U.S. patent application number 10/439632 was filed with the patent office on 2004-03-18 for methods of treating hepatitis.
Invention is credited to Choi, Augustine M. K., Otterbein, Leo E., Zuckerbraun, Brian.
Application Number | 20040052866 10/439632 |
Document ID | / |
Family ID | 29550139 |
Filed Date | 2004-03-18 |
United States Patent
Application |
20040052866 |
Kind Code |
A1 |
Otterbein, Leo E. ; et
al. |
March 18, 2004 |
Methods of treating hepatitis
Abstract
The present invention relates to a method of treating hepatitis
in a patient, which includes administering a pharmaceutical
composition that includes carbon monoxide to the patient.
Inventors: |
Otterbein, Leo E.; (New
Kensington, PA) ; Choi, Augustine M. K.; (Pittsburgh,
PA) ; Zuckerbraun, Brian; (Pittsburgh, PA) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Family ID: |
29550139 |
Appl. No.: |
10/439632 |
Filed: |
May 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60381527 |
May 17, 2002 |
|
|
|
Current U.S.
Class: |
424/699 |
Current CPC
Class: |
A61P 31/14 20180101;
A61K 33/00 20130101; A61P 31/12 20180101; A61K 45/06 20130101; A61P
1/16 20180101; Y02A 50/463 20180101; Y02A 50/30 20180101; A61K
33/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/699 |
International
Class: |
A61K 033/00 |
Goverment Interests
[0002] This invention was made with Government support under
National Institutes of Health Grant Nos. R01-GM-44100, HL 58688,
HL55330, HL60234, and AI42365. The Government has certain rights in
this invention.
Claims
What is claimed is:
1. A method of treating hepatitis in a patient, comprising:
identifying a patient diagnosed as suffering from hepatitis; and
administering to the patient a pharmaceutical composition
comprising an amount of carbon monoxide effective to treat
hepatitis in the patient.
2. The method of claim 1, wherein the pharmaceutical composition is
in gaseous form and is administered to the patient by
inhalation.
3. The method of claim 1, wherein the pharmaceutical composition is
in liquid form and is administered to the patient orally.
4. The method of claim 1, wherein the pharmaceutical composition is
administered directly to the abdominal cavity of the patient.
5. The method of claim 1, wherein the patient is infected with a
virus selected from the group consisting of: hepatitis A virus;
hepatitis B virus; hepatitis C virus; hepatitis D virus; hepatitis
E virus; and hepatitis G virus.
6. The method of claim 1, wherein the patient is an alcoholic.
7. The method of claim 1, further comprising administering to the
patient a treatment selected from the group consisting of:
withholding or reducing administration of hepatitis-inducing drugs;
and administering corticosteroids or antiviral agents to the
patient.
8. The method of claim 1, wherein the pharmaceutical composition is
administered by artificial lung.
9. The method of claim 1, wherein the pharmaceutical composition is
administered by an extracorporeal membrane gas exchange device.
10. The method of claim 1, wherein the hepatitis is caused by
exposure to a hepatotoxic agent.
11. A method of treating hepatitis in a patient, comprising: (a)
identifying a patient suffering from or at risk for hepatitis; (b)
providing a vessel containing a pressurized gas comprising carbon
monoxide gas; (c) releasing the pressurized gas from the vessel, to
form an atmosphere comprising carbon monoxide gas; and (d) exposing
the patient to the atmosphere, wherein the amount of carbon
monoxide in the atmosphere is sufficient to treat hepatitis in the
patient.
12. A method of administering a hepatotoxic drug to a patient,
comprising: (a) administering the hepatotoxic drug to the patient;
and (b) before, during, or after step (a), administering to the
patient a pharmaceutical composition comprising carbon monoxide in
an amount effective to treat hepatitis in the patient.
13. The method of claim 12, wherein carbon monoxide is administered
before step (a).
14. The method of claim 12, wherein carbon monoxide is administered
during step (a).
15. The method of claim 12, wherein carbon monoxide is administered
after step (a).
16. The method of claim 12, wherein the hepatotoxic drug is
selected from the group consisting of: isoniazid, methyldopa,
acetaminophen, amiodarone, and nitrofurantoin.
17. A vessel comprising medical grade compressed carbon monoxide
gas, the vessel bearing a label indicating that the gas can be used
to treat hepatitis in a patient.
18. The vessel of claim 17, wherein the carbon monoxide gas is in
admixture with an oxygen-containing gas.
19. The vessel of claim 17, wherein the carbon monoxide gas is
present in the admixture at a concentration of at least about
0.025%.
20. The vessel of claim 17, wherein the carbon monoxide gas is
present in the admixture at a concentration of at least about
0.05%.
21. The vessel of claim 17, wherein the carbon monoxide gas is
present in the admixture at a concentration of at least about
0.10%.
22. The vessel of claim 17, wherein the carbon monoxide gas is
present in the admixture at a concentration of at least about
1.0%.
23. The vessel of claim 17, wherein the carbon monoxide gas is
present in the admixture at a concentration of at least about
2.0%.
24. The vessel of claim 17, wherein the label further indicates
that the gas can be administered to a patient in conjunction with
administration of a hepatotoxic drug.
25. A vessel comprising medical grade compressed carbon monoxide
gas, the vessel bearing a label indicating that the gas can be
administered to a patient in conjunction with administration of a
hepatotoxic drug.
26. A method of treating hepatitis in a patient, the method
comprising: identifying a patient suffering from or at risk for
hepatitis not caused by surgery or endotoxin; and administering to
the patient a pharmaceutical composition comprising an amount of
carbon monoxide effective to treat hepatitis in the patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/381,527 filed May 17, 2002, which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0003] This invention relates to the treatment of hepatitis.
BACKGROUND
[0004] 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. 47:195-201, 1991;
Christodoulides et al., Circulation 97:2306-9, 1995) and inhibits
platelet aggregation (Mansouri et al., Thromb Haemost. 48:286-8,
1982). Inhalation of low levels of carbon monoxide (CO) has been
shown to have anti-inflammatory effects in some models.
[0005] 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.sup.th Edition, Section 4, Chapter 42).
SUMMARY
[0006] The present invention is based, in part, on the discovery
that administration of CO can protect against the development of
hepatitis.
[0007] Accordingly, 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.
[0008] 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. In one embodiment of the
present invention, the pharmaceutical composition is administered
to the patient by inhalation. In another embodiment, the
pharmaceutical composition is administered to the patient orally.
In still another embodiment, the pharmaceutical composition is
administered directly to the abdominal cavity of the patient. In
yet another embodiment, the pharmaceutical composition is
administered by an extracorporeal membrane gas exchange device or
an artificial lung. In another embodiment, the patient is an
alcoholic.
[0009] The patient can be an animal, human or non-human. For
example, 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.
[0010] In another embodiment, 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. Also contemplated is use of CO and any of the
above-listed agents in the preparation of a medicament for
treatment or prevention of hepatitis.
[0011] In another embodiment, 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.
[0012] In another aspect, 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.
[0013] In still another aspect, 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. Also contemplated is use of CO in the preparation
of a medicament, e.g., a gaseous or liquid medicament, for use in
the treatment or prevention of hepatitis.
[0014] 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.
[0015] Also within the invention is a method of administering a
hepatitis-inducing drug (i.e., a hepatotoxic drug, e.g., isoniazid,
methyldopa, acetaminophen, amiodarone, or nitrofurantoin) to a
patient. 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.
[0016] In another aspect, 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. Alternatively or in addition, 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. 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%.
[0017] Also within the invention is the use of 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.
[0018] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Suitable
methods and materials are described below, although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present invention. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. The materials, methods, and examples are
illustrative only and not intended to be limiting.
[0019] The details of one or more embodiments of the invention are
set forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a bar graph illustrating that induction of HO-1
protects mouse hepatocytes from TNF-.alpha./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.
[0021] 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-.kappa.B activation-dependent manner.
CO=carbon monoxide; Air=room air; TNF=tumor necrosis factor alpha;
BAY=BAY 11-7082 (inhibits NF-KB activation);
I.kappa.B=I.kappa.B.alpha. (prevents NF-.kappa.B activation);
ODQ=1H-[1,2,4]Oxadiazolo[4,3-a]quinoxa- lin-1-one (a selective
guanylyl cyclase inhibitor); Lac-Z=pIEP-Lac-Z (adenoviral control).
Results shown are the mean.+-.SD of triplicate wells from four
independent experiments (*p<0.01).
[0022] FIG. 3 is a bar graph illustrating that exogenous CO
protects human hepatocytes against TNF-.alpha./Actinomycin-D
(ActD)--induced cell death. CO=carbon monoxide; Air=room air;
TNF=TNF-.alpha./ActD. Results are mean.+-.SD of triplicate wells
from 3 independent experiments. *p<0.05.
[0023] FIG. 4 is a bar graph illustrating that exogenous CO causes
an increase in NF-.kappa.B activation in hepatocytes. CO=carbon
monoxide; Air=room air; BAY=BAY 11-7082; CM=cytokine mixture
(TNF-.alpha. (500 U/ml), IL-1.beta. (100 U/ml), and IFN-.delta.
(100 U/ml)). Results shown are the mean.+-.SE of triplicate wells
from three independent experiments. *p<0.001 versus Air.
[0024] FIG. 5 is a picture of a polyacrylamide gel illustrating
that exogenous CO induces an increase in NF-.kappa.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.
[0025] FIGS. 6A-6C 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-.alpha. (500 U/ml), IL-1 (100 U/ml), and
IFN-.delta. (100 U/ml)). FIG. 6C: CO-exposed hepatocytes. Images
are representative of 6 different fields. Bar represents 10
.mu.m.
[0026] FIG. 7 is a bar graph illustrating that exogenous CO-induced
protection of hepatocytes involves NF-.kappa.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.
[0027] 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-.alpha. alone. iNOS=inducible nitric oxide synthase; CO=carbon
monoxide; Air=room air; TNF=TNF-.alpha./ActD; .beta.-Actin=control
protein. The immunoblot is representative of 3 independent
experiments.
[0028] FIG. 9 is a bar graph illustrating that iNOS
activity-deficient mouse (inos.sup.-/-) hepatocytes are not
protected by CO against TNF-a-induced cell death. CO=carbon
monoxide; Air=room air; TNF=TNF-.alpha./ActD; inos.sup.-/-=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.
[0029] FIG. 10 is a bar graph illustrating that
exogenously-administered CO prevents TNF-.alpha./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.
[0030] FIGS. 11A-11H are photomicrographs of liver samples
illustrating that exogenously-administered CO prevents
TNF-.alpha./D-Gal-induced liver injury in mice. FIGS. 11A and 11B:
liver samples from mice exposed to room air and CO, respectively,
and stained with hematoxylin & eosin (H & E). FIGS. 11C and
11D: liver samples from TNF-WD-Gal-treated mice exposed to room air
and CO, respectively, and stained with H & E. FIGS. 11E and
11F: liver samples from TNF-.alpha./D-Gal-treated mice exposed to
room air and CO, respectively, and stained to detect activated
caspase-3. FIGS. 11G and 11H: liver samples from
TNF-.alpha./D-Gal-treated mice exposed to room air and CO,
respectively, and stained using terminal deoxynucleotidyl
transferase mediated dUTP nick end labeling (TUNEL). Images are
representative sections from 15-20 sections/liver from 3-4
individual mice/group. Bar represents 20 .mu.m.
[0031] FIG. 12 is a picture of a Western blot illustrating that
livers of mice exposed to TNF-.alpha./D-Gal and treated with
inhaled CO display increased iNOS protein levels. Wild type=wild
type mice; iNOS.sup.-/-=iNOS deficient mice; CO=carbon monoxide;
Air=room air; TNF=TNF-.alpha./D-Gal; .beta.-Actin=control
protein.
[0032] FIGS. 13A-13D are photomicrographs of liver samples
illustrating that the livers of mice exposed to TNF-.alpha./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-.alpha./D-Gal and room air. FIG. 13D: liver sample
from mouse exposed to TNF-.alpha./D-Gal and CO. Images are
representative of 6 separate animals and 6-10 different
sections/liver sample. Bar represents 20 .mu.m.
[0033] FIG. 14 is a bar graph illustrating that CO does not protect
against liver damage in the absence of iNOS function/expression.
L-NIL=L-N6-(1-iminoethyl)-lysine-dihydrochloride (a selective
inhibitor of iNOS); CO=carbon monoxide; Air=room air;
TNF=TNF-.alpha./D-Gal. Results are mean.+-.SD of 6-8 animals/group.
*p<0.01 versus CO/TNF-a/D-gal and air and CO controls.
[0034] 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-.alpha./D-Gal. CO=carbon
monoxide; Air=room air; TNF=TNF-60 /D-Gal; .beta.-Actin=control
protein. Blot is representative of 2 independent experiments.
[0035] 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-.alpha./D-Gal if iNOS is
inhibited using L-NIL. CO=carbon monoxide; Air=room air;
TNF=TNF-.alpha./D-Gal; .beta.-Actin=control protein;
L-NIL=L-N6-(1-iminoethyl)-lysine-dihydrochl- oride (a selective
inhibitor of iNOS). Blot is representative of 2 independent
experiments.
[0036] FIG. 17 is a bar graph illustrating that CO-induced HO-1 is
protective against TNF-.alpha.-induced liver damage in mice.
ALT=serum alanine aminotransferase; Air=room air;
TNF=TNF-.alpha./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.
[0037] FIG. 18 is a bar graph illustrating that induction of HO-1
is protective against TNF-.alpha.-induced liver injury independent
of iNOS activity. ALT=serum alanine aminotransferase; Air=room air;
TNF=TNF-.alpha./D-Gal;
L-NIL=L-N6-(i-iminoethyl)-lysine-dihydrochloride (a selective
inhibitor of iNOS); CoPP=cobalt protoporphyrin (an inducer of
HO-1); iNOS.sup.-/-=iNOS deficient mice. Results are mean.+-.SD of
6-8 mice/group. *p<0.001 versus Air/TNF and L-NIL/TNF.
[0038] 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)=hepatocytes
isolated from wild type C.sub.57BL/6J mice; hmox-1.sup.-/- (white
bars)=hepatocytes isolated from HO-1 null mice; CO=carbon monoxide;
Air=room air; TNF-.alpha.=TNF-.alpha.- /ActD. *p<0.01 versus
non-TNF-.alpha./ActD treated cells and versus
TNF-.alpha./ActD-treated cells that were also treated with CO.
[0039] FIG. 20 is bar graph illustrating that HO-1 expression is
required for NO-induced protection of mouse hepatocytes from
TNF-.alpha./ActD-induced cell death. Wild type (black
bars)=hepatocytes isolated from wild type C57BL/6J mice;
hmox-1.sup.-/- (white bars)=hepatocytes isolated from HO-1 null
mice; SNAP=s-nitroso-N-acetyl-p- enicillaamine (an NO donor);
Air=room air; TNF-A=TNF-.alpha./ActD. *p<0.01 versus
non-TNF-.alpha./ActD treated cells and versus
TNF-.alpha./ActD-treated cells that were also treated with NO.
[0040] 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.
DETAILED DESCRIPTION
[0041] The term "carbon monoxide" (or "CO") as used herein
describes molecular carbon monoxide in its gaseous state,
compressed into liquid form, or dissolved in aqueous solution. The
terms "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. 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.
[0042] The terms "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.
[0043] For gases, 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%. For liquid solutions of CO, 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.
[0044] The term "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. The term
"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.
[0045] The term "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). 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; .beta.-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., zafirlukast;
anti-tuberculous drugs, e.g., rifampicin and pyrazinamide;
antifungal agents, e.g., ketoconazole, terbinafine, fluconazole,
and itraconazole; antidiabetic drugs, e.g., thiazolidinediones,
e.g., troglitazone and rosiglitazone; drugs used in neurologic
disorders, e.g., neuroleptic agents, antidepressants (e.g.,
fluoxetine, paroxetine, venlafaxine, trazodone, tolcapone, and
nefazodone), hypnotics (e.g., alpidem, zolpidem, and bentazepam),
and other drugs, e.g., tacrine, dantrolene, riluzole, tizanidine,
and alverine; nonsteroidal anti-inflammatory drugs, e.g.,
bromfenac; COX-2 inhibitors; cyproterone acetate; leflunomide;
antiviral agents, e.g., fialuridine, didanosine, zalcitabine,
stavudine, lamivudine, zidovudine, abacavir; anticancer drugs,
e.g., tamoxifen and methotrexate; recreational drugs, e.g.,
cocaine, phencyclidine, and
5-methoxy-3,4-methylenedioxymethamphetamine; L-asparaginase;
amodiaquine; hycanthone; anesthetic agents; e.g., halothane,
enflurane, and isoflurane; vitamins e.g., vitamin A; and dietary
and/or environmental toxins, e.g., pyrrolizidine alkaloids, toxin
from Amanita phalloides or other toxic mushrooms, aflatoxin,
arsenic, Bordeaux mixture (copper salts and lime), vinyl chloride
monomer; carbon tetrachloride, beryllium, dimethylformamide,
dimethylnitrosamine, methylenedianiline, phosphorus, chlordecone
(Kepone), 2,3,7,8-tetrachloro-dibenzo p-dioxin (TCDD),
tetrachloroethane, tetrachloroethylene, 2,4,5-trinitrotoluene,
1,1,1-trichloroethane, toluene, and xylene, and known "herbal
remedies," e.g., ephedrine and eugenol.
[0046] 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.sup.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.
[0047] Skilled practitioners will appreciate that 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.
[0048] 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.
[0049] 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. In a given day, 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.
[0050] If the patient needs to be treated with a hepatotoxic drug
(e.g., because prescribed by a physician), the patient can be
treated with CO (e.g., a gaseous CO composition) before, during,
and/or after administration of the drug. For example, 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. Alternatively or
in addition, CO can be administered to the patient concurrent with
administration of the drug. Alternatively or in addition, 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).
[0051] Preparation of Gaseous Compositions
[0052] 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.
For example, 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.sub.2, O.sub.2, N.sub.2) are in
the same vessel, except that NO and O.sub.2 cannot be stored
together. Optionally, the methods of the present invention can be
performed using multiple vessels containing individual gases. For
example, 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.
[0053] 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. Preferably, the
amount of nitrogen in the gaseous composition is about 79% by
weight, the amount of oxygen is about 21% by weight and 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. It is noted that gaseous
carbon monoxide compositions having concentrations of carbon
monoxide greater than 0.3% (such as 1% or greater) may be used for
short periods (e.g., one or a few breaths), depending upon the
application.
[0054] 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. Alternatively, 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.
[0055] 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. Further, it
is known in the art that 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.
[0056] Preparation of Liquid Compositions
[0057] 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. For example, the liquid can be placed
in a so-called "CO.sub.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. As another example, 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. As still another example, 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.
[0058] It is likely that such a liquid composition intended to be
introduced into a living animal will be at or about 37.degree. C.
at the time it is introduced into the animal.
[0059] 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)). In general, the liquid will be an aqueous
solution. Examples of solutions include Phosphate Buffered Saline
(PBS), Celsior.TM., Perfadex.TM., Collins solution, citrate
solution, and University of Wisconsin (UW) solution (Oxford
Textbook of Surgery, Morris and Malt, Eds., Oxford University Press
(1994)). In one embodiment of the present invention, the liquid is
Ringer's Solution, e.g., lactated Ringer's Solution, or any other
liquid that can be used infused into a patient. In another
embodiment, the liquid includes blood, e.g., whole blood.
[0060] Any suitable liquid can be saturated to a set concentration
of carbon monoxide via gas diffusers. Alternatively, 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.
[0061] Treatment of Patients with Carbon Monoxide Compositions
[0062] 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).
[0063] Systemic Delivery of Carbon Monoxide
[0064] 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. The 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. It is to be further understood that for any particular
subject, specific dosage regimens should be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
compositions, and that the concentration ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the claimed composition. Treatments can be monitored
and CO dosages can be adjusted to ensure optimal treatment of the
patient. Acute, sub-acute and chronic administration of carbon
monoxide are contemplated by the present invention, depending upon,
e.g., the severity or persistence of hepatitis in the patient.
Carbon monoxide can be delivered to the patient for a time
(including indefinitely) sufficient to treat the condition and
exert the intended pharmacological or biological effect.
[0065] The following are examples of some methods and devices that
can be utilized to administer gaseous carbon monoxide compositions
to patients.
[0066] Ventilators
[0067] 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.sub.2, 79% N.sub.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. This is determined by pulmonary graphics (i.e.,
respiratory rate, tidal volumes etc.). 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. 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.sub.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.sub.2. Carbon monoxide can also be mixed with any level of
O.sub.2 to provide therapeutic delivery of carbon monoxide without
consequential hypoxic conditions.
[0068] 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.sub.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.
[0069] Portable Inhaler
[0070] 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.
[0071] Intravenous Artificial Lung
[0072] An artificial lung (a catheter device for gas exchange in
the blood) designed for O.sub.2 delivery and CO.sub.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)).
[0073] Normobaric Chamber
[0074] In certain instances, it would be desirable to expose the
whole patient to carbon monoxide. 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.
Upon completion of the exposure, the chamber could be flushed with
air (e.g., 21% O.sub.2, 79% N.sub.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.
[0075] Systemic Delivery of Liquid CO Compositions
[0076] The present invention further contemplates that 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. For example, liquid CO
compositions, such as CO-saturated Ringer's Solution, can be
infused into a patient suffering from or at risk for hepatitis.
Alternatively or in addition, CO-partially or completely saturated
whole (or partial) blood can be infused into the patient.
[0077] 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).
[0078] Topical Treatment of Organs with Carbon Monoxide
[0079] Alternatively or in addition, 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. For example, gases, e.g.,
carbon dioxide, 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)). The skilled practitioner will
appreciate that similar procedures could be used to administer
carbon monoxide compositions directly to the liver of a
patient.
[0080] 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. As with gaseous
compositions, aqueous compositions can be applied directly to the
liver. For example, liquids, e.g., saline solutions containing
dissolved CO, can be injected into the abdominal cavity of patients
during laproscopic procedures. The skilled practitioner will
appreciate that similar procedures could be used to administer
liquid carbon monoxide compositions directly to the liver of a
patient. Further, 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)).
[0081] Use of Hemoxygenase-1, Other Compounds, and Other Treatments
for Hepatitis
[0082] Also contemplated by the present invention is the induction
or expression of hemeoxygenase-1 (HO-1) in conjunction with
administration of CO. For example, HO-1 can be induced in a patient
suffering from or at risk for hepatitis. As used herein, the term
"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.
[0083] HO-1 can be induced in a patient by any method known in the
art. For example, production of HO-1 can be induced by hemin, by
iron protoporphyrin, or by cobalt protoporphyrin. A variety of
non-heme agents including heavy metals, cytokines, hormones, NO,
COCl.sub.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.
[0084] HO-1 expression in a cell can be increased via gene
transfer. As used herein, 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) encoding HO-1 or ferritin would be administered to
a patient suffering from or at risk for hepatitis, by mouth, by
inhalation, or by injection into the liver. Similarly, plasmid
vectors encoding HO-1 or apoferritin can be administered, e.g., as
naked DNA, in liposomes, or in microparticles.
[0085] Further, 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).
[0086] Alternatively or in addition, any of the products of
metabolism by HO-1, e.g., bilirubin, biliverdin, iron, and/or
ferritin, can be administered to a patient in conjunction with CO
in order to prevent or treat hepatitis. Further, the present
invention contemplates that iron-binding molecules other than
ferritin, e.g., desferoxamine (DFO), iron dextran, and/or
apoferritin, can be administered to the patient. Further still, the
present invention contemplates that enzymes (e.g., biliverdin
reductase) 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] The invention is illustrated in part by the following
examples, which are not to be taken as limiting the invention in
any way.
EXAMPLE 1
Carbon Monoxide Attenuates Liver Injury
[0091] Animals
[0092] Male C57BL/6J (Charles Rivers Laboratories, Bar Harbor,
Me.), 8-12-wk-old inos.sup.-/- mice and wild type littermates
(bred/maintained at the University of Pittsburgh) were used for in
vivo experiments.
[0093] Acute Hepatic Injury Models
[0094] Groups of mice were administered TNF-.alpha./D-gal (0.3
.mu.g/8 mg/mouse, i.p., respectively). Depending on the
experimental condition, some mice received CO (250 ppm), the
selective NO donor O.sub.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). Additionally, the selective inhibitor of iNOS
L-N6-(1-iminoethyl)-lysine-dihydrochloride (L-NIL; 5 mg/kg, i.p.,
Alexis Biochemicals) or the HO-1 inhibitor tin protoporphyrin
(SnPP; 50 .mu.mol/kg, i.p., Frontier Scientific) was administered
when specified. Where indicated, acetaminophen (Sigma Chem. Co.; St
Louis, Mo.) was administered (500 mg/kg, i.p.).
[0095] Hepatocyte Cell Culture.
[0096] Mouse primary hepatocytes were harvested from C57BL/6J,
mkk3.sup.-/-, inos.sup.-/- (in-house breeding colony), or
hmox-1.sup.-/- mice as described in Kim et al. (J. Biol. Chem. 272:
1402-1411 (1997)). Hepatocytes were used on days 1-3 following
harvest.
[0097] Induction of Hepatocyte Death/Apoptosis
[0098] Cells were treated with TNF-.alpha. (10 ng/ml) and
actinomycin-D (Act-D; 200 ng/ml, Sigma Chemical Co. St. Louis, Mo.)
to induce cell death. TNF-.alpha./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 .mu.M), and/or
additional pharmacologic agents where indicated. Twelve hours after
TNF-.alpha./ActD treatment, cells were washed and stained with
crystal violet to determine viability as previously described
(Id.). Where indicated, the selective in vitro inhibitor of iNOS,
L-N5-(1-iminoethyl)-ornithine-2HCl (LNIO;1-2 MM; Calbiochem, San
Diego, Calif.) was administered.
[0099] Gene Transfer/Plasmids.
[0100] In some experiments, gene transfer of an I.kappa.B.alpha.
superrepressor (Hellerbrand et al., Hepatology 27:1285-1295 (1998))
or .beta.-galactosidase using adenoviral vectors (10 pfu/cell) was
performed 12 hours prior to TNF-.alpha./ActD treatment. NF-.kappa.B
activation was evaluated 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-.kappa.B reporter
constructs (pGL3-kappa.beta. luciferase, 100 ng/well; and
pIEP-Lac-z 0.5 .mu.g/well) using Lipofectin.TM. (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 .mu.g/well) and
pIEP-LacZ (0.5 .mu.g/well) as described above.
[0101] Luciferase Reporter Assays
[0102] 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.
[0103] Electrophoretic Mobility Shift Assay
[0104] Nuclei were extracted from hepatocytes following treatment.
A double-stranded DNA NF-.kappa.B consensus sequence (GGGGACTTTCCC
(SEQ ID NO: 1)); Santa Cruz Biotechnology, Santa Cruz, Calif.) was
labelled with [.delta.-.sup.32P]-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)).
[0105] Immunoblot Analysis
[0106] Western blot analysis was performed on primary hepatocytes
in culture or from liver homogenates with antibodies to iNOS
(Transduction Laboratories, Lexington, Ky.; 1:1000), HO-1
(Calbiochem; 1:2000), or .beta.-actin (Sigma Chemical; 1:5000).
Thirty .mu.g protein in cell culture experiments or 100 .mu.g
protein from liver homogenates was loaded per well for
SDS-PAGE.
[0107] Histology/Immunohistochemistry
[0108] For histology and immunohistochemistry, 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.
[0109] CO Exposure
[0110] 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.sup.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.
[0111] HO-1 Protects Against Liver Injury.
[0112] 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-.alpha./D-gal (0.3 .mu.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-.alpha./D-gal. Induction of HO-1
prevented liver injury as measured by serum ALT levels.
[0113] Exogenous CO Protects Hepatocytes
[0114] Whether exogenous CO is protective against hepatocyte cell
death in vitro was investigated. The results are presented in FIGS.
2 and 3. To generate the data presented in FIG. 2, mouse
hepatocytes were pre-incubated with CO (250 ppm) for 1 hr (standard
pre-treatment time for all experiments) prior to addition of
TNF-.alpha./Act-D (10 ng/200 ng/ml respectively). Cells were
maintained in CO for the duration of the experiment. Twelve hours
afterward, cell viability was measured as described in Kim et al.
(J. Biol. Chem. 272: 1402-1411 (1997)). Adenoviral experiments
involved incubating hepatocytes overnight with 10 pfu/cell of the
adenovirus prior to addition of TNF-.alpha./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. To evaluate the role of cGMP and confirm the role of
NF-.kappa.B, 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
.mu.M) or the NF-.kappa.B inhibitor BAY 11-7082, (10 .mu.M). Cells
were treated with the inhibitors for 1 hour prior to the 1 hour
pretreatment with CO. TNF-.alpha./ActD was then added and the cells
tested for viability 12 hours later. NF-.kappa.B activation was
critical to the protection elicited by CO while cGMP was not
involved. Exposure to CO led to significantly less cell death
(*p<0.01) than without CO.
[0115] To generate the data presented in FIG. 3, human primary
hepatocytes obtained from a donor liver resection were treated with
CO and TNF-.alpha./ActD as described above.
[0116] Exposure of primary mouse, rat, and human hepatocytes to CO
inhibited TNF-a induced apoptosis. Inhibition of hepatocyte
apoptosis was independent of cGMP generation, as the selective
guanylyl cyclase inhibitor ODQ did not reverse the protection
provided by CO (FIG. 2). Additionally, CO treatment inhibited cell
death both in the presence of SB203580 (3-30 .mu.M, Calbiochem), a
selective inhibitor of p38 MAPK activation, and in hepatocytes from
mkk3.sup.-/- mice, the dominant upstream kinase for p38 (data not
shown). Thus, the effects of CO were independent of the cGMP/p38
MAPK pathway. In these experiments, hepatocytes were pre-treated
with CO for one hour prior to addition of TNF-.alpha./ActD to the
medium. If CO treatment was initiated after addition of
TNF-.alpha., less protection was observed (data not shown).
[0117] The Role of NF-.kappa.B in CO Protection
[0118] Whether CO-induced protection of hepatocytes depends upon
NF-.kappa.B was investigated. FIGS. 4, 5, and 6A-6C present data
illustrating that that CO induced an increase in NF-.kappa.B
nuclear translocation and DNA binding in mouse hepatocytes as
measured by NF-.kappa.B luciferase reporter assay activity, EMSA,
and immunostaining for RelA/p65 nuclear translocation,
respectively.
[0119] To generate the data presented in FIG. 4, evaluation of
NF-.kappa.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 .mu.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-.alpha. (500 U/ml), IL-1.beta. (100 U/ml), and
IFN-.delta. (100 U/ml), which was used as a positive control for
NF-.kappa.B activation. Results were corrected for transfection
efficiency and protein concentration.
[0120] To generate the data in FIG. 5, NF-KB DNA binding was
evaluated using EMSA in hepatocytes treated with CO (250 ppm). Note
the time-dependent increase in NF-.kappa.B binding (total) with
expression peaking at one hr (Lanes 1, 4, 7). Extracts were then
supershifted to identify the different NF-.kappa.B dimers using
antibodies against p50 (Lanes 2, 5, 8) and p65 (Lanes 3, 6, 9).
[0121] To generate the data in FIGS. 6A-6C, primary hepatocytes
were immunostained for nuclear p65 localization following exposure
to 1 hr CO (250 ppm). Images depict nuclear translocation of
NF-.kappa.B (arrows pointing to green nuclei that depict the
translocation of NF-.kappa.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).
[0122] NF-.kappa.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). These data combined with
the positive immunostaining and EMSA results support the notion
that CO induces a moderate increase in NF-KB that in itself may in
part result in selective gene expression. To evaluate whether
NF-.kappa.B activity is needed for protection mediated by CO,
adenoviral gene transfer of I.kappa.B.alpha. was utilized to
prevent NF-.kappa.B translocation and BAY 11-7082 (1-10 mM,
Calbiochem) was used to inhibit NF-.kappa.B activation. The
protective effects of CO were abrogated by inhibition of
NF-.kappa.B activation.
[0123] The Role of NF-.kappa.B-Dependent iNOS Expression in CO
Protection
[0124] Whether CO-mediated protection of hepatocytes requires
expression of iNOS and generation of NO was investigated. The
results are presented in FIGS. 7, 8, and 9.
[0125] To generate the data in FIG. 7, 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 an
iNOS promoter reporter construct and pIEP-LacZ 24 hr prior to
exposure to BAY 11-7082 (10 .mu.M) or vehicle. Cells were incubated
with BAY 1 hr prior to exposure to CO (250 ppm). Luciferase
activity (reported as arbitrary units; A.U.) was assayed as above.
Cytokine mixture (CM; see above) was used as a positive control to
induce iNOS expression, and results were corrected for transfection
efficiency and protein concentration.
[0126] To generate the data in FIG. 8, expression of iNOS protein
was evaluated using immunoblotting techniques. Briefly, cell
extracts from hepatocytes were treated with TNF-.alpha./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-.alpha. induces
iNOS expression minimally, while those cells treated with
TNF-.alpha. in the presence of CO show a significantly greater
induction in iNOS protein.
[0127] To generate the data presented in FIG. 9, mouse hepatocytes
were isolated from inos.sup.-/- 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. Those groups exposed to CO
received a one-hour pretreatment prior to addition of
TNF-.alpha./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.
[0128] 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-.kappa.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). Using
hepatocytes from iNOS knockout mice (inos.sup.-/-) and wild type
hepatocytes treated with the selective iNOS inhibitor L-NIO (1 mM,
Calbiochem), applicants investigated whether CO could protect
against TNF-.alpha.-induced death in the absence of iNOS activity.
Hepatocytes lacking iNOS activity were not protected by CO from
TNF-.alpha.-induced cell death while wild type hepatocytes were
protected (FIG. 9). Taken together, these data show that CO
requires NF-.kappa.B activation and iNOS expression to protect
hepatocytes from cell death in vitro.
[0129] Inhaled CO is Protective Against Liver Failure
[0130] Whether inhaled CO protects mice against liver injury in a
TNF-.alpha./D-gal model of fulminant hepatic failure was
investigated. The results are presented in FIGS. 10 and
11A-11H.
[0131] To generate the data presented in FIG. 10, mice were
pre-treated with CO (250 ppm) for one hour prior to receiving
TNF-.alpha./D-gal (0.3 .mu.g/8 mg/mouse; i.p., respectively). After
receiving TNF-.alpha./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.
[0132] To generate the data presented in FIGS. 11A-11H, liver
samples from mice treated with TNF-.alpha./D-gal in the presence
and absence of CO (250 ppm) for 8 hr 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-.alpha./D-gal-induced liver damage as assessed by
H&E staining. Livers from mice exposed to CO also displayed
fewer TUNEL positive cells, displayed less staining of activated
caspase-3, and had normal architecture. Air-exposed control mice
that received TNF-.alpha./D-gal showed marked hepatic inflammation,
edema, hemorrhage and loss of architecture.
[0133] Results discussed above were confirmed using
lipopolysaccharide (LPS, also referred to as endotoxin) in place of
TNF. In these confirmatory studies, 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. In mice pretreated with 250 ppm CO, the
increase in ALT was reduced by >75%, to 250+/-75 IU/ml. To
further characterize the effects observed with CO in this model,
serum interleukin-6 was measured, and found to be reduced 65% in
animals breathing CO vs air-breathing controls (data not shown).
Tissue histopathology of the livers from these mice was similar to
that demonstrated using TNF/D-Gal. Untreated and CO-treated mice
(no LPS/D-Gal) had no signs of injury while those treated with air
and LPS/D-Gal showed marked injury including edema, hemorrhage,
neutrophil infiltration and an overall destruction of normal
morphology and architecture. In contrast, livers from mice treated
with CO and LPS/D-Gal were protected to the same extent as mice
treated with CO and TNF/D-Gal. Few changes in the markers of
inflammation (edema, hemorrhage, neutrophil infiltration) were
observed. Architecture was maintained and appeared grossly similar
to untreated and CO (in the absence of LPS/D-Gal)-treated mice.
Overall, the use of LPS/D-Gal to induce acute hepatitis paralleled
and confirmed data generated using TNF/D-Gal treatment.
[0134] The Role of iNOS in CO Protection Against Liver Damage
[0135] Whether hepatic iNOS protein levels were increased in the
livers of CO-exposed mice after treatment with TNF-.alpha./D-gal
was investigated using immunoblotting techniques and
immunohistochemistry. Further, whether CO would protect
inos.sup.-/- mice or wild type mice treated with the selective iNOS
inhibitor L-NIL (10 mg/kg, i.p; dosed every 2 hours) was
investigated to determine whether iNOS expression has a functional
role. The results are provided in FIGS. 12, 13A-13D, and 14.
[0136] To generate the data presented in FIG. 12, male C57BL/6J
mice were treated with air or CO (250 ppm) 1 hr prior to
TNF-.alpha./D-gal (0.3 .mu.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-.alpha./D-gal-treated
mice, but was markedly increased in mice treated with
TNF-.alpha./D-gal and CO. As expected, inos.sup.-/- mice showed no
expression of iNOS protein.
[0137] To generate the data in FIGS. 13A-13D, mouse liver sections
were immunostained for iNOS expression. The liver sections were
obtained from mice treated with TNF-.alpha./D-gal in the presence
or absence of CO, and from air and CO controls that received no
TNF-.alpha./D-gal. Livers from mice exposed to CO and not receiving
TNF-.alpha./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-.alpha./D-gal. The
increased expression appeared to be localized around blood
vessels.
[0138] To generate the data in FIG. 14, the efficacy of CO-induced
protection was tested in the absence of iNOS activity using
inos.sup.-/- and wild type mice that were treated with L-NIL, the
selective inhibitor of iNOS (L-NIL; 5 mg/kg, i.p. dosed every two
hours). L-NIL was administered 2 hr prior to CO. CO-treated animals
were then pre-treated (250 ppm) for 1 hr prior to
TNF-.alpha./D-gal. In the absence of iNOS function/expression, CO
is unable to protect against liver damage as assessed by serum ALT
levels and histopathology (data not shown).
[0139] Thus, it appears the protective effect of inhaled CO in
TNF-.alpha.-induced liver failure is dependent upon iNOS
activity.
[0140] The Role of HO-1 in CO Protection Against Acute Liver
Failure
[0141] Whether CO and NO exert protection against acute liver
failure through an HO-1dependent mechanism was investigated. The
data are presented in FIGS. 15, 16, 17, and 18.
[0142] To generate the data presented in FIG. 15, immunoblotting
was performed to observe HO-1 expression in the livers of mice that
received TNF-.alpha./D-gal in the presence and absence of CO (250
ppm). CO-treated mice showed a significant increase in HO-1
expression in both the presence and absence of
TNF-.alpha./D-gal.
[0143] To assess the role of iNOS on TNF-.alpha./D-gal-induced HO-1
expression in the liver (data presented in FIG. 16), 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.
[0144] To test the protective role of CO-induced HO-1 (data
presented in FIG. 17), mice were given SnPP (50 .mu.mol/kg, s.c.),
the selective inhibitor of HO-1, 5 hr prior to CO. Alternatively,
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-.alpha./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.
[0145] To generate the data presented in FIG. 18, wild type
C57BL/6J mice were pretreated for 24 hr with L-NIL in the drinking
water (4.5 mM) as described in Stenger et al. (J. Exp. Med. 183:
1501-1514 (1996)). These mice and inos.sup.-/- mice were then
administered CoPP. L-NIL was maintained in the water throughout the
experiment. Control and inos.sup.-/- mice received normal drinking
water. Twenty-four hr after administration of CoPP,
TNF-.alpha./D-gal was administered and serum ALT determined 6-8 hr
later. Note in FIG. 18 that induction of HO-1 provides protection
regardless of the presence of iNOS.
[0146] Immunoblotting of liver extracts from mice treated with CO
in the presence or absence of TNF-.alpha./D-gal showed
up-regulation of HO-1 (FIG. 15). The addition of the iNOS inhibitor
L-NIL to these above groups, which abrogated the protection (FIG.
17), also prevented up-regulation of HO-I (FIG. 16). To determine
whether HO-1 was central to CO-elicited hepatoprotection, tin
protoporphyrin-IX (SnPP, 50 .mu.mol/kg, s.c., Frontier Scientific)
was used as a selective inhibitor of HO-1 activity. SnPP
significantly diminished the protective effects of CO in this model
(FIG. 17). SnPP administration in the absence of TNF-.alpha./D-gal
had no deleterious or protective effects (data not shown). These
results suggest that up-regulation of HO-1 is important to the
protective effects of CO.
[0147] To determine if up-regulation of HO-1 would also be needed
if protection was initiated by NO, 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-.alpha./D-gal
administration. Mice were randomized and treated with
TNF-.alpha./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.
[0148] Because these data suggest that CO and NO require HO-1
activity to protect against TNF-a-induced hepatocyte death, whether
protection mediated by HO-1 requires iNOS activity was
investigated. Using inos.sup.-/- mice, HO-1 was induced via
administration of CoPP. TNF-.alpha./D-gal was injected 24 hr
thereafter, at the peak of HO-1 expression, and liver damage was
assessed 6-8 hr later. The results show that induction of HO-1 was
able to significantly prevent liver injury independently of iNOS
activity with a >50% reduction in serum ALT (FIG. 18). These
results were confirmed using L-NIL. Mice were pre-treated with
drinking water containing L-NIL (4.5 mM) for 24 hours. This method
effectively inhibits NOS activity. Control mice received normal
water. Subsequently, CoPP was administered to induce HO-1
expression and 24 hours thereafter mice were challenged with
TNF-.alpha./D-gal. L-NIL treatment alone did not change the
severity of injury induced in this model. All animals receiving
CoPP (with and without L-NIL) were protected from liver injury
(FIG. 18).
[0149] Whether HO-1 expression is required for CO- or NO-induced
protection from TNF-.alpha./ActD-induced hepatocyte cell death was
investigated. The data are presented in FIGS. 19 and 20.
[0150] To generate the data presented in FIG. 19, mouse hepatocytes
were isolated from HO-1 null mice (hmox-1.sup.-/-) and wild type
(C57BL/6J) littermates, pretreated for 1 hour with CO (250 ppm),
and treated with TNF-.alpha./ActD. Viability was assayed as
described above. CO significantly protected wild type hepatocytes,
but was unable to protect hepatocytes isolated from hmox-1-/-
mice.
[0151] To generate the data presented in FIG. 20, mouse hepatocytes
were isolated from HO-1 null mice (hmox-1.sup.-/-) and wild type
(C57BL/6J) littermates, pretreated with the NO donor SNAP (500
.mu.M), and then treated with TNF-.alpha./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.
[0152] As discussed above, air-treated wild type and hmox-1.sup.-/-
cells exposed to TNF-.alpha./ActD underwent cell death as expected,
while CO- or NO-treated wild type cells were protected in the
presence of TNF-.alpha./ActD (FIGS. 19 and 20). The protection
conferred by CO and NO was lost in cells lacking functional HO-1
(hmox-1.sup.-/-). Thus, it appears that HO-1 can provide protection
in this model without the involvement of iNOS, suggesting that HO-1
or one or more of its catalytic products can, in part, exert
cytoprotective effects in this model.
[0153] Inhaled CO is Protective Against Acetaminophen-Induced
Hepatitis
[0154] Whether inhaled CO is protective against acetaminophen
(APAP)-induced hepatitis was investigated. The data are presented
in FIG. 21.
[0155] To generate the data in FIG. 21, 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.
[0156] The results discussed in this Example demonstrate that a low
concentration of CO can protect against TNF-.alpha./D-gal-induced
fulminant hepatitis and illustrate a unique and previously
unrecognized dependence on both HO-1 and iNOS in the CO-induced
protection of livers from damage by TNF-.alpha./D-gal.
[0157] Without intending to be bound by theory, it is possible that
CO mediated protection operates by activating NF-.kappa.B, which in
the presence of an inflammatory stimulus leads to the up-regulation
of iNOS with the consequent production of NO. In addition to the
induction of iNOS, other NF-.kappa.B dependent
antiapoptotic/protective genes may be induced. During the 1 hour
pre-treatment with CO and before the cells are treated with
TNF-.alpha., significant activation of NF-.kappa.B was present,
which could be part of the priming of the cellular apparatus
discussed above. The activation of NF-.kappa.B by CO may in part
result from a mild increase in reactive oxygen species generation
originating from the mitochondria (preliminary observations). One
hour might also permit time for expression of NF-.kappa.B-dependent
anti-apoptotic genes. The next step in such a hypothetical model
might lead to NO production following the up-regulation of iNOS. NO
leads to up-regulation of HO-1, the activity of which confers
protective effects. The protective effect of HO-1 could be due to
removal of heme or to any one or more of its three products: CO,
biliverdin/bilirubin or iron/ferritin. Given that exogenous CO was
administered throughout the duration of the experiments, it appears
unlikely that endogenously-produced CO alone mediates HO-1
protection. However, the combination of CO with other products of
HO-1 or these other products acting individually might be
involved.
[0158] In a study described above, CO was administered in a
clinically-relevant model of acetaminophen (APAP)-induced hepatitis
that has a time course that is similar to the development of acute
hepatitis in humans. The data demonstrate that exposure to CO 4
hours after administration of APAP (500 mg/kg, i.p.) resulted in a
62% reduction in liver injury (FIG. 21). In this model of
APAP-induced liver injury, mice show signs of hepatitis as early as
2-4 hours after APAP administration and lethality occurs by 24-48
hours. Thus, CO was administered after the initiation of liver
injury. 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).
[0159] In summary, employing a model of liver injury driven
principally by TNF-.alpha.-induced apoptosis, the following was
demonstrated: first, inhaled CO can prevent hepatitis in this
model; second, protection by CO requires generation of a second
gaseous molecule, NO; third, NO exerts its beneficial effects, at
least in part, via upregulation of HO-1; and fourth, up-regulation
of HO-1 is protective without a need for iNOS/NO activity, i.e.,
without an obligate continuation of the cycle.
EXAMPLE 2
Protocol for the Treatment of Hepatitis
[0160] 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.
[0161] Treatment of Patients
[0162] 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.
For example, 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). Further, 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.
[0163] Liver Transplant Procedures
[0164] Treatment of a Liver Donor
[0165] 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.
[0166] For non-human animals (e.g., pigs) to be used as
xenotransplantation donors, 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. For
example, one could use levels greater than 500 ppm (e.g., 1000 ppm
or higher, and up to 10,000 ppm, particularly for brief times).
[0167] Treatment of the Liver In Situ
[0168] 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. 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).
[0169] Treatment of the Liver Ex Vivo
[0170] 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.
[0171] Treatment of a Liver Recipient
[0172] 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. For example, the patient can inhale a
concentration of carbon monoxide, e.g., 3000 ppm, for three
consecutive 10 second breath holds. Alternatively, 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.
[0173] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
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