U.S. patent application number 11/855870 was filed with the patent office on 2008-09-25 for pulse drug nebulization systems, formulations therefore, and methods of use.
This patent application is currently assigned to BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM. Invention is credited to Perenlei Enkhbaatar, Edward R. Kraft, Gabriela A. Kulp, Daniel L. Traber.
Application Number | 20080230053 11/855870 |
Document ID | / |
Family ID | 39615684 |
Filed Date | 2008-09-25 |
United States Patent
Application |
20080230053 |
Kind Code |
A1 |
Kraft; Edward R. ; et
al. |
September 25, 2008 |
PULSE DRUG NEBULIZATION SYSTEMS, FORMULATIONS THEREFORE, AND
METHODS OF USE
Abstract
Liquid nebulizer apparatus, systems, and formulation
compositions, as well as systems for the nebulized, aerosol
delivery of such compositions, for the administration and
insufflation of medicinal aerosols into the pulmonary system of a
mammal are described. The nebulizing apparatus and system can
effectively aerosolize a variety of viscosities of medicinal liquid
drug carriers, including those made up of oil, water, or emulsions
of oil and water. Drugs dissolved or suspended in the compositions
and formulations described and adapted for use herein are not
damages or denatured by the nebulization process when the nebulizer
described is used. Further, the nebulization system itself can be
adapted for use with both mechanically assisted pulmonary
ventilation systems as well as hand-held inhalers and nose/mouth
face masks for use in pulmonary drug delivery.
Inventors: |
Kraft; Edward R.;
(Galveston, TX) ; Enkhbaatar; Perenlei;
(Galveston, TX) ; Traber; Daniel L.; (Galveston,
TX) ; Kulp; Gabriela A.; (Santa Fe, TX) |
Correspondence
Address: |
LOCKE LORD BISSELL & LIDDELL LLP;ATTN: IP DOCKETING
600 TRAVIS, SUITE 3400
HOUSTON
TX
77002-3095
US
|
Assignee: |
BOARD OF REGENTS, THE UNIVERSITY OF
TEXAS SYSTEM
Austin
TX
|
Family ID: |
39615684 |
Appl. No.: |
11/855870 |
Filed: |
September 14, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60845087 |
Sep 15, 2006 |
|
|
|
60891128 |
Feb 22, 2007 |
|
|
|
Current U.S.
Class: |
128/200.23 ;
128/200.14; 514/458 |
Current CPC
Class: |
A61M 15/00 20130101;
B05B 7/0876 20130101; A61M 2202/0007 20130101; B05B 7/0892
20130101; A61M 15/0021 20140204; B05B 7/066 20130101; A61M 15/08
20130101; A61M 15/002 20140204; A61M 15/009 20130101; A61M 11/06
20130101; A61M 11/04 20130101; A61M 16/06 20130101 |
Class at
Publication: |
128/200.23 ;
128/200.14; 514/458 |
International
Class: |
A61M 11/00 20060101
A61M011/00; A61K 31/355 20060101 A61K031/355 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The government may own rights in the present invention
pursuant to grant number 1 P01 GM066312-01A2 from the National
Institute of Health.
Claims
1. A nebulization system comprising: a nozzle; an outer
gas-delivery tube; and an inner microchannel delivery tube having a
central fluid channel; wherein the outer delivery tube is
concentrically configured around the inner delivery tube, such
concentric configuration forming an annular intermediate space
between the two tubes, and wherein the intermediate air space
between the two tubes is the free air opening value, the value of
which is the internal diameter of the outer gas-delivery tube minus
the total inside diameter of the inner microchannel delivery
tube.
2. The nebulization system of claim 1, wherein the nozzle has an
air volume to nebulized droplet volume ratio less than about
60,000:1.
3. The nebulization system of claim 1, wherein the value of the
intermediate air space between the two tubes ranges from about
0.00000259 in.sup.2 to about 0.001 in.sup.2.
4. The nebulization system of claim 1, wherein the outer gas
delivery tube has an inner diameter ranging from about 0.01 inches
to about 0.05 inches.
5. The nebulization system of claim 1, wherein the inner
microchannel delivery tube has an outer diameter ranging from about
0.01 inches to about 0.04 inches.
6. The nebulization system of claim 1, wherein the system is
capable of producing aerosol droplets of compositions comprising a
fatty acid or lipids having particle sizes ranging from about 2
.mu.m to about 12 .mu.m.
7. A pulmonary drug delivery system capable of nebulizing a
composition comprising a water-insoluble or substantially
water-insoluble drug and a fatty acid or lipid, the system
comprising: a reservoir for containing the drug composition; a
reservoir for containing a propellant gas; a mechanical control
valve capable of regulating the flow of the propellant gas and the
drug composition; and a nebulizing nozzle adapted to receive both
the drug composition and the propellant gas; wherein the nebulizing
nozzle can produce aerosol droplets having a particle size ranging
from about 2 .mu.m to about 12 .mu.m in median mass aerodynamic
size.
8. The pulmonary drug delivery system of claim 7, further
comprising an assembly fitted into the air stream of a mammalian
patient.
9. The pulmonary drug delivery system of claim 7, wherein the
nebulizing nozzle can produce aerosol droplets having a particle
size ranging from about 2 .mu.m to about 5 .mu.m.
10. A non-aqueous medicinal aerosol composition, comprising: a
therapeutically effective amount of a pulmonary medicament or a
derivative, metabolite, solvate, hydrate, prodrug, or polymorph
thereof; a lipid or fatty acid; and a fluid propellant carrier.
11. The composition of claim 10, wherein the pulmonary medicament
is gamma-tocopherol.
12. The medicament of claim 11, wherein the gamma-tocopherol is in
either the d-, l-, or dl-conformation, or a combination
thereof.
13. The composition of claim 10, wherein the therapeutically
effective amount of the pulmonary medicament ranges from about 1%
(w/w) to about 99% (w/w).
14. The composition of claim 10, wherein the lipid or fatty acid is
present in an amount ranging from about 1% (w/w) to about 99%
(w/w).
15. The composition of claim 10, further comprising an antioxidant
for use in the preparation of a medicament for the treatment of
disease or disorder of the pulmonary system, including but not
limited to acute lung injury.
16. The composition of claim 15, wherein the antioxidant is a
vitamin selected form the group consisting of Vitamin E, Vitamin C,
riboflavin, niacin, vitamin K3, ascorbic acid, coenzyme-Q and
.beta.-carotene.
17. The composition of claim 10, wherein the fatty acid is selected
from the group consisting of flax seed oil, sunflower oil, saffron
oil, and canola oil.
18. The composition of claim 10, further comprising a
surfactant.
19. The medicinal composition of claim 10, wherein the
therapeutically effective amount ranges from about 1 mg/kg/d to
about 1,000 mg/kg/d.
20. A drug nebulizing apparatus adapted for pulmonary inhalation
and delivery of aerosolized medicaments into a mammalian patient,
the apparatus comprising: a reservoir containing a drug
formulation; and a nebulizing nozzle adapted to be fit to a
breathing circuit and further attached to a face mask; wherein the
nebulizing nozzle produces aerosolized droplets sized for
pulmonary, inhaled drug delivery of the drug formulation.
21. The drug nebulizing apparatus of claim 19, wherein the drug
formulation is in the form of a mixture, a solution, a suspension,
or an emulsion.
22. The drug nebulizing apparatus of claim 19, wherein the face
mask is capable of being fit to the patient's nose, mouth, or both
the nose and the mouth.
23. The drug nebulizing apparatus of claim 19, wherein the droplets
range in size from about 2 .mu.m to about 10 .mu.m.
24. The drug nebulizing apparatus of claim 22, wherein the droplets
range in size from about 2 .mu.m to about 5 .mu.m.
25. The drug nebulizing apparatus of claim 19, wherein the
nebulizing nozzle comprises a fluid micro-tube with an air delivery
tube capable of nebulizing a fluid from the micro-tube into
droplets into droplets sized for inhaled drug delivery.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Patent Application Ser. No. 60/845,087, filed Sep. 15, 2006, and
U.S. Provisional Patent Application Ser. No. 60/891,128, filed Feb.
22, 2007, the contents of all of which are incorporated herein by
reference in their entirety.
REFERENCE TO APPENDIX
[0003] Not applicable.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] The present invention relates to compositions, formulations
and methods for the treatment and prevention of cytoxicity, cell
damage and inflammation in the pulmonary system of patients, as
well as systems for use in delivering such compositions and
formulations to the patient. More particularly, the present
invention relates to tocopherol compositions and formulations for
use in the treatment of pulmonary disorders in patients, methods
for their use, and systems for the delivery of such
tocopherol-containing compositions and formulations to the
pulmonary region of a patient.
[0006] 2. Description of the Related Art
[0007] Burn inhalation injury of the lungs increases morbidity and
mortality, with 70% of victims of smoke inhalation dying within the
first 12 hours of [Shirani, K. Z., et al., Ann. Surg., Vol. 205(1):
pp. 82-87 (1987)]. Lung injury is traumatic, and is typically
caused by heat and chemical irritation, with chemical injury being
the leading lethal cause of smoke inhalation injury. Similarly,
thermally injured patients who sustain inhalation injury have a 20
fold increase in mortality [see, Saffle, J. R., et al., J. Burn
Care Rehabil., Vol. 16(3, pt. 1); pp. 219-232 (1995)].
[0008] Injury from burn and smoke inhalation has been demonstrated
to produce a systemic inflammatory response and increase levels of
reactive oxygen species (ROS) [Traber, D. L., et al., Burns Inc.
Therm. Inj., Vol. 14(5): pp. 357-364 (1988)]. ROS produces an
increase in pulmonary microvasculature and pulmonary edema
accompanied by increased lipid peroxidation in lung tissue.
Inhibition of lipid peroxidation has been demonstrated to reduce
these symptoms in animals subjected to hot sawdust smoke by Z. Min,
et al., [J. Med. Cell. PLA, Vol. 5(2): pp. 176-180 (1990)].
[0009] Antioxidants are compounds that reduce oxidation products
and have been demonstrated to reduce cytotoxicity in smoke
inhalation-lung injury, adult respiratory distress syndrome,
emphysema and asthma. More recently it has been reported that the
use of antioxidants such as vitamin E may be beneficial in the
treatment of victims of fire accidents who sustain both thermal
injury to the skin and smoke inhalation and exhibit evidence of
oxidant injury [Morita, N., et al., Shock, Vol. 25(3): pp. 277-282
(2006)]. For example, vitamin C and vitamin E (alpha-tocopherol and
gamma tocopherol) are antioxidants in vivo which may act together
to scavenge ROS to produce non-reactive compounds within the human
body. One of the important chemical features of the tocopherols is
that they are redox agents which act under certain circumstances as
antioxidants. In acting as an antioxidant, tocopherols presumably
prevent the formation of toxic oxidation products, such as
perioxidation products formed from unsaturated fatty acids.
Further, it has recently been discovered that individual members in
the class of tocopherols may exhibit different biological
properties from one another despite their structural similarity.
Some investigators, for example, believe that .gamma.-tocopherol,
unlike .alpha.-tocopherol, acts in vivo as a trap for
membrane-soluble electrophilic nitrogen oxides and other
electrophilic mutagens [Christen, S., et al. Proc. Natl. Acad. Sci.
94: 3217-3222 (1997]. Vitamin E is remarkably safe, and falls
within a class of compounds that are "generally regarded as safe"
or "GRAS". Vitamin E is available in several forms that present
varied activities between them. Whereas alpha-tocopherol has been
widely investigated for therapeutic uses, until recently
gamma-tocopherol (a form of "des-methyl tocopherol") has received
much less attention in science. However, gamma-tocopherol presents
a variety of beneficial advantages over alpha-tocopherol in various
considerations. In one particular regard, gamma-tocopherol has been
characterized to exhibit much more potent anti-oxidant qualities,
resulting in a unique anti-inflammatory activity not shared with
the alpha-tocopherol. In addition, gamma-tocopherol is believed to
enhance outcomes of therapy when combined with certain other
bioactive agents or drugs. Antioxidants, and in particular, gamma
tocopherol is capable of preserving the elastase inhibitor capacity
of the lower respiratory tract fluid of mammals exposed to harmful,
chemical gases. Thus, it is believed that the direct delivery of
antioxidants such as vitamin E, and particularly, gamma tocopherol
directly to the airways of mammals, may reduce or treat the injury
resulting from burn and smoke inhalation, as well as other
pulmonary disorders.
[0010] Inhalation-based therapies have been extensively evaluated
as site-specific method to treat pulmonary disorders due to their
ability to rapidly and selectively deposit agents in the lung in
greater amounts than can be readily achieved by other methods [see:
Kuhn, R. J., Pharmacotherapy, Vol. 22: pp. 80S-85S (2002)].
Consequently, a variety of aerosolized compounds have been
researched and their aerosolization attempted, including
recombinant proteins, glutathione, and vitamins such as Vitamin E
[Hybertson, B. M., et al., Free Radic. Biol. Med., Vol. 18: pp.
537-542 (1995)]. However, these attempts have been largely
unsuccessful due to the substantial insolubility of these
therapeutic agents and potential therapeutic agents in carrier
systems that are suitable for use in aerosol therapeutic delivery
systems. For example, many of the pharmaceutical compounds,
vitamins, and biological agents that exhibit promise in the
treatment of pulmonary diseases, disorders and damage that result
from smoke inhalation are insoluble in water and other the carriers
for aerosol formulation and for use in nebulizers. Further, these
same compounds which exhibit potential therapeutic applicability in
the pulmonary region of patients are often soluble only in
oil-based solvents or compounds, and as such they are unable to be
aerosolized by the current nebulizer systems available and in use.
In the event that such oil-based formulations can be realized, and
they can be transformed into an aerosol by a nebulizer, the
exceedingly high flow rate required to aerosolize them, and the
resultant particle size of the aerosols makes the formulations
unsuitable for use in treatment. Consequently, not only are new
formulations necessary, but a new nebulizer design is required in
order to convert formulations of such therapeutic agents into
aerosols having the desired particle size in the desired range of 2
.mu.m to 12 .mu.m.
[0011] The present invention meets these needs by providing novel,
pharmaceutical compositions of tocopherols, such as gamma
tocopherol, and tocopherol derivatives which are demonstrated
herein to protect animals from cytotoxic injury and death,
pulmonary injury, as well as other injuries and disease conditions,
including inflammatory diseases, as well as methods and systems for
delivering these compositions by way of nebulizing such
water-insoluble drug formulations.
BRIEF SUMMARY OF THE INVENTION
[0012] In one aspect of the present disclosure, an apparatus for
nebulizing a non-aqueous liquid is described.
[0013] In accordance with another aspect of the present disclosure,
a nebulization system comprising a nebulizing nozzle capable of
nebulizing a composition comprising fatty acids or lipids is
described, wherein the nebulizing nozzle comprises an outer
gas-delivery tube and an inner microchannel delivery tube having a
central fluid channel, wherein the outer delivery tube is
concentrically configured around the inner delivery tube, such
concentric configuration forming an annular intermediate space
between the two tubes, and wherein the intermediate air space
between the two tubes is the free air opening value, the value of
which is the internal diameter of the outer gas-delivery tube minus
the total inside diameter of the inner microchannel delivery
tube.
[0014] In accordance with another aspect of the present disclosure,
a pulmonary drug delivery system capable of nebulizing a
composition comprising a water-insoluble or substantially
water-insoluble drug and a fatty acid or lipid is described,
wherein the system comprises a reservoir for containing the drug
composition; a reservoir for containing a propellant gas; a
mechanical control valve capable of regulating the flow of the
propellant gas and the drug composition; and a nebulizing nozzle
adapted to receive both the drug composition and the propellant
gas, wherein the nebulizing nozzle can produce aerosol droplets
having a particle size ranging from about 2 .mu.m to about 12 .mu.m
in median mass aerodynamic size.
[0015] In another aspect of the present disclosure, formulations
for nebulization of water-insoluble drugs are provided.
[0016] In another aspect of the present disclosure, methods of
delivering formulations of water-insoluble drugs via nebulizer are
provided.
[0017] In one aspect of the present disclosure, a pharmaceutical
kit for aerosol administration of a medicament is described.
[0018] In a further aspect of the present disclosure, methods for
preparing formulations for nebulization are described.
[0019] In yet another aspect of the present disclosure, the use of
antioxidants in combination with one or more fatty acids or lipids
in the preparation of a medicament comprising gamma tocopherol for
the treatment of pulmonary disorders is described.
[0020] In another aspect of the present disclosure, the use of
compositions comprising gamma-tocopherol are described for use in
reducing the levels of reactive oxygen species in the pulmonary
microvasculature of a patient.
[0021] In a further aspect of the present disclosure, the use of
alpha or gamma-tocopherol in the manufacture of a medicament for
the inhibition of lipid peroxidation in the pulmonary system of a
patient is described.
[0022] In further aspects of the present disclosure, a non-aqueous
medicinal aerosol composition is described, the composition
comprising a therapeutically effective amount of an inhibitor of
c-GMP-specific phosphodiesterase (PDE) type IV or type V, or a
derivative, metabolite, solvate, prodrug, or polymorph thereof, and
a lipid or fatty acid.
[0023] In accordance with this aspect of the present disclosure,
the inhibitor of c-GMP PDE IV or PDE V may be sildenafil, or a
derivative, metabolite, prodrug, polymorph, or solvate thereof, in
a therapeutically-effective amount ranging from about 1 mg/kg/day
to about 1,000 mg/kg/day.
[0024] In accordance with further aspects of the present
disclosure, a drug nebulizing apparatus adapted for pulmonary
inhalation and delivery of aerosolized medicaments into a mammalian
patient is described, wherein the apparatus comprises a reservoir
containing a drug formulation and a nebulizing nozzle adapted to be
fit to a breathing circuit and further attached to a face mask,
wherein the nebulizing nozzle produces aerosolized droplets sized
for pulmonary, inhaled drug delivery of the drug formulation, and
wherein the nebulizing nozzle comprises a fluid micro-tube with an
air delivery tube capable of nebulizing a fluid from the micro-tube
into droplets into droplets sized for inhaled drug delivery. In
accordance with this aspect of the disclosure, the drug formulation
may be in the form of a mixture, a solution, a suspension, or an
emulsion, and the droplets may range in size from about 2 .mu.m to
about 10 .mu.m, including from about 2 .mu.m to about 5 .mu.m. In
further accordance with this aspect of the disclosure, the face
mask is typically capable of being fit to the patient's nose,
mouth, or both the nose and the mouth.
[0025] In accordance with yet another aspect of the present
disclosure, a face mask for use in a pressurized drug delivery
system is provided, the face mask comprising an at least partially
deformable body having a surface for placement against a face of a
patient and a nose bridge section formed in an upper section of the
body, a vent to the atmosphere outside of the face mask, and a
connector integral to a portion of the mask, the connector defining
a fluid pathway into an interior portion of the face mask and
constructed to receive, under pressure, an aerosolized drug
composition. In association with this aspect, the face mask may be
coupled to a nebulizer drug delivery system for delivering an
aerosolized drug through the face mask. Further, the body of the
face mask may include a bottommost surface for contacting the face
when the face mask is applied against the face and the body at
least partially deforms.
[0026] In one embodiment, the pharmaceutical compositions of the
present invention comprises gamma tocopherol measured at about 5%
to about 10% (w/v). In one embodiment, pharmaceutical compositions
of the present invention comprise gamma tocopherol measured at
about 10% to about 15% (w/v). In one embodiment, pharmaceutical
compositions of the present invention comprise gamma tocopherol
measured at about 15% to about 20% (w/v). In one embodiment,
pharmaceutical compositions of the present invention comprise gamma
tocopherol measured at about 20% to about 25% (w/v). In a preferred
embodiment, the pharmaceutical compositions of the present
invention comprise gamma tocopherol measured at about 10%
(w/v).
[0027] In one embodiment, methods of preparing pharmaceutical
compositions of the present invention further comprise adjusting
the osmolarity of the pharmaceutical composition to an osmolarity
in the range from about 200 to about 400 mOsmol/L. In one
embodiment, the osmolarity of the pharmaceutical composition is in
the range from about 240 to about 360 mOsmol/L or an isotonic
range.
[0028] In one embodiment of the present disclosure, the pH of the
tocopherol compositions, in particular the gamma tocopherol
composition, is in the range from about 2 to about 9, while in
other embodiments, the pH may be in the range from about 3 to about
8. The pH of the pharmaceutical composition may be adjusted to a
physiologically compatible range. For example, in one embodiment,
the pH of the pharmaceutical compositions described herein may be
in the range from about 3.0 to about 7.5. In another embodiment,
the pharmaceutical compositions of the present invention may have a
pH in the range from about 3.5 to about 7.5.
[0029] In one embodiment, storage of the gamma tocopherol
pharmaceutical composition is about three months, and the storage
temperature is in the range from about 15.degree. C. to about
30.degree. C., and more preferably in the range from about
20.degree. C. to about 25.degree. C. In another embodiment, storage
of the gamma tocopherol pharmaceutical composition is about six
months, and the storage temperature is in the range from about
15.degree. C. to about 30.degree. C., and more preferably in the
range from about 20.degree. C. to about 25.degree. C. In another
embodiment, storage of the gamma tocopherol pharmaceutical
composition is about twelve months, and the storage temperature is
in the range from about 15.degree. C. to about 30.degree. C., and
more preferably in the range from about 20.degree. C. to about
25.degree. C.
[0030] The present invention further includes kits comprising
tocopherol and tocopherol compositions in accordance with the
present disclosure. In accordance with further aspects of this
embodiment, the present disclosure contemplates and includes kits
comprising gamma tocopherol and .gamma.-tocopherol pharmaceutical
compositions of the present invention. In certain embodiments, such
kits may comprise one or more containers to store the gamma
tocopherol pharmaceutical compositions.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0031] The following figures form part of the present specification
and are included to further demonstrate certain aspects of the
present invention. The invention may be better understood by
reference to one or more of these figures in combination with the
detailed description of specific embodiments presented herein.
[0032] FIG. 1A. illustrates a cross-sectional view of a
micro-channel nebulizing nozzle in accordance with an aspect of the
present disclosure.
[0033] FIG. 1B. illustrates a partial cut-away view of the proximal
end of the nebulizing nozzle illustrated in FIG. 1A.
[0034] FIG. 1C illustrates an end-view of the proximal end of the
nebulizing nozzle of FIG. 1B, taken along line 1-1.
[0035] FIG. 2A illustrates a schematic side view of a portion of a
nebulizer in accordance with an aspect of the present
disclosure.
[0036] FIG. 2B illustrates a cross-sectional schematic view of the
nebulizer of FIG. 2A, taken along line 2-2.
[0037] FIG. 3 illustrates a multiple nebulizer nozzle
configuration, in accordance with an aspect of the present
disclosure.
[0038] FIG. 4 illustrates a schematic view of a nebulizing system
of the present disclosure for use in association with a mechanical
ventilator.
[0039] FIG. 5 illustrates a hand-held micro-channel nebulizing
system in accordance with an aspect of the present disclosure.
[0040] FIG. 6A illustrates a micro-channel nebulizer configured to
operate continuously using any standard air/oxygen source commonly
available for use in respiratory therapy, for use in association
with aspects of this disclosure.
[0041] FIG. 6B illustrates an exemplary manner in which the device
of FIG. 6A may be adapted to a semi-closed passive breathing
circuit.
[0042] FIG. 7A illustrates the effect of alpha- and
gamma-tocopherol on pulmonary gas exchange evaluated by measuring
the PaO.sub.2/FiO.sub.2 ratio.
[0043] FIG. 7B illustrates the effect of alpha- and
gamma-tocopherol on changes in lung lymph flow over time.
[0044] FIG. 7C illustrates the effect of alpha- and
gamma-tocopherol as delivered via the nebulizers of the present
disclosure on the pulmonary vascular permeability of a shunt
fraction of animals tested, as evaluated by measuring the pulmonary
shunt fraction (Qs/Qt) over time.
[0045] FIG. 7D illustrates the effect of formulations administered
in accordance with the present disclosure on peak airway pressures
over time.
[0046] FIG. 8 illustrates nebulized fatty acid droplets generated
using systems and methods of the present disclosure, impacted on a
counting slide.
[0047] FIG. 9 illustrates the exemplary gamma-tocopherol
concentration in lung tissue.
[0048] FIG. 10A illustrates the effect of gamma-tocopherol
(.gamma.-T) nebulization on PaO.sub.2/FiO.sub.2.
[0049] FIG. 10B illustrates the effect of gamma-tocopherol
(.gamma.-T) nebulization on pulmonary shunt fractions.
[0050] FIG. 11 illustrates the effect of .gamma.-T nebulization on
lung lymph flow (pulmonary transvascular fluid flux).
[0051] FIG. 12A illustrates the effect of .gamma.-T nebulization on
lung wet-to-dry ratio.
[0052] FIG. 12B illustrates the effect of .gamma.-T nebulization on
airway obstructions.
[0053] FIGS. 13A-13B illustrate the effect of .gamma.-tocopherol
(.gamma.-T) nebulization on malondialdehyde (MDA) level (A) and
3-nitrotyrosine level (B) in lung tissue.
[0054] FIGS. 14A-14B illustrate the effect of .gamma.-tocopherol
(T) nebulization on poly(ADP-ribose) polymerase activity in lung
tissue.
[0055] FIG. 15A illustrates the effect of .gamma.-tocopherol
(.gamma.-T) nebulization on IL-8.
[0056] FIG. 15B illustrates the effect of .gamma.-tocopherol
(.gamma.-T) on mRNA in lung tissue.
[0057] While the inventions disclosed herein are susceptible to
various modifications and alternative forms, only a few specific
embodiments have been shown by way of example in the drawings and
are described in detail below. The figures and detailed
descriptions of these specific embodiments are not intended to
limit the breadth or scope of the inventive concepts or the
appended claims in any manner. Rather, the figures and detailed
written descriptions are provided to illustrate the inventive
concepts to a person of ordinary skill in the art and to enable
such person to make and use the inventive concepts.
DEFINITIONS
[0058] The following definitions are provided in order to aid those
skilled in the art in understanding the detailed description of the
present invention.
[0059] The phrase "pharmaceutical composition" refers to a
formulation of a compound and a medium generally accepted in the
art for the delivery of the biologically active compound to
mammals, e.g., humans. Such a medium includes all pharmaceutically
acceptable carriers, diluents or excipients therefore.
[0060] The phrase "pharmaceutically acceptable carrier, diluent or
excipient" as used herein includes without limitation any adjuvant,
carrier, excipient, glidant, sweetening agent, diluent,
preservative, dye/colorant, flavor enhancer, surfactant, wetting
agent, dispersing agent, suspending agent, stabilizer, isotonic
agent, solvent, or emulsifier which has been approved by the United
States Food and Drug Administration as being acceptable for use in
humans or domestic animals.
[0061] The term "therapeutically effective amount", as used herein,
the dose administered to an animal, such as a mammal, in particular
a human, should be sufficient to prevent the targeted disease or
disorder, e.g., cancer, delay its onset, slow its progression, or
treat the disease or disorder (e.g., reverse or negate the
condition). One skilled in the art will recognize that dosage will
depend upon a variety of factors including the strength of the
particular composition employed, as well as the age, species,
condition, and body weight of the animal. The size of the dose will
also be determined by the route, timing, and frequency of
administration as well as the existence, nature, and extent of any
adverse side-effects that might accompany the administration of a
particular composition and the desired physiological effect.
[0062] "Biological active agent", as used herein, refers to any
amino acid, peptide, protein, or antibody, natural or synthetic,
which exhibits a therapeutically useful effect.
[0063] Such biologically active agents may include recombinant
proteins, enzymes, peptoids, or PNAs, as well as combinations of
such agents.
[0064] The phrase "pharmaceutically acceptable" or
"pharmacologically-acceptable" refers to compositions that do not
produce an allergic or similar unexpected reaction when
administered to a human or animal in a medical or veterinary
setting.
[0065] The compositions of the present invention may be prepared
for pharmaceutical administration by methods and with excipients
generally known in the art, such as described in Remington's
Pharmaceutical Sciences [Troy, David B., Ed.; Lippincott, Williams
and Wilkins; 21st Edition, (2005)].
[0066] "Treating" or "treatment" as used herein covers the
treatment of the disease or condition of interest, e.g., tissue
injury, in a mammal, preferably a human, having the disease or
condition of interest, and includes: (i) preventing the disease or
condition from occurring in a mammal, in particular, when such
mammal is predisposed to the condition but has not yet been
diagnosed as having it; (ii) inhibiting the disease or condition,
i.e., arresting its development; (iii) relieving the disease or
condition, i.e., causing regression of the disease or condition; or
(iv) relieving the symptoms resulting from the disease or
condition.
[0067] As used herein, the terms "disease," "disorder," and
"condition" may be used interchangeably or may be different in that
the particular malady or condition may not have a known causative
agent (so that etiology has not yet been worked out) and it is
therefore not yet recognized as a disease but only as an
undesirable condition or syndrome, wherein a more or less specific
set of symptoms have been identified by clinicians.
[0068] The term "water-insoluble" encompasses the terms sparingly
water-soluble, slightly or very slightly water-soluble, as well as
practically or totally water-insoluble compounds [see, Remington:
The Science and Practice of Pharmacy, vol. I, 194-195 (Gennaro,
ed., 1995)]. As used herein, a compound is water-insoluble for the
purposes of this invention if it requires at least 30 parts solvent
(e.g., water or saline) to dissolve one part solute (Id.). In
accordance with the present disclosure, the term "water-insoluble"
also encompasses oil- or lipid-soluble, as well as substantially
oil- or lipid soluble.
[0069] As used herein, the term "tocopherol" includes all such
natural and synthetic tocopherol or Vitamin E compounds having the
general structure as shown below, including all 10 isomers (five
tocopherols (.alpha.-, .beta.-, .gamma.-, .delta.-, .zeta..sub.2-)
and five tocotrienols (.alpha./.zeta..sub.1-, .beta./.epsilon.-,
.gamma.-, .delta.-, .eta.-), as well as combinations thereof,
including but not limited to .alpha.-tocopherol (alpha
tocopherol)(2,5,7,8-tetramethyl-2-(4',8',12'-trimethyldecyl)-6-chromanole-
), .beta.-tocopherol (beta-tocopherol), .gamma.-tocopherol (gamma
tocopherol), .delta.-tocopherol (delta tocopherol), and
.zeta..sub.2-tocopherol, as well as the d, l and dl [also referred
to equivalently as the (+), (-), and (.+-.) forms] enantiomers,
prodrugs, esters, solvates, and/or polymorphs thereof, or mixtures
of any of these compounds. These can be represented generally by
the structure (I) below,
##STR00001##
wherein, R.sub.1, R.sub.2 and R.sub.3 may each alternatively be
selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,
aralkyl, and heteroaralkyl moieties, any of which can be
unsubstituted or substituted with one or more of the same or
different substituents, which are typically selected from --X,
--R', .dbd.O, --OR', --SR', .dbd.S, --NR'R', --NR'R'R'.sup.+,
.dbd.NR', --CX.sub.3, --CN, --OCN, --SCN, --NCO, --NCS, --NO,
--NO.sub.2, .dbd.N.sub.2, --N.sub.3, --S(O).sub.2O.sup.-,
--S(O).sub.2OH, --S(O).sub.2R', --C(O)R', --C(O)X, --C(S)R',
--C(S)X, --C(O)OR', --C(O)O.sup.-, --C(S)OR', --C(O)SR', --C(S)SR',
--C(O)NR'R', --C(S)NR'R' and --C(NR)NR'R', where each X is
independently a halogen (F, Cl, Br, or I, preferably F or Cl) and
each R' is independently hydrogen, alkyl, alkenyl, or alkynyl;
wherein the wavy line, , represents that the stereochemistry at
this point may be in the form of the E- or Z-isomer; and ``
represents that the carbon-carbon bond may be a single or double
(olefinic) bond.
[0070] While practical size limits for the various substituent
groups will be apparent to those skilled in the art, generally
preferred are the alkyl, alkenyl, alkynyl, aryl, heteroaryl,
aralkyl, and heteroaralkyl moieties containing up to about 40
carbon atoms, more preferably up to about 20 carbon atoms and most
preferably up to about 10 carbon atoms.
[0071] As to any of the above groups that contain one or more
substituents, it is understood, of course, that such groups do not
contain any substitution or substitution patterns which are
sterically impractical and/or synthetically non-feasible. In
addition, the compounds of this invention include all
stereochemical isomers and mixtures thereof arising from the
substitution of these compounds.
[0072] Except as otherwise specifically provided or clear from the
context, the term "compounds" of the invention should be construed
as including the "pharmaceutically acceptable salts" thereof (which
expression has been eliminated in certain instances for the sake of
brevity).
[0073] The term "gamma-tocopherol" or ".gamma.-tocopherol", as used
herein, refers to
2,7,8-trimethyl-(4,8,12-trimethyltridecyl)chroman-6-ol, alternately
and equally acceptably referred to as d-gamma-tocopherol,
RRR-gamma-tocopherol, 2R, 4'R, 8'R-gamma-tocopherol, gamma-TOH,
gamma-T and gamma-TH, and having the CAS registry number
[54-28-4].
[0074] As used herein, the term "vitamin" refers to those compounds
which are considered to be nutrients required for essential
metabolic reactions within the body, and which are capable of
acting both as catalysts and participants in chemical reactions
within the body of mammals [Kutsky, R. J. Handbook of Vitamins and
Hormones. Van Nostrand Reinhold, N.Y. (1973); Kirk-Othmer,
Encyclopedia of Chemical Technology, Third Edition, John Wiley and
Sons, NY, Vol. 24:104 (1984)].
[0075] As used herein, the term "%" when used without qualification
(as with w/v, v/v, or w/w) means % weight-in-volume for solutions
of solids in liquids (w/v), % weight-in-volume for solutions of
gases in liquids (w/v), % volume-in-volume for solutions of liquids
in liquids (v/v) and weight-in-weight for mixtures of solids and
semisolids (w/w), such as described in Remington's Pharmaceutical
Sciences [Troy, David B., Ed.; Lippincott, Williams and Wilkins;
21st Edition, (2005)].
[0076] The terms "patient" and "subject", as used herein, are used
interchangeably and refer generally to a mammal, and more
particularly to human, ape, monkey, rat, pig, dog, rabbit, cat,
cow, horse, mouse, sheep and goat. In accordance with this
definition, lung surfaces or membranes described and referenced in
accordance with this disclosure refer to those of a mammal,
preferably a human or an animal test subject, such as a sheep.
[0077] The term "particle size" or "droplet size" is used in the
context of the present disclosure to refer to the average diameter
of particles, e.g., drug, lipid vesicles, in a suspension, and is
defined herein as the "Mass Median Aerodynamic Diameter" (MMAD)
which is referenced from an equivalent aqueous solution with a
density of 1.0 g/ml. As the fluid density decreases the real
droplet diameter/volume increases and conversely. Lung deposition
of a particle or droplet is primarily dependent on the MMAD of the
individual particle or droplet.
[0078] The term "spray dry" refers to a nebulization method that
allows for the evaporation of a solvent in part of the nebulized
formulation that results in a smaller droplet after a time when a
portion of the droplet has evaporated. Within the context of this
disclosure spray drying is an essential part of the operation of
handheld inhalers as well as the operation of pharmaceutical
manufacturing methods and column injection in analytical chemistry
application especially in gas chromatography.
[0079] The term "droplet" (or a tiny drop) is an individual
particle from a fine spray of liquid that was nebulized into an
aerosol.
[0080] "Insufflation" as used herein refers to blowing or inhaling
a medicinal powder, solution or formulation into the lungs of a
patient.
[0081] The term "hygroscopic" generally refers herein to a
condition whereby a nebulized droplet composition absorbs water
from the humidity in the droplet air stream and causes the droplet
to expand and grow causing the MMAD to increase in size.
[0082] The term "drug" as used in conjunction with the present
disclosure means any compound which is biologically active, e.g.,
exhibits or is capable of exhibiting a therapeutic or prophylactic
effect in vivo, or a biological effect in vitro.
DETAILED DESCRIPTION
[0083] One or more illustrative embodiments incorporating the
invention disclosed herein are presented below. Not all features of
an actual implementation are described or shown in this application
for the sake of clarity. It is understood that in the development
of an actual embodiment incorporating the present invention,
numerous implementation-specific decisions must be made to achieve
the developer's goals, such as compliance with system-related,
business-related, government-related and other constraints, which
vary by implementation and from time to time. While a developer's
efforts might be complex and time-consuming, such efforts would be,
nevertheless, a routine undertaking for those of ordinary skill the
art having benefit of this disclosure.
[0084] In general terms, Applicants have created nebulizer
assemblies, nebulizer fluid nozzle assemblies, and methods of using
such assemblies to deliver aerosols comprising one or more
water-insoluble or oil-soluble drugs to the pulmonary region of a
patient, for the purpose of delivering a therapeutically effective
amount of the drug to the pulmonary region so as to treat a disease
or disorder of the pulmonary system.
I. Nebulizer Assembly and Design
[0085] Turning now to the figures, FIG. 1A provides a nebulizer 10
in accordance with an aspect of the present disclosure. Nebulizer
fluid nozzle assembly 10 comprises an outer gas-delivery tube 12
and an inner microtube 14, each of which are concentrically
configured and positioned along a central axis 15, and each having
a free end at the proximal end 11 of the assembly 10. Intermediate
between the outer gas-delivery tube 12 and the inner microtube 14
is an annular intermediate space, which serves to convey the
nebulizing carrier gas via gas entry port 5 through the nebulizer
needle from the distal to the proximal end, whereupon it acts to
nebulize the liquid within inner microtube 14 into an aerosol
having an aerosolized particle size ranging from about 1 mm to
about 10 mm. In accordance with the present disclosure, the
particle size may be controlled by the spatial relationship of
tubes 12 and 14 to each other, and the flow rate of the carrier
gas. In typical operation, generally speaking, drug emulsion 19
enters the inner microtube 14 of nebulizing assembly 10 via port 4
at the distal end 13, is propelled down the fluid microchannel 18
via an appropriate gas, and is aerosolized into particles of the
desired size at the proximal end 11 of the assembly. Appropriate
gases for use in nebulization in accordance with the present
disclosure include oxygen, oxygen mixtures, nitrogen, argon,
helium, and purified air, as well as combinations of these gases in
various proportions (e.g., 70% oxygen, 30% nitrogen). While the
outer tube 12 and the inner microtube 14 are illustrated to be
substantially cylindrical in shape, those of skill in the art will
appreciate that they can also be of any appropriate shape,
providing such shape provides the same advantageous flow rates and
particle sizes as the illustrated arrangement. Additionally, while
the nebulizer 10 is illustrated to comprise inner and outer tubes
which are substantially blunt at the proximal end 11 of the
assembly, it is equally acceptable for either outer tube 12, inner
microtube 14, or both to have an outer lip comprising an annular
bevel (not shown), the angle of such a bevel ranging from about
5.degree. to about 88.degree..
[0086] FIG. 2A illustrates a cross-sectional view of the proximal
end 11 of nebulizer fluid nozzle assembly 10. As is apparent
therein, the spacing between the outer surface of inner-microtube
14 and the interior surface of outer gas-delivery tube 12 has a
diameter d.sub.3, and this has a value proportional to the outer
diameter D.sub.1 of inner-microtube 14. The intermediate air space
16 between the two tubes 12 and 14 may generally be described to be
the free air opening value, the value of which is the internal
diameter of the outer gas-delivery tube, D.sub.2, minus the total
outside diameter of the inner microchannel delivery tube, D.sub.1.
In accordance with aspects of the present disclosure, the outer gas
delivery tub 12 has an inner diameter D.sub.2 ranging from about
0.01 inches to about 0.05 inches, and the inner microchannel
delivery tube 14 has an outer diameter ranging from about 0.01
inches to about 0.04 inches. The value of the intermediate air
space between the two tubes ranges from about 0.000009 in.sup.2 to
about 0.001 in.sup.2, and more preferably from about 0.00000259
in.sup.2 to about 0.001 in.sup.2.
[0087] In accordance with further aspects of this disclosure, the
nebulizing nozzle 11 preferably has an air volume to nebulized
droplet volume ratio less than about 60,000:1, and more preferably
an air volume to nebulized droplet volume ratio less than about
15,000:1.
[0088] The micro-channel nebulizer assemblies described herein are
ideally suited for delivery of aerosols of formulation compositions
comprising oil, bound water, or emulsion formulations via
nebulization in single or multi-phase water-in-oil or oil-in-water
droplets formation. Accordingly, the nebulizer assemblies described
herein, such as nozzle assembly 10, are capable of generating
aerosol droplets having a particle size ranging from about 2 .mu.m
to about 20 .mu.m, preferably from about 2 .mu.m to about 12 .mu.m,
and more preferably from about 5 .mu.m to about 10 .mu.m. Such
aerosol droplet particle sizes include about 3 .mu.m, about 4
.mu.m, about 5 .mu.m, about 6 .mu.m, about 7 .mu.m, about 8 .mu.m,
about 9 .mu.m, about 10 .mu.m, about 11 .mu.m, about 12 .mu.m,
about 13 .mu.m, about 14 .mu.m, about 15 .mu.m, about 16 .mu.m,
about 17 .mu.m, about 18 .mu.m, about 19 .mu.m, and about 20 .mu.m,
as well as ranges between any two of these values, such as from
about 4 .mu.m to about 11 .mu.m.
[0089] An alternative aspect of the present disclosure is
illustrated in FIG. 3, wherein the proximal end 71 of nebulizing
nozzle 70 is shown. In accordance with this aspect, nebulizing
nozzle 70 comprises a plurality of individual micro-channel nozzles
74a-74f within a larger mass air channel/annular intermediate space
76 within outer air-delivery tube 72. As shown therein, the
plurality of individual micro-channel nozzles 74 is preferably
arranged around a central, longitudinal axis 75 extending through
outer tube 72. The plurality of individual micro-channel nozzles 74
can range from about 2 to about 12 nozzles, including 3, 4, 5, 6,
7, 8, 9, 10, 11 and 12 individual nozzles.
[0090] Nebulizing nozzles and assemblies, in accordance with the
present disclosure, may be made from any number of appropriate
materials, including but not limited to metals of any appropriate
gauge, including stainless steel, metal alloys, coated metals, and
polymeric materials, both natural and synthetic, as well as
co-polymers, homo-polymers, and ter-polymers of such polymeric
materials. Such polymeric materials may include polyethylenes,
polyurethanes, polyacryaltes, polystyrenes, polymethacrylates,
amino-based polymers, cellulosic-based polymers, phenolic-based
polymers, and combinations thereof. Coatings suitable for use with
the materials for manufacture of the nebulizers and nebulizing
assemblies described herein include both natural and synthetic
coating materials, and are optionally included on the assemblies so
as to enhance the flow rates, protect the outer surface of the
materials, or both.
[0091] Turning to FIG. 4, another aspect of the present disclosure
is illustrated generally therein. In the embodiment shown in FIG.
4, a nebulizer system in accordance with the present disclosure may
be adapted to work in conjunction with a mechanical ventilator 124
and has been configured to synchronize the cyclic nebulization of
drug droplet aerosol mist generation at the nebulizing nozzle 111A
and the injection of that drug mist at the beginning of the
inspiration cycle of the ventilator and inject the drug aerosol
mist directly into the ventilator circuit at the endotracheal tube
or tracheal tube 132, and directly into the pulmonary region of the
patient 114 via air tube 113. This configuration reduces material
loss (e.g., aerosol drug loss) in the ventilator circuit
tubing.
[0092] The nebulizer nozzle with a "pulse jet" cycle operation in
accordance with the present disclosure is configured using the
ventilator breath cycle signal output 118 signaling the nebulizer
controller 121, having cycle timers 119 and 120. The nebulization
of the drug 133 in a oil-based solution in accordance with aspects
of the present disclosure and contained within aspirator 129 is
commenced at the leading edge of the inspiration cycle and operates
for a short period of time at the beginning of inspiration. The
output of the nebulizer is synchronized with the inspiration start
trigger generated by the existing electronic control outputs of the
ventilator. With continued reference to FIG. 4, the ventilator
electronic output signal 118 then signals cycle timers 119 and 120
that control electric solenoid valves on the air supply 130 and
fluid supply 131 to the nebulizer nozzle 111A, respectively. The
nebulization process is substantially instantaneous and independent
of the tidal volume inspiration flow volume. The nebulized
particles are then injected directly into the ventilator air stream
113 ahead of the bulk of the inspired tidal volume. The output of
the nebulizing nozzle 111A may be connected directly into the end
of the ventilator tubing "Y" 123 at the ET tube connector. The
existing ventilator O.sub.2 mixer 125 output may be tapped via
connection 126 to provide the nebulizer atomizing gas supply, thus
providing nebulizer gas at the same O.sub.2 concentration as the
ventilator inspired oxygen concentration (FiO.sub.2). The nebulizer
cycle time, about 1.2 seconds, for the air flow is slightly longer
than the fluid flow cycle time, which is about 0.4 seconds. The
fluid flow may be delayed about 0.2 seconds from the start of the
cycle and ends before the end of the air flow. This dual cycle
clears the nozzle with air so the nozzle does not drip. Total drug
volume nebulized and delivered with each breath is a function of
the fluid cycle duration fluid viscosity/fluid micro-channel size
and the number of individual nebulizing nozzles.
[0093] The devices described and illustrated in FIG. 4 may be
readily manufactured from existing technologies and electronic
control configurations. The system uses existing signal generations
from the various mechanical ventilation devices as the controller
for the nebulizing device. The entire device simply adapts with the
existing mechanical ventilating equipment.
[0094] In a further aspect of the present disclosure, a portable
handheld nebulizing or inhaler device 50 is illustrated generally
in FIG. 5, comprising a lower equalization chamber portion 43 and
an upper pressure vessel portion 35, wherein the inhaler device 50
is capable of delivering metered doses of a medicament to a
patient. In this arrangement, the medicinal formulation 38
comprising the medicament, such as described in more detail below,
is retained within a bladder 37, separated from the propellant gas
36 contained within a pressure vessel 35. In accordance with this
structural relationship, the medicinal composition within bladder
37 is not emulsified with the propellant gas 36, thus allowing many
different propellant gases to be used in this configuration
including those gasses that may be difficult or impossible to
solubilize with the drug formulation. Bladder 37 is attached to
stem 45 by an appropriate attachment means, including mechanical
attachment means such as a clip, chemical attachment means such as
medically-acceptable glues and adhesives, and combinations thereof.
The drug formulation flow and gas flow is actuated mechanically.
Ports 39, 40 in the actuating valve assembly 41 allow the drug
composition 38 and the liquefied gas 36 to enter into the valve
assembly 41 and on to the nebulizing nozzle 52. Valve assembly 41
acts to not only transfer the drug composition from the bladder 37
to the nebulizing nozzle 52, but also acts to connect pressure
vessel 35 with The pressurized drug composition flows to the end of
the micro-fluid channel 49 and then is disrupted into aerosol mist
42 by the expanding propellant gas. In one aspect of the present
disclosure, it is preferable to use a liquefied propellant gas,
such as fluorinated hydrocarbon, in this configuration in order to
provide enough gas volume for multiple actuations and also to adapt
the gas vessel for a lower operating pressure. In accordance with
another aspect of the present disclosure, it is preferable to use a
non-liquified gas as the propellant gas, such as carbon dioxide,
nitrogen, or nitrous oxide. Preferably, in accordance with this
aspect, the gas is nitrogen gas, owing to its oderless and
tasteless characteristics, and its substantial insolubility in
product formulations.
[0095] In yet another aspect of the present disclosure, the
formulations and devices described herein can provide for medicinal
droplets that do not substantially evaporate. As such, a mist of
persistent and size stable droplets containing medicine can be
generated into a large vessel. In general, droplets in the 2-5
.mu.m are well known to be the optimum size for pulmonary drug
delivery and are well known to stay suspended in air with only
minor movement of air currents within the vessel. Importantly, in
this embodiment, an environment of medicinal droplets is produced
in a space and remains suspended and persist as a stable aerosol in
the air within the environment until inhaled. The method described
would be suitable for passive inhaled drug delivery for one or more
persons. More particularly, the embodiment would provide for drug
delivery for many persons as in a mass casualty situation where a
medicinal nebulizer could be shared with one or more persons. This
embodiment would be useful to minimize equipment and personnel for
mass inhalation pulmonary drug delivery.
[0096] Still, in yet another embodiment, the devices and methods
described herein may be useful in industrial applications where the
generation of an aerosol from a viscose fluid or emulsion is
required. These applications shall include pharmaceutical
manufacture, oil micro-droplet lubrication and fuel injection.
II. Nebulizing Mask Adaptations
[0097] In accordance with further aspects of the present
disclosure, and as illustrated in FIG. 6A and FIG. 6B, the
micro-channel nebulizer systems described above may be adapted for
continuous or semi-continuous nebulization and delivery of
inspirable droplets via a semi-closed face mask breathing circuit
62. The drug is contained in a flexible membrane within a
pressurized housing, which allows for the nebulizer to operate in
various positions unaffected by gravity. Humidified air is provided
by a separate standard mask humidifier which is existing equipment
for face mask delivery of oxygen. The mask and lipid nebulizer is
intended to be adaptable to existing equipment.
[0098] There are certain instances wherein it is desirable to
provide medicines by nebulization and subsequent inhalation by a
spontaneously breathing person. In this application, the face mask
may be coupled to a nebulizer drug delivery system for delivering
an aerosolized drug through the face mask, such that medicines are
continuously nebulized into a space that holds the medicinal
droplets suspended in a contained inspirable air flow which is in
proximity to the mouth and nose of the person.
[0099] The nebulizer continuously generates droplets into the air
volume in a tube connected to a close fitting mask coving the mouth
and nose of the person. As the person inhales the medicine/air
mixture, the medicine enters the lungs via the mouth and nose. On
exhale, a valve on the mask opens and allows the exhaled air to
flow out of the system.
[0100] FIG. 6A generally illustrates a micro-channel nebulizer
configured to operate continuously using any standard (e.g., 50
psi) air/oxygen source commonly available for use in respiratory
therapy, for use in association with this aspect of the disclosure.
A micro-channel nebulizing nozzle 1A may be pressed into a plastic
block containing air channels and a pressurized medicament chamber
49. Air/oxygen is supplied through a hose 45 connected to the ports
of the air channel 51. Air is allowed to flow through the channel
51 and on to the micro-channel air delivery tube 8. A portion of
the supplied air is regulated by a spring loaded flow valve 46 and
allowed to flow into and pressurize the medicine reservoir chamber
46 containing a medicine reservoir assembly 52. Another spring
loaded blow-off valve 47 regulates the pressure in medicament
reservoir 49. Excess pressure, as regulated by blow-off valve 47,
is vented through a port 48. The pressure in the reservoir exerts
pressure through ports 53 on the medicament bladder 37 and forces
the medicine in the bladder 38 into the micro-channel liquid
delivery tube 9.
[0101] FIG. 6B illustrates an exemplary manner in which the
continuous nebulizing device in FIG. 6A may be adapted to a
semi-closed passive breathing circuit. As illustrated therein, a
face mask 62 is included, wherein the face mask is contoured to
cover the nose and mouth of a patient. Humidified air 61 and
nebulized medicine is combined in a "Y" adapter 60. The humidified
air/medicinal mist is then fed into the face mask through port 63.
The person inhales the medicine/air mixture during the course of
normal respiration, and then exhales. The person's exhaled breath
may then be vented through one or more one-way valves 64 on mask
62, allowing the exhaled breath to exit the breathing circuit
without inhibiting the flow of nebulized medicine.
II. Formulation of Water-Insoluble Drugs
[0102] Compositions comprise water-insoluble or substantially
water-insoluble drugs or biologically active substances, as well as
oil-soluble or lipid-soluble drugs and biologically active
substances, in combination with one or more lipids or fatty acids,
including naturally-occurring fats and oils. In accordance with one
aspect of the present disclosure, the compositions can comprise a
water-insoluble or substantially water-insoluble drug or
biologically active substance and one or more fatty acids. In a
further aspect, the compositions can comprise a water-insoluble or
substantially water-insoluble drug or biologically active
substance, one or more fatty acids, and one or more surfactants. In
yet another aspect, the compositions suitable for use with the
nebulizer assemblies of the present disclosure can comprise a
water-insoluble or substantially water-insoluble drug or
biologically active substance, water, and at least one gelling
agent.
[0103] The compositions, according to the invention, may be
comprised of a drug itself or any mixture of a biologically active
substance with a solvent, and oil, a gelling agent, a carrier or
adjuvant, emulsifier, one or more different drugs, polymers,
excipients, coatings and combinations thereof. In essence, the
drug(s) or substances can be combined with any combination of
pharmaceutically acceptable components to be delivered to the
cellular surfaces within the pulmonary system by the method
described herein, e.g., pulmonary drug delivery. The drug(s) does
not have to be dissolved in a drug delivery medium solvent but can
be suspended or emulsified in a solvent or medium. The delivery
medium can take the form of an aqueous mixture, oil, or an organic
liquid. The delivery media solution can also comprise microspheres
or nanospheres of biologically active substances.
[0104] The compounds useful in the formulations and
therapeutically-useful compositions of the present invention can be
used in the form of pharmaceutically acceptable salts derived from
inorganic or organic acids. The term "pharmaceutically acceptable
salt" as used herein is meant to refer to those salts which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of humans and lower animals without undue
toxicity, irritation, allergic response and the like and are
commensurate with a reasonable benefit/risk ratio. Pharmaceutically
acceptable salts are well-known in the art. For example, P. H.
Stahl, et al. describe pharmaceutically acceptable salts in detail
in "Handbook of Pharmaceutical Salts: Properties, Selection, and
Use" (Wiley VCH, Zunch, Switzerland: 2002). The salts can be
prepared in situ during the final isolation and purification of the
compounds of the present invention or separately by reacting a free
base function with a suitable organic acid. Representative acid
addition salts include, but are not limited to acetate, adipate,
alginate, citrate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, camphorate, camphorsulfonate, digluconate,
glycerophosphate, hemisulfate, heptanoate, hexanoate, flimarate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate
(isethionate), lactate, maleate, methanesulfonate, nicotinate,
2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,
3-phenylpropionate, picrate, pivalate, propionate, succinate,
tartrate, thiocyanate, phosphate, glutamate, bicarbonate,
p-toluenesulfonate and undecanoate.
[0105] Also, the basic nitrogen-containing groups can be
quaternized with such agents as lower alkyl halides such as methyl,
ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl
sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long
chain halides such as decyl, lauryl, myristyl and stearyl
chlorides, bromides and iodides; arylalkyl halides like benzyl and
phenethyl bromides and others. Water or oil-soluble or dispersible
products are thereby obtained. Examples of acids which can be
employed to form pharmaceutically acceptable acid addition salts
include such inorganic acids as hydrochloric acid, hydrobromic
acid, sulphuric acid and phosphoric acid and such organic acids as
oxalic acid, maleic acid, succinic acid and citric acid.
[0106] Basic addition salts can be prepared in situ during the
final preparation, formulation, or purification of the
therapeutically-useful compounds, substantially water-insoluble
compounds described for use in aspects of this disclosure by
reacting a carboxylic acid-containing moiety with a suitable base
such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically acceptable metal cation or with ammonia or an
organic primary, secondary or tertiary amine. Pharmaceutically
acceptable salts include, but are not limited to, cations based on
alkali metals or alkaline earth metals such as lithium, sodium,
potassium, calcium, magnesium and aluminum salts and the like and
nontoxic quaternary ammonia and amine cations including ammonium,
tetramethylammonium, tetraethylammonium, methylamine,
dimethylamine, trimethylamine, triethylamine, diethylamine,
ethylamine and the like. Other representative organic amines useful
for the formation of base addition salts include ethylenediamine,
ethanolamine, diethanolamine, piperidine, piperazine and the
like.
[0107] Pharmaceutically acceptable salts of compounds which may be
used in formulations and systems described herein may be obtained
using standard procedures well known in the art, for example by
reacting a sufficiently basic compound such as an amine with a
suitable acid affording a physiologically acceptable anion. Alkali
metal (for example, sodium, potassium or lithium) or alkaline earth
metal (for example calcium or magnesium) salts of carboxylic acids
can also be made.
[0108] The formulations described for use herein may conveniently
be presented in unit dosage form and may be prepared by any of the
methods well known in the art of pharmacy. All methods include the
step of bringing into association one or more of the compounds
described herein, or a pharmaceutically acceptable salt or solvate
thereof ("active ingredient"), with a carrier which constitutes one
or more accessory ingredients. In general, the formulations are
prepared by uniformly and intimately bringing into association the
active ingredient with liquid carriers or finely divided solid
carriers or both and then, if necessary, shaping the product into
the desired formulation.
[0109] The compound or a pharmaceutically acceptable ester, salt,
solvate or prodrug thereof, such as a pharmaceutically acceptable
ester, salt, solvate, or prodrug of alpha-, beta-, or
gamma-tocopherol, can be mixed with other active materials that do
not impair the desired action, or with materials that supplement
the desired action, including other drugs against inflammatory
disease or lung injury.
[0110] As recited above, the present invention uses (1) a novel
nebulizing nozzle configuration that requires limited energy from a
compressed gas to cause droplet nebulization from a mass fluid into
droplets of a suitable size; and (2) a drug carrier(s) that is
based on essential fatty acid oils, lipids, gelled aqueous
solutions, emulsions, or combinations thereof that are harmlessly
absorbed by the lung tissues and are metabolized or expired.
[0111] Water-insoluble, substantially water-insoluble drugs, or
sparingly water soluble drugs suitable for use in the medicament
compositions of the present disclosure include, but are not limited
to, vitamins, antioxidants, anti-bronchitus agents, anti-pneumonia
agents, pulmonary anti-cancer agents, antianginal agents,
antihypertensive agents, and combinations thereof. In accordance
with one aspect of the present disclosure, the preferred drug is a
vitamin or a combination of two or more vitamins. Suitable vitamins
for use herein include but are not limited to Vitamin A, Vitamin B
(including Vitamin B.sub.12), Vitamin C, Vitamin D, Vitamin E,
Vitamin K3 (menadione; 1,4-dehydro-1,4-dioxo-2-methyl-naphthalene,
MNQ), retinol, riboflavin, niacin, ascorbic acid, .beta.-carotene,
and Coenzyme Q, including various derivatives of Coenzyme Q having
various isoprenoid side chains, including but not limited to QH,
QH.sub.2, Q.sub.3 and Q.sub.10.
[0112] In accordance with a further aspect of the present
disclosure, the formulation composition suitable for nebulization
using the assemblies described herein comprises Vitamin E, also
known as tocopherol, or a derivative, metabolite, ester, hydrate,
solvate, prodrug, or polymorph thereof. Tocopherols suitable for
use in the compositions herein include all those tocopherols within
the range of natural and synthetic compounds known by the generic
term Vitamin E. In accordance with one aspect of the present
disclosure, the tocopherol may be selected from the group
consisting of alpha-tocopherols, beta-tocopherols,
gamma-tocopherols, and delta tocopherols, as well as combinations
thereof. Tocopherols occur in a number of isomeric forms, the D and
DL forms being most widely available, all of which are suitable for
use herein. In accordance with one aspect of the present
disclosure, the tocopherols are preferably gamma-tocopherols.
[0113] Tocopherols suitable for use in accordance with these
aspects of the present disclosure may be obtained from plants, such
as by extracted from plants using known procedures, or prepared
synthetically using known organic synthetic methods, all of which
are in accordance with the present disclosure. Additionally, and as
suggested above, any of the forms or isomers of tocopherols and
their derivatives, eg. esters may be used according to the present
invention. Thus for example, gamma-tocopherol can be used as such,
or in the form of its esters such as gamma-tocopherol acetate,
linoleate, nicotinate or hemi succinate-ester, many of which are
available commercially or through known synthetic routes.
[0114] In accordance with further aspects and embodiments of the
present disclosure, the compositions may comprise antianginal
and/or antihypertensive drugs or biologically active agents which
are insoluble or substantially insoluble in water, or exhibit poor
water solubility (e.g., less than about 5 mg/mL). Compounds of
these types suitable for use herein include inhibitors of cAMP
(3',5'-cyclic adenosine monophosphate), cGMP (3',5'-cyclic
guanosine monophosphate), inhibitors of cGMP-specific
phosphodiesterase type IV (PDE IV), inhibitors of c-GMP-specific
phosphodiesterase type V (PDE V), drugs that may exhibit
anti-anginal effects, including drugs which exhibit therapeutic
effects on angina pectoris, and compounds which can enhance the
natriuretic effect of atrial natriuretic peptide (ANP). Suitable
examples of such drugs include but are not limited to atenolol,
amlodipine, diltiazem, eplerenone, naphthyriclin-4-one derivatives,
griseolic acid, dihydrodesoxygriseolic acid, derivatives of
griseolic acid and dihydrodesoxygriseolic acid, angiotensin
converting enzyme inhibitors, todalafil, vardenafil, ranolazine
[see, Tafreshi, M. J., et al., Ann. Pharmacother., Vol. 40(4): pp.
689-693 (2006)], sildenafil, sildenafil citrate (Viagra.RTM.;
Pfizer, Inc., New York), N-desmethyl sildenafil, and T-1032
(methyl-2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4-
,5-trimethoxyphenyl)-3-isoquionoline carboxylate sulfate; see:
Noto, T., et al., J. Pharm. Exp. Ther., Vol. 294(3): pp. 870-875
(2000)], as well as derivatives, solvates, prodrugs, and polymorphs
thereof. In accordance with one aspect of this embodiment, the drug
is sildenafil. Such drugs may be used in a therapeutically
effective amount ranging from about 1 mg/kg/d to about 1,000
mg/kg/d, as well as therapeutically effective amounts within this
range.
[0115] "Naturally-occurring fats and oils" as used herein refers to
the glyceryl esters of fatty acids (i.e., triglycerides) normally
found in animal or plant tissues, including those which have been
hydrogenated to reduce or eliminate unsaturation. Naturally
occurring fats and oils include vegetable oils such as linseed oil,
soybean oil, sunflower seed oil, corn oil, sesame oil, olive oil,
castor oil, coconut oil, palm oil, peanut oil, jojoba oil, neem
oil, and macadamia nut oil.
[0116] Selected naturally-occurring fats and oils suitable for use
in formulations of the present disclosure include, but are not
limited to, the following compounds: Adansonla Digitata Oil;
Apricot (Prunus armeniaca) Kernel Oil; Argania Spinosa Oil;
Argemone Mexicana Oil; Avocado (Persea gratissima) Oil; Babassu
(Orbignya olelfera) Oil; Balm Mint (Melissa officinalis) Seed Oil;
Bitter Almond (Prunus amygdalus amara) Oil; Bitter Cherry (Prunus
cerasus) Oil; Black Currant (Ribes nigrum) Oil; Borage (Borago
officinalis) Seed Oil; Brazil (Bertholletia excelsa) Nut Oil;
Burdock (Arctium lappa) Seed Oil; Butter; C12-18 Acid Triglyceride;
Calophyllum Tacamahaca Oil; Camellia Kissi Oil; Camellia Oleifera
Seed Oil; Canola Oil; Caprylic/Capric/Liuric Triglyceride;
Caprylic/Capric/Linoleic Triglyceride;
Caprylic/Capric/Myristic/Stearic Triglyceride;
Caprylic/Capric/Stearic Triglyceride; Caprylic/Capric Triglyceride;
Caraway (Carum carvi) Seed Oil; Carrot (Daucus Carota Sativa) Oil;
Cashew (Anacardium occidentale) Nut Oil; Castor Oil Benzoate;
Castor (Ricinus communis) Oil; Cephalins; Chaulmoogra (Taraktogenos
kurzii) Oil, Chia (Salvia hispanica) Oil; Cocoa (Theobrama cocao)
Butter; Coconut (Cocos nucifera) Oil; Cod Liver Oil; Coffee (Coffea
arabica) Oil; Corn (Zea mays) Germ Oil; Corn (Zea mays) Oil;
Cottonseed (Gossypium) Oil; C10-18 Triglycerides; Cucumber (Cucumis
sativus) Oil; Dog Rose (Rosa canina) Hips Oil; Egg Oil; Emu Oil;
Epoxidized Soybean Oil; Evening Primrose (Oenothera biennis) Oil;
Fish Liver Oil; Gevuina Avellana Oil; Glyceryl Triacetyl
Hydroxystearate; Glyceryl Triacetyl Ricinoleate; Glycolipids;
Glycosphingolipids; Goat Butter; Grape (Vitis vinifera) Seed Oil;
Hazel (Croylus americana) Nut Oil; Hazel (Corylus aveilana) Nut
Oil; Human Placental Lipids; Hybrid Safflower (Carthamus
tinctorius) Oil; Hybrid Sunflower (Helianthus annuus) Seed Oil;
Hydrogenated Canola Oil; Hydrogenated Castor Oil; Hydrogenated
Castor Oil Laurate; Hydrogenated Castor Oil Triisostearate;
Hydrogenated Coconut Oil; Hydrogenated Cottonseed Oil; Hydrogenated
C12-18 Triglycerides; Hydrogenated Fish Oil; Hydrogenated Lard;
Hydrogenated Menhaden Oil; Hydrogenated Milk Lipids; Hydrogenated
Mink Oil; Hydrogenated Olive Oil; Hydrogenated Orange Roughy Oil;
Hydrogenated Palm Kernel Oil; Hydrogenated Palm Oil; Hydrogenated
Peanut Oil; Hydrogenated Rapeseed Oil; Hydrogenated Shark Liver
Oil; Hydrogenated Soybean Oil; Hydrogenated Tallow; Hydrogenated
Vegetable Oil; Isatis Tinctoria Oil; Job's Tears (Coix
Lacryma-Jobi) Oil; Jojoba Oil; Kiwi (Actinidia chinensis) Seed Oil;
Kukui (Aleurites Moluccana) Nut Oil; Lard; Lauric/Palmitic/Oleic
Triglyceride; Linseed (Linum usitatissiumum) Oil; Lupin (Lupinus
albus) Oil; Macadamia Nut Oil; Macadamia Ternifolia Seed Oil;
Macadamia Integrifolia Seed Oil; Maleated Soybean Oil; Mango
(Mangifera indica) Seed Oil; Marmot Oil; Meadowfoam
(Limnanthesfragra alba) Seed Oil; Menhaden Oil; Milk Lipids; Mink
Oil; Moring a Pterygosperma Oil; Mortierella Oil; Musk Rose (Rosa
moschata) Seed Oil; Neatsfoot Oil; Neem (Melia azadirachta) Seed
Oil; Oat (Avena sativa) Kernel Oil; Oleic/Linoleic Triglyceride;
Oleic/Palmitic/Lauric/Myristic/Linoleic Triglyceride; Oleostearine;
Olive (Olea europaea) Husk Oil; Olive (Olea europaea) Oil; Omental
Lipdis; Orange Roughy Oil; Ostrich Oil; Oxidized Corn Oil; Palm
(Elaeis guineensis) Kernel Oil; Palm (Elaeis guineensis) Oil;
Passionflower (Passiflora edulis) Oil; Peach (Prunus persica)
Kernel Oil; Peanut (Arachis hypogaea) Oil; Pecan (Caiya
illinoensis) Oil; Pengawar Djambi (Cibotium barometz) Oil;
Phospholipids; Pistachio (Pistacia vera) Nut Oil; Placental Lipids;
Poppy (Papaver orientale) Oil; Pumpkin (Cucurbita pepo) Seed Oil;
Quinoa (Chenopodium Quinoa) Oil; Rapeseed (Brassica campestris)
Oil; Rice (Oryza sativa) Bran Oil; Rice (Oryza sativa) Germ Oil;
Safflower (Carthamus tinctorius) Oil; Salmon Oil; Sandalwood
(Santalum album) Seed Oil; Seabuchthorn (Hippophae rhamnoides) Oil;
Sesame (Sesamum indicum) Oil; Shark Liver Oil; Shea Butter
(Butyrospermum parkii); Silk Worm Lipids; Skin Lipids; Soybean
(Glycine soja) Oil; Soybean Lipid; Sphingolipids; Sunflower
(Helianthus annuus) Seed Oil; Sweet Almond (Prunus amygdalus
dulcis) Oil; Sweet Cherry (Prunus avium) Pit Oil; Tali Oil; Tallow;
Tea Tree (Melaleuca alternifolia) Oil; Telphairia Pedata Oil;
Tomato (Solanum lycopersicum) Oil; Triarachidin; Tiibehenin;
Tricaprin; Tricaprylin; Trichodesma Zeylanicum Oil; Trierucin;
Triheptanoin; Triheptylundecanoin; Trihydroxymethoxystearin;
Trihydroxystearin; Triisononanoin; Triisopalmitin; Triisostearin;
Trilaurin; Trilinolein; Trilinolenin; Trimyristin; Trioctanoin;
Triolein; Tripalmitin; Tripalmitolein; Triricinolein; Trisebacin;
Tristearin; Triundecanoin; Tuna Oil; Vegetable Oil; Walnut (Juglans
regia) Oil; Wheat Bran Lipids; Wheat (Triticum vulgare) Germ Oil,
and combinations of such fatty acid oils. In accordance with one
preferred aspect of this embodiment, the formulation composition
comprises Linseed Oil, which is also known as flaxseed oil, as well
as fatty acids found therein, including but not limited to
linolenic acid (LA), linoleic acid, oleic acid, stearic acid,
palmitic acid, alpha-linolenic acid (LNA), and gamma-linolenic acid
(GLA), any of which may be saturated or unsaturated as appropriate.
In accordance with further aspects of the present disclosure, the
formulations may also comprise hylauronic acid in an amount
suitable to minimize water from transpiring across the droplets
formed by the nebulizer.
[0117] Tocopherol, and in particular gamma tocopherol, compositions
of the present invention may further optionally comprise
preservatives. As used herein, the term "preservative" is intended
to mean a compound used to prevent the growth of microorganisms.
Such preservatives may be used in the tocopherol and gamma
tocopherol pharmaceutical compositions described herein at typical
concentrations in accordance with current pharmaceutical practices
as described. [see: The United States Pharmacopeia-National
Formulary, 29th Edition, (2006) Rockville, Md.; and, Remington's
Pharmaceutical Sciences, 21st Edition, Troy, D B, Ed. Lippincott,
Williams and Wilkins; (2005)]. Exemplary preservatives which may be
used with the compositions and systems of the present disclosure
include but are not limited to antifungal and antimicrobial
preservatives, such as benzoic acid, hydroxy benzoate and its
derivatives, butylparaben, ethylparaben, methylparaben,
propylparaben, sodium benzoate, benzalkonium chloride, benzethonium
chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol,
phenol, phenylethyl alcohol, phenylmercuric nitrate and thimerosal;
and antioxidants, such as ascorbic acid, ascorbyl palmitate,
butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorus
acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium
bisulfite, sodium formaldehyde sulfoxylate, and sodium
metabisulfite, as well as combinations of two or more of the these
preservatives.
[0118] Tocopherol and gamma tocopherol pharmaceutical compositions
and formulations of the present invention may further comprise one
or more pH modifying agents (buffering agents), in order to
maintain the pH of the composition in the desired range, e.g., from
a pH of from about 3.5 to about 8. pH modifying agents suitable for
use herein, include, but are not limited to, inorganic salts,
alkali earth and/or alkali rare earth hydroxides (e.g., NaOH, KOH,
or CsOH); carbonate or bicarbonate of any appropriate alkali or
alkali rare earth metal (e.g., Na.sub.2CO.sub.3, K.sub.2CO.sub.3,
NaHCO.sub.3, and KHCO.sub.3); phosphates, such as calcium hydrogen
phosphate, potassium metaphosphate, and potassium phosphate
monobasic; inorganic acids such as hydrochloric acid (HCl), and
organic acids such as acetic acid, citric acid, succinic acid,
fumaric acid, malic acid, maleic acid, glutaric acid or lactic
acid, as well as combinations thereof, any of which may be
water-soluble or water-insoluble, anhydrous or hydrated (e.g.,
dehydrate or semihydrate), as appropriate.
[0119] Others components which may be included in the
therapeutically useful compositions of the present disclosure
include but are not limited to binding materials (e.g., block
polymers, natural and synthetic rubber, polyacrylates,
polyurethanes, silicones and styrene-butadiene copolymers);
colorants, including but not limited to FD & C yellow # 6, FD
& C red # 40, FD & C blue # 2, and FD & C violet # 1,
as well as any other appropriate dye or combination of dyes; and,
UV inhibitors, to inhibit UV decomposition or isomerization of the
therapeutic compositions.
[0120] Other pharmaceutically acceptable formulation excipients may
also be used in accordance with the formulation compositions
described and disclosed herein, including but not limited to
coatings, stabilizers, emulsifiers, and the like, such as those
described in "The Handbook of Pharmaceutical Manufacturing
Formulations" [Niazi, S. K., CRC Press (2004)]. Additionally, and
in accordance with aspects of the present disclosure, one or more
surface active agents (surfactants) may be added to the formulation
compositions as appropriate. Although not required, incorporation
of a compatible surfactant can improve the stability of the instant
respiratory dispersions, increase pulmonary deposition and
facilitate the preparation of the suspension. Moreover, by altering
the components, the density of the particle or structural matrix
may be adjusted to approximate the density of the surrounding
medium and further stabilize the dispersion. Any suitable surface
active agent (surfactant) may be used in the context of the present
invention, provided that the surfactant is preferably
physiologically acceptable. Physiologically acceptable surfactants
are generally known in the art and include various detergents and
phospholipids, as discussed in more detail below. In accordance
with one aspect, it is preferred that the surfactant is a
phospholipid including, but not limited to, an extract of a natural
surfactant such as any number of known pulmonary surfactants,
including bovine- and calf-lung surfactant extracts, an egg
phospholipid, a vegetable oil phospholipid such as a soybean
phospholipid, or phosphatidylcholine. Preferably, in accordance
with aspects of the present disclosure, the surfactant suitable for
use with the therapeutic compositions of the present disclosure is
an extract of a natural surfactant, an egg phospholipid, or
combinations thereof. More preferably, the compositions may any one
or more of a number of biocompatible materials as surfactants, such
as surfactants comprising phospholipids.
[0121] In a broad sense, surfactants suitable for use in the
present invention include any compound or composition that aids in
the formation and maintenance of the stabilized
respiratory/pulmonary dispersions by forming a layer at the
interface between the particles of therapeutic compound (e.g.,
tocopherol or .gamma.-tocopherol) and the suspension medium. The
surfactant may comprise a single compound or any combination of
compounds, such as in the case of co-surfactants. In accordance
with certain aspects of the present disclosure, depending upon the
specific therapeutic composition or formulation, preferred
surfactants include but are not limited to those surfactants that
are substantially insoluble in the medium, nonfluorinated, and
selected from the group consisting of saturated and unsaturated
lipids, especially those that are obtained or extracted from
natural sources, nonionic detergents, nonionic block copolymers,
ionic surfactants, and combinations of such agents. It should be
emphasized that, in addition to the aforementioned surfactants,
suitable (i.e. biocompatible) fluorinated surfactants are
compatible with the teachings herein and may be used to provide the
desired stabilized therapeutic preparations.
[0122] Lipids, including phospholipids, from both natural and
synthetic sources are particularly compatible with the present
inventions and may be used in varying concentrations to form the
particle or structural matrix useful in the final, therapeutic
compositions. Generally compatible lipids include those that have a
gel to liquid crystal phase transition greater than about
40.degree. C. Preferably, and as described in more detail below,
the incorporated lipids are relatively long chain (i.e.
C.sub.16-C.sub.22) saturated lipids and preferably comprise one or
more phospholipids. Exemplary phospholipids useful in the disclosed
stabilized preparations of the present invention comprise egg
phosphatidylcholine, dilauroylphosphatidylcholine,
dioleylphosphatidylcholine, dipalmitoylphosphatidyl-choline,
disteroylphosphatidylcholine, short-chain phosphatidylcholines,
phosphatidylethanolamine, dioleylphosphatidylethanolamine,
phosphatidylserine, phosphatidylglycerol, phosphatidylinositol,
glyco lipids, ganglioside GM1, sphingomyelin, phosphatidic acid,
cardiolipin; lipids bearing polymer chains such as polyethylene
glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids
bearing sulfonated mono-, di-, and polysaccharides; fatty acids
such as palmitic acid, stearic acid, and oleic acid; cholesterol,
cholesterol esters, and cholesterol hemisuccinate. Due to their
excellent biocompatibility characteristics, phospholipids and
combinations of phospholipids and poloxamers are particularly
suitable for use in the stabilized dispersions disclosed herein.
With regard to biologically-compatible surfactants comprising
phospholipids, in accordance with certain aspects of the present
disclosure, it is preferable that such biologically-compatible
surfactants for use with the therapeutic compositions described
herein, especially those comprising tocopherol and/or
gamma-tocopherol, include those surfactants that are extracts of
natural surfactants, in particular pulmonary surfactants, and
synthetic synthesized mixtures of pulmonary surfactants in order to
mimic natural lung surfactant. Exemplary surfactants suitable for
use with the present compositions of tocopherol and
gamma-tocopherol include but are not limited to SURVANTA.RTM.
(beractant, available from the Ross Products Division of Abbott
Laboratories), a bovine-lung pulmonary surfactant comprising
phospholipids, neutral lipids, fatty acids, and
surfactant-associated proteins; SURFAXIN.RTM. (lucinactant,
available from Discovery Laboratories, Inc.); INFASURF.RTM.
(calfactant, available from Forest Pharmaceuticals, Inc., St.
Louis, Mo.), an extract of natural surfactant from calf lung which
includes phospholipids, neutral lipids, and hydrophobic
surfactant-associated proteins B and C(SP-B and SP-C);
CUROSURF.RTM. (poractant alpha, available from Chiesi Farmaceutici,
S.p.A., Parma, Italy), a non-pyrogenic pulmonary surfactant that is
an extract of natural porcine lung comprising polar lipids (mainly
phospholipids) and hydrophobic low molecular weight proteins
(surfactant associated proteins SP-B and SP-C); and ALVEOFACT.RTM.
(available from Boehringer Ingelheim Pharma, Ingelheim, Germany), a
natural bovine extract/surfactant comprising bovine lung
phospholipids; as well as the synthetic pulmonary surfactants
EXOSURF.RTM., VENTICUTE.RTM., ADSURF.RTM. (Pumactant.TM.), and
KL-4, all of which synthetic surfactants comprise the phospholipid
dipalmitoylphosphatidylcholine (DPPC).
[0123] Compatible nonionic detergents for use in the formulations
of the present disclosure comprise, without limitation, sorbitan
esters including sorbitan trioleate (SPAN.TM. 85), sorbitan
sesquioleate, sorbitan monooleate, sorbitan monolaurate,
polyoxyethylene (20) sorbitan monolaurate, and polyoxyethylene (20)
sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl
polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether,
glycerol esters, and sucrose esters. Other suitable nonionic
detergents include any of those which can be easily identified
using "McCutcheon's Emulsifiers and Detergents" (McPublishing Co.,
Glen Rock, N.J.) which is incorporated herein in its entirety.
Preferred block copolymers include but are not limited to diblock
and triblock copolymers of polyoxyethylene and polyoxypropylene,
including poloxamer 188 (PLURONIC.TM. F-68), poloxamer 407
(PLURONIC.TM. F-127), and poloxamer 338. Ionic surfactants such as
sodium sulfosuccinate, and fatty acid soaps may also be utilized.
In accordance with certain aspects and embodiments of the present
disclosure, the therapeutic compositions described herein may
comprise oleic acid or its alkali salt.
[0124] Those skilled in the art will further appreciate that, a
wide range of surfactants, including those not listed above, may
optionally be used in conjunction with the present invention.
Moreover, the optimum surfactant, or combination thereof, for a
given application can readily be determined by empirical studies
that do not require undue experimentation. It will further be
appreciated that, the preferred insolubility of any incorporated
surfactant in the suspension medium will dramatically decrease the
associated surface activity. As such, it is arguable as to whether
these materials have surfactant-like character prior to contracting
an aqueous bioactive surface (e.g. the aqueous hypophase in the
lung).
[0125] On a weight to weight basis, the instant formulations and
compositions of the therapeutic compositions comprising tocopherols
such as gamma-tocopherol may comprise varying levels of surfactant.
In this regard, the compositions and formulations described herein
which include one or more surfactants will preferably comprise
greater than about 0.1%, about 1%, about 5%, about 10%, about 15%,
about 18%, or even about 20% w/w % surfactant. In accordance with a
further aspect of the present disclosure, the therapeutic
compositions and formulations described herein may comprise greater
than about 25%, about 30%, about 35%, about 40%, about 45%, or
about 50% w/w surfactant. Still other exemplary embodiments of the
present disclosure will include therapeutic compositions and
formulations as described herein, further comprising one or more
surfactants, wherein the surfactant or surfactants are present at
greater than about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90% or even about 95% w/w.
[0126] In certain embodiments, the present invention employs a
novel composition comprising one or more lipids associated with at
least one drug. A lipid as referred to herein is a substance that
is characteristically insoluble in water and extractable with an
organic solvent. Lipids include, for example, the substances
comprising the fatty droplets that naturally occur in the cytoplasm
as well as the class of compounds which are well known to those of
skill in the art which contain long-chain aliphatic hydrocarbons
and their derivatives, such as fatty acids, alcohols, amines, amino
alcohols, and aldehydes. Of course, compounds other than those
specifically described herein that are understood by one of skill
in the art as lipids are also encompassed by the compositions and
methods of the present invention.
[0127] A lipid for use with the present disclosure may be naturally
occurring or synthetic (i.e., designed or produced by man).
However, a lipid is typically a biological substance. Biological
lipids are well known in the art, and include for example and
without limitation, neutral fats, phospholipids, phosphoglycerides,
steroids, terpenes, lysolipids, glycosphingolipids, glycolipids,
sulphatides, lipids with ether and ester-linked fatty acids and
polymerizable lipids, and combinations thereof.
[0128] A. Lipid Types
[0129] A neutral fat may comprise a glycerol and a fatty acid. A
typical glycerol is a three carbon alcohol. A fatty acid generally
is a molecule comprising a carbon chain with an acidic moeity
(e.g., carboxylic acid) at an end of the chain. The carbon chain
may of a fatty acid may be of any length, however, it is preferred
that the length of the carbon chain be of from about 2, about 3,
about 4, about 5, about 6, about 7, about 8, about 9, about 10,
about 11, about 12, about 13, about 14, about 15, about 16, about
17, about 18, about 19, about 20, about 21, about 22, about 23,
about 24, about 25, about 26, about 27, about 28, about 29, to
about 30 or more carbon atoms, and any range derivable therein.
However, a preferred range is from about 14 to about 24 carbon
atoms in the chain portion of the fatty acid, with about 16 to
about 18 carbon atoms being particularly preferred in certain
embodiments. In certain embodiments the fatty acid carbon chain may
comprise an odd number of carbon atoms, however, an even number of
carbon atoms in the chain may be preferred in certain embodiments.
A fatty acid comprising only single bonds in its carbon chain is
called saturated, while a fatty acid comprising at least one double
bond in its chain is called unsaturated.
[0130] Specific fatty acids include, but are not limited to,
linoleic acid, oleic acid, palmitic acid, linolenic acid, stearic
acid, lauric acid, myristic acid, arachidic acid, palmitoleic acid,
arachidonic acid ricinoleic acid, tuberculosteric acid,
lactobacillic acid. An acidic group of one or more fatty acids is
covalently bonded to one or more hydroxyl groups of a glycerol.
Thus, a monoglyceride comprises a glycerol and one fatty acid, a
diglyceride comprises a glycerol and two fatty acids, and a
triglyceride comprises a glycerol and three fatty acids.
[0131] A phospholipid generally comprises either glycerol or an
sphingosine moiety, an ionic phosphate group to produce an
amphipathic compound, and one or more fatty acids. Types of
phospholipids include, for example, phophoglycerides, wherein a
phosphate group is linked to the first carbon of glycerol of a
diglyceride, and sphingophospholipids (e.g., sphingomyelin),
wherein a phosphate group is esterified to a sphingosine amino
alcohol. Another example of a sphingophospholipid is a sulfatide,
which comprises an ionic sulfate group that makes the molecule
amphipathic. A phopholipid may, of course, comprise further
chemical groups, such as for example, an alcohol attached to the
phosphate group. Examples of such alcohol groups include serine,
ethanolamine, choline, glycerol and inositol. Thus, specific
phosphoglycerides include a phosphatidyl serine, a phosphatidyl
ethanolamine, a phosphatidyl choline, a phosphatidyl glycerol or a
phosphatidyl inositol. Other phospholipids include a phosphatidic
acid or a diacetyl phosphate. In one aspect, a phosphatidylcholine
comprises a dioleoylphosphatidylcholine (a.k.a. cardiolipin), an
egg phosphatidylcholine, a dipalmitoyl phosphalidycholine, a
monomyristoyl phosphatidylcholine, a monopalmitoyl
phosphatidylcholine, a monostearoyl phosphatidylcholine, a
monooleoyl phosphatidylcholine, a dibutroyl phosphatidylcholine, a
divaleroyl phosphatidylcholine, a dicaproyl phosphatidylcholine, a
diheptanoyl phosphatidylcholine, a dicapryloyl phosphatidylcholine
or a distearoyl phosphatidylcho line.
[0132] A glycolipid is related to a sphinogophospholipid, but
comprises a carbohydrate group rather than a phosphate group
attached to a primary hydroxyl group of the sphingosine. A type of
glycolipid called a cerebroside comprises one sugar group (e.g., a
glucose or galactose) attached to the primary hydroxyl group.
Another example of a glycolipid is a ganglioside (e.g., a
monosialoganglioside, a GM1), which comprises about 2, about 3,
about 4, about 5, about 6, to about 7 or so sugar groups, that may
be in a branched chain, attached to the primary hydroxyl group. In
other embodiments, the glycolipid is a ceramide (e.g.,
lactosylceramide).
[0133] A steroid is a four-membered ring system derivative of a
phenanthrene. Steroids often possess regulatory functions in cells,
tissues and organisms, and include, for example, hormones and
related compounds in the progestagen (e.g., progesterone),
glucocoricoid (e.g., cortisol), mineralocorticoid (e.g.,
aldosterone), androgen (e.g., testosterone) and estrogen (e.g.,
estrone) families. Cholesterol is another example of a steroid, and
generally serves structural rather than regulatory functions.
Vitamin D is another example of a sterol, and is involved in
calcium absorption from the intestine.
[0134] A terpene is a lipid comprising one or more five carbon
isoprene groups. Terpenes have various biological functions, and
include, for example and without limitation, vitamin A, coenyzme Q
and carotenoids (e.g., lycopene and 1-carotene).
[0135] B. Charged and Neutral Lipid Compositions
[0136] In certain embodiments, a lipid component of a composition
in accordance with the present disclosure may be uncharged or
primarily uncharged. In one embodiment, a lipid component of a
composition comprises one or more neutral lipids. In another
aspect, a lipid component of a composition may be substantially
free of anionic and cationic lipids, such as certain phospholipids
(e.g., phosphatidyl choline) and cholesterol.
[0137] In certain aspects, a lipid component of an uncharged or
primarily uncharged lipid composition comprises about 50%, about
55%, about 60%, about 65%, about 70%, about 75%, about 80%, about
85%, about 95%, about 96%, about 97%, about 98%, about 99% or 100%
lipids without a charge, substantially uncharged lipid(s), and/or a
lipid mixture with equal numbers of positive and negative
charges.
[0138] In other aspects, a lipid composition may be charged. For
example, charged phospholipids may be used for preparing a lipid
composition according to the present invention and can carry a net
positive charge or a net negative charge. In a non-limiting
example, diacetyl phosphate can be employed to confer a negative
charge on the lipid composition, and stearylamine can be used to
confer a positive charge on the lipid composition.
[0139] C. Making Lipids
[0140] Lipids can be obtained from natural sources, commercial
sources or chemically synthesized, as would be known to one of
ordinary skill in the art. For example, phospholipids can be from
natural sources, such as egg or soybean phosphatidylcholine, brain
phosphatidic acid, brain or plant phosphatidylinositol, heart
cardiolipin and plant or bacterial phosphatidylethanolamine. In
another example, lipids suitable for use according to the present
invention can be obtained from commercial sources. For example,
dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma
Chemical Co., dicetyl phosphate ("DCP") may be obtained from K
& K Laboratories (Plainview, N.Y.); cholesterol ("Chol") may be
obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol
("DMPG") and other lipids known to those of skill in the art may be
obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). In
certain embodiments, stock solutions of lipids in chloroform or
chloroform/methanol can be stored at about -20.degree. C.
Preferably, chloroform is used as the only solvent since it is more
readily evaporated than methanol, allowing for more expedient lipid
recovery.
[0141] D. Lipid Composition Structures
[0142] In one preferred embodiment of the invention, the drugs may
be associated with one or more lipids, instead of or in addition
to, the fatty-acid. In accordance with this aspect of the
disclosure, a drug associated with a lipid may be dispersed in a
solution containing a lipid, dissolved with a lipid, emulsified
with a lipid, mixed with a lipid, combined with a lipid, covalently
bonded to a lipid, contained as a suspension in a lipid, contained
or complexed with a micelle or liposome, or otherwise associated
with a lipid or lipid structure. A lipid or lipid/chimeric
polypeptide associated composition of the present invention is not
limited to any particular structure. For example, they may also
simply be interspersed in a solution, possibly forming aggregates
which are not uniform in either size or shape. In another example,
they may be present in a bilayer structure, as micelles, or with a
"collapsed" structure. In another non-limiting example, a
lipofectamine (Gibco BRL)-chimeric polypeptide or Superfect
(Qiagen)-chimeric polypeptide complex is also contemplated.
[0143] In accordance with certain aspects of the present
disclosure, a fatty acid- or lipid-containing composition may
comprise about 1%, about 2%, about 3%, about 4% about 5%, about 6%,
about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,
about 13%, about 14%, about 15%, about 16%, about 17%, about 18%,
about 19%, about 20%, about 21%, about 22%, about 23%, about 24%,
about 25%, about 26%, about 27%, about 28%, about 29%, about 30%,
about 31%, about 32%, about 33%, about 34%, about 35%, about 36%,
about 37%, about 38%, about 39%, about 40%, about 41%, about 42%,
about 43%, about 44%, about 45%, about 46%, about 47%, about 48%,
about 49%, about 50%, about 51%, about 52%, about 53%, about 54%,
about 55%, about 56%, about 57%, about 58%, about 59%, about 60%,
61%, about 62%, about 63%, about 64%, about 65%, about 66%, about
67%, about 68%, about 69%, about 70%, about 71%, about 72%, about
73%, about 74%, about 75%, about 76%, about 77%, about 78%, about
79%, about 80%, about 81%, about 82%, about 83%, about 84%, about
85%, about 86%, about 87%, about 88%, about 90%, about 91%, about
92%, about 93%, about 94%, about 95%, about 96%, about 97%, about
98%, about 99%, about 100%, or any range derivable therein, of a
particular lipid, lipid type or fatty acid, in combination with one
or more therapeutic components such as a drug, biologic agent, or
other therapeutic material disclosed herein or as would be known to
one of skill in the art. In a non-limiting example, a lipid
composition may comprise about 10% to about 20% neutral lipids, and
about 13% to about 84% of a tocopherol such as gamma-tocopherol,
and about 1% cholesterol. Thus, it is contemplated that lipid
compositions of the present invention may comprise any of the
lipids, lipid types or other components in any combination or
percentage range.
[0144] The compositions according to the present disclosure may
also comprise antibiotics as the drug, or in combination with one
or more drugs, e.g., in combination with tocopherol. Antibiotics
suitable for use according to the invention are selected from the
group including but not limited to amoxycillin, ampicillin,
penicillin, clavulanic acid, aztreonam, imipenem, streptomycin,
gentamicin, vancomycin, clindamycin, ephalothin, erythromycin,
polymyxin, bacitracin, amphotericin, nystatin, rifampicin,
tetracycline, coxycycline, chloramphenicol, and zithromycin.
[0145] Compositions according to the invention may also contain a
"gelling agent" in combination with the drug or biologically active
agent and lipid. The gelling agent may be selected from the group
including but not limited to hydroxyethyl cellulose (HEC),
hydroxymethylcellulose (HMC), Natrasol.RTM., pectines, agar,
alginic acid and its salts, guar gum, pectin, polyvinyl alcohol,
polyethylene oxide, cellulose and its derivatives, propylene
carbonate, polyethylene glycol, hexylene glycol sodium
carboxymethylcellulose, polyacrylates,
polyoxyethylene-polyoxypropylene block copolymers, pluronics, wood
wax alcohols, tyloxapol (a nonionic surfactant oligomer), proteins
and sugars.
IV. Therapeutic Treatment
[0146] The formulations of water-insoluble and substantially
water-insoluble compounds may be used, in combination with the
nebulizer systems described herein, in order to administer a
therapeutically effective amount of one or more drugs or biological
agents as an aerosolized mixture. Preferably, and in accordance
with an aspect of the present disclosure, the formulations
comprising one or more water-insoluble compounds and a lipid can be
administered using a nebulizer as described herein for the
treatment of one or more pulmonary diseases. Pulmonary diseases and
disorders which may be treatable using the compositions,
formulations, methods, and apparatus/systems of the present
disclosure include but are not limited to asthma; alpha-1
antitrypsin deficiency (AAT Deficiency); dust-related pulmonary and
lung diseases and disorders, including asbestosis; avian flu;
bronchitis, including acute bronchitis; bronchiectasis;
bronchopulmonary dysplasia (BPD); chronic cough; chronic
obstructive pulmonary diseases and disorders; the common cold;
chronic obstructive pulmonary disorder (COPD); croup; cystic
fibrosis (CF); emphysema; farmer's lung; influenza; hantavirus;
rhinitis (hay fever); histoplasmosis; interstitial lung disease;
legionellosis (Legionnaire's disease); lung cancer (including both
small cell, large cell and mixed small cell/large cell carcinoma);
lung damage resultant from inhalation of smoke and heat;
inflammation and lung damage resultant from inhalation of
chemicals; lymphangioleiomyomatosis (LAM); occupational lung
disease; pleurisy; pneumonia; pneumothorax; pulmonary embolus;
pulmonary fibrosis; pulmonary hypertension; respiratory distress
syndrome; respiratory syncytial virus (RSV); sarcoidosis; severe
acute respiratory syndrome (SARS); sleep apnea; and tuberculosis,
as well as two or more such diseases or disorders exhibiting
themselves simultaneously. In accordance with one embodiment of the
present disclosure, the preferred pulmonary disorder to be treated
is lung damage resultant from inhalation of heat and smoke. In
accordance with a further embodiment of the present disclosure, the
pulmonary disorder to be treated is bronchitis.
[0147] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the scope of the
invention.
EXAMPLES
Example 1
Comparative Example
[0148] Two ultrasound nebulizers, DeVilbiss.RTM. Ultra-Neb 99.RTM.
(available from Sunrise Medical, Respiratory Products Div.,
Somerset, Pa.) and Aeroneb-Pro.RTM. (available from Aerogen, Inc.,
now Nektar Therapeutics, Mountain View, Calif.) and an AirLife.TM.
jet nebulizer (Cardinal Health, Inc., Dublin, Ohio) were selected
for testing tocopherol nebulization. The viscose tocopherol
preparation (neat tocopherol) would not nebulize with these
devices, regardless of their manipulation or air flow adjustment.
Tocopherol was then dissolved into an essential fatty acid rich
flax seed oil (linseed oil) preparation (comprising a variety of
fatty acids, including linolenic acid, linoleic acid, oleic acid,
stearic acid, and palmitic acid) at a concentration of 8.3% w/w.
This much less viscose mixture was then introduced into the
selected nebulizers above and again tested. As before, the
tocopherol-fatty acid mixture could not be nebulized using these
commercially-available devices. It is apparent from these tests
that the existing, commercially available nebulizing apparatus are
not designed for, and are largely incapable of, nebulizing viscose
liquids into droplets of an appropriate size suitable for pulmonary
delivery.
Example 2
Measured Air Flow of Nebulizer Nozzles of the Present
Disclosure
[0149] Table 1 demonstrates the measured air flow of selected
micro-channel nozzle configurations whereby a selected fluid nozzle
14 is surrounded by an air delivery tube 12. Nebulizing air in this
example is delivered at 50 psi. The free air space surrounding the
fluid delivery tube 14 enclosed by the air delivery tube 12
provides for the calculated "SQ inch Neb Free Air Opening". The
actual nebulized air flow rate "Neb Air Flow cc/sec" is related to
the free air opening size however, the amount of air flowing
through a given free air space is also influenced by the resistance
offered by the surfaces of the delivery tubes 14 and 12 in contact
with the air and may not be linier in function.
TABLE-US-00001 TABLE 1 Approximate Air flow rates with various
nozzle combination configurations Fluid Neb Air SQ inch Neb *Neb
Air Gauge Size O.D. I.D. Free Air Flow Config. Fluid/Air "1 O.D."
"2 I.D." Opening cc/sec A 22/16 .0280 .0470 .00111920 175 B 22/18
.0280 .0330 .00023955 30 C 25/16 .0200 .0470 .00142079 267 D 25/18
.0200 .0330 .00054114 110 E 25/20 .0200 .0230 .00001013 40 F 27/16
.0160 .0470 .00153389 300 G 27/18 .0160 .0330 .00065424 140 H 27/20
.0160 .0230 .00021441 60 I 27/22 .0160 .0170 .00000259 15 J 30/16
.0120 .0470 .00162185 320 K 30/18 .0120 .0330 .00074220 110 L 30/20
.0120 .0230 .00030238 95 M 30/22 .0120 .0160 .00008796 45 *Air
pressure at 50 PSI
[0150] Table 2 demonstrates the fluid delivery rates of various
viscosity fluids though various fluid delivery tubes 14 (FIG.
1A-1C) at various fluid pressures. The actual fluid flow rate is
related to the delivery tube internal diameter size however, the
amount of fluid flowing through a given fluid opening is also
influenced by the resistance offered by the surfaces of the
delivery tube 14 in contact with the fluid as well as the surface
tension of the fluid to be delivered and may not be linier in
function. Configurations are based on the nozzle configurations
described in Table 1, above.
TABLE-US-00002 TABLE 2 Approximate Fluid Flow Rates at Various
Fluid Pressures With Various Fluid Nozzles. Inter- Fluid Fluid nal
Pres- Flow rate Flow Rate Flow Rate Con- Nozzle dia- sure 100% ETOH
0.9% Saline Flax Oil fig. G Size meter PSI cc/sec cc/sec cc/sec A,
B 22 .0160 5 0.125 0.110 0.0034722 A, B 22 .0160 10 0.333 0.300
0.0073529 A, B 22 .0160 20 0.435 0.400 0.0195744 A, B 22 .0160 30
1.000 0.857 0.0241935 C-E 25 .0100 5 0.0233 0.0221 C-E 25 .0100 10
0.050 0.0487 C-E 25 .0100 20 0.111 0.1071 C-E 25 .0100 30 0.200
0.1866 0.0038265 F-I 27 .0080 5 0.010 0.0092 F-I 27 .0080 10 0.023
0.0189 F-I 27 .0080 20 0.045 0.0396 F-I 27 .0080 30 0.68 0.0618
0.0023585 J-M 30 .0060 5 0.00336 0.003195 J-M 30 .0060 10 0.00698
0.006662 J-M 30 .0060 20 0.01376 0.013102 J-M 30 .0060 30 0.020027
0.019032 0.0006527
[0151] Table 2 demonstrates the measured air flow of selected
micro-channel nozzle configurations whereby a selected fluid nozzle
14 is surrounded by an air delivery tube 12. Nebulizing air in this
example is delivered at about 50 psi. The free air space 16
surrounding the fluid delivery tube 14 enclosed by the air delivery
tube 12 provides for the calculated "SQ inch Neb Free Air Opening".
The actual nebulized air flow rate "Neb Air Flow cc/sec" is related
to the free air opening size however, the amount of air flowing
through a given free air space is also influenced by the resistance
offered by the surfaces of the delivery tubes 14 and 12 in contact
with the air and may not be linier in function.
Example 3
[0152] In this example, and as illustrated in Table 3, the
preferred configurations derived from Tables 1 and 2 are
demonstrated. This is by way to show the preferred nebulizing air
flow rates of the combinations sited but does not limit the
arrangements of various combinations of fluid and air tube sizes
that achieve the preferred embodiment. The preferred free
nebulizing air open area ranges from about 0.000009 to 0.001 square
inches. Nebulizing air pressures can be lowered in instances were
there is a larger free air opening in order to bring the nebulizing
air flow rate into the preferred range of .gtoreq.100 cc/second.
Nebulizing air pressures, fluid pressures, micro-channel fluid tube
size and the viscosity of the fluid to be nebulized are selected
from a range of components of fluid delivery tubes 1 and air
delivery tubes 2 to achieve the preferred air and fluid flow rates
and the nebulized droplet size within the preferred air volume to
viscous liquid volume ratio of less than about 60, 000:1.
TABLE-US-00003 TABLE 3 Preferred Nozzle Configurations *Neb Air
Gauge Size Flow Config. Fluid/Air cc/sec B 22/18 30 D 25/18 110 E
25/20 40 H 27/20 60 K 30/18 110 L 30/20 95 M 30/22 45 *Air pressure
at 50 PSI
Example 4
[0153] Animals: Adult female sheep were cared for in the
Investigative Intensive Care Unit at the University of Texas,
Galveston Branch. The experimental procedure was approved by the
Animal Care and Use Committee of the University of Texas Medical
Branch. The National Institutes of Health and American
Physiological Society guidelines for animal care were strictly
followed. The Investigative Intensive Care unit is accredited by
The Association for the Assessment and Accreditation of Laboratory
Animal Care International.
[0154] Animal model. Sheep (30-40 kg) were surgically prepared, as
described by Enkhbaatar, P. K., et al. [Am. J. Physiol. Regul.
Inter. Comp. Physiol., Vol. 285(2): R366-R372 (2003)]. A Swan-Ganz
thermal dilution catheter (model 93A-1317-F, Edwards Critical Care
Division, Irvine, Calif.) was inserted through the right external
jugular vein for the measurement of the core body temperature to
evaluate blood gas and the fluid resuscitations. An arterial
catheter was inserted into the right femoral artery (16 gauge, 24
in., Intracath, Becton Dickinson, Sandy, Utah) for the measurement
of arterial blood gas. To evaluate changes in lung lymph flow, an
efferent lymph vessel from the caudal mediastinal lymph node was
cannulated (Silastic catheter 0.025-in ID, 0.047-in OD; Dow
Corning, Midland, Mich.) according to a modification of the
technique described by Staub and colleagues [Staub, N., et al., J.
Surg. Res., Vol. 19, pp. 315-320 (1975); Traber, D., et al., J.
Appl. Physiol., Vol. 54, pp. 1167-1171 (1983)]. After a 7-day
recovery period, the sheep were deeply anesthetized with halothane
and were given a burn (40% total body surface area [TBSA], third
degree) and inhalation injury (48 breaths of cotton smoke,
<40.degree. C.). After burn/smoke injury, all sheep were placed
on a ventilator with positive end-expiratory pressure set to 5 cm
H.sub.2O and tidal volume maintained at 15 mL/kg. The latter tidal
volume is equal to about 10 ml/kg in humans due to the large dead
space of sheep [Melo, V., et al., Anesthesiology, Vol. 97, pp.
671-681 (2002)]. All animals were given fluid resuscitation with
Ringer's solution strictly according to the Parkland formula (4
mL/kg % TBSA burned/24 hr). The experiment was continued for 48
hr.
[0155] Animal grouping: The animals were randomized into 3 groups
as follows: (1) a Vitamin E nebulization group (B&S, Vitamin E,
n=6). To achieve the desired particle size, 1 gram mixed
tocopherols [1000 mg Decanox.TM. MTS-90G (94 mg/g alpha tocopherol,
15 mg/g beta-tocopherol, 604 mg/g gamma-tocopherol, 201 mg/g delta
tocopherol for a total of 914 mg/g total mixed tocopherols),
purchased from Daniels Midland Co., Decatur, Ill.] was added to 11
grams of linseed oil (flax oil) and mixed for 3 hours to make an
8.3% (w/w) solution of tocopherol in linseed oil before starting
the nebulization, using a nebulizer as described with the present
disclosure. Nebulization was started 1 hour after the combined burn
and smoke inhalation injury, and repeated every 12 h. (2) A saline
nebulization group (B&S, Saline, n=6): injured and nebulized
with 15 mL saline at the same intervals as the vitamin E treatment.
(3) A sham group (Sham, n=5): cared for similarly to the other
groups, that is, given the same amount of fluid as burned animals,
placed on a ventilator, and studied for 48 h but not injured or
treated with saline or tocopherol.
[0156] Burn and smoke inhalation injury: The protocol followed was
similar to that described in the art [Kimura, R., et al., J. Appl.
Physiol., Vol. 64(3): pp. 1107-1113 (1988); Enkhbaatar, P., et al.,
Am. J. Physiol. Regul. Integr. Comp. Physiol., Vol. 285(2): pp.
R366-R372 (2003)]. Briefly, under induction of anesthesia with 10
mg/kg ketamine (Ketalar, Parke-Davis, Morris Plains, N.J.), a
tracheotomy was performed, and a cuffed tracheostomy tube (10-mm
diameter; Sheiley, Irvine, Calif.) was inserted. Anesthesia was
maintained with halothane. Using a Bunsen burner, a third-degree
flame burn of 20% of the total body surface area was made on 1
flank of the subject. Thereafter, inhalation injury was induced
while the sheep was in the prone position as described previously
[id.]. A modified bee smoker was filled with 50 g burning cotton
toweling and was connected to the tracheostomy tube via a modified
endothoracheal tube containing an indwelling thermistor from a
Swan-Ganz catheter. During the insufflation procedures, the
temperature of the smoke did not exceed 40.degree. C. The sheep
were insufflated with a total of 48 breaths of cotton smoke. After
smoke insufflation, another 20% total body surface area,
third-degree burn, was made on the contralateral flank.
[0157] Resuscitation protocol: The protocol for subject
resuscitation has been described previously in the art [id.].
Briefly, immediately following the injury, anesthesia was
discontinued and the animals were allowed to awaken and were
mechanically ventilated with a Servo ventilator (model 900C,
Simens-Elena, Solna, Sweden) throughout the next 48 h experimental
period. Ventilation was performed with a positive end-expiratory
pressure (PEEP) of 5 cm H.sub.2O and a tidal volume of 15 mL/kg.
The respiratory rate was set to maintain normocapnia. For the first
3 h post-injury, all animals received an inspired oxygen
concentration (FiO.sub.2) of 100% to expedite the removal of carbon
monoxide (CO); thereafter, the FiO.sub.2 was adjusted to maintain
the arterial oxygen saturation to be greater than 90%. These
respiratory settings allowed rapid carboxyhemoglobin clearance
after smoke inhalation.
[0158] During the experiment, fluid resuscitation was performed
with Ringer lactate solution following the formula (4 mL/% burn
surface area/kg body weight for the first 24 h and 2 mL/% burned
surface area/kg body weight per day for the next 48 hours). During
this experimental period, the animals were allowed free access to
food, but not to water, to allow accurate determination of fluid
balance.
[0159] Concurrent cutaneous burn and smoke inhalation (B&S)
injuries have gross evidence of lung oxidant injury. Additionally,
inflammatory blood cells, neutrophil granulocytes, infiltrate the
lung tissues causing edema, swelling and additional tissue damage
from products of the inflammatory cells. Neutrophil infiltrate is
an integral part of the formation or obstructive airway cast
formation following inhalation injury. Obstructive airway casts is
the major cause of pulmonary obstruction following inhalation
injury. Pulmonary obstruction reduces the flow of air in and out of
the lung and is the primary cause of death following inhalation
injury. Therefore, we hypothesized that direct lung insufflation by
nebulization of gamma-tocopherol (.gamma.-T), a potent reactive
oxygen and nitrogen scavenger, would attenuate this injury by
reducing oxidative stress. Additionally, flax seed oil as used in
the present examples is made up of about 69% short chain essential
fatty acids, which also have been shown to exhibit
anti-inflammatory properties, and was used as a carrier for an 8.3%
solution of mixed tocopherols.
[0160] Acute lung injury was induced in adult female sheep by smoke
inhalation (48 breaths of cotton smoke, <40.degree. C.) with
concurrent 40% TBSA 3rd degree cutaneous burn under deep
anesthesia. Sheep (35.4.+-.1.0 kg) were divided into 4 groups: 1)
sham (not injured, nebulized flax oil (FO)) n=4; 2) saline
(injured, nebulized saline, n=6); 3) FO (injured, nebulized FO,
n=4); and 4) gamma-tocopherol+FO (injured, nebulized FO-gT, n=4).
Insufflation by nebulization was started 1 h post-injury and 22 ml
FO with or without g-T (mixed natural tocopherols, 528 mg/48 h) was
continuously delivered into the pulmonary system for 48 hours using
the subject lipid nebulization system using the control system as
shown in FIG. 4 with a nebulizing nozzle in accordance with FIGS.
1A-1C, and configured as described in Table 1, Configuration H, of
Example 1.
[0161] Cardiopulmonary variables were unchanged in sham animals
receiving flax seed oil over 48 hours and flax seed oil with 8.3%
mixed tocopherols (FIG. 7A). Combined burn and smoke inhalation
injury caused severe pulmonary dysfunction evidenced by
deteriorated pulmonary gas exchange (FIG. 7A), increased pulmonary
vascular permeability (FIGS. 7B and 7C), and increased ventilatory
pressures (FIG. 7D) were observed. These changes were associated
with decreased lung gT levels. FO nebulization significantly
improved the pathological changes seen in the saline group (FIGS.
7A-D). Gamma-tocopherol addition further improved pulmonary
function (FIGS. 6A-D) along with 100-fold elevation in the lung
.gamma.-T concentrations (18.0.+-.7.6 vs. 0.017.+-.0.005 in saline
group).
[0162] Pulmonary administration by nebulization of flax seed oil
did not interfere with gas exchange or other pulmonary function
when administered at a rate of .ltoreq.0.5 cc per hour in uninjured
sham animals. All (6/6) saline nebulized control animals (FIG. 7A)
deteriorated into respiratory distress (ARDS defined as a blood
PO.sub.2 ratio to inspired O.sub.2 concentration of .ltoreq.200;
PO.sub.2:FiO.sub.2 or P/F Ratio.ltoreq.200) within 24 hours of the
combined burn and smoke inhalation injury. None (0/4) of the
animals receiving nebulized flax oil with 8.3% mixed tocopherols
administered at 0.3 to 0.5 cc/hour deteriorated into ARDS. All
physiological parameters (FIGS. 7A-D) were significantly improved
in the insufflated flax oil/tocopherol group as compared with the
saline nebulization group.
[0163] Histological examination of lung and burn wound tissues were
performed during animal sacrifice necropsy at 48 hours post injury.
Profound inflammatory cell infiltrate of neutrophils was noted in
the B&S Saline control group in both the lung and burn wound
tissues. Neutrophil infiltrate was noted in airway casts following
smoke inhalation injury in the B&S Saline control group. A
marked reduction in inflammatory cell neutrophil infiltrate was
noted in the flax (FO) group and the gamma-tocopherol+flax (FO-gT)
group. These observations along with the physiological measurements
demonstrate that inspired flax (FO) and the gamma-tocopherol+flax
(FO-gT) reduces the local and systemic inflammatory response
following a smoke inhalation injury.
Example 5
Comparison of Lipid Carrier Compositions
[0164] Two medicinal lipid compositions were prepared, and compared
using the techniques described in the examples above. The first
composition comprised tocopherol and flax seed oil as a carrier,
wherein the flax seed oil comprised at least five (5) fatty acids,
as shown in Table 4, below. The second, comparative composition
comprised tocopherol and a "special" purified fatty-acid mixture
(available from Nu-Chek Prep, Inc., Elysin, Minn.) having a high
(greater than about 50 wt. %) linolenic acid content. The
comparative compositions are shown in Table 4. Initial tests seem
to suggest that the use of the "special" mixture with the high
omega-3-linolenic acid content may be even more advantageously
beneficial in reducing inflammatory response in the lungs of a
patient, following lung injury.
TABLE-US-00004 TABLE 4 Comparison of standard Flax Seed oil and
Purified "Special" Oil from Nu-Chek Prep., Inc. Linolenic Linoleic
Oleic Stearic Palmitic Acid Acid Acid Acid Acid Oil (C-18) (C-18)
(C-18) (C-18) (C-16) Flax Seed Oil 47 wt. % 24 wt. % 19 wt. % 3 wt.
% 6 wt. % (Linseed) Purified 68 wt. % 29 wt. % 3 wt. % -- --
"Special" Oil
Example 6
Comparison of Lipid Carrier Compositions
[0165] In this example, nebulized fatty acid droplets (nebulized in
accordance with aspects and systems of the present disclosure) were
impacted on a counting slide and examined, as illustrated in FIG.
8. The pictured flattened, semi-hemispherical droplets in FIG. 8
are more than twice the diameter of the spherical droplets before
impact. Oils containing large amounts of linolenic and linoleic
fatty acids tend to "flow out" as these fatty acids are natural
wetting agents. The droplet population is within the target size
distribution of 1-5 .mu.m Mass Median Aerodynamic Diameter (MAMD).
The small in-flight droplet size creates imaging difficulties with
regard to depth of field at required magnifications for
visualization. It is worth noting that the difficulty in
measurement of droplets.ltoreq.5 .mu.m has been acknowledged by
many others in the art.
Example 7
Additional Aerosol Formulation Delivery Studies
Animals/Animal Model
[0166] Twenty-four adult female sheep were cared for, prepared, and
maintained as described in Example 4, above.
Experimental Design
[0167] The sheep were randomly assigned to one of the following
four groups: 1) Sham and nebulized with flax oil (FO) (not injured,
FO-nebulized, n=6); 2) Saline (injured, saline-nebulized, n=6); 3)
FO (injured, FO-nebulized, n=6); 4) .gamma.-T+FO (injured,
FO+.gamma.-T-nebulized, n=6). Sham animals received no injury but
were surgically prepared like treated animals, placed on a
ventilator, and given fluid resuscitation and nebulized with 24 ml
FO over 47 hr. Saline animals were nebulized with 24 ml of 0.9%
NaCl over 47 hr after injury. FO animals were nebulized 24 ml FO
over 47 hr after injury. .gamma.-T+FO animals were nebulized 24 ml
.gamma.-T+FO mixture solution (.gamma.-T:1220 mg) over 47 hr after
injury.
Aerosol Delivery & Material.
[0168] The novel, viscous lipid formulation nebulization nozzle and
control system described herein was adapted to a Siemans.RTM. 900c
servo ventilator (Siemans-Elema AB, Sweden), as shown and discussed
generally in relation to FIGS. 1-6B, above. Briefly, for purposes
of this particular experiment, the nebulizing nozzle (FIG. 1B) was
fabricated from hypodermic needle stock material with a center
fluid delivery tube and an outer air delivery tube, in accordance
with aspects of the present disclosure. For the purpose of
calibration, calibrated blood counting slides were waved through
the ventilator inspiratory airflow containing nebulized flax oil
formulations allowing droplets to impact onto the slide. The slides
were then observed under light microscopy (200.times.) and sized
visually and counted. The vast majority (100:1) of observed
impacted deformed and flattened droplets larger than 2 .mu.m and
smaller than 10 .mu.m in diameter were conservatively considered to
be in the 2-5 .mu.m spherical range.
[0169] The output end of the nebulizing nozzle is positioned in the
center of flow within the "Y" connector of the ventilator circuit
immediately adjacent to and directed toward the tracheostomy tube
connector. The nozzle is fed from an air/liquid flow control system
adapted to and controlled by the electronic output of the 900c
ventilator. The oxygen-air mixer blending the inspired air for the
ventilator is tapped to provide the nebulizing air supply providing
the nebulization air at the same FiO.sub.2 as the ventilator air
FiO.sub.2. The control cycle timers are programmed to provide 1.0
second of air flow and 0.4 seconds of fluid flow with each
inspiration cycle. The fluid flow and subsequent nebulization is
configured to occur in the first 0.6 seconds of inspiration thereby
providing the nebulized droplets at the beginning of the inspired
air flow into the lungs. This nebulizer configuration provides an
additional inspired air flow volume of 50 mL/second/breath which
was deducted from the total tidal volume.
[0170] Cold pressed, filtered flax seed oil (Spectrum Organic
Products LLC, Melville, N.Y.) was used alone and as a carrier for
8.3% solution w/w of mixed tocopherols (Decanox.TM. MTS-90G, a gift
from Dr. Brent Flickinger, Archer Daniels Midland Co., Decatur,
Ill.) which was measured to contain .gamma.-T (610 mg/g) and,
.alpha.-T (91 mg/g). The flax oil mixtures were sterile filtered
through a 0.22 .mu.m pore filter prior to use. Flax oil alone or
flax oil containing 8.3% Decanox.TM. was administered by continuous
pulse nebulization synchronized with the inspiration cycle at a
rate of about 0.45-0.5 ml/hour or 11-12 ml 24 hours. Direct lung
tissue .gamma.-T concentration measurements (FIG. 9) demonstrated
that the aerosolized material was deposited into the lung.
Measured Variables
[0171] Arterial and mixed venous blood samples were taken at
different time points for measurement of blood gases (IL GEM
Premier 3000 Blood Gas Analyzer; GMI, Minnesota).
PaO.sub.2/F.sub.iO.sub.2 ratio was measured to help assess
pulmonary gas exchange. The pulmonary microvascular fluid flux was
evaluated by measuring the lung lymph flow. Sheep were sacrificed
under deep halothane anesthesia 48 hr after injury. The right lung
was then removed, and a 1-cm-thick section was taken from the
middle of the lower lobe, injected with 10% formalin, and immersed
in formalin. Four tissue samples were taken at predetermined sites
for histological examination. Fixed samples were embedded in
paraffin, sectioned at 4 .mu.m, and stained with hematoxylin and
eosin. A pathologist without knowledge of the group assignments
evaluated the lung histology. Levels of airway obstruction were
obtained with a standardized protocol. Fifteen bronchi were
investigated, and the percentage of area obstructed by the cast was
estimated (0%-100%). The remaining lower one-half of the right
lower lobe was used for the determination of bloodless wet-to-dry
weight ratio. Pulmonary shunt fraction (Qs/Qt) was calculated using
standard equations.
.gamma.-Tocopherol Measurement
[0172] A modification of the method by Podda and colleagues [Podda,
M., et al., J. Lipid Res., Vol. 37, pp. 893-901 (1996)] was used
for .alpha.- and .gamma.-T analyses, as described previously
[Morita, N., et al., SHOCK, Vol. 25(3), pp. 277-282 (2006)].
Briefly, tissue (.about.50 mg) or plasma (100 .mu.L) was saponified
with alcoholic KOH, extracted with hexane, the extract dried under
nitrogen, the residue resuspended in 1:1 ethanol-methanol, then
injected into an HPLC system. Tocopherols were detected using an
electrochemical detector, and quantitated by comparison to
authentic standards. Tissue .gamma.-T levels were adjusted by the
wet and dry ratio to correct for the weight change caused by the
inflammation and edema.
Malondialdehyde Measurement
[0173] Malondialdehyde (MDA) concentrations were utilized to
estimate the lipid peroxidation in the lung and were measured as
thiobarbituric acid reactive material. Lung tissue MDA levels were
quantified with a commercially available assay (Northwest Life
Science Specialties, Vancouver, Wash.). The level of lipid
peroxides is expressed as MDA per milligram protein, measured using
a commercially available assay (Fluka BioChemika, Buchs,
Switzerland).
3-Nitotyrosine Measurement
[0174] 3-Nitrotyrosine concentration, an index of the nitrosylation
of proteins, was determined by enzyme-linked immunosorbent assay
(ELISA). After the study, samples of lung tissue were collected and
homogenates prepared after adding 2 mL of the 10.times. diluted
halogenations buffer (1:10; Cayman Chemical, Ann Arbor, Mich.)
containing 250 mM Tris-HCL (pH 7.4), 10 mM EDTA, and 10 mM
ethyleneglycol-bis(-aminoethylether)-N,N,N',N'-tetraacetic acid
(EGTA). The supernatant was obtained by centrifugation
(10,000.times.g at 4.degree. C.) for 15 min and the supernatant
used for assessment. We used the Hycut biotechnology 3-NT solid
phase ELISA (Cell Sciences Inc., Canton, Mass.) according to the
manufacturer's protocol.
Immunohistochemistry of PARP Activity
[0175] For the immunohistochemical detection of poly(ADP-ribose),
mouse monoclonal anti-PAR antibody (Calbiochem, San Diego, Calif.,
USA) (1:1000, overnight, 4.degree. C.) was used after antigen
retrieval. Secondary labeling was achieved by using biotinylated
horse anti-mouse antibody (Vector Laboratories, Burlingame, Calif.,
USA) (30 min room temperature). Horseradish peroxidase-conjugated
avidin (30 min, room temperature) and brown colored
diaminobenzidine (6 min, room temperature) was used to visualize
the labeling (Vector Laboratories, Burlingame, Calif., USA). The
sections were counterstained with hematoxilin (blue color).
[0176] The intensity of PAR staining of individual sections was
determined by a blinded experimenter according to a
semiquantitative PAR-positivity score from 1-10. (1: no staining,
2: light cytoplasmic staining, 3: few positive nuclei, 4: light
nuclear staining in approximately 10% of cells, 5: light nuclear
staining in approximately 25% of cells, 6: light nuclear staining
in approximately 50% of cells, 7: strong nuclear staining in
approximately 50% of cells, 8: approximately 75% of the nuclei are
positive, 9: approximately 90% of the nuclei are positive, 10: few
negative cells).
IL-8 and IL-6 mRNA Measurement
[0177] Lung tissue was excised at the time of sacrifice and
immersed in liquid N.sub.2. Total RNA is obtained using a
commercially available total RNA purification kit, Purescript.TM.
(Gentra Systems, Inc., Minneapolis, Minn.). Briefly 100 mg of the
freshly frozen lung was lysed and homogenized using a mortar and
pestle with 3 ml of lysis buffer containing EDTA, citric acid, and
SDS according to the manufacturer's protocol, except that the
homogenized tissue was incubated overnight at room temperature in
the lysis buffer. Precipitation buffer was added and incubated 10
min on ice to precipitate protein and DNA and centrifuged at 3,000
g. The supernatant was placed in 3 ml of isopropanol and
centrifuged at 3,000 g for 5 min. The pellet was washed with 3 ml
of 70% ethanol, centrifuged again and air-dried for 10 min. The
pellet was resuspended in DEPC-treated water. Total RNA was
quantitated spectrophotometrically at 260 nm. Quality of the
isolated RNA was controlled by measuring the ratio of 28 s/18 s
rRNA. Messenger RNA was isolated from the total RNA by the Straight
A's TM mRNA Isolation System (Novagen, Madison, Wis.) purification
procedure in which mRNA was first hybridized to oligo dT coupled
magnetic beads, washed, and then eluted to obtain polyadenylated
mRNA according to the manufacturer's protocol. First strand cDNA
was synthesized by reverse transcription of the mRNA samples using
MMLV-derived reverse transcriptase (Perkin Elmer, Branchburg, N.J.)
and random hexamers for priming according to standard techniques
[Sambrook, J., Fritsch, E., and Maniatis, T., "Molecular Cloning: A
Laboratory Manual.", Cold Spring Harbor Laboratory Press, New York:
(1989)]. The cDNA was then used as a template for real-time PCR.
Primers and probes were designed using a commercial online primer
design program (Biosearch Technologies, Inc., Novato, Calif.)
(Table 5) and purchased from the same company. QPCR was performed
with a Rotor-Gene.TM. 3000 (Corbett Research, San Francisco,
Calif.).
TABLE-US-00005 TABLE 5 GAPDH, IL-8 and IL-6 Primers and Probes.
Probe Conc. Primer Sequence Ov GAPDH Forward 0.5 .mu.M 5'
CGCTCCCATGTTTGTG 3' Ov GAPDH Reverse 0.5 .mu.M 5' GAGGCATTGCTGACAA
3' Ov GAPDH Probe 62.5 .mu.M 5' dTGGGCGTGAACCACGAGAAGTA TA 3' Ov
IL-8 Forward 0.5 .mu.M 5' GCAACCCTAGACTGCT 3' Ov IL-8 Reverse 0.5
.mu.M 5' CCAGTGAAGAATAAAGAAATCG 3' Ov IL-8 Probe 62.5 .mu.M 5'
TCACGAGTTCCTGTTAACTGTGC 3' Ov IL-6 Forward 0.5 .mu.M 5'
TTGAGGGAAATCAGGAAA 3' Ov IL-6 Reverse 0.5 .mu.M 5'
GCTGGAGTGGTTATTAGAC 3' OV IL-6 Probe 62.5 .mu.M 5'
TCATGGAGTTGCAGAGCAGTATC A 3'
[0178] The reaction mixtures consisted of dilutions of cDNA from
256 ng of RNA, primer and probe concentrations as indicated (Table
5), and subjected to amplification using a final, optimized
concentration of MgCl.sub.2, 0.375 U of Taq polymerase
(AmpliTaq.RTM., Perkin-Elmer) and 0.2 mM dTP's in a reaction volume
of 15 .mu.l. The mixtures were amplified for 40 cycles at a melting
temperature of 95.degree. C. for 10 min, an annealing temperature
of 55.degree. C. for 10 s, and extension at 60.degree. C. for 45 s.
The threshold amplifications (Ct) for each dilution, and reaction
efficiencies were determined for each analyte using Rotor-Gene.TM.
software (Corbett Research). The copy numbers were normalized
between samples using GAPDH copy numbers obtained by determination
of GAPDH copy number using an external standard constructed from
the v-erb gene. All results were expressed as copy numbers per
.mu.g of total RNA.
Statistical Analysis
[0179] Summary statistics of data are expressed as means
.+-.standard error of the mean. Statistical significance was
determined using a two-factor analysis of variance with repeated
measures. The two factors were treatment and time. Fisher's least
significant difference procedure with Bonferoni's adjustment for
number of comparisons is used for the multiple comparisons (or
post-hoc analysis). Effects and interactions were assessed at the
p<0.05 level of significance.
Results
[0180] All animals survived the 48 h experimental period after the
combined injury with 40% TBSA burn and smoke inhalation. There were
no statistically significant differences in the mean arterial
carboxyhemoglobin levels measured immediately after smoke exposure
between the saline, FO and .gamma.-T+FO groups (67.4%.+-.6%,
77.0%.+-.5% and 68% 7%, respectively). Since vitamin E may decrease
platelet adhesion, the clotting time was evaluated. The .gamma.-T
treatment did not result in a bleeding tendency in any of the
groups. The activated clotting time was 144.+-.14 s at baseline,
163.+-.3 s at 24 hr, and 160.+-.10 s at 48 hr in the .gamma.-T+FO
group and 158.+-.3 s at baseline, 177.+-.10 s at 24 hr, and
183.+-.12 s at 48 hr in the nebulized saline group.
[0181] The lung contains primarily .alpha.-T and relatively low
.gamma.-T concentrations in sheep. Lung .gamma.-T concentrations
were low, and as shown previously, burn and smoke inhalation injury
further reduced both .alpha.- and .gamma.-T concentrations in lung
tissue. However, the nebulization significantly increased .gamma.-T
concentrations in lungs of sheep in the .gamma.-T+FO group. No
increases were found in plasma .gamma.-T (data not shown)
documenting that the .gamma.-T administration is confined to the
lung.
[0182] Table 6 shows a comparison of effects of FO or .gamma.-T+FO
nebulization on pulmonary gas exchange (PaO.sub.2/FiO.sub.2 ratio
and Qs/Qt) and pulmonary transvascular fluid flux (lung lymph
flow). There was a significant decrease in PaO.sub.2/FiO.sub.2 and
an increase in pulmonary shunt fraction and lung lymph flow in the
saline group resulting from the combined burn and smoke inhalation
injury as compared with the sham group at 24, 36, and 48 hr. FO
nebulization had a tendency to attenuate the changes seen in
animals with the saline group. As compared with the FO group, the
use of nebulized .gamma.-T+FO significantly improved the
PaO.sub.2/FiO.sub.2 at 36 hr and in lung lymph flow at 24 hr and 48
hr. PaO.sub.2/FiO.sub.2 ratio (FIG. 10A) was markedly decreased in
animals that were nebulized with saline (injured) as compared with
sham animals (uninjured). Nebulization of .gamma.-T+FO attenuated
the decrease in this variable. Statistically significant
differences were observed at 24, 30, 36, 42 and 48 hr compared with
the saline group and at 30, 36, and 42 hr compared with FO group.
An increase in pulmonary shunt fraction (FIG. 10B) seen in the
saline group was significantly attenuated by FO nebulization at 48
hr and .gamma.-T+FO nebulization at 36, 42 and 48 hr after the
combined injury.
TABLE-US-00006 TABLE 6 Pulmonary Gas Exchange and Transvascular
Fluid Flux Results. Time (hours, h) 0 6 12 24 36 48
PaO.sub.2/FiO.sub.2 Sham 487 .+-. 14 575 .+-. 24 560 .+-. 19 575
.+-. 14 558 .+-. 15 560 .+-. 11 Saline 492 .+-. 16 467 .+-. 63 443
.+-. 35 174 .+-. 31* 117 .+-. 30* 81 .+-. 17* FO 486 .+-. 16 525
.+-. 28 416 .+-. 59 290 .+-. 65* 194 .+-. 40* 137 .+-. 24*
.gamma.-T + FO 490 .+-. 13 523 .+-. 41 440 .+-. 64 .sup. 415 .+-.
55*.sup..dagger. .sup. 349 .+-. 48*.sup..dagger..dagger-dbl. .sup.
270 .+-. 48*.sup..dagger. Qs/Qt Sham 0.19 .+-. 0.02 0.12 .+-. 0.01
0.15 .+-. 0.01 0.14 .+-. 0.01 0.13 .+-. 0.01 0.14 .+-. 0.01 Saline
0.19 .+-. 0.04 0.14 .+-. 0.03 0.15 .+-. 0.02 0.33 .+-. 0.05* 0.41
.+-. 0.07* 0.49 .+-. 0.07 FO 0.17 .+-. 0.09 0.14 .+-. 0.02 0.18
.+-. 0.03 0.24 .+-. 0.04 0.30 .+-. 0.06* .sup. 0.33 .+-.
0.06*.sup..dagger. .gamma.-T + FO 0.18 .+-. 0.01 0.14 .+-. 0.01
0.20 .+-. 0.03 0.23 .+-. 0.05 .sup. 0.23 .+-. 0.03.sup..dagger.
.sup. 0.26 .+-. 0.03.sup..dagger. Lymph Sham 2.8 .+-. 0.7 3.5 .+-.
0.6 3.1 .+-. 0.6 3.7 .+-. 1.4 2.8 .+-. 0.8 3.2 .+-. 0.7 Saline 4.8
.+-. 0.9 11.8 .+-. 1.8 19.6 .+-. 5.1 42.7 .+-. 6.0* 46.5 .+-. 4.1*
48.2 .+-. 1.9* FO 6.8 .+-. 2.0 9.4 .+-. 2.2 14.4 .+-. 3.4 .sup.
23.5 .+-. 7.5*.sup..dagger. .sup. 23.5 .+-. 7.1*.sup..dagger. .sup.
26.9 .+-. 5.9*.sup..dagger. .gamma.-T + FO 4.1 .+-. 1.17 9.9 .+-.
4.3 9.24 .+-. 3.3 .sup. 8.6 .+-. 2.8.sup..dagger..dagger-dbl. .sup.
9.1 .+-. 2.5.sup..dagger. 10.5 .+-. 2.2.sup..dagger..dagger-dbl.
Data are expressed as means .+-. SEM. *P < 0.05 vs. Sham.
.sup..dagger.P < 0.05 vs. Saline; .sup..dagger-dbl.P < 0.05
vs. FO.
[0183] Lung lymph flow, a characteristic of pulmonary transvascular
fluid flux, was markedly increased in injured, saline nebulized
animals compared with the sham group (FIG. 11). The lymph flow
began to increase 12 hr after the insult and a peak was observed at
42 hr. However, nebulization of .gamma.-T+FO reversed this increase
in pulmonary transvascular fluid flux and significant differences
were observed between .gamma.-T+FO and Saline groups at 18, 24, 30,
36, 42 and 48 hr, and .gamma.-T+FO and FO groups at 24 and 48 hr
after the combined injury. Lung bloodless wet-to-dry weight ratio,
a measure of lung water content, was significantly increased at 48
hr after insult in the saline group as compared with the sham group
(FIG. 12A). However, the nebulization of .gamma.-T+FO significantly
reduced this increase.
[0184] The airway obstruction score revealed a significant increase
in mean obstruction of bronchi (FIG. 12B) in the saline group as
compared with the sham group. Treatment with .gamma.-T+FO
nebulization significantly reduced the obstruction score.
[0185] FIG. 13A illustrates the effect of .gamma.-T+FO nebulization
on malondialdehyde concentration which is an index of lipid
peroxidation (ROS) in lung tissue.
[0186] Malondialdehyde concentration was significantly increased in
the saline group as compared with the sham group. Malondialdehyde
levels did not markedly increase in animals treated with
.gamma.-T+FO nebulization.
[0187] 3-Nitrotyrosine is a marker of nitrosative stress, resulting
from reactive nitrogen species (RNS) such as peroxynitrite. Burn
and smoke injury caused a marked increase in lung 3-nitrotyrosine
48 h after the insults. .gamma.-T+FO nebulization significantly
prevented the increase in 3-nitrotyrosine (FIG. 13B).
[0188] After burn and smoke injury, there was a marked increase in
poly (ADP-ribose) reactivity in the Saline and FO groups (FIGS.
14A-14B). Treatment with .gamma.-T+FO nebulization prevented this
increase in activity (FIG. 14A). FIG. 14B shows the PAR-positivity
score graph which quantified the degree of poly (ADP-ribose)
histochemical stain. Burn and smoke injury caused a significant
increase in lung poly (ADP-ribose) polymerase activity. However,
.gamma.-T+FO nebulization significantly prevented the increase in
lung poly (ADP-ribose) polymerase activity.
[0189] To determine the pro-inflammatory chemokines, IL-8 and IL-6
mRNA were measured in lung tissue (FIG. 15). Burn and smoke injury
caused a marked increase in lung IL-8 and IL-6 mRNA 48 hr after the
insults. .gamma.-T+FO nebulization prevented the increase in IL-8
and IL-6 mRNA (FIG. 15).
[0190] The invention has been described in the context of preferred
and other embodiments and not every embodiment of the invention has
been described. Obvious modifications and alterations to the
described embodiments are available to those of ordinary skill in
the art. The disclosed and undisclosed embodiments are not intended
to limit or restrict the scope or applicability of the invention
conceived of by the Applicants, but rather, in conformity with the
patent laws, Applicants intends to protect all such modifications
and improvements to the full extent that such falls within the
scope or range of equivalent of the following claims.
[0191] All of the methods, processes and/or apparatus disclosed and
claimed herein can be made and executed without undue
experimentation in light of the present disclosure. While the
methods, apparatus and processes of this invention have been
described in terms of preferred embodiments, it will be apparent to
those of skill in the art that variations may be applied to the
methods, apparatus and/or processes and in the steps or in the
sequence of steps of the methods described herein without departing
from the concept and scope of the invention. For example, while
objects of the present invention have been described as being in
specific spatial relationships such as "parallel to" and
"horizontal to", it is envisioned that such objects can also be at
a variety of angles (e.g., acute, obtuse, or oblique angles) with
respect to one another without departing from the scope of the
present invention. More specifically, it will be apparent that
certain features which are both mechanically and functionally
related can be substituted for the features described herein while
the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those skilled in the art
are deemed to be within the scope and concept of the invention.
[0192] Further, any documents to which reference is made in the
application for this patent as well as all references listed in any
list of references filed with the application are hereby
incorporated by reference. However, to the extent statements might
be considered inconsistent with the patenting of this invention
such statements are expressly not to be considered as made by the
applicant(s).
* * * * *