U.S. patent application number 14/382829 was filed with the patent office on 2015-02-12 for inhalation of nitric oxide for treating respiratory diseases.
The applicant listed for this patent is Advanced Inhalation Therapies (AIT) Ltd.. Invention is credited to Yossef Av-Gay, David Greenberg, Christopher C. Miller.
Application Number | 20150044305 14/382829 |
Document ID | / |
Family ID | 49116038 |
Filed Date | 2015-02-12 |
United States Patent
Application |
20150044305 |
Kind Code |
A1 |
Av-Gay; Yossef ; et
al. |
February 12, 2015 |
INHALATION OF NITRIC OXIDE FOR TREATING RESPIRATORY DISEASES
Abstract
A method of treating a human subject which is effected by
intermittent inhalation of gaseous nitric oxide at a concentration
of at least 160 ppm is disclosed. The method can be utilized for
treating a human subject suffering from, or prone to suffer from, a
disease or disorder that is manifested in the respiratory tract, or
from a disease or disorder that can be treated via the respiratory
tract. The disclosed method can be effected while monitoring one or
more of on-site and off-site parameters such as vital signs,
methemoglobin levels, pulmonary function parameters, blood
chemistry and hematological parameters, blood coagulation
parameters, inflammatory marker levels, liver and kidney function
parameters and vascular endothelial activation parameters, such
that no substantial deviation from a baseline in seen in one or
more of the monitored parameters.
Inventors: |
Av-Gay; Yossef; (Vancouver,
CA) ; Miller; Christopher C.; (N. Vancouver, CA)
; Greenberg; David; (Omer, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Advanced Inhalation Therapies (AIT) Ltd. |
Beer-Yaacov |
|
IL |
|
|
Family ID: |
49116038 |
Appl. No.: |
14/382829 |
Filed: |
March 7, 2013 |
PCT Filed: |
March 7, 2013 |
PCT NO: |
PCT/IL13/50211 |
371 Date: |
September 4, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61607686 |
Mar 7, 2012 |
|
|
|
Current U.S.
Class: |
424/718 |
Current CPC
Class: |
A61K 33/00 20130101;
A61M 2202/0275 20130101; A61K 9/007 20130101; A61B 5/14507
20130101; A61M 16/122 20140204; A61M 2230/205 20130101; A61B
5/14542 20130101; A61P 31/00 20180101; A61B 5/0833 20130101; A61P
11/00 20180101; A61M 2205/3303 20130101; A61M 2230/202 20130101;
A61B 5/0836 20130101; A61M 16/0003 20140204; A61P 11/08 20180101;
A61M 16/12 20130101; A61M 2230/207 20130101; A61B 5/4244 20130101;
A61M 2202/0007 20130101; A61M 2230/432 20130101; A61M 2202/0275
20130101; A61M 2202/0007 20130101 |
Class at
Publication: |
424/718 |
International
Class: |
A61K 33/00 20060101
A61K033/00; A61K 9/00 20060101 A61K009/00 |
Claims
1. A method of treating a human subject in need of inhalation of
gaseous NO (gNO), the method comprising subjecting the subject to
intermittent inhalation of gNO at a concentration of at least 160
ppm, while monitoring at least one on-site parameter selected from
the group consisting of: a methemoglobin level (SpMet); an oxygen
saturation level (SpO.sub.2); and an end tidal CO.sub.2 level
(ETCO.sub.2); in the subject, wherein a change in at least one of
said on-site parameters following said subjecting is less than 2
acceptable deviation units from a baseline.
2. The method of claim 1, wherein said monitoring is of at least
two of said on-site parameters.
3. The method of claim 1, wherein said monitoring is of all of said
on-site parameters.
4. The method of claim 2, wherein a change in said at least two
on-site parameters following said subjecting is less than 2
acceptable deviation units from a baseline.
5. The method of claim 3, wherein a change in any of said on-site
parameters following said subjecting is less than 2 acceptable
deviation units from a baseline.
6. The method of claim 1, further comprising monitoring at least
one off-site parameter selected from the group consisting of: a
serum nitrite/nitrate level (NO.sub.2.sup.-/NO.sub.3.sup.-); and an
inflammatory cytokine plasma level.
7. The method of claim 6, wherein said cytokine is selected from
the group consisting of (TNF).alpha., (IL)-1.beta., IL-6, IL-8,
IL-10 and IL-12p70.
8. The method of claim 1, wherein a change in one or both of said
off-site parameters following said subjecting is less than 2
acceptable deviation units from a baseline.
9. The method of claim 1, further comprising monitoring urine
nitrite level in the subject.
10. The method of claim 9, wherein a change in said urine nitrite
level following said subjecting is less than 2 acceptable deviation
units from a baseline.
11. The method of claim 1, further comprising monitoring in the
subject at least one off-site parameter selected from the group
consisting of: a hematological marker a vascular endothelial
activation factor; a coagulation parameter; a serum creatinine
level; and a liver function marker.
12. The method of claim 11, wherein said hematological marker is
selected from the group consisting of a hemoglobin level, a
hematocrit ratio, a red blood cell count, a white blood cell count,
a white blood cell differential and a platelet count in the
subject.
13. The method of claim 11, wherein a change in at least one of
said off-site parameters following said subjecting is less than 2
acceptable deviation units from a baseline.
14. The method of claim 11, wherein a change in any of said
off-site parameters following said subjecting is less than 2
acceptable deviation units from a baseline.
15. The method of claim 1, further comprising monitoring in the
subject at least one on-site parameter selected from the group
consisting of: a vital sign; and a pulmonary function.
16. The method of claim 15, wherein no deterioration in at least
one of said parameters is observed during and following said
subjecting.
17. The method of claim 1, wherein said intermittent inhalation
comprises at least one cycle of continuous inhalation of said gNO
for a first time period, followed by inhalation of no gNO for a
second time period.
18. The method of claim 17, wherein said first time period is about
30 minutes.
19. The method of claim 17, wherein said second time period ranges
from 3 to 5 hours.
20. The method of claim 18, wherein said second time period ranges
from 3 to 5 hours.
21. The method of claim 17, wherein said inhalation comprises from
1 to 6 of said cycles per day.
22. The method of claim 21, wherein said inhalation comprises 5 of
said cycles per day.
23. The method of claim 17, wherein during said first time period,
said concentration of gNO in said mixture deviates from said
concentration of at least 160 ppm by less than 10%.
24. The method of claim 17, wherein during said first time period,
a concentration of NO.sub.2 in said mixture is less than 5 ppm.
25. The method of claim 17, wherein during said first time period,
a concentration of O.sub.2 in said mixture ranges from 20% to
25%.
26. The method of claim 17, wherein during said first time period,
a fraction of inspired oxygen level (FiO.sub.2) in said mixture
ranges from 21% to 100%.
27. The method of claim 1, wherein said parameter is ETCO.sub.2 and
during and following said subjecting, said ETCO.sub.2 is less than
60 mmHg.
28. The method of claim 1, wherein said parameter is SpMet and
during and following said subjecting, said SpMet is increased by
less than 5%.
29. The method of claim 1, wherein said parameter is SpO.sub.2 and
during said subjecting, a level of said SpO.sub.2 is higher than
89%.
30. The method of claim 1, wherein said intermittent inhalation is
effected during a time period that ranges from 1 to 7 days.
31. The method of claim 30, wherein said intermittent inhalation is
effected during a time period of 5 days.
32. The method of claim 1, wherein said human subject is suffering
from a disease or disorder that is manifested in the respiratory
tract or from a disease or disorder that can be treated via the
respiratory tract.
33. The method of claim 1, wherein said human subject is suffering
from a disease or disorder of an otolaryngological and/or an upper
respiratory tract and/or a lower respiratory system.
34. The method of claim 32, wherein said disease or disorder is
selected from the group consisting of a heparin-protamine reaction,
a traumatic injury, a traumatic injury to the respiratory tract,
acidosis or sepsis, acute mountain sickness, acute pulmonary edema,
acute pulmonary hypertension, acute pulmonary thromboembolism,
adult respiratory distress syndrome, an acute pulmonary
vasoconstriction, aspiration or inhalation injury or poisoning,
asthma or status asthmaticus, bronchopulmonary dysplasia, hypoxia
or chronic hypoxia, chronic pulmonary hypertension, chronic
pulmonary thromboembolism, cystic fibrosis (CF), fat embolism of
the lung, haline membrane disease, idiopathic or primary pulmonary
hypertension, inflammation of the lung, perinatal aspiration
syndrome, persistent pulmonary hypertension of a newborn, and post
cardiac surgery.
35. The method of claim 32, wherein said disease or disorder is
selected from the group consisting of a bacterial-, viral- and/or
fungal bronchiolitis, a bacterial-, viral- and/or fungal
pharyngitis and/or laryngotracheitis, a bacterial-, viral- and/or
fungal pneumonia, a bacterial-, viral- and/or fungal sinusitis, a
bacterial-, viral- and/or fungal upper and/or lower respiratory
tract infection, a bacterial-, viral- and/or fungal-exacerbated
asthma, a respiratory syncytial viral infection, bronchiectasis,
bronchitis, chronic obstructive lung disease (COPD), cystic
fibrosis (CF), emphysema, otitis, otitis media, primary ciliary
dyskinesia (PCD), and pulmonary aspergillosis (ABPA) and
cryptococcosis.
36. The method of claim 32, wherein said disease or disorder is
associated with a pathogenic microorganism.
37. The method of claim 36, wherein said pathogenic microorganism
is selected from the group consisting of a Gram-negative bacterium,
a Gram-positive bacterium, a virus, a fungus and a parasite.
38. The method of claim 36, wherein said disease or disorder is
selected from the group consisting of a bacterial-, viral- and/or
fungal bronchiolitis, a bacterial-, viral- and/or fungal
pharyngitis and/or laryngotracheitis, a bacterial-, viral- and/or
fungal sinusitis, a bacterial-, viral- and/or fungal upper and/or
lower respiratory tract infection, a bacterial-, viral- and/or
fungal-exacerbated asthma, a bacterial-, viral-, fungal- and/or
parasitic pneumonia, a common cold, a cystic fibrosis related
infection, a respiratory syncytial viral infection, acidosis or
sepsis, an oral fugal infection, aspergilloma, bronchitis,
candidiasis of the oral cavity (thrush), canker sores, epiglottitis
(supraglottitis), halitosis, herpes, laryngitis, laryngotracheitis,
nasopharyngitis, otitis and otitis media, pharyngitis, pulmonary
aspergillosis (ABPA), cryptococcosis, respiratory syncytial virus
infection, a bacterial-, viral- and/or fungal conjunctivitis and
uveitis, rhinitis, rhinopharyingitis, rhinosinusitis, stomatitis,
tonsillitis, tracheitis, tuberculosis, tympanitis, and preemptive,
preventative and prophylactic treatment thereof.
39. The method of claim 1, wherein said human subject is suffering
from a disease or disorder selected from the group consisting of a
bacterial-, viral- and/or fungal bronchiolitis, a bacterial-,
viral- and/or fungal pharyngitis and/or laryngotracheitis, a
bacterial-, viral- and/or fungal pneumonia, a bacterial-, viral-
and/or fungal sinusitis, a bacterial-, viral- and/or fungal upper
and/or lower respiratory tract infection, a bacterial-, viral-
and/or fungal-exacerbated asthma, a respiratory syncytial viral
infection, bronchiectasis, bronchitis, chronic obstructive lung
disease (COPD), cystic fibrosis (CF), aspergilloma, emphysema,
otitis, otitis externa, otitis media, primary ciliary dyskinesia
(PCD), pulmonary aspergillosis (ABPA) and cryptococcosis.
40. The method of claim 1, wherein said human subject is suffering
from bronchiolitis.
41. The method of claim 40, wherein said bronchiolitis is
associated with a virus.
42. The method of claim 41, wherein said virus is selected from the
group consisting of a respiratory syncytial virus (RSV), a
rhinovirus, a coronavirus, an enterovirus, an influenza A and/or B
virus, a parainfluenza 1, 2 and/or 3 virus, a bocavirus, a human
metapneumovirus, SARS and an adenovirus.
43. The method of claim 1, wherein said human subject is suffering
from asthma.
44. The method of claim 1, wherein said human subject is suffering
from cystic fibrosis.
45. The method of claim 1, wherein said human subject is infected
by an influenza virus.
46. The method of claim 1, wherein said human subject is suffering
from COPD.
47. The method of claim 1, wherein said human subject is suffering
from a disease or disorder selected from the group consisting of an
acute respiratory disease or disorder, a chronic respiratory
disease or disorder, an obstructive respiratory disease or
disorder, an intrinsic or extrinsic restrictive respiratory disease
or disorder, a pulmonary vascular disease or disorder, an
infectious respiratory disease or disorder, an inflammatory
respiratory disease or disorder, a pleural cavity disease or
disorder, and a neonatal respiratory disease or disorder.
48. The method of claim 1, wherein said human subject is an
immuno-compromised human subject.
49. The method of claim 48, wherein said immune-compromised human
subject is selected from the group consisting of a subject
suffering from HIV, a subject suffering from cancer, a subject
undergoing or which underwent chemotherapy, and a subject
undergoing or which underwent transplantation.
50. The method of claim 1, wherein said human subject is prone to
suffer from a disease or disorder that is manifested in the
respiratory tract or from a disease or disorder that can be treated
via the respiratory tract.
51. The method of claim 50, wherein said human subject is selected
from the group consisting of an immune-compromised subject, a
subject suffering from chronic asthma, a subject suffering from
chronic sinusitis, a subject exposed to an infectious respiratory
tract disease or disorder and a subject exposed to a pathogen.
52. The method of claim 51, wherein said immune-compromised human
subject is selected from the group consisting of a subject
suffering from HIV, a subject suffering from cancer, a subject
undergoing or which underwent chemotherapy, and a subject
undergoing or which underwent transplantation.
53. A method of treating a human subject suffering from a disease
or disorder that is manifested in the respiratory tract or a
disease or disorder that can be treated via the respiratory tract,
the method comprising subjecting the subject to intermittent
inhalation regimen, gNO at a concentration of at least 160 ppm,
thereby treating the disease or disorder, wherein said subjecting
is effected while monitoring at least one on-site parameter
selected from the group consisting of: a methemoglobin level
(SpMet); an oxygen saturation level (SpO.sub.2); and an end tidal
CO.sub.2 level (ETCO.sub.2); in the subject, wherein a change in at
least one of said on-site parameters following said subjecting is
less than 2 acceptable deviation units from a baseline.
54. A method of treating a human subject prone to suffer from a
disease or disorder that is manifested in the respiratory tract or
a disease or disorder that can be treated via the respiratory
tract, the method comprising subjecting the subject to intermittent
inhalation regimen, gNO at a concentration of at least 160 ppm,
thereby treating the disease or disorder, wherein said subjecting
is effected while monitoring at least one on-site parameter
selected from the group consisting of: a methemoglobin level
(SpMet); an oxygen saturation level (SpO.sub.2); and an end tidal
CO.sub.2 level (ETCO.sub.2); in the subject, wherein a change in at
least one of said on-site parameters following said subjecting is
less than 2 acceptable deviation units from a baseline.
Description
FIELD AND BACKGROUND OF THE INVENTION
[0001] The present invention, in some embodiments thereof, relates
to therapy, and more particularly, but not exclusively, to methods
and devices for treating respiratory diseases by inhalation of
gaseous nitric oxide.
[0002] Nitric oxide (NO) is a small lipophilic signaling molecule
with a small stokes radius and a molecular weight of 30 grams/mol
that enables it to cross the glycolipid cell plasma membrane into
the cytosol readily and rapidly. NO has an unpaired electron
available in its outer orbit that characterizes it as a free
radical. NO has been shown to play a critical role in various
bodily functions, including the vasodilatation of smooth muscle,
neurotransmission, regulation of wound healing and immune responses
to infections such as caused by bactericidal action directed toward
various organisms. NO has been demonstrated to play an important
role in wound healing through vasodilatation, angiogenesis,
anti-inflammatory and antimicrobial action.
[0003] NO is a common air pollutant and is present in
concentrations of 150-650 ppm in cigarette smoke and up to 1200 ppm
in cigar and pipe smoke. The National Institute for Occupational
Safety and Health (OSHA) and the Environmental Protection Agency
have given an inhalation threshold limit value (TLV) as a
time-weighted average (TWA) of 25 ppm for NO. The TLV-TWA is the
concentration to which a person's respiratory system may be exposed
continuously throughout a normal work week without adverse effects
and, when represented in ppm hours units, is calculated to be 200
ppm hours. This level is a time-weighted average, that is, the
average level of NO should be less than 25 ppm; however, brief
exposures to higher concentrations are allowed.
[0004] NO is produced by the innate immune response in organs and
cells exposed to bacterial and viral infections. These include,
among others, the nasopharyngeal airway, lungs and circulating
neutrophils and macrophages. NO is also a highly reactive
microbicidal free radical that possesses antimicrobial activity
against broad range of bacteria, parasites, fungi and viruses. The
pore diameter in the cell walls of the microorganisms through which
the NO molecule must pass to affect these pathogens is
approximately five times wider so that there are few barriers to NO
cell penetration.
[0005] NO is therefore an essential part of the innate immune
response. In addition, NO is one of the smallest, yet one of the
most important, biological signaling molecules in mammals.
[0006] Other than being a well-established direct antimicrobial
agent, it has been hypothesized that the antimicrobial and cellular
messenger regulatory properties of NO, delivered in an exogenous
gaseous form, might easily enter the pulmonary milieu and be useful
in optimizing the treatment of uncontrolled pulmonary disease with
specific actions directed at reducing bacterial burden, reducing
inflammation and improving clinic al symptoms.
[0007] Some respiratory disorders and physiological conditions can
be treated by inhalation of gaseous nitric oxide (gNO). The use of
gNO by inhalation can prevent, reverse, or limit the progression of
disorders such as acute pulmonary vasoconstriction, traumatic
injury, aspiration or inhalation injury, fat embolism in the lung,
acidosis, inflammation of the lung, adult respiratory distress
syndrome, acute pulmonary edema, acute mountain sickness, post
cardiac surgery, acute pulmonary hypertension, persistent pulmonary
hypertension of a newborn, perinatal aspiration syndrome, haline
membrane disease, acute pulmonary thromboembolism,
heparin-protamine reactions, sepsis, asthma and status asthmaticus
or hypoxia. Inhaled gNO can also be used to treat cystic fibrosis
(CF), chronic pulmonary hypertension, bronchopulmonary dysplasia,
chronic pulmonary thromboembolism and idiopathic or primary
pulmonary hypertension or chronic hypoxia.
[0008] From the toxicological aspect, NO has a half-life in the
body of less than 6 seconds and a radius of action of approximately
200 microns from its site of origin, beyond which it is inactivated
through binding to sulfhydryl groups of cellular thiols or by
nitrosylation of the heme moieties of hemoglobin to form
methemoglobin (MetHb).
[0009] MetHb reductase reduces NO to nitrates in the blood serum.
Nitrate has been identified as the predominant nitric oxide
metabolite excreted in the urine, accounting for more than 70% of
the nitric oxide dose inhaled. Nitrate is cleared from the plasma
by the kidney at rates approaching the rate of glomerular
filtration. Blood levels of MetHb in healthy humans are typically
less than 2%.
[0010] Potential side effects of high dose NO treatment hence
include the binding of NO to hemoglobin and the formation of MetHb,
which could lead to decreased oxygen transport, and the capacity of
NO to act as a nitrosylating agent on proteins and other cell
constituents. Formation of MetHb and increased levels thereof have
been observed in previous studies of gNO inhalation by healthy
human individuals, wherein inhalation of gNO at 128 ppm for 3 hours
and at 512 ppm for 55 minutes has been reported to drive the levels
of MetHb over the safe threshold of 5% [Borgese N. et al., J. Clin.
Invest., 1987, 80, 1296-1302; Young J. D. et al., Intensive Care
Med., 1994, 20, 581-4 and Young J. D. et al., Brit. J. Anaesthesia,
1996, 76, 652-656].
[0011] Thus, concerns have been raised regarding the potential use
of NO as a therapeutic agent in various clinical scenarios. To
date, studies indicate that acute to pulmonary injury, pulmonary
edema, hemorrhage, changes in surface tension of surfactant,
reduced alveolar numbers and airway responsiveness may be caused by
high airway levels of NO, NO.sub.2 and other oxides of nitrogen
[Hurford W., Resp. Care, 2005, 50, 1428-9].
[0012] Several animal studies conducted in order to evaluate the
safety window for gNO exposure were reported on the Primary Medical
Review of NDA 20-845 (INOmax nitric oxide gas). Included in these
reports is the study referred to as RDR-0087-DS, wherein groups of
10 rats each were exposed to room air or to 80, 200, 300, 400 or
500 ppm gNO for 6 continuous hours per day for up to 7 days. It is
reported that all of the animals died on the first day of exposure
to 400 and 500 ppm gNO with MetHb levels of 72.5 and 67 percents
respectively. Six of the animals treated with 300 ppm gNO died
during the first 1-2 days. All deaths were attributed to
methemoglobinemia.
[0013] In additional studies, rats were exposed continuously to
room air, 40, 80, 160, 200 and 250 ppm gNO for 6 hours/day for 28
days. No deaths occurred at gNO concentrations below 200 ppm.
[0014] At present, inhalation of gaseous nitric oxide (gNO) as a
selective, short acting vasodilator is approved only at 80 ppm for
use in full term infants with hypoxic respiratory failure
associated with pulmonary hypertension. However, other studies have
shown that at such low concentration of inhaled gNO, treatment of
adults' respiratory diseases is limited, and the use of higher
doses of gNO for treating various medical conditions by inhalation
requires in-depth safety studies in humans.
[0015] Miller et al. reported the effect of 1,600 ppm hours gNO
against five planktonic (suspended in a liquid) species of
methicillin resistant S. aureous (MRSA). An in vitro biofilm MRSA
model was also used to compare gNO to the antibiotic vancomycin as
an antibacterial agent. For the biofilm experiment, a drip flow
reactor was used to grow a MRSA biofilm which was then exposed for
eight hours to Ringers lactate, 200 ppm gNO (1,600 ppm hours), air
or vancomycin (100-times MIC level). A reduction in the population
of all five MRSA planktonic strains was observed after exposure to
1,600 ppm hours of gNO. In the biofilm experiment gNO was also
shown to reduce MRSA.
[0016] Additional animal studies have shown that gNO at 160-200 ppm
can exert potent antimicrobial effects against a broad range of
microbes in vitro, ex vivo and in animal models [Kelly T. J. et
al., J. Clin. Invest., 1998, 102, 1200-7; McMullin B. et al., Resp.
Care., 2005, 50, 1451-6; Ghaffari A. et al., Nitric Oxide, 2005,
12, 129-40; Ghaffari A. et al., Wound Repair Regen., 2007, 15,
368-77; Miller C. C. et al., J. Cutan. Med. Surg. 2004, 8, 233-8;
Miller C. C. et al., Nitric Oxide, 2009, 20, 16-23], further
suggesting its use as an antimicrobial agent in appropriate
concentrations.
[0017] Studies conducted in a rat model of Pseudomonas aeruginosa
pneumonia tested the antimicrobial effect of a gNO inhaled delivery
regimen of intermittent 30 minute exposures of 160-200 ppm gNO, and
revealed that 160 ppm gNO in that regiment is effective to reduce
the pulmonary bioburden and leukocyte infiltration [Hergott C. A.
et al., Am. J. Resp. Crit. Care Med., 2006, 173, A135]. This
treatment was also shown to decrease the clinical symptoms of
bovine respiratory disease in cattle [Schaefer A. L. et al., Online
J. Vet. Res., 2006, 10, 7-16].
[0018] Miller, C. C. et al. [J. Cutan. Med. Surg., 2004, 8(4),
233-8] reported on topical treatment of a subject who had a
chronic, non-healing wound and presence of a reoccurring biofilm
with gNO at a treatment concentration of 200 ppm for two weeks.
[0019] Within the first three days of treatment, the subject's
biofilm was no longer visibly present and at one week, the wound
size was reduced by 42%. The subject's ulcer continued to heal
following the cessation of nitric oxide exposure.
[0020] WO 2005/110441 teaches a method and a corresponding device
for combating microbes and infections by delivering intermittent
high doses of 160-400 ppm gNO to a mammal for a period of time
which cycles between high and low concentration of nitric oxide
gas. The regimen involves delivery of 160 ppm gNO for 30 minutes
every four hours with 0-20 ppm delivered for the 3.5 hours between
the higher concentration deliveries. No experimental data are
presented in this publication.
[0021] U.S. Pat. No. 7,122,018 teaches topical intermittent
exposure to high concentration of nitric oxide ranging 160-400 ppm,
for treatment of infected wounds and respiratory infections by a
regimen of 4-hour sessions interrupted by 1 hour of rest while
monitored methemoglobin blood levels.
[0022] U.S. Pat. No. 7,516,742 teaches intermittent high-low dosing
by inhalation of gNO to overcome gNO-related toxicity, wherein the
high concentration of gNO ranges from 80 to 300 ppm and the low
concentration ranges from 0 to 80 ppm, while the regimen may be 160
ppm for 30 minutes every four hours with 20 ppm delivered for the
3.5 hours between the higher concentration deliveries while
monitoring the concentration of O.sub.2, NO and NO.sub.2.
[0023] U.S. Pat. No. 7,520,866 teaches topical exposure of wounds
to gNO at a high concentration ranging 160-400 ppm with a regime of
two 4-hour sessions, interrupted by 1 hour of rest, wherein after a
first treatment period with high concentration of gNO, a second
treatment period at a lower concentration of 5-20 ppm may be
provided to restore the balance of nitric oxide and induce collagen
expression to aid in the closure of the wound.
[0024] U.S. Pat. No. 7,955,294 teaches a method and a corresponding
device for topical and inhaled intermittent delivery high-low doses
of gNO for a period of time which cycles between high and low
concentration, with an exemplary cycle regimen of 160-200 ppm for
30 minutes followed by 0-80 ppm 3.5 hours wherein the cycling
regimen can span 1-3 days.
[0025] Additional background art includes U.S. Pat. Nos. 8,083,997,
8,079,998, 8,066,904, 8,057,742, 7,531,133, 6,432,077, U.S. Patent
Application Nos. 2011/0262335, 2011/0259325, 2011/0240019,
2011/0220103 and 2010/0331405, 2011/0112468, 2008/028786,
2007/0116785, 2007/0088316, 2007/0065473, 2007/0014688,
2006/0207594, 2005/0191372 and WO 2006/071957, WO 2006/110923, WO
2006/110923, WO 2007/057763, WO 2007/057763, WO 2000/30659 and EP
0692984; Miller C. C. et al., Antimicrobial Agents And
Chemotherapy, 2007, 51(9), 3364-3366; and Miller C. C. et al.,
[Resp Care, 2008, 53(11), 1530].
SUMMARY OF THE INVENTION
[0026] The present inventors have studied the effect of
intermittent inhalation of gaseous nitric oxide at a concentration
of 160 ppm or more by human subjects and have shown that such
intermittent inhalation protocol do not result in substantial
changes in various physiological parameters of the human subject.
Exemplary such parameters are those obtainable on-site in
real-time, such as methemoglobin level, end-tidal CO.sub.2 level,
and oxygenation, and parameters which are obtainable off-site in
the laboratory, such as blood nitrite level, urine nitrite level,
and inflammatory markers' level. The present inventors have
therefore demonstrated that such a method can be effected
safely.
[0027] Embodiments of the present invention therefore relate to
methods of administering gaseous nitric oxide to human subjects in
need thereof, while these parameters remain substantially
unchanged. The disclosed administration can be used in methods of
treating and/or preventing various medical conditions, which are
manifested in the respiratory tract, or which can be treated via
the respiratory tract, by subjecting a human subject to
intermittent inhalation of gaseous nitric oxide at a concentration
of 160 ppm or more.
[0028] According to an aspect of some embodiments of the present
invention there is provided a method of treating a human subject in
need of inhalation of gaseous NO (gNO), the method comprising
subjecting the subject to intermittent inhalation of gNO at a
concentration of at least 160 ppm, while monitoring at least one
on-site parameter selected from the group consisting of:
[0029] a methemoglobin level (SpMet);
[0030] an oxygen saturation level (SpO.sub.2); and
[0031] an end tidal CO.sub.2 level (ETCO.sub.2);
[0032] in the subject, wherein a change in at least one of the
on-site parameters following the subjecting is less than 2
acceptable deviation units from a baseline.
[0033] According to some embodiments of the present invention, the
monitoring is of at least two of the on-site parameters.
[0034] According to some embodiments of the present invention, the
monitoring is of all of the on-site parameters.
[0035] According to some embodiments of the present invention, a
change in the at least two on-site parameters following the
subjecting is less than 2 acceptable deviation units from a
baseline.
[0036] According to some embodiments of the present invention, a
change in any of the on-site parameters following the subjecting is
less than 2 acceptable deviation units from a baseline.
[0037] According to some of any of the embodiments described
herein, the method further comprises, or is effected while,
monitoring at least one off-site parameter selected from the group
consisting of:
[0038] a serum nitrite/nitrate level
(NO.sub.2.sup.-/NO.sub.3.sup.-); and
[0039] an inflammatory cytokine plasma level, as described
herein.
[0040] According to some embodiments of the present invention, the
cytokine is selected from the group consisting of (TNF).alpha.,
(IL)-1.beta., IL-6, IL-8, IL-10 and IL-12p70.
[0041] According to some embodiments of the present invention, a
change in one or both of the off-site parameters following the
subjecting is less than 2 acceptable deviation units from a
baseline.
[0042] According to some of any of the embodiments described
herein, the method further comprises, or is effected while,
monitoring urine nitrite level in the subject.
[0043] According to some embodiments of the present invention, a
change in the urine nitrite level following the subjecting is less
than 2 acceptable deviation units from a baseline.
[0044] According to some of any of the embodiments described
herein, the method further comprises, or is effected while,
monitoring in the subject at least one off-site parameter selected
from the group consisting of:
[0045] a hematological marker
[0046] a vascular endothelial activation factor;
[0047] a coagulation parameter;
[0048] a serum creatinine level; and
[0049] a liver function marker, as described herein for any of
these parameters.
[0050] According to some embodiments of the present invention, the
hematological marker is selected from the group consisting of a
hemoglobin level, a hematocrit ratio, a red blood cell count, a
white blood cell count, a white blood cell differential and a
platelet count in the subject.
[0051] According to some embodiments of the present invention, a
change in at least one of the off-site parameters following the
subjecting is less than 2 acceptable deviation units from a
baseline.
[0052] According to some embodiments of the present invention, a
change in any of the off-site parameters following the subjecting
is less than 2 acceptable deviation units from a baseline.
[0053] According to some of any of the embodiments described
herein, the method further comprises, or is effected while,
monitoring in the subject at least one on-site parameter selected
from the group consisting of:
[0054] a vital sign; and
[0055] a pulmonary function, as described herein for any of these
parameters.
[0056] According to some embodiments of the present invention, no
deterioration in at least one of the parameters is observed during
and following the subjecting.
[0057] According to some embodiments of the present invention, the
intermittent inhalation comprises at least one cycle of continuous
inhalation of the gNO for a first time period, followed by
inhalation of no gNO for a second time period.
[0058] According to some embodiments of the present invention, the
first time period is about 30 minutes.
[0059] According to some embodiments of the present invention, the
second time period ranges from 3 to 5 hours.
[0060] According to some embodiments of the present invention, the
second time period ranges from 3 to 5 hours.
[0061] According to some embodiments of the present invention, the
inhalation comprises from 1 to 6 of the cycles per day.
[0062] According to some embodiments of the present invention, the
inhalation comprises 5 of the cycles per day.
[0063] According to some embodiments of the present invention,
during the first time period, the concentration of gNO in the
mixture deviates from the concentration of at least 160 ppm by less
than 10%.
[0064] According to some embodiments of the present invention,
during the first time period, a concentration of NO.sub.2 in the
mixture is less than 5 ppm.
[0065] According to some embodiments of the present invention,
during the first time period, a concentration of O.sub.2 in the
mixture ranges from 20% to 25%.
[0066] According to some embodiments of the present invention,
during the first time period, a fraction of inspired oxygen level
(FiO.sub.2) in the mixture ranges from 21% to 100%.
[0067] According to some of any of the embodiments of the present
invention, the parameter is ETCO.sub.2 and during and following the
subjecting, the ETCO.sub.2 is less than 60 mmHg.
[0068] According to some of any of the embodiments of the present
invention, the parameter is SpMet and during and following the
subjecting, the SpMet is increased by less than 5%.
[0069] According to some of any of the embodiments of the present
invention, the parameter is SpO.sub.2 and during the subjecting, a
level of the SpO.sub.2 is higher than 89%.
[0070] According to some of any of the embodiments of the present
invention, the intermittent inhalation is effected during a time
period that ranges from 1 to 7 days.
[0071] According to some of any of the embodiments of the present
invention, the intermittent inhalation is effected during a time
period of 5 days.
[0072] According to some of any of the embodiments of the present
invention, the human subject is suffering from a disease or
disorder that is manifested in the respiratory tract or from a
disease or disorder that can be treated via the respiratory
tract.
[0073] According to some of any of the embodiments of the present
invention, the human subject is suffering from a disease or
disorder of an otolaryngological and/or an upper respiratory tract
and/or a lower respiratory system.
[0074] According to some embodiments of the present invention, the
disease or disorder is selected from the group consisting of a
heparin-protamine reaction, a traumatic injury, a traumatic injury
to the respiratory tract, acidosis or sepsis, acute mountain
sickness, acute pulmonary edema, acute pulmonary hypertension,
acute pulmonary thromboembolism, adult respiratory distress
syndrome, an acute pulmonary vasoconstriction, aspiration or
inhalation injury or poisoning, asthma or status asthmaticus,
bronchopulmonary dysplasia, hypoxia or chronic hypoxia, chronic
pulmonary hypertension, chronic pulmonary thromboembolism, cystic
fibrosis (CF), fat embolism of the lung, haline membrane disease,
idiopathic or primary pulmonary hypertension, inflammation of the
lung, perinatal aspiration syndrome, persistent pulmonary
hypertension of a newborn, and post cardiac surgery.
[0075] According to some embodiments of the present invention, the
disease or disorder is selected from the group consisting of a
bacterial-, viral- and/or fungal bronchiolitis, a bacterial-,
viral- and/or fungal pharyngitis and/or laryngotracheitis, a
bacterial-, viral- and/or fungal pneumonia, a bacterial-, viral-
and/or fungal sinusitis, a bacterial-, viral- and/or fungal upper
and/or lower respiratory tract infection, a bacterial-, viral-
and/or fungal-exacerbated asthma, a respiratory syncytial viral
infection, bronchiectasis, bronchitis, chronic obstructive lung
disease (COPD), cystic fibrosis (CF), emphysema, otitis, otitis
media, primary ciliary dyskinesia (PCD), and pulmonary
aspergillosis (ABPA) and cryptococcosis.
[0076] According to some embodiments of the present invention, the
disease or disorder is associated with a pathogenic
microorganism.
[0077] According to some embodiments of the present invention, the
pathogenic microorganism is selected from the group consisting of a
Gram-negative bacterium, a Gram-positive bacterium, a virus, a
fungus and a parasite.
[0078] According to some embodiments of the present invention, the
disease or disorder is selected from the group consisting of a
bacterial-, viral- and/or fungal bronchiolitis, a bacterial-,
viral- and/or fungal pharyngitis and/or laryngotracheitis, a
bacterial-, viral- and/or fungal sinusitis, a bacterial-, viral-
and/or fungal upper and/or lower respiratory tract infection, a
bacterial-, viral- and/or fungal-exacerbated asthma, a bacterial-,
viral-, fungal- and/or parasitic pneumonia, a common cold, a cystic
fibrosis related infection, a respiratory syncytial viral
infection, acidosis or sepsis, an oral fugal infection,
aspergilloma, bronchitis, candidiasis of the oral cavity (thrush),
canker sores, epiglottitis (supraglottitis), halitosis, herpes,
laryngitis, laryngotracheitis, nasopharyngitis, otitis and otitis
media, pharyngitis, pulmonary aspergillosis (ABPA), cryptococcosis,
respiratory syncytial virus infection, a bacterial-, viral- and/or
fungal conjunctivitis and uveitis, rhinitis, rhinopharyingitis,
rhinosinusitis, stomatitis, tonsillitis, tracheitis, tuberculosis,
tympanitis, and preemptive, preventative and prophylactic treatment
thereof.
[0079] According to some of any of the embodiments of the present
invention, the human subject is suffering from a disease or
disorder selected from the group consisting of a bacterial-, viral-
and/or fungal bronchiolitis, a bacterial-, viral- and/or fungal
pharyngitis and/or laryngotracheitis, a bacterial-, viral- and/or
fungal pneumonia, a bacterial-, viral- and/or fungal sinusitis, a
bacterial-, viral- and/or fungal upper and/or lower respiratory
tract infection, a bacterial-, viral- and/or fungal-exacerbated
asthma, a respiratory syncytial viral infection, bronchiectasis,
bronchitis, chronic obstructive lung to disease (COPD), cystic
fibrosis (CF), aspergilloma, emphysema, otitis, otitis externa,
otitis media, primary ciliary dyskinesia (PCD), pulmonary
aspergillosis (ABPA) and cryptococcosis.
[0080] According to some embodiments of the present invention, the
human subject is suffering from bronchiolitis.
[0081] According to some embodiments of the present invention, the
bronchiolitis is associated with a virus.
[0082] According to some embodiments of the present invention, the
virus is selected from the group consisting of a respiratory
syncytial virus (RSV), a rhinovirus, a coronavirus, an enterovirus,
an influenza A and/or B virus, a parainfluenza 1, 2 and/or 3 virus,
a bocavirus, a human metapneumovirus, SARS and an adenovirus.
[0083] According to some embodiments of the present invention, the
human subject is suffering from asthma.
[0084] According to some embodiments of the present invention, the
human subject is suffering from cystic fibrosis.
[0085] According to some embodiments of the present invention, the
human subject is infected by an influenza virus.
[0086] According to some embodiments of the present invention, the
human subject is suffering from COPD.
[0087] According to some embodiments of the present invention, the
human subject is suffering from a disease or disorder selected from
the group consisting of an acute respiratory disease or disorder, a
chronic respiratory disease or disorder, an obstructive respiratory
disease or disorder, an intrinsic or extrinsic restrictive
respiratory disease or disorder, a pulmonary vascular disease or
disorder, an infectious respiratory disease or disorder, an
inflammatory respiratory disease or disorder, a pleural cavity
disease or disorder, and a neonatal respiratory disease or
disorder.
[0088] According to some embodiments of the present invention, the
human subject is an immuno-compromised human subject.
[0089] According to some embodiments of the present invention, the
immune-compromised human subject is selected from the group
consisting of a subject suffering from HIV, a subject suffering
from cancer, a subject undergoing or which underwent chemotherapy,
and a subject undergoing or which underwent transplantation.
[0090] According to some embodiments of the present invention, the
human subject is prone to suffer from a disease or disorder that is
manifested in the respiratory tract or from a disease or disorder
that can be treated via the respiratory tract.
[0091] According to some embodiments of the present invention, the
human subject is selected from the group consisting of an
immune-compromised subject, a subject suffering from chronic
asthma, a subject suffering from chronic sinusitis, a subject
exposed to an infectious respiratory tract disease or disorder and
a subject exposed to a pathogen.
[0092] According to some embodiments of the present invention, the
immune-compromised human subject is selected from the group
consisting of a subject suffering from HIV, a subject suffering
from cancer, a subject undergoing or which underwent chemotherapy,
and a subject undergoing or which underwent transplantation.
[0093] According to an aspect of some embodiments of the present
invention there is provided a method of treating a human subject
suffering from a disease or disorder that is manifested in the
respiratory tract or a disease or disorder that can be treated via
the respiratory tract, as described herein, the method comprising
subjecting the subject to intermittent inhalation regimen, gNO at a
concentration of at least 160 ppm, thereby treating the disease or
disorder, wherein the subjecting is effected while monitoring at
least one on-site parameter selected from the group consisting
of:
[0094] a methemoglobin level (SpMet);
[0095] an oxygen saturation level (SpO.sub.2); and
[0096] an end tidal CO.sub.2 level (ETCO.sub.2);
[0097] in the subject, as described herein, wherein a change in at
least one of the on-site parameters following the subjecting is
less than 2 acceptable deviation units from a baseline.
[0098] According to an aspect of some embodiments of the present
invention there is provided a method of treating a human subject
prone to suffer from a disease or disorder that is manifested in
the respiratory tract or a disease or disorder that can be treated
via the respiratory tract, as described herein, the method
comprising subjecting the subject to intermittent inhalation
regimen, gNO at a concentration of at least 160 ppm, as described
herein, thereby treating the disease or disorder, wherein the
subjecting is effected while monitoring at least one on-site
parameter selected from the group consisting of:
[0099] a methemoglobin level (SpMet);
[0100] an oxygen saturation level (SpO.sub.2); and
[0101] an end tidal CO.sub.2 level (ETCO.sub.2),
[0102] in the subject, as described herein, wherein a change in at
least one of the on-site parameters following the subjecting is
less than 2 acceptable deviation units from a baseline, as
described herein.
[0103] According to some of any of the embodiments described
herein, the subjecting is effected by an inhalation device selected
from the group consisting of stationary inhalation device, a
portable inhaler, a metered-dose inhaler, an atmospherically
controlled enclosure and an intubated inhaler.
[0104] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0105] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0106] In the drawings:
[0107] FIGS. 1A-B present background art bar graphs showing the gNO
dosage curve as measured for S. aureus (FIG. 1A) and P. aeruginosa
(FIG. 1B) grown on solid media, wherein relative percentage of
growth of colony forming units (CFU) at 50, 80, 120 and 160 parts
per million (ppm) of gaseous nitric oxide (gNO) compared with
growth of CFU in medical air (100%);
[0108] FIGS. 2A-C present background art comparative plots showing
the viral plaque formation in tissue as a function of time as
measured for influenza A/victoria/H3N2 virions after exposure to
nitric oxide 160 ppm and 800 ppm continuously for 4 hours (FIG.
2A), the same virions after being exposed to one gNO dose over 30
minute as compared to three 30 minute treatments Q4 h (FIG. 2B),
and the effect of continuous exposure to gNO at a concentration of
160 ppm for 3 hours of the highly pathogenic Avian Influenza H7N3
(as presented in US 2007/0116785);
[0109] FIGS. 3A-D present images showing tissue culture samples
harboring human rgRSV30 a common viral lung virus and the causative
agent of Broncheolitis, coupled to a green fluorescent protein, and
having a starting viral level of 2000 PFU (FIG. 3A), 1000 PFU (FIG.
3B) and 500 PFU (FIG. 3C), upon exposure to 160 ppm gNO for 30
minutes, and a comparative bar plot presenting the plaque reduction
in the tested samples to control samples exposed to ambient
air;
[0110] FIGS. 4A-B present of the data obtained while monitoring
methemoglobin (MetHb) levels before, during and after inhalation of
160 ppm of gaseous nitric oxide by 10 healthy human individuals,
undergone 5 courses of gNO administration by inhalation daily, each
lasting 30 minutes, for 5 consecutive days, while methemoglobin
levels were measured using a pulse oximeter, wherein FIG. 4A is a
plot of methemoglobin levels by percents as a function of time as
measured before (time point j), during 250 individual 30 minutes
gNO administration courses (time interval of 0 to 30 minutes),
after the courses (time interval of 30 to 60 minutes) and at 120
minutes, 180 minutes and 240 minutes after gNO administration was
discontinued, and FIG. 4B is a plot of methemoglobin levels by
percents as a function of time as measured at the beginning and end
of 30 minutes gNO administration courses given over the course of 5
days, and followed 8, 12 and 26 days after gNO administration was
discontinued;
[0111] FIGS. 5A-F present the data obtained while monitoring
pulmonary function before, during and after inhalation of 160 ppm
of gaseous nitric oxide by 10 healthy human individuals, wherein
baseline values of pulmonary function tests were obtained within 7
days prior to gNO administration, and values during gNO
administration were obtained on day 2 of the 5-days treatment and
other data were obtained after the final gNO administration on day
5 and on days 8, 12 and 26, wherein FIG. 5A presents forced
expiratory volume in 1 second (FEV1) in percents (FEV.sub.1), FIG.
5B presents maximum mid-expiratory flow (MMEF), FIG. 5C presents
carbon monoxide diffusing capacity (DLCO), FIG. 5D presents forced
vital capacity (FVC), FIG. 5E presents total lung capacity (TLC)
and FIG. 5F presents residual volume (RV), while all data are
presented as means of all ten subjects and absolute differences
compared to baseline prior to gNO administration, and statistical
differences were assessed by Mann-Whitney test;
[0112] FIGS. 6A-F present blood levels of various cytokines before
and after inhalation of 160 ppm gaseous nitric oxide by 10 healthy
human individuals, as measured from blood samples collected within
7 days prior to gNO administration, each day during the treatment
and 8, 12 and 26 days thereafter, wherein FIG. 6A presents the
plasma levels of tumor necrosis factor (TNF).alpha., interleukin
(IL)-1.beta. data is presented in FIG. 6B, IL-6 in FIG. 6C, IL-8 in
FIG. 6D, IL-10 in FIG. 6E and IL-12p70 in FIG. 6F, as determined by
a cytometric bead array while statistical differences are compared
by repeated measures ANOVA with Bonferroni post test for parametric
data (IL-6, IL-8, IL-10, IL-12p70), or Friedman test with Dunn's
post test for non-parametric data (TNF and IL-1b); and
[0113] FIGS. 7A-C present plasma levels of angiopoietin (Ang)-1 and
Ang-2 before and after inhalation of 160 ppm gaseous nitric oxide
by 10 healthy human individuals, as measured in blood sample
collected within 7 days prior to gNO inhalation, each day during
gNO administration and 8, 12 and 26 days thereafter, wherein plasma
levels of Ang 1 are shown in FIG. 7A, Ang-2 in FIG. 7B, and
Ang-2/Ang-1 ratios in FIG. 7C, as determined by using a cytometric
bead array while statistical differences were assessed compared by
Friedman test with Dunn's post test.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0114] The present invention, in some embodiments thereof, relates
to medical treatment of respiratory diseases in human subjects, and
more particularly, but not exclusively, to medical procedures based
on inhalation of gaseous nitric oxide and devices for effecting the
same.
[0115] The principles and operation of the present invention may be
better understood with reference to the figures and accompanying
descriptions.
[0116] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0117] As discussed hereinabove, inhalation of gaseous nitric oxide
(gNO) has been shown to be a highly effective broad-spectrum
antimicrobial therapy; however, at effective antimicrobial
concentration gNO may present serious adverse effects on humans. As
shown in previous studies, the currently approved dose of 80 ppm
gNO is presumably too low to exert sufficient antimicrobial
effects.
[0118] As further discussed hereinabove, intermittent dosing and
delivery by inhalation of gNO, cycling between high concentrations
of gNO for a relatively short period of time and longer periods of
no or low concentration of gNO has been suggested for overcoming
the problems of NO toxicity. It has been suggested that the high
concentration of gNO, delivered according to an intermittent
regimen, would be effective in overwhelming the nitric oxide
defense mechanisms of pathogens.
[0119] It has been further suggested in the art that the high
concentration of gNO may be delivered at a concentration of between
80 ppm to 300 ppm, and that the time periods for delivering the
high concentration should afford a daily delivery of 600 to 1000
ppm hours.
[0120] However, to date, a regimen of intermittent inhalation of
gNO, cycling between high concentrations of gNO for a relatively
short period of time and longer periods of no or low concentration
of gNO has not been applied on humans. Studies demonstrating safety
and efficacy of such protocols have never been conducted in human
subjects and no protocols were provided for monitoring safety
parameters and/or for treating human patients in need of gNO
inhalation above the approved dose of 80 ppm.
[0121] In the course of devising and practicing novel methods of
treating various bacterial, viral and protozoal infections, the
present inventors have conducted studies in human subjects, and
compiled suitable protocols for safe and effective treatment of a
human subject by intermittent inhalation of high concentrations of
gNO. The present inventors have demonstrated that short durations
of high concentrations of gNO do not cause lung injury or other
signs of adverse effects in humans and even improve some vital
effects such as lung function and heart rate.
[0122] Specifically, the present inventors have conducted a
prospective phase I open label safety study in healthy adults, who
inhaled 160 ppm gNO for 30 minutes, five times a day, for five
consecutive days. Neither significant adverse events nor adverse
events attributable to gNO inhalation occurred and all individuals
tolerated the gNO treatment courses well. Forced expiratory volume
in 1 sec (FEV.sub.1) percentage and other lung function parameters
were improved and serum nitrites/nitrates, prothrombin,
pro-inflammatory cytokine and chemokine levels, did not differ
between baseline and day 5, while methemoglobin levels increased
during the study period to a tolerated and accepted level of 0.9%.
It was thus demonstrated that inhalation of 160 ppm gNO or more for
30 minutes, about 5 times daily, for 2-7 consecutive days, is safe
and well tolerated in healthy individuals.
[0123] The present invention, in some embodiments thereof,
therefore provides methods of treating human subjects by
intermittent inhalation of high concentration of gNO. In some
embodiments, the methods disclosed herein are effected while
monitoring various parameters relevant for maintaining the desired
dosage and regimen, relevant to the safety of the procedure and
relevant for efficacy of the treatment.
[0124] According to an aspect of some embodiments of the present
invention, there is provided a method of treating a human subject
in need of inhalation of gaseous NO (gNO), which is effected by
subjecting the human subject to intermittent inhalation of gNO at a
concentration of at least 160 ppm.
[0125] In some embodiments, the method is effected while monitoring
various physiological parameters in the subject, as described
herein.
[0126] According to some embodiments of the invention, subjecting
the human subject to gNO intermittent inhalation is effected by
intermittently subjecting the human subject to a gaseous mixture
which contains gNO at the indicated concentration (a gNO-containing
gaseous mixture).
[0127] The human subject can be subjected to the inhalation by
active or passive means.
[0128] By "active means" it is meant that the gaseous mixture is
administered or delivered to the respiratory tract of the human
subject. This can effected, for example, by means of an inhalation
device having a delivery interface adapted for human respiratory
organs. For example, the delivery interface can be placed
intermittently on the human subject's respiratory organs, whereby
when it is removed, the subject breaths ambient air or any other
gaseous mixture that is devoid of gNO, as defined herein.
[0129] By "passive means" it is meant that the human subject
inhales a gaseous mixture containing the indicated dose of gNO
without devices for delivering the gaseous mixture to the
respiratory tract.
[0130] For example, the subject can be subjected to 160 ppm or more
gNO in an intermittent regimen by entering and exiting an
atmospherically controlled enclosure filled with the gNO-containing
mixture of gases discussed herein, or by filling and evacuating an
atmospherically controlled enclosure which is in contact with a
subject's respiratory tract.
[0131] The term "intermittent" is used herein and in the art as an
antonym of "continuous", and means starting and ceasing an action
and/or performing an action in intervals.
[0132] By "intermittent inhalation" it is meant that the subject is
subjected to a gaseous mixture that contains the indicated
concentration of gNO intermittently, and thus inhales such a
gNO-containing gaseous mixture two or more times with intervals
between each inhalation. The subject therefore inhales the
gNO-containing gaseous mixture, then stops inhaling a
gNO-containing gaseous mixture and inhales instead a gaseous
mixture that does not contain the indicated concentration of gNO
(e.g., air), then inhales again the gNO-containing gaseous mixture,
and so on and so forth.
[0133] Hereinthroughout, "a gNO-containing gaseous mixture" is
used, for simplicity, to describe a gaseous mixture that contains
at least 160 ppm gNO. The gNO-containing mixture can comprise 160
ppm, 170 ppm, 180 ppm, 190 ppm, 200 ppm and even higher
concentrations of gNO. Other gaseous mixtures mentioned herein
include less than 160 ppm gNO or are being essentially devoid of
gNO, as defined herein.
[0134] By "essentially devoid of gNO" it is meant no more than 50
ppm, no more than 40 ppm, no more than 30 ppm, no more than 20 ppm,
no more than 10 ppm, no more than 5 ppm, no more than 1 ppm and no
more than ppb, including absolutely no gNO.
[0135] In some embodiments, the method is carried out while
maintaining a controlled mixture of inhaled and exhaled gases by
standard means for monitoring and controlling, on-site, the
contents and/or flow of the mixture to which the subject is
subjected to, or that which is delivered through a delivery
interface, and/or while monitoring on-site exhaled gases and
controlling the intake by feedback in real-time. In some
embodiments, the method is effected while monitoring the
concentration of gNO, FiO.sub.2/O.sub.2, ETCO.sub.2, and NO.sub.2
in the gaseous mixture to which the subject is exposed or by
monitoring other bodily systems non-invasively, such as blood
oxygen saturation (SpO.sub.2/SaO.sub.2/DO) and the presence of
methemoglobin in the blood (SpMet).
[0136] In some embodiments, the concentration of gNO in the
gNO-containing gaseous mixture is controlled so as not to deviate
from a predetermined concentration by more than 10%. For example,
the method is carried out while the concentration of gNO, set to
160 ppm, does not exceed margins of 144 ppm to 176 ppm.
[0137] Similarly, the NO.sub.2 content in a gNO-containing gaseous
mixture is controlled such that the concentration of NO.sub.2 is
maintained lower than 5 ppm.
[0138] Further, oxygen level in the gNO-containing gaseous mixture
is controlled such that the concentration of O.sub.2 in the mixture
ranges from about 20% to about 25%.
[0139] Alternatively or in addition, the oxygen level in the
gNO-containing gaseous mixture is controlled such that the fraction
of inspired oxygen (FiO.sub.2) ranges from about 20% to about
100%.
[0140] The phrase "fraction of inspired oxygen" or "FiO.sub.2", as
used herein, refers to the fraction or percentage of oxygen in a
given gas sample. For example, ambient air at sea level includes
20.9% oxygen, which is equivalent to FiO.sub.2 of 0.21.
Oxygen-enriched air has a higher FiO.sub.2 than 0.21, up to 1.00,
which means 100% oxygen. In the context of embodiments of the
present invention, FiO.sub.2 is kept under 1 (less than 100%
oxygen)
[0141] The phrase "end tidal CO.sub.2" or "ETCO.sub.2", as used
herein, refers to the partial pressure or maximal concentration of
carbon dioxide (CO.sub.2) at the end of an exhaled breath, which is
expressed as a percentage of CO.sub.2 or the pressure unit mmHg
Normal values for humans range from 5% to 6% CO.sub.2, which is
equivalent to 35-45 mmHg.
[0142] Since CO.sub.2 diffuses out of the lungs into the exhaled
air, ETCO.sub.2 values reflect cardiac output (CO) and pulmonary
blood flow as the gas is transported by the venous system to the
right side of the heart and then pumped to the lungs by the right
ventricles.
[0143] A device called capnometer measures the partial pressure or
maximal concentration of CO.sub.2 at the end of exhalation. In the
context of embodiments of the present invention, a capnometer is
used and ETCO.sub.2 levels are monitored so as to afford a warning
feedback when ETCO.sub.2 is more than 60 mmHg
[0144] Levels of respiratory NO, NO.sub.2 and O.sub.2 concentration
levels (both inhaled and exhaled; inspiratory and expiratory gases)
are typically monitored continuously by sampling from a mouthpiece
sample port located in an inhalation mask NO, NO.sub.2 and O.sub.2
equipped with an electrochemical analyzer. In the context of
embodiments of the present invention, safety considerations
requires the absolute minimization of the number of occasions in
which NO.sub.2 levels exceed 5 ppm, gNO concentration variations
exceeding 10%, and FiO.sub.2/O.sub.2 levels drop below 20% during
gNO administration.
[0145] According to some embodiments of the present invention, the
intermittent inhalation includes one or more cycles, each cycle
comprising continuous inhalation of a gaseous mixture containing
gNO at the specified high concentration (e.g., at least 160 ppm)
for a first time period, followed by inhalation of a gaseous
mixture containing no gNO for a second time period. According to
some embodiments of the present invention, during the second period
of time the subject may inhale ambient air or a controlled mixture
of gases which is essentially devoid of gNO, as defined herein.
[0146] In some embodiments, the first time period spans from 10 to
45 minutes, or from 20 to 45 minutes, or from 20 to 40 minutes, and
according to some embodiments, spans about 30 minutes.
[0147] According to some embodiments of the present invention, the
second time period ranges from 3 to 5 hours, or from 3 to 4 hours,
and according to some embodiments the second time period spans
about 3.5 hours.
[0148] According to some embodiments of the present invention, this
inhalation regimen is repeated 1-6 times over 24 hours, depending
on the duration of the first and second time periods.
[0149] In some embodiments, a cycle of intermittent delivery of
gNO, e.g., 160 ppm for 30 minutes followed by 3.5 hours of
breathing no gNO, is repeated from 1 to 6 times a day. According to
some embodiments, the cycles are repeated 5 times a day.
[0150] According to some embodiments of the present invention, the
regimen of 1-5 cycles per day is carried out for 1 to 7 days, or
from 2 to 7 days, or from 3 to 7 days.
[0151] According to some embodiments of the present invention, the
intermittent inhalation is effected during a time period of 5 days.
However, longer time periods of intermittent gNO administration as
described herein, are also contemplated.
[0152] In some embodiments, the method is effected while monitoring
one or more physiological parameters in the subject and while
assuring that no substantial change is effected in the monitored
parameters (as demonstrated herein).
[0153] In some embodiments, monitoring the one or more
physiological parameters is effected by noninvasive measures and/or
mild invasive measures.
[0154] In some embodiments, monitoring the physiological
parameter(s) in the subject is effected by on-site measurement and
analysis techniques based on samples collected sporadically,
continuously or periodically from the subject on-site in real-time
at the subject's bed-side, and/or off-site measurement and analysis
techniques based on samples collected sporadically or periodically
from the subject which are sent for processing in a off-site which
provides the results and analysis at a later point in time.
[0155] In the context of some embodiments of the present invention,
the phrase "on-site measurement and analysis techniques" or
"on-site techniques", refers to monitoring techniques that inform
the practitioner of a given physiological parameter of the subject
in real-time, without the need to send the sample or raw data to an
off-site facility for analysis. On-site techniques are often
noninvasive, however, some rely on sampling from an invasive
medical device such as a respiratory tubus, a drainer tube, an
intravenous catheter or a subcutaneous port or any other
implantable probe. Thus, the phrase "on-site parameters", as used
herein, refers to physiological parameters which are obtainable by
online techniques.
[0156] Other that the trivial advantage of real-time on-site
determination of physiological parameters, expressed mostly in the
ability of a practitioner to respond immediately and manually to
any critical change thereof, the data resulting from real-time
online determination of physiological parameters can be fed into
the machinery and be used for real-time feedback controlling of the
machinery. In the context of embodiments of the present invention,
the term "real-time" also relates to systems that update
information and respond thereto substantially at the same rate they
receive the information. Such real-time feedback can be used to
adhere to the treatment regimen and/or act immediately and
automatically in response to any critical deviations from
acceptable parameters as a safety measure.
[0157] Hence, according to embodiments of the present invention,
the term "on-site parameter" refers to physiological and/or
mechanical and/or chemical datum which is obtainable and can be put
to use or consideration at or near the subject's site (e.g.,
bed-side) in a relatively short period of time, namely that the
time period spanning the steps of sampling, testing, processing and
displaying/using the datum is relatively short. An "on-site
parameter" can be obtainable, for example, in less than 30 minutes,
less than 10 minutes, less than 5 minutes, less than 1 minute, less
than 0.5 minutes, less than 20 seconds, less than 10 seconds, less
than 5 seconds, or less than 1 second from sampling to use. For
example, the time period required to obtain on-site parameters by a
technique known as pulse oximetry is almost instantaneous; once the
device is in place and set up, data concerning, e.g., oxygen
saturation in the periphery of a subject, are available in less
than 1 second from sampling to use.
[0158] In the context of some embodiments of the present invention,
the phrase "off-site measurement and analysis techniques" or
"off-site techniques", refers to techniques that provide
information regarding a given physiological parameter of the
subject after sending a sample or raw data to an offline, and
typically off-site facility, and receiving the analysis offline,
sometimes hours or days after the sample had been obtained.
Off-site techniques are oftentimes based on samples collected by
mild invasive techniques, such as blood extraction for monitoring
inflammatory cytokine plasma level, and invasive techniques, such
as biopsy, catheters or drainer tubus, however, some off-site
techniques rely on noninvasive sampling such as urine and stool
chemistry offline and off-site analyses. The phrase "off-site
parameters", as used herein, refers to physiological parameters
which are obtainable by off-site laboratory techniques.
[0159] Hence, according to embodiments of the present invention,
the term "off-site parameter" refers to physiological and/or
mechanical and/or chemical datum which is obtain and can be put to
use or consideration in a relatively long period of time, namely
that the time period spanning the steps of sampling, testing,
processing and displaying/using the datum is long compared to
on-site parameters. Thus, an "off-site parameter" is obtainable in
more than 1 day, more than 12 hours, more than 1 hour, more than 30
minutes, more than 10 minutes, or more than 5 minutes from sampling
to use.
[0160] An "off-site parameter" is typically obtainable upon
subjecting a sample to chemical, biological, mechanical or other
procedures, which are typically performed in a laboratory and hence
are not performed "on-site", namely by or near the subject's
site.
[0161] Noninvasive measures for monitoring various physiological
parameters include, without limitation, pulse oximetry,
nonintubated respiratory analysis and/or capnometry. Mild invasive
measures for monitoring various physiological parameters include,
without limitation, blood extraction, continuous blood gas and
metabolite analysis, and in some embodiments intubated respiratory
analysis and transcutaneous monitoring measures.
[0162] The term "pulse oximetry" refers to a noninvasive and
on-site technology that measures respiration-related physiological
parameters by following light absorption characteristics of
hemoglobin through the skin (finger, ear lobe etc.), and on the
spectroscopic differences observed in oxygenated and deoxygenated
species of hemoglobin, as well as hemoglobin species bound to other
molecules, such as carbon monoxide (CO), and methemoglobin wherein
the iron in the heme group is in the Fe.sup.3+ (ferric) state.
Physiological parameters that can be determined by pulse oximetry
include SpO.sub.2, SpMet and SpCO.
[0163] The phrase "nonintubated respiratory analysis", as used
herein, refers to a group of noninvasive and on-site technologies,
such as spirometry and capnography, which provide measurements of
the physiological pulmonary mechanics and respiratory gaseous
chemistry by sampling the inhaled/exhaled airflow or by directing
subject's breath to a detector, all without entering the subject's
respiratory tract or other orifices nor penetrating the skin at any
stage.
[0164] The term "spirometry" as used herein, refers to the battery
of measurements of respiration-related parameters and pulmonary
functions by means of a noninvasive and on-site spirometer.
Following are exemplary spirometry parameters which may be used in
the context of some embodiments of the present invention:
[0165] The spirometric parameter Tidal volume (TV) is the amount of
air inhaled and exhaled normally at rest, wherein normal values are
based on person's ideal body weight.
[0166] The spirometric parameter Total Lung Capacity (TLC) is the
maximum volume of air present in the lungs.
[0167] The spirometric parameter Vital Capacity (VC) is the maximum
amount of air that can expel from the lungs after maximal
inhalation, and is equal to the sum of inspiratory reserve volume,
tidal volume, and expiratory reserve volume.
[0168] The spirometric parameter Slow Vital Capacity (SVC) is the
amount of air that is inhaled as deeply as possible and then
exhaled completely, which measures how deeply a person can
breathe.
[0169] The spirometric parameter Forced Vital Capacity (FVC) is the
volume of air measured in liters, which can forcibly be blown out
after full inspiration, and constitutes the most basic maneuver in
spirometry tests.
[0170] The spirometric parameter Forced Expiratory Volume in the
1st second (FEV1) is the volume of air that can forcibly be blown
out in one second, after full inspiration.
[0171] Average values for FEV1 in healthy people depend mainly on
sex and age, whereas values falling between 80% and 120% of the
average value are considered normal. Predicted normal values for
FEV1 can be calculated on-site and depend on age, sex, height,
weight and ethnicity as well as the research study that they are
based on.
[0172] The spirometric parameter FEV1/FVC ratio (FEV1%) is the
ratio of FEV1 to FVC, which in healthy adults should be
approximately 75-80%. The predicted FEV1% is defined as FEV1% of
the patient divided by the average FEV1% in the appropriate
population for that person.
[0173] The spirometric parameter Forced Expiratory Flow (FEF) is
the flow (or speed) of air coming out of the lung during the middle
portion of a forced expiration. It can be given at discrete times,
generally defined by what fraction remains of the forced vital
capacity (FVC), namely 25% of FVC (FEF25), 50% of FVC (FEF50) or
75% of FVC (FEF75). It can also be given as a mean of the flow
during an interval, also generally delimited by when specific
fractions remain of FVC, usually 25-75% (FEF25-75%).
[0174] Measured values ranging from 50-60% up to 130% of the
average are considered normal, while predicted normal values for
FEF can be calculated on-site and depend on age, sex, height,
weight and ethnicity as well as the research study that they are
based on. Recent research suggests that FEF25-75% or FEF25-50% may
be a more sensitive parameter than FEV1 in the detection of
obstructive small airway disease.
[0175] However, in the absence of concomitant changes in the
standard markers, discrepancies in mid-range expiratory flow may
not be specific enough to be useful, and current practice
guidelines recommend continuing to use FEV1, VC, and FEV1/VC as
indicators of obstructive disease.
[0176] The spirometric parameter Negative Inspiratory Force (NIF)
is the greatest force that the chest muscles can exert to take in a
breath, wherein values indicate the state of the breathing
muscles.
[0177] The spirometric parameter MMEF or MEF refers to maximal
(mid-)expiratory flow and is the peak of expiratory flow as taken
from the flow-volume curve and measured in liters per second. MMEF
is related to peak expiratory flow (PEF), which is generally
measured by a peak flow meter and given in liters per minute.
[0178] The spirometric parameter Peak Expiratory Flow (PEF) refers
to the maximal flow (or speed) achieved during the maximally forced
expiration initiated at full inspiration, measured in liters per
minute.
[0179] The spirometric parameter diffusing capacity of carbon
monoxide (D.sub.LCO) refers to the carbon monoxide uptake from a
single inspiration in a standard time (usually 10 sec). On-site
calculators are available to correct D.sub.LCO for hemoglobin
levels, anemia, pulmonary hemorrhage and altitude and/or
atmospheric pressure where the measurement was taken.
[0180] The spirometric parameter Maximum Voluntary Ventilation
(MVV) is a measure of the maximum amount of air that can be inhaled
and exhaled within one minute. Typically this parameter is
determined over a 15 second time period before being extrapolated
to a value for one minute expressed as liters/minute. Average
values for males and females are 140-180 and 80-120 liters per
minute respectively.
[0181] The spirometric parameter static lung compliance (Cst)
refers to the change in lung volume for any given applied pressure.
Static lung compliance is perhaps the most sensitive parameter for
the detection of abnormal pulmonary mechanics. Cst is considered
normal if it is 60% to 140% of the average value of a commensurable
population.
[0182] The spirometric parameter Forced Expiratory Time (FET)
measures the length of the expiration in seconds.
[0183] The spirometric parameter Slow Vital Capacity (SVC) is the
maximum volume of air that can be exhaled slowly after slow maximum
inhalation.
[0184] Static intrinsic positive end-expiratory pressure (static
PEEPi) is measured as a plateau airway opening pressure during
airway occlusion.
[0185] The spirometric parameter Maximum Inspiratory Pressure (MIP)
is the value representing the highest level of negative pressure a
person can generate on their own during an inhalation, which is
expresented by centimeters of water pressure (cmH.sub.2O) and
measured with a manometer and serves as n indicator of diaphragm
strength and an independent diagnostic parameter.
[0186] The term "capnography" refers to a technology for monitoring
the concentration or partial pressure of carbon dioxide (CO.sub.2)
in the respiratory gases. End-tidal CO.sub.2, or ETCO.sub.2, is the
parameter that can be determined by capnography.
[0187] Gas detection technology is integrated into many medical and
other industrial devices and allows the quantitative determination
of the chemical composition of a gaseous sample which flows or
otherwise captured therein. In the context of embodiments of the
present invention, such chemical determination of gases is part of
the on-site, noninvasive battery of tests, controlled and monitored
activity of the methods presented herein. Gas detectors, as well as
gas mixers and regulators, are used to determine and control
parameters such as fraction of inspired oxygen level (FiO.sub.2)
and the concentration of nitric oxide in the inhaled gas
mixture.
[0188] According to some embodiments of the present invention, the
measurement of vital signs, such as heart rate, blood pressure,
respiratory rate and a body temperature, is regarded as part of a
battery of on-site and noninvasive measurements.
[0189] The phrase "integrated pulmonary index", or IPI, refers to a
patient's pulmonary index which uses information on inhaled/exhaled
gases from capnography and on gases dissolved in the blood from
pulse oximetry to provide a single value that describes the
patient's respiratory status. IPI, which is obtained by on-site and
noninvasive techniques, integrates four major physiological
parameters provided by a patient monitor (end-tidal CO.sub.2 and
respiratory rate as measured by capnography, and pulse rate and
blood oxygenation SpO.sub.2 as measured by pulse oximetry), using
this information along with an algorithm to produce the IPI score.
IPI provides a simple indication in real time (on-site) of the
patient's overall ventilatory status as an integer (score) ranging
from 1 to 10. IPI score does not replace current patient
respiratory parameters, but used to assess the patient's
respiratory status quickly so as to determine the need for
additional clinical assessment or intervention.
[0190] According to some of any of the embodiments described
herein, the monitored physiological or chemical parameters include
one or more of the following parameters:
[0191] a methemoglobin level (SpMet) (an on-line parameter);
[0192] an end-tidal CO.sub.2 level (ETCO.sub.2) (an on-line
parameter);
[0193] an oxygenation level/FIO2 or oxygen saturation level
(SpO.sub.2) (an on-line parameter);
[0194] an inflammatory cytokine plasma level (an off-line
parameter); and
[0195] a serum nitrite/nitrate level
(NO.sub.2.sup.-/NO.sub.3.sup.-) (an off-line parameter);
[0196] According to some of any of the embodiments described
herein, the monitored physiological or chemical parameters further
include one or more of the following parameters:
[0197] a urine level of nitrogen dioxide (urine nitrite level) (an
off-line parameter);
[0198] a vital sign selected from the group consisting of a heart
rate, a blood pressure, a respiratory rate and a body temperature
(an on-line parameter);
[0199] a pulmonary function (spirometric parameter) (an on-line
parameter) such as, but not limited to, forced expiratory volume
(FEV.sub.1), maximum mid-expiratory flow (MMEF), diffusing capacity
of the lung for carbon monoxide (D.sub.LCO), forced vital capacity
(FVC), total lung capacity (TLC) and residual volume (RV);
[0200] a hematological marker (an off-line parameter), such as, but
not limited to, a hemoglobin level, a hematocrit ratio, a red blood
cell count, a white blood cell count, a white blood cell
differential and a platelet count;
[0201] a coagulation parameter (an off-line parameter) such as, but
not limited to, a prothrombin time (PT), a prothrombin ratio (PR)
and an international normalized ratio (INR);
[0202] a serum creatinine level (an off-line parameter);
[0203] a liver function marker (an off-line parameter) selected
from the group consisting of a aspartate aminotransferase (AST)
level, a serum glutamic oxaloacetic transaminase (SGOT) level, an
alkaline phosphatase level, and a gamma-glutamyl transferase (GGT)
level;
[0204] a vascular endothelial activation factor (an off-line
parameter) selected from the group consisting of Ang-1, Ang-2 and
Ang-2/Ang-1 ratio.
[0205] Non-limiting examples of inflammatory cytokines include
(TNF).alpha., (IL)-1.beta., IL-6, IL-8, IL-10 and IL-12p70.
[0206] According to some embodiments of the present invention, the
method as disclosed herein is such that no substantial change in at
least one of the monitored parameters is observed.
[0207] In the context of the present embodiments, a change in a
parameter is considered substantial when a value of an observation
(measurement, test result, reading, calculated result and the
likes) or a group of observations falls notably away from a normal
level, for example falls about twice the upper limit of a normal
level.
[0208] A "normal" level of a parameter is referred to herein as
baseline values or simply "baseline". In the context of the present
embodiments, the term "baseline" is defined as a range of values
which have been determined statistically from a large number of
observations and/or measurements which have been collected over
years of medical practice with respect to the general human
population, a specific sub-set thereof (cohort) or in some cases
with respect to a specific person. A baseline is a
parameter-specific value which is generally and medically accepted
in the art as normal for a subject under certain physical
conditions. These baseline or "normal" values, and means of
determining these normal values, are known in the art.
Alternatively, a baseline value may be determined from or in a
specific subject before effecting the method described herein using
well known and accepted methods, procedures and technical means. A
baseline is therefore associated with a range of tolerated values,
or tolerance, which have been determined in conjunction with the
measurement of a parameter. In other words, a baseline is a range
of acceptable values which limit the range of observations which
are considered as "normal". The width of the baseline, or the
difference between the upper and lower limits thereof are referred
to as the "baseline range", the difference from the center of the
range is referred to herein as the "acceptable deviation unit" or
ADU. For example, a baseline of 4-to-8 has a baseline range of 4
and an acceptable deviation unit of 2.
[0209] In the context of the present embodiments, a significant
change in an observation pertaining to a given parameter is one
that falls more than 2 acceptable deviation unit (2 ADU) from a
predetermined acceptable baseline. For example, an observation of
10, pertaining to a baseline of 4-to-8 (characterized by a baseline
range of 4, and an acceptable deviation unit of 2), falls one
acceptable deviation unit, or 1 AUD from baseline. Alternatively, a
change is regarded substantial when it is more than 1.5 ADU, more
than 1 ADU or more than 0.5 ADU.
[0210] In the context of the present embodiments, a "statistically
significant observation" or a "statistically significant deviation
from a baseline" is such that it is unlikely to have occurred as a
result of a random factor, error or chance.
[0211] It is noted that in some parameters or groups of parameters,
the significance of a change thereof may be context-dependent,
biological system-dependent, medical case-dependent, human
subject-dependent, and even measuring machinery-dependent, namely a
particular parameter may require or dictate stricter or looser
criteria to determine if a reading thereof should be regarded as
significant. It is noted herein that in specific cases some
parameters may not be measurable due to patient condition, age or
other reasons. In such cases the method is effected while
monitoring the other parameters.
[0212] A deviation from a baseline is therefore defined as a
statistically significant change in the value of the parameter as
measured during and/or following a full term or a part term of
administration the regimen described herein, compared to the
corresponding baseline of the parameter. It is noted herein that
observations of some parameters may fluctuate for several reasons,
and a determination of a significant change therein should take
such events into consideration and correct the appropriate baseline
accordingly.
[0213] Monitoring methemoglobin and serum nitrite levels has been
accepted in the art as a required for monitoring the safety of gNO
inhalation in a subject. Yet, to date, no clear indication that
methemoglobin and serum nitrite levels remain substantially
unchanged upon gNO inhalation by a human subject.
[0214] According to some embodiments of the present invention, the
method comprises monitoring at least one of the parameters
described hereinabove.
[0215] According to some embodiments, the monitored parameter is
methemoglobin level.
[0216] As methemoglobin levels can be measured using noninvasive
measures, the parameter of percent saturation at the periphery of
methemoglobin (SpMet) is used to monitor the stability, safety and
effectiveness of the method presented herein. Hence, according to
some embodiments of the present invention, the followed parameter
is SpMet and during and following the administration, the SpMet
level does not exceed 5%, and preferably does not exceed 1%. As
demonstrated in the Examples section that follows, a SpMet level of
subjects undergoing the method described herein does not exceed
1%.
[0217] According to some embodiments, the monitored parameter is
serum nitrate/nitrite level.
[0218] High nitrite and nitrate levels in a subject's serum are
associated with NO toxicity and therefore serum nitrite/nitrate
levels are used to detect adverse effects of the method presented
herein. According to some embodiments of the present invention, the
tested parameter is serum nitrite/nitrate, which is monitored
during and following the treatment and the acceptable level of
serum nitrite is less than 2.5 micromole/liter and serum nitrate is
less than 25 micromole/liter.
[0219] According to some embodiments, the monitored parameter is
level of inflammatory markers.
[0220] An elevation of inflammatory markers is associated with a
phenomenon called "cytokine storm", which has been observed in
subjects undergoing gNO inhalation treatment.
[0221] Monitoring inflammatory markers while performing the method
as described herein has never been taught heretofore. Moreover,
methods involving gNO inhalation at a regimen in which no
significant change in inflammatory markers is observed have never
been taught heretofore.
[0222] According to some embodiments, the method comprises
monitoring at least two of the above-mentions parameters.
[0223] In some of these embodiments, the monitored parameters are
two or all of methemoglobin level, serum nitrite level and
inflammatory markers.
[0224] While changes in methemoglobin level, serum nitrite level
and inflammatory markers are typically observed in subjects
subjected to gNO inhalation, the findings that no substantial
change in these parameters has been observed in human subjects
undergoing the disclosed regimen are surprising.
[0225] Hence, according to some embodiments of the present
invention, the method as disclosed herein is carried out while
monitoring the methemoglobin level (SpMet), the serum nitrite level
(NO.sub.2.sup.-) and a group of inflammatory cytokine plasma level,
such as, but not limited to, (TNF).alpha., (IL)-1.beta., IL-6,
IL-8, IL-10 and IL-12p70 serum levels in the subject, wherein a
change in at least one of these parameters is less than 2
acceptable deviation units from a baseline.
[0226] According to some of any of the embodiments described
herein, the method is effected while monitoring at least one, at
least two, or all on-site parameters which include SpMet, SpO.sub.2
and ETCO.sub.2, and/or monitoring at least one or all off-site
parameters which include serum nitrite/nitrate level and
inflammatory cytokines in the plasma.
[0227] For example, the method is effected while monitoring SpMet
as an on-site parameter. Alternatively, the method is effected
while monitoring SpMet and ETCO.sub.2 as on-site parameters.
Alternatively, the method is effected while monitoring SpMet,
ETCO.sub.2 and SpO.sub.2 as on-site parameters.
[0228] Further alternatively, the method is effected while
monitoring SpMet as one on-site parameter, and inflammatory
cytokines in the plasma as one off-site parameter.
[0229] Alternatively, the method is effected while monitoring SpMet
and ETCO.sub.2 as on-site parameters, and serum nitrite/nitrate
level as one off-site parameter. Alternatively, the method is
effected while monitoring SpMet as one on-site parameter, and
inflammatory cytokines in the plasma and serum nitrite/nitrate
level as off-site parameters. Alternatively, the method is effected
while monitoring ETCO.sub.2 as one on-site parameter, and
inflammatory cytokines in the plasma and serum nitrite/nitrate
level as off-site parameters. Alternatively, the method is effected
while monitoring SpO.sub.2 as one on-site parameter, and
inflammatory cytokines in the plasma and serum nitrite/nitrate
level as off-site parameters.
[0230] Further alternatively, the method is effected while
monitoring SpMet, ETCO.sub.2 and SpO.sub.2 as on-site parameters,
and inflammatory cytokines in the plasma and serum nitrite/nitrate
level as off-site parameters.
[0231] According to some of any of the embodiments described
herein, the method is effected while monitoring at least one, at
least two, or all on-site parameters which include SpMet, SpO.sub.2
and ETCO.sub.2, and/or monitoring at least one or all off-site
parameters which include serum nitrite/nitrate level and
inflammatory cytokines in the plasma, and further monitoring one or
more and in any combination of:
[0232] a urine NO.sub.2 level (an off-line parameter);
[0233] a vital sign (an on-line parameter);
[0234] a pulmonary function (an on-line parameter);
[0235] a hematological marker (an off-line parameter);
[0236] a coagulation parameter (an off-line parameter);
[0237] a serum creatinine level (an off-line parameter);
[0238] a liver function marker (an off-line parameter);
[0239] a vascular endothelial activation factor (an off-line
parameter).
[0240] According to some of any of the embodiments described
herein, the method is effected while monitoring at least one, at
least two, or all on-site chemical parameters in the inhaled gas
mixture, such as FiO.sub.2 and NO.sub.2.
[0241] It is noted herein that for any of the abovementioned
embodiments, that the method is effected while no substantial
change is observed in any one or more than one or all of the
monitored parameters described herein.
[0242] According to some embodiments of the present invention, the
method is effected while monitoring urine nitrite levels, such that
the urine nitrite level is substantially unchanged during and
subsequent to carrying out the method as presented herein. It is
noted herein that urine nitrite levels may fluctuate for several
known reasons, and a determination of a significant change therein
should take such events into consideration and correct the
appropriate baseline accordingly.
[0243] It is noted that urine nitrite level is indicative for the
safety of gNO inhalation, yet, has never been monitored heretofore
in the context of gNO inhalation in general and in the context of
intermittent gNO inhalation as disclosed herein.
[0244] According to some embodiments of the present invention,
hematological markers, such as the hemoglobin level, the hematocrit
ratio, the red blood cell count, the white blood cell count, the
white blood cell differential and the platelet count, are
substantially unchanged during and subsequent to carrying out the
method as presented herein.
[0245] According to some embodiments of the present invention,
vascular endothelial activation factors, such as Ang-1, Ang-2 and
Ang-2/Ang-1 ratio, as well as the serum creatinine level and
various liver function markers, such as the aspartate
aminotransferase (AST) level, the serum glutamic oxaloacetic
transaminase (SGOT) level, the alkaline phosphatase level, and the
gamma-glutamyl transferase (GGT) level, are substantially unchanged
during and subsequent to carrying out the method as presented
herein.
[0246] Oxygenation of the subject can be assessed by measuring the
subject's saturation of peripheral oxygen (SpO.sub.2). This
parameter is an estimation of the oxygen saturation level, and it
is typically measured using noninvasive measures, such as a pulse
oximeter device. Hence, according to some embodiments of the
present invention, the followed parameter during and following the
administration is SpO.sub.2, and the level of SpO.sub.2 is higher
than about 89%.
[0247] According to some embodiments of the present invention,
various vital signs, such as the heart rate, the blood pressure,
the respiratory rate and the body temperature; and/or various
pulmonary functions (spirometric parameter), such as forced
expiratory volume (FEV.sub.1), maximum mid-expiratory flow (MMEF),
diffusing capacity of the lung for carbon monoxide (D.sub.LCO),
forced vital capacity (FVC), total lung capacity (TLC) and residual
volume (RV); and various coagulation parameters, such as the
prothrombin time (PT), the prothrombin ratio (PR) and the
international normalized ratio (INR), are substantially unchanged
during and subsequent to carrying out the method as presented
herein. It is noted that these parameters are regarded as an
indication that the general health of the subject is not
deteriorating as a result of the medical condition and/or the
treatment.
[0248] According to some embodiments, the aforementioned general
health indicators show an improvement during and subsequent to
carrying out the method as presented herein, indicating that the
treatment is beneficial to the subject.
[0249] Thus, according to some embodiments of the present
invention, the method as disclosed herein is effected such that
general health indicators as described herein are at least remained
unchanged or are improved.
[0250] According to some embodiments of the present invention, a
human subject in need of gNO inhalation treatment is a human that
suffers from a disease or disorder of the respiratory tract.
[0251] As used herein, the phrase "respiratory tract" encompasses
all organs and tissues that are involved in the process of
respiration in a human subject or other mammal subject, including
cavities connected to the respiratory tract such as ears and
eyes.
[0252] A respiratory tract, as used herein, encompasses the upper
respiratory tract, including the nose and nasal passages, prenasal
sinuses, pharynx, larynx, trachea, bronchi, and nonalveolar
bronchioles; and the lower respiratory tract, including the lungs
and the respiratory bronchioles, alveolar ducts, alveolar sacs, and
alveoli therein.
[0253] Respiratory diseases and disorders which are treatable by
any of the methods presented herein, can be classified as:
Inflammatory lung disease; Obstructive lung diseases such as COPD;
Restrictive lung diseases; Respiratory tract infections, such as
upper/lower respiratory tract infections, and malignant/benign
tumors; Pleural cavity diseases; pulmonary vascular diseases; and
Neonatal diseases.
[0254] According to embodiments of the present invention,
restrictive diseases include intrinsic restrictive diseases, such
as asbestosis caused by long-term exposure to asbestos dust;
radiation fibrosis, usually from the radiation given for cancer
treatment; certain drugs such as amiodarone, bleomycin and
methotrexate; as a consequence of another disease such as
rheumatoid arthritis; hypersensitivity pneumonitis due to an
allergic reaction to inhaled particles; acute respiratory distress
syndrome (ARDS), a severe lung condition occurring in response to a
critical illness or injury; infant respiratory distress syndrome
due to a deficiency of surfactant in the lungs of a baby born
prematurely; idiopathic pulmonary fibrosis; idiopathic interstitial
pneumonia, of which there are several types; sarcoidosis;
eosinophilic pneumonia; lymphangioleiomyomatosis; pulmonary
Langerhans' cell histiocytosis; pulmonary alveolar proteinosis;
interstitial lung diseases (ILD) such as inhaled inorganic
substances: silicosis, asbestosis, berylliosis, inhaled organic
substances: hypersensitivity pneumonitis, drug induced:
antibiotics, chemotherapeutic drugs, antiarrhythmic agents, to
statins, connective tissue disease: Systemic sclerosis,
polymyositis, dermatomyositis, systemic lupus erythematosus,
rheumatoid arthritis, infection, atypical pneumonia, pneumocystis
pneumonia (PCP), tuberculosis, chlamydia trachomatis, RSV,
idiopathic sarcoidosis, idiopathic pulmonary fibrosis, Hamman-Rich
syndrome, antisynthetase syndrome, and malignant lymphangitic
carcinomatosis; and extrinsic restrictive diseases, such as
neuromuscular diseases, including Myasthenia gravis and Guillain
bane; nonmuscular diseases of the upper thorax such as kyphosis and
chest wall deformities; diseases restricting lower
thoracic/abdominal volume due to obesity, diaphragmatic hernia, or
the presence of ascites; and pleural thickening.
[0255] According to embodiments of the present invention,
obstructive diseases include asthma, COPD, chronic bronchitis,
emphysema, bronchiectasis, CF, and bronchiolitis.
[0256] Respiratory diseases and disorders which are treatable by
any of the methods presented herein, can also be classified as
acute or chronic; caused by an external factor or an endogenous
factor; or as infectious or noninfectious respiratory diseases and
disorders.
[0257] Diseases and disorders of the respiratory tract include
otolaryngological and/or an upper respiratory tract and/or a lower
respiratory system diseases and disorders, and are also referred to
herein as "respiratory diseases" or "respiratory diseases and
disorders".
[0258] Exemplary, and most common, diseases and disorders of the
respiratory tract include acute infections, such as, for example,
sinusitis, bronchiolitis, tuberculosis, pneumonia, bronchitis, and
influenza, and chronic conditions such as asthma, CF and chronic
obstructive pulmonary disease.
[0259] According to some embodiments of the present invention,
subject in need of gNO inhalation treatment is a human subject that
suffers from a disease or disorder that is manifested in the
respiratory tract, as defined herein.
[0260] In any of the embodiments described herein a human subject
includes any living human at any age, from neonatals and newborns,
to adults and elderly people, at any weight, height, and any other
physical state.
[0261] A disease or disorder that is manifested in the respiratory
tract encompasses also any disease or disorder that is not caused
by an infection or airway obstruction in the respiratory tract,
rather, is caused by another factor yet can be manifested by an
infection or airway obstruction in the respiratory tract.
[0262] An exemplary such condition is cystic fibrosis (CF). CF is a
genetic disorder in which mutations in the epithelial chloride
channel, CF transmembrane conductance regulator (CFTR), impairs
various mechanism of innate immunity. Chronic microbial lung
infections are the leading cause of morbidity and mortality in CF
patients. Early antibiotic eradication treatment of CF patients for
the most prevalent bacterial pathogen, Pseudomonas aeruginosa, has
considerably increased the life expectancy in CF, however still the
vast majority of adult CF patients suffer from chronic P.
aeruginosa lung infections which are difficult to treat due to
biofilm formation and the development of antibiotic resistant
strains of the virulent. Other species found in CF airways include
antibiotic resistant strains such as methicillin-resistant S.
aureus (MRSA), members of the Burkholderia cepacia complex,
Haemophilus influenzae, Stenotrophomonas maltophilia, Achromobacter
xylosoxidans, non-tuberculous mycobacteria (NTM) species and
various strict anaerobic bacteria.
[0263] According to some embodiments of the present invention, a
human subject in need of gNO inhalation treatment is a human
subject that is prone to suffer from a respiratory tract disease or
disorder. By "prone to suffer" it is meant that the human subject
is at a higher risk of suffering from the disease or disorder
compared to a normal subject.
[0264] Such human subjects include, for example, immuno-compromised
subjects such as subjects having HIV, cancer patients undergoing or
which underwent chemotherapy, cancer and other patients undergoing
or which underwent transplantation, including bone marrow
transplantation and transplantation of a solid organ, subjects with
chronic asthma or sinusitis, and subjects which were in contact
with subject(s) afflicted by an infectious respiratory tract
disease or disorder, or which have otherwise been exposed to a
pathogen. It is noted herein that subjecting a human subject prone
to suffer from a respiratory tract disease or disorder to the gNO
inhalation treatment presented herein, can be regarded as a
preventative treatment, preventive care, or as a prophylactic
medical treatment.
[0265] Alternatively, a human subject in need of gNO treatment is
an immuno-compromised subject such as subjects having HIV, cancer
patients undergoing or which underwent chemotherapy, cancer and
other patients undergoing or which underwent transplantation,
including bone marrow transplantation and transplantation of a
solid organ, which have been infected or otherwise suffer from a
respiratory disease or disorder as described herein.
[0266] Exemplary diseases or disorders of such immune-compromised
subjects are described in more detail hereinbelow.
[0267] According to some embodiments of the present invention, a
human subject in need of gNO inhalation treatment is a human
subject that suffers from a disease or disorder that is treatable
via the respiratory tract.
[0268] Since inhaled gNO is absorbed in the lungs, it contacts the
blood system and hence can reach other tissues and organs in the
biological system. Thus, diseases and disorders that are not
associated directly to the respiratory tract, yet can be treated by
inhalation of agents that show therapeutic effect on such diseases
and disorders, can be treated according to embodiments of the
present invention. Exemplary such diseases and disorders include,
but are not limited to, acidosis, sepsis, leishmaniasis, and
various viral infections.
[0269] The parasite family, Leishmania, has been extensively
studied in the literature which shows that gNO kills the parasite
directly. Leishmania parasites preferentially infect macrophages.
Infection by Leishmania causes the macrophage to produce IFN-gamma
which induces the production of iNOS, an enzyme responsible for the
production of nitric oxide. However, certain presentations of
Leishmania cause the macrophage to also produce IL-10 and TGF-Beta
which both minimize the induction of iNOS. The decrease in NO
levels is a key factor allowing the infection to continue. It would
therefore be highly beneficial to determine if treatment with gNO
inhalation circumvents the defense system of the parasite.
Nonetheless, gNO administered by inhalation at any concentration
has not been demonstrated as safe or effective against
leishmaniasis hitherto.
[0270] Additional such diseases and disorders include viral
infections. Viruses have been and most likely will stay a
challenging "moving target" for modern medicinal methodologies.
Without cell walls and thiol based detoxification pathways, viruses
may be inherently more susceptible to nitrosative stress. Several
in-vitro studies, using NO donors, as oppose to gNO, have
demonstrated that NO inhibits viral ribonucleotide reductase, a
necessary constituent enzyme of viral DNA synthesis and therefore
inhibit viral replication. It has been demonstrated that NO
inhibits the replication of viruses early during the replication
cycle, involving the synthesis of vRNA and mRNA encoding viral
proteins. Other direct mechanisms could also account for the
viricidal effects through viral DNA deamination. Nonetheless, gNO
administered by inhalation has not been demonstrated as safe or
effective against acute viral infections or as a prophylactic viral
treatment hitherto.
[0271] The present inventors have demonstrated that the use of
supraphysiologic concentrations of gNO administered by inhalation
may provide a broad spectrum, non-specific antiviral activity to be
used at various stages of infection. The present inventors have
tested two strains of human influenza (influenza A/victoria H3N2)
and one strain of highly pathogenic avian influenza (H7N2), as well
as human respiratory syncytial virus (rgRSV30), using the
traditional plaque or fluorescence assays, and demonstrated that
treating RSV and influenza with 160 ppm exogenous gaseous NO
reduced their infectivity.
[0272] According to some embodiments of the present invention, a
human in need of gNO inhalation is a human afflicted by a disease
or disorder that is treatable by gNO.
[0273] The range of treatable diseases and disorders spans
ophthalmological, otolaryngological and/or an upper respiratory
tract and/or a lower respiratory system diseases and disorders, as
well as systemic medical conditions.
[0274] Exemplary diseases and disorders treatable by gNO include,
without limitation, a heparin-protamine reaction, a traumatic
injury, a traumatic injury to the respiratory tract, acidosis or
sepsis, acute mountain sickness, acute pulmonary edema, acute
pulmonary hypertension, acute pulmonary thromboembolism, adult
respiratory distress syndrome, an acute pulmonary vasoconstriction,
aspiration or inhalation injury or poisoning, asthma or status
asthmaticus, bronchopulmonary dysplasia, hypoxia or chronic
hypoxia, chronic pulmonary hypertension, chronic pulmonary
thromboembolism, cystic fibrosis (CF), Aspergilosis, aspergilloma,
Cryptococcosis, fat embolism of the lung, haline membrane disease,
idiopathic or primary pulmonary hypertension, inflammation of the
lung, perinatal aspiration syndrome, persistent pulmonary
hypertension of a newborn and post cardiac surgery.
[0275] According to some embodiments of the present invention,
exemplary treatable diseases or disorders include, without
limitation, a bacterial-, viral- and/or fungal bronchiolitis, a
bacterial-, viral- and/or fungal pharyngitis and/or
laryngotracheitis, a bacterial-, viral- and/or fungal pneumonia, a
bacterial-, viral- and/or fungal pulmonary infection, a bacterial-,
viral- and/or fungal sinusitis, a bacterial-, viral- and/or fungal
upper and/or lower respiratory tract infection, a bacterial-,
viral- and/or fungal-exacerbated asthma, a respiratory syncytial
viral infection, bronchiectasis, bronchitis, chronic obstructive
lung disease (COPD), cystic fibrosis (CF), Aspergilosis,
aspergilloma, Cryptococcosis, emphysema, otitis, a bacterial-,
viral- and/or fungal otitis externa, otitis media, conjunctivitis,
uveitis primary ciliary dyskinesia (PCD) and pulmonary
aspergillosis (ABPA).
[0276] According to some embodiments of the present invention, the
disease or disorder treatable by gNO is associated with a
pathogenic microorganism. The pathogenic microorganisms, according
to some embodiments of the present invention, can be, for example,
Gram-negative bacteria, Gram-positive bacteria, viruses and viable
virions, fungi and parasites.
[0277] Exemplary pathogenic microorganisms include, but are not
limited to, Acinetobacter baumarmii, Aspergillus niger, Bacteroides
vufgatus, Burkhofderia cepacia, Candida albicans, Clostridium
perfringes, Enteric Group 137, Enterococcus faecium, Enterohacter
aerogenes, Escherichia cofi, Klebsiella pneumoniae, Klebsiella
pneumoniae, Klebsiella pneumoniae, Mycobacteria tuberculosis,
Pasteurella muftocida, Propbnibacterium acnes,
Propbnibacteriumgranulosum, Proteus mirabilis, Providencia
rusfigianii, Pseudomonas aeruginosa, Pseudomonas sp., Serratia
marcesecens, Staphylococcus aureus, Staphylococcus aureus (FVL
positive), Staphylococcus aureus (VNL positive), Staphylococcus
aureus MRSA, Staphylococcus aureus MRSA, Staphylococcus aureus
MRSA, Streptococci Group B, Streptococci Group D, Streptococci
Group G, Streptococcipyrogenes rosenbach Group A, Streptococcus
pneumoniae, Trichophyton meriagrophytes, Trichophyton rubrum, and
Vibrio vuMucus.
[0278] Exemplary Gram-negative bacteria include, but are not
limited to, Proteobacteria, Enterobacteriaceae, Acinetobacter
baumannii, Bdellovibrio, Cyanobacteria, Enterobacter cloacae,
Escherichia coli, Helicobacter, Helicobacter pylori, Hemophilus
influenza, Klebsiella pneumonia, Legionella, Legionella
pneumophila, Moraxella, Moraxella catarrhalis, Neisseria
gonorrhoeae, Neisseria meningitides, Proteus mirabilis,
Pseudomonas, Pseudomonas aeruginosa, Salmonella, Salmonella
enteritidis, Salmonella typhi, Serratia marcescens, Shigella,
Spirochaetes and Stenotrophomonas.
[0279] Exemplary Gram-positive bacteria include, but are not
limited to, Bacillus species such as B. alcalophilus, B. alvei, B.
aminovorans, B. amyloliquefaciens, B. aneurinolyticus, B.
anthracis, B. aquaemaris, B. atrophaeus, B. boroniphilus, B.
brevis, B. caldolyticus, B. centrosporus, B. cereus, B. circulans,
B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B.
globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B.
licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B.
mycoides, B. natto, B. pantothenticus, B. polymyxa, B.
pseudoanthracis, B. pumilus, B. schlegelii, B. sphaericus, B.
sporothermodurans, B. stearothermophilus, B. subtilis, B.
thermoglucosidasius, B. thuringiensis, B. vulgatis and B.
weihenstephanensis, Clostridium species such as C. acetobutylicum,
C. aerotolerans, C. argentinense, C. baratii, C. beijerinckii, C.
bifermentans, C. botulinum, C. butyricum, C. cadaveris, C.
cellulolyticum, C. chauvoei, C. clostridioforme, C. colicanis, C.
difficile, C. estertheticum, C. fallax, C. feseri, C.
formicaceticum, C. histolyticum, C. innocuum, C. kluyveri, C.
lavalense, C. ljungdahlii, C. novyi, C. oedematiens, C.
paraputrificum, C. perfringens, C. phytofermentans, C. piliforme,
C. ragsdalei, C. ramosum, C. scatologenes, C. septicum, C.
sordellii, C. sporogenes, C. sticklandii, C. tertium, C. tetani, C.
thermocellum, C. the rmosaccharolyticum, C. tyrobutyricum,
Corynebacterium species such as C. accolens, C. afermentans, C.
amycolatum, C. aquaticum, C. argentoratense, C. auris, C. bovis, C.
diphtheriae, C. equi, C. flavescens, C. glucuronolyticum, C.
glutamicum, C. granulosum, C. haemolyticum, C. halofytica, C.
jeikeium, C. macginleyi, C. matruchotii, C. minutissimum, C.
parvum, C. propinquum, C. pseudodiphtheriticum, C.
pseudotuberculosis, C. pyogenes, C. renale, C. spec, C. striatum,
C. tenuis, C. ulcerans, C. urealyticum, C. urealyticum and C.
xerosis, Listeriai species such as L. grayi, L. innocua, L.
ivanovii, L. monocytogenes, L. murrayi, L. seeligeri and L.
welshimeri, Staphylococcus species such as S. arlettae, S. aureus,
S. auricularis, S. capitis, S. caprae, S. carnosus, S. chromogenes,
S. cohnii, S. condimenti, S. delphini, S. devriesei, S.
epidermidis, S. equorum, S. felis, S. fleurettii, S. gallinarum, S.
haemolyticus, S. hominis, S. hyicus, S. intermedius, S. kloosii, S.
leei, S. lentus, S. lugdunensis, S. lutrae, S. massiliensis, S.
microti, S. muscae, S. nepalensis, S. pasteuri, S. pettenkoferi, S.
piscifermentans, S. pseudintermedius, S. pseudolugdunensis, S.
pulvereri, S. rostri, S. saccharolyticus, S. saprophyticus, S.
schleiferi, S. sciuri, S. simiae, S. simulans, S. stepanovicii, S.
succinus, S. vitulinus, S. warneri and S. xylosus, and
Streptococcus species such as S. agalactiae, S. anginosus, S.
bovis, S. canis, S. constellatus, S. dysgalactiae, S. equinus, S.
iniae, S. intermedius, S. mitis, S. mutans, S. oralis, S.
parasanguinis, S. peroris, S. pneumoniae, S. pyogenes, S. ratti, S.
salivarius, S. sanguinis, S. sobrinus, S. suis, S. thermophilus, S.
uberis, S. vestibularis, S. viridians and S. zooepidemicus.
[0280] As discussed hereinabove, and demonstrated in the Examples
section that follows below, the disease or disorder which can be
treated by effecting the method presented herein to a human
subject, includes bacterial-, viral- and/or fungal bronchiolitis,
bacterial-, viral- and/or fungal pharyngitis and/or
laryngotracheitis, bacterial-, viral- and/or fungal sinusitis,
bacterial-, viral- and/or fungal upper and/or lower respiratory
tract infection, bacterial-, viral- and/or fungal-exacerbated
asthma, bacterial-, viral-, fungal- and/or parasitic pneumonia, the
common cold, cystic fibrosis related infections, aspergillosis,
aspergilloma, respiratory syncytial viral infections, acidosis or
sepsis, oral fugal infections, bronchitis, candidiasis of the oral
cavity (thrush), canker sores, epiglottitis (supraglottitis),
halitosis, herpes, laryngitis, laryngotracheitis, nasopharyngitis,
otitis externa and otitis media, conjunctivitis, uveitis (and other
eye infections) pharyngitis, pulmonary aspergillosis (ABPA),
respiratory syncytial virus infections, rhinitis,
rhinopharyingitis, rhinosinusitis, stomatitis, tonsillitis,
tracheitis, tuberculosis, cryptococcosis and tympanitis.
[0281] According to some embodiments of the present invention, a
human subject in need of gNO inhalation is a human subject in need
of preemptive, preventative and prophylactic treatment of a disease
or disorder as described herein. Hence, a subject not suffering
from any current or manifested disease, and/or a subject that is
suspected of being exposed to a pathogen, and/or a subject that
suffers from one disease, is treated by the method(s) presented
herein in order to prevent the occurrence of another disease or
disorder.
[0282] As presented in the Examples section that follows below, the
present inventors have contemplated treating bronchiolitis as this
condition is defined hereinbelow.
[0283] Hence, according to an aspect of some embodiments of the
present invention, there is provided a method of treating a human
subject suffering from bronchiolitis, which is effected by
subjecting the subject to intermittent inhalation regimen, gNO at a
concentration of at least 160 ppm, thereby treating
bronchiolitis.
[0284] It is noted herein that the treatable bronchiolitis,
according to some embodiments of the present invention, can be
associated with a pathogenic microorganism or not associated
therewith. It is therefore noted that the method presented herein
can be used to treat idiopathic bronchiolitis, bacterial- and/or
viral-induced bronchiolitis and/or bronchiolitis that is associated
with other medical conditions such as, but not limited to, immune
deficiency.
[0285] In some embodiments, the bronchiolitis is a viral-induced
bronchiolitis.
[0286] Exemplary viral infections that are known to be manifested
by bronchiolitis include, but not limited to, respiratory syncytial
viruses (RSV), rhinoviruses, coronaviruses, enteroviruses,
influenza A and/or B viruses, parainfluenza 1, 2 and/or 3 viruses,
bocaviruses, human metapneumoviruses, SARS and adenoviruses.
However, infections caused by any other viruses are also
contemplated.
[0287] The findings that high concentration of inhaled gNO, which
was shown to exhibit a therapeutic effect against a variety of
conditions associated with pathogenic microorganisms, can be safely
used in human subjects indicate that the disclosed intermittent
inhalation of gNO can be efficiently utilized for treating such
conditions, as well as any other conditions that are treatable by
gNO when contacting the respiratory tract.
[0288] Hence, according to an aspect of some embodiments of the
present invention, there is provided a method of treating a human
subject suffering from a disease or a disorder which is associated,
directly or indirectly, with a pathogenic microorganism, as
described herein. The method is effected by subjecting the subject
to intermittent inhalation regimen of gNO at a concentration of at
least 160 ppm, as described in any of the present embodiments.
[0289] According to another aspect of some embodiments of the
present invention, there is provided a method of treating a human
subject suffering from a disease or disorder that is manifested in
the respiratory tract or a disease or disorder that can be treated
via the respiratory tract, which is effected by subjecting the
subject to intermittent inhalation regimen, gNO at a concentration
of at least 160 ppm, as described in any of the present
embodiments.
[0290] According to another aspect of some embodiments of the
present invention, there is provided a method of treating a human
subject prone to suffer from a disease or disorder that is
manifested in the respiratory tract or a disease or disorder that
can be treated via the respiratory tract, as described herein,
which is effected by subjecting the subject to intermittent
inhalation regimen, gNO at a concentration of at least 160 ppm, as
described in any of the present embodiments. Such a method can be
regarded as a preventive or prophylaxis treatment of the
subject.
[0291] According to another aspect of some embodiments of the
present invention, there is provided a method of treating a human
subject suffering from an ophthalmological, otolaryngological
and/or upper respiratory tract disease or disorder, as described
herein, which is effected by subjecting the subject to intermittent
inhalation regimen, gNO at a concentration of at least 160 ppm, as
described in any of the present embodiments.
[0292] According to some embodiments of the present invention, the
otolaryngological and/or upper respiratory tract disease and
disorder involves an infection or an inflammation of a bodily site
selected from the group consisting of an ear cavity, a nasal
cavity, a sinus cavity, an oral cavity, a pharynx, a epiglottis, a
vocal cord, a trachea, an apex and an upper esophagus.
[0293] According to some embodiments of the present invention, the
ophthalmological, otolaryngological and/or upper respiratory tract
diseases and disorders include, without limitation, the common
cold, a stomatognathic disease, amigdalitis, an oral fugal
infection, bacterial-, viral- and/or fungal sinusitis, bronchitis,
candidiasis of the oral cavity (thrush), canker sores, epiglottitis
(supraglottitis), halitosis, herpes, laryngitis, laryngotracheitis,
nasopharyngitis, otitis (externa and media), conjunctivitis,
uveitis and other eye infections, pharyngitis, rhinitis,
rhinopharyingitis, rhinosinusitis, stomatitis, tonsillitis,
tracheitis, tracheitis and tympanitis.
[0294] According to another aspect of some embodiments of the
present invention, there is provided a method of treating a human
subject suffering from a disease or disorder of the lower
respiratory system, as described herein, by intermittent inhalation
regimen, gNO at a concentration of at least 160 ppm, as described
in any of the embodiments herein.
[0295] According to some embodiments of the present invention,
diseases and disorders of the lower respiratory system include,
without limitation, an obstructive condition, a restrictive
condition, a vascular disease and an infection, an inflammation due
to inhalation of foreign matter and an inhaled particle
poisoning.
[0296] According to some embodiments of the present invention, the
obstructive condition includes, without limitation, a chronic
obstructive lung disease (COPD), emphysema, bronchiolitis,
bronchitis, asthma and viral, bacterial and fungal exacerbated
asthma; the restrictive condition includes, without limitation,
fibrosis, cystic fibrosis, sarcoidosis, alveolar damage and pleural
effusion; the vascular disease includes, without limitation,
pulmonary edema, pulmonary embolism and pulmonary hypertension; the
infection includes, without limitation, respiratory syncytial virus
infection, tuberculosis, a viral-, bacterial-, fungal-, and/or
parasitic pneumonia, idiopathic pneumonia; and the inflammation due
to inhalation of foreign matter and an inhaled particle poisoning
includes, without limitation, smoke inhalation, asbestosis and
exposure to particulate pollutants and fumes.
[0297] According to some embodiments of the present invention, any
of the methods of treating or preventing a subject as described
herein encompasses all of the conditions, disease and disorders
described hereinabove for subjects in need of gNO inhalation.
[0298] It is noted herein that any of the methods described herein
can be used beneficially to treat bronchiolitis, which occurs in
infants and children. Administration by inhalation is considered to
be a preferred method of for young patients and more so when
invasive techniques are avoided.
[0299] Influenza of all sorts and types is also treatable by the
methods presented herein, and where some embodiments being based on
a relatively simple and noninvasive techniques, these methods are
particularly preferred in complicated and severe cases of
influenza.
[0300] The methods presented herein are effective in treating
asthma in children and adults, as well as treating COPD and CF.
[0301] The methods presented herein are fast and effective in
treating a resent medical condition, disease or disorder. Moreover,
the methods presented herein are effective in preventing the
disease or disorder from taking hold in a subject which is prone to
suffer from, contract or develop a disease or disorder which is
associated with the respiratory tract. According to some
embodiments, some methods of gNO inhalation are particularly useful
in preventing a disease or disorder, while other methods are
particularly effective in treating an existing disease or
disorder.
[0302] According to some embodiments of the present invention, any
of the methods described herein can be used in the context of the
following conditions:
[0303] Any of the methods presented herein can be used effectively
to treat respiratory diseases or disorders that occur in humans
which are diagnosed with medical conditions that adversely affect
their innate immune system. Humans which are diagnosed with such
medical conditions are said to be immuno-compromised or
immuno-suppressed. It is noted herein that immuno-suppression may
be a direct result of a pathogen, such as an HIV infection, or an
indirect result such as immuno-suppression that occurs in cancer
patients being treated with chemotherapeutic agents. Hence,
according to some embodiments of the present invention, the methods
presented herein are used to treat a present respiratory disease or
disorder in immuno-compromised human subject.
[0304] Immuno-compromised or immuno-suppressed human subjects are
intrinsically more susceptible to opportunistic infections,
rendering them prone to suffer from respiratory diseases or
disorders. Other incidents and conditions that render a human more
susceptible to infections are associated with location, occupation,
age, living and environmental conditions, close contact with large
groups of people and livestock, close contact with sick people and
the likes, all of which are encompassed in the context of the
present invention as rendering a human subject prone to suffer from
a respiratory disease or disorder.
[0305] According to some embodiments of the present invention, any
of the methods presented herein are used to treat opportunistic
infections in a human subject.
[0306] Exemplary opportunistic infections, which occur in human
suffering from HIV, and can be treated or prevented by the methods
presented herein include, without limitation pneumocystis jiroveci
infection, pneumocystis carinii infection and pneumocystis
pneumonia (a form of pneumonia caused by the yeast-like
fungus).
[0307] Exemplary medical conditions which are associated with
immunosuppression include AIDS, cancer, primary ciliary dyskinesia
(PCD, also known as immotile ciliary syndrome or Kartagener
Syndrome).
[0308] According to some embodiments of the present invention, any
of the methods presented herein is used to treat a human subject
suffering from AIDS.
[0309] According to some embodiments of the present invention, any
of the methods presented herein are used to treat a human subject
suffering from cancer.
[0310] According to some embodiments of the present invention, any
of the methods presented herein can be used to treat or prevent an
infection associated with immune deficiency. These include
prevention/pre-emptive treatment and treatment of infections in
oncology patients.
[0311] According to some embodiments of the present invention, in
any of the methods presented herein the human subject is at risk of
suffering from a nosocomial infection.
[0312] Exemplary groups of human subject which are prone to suffer
respiratory disease or disorder due to general, environmental and
occupational conditions include, without limitation, elderly
people, medical staff and personnel (doctors, nurses, caretakers
and the likes) of medical facilities and other care-giving homes
and long-term facilities, commercial airline crew and personnel
(pilots, flight attendants and the likes), livestock farmers and
the likes.
[0313] According to some embodiments, the methods presented herein
are used to treat or prevent nosocomial infections, such as
infections stemming from direct-contact transmission,
indirect-contact transmission, droplet transmission, airborne
transmission, common vehicle transmission and vector borne
transmission. Exemplary nosocomial infections are caused by
antibiotic resistant bacteria such as carbapenem-resistant
Klebsiella (KPC) or other Enterobacteriaceae, MRSA methicillin
resistance Staph. Aureus, Group A Streptococcus, Staphylococcus
aureus (methicillin sensitive or resistance), Neisseria
meningitides of any serotype and the likes.
[0314] Hence, according to embodiments of the present invention,
the methods presented herein can be used to prevent carriage,
transmission and infection of pathogenic bacteria and antibiotic
resistant pathogenic microorganisms.
[0315] According to some embodiments of the present invention, any
of the methods of treatment presented herein further includes
monitoring, during and following administration gNO, one or more of
the parameters as described in any of the embodiments
hereinabove.
[0316] In some embodiments, the methods are effected while
monitoring one, two, etc., or all of:
[0317] a methemoglobin level (SpMet) (an on-line parameter);
[0318] an end-tidal CO.sub.2 level (ETCO.sub.2) (an on-line
parameter);
[0319] an oxygenation level or oxygen saturation level (SpO.sub.2)
(an on-line parameter);
[0320] an inflammatory cytokine plasma level (an off-line
parameter); and
[0321] a serum nitrite/nitrate level
(NO.sub.2.sup.-/NO.sub.3.sup.-) (an off-line parameter).
[0322] In some embodiments, no significant deviation from baseline,
as described herein, is shown in at least one, two, three, four or
all of the above parameters, when monitored, as described
herein
[0323] Other parameters and markers may be monitored as well, as
presented hereinabove, while showing significant deviation from a
baseline, and various general health indicators show no change to
the worse, or an improvement, as presented hereinabove.
[0324] According to some embodiments of the present invention, in
any of the methods of treatment presented herein, the gNO
administration can be effected by an inhalation device which
includes, without limitation, a stationary inhalation device, a
portable inhaler, a metered-dose inhaler and an intubated
inhaler.
[0325] An inhaler, according to some embodiments of the present
invention, can generate spirometry data and adjust the treatment
accordingly over time as provided, for example, in U.S. Pat. No.
5,724,986 and WO 2005/046426. The inhaler can modulate the
subject's inhalation waveform to target specific lung sites.
According to some embodiments of the present invention, a portable
inhaler can deliver both rescue and maintenance doses of gNO at
subject's selection or automatically according to a specified
regimen.
[0326] According to some embodiments of the present invention, an
exemplary inhalation device may include a delivery interface
adaptable for inhalation by a human subject.
[0327] According to some embodiments of the present invention, the
delivery interface includes a mask or a mouthpiece for delivery of
the mixture of gases containing gNO to a respiratory organ of the
subject.
[0328] According to some embodiments of the present invention, the
inhalation device further includes a gNO analyzer positioned in
proximity to the delivery interface for measuring the concentration
of gNO, oxygen and nitrogen dioxide flowing to the delivery
interface, wherein the analyzer is in communication with the
controller.
[0329] According to some embodiments of the present invention,
subjecting the subject to the method described herein is carried
out by use of an inhalation device which can be any device which
can deliver the mixture of gases containing gNO to a respiratory
organ of the subject. An inhalation device, according to some
embodiments of the present invention, includes, without limitation,
a stationary inhalation device comprising tanks, gauges, tubing, a
mask, controllers, values and the likes; a portable inhaler
(inclusive of the aforementioned components), a metered-dose
inhaler, a an atmospherically controlled enclosure, a respiration
machine/system and an intubated inhalation/respiration
machine/system. An atmospherically controlled enclosure includes,
without limitation, a head enclosure (bubble), a full body
enclosure or a room, wherein the atmosphere filling the enclosure
can be controlled by flow, by a continuous or intermittent content
exchange or any other form of controlling the gaseous mixture
content thereof.
[0330] It is expected that during the life of a patent maturing
from this application many relevant medical procedures involving
inhalation of gNO will be developed and the scope of the term
treatment by inhalation of gNO is intended to include all such new
technologies a priori.
[0331] As used herein the term "about" refers to .+-.10%.
[0332] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0333] The term "consisting of" means "including and limited
to".
[0334] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0335] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0336] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0337] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
[0338] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0339] As used herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical or aesthetical
symptoms of a condition, and substantially preventing the
appearance of clinical or aesthetical symptoms of a condition,
namely preemptive, preventative and prophylactic treatment.
[0340] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0341] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental or calculated support in the following
examples.
EXAMPLES
[0342] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion.
Example 1
Background Art
Determination of Effective Antimicrobial Level of gNO
[0343] The direct effect of gNO on bacteria was studied by
determining the concentration of gNO which is lethal for microbes.
Once an optimal dose was estimated, timing study was conducted to
optimize the duration of exposure of the microbes to gNO.
[0344] For these initial studies, highly dense inoculums of P.
aeruginosa and S. aureus suspensions (10.sup.8 chum) were plated
onto agar plates. These plates were then exposed to various
concentrations of gNO in an exposure device in order to evaluate
the effect on colony growth.
[0345] FIGS. 1A-B present bar-plot showing the gNO dosage curve on
as measured for S. aureus (FIG. 1A) and P. aeruginosa (FIG. 1B)
grown on solid media, wherein relative percentage of growth of
colony forming units (CFU) at 50, 80, 120 and 160 parts per million
(ppm) of gaseous nitric oxide (gNO) compared with growth of CFU in
medical air (100%).
[0346] As can be seen in FIGS. 1A-B, the results confirmed that gNO
has an inhibitory effect on P. aeruginosa and S. aureus growth. The
data provided preliminary evidence that there was a time and dose
relationship trend, with the amount of bactericidal (antibiotic)
activity increasing with increased time of exposure and
concentration of gNO. As the concentration of gNO increased, the
number of colonies growing on the plates decreased. Although there
was a downward bactericidal trend towards 5-10% survival, none of
the data showed a 100% bactericidal effect. Some bacteria may have
survived because the materials and chemicals in the agar may have
reacted with the gNO and buffered the effect.
[0347] It is noted 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 observation suggested
that gNO exposure prevented the growth of the bacteria, and may
have killed the bacteria at some point during the gNO exposure.
[0348] These results demonstrated that gNO had a bacteristatic
effect on both bacterial strains, and as a result, subsequent
studies were designed to further study the bactericidal effects of
gNO. The studies demonstrated that levels of gNO greater than 120
ppm reduced the colony formation by greater than 90%. Studies then
followed indicating that the time required to achieve this effect
occurred between 8-12 hours.
[0349] A similar procedure was used to determine the time required
to induce an effective bactericidal effect with 200 parts per
million gNO, a concentration just above the dose used in the
dose-ranging study presented hereinabove, on a representative
collection of drug resistant gram-positive and gram-negative
strains of bacteria associated with clinical infection.
[0350] A successful bactericidal 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 evaluate if
yeasts, a multi-drug resistant strain of bacteria and actinomycetes
have a similar response. These bacteria represent a comprehensive
variety of drug resistant bacterial pathogens that contribute to
both respiratory and wound infections. The results from these
studies laid the foundation for use of gNO at a concentration
higher than 160 ppm as an antibacterial agent, specifically for use
against bacteria associated with clinical infections.
[0351] For this study, saline was selected as a suspension media
because it would not mask the direct effect of gNO as a
bactericidal, 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 better represents
the hostile host environment that bacteria typically are exposed to
in vivo. In saline, the colonies were static but remained viable.
These conditions are similar to the approach previously used in
animal models.
[0352] Table 1 present the results of this study of the effect of
200 ppm gNO on a variety of microbes.
TABLE-US-00001 TABLE 1 Latent Gram Period -2.5 Log.sub.10
LD.sub.100 Bacteria Staining (hours) (hours) (hours) S. aureus
(ATCC) Positive 3 3.3 4 P. aeruginosa (ATCC) Negative 1 2.1 3 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 Streptococci
Positive 1 1.5 2 Mycobacterium Positive 7 9.2 10 Average 2.77 3.82
4.77 SD 1.01 1.17 1.3
[0353] As can be seen in Table 1, this study showed that gNO at 200
ppm had a complete bactericidal effect on all microorganisms
tested. Without exception, every bacteria challenged with 200 ppm
gNO had at least a 3 log.sub.10 reduction in CFU/mL.
[0354] Furthermore, every test resulted in a complete and total
cell death of all bacteria.
[0355] These results were characterized by a period of latency when
it appeared that the bacteria were unaffected by gNO exposure. 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 all were susceptible to
200 ppm gNO. It is noted that the two drug resistant bacteria
strains were also susceptible to treatment with gNO at 200 ppm.
[0356] These results indicate to 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.
Example 2
Determination of Effective Antiviral Level of gNO
[0357] The efficacy of treating human influenza A with gNO has been
studied. Two strains (H3N2 and H7N3) of the virus were studied and
showed that treating influenza virions or incubated cells with 160
ppm exogenous gNO reduced not only viral replication but also their
infectivity in a Madin-Darby Canine Kidney (MDCK) cell model of
infection. gNO has been demonstrated as an effective anti-viral
agent in both human Influenza A and highly pathogenic avian
influenza.
[0358] The viruses used for the following experiments were from
freezer stocks containing 1.times.10.sup.6-1.times.10.sup.7
pfu's/ml.
[0359] A standard plaque assay was used for the study. Frozen stock
solutions of virions were diluted 1:10 in PBS and 3 ml were placed
in each well of six well trays.
[0360] The samples were either exposed to 160 ppm gNO or medical
air at 37.degree. C. Following exposure 0.5 ml was inoculated onto
confluent MDCK cells, grown in six well trays, and incubated at
37.degree. C. for 1 hour. The inoculums were then removed and 1:1
mixture of 2.times.DMEM and agar, with 2% trypsin, was added to
each well and then incubated at 37.degree. C. After 2 days the
trays were fixed with 3.7% formaldehyde and the agar was removed
from each well. The wells were then stained with crystal violet
revealing the plaques.
[0361] A standard plaque assay was used for a hemagglutination
assay. Frozen stocks of virions were diluted 1:10 in PBS and 3 ml
were placed in each well of six well trays.
[0362] The samples were either exposed to 160-20,000 ppm gNO or
medical air at 37.degree. C. For measure the effect of gNO on pH,
when a large concentration of NO is added to saline the pH falls,
therefore, a standard acid/base buffers were used to match the pH
in the control to that of the treated. Following exposure the
samples were diluted 1:2 in round bottom 96 well trays. Guinea pig
red blood cells were added and agglutination was measured according
to standard procedures.
[0363] FIGS. 2A-C present plot of viral growth as a function of
time measured for influenza A/victoria/H3N2 virions after exposure
to nitric oxide 160 ppm and 800 ppm continuously for 4 hours (FIG.
2A), the same virions after being exposed to one gNO dose over 30
minute as compared to three 30 minute treatments Q4 h (FIG. 2B),
and the effect of continuous exposure to gNO at a concentration of
160 ppm for 3 hours of the highly pathogenic Avian Influenza
H7N3.
[0364] As can be seen in FIGS. 2A-C, gNO has been shown as capable
of reducing the infectivity of 2 strains of human influenza
A/Victoria/H3N2 and HPAI/H7N2 viruses, and that the anti-viral
effect of exposure to 160 ppm gNO is more evident in the
intermittent form of exposure.
[0365] The efficacy of treating viral infection by respiratory
syncytial virus by the methods presented herein was tested by
exposure for 30 minutes of human respiratory syncytial virus
(rgRSV30) to a gas mixture containing 160 ppm nitric oxide, using
standard plaque assay as described hereinabove.
[0366] FIGS. 3A-D present the data obtained in the experiment using
tissue culture samples harboring human rgRSV30, coupled to a green
fluorescent protein, wherein the control experiment the samples
were exposed to a ambient air (data not shown), and the tested
samples having a starting viral level of 2000 PFU (FIG. 3A), 1000
PFU (FIG. 3B) and 500 PFU (FIG. 3C), were exposed to 160 ppm gNO
for 30 minutes, whereas FIG. 3D presents a comparative bar plot
comparing the control to test results.
[0367] As can be seen in FIGS. 3A-D, when the starting
plaque-forming unit (PFU) of RSV was 2000 and 1000 PFU, a single
exposure of 30 minutes to 160 ppm gNO reduced the virus viability
by a factor of bout 10, and at a starting evel of 500 PFU, viral
viability was substantially nullified.
Example 3
Administration of gNO to Healthy Human Subjects
[0368] Cohort:
[0369] 10 healthy adult volunteer subjects (5 males, 5 females),
aged 20 to 62 years, were enrolled in the study after screening
their medical history, a physical examination, pulmonary function
tests and blood values. Exclusion criteria included individuals
less than 19 years of age, pregnant females and unwilling to
practice birth control during the study, diagnosed with pulmonary
disease, epistaxis, hemoptysis, methemoglobinemia, organ transplant
recipient or receiving antibiotic therapy.
[0370] Regimen and Post-Treatment:
[0371] After obtaining informed consent, treatment was initiated
within 5 days of enrollment. Subjects were housed in a hospital
ward and received 160 ppm gNO for 30 minutes every four hours (Q4
h), five times a day, for five consecutive days by inhalation.
Subjects returned for follow-up evaluations 3, 7 and 21 days after
the final gNO administration. Subject safety was determined by
monitoring vital signs, methemoglobin levels, lung function, blood
chemistry, hematology, prothrombin time, inflammatory
cytokine/chemokines levels and endothelial activation.
[0372] These parameters were compared to baseline and at various
time-points during and after gNO administration.
[0373] Device:
[0374] Subjects were administered gNO through a modified disposable
mouthpiece to maximize mixing. Inspiration was spontaneously
initiated by the subject from a conventional intermittent positive
pressure breathing respirator (Mark-7, Carefusion, USA) in fixed
flow mode delivering 48 liters per minute (LPM). Flows of gas were
verified with a calibrated mass flow meter (TSI, USA). Gaseous
nitric oxide (gNO, obtained at INOmax, Ikaria, USA) at a
concentration of 800 ppm delivered at a flow of 12 LPM was titrated
into a distal delivery port on the mouth piece connected to the
respirator during inspiratory phase only (pressure switch). The
Mark 7 respirator was supplied by an air/oxygen blender (Bird
Sentry, Carefusion, USA) set to deliver 26% oxygen.
[0375] All components of the gNO delivery system were approved by
the Therapeutic Product Directorate of Health Canada.
[0376] Monitoring of Chemicals and Physiological Parameters During
Administration:
[0377] The levels of gNO, NO.sub.2, O.sub.2 and methemoglobin were
monitored during the administration of gNO. The target gas mixture
was 160 ppm gNO with a nitrogen dioxide (NO.sub.2) level of less
than 5 ppm and an oxygen (O.sub.2) level ranging from 21% to 25%.
Inspiratory NO, NO.sub.2 and O.sub.2 levels were continuously
monitored by sampling from the mouthpiece sample port located about
6 millimeters from the mouth of the subject with an AeroNOx
(Pulmonox, AB, Canada) NO, NO.sub.2 and O.sub.2 electrochemical
analyzer. Delivery safety was determined by the number of occasions
that NO.sub.2 exceeded 5 ppm, gNO exceeding 10% variation and
O.sub.2 dropping below 20% during gNO administration. A
commercially available noninvasive pulse oximeter (Rad 57, Masimo
Corporation, USA) was used to measure saturation levels at the
periphery of methemoglobin (SpMet).
[0378] These parameters were measured continuously during every gNO
administration course and for 3.5 hours after the first treatment
of the day. Daily serum samples were collected and frozen at
-80.degree. C. and the serum nitrite/nitrite level was measured
using the Griess reagent.
[0379] Subjects underwent full pulmonary function tests (PFT),
including lung diffusing capacity (DLCO) by a trained technician
utilizing a calibrated pulmonary function system (Jaeger
MasterScreen, VIASYS Healthcare, USA) on screening and days 2, 8,
12 and 26. Spirometry test (Microloop by Micro Medical, England)
was performed on days 1, 3 and 4. Effect of gNO on lung function
and DLCO was determined by changes from baseline, treatment days
and follow up days.
[0380] General medical examinations were performed by a pulmonary
physician on screening and on days 8, 12 and 26 to obtain
oxygenation and vital sign measurements.
[0381] Abbreviated physical examination by a registered nurse was
carried out each day prior to initiation of treatments on days 1-5.
Oxygenation was measured with a pulse oximeter (Rad 57, Masimo
Corporation, USA) which was used according to manufacturer's
guidelines to measure functional oxygen saturation of arterial
hemoglobin (SpO.sub.2) and heart rate. These parameters were
measured continuously during every gNO administration and for 3.5
hours after the first treatment of the day.
[0382] Cardiovascular status was determined by monitoring heart
rate, blood pressure, respiratory rate and temperature. Values were
recorded prior to the start of each gNO administration, following a
5 minute rest. During treatments, vital signs (except temperature)
were also performed 15 minutes after the start of the treatment and
at the end of gNO administration and recorded. After the first
treatment each day, vital signs were recorded at every 30 minutes
until the start of the second gNO administration of the day.
[0383] Hematological assessment included a complete blood count and
differentials (hemoglobin, hematocrit, red blood cell count, white
blood cell count, white blood cell differential, and platelet
count) were obtained in order to monitor blood chemistry,
hematology and inflammation measurements. The blood chemistry
profile included serum creatinine, and liver function tests such as
aspartate aminotransferase (AST) serum glutamic oxaloacetic
transaminase (SGOT), alkaline phosphatase, and gamma-glutamyl
transferase (GGT). The effect of gNO on coagulation was determined
by the prothrombin time (PT) and its derived measures of
prothrombin ratio (PR) and international normalized ratio (INR).
Heparinized plasma was collected at baseline and on days 1, 2, 4,
and 5 of gNO administration, and on follow-up days 3, 7 and 21 and
frozen at -80.degree. C. Plasma cytokine levels were assessed using
the human inflammation cytokine bead array kit (BD Bioscience,
Canada). Plasma levels of angiopoietin Ang-1 and Ang-2 were
determined by ELISA (R&D Systems, USA).
[0384] A total of 750 measurements of gNO were recorded during the
study. The average inspired gNO was 163.3 ppm (SD=4.0). The highest
gNO concentration recorded was 177 ppm. The highest NO.sub.2 level
recorded during the treatments was 2.8 ppm (mean: 2.32; 95%
confidence level: 2.17-2.47 ppm) and none of the subjects
experienced a NO.sub.2 level higher than 5 ppm. This was consistent
with the performance specifications provided by the manufacturer of
the apparatus of 1.56 ppm (SD=0.3). Of the 300 recorded oxygen
values, the average oxygen level was 22.0% (SD=0.22%).
[0385] Data Analysis:
[0386] Descriptive statistical characteristics of the subjects
prior to, during, and at the end of the study were tabulated and
expressed as mean.+-.standard deviation (SD).
[0387] Differences in continuous variables (methemoglobin, serum
nitrites/nitrates and SpO.sub.2 levels) over the course of the
study were analyzed utilizing repeated measures analysis of
variance. Categorical events (number of subjects with a particular
adverse event) were determined by constructing 95% confidence
limits for their incidence.
[0388] Differences between continuous variables at two specific
times were evaluated with the paired t-test. Categorical events
such as clinical pulmonary function and lung diffusion changes,
changes in serum inflammatory markers, hematology, clinical
chemistry and incidence of adverse events were analyzed by
constructing 95% confidence limits for their incidence.
[0389] The data were analyzed using the unpaired Mann-Whitney test
for comparison between any two groups and ANOVA for repeated
measures of variance. Baseline comparisons were analyzed by
repeated measures ANOVA with Bonferroni post test for parametric
data, or Friedman test with Dunn's post test for non-parametric
data.
[0390] Data analysis and graphical presentation were done using a
commercial statistics package (Graphpad-Prism V 3.0, GraphPad
Software Inc., USA).
[0391] Unless otherwise specified, p<0.05 indicated statistical
significance. Results were represented by mean.+-.SD from at least
three independent measurements.
[0392] Results of Safety Studies:
[0393] Medical observation of adverse effects and general safety
issues, concerning the repeated delivery of gNO at a concentration
of 160 ppm into the airways of 10 healthy adult individuals, was
effected by monitoring excessive NO.sub.2 levels, while maintaining
acceptable arterial hemoglobin oxygen saturation (SpO.sub.2). A
total of 250 gNO administration procedures were conducted to 10
subjects during the study period. All treatments were well
tolerated and no significant adverse events were observed. Three
minor adverse events were reported: One subject reported bruising
of the arm from multiple attempts to successfully draw blood, while
two other subjects reported a numbing sensation of the tongue
during gNO administration. This was resolved by instructing the
subject to relax and reposition the mouth piece.
[0394] During and after gNO administration, all vital signs
remained within normal limits for age and with respect to baseline
values. Specifically, there was no drop in blood pressure (which
could potentially occur due to the vasodilator effect of gNO
administration) during or after gNO administration. No sudden
incidences of hypoxemia (less than 85% SpO.sub.2) were observed
during or after gNO administration.
[0395] The lowest observed SpO.sub.2 was 93%. SpO.sub.2 levels over
time decreased slightly between the pretreatment and post treatment
but neither differed significantly statistically nor clinically.
ANOVA analysis ruled out that this decrease was associated with the
five repeated exposures to gNO over the course of the same day.
[0396] FIGS. 4A-B present results of monitoring methemoglobin
levels before, during and after inhalation of 160 ppm of gaseous
nitric oxide by 10 healthy human individuals, undergone 5 gNO
administration courses daily, each lasting 30 minutes, for 5
consecutive days, while methemoglobin levels were measured using a
pulse oximeter, wherein FIG. 4A is a plot of methemoglobin levels
by percents as a function of time as measured before (time point
0), during 250 individual 30 minutes gNO administration courses
(time interval of 0 to 30 minutes), after the courses (time
interval of 30 to 60 minutes) and at 120 minutes, 180 minutes and
240 minutes after gNO administration was discontinued, and FIG. 4B
is a plot of methemoglobin levels by percents as a function of time
as measured at the beginning and end of 30 minutes gNO
administration courses given over the course of 5 days, and
followed 8, 12 and 26 days after gNO administration was
discontinued.
[0397] As can be seen in FIG. 4A, all 930 recorded methemoglobin
percent levels (SpMet) remained below the acceptable maximal level
of 5%. The initial baseline SpMet was 0.16 (SD=0.10) percent. The
highest SpMet was observed at the end of the minutes treatment and
was 2.5% with an average increase of 0.9% (SD=0.08).
[0398] SpMet increased as predicted by about 1% between
pretreatment and post treatment (p<0.001) and returned to
baseline after 3.5 hours prior to the next gNO administration.
[0399] As can be seen in FIG. 4B, ANOVA analysis ruled out that
this increase was associated with repeated treatments on the same
day, as there was no accumulative or lingering effect on SpMet
after five daily treatments for five consecutive days. Follow-up
SpMet measurements on 3, 7 and 21 days after the final exposure to
gNO on day 5 did not show any residual increase in SpMet.
[0400] Methemoglobin is reduced by an enzymatic reductase resulting
theoretically in an increase in blood nitrite/nitrate levels.
However, no significant differences in serum nitrite/nitrate levels
from baseline were observed during the trial. One subject had
significantly higher peak nitrite and nitrate values (p<0.001)
which was also slightly different at baseline (p=0.038) compared to
the other subjects.
[0401] There were no statistically, nor clinically significant
changes in blood coagulation parameters, clinical chemistry and
hematological parameters from baseline to completion of day 5.
Although eosinophil cell numbers decreased during the study
(baseline 0.15 giga/L; SD=0.12; end of study: 0.19 giga/L
(SD=0.19), this difference was not significant (p=0.104). A 1%
increase in neutrophil cell numbers from a baseline value of zero
to 0.01 giga/L at the end of study was found, which also did not
reach statistical nor clinical significance (p=0.169).
[0402] FIGS. 5A-F present various results of monitoring pulmonary
function before, during and after inhalation of 160 ppm of gaseous
nitric oxide by 10 healthy human individuals, wherein baseline
values of pulmonary function tests were obtained within 7 days
prior to gNO administration, and values during gNO administration
were obtained on day 2 of the 5-days treatment and other data were
obtained after the final gNO administration on day 5 and on days 8,
12 and 26, wherein FIG. 5A presents forced expiratory volume in 1
second in percents (FEV.sub.1), FIG. 5B presents maximum
mid-expiratory flow (MMEF), FIG. 5C presents carbon monoxide
diffusing capacity (DLCO), FIG. 5D presents forced vital capacity
(FVC), FIG. 5E presents total lung capacity (TLC) and FIG. 5F
presents residual volume (RV), while all data are presented as
means of all ten subjects and absolute differences compared to
baseline prior to gNO administration, and statistical differences
were assessed by Mann-Whitney test.
[0403] As can be seen in FIGS. 5A-F, pulmonary function tests did
not reveal any abnormalities for any subjects during and after gNO
administration treatments.
[0404] Specifically, airflow as measured by FEV.sub.1 and maximum
mid-expiratory flow (MMEF) did not differ from baseline during the
course of the study. Other lung function measurements such as DLCO,
forced vital capacity (FVC), total lung capacity (TLC) and residual
volume (RV) also did not change from baseline measurement.
[0405] To assess whether gNO inhalation may cause inflammation or
endothelial activation cytokines and the vascular endothelium
activation factors Ang-1 and Ang-2 were quantified in peripheral
plasma at baseline at various time points thereafter.
[0406] FIGS. 6A-F present blood levels of various cytokines before
and after inhalation of 160 ppm gaseous nitric oxide by 10 healthy
human individuals, as measured from blood samples collected within
7 days prior to gNO administration, each day during the treatment
and 8, 12 and 26 days thereafter, wherein FIG. 6A presents the
plasma levels of tumor necrosis factor (TNF).alpha., interleukin
(IL)-1.beta. data is presented in FIG. 6B, IL-6 in FIG. 6C, IL-8 in
FIG. 6D, IL-10 in FIG. 6E and IL-12p70 in FIG. 6F, as determined by
a cytometric bead array while statistical differences are compared
by repeated measures ANOVA with Bonferroni post test for parametric
data (IL-6, IL-8, IL-10, IL-12p70), or Friedman test with Dunn's
post test for non-parametric data (TNF and IL-1b).
[0407] As can be seen in FIGS. 6A-F, cytokine levels of TNF, IL-6,
IL-8, IL-10, IL-1b and IL-12p70 were unaffected by inhalation of
gNO as compared to baseline.
[0408] Comparisons between baseline cytokine levels and levels at
each of the sampling time points for all 10 human participants
resulted in no significant differences, compared by repeated
measures ANOVA with Bonferroni post test for parametric data, or
Friedman test with Dunn's post test for non-parametric data.
[0409] FIGS. 7A-C present plasma levels of angiopoietins Ang-1 and
Ang-2 before and after inhalation of 160 ppm gaseous nitric oxide
by 10 healthy human individuals, as measured in blood sample
collected within 7 days prior to gNO inhalation, each day during
gNO administration and 8, 12 and 26 days thereafter, wherein plasma
levels of Ang 1 are shown in FIG. 7A, Ang-2 in FIG. 7B, and
Ang-2/Ang-1 ratios in FIG. 7C, as determined by using a cytometric
bead array while statistical differences were assessed compared by
Friedman test with Dunn's post test.
[0410] As can be seen in FIGS. 7A-C, Ang-2 and Ang-2/Ang-1 ratios
were not affected in this study. Outlier data in FIGS. 4A-C did not
show any correlation with changes in any of the other parameters,
and thus appears to be isolated findings of unknown
significance.
[0411] Conclusions:
[0412] The safety of a treatment of human by inhalation of gNO at a
concentration of 160 ppm, has been demonstrated and presented
herein. It has been shown herein that 160 ppm gNO can be safely
delivered to healthy human lungs in a pulsed manner for five
consecutive days, showing no significant adverse events. All vital
signs remained well within acceptable clinical margins during and
several days after gNO administration at 160 ppm.
[0413] At least with regards to methemoglobin and NO.sub.2 levels,
the findings presented herein are superior to findings obtained for
continuous inhalation of 80 ppm gNO, which is the currently
approved gNO dose for inhalational use in full term infants,
presumably due to the intermittent dosing strategy utilized herein.
While continuous delivery of 80 ppm gNO has been reported to cause
at least 5% increase of SpMet levels, with 35% of the subjects
exceeding 7%, the results presented hereinabove (all 930 recorded
SpMet levels) remained below 5%.
[0414] While the expected increase in methemoglobin levels during
one treatment course was estimated at 1%, the observed average rise
of 0.9% methemoglobin for the ten individuals in a single treatment
course was consistent with first order pharmacokinetics model
estimates, considering the .+-.1% absolute accuracy of the pulse
oximeter. The study established that 3.5 hour interim period
allowed the methemoglobin concentration to return to baseline,
thereby allowing five daily cycles for five days without a
significant clinical increase in methemoglobin concentrations.
[0415] Taken together, it has been shown herein that intermittent
gNO dosing strategy is safe for humans with regard of methemoglobin
production and metabolic burden.
[0416] Similarly, the mean peak concentrations of NO.sub.2 level
shown hereinabove (2.8 ppm) is comparable with that observed during
continuous delivery of 80 ppm (2.6 ppm) of previous studies. The
limitations of this and other studies with regard to gNO delivery
are that the NO and NO.sub.2 levels are only known at the entry
point into the subjects' respiratory tract and the actual resulting
levels of oxides of nitrogen in the lung are unknown. Despite this
resilience to nitrosative stress, it may well be prudent in future
studies to screen subjects for thiol and methemoglobin reductase
deficiencies.
[0417] The study presented hereinabove also demonstrates that 160
ppm of gNO, delivered as outlined, impacts lung function only
minimally, and acute airway inflammation, measured by determining
flow rates, was not detectable. Possibly, potential deleterious
airway reactivity could be masked or prevented by the ameliorative
smooth muscle relaxation that is known to be exerted by gNO. In
patients with pulmonary infection, high NO delivery might cause an
increase in airway reactivity.
[0418] However, the vasodilatory activity of NO may benefit the
patient in addition to the antimicrobial activity of NO.
[0419] The delivery of 160 ppm NO to humans shown herein did not
cause lung parenchymal injury, as measured by different lung
function parameters. Likewise, plasma inflammatory cytokine levels,
the earliest host responses to lung injury, and levels of
eosinophils and neutrophils remained constant during and days after
gNO inhalation. In addition, the vascular endothelial activation
factors Ang-1, Ang-2 and the Ang-2/Ang-1 ratio were unaffected by
gNO administration by inhalation.
[0420] Pulmonary function mechanics and inflammatory markers
remained unchanged compared to baseline values in measurements
three days and 28 days post treatment by gNO administration. While
it cannot be exclude that some longer term change may occur in lung
function, the absence of any sign of inflammation in the post
treatment period shown hereinabove makes this unlikely. If serum
inflammatory markers may prove insensitive to measure acute or even
chronic changes in the lungs, inflammatory markers from
bronchoalveolar lavage (BAL) fluids could be sampled.
Example 4
Treatment of Bronchiolitis in Infants Using gNO
[0421] The following is a protocol for testing the efficacy and for
treating infants suffering from viral bronchiolitis. Each patient
is enrolled by the research physician, and a parental informed
consent signature is obtained in an official document. A detailed
questionnaire is filled by the physician and blood and
nasopharyngeal samples for respiratory viruses are obtained.
[0422] Indication and Prognosis:
[0423] Bronchiolitis is defined as an infection of the small
airways. It is also one of the most common manifestations of acute
lower respiratory system infection in early infancy, and is the
leading cause of global child mortality. In 2005 it has been
estimated that 2.8 to 4.3 million young children worldwide
developed RSV-associated severe ALRI necessitating hospital
admission. Hospitalization for bronchiolitis is expensive with US
hospital charges alone exceeding $1 billion in 2006. These charges
in part reflect length of stay (LOS) in the hospital. The mean LOS
for bronchiolitis in the United States is 3.3 days.
[0424] Bronchiolitis, which includes conditions associated with
pathogenic viruses, bacteria, fungi or other irritants, is
currently the most common reason for pediatric hospital admission
in the United States, accounting for almost 20% of all-cause infant
hospitalizations. Viral etiology is the main cause and among the
respiratory viruses, respiratory syncytial virus (RSV) is believed
to be the most important viral pathogen causing acute lower
respiratory infection (ALRI) in young children. It is estimated
that 60,000 to 199,000 children younger than 5 years die yearly
from RSV-associated ALRI, with 99% of these deaths occurring in
developing countries. The disease is common mainly in the first
year of life. The clinical signs and symptoms are consistent with
hypoxia, difficulty in breathing, coryza, poor feeding, cough,
wheeze and crepitations on auscultation and in some cases
respiratory failure.
[0425] Current Treatment of Acute Bronchiolitis:
[0426] No specific treatment is available hitherto for the viral
infection and only supportive treatment such as oxygen and
inhalations of hypertonic saline or steroids with or without beta
agonist drugs are being used to date.
[0427] Infants with hypoxemia are admitted for oxygen
supplementation and supportive treatment. The administration of
oxygen and fluids are the cornerstone of the treatment of acute
viral bronchiolitis. To date, all other interventions, including
inhaled bronchodilators, corticosteroids, chest physiotherapy,
anti-viral agents, and antibiotics are not proven to be effective,
and are not routinely recommended for the treatment of acute viral
bronchiolitis.
[0428] Disease Related Conditions:
[0429] Respiratory viruses are often responsible for the
bronchiolitis manifestation, which is caused, exacerbated thereby
or otherwise associated therewith. Among them the most common are
respiratory syncytial virus (RSV), rhinovirus, coronavirus,
enterovirus, influenza A and B, parainfluenza 1, 2 and 3,
bocavirus, human metapneumovirus, SARS and adenovirus. However,
other viruses and other pathogens often cause infections that are
manifested by bronchiolitis, and, in addition, bronchiolitis can
occur as a result of conditions which are not associated with any
pathogen (e.g., cystic fibrosis complication complications, cancer
related immunosuppression, and various lung disease, etc.).
[0430] Safety and Adverse Effects:
[0431] The observational objectives of the treatment with gNO of
infants 2-12 months old suffering from bronchiolitis include:
[0432] Assessment of clinical outcome;
[0433] Assessment of off-site parameters such as white blood cells
counts; and
[0434] Assessment of respiratory viral load in the nasopharynx.
[0435] Any adverse event is documented and serious adverse events
are addressed according to established protocols, the gNO treatment
is ceased and the event is reported to the relevant party.
[0436] Cohort Definition:
[0437] The cohort (case) definition fulfills the following
criteria:
[0438] Infants 2-12 months old;
[0439] Diagnosed with bronchiolitis (respiratory distress with
hypoxia);
[0440] No concomitant diseases such as pneumonia or otitis
media;
[0441] No antibiotic treatment has been prescribed or needed;
[0442] Clinical score between 6-10 (see detailed description
hereinbelow);
[0443] No underlying diseases; and
[0444] Documented informed parental consent.
[0445] An underlying disease is one such as genetics disorders or
chronic lung diseases.
[0446] Regimen:
[0447] Infants 2-12 months old which are admitted to the pediatric
ward due to bronchiolitis are subjected to gNO treatment according
to the regimen described in Example 3 hereinabove, namely, to
inhalation of 163.3 ppm (SD of 4.0) gNO for 30 minutes, 5 times
daily, for 5 consecutive days or until discharged, which occurs
first.
[0448] The enrolled infants can be randomized in a 1:1 or 1:2 ratio
to receive gNO with O.sub.2 or Placebo (air) with O.sub.2.
[0449] Clinical Score:
[0450] Table 3 presents the various criteria and scoring attributed
to each observation, which is then summed up to obtain a clinical
score.
TABLE-US-00002 TABLE 3 Use of SaO.sub.2 (room accessory score <6
months .gtoreq.6 months Wheezing air) muscle 0 40 30 None .sup.
.gtoreq.95% 1 41-55 31-45 End 92-94% + expiration audible by
stethoscope 2 56-70 46-60 Inspiration & 90-92% ++ expiration
audible by stethoscope 3 >70 >60 Audible .sup. .ltoreq.89%
+++ without stethoscope.sup.2
[0451] Clinical score is considered mild if <5; moderate at 6-7;
and severe at 11-12. If wheezes is not audible due to a minimal air
entry, it is attributed a score of 3.
[0452] Monitored Parameters:
[0453] Clinical monitoring is carried our by recording a clinical
score by a physician twice daily. Oxygen saturation in room air is
recorded three times a day.
[0454] Off-site laboratory monitoring is performed before and
during treatment and includes blood levels of methemoglobin, serum
nitrites/nitrates, prothrombin, pro-inflammatory cytokines and 18
chemokines.
[0455] Table 4 presents the schedule for various protocol
activities.
TABLE-US-00003 TABLE 4 Treatment day count Activity 0 1 2 3 4 5 6
10 30 Enrollment and informed + consent Questionnaire + + gNO
inhalation regimen + + + + + + Blood tests for WBC count, + + + +
CRP, serum nitrites/nitrates and prothrombin, Blood levels of + + +
+ + + + + methemoglobin Nasopharyngeal wash for + + respiratory
viruses Blood levels for pro- + + + + inflammatory cytokines and
chemokines Clinical score assessment + + + + + + + + +
[0456] Criteria for Efficacy:
[0457] The treatment is assessed by determining the rate of
improvement of the clinical score, reduction of the length of
hospitalization, rate of improvement of O.sub.2 saturation, and
rate of referral to a pediatric intensive care unit.
[0458] In addition, a reduction in viral load in the nasopharynx,
determined by RT-PCR, is used to assess efficacy.
Example 5
Treatment of Bronchiolitis in Infants Using gNO-Clinical Study
Protocol
[0459] The following is an exemplary protocol for clinical studies
based on the methods according to embodiments of the present
invention, aimed at treatment of bronchiolitis in humans.
Specifically, the topic of the study is a randomized double blind
evaluation of efficacy, safety and tolerability of nitric oxide
given intermittently via inhalation to subjects with
bronchiolitis.
[0460] The objectives of the study include the assessment of safety
and tolerability of gNO intermittent inhalation treatment in 2-12
month old infants suffering from bronchiolitis. Other objectives
include the assessment of efficacy of gNO intermittent inhalation
treatment compared with standard treatment using O.sub.2 in a group
of similar subjects.
[0461] Equipment:
[0462] An improvised inhalation device is based on standard
hospital equipment and hospital oxygen source. For example, oxygen
is supplied from the main hospital oxygen system via an oxygen
blender, such as for example, Bird model 03800, followed by a
hospital's oxygen mass flow meter. The oxygen-rich air is monitored
so as to reach a maximal final concentration of about 40% O.sub.2.
The blended air/oxygen is supplied to the subject via a Y-shape
connector attached next to a standard hospital face mask such as
for example, a Hospiltak mask by Unomedical Inc.
[0463] gNO flow, tapped from a tank containing 800 ppm nitric oxide
in 99.999% pure N.sub.2, supplied by an authorized gas provider in,
for example, 50 or 30 litter containers with 120 bar or 150 bar
respectively, is adjusted by passing through a standard hospital's
regulator, such as for example C are Fusion.TM. model 400, and the
hospital mass flow meter, such as a C are Fusion.TM. model
77063.
[0464] NO accountability cannot be checked directly since several
subjects may receive NO from the same container, and the net amount
of NO used for each inhalation is minimal comparing to the total
container weight. The amount of NO given per treatment is therefore
evaluated based on pressure changes in the container (a rough
evaluation).
[0465] The gNO flow is adjusted before each inhalation cycle based
on the gNO concentration detected in the subject's mask. After a
fresh system calibration, the regulator and the mass flow meter are
adjusted to deliver 160 ppm of gNO at 5 to 15 liters per minute.
gNO is supplied to the subject via the second arm of the Y shaped
connector (specified above) attached next to the face mask. Mask
and tubing ports are used to monitor continuously gNO and NO.sub.2
concentrations and FiO.sub.2 values delivered to the subject.
[0466] Halitus (exhalation) is monitored by, for example, end-tidal
CO.sub.2 (EtCO.sub.2).
[0467] EtCO.sub.2 is monitored using standard equipment such as
Microcap.RTM. Portable Capnography Monitor, Cat.# CS04179 by
Oridion, Israel, with nasal prongs such as (Infant Neonate
Cat.#008179 by Oridion, Israel.
[0468] Methemoglobin (SpMet) and oxygen saturation or dissolved
oxygen (SpO.sub.2) levels are monitored continuously using a
dedicated monitor such as for example RAD 57 by Masimo.
[0469] Cohort:
[0470] The population for the study is 44 children of 2-12 months
old diagnosed with bronchiolitis, whom required hospitalization
(expected dropout rate is 10%). The population is split into two
groups: Group 1, referred to as the treatment group, receives
intermittent (5.times.30 minutes, a day) inhalation of 160 ppm gNO
in addition to the standard treatment of O.sub.2 administration,
for up to 5 days. Between gNO inhalations, the subjects continue to
receive the standard inhalation treatment (O.sub.2). Group 2,
referred to as the control group, receives continuous inhalation of
the standard treatment (O.sub.2).
[0471] Table 5 presents clinical score calculation, wherein a score
lower than 5 is mild; 6-10 is moderate, and 11-12 is severe, 11-12.
In a score of 2, if wheezes are not audible due to a minimal air
entry, it is considered a score of 3.
TABLE-US-00004 TABLE 5 SaO.sub.2 Accessory (room muscle Score <6
months .gtoreq.6 months Wheezing air) use 0 40 30 None .sup.
.gtoreq.95% None 1 41-55 31-45 End expiration 92-94% + With
Stethoscope 2 56-70 46-60 Insp. & 90-92% ++ Expiration With
stethoscope 3 >70 >60 Audible without .sup. .ltoreq.89% +++
Stethoscope
[0472] Inclusion criteria are defined as male or female 2-12 months
old, diagnosed with bronchiolitis at a clinical score of less than
10 (see below), and informed consent by parents/legal guardian.
[0473] Exclusion criteria include: diagnosis of concomitant
diseases such as pneumonia, urinary tract infection or otitis
media; prematurity of less than 36 weeks gestational age; subject
receiving RSV immunoglobulin prophylaxis; diagnosis of
methemoglobinemia, chronic lung disease, immunodeficiency or heart
disease; subject use of an investigational drug within 30 days
before enrolment and not expected to participate in a new study
within 30 days; history of frequent epistaxis of more than 1
episode per month; significant hemoptysis within 30 days of more
than 5 ml of blood in one coughing episode or more than 30 ml of
blood in a 24 hour period; methemoglobin of more than 3% at
screening; inability to fulfill the study design; presence of a
condition or abnormality that in the opinion of the investigator
would compromise the safety of the subject or the quality of the
data; underlying diseases such as genetic disorders, such as cystic
fibrosis or Down syndrome or chronic lung diseases such as
bronchopulmonary dysplasia, primary ciliary diskynesia,
bronchiolitis obliterans, hypotonia or congenital heart
disease.
[0474] Regime, Route and Dosage Form of Administration:
[0475] The duration of the study for each subject is 30 (+5) days
from admission to the department, through the treatment, including
the follow up period. All subjects show up for follow up visits on
day 14 (+5 days) and are contacted on day 30 (+5 days) from day of
admission.
[0476] Table 6 presents an exemplary study's assessment activity
schedule, wherein (*) denotes treatment for 5 days or until subject
discharge (whichever comes first), (**) denotes treatment on day 5
or at subject discharge (whichever comes first), in the morning,
(***) denotes treatment by blinded study physician, and (****)
denotes treatment in case the 5th treatment day is the 6th day from
admission to the study.
TABLE-US-00005 TABLE 6 Day 1 - 1.sup.st Day Day Day Study Day 14 21
30 Day 1- inhalation Day 2 Day 3 Day 4 Day 5 **** 6 (+5) (+5) (+5)
Admission + + +* +* +* +* +* Screening, Within 4 signing hours
informed from consent and admission Enrollment Randomization +
Physical + +* +* +* +* +* + + exam*** Clinical score + + +* +* +*
+* +* + + assessment (twice a day morning + evening)*** Vital + +
+* +* +* +* +* + + signs(once per shift) Study + +* +* +* +* +*
(NO/control) treatment Blood tests: % + + +* +* +* +* +*
methemoglobin; % oxyhemoglobin; Heart rate (every inhalation
treatment .times. 5/24 hours)* NO, NO.sub.2, + + +* +* +* +* +*
FiO2 levels from the mask ETCO2 level + + +* +* +* +* +* from the
nasal prongs located in the subject nostril Nasopharyngeal + +* +*
+* +* +* + + and oropharyngeal swabs for Streptococcus pneumoniae,
Haemophilus influenzae and Staphylococcus aureus Nasopharyngeal +
wash for respiratory viruses + PCR Questionnaire + + + + + + + + +
+/phone for all adverse effects Concomitant + + +* +* +* +* +* + +
+ medication Document all + + +* +* +* +* +* + + + lab tests done
for medical reasons
[0477] Table 7 presents the gNO administration and assessment
schedule, wherein (*) denotes activity on the first inhalation on
the first day of treatment.
TABLE-US-00006 TABLE 7 Study Study Pre- Treatment During Study
Treatment treatment start treatment end Time 0-min 30 min 60* min
90* min 120* min 180* min 210* min Subject + eligibility NO
cylinder + + pressure Standard O.sub.2 + + + + + + + + + treatment
NO Start ongoing End treatment (Group 1) OxyHem + + + Ongoing, + +
+ + + + MethHem record the measure-% value observed in case
exceeding the approved range Heart Rate + 15 min after + + + + + +
the start of the treatment NO + NO.sub.2, + + Ongoing, + FiO2,
levels record the taken from value observed the mask in case
exceeding the approved range ETCO.sub.2 level + + taken from the
nasal prongs located in the subject nostril all adverse + + + + + +
+ + + effects Concomitant + + + + + + + + + medication
[0478] Treatment blindness is kept by separating between un-blinded
team members giving the actual treatment and blinded team members,
and by hiding the NO container/source and all study related
equipment behind a curtain.
[0479] Group 1 (treatment group) receives the standard treatment
(O.sub.2) combined with the inhalation via face mask of 0.08%
gaseous nitric oxide (gNO, 800 ppm) administered for 30 minutes
every four hours, keeping a minimum of 3 hours between the end of
one gNO inhalation cycle and the beginning of the next cycle, five
times a day for five consecutive days or until a decision to
discontinue therapy. The maximal cumulative exposure to nitric
oxide is estimate at 2,000 ppm hours.
[0480] Group 2 (control group) receives standard treatment of
O.sub.2 inhalation with the identical equipment as used to
administer gNO to members of Group 1.
[0481] The end of study treatment for both groups is assessed by a
"blinded" study physician based on clinical assessment. Subject
improvement may lead to a decision of subject discharge from the
study.
[0482] Considering an expected dropout rate of approximately 10%,
44 subjects are recruited, in order to have a sample size of at
least 40 (20 per group) subjects who completed the study.
[0483] Study End Points:
[0484] Primary safety end points include determining the
methemoglobin (MetHb) percentage associated with inhaled gNO, and
determining adverse events associated with inhaled gNO.
[0485] Primary tolerability end points include proportion (%) of
subjects whom prematurely discontinued the study for any reason,
and proportion (%) of subjects whom prematurely discontinued the
study due to adverse effects.
[0486] Secondary efficacy end points include the comparison of the
length of hospital stay (LOS) in days of subjects 2-12 months old
diagnosed with bronchiolitis which were treated with gNO and
standard treatment versus subjects treated with standard treatment;
the comparison of the rate of clinical score improvement of
subjects 2-12 months old diagnosed with bronchiolitis which were
treated with gNO and standard treatment versus subjects treated
with standard treatment; and the comparison of the length of oxygen
treatment in hours of subjects 2-12 months old diagnosed with
bronchiolitis whom been treated with gNO and standard treatment
versus subjects treated with standard treatment.
[0487] Observational end points include observation of the number
of subjects with MetHb level higher than about 5% at any time
point; observation of the change in the mean neutrophil and
eosinophil counts of subjects 2-12 months old whom been treated
with gNO for bronchiolitis versus subjects treated with standard
treatment, observation of the number of subjects with study drug
related bleeding at any time point; observation of the reduction of
bacterial carriage in the nasopharynx of S. pneumoniae, H.
influenzae, S. Aureus and the assessment of clinical outcome during
21 days from admission to the study of subjects 2-12 months old
treated with gNO for bronchiolitis; and the assessment of off-site
parameters such as white blood cell counts in subjects 2-12 months
old treated with gNO for bronchiolitis versus subjects treated with
standard treatment.
[0488] Criteria for treatment end due to subject improvement are
based on the subject's improvement that leads to a decision of
subject discharge from the hospital.
[0489] Criteria for early temporary treatment discontinuation
include:
Blood methemoglobin more than 5%; NO.sub.2 level measured near
subject's mouth more than 5 ppm; SpO.sub.2 during treatment more
than 90%; ETCO.sub.2 of more than 60 mmHg; and Adverse effects that
are suspected to be NO related, according to physician
discretion.
[0490] In any event that a measured parameter exceeds the approved
level, the current study treatment inhalation is be stopped. A
repeat level is then measured 30 minutes later and the final
measurement is recorded. The next inhalation starts according to
study protocol.
[0491] Criteria for early permanent treatment discontinuation
include:
A second episode of blood methemoglobin more than 5%; and A
clinical score of more than 10.
[0492] A subject whose treatment was discontinued for any reason
except voluntary parent/legal guardian consent withdrawal,
completes all study assessment including the follow up visits.
[0493] Concomitant medication (i.e. antibiotics or steroids) given
during the study are not be held as a reason for treatment early
discontinuation.
[0494] Criteria for early study withdrawal include:
Subject's parent/legal guardian withdrew consent; Study management
requested subject to be withdrawn; Investigator's discretion;
Protocol violation/non-compliance; Loss to follow-up/failure to
return; Adverse event or serious adverse event; and Death; in which
case the study is stopped for all cohort member for medical
re-evaluation.
[0495] Treatment Procedures:
[0496] Day 1:
[0497] Up to 5 inhalations depending on time of admission.
[0498] Pretreatment includes monitoring and recording levels of
oxyhemoglobin (%), methemoglobin (%), EtCO.sub.2, FiO.sub.2, NO and
NO.sub.2. A baseline for gas flow is established to match patient's
minute ventilation (5-15 Lpm), and the gNO flow is validated to
achieve 160 ppm in the inhalation mask, with fluctuating not
exceeding 15 ppm. If gNO is fluctuating, baseline gas flow is
increased to meet patient's minute ventilation and flow demands. At
the end of the pretreatment stage the mask is placed on the
subject's face.
[0499] At treatment start the following parameters are
recorded:
Start time and cylinder pressure; heart rate; Oxyhemoglobin (%) and
methemoglobin (%); and FiO.sub.2, NO and NO.sub.2 levels.
[0500] During treatment the following parameters are monitored and
recorded continuously:
Oxyhemoglobin (%) and methemoglobin (%); FiO.sub.2, NO and NO.sub.2
levels taken from the mask; Any form of adverse effects; and heart
rate 15 min after the start of the treatment.
[0501] Treatment ends 30 minutes after the start of treatment, and
the following is recorded:
[0502] Stop treatment time and cylinder pressure;
[0503] Any form of adverse effects;
FiO.sub.2, NO and NO.sub.2 levels; Heart rate; Oxyhemoglobin (%)
and methemoglobin (%) levels; Only after the first inhalation of
the first day for both groups, the following is recorded:
Oxyhemoglobin (%), methemoglobin (%) and heart rate 60, 90, 120,
150, 180 and 210 minutes after the start of treatment;
[0504] Questionnaire including all adverse effects is completed by
the study coordinator and study physician, and vital signs are
measured once per shift.
[0505] Days 2-5 (or Day 6):
[0506] The last day of treatment is the 6th day from admission.
[0507] On Day 5 (or Day 6, if the 5th treatment day ends on the 6th
day from admission or discharge day/last day of study treatment) up
to 5 inhalations, according subject clinical status.
[0508] Once a day in morning physical examination is performed
vital signs are recorded (including temperature, pulse, respiratory
rate, blood pressure etc.);
Assessment of clinical score by a blinded physician; and
[0509] Detection of bacteria by culture from nasal swab
(Nasopharyngeal and oropharyngeal swabs for Streptococcus
pneumoniae, Haemophilus influenzae and Staphylococcus aureus)
[0510] On a routine basis questionnaire including all adverse
effects is completed by the study coordinator and study physician,
and the subject is observed so as to meet inclusion and exclusion
criteria.
[0511] Pretreatment, treatment start, during treatment and end of
treatment procedures are conducted as in Day 1.
[0512] End of treatment assessments are made 2-4 hours after last
inhalation treatment, and include assess clinical score, vital
signs by a blinded physician, oxyhemoglobin (%) and methemoglobin
(%), detection of bacteria by culture from nasal swab.
[0513] Scheduled Follow Up Visits:
[0514] Days 14+5 and 21+5:
Questionnaire including any adverse effects is completed by the
study Physician; Review and document concomitant medications;
Physical examination by the study physician; Assess clinical score
by a blinded physician; Perform detection of bacteria by culture
from nasal swab; Record in subject study file any lab tests (i.e.,
blood tests and chest x-ray) if taken for clinical reasons.
[0515] Day 30+5:
Questionnaire including any adverse effects is completed by the
study physician; Review and document concomitant medications.
[0516] Medical/Clinical Assessment:
[0517] Initial demography/medical history form is filled, and a
physical examination is performed. A physical examination is
performed by a physician at the screening day and during follow up
visits. Body systems which are examined grossly include general,
skin, lymph nodes, head, eyes, ears, nose and throat, respiratory,
cardiovascular, gastrointestinal, neurologic and muscoskeletal. New
abnormal findings are documented and followed by a physician at the
next scheduled visit.
[0518] An abbreviated physical examination is performed as deemed
necessary by the investigators. In addition to vital signs, body
systems to be examined grossly. Vital signs measurements after
resting for 5 minutes, including heart rate, blood pressure,
respiration rate and temperature, are performed and recorded three
times a day.
[0519] Oxygen saturation (SpO.sub.2) is measured by pulse oximetry
(using e.g., RAD 57) during each treatment and recorded before,
during and after NO treatment. In any event that SpO.sub.2 level
during treatment is less than about 89%, the current study
treatment inhalation is stopped. A repeat level is measured 30
minutes later and the final measurement should be recorded. The
next inhalation starts according to study protocol.
[0520] Methemoglobin levels are measured non-invasively and
continuously using a pulse methemoglobinometer during treatment and
recorded before, during and after on Day 1 through Day 5. Any
methemoglobin level of more than about 5% requires a repeat
measurement 30 minutes later, and the final measurement is
recorded. The next inhalation starts according to study
protocol.
[0521] End-tidal CO.sub.2 (EtCO.sub.2) is measured using, e.g.,
MicroCap Capnograph and recorded before and after treatment on Day
1 through Day 5.
[0522] Information regarding occurrence of adverse effects or
events is documented throughout the study and until 30 day subjects
follow up is complete. Event duration (start and stop dates and
times), severity, outcome, treatment and relation to study
medication (causality) and if the event is regarded as a severe
adverse event, is recorded in the case report form. Adverse effect
or events are monitored until day 30 follow up visit/call.
[0523] Concomitant medication given during the study does not lead
to study treatment discontinuation. All concomitant medications and
concurrent therapies are documented throughout the study until day
30 follow up visit/call. The following information is recorded:
dose, route, unit frequency of administration, and indication (if
deemed relevant) for administration of medication. The reason for
administration of concomitant medications is considered as an
adverse effect or event unless it was scheduled prior to study
start.
[0524] Any laboratory tests results received during subject's
hospitalization or study follow up are recorded in the subject file
and used for observational analysis.
[0525] Nasal wash for determination of viral shedding is collected
in a sterile specimen cup. Nasal swab (nasopharyngeal and
oropharyngeal) for determination of bacteria (Streptococcus
pneumoniae, Haemophilus influenzae and Staphylococcus aureus) is
collected in a sterile specimen cup.
[0526] Adverse Effects or Events:
[0527] Adverse events are recorded from the date of subject's
signed informed consent form and throughout the study, including
the follow-up period. Adverse events should be reviewed and updated
at each subsequent visit and during any phone contact with the
subject. The intensity or severity of adverse event is
characterized as mild if it is easily tolerated, moderate if it is
sufficiently discomforting to interfere with daily activity, and
severe if it prevents normal daily activities.
[0528] The causality of the adverse event is assessed as:
[0529] Unrelated when the adverse event is clearly and
incontrovertibly due to extraneous causes (disease, environment,
etc.);
[0530] Unlikely related if the adverse event meets at least two of
the following criteria:
[0531] it does not follow a reasonable temporal sequence from
study-drug administration; it could readily have been produced by
the subject's clinical state, environmental or toxic factors or
other modes of therapy administered to the subject; it does not
follow a known pattern of response to the study-drug, and does not
reappear or worsen when the drug is re-administered;
[0532] Possibly related if the adverse event meets at least two of
the following criteria: it follows a reasonable temporal sequence
from study-drug administration; a causal relationship to the
experimental treatment cannot necessarily be reasonably excluded
and an alternative explanation (e.g., concomitant drug or
concomitant disease) cannot be reasonably suggested as causing the
SAE; and it follows a known pattern of response to the
study-drug;
[0533] Probably related if the adverse event meets at least three
of the following criteria: it follows a reasonable temporal
sequence from study-drug administration; it cannot be reasonably
explained by the known characteristics of the subject's clinical
state, environmental or toxic factors or other modes of therapy
administered to the subject; it disappears or decreases on
cessation or reduction of the drug dose, and it follows a known
pattern of response to the study-drug.
[0534] Potentially NO related adverse events are those which are
associated with methemoglobinemia (% methemoglobin elevation) of
more than 5%, and NO.sub.2 elevation of more than 5 ppm.
[0535] Data Processing and Statistical Considerations:
[0536] For categorical variables summary tables are created, giving
sample size, absolute and relative frequency and 95% confidence
interval for proportions by study group.
[0537] For continuous variables summary tables are provided, giving
sample size, arithmetic mean, standard deviation, coefficient of
variation (if appropriate), median, minimum and maximum,
percentiles and 95% confidence interval by study group for means of
variables.
[0538] Chi-square test or Fisher's Exact test are applied for
testing difference in proportions between the study groups.
[0539] Adverse events are coded according to coding dictionaries
such as Medical Dictionary for Regulatory Activities (MedDRA
version 14.0 or higher) and presented in tables by System Organ
Class (SOC) and Preferred Term (PT).
[0540] 95% confidence interval (CI) is calculated for the
proportion of subjects having adverse effects associated with
inhaled gNO.
[0541] The Paired T-test or Signed rank test for two means are
applied for analyzing changes in continuous parameters within each
study group.
[0542] The two-sample T-test or Non-parametric
Wilcoxon-Mann-Whitney Rank sum test for independent samples are
applied for analyzing differences in continuous parameters between
the study groups.
[0543] All tests are two-tailed, and a p-value of 5% or less is
considered statistically significant.
[0544] The data are analyzed using data processing software such as
the SAS.RTM. version 9.1 (SAS Institute, Cary N.C.).
[0545] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0546] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
* * * * *