US20040052866A1 - Methods of treating hepatitis - Google Patents

Methods of treating hepatitis Download PDF

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US20040052866A1
US20040052866A1 US10/439,632 US43963203A US2004052866A1 US 20040052866 A1 US20040052866 A1 US 20040052866A1 US 43963203 A US43963203 A US 43963203A US 2004052866 A1 US2004052866 A1 US 2004052866A1
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Prior art keywords
patient
carbon monoxide
hepatitis
administered
gas
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Leo Otterbein
Augustine Choi
Brian Zuckerbraun
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Yale University
University of Pittsburgh
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Priority to US10/439,632 priority Critical patent/US20040052866A1/en
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Assigned to YALE UNIVERSITY reassignment YALE UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHOI, AUGUSTINE M. K., OTTERBEIN, LEO E.
Assigned to PITTSBURGH OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION, UNIVERSITY OF reassignment PITTSBURGH OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION, UNIVERSITY OF ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ZUCKERBRAUN, BRIAN
Priority to US13/544,701 priority patent/US20120276220A1/en
Priority to US13/803,330 priority patent/US20130202720A1/en
Priority to US14/094,140 priority patent/US9522163B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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

  • This invention relates to the treatment of hepatitis.
  • Carbon monoxide gas is poisonous in high concentrations. However, it is now recognized as an important signaling molecule (Verma et al., Science 259:381-384, 1993). It has also been suggested that carbon monoxide acts as a neuronal messenger molecule in the brain (Id.) and as a neuro-endocrine modulator in the hypothalamus (Pozzoli et al., Endocrinology 735:2314-2317, 1994). Like nitric oxide (NO), carbon monoxide is a smooth muscle relaxant (Utz et al., Biochem Pharmacol.
  • Hepatitis is a disease characterized by inflammation of the liver.
  • the inflammation can be characterized by diffuse or patchy necrosis affecting acini.
  • Causative agents of hepatitis include, for example, viruses, e.g., specific hepatitis viruses, e.g., hepatitis A, B, C, D, E, and G viruses; alcohol; and other drugs (e.g., isoniazid, methyldopa, acetaminophen, amiodarone, and nitrofurantoin) (see The Merck Manual of Diagnosis and Therapy, 17 th Edition, Section 4, Chapter 42).
  • the present invention is based, in part, on the discovery that administration of CO can protect against the development of hepatitis.
  • the present invention features a method of treating, preventing, or reducing the risk of, hepatitis in a patient.
  • the method includes identifying a patient diagnosed as suffering from or at risk for hepatitis (e.g., a patient diagnosed as suffering from or at risk for hepatitis), and administering to the patient a pharmaceutical composition comprising an amount of carbon monoxide effective to treat hepatitis in the patient.
  • the pharmaceutical composition can be administered to the patient by any method known in the art for administering gases and/or liquids to patients, e.g., via inhalation, insufflation, infusion, injection, and/or ingestion.
  • the pharmaceutical composition is administered to the patient by inhalation.
  • the pharmaceutical composition is administered to the patient orally.
  • the pharmaceutical composition is administered directly to the abdominal cavity of the patient.
  • the pharmaceutical composition is administered by an extracorporeal membrane gas exchange device or an artificial lung.
  • the patient is an alcoholic.
  • the patient can be an animal, human or non-human.
  • the patient can be any mammal, e.g., humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats.
  • the hepatitis can be the result of, or a person may be considered at risk for hepatitis because of, any of a number of factors, e.g., infections, e.g., viral infections, e.g., infection with hepatitis A, B, C, D, E and/or G virus; alcohol use (e.g., alcoholism); drug use (e.g., one or more drugs described herein, e.g., acetaminophen, anesthetics, anti-tuberculous drugs, antifungal agents, antidiabetic drugs, neuroleptic agents, and drugs used to treat HIV infection and AIDS); autoimmune conditions (e.g., autoimmune hepatitis); and/or surgical procedures.
  • the pharmaceutical composition can be in any form, e.g., gaseous or liquid form.
  • the method further includes administering to the patient at least one of the following treatments: inducing HO-1 or ferritin in the patient; expressing recombinant HO-1 or ferritin in the patient; and administering a pharmaceutical composition comprising HO-1, bilirubin, biliverdin, ferritin, or apoferritin, iron, desferoxamine, or iron dextran to the patient.
  • a pharmaceutical composition comprising HO-1, bilirubin, biliverdin, ferritin, or apoferritin, iron, desferoxamine, or iron dextran to the patient.
  • CO CO and any of the above-listed agents in the preparation of a medicament for treatment or prevention of hepatitis.
  • the hepatitis (or the risk for hepatitis) is not caused by surgery (e.g., abdominal or transplant surgery), bacterial endotoxin, septic shock, and/or systemic inflammation.
  • the invention features a method of treating or preventing hepatitis in a patient, which includes identifying a patient suffering from or at risk for hepatitis (e.g., a patient diagnosed as suffering from or at risk for hepatitis), providing a vessel containing a pressurized gas comprising carbon monoxide gas, releasing the pressurized gas from the vessel to form an atmosphere comprising carbon monoxide gas, and exposing the patient to the atmosphere, wherein the amount of carbon monoxide in the atmosphere is sufficient to treat hepatitis in the patient.
  • a patient suffering from or at risk for hepatitis e.g., a patient diagnosed as suffering from or at risk for hepatitis
  • the invention features a method of performing abdominal surgery, e.g., liver transplantation, on a patient, which includes identifying a patient in need of abdominal surgery, wherein hepatitis is a risk of the abdominal surgery; performing abdominal surgery on the patient, and before, during, or after the performing step, causing the patient to inhale an amount of carbon monoxide gas sufficient to reduce the risk of hepatitis in the patient.
  • a medicament e.g., a gaseous or liquid medicament, for use in the treatment or prevention of hepatitis.
  • the invention also features a method of treating hepatitis in a patient suffering from or at risk for hepatitis not caused by surgery and/or endotoxin, e.g., hepatitis caused by any factor described herein other than surgery and/or endotoxin.
  • the method includes identifying a patient suffering from or at risk for hepatitis not caused by surgery and/or endotoxin and administering to the patient a pharmaceutical composition comprising an amount of carbon monoxide effective to treat hepatitis in the patient.
  • a hepatitis-inducing drug i.e., a hepatotoxic drug, e.g., isoniazid, methyldopa, acetaminophen, amiodarone, or nitrofurantoin
  • the method includes administering the drug to the patient, and before, during, and/or after administering the drug, administering to the patient a pharmaceutical composition comprising carbon monoxide in an amount effective to treat hepatitis in the patient.
  • the invention provides a vessel comprising medical grade compressed CO gas.
  • the vessel can bear a label indicating that the gas can be used to treat hepatitis in a patient.
  • the vessel can bear a label indicating that the gas can be administered to a patient in conjunction with administration of a hepatitis-inducing drug (i.e., a hepatotoxic drug), e.g., acetaminophen.
  • a hepatitis-inducing drug i.e., a hepatotoxic drug
  • the CO gas can be in an admixture with nitrogen gas, with nitric oxide and nitrogen gas, or with an oxygen-containing gas.
  • the CO gas can be present in the admixture at a concentration of at least about 0.025%, e.g., at least about 0.05%, 0.10%, 0.50%, 1.0%, 2.0%, 10%, 50%, or 90%.
  • CO in the manufacture of a medicament for treatment or prevention of hepatitis.
  • the medicament can be used in a method for treating hepatitis in a patient suffering from or at risk for hepatitis in accordance with the methods described herein.
  • the medicament can be in any form described herein, e.g., a liquid or gaseous CO composition.
  • FIG. 1 is a bar graph illustrating that induction of HO-1 protects mouse hepatocytes from TNF- ⁇ /D-gal-induced cell death.
  • CoPP cobalt protoporphyrin
  • ALT serum alanine aminotransferase
  • TNF tumor necrosis factor alpha. Results are the mean ⁇ SD of 6-8 mice/group *p ⁇ 0.005.
  • FIG. 2 is a bar graph illustrating that exogenous CO protects hepatocytes against TNF-A induced cell death in a cGMP/p38 pathway-independent and an NF- ⁇ B activation-dependent manner.
  • FIG. 3 is a bar graph illustrating that exogenous CO protects human hepatocytes against TNF- ⁇ /Actinomycin-D (ActD)—induced cell death.
  • FIG. 4 is a bar graph illustrating that exogenous CO causes an increase in NF- ⁇ B activation in hepatocytes.
  • CO carbon monoxide
  • Air room air
  • BAY BAY 11-7082
  • CM cytokine mixture (TNF- ⁇ (500 U/ml), IL-1 ⁇ (100 U/ml), and IFN- ⁇ (100 U/ml)). Results shown are the mean ⁇ SE of triplicate wells from three independent experiments. *p ⁇ 0.001 versus Air.
  • FIG. 5 is a picture of a polyacrylamide gel illustrating that exogenous CO induces an increase in NF- ⁇ B nuclear translocation and DNA binding as measured by electrophoretic mobility shift assay (EMSA).
  • FP free probe (no nuclear protein, thus no DNA binding);
  • TOTAL NFkB bands without antibody supershifting.
  • FIGS. 6 A- 6 C are photomicrographs of primary hepatocytes immunostained to detect nuclear p65 localization, illustrating that exogenous CO causes an increase in NF-KB activation in hepatocytes.
  • FIG. 6A air-exposed hepatocytes.
  • FIG. 6B hepatocytes exposed to cytokine mixture (TNF- ⁇ (500 U/ml), IL-1 (100 U/ml), and IFN- ⁇ (100 U/ml)).
  • FIG. 6C CO-exposed hepatocytes. Images are representative of 6 different fields. Bar represents 10 ⁇ m.
  • FIG. 7 is a bar graph illustrating that exogenous CO-induced protection of hepatocytes involves NF- ⁇ B-dependent iNOS expression.
  • CO carbon monoxide
  • Air room air
  • BAY BAY 11-7082
  • CM cytokine mixture. Results shown are the mean ⁇ SE of triplicate wells from four independent experiments. *p ⁇ 0.001 versus air and air/BAY-treated cells.
  • FIG. 8 is a picture of a Western blot illustrating that iNOS protein expression in hepatocytes is markedly increased by exposure to TNF-a in the presence of CO as compared to exposure to TNF- ⁇ alone.
  • iNOS inducible nitric oxide synthase
  • CO carbon monoxide
  • Air room air
  • TNF TNF- ⁇ /ActD
  • ⁇ -Actin control protein.
  • the immunoblot is representative of 3 independent experiments.
  • FIG. 9 is a bar graph illustrating that iNOS activity-deficient mouse (inos ⁇ / ⁇ ) hepatocytes are not protected by CO against TNF-a-induced cell death.
  • CO carbon monoxide
  • Air room air
  • TNF TNF- ⁇ /ActD
  • inos ⁇ / ⁇ iNOS knockout mice
  • L-NIO L-N5-(1-iminoethyl)-ornithine-2HCl. Results shown are the mean ⁇ SE of triplicate wells from four independent experiments. *p ⁇ 0.01 versus non-TNF/ActD and CO/TNF/ActD-treated cells.
  • FIG. 10 is a bar graph illustrating that exogenously-administered CO prevents TNF- ⁇ /D-Gal-induced liver injury in mice.
  • ALT serum alanine aminotransferase
  • CO carbon monoxide
  • Air room air. Results presented as mean ⁇ SD of 18-20 mice. *p ⁇ 0.001 versus air-treated.
  • FIGS. 11 A- 11 H are photomicrographs of liver samples illustrating that exogenously-administered CO prevents TNF- ⁇ /D-Gal-induced liver injury in mice.
  • FIGS. 11 A and 11 B liver samples from mice exposed to room air and CO, respectively, and stained with hematoxylin & eosin (H & E).
  • FIGS. 11 C and 11 D liver samples from TNF-WD-Gal-treated mice exposed to room air and CO, respectively, and stained with H & E.
  • FIGS. 11 E and 11 F liver samples from TNF- ⁇ /D-Gal-treated mice exposed to room air and CO, respectively, and stained to detect activated caspase-3.
  • FIGS. 11 A and 11 B liver samples from mice exposed to room air and CO, respectively, and stained with hematoxylin & eosin (H & E).
  • FIGS. 11 C and 11 D liver samples from TNF-WD-Gal-treated mice exposed to room air and CO, respectively,
  • FIG. 12 is a picture of a Western blot illustrating that livers of mice exposed to TNF- ⁇ /D-Gal and treated with inhaled CO display increased iNOS protein levels.
  • FIGS. 13 A- 13 D are photomicrographs of liver samples illustrating that the livers of mice exposed to TNF- ⁇ /D-Gal and treated with inhaled CO display increased iNOS protein levels.
  • FIG. 13A liver sample from room air-exposed mouse.
  • FIG. 13B liver sample from CO-exposed mouse.
  • FIG. 13C liver sample from mouse exposed to TNF- ⁇ /D-Gal and room air.
  • FIG. 13D liver sample from mouse exposed to TNF- ⁇ /D-Gal and CO. Images are representative of 6 separate animals and 6-10 different sections/liver sample. Bar represents 20 ⁇ m.
  • FIG. 14 is a bar graph illustrating that CO does not protect against liver damage in the absence of iNOS function/expression.
  • FIG. 15 is a picture of a Western blot illustrating that the livers of CO-treated mice displayed increased expression of HO-1 in both the presence and absence of TNF- ⁇ /D-Gal.
  • CO carbon monoxide
  • Air room air
  • TNF TNF- 60 /D-Gal
  • ⁇ -Actin control protein. Blot is representative of 2 independent experiments.
  • FIG. 16 is a picture of a Western blot illustrating that the livers of CO-treated mice do not display increased expression of HO-1 in the presence or absence of TNF- ⁇ /D-Gal if iNOS is inhibited using L-NIL.
  • CO carbon monoxide
  • Air room air
  • TNF TNF- ⁇ /D-Gal
  • ⁇ -Actin control protein
  • L-NIL L-N6-(1-iminoethyl)-lysine-dihydrochloride (a selective inhibitor of iNOS). Blot is representative of 2 independent experiments.
  • FIG. 17 is a bar graph illustrating that CO-induced HO-1 is protective against TNF- ⁇ -induced liver damage in mice.
  • ALT serum alanine aminotransferase
  • Air room air
  • TNF TNF- ⁇ /D-Gal
  • Sn tin protoporphyrin (an inhibitor of HO-1)
  • VP V-PYRRO (a nitric oxide donor). Results are expressed as mean ⁇ SD of 8-10 mice/group. *p ⁇ 0.05 versus CO/TNF/D-gal-treated mice.
  • FIG. 18 is a bar graph illustrating that induction of HO-1 is protective against TNF- ⁇ -induced liver injury independent of iNOS activity.
  • ALT serum alanine aminotransferase
  • Air room air
  • TNF TNF- ⁇ /D-Gal
  • L-NIL L-N6-(i-iminoethyl)-lysine-dihydrochloride (a selective inhibitor of iNOS)
  • CoPP cobalt protoporphyrin (an inducer of HO-1);
  • iNOS ⁇ / ⁇ iNOS deficient mice. Results are mean ⁇ SD of 6-8 mice/group. *p ⁇ 0.001 versus Air/TNF and L-NIL/TNF.
  • FIG. 19 is bar graph illustrating that HO-1 expression is required for CO-induced protection of mouse hepatocytes from TNF-a/ActD-induced cell death.
  • Wild type black bars
  • hmox-1 ⁇ / ⁇ white bars
  • CO carbon monoxide
  • Air room air
  • TNF- ⁇ TNF- ⁇ /ActD.
  • FIG. 20 is bar graph illustrating that HO-1 expression is required for NO-induced protection of mouse hepatocytes from TNF- ⁇ /ActD-induced cell death.
  • Wild type black bars
  • hmox-1 ⁇ / ⁇ white bars
  • SNAP s-nitroso-N-acetyl-penicillaamine (an NO donor)
  • Air room air
  • TNF-A TNF- ⁇ /ActD. *p ⁇ 0.01 versus non-TNF- ⁇ /ActD treated cells and versus TNF- ⁇ /ActD-treated cells that were also treated with NO.
  • FIG. 21 is a bar graph illustrating that CO-exposed mice were protected from acetaminophen-induced liver injury.
  • ALT serum alanine aminotransferase
  • Air room air
  • APAP acetaminophen. Results are mean ⁇ SD of 4-8 mice/group.
  • carbon monoxide (or “CO”) as used herein describes molecular carbon monoxide in its gaseous state, compressed into liquid form, or dissolved in aqueous solution.
  • carbon monoxide composition and “pharmaceutical composition comprising carbon monoxide” is used throughout the specification to describe a gaseous or liquid composition containing carbon monoxide that can be administered to a patient and/or an organ, e.g., the liver.
  • an organ e.g., the liver.
  • the skilled practitioner will recognize which form of the pharmaceutical composition, e.g., gaseous, liquid, or both gaseous and liquid forms, is preferred for a given application.
  • effective amount and “effective to treat,” as used herein, refer to an amount or concentration of carbon monoxide utilized for period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome.
  • Effective amounts of carbon monoxide for use in the present invention include, for example, amounts that prevent hepatitis, reduce the risk of hepatitis, reduce the symptoms of hepatitis, or improve the outcome of other hepatitis treatments.
  • effective amounts of carbon monoxide generally fall within the range of about 0.0000001% to about 0.3% by weight, e.g., 0.0001% to about 0.25% by weight, preferably at least about 0.001%, e.g., at least 0.005%, 0.010%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.10%, 0.15%, 0.20%, 0.22%, or 0.24% by weight carbon monoxide.
  • Preferred ranges include, e.g., 0.001% to about 0.24%, about 0.005% to about 0.22%, about 0.005% to about 0.05%, about 0.010% to about 0.20%, about 0.02% to about 0.15%, about 0.025% to about 0.10%, or about 0.03% to about 0.08%, or about 0.04% to about 0.06%.
  • effective amounts generally fall within the range of about 0.0001 to about 0.0044 g CO/100 g liquid, e.g., at least 0.0001, 0.0002, 0.0004, 0.0006, 0.0008, 0.0010, 0.0013, 0.0014, 0.0015, 0.0016, 0.0018, 0.0020, 0.0021, 0.0022, 0.0024, 0.0026, 0.0028, 0.0030, 0.0032, 0.0035, 0.0037, 0.0040, or 0.0042 g CO/100 g aqueous solution.
  • Preferred ranges include, e.g., about 0.0010 to about 0.0030 g CO/100 g liquid, about 0.0015 to about 0.0026 g CO/100 g liquid, or about 0.0018 to about 0.0024 g CO/100 g liquid. A skilled practitioner will appreciate that amounts outside of these ranges may be used, depending upon the application.
  • patient is used throughout the specification to describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided.
  • Veterinary applications are contemplated by the present invention.
  • the term includes but is not limited to mammals, e.g., humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats.
  • treat(ment),” is used herein to describe delaying the onset of, inhibiting, or alleviating the effects of a condition, e.g., hepatitis, in a patient.
  • hepatitis is an art-recognized term and is used herein to refer to a disease of patients characterized in part by inflammation of the liver.
  • Causative agents of hepatitis include, for example, infections, e.g., infection with specific hepatitis viruses, e.g., hepatitis A, B, C, D, E, and G viruses; or hepatotoxic agents, e.g., hepatotoxic drugs (e.g., isoniazid, methyldopa, acetaminophen, amiodarone, and nitrofurantoin), and toxins (e.g., endotoxin or environmental toxins).
  • infections e.g., infection with specific hepatitis viruses, e.g., hepatitis A, B, C, D, E, and G viruses
  • hepatotoxic agents e.g., hepatotoxic drugs (e.g., isoniazid, methyld
  • Hepatitis may occur postoperatively in liver transplantation patients. Further examples of drugs and toxins that may cause hepatitis (i.e., hepatotoxic agents) are described in Feldman: Sleisenger & Fordtran's Gastrointestinal and Liver Disease, 7th ed., Chapter 17 (Liver Disease Caused by Drugs, Anesthetics, and Toxins), the contents of which are expressly incorporated herein by reference in their entirety.
  • Such examples include, but are not limited to, methyl dopa and phenytoin, barbiturates, e.g., phenobarbital; sulfonamides (e.g., in combination drugs such as co-trimoxazole (sulfamethoxazole and trimethoprim); sulfasalazine; salicylates; disulfiram; ⁇ -adrenergic blocking agents e.g., acebutolol, labetalol, and metoprolol); calcium channel blockers, e.g., nifedipine, verapamil, and diltiazem; synthetic retinoids, e.g., etretinate; gastric acid suppression drugs e.g., oxmetidine, ebrotidine, cimetidine, ranitidine, omeprazole and famotidine; leukotriene receptor antagonists, e.g.,
  • Symptoms of hepatitis can include fatigue, loss of appetite, stomach discomfort, and/or jaundice (yellowing of the skin and/or eyes). More detailed descriptions of hepatitis are provided, for example, in the The Merck Manual of Diagnosis and Therapy, 17 th Edition, Section 4, Chapter 42, Section 4, Chapter 44, and Section 4, Chapter 40, the contents of which are expressly incorporated herein by reference in their entirety.
  • a patient can be diagnosed by a physician as suffering from hepatitis by any method known in the art, e.g., by assessing liver function, e.g., using blood tests for serum alanine aminotransferase (ALT) levels, alkaline phosphatase (AP), or bilirubin levels.
  • ALT serum alanine aminotransferase
  • AP alkaline phosphatase
  • Individuals considered at risk for developing hepatitis may benefit particularly from the invention, primarily because prophylactic treatment can begin before there is any evidence of hepatitis.
  • Individuals “at risk” include, e.g., patients infected with hepatitis viruses, or individuals suffering from any of the conditions or having the risk factors described herein (e.g., patients exposed to hepatotoxic agents). The skilled practitioner will appreciate that a patient can be determined to be at risk for hepatitis by a physician's diagnosis.
  • Amounts of CO effective to treat hepatitis can be administered to a patient on the day the patient is diagnosed as suffering from hepatitis or any condition associated with hepatitis, or as having any risk factor associated with an increased likelihood that the patient will develop hepatitis (e.g., that the patient has recently been, is being, or will be exposed to a hepatotoxic agent, e.g., a hepatotoxic drug such as acetaminophen).
  • Patients can inhale CO at concentrations ranging from 10 ppm to 1000 ppm, e.g., about 100 ppm to about 800 ppm, about 150 ppm to about 600 ppm, or about 200 ppm to about 500 ppm.
  • Preferred concentrations include, e.g., about 30 ppm, 50 ppm, 75 ppm, 100 ppm, 125 ppm, 200 ppm, 250 ppm, 500 ppm, 750 ppm, or about 1000 ppm.
  • CO can be administered to the patient intermittently or continuously.
  • CO can be administered for about 1, 2, 4, 6, 8, 10, 12, 14, 18, or 20 days, or greater than 20 days, e.g., 1 2, 3, 5, or 6 months, or until the patient no longer exhibits symptoms of hepatitis, or until the patient is diagnosed as no longer being at risk for hepatitis.
  • CO can be administered continuously for the entire day, or intermittently, e.g., a single whiff of CO per day (where a high concentration is used), or for up to 23 hours per day, e.g., up to 20, 15, 12, 10, 6, 3, or 2 hours per day, or up to 1 hour per day.
  • the patient can be treated with CO (e.g., a gaseous CO composition) before, during, and/or after administration of the drug.
  • CO e.g., a gaseous CO composition
  • CO can be administered to the patient, intermittently or continuously, starting 0 to 20 days before the drug is administered (and where multiple doses are given, before each individual dose), e.g., starting at least about 30 minutes, e.g., about 1, 2, 3, 5, 7, or 10 hours, or about 1, 2, 4, 6, 8, 10, 12, 14, 18, or 20 days, or greater than 20 days, before the administration.
  • CO can be administered to the patient concurrent with administration of the drug.
  • CO can be administered to the patient after administration of the drug, e.g., starting immediately after administration, and continuing intermittently or continuously for about 1, 2, 3, 5, 7, or 10 hours, or about 1, 2, 5, 8, 10, 20, 30, 50, or 60 days, indefinitely, or until a physician determines that administration of CO is no longer necessary (e.g., after the hepatotoxic drug is eliminated from the body or can no longer cause damage to the liver).
  • a carbon monoxide composition may be a gaseous carbon monoxide composition.
  • Compressed or pressurized gas useful in the methods of the invention can be obtained from any commercial source and in any type of vessel appropriate for storing compressed gas.
  • compressed or pressurized gases can be obtained from any source that supplies compressed gases, such as oxygen, for medical use.
  • the term “medical grade” gas, as used herein, refers to gas suitable for administration to patients as defined herein.
  • the pressurized gas including CO used in the methods of the present invention can be provided such that all gases of the desired final composition (e.g., CO, He, NO, CO 2 , O 2 , N 2 ) are in the same vessel, except that NO and O 2 cannot be stored together.
  • the methods of the present invention can be performed using multiple vessels containing individual gases.
  • a single vessel can be provided that contains carbon monoxide, with or without other gases, the contents of which can be optionally mixed with room air or with the contents of other vessels, e.g., vessels containing oxygen, nitrogen, carbon dioxide, compressed air, or any other suitable gas or mixtures thereof.
  • Gaseous compositions administered to a patient according to the present invention typically contain 0% to about 79% by weight nitrogen, about 21% to about 100% by weight oxygen and about 0.0000001% to about 0.3% by weight (corresponding to about 1 ppb or 0.001 ppm to about 3,000 ppm) carbon monoxide.
  • the amount of nitrogen in the gaseous composition is about 79% by weight
  • the amount of oxygen is about 21% by weight
  • the amount of carbon monoxide is about 0.0001% to about 0.25% by weight, preferably at least about 0.001%, e.g., at least about 0.005%, 0.010%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.10%, 0.15%, 0.20%, 0.22%, or 0.24% by weight.
  • Preferred ranges of carbon monoxide include about 0.005% to about 0.24%, about 0.01% to about 0.22%, about 0.015% to about 0.20%, about 0.08% to about 0.20%, and about 0.025% to about 0.1% by weight.
  • gaseous carbon monoxide compositions having concentrations of carbon monoxide greater than 0.3% may be used for short periods (e.g., one or a few breaths), depending upon the application.
  • a gaseous carbon monoxide composition may be used to create an atmosphere that comprises carbon monoxide gas.
  • An atmosphere that includes appropriate levels of carbon monoxide gas can be created, for example, by providing a vessel containing a pressurized gas comprising carbon monoxide gas, and releasing the pressurized gas from the vessel into a chamber or space to form an atmosphere that includes the carbon monoxide gas inside the chamber or space.
  • the gases can be released into an apparatus that culminates in a breathing mask or breathing tube, thereby creating an atmosphere comprising carbon monoxide gas in the breathing mask or breathing tube, ensuring the patient is the only person in the room exposed to significant levels of carbon monoxide.
  • Carbon monoxide levels in an atmosphere can be measured or monitored using any method known in the art. Such methods include electrochemical detection, gas chromatography, radioisotope counting, infrared absorption, colorimetry, and electrochemical methods based on selective membranes (see, e.g., Sunderman et al., Clin. Chem. 28:2026-2032, 1982; Ingi et al., Neuron 16:835-842, 1996). Sub-parts per million carbon monoxide levels can be detected by, e.g., gas chromatography and radioisotope counting.
  • carbon monoxide levels in the sub-ppm range can be measured in biological tissue by a midinfrared gas sensor (see, e.g., Morimoto et al., Am. J. Physiol. Heart. Circ. Physiol 280:H482-H488, 2001). Carbon monoxide sensors and gas detection devices are widely available from many commercial sources.
  • a carbon monoxide composition may also be a liquid carbon monoxide composition.
  • a liquid can be made into a carbon monoxide composition by any method known in the art for causing gases to become dissolved in liquids.
  • the liquid can be placed in a so-called “CO 2 incubator” and exposed to a continuous flow of carbon monoxide, preferably balanced with carbon dioxide, until a desired concentration of carbon monoxide is reached in the liquid.
  • carbon monoxide gas can be “bubbled” directly into the liquid until the desired concentration of carbon monoxide in the liquid is reached. The amount of carbon monoxide that can be dissolved in a given aqueous solution increases with decreasing temperature.
  • an appropriate liquid may be passed through tubing that allows gas diffusion, where the tubing runs through an atmosphere comprising carbon monoxide (e.g., utilizing a device such as an extracorporeal membrane oxygenator).
  • the carbon monoxide diffuses into the liquid to create a liquid carbon monoxide composition.
  • the liquid can be any liquid known to those of skill in the art to be suitable for administration to patients (see, for example, Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press (1994)).
  • the liquid will be an aqueous solution.
  • solutions include Phosphate Buffered Saline (PBS), CelsiorTM, PerfadexTM, Collins solution, citrate solution, and University of Wisconsin (UW) solution (Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press (1994)).
  • the liquid is Ringer's Solution, e.g., lactated Ringer's Solution, or any other liquid that can be used infused into a patient.
  • the liquid includes blood, e.g., whole blood.
  • Any suitable liquid can be saturated to a set concentration of carbon monoxide via gas diffusers.
  • pre-made solutions that have been quality controlled to contain set levels of carbon monoxide can be used. Accurate control of dose can be achieved via measurements with a gas permeable, liquid impermeable membrane connected to a carbon monoxide analyzer. Solutions can be saturated to desired effective concentrations and maintained at these levels.
  • a patient can be treated with a carbon monoxide composition by any method known in the art of administering gases and/or liquids to patients.
  • Carbon monoxide compositions can be administered to a patient diagnosed with, or determined to be at risk for, hepatitis.
  • the present invention contemplates the systemic administration of liquid or gaseous carbon monoxide compositions to patients (e.g., by inhalation and/or ingestion), and the topical administration of the compositions to the patient's liver (e.g., by introduction into the abdominal cavity).
  • Gaseous carbon monoxide compositions can be delivered systemically to a patient, e.g., a patient diagnosed with, or determined to be at risk for hepatitis. Gaseous carbon monoxide compositions are typically administered by inhalation through the mouth or nasal passages to the lungs, where the carbon monoxide is readily absorbed into the patient's bloodstream.
  • concentration of active compound (CO) utilized in the therapeutic gaseous composition will depend on absorption, distribution, inactivation, and excretion (generally, through respiration) rates of the carbon monoxide as well as other factors known to those of skill in the art.
  • Medical grade carbon monoxide (concentrations can vary) can be purchased mixed with air or another oxygen-containing gas in a standard tank of compressed gas (e.g., 21% O 2 , 79% N 2 ). It is non-reactive, and the concentrations that are required for the methods of the present invention are well below the combustible range (10% in air). In a hospital setting, the gas presumably will be delivered to the bedside where it will be mixed with oxygen or house air in a blender to a desired concentration in ppm (parts per million). The patient will inhale the gas mixture through a ventilator, which will be set to a flow rate based on patient comfort and needs.
  • a ventilator which will be set to a flow rate based on patient comfort and needs.
  • Fail-safe mechanism(s) to prevent the patient from unnecessarily receiving greater than desired amounts of carbon monoxide can be designed into the delivery system.
  • the patient's carbon monoxide level can be monitored by studying (1) carboxyhemoglobin (COHb), which can be measured in venous blood, and (2) exhaled carbon monoxide collected from a side port of the ventilator.
  • COHb carboxyhemoglobin
  • Carbon monoxide exposure can be adjusted based upon the patient's health status and on the basis of the markers. If necessary, carbon monoxide can be washed out of the patient by switching to 100% O 2 inhalation.
  • Carbon monoxide is not metabolized; thus, whatever is inhaled will ultimately be exhaled except for a very small percentage that is converted to CO 2 .
  • Carbon monoxide can also be mixed with any level of O 2 to provide therapeutic delivery of carbon monoxide without consequential hypoxic conditions.
  • Face Mask and Tent A carbon monoxide-containing gas mixture is prepared as above to allow passive inhalation by the patient using a facemask or tent.
  • the concentration inhaled can be changed and can be washed out by simply switching over to 100% O 2 .
  • Monitoring of carbon monoxide levels would occur at or near the mask or tent with a fail-safe mechanism that would prevent too high of a concentration of carbon monoxide from being inhaled.
  • Compressed carbon monoxide can be packaged into a portable inhaler device and inhaled in a metered dose, for example, to permit intermittent treatment of a recipient who is not in a hospital setting.
  • Different concentrations of carbon monoxide could be packaged in the containers.
  • the device could be as simple as a small tank (e.g., under 5 kg) of appropriately diluted CO with an on-off valve and a tube from which the patient takes a whiff of CO according to a standard regimen or as needed.
  • An artificial lung (a catheter device for gas exchange in the blood) designed for O 2 delivery and CO 2 removal can be used for carbon monoxide delivery.
  • the catheter when implanted, resides in one of the large veins and would be able to deliver carbon monoxide at given concentrations either for systemic delivery or at a local site.
  • the delivery can be a local delivery of a high concentration of carbon monoxide for a short period of time at the site of the procedure, e.g., in proximity to the liver (this high concentration would rapidly be diluted out in the bloodstream), or a relatively longer exposure to a lower concentration of carbon monoxide (see, e.g., Hattler et al., Artif. Organs 18(11):806-812 (1994); and Golob et al., ASAIO J., 47(5):432-437 (2001)).
  • the patient would be inside an airtight chamber that would be flooded with carbon monoxide (at a level that does not endanger the patient, or at a level that poses an acceptable risk without the risk of bystanders being exposed.
  • the chamber could be flushed with air (e.g., 21% O 2 , 79% N 2 ) and samples could be analyzed by carbon monoxide analyzers to ensure no carbon monoxide remains before allowing the patient to exit the exposure system.
  • aqueous solutions comprising carbon monoxide can be created for systemic delivery to a patient, e.g., for oral delivery and/or by infusion into the patient, e.g., intravenously, intra-arterially, intraperitoneally, and/or subcutaneously.
  • liquid CO compositions such as CO-saturated Ringer's Solution
  • CO-partially or completely saturated whole (or partial) blood can be infused into the patient.
  • the present invention also contemplates that agents capable of delivering doses of gaseous CO compositions or liquid CO compositions can be utilized (e.g., CO-releasing gums, creams, ointments, lozenges, or patches).
  • agents capable of delivering doses of gaseous CO compositions or liquid CO compositions can be utilized (e.g., CO-releasing gums, creams, ointments, lozenges, or patches).
  • carbon monoxide compositions can be applied directly to the liver, e.g., to the entire liver, or to any portion thereof.
  • a gaseous composition can be directly applied to the liver of a patient by any method known in the art for insufflating gases into a patient.
  • gases e.g., carbon dioxide
  • gases are often insufflated into the abdominal cavity of patients to facilitate examination during laproscopic procedures (see, e.g., Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press (1994)).
  • gases e.g., carbon dioxide
  • the skilled practitioner will appreciate that similar procedures could be used to administer carbon monoxide compositions directly to the liver of a patient.
  • Aqueous carbon monoxide compositions can also be administered topically to the liver of a patient.
  • Aqueous forms of the compositions can be administered by any method known in the art for administering liquids to patients.
  • aqueous compositions can be applied directly to the liver.
  • liquids e.g., saline solutions containing dissolved CO
  • saline solutions containing dissolved CO can be injected into the abdominal cavity of patients during laproscopic procedures.
  • similar procedures could be used to administer liquid carbon monoxide compositions directly to the liver of a patient.
  • an in situ exposure can be carried out by flushing the liver or a portion thereof with a liquid carbon monoxide composition (see Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press (1994)).
  • HO-1 hemeoxygenase-1
  • induction or expression of hemeoxygenase-1 (HO-1) in conjunction with administration of CO can be induced in a patient suffering from or at risk for hepatitis.
  • induce(d) means to cause increased production of a protein, e.g., HO-1, in isolated cells or the cells of a tissue, organ or animal using the cells' own endogenous (e.g., non-recombinant) gene that encodes the protein.
  • HO-1 can be induced in a patient by any method known in the art.
  • production of HO-1 can be induced by hemin, by iron protoporphyrin, or by cobalt protoporphyrin.
  • non-heme agents including heavy metals, cytokines, hormones, NO, COCl 2 , endotoxin and heat shock are also strong inducers of HO-1 expression (Choi et al., Am. J. Respir. Cell Mol. Biol. 15:9-19, 1996; Maines, Annu. Rev. Pharmacol. Toxicol. 37:517-554, 1997; and Tenhunen et al., J. Lab. Clin. Med. 75:410-421, 1970).
  • HO-1 is also highly induced by a variety of agents causing oxidative stress, including hydrogen peroxide, glutathione depletors, UV irradiation, endotoxin and hyperoxia (Choi et al., Am. J. Respir. Cell Mol. Biol. 15:9-19, 1996; Maines, Annu. Rev. Pharmacol. Toxicol. 37:517-554, 1997; and Keyse et al., Proc. Natl. Acad. Sci. USA 86:99-103, 1989).
  • a “pharmaceutical composition comprising an inducer of HO-1” means a pharmaceutical composition containing any agent capable of inducing HO-1 in a patient, e.g., any of the agents described above, e.g., NO, hemin, iron protoporphyrin, and/or cobalt protoporphyrin.
  • HO-1 expression in a cell can be increased via gene transfer.
  • the term “express(ed)” means to cause increased production of a protein, e.g., HO-1 or ferritin, in isolated cells or the cells of a tissue, organ or animal using an exogenously administered gene (e.g., a recombinant gene).
  • the HO-1 or ferritin is preferably of the same species (e.g., human, mouse, rat, etc.) as the recipient, in order to minimize any immune reaction.
  • Expression could be driven by a constitutive promoter (e.g., cytomegalovirus promoters) or a tissue-specific promoter (e.g., milk whey promoter for mammary cells or albumin promoter for liver cells).
  • An appropriate gene therapy vector e.g., retrovirus, adenovirus, adeno associated virus (AAV), pox (e.g., vaccinia) virus, human immunodeficiency virus (HIV), the minute virus of mice, hepatitis B virus, influenza virus, Herpes Simplex Virus-1, and lentivirus
  • a gene therapy vector e.g., retrovirus, adenovirus, adeno associated virus (AAV), pox (e.g., vaccinia) virus, human immunodeficiency virus (HIV), the minute virus of mice, hepatitis B virus, influenza virus, Herpes Simplex Virus-1, and lentivirus
  • exogenous HO-1 protein can be directly administered to a patient by any method known in the art.
  • Exogenous HO-1 can be directly administered in addition, or as an alternative, to the induction or expression of HO-1 in the patient as described above.
  • the HO-1 protein can be delivered to a patient, for example, in liposomes, and/or as a fusion protein, e.g., as a TAT-fusion protein (see, e.g., Becker-Hapak et al., Methods 24:247-256, 2001).
  • any of the products of metabolism by HO-1 e.g., bilirubin, biliverdin, iron, and/or ferritin
  • HO-1 e.g., bilirubin, biliverdin, iron, and/or ferritin
  • iron-binding molecules other than ferritin e.g., desferoxamine (DFO), iron dextran, and/or apoferritin
  • DFO desferoxamine
  • the present invention contemplates that catalyze the breakdown any of these products can be inhibited to create/enhance the desired effect. Any of the above can be administered, e.g., orally, intravenously, intraperitoneally, or by direct administration to the liver.
  • the present invention contemplates that compounds that release CO into the body after administration of the compound (e.g., CO-releasing compounds, e.g., photoactivatable CO-releasing compounds), e.g., dimanganese decacarbonyl, tricarbonyldichlororuthenium (II) dimer, and methylene chloride (e.g., at a dose of between 400 to 600 mg/kg, e.g., about 500 mg/kg), can also be used in the methods of the present invention, as can carboxyhemoglobin and CO-donating hemoglobin substitutes.
  • CO-releasing compounds e.g., photoactivatable CO-releasing compounds
  • dimanganese decacarbonyl e.g., tricarbonyldichlororuthenium (II) dimer
  • methylene chloride e.g., at a dose of between 400 to 600 mg/kg, e.g., about 500 mg/kg
  • the above can be administered to a patient in any way, e.g., by oral, intraperitoneal, intravenous, or intraarterial administration. Any of the above compounds can be administered to the patient locally and/or systemically, and in any combination.
  • the present invention further contemplates treating/preventing hepatitis by administering CO to the patient in combination with any other known methods or compounds for treating hepatitis, e.g., cessation or reducing administration of causative drugs; administering corticosteroids and/or a-interferon or other antiviral agents to the patient; and/or performing surgery on the patient, e.g., liver transplantation.
  • any other known methods or compounds for treating hepatitis e.g., cessation or reducing administration of causative drugs
  • administering corticosteroids and/or a-interferon or other antiviral agents e.g., a-interferon or other antiviral agents
  • mice Male C57BL/6J (Charles Rivers Laboratories, Bar Harbor, Me.), 8-12-wk-old inos ⁇ / ⁇ mice and wild type littermates (bred/maintained at the University of Pittsburgh) were used for in vivo experiments.
  • mice were administered TNF- ⁇ /D-gal (0.3 ⁇ g/8 mg/mouse, i.p., respectively).
  • some mice received CO (250 ppm), the selective NO donor O 2 -vinyl 1-(pyrrolidin-1-yl) diazen-1-ium-1,2-diolate (V-PYRRO; 10 mg/kg subcutaneously (s.c.), Alexis Biochem., San Diego, Calif.) or cobalt protoporphyrin (CoPP, 5 mg/kg, intraperitoneally (i.p.), Frontier Scientific, Logan, Utah).
  • V-PYRRO the selective NO donor O 2 -vinyl 1-(pyrrolidin-1-yl) diazen-1-ium-1,2-diolate
  • CoPP cobalt protoporphyrin
  • L-NIL L-N6-(1-iminoethyl)-lysine-dihydrochloride
  • SnPP HO-1 inhibitor tin protoporphyrin
  • acetaminophen Sigma Chem. Co.; St Louis, Mo. was administered (500 mg/kg, i.p.).
  • Mouse primary hepatocytes were harvested from C57BL/6J, mkk3 ⁇ / ⁇ , inos ⁇ / ⁇ (in-house breeding colony), or hmox-1 ⁇ / ⁇ mice as described in Kim et al. (J. Biol. Chem. 272: 1402-1411 (1997)). Hepatocytes were used on days 1-3 following harvest.
  • TNF- ⁇ 10 ng/ml
  • Act-D actinomycin-D
  • TNF- ⁇ /ActD treatment has been demonstrated to induce cell death, specifically apoptosis, in primary hepatocytes (see, e.g., Kim et al. (J. Biol. Chem. 272: 1402-1411 (1997)).
  • Hepatocytes were treated with CO, the NO donor s-nitroso-N-acetyl-penicillamine (SNAP; 250-750 ⁇ M), and/or additional pharmacologic agents where indicated.
  • SNAP NO donor s-nitroso-N-acetyl-penicillamine
  • hepatocytes were co-transfected with NF- ⁇ B reporter constructs (pGL3-kappa ⁇ luciferase, 100 ng/well; and pIEP-Lac-z 0.5 ⁇ g/well) using LipofectinTM (Invitrogen, Carlsbad, Calif.) as instructed by the manufacturer. Evaluation of iNOS expression was performed using a luciferase reporter assay as described in Lowenstein et al. (Proc. Natl. Acad. Sci. U.S.A 90: 9730-9734 (1993)). Briefly, hepatocytes were co-transfected with iNOS promoter reporter constructs (pXP2; 1 ⁇ g/well) and pIEP-LacZ (0.5 ⁇ g/well) as described above.
  • Hepatocytes were transfected with plasmids as described above and treated with various stimuli 24 hours after transfection.
  • Luciferase activity (reported as arbitrary units; A.U.) was assayed 6 hours after initiation of treatment, using a luciferase assay kit (Promega, Madison, Wis.) and a Berthold Luminometer. Results were corrected for transfection efficiency and protein concentration.
  • Nuclei were extracted from hepatocytes following treatment.
  • a double-stranded DNA NF- ⁇ B consensus sequence (GGGGACTTTCCC (SEQ ID NO: 1)); Santa Cruz Biotechnology, Santa Cruz, Calif.) was labelled with [ ⁇ - 32 P]-ATP and incubated with 5 mg of total nuclear protein. Some incubations were performed in the presence of antibodies against p65/RelA or p50 (Santa Cruz Biotech) to evaluate for supershift.
  • Electrophoretic mobility shift assay (EMSAs) were performed as described in Taylor et al. (J. Biol. Chem. 273:15148-15156 (1998)).
  • livers were fixed in 2% paraformaldehyde and then snap frozen in liquid nitrogen. Livers were then sectioned (7 microns thick) and stained with hematoxylin and eosin (H&E). Liver sections were also stained for TUNEL and activated caspase-3 using kits according to the manufacturer's instructions (Promega). Sections for iNOS immunocytochemistry were blocked with 5% goat serum containing 0.2% bovine serum albumin. Thereafter, sections were incubated for 1 hour at room temperature with anti-iNOS antibody (Transduction Laboratories; 1:300), then washed and probed with a secondary antibody conjugated to Alexa-488 (Molecular Probes, Eugene, Oreg.).
  • Nuclei were stained with Hoechst dye. Images were acquired using an Olympus Provus microscope. Hepatocytes in culture were plated on gelatinized coverslips, stimulated as indicated, and then fixed in 2% paraformaldehyde containing 0.1% Triton X-100. Blocking and staining was similar to liver sections except anti-p65/RelA antibody (Santa Cruz Biotechnology; 1:350) was utilized.
  • the animals were exposed to CO at a concentration of 250 ppm. Briefly, 1% CO in air was mixed with air (21% oxygen) in a stainless steel mixing cylinder and then directed into a 3.70 ft 3 glass exposure chamber at a flow rate of 12 L/min. A CO analyzer (Interscan, Chatsworth, Calif.) was used to measure CO levels continuously in the chamber. CO concentrations were maintained at 250 ppm at all times. Mice were placed in the exposure chamber as required.
  • HO-1 Whether HO-1 is protective against acute hepatic failure was investigated. The results are presented in FIG. 1. Cobalt protoporphyrin (5 mg/kg, i.p.) was administered to male C57BL/6J mice. Twenty-four hours later, TNF- ⁇ /D-gal (0.3 ⁇ g/8 mg/mouse, i.p., respectively) was administered to the mice. Serum alanine aminotransferase (ALT) levels in the mice were measured 8 hours after administration of TNF- ⁇ /D-gal. Induction of HO-1 prevented liver injury as measured by serum ALT levels.
  • Adenoviral experiments involved incubating hepatocytes overnight with 10 pfu/cell of the adenovirus prior to addition of TNF- ⁇ /ActD, and then assaying for viability using crystal violet.
  • the roles of signaling molecules guanylyl cyclase and p38 MAPK were also investigated in this model.
  • hepatocytes were treated separately with the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Calbiochem; 2-10 ⁇ M) or the NF- ⁇ B inhibitor BAY 11-7082, (10 ⁇ M).
  • sGC soluble guanylate cyclase
  • FIGS. 4, 5, and 6 A- 6 C present data illustrating that that CO induced an increase in NF- ⁇ B nuclear translocation and DNA binding in mouse hepatocytes as measured by NF- ⁇ B luciferase reporter assay activity, EMSA, and immunostaining for RelA/p65 nuclear translocation, respectively.
  • NF- ⁇ B activation was performed using a luciferase reporter assay as described in Chow et al. (J. Biol. Chem. 274: 10689-10692 (1999)). Briefly, hepatocytes were co-transfected with NF-KB reporter constructs and pIEP-Lac-z 24 hr prior to addition of BAY 11-7082 (10 ⁇ M) or vehicle. Cells were incubated for 1 hr prior to CO (250 ppm).
  • Luciferase activity (reported as arbitrary units; A.U.) was assayed 6 hr after exposure to CO or a cytokine mixture (CM) composed of TNF- ⁇ (500 U/ml), IL-1 ⁇ (100 U/ml), and IFN- ⁇ (100 U/ml), which was used as a positive control for NF- ⁇ B activation. Results were corrected for transfection efficiency and protein concentration.
  • CM cytokine mixture
  • NF-KB DNA binding was evaluated using EMSA in hepatocytes treated with CO (250 ppm). Note the time-dependent increase in NF- ⁇ B binding (total) with expression peaking at one hr (Lanes 1, 4, 7). Extracts were then supershifted to identify the different NF- ⁇ B dimers using antibodies against p50 (Lanes 2, 5, 8) and p65 (Lanes 3, 6, 9).
  • FIGS. 6 A- 6 C To generate the data in FIGS. 6 A- 6 C, primary hepatocytes were immunostained for nuclear p65 localization following exposure to 1 hr CO (250 ppm). Images depict nuclear translocation of NF- ⁇ B (arrows pointing to green nuclei that depict the translocation of NF- ⁇ B) in both CM (used as a positive control) and CO-treated cells versus no localization in air treated cells (arrows pointing to blue nuclei).
  • NF- ⁇ B luciferase reporter assay activity peaked one hour after placing cells in the CO atmosphere.
  • a cytokine mixture (CM) was included in the treatment groups as a positive signal as well as a standard for maximum reporter activity by which to evaluate the effects of CO.
  • Transfection efficiency in primary hepatocytes is difficult, but the reporter activity was very significant (*p ⁇ 0.001 versus control).
  • NF- ⁇ B activity is needed for protection mediated by CO
  • adenoviral gene transfer of I ⁇ B ⁇ was utilized to prevent NF- ⁇ B translocation and BAY 11-7082 (1-10 mM, Calbiochem) was used to inhibit NF- ⁇ B activation.
  • the protective effects of CO were abrogated by inhibition of NF- ⁇ B activation.
  • iNOS protein was evaluated using immunoblotting techniques. Briefly, cell extracts from hepatocytes were treated with TNF- ⁇ /ActD for 6-8 hr in the presence and absence of CO (250 ppm). Control cells received air or CO alone. Note in FIG. 8 that TNF- ⁇ induces iNOS expression minimally, while those cells treated with TNF- ⁇ in the presence of CO show a significantly greater induction in iNOS protein.
  • mouse hepatocytes were isolated from inos ⁇ / ⁇ or from wild type C57BL/6J mice, which were then pre-treated for 1 hr with L-NIO (1 mM) to inhibit iNOS prior to CO administration.
  • L-NIO 1 mM
  • Those groups exposed to CO received a one-hour pretreatment prior to addition of TNF- ⁇ /ActD and were then returned to CO exposure.
  • CO did not provide protection against cell death, as evaluated via crystal violet exclusion 12 hr later, in cells where iNOS expression was absent or inhibited.
  • FIG. 7 Exposure of hepatocytes to CO produced a highly significant increase in activity in an iNOS luciferase reporter assay (FIG. 7). Again, a cytokine mixture was used as both a positive control in these low efficiency transfections and as a standard by which to evaluate the effects of CO. Consistent with the NF- ⁇ B dependence of iNOS expression, decreased reporter activity was observed in hepatocytes treated with BAY 11-7082 (FIG. 7). Additionally, iNOS protein was markedly increased in response to TNF-a in the presence of CO compared to TNF-A alone (FIG. 8).
  • mice were pre-treated with CO (250 ppm) for one hour prior to receiving TNF- ⁇ /D-gal (0.3 ⁇ g/8 mg/mouse; i.p., respectively). After receiving TNF- ⁇ /D-gal, mice were returned to the CO exposure chamber and their serum was analyzed for ALT levels 6-8 hr later. Without exposure to CO, liver failure occurred in 6-8 hr driven primarily by apoptosis of hepatocytes as in the in vitro model described above. Serum ALT in mice treated with CO was 74% lower than in air-exposed mice.
  • liver samples from mice treated with TNF- ⁇ /D-gal in the presence and absence of CO 250 ppm were sectioned and stained for hematoxylin & eosin (H&E), activated caspase 3 (as indicated by an increase in red intensity), and for TUNEL positive cells (as demarcated by the increased green cellular staining; a marker of cell death). Nuclei stained blue. Exposure to CO markedly reduced TNF- ⁇ /D-gal-induced liver damage as assessed by H&E staining.
  • LPS lipopolysaccharide
  • endotoxin lipopolysaccharide
  • results discussed above were confirmed using lipopolysaccharide (LPS, also referred to as endotoxin) in place of TNF.
  • LPS/D-Gal administration resulted in an increase in serum ALT levels from a control level of 20+/ ⁇ 5 IU/ml to >1000 IU/ml, as measured 8 hours following LPS/D-Gal administration.
  • the increase in ALT was reduced by >75%, to 250+/ ⁇ 75 IU/ml.
  • serum interleukin-6 was measured, and found to be reduced 65% in animals breathing CO vs air-breathing controls (data not shown).
  • mice Male C57BL/6J mice were treated with air or CO (250 ppm) 1 hr prior to TNF- ⁇ /D-gal (0.3 ⁇ g/8 mg/mouse, i.p., respectively) administration. Six hours later, livers were harvested to evaluate iNOS expression by immunoblotting. Results show that iNOS expression was increased modestly in air/TNF- ⁇ /D-gal-treated mice, but was markedly increased in mice treated with TNF- ⁇ /D-gal and CO. As expected, inos ⁇ / ⁇ mice showed no expression of iNOS protein.
  • mice liver sections were immunostained for iNOS expression.
  • the liver sections were obtained from mice treated with TNF- ⁇ /D-gal in the presence or absence of CO, and from air and CO controls that received no TNF- ⁇ /D-gal. Livers from mice exposed to CO and not receiving TNF- ⁇ /D-gal displayed a modest increase in iNOS expression. However, a significantly greater increase in expression (indicated by an increase in green-stained cells) was observed in livers from mice that were exposed to CO and received TNF- ⁇ /D-gal. The increased expression appeared to be localized around blood vessels.
  • mice were administered L-NIL (5 mg/kg, i.p.) 2 hr prior to pre-treatment with CO (250 ppm) and every 2 hr thereafter. Control mice received L-NIL and remained in room air. Note in FIG. 16 that CO increased HO-1 expression in vehicle-treated mice, but was unable to induce expression when iNOS was inhibited. L-NIL treatment alone had a minimal effect on HO-1 expression.
  • mice were given SnPP (50 ⁇ mol/kg, s.c.), the selective inhibitor of HO-1, 5 hr prior to CO.
  • the mice were given VPYRRO (VP), an NO donor (10 mg/kg, s.c.).
  • VP was selectively designed to deliver NO directly to the liver.
  • One hour after the initial VP dose the animals were exposed to CO for 1 hr prior to administration of TNF- ⁇ /D-gal (see above). Serum ALT levels were determined 6-8 hr later. Note that CO was not able to provide protection in animals where HO-1 activity was blocked.
  • VP when administered 2 hr prior and then every 2 hr thereafter, provided protection against injury as determined 8 hour later by serum ALT measurements.
  • mice were treated with the pharmacological NO donor V-PYRRO/NO. This agent is metabolized by the liver, resulting in release of NO by hepatocytes.
  • V-PYRRO/NO also provides protection following LPS/D-gal or TNF- ⁇ /D-gal administration. Mice were randomized and treated with TNF- ⁇ /D-gal with or without SnPP to evaluate the role of HO-1.
  • V-PYYRO/NO was protective, as assayed by serum ALT. However, SnPP abrogated the ability of this NO donor to protect against liver damage (FIG. 17). Thus, it appears that CO- or NO-initiated hepatoprotection is at least partially dependent on HO-1.
  • mouse hepatocytes were isolated from HO-1 null mice (hmox-1 ⁇ / ⁇ ) and wild type (C57BL/6J) littermates, pretreated for 1 hour with CO (250 ppm), and treated with TNF- ⁇ /ActD. Viability was assayed as described above. CO significantly protected wild type hepatocytes, but was unable to protect hepatocytes isolated from hmox-1 ⁇ / ⁇ mice.
  • mouse hepatocytes were isolated from HO-1 null mice (hmox-1 ⁇ / ⁇ ) and wild type (C57BL/6J) littermates, pretreated with the NO donor SNAP (500 ⁇ M), and then treated with TNF- ⁇ /ActD 1 hour later.
  • NO donor SNAP 500 ⁇ M
  • TNF- ⁇ /ActD TNF- ⁇ /ActD 1 hour later.
  • SNAP has been demonstrated to protect hepatocytes in this model.
  • SNAP significantly protected against cell death in wild type hepatocytes but did not provide significant protection against cell death in hepatocytes isolated from hmox-1 ⁇ / ⁇ mice.
  • mice Male C57BL/6J mice were exposed to CO (250 ppm) either 1 hr prior or 4 hr post administration of APAP (500 mg/kg, i.p.). The mice were then maintained in CO for the duration of the experiment. Serum ALT levels were determined 20 hr after APAP administration. Control mice received APAP and were maintained in air. This protocol was designed to allow hepatitis to develop for four hours before administering CO. CO significantly reduced damage to the liver as assessed by serum ALT (622 ⁇ 44 vs 175 ⁇ 137, p ⁇ 0.01 as compared to controls). This protection was similar to that observed in a separate group of animals that had been pre-treated with CO prior to APAP. These data support the therapeutic use of CO in a clinically relevant situation where treatment would begin after the initiation of hepatitis.
  • CO mediated protection operates by activating NF- ⁇ B, which in the presence of an inflammatory stimulus leads to the up-regulation of iNOS with the consequent production of NO.
  • NF- ⁇ B dependent antiapoptotic/protective genes may be induced.
  • significant activation of NF- ⁇ B was present, which could be part of the priming of the cellular apparatus discussed above.
  • the activation of NF- ⁇ B by CO may in part result from a mild increase in reactive oxygen species generation originating from the mitochondria (preliminary observations).
  • Consistent with the data in the APAP model are the results in a murine model of hemorrhagic shock where the therapeutic initiation of inhaled CO during resuscitation following a 2.5 hour shock phase resulted in protection against liver injury (>65% reduction in serum ALT at 24 hr p ⁇ 0.01; n 6-10/group).
  • the following example illustrates protocols for use in treating a patient diagnosed as suffering from hepatitis.
  • the example also illustrates protocols for treating patients before, during, and/or after surgical procedures, e.g., a surgical procedure to transplant a liver.
  • Skilled practitioners will appreciate that any protocol described herein can be adapted based on a patient's individual needs, and can be adapted to be used in conjunction with any other treatment for hepatitis.
  • Treatment of a patient with CO can begin on the day the patient is diagnosed as suffering from hepatitis, for example, hepatitis caused by viral infection and/or alcohol abuse.
  • the patient can be diagnosed by a physician using any art-known method.
  • a physician may make such a diagnosis using data obtained from blood tests, e.g., tests to determine serum ALT levels and tests to determine whether a patient is infected with a particular virus (e.g., any known hepatitis virus).
  • a physician may consider a patient's medical history in making such a diagnosis (e.g., by considering whether a patient is an alcoholic or a chronic drug user).
  • the patient can inhale CO at concentration of about 250 to 500 ppm for one hour per day. This treatment can continue for about 30 days, or until the patient is diagnosed as no longer having or being at risk for hepatitis.
  • the donor Prior to harvesting a liver or portion thereof, the donor can be treated with inhaled carbon monoxide (250 ppm) for one hour. Treatment can be administered at doses varying from 10 ppm to 1000 ppm for times varying from one hour to six hours, or for the entire period from the moment when it becomes possible to treat a brain-dead (cadaver) donor to the time the organ is removed. For a human donor, treatment should start as soon as possible following the declaration that brain death is present. In some applications, it may be desirable to begin treatment before brain death.
  • inhaled carbon monoxide 250 ppm
  • Treatment can be administered at doses varying from 10 ppm to 1000 ppm for times varying from one hour to six hours, or for the entire period from the moment when it becomes possible to treat a brain-dead (cadaver) donor to the time the organ is removed.
  • cadaver brain-dead
  • treatment should start as soon as possible following the declaration that brain death is present. In some applications, it may be desirable to begin
  • the live donor animal can be treated with relatively high levels of inhaled carbon monoxide, as desired, so long as the carboxyhemoglobin so produced does not compromise the viability and function of the organ to be transplanted.
  • levels greater than 500 ppm e.g., 1000 ppm or higher, and up to 10,000 ppm, particularly for brief times.
  • a liver Before a liver is harvested from a donor, it can be flushed or perfused with a solution, e.g., a buffer or medium, while it is still in the donor.
  • a solution e.g., a buffer or medium
  • the intent is to flush the liver with a solution saturated with carbon monoxide and maintained in a carbon monoxide atmosphere so that the carbon monoxide content remains at saturation. Flushing can take place for a time period of at least 10 minutes, e.g., 1 hour, several hours, or longer.
  • the solution should ideally deliver the highest concentration of carbon monoxide possible to the cells of the liver (or portion thereof).
  • a liver can be preserved in a medium that includes carbon monoxide from the time it is removed from the donor to the time it is transplanted to the recipient. This can be performed by maintaining the liver in the medium comprising CO, or by perfusing it with such a medium. Since this occurs ex vivo rather than in an animal, very high concentrations of CO gas can be used (e.g., 10,000 ppm) to keep the medium saturated with CO.
  • Treatment of the recipient with CO can begin on the day of transplantation at least 30 minutes before surgery begins. Alternatively, it could begin at least 30 minutes before reperfusion of the organ in the recipient. It can be continued for at least 30 minutes, e.g., 1 hour.
  • Carbon monoxide doses between 10 ppm and 3000 ppm can be delivered for varying times, e.g., minutes or hours, and can be administered on the day of and on days following transplantation.
  • the patient can inhale a concentration of carbon monoxide, e.g., 3000 ppm, for three consecutive 10 second breath holds.
  • a lower concentration of the gas can be delivered intermittently or constantly, for a longer period of time, with regular breathing rather than breath holding.
  • Carboxyhemoglobin concentrations can be utilized as a guide for appropriate administration of carbon monoxide to a patient. Usually, treatments for recipients should not raise carboxyhemoglobin levels above those considered to pose an acceptable risk for a patient in need of a transplant.

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US13/803,330 US20130202720A1 (en) 2002-05-17 2013-03-14 Methods of treating hepatitis
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