WO2012050874A2 - Ciblage de l'hème pour le traitement de maladies inflammatoires à médiation immunitaire - Google Patents

Ciblage de l'hème pour le traitement de maladies inflammatoires à médiation immunitaire Download PDF

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WO2012050874A2
WO2012050874A2 PCT/US2011/053634 US2011053634W WO2012050874A2 WO 2012050874 A2 WO2012050874 A2 WO 2012050874A2 US 2011053634 W US2011053634 W US 2011053634W WO 2012050874 A2 WO2012050874 A2 WO 2012050874A2
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heme
mice
hmoxl
free
sad
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WO2012050874A3 (fr
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Miguel P. Soares
Bernat Olle
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Soares Miguel P
Bernat Olle
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    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is generally in the fields of inflammation and infection and relates in particular to methods and compositions for rendering an organism resistant to immune and inflammatory damage.
  • Heme iron protoporphyrin IX
  • Hb hemoglobin
  • RBC red blood cells
  • Hb a tetrameric protein that accounts for 97% of the total RBC dry content.
  • ROS/RNS reactive oxygen/nitrogen species
  • Free heme (a heme molecule that is not contained within the heme pocket of a hemoprotein) can catalyze the production of free radicals through Fenton chemistry (Fenton HJH., J. Chem. Soc. (Lond.) 1894, 65:899-910).
  • a heme pocket of a protein is defined as an area of that protein, which normally protects the iron contained inside the heme prosthetic group against oxidation, despite the fact that oxygen is being carried at this site. Under homeostasis this pro-oxidant effect is tightly controlled by the insertion of heme into the heme pockets of hemoproteins, which control the rate of electron exchange between Fe-heme and a variety of ligands.
  • Hb heme oxygenase- 1
  • HO-1 heme oxygenase- 1
  • TNF Tumor Necrosis Factor
  • Fas peroxinitrate of hydrogen peroxide
  • HO-1 is protective against a variety of immune -mediated inflammatory diseases (Soares MP, Bach FH. Trends Mol. Med. 2009. 15:50-58; Seixas E, Gozzelino R, Chora A, Ferreira A, Silva G, et al. Proc. Natl. Acad. Sci. USA. 2009.106: 15837-42). Deletion of the Hmoxl gene (encoding HO-1) has been shown to exacerbate a number of pathologies in mice, including among others severe sepsis (Chung, SW Liu X, Macias AA,
  • heme release from oxidized cell-free hemoglobin can contribute to the pathogenesis of severe forms of malaria (the disease caused by Plasmodium infection) in mice, and the onset of this experimental malaria in mice is associated with higher concentration of free heme in the plasma (Seixas E, Gozzelino R, Chora A, Ferreira A, Silva G, Larsen R, Rebelo S, Penido C, Smith NR, Coutinho A, Soares MP., Proc. Natl. Acad. Sci. USA. 2009. 106: 15837-42).
  • Upregulation of HO can also be detrimental to cells: the heme depletion and accumulation of CO or bilirubin but in particular the labile Fe (Suttner D, Dennery PA., FASEB Journal, 1999, 13, 1800-1809) it causes are potentially toxic.
  • Properly titrating the dose of agents that upregulate HO would be complicated because toxicity has to be defined in terms of each of the main products of heme metabolism.
  • sensitivity to the effects of these products is likely to be tissue- or cell-type specific (Mancuso C, Barone E. Current Drug Metabolism, 2009, 10, 579-594 579).
  • HO-1 responds to oxidative stress, and as such, inducers of HO-1 are molecules that cause undesirable sub-lethal levels of cell injury.
  • N-acetylcysteine an antioxidant molecule that limits the accumulation of free radicals, a downstream effect of heme release, has been explored in sepsis but shown to aggravate rather than ameliorate cardiovascular failure (Paterson RL, Galley HF, Webster NR., Crit. Care Med., 2003, 31 :2574), a hallmark of severe sepsis. Malaria remains one of the main causes of mortality worldwide, despite the development of several antimalarial drugs, and emergence of resistance to several of those antimalarial drugs has already been reported.
  • the method of treatment involves administration of a pharmaceutical composition
  • a pharmaceutical composition comprising as an active agent a DNA sequence, a protein, lipid, or any synthetic compound that binds to free heme ("heme scavenger") and interferes with and/or inactivates the pro-oxidant properties thereof.
  • active agents of the invention bind the iron in the heme.
  • examples of such agents include heme-binding proteins, such as Hemopexin (HPX), Albumin, or alpha 1 -microglobulin, or the heme-neutralizing protein Histidine-rich protein-2 (HRP-2), Peroxiredoxin 1 , heme-specific antibodies or a fragment or variant thereof.
  • HPX Hemopexin
  • Albumin or alpha 1 -microglobulin
  • HRP-2 heme-neutralizing protein Histidine-rich protein-2
  • Peroxiredoxin 1 heme-specific antibodies or a fragment or variant thereof.
  • the agents can be isolated from samples of human
  • the method of treatment involves administration of a
  • composition comprising as an active agent the hemoglobin-binding protein Haptoglobin (HPT), wherein HPT has been isolated from samples of human or animal blood, or recombinantly produced.
  • HPT hemoglobin-binding protein
  • the pharmaceutical composition may comprise an active agent fragment or variant of HPT that retains its Hb-neutralizing activity.
  • administration of a heme scavenger is used therapeutically against an infection and prevent the cytotoxic effects of free heme leading to the development of tissue damage and causing disease.
  • this protective effect may be associated with modulation of pathogen load.
  • administration of the heme- binding protein HPX is used to prevent the development of severe forms of malaria caused by Plasmodium infection, tuberculosis (TB) caused by Mycobacterium tuberculosis or severe sepsis, e.g., caused by polymicrobial infections.
  • a method of treatment of a free heme-mediated pathology comprising
  • an agent that suppresses extracellular release of, or inhibits, endogenous pro-inflammatory ligands and adjuvants thereof including but not limited to, High- mobility group protein Bl(HMGBl), uric acid, Fas ligand, H 2 0 2 , ONOO-, and heat shock proteins, either alone or in combination with administration of a heme-binding protein, is also provided.
  • HMGBl High- mobility group protein Bl(HMGBl)
  • uric acid including Fas ligand, H 2 0 2 , ONOO-, and heat shock proteins
  • blood taken from an individual with a blood-borne infection can be contacted with at least one capture agent against one or more heme-binding proteins, e.g. a heme-specific antibody, thus obtaining a measurement of free heme as well as the levels of one or more heme-binding proteins in blood, and comparing said measurement with baseline values of the concentration of said one or more less free heme or more heme-binding proteins in a healthy individual.
  • concentration of cell-free Hb, total plasma heme, or free heme may also be measured.
  • said measurements can provide a prognosis for the outcome of a disease.
  • said measurements can be used to stratify patients according to their likelihood of responding positively to an intervention.
  • said measurements are used to monitor the response of a patient to an approved or
  • said measurements are used for the diagnosis, prognosis, stratification, and monitoring of a disease associated with free heme-mediated pathological damage, which is not caused by an infectious agent.
  • FIG's. 1A, IB, 1C, ID, IE, and IF collectively show that HO-1 affords host protection against polymicrobial infection in mice without interfering with the host pathogen load, that is it confers tolerance against polymicrobial infection.
  • (A) Hmoxl mRNA expression in peritoneal leukocytes (Perit. leu.), lung, liver, and kidney after low- grade CLP (Cecal Ligation and Puncture, a standard model for the study of sepsis) in BALB/c mice, as determined by quantitative RT-PCR. Data are shown as mean ⁇ SD (n 3 per group).
  • IU international units (D) Representative examples of hematoxylin and eosin (H&E) stained liver, kidney, and heart tissues from Hmoxl +/+ and Hmoxl-/- mice after low-grade CLP. Magnifications are 400x. Arrows indicate red blood cells (RBC) associated with vascular congestion and/or thrombosis.
  • CV Coronary vessel
  • M M
  • E Bacterial load (CFU) in the peritoneum and blood of mice subjected to low-grade CLP (12 h after CLP). Circles represent individual mice. Bars represent median values, ns, a non-statistically significant difference.
  • FIG's. 2A, 2B, 2C, 2D, 2E, 2F, and 2G collectively show that HO-1 prevents heme-driven severe sepsis.
  • Protoporphyrins were administered (i.p.; 15 mg/kg) at 2, 12, and 24 h after CLP. Dotted line shows statistical comparison of vehicle- and FePPIX-treated animals.
  • FIG's. 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H collectively show that free heme promotes the pathogenesis of severe sepsis.
  • B Hemoglobin,
  • C haptoglobin,
  • D free heme, or
  • F Survival of wild-type
  • mice subjected to high-grade CLP.
  • G Serological markers of organ injury in mice treated as in (F). Measurements were made in serum from IgG-treated mice at the time of death (36 h) and in HPX-treated mice at the end of the experiment (day 11).
  • H Representative H&E staining (400x magnification) in mice treated as in (F). Samples are representative of 3 mice. CV: Coronary vessel; G: glomerulus. M: Myocardium. Samples are representative results of 3 mice in each group. HPX-treated mice in panels G and H were analyzed 11 days after CLP. Control IgG-treated mice in panels G and H were analyzed 24 to 36 h after CLP (time of death). *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ns: non-significant.
  • FIG's. 4A, 4B, and 4C collectively show that the oxidative effect of free heme sensitizes hepatocytes to programmed cell death.
  • A Primary BALB/c hepatocytes were either untreated (NT) or exposed to heme (5 ⁇ , lh) plus mouse recombinant TNF (5 ng/ml, for 16 h), anti-Fas antibody (0.5 ⁇ g/ml, for 4 h), H202 (125 ⁇ , for 8 h), or the ONOO- donor 3- morpholino-sydnonimine (SIN-1) (100 ⁇ , for 24 h). Production of free radicals was determined by flow cytometry using CM-H2DCFDA.
  • 2nd signal refers to TNF; anti- Fas; H202 or ONOO-, as specified for each panel.
  • B Percentage of cell death in primary hepatocytes treated as in (A). When indicated (+), hepatocytes were pretreated with the antioxidant N-acetyl-cysteine (NAC; 10 mM, for 4 h). Cell viability was determined by crystal violet staining.
  • FIG's. 5A, 5B, 5C, 5D, 5E, 5F, and 5G collectively show that free heme triggers the release of HMGB1 from hepatocytes.
  • HMGB1 red
  • DNA blue
  • HMGB1 red
  • DNA blue
  • Magnifications are 400x. Images are representative of 3 independent experiments. One nucleus per field is outlined (dotted line).
  • HMGB1 was measured by western (immuno) blotting of proteins in the supernatants of primary mouse (BALB/c) hepatocytes that were exposed to heme (5 ⁇ , for 1 h) and TNF (5 ng/ml; for 16 h) in culture. A representative result from 2 independent experiments is shown.
  • NS indicates a non-specific band
  • C HMGB 1 was measured by western (immuno) blotting of proteins in the supernatants of mouse Hepal-6 hepatocytes exposed to heme and TNF as described in (A). When indicated, cells were pretreated with the antioxidant NAC (10 mM; for 4h). A representative result from 2 independent experiments is shown.
  • HMGB1 was measured by western (immuno) blotting of proteins in the supernatants of mouse Hepal-6 hepatocytes treated with heme and TNF as in (A) and either not transduced or transduced with LacZ or Hmoxl Rec.Ad. Blots are representative of 2 independent experiments.
  • E HMGB1 staining in the liver and kidney from Hmoxl+/+ and Hmoxl-I- mice 24 h after CLP. One out of three representative samples are shown. Samples were counterstained with hematoxylin.
  • FIG's. 6A, 6B, 6C, 6D, 6E, 6F, 6G, and 6H collectively show that HPX suppresses the cytotoxic effect of free heme.
  • A Primary BALB/c hepatocytes were untreated (NT) or exposed to heme (5 ⁇ ), or HPX-heme complexes (5 ⁇ , for 1 h), and TNF (5 ng/ml; for 16 h). Production of free radicals was determined by flow cytometry using the broad free radical probe CM-H2DCFDA.
  • HMGB1 was measured by western blotting of proteins in the supernatants of primary mouse (BALB/c) hepatocytes treated as in (A). Blots are representative of 2 independent experiments.
  • E HMGB1 measured by western blotting in the supernatants of primary human hepatocytes treated as in (C).
  • Solid line refers to the expected median survival time as function of HPX serum concentration at the time of septic shock diagnosis, predicted by the best model for survival time (based on Lognormal distribution).
  • Grey circles represent individuals that succumbed during hospitalization (non- survivors).
  • White circles represent individuals that survived septic shock and left the hospital at the times indicated. P ⁇ 0.05 for the respective effect.
  • FIG.7 shows the role of free heme in the pathogenesis of severe sepsis.
  • the pathogenesis of severe sepsis is associated with hemolysis, which involves the release of hemoglobin (Hb) from red blood cells (RBC). Oxidation of cell free Hb leads to the release of its prosthetic heme groups.
  • This pathological event can be prevented by the acute phase protein haptoglobin (HPT) while free heme can be captured by the acute phase protein hemopexin (HPX).
  • HPT acute phase protein haptoglobin
  • HPX acute phase protein hemopexin
  • heme oxygenase- 1 heme oxygenase-1 (HO-1) in parenchymal cells affords cytoprotection against free heme thus suppressing tissue damage and ultimately multiple organ dysfunction/failure.
  • FIG. 8 shows the effect of HO-1 on bacterial load.
  • FIG.'s 9A, 9B, 9C, 9D, 9E, 9F, 9G, 9H, 91 collectively show modulation of cytokine production by HO-1.
  • A TNF concentrations in peritoneal fluid following low- grade CLP.
  • B TNF concentrations in supernatants of peritoneal leukocytes simulated with LPS (6h).
  • C TNF concentrations in supernatants of bone marrow-derived Mo exposed to live Gram positive (G pos ; Enterococcus subspecies) or Gram negative (G neg ; Escherichia coli) bacteria (8h).
  • D IL-6 concentrations in peritoneal fluid following low- grade CLP.
  • E IL-6 concentrations in supernatants of peritoneal leukocytes simulated with LPS (24h).
  • F IL-6 concentrations in supernatants of bone marrow-derived Mo exposed to live bacteria, as in (C).
  • G IL-10 concentrations in peritoneal fluid following low grade CLP.
  • H IL-10 concentrations in supernatants of peritoneal leukocytes simulated with LPS (24h).
  • I IL-10 concentrations in supernatants of bone marrow derived Mo exposed to live bacteria, as in (C). Cytokine concentrations were quantified by ELISA.
  • iNOS Inducible nitric oxide synthase
  • HO-1 HO-1
  • a- Tubulin were detected by western blot in peritoneal leukocytes from Hmoxl+/+
  • FIG. 11 shows red blood cell morphology in mice subjected to CLP.
  • FIG.'s 12A, 12B, 12C, 12D, 12E shows HO-1 modulates PMN
  • E Number of peritoneal infiltrating PMN (GRl hlgh ) cells in BALB/c mice receiving heme (80 nM, i.p.) or vehicle (PBS) and subjected or not to low-grade CLP. Peritoneal infiltrates were collected 16h post-CLP. Notice that while the production of free radicals was similar in PMN cells and Mo from Hmoxl-I- vs. Hmoxl+/+ mice (C) the net "oxidative power" of peritoneal infiltrating leukocytes was significantly higher in Hmoxl-I- vs. Hmoxl+/+ mice, as assessed in vitro by the oxidation of purified hemoglobin (D).
  • FIG. 13 shows infiltrating leukocytes following CLP.
  • FIG. 14 shows Adenoviral over expression of HO-1 in hepatocytes. Detection of HO-1 and a-tubulin by western blotting, in Hepal-6 hepatocytes, transfected with and without LacZ and Hmoxl Rec. Ad.
  • EC endothelial cell
  • PVC perivascular compartment
  • GL glia limitans (dotted line)
  • RBC red blood cells
  • iRBC infected RBC. Magnification: lOOx.
  • FIG.'s 16A, 16B, 16C, 16D, 16E, 16F show that HO-1 mediates the protective effect of HbSAD against ECM.
  • a) Mean ratio of Hmoxl vs. hypoxanthine-guanine phosphoribosyltransferase (Hprt) mRNA molecules in naive Hb M and Hb SAD mice ⁇ standard deviation (n 4/group).
  • Hprt hypoxanthine-guanine phosphoribosyltransferase
  • Hmoxl +1+ and Hb SAD Hmoxl +/- mice at the time of ECM onset in Hb SAD Hmoxl +/- mice.
  • FIG.'s 19A, 19B, 19C, 19D show that Sickle human Hb prevents the onset of ECM via the induction of Ho-1 expression by Nrf2.
  • a) Mean ratio of Hmoxl vs. hypoxanthine- guanine phosphoribosyltransferase (Hprt) mRNA molecules in peripheral blood mononuclear cells of na ' ive HbwtNrf2+/+, HbwtNrf2+/-, Hb SAD Nrf2+/+ and Hb SAD Nrf2+/- mice ⁇ standard deviation (n 6-8/group).
  • Hprt hypoxanthine- guanine phosphoribosyltransferase
  • FIG.'s 20A, 20B, 20C, 20D, 20E, 20F, 20G, 20H, 201 show that H ⁇ inhibits free heme accumulation via the production of CO.
  • FIG.'s 21 A, 21B, 21C show that Hb ⁇ expressing a wild type ?-chain of human Hb are not protected against ECM.
  • Brain edema was measured by Evans blue (EB) accumulation in brains of naive vs.
  • FIG.'s 22A, 22B, 22C show modulation of HO-1 expression in different experimental settings.
  • Hprt phosphoribosyltransferase
  • FIG.'s 24A, 24B, 24C, 24D show that Hb SAD allele inhibits neuroinflammation.
  • FIG. 25 shows that the Hb SAD allele inhibits neuroinflammation (continued)
  • Recipients were lethally irradiated before the adoptive transfer.
  • Grey shading indicates expected time of ECM.
  • FIG.'s 27A, 27B, 27C, 27D show that Parasite load is not modulated by heme or CO
  • Hb SAD Hmoxl +/ ⁇ mice exposed to CO 250 ppm, days 4-7 post-infection
  • FIG. 28 shows that albumin stops the cytotoxic effect of free heme.
  • the methods used to generate the data in the FIGure are explained in Example 16.
  • the Y axis shows the percent toxicity to hepatocytes
  • the X axis shows the different solutions that hepatocytes were exposed to (denoted by the symbol +), or not exposed to (denoted by the symbol -).
  • FIG.'s 29A, 29B, 29C collectively show that modulation of transporter molecules involved in regulation of the intracellular heme content can suppress the cytotoxic effects of free heme.
  • FIG.'s 30A, 30B, 30C, 30D, 30E collectively show the levels of cell-free hemoglobin, haptoglobin, total free heme (total plasma heme), hemopexin, and free heme, in a cohort of human patients diagnosted with malaria described in Example 18.
  • FIG.'s 31 A, 3 IB, 31C collectively show the serum concentrations of total plasma heme, free heme, and hemopexin in a cohort of human patients diagnosed with sepsis described in Example 7 and Example 19. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • heme is used to refer to a prosthetic group that consists of an iron atom contained in the center of a porphyrin. Heme is a component of a number of hemoproteins.
  • free heme is used to refer to heme molecules that are not contained within the heme pockets of hemoproteins, that is an area of the protein, which normally protects the iron contained inside the heme prosthetic group against oxidation, despite the fact that oxygen is being carried at this site. Heme molecules associated with proteins or lipids in a manner that does not control its pro-oxidant activity are intended to be included in this definition.
  • total plasma heme or “total heme in plasma” are used to refer to free heme plus heme complexed with hemoproteins, such as hemopexin and haptoglobin.
  • heme scavenger is used to refer to a molecule that binds and/or inhibits, reduces or neutralizes, heme and its cytotoxic effects.
  • heme oxygenase- 1 (HO-1) is used to refer to the enzyme that catabolizes free heme into equimolar amounts of Fe2+ (labile Fe), carbon monoxide (CO), and biliverdin.
  • labile Fe or "labile iron” is used to refer to a product of heme catabolism by HO-1, resulting from release of Fe from the protoporphyrin IX ring. Fe produced in this manner can catalyze the production of free radicals through the Fenton chemistry and thus act as a cytotoxic pro-oxidant.
  • HPX hemopexin
  • HPT haptoglobin
  • hemoprotein'Or heme -binding protein
  • tolerance (not to be confused with immunological tolerance), is used to refer to a strategy used by an infected organism to minimize the tissue damage inflicted under a given pathogen burden, without targeting the pathogen itself (as opposed to "resistance", a defense strategy used by organisms to directly reduce or eliminate an invading pathogen by mounting an inflammatory and immune response capable of reducing the pathogen load).
  • HMGB1 High-mobility group protein Bl
  • HMGB1 High-mobility group protein Bl
  • free heme-mediated pathological damage is used to refer to damage to tissues caused by free heme. Without wishing to be bound by theory, this damage is thought to be mediated by the production of free radicals which, in the presence of other proinflammatory agonists, such as Tumor Necrosis Factor a, can induce a variety of cell types to undergo programmed cell death.
  • proinflammatory agonists such as Tumor Necrosis Factor a
  • terapéuticaally effective amount of a compound is used to refer to an amount sufficient to cure, reduce, alleviate or partially arrest the clinical manifestations of a given disease and its complications. Effective amounts will depend on a number of factors, such as the severity of the disease, weight, sex, and age of the subject to be treated.
  • CLP Cosmetic Ligation and Puncture
  • neutralization of free heme by heme scavengers offers the advantage of directly targeting the pro-oxidant iron atom of heme (which is ultimately responsible for the deleterious effects of hemolysis) as opposed to downstream mediators and products of heme catabolism (such as HO- 1 , CO, biliverdin, or bilirubin).
  • free heme sensitizes cells to undergo apoptosis or programmed cell death in the presence of pro-inflammatory agonists. For example, it has been discovered that free heme synergizes with Fas ligand, H 2 0 2 , ONOO-, and other proinflammatory ligands to cause cell death. In contrast, heme alone, or a pro-inflammatory ligand alone, do not cause a significant amount of cell death.
  • the invention proposes heme scavengers that directly bind the Fe atom in heme, thus inhibiting its pro-oxidant properties.
  • the invention discloses heme scavengers that do not cause significant iron deficiency or anemia.
  • the heme scavengers of the invention form complexes with heme in a way that neutralizes its pro-oxidant effect and/or that facilitates re-uptake or recycling of the heme into cells, where heme can be catabolized by heme oxygenases and the iron product reutilized by the body.
  • the pharmaceutical compositions of the invention include HPX.
  • HPX is a glycoprotein belonging to the family of the acute-phase proteins, whose synthesis is induced after an inflammatory event. It participates in maintaining and recycling the iron pool by virtue of its high binding affinity toward heme.
  • the use of HPX is preferred because of its very high affinity for heme Kd ⁇ 1 pM, and its ability to preserve iron homeostasis by transporting heme back into cells (via recognition and internalization of the HPX-heme complexes by CD91 receptors). It has been discovered that administration of HPX results in scavenging of heme, as well as inhibition of its pro- oxidant effects in cells.
  • binding of heme by HPX inhibits the production of free radicals in response to pro-inflammatory agonists (see for example FIG. 6 A), and inhibits the ability of free heme to sensitize a variety of cell types, including hepatocytes to undergo programmed cell death in response to pro-inflammatory agonists (see for example FIG.'S 6B, 6C). Furthermore, it has also been found that HPX binding of heme also suppresses extracellular release of endogenous pro-inflammatory ligands, such as HMGB1 (see for example FIG.'s 6D, 6E).
  • the pharmaceutical compositions of the invention may include other human proteins that act as heme scavengers, such as albumin (which has a lower affinity for heme than HPX, but on the other hand can be administered in higher amounts than HPX because its typical physiological concentration in human blood, of 35-55g/L, is 30 to 100 times higher than typical physiological concentrations of HPX). Both HPX and albumin scavenge heme and prevent its pro-oxidant effects (see for example FIG. 28).
  • albumin which has a lower affinity for heme than HPX, but on the other hand can be administered in higher amounts than HPX because its typical physiological concentration in human blood, of 35-55g/L, is 30 to 100 times higher than typical physiological concentrations of HPX.
  • the pharmaceutical compositions may also include alpha 1 -microglobulin, which is also known to bind heme.
  • the pharmaceutical compositions may also include peroxiredoxin 1 , a protein known to chelate heme inside cells, which is preferably administered with any delivery system suitable for intracellular protein delivery known in the art (see for example Siprashvili Z, Reuter JA, Khavari PA, Molecular Therapy, 2004, 9, 721-728).
  • compositions of the invention may include HPX, Albumin, alpha 1 -microglobulin, or peroxiredoxin 1 proteins from non-human animals.
  • the pharmaceutical composition includes antibodies and fragments thereof that recognize specifically free heme.
  • Preferred antibodies bind free heme and do not bind heme bound in the heme pockets of hemoproteins.
  • the antibodies of the invention preferably neutralize the pro oxidant and/or cytotoxic effects of free heme via neutralization of its iron atom.
  • DNA sequences encoding the antibodies (or fragments thereof) can optionally be fused to DNA sequences encoding specific protein domains modulating the effector function of the heme binding domain of the VL peptide.
  • the pharmaceutical composition includes Histidine Rich Protein-2 (HRP-2), or a fragment thereof.
  • HRP-2 Histidine Rich Protein-2
  • This protein is naturally synthesized by the Plasmodium parasite, and can bind up to 19 molecules of heme.
  • This protein has been described in the art (see for example Pandey AV, Babbarwal VK, Okoyeh TN, Joshi RM, Puri SK, Singh, Chauhan VS. Biochemical and Biophysical Research Communications, 2003, 308, 736-743). It neutralizes the pro-oxidant activity of heme (Mashima R, Tilley L, Siomos M, Papalexis V, Raftery MJ, Stacker R. Journal of Biological Chemistry, 2002, 277 (17), 14514-20).
  • the DNA sequence encoding the heme binding domain of HRP2 can be fused to DNA sequences encoding specific protein domains modulating the effector function of the HRP2 heme binding domain.
  • the pharmaceutical composition includes Rhodnius Heme Binding Protein, a protein derived from the organism Rhodnius prolixus which has been characterized as heme-binding (Graca-Souza AV, Paiva-Silva G, Oliveira MF,
  • the pharmaceutical composition includes Heme
  • HeLp Lipoprotein
  • the pharmaceutical composition includes Heme-Binding
  • HbpA Lipoprotein derived from the organism Haemophilus influenza, which has been characterized in the art (Morton DJ, Madore LL, Smith A, Van Wagoner TM, Seale TW, Whitby PW, Stull TL, FEMS Microbiology Letters, 2005, 253, 193-199).
  • the pharmaceutical composition includes a heme-binding Strep-pneumonniae antigen described in US Patent 5,474,905.
  • the non-human-derived heme-binding proteins of the invention may be humanized or made less immunogenic by using a number of protein engineering methods known in the art (see for example Hurle MR, Gross M. Curr Opin BiotechnoL, 1994, 5(4):428-33, US Patent 6,992,174, US Publication 2004/0230380 Al).
  • the heme-binding proteins may be naturally immunogenic, or modified to increase their immunogenicity to facilitate clearance of protein-heme complexes in circulation.
  • Lipoprotein, or the heme-binding Strep-pneumonniae antigen may be fused to an HPX domain that is recognized by the hemopexin receptor on the surface of liver cells.
  • the pharmaceutical composition includes Low-Density Lipoprotein (LDL) and High-Density Lipoprotein (HDL).
  • LDL Low-Density Lipoprotein
  • HDL High-Density Lipoprotein
  • synthetic liposomes may be used to scavenge heme.
  • Lipids derived from non-human organisms may also be used, such as Drosophila retinoid- and fatty acid-binding glycoprotein (RFABG), which is known to bind heme (Duncan T, Osawa Y, Kutty RK, Wiggert B., The Journal of Lipid Research, 1999, 40, 1222-1228).
  • RABG Drosophila retinoid- and fatty acid-binding glycoprotein
  • the pharmaceutical composition includes artemisinin and/or a heme-binding quinoline (e.g. the 4-aminoquinoline chloroquine, the 8- aminoquinoline primaquine, isoquinoline, and the quinolinemethanols mefloquine and quinine), which are drugs that been shown in the art to bind heme (Pandey AV, Babbarwal VK, Okoyeh JN, Joshi RM, Puri SK, Singh RL, Chauhan VS. Biochemical and
  • the pharmaceutical composition includes a heme-binding aptamer (see some examples of heme-binding aptamers in Niles JC, DeRisi JL, Marietta MA. Proc. Nat. Ac. Sci. USA., 2009, 106(32), 13266-13271).
  • the heme- binding aptamer binds directly the Fe atom and inhibits the pro-oxidant effects of heme.
  • the pharmaceutical composition further comprises the heme chelator sodium cyanide.
  • the pharmaceutical composition comprises a synthetic histidine-rich protein.
  • the heme-binding lipids and synthetic heme chelators of the invention are linked to molecules that preferentially deliver them to macrophages.
  • molecules that preferentially deliver them to macrophages include Dou H, Destache CJ, Morehead JR, Mosley RL, Boska MD, Kingsley J, Gorantla S, Poluektova L, Nelson A, Chaubal M, Werling J, Kipp J, Rabinow BE, Gendelman HE. Blood. 2006, 108(8): 2827-2835).
  • the heme-binding lipids and synthetic heme chelators of the invention are fused or chemically linked to an HPX domain that is recognized by the hemopexin receptor (CD91) on the surface of liver cells.
  • CD91 hemopexin receptor
  • Haptoglobin also belongs to the acute-phase proteins, and is found in human plasma at 1-3 g/L. It binds hemoglobin and prevents loss of iron through the kidneys. Humans are polymorphic for haptoglobin, with three major phenotypes. Hp 1-1 is the most common, and the most effective in binding free hemoglobin. Hp 2-2 is the least effective. (Kasvosve I, Speeckaert MM, Speeckaert R, Masukume G, Delanghe JR. Adv Clin Chem, 2010, 50, 23; Van Vlierberghe H, Langlois M, Delanghe, J. Clin Chim Acta., 2004, 345, 35). E. Isolation or recombinant expression of Heme-Binding Proteins
  • Human Hemopexin can be obtained from human blood samples following methods described in the art (Aisen P., Leibman A., and Harris D., The Journal of Biological Chemistry, 1974, vol 249, no 21, pp 6824-6827; Tsutsui K., and Mueller G. C, Anal. Biochem., 1982, vol. 121, pp: 244-250), which generally involve combinations of buffering, ion exchange, ultrafiltration, and chromatography steps.
  • Human Haptoglobin can be purified from human blood following methods described in the art such as Cohn fractionation, salt-fractionation (Jayle M.F., Boussier G., and Tonnelat J., 1956, Bull. Soc. Chim. Biol, Paris, vol 38, pp 434); preparative electrophoresis (Boussier G., C.R. Acad. Sci., Paris, 1958, vol 246, p 1769), anionic exchange (US Publication No.
  • hemopexin and haptoglobin can also be obtained from bovine, canine, mouse, rat, porcine, goat, or rabbit serum (Smith A., Morgan W.T., Biochem Biophys Res Commun, 1978, 84,151-157).
  • Human Albumin can be obtained in large volumes from human blood using, for example, the traditional Cohn method, heat shock methods, or liquid chromatography (see for example Tanaka K, Shigueoka EM, Sawatani E, Dias GA, Arashiro F, Campos TC, Nakao HC, Braz, J Med Biol Res., 1998, 31(11): 1383-8). Expression of Heme-Binding Proteins in Recombinant and Transgenic Hosts
  • Recombinant human hemopexin can be obtained from commercial providers by expression in recombinant hosts, including NSO hybridoma cells (US Biological) and HEK293 cells (Reprokine Ltd.), and its expression in Pichia Pastoris has also been described (Bakker W.W. Borghuis T. Harmsen M.C., van den Berg A. Kema LP., Niezen K.E., Kapojos J.J., Kidney Int., 2005; 68(2): pp: 603-610).
  • Recombinant human haptoglobin can be expressed in recombinant hosts such as Baculovirus insect cells (Heinderyckx M., Jacobs P., and Bollen A., Mol Biol Reports, 1989, vol 13 (4), pp: 225- 232), or E. coli (available commercially from Prospec). It is also within the scope of the present invention to use transgenic animals to produce hemopexin and haptoglobin.
  • Heterologous DNA sequences encoding hemopexin and haptoglobin can be introduced into a transgenic animal.
  • hemopexin and haptoglobin can be expressed in the mammary glands of a non-human female animal such as a goat, sheep, or cattle.
  • General procedures for generating transgenic animals are known in the art, for instance see Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986.
  • HbpA Heme-Binding Lipoprotein
  • the amino acid sequence or the glycan moieties of hemopexin, haptoglobin, Albumin, Alpha- 1 microglobulin, Peroxiredoxin 1, and other heme scavengers of the invention can be modified. Methods known in the art such as site-directed mutagenesis, or insertional mutagenesis may be used to alter the sequence of the proteins. Addition of groups, e.g. N- or C-terminal tags, such as 6His-tags, may be used to aid in purification.
  • the heme scavengers may also be linked to a molecule that alters solubility or hal -life in circulation, such as a Polyethylene Glycol moiety.
  • the heme scavengers of the invention may also be expressed as fusion proteins.
  • recombinant hemopexin or haptoglobin may be fused to another protein, such as an. antibody, antibody fragment, or to a cell-surface molecule in order to facilitate targeting to a type of cel l or tissue, or modify half life in circulation. They may also be fused to a molecule that further enhances the ability to combat free heme-mediated tissue damage, for example an anti-TNF antibody, an anti-HMGBl antibody, or an anti-Fas ligand antibody.
  • Other heme scavengers of the invention may be fused or chemically linked to HPX domains that target them to the hemopexin receptor on li er cells.
  • a hemopexin protein is modified by altering amino acid residues responsible for protease activity (Bakker W.W. Borghuis T. Harmsen M.C., van den Berg A. Kema LP., Niezen K.E., Kapojos J.J., Kidney Int., 2005; 68(2): pp: 603-610) which may be an undesirable, potentially toxic activity for certain therapeutic applications.
  • Said protease activity may be modified using mutagenesis methods to generate and select variants with reduced or abrogated protease activity but impaired free heme-binding activity.
  • hemopexin and/or haptoglobin are non human-derived, they may be humanized by substi tution of key amino acids so that specific epitopes appear to the human immune system to be human rather than foreign (see for example Hurle MR, Gross M. Curr Opin BiotechnoL, 1994, 5(4):428-33) engineered variants of hemopexin or haptoglobin, may be designed to be non-immunogenic by elimination of MHC-binding epitopes in the protein sequence and alteration of the protein sequence to increase stability to aggregation by methods known in the art.
  • the amino acid sequence can be optimized to improve or alter properties of the proteins such as size, binding affinity, increase serum half-life, stability to temperature and proteolysis, or to facilitate purification.
  • the pharmaceutical composition may comprise fragments from hemopexin and haptoglobin, that retain their free-heme, and Hb-neutralizing activities, respectively.
  • the pharmaceutical composition may comprise an
  • Albumin variant that has been modified to increase its affinity for free heme.
  • the heme scavengers of the invention may be administered by conventional methods, for example parenteral, e.g. subcutaneous or intramuscular injection, intravenous infusion, or infusion by means of a pump.
  • one or more hemoproteins of the invention (for example, hemopexin, haptoglobin or albumin) are administered in salme by mtra venous infusion.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • the heme scavengers can be administered alone, but it is preferable to formulate them in pharmaceutically acceptable carriers.
  • accepted carrier is used to refer to carriers compatible with the heme scavengers, and not deleterious to the recipients.
  • the carriers will be water or saline, sterile and pyrogen free.
  • Pharmaceutical compositions of heme scavengers may be formulated in aqueous form well in advance of being administered, for example, weeks or months or before being administered.
  • the pharmaceutical compositions of hemopexin, haptoglobin, or other human hemoproteins will typically be non-immuriogenic, since in general they will be unmodified human proteins.
  • compositions used for therapeutic administration can be sterilized by filtration through sterile filtration membranes (for example, 0.2 micron membranes).
  • compositions can be administered orally, rectally, parenterally, intra vaginally, mtrapeiitoneally, topically, bucaliy, or as an oral or nasal spray.
  • parenteral administration is used.
  • Formulations suitable for parenteral administration include aqueous (for example, water or saline) and non-aqueous sterile suspensions and injection solutions.
  • the carrier is a parenteral earner, more preferably a solution that is isotonic with the blood of the recipient.
  • carrier vehicles include water, saline, Ringer's solution, and dextrose solution.
  • Non-aqueous vehicles such as fixed oils, ethyl oleate, and liposomes, may also be used.
  • the carrier may contain additives that enhance isotonic! ty and chemical stability, or generally render the formulation appropriate for use in a subject.
  • Such materials are non-toxic to recipients at she dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or
  • immunoglobulins include hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, di saccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as manmtol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
  • hydrophilic polymers such as polyvinylpyrrolidone
  • amino acids such as glycine, glutamic acid, aspartic acid, or arginine
  • monosaccharides include cellulose or its derivatives, glucose, mannose, or dextrins
  • chelating agents such as EDTA
  • sugar alcohols such as manmtol or sorbitol
  • counterions such as sodium
  • nonionic surfactants such as
  • composition comprises the following carriers: 0.01 M sodium phosphate, 0.15 mM sodium chloride, 15 ug/ml polysorbate 80, pH 7.2.
  • the pH and buffer are selected to match physiological conditions and the salt is added as a tonicifier.
  • Polysorbate is added to lower the surface tension of the solution and to lower non-specific adsorption of the hemoproteins to the container closure system.
  • the pharmaceutical compositions may be presented in unit-dose or multi-dose containers, for example sealed ampules, vials, syringes, and intravenous solution bags. These containers may have a sterile access port, or a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical compositions may also be stored in a freeze-dried (lyophilised) condition requiring the addition of the sterile liq uid carrier, for example, water, immediately prior to use.
  • a lyophilized formulation vials are filled with sterile-filtered aqueous hemopexin (or another hemoprotein) solution and the resulting mixture is lyophilized.
  • the infusion solution can be prepared by reconstituting the lyophilized hemopexin (or another hemoprotein) using bacteriostatic Water-for- Injection.
  • the required dose of heme scavenger may be calculated on the basis of blood concentrations of free heme in the subject to be treated. For example, a stoichiometric amount of hemopexin to free heme may be used to obtain an initial estimation of the dose required (one mole of hemopexin per mole of free heme in blood).
  • Total plasma heme may typically be encountered in human blood in concentrations ranging from, undetectable to 20 ⁇ under homeostasis (the upper limit corresponding to subjects with sickle ceil conditions), and in concentrations of up to 50 ⁇ in patients affected by severe sepsis or severe malaria. Assuming typical blood volumes for a human of 5 L, the total
  • hemopexin dosed parenterally to scavenge 50 ⁇ heme can be up to 1.5g (or 0.2g/kg for a 70kg subject). More typically, a patient with severely reduced levels of hemopexin (e.g. close to Og/L), may be administered approximately lOg of hemopexin (approximate amount needed to obtain a blood concentration of 2g/ ' L in an HPX-depleted subject with 5L of blood). More preferably, the selected hemopexin dose is at least 1 mg/ ' kg, and most preferably for humans between about 20mg/kg and 2g/kg.
  • free hemoglobin in plasma is typically encountered at .5-2g/L under homeostasis, corresponding to 7.8-30uM, and can increase up to lOg/L during severe hemolytic episodes.
  • a typical amount of haptoglobin to scavenge 30 ⁇ hemoglobin can be 15g (assuming 5L of blood and use of an isoform of haptoglobin with molecu lar weight of 98kDa), corresponding to 0.2g/kg for a 70kg subject.
  • the exact dose will be subject to therapeutic discretion. More preferably, this dose is a least 1 mg/kg, and most preferably for humans of about 20mg/kg or more.
  • the proteins may be typically administered using an intravenous bag solution or by continuous subcutaneous infusions, for example, using a mini-pump.
  • the length of treatment needed to observe a reduction of free heme levels and improvement of pathological outcomes may vary as a function of inter- individual differences and the disease being treated.
  • the amount of the pharmaceutical compositions which will be therapeutically effective can be determined by standard clinical techniques. In vitro and animal model assays such as those described in the Examples herein may optionally be employed to help identify optimal dosage ranges. Effective doses may be extrapolated from dose-response curves derived from such in vitro or animal model assays.
  • the dosage of the compositions to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. The precise dose to be employed in the pharmaceutical composition will depend on factors such as the route of administration, the seriousness of the condition, the age, weight, and response of the individual patient, and the severity of the patient's symptoms, and should be decided according to the judgment of the practitioner.
  • the dosage frequency can be, for example, once monthly, once weekly, twice weekly, once daily, twice daily, 4 times daily, or continuous infusion in certain cases of patients in severe condition.
  • the levels of administered protein in blood may be monitored so they are maintained within desirable physiological ranges (for example i-2g/L for HPX, more preferably 1.5-2g L, and ,5-1.5g/L for HPT, more preferably 1-L5g/ ' L).
  • the heme scavengers of the invention may optionally be administered by drug delivery systems known in the art in order to control their rate of release in circulation, enhance stability, increase solubility, or decrease adverse effects such as undesired proteolytic activity or immunogenicity.
  • delivery systems include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of nanoparticlesor or microcapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).
  • Drug delivery matrices may include polylactides (U.S. Pat. No.
  • Polynucleotides encoding HPX or other hemoproteins may be delivered by known gene delivery systems consisting of viral or non- viral vectors known in the art (for example, see Gilboa, J. Virology, 44:845 (1982); Hocke, Nature, 320:275 (1986); Wilson, et al, Proc. Natl. Acad. Sci. U.S.A., 85:3014; Chakrabarty et al, Mol. Cell Biol, 5:3403 (1985); Yates et al, Nature, 313:812 (1985)).
  • heme transporters include the mitochondrial adenosine triphosphate-binding cassette family member (Abcb6) and the adenine nucleotide translocator (ANT) (both of which can regulate heme synthesis by controlling the access of heme precursors to the mitochondria), the heme -responsive gene-1 (HRG-1) (which encodes a transmembrane protein that facilitates heme transport into the cytoplasm), the heme carrier protein- 1 (HCPl) (which can promote the import of extracellular free heme), the adenosine triphosphate-binding cassette Abcg2/Bcrp (which regulates heme export), and the feline leukemic virus receptor (FLVCR) (which also controls intracellular heme export).
  • Abcb6 mitochondrial adenosine triphosphate-binding cassette family member
  • ANT adenine nucleotide translocator
  • HRG-1 which encodes a transmembrane protein that facilitates hem
  • induction of programmed cell death in hepatocytes exposed to heme and pro-inflammatory agonists can be suppressed by administration of recombinant viruses encoding shRNAs targeting heme transporters, for example bcrp/Abcg2 and Abcb6 (See FIG.'S 29A, 29B, and Example 17).
  • compositions of the invention that modulate heme transporters may comprise a polynucleotide, a peptide, a protein, or a small molecule.
  • the heme transporters HCPl, HRG-1, Abcb6, and ANT can be inhibited with the antibodies ab25134, ab6780, ab47837, ab54418, respectively.
  • compositions may further comprise additional compounds that regulate the levels of free heme in circulation.
  • additional compounds that regulate the levels of free heme in circulation. These may include proteins that catabolize free heme, compounds that modulate proteins that regulate the levels of free heme, or products of the cataboiism of free heme, among others.
  • compositions further comprise
  • the pharmaceutical compositions further comprise a therapeutically effective amount of one or more compounds selected from morphine, a glucocorticoid, a nonsteroidal-antiinflammatory drug, a salicylate such as aspirin, doxorubicin, metoprolol, salbutamoL isoprotere ol, dobutamine, noradrenaline, flunarizine, pentaerithrityl trinitrate, pentaerithrityl tetranitrate, simvastatin, lovastatin, atorvastatin, rosuvastatin, venlafaxine, chlorpromazine, quetiapine, cyclosporine A, rapamycin, tranilast, sildenafil citrate, cytokine interleukin-10, 15-deoxy-12, 14- prostaglandin J2, vascular endothelial growth factor, stromal cell-derived factor 1, nitric oxide (NO), and nerve growth factor.
  • the pharmaceutical compositions further comprise a therapeutically effective amount of a natural substance selected from curcumin, resveratrol, ferulic acid, and L-carnitine. Without wishing to be bound by theory, these natural substances may regulate HO-1.
  • the pharmaceutical compositions further comprise, or are administered in conjunction with, products of the catabolism of free heme such as CO, biliverdin, or bilirubin.
  • the pharmaceutical compositions are administered preceded by, at the same time, or followed by inhalation of carbon monoxide. Carbon monoxide can be administered and dosed as described in US Patent No. 7,238,469.
  • the pharmaceutical compositions further comprise therapeutical ly effective amounts of biliverdin and/or bilirubin, which may optionally be administered in conjunction with carbon monoxide inhalation. Additionally or
  • modulators of HPX, HPT, and/or Albumin production can be administered, as well as modulators of biliverdin reductase.
  • the pharmaceutical compositions comprise a molecule that prevents release of heme from hemoproteins. It has been discovered that Sickle human Hb suppresses the onset of Experimental Cerebral Malaria via induction of HO-1 and production of CO, which inhibits the accumulation of free heme by stopping release of free heme from hemoproteins, thus affording tolerance against Plasmodium infection. (See FIG.'s 20A-1 and Example 15).
  • the pharmaceutical compositions comprise a hydrophilic CO-releasing molecule, for example a metal carbonyl (see for example US Patent Application Serial No. 10/535,508). Without wishing to be bound by theory, these molecules may prevent release of heme bound to Hb while being non-toxic to cells by virtue of their limited cell permability.
  • the pharmaceutical compositions further comprise at least one additio al compound selected from a labile Fe chelator and a molecule that directly degrades heme.
  • Fe chelators include chemicals such as Desferal (also kno wn as desferroxiamine), deferiprone, Emylenediammetetraaeetie acid (EDTA) and
  • DETAPAC Diethyienetriaminepentaacetic Acid
  • salicyialdehyde isonicotinoyl isonicotinoyl
  • S IH hydrazone
  • OSH reduced glutathione
  • xanthine oxidase xanthine oxidase
  • ADPH-cytochrome P-450 reductase xanthine oxidase
  • combination of the heme scavengers of the invention with a labile Fe chelator and/or a molecule that degrades heme may enhance the efficacy of the
  • compositions by counteracting the toxicity of both free heme as well as labile Fe, thus addressing a limitation of existing iron chelation therapies, which target labile Fe but not Fe inside protoporphyrins (e.g., heme).
  • the pharmaceutical compositions further comprise at least one antioxidant.
  • the antioxidant is N -acetylcysteine, reduced glutathione, urate, or butyl ated hydroxyanisole.
  • Antioxidants that act on lipid phases are particularly useful since heme is a hydrophobic molecule.
  • the pharmaceutical compositions further comprise at least one additional compound that suppresses extracellular release of, or inhibits, endogenous pro-inflammatory ligands.
  • additional compound that suppresses extracellular release of, or inhibits, endogenous pro-inflammatory ligands For example, antibodies against HMGB1, TNF, Fas ligand, or heat shock proteins (for example anti-HSP70 or anti-HSP60) or compounds that suppress the extracellular release or crystallization of uric acid, may be administered in conjunction with heme scavengers.
  • free heme is administered at slightly above normal concentration to subjects at high risk for a heme -mediated pathology, for example subjects at high risk of being infected with Plasmodium.
  • Prophylactic administration of low levels of free heme optionally in combination with a heme scavenger, chelator or binding agent, can promote tolerance against free-heme mediated pathologies.
  • potent and non-toxic heme scavengers are selected by screening a library of compounds in an in vitro cellular assay.
  • a single cell or a colony of cells in a well for example, primary BALB/c mouse hepatocytes or human hepatocytes
  • a pro-inflammatory agonist for example TNF, lipopolysaccharide (LPS), a Fas crosslinker, hydrogen peroxide (H202), or peroxinitrite (ONOO-)
  • a pro-inflammatory agonist for example TNF, lipopolysaccharide (LPS), a Fas crosslinker, hydrogen peroxide (H202), or peroxinitrite (ONOO-)
  • the cell or colony of cells is exposed to 5 uM heme for a period of lh, and one pro-inflammatory agonist selected from TNF (5 ng/ml, for 16 h), anti-Fas antibody (0.5 ⁇ g/ml, for 4 h), H202 (125 uM, for 8 h), or the ONOO- donor 3- morpholino-sydnonimine (SIN-1) (100 ⁇ , for 24 h).
  • a cell or colony of cells is pre-incubated with a candidate heme scavenger, and later both heme and a pro-inflammatory agonist are co-administered at the same concentrations previously used in the positive control.
  • Percentage of cell death after administration of heme and pro-inflammatory agonist is measured to establish the efficacy and potency of the candidate heme scavenger.
  • Cell viability can be determined by crystal violet staining.
  • production of free radicals as determined, for example, by flow cytometry using a fluorescent dye (for example CM-H2DCFDA) can be measured to establish the efficacy and potency of the candidate heme scavenger.
  • Each experiment can optionally be run in triplicate, using cells isolated from different mice.
  • the statistical significance of the difference in cell survival or production of free radicals with respect to the control may be established at the 90%, 95%, or 99% confidence level.
  • High throughput multi-well formats (for example 96-, 384-, 1536-well formats) can be used to screen large libraries of compounds.
  • potent and non-toxic heme scavengers are selected by screening compounds in an in vivo model of sepsis (for example, a BALB/c mouse).
  • an in vivo model of sepsis for example, a BALB/c mouse.
  • mice are subjected to Cecal ligation and puncture (CLP) and the percentage survival and time to death are measured.
  • CLP Cecal ligation and puncture
  • mice are subjected to CLP and administered a candidate heme scavenger or a vehicle afterwards (for example, 2, 12, 24, and 36 hours after CLP, intraperitoneally).
  • Animal survival and/or serological markers of organ injury for example, aspartate amino transferase (AST), blood urea nitrogen (BUN), and creatine - phosphokinase (CPK)
  • AST aspartate amino transferase
  • BUN blood urea nitrogen
  • CPK creatine - phosphokinase
  • potent and non-toxic heme scavengers are selected by screening compounds in an in vivo model of induced liver injury.
  • mice are administered a dose of heme (to sensitize them to subsequent TNF-mediated apoptosis), for example, a lmg dose, and subsequently liver injury and death is induced by administration of an intravenous injection of the molecule concanavalin A (ConA).
  • ConA concanavalin A
  • a 1.5mg/kg dose of ConA or greater can be given.
  • Liver injury can be assessed by transaminase release within hours after ConA is given.
  • mice are pretreated with a mixture of heme and a candidate heme scavenger, and subsequently administered ConA to induce liver injury.
  • Animal survival and/or serological markers of organ injury are measured after a period of time, for example 6 hours, 12 hours, or 1, 3, 5, 7, 9, 11, or 15 days. Effective heme scavengers prevent death of the mice or extend survival.
  • potent and non-toxic heme scavengers are selected by screening compounds in an in vivo model of microbial challenge.
  • mice are administered a dose of heme (to sensitize them to proinflammatory agonist-mediated apoptosis), for example, a lmg dose, and subsequently mice are challenged with heat-killed bacteria (or alternatively, with Lypopolysaccharide (LPS)), and mortality is measured.
  • mice are pretreated with a mixture of heme and a candidate heme scavenger, and subsequently challenged with heat-killed bacteria or LPS. Animal survival and/or serological markers of organ injury are measured after a period of time, for example 1, 3, 5, 7, 9, 11, or 15 days. Effective heme scavengers reduce mortality or extend survival.
  • HPX and optionally total plasma heme, free heme, free Hemoglobin, and/or HPT, are measured in a patient potentially affected by a free-heme mediated pathology, for example sepsis or malaria.
  • the measurements are first made at the time of patient intake to a clinical care center, or at the time of patient inclusion, in the intensive care unit.
  • the severity of the condition may dictate the frequency of measurements. For example, patients at risk of imminent death from severe sepsis or severe malaria may be tested every day, or before and after treatment by administration of a heme scavenger or before and after treatment by any other intervention known in the art for such conditions.
  • Patients affected by free-heme mediated pathologies of a chronic nature may be tested less frequently, for example weekly, monthly, or yearly.
  • Samples can be obtained for testing using standard techniques known in the art. Typically samples are obtained by blood draw. Blood is a preferred biological sample. The preferred blood constituent that is analyzed is either plasma, or serum, more preferably serum. The sample may be pretreated as necessary by dilution in an appropriate buffer solution, or concentrated. Standard buffer solutions such as phosphate may be used. Means of preparing blood piasrna and serum samples are known in the art and typically involve centrif ligation and filtration. The samples may be stored for up to 24 hours at 2°-8°C, or at -20°C or lower for longer periods, prior to measurement.
  • samples can be obtained from other biofluids, such as urine, or tissue biopsies.
  • the diagnostic hemoprotem analyt.es of the invention may be detected and quantified by a number of methods known in the art. Typical diagnostic methods focusing on protein detection include binding techniques such as ELISA, immunoMstoehemistry, microarray and functional techniques such as enzymatic assays among others.
  • HP and BPT are detected and quantified using an immunological binding assay (see for example US Patent No's: 4,366,241;
  • Immunological binding assays utilize a capture agent to specifically bind to the anaiyte (for example HPX or HPT).
  • the capture agent is an antibody against HPX, HPT, or isoforms thereof.
  • Antibodies for detecting HPX and HPT are commercial ly available (for example, anti-HPX antibodies ab27710, and ab27711, and anti-HPT antibodies ab8968, ab4248, and ab 13429).
  • the immunological assay may also use a labeling agent to label the binding complex formed by the capture agent and the anaiyte, such as another antibody, bearing a label, that that binds to the antibody- PX or the antibody-HPT complex.
  • a labeling agent to label the binding complex formed by the capture agent and the anaiyte, such as another antibody, bearing a label, that that binds to the antibody- PX or the antibody-HPT complex.
  • Standard ELISA kits for detection of HPX and HPT can be obtained from commercial vendors (GenWay, Alpco Diagnostics, Kamiya Medical Company).
  • HP and BPT may also be quantified using a radioimmunoassay (see for example
  • the present invention provides methods to measure and quantify free heme using a capture agent that specifically binds to free heme (but not to heme bound to hemoproteins or other blood constituents).
  • the capture agent may be an antibody against heme, an antibody fragment, or any other type of protein, peptide, or nucleic-acid based binder.
  • phage display (US Patent 5,395,750), bacterial display (US Patent 5,348,867), yeast display (US Patent 6,300,065), ribosome display, tnRNA display, mammalian cell display, or hybridoma technology, among others, may be used to select high affinity binders from large polypeptide libraries.
  • bacterial display US Patent 5,348,867
  • yeast display US Patent 6,300,065
  • ribosome display tnRNA display
  • mammalian cell display or hybridoma technology, among others
  • the assays of this inven tion may be scored according to standard statistical methods known in the art. Assays will generally be scored as positive where the levels of the assayed anaiytes experience a detectable change compared to a baseline value (for example the levels of the same analyte in a typical healthy patient; or the levels of the same analyte in the same patient, at the time of patient intake to a clinical care center, or at the time of inclusion in the intensive care unit).
  • the change is a statistically significant change, as determined using known statistical tests such as a t-test, analysis of variance (AN OVA), serniparametric techniques, non-parametric techniques (e.g.,
  • the statistically significant change is significant at least at the 85%, more preferably at least at the 95%, and most preferably at the 99% confidence level ), in certain embodiments, the change is at least a 10% decrease, preferably at least a 20% decrease, and more preferably at least a 50% decrease. Kits
  • this invention contemplates kits for performing one or more of the assays described herein.
  • Typical iy such kits will inciude reagents for the detection of one or more of I IPX.
  • the ki s may include reagents for the detection of anaiytes in blood or in urine.
  • Such reagents may include, but are not limited to, antibodies specific for HPX, HPT, Al bumin, b, and free heme.
  • the kits can optional iy contain additional materials for the collection of blood or urine.
  • the kits can also include instructional materials containing protocols for the practice of the assays of this invention.
  • the materials may be in the form of any suitable storage media, including but not limited to printed material, electronic, or optical storage media.
  • the therapeutic agents of the invention are suited for the treatment of pathologies associated with the accumulation of free heme in plasma or locally in tissues. In most cases this is associated with hemolysis, such as hemolysis caused by trauma driven from, bums or hemorrhage; a red-blood cell disorder, such as paroxysomal nocturnal hemoglobinuria, hereditary spherocytosis, sickle ceil disease, thalassemia., or pyruvate kinase deficiency; hemodynamic stress, such as microangiopathy, aortic stenosis, disseminated intravascular coagulation, or wherein, said hemodynamic stress is caused by a prosthetic heart valve or by extracorporeal circulation during surgery; an infection, such as malaria, dengue hemorrhagic fever, Chagas disease, or sepsis, or an infection by other pathogens that cause hemolysis, for example by releasing hemolysins, for example hemolysin-releasing bacteria such as Streptococcus
  • an antibody such as acute hemolytic transfusion reactions or paroxysomal cold hemoglobinuria
  • a chemical such as lead poisoning, potassium dichromate poisoning, or arsenic poisoning.
  • Heme release from myoglobin can act in a toxic manner and can be treated according to the invention.
  • the agents can also be used to prevent or treat pathological outcomes of pregnancy, ischemia-reperfusion injury, allograft rejection, neuroinflammation, endotoxic shock, autoimmune neuroinflammation, restenosis, myocardial infarction, or
  • HO-1 affords host tolerance against polymicrobial infection: Severe sepsis was produced in BALB/c mice by low-grade polymicrobial infection induced by cecal ligation and puncture (CLP). Using quantitative reverse transcription-polymerase chain reaction (RT-PCR), we measured expression of the Hmoxl gene and found that it was induced in peritoneal infiltrating leukocytes, liver, lung, and kidney at various time points after CLP (FIG. 1A). Mortality increased from 13% in wild type (Hmoxl+/+) mice to 80% in Hmoxl -deficient (Hmoxl-/-) mice when both were subjected to low-grade CLP (FIG. IB).
  • CLP cecal ligation and puncture
  • Hmoxl-/- versus Hmoxl +/+ mice did not result from higher pathogen (bacterial) load, as assessed by comparing the number of colony forming units (CFU) in the peritoneum and blood (FIG. IE) as well as in the liver, spleen, kidneys and lungs (FIG. 8). Hmoxl-/- mice also succumbed when challenged with heat-killed bacteria (60% mortality), whereas Hmoxl +/- and Hmoxl +/+ mice did not (0% mortality) (FIG. IF).
  • CFU colony forming units
  • HO-1 affords tolerance against polymicrobial infection independently of its previously reported antimicrobial activity.
  • Production of several cytokines involved in the pathogenesis of severe sepsis, [for example, TNF, interleukin-6 (IL-6), and IL-10] was similar in Hmoxl-/- versus Hmoxl+I- or Hmoxl+/+ mice subjected to low-grade CLP (FIG. 9A, D and G).
  • peritoneal or bone marrow-derived monocytes/macrophages (Mo) from Hmoxl-/- versus Hmoxl+/+ mice produced similar amounts of IL-6 when exposed in vitro to bacterial lipopolysaccharide (LPS) or to live bacteria (FIG. 9E and 9F), while producing slightly but significantly higher amounts of TNF when exposed to LPS (FIG. 9B) but not to live bacteria (FIG. 9C). Higher production of IL-10 also occurred in Hmoxl-/- versus Hmoxl+/+ Mo exposed to LPS or to live bacteria (FIG. 9H and I).
  • HO-1 regulates the expression of a subset of cytokines, (for example IL-10) (FIG. 9H and 91) in response to bacterial agonists (LPS) (FIG. 9H) or live bacteria (FIG. 91), we cannot exclude that this effect might contribute to the protective mechanism via which HO-1 suppresses the pathogenesis of severe sepsis.
  • LPS bacterial agonists
  • FPN- ⁇ interferon- ⁇
  • peritoneal Mo from naive Hmoxl-/- mice produced slightly but significantly higher amounts of nitric oxide (NO) than did Hmoxl+/+ peritoneal Mo (FIG. 10A). Whether reduced NO production contributes to the protective action of HO-1 remains to be established.
  • EXAMPLE 2 Whether reduced NO production contributes to the protective action of HO-1 remains to be established.
  • HO-1 prevents free heme from eliciting severe sepsis: Free heme, the substrate of HO-1 activity, is cytotoxic to red blood cells and causes hemolysis. This produces cell-free hemoglobin and eventually more free heme (that is, heme not contained within the heme pockets of hemoglobin). This definition of free heme does not preclude the association of heme with other proteins or lipids in a manner that does not control its ability to induce oxidative stress.
  • FIG. 2C characterized by multiple end-stage organ failure, as revealed serologically by increased AST, BUN and CPK plasma concentrations (FIG. 2D). Organ damage was confirmed histologically (FIG. 2E). Heme administration to naive wild-type (Hmoxl+/+) mice, while not lethal per se (0% mortality), elicited kidney, but not liver or cardiac, damage (FIG. 2D). Heme administration was also not lethal in mice subjected to "sham laparotomy" (0% mortality). Moreover, "iron-free" protoporphyrin IX failed to cause organ damage or to precipitate severe sepsis when administered to mice subjected to low- grade CLP (0% mortality) (FIG. 2C).
  • CD45+CD1 lb+GRl+ PMN cells in Hmoxl-/- mice subjected to low-grade CLP was two to threefold higher than those in Hmoxl +/- and Hmoxl+/+ mice (FIG. 12 and FIG. 13A and 13B). This was not the case for peritoneal NK, T, or B cells (FIG. 13C, D, and E). Expression of the phagocytic NADPH oxidase gp91phox in peritoneal infiltrating leukocytes was also higher in Hmoxl-/- vs. Hmoxl+/+ mice (FIG. 12B).
  • Free heme is a critical component in the pathogenesis of severe sepsis: When subjected to high-grade CLP (>90% mortality) (FIG. 3A), wild type (Hmoxl+/+) mice displayed abnormal red blood cell morphology (poikilocytosis) (FIG. 11). This was associated with the accumulation of cell-free hemoglobin in plasma (FIG. 3B), as compared to mice subjected to low-grade CLP ( ⁇ 20%> mortality) (FIG. 3A, 3B and 11). Moreover, there was a decrease in haptoglobin plasma concentrations in Hmoxl+/+ mice subjected to high-grade CLP, as compared to mice subjected to lowgrade CLP (FIG.
  • mice that received a non- heme-binding protein (namely IgG) after high-grade CLP succumbed to liver, cardiac, and kidney failure, as assessed by AST, BUN and CPK plasma concentrations, respectively (FIG. 3G).
  • IgG non-heme-binding protein
  • Free heme elicits programmed cell death We have previously shown that free heme can promote programmed cell death in response to TNF (Seixas E, Gozzelino R, Chora A, Ferreira A, Silva G, et al, Proc. Natl. Acad. Sci. USA., 2009, 106: 15837-42). We asked whether this effect is extended to other agonists involved in the pathogenesis of severe sepsis. Because hepatic failure is a central component of severe sepsis, we tested whether free heme induces oxidative stress and TNF -mediated programmed cell death in primary mouse hepatocytes in vitro.
  • hepatocytes When exposed to free heme, hepatocytes did not produce significant amounts of intracellular free radicals, as assessed by flow cytometry using a broad free-radical probe (FIG. 4A). However, when exposed to free heme and TNF (FIG. 4A) or free heme plus Fas crosslinking (which activates the Fas signaling transduction pathway), hepatocytes produced high amounts of intracellular free radicals (FIG. 4A). This effect was not observed when hepatocytes were exposed to free heme and oxidizing agents such as hydrogen peroxide (H202) or peroxynitrite (ONOO-), which are sufficient per se to cause free radical accumulation in hepatocytes (FIG. 4A).
  • H202 hydrogen peroxide
  • ONOO- peroxynitrite
  • HMGB1 high mobility group box 1
  • HMGB1 an endogenous pro-inflammatory ligand involved in the pathogenesis of severe sepsis and previously linked to HO-1.
  • HMGB1 expression was mainly restricted to the nucleus (FIG. 5A).
  • HMGB1 was translocated from the nucleus to the cytoplasm (FIG. 5A) and released extracellularly (FIG. 5B). This was not the case when hepatocytes were exposed to either free heme or TNF alone (FIG. 5, A and B). Extracellular HMGB1 release was suppressed by the antioxidant NAC (FIG. 5C), as well as by HO-1
  • the ability of free heme to sensitize hepatocytes in vitro such that they produce high levels of free radicals in response to TNF was inhibited when heme was bound to HPX (FIG. 6A).
  • the ability of free heme to sensitize hepatocytes to undergo programmed cell death in response to TNF was also inhibited once heme was bound to HPX (FIG. 6B). Similar results were obtained using primary human hepatocytes in that HPX prevented heme sensitization to programmed cell death in response to TNF (FIG. 6C). Accordingly, HPX-bound heme also failed to promote HMGB1 release from primary mouse
  • HPX serum concentration is associated with organ dysfunction and fatal outcome in septic shock patients: Given that HPX plasma concentration is reduced in mice that succumb to severe sepsis (FIG. 3E) we asked whether this would also be the case in patients that succumb to septic shock. In a cohort of 52 patients (Table 1), HPX serum concentration within 48h of presentation with septic shock was positively associated with patient survival time (FIG. 6F). That is, patients presenting lower HPX serum
  • mice and genotyping BALB/c, BALB/c.SCID, BA B/cHmoxl +/-,
  • BALB/c.SCID.Hmoxi+/- mice were maintained under specific pathogen-free conditions, according to the Animal Care Committee of the Instituto Gulbenkian de Ciencia. All animal protocols were approved by the "Direccao Geral de Veterinaria" of the Portuguese government.
  • BALB/c. Hmoxl+I- were generated originally by Shaw-Fang Yet (Pulmonary and Critical Care Division, Brigham and Women's Hospital, Boston, MA, 02115, USA) by disruption of exon 3 in the Hmoxl locus(l). Mice were backcrossed 10 times into the BALB/c background.
  • Heterozygous (Hmoxl+I-) breeding pairs yield about -8% viable and otherwise healthy homozygous HO- 1 -deficient mice. Littermate Hmoxl- 1+ and Hmoxl+/+ mice were used as controls. Mice were genotyped by PCR. Briefly, a 400 bp PCR product spanning the 5 ' flanking region of the neomycin cDNA in the Hmoxl locus was amplified from genomic DNA. For the endogenous Hmoxl locus, a 456 bp product was amplified as well. PCRs were repeated at least 2 times before experiments were performed and were carried out after experiments to confirm genotypes.
  • Cell culture Primary mouse peritoneal leukocytes were obtained by peritoneal "lavage" using ice cold apyrogen PBS (Sigma). Briefly, leukocytes were washed in PBS and resuspended in RPMI 1640 Glutamax I (Gibco), supplemented with 5% fetal bovine serum, 50 U/ml penicillin and 50 ⁇ g/ml streptomycin (Life Technologies). For cytokine measurements, cells (2.5xl0 4 ) were plated in flat-bottom 96-well microtiter plates (Techno Plastic Products AG)(100 ⁇ , 2h, 37°C), non-adherent cells were removed and adherent cells, i.e.
  • Bone marrow cells were incubated for 6 days in RPMI 1640 Glutamax I (Gibco), 10% FCS, 30% L929 supernatant (as macrophage colony-stimulating factor, M-CSF, source).
  • the bone marrow derived macrophages (BMDM) were seeded (16h) in 6 well plates (3xl0 5 cells/well) in RPMI, 3.3% FCS, 5% L929 supernatant.
  • BMDM bone marrow derived macrophages
  • Protoporphyrins Heme (iron-protoporphyrin; FePPIX; Frontier Scientific) and protoporphyrin IX (protoporphyrin IX di-sodium salt; NaPPIX; Frontier Scientific) were dissolved in 0.2 M HC1 and adjusted to pH 7.4 using sterile 0.2 M NaOH.
  • Primary hepatocytes Primary mouse hepatocytes were isolated. Briefly, livers from naive BALB/c mice were perfused through the portal vein (5ml/min, 10 min, 37°C) with liver perfusion medium (Invitrogen) and the tissue was disrupted.
  • Cells were filtered ( ⁇ ), washed (William's-E medium; 4% FCS) (Invitrogen), pelleted (lOOg; 30 sec; 20°C) and re-suspended (William's-E medium, 4% FCS).
  • Hepatocytes were isolated in a Percoll gradient (1.06/1.08/1.12 g/ml; 750g; 20 min.; 20°) (GE Healthcare), re-suspended (William's-E medium; 4% FCS), centrifuged (2x200g; 10 min.; 4°C), re-suspended (William's-E medium; 4% FCS) and seeded onto gelatin (0.2%)-coated plates. Medium was replaced after 4h and experiments performed 24- 48 h thereafter. Primary human hepatocytes were cultured in Hepatocyte Culture Medium (Lonza) as detailed by the supplier (Lonza).
  • Hepatocytes were seeded and exposed to heme (5 ⁇ , lh) in Hanks Balanced Salt Solution (HBSS; Invitrogen), without serum, to avoid potential heme-scavenging by serum proteins.
  • HBSS Hanks Balanced Salt Solution
  • hepatocytes were washed (PBS), and challenged in DMEM, 10% FCS (Hepal-6) or 4% FCS (primary hepatocytes), with human recombinant TNF (5-40 ng/ml, 3-16h; R&D Systems), Fas ligand (Jo2 anti CD95 antibody; 0.5 ⁇ g/ml, 4h; BD Biosciences), H202 (125 ⁇ , 8h; Sigma), or 3- morpholinosydnonimine (SIN-1; 100 ⁇ , 24h; Sigma). Cell viability was assessed by crystal violet assay, as described.
  • Heme iron-protoporphyrin; FePPIX; Frontier Scientific
  • FePPIX iron-protoporphyrin di-sodium salt
  • FePPIX iron-protoporphyrin di-sodium salt
  • TNF-a, IL-6 and IL-10 were quantified by ELISA, according to manufacturer's instructions (Becton Dickinson).
  • Nitric oxide (NO) was measured using a Griess colorimetric assay.
  • CLP was performed as described elsewhere (see for example Wictherman KA, Baue AE, Chaudry IH., Journal of Surgical Research, 1980. 29, 189-201). Briefly, mice were anesthetized (ketamine/xylazine; 120/16 mg/kg, i.p.). Under sterile conditions, a 1 cm incision was made parallel to the midline, the cecum was exteriorized and ligated (sterile 3-0 Mersilk sutures; Ethicon) immediately distal to the ileocecal valve (reducing the lumen 50-60% for low-grade CLP and 80-90%) for high-grade CLP).
  • Cecum was punctured once with a 23-gauge needle (low-grade CLP) or twice with a 21 -gauge needle (high-grade CLP), its content extruded by applying pressure and re -inserted into the abdominal cavity.
  • the peritoneal wall was sutured with sterile 3-0 Dafilon sutures (Braun) and the skin was closed with a surgical staple (Autoclip 9mm; Becton Dickinson).
  • a single dose of saline was injected subcutaneous ly (1 ml/animal) for fluid resuscitation. After the surgical procedure, animals were maintained at 37°C (30 min) and received antibiotics (Imipenem/Cilastine; Tienam; MSD; 0.5 mg/sc/animal), 2 hours after the surgical procedure and every 12h during 72h.
  • Colony- forming units Peritoneal fluid was obtained by "peritoneal lavage" with 5 ml of sterile PBS (Sigma). Organs were weighed and homogenized under sterile conditions in 0.5 ml PBS using dounce tissue grinders (Sigma). Serial dilutions of blood, peritoneal lavage and homogenized organs, were immediately plated on TRYPTICASETM Soy Agar II plates supplemented with 5% Sheep Blood (Becton Dickinson). CFU were counted after 24h incubation at 37°C.
  • Flow cytometry Leukocytes were washed and blocked in calcium and magnesium- free PBS containing 2% FCS (v/v). After incubation (30 min, 4°C) with fluorochrome- conjugated monoclonal antibodies directed against CD1 lb (clone Ml/70), IAd (clone AMS- 32.1), GR1 (clone 1A8), CD49b (clone DX5), a/ TCR (clone H57-597) or CD19 (clone 1D3)(BD Biosciences, Pharmingen, San Diego, CA), cells were washed twice with PBS, 2% FCS (v/v) and acquired in a FACScan or FACSCalibur, using CellQuest software (BD Biosciences).
  • Dead cells were excluded from the analysis using propidium iodide. Analysis was done using Flow Jo software (Tree Star Inc.) Cellular free radical generation was determined by incubating cells (10 ⁇ ; 15 min; 37°C, 95% humidity, 5% C02) with the broad free radical probe 5-(and-6)- chloromethyl-2'7'- dichlorodihydrofluoscein diacetate acetyl ester (CM-H 2 DCFDA; Molecular Probes).
  • CM-H 2 DCFDA chloromethyl-2'7'- dichlorodihydrofluoscein diacetate acetyl ester
  • Alexa568-conjugated goat anti-rabbit IgG (Invitrogen; 5 ⁇ g/ml) was used as secondary antibody, and nuclear DNA was stained with Hoechst 33342 (10 ⁇ g/ml, PBS, 20 min; Invitrogen), and cells were mounted in Vectashield (Vector Laboratories). Images were captured with a fluorescence microscope (Leica, DMRA2), equipped with UV light and Evolution MP 5.0 Color Camera (Media Cybernetics, Canada). Images were analyzed using ImageJ software (NIH, USA).
  • HMGBl was detected in paraffin embedded, formalin- fixed sections (5 ⁇ ) after microwave antigen retrieval (0.01 M citrate buffer, pH 6.0, 20 min), using rabbit anti-human HMGBl (Becton Dickinson, 556528)(0 ⁇ g/ml, 4°C, overnight). Rabbit IgG was detected using biotin-conjugated donkey anti-rabbit secondary antiserum (1 : 1000; Jackson Immunoresearch) and streptavidin-conjugated horseradish peroxidase amplification kit (Vectastain Elite ABC Kit, Vector Labs). Signal was revealed with 3,3'- diaminobenzidine (DAB). Sections were counterstained with Harris hematoxylin.
  • DAB 3,3'- diaminobenzidine
  • Negative controls were performed by omitting the primary antibody or using a nonspecific rabbit polyclonal antibody. Images were obtained and analyzed as described above.
  • Serum biochemistry Blood was collected in tubes with heparin after cardiac puncture, centrifuged (2x 5 min, 1600g). Aspartate amino transferase (AST), blood urea nitrogen (BUN) and creatine-phosphokinase (CPK) were measured according to the protocols of the International Federation of Clinical Chemistry, as described (10-12), by
  • Purified HPX binds heme as assessed by absorbance and circular dichroism spectroscopy of the apoprotein or the oxidized and reduced heme-HPX complexes; the concentration of the protein and equimolar heme binding were quantified using published procedures and extinction coefficients. Neither the apo-HPX nor the heme-HPX complex are toxic for cells in vitro even at high concentrations. Mice received purified HPX by intraperitoneal injection, (50mg/kg) at 2, 12, 24 and 36 h after CLP.
  • HO-1 was detected using a rabbit anti-human HO-1 polyclonal antibody (1 :2.500; SPA-895, StressGen). Monoclonal antibodies were used to detect a-tubulin (T9026, 1 :5.000 dilution; Sigma) and iNOS (Becton Dickinson). Primary antibodies were detected using horseradish peroxidase-conjugated donkey anti-rabbit, goat anti-mouse or rabbit anti-mouse IgG secondary antibodies (Pierce, Rockford).
  • Peroxidase activity was visualized using the SuperSignal chemiluminescent detection kit (Pierce), according to manufacturer's instructions and stored in the form of photoradiographs (BiomaxTMMS, Eastman Kodak) or using the Image Station 440CF (Kodak). Digital images were obtained using an image scanner equipped with Adobe Photoshop software. Septic Shock Patients We analyzed the plasma concentration of HPX in 52 patients undergoing septic shock, as defined by the American College of Chest Physicians
  • Y a+b!genotype+c!heterozygote, with 79 denoting the variable under analysis, using logarithmic transformation when appropriate, a is the base-line referring to the Hmoxl-I- mean, b is the mean effect of adding a Hmoxl+/+ allele in the genotype (additive effect), c is the deviation of heterozygote mean from a single additive effect, genotype is a explanatory variable denoting the genotype coded as 0, 1, and 2 (0, 1, 2 Hmoxl+/+ alleles, respectively) and Hmoxl+/- is the binary variable indicating the heterozygote genotype.
  • Model validation was done by a thorough residual analysis, which included testing normality of the residuals and visual inspection of any trend in the residuals across genotypes.
  • Statistical significance refers to additive effects in the regression analysis. Kolmogorov-Smirnov and Shapiro-Wilk tests were performed to infer whether or not data could come from normal distributions. All statistical tests were done at 5% significance level, using InStat and R software. All statistical tests were performed at a 5% significance level, using InStat and R software.
  • a survival analysis was performed using the package "survival" available in the R software. For each patient, survival time was computed by the difference between the time of patient inclusion in the intensive care unit and the respective closing date of the hospital record.
  • mice C57BL/6 Hmoxl 1' mice were provided originally by Shaw-Fang Yet (Pulmonary and Critical Care Division, Brigham and Women's Hospital, Boston).
  • Hb SAD Nrf2 +l' mice were generated from Hb SAD Nrf2 +l' x Hb wt Nr 2 +/" breeding.
  • C57BL/6 Hb SAD mice (expressing 19% Hb SAD , i.e. ⁇ ⁇ 2 ⁇ 2 5 ⁇ ) were provided originally by Annie Henri (INSERM U733 IUH Hopital Saint-Louis, Paris). While hemizygous Hb SAD can develop typical complications of sickle cell disease, e.g.
  • Hb SAD Hmoxl +/ ⁇ mice were generated from Hb SAD Hmoxl +/ ⁇ x Hb wt Hmoxl 1' breeding.
  • Hb A/a mice expressing only one copy of the human Hb alpha, gamma-beta chain alleles and one copy of the endogenous alleles of the mouse Hb chains were produced by breeding the Hb A/A with C57BL/6 Hb wt mice. Interbreeding of Hb A/a mice produced, among other genotypes, Hb A/a mice and Hb a/a mice, expressing only the endogenous alleles of the mouse Hb chains. Mice were genotyped by PCR (Hmoxl and Nrf2) and isoelectric focusing ⁇ Hb), as described elsewhere (Pamplona, A. et al. Nat. Med. 2007. 13, 703-710).
  • Bone Marrow chimeras were generated in Hmoxl , Hmoxl " mice expressing or not the Hb SAD allele (8-10 weeks). Mice (recipients) were lethally irradiated (900 rad, 2.35 minutes, 137Cs source)(Gammacell 2000, Molsgaard Medical, Denmark) and
  • mice were infected by intraperitoneal
  • Iron-protoporphyrin IX FePPIX; heme
  • zinc-protoporphyrin IX ZnPPIX
  • CO treatment Mice were placed in a gastight 60 L capacity chamber and exposed continuously between days 4-7 post-infection to CO at a flow rate of ⁇ 12 L/min (final concentration of 250 parts per million; ppm), as described (Pamplona, A. et al. Nat. Med. 2007. 13, 703-710). CO concentration was monitored using a CO analyzer (InterScan Corporation, Chats worth).
  • Leukocyte brain infiltration Leukocytes were isolated from the brain of P.
  • mice when clinical symptoms of ECM were detectable in control groups.
  • Mice were perfused with PBS in toto, brains were collected, homogenized, digested (30 min, 37°C) in Hanks-balanced salt solution (HBSS; Life Technologies) supplemented with 0.2 mg/ml collagenase VIII (Sigma-Aldrich), strained (100 ⁇ ) (Becton Dickinson) and centrifuged (1200 g; 10 min). Brain leukocyte infiltration was quantified by flow cytometry.
  • HBSS Hanks-balanced salt solution
  • RNeasy Plus Mini Kit (Quiagen), according to manufacturers recommendation.
  • RNeasy Protect Animal Blood Kit (Quiagen) was used for the extraction of RNA from whole blood.
  • cDNA was synthesized as described (Pamplona, A. et al. Nat. Med. 2007. 13, 703- 710).
  • Hmoxl mRNA was quantified by qRT-PCR (Roche System) as described
  • TMB tetramethylbenzidine peroxidase assay
  • Sickle human Hb confers a survival advantage against malaria in mice: Inoculation of C57BL/6 mice (Hb wt ) with P. berghei ANKA infected red blood cells (RBC) led within 6 to 12 days to the development of clinical signs of ECM, i.e. head deviation, tendency to roll-over upon stimulation, paraplegia, ataxia, convulsions and ultimately to death (FIG. 15 A). Incidence of ECM was significantly reduced in hemizygous C57BL/6 Hb SAD mice (FIG.
  • Hb SAD mice present a very mild sickle cell syndrome, which does not lead to anemia (Table 3), similar to the asymptomatic human A/S sickle cell trait that affords protection against malaria.
  • the protective effect of Hb SAD against ECM is consistent with previous observations in other rodent models of sickle cell disease.
  • Hb wt C51B /6 (FIG. 15a) mice is expected given that the development of ECM is strongly favored in the C57BL/6 genetic background.
  • Hb SAD mice that did not succumb within 6-12 days post-infection also did not develop the pathologic hallmarks of ECM or those associated with human CM), including blood brain barrier (BBB) disruption (FIG. 15b,c), perivascular RBC accumulation in brain (FIG. 15b) and brain edema (FIG. 15c).
  • BBB blood brain barrier
  • FIG. 15b,c perivascular RBC accumulation in brain
  • FIG. 15c brain edema mice
  • Hb SAD mice develop a mild form of sickle cell disease.
  • Hb SAD mice developed a relatively more severe form of anemia with concomitant increase of circulating leukocytes, lymphocytes, monocytes and platelets, as compared to infected Hb wt mice.
  • MCV Mean corpuscular volume
  • MCHC Mean cell hemoglobin concentration. *P ⁇ 0.05 in Hb SAD vs. Hb wt mice, compared among non-infected or infected groups.
  • Leukocytes ( ⁇ 10 3 / ⁇ 1) 9.03 ⁇ 3.51 13.17 ⁇ 2.13 1.64 ⁇ 0.50 3.52 ⁇ 1.20*
  • Lymphocytes ( ⁇ 10 3 / ⁇ 1) 6.0 ⁇ 1.69 10.81 ⁇ 1.54* 0.89 ⁇ 0.37 2.51 ⁇ 0.86* Monocytes ( ⁇ 10 3 / ⁇ 1) 0.96 ⁇ 0.44 1.14 ⁇ 0.42 0.20 ⁇ 0.10 0.41 ⁇ 0.15*
  • Hb SAD mice against ECM was not associated with reduction of pathogen load, as assessed by the percentage of infected RBC, i.e. parasitemia (FIG. 15e) as well as by the number of circulating infected RBC (FIG. 15f) vs. control Hb wt (FIG. 15e,f) or Hb A/a mice (FIG. 21c).
  • Sickle human Hb induces the expression of HO-1 that confers tolerance against
  • Plasmodium infection Humans and rodents carrying the HbS mutation express high levels of HO-1 in the hematopoietic compartment (Belcher, J.D., Mahaseth, H., Welch, T.E., Otterbein, L.E., Hebbel, R.P., and Vercellotti, G.M. (2006). J Clin Invest 116, 808- 816). Consistent with this, naive Hb SAD mice also express high levels of Hmoxl mRNA in the bone marrow and peripheral blood, as compared to naive Hb wt mice (FIG. 16a).
  • Hb A/a mice that expressed similar levels of Hmoxl mRNA in the bone marrow and peripheral blood vs. littermate control Hb a/a mice (FIG. 22b).
  • Hb a/a mice This demonstrates that expression of a p s - related variant but not a normal ⁇ -globin chain is required to induce Hmoxl expression.
  • Na ' ive Hb SAD mice also expressed higher levels of Hmoxl mR A in the kidneys (FIG. 22a), which is consistent with the chronic
  • Hb SAD mice expressed similar levels of Hmoxl mRNA in the liver, heart, lung and spleen (FIG. 22a), as compared to Hb wt mice.
  • Hb SAD mice Given that HO-1 is protective against severe forms of malaria in mice ( Pamplona, A. et al. Nat. Med. 2007. 13, 703-710) Pamplona et al, 2007; Seixas et al, 2009), we asked whether its induction in Hb SAD mice (FIG. 12a) is required to suppress the development of ECM (FIG. 15a). Deletion of one Hmoxl allele ⁇ Hmoxl 1 ) reduced Hmoxl mRNA expression in bone marrow and whole blood leukocytes of Hb SAD mice (FIG.
  • Hb SAD Hmoxl +/ - vs. Hb SAD Hmoxl +l+ mice (FIG. 16f), which is consistent with the notion that induction of HO-1 expression by sickle human Hb confers tolerance against
  • Hb SAD Hmoxl +/ ⁇ mice survival was slightly but significantly higher than that of Hb wt Hmoxl +/ ⁇ mice (FIG. 16b). This suggests that residual HO-1 expression in Hb SAD Hmoxl +/ - mice (FIG. 22c) might account for this effect or alternatively that sickle human Hb might act, to a limited extent, independently of HO-1 to afford protection against ECM.
  • breeding of Hmoxl 1' mice is lethal and does not yield viable progeny (Yet, S.F., Perrella, M.A., Layne, M.D., Hsieh, CM., Maemura, K., Kobzik, L., Wiesel, P., Christou, H., Kourembanas, S., and Lee, M.E. (1999). Journal of Clinical Investigation 103, R23-29). NS: not significant.
  • Hb SAD vs. Hb wt mice revealed by a significant reduction in the frequency of Hb SAD vs. Hb wt homozygous Hmoxl deficient ⁇ Hmoxl ' ' ) offspring obtained from heterozygous-deficient (Hb wt Hmoxl +/' ) breeding.
  • Hb wt Hmoxl +/' breeding was similar to that obtained from Hb SAD Hmoxl +/' x Hb wt Hmoxl +/' breeding ( ⁇ 2 ; p>0.05).
  • Hb SAD Hmoxl +/+ neonates exhibit transient anemia at delivery, related to hemolysis caused by Hb SAD polymerization, most probably due to transient hypoxia associated with late fetal development and delivery.
  • Hb values in Hb SAD mice return to normal levels shortly after weaning (Trudel et al., 1991).
  • Leukocytes ( ⁇ 10 3 / ⁇ 1) 13.17 ⁇ 2.13 12.27 ⁇ 3.86 3.52 ⁇ 1.20 2.65 ⁇ 1.52
  • Lymphocytes ( ⁇ 10 3 / ⁇ 1) 10.8 ⁇ 1.54 12.01 ⁇ 3.37 2.51 ⁇ 0.86 2.35 ⁇ 0.81
  • chemokines involved in the pathogenesis of ECM Several chemokines can contribute to the pathogenesis of ECM and presumably to that of human CM.
  • Expression of mRNA encoding Ccl2 (Mcp-1), Ccl3 (MlPIa), Ccl5 (Rantes) and CxcllO (Ip-10) were decreased in the brain of Hb SAD mice that did not develop ECM vs. Hb wt mice that succumbed to ECM (FIG. 17a).
  • mRNA encoding the chemokine Cell 9 (MIPS a) and the chemokine receptor Ccr7 was not modulated by Hb SAD and/or by HO-1 (FIG. 17c). This was also the case for several other genes previously involved or not in the pathogenesis of ECM (FIG. 18 and FIG. 25).
  • Sickle human Hb confers tolerance against Plasmodium infection via HO-1 expression in bone marrow and blood cells: We asked whether the protective effect of Hb SAD against ECM requires the expression of HO-1 in the hematopoietic or
  • Hb SAD Hmoxl +l+ or Hb SAD Hmoxl +/ ⁇ mice were performed syngenic bone marrow transplants from Hb SAD Hmoxl +l+ or Hb SAD Hmoxl +/ ⁇ mice into lethally irradiated Hb wt Hmoxl +/+ or Hb wt Hmoxl +/ ⁇ mice to generate chimeric Hb SAD mice in which one Hmoxl allele is deleted in the hematopoietic (Hb SAD Hmoxl +/ ⁇ - Hb wt Hmoxl +/+ ) or non-hematopoietic
  • Hb SAD Hmoxl +/+ - Hb wt Hmoxl +/ ⁇ Chimeric Hb SAD mice carrying two functional Hmoxl alleles in the hematopoietic and in the non-hematopoietic compartments (Hb SAD Hmoxl +l+ ⁇ Hb wt Hmoxl +l+ ) did not succumb to ECM (FIG. 18a) or developed brain edema (FIG. 18b) in response to P. berghei ANKA infection.
  • Sickle human Hb induces the expression of HO-1 via the transcription factor Nrf2:
  • Nrf2 plays a central role in the regulation of HO-1 expression. Therefore we asked whether induction of HO-1 expression in whole blood leukocytes of naive Hb SAD mice (FIG. 16a) involved the transcription factor Nrf2. We found that this is the case since deletion of one Nrf2 allele in Hb mice
  • Sickle human Hb confers tolerance against Plasmodium infection via CO produced through heme catabolism by HO-1 : Consistent with similar observations in individuals carrying the HbS mutation in the homozygous or heterozygous form (Muller-Eberhard, U., Javid, J., Liem, H.H., Hanstein, A., and Hanna, M. (1968). Blood 32, 811-815), na ' ive Hb mice had significantly higher concentration of free heme in plasma, as compared to age-matched control naive Hb wt mice (FIG. 20a).
  • Plasma free heme concentration increased significantly following P. berghei ANKA infection in Hb wt mice (FIG. 20a), an effect we have previously shown to contribute in a critical manner to the pathogenesis of ECM (Pamplona, A. et al. Nat. Med. 2007. 13, 703-710), (reviewed in Gozzelino R, Jeney V, Soares MP. Mechanisms of Cell Protection by Heme Oxygenase-1, in Annual Review of Pharmacology and Toxicology. 2010. 50: 323-354). Albeit less pronounced, this increase was also observed in Hb SAD mice (FIG. 20a). When challenged with free heme after infection, Hb SAD succumbed to ECM (FIG.
  • Hb SAD Hmoxl +l - mice receiving CO i.e. 0 % mortality.
  • Hepatocytes Heap cells
  • heme 40 mM, lh
  • HBSS Hanks Balanced Salt Solution
  • Hepatocytes were washed (PBS), and challenged in DMEM, 10% FCS, with mouse recombinant TNF (5-40 ng/ml, 3-16h; R&D Systems). Cell viability was assessed by crystal violet assay. Heme (iron-protoporphyrin; FePPIX; Frontier Scientific) was dissolved in DMSO. Heme/HSA was prepared as follows: Human Serum Albumin (HSA) (lmM in PBS) was incubated with heme (2mM; DMSO); 4h/4C. Heme/HSA was separated from excess free heme and free albumin by PD-10 column.
  • HSA Human Serum Albumin
  • Binding was confirmed by electrophoresis of albumin in a native gel as well as by absorption spectrophotometry. Heme and heme/HSA were used at 40 ⁇ heme equivalents. As illustrated in FIG. 28, albumin blocked nearly 100% of the cytotoxicity otherwise observed when cells were exposed to heme and TNF.
  • Hepatocytes (Hepa cells) were cultured in 96 well plates (5xl0 3 cells/well) and transduced the day after with recombinant lentivirus (1-5) encoding shRNA targeting heme transporters. Cells were selected (2 days) under Puromycin, washed and exposed to heme (40 ⁇ , lh) in Hanks Balanced Salt Solution (HBSS; Invitrogen), without serum, to avoid potential heme-scavenging by serum proteins.
  • HBSS Hanks Balanced Salt Solution
  • hepatocytes were washed with phosphate buffered saline (PBS), and challenged in DMEM, 10% foetal calf serum (FCS), with mouse recombinant TNF (50 ng/ml, 4-6h; R&D Systems). Cell viability was assessed by crystal violet assay. The results are shown in FIGS. 29A, 29B, and 29C.
  • Cerebral Malaria was defined according to the WHO criteria: a coma score ⁇ 3 in Blantyre Scale for children ⁇ 60 months or a coma score ⁇ 7 in Glasgow Scale for children > 60 months. Meningitis and encephalitis were ruled out by cerebrospinal fluid analysis after lumbar puncture.
  • the severe non-cerebral malaria group included patients with severe malaria anemia (hemoglobin ⁇ 5 g/dl or hematocrit ⁇ 15%) and/or hyperparasitemia (> 100 red blood cells parasitized by one high-power microscopic field).
  • the uncomplicated malaria (UM) group represents patients with malaria diagnosis and febrile illness without any clinical finding suggestive of other causes of infection and with no manifestations of severe malaria. All the uncomplicated malaria patients were outpatients. Serum
  • Plasma HPX and haptoglobin were determined by ELISA (Life Diagnostics). Plasma hemoglobin was determined by spectroscopy at I577. Total plasma heme was measured using the 3,3', 5,5' tetramethylbenzidine (TMB) peroxidase assay (BD
  • FIG.'S 30A-E summarize the results of the study. HPX and HPT levels were reduced in malaria patients compared to asymptomatic patients, and total heme and free heme levels were increased in malaria patients compared to asymptomatic patients. EXAMPLE 19
  • Levels of levels of total plasma heme, free heme, and hemopexin in a cohort of human patients diagnosed with sepsis The same cohort of 52 patients described in EXAMPLE 7 was analyzed for serum concentrations of HPX, Total heme, and free heme. HPX levels were reduced in non-survivors, and total heme and free heme levels were increased in non-survivors.

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Abstract

La présente invention concerne des procédés destinés à limiter les effets nuisibles de l'hème libre pour le traitement de pathologies inflammatoires et infectieuses, comprenant l'administration de piégeurs de l'hème tels que des hémoprotéines humaines, et des procédés de diagnostic, de pronostic, de classification, et de surveillance de maladies associées à une lésion pathologique médiée par l'hème.
PCT/US2011/053634 2010-09-28 2011-09-28 Ciblage de l'hème pour le traitement de maladies inflammatoires à médiation immunitaire WO2012050874A2 (fr)

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