WO2012050874A2 - Targeting heme for the treatment of immune mediated inflammatory diseases - Google Patents

Targeting heme for the treatment of immune mediated inflammatory diseases 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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PCT/US2011/053634
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WO2012050874A3 (en
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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.

Abstract

The present invention relates to methods of limiting the deleterious effects of free heme for the treatment of inflammatory and infectious pathologies, including administration of heme scavengers such as human hemoproteins, and to methods for the diagnosis, prognosis, stratification, and monitoring of diseases associated with free heme- mediated pathological damage.

Description

TARGETING HEME FOR THE TREATMENT OF IMMUNE MEDIATED
INFLAMMATORY DISEASES RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application Serial No. 61/387,395 filed September 28, 2010. The entire contents of the above -referenced application are incorporated by reference herein. FIELD OF THE APPLICATION
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. BACKGROUND OF THE INVENTION
Heme (iron protoporphyrin IX) is a prosthetic group that consists of an iron atom contained in the center of a porphyrin ring. Heme is a component of a number of hemoproteins that are essential to support aerobic life. One of the largest pools of hemoproteins is the hemoglobin (Hb) contained inside red blood cells (RBC). Hb is a tetrameric protein that accounts for 97% of the total RBC dry content. When confined inside RBC, Hb tetramers are maintained in a reduced (Fe++) state. However, if released from RBC, Hb dissociates into dimers, which react avidly with reactive oxygen/nitrogen species (ROS/RNS). This results in Hb oxidation, from ferrous (Fe++) to ferric (Fe+++) Hb, that releases its heme prosthetic groups.
"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. Under oxidative stress, however, some hemoproteins such as Hb can release their prosthetic heme groups, producing free heme that can catalyze the production of free radicals in an unfettered manner. Under oxidative stress, cells can avoid the pro-oxidant effects of free heme by rapidly inducing the expression of the heme oxygenase- 1 (HO-1) isoenzyme, which increases the rate of free heme catabolism, preventing it from inducing programmed cell death in response to proinflammatory agonists such as Tumor Necrosis Factor (TNF), Fas, peroxinitrate of hydrogen peroxide, among others (Seixas E, Gozzelino R, Chora A, Ferreira A, Silva G, et al. Proc. Natl. Acad. Sci. USA. 2009, 106: 15837-42).
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,
Baronand RM, Perrella MA., J. Clin.Invest. 2008. 118, 239-247), experimental cerebral malaria (Pamplona, A. et al, Nat. Med. 2007. 13, 703-710), non cerebral forms of severe malaria (Seixas E, Gozzelino R, Chora A, Ferreira A, Silva G, et al. Proc. Natl. Acad. Sci. USA. 2009.106:15837-42), hemolytic diseases such as caused by sickle hemoglobin, ischemia-reperfusion injury (Fujita, T. Toda K, Karimova A, Yan S, Naka Y, Yet S, Pinsky DJ,. Nat.Med. 2001. 7, 598-604), allograft rejection (Yamashita K, Ollinger R, McDaid J, Sakahama H, Wang H, Tyagi S, Csizmadia E, Smith NR, Soares MP, Bach FH., FASEB J. 2006. 20, 776-778), neuroinflammation (Chora AA, Fontoura P, Cunha A, Pais TF, Cardoso S, Ho PP, Lee LY, Sobel RA, Steinman L, Soares MP, J. Clin. Invest. 2007. 117, 438-447), atherosclerosis (Shaw-Fang et al), and pathological outcomes of pregnancy (Zenclussen ML, Casalis PA, El-Mousleh T, Rebelo S, Langwisch S, Linzke N, Volk HD, Fest S, Soares MP, Zenclussen AC. J Pathol. 2011 Oct;225(2):293-304. doi: 10.1002/path.2946. Epub 2011 Jul 8). Pharmacological induction of HO-1 or
administration of the end products of heme catabolism (Carbon monoxide (CO), biliverdin, bilirubin) can exert therapeutic effects on some of these diseases, in mice (Soares MP, Bach FH., Trends Mol. Med. 2009. 15:50-58).
The molecular mechanisms that mediate the protective effects of HO-1, and ways to exploit them for therapeutic purposes, have been discussed in the literature (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). For example, it has been shown that 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). The same is true for severe sepsis as well as pathological outcomes of pregnancy (Zenclussen ML, Casalis PA, El-Mousleh T, Rebelo S, Langwisch S, Linzke N, Volk HD, Fest S, Soares MP, Zenclussen AC. J Pathol. 2011 Oct;225(2):293-304. doi: 10.1002/path.2946. Epub 2011 Jul 8). It has also been shown that induction of HO-1 expression in response to stress caused by microbial infection suppresses the development of severe sepsis in mice (Chung SW, Liu X, Macias AA, Baron and, RM, Perrella MA., J. Clin. Invest., 2008, 118, 239-247).
The potential clinical applications of HO-1 induction or administration of the end products of heme catabolism have been extensively discussed. However, these approaches have several limitations. There are well established human polymorphisms in the HMOXl promoter, which regulates the level of HO-1 expression in response to many stimuli; individuals with certain polymorphisms may mount a poor HO-1 response to HO-1 activating agents (Soares MP, Bach FH, Trends in Molecular Medicine, 2009, 15 (2): 50-8, 60). It is also not clear whether HO-1 could be induced to a level at which there is an optimal, or sufficient, amount of CO and biliverdin or bilirubin produced at the site of pathology. 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. In addition, 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). Furthermore, HO-1 responds to oxidative stress, and as such, inducers of HO-1 are molecules that cause undesirable sub-lethal levels of cell injury.
Direct administration of CO has been described in the art (US Patent 7,238,469) and explored clinically, but CO is a highly toxic molecule that naturally combines with hemoglobin with an affinity that is 200 times higher to that of oxygen and renders it ineffective for delivering oxygen to bodily tissues, and thus direct administration of CO could be toxic. Furthermore, there is currently no practical approach for administering biliverdin or bilirubin in amounts that would be needed if they were to be used broadly (Ollinger R, Yamashita K, Bilban M, Erat A, Kogler P, Thomas M, Csizmadia E, Usheva A, Margreiter R, Bach FH., Cell Cycle. 2007, 6(l):39-43). In addition, bilirubin and biliverdin are non-specific antioxidants, and in excessive amounts, can cause pathologies such as newborn jaundice. Some of the diseases, which have been targeted
pharmacologically by induction of HO-1 or administration of end products of heme catabolism constitute large unmet needs. Severe sepsis, for example, is a disease with limited treatment options that kills more than half a million individuals per year, in the United States alone. N-acetylcysteine (NAC), 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.
It is therefore an object of the present invention to provide treatments that limit the deleterious effects that free heme can have on tissues while bypassing the limitations of known therapeutic approaches. It is also an object of the present invention to provide methods for the diagnosis, prognosis, and monitoring of free-heme associated pathologies.
SUMMARY OF THE INVENTION
Methods to reduce the deleterious effects of free heme for the treatment of immune mediated inflammatory diseases, including infectious pathologies, have been developed. It has been discovered that the accumulation of free heme in the circulation of humans is strongly associated with the development of severe forms of malaria and sepsis, and that plasma concentration of heme-binding hemoproteins are reduced in human patients that succumb vs. those that survive severe inflammatory conditions mediated by free heme, such as severe sepsis and malaria. It has also been discovered that administration of heme- binding hemoproteins can increase tolerance (ameliorate health without targeting the pathogen) against infection by preventing inflammatory tissue damage, and thus increase survival of an infected host. Therefore, the present invention discloses that targeting free heme is of therapeutic use in the prevention of a range of immune mediated inflammatory diseases, including infectious pathologies. More recently targeting heme has also been shown to provide protection against tuberculosis.
In one aspect, the method of treatment involves administration of 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. Preferred 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. The agents can be isolated from samples of human or animal blood, or recombinantly or synthetically produced. Alternatively, the pharmaceutical composition may comprise an active agent fragment or variant of HPX as well as heme- antibodies or heme-binding fragments thereof.
In another aspect, the method of treatment involves administration of a
pharmaceutical 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. Alternatively, the pharmaceutical composition may comprise an active agent fragment or variant of HPT that retains its Hb-neutralizing activity.
In one aspect, 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. In some instances, this protective effect may be associated with modulation of pathogen load. For example, 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
administering 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, H202, ONOO-, and heat shock proteins, either alone or in combination with administration of a heme-binding protein, is also provided.
Methods for the diagnosis, prognosis, stratification, and monitoring of diseases associated with free heme-mediated pathological damage are also provided. In one aspect, blood taken from an individual with a blood-borne infection (or suspected of 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. Optionally, concentration of cell-free Hb, total plasma heme, or free heme may also be measured. In another aspect, said measurements can provide a prognosis for the outcome of a disease. In another aspect, said measurements can be used to stratify patients according to their likelihood of responding positively to an intervention. In yet another aspect, said measurements are used to monitor the response of a patient to an approved or
experimental therapy. In another aspect, 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.
BRIEF DESCRIPTION OF THE DRAWINGS
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). (B) Survival of Hmoxl+/+ (n=15), Hmoxl+/- (n=12), Hmoxl-/- (n=10), SCID.Hmoxl+/+ (n=5), and SCID.Hmoxl-/- (n=5) BALB/c mice after low-grade CLP. (C) Serological markers of organ injury in Hmoxl+/+ (n=15 to 17), Hmoxl+/- (n=10 to 13), and Hmoxl-/- (n=6) BALB/c mice, 24 h after low-grade CLP. Data are shown as mean ± SD. Dashed lines indicate basal plasma concentrations in naive wild-type BALB/c mice. 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:
Myocardium; PV: Portal vein; G: glomerulus (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. (F) Survival of Hmoxl+/+ (n=7), Hmoxl+/- (n=6), and Hmoxl-/- (n=5) BALB/c mice after administration (i.p.) of heat-killed bacteria (Bact.). *P<0.05;
**P<0.01; ***P<0.001; n: non-significant.
FIG's. 2A, 2B, 2C, 2D, 2E, 2F, and 2G collectively show that HO-1 prevents heme-driven severe sepsis. (A) Hemoglobin and haptoglobin plasma concentrations in Hmoxl+/+ (n=10), Hmoxl+/- (n=l 1), and Hmoxl-/- (n=6) BALB/c mice, 12 h after low- grade CLP. (B) Free heme and HPX plasma concentrations in Hmoxl +/+ (n=10),
Hmoxl +/- (n=l 1), and Hmoxl-/- (n=6) BALB/c mice, 12 h after low-grade CLP. (C) Survival of Hmoxl+/+ BALB/c mice after lowgrade CLP. When indicated, mice received vehicle (n=6), protoporphyrin IX (NaPPIX; n=8), or heme (FePPIX; n=13).
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. (D)
Measurement of serum AST, BUN, and CPK. Mice were treated as in (C), and serum biochemistry was analyzed 24 h after low-grade CLP. Data are shown as mean ± SD (8 to 9 mice per group). IU: international units. (E) Representative examples of H&E staining (400x magnifications) of tissue samples taken 24 h after low-grade CLP from mice that received heme described as in (C). Arrows indicate red blood cells (RBC). PV: Portal vein; G: glomerulus. Samples are representative results of 3 mice in each group. (F) Bacterial load in the peritoneum and blood of mice (n=12 per group) treated as described in (C), 12 to 24 h after CLP. Veh, vehicle; Heme, FePPIX. (G) Survival of BALB/c wild- type (Hmoxl+/+) mice after administration of a sublethal bolus (i.p.) of heat-killed bacteria (E. coli) followed by heme administration as in (C) (8 mice per group). Circles represent individual mice. Bars represent median values. *P<0.05; **P<0.01; ***P<0.001; ns, non-significant.
FIG's. 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H collectively show that free heme promotes the pathogenesis of severe sepsis. (A) Survival of wildtype (Hmoxl +/+)
BALB/c mice subjected to 'sham' laparotomy (n=3), low-grade (LG) (n=15) CLP, or high-grade (HG) (n=l 1) CLP. (B) Hemoglobin, (C) haptoglobin, (D) free heme, or (E) HPX plasma concentrations in na'ive (n=6 to 8) mice or 12 h after 'sham' laparotomy (n=10), low-grade (LG) CLP (n=6), or high-grade (HG) CLP (n=l 1 to 12). Circles represent individual mice. Bars represent median values. (F) Survival of wild-type
(Hmoxl+/+) BALB/c mice subjected to high-grade CLP. Mice received purified rabbit HPX (i.p., 50mg/kg; n=9), purified rabbit polyclonal IgG (i.p., 50mg/kg; n=16), or vehicle (i.p., PBS; n=7) at 2, 12, 24, and 36 h after 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).
Results shown are the mean ± SD (n=5 to 6 mice per group). (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 μΜ, lh) plus mouse recombinant TNF (5 ng/ml, for 16 h), anti-Fas antibody (0.5 μg/ml, for 4 h), H202 (125 μΜ, for 8 h), or the ONOO- donor 3- morpholino-sydnonimine (SIN-1) (100 μΜ, 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. (C) Percentage of cell death in primary hepatocytes treated as in (A). When indicated (+), hepatocytes were transduced with a LacZ- or Hmoxl -encoding Rec.Ad. Cell viability was determined as in (B). Data are representative of three independent experiments, using hepatocytes isolated from different mice. Bars indicate mean ± SD of n=5 to 6 independent samples per group. *P<0.05; **P<0.01; ***P<0.001.
FIG's. 5A, 5B, 5C, 5D, 5E, 5F, and 5G collectively show that free heme triggers the release of HMGB1 from hepatocytes. (A) HMGB1 (red) and DNA (blue) in mouse Hepal-6 hepatocytes exposed to vehicle (Untreated), free heme (40 μΜ; for lh), TNF (50 ng/ml, for 3 h), or heme (40 μΜ; for lh) plus TNF (50 ng/ml; for 3h). Magnifications are 400x. Images are representative of 3 independent experiments. One nucleus per field is outlined (dotted line). (B) HMGB1 was measured by western (immuno) blotting of proteins in the supernatants of primary mouse (BALB/c) hepatocytes that were exposed to heme (5 μΜ, 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. (D) 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.
Magnifications are 400x. Arrows indicate representative nuclei from which HMGB1 underwent full translocation from the nucleus to the cytoplasm and extracellular space. (F and G) HMGB1 and Ig heavy chain (IgH) were detected by western blot in the peritoneal cavity (F) or plasma (G) of Hmoxl+/+, Hmoxl+I-, or Hmoxl-I- mice, 12 h after low-grade CLP. Numbers indicate individual animals (n=3 per genotype).
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 μΜ), or HPX-heme complexes (5μΜ, 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. (B) Primary BALB/c hepatocytes were treated as in (A) exposed to heme (5 μΜ), HPX (5 μΜ), or heme-HPX complexes (5 μΜ, for 1 h) and, when indicated, to TNF (5 ng/ml; for 16 h). Cell viability was determined by crystal violet staining. Results shown are the mean ± SD from six samples in one out of two
independent experiments using hepatocytes pooled from 3 mice. (C) Primary human hepatocytes were exposed to heme (10 μΜ; 1 h) or heme-HPX complexes (10 μΜ; 1 h), and then to TNF (10 ng/ml; 8 h). Cell viability was determined by crystal violet staining. Results shown are the mean ± SD from six samples in one experiment representative of four independent experiments. (D) 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). (F) Survival time (see Material and Methods) of patients developing septic shock versus prediction of best fitted model. 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. (G) Expected mortality probability at day 28, plotted 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). (H) Box-plot representation of hemopexin (HPX) serum concentration at the time of septic shock diagnosis in a cohort of 52 patients, including survivors (n=34) and non-survivors (n=18). Data represents mean (IQR 25- 75%). *P<0.05.
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). Once the concentration of HPT and/or HPX in serum decreases bellow a certain threshold level, free heme accumulates in plasma and can sensitize cells in parenchymal tissues to undergo programmed cell death in response to a variety of proinflammatory agonists. This leads to the release of endogenous proinflammatory ligands from damaged tissues, e.g. HMGB1. Expression of the stress responsive enzyme 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. CFU in Hmoxl+/+(n=5),
Hmoxl+I- (n=4) and Hmoxl-I- (n=5) mice, 12h after low-grade CLP. Each circle represents an individual mouse and bars indicate median values. For each organ, a regression analysis was performed to compare data across genotypes, n: nonsignificant.
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 (Gpos; Enterococcus subspecies) or Gram negative (Gneg; 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. Cytokine concentration in (A, D, G) was measured in Hmoxl+/+ (n=13 at 6h and n=6 at 12h), Hmoxl+I- (n=6 at 6h and n=4 at 12h) and Hmoxl-I- (n=6 at 6h and n=3 at 12h) mice. Circles represent individual mice. Bars represent median values. Data in (B, C, E, F, H and I) is shown as mean ± SD from one representative experiment out of three. Regression analysis was used to describe data across genotypes in (A, B, D, E, G, H) by fitting a regression model for each time point or LPS concentration. Mann- Whitney test was used for pair wise comparisons in (C, F, I). *P<0.05; **P<0.01; ***P<0.001; n: nonsignificant.
FIG's 10A and 10B show that HO-1 regulates the production of NO in Mo (A) NO concentration in supernatants of peritoneal leukocytes of naive Hmoxl+/+ (n=3),
Hmoxl+I- (n=3) and Hmoxl-I- (n=3) mice stimulated in vitro with bacterial LPS plus IFN- % (48h). Results are shown as mean ± SD from one representative experiment using 3 animals. For each LPS concentration, a regression analysis was performed to compare data across genotypes. ***P<0.001. (B) Inducible nitric oxide synthase (iNOS), HO-1, and a- Tubulin were detected by western blot in peritoneal leukocytes from Hmoxl+/+,
Hmoxl+I- or Hmoxl-I- mice, 12h after low-grade CLP. Numbers indicate individual mice (n=3 per genotype). Notice that NOS2/iNOS expression, the major source of NO in LPS/IFN-γ activated Mo, was similar in Hmoxl-I- vs. Hmoxl+/+ peritoneal Mo, suggesting that HO-1 modulates NOS2/iNOS activity but not its level of expression.
FIG. 11 shows red blood cell morphology in mice subjected to CLP. Blood smears from (A) na'ive BALB/c mice, (B) BALB/c mice 48 h after low-grade CLP or (C) BALB/c mice 48 h after high-grade CLP (Giemsa staining, lOOOx). Note the appearance of schistocytes (1), acanthocytes (2) and stomatocytes (3) as well as "ghost" cells (4), indicative of disseminated intravascular coagulation and hemolysis.
FIG.'s 12A, 12B, 12C, 12D, 12E shows HO-1 modulates PMN
(Polymorphonuclear) cell activation in response to CLP. (A) Number of peritoneal PMN (GRlhlgh) cells in na'ive BALB/c mice (Oh) vs. BALB/c mice subjected to low-grade CLP (6h and 12h). Circles represent individual mice and bars are median values for each genotype. Hmoxl+/+ (n=7 at Oh, n=13 at 6h and n=5 at 12h), Hmoxl+I- (n=5 at Oh, n=6 at 6h and n=5 at 12h) and Hmoxl-I- (n=7 at Oh, n=6 at 6h and n=3 at 12h). Data for each time point, was compared by regression analysis across genotypes. (B) Expression of the NADPH oxidase gp91phox subunit, HO-1 and a-tubulin, determined by western blotting in whole cell extracts from peritoneal leukocytes from Hmoxl+/+(n=3), Hmoxl+I- (n=3) and Hmoxl-I- (n=3) mice subjected to low-grade CLP (12h). Numbers indicate individual mice. (C) Representative plots and histograms for free radical production in peritoneal infiltrating PMN cells (CD1 lb+Ly6Ghigh) and Mo (CD1 lb+Ly6Glow) from Hmoxl+l+ (n=4), Hmoxl+I- (n=4) or Hmoxl-I- (n=5), 12h after low-grade CLP. Free radicals were detected using the broad-spectrum probe CM-H2DCFDA. (D) In vitro conversion of purified hemoglobin (Fe[II]) into met-hemoglobin (Fe[III]), determined
spectrophotometrically after co-incubation of purified hemoglobin (Fe[II]) with peritoneal infiltrating leukocytes (3-5.106 cells) isolated from Hmoxl+/+, Hmoxl+I- or Hmoxl-I- mice 12h after low-grade CLP. Circles represent data obtained with cells isolated from individual mice and bars are median values for each genotype (n=9 per genotype). A regression model was used to compare data across genotypes. (E) Number of peritoneal infiltrating PMN (GRlhlgh) 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). Rather than a consequence of increased free radical production in a per-cell basis, this pro-oxidant effect was due to the relative increase in the proportion of peritoneal infiltrating PMN cells, i.e. -75% vs. 55%, in Hmoxl-I- vs. Hmoxl+/+ mice, respectively. ANOVA and t-tests were applied to compare different treatment conditions using Bonferroni correction for pair wise comparisons. *P<0.05; **P<0.01; ***P<0.001; n: non-significant.
FIG. 13 shows infiltrating leukocytes following CLP. Number of (A) peritoneal leukocytes, (B) CD1 lb+ (PMN and Mo), (C) NK cells, (D) T cells and (E) B cells were quantified by flow cytometry, before (Oh) and after (6h and 12h) low grade CLP in Hmoxl+/+ (n=7 at Oh, n=13 at 6h and n=5 at 12h), Hmoxl+I- (n=5 at Oh, n=6 at 6h and n=5 at 12h) and Hmoxl-I- (n=7 at Oh, n=6 at 6h and n=3 at 12h) mice. Circles represent individual animals. Bars represent median values in each genotype. A regression analysis was performed to describe CD1 lb+, NK, and B-cell data. T-cell samples did not follow a Normal distribution and was analyzed by Kruskal-Wallis and Mann- Whitney tests.
*P<0.05; **P<0.01; ***P<0.001; n: non-significant.
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.
FIG.'s 15A, 15B, 15C, 15D, 15E, 15F show that HbSAD prevents Experimental Cerebral Malaria (ECM) onset, a) Survival of P. berghei ANKA infected Hbwt (n=9l) and HbSAD (n=76) mice. Results shown are the summary of 10 independent experiments. Survival advantage for each experiment, P<0.05. Grey shading indicates expected time of ECM. b) Representative H&E stained microvessel in the (blood-brain barrier) BBB of infected HbM and HbSAD mice, at ECM onset in HbM mice (n=3/group). EC: endothelial cell; PVC: perivascular compartment; GL: glia limitans (dotted line); RBC: red blood cells; iRBC: infected RBC. Magnification: lOOx. c) Mean brain edema in naive vs. infected HbM and HbSAD mice ± standard deviation (n=4/group), at the time of ECM onset in HZr'mice. ns: not significant, d) Brain leukocyte sequestration in naive vs. infected HbM and HbSAD mice, at the time of ECM onset in Hbw'micQ. Dots represent single mice (n=4-14/group). Lines represent mean values, nd: not determined, e) Mean percentage of infected RBC in Hbwt (n=91) and HbSAD (n=76) mice ± standard deviation. Same mice as in (a). ) Mean number of infected RBC in HbM (n=7) and HbSAD (n=9) mice ± standard deviation at the time of ECM onset in HbM mice.
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 HbM and HbSAD mice ± standard deviation (n=4/group). b) Survival of P. berghei ANKA infected HbwtHmoxl+i+ (n=19), HbSAD Hmoxl +/+ (n=13), HbwtHmoxl+i- (n=15) and HbsADHmoxl+i- (n=15) mice. Results shown are the summary of 3 independent experiments. Survival advantage for each experiment, P<0.05. Grey shading indicates expected time of ECM. c) Representative H&E stained micro-vessel in the brain of infected HbSAD Hmoxl +/+ and HbSAD Hmoxl +/- mice, at ECM onset in HbsADHmoxl+/- mice (n=3/group). EC: endothelial cells; PVC:
perivascular compartment; GL: glia limitans (dotted line); RBC: red blood cells.
Magnification: lOOx. d) Mean brain edema in naive vs. infected HbSAD Hmoxl +/+ and HbSAD Hmoxl+i- mice, at ECM onset in HbSAD Hmoxl+i- mice ± standard deviation (n=3-4/group). e) CD8+ T cells and activated GrB+CD8+ T cells in brains of naive vs. infected HbSAD
Hmoxl +1+ and HbSAD Hmoxl +/- mice, at the time of ECM onset in HbSAD Hmoxl +/- mice. Dots represent single mice (n=4-5/group). Lines represent mean values, nd: not determined./) Mean percentage of infected RBC (parasitemia) in the same mice as in (b).
FIG.'s 17A, 17B, 17C show induction of HO-1 by HbSAD inhibits chemokine production in the brain. Quantification of mRNA encoding chemokines and chemokine receptors in the brains of naive (-) and P. berghei ANKA infected (+) mice carrying one (-) or two (+) functional Hmoxl alleles and expressing (+)HbSAD or not (-). Results are shown as mean fold induction over naive Hbwt Hmoxl+/+ mice ± standard deviation (n=4- 8/group), analyzed at ECM onset in Hbwt or HbSADHmoxl+i- control groups. Panel (a): genes inhibited by HbsAD under the control of HO-1. Panel (b): genes inhibited by HbsAD, presumably not under the control of HO-1. Panel (c): genes not regulated by HbsAD.
FIG.'s 18A, 18B, 18C show that the protective effect of sickle Hb against ECM is mediated by HO-1 expression in blood and hematopoietic cells, a) Survival of P. berghei ANKA infected chimeric mice resulting from the adoptive transfer of bone marrow from HbSADHmoxl+i+ mice into HbwtHmoxl+i+ recipients (n=6); from HbwtHmoxl+i+ mice into HbSADHmoxl+/+ recipients (n=5); from HbsADHmoxl+/+ mice into HbwtHmoxl+i- recipients (n=8) and from HbSADHmoxl+i- into HbwtHmoxl+i+ recipients (n=7). Recipients were lethally irradiated before the adoptive transfer. Grey shading indicates expected time of ECM. Results shown were obtained in four independent experiments, b) Mean brain edema ± standard deviation (n=3/group) in chimeric mice, produced as in (a), c) Mean percentage of infected RBC in chimeric mice ± standard deviation, same mice as in (a).
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+/-, HbSADNrf2+/+ and HbSADNrf2+/- mice ± standard deviation (n=6-8/group). b) Survival of P. berghei ANKA-infected HbwtNrf2+/+ (n=6), HbwtNrf2+/- (n=13), HbSADNrf2+/+ (n=10) and HbSADNrf2+/- (n=14) mice. Results shown are the summary of 3 independent experiments. Grey shading indicates expected time of ECM. c) Brain edema was measured by Evans blue (EB) accumulation in brains of infected HbwtNrf2+/+, HbwtNrf2+/-, HbSADNrf2+/+ and HbSADNrf2+/- mice, at the time of ECM onset. Mean ± standard deviation (n=4- 5/group). d) Mean percentage of infected RBC (parasitemia) ± standard deviation, same mice as in (b).
FIG.'s 20A, 20B, 20C, 20D, 20E, 20F, 20G, 20H, 201 show that H^ inhibits free heme accumulation via the production of CO. a) Mean plasma free heme concentration in na'ive vs. P. berghei ANKA infected Hbwt and HbsAD mice at ECM onset in Hbwt mice ± standard deviation (n=4-l 5/group). b) Survival of infected Hbwt mice receiving vehicle (n=10) or heme (35-40 mg/kg, every 48h, day 2 pre-infection to 4 post-infection)(n=8). Results shown were obtained in two independent experiments, with similar results, c) Survival of infected HbsAD mice receiving vehicle (n=8) or heme (20 mg/kg, every 12h, day 4-7 post-infection)(n=8). Results shown were obtained in two independent
experiments, with similar results, d) Mean brain edema in HbSAD mice treated as in (c) at ECM onset in heme-treated HbSAD raicQ ± standard deviation (n=4/group). e) Survival of infected HbSADHmoxl+/- mice exposed to air (n=8) or CO (250 ppm, days 4-7 post infection)(n=12). Results shown were obtained in three independent experiments, with similar results./) Mean brain edema in HbSADHmoxl+i- mice treated as in (e), at ECM onset in air-treated mice ± standard deviation (n=3-4/group). g) Mean free heme in plasma of HbSADHmoxl+i -mice treated as in (e) ± standard deviation (n=4-6/group). h) Survival of infected HbsADHmoxl+i- mice exposed to CO (250ppm; days 4-7 post-infection) and receiving vehicle (n=8) or heme (20 mg/kg, every 12h, days 4-7 post-infection)(n=8). Results shown were obtained in two independent experiments, with similar results, i) Mean brain edema in HbSADHmoxl+i- mice treated as in (h), at ECM onset in heme-treated mice ± standard deviation (n=3-4/group). Grey shading in (b, c, e and h) indicates expected time of ECM.
FIG.'s 21 A, 21B, 21C show that Hb^ expressing a wild type ?-chain of human Hb are not protected against ECM. a) Survival of P. berghei ANKA-infected HbA/a (n=15) and littermate control Hba/a (n=19) mice. Results shown are the summary of 3 independent experiments. Survival advantage for each experiment, P<0.05. Grey shading indicates expected time of ECM. b) Brain edema was measured by Evans blue (EB) accumulation in brains of naive vs. infected HbA/a and Hba/a mice ± standard deviation (n=3-4/group), at the time of ECM onset in HbA/a mice, c) Mean percentage of infected RBC in HbA/a and littermate control Hba/a mice ± standard deviation, same mice as in (a). Notice that the HbA/a and Hba/a mice used in these experiments are in a mixed C57BL/6.SV129 genetic background, produced by breeding HbA/a mice. Of importance, expression of a non- mutated ?-chain of human Hb in the HbA/a mice does not protect against ECM, as compared to littermate control Hba/a mice. This supports the notion that protection of HbSAD mice against ECM is afforded by the mutations of ?-chain of human Hb and not by the ?-chain of human Hb itself.
FIG.'s 22A, 22B, 22C show modulation of HO-1 expression in different experimental settings. Mean ratio of Hmoxl vs. hypoxanthine-guanine
phosphoribosyltransferase (Hprt) mRNA molecules in a) different organs of naive Hbwt and HbSAD mice ± standard deviation (n=4/group), b) bone marrow and peripheral blood leukocytes of naive HbA/a and Hba/a mice ± standard deviation (n=4) and c) bone marrow and peripheral blood leukocytes of naive HbSADHmoxl+/+ and HbSADHmoxl+/- mice ± standard deviation (n=4). Notice in (b) the lack of up-regulation of Hmoxl mRNA expression in HbA/a vs. Hba/a mice. Notice in (c) the significant decrease in the levels of Hmoxl mRNA expression in HbSAD mice with only one (Hmoxl 1 ) vs. two (Hmoxl 1 ) functional Hmoxl alleles. FIG.'s 23A, 23B show pharmacologic inhibition of HO activity negates the protective effect of sickle Hb against ECM. a) Survival of P. berghei ANKA-infected HbSAD mice receiving vehicle (n=8) or the HO enzymatic inhibitor ZnPPIX (5
mg/kg/every 12 hours, from day 2 pre -infection to day 7 post-infection)(n=8).
Vehicle-treated P. berghei ANKA-infected Hbwt mice were used as controls (n=8). Grey shading indicates expected time of ECM. Results shown were pooled from 2 independent experiments with similar results, b) Mean percentage of infected RBC (parasitemia) in infected HbSAD mice receiving vehicle (n=8) or ZnPPIX (n=8) and in vehicle-treated Hbwt mice (n=8) ± standard deviation.
FIG.'s 24A, 24B, 24C, 24D show that HbSAD allele inhibits neuroinflammation.
Quantification of mRNA encoding (a) cytokines, (b) molecules expressed by leukocyte, (c) co-stimulatory molecules expressed by antigen presenting cells and (d) diverse set of molecules, in the brains of naive (-) vs. berghei ANKA-infected infected (+) mice carrying one (-) or two (+) functional Hmoxl alleles and expressing (+) HbSAD or not (-). Results are shown as mean ± standard deviation (n=4-8/group), analyzed at the time of ECM onset. Notice that inhibition of pro-inflammatory gene expression in P. berghei ANKA-infected HbSAD vs. Hbwt mice is similar whether the Hb mice have one or two functional Hmoxl alleles. This suggests that the HbSAD allele might exert protective effects independently of HO-1.
FIG. 25 shows that the HbSAD allele inhibits neuroinflammation (continued)
Quantification of mRNA encoding a diverse set of molecules, in the brains of naive (-) vs. berghei ANKA-infected infected (+) mice carrying one (-) or two (+) functional Hmoxl alleles and expressing (+) HbSAD or not (-). Results are shown as mean ± standard deviation (n=4-8/group), analyzed at the time of ECM onset. Notice that inhibition of pro-inflammatory gene expression in P. berghei ANKA-infected HbSAD vs. Hbwt mice is similar whether the HbSAD mice have one or two functional Hmoxl alleles, suggesting that the HbSAD allele might exert protective effects independently of HO-1.
FIG.'s 26A, 26B, 26C show that the protective effect of sickle Hb against ECM is mediated by the expression of HO-1 in hematopoietic cells, a) Survival of P. berghei ANKA-infected chimeric HbSAD mice resulting from the adoptive transfer of bone marrows from HbSADHmoxl+/+ mice into HbSADHmoxl+/+ recipients (n=6); from
HbSADHmoxl+/+ mice into HbSADHmoxl+/~ recipients (n=7) and from HbSADHmoxl+/~ mice into HbSADHmoxl+/+ recipients (n=8). Recipients were lethally irradiated before the adoptive transfer. Grey shading indicates expected time of ECM. Results shown were pooled from 5 independent experiments, b) Brain edema was measured by Evans blue (EB) accumulation in brains of chimeric mice, produced as in (a). Results are shown as mean ± standard deviation (n=3/group). c) Mean percentage of infected RBC in chimeric mice, produced as in (a). Results are shown as mean ± standard deviation. Same mice as in (a).
FIG.'s 27A, 27B, 27C, 27D show that Parasite load is not modulated by heme or CO a) Mean percentage of P. berghei ANKA-infected RBC (parasitemia) in infected Hbwt mice receiving vehicle (n=10) or heme (50 mg/kg, every second day; from day 2 pre-infection to day 7 post-infection)(n=8) ± standard deviation, b) Mean percentage of infected RBC (parasitemia) in HbSAD mice receiving vehicle (n=8) or heme (days 4-7 post-infection, 30 mg/kg, every 12 h)(n=8) ± standard deviation, c) Mean percentage of P. berghei ANKA-infected RBC (parasitemia) in infected HbSADHmoxl+/~ mice exposed to air (n=8) or CO (250 ppm, days 4-7 post-infection)(n=12) ± standard deviation, d) Mean percentage of P. berghei ANKA-infected RBC (parasitemia) in infected
HbSADHmoxl+/~ mice exposed to CO (250 ppm, days 4-7 post-infection) and receiving vehicle or heme (days 4-7 post-infection, 25 mg/kg, every 12 h)(n=8) ± standard deviation.
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, and 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. A) Induction of programmed cell death in hepatocytes exposed to heme and TNF. B) Suppression of heme cytotoxicity with recombinant viruses encoding shRNAs targeting bcrp/Abcg2. C) Suppression of heme cytotoxicity with recombinant viruses encoding shRNAs targeting Abcb6.
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
A description of preferred embodiments of the invention follows. It will be understood that the particular embodiments of the invention are shown by way of illustration and not as limitations of the invention. At the outset, the invention is described in its broadest overall aspects, with a more detailed description following. The features and other details of the compositions and methods of the invention will be further pointed out in the claims. I. Definitions:
The term "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. The term "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.
The terms "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.
The term "heme scavenger" is used to refer to a molecule that binds and/or inhibits, reduces or neutralizes, heme and its cytotoxic effects.
The term "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.
The term "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.
The terms "hemopexin" (HPX) and "haptoglobin" (HPT) are used to refer to the proteins which, in mammals, including but not limited to humans, are encoded by the HPX and HP genes, respectively, which bind heme and hemoglobin, respectively.
The terms "hemoprotein'Or "heme -binding protein" are used to refer to
metalloproteins containing a heme prosthetic group bound to the protein. The term "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).
The term "High-mobility group protein Bl" (HMGB1) is used to refer to a protein that, in mammals, such as humans, is encoded by the HMGB1 gene.
The term "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.
The term "therapeutically 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.
The term "Cecal Ligation and Puncture" (CLP) refers to a standard animal model for the study of sepsis.
II. Free heme as a therapeutic target in the treatment of inflammatory and infectious pathologies
It has been discovered that the accumulation of free heme in the circulation of humans is strongly associated with the development of severe forms of malaria, and that plasma concentration of heme-binding hemoproteins is reduced in human patients that succumb vs. those that survive severe inflammatory conditions mediated by free heme, such as severe sepsis and malaria. It has also been discovered that administration of heme- binding proteins can increase tolerance against infection and inflammatory tissue damage, and promote host survival. Together, these findings strongly indicate that targeting free heme is of therapeutic use in the prevention of a range of infectious and inflammatory conditions characterized by hemolysis (release of Hb from red blood cells) and consequent production of extracellular hemoglobin associated with release of free heme. While previous attempts to prevent or ameliorate the deleterious effects of hemolysis (for example, production of free radicals and induction of programmed cell death) focused on enhancing catabolism of free heme by the enzyme HO-1, or relied on administration of the end products of heme catabolism (Carbon monoxide (CO), biliverdin, bilirubin), the present invention discloses neutralization of free heme by heme scavengers as a therapeutic alternative that addresses several limitations of the previous approaches. In particular, 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).
It has also been discovered that 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, H202, 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. This newly discovered phenomena strongly indicates that approaches to scavenge heme that simply bind heme but do not suppress its pro-oxidant activity (for example, proteins or antibodies that bind the protoporphyrin ring of heme but do not directly shelter the Fe atom in heme) will be ineffective at limiting the deleterious effects of heme. To address this limitation, the invention proposes heme scavengers that directly bind the Fe atom in heme, thus inhibiting its pro-oxidant properties.
Furthermore, 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.
III. Therapeutic Heme Scavengers
A. Human-derived heme -binding proteins
In a preferred embodiment, 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. In particular, 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).
Alternatively, 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).
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).
B. Non-human-derived heme-binding proteins
The pharmaceutical compositions of the invention may include HPX, Albumin, alpha 1 -microglobulin, or peroxiredoxin 1 proteins from non-human animals.
In one embodiment 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.
In one embodiment, the pharmaceutical composition includes Histidine Rich Protein-2 (HRP-2), or a fragment thereof. 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.
In another embodiment, 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,
"Hematophagy and heme toxicity: what can we learn from natural-born vampires?" Chapter III, In: Heme Oxygenase, Editors Otterbeing LE et al. 2005. pp 55-76, Nova Science Publishers).
In another embodiment, the pharmaceutical composition includes Heme
Lipoprotein (HeLp) derived from the organism Boophilus microplus (a genus of ticks), which has been characterized as being able to bind several molecules of heme per molecule of protein (Sorgine MH, Logullo C, Zingali RB, Paiva-Silva GO, Juliano L, Oliveira PL., J Biol Chem., 275(37):28659-65).
In another embodiment, the pharmaceutical composition includes Heme-Binding
Lipoprotein (HbpA) 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).
In another embodiment, 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). Alternatively, the heme-binding proteins may be naturally immunogenic, or modified to increase their immunogenicity to facilitate clearance of protein-heme complexes in circulation.
Heme-specific antibodies, HRP-2, Rhodnius Heme Binding Protein, Heme
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. C. Heme -binding lipids and synthetic heme chelators
In another embodiment, the pharmaceutical composition includes Low-Density Lipoprotein (LDL) and High-Density Lipoprotein (HDL). The ability to bind heme of both these lipids has been characterized in the art. Alternatively, 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).
In a preferred embodiment, 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
Biophysical Research Communications, 2003, 308, 736-743).
In another embodiment, 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). Preferably, the heme- binding aptamer binds directly the Fe atom and inhibits the pro-oxidant effects of heme.
In another embodiment, the pharmaceutical composition further comprises the heme chelator sodium cyanide.
In another embodiment, the pharmaceutical composition comprises a synthetic histidine-rich protein.
Preferably, the heme-binding lipids and synthetic heme chelators of the invention are linked to molecules that preferentially deliver them to macrophages. Several systems for specific delivery of molecules to macrophages have been described in the art (see for example 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).
In a preferred embodiment, 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. D. Hemoglobin-binding proteins
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
Isolation of Heme-Binding Proteins from Human or Animal Blood
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. 20090281282; Connell G.E., Smithies O., Biochem J, 1958, vol 72, pp 115-121), or monoclonal antibody affinity chromatography (Tseng C.F., Huang H.Y., Yang Y.T., Mao S.J., Protein Expr Purif., 2004;33(2), pp 265-273).
Alternatively, 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. In particular, 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.
Recombinant production of Albumin has been described in the art (see for example US Patent Application Serial No. 11/721813 and US Patent No. 5,962,649). Similarly, recombinant production of alpha- 1 microglobulin has been described (Kwasek A, Osmark P, Allhorn M, Lindqvist A, Akerstrom B, Wasylewski Z. Protein Expr Purif, 2007, May; 53(l): 145-52).
Recombinant production of HRP-2 in E. coli strain BL21(DE3) has been described in the art (Pandey AV, Babbarwal VK, Okoyeh JN, Joshi RM, Puri SK, Singh, Chauhan VS. Biochemical and Biophysical Research Communications, 2003, 308, 736-743). The process involves transformation of a plasmid containing the gene encoding P. falciparum HRP-2 into Escherichia coli strain BL21(DE3), and growth at 37 C followed by purification of HRP- 2 by metal chelate chromatography.
Recombinant production of Heme-Binding Lipoprotein (HbpA) derived from the organism Haemophilus influenza has been described in the art (Morton DJ, Madore LL, Smith A, VanWagoner TM, Seale TW, Whitby PW, Stall TL, FEMS Microbiology Letters, 2005. 253, 193-199).
F. Engineered variants of heme scavengers
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. For instance, 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.
In a preferred embodiment, 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.
In some cases, wherein 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.
In other embodiments, 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.
In another embodiment, the pharmaceutical composition may comprise fragments from hemopexin and haptoglobin, that retain their free-heme, and Hb-neutralizing activities, respectively. In another embodiment, the pharmaceutical composition may comprise an
Albumin variant that has been modified to increase its affinity for free heme.
IV. Pharmaceutical Compositions
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. In a preferred embodiment, 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. The term "acceptable" carrier is used to refer to carriers compatible with the heme scavengers, and not deleterious to the recipients. Typically, 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.
The pharmaceutical compositions used for therapeutic administration can be sterilized by filtration through sterile filtration membranes (for example, 0.2 micron membranes).
A. Route of administration
The pharmaceutical compositions can be administered orally, rectally, parenterally, intra vaginally, mtrapeiitoneally, topically, bucaliy, or as an oral or nasal spray.
Preferably, parenteral administration is used. Formulations suitable for parenteral administration include aqueous (for example, water or saline) and non-aqueous sterile suspensions and injection solutions. B. Carriers
Preferably the carrier is a parenteral earner, more preferably a solution that is isotonic with the blood of the recipient. Examples of such 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; 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.
In a preferred embodiment, an HPX-, HPT-, or Albumin-containing
pharmaceutical 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.
C. Dosage
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. As an example of 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 20μΜ under homeostasis (the upper limit corresponding to subjects with sickle ceil conditions), and in concentrations of up to 50μΜ in patients affected by severe sepsis or severe malaria. Assuming typical blood volumes for a human of 5 L, the total
pharmaceutically effective amount of hemopexin dosed parenterally to scavenge 50μΜ 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.
Similarly, 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 30μ 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. If given continuously, 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).
D, Del very Systems
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. Examples of 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. 3,773,919, EP 0058481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al, Biopolymers, 22:547-556 (1983)), poly (2- hydroxyethyl metfiacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988), or liposomes (see Langer, Science, 249: 1527-1533 (1990).
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)).
E. Modulators of Heme Transporters
It has been discovered that modulation of transporter molecules involved in regulation of the intracellular heme content can suppress the cytotoxic effects of free heme. Among these cellular heme transporters are 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).
In particular, it has been shown that 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).
The pharmaceutical compositions of the invention that modulate heme transporters may comprise a polynucleotide, a peptide, a protein, or a small molecule. For example, the heme transporters HCPl, HRG-1, Abcb6, and ANT, can be inhibited with the antibodies ab25134, ab6780, ab47837, ab54418, respectively.
F. Combination Therapies
The pharmaceutical compositions may further comprise 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.
In one embodime t, the pharmaceutical compositions further comprise
therapeutical iy effective amounts of protein HO- lor a polynucleotide encoding HO-.1.
In another embodiment, 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. Without wishing to be bound by theory, these compounds may regulate HO-1.
In another embodiment, 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. In another embodiment, the pharmaceutical compositions further comprise, or are administered in conjunction with, products of the catabolism of free heme such as CO, biliverdin, or bilirubin. In a preferred embodiment 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. In another embodiment, 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
alternatively, modulators of HPX, HPT, and/or Albumin production can be administered, as well as modulators of biliverdin reductase.
In another embodiment, 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). In a preferred embodiment, 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.
In another embodiment, the pharmaceutical compositions further comprise at least one additio al compound selected from a labile Fe chelator and a molecule that directly degrades heme. Examples of Fe chelators include chemicals such as Desferal (also kno wn as desferroxiamine), deferiprone, Emylenediammetetraaeetie acid (EDTA) and
Diethyienetriaminepentaacetic Acid (DETAPAC), and salicyialdehyde isonicotinoyl.
hydrazone (S IH). Examples of molecules that degrade heme involve reduced glutathione (OSH), xanthine oxidase, and ADPH-cytochrome P-450 reductase. Without wishing to be bound by theor/, 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
pharmaceutical 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).
In another embodiment, the pharmaceutical compositions further comprise at least one antioxidant. Preferably, 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.
In another embodiment, the pharmaceutical compositions further comprise at least one 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. G. Administration of low levels of heme for prophylactic treatment of free-heme mediated conditions
It has been discovered that administration of low levels of free heme, despite the cytotoxicity of free heme, exerts a protective effect in animal models of malaria against a subsequent heme challenge (see FIG.'s 20, 27, and Example 15). In a preferred embodiment, 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.
H. Methods to screen for potent heme scavengers
Methods for the selection of potent and non-toxic heme scavengers are also provided.
In one embodiment, potent and non-toxic heme scavengers are selected by screening a library of compounds in an in vitro cellular assay. In a first step, and in order to establish a positive control, a single cell or a colony of cells in a well (for example, primary BALB/c mouse hepatocytes or human hepatocytes) is exposed to both heme and a pro-inflammatory agonist (for example TNF, lipopolysaccharide (LPS), a Fas crosslinker, hydrogen peroxide (H202), or peroxinitrite (ONOO-)) in a concentration sufficient to cause cell death. For example, 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 μg/ml, for 4 h), H202 (125 uM, for 8 h), or the ONOO- donor 3- morpholino-sydnonimine (SIN-1) (100 μΜ, for 24 h). In a second step, 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. Alternatively, 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.
In another embodiment, potent and non-toxic heme scavengers are selected by screening compounds in an in vivo model of sepsis (for example, a BALB/c mouse). In a first experiment, and in order to establish a positive control, mice are subjected to Cecal ligation and puncture (CLP) and the percentage survival and time to death are measured. In a second experiment, 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)), are measured after a period of time, for example 3, 5, 7, 9, 11 , or 15 days after CLP.
In another embodiment, potent and non-toxic heme scavengers are selected by screening compounds in an in vivo model of induced liver injury. In a first experiment, and in order to establish a positive control, 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). For example, 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. In a second experiment, 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.
In another embodiment potent and non-toxic heme scavengers are selected by screening compounds in an in vivo model of microbial challenge. In a first experiment, and in order to establish a positive control, 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. In a second experiment, 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.
V. Diagnostic assays and kits
Methods of obtaining a diagnosis, prognosis, or for monitoring a free-heme mediated pathology have been developed based on the discovery that HPX blood concentrations are altered in patients affected by a free-heme mediated pathology. It has beeri discovered thai low HPX serum concentration is associated with organ dysfunction and fatal outcome, and that HPX serum concentration soon after events that trigger hemolysis, such as septic shock, is positively associated with patient survival time. That is, patients presenting lower HPX serum concentrations succumbed at earlier time points, as compared to patients with higher HPX serum concentrations.
In one embodiment, 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. In a preferred embodiment, 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. A. Samples to be analyzed
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.
Alternatively, samples can be obtained from other biofluids, such as urine, or tissue biopsies.
B. Methods of analysis
Measurement of blood proteins
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.
In a preferred embodiment, HP and BPT are detected and quantified using an immunological binding assay (see for example US Patent No's: 4,366,241;
4,376,1 10; 4,517,288 for a number of assays known in the art). Immunological binding assays utilize a capture agent to specifically bind to the anaiyte (for example HPX or HPT). Preferably 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. 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
Sambrook et al. (1989) Molecular Cloning— A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY) or a Western blot. Measurement of Heme
Total plasma heme (free heme in circulation plus heme conjugated to
hemoproteins) can be measured using methods known in the art (see Bal i j, Proc Nat Ac Sci 1993, vol 90, pp: 9285-9289). For example, the 3,3', 5,5' tetranK^thylben.zidme (TMB) peroxidase assay (BD Biosciences), at λ = 655 nm can be used. Purified hemoglobin can be used as standard for plasma hemoglobin and heme measurements. Plasma hemoglobin can be determined by spectroscopy at λ =577.
Known methods to measure heme are limited to measurement of total plasma heme, and do not provide means for measuring free heme (the fraction of total plasma heme that is not bound to hemoproteins) 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. Techniques well know in the art, such as 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. Statistical methods
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). Preferably , 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.,
VVilcoxon Mann- Whitney Test). Preferably 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
In certain embodiments, 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. Albumin, HPT, I lb. total plasma, heme, and/or tree heme. 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.
VI. Diseases and conditions to be treated with heme-binding proteins
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, vancomycin-resistant enterococci, E. coli, Staphylococcus Aureus, Pseudomonas, Serratia spp., Proteus spp., Listeria spp.,
Bacillus cereiis, and Clostridium tetani, or hemolysin-releasing fungi such as Aspergillus fumigasus, Staeybotrys chartarum, Candida albicans, or Penicillinum ehysogenum;
administration of an antibody, such as acute hemolytic transfusion reactions or paroxysomal cold hemoglobinuria; or by exposure to a. chemical, such as lead poisoning, potassium dichromate poisoning, or arsenic poisoning.
Diseases associated with rhabdomyolysis, a condition associated with damaged skeletal muscle tissue and the release of the hemoprotein myoglobin can also be treated. 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
complications associated with transplantation.
The following examples are put forth so as to provide those of ordinary skil l in the art with a complete disclosure and description of how the methods and compositions claimed herein are performed, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.
Other Embodiments
All publications, patents, and patent applications mentioned in tills specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
While the invention has been described in co nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is i tended to cover a y variations, uses, or adaptations of the i ve tion following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
Other embodiments are within the claims.
EXAMPLES
EXAMPLE 1.
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). Similar results were obtained in BALB/c SCID mice lacking B and T lymphocytes (FIG. IB), demonstrating that the protective effect of HO-1 is not dependent on adaptive immunity. Mortality of heterozygous Hmoxl+/- mice was similar to Hmoxl+/+ mice (FIG. IB). The higher mortality of Hmoxl-/- versus Hmoxl+/+ mice was not attributable to the surgical procedure per se, since Hmoxl-/- mice did not succumb to sham
laparotomy, which mimics CLP without polymicrobial infection. Mortality of Hmoxl -I - mice after CLP was associated with the development of multiple end-stage organ failure, a hallmark of severe sepsis (2). Plasma concentrations of aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine phosphokinase (CPK), markers of liver, kidney, and muscle dysfunction, respectively, were significantly increased in infected Hmoxl -I - versus Hmoxl+/+ mice (FIG. 1C). Liver, kidney, and cardiac damage were confirmed by histological detection of centrolobular necrosis, tubular epithelial necrosis, and myocardial necrosis, respectively (FIG. ID). This demonstrates that induction of HO-1 expression in response to polymicrobial infection limits tissue damage and the development of severe sepsis. Exacerbated mortality of 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). This demonstrates that 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). Likewise, 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). Since 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. When exposed to LPS and interferon-γ (IFN- γ), 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.
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. We asked whether increased mortality of Hmoxl-I- mice subjected to polymicrobial infection was associated with increased hemolysis as well as with the accumulation of cell-free hemoglobin and/or free heme in plasma. When subjected to low- grade CLP, Hmoxl-I- mice, but not Hmoxl+/+ mice, accumulated extracellular hemoglobin (FIG. 2A) and free heme in plasma (FIG. 2B), while plasma concentrations of the hemoglobin-binding protein haptoglobin (FIG. 2A) and the heme -binding protein HPX were decreased (FIG. 2B). We then asked whether accumulation of free heme in plasma contributes to the pathogenesis of severe sepsis. Heme administration to wild type (Hmoxl+/+) mice subjected to low-grade CLP lead to severe sepsis (77% mortality) (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). These observations demonstrate that free heme can precipitate the onset of severe sepsis in mice subjected to an otherwise benign (non-lethal) polymicrobial infection. They also reveal that the kidney is particularly vulnerable to the damaging effects of free heme. The number of CFU in peritoneum and blood was similar in mice subjected to low-grade CLP and receiving or not heme thereafter (FIG. 2F). This demonstrates that the ability of free heme to precipitate severe sepsis in mice (FIG. 2C) was not associated with increased pathogen load (FIG. 2F), thus revealing that free heme compromises host tolerance against polymicrobial infection. This notion is strongly supported by the observation that administration of free heme to wild type (Hmoxl+/+) mice subjected to a sub-lethal dose of heat-killed bacteria lead to 100% mortality, as compared to 12.5% mortality in control mice receiving vehicle (FIG. 2G). We then asked whether the deleterious effect of free heme could be attributed to its previously described action on polymorphonuclear (PMN) cells. The number of peritoneal-infiltrating
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). This effect was attributed to the increased numbers of PMN cells in Hmoxl-/- versus Hmoxl+/+ mice and was associated with enhanced oxidative activity in peritoneal leukocytes from Hmoxl -I - mice relative to Hmoxl+/+ mice (FIG. 12C and 12D). While heme administration to naive Hmoxl+/+ mice can elicit peritoneal PMN cell infiltration (FIG. 12E), this effect was negligible when heme was administered to mice subjected to low-grade CLP (FIG. 12E). While this data suggests that hemedriven PMN cell activation does not play a major role in the pathogenesis of severe sepsis, we cannot exclude that other putative effects of free heme on PMN cells, (for example degranulation), might act in a detrimental manner to promote the pathogenesis of severe sepsis.
EXAMPLE 3.
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. 3C), confirming that hemolysis occurs in response to high-grade, but not low-grade infection. Similarly, the concentration of free heme in plasma increased (FIG. 3D) while HPX plasma concentration decreased (FIG. 3E), in mice subjected to high-grade relative to low- grade CLP. Given that mortality in response to polymicrobial infection is associated with high concentrations of free heme and low concentrations of HPX in plasma, we hypothesized that one might be able to prevent the onset of severe sepsis by restoring HPX plasma concentration, so that HPX is available to neutralize the rising amounts of free heme. Administration of purified HPX to wild-type (Hmoxl+/+) mice subjected to high- grade CLP reduced the mortality level to 22%, as compared to 86% and 69% in control mice that received PBS (the HPX vehicle) or IgG, respectively (FIG. 3F). The protective effect of HPX was associated with the return of liver, kidney, and cardiac function to homeostatic levels, as assessed by AST, BUN and CPK plasma concentrations, respectively (FIG. 3G) and as confirmed histologically (FIG. 3H). In contrast, control 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).
EXAMPLE 4.
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. 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). Primary hepatocytes did not undergo programmed cell death when exposed to TNF, Fas crosslinking, H202, or ONOO- (FIG. 4B), while programmed cell death was readily induced in cells treated with free heme first and then TNF, Fas crosslinking, H202, or ONOO- (FIG. 4B). These observations suggest that the redox activity of the heme-Fe atom underlies its cytotoxicity, presumably by catalyzing the production of free radicals through Fenton chemistry. Consistent with this hypothesis, the anti-oxidant Nacetyl- cysteine (NAC) protected hepatocytes from programmed cell death in the presence of free heme and either TNF, Fas ligand, H202, or ONOO- (FIG. 4B). These observations reveal that the pathological effects of free heme, namely its ability to synergize with other cytotoxic agonists to cause tissues damage, can be extended to a variety of agonists other than TNF, including some previously involved in pathogenesis of severe sepsis. Transduction of hepatocytes with a recombinant adenovirus (Rec.Ad.) that expresses HO-1 (FIG 14) was protective against programmed cell death in the presence of free heme and either TNF, Fas crosslinking, H202, or ONOO-, when compared with control hepatocytes transduced with a Rec.Ad that expresses LacZ (FIG. 4C). The cytoprotective effect of HO-1 was associated with the inhibition of free radical production (unpublished data), suggesting that HO-1 acts as an antioxidant to suppress the cytotoxic effects of free heme.
EXAMPLE 5.
Heme triggers HMGB1 release in vitro and in vivo: We reasoned that the cytotoxic effect of free heme might precipitate severe sepsis by eliciting tissue damage per se as well as by promoting the release of high mobility group box 1 (HMGB1), an endogenous pro-inflammatory ligand involved in the pathogenesis of severe sepsis and previously linked to HO-1. In untreated primary hepatocytes, HMGB1 expression was mainly restricted to the nucleus (FIG. 5A). However, when hepatocytes were exposed in vitro to free heme plus TNF, 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
overexpression 11 (FIG. 5D). These observations reveal that the oxidative effect of free heme promotes HMGB1 release from hepatocytes, an effect suppressed by HO-1. Next, we asked whether HO-1 would prevent HMGB1 release from damaged tissue in vivo, as suggested in previous studies. In Hmoxl-I- mice subjected to low-grade CLP there was translocation of HMGB1 into the cytoplasm, as assed in the liver and kidney (FIG. 5E), while translocation was less pronounced in wild-type (Hmoxl+/+) mice (FIG. 5E). This effect was associated with the systemic release of HMGB1 into the peritoneum and plasma of Hmoxl-I- relative to Hmoxl+/+ mice (FIG. 5, F and G). The relative amount of peritoneal or plasma IgG was unchanged in Hmoxl-I- versus Hmoxl+/+ mice (FIG. 5, F and G). EXAMPLE 6.
HPX neutralizes the cytotoxic effect of free heme: We next sought to determine whether HPX, which binds tightly to heme (Kd < 1 pM) in a manner that dampens its pro- oxidant activity, can also prevent heme -mediated tissue damage (FIG. 3F, G and H). 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). Moreover, 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
hepatocytes in vitro (FIG. 6D). Similarly, HPX-bound heme also failed to promote HMGB1 release from primary human hepatocytes, in response to TNF (FIG. 6E). The increased molecular weight of the cell-free HMGB1 released from primary human hepatocytes is most probably attributed to post-transcriptional HMGB1 modifications, (for example phosphorylation). Taken together, these observations show that HPX suppresses the cytotoxic effects of free heme in both mouse and human hepatocytes.
EXAMPLE 7.
Low 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
concentrations succumbed at earlier time points, as compared to patients with higher HPX serum concentrations (FIG. 6F). This observation allows to extrapolate the probability of survival/mortality as a function of HPX serum concentration (FIG. 6G), which is in keeping with the observation that HPX serum concentration within 48h of septic shock diagnosis was higher in patients that survived septic shock, as compared to non-survivors (FIG. 6H). Finally, there was an inverse correlation between HPX serum concentration and severity of organ dysfunction, as defined using the sequential organ failure assessment (SOFA) score (See Moreno R, Vincent JL, Matos R, Mendonca A, Cantraine F, Thijs, Takala J, C. Sprung C, Antonelli M, Bruining H,Willatts S. Intensive Care Med., 1999. 25, 686-696) (Table 2). Overall these observations support the notion that HPX serum concentration defines the extent of tissue damage (organ dysfunction) and hence the outcome of septic shock in humans. TABLE 1 :
Characteristics of septic shock patients. Severity of organ dysfunction was defined using the Sequential Organ Failure Assessment (SOFA) score. Ranges are indicated between brackets.
Figure imgf000047_0001
TABLE 2:
Correlations between HPX serum concentration and SOFA. The correlations are based on Spearman coefficient calculated between HPX and SOFA at the days indicated. P-values are given in parentheses.
Figure imgf000047_0002
EXAMPLE 8.
Experimental Methods Used in Examples 1 to 7
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 μg/ml streptomycin (Life Technologies). For cytokine measurements, cells (2.5xl04) were plated in flat-bottom 96-well microtiter plates (Techno Plastic Products AG)(100 μΐ, 2h, 37°C), non-adherent cells were removed and adherent cells, i.e. Mo, were activated with bacterial LPS (Sigma, E. coli serotype 0127:B8) for 6 or 24h. 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 (3xl05 cells/well) in RPMI, 3.3% FCS, 5% L929 supernatant. BMDM were incubated with live Gram-positive (Enterococcus subsp. isolated from mouse intestine) or Gram-negative (Escherichia coli DH5a) bacteria (8h), after which cell culture supernatant was collected and centrifuged (5 min, 1200 rpm, 4°C) to remove cells, and bacteria (5 min, 10.000 rpm, 4°C). Cell-free supernatants were stored at -80°C until used. Hepal-6 cells (C57L mouse hepatoma; ATCC) were seeded in DMDM (Invitrogen), 10% FCS, 2 U/ml penicillin, 20 U/ml streptomycin (Invitrogen). All cells were incubated at 37°C, 95%> humidity, 5% C02.
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 (ΙΟΟμιη), 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).
Heme sensitization assays: Hepatocytes were seeded and exposed to heme (5 μΜ, lh) in Hanks Balanced Salt Solution (HBSS; Invitrogen), without serum, to avoid potential heme-scavenging by serum proteins. Subsequently, 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 μg/ml, 4h; BD Biosciences), H202 (125 μΜ, 8h; Sigma), or 3- morpholinosydnonimine (SIN-1; 100 μΜ, 24h; Sigma). Cell viability was assessed by crystal violet assay, as described. Heme (iron-protoporphyrin; FePPIX; Frontier Scientific) was dissolved in sterile 0.2M NaOH at alkaline pH, and adjusted to pH 7.4 using sterile 0.2M HC1. Iron-free protoporphyrin (protoporphyrin di-sodium salt; NaPPIX; Frontier Scientific) was dissolved in sterile 0.2M HC1 at acid pH, and adjusted to pH 7.4 using sterile 0.2M NaOH. Aliquots were stored at -80°C until use.
Cytokines and nitric oxide measurements: 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: 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 (CFU): 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 TRYPTICASE™ Soy Agar II plates supplemented with 5% Sheep Blood (Becton Dickinson). CFU were counted after 24h incubation at 37°C.
cDNA synthesis and Light Cycler analysis: Total RNA was extracted using RNeasy" Protect Mini Kit (Qiagen) and reverse transcribed (SuperScriptll RNase H- reverse transcriptase; Invitrogen) using random hexamer primers (Invitrogen) as follows, 70°C/10 min, 37°C/50 min and 95°C/5 min (Robocycler Stratagene). The reaction was carried out using 1 μΐ of cDNA with 3 pmol of each primer, 2 mM MgC12 and IX
FastStart DNA SYBR Green I mix (Roche). Thermal cycler program comprised 1 cycle at 95°C/10 min, 45 cycles at 95°C/15 sec 58°C/5 sec, 72°C/16 sec, with transition rates of 20°C/s. PCR products were quantified by light cycler real-time quantitative PCR software (Roche). Cycle numbers in the log-linear phase were plotted against the logarithm of template DNA. External standardization was performed using full-length HO-1 cDNA. Hypoxanthine-guanine-phosphoribosyl transferase (HPRT) was used to normalize cDNA levels.
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 μΜ; 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-H2DCFDA; Molecular Probes). Immunofluorescence: Hepal-6 cells, seeded and treated (as described above) on glass cover slips (Paul Marienfeld GmbH & Co.), were fixed (4% paraformaldehyde, 30 min), permeabilized (0.1% Triton X-100, 20 min), blocked (PBS, 10%> goat serum, 20 min), and incubated overnight at 4°C with rabbit anti-human HMGBl antibody (Abeam; ab 18256; 0.5 μg/ml) or control rabbit IgG (Sigma), in PBS, 10%> goat serum. Alexa568-conjugated goat anti-rabbit IgG (Invitrogen; 5 μg/ml) was used as secondary antibody, and nuclear DNA was stained with Hoechst 33342 (10 μg/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).
Histology and Immunohistology: Tissue samples were processed and stained essentially as described. HMGBl was detected in paraffin embedded, formalin- fixed sections (5 μιη) 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.
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
spectrophotometric analysis (modular DP; Roche-Hitachi; Echevarne Laboratories, Barcelona, Spain). Plasma HPX and haptoglobin were determined by ELISA (Life Diagnostics). Plasma hemoglobin were determined by spectroscopy at λ=577. Total plasma heme was measured using the 3,3', 5,5' tetramethylbenzidine (TMB) peroxidase assay (BD Biosciences), at λ= 655 nm. Purified hemoglobin was used as standard for plasma hemoglobin and heme measurements. Blood smears were fixed in methanol and stained with Giemsa stain, and images were obtained and analyzed as described above. HPX Intact apo-HPX was isolated from rabbit serum as described. 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.
Western blotting Proteins were prepared and subjected to electrophoresis, essentially as described before. For HMGB1 detection in peritoneal fluid and in serum, samples were ultra-filtered with Centricon 100 columns (Millipore) and precipitated with trichloroacetic acid (TCA), washed in acetone (twice), dried, dissolved in urea (8 M), and added to SDS-PAGE loading buffer. Primary hepatocyte and Hepal-6 culture supematants were concentrated on Vivaspin 500 columns (10 kDa MWCO; Vivascience AG) resulting in approx. 10-fold up-concentration. HMGB1 was detected using polyclonal antibody (Abeam; ab 18256; 0.1 μg/ml). 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
(ACCPySociety of Critical Care MedicineSCCM) consensus criteria. Patients were treated according to standard recommendations, including aggressive fluid resuscitation, broad- spectrum antibiotic therapy over the first 24 hours, vasoactive agents and at least one intravenous dose of hydrocortisone. Blood samples were collected on the 1st, 2nd, 3rd, 5th and 7th day after septic shock diagnosis. Blood was collected on ice between 10:00h and 12:00h using an arterial line or a peripheral vein, and plasma was collected by centrifugation (800 g; 15 min; 4°C), aliquoted and stored (-70°C) until analysis. Organ dysfunction was defined using the Sequential Organ Failure Assessment (SOFA) score on the basis of daily measurements). The outcome analyzed were 28th day hospital mortality. The study protocol was approved by the institutional review board (IRB) of each participating center (University Hospital of Federal University of Rio de Janeiro, Hospital Quinta D'Or, Casa de Saiide Sao Jose, Rio de Janeiro, Brazil). All patients, or their legal surrogates, gave written informed consent before any study-related procedures.
Statistical analysis: The comparison of two independent samples was assessed by the Student's t-test and the Mann- Whitney test for gaussian and non-gaussian distributed samples, respectively. To compare more than two samples, ANOVA or Kruskal-Wallis tests were performed for gaussian and non-gaussian distributed samples, respectively. Comparison of different survival curves for the variously treated animals was done by the non-parametric Log-rank test. For pairwise comparisons, the Bonferroni correction was used to warrant the overall significance level. Regression models were applied to describe genotype-based data, and statistical significance presented throughout the paper refers to additive effects. Kolmogorov-Smirnov and Shapiro- Wilk tests were performed to assess the normality of the samples under analysis. Regression models were applied to describe genotype-based data. In all data sets, model equation applied was the following:
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. For human data, 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. Patients that left hospital after treatment were considered as right censored observations for the respective survival time. Since the survival time could be approximated by a Lognormal distribution, several survival regression models based on such probability distributions were fitted to the data. HPX was included in the models as an explanatory variable using either the first or the last time point measure available for a patient. The statistical significance of this explanatory variable in the models was assessed by the traditional z-score tests. A correlation analysis between SOFA score and HPX at different time points was also performed using
Spearman's coefficient. Experimental Methods Used in Examples 9 to 15
Mice: C57BL/6 Hmoxl 1' mice were provided originally by Shaw-Fang Yet (Pulmonary and Critical Care Division, Brigham and Women's Hospital, Boston).
C57BL/6 Nrft'1' mice were obtained from the RIKEN BioResource Center (Koyadai, Tsukuba, Ibaraki, Japan). HbSADNrf2+l' mice were generated from HbSADNrf2+l' x Hbwt Nr 2+/" breeding. C57BL/6 HbSAD mice (expressing 19% HbSAD, i.e. αΗ 2β2 5ΑΟ) were provided originally by Annie Henri (INSERM U733 IUH Hopital Saint-Louis, Paris). While hemizygous HbSAD can develop typical complications of sickle cell disease, e.g.
generalized congestion and microvascular occlusions, occasionally with thrombosis and infarctions of lung, kidneys, penis and myocardium these occur only when HbSAD mice are exposed to hypoxia or upon extensive aging, i.e. 38-75 weeks (Trudel et al, 1994).
Hemizygous HbSAD mice used in the experiments described hereby were under the age of 18 weeks presenting no overt hematological changes or complications of sickle cell disease. HbSADHmoxl+/~ mice were generated from HbSADHmoxl+/~ x Hbwt Hmoxl 1' breeding. C57BL/6.Svl29 mice, expressing the human Hb alpha, gamma-beta chain alleles without the endogenous alleles of the mouse Hb chains, i.e. HbA/A mice, were provided originally by Tim Townes (University of Alabama at Birmingham, USA). HbA/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 HbA/A with C57BL/6 Hbwt mice. Interbreeding of HbA/a mice produced, among other genotypes, HbA/a mice and Hba/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). Experimental protocols were approved by the "Instituto Gulbenkian de Ciencia animal care committee". Bone Marrow chimeras were generated in Hmoxl , Hmoxl " mice expressing or not the HbSAD allele (8-10 weeks). Mice (recipients) were lethally irradiated (900 rad, 2.35 minutes, 137Cs source)(Gammacell 2000, Molsgaard Medical, Denmark) and
reconstituted 4 hours thereafter with 5xl06 total bone marrow cells from Hmoxl+/+, Hmoxl+/~ expressing or not the HbSAD allele (6 weeks). Chimerism was assessed 8-10 weeks thereafter by RT-PCR, as described (Seixas E, Gozzelino R, Chora A, Ferreira A, Silva G, et al. Proc. Natl. Acad. Sci. USA. 2009.106: 15837-42). Flow cytometry was used to assess percent and total number of circulating cells in reconstituted mice as compared to control non-chimeric mice.
Parasites, infection and disease assessment: Mice were infected by intraperitoneal
(i.p.) inoculation of 105 RBCs infected with (GFP)- . berghei ANKA. Parasitemias were determined by flow cytometry. Infected mice were monitored twice daily for clinical symptoms of ECM including hemi- or paraplegia, head deviation, tendency to roll-over on stimulation, ataxia and convulsions. All experiments were performed in mice sacrificed under C02 and perfused with PBS, at the time of ECM in control mice.
Protoporphyrins. Iron-protoporphyrin IX (FePPIX; heme) and zinc-protoporphyrin IX (ZnPPIX) were dissolved in 0.2 M NaOH, neutralized (pH 7.4) with 0.2 M HC1 and administered (i.p.), as described (Pamplona, A. et al. Nat. Med. 2007. 13, 703-710).
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).
Histology: Brains were harvested, when clinical signs of ECM were noticed in control mice. Tissue was fixed in buffered 4% (vol/vol) paraformaldehyde and histological analysis was performed on perfusion-fixed tissues.
BBB permeability: Mice were injected intravenously (i.v.) with 0.1 ml of 2% Evans Blue (Sigma) when clinical symptoms of ECM were noticed in control mice. Mice were sacrificed lh thereafter. Brains were weighted and placed in formamide (Merck) (37°C, 48h) to extract Evans Blue dye. Absorbance was measured at λ=620 nm (Spectronic Unicam, He ios β). Evans Blue concentration was calculated from a standard curve of Evans Blue and is expressed as μg of Evans Blue per g of brain tissue (Pamplona, A. et al. Nat. Med. 2007. 13, 703-710). Leukocyte brain infiltration: Leukocytes were isolated from the brain of P. berghei ANKA infected 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 μιη) (Becton Dickinson) and centrifuged (1200 g; 10 min). Brain leukocyte infiltration was quantified by flow cytometry.
Quantitative real-time reverse transcription PCR (qRT-PCR): Mice were sacrificed at day ECM onset in Hbwt mice. RNA was isolated from brain, liver, kidney, heart, bone marrow, spleen and lungs using Trizol Reagent (Invitrogen, Life technologies) and
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
(Pamplona, A. et al. Nat. Med. 2007. 13, 703-710). TAQMAN® Gene Signature Mouse Immune Array (Applied Biosystems) was used to quantify all other mRNAs (7900HT ABI system), according to manufactures recommendations.
Serum biochemistry. Blood was collected in heparinized tubes by cardiac puncture, centrifuged (2x5min, 1600g). Hematograms were measured by focused flow technology (Hemavet Multispecies Hematology System, HV950FS, Drew Scientific Inc., CD VET Lab, Centra Diagnostico Veterinario, Lisboa, Portugal). Plasma Hb was determined by spectroscopy at λ=577. Total plasma heme was measured using the 3,3', 5,5'
tetramethylbenzidine (TMB) peroxidase assay (BD Biosciences), at λ=655 nm. Purified Hb was used as standard for plasma Hb and heme measurements.
Statistical analysis: Non-parametric Mann- Whitney U test was used to assess statistical significance between averages in different samples in which n<5. In samples with n>5 the unpaired Student's t-test for unequal variances was used. Normal
distributions were confirmed using the Kolmogorov-Smirnov test. Significant differences in survival were evaluated by the generation of Kaplan-Meier plots and by performing log- rank analysis for all experiments in which survival was assessed as an end-point.
Statistical analysis for the progeny-expected ratios was performed using Pearson's chi- squared tests. * <0.05 or ** <0.01 were considered statistically significant. EXAMPLE 9.
Sickle human Hb confers a survival advantage against malaria in mice: Inoculation of C57BL/6 mice (Hbwt) 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 HbSAD mice (FIG. 15 A) expressing a β-chain of human Hb carrying the p6Glu->Val (HbS) mutation as well as two additional mutations, p23Ala->Ile (Antilles231) and p121Asp->Gln
(D-Punjab121¾ known to enhance HbS polymerization in humans and mice. Naive HbSAD 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 HbSAD against ECM is consistent with previous observations in other rodent models of sickle cell disease. HbSAD mice that did not develop ECM, succumbed 20-25 days post-infection from hyperparasitemia-induced anemia, a condition unrelated to ECM.
In a similar manner to C57BL/6 Hbwt mice expressing only the endogenous mouse Hb, when infected with P. berghei ANKA C57BL/6.Sv/129 HbA/a mice expressing normal human Hb as well as the endogenous mouse Hb, developed clinical signs of ECM, succumbing 6 to 12 days after infection (FIG. 21a). Littermate control C57BL/6.Sv/129 Hba/a mice expressing only the endogenous mouse Hb also developed ECM but with lower incidence, as compared to HbA/a mice (FIG. 21a). The relative lower incidence of ECM in Hba/a C57BL/6.Sv/129 (FIG. 21a) vs. Hbwt C51B /6 (FIG. 15a) mice is expected given that the development of ECM is strongly favored in the C57BL/6 genetic background. These observations demonstrate that the protective effect of HbSAD against ECM is due to the mutations in the human β-globin chain, and should not be attributed to a putative effect of the human Hb per se. This mimics the protective effect of the human A/S sickle cell trait against the development of severe forms of malaria, including CM.
HbSAD 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). These pathologic features were present in Hbwt (FIG. 15b,c) and HbA/a mice (FIG. 21b).
Given that BBB disruption and brain edema develop in P. berghei ANKA infected C57BL/6 mice via a CD8+ T cell-dependent mechanism, we asked whether the protective effect of Hb against ECM was associated with modulation of CD 8 T cell activation and/or brain sequestration. The number of CD45hlgh leukocytes and CD8+ T cells, including activated/effector granzyme B-positive (GrB+) CD8+ T cells, was significantly reduced in P. berghei ANKA infected HbSAD, as compared to Hbwt mice at ECM onset (FIG. 15d). This effect was paralleled by a similar, albeit less pronounced, reduction in the number of activated/effector GrB+CD8+ T cells in the spleen of infected HbSAD versus Hbwt mice, suggesting that HbSAD inhibits overt CD8+ T cell activation in response to
Plasmodium infection.
HbSAD mice develop a mild form of sickle cell disease. Blood was harvested from naive or P. berghei ANKA-infected Hbwt and HbSAD mice and analyzed for different hematological parameters, as specified. Data is shown as mean ± standard deviation (n=7- 9/group), pooled from 2 independent experiments with similar results. Notice a mild increase in total leukocytes and lymphocytes of na'ive as well as infected HbSAD vs. Hbwt mice. Notice as well a mild decrease in RBC numbers, Hb, hematocrit and MCV of HbSAD vs. Hbwt mice after P. berghei ANKA-infection. This data suggests that na'ive HbSAD develop a very mild form of sickle cell disease that resembles that observed in
heterozygous individuals carrying one HbS allele Notice as well that when infected with P. berghei ANKA, HbSAD mice developed a relatively more severe form of anemia with concomitant increase of circulating leukocytes, lymphocytes, monocytes and platelets, as compared to infected Hbwt mice. MCV: Mean corpuscular volume; MCHC: Mean cell hemoglobin concentration. *P<0.05 in HbSAD vs. Hbwt mice, compared among non-infected or infected groups.
TABLE 3:
Not infected Infected
mSAD mSAD
Hbwt Hbwt
RBC (106/ml) 8.24±0.43 8.04±0.38 7.59±1.70 6.22±0.84*
Hb (g/dl) 12.7±0.7 12.3±0.8 10.9±3.3 9.8±2.5
Hematocrit (%) 34.2±1.9 32.0±1.4* 32.4±11.3 27.7±7.1 *
MCV (fl) 41.5±1.3 39.9±0.8* 42.2±10.7 44.4±8.9
MCHC (%) 37.1±1.0 38.5±2.1 34.2±2.9 35.4±3.0
Leukocytes (χ103/μ1) 9.03±3.51 13.17±2.13 1.64±0.50 3.52±1.20*
Neutrophils (χ103/μ1) 1.81±1.37 2.06±0.98 0.47±0.19 0.55±0.23
Lymphocytes (χ103/μ1) 6.0±1.69 10.81±1.54* 0.89±0.37 2.51±0.86* Monocytes (χ103/μ1) 0.96±0.44 1.14±0.42 0.20±0.10 0.41±0.15*
Eosonophils (χ103/μ1) 0.27±0.27 0.24±0.22 0.06±0.04 0.05±0.04
Basophils/μΐ 76.3±91.2 55.0±86.2 10.0±22.4 0.0±0.0
Platelets (χ103/μ1) 0.47±0.18 0.56±0.1 0.13±0.03 0.23±0.01 *
EXAMPLE 10.
Sickle human Hb confers tolerance against Plasmodium infection in mice:
Protection of HbSAD 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 Hbwt (FIG. 15e,f) or HbA/a mice (FIG. 21c). While the protective effect of the human sickle cell trait against malaria has been associated with decreased pathogen load, there are several instances where this does not appear to be the case (Crompton, P.D., Traore, B., Kayentao, K., Doumbo, S., Ongoiba, A., Diakite, S.A., Krause, M.A., Doumtabe, D., Kone, Y., Weiss, G., et al. J Infect Dis. 2008. 198, 1265-1275.), which is in keeping with the observation that HbSAD confer protection against ECM without interfering with pathogen load. These observations suggest that mutations in the β-chain of human Hb, such as those in HbSAD can afford tolerance against Plasmodium infection, a host defense strategy that limits disease severity by preventing tissue damage, without targeting the pathogen. This contrasts to resistance against infection, the well-recognized host defense strategy that limits disease severity by decreasing pathogen load (Raberg, L., Graham, A.L., and Read, A.F. (2009). Philos Trans R Soc Lond B Biol Sci 364, 37-49).
EXAMPLE 11.
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 HbSAD mice also express high levels of Hmoxl mRNA in the bone marrow and peripheral blood, as compared to naive Hbwt mice (FIG. 16a). This was not the case in HbA/a mice that expressed similar levels of Hmoxl mRNA in the bone marrow and peripheral blood vs. littermate control Hba/a mice (FIG. 22b). This demonstrates that expression of a ps - related variant but not a normal β-globin chain is required to induce Hmoxl expression. Na'ive HbSAD mice also expressed higher levels of Hmoxl mR A in the kidneys (FIG. 22a), which is consistent with the chronic
development of kidney injury, revealed clinically only after extensive aging, (De Paepe, M.E., and Trudel, M. (1994). Kidney Int 46, 1337-1345). HbSAD mice expressed similar levels of Hmoxl mRNA in the liver, heart, lung and spleen (FIG. 22a), as compared to Hbwt 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 HbSAD 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 HbSAD mice (FIG. 22c), without causing overt post-natal lethality (Table 4) (Wiesel, P., Patel, A.P., DiFonzo, N., Marria, P.B., Sim, C.U., Pellacani, A., Maemura, K., LeBlanc, B.W., Marino, K., Doerschuk, CM., et al. (2000). Circulation 102, 3015-3022). When challenged by P. berghei ANKA infection, HbSADHmoxl+/~ mice succumbed to ECM (FIG. 16b), with concomitant development of BBB disruption (FIG. 16c), brain edema (FIG. 16d) and sequestration of CD45hlgh leukocytes, CD8+ T cells and activated
GrB+CD8+ T cells in the brain (FIG. 16e) but without noticeable hematological changes (Table 5). Moreover, ECM was not associated with increased parasite load in
HbSADHmoxl+/- vs. HbSADHmoxl+l+ mice (FIG. 16f), which is consistent with the notion that induction of HO-1 expression by sickle human Hb confers tolerance against
Plasmodium infection.
Survival of infected HbSADHmoxl+/~ mice was slightly but significantly higher than that of HbwtHmoxl+/~ mice (FIG. 16b). This suggests that residual HO-1 expression in HbSADHmoxl+/- 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.
Pharmacologic inhibition of HO activity, by zinc protoporphyrin IX
(ZnPPIX)(Pamplona, A. et al. Nat. Med. 2007. 13, 703-710, Pamplona et al, 2007), increased the incidence of ECM in HbSAD mice vs. vehicle-treated controls (FIG. 23a). This effect was not associated with modulation of parasitemia (FIG. 23b), suggesting that heme catabolism by HO-1 per se and/or via the production of the end-products of heme catabolism, i.e. CO, biliverdin and/or iron, confers tolerance against Plasmodium infection in HbSAD mice. Enhanced mortality of Hmoxl homozygous deletion in Hb mice. Offspring of
HbSADHmoxl+/~ x HbwtHmoxl+/~ breeding vs. HbwtHmoxl+/~ x HbwtHmoxl+/~ breeding. Notice that 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. Notice as well that the early lethality associated with Hmoxl deletion was significantly (χ2; p<0.00001) increased in HbSAD vs. Hbwt mice, revealed by a significant reduction in the frequency of HbSAD vs. Hbwt homozygous Hmoxl deficient {Hmoxl' ') offspring obtained from heterozygous-deficient (HbwtHmoxl+/') breeding. Frequency of Hbw f Hmoxl' ' offspring obtained from HbwtHmoxl+/' x
HbwtHmoxl+/' breeding was similar to that obtained from HbSADHmoxl+/' x HbwtHmoxl+/' breeding (χ2; p>0.05). HbSADHmoxl+/+ neonates exhibit transient anemia at delivery, related to hemolysis caused by HbSAD polymerization, most probably due to transient hypoxia associated with late fetal development and delivery. Hb values in HbSAD mice return to normal levels shortly after weaning (Trudel et al., 1991). These observations reveal that expression of HO-1 in the HbSAD fetus and/or neonate is required to counter the toxicity associated with the hemolysis-related free Hb and free heme, thus preventing early mortality.
TABLE 4:
Figure imgf000061_0001
Number 148 10.90 320
Percentage 23.56 1 0.07
HbwtHmoxl+/+ HbwtHmoxl+/' HbwtHmoxl'''
Number 293 575 21
Percentage 21.58 42.34 1.55
HbwtHmoxl+/' x Hbwt Hmoxl +/'
Hmoxl+/+ Hmoxl ' Hmoxl'
Number 193 36.83 323
Percentage 61.64 8 1.53
Hematological effect associated with deletion of the Hmoxl allele in Hb mice: Blood was harvested from na'ive or P. berghei ANKA-infected HbSAD mice carrying two (Hmoxl ) or one (Hmoxl ") functional Hmoxl alleles. Blood was analyzed for different parameters, as specified. Data is shown as mean ± standard deviation (n=6-9/group), pooled from 2 independent experiments. Notice the lack of significant changes in the different parameters analyzed with exception of an increase in the number of platelets in non- infected HbSADHmoxl+'~ vs. HbSADHmoxl+'~ mice. MCV: Mean corpuscular volume; MCHC: Mean cell hemoglobin concentration.
TABLE 5:
Not infected Infected
Hmoxl +^+ Hmoxl 1' Hmox+7+ Hmoxl
RBC (106/ml) 8.04±0.38 8.06±0.38 6.22±0.84 6.87±0.94
Hb (g/dl) 12.3±0.8 12.2±0.5 9.8±2.5 12.8±1.5
Hematocrit (%) 32.0±1.4 32.1±1.0 27.7±7.1 35.8±7.8
MCV (fl) 39.9±0.8 39.8±0.9 44.4±8.9 39.9±0.2
MCHC (%) 38.5±2.1 38.1±1.1 35.4±3.0 34.7±0.8
Leukocytes (χ103/μ1) 13.17±2.13 12.27±3.86 3.52±1.20 2.65±1.52
Neutrophils (χ103/μ1) 2.06±0.98 2.76±1.63 0.55±0.25 0.44±0.18
Lymphocytes (χ103/μ1) 10.8±1.54 12.01±3.37 2.51±0.86 2.35±0.81
Monocytes (χ103/μ1) 1.14±0.42 1.33±0.06 0.41±0.15 0.45±0.26
Eosonophils (χ103/μ1) 0.24±0.22 0.36±0.34 0.05±0.04 0.05±0.04
Basophils/μΐ 55.0±86.2 13.8±38.9 0.0±0.0 6.7±16.3
Platelets (χ103/μ1) 0.56±0.06 0.80±0.09* 0.23±0.09 0.17±0.05 EXAMPLE 12.
Induction of HO-1 by sickle human Hb inhibits the production of 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 HbSAD mice that did not develop ECM vs. Hbwt mice that succumbed to ECM (FIG. 17a). This inhibitory effect was mediated by HO-1, since expression of mRNA encoding these chemokines was significantly increased in the brain of infected HbSADHmoxl+/~ vs. HbSADHmoxl+/+ (FIG. 17a). The functional involvement of
CXCLlO/IP-10 in the pathogenesis of ECM (Campanella, G.S., Tager, A.M., El Khoury, J.K., Thomas, S.Y., Abrazinski, T.A., Manice, L.A., Colvin, R.A., and Luster, A.D.
(2008). Proc Natl Acad Sci USA 105, 4814-4819) suggests that its inhibition by HO-1 might contribute in a critical manner to the protective effect afforded by HbSAD against ECM. Expression of mRNAs encoding other chemokines, such as Cxclll (Ip-9) or the chemokine receptors Ccr2 and CxcrS was also inhibited by HbSAD but in a manner that was not impaired in HbSADHmoxl+'~ vs. HbSADHmoxl+l+ mice (FIG. 17b). This suggests that the inhibitory effect of HbSAD over the expression of these genes is probably not mediated by HO-1. Expression of mRNA encoding the chemokine Cell 9 (MIPS a) and the chemokine receptor Ccr7 was not modulated by HbSAD 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).
EXAMPLE 13.
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 HbSAD against ECM requires the expression of HO-1 in the hematopoietic or
non-hematopoietic cell compartment. We performed syngenic bone marrow transplants from HbSADHmoxl+l+ or HbSADHmoxl+/~ mice into lethally irradiated HbwtHmoxl+/+ or HbwtHmoxl+/~ mice to generate chimeric HbSAD mice in which one Hmoxl allele is deleted in the hematopoietic (HbSADHmoxl+/~- HbwtHmoxl+/+) or non-hematopoietic
(HbSADHmoxl+/+- HbwtHmoxl+/~) compartment. Chimeric HbSAD mice carrying two functional Hmoxl alleles in the hematopoietic and in the non-hematopoietic compartments (HbSADHmoxl+l+^HbwtHmoxl+l+) did not succumb to ECM (FIG. 18a) or developed brain edema (FIG. 18b) in response to P. berghei ANKA infection. Control chimeric mice in which the bone marrow of HbwtHmoxl+/+ mice was transferred into lethally irradiated HbSADHmoxl+l+ mice, developed ECM (FIG. 18a) and brain edema (FIG. 18b), confirming that cells derived from the hematopoietic compartment confer the protective effect of HbSAD.
Chimeric HbSAD mice carrying a single functional Hmoxl allele in hematopoietic cells (HbSADHmoxl+' HbwtHmoxl+l+) succumbed to ECM (FIG. 18a), developing brain edema (FIG. lb). The reverse was not true in that deletion of a single Hmoxl allele in non-hematopoietic cells (HbSADHmoxl+/+- HbwtHmoxl+/~) did not impair the protective effect ofHbSAD against ECM (FIG. 18a), confirmed by lack of brain edema (FIG. 18b). Similar results were obtained when transferring bone marrows from HbSADHmoxl+l+ or HWAUHmoxf~ mice into HWAUHmoxf o HWAUHmoxf~ mice (FIG. 26). Lethality after day 12 post-infection (FIG. 18a) was most probably due to the development of a
"composite disease" in which high levels of parasitemia (>20%) synergize with sickle human Hb to cause death, without overt clinical or pathological signs of ECM. These observations reveal that the protective effect of HbSAD requires that the induction of HO-1 expression in the hematopoietic cell compartment, consistent with the observed induction of HO-1 expression in blood and bone marrow cells of naive HbSAD mice (FIG. 16a). The protective effect of HO-1 expression in the hematopoietic compartment was not associated with modulation of pathogen load (FIG. 18c and FIG. 26c), confirming that HO-1 affords tolerance against Plasmodium infection.
EXAMPLE 14.
Sickle human Hb induces the expression of HO-1 via the transcription factor Nrf2: The transcription factor 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 HbSAD mice (FIG. 16a) involved the transcription factor Nrf2. We found that this is the case since deletion of one Nrf2 allele in Hb mice
was sufficient to reduce the level of Hmoxl mR A expression in whole blood leukocytes to those of naive HbwtNrft+/+ mice (FIG. 19a). This suggests that sickle human Hb induces Hmoxl transcription and ultimately expression via a mechanism involving the
transcription factor Nrf2. Incidence of ECM increased significantly in P. berghei ANKA infected HbSADNrf2+/~ vs. HbSADNrf2+/+ mice (FIG. 19b), confirmed by the development of brain edema (FIG. 19c). A similar effect was observed in HbSAD mice lacking two Nrf2 alleles, i.e. Hb^Nrf ^ mice (n=3; 33% survival). This loss of protection against ECM observed in HbSADNrf2+/~ or HbSADNrfi- mice vs. HbSADNrf2+/+ was not due to increased parasite load (FIG. 19d), which is consistent with the notion that induction of HO-1 expression by the transcription factor Nrf2 confers tolerance against Plasmodium infection. EXAMPLE 15.
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 Hbwt mice (FIG. 20a). We refer to free heme as heme not contained within the "heme pockets" of hemoglobin, which does not preclude its association with other proteins or lipids, presumably in a manner that does not control its pro-oxidant activity (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). We asked whether, despite its cytotoxicity, when pre-exposed at low levels to cells, free heme would exert a protective effect in vivo against a subsequent heme challenge. Administration of free heme to Hbwt mice prior to P. berghei ANKA infection suppressed the incidence of ECM, as compared to vehicle -treated Hbwt mice (FIG. 20b). This protective effect was not associated with modulation of parasitemia (FIG. 27a), suggesting that low concentration of free heme in the plasma of na'ive HbSAD mice (FIG. 20a) contributes to the induction of tolerance against Plasmodium infection.
Plasma free heme concentration increased significantly following P. berghei ANKA infection in Hbwt 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 HbSAD mice (FIG. 20a). When challenged with free heme after infection, HbSAD succumbed to ECM (FIG. 20c), confirmed by the occurrence of brain edema (FIG. 20d). This reveals that free heme has a dual effect in the control of ECM onset, being protective when present at slightly above normal concentration before infection (FIG. 20b) while highly deleterious after infection (FIG. 20c). Free heme did not interfere with pathogen load (FIG. 27a and b), revealing that when present at slightly above normal concentration before infection free heme promotes tolerance against malaria, while impairing tolerance against malaria when present at higher concentrations after infection. Heme administration at the same dosage and schedule to na'ive Hbwt or HbSAD mice did not result in lethality.
When applied via inhalation to wild type mice, CO suppresses the pathogenesis of ECM via a mechanism that relies on the ability of this gasotransmitter to inhibit heme release from Hb (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). We asked whether the protective effect of HbSAD against ECM was mediated via this mechanism. Inhaled CO suppressed the incidence of ECM in Hb Hmoxl ' mice (FIG. 20e), confirmed by the lack of brain edema (FIG. 20f). CO did not modulate parasitemia (Suppl. FIG. 7c). Instead, the protective effect of CO was associated with a significant reduction of plasma free heme concentration, below that of naive HbSADHmoxl+/~ mice (FIG. 20g). Administration of free heme to infected HbSADHmoxl+/~ mice abrogated the protective effect of CO, restoring ECM incidence (FIG. 20h), confirmed by brain edema (FIG. 20i). Heme was not toxic when administered at the same dosage and schedule to naive
HbSADHmoxl+l- mice receiving CO, i.e. 0 % mortality. These observations demonstrate that sickle human Hb suppresses the onset of ECM via the induction of HO-1 and the production of CO, which inhibits the accumulation of free heme thus affording tolerance against Plasmodium infection. While it has been previously shown that this mechanism suppresses the onset of ECM in wild type mice (Pamplona, A. et al. Nat. Med. 2007. 13, 703-710), this is the first demonstration that such a mechanism operates under
experimental conditions mimicking those affording natural protection against Plasmodium infection in human populations.
EXAMPLE 16.
Albumin stops the cytotoxic effect of free heme: Hepatocytes (Heap cells) were seeded and exposed to heme (40 mM, lh) in Hanks Balanced Salt Solution (HBSS;
Invitrogen), without serum, to avoid potential heme-scavenging by serum proteins.
Subsequently, 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. Binding was confirmed by electrophoresis of albumin in a native gel as well as by absorption spectrophotometry. Heme and heme/HSA were used at 40μΜ heme equivalents. As illustrated in FIG. 28, albumin blocked nearly 100% of the cytotoxicity otherwise observed when cells were exposed to heme and TNF.
EXAMPLE 17.
Modulation of heme transporters can suppress the cytotoxic effects of heme:
Hepatocytes (Hepa cells) were cultured in 96 well plates (5xl03 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 μΜ, lh) in Hanks Balanced Salt Solution (HBSS; Invitrogen), without serum, to avoid potential heme-scavenging by serum proteins. Subsequently, 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.
EXAMPLE 18
Levels of cell-free hemoglobin, haptoglobin, total free heme (total plasma heme), hemopexin, and free heme, in a cohort of human patients diagnosed with malaria: The population in this study has been described in a publication (Sambo, MR, Trovoada MJ, Benchimol C, Quinhentos C, Goncalves L, Velosa R, Marques MI, Sepiilveda N, Clark TG, Mustafa S. Plos One. 2010). A total of 749 children, living in Luanda, Angola and ranging from 6 months to 13 years of age were enrolled in the study. Ethical permission was granted by the Ethical Committee of the "Hospital Pediatrico David Bernardino" in Luanda, Angola appointed by the Angolan Ministry of Health. Written, informed consent was obtained from the parents or guardians of each child. Sample collection was carried out from February 2005 to May 2007 and comprised 130 children that developed cerebral malaria, 158 patients with severe non-cerebral malaria, 142 patients with uncomplicated malaria and 319 uninfected controls. The mean age in months was 54.2 for cerebral malaria cases, 45.9 for severe non-cerebral malaria patients, 50.3 for uncomplicated malaria patients and 60.9 for uninfected controls. Malaria was diagnosed on the basis of a positive asexual parasitaemia detected on a Giemsa-stained thick smear. 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
biochemistry was performed in a sub-group of these pathients for which plasma was available. 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
Biosciences), at 1655 nm. Purified hemoglobin was used as standard for plasma hemoglobin and heme measurements. 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.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS What is claimed:
1. A method for treating a heme-mediated disease in a mammal in need thereof comprising administering an agent that binds free heme and inhibits heme-mediated damage or oxidation.
2. The method of claim 1, wherein the agent binds heme iron.
3. The method of claim 1, wherein the agent results in re -uptake or recycling of heme into cells.
4. The method of claim 1, wherein the agent degrades heme.
5. The method of claim 1, wherein the agent is selected from the group consisting of heme-specific antibodies, Hemopexin, Haptoglobin, Albumin, Alpha- 1 -microglobulin, Peroxiredoxin 1 , Rhodnius Heme Binding Protein, Boophilus microplus Heme
Lipoprotein, Haemophilus influenza Heme-Binding Lipoprotein, Low Density
Lipoprotein, High Density Lipoprotein, Drosophila retinoid- and fatty acid-binding glycoprotein, and fragments and variants thereof, in an amount sufficient to reduce the levels of free heme.
6. The method of claim 1, wherein the agent is selected from the group consisting of artemisinin, the 4-aminoquinoline chloroquine, the 8-aminoquinoline primaquine, isoquinoline, and the quinolinemethanols mefloquine and quinine, and wherein the disease is not malaria or sepsis.
7. The method of claim 1, wherein the agent is selected from the group consisting of a heme -bound helical bundle peptide, a peptide covalently attached to the heme periphery, and a disulfide-dimer peptide coordinated to exchange-inert metalloporphyrins.
8. The method of claim 1, wherein the agent is selected from the group consisting a DNA sequences, protein or peptide.
9. A method of claim 1 , further comprising administering a therapeutically effective amount of at least one heme-binding protein and at least one compound selected from protein heme-oxygenase 1 , a polynucleotide encoding heme -oxygenase 1, carbon monoxide, morphine, a glucocorticoid, a nonsteroidal-antiinflammatory drug, a salicylate, doxorubicin, metoprolol, salbutarnol, isoproterenol, 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), nerve growth factor, curcumin, resveratrol, ferulic acid, L-carnitine, carbon monoxide, biliverditt, bilirubin, desferal, deferiprone ethyienedia metetraacetic acid, diethylenetriaminepentaacetic acid, salicylaldehyde isonicotinoyl hydrazone, reduced glutatliione, xanthine oxidase, NADPH-cytochrome P-450 reductase, N-acetylcysteine, urate, butyiated hydroxyanisole, sodium cyanide, an anti-TNF antibody, an anti- MGBl antibody, and a Fas crosskinking inhibitor.
10. A method for treating a heme-mediated disease in a mammal in need thereof comprising administering an agent that modulates a heme transporter. 1 I . The method of any one of claims 1 to 8, wherein said disease is selected from the group consisting of burns, hemorrhage, sepsis, malaria, tuberculosis, paroxysomal nocturnal hemoglobinuria, hereditary spherocytosis, sickle cell disease, thalassemia, pyruvate kinase deficiency, microangiopathy, aortic stenosis, disseminated intravascular coagulation, hemodynamic stress caused by a prosthetic heart valve or by extracorporeal circulation during surgery, malaria, dengue hemorrhagic fever, sepsis, Chagas disease, an infection by Streptococcus, vancomycin-resistant enterococci, E. coli, Staphylococcus Aureus, Pseudomonas, Serratia spp., Proteus spp., Listeria spp., Bacillus cereus,
Clostridium tetani, Aspergillus fumigasus, Stacybotiys chartarum, Candida albicans, or PemofJlinum cb.ysogenu.rn, an acute hemolytic transfusion reaction, paroxysomal cold hemoglobinuria, lead poisoning, potassium dichromate poisoning, arsenic poisoning, ischemia-reperfusion injury, allograft rejection, neuroinflammation, endotoxic shock, restenosis, myocardial infarction, and complications associated with transplantation, pathological outcomes associated with pregnancy.
12. A method of identifying a candidate compound that reduces the levels of free heme in a mammal, said method comprising: (i) incubating a single cell or a colony of cells with a candidate compound, (ii) contacting said cell or cell colony with free heme, (iii) contacting said cell or cell colony with a pro-inflammatory agonist, and (iv) determining whether administration of the candidate compound results in a reduction in the percentage of cell death or in a reduction in the amounts of free radicals generated by the cell relative to a control cell or cell colony contacted with free heme and pro-inflammatory agonist but not contacted with said candidate compound.
13. A method of identifying a candidate compound that reduces the levels of free heme in a mammal, said method comprising: (i) pretreating a mammal with a candidate compound or with vehicle, (ii) administering a dose of heme to the mammal, (iii) administering a dose of concanavalin A to the mammal, and (iv) determining whether pretreatment with the candidate compound results in increase in percentage animal survival, an increase in animal survival time, or a decrease in serological markers of organ injury relative to an animal administered a vehicle control compound but not administered said candidate compound.
14. A method of determining the status of a free-heme associated condition in an individual, comprising:
(a) obtaining a first biomarker profile from a first biological sample taken from the individual; and
(b) comparing the concentrations of hemopexin and free heme and, optionally, haptoglobin, cell-freehemoglobin, and total plasma heme in the individual's biomarker profile to a reference biomarker profile obtained from a reference population;
wherein such comparison is capable of classifying the individual as belonging to or not belonging to the reference population, and wherein the comparison determines the status of a free-heme associated condition in the individual.
15. A method of treating a patient comprising administering free heme in a therapeutically effective amount whereby the patient is protected against a heme-mediated disease.
16. A method of treating a patient comprising administering free heme in combination with a heme chelator in a therapeutically effective amount whereby the patient is protected against a heme-mediated disease.
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