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WO2024238337A1 - Peptide à double domaine pour appauvrir hmgb1 de la circulation par l'intermédiaire d'un système hspg du foie - Google Patents

Peptide à double domaine pour appauvrir hmgb1 de la circulation par l'intermédiaire d'un système hspg du foie Download PDF

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WO2024238337A1
WO2024238337A1 PCT/US2024/028808 US2024028808W WO2024238337A1 WO 2024238337 A1 WO2024238337 A1 WO 2024238337A1 US 2024028808 W US2024028808 W US 2024028808W WO 2024238337 A1 WO2024238337 A1 WO 2024238337A1
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disease
mice
hmgbl
hmgb1
lung
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JR. Kirkwood Arthur PRITCHARD
Hao Xu
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Medical College of Wisconsin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/02—Oxidoreductases acting on a peroxide as acceptor (1.11) with H2O2 as acceptor, one oxygen atom of which is incorporated into the product (1.11.2)
    • C12Y111/02002—Myeloperoxidase (1.11.2.2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • This invention relates generally to a small end-capped peptide useful for the management of vasculopathy and chronic lung disease in, e.g., sickle cell anemia (SCA) patients.
  • this invention is directed to a dual-domain peptide designed to bind and deplete high mobility group box-1 (HMGB1) from the circulation via uptake by the liver’s heparin sulfate proteoglycan (HSPG) system and can be used in a titratable fashion appropriate for the disease condition.
  • HMGB1 high mobility group box-1
  • HSPG heparin sulfate proteoglycan
  • Sickle cell disease is a chronic, devastating family of closely related disease conditions affecting nearly 100,000 people in the USA and an estimated 20 million people worldwide.
  • the most severe form of SCD is homozygous sickle cell anemia (SCA), the most common SCD condition.
  • SCA sickle cell anemia
  • SCA the most common SCD condition.
  • SCA sickle cell anemia
  • childhood mortality from SCD in high-income countries is similar to the mortality rates in the general population, and although the median survival of adults with SCD is approaching more than 60 years, this notable achievement is still 16-17 years shorter than the average life expectancy at birth for all people in the USA.
  • Vasculopathy and chronic lung disease are progressive sequelae that contribute to the morbidity and mortality of individuals who have sickle cell anemia (SCA).
  • Sickle red blood cells are more fragile, oxidatively damaged, and rigid (due to hemoglobin S polymerization) than normal red blood cells (RBC).
  • Sickle red blood cells (sRBC) can become trapped in the microcirculation, impairing blood flow, and increasing the risk of rupture resulting in the intravascular release of cell-free hemoglobin (cf-Hb) that, after oxygenation, generates superoxide anion (O2-»).
  • the latter scavenges nitric oxide (»NO) and is the mechanistic basis for SCA’s increased resistance to *NO donors, such as sodium nitrite.
  • the pathobiology of SCD is considered to be mediated by a vicious cycle composed of four interconnected processes; 1) hemoglobin (Hb) polymerization, 2) altered red blood cell rheology and adhesion-mediated vaso-occlusion, 3) hemolysis-mediated endothelial dysfunction, and 4) concerted activation of sterile inflammation.
  • Vasculopathy is often considered to precede chronic organ injury.
  • pathogenic steps combine to form the systemic and pathophysiological processes used to characterize SCA, such as chronic oxidative stress and inflammation, hypercoagulation, and repeated bouts of ischemia-reperfusion injury. Therapeutic targeting of the mechanisms mediating these processes underpins current SCD drug development.
  • the term “sickle cell disease” or “SCD” is used when discussing the vasculopathy and chronic organ disease that develops in Townes sickle cell mice, the humanized strain that most closely replicates human SCA.
  • SCD smooth muscle cell disease
  • SCA smooth muscle cell anemia
  • the hE-HB- B10 peptide was based on prior work linking the arginine-rich heparin-binding domain of apolipoprotein E (hE, LRKLRKRLLR) to 18A, an amphipathic a-helical domain from apolipoprotein A-l, to generate an hE-dual -domain peptide or apo E mimetic chimeric peptide called hE18A or Ac-hE18A-NH2 (indicating the said peptide is end capped).
  • HSPG heparin sulfate proteoglycan
  • the present invention provides a composition for treating a symptom or disorder associated with excess recruitment of myeloid cells that express myeloperoxidase/peroxidase thus indirectly reducing peroxidase activity in the subject comprising: an effective amount of an end-capped (N-acetylated, C-amidated) dualdomain peptide hE-HMGBl-BP (Ac-LRKLRKRLLR-GG-AHSANNFDVKGI-NH 2 , acetate salt) configured to bind and deplete HMGB1 from circulation via uptake by the liver’s heparin sulfate proteoglycan (HSPG) system; and an acceptable carrier.
  • an end-capped (N-acetylated, C-amidated) dualdomain peptide hE-HMGBl-BP Ac-LRKLRKRLLR-GG-AHSANNFDVKGI-NH 2 , acetate salt
  • HSPG heparin sulfate proteoglycan
  • composition may further comprise an effective amount of N-acetyl- lysyltyrosylcysteine amide (KYC) designed to inhibit myeloperoxidase (MPO)-initiated and HMGB1 -propagated inflammatory pathway.
  • KYC N-acetyl- lysyltyrosylcysteine amide
  • the end capped dual-domain peptide hE-HMGBl-BP may contain a GG bridge between 12mer HMGB1-BP peptide domain and apoE receptor binding peptide domain (hE).
  • the 12mer peptide identified by phage display is designed to bind to HMGB1 and to be removed from the circulation by uptake by the liver through the HSPG system or the apolipoprotein E receptor domain.
  • the composition may be in a unit dosage form selected from the group consisting of a tablet, a capsule, a solution, a suspension, a syrup, a beverage, an oral or ophthalmic formulation and an injection.
  • the present invention provides a method for treating a symptom or disorder associated with excess recruitment of myeloid cells that express myeloperoxidase/peroxidase thus indirectly reducing peroxidase activity in the subject comprising administering to a subject in need of such treatment an effective amount of endcapped (N-acetylated, C-amidated) dual-domain peptide hE-HMGBl-BP (Ac-LRKLRKRLLR- GG-AHSANNFDVKGI-NH _, 2, acetate salt) designed to bind and deplete HMGB1 from circulation via uptake by the liver’s heparin sulfate proteoglycan (HSPG) system.
  • endcapped (N-acetylated, C-amidated) dual-domain peptide hE-HMGBl-BP Ac-LRKLRKRLLR- GG-AHSANNFDVKGI-NH _, 2, acetate salt
  • the method may further comprise administering to the subject in need of such treatment an effective amount of N-acetyl-lysyltyrosylcysteine amide (KYC) designed to inhibit the myeloperoxidase (MPO)-initiated and HMGB 1 -propagated inflammatory pathway in bronchopulmonary dysplasia.
  • KYC N-acetyl-lysyltyrosylcysteine amide
  • MPO myeloperoxidase
  • HMGB 1 myeloperoxidase
  • the method may further comprise administering to the subject in need of such treatment an effective amount of N6022 (l-[4-(aminocarbonyl)-2-methylphenyl]-5-[4-(lH- imidazol-l-yl)phenyl]-lH-pyrrole-2 -propanoic acid) designed to inhibit GSNOR.
  • hE-HMGB 1 -BP defined by an end-capped (N-acetylated, C- amidated) dual-domain peptide to said subject may improve vascular function, decrease pulmonary inflammation, and/or increase cardioprotection in the subject.
  • the symptom or disorder may be associated with excess recruitment of myeloid cells that express myeloperoxidase/peroxidase thus indirectly reducing peroxidase activity in the subject is at least one of wound inflammation, hypersensitivity, digestive disease, cardiovascular disease, neuronal disease, lung disease, autoimmune disease, degenerative neurological disease, degenerative muscle disease, infectious disease, disease associated with graft transplantation, allergic disease, musculo-skeletal inflammation, and sepsis.
  • the symptom or disorder associated with excess recruitment of myeloid cells that express myeloperoxidase/peroxidase thus indirectly reducing peroxidase activity in the subject may be at least one of hypertension, peripheral vascular disease, pulmonary inflammation, asthma, atherosclerosis, diabetes, persistent pulmonary hypertension, sickle cell disease, neurodegenerative disease, multiple sclerosis, Alzheimer's disease, lung cancer, lupus, ischemic heart disease, Parkinson's disease, Crohn's disease, inflammatory bowel disease, necrotizing enterocolitis, arthritis, polymyocytis, cardiomyopathy, psoriasis, amyotrophic lateral sclerosis, muscular dystrophy, cystic fibrosis, attention deficiency hyperactive disorder, acute lung injury, acute respiratory distress syndrome, flu (including HINT), heart failure, chemotherapy -induced heart failure, arthritis, rheumatoid arthritis, acute myocardial infarction, traumatic brain injury (TBI), chronic traumatic encephalopathy (
  • the present invention provides use of an end-capped (N-acetylated, C-amidated) dual-domain peptide hE-HMGB 1 -BP (Ac- LRKLRKRLLR-GG-AHSANNFDVKGI-NH-2, acetate salt) for the manufacture of a pharmaceutical composition for alleviating a symptom associated with excess recruitment of myeloid cells that express myeloperoxidase/peroxidase thus indirectly reducing peroxidase activity in the subject.
  • the pharmaceutical composition may be formulated as an oral dose comprising the end capped dual -domain peptide hE-HMGBl-BP and a carrier.
  • the present invention provides an endcapped (N-acetylated, C-amidated) dual-domain peptide hE-HMGBl-BP (Ac-LRKLRKRLLR- GG-AHSANNFDVKGI-NH _, 2, acetate salt) for use in alleviating a symptom associated with excess recruitment of myeloid cells that express myeloperoxidase/peroxidase thus indirectly reducing peroxidase activity in the subject.
  • the end capped dual-domain peptide hE-HMGBl-BP may be formulated as an oral dose comprising the dual -domain peptide hE-HMGBl-BP and a carrier.
  • the present invention shows that SCA causes vasculopathy and chronic lung disease by an HMGB1- and GSNOR-dependent mechanism where HMGB1 induces GSNOR.
  • SCD induces vasculopathy and chronic lung injury by increasing HMGB 1 and GSNOR.
  • HMGB1 increases GSNOR, impairing vasodilation and increasing chronic lung injury.
  • HMGB1 and GSNOR reduce vascular and pulmonary nitric oxide bioavailability.
  • Figures 1A-1E Effects of hE-HMGBl-BP on Vasodilation and Plasma HMGB1 in SS Mice.
  • Figures 2A-2B Recombinant HMGB1 Increases Endothelial GSNOR, and hE- HMGB1-BP Treatment of SS Mice Reduces GSNOR Protein in Facialis Arteries.
  • Figure 2A Representative chemiluminescence images of GSNOR immunoblots and total protein blots of facialis artery isolates and extracts from PBS- and hE-HMGBl -BP-treated SS mice.
  • Facialis artery isolates from SS mice treated with hE-HMGBl -BP (2.72 mg/kg/d, 3 weeks) (n 9) contain -20% less GSNOR protein than facialis artery isolates from SS mice treated with PBS.
  • Figures 3A-3D Effects of N6022 and KYC on Vasodilation in SS Mice.
  • Figure 3B Line graph showing the effects of KYC treatment (sterile PBS [1% DMSO]; 3 mg/kg/d, 3 weeks) on ACh- induced vasodilation ⁇ L-NAME (100 pM) of facialis arteries from SS mice.
  • FIGS 4A-4B SCA Decreases Protein SNOs and Increases GSNOR in Lungs.
  • Figure 4A Upper Left Panel: Chemiluminescence immunoblots showing the images of protein SNOs and P-actin per lane of lung homogenates prepared from AA and SS mice.
  • Band-Intensity Ratio (BIR) data from total anti-iodoTMT antibody band intensities per lane were divided by their corresponding P-actin band intensity, and BIR ratios normalized to the mean of AA mice (1.0) and plotted as scatter plots in bars (mean ⁇ SD).
  • BIR Band-Intensity Ratio
  • BIR Band-Intensity Ratio
  • Figures 5A-5D Effects of SCA and hE-HMGBl-BP and KYC Treatments on Protein SNOs and GSNOR in Lungs.
  • Figure 5 A AA and PBS-treated SS mouse lung protein SNOs. Representative chemiluminescence immunoblots of protein SNOs and P-actin from AA and SS mice treated with PBS, hE-HMGBl-BP, or KYC. Test group and mouse ID numbers are provided for each lane.
  • Figure 5B AA and PBS-treated SS mouse lung GSNOR.
  • FIG. 6A-6D Effects of SCA and hE-HMGB 1 -BP and KYC Treatments on CD31 and GSNOR Lung Expression.
  • Figure 6A Lung Sections from AA and SS mice immunostained for CD31 and GSNOR.
  • Representative merged lung sections (three each) from AA and SS mice treated with PBS, hE-HMGB 1-BP, or KYC test groups. Sections were immunostained for CD31 (green), GSNOR (red), and nuclei (blue - DAPI).
  • Figure 6B Relative fluorescence intensities for CD31 were randomly captured from five to four regions. CD31 fluorescence intensities were determined using NIH Image J, tested for outliers, and averaged. Average intensities for CD31 in each lung were plotted as scatter plots in bars (mean ⁇ SD) for AA and SS mice treated with PBS, hE-HMGBl-BP, and KYC.
  • Average intensities for GSNOR in each lung were plotted as scatter plots in bars (mean ⁇ SD) for AA, SS mice treated with PBS, hE-HMGBl-BP, and KYC, showing the mean ⁇ SD for each test group.
  • GSNOR dehydrogenase activity in lung lysates was determined from the decrease in NADH absorbance at 340 nm after the addition of GSNO per Liu et al.
  • the decrease in absorbance at 340 nm was calculated by subtracting the values of the negative controls from the absorbance in duplicate samples.
  • the resulting GSNOR activity was expressed as a ratio of the absorbance decrease in individual samples to the average absorbance decrease in AA samples and plotted as scatter plots in bars (mean ⁇ SD).
  • Figures 7A-7C Effects of SCA and hE-HMGBl-BP and KYC Treatments Lung Simplification and Acinar Airspace in SS Mice.
  • FIG. 8 A diagram illustrating the SCA inflammatory pathway of the present invention.
  • FIG. 9 Biolayer Interferometer association and dissociation curves for HMGB 1 -BP and rh-HMGBl.
  • the raw data from the kinetic studies were analyzed with Octet Data Analysis 9.0.
  • the baseline was subtracted from the wavelength shift that occurs in response to increasing concentrations of HMGB 1 binding to HMGB 1 -BP immobilized on the biosensors. Based on these shifts in wavelengths to the different HMGB1 concentrations, the Kd of HMGB 1 -BP for HMGB1 is estimated to be 170 ⁇ 36 nM.
  • FIG. 10 HMGB1 immunoblots showing the association of biotin-labeled HMGB1- BP (Biotin- Ahx-AHSANNFDVKGI-amide) and biotin-Ahx-scrambled-HMGBl-BP with plasma spiked with rHMGBl.
  • the immunoblots show that beads alone have little affinity for HMGB1.
  • biotin Ahx-HMGBl-BP has a high specific affinity for r- HMGB1.
  • the affinity of biotin- AhxscHMGBl -BP is greatly reduced, being reduced to random ionic and lipophilic interactions rather than the site-specific interaction with HMGB1- BP.
  • FIG 11 through Figure 14 SI-Gels 1-4 Protein SNO Protein SNOs and intensities for AA and SS mice treated with PBS, hE-HMGBl-BP, or KYC were divided by their corresponding [3-actin band intensities and normalized by the mean of AA mice (1.0).
  • Figure 16 The bar scatter plots show that SCA increases lung GSNOR and that hE- HMGBl-BP and KYC treatment decreases GSNOR in the lungs of SS mice to levels that are essentially equivalent to genetic control mice.
  • subject means mammals and non-mammals.
  • “Mammals” means any member of the class Mammalia including, but not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and the like.
  • non-mammals include, but are not limited to, birds, and the like.
  • the term "subject” does not denote a particular age or sex.
  • administering includes any means for introducing a compound of the present invention into the body, preferably into the systemic circulation. Examples include but are not limited to oral, buccal, sublingual, pulmonary, transdermal, transmucosal, as well as subcutaneous, intraperitoneal, intravenous, and intramuscular injection.
  • the phrase "therapeutically effective amount” means the amount of a compound that, when administered to a subject for treating a disease or disorder, is sufficient to affect such treatment for the disease or disorder.
  • the “therapeutically effective amount” can vary depending on the compound, the disease or disorder and its severity, and the age, weight, etc., of the subject to be treated.
  • the term “treating” or “treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treating” or “treatment” refers to delaying the onset of the disease or disorder, or even preventing the same.
  • peptide refers to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • peptide specifically includes the non-genetically coded amino acids that either occur naturally or are chemically synthesized including, but not limited to synthetic .alpha.- and .beta.-amino acids known to one of skill in the art.
  • the nature of the invention is a small end-capped peptide.
  • the physical and biochemical nature of the invention is an end-capped (N-acetylated, C-amidated) dual-domain peptide.
  • High mobility group box-1 (HMGB1) is a predominant mediator of TLR4-reporter cell activity in the plasma from SS mice and humans.
  • the present inventors designed and made a second hE-dual-domain peptide to target HMGB1, called hE-HMGBl-BP, using the same protocols previously used to make hE-HB-BlO and as described in M.S. Hanson, H. Xu, T.C. Flewelen, S.L. Holzhauer, D. Retherford, D.W. Jones, A.C. Frei, K.A. Pritchard, Jr., C.A. Hillery, N. Hogg, N.J.
  • SS mice were treated with hE-HMGB 1-BP and the effects of targeting extracellular HMGB1 on vasodilation and lung morphometries was determined.
  • SCA myeloperoxidase
  • MPO myeloperoxidase
  • SS mice were also treated with N-acetyl-lysyltyrosylcysteine amide (KYC).
  • KYC is a systems pharmacology agent that inhibits the MPO-initiated and HMGB 1 -propagated inflammatory pathway in bronchopulmonary dysplasia.
  • HMGB 1 increases vascular inflammation by inducing S-nitrosoglutathione reductase (GSNOR) activity, and dysregulated GSNOR impairs vascular endothelial cell function
  • SS mice were treated with N6022 (l-[4-(aminocarbonyl)-2-methylphenyl]-5-[4-(lH- imidazol-l-yl)phenyl]-lH-pyrrole-2 -propanoic acid) to inhibit GSNOR.
  • N6022 l-[4-(aminocarbonyl)-2-methylphenyl]-5-[4-(lH- imidazol-l-yl)phenyl]-lH-pyrrole-2 -propanoic acid
  • the present invention shows the inflammatory roles of HMGB 1 in SCD pathogenesis. Given previous reports linking HMGB1 to impaired vasodilation, a similar effect in SCD should be anticipated. However, surprisingly, it has been found that HMGB1 induces vascular and pulmonary S-nitrosoglutathione reductase (GSNOR), a novel mediator that decreases *NO bioavailability. Therefore, the present invention illustrates the mechanisms by which SCD induces vasculopathy and chronic organ injury by increasing sterile inflammation.
  • GSNOR S-nitrosoglutathione reductase
  • hE-HMGBl-BP A dual-domain or chimeric peptide called hE-HMGBl-BP (Ac-LRKLRKRLLR-GG- AHSANNFDVKGI-NH2, acetate salt) was designed to bind and deplete high mobility group box-1 (HMGB1) from the circulation via uptake by the liver’s heparin sulfate proteoglycan (HSPG) system.
  • HMGB1 high mobility group box-1
  • HSPG heparin sulfate proteoglycan
  • the dual-domain peptide contains the hE or human apolipoprotein E receptor domain (LRKLRKRLLR, denoted hE; residues 141-150) and a 12mer peptide identified by the combination of phage display and Biolayer Interferometry to determine which phage 12mer had the highest affinity for recombinant human HMGB1.
  • hE human apolipoprotein E receptor domain
  • HMGB1-BP HMGB1 binding peptide
  • the purpose of the dual-domain peptide is to bind and remove HMGB 1 from the circulation via uptake of the liver’s HSPG system.
  • the operation of the dual -domain peptide is the 12mer peptide identified by phage display binds to HMGB1 with an apparent Kd of 170 ⁇ 36 nM and is removed from the circulation by uptake by the liver through the HSPG system or the apolipoprotein E receptor domain.
  • MPO myeloperoxidase
  • HMGB1 is a potent damage-associated molecular pattern (DAMP) molecule that binds and induces Toll-like receptor 4 (TLR4)-dependent inflammation, increases vascular permeability and recruitment of neutrophils and mononuclear cells, all the while impairing vasodilation and increasing apoptotic cell death.
  • DAMP damage-associated molecular pattern
  • TLR4 Toll-like receptor 4
  • MPO and HMGB1 are both components in sterile inflammation, where MPO initiates oxidative vascular and organ injury and HMGB1 propagates the cycle by recruiting innate immune cells to sites of injury.
  • compositions for simultaneously binding and removing HMGB1 inflammatory activity and targeting other disease-related pathways containing one or more of the peptides described above and a pharmaceutically acceptable carrier.
  • these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampules, auto-injector devices or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation.
  • the peptides of the present invention may be incorporated into transdermal articles designed to deliver the appropriate amount of peptide in a continuous fashion.
  • an inhibitor according to the invention is administered directly to HMGB1, to a tissue comprising HMGB1, a body fluid that contacts HMGB1, or a body location from which the inhibitor can diffuse or be transported to the HMGB1. It is sufficient that the inhibitor is administered to the subject in an amount and by a route whereby an amount of the inhibitor sufficient to bind HMGB1 arrives, directly or indirectly to HMGB1.
  • the liquid forms in which the novel compositions of the present invention may be incorporated for administration by injection include aqueous solutions and similar pharmaceutical vehicles.
  • the peptides of the invention will be provided as pharmaceutically acceptable salts.
  • Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts which may, for example, be formed by mixing a solution of the peptide according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.
  • a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.
  • suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts, alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.
  • alkali metal salts e.g., sodium or potassium salts
  • alkaline earth metal salts e.g., calcium or magnesium salts
  • suitable organic ligands e.g., quaternary ammonium salts.
  • Suitable dosage levels are provided for binding HMGB1 and removing HMGB1 from circulation thus preventing HMGB1 from binding to proinflammatory receptors such as TLR4, RAGE or receptors that send out signals to recruit neutrophils and other innate immune cells.
  • suitable dosage levels for reducing the recruitment of myeloid cells that express myeloperoxidase/peroxidase and indirectly reducing peroxidase activity in a human subject i.e., an effective therapeutic amount to bind to HMGB1 and reduce the excess recruitment of myeloid cells thus indirectly reducing peroxidase activity
  • an effective therapeutic amount to bind to HMGB1 and reduce the excess recruitment of myeloid cells thus indirectly reducing peroxidase activity is about 0.01-1000 mg/kg, per day, preferably about 0.1-500 mg/kg per day, and especially about 0.1-50 mg/kg per day.
  • the invention provides a method of treating a disease or condition in a subject that is associated with excess recruitment of myeloid cells that express myeloperoxidase/peroxidase thus indirectly reducing peroxidase activity.
  • the method includes the step of administering to a subject in need of such therapy one or more of the peptides as described above.
  • the subject is a human or a non-human mammal.
  • the method includes the additional step of mixing the peptide with a pharmaceutically acceptable carrier before the peptide is administered.
  • the method is carried out to improve vascular function, decrease pulmonary inflammation, and/or increase cardioprotection in the subject.
  • inventive peptides act on a molecular process common to a plethora of medical diseases and conditions.
  • the present peptides are envisioned to be useful in treating a wide range of diseases and conditions attributable to excess recruitment of myeloid cells that express myeloperoxidase or eosinophil peroxidase and indirectly associated with aberrant peroxidase activity, including but not limited to, wound inflammation, hypersensitivity, digestive disease, cardiovascular disease, neuronal disease, lung disease, autoimmune disease, degenerative neurological disease, degenerative muscle disease, infectious disease, disease associated with graft transplantation, allergic disease, skeletal inflammation, and sepsis.
  • Methods of the invention are further envisioned to be useful in treating hypertension, peripheral vascular disease, pulmonary inflammation, asthma, atherosclerosis, diabetes, persistent pulmonary hypertension, sickle cell disease, neurodegenerative disease, multiple sclerosis, Alzheimer’s disease, lung cancer, lupus, ischemic heart disease, Parkinson’s disease, Crohn’s disease, inflammatory bowel disease, necrotizing enterocolitis, arthritis, polymyocytis, cardiomyopathy, psoriasis, amyotrophic lateral sclerosis, muscular dystrophy, cystic fibrosis, attention deficiency hyperactive disorder, acute lung injury, acute respiratory distress syndrome, flu (including H1N1), heart failure, chemotherapy-induced heart failure, arthritis, rheumatoid arthritis, acute myocardial infarction, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), ischemic or hemorrhagic stroke, or bronchopulmonary dysplasia.
  • TBI traumatic brain injury
  • CTE
  • the disclosed methods will find use in the promotion of angiogenesis in tissues of a subject, or the promotion of angiogenesis impaired by persistent pulmonary hypertension, peripheral vascular disease or vascular disease in the myocardium in the subject, or the treatment of a disease or condition associated with abnormal, excessive blood vessel development in the subject.
  • the disclosed methods are additionally useful in treating subjects for the reduction and/or prevention of ischemic injury to a subject's heart following an ischemic event or insult.
  • the disclosure also encompasses the use of a peptide as described herein for the manufacture of a medicament for reducing the excess recruitment of myeloid cells that express myeloperoxidase/peroxidase thus indirectly reducing peroxidase activity and subsequently targeting a second pathway in a subject.
  • Such methods include the steps of (a) providing a peptide as described herein, and (b) mixing the peptide with a pharmaceutically acceptable carrier.
  • the invention encompasses the manufacture and use of medicaments specifically purposed for treatment of one or more of the diseases/conditions described above.
  • Example 1- HMGB1 and GSNOR Induce Vasculopathy and Chronic Lung Injury in SCD
  • mice are phenotypically normal.
  • Townes Hbatml(HBA)Tow/Hbbtm2(HBGl,HBB*)Tow mice express sickle beta-hemoglobin, have murine SCD and are referred to as SCD mice or SS mice.
  • Untreated AA mice were used as genetic controls for SS mice. All mice were housed until they were 8-9 months old. Mice were cared for according to the Association for Assessment and Accreditation of Laboratory Animal Care specifications. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC). Both male and female mice were used, and data were pooled for some experiments.
  • IACUC Institutional Animal Care and Use Committee
  • hE-HMGBl-BP Design and Validation.
  • the hE-HMGBl-BP was designed and developed using the same strategy and phage display protocols used to design and develop hE- Hb-BlO to reduce cf-Hb in the circulation of mice as described in M.S. Hanson, H. Xu, T.C. Flewelen, S.L. Holzhauer, D. Retherford, D.W. Jones, A.C. Frei, K.A. Pritchard, Jr., C.A. Hillery, N. Hogg, N.J.
  • hE-Hb-BlO dualdomain peptide is that hE-HMGBl-BP (Ac-LRKLRKRLLR-GG-AHSANNFDVKGLNH-2, acetate salt) contains a GG bridge between the 12mer HMGB1-BP peptide domain and the apoE receptor binding peptide domain (hE).
  • HUVEC cultures were maintained in HUVEC media [DMEM (Catalog #1001, ScienCell, Carlsbad, CA) + L-glutamine, 10% FBS, 1% antibiotic/antimycotics] until confluent.
  • HUVEC cultures were treated with fresh media without or with r-HMGBl (20 ng/mL) each day for five days. HUVEC cultures were washed three times with serum-free DMEM media (#09221, ScienCell, Carlsbad, CA) supplemented with L-glutamine and 1% antibiotic/antimycotic) and cell proteins isolated using MOPS lysis buffer (20 mM MOPS, 2 mM EGTA, 5 mM EDTA, 30 mM NaF, 10 mM 0- glycerophosphate, 10 mM Na pyrophosphate, 2 mM Na orthovanadate, 1 mM PMSF, 0.5% NP- 40, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktails 2 and 3, pH 7.0) and immunoblotted for GSNOR using a rabbit GNSOR antibody GTX89762 (Genetex, Irvine, CA) and 0-actin using a mouse 0-actin antibody (A2228, Sigma-Ald
  • mice Effects of hE-HMGBl-BP on vasodilation, eNOS-dependent vasodilation, and plasma HMGB1 Townes SS mice were treated daily with hE-HMGBl-BP (Biomatik Corp., Kitchener, Ontario; 2.72 mg/kg/d, subcutaneous injection) for three weeks.
  • mice On the day of the vasodilation studies, mice were anesthetized with isoflurane (1-3%), subjected to thoracotomy, and euthanized by exsanguination. A hypodermic needle was inserted into the heart’s right side, and blood was drawn into EDTA. Plasma was separated by centrifugation.
  • the plasma was divided into aliquots and stored at -80oC until analysis for HMGB 1 using ELISA kits from Tecan (30164033, Mannedorf, Switzerland) as described. Facialis arteries were isolated by microdissection, cannulated, and pressurized to 60 mmHg.
  • MOPS physiological buffer [CaC12 2H2O (2.0 mM), EDTA (0.02 mM), glucose (5.0 mM), KC1 (4.7 mM), MgSO4 «7H2O (1.17 mM), MOPS (3.0 mM), NaCl (145 mM), NaH2PO4OH2O (1.2 mM), pyruvic acid (2.0 mM)] was replaced with 37oC MOPS physiological buffer containing L- NAME (100 pM) and Ach-induced relaxation responses determined once again, as above.
  • eNOS-dependent vasodilation was determined from the area between curves (ABC). The area between vasodilation curves in the absence and presence of L-NAME was calculated using the area under the curve function in Prism GraphPad 9.5.0 after correction of paired data for negative relaxation responses.
  • vasodilation or endothelial-dependent is defined as the relaxation response of a pressurized facialis artery, preconstricted with U46619 and stimulated with increasing concentrations of Ach added to the physiological bath.
  • eNOS-dependent vasodilation is the difference in ABC of the relaxation responses in the absence and presence of L-NAME in response to accumulating doses of Ach added to the bath as above.
  • the homogenate was then concentrated by centrifugation using a spin column with a molecular cut-off of lOkDa (BioVision, Milpitas, CA; catalog# 1997-25). Proteins in concentrated facialis artery lysates were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and membranes immunoblotted with anti -GSNOR/ ADH5 rabbit polyclonal antibody (Proteintech Group Inc., Rosemont, IL; catalog#16379-l-AP) to determine GSNOR expression. GSNOR band intensities were normalized to total protein visualized with TGX Stain-Free gel technology (BioRad, Hercules, CA; catalog# 4568094).
  • SS mice were treated with phosphate-buffered saline (PBS) or N6022 (l-[4-(aminocarbonyl)-2-methylphenyl]-5-[4- (lH-imidazol-l-yl)phenyl]-lH-pyrrole-2-propanoic acid; Cayman Chemical, Ann Arbor, MI; subcutaneous injection; 1 mg/kg/d, 3 weeks).
  • PBS phosphate-buffered saline
  • N6022 l-[4-(aminocarbonyl)-2-methylphenyl]-5-[4- (lH-imidazol-l-yl)phenyl]-lH-pyrrole-2-propanoic acid
  • Cayman Chemical Ann Arbor, MI
  • subcutaneous injection 1 mg/kg/d, 3 weeks.
  • DMSO dimethyl sulfoxide
  • the right main bronchus was ligated with silk at the carina and snap-frozen in liquid nitrogen for immunoblot analysis after resection below the ligature.
  • the trachea was then cannulated with an Instech Solomon (20G) stainless steel feeding tube (Plymouth Meeting, PA).
  • the left lobe was inflated with 10% neutral buffered formalin at 25 cm-H20 (2.4 kPa).
  • the fixed lung was stored in 10% buffered formalin for 24 h and then embedded in paraffin for histology.
  • Lung sections (5 pm) were mounted on SuperFrost Plus-coated slides (Denville Scientific, Metuchen, NJ).
  • Endothelial-dependent vasodilation in AA mice is robust achieving relaxation responses to Ach greater than 50% of the Dmax of the pressurized vessel ( Figure 1).
  • endothelial-dependent vasodilation in SS mice was impaired by more than 75% of the vasodilation in AA mice (p ⁇ 0.0001).
  • N6022 requires 1% DMSO to be added to injection PBS buffer to ensure complete dissolution.
  • SS mice were treated with PBS+1% DMSO as injection control for the DMSO in the N6022 injection buffer.
  • SS+PBS mice had little, if any eNOS-dependent vasodilation (Figure 3A, ns), which contrasts with the vasodilation in facialis arteries isolated from SS mice treated with neat PBS as an injection control ( Figure 1A).
  • FIG. 5A and 5B Representative protein SNOs and GSNOR immunoblots can be seen in Figures 5A and 5B, respectively.
  • the immunoblots for protein SNOs and GSNOR for all mice in the test groups can be found in Figure 11 to Figure 15.
  • Band intensities integrated for the whole lane revealed decreased lung protein SNOs in PBS-treated SS mice (p ⁇ 0.01).
  • CD31 and GSNOR resulted in marked increases of yellow, or purple immunofluorescence. These colors are best explained as the result of GSNOR (red) co-localizing with CD31+ cells (green) to make yellow, or co-localizing with cell nuclei (blue) to make purple in lung sections prepared from SS+PBS mice.
  • CD31 is expressed on most non-erythroid cells of hematopoietic lineage that includes platelets, monocytes, neutrophils, human T cells, and human and mouse B cell subsets.
  • the notable increase in yellow staining in sections of lungs from SS mice could be attributed to any combination of CD31+ cells, like endothelial cells or innate immune cells (neutrophils, platelets). Previous reports indicate that CD31+ cells often form mixed aggregates in SCD.
  • CD31 + cell staining in the lungs of SS mice treated with KYC decreased by 40% compared to levels of CD31+ cell staining in sections of lungs from SS mice treated with PBS ( Figure 6B, p ⁇ 0.05), which was within 20% of the CD31+ cell staining in sections of lungs from untreated AA mice.
  • GSNOR staining (red) in sections of lungs from SS mice treated with PBS increased by 9.3-fold compared to staining sections of lungs from untreated AA mice ( Figure 6C, p ⁇ 0.05).
  • GSNOR staining in sections of lungs from SS mice treated with hE-HMGB l-BP decreased by 64%
  • GSNOR staining in sections of lungs from SS mice treated with KYC decreased by 85%.
  • the present inventors also measured GSNOR dehydrogenase activity in lung homogenates from these mice following previous established protocols described by others. The studies showed that SCD increased lung GSNOR dehydrogenase activity by nearly 20% ( Figure 6D, p ⁇ 0.05).
  • GSNOR dehydrogenase activity decreased by approximately 18% in lung homogenates from SS mice treated with hE-HMGBl-BP or KYC ( Figure 6D, p ⁇ 0.05), to levels that were essentially the same as the GSNOR dehydrogenase activity in lung homogenates from AA mice ( Figure 6D, ns).
  • GSNOR immunostaining in blood vessels and airway smooth muscle cells appeared more prominent than in other types of lung cells, confirming observations by Raffay et al..
  • GSNOR staining in alveolar microvessels in SS+hE-HMGBl-BP and SS+KYC mice were not reduced to the levels observed in these same microvessels in untreated AA mice.
  • SS+hE-HMGBl-BP and SS+KYC RAC numbers were intermediate to the RAC numbers in AA and SS+PBS lungs (p ⁇ 0.01).
  • SS+PBS lung MLI were increased compared to the MLI in AA lungs ( Figure 7C, p ⁇ 0.0001).
  • MLI in SS+hE-HMGBl-BP and SS+KYC mice were shorter that MLI in SS+PBS mice and were found to be essentially indistinguishable from MLI line lengths in lungs from AA mice ( Figure 7C, p ⁇ 0.01, ns, respectively).
  • Example 1 demonstrates that SCD impairs vasodilation and induces chronic lung disease by an HMGB1- and GSNOR-dependent mechanism. Specifically, SCD impairs endothelial- and eNOS-dependent vasodilation and worsens lung morphometries by HMGB1- mediated processes. Decreasing plasma HMGB1 levels with hE-HMGBl-BP significantly improves vasodilation and lung morphometries in SS mice compared to levels in untreated control AA mice.
  • HMGB 1 increases both GSNOR expression and activity to reduce vascular and pulmonary intracellular *NO bioavailability, resulting in impaired vasodilation and worsening of lung morphometries in SS mice.
  • Treating SS mice with N6022 to inhibit vascular GSNOR activity provides additional support for the idea that dysregulated GSNOR activity plays a causal role in how SCD impairs vasodilation in mice.
  • HMGB1 is involved in arteriopathy in SCD.
  • the present invention shows that targeting HMGB1 not only improves vasodilation but also reduces chronic lung injury, confirming HMGBl ’s role in these two mechanisms in sterile inflammation pathogenesis.
  • HMGB1 is likely more important in SCD than currently recognized and the findings bring a new perspective. It is clear HMGB1 impairs endothelial function and has greater impact on chronic lung injury than cf-Hb.
  • the present studies do not downplay cf-Hb’ s role in SCD. Rather, they position HMGB1 as a mechanism in sterile inflammation as an equally or more significant factor in SCD. This viewpoint is vital for expanding the understanding of the mechanisms mediating SCD and highlights the need for unbiased and comprehensive experiments that address SCD’s complex multifactorial nature.
  • HMGB1 and cf-Hb add additional complexity to understanding how these two agents impair vascular function and induce chronic lung disease in SCD.
  • HMGB1 and cf-Hb form a complex that is reported to synergistically activate TLR4. Targeting HMGB1 seems more effective at improving vasodilation in SS mice than targeting cf- Hb.
  • MPO, HMGB1, and GSNOR are all participants in sterile inflammation.
  • MPO, HMGB1, and GSNOR are sufficient to impair vasodilation and induce chronic lung injury in SS mice.
  • Treatment strategies should account for the combined effects of the mechanisms and pathways to account for SCD complexity.
  • Multi -targeted approaches should be developed, combining HMGB1 and GSNOR inhibitors with other treatments for delineating different aspects of SCD pathogenesis.
  • the findings enhance the understanding of SCD and demonstrate that targeting mechanisms with KYC is more effective than targeting HMGB1 with hE-HMGBl-BP.
  • HMGB1 increases myeloid cell recruitment, vascular inflammation and vascular leakage, and GSNOR decreases intracellular GSNO and therefore protein SNOs
  • therapeutic strategies must be designed to target both mechanistic agents or therapy will be incomplete.
  • hE-HMGBl-BP was unable to restore protein SNOs despite reducing GSNOR levels and activity in SS mice.
  • KYC effectively restored protein SNOs and did reduce GSNOR levels and activity.
  • the present invention improves the understanding of SCD pathophysiology and identifies HMGB 1 and GSNOR as key mechanistic agents in the onset and progression of vasculopathy and chronic lung injury.
  • the findings point toward the need for comprehensive, multi -targeted therapeutic strategies to target SCD complexity more effectively.
  • HMGB1 Human HMGB1 (10 nM, Sigma- Aldrich, St. Louis, MO) was added into a 96-well plate and incubated overnight at 4°C with gentle agitation for target coating. After pouring off the coating solution, the plate was incubated with a blocking buffer for 1 hour at 4°C. After washing with TBST, the phage prepared using PhD-12 Phage Display Peptide Library Kit (NEB, Ipswich, MA) were added to the plate and rocked gently for 1 h at room temperature.
  • NEB PhD-12 Phage Display Peptide Library Kit
  • the plate was subjected to 3 rounds of washes with increasingly stringent conditions containing 0.1, 0.2, and 0.3% Tween-20 at each successive round. After the last wash, bound phage with high binding affinity were harvested by elution with 1 pM HMGB1 and amplified for further screening. The amplified phage from single plaques were identified by DNA sequencing. The HMGB 1 -binding was verified by ELISA using 105 to 1012 phage virions and HRP -conjugated anti-M13 antibody. Peptide HMGB1-BP (AHSANNFDVKGI) was selected as the lead peptide.
  • Biomatik then synthesized the LRKLRKRLLR-GG-HMGB 1 -BP to create hE-HMGBl-BP N-acetyl- LRKLRKRLLR-GG-AHSANNFDVKGI-amide as was done to to design and make hE-Hb-BlOl except a -GG- linker was added to increase the distance between the hE domain and HMGB 1 -BP domain to separate the HMGB 1 -BP domain from the hE domain.
  • hEHMGBl-BP and biotin- labeled HMGB1-BP were synthesized, purified, and verified by LC-MS/MS (Biomatik, Kitchener, Ontario, Canada).
  • streptavidin-agarose beads (Sigma- Aldrich, St. Louis, MO) were blocked with 5% BSA in PBS at 4°C overnight.
  • Biotinylated HMGB1-BP or biotinylated scrambled HMGB1-BP Biomatik, Wilmington, DE
  • the Streptavidin-agarose beads were added to the mixture and incubated at 4°C for 2 h.
  • the beads were centrifuged to pellet the beads, washed with PBS 3 times, and boiled in Laemmli Sample Buffer (Bio-Rad, Hercules, CA).
  • the proteins in the pulldown were immunoblotted for r-HMGBl using an anti-HMGBl antibody (Abeam, Waltham, MA) using established protocols.
  • mice 8-9 months old received daily subcutaneous (SQ) injections of hE- HMGBl-BP (2.72 mg/kg/d, [Ac-LRKLRKRLLR-GGAHSANNFDVKGI-amide (acetate salt)] Biomatik Corp., Kitchener, Ontario) for three weeks.
  • SQ subcutaneous
  • mice were fully anesthetized with 1-3% isoflurane, underwent thoracotomy, and were euthanized by exsanguination. Blood was drawn from the heart’s right side into EDTA using a hypodermic needle, and plasma separated following centrifugation. Aliquots of plasma were stored at -80°C until analysis.
  • Facialis arteries were isolated by microdissection, cannulated, and pressurized to 60 mmHg.
  • Vasodilation changes in U46619-pre-constricted, pressurized facialis arteries were determined via videomicroscopy in response to acetylcholine (Ach,10-7-10-4M) as described.
  • the MOPS buffer [CaC12 «2H2O (2.0 mM), EDTA (0.02 mM), Glucose (5.0 mM), KC1 (4.7 mM), MgSO4-7H2O (E17 mM), MOPS (3.0 mM), NaCl (145 mM), NaH2PO4DH2O (1.2 mM), Pyruvic acid (2.0 mM)] was replaced with 37°C MOPS buffer containing L-NAME (100 pM) and Ach-induced relaxation responses determined as before.
  • eNOS-dependent vasodilation was calculated from the area between curves (ABC) ⁇ L-NAME using Prism GraphPad 9.5.0.3 Plasma HMGB1 was determined by ELISA (Tecan ELISA kits, 30164033, Mannedorf, Switzerland) as described.
  • Example 6- Effects of hE-HMGBl-BP on facialis artery GSNOR in SS mice [0124] SS mice were treated with hE-HMGB 1-BP (2.72 mg/kg/d). After three weeks, the mice were fully anesthetized, euthanized by exsanguination, facialis arteries isolated, snap- frozen in liquid nitrogen, and proteins were extracted using 200 pL of T-PER lysing buffer (Tissue Protein Extraction Reagent, ThermoFisher Scientific, Milwaukee, WI; catalog #78510) in a bead lysis microcentrifuge tube (NextAdvance, Inc, Troy, NY; catalog # PINKE1-RNA).
  • T-PER lysing buffer T-PER lysing buffer
  • the facialis artery homogenates were concentrated by centrifugation using a spin column with a lOkDa molecular cut-off (BioVision, Milpitas, CA; catalog# 1997-25). Proteins in the concentrated facialis artery lysates were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and immunoblotted with anti- GSNOR/ ADH5 rabbit polyclonal antibody (Proteintech Group Inc., Rosemont, IL; catalog#16379-l-AP) to determine GSNOR expression. GSNOR band intensities were normalized to total protein visualized with TGX Stain-Free gel technology (BioRad, Hercules, CA; catalog# 4568094).
  • HUVEC cultures were maintained in HUVEC media [DMEM (Catalog #1001,
  • HUVEC cultures were used for studies at passages three to four, and no studies were performed with cultures at or beyond passage five. HUVEC cultures were treated with fresh media ⁇ r- HMGB1 (20 ng/mL) daily for five days.
  • R-HMGBl was from Acrobiosystems (cat# HM1- H5220, Newark, DE) or GenScript (cat# Z028030, Piscataway, NJ) and tested for endotoxin.
  • HUVEC cultures were washed 3x with serum-free DMEM media (#09221, ScienCell, Carlsbad, CA) supplemented with L-glutamine and 1% antibiotics/antimycotics).
  • Cell proteins were isolated using MOPS lysis buffer (20 mM MOPS, 2 mM EGTA, 5 mM EDTA, 30 mM NaF, 10 mM P-glycerophosphate, 10 mM Na pyrophosphate, 2 mM Na orthovanadate, 1 mM PMSF, 0.5% NP-40, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktails 2 and 3, pH 7.0) and immunoblotted for GSNOR using a rabbit GNSOR antibody (GTX89762, Genetex, Irvine, CA) and P-actin using a mouse P-actin antibody (A2228, Sigma-Aldrich, St Louis, MO) using established protocols.
  • MOPS MOPS lysis buffer
  • GTX89762 rabbit GNSOR
  • Example 8 Effects of inhibiting the inflammatory pathway and GSNOR on facialis artery vasodilation
  • SS mice were treated with phosphate-buffered saline (PBS), KYC (Nacetyl- lysyltyrosylcysteine amide (Biomatik Corp., Kitchener, Ontario); subcutaneous injection, 3 mg/kg/d, 3 weeks) or N6022 (l-[4-(aminocarbonyl)-2-methylphenyl]-5-[4- (IH-imidazol-l- yl)phenyl]-lH-pyrrole-2-propanoic acid (Cayman Chemical, Ann Arbor, MI); SQ; 1 mg/kg/d, 3 weeks).
  • PBS phosphate-buffered saline
  • KYC Nacetyl- lysyltyrosylcysteine amide
  • subcutaneous injection 3 mg/kg/d, 3 weeks
  • N6022 l-[4-(aminocarbonyl)-2-methylphenyl]-5-[4- (IH-imidazol-
  • DMSO dimethyl sulfoxide
  • Protein SNOs were normalized to P-actin, and the P-actin normalized protein SNOs from SS mice groups were normalized to the mean of the P- actin-normalized protein SNOs for AA mice.
  • GSNOR levels were determined by immunoblotting with anti-GSNOR/ADH5 rabbit polyclonal antibody (Proteintech Group Inc., Rosemont, IL; catalog#16379-l-AP). The normalization strategy used for protein SNOs was also used for GSNOR.
  • Example 10- Effects of inhibiting the inflammatory pathway or GSNOR on lung protein SNOs and GSNOR
  • SS mice were treated with PBS, KYC (3 mg/kg/d), and hE-HMGBl-BP (2.72 mg/kg/d) subcutaneously as before. After three weeks, untreated AA mice and treated SS mice were fully anesthetized, euthanized by exsanguination, and lungs perfused in situ with cold PBS as above.
  • the right main lung bronchus was ligated with silk at the carina and snap-frozen in liquid nitrogen for immunoblot analysis after resection below the ligature.
  • the trachea was cannulated using an Instech Solomon (20G) stainless steel feeding tube (Plymouth Meeting, PA). After securely ligating the trachea with silk, the left lobe was inflated with 10% neutral buffered formalin at 25 cm-H20 (2.4 kPa). After one hour, the fixed lung was stored in 10% buffered formalin for 24 h and then embedded in paraffin for histology. Lung sections (5pm) were mounted on SuperFrost Plus-coated slides (Denville Scientific, Metuchen, NJ).
  • Lung CD31 and GSNOR immunofluorescent images were captured using a Zeiss Axioimager Z1 microscope with Zeiss axiocam HRC 13megapixel camera (2/3” CCD sensor). 8-bit images were collected with fixed exposure times for each channel.
  • the filter specifications are DAPI filter - (ET395/25(Ex), T425(BS), ET460/50 (Em)), Green fluorophore filter - (ET470/40(Ex), T495(BS), ET525/50 (Em)), Red fluorophore filter - (ET546/22(Ex), T565(BS), ET590/33 (Em)). Images were captured without binning at 1760x1760 pixels and saved as Tif images.
  • CD31 and GSNOR Single-color images are merged using the Zeiss Axiovison acquisition software (V4.9). Fluorescent intensities for CD31 and GSNOR were estimated separately using NIH Image J. The mean of four to five immunofluorescence intensities of CD31 and GSNOR from random locations in lung sections for each mouse and all mean intensities from lungs of SS mice treated with PBS, hE-HMGBl-BP, and KYC were normalized to the mean of CD31 and GSNOR immunofluorescence in sections from AA mice as above. CD31 and GSNOR immunofluorescence data represent the mean of four to five random images per section, tested for outliers for each mouse.
  • GSNOR dehydrogenase activity was determined from NADH consumption per Liu et al. Briefly, frozen mouse lungs were lysed in Tris buffer (20 mM, pH 8.0) containing EDTA (0.5 mM) and protease inhibitors. Lysates were centrifuged (4°C, 16,000g, 10 min), and supernatant protein was determined with Pierce BCA protein assay kit (ThermoFisher 23225). Lysate aliquots (0.5 mg/ml) and 200 pM NADH (Sigma N8129) were added in duplicate to a 96-well UV transparent plate (ThermoFisher 8404).
  • Lung morphometries consisting of radial alveolar counts (RAC) and mean linear intercept (MLI) lengths were determined as described.
  • HMGB1 induces human lung endothelial cell cytoskeletal rearrangement and barrier disruption, Microvasc Res 81(2) (2011) 189-97.
  • H. Xu, Y. Yao, Z. Su, Y. Yang, R. Kao, C.M. Martin, T. Rui, Endogenous HMGB1 contributes to ischemia-reperfusion-induced myocardial apoptosis by potentiating the effect of TNF-a/JNK, American Journal of Physiology -Heart and Circulatory Physiology 300(3) (2011) H913-H921.
  • HMGB1 High-mobility group box 1 protein

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Abstract

Un peptide à double domaine à coiffe terminale (N-acétylé, C-amidé) appelé hE-HMGBl-BP a été conçu pour se lier et appauvrir la HMGB1 de la circulation par l'intermédiaire de l'absorption par le système protéoglycane à sulfate d'héparine (HSPG) du foie.
PCT/US2024/028808 2023-05-17 2024-05-10 Peptide à double domaine pour appauvrir hmgb1 de la circulation par l'intermédiaire d'un système hspg du foie Pending WO2024238337A1 (fr)

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