Dual-domain peptide for depleting hmgb1 from the circulation via liver hspg system

Abstract

An end-capped (N-acetylated, C-amidated) dual-domain peptide called hE-HMGBl-BP was designed to bind and deplete HMGB1 from circulation via uptake by the liver's heparin sulfate proteoglycan (HSPG) system.

Description

DUAL-DOMAIN PEPTIDE FOR DEPLETING HMGB1 FROM THE CIRCULATIONVIA LIVER HSPG SYSTEMCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional patent application serial number 63 / 467,104 filed May 17, 2023, and U.S. provisional patent application serial number 63 / 530,309 filed August 2, 2023, the entire disclosures of which are hereby incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under HL012783 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] 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. In particular, 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.BACKGROUND OF THE INVENTION

[0004] Sickle cell disease (SCD) 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. Each year, approximately 300,000 infants are born with SCA, a number that could reach 500,000 by 2050. Interestingly, 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.

[0005] 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) thannormal 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.

[0006] Thus, 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. These 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.

[0007] Hemolysis and the resulting release of cell-free Hb (cf-Hb) in SCD have long been considered one of the primary mechanisms for impairing vascular function by preventing vasodilation, enhancing vascular adhesion, and increasing vasculopathy. Sickled RBCs are more rigid and oxidized than biconcave RBCs, making them more prone to being trapped in microvessels. These blockages not only impede microvascular blood flow but also increase the risk of intravascular hemolysis. Mechanistically, cf-Hb undergoes dioxygenation reactions resulting in superoxide anion (02*-) generation. Superoxide anion scavenges nitric oxide (*NO) at the rate of diffusion to form peroxynitrite (ONOO-), a potent oxidant that can induce severe cellular injury and damage. These biochemical and radical reactions are considered foundational to the mechanisms by which cf-Hb increases oxidative stress and how SCD increases «NO consumption, and resistance to »NO donors and reduces *NO bioavailability.

[0008] Despite many reports describing how cf-Hb initiates and propagates SCD pathophysiology, some have argued that hemolysis does not account for all the observed complications in SCD.

[0009] In this application, 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. However, “sickle cell disease” or“SCD” and “sickle cell anemia” or “SCA” are often used interchangeably and are used interchangeably herein.BRIEF SUMMARY OF THE INVENTION

[0010] To investigate whether other contributory factors other than cf-Hb may also impair endothelial function in SCA, the present inventors designed a small dual-domain peptide called hE-HB-BlO that would bind and deplete cf-Hb from the circulation of treated mice. 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). Treating hypercholesterol emic mice and rabbits with hE18A effectively depleted atherogenic apolipoprotein B-containing particles from the circulation via uptake by the liver’s heparin sulfate proteoglycan (HSPG) system.

[0011] The present inventors hypothesized that if endothelial-dependent vasodilation did not significantly improve after treating mice with hE-HB-BlO, this would suggest other negative mediators of endothelial cell function were present. The hE-Hb-BlO peptide made it possible to assess vascular endothelial function in control and SS mice under conditions where cf-Hb was essentially eliminated as a variable in much the same way that inhaled »NO was used to eliminate cf-Hb as an oxidative source O2-*.

[0012] These vasodilation studies, which focused on endothelial function in isolated facialis arteries showed that reducing cf-Hb in the circulation in SCD mice to levels approaching controls had little to no effect on endothelial-dependent vasodilation but did switch the mechanism of vasodilation from non-eNOS-dependent to eNOS-dependent in the facialis arteries isolated from SCD mice. In addition, reducing cf-Hb from the circulation inf SCD mice reduced liver injury-based on significant reductions in plasma alanine transaminase (ALT) levels. These studies suggest that cf-Hb has little to no effect on endothelial-dependent vasodilation, a minimal inhibitory effect on eNOS-dependent vasodilation and a modest effect on liver injury in SCD mice. More importantly, if effectively reducing cf-Hb did not significantly improve endothelial- dependent vasodilation, then other pathways and mechanistic inhibitors of vascular function must exist in SCD mice besides Hb.

[0013] In one embodiment of the present invention, 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-NH2, 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.

[0014] The composition may further comprise an effective amount of N-acetyl- lysyltyrosylcysteine amide (KYC) designed to inhibit myeloperoxidase (MPO)-initiated and HMGB1 -propagated inflammatory pathway.

[0015] 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).

[0016] 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.

[0017] 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.

[0018] In one embodiment of the present invention, 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.

[0019] 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.

[0020] 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.

[0021] The administration of 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.

[0022] 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.

[0023] 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 (CTE), ischemic or hemorrhagic stroke, or bronchopulmonary dysplasia.

[0024] In one embodiment of the present invention, 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.

[0025] The pharmaceutical composition may be formulated as an oral dose comprising the end capped dual -domain peptide hE-HMGBl-BP and a carrier.

[0026] In one embodiment of the present invention, 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.

[0027] 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.

[0028] In summary, the present invention shows that SCA causes vasculopathy and chronic lung disease by an HMGB1- and GSNOR- dependent mechanism where HMGB1 induces GSNOR.

[0029] The present inventors have found that:1. SCD induces vasculopathy and chronic lung injury by increasing HMGB 1 and GSNOR.2. HMGB1 increases GSNOR, impairing vasodilation and increasing chronic lung injury.3. HMGB1 and GSNOR reduce vascular and pulmonary nitric oxide bioavailability.4. Targeting HMGB 1 and GSNOR restores vasodilation and improves lung morphometries.5. Sterile inflammation profoundly impacts vascular function and lung morphometries.

[0030] Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The disclosure will be better understood and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description. Such detailed description makes reference to the following drawings.

[0032] Figures 1A-1E. Effects of hE-HMGBl-BP on Vasodilation and Plasma HMGB1 in SS Mice. (Figure 1A) Line graph showing ACh-induced vasodilation of isolated facialis arteriesfrom untreated Townes AA mice in the absence (-) (black, closed diamonds, n = 8) and presence (+) of L-NAME (100 pM, white, open diamonds, n = 8). Differences in line curves were determined using two-way ANOVA for significance with respect to concentration and test group (**** = pO.OOOl). (Figure IB) Line graphs showing 1) ACh-induced vasodilation of isolated facialis arteries from PBS-treated Townes SS mice in the absence (black, closed circles, n=8) and presence of L-NAME (100 pM, white, open circles, n = 8); 2) ACh-induced vasodilation of facialis arteries from hE-HMGBl -BP-treated (2.72 mg / kg / d, 3 weeks) Townes SS mice in the absence (black, closed squares, n = 8) and presence of L-NAME (100 pM, white, open square, n = 8). Differences in line curves were determined using two-way ANOVA for significance with respect to concentration and test group (**** = p<0.0001). (Figure 1C) Bar graph showing eNOS-dependent vasodilation: Scatter plot bar graph showing mean ± SD and individual data points for Area Between Curves ± L-NAME (ABC, arbitrary units) for AA mice, PBS-treated SS mice, and hE-HMGBl -BP-treated SS mice. Differences between means were analyzed for significance using ANOVA (** = p<0.007, *** = p<0.0001). (Figure ID) Effects of hE- HMGB1-BP on plasma HMGB1 concentrations: Bar chart showing plasma HMGB1 concentrations in AA mice (n = 17, white open bar; white open circles), PBS-treated SS mice (n = 14, black, closed bar; white, open squares), and hE-HMGBl -BP-treated SS mice (n = 15, light gray, closed bar; white, open upright triangles). Data were analyzed by ANOVA with the Tukey, non-parametric posthoc test. Differences between means were analyzed for significance using ANOVA (** = p<0.003, **** = p<0.0005). (Figure IE) Scatter plot of eNOS-dependent vasodilation as a function of plasma HMGB1 : This scatter plot shows data points for AA (n=3, filled circle), SS+PBS (n=4, filled square) and SS+hE-HMGBl-BP (n=6, open square) mice that were tested for eNOS-dependent vasodilation and plasma HMGB1. The scatter plots show the equation, the 95% confidence limits, and the p value for this mixed population's slope being different from zero (n=14, p<0.0094).

[0033] 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.The mean of the GSNOR band intensities corrected for total protein (per lane) for PBS-treated SS mice was used to normalize band intensity ratios. Individual data points plotted as scatter plots in bars (mean ± SD, PBS, n = 10; hE-HMGBl-BP, n = 9 * = p < 0.05). (Figure 2B) Chemiluminescence images from GSNOR and P-actin immunoblots showing that r-HMGBl (20 ng / mL / d, fresh each day for 5 days) increases human umbilical vein endothelial cell (HUVEC) GSNOR protein expression by 80%. Data are plotted as scatter plots in bars (mean ± SD, n = 4, ** = p < 0.01).

[0034] Figures 3A-3D. Effects of N6022 and KYC on Vasodilation in SS Mice. (Figure 3 A) Line graph showing the effects of PBS treatment (sterile PBS [1% DMSO], 3 weeks) on ACh- induced vasodilation of facialis arteries ± L-NAME (100 pM) from SS mice. Differences between curves were determined using two-way ANOVA (ns, n = 9). (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. Differences between curves were determined using two-way ANOVA (** = p<0.009, n = 7). (Figure 3C) Line graph showing the effects of N6022 treatment (sterile PBS [1% DMSO]; 1 mg / kg / d, 3 weeks) on ACh-induced vasodilation ± L-NAME (100 pM) of facialis arteries from SS mice. Differences between curves were determined using two-way ANOVA (**** = p<0.0001, n = 6). (Figure 3D) Bar graphs showing the mean of the area between the curves (ABC) calculated from the vasodilation curves (± L-NAME) from line graphs in A, B, and C. These bar graphs demonstrated that inhibiting the inflammatory pathway in SS mice with KYC or GSNOR with N6022 improved eNOS-dependent vasodilation. Statistical analysis was not performed on the differences in eNOS-dependent vasodilation resulting from the different treatments because DMSO oxidizes protein thiols and improves vasodilation in an unclear mechanism, making it difficult to estimate the contribution of any agent accurately.

[0035] Figures 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). Upper Right Panel: Bar chart showing male (closed) and female (open) circles for AA mice and male (closed) and female (open)squares for SS mice. SCA decreased lung protein SNOs in SS mice (n = 6) by 35% compared with levels in AA mice (n = 6, * = p <0.05). (Figure 4B) Lower Left Panel: Chemiluminescence immunoblots showing the images of GSNOR and P-actin per lane of lung homogenates prepared from AA and SS mice. Band-Intensity Ratio (BIR) data from GSNOR band intensities 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). Lower Right Panel: Bar chart showing male (closed) and female (open) circles for AA mice and male (closed) and female (open) squares for SS mice. SCA increased GSNOR in SS mice (n = 6) by 28% compared with levels in AA mice (n= 6, * = p <0.05).

[0036] 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.Representative chemiluminescence immunoblots of GSNOR and P-actin from AA and SS mice treated with PBS, hE-HMGBl-BP, or KYC. Test group and mouse number are provided for each lane. (Figure 5C) Protein SNO band intensities for AA and SS mice treated with PBS, hE- HMGBl-BP, or KYC were divided by their corresponding P-actin band intensities and normalized by the mean of AA mice (1.0). Normalized BIR data were plotted as scatter plots in bars (mean ± SD) for the AA and SS mice treated with PBS, hE-HMGBl-BP, or KYC test groups. ANOVA was used to analyze normalized BIR data with an appropriate posthoc test (n = 14 for each test group; * = p < 0.05). The bar scatter plots show that SCA decreases lung protein SNOs and that KYC treatment increases lung protein SNOs in SS mice. (Figure 5D) GSNOR band intensities for AA and SS mice treated with PBS, hE-HMGBl-BP, or KYC were divided by their corresponding P-actin band intensities and normalized by the mean of AA mice (1.0). Normalized BIR data were plotted as scatter plots in bars (mean ± SD) for the AA and SS mice treated with PBS, hE-HMGBl-BP, or KYC test groups. ANOVA analyzed normalized BIR data with appropriate post hoc test (n = 14 for each test group; * = p < 0.05). 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. Additional SNOs and GSNOR immunoblots for these studies are available in Figure 16.

[0037] Figures 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. ANOVA was used to analyze, with an appropriate post hoc test, differences between means (AA, n = 4; SS+PBS, n = 4; SS+hE-HMGBl-BP, n = 4; and, SS+KYC, n = 3, *** = p < 0.001, ** = p < 0.05). (Figure 6C) Relative fluorescence intensities for GSNOR were randomly captured from five to four regions. GSNOR fluorescence intensities were determined using NIH Image J, tested for outliers, and averaged. 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. ANOVA was used to analyze differences between means with an appropriate post hoc test (AA, n = 4; SS+PBS, n = 4; SS+hE-HMGBl-BP, n = 4; and SS+KYC, n = 3, ** = p < 0.05, * = p < 0.05). (Figure 6D) 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). ANOVA was used to analyze differences between means with an appropriate post hoc test (AA, SS+PBS, SS+hE-HMGBl-BP, and SS+KYC: n = 9 each test group, * = p < 0.05).

[0038] Figures 7A-7C. Effects of SCA and hE-HMGBl-BP and KYC Treatments Lung Simplification and Acinar Airspace in SS Mice. (Figure 7A) Representative H&E images of lung sections showing alveolar and vascular structures in untreated Townes AA (n = 10), PBS-treated- (n = 9), KYC-treated- (n = 7), and hE-HMGB 1 -BP-treated SS mice (n = 6). (Figure 7B) Scatter plots in bars showing the mean ± SD of the individual RAC values in lung sections from AA (n = 10), and the PBS-treated- (n = 9), KYC-treated- (n = 7), and hE-HMGB 1 -BP-treated- (n = 6)SS mice. SCA decreases RAC values in the lungs of SS mice compared to RAC values in AA mice (**** = p <0.0001). Treatment of SS mice with KYC and hE-HMGBl-BP increased RAC values to numbers that are intermediate to RAC values in AA and PBS-treated SS mice (** = p < 0.01). (Figure 7C) Scatter plots in bar showing the mean ± SD for the individual MLI values in lung sections from Townes AA (n = 10), and PBS-treated- (n=9), KYC-treated- (n = 7), and hE- HMGB1 -BP-treated SS mice (n = 6). SCA increases MLI values in PBS-treated SS mice compared to AA mice (** = p <0.0001). Treatment of SS mice with KYC- and hE-HMGBl-BP decreased MLI values to numbers indistinguishable from the MLI values in AA mice (** = p < 0.002).

[0039] Figure 8. A diagram illustrating the SCA inflammatory pathway of the present invention.

[0040] Figure 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.

[0041] Figure 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. In contrast, in plasma, biotin Ahx-HMGBl-BP has a high specific affinity for r- HMGB1. In contrast, 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.

[0042] Figure 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).Normalized BIR data were plotted as scatter plots in bars (mean ± SD) for the AA and SS mice treated with PBS, hE-HMGBl-BP, or KYC test groups. ANOVA was used to analyze normalized BIR data with an appropriate posthoc test (n = 14 for each test group; * = p < 0.05).The bar scatter plots show that SCA decreases lung protein SNOs and that KYC treatment increases lung protein SNOs in SS mice.

[0043] Figure 15. GSNOR band intensities for AA and SS mice treated with PBS, hE- HMGB1-BP, or KYC were divided by their corresponding P-actin band intensities and normalized by the mean of AA mice (1.0). Normalized BIR data were plotted as scatter plots in bars (mean ± SD) for the AA and SS mice treated with PBS, hE-HMGBl-BP, or KYC test groups. ANOVA analyzed normalized BIR data with appropriate post hoc test (n = 14 for each test group; * = p < 0.05).

[0044] 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.

[0045] All data are presented as the mean (x) ± SD, n = number. The student's t-test was used to determine the differences between the means of two test groups. ANOVA and appropriate post-hoc tests were used to determine the significance between three or more test groups. Two- way ANOVA was used to determine differences between vasodilation curves.

[0046] While the invention is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and are herein described in detail. The description of specific embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.DETAILED DESCRIPTION OF THE INVENTION

[0047] I. IN GENERAL

[0048] This invention is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the language of the appended claims.

[0049] As used in this disclosure and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. The terms “a”(or “an”), “one or more” and “at least one” can be used interchangeably. The terms “comprising”, “including”, and “having” can also be used interchangeably.

[0050] As used herein, "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. Examples of non-mammals include, but are not limited to, birds, and the like. The term "subject" does not denote a particular age or sex.

[0051] As used herein, “administering” or “administration” 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.

[0052] As used herein, 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.

[0053] As used herein, 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.

[0054] As used herein, the term "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 naturallyoccurring amino acid polymers. For example, "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.

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Chemical compound names that are commonly used and recognized in the art are used interchangeably with the equivalent IUPAC name. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patents specifically mentioned herein are incorporated by reference for all purposes including describing and disclosing the chemicals, instruments, statistical analysis, and methodologies which are reported in the publications which might be used in connection with the invention. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0056] II. THE INVENTION

[0057] 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.Background

[0058] High mobility group box-1 (HMGB1) is a predominant mediator of TLR4-reporter cell activity in the plasma from SS mice and humans.

[0059] 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. Wandersee, A Novel Hemoglobin-Binding Peptide Reduces Cell-Free Hemoglobin in Murine Hemolytic Anemia, Am J Physiol Heart Circ Physiol 304(2) (2013) H328-36, hereby incorporated by reference.

[0060] SS mice were treated with hE-HMGB 1-BP and the effects of targeting extracellular HMGB1 on vasodilation and lung morphometries was determined. However, as SCA also impairs vasodilation by myeloperoxidase (MPO) dependent mechanisms and MPO generatestoxic oxidants that initiate a proinfl am m at ory pathway mediated, in part, by HMGB, 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.

[0061] Finally, if 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. By selectively inhibiting HMGB1, the inflammatory pathway, and GSNOR, the present invention provides additional mechanistic insight into their respective roles in SCA-induced vasculopathy and chronic lung injury.

[0062] 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.Dual Domain Peptide

[0063] 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.

[0064] 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. In this case the two domains (hE) and the HMGB1 binding peptide (HMGB1-BP) are covalently linked by a -GG- or -gly-gly- bridge.

[0065] 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 ± 36nM and is removed from the circulation by uptake by the liver through the HSPG system or the apolipoprotein E receptor domain.

[0066] The present inventors further investigated the role of myeloperoxidase (MPO) in the impairment of vascular function and chronic organ injury in SCD. It was found that MPO generates toxic oxidants (HOC1, NO2) that damage the liver and lungs of SCD mice. As neutrophil-derived MPO activity increases cell death, and dead and dying cells passively release high mobility group box-1 (HMGB1), the present inventors consider MPO something more than a simple biomarker of neutrophil recruitment. 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. Importantly, 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.

[0067] The disclosure also includes pharmaceutical 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. Preferably 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. It is also envisioned that the peptides of the present invention may be incorporated into transdermal articles designed to deliver the appropriate amount of peptide in a continuous fashion.

[0068] It is not critical whether 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.

[0069] 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.

[0070] In certain embodiments, the peptides of the invention will be provided as pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their 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. Furthermore, where the compounds of the invention carry an acidic moiety, 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.

[0071] 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. Thus, 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) 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.

[0072] In another aspect, 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.

[0073] The method includes the step of administering to a subject in need of such therapy one or more of the peptides as described above. In certain preferred embodiments, the subject is a human or a non-human mammal.

[0074] Preferably, the method includes the additional step of mixing the peptide with a pharmaceutically acceptable carrier before the peptide is administered.

[0075] In preferred embodiments of the invention, the method is carried out to improve vascular function, decrease pulmonary inflammation, and / or increase cardioprotection in the subject. However, it can be appreciated that the inventive peptides act on a molecular processcommon to a plethora of medical diseases and conditions. Therefore, 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.

[0076] 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.

[0077] Further, 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.

[0078] 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 acceptablecarrier. As well, the invention encompasses the manufacture and use of medicaments specifically purposed for treatment of one or more of the diseases / conditions described above.

[0079] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims.

[0080] III. EXAMPLES

[0081] Example 1- HMGB1 and GSNOR Induce Vasculopathy and Chronic Lung Injury in SCD

[0082] Materials and Methods

[0083] Mice: Townes Hbatml(HBA)Tow / Hbbtm3(HBGl,HBB*)Tow AA 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.

[0084] 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. Wandersee, A Novel Hemoglobin-Binding Peptide Reduces Cell-Free Hemoglobin in Murine Hemolytic Anemia, Am J Physiol Heart Circ Physiol 304(2) (2013) H328-36, hereby incorporated by reference. Biolayer interferometry studies revealed the Kd of HMGB1-BP for r-HMGBl to be estimated at 170 ± 36 nM, which is typical for a phage display 12mer peptide. A minor difference in the design of the hE-HMGB l-BP vs. the 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).

[0085] Effects of r-HMGBl on GSNOR expression in HUVEC cultures 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-Aldrich, St. Louis, MO) and previously published protocols.

[0086] 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. 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. Changes in vasodilation of (5Z)-7- [(lR,4S,5S,6R)-6-[(lE,3S)-3-hydroxy-l-octenyl]-2-oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid (U46619)-preconstricted, pressurized facialis arteries were determined by video microscopy in response to acetylcholine (Ach) (10-7-10-4 M) as before. After determining vasodilation in the absence of N(G)-nitro-L-arginine methyl ester (L-NAME), the bath’s 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 thearea under the curve function in Prism GraphPad 9.5.0 after correction of paired data for negative relaxation responses. In this application, 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. Likewise, 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.

[0087] Effects of hE-HMGBl-BP on facialis artery GSNOR in SS mice. SS mice were treated with hE-HMGBl-BP (2.72 mg / kg / d). After three weeks, SS mice were fully anesthetized, euthanized by exsanguination, and facialis arteries isolated by microdissection. The facialis arteries were snap frozen in liquid nitrogen, and proteins were isolated by homogenization using 200 pL of T-PER lysing buffer (Tissue Protein Extraction Reagent, Thermo Fisher Scientific, Milwaukee, WI; catalog #78 10) in a bead lysis microcentrifuge tube (NextAdvance, Inc, Troy, NY; catalog # PINKE1-RNA). 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).

[0088] Effects of inhibiting GSNOR on vasodilation in SS mice. 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). N6022 requires dimethyl sulfoxide (DMSO) for dissolution, so DMSO (Sigma-Aldrich, St. Louis, MO; 1% v / v) was added to the PBS solutions to control for the influence of DMSO on vasodilation. After three weeks, the SS mice were fully anesthetized, euthanized by exsanguination, and facialis arteries isolated as described above. Relaxation responses to Ach (10-7 to 10 4 M) were determined in MOPS physiological buffer in the absence and presence of L-NAME by video microscopy as described.

[0089] Effects of hE-HMGBl-BP, and KYC treatment on lung protein SNOs and GSNOR in SS mice. Two separate experiments were performed to determine the effects of SCDon protein SNOs and GSNOR in the lung. In the first experiment, untreated AA mice were used as genetic controls for untreated SS mice. Untreated AA and SS mice were fully anesthetized, euthanized by exsanguination, and lungs perfused in situ with cold PBS to remove non-adherent blood elements and lungs were homogenized and used for protein SNOs and GSNOR immunoblots. In the second experiment, untreated AA mice were used as genetic controls, and SS mice were treated with PBS, hE-HMGBl-BP (2.72 mg / kg / d) and KYC (3 mg / kg / d). After three weeks, untreated AA mice and treated SS mice were fully anesthetized, euthanized by exsanguination, and lungs perfused in situ with cold PBS to remove non-adherent blood elements.

[0090] 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). After the trachea was securely ligated with silk, the left lobe was inflated with 10% neutral buffered formalin at 25 cm-H20 (2.4 kPa). After 1 h, 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). Slides were deparaffinized, and sections stained with hematoxylin and eosin (H&E; Sigma, St. Louis, MO; catalog# MHS16 and HT110116) or immunostained for CD31 (R&D Systems, Minneapolis, MN; Catalog# AF3628) and GSNOR (Proteintech Group, Rosemont, IL; Catalog# 11051-1-AP) for co-localization studies. Four to five immunofluorescence images were randomly captured for each lung section, the fluorescence intensities were analyzed for outliers and then averaged, and the average intensity was plotted as a single data point for each mouse. Protein SNOs were analyzed by Pierce S-Nitrosylation Western Blot Kit (Thermo Fisher, Milwaukee, WI; Catalog# 90105). All other mentioned reagents and chemicals were purchased from Thermo Fisher (Milwaukee, WI).

[0091] Statistics

[0092] All data are presented as x ± SD, n = number. The student’s t-test was used to determine differences between the means of two test groups. ANOVA, with the appropriate post hoc test, was used to determine differences between three or more test groups. Differences in vasodilation were determined using 2-way ANOVA with multiple comparisons and the appropriate post hoc tests.

[0093] Results

[0094] Effects of hE-HMGB 1 -BP on Endothelial- and eNOS-dependent-Vasodilation, andPlasma HMGB1 Concentrations. Endothelial-dependent vasodilation in AA mice (closed right triangle) is robust achieving relaxation responses to Ach greater than 50% of the Dmax of the pressurized vessel (Figure 1). In contrast, endothelial-dependent vasodilation in SS mice (closed square) was impaired by more than 75% of the vasodilation in AA mice (p < 0.0001). Interestingly, treating SS mice with hE-HMGB 1 -BP (closed circle) increased endothelial- dependent vasodilation by more than 2.5 times the vasodilation in SS+PBS mice (p < 0.0001). Endothelial-dependent vasodilation in SS+hE-HMGBl-BP mice (closed circle) was essentially equivalent to the endothelial-dependent vasodilation in AA mice (closed upright triangle, ns, p = 0.1071).

[0095] The summary data demonstrating the effects of hE-HMGB 1 -BP on eNOS-dependent vasodilation in SS mice can be seen in Figure IB. In untreated AA mice, eNOS-dependent vasodilation was calculated to 83.2 ± 37.7, while in SS+PBS mice eNOS-dependent vasodilation was reduced to 24.7 ± 15.4 (Figure IB, p < 0.001), whereas in SS+hE-HMGBl-BP mice, eNOS- dependent vasodilation was increased substantially to 90.5 ± 19.3 (Figure IB, p < 0.01). Interestingly, comparing eNOS-dependent vasodilation (mean ± SD) to plasma HMGB1 (mean ± SD) for the same three test groups demonstrates that eNOS-dependent vasodilation shares a reciprocal relationship with plasma HMGB1. Plasma HMGB1 concentrations in untreated AA mice were 11.0 ± 4.6 ng / mL, whereas they were significantly greater in SS+PBS mice at 26.6 ± 14.7 ng / mL (Figure 1C, p < 0.0001). In contrast, plasma HMGB1 concentrations in SS+hE- HMGBl-BP mice were significantly reduced to 14.2 ± 6.0 ng / mL (Figure 1C, p < 0.01). These data demonstrate that therapeutic targeting of HMGB1 restores endothelial- and eNOS- dependent vasodilation and when examined in the context of plasma HMGB1 reveals that endothelial and eNOS-dependent vasodilation share a reciprocal relation with plasma HMGBl.

[0096] Effects of hE-HMGBl-BP on Facialis Artery GSNOR in SS Mice and r-HMGBl on HUVEC GSNOR Expression. The representative immunoblot in Figure 2A shows that treating SS mice with hE-HMGBl-BP reduces GSNOR protein by 20% compared to GSNOR levels in facialis arteries from PBS-treated SS mice (p < 0.05). The effects of r-HMGBl on HUVEC GSNOR expression can be seen in Figure 2B. The immunoblots show that r-HMGBl induces GSNOR expression in HUVEC cultures by 80 % (p < 0.05). These findings areconsistent with the hypothesis that SCD increases vascular GSNOR expression via an HMGB1 - dependent mechanism.

[0097] Effects of PBS and N6022 on Vasodilation in SS Mice: 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). In contrast to the lack of vasodilation in SS mice treated with PBS+1% DMSO, SS mice treated with N6022+l% DMSO had markedly increased endothelial- and eNOS-dependent vasodilation, i.e., the area between curves (ABC) (Figure 3B, p < 0.001). These data are consistent with the hypothesis that SCD impairs endothelial- and eNOS- dependent vasodilation in SS mice by a GSNOR-dependent mechanism.

[0098] Effects of hE-HMGBl-BP and KYC on Protein SNOs and GSNOR in Lung Homogenates from SS mice. SCD decreased protein SNOs in lung homogenates from untreated SS mice by 35% relative to the protein SNOs in lung homogenates from untreated AA mice (Figure 4A, p < 0.05). Importantly, lungs from untreated SS mice contained 28% more GSNOR protein than lung homogenates from untreated AA mice (Figure 4B, p < 0.05). These immunoblot data are consistent with the hypothesis that SCD decreases lung protein SNOs by a GSNOR-dependent mechanism.

[0099] 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). Even though treating SS mice with hE-HMGBl-BP tended to improve lung protein SNOs, the difference in means for protein SNOs between SS mice treated with PBS and hE-HMGBl-BP did not achieve significance (ns). In contrast, treating SS mice with KYC markedly increased lung protein SNOs compared to protein SNOs in lungs from SS mice treated with PBS (Figure 5C, p < 0.05). In contrast to the lack of an effect on lung protein SNOs in SS mice treated with hE-HMGBl-BP, treating SS mice with hE-HMGBl-BP or KYC markedly decreased lung GSNOR (Figure 5D, p < 0.05). These data are consistent with the hypothesis that SCD decreases lung protein SNOs by HMGB1- and GSNOR-dependent mechanism and that protein SNO balance in the lung is likelymediated by a factor or factors beyond HMGB1 . Support for this conclusion comes from immunoblots showing that protein SNOs and GSNOR can be fully restored SS mice with advanced SCD by treating SS mice with KYC, a systems chemico-pharmacology agent, but not with hE-HMGBl-BP (Figure 5C and 5D, p < 0.05), a classical test agent with only one target.

[0100] Effects of hE-HMGBl-BP and KYC on CD31 (PECAM-1) and GSNOR Expression in Lung Sections and on GSNOR Activity in Lung homogenates: Merged images from sections of lungs were examined for differences in immunostaining for CD31 (green) to identify endothelial cell-lined vessels, GSNOR (red) in lung tissues, and lung cell nuclei (stained with DAPI, blue) in untreated AA, and SS mice treated with PBS, hE-HMGBl-BP, or KYC. Figure 6A shows the variation in immunostaining from lung sections from the four different test groups: AA, SS+PBS, SS+hE-HMGBl-BP and SS+KYC mice. Immunostaining for GSNOR (red) in sections of lungs from AA mice was above background suggesting AA mice express low levels of GSNOR in lung parenchyma. DAPI staining of cell nuclei is bright blue. The intent for immunostaining for CD31 was to identify vascular endothelial cells lining blood vessels in lung sections. CD31 immunostaining is bright green and can be seen lining the lumen of blood vessels in sections of lungs from AA mice (Figure 6A, AA: image 2 and 3). In contrast to lung sections from AA mice, sections from SS+PBS mice appear bright yellow, red, and at times even purple (Figure 6A, SS+PBS: images 1-3). Immunostaining for 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.

[0101] Analysis of fluorescence intensities for CD31 and GSNOR in immunostained lung sections revealed that CD31+ cells in lungs from SS+PBS mice increased by 100% compared to CD31+ cell staining in lungs from AA mice (Figure 6B, p < 0.001). CD31+ staining in sections of lung from SS mice treated with hE-HMGBl-BP was reduced by 41% compared to CD31 + cell staining in the sections of lungs from SS mice treated with PBS (Figure 6B, p < 0.001).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%, while GSNOR staining in sections of lungs from SS mice treated with KYC decreased by 85%. To better understand how SCD, hE-HMGBl-BP and KYC modulate lung GSNOR activity, 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). Interestingly, 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). As a way of further characterizing lung GSNOR, it was observed that 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.. In addition, 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.

[0102] Effects hE-HMGBl-BP, and KYC on Lung Radial Alveolar Counts (RAC) and Mean Linear Intercept (MLI) in SS Mice. H&E-stained slides prepared from lungs of AA mice show typical lung blood vessels, airways, microvessels, and alveoli where the number of adherent cells in lung parenchyma are low in number, and that alveoli counts are at an age- appropriate level for 8-9-month-old healthy control mice (Figure 7A). In contrast, lungs of SS+PBS mice appear to have a greater number of adherent cells, fewer alveoli, and larger open spaces than the lungs from AA mice. Such differences in lung morphometric structure suggest SS+PBS lungs are less complex (Figure 7A, second panel) than AA lungs (Figure 7A, first panel). Morphometric measurements reveal that lungs from SS+PBS mice have fewer RAC and longer MLI than lungs from AA mice (Figure 7B and 7C, p<0.0001, and p<0.0001, respectively). In contrast, lungs in SS-hE-HMGBl-BP and SS+KYC mice were more complex than lungs from SS+PBS mice (Figure 7A third and fourth panels vs. second panel, Figure 7Band 7C, p<0.01 and p<0.001, respectively). 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). In contrast, 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). These morphometric data are consistent with the hypothesis that SCD induces chronic lung disease by an HMGB1- and GSNOR-dependent mechanism. Moreover, this mechanism can be effectively inhibited by reducing HMGB1 to control levels or therapeutically targeting MPO, HMGB1 and GSNOR in the SCD inflammatory pathway.

[0103] Discussion

[0104] 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.

[0105] Additionally, the example reveals a new link between HMGB1 and GSNOR. The present invention demonstrates that 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.

[0106] The present inventors observed significant improvements in lung morphometries from treating SS mice with either hE-HMGBl-BP or KYC (Figure 7A-7C). Both treatments reduce lung GSNOR levels, while only KYC increased lung protein SNOs (Figures 5A-5D and 6A-6D). These findings suggest that the mechanisms that HMGB1 uses to induce lung injury are different from the mechanisms KYC uses to improve lung morphometries. In the case of inhibiting / reducing GSNOR levels and activity, this appears to be an effective therapeutic strategy for improving ONO bioavailability (Figures 1A-1E, 3A-3D, and 5A-5D). Such findings have been demonstrated and reported in other disease states. Lung morphometric studies show that treating SS mice with hE-HMGBl-BP or KYC significantly improves RAC and MLI,compared to PBS-treated SS mice (Figure 7A-7C). These data indicate that both HMGB1 and GSNOR mediate chronic lung injury in SS mice and targeting one or both holds promise as therapeutic strategies in SCD.

[0107] The use of hE-HMGBl-BP, which was designed to specifically target extracellular HMGB1, not only enhances endothelial-dependent vasodilation but also reduces chronic lung injury. Similarly, it was shown that KYC modulates GSNOR levels and activity, plus restores lung protein SNOs (Figure 5A-5D). These observations indicate that targeting GSNOR with KYC or improving vasodilation with N6022 are both effective therapeutic approaches for reducing the severity of SCD (Figures 1 A-1E, 5A-5D, 6A-6D and 7A-7C) as has been shown for other diseases.

[0108] Cell-free Hb’s involvement in SCD pathogenesis has found widespread and continued support for over the past 20 years. However previously, it was suggested that HMGB1 was the predominant plasma mediator of TLR4-receptor reporter cell activity in baseline and crisis SCD. The findings strongly support the premise that HMGB1 is a critical mediator of inflammation in SCD. Murine SCD drastically impairs endothelial- and eNOS-dependent vasodilation (Figure 1A-1E). Lowering plasma HMGB1 levels with hE-HMGBl-BP markedly improves vasodilation (Figure 1A-1E) while lowering plasma levels of cf-Hb has minimal effects on endothelial- dependent vasodilation. Findings are consistent that 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.

[0109] The data presented help clarify HMGBl’s importance to SCD pathogenesis, especially when compared to the traditional role of cf-Hb. 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.

[0110] Potential interactions between 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.

[0111] The research highlights the pathogenic roles of MPO, HMGB1, and GSNOR in sterile inflammation and vasculopathy in SCD. If SCD is mediated by a vicious cycle of four processes starting with Hb polymerization, impaired RBC rheology, hemolysis and vascular endothelial adhesion and sterile inflammation, then MPO, HMGB1 and GSNOR should also be examined in the context of the vicious cycle. The results and those of others suggest that MPO, HMGB1 and GSNOR are all participants in sterile inflammation. In addition, MPO, HMGB1, and GSNOR are sufficient to impair vasodilation and induce chronic lung injury in SS mice.

[0112] The multifactorial nature of sterile inflammation in SCD requires consideration of efficient therapeutic approaches. The data reported in this application support the idea that using a systems pharmacology agent to treat SCD is more effective than a monotherapy drug, such as hE-HMGBl-BP in managing SCD. The complex interactions among HMGB1, cf-Hb, and eNOS in SCD’s pathophysiology suggest that focusing on just one pathway might be insufficient.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. As 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. These results suggest that multimodal therapies should be more effective than monomodal agents.

[0113] 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. By focusing on these novel targets and embracing multimodal approaches, it may be possible to reduce the detrimental effects of sterile inflammation in SCD on vasculopathy and chronic lung diseases, and in so doing ultimately improve patient outcomes.

[0114] In conclusion, as illustrated in Figure 8, the results suggest SC A impairs vasodilation and induces chronic lung disease through a mechanism involving HMGB 1 -induced increases in GSNOR expression and activity. Dysregulating GSNOR activity reduces *NO bioavailability and protein SNOs essential for maintaining and preserving vascular function, preventing chronic lung injury, and restoring lung repair and regeneration.

[0115] Example 2- HMGB1-BP Validation and Design and Development of hE- HMGB1-BP: Peptide Identification, Dual-Domain Design, and Synthesis.

[0116] Peptide identification and design of the hE-dual-domain peptide were performed. 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. 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).

[0117] Example 3- Binding kinetics

[0118] Referring to Figure 9, streptavidin biosensors (Sartorius, Bohemia, NY) were equilibrated in kinetic buffer and then dipped into 100 nM biotinylated-Ahx-HMGBl-BP (Biomatik, Kitchener, Ontario, Canada) for loading. After baseline recording on Octet RED96 (ForteBio, Menlo Park, CA), the biosensors were transferred to serial concentrations of r- HMGB1 ranging from 12.5 to 400 nM, and rates of association of HMGB1-BP and rHMGBl recorded in parallel. Following the rHMGBl association, the biosensors were placed into kinetic buffer rates of dissociation of rHMGBl from HMGB1-BP recorded in parallel. The dissociation constant (Kd) was calculated from the association and dissociation rates using Octet’s Data Analysis software (version 9.0).

[0119] Example 4- Affinity Pulldown

[0120] Referring to Figure 10, 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) was incubated with mouse plasma supplemented with 30 pg / ml recombinant human HMGB 1 (GenScript, Piscataway, NJ) at 4°C overnight. 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.

[0121] Example 5- Effects of hE-HMGBl-BP on endothelial-, non-eNOS- and eNOS- dependent vasodilation, and plasma HMGB1

[0122] Townes SS 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. On the day of vasodilation studies, 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. After vasodilation studies were performed in the absence of N(G)-nitro-L-arginine methyl ester(L-NAME), 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.

[0123] 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). 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).

[0125] Example 7- Effects of r-HMGBl on GSNOR expression in HUVEC cultures

[0126] 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 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.

[0127] Example 8- Effects of inhibiting the inflammatory pathway and GSNOR on facialis artery vasodilation

[0128] 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). Since N6022 requires dimethyl sulfoxide (DMSO) for dissolution, DMSO (Sigma- Aldrich, St. Louis, MO; 1% v / v) was added to PBS and KYC injection buffers to control for nonspecific effects of DMSO on vasodilation. After three weeks, the SS mice were fully anesthetized, euthanized by exsanguination, and facialis arteries isolated as described above. Relaxation responses of cannulated and pressurized facialis arteries to Ach (10-7-10-4M) were determined as described above.

[0129] Example 9- Effects of SCA on lung protein SNOs and GSNOR

[0130] Untreated AA and SS mice, 8-9-months-old, were fully anesthetized, euthanized by exsanguination, and lungs perfused in situ with cold PBS to remove blood elements. Lungs were isolated, portioned, and portions snapped frozen in liquid nitrogen. The frozen lung portions were homogenized on the analysis day, and lysates were prepared as previously described. Protein SNOs were determined using the Pierce S -Nitro sylati on Western Blot Kit (ThermoFisher, Milwaukee, WI; Catalog# 90105). 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.

[0131] Example 10- Effects of inhibiting the inflammatory pathway or GSNOR on lung protein SNOs and GSNOR

[0132] 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.

[0133] 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). Slides were deparaffinized, and sections stained with hematoxylin and eosin (H&E; Sigma, St. Louis, MO; catalog# MHS16 and HT110116) for morphometric studies or immunostained for CD31 with a polyclonal goat IgG antibody (R&D Systems, Minneapolis, MN; Catalog# AF3628) and visualized with Alexa Fluor Plus 488-conjugated donkey anti-goat IgG (ThermoFisher, Milwaukee, WI; Catalog# A32814TR) and for GSNOR with a rabbit anti-GSNOR polyclonal antibody (Proteintech Group, Rosemont, IL; Catalog# 11051-1-AP) and visualized with an Alexa Fluor 546-conjugated donkey anti-rabbit IgG (ThermoFisher, Milwaukee, WI; Catalog# Al 0040) for co-localization studies. 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. 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 asabove. CD31 and GSNOR immunofluorescence data represent the mean of four to five random images per section, tested for outliers for each mouse.

[0134] Example 11- Lung GSNOR Dehydrogenase Activity

[0135] 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 GSNOR Enzyme activity was started by adding GSNO (Sigma 487920) to the assay mix (final concentration = 400 pM in 200 pL). Tris buffer and 10 pg / mL recombinant GSNOR (ProSpec ENZ-595) were used as negative and positive controls, respectively. The plate was incubated at room temperature for 30 min, and absorbance at 340 nm was measured using a ThermoFisher UV / Vis microplate reader. The decrease in absorbance at 340 nm was calculated by subtracting the values of the negative controls from those of the 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.

[0136] Example 12- Lung morphometriesDeparaffmized lung sections were stained with hematoxylin and eosin (H&E; Sigma, St. Louis, MO; catalog# MHS16 and HT110116). Lung morphometries consisting of radial alveolar counts (RAC) and mean linear intercept (MLI) lengths were determined as described.

[0137] References

[0138] Gladwin, Sickle cell disease and pulmonary hypertension in Africa: a global perspective and review of epidemiology, pathophysiology, and management, Am J Hematol 83(1) (2008) 63-70.

[0139] F.B. Piel, S.I. Hay, S. Gupta, D.J. Weatherall, T.N. Williams, Global burden of sickle cell anaemia in children under five, 2010-2050: modelling based on demographics, excess mortality, and interventions, PLoS Med 10(7) (2013) el001484.

[0140] P.Q. Le, B. Gulbis, L. Dedeken, S. Dupont, A. Vanderfaeillie, C. Heijmans, S. Huybrechts, C. Devalck, A. Efira, M.-F. Dresse, L. Rozen, F.S. Benghiat, A. Ferster, Survival among children and adults with sickle cell disease in Belgium: Benefit from hydroxyureatreatment: Survival in Sickle Cell Disease in Belgium, Pediatric Blood & Cancer 62(1 1) (2015) 1956-1961.

[0141] E. Arias, J. Xu, B. Tejada-Vera, S. Murphy, B. Bastian, U.S. State Life Tables, 2020, National Vital Statistics Reports, Centers for Disease Control and Prevention, 2022, pp. 1-18.

[0142] L.C. Wu, C.W. Sun, T.M. Ryan, K.M. Pawlik, J. Ren, T.M. Townes, Correction of sickle cell disease by homologous recombination in embryonic stem cells, Blood 108(4) (2006) 1183-8.

[0143] P. Sundd, M.T. Gladwin, E.M. Novelli, Pathophysiology of Sickle Cell Disease, Annual Review of Pathology: Mechanisms of Disease 14(1) (2019) 263-292.

[0144] A.A. Kassim, M.R. DeBaun, Sickle cell disease, vasculopathy, and therapeutics, Annu Rev Med 64 (2013) 451-66.

[0145] C D. Reiter, R.J. Teng, J.S. Beckman, Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite, J Biol Chem 275(42) (2000) 32460-6.

[0146] R. Radi, Nitric oxide, oxidants, and protein tyrosine nitration, Proc Natl Acad Sci U S A 101(12) (2004) 4003-8.

[0147] C D. Reiter, X. Wang, J.E. Tanus-Santos, N. Hogg, R.O. Cannon, 3rd, AN.Schechter, M.T. Gladwin, Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease, Nat Med 8(12) (2002) 1383-9.

[0148] P.C. Minneci, K.J. Deans, H. Zhi, P.S. Yuen, R.A. Star, S.M. Banks, A.N. Schechter, C. Natanson, M.T. Gladwin, S B. Solomon, Hemolysis-associated endothelial dysfunction mediated by accelerated NO inactivation by decompartmentalized oxyhemoglobin, J Clin Invest 115(12) (2005) 3409-17.

[0149] R.P. Hebbel, Reconstructing sickle cell disease: a data-based analysis of the "hyperhemolysis paradigm" for pulmonary hypertension from the perspective of evidence-based medicine, Am J Hematol 86(2) (2011) 123-54.

[0150] G.M. Anantharamaiah, D.W. Garber, D. Goldberg, E. Morrel, G. Datta, M.N. Palgunachari, T.C. Register, S.E. Appt, C.R. White, Novel fatty acyl apoE mimetic peptides have increased potency to reduce plasma cholesterol in mice and macaques, J Lipid Res 59(11) (2018) 2075-2083.

[0151] 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. Wandersee, A Novel Hemoglobin-Binding Peptide Reduces Cell-Free Hemoglobin in Murine Hemolytic Anemia, Am J Physiol Heart Circ Physiol 304(2) (2013) H328-36.

[0152] H. Zhang, H. Xu, D. Weihrauch, D.W. Jones, X. Jing, Y. Shi, D. Gourlay, K.T. Oldham, C.A. Hillery, K.A. Pritchard, Jr., Inhibition of myeloperoxidase decreases vascular oxidative stress and increases vasodilatation in sickle cell disease mice, J Lipid Res 54(11) (2013) 3009-3015.

[0153] K. Xie, Y. Yu, Y. Pei, L. Hou, S. Chen, L. Xiong, G. Wang, Protective Efects of Hydrogen Gas on Murine Polymycrobial Sepsis via Reducing Oxidative Stress and HMGB1 Release, Shock 34(1) (2010) 90-97.

[0154] H. Yang, H. Wang, U. Andersson, Targeting Inflammation Driven by HMGB1, Front Immunol 11 (2020) 484.

[0155] R.K. Wolfson, E.T. Chiang, J.G. Garcia, HMGB1 induces human lung endothelial cell cytoskeletal rearrangement and barrier disruption, Microvasc Res 81(2) (2011) 189-97.

[0156] Z. Zhu, X. Peng, X. Li, T. Tu, H. Yang, S. Teng, W. Zhang, Z. Xing, J. Tang, X. Hu, Z. Fang, S. Zhou, HMGB1 impairs endothelium-dependent relaxation in diabetes through TLR4 / eNOS pathway, Faseb j 34(6) (2020) 8641-8652.

[0157] Y. Zhang, X. Li, A.L. Pitzer, Y. Chen, L. Wang, P.L. Li, Coronary endothelial dysfunction induced by nucleotide oligomerization domain-like receptor protein with pyrin domain containing 3 inflammasome activation during hypercholesterolemia: beyond inflammation, Antioxid Redox Signal 22(13) (2015) 1084-96.

[0158] L.K. Gadanec, U. Andersson, V. Apostolopoulos, A. Zulli, Glycyrrhizic Acid Inhibits High-Mobility Group Box-1 and Homocysteine-Induced Vascular Dysfunction, Nutrients 15(14) (2023).

[0159] 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.

[0160] R.J. Teng, X. Jing, D.P. Martin, N. Hogg, A. Haefke, G.G. Konduri, B.W. Day, S. Naylor, K.A. Pritchard, Jr., N-AcetyLLysyltyrosylcysteine Amide, a Novel Systems Pharmacology Agent, Reduces Bronchopulmonary Dysplasia in Hyperoxic Neonatal Rat Pups, Free Radic Biol Med 166 (2021) 73-89.

[0161] A. Guellouz, M. Valerio-Lepiniec, A. Urvoas, A. Chevrel, M. Graille, Z. Fourati- Kammoun, M. Desmadril, H. van Tilbeurgh, P. Minard, Selection of specific protein binders for pre-defined targets from an optimized library of artificial helicoidal repeat proteins (alphaRep), PLoS One 8(8) (2013) e71512.

[0162] R J. Teng, X. Jing, T. Michalkiewicz, A.J. Afolayan, T.J. Wu, G.G. Konduri, Attenuation of endoplasmic reticulum stress by caffeine ameliorates hyperoxia-induced lung injury, Am J Physiol Lung Cell Mol Physiol 312(5) (2017) L586-L598.

[0163] H. Xu, N.J. Wandersee, Y. Guo, D.W. Jones, S.L. Holzhauer, M.S. Hanson, E. Machogu, D.C. Brousseau, N. Hogg, J.C. Densmore, S. Kaul, C.A. Hillery, K.A. Pritchard, Jr., Sickle cell disease increases high mobility group box 1: a novel mechanism of inflammation, Blood 124(26) (2014) 3978-81.

[0164]

[0165] J.R. Privratsky, P.J. Newman, PECAM-1 : regulator of endothelial junctional integrity, Cell Tissue Res 355(3) (2014) 607-19.

[0166]

[0167] V.M. Dominical, L. Samsel, J.S. Nichols, F.F. Costa, J.P. McCoy, Jr., N. Conran, G.J. Kato, Prominent role of platelets in the formation of circulating neutrophil-red cell heterocellular aggregates in sickle cell anemia, Haematologica 99(11) (2014) e214-7.

[0168] N. Conran, J.D. Belcher, Inflammation in sickle cell disease, Clin Hemorheol Microcirc 68(2-3) (2018) 263-299.

[0169] L. Liu, A. Hausladen, M. Zeng, L. Que, J. Heitman, J.S. Stamler, A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans, Nature 410(6827) (2001) 490-4.

[0170] H.J. Einisman, B. Gaston, C. Wijers, L.A. Smith, T.H. Lewis, S.J. Lewis, T.M.Raffay, Tracheomalacia in bronchopulmonary dysplasia: Trachealis hyper-relaxant responses to S-nitrosoglutathione in a hyperoxic murine model, Pediatr Pulmonol 54(12) (2019) 1989-1996.

[0171] T.M. Raffay, K. Bonilla-Fernandez, A. Jafri, R.B. Sopi, L.A. Smith, F. Cui, M. O'Reilly, R. Zhang, C.A. Hodges, P.M. MacFarlane, G. Deutsch, R.J. Martin, B. Gaston, Bronchopulmonary Dysplasia and Pulmonary Hypertension. The Role of Smooth Muscle adh5, Am J Respir Cell Mol Biol 65(1) (2021) 70-80.

[0172] L.G. Que, Z. Yang, N.L. Lugogo, R.K. Katial, S.A. Shoemaker, J.M Troha, D.M.Rodman, R.M. Tighe, M. Kraft, Effect of the S-nitrosoglutathione reductase inhibitor N6022 on bronchial hyperreactivity in asthma, Immun Inflamm Dis 6(2) (2018) 322-331.

[0173] N. Saxena, J. Won, S. Choi, A.K. Singh, I. Singh, S-nitrosoglutathione reductase (GSNOR) inhibitor as an immune modulator in experimental autoimmune encephalomyelitis, Free Radic Biol Med 121 (2018) 57-68.

[0174] M. Khan, P. Kumar, F. Qiao, S.M.T. Islam, A.K. Singh, J.S. Won, W. Feng, I. Singh, Targeting GSNOR for functional recovery in a middle-aged mouse model of stroke, Brain Res 1741 (2020) 146879.

[0175] G. Yu, Y. Liang, Z. Huang, D.W. Jones, K.A. Pritchard, Jr., H. Zhang, Inhibition of myeloperoxidase oxidant production by N-acetyl lysyltyrosylcysteine amide reduces brain damage in a murine model of stroke, J Neuroinflammation 13(1) (2016) 119.

[0176] H. Zhang, A. Ray, N.M. Miller, D. Hartwig, K.A. Pritchard, Jr., B.N. Dittel, Inhibition of Myeloperoxidase at the Peak of Experimental Autoimmune Encephalomyelitis Restores Blood-Brain-Barrier Integrity and Ameliorates Disease Severity, J Neurochem (2016) 826-36.

[0177] D. Weihrauch, D.P. Martin, D. Jones, J. Krolikowski, J. Struve, S. Naylor, K.A. Pritchard, Jr., Inhibition of myeloperoxidase increases revascularization and improves blood flow in a diabetic mouse model of hindlimb ischaemia, Diab Vase Dis Res 17(3) (2020) 1479164120907971.

[0178] G.J. Kato, M.T. Gladwin, M.H. Steinberg, Deconstructing sickle cell disease: reappraisal of the role of hemolysis in the development of clinical subphenotypes, Blood Rev 21(1) (2007) 37-47.

[0179] R.P. Hebbel, G.M. Vercellotti, Multiple inducers of endothelial NOS (eNOS) dysfunction in sickle cell disease, Am J Hematol 96(11) (2021) 1505-1517.

[0180] T. Babikian, T. Merkley, R.C. Savage, C.C. Giza, H. Levin, Chronic Aspects of Pediatric Traumatic Brain Injury: Review of the Literature, Journal of Neurotrauma 32(23) (2015) 1849-1860.

[0181] U. Andersson, W. Ottestad, K.J. Tracey, Extracellular HMGB1 : a therapeutic target in severe pulmonary inflammation including COVID-19?, Mol Med 26(1) (2020) 42.

[0182] M.T. Lotze, K.J. Tracey, High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal, Nature Reviews Immunology 5(4) (2005) 331-342.

[0183] K.M. Casin, J. Fallica, N. Mackowski, R.J. Veenema, A. Chan, A. St Paul, G. Zhu, D. Bedja, S. Biswal, M.J. Kohr, S-Nitrosoglutathione Reductase Is Essential for Protecting the Female Heart From Ischemia-Reperfusion Injury, Circ Res 123(11) (2018) 1232-1243.

Claims

CLAIMSWe claim:

1. 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 a subject comprising: an effective amount of an end-capped (N-acetylated, C-amidated) dual -domain peptide hE-HMGBl-BP (Ac-LRKLRKRLLR-GG-AHSANNFDVKGI-NH-2, acetate salt) configured to bind and deplete HMGB1 from circulation via uptake by a liver’s heparin sulfate proteoglycan (HSPG) system; and an acceptable carrier.

2. The composition according to claim 1, further comprising an effective amount of N- acetyl-lysyltyrosyl cysteine amide (KYC) designed to inhibit myeloperoxidase (MPO)-initiated and HMGB1 -propagated inflammatory pathway.

3. The composition according to claim 1, wherein the end capped dual-domain peptide hE-HMGBl-BP contains a GG bridge between 12mer HMGB1-BP peptide domain and an apoE receptor binding peptide domain (hE).

4. The composition according to claim 3, wherein 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 an apolipoprotein E receptor domain5. The composition according to claim 1, wherein said composition is 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.

6. 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 a subject comprisingadministering to the subject in need of such treatment an effective amount of end-capped (N-acetylated, C-amidated) dual-domain or chimeric peptide hE-HMGBl-BP (Ac- LRKLRKRLLR-GG-AHSANNFDVKGI-NH-2, acetate salt) designed to bind and deplete HMGB1 from circulation via uptake by a liver’s heparin sulfate proteoglycan (HSPG) system.

7. The method according to claim 5, further comprising administering to the subject in need of such treatment an effective amount of N-acetyl-lysyltyrosyl cysteine amide (KYC) designed to inhibit myeloperoxidase (MPO)-initiated and HMGB 1 -propagated inflammatory pathway in bronchopulmonary dysplasia.

8. The method according to claim 5, further comprising 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 S- nitrosoglutathione reductase (GSNOR).

9. The method according to claim 5, wherein administration of hE-HMGB 1 -BP defined by the end-capped (N-acetylated, C-amidated) dual-domain peptide to said subject improves vascular function, decreases pulmonary inflammation, and / or increases cardio protection in the subject.

10. The method according to claim 5, wherein the symptom or disorder 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.

11. The method according to claim 5, wherein the symptom or disorder associated with excess recruitment of myeloid cells that express myeloperoxidase / peroxidase thus indirectly reducing peroxidase activity in the subject is at least one of hypertension, peripheral vasculardisease, 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, and bronchopulmonary dysplasia.

12. Use of an end-capped (N-acetylated, C-amidated) dual-domain peptide hE-HMGBl- 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 a subject.

13. The use according to claim 12, wherein the pharmaceutical composition is formulated as an oral dose comprising the end capped dual-domain peptide hE-HMGBl-BP and a carrier.

14. The use according to claim 12, wherein the symptom 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.

15. The use according to claim 12, wherein the symptom associated with excess recruitment of myeloid cells that express myeloperoxidase / peroxidase thus indirectly reducing peroxidase activity in the subject is 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, lungcancer, 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, and bronchopulmonary dysplasia.

16. An end-capped (N-acetylated, C-amidated) dual-domain peptide hE-HMGBl-BP (Ac-LRKLRKRLLR-GG-AHS ANNFDVKGI-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 a subject.

17. The end-capped (N-acetylated, C-amidated) dual-domain peptide hE-HMGBl-BP (Ac-LRKLRKRLLR-GG-AHSANNFDVKGI-NH-2, acetate salt) of claim 16, wherein the end capped dual-domain peptide hE-HMGBl-BP is formulated as an oral dose comprising the dualdomain peptide hE-HMGBl-BP and a carrier.

18. The end-capped (N-acetylated, C-amidated) dual-domain peptide hE-HMGBl-BP ( Ac-LRKLRKRLLR-GG-AHS ANNFDVKGI-NH-2, acetate salt) of claim 16, wherein the symptom 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.

19. The end-capped (N-acetylated, C-amidated) dual-domain peptide hE-HMGBl-BP ( Ac-LRKLRKRLLR-GG-AHS ANNFDVKGLNH-2, acetate salt) of claim 16, wherein the symptom associated with excess recruitment of myeloid cells that express myeloperoxidase / peroxidase thus indirectly reducing peroxidase activity in the subject is at leastone 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 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, and bronchopulmonary dysplasia.

Images