WO2024112490A2 - Formulations de nitrosyle-hème pour le traitement d'affections cardiovasculaires, de l'hémolyse, et la stabilisation de molécules d'hème acellulaires - Google Patents

Formulations de nitrosyle-hème pour le traitement d'affections cardiovasculaires, de l'hémolyse, et la stabilisation de molécules d'hème acellulaires Download PDF

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WO2024112490A2
WO2024112490A2 PCT/US2023/078075 US2023078075W WO2024112490A2 WO 2024112490 A2 WO2024112490 A2 WO 2024112490A2 US 2023078075 W US2023078075 W US 2023078075W WO 2024112490 A2 WO2024112490 A2 WO 2024112490A2
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heme
containing compound
sulfide
thiol
composition
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WO2024112490A3 (fr
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Anthony W. DeMartino
Daniel B. Kim-Shapiro
Jason J. Rose
Jesus TEJERO BRAVO
Laxman POUDEL
Mark Thomas Gladwin
Matthew R. DENT
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Wake Forest University
University of Pittsburgh
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University of Pittsburgh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/42—Haemoglobins; Myoglobins

Definitions

  • compositions and methods including stabilized nitric oxide, such as a NO- heme complex and a thiol or sulfide compound.
  • the compositions and methods disclosed herein can induce vasodilation or treat a hemolytic condition.
  • NO synthases It is canonically produced by NO synthases. NO generated in the endothelium freely diffuses to smooth muscle where it binds soluble guanylyl cyclase (sGC), ultimately stimulating vasorelaxation.
  • sGC soluble guanylyl cyclase
  • other sources of NO also regulate vascular tone, such as inorganic nitrite, which serves as a stable vascular pool of NO. Hemoglobin reduces nitrite to NO in red blood cells, but how this erythrocytic NO escapes the red blood cell without capture by abundant hemoglobin to reach sGC in the smooth muscle has not yet been elucidated.
  • a composition that includes a nitric oxide (NO)-heme complex (or both a NO source and a heme source, which form a NO-heme complex), a heme-solubilizing agent (such as albumin, a lipid-nanoparticle, a lipid-microparticle, a lipid emulsion, a micelle, a ghost red blood cell, a cell membrane component, or a cell membrane lysate), and a thiol-containing compound (such as 8123-109216-02 glutathione, cysteine, N-acetylcysteine, cysteinyl glycine, a reducing agent, an electron donor compound, a low molecular weight thiol, or a high molecular weight thiol) or a sulfide-containing compound (such as hydrogen sulfide, an inorganic sulfide, a reducing agent, an electron donor compound,
  • NO nitric oxide
  • the composition further includes a pharmaceutically acceptable carrier.
  • the composition can be used, for example, to induce vasodilation in a subject, such as a subject with a cardiovascular condition, respiratory failure, metabolic syndrome, diabetes, a lipid disorder, inflammation, aging, or an infectious disease.
  • compositions that includes a thiol-containing compound (such as glutathione, cysteine, N-acetylcysteine, cysteinyl glycine, a reducing agent, an electron donor compound, a low molecular weight thiol, or a high molecular weight thiol) or a sulfide-containing compound (such as hydrogen sulfide, an inorganic sulfide, a reducing agent, an electron donor compound, a lower molecular weight sulfide, or a high molecular weight sulfide); a NO source (such as a NO-donor or NO-generating agent, e.g., nitrite); and a heme-solubilizing agent (such as albumin, a lipid-nanoparticle, a lipid-microparticle, a lipid emulsion, a micelle, a ghost red blood cell, a cell membrane component, or a cell
  • the composition further includes a pharmaceutically acceptable carrier.
  • the composition can be used, for example, to treat a hemolytic condition in a subject, such as an inherited hemolytic condition (e.g., sickle cell anemia or a thalassemia) or an acquired hemolytic condition.
  • the acquired hemolytic condition results from an infection (such as a viral or bacterial infection), a medication (such as penicillin, antimalarial medication, sulfa or acetaminophen), a blood cancer, an autoimmune disorder (such as lupus, rheumatoid arthritis, or ulcerative colitis), a tumor, hypersplenism, a mechanical heart valve or a blood transfusion reaction.
  • composition that includes a cell-free heme protein, a NO-heme complex (or both a NO source and a heme source, which form a NO-heme complex), and a thiol- containing compound (such as glutathione, cysteine, N-acetylcysteine, cysteinyl glycine, a reducing agent, an electron donor compound, a lower molecular weight sulfide, or a high molecular weight sulfide) or a sulfide-containing compound (such as hydrogen sulfide, an inorganic sulfide, a reducing agent, an electron donor compound, a lower molecular weight sulfide, or a high molecular weight sulfide).
  • a thiol- containing compound such as glutathione, cysteine, N-acetylcysteine, cysteinyl glycine
  • a reducing agent such as glutathione, cysteine
  • the composition further includes a pharmaceutically acceptable carrier.
  • the composition can be used, for example, for ameliorating the effects of heme-protein interactions with nitric oxide (such as the vasoconstrictive effect of heme-protein interactions with nitric oxide) in a subject.
  • Also provided herein is a method (such as an in vitro, in silico, ex vivo or in vivo method) of producing a stable nitric oxide (NO)-heme complex by contacting NO, a NO donor, nitrite or nitrate with a ferric or ferrous heme in the presence of a thiol-containing compound (such as glutathione, cysteine, N-acetylcysteine, cysteinyl glycine, a reducing agent, an electron donor compound, a low 8123-109216-02 molecular weight thiol, or a high molecular weight thiol) or a sulfide-containing compound (such as hydrogen sulfide, an inorganic sulfide, a reducing agent, an electron donor compound, a lower molecular weight sulfide, or a high molecular weight sulfide).
  • a thiol-containing compound such as glutathione,
  • FIGs.1A-1G depict basis spectra and reaction kinetics of glutathione (GSH)-assisted NO- ferroheme formation in anaerobic MeOH:PBS buffer.
  • FIG.1A shows representative reductive nitrosylation of 12.5 ⁇ M ferric hemin by 1250 ⁇ M NO showing spectral changes (top) with initial spectrum in light gray, and kinetics at 570 and 611 nm (bottom).
  • FIG.1B shows GSH-catalyzed reductive nitrosylation of 12.5 ⁇ M ferric hemin with 125 ⁇ M GSH and 125 ⁇ M NO showing same regions as FIG.1A with arrows showing spectral evolution.
  • FIG.1C shows addition of 250 ⁇ M NO to 12.5 ⁇ M ferrous heme (from ⁇ 100 ⁇ M sodium dithionite) results in NO-ferroheme spectrum (hashed line) that is identical to GSH-generated species (solid line) observed in both FIG.1A and FIG.1B, except with the characteristic dithionite peak at 315 nm.
  • FIG.1D shows the spectra of 25 ⁇ M ferric heme (hashed line) and 25 ⁇ M ferric heme with 250 ⁇ M GSH (solid line) are identical.
  • FIG.1E 12.5 ⁇ M ferric heme with 125 ⁇ M GSH (black line) reacted with ⁇ 100 ⁇ M dithionite, yielding ferrous heme (grey line). All reaction kinetics were completed at 23°C.
  • FIG.1F observed pseudo 1st-order rate constants vs concentration of either GSH (open circle, [NO] at 250 ⁇ M) or NO (open squares, [GSH] at 250 ⁇ M) resulting in observed 2nd-order rate constants of 7000 and 2300 M-1s-1, respectively.
  • FIG.1G stoichiometry of the thiol- catalyzed reaction in MeOH:PBS buffer determined by NO quantification using a chemiluminescent NO analyzer.20 ⁇ M NO-ferroheme solution injections (based on heme) were injected into ferricyanide solution (open squares, untreated) yielding 17.9 ⁇ 1.9 ⁇ M NO and into Cu/Cys (2C assay), yielding 16.0 ⁇ 2.0 ⁇ M NO (circles, untreated).
  • DMPO 5,5-dimethyl-1- pyrroline-N-oxide
  • FIGs.2A-2F show that GSH accelerates the reaction of NO and ferric heme in suspended red blood cell (RBC) membrane white ghosts under anaerobic conditions.
  • FIG.2B 50 ⁇ M NO with 25 ⁇ M ferric heme solubilized in the RBC membranes results in reductive nitrosylation in the membranes over time. The time course follows the formation of NO-ferroheme at 570 nm.
  • FIG.2C electron paramagnetic resonance (EPR) measurements of the final NO-ferroheme product made using RBC membranes: without GSH (b, black line, 5.2 ⁇ M NO- ferroheme) and with GSH added (a, gray line, 13.5 ⁇ M NO-ferroheme).
  • FIG.2E top, EPR spectra of 25 ⁇ M ferric heme, 50 ⁇ M GSH, and 50 ⁇ M NO reacted in RBC membranes at three different powers: 0.1, 1, and 10 mW.
  • FIGs.3A-3E show NO-ferroheme formation via GSH-catalyzed reductive nitrosylation of ferric heme in serum albumin.
  • FIG.3A Spectral changes of 25 ⁇ M ferric heme solubilized by 500 ⁇ M albumin in PBS with 250 ⁇ M NO and 250 ⁇ M GSH under anaerobic conditions at 22°C.
  • FIG. 3B Rates of NO-ferroheme generation in albumin increase with GSH concentration.
  • FIG.3C The EPR signature of NO-ferroheme in albumin is consistent with a pentacoordinate NO-ferroheme species. Experimental data are shown together with the theoretical simulation used to obtain g-values and hyperfine tensors, as described in the methods.
  • FIG.3D Transfer of NO-ferroheme from membranes to serum albumin.25 ⁇ M ferric heme, 50 ⁇ M glutathione, and 50 ⁇ M NO were added to RBC membrane ghosts under deaerated conditions. Addition of 75 ⁇ M serum albumin resulted in the hashed black spectrum, which exhibits typical light scattering due to turbidity from insoluble membranes.
  • FIG.3E Transfer of NO-ferroheme from albumin to apo-myoglobin in 1:1 ratio of heme to apo-myoglobin. Peaks characteristic of nitrosyl- myoglobin are observed rapidly following addition of apo-myoglobin to NO-ferroheme solubilized with albumin. Observation of isosbestic points indicate direct NO-ferroheme transfer.
  • FIGs.4A-4C depict the effects of NO-ferroheme albumin on platelet activation.
  • ADP adenosine diphosphate
  • FIG.4C Establishment of sGC activation by NO-ferroheme via cGMP detection. All controls without NO or NO-ferroheme exhibited basal cGMP production.
  • FIGs.5A-5E depict changes in murine mean arterial pressure (MAP) by NO-ferroheme in albumin from glutathione-catalyzed reductive nitrosylation.
  • FIG.5A NO-ferroheme induced vasodilation at different concentrations under 10% oxygenated breathing.
  • FIG.5C Maximum changes in MAP under hypoxia.
  • FIG.5D Maximum changes in MAP under normoxia.
  • NO-ferroheme solution open squares
  • FIG.5E Comparison between maximum MAP responses toward NO-ferroheme at each concentration under hypoxia (10% oxygen, gray-filled circles) and normoxia (21% oxygen, open squares).
  • FIGs.5A and 5B show changes in murine mean arterial pressure (MAP) by NO-ferroheme prepared via ferrous heme generation by sodium dithionite and NO addition in albumin.
  • FIG.6A Observed MAP changes from NO-ferroheme made from ferrous heme.
  • hypoxia 10% oxygen
  • These regimens were administered in doses 10 minutes apart giving estimated blood concentration in the mouse of 7.5 nM, 75 nM, 0.75 ⁇ M, and 7.5 ⁇ M of each preparation.
  • FIG.6B ⁇ MAP max quantification for each injected species described in FIG.6A.
  • FIG.7 shows stability of NO-ferroheme in serum albumin mixed with oxyhemoglobin.
  • FIGs.8A-8G show example NO traces from NO chemiluminescence analyzer.
  • FIG.8A NO response from nitrite calibration traces.
  • FIG.8B Example calibration curve using known quantities of sodium nitrite at 25°C.
  • each point of the curve represents an average of 3 injections at one nitrite concentration.
  • FIG.8C Example injections of NO-ferroheme prepared in MeOH:PBS buffer using GSH into a chemiluminescence analyzer purge vessel containing ferricyanide solution at 25°C.
  • FIG.8D Example NO response from 2C (copper/cysteine) calibration traces.
  • FIG.8E Example calibration curve using known quantities of fresh S-nitrosoglutathione at 54°C. Each point represents one injection in one experiment.
  • FIG.8F Example injections of the same NO-ferroheme solution into a chemiluminescence analyzer purge vessel containing the 2C solution at 54°C.
  • FIG.8G Same as in FIG.8E, except the solution was first pretreated with excess DMPO to block GSNO formation before synthesis of NO-ferroheme: no NO originating from S-nitrosothiols is detectable. In all cases, solutions are purged briefly ( ⁇ 1-2 min) with nitrogen to remove dissolved free NO before addition to the chemiluminescence analyzer.
  • FIGs.9A-9C show formation of NO-ferroheme via GSH-catalyzed reductive nitrosylation of ferric heme in albumin at different NO concentrations.
  • FIG.9A Observed pseudo first-order rate constants vs GSH concentration in reaction of ferric heme, NO, and GSH in solution containing albumin.
  • the reaction exhibits two phases, that each comprise roughly 50% of the reaction at each GSH concentration (right y-axis, open squares).
  • the fast phase (left y-axis, open diamonds) exhibits an observed 2 nd -order rate constant of 700 M -1 s -1 , while the second (left y-axis, closed circles) is 20 M- 1 s -1 .
  • the two phases are linear under pseudo first-order conditions, indicating they are both GSH dependent. Reactions were conducted at 22 °C. Each k obs represents an average of at least 3 trials.
  • FIG.9B Determined stoichiometry of this reaction in albumin using the chemiluminescence NO detection with the ferricyanide test (closed circles) and the copper/cysteine (2C) assay (open squares), as described in FIG.1.
  • 25 ⁇ M ferric heme was mixed with 50 ⁇ M GSH and a variable amount of NO in 75 ⁇ M albumin under anaerobic or aerobic (21% O 2 ) conditions. Slightly more GSNO is produced under aerobic conditions and slightly more NO-ferroheme is generated in anaerobic conditions.
  • FIG.9C NO-ferroheme formation is observed via EPR (approaching the limit of detection) at 2 ⁇ M NO as described in FIG. 9B under anaerobic conditions.
  • the experimental (solid line) and simulated data (hashed line) are 8123-109216-02 consistent with formation of 5-coordinate nitrosyl heme, as observed in FIG.3. All data – where appropriate – are presented as mean values ⁇ SD.
  • FIGs.10A-10B show other transfers of NO-ferroheme.
  • FIG.10A Directionality of NO- ferroheme binding: NO-ferroheme in albumin incubated with membranes does not transfer to the membranes, but rather remains bound to albumin.25 ⁇ M ferric heme, 50 ⁇ M glutathione, and 50 ⁇ M NO were added to 75 ⁇ M albumin under deaerated conditions. Addition of RBC ghost membranes results in typical light scattering due to turbidity from insoluble membranes. The mixture was centrifuged at 30,000 g for 2 hours, leaving behind NO-ferroheme in the albumin (hashed line), indicating little transfer to the membranes.
  • FIG.10B Direct formation of NO-myoglobin from a mixture of RBC ghost membrane-solubilized NO-ferroheme (from 15 ⁇ M hemin, 50 ⁇ M NO, and 150 ⁇ M GSH) and 20 ⁇ M apo-myoglobin. Transfer experiments were conducted at 21°C.
  • FIG.11 shows NO-ferroheme albumin stability in the presence of an equivalent of oxyhemoglobin.25 ⁇ M NO-ferroheme in 75 ⁇ M albumin was added to 25 ⁇ M oxyhemoglobin in an aerobic atmosphere and monitored via UV-Visible spectroscopy over the course of 5 hours.
  • Dissociation of NO from NO-ferroheme albumin should result in NO dioxygenation with concomitant oxyhemoglobin oxidation to methemoglobin, which has a characteristic absorption peak around 630 nm (3.9 mM -1 cm -1 ). Roughly 7 ⁇ M of methemoglobin is generated, corresponding to a rate constant for NO loss from NO-ferroheme of 2.1 x 10 -5 s -1 . Spectra were collected at 23 °C.
  • FIG.12 shows other relevant platelet control experiments. Experimental conditions for platelet-rich plasma activation are described in the Examples.2 ⁇ M adenosine diphosphate (ADP) was added to activate platelets.
  • ADP adenosine diphosphate
  • FIG.13A-13C show elimination of nitrite as potential vasodilating species during administration of NO-ferroheme albumin solution.
  • FIG.13A Analysis of NO-yielding species in the primary stock solution of NO-ferroheme albumin administered to mice.
  • NO-ferroheme, GSNO, and nitrite were quantified using the tri-iodide assay and nitrite standard solutions for the calibration curve.
  • GSNO and nitrite were isolated from albumin-conjugated NO-ferroheme via elution using a 10 kDa molecular weight cut-off (MWCO) spin filter.
  • MWCO molecular weight cut-off
  • Nitrite signals were differentiated from that of GSNO using acidified sulfanilamide, which consumes nitrite but not GSNO.
  • 50 ⁇ 12 ⁇ M nitrite was found in the stock formulations of 300 ⁇ M NO-ferroheme, or roughly 1.25 ⁇ M nitrite in blood concentration at the highest dose.
  • FIGs.14A-14B show typified flow cytometry gating for platelet sorting and platelet activation determination.
  • FIG.14A shows events plotted by side scatter (SSC-H) vs forward scattering (FSC-H) which is used to separate red blood cells from platelets by size. These events are then plotted by CP-CD61 fluorescence on the x-axis (FIG.14B) vs PAC1-FITC fluorescence on the y-axis indicating activated platelets (upper right corner).
  • FIGs.15A-15C show reaction of heme with NO in the red cell ghost system.
  • FIG.15A Reaction of 25 ⁇ M heme and 100 ⁇ M NO in the red cell ghost system under anoxic conditions at room temperature in the absence of any thiol species. Heme was first solubilized in 10% red cell ghosts (v/v) in PBS, and the kinetics of its reaction with NO was studied in a UV-Vis spectrometer with an integrating sphere (Cary 100) for one hour, scanning every minute. The product of the reaction is NO-ferroheme and has a calculated half-life of about 4 minutes for first-order kinetics. The dotted line represents the heme absorption in membranes. Inset: Absorption change at 570 nm. FIG.
  • FIG.15C Reaction of 25 ⁇ M heme with 100 ⁇ M NO in red cell ghost system in the presence of 250 ⁇ M sodium sulfide. Sulfide was added to the pre-solubilized heme in membranes before adding NO and the reaction kinetics was studied for 30 minutes in Cary 100. All reaction conditions were kept the same as those in the reaction without a thiol. The dotted line represents the spectrum of the heme-thiol-membrane system before adding NO. The presence of sulfide completes the NO-ferroheme reaction within the first scan. Inset: Absorption change at 570 nm. FIG.16 depicts an exemplary mechanism by which NO-ferroheme albumin may cause vasodilation.
  • FIG.17 depicts exemplary electron transfer mechanisms for reactions described herein.
  • SEQUENCES The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as 8123-109216-02 defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NO: 1 is an exemplary amino acid sequence for human serum albumin.
  • SEQ ID NO: 4 is an exemplary amino acid sequence for human hemoglobin subunit beta.
  • Nitric oxide is an endogenously produced signaling molecule that regulates blood flow and platelet activation.
  • NO is an endogenously produced signaling molecule that regulates blood flow and platelet activation.
  • intracellular and intravascular diffusion of NO are limited by scavenging reactions with several hemoproteins.
  • NO can be stabilized as a labile ferrous heme-nitrosyl complex (Fe 2+ -NO, NO-ferroheme or NO-heme).
  • a reaction between NO, labile ferric heme (Fe 3+ ), and reduced thiols yields NO-ferroheme and a thiyl radical.
  • This thiol- catalyzed reductive nitrosylation occurs when heme is solubilized in lipophilic environments, such as red blood cell membranes or bound to serum albumin.
  • the resulting NO-ferroheme resists oxidative inactivation, is soluble in cell membranes, and is transported intravascularly by albumin to promote 8123-109216-02 potent vasodilation.
  • This provides an alternative route for NO delivery from erythrocytes and blood via transfer of NO-ferroheme and activation of apo-sGC.
  • the iron-containing prosthetic group heme (iron protoporphyrin IX) is ubiquitous in biology. In blood, heme associates with proteins, including hemoglobin in red blood cells (RBCs), along with hemopexin, apolipoproteins, and albumin in plasma.
  • Heme also soluble in membrane lipids, is present in the erythrocyte membrane and is actively transported between cells via specific heme transporters.
  • NOS nitric oxide synthase
  • sGC soluble guanylate cyclase
  • K d 5 x 10 -8 – 4 x 10 -12 M.
  • Deoxygenating hemoglobin also produces NO and stimulates vasodilation via nitrite reduction, with NO bound to hemoglobin (called iron nitrosyl hemoglobin) forming during physiological artery-to-vein deoxygenation.
  • RBCs themselves contain active endothelial NOS (eNOS). 9,10 NO is scavenged by hemoproteins such as oxygenated hemoglobin and myoglobin at almost diffusion-limited rates ( ⁇ 6-8 x 10 7 M -1 s -1 at 20°C), 11 and reacts with deoxygenated hemoglobin and myoglobin at similarly high reaction rates to form iron-nitrosyl globins. Both reaction processes limit NO bioavailability to activate sGC.
  • eNOS active endothelial NOS
  • NO signaling in hemoprotein-rich environments may involve other NO-derived species such as S-nitrosothiols 17–19 or other nitrogen oxides such as N2O3, 20–24 as such species cannot undergo NO autocapture and dioxygenation by hemoproteins.
  • NO- ferroheme nitrosyl heme
  • 25–27 Nitrosyl heme can directly activate heme-free (apo-) sGC in vitro. 28 Further, labile heme is a signaling molecule. 29–31 Heme transporters are found on the membranes of many cells, including RBCs.
  • EPR electron paramagnetic resonance
  • 1,3,35 NO reversibly binds ferric heme with much lower affinity than for ferrous heme.
  • a nitrosyl-ferric heme can then react with 8123-109216-02 hydroxide to form nitrite and a ferrous heme, which rapidly binds any excess NO to form NO- ferroheme in a process known as reductive nitrosylation.
  • 37,38 The reduction step is rate-limiting, very slow, and requires two molecules of NO to form one NO-ferroheme. 37 This process is therefore an inefficient source of nitrosyl-ferrous heme in vivo.
  • Disclosed herein is an alternative mechanism for rapid formation of a stable iron-nitrosylated heme using one NO molecule.
  • NO-ferroheme is readily taken up by RBC membranes and serum albumin, is stable in the presence of oxyhemoglobin, and exhibits potent vasodilatory responses in vivo. II.
  • EPR electron paramagnetic resonance GSH reduced glutathione GSNO S-nitrosoglutathione H2S hydrogen sulfide Hb hemoglobin HBOC hemoglobin-based oxygen carrier L-NAME L-N G -nitroarginine methyl ester
  • MAP mean arterial pressure MWCO molecular weight cut-off NO nitric oxide
  • PDE phosphodiesterase RBC red blood cell sGC soluble guanylyl cyclase SP systolic blood pressure III. Summary of Terms Unless otherwise noted, technical terms are used according to conventional usage.
  • an antigen includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.”
  • the term “comprises” 8123-109216-02 means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
  • Administration To provide or give a subject an agent, such as a therapeutic composition, by any effective route.
  • routes of administration include, but are not limited to, injection or infusion (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intravenous, intracerebroventricular, intrastriatal, intracranial and into the spinal cord), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • Albumin A family of globular transport proteins that generally include three homologous domains that assemble to form a heart-shaped protein.
  • Exemplary albumins include human serum albumin, alpha-fetoprotein, vitamin D-binding protein, afamin, extracellular matrix protein 1, bovine serum albumin, and porcine serum albumin. Further exemplary albumins include sterile solutions of human albumin in aqueous diluent at a defined percentage, such as 5% or 25%. Other exemplary albumins include AlbukedTM 5, AlbukedTM 25, Albumin-ZLBTM, AlbuminexTM, AlbuRx;TM, AlbuteinTM, FlexbuminTM, Human Albumin GrifolsTM, KedbuminTM, Plasbumin-25TM, Plasbumin- 5TM, Alburex-25TM, Alburex-5TM, OctalbinTM, and Plasbumin-5TM.
  • Human serum albumin is a transport protein found in human blood. An exemplary amino acid sequence for human serum albumin is set forth herein as SEQ ID NO: 1 (see also NCBI Ref Seq: NM_000477.7). “Bovine serum albumin” is a transport protein found in bovine blood. An exemplary amino acid sequence for bovine serum albumin is set forth herein as SEQ ID NO: 2 (see also GenBank: CAA76847.1).
  • Anemia A deficiency of red blood cells and/or hemoglobin. Anemia is a common disorder of the blood, and it results in a reduced ability of blood to transfer oxygen to the tissues. Since all human cells depend on oxygen for survival, varying degrees of anemia can have a wide range of clinical consequences.
  • anemia refers to all types of clinical and subclinical anemia, including but not limited to: blood-loss anemia, hemorrhagic shock, ineffective hematopoiesis from primary bone marrow failure, ineffective hematopoiesis from hematologic malignancy or malignant invasion of the bone marrow, drug-induced ineffective hematopoiesis, chemotherapy-induced ineffective hematopoiesis, radiation-induced drug-induced ineffective hematopoiesis, microcytic anemia, iron deficiency anemia, hemoglobinopathies, heme synthesis defect, globin synthesis defect, sideroblastic 8123-109216-02 defect, normocytic anemia, anemia of chronic disease, aplastic anemia
  • a blood transfusion may be administered.
  • Clinicians may use any of a number of clinically accepted criteria to determine that a blood transfusion is appropriate to treat a subject with anemia. For instance, the Rivers protocol for early goal-directed therapy for sepsis requires keeping the hematocrit above 30.
  • Bleeding disorder A general term for a wide range of medical problems that lead to poor blood clotting and continuous bleeding. Clinicians also refer to bleeding disorders by terms such as, for example, coagulopathy, abnormal bleeding, and clotting disorders. Bleeding disorders include any congenital, acquired, or induced defect that results in abnormal (or pathological) bleeding.
  • Examples include, but are not limited to, disorders of insufficient clotting or hemostasis, such as hemophilia A (a deficiency in Factor VIII), hemophilia B (a deficiency in Factor IX), hemophilia C (a deficiency in Factor XI), other clotting factor deficiencies (such as Factor VII or Factor XIII), abnormal levels of clotting factor inhibitors, platelet disorders, thrombocytopenia, vitamin K deficiency and von Willebrand’s disease.
  • Blood The fluid that circulates through the heart, arteries, capillaries, and veins (that is, the circulatory system), and is the primary transport mechanism in the body.
  • Blood transports oxygen from the lungs to the body tissues and carbon dioxide from the tissues to the lungs. Blood also transports nutritive substances and metabolites to the tissues and removes waste products to the kidneys and other organs for excretion. In addition, blood plays a critical role in maintenance of fluid balance. Blood has two primary parts – plasma (the fluid portion) and formed elements (the solid components). The solid components of blood include erythrocytes (red blood cells), leukocytes (white blood cells) and platelets. As used herein, “whole blood” refers to blood that has not had any components removed (blood that contains both the fluid and solid components). A “blood product” refers to one or more components of the blood, such as red blood cells, serum, or plasma.
  • Cardiogenic shock A medical condition that results from the heart being unable to pump a sufficient amount of blood to the brain and other vital organs.
  • Cerebral ischemia or ischemic stroke A condition that occurs when an artery to or in the brain is partially or completely blocked such that the oxygen demand of the tissue exceeds the oxygen supplied. Deprived of oxygen and other nutrients following an ischemic stroke, the brain suffers damage as a result of the stroke. Ischemic stroke can be caused by several different kinds of diseases. The most common problem is narrowing of the arteries in the neck or head. This is most often caused by atherosclerosis, or gradual cholesterol deposition. If the arteries become too narrow, blood cells may collect in them 8123-109216-02 and form blood clots (thrombi).
  • blood clots can block the artery where they are formed (thrombosis), or can dislodge and become trapped in arteries closer to the brain (embolism).
  • Another cause of stroke is blood clots in the heart, which can occur as a result of irregular heartbeat (for example, atrial fibrillation), heart attack, or abnormalities of the heart valves. While these are the most common causes of ischemic stroke, there are many other possible causes. Examples include use of street drugs, traumatic injury to the blood vessels of the neck, or disorders of blood clotting.
  • Contacting Placement in direct physical association; includes both in solid and liquid form. When used in the context of an in vivo method, “contacting” also includes administering. Control: A reference standard.
  • control is a negative control. In other aspects, the control is a positive control.
  • a control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients diagnosed with a disease or condition, for example a hemolytic condition, that have a known prognosis or outcome, or a group of samples that represent baseline or normal values).
  • the control may be a subject not receiving treatment with an agent (e.g., a NO-heme complex) or receiving an alternative treatment, or a baseline reading of the subject prior to treatment with an agent.
  • Cysteine A thiol-containing non-essential amino acid that is oxidized to form cystine.
  • Glutathione A tripeptide with a gamma peptide linkage between the carboxyl group of the glutamate side chain and cysteine.
  • Molecular Formula C 10 H 17 N 3 O 6 S. PubChem CID: 124886.
  • IUPAC (2S)-2-amino-5-[[(2R)-1-(carboxymethylamino)-1-oxo-3-sulfanylpropan-2-yl]amino]-5- oxopentanoic acid.
  • Heart failure An acute or chronic condition characterized by the inability of the heart to pump sufficient blood for the body’s needs.
  • Heme A cofactor including an iron ion contained in the center of a porphyrin.
  • a “heme protein” or “hemoprotein” is a metalloprotein containing a heme prosthetic group. Heme-containing proteins include, but are not limited to, hemoglobin, myoglobin, cytoglobin, neuroglobin and cytochromes.
  • heme includes heme A, heme B, heme C, heme D, heme O, heme I, heme m, or heme S (see, e.g., Lin, Biochim Biophys Acta 1854(8):844-859, 2015; Ajioka et al., Biochim Biophys Acta 1763(7):723-736, 2006; Caughey et al., J Biol Chem 250:7602-7622, 1975; Kleingardner and Bren, Acc Chem Res 48(7):1845-1852, 2015; Bali et al., Cell Mol Life Sci 71(15):2837-2863, 2014; Cheesman et al., J Am Chem Soc 126: 4157-4166, 2004).
  • a “cell-free heme” is not bound or associated with a cell.
  • Cell-free heme proteins can generate reactive oxygen species, release free heme/iron, bind to or scavenge nitric oxide, and trigger inflammation.
  • Cell free- heme is also inclusive of synthetic heme molecules and/or artificial porphyrin molecules.
  • cell free-heme includes HemoCD. See Tejero, PNAS (2023) 120:e2301732120; see also Mao et al., PNAS (2023) 120:e2209924120.
  • Heme-solubilizing agent A carrier agent which covalently bonds to, or otherwise associate with heme to increase its solubility in solution.
  • the heme-solubilizing agent solubilizes heme in a saline solution at physiological pH. In another example, the heme-solubilizing agent solubilizes heme in whole blood.
  • Exemplary heme-solubilizing agents include albumin, heme- binding proteins, micelles, cellular membranes, nanoparticles, microparticles, lipid nanoparticles, lipid microparticles.
  • Hemoglobin (Hb) The iron-containing oxygen-transport metalloprotein in the red blood cells of the blood in vertebrates and other animals. In humans, the hemoglobin molecule is an assembly of four globular protein subunits.
  • Each subunit is composed of a protein chain tightly associated with a non-protein heme group.
  • Each protein chain arranges into a set of alpha-helix structural segments connected together in a globin fold arrangement, so called because this arrangement is the same folding motif used in other heme/globin proteins.
  • This folding pattern of each subunit contains a pocket which strongly binds the heme group.
  • An exemplary amino acid sequence for human hemoglobin subunit alpha is set forth as SEQ ID NO: 3.
  • An exemplary amino acid sequence for human hemoglobin subunit beta is set forth as SEQ ID NO: 4.
  • HBOC Hemoglobin-based oxygen carrier
  • HBOCs include, but are not limited to, DCLHb (HEMASSIST TM ; Baxter), MP4 (HEMOSPAN TM ; Sangart), pyridoxylated Hb POE – conjugate (PHP) + catalase & SOD (Apex Biosciences), O-R- PolyHbA0 (HEMOLINK TM ; Hemosol), PolyBvHb (HEMOPURE TM ; Biopure), PolyHb (POLYHEME TM ; Northfield), rHb1.1 (OPTRO TM ; Somatogen), PEG-Hemoglobin (Enzon), OXYVITA TM and HBOC-201 (Greenburg and Kim, Crit Care 8(Suppl 2):S61-S64, 2004; te Lintel Hekkert et al., Am J Physiol Heart Circ Physiol 298:H1103-H1113, 2010; Eisenach, Anesthesiology 111:946-963, 2009
  • Hemolysis The breaking open of red blood cells and the release of hemoglobin into the surrounding fluid. 8123-109216-02 Hemolysis can be caused by an inherited hemolytic condition, such as sickle cell anemia, thalassemia, a red cell membrane disorder (such as hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikliocytosis, hereditary stomatocytosis, or hereditary exocytosis), pyruvate kinase deficiency, paroxysmal nocturnal hemoglobinuria, or glucose-6-phosphate dehydrogenase deficiency.
  • an inherited hemolytic condition such as sickle cell anemia, thalassemia, a red cell membrane disorder (such as hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikliocytosis, hereditary stomatocytosis, or
  • Hemolysis can also be acquired, such as by an infection (such as a viral infection, a fungal infection, a parasitic infection or a bacterial infection), a medication (such as penicillin, a sulfa drug, a chemotherapeutic agent, quinine, an antimalarial medicament, an immunosuppressive medicament, an anti-inflammatory medicament, levodopa, or acetaminophen), a blood cancer, an autoimmune disorder (such as lupus, rheumatoid arthritis, vasculitis, Crohn’s disease, or ulcerative colitis), a tumor, oncologic induced anemia, hypersplenism, hepatitis, a mechanical heart valve, heart-lung bypass machine, extracorporeal membrane oxygenation, hemodialysis, preclampsia, eclampsia, malignant hypertension, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, as a sequelae of strenuous
  • Heterologous A heterologous protein or polypeptide refers to a protein or polypeptide derived from a different source or species.
  • Hypertension Abnormally elevated blood pressure.
  • Ischemia A vascular phenomenon in which a decrease in the blood supply to a bodily organ, tissue, or part is caused, for instance, by constriction or obstruction of one or more blood vessels. Ischemia sometimes results from vasoconstriction or thrombosis or embolism. Ischemia can lead to direct ischemic injury, tissue damage due to cell death caused by reduced oxygen supply.
  • Ischemia/reperfusion injury In addition to the immediate injury that occurs during deprivation of blood flow, ischemic/reperfusion injury involves tissue injury that occurs after blood flow is restored.
  • MAP Mean arterial pressure
  • SP systolic blood pressure
  • a treating clinician can measure MAP using, among other methods, a sphygmomanometer, oscillometric blood pressure device, arterial catheters, or central venous catheters. See DeMers D, Wachs D. Physiology, Mean Arterial Pressure. In: StatPearls. StatPearls Publishing 2023.
  • N-acetylcysteine The N-acetylated derivative of the natural amino acid L-cysteine. Molecular Formula: C5H9NO3S. PubChem CID: 12035. IUPAC: (2R)-2-acetamido-3- sulfanylpropanoic acid.
  • NO-heme complex Niric oxide (NO) covalently bound to the iron complexed by a heme group. Examples include NO-ferroheme (which can be labile in some examples). Further examples include NO-ferroheme (which can be complexed to albumin in some examples), NO-ferriheme, 8123-109216-02 nitrosyl heme, and NO-ferric heme.
  • Nitrite The inorganic anion -NO2- or a salt of nitrous acid (NO2-). Nitrites are often highly soluble, and can be oxidized to form nitrates or reduced to form nitric oxide or ammonia. Nitrite may form salts with alkali metals, such as sodium (NaNO 2 , also known as nitrous acid sodium salt), potassium and lithium, with alkali earth metals, such as calcium, magnesium, and barium, with organic bases, such as amine bases, for example, dicyclohexylamine, pyridine, arginine, lysine and the like.
  • alkali metals such as sodium (NaNO 2 , also known as nitrous acid sodium salt)
  • alkali earth metals such as calcium, magnesium, and barium
  • organic bases such as amine bases, for example, dicyclohexylamine, pyridine, arginine, lysine and the like.
  • Oxygen carrier Molecules or compounds that are capable of binding, transporting and releasing oxygen in the body. Oxygen carriers include natural proteins, such as hemoglobin, myoglobin and hemocyanin, as well as artificial oxygen carriers, including hemoglobin-based oxygen carriers (HBOCs), perfluorocarbons (PFCs), liposome-encapsulated hemoglobin, regulator of CO metabolism (RcoM), and porphyrin metal complexes. See U.S. App. No.16/302,324, incorporated by reference herein in its entirety.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • ProliNONOate (or PROLI NONOate): 1-(hydroxy-NNO-azoxy)-L-proline, A NO-donor and NO-generating agent with the molecular formula (C5H7N3O4) 2- .
  • ProliNONOate includes a disodium salt: Na2(C5H7N3O4) or C5H7N3O4 , 2Na; 1-(hydroxy-NNO- azoxy)-L-proline, disodium salt.
  • PubChem CID 135438445.
  • Pulmonary hypertension Abnormally elevated blood pressure in the pulmonary circulation. Pulmonary hypertension affects the arteries in the lungs and right side of the heart.
  • a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell.
  • a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.
  • Substantial purification denotes purification from other proteins or cellular components.
  • a substantially purified protein is at least 60%, 70%, 80%, 90%, 8123-109216-02 95% or 98% pure.
  • a substantially purified protein is 90% free of other proteins or cellular components.
  • Reducing agent An element or compound that loses (or "donates") an electron to another chemical species in a redox chemical reaction.
  • a reducing agent is typically in one of its lower possible oxidation states, and is known as the electron donor.
  • a reducing agent is oxidized, because it loses electrons in the redox reaction.
  • Exemplary reducing agents include, but are not limited to, sodium dithionite, ascorbic acid, N-acetylcysteine, methylene blue, glutathione, cytochrome b5/b5- reductase, hydralazine, earth metals, formic acid, and sulfite compounds.
  • Respiratory failure A condition that occurs when not enough oxygen passes from the lungs into the blood. Respiratory failure can be either acute or chronic. Diseases and disorders that impair breathing can lead to respiratory failure. Examples include chronic obstructive lung disease (COPD), pneumonia, acute respiratory distress syndrome, interstitial lung disease, pulmonary embolism, cystic fibrosis, asthma, acute lung injury, inhalational lung injury.
  • COPD chronic obstructive lung disease
  • Sequence identity/similarity The identity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods.
  • Biol.215:403- 10, 1990) is available from several sources, including the National Center for Biological Information (NCBI) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
  • NCBI National Center for Biological Information
  • Sickle cell anemia A group of genetic disorders caused by sickle hemoglobin. In many forms of the disease, the red blood cells change shape upon deoxygenation because of polymerization 8123-109216-02 of the abnormal sickle hemoglobin. This process damages the red blood cell membrane, and can cause the cells to become stuck in blood vessels. This deprives the downstream tissues of oxygen and causes ischemia and infarction, which may cause organ damage, such as stroke.
  • Sulfide Inclusive of inorganic sulfides and organic sulfides.
  • An “inorganic sulfide” is an inorganic anion of sulfur with the chemical formula S 2 ⁇ or a compound containing one or more S 2 ⁇ ions.
  • Exemplary inorganic sulfides include lead(II) sulfide, hydrogen sulfide (H2S), and bisulfide (SH ⁇ ).
  • An “organic sulfide” is an organosulfur functional group with the connectivity R ⁇ S ⁇ R'.
  • Exemplary organic sulfides include diethyl sulfide, ethyl phenyl sulfide, and 3- (methylthio)cyclopentene.
  • a sulfide-containing compound, inorganic sulfide-containing compound, or an organic sulfide-containing compound are inclusive of single compound species or polymeric compound species.
  • the sulfide includes a sulfur covalently bound to a hydrogen.
  • Synthetic Produced by artificial means in a laboratory, for example a synthetic polypeptide can be chemically synthesized in a laboratory.
  • Thalassemia An inherited autosomal recessive blood disease.
  • Therapeutically effective amount A quantity of compound or composition sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount of a composition necessary to increase vasodilation in a subject, or to treat a hemolytic condition in a subject. In some aspects herein, a therapeutically effective amount of a disclosed composition increases vasodilation, such as measured by blood vessel size.
  • Thiol An organosulfur compound of the form R ⁇ SH, where R represents an alkyl or other organic substituent.
  • exemplary thiols include glutathione, cysteine, N-acetylcysteine, cysteinyl glycine, propanethiol, cyclopentanethiol, and o-miercaptobenzoic acid.
  • a thiol-containing compound is inclusive of single compound species or polymeric compound species.
  • Vasospasm One cause of stroke; occurs secondary to spasm of blood vessels supplying the brain. This type of stroke typically follows a subarachnoid aneurismal hemorrhage with a delayed development of vasospasm within 2-3 weeks of the bleeding event.
  • Vasodilation A state of increased caliber of the blood vessels, or the act of dilation of a blood vessel, for instance dilation of arterioles leading to increased blood flow to a body part. In some examples, vasodilation is measured by the diameter of the blood vessel. 8123-109216-02 IV. Overview A fast chemical route to a NO signaling molecule that is stable in the presence of oxyhemoglobin is identified herein. Under standard reductive nitrosylation conditions, labile ferric heme consumes two equivalents of NO to slowly form nitrosyl ferrous heme.
  • addition of a reduced thiol greatly accelerates the formation of NO-ferroheme with a more than 500-fold increase in kobs even with tenfold less NO under otherwise analogous conditions.
  • NO-ferroheme is stabilized when solubilized by cell membranes or serum albumin, has a long half-life in the presence of NO-scavenging oxyhemoglobin, and exhibits potent NO signaling effects of platelet inhibition and vasodilation.
  • glutathione serves as a reducing partner for ferric heme and accelerates the formation of ferrous nitrosyl.
  • Membrane-solubilized NO-ferroheme may be directly transferred to extracellular serum albumin and may thus represent a feasible export route from circulating RBCs.
  • loss of NO-ferroheme directly from NO-hemoglobin may explain previously unexplained artery-to-vein gradients in NO-hemoglobin during the inhalation of NO gas in humans, an observation that was surprising given the slow off-rate of NO from ferrous hemoglobin.
  • 33 At any given time there is 1.5 – 50 ⁇ M heme-albumin found in human circulation. 49 NO- ferroheme can form from ferric heme albumin in the presence of glutathione and NO.
  • NO-ferroheme can be shuttled, both from membranes to albumin and from albumin to apo-hemoproteins.
  • the transfer is unidirectional to a stable heme binding partner, and heme import into or export out of cells may be facilitated by other heme binding partners with low-to-moderate affinity such as Hsp90 or GAPDH, both of which shuttle heme. 4 NO-ferroheme may be more readily taken up by cells compared to heme alone and facilitates heme mobilization.
  • vasodilation and platelet inhibition indicate NO derived from NO-ferroheme, or NO-ferroheme itself, directly binds and activates sGC in smooth muscle and platelets.
  • NO-ferroheme in albumin is protected against NO scavenging via the dioxygenation reaction with oxyhemoglobin, increasing the effective lifetime of NO in a physiological environment like blood by several orders of magnitude.
  • the NO koff from NO-ferroheme in the albumin binding pocket has been reported to be 1.4 x 10 -4 s -1 , 71,72 but surprisingly a rapid and robust vasodilatory response is observed, conceivably through direct NO-ferroheme shuttling.
  • NO release from NO-ferroheme albumin may be triggered upon cellular import; the rate of NO dissociation from heme-albumin is accelerated by allosteric binding of small molecules, including ibuprofen and warfarin, which triggers His146 to replace NO as an axial heme ligand. 49,71 NO-ferroheme itself may directly activate apo-sGC, 25,28 once imported into the endothelium and smooth muscle. It is demonstrated herein that glutathione facilitates the rapid reductive nitrosylation of ferric heme, resulting in the formation of NO-ferroheme and a thiyl radical.
  • compositions including a NO-heme complex and/or a NO source and a heme source, a thiol-containing compound and/or a sulfide-containing compound, and a heme- 8123-109216-02 solubilizing agent.
  • the composition further includes a pharmaceutically acceptable carrier.
  • the thiol-containing compound includes glutathione, cysteine, N- acetylcysteine, cysteinyl glycine, a reducing agent, an electron donor compound, a low molecular weight thiol, and/or a high molecular weight thiol.
  • the sulfide-containing compound includes hydrogen sulfide, an inorganic sulfide, a reducing agent, an electron donor compound, a lower molecular weight sulfide, and/or a high molecular weight sulfide.
  • the heme-solubilizing agent includes albumin, a lipid-nanoparticle, a lipid-microparticle, a lipid emulsion, a micelle, a ghost red blood cell, a cell membrane component, and/or a cell membrane lysate.
  • the NO source includes a NO-donor, a NO-generating agent, NO, NO gas, nitrite, nitrate, a NONOate, spermine NONOate, DETANONOate, DEA NONOate, and/or PROLI NONOate.
  • the heme source includes ferric heme, ferrous heme, Heme A, Heme B, Heme C, Heme O, Heme I, Heme m, Heme D, Heme S, and/or a synthetic porphyrin.
  • the synthetic porphyrin includes iron meso-tetraphenylporphine (FeTPP), iron 5,10,15,20- tetrakis(4-sulfonatophenyl)porphyrin (FeTPPS, F) salts, and/or HemoCD.
  • FeTPPS, F includes two methyl- ⁇ -cyclodextrin (CD) dimers linked by pyridine (Py3CD, P) and/or imidazole (Im3CD, I).
  • the composition includes a NO-heme complex, a glutathione, and albumin.
  • the composition includes the NO-heme complex and the NO source, and the molar ratio of NO-heme complex to the NO source is greater than 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 1000:1, or 10,000:1.
  • the composition includes the NO-heme complex and the NO source, and the molar ratio of NO-heme complex to the NO source is greater than 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 1000:1, or 10,000:1.
  • the composition includes the NO-heme complex and the heme source, and the molar ratio of NO-heme complex to the heme source is greater than 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 1000:1, or 10,000:1.
  • the molar ratio of the NO-heme complex to the thiol-containing compound is 10:1 to 1000:1 or about 10:1; 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 1000:1.
  • the molar ratio of the NO-heme complex to the sulfide-containing compound is 10:1 to 1000:1 or about 10:1; 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 1000:1.
  • the heme- solubilizing agent is albumin.
  • the molar ratio of the NO-heme complex to the heme-solubilizing agent is 1:5 to 5:1 or about 3:5000, 3:5, or 1:1.
  • the subject is suspected of being in need of vasodilation. In some examples, the subject has, or is suspected of having, a cardiovascular condition, respiratory failure, metabolic syndrome, diabetes, a lipid disorder, inflammation, aging, and/or an infectious disease.
  • the cardiovascular condition includes hypertension, hypertensive 8123-109216-02 emergency, hypertensive urgency, malignant hypertension, pulmonary hypertension, primary pulmonary hypertension, secondary pulmonary hypertension, group 1 pulmonary hypertension, group 2 pulmonary hypertension, group 3 pulmonary hypertension, group 4 pulmonary hypertension, group 5 pulmonary hypertension, myocardial infarction, cerebrovascular accident or stroke, ischemia- reperfusion injury, ischemia-reperfusion derangement from organ transplant, ischemia-reperfusion derangement from vascular bypass surgery, heart failure, cardiogenic shock, right ventricular failure, arterial vasospasm, venous vasospasm, subarachnoid hemorrhage, atrial fibrillation, deep vein thrombosis, pulmonary embolism, valvular heart disease, artificial heart valves, transient ischemic attack, venous thromboembolism, antiphospholipid syndrome, vasospasm, cerebral vasospasm, cardiac vaso
  • the respiratory failure results from inhalational lung injury, acute lung injury, acute respiratory distress syndrome, pneumonia, pulmonary infection, interstitial lung disease, granulomatous lung disease, environmental lung disease, occupational lung disease, chronic obstructive pulmonary disease, asthma, and/or cystic fibrosis.
  • infectious disease is a bacterial, viral, parasitic, and/or fungal disease.
  • the subject’s mean MAP decreases. In some examples the MAP decreases by at least 80, 70, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1 mmHg.
  • MAP decreases within 3, 2, or 1 hours, within 45, 30, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minutes, or within 50, 40, 30, 20, 10, or 5 seconds of the administering.
  • the MAP returns to at least 100%, 95%, 90%, 85%, 80%, 75%, or 70% of the preadministration MAP within 50, 45, 40, 35, 30, 25, 20, 15, or 10 minutes of the administering.
  • the composition is not purified, filtered, or refined. Also disclosed herein is a method of making the compositions disclosed herein. In some examples, this includes contacting the NO source, the heme, the thiol-containing compound or the sulfide-containing compound, and the heme-solubilizing agent.
  • this includes contacting the NO-heme complex, the thiol-containing compound or the sulfide-containing compound, and the heme-solubilizing agent.
  • the NO source includes PROLI NONOate
  • the heme includes ferric heme
  • the thiol-containing compound includes glutathione
  • the heme-solubilizing agent includes albumin.
  • a kit including a NO-heme complex, and a thiol-containing compound or a sulfide-containing compound. This kit may be used according to the methods disclosed above.
  • kits including a NO source, heme, and a thiol-containing compound or a sulfide-containing compound.
  • This kit may also be used according to the methods disclosed above.
  • the kits further include a heme-solubilizing agent and/or a pharmaceutically acceptable carrier.
  • a composition including a thiol-containing compound or a sulfide- containing compound, a NO source, and a heme-solubilizing agent.
  • the 8123-109216-02 composition further includes a pharmaceutically acceptable carrier.
  • the thiol- containing compound includes glutathione, cysteine, N-acetylcysteine, cysteinyl glycine, a reducing agent, an electron donor compound, a low molecular weight thiol, and/or a high molecular weight thiol.
  • the sulfide-containing compound includes hydrogen sulfide, an inorganic sulfide, a reducing agent, an electron donor compound, a lower molecular weight sulfide, and/or a high molecular weight sulfide.
  • the NO source includes a NO-donor, a NO-generating agent, NO, NO gas, nitrite, nitrate, a NONOate, spermine NONOate, DETANONOate, DEA NONOate, and/or PROLI NONOate.
  • the heme-solubilizing agent includes albumin, a lipid-nanoparticle, a lipid-microparticle, a lipid emulsion, a micelle, a ghost red blood cell, a cell membrane component, and/or a cell membrane lysate.
  • the composition includes glutathione, nitrite, and albumin.
  • the hemolytic condition includes an inherited hemolytic condition.
  • the inherited hemolytic condition includes sickle cell anemia, a thalassemia, a red cell membrane disorder, pyruvate kinase deficiency, paroxysmal nocturnal hemoglobinuria, and/or glucose-6-phosphate dehydrogenase deficiency.
  • the red cell membrane disorder includes hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikliocytosis, hereditary stomatocytosis, and/or hereditary exocytosis.
  • the hemolytic condition includes an acquired hemolytic condition.
  • the acquired hemolytic condition results from an infection, a medication, a blood cancer, an autoimmune disorder, a tumor, oncologic induced anemia, hypersplenism, hepatitis, a mechanical heart valve, heart-lung bypass machine, extracorporeal membrane oxygenation, hemodialysis, preclampsia, eclampsia, malignant hypertension, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, as a sequelae of strenuous activity, and/or a blood transfusion reaction.
  • the infection includes a viral infection, a fungal infection, a parasitic infection and/or a bacterial infection.
  • the medication includes penicillin, a sulfa drug, a chemotherapeutic agent, quinine, an antimalarial medicament, an immunosuppressive medicament, an anti-inflammatory medicament, levodopa, and/or acetaminophen.
  • the autoimmune disorder incldues lupus, rheumatoid arthritis, vasculitis, Crohn’s disease, and/or ulcerative colitis.
  • the subject’s free heme detoxifies by at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90%, or 95%, relative to a control.
  • the composition is not purified, filtered, or refined. Also disclosed herein is a method of making the previously disclosed compositions. In some examples, this includes contacting the thiol-containing compound or the sulfide-containing compound, the NO source, and the heme-solubilizing agent. Further disclosed is a kit, which can be used according to the methods disclosed above. In some examples, the kit includes a thiol-containing compound or a sulfide-containing compound and a 8123-109216-02 NO source. In some examples, the kit further includes a heme-solubilizing agent and/or a pharmaceutically acceptable carrier.
  • kits further includes a blood product, such as a blood product including red cells, platelets, plasma, antihemophilic factor, and/or granulocytes.
  • the composition is cell-free.
  • the composition further includes a pharmaceutically acceptable carrier.
  • the cell-free heme protein includes hemoglobin, an artificial oxygen carrier, a hemoglobin-based oxygen carriers, neuroglobin, cytoglobin, a microbially derived heme protein, stroma-free hemoglobin, 2,3 DPG-free hemoglobin, 2,3 DPG-reduced hemoglobin, synthetic porphyrin, hemoglobin glutamer-200, HBOC-201, polymerized hemoglobin, recombinant Hemoglobin 1.1 (rHb1.1), diasparin-cross linked hemoglobin (DCLHb), poly(ethylene) glycol conjugated hemoglobin, and/or an o-raffinose cross-linked haemoglobin-based oxygen carrier.
  • the synthetic porphyrin includes iron meso-tetraphenylporphine (FeTPP), iron 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (FeTPPS, F) salts, and/ HemoCD.
  • FeTPPS, F includes two methyl- ⁇ -cyclodextrin (CD) dimers linked by pyridine (Py3CD, P) and/or imidazole (Im3CD, I).
  • the thiol-containing compound includes glutathione, cysteine, N-acetylcysteine, cysteinyl glycine, a reducing agent, an electron donor compound, a low molecular weight thiol, and/or a high molecular weight thiol.
  • the sulfide-containing compound includes hydrogen sulfide, an inorganic sulfide, a reducing agent, an electron donor compound, a lower molecular weight sulfide, and/or a high molecular weight sulfide.
  • the NO source includes a NO-donor, a NO-generating agent, NO gas, nitrite, nitrate, a NONOate, spermine NONOate, DETANONOate, DEA NONOate, and/or proliNONOate.
  • the heme includes ferric heme and/or ferrous heme.
  • the composition includes the NO-heme complex, 2,3 DPG free hemoglobin, and glutathione. Also disclosed herein is a method of administering cell-free heme to a subject. In some examples, the method includes administering to the subject a therapeutically effective amount of the previously described compositions.
  • the composition ameliorates the vasoconstrictive effect of heme-protein interactions with NO.
  • the vasoconstrictive effect is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%, relative to a control.
  • the subject has a hemolytic condition, or the subject is in need of or is suspected of being in need of a blood transfusion.
  • the hemolytic condition includes an inherited hemolytic condition.
  • the inherited hemolytic condition includes sickle cell anemia, a thalassemia, a red cell membrane disorder, pyruvate kinase deficiency, paroxysmal nocturnal hemoglobinuria, and/or glucose-6-phosphate dehydrogenase deficiency.
  • the red cell membrane disorder includes hereditary 8123-109216-02 spherocytosis, hereditary elliptocytosis, hereditary pyropoikliocytosis, hereditary stomatocytosis, and/or hereditary exocytosis.
  • the hemolytic condition includes an acquired hemolytic condition.
  • the acquired hemolytic condition results from an infection, a medication, a blood cancer, an autoimmune disorder, a tumor, oncologic induced anemia, hypersplenism, hepatitis, a mechanical heart valve, a heart-lung bypass machine, extracorporeal membrane oxygenation, hemodialysis, preclampsia, eclampsia, malignant hypertension, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, a sequelae of strenuous activity, and/or a blood transfusion reaction.
  • the infection includes a viral infection, a fungal infection, a parasitic infection and/or a bacterial infection.
  • the medication includes penicillin, an antimalarial medicament, a sulfa drug, a chemotherapeutic agent, quinine, an immunosuppressive medicament, an anti-inflammatory medicament, levodopa, and/or acetaminophen.
  • the autoimmune disorder includes lupus, rheumatoid arthritis, vasculitis, Crohn’s disease, and/or ulcerative colitis.
  • the composition is not purified, filtered, or refined. Also disclosed herein is a method of making the previously disclosed compositions. In some examples, this includes contacting the cell-free heme protein, the NO-heme complex, and the thiol- containing compound or the sulfide-containing compound.
  • the method includes contacting the cell-free heme protein, a NO source, heme, and the thiol-containing compound or the sulfide-containing compound.
  • a kit including a cell-free heme protein, a NO-heme complex, and a thiol-containing compound or a sulfide-containing compound This kit can be used according to the previously disclosed methods.
  • a kit including a cell-free heme protein, a NO source, heme, and a thiol-containing compound or a sulfide-containing compound This kit can also be used according to the previously disclosed methods.
  • kits above further include a heme-solubilizing agent, a pharmaceutically acceptable carrier and/or a blood product (such as red cells, platelets, plasma, antihemophilic factor, and/or granulocytes).
  • a method of producing a stable NO-heme complex including contacting a NO source and heme in the presence of a thiol-containing compound or a sulfide- containing compound, thereby producing a stable NO-heme complex.
  • the NO source includes a NO-donor, a NO-generating agent, NO, NO gas, nitrite, nitrate, a NONOate, spermine NONOate, DETANONOate, DEA NONOate and/or PROLI NONOate.
  • the heme includes ferric heme, ferrous heme, Heme A, Heme B, Heme C, Heme O, Heme I, Heme m, Heme D, Heme S, and/or a synthetic porphyrin.
  • the synthetic porphyrin includes iron meso-tetraphenylporphine (FeTPP), iron 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (FeTPPS, F) salts, and/or HemoCD.
  • FeTPPS, F includes two methyl- ⁇ - cyclodextrin (CD) dimers linked by pyridine (Py3CD, P) and/or imidazole (Im3CD, I).
  • the thiol-containing compound includes glutathione, cysteine, N-acetylcysteine, cysteinyl 8123-109216-02 glycine, a reducing agent, an electron donor compound, a low molecular weight thiol, and/or a high molecular weight thiol.
  • the sulfide-containing compound includes hydrogen sulfide, an inorganic sulfide, a reducing agent, an electron donor compound, a lower molecular weight sulfide, and/or a high molecular weight sulfide.
  • the molar ratio of the NO source to the heme is 1000:1 to 1:1000, 1:1 to 1:10 or about 2:1.
  • the molar ratio of the NO source to the thiol-containing compound is 1:1 to 1:100,000 or about 1:5. In some examples, the molar ratio of the NO source to the sulfide-containing compound is 1:1 to 1:100,000 or about 1:5. In some examples, the molar ratio of the heme to the thiol-containing compound is 10:1 to 1:10,000, 1:5 to 1:1000, or about 1:10. In some examples, the molar ratio of the heme to the sulfide-containing compound is 10:1 to 1:10,000, 1:5 to 1:1000, or about 1:10. In some examples, the method further includes purifying the stable NO-heme complex.
  • the purifying the stable NO- heme complex includes centrifugation and/or filtration. In some examples, the purifying includes removing nitrite, S-nitrosoglutathione (GSNO), and/or dithionite.
  • the contacting step occurs in the presence of a heme-solubilizing agent, such as albumin, a lipid-nanoparticle, and/or a ghost red blood cell. In some examples, the contacting step occurs in the presence of a cell-free heme protein such as hemoglobin, an artificial oxygen carrier, a hemoglobin-based oxygen carriers, neuroglobin, cytoglobin, hemoglobin glutamer-200, or HBOC-201.
  • the contacting step occurs in the presence of a pharmaceutically acceptable carrier.
  • the method is an in vitro, in silico, ex vivo or in vivo method.
  • Pharmaceutical Compositions The compositions described herein can be administered as isolated compounds, or as part of a pharmaceutical composition. Accordingly, provided herein are pharmaceutical compositions that include a nitric oxide (NO)-heme complex, a NO source, a heme source, a thiol-containing compound, a sulfide-containing compound, a heme-solubilizing agent, and/or a cell-free heme protein as well as one or more pharmaceutically acceptable excipients, and optionally one or more other active (therapeutic) ingredients.
  • NO nitric oxide
  • excipient(s) are "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Proper formulation of the pharmaceutical composition is dependent upon several factors, such as the route of administration chosen.
  • the pharmaceutical compositions disclosed herein can be manufactured by means including but not limited to: conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.
  • the composition includes one or more of the following excipients: N- acetyl cysteine, sodium citrate, glycine, histidine, glutamic acid, sorbitol, maltose, mannitol, trehalose, lactose, glucose, raffinose, dextrose, dextran, ficoll, gelatin, hydroxyethyl starch, benzalkonium chloride, benzethonium chloride, benzyl alcohol, chlorobutanol, m-cresol, myristyl gamma- picolinium chloride, paraben methyl, paraben propyl, 2-penoxythanol, phenyl mercuric nitrate, 8123-109216-02 thimerosal, acetone sodium bisulfite, argon, ascorbyl palmitate, ascorbate (sodium/acid), bisulfite sodium, butylated hydroxy anisole (BHA)
  • compositions disclosed herein is pegylated, polymerized or cross-linked.
  • the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein can be pegylated, polymerized or cross-linked.
  • the pharmaceutical composition further includes a native or recombinant globin molecule, such as a native or recombinant hemoglobin or neuroglobin, or includes a hemoglobin-based oxygen carrier (HBOC).
  • the HBOC includes DCLHb (HEMASSIST TM ; Baxter), MP4 (HEMOSPAN TM ; Sangart), pyridoxylated Hb POE – conjugate (PHP) + catalase & SOD (Apex Biosciences), O-R-PolyHbA0 (HEMOLINK TM ; Hemosol), PolyBvHb (HEMOPURE TM ; Biopure), PolyHb (POLYHEME TM ; Northfield), rHb1.1 (OPTRO TM ; Somatogen), PEG-Hemoglobin (Enzon), OXYVITA TM or HBOC-201, or any combination thereof.
  • DCLHb HMASSIST TM ; Baxter
  • MP4 HEMOSPA
  • the composition further includes cytoglobin, a microbially derived heme protein, stroma- free hemoglobin, 2,3 DPG-free hemoglobin, 2,3 DPG-reduced hemoglobin, a synthetic porphyrin (such as iron meso-tetraphenylporphine (FeTPP), iron 5,10,15,20-tetrakis(4- sulfonatophenyl)porphyrin (FeTPPS, F) salts (such as two methyl- ⁇ -cyclodextrin (CD) dimers linked by pyridine (Py3CD, P) or imidazole (Im3CD, I)), or HemoCD), hemoglobin glutamer-200, polymerized hemoglobin, poly(ethylene) glycol conjugated hemoglobin, or an o-raffinose cross- linked haemoglobin-based oxygen carrier
  • the pharmaceutical compositions disclosed herein can be administered by a variety of routes, depending upon whether local
  • compositions include those suitable for parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), or intraperitoneal administration, although the most suitable route may depend upon for example the condition and disorder of the recipient.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular or injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump.
  • Pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • the compounds can be contained in such pharmaceutical compositions with pharmaceutically acceptable 8123-109216-02 diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers, preservatives and the like.
  • a pharmacologic reference can be used for guidance, for example Modern Pharmaceutics, 5th Edition, Banker & Rhodes, CRC Press (2009); and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, 13th Edition, McGraw Hill, New York (2018) can be consulted.
  • the compositions can conveniently be presented in unit dosage form.
  • these methods include the step of bringing into association the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol- containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell- free heme protein with the carrier which constitutes one or more accessory ingredients.
  • the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired composition.
  • compositions including the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein can be formulated for parenteral administration by injection or infusion.
  • Compositions for injection or infusion may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the pharmaceutical compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use.
  • sterile liquid carrier for example, saline or sterile pyrogen-free water
  • Extemporaneous injection or infusion solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.
  • compositions for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compounds which can contain antioxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • exemplary lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension can also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the pharmaceutical compositions described above can include other agents having regard to the type of pharmaceutical composition in question, for example those for oral administration can include flavoring agents. 8123-109216-02
  • Unit dosage pharmaceutical compositions are those containing an effective dose, as hereinbelow recited, or an appropriate fraction thereof, of an active ingredient.
  • the term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • compositions including the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein can be effective over a wide dosage range and can be generally administered in a therapeutically effective amount.
  • the amount of the compound administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual composition or formulation administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • the amount of active ingredient that is combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • the pharmaceutical compositions include one or more of the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, or the cell-free heme protein in combination with one or more pharmaceutically acceptable carriers (excipients).
  • the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein constitute about 0.01% to about 50% of the pharmaceutical composition.
  • the nitric oxide (NO)-heme complex constitute about 0.01% to about 50%, about 0.01% to about 45%, about 0.01% to about 40%, about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 10%, about 0.01% to about 5%, about 0.05% to about 50%, about 0.05% to about 45%, about 0.05% to about 40%, about 0.05% to about 30%, about 0.05% to about 20%, about 0.05% to about 10%, about 0.1% to about 50%, about 0.1% to about 45%, about 0.1% to about 40%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 10%, about 0.1% to about 50%, about 0.1% to about 45%, about 0.1% to about 40%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.5% to about 50%, about 0.5% to about 45%, about 0.5% to about 40%, about 0.5% to about 0.5% to about 40%, about 0.1% to about 30%
  • Specific non-limiting examples include about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, or a range between any two of these values.
  • compositions can be administered to a patient already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications.
  • the pharmaceutical compositions can be sterilized or may be sterile filtered.
  • Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • the pH of preparations incorporating the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein is about 3 to about 11, about 5 to about 9, about 5.5 to about 6.5, or about 5.5 to about 7.5.
  • the composition includes the (i) NO-heme complex and (ii) the NO source, the heme source, the thiol-containing compound, or the sulfide-containing compound, with a molar ratio of the NO-heme complex to at least one member of (ii) being about 1:10 to about 100,000:1.
  • This can be the molar ratio of the NO-heme complex to the NO source, the molar ratio of the NO- heme complex to the heme source, the molar ratio of the NO-heme complex to the thiol-containing compound, the molar ratio of the NO-heme complex to the sulfide-containing compound, or the molar ratio of the NO-heme complex to some combination of the preceding.
  • the molar ratio of the NO-heme complex to at least one member of (ii) is about 1:1 to about 2:1, about 1:1 to about 3:1, about 1:1 to about 4:1, about 1:1 to about 5:1, about 1:1 to about 6:1, about 1:1 to about 7:1, about 1:1 to about 8:1, about 1:1 to about 9:1, about 1:1 to about 10:1, about 1:1 to about 20:1, about 1:1 to about 30:1, about 1:1 to about 40:1, about 1:1 to about 50:1, about 1:1 to about 60:1, about 1:1 to about 70:1, about 1:1 to about 80:1, about 1:1 to about 90:1, about 1:1 to about 100:1, about 1:1 to about 1000:1, about 1:1 to about 10,000:1, about 2:1 to about 3:1, about 2:1 to about 4:1, about 2:1 to about 5:1, about 2:1 to about 6:1, about 2:1 to about 7:1, about 2:1 to about 8:1, about 2:1 to about 9:1, about 2:1 to about 10:1, about 2:1 to about 20:1, about 2:1 to about 30:1, about 2:1 to about 40:1, about 2:1 to about 50:1, about 2:1 to about 60:1, about 1:1 to about 9
  • compositions include about 1:10, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 1000:1, about 10,000:1, or about 100,000:1, or an amount greater than any of the forgoing examples.
  • the composition includes the NO-heme complex and the heme-solubilizing agent, such as albumin with the molar ratio of the NO-heme complex to heme-solubilizing agent being about 1:10 to about 10:1.
  • the molar ratio of the NO-heme complex to the heme-solubilizing agent is about 1:5 to about 5:1, about 1:2 to about 5:1, about 1:1 to about 5:1, about 2:1 to about 5:1,about 1:5 to about 2:1, about 1:2 to about 2:1, about 1:1 to about 2:1, about 1:5 to about 1:1, about 1:2 to about 1:1, about 1:5 to about 1:2, or a value within one of these ranges.
  • the molar ratio of the NO-heme complex to the heme-solubilizing agent being about 1:1, about 1:2, about 1:5, about 1:10, about 1:100, or 1:1000, about 1:10,000, about 3:5000, or about 3:5, an amount less than any of the forging examples, or a range between any two of these values.
  • the composition includes the NO source and the heme with the molar ratio of the NO source to the heme being about 1000:1 to about 1:1000.
  • the molar ratio of the NO source to the heme is about 1000:1 to about 1:1000, about 100:1 to about 1:1000, about 10:1 to about 1:1000, about 1:1 to about 1:1000, about 1:10 to about 1:1000, about 1:100 to about 1:1000, about 1000:1 to about 1:100, about 100:1 to about 1:100, about 10:1 to about 1:100, about 1:1 to about 1:100, about 1:10 to about 1:100, about 1000:1 to about 1:10, about 100:1 to about 1:10, about 10:1 to about 1:10, about 1:1 to about 1:10, about 1000:1 to about 1:1, about 100:1 to about 1:1, about 10:1 to about 1:1, about 1000:1 to about 10:1, about 100:1 to about 10:1, about 1000:1 to about 100:1, or a value within one of these ranges.
  • the composition includes the NO source and the thiol-containing compound with the molar ratio of the NO source to the thiol-containing compound being about 1:1 to about 1:100,000.
  • the molar ratio of the NO source to the thiol-containing compound is about 1:1 to about 1:5, about 1:1 to about 1:10, about 1:1 to about 1:100, about 1:1 to 8123-109216-02 about 1:1000, about 1:1 to about 1:10,000, about 1:1 to about 1:100,000, about 1:5 to about 1:10, about 1:5 to about 1:100, about 1:5 to about 1:1000, about 1:5 to about 1:10,000, about 1:5 to about 1:100,000, about 1:10 to about 1:100, about 1:10 to about 1:1000, about 1:10 to about 1:10,000, about 1:10 to about 1:100,000, about 1:100 to about 1:1000, about 1:100 to about 1:10,000, about 1:100 to about 1:100,000, about 1:1000 to about 1:10,000, about 1:100 to about 1:100,000, about 1:1000 to about 1:10,000, about 1:1000 to about 1:100,000, about 1:10,000 to about 1:100,000, or a value within one of these ranges.
  • the composition includes the NO source and the sulfide-containing compound with the molar ratio of the NO source to the sulfide-containing compound being about 1:1 to about 1:100,000.
  • the molar ratio of the NO source to the sulfide-containing compound is about 1:1 to about 1:5, about 1:1 to about 1:10, about 1:1 to about 1:100, about 1:1 to about 1:1000, about 1:1 to about 1:10,000, about 1:1 to about 1:100,000, about 1:5 to about 1:10, about 1:5 to about 1:100, about 1:5 to about 1:1000, about 1:5 to about 1:10,000, about 1:5 to about 1:100,000, about 1:10 to about 1:100, about 1:10 to about 1:1000, about 1:10 to about 1:10,000, about 1:10 to about 1:100,000, about 1:100 to about 1:1000, about 1:100 to about 1:10,000, about 1:100 to about 1:100,000, about 1:1000 to about 1:10,000, about 1:100 to about 1:100,000, about 1:1000 to about 1:10,000, about 1:1000 to about 1:100,000, about 1:10,000 to about 1:100,000, or a value within one of these ranges.
  • the composition includes the heme and the thiol or sulfide-containing compound, with the molar ratio of the heme to the thiol or sulfide-containing compound being about 10:1 to about 1:10,000.
  • the molar ratio of the heme to the thiol or sulfide- containing compound is about 10:1 to about 1:10,000, about 1:1 to about 1:10,000, about 1:5 to about 1:10,000, about 1:10 to about 1:10,000, about 1:100 to about 1:10,000, about 1:1000 to about 1:10,000, about 10:1 to about 1:1000, about 1:1 to about 1:1000, about 1:5 to about 1:1000, about 1:10 to about 1:1000, about 1:100 to about 1:1000, about 10:1 to about 1:100, about 1:1 to about 1:100, about 1:5 to about 1:100, about 1:10 to about 1:100, about 10:1 to about 1:10, about 1:1 to about 1:10, about 1:5 to about 1:10, about 10:1 to about 1:5, about 1:1 to about 1:5, about 10:1 to about 1:1, or a value within one of these ranges.
  • compositions disclosed herein can include a cell-free heme protein, such as hemoglobin and/or a heme-solubilizing agent, such as albumin.
  • a cell-free heme protein such as hemoglobin
  • a heme-solubilizing agent such as albumin.
  • An exemplary amino acid sequence for hemoglobin is given as SEQ ID NO: 1.
  • An exemplary amino acid sequence for albumin is given as SEQ ID NO: 2.
  • compositions disclosed herein can include proteins having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at 8123-109216-02 least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9% or 100% sequence identity with respect to SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the pharmaceutical composition can include a colloid plasma volume expander.
  • the pharmaceutical composition can include an oxygen carrier.
  • the pharmaceutical composition can include an artificial oxygen carrier. VI.
  • compositions include a nitric oxide (NO)-heme complex, a NO source, a heme source, a thiol-containing compound, a sulfide-containing compound, a heme-solubilizing agent, and/or a cell-free heme protein as well as one or more pharmaceutically acceptable excipients, and optionally one or more other active (therapeutic) ingredients.
  • NO nitric oxide
  • the method includes administering to the subject a therapeutically effective amount of a composition including the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein, or a pharmaceutical composition containing the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme- solubilizing agent, and/or the cell-free heme protein.
  • a composition including the nitric oxide (NO)-heme complex the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein
  • a pharmaceutical composition containing the nitric oxide (NO)-heme complex the NO source, the
  • the method includes administering a composition, including (i) a nitric oxide (NO)-heme complex or (ii) a NO source and a heme source; a thiol-containing compound or a sulfide-containing compound; and a heme- solubilizing agent, and optionally, a pharmaceutically acceptable carrier.
  • a composition including (i) a nitric oxide (NO)-heme complex or (ii) a NO source and a heme source; a thiol-containing compound or a sulfide-containing compound; and a heme- solubilizing agent, and optionally, a pharmaceutically acceptable carrier.
  • the method includes selecting a subject in need of vasodilation, or suspected of being in need of vasodilation prior to administration of the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein or pharmaceutical composition including the aforementioned.
  • NO nitric oxide
  • Subjects in need of vasodilation may include those with, or suspected of having a cardiovascular condition (such as hypertension, hypertensive emergency, hypertensive urgency, malignant hypertension, pulmonary hypertension, primary pulmonary hypertension, secondary pulmonary hypertension, group 1 pulmonary hypertension, group 2 pulmonary hypertension, group 3 pulmonary hypertension, group 4 pulmonary hypertension, group 5 pulmonary hypertension, myocardial infarction, cerebrovascular accident or stroke, ischemia-reperfusion injury, ischemia-reperfusion derangement from organ transplant, ischemia-reperfusion derangement from vascular bypass surgery, heart failure, cardiogenic shock, right ventricular failure, arterial vasospasm, venous vasospasm, subarachnoid hemorrhage, atrial fibrillation, deep vein thrombosis, pulmonary embolism, valvular 8123-109216-02 heart disease, artificial heart valves, transient ischemic attack, venous thromboembolism, antiphospholipid syndrome
  • the subject’s MAP decreases following the administering to the subject a therapeutically effective amount of a composition including the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein.
  • MAP decreases by about 80 to about 1 mmHG.
  • MAP decreases by about 80 to about 1, about 50 to about 1, about 30 to about 1, about 10 to about 1, about 80 to about 10, about 50 to about 10, about 30 to about 10, about 80 to about 30, about 50 to about 30, about 80 to about 50 mmHG, or a value within one of these ranges.
  • MAP decreases by at least about 80, about 70, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, about 5, or about 1 mmHG.
  • Specific non-limiting examples include a decrease of about 80, about 70, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 10, about 5, or about 1 mmHg, or a range between any two of these values. The foregoing all representing decreases in MAP measured in mmHG.
  • MAP decreases by a percentage relative to a control, such as a historical control, or such as preadministration MAP, by about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, an amount greater than any of the forgoing examples, or a range between any two of these values.
  • the subject’s MAP decreases after administering the composition including the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein.
  • NO nitric oxide
  • the subject’s MAP decreases within about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour, within about 45 minutes, about 30 minutes, about 15 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, or about 1 minute, or within about 50 seconds, about 40 seconds, about 30 seconds, about 20 seconds, about 10 seconds, or about 5 seconds, a range between any two of these values, or within an amount less than any of the forgoing examples.
  • the subject’s MAP returns to a percentage of the pre- administration MAP after decreasing, following the administering the composition including the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein.
  • the subject’s MAP returns to at least about 100%, about 99%, about 98%, about 97%, about 96%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, a range between any two of these values, or at a value that is at least any of the forgoing examples.
  • the subject’s MAP returns within about 50 minutes, about 45 minutes, about 40 minutes, about 35 minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 15 minutes, about 10 minutes, a range between any two of these values, or within an amount less than any of the forgoing examples following the administering.
  • the subject’s blood vessel increases in diameter relative to a control, such as a pre-administration control, or a historical control, following the administering to the subject a therapeutically effective amount of a composition including the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein.
  • a control such as a pre-administration control, or a historical control
  • the blood vessel increases by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, or at least 200%, or a range between any two of these values. Also provided herein are methods of creating a stable NO-heme complex in vivo or in vitro.
  • these methods can reduce or eliminate the deleterious effects of free heme release in hemolytic conditions.
  • the method includes administering to the subject a therapeutically effective amount of a composition including the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein, or a pharmaceutical composition containing the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein.
  • a composition including the nitric oxide (NO)-heme complex the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein.
  • the method includes administering a composition including a thiol- containing compound or a sulfide-containing compound, a NO source, and a heme-solubilizing agent.
  • the method includes selecting a subject with or suspected of having a hemolytic condition prior to administration of the composition including the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein.
  • NO nitric oxide
  • the hemolytic condition is an inherited hemolytic condition such as sickle cell anemia, a thalassemia, a red cell membrane disorder, pyruvate kinase deficiency, paroxysmal nocturnal hemoglobinuria, or glucose-6- phosphate dehydrogenase deficiency.
  • red cell membrane disorders include hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikliocytosis, hereditary stomatocytosis, or hereditary exocytosis.
  • the hemolytic condition is an acquired hemolytic condition such as one resulting from an infection, a medication, a blood cancer, an autoimmune disorder, a 8123-109216-02 tumor, oncologic induced anemia, hypersplenism, hepatitis, a mechanical heart valve, heart-lung bypass machine, extracorporeal membrane oxygenation, hemodialysis, preclampsia, eclampsia, malignant hypertension, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, as a sequelae of strenuous activity, or a blood transfusion reaction.
  • the infection includes a viral infection, a fungal infection, a parasitic infection, or a bacterial infection.
  • the medication includes penicillin, an antimalarial medication, a sulfa drug, a chemotherapeutic agent, quinine, an immunosuppressive medicament, an anti-inflammatory medicament, levodopa, or acetaminophen.
  • the autoimmune disorder includes lupus, rheumatoid arthritis, vasculitis, Crohn’s disease, or ulcerative colitis.
  • administering the therapeutically effective amount of a composition including the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein reduces or eliminates the deleterious effects of free heme release in hemolytic conditions.
  • NO nitric oxide
  • the administering detoxifies the subject’s free heme by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, a range between any two of these values, or by at least as much as any of the forgoing examples, relative to a control, such as a healthy control. Further provided herein are methods of administering cell free heme to a subject.
  • the method includes administering to the subject a therapeutically effective amount of a composition including the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein, or a pharmaceutical composition containing the NO-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme- solubilizing agent, and/or the cell-free heme protein.
  • a composition including the nitric oxide (NO)-heme complex the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein
  • a pharmaceutical composition containing the NO-heme complex the NO source, the heme source, the thiol-containing compound,
  • the method includes administering a composition including a cell free heme protein, a NO-heme complex or a NO source and a heme source, and a thiol-containing compound or a sulfide-containing compound.
  • the method further includes administering a blood product, such as red cells, platelets, plasma, antihemophilic factor, and/or granulocytes.
  • the blood product is intermixed with the composition including the NO-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein.
  • the composition including the NO-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein is administered separately from the blood product.
  • whole blood is administered to the subject.
  • the method includes selecting a subject with a hemolytic condition, or suspected of having a hemolytic condition.
  • the hemolytic condition is, or is suspected of being an inherited 8123-109216-02 hemolytic condition (such as sickle cell anemia, a thalassemia, a red cell membrane disorder, pyruvate kinase deficiency, paroxysmal nocturnal hemoglobinuria, or glucose-6-phosphate dehydrogenase deficiency).
  • the red cell membrane disorder is or is suspected of being hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikliocytosis, hereditary stomatocytosis, or hereditary exocytosis.
  • the hemolytic condition is or is suspected of being an acquired hemolytic condition, such as resulting from an infection (such as a viral infection, a fungal infection, a parasitic infection or a bacterial infection), a medication (such as penicillin, an antimalarial medicament, a sulfa drug, a chemotherapeutic agent, quinine, an immunosuppressive medicament, an anti-inflammatory medicament, levodopa, or acetaminophen), a blood cancer, an autoimmune disorder (such as lupus, rheumatoid arthritis, vasculitis, Crohn’s disease, or ulcerative colitis), a tumor, oncologic induced anemia, hypersplenism, hepatitis, a mechanical heart valve, a heart-lung bypass machine, extracorporeal membrane oxygenation, hemodialysis, preclampsia, eclampsia, malignant hypertension, disseminated intravascular coagulation, thrombotic thrombocytopenic
  • the method includes selecting a subject in need of, or suspected of being in need of a blood transfusion.
  • administering the therapeutically effective amount of a composition including the nitric oxide (NO)-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein ameliorates the vasoconstrictive effect of heme-protein interactions with NO.
  • Specific non- limiting examples include administering the composition in an amount that ameliorates the vasoconstrictive effect by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%, or an amount greater than any of the forgoing examples, or a range between any two of these values, relative to a control, such as a healthy control.
  • the NO-heme complex is a stable NO-heme complex.
  • the method involves contacting at least two, at least three, at least four, at least five, at least six, or all of: a NO-heme complex, a NO source, a heme source, a thiol-containing compound, a sulfide-containing compound, a heme-solubilizing agent, or a cell-free heme protein.
  • the method includes contacting a NO source, heme, and a thiol-containing compound or a sulfide-containing compound.
  • the method includes contacting a NO source and heme in the presence of a thiol-containing compound or a sulfide-containing compound.
  • the NO-heme complex, the NO source, the heme source, the thiol- 8123-109216-02 containing compound, the sulfide-containing compound, the heme-solubilizing agent, and the cell-free heme protein can be provided at, or result in, any suitable molar ratio, such as the molar ratios described under section V above.
  • At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.99% of the NO-heme complex product forms within about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 1 minute, 2 minutes, 3 minutes, 4 minutes 5 minutes, 10 minutes, 15 minutes 20 minutes, or 30 minutes.
  • the heme is one or more of ferric heme, ferrous heme, Heme A, Heme B, Heme C, Heme O, Heme I, Heme m, Heme D, Heme S, or a synthetic porphyrin (such as iron meso- tetraphenylporphine (FeTPP), iron 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (FeTPPS, F) salts (such as two methyl- ⁇ -cyclodextrin (CD) dimers linked by pyridine (Py3CD, P) or imidazole (Im3CD, I)), or HemoCD).
  • a synthetic porphyrin such as iron meso- tetraphenylporphine (FeTPP), iron 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (FeTPPS, F) salt
  • the thiol-containing compound is one or more of glutathione, cysteine, N-acetylcysteine, cysteinyl glycine, a reducing agent, an electron donor compound, a low molecular weight thiol, and/or a high molecular weight thiol.
  • the sulfide-containing compound is one or more of hydrogen sulfide, a reducing agent, an electron donor compound, a lower molecular weight sulfide, and/or a high molecular weight sulfide.
  • the method includes purifying the NO-heme complex, or purifying a product of the reaction, or a remaining reactant.
  • the method includes purifying the NO-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide- containing compound, the heme-solubilizing agent, and/or the cell-free heme protein.
  • the method includes purifying a product of the reaction, such as the NO-heme complex, by removing one or more of the NO-heme complex, the NO source, the heme source, the thiol-containing compound, the sulfide-containing compound, the heme-solubilizing agent, and/or the cell-free heme protein from a mixture including the products of the reaction.
  • the method includes removing nitrite, S-nitrosoglutathione (GSNO), or dithionite.
  • the method includes incorporating a buffer, an acid, or a base into the reaction, to adjust the pH to about 3 to about 11, about 5 to about 9, about 5.5 to about 6.5, or about 5.5 to about 7.5.
  • the purification includes centrifugation and/or filtration.
  • the method can be carried out in vitro (such as in a lab) in silico (such as automated) ex vivo, or in vivo (such as by administering two reagents which form the NO-heme complex in the subject). VII. Kits Also provided are compositions and kits that can be used with the disclosed methods.
  • the kit includes one or more of, such as at least one, at least two, at least three, at least four, at least five, at least six, or all of: a NO-heme complex, a NO source, a heme source, a thiol- containing compound, a sulfide-containing compound, a heme-solubilizing agent, and/or a cell-free 8123-109216-02 heme protein.
  • the kit includes a NO-heme complex, and a thiol-containing compound or a sulfide-containing compound.
  • the kit includes a NO source, heme, and a thiol-containing compound or a sulfide-containing compound.
  • the kit includes a thiol-containing compound or a sulfide-containing compound and a NO source.
  • the kit further includes a pharmaceutically acceptable carrier, a heme-solubilizing agent, a blood product, such as red blood cells in an IV bag.
  • the aspects of the kit can be provided in any suitable ratio or result in any suitable ratio when combined, such as the molar ratios described under section V above. In some examples, the aspects of the kit are provided in separate containers.
  • kits are provided premixed in a single container, or are provided in a container with a breakable divider.
  • instructions for mixing or administering the aspects of the kit are provided in the kit.
  • the kit includes an aspect for purifying a product obtained by reacting the elements of the kit, such as a microcentrifuge, or a filtration device, such as filter paper, a vacuum filtration apparatus, or a column.
  • the kit includes a buffer, an acid, or a base.
  • the kit includes a blood pressure monitoring device, or an aspect of such a device, such as a blood pressure cuff.
  • the kit includes a further active (therapeutic) ingredient, such as a medicament for high or low blood pressure.
  • a further active (therapeutic) ingredient such as a medicament for high or low blood pressure.
  • the aspects of the kit can be mixed and immediately administered to a subject without purifying the products of a reaction.
  • the aspects of the kit are mixed in the process of being administered to a subject, such as two IV bags leading into a single IV line.
  • the aspects of the kits are administered separately, and mix in the subject, such as by providing an infusion in each arm, or such as by providing two subsequent infusions. VIII. Additional Aspects Clause 1. A composition comprising a nitric oxide (NO)-heme complex, albumin and a thiol in a pharmaceutically acceptable carrier. Clause 2.
  • NO nitric oxide
  • composition of clause 1, wherein the thiol comprises glutathione, cysteine, N-acetylcysteine, a reducing agent, or an electron donor compound.
  • Clause 3. A method of inducing vasodilation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of clause 1 or clause 2.
  • Clause 4. The method of clause 3, wherein the subject has a cardiovascular condition, respiratory failure, metabolic syndrome, diabetes, a lipid disorder, inflammation, aging, or an infectious disease.
  • the cardiovascular condition comprises hypertension, hypertensive emergency, hypertensive urgency, pulmonary hypertension, myocardial 8123-109216-02 infarction, cerebrovascular accident or stroke, ischemia-reperfusion injury, heart failure, or cardiogenic shock.
  • the respiratory failure results from inhalational lung injury, acute lung injury, acute respiratory distress syndrome, pneumonia, pulmonary infection, interstitial lung disease, chronic obstructive pulmonary disease, asthma or cystic fibrosis.
  • the infectious disease is a bacterial, viral or fungal disease.
  • a composition comprising a thiol agent; a NO-donor or NO-generating agent; albumin; and a pharmaceutically acceptable carrier.
  • the thiol agent comprises glutathione, cysteine, N-acetylcysteine, a reducing agent or an electron donor compound.
  • the NO-generating agent is nitrite.
  • the inherited hemolytic condition comprises sickle cell anemia or a thalassemia.
  • Clause 14 The method of clause 11, wherein the subject suffers from an acquired hemolytic condition.
  • Clause 15. The method of clause 14, wherein the acquired hemolytic condition results from an infection (such as a viral or bacterial infection), a medication (such as penicillin, antimalarial medication, sulfa or acetaminophen), a blood cancer, an autoimmune disorder (such as lupus, rheumatoid arthritis or ulcerative colitis), a tumor, hypersplenism, a mechanical heart valve or a blood transfusion reaction.
  • an infection such as a viral or bacterial infection
  • a medication such as penicillin, antimalarial medication, sulfa or acetaminophen
  • a blood cancer such as an autoimmune disorder (such as lupus, rheumatoid arthritis or ulcerative colitis)
  • a tumor such as a mechanical heart valve or a blood transfusion
  • a composition comprising a cell-free heme protein, a NO-heme complex and a thiol in a pharmaceutically acceptable carrier.
  • Clause 17. A method of ameliorating the effects of heme-protein interactions with nitric oxide in a subject, comprising administering to the subject a therapeutically effective amount of the composition of clause 16.
  • Clause 18. The method of clause 17, wherein the vasoconstrictive effect of heme-protein interactions with nitric oxide is ameliorated. 8123-109216-02
  • a method of producing a stable nitric oxide (NO)-heme complex comprising contacting NO, a NO donor, nitrite or nitrate with a ferric or ferrous heme in the presence of a thiol agent.
  • Clause 20 The method of clause 19, wherein the thiol agent comprises glutathione, cysteine, N-acetylcysteine, a reducing agent or an electron donor compound.
  • Clause 21 The method of clause 19 or clause 20, wherein the contacting step occurs in the presence of a carrier protein or a lipid-nanoparticle carrier.
  • Clause 22 The method of any one of clauses 19-21, which is an in vitro, in silico, ex vivo or in vivo method.
  • nitric oxide donor ProliNONOate was purchased from Cayman Chemical (Ann Arbor, MI), and all stock solutions were prepared anaerobically in NaOH.
  • Nitric oxide gas was obtained from Matheson Tri-Gas, Inc. (Irving, TX) and was bubbled through 1 M NaOH solution before use.
  • Methanol solvent HPLC grade
  • L-glutathione was obtained from Alexis Biochemicals (San Diego, CA). S-nitrosoglutathione was both purchased and made as previously described.
  • 77 Buffers were prepared with 0.1 to 0.5 mM EDTA where appropriate.
  • UV-Visible Spectroscopy and Kinetics All individual spectra and pseudo-first-order kinetics were performed using a thermostatted (23/37°C ⁇ 0.2) Cary 50, Cary 100, or HP8453 UV-Visible spectrophotometer (Agilent Technologies) running either Cary WinUV Scan (v 5.23.1042, Agilent Technologies), Cary WinUV Scanning 8123-109216-02 Kinetics (v3.00(183), Agilent Technologies), or UV-Visible ChemStation (vB.04.01[61], Agilent Technologies) for reactions ferric heme reactions with NO and without glutathione in PBS and MeOH/PBS buffer (1 part PBS in 5 parts methanol) unless otherwise noted.
  • Cary WinUV Scan v 5.23.1042, Agilent Technologies
  • Cary WinUV Scanning 8123-109216-02 Kinetics v3.00(183), Agilent Technologies
  • UV-Visible ChemStation vB.04.
  • the concentration of hemin was precisely determined by absorption spectroscopy using the extinction coefficient at 385 nm of 58.4 mM -1 cm -1 .
  • Reduced glutathione stocks (25 - 100 mM) were made regularly in deaerated PBS and sealed in septum capped vials. Methanol was deaerated separated before mixing in a 5:1 ratio with deaerated PBS to make MeOH:PBS buffer for spectroscopy experiments.
  • the precise concentration of NO from bubbling or from ProliNONOate was determined either by titrating with excess oxyhemoglobin and calculating the amount of methemoglobin produced by fitting absorption spectra to normalized basis spectra or using an ozone- based chemiluminescent analyzer. Stock concentrations of ProliNONOate were also verified through absorption spectroscopy, using the extinction coefficient at 248 nm of 8.4 mM -1 cm -1 .
  • a sample stopped-flow experiment was performed as follows: 25 ⁇ M hemin and 250 ⁇ M hemin were loaded into one syringe, and a range of NO concentrations between 125 ⁇ M and 2.5 mM were loaded in the other syringe and the syringe contents were mixed 1:1 in the stoppled flow.
  • the above reaction conditions were replicated plus the addition of 50 mM DMPO in deaerated MeOH:PBS solution.
  • Chemiluminescent assays of NO-ferroheme and S-nitrosothiols A Zysense Nitric Oxide Analyzer 280i or an ECO PHYSICS nCLD 88 liquid NO system was used to detect NO congeners, with data from the former recorded using SIEVERS Windows 95/98/NT (v3.21 PNN) and the latter using eDAQ Chart (v5.5.27). Measurements of nitrosothiols was made using Cu/Cys assay (2C assay) or tri-iodide assay. 40 NO-ferroheme was measured by injecting an amount each sample into the NOA purge vessel containing 50 mM potassium ferricyanide in PBS at 25 or 37°C.
  • the direct method involved adding ferric heme solution and glutathione directly to albumin in an argon- or nitrogen-filled, septum-sealed vial, followed by NO addition. Solutions were used after five minutes.
  • the indirect method involved NO addition to heme and GSH in PBS before then adding albumin after five minutes. The NO-ferroheme was then incubated with albumin for thirty minutes.
  • Preparation of red blood cell membrane ghosts Red cells were separated from the whole blood by sedimentation via centrifugation at 1000 g for 10 minutes. These packed red cells were then hemolyzed using 5 mM phosphate buffer, pH 8 (4.674 mM Na 2 HPO 4 + 0.326 mM NaH 2 PO 4 ) in the volume ratio of 1:40 for an hour.
  • EPR electron paramagnetic resonance
  • the sample was incubated for another 30 minutes at 37°C after adding 75 ⁇ M albumin. After incubation, the sample was scanned for absorption in the Cary 50 UV-Vis spectrometer. The sample was subsequently spun at 30,000 g for 8123-109216-02 two hours in a Sorvall R RC-5B centrifuge and the absorption spectrum of the supernatant was measured. NO-ferroheme transfer to apo-myoglobin NO-ferroheme in albumin was made using 25 ⁇ M heme and 50 ⁇ M NO mixed for five minutes under anaerobic conditions, in pH 7.4 PBS buffer, in the presence of GSH (50, 125, 250 ⁇ M) before adding albumin.
  • GSH 50, 125, 250 ⁇ M
  • PRP was diluted 1:7 using the mixture of NO-ferroheme at concentrations described above, 1 ⁇ M ODQ (Cayman Chemical), 10 nM PDE inhibitor (Tadalafil, Cayman Chemical), or 2 ⁇ M NO. The final 500 ⁇ L volumes were adjusted with argon-degassed buffer. Samples with ODQ were incubated with platelets for 10 minutes at room temperature prior to any further additions. All samples were incubated in a 37°C water bath for 15 minutes and then fixed using a 1:4 dilution of ice-cold 99.5% ethanol. The supernatants were collected after centrifuging at 1,500 g for 10 minutes followed by hours of incubation for the evaporation of ethanol.
  • ELISA buffer 500 ⁇ L was then added to the ethanol-free samples and used in the ELISA 96-well plate for triplicate readings. The non-acetylated approach was performed following the manufacturer’s instructions. The plate was analyzed at 412 nm wavelength at room temperature by a Molecular Devices SpectraMax 340 PC microplate reader. SoftMax Pro (v4.8) was used to collect and analyze ELISA data. Platelet activation Samples were prepared using heme (25 ⁇ M), NO (20 ⁇ M), albumin (75 ⁇ M) with and without GSH (250 ⁇ M) under normoxic or anoxic conditions.
  • PRP platelet rich plasma
  • PAC-1 FITC BDTM FITC Mouse Anti-Human PAC-1, 25 ⁇ g/mL (1 mL); catalog no.340507; clone PAC-1 RUO (GMP)) – which labels activated platelets – and CD61 (BDTM PerCP Mouse Anti-Human CD61, 3 ⁇ g/mL (1 mL); catalog no.340506; clone 8123-109216-02 RUU-PL7F12 RUO (GMP)) – which labels all platelets – and incubated at room temperature in the dark for 20 minutes and then fixed in 1% buffered formaldehyde.
  • PAC-1 FITC BDTM FITC Mouse Anti-Human PAC-1, 25 ⁇ g/mL (1 mL); catalog no.340507; clone PAC-1 RUO (GMP)
  • CD61 BDTM PerCP Mouse Anti-Human CD61, 3 ⁇ g/mL (1 mL); catalog no.34050
  • Platelets were sorted using a BD FACSCalibur Analyzer. Data were collected and analyzed using BD CellQuest Pro (v6.0). Gating strategy was based on side and forward scattering compared to red blood cells and then also for activated platelets. Platelets were labelled along the x-axis and activation on the y-axis. Activation was taken as the upper right quadrant divided by the sum of the two right quadrants (FIG.14). All antibodies were verified with positive a negative controls, 82 and a platelet sample was not used where these positive or negative controls did not work.
  • a tracheal tube was introduced and connected to a rodent ventilator (Model 849, MidiVent). Upon completion, in some animals, the fraction of inspiratory O2 was decreased from 21% to 10% to induce hypoxia.75 ⁇ L of L-NAME was administered intravenously in a bolus at a dose of 10 mg/kg bodyweight. Ten minutes after L-NAME injection, animals were injected with 50 ⁇ L of NO-ferroheme, normal saline (NS), NO control, or freshly prepared GSNO 77 control for 4 times at 10-minute intervals.
  • NS normal saline
  • GSNO 77 control freshly prepared GSNO 77 control for 4 times at 10-minute intervals.
  • An NO-ferroheme in albumin stock was prepared via a concentrated version of the direct method as described above: 300 ⁇ M ferric heme was mixed anaerobically on a Schlenk line with 3 mM GSH and 600 ⁇ M NO in 500 ⁇ M albumin using air-tight Hamilton syringes. NO controls were generated similarly and comprised 300 ⁇ M dissolved NO with 3 mM GSH in anaerobic albumin solution. Likewise, GSNO stocks comprised 300 ⁇ M GSNO with 3 mM GSH in anaerobic albumin solution.
  • MAP Mean arterial pressure
  • Spectra showed isosbestic points at 503 and 594 nm (e.g., FIG.1B, top), and traces fit well to single exponential kinetic analyses (e.g., FIG.1B, bottom), together indicating a two-species rate-limiting reaction without significant formation of intermediates to generate NO-ferroheme.
  • the identity and stoichiometry of the reaction products support a mechanism involving thiol- catalyzed reductive nitrosylation of ferric heme to form NO-ferroheme and a thiyl radical, which subsequently reacts with excess NO to form secondary S-nitrosoglutathione (GSNO).
  • GSNO secondary S-nitrosoglutathione
  • NO-ferroheme and GSNO were identified and quantified by reductive chemiluminescence with NO detection in the chemiluminescent nitric oxide analyzer (FIG.1G, Untreated bars). Detection of NO specifically derived from NO-ferroheme was achieved through chemical oxidation with potassium ferricyanide, which oxidizes ferrous nitrosyl species and liberates NO without degrading GSNO (FIG. 8123-109216-02 8A).
  • the thiol may initially bind free ferriheme or pre- formed NO-ferriheme, though this order of addition cannot readily be determined from the reaction kinetics or stoichiometry.
  • the traditional reductive nitrosylation mechanism does not yield a radical intermediate and, as it is base-driven, 8123-109216-02 should also result in a considerable amount of nitrite as well as sub-stoichiometric GSNO.
  • DMPO completely abrogated the formation of GSNO (0.1 ⁇ 0.06 ⁇ M, FIG.8G) while preserving NO-ferroheme formation (18.1 ⁇ 1.5 ⁇ M), providing strong evidence for the thiyl radical hypothesis and thiol-catalyzed reductive nitrosylation (FIG 1G, + DMPO bars).
  • NO-Ferroheme Formation in Hemoglobin-Depleted RBC Membranes Heme is hydrophobic and can be solubilized in organic solvents like methanol. In vivo, heme is solubilized in cell lipid bilayers and is particularly abundant in erythrocyte membranes, where it may facilitate cytotoxic lipid peroxidation.
  • Heme binding to albumin is well-characterized, 48,49 and heme-nitrosyl species have been observed in human plasma, especially after the inhalation of NO gas. 50 Thiol- catalyzed NO-ferroheme formation was observed using serum albumin to solubilize ferric heme and NO-ferroheme product (FIG.3A). As with the MeOH:PBS system and RBC ghosts, GSH accelerates the rate of NO-ferroheme formation in a dose-dependent manner in 500 ⁇ M serum albumin (FIG. 3B), which approximates mammalian plasma concentrations.
  • this reaction exhibits biphasic kinetics under pseudo first-order conditions, and may indicate two different environments for the heme (FIGs.3B and 9A).
  • Example 5 NO-Ferroheme Transfer As labile heme (and thus potentially NO-ferroheme) can be found in membranes, the transfer of NO-ferroheme formed in RBC ghosts to albumin was qualitatively characterized. Ferric heme (25 ⁇ M), 50 ⁇ M glutathione, and 50 ⁇ M NO were added to RBC ghosts under anaerobic conditions at 37°C (FIG.3D). After subsequent incubation with albumin, the mixture was centrifuged at 30,000 x g for 2 hours to remove membranes. The clarified supernatant contained NO-ferroheme solubilized in albumin, demonstrating transfer from membranes to albumin.
  • the isosbestic points at 397, 522, and 593 nm indicate direct transfer without formation of any other species or intermediates.
  • Direct formation of NO-myoglobin from apo-myoglobin with NO-ferroheme in membranes is also readily observed (FIG.10B).
  • Incubation of NO-myoglobin with albumin or membranes did not result in loss of NO-myoglobin signal, implying the transfer from albumin to apo-myoglobin is favored, not vice versa.
  • direct transfer of NO-ferroheme from solubilizing membranes or albumin to another apo- hemoprotein, like sGC, is feasible.
  • Example 6 Stability of NO-Ferroheme in the Presence of Oxyhemoglobin Stable NO-ferroheme can perform a signaling function. Stability can be with respect to oxidation and premature NO release. While free NO is rapidly oxidized by oxyhemoglobin to form nitrate (NO dioxygenation), 11,53 NO complexed into NO-ferroheme should be stable under oxygenated conditions until the NO dissociates away from the heme, as observed with other nitrosyl- hemoproteins such as myoglobin.
  • sGC can then be stimulated, either via NO release or direct NO-ferroheme binding to apo-sGC.
  • PRP platelet-rich plasma
  • ADP adenosine diphosphate
  • cGMP was detected with and without ODQ, a sGC inhibitor, and tadalafil, a phosphodiesterase (PDE) inhibitor (FIG.4C).
  • Example 8 In Vivo Signaling Properties of NO-Ferroheme Albumin
  • MAP mean arterial blood pressure
  • FOG.5 normoxia and hypoxia
  • catheters were inserted into the right carotid artery and jugular vein for monitoring MAP and for dose injections, respectively.
  • Fresh NO-ferroheme stock solutions were generated in an anaerobic environment by mixing 300 ⁇ M hemin, 3 mM glutathione, 600 ⁇ M NO, and 500 ⁇ M bovine serum albumin in PBS.
  • mice were first treated with the NOS inhibitor L-N G -nitroarginine methyl ester (L-NAME).
  • L-NAME the NOS inhibitor L-N G -nitroarginine methyl ester
  • Four doses of NO-ferroheme were administered at estimated blood concentrations of 7.5 nM, 75 nM, 0.75 ⁇ M, and 7.5 ⁇ M.
  • Doses were injected intravenously every ten minutes, long enough for the blood pressure to stabilize.
  • Injections of NO-ferroheme solution in hypoxic animals were characterized by a rapid, transient relaxation, as monitored by changes in MAP. The MAP almost completely returned to the L- NAME induced baseline after ten minutes when compared to normal saline controls (FIG.5A).
  • controls for the maximum amount of GSNO theoretically generated were made (300 ⁇ M GSNO, 3 mM glutathione, and 500 ⁇ M albumin).
  • GSNO GSNO also did not elicit a large change in MAP (FIG.5A).
  • nitrite whose potency is increased >100 fold under hypoxia due to reduction of nitrite by deoxygenated hemoproteins, 62 NO-ferroheme elicits similar vasodilation under hypoxia and normoxia.
  • Example 9 Identification of NO-Ferroheme as the Vasodilating Species
  • GSH presents a physiological and kinetically viable route to NO-ferroheme from labile ferric heme, generating this compound in vitro results in a mixture of vasodilating species, including GSNO.
  • NO-ferroheme albumin was synthesized using sodium dithionite to avoid GSNO formation.
  • Example 10 Reactions with Cysteinyl Glycine and Sodium Sulfide 25 ⁇ M heme and 100 ⁇ M NO were reacted in the red cell ghost system under anoxic conditions at room temperature in the absence of any thiol species. Heme was first solubilized in 10% red cell ghosts (v/v) in PBS, and the kinetics of its reaction with NO was studied in a UV-Vis spectrometer with an integrating sphere (Cary 100) for one hour, scanning every minute.
  • FIG.15A shows the results of this reaction. 25 ⁇ M heme with 100 ⁇ M NO were reacted in the red cell ghost system in the presence of 250 ⁇ M cysteinyl glycine (Sigma Aldrich).
  • Cysteinyl glycine was added to the pre-solubilized heme in membranes before adding NO and the reaction kinetics was studied for 35 minutes in Cary 100. All reaction conditions were kept the same as those in the reaction without a thiol.
  • FIG.15B shows that the presence of cysteinyl glycine completes the NO-ferroheme reaction within the first scan. 25 ⁇ M heme with 100 ⁇ M NO (Matheson) were reacted in the red cell ghost system in the presence of 250 ⁇ M sodium sulfide. Sulfide was added to the pre-solubilized heme in membranes before adding NO and the reaction kinetics was studied for 30 minutes in Cary 100.
  • Transferrin receptor 1 is a cellular receptor for human heme-albumin. Commun Biol 3, 1–13 (2020). 74. Shvartsman, M., Bilican, S. & Lancrin, C. Iron deficiency disrupts embryonic haematopoiesis but not the endothelial to haematopoietic transition. Sci Rep 9, 6414 (2019). 75. Wu, S. M. et al. Developmental Origin of a Bipotential Myocardial and Smooth Muscle Cell Precursor in the Mammalian Heart. Cell 127, 1137–1150 (2006). 76. Vogel, S. M., Minshall, R. D., Pilipovi ⁇ , M., Tiruppathi, C.

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Abstract

L'invention concerne des compositions et des procédés pour induire une vasodilatation et/ou traiter une condition hémolytique chez un sujet. L'invention concerne également des procédés pour améliorer les effets (tels que des effets vasoconstrictifs) d'interactions hème-protéine avec de l'oxyde nitrique et des procédés de production d'un complexe d'oxyde nitrique (NO)-hème stable.
PCT/US2023/078075 2022-10-27 2023-10-27 Formulations de nitrosyle-hème pour le traitement d'affections cardiovasculaires, de l'hémolyse, et la stabilisation de molécules d'hème acellulaires Ceased WO2024112490A2 (fr)

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