U.S. patent application number 11/982430 was filed with the patent office on 2008-11-20 for device and method for treatment of wounds with nitric oxide.
Invention is credited to Christopher C. Miller, Alex Stenzler.
Application Number | 20080287861 11/982430 |
Document ID | / |
Family ID | 36615266 |
Filed Date | 2008-11-20 |
United States Patent
Application |
20080287861 |
Kind Code |
A1 |
Stenzler; Alex ; et
al. |
November 20, 2008 |
Device and method for treatment of wounds with nitric oxide
Abstract
Topical exposure of nitric oxide gas to wounds such as chronic
non-healing wounds may be beneficial in promoting healing of the
wound and in preparing the wound bed for further treatment and
recovery. Nitric oxide gas may be used, for example, to reduce the
microbial infection and burden on these wounds, manage exudate
secretion by reducing inflammation, upregulate expression of
endogenous collagenase to locally debride the wound, and regulate
the formation of collagen. High concentration of nitric oxide
ranging from about 160 to 400 ppm may be used without inducing
toxicity in the healthy cells around a wound site. Additionally,
exposure to the high concentration for a first treatment period
reduces the microbial burden and inflammation at the wound site and
increase collagenase expression to debride necrotic tissue at the
wound site. After a first treatment period with high concentration
of nitric oxide, a second treatment period at a lower concentration
of nitric oxide preferably ranging from about 5-20 ppm may to
provided to restore the balance of nitric oxide and induce collagen
expression to aid in the closure of the wound.
Inventors: |
Stenzler; Alex; (Long Beach,
CA) ; Miller; Christopher C.; (North Vancouver,
CA) |
Correspondence
Address: |
Sidley Austin LLP
555 West 5th Street, Suite 4000
Los Angeles
CA
91723
US
|
Family ID: |
36615266 |
Appl. No.: |
11/982430 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11487600 |
Jul 13, 2006 |
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11982430 |
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11021109 |
Dec 23, 2004 |
7122018 |
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11487600 |
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10944479 |
Sep 17, 2004 |
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11021109 |
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10615546 |
Jul 8, 2003 |
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10944479 |
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10172270 |
Jun 14, 2002 |
6793644 |
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10944479 |
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09749022 |
Dec 26, 2000 |
6432077 |
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10172270 |
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60431876 |
Dec 9, 2002 |
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60409400 |
Sep 10, 2002 |
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60394690 |
Jul 9, 2002 |
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Current U.S.
Class: |
604/23 |
Current CPC
Class: |
A61B 90/40 20160201;
A61L 15/44 20130101; A61L 2300/114 20130101; A61M 13/003 20130101;
A61M 35/30 20190501; A61L 2300/602 20130101; A61L 15/18 20130101;
A61M 1/0088 20130101; A61H 33/14 20130101; A61K 2300/00 20130101;
A61L 2/0094 20130101; A61M 1/0031 20130101; A61P 31/00 20180101;
A61P 31/02 20180101; A61M 2202/0275 20130101; A61M 2205/3334
20130101; A61M 13/006 20140204; A61K 33/00 20130101; A61P 17/02
20180101; A61K 33/00 20130101 |
Class at
Publication: |
604/23 |
International
Class: |
A61M 37/00 20060101
A61M037/00 |
Claims
1. A method for debriding necrotic tissue located at a wound site
of a subject, comprising exposing the wound site to exogenous
nitric oxide gas at a concentration and for a period of time
sufficient to increase expression of endogenous collagenase at the
wound site.
2. The method of claim 1, wherein the period of time and
concentration of exogenous nitric oxide gas are sufficient to
increase expression of endogenous gelatinase at the wound site.
3. The method of claim 1, wherein the period of time and
concentration of exogenous nitric oxide gas do not induce
significant toxicity to healthy cells surrounding the wound
site.
4. The method of claim 1, wherein the concentration of exogenous
nitric oxide gas is from about 120 ppm to about 400 ppm.
5. The method of claim 1, wherein the concentration of exogenous
nitric oxide gas is from about 200 ppm to about 250 ppm.
6. The method of claim 1, wherein the period of time is from about
5 hours to about 72 hours.
7. The method of claim 1, additionally comprising mechanically
debriding necrotic tissue located at the wound site following
exposure to the exogenous nitric oxide gas.
8. The method of claim 1, additionally comprising monitoring the
expression of endogenous collagenase at the wound site during
exposure to the exogenous nitric oxide gas.
9. A method for debriding necrotic tissue located at a wound site
of a subject, comprising: providing a flow-controlled source of gas
containing exogenous nitric oxide; and exposing the wound site to
exogenous nitric oxide gas from the flow-controlled source of gas
at a concentration and for a period of time sufficient to increase
expression of endogenous collagenase at the wound site; wherein the
increased expression of endogenous collagenase at the wound site
contributes to debriding of the necrotic tissue.
10. The method of claim 9, wherein the period of time and
concentration of exogenous nitric oxide gas are sufficient to
increase expression of endogenous gelatinase at the wound site.
11. The method of claim 9, wherein the period of time and
concentration of exogenous nitric oxide gas do not induce
significant toxicity to healthy cells surrounding the wound
site.
12. The method of claim 9, wherein the concentration of exogenous
nitric oxide gas is from about 120 ppm to about 400 ppm.
13. The method of claim 9, wherein the concentration of exogenous
nitric oxide gas is from about 200 ppm to about 250 ppm.
14. The method of claim 9, wherein the period of time is from about
5 hours to about 72 hours.
15. The method of claim 9, additionally comprising mechanically
debriding necrotic tissue located at the wound site following
exposure to the exogenous nitric oxide gas.
16. The method of claim 9, additionally comprising monitoring the
expression of endogenous collagenase at the wound site during
exposure to the exogenous nitric oxide gas.
17. The method of claim 9, additionally comprising grafting skin
onto the wound site.
18. A method of managing exudate from necrotic tissue at a wound
site of a subject, comprising exposing the wound site to exogenous
nitric oxide gas at a concentration and for a period of time
sufficient to reduce inflammation at the wound site.
19. The method of claim 18, additionally comprising providing a
flow-controlled source of gas containing exogenous nitric
oxide.
20. The method of claim 18, additionally comprising cleaning the
wound site prior to exposing the wound site to the exogenous nitric
oxide gas.
21. The method of claim 18, additionally comprising applying a gas
permeable dressing to the wound site prior to exposing the wound
site to the exogenous nitric oxide gas.
22. The method of claim 18, wherein the period of time and
concentration of exogenous nitric oxide gas do not induce
significant toxicity to healthy cells surrounding the wound
site.
23. The method of claim 18, wherein the concentration of exogenous
nitric oxide gas is from about 120 ppm to about 400 ppm.
24. The method of claim 18, wherein the concentration of exogenous
nitric oxide gas is from about 200 ppm to about 250 ppm.
25. The method of claim 18, wherein the period of time is from
about 5 hours to about 72 hours.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 11/487,600, filed Jul. 13, 2006.
[0002] U.S. application Ser. No. 11/487,600 is a continuation of
U.S. application Ser. No. 11/021,109, filed Dec. 23, 2004 and
issued as U.S. Pat. No. 7,122,018, which in turn is a C.I.P. of
U.S. application Ser. No. 10/944,479, filed Sep. 17, 2004, and U.S.
application Ser. No. 10/615,546, filed Jul. 8, 2003.
[0003] U.S. application Ser. No. 10/944,479 is a continuation of
U.S. application Ser. No. 10/172,270, filed Jun. 14, 2002 and
issued as U.S. Pat. No. 6,793,644, which in turn is a continuation
of U.S. application Ser. No. 09/749,022, filed on Dec. 26, 2000 and
issued as U.S. Pat. No. 6,432,077.
[0004] U.S. application Ser. No. 10/615,546 claims priority to U.S.
Provisional Application No. 60/431,876, filed Dec. 9, 2002,
60/409,400, filed Sep. 10, 2002, and 60/394,690, filed Jul. 9,
2002.
[0005] The above patents and patent applications are incorporated
by reference as if set forth fully herein.
FIELD OF THE INVENTION
[0006] The field of the invention relates to devices and methods
for treating wounds and infections, and more specifically, the
treatment of wounds and infections with nitric oxide.
BACKGROUND OF THE INVENTION
[0007] The treatment of infected surface or subsurface lesions in
patients has typically involved the topical or systemic
administration of anti-infective agents to a patient. Antibiotics
are one such class of anti-infective agents that are commonly used
to treat an infected abscess, lesion, wound, or the like.
Unfortunately, an increasingly number of infective agents such as
bacteria have become resistant to conventional antibiotic therapy.
Indeed, the increased use of antibiotics by the medical community
has led to a commensurate increase in resistant strains of bacteria
that do not respond to traditional or even newly developed
anti-bacterial agents.
[0008] For example, Staphylococci are known to be significant
pathogens that cause severe infections in humans, including
endocarditis, pneumonia, sepsis and toxic shock. Methicillin
resistant S. aureus (MRSA) is now one of the most common causes of
nosocomial infections worldwide, causing up to 89.5% of all
staphylococci infection. Community outbreaks of MRSA have also
become increasingly frequent. The main treatment for these
infections is the administration of glycopeptides (Vancomycin and
Teicoplanin). MRSA have been reported for two decades, but
emergence of glycopeptide-resistance in S. aureus-namely
glycopeptide intermediate (GISA) has been reported only since
1997..sup.22 The glycopeptides are given only parenterally, and
have many toxic side effects. The recent isolation of the first
clinical Vancomycin-resistant strains (VRSA) from a patient in USA
has heightened the importance and urgency of developing new agents.
Even when new anti-infective agents are developed, these agents are
extremely expensive and available only to a limited patient
population.
[0009] P. aeruginosa is another problematic pathogen that is
difficult to treat because of its resistance to antibiotics. It is
often acquired in the hospital and causes severe respiratory tract
infections. P. aeruginosa is also associated with high mortality in
patients with cystic fibrosis, severe burns, and in AIDS patients
who are immunosuppressed. The clinical problems associated with
this pathogen are many, as it is notorious for its resistance to
antibiotics due to the permeability barrier afforded by its outer
membrane lipopolysaccharide (LPS). The tendency of P. aeruginosa to
colonize surfaces in a biofilm phenotype makes the cells impervious
to therapeutic concentrations of antibiotics.
[0010] Another problem with conventional anti-infective agents is
that some patients are allergic to the very compounds necessary to
their treat their infection. For these patients, only few drugs
might be available to treat the infection. If the patient is
infected with a strain of bacteria that does not respond well to
substitute therapies, the patient's life can be in danger.
[0011] A separate problem related to conventional treatment of
surface or subsurface infections is that the infective agent
interferes with the circulation of blood within the infected
region. It is sometimes the case that the infective agent causes
constriction of the capillaries or other small blood vessels in the
infected region which reduces bloodflow. When bloodflow is reduced,
a lower level of anti-infective agent can be delivered to the
infected region. In addition, the infection can take a much longer
time to heal when bloodflow is restricted to the infected area.
This increases the total amount of drug that must be administered
to the patient, thereby increasing the cost of using such drugs.
Topical agents may sometimes be applied over the infected region.
However, topical anti-infective agents do not penetrate deep within
the skin where a significant portion of the bacteria often reside.
Topical treatments of anti-infective agents are often less
effective at eliminating infection than systemic administration
(i.e., oral administration) of an anti-infective
pharmaceutical.
[0012] In addition, despite recent advances in chronic wound care,
many lower extremity ulcers do not heal. Chronic ulcers of the
lower extremities are a significant public health problem. Besides
the large financial burden placed on the health care system for
their treatment, they cause a heavy toll in human suffering. As the
population ages and with the current obesity crisis in North
America, venous, diabetic, and pressure ulcers are likely to become
ever more common. Approximately 4 million (1% of population) people
in the United States develop chronic lower leg ulcers, the majority
classified as diabetic or venous leg ulcers, and this number can
climb to 4%-5% in older (>80 years of age) patients.
[0013] Aside from infection, a variety of factors can potentially
influence wound healing of chronic ulcers. These include excessive
exudate, necrotic tissue, poor tissue handling, and impaired tissue
perfusion, as well as from clinical conditions such as advanced
age, diabetes, and steroid administration.
[0014] Exudate is a clear, straw colored liquid produced by the
body in response to tissue damage. Although exudate is primarily
water, it also contains cellular materials, antibodies, nutrients
and oxygen. In the immediate response to an injury, exudate is
produced by the body to flush away any foreign materials from the
site. It then is the carrier for polymorphs and monocytes so that
they may ingest bacteria and other debris. Exudate also enables the
movement of these phagocytic cells within the wound to help clean
it as well as enables the migration of epithelial cells across the
wound surface.
[0015] While exudate is an important component of wound healing,
too much of it in response to chronic inflammation can worsen a
wound as the enzymes in the fluid can attack healthy tissues. This
may exacerbate the failure of the wound to close as well as place
additional psychological pressure on the patient. Chronic wounds
frequently have excessive exudate, usually associated with a
chronic infection and/or biofilm that has upregulated the
inflammatory cells of the body. This may be a local response or may
include a systemic increase in inflammatory markers and circulating
cytokines.
[0016] Chronic wounds also lead to the formation of necrotic
tissue, which in turn lead to growth of microbes. Debridement of
necrotic tissue is deemed as an important wound bed preparation for
successful wound healing. Sharp and surgical debridement rapidly
remove necrotic tissue and reduce the bacterial burden, but also
carry the greatest risk of damage to viable tissue and require high
levels of technical skill. Chemical, mechanical and autolytic
debridement are frequently regarded as safer options, although the
risk to the patient of ongoing wound complications is greater.
[0017] Additionally, the collagenase family of Metalloproteinases
(MMP's) are a class of enzymes which are able to cleave native
collagen into fragments. These fragments may then spontaneously
denature into gelatin. Gelatin peptides are further cleaved by
gelatinases such as MMP-2. Since the dry weight of skin is composed
of 70-80% collagen, and since necrotic tissue is anchored to the
wound bed by collagen fibers, enzymes which cleave collagen may be
beneficial and assist in the debridement of this tissue. However,
in chronic non-healing wounds, the levels and activity of
collagenases are insufficient for the removal of necrotic tissue.
Jung K, Knoll A G, Considerations for the use of Clostridial
collagenase in clinical practice. Clin Drug Invest 1998;
15:245-252. Also, wound fluid from diabetics, for example, may have
decreased MMP-2 activity. Furthermore, while exogenous application
of collagenase has been proposed, its application suffers from the
drawback of not being selective and risk the cleavage of collagen
anchoring healthy cells in addition to necrotic tissue.
[0018] In the 1980's, it was discovered by researchers that the
endothelium tissue of the human body produced nitric oxide (NO),
and that NO is an endogenous vasodilator, namely, an agent that
widens the internal diameter of blood vessels. NO is most commonly
known as an environmental pollutant that is produced as a byproduct
of combustion. At low concentrations such as less than 100 ppm,
researchers have discovered that inhaled NO can be used to treat
various pulmonary diseases in patients. For example, NO has been
investigated for the treatment of patients with increased airway
resistance as a result of emphysema, chronic bronchitis, asthma,
adult respiratory distress syndrome (ARDS), and chronic obstructive
pulmonary disease (COPD).
[0019] While NO has shown promise with respect to certain medical
applications, delivery methods and devices must cope with certain
problems inherent with gaseous NO delivery. First, exposure to high
concentrations of NO may be toxic, especially exposure to NO in
concentrations over 1000 ppm. Even lower levels of NO, however, can
be harmful if the time of exposure is relatively high. For example,
the Occupational Safety and Health Administration (OSHA) has set
exposure limits for NO in the workplace at 25 ppm time-weighted
averaged for eight (8) hours. It is extremely important that any
device or system for delivering NO include features that prevent
the leaking of NO into the surrounding environment. If the device
is used within a closed space, such as a hospital room or at home,
dangerously high levels of NO can build up in a short period of
time. One concern over NO toxicity is the binding of NO, when
absorbed into the circulation system such as through inhalation, to
hemoglobin that give rise to methemoglobin
[0020] Another problem with the delivery of NO is that NO rapidly
oxidizes in the presence of oxygen to form NO.sub.2, which is
highly toxic, even at low levels. If the delivery device contains a
leak, unacceptably high levels NO.sub.2 of can develop. In
addition, to the extent that NO oxidizes to form NO.sub.2, there is
less NO available for the desired therapeutic effect. The rate of
oxidation of NO to NO.sub.2 is dependent on numerous factors,
including the concentration of NO, the concentration of O.sub.2,
and the time available for reaction. Since NO will react with the
oxygen in the air to convert to NO.sub.2, it is desirable to have
minimal contact between the NO gas and the outside environment.
[0021] Accordingly, there is a need for a device and method for the
treatment of surface and subsurface infections and wounds by the
topical application of NO. The device is preferably leak proof to
the largest extent possible to avoid a dangerous build up of NO and
NO.sub.2 concentrations. In addition, the device should deliver NO
to the infected region of the patient without allowing the
introduction of air that would otherwise react with NO to produce
NO.sub.2. The application of NO to the infected region preferably
decreases the time required to heal the infected area by reducing
pathogen levels. The device preferably includes a NO and NO.sub.2
absorber or scrubber that will remove or chemically alter NO and
NO.sub.2 prior to discharge of the air from the delivery
device.
SUMMARY OF THE INVENTION
[0022] It has been discovered that NO will interfere with or kill
the growth of bacteria grown in vitro and has been investigated for
its use as a sterilizing agent. PCT International Application No.
PCT/CA99/01123 published Jun. 2, 2000, by one of the named
inventors of the present application, discloses a method and
apparatus for the treatment of respiratory infections by NO
inhalation.
[0023] Topical exposure of nitric oxide gas to wounds such as
chronic non-healing wounds may be beneficial in promoting healing
of the wound and in preparing the wound bed for further treatment
and recovery. Nitric oxide gas may be used, for example, to reduce
the microbial infection and burden on these wounds, manage exudate
secretion by reducing inflammation, upregulate expression of
endogenous collagenase to locally debride the wound, and regulate
the formation of collagen.
[0024] In a first aspect of the invention, a device for the topical
delivery of nitric oxide gas to an infected area of skin includes a
source of nitric oxide gas, a bathing unit, a flow control valve,
and a vacuum unit. The bathing unit is in fluid communication with
the source of nitric oxide gas and is adapted for surrounding the
area of infected skin and forming a substantially air-tight seal
with the skin surface. The flow control valve is positioned
downstream of the source of nitric oxide and upstream of the
bathing unit for controlling the amount of nitric oxide gas that is
delivered to the bathing unit. The vacuum unit is positioned
downstream of the bathing unit for withdrawing gas from the bathing
unit.
[0025] In a second aspect of the invention, the device according to
the first aspect of the invention includes a controller for
controlling the operation of the flow control valve and the vacuum
unit.
[0026] In a third aspect of the invention, the device according to
the first aspect of the invention further includes a source of
diluent gas and a gas blender. The diluent gas and the nitric oxide
gas are mixed by the gas blender. The device also includes a nitric
oxide gas absorber unit that is positioned upstream of the vacuum
unit. The device also includes a controller for controlling the
operation of the flow control valve and the vacuum unit.
[0027] In a fourth aspect of the invention, a method of delivering
an effective amount of nitric oxide to an infected area of skin
includes the steps of providing a bathing unit around the infected
area of skin, the bathing unit forming a substantially air-tight
seal with the skin. Gas containing nitric oxide is then transported
to the bathing unit so as to bathe the infected area of skin with
gaseous nitric oxide. Finally, at least a portion of the nitric
oxide gas is evacuated from the bathing unit.
[0028] In a fifth aspect of the invention a method of treating
infected tissue with topical nitric oxide exposure includes the
steps of providing a source of nitric oxide containing gas and
delivering the nitric oxide containing gas to a skin surface
containing infected tissue so as to bathe the infected tissue with
nitric oxide.
[0029] In a sixth aspect of the invention, a method of treating
wounds with topical nitric oxide exposure includes the steps of
providing a source of nitric oxide containing gas and delivering
the nitric oxide containing gas to the wound so as to bathe the
wound with nitric oxide. Preferably, the treatment method includes
continuous exposure of the wound to a sufficiently high
concentration of nitric oxide gas for a sufficient amount of time
to kill or effect a 2-3 log.sub.10 reduction in the microorganism
population at the wound site, without significant toxicity to the
subject or the host cells of the treated subject. For example, the
high concentration of nitric oxide gas may range from about 120 ppm
to about 400 ppm, and more preferably at about 200 ppm to 250 ppm.
The amount of time for the exposure of nitric oxide may also range
from 5 hours to 96 hours, yet optimal exposure time and
concentration can be determined based on the individual condition
of the subject as prescribed by a physician. In another embodiment,
the treatment method may also include a second treatment period,
subsequent to the first treatment period with high concentration of
nitric oxide gas, in which the wound is treated with a lower
concentration of nitric oxide gas. Preferably, the lower
concentration of nitric oxide gas ranges from 1 ppm to 80 ppm, and
more preferably ranges from 5 ppm to 20 ppm. The exposure time for
the second treatment period may also range from 5 hours to 96
hours, depending on the individual condition of the treated
subject. In another embodiment, the wound is exposed to 200 ppm of
nitric oxide gas for about 7-8 hours preferably during the night
while the patient sleeps, and nitric oxide exposure may be
withdrawn during the day time, or provided at a low concentration
(e.g., 5 ppm to 20 ppm) for about 5-16 hours.
[0030] In a seventh aspect of the invention, a method of managing
exudate secretion in a wound with topical exposure of nitric oxide
includes the steps of removing excess exudate, dressing the wound
with a gas permeable dressing, providing a source of nitric oxide
containing gas, and delivering the nitric oxide containing gas to
the wound so as to bathe the wound with nitric oxide.
[0031] In an eighth aspect of the invention, a method of debriding
a wound with topical exposure of nitric oxide includes the steps of
providing a source of nitric oxide containing gas, and delivering
the nitric oxide containing gas to the wound so as to upregulate
the expression of endogenous enzymes such as collagenase and
gelatinase by the host cells located locally at the wound site of
the treated subject. Preferably, the treatment method includes
exposure of the wound to a sufficiently high concentration of
nitric oxide gas for a sufficient amount of time to upregulate
expression of endogenous collagenase without significant toxicity
to the host cells of the treated subject. For example, the high
concentration of nitric oxide gas may range from 120 ppm to 400
ppm, and more preferably at about 200 ppm-250 ppm. The amount of
time for the exposure of nitric oxide may also range from 5 hours
to 72 hours, yet optimal exposure time and concentration can be
determined based on the individual condition of the subject as
prescribed by a physician. Preferably, the expression of
collagenase in the host cells may be monitored by taking biopsies
and analyzing the expression of collagenase mRNA or protein through
a various of techniques available in the art, such as Northern
blot, RT-PCR, quantitative RT-PCR, immunostaining,
immunoprecipitation, or ELISA. Additionally, after the treatment
period with the high concentration of nitric oxide gas, the wound
may also be exposed to a lower concentration for a second treatment
period so as to reduce collagenase expression and increase collagen
expression.
[0032] In a ninth aspect of the invention, a method for wound bed
preparation with topical nitric oxide exposure includes the steps
of providing a source of nitric oxide gas and delivering the nitric
oxide containing gas to the wound.
[0033] In a tenth aspect of the invention, a method of reducing
scarring in the healing process of a wound with topical nitric
oxide exposure includes providing a source of nitric oxide gas,
exposing the wound to a high concentration of exogenous nitric
oxide gas for a treatment period without inducing toxicity to the
subject or to healthy cells surrounding the wound, exposing the
wound to a decreased concentration of exogenous nitric oxide gas
for a second treatment period sufficient to increase the expression
of collagen mRNA; and exposing the wound to a third concentration
of exogenous nitric oxide gas for a third treatment period, wherein
the third concentration is between the high concentration and the
decreased concentration. The high concentration preferably ranges
from about 200 ppm to 400 ppm, the decreased concentration
preferably ranges from about 5-20 ppm, and the third concentration
ranges from about 20 ppm to 200 ppm. Also, the first treatment
period is preferably at least seven hours in a day, and the second
and third treatment periods, each preferably ranges from about 5-12
hours in a day. The three step treatment may also be provided for
multiple days, and preferably for at least 3-14 days.
[0034] It is an object of the invention to provide a delivery
device for the topical delivery of a NO-containing gas to any
exposed wounds on the skin surface or subsurface, or any exposed
surface of the body such as the eye, or any exposed internal organs
of the body. It is a further object of the device to prevent the
NO-containing gas from leaking from the delivery device. The method
of delivering an effective amount of nitric oxide gas to the
infected or wounded area kills bacteria and other pathogens and
promotes the healing process.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 illustrates a schematic representation of the NO
delivery device according to one aspect of the invention.
[0036] FIG. 2 illustrates a bathing unit surrounding the foot of a
patient.
[0037] FIG. 3 illustrates a bathing unit surrounding the hand of a
patient.
[0038] FIG. 4 illustrates a bathing unit including an agitator
located therein.
[0039] FIG. 5 shows a specialized gaseous nitric oxide (gNO)
incubation chamber designed to conduct in vitro studies on the
effects of gNO exposure on mammalian cell cultures as well as
microbial cells under optimal growth conditions.
[0040] FIG. 6 depicts a S. aureus dosage curve for exposure to
gaseous NO (gNO) with bacteria grown on solid media. Relative
percentages of growth of S. aureus colony forming units (cfu) at
50, 80, 120 and 160 parts per million (ppm) of nitric oxide
compared with growth of S. aureus cfu in medical air (100%) are
shown.
[0041] FIG. 7 depicts a Pseudomonas aeruginosa dosage curve for
exposure to NO gas with bacteria grown on solid media. Relative
percentages of growth of P. aeruginosa colony forming units (cfu)
at 50, 80, 120 and 160 parts per million (ppm) of nitric oxide
compared with growth of P. aeruginosa cfu in medical air (100%) are
shown.
[0042] FIG. 8a-8m depict the bacteriocidal effect of 200 ppm gNO on
a variety of microbes.
[0043] FIG. 9 illustrates wound bacterial content following topical
application of 200 ppm gNO in a full thickness infected wound model
in rabbits.
[0044] FIG. 10 shows wound bacterial content following topical
application of 400 ppm gNO in a full thickness infected wound model
in rabbits.
[0045] FIG. 11 shows rabbit blood serum NOx (NO.sub.2 &
NO.sub.3) levels following topical application of 400 ppm gNO.
[0046] FIG. 12 illustrates rabbit blood methemoglobin levels
following topical application of 400 ppm gNO on a full thickness
infected wound model.
[0047] FIG. 13 illustrates histology analysis of full thickness
infected wound exposed to 200 ppm gNO for 24 hours.
[0048] FIG. 14 shows mRNA expression for collagen and collagenase
following exposure to 200 ppm gNO for 24 hours and 48 hours.
[0049] FIG. 15 illustrates the morphology of fibroblast cells
exposed inside gNO chamber to less than 200 ppm NO versus control
group inside conventional tissue culture incubator.
[0050] FIG. 16 illustrates increase in fibroblast cell
proliferation following exposure to 200 ppm of NO in comparison
with control.
[0051] FIG. 17 illustrates cell attachment capacity of human
fibroblasts following exposure to 160 ppm of gNO.
[0052] FIG. 18 shows the results of fibroblasts grown in a 3D
matrix and exposed to 200 ppm NO for 8 hours per day for 3 days
compared with control cells in air or conventional incubator.
[0053] FIG. 19 shows the amount of proliferation of fibroblasts
grown in a 3D matrix and exposed to 200 ppm NO for 8 hours per day
for 3 days compared with control cells in air or conventional
incubator.
[0054] FIG. 20 shows the amount of tube formation in human
endothelial cells grown in matrigel and exposed to air (top panels)
or 200 ppm NO (bottom panels) for 24 hours. Left panels at 8 hours
of exposure. Right panels at 24 hours of exposure.
[0055] FIG. 21 shows an increased collagen mRNA expression in
fibroblast exposed to 5 ppm of NO.
[0056] FIG. 22 shows various photographs of a human non-healing leg
ulcers at various stages of treatment with nitric oxide gas.
[0057] FIG. 23 shows the reduction in wound size in the human
non-healing ulcer of FIG. 23 following nitric oxide gas treatment.
Significant decrease in area was observed following 3 and 14 days
of gNO application to the wound (*p=0.019 vs. day 0; ** p=0.014 vs.
day 3). Wound status did not deteriorate after removal of treatment
(arrow, day 14). Wound was completely healed following 26 weeks
(186 days; ***p<0.01 vs. day 3). Values are means and standard
deviations.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0058] Referring now to FIG. 1, a NO delivery device 2 is shown
connected to a patient 4. In its most general sense, the NO
delivery device 2 includes a bathing unit 6 that is fluidically
connected to a NO gas source 8, a flow control valve 22, and a
vacuum unit 10. FIG. 1 illustrates one preferred embodiment of the
invention.
[0059] In FIG. 1, the NO gas source 8 is a pressurized cylinder
containing NO gas. While the use of a pressurized cylinder is the
preferred method of storing the NO-containing gas source 8, other
storage and delivery means, such as a dedicated feed line (wall
supply) can also be used. Typically, the NO gas source 8 is a
mixture of N.sub.2 and NO. While N.sub.2 is typically used to
dilute the concentration of NO within the pressurized cylinder, any
inert gas can also be used. When the NO gas source 8 is stored in a
pressurized cylinder, it is preferable that the concentration of NO
in the pressurized cylinder fall within the range of about 800 ppm
to about 2500 ppm. Commercial nitric oxide manufacturers typically
produce nitric oxide mixtures for medical use at around the 1000
ppm range. Extremely high concentrations of NO are undesirable
because accidental leakage of NO gas is more hazardous, and high
partial pressures of NO tends to cause the spontaneous degradation
of NO into nitrogen. Pressurized cylinders containing low
concentrations of NO (e.g., less than 100 ppm NO) can also be used
in accordance with the device and method disclosed herein. Of
course, the lower the concentration of NO used, the more often the
pressurized cylinders will need replacement.
[0060] FIG. 1 also shows source of diluent gas 14 as part of the NO
delivery device 2 that is used to dilute the concentration of NO.
The source of diluent gas 14 can contain N.sub.2, O.sub.2, Air, an
inert gas, or a mixture of these gases. It is preferable to use a
gas such as N.sub.2 or an inert gas to dilute the NO concentration
since these gases will not oxidize the NO into NO.sub.2 as would
O.sub.2 or air. The source of diluent gas 14 is shown as being
stored within a pressurized cylinder. While the use of a
pressurized cylinder is shown in FIG. 1 as the means for storing
the source of diluent gas 14, other storage and delivery means,
such as a dedicated feed line (wall supply) can also be used.
[0061] The NO gas from the NO gas source 8 and the diluent gas from
the diluent gas source 14 preferably pass through pressure
regulators 16 to reduce the pressure of gas that is admitted to the
NO delivery device 2. The respective gas streams pass via tubing 18
to an optional gas blender 20. The gas blender 20 mixes the NO gas
and the diluent gas to produce a NO-containing gas that has a
reduced concentration of NO. Preferably, the NO-containing gas that
is output from the gas blender 20 has a concentration that is less
than about 400 ppm and more preferably about 200 ppm. Depending on
the concentration needed for the specific application, the
concentration of NO-containing gas that is output from the gas
blender 20 can also be regulated to less than about 100 ppm or less
than about 40 ppm, if desired.
[0062] The NO-containing gas that is output from the gas blender 20
travels via tubing 18 to a flow control valve 22. The flow control
valve 22 can include, for example, a proportional control valve
that opens (or closes) in a progressively increasing (or decreasing
if closing) manner. As another example, the flow control valve 22
can include a mass flow controller. The flow control valve 22
controls the flow rate of the NO-containing gas that is input to
the bathing unit 6. The NO-containing gas leaves the flow control
valve 22 via flexible tubing 24. The flexible tubing 24 attaches to
an inlet 26 in the bathing unit 6. The inlet 26 might include an
optional one way valve 64 (see FIG. 3) that prevents the backflow
of gas into the tubing 24.
[0063] Still referring to FIG. 1, the bathing unit 6 is shown
sealed against the skin surface of a patient 4. The infected area
30 which can be an abscess, lesion, wound, or the like, is enclosed
by the bathing unit 6. The bathing unit 6 preferably includes a
seal portion 32 that forms a substantially air-tight seal with the
skin of the patient 4, or any other exposed surface of the body
(e.g., eye) or exposed internal organs desired to be treated.
Substantially air-tight is meant to indicate that the NO-containing
gas does not leak out of the bathing unit 6 in significant amounts
(i.e., no more than about 5% of the NO-containing gas delivered to
the bathing unit 6). The seal portion 32 may comprise an inflatable
seal 61, such as that shown in FIGS. 2 and 3, or alternatively the
seal portion 32 may comprise a flexible skirt or the like that
confirms to the surface of the patient 4. The seal portion 32 also
might include an adhesive portion that adheres to the skin surface
of a patient 4. In other envisioned embodiments, the sealing
portion 32 may merely comprise the interface of the bathing unit 6
with the surface of the patient's 4 skin.
[0064] The bathing unit 6 can be made of a virtually limitless
number of shapes and materials depending on its intended use. The
bathing unit 6 might be formed as a rigid structure, such as that
shown in FIG. 1, that is placed over the infected area 30.
Alternatively, the bathing unit 6 can be formed of a flexible,
bag-like material that is inflatable over the infected area 30.
FIG. 2 shows such a structure in the shape of a boot that is placed
over the patient's 4 foot. FIG. 3 shows another inflatable bathing
unit 6 that is formed in the shape of a mitten or glove that is
worn over the patient's 4 hand.
[0065] In one preferred embodiment of the invention, the bathing
unit 6 includes an NO sensor 34 that measures the concentration of
NO gas within the bathing unit 6. The NO sensor 34 preferably
reports this information to a controller 36 via signal line 38. An
optional NO.sub.2 sensor 40 can also be included within the bathing
unit 6. The NO.sub.2 sensor 40 preferably reports the concentration
of NO.sub.2 to the controller 36 via signal line 42. The sensors
40, 42 can be a chemilluminesense-type, electrochemical cell-type,
or spectrophotometric-type sensor.
[0066] The bathing unit 6 also includes an outlet 44 that is used
to remove gas from the bathing unit 6. The outlet 44 is preferably
located away from the gas inlet 26 such that NO gas does not
quickly enter and exit the bathing unit 6. Preferably, the inlet 26
and outlet 44 are located in areas of the bathing unit 6 such that
the NO gas has a relatively long residence time. Flexible tubing 46
is connected to the outlet 44 and provides a conduit for the
removal of gases from the bathing unit 6.
[0067] In one preferred embodiment of the invention, the flexible
tubing 46 is in fluid communication with an absorber unit 48. The
absorber unit 48 preferably absorbs or strips NO from the gas
stream that is exhausted from the bathing unit 6. It is also
preferable for the absorber unit 48 to also absorb or strip
NO.sub.2 from the gas stream that is exhausted from the bathing
unit 6. Since these gases are toxic at high levels, it is
preferable that these components are removed from the delivery
device 2 prior to the gas being vented to the atmosphere. In
addition, these gases can react with the internal components of the
vacuum unit 10 and interfere with the operation of the delivery
device 2.
[0068] The now clean gas travels from the absorbing unit 48 to the
vacuum unit 10 via tubing 50. The vacuum unit 10 provides a
negative pressure within the tubing 50 so as to extract gases from
the bathing unit 6. The vacuum unit 10 is preferably controllable
with respect to the level of vacuum or suction supplied to the
tubing 50 and bathing unit 6. In this regard, in conjunction with
the flow control valve 22, the amount of NO gas within the bathing
unit 6 can be regulated. Preferably, the vacuum unit 10 is coupled
with the controller 36 via a signal line 52. The controller 36, as
discussed below, preferably controls the level of output of the
vacuum unit 10. The gas then passes from the vacuum unit 10 to a
vent 54 that is open to the atmosphere.
[0069] It should be understood that the absorbing unit 48 is an
optional component of the delivery device 2. The gas laden with NO
and NO.sub.2 does not have to be removed from the gas stream if
there is no concern with local levels of NO and NO.sub.2. For
example, the gas can be exhausted to the outside environment where
high concentrations of NO and NO.sub.2 will not develop.
Alternatively, a recirculation system (not shown) might be used to
recycle NO with the bathing unit 6.
[0070] Still referring to FIG. 1, the delivery device 2 preferably
includes a controller 36 that is capable of controlling the flow
control valve 22 and the vacuum unit 10. The controller 36 is
preferably a microprocessor-based controller 36 that is connected
to an input device 56. The input device 56 is used by an operator
to adjust various parameters of the delivery device such as NO
concentration, residence or exposure time of NO, pressure within
the bathing unit 6, etc. An optional display 58 can also be
connected with the controller 36 to display measured parameters and
settings such as the set-point NO concentration, the concentration
of NO within the bathing unit 6, the concentration of NO.sub.2
within the bathing unit 6, the flow rate of gas into the bathing
unit 6, the flow rate of gas out of the bathing unit 6, the total
time of delivery, and the like.
[0071] The controller 36 preferably receives signals from sensors
34, 40 regarding gas concentrations if such sensors 34, 40 are
present within the delivery device 2. Signal lines 60, 52 are
connected to the flow control valve 22 and vacuum unit 10
respectively for the delivery and receipt of control signals.
[0072] In another embodiment of the invention, the controller 36 is
eliminated entirely. In this regard, the flow rate of the gas into
the bathing unit 6 and the flow rate of the gas out of the bathing
unit 6 are pre-set or adjusted manually. For example, an operator
can set a vacuum output that is substantially equal to the flow
rate of the gas delivered to the bathing unit 6 via the flow
control valve 22. In this manner, NO gas will be able to bathe the
infected area 30 without any build-up or leaking of NO or NO.sub.2
gas from the delivery device 2.
[0073] FIG. 2 illustrates a bathing unit 6 in the shape of a boot
that is used to treat an infected area 30 located on the leg of the
patient 4. The bathing unit 6 includes an inflatable seal 61 that
surrounds the leg region to make a substantially air-tight seal
with the skin of the patient 4. This embodiment shows a nozzle 62
that is affixed near the inlet 26 of the bathing unit 6. The nozzle
62 directs a jet of NO gas onto the infected area 30. The jet of
gaseous NO aids in penetrating the infected area 30 with NO to kill
or inhibit the growth of pathogens. FIG. 3 shows another embodiment
of the bathing unit 6 in the shape of a mitten or glove. The
bathing unit 6 is also inflatable and contains an inflatable seal
61 that forms a substantially air-tight seal around the skin of the
patient 4. FIG. 3 also shows an optional one way valve 64 located
in the inlet 26. As seen in FIGS. 3 and 4, the inlet 26 and outlet
44 are located away from one another, and preferably on opposing
sides of the treated area such that freshly delivered NO gas is not
prematurely withdrawn from the bathing unit 6.
[0074] For treatment of an infected area 30, the bathing unit 6 is
placed over the infected area 30. An air-tight seal is then formed
between the skin of the patient 4 and the bathing unit 6. If the
bathing unit 6 has an inflatable construction, the bathing unit 6
must be inflated with gas. Preferably, the bathing unit 6 is
initially inflated only with the diluent gas to prevent the leaking
of NO and NO.sub.2 from the device 2. Once an adequate air-tight
seal has been established, the operator of the device initiates the
flow of NO from the NO gas source 8 to the bathing unit 6. As
described above, this may be accomplished manually or via the
controller 36.
[0075] Once the bathing unit 6 has started to fill with NO gas, the
vacuum unit 10 is turned on and adjusted to the appropriate output
level. For an inflatable bathing unit 6, the output level (i.e.,
flow rate) of the vacuum unit 10 should be less than or equal to
the flow rate of NO gas entering the bathing unit 6 to avoid
deflating the bathing unit 6. In embodiments of the device where
the bathing unit 6 is rigid, the vacuum unit 10 can be set to
create a partial vacuum within the bathing unit 4. In this regard,
the partial vacuum helps to form the air-tight seal between the
skin of the patient 4 and the bathing unit 6. Of course, the vacuum
unit 10 can also be set to withdraw gas at a substantially equal
rate as the gas is delivered to the bathing unit 6. An effective
amount of NO is delivered to the bathing unit 6 to kill pathogens
and/or reduce the growth rate of the pathogens in the infected area
30. Pathogens include bacteria, viruses, and fungi.
[0076] FIG. 4 shows another embodiment of the invention in which
the bathing unit 6 includes an agitator 66 that is used to create
turbulent conditions inside the bathing unit 6. The agitator 66
preferably is a fan-type of mechanism but can include other means
of creating turbulent conditions within the bathing unit 6. The
agitator 66 aids in refreshing the infected area 30 with a fresh
supply of NO gas.
Examples of Nitric Oxide Applications
[0077] In chronic non-healing wound such as in patients suffering
from diabetic lesions, a variety of factors can potentially
influence wound healing, including infections, excessive exudate,
necrotic tissue, poor tissue handling, and impaired tissue
perfusion. Nitric oxide gas can be used to reduced the infection or
microbial burden on the wound. While the examples discussed below
are applications of nitric oxide to the skin, nitric oxide can also
be topically applied to other surfaces of the body such as the eye,
or any other exposed surface such as muscle, ligaments, tendons,
and internal organs of the body that may be exposed, for example,
due to cut, tear, or wound.
[0078] To study the effects of gaseous nitric oxide on potential
pathogens, a custom gas exposure incubator was designed and
validated for temperature, humidity, and gas concentrations,
providing an environment that matches that of a microbiologic
incubator, while enabling controlled exposure of precise
concentrations of the gas. FIG. 5 shows a specialized gaseous
nitric oxide (gNO) incubation chamber designed to conduct in vitro
studies on the effects of gNO exposure on mammalian cell cultures
as well as microbial cells under optimal growth conditions. The gNO
chamber allowed control and adjustment of following factors in all
in vitro studies: gNO dose, total air flow, NO.sub.2 levels,
O.sub.2 levels, CO.sub.2 levels, temperature, and humidity.
[0079] For the initial pilot studies, two strains of bacterial
pathogen were selected based on two proposed clinical applications
of gNO for respiratory infections and topical application. P.
aeruginosa is associated primarily with pulmonary disease, but may
also be associated with skin infection such as in severe burns. S.
aureus is associated with surface wound infections. Both of these
micro-organisms were chosen for the pilot study.
[0080] The first step in the process of evaluating the direct
effect of gNO on bacteria was to design a simple study to determine
what dose, if any, would be an approximate lethal concentration
level for microbes. Once an optimal dose was estimated, then a
timing study would be conducted. For these initial studies, highly
dense inoculums of P. aeruginosa and S. aureus suspensions
(10.sup.8 cfu/ml) were plated onto agar plates. These plates were
then exposed to various concentrations of gNO in the exposure
device in order to evaluate the effect on colony growth.
[0081] FIGS. 6 and 7 demonstrate that levels of gNO greater than
120 ppm reduced the colony formation of the bacteria by greater
than 90%. Further studies indicated that the time required to
achieve this affect occurred between 8-12 hours. These results
confirm that gNO has an inhibitory effect on P. aeruginosa and S.
aureus growth. Additionally, the data provide preliminary evidence
that there is a time and dose relationship trend, with the amount
of bacteriocidal activity increasing with increased time of
exposure and concentration of gNO. That is, as the concentration of
gNO increases, the number of colonies growing on the plates
decreases.
[0082] Although there was a downward bacteriocidal trend towards
5-10% survival with increasing gNO to 120 ppm, none of the initial
data showed a 100% bacteriocidal effect. Some bacteria may have
survived because the materials and chemicals in the agar may have
reacted with the gNO and buffered the effect. Of significance, was
the observation that bacterial colonies remained the same in size
and number after being transferred to a conventional incubator for
24 hours whereas controls increased in number and size to the
degree that they could not be counted. This strongly suggested that
gNO exposure prevented the growth of the bacteria, and may have
killed the bacteria at some point during the gNO exposure.
Accordingly, subsequent studies were designed to further study the
bacteriocidal effects of gNO.
[0083] Following the dose and time ranging studies, a series of
experiments were performed to determine the time required to
effectively induce a bacteriocidal effect with 200 parts per
million of gNO, a concentration just above the dose used in the
dose-ranging study, on a representative collection of drug
resistant gram-positive and gram-negative strains of bacteria
associated with clinical infection. A successful bacteriocidal
effect was defined as a decrease in bacteria greater than 3
log.sub.10 cfu/ml. Further, C. albicans, Methicillin Resistant S.
aureus (MRSA), a particularly resistant strain of P. aeruginosa
from a cystic fibrosis patient, Group B Streptococcus, and M.
smegmatis were also included to see if yeast, multi-drug resistant
strains of bacteria, and actinomycetes have a similar response. The
drug-resistant bacteria represent a variety of pathogens that
contribute to both respiratory and wound infections.
[0084] For these experiments, saline was selected as a suspension
media because it would not mask the direct effect of gNO as a
bacteriocidal, whereas fully supplemented growth medium might
introduce external variables (e.g., buffer or react with gNO).
Other media might also provide metabolites and replenish nutrients
that produce enzymes that protect bacteria from oxidative and
nitrosative damage, thereby masking the effect of gNO. Furthermore,
it has been suggested that a saline environment more realistically
represents the hostile host environment to which bacteria are
typically exposed in vivo. In saline, the colonies were static but
remained viable. This is similar to the approach of Webert and
Jean's use of animal models. Webert K E, et al (2000), Effects of
inhaled nitric oxide in a rat model of Pseudomonas aeruginosa
pneumonia, Crit Care Med, 28(7):2397-2405 and Jean D, et al.,
(2002) Beneficial effects of nitric oxide inhalation on pulmonary
bacterial clearance, Critical Care Medicine. 30(2):442-7.
[0085] FIG. 8 shows the results of these experiments with the line
plotted by square-shaped points representing survival curves of the
control exposure microorganisms and the line plotted in
triangle-shaped points representing the survival curves of the NO
exposed microorganisms. These studies showed that gNO at 200 ppm
had a completely bacteriocidal effect on all microorganisms tested.
Without exception, every bacteria challenged with 200 ppm gNO had
at least a three log.sub.10 reduction in cfu/ml and every test
resulted in a complete and total cell death of all bacteria. These
results were also characterized by a period of latency when it
appeared that the bacteria were unaffected by gNO exposure (Table
1). The latent period was then followed by an abrupt death of all
cells. Gram negative and gram positive bacteria, antibiotic
resistant bacterial strains, yeast and mycobacteria were all
susceptible to 200 ppm gNO. Of importance, is the observation that
the two drug resistant bacteria strains were also susceptible.
Accordingly, these results show that gNO directly exhibits a
non-specific lethal effect on a variety of potentially pathogenic
microorganisms.
[0086] The study also indicates a significant difference in the lag
period for mycobacteria compared to all other organisms. The lag
period suggests that mycobacteria may have a mechanism that
protects the cell from the cytotoxicity of gNO for a longer period
than other bacteria. Applicants believe that there is a dose-time
dependent gNO threshold reached within the cell at which point
rapid cell death occurs. It is possible that this threshold occurs
when the normal NO detoxification pathways of the bacteria are
overwhelmed. These studies indicate and confirm that
supraphysiologic levels of NO (provided exogenously, for example,
via delivery of 120 ppm to 400 ppm exogenous NO) may be
bacteriocidal on representative strains of drug resistant bacteria
and the effect appears to be abrupt, lethal and non-specific on
these bacteria.
TABLE-US-00001 TABLE 1 Gram Latent Period -2.5 Log.sub.10
LD.sub.100 Bacteria staining (hrs) (hrs) (Hrs) S. aureus (ATCC)
Positive 3 3.3 4 P. aeruginosa Negative 1 2.1 3 (ATCC) MRSA
Positive 3 4.2 5 Serracia sp. Negative 4 4.9 6 S. aureus (Clinical)
Positive 3 3.7 4 Klebsiella sp. #1 Negative 3 3.5 6 Klebsiella sp.
#2 Negative 2 4.1 5 Klebsiella sp. #3 Negative 3 5.1 6 S.
maltophilia Negative 2 2.8 4 Enterobacter sp. Negative 4 5.3 6
Acinetobacter sp. Negative 4 5 6 E. coli Negative 3 4.2 5 Group B
Positive 1 1.5 2 Streptococci Average N/A 2.77 3.82 4.77 SD N/A
1.01 1.17 1.30 Mycobacterium Positive 7 9.2 10
[0087] To achieve a lethal effect over a broad range of microbes,
200 ppm of nitric oxide gas is preferably exposed to the wound site
for at least 7 hours continuously such as when the patient is
asleep at night. Shorter times may be used with higher
concentration such as 400 ppm. Longer treatment options may also be
provided that span days. Depending on the subject, periods of
breaks in between treatment may also be arranged.
[0088] In vivo studies in animal models have further shown the
beneficial effects of nitric oxide gas. In an animal model,
full-thickness cutaneous wounds (Set A: four rabbits with eight 8.0
mm punch biopsies & Set B: 4 rabbits with two 50.times.15 mm
wounds) were made on each side of dorsal midline and infected with
equal volume of Staphylococcus aureus suspension on day zero. On
day one, treated groups in A and B were respectively exposed to 200
and 400 ppm gNO for total of three days. Set A was exposed for two
4-hour sessions, interrupted by 1-hour of rest, inside a
specialized restraining exposure chamber. A 24-hour continuous
delivery model was used for animals in Set B by design of a
specialized wound patch. Control groups were only exposed to
medical grade air with corresponding flow rate. Four random sample
punch biopsies (8.0 mm) were collected on post wounding days 3 and
analyzed for bacterial content. Another four punch biopsies from
both wound and normal skin tissue were collected for fibroblast
viability analysis and toxic effects of gNO.
[0089] FIG. 9 reveals data from the animal study on bacterial
content of the wounds exposed to 200 ppm gNO continuously for 72
hours when compared to control group only exposed to medical air. A
significant bacterial reduction is observed in treated wounds.
Rabbits appeared comfortable and at ease during the therapy and no
toxic effect or damage were observed in the skin of treated animals
when compared to the control. NO.sub.2 did not exceed safety
limits, at any point of the study, set by Occupational Safety and
Health Administration (<4.3.+-.0.3 ppm). FIG. 10 shows similar
set of data as seen in FIG. 9, but where animal wounds were exposed
to 400 ppm of gNO therapy. On average well over 10 fold drop
(p<0.05) in bacterial content is observed in comparison between
control and treated groups.
[0090] FIG. 11 demonstrates that nitrogen oxides levels (NO.sub.2
and NO.sub.3), one of end products of nitric oxide metabolism,
measured in blood serum collected from the animals following
exposure to 200 ppm gNO intermittently for 6 days. None of the
samples show an increased level of NOx due to exposure to gNO
indicating the fact that exposing full thickness wounds (8 at 8.0
mm in diameter) will not increase the nitric oxide level in
animal's circulation system.
[0091] FIG. 12 indicates the level of methemoglobin (MetHb) in
animal's blood following 6 day intermittent exposure to 200 ppm
gNO. Animals in the treated group did not show an increase level of
MetHB in comparison with the control group exposed to air. This
further supports the data presented in FIG. 11 to the fact that
topical application of gNO on open wounds did not contribute to an
increase level of nitric oxide in the circulation and that the
topical application of an open wound to about 200 ppm poses no
significant toxicity concerns over the formation of
methemoglobin.
[0092] FIG. 13 presents histological analysis of tissue blocks
prepared on wound punch biopsies from animals in treated and
control groups. Samples from the control group show more advanced
neutrophil infiltration and so a higher degree of inflammatory
reaction. A lower level of neutrophil concentration is seen in
wounds treated with gNO. Wounds treated with gNO also show a layer
of scab closing on the wound, but control wounds remain open for
longer period of time. Overall, a healthier healing process is
observed in the wounds treated with gNO. No toxic effects (cellular
debris due to apoptosis) can be seen in gNO treated group.
[0093] While the inflammatory response is integral to wound
healing, an aberrant inflammatory response is believed to be one
causal factor in chronic wounds and excess exudate. NO inhibits
platelet aggregation, assists in maintaining vascular tone, and
inhibits mast cell degranulation. Delledonne M, et al., (2003) The
functions of nitric oxide-mediated signaling and changes in gene
expression during the hypersensitive response, Antioxid Redox
Signal, 5:33-41. and Hickey M J., (2001), Role of inducible nitric
oxide synthase in the regulation of leukocyte recruitment, Clin Sci
(Lond), 100:1-12. NO produced constitutively by endothelial cells
has been shown to have an on-going anti-inflammatory effect. Id.
This may in part be due to its effect on platelet aggregation. iNOS
is upregulated during the inflammatory response. Studies have shown
that iNOS derived NO may also have anti-inflammatory
characteristics. Id. Collectively, by maintaining vascular tone,
promoting angiogenesis, moderating inflammation and inhibiting mast
cell degranulation, NO can be viewed as an important molecule for
exudate management. Accordingly, exogenously applied nitric oxide
may duplicate and supplement the actions of endogenous nitric oxide
to reduce the local inflammatory response as well as down regulate
the message that the systemic inflammatory response system had been
receiving to increase the sending of inflammatory cells. This
eventually may lead to a healthy level of exudate production.
[0094] FIG. 14 shows that expression of collagenase mRNA is
increased as the exposure time to high concentration of gNO (at 200
ppm) increases. This suggests that high concentration of nitric
oxide upregulate collagenase that may lead to the enzymatic
cleavage of collagen. An independent study by Witte et al (2002)
found that MMP-2 activity was also upregulated by NO donors. Witte
M B, et al, (2002) Nitric oxide enhances investigational wound
healing in diabetes, Br J Surg., 89:1594-601. Thus, Applicants
believe that NO may upregulate expression of both collagenase
(MMP-1) and gelatinase (MMP-2), which may be important in keeping
the wound clean from necrotic tissue while not prolonging the
inflammatory phase.
[0095] Rather than applying exogenous collagenase for enzymatic
debridement of necrotic tissue, exposing a wound with necrotic
tissue to exogenous NO gas to upregulate endogenous collagenase may
be more beneficial. When endogenous collagenase is released by the
cell, it automatically releases TIMP's (tissue inhibitor of
metalloproteinase). This ensures that the matrix degradation is
coordinated and allows the establishment of sharp geographical
boundaries of collagenolytic activity and the protection of areas
of connective tissue from the activity of the enzyme. In contrast,
use of exogenous collagenase material to debride a wound confers no
protection to specific areas of the wound as it is active on every
cell that comes in contact with it whether or not the effect is
desired. The ability of nitric oxide to debride a wound is further
supported by the possible inhibition of collagen expression due to
high concentration of exogenous nitric oxide applied to the wound,
as seen in FIG. 14 (left panel).
[0096] Preferably, after the exposure of the wound to high
concentration of nitric oxide gas for a first treatment period
(e.g., 5-8 hours per day), the necrotic tissue may be mechanically
removed easily and the concentration of nitric oxide gas can be
decreased for a second treatment period. The low concentration of
nitric oxide gas (e.g., at 5-20 ppm) delivered for the second
treatment period may upregulate the expression of collagen mRNA
leading to synthesis of new collagen to aid in the closure of the
wound. For example, FIG. 22 shows an increased collagen mRNA
expression in fibroblast exposed to 5 ppm of NO. The second
treatment period may be for a period 7-16 hours per day. Further,
the treatment with high and low concentration of nitric oxide gas
can be repeated for several days.
[0097] For chronic non-healing ulcers on the skin, it is also
possible to graft natural skin tissue or synthetically produced
skin tissue onto the ulcer after the wound has been prepared. Wound
bed preparation may include the reduction of microbial load,
debridement, and the management of exudate.
[0098] It is believed that the body's natural response to injury is
to increase the amount of nitric oxide in order to reduce bacterial
count at the injury site, help remove dead cells and then promote
healing. The message sent by the injury site has more than just the
cells at the injury site producing nitric oxide and this circulates
NO around the body in the blood stream. After a few days of this
preparation for healing, the body decreases the nitric oxide it
produces to a new level that will promote healing. If a wound fails
to heal or becomes infected, the body maintains the circulating
nitric oxide at a high level and the wound is then caught with a
concentration of nitric oxide that may prevent it from healing. It
becomes the "Catch 22" of wound healing. Bathing the injury site to
high concentration of nitric oxide gas (e.g., 120 ppm to 400 ppm)
sends a message to the body that there is enough nitric oxide at
the injury site and therefore the body can shut down the extra
production by other cells. This enables the local site to heal
while it receives the appropriate supraphysiological concentration
of nitric oxide gas to inhibit microbial growth.
Additional Safety Studies
[0099] In addition to the above study showing no toxicity of in
vivo exposure of 200 ppm of nitric oxide gas in an animal model for
an open wound, studies to confirm the viability of normal host
cells exposed to gNO were performed on fibroblasts, endothelial
cells, keratinocytes, alveolar epithelial cells, macrophages, and
monocytes, in both flat plate and 3-D growth models for some
studies. These experiments looked at viability, proliferation,
migration, attachment, expression and tube formation in the
appropriate models.
[0100] Fibroblast cells obtained from adult patients undergoing
elective reconstructive surgery were cultured in Dulbeco's Modified
Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum
(FBS) and antibiotic-antimycotic preparation and divided into ten
25 cm.sup.2 vented culture flasks (COSTAR). Four of these flasks
(treated group) were exposed to 20 or 200 ppm humidified gNO inside
a specialized NO incubation chamber at 37.degree. C. for 24 and 48
hours. The NO exposure chamber was validated prior to the study to
eliminate extraneous variables and ensure optimal conditions for
fibroblast cell growth. Another four flasks (control group) were
placed inside conventional culture incubator and exposed only to
ambient humidified air at 37.degree. C. Two flasks were separately
harvested and counted as the number of cells at zero time.
Following the treatment, fibroblast cells were harvested and
evaluated for morphology, cell count, capacity to proliferate and
medium pH. The results from these experiments show that exposure to
around 200 ppm of gNO did not have harmful effects on the
fibroblast.
[0101] FIG. 15 shows morphology of fibroblast cells from the
viability study, where cultured human fibroblast cells were exposed
to various gNO concentrations less than 200 ppm continuously for 48
hours. Morphological appearance and attachment capacity of control
and treated dermal fibroblasts cells following 48 hours period were
quite comparable. Cells under gNO appeared healthy and attached to
the culture plates. No toxic effect due to exposure to gNO was
observed.
[0102] FIG. 16 shows that, in addition to a lack of toxicity to
fibroblast cells, exposure to 200 ppm NO may also have positive
effect of increasing proliferation of fibroblast cells that may
further aid in the wound healing process.
[0103] FIG. 17 shows results from cell attachment capacity from the
fibroblast cells exposed to 160 ppm of gNO. Capability of cells to
reattach to the culture plates within a specified time limit is
commonly used as an indication of viability of cells in culture.
Both the control and treated groups show a 70% attachment capacity
within 1 hour of culturing. This result in conjunction with cell
morphology and count support the safety of gNO therapy for topical
applications on mammalian skin tissue at least at a range between
100 to 200 ppm of gNO.
[0104] FIG. 18 shows the amount of migration of fibroblasts grown
in a 3D matrix and exposed to 200 ppm NO for 8 hours per day for 3
days compared with control cells in air or conventional incubator.
As seen from these results, NO does not appear to affect (or more
specifically does not interfere with) the migration of these
fibroblasts under these conditions.
[0105] FIG. 19 shows the amount of proliferation of fibroblasts
grown in a 3D matrix and exposed to 200 ppm NO for 8 hours per day
for 3 days compared with control cells in air or conventional
incubator. Again, NO does not appear to interfere with the
proliferation of fibroblasts under these conditions.
[0106] FIG. 20 shows the tube formation in human endothelial cells
grown in matrigel and exposed to air (top panels) or 200 ppm NO
(bottom panels) for 8 hours (left panels) or 24 hours (right
panels). Again, no significant difference between exposure to air
and 200 ppm can be discerned.
Human Case Study
[0107] This case study involved a 55-year-old man with a 30 year
history of severe venous disease, both deep and superficial,
related to deep vein thrombophlebitis. Initially, while in his
twenties, the patient developed bilateral non-healing venous leg
ulcers that were surgically treated. The surgical sites healed but
the ulcers continued to recur. Initially, the patient presented
with a small ulcer located just below the medial malleolus of the
left ankle. Although not increasing in size, this ulcer did not
completely heal with two years of standard of care therapy.
[0108] Most of the time the wound base was covered with a
biofilm--a tenacious, yellow-colored, gel-like material. Edema
control was maintained by using graduated compression stockings.
Antimicrobial dressings were tried including Manuka Honey, a starch
iodine preparation (Iodosorb, Smith & Nephew, Largo, Fla.,
USA), and colloidal silver (Aquacel AG, ConvaTec, Princeton, N.J.,
USA). His wound was frequently debrided in order to physically
remove the biofilm. This was generally ineffective as the biofilm
was frequently noted to be present again at the next visit. Twenty
percent benzyol peroxide lotion was applied every few days in order
to trigger the development of granulation tissue; however, this was
ineffective as well. At times there would be improvement as the
ulcer would appear to become covered with new skin only to break
down weeks later. This poor progress to complete closure was noted
despite wound care that addressed proper moisture balance, wound
bed preparation, and treatment of the underlying disease.
[0109] This failure of his wound to close had a significant impact
on quality of life for this patient. He made clinic office visits
at least once a month for the entire two years. The cost of the
treatment, including the surgeon s time and treatment materials
(several thousand dollars), put pressure on the health care system
as well as on the patient, with him having to travel several hours
each visit for treatment. As previous treatments proved
ineffective, the patient was invited to participate in this
experimental study. Following a discussion of the experimental
therapy and potential risks, an informed consent was obtained.
[0110] The patient was seen at the clinic where the wound was
assessed and photographed (FIG. 22). The treatment regimen was
explained and the use of the CidaNOx Delivery System and boot was
demonstrated. Arrangements were made to meet at the patient's home
the following day to set up the equipment and for him to have a
repeat training on the use of the treatment system. Training
included use of the system as well as safety information on using
the gas equipment.
[0111] Nitric oxide gas (ViaNOx-H, VIASYS Healthcare, Yorba Linda,
Calif., USA) was applied to the lower extremity with use of a
gas-diluting delivery system (CidaNOx Delivery System) designed
specifically for the study (PulmoNOx Medical Inc., Edmonton,
Alberta, Canada). This CidaNOx delivery system contains an internal
air pump for dilution of the gNO and a flow control circuit to
dilute the 800 parts per million (ppm) in the NO source cylinder
down to the therapeutic level of 200 ppm. The total flow from the
system was 1.0 L/min and included one-quarter of a liter per minute
(250 ml/min) flow of gNO. Several internal pressure sensors assure
the dilution flow is operational and monitor the system. The flow
of nitric oxide was limited to 250 ml/min by a mechanically set
pressure regulator and a mechanical flowmeter that have no external
controls that could be changed by the patient. The concentration of
nitric oxide delivered was assured by measurement of the CidaNOx
output with a calibrated nitric oxide analyzer (AeroNOx, Pulmonox
Medical Inc.) that is approved for monitoring inhaled NO in human
patients.
[0112] The 200 ppm gNO from the CidaNOx Delivery System flowed out
to a single patient use plastic boot that covered the patient's
lower extremity. The boot had an inflatable cuff near the top that
provided a low-pressure seal. A secondary air outlet from the
CidaNOx unit managed the inflation of the cuff. The patient
connected the pump outlet to the cuff connector until it was
inflated and then the connector was sealed closed with the provided
clamp. The gNO flow was then connected to the inlet connector near
the toe of the boot and the return line to the connector near the
top of the boot. The return line passed through the CidaNOx unit
and then out through a scavenger consisting of charcoal and
potassium permanganate that absorbs the nitrogen oxides. The
CidaNOx Delivery System had two toggle positions, one for delivery
of gNO and the other for delivery of air only. At the end of the
treatment period, the patient switched the delivery flow to air
only so as to clear the boot of remaining gNO before taking the
boot off.
[0113] The patient was instructed to continue wearing supportive
stockings and to use a hydrofiber dressing (Aquacel, Convatec) on
the wound when not receiving gNO treatment. During the gNO
treatment, he removed the supportive stocking and replaced the
Aquacel dressing with a porous, low adherence dressing (ETE,
Molnlycke Health Care, Sweden), which had previously been shown to
allow the diffusion of gNO through it (data not shown).
[0114] To explore the potential for wound bed preparation and
accelerated wound healing from prolonged use, treatment regimen
beyond three days was chosen and which was stopped at 14 days to
evaluate the short-term effects and explore the possibility that
the short-term effects would improve the longer-term outcome. The
patient was encouraged to wear the gNO boot as often as possible
during each 24-hour period. As the patient worked during the day,
it was decided that it would be most practical to wear the boot and
receive the gNO treatments only while in bed at night. The patient
recorded the date, time, and duration of each treatment period on a
data sheet, and any significant observations related to the wound,
treatment, or equipment. The wound size (cm.sup.2) was measured
using digital photography and densitometry technique (Scion Image
-4.02, Scion Corp., Frederick Md., USA).
[0115] The patient self-administered the treatment for 14
consecutive nights. The nocturnal treatment duration varied from
6.5 to 9.75 hour per treatment. The cumulative wound exposure to
200 ppm gNO during the 14 treatment periods was 105.25 hour. The
wound was assessed and photographed on day 0 (FIG. 22A,
pretreatment), day 3 (FIG. 22B, following accumulative 24 hour of
gNO exposure), and day 14 (FIG. 22C). The wound was also assessed
and photographed ten days following the completion of the 14-day
treatment (FIG. 22D) and in the 6th and 26th week following the
completion of the treatment (FIGS. 22E and 22F, respectively).
[0116] During the active treatment period, the subject was assessed
with respect to the use of the CidaNOx system. The subject found
the system easy to use in a fixed location, found the application
of the bag comfortable, and never reported any pain associated with
its use. He suffered no bleeding episodes. FIG. 23A shows the
initial presentation of the ulcer prior to use of the gNO. The
wound base was covered by a biofilm and there was little healthy
granulation tissue present and there was no evidence of new skin
growth from the edges. The wound was malodorous.
[0117] After 24 hours of NO exposure (3 days at 8 hours day), for
the first time there was healthy granulation tissue noted in the
ulcer base. There was also early evidence of new skin growth from
the edges observed. The malodorous odor was also absent.
Concomitantly, there was less biofilm present (FIG. 22B). At 14
days of therapy (FIG. 22C) the ulcer clearly had diminished in
size. By then it had almost completely epithelialized. Significant
wound size reduction was observed as early as day 3 of gNO
treatment (p=0.014), with approximately 75% reduction in wound area
by the end of gNO therapy at day 14 (FIG. 23). The wound was
further assessed 10 days after cessation of gNO treatment (FIG.
22D). There did not appear to be any deterioration of the wound
during this time, although the ulcer was judged to be incompletely
healed. No significant deterioration in wound size was observed
compared to the last day of gNO treatment (FIG. 23). Six weeks
later the wound was judged to be about 90% healed with no
deterioration in wound size or epithelialization (FIG. 22E and FIG.
23). At 26 weeks post NO discontinuation, the ulcer was noted to be
completely healed and reepithelialized (FIG. 22F). Over the entire
post-treatment time, there were no changes to the dressing regimen
and no other anti-microbials or antibiotics were used.
[0118] The average time for ulcers that result from venous stasis
disease to heal under optimal care ranges from 12 to 16 weeks. Our
patient, who had a nonresponsive ulcer for more than two years,
exhibited a positive response to a brief exposure to gaseous nitric
oxide. His wound decreased in size, a granular base was
established, and the malodorous smell was eradicated during this
two-week period. Further studies and randomized controlled trials
will be able to answer whether a longer exposure or a different
concentration, once the biofilm was eliminated, would have made a
difference in the closure of the lesions.
[0119] While embodiments of the present invention have been shown
and described, various modifications may be made without departing
from the scope of the invention. For example, the types of tissue
that may have wounds to be treated using the methods described
herein may include, without limitation, the skin, muscle, tendon,
ligament, mucosa, bone, cartilage, cornea, and exposed internal
organs. The tissue may be damaged by surgical incisions, trauma
(mechanical, chemical, viral, bacterial, or thermal in nature), or
other endogenous pathological processes. The invention, therefore,
should not be limited, except to the following claims, and their
equivalents.
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