Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4747—Apoptosis related proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
  • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
  • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
  • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag

Definitions

• the process of tissue repair as a part of would healing involves two phases.
• the first phase is the regenerative phase, in which injured cells are replaced by cells of the same type.
• the second phase is the formation of fibrous tissues, also called fibroplasia or fibrosis, in which connective tissue replaces normal parenchymal tissues.
• fibrous tissues also called fibroplasia or fibrosis, in which connective tissue replaces normal parenchymal tissues.
• the tissue repair process can become pathogenic if the firbrosis phase continues unchecked, leading to extensive tissue remodeling and the formation of permanent scar tissue.
• Liver fibrosis is characterized by an excessive accumulation of extracellular matrix proteins including collagen.
• damaged and dead hepatocytes from liver injuries recruit Kupffer cells at their lesion sites.
• Kupffer cells secrete substantial quantities of cytokines, including transforming growth factor b ⁇ (TGF- b ⁇ ) to control liver inflammation.
• TGF-b ⁇ transforming growth factor b ⁇
• the elevated TGF-b! causes the activation of quiescent hepatic stellate cells (HSCs) that proliferate and become extracellular matrix (ECM)- producing myofibroblast-like cells.
• HSCs quiescent hepatic stellate cells
• ECM extracellular matrix
• Liver fibrosis results from chronic inflammatory liver diseases or iterative liver damages.
• causes for iterative liver injury and fibrosis include viral infections (hepatitis B and C), alcohol abuse, and nonalcoholic steatohepatitis (NASH).
• Liver fibrosis has the potential to develop into liver cirrhosis and cancer with higher mortality rates than those of other major cancers (lung, colorectal, stomach, or breast cancer). Accordingly, liver fibrosis remains a major cause of death with few therapeutic strategies, and there is a great need for treatment to reduce and prevent fibrosis.
• the present invention is based, at least in part, on the discovery that a fragment of MFG-E8, hereinafter NP-011, is surprisingly effective in treating and/or preventing diseases including but not limited to fibrosis, cirrhosis, steatosis, and nonalcoholic steatohepatitis (NASH).
• diseases including but not limited to fibrosis, cirrhosis, steatosis, and nonalcoholic steatohepatitis (NASH).
• the polypeptide for treating and/or preventing diseases including but not limited to fibrosis, cirrhosis, steatosis, and NASH.
• the polypeptide comprises an MFG-E8 polypeptide comprising an epidermal growth factor (EGF)-like domain, a Cl domain, and optionally a signal peptide, but lacking a functional C2 domain.
• the polypeptide comprises an MFG-E8 polypeptide comprising an epidermal growth factor (EGF)-like domain, a Cl domain, and optionally a signal peptide, but lacking a medin polypeptide or a fragment thereof.
• said MFG-E8 polypeptide lacks at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 amino acids within the C-terminal domain comprising amino acids 226-335 of the MFG-E8 polypeptide.
• the MFG-E8 polypeptide comprises at least 180, 190, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, or 280 amino acids of the MFG-E8 polypeptide.
• the MFG-E8 polypeptide comprises at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acids 1-225 of SEQ ID NO: 10 or SEQ ID NO: 12. In some embodiments, the MFG-E8 polypeptide comprises at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acids 24-225 of SEQ ID NO: 10 or SEQ ID NO: 12.
• the MFG-E8 polypeptide is not glycosylated.
• said polypeptide further comprises a heterologous sequence, e.g., a FLAG tag, a HIS tag, and/or a Fc portion of an immunoglobulin.
• a heterologous sequence may extend the half life of the polypeptide in vivo.
• the polypeptide is capable of one or more biological activities including but not limited to: decreasing the expression level of TGF-b; decreasing TGF-b signaling; decreasing phosphorylation of SMAD, e.g., SMAD2, ERK, and/or any other phosphorylated proteins downstream of TGF-b; increasing NOTCH signaling; decreasing fibrosis-related gene expression including but not limited to Collal, Colla2, or Acta2; decreasing interaction between TGF-b and one or more integrins, e.g., integrin b3 and/or integrin b5; decreasing proliferation of hepatic stellate cells (HSC); decreasing the expression level of matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 12 (MMP12), TMP2, ERK, and/or SMAD2; increasing collagenase activity; and/or increasing collagen uptake by macrophages.
• MMP2 matrix metallopeptidase 2
• MMP12 matrix metallopeptid
• the polypeptide is in a pharmaceutical composition comprising the polypeptide and one or more pharmaceutically acceptable carriers and/or diluents.
• kits comprising the polypeptide.
• nucleic acid molecule encoding said polypeptide.
• the nucleic acid comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence identity to nucleotides 61-735 of SEQ ID NO: 9 or SEQ ID NO: 11.
• the nucleic acid comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence identity to nucleotides 130-735 of SEQ ID NO: 9 or SEQ ID NO: 11.
• a vector comprising the nucleic acid molecule, optionally wherein the vector is an expression vector or a viral vector.
• the nucleic acid molecule is operatively linked to a promoter and/or other regulatory sequences.
• the nucleic acid molecule and/or the vector are in a pharmaceutical composition suitable for administering to a subject.
• Such pharmaceutical composition may comprise one or more agents (e.g., liposomes, polymers) that aid the endocytosis and/or longer half-life in vivo , and/or reduce immune reactions against the composition in the subject.
• a viral particle comprising said vector.
• the viral particle is AAV.
• the AAV comprising the viral vector is administered to a subject, e.g., a human, to treat and/or prevent diseases such as fibrosis, cirrhosis, steatosis, NASH, myocardial infarction, lung fibrosis, idiopathic pulmonary fibrosis, and/or Alzheimer’s Disease.
• a host cell comprising said vector.
• the host cell is a mammalian cell.
• the host cell is a yeast cell, e.g., Pichia pastoris.
• a method of producing a polypeptide by culturing the host cell in a culture medium, e.g., wherein the host cell secretes the polypeptide into the medium.
• the method further comprises isolating the polypeptide from the medium and optionally further purifying the polypeptide to generate substantially pure polypeptide.
• provided herein is a method of decreasing or inhibiting fibrosis in a subject by contacting a cell of the subject with the polypeptide. Also provided herein is a method of decreasing or inhibiting proliferation of HSC by contacting the HSC with the polypeptide. Additionally provided herein is a method of decreasing or inhibiting steatosis in a subject by contacting a cell of the subject with the polypeptide.
• the subject in these methods is a mammal, such as a rat, mouse, or human, preferably a human.
• a method of increasing the activity of a macrophage e.g., an uptake of collagens or fibrotic tissues
• a macrophage e.g., an uptake of collagens or fibrotic tissues
• Such increase in the activity of a macrophage contributes to reversal of fibrosis in various tissues (e.g., liver, lung, etc.).
• provided herein is a method of treating or preventing a disorder in a subject in need thereof by administering to the subject the polypeptide.
• the disorder is fibrosis (chronic or acute), cirrhosis, steatosis, NASH, and/or lung fibrosis.
• the polypeptide of present disclosure treats or prevents idiopathic pulmonary fibrosis (IPF).
• IPF idiopathic pulmonary fibrosis
• the polypeptide may also alter the expression level of IPF biomarkers.
• the polypeptide may decrease the the expression level of at least one biomarker selected from aSMA, collagen (Collal), TMP2, MMP2, MMP12, phosphorylated ERK, ERK, phosphorylated SMAD2, and SMAD2.
• the polypeptide of present disclosure treats or prevents myocardial infarction.
• the polypeptide may improve or increase the function of a heart following myocardial infarction, e.g., relative to improvement seen in a corresponding untreated subject.
• the polypeptide may increase left ventricular ejection fraction and/or fraction shortening.
• the polypeptide may inhibit or decrease the fibrosis associated with myocardial infarction.
• the present invention also contemplates a method of treating or preventing Alzheimer’s Disease (AD).
• the subject of treatment may have a genetic mutation associated with AD.
• the subject may have (i) at least one mutation in amyloid precursor protein (APP) selected from K670N/M671L, I716V, and V717I; and/or (ii) at least one mutation in PSEN1 selected from M146L and L286V.
• APP amyloid precursor protein
• the polypeptide of present disclosure may improve the memory loss and/or behaviors associated with Alzheimer’s disease.
• the polypeptide may alter the physiological and histopathological changes in the brain.
• the polypeptide may decrease the amount of amyloid plaques, the amount of amyloid beta, the number of microglia, the level of neuroinflammation (or inflammation of brain), and/or the amount of glial fibrillary acidic protein (GFAP) in the brain of the subject, preferably in hippocampus and/or cortex of the brain of the subject.
• GFAP glial fibrillary acidic protein
• the subject may also be treated with an additional agent that treats the disorder.
• the method does not induce amyloid formation in the subject.
• the polypetide may be administered to the subject via any of various routes, e.g., an intravenous, subcutaneous, intra-arterial, intraperitoneal, or intramuscular route.
• routes e.g., an intravenous, subcutaneous, intra-arterial, intraperitoneal, or intramuscular route.
• the subject in these methods is a mammal, e.g., a rat, mouse, or human, preferably a human.
• Fig. IA-Fig. IF show that NP-011 inhibits liver fibrosis and advantages of NP-011 for treating liver disease.
• FIG. 1A Overall schematic schedule for testing efficacy of the human recombinant proteins in liver fibrosis model.
• Fig. IB Representative images of histological analysis (H&E and Sirius red staining) for the liver of normal mice, TAA- induced liver fibrosis model (Sham), and protein (MFG-E8, NP-011, NP-012, and NP-013) administered liver fibrosis model. Scale bar, 200 pm.
• FIG. 1C Comparison of quantitative fibrotic area in the livers of the mice. Bars represent the means ⁇ SD from three replicates in each group.
• FIG. ID Comparison of mRNA expression for fibrotic markers ( CollaJ Colla2 , and Acta2) in the liver of normal mice, TAA-induced liver fibrosis model (Sham), and protein (MFG-E8, NP-011, NP-012, and NP-013) administered liver fibrosis model. Bars represent the means ⁇ SD from three replicates in each group. *P ⁇ 0.05, **P ⁇ 0.01. (Fig. ID).
• IE Luciferase reporter assay for confirming TGF-b signaling pathway transcriptional activity from human HEK-293FT cells after 0.5 h, 1 h, 2 h treatment of NP- 011 (500 ng/mL) or NP-013 (500 ng/mL). Bars represent the means ⁇ SD from three replicates in each group. *P ⁇ 0.05, **P ⁇ 0.01. (Fig. IF) Gene Set Enrichment Assay (GSEA) data showing the significant enrichment of Notch signaling gene sets in NP-011 administered group, but not in MFG-E8 administered group, compared to Sham group.
• GSEA Gene Set Enrichment Assay
• Fig. 2A-Fig. 2F show that NP-011 inhibits fibrosis at low dosage. Resolution of fibrosis by NP-011 at low dosage.
• FIG. 2A Overall schematic schedule for testing efficacy of the NP-011 in liver fibrosis model.
• FIG. 2B Representative images for comparing fibrotic areas (Sirius red stained areas) in the liver of normal mice, TAA-induced liver fibrosis model and NP-011 -administered liver fibrosis model at various dose ranges (20 pg/kg - 160 pg/kg). Scale bar, 200 pm.
• Fig. 2C Quantitative analysis of the fibrotic areas in the livers of tested mice in Fig. 2B.
• Fig. 2D Comparison of a-SMA (. Acta2 ) expression in the liver of normal mice, TAA- induced liver fibrosis model, and NP-011 -administered liver fibrosis model by quantitative PCR and immunostaining, respectively. Scale bar, 100 pm. Bars represent the means ⁇ SD from three replicates in each group. **P ⁇ 0.01.
• Fig. 2E Heatmap data shows fibrosis relevant genes observed in livers from normal mice, TAA-induced mice, and NP-011- administered liver fibrosis mice.
• Fig. 2F Comparison of TGF-bI mRNA expression in advanced liver fibrosis model. Bars represent the means ⁇ SD from three replicates in each group. **P ⁇ 0.01.
• Fig. 3A-Fig. 3E show that NP-011 inhibits and prevents liver fibrosis. Therapeutic efficacy of minimal dosage of NP-011 in various model inductions.
• Fig. 3A Overall schematic schedule fortesting efficacy of NP-011 in advanced liver fibrosis model.
• Fig. 3B Representative images for analyzing fibrotic areas in livers of normal mice, TAA-induced liver fibrosis model, and 40 pg/kg NP-011 -administered liver fibrosis model with different frequencies of administrations (1, 2, 4, and 6 times). Scale bar, 50 pm.
• Fig. 3C Overall schematic schedule for testing efficacy of NP-011 in progressing liver fibrosis model.
• Fig. 3D Representative images for analyzing fibrotic areas in livers of normal mice, TAA-induced liver fibrosis model, and TAA-injected liver fibrosis model with co-administrations of NP-011. Scale bar, 100 pm.
• FIG. 3E Left panel: Graphical description represents human liver fibrosis model using human embryonic stem cell (hES)-derived hepatocytes and human primary HSCs treated with APAP. Middle panel: Representative images for analyzing hepatocytes and activated myofibroblasts with ALB (albumin; red) and a-SMA (a biomarker for fibrosis Acta2, green). Scale bar, 50 pm.
• Right panel Quantitative analysis of the percentage of a-SMA positive cells in human liver fibrosis model. Bars represent the means ⁇ SD from five replicates in each group. **P ⁇ 0.01.
• Fig. 4A-Fig. 4D show that NP-011 inhibits and prevents liver cirrhosis. Efficacy test in DMN-induced cirrohosis model in rat.
• FIG. 4A Overall schematic presentation of efficacy test of NP-011 in DMN-induced liver cirrhosis model.
• FIG. 4B Survival curves of DMN- induced cirrhosis model are shown for 10 rat per group.
• FIG. 4C Representative images for H&E staining and Immunohistochemistry analysis of rat liver tissues labelling alpha smooth muscle actin.
• Fig. 5A-Fig. 5D show that NP-011 inhibits and prevents NASH. Efficacy test in MCD diet NASH model.
• FIG. 5A Overall schematic presentation of efficacy test of NP-011 in MCD diet NASH model.
• FIG. 5B Representative images for Oil red O staining and
• FIG. 5C the quantification of the relative Oil red O positive areas in the liver of normal mice, MCD diet NASH model, and NP-011 administrated liver NASH model show the significant therapeutic efficacy of secretory NP-011.
• Fig. 5D Quantification of the degree of macrovesicular steatosis and microvesicular steatosis and hypertrophy in the livers of each group. Scale bar, 100 pm. Bars represent the means ⁇ SD from five mice in each group. **P ⁇ 0.01, ANOVA followed by Tukey’s multiple comparison test.
• Fig. 6 shows the interaction of NP-011 with activated HSCs and macrophages.
• Representative images of immunostaining for human MFG-E8 (NP-011), mouse a-SMA, and mouse F4/80 (a marker for macrophages) in the liver tissues of NP-011 administrated TAA- induced liver fibrosis model. Scale bar 20pm.
• Fig. 7A-Fig. 7D show thatNP-Ol 1 inhibits proliferation of HSCs. Actions of NP-011 on HSCs.
• FIG. 7A and Fig. 7B PLA assay for studying physical interaction between TGFBRI and integrin anb3/anb5. Red signals (white arrow heads) indicates the interaction between TGFBRI and Integrin b3 (Fig. 7A), and
• Fig. 8A-Fig. 8C show that NP-01 1 decreases MMP2 expression and increases collagenase activity. Regulating pro-fobrotic MMP2 and collagenase activity in HSCs.
• FIG. 7A Comparison of MMP2 mRNA expression in human HSCs in the presence of TGF-b!, NP-011. Bars represent the means ⁇ SD from three replicates in each group. **P ⁇ 0.01.
• FIG. 7B and Fig. 7C Collagenase activity assay of cell lysates (Fig. 7B) and conditioned media (Fig. 7C) from the culture of control HSCs and TGF-b! -treated HSCs with/without NP-011 treatment. Bars represent the means ⁇ SD from three replicates in each group. **P ⁇ 0.01.
• Fig. 9A-Fig. 9E show that NP-011 increases macrophage-mediated collagen uptake.
• FIG. 9A Overall schematic schedule for macrophage differentiation from monocyte by treatment of PMA.
• FIG. 9C Flow cytometry results showed that the FITC beads were uptaken by differentiated macrophages.
• FIG. 9D Graphical description of fibrotic collagen uptake assay by differentiated macrophages with/without NP-011.
• FIG. 9E Quantitative analysis of the relative CUI (collagen uptake index) for control and NP-011 treatment. Bars represent the means ⁇ SD from three replicates in each group. **P ⁇ 0.01.
• Fig. 10 shows that NP-011 interacts with activated HSCs and macrophages.
• Arrow heads indicates the engulfment of collagen by macrophage (F4/80-positive cells).
• Fig. llA-Fig. 11C show biodistribution and safety profile of NP-011.
• Fig. 11 A The organ distribution in healthy male mice at 30 minutes and 60 minutes after intravenously injection of NP-011 (160 pg/kg).
• Fig. 11B and Fig. 11C Quantitative analysis for the number of inflammatory relevant cells and blood biochemistry in male rat (Fig. 11B) and female rat (Fig. 11C) compared between normal and two NP-011 administration group.
• FIG. 12 shows a diagram of a model for idiopathic pulmonary fibrosis (IPF).
• FIG. 13 shows the images of lung tissues that were stained to show distribution of collagen and aSMA at day 3 (D3), day 5 (D5), day 7 (D7), and day 14 (D14) after treatment with bleomycin.
• FIG. 14 shows the changes in expression levels of IPF biomarkers (Collal, MMP2, MMP12, and TIMP1) at day 3 (D3), day 5 (D5), day 7 (D7), and day 14 (D14) after treatment with bleomycin.
• FIG. 15 shows the changes in expression levels of the subfactors of the signaling pathway related to IPF at at day 3 (D3), day 5 (D5), day 7 (D7), and day 14 (D14) after treatment with bleomycin.
• the subfactors include aSMA, pERK, tERK, pSMAD2 (phosphorylated SMAD2), and tSMAD2 (total SMAD2). Actin is shown as a loading control.
• FIG. 16A-FIG. 16B show diagrams of a model for IPF for testing in vivo efficacy of NP-011. Two cases with different time points are presented: Case 1 (FIG. 16A) and Case 2 (FIG. 16B).
• FIG. 17 shows that NP-011 is efficacious against IPF.
• the lung tissues were stained to show distribution of collagen and aSMA after injection of NP-011.
• Mice were injected with NP-Ol l at day 3 (Case 1 of FIG. 16A).
• FIG. 18 shows that NP-011 is efficacious against IPF.
• the lung tissues were stained to show distribution of collagen and aSMA after injection of NP-011.
• Mice were injected with NP-011 at day 5 (Case 2 of FIG. 16B).
• FIG. 19 shows that NP-011 is efficacious against IPF.
• NP-011 reverses the bleomycin-induced changes in expression levels of the subfactors of the signaling pathway related to IPF.
• Mice were injected with NP-011 at day 3 (Case 1 of FIG. 16A).
• the subfactors include pERK, tERK, pSMAD2, and tSMAD2.
• Actin is shown as a loading control.
• the histogram in the right panel shows quantification of the expression level of pSMAD2 normalized to the actin level.
• FIG. 20 shows that NP-Ol l is efficacious against IPF.
• NP-011 completely reverses the bleomycin-induced changes in expression levels of the subfactors of the signaling pathway related to IPF.
• Mice were injected with NP-011 at day 5 (Case 1 of FIG. 16B).
• the subfactors include aSMA, pERK, and tERK.
• Actin is shown as a loading control.
• the histogram in the right panel shows quantification of the expression level of pERK normalized to the actin level.
• FIG. 21 shows that NP-011 is efficacious against IPF.
• NP-011 alters the expression levels of IPF biomarkers (e.g., as shown herein collagen (Colal), MMP2, MMP12, and TIMP1). Specifically, NP-011 reverses the bleomycin-induced increase in expression levels of IPF biomarks.
• FIG. 22 shows a diagram of LAD-ligation model for myocardial infarction.
• the left anterior descending artery (LAD) of a rat is ligated with one single stitch, forming an ischemia that can be seen almost immediately.
• LAD left anterior descending artery
• This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia (e.g., Kolk et al. (2009) J Vis Exp , 21 :pii 1438).
• FIG. 22 also presents details of the studies presented in FIG. 23-FIG. 26.
• FIG. 23A-FIG. 23C show that NP-011 reverses the symptoms of myocardial infarction, and increases heart function.
• Measurements of left ventricular ejection fraction (EF) and fraction shortening (FS) are shown for mice at 2 weeks (FIG. 23 A), 4 weeks (FIG. 23B), or 8 weeks (FIG. 23 C) after LAD ligation.
• Measurements are shown for mice treated with none (AMI; acute myocardial infarction), PBS (phosphate buffered saline), MFG-E8, or NP-011.
• FIG. 24A-FIG. 24B shows that NP-Ol 1 reverses symptoms of myocardial infarction, and increases the heart function.
• Time course analysis of the heart from day 1 to day 56 after LAD ligation is shown. Specifically, functional improvement of heart is assessed by measuring left ventricular ejection fraction (EF; FIG. 24 A) and fraction shortening (FS; FIG. 24B).
• EF left ventricular ejection fraction
• FS fraction shortening
• FIG. 25 shows a change in body weight after time. No significant change in body weight is observed from day 1 to day 56.
• FIG. 26A-FIG. 26B show that NP-011 reverses symptoms of myocardial infarction. NP-011 reverses the fibrosis associated with myocardial infarction.
• FIG. 26A shows the images of H&E-stained cross-sectioned hearts of rats treated with with none (AMI), PBS, MFG-E8, or NP-011.
• FIG. 26B shows the measurement of fibrosis area compared to overall cross-sectional area of hears from FIG. 26A.
• FIG. 27A-FIG. 27E show that NP-011 reverses the behaviors and memory loss associated with Alzheimer’s Disease.
• FIG. 27A shows a diagram of Alzheimer’s Disease (AD) model 5XFAD mice and details of the in vivo studies. 5XFAD mice express human APP and PSEN1 transgenes with a total of five AD-linked mutations: the Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations in APP, and the M146L and L286V mutations in PSEN1. Behavioral and memory tests were performed after injection of NP-011 to 5XFAD.
• FIG. 1 shows a diagram of Alzheimer’s Disease (AD) model 5XFAD mice and details of the in vivo studies. 5XFAD mice express human APP and PSEN1 transgenes with a total of five AD-linked mutations: the Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutation
• FIG. 27B shows the behavioral analysis of the AD model immediately after injection with NP-011 (at 3.5 months from the initiation of the study as shown in FIG. 27A).
• FIG. 27C shows the behavioral analysis of the AD model 3 months after injection with NP-011 (at 6.5 months from the initiation of the study as shown in FIG. 27A).
• FIG. 27D shows the result of the passive avoidance test of the AD model immediately after injection with NP-011 (at 3.5 months from the initiation of the study as shown in FIG. 27A).
• FIG. 27E shows the result of the passive avoidance test of the AD model 3 months after injection with NP-011 (at 6.5 months from the initiation of the study as shown in FIG. 27A).
• FIG. 28A-FIG. 28B show that NP-Ol l reverses the neuropathological alterations in the brains of Alzheimer’s Disease. Specifically, NP-011 decreases the amount of amyloid plaques in AD.
• FIG. 28A shows hippocampus and cortex of AD brains stained with thioflavin S that detects amyloid plaques
• FIG. 28B shows quantification of the amyloid plaques as stained by thioflavin S.
• FIG. 29A-FIG. 29B show that NP-011 decreases the amount of amyloid beta in Alzheimer’s Disease.
• FIG. 29A shows hippocampus and cortex of AD brains stained with the 6E10 antibody that specifically recognizes amyloid beta
• FIG. 29B shows quantification of the amyloid beta in hippocampus and cortex of AD brains.
• FIG. 30A-FIG. 30B show that NP-011 decreases the number of microglial cells in Alzheimer’s Disease.
• FIG. 30A shows hippocampus and cortex of AD brains stained with an IBA-l antibody that recognizes microglia
• FIG. 30B shows quantification of the microglial cells in hippocampus and cortex of AD brains.
• FIG. 31A-FIG. 31B show that NP-011 decreases the amount of glial fibrillary acidic protein (GFAP) that is upregulated in astrocytes of Alzheimer’s Disease.
• FIG. 31A shows hippocampus and cortex of AD brains stained for GFAP, and
• FIG. 30B shows quantification of GFAP in hippocampus and cortex of AD brains.
• the present invention relates, in part, to compositions and methods for treating and/or preventing diseases related to fibrosis.
• coding region refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues
• noncoding region refers to regions of a nucleotide sequence that are not translated into amino acids (e.g ., 5' and 3' untranslated regions).
• nucleic acid strand refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
• a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
• the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
• prophylactic and/or therapeutic treatments includes prophylactic and/or therapeutic treatments.
• prophylactic or therapeutic treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g ., disease or other unwanted state of the host animal), then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition); whereas, if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
• the unwanted condition e.g ., disease or other unwanted state of the host animal
• preventing is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.
• a condition such as a local recurrence (e.g., pain)
• a disease such as cancer
• a syndrome complex such as heart failure or any other medical condition
• prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
• the amount (e.g., of expression of TGF-b, proliferation of HSC, NOTCH signaling) is“higher” or“lower” or“increased” or“decreased” than the amount of the control, if the amount is greater or less, respectively, than the control level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or than that amount.
• Such“significance” can be assessed from any desired or known point of comparison, such as a particular post-treatment versus pre-treatment measurement ratio (e.g ., l-fold, l . l-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, and the like).
• the amount of the growth factor in the subject can be considered “significantly” higher or lower than the control amount if the amount is at least about two, three, four, or five times, higher or lower, respectively, than the normal amount of the biomarker.
• Such“significance” can also be applied to any other measured parameter described herein.
• A“therapeutically effective amount” of e.g., a polypeptide is an amount capable of producing a medically desirable result in a treated patient, e.g., decrease in fibrosis, steatosis, decrease in expression of TGF-b, with an acceptable benefit: risk ratio, preferably in a human or non-human mammal.
• subject refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
• Mammalian subjects include but are not limited to humans, non-human primates, domestic animals, farm animals, rodents, and the like, which is to be the recipient of a particular treatment.
• By“increase in serum half life” or“increase in half life in vivo'’ means the positive change in circulating half-life of a modified biologically active molecule relative to its non- modified form.
• serum half-life is measured by taking blood samples at various time points after administration of the biologically active molecule, and determining the concentration of that molecule in each sample. Measuring the change in serum concentration with time allows calculation of the serum half-life.
• the relative increase in serum half-life or ti/2 may be determined.
• A“kit” is any manufacture (e.g., a package or container) comprising at least one reagent, e.g., a polypeptide described herein.
• the kit may also comprise additional reagents, carriers, or diluents.
• the kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.
• the kit may comprise one or more reagents necessary to express a composition useful in the methods of the present invention.
• the kit may further comprise a reference standard.
• Reagents in the kit may be provided in individual containers or as mixtures of two or more reagents in a single container.
• instructional materials which describe the use of the compositions within the kit can be included.
• the polypeptides provided herein for treatment of various diseases include a MFG- E8 polypeptide comprising an epidermal growth factor (EGF)-like domain, a Cl domain, and optionally a signal peptide, but lacking a functional C2 domain and/or a medin polypeptide or a fragment thereof.
• the MFG-E8 polypeptide may lack at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 amino acids within the C- terminal domain of MFG-E8 comprising amino acids 226-335.
• the MFG-E8 polypeptide may comprise at least 180, 190, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, or 280 amino acids.
• the MFG-E8 polypeptide may have at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acids 1-225 of SEQ ID NO: 10.
• the polypeptide may also have at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acids 2-225 of SEQ ID NO: 10.
• the MFG-E8 polypeptide may be of human or of any orthologs, e.g., mouse, rat, chimpanzee, horse, etc.
• MFG-E8 is also called lactadherin.
• Representative human MFG-E8 cDNA and human BRD7 protein sequences are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI).
• NCBI National Center for Biotechnology Information
• Human MFG-E8 isoform B (NP 001108086.1) is encodable by the transcript variant 2 (NM_00l 114614.3), which lacks an alternate in-frame exon compared to variant 1.
• the resulting isoform B has the same N- and C-termini but is shorter compared to isoform A.
• Human MFG-E8 isoform D (NP 001297248.1) is encodable by transcript variant 4 (NM_00l310319.2), which lacks an alternate in-frame exon in the 5’ coding region compared to transcript variant 1.
• the encoded isoform D has the same N- and C-termini but is shorter compared to isoform A.
• Human MFG-E8 isoform C (NP_00l297249. l) is encodable by the transcript variant 3 (NM_001310320.2), which includes an alternate internal exon resulting in the use of an alternate translation start site, compared to variant 1.
• the encoded isoform C has a shorter distinct N-terminus compared to isoform A.
• Human MFG-E8 isoform E (NP 001297250.1) is encodable by the transcript variant 5 (NM_00l310321.2), which includes an alternate 5’ terminal exon resulting in the use of a downstream in-frame translation start site, compared to variant 1.
• the encoded isoform E has a shorter N-terminus than isoform A.
• NR_005919.2 is encodable by the transcript variant 1 (NM_005928.4), which encodes the longest isoform A.
• Nucleic acid and polypeptide sequences of MFG-E8 orthologs in organisms other than human are well know and include for example, chimpanzee MFG-E8
• polypeptides are NP-011.
• NP-011 and other polypeptides described herein have many biological activities that are useful in inhibiting fibrosis.
• polypeptides, such as NP-011, described herein decrease the expression of many genes including TGF-b!, MMP2, and/or fibrosis-related genes, e.g., Collal, Colla2,
• the polypeptides described herein modulate the signaling pathway, e.g., decreases TGF- b signaling or increases NOTCH signaling.
• TGF- b signaling controls a diverse set of cellular processes, including cell proliferation, recognition, differentiation, apoptosis, and specification of developmental fate, during embryogenesis as well as in mature tissues, in species ranging from flies and worms to mammals.
• a TGF-b ligand initiates signaling by binding to and bringing together type I and type II receptor serine/threonine kinases on the cell surface. This allows receptor II to phosphorylate the receptor I kinase domain, which then propagates the signal through phosphorylation of the Smad proteins.
• the activated Smad complexes are translocated into the nucleus and, in conjunction with other nuclear cofactors, regulate the transcription of target genes.
• TGF- b and its signaling pathway regulate cell proliferation.
• damaged and dead hepatocytes from liver injuries recruit Kupffer cells at their lesion sites.
• Kupffer cells secrete substantial quantities of cytokines, including transforming growth factor b ⁇ (TGF-b! to control liver inflammation.
• TGF-b! causes the activation of quiescent HSCs that proliferate and become extracellular matrix (ECM)-producing myofibroblast-like cells.
• ECM extracellular matrix
• TGF-b! or decreasing TGF- b signaling inhibits activation and/or proliferation of HSCs, and results in inhibition of fibrosis.
• NOTCH signaling is important for liver regeneration, and increase in NOTCH signaling demonstrates NP-01 l’s role in promoting liver regeneration.
• TGF- b signaling may be monitored via many ways known in the art.
• the level of expression of downstream target genes e.g., ATF4, CDKN1A (p2lCIPl, WAF1), CDKN1B (P27KIP1), CDKN2B (pl5INK4b), COL1A1, COL1A2, DCN, EMP1, FOS, GADD45B, GSC, HERPUD1, IFRD1, IGF1, IGFBP3, IL6, JUN, JUNB, MYC, PDGFB, SERPINE1 (PAI-l), TGFB 1I1, TNFSF10 (TRAIL), TSC22D1 (TGFB1I4), TGFBI, TGIF1) and/or phosphorylation of downstream proteins (e.g., SMAD) may be monitored.
• ATF4 CDKN1A
• CDKN1B P27KIP1
• CDKN2B pl5INK4b
• kits that allow monitoring of the TGF-b signaling pathway, e.g., Qiagen Cat# PAHS-035Z, PAMM-035Z, PARN-035Z, PAHS-235Z, PAMM-235Z, PARN-235Z, CRHS-00035Z-100, CRHS-00245Z-100,
• CRMM-00035Z-100 CRMM-00235Z-100, CCS-017L, CCS-017G, CLS-017L, EAHS- 251Z, GH-035A, SEH00508A, SEM02991A.
• downstream target genes e.g., CDKN1A (p2lCIPl, WAF1), CFLAR (Casper), FOSL1 (fira- 1), ID1, IL2RA (CD25), NFKB1, PTCRA, CD44, ERBB2 (HER-2, NEU), DTX1, HES1, HES5, HEY1, HEY2, HEYL, JAG1, KRT1, LFNG, LOR, NOTCH1, PPARG, CHUK (IKKa), IFNG, IL17B, IL2RA (CD25), NFKB1, NFKB2, STAT6) and/or phosphorylation of the downstream protein may be monitored to assess the Notch signaling pathway.
• downstream target genes e.g., CDKN1A (p2lCIPl, WAF1), CFLAR (Casper), FOSL1 (fira- 1), ID1, IL2RA (CD25), NFKB1, PTCRA, CD44, ERBB2 (HER-2, NEU),
• the polypeptides described herein decrease the phosphorylation of SMAD, a protein in the TGF-b signaling pathway.
• the polypeptides described herein disrupt and/or decrease interaction between TGF-b receptor 1 (TGFBR1) and one or more integrins, e.g., integrin b3 and/or integrin b5.
• TGFBR1 TGF-b receptor 1
• integrins e.g., integrin b3 and/or integrin b5.
• polypeptides described herein decreases proliferation of
• the polypeptides described herein increases collagenase activity and/or collagen uptake by macrophages.
• Certain polypeptides provided herein lack a medin polypeptide or a fragment thereof, which represents about 50 amino acids of the C2 domain.
• Medin is a major constituent of amyloid found in the aorta. Medin and the amyloid have been implicated in etiology of diseases including Alzheimer’s disease and type 2 diabetes. Repeated administration of a polypeptide comprising the medin polypeptide may increase the risk of inducing amyloid formation.
• the polypeptides provided herein that lack the medin polypeptide or a fragment thereof reduce the risk of inducing amyloid formation.
• the present invention further provides a modified polypeptide wherein the polypeptide described herein is fused in frame with a heterologous polypeptide and/or one or more chemical moieties.
• modification may be useful, for example, in extending the serum half-life of the polypeptide, serving as a tag during protein purification, or enhancing immunogenicity during production of antibodies to the polypeptide.
• a“chimeric protein” or“fusion protein” comprises all or part (preferably a biologically active part) of a polypeptide of the present invention linked to a heterologous polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the respective polypeptide.
• the polypeptide of the present invention and the heterologous polypeptide are preferably fused to each other in such a way as to preserve their respective functions exhibited when expressed independently of the fusion.
• the heterologous polypeptide can be fused to the amino- terminus or the carboxyl-terminus of the polypeptide of the present invention.
• the fusion protein contains a heterologous signal sequence, immunoglobulin fusion protein, tag (e.g., FLAG, GST, etc.), toxin, or other useful protein sequence.
• Chimeric and fusion proteins of the present invention can be produced by standard recombinant DNA techniques.
• the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
• PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence.
• anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence.
• many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
• a nucleic acid encoding a polypeptide of the present invention can be cloned into such an expression vector so that the fusion moiety is linked in-frame to the polypeptide of the present invention.
• the heterologous peptide may optionally correspond to a moiety that alters the solubility, affinity, stability or valency of the polypeptide (e.g., NP-011).
• a polypeptide such as NP-011 may be operably linked to an immunoglobulin constant region such as a human Oyl domain or Cy4 domain (e.g, the hinge, CH2 and CH3 regions of human IgOyl, or human IgOy4, see e.g, Capon et al. U.S. Patents 5,116,964; 5,580,756; 5,844,095 and the like, incorporated herein by reference).
• constant regions may retain regions which mediate effector function (e.g, Fc receptor binding) or may be altered to reduce effector function. Such constant regions may also extend the serum half-life of the fusion protein.
• a resulting fusion protein may have altered solubility, binding affinity, stability and/or valency (i.e., the number of binding sites available per polypeptide) as compared to the independently expressed first peptide, and may increase the efficiency of protein purification.
• the fusion protein retains the biological activity of the polypeptide.
• a polypeptide may be modified covalently or non-covalently with one or more chemical moieties, e.g., polyethylene glycol (PEG), lipids, or PEG- modified lipids.
• PEG polyethylene glycol
• Such modification may increase the solubility, stability, and/or serum-half life of the polypeptide.
• compositions and methods provided herein are particularly useful in treating and/or preventing the disorders including those presented below.
• hepatic fibrosis In hepatic fibrosis, excessive connective tissue accumulates in the liver; this tissue represents scarring in response to chronic, repeated liver cell injury. Commonly, fibrosis progresses, disrupting hepatic architecture and eventually function, as regenerating hepatocytes attempt to replace and repair damaged tissue. When such disruption is widespread, cirrhosis is diagnosed.
• Various types of chronic liver injury can cause fibrosis.
• Self-limited, acute liver injury e.g., acute viral hepatitis A
• acute viral hepatitis A does not necessarily distort the scaffolding architecture and hence does not cause fibrosis, despite loss of hepatocytes.
• hepatic fibrosis can regress if the cause is reversible (eg, with viral clearance).
• fibrosis becomes permanent. Fibrosis develops even more rapidly in mechanical biliary obstruction.
• hepatic perivascular stellate cells which store fat initiates fibrosis. These and adjacent cells proliferate, becoming contractile cells termed myofibroblasts. These cells produce excessive amounts of abnormal matrix (consisting of collagen, other glycoproteins, and glycans) and matricellular proteins. Kupffer cells (resident macrophages), injured hepatocytes, platelets, and leukocytes aggregate. As a result, reactive oxygen species and inflammatory mediators (e.g., platelet-derived growth factor, transforming growth factors, connective tissue growth factor) are released. Thus, stellate cell activation results in abnormal extracellular matrix, both in quantity and composition.
• Ito cells hepatic perivascular stellate cells
• Myofibroblasts stimulated by endothelin-l, contribute to increased portal vein resistance and increase the density of the abnormal matrix.
• Fibrous tracts join branches of afferent portal veins and efferent hepatic veins, bypassing the hepatocytes and limiting their blood supply.
• fibrosis contributes both to hepatocyte ischemia (causing hepatocellular dysfunction) and portal hypertension.
• the extent of the ischemia and portal hypertension determines how the liver is affected.
• congenital hepatic fibrosis affects portal vein branches, largely sparing the parenchyma. The result is portal hypertension with sparing of hepatocellular function.
• Cirrhosis is a late stage of hepatic fibrosis that has resulted in widespread distortion of normal hepatic architecture. Cirrhosis is characterized by regenerative nodules surrounded by dense fibrotic tissue. Symptoms may not develop for years and are often nonspecific (e.g., anorexia, fatigue, weight loss). Late manifestations include portal hypertension, ascites, and, when decompensation occurs, liver failure. Diagnosis often requires liver biopsy. Cirrhosis is usually considered irreversible. Treatment is supportive.
• Cirrhosis is the seventh leading cause of death in the United States, according to the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Cirrhosis is defined pathologically by the loss of normal microscopic lobular architecture with fibrosis (i.e., the growth of scar tissue due to infection, inflammation, injury, or even healing) and nodular regeneration. Because of chronic damage to the liver, scar tissue slowly replaces normal functioning liver tissue resulting in progressively diminishing blood flow through the liver. As the normal liver tissue is lost, nutrients, hormones, drugs and poisons are not processed effectively by the liver. In addition, protein production and other substances produced by the liver are inhibited.
• NIDDK National Institute of Diabetes and Digestive and Kidney Diseases
• Symptoms of cirrhosis vary, depending on severity and individuals. Symptoms may include abnormal nerve function, ascites (build-up of fluid in the abdominal cavity), breast enlargement in men, coughing up or vomiting blood, curling of fingers (Dupuytren contracture of the palms), gallstones, hair loss, itching, jaundice, kidney failure, liver encephalopathy, muscle loss, poor appetite, portal hypertension, redness of palms, salivary gland enlargement in cheeks, shrinking of testes, small spider-like veins in skin, weakness, weight loss, etc.
• the symptoms of cirrhosis may resemble other conditions or medical problems. Mild cirrhosis may not exhibit any symptoms at all.
• Cirrhosis is a progressive liver disease, and the damage sustained by the liver is irreversible. However, with proper nutrition, avoidance of certain toxins (i.e., alcohol), vitamin supplementation, and management of cirrhosis complications, further liver damage can often be delayed or stopped. In severe cases of cirrhosis, liver transplantation may be considered.
• HCV hepatitis and other viruses
• drugs chemical exposure, bile duct obstruction, autoimmune diseases, obstruction of outflow of blood from the liver (i.e., Budd-Chiari syndrome), heart and blood vessel disturbances, alphal- antitrypsin deficiency, high blood galactose levels, high blood tyrosine levels, glycogen storage disease, diabetes, malnutrition, hereditary accumulation of too much copper (Wilson Disease) or iron (hemochromatosis).
• Cirrhosis is a progressive liver disease, and the damage sustained by the liver is irreversible. However, with proper nutrition, avoidance of certain toxins (i.e., alcohol),
• Non-alcoholic steatohepatitis also known as non-alcoholic fatty liver disease, describes a hepatic disorder typically characterized by an alcoholic pathogenesis without alcohol consumption.
• the fat deposit in liver cells is mostly triglyceride, and the severity of NASH is directly related to the amount of fat in the liver. Histologically, if 50% of liver cells had steatosis (fatty liver accumulation), or if the total weight of fat is greater than 5% of the entire liver, then steatohepatitis can be diagnosed.
• NASH is further characterized by elevated serum aminotransferase activities with hepatic steatosis, inflammation, and occasionally fibrosis that may progress to cirrhosis.
• NASH Newcastle disease virus
• Fibrosis related disorders that may be amenable to treatment with the methods provided herein include, but are not limited to, collagen disease, interstitial lung disease, human fibrotic lung disease (e.g., obliterative bronchiolitis, idiopathic pulmonary fibrosis, pulmonary fibrosis from a known etiology, tumor stroma in lung disease, systemic sclerosis affecting the lungs, Hermansky-Pudlak syndrome, coal worker's pneumoconiosis, asbestosis, silicosis, chronic pulmonary hypertension, AIDS-associated pulmonary hypertension, sarcoidosis, moderate to severe asthma and the like), fibrotic vascular disease, arterial sclerosis, atherosclerosis, varicose veins, coronary infarcts, cerebral infarcts, myocardial fibrosis, musculoskeletal fibrosis, post-surgical adhesions, human kidney disease (e.g., nephritic syndrome,
• the fibrosis related disorder is selected from systemic or local scleroderma, keloids, hypertrophic scars, atherosclerosis, restenosis, pulmonary inflammation and fibrosis, idiopathic pulmonary fibrosis, liver cirrhosis, fibrosis as a result of chronic hepatitis B or C infection, kidney disease, heart disease resulting from scar tissue, macular degeneration, and retinal and vitreal retinopathy.
• the fibrosis related disorder results from chemotherapeutic drugs, radiation-induced fibrosis, and injuries and burns.
• Wilson hereditary hemochromatosis
• non-HFE hereditary hemochromatosis ferroportin Transferrin receptor 2
• Hepcidin Hemojuvelin
• Sitosterolemia/hepatobiliary cholesterol transporter 5 and 8 progressive familial intrahepatic cholestasis type 3, hereditary fructose intolerance, tyrosinemia type I, argininosuccinate lyase deficiency, citrin deficiency, cholesteryl ester storage disease and Wolman disease, anti-l antitrypsin deficiency, cystic fibrosis, Alstrom syndrome, and congenital hepatic fibrosis.
• Lung fibrosis (“scarring of the lungs”) is a respiratory disease in which scars are formed in the lung tissues, leading to serious breathing problems. Scar formation, the accumulation of excess fibrous connective tissue (the process called fibrosis), leads to thickening of the walls, and causes reduced oxygen supply in the blood. A consequence is a perpetual shortness of breath.
• Lung fibrosis involves gradual exchange of normal lung parenchyma with fibrotic tissue.
• the replacement of normal lung with scar tissue causes irreversible decrease in oxygen diffusion capacity, and the resulting stiffness or decreased compliance makes lung fibrosis a restrictive lung disease.
• Lung fibrosis is perpetuated by aberrant wound healing, rather than chronic inflammation. It is the main cause of restrictive lung disease that is intrinsic to the lung parenchyma. In some cases, lung fibrosis has been associated with cigarette smoking.
• Treatment option for lung fibrosis is limiting, and includes immunosuppressive therapy such as corticosteroids.
• Idiopathic pulmonary fi brosis the most common form of the idiopathic interstitial pneumonias, is a chronic, progressive, irreversible, and usually lethal lung disease of unknown cause. IPF occurs in middle-aged and elderly adults (median age at diagnosis 66 years, range 55-75 years), is limited to the lungs, and is associated with a histopathological or radiological pattern typical of usual interstitial pneumonia.
• the main histopathological features of usual interstitial pneumonia is a heterogeneous appearance with areas of subpleural and paraseptal fi brosis and honeycombing (i.e., cystic fibrotic airspaces lined by bronchiolar epithelium and often fi lled by mucin and variable numbers of infl ammatory cells) alternating with areas of less aff ected or normal parenchyma (spatial heterogeneity).
• Small areas of active fibrosis are present in the background of collagen deposition, and they reflect the temporal heterogeneity of the process and indicate current ongoing disease.
• Inflammation is usually mild and consists of a patchy lymphoplasmacytic interstitial infiltrate.
• the presence of a usual-interstitial-pneumonia pattern on high-resolution CT is characterised by reticular opacities, often associated with traction bronchiectasis, with little or no ground-glass opacifications.
• Honeycombing manifested as subpleural, clustered cystic airspaces with well-defi ned walls (typically 3-10 mm in diameter), is common and is critical for making a defi nite diagnosis.
• IPF Intrapulmonary disease
• a chronic and progressive exertional dyspnoea and cough Bibasilar inspiratory crackles are heard on chest auscultation and frequently fi nger clubbing is found.
• the natural history of IPF has been characterised as a steady or slowly progressive lung disorder, and most patients follow this pattern.
• recent findings indicate that IPF is a heterogeneous disease and new clinical phenotypes with distinct patterns of survival are being described.
• the pathogenic mechanisms are unclear, but a growing body of evidence indicates that the disease is the result of an abnormal behaviour of the alveolar epithelial cells that provoke the migration, proliferation, and activation of mesenchymal cells, with the formation of fibroblast and myofibroblast foci.
• Activated myofibroblasts secrete exaggerated amounts of extracellular matrix molecules with the subsequent destruction of the lung architecture. No therapy has been clearly shown to prolong survival.
• a myocardial infarction commonly known as a heart attack, occurs when a portion of the heart is deprived of oxygen due to blockage of a coronary artery.
• Coronary arteries supply the heart muscle (myocardium) with oxygenated blood. Without oxygen, muscle cells served by the blocked artery begin to die (infarct).
• Atherosclerotic plaques are often present for decades before they result in symptoms.
• Atherosclerosis is characterized by progressive inflammation of the walls of the arteries. Inflammatory cells, particularly macrophages, move into affected arterial walls. Over time, they become laden with cholesterol products, particularly LDL, and become foam cells. A cholesterol core forms as foam cells die. In response to growth factors secreted by macrophages, smooth muscle and other cells move into the plaque and act to stabilize it. A stable plaque may have a thick fibrous cap with calcification. If there is ongoing inflammation, the cap may be thin or ulcerate. Exposed to the pressure associated with blood flow, plaques, especially those with a thin lining, may rupture and trigger the formation of a blood clot (thrombus). ALZHEIMER’S DISEASE
• Alzheimer's Disease is a dementing disorder characterized by progressive impairments in memory and cognition. It typically occurs in later life; and is associated with a multiplicity of structural, chemical and functional abnormalities involving brain regions concerned with cognition and memory. Epidemiologic studies suggest that the dementia presently occurs in up to 10% of individuals over the age of 65 and it is estimated that in the United States alone, as many as 4 million individuals may be affected with Alzheimer's Disease. The cost of caring for such individuals is well over 80 billion dollars annually and increasing rapidly.
• Alzheimer's disease The cause of Alzheimer's disease is poorly understood. About 70% of the risk is believed to be inherited from a person's parents with many genes usually involved. Other risk factors include a history of head injuries, depression, and hypertension. The disease process is associated with plaques and neurofibrillary tangles in the brain. A probable diagnosis is based on the history of the illness and cognitive testing with medical imaging and blood tests to rule out other possible causes. Initial symptoms are often mistaken for normal ageing. Examination of brain tissue is needed for a definite diagnosis. There are no medications or supplements that have been shown to decrease risk.
• extracellular amyloid beta (Ap) deposits are the fundamental cause of the disease. Support for this postulate comes from the location of the gene for the amyloid precursor protein (APP) on chromosome 21, together with the fact that people with trisomy 21 (Down Syndrome) who have an extra gene copy almost universally exhibit at least the earliest symptoms of AD by 40 years of age. Also, a specific isoform of apolipoprotein, APOE4, is a major genetic risk factor for AD. While apolipoproteins enhance the breakdown of beta amyloid, some isoforms are not very effective at this task (such as APOE4), leading to excess amyloid buildup in the brain. Further evidence comes from the finding that transgenic mice that express a mutant form of the human APP gene develop fibrillar amyloid plaques and Alzheimer's-like brain pathology with spatial learning deficits.
• APP amyloid precursor protein
• compositions provided herein may be administered to a subject in combination with any known agents that treat the disorder, e.g., liver fibrosis.
• the methods provided herein may administer the subject any known agents that treat the disorder in combination with the compositions (e.g., NP-011) disclosed herein.
• NP-011 a known agent that treat the disorder in combination with the compositions
• Various treatments for fibrosis-related disorders are known to those skilled in the art.
• Treatments for fibrotic disorders include anti-inflammatory agents, corticosteroids, penicillamine, and colchicine. See e.g., The Merck Manual. 20th ed. Merck Research Laboratories, 2018.
• fibrosis represents a response to hepatic damage
• primary treatment should focus on the cause (removing the basis of the liver injury).
• Such treatment may include eliminating hepatitis B virus or hepatitis C virus in chronic viral hepatitis, abstaining from alcohol in alcoholic liver disease, removing heavy metals such as iron in hemochromatosis or copper in Wilson disease, and decompressing bile ducts in biliary obstruction.
• Such treatments may stop the fibrosis from progressing and, in some patients, also reverse some of the fibrotic changes.
• Treatments aimed at reversing the fibrosis are usually too toxic for long-term use (e.g., corticosteroids, penicillamine) or have no proven efficacy (eg, colchicine).
• Other antifibrotic treatments are under study.
• Silymarin, present in milk thistle, is a popular alternative medicine used to treat hepatic fibrosis. It appears to be safe (except when combined with certain drugs to treat hepatitis C) but lacks efficacy.
• anti-fibrotic therapy includes administration of profibrotic factor antagonists and/or anti-fibrotic agents.
• Anti-fibrotic therapy encompasses agents that inhibit or antagonize profibrotic factors, such as agents that antagonize one or more growth factors or cytokines involved in the formation and maintenance of fibrotic tissue.
• anti-fibrotic therapy targets fibrocyte, fibrocyte precursor, myofibroblast precursor, and/or hematopoetic monocyte precursor differentiation and fibrotic tissue formation and maintenance.
• Pro fibrotic factors that may be targeted with antagonists as part of the therapies of the present invention include, without limitation, a transforming growth factor type b (TGF- b, including TGF- b1-5), VEGF, EGF, RANTES, members of the interleukin family (e.g., IL-l, IL-4, IL-5, IL-6, IL-8 and IL-13), tumor necrosis factor type alpha (TNF-a), platelet- derived growth factor (PDGF), basic fibroblast growth factor (bFGF), monocyte chemoattractant protein type 1 (MCP-l), macrophage inflammatory protein (e.g., MIP-la, MIP-2), connective tissue growth factor (CTGF), endothelin-l, angiotensin-l 1, rennin, leptin, chemokines (e.g., CCL2, CCL12, CXCL12, CXCR4, CCR3, CCR5, CCR7), SLC/CCL21 and other
• Anti-fibrotic therapy may include antagonists of the corresponding receptor of one or more of the pro fibrotic factors.
• Such antagonists may include inactive forms of one or more of the pro fibrotic factors and/or cytokines, such as fragments thereof. Such forms in suitable concentrations may compete with its corresponding profibrotic factors and/or cytokines for binding to its receptor.
• certain antibodies to the receptor may be used to interfere with or prevent binding thereto of the corresponding pro fibrotic factors and/or cytokines.
• Anti-fibrotic therapy may also include soluble forms of the receptor of one or more of the pro fibrotic factors and/or cytokines, such that the soluble receptor competes with its corresponding native cellular receptor for the target ligand.
• the therapy may further include compounds that compete with or otherwise interfere with binding of one or more of the profibrotic factors and/or cytokines with its receptor.
• the proteoglycan decorin is known to bind to TGF-b, thereby reducing its availability for binding to its receptor.
• Mannose-6-phosphate is also known to compete with TGF-b for binding to its corresponding receptor.
• Other known binding inhibitors ofTGF-b include latent transforming growth factor- b binding protein (LTBP) and latency associated peptide (LAP), both of which natively bind to the intracellular precursor of TGF-b.
• LTBP latent transforming growth factor- b binding protein
• LAP latency associated peptide
• anti-fibrotic therapy may include one or more oligoribonucleotides that contain at least one sequence that is antisense with respect to one or more of the profibrotic factors and/or cytokines. Such components may also include one or more expression plasmids having suitable transcriptional control sequences that yield antisense sequences. In other selected embodiments, anti-fibrotic therapy may include one or more double-stranded oligoribonucleotides, or expression plasmids encoding thereof, that are suitable for degrading transcripts of one or more of the profibrotic factors and/or cytokines via RNAmediated interference.
• anti-fibrotic therapy may include one or more single-stranded oligonucleotide aptamers, or expression plasmids encoding therof, that are suitable for inhibiting or interfering with the binding of pro fibrotic factors to their cognate receptors.
• a suitable profibrotic factor antagonist may include components known to inhibit, attenuate, or interfere with one or more components of the intracellular signaling pathways activated by one or more of the pro fibrotic factors upon binding to its corresponding receptor.
• anti -fibrotic therapy may include components that inhibit or attenuate downstream signal pathway molecules such as SMAD family members and SARA.
• a suitable anti-fibrotic therapy may include one or more molecules that are suitable for inhibiting or interfering with the cellular adhesions require for fibrosis.
• a suitable component may include interfering antibodies to the ICAM -1 and/or CD 11, CD 49 or CD 18 molecules, thereby interfering with the adhesion interaction there between.
• a suitable profibrotic factor antagonist may include inhibitors of collagen synthesis, such as proline analogs that interfere with post-translation processing of collagen precursors.
• Pirfenidone for example, is an orally active small molecule drug that may inhibit collagen synthesis, downregulate production of multiple cytokines and block fibroblast proliferation.
• TGF-b transforming growth factor beta family play a central role in wound healing and in tissue repair, and are found in all tissues.
• TGF-b is produced by many parenchymal cell types, as well as infiltrating cells such as lymphocytes, monocytes/macrophages, and platelets. Following wounding or inflammation, such cells such are potential sources of TGF-b.
• TGF-b stimulates the production of various extracellular matrix proteins, inhibits the degradation of these matrix proteins, and promotes tissue fibrosis, all of which contribute to the repair and restoration of the affected tissue. In many diseases, excessive TGF-b contributes to a pathologic excess of tissue fibrosis that can compromise normal organ function.
• TGF-b antagonists include, but are not limited to: monoclonal and polyclonal antibodies directed against one or more isoforms ofTGF-b (Dasch et ak, U.S. Pat. No. 5,571,714; see, also, WO 97/13844 and WO 00/66631); TGF-b receptors, soluble forms of such receptors (preferably soluble TGF-P type III receptor), or antibodies directed against TGF-b receptors (Segarini et ak, U.S. Pat. No. 5,693,607; Lin et ak, U.S. Pat. No. 6,001,969, U.S. Pat. No. 6,010,872, U.S. Pat. No.
• the TGF-b antagonist is a human or humanized monoclonal antibody that blocks TGF-b binding to its receptor (or fragments thereof such as F(ab)2 fragments, Fv fragments, single chain antibodies and other forms or fragments of antibodies that retain the ability to bind to TGF-b, e.g., a monoclonal antibody from hybridoma 1 D 11.16 (ATCC Accession No. HB 9849 described in Dasch et al., U.S. Pat. No. 5,783, 185).
• the profibrotic factor antagonists can be replaced with, or augmented with, a cytokine known to have anti-fibrotic effects, such as IL-12, IL-10, IFN-g or BMP-7 (OP-l).
• a cytokine known to have anti-fibrotic effects such as IL-12, IL-10, IFN-g or BMP-7 (OP-l).
• the nucleic acid sequences encoding IFN-y polypeptides may be accessed from public databases, e.g. Genbank, journal publications, etc. While various mammalian IFN-g polypeptides are of interest, for the treatment of human disease, generally the human protein will be used. Human IFN-g coding sequence may be found in Genbank, accession numbers P01579 and CAA00375. The corresponding genomic sequence may be found in Genbank, accession numbers 100219; M37265; and V00536.
• IFN-glb (Actimmune®; human interferon) is a single-chain polypeptide of 140 amino acids. It is made recombinantly in E. coli and is unglycosylated. Rinderknecht et al. (1984) J. Biol. Chern. 259:6790-6797.
• the IFN-g to be used in anti-fibrotic therapy may be any of natural IFN-g, recombinant IFN-g and the derivatives thereof so far as they have a IFN-g activity, particularly human IFN-g activity.
• Anti-fibrotic therapy comprises one or more Serum amyloid P (SAP) agonists, one or more C-reative protein (CRP) antagonists, or a combination thereof.
• SAP agonists are useful in the treatment of various fibrotic disorders.
• SAP agonists encompass all compounds and compositions that increase or otherwise mimic endogenous SAP signaling, including compounds that increase SAP activity.
• Serum amyloid P or pentraxin-2, a member of the pentraxin family, is a 27- kDa protein that is produced by the liver, secreted into the blood, and circulates as stable 135- kDa pentamers.
• SAP reduces neutrophil adhesion to ECM proteins, inhibits the differentiation of monocytes into fibrocytes, decreases profibrotic macrophages, activates the complement pathway, and promotes phagocytosis of cell debris.
• SAP reduces bleomycin- induced lung fibrosis.
• Representative agonists of SAP includes human SAP protein or an active fragment thereof, anti-FcyR antibodies, aggregated antibodies, SAP peptidomimetic, FcyR crosslinkers, and aptamers.
• CRP antagonists encompass all compounds and compositions that decrease, block, or inhibit CRP signaling.
• Representative antagonists include an anti-sense nucleic acid that targets the expression of CRP, RNA interference molecules, e.g., short interfereing RNA, dsRNA, and Locked Nucleic Acids (LNAs).
• RNA interference molecules e.g., short interfereing RNA, dsRNA, and Locked Nucleic Acids (LNAs).
• Nucleic acid molecules that encode the polypeptides disclosed herein, e.g., the NP- 011 protein, are provided.
• Nucleic acid molecules disclosed herein can be in the form of RNA or in the form of DNA.
• DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand/or non-coding (anti-sense) strand.
• the nucleic acid molecule is isolated.
• a nucleic acid molecule is substantially pure.
• the nucleic acid is cDNA or is derived from cDNA.
• the nucleic acid is recombinantly produced.
• a nucleic acid molecule of the present invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
• the nucleic acid molecules so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
• oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the present invention can be prepared by standard synthetic techniques, e.g ., using an automated DNA synthesizer.
• the nucleic acid molecule comprises a protein coding sequence operably linked to a control sequence that controls the expression of the coding sequence in a host cell or in vitro (see below).
• the coding sequence is a cDNA.
• the disclosure also relates to vectors containing nucleic acid molecules that comprise a protein (e.g., NP-011) coding sequence operably linked to a control sequence that controls the expression of the coding sequence in a host cell or in vitro.
• the nucleic acid molecule comprises a coding sequence for a protein that is fused in the same reading frame to a heterologous polynucleotide sequence.
• the heterologous polynucleotide sequence encodes a leader peptide sequence that facilitates the secretion of the expressed protein from the host cell transformed with the polypeptide (e.g., NP-011) encoding nucleic acid molecule(s).
• a protein containing a leader sequence is referred to as a preprotein and can have the leader sequence cleaved by the host cell to form the processed form of the protein.
• Such leader peptide sequences and their use facilitating the secretion of recombinant proteins in host cells is generally known in the art.
• the heterologous polynucleotide sequence encodes additional 5' amino acid residues that can function for example, to facilitate purification, add or improve protein stability and/or therapeutic properties of the recombinantly expressed protein.
• NP-01 1 and/or similar proteins are secreted from the host cells.
• NP-011 and/or similar proteins lack the N-terminal methionine and do not comprise a heterologous sequence.
• NP-011 comprises a heterologous sequence that extends the half-life in vivo.
• a nucleic acid sequence encoding the protein or a vector comprising said nucleic acid sequence is constructed by chemical synthesis using an oligonucleotide synthesizer.
• oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and codon optimization based on the host cell preferences. Standard methods can routinely be applied to synthesize and isolate polynucleotide sequences encoding the polypeptide (e.g., NP-011).
• the vector may be a construct, which is capable of delivering, and in some embodiments, expressing, one or more gene(s) or sequence(s) of interest in a host cell.
• vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
• the nucleic acid sequences encoding the polypeptide can routinely be operably linked to a control sequence appropriate for expression in a desired host.
• the nucleic acid sequence(s) encoding the polypeptide is inserted into one or more expression vectors and operably linked to a control sequence(s) appropriate for expression of the protein in a desired host.
• the coding sequence can be operably linked to or associated with transcriptional and translational expression control sequences that are functional in the chosen expression host.
• recombinant expression vectors are used to amplify and express DNA encoding the polypeptide.
• Recombinant expression vectors are replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding the polypeptide (e.g., NP-011) operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes.
• a transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences, as described in detail below.
• Such regulatory elements can include an operator sequence to control transcription.
• the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated.
• DNA regions are operably linked when they are functionally related to each other.
• DNA for a signal peptide secretory leader
• a promoter is operably linked to a coding sequence if it controls the transcription of the sequence
• a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
• Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell.
• the protein can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final protein.
• the disclosure provides a composition, e.g., a pharmaceutical composition, comprising a nucleic acid or vector of as described above or elsewhere herein, optionally further comprising one or more carriers, diluents, excipients, or other additives.
• a host cell transformed with the nucleic acid molecule or cDNA molecules and/or the vectors disclosed herein.
• the disclosure also provides host cells transformed with the disclosed nucleic acid molecule or molecules operably linked to a control sequence and optionally inserted into a vector.
• the disclosure provides a method of making the polypeptides provided herein comprising culturing a transformed host cell herein under suitable conditions for producing the the polypeptide.
• the disclosure optionally provides isolating the polypeptide secreted from the host cell.
• the disclosure also optionally provides the polypeptide produced using this method and pharmaceutical compositions comprising the the polypeptide and a pharmaceutically acceptable carrier.
• Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus.
• Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli , including pCRl, pBR322, pMB9 and their derivatives, and also wider host range plasmids, such as M13 and filamentous single-stranded DNA phages.
• a host cell may be a cell or a population of cells harboring or capable of harboring a recombinant nucleic acid.
• Host cells can be prokaryotic (e.g, E. coli), or eukaryotic. Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli.
• the host cells can be fungal cells including yeast such as Saccharomyces cerevisiae, Pichia pastoris , or Schizosaccharomyces pombe.
• the host cells also be any of various animal cells, such as insect cells ( e.g ., Sf-9) or established cell lines of mammalian origin.
• suitable mammalian host cell lines include HEK-293, HEK293F, and HEK-293T, the COS- 7 lines of monkey kidney cells, described by Gluzman (i Cell 23 : 175 (1981)), and other cell lines including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines.
• Mammalian expression vectors can comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
• Baculovirus systems for production of heterologous proteinsln sect cells are reviewed by Luckow and Summers, BioTechnology 6:47 (1988). Cell-free translation systems could also be employed.
• the host cell is Pichia pastor is.
• the nucleic acid molecules, the vectors, and host cells are provided in pharmaceutical compositions.
• a component of pharmaceutical compositions aid delivery of nucleic acid into the intended recipient.
• the DNA constructs are delivered to cells by transfection, i.e., by delivery of“naked” DNA or in a complex with a colloidal dispersion system.
• a colloidal system includes macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
• the preferred colloidal system of this invention is a lipid-complexed or liposome-formulated DNA.
• a plasmid containing a transgene bearing the desired DNA constructs may first be experimentally optimized for expression (e.g, inclusion of an intron in the 5' untranslated region and elimination of unnecessary sequences (Felgner, et al., Ann NY Acad Sci 126-139, 1995).
• Formulation of DNA e.g. with various lipid or liposome materials, may then be effected using known methods and materials and delivered to the recipient mammal.
• the targeting of liposomes can be classified based on anatomical and mechanistic factors.
• Anatomical classification is based on the level of selectivity, for example, organ- specific, cell-specific, and organelle-specific.
• Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs, which contain sinusoidal capillaries.
• RES reticulo-endothelial system
• Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
• a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
• the surface of the targeted delivery system may be modified in a variety of ways.
• lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
• Various linking groups can be used for joining the lipid chains to the targeting ligand. Naked DNA or DNA associated with a delivery vehicle, e.g ., liposomes, can be administered to several sites in a subject.
• Nucleic acids can be delivered in any desired vector. These include viral or non-viral vectors, including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus). Nucleic acids can be administered in any desired format that provides sufficiently efficient delivery levels, including in virus particles, in liposomes, in nanoparticles, and complexed to polymers.
• viral or non-viral vectors including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine
• the nucleic acids encoding a protein or nucleic acid of interest may be in a plasmid or viral vector, or other vector as is known in the art. Such vectors are well known and any can be selected for a particular application.
• the gene delivery vehicle comprises a promoter and a demethylase coding sequence.
• Preferred promoters are tissue-specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidylate synthase promoters.
• promoters which are activatable by infection with a virus such as the a- and b-interferon promoters, and promoters which are activatable by a hormone, such as estrogen.
• promoters which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter.
• a promoter may be constitutive or inducible.
• naked polynucleotide molecules are used as gene delivery vehicles, as described in WO 90/11092 and U.S. Patent 5,580,859.
• Such gene delivery vehicles can be either growth factor DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curiel et ah, Hum. Gene. Ther.
• a gene delivery vehicle can optionally comprise viral sequences such as a viral origin of replication or packaging signal. These viral sequences can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus.
• the growth factor gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et ah, Cell 33 : 153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci.
• Herpes virus e.g ., Herpes Simplex Virus (U.S. Patent No. 5,631,236 by Woo et al., issued May 20, 1997 and WO 00/08191 by Neurovex), vaccinia virus (Ridgeway (1988) Ridgeway,“Mammalian expression vectors,” In: Rodriguez R L, Denhardt D T, ed.
• Vectors A survey of molecular cloning vectors and their uses.
• RNA viruses include an alphavirus, a poxivirus, an arena virus, a vaccinia virus, a polio virus, and the like.
• vectors comprising nucleic acids encoding the polypeptide may be transfected into host cells.
• the host cells may be cultured in a culture medium to produce the polypeptide.
• the polypeptide is secreted into the medium.
• the polypeptide may be isolated from the medium using any suitable technique.
• polypeptides of the present invention (e.g., NP-011) produced by a transformed host cell can be purified according to any suitable method.
• suitable methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
• Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column.
• the polypeptide can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography.
• supernatants from systems that secrete the polypeptides into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix.
• a suitable purification matrix for example, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
• the matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
• a cation or anion exchange step can be employed.
• Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
• RP-HPLC reversed-phase high performance liquid chromatography
• hydrophobic RP-HPLC media e.g, silica gel having pendant methyl or other aliphatic groups
• HPLC high performance liquid chromatography
• an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
• the language “substantially free of cellular material” includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
• protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a“contaminating protein”).
• a“contaminating protein” also referred to herein as a“contaminating protein”.
• culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.
• the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.
• the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of the polypeptide disclosed herein formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
• the pharmaceutical compositions of the present invention may be specially formulated for storage and use by combining a purified agent of the present inventionencompassed by the present invention with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient).
• a pharmaceutically acceptable vehicle e.g., a carrier or excipient.
• Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
• Examples of pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15
• compositions comprising the polypeptide of present invention.
• the salts may be relatively non-toxic, inorganic and organic acid addition to the formulation.
• the salts may be added to the polypeptide at any stage of the purification (including after purification).
• Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19).
• antioxidants such as sodium lauryl sulfate and magnesium stearate, as well as release agents, preservatives and antioxidants can also be present in the compositions.
• pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
• water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium
• compositions of this invention suitable for parenteral administration may comprise one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
• microorganisms Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
• antibacterial and antifungal agents for example, paraben, chlorobutanol, phenol sorbic acid, and the like.
• isotonic agents such as sugars, sodium chloride, and the like into the compositions.
• prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
• pharmaceutical formulations include polypeptide complexed with liposomes.
• Methods to produce liposomes are known to those of skill in the art.
• some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).
• PEG-PE PEG-derivatized phosphatidylethanolamine
• sustained-release preparations comprising the polypeptide of present invention can be produced.
• suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the polypeptide, where the matrices are in the form of shaped articles (e.g., films or microcapsules).
• sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl- methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LEIPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly- D-(-)-3-hydroxybutyric acid.
• polyesters such as poly(2-hydroxyethyl- methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copoly
• compositions described herein may be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal.
• Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
• the compositions can be suitably administered by pulse infusion.
• polypeptides of present invention can be conjugated or modified according to well-known pharmacological methods in the art (e.g., pegylation, glycosylation, oligomerization, etc.) in order to further enhance desirable biological activities, such as increased bioavailability and decreased proteolytic degradation.
• pharmacological methods e.g., pegylation, glycosylation, oligomerization, etc.
• the polypeptide is lyophilized and/or stored in a lyophilized form. In some embodiments, a formulation comprising the polypeptide described herein is lyophilized.
• A“kit” is any manufacture (e.g. a package or container) comprising at least one composition, e.g. a polypeptide, of the present invention.
• the kit may also comprise additional diluents.
• the kit may comprise one or more agents necessary to resuspend the lyophilized composition and/or a filtration unit to remove aggregates.
• the kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.
• the kit may comprise one or more reagents necessary to express a composition useful in the methods of the present invention. Reagents in the kit may be provided in individual containers or as mixtures of two or more reagents in a single container.
• instructional materials which describe the use of the compositions within the kit may be included.
• sequence identity or homology refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g ., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position.
• the percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity.
• the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity.
• a comparison is made when two sequences are aligned to give maximum homology.
• “loop out regions”, e.g. those arising from, from deletions or insertions in one of the sequences are counted as mismatches.
• the comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm.
• allelic variants of a nucleic acid molecule of the present invention can exist in the population, the skilled artisan will further appreciate that sequence changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein encoded thereby.
• sequence changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein encoded thereby.
• nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity.
• amino acid residues that are not conserved or only semi-conserved among homologs of various species may be non-essential for activity and thus would be likely targets for alteration.
• amino acid residues that are conserved among the homologs of various species e.g ., murine and human
• amino acid residues that are conserved among the homologs of various species may be essential for activity and thus would not be likely targets for alteration.
• nucleic acid molecules encoding a polypeptide of the present invention that contain changes in amino acid residues that are not essential for activity.
• polypeptides differ in amino acid sequence from the naturally-occurring proteins which correspond to the markers of the present invention, yet retain biological activity.
• a polypeptide has an amino acid sequence that is at least about 40% identical, 50%, 60%, 70%, 75%, 80%, 83%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or identical to the amino acid sequence of a polypeptide described herein.
• Nucleic acid variants may contain alterations in the coding regions, non-coding regions, or both. In some embodiments, the nucleic acid variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, the nucleic acid variants are produced by silent substitutions due to the degeneracy of the genetic code. Nucleic acid variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli). Vectors and cells comprising the nucleic acids described herein are also provided.
• An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of nucleic acids of the present invention, such that one or more amino acid residue substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
• amino acids with basic side chains e.g, lysine, arginine, histidine
• acidic side chains e.g, aspartic acid, glutamic acid
• uncharged polar side chains e.g, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
• non-polar side chains e.g, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
• beta-branched side chains e.g, threonine, valine, isoleucine
• aromatic side chains e.g ., tyrosine, phenylalanine, tryptophan, histidine
• mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.
• the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
• Non-conservative substitutions include those in which (a) a residue having an electropositive side chain (e.g., Arg, His, or Lys) is substituted for, or by, an electronegative residue (e.g, Glu or Asp), (b) a hydrophilic residue (e.g, Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g, Ala, Leu, Ile, Phe, or Val), (c) a Cys or Pro is substituted for, or by, any other residue, or (d) a residue having a bulky hydrophobic or aromatic side chain (e.g, Val, His, Ile, or Trp) is substituted for, or by, one having a smaller side chain (e.g, Ala or Ser) or no side chain (e.g, Gly).
• an electropositive side chain e.g., Arg, His, or Lys
• an electronegative residue e.g, Glu or Asp
• a hydrophilic residue e
• substitutions can be readily identified.
• a substitution can be taken from any one of D-Ala, Gly, beta-Ala, L-Cys and D-Cys.
• a replacement can be any one of D-Lys, Arg, D-Arg, homo-Arg, Met, D-Met, ornithine, or D-omithine.
• substitutions in functionally important regions that can be expected to induce changes in the properties of isolated polypeptides are those in which (a) a polar residue (e.g, Ser or Thr) is substituted for (or by) a hydrophobic residue (e.g, Leu, Ile, Phe, or Ala); (b) a Cys residue is substituted for (or by) any other residue; (c) a residue having an electropositive side chain (e.g, Lys, Arg, or His), is substituted for (or by) a residue having an electronegative side chain (e.g, Glu or Asp); or (d) a residue having a bulky side chain (e.g, Phe) is substituted for (or by) one not having such a side chain (e.g, Gly).
• a polar residue e.g, Ser or Thr
• a hydrophobic residue e.g, Leu, Ile, Phe, or Ala
• Cys residue is substituted for (or by) any other residue
• Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT
• Glycine Gly, G
• GGC GGG, GGT
• Isoleucine (lie, I) ATA, ATC, ATT
• Serine (Ser, S) AGC, AGT , TCA, TCC, TCG, TCT
• nucleotide triplet An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.
• nucleotide sequence of a DNA or RNA coding for a polypeptide of the invention can be used to derive the fusion protein or polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence.
• corresponding nucleotide sequences that can encode the fusion protein or polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence).
• description and/or disclosure herein of a nucleotide sequence which encodes a fusion protein or polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence.
• description and/or disclosure of a fusion protein or polypeptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
• nucleic acid and amino acid sequence information for nucleic acid and polypeptide molecules useful in the present invention are well known in the art and readily available on publicly available databases, such as the National Center for Biotechnology Information (NCBI).
• NCBI National Center for Biotechnology Information
• exemplary nucleic acid and amino acid sequences derived from publicly available sequence databases are provided in Table 1 below.
• SEQ ID NO: 1 Human milk fat globule-EGF factor 8 protein (MFGE8L transcript variant 2 cDNA PMM 001114614.3 CDS from 61 to 10683
• SEQ ID NO: 5 Human milk fat globule-EGF factor 8 protein (MFGE8). transcript variant 3 cDNA PMM 001310320.2 CDS from 138 to 1863
• amino acids 24-225 amino acids 24-225 that corresponds to NP-011(a) is underlined.
• Amino acids 1-23 corresponds to the signal peptide.
• Amino acids 1-23 corresponds to the signal peptide.
• RNA nucleic acid molecules e.g ., thymines replaced with uridines
• nucleic acid molecules encoding orthologs of the encoded proteins e.g ., thymines replaced with uridines
• MFG-E8 NP-011, NP-012, and NP-013
• TAA thioacetamide, Sigma- Aldrich, St. Louis, MO, ETSA
• n 4 for each group.
• the mice were sacrificed three days after the last NP-Ol l administration; all experimental procedures were approved by the Institutional Animal Care and ETse Committee of Korea ETniversity (KOREA-2016- 0254).
• liver cirrhosis Animal model for liver cirrhosis was designed and conducted by National Center of Efficacy Evaluation for the Development of Health Products Targeting Digestive Disorders (NCEED, Incheon, Korea). Liver cirrhosis was induced by intraperitoneal injection of dimethylnitrosamine (DMN in saline, 10 mg/ml/kg) for 3 consecutive days each week during a 6-week period using male 6-week-old Sprague-Dawley rats (Orient Bio, Gapyeong, Korea).
• DN in saline 10 mg/ml/kg
• Administration of NP-011 was initiated from the week 3 using intravenous (G3-G6) with 40 pg/kg intravenously. General observation was conducted every day and body weight was measured twice for a week.
• mice 6-weeks-old male C57BL/6 mouse fed standard chow or Methionine and choline- deficient (MCD) diet for 10 weeks.
• MCD Methionine and choline- deficient
• the procedures employed for the handling and care of the animals were approved by the Korea University Institutional Animal Care and Use Committee (KUIACUC-2018-3).
• liver tissues were fixed in 4% paraformaldehyde (PFA) and dehydrated in a graded ethanol series. The tissues were then cleared in xylene and embedded in paraffin. Paraffin-embedded tissue sections were stained with Sirius red (American MasterTech Scientific, Lodi, CA, USA) for the evaluation of liver fibrosis. For immunofluorescence staining, sectioned tissues underwent antigen retrieval with citric acid and tissues blocked with 10% donkey serum containing PBS were probed with the primary antibody against alpha-smooth muscle actin (a-SMA) or albumin (ALB) at 4 °C overnight.
• a-SMA alpha-smooth muscle actin
• ALB albumin
• the sections were washed with 0.1% bovine serum albumin (BSA) containing phosphate buffered saline (PBS) and stained with fluorescently labeled secondary antibodies (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA, USA). Digital images were captured using a microscope (Nikon, Tokyo, Japan) and analyzed using ImageJ software.
• BSA bovine serum albumin
• PBS phosphate buffered saline
• Liver tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) according to a standard procedure. Histological scoring of the liver lesions was assessed by grading system for rodent NAFLD, as described (1): Macrovesicular steatosis (0-3), microvesicular steatosis (0-3), hypertrophy (0-3), numbers of inflammatory foci/field (0-3). All cells of randomly picked five images were counted for each histological feature and calculated.
• OCT-embedded frozen livers were sectioned at 4pm, and stained with Oil Red O staining kit (Lifeline cell technology, Frederick, MD, USA, Cat.LL-0052). Oil Red O stained area were measured by Image J.
• OCT-embedded frozen livers were sectioned at 4pm, slides were permeabilized and masking by 0.1% Triton X-100 and 10% donkey serum. The liver sections were incubated with primary and secondary antibodies specified in Table 2.
• the luciferase signaling reporter array was performed according to the manufacturer’s protocol (Qiagen, Hilden, Germany). The detailed methods for whole transcriptome analysis was described in supplementary materials and methods.
• Quantitative polymerase chain reaction was performed using the CFX96 real- time PCR detection system (Bio-Rad, Hercules, CA, USA) with iQTM SYBR® Green Supermix (Bio-Rad). The specific primers used are provided in Table 3. The mRNA levels were normalized to the level of GAPDH (glyceraldehyde-3 -phosphate dehydrogenase).
• the human hepatic stellate cell (HSC) line, hTERT-HSC was cultured in Dulbecco’s modified Eagle’s medium (DMEM; GE Healthcare Life Sciences, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco/Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco).
• DMEM Dulbecco’s modified Eagle’s medium
• FBS Gibco/Thermo Fisher Scientific, Waltham, MA, USA
• streptomycin Gibco
• the human HEK- 293FT cells kindly provided from Prof.
• Hyunggee Kim (Korea university) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GE Healthcare Life Sciences, IL, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA), 100 U/mL of penicillin and 100 mg/mL of streptomycin (Gibco).
• DMEM Dulbecco modified Eagle’s medium
• FBS fetal bovine serum
• penicillin 100 mg/mL of penicillin
• streptomycin streptomycin
• Protein samples were prepared by solubilizing HSCs in RIPA lysis buffer (LPS solution) containing proteinase inhibitors (Roche, Basel, Switzerland). A total of 40 pg of protein from cells was separated by SDS-PAGE (Bio-Rad) and transferred to PVDF transfer membranes (Pall Corporation, Port Washington, NY, USA). The membranes were incubated for 60 min with 5% skim milk in TBS-T (10 mM Tris-HCl pH 7.9, 150 mM NaCl, and 0.05% Tween-20) to block nonspecific antibody binding sites. After blocking, the membranes were immunoblotted with primary antibodies overnight at 4 °C. Antibodies used in the present study are provided in Table 2.
• IP immunoprecipitation
• liver spheroids were formed by mixture of hepatocytes (Hepatosight-S ® , NEXEL, Seoul, South Korea) and hTert-HSCs (kindly provided from Dr. David Brenner, ETniversity of California at San Diego, Ca, ETSA) in ultra-low attachment 96 well plate (Corning) with the cell density ratio 2 to 1, respectively.
• the liver spheroids were cultured for 21 days, and 50 mM of acetaminophen (APAP) was treated to induce the fibrosis.
• APAP acetaminophen
• 500 ng/ml of NP-011 was treated for 48 hr.
• HSCs human HSC lines
• DMEM fetal calf serum
• FBS fetal bovine serum
• the serum-starved HSCs were pre treated with 10 ng/mL TGF-b! for 1 hour, and the HSCs were exposed to 100-1500 ng/mL NP-011 for 6 hours.
• the HSCs were pre-treated with 1 mM CT (Selleck Chemicals, Houston, TX, USA) for 2 hours before treating with TGF- b ⁇ .
• the activation and deactivation of HSCs was quantitatively determined by 5-ethynyl-2'- deoxyuridine (EdU) assay.
• PKA Proximity ligation assay
• PLA incorporation assay human HSCs were seeded at 2 c 10 4 cells per well on the 18 mm circular cover glass in l2-well plates and cultured for 24 hours. After serum starvation for 24 hours, the cells were treated with 10 ng/ml of TGF-b! and/or 500 ng/ml of NP-011, and then, further incubated for 30 minutes. The PLA incorporation was accessed using the Duolink In Situ Red Starter Kit (Merck) according to the manufacturer’s instructions. Digital images of PLA-positive cells were captured using a microscope (Nikon) and analyzed using ImageJ software ⁇ https://imagej .nih.gov/ij/>. EdU incorporation assays
• human HSCs were seeded at 2 c 10 4 cells per well in l2-well plates and cultured for 24 hr. After treatment with TGF-b I and/or NP-011, the serum-starved HSCs were incubated with EdU (10 mM) for an additional 6 hours and EdU incorporation was accessed using the Click-iT EdU Imaging Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Digital images of EdU-positive cells were captured using a microscope (Nikon) and analyzed using ImageJ software.
• the collagenase activity assay was performed according to the manufacturer’s protocol (Chondrex, WA, USA). Enzyme sources were mixed with 180m1 of solution B. Enzymatic reaction was initiated by mixing with 200m1 of 1.0 mg/mL FITC-labeled bovine collagen I substrate, followed by incubation at 37°C for lhour. For negative controls, the enzymatic reaction was performed either without the collagen substrate or the enzyme source. To stop the enzymatic reaction, 10m1 of lOmM o-phenanthroline and 10m1 of 38.5 mM elastase were added to every sample, followed by incubation at 37°C for 10 mins.
• THP-1 differentiation and fluorescent bead phagocytosis assay THP-1 differentiation and fluorescent bead phagocytosis assay.
• THP-l cells were purchased from ATCC and cultured in RPMI-1640 (Gibco, 31800- 022) containing 10% of heat inactivated fetal bovine serum (gibco, 16000-044) and 50 mM b-mercaptoethanol in 37 °C, 5% CO2 incubator.
• THP-l cells were differentiated into macrophages using 200 ng/ml of phorbol l2-myristate l3-acetate (PMA, Sigma, P8139) for 48 hours followed by 72 hours in PMA-free medium. In phagocytosis assay, THP-l cells were seeded at 1.05 c 10 5 cells/cm 2 .
• Carboxylate-Modified Microspheres 2.0 pm, yellow- green fluorescent beads (Invitrogen, F8827) were washed in THP-l cell culture media and resuspended at a final dilution of 1 : 500 in serum free RPMI-1640. Cells were incubated with fluorescent beads for 4 hours in 37 °C, 5% CO2 incubator. Cells were detached with TrypLE (Gibco, 12604-021) and measured by flow cytometry (Accuri C6 Plus). Biodistribution study
• mice 6-weeks-old, male C57BL/6 mouse were randomized into two groups, the control group (Saline) and the NP-011 treated group (NP-011, 160 pg/kg body weight in saline) with 3 animals for each time-point.
• Mice were injected intravenously and maintained for different time-points. Mice were sacrificed by CO2 inhalation, and the organs (brain, heart, lung, liver, kidney, and spleen) were collected directly in PBS to remove the fat and blood traces. The organs stored at -80 °C immediately. The procedures employed for the handling and care of the animals were approved by the Korea University Institutional Animal Care and Use Committee (KUIACUC-2018-0027 and KUIACUC-2018-0040).
• NP-011 4-week repeated toxicity study of NP-011 was conducted by ChemOn Inc. (Non- clinical CRO company, Korea, Gyeonggi-DO). Total 30 SD rats (15 males and 15 females, 6 weeks-old) were randomly divided into 3 groups; Gl (vehicle control), G2 (NP-011 0.2 mg/kg, daily), and G3 (NP-011 2.2 mg/kg, daily), each 5 male rats and 5 female rats per group. The vehicle and NP-011 were administrated via tail vein of rates. The body weight and consumption of food and water were measured once before the start and every week during the test in all groups.
• Example 2 NP-011 Inhibits Liver Fibrosis
• Human MFG-E8 contains three domains: a signal peptide at the N-terminus, an epidermal growth factor (EGF)-like domain that bears an arginine-glycine-aspartate (RGD) motif, and C domains (Cl and C2). Although it is well known that MFG-E8 modulates inflammatory responses by RGD motif-mediated binding to immune cells and engulfing phosphatidylserine (PS)-expressing apoptotic cells, it is unclear how MFG-E8 is responsible for the anti-fibrotic effect.
• EGF epidermal growth factor
• RGD arginine-glycine-aspartate
• Cl and C2 C domains
• Fig. 1A A subsequent efficacy test in a thioacetamide (TAA)-induced liver fibrosis mouse model (Fig. 1A) revealed that the administration of commercial MFG-E8 or NP-013 effectively reduced the fibrotic area (Sirius red-stained area, Fig. IB and Fig. 1C) and downregulated the expression level of liver fibrosis-related genes ( CollaJ Colla2 , and Acta2 ) (Fig. ID). However, it is compelling that the administration of NP-011 eliminated the fibrotic area more substantially and downregulated the expression level of fibrotic genes in the injured liver more significantly than the administration of MFG-E8 and NP-013 (Fig. IB to Fig. 1C).
• TAA thioacetamide
• NP-011 lacks the C2 domain that was thought to be important for the function of MFG-E8, including the anti-fibrotic effect of MFG-E8.
• the administration of NP-012 was less efficacious in reducing the fibrotic area than MFG-E8 and NP-013 (Fig. IB and Fig. 1C) and significantly upregulated the expression of Acta2 (alpha smooth muscle actin, a-SMA) in the injured liver (Fig. ID).
• Acta2 alpha smooth muscle actin, a-SMA
• GSEA further identified that the administration of NP-011 significantly enriched the Notch signaling in whole transcriptome analysis of injured liver (Fig. IF) which is associated with the regeneration processes of injured liver (15).
• Example 3 NP-011 Significantly Reverses Liver Fibrosis at Minimal Dosage
• NP-011 different doses of NP-011 (20 pg/kg, 40 pg/kg, 80 pg/kg, and 160 pg/kg) were administered in a TAA-induced liver fibrosis model and analyzed the fibrotic factors 3 days after NP-011 administration (Fig. 2A).
• the administration of TAA significantly increased the fibrosis area in the mouse liver (Fig. 2B and Fig. 2C, Sham).
• all the NP-011 -administrated groups ranging from 20 to 160 ug/kg showed remarkably diminished fibrosis area (Fig. 2B and Fig. 2C).
• a-SMA a marker for myofibroblast-like cells differentiated from HSCs
• a-SMA a marker for myofibroblast-like cells differentiated from HSCs
• NP-011 shows great therapeutic efficacy at low dosage ranges; most effectively, the administration of 40 pg/kg of NP-011 might be the minimum efficacious dose, resulting in a constant reduction of TGF-b expression in injured liver.
• Example 4 The Minimum Dose of NP-011 Shows Therapeutic Efficacy in Different Models Associated with Fibrosis
• NP-011 The minimum efficacious dose of NP-011 (40 pg/kg) was further tested in different liver fibrosis models. Firstly, the efficacy of repeated administrations of NP-011 was tested in chronic model of liver fibrosis. For this, TAA injections were extended from 8 to 12 weeks (3 times a week), and then 40 pg/kg of NP-011 was administered to the mice one to six times with 5-day intervals (Fig. 3A). The l2-week TAA injections resulted in the development of sustained fibrosis areas in the liver despite the TAA injections were halted 30 days before sacrificing the mice (Fig. 3B, sham). In contrast, the administration of NP-011 significantly resolved the fibrotic areas in injured livers and the fibrotic regression was positively correlated with the times of administration (Fig. 3B).
• Liver fibrosis is a progressive disease and thus evaluation of the efficacy of NP-011 on progressing liver fibrosis may provide a better translation of the animal study to the clinical realm.
• the NP-011 40 pg/kg, three times a week
• TAA TAA for last 4 weeks of model induction
• the concurrent administrations of TAA and NP-011 markedly diminished the fibrosis areas in the livers, compared with the liver that received TAA only (Fig. 3D).
• NP-011 significantly reduced APAP-induced HSC activation in 3D hepatic spheroids consisting of Hepatocytes and hepatic stellate cells (Fig. 3E).
• NP-011 was further tested in dimethylnitrosamine (DMN)-induced liver cirrhosis model (Fig. 4A) (16).
• the DMN injection for 6 weeks in rats resulted the 75% lethality with severe fibrosis in the liver (Fig. 4B and Fig. 4C).
• Histopathological analysis showed that significant necrotic (piecemeal and lobular) and fibrotic events occurred in the liver of DMN-induced cirrhosis model (Fig. 4D).
• daily administrations of NP- 011 reduced the lethality of animals down to 50% with profound attenuation of the liver fibrosis (Fig. 4B and Fig. 4C), and also reduced piecemeal necrosis ( ⁇ 20%), lobular necrosis ( ⁇ 30%), fibrosis score (-24.3%) and HSC activation (-17.7%) (Fig. 4D).
• NP-011 methionine-choline deficient (MCD) diet-induced NASH model
• MCD methionine-choline deficient
• Fig. 5A methionine-choline deficient (MCD) diet-induced NASH model
• HSCS Hepatic Stellate Cells
• NP-01 l rapid and effective suppression TGF-b signaling observed in diseased liver (Fig. IE and 2F).
• NP-011 preferentially bound to a- SMA-positive HSCs, and some of NP-011 interacted with macrophages in the liver (Fig. 6).
• Integrin anb3 and anb5 on the surface of HSCs have been previously implicated in regulating fibrosis.
• the crosstalk between integrin and TGF-b signaling may be important in the regulation of pathological epithelial to mesenchymal transition (EMT) and myofibroblast differentiation.
• EMT pathological epithelial to mesenchymal transition
• Proximity Ligation Assay revealed direct physical interactions between TGFBRI and integrin b3/b5, and the interactions became stronger upon TGF-b! treatment in HSCs (Fig. 7A and Fig. 7B).
• NP-011 treatment in TGF-b!- treated HSCs significantly decreased the interactions between TGFBRI and integrin b3/b5 (Fig. 7A and Fig. 7B).
• Western blot analysis showed that NP-011 treatment significantly attenuated the phosphorylation of a downstream molecule of TGF-b signaling, Smad2, in TGF-b! -treated HSCs, and the interactions between TGFBRI and integrin b3/b5 were further confirmed by immunoprecipitation assay (Fig. 7C).
• NP-011 in the expression of pro-fibrotic MMP2 and collagenase activity in HSCs were further tested based on the previous finding that showed secretion of pro-fibrotic MMP2 and expression of collagenase mRNA in rat HSCs.
• Expression of MMP2 mRNA in HSCs was increased in HSCs after TGF-b! treatment.
• NP-011 treatment in TGF- b ⁇ -treated HSCs significantly down-regulated the increased expression of MMP2 (Fig. 8A).
• the collagenase activity was down-regulated in HSCs after TGF-b! treatment, and the decreased collagenase activity was recovered in TGF-b!
• NP-011 Bio-distribution and safety test of NP-011 were further assessed in a rodent model.
• the administrated NP-011 was preferentially delivered into liver, and then about 48% and 58% of administrated NP-011 were detected in the liver within 30 min and 60 min, respectively (Fig. 11).
• no adverse effects were observed in hematology and biochemistry analysis of blood serum both in male and female rat when 0.2 mg/kg and 2 mg/kg of NP-011 was intravenously administrated into rats for 4 weeks daily (Fig. 11).
• NP-011 preferentially targets liver after its administration and has excellent safety profiles.
• NP-011 shows a powerful anti-fibrotic effect for inhibiting, preventing, and reversing liver fibrosis.
• NP-011 is superior for clinical application over the full length MFG-E8 (lactadherin) by truncation of the C2 domain for the following reasons: 1) Medin site is eliminated in NP-011 by structural truncation of MFG-E8.
• Medin is aortic medial amyloid that occurs in people older than 60 years, and accumulated medin is engaged in Alzheimer’s disease and type 2 diabetes.
• MFG-E8 (Lactadherin) has the medin in its component, and it is positioned within C2 domain.
• NP-011 eliminates the concern for inducing amyloid formation in patients upon administration, and the concern for side effects that may cause Alzheimer’s disease and diabetes upon repeated administrations into patients. Because the most common liver disease, non-alcoholic fatty liver disease (NAFLD), is closely related with type 2 diabetes, it is a crucial factor for treating liver disease. 2) NP-011 exhibits better binding affinity against collagen by removing the glycosylation sites of MFG-E8. MFG-E8 contains glycosylation sites in the C2 domain.
• NASH-E8 non-alcoholic fatty liver disease
• NP-011 By removing the C2 domain, NP-011 has no glycosylation that might interfere with the binding of the discoidin domain (Cl domain) of MFG-E8 to the accumulated collagen in liver fibrosis.
• the well-known discoidin domain receptor (DDR) has discoidin domain, stalk region, and transmembrane domain in its structure. Although the mechanism of collagen binding of DDR has not been identified, binding of discoidin domain of DDR to collagen has been reported.
• discoidin domain of DDR has no glycosylation (glycosylation sites are only located at stalk region of DDR), therefore, discoidin domain of NP-011 might be similar to the discoidin domain of DDR, and may facilitate binding of NP-011 to accumulated collagen in the liver.
• NP-011 has therapeutic efficacy in various liver disease models including nonalcoholic steatohepatitis (NASH).
• NASH nonalcoholic steatohepatitis
• NASH nonalcoholic steatohepatitis
• the therapeutic capacity of NP-011 in both NASH and fibrosis represent a novel therapy for inhibiting and reversing complex and progressive liver diseases.
• NP-011 for clinical application.
• NP- 011 was produced using a yeast system, and this system has several advantages including facile genetic manipulation and rapid growth, as well as eukaryotic features including a secretory pathway for correct protein processing and post-translational modification.
• the production yield of NP-011 from yeast resulted 40 mg/L, and this yield provides reliable manufacturing for clinical applications.
• GMP General Good Manufacturing Practice
• NP- 011 is produced as a secreted protein (authentic NP-011) without a methionine at the N- terminus of the protein, any tag (e.g., FLAG-tag or his-tag), or random glycosylations, which could induce significant problems upon administration of medications.
• NP-011 provides a highly effective and reliable new protein therapy for treating, inhibiting, preventing, and reversing liver fibrosis as well as other liver diseases disclosed herein.
• Example 7 NP-011 Reverses, Inhibits, and Prevents Idiopathic Pulmonary Fibrosis (IPF)
• NP-011 is highly efficacious against IPF, and reverses and inhibits IPF in vivo.
• a model for IPF was induced by injection of bleomycin. Reversal of IPF by NP-011 is evident in histological studies of the lung tissues (FIG. 17 and FIG. 18, as compared to FIG. 13).
• NP-011 reverses the bleomycin-induced changes in the expression level of IPF biomarkers such as aSMA, collagen (Collal), TMP2, MMP2, MMP12, pERK, tERK, pSMAD2 (phosphorylated SMAD2), and tSMAD2 (total SMAD2) (FIG. 12-FIG. 21).
• IPF biomarkers such as aSMA, collagen (Collal), TMP2, MMP2, MMP12, pERK, tERK, pSMAD2 (phosphorylated SMAD2), and tSMAD2 (total SMAD2)
• Example 8 NP-011 Reverses, Inhibits, and Prevents Symptoms of Myocardial Infarction and Improves Heart Function
• NP-011 is highly efficacious against myocardial infarction in vivo.
• a rat model for myocardial infarction was induced by LAD ligation.
• Injection of NP-011 increases the heart function as evidenced by increased left ventricular ejection fraction (EF) and fraction shortening (FS) in rats suffering from myocardial infarction (FIG. 23A-24B).
• EF left ventricular ejection fraction
• FS fraction shortening
• Similar body weight for rats injected with NP-011 compared with control rats demonstrate the safety of NP-011 (FIG. 25).
• Histological studies of cross- sectioned hearts demonstrate that NP-01 1 also reverses and inhibits the fibrosis associated with myocardial infarction (FIG. 26A-FIG. 26B).
• NP-011 provides a highly effective therapy for treating, inhibiting, preventing, and reversing myocardial infarction.
• Example 9 NP-011 Reverses, Inhibits, and Prevents Alzeheimer’s Disease
• NP-011 is highly efficacious against Alzheimer’s Disease in vivo.
• NP-011 reverses the behaviors and memory loss associated with Alzheimer’s Disease (FIG. 27A-FIG. 27E).
• NP-01 1 also reverses and inhibits the neuropathological alteration in AD brains.
• NP-011 specifically decreases the amount of amyloid plaques (FIG. 28A-FIG. 28B) and amyloid beta (FIG. 29A- FIG. 29B).
• NP-011 decreases the number of microglial cells, the brain's innate immune system, the presence of which impies neuroinflammation (FIG. 30A-FIG. 30B).
• NP-011 decreases the amount of glial fibrillary acidic protein (GFAP) that is upregulated in astrocytes of Alzeheimer’s Disease (FIG. 31A-FIG. 31B).
• GFAP glial fibrillary acidic protein
• NP- 011 provides a highly effective therapy for treating, inhibiting, preventing, and reversing Alzheimer’s Disease.
• Troeger JS Mederacke I, Gwak GY, Dapito DH, Mu X, Hsu CC, Pradere JP, Friedman RA, Schwabe RF. Deactivation of hepatic stellate cells during liver fibrosis resolution in mice. Gastroenterology 2012;143: 1073-1083.
• Integrins anb5 and anb3 promote latent TGF-b! activation by human cardiac fibroblast contraction. Cardiovasc Res 2014; 102:407-417.

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