Note: Descriptions are shown in the official language in which they were submitted.
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CARBON MONOXIDE AS A BIOMARKER AND
THERAPEUTIC AGENT
TECHNICAL FIELD
The present invention relates to the use of carbon monoxide (CO) as a
biomarker and therapeutic agent of heart, lung, liver, spleen, brain, skin and
kidney
diseases and other conditions and disease states including, for example,
asthma,
emphysema, bronchitis, adult respiratory distress syndrome, sepsis, cystic
fibrosis,
pneumonia, interstitial lung diseases, idiopathic pulmonary diseases, other
lung
diseases including primary pulmonary hypertension, secondary pulmonary
1o hypertension, cancers, including lung, larynx and throat cancer, arthritis,
wound
healing, Parkinson's disease, Alzheimer's disease, peripheral vascular disease
and
pulmonary vascular thrombotic diseases such as pulmonary embolism. CO may be
used to provide anti-inflammatory relief in patients suffering from oxidative
stress and
other conditions especially including sepsis and septic shock. In addition, CO
may be
~5 used to store organs prior to transplantation. In addition, carbon monoxide
may be used
as a biomarker or therapeutic agent for reducing respiratory distress in lung
transplant
patients, to reduce or inhibit oxidative stress, inflammation or rejection of
transplants in
transplant patients.
BACKGROUND
2o Heme oxygenase (HO) catalyzes the first and rate limiting step in the
degradation of heme to yield equimolar quantities of biliverdin IXa, carbon
monoxide
(CO), and iron (Choi et al., Am. J. Respir. Cell Mol. Biol. 15: 9-19; and
Maines, Annu.
Rev. Pharmacol. Toxicol. 37: 517-554). Three isoforms of HO exist; HO-1 is
highly
inducible while HO-2 and HO-3 are constitutively expressed (Choi et al.,
supra,
25 Maines, supra and McCoubrey et al., E. J. Bioch. 247: 725-732). Although
heme is the
major substrate of HO-1, a variety of non-heme agents including heavy metals,
cytokines, hormones, endotoxin and heat shock are also strong inducers of HO-1
expression (Choi et al., supra, Maines, supra and Tenhunen et al., J. Lab.
Clin. Med.
75: 410-421). This diversity of HO-1 inducers has provided further support for
the
3o speculation that HO-1, besides its role in heme degradation, may also play
a vital
function in maintaining cellular homeostasis. Furthermore, HO-1 is highly
induced by
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a variety of agents causing oxidative stress including hydrogen peroxide,
glutathione
depletors, UV irradiation, endotoxin and hyperoxia (Choi, et al. supra,
Maines, su ra
and Keyse, et al. Proc. Natl. Acad. Sci. USA . 86: 99-103 ). One
interpretation of this
finding is that HO-1 can serve as a key biological molecule in the adaptation
and/or
defense against oxidative stress (Choi, et al., supra" Lee, et al., Proc Natl
Acad Sci USA
93: 10393-10398; Otterbein et al., Am. J. J. Respir. Cell Mol .Biol. 13: 595-
601; Poss
et al., Proc. Natl. Acad. Sci. USA. 94: 10925-10930; Vile, et al., Proc. Natl.
Acad. Sci.
91: 2607-2610; Abraham et al., Proc. Natl. Acad. Sci. USA. 92: 6798-6802; and
Vile
and Tyrrell, J. Biol. Chem. 268: 14678-14681. Our laboratory and others have
shown
io that induction of endogenous HO-1 provides protection both in vivo and in
vitro against
oxidative stress associated with hyperoxia and lipopolysaccharide-induced
tissue injury
(Lee et al., su ra, Otterbein, et al., supra and Taylor et al., Am. J.
Physiol. 18: L582-
L591). We have also shown that exogenous administration of HO-1 via gene
transfer
can provide protection against oxidant tissue injury and elicit tolerance to
hyperoxic
stress (Otterbein, et al., Am. J. Resp. Crit. Care Med. 157: A565 (Abstr)).
Carbon monoxide (CO) is a gaseous molecule with known toxicity and lethality
to living organisms (Haldane, Biochem. J. 21: 1068- 1075; and Chance, et al.,
1970,
Ann. NY Acad Sci. 174: 193-204.). However, against this known paradigm of CO
toxicity, there has been renewed interest in recent years in CO behaving as a
regulatory
2o molecule in cellular and biological processes based on several key
observations.
Mammalian cells have the ability to generate endogenous CO primarily through
the
catalysis of heme by the enzyme heme oxygenase (HO) (Choi et al., supra and
Maines,
supra). The total cellular production of CO is generated primarily via heme
degradation by HO (Marilena, Biochem. Mol. Med. 61: 136-142 and Verma, et al.,
1993, Science 259: 381-384). Moreover, CO, akin to the gaseous molecule nitric
oxide, plays important roles in mediating neuronal transmission (Verma et al.,
supra
and Xhuo, et al., Science 260: 1946-1950) and in the regulation of vasomotor
tone
(Morita, and Kourembanas, 1995, J. Clin. Invest. 96: 2676-2682.; Morita et
al., 1995
Proc. Natl. Acad. Sci. USA 92: -1479; and Goda et al., 1998, J. Clin. Inv.
101: 604-12).
so Other publication relating to the biological actions of CO include Pinsky
et al., U.S.
Patent No. 6,316,403, Sato et al., J. Immunol. 166: 4185-4194 (2001); Fujita
et al., Nat.
Med. 7(5): 598-604 (2001); Nachar et al., High Altitude Medicine & Biology
2:377-385
(2001); Vassalli et al., Crit. Care. Med. 29: 359-366 (2001); Otterbein et
al., Am. J.
2
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Physiol. Lung Cell Mol. Physiol. 276: L688-L694 (1999); Cardell et al., Brit.
J.
Pharmacol. 124: 1065-1068 (1998); Otterbein et al., Nat. Med. 6(4): 422-428
(2000);
and Otterbein et al., Am. J. Physiol Lung Cell Mol. Physiol, 279: L1029-L1037
(2000).
Septic shock and sepsis syndrome, resulting from excessive stimulation of
immune cells, particularly monocytes and macrophages, remains one of the
leading
causes of death in hospitalized patients. Parillo, et al., Ann. Intern. Med.
113, 991-992
(1992). The pathophysiological alterations observed in sepsis are often not
due to the
infectious organism itself, but rather to the uncontrolled production of pro-
1o inflammatory cytokines and chemokines including TNF-a, IL-1, and MIP-1 that
leads
to leukocyte recruitment, capillary leak and ultimately participates in the
lethality of
sepsis. Beutler, et al., 232, 977-980 (1986); Netea, et al., Immunology 94,
340-344
(1998); and Wolpe, et al., J. Exp. Med. 167, 570-581 (1988).
Lipopolysaccharide
(LPS), a constituent of the gram negative bacterial cell wall, is the leading
cause of
is sepsis, and when administered experimentally to macrophages or mice, mimics
the
same inflammatory response. Following LPS administration, there is a rapid but
transient increase in these pro-inflammatory mediators which are subsequently
down-
modulated by a battery of anti-inflammatory cytokines including interleukin-10
(IL-10)
and interleukin-4 (IL-4), which inhibit the synthesis of the pro-inflammatory
cytokines
2o and chemokines. J. Exp. Med. 177, 1205-1208 (1993). LPS initially binds to
the CD14
and toll-like receptor (TLR) 2 (or 4) at the cell surface, (Yang, et al.,
Nature. 395 : 284-
288 (1998) and Chow, et al., J. Biol. Chem. 274 : 10689-10692 (1999) and has
then
been shown to activate the mitogen activated protein (MAP) kinase pathways
including
p38, p42/p44 ERK and JNK (MAP) kinases. Liu" et al., J. Immunol. 153, 2642-
2652
2s (1994); Hambleton" et al., Proc. Natl. Acad. Sci. USA. 93, 2274-2778
(1996); Han" et
al., J. Biol. Chem. 268, 25009-25014 (1993); Han" et al., Science 265, 808-811
(1994); Sanghera" et al., J. Immunol. 156, 4457-4465 (1996), and Raingeaud" et
al., J.
Biol. Chem. 270, 7420-7426 (1995). The relationship between the activation of
these
signaling molecules, downstream cytokine expression, and physiologic function
so represents an active line of investigation.
The United States Government has provided support for research which led to
the present invention under one or more of NIH grant numbers HL60234, AI42365
and
HL55330. Consequently, the government retains certain rights in the invention.
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SUMMARY
The present invention relates to novel pharmaceutical compositions for
delivering to patients suffering from the effects of oxidative stress, the
compositions
comprising effective concentrations of carbon monoxide in a gaseous mixture
comprising oxygen and optionally, nitrogen gas (as well as other minor
optional
gaseous components). An additional aspect of the present invention is directed
to a
method for delaying the onset of, inhibiting or alleviating the effects of
oxidative stress,
the method comprising delivering a therapeutic gas comprising carbon monoxide
in an
amount and for a time effective to delay the onset of, inhibit or alleviate
the effects of
oxidative stress in the patient. It has unexpectedly been discovered that the
delivery of
a therapeutic gas comprising low concentrations (i.e., concentrations ranging
from
about 1 ppb (part per billion) to about 3,000 ppm (preferably above about 0.1
ppm
within this range) of the gas, preferably about 1 ppm to about 2,800 ppm, more
preferably about 25 ppm to about 750 ppm, even more preferably about 50 ppm to
about 500 ppm, e.g., about 250 ppm) of carbon monoxide is an extremely
effective
method for delaying the onset of, inhibiting or reversing the effects of
oxidative stress
in a patient. This is an unexpected result. It is noted here that in the
method of the
present invention, an amount of carbon monoxide in the therapeutic gaseous
composition which is in excess of 0.3% may sometimes be used, depending upon
the
2o condition or disease state to be treated.
In another aspect, the present invention is directed to the use of carbon
monoxide gas in the preparation of a medicament for use in treating a patient
suffering
from or at risk for emphysema, bronchitis, cystic fibrosis, pneumonia,
interstitial lung
disease, wound healing, arthritis, Parkinson's disease, and/or Alzheimer's
disease.
In yet another aspect, the present invention is directed to the use of carbon
monoxide gas in the preparation of a medicament for use in treating a patient
suffering
from or at risk for localized inflammation of the kidney, spleen, and/or skin.
The present invention also provides methods for using carbon monoxide as a
biomarker for determining that a patient is suffering from oxidative stress or
is at risk
3o for or is suffering from a number of conditions or disease states that are
secondary to or
result in oxidative stress, for example, asthma, emphysema, bronchitis, adult
respiratory
distress syndrome, sepsis, cystic fibrosis, pneumonia, interstitial lung
diseases,
idiopathic pulmonary diseases, other lung diseases including primary pulmonary
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hypertension, secondary pulmonary hypertension, cancers, including lung,
larynx and
throat cancer, arthritis, wound healing, Parkinson's disease, Alzheimer's
disease,
peripheral vascular disease and pulmonary vascular thrombotic diseases such as
pulmonary embolism, among others. The method comprises detecting carbon
monoxide in a patient's breath to determine whether detectable levels of
carbon
monoxide occur in the breath. If detectable levels of carbon monoxide appear
in the
patient's breath, the patient may be diagnosed with oxidative stress or for
being at risk
for oxidative stress. The manifestations of oxidative stress may take the form
of one or
more of the above-referenced conditions or disease states. Appropriate
therapeutic
io steps or other steps may be taken after such diagnosis to alleviate or
treat the condition
that is responsible for the oxidative stress in the patient. In one
embodiment, the
method includes measuring carbon monoxide in breath exhaled by a patient,
wherein an
amount of carbon monoxide of at least about 1 ppm in the breath is indicative
that the
patient is at risk for sepsis or septic shock.
Another aspect of the present invention relates to the finding that in certain
patients, the administration of carbon monoxide in effective amounts to the
patient may
be used to induce HO-1 enzyme in the patient and prevent or limit oxidative
stress in
the patient, especially oxidative stress caused by hyperoxia or sepsis. HO-1
enzyme is
implicated in maintaining homeostasis in the cells of the patient.
2o Still another aspect of the present invention relates to the use of carbon
monoxide to delay the onset of, inhibit or alleviate the effects of oxidative
stress which
occur in transplant patients, in particular, organ transplant patients,
especially, but not
exclusively lung transplant patients.
Another aspect of the present invention relates to a method for inhibiting the
production of pro-inflammatory cytokines such as TNF-a, IL-1(3, IL-6, and MIP-
1(3
and augmenting the production (expression) of the anti-inflammatory cytokine
IL-10
and IL-4 in a patient, the method comprising administering to the patient an
effective
amount of CO.
Still another aspect of the present invention relates to a method to preserve
so organs or tissue for transplants comprising adding to media in which the
organs or
tissue are stored a preservative effective amount or concentration of carbon
monoxide.
In this aspect of the present invention, the inclusion of carbon monoxide in
effective
amounts reduces, inhibits or alleviates the formation of reactive oxygen in
the stored
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organ or tissue, thus extending the period in which organ transplants can be
effectively
stored without suffering oxidative damage. In one embodiment, the method
includes
providing a medium comprising carbon monoxide and
storing the organ in the medium, wherein the carbon monoxide is present in the
medium in an amount sufficient to enhance storage stability of the organ.
Another aspect of the present invention relates to a method to prevent or
reduce
the likelihood of damage caused by oxidative stress associated with hyperoxia
in a
patient comprising administering an effective amount of carbon monoxide to a
hyperoxic patient.
io 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. Although suitable methods and materials for the practice or
testing
of the present invention are described below, other methods and materials
similar or
equivalent to those described herein, which are well known in the art, can
also be used.
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. In addition, the materials, methods, and
examples
are illustrative only and not intended to be limiting.
DETAILED DESCRIPTION
2o The following definitions are used to describe the present invention.
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 term "carbon monoxide composition" or "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. 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.
The terms "effective amount" and "effective to treat," as used herein, refer
to
3o the administration of carbon monoxide in an amount or concentration and 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
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intended effect or physiological outcome. 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 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 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
1o about 0.0001 to about 0.0044 g CO/100 g liquid, e.g., at least about
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. An effective amount of carbon
monoxide within the context of reducing the production or effect of
inflammatory
cytokines can be, for example, an amount sufficient to inhibit production
and/or effect
of TNF-oc, IL-l, IL-6 and MIP-1, among others. Alternatively, it can be an
amount
sufficient to induce or increase production of anti-inflammatory cytokines
such as IL-
10, among others. Within the context of transplant patients, an effective
amount of
carbon monoxide is that amount administered to the transplant patient to
reduce the
likelihood of rejection through an unfavorable immunological reaction. Within
the
context of preserving stored organs to be used for transplantation, an
effective amount
of carbon monoxide can be an amount that is added, e.g., bubbled, into the
medium in
which the transplant organs are stored in order to enhance preservation of the
organ and
reduce the likelihood that the organ will be subject to some measure of
oxidative
damage. A skilled practitioner will appreciate that amounts outside of the
ranges
described above may be used depending upon the application.
The term "patient" is used throughout the specification to describe an animal,
so human or non-human, to whom treatment according to the methods of the
present
invention is provided. Veterinary applications are clearly anticipated by the
present
invention. The term includes but is not limited to mammals, e.g., humans,
other
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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., emphysema,
bronchitis,
arthritis, cystic fibrosis, pneumonia, interstitial lung disease, Parkinson's
disease,
Alzheimer's disease, or inflammation of the kidneys, spleen, or skin. In the
case of
wound healing, the term describes the promotion of wound healing, e.g., the
promotion
of skin wound healing. Individuals considered at risk for developing a
condition
described herein may benefit particularly from the invention, primarily
because
1o prophylactic treatment can begin before there is any evidence of the
condition. The
skilled practitioner will appreciate that a patient can be determined to be at
risk for any
of the conditions described herein by any method known in the art, e.g., by a
physician's diagnosis.
The term "biomarker" is used to describe carbon monoxide produced in the
15 breath of a patient in minor, detectable amounts, and which provides
evidence that the
patient is at risk for, is in the early stages of, or is suffering from
oxidative stress is at
risk for or is suffering from one or more of the conditions or disease states
that are
secondary to or that may result in oxidative stress. The amount of carbon
monoxide in
the breath of a patient that may function as a biomarker may be as low as
0.001 ppm,
2o but is generally at least about 0.1 ppm.
Preparation of Gaseous Carbon Monoxide Compositions
A carbon monoxide composition may be a gaseous carbon monoxide
composition.~Compressed or pressurized gas useful in the methods of the
invention can
25 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
pressurized gas including carbon monoxide used in the methods of the present
invention can be provided such that all gases of the desired final composition
(e.g., CO,
so COZ, O2, NZ) are mixed in the same vessel. 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
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with the contents of other vessels, e.g., vessels containing oxygen, nitrogen,
carbon
dioxide, helium, 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.
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 of carbon monoxide. Preferred
ranges include 0.005% to about 0.24%, about 0.01% to about 0.22%, and about
0.08%
to about 0.20%. 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.
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
2o 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.
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.,
so 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,
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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.
s Preparation of Liquid Carbon Monoxide Compositions
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 "COZ 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
~s 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.
2o In one embodiment, the liquid can be any liquid known to those of skill in
the
art to be suitable administration to a patient (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 appropriate solutions include
Phosphate
Buffered Saline (PBS), CelsiorTM, PerfadexTM, Collins solution, citrate
solution, and
25 University of Wisconsin (UW) solution (Oxford Textbook of Surgery, Morris
and Malt,
Eds., Oxford University Press (1994)).
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
so 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.
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Treatment of Patients with Carbon Monoxide Compositions
A patient can be treated with a carbon monoxide composition by any method
known in the art of administering gases and/or liquids to patient. Carbon
monoxide
compositions can be administered to a patient diagnosed with, or determined to
be at
risk, for example, for emphysema, bronchitis, arthritis, cystic fibrosis,
pneumonia,
interstitial lung disease, Parkinson's disease, Alzheimer's disease, or
inflammation of
the kidneys, spleen, or skin; or to promote wound healing, e.g., healing of
skin wounds
not associated with surgery. The invention contemplates the systemic
administration of
liquid or gaseous carbon monoxide compositions to patients (e.g., by
inhalation and/or
1o ingestion), and the topical administration of the compositions to the
patient's lungs
(e.g., by inhalation or intratracheal administration), joints (e.g., by
infusion or
transdermal administration) skin (e.g., by injection or by applying the
composition to
the surface of the skin), and other organs (e.g., by ingestion, insufflation,
and/or
introduction into the abdominal cavity).
Systemic Delivery of Carbon Monoxide
Gaseous carbon monoxide compositions can be delivered systemically to a
patient. Gaseous carbon monoxide compositions are typically administered by
inhalation through the mouth or nasal passages to the lungs, where the carbon
2o monoxide may exert its effect directly or be 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. Acute, sub-acute and chronic administration of carbon
3o monoxide are contemplated by the present invention, depending upon, e.g.,
the severity
or persistence of the condition 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.
m
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The following are examples of some methods and devices that can be utilized to
administer gaseous carbon monoxide compositions to patients.
Ventilators
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% OZ, 79% N2). 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
1o 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 mechanisms)
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
2o washed out of the patient by switching to 100% 02 inhalation. Carbon
monoxide is not
metabolized; thus, whatever is inhaled will ultimately be exhaled except for a
very
small percentage that is converted to COZ. Carbon monoxide can also be mixed
with
any level of OZ 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% OZ.
3o 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.
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Portable inhaler
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
s 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.
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Intravenous Artificial Lung
An artificial lung (a catheter device for gas exchange in the blood) designed
for
OZ delivery and COZ 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
s 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 spleen
or kidney
(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.,
1o Artif. Organs 18(11):806-812 (1994); and Golob et al., ASAIO J., 47(5):432-
437
(2001 )).
Normobaric chamber
In certain instances, it would be desirable to expose the whole patient to
carbon
15 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 t'he risk of bystanders' being exposed. Upon
completion of
the exposure, the chamber could be flushed with air (e.g., 21°Io OZ,
79% NZ), and
samples could be analyzed by carbon monoxide analyzers to ensure no carbon
2o monoxide remains before allowing the patient to exit the exposure system.
Liquid Compositions
The present invention further contemplates that a liquid composition
comprising
carbon monoxide can be created for systemic delivery to a patient, e.g., for
oral
25 delivery and/or by injection into the body, e.g., intravenously, intra-
arterially,
intraperitoneally, and/or subcutaneously.
Topical Treatment of Organs with Carbon Monoxide
Alternatively or in addition, carbon monoxide compositions can be applied
3o directly to organs, e.g., the skin, spleen, lung, and/or kidney(s). Gaseous
compositions
can be applied directly to the interior and/or exterior of the patient's body
to treat the
patient's organs. A gaseous composition can be directly applied to the
internal organs
of a patient by any method known in the art for insufflating gases into a
patient. For
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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 an internal organ of a patient. The
skin and
underlying joints can be treated topically with a gaseous composition by, for
example,
exposing the affected skin to the gaseous composition in a normobarometric
chamber
(described above), and/or by blowing the carbon monoxide composition directly
onto
the skin.
1o Liquid carbon monoxide compositions can also be administered topically to a
patient's organs. Liquid forms of the compositions can be administered by any
method
known in the art for administering liquids to patients. As with gaseous
compositions,
liquid compositions can be applied directly to the interior and/or exterior of
the body to
treat a patient's organs. For example, the liquid compositions can be
administered
~5 orally, e.g., by causing the patient to ingest an encapsulated or
unencapsulated dose of
the aqueous carbon monoxide composition. As another example, liquids, e.g.,
saline
solutions containing dissolved CO, can be injected into the abdominal cavity
of patients
during laproscopic procedures. Alternatively or in addition, in situ exposures
or
organs, e.g., kidney(s) and spleen, can be performed by any method known in
the art,
2o e.g., by in situ flushing of the organ with a liquid carbon monoxide
composition (see
Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford University Press
(1994)).
The skin can be treated topically with a liquid composition by, for example,
injecting
the liquid compositions into the skin. As a further example, the skin and
underlying
joints can be treated topically by applying the liquid composition directly to
the surface
25 of the skin, e.g., by pouring or spraying the liquid onto the skin and/or
by submerging
the skin in the liquid composition.
Disorders and Conditions
Carbon monoxide gas can be used in the preparation of a medicament for use in
so treating conditions or disease states such as asthma, emphysema,
bronchitis, adult
respiratory distress syndrome, sepsis, cystic fibrosis, pneumonia,
interstitial lung
diseases, idiopathic pulmonary diseases, other lung diseases including primary
pulmonary hypertension, secondary pulmonary hypertension, cancers, including
lung,
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larynx and throat cancer, arthritis, Parkinson's disease, Alzheimer's disease,
peripheral
vascular disease and pulmonary vascular thrombotic diseases such as pulmonary
embolism; and in treating a patient suffering from or at risk for localized
inflammation
of organs, e.g., the kidney, spleen, and/or skin. The present invention can
also be used
to aid in wound healing, e.g., skin wound healing. Of particular interest is
treatment of
wounds not caused by surgery.
The present invention may also be used to delay the onset of, or alleviate the
effects of oxidative stress in transplant patients, in particular organ
transplant patients,
especially lung transplant patients. The carbon monoxide compositions may also
be
1o used to treat inflammatory conditions of the lungs or inflammation that
occurs
secondary to sepsis or rejection in transplant patients. While not being
limited by way
of theory, low dosage CO is believed to act as an anti-inflammatory agent by
inhibiting
the production and/or effect of pro-inflammatory cytokines such as TNF-oc, IL-
1, IL-6,
MIP-1, and/or by inducing or promoting the action of anti-inflammatory
cytokines IL-4
~5 and IL-10.
The term "oxidative stress" is used to describe a condition resulting from the
overwhelming production of reactive oxygen which cannot be quenched by
endogenous
antioxidants. Oxidative stress may result in permanent tissue damage caused by
the
action of the reactive oxygen species on the tissue. The physiological
manifestation of
20 oxidative stress take the form of or occurs during various conditions or
disease states
that include asthma, emphysema, bronchitis, adult respiratory distress
syndrome, sepsis
or septic shock, cystic fibrosis, pneumonia, interstitial lung diseases,
idiopathic
pulmonary diseases, other lung diseases including primary pulmonary
hypertension,
secondary pulmonary hypertension, lung cancer and pulmonary vascular
thrombotic
25 diseases such as pulmonary embolism or any inflammatory disease of the
lungs.
The term "sepsis" is used to describe the presence of various pus-forming and
other pathogenic organisms or their toxins (generally, lipopolysaccharides or
LPS
bacterial cell walls) in the blood tissues. Sepsis will often result in
oxidative stress in
those tissues exposed to the pathogens or their toxins. Sepsis often manifests
itself in
3o the production of pro-inflammatory cytokines such as TNF-cc, IL-l, IL-6 and
MIP-1,
the production of which is reduced or reversed by the administration of
effective
amounts of carbon monoxide.
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The present invention may be used to treat inflammation. The term
"inflammation" is used to describe the fundamental pathological process
consisting of a
dynamic complex of cytologic and histologic reactions that occur in the
affected blood
vessels and adjacent tissues in response to an injury or abnormal stimulation
caused by
a physical, chemical or biologic agent, including the local reactions and
resulting
morphologic changes, the destruction or removal of the injurious material, and
the
responses that lead to repair and healing. The term includes various types of
inflammation such as acute, allergic, alterative (degenerative), atrophic,
catarrhal (most
frequently in the respiratory tract), croupous, fibrinopurulent, fibrinous,
immune,
hyperplastic or proliferative, subacute, serous and serofibrinous.
Inflammation
localized in the liver, heart, skin (e.g., dermatitis, inflammation due to
bacterial, fungal,
or viral infections and/or allergic or autoimmune reactions), spleen, brain,
kidney (e.g.,
bacterial pyelonephritis, interstitial nephritis, and/or glomerulonephritis)
and
pulmonary tract, especially the lungs, and that associated with sepsis or
septic shock is
favorably treated by the methods according to the present invention.
The term "cancer" is used as a general term to describe any of various types
of
malignant neoplasms, most of which invade surrounding tissues, may metastasize
to
several sites and are likely to recur after attempted removal and to cause
death of the
patient unless adequately treated. Cancers which may be treated using the
present
2o compositions and methods include, for example, stomach, colon, rectal,
liver,
pancreatic, lung, breast, cervix uteri, corpus uteri, ovary, prostate, testis,
bladder, renal,
brain/CNS, head and neck, throat, Hodgkins disease, non-Hodgkins leukemia,
skin
melanoma, various sarcomas, small cell lung cancer, choriocarcinoma,
mouth/pharynx,
oesophagus, larynx, melanoma, kidney and lymphoma, among others.
25 The present invention can be used to treat conditions or disorders that
involve
the respiratory system, e.g., emphysema, bronchitis, cystic fibrosis,
pneumonia, and
interstitial lung disease. The term "emphysema" as used herein refers to a
lung disease
characterized by an enlargement of lung alveoli. In this condition, alveolar
walls are
destroyed, causing bronchioles to lose structural support and collapse during
exhaling
30 (see, e.g., The Merck Manual of Diagnosis and Therapy, 17'h Edition,
Section 6,
Chapter 68). The term "bronchitis" refers to a lung disease characterized by
inflammation of the tracheobronchial tree. Bronchitis may develop after
infections,
e.g., viral infections, e.g., the common cold; or bacterial infections, or
after exposure to
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an irntant (see, e.g., The Merck Manual of Diagnosis and Therapy, 17'"
Edition,
Section 6,~Chapter 69). The term "cystic fibrosis" refers to a genetic disease
of the
exocrine glands. The disease primarily affects the gastrointestinal tract and
respiratory
systems, and usually is characterized by chronic obstructive pulmonary disease
(COPD) (see, e.g., The Merck Manual of Diagnosis and Therapy, 17'" Edition,
Section
19, Chapter 267). The term "pneumonia" refers to a lung disease affecting the
parenchyma, and includes, e.g., bacterial, viral, and aspiration pneumonia
(see, e.g.,
The Merck Manual of Diagnosis and Therapy, 17'" Edition, Section 6, Chapter
73).
The terms "interstitial lung disease" or "idiopathic interstitial lung
disease" refer to a
1o group of lung diseases of unknown etiology, which produce difFuse
pathologic changes
usually involving the interalveolar interstitial tissue (see, e.g., The Merck
Manual of
Diagnosis and Therapy, 17'~ Edition, Section 6, Chapter 78).
As used herein, the term "arthritis" refers to a condition characterized by
inflammation of the joints, and includes, for example, rheumatoid arthritis
(RA) (a
is chronic inflammatory polyarthritis that often leads to destruction of the
joints), psoriatic
arthritis (inflammatory arthritis associated with psoriasis), ankylosing
spondylitis
(inflammation of the axial skeleton and large peripheral joints), and
ankylosis
(immobility or fusion of the joint) (see, e.g., The Merck Manual of Diagnosis
and
Therapy, 17'" Edition, Section 5, Chapter 50; Section 5, Chapter 51; Section
5, Chapter
20 51; and Section 9, Chapter 108, respectively).
The term "Parkinson's disease" refers to "an idiopathic, slowly progressive,
degenerative CNS disorder characterized by slow and decreased movement,
muscular
rigidity, resting tremor, and postural instability." (The Merck Manual of
Diagnosis and
Therapy, 17'" Edition, Section 14, Chapter 179). The term "Alzheimer's
disease"
25 refers to a disease characterized by "a progressive, inexorable loss of
cognitive function
associated with an excessive number of senile plaques in the cerebral cortex
and
subcortical gray matter, which also contains b-amyloid and neurofibrillary
tangles
consisting of tau protein," (The Merck Manual of Diagnosis and Therapy, 17'"
Edition,
Section 14, Chapter 171).
so Low dosage CO may also be used in the present invention to induce HO-1
enzyme in patients and prevent or limit oxidative stress, especially oxidative
stress
caused by hyperoxia or sepsis. Induced HO-1 is implicated in maintaining
homeostasis
in the cells of the patient.
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Treatment of Organs and Tissues to Enhance Stora,~e Stability.
The present invention also relates to the use of CO as a preservative for
storing
organs or tissues to be used in transplants. It is an unexpected result that
the inclusion
s of low dosage CO in the mediumin which organs to be transplanted are stored
will
substantially reduce the likelihood of oxidative damage to the organs during
storage
and substantially enhance the storage time that organs to be transplanted may
be safely
stored without suffering irreversible oxidative damage. Thus, in one
embodiment of
the present invention, an effective amount of CO is bubbled into storage
medium either
1o before or preferably when an organ is first placed in the media or shortly
thereafter.
CO may also be used to enhance the storage stability of organs which have been
scored
for some time in media, but in those instances, oxidative damage may have
become
irreversible, thus limiting the intended effect.
Accordingly, the present invention provides a method for enhancing the storage
is stability of an organ or tissue. The storage stability is enhanced by
exposing the organ
or tissue to liquid and/or gaseous carbon monoxide compositions. Exposure of
an
organ or tissue to gaseous carbon monoxide compositions can be performed in
any
chamber or area suitable for creating an atmosphere that includes appropriate
levels of
carbon monoxide gas. Such chambers include, for example, incubators, and
chambers
2o built for the purpose of accommodating an organ in a preservation solution.
As another
example, an appropriate chamber may be a chamber wherein only the gases fed
into the
chamber are present in the internal atmosphere, such that the concentration of
carbon
monoxide can be established and maintained at a given concentration and
purity, e.g.,
where the chamber is airtight. For example, a COZ incubator may be used to
expose an
2s organ to a carbon monoxide composition, wherein carbon monoxide gas is
supplied in a
continuous flow from a vessel that contains the gas.
With respect to liquid carbon monoxide compositions, the exposure may be
performed in any chamber or space having sufficient volume for submerging the
organ
or tissue, completely or partially, in the carbon monoxide composition. In one
3o embodiment of the present invention, the organ may be exposed to a carbon
monoxide
composition by placing the organ in any suitable container, and causing the
carbon
monoxide composition to "wash over" the organ, such that the organ is exposed
to a
continuous flow of the carbon monoxide composition. In another embodiment, the
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organ is perfused with a carbon monoxide composition. The term "perfusion" is
an art
recognized term, and relates to the passage of a liquid, e.g., a carbon
monoxide
composition, through the blood vessels of an organ or tissue. Methods for
perfusing
organs ex vivo and in situ are well known in the art. An organ can be perfused
with a
carbon monoxide composition ex vivo, for example, by continuous hypothermic
machine perfusion (see Oxford Textbook of Surgery, Morris and Malt, Eds.,
Oxford
University Press (1994)). Optionally, the organ can be perfused with a wash
solution,
e.g., UW solution without carbon monoxide, prior to perfusion with the carbon
monoxide composition to remove the donor's blood from the organ. Such a
process
io could be performed to avoid competition for carbon monoxide by the donor's
hemoglobin. As another option, the wash solution can be a carbon monoxide
composition. As still another example, an appropriate liquid may be passed
through
tubing that allows gas diffusion, which runs through an atmosphere comprising
carbon
monoxide (e.g., through a chamber, such as with extracorporeal membrane
oxygenation), to create a liquid carbon monoxide composition, which may then
be
passed into an organ (e.g., perfused into the organ by connecting the tubing
to the
organ).
As another example, the organ may be placed, e.g., submerged, in a medium or
solution that does not include carbon monoxide, and placed in a chamber such
that the
2o medium or solution can be made into a carbon monoxide composition via
exposure to a
carbon monoxide-containing atmosphere as described herein. As still another
example,
the organ may be submerged in a liquid that does not include carbon monoxide,
and
carbon monoxide may be "bubbled" into the liquid.
The present invention contemplates that any or all of the above methods for
exposing an organ to a liquid carbon monoxide composition, e.g., washing,
submerging, or perfusing, can be used in a given procedure, e.g., used in a
single
procedure for enhancing the storage stability of an organ or tissue.
Carbon Monoxide as a Diagnostic Tool
3o In addition to using CO as a therapeutic agent, the measurement of CO may
be a
useful diagnostic tool, e.g., a biomarker, to determine whether a patient is
in oxidative
stress or has a condition or a disease state where CO may be implicated, e.g.,
sepsis or
septic shock. In general, a patient suspected of being in oxidative stress or
at risk for
CA 02484770 2004-11-04
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oxidative stress is monitored to determine whether detectable levels of carbon
monoxide may be measured in the exhaled breath of the patient. If detectable
levels of
carbon monoxide are seen (i.e., an amount of carbon monoxide of at least about
0.01
ppm in the patient's breath), then the attending physician or caregiver may
then begin
s to administer therapeutic doses of carbon monoxide to treat oxidative stress
or any one
or more of the conditions or disease states which are secondary to or result
in oxidative
stress.
In one embodiment, a patient will have his or her exhaled breath analyzed for
the presence of CO. CO content in a patient's breath is measured by a CO
monitor (for
example, using a Logan LR2000) which is sensitive to the detection of CO from
0 to
about 1000 ppm (with a sensitivity as low as 1 ppb). In this method, the
subjects
exhale slowly from functional FVC into the breath analyzer with a constant
flow (5-6
1/m) over a 20-30 second interval. Two successful recordings are made and mean
values will be used for all calculations. Ambient CO levels are recorded
before each
breath in order to provide control or background values. While any elevation
in CO
levels from background numbers may implicate an actual or incipient state of
oxidative
stress, an amount of CO of at least about 1 ppm provides a clear indication
that the
patient is in or is about to suffer oxidative stress.
A number of embodiments of the invention have been described. Nevertheless,
2o 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.
21
