning and propagation of the expression constructs including (but not limited to) plasmids, cosmids, phage vectors, viral vectors, and yeast artificial chromosomes.
• Expression constructs of the present invention may comprise vector sequences that facilitate the cloning and propagation of the expression constructs.
• vectors including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic host cells.
• Standard vectors useful in the current invention are well known in the art and include (but are not limited to) plasmids, cosmids, phage vectors, viral vectors, and yeast artificial chromosomes.
• the vector sequences may contain a replication origin for propagation in E.
• coli coli
• SV40 origin of replication an ampicillin, neomycin, or puromycin resistance gene for selection in host cells; and/or genes (e.g., dihydrofolate reductase gene) that amplify the dominant selectable marker plus the gene of interest.
• ampicillin, neomycin, or puromycin resistance gene for selection in host cells
• genes e.g., dihydrofolate reductase gene
• express and expression mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence.
• a DNA sequence is expressed in or by a cell to form an "expression product” such as a protein.
• the expression product itself e.g., the resulting protein, may also be said to be “expressed” by the cell.
• An expression product can be characterized as intracellular, extracellular or secreted.
• intracellular means something that is inside a cell.
• extracellular means something that is outside a cell.
• a substance is "secreted” by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.
• the term "isolated” means that the material being referred to has been removed from the environment in which it is naturally found, and is characterized to a sufficient degree to establish that it is present in a particular sample. Such characterization can be achieved by any standard technique, such as, e.g., sequencing, hybridization, immunoassay, functional assay, expression, size determination, or the like. Thus, a biological material can be “isolated” if it is free of cellular components, i.e., components of the cells in which the material is found or produced in nature.
• an isolated nucleic acid molecule (or isolated polynucleotide molecule), or an isolated oligonucleotide, can be a PCR product, an mRNA transcript, a cDNA molecule, or a restriction fragment.
• a nucleic acid molecule excised from the chromosome that it is naturally a part of is considered to be isolated. Such a nucleic acid molecule may or may not remain joined to regulatory, or non-regulatory, or non-coding regions, or to other regions located upstream or downstream of the gene when found in the chromosome.
• Nucleic acid molecules that have been spliced into vectors such as plasmids, cosmids, artificial chromosomes, phages and the like are considered isolated.
• Isolated nucleic acid molecules of the present invention do not encompass uncharacterized clones in man-made genomic or cDNA libraries.
• a protein that is associated with other proteins and/or nucleic acids with which it is associated in an intact cell, or with cellular membranes if it is a membrane-associated protein, is considered isolated if it has otherwise been removed from the environment in which it is naturally found and is characterized to a sufficient degree to establish that it is present in a particular sample.
• a protein expressed from a recombinant vector in a host cell, particularly in a cell in which the protein is not naturally expressed, is also regarded as isolated.
• An isolated organelle, cell, or tissue is one that has been removed from the anatomical site (cell, tissue or organism) in which it is found in the source organism.
• An isolated material may or may not be “purified”.
• purified refers to a material (e.g., a nucleic acid molecule or a protein) that has been isolated under conditions that detectably reduce or eliminate the presence of other contaminating materials. Contaminants may or may not include native materials from which the purified material has been obtained.
• a purified material preferably contains less than about 90%, less than about 75%, less than about 50%, less than about 25%, less than about 10%, less than about 5%, or less than about 2% by weight of other components with which it was originally associated.
• nucleic acid molecules can be purified by precipitation, chromatography (including preparative solid phase chromatography, oligonucleotide hybridization, and triple helix chromatography), ultracentrifugation, and other means.
• Polypeptides can be purified by various methods including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reverse-phase HPLC, gel filtration, affinity chromatography, ion exchange and partition chromatography, precipitation and salting-out chromatography, extraction, and counter-current distribution.
• Cells can be purified by various techniques, including centrifugation, matrix separation (e.g., nylon wool separation), panning and other immunoselection techniques, depletion (e.g., complement depletion of contaminating cells), and cell sorting (e.g., fluorescence activated cell sorting (FACS)). Other purification methods are possible.
• the term “substantially pure” indicates the highest degree of purity that can be achieved using conventional purification techniques currently known in the art.
• FACS fluorescence activated cell sorting
• substantially free means that contaminants, if present, are below the limits of detection using current techniques, or are detected at levels that are low enough to be acceptable for use in the relevant art, for example, no more than about 2-5% (w/w).
• the term “substantially pure” or “substantially free” means that the purified material being referred to is present in a composition where it represents 95% (w/w) or more of the weight of that composition. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, or any other appropriate method known in the art. [092] As used herein, the terms “mutant” and “mutation” refer to any detectable change in genetic material (e.g., DNA) or any process, mechanism, or result of such a change.
• mutating refers to a process of creating a mutant or mutation.
• nucleic acid hybridization refers to anti-parallel hydrogen bonding between two single-stranded nucleic acids, in which A pairs with T (or U if an RNA nucleic acid) and C pairs with G.
• Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters.
• Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under “low stringency” conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid). See Molecular Biology of the Cell, Alberts et al, 3rd ed., New York and London: Garland Publ, 1994, Ch. 7.
• hybridization of two strands at high stringency requires that the sequences exhibit a high degree of complementarity over an extended portion of their length.
• high stringency conditions include: hybridization to filter-bound DNA in 0.5 M NaHPC-4, 7% SDS, 1 mM EDTA at 65°C, followed by washing in O.
• lx SSC/0.1% SDS at 68°C (where lx SSC is 0.15M NaCl, 0.15M Na citrate) or for oligonucleotide molecules washing in 6xSSC/0.5% sodium pyrophosphate at about 37°C (for 14 nucleotide-long oligos), at about 48°C (for about 17 nucleotide-long oligos), at about 55°C (for 20 nucleotide-long oligos), and at about 60°C (for 23 nucleotide-long oligos)).
• high stringency hybridization refers to a combination of solvent and temperature where two strands will pair to form a "hybrid" helix only if their nucleotide sequences are almost perfectly complementary (see Molecular Biology of the Cell, Alberts et al, 3rd ed., New York and London: Garland Publ, 1994, Ch. 7).
• Conditions of intermediate or moderate stringency such as, for example, an aqueous solution of 2XSSC at 65°C; alternatively, for example, hybridization to filter-bound DNA in 0.5 M NaHP0 4 , 7% SDS, 1 mM EDTA at 65°C, and washing in 0.2 x SSC/0.1% SDS at 42°C
• low stringency such as, for example, an aqueous solution of 2XSSC at 55°C
• standard hybridization conditions refers to hybridization conditions that allow hybridization of sequences having at least 75% sequence identity. According to a specific embodiment, hybridization conditions of higher stringency may be used to allow hybridization of only sequences having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity.
• Nucleic acid molecules that "hybridize" to any desired nucleic acids of the present invention may be of any length. In one embodiment, such nucleic acid molecules are at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, and at least 70 nucleotides in length. In another embodiment, nucleic acid molecules that hybridize are of about the same length as the particular desired nucleic acid.
• sequence similarity generally refers to the degree of identity or correspondence between different nucleotide sequences of nucleic acid molecules or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al, supra). Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin), etc.
• the term "about” or “approximately” means within an acceptable range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system.
• “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value.
• the term can mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2-fold, of a value.
• the term 'about' means within an acceptable error range for the particular value.
• Treating" or “treatment” of a state, disorder or condition includes:
• the benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
• fibrosis afflicting e.g., the liver, pancreas, lung, heart, nervous system, kidneys, bone marrow, lymph nodes, endomyocardium, and retroperitoneum can be treated.
• diseases, disorders and conditions such as cirrhosis, diffuse parenchymal lung disease , post-vasectomy pain syndrome, tuberculosis, sickle-cell anemia, rheumatoid arthritis, progressive massive fibrosis, idiopathic pulmonary fibrosis (and other fibrosing conditions of the lung), injection fibrosis, myelofibrosis, cardiac fibrosis, pancreatic fibrosis, skin fibrosis, scleroderma, intestinal fibrosis or strictures, vascular fibrosis (including atherosclerosis and phlebosclerosis), gliosis of the brain, and mediastinal fibrosis can be treated using the compositions and methods of the present invention.
• diseases, disorders and conditions such as cirrhosis, diffuse parenchymal lung disease , post-vasectomy pain syndrome, tuberculosis, sickle-cell anemia, rheumatoid arthritis, progressive massive fibrosis, idiopathic
• Patient or “subject” refers to mammals and includes human and veterinary subjects.
• compositions provided by the present invention for therapy as is, it may be preferable to administer it in a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient, diluent, or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
• a pharmaceutical composition or formulation comprising at least one active composition, or a pharmaceutically acceptable derivative thereof, in association with a pharmaceutically acceptable excipient, diluent, and/or carrier.
• the excipient, diluent and/or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• the phrase "pharmaceutically acceptable” refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
• the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant.
• a binder for compressed pills
• a glidant for compressed pills
• an encapsulating agent for a glidant
• a flavorant for a flavorant
• a colorant for a colorant for injectable solutions.
• suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
• pharmaceutically acceptable derivative means any pharmaceutically acceptable salt, solvate or prodrug, e.g. ester, of a compound of the invention, which upon administration to the recipient is capable of providing (directly or indirectly) a compound of the invention, or an active metabolite or residue thereof.
• Such derivatives are recognizable to those skilled in the art, without undue experimentation. Nevertheless, reference is made to the teaching of Burger's Medicinal Chemistry and Drug Discovery, 5th Edition, Vol 1 : Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives.
• Preferred pharmaceutically acceptable derivatives are salts, solvates, esters, carbamates, and phosphate esters. Particularly preferred pharmaceutically acceptable derivatives are salts, solvates, and esters. Most preferred pharmaceutically acceptable derivatives are salts and esters.
• the terms “therapeutically effective” and “effective amount”, used interchangeably, applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to an animal in need thereof.
• the term “therapeutically effective” refers to that quantity of a compound or pharmaceutical composition that is sufficient to reduce or eliminate at least one symptom of a disease specified herein. Note that when a combination of active ingredients is administered the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually.
• the dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the patient's medical history, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like.
• the initial dose may be larger, followed by smaller maintenance doses.
• the dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level.
• Therapeutically effective dosages according to the present invention can be determined stepwise by combinations of approaches such as (i) characterization of effective doses of the compound in in vitro assays using protein binding as a readout followed by (ii) characterization in cell cultures using autophagy inhibition or inhibition of HSC activation as a readout followed by (iii) characterization in animal studies using enhanced fibrosis regression or reduced development of fibrosis or related tissue injury as a readout, followed by (iv) characterization in human trials using enhanced fibrosis regression or reduced development of fibrosis or related tissue injury as a readout.
• compositions of the invention can be formulated for administration in any convenient way for use in human or veterinary medicine.
• the one or more exogenous factors, i.e., active ingredient
• Autophagy inhibitors of the invention can be targeted to specific cells, e.g. stellate cells, such as, but not limited to, hepatic stellate cells, according to methods known in the art. See, e.g., Sato, Y. et al. "Resolution of liver cirrhosis using vitamin A- coupled liposomes to deliver siRNA against a collagen-specific chaperone” (2008) Nature Biotechnology; 26(4)431-442; Friedman, S. “Targeting siRNA to arrest fibrosis” (2008) Nature Biotechnology; 26(4)399-400; and Moreno, M. et al. "Reduction of Advanced Liver Fibrosis by Short-Term Targeted Delivery of an Angiotensin Receptor Blocker to Hepatic Stellate Cells in Rats” (2010) Hepatology; 51 :942-952.
• the effective amounts of compounds of the present invention include doses that partially or completely achieve the desired therapeutic, prophylactic, and/or biological effect.
• the actual amount effective for a particular application depends on the condition being treated and the route of administration.
• the effective amount for use in humans can be determined from animal models. For example, a dose for humans can be formulated to achieve circulating and/or gastrointestinal concentrations that have been found to be effective in animals.
• PCR Polymerase chain reaction
• RT-PCR reverse transcriptase polymerase chain reaction
• q-RT-PCR quantitative reverse transcriptase polymerase chain reaction
• the present invention provides compositions that are useful for the treatment of fibrosis.
• the present compositions inhibit autophagy, which is presently discovered to be linked to HSC activation.
• HSC activation is a hallmark of fibrosis, and inhibition of HSC activation is critical to developing new therapeutic compositions for the treatment of fibrosis, including in a preferred embodiment, hepatic fibrosis.
• compositions include small molecule inhibitors of signaling molecules in the autophagy signaling pathway.
• Chemical agents referred to in the art as "small molecule” compounds are typically organic, non-peptide molecules, having a molecular weight less than 10,000 Da, preferably less than 5,000 Da, more preferably less than 1,000 Da, and most preferably less than 500 Da.
• This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified utilizing the screening methods described below.
• Alternative appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for p53/RPL26/nuclolin- modulating activity. Methods for generating and obtaining small molecules are well known in the art (Schreiber, Science 2000; 151 : 1964-1969; Radmann et al, Science 2000; 151 : 1947- 1948).
• autophagy inhibitors are nucleic acid based inhibitors, such as antisense oligonucleotides, ribozymes, and TFOs.
• nucleic acid molecules are designed to target signaling molecules in the autophagy signaling pathway, such as, but not limited to, Atg5 and Atg7.
• antisense oligonucleotides provided by the present invention which inhibit autophagy include R Ai molecules targeted to ATG5 or ATG7 , including ATG5 shRNA sequences:
• compositions comprising inhibitors
• autophagy molecules including but not limited to VPS34, Beclin 1, Atg3, Atg4, Atg5, Atg7, Atg8, Atgl2, Atgl4 and Atgl6 .
• VPS34 Beclin 1
• Atg3, Atg4, Atg5, Atg7, Atg8, Atgl2, Atgl4 and Atgl6 The nucleic and amino acid sequences of those target signaling molecules are known in the art. The skilled artisan can readily design suitable inhibitors that target those autophagy molecules based on their known nucleic and amino acid sequences.
• nucleic acid sequence of ATG5 has
• GenBank® Accession No. NM_004849 SEQ ID NO: 4
• the nucleic acid sequence of ATG7 has GenBank® Accession No. NM_006395 (SEQ ID NO: 6) and the amino acid sequence has GenBank® Accession No. NP_006386 (SEQ ID NO: 7)
• the nucleic acid sequence of phosphoinositide-3 -kinase, class 3 (VPS34) has GenBank® Accession number NM_002647 (SEQ ID NO: 1 1) and the amino acid sequence has GenBank® Accession No.
• NP_002638 (SEQ ID NO: 12); the nucleic acid sequence of Beclin 1 has GenBank® Accession number NM_003766 (SEQ ID NO: 13) and the amino acid sequence has GenBank® Accession number NP_003757 (SEQ ID NO: 14); the nucleic acid sequence of ATG3 has GenBank® Accession number NM_022488 (SEQ ID NO: 15) and the amino acid sequence has GenBank® Accession number NP_071933 (SEQ ID NO: 16).
• the nucleic acid sequence of ATG4 has GenBank® Accession number NM_052936 (SEQ ID NO: 17) and the amino acid sequence has GenBank® Accession number NP_443168 (SEQ ID NO: 18).
• the nucleic acid sequence of ATG8 has GenBank® Accession number NM_007278 (SEQ ID NO: 19) and the amino acid sequence has GenBank® Accession number NP_009209 (SEQ ID NO: 20).
• the Atg8 gene family encodes ubiquitin-like proteins that share a similar structure consisting of two amino-terminal helices and a ubiquitin-like core. Whereas yeast has a single Atg8 gene, many other eukaryotes contain multiple Atg8 orthologs.
• Atg8 genes of multicellular animals can be divided, by sequence similarities, into three subfamilies: microtubule-associated protein 1 light chain 3 (MAP1LC3 or LC3), ⁇ -aminobutyric acid receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE- 16), which are present in sponges, cnidarians (such as sea anemones, corals and hydras) and bilateral animals.
• MAP1LC3 or LC3 microtubule-associated protein 1 light chain 3
• GABARAP ⁇ -aminobutyric acid receptor-associated protein
• GATE- 16 Golgi-associated ATPase enhancer of 16 kDa
• the nucleic acid sequence of ATG12 has GenBank® Accession number NM_004707 (SEQ ID NO: 21) and the amino acid sequence has GenBank® Accession number NP_004698 (SEQ ID NO: 22); the nucleic acid sequence of ATG14 has GenBank® Accession number NM_014924 (SEQ ID NO: 23) and the amino acid sequence has GenBank® Accession number NP_055739 (SEQ ID NO: 24); the nucleic acid sequence of ATG16 has GenBank® Accession number NM_030803 (SEQ ID NO: 25) and the amino acid sequence has GenBank® Accession number NP_110430 (SEQ ID NO: 26).
• nucleic acid and amino acid sequences for human autophagy genes and polypeptides are provided herein. It is to be understood that variants, including mutants and/or other mammalian species and/or homologs of the exemplary sequences are also encompassed by the present invention. Such sequences are well known in the art.
• the invention provides nucleic acids encoding human autophagy proteins, such as those described herein (e.g., VPS34, Beclin-1, Atg3, Atg4, Atg5, Atg7, Atg8, Atgl2, Atgl4 and Atgl6).
• the nucleic acids encode human Atg5 and human Atg7.
• the invention further provides nucleic acids encoding their fragments, mutants and/or tagged versions thereof.
• these fragments, mutants and tagged versions can either have the same or similar activity as whole-length protein (which makes them useful as detectable alternatives), can have lower or no activity (which makes them useful as negative controls), or can have a dominant-negative activity (which makes them useful as inhibitors/competitors).
• autophagy inhibitors include, for example,
• the invention further provides that fragments of autophagy signaling molecules (e.g., Beclin-1, VPS34, Atg5 or Atg7 fragments), can be used to inhibit autophagy for treating fibrosis.
• autophagy signaling molecules e.g., Beclin-1, VPS34, Atg5 or Atg7 fragments
• the invention further provides that fragments of autophagy signaling molecules (i.e., polypeptides that do not comprise the full-length amino acid sequence of the autophagy signaling molecule) that have dominant-negative effects on autophagy (e.g., mutant fragments) can be used to inhibit autophagy in vivo for treatment of fibrosis, and diseases associated with fibrosis, such as, but not limited to, cirrhosis, diffuse parenchymal lung disease, post-vasectomy pain syndrome, tuberculosis, sickle-cell anemia, rheumatoid arthritis, progressive massive fibrosis, idiopathic pulmonary fibrosis (and other fibrosing conditions of the lung), injection fibrosis, myelofibrosis, cardiac fibrosis, pancreatic fibrosis, skin fibrosis, scleroderma, intestinal fibrosis or strictures, vascular fibrosis (including atherosclerosis and phlebosclerosis), gliosis
• the present invention also provides methods of screening for a compound capable of inhibiting autophagy in a cell.
• the invention provides a method for identifying a candidate compound useful for inhibiting autophagy in a cell, which method comprises: (a) contacting a first cell with a test compound under conditions sufficient to allow the cell to respond to said contact with the test compound; (b) determining in the cell prepared in step (a) an expression level of a predetermined marker of autophagy; and (c) comparing the expression level of a predetermined marker of autophagy determined in step (b) to the expression level of a predetermined marker of autophagy in a second (control) cell that has not been contacted with the test compound; wherein a detectable change in the expression level of a predetermined marker of autophagy in the first cell in response to contact with the test compound compared to the expression level of a predetermined marker of autophagy in the second cell that has not been contacted with the test compound, indicates that the test compound can inhibit autophagy and is a
• the predetermined marker of autophagy is the LC3B-II protein, wherein autophagy is inhibited if the expression level of LC3B-II protein is decreased.
• the predetermined marker of autophagy is the accumulation of intracellular lipid droplets, wherein autophagy is inhibited if the number of lipid droplets in a cell is increased.
• the predetermined marker of autophagy is the relative number of autophagolysosomes, wherein autophagy is inhibited if the number of autophagolysosomes in a cell is decreased.
• the candidate compound is additionally tested to confirm that the compound inhibits stellate cell (e.g., hepatic stellate cell) activation.
• the method further comprises: (a) contacting a first cell with the candidate compound under conditions sufficient to allow the cell to respond to said contact with the candidate compound; (b) determining in the cell prepared in step (a) an expression level of a predetermined marker of hepatic stellate cell (HSC) activation; and (c) comparing the expression level of the predetermined marker of HSC activation determined in step (b) to the expression level of a predetermined marker of HSC activation in a second (control) cell that has not been contacted with the candidate compound; wherein a detectable change in the expression level of a predetermined marker of HSC activation in the first cell in response to contact with the candidate compound compared to the expression level of a predetermined marker of HSC activation in the second cell that has not been contacted with the candidate compound, indicates that the
• the predetermined marker of HSC activation is a fibrogenic gene or protein such as collagen led, collagen 1 ⁇ 2, Tgfl31, Type I Tgfp-R, ⁇ -Pdgf-R, Timp 1 & 2, alpha SMA or MMP-2, wherein HSC activation is inhibited if expression of one or more of these fibrogenic genes or proteins is reduced.
• the predetermined marker of HSC activation is ADRP, wherein HSC activation is inhibited if ADRP gene or protein expression is increased.
• test compound useful in the screening assays can be, without limitation, a small organic or inorganic molecule, a polypeptide (including an antibody, antibody fragment, or other immunospecific molecule), an oligonucleotide molecule, a polynucleotide molecule, or a chimera or derivative thereof.
• Screening assays of the invention can be performed in high- throughput and array formats.
• High-throughput screening (HTS) assays useful in the screening methods of the present invention include cell-based and cell-free assays, directed against individual protein targets.
• Several methods of automated assays that have been developed in recent years enable the screening of tens of thousands of compounds in a short period of time (see, e.g., U.S. Patent Nos. 5,585,277, 5,679,582, and 6,020,141). Such HTS methods are particularly preferred.
• Candidate compounds to be tested as modulators can be selected from chemical compounds, including libraries of chemical compounds. There are a number of different libraries that can be used for the identification of small molecule modulators, including: (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules. For reviews, see Science 1998; 282:63-68 and Myers, Curr. Opin. Biotechnol. 1997;8:701-707.
• Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind almost any molecule.
• the systematic evolution of ligands by exponential enrichment (SELEX) process (Ellington et al, Nature 1990;346:818-22; Tuerk et al, Science 1990;249:505-10) can be used to find such aptamers.
• Small molecules that can act as autophagy inhibitors can be further optimized using classical ligand-based design approaches (such as, e.g., virtual screening, pharmacophore modeling, quantitative structure-activity relationship (QSAR), etc.) as well as by synthesizing combinatorial libraries (for review see, e.g., Klabunde et al, Chembiochem. 2002;3:928-44).
• classical ligand-based design approaches such as, e.g., virtual screening, pharmacophore modeling, quantitative structure-activity relationship (QSAR), etc.
• Cell-based arrays combine the technique of cell culture in conjunction with the use of fluidic devices for measurement of cell response to test compounds in a sample of interest, screening of samples for identifying molecules that induce a desired effect in cultured cells, and selection and identification of cell populations with novel and desired characteristics.
• High-throughput screens can be performed on fixed cells using fluorescent-labeled antibodies, biological ligands and/or nucleic acid hybridization probes, or on live cells using multicolor fluorescent indicators and biosensors. The choice of fixed or live cell screens depends on the specific cell-based assay required.
• Transfected cell microarrays are a complementary technique in which array features comprise clusters of cells overexpressing defined cDNAs. Complementary DNAs cloned in expression vectors are printed on microscope slides, which become living arrays after the addition of a lipid transfection reagent and adherent mammalian cells (Bailey et al, Drug Discov. Today 2002;7(18 Suppl):Sl 13-8).
• Cell-based arrays are described in detail in, e.g., Beske, Drug Discov. Today 2002;7(18 Suppl):S 131-5; Sundberg et al, Curr. Opin. Biotechnol. 2000; 11 :47-53; Johnston et al, Drug Discov. Today 2002;7:353-63; U.S. Patents No. 6,406,840 and 6,103,479, and U.S. published patent application No. 2002/0197656.
• Protein arrays are solid-phase, ligand binding assay systems using immobilized proteins on surfaces that are selected from glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles.
• the ligand binding assays using these arrays are highly parallel and often miniaturized. Their advantages are that they are rapid, can be automated, are capable of high sensitivity, are economical in their use of reagents, and provide an abundance of data from a single experiment.
• Automated multi-well formats are the best-developed HTS systems. Automated 96-well plate-based screening systems are the most widely used. The current trend in plate based screening systems is to reduce the volume of the reaction wells further, thereby increasing the density of the wells per plate (96 wells to 384 wells, and 1,536 wells per plate). The reduction in reaction volumes results in increased throughput, dramatically decreased bioreagent costs, and a decrease in the number of plates that need to be managed by automation.
• protein arrays that can be used for HTS, see, e.g., U.S. Patents No. 6,475,809; 6,406,921 ; and 6, 197,599; and International Publications No. WO 00/04389 and WO 00/07024.
• sources of proteins include cell-based expression systems for recombinant proteins, purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for peptides.
• proteins are correctly folded and functional. This is not always the case, e.g., where recombinant proteins are extracted from bacteria under denaturing conditions, whereas other methods (isolation of natural proteins, cell free synthesis) generally retain functionality.
• arrays of denatured proteins can still be useful in screening antibodies for cross-reactivity, identifying auto-antibodies, and selecting ligand binding proteins.
• the immobilization method used should be reproducible, applicable to proteins of different properties (size, hydrophilic, hydrophobic), amenable to high throughput and automation, and compatible with retention of fully functional protein activity. Both covalent and non-covalent methods of protein immobilization can be used.
• Substrates for covalent attachment include, e.g., glass slides coated with amino- or aldehyde- containing silane reagents (Telechem).
• Telechem amino- or aldehyde- containing silane reagents
• reversible covalent coupling is achieved by interaction between the protein derivatized with phenyldiboronic acid, and salicylhydroxamic acid immobilized on the support surface.
• Covalent coupling methods providing a stable linkage can be applied to a range of proteins. Non-covalent binding of unmodified protein occurs within porous structures such as HydroGelTM (PerkinElmer), based on a 3 -dimensional polyacrylamide gel.
• Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
• Labels useful in the present invention include, but are not limited to, biotin for staining with labeled avidin or streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, fluorescein-isothiocyanate (FITC), Texas red, rhodamine, green fluorescent protein, enhanced green fluorescent protein, lissamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX [Amersham], SyBRTM Green I & II [Molecular Probes], and the like), radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), enzymes (e.g., hydrolases, particularly phosphatases such as alkaline phosphatase, esterases and glycosidases, or oxidoreductases, particularly peroxidases such as horse radish peroxidase, and the like), substrates, cofactors
• radiolabels and chemilluminescent labels can be detected using photographic film or scintillation counters; fluorescent markers can be detected using a photo-detector to detect emitted light (e.g., as in fluorescence-activated cell sorting); and enzymatic labels can be detected by providing the enzyme with a substrate and detecting, e.g., a colored reaction product produced by the action of the enzyme on the substrate.
• the screening methods of the present invention can be used to identify a candidate compounds useful to treat a condition that can be treated by inhibiting autophagy, and in particular, for treating fibrosis.
• the present invention provides novel methods for treating fibrosis comprising administering to a patient in need of such treatment an effective amount of at least one inhibitor of autophagy.
• the patient is administered a nucleic acid-based inhibitor of autophagy, such as an antisense oligonucleotide, ribozyme, TFO, small molecule or aptamer.
• a patient afflicted with fibrosis is administered an shRNA molecule that targets ATG5, wherein the shRNA molecule is administered in an amount effective for decreasing the expression level of ATG5 in the patient.
• the expression level of ATG5 may be partially reduced or completely abolished, provided that the level of reduction is sufficient to inhibit autophagy.
• An exemplary ATG5 shRNA molecule has the nucleic acid sequence: GTGAUCAACGAAAUGCAGA- UUCAAGAGA-UCUGCAUUUCGUUGAUCAC (SEQ ID NO: 8) or CUUUCUUCAUAUUAGCAU-UUCAAGAGA-AAUGCUAAUAUGAAGAAAG (SEQ ID NO: 9).
• the patient is afflicted with hepatic fibrosis, and inhibition of autophagy results in reduced HSC activation.
• a patient afflicted with fibrosis is administered an shRNA molecule that targets ATG7, wherein the shRNA molecule is administered in an amount effective for decreasing the expression level or activity of ATG7 in the patient.
• the expression level or activity of ATG7 may be partially reduced or completely abolished, provided that the level of reduction is sufficient to inhibit autophagy.
• An exemplary ATG7 shRNA molecule has the nucleic acid sequence: GCAGCUCAUUGAUAACCAU-UUCAAGAGA-AUGGUUAUCAAUGAGCUGC (SEQ ID NO: 10).
• the patient is afflicted with hepatic fibrosis, and inhibition of autophagy results in reduced HSC activation.
• a patient afflicted with fibrosis is administered a composition comprising chloroquine, 3 -OH chloroquine, 3-methyladenine (3 MA), Wortmannin, a small molecule or bafilomycin Al in an amount effective for inhibiting autophagy in the patient.
• the patient is afflicted with hepatic fibrosis, and inhibition of autophagy results in reduced HSC activation.
• Fibroses which can be treated according to the methods of the present invention include fibroses afflicting an organ or tissue that is a member selected from the group consisting of liver, pancreas, lung, heart, nervous system, kidneys, bone marrow, lymph nodes, endomyocardium, and retroperitoneum.
• fibroses of other organs and tissues is also possible.
• the fibrosis is hepatic fibrosis.
• Diseases, disorders and conditions which may be treated according to the present methods include by way of example and without limitation, cirrhosis, diffuse parenchymal lung disease, post-vasectomy pain syndrome, tuberculosis, sickle-cell anemia, rheumatoid arthritis, progressive massive fibrosis, idiopathic pulmonary fibrosis (and other fibrosing conditions of the lung), injection fibrosis, myelofibrosis, cardiac fibrosis, pancreatic fibrosis, skin fibrosis, scleroderma, intestinal fibrosis or strictures, vascular fibrosis (including atherosclerosis and phlebosclerosis), gliosis of the brain, and mediastinal fibrosis.
• cirrhosis diffuse parenchymal lung disease
• post-vasectomy pain syndrome tuberculosis
• sickle-cell anemia rheumatoid arthritis
• progressive massive fibrosis idiopathic pulmonary fibrosis
• the disease or disorder is hepatic fibrosis and/or cirrhosis, or kidney or lung fibrosis.
• two or more autophagy inhibitors can be used in combination. These two or more inhibitors may be administered at the same or different times, at the same or different sites, and at the same or different frequencies and dosages.
• the invention further provides for using combination therapy to enhance a patient's recovery from fibrosis.
• combination therapy includes administering an autophagy inhibitor according to the invention and one or more therapeutic reagents in amounts sufficiently effective to increase the rate of fibrotic regression.
• Other drugs in combination could include, but are not limited to angiotensin receptor blockers (e.g., Losartan)(Colmenero J, et al. Effects of losartan on hepatic expression of non-phagocytic NADPH oxidase and fibrogenic genes in patients with chronic hepatitis C.
• tyrosine kinase inhibitors e.g., Gleevec, sorafenib
• STI-571 Imatinib mesylate
• HGF hepatocyte growth factor
• HGF mimetics Inagaki Y, et al. Hepatocyte growth factor suppresses profibrogenic signal transduction via nuclear export of Smad3 with galectin-7. Gastroenterology 2008; 134: 1 180-1 190
• cannabinoid receptor type 1 antagonists Teixeira-Clerc F, et al. CBl cannabinoid receptor antagonism: a new strategy for the treatment of liver fibrosis. Nat Med 2006; 12:671-676.
• Antagonists of transforming growth factor beta or its receptors or signaling molecules Liu Y, et al.
• chemokine or chemokine receptor antagonists Rosenker E, et al. Pharmacological inhibition of integrin alphavbeta3 aggravates experimental liver fibrosis and suppresses hepatic angiogenesis.
• the present invention also provides pharmaceutical compositions for the treatment of fibrosis comprising inhibitors of autophagy.
• Such molecules capable of inhibiting autophagy can be advantageously formulated in a pharmaceutical composition with a pharmaceutically acceptable carrier.
• the candidate compound may be designated as an active ingredient or therapeutic agent for the treatment of fibrosis, such as, but not limited to hepatic fibrosis.
• these compositions are useful for the treatment of cirrhosis, diffuse parenchymal lung disease , post-vasectomy pain syndrome, tuberculosis, sickle-cell anemia, rheumatoid arthritis, progressive massive fibrosis, idiopathic pulmonary fibrosis (and other fibrosing conditions of the lung), injection fibrosis, myelofibrosis, cardiac fibrosis, pancreatic fibrosis, skin fibrosis, scleroderma, intestinal fibrosis or strictures, vascular fibrosis (including atherosclerosis and phlebosclerosis), gliosis of the brain, and mediastinal fibrosis.
• the concentration of the active ingredient depends on the desired dosage and administration regimen. Suitable dose ranges of the active ingredient are from about 0.01 mg/kg to about 1500 mg/kg of body weight per day.
• the autophagy inhibitors of the invention can be formulated according to known methods used to prepare pharmaceutically useful compositions.
• RNAi molecules e.g., siRNAs or shRNAs
• specific for (targeted to) ATG5 or ATG7 can be combined in admixture, either as the sole active material or with other known active materials (e.g. therapeutic reagents for combination therapies), with pharmaceutically suitable diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers.
• diluents e.g., Tris-HCl, acetate, phosphate
• preservatives e.g., Thimerosal, benzyl alcohol, parabens
• emulsifiers e.g., solubilizers, adjuvants and
• compositions can contain siRNAs or shRNAs (or other autophagy inhibitors of the invention) complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
• PEG polyethylene glycol
• metal ions or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc.
• liposomes such as polyacetic acid, polyglycolic acid, hydrogels, etc.
• Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of the autophagy inhibitors.
• the active ingredient can be delivered in a vesicle, particularly a liposome or a viral vector (e.g., retrovirus, lentivirus, adenovirus, or adeno-associated virus-based vectors).
• the therapeutic agent can be delivered in a controlled release manner.
• a therapeutic agent can be administered using intravenous infusion with a continuous pump, in a polymer matrix such as poly-lactic/glutamic acid (PLGA), in a pellet containing a mixture of cholesterol and the active ingredient (SilasticRTM; Dow Corning, Midland, MI; see U.S. Patent No. 5,554,601), by subcutaneous implantation, or by transdermal patch.
• PLGA poly-lactic/glutamic acid
• the invention relates to a kit comprising an effective amount of a pharmaceutical composition comprising at least one autophagy inhibitor, and is useful for the treatment of fibrosis and is packaged in a manner suitable for administration to a patient.
• the kits also include instructions teaching one or more of the methods described herein.
• Autophagy inhibitors of the invention can be administered topically, parenterally, or by inhalation.
• parenteral includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques.
• the autophagy inhibitors of the invention are administered intravenously or subcutaneously.
• the autophagy inhibitors of the invention are administered orally.
• the compositions of the invention will typically contain an effective amount of the autophagy inhibitor, alone or in combination with an effective amount of any other active material, e.g., those described above.
• Effective amounts, or dosages, and desired concentrations of autophagy inhibitors and active compounds contained in the compositions may vary depending upon many factors, including the intended use, patient's body weight and age, and route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration can be performed according to art- accepted practices.
• nucleic acid-based inhibitor of the invention can range from about 0.01 mg/kg/day to about 1500 mg/kg/day.
• typical dosages of chloroquine which is an approved drug for human treatment in several diseases such as malaria and some autoimmune disorders, range from about 5 mg/kg/day to about 350 mg/kg/day.
• typical dosages of 3MA and other chemical inhibitors range from about 0.001 mg/kg/day to about 500 mg/kg/day.
• typical dosages of a small molecule autophagy inhibitor of the invention range from about 100 mg/kg/day to about 1000 mg/kg/day.
• the dosages may be more or less than those disclosed, and should be determined by the physician or other healthcare provider administering the composition to a patient.
• HSC mouse hepatic stellate cell
• JS mouse hepatic stellate cell
• DMEM Dulbecco's modified Eagle medium
• fetal bovine serum 10% fetal bovine serum
• siAtgS shRNAs were: GTGAUCAACGAAAUGCAGA-UUCAAGAGA-UCUGCAUUUCGUUGAUCAC (SEQ ID NO: 8) and CUUUCUUCAUAUUAGCAU-UUCAAGAGA-
• AAUGCUAAUAUGAAGAAAG (SEQ ID NO: 9).
• the sequence for the siAtg? shRNA was GC AGCU C AUUGAUAAC C AU-UUCAAGAGA- AUGGUUAU C AAUGAGCUGC (SEQ ID NO: 10).
• the dashes in the sequences separate the sense, loop and antisense portions, respectively, of the sequences in each shRNA sequence. Knockdown and autophagy inhibition was optimum at 8 days of transduction, which was the time point used for all the experiments.
• liver samples were formalin-fixed, paraffin-embedded, sectioned at 4 um and processed routinely for Hematoxylin and eosin staining.
• Sirius red (Sigma) was used to determine type 1 collagen deposition. Ten random areas per slide were scored for fibrosis by a blinded pathologist. Immunohistochemical staining of ASMA was performed with a rabbit polyclonal antibody (Abeam). Sections were stained with Sirius Red solution (saturated picric acid containing 0.1% Direct Red 80 and 0.1% Fast Green FCF) to visualize type 1 collagen deposition.
• FVI and C57BL6 mice were purchased from Charles River
• Atg7 F/F mice previously described in Komatsu, M., et al. "Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration” (2007) Proc Natl Acad Sci U S A 104: 14489-14494, were crossed with a transgenic line expressing ere recombinase under the control of the GFAP promoter (GFAP-cre) to generate Atg7 F/F -GFAP-cre mice with a stellate cell-specific knockout of Atg7 (see, Yang, L., et al.
• mice were used to induce hepatic fibrosis.
• mice received three intraperitoneal (IP) injections of 10% CC14 (diluted in corn oil) at a dose of 0.5 ⁇ 1/g body weight.
• IP intraperitoneal
• mice received IP injections of 10% CC14 (diluted in 1 :3 in corn oil) at a dose of 0.5 ⁇ 1/g body weight three times per week for 6 weeks.
• TAA treatment was administered by 3 IP injections (100 mg/body weight) every other day. Under ketamine/xylazine anesthesia, animals were sacrificed 48 h after the last dose.
• Proteins were extracted from cell samples and subjected to SDS- polyacrylamide gel electrophoresis. Membranes were incubated with the following primary antibodies: rabbit anti-Atg7, rabbit anti-LC3, rabbit anti-Atg5 (Cell Signaling Technology, Danvers, MA), chicken anti-ADRP (Millipore, Billerica, MA), rabbit anti-GAPDH (Sigma- Aldrich), rabbit anti-type I collagen (Rockland Inc., Gilbertsville, PA), rabbit anti-aSMA (Millipore), rabbit anti-P-PDGFR (Santa Cruz Biotechnology®, Inc., Santa Cruz, CA), rabbit anti-MMP2 (Abeam, Cambridge, MA) mouse anti-Tubulin (Sigma-Aldrich), rabbit anti-P62 (Enzo Biochem, New York, NY), mouse anti-cre recombinase (Abeam). The reactions were detected with horse radish peroxidase (HRP)-conjugated secondary antibodies. Blots were developed using
• Morphometric analysis was performed to identify autophagic vacuoles characterized by the presence of a double membrane (complete or at least partially visible), absence of ribosomes attached to the cytosolic side of the membrane, luminal density similar to cytosol, and identifiable organelles or regions of organelles in their lumen using standard methods (Watson et al. 2009. Analysis of knockout mice suggests a role for VGF in the control of fat storage and energy expenditure. BMC Physiol. 9: 19). Cytoplasmic lipid droplets were also identified.
• Serum AST and ALT quantitative determinations were performed using spectrophotometer analysis (Pointe Scientific, Inc., Canton, MI).
• Example 1 Inhibition of autophagy in hepatic stellate cells reduced conversion of LC3B-I to LC3B-II
• Chloroquine is a well-characterized inhibitor of autophagy, and functions by blocking the late stage fusion of lysosomes with autophagosomes. As a result, autophagic flow is inhibited and there is accumulation of LC3B-II following its conversion from LC3B-I.
• JS 1 mHSCs were either incubated with 3MA or infected with lentiviral siAtg7 or a vector control shRNA for four (4) days. Cells were then harvested for immunoblot. In cultured, activated mHSCs, there is conversion of LC3B-I to LC3B-II, indicating autophagy ( Figure 2). Knockdown of Atg7 using siAtg7 specifically inhibited LC3B conversion, demonstrating that Atg7 shRNA specifically inhibited autophagy. mRNA levels of the fibrogenic genes and ADRP were normalized to GAPDH and fold change was calculated comparing treated to control cells.
• JS1 cells were transduced with a lentiviral vector expressing shRNA to the Atg7 (siAtg7 cells), Atg5 (siAtg5 cells) or with an empty lentivirus control ( Figures 6A and 6B)). Knockdown of Atg7 and Atg5 was confirmed by immunoblot on day 4, 6, or 8 ( Figures 6A and 6B), and at day 8 led to significant decreases in LC3B-II ( Figures 6A and 6B) and numbers of AV, as shown for siAtg7 ( Figure 6C).
• Example 2 Autophagic function is increased with hepatic stellate cell activation in vivo and in human cell lines
• HSC human hepatic stellate cell line LX-2 were seeded in 6 well plates and treated with or without (control) 5 M 3- methyladenine (3MA) for 24 h. Immunoblot analysis was performed and expression of collagen led, ⁇ -PDGFR, and MMP9 was determined. As shown in Figure 11A, treatment of human LX-2 cells with the autophagy inhibitor 3MA decreased expression of those fibrogenic proteins. GAPDH was used as a loading control.
• RNA levels of the fibrogenic genes were normalized to GAPDH and fold change was calculated comparing treated cells to control (untreated) cells.
• Figure 1 1B 3MA treatment of LX-2 cells downregulated the mRNA levels of the fibrogenic genes collagen led, ⁇ -Pdgfr, Timpl, Timp2 and Mmp9, indicating that 3MA treatment decreased fibrotic genes.
• Example 3 BDL provoked autophagy in HSCs in vivo
• BDL bile duct ligation
• HSC autophagy in vivo In order to establish that autophagy is a generalized feature of HSC activation in vivo and not unique to a specific etiology, isolated rat HSCs were analyzed by immunoblot 10 days after BDL. BDL provoked autophagy in HSCs, as increased LC3B-II expression and conversion was observed ( Figure 12). Protein expression was normalized to GAPDH.
• Example 4 Inhibition of autophagy in stellate cells attenuated liver fibrosis in vivo
• a transgenic mouse line was generated in which expression of
• Atg7 was specifically attenuated in stellate cells specifically within liver by crossing Atg7 F/F with animals expressing ere recombinase under the glial fibrillary acidic protein promoter (GFAP-cre).
• GFAP-cre glial fibrillary acidic protein promoter
• Stellate cells isolated from Atg7 F/F -GFAP-cre contained reduced LC3B-II levels, a compensatory increase in LC3B-I levels, and P62 accumulation (Figure 14A).
• EM of these isolated cells displayed a significantly reduced number of AV compared with their Atg7 F/F littermates ( Figure 15).
• the decrease in autophagy levels was specific to stellate cells, as there were no changes in AV number or Atg7 expression in other cell types as assessed by whole liver EM and Atg7 immunohistochemistry.
• mice with autophagy-defective hepatic stellate cells displayed a normal liver architecture, and stellate cell ultrastructure was intact.
• Atg7 F/F -GFAP -ere mice had attenuated liver fibrosis.
• Chronic fibrosis was induced by 6 weeks of CC14 in Atg7 F/F -GFAP-cre and Atg7 F/F mice.
• Collagen accumulation as assessed by Sirius Red morphometry, was significantly reduced in Atg7 F/F -GFAP-cre mice.
• Total a- SMA protein in HSC lysates was also significantly reduced (Figure 16).
• Example 6 Autophagy provides cellular energy
• Lipid droplets are catabolized into free fatty acids (FFA) by intracellular lipases to undergo mitochondrial ⁇ -oxidation, generating ATP. Because autophagy metabolizes lipid and provides substrates for ⁇ -oxidation and energy production, impact of inhibiting autophagy on ATP production in stellate cells was assessed. Inhibition of autophagy by 3MA led to a decrease in ATP levels ( Figure 28). Moreover, treatment with etomoxir, an inhibitor of fatty acid oxidation, provoked accumulation of LD in stellate cells and a decrease in collagen lal, a-SMA, Mmp2 and ⁇ -Pdgfr expression ( Figure 29), without affecting autophagic activity.
• FFA free fatty acids
• Quiescent hepatic stellate cells contain retinyl esters and triglycerides at similar concentrations, which together account for 75% of the total lipid content within cytoplasmic LD. Loss of these droplets is a characteristic feature of stellate cell activation in liver injury, yet its functional link to this response has been obscure.
• autophagy contributes to the intracellular catabolism of lipids in hepatocytes, fibroblasts and neurons, but a link to the broader tissue context has been lacking.
• pharmacological or genetic inhibition of autophagy in hepatocytes leads to reduced rates of B- oxidation and marked lipid accumulation in cytosolic LD.
• Autophagy is an ATP-dependent process and autophagy inhibition reduces mitochondrial B-oxidation rates and energy production.
• the role of LD in providing energy to support stellate cell activation has not previously been examined.
• autophagy-deficient stellate cells may be unable to process LD by acid lipases, resulting in lipid droplet accumulation and decreased FFA availability, leading to decreased mitochondrial B-oxidation and ATP production.
• autophagy-independent inhibition of B-oxidation with etomoxir mimics the effect of blocking autophagy on activation, fibrogenesis and lipid accumulation, implicating B-oxidation and ATP production in stellate cell activation.
• compositions and methods of this invention have been described in terms of specific embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention as defined by the appended claims.

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