WO2021024265A1 - Methods of treating non-infectious inflammatory disorders - Google Patents

Methods of treating non-infectious inflammatory disorders Download PDF

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WO2021024265A1
WO2021024265A1 PCT/IL2020/050866 IL2020050866W WO2021024265A1 WO 2021024265 A1 WO2021024265 A1 WO 2021024265A1 IL 2020050866 W IL2020050866 W IL 2020050866W WO 2021024265 A1 WO2021024265 A1 WO 2021024265A1
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seq
functional analog
opn
amino acid
protein
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Jonathan Leor
Tal KONFINO
Itai ROTEM
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Ramot at Tel Aviv University Ltd
Tel HaShomer Medical Research Infrastructure and Services Ltd
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Ramot at Tel Aviv University Ltd
Tel HaShomer Medical Research Infrastructure and Services Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to methods of treating non-infectious inflammatory disorders, such as disorders involved with the cardiovascular function.
  • cardiomyocytes are an effective way to repair an injured heart, a process that while preset in lower vertebrates, is absent in adult mammals.
  • Studies aimed at regeneration of cardiomyocytes revealed various factors that can promote cardiomyocyte proliferation during cardiac development and also promote cardiomyocyte regeneration after cardiac injury in the adult heart [1].
  • Macrophages are essential for infarct healing, repair and regeneration in the adult and neonatal heart [2, 3].
  • an Osteopontin (OPN) protein or a functional analog thereof for use in the treatment of a non-infectious inflammatory disorder.
  • the present disclosure provides in accordance with some other aspects, a method for treating a non-infectious inflammatory disorder in a subject, the method comprising administrating a subject in need an amount of an OPN protein or a functional analog thereof, the amount being effective to treat the non-infectious inflammatory disorders.
  • compositions for the treatment of non-infectious inflammatory disorders comprising as active ingredient an amount of an OPN protein or functional analog thereof, the amount being effective to treat the non-infectious inflammatory disorders.
  • kits comprising an OPN protein or functional analog thereof and instructions for use of the OPN protein or functional analog thereof for the treatment of non-infectious inflammatory disorders.
  • An Osteopontin (OPN) protein or a functional analog thereof for use in the treatment of a non-infectious inflammatory disorder.
  • OPN protein or a functional analog thereof for use of Embodiment No. 1 wherein said OPN protein is or comprises at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 11.
  • said functional analog is at least one of small molecule, an antisense, an siRNA, a polypeptide or a peptide.
  • OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 3, wherein said functional analog is a polypeptide or a peptide.
  • OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 4, wherein said functional analog is a derivative or a fragment of the OPN protein.
  • OPN protein or a functional analog thereof for use any one of Embodiments No. 1 to 5, wherein said functional analog is or comprises an amino acid sequence having at least 75% similarity to at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:11.
  • OPN protein or a functional analog thereof for use any one of Embodiments No. 1 to 6, wherein said functional analog is or comprises an amino acid sequence that is at least 75% identical to at least one amino acid sequence denoted by SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 11.
  • OPN protein or a functional analog thereof for use any one of Embodiments 1 to 5, wherein said functional analog is or comprises an amino acid sequence denoted by at least one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21.
  • OPN protein or a functional analog thereof for use of Embodiment 8 wherein said functional analog is or comprises an amino acid sequence that is at least 75% similar to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21.
  • the OPN protein or a functional analog thereof for use of Embodiment 8 wherein said functional analog is or comprises an amino acid sequence that is at least 75% identical to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21.
  • OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 13, being an isolated, a purified or a synthetic OPN protein or a functional analog thereof.
  • OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 14, for use in combination with a pharmaceutical carrier.
  • OPN protein or a functional analog thereof for use of Embodiment No. 15 said pharmaceutical carrier is a liposome or an exosome.
  • OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 16, suitable for parental administration or local administration into a target tissue.
  • i.v intravenous
  • i.c intracoronary
  • i.m intramuscular
  • i.p intraperitoneal
  • the OPN protein or a functional analog thereof for use of Embodiment No. 17, said local administration comprises intramyocardial administration, intracoronary administration, trans coronary administration or retrograde coronary sinus administration.
  • OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 20, wherein the non-infectious inflammatory disorder is involved with a condition selected from the group consisting tissue injury, infarct, trauma, necrosis, sterile inflammatory disorder.
  • OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 20, wherein the non-infectious inflammatory disorder is a cardiovascular disorder.
  • Embodiment No. 22 The OPN protein or a functional analog thereof for use of Embodiment No. 22, wherein the cardiovascular disorder is selected from or associated with myocarditis, heart failure, atherosclerosis and myocardial infarction.
  • a method for treating a non-infectious inflammatory disorder in a subject comprising administrating a subject in need an amount of an OPN protein or a functional analog thereof, the amount being effective to treat the non-infectious inflammatory disorders.
  • OPN protein is or comprises at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:11.
  • Embodiment 35 wherein said functional analog is or comprises an amino acid sequence that is at least 85% identical to at least one amino acid sequence denoted by SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • said OPN protein or a functional analog thereof being an isolated, a purified or a synthetic OPN protein or a functional analog thereof.
  • Embodiment 39 The method of Embodiment 38, comprising administration of said OPN or functional analog thereof with a pharmaceutical carrier.
  • said pharmaceutical carrier is a liposome or an exosome.
  • Embodiment 41 The method of Embodiment 41, said administration comprising at least one of intravenous (i.v) injection, intracoronary (i.c), intramuscular (i.m), intraperitoneal (i.p) injection.
  • i.v intravenous
  • i.c intracoronary
  • i.m intramuscular
  • i.p intraperitoneal
  • said local administration comprises intramyocardial administration, intracoronary administration, trans coronary administration or retrograde coronary sinus administration.
  • Embodiment 46 The method of Embodiment 45, wherein the non-infectious inflammatory disorder is a cardiovascular disorder.
  • Embodiment 48 The method of Embodiment 47, wherein the non-infectious inflammatory disorder comprises myocardial infarction.
  • 49. A CD44 receptor agonist for use in the treatment of a non-infectious inflammatory disorder.
  • the CD44 receptor agonist for use of Embodiment No. 49 being is at least one of small molecule, an antisense, an siRNA, a polypeptide or a peptide.
  • the CD44 receptor agonist for use of any one of Embodiments No. 49 to 51 , being an OPN protein or a functional analog thereof.
  • Embodiment No. 52 The CD44 receptor agonist for use of Embodiment No. 52, wherein said OPN protein or functional analog thereof are as defined in any one of Embodiments 2 to 14.
  • the CD44 receptor agonist for use of any one of Embodiments No. 49 to 53, for use in combination with a pharmaceutical carrier.
  • the CD44 receptor agonist for use of Embodiment No. 54, the pharmaceutical carrier is a liposome or an exosome.
  • the CD44 receptor agonist for use of any one of Embodiments No. 49 to 55, suitable for parental administration or local administration into a target tissue.
  • the CD44 receptor agonist for use of Embodiment No. 56 comprising at least one of intravenous (i.v) injection, intracoronary (i.c), intramuscular (i.m), intraperitoneal (i.p) injection.
  • the CD44 receptor agonist for use of Embodiment No. 56 said local administration comprises intramyocardial administration, intracoronary administration, trans coronary administration or retrograde coronary sinus administration.
  • the CD44 receptor agonist for use of any one of Embodiments No. 49 to 58, wherein the non-infectious inflammatory disorders is an acute inflammation.
  • Embodiment No. 61 The CD44 receptor agonist for use of Embodiment No. 61, wherein the cardiovascular disorder is selected from or associated with myocarditis, heart failure, atherosclerosis and myocardial infarction.
  • a method for treating a non-infectious inflammatory disorder in a subject comprising administrating a subject in need an amount of a CD44 receptor agonist, the amount being effective to treat the non-infectious inflammatory disorders.
  • Embodiment 65 The method of Embodiment 64, wherein said CD44 receptor agonist is as defined in any one of Embodiments 49 to 53.
  • Embodiment 64 or 65 comprising administration of said CD44 receptor agonist with a pharmaceutical carrier.
  • said pharmaceutical carrier is a liposome or an exosome.
  • Embodiments 65 to 67 comprising administration of said CD44 receptor agonist by parental administration or local administration into a target tissue.
  • Embodiment 68 The method of Embodiment 68, said administration comprising at least one of intravenous (i.v) injection, intracoronary (i.c), intramuscular (i.m), intraperitoneal (i.p) injection.
  • i.v intravenous
  • i.c intracoronary
  • i.m intramuscular
  • i.p intraperitoneal
  • said local administration comprises intramyocardial administration, intracoronary administration, trans coronary administration or retrograde coronary sinus administration.
  • Embodiment 72 The method of Embodiment 72, wherein the non-infectious inflammatory disorder is a cardiovascular disorder.
  • Embodiment 75 The method of Embodiment 74, wherein the non-infectious inflammatory disorder comprises myocardial infarction.
  • a pharmaceutical composition for the treatment of non-infectious inflammatory disorders comprising as active ingredient an amount of an OPN protein or functional analog thereof as defined in any one of Embodiments 1 to 21, the amount being effective to treat the non-infectious inflammatory disorders.
  • a pharmaceutical composition for the treatment of non-infectious inflammatory disorders comprising as active ingredient an amount of at least one CD44 receptor agonist as defined in any one of Embodiments 49 to 63, the amount being effective to treat the non-infectious inflammatory disorders.
  • kits comprising an OPN protein or functional analog thereof as defined in any one of Embodiments 1 to 24 and instructions for use of the OPN protein or functional analog thereof for the treatment of non-infectious inflammatory disorders.
  • a kit comprising at least one CD44 receptor agonist as defined in any one of Embodiments 49 to 63 and instructions for use of at least one CD44 receptor agonist for the treatment of non-infectious inflammatory disorders.
  • Figures 1A-1C Macrophages accumulated at the site of Apex Resected (AR) in neonatal hearts
  • MI myocardial infraction
  • FIGS. 2A-2I CCR2 + monocytes are essential for neonatal heart regeneration
  • Fig. 2A is a bar graph showing flow cytometry analysis of CCR2 + F4/80 macrophages accumulation at day 1 and day 3 after AR.
  • Fig.2B is a bar graph showing flow cytometry analysis of processed hearts, 3 days after AR
  • Fig. 2C is a graph showing the 28 days mortality rate after AR and CCR2 inhibition vs. AR only, the differences between groups were calculated by Fischer’s exact test
  • Figs.2D and 2E are graphs showing transthoracic echocardiography of LV fractional shortening (FS) at one day after AR (Fig. 2D) and 24 days after AR (Fig. 2E), the differences between groups were calculated by non- parametric Mann- Whitney test
  • Fig. 2A is a bar graph showing flow cytometry analysis of CCR2 + F4/80 macrophages accumulation at day 1 and day 3 after AR.
  • Fig.2B is a bar graph showing flow cytometry analysis of processed hearts, 3 days after AR
  • Fig. 2C is
  • FIG. 2F is an image showing granulation tissue formation after AR and CCR2 antibody treatment, black circle show macrophage depletion, scale bar: 100 mm;
  • Fig. 2G is an image showing thinning of the resection area and impaired healing at day 7 after AR and CCR2 + monocyte/macrophages inhibition are shown by the black circle), scale bar: 200 mm;
  • Fig. 2H is an image obtained at day 24 following AR and CCR2 monocyte/macrophage inhibition (black circle), scale bar: 500 mm;
  • Fig. 21 is an image showing complete apical regeneration at day 21 following AR, without CCR2 inhibition, scale bar: 500 mm.
  • Figures 3A-3E The effect of cardiac injury on cytokine secretion
  • Figs. 3A to 3E are bar graphs showing representative cytokine secretion from macrophages, 3 days after cardiac injury in neonatal hearts after AR, MI or sham operation and adult hearts after MI, Fig. 3A -interleukin 6 (IL6), Fig. 3B - IL10, Fig. 3C -IL13, Fig. 3D - Vascular endothelial growth factor (VEGF) and Fig. 3E - Osteopontin (OPN), hearts that were harvested 3 days after the procedure, isolated macrophages were cultured for 24h and conditioned media was analyzed by Qu antibody array, statistical analysis was performed using one-way ANOVA, with Tukey’s multiple comparison post- test. Differences were considered significant at p ⁇ 0.05.
  • IL6 interleukin 6
  • Fig. 3B - IL10 Fig. 3C -IL13
  • Fig. 3D Vascular endothelial growth factor
  • OPN Osteopontin
  • FIGS 4A-40 OPN increase neonatal tissue outgrowth via cardiomyocyte and non-cardiomyocyte proliferation
  • Figs. 4A to 4F are representative images of organ culture, taken from neonatal mouse heart, treated with 0 (Figs. 4A and 4B), 400ng/ml of OPN (Figs. 4C and 4D) or 800ng/ml of OPN (Figs. 4E and 4F) for 72 hours, the arrows pointing to cells that were seen budding from the heart;
  • Figs. 4B, 4D and 4F are imaging after DAPI staining;
  • Fig. 4G is a bar graph showing quantification of the outgrowth, statistical analysis was performed using Kruskal-Wallis test, with Dunn’s multiple comparisons post-test; Fig.
  • FIG. 4H and 41 are images showing mouse neonatal cardiomyocytes that were treated with 800ng/ml OPN for 24h (Fig. 41) or without (Fig. 4H) , arrows indicating the presence of cardiomyocytes (using pH3);
  • FIG. 4J and 4K are bar graphs showing the number of PH3/DAPI positive cells in neonatal cardiomyocyte (Fig. 4J) and non-cardiomyocyte cells (Fig. 4K), numbers were normalized to untreated cells, statistical analysis was performed using unpaired t test;
  • Fig. 4L is an image of hears stained for the OPN receptor CD44 (white arrow); Fig.
  • FIG. 4M is a bar graph showing the effect of OPN on cell death in neonatal cardiomyocytes, statistical analysis was performed using unpaired t test
  • Fig. 4N and 40 are bar graphs showing the viability of mouse neonatal cardiomyocytes treated with or without 800ng/ml OPN for 24h was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Fig. 4N) and RealTime-GloTM MT Cell Viability Assay (Fig. 40);
  • Statistical analysis was performed using two-way repeated measures ANOVA and Holm-Sidack’s multiple comparison post-test.
  • Figures 5A-5P OPN stimulated YAP signaling in neonatal cardiomyocytes via CD44.
  • Fig. 5A is an exemplary western blot gel using antibodies against YAP1, pYAPl-127, LATS1/2, pLATS 1/2, ERK and pERK;
  • Fig. 5B is a bar graph showing the western blot results after calculating the ratio between phosphorylated and unphosphorylated proteins;
  • Figs. 5C to 5E are fluorescent images of cells showing YAP translocation from the cytoplasm into the nucleus following OPN treatment; YAP stating is shown in white arrows, Fig. 5C is control showing YAP (white arrows) was located to the cytoplasm;
  • Fig. 5D cells treated with 800ng OPN showing YAP (white arrows) was translocated into the nucleus;
  • Fig. 5A is an exemplary western blot gel using antibodies against YAP1, pYAPl-127, LATS1/2, pLATS 1/2, ERK and pERK;
  • Fig. 5B is a bar
  • Fig. 5E cells treated with 800ng OPN and with an CD44 inhibitor showing that the YAP (white arrows) translocation into the nucleus was prevented;
  • Fig. 5F to 5P are bar graphs showing gene expression obtained by real time PCR AACT using GAPDH as an endogenous control, gene expression analysis, using real time PCR was performed in neonatal cardiomyocytes treated with or without 800ng/ml OPN after 24h, Fig. 5F showing the expression of the SPP-1 (OPN gene) that was up-regulated by OPN;
  • Fig. 5G showing the expression of one of the OPN receptors CD44;
  • Fig. 5H showing the expression of cell cycle genes including the transcriptional enhancer factor TEF-1 (TEAD1) which interacts with YAP1;
  • CTGF connective tissue growth factor
  • Fig. 5J showing the expression of the cyclin-dependent kinase 1 (CDK1) which are YAP downstream targets
  • Fig. 5K showing the expression of the cyclin B1 (CCNB1) regulator of the mitotic (M phase), that is upregulated by OPN
  • Fig. 5L showing the expression of cyclin D1 (CCND1), which encoded cyclin Dl
  • Fig. 5M showing the expression of matrix metalloproteinase (MMP)-12
  • Fig. 5N showing the expression of VEGF
  • Fig. 5N showing the expression of VEGF
  • Fig. 5P showing the expression of insulin-like growth factor -1 (IGF-1);
  • IGF-1 insulin-like growth factor -1
  • Figures 6A-6B OPN improved wound healing and angiogenesis.
  • Fig. 6A is a bar graph showing the effect of OPN on gap closure in neonatal cardiomyocytes; data shows that OPN accelerated the gap closure in neonatal cardiomyocytes compared with control untreated cardiomyocytes and the addition of CD44 Abs to the culture medium eliminated the favourable effects of OPN, suggesting that the effects of OPN on cardiomyocytes are mediated by CD44;
  • Fig. 6B is a bar graph showing the effect of OPN on gap closure in neonatal cardiac fibroblasts that; data shows that OPN stimulate proliferation and migration of fibroblasts.
  • Figure 7 OPN improved angiogenesis
  • Fig. 7 is a bar graph showing the effect of OPN on number of polygons in human umbilical vein endothelial cells (HUVEC); development of tube formation was monitored by confocal microscopy.; statistical analysis was performed using two-way ANOVA and Holm-Sidak's multiple comparison test.
  • Figures 8A-8M Single injection of OPN into infarcted heart decreased LV remodeling and improved cardiac function 28 days after MI.
  • Figs. 8A to 8M are bar graphs showing echocardiography parameters; Fig. 8 A shows anterior wall (AW) diastole (dia); Fig. 8B shows anterior wall (AW) systole (sys); Fig. 8C shows Posterior Wall (PW) diastole (dia); Fig. 8D shows Posterior Wall (PW) systole (sys); Fig. 8E shows Left Ventricular End Diastolic Diameter (LVEDD); Fig. 8F shows Left Ventricular End Systolic Diameter (LVESD); Fig. 8G shows Left Ventricular End Diastolic (LVED) area; Fig.
  • FIG. 8H shows Left Ventricular End Systolic (LVES); Fig. 81 shows Left Ventricular Volume Diastolic (LV Vol;d); Fig. 8J shows Left Ventricular Volume Systolic (LV Vol;s); Fig. 8K shows % Fractional Shortening (FS); Fig. 8L shows Endocardial Fractional Area Change (FAC); Fig. 8M shows % Ejection Fraction (EF), statistical analysis was performed using two-way ANOVA and Holm's-Sidak’s multiple comparison test.
  • FIGS 9A-9P Single injection of OPN into the infarcted heart improved regional cardiac function.
  • Fig. 9A is a representation of mapping LV segments in a short axis view
  • Figs. 9B to 9H are graphs showing the effect of OPN on regional and global radial strain of the infarcted as well as the non-infarcted segments
  • Fig. 9B is anterior septum
  • Fig. 9C is anterior free segment
  • Fig. 9D is posterior septal segment
  • Fig. 9E is interior free segment
  • Fig. 9F is posterior segment
  • Fig. 9G is lateral segment
  • Fig. 9H is average curve-short axis
  • Fig. 91 is a representation of mapping LV segments in a long axis view
  • Fig. 9J is anterior apex
  • Fig. 9K is anterior mid
  • Fig. 9L is anterior base
  • Fig. 9M is posterior apex
  • Fig. 9N is posterior mid
  • Fig. 90 is posterior base
  • Fig. 9P is average curve-long axis.
  • Figs. 10A to 10F are representative pictures of mice hearts, with OPN treatment (Figs. 10A, 10C and 10E) or as control (Figs. 10B, 10D and 10F), 28-days after MI; Hearts were stained for Piero Sirius Red staining for collagen shown in black circle, data shows that OPN increased scar thickening and reduced scar length.
  • the present invention is based on the findings that an Osteopontin protein can be used for the treatment of non-infectious inflammatory disorders, specifically, non-infectious inflammatory disorders associated with cardiovascular conditions, such as myocardial infraction.
  • Example 3 an up-regulated expression of Sppl gene that encodes the Osteopontin protein was observed in the neonatal heart after AR and MI but not in sham operation.
  • Example 4 neonatal macrophages secreted higher amounts of Osteopontin protein after MI and after AR.
  • Example 5, 7 and 8 Osteopontin protein improved the cardiac conditions and function.
  • an Osteopontin (OPN) protein or a functional analogue thereof for use in the treatment of a non-infectious inflammatory disorder.
  • Osteopontin denoted as bone sialoprotein I (BSP-1 or BNSP), early T-lymphocyte activation (ETA-1), secreted phosphoprotein 1 (SPP1), 2ar and Rickettsia resistance (Ric), refers to a secreted phosphorylated extracellular matrix (ECM) protein.
  • OPN protein was suggested to be mediated through various receptors, including for example a CD44 receptor as well as a variety of integrin receptors, such as avb1, avb3, avb5, a5b1, a8b1, a4b1, a9b1, or a9b4.
  • the OPN protein in accordance with the present discolored encompasses OPN protein isoforms and specifically human isoforms of the OPN protein.
  • isoform refers to two or more proteins, typically similar proteins, that originate from a single gene or gene family and are the result of genetic differences.
  • the isoforms can be encoded by different genes or by RNA transcripts from the same gene which have had different exons removed.
  • a set of protein isoforms may be formed from alternative splicing, variable promoter usage, or other post-transcriptional modifications of a single gene. For example, through RNA splicing mechanisms, mRNA has the ability to select different protein-coding segments (exons) of a gene, or even different parts of exons from RNA to form different mRNA sequences such that each unique sequence produces a specific form of a protein.
  • the protein isoforms may have same function.
  • OPN isoforms may be, for example, transcriptionally spliced OPN isoforms, multiple post- translational modifications (phosphorylation, glycosylation and sulfation) isoforms and multiple functionally-active proteolytic forms cleaved by various proteins such as thrombin, MMPs, plasmin and cathepsin DOPN protein.
  • OPN isoforms are denoted as isoform A, isoform B, isoform C, isoform D, OPN5, isoform 4 or isoform 5.
  • the OPN protein is or comprises at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9 or SEQ ID NO: 11.
  • the OPN protein consists of at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9 or SEQ ID NO: 11.
  • human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 314 amino acid residues as denoted by Accession Number NP_001035147.1.
  • human OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO:1.
  • human OPN protein provided by Accession Number NP_001035147.1 is encoded by a nucleic acid sequence denoted by Accession Number NM_001040058.2.
  • the OPN gene comprises a nucleic acid sequence as denoted by SEQ ID NO:2.
  • the gene encoding the OPN protein is denoted at times as SPP1.
  • the OPN protein provided by SEQ ID NO:1 is at times denoted as OPN isoform A.
  • human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 300 amino acid residues as denoted by Accession Number NP_000573.1. In some embodiments, human OPN protein refers to an amino acid sequence of 300 amino acid residues as denoted by number UniProt No. P10451-5. In some other embodiments, human OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO:3 In some other embodiments, human OPN protein denoted by Accession Number NP_000573.1 is encoded by a nucleic acid sequence denoted by Accession Number NM_000582.2. In some other embodiments, human OPN protein is encoded by a nucleic acid sequence provided herein below by SEQ ID NO:4. The OPN protein provided by SEQ ID NO:3 is at times denoted as OPN isoform B or isoform 5.
  • human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 287 amino acid residues as denoted by Accession Number NP_001035149.1. In some other embodiments, human OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO:5. In some other embodiments, human OPN protein provided by Accession Number NP_001035149.1 is encoded by a nucleic acid sequence denoted by Accession Number NM_001040060.1. In some other embodiments, human OPN protein is encoded by a nucleic acid sequence provided herein below by SEQ ID NO:6. The OPN protein provided by SEQ ID NO:5 is at times denoted as OPN isoform C.
  • human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 292 amino acid residues as denoted by UniProt No. P10451-4. In some other embodiments, human OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO:7 The OPN protein provided by SEQ ID NO:7 is at times denoted as OPN isoform D. In some embodiments, human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 300 amino acid residues as denoted by UniProt No. P10451-2. In some specific embodiments, the OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO:8 The OPN protein provided by SEQ ID NO:8 is at times denoted as OPN isoform B.
  • human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 273 amino acid residues as denoted by Accession Number NP_001238758.1.
  • human OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO:9.
  • human OPN protein denoted by Accession Number NP_001238758.1 is encoded by a nucleic acid sequence denoted by Accession Number NM_001251829.1.
  • human OPN protein is encoded by a nucleic acid sequence provided herein below by SEQ ID NO: 10.
  • the OPN protein provided by SEQ ID NO:9 is at times denoted as OPN isoform 4.
  • human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 327 amino acid residues as denoted by Accession Number NP_001238759.1. In some other embodiments, human OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO: 11. In some other embodiments, human OPN protein denoted by Accession Number NP_001238759.1is encoded by a nucleic acid sequence denoted by Accession Number NM_001251830.1. In some other embodiments, human OPN protein is encoded by a nucleic acid sequence provided herein below by SEQ ID NO: 12. The OPN protein provided by SEQ ID NO: 11 is at times denoted as OPN isoform 5 or variant 6.
  • the present disclosure also encompasses a functional analog of OPN.
  • the term “functional analogue ” thereof refers to a chemical/biological compound (entity) that has or may produce similar physical, chemical, biochemical, or pharmacological properties as a reference compound.
  • the functional analogue has or can produce the same activity, e.g. biological, cellular or therapeutic activity of OPN protein or at least one of the OPN protein isoforms.
  • the functional analogue in accordance with the present disclosure is or comprises at least one of a small molecule, an antisense, an siRNA, a polypeptide or a peptide.
  • the functional analogue is capable of treating a non- infectious inflammatory disorder as described herein.
  • the functional analogue has a similar mechanism of action as OPN protein.
  • the functional analogue may be selected such that it binds to a similar receptor(a) or activate a similar receptor(s) as the OPN protein to initiate a physiological/biological/cellular/biochemical response.
  • the effect of OPN was reduced or eliminated after addition of CD44 receptor inhibitor (CD44 antibody).
  • CD44 receptor inhibitor CD44 antibody
  • the effect of the OPN protein may be mediated, at least in part, via a CD44 receptor.
  • the functional analogue may be selected such that it binds to CD44 receptor.
  • the CD44 receptor refers to a multistructural and multifunctional cell surface molecule involved in cell proliferation, cell differentiation, cell migration, angiogenesis, presentation of cytokines, chemokines, and growth factors to the corresponding receptors, and docking of proteases at the cell membrane, as well as in signaling for cell survival.
  • the functional analogue is an agonist of CD44 receptor. In some embodiments, the functional analogue is an agonist of at least one integrin receptor.
  • the functional analog may be at least one of a full agonist, a co-agonist, a selective agonist, an inverse agonist.
  • the functional analogue as described herein may be at least one of a small molecule, an antisense, an siRNA, a polypeptide or a peptide.
  • the functional analogue is a structural analog of the OPN protein.
  • a structural analog as used herein refers to a functional analog that has a similar structure as a reference compound.
  • a structural analog having a similar structure as of OPN protein is characterized by comprising an amino acid sequence.
  • the functional analog is a polypeptide or a peptide.
  • polypeptide or “ peptide ” as used herein refers to a chain of amino acid residues connected by peptide bonds.
  • a peptide typically includes one or more amino acids, and in some embodiments between 2 and 50 amino acids, between 2 and 20 amino acids, between 2 to 10 amino acid.
  • a polypeptide typically refers to an amino acid sequence that do not fold to a specific structure as a protein.
  • the term polypeptide is interchangeably used with the term protein and both are used define an amino acid sequence.
  • the amino acid sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group.
  • the peptides or polypeptides of the present disclosure can be modified at one or more of its amino acid residues, for example by manosylation, glycosylation, amidation (for example C-terminal amides), carboxylation or phosphorylation.
  • the OPN protein or functional analog thereof may be an isolated protein or functional analog thereof, a purified protein or functional analog thereof or a synthesized protein or functional analog thereof.
  • isolated refers to molecules, such as the amino acid sequences described herein, peptides or polypeptides that are removed from their natural environment, isolated, or separated.
  • the synthesized protein or functional analog thereof may be produced through genetic engineering methods, expression in a host cell, or through any other suitable means as known in the art. Methods for producing peptides or polypeptides are well known in the art.
  • the functional analog encompasses derivatives of OPN protein or the OPN protein isoforms.
  • the term "derivative” is used to define an amino acid sequence (e.g. peptide or polypeptide), that is different in at least one amino acid residue from the amino acid sequence of OPN protein or the OPN protein isoforms.
  • Such modification includes insertions, deletions, substitutions and combination thereof.
  • the modifications do not alter the activity of the OPN protein and any derivative in accordance with the present disclosure is capable of treating a non-infectious inflammatory disorder as described herein.
  • Such derivatives may be selected such that they maintain or even improve (i.e. do not reduce) the activity of the OPN protein.
  • a derivative of OPN protein may be a result of alternative splicing or polymorphism of the encoding gene.
  • the functional analog comprises a derivative of at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 11.
  • the functional analog is or comprises an amino acid sequence having at least 50%, at times having at least 60%, at times having at least 70%, at times having at least 75%, at times having at least 80%, at times having at least 85%, at times having at least 90%, at times having at least 95%, at times having at least 97%, at times having at least 98% similarity to an amino acid sequence denoted by at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 11.
  • Sequence similarity also denoted as sequence homology as used herein refers to the amount (%) of amino acids that are conserved with similar physicochemical properties (e.g. leucine and isoleucine), over a specified region, when compared and aligned for maximum correspondence over a comparison window.
  • the functional analog is or comprises an amino acid sequence having at least 60%, at times having at least 70%, at times having at least 75%, at times having at least 80%, at times having at least 85%, at times having at least 90%, at times having at least 95%, at times having at least 97%, at times having at least 98% identity to an amino acid sequence denoted by at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 11.
  • Sequence identity refers to the amount (%) of amino acids that the same (identical) over a specified region, when compared and aligned for maximum correspondence over a comparison window. In determining the sequence identity, it is possible to not count the gaps such that the sequence identity is relative to the shorter sequence of the two compared sequences.
  • the OPN protein derivative may include any modification including insertions, deletions, substitutions and combination thereof. As also indicted herein, such modifications may be a result of alternative splicing or polymorphism of a gene encoding an OPN protein (including all isoforms). Alternatively or additionally, such modifications may be obtained by synthetic methods. It should be appreciated that by the terms "insertions” or “deletions”, as used herein it is meant any addition or deletion, respectively, of amino acid residues to the polypeptides or peptides being derivatives or fragments used by the invention, of between 1 to 50 amino acid residues, between 1 to 20 amino acid residues, between 1 to 10 amino acid residues or between 1 to 5 amino acid residues.
  • insertions or deletions may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. It should be noted that the insertions or deletions encompassed by the invention may occure in any position of the modified peptide, as well as in any of the N' or C termini thereof.
  • amino acid “ substitutions ” are the result of replacing one amino acid with another amino acid, for example with another amino acid that has similar structural and/or chemical properties (conservative amino acid replacements). This may be being due to point mutation in DNA sequence caused by nonsynonymous missense mutation which alters the codon sequence to code other amino acid instead of the references. Alternatively, amino acid substitution may be a results of analysis and/or rationale replacement.
  • the amino acid substitutions may be conservative replacement. Such a replacement encompasses a change in an amino acid into another amino acid exhibiting similar properties.
  • Conservative amino acid replacements also denoted as conservative amino acid “substitutions” or conservative amino acid mutations is an amino acid replacement in a protein that changes a given amino acid to a different amino acid with similar biochemical, structural and/or chemical properties as described herein.
  • amino acids may be sorted into six main classes on the basis of their structure and the general chemical characteristics of their side chains (R groups): Aliphatic: Isoleucine (I), Leucine (L), Glycine (G), Alanine (A), Valine (V); Hydroxyl or sulfur/selenium-containing: Serine (S), Cysteine (C) , Threonine (T) , Methionine (M); Cyclic: Proline (P); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Basic: Histidine (H), Lysine (K), Arginine (R); Acidic and their amides: Aspartate (D), Glutamate (E), Asparagine (N), Glutamine (Q).
  • R groups Aliphatic: Isoleucine (I), Leucine (L), Glycine (G), Alanine (A), Valine (V); Hydroxyl or sulfur/selenium-containing: Serine (S), Cysteine (C
  • each of the following groups contains other exemplary amino acids that are conservative substitutions for one another: 1) Very small: Alanine (A), Glycine (G); 2) Negative charge: Aspartic acid (D), Glutamic acid (E); 3) Polar (amidated carboxyl side chain): Asparagine (N), Glutamine (Q); 4) Positively charged: Arginine (R), Lysine (K); 6) Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W), and occasionally also Histidine (H); 7) Small polar: Serine (S), Threonine (T); 8) Sulfur-containing: Cysteine (C), Methionine (M); 9) Small: Ala (A), Glycine (G), Serine (S); 10) Beta-branched: Valine (V), Isoleucine (I) and occasionally also Threonine (T); 11) Polar: Asparagine (N), Glutamine (Q), Serine (S), Thre
  • nonpolar “hydrophobic” amino acids are selected from the group consisting of Valine (V), Isoleucine (I), Leucine (L), Methionine (M), Phenylalanine (F), Tryptophan (W), Cysteine (C), Alanine (A), Tyrosine (Y), Histidine (H), Threonine (T), Serine (S), Proline (P), Glycine (G), Arginine (R) and Lysine (K); “polar” amino acids are selected from the group consisting of Arginine (R), Lysine (K), Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q); “positively charged” amino acids are selected form the group consisting of Arginine (R), Lysine (K) and Histidine (H) and “acidic” amino acids are selected from the group consisting of Aspartic acid (D), Asparagine (N), G
  • the replacement is a radical replacement.
  • a radical replacement substitution is an exchange of an amino acid into another amino acid with different properties.
  • Amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • amino acid residue encompasses both naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g-carboxyglutamate, and O-phosphoserine.
  • amino acid analogs refers to compounds that have the same fundamental chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups or modified peptide backbones but retain the same basic chemical structure as a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • the functional analog is a derivative of OPN protein as described herein.
  • the OPN protein derivative includes at least one amino acid modification.
  • the OPN protein derivative includes an amino acid modification (substitution) of the amino acid sequence denoted in SEQ ID NO:1.
  • modification can be in at least one amino acid residue number 58, 188, 224, 237, 275, 276, 277, 301.
  • the functional analog is or comprises an amino acid sequence denoted by at least one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16 or SEQ ID NO:17.
  • the functional analog consists of an amino acid sequence denoted by at least one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16 or SEQ ID NO:17.
  • the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 13.
  • the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 14.
  • the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 15.
  • the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 16.
  • the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 17.
  • the functional analog is a derivative that includes an amino acid modification of the amino acid sequence denoted in SEQ ID NO:1 in which at least one amino acid was deleted.
  • functional analogue comprises at least one of fragment of OPN protein or isoforms thereof.
  • fragment refers to any peptide or polypeptide which is at least one amino acid shorter than the OPN protein or OPN isoforms (amino acid sequence) which is obtained by deletion of at least one amino acid residue from the OPN or isoforms amino acid sequence in accordance with the present disclosure.
  • fragments of the present disclosure are functional fragment, i.e. have the same activity and/or mechanism of action as the proteins/polypeptide of the present disclosure.
  • the fragments of the present disclosure may be selected such that they maintain or even improve (do not reduce) the activity of the OPN protein. As described herein, the fragments may be due to alternative splicing or polymorphism occurring in a gene encoding the OPN protein. Alternatively or additionally, the fragments may be synthesized.
  • the functional analog comprises a fragment of an amino acid sequence denoted by at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 11.
  • the functional analog comprises a fragment of an amino acid sequence denoted by at least one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16 or SEQ ID NO:17.
  • the functional analog comprises a fragment of an amino acid sequence denoted by at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17.
  • the functional analog is or comprises an amino acid sequence denoted by at least one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21.
  • the functional analog an amino acid sequence denoted by at least one of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.
  • the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 18. In some specific embodiments, the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 19.
  • the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO:20.
  • the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO:21.
  • the functional analog is or comprises an amino acid sequence denoted by at least one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the functional analog consists an amino acid sequence denoted by at least one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the fragment comprises an amino acid sequence comprising amino acid residues 159-161 of OPN protein as denoted by SEQ ID NO:1.
  • the fragment comprises an amino acid sequence comprising RGD.
  • the amino acid sequence RGD is denoted by SEQ ID NO:22.
  • the fragment comprises an amino acid sequence comprising amino acid residues 158-162 of OPN protein as denoted by SEQ ID NO:1.
  • the fragment comprises an amino acid sequence GRGDS.
  • the amino acid sequence GRGDS is denoted by SEQ ID NO:23.
  • the fragment comprises an amino acid sequence comprising amino acid residues 131-143 of OPN protein as denoted by SEQ ID NO:1.
  • the fragment comprises an amino acid sequence ELVTDFPTDLPAT.
  • the amino acid sequence ELVTDFPTDLPAT is denoted by SEQ ID NO:24.
  • the fragment comprises an amino acid sequence comprising amino acid residues 162-167 of OPN protein as denoted by SEQ ID NO:1.
  • the fragment comprises an amino acid sequence SVVYGLR.
  • the amino acid sequence SVVYGLR is denoted by SEQ ID NO:25.
  • the fragment comprises an amino acid sequence comprising amino acid residues 290-305 of OPN protein as denoted by SEQ ID NO:1.
  • the fragment comprises an amino acid sequence KSKEEDKHLKFRISHE.
  • the amino acid sequence KSKEEDKHLKFRISHE is denoted by SEQ ID NO:26.
  • the fragment comprises an amino acid sequence comprising amino acid residues 167-206 of OPN protein as denoted by SEQ ID NO:1.
  • the fragment comprises an amino acid sequence LRSKSKKFRRPDIQYPDATDEDITSHMESEELNGAYKAIP.
  • the amino acid sequence LRSKSKKFRRPDIQYPDATDEDITSHMESEELNGAYKAIP is denoted by SEQ ID NO:27.
  • the functional analog is or comprises at least one amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the functional analog consists of at least one amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the functional analog is or comprises at least one amino acid sequence denoted by SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the functional analog consists of at least one amino acid sequence denoted by SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the functional analog is or comprises at least one amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the functional analog consists of at least one amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the present invention also encompasses derivatives or fragments of at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the present invention also encompasses derivatives or fragments of at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
  • the present invention also encompasses derivatives or fragments of at least one amino acid sequence denoted by SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21.
  • the present invention also encompasses derivatives or fragments of at least one amino acid sequence denoted by SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the peptides derivatives and/or fragments in some examples may be determined by peptidomimetics methods or computational analysis.
  • the functional analog is or comprises an amino acid sequence having at least 50%, at times having at least 60%, at time having at least 70%, at times having at least 75%, at times having at least 80%, at times having at least 85%, at times having at least 90%, at times having at least 95%, at times having at least 97% similarity to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the functional analog is or comprises an amino acid sequence having at least 85% similar to at least one amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the functional analog is or comprises an amino acid sequence having at least 50%, at times having at least 60%, at time having at least 70%, at times having at least 75%, at times having at least 80%, at times having at least 85%, at times having at least 90%, at times having at least 95%, at times having at least 97% identity to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • the functional analog is or comprises an amino acid sequence having an amino acid sequence that is at least 85% identical to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
  • a derivative is different in at least one amino acid residue from an amino acid sequence of OPN protein as well as from any fragment thereof, such modification includes insertions, deletions, substitutions and combination thereof.
  • the OPN protein or functional analog thereof as used herein also encompasses in accordance with some examples, products of gene therapy.
  • the gene therapy in the context of the present application may modify, for example, by amplification a gene of the OPN protein or a functional analog thereof to treat a non- infectious inflammatory disorder as described herein.
  • Examples of gene therapy include plasmid DNA, miRNA, viral vectors, bacterial vectors, human gene editing technology and the like.
  • the OPN protein or the functional analog thereof may be formulated into a pharmaceutical formulation with at least one pharmaceutically acceptable carrier/s, diluent/s, excipient/s.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art.
  • the carrier is a pharmaceutical buffering agent (buffers). Buffers for use in pharmacy are well known in the art and include, inter alia, phosphate buffer solution (PBS), dextrose, etc.
  • PBS phosphate buffer solution
  • dextrose dextrose
  • the OPN protein or the functional analog thereof of the invention is associated with at least one of pharmaceutically acceptable carrier in the composition to form a delivery system.
  • physiologically acceptable carrier also encompasses a carrier that is useful in preparing a delivery system for the OPN protein or functional analog thereof of the invention that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and acceptable for veterinary use as well as human pharmaceutical use.
  • association it is to be understood as being carried by the physiologically acceptable carrier by any means of suspension, emulsion, dissolution, embedment, attachment, entrapment, encapsulation, chemical bonding, adsorption, and the like and contemplates any manner by which the OPN protein or functional analog thereof of the invention is held to form the integral delivery system.
  • the delivery system in accordance with some embodiments, comprises a matrix, e.g. in which the OPN protein or functional analog thereof is entrapped.
  • the matrix is a biodegradable matrix.
  • the delivery system comprises an organized collection of lipids, such as liposomes or micelles.
  • lipids such as liposomes or micelles.
  • the OPN protein or the functional analog thereof may be associated with liposomes.
  • the OPN protein or the functional analog thereof is encapsulated within the intraliposomal core of the liposomes. In some embodiments, the OPN protein or the functional analog thereof is present within a vesicle. In some embodiments, the OPN protein or the functional analog thereof is encapsulated within exosomes.
  • Exosomes as used herein refer to extracellular vesicles (EVs) that are produced in the endosomal compartment of eukaryotic cells and composed of cellular membranes with adhesive proteins on their surface.
  • EVs extracellular vesicles
  • the liposomes, exosomes or any other delivery system can be modified to include on it’s surface specific residues to enhance localization to a target tissue, such as a cardiovascular tissue.
  • composition of the invention in accordance with the present disclosure is administered with an effective amount of the OPN protein or functional analog thereof.
  • effective amount is intended to mean that amount of the OPN protein or functional analog thereof is sufficient to cause a beneficiary change.
  • the amount of the OPN protein or functional analog in the formulation being the effective amount is typically determined in appropriately designed clinical trials (dose range studies) to achieve a desired effect and the person versed in the art will know how to properly conduct such trials in order to determine the amount.
  • an effective amount depends on a variety of factors including the distribution profile of the nanoparticles within the body, a variety of pharmacological parameters such as half life in the body, undesired side effects, if any, on factors such as age and gender of the treated individual etc.
  • the amount must be effective to achieve a desired therapeutic effect such as rapid recovery of the treated subject, or improvement or elimination of symptoms and other indicators associated with the inflammation under treatment, selected as appropriate measures by those skilled in the art.
  • the OPN protein or the functional analog is administered and dosed taking into account the clinical condition of the individual, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the dosage form may be single dosage form or a multiple dosage form to be provided over a period of several days.
  • the schedule of treatment with the OPN protein or functional analog generally has a length proportional to the length of the non-infectious inflammatory process, the parameters of the individual to be treated (e.g. age and gender).
  • the OPN protein or the functional analog is suitable for systemic administration.
  • systemic administration means the administration of the OPN protein or the functional analog directly into the circulatory system so that the entire body is affected by the administration.
  • Systemic administration includes enteral administration (i.e. absorption of the drug through the gastrointestinal tract) or parenteral administration (i.e. generally injection, infusion, or implantation).
  • the OPN protein or the functional analog is suitable for parental administration.
  • parenteral administration means modes of administration other than enteral and local administration.
  • the OPN protein or the functional analog thereof is suitable for administration by at least one o intravenous (i.v) injection, intracoronary (i.e), intramuscular (i.m), intraperitoneal (i.p) injection.
  • i.v intravenous
  • i.e intracoronary
  • i.m intramuscular
  • i.p intraperitoneal
  • the OPN protein or the functional analog thereof is suitable is administered by intravenous injection or intra-arterial.
  • the OPN protein or the functional analog is suitable for administration to the vascular system.
  • the OPN protein or the functional analog may be applicable for intravenous injection or intravenous infusion.
  • the OPN protein or the functional analog are suitable for local administration to the target site, e.g. of inflammation site.
  • target site is used to denote tissue or organ that is either inflamed (non-infectious inflammation); or tissue that is injured for example an ischemic tissue.
  • the OPN protein or the functional analog is suitable for administration into a cardiac tissue/myocardium.
  • the OPN protein or the functional analog can be administered by at least one of an intramyocardial administration, an intracoronary administration, a transvenous coronary sinus administration or a retrograde coronary sinus administration.
  • the OPN protein or the functional analog is suitable for intramyocardial administration.
  • Intramyocardial administration as used herein means direct administration into the heart muscles or ventricles. Intramyocardial administration includes for example epicardial and transendocardial administration.
  • Epicardial administration refers to administration into the inner layer of the pericardium, a conical sac of fibrous tissue that surrounds the heart and the roots of the great blood vessel.
  • Transendocardial administration refers to administration through or across the endocardium.
  • OPN protein or the functional analog is suitable for intracoronary administration.
  • Intracoronary administration as used herein means direct administration into to or within the coronary artery or the heart. Intracoronary administration encompasses direct injection or infusion and provides an advantage of direct local delivery.
  • the OPN protein or the functional analog is suitable for transvenous coronary sinus administration.
  • Transvenous coronary administration as used herein means administration directly across or through the heart.
  • the OPN protein or the functional analog is suitable for retrograde coronary sinus administration.
  • Retrograde coronary sinus administration as used herein means administration via the coronary sinus and vein.
  • Direct administration may be done by devices dedicated for such an administration.
  • direct administration is applicable by using a catheter, microcatheter or injection by needle.
  • direct administration is applicable by using a balloon such as an inflatable balloon. The balloon or any other suitable device may be introduced, for example, by a catheter.
  • OPN was suggested to stimulate neonatal cardiomyocyte proliferation and significantly improve LV remodeling and function after MI.
  • Endogenous tissue engineering by OPN reduces LV wall stress by increasing scar thickness and stabilizing chamber size. By thickening the scar, wall stress is reduced (Laplace law) and the degree of outward motion of the infarct that occurs during systole (dyskinesis) is also reduced. This effect may be considered to be analogous to an external device that constrains the myocardium from expanding, thereby limiting adverse LV remodeling and prevent mechanical complications after MI.
  • the OPN protein including all isoforms of OPN and functional analog including for example naturally occurring derivatives or fragments as well as OPN-derived synthetic peptide or OPN agonists can be delivered into the infarcted myocardium immediately or a few days after coronary reperfusion.
  • OPN can be combined with scaffolds, injectable biomaterials or encapsulated into liposomes to improve delivery and retention in the infarcted heart
  • OPN can be delivered intra-coronary by a catheter-based approach, by IV infusion of liposomes or other nanocarriers that target the infarcted myocardium or by catheter-based direct injection into the infarct.
  • the OPN protein or the functional analog thereof may be applicable for treating non-infectious inflammatory disorders as further detailed herein.
  • a further aspect of the invention relates to a method of treating or preventing a non-infectious inflammatory disorder or a condition associated therewith.
  • the method of the invention may comprise the step of administering to a subject in need thereof an effective amount of the OPN protein or the functional analog thereof, or of any composition comprising the same.
  • the method of the invention may be applicable for the treatment, prevention or inhibition of non-infectious inflammatory disorder or a condition associated therewith in a subject in need thereof, the method comprising the step of administering to said subject a therapeutically effective amount of the OPN protein, or functional analog thereof or of any composition or the delivery system comprising the same.
  • non-infectious inflammatory disorders used herein is to be understood as encompassing any immune response that is not related to activation of the immune system, e.g. by an infection.
  • Such non-infectious inflammatory disorders denote any disorder which the activation of macrophages or activated macrophages play a role such as auto-immune disorders and inflammatory disorders which are not infection related, i.e. non-pathogenic, caused by other than an infectious agent (e.g. auto-antigen, hypersensitivity, wound).
  • Non-limiting examples of activated macrophage-related, non-infectious inflammatory disorders are inflammatory diseases of the gastrointestinal tract such as Crohn's disease, inflammatory bowel disease, gastritis, colitis, ulcerative colitis, colon irritable, gastric ulcer and duodenal ulcer, inflammatory diseases of the skin such as psoriasis, inflammatory diseases of the respiratory system such as asthma, allergic rhinitis or chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, sarcoidosis, inflammatory diseases of the musculoskeletal system such as rheumatoid arthritis, osteomyelitis, osteoporosis, or neuritis, systemic sclerosis, inflammatory diseases of the kidneys such as glomerulonephritis, renal ischemia, or renal inflammation; inflammatory diseases of the nervous system such as multiple sclerosis, Alzheimer's disease and H1V- 1 -associated dementia; autoimmune diseases such as diabetes, type 1 and 2 diabetes mellitus and graft
  • the non-infectious inflammatory disorders also denote tissue healing and repair.
  • the tissue may be injured or damaged as a result of ischemia or necrosis.
  • Non-limiting examples of tissue damage may include infracted myocardium, myositis, myocarditis, myocardial fibrosis, glumerulonephritis, diabetic nephropathy, kidney infarct, glomerular sclerosis, stroke, liver injury, brain injury, pulmonary fibrosis, ischemic limb, athrosclerosis vascular disease, chronic ulcer.
  • the non-infectious inflammatory disorders may be acute non-infectious inflammatory disorders or chronic non-infectious inflammatory disorders. It is specifically noted that the non-infectious inflammatory disorders exclude any phatogenic, e.g. bacterial, mediated inflammation (e.g. due to pathogenic infection).
  • the non-infectious inflammatory disorders may be involved in a condition selected with tissue injury, infarct, trauma, and necrosis. According to some embodiments, the non-infectious inflammatory disorders are associated with a damaged tissue condition, for example healing a wounded tissue.
  • the non-infectious inflammatory disorders involve at least one of the following conditions tissue injury, infarct, trauma, necrosis, sterile inflammatory disorder.
  • the sterile inflammatory disorder may be in some examples an autoimmune sterile inflammatory disorder.
  • the non-infectious inflammatory disorder is associated with a cardiovascular disorder.
  • a cardiovascular disorder As shown in the Examples below and specifically in Examples 1 and 2, accumulation of monocytes and macrophages was observed at a heart resection site and specifically CCR2+ monocytes.
  • Cardiovascular disorders as used herein encompasses a range of conditions that affect the heart structure and/or heart activity of a subject. Such disorders generally involve narrowed or blocked blood vessels.
  • the methods of the invention may be applicable for treating a cardiovascular disorder selected from or associated with myocarditis, heart failure, atherosclerosis and myocardial infarction.
  • the methods of the invention may be applicable for subjects suffering from a cardiovascular disorder, which may be a genetic disorder or an acquired disorder.
  • the methods of the invention may be applicable for treating acquired cardiovascular disorders.
  • Acquired cardiovascular disorders may result in a variety of factors affected by a subject’s lifestyle and can results from factors such as exposure to toxic substances such as toxins, radiation or chemotherapy
  • the acquired cardiovascular disorder may be at least one of coronary artery disease (CAD), myocardial infarction (MI), heart failure, cardiomyopathy, myocarditis.
  • CAD coronary artery disease
  • MI myocardial infarction
  • heart failure heart failure
  • cardiomyopathy myocarditis
  • the methods of the invention are applicable for the treatment, inhibition or delaying the cardiovascular disorder associated with coronary artery disease.
  • the methods of the invention are applicable for subjects having a tendency to develop a coronary artery disease.
  • Coronary Artery Disease is a type of cardiovascular disease that involves atherosclerosis, i.e. hardening and narrowing of the coronary arteries, producing blockages in the vessels that carry blood to the heart.
  • Atherosclerosis is a condition that may develop with time such that it is slowing causes to blocking arteries and eventually restricting blood flow to the heart and may ultimately cause heart attack, stroke, and peripheral vascular disease.
  • the methods of the invention are applicable for the treatment, inhibition or delaying the cardiovascular disorder associated with heart failure. Specifically, the methods of the invention are applicable for subjects having a tendency to develop heart failure.
  • Heart Failure relates to a condition at which the heart’s ability to pump is weaker than normal and hence blood moves through the heart and body at a slower rate, pressure increases in the heart, and the heart can’t supply enough blood and oxygen to the body’s cells, resulting in fatigue and shortness of breath.
  • the methods of the invention may be applicable, in accordance with some embodiments, for the treatment, inhibition or delaying the cardiovascular disorder associated with a heart failure which is an ischemic heart failure (IHF).
  • IHF ischemic heart failure
  • Ischemic heart failure relates to a cardiac condition characterized by systolic dysfunction and reduced cardiac output typically resulting from an imbalance between myocardial oxygen demand and supply.
  • the methods of the invention are applicable for the treatment, inhibition or delaying the cardiovascular disorder associated with cardiomyopathy.
  • the methods of the invention are applicable for subjects having a tendency to develop cardiomyopathy.
  • Cardiomyopathy relates to a progressive disease that causes the heart to become abnormally enlarged, thickened, and/or stiffened, cardiomyopathy (also known as heart muscle disease) limits the heart muscle’s ability to pump blood effectively, often leading to other heart conditions such as heart failure or arrhythmia. Cardiomyopathy may result from exposure to toxins, chemotherapy, radiation due to autoimmune conditions.
  • the methods of the invention are applicable for the treatment, inhibition or delaying the cardiovascular disorder associated with myocardial infarction. Specifically, the methods of the invention are applicable for subjects having a tendency to develop myocardial infarction.
  • MI Myocardial Infarction
  • plaque a disorder that takes place when blood flow to the heart is severely reduced or cut off, due to the hardening and narrowing of the coronary arteries from the build-up of fat, cholesterol, and other substances, known together as "plaque.”
  • plaque Such a blood clot forms around the plaque, blocking blood flow and may result in permanent damage or death of part of the heart muscle.
  • the methods of the invention are applicable for the treatment, inhibition or delaying the cardiovascular disorder associated with myocarditis. Specifically, the methods of the invention are applicable for subjects having a tendency to develop a myocarditis.
  • Myocarditis is an inflammation of the heart muscle (myocardium) and may affect the heart muscle and the heart's electrical system, reducing the heart's ability to pump and causing rapid or abnormal heart rhythms (arrhythmias) It should be noted that myocarditis can damage the heart muscle, at times even permanently and ultimately causing heart failure, heart attack or heart stroke or arrhythmias.
  • OPN As detailed herein and shown in Example 5, OPN stimulated cardiac cell proliferation in neonatal heart and the proliferation was OPN-concentration dependent. Further, as shown in Example 6 below, OPN stimulated cell cycle in cardiomyocytes an effect which was suggested to be mediated via CD44 receptor. Further and as shown in Example 8 below, OPN simulated wound heating by stimulating both proliferation and migration of both cardiomyocytes and fibroblasts. Finally, as shown in Example 10 below, OPN significantly improve LV remodeling and function after MI and reduces LV wall stress by increasing scar thickness and stabilizing chamber size.
  • the methods of the present invention may be applicable for restoring damaged myocardium or for regeneration of myocardial.
  • the methods of the invention are applicable for the restoring permanently damaged myocardium.
  • the damaged myocardium may be transiently damaged or permanently damaged.
  • the methods of the invention are applicable for the regeneration of myocardium.
  • the regeneration of myocardium comprises angiogenesis and myogenesis.
  • OPN protein or a functional derivative thereof may be beneficial to a subject having reduced expression of OPN or a defected expression of OPN.
  • the former may be considered in some examples as an OPN-deficient disorder and may be treated with the OPN protein or functional analog as described herein.
  • the administration of the OPN protein or the functional analog thereof may be in combination with one or more other active agents, such as one or more immunomodulatory agents or anti-inflammatory agents.
  • An immunomodulatory agent as used herein refers to a substance that stimulates or suppresses the immune system and can be used as immunotherapy.
  • An immunomodulatory agent can be recombinant, synthetic, or natural agent.
  • immunomodulatory agents that can be administrated with the OPN protein or a functional analog thereof include immunomodulatory imide drugs (IMiDs) such as thalidomide and its analogues (lenalidomide, pomalidomide, iberdomide, and apremilast).
  • IiDs immunomodulatory imide drugs
  • anti-inflammatory agents which may be combined with the OPN protein or functional analog thereof include, without being limited thereto, steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs (NSAID).
  • the additional agent may be administered to the subject before, concomitant or after administration of the OPN protein or the functional analog thereof.
  • the two (or more) may be in the same formulation or formulated in two different formulations.
  • the OPN protein or the functional analog thereof as defined herein may form part of a therapeutic kit for use by a practitioner, e.g. a medical doctor, a nurse, or by a subject in need of the treatment.
  • the kit may comprise the OPN protein or the functional analog thereof within a physiologically acceptable carrier, and instructions for use of OPN protein or the functional analog thereof for administering to the subject, typically by injection.
  • the kit may comprise a single dosage unit of the OPN protein or the functional analog thereof for single administration or may comprise multiple dosages, e.g. for multiple, sequential administrations according to a predefined schedule of treatment.
  • disease As used herein, “disease”, “disorder”, “condition” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms. It is understood that the interchangeably used terms "associated” and “related”, when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder, condition or pathology causes a second disease, disorder, condition or pathology.
  • treatment refers to the administering of a therapeutic amount of the formulation of the present invention which is effective to improve one or more undesired symptoms associated with a disease or condition as described herein.
  • the method of the invention involves the administration of a therapeutically effective amount of the OPN protein or functional analog thereof of the invention.
  • the "effective amount” or “therapeutically effective” for purposes disclosed herein indicates that the amount of the OPN protein or functional analog thereof is effective to treat, inhibit or delay one or more symptoms of a disease as described herein.
  • subject refers to a living organism that is treated with the formulation as described herein, including, but not limited to, any mammal, such as a human.
  • N a neonatal mouse model of apical resection (AR) and left anterior descending (LAD) ligation MI were used.
  • newborn mice were anaesthetized by inhalation of 2% isoflurane and 100% oxygen.
  • the mice were cooled down on an ice bed for 4 minutes, thereby causing asystole and reversible apnea, and preventing excessive blood loss during surgery.
  • the cooling down period also provides additional anesthesia.
  • the chest was opened by left thoracotomy, and iridectomy scissors were used to carefully and gradually resect thin segments from the LV apex, as previously described, [4] and the thoracic skin was closed using adhesive tissue glue.
  • mice were anaesthetized with 1-3% isoflurane, intubated and ventilated with 100% oxygen. The chest was shaved and opened by left thoracotomy, and LAD ligation was performed using an 6-0 prolene suture (Ethicon, Cornelia, GA, USA) to permanently occlude the LAD coronary artery. Immediately after the occlusion, 40ml of PBS with or without 200 ng of OPN were injected to the infarcted area.
  • macrophage reporter mice was used [5].
  • the inventors used crossed Csflr-icre mice with the cre- reporter 35 Rosa mT/mG in which CsflR-positive cells are GFP positive.
  • cardiac macrophages total heart cells were isolated using an enzymatic digestion mixture, [7] which was added to the heart for three 15-minute cycles in a rotating water bath warmed to 37°C. Macrophages were purified based on plastic adherence, as previously described. Briefly, cells were incubated for 2 hours at 37°C in humid air with 5% CO2 on 6 well plates supplemented with RPMI (Biological Industries, Beit-Haemek, Israel) with 10% fetal bovine serum (FBS) (Biological Industries, Beit-Haemek, Israel) and 1% streptomycin.
  • RPMI Biological Industries, Beit-Haemek, Israel
  • FBS fetal bovine serum
  • non-adherent cells were washed, and a 3-minute trypsin- EDTA treatment was added to the remaining cells [3]. The trypsin was then blocked with fresh medium and washed, and the intact adherent cells were considered to be cardiac macrophages.
  • mice One-day-old neonatal mice were subjected to AR. Following recovery, the mice were treated with 20ml of CCR2 Ab injected IP (clone:MC21) [8]. every 48 hours, 3 times in total, to achieve CCR2 + monocyte blockade for the first week after AR.
  • CCR2 Ab injected IP clone:MC21
  • the cells were stained with the macrophage marker F4/80, and displayed with > 90% positive staining for F4/80 in culture (data not shown).
  • Cardiac macrophages were grown for 24 hours for conditioned media collection.
  • One hundred micro-liters of cell culture supernatant (for each mouse, per group) was used for cytokine and chemokine detection and quantification by a custom-made mouse Quantibody Array kit (RayBiotech Inc. Insight Biotechnology Ltd, Wembley, UK), according to the manufacturer’s instructions.
  • the array was designed to quantitatively detect 14 cytokines, chemokines, and growth factors simultaneously: Interleukin (IL)-lb, IL-4, IL-6, IL-10, IL-12 p40/p70, IL-17, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF) -a, stromal cell-derived factor (SDF) -1, IL -13, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and OPN.
  • IL Interleukin
  • IL-4 Interleukin
  • IL-6 interleukin-10
  • IL-12 p40/p70 interleukin-17
  • MCP monocyte chemoattractant protein
  • TNF tumor necrosis factor
  • SDF stromal cell-derived factor
  • VEGF vascular endothelial growth factor
  • IGF insulin-like growth factor
  • HGF hepatocyte growth factor
  • the signals (green fluorescence, Cy3 channel, 555 nm excitation, and 565 nm emission) were captured using a GenePix 4000B laser scanner (Bio-Rad Laboratories, Hercules, CA, USA), and extraction with GenePix Pro 6.0 microarray analysis software. Quantitative data analysis was performed using RayBiotech mouse Array software (Q- Analyzer Software for QAM-INF-1, Norcross, GA). Sample concentrations (pg/ml) were determined from mean fluorescent intensities (median values) compared with five- parameter linear regression standard curves, generated from standards provided by the manufacturer.
  • a series of isochronal maps reflecting the cytokine/chemokine concentrations detected by the microarrays were constructed by Matlab software (R2009a, MathWorks, Inc., Natick, MA, USA), in order to transform numerical data into simplex graphical patterns.
  • Hearts were harvested from 1 -day-old, neonatal mice. The atrial area was removed and the ventricular area was sliced into 2 transverse sections, which were cultured on a coated 48 well dish with cardiomyocyte growth media for 48 hours in order to achieve adhesion. Following this, tissues were treated with 800ng/ml OPN (Sigma-Aldrich, St.Louis, MO, USA), 400ng/ml or control culture media without OPN (6 wells in each group), for 72 hours [9]. Cardiac tissue growth was then assessed by cell budding (outgrowth) from the cultured heart slices using SigmaScan Pro (Systat Software San Jose, CA, USA).
  • the outgrowth was stained with antibodies against a-cardiac actin (Santa Cruz Biotechnology, Dallas, TX, USA), PH3 (S10 Epitomics, Burlingame, CA, USA), CD31 (Santa Cruz Biotechnology, Dallas, TX, USA) and F4/80 (BioLegend, San Diego, CA, USA).
  • Isolation and culture of neonatal mouse cardiomyocytes was done as previously described [10]. Briefly, 10 hearts were extracted from1-day old mice into a bacterial dish containing sterile, 4°C, PBS. After removing unwanted tissue, the hearts were transferred into a drop of isolation medium (BDM: Sigma-Aldrich, B-0753; HBSS: BI,02-017-1B; trypsin 0.25%, Gibco) and minced to small pieces. The pieces were then transferred to a conical tube containing 10ml of isolation medium and incubated overnight with gentle agitation at 4°C.
  • isolation medium BDM: Sigma-Aldrich, B-0753; HBSS: BI,02-017-1B; trypsin 0.25%, Gibco
  • the isolation medium was replaced by digestion medium (collagenase/dispase: Sigma-Aldrich, 10269638001; L-15: BI, 01-115-1 A, BDM, HBSS) and the conical tube was transferred to a 37 °C water bath for 20 min with gentle agitation. Then, when the digestive tissue sank to the bottom, supernatant-containing cells were transferred to a fresh conical tube, and filled with medium-free collagenase/dispase, through a sterile cell-strainer (100 mm nylon mesh).
  • digestion medium collagenase/dispase: Sigma-Aldrich, 10269638001; L-15: BI, 01-115-1 A, BDM, HBSS
  • DMEM high glucose M-199: BI, 01-080-1B
  • Horse serum/HS, FBS, pen/strep horse serum/HS, FBS, pen/strep
  • DMEM low glucose M-199, pen/strep
  • Mouse neonatal cardiomyocytes were treated with or without 800ng/ml OPN (02260, Sigma-Aldrich) for 24h. Then, the cells were washed and fixed using 4% formalin. Blocking was performed using CAS-Block (008120, Life Technologies, Freseric, MD, USA) for 10 min and M.O.M/ Mouse IgG blocking Reagent (MKB-2213, VECTOR LABORATORIES, Burlingame, CA, USA) for 20 min.
  • RNA Extraction and Quantitative RT-PCR were added against actinin (1:200, A7811, Sigma-Aldrich) and pH3 (1:100, S10, ab47297, Abeam) or CD44i (1:100, KM81, abll2178, Abeam) or Yes- Associated Protein (YAP) (1:50, LS-C331201, LSBio) for overnight in 4°C.
  • secondary antibodies Alexa Fluor 488 (1:200, Jackson ImmunoResearch
  • Texas Red 1:200, Jackson ImmunoResearch
  • DAPI BAR-NAOP
  • neonatal cardiomyocytes were treated with or without 800ng/ml OPN (02260, Sigma- Aldrich) and 10mg/ml CD44i (KM81, Abeam), an antibody against CD44, for 24h. Then, the cells were exposed to 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay according to the manufacturer’s protocol (ab211091). The number of cells was determined via spectrophotometer using a wavelength of 595 nm.
  • the Hippo signaling pathway is an evolutionarily ancient cascade of kinases and effector proteins that control cell proliferation and organ size.
  • the inventors isolated and cultured mouse neonatal cardiomyocytes in starvation medium for 24 hours prior to lysis. Proteins were extracted and quantified using the Bradford method and 15mg protein was loaded to electrophoresis gel.
  • Immunolabeling was done by antibodies directed against the following: c-terminal of EEK2 (#sc-154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated ERK1/2 (Sigma), LATS1 (1:250; #3477; CST), LATS1 (Thrl079) (1:500; #bs-7913R; Bioss ANTIBODIES), YAP (1:500; #4912; CST), phosphorylated-YAPl (p-YAP; 1:500; #4911; CST).
  • Cardiac fibroblasts were extracted from 4 neonatal hearts using an enzymatic digestion mixture containing 0.25% trypsin-EDTA (Gibco-Invitrogen, Carlsbad, CA, USA) and 2.4 U/ml dispase II, (Sigma-Aldrich, St. Louis, MO, USA). 44 Next, the cells were counted using trypan blue and plated with fibroblasts medium (DMEM high- glucose, FBS, pen/strep, glutamine and b-mercaptoethanol) to 2xl0 5 /well in a 96-well plate. After 24h, the plated fibroblast medium was replaced with a new, low-glucose, DMEM medium.
  • DMEM fibroblasts medium
  • the scratch assay employed a "scratch" across the middle of the well, using a sterile I0ml pipette tip. The rate of gap closure was monitored by serial microscope images. The images were then compared to quantify the migration rate of the cells up to 4 days.
  • hearts were harvested at different time points after the procedure, washed with PBS and then fixed in 4% paraformaldehyde overnight. Adjacent blocks were embedded in paraffin and sectioned into 5 mm slices. Hematoxylin/eosin and Masson's Trichrome staining (to detect scarring and fibrosis) were performed according to standard procedure.
  • HE hematoxylin and eosin
  • MI myocardial infraction
  • sham operation on one-day-old newborn mice were induced.
  • Hearts were harvested at 1, 3, 5 and 7 days after injury and analyzed by flow cytometry.
  • Both AR and MI robustly induced macrophage (F4/80) accumulation in the injured heart (Fig. 1C).
  • the percentage of cardiac macrophages was 0.4%-1.4% of the total number of neonatal heart cells in the first 7 days after birth.
  • macrophages accumulated in the injured neonatal heart 1 to 5 days after AR, and 1 to 3 days after MI.
  • Cardiac macrophages may be categorized by the expression of the chemokine (MCP-1) receptor CCR2 [12].
  • MCP-1 chemokine receptor CCR2
  • a subgroup analysis after AR vs. sham operation revealed that the infiltrate was dominated by CCR2 + macrophages, on the first day after injury (Fig. 2A). As can be seen in Fig. 2A, a sharp rise in the percentage of CCR2 + F4/80 macrophages was observed at day one after AR.
  • Statistical analysis was performed using 2-way-ANOVA, with Holm- Sidack’ s multiple comparison post-test.
  • CCR2 abs 20m1 were injected, 48 and 96 hours after AR, to achieve CCR2 + monocyte inhibition during the first week after injury.
  • CCR2 Abs or saline control were treated with CCR2 Abs or saline control, such that CCR2 Abs (20ml) were injected three times: immediately after AR, 48 and 96 hours after injury.
  • CCR2 Abs 20ml
  • CCR2 Ab treatment significantly depleted F4/80 monocyte/macrophage accumulation in the injured heart and the number of cardiac F4/80 macrophages (Fig. 2B).
  • Mice treated by CCR2 Abs died within a day after the first echocardiography study (day 1).
  • FIG. 2G On day 7 (Fig. 2G) scar thinning and early deposition of collagen were observed. By days 24-25 following AR and CCR2 Ab therapy, an apical scar was observed (Fig. 2H). Analysis was done by H&E staining showing apical scar and collagen deposition by Masson Trichrome staining.
  • the top upregulated genes were glycoprotein non-metastatic melanoma protein B (GPNMB), encoding a transmembrane glycoprotein involve in cell proliferation, and Sppl encoding Osteopontin (OPN), a matricellular protein involved in tissue repair, remodeling, chemotaxis, and immunomodulation [13].
  • GPNMB glycoprotein non-metastatic melanoma protein B
  • OPN Osteopontin
  • Table 1 Representative up-regulated genes 3 days after AR, compared with sham operation. Table 1 (Cont.):
  • Table 1 shows that the up-regulated genes were mostly related to inflammation, particularly macrophage activation, and ECM deposition
  • Table 2 Gene Expression in Neonatal Hearts after MI Compared with Sham- Operated Hearts.
  • up-regulated genes were mostly related to inflammation and ECM deposition where as down-regulated genes mostly involved cell maintenance.
  • Macrophages were collected from neonatal hearts following AR, MI, or sham-operation, 3 days after surgery.
  • macrophages were isolated from adult hearts, 3 days after MI.
  • the rational of this specific time was based on macrophage kinetic studies in the present ( Figure 1C) and previous studies [3]. Quantibody array was used (RayBiotech Inc.
  • cytokine secretion characteristics of 18 known cytokines Interleukin (IL)-lb, IL-4, IL-6, IL-10, IL-12 p40/p70, IL-17, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF) -a, Stromal cell-derived factor (SDF) -1, IL -13, Vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and OPN.
  • IL Interleukin
  • IL-4 Interleukin
  • IL-6 interleukin-10
  • IL-12 p40/p70 interleukin-17
  • MCP monocyte chemoattractant protein
  • TNF tumor necrosis factor
  • SDF Stromal cell-derived factor
  • VEGF Vascular endothelial growth factor
  • IGF insulin-like growth factor
  • HGF hepatocyte growth factor
  • cytokine secretion profiles were observed (normalized to macrophage numbers) (Figs. 3A-3E).
  • IL-6 a pleiotropic cytokine involved in both inflammation and regeneration, was increased after injury, particularly after MI in the neonatal heart, compared with sham-operated neonatal hearts (Fig. 3A).
  • the amount of secreted IL-6 was greater in neonatal than adult macrophages, after myocardial injury.
  • Macrophage secretion of IL-10, an anti-inflammatory, pro-fibrotic cytokine, and IL-13, an anti-inflammatory, regenerative cytokine were lower, 3 days after cardiac injury, compared with sham operation in neonatal hearts (Figs. 3B and 3C).
  • Macrophage secretion of VEGF was the highest in adult macrophages (Fig. 3D), 3 days after MI. Surprisingly, neonatal macrophages secreted low amounts of VEGF (normalized to macrophage number) after AR, MI, and sham operation. This finding suggests that the reparative effects of neonatal macrophages are independent of VEGF.
  • OPN was robustly up regulated after myocardial injury in the neonatal hearts (Tables 1 and 2).
  • OPN is a component of the ECM with immunomodulatory, reparative properties. 22
  • neonatal macrophages secreted higher amounts of OPN after MI, than after AR or adult macrophages after MI (Fig.3E).
  • Fig.3E the data suggested that macrophage secretion of IL-6 and OPN were significantly increased after myocardial injury, particularly in neonatal hearts.
  • OPN may mediate the reparative and regenerative effects of neonatal macrophages.
  • Figs. 4A-4F are microscopic images and Figs. 4B, 4D and 4F are fluorescence images after DAPI staining, with the arrows in these figures indicating cells budding from the heat.
  • Myocardial regeneration in the neonatal heart involves proliferation of resident cardiomyocytes [4].
  • OPN can stimulate cardiomyocyte proliferation
  • 1 -day-old neonatal cardiomyocytes were subjected to 800ng/ml of OPN.
  • Staining for phosphohistone (pH)3, a marker of nuclei division showed a higher expression of pH3 in one-day-old cardiomyocyte and non-cardiomyocyte cells treated with OPN, compared with controls (Figs. 4H-4K).
  • Figs. 4J and 4K are quantitative analysis showing the number of PH 3 /DAPI positive cells in neonatal cardiomyocyte (Fig.4J) and non-cardiomyocyte cells (Fig.4K), numbers were normalized to untreated cells. The results show that OPN increased the number pf pH3 positive cardiac cells, statistical analysis was performed using unpaired t test.
  • CD44 receptor is known as one of the receptors to which OPN binds.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • colorimetric assay to assess the number of cultured cardiomyocytes in low-glucose medium. While the number of un-treated cells significantly decreased, OPN preserved the cardiomyocyte count in a low glucose medium for 24 hours (Fig. 4N). Notably, antibodies that block the OPN receptor CD44 eliminate the protective effect of OPN on the number of cardiomyocyte (Fig. 4N).
  • the Hippo signaling pathway is an evolutionarily ancient cascade of kinases and proteins that control cell proliferation and organ size [14]. Inactivation of the Hippo pathway or activation of its downstream effector, the Yes- Associated Protein (YAP) transcription co-activator, stimulates myocardial regeneration [15].
  • YAP Yes- Associated Protein
  • YAP is transcriptionally active when it is localized to the nucleus. Histological staining confirmed the effects of OPN on YAP nuclear localization. While YAP in untreated cardiomyocytes was located in the cytoplasm (Figs. 5C), OPN treatment translocated YAP into the nuclei (Fig. 5D). Moreover, blocking OPN signaling, by CD44 Abs, prevented the nuclear localization of YAP (Fig. 4E). Together, the data indicate that OPN activates YAP, together with nuclear localization, via CD44.
  • OPN up-regulated the expression of the SPP-1 (OPN gene) (Fig. 5F) and one of the OPN receptors, CD44 receptor (Fig. 5G), as well as cell cycle genes including the transcriptional enhancer factor TEF-1 (TEAD1) (Fig. 5H), which interacts with YAP1, connective tissue growth factor (CTGF) (Fig. 51) and cyclin-dependent kinase 1 (CDK1) (Fig. 5J), which are downstream targets of YAP.
  • TEF-1 transcriptional enhancer factor TEF-1
  • CTGF connective tissue growth factor
  • CDK1 cyclin-dependent kinase 1
  • OPN significantly up- regulated cyclin B1 (Fig. 5K), a regulator of the mitotic (M) phase, and CCND1, (Fig. 5L) which encode cyclin Dl.
  • OPN up-regulated other reparative genes such as MMP-12 (Fig.5M) VEGF (Fig. 5N), and CCL2 (Fig. 50), which regulates macrophage recruitment via monocyte chemoattractant protein 1 (MCP1), and insulin-like growth factor -1 (Fig. 5P).
  • MMP-12 Fig.5M
  • VEGF Fig. 5N
  • CCL2 Fig. 50
  • MCP1 monocyte chemoattractant protein 1
  • Fig. 5P insulin-like growth factor -1
  • Example 8 OPN Stimulated “Wound Healing” in Neonatal Cardiomyocytes and Fibroblasts
  • Angiogenesis is critical element in myocardial regeneration and repair [2]. To determine the effect of OPN on angiogenesis, an EC tube formation assay was used. An incremental dose of OPN stimulated the generation of EC tubes, as assessed by the number of polygons (Fig. 7). Thus, OPN can stimulate angiogenesis.
  • Example 10 OPN Improved LV Remodeling and Function After MI in Adult Mouse
  • OPN therapy prevented diastolic thinning and improved systolic thickening of the anterior wall (infarcted heart segments) (Figs. 8A, B), posterior wall (Figs. 8C, D), reduced LV dilatation (Figs. 8E-J), and improved LV contractility as reflected by fractional shortening FS, fractional area change FAC and LVEF (Figs.
  • OPN significantly increased scar thickness and reduced the length of the scar (Figs. 10A-10F). By thickening the scar, wall stress is reduced (Laplace law) and the degree of outward motion of the infarct that occurs during systole (dyskinesis) is also reduced.
  • Table 4 discloses the list of sequences of the present application.

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Abstract

The present invention relates to an Osteopontin protein or a functional analog thereof, compositions and kits comprising the same and methods for the treatment of non-infectious inflammatory disorders.

Description

METHODS OF TREATING NON-INFECTIOUS INFLAMMATORY
DISORDERS
TECHNOLOGICAL LIELD
The present invention relates to methods of treating non-infectious inflammatory disorders, such as disorders involved with the cardiovascular function.
BACKGROUND ART
References considered to be relevant as background to the presently disclosed subject matter are listed below:
[1] Cui M, Wang Z, Bassel-Duby R and Olson EN. Development. 2018; 145.
[2] Aurora AB, Porrello ER, Tan W, Mahmoud AI, Hill JA, Bassel-Duby R, Sadek HA and Olson EN. J Clin Invest. 2014;124:1382-92.
[3] Ben-Mordechai T, Holbova R, Landa-Rouben N, Harel-Adar T, Feinberg MS, Abd Elrahman I, Blum G, Epstein FH, Silman Z, Cohen S and Leor J. J Am Coll Cardiol. 2013;62:1890-901.
[4] Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, Olson EN and Sadek HA. Science. 2011;331:1078-80.
[5] Palevski D, Levin-Kotler LP, Kain D, Naftali-Shani N, Landa N, Ben-Mordechai T, Konfino T, Holbova R, Molotski N, Rosin- Arbesfeld R, Lang RA and Leor J. J Am Heart Assoc. 2017; 6.
[6] Konfino T, Landa N, Ben-Mordechai T and Leor J. Journal of the American Heart Association. 2015;4.
[7] Itzhaki-Alfia A, Leor J, Raanani E, Sternik L, Spiegelstein D, Netser S, Holbova R, Pevsner-Fischer M, Lavee J and Barbash IM. Circulation. 2009;120:2559-66.
[8] Mack M, Cihak J, Simonis C, Luckow B, Proudfoot AE, Plachy J, Bruhl H, Frink M, Anders HJ, Vielhauer V, Pfirstinger J, Stangassinger M and Schlondorff D. J Immunol. 2001;166:4697-704. [9] Sharon Y, Raz Y, Cohen N, Ben-Shmuel A, Schwartz H, Geiger T and Erez N. Cancer Res. 2015;75:963-973.
[10] Ehler E, Moore-Morris T and Lange S. Journal of visualized experiments: JoVE. 2013.
[11] Liang CC, Park AY and Guan JL. Nature protocols. 2007;2:329-33.
[12] Lavine KJ, Epelman S, Uchida K, Weber KJ, Nichols CG, Schilling JD, Ornitz DM, Randolph GJ and Mann DL. Proc Natl Acad Sci USA. 2014; 111 : 16029-34.
[13] Lok ZSY and Lyle AN. Arterioscler Thromb Vase Biol. 2019;39:613-622.
[14] Zhao B, Tumaneng K and Guan KL. Nat Cell Biol. 2011;13:877-83.
[15] Xin M, Kim Y, Sutherland LB, Murakami M, Qi X, Me Anally J, Porrello ER, Mahmoud AI, Tan W, Shelton JM, Richardson JA, Sadek HA, Bassel-Duby R and Olson EN. Proc Natl Acad Sci USA. 2013;110:13839-44.
[16] Murry CE, Giachelli CM, Schwartz SM and Vracko R .Am J Pathol. 1994; 145: 1450- 62.
Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.
BACKGROUND
The regeneration of cardiomyocytes is an effective way to repair an injured heart, a process that while preset in lower vertebrates, is absent in adult mammals. Studies aimed at regeneration of cardiomyocytes revealed various factors that can promote cardiomyocyte proliferation during cardiac development and also promote cardiomyocyte regeneration after cardiac injury in the adult heart [1].
Macrophages are essential for infarct healing, repair and regeneration in the adult and neonatal heart [2, 3]. The ability of macrophages to penetrate tissue sites, integrate environmental input and transmit regenerative signals, position these cells as central orchestrators of the regenerative response. GENERAL DESCRIPTION
The present disclosure provides in accordance with some aspects, an Osteopontin (OPN) protein or a functional analog thereof for use in the treatment of a non-infectious inflammatory disorder.
The present disclosure provides in accordance with some other aspects, a method for treating a non-infectious inflammatory disorder in a subject, the method comprising administrating a subject in need an amount of an OPN protein or a functional analog thereof, the amount being effective to treat the non-infectious inflammatory disorders.
The present disclosure provides in accordance with some further aspects, a pharmaceutical composition for the treatment of non-infectious inflammatory disorders, the composition comprising as active ingredient an amount of an OPN protein or functional analog thereof, the amount being effective to treat the non-infectious inflammatory disorders.
The present disclosure provides in accordance with yet some further aspects, a kit comprising an OPN protein or functional analog thereof and instructions for use of the OPN protein or functional analog thereof for the treatment of non-infectious inflammatory disorders.
EMBODIMENTS
Some embodiments of this disclosure will now be described in the following numbered paragraph. The following description intends to add on the above general description and not limit it in any manner.
1. An Osteopontin (OPN) protein or a functional analog thereof for use in the treatment of a non-infectious inflammatory disorder.
2. The OPN protein or a functional analog thereof for use of Embodiment No. 1 , wherein said OPN protein is or comprises at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 11. 3. The OPN protein or a functional analog thereof for use of Embodiment No. 1 or 2, wherein said functional analog is at least one of small molecule, an antisense, an siRNA, a polypeptide or a peptide.
4. The OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 3, wherein said functional analog is a polypeptide or a peptide.
5. The OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 4, wherein said functional analog is a derivative or a fragment of the OPN protein.
6. The OPN protein or a functional analog thereof for use any one of Embodiments No. 1 to 5, wherein said functional analog is or comprises an amino acid sequence having at least 75% similarity to at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:11.
7. The OPN protein or a functional analog thereof for use any one of Embodiments No. 1 to 6, wherein said functional analog is or comprises an amino acid sequence that is at least 75% identical to at least one amino acid sequence denoted by SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 11.
8. The OPN protein or a functional analog thereof for use any one of Embodiments 1 to 5, wherein said functional analog is or comprises an amino acid sequence denoted by at least one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21.
9. The OPN protein or a functional analog thereof for use of Embodiment 8, wherein said functional analog is or comprises an amino acid sequence that is at least 75% similar to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21.
10. The OPN protein or a functional analog thereof for use of Embodiment 8, wherein said functional analog is or comprises an amino acid sequence that is at least 75% identical to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21. 11. The OPN protein or a functional analog thereof for use any one of Embodiments 1 to 5, wherein said functional analog is or comprises an amino acid sequence denoted by at least one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
12. The OPN protein or a functional analog thereof for use of Embodiment 11, wherein said functional analog is or comprises an amino acid sequence that is at least 85% similar to at least one amino acid sequence denoted by SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
13. The OPN protein or a functional analog thereof for use of Embodiment 11, wherein said functional analog is or comprises an amino acid sequence that is at least 85% identical to at least one amino acid sequence denoted by SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
14. The OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 13, being an isolated, a purified or a synthetic OPN protein or a functional analog thereof.
15. The OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 14, for use in combination with a pharmaceutical carrier.
16. The OPN protein or a functional analog thereof for use of Embodiment No. 15, said pharmaceutical carrier is a liposome or an exosome.
17. The OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 16, suitable for parental administration or local administration into a target tissue.
18. The OPN protein or a functional analog thereof for use of Embodiment No. 17, said administration comprising at least one of intravenous (i.v) injection, intracoronary (i.c), intramuscular (i.m), intraperitoneal (i.p) injection.
19. The OPN protein or a functional analog thereof for use of Embodiment No. 17, said local administration comprises intramyocardial administration, intracoronary administration, trans coronary administration or retrograde coronary sinus administration. 20. The OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 19, wherein the non-infectious inflammatory disorders is an acute inflammation.
21. The OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 20, wherein the non-infectious inflammatory disorder is involved with a condition selected from the group consisting tissue injury, infarct, trauma, necrosis, sterile inflammatory disorder.
22. The OPN protein or a functional analog thereof for use of any one of Embodiments No. 1 to 20, wherein the non-infectious inflammatory disorder is a cardiovascular disorder.
23. The OPN protein or a functional analog thereof for use of Embodiment No. 22, wherein the cardiovascular disorder is selected from or associated with myocarditis, heart failure, atherosclerosis and myocardial infarction.
24. The OPN protein or a functional analog thereof for use of Embodiment No. 22, wherein said cardiovascular disorder comprises myocardial infarction.
25. A method for treating a non-infectious inflammatory disorder in a subject, the method comprising administrating a subject in need an amount of an OPN protein or a functional analog thereof, the amount being effective to treat the non-infectious inflammatory disorders.
26. The method of Embodiment 25, wherein said OPN protein is or comprises at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:11.
27. The method of Embodiment 25 or 26, wherein said functional analog is at least one of small molecule, an antisense, an siRNA, a polypeptide or a peptide.
28. The method of any one of Embodiments 25 to 27, wherein said functional analog is a polypeptide or a peptide.
29. The method of any one of Embodiments 25 to 28, wherein said functional analog is a derivative or a fragment of the OPN protein.
30. The method of any one of Embodiments 25 to 29, wherein said functional analog is or comprises an amino acid sequence having at least 75% similarity to at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 11.
31. The method of any one of Embodiments 25 to 30, wherein said functional analog is or comprises an amino acid sequence that is at least 75% identical to at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 11.
32. The method of any one of Embodiments 25 to 29, wherein said functional analog is or comprises at least one amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21.
33. The method of Embodiment 32, wherein said functional analog is or comprises an amino acid sequence that is at least 75% similar to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.
34. The method of Embodiment 32, wherein said functional analog is or comprises an amino acid sequence that is at least 75% identical to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.
35. The method of Embodiments 25 to 29, wherein said functional analog is or comprises an amino acid sequence denoted by at least one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
36. The method of Embodiment 35, wherein said functional analog is or comprises an amino acid sequence that is at least 85% similar to at least one amino acid sequence denoted by SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
37. The method of Embodiment 35, wherein said functional analog is or comprises an amino acid sequence that is at least 85% identical to at least one amino acid sequence denoted by SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27. 38. The method of any one of Embodiments 25 to 37, wherein said OPN protein or a functional analog thereof being an isolated, a purified or a synthetic OPN protein or a functional analog thereof.
39. The method of Embodiment 38, comprising administration of said OPN or functional analog thereof with a pharmaceutical carrier.
40. The method of Embodiment 38, said pharmaceutical carrier is a liposome or an exosome.
41. The method of any one of Embodiments 25 to 40, comprising administration of said OPN or functional analog thereof by parental administration or local administration into a target tissue.
42. The method of Embodiment 41, said administration comprising at least one of intravenous (i.v) injection, intracoronary (i.c), intramuscular (i.m), intraperitoneal (i.p) injection.
43. The method of Embodiment 41, said local administration comprises intramyocardial administration, intracoronary administration, trans coronary administration or retrograde coronary sinus administration.
44. The method of any one of Embodiments 25 to 43, wherein the non-infectious inflammatory disorders is an acute inflammation.
45. The method of any one of Embodiments 25 to 44, wherein the non-infectious inflammatory disorder is involved with a condition selected from the group consisting tissue injury, infarct, trauma, necrosis, sterile inflammatory disorder.
46. The method of Embodiment 45, wherein the non-infectious inflammatory disorder is a cardiovascular disorder.
47. The method of any one of Embodiments 25 to 46, wherein the non-infectious inflammatory disorders is selected from or associated with myocarditis, heart failure, atherosclerosis and myocardial infarction.
48. The method of Embodiment 47, wherein the non-infectious inflammatory disorder comprises myocardial infarction. 49. A CD44 receptor agonist for use in the treatment of a non-infectious inflammatory disorder.
50. The CD44 receptor agonist for use of Embodiment No. 49, being is at least one of small molecule, an antisense, an siRNA, a polypeptide or a peptide.
51. The CD44 receptor agonist for use of any one of Embodiment No. 49 or 50, being a polypeptide or a peptide.
52. The CD44 receptor agonist for use of any one of Embodiments No. 49 to 51 , being an OPN protein or a functional analog thereof.
53. The CD44 receptor agonist for use of Embodiment No. 52, wherein said OPN protein or functional analog thereof are as defined in any one of Embodiments 2 to 14.
54. The CD44 receptor agonist for use of any one of Embodiments No. 49 to 53, for use in combination with a pharmaceutical carrier.
55. The CD44 receptor agonist for use of Embodiment No. 54, the pharmaceutical carrier is a liposome or an exosome.
56. The CD44 receptor agonist for use of any one of Embodiments No. 49 to 55, suitable for parental administration or local administration into a target tissue.
57. The CD44 receptor agonist for use of Embodiment No. 56, said administration comprising at least one of intravenous (i.v) injection, intracoronary (i.c), intramuscular (i.m), intraperitoneal (i.p) injection.
58. The CD44 receptor agonist for use of Embodiment No. 56 said local administration comprises intramyocardial administration, intracoronary administration, trans coronary administration or retrograde coronary sinus administration.
59. The CD44 receptor agonist for use of any one of Embodiments No. 49 to 58, wherein the non-infectious inflammatory disorders is an acute inflammation.
60. The CD44 receptor agonist for use of any one of Embodiments No. 49 to 59, wherein the non-infectious inflammatory disorder is involved with a condition selected from the group consisting tissue injury, infarct, trauma, necrosis, sterile inflammatory disorder. 61. The CD44 receptor agonist for use of any one of Embodiments No. 49 to 60, wherein the non-infectious inflammatory disorder is a cardiovascular disorder.
62. The CD44 receptor agonist for use of Embodiment No. 61, wherein the cardiovascular disorder is selected from or associated with myocarditis, heart failure, atherosclerosis and myocardial infarction.
63. The CD44 receptor agonist for use of Embodiment No. 61, wherein said cardiovascular disorder comprises myocardial infarction.
64. A method for treating a non-infectious inflammatory disorder in a subject, the method comprising administrating a subject in need an amount of a CD44 receptor agonist, the amount being effective to treat the non-infectious inflammatory disorders.
65. The method of Embodiment 64, wherein said CD44 receptor agonist is as defined in any one of Embodiments 49 to 53.
66. The method of Embodiment 64 or 65, comprising administration of said CD44 receptor agonist with a pharmaceutical carrier.
67. The method of Embodiment 66, said pharmaceutical carrier is a liposome or an exosome.
68. The method of any one of Embodiments 65 to 67, comprising administration of said CD44 receptor agonist by parental administration or local administration into a target tissue.
69. The method of Embodiment 68, said administration comprising at least one of intravenous (i.v) injection, intracoronary (i.c), intramuscular (i.m), intraperitoneal (i.p) injection.
70. The method of Embodiment 68, said local administration comprises intramyocardial administration, intracoronary administration, trans coronary administration or retrograde coronary sinus administration.
71. The method of any one of Embodiments 64 to 70, wherein the non-infectious inflammatory disorders is an acute inflammation. 72. The method of any one of Embodiments 64 to 71, wherein the non-infections inflammatory disorder is involved with a condition selected from the group consisting tissue injury, infarct, trauma, necrosis, sterile inflammatory disorder.
73. The method of Embodiment 72, wherein the non-infectious inflammatory disorder is a cardiovascular disorder.
74. The method of any one of Embodiments 64 to 73, wherein the non-infectious inflammatory disorders is selected from or associated with myocarditis, heart failure, atherosclerosis and myocardial infarction.
75. The method of Embodiment 74, wherein the non-infectious inflammatory disorder comprises myocardial infarction.
76. A pharmaceutical composition for the treatment of non-infectious inflammatory disorders, the composition comprising as active ingredient an amount of an OPN protein or functional analog thereof as defined in any one of Embodiments 1 to 21, the amount being effective to treat the non-infectious inflammatory disorders.
77. A pharmaceutical composition for the treatment of non-infectious inflammatory disorders, the composition comprising as active ingredient an amount of at least one CD44 receptor agonist as defined in any one of Embodiments 49 to 63, the amount being effective to treat the non-infectious inflammatory disorders.
78. A kit comprising an OPN protein or functional analog thereof as defined in any one of Embodiments 1 to 24 and instructions for use of the OPN protein or functional analog thereof for the treatment of non-infectious inflammatory disorders.
79. A kit comprising at least one CD44 receptor agonist as defined in any one of Embodiments 49 to 63 and instructions for use of at least one CD44 receptor agonist for the treatment of non-infectious inflammatory disorders.
BRIEF DESCRIPTION OF THE DRAWINGS
In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: Figures 1A-1C: Macrophages accumulated at the site of Apex Resected (AR) in neonatal hearts,
Figs. 1A and IB are images taken three days after AR, Fig. 1A - white circle shows macrophages (MF) accumulated in the injured area, LV= left ventricular, scale bar: 500 mm; Fig. IB - higher magnification of image of Fig. 1A, showing macrophages (white circle) accumulated at the border zone and AR site, but not in remote areas, scale bar: 200 mm; Fig. 1C is a bar graph showing flow cytometry analysis of hearts harvested from neonatal hearts after AR, myocardial infraction (MI) or sham operation, at different time points, the Y-axis shows analysis of F4/80 positive cells that classify monocytes/macrophages .
Figures 2A-2I: CCR2+ monocytes are essential for neonatal heart regeneration,
Fig. 2A is a bar graph showing flow cytometry analysis of CCR2+ F4/80 macrophages accumulation at day 1 and day 3 after AR.; Fig.2B is a bar graph showing flow cytometry analysis of processed hearts, 3 days after AR; Fig. 2C is a graph showing the 28 days mortality rate after AR and CCR2 inhibition vs. AR only, the differences between groups were calculated by Fischer’s exact test; Figs.2D and 2E are graphs showing transthoracic echocardiography of LV fractional shortening (FS) at one day after AR (Fig. 2D) and 24 days after AR (Fig. 2E), the differences between groups were calculated by non- parametric Mann- Whitney test; Fig. 2F is an image showing granulation tissue formation after AR and CCR2 antibody treatment, black circle show macrophage depletion, scale bar: 100 mm; Fig. 2G is an image showing thinning of the resection area and impaired healing at day 7 after AR and CCR2+ monocyte/macrophages inhibition are shown by the black circle), scale bar: 200 mm; Fig. 2H is an image obtained at day 24 following AR and CCR2 monocyte/macrophage inhibition (black circle), scale bar: 500 mm; Fig. 21 is an image showing complete apical regeneration at day 21 following AR, without CCR2 inhibition, scale bar: 500 mm.
Figures 3A-3E: The effect of cardiac injury on cytokine secretion
Figs. 3A to 3E are bar graphs showing representative cytokine secretion from macrophages, 3 days after cardiac injury in neonatal hearts after AR, MI or sham operation and adult hearts after MI, Fig. 3A -interleukin 6 (IL6), Fig. 3B - IL10, Fig. 3C -IL13, Fig. 3D - Vascular endothelial growth factor (VEGF) and Fig. 3E - Osteopontin (OPN), hearts that were harvested 3 days after the procedure, isolated macrophages were cultured for 24h and conditioned media was analyzed by Qu antibody array, statistical analysis was performed using one-way ANOVA, with Tukey’s multiple comparison post- test. Differences were considered significant at p<0.05.
Figures 4A-40: OPN increase neonatal tissue outgrowth via cardiomyocyte and non-cardiomyocyte proliferation
Figs. 4A to 4F are representative images of organ culture, taken from neonatal mouse heart, treated with 0 (Figs. 4A and 4B), 400ng/ml of OPN (Figs. 4C and 4D) or 800ng/ml of OPN (Figs. 4E and 4F) for 72 hours, the arrows pointing to cells that were seen budding from the heart; Figs. 4B, 4D and 4F are imaging after DAPI staining; Fig. 4G is a bar graph showing quantification of the outgrowth, statistical analysis was performed using Kruskal-Wallis test, with Dunn’s multiple comparisons post-test; Fig. 4H and 41 are images showing mouse neonatal cardiomyocytes that were treated with 800ng/ml OPN for 24h (Fig. 41) or without (Fig. 4H) , arrows indicating the presence of cardiomyocytes (using pH3); Fig. 4J and 4K are bar graphs showing the number of PH3/DAPI positive cells in neonatal cardiomyocyte (Fig. 4J) and non-cardiomyocyte cells (Fig. 4K), numbers were normalized to untreated cells, statistical analysis was performed using unpaired t test; Fig. 4L is an image of hears stained for the OPN receptor CD44 (white arrow); Fig. 4M is a bar graph showing the effect of OPN on cell death in neonatal cardiomyocytes, statistical analysis was performed using unpaired t test, Fig. 4N and 40 are bar graphs showing the viability of mouse neonatal cardiomyocytes treated with or without 800ng/ml OPN for 24h was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Fig. 4N) and RealTime-Glo™ MT Cell Viability Assay (Fig. 40); Statistical analysis was performed using two-way repeated measures ANOVA and Holm-Sidack’s multiple comparison post-test.
Figures 5A-5P: OPN stimulated YAP signaling in neonatal cardiomyocytes via CD44.
Fig. 5A is an exemplary western blot gel using antibodies against YAP1, pYAPl-127, LATS1/2, pLATS 1/2, ERK and pERK; Fig. 5B is a bar graph showing the western blot results after calculating the ratio between phosphorylated and unphosphorylated proteins; Figs. 5C to 5E are fluorescent images of cells showing YAP translocation from the cytoplasm into the nucleus following OPN treatment; YAP stating is shown in white arrows, Fig. 5C is control showing YAP (white arrows) was located to the cytoplasm; Fig. 5D cells treated with 800ng OPN showing YAP (white arrows) was translocated into the nucleus; Fig. 5E cells treated with 800ng OPN and with an CD44 inhibitor, showing that the YAP (white arrows) translocation into the nucleus was prevented; Fig. 5F to 5P are bar graphs showing gene expression obtained by real time PCR AACT using GAPDH as an endogenous control, gene expression analysis, using real time PCR was performed in neonatal cardiomyocytes treated with or without 800ng/ml OPN after 24h, Fig. 5F showing the expression of the SPP-1 (OPN gene) that was up-regulated by OPN; Fig. 5G showing the expression of one of the OPN receptors CD44; Fig. 5H showing the expression of cell cycle genes including the transcriptional enhancer factor TEF-1 (TEAD1) which interacts with YAP1; Fig. 51 showing the expression of the connective tissue growth factor (CTGF); Fig. 5J showing the expression of the cyclin-dependent kinase 1 (CDK1) which are YAP downstream targets; Fig. 5K showing the expression of the cyclin B1 (CCNB1) regulator of the mitotic (M phase), that is upregulated by OPN; Fig. 5L showing the expression of cyclin D1 (CCND1), which encoded cyclin Dl; Fig. 5M showing the expression of matrix metalloproteinase (MMP)-12; Fig. 5N showing the expression of VEGF; Fig. 50 showing the expression of chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemoattractant protein 1 (MCP1) which regulates macrophage recruitment; Fig. 5P showing the expression of insulin-like growth factor -1 (IGF-1); Statistical analysis was performed using unpaired t test.
Figures 6A-6B: OPN improved wound healing and angiogenesis.
Fig. 6A is a bar graph showing the effect of OPN on gap closure in neonatal cardiomyocytes; data shows that OPN accelerated the gap closure in neonatal cardiomyocytes compared with control untreated cardiomyocytes and the addition of CD44 Abs to the culture medium eliminated the favourable effects of OPN, suggesting that the effects of OPN on cardiomyocytes are mediated by CD44; Fig. 6B is a bar graph showing the effect of OPN on gap closure in neonatal cardiac fibroblasts that; data shows that OPN stimulate proliferation and migration of fibroblasts. Figure 7: OPN improved angiogenesis
Fig. 7 is a bar graph showing the effect of OPN on number of polygons in human umbilical vein endothelial cells (HUVEC); development of tube formation was monitored by confocal microscopy.; statistical analysis was performed using two-way ANOVA and Holm-Sidak's multiple comparison test.
Figures 8A-8M: Single injection of OPN into infarcted heart decreased LV remodeling and improved cardiac function 28 days after MI.
Figs. 8A to 8M are bar graphs showing echocardiography parameters; Fig. 8 A shows anterior wall (AW) diastole (dia); Fig. 8B shows anterior wall (AW) systole (sys); Fig. 8C shows Posterior Wall (PW) diastole (dia); Fig. 8D shows Posterior Wall (PW) systole (sys); Fig. 8E shows Left Ventricular End Diastolic Diameter (LVEDD); Fig. 8F shows Left Ventricular End Systolic Diameter (LVESD); Fig. 8G shows Left Ventricular End Diastolic (LVED) area; Fig. 8H shows Left Ventricular End Systolic (LVES); Fig. 81 shows Left Ventricular Volume Diastolic (LV Vol;d); Fig. 8J shows Left Ventricular Volume Systolic (LV Vol;s); Fig. 8K shows % Fractional Shortening (FS); Fig. 8L shows Endocardial Fractional Area Change (FAC); Fig. 8M shows % Ejection Fraction (EF), statistical analysis was performed using two-way ANOVA and Holm's-Sidak’s multiple comparison test.
Figures 9A-9P: Single injection of OPN into the infarcted heart improved regional cardiac function.
Fig. 9A is a representation of mapping LV segments in a short axis view; Figs. 9B to 9H are graphs showing the effect of OPN on regional and global radial strain of the infarcted as well as the non-infarcted segments; Fig. 9B is anterior septum; Fig. 9C is anterior free segment; Fig. 9D is posterior septal segment; Fig. 9E is interior free segment; Fig. 9F is posterior segment; Fig. 9G is lateral segment; Fig. 9H is average curve-short axis; Fig. 91 is a representation of mapping LV segments in a long axis view; Fig. 9J is anterior apex; Fig. 9K is anterior mid; Fig. 9L is anterior base; Fig. 9M is posterior apex; Fig. 9N is posterior mid; Fig. 90 is posterior base; Fig. 9P is average curve-long axis.
Figures 10A to 10F : OPN increased scar thickness
Figs. 10A to 10F are representative pictures of mice hearts, with OPN treatment (Figs. 10A, 10C and 10E) or as control (Figs. 10B, 10D and 10F), 28-days after MI; Hearts were stained for Piero Sirius Red staining for collagen shown in black circle, data shows that OPN increased scar thickening and reduced scar length.
DETAILED DESCRIPTION OF EMBODIMENTS
The present invention is based on the findings that an Osteopontin protein can be used for the treatment of non-infectious inflammatory disorders, specifically, non-infectious inflammatory disorders associated with cardiovascular conditions, such as myocardial infraction.
It was surprisingly found by the inventors that administration of Osteopontin protein induced proliferation of cardiomyocyte, increased cardiac tissue growth and induced angiogenesis as well as increased scar thickness and overall significantly improved cardiac function and.
Specifically, and as shown in the Examples below, for example in Example 3, an up-regulated expression of Sppl gene that encodes the Osteopontin protein was observed in the neonatal heart after AR and MI but not in sham operation. In addition, as shown in Example 4, neonatal macrophages secreted higher amounts of Osteopontin protein after MI and after AR. Further, as shown in Examples 5, 7 and 8, Osteopontin protein improved the cardiac conditions and function.
As the human heart has a very limited capacity to regenerate the heart muscle cells, and any major muscle damage may lead to heart failure, these surprising findings of the inventors are highly valuable as they can lead to the development of new reparative therapeutic strategies for non-infectious inflammatory disorders, and specifically those which are associated with cardiovascular disorders.
Thus, the present disclosure provides, in accordance with its first aspect, an Osteopontin (OPN) protein or a functional analogue thereof for use in the treatment of a non-infectious inflammatory disorder.
In the following text, when referring to the OPN protein or a functional analogue thereof it is to be understood as also referring to the compositions, kits and methods disclosed herein. Thus, whenever providing a feature with reference to the OPN protein or a functional analogue thereof, it is to be understood as defining the same feature with respect to the compositions, kits or methods, mutatis mutandis. As used herein Osteopontin (OPN) denoted as bone sialoprotein I (BSP-1 or BNSP), early T-lymphocyte activation (ETA-1), secreted phosphoprotein 1 (SPP1), 2ar and Rickettsia resistance (Ric), refers to a secreted phosphorylated extracellular matrix (ECM) protein. The activity of OPN protein was suggested to be mediated through various receptors, including for example a CD44 receptor as well as a variety of integrin receptors, such as avb1, avb3, avb5, a5b1, a8b1, a4b1, a9b1, or a9b4.
The OPN protein in accordance with the present discolored encompasses OPN protein isoforms and specifically human isoforms of the OPN protein.
The term “ isoform ” refers to two or more proteins, typically similar proteins, that originate from a single gene or gene family and are the result of genetic differences. The isoforms can be encoded by different genes or by RNA transcripts from the same gene which have had different exons removed. A set of protein isoforms may be formed from alternative splicing, variable promoter usage, or other post-transcriptional modifications of a single gene. For example, through RNA splicing mechanisms, mRNA has the ability to select different protein-coding segments (exons) of a gene, or even different parts of exons from RNA to form different mRNA sequences such that each unique sequence produces a specific form of a protein. The protein isoforms may have same function. OPN isoforms may be, for example, transcriptionally spliced OPN isoforms, multiple post- translational modifications (phosphorylation, glycosylation and sulfation) isoforms and multiple functionally-active proteolytic forms cleaved by various proteins such as thrombin, MMPs, plasmin and cathepsin DOPN protein. At times, OPN isoforms are denoted as isoform A, isoform B, isoform C, isoform D, OPN5, isoform 4 or isoform 5.
In some embodiments, the OPN protein is or comprises at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9 or SEQ ID NO: 11.
In some embodiments, the OPN protein consists of at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9 or SEQ ID NO: 11.
In some embodiments, human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 314 amino acid residues as denoted by Accession Number NP_001035147.1. In some other embodiments, human OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO:1. In some other embodiments, human OPN protein provided by Accession Number NP_001035147.1 is encoded by a nucleic acid sequence denoted by Accession Number NM_001040058.2. In some embodiments, the OPN gene comprises a nucleic acid sequence as denoted by SEQ ID NO:2. The gene encoding the OPN protein is denoted at times as SPP1. The OPN protein provided by SEQ ID NO:1 is at times denoted as OPN isoform A.
In some embodiments, human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 300 amino acid residues as denoted by Accession Number NP_000573.1. In some embodiments, human OPN protein refers to an amino acid sequence of 300 amino acid residues as denoted by number UniProt No. P10451-5. In some other embodiments, human OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO:3 In some other embodiments, human OPN protein denoted by Accession Number NP_000573.1 is encoded by a nucleic acid sequence denoted by Accession Number NM_000582.2. In some other embodiments, human OPN protein is encoded by a nucleic acid sequence provided herein below by SEQ ID NO:4. The OPN protein provided by SEQ ID NO:3 is at times denoted as OPN isoform B or isoform 5.
In some embodiments, human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 287 amino acid residues as denoted by Accession Number NP_001035149.1. In some other embodiments, human OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO:5. In some other embodiments, human OPN protein provided by Accession Number NP_001035149.1 is encoded by a nucleic acid sequence denoted by Accession Number NM_001040060.1. In some other embodiments, human OPN protein is encoded by a nucleic acid sequence provided herein below by SEQ ID NO:6. The OPN protein provided by SEQ ID NO:5 is at times denoted as OPN isoform C.
In some embodiments, human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 292 amino acid residues as denoted by UniProt No. P10451-4. In some other embodiments, human OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO:7 The OPN protein provided by SEQ ID NO:7 is at times denoted as OPN isoform D. In some embodiments, human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 300 amino acid residues as denoted by UniProt No. P10451-2. In some specific embodiments, the OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO:8 The OPN protein provided by SEQ ID NO:8 is at times denoted as OPN isoform B.
In some embodiments, human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 273 amino acid residues as denoted by Accession Number NP_001238758.1. In some other embodiments, human OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO:9. In some other embodiments, human OPN protein denoted by Accession Number NP_001238758.1is encoded by a nucleic acid sequence denoted by Accession Number NM_001251829.1. In some other embodiments, human OPN protein is encoded by a nucleic acid sequence provided herein below by SEQ ID NO: 10. The OPN protein provided by SEQ ID NO:9 is at times denoted as OPN isoform 4.
In some embodiments, human OPN protein refers to a protein (polypeptide chain) having an amino acid sequence of 327 amino acid residues as denoted by Accession Number NP_001238759.1. In some other embodiments, human OPN protein consists or comprises the amino acid sequence as denoted by SEQ ID NO: 11. In some other embodiments, human OPN protein denoted by Accession Number NP_001238759.1is encoded by a nucleic acid sequence denoted by Accession Number NM_001251830.1. In some other embodiments, human OPN protein is encoded by a nucleic acid sequence provided herein below by SEQ ID NO: 12. The OPN protein provided by SEQ ID NO: 11 is at times denoted as OPN isoform 5 or variant 6.
The present disclosure also encompasses a functional analog of OPN.
The term “functional analogue ” thereof refers to a chemical/biological compound (entity) that has or may produce similar physical, chemical, biochemical, or pharmacological properties as a reference compound. In the context of the present disclosure, the functional analogue has or can produce the same activity, e.g. biological, cellular or therapeutic activity of OPN protein or at least one of the OPN protein isoforms.
The functional analogue in accordance with the present disclosure is or comprises at least one of a small molecule, an antisense, an siRNA, a polypeptide or a peptide. In accordance with some embodiments, the functional analogue is capable of treating a non- infectious inflammatory disorder as described herein.
In accordance with some examples, the functional analogue has a similar mechanism of action as OPN protein. For example, the functional analogue may be selected such that it binds to a similar receptor(a) or activate a similar receptor(s) as the OPN protein to initiate a physiological/biological/cellular/biochemical response.
As shown in the Examples below and specifically in Examples 6 and 8, the effect of OPN was reduced or eliminated after addition of CD44 receptor inhibitor (CD44 antibody). Without being bound by theory, it was suggested by the inventors that the effect of the OPN protein may be mediated, at least in part, via a CD44 receptor. This, in some examples, the functional analogue may be selected such that it binds to CD44 receptor. The CD44 receptor refers to a multistructural and multifunctional cell surface molecule involved in cell proliferation, cell differentiation, cell migration, angiogenesis, presentation of cytokines, chemokines, and growth factors to the corresponding receptors, and docking of proteases at the cell membrane, as well as in signaling for cell survival.
In some embodiments, the functional analogue is an agonist of CD44 receptor. In some embodiments, the functional analogue is an agonist of at least one integrin receptor. The functional analog may be at least one of a full agonist, a co-agonist, a selective agonist, an inverse agonist.
The functional analogue as described herein may be at least one of a small molecule, an antisense, an siRNA, a polypeptide or a peptide.
In some embodiments, the functional analogue is a structural analog of the OPN protein. A structural analog as used herein refers to a functional analog that has a similar structure as a reference compound. In the context of the present invention, a structural analog having a similar structure as of OPN protein is characterized by comprising an amino acid sequence. In accordance with some embodiments, the functional analog is a polypeptide or a peptide.
The term “ polypeptide ” or “ peptide ” as used herein refers to a chain of amino acid residues connected by peptide bonds. A peptide typically includes one or more amino acids, and in some embodiments between 2 and 50 amino acids, between 2 and 20 amino acids, between 2 to 10 amino acid. A polypeptide typically refers to an amino acid sequence that do not fold to a specific structure as a protein. In the context of the present disclosure, the term polypeptide is interchangeably used with the term protein and both are used define an amino acid sequence. The amino acid sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group.
The peptides or polypeptides of the present disclosure can be modified at one or more of its amino acid residues, for example by manosylation, glycosylation, amidation (for example C-terminal amides), carboxylation or phosphorylation.
The OPN protein or functional analog thereof may be an isolated protein or functional analog thereof, a purified protein or functional analog thereof or a synthesized protein or functional analog thereof.
The term "isolated" refers to molecules, such as the amino acid sequences described herein, peptides or polypeptides that are removed from their natural environment, isolated, or separated.
The synthesized protein or functional analog thereof may be produced through genetic engineering methods, expression in a host cell, or through any other suitable means as known in the art. Methods for producing peptides or polypeptides are well known in the art.
In some embodiments, the functional analog encompasses derivatives of OPN protein or the OPN protein isoforms. The term "derivative" is used to define an amino acid sequence (e.g. peptide or polypeptide), that is different in at least one amino acid residue from the amino acid sequence of OPN protein or the OPN protein isoforms. Such modification includes insertions, deletions, substitutions and combination thereof. As noted herein, the modifications do not alter the activity of the OPN protein and any derivative in accordance with the present disclosure is capable of treating a non-infectious inflammatory disorder as described herein. Such derivatives may be selected such that they maintain or even improve (i.e. do not reduce) the activity of the OPN protein. In some examples, a derivative of OPN protein may be a result of alternative splicing or polymorphism of the encoding gene. In some embodiments, the functional analog comprises a derivative of at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 11.
In some embodiments, the functional analog is or comprises an amino acid sequence having at least 50%, at times having at least 60%, at times having at least 70%, at times having at least 75%, at times having at least 80%, at times having at least 85%, at times having at least 90%, at times having at least 95%, at times having at least 97%, at times having at least 98% similarity to an amino acid sequence denoted by at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO: 11.
Sequence similarity also denoted as sequence homology as used herein refers to the amount (%) of amino acids that are conserved with similar physicochemical properties (e.g. leucine and isoleucine), over a specified region, when compared and aligned for maximum correspondence over a comparison window.
In some embodiments, the functional analog is or comprises an amino acid sequence having at least 60%, at times having at least 70%, at times having at least 75%, at times having at least 80%, at times having at least 85%, at times having at least 90%, at times having at least 95%, at times having at least 97%, at times having at least 98% identity to an amino acid sequence denoted by at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 11.
Sequence identity as used herein refers to the amount (%) of amino acids that the same (identical) over a specified region, when compared and aligned for maximum correspondence over a comparison window. In determining the sequence identity, it is possible to not count the gaps such that the sequence identity is relative to the shorter sequence of the two compared sequences.
As indicated herein, the OPN protein derivative may include any modification including insertions, deletions, substitutions and combination thereof. As also indicted herein, such modifications may be a result of alternative splicing or polymorphism of a gene encoding an OPN protein (including all isoforms). Alternatively or additionally, such modifications may be obtained by synthetic methods. It should be appreciated that by the terms "insertions" or "deletions", as used herein it is meant any addition or deletion, respectively, of amino acid residues to the polypeptides or peptides being derivatives or fragments used by the invention, of between 1 to 50 amino acid residues, between 1 to 20 amino acid residues, between 1 to 10 amino acid residues or between 1 to 5 amino acid residues. For example, insertions or deletions may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. It should be noted that the insertions or deletions encompassed by the invention may occure in any position of the modified peptide, as well as in any of the N' or C termini thereof.
As known in the art, amino acid “ substitutions ” are the result of replacing one amino acid with another amino acid, for example with another amino acid that has similar structural and/or chemical properties (conservative amino acid replacements). This may be being due to point mutation in DNA sequence caused by nonsynonymous missense mutation which alters the codon sequence to code other amino acid instead of the references. Alternatively, amino acid substitution may be a results of analysis and/or rationale replacement.
In some embodiments, the amino acid substitutions may be conservative replacement. Such a replacement encompasses a change in an amino acid into another amino acid exhibiting similar properties. Conservative amino acid replacements (also denoted as conservative amino acid “substitutions” or conservative amino acid mutations) is an amino acid replacement in a protein that changes a given amino acid to a different amino acid with similar biochemical, structural and/or chemical properties as described herein. For example, amino acids may be sorted into six main classes on the basis of their structure and the general chemical characteristics of their side chains (R groups): Aliphatic: Isoleucine (I), Leucine (L), Glycine (G), Alanine (A), Valine (V); Hydroxyl or sulfur/selenium-containing: Serine (S), Cysteine (C) , Threonine (T) , Methionine (M); Cyclic: Proline (P); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Basic: Histidine (H), Lysine (K), Arginine (R); Acidic and their amides: Aspartate (D), Glutamate (E), Asparagine (N), Glutamine (Q).
In alternative or additional examples, each of the following groups contains other exemplary amino acids that are conservative substitutions for one another: 1) Very small: Alanine (A), Glycine (G); 2) Negative charge: Aspartic acid (D), Glutamic acid (E); 3) Polar (amidated carboxyl side chain): Asparagine (N), Glutamine (Q); 4) Positively charged: Arginine (R), Lysine (K); 6) Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W), and occasionally also Histidine (H); 7) Small polar: Serine (S), Threonine (T); 8) Sulfur-containing: Cysteine (C), Methionine (M); 9) Small: Ala (A), Glycine (G), Serine (S); 10) Beta-branched: Valine (V), Isoleucine (I) and occasionally also Threonine (T); 11) Polar: Asparagine (N), Glutamine (Q), Serine (S), Threonine (T);
In alternative or additional examples, a conservation analysis is based on the following, nonpolar “hydrophobic” amino acids are selected from the group consisting of Valine (V), Isoleucine (I), Leucine (L), Methionine (M), Phenylalanine (F), Tryptophan (W), Cysteine (C), Alanine (A), Tyrosine (Y), Histidine (H), Threonine (T), Serine (S), Proline (P), Glycine (G), Arginine (R) and Lysine (K); “polar” amino acids are selected from the group consisting of Arginine (R), Lysine (K), Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q); “positively charged” amino acids are selected form the group consisting of Arginine (R), Lysine (K) and Histidine (H) and “acidic” amino acids are selected from the group consisting of Aspartic acid (D), Asparagine (N), Glutamic acid (E) and Glutamine (Q).
In some embodiments, the replacement is a radical replacement. A radical replacement (substitution) is an exchange of an amino acid into another amino acid with different properties.
Amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
It should be noted that the term amino acid residue as used herein encompasses both naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g-carboxyglutamate, and O-phosphoserine. “Amino acid analogs” refers to compounds that have the same fundamental chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups or modified peptide backbones but retain the same basic chemical structure as a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
In some embodiments, the functional analog is a derivative of OPN protein as described herein.
In some embodiments, the OPN protein derivative includes at least one amino acid modification.
For examples, the OPN protein derivative includes an amino acid modification (substitution) of the amino acid sequence denoted in SEQ ID NO:1. Such modification can be in at least one amino acid residue number 58, 188, 224, 237, 275, 276, 277, 301.
In some embodiments, the functional analog is or comprises an amino acid sequence denoted by at least one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16 or SEQ ID NO:17.
In some embodiments, the functional analog consists of an amino acid sequence denoted by at least one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16 or SEQ ID NO:17.
In some specific embodiments, the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 13.
In some specific embodiments, the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 14.
In some specific embodiments, the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 15.
In some specific embodiments, the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 16.
In some specific embodiments, the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 17.
In some embodiments, the functional analog is a derivative that includes an amino acid modification of the amino acid sequence denoted in SEQ ID NO:1 in which at least one amino acid was deleted. In some further embodiments, functional analogue comprises at least one of fragment of OPN protein or isoforms thereof.
The term "fragment ” as herein defined refers to any peptide or polypeptide which is at least one amino acid shorter than the OPN protein or OPN isoforms (amino acid sequence) which is obtained by deletion of at least one amino acid residue from the OPN or isoforms amino acid sequence in accordance with the present disclosure. As noted herein, the fragments of the present disclosure are functional fragment, i.e. have the same activity and/or mechanism of action as the proteins/polypeptide of the present disclosure.
The fragments of the present disclosure may be selected such that they maintain or even improve (do not reduce) the activity of the OPN protein. As described herein, the fragments may be due to alternative splicing or polymorphism occurring in a gene encoding the OPN protein. Alternatively or additionally, the fragments may be synthesized.
In some embodiments, the functional analog comprises a fragment of an amino acid sequence denoted by at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO: 11.
In some embodiments, the functional analog comprises a fragment of an amino acid sequence denoted by at least one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16 or SEQ ID NO:17.
In some embodiments, the functional analog comprises a fragment of an amino acid sequence denoted by at least one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17.
In some embodiments, the functional analog is or comprises an amino acid sequence denoted by at least one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21.
In some embodiments, the functional analog an amino acid sequence denoted by at least one of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.
In some specific embodiments, the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 18. In some specific embodiments, the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO: 19.
In some specific embodiments, the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO:20.
In some specific embodiments, the functional analog is or comprises the amino acid sequence as denoted by SEQ ID NO:21.
In some embodiments, the functional analog is or comprises an amino acid sequence denoted by at least one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
In some embodiments, the functional analog consists an amino acid sequence denoted by at least one of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
In some embodiments, the fragment comprises an amino acid sequence comprising amino acid residues 159-161 of OPN protein as denoted by SEQ ID NO:1. In some embodiments, the fragment comprises an amino acid sequence comprising RGD. The amino acid sequence RGD is denoted by SEQ ID NO:22.
In some embodiments, the fragment comprises an amino acid sequence comprising amino acid residues 158-162 of OPN protein as denoted by SEQ ID NO:1. In some embodiments, the fragment comprises an amino acid sequence GRGDS. The amino acid sequence GRGDS is denoted by SEQ ID NO:23.
In some embodiments, the fragment comprises an amino acid sequence comprising amino acid residues 131-143 of OPN protein as denoted by SEQ ID NO:1. In some embodiments, the fragment comprises an amino acid sequence ELVTDFPTDLPAT. The amino acid sequence ELVTDFPTDLPAT is denoted by SEQ ID NO:24.
In some embodiments, the fragment comprises an amino acid sequence comprising amino acid residues 162-167 of OPN protein as denoted by SEQ ID NO:1. In some embodiments, the fragment comprises an amino acid sequence SVVYGLR. The amino acid sequence SVVYGLR is denoted by SEQ ID NO:25. In some embodiments, the fragment comprises an amino acid sequence comprising amino acid residues 290-305 of OPN protein as denoted by SEQ ID NO:1. In some embodiments, the fragment comprises an amino acid sequence KSKEEDKHLKFRISHE. The amino acid sequence KSKEEDKHLKFRISHE is denoted by SEQ ID NO:26.
In some embodiments, the fragment comprises an amino acid sequence comprising amino acid residues 167-206 of OPN protein as denoted by SEQ ID NO:1. In some embodiments, the fragment comprises an amino acid sequence LRSKSKKFRRPDIQYPDATDEDITSHMESEELNGAYKAIP. The amino acid sequence LRSKSKKFRRPDIQYPDATDEDITSHMESEELNGAYKAIP is denoted by SEQ ID NO:27.
In some embodiments, the functional analog is or comprises at least one amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
In some embodiments, the functional analog consists of at least one amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
In some embodiments, the functional analog is or comprises at least one amino acid sequence denoted by SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
In some embodiments, the functional analog consists of at least one amino acid sequence denoted by SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
In some embodiments, the functional analog is or comprises at least one amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
In some embodiments, the functional analog consists of at least one amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
The present invention also encompasses derivatives or fragments of at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
The present invention also encompasses derivatives or fragments of at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
The present invention also encompasses derivatives or fragments of at least one amino acid sequence denoted by SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21.
The present invention also encompasses derivatives or fragments of at least one amino acid sequence denoted by SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
The peptides derivatives and/or fragments in some examples may be determined by peptidomimetics methods or computational analysis.
In some embodiments, the functional analog is or comprises an amino acid sequence having at least 50%, at times having at least 60%, at time having at least 70%, at times having at least 75%, at times having at least 80%, at times having at least 85%, at times having at least 90%, at times having at least 95%, at times having at least 97% similarity to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27. In some embodiments, the functional analog is or comprises an amino acid sequence having at least 85% similar to at least one amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
In some embodiments, the functional analog is or comprises an amino acid sequence having at least 50%, at times having at least 60%, at time having at least 70%, at times having at least 75%, at times having at least 80%, at times having at least 85%, at times having at least 90%, at times having at least 95%, at times having at least 97% identity to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
In some embodiments, the functional analog is or comprises an amino acid sequence having an amino acid sequence that is at least 85% identical to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
As described herein, a derivative is different in at least one amino acid residue from an amino acid sequence of OPN protein as well as from any fragment thereof, such modification includes insertions, deletions, substitutions and combination thereof.
The OPN protein or functional analog thereof as used herein also encompasses in accordance with some examples, products of gene therapy. It should be noted that the gene therapy in the context of the present application may modify, for example, by amplification a gene of the OPN protein or a functional analog thereof to treat a non- infectious inflammatory disorder as described herein. Examples of gene therapy include plasmid DNA, miRNA, viral vectors, bacterial vectors, human gene editing technology and the like. The OPN protein or the functional analog thereof may be formulated into a pharmaceutical formulation with at least one pharmaceutically acceptable carrier/s, diluent/s, excipient/s.
As used herein “ pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. In some examples, the carrier is a pharmaceutical buffering agent (buffers). Buffers for use in pharmacy are well known in the art and include, inter alia, phosphate buffer solution (PBS), dextrose, etc. The pharmaceutically acceptable carrier does not typically interfere with the effectiveness of the biological activity of the active ingredients and is not toxic to the host or patient.
In accordance with some embodiments, the OPN protein or the functional analog thereof of the invention is associated with at least one of pharmaceutically acceptable carrier in the composition to form a delivery system. Hence, in accordance with such embodiments, the term "physiologically acceptable carrier” also encompasses a carrier that is useful in preparing a delivery system for the OPN protein or functional analog thereof of the invention that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and acceptable for veterinary use as well as human pharmaceutical use.
By the use of the term “ associated ’ or "association" it is to be understood as being carried by the physiologically acceptable carrier by any means of suspension, emulsion, dissolution, embedment, attachment, entrapment, encapsulation, chemical bonding, adsorption, and the like and contemplates any manner by which the OPN protein or functional analog thereof of the invention is held to form the integral delivery system.
The delivery system in accordance with some embodiments, comprises a matrix, e.g. in which the OPN protein or functional analog thereof is entrapped. In some examples, the matrix is a biodegradable matrix.
In some embodiments, the delivery system comprises an organized collection of lipids, such as liposomes or micelles. The OPN protein or the functional analog thereof may be associated with liposomes.
In some embodiments, the OPN protein or the functional analog thereof is encapsulated within the intraliposomal core of the liposomes. In some embodiments, the OPN protein or the functional analog thereof is present within a vesicle. In some embodiments, the OPN protein or the functional analog thereof is encapsulated within exosomes. Exosomes as used herein refer to extracellular vesicles (EVs) that are produced in the endosomal compartment of eukaryotic cells and composed of cellular membranes with adhesive proteins on their surface. As appreciated, the liposomes, exosomes or any other delivery system can be modified to include on it’s surface specific residues to enhance localization to a target tissue, such as a cardiovascular tissue.
The composition of the invention in accordance with the present disclosure is administered with an effective amount of the OPN protein or functional analog thereof. The term “effective amount ” is intended to mean that amount of the OPN protein or functional analog thereof is sufficient to cause a beneficiary change. The amount of the OPN protein or functional analog in the formulation being the effective amount is typically determined in appropriately designed clinical trials (dose range studies) to achieve a desired effect and the person versed in the art will know how to properly conduct such trials in order to determine the amount. As generally known, an effective amount depends on a variety of factors including the distribution profile of the nanoparticles within the body, a variety of pharmacological parameters such as half life in the body, undesired side effects, if any, on factors such as age and gender of the treated individual etc. The amount must be effective to achieve a desired therapeutic effect such as rapid recovery of the treated subject, or improvement or elimination of symptoms and other indicators associated with the inflammation under treatment, selected as appropriate measures by those skilled in the art.
The OPN protein or the functional analog is administered and dosed taking into account the clinical condition of the individual, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The dosage form may be single dosage form or a multiple dosage form to be provided over a period of several days. The schedule of treatment with the OPN protein or functional analog generally has a length proportional to the length of the non-infectious inflammatory process, the parameters of the individual to be treated (e.g. age and gender). In some embodiments, the OPN protein or the functional analog is suitable for systemic administration.
The phrase "systemic administration ” as used herein mean the administration of the OPN protein or the functional analog directly into the circulatory system so that the entire body is affected by the administration.
Systemic administration includes enteral administration (i.e. absorption of the drug through the gastrointestinal tract) or parenteral administration (i.e. generally injection, infusion, or implantation).
In some embodiments, the OPN protein or the functional analog is suitable for parental administration.
The phrase "parenteral administration" as used herein means modes of administration other than enteral and local administration.
In some embodiments, the OPN protein or the functional analog thereof is suitable for administration by at least one o intravenous (i.v) injection, intracoronary (i.e), intramuscular (i.m), intraperitoneal (i.p) injection.
In some other embodiments, the OPN protein or the functional analog thereof is suitable is administered by intravenous injection or intra-arterial.
In some embodiments, the OPN protein or the functional analog is suitable for administration to the vascular system. Specifically, the OPN protein or the functional analog may be applicable for intravenous injection or intravenous infusion.
In some other embodiments, the OPN protein or the functional analog are suitable for local administration to the target site, e.g. of inflammation site. In the context of the present invention, the term “ target site ” is used to denote tissue or organ that is either inflamed (non-infectious inflammation); or tissue that is injured for example an ischemic tissue.
Specifically, the OPN protein or the functional analog is suitable for administration into a cardiac tissue/myocardium.
In some embodiments, the OPN protein or the functional analog can be administered by at least one of an intramyocardial administration, an intracoronary administration, a transvenous coronary sinus administration or a retrograde coronary sinus administration.
In some embodiments, the OPN protein or the functional analog is suitable for intramyocardial administration.
Intramyocardial administration as used herein means direct administration into the heart muscles or ventricles. Intramyocardial administration includes for example epicardial and transendocardial administration. Epicardial administration refers to administration into the inner layer of the pericardium, a conical sac of fibrous tissue that surrounds the heart and the roots of the great blood vessel. Transendocardial administration refers to administration through or across the endocardium.
In some embodiments, OPN protein or the functional analog is suitable for intracoronary administration.
Intracoronary administration as used herein means direct administration into to or within the coronary artery or the heart. Intracoronary administration encompasses direct injection or infusion and provides an advantage of direct local delivery.
In some embodiments, the OPN protein or the functional analog is suitable for transvenous coronary sinus administration.
Transvenous coronary administration as used herein means administration directly across or through the heart.
In some embodiments, the OPN protein or the functional analog is suitable for retrograde coronary sinus administration.
Retrograde coronary sinus administration as used herein means administration via the coronary sinus and vein.
Direct administration may be done by devices dedicated for such an administration. In some embodiments, direct administration is applicable by using a catheter, microcatheter or injection by needle. In some embodiments, direct administration is applicable by using a balloon such as an inflatable balloon. The balloon or any other suitable device may be introduced, for example, by a catheter.
As shown herein in the examples, OPN was suggested to stimulate neonatal cardiomyocyte proliferation and significantly improve LV remodeling and function after MI. “Endogenous tissue engineering” by OPN reduces LV wall stress by increasing scar thickness and stabilizing chamber size. By thickening the scar, wall stress is reduced (Laplace law) and the degree of outward motion of the infarct that occurs during systole (dyskinesis) is also reduced. This effect may be considered to be analogous to an external device that constrains the myocardium from expanding, thereby limiting adverse LV remodeling and prevent mechanical complications after MI. Hence, the OPN protein including all isoforms of OPN and functional analog including for example naturally occurring derivatives or fragments as well as OPN-derived synthetic peptide or OPN agonists can be delivered into the infarcted myocardium immediately or a few days after coronary reperfusion. In addition, OPN can be combined with scaffolds, injectable biomaterials or encapsulated into liposomes to improve delivery and retention in the infarcted heart OPN can be delivered intra-coronary by a catheter-based approach, by IV infusion of liposomes or other nanocarriers that target the infarcted myocardium or by catheter-based direct injection into the infarct.
These findings suggest a new reparative therapy for treating non-infectious inflammatory disorders such as acute MI as well as other acute myocardial injuries such as myocarditis.
Thus, the OPN protein or the functional analog thereof may be applicable for treating non-infectious inflammatory disorders as further detailed herein. In line with the above, a further aspect of the invention relates to a method of treating or preventing a non-infectious inflammatory disorder or a condition associated therewith.
Specifically, the method of the invention may comprise the step of administering to a subject in need thereof an effective amount of the OPN protein or the functional analog thereof, or of any composition comprising the same.
In some embodiments, the method of the invention may be applicable for the treatment, prevention or inhibition of non-infectious inflammatory disorder or a condition associated therewith in a subject in need thereof, the method comprising the step of administering to said subject a therapeutically effective amount of the OPN protein, or functional analog thereof or of any composition or the delivery system comprising the same. As used herein, the term " non-infectious inflammatory disorders” used herein is to be understood as encompassing any immune response that is not related to activation of the immune system, e.g. by an infection. Such non-infectious inflammatory disorders denote any disorder which the activation of macrophages or activated macrophages play a role such as auto-immune disorders and inflammatory disorders which are not infection related, i.e. non-pathogenic, caused by other than an infectious agent (e.g. auto-antigen, hypersensitivity, wound).
Non-limiting examples of activated macrophage-related, non-infectious inflammatory disorders are inflammatory diseases of the gastrointestinal tract such as Crohn's disease, inflammatory bowel disease, gastritis, colitis, ulcerative colitis, colon irritable, gastric ulcer and duodenal ulcer, inflammatory diseases of the skin such as psoriasis, inflammatory diseases of the respiratory system such as asthma, allergic rhinitis or chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, sarcoidosis, inflammatory diseases of the musculoskeletal system such as rheumatoid arthritis, osteomyelitis, osteoporosis, or neuritis, systemic sclerosis, inflammatory diseases of the kidneys such as glomerulonephritis, renal ischemia, or renal inflammation; inflammatory diseases of the nervous system such as multiple sclerosis, Alzheimer's disease and H1V- 1 -associated dementia; autoimmune diseases such as diabetes, type 1 and 2 diabetes mellitus and graft versus host reaction; infectious disease such as nephritis, sepsis, septic shock, endotoxic shock, adult respiratory distress syndrome; inflammatory conditions of the cardiovascular system, such as myocardial infarction, myocarditis, atherosclerosis, hypertensive cardiomyopathy, atheroma, intimal hyperplasia or restenosis.
The non-infectious inflammatory disorders also denote tissue healing and repair. The tissue may be injured or damaged as a result of ischemia or necrosis.
Non-limiting examples of tissue damage may include infracted myocardium, myositis, myocarditis, myocardial fibrosis, glumerulonephritis, diabetic nephropathy, kidney infarct, glomerular sclerosis, stroke, liver injury, brain injury, pulmonary fibrosis, ischemic limb, athrosclerosis vascular disease, chronic ulcer.
The non-infectious inflammatory disorders may be acute non-infectious inflammatory disorders or chronic non-infectious inflammatory disorders. It is specifically noted that the non-infectious inflammatory disorders exclude any phatogenic, e.g. bacterial, mediated inflammation (e.g. due to pathogenic infection).
In some embodiment, the non-infectious inflammatory disorders may be involved in a condition selected with tissue injury, infarct, trauma, and necrosis. According to some embodiments, the non-infectious inflammatory disorders are associated with a damaged tissue condition, for example healing a wounded tissue.
In some embodiment, the non-infectious inflammatory disorders involve at least one of the following conditions tissue injury, infarct, trauma, necrosis, sterile inflammatory disorder. The sterile inflammatory disorder may be in some examples an autoimmune sterile inflammatory disorder.
In some embodiments, the non-infectious inflammatory disorder is associated with a cardiovascular disorder. As shown in the Examples below and specifically in Examples 1 and 2, accumulation of monocytes and macrophages was observed at a heart resection site and specifically CCR2+ monocytes.
Cardiovascular disorders as used herein encompasses a range of conditions that affect the heart structure and/or heart activity of a subject. Such disorders generally involve narrowed or blocked blood vessels.
In some embodiments, the methods of the invention may be applicable for treating a cardiovascular disorder selected from or associated with myocarditis, heart failure, atherosclerosis and myocardial infarction.
In certain embodiments, the methods of the invention may be applicable for subjects suffering from a cardiovascular disorder, which may be a genetic disorder or an acquired disorder.
In some embodiments, the methods of the invention may be applicable for treating acquired cardiovascular disorders.
Acquired cardiovascular disorders may result in a variety of factors affected by a subject’s lifestyle and can results from factors such as exposure to toxic substances such as toxins, radiation or chemotherapy
The acquired cardiovascular disorder may be at least one of coronary artery disease (CAD), myocardial infarction (MI), heart failure, cardiomyopathy, myocarditis. In some embodiments the methods of the invention are applicable for the treatment, inhibition or delaying the cardiovascular disorder associated with coronary artery disease.
Specifically, the methods of the invention are applicable for subjects having a tendency to develop a coronary artery disease.
Coronary Artery Disease (CAD) is a type of cardiovascular disease that involves atherosclerosis, i.e. hardening and narrowing of the coronary arteries, producing blockages in the vessels that carry blood to the heart. Atherosclerosis is a condition that may develop with time such that it is slowing causes to blocking arteries and eventually restricting blood flow to the heart and may ultimately cause heart attack, stroke, and peripheral vascular disease.
In some embodiments the methods of the invention are applicable for the treatment, inhibition or delaying the cardiovascular disorder associated with heart failure. Specifically, the methods of the invention are applicable for subjects having a tendency to develop heart failure.
Heart Failure relates to a condition at which the heart’s ability to pump is weaker than normal and hence blood moves through the heart and body at a slower rate, pressure increases in the heart, and the heart can’t supply enough blood and oxygen to the body’s cells, resulting in fatigue and shortness of breath.
The methods of the invention may be applicable, in accordance with some embodiments, for the treatment, inhibition or delaying the cardiovascular disorder associated with a heart failure which is an ischemic heart failure (IHF).
Ischemic heart failure relates to a cardiac condition characterized by systolic dysfunction and reduced cardiac output typically resulting from an imbalance between myocardial oxygen demand and supply.
In some embodiments the methods of the invention are applicable for the treatment, inhibition or delaying the cardiovascular disorder associated with cardiomyopathy. Specifically, the methods of the invention are applicable for subjects having a tendency to develop cardiomyopathy. Cardiomyopathy relates to a progressive disease that causes the heart to become abnormally enlarged, thickened, and/or stiffened, cardiomyopathy (also known as heart muscle disease) limits the heart muscle’s ability to pump blood effectively, often leading to other heart conditions such as heart failure or arrhythmia. Cardiomyopathy may result from exposure to toxins, chemotherapy, radiation due to autoimmune conditions.
In some embodiments the methods of the invention are applicable for the treatment, inhibition or delaying the cardiovascular disorder associated with myocardial infarction. Specifically, the methods of the invention are applicable for subjects having a tendency to develop myocardial infarction.
Myocardial Infarction (MI) also called heart attack is a disorder that takes place when blood flow to the heart is severely reduced or cut off, due to the hardening and narrowing of the coronary arteries from the build-up of fat, cholesterol, and other substances, known together as "plaque." Such a blood clot forms around the plaque, blocking blood flow and may result in permanent damage or death of part of the heart muscle.
In some further embodiments the methods of the invention are applicable for the treatment, inhibition or delaying the cardiovascular disorder associated with myocarditis. Specifically, the methods of the invention are applicable for subjects having a tendency to develop a myocarditis.
Myocarditis is an inflammation of the heart muscle (myocardium) and may affect the heart muscle and the heart's electrical system, reducing the heart's ability to pump and causing rapid or abnormal heart rhythms (arrhythmias) It should be noted that myocarditis can damage the heart muscle, at times even permanently and ultimately causing heart failure, heart attack or heart stroke or arrhythmias.
As detailed herein and shown in Example 5, OPN stimulated cardiac cell proliferation in neonatal heart and the proliferation was OPN-concentration dependent. Further, as shown in Example 6 below, OPN stimulated cell cycle in cardiomyocytes an effect which was suggested to be mediated via CD44 receptor. Further and as shown in Example 8 below, OPN simulated wound heating by stimulating both proliferation and migration of both cardiomyocytes and fibroblasts. Finally, as shown in Example 10 below, OPN significantly improve LV remodeling and function after MI and reduces LV wall stress by increasing scar thickness and stabilizing chamber size.
It should be apricated that by the treatment, inhibition or delaying a cardiovascular disorder in accordance of the present invention, an improvement in the subject health is obtained. For example, the methods of the present invention may be applicable for restoring damaged myocardium or for regeneration of myocardial.
In some embodiments the methods of the invention are applicable for the restoring permanently damaged myocardium. The damaged myocardium may be transiently damaged or permanently damaged.
In some embodiments the methods of the invention are applicable for the regeneration of myocardium. The regeneration of myocardium comprises angiogenesis and myogenesis.
It should be noted that the methods of the disclosure comprising administration of OPN protein or a functional derivative thereof may be beneficial to a subject having reduced expression of OPN or a defected expression of OPN. The former may be considered in some examples as an OPN-deficient disorder and may be treated with the OPN protein or functional analog as described herein.
The administration of the OPN protein or the functional analog thereof may be in combination with one or more other active agents, such as one or more immunomodulatory agents or anti-inflammatory agents. An immunomodulatory agent as used herein refers to a substance that stimulates or suppresses the immune system and can be used as immunotherapy. An immunomodulatory agent can be recombinant, synthetic, or natural agent. Examples of immunomodulatory agents that can be administrated with the OPN protein or a functional analog thereof include immunomodulatory imide drugs (IMiDs) such as thalidomide and its analogues (lenalidomide, pomalidomide, iberdomide, and apremilast). Examples of anti-inflammatory agents which may be combined with the OPN protein or functional analog thereof include, without being limited thereto, steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs (NSAID). The additional agent may be administered to the subject before, concomitant or after administration of the OPN protein or the functional analog thereof. When administered together with the OPN protein or the functional analog thereof, the two (or more) may be in the same formulation or formulated in two different formulations.
The OPN protein or the functional analog thereof as defined herein may form part of a therapeutic kit for use by a practitioner, e.g. a medical doctor, a nurse, or by a subject in need of the treatment. The kit may comprise the OPN protein or the functional analog thereof within a physiologically acceptable carrier, and instructions for use of OPN protein or the functional analog thereof for administering to the subject, typically by injection. The kit may comprise a single dosage unit of the OPN protein or the functional analog thereof for single administration or may comprise multiple dosages, e.g. for multiple, sequential administrations according to a predefined schedule of treatment.
As used herein, “disease”, “disorder”, “condition” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms. It is understood that the interchangeably used terms "associated" and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder, condition or pathology causes a second disease, disorder, condition or pathology.
As noted above, the invention provides methods for treating disorders specified above. The term “treatment” as used herein refers to the administering of a therapeutic amount of the formulation of the present invention which is effective to improve one or more undesired symptoms associated with a disease or condition as described herein.
As indicated above, the method of the invention involves the administration of a therapeutically effective amount of the OPN protein or functional analog thereof of the invention. The "effective amount" or “therapeutically effective” for purposes disclosed herein indicates that the amount of the OPN protein or functional analog thereof is effective to treat, inhibit or delay one or more symptoms of a disease as described herein.
As used herein, the term “ subject ’ refers to a living organism that is treated with the formulation as described herein, including, but not limited to, any mammal, such as a human.
The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range.
Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.
It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise “a” may be considered as “at least one”.
Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “ comprises ” and “comprising” , will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.
NON-LIMITING EXAMPLES MATERIALS AND METHODS
The study has been approved by the Sheba Medical Center Internal Review Board Committee and was performed in accordance with the guidelines of The Animal Care and Use Committee of the Sheba Medical Center, Tel Aviv University. Neonatal Mouse Model of Apical Resection (AR) or Myocardial Infarction (MI)
To determine the role of macrophages in myocardial regeneration and repair, a neonatal mouse model of apical resection (AR) and left anterior descending (LAD) ligation MI were used. One-day-old neonatal ICR mice (Harlan Laboratories, Jerusalem, Israel) were subjected to AR, MI or sham operation (n=30). To avoid pain and stress, newborn mice were anaesthetized by inhalation of 2% isoflurane and 100% oxygen. The mice were cooled down on an ice bed for 4 minutes, thereby causing asystole and reversible apnea, and preventing excessive blood loss during surgery. The cooling down period also provides additional anesthesia. For AR, the chest was opened by left thoracotomy, and iridectomy scissors were used to carefully and gradually resect thin segments from the LV apex, as previously described, [4] and the thoracic skin was closed using adhesive tissue glue.
For MI induction, the same anesthesia protocol was used. Permanent occlusion of the mid LAD coronary artery was performed using an 8-0 prolene suture (Ethicon, Cornelia, GA, USA). Finally, animals were placed on a heating pad (37°C), allowed to recover and return to their mothers. Sham-operated mice underwent the same procedure except for resection or LAD ligation. Average survival rate after AR and MI was 70 and 40%, respectively. Survival after sham operation was 95%.
MI in Adult Mouse and OPN Injection
To determine the role of macrophage-derived cytokines in infarct repair, the mouse model of MI in female 12-week-old ICR or C57BL/6 mice was used (Harlan Laboratories, Jerusalem, Israel) [3, 5, 6]. Mice were anaesthetized with 1-3% isoflurane, intubated and ventilated with 100% oxygen. The chest was shaved and opened by left thoracotomy, and LAD ligation was performed using an 6-0 prolene suture (Ethicon, Cornelia, GA, USA) to permanently occlude the LAD coronary artery. Immediately after the occlusion, 40ml of PBS with or without 200 ng of OPN were injected to the infarcted area. The chest was closed and the skin sutured with a 5-0 prolene suture (Ethicon, Cornelia, GA, USA). Mice were placed on a heating pad (37°C) until recovery. Average survival rate after MI was 80%, and average survival rate after sham operation was 95%. Gene Expression Analysis in Neonatal Heart
To search for differentially regulated gene networks in the neonatal heart after AR and MI, a comprehensive gene analysis was performed by hybridizing microarray chips with RNA probes prepared from neonatal mouse hearts 3 days after resection, MI or sham operation. Briefly, total RNA was extracted from these hearts (n=4 per group), using EZ- RNA (total RNA isolation kit, Biological Industries, Beit Haemek, Israel) according to the manufacturer's instructions. Total RNA was quantified by using a spectrophotometer and confirmed by Qubit Fluorometric Quantitation (Life Technologies, Grand Island, NY, USA). Gene array was performed using an Affymetrix Mouse Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA). For raw data generation, we used a gene level RNA sketch algorithm (Affymetrix Expression Console and Partek Genomics Suite 6.2.). Genes were filtered and analyzed using fold change calculations and unsupervised hierarchical cluster analysis (SpotfireDecisionSite for Functional Genomics; Somerville, MA, USA). Further processing included functional analysis and over-representation calculations based on Gene Ontology and publication data: DAVID (http://appsl.niaid.nih.gov/David/upload.asp). Finally, we used the database for Annotation (GO), Visualization, and Integrated Discovery.
Rosamt/mg xCsf-lR-icre Neonatal Mice
To assess monocyte/macrophage accumulation in the neonatal heart, macrophage reporter mice was used [5]. The inventors used crossed Csflr-icre mice with the cre- reporter 35 RosamT/mG in which CsflR-positive cells are GFP positive.
Cardiac Macrophage Isolation, Culture and Flow Cytometry
To obtain neonatal cardiac macrophages, hearts were harvested 1,3,5 and 7 days after AR, MI and sham (n=5 in each group at each time point). To obtain cardiac macrophages, total heart cells were isolated using an enzymatic digestion mixture, [7] which was added to the heart for three 15-minute cycles in a rotating water bath warmed to 37°C. Macrophages were purified based on plastic adherence, as previously described. Briefly, cells were incubated for 2 hours at 37°C in humid air with 5% CO2 on 6 well plates supplemented with RPMI (Biological Industries, Beit-Haemek, Israel) with 10% fetal bovine serum (FBS) (Biological Industries, Beit-Haemek, Israel) and 1% streptomycin. Then, to further ensure macrophage enrichment, non-adherent cells were washed, and a 3-minute trypsin- EDTA treatment was added to the remaining cells [3]. The trypsin was then blocked with fresh medium and washed, and the intact adherent cells were considered to be cardiac macrophages.
To assess the amount and distribution of macrophages, isolated heart cells were analyzed by flow cytometry as previously described [3]. The phenotype of neonatal cardiac macrophages was analyzed by flow cytometry, using the following fluorescent anti-mouse antibodies: F4/80-APC, CD192 (CCR2)-PE, APC- k isotype Ctrl, PE- k isotype Ctrl (all from BioLegend, San-Diego, CA, USA). Labeled cells (0.5 x 106) from each sample were acquired and analyzed using Flow Cytometer Calibur Cytofluorimeter (Cyteck Development, Fremont, CA, USA) with Flowjo software (Tree Star, Ashland, OR, USA).
CCR2 Inhibition
One-day-old neonatal mice were subjected to AR. Following recovery, the mice were treated with 20ml of CCR2 Ab injected IP (clone:MC21) [8]. every 48 hours, 3 times in total, to achieve CCR2+ monocyte blockade for the first week after AR.
Cytokine and Chemokine Quantification
To determine cytokine and chemokine profiles in macrophages macrophages were isolated from various hearts as previously described [3]. To obtain cardiac macrophages, neonatal hearts were harvested 3 days after AR, MI and sham, as were adult hearts 3 days after MI (n=4 in each group). Total heart cells were isolated using an enzymatic digestion mixture, which was added to the heart for three 15-minute cycles in a rotating water bath warmed to 37°C, as previously described. [7]. Macrophages were purified based on plastic adherence, as previously described. Briefly, cells were incubated for 2 hours at 37°C in humid air with 5% CO2 on 6 well plates supplemented with RPMI (Biological Industries, Beit-Haemek, Israel) with 10% FBS (Biological Industries, Beit-Haemek, Israel) and 1 % streptomycin. Then, non-adherent cells were washed, and to further ensure macrophage enrichment, a 3-minute trypsin-EDTA treatment was added to the remaining cells. The trypsin was then blocked with fresh medium and washed, and the intact adherent cells were considered to be cardiac macrophages. To determine purification efficiency, the cells were stained with the macrophage marker F4/80, and displayed with > 90% positive staining for F4/80 in culture (data not shown). Cardiac macrophages were grown for 24 hours for conditioned media collection. One hundred micro-liters of cell culture supernatant (for each mouse, per group) was used for cytokine and chemokine detection and quantification by a custom-made mouse Quantibody Array kit (RayBiotech Inc. Insight Biotechnology Ltd, Wembley, UK), according to the manufacturer’s instructions. The array was designed to quantitatively detect 14 cytokines, chemokines, and growth factors simultaneously: Interleukin (IL)-lb, IL-4, IL-6, IL-10, IL-12 p40/p70, IL-17, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF) -a, stromal cell-derived factor (SDF) -1, IL -13, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and OPN.
The signals (green fluorescence, Cy3 channel, 555 nm excitation, and 565 nm emission) were captured using a GenePix 4000B laser scanner (Bio-Rad Laboratories, Hercules, CA, USA), and extraction with GenePix Pro 6.0 microarray analysis software. Quantitative data analysis was performed using RayBiotech mouse Array software (Q- Analyzer Software for QAM-INF-1, Norcross, GA). Sample concentrations (pg/ml) were determined from mean fluorescent intensities (median values) compared with five- parameter linear regression standard curves, generated from standards provided by the manufacturer. A series of isochronal maps reflecting the cytokine/chemokine concentrations detected by the microarrays were constructed by Matlab software (R2009a, MathWorks, Inc., Natick, MA, USA), in order to transform numerical data into simplex graphical patterns.
Organ Culture of Neonatal Heart
To further determine the effect of OPN on neonatal heart an organ culture was used. Hearts were harvested from 1 -day-old, neonatal mice. The atrial area was removed and the ventricular area was sliced into 2 transverse sections, which were cultured on a coated 48 well dish with cardiomyocyte growth media for 48 hours in order to achieve adhesion. Following this, tissues were treated with 800ng/ml OPN (Sigma-Aldrich, St.Louis, MO, USA), 400ng/ml or control culture media without OPN (6 wells in each group), for 72 hours [9]. Cardiac tissue growth was then assessed by cell budding (outgrowth) from the cultured heart slices using SigmaScan Pro (Systat Software San Jose, CA, USA). The outgrowth was stained with antibodies against a-cardiac actin (Santa Cruz Biotechnology, Dallas, TX, USA), PH3 (S10 Epitomics, Burlingame, CA, USA), CD31 (Santa Cruz Biotechnology, Dallas, TX, USA) and F4/80 (BioLegend, San Diego, CA, USA).
Isolation and Culture of Neonatal Cardiomyocytes
Isolation and culture of neonatal mouse cardiomyocytes was done as previously described [10]. Briefly, 10 hearts were extracted from1-day old mice into a bacterial dish containing sterile, 4°C, PBS. After removing unwanted tissue, the hearts were transferred into a drop of isolation medium (BDM: Sigma-Aldrich, B-0753; HBSS: BI,02-017-1B; trypsin 0.25%, Gibco) and minced to small pieces. The pieces were then transferred to a conical tube containing 10ml of isolation medium and incubated overnight with gentle agitation at 4°C. Next, the isolation medium was replaced by digestion medium (collagenase/dispase: Sigma-Aldrich, 10269638001; L-15: BI, 01-115-1 A, BDM, HBSS) and the conical tube was transferred to a 37 °C water bath for 20 min with gentle agitation. Then, when the digestive tissue sank to the bottom, supernatant-containing cells were transferred to a fresh conical tube, and filled with medium-free collagenase/dispase, through a sterile cell-strainer (100 mm nylon mesh). After centrifugation, cells taken from the precipitate were re-suspended with a plating medium (DMEM high glucose; M-199: BI, 01-080-1B; Horse serum/HS, FBS, pen/strep) and seeded on 3.5cm bacterial dish for 2.5h. Next, the cells were counted using trypan blue to 2xl04/well in 96-well plate, pre- coated with collagen 0.01%. The cells were plated for 18h and the plating medium was replaced by medium without serum (DMEM low glucose, M-199, pen/strep).
Neonatal Cardiomyocytes Staining
Mouse neonatal cardiomyocytes were treated with or without 800ng/ml OPN (02260, Sigma-Aldrich) for 24h. Then, the cells were washed and fixed using 4% formalin. Blocking was performed using CAS-Block (008120, Life Technologies, Freseric, MD, USA) for 10 min and M.O.M/ Mouse IgG blocking Reagent (MKB-2213, VECTOR LABORATORIES, Burlingame, CA, USA) for 20 min. Next, primary antibodies were added against actinin (1:200, A7811, Sigma-Aldrich) and pH3 (1:100, S10, ab47297, Abeam) or CD44i (1:100, KM81, abll2178, Abeam) or Yes- Associated Protein (YAP) (1:50, LS-C331201, LSBio) for overnight in 4°C. Then, secondary antibodies Alexa Fluor 488 (1:200, Jackson ImmunoResearch), Texas Red (1:200, Jackson ImmunoResearch) and DAPI (BAR-NAOP) were added. RNA Extraction and Quantitative RT-PCR (qRT-PCR)
Total RNA was extracted from neonatal cardiomyocytes using RNeasy mini kit according to the manufacturer’s protocol (Cat: 74134, Qiagen, Hilden, Germany ). The cDNA was generated from total RNA using Verso cDNA kit (Cat: AB1453B, ABgene, Epsom, Surrey, UK ). qRT-PCR was performed in triplicate using absolute blue SYBR Green (Thermo, Waltham, MA, USA). DDCt values were normalized to GAPDH. Statistical analysis was performed using unpaired t test. Primers were designed with PubMed, Primer 3 input, and UCSC In-Silico PCR.
Figure imgf000050_0001
MTT
To estimate the number of cardiac cells in culture, neonatal cardiomyocytes were treated with or without 800ng/ml OPN (02260, Sigma- Aldrich) and 10mg/ml CD44i (KM81, Abeam), an antibody against CD44, for 24h. Then, the cells were exposed to 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay according to the manufacturer’s protocol (ab211091). The number of cells was determined via spectrophotometer using a wavelength of 595 nm.
RealTime-glo™ Assay
To estimate the effect of OPN on cell number and viability, Promega's RealTime- Glo™ MT Cell Viability Assay protocol (Cat. #G9711) was used. Briefly, RealTime-Glo reagents were added to neonatal cardiomyocytes with a low glucose medium without serum and incubated at 37°C and 5% CO2 for 1 hour. Then, at time Oh, luminescence was measured using luminometer with integration time of 0.3 second per well. After adding 800ng/m l/well of OPN, the cells were returned to the cell culture incubator for 24h and luminescence was measured again.
Western Blot Analysis
The Hippo signaling pathway is an evolutionarily ancient cascade of kinases and effector proteins that control cell proliferation and organ size. To determine whether the effect of OPN on proliferation of neonatal cardiomyocytes is mediated by the Hippo- YAP pathway, the inventors isolated and cultured mouse neonatal cardiomyocytes in starvation medium for 24 hours prior to lysis. Proteins were extracted and quantified using the Bradford method and 15mg protein was loaded to electrophoresis gel. Immunolabeling was done by antibodies directed against the following: c-terminal of EEK2 (#sc-154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated ERK1/2 (Sigma), LATS1 (1:250; #3477; CST), LATS1 (Thrl079) (1:500; #bs-7913R; Bioss ANTIBODIES), YAP (1:500; #4912; CST), phosphorylated-YAPl (p-YAP; 1:500; #4911; CST).
Isolation and Culture of Neonatal Cardiac Fibroblasts
Cardiac fibroblasts were extracted from 4 neonatal hearts using an enzymatic digestion mixture containing 0.25% trypsin-EDTA (Gibco-Invitrogen, Carlsbad, CA, USA) and 2.4 U/ml dispase II, (Sigma-Aldrich, St. Louis, MO, USA).44 Next, the cells were counted using trypan blue and plated with fibroblasts medium (DMEM high- glucose, FBS, pen/strep, glutamine and b-mercaptoethanol) to 2xl05/well in a 96-well plate. After 24h, the plated fibroblast medium was replaced with a new, low-glucose, DMEM medium.
“Wound Healing” Scratch Assay with Neonatal Cardiomyocytes
To determine the effect of OPN (800 ng/mL) on cardiomyocyte or fibroblast migration and “wound healing”, we used the scratch assay [11]. The assay employed a "scratch" across the middle of the well, using a sterile I0ml pipette tip. The rate of gap closure was monitored by serial microscope images. The images were then compared to quantify the migration rate of the cells up to 4 days.
Endothelial Cell (EC) Tube Formation Assay
To determine the effect of OPN on angiogenesis, we used an EC tube formation assay. Basement membrane matrix (Matrigel®, BD Biosciences) was added to 96-well plate and solidified by incubation at 37°C for 30 minutes. 1 x 103 human umbilical vein endothelial cells (HUVECs) were plated onto each Matrigel-containing well with endothelial cell growth medium and with or without 200, 400 or 800ng/ml/well OPN. The tube structures were evaluated by confocal microscopy. Echocardiography to Evaluate Cardiac Remodeling and Function
To assess LV remodeling and function after myocardial injury in neonatal heart, we used a special small animal echocardiography system (Vevo 2100 Imaging System; VisualSonics, Toronto, Ontario, Canada) equipped with a 22- to 55-MHz linear-array transducer (MS550D MicroScan Transducer (VisualSonics, Toronto, Ontario, Canada). Echocardiographic studies were done before, at day 1 and day 7 or 28 after injury or sham-operation. Light anesthesia was induced by inhalation of 2% isoflurane/98% O2 and subsequently maintained by 0.5% to 1% isoflurane. We controlled the isoflurane flow to maintain the heart rate at >400 bpm (mean 428+8 bpm). All measurements were averaged for 3 consecutive cardiac cycles and performed by an experienced technician who was blinded to the treatment groups. Values were calculated for each animal as follows: ejection fraction = [(LV vol d-LV vol s)/LV vol d] xl00; fractional shortening = [(LVDD-LVSD)/LVDD] xl00.
To improve the sensitivity to detect regional wall motion abnormalities in adult mouse after MI, we used speckle-tracking based strain analysis [3] for strain quantification in the longitudinal and radial axes. Echocardiographic parasternal long- axis images were acquired at a frame rate of 280 frames per second. Three consecutive cardiac cycles were selected and their endocardium and epicardium borders were traced. If needed, borders were corrected in order to preserve as precise tracking as possible throughout each cine loop. Each LV image in long axis was divided into six segments for regional speckle-tracking based strain analysis: anterior base, anterior mid, anterior apex, posterior apex, posterior-mid and posterior base. Peak strain data were recorded from each segment for regional speckle-tracking based strain analysis. Global strain of the LV was calculated as the averaged peak strain obtained from all six segments [3].
Histological Analysis
To assess myocardial injury, healing, repair, and regeneration after AR, MI, and sham operation, hearts were harvested at different time points after the procedure, washed with PBS and then fixed in 4% paraformaldehyde overnight. Adjacent blocks were embedded in paraffin and sectioned into 5 mm slices. Hematoxylin/eosin and Masson's Trichrome staining (to detect scarring and fibrosis) were performed according to standard procedure. To identify neonatal cardiomyocytes, cell proliferation, and macrophages, heart sections were stained with hematoxylin and eosin (HE) and antibodies against a- cardiac actin (Santa Cruz Biotechnology, Dallas, TX, USA), actinin ( Sigma-Aldrich, St.Louis, MO, USA) and phospho-histone 3 (PS10 Epitomics, Burlingame, CA, USA.).
For the CCR2 inhibition experiment, hearts were harvested at days 3 and 21 after AR, and treated according to the previously described protocol. To identify macrophages, heart sections were stained with antibodies against F4/80 (BioLegend, San Diego, CA, USA).
Statistical Analysis
Statistical analysis was performed with GraphPad Prism version 8.00 (GraphPad Software). Variables were expressed as mean+SEM. Specific statistical tests are detailed in the figure legends. In brief, differences between values were tested by unpaired t test or 1-way analysis of variance (ANOVA) test (>2 groups) followed by Tukey multiple comparison test. If values were not normally distributed (tested by the D’Agostino- Pearson omnibus normality test), we used the nonparametric Mann- Whitney test or Kruskal-Wallis test (>2 groups), followed by a Dunn multiple comparison post-test. To assess differences in cardiac function along time, with and without OPN therapy, we used 2-way repeated-measures ANOVA followed by Holm-Sidak’s multiple comparison test to assess the significance of predefined comparisons at specific time points
RESULTS
Example 1: Macrophages in Regeneration and Repair of Neonatal Heart
To investigate the role of monocytes/macrophages in neonatal heart regeneration, the apex resected (AR) Rosamt/mg x Csf-lR-icre newborn transgenic mice was used, in which monocytes and macrophages expressed GFP [5]. Three and four days after AR, microscopic examination revealed a robust accumulation of monocytes and macrophages at the resection site, as well at the blood clots and in the granulation tissue (Figs. 1A, IB). Cardiomyocytes expressed tomato red, and nuclei were stained with blue with DAPI. These findings are compatible with previous report of the group [6] and those of others on the essential role of macrophages in myocardial regeneration.
To determine temporal changes in macrophage accumulation after AR in neonatal heart, myocardial infraction (MI) and sham operation on one-day-old newborn mice were induced. Hearts were harvested at 1, 3, 5 and 7 days after injury and analyzed by flow cytometry. Both AR and MI robustly induced macrophage (F4/80) accumulation in the injured heart (Fig. 1C). The percentage of cardiac macrophages was 0.4%-1.4% of the total number of neonatal heart cells in the first 7 days after birth. Following injury, macrophages accumulated in the injured neonatal heart 1 to 5 days after AR, and 1 to 3 days after MI. Compared with sham operation, myocardial injury provoked macrophage response and increased macrophage fraction by 16.6 fold after MI and by 7.7-fold after AR, on the first day after injury. Subsequently, the % of macrophages sharply decreased after MI and gradually decreased after AR, to the level of sham operated hearts, within 7 days.
Example 2: Myocardial Regeneration is Impaired after Inhibition of CCR2+ Monocytes/ Macrophages
Cardiac macrophages may be categorized by the expression of the chemokine (MCP-1) receptor CCR2 [12]. A subgroup analysis after AR vs. sham operation revealed that the infiltrate was dominated by CCR2+ macrophages, on the first day after injury (Fig. 2A). As can be seen in Fig. 2A, a sharp rise in the percentage of CCR2+ F4/80 macrophages was observed at day one after AR. Statistical analysis was performed using 2-way-ANOVA, with Holm- Sidack’ s multiple comparison post-test.
To determine the role of CCR2+ monocytes/macrophages on neonatal heart regeneration, one-day-old mice were subjected to AR and a subsequent injection of CCR2 Abs (antibodies) [8]. CCR2 abs 20m1 were injected, 48 and 96 hours after AR, to achieve CCR2+ monocyte inhibition during the first week after injury. To inhibit CCR2+ monocyte migration from the circulation into the injured heart, neonatal mice were treated with CCR2 Abs or saline control, such that CCR2 Abs (20ml) were injected three times: immediately after AR, 48 and 96 hours after injury. To determine the efficacy of CCR2 Abs, a preliminary experiment was done, and the amount of macrophages within the heart tissue by flow cytometry was assessed. Compared with control, CCR2 Ab treatment significantly depleted F4/80 monocyte/macrophage accumulation in the injured heart and the number of cardiac F4/80 macrophages (Fig. 2B). These findings suggested that CCR2+ monocytes are the major source of macrophages in the injured neonatal heart.
Significantly, postoperative mortality was higher in mice treated with CCR2 Abs, compared with control animals (14 of 19 vs. 1 of 5; p=0.02; Fig. 2C). Mice treated by CCR2 Abs died within a day after the first echocardiography study (day 1). Specifically, these results indicated that the mortality was higher in mice treated with CCR2 Abs, compared with untreated animals (14 of 19 vs. 1 of 5; p=0.02).
As a result of this early mortality in the CCR2-Ab-treated group, echocardiography measurements in the CCR2 Ab-treated animals were collected from two groups, each having undergone one echocardiography study: baseline echo (day 1) in one group (n=9), and the final echo (24-25 days) in the second group (n=4). Inhibition of CCR2+ monocytes and subsequent early depletion of cardiac macrophages compromised cardiac function at day 1 after AR (Fig. 2D). However, at day 24, LV fractional shortening (FS) was similar in the CCR2+ -depleted and the control group (Figure 2E). Histologic examinations revealed loose granulation tissue at the AR site, at day 3 after CCR2 Ab injections (Fig.2F). As can be seen in Fig. 2F using immunostaining for macrophage F4/80, macrophage depletion was observed on day 3 after AR as indicated by the black circle.
On day 7 (Fig. 2G) scar thinning and early deposition of collagen were observed. By days 24-25 following AR and CCR2 Ab therapy, an apical scar was observed (Fig. 2H). Analysis was done by H&E staining showing apical scar and collagen deposition by Masson Trichrome staining.
In contrast, after AR, control neonatal hearts regenerated with minimal superficial scaring (Fig. 21). Together, these findings suggest that CCR2+ monocytes are the major source of cardiac macrophages after AR and that CCR2+ macrophages are essential for neonatal heart regeneration.
Example 3: Gene Expression Profiling of Neonatal Hearts Following AR, MI and Sham Operation
To search for differentially-regulated gene networks in the neonatal heart after AR and MI, a comprehensive gene analysis was performed by hybridizing microarray chips with RNA probes prepared from neonatal mouse heart 3 days after AR, MI or sham operation. The experiments were repeated 3 times to confirm reproducibility. After AR, the highest up-regulated genes were related to inflammation, particularly macrophage activation, and ECM deposition (Table 1). The top up-regulated genes were related to macrophage activation and function. Up-regulated genes were mostly related to inflammation, particularly macrophage activation, and ECM deposition. The top upregulated genes were glycoprotein non-metastatic melanoma protein B (GPNMB), encoding a transmembrane glycoprotein involve in cell proliferation, and Sppl encoding Osteopontin (OPN), a matricellular protein involved in tissue repair, remodeling, chemotaxis, and immunomodulation [13].
Table 1: Representative up-regulated genes 3 days after AR, compared with sham operation.
Figure imgf000057_0001
Table 1 (Cont.):
Representative up-regulated genes 3 days after AR, compared with sham operation.
Figure imgf000058_0001
Table 1 shows that the up-regulated genes were mostly related to inflammation, particularly macrophage activation, and ECM deposition
Next, myocardial gene expression was compared 3 days after MI vs. sham operation. Upregulated genes were mostly related to inflammation and ECM deposition (Table 2). Down-regulated genes mostly involved cell maintenance. Again, the top upregulated genes were OPN and GPNMB.
Table 2: Gene Expression in Neonatal Hearts after MI Compared with Sham- Operated Hearts.
Figure imgf000059_0001
Table 2 (cont.): Gene Expression in Neonatal Hearts after MI Compared with Sham- Operated Hearts.
Figure imgf000060_0001
As shown in Table 2, up-regulated genes were mostly related to inflammation and ECM deposition where as down-regulated genes mostly involved cell maintenance.
Example 4: Cytokine Profile of Neonatal Cardiac Macrophages
To further explore the role of macrophages in myocardial regeneration, the profile of neonatal macrophage-derived cytokines was analyzed. Macrophages were collected from neonatal hearts following AR, MI, or sham-operation, 3 days after surgery. In addition, macrophages were isolated from adult hearts, 3 days after MI. To obtain the greatest amount of macrophages from the injured heart, we collected macrophages 3 days after injury. The rational of this specific time was based on macrophage kinetic studies in the present (Figure 1C) and previous studies [3]. Quantibody array was used (RayBiotech Inc. Insight Biotechnology Ltd, Wembley, UK.) to determine cytokine secretion characteristics of 18 known cytokines: Interleukin (IL)-lb, IL-4, IL-6, IL-10, IL-12 p40/p70, IL-17, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF) -a, Stromal cell-derived factor (SDF) -1, IL -13, Vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and OPN.
Several differences in cytokine secretion profiles were observed (normalized to macrophage numbers) (Figs. 3A-3E). For example, IL-6, a pleiotropic cytokine involved in both inflammation and regeneration, was increased after injury, particularly after MI in the neonatal heart, compared with sham-operated neonatal hearts (Fig. 3A). Notably, the amount of secreted IL-6 was greater in neonatal than adult macrophages, after myocardial injury.
Macrophage secretion of IL-10, an anti-inflammatory, pro-fibrotic cytokine, and IL-13, an anti-inflammatory, regenerative cytokine were lower, 3 days after cardiac injury, compared with sham operation in neonatal hearts (Figs. 3B and 3C).
Macrophage secretion of VEGF was the highest in adult macrophages (Fig. 3D), 3 days after MI. Surprisingly, neonatal macrophages secreted low amounts of VEGF (normalized to macrophage number) after AR, MI, and sham operation. This finding suggests that the reparative effects of neonatal macrophages are independent of VEGF.
OPN was robustly up regulated after myocardial injury in the neonatal hearts (Tables 1 and 2). OPN is a component of the ECM with immunomodulatory, reparative properties.22 Notably, neonatal macrophages secreted higher amounts of OPN after MI, than after AR or adult macrophages after MI (Fig.3E). Taken together, the data suggested that macrophage secretion of IL-6 and OPN were significantly increased after myocardial injury, particularly in neonatal hearts. Thus, OPN may mediate the reparative and regenerative effects of neonatal macrophages.
Example 5: OPN Stimulated Cardiac Cell Proliferation in Neonatal Heart
To further understand the relevance of the above findings to myocardial regeneration in the neonatal heart, and to determine the role of OPN in myocardial regeneration, an neonatal heart organ culture technique was used, The inventors harvested 1-day-old mouse hearts, sectioned them into 0.5-1 mm pieces and then cultured the sections. Cultured heart explants were treated for 72 hours in cardiomyocyte culture media enriched with OPN at a concentration of 400 and 800ng/ml, and without OPN, as a control group (n=6 in each group). After 72 hours budding of cells from the cultured heart tissues and quantified the outgrowth of cells were observed (Figs. 4A-4F). Figs. 4A, 4C and 4E are microscopic images and Figs. 4B, 4D and 4F are fluorescence images after DAPI staining, with the arrows in these figures indicating cells budding from the heat.
The data showed that 800 ng/ml of OPN significantly increased cardiac cell outgrowth. The outgrowth of cardiac cells was significantly higher in cultures treated with 800ng/ml OPN, than 400ng/ml of OPN and control medium (Fig. 4G). Example 6: OPN Stimulated Cell Cycle in Cardiomyocytes
Myocardial regeneration in the neonatal heart involves proliferation of resident cardiomyocytes [4]. To determine whether OPN can stimulate cardiomyocyte proliferation, 1 -day-old neonatal cardiomyocytes were subjected to 800ng/ml of OPN. Staining for phosphohistone (pH)3, a marker of nuclei division, showed a higher expression of pH3 in one-day-old cardiomyocyte and non-cardiomyocyte cells treated with OPN, compared with controls (Figs. 4H-4K).
Figs. 4J and 4K are quantitative analysis showing the number of PH3/DAPI positive cells in neonatal cardiomyocyte (Fig.4J) and non-cardiomyocyte cells (Fig.4K), numbers were normalized to untreated cells. The results show that OPN increased the number pf pH3 positive cardiac cells, statistical analysis was performed using unpaired t test.
To test the mechanism of action for the observed effects of OPN, the expression of CD44 receptor was tested. A positive expression of CD44 in neonatal cardiomyocytes was confirmed (Fig. 4L). CD44 receptor is known as one of the receptors to which OPN binds.
Next to determine the effect of OPN on the rate of cell death in cardiomyocytes cultured in low glucose, the cells were stained with Propidium Iodide. The percentage of dead cells was lower in cardiomyocytes treated with OPN, than in controls (Fig. 4M). Thus, the results show that OPN did not affected (killed) the number of dead cells in culture.
To further assess cardiomyocyte proliferation, the inventors used tetrazolium dye
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colorimetric assay to assess the number of cultured cardiomyocytes in low-glucose medium. While the number of un-treated cells significantly decreased, OPN preserved the cardiomyocyte count in a low glucose medium for 24 hours (Fig. 4N). Notably, antibodies that block the OPN receptor CD44 eliminate the protective effect of OPN on the number of cardiomyocyte (Fig. 4N).
To confirm the effect of OPN on cultured cardiomyocyte viability and quantity, another cell viability assay RealTime-Glo™ was used. Fluorescent analysis revealed that OPN significantly increased the number of cardiomyocytes, compared with control (Fig. 40).
Taken together, these results show that OPN significantly increased the number of viable cells, compared with controls.
Example 7: OPN Stimulated YAP Signalling and Upregulated Reparative Genes
The Hippo signaling pathway is an evolutionarily ancient cascade of kinases and proteins that control cell proliferation and organ size [14]. Inactivation of the Hippo pathway or activation of its downstream effector, the Yes- Associated Protein (YAP) transcription co-activator, stimulates myocardial regeneration [15].
To determine whether the mechanism by which OPN stimulate cardiomyocyte proliferation and whether the Hippo- Yap pathway is involved, we stimulated neonatal cardiomyocytes with and without OPN (800 ng/ml) for 24 hours. Then, we performed immunoblotting with antibodies against YAP1, pY API-127, LATS1/2 and calculated the ratio between phosphorylated and unphosphorylated proteins. To determine whether the Hippo- YAP pathway mediates the proliferative effects of OPN, immunoblotting with and without OPN was performed. Indeed, OPN induced phosphorylation of YAP 1 in neonatal cardiomyocytes, and the extracellular signal-regulated kinase (ERK)2, while it decreased the phosphorylation of the large tumor-suppressor kinase (LATS)l/2, a YAP1 inhibitor and member of the Hippo signaling pathway (Figs. 5A, B).
YAP is transcriptionally active when it is localized to the nucleus. Histological staining confirmed the effects of OPN on YAP nuclear localization. While YAP in untreated cardiomyocytes was located in the cytoplasm (Figs. 5C), OPN treatment translocated YAP into the nuclei (Fig. 5D). Moreover, blocking OPN signaling, by CD44 Abs, prevented the nuclear localization of YAP (Fig. 4E). Together, the data indicate that OPN activates YAP, together with nuclear localization, via CD44.
To further investigate the effects of OPN on cell cycle, gene expression in cardiomyocytes, with and without OPN exposure was analysed. Analysis revealed that OPN up-regulated the expression of the SPP-1 (OPN gene) (Fig. 5F) and one of the OPN receptors, CD44 receptor (Fig. 5G), as well as cell cycle genes including the transcriptional enhancer factor TEF-1 (TEAD1) (Fig. 5H), which interacts with YAP1, connective tissue growth factor (CTGF) (Fig. 51) and cyclin-dependent kinase 1 (CDK1) (Fig. 5J), which are downstream targets of YAP. Furthermore, OPN significantly up- regulated cyclin B1 (Fig. 5K), a regulator of the mitotic (M) phase, and CCND1, (Fig. 5L) which encode cyclin Dl.
OPN up-regulated other reparative genes such as MMP-12 (Fig.5M) VEGF (Fig. 5N), and CCL2 (Fig. 50), which regulates macrophage recruitment via monocyte chemoattractant protein 1 (MCP1), and insulin-like growth factor -1 (Fig. 5P).The above genes are important for infarct repair.
Overall, these findings indicate that OPN stimulates cell-cycle activity and expression of reparative genes in neonatal cardiomyocytes through YAP1 activity. Because macrophages are a major source of OPN after injury [16], these findings provide another mechanism by which macrophages may contribute to neonatal heart regeneration and repair.
Example 8: OPN Stimulated “Wound Healing” in Neonatal Cardiomyocytes and Fibroblasts
To determine the effect of OPN on cell proliferation and migration, and to mimic myocardial regeneration and repair in vitro, cultured neonatal cardiomyocytes or cardiac fibroblasts were exposed to ’’wound healing” scratch assay [11] and subsequent OPN (800ng/ml) treatment. OPN significantly improved the rate of gap closure in neonatal cardiomyocytes compared with control untreated cardiomyocytes (and addition of CD44 antibodies (Abs) to the culture medium eliminated the favourable effects of OPN (Fig. 6A), suggesting that the effects of OPN on cardiomyocytes are mediated by CD44. Next, the scratch assay was repeated with neonatal cardiac fibroblasts.
The gap was completely closed within 12 hours, 30 time faster than cardiomyocytes (Fig. 6B). Nevertheless, adding OPN into the culture medium enhanced the rate of gap closure. Together, these functional in vitro studies suggest that OPN stimulate proliferation and migration of both neonatal cardiomyocytes and fibroblasts.
Example 9: OPN Stimulates Endothelial Cells (EC) Tube Formation
Angiogenesis is critical element in myocardial regeneration and repair [2]. To determine the effect of OPN on angiogenesis, an EC tube formation assay was used. An incremental dose of OPN stimulated the generation of EC tubes, as assessed by the number of polygons (Fig. 7). Thus, OPN can stimulate angiogenesis. Example 10: OPN Improved LV Remodeling and Function After MI in Adult Mouse
To determine whether the favorable effects of OPN in the neonatal heart are applicable to the adult heart, 12-week-old female mice were allocated to MI and single intra-myocardial injection of OPN (200ng) or control. LV remodeling and global and regional function were assessed by echocardiography and speckle-tracking based strain imaging (Table 3). Significantly, OPN therapy prevented diastolic thinning and improved systolic thickening of the anterior wall (infarcted heart segments) (Figs. 8A, B), posterior wall (Figs. 8C, D), reduced LV dilatation (Figs. 8E-J), and improved LV contractility as reflected by fractional shortening FS, fractional area change FAC and LVEF (Figs. 8K- M), 30 days after ML Taken together, the data showed that significantly, OPN therapy prevented diastolic thinning and improved systolic thickening of the anterior wall (infarcted heart segments), posterior wall, reduced LV dilatation, and improved LV contractility as reflected by FS, FAC and LVEF, 30 days after ML
Compared with conventional echocardiographic imaging, speckle-tracking based strain echocardiography efficiently detects delicate regional changes in cardiac performance after MI, and also identifies early differences in response to treatment [3]. The favorable effects of OPN on myocardial function after MI were supported by LV speckle-tracking-based radial strain analysis in short- and long-axis views. OPN improved regional and global radial strain of the infarcted as well as non-infarcted segments in the short-axis view (Figs. 9A-9H). This favorable effect appeared within one day after MI. These findings were confirmed by the radial strain imaging at the long-axis view (Figs. 9I-9P), particularly at the infarcted apical segment, which is prone to dyskinesis and aneurysm formation (Figs. 9J, 9K). Overall, OPN therapy improved regional and global LV function after MI. Taken together, the data showed that OPN therapy improved regional and global LV function after MI.
Assessment of myocardial fibrosis by picrosirius red staining provided further insight into the mechanism, by which OPN improved cardiac function. OPN significantly increased scar thickness and reduced the length of the scar (Figs. 10A-10F). By thickening the scar, wall stress is reduced (Laplace law) and the degree of outward motion of the infarct that occurs during systole (dyskinesis) is also reduced.
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
The following Table 4 discloses the list of sequences of the present application.
Table 4: Sequences
Figure imgf000069_0001

Claims

CLAIMS:
1. An Osteopontin (OPN) protein or a functional analog thereof for use in the treatment of a non-infectious inflammatory disorder.
2. The OPN protein or a functional analog thereof for use of claim 1 , wherein said OPN protein is or comprises at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:11.
3. The OPN protein or a functional analog thereof for use of claim 1 or 2, wherein said functional analog is at least one of small molecule, an antisense, an siRNA, a polypeptide or a peptide.
4. The OPN protein or a functional analog thereof for use of any one of claims 1 to
3, wherein said functional analog is a polypeptide or a peptide.
5. The OPN protein or a functional analog thereof for use of any one of claims 1 to
4, wherein said functional analog is a derivative or a fragment of the OPN protein.
6. The OPN protein or a functional analog thereof for use any one of claims 1 to 5, wherein said functional analog comprises an amino acid sequence having at least 75% similarity to at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:11.
7. The OPN protein or a functional analog thereof for use any one of claims 1 to 6, wherein said functional analog comprises an amino acid sequence that is at least 75% identical to at least one amino acid sequence denoted by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:11.
8. The OPN protein or a functional analog thereof for use any one of claims 1 to 7, wherein said functional analog is or comprises an amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.
9. The OPN protein or a functional analog thereof for use of claim 8, wherein said functional analog comprises an amino acid sequence that is at least 85% similar to at least one amino acid sequence denoted by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID NO:20, or SEQ ID NO:21.
10. The OPN protein or a functional analog thereof for use of claim 8, wherein said functional analog comprises an amino acid sequence that is at least 85% identical to at least one amino acid sequence denoted by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, or SEQ ID NO:21.
11. The OPN protein or a functional analog thereof for use any one of claims 1 to 7, wherein said functional analog comprises an amino acid sequence denoted by SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
12. The OPN protein or a functional analog thereof for use of claim 11 , wherein said functional analog comprises an amino acid sequence that is at least 85% similar to at least one amino acid sequence denoted by SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
13. The OPN protein or a functional analog thereof for use of claim 11 , wherein said functional analog comprises an amino acid sequence that is at least 85% identical to at least one amino acid sequence denoted by SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.
14. The OPN protein or a functional analog thereof for use of any one of claims 1 to
13, being an isolated, a purified or a synthetic OPN protein or a functional analog thereof.
15. The OPN protein or a functional analog thereof for use of any one of claims 1 to
14, for use in combination with a pharmaceutical carrier.
16. The OPN protein or a functional analog thereof for use of any one of claims 1 to
15, said pharmaceutical carrier is a liposome or an exosome.
17. The OPN protein or a functional analog thereof for use of any one of claims 1 to
16, suitable for parental administration or local administration into a target tissue.
18. The OPN protein or a functional analog thereof for use of claim 17, said administration comprising at least one of intravenous (i.v) injection, intracoronary (i.c), intramuscular (i.m), intraperitoneal (i.p) injection.
19. The OPN protein or a functional analog thereof for use of claim 17, said local administration comprises intramyocardial administration, intracoronary administration, trans coronary administration or retrograde coronary sinus administration.
20. The OPN protein or a functional analog thereof for use of any one of claims 1 to 19, wherein the non-infectious inflammatory disorders is an acute inflammation.
21. The OPN protein or a functional analog thereof for use of any one of claims 1 to 19, wherein the non-infectious inflammatory disorder is involved with a condition selected from the group consisting tissue injury, infarct, trauma, necrosis, sterile inflammatory disorders.
22. The OPN protein or a functional analog thereof for use of any one of claims 1 to 21, wherein the non-infectious inflammatory disorder is a cardiovascular disorder.
23. The OPN protein or a functional analog thereof for use of claim 22, wherein the cardiovascular disorder is selected from or associated with myocarditis, heart failure, atherosclerosis and myocardial infarction.
24. The OPN protein or a functional analog thereof for use of claim 22, wherein said cardiovascular disorder comprises myocardial infarction.
25. A method for treating a non-infectious inflammatory disorder in a subject, the method comprising administrating a subject in need an amount of an OPN protein or a functional analog thereof, the amount being effective to treat the non-infectious inflammatory disorders.
26. The method of claim 22, wherein said OPN protein and said functional analog is as defined in any one of claims 2 to 14.
27. The method of claim 25 or 26, comprising administration of said OPN or functional analog thereof with a pharmaceutical carrier.
28. The method of claim 24, said pharmaceutical carrier is a liposome.
29. The method of any one of claims 25 to 28, comprising administration of said OPN or functional analog thereof by parental administration or local administration into a target tissue.
30. The method of claim 29, said administration comprising at least one of intravenous (i.v) injection, intracoronary (i.c), intramuscular (i.m), intraperitoneal (i.p) injection.
31. The method of claim 30, said local administration comprises intramyocardial administration, intracoronary administration, trans coronary administration or retrograde coronary sinus administration.
32. The method of any one of claims 25 to 31, wherein the non-infectious inflammatory disorders is an acute inflammation.
33. The method of any one of claims 25 to 32, wherein the non-infectious inflammatory disorder is involved with a condition selected from the group consisting tissue injury, infarct, trauma, necrosis, sterile inflammatory disorder.
34. The method of claim 33, wherein the non-infectious inflammatory disorder is a cardiovascular disorder.
35. The method of any one of claims 25 to 34, wherein the non-infectious inflammatory disorders is selected from or associated with myocarditis, heart failure, atherosclerosis and myocardial infarction.
36. The method of claim 35, wherein the non-infectious inflammatory disorder comprises myocardial infarction.
37. A pharmaceutical composition for the treatment of non-infectious inflammatory disorders, the composition comprising as active ingredient an amount of an OPN protein or functional analog thereof as defined in any one of claims 1 to 24, the amount being effective to treat the non-infectious inflammatory disorders.
38. A kit comprising an OPN protein or functional analog thereof as defined in any one of claims 1 to 24 and instructions for use of the OPN protein or functional analog thereof for the treatment of non-infectious inflammatory disorders.
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