U.S. patent application number 09/729468 was filed with the patent office on 2002-08-08 for carbon dioxide enhancement of inhalation therapy.
Invention is credited to Knight, J. Vernon, Koshkina, Nadezhda, Waldrep, J. Clifford.
Application Number | 20020106330 09/729468 |
Document ID | / |
Family ID | 22614028 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020106330 |
Kind Code |
A1 |
Waldrep, J. Clifford ; et
al. |
August 8, 2002 |
CARBON DIOXIDE ENHANCEMENT OF INHALATION THERAPY
Abstract
The present invention provides a method of increasing the
deposition of aerosolized drug in the respiratory tract of an
individual or animal, comprising the step of administering said
aerosolized drug in an air mixture containing up to about 10%
carbon dioxide gas.
Inventors: |
Waldrep, J. Clifford; (The
Woodlands, TX) ; Knight, J. Vernon; (Houston, TX)
; Koshkina, Nadezhda; (Houston, TX) |
Correspondence
Address: |
Dr. Benjamin Adler
McGREGOR & ADLER, LLP
8011 Candle Lane
Houston
TX
77071
US
|
Family ID: |
22614028 |
Appl. No.: |
09/729468 |
Filed: |
December 4, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60169038 |
Dec 4, 1999 |
|
|
|
Current U.S.
Class: |
424/43 |
Current CPC
Class: |
A61K 48/00 20130101;
A61K 9/1271 20130101; A61K 9/0078 20130101; Y10S 977/926 20130101;
A61K 9/124 20130101; Y10S 977/773 20130101; A61P 43/00 20180101;
Y10S 977/801 20130101; A61P 11/00 20180101; Y10S 977/915 20130101;
A61K 9/127 20130101; A61K 9/1272 20130101; Y10S 977/907
20130101 |
Class at
Publication: |
424/43 |
International
Class: |
A61K 009/00 |
Claims
What is claimed is:
1. A method of increasing the deposition of aerosolized drug in the
respiratory tract of an individual or animal, comprising the step
of: administering said aerosolized drug in an air mixture
containing up to about 10% carbon dioxide gas.
2. The method of claim 1, wherein said air mixture contains 2.5%
carbon dioxide gas.
3. The method of claim 1, wherein said air mixture contains 5%
carbon dioxide gas.
4. The method of claim 1, wherein said air mixture contains 7.5%
carbon dioxide gas.
5. The method of claim 1, wherein said aerosol is administered for
a period of time from about 1 minute to about 30 minutes.
6. The method of claim 1, wherein said drug is aerosolized by a jet
nebulizer.
7. The method of claim 1, wherein said drug is a water soluble or
buffer soluble drug.
8. The method of claim 7, wherein said water soluble or buffer
soluble drug is selected from the group consisting of an
antibiotic, a muclolytic, a bronchodilator, a parasympathetic
agent, an enzyme and an anti-viral.
9. The method of claim 1, wherein said drug is an insoluble drug
delivered via a carrier.
10. The method of claim 9, wherein said carrier is selected from
the group consisting of a liposome, a slow release polymer and a
polycationic polymer.
11. The method of claim 10, wherein said liposome is a conventional
liposome or a sterically stabilized liposome.
12. The method of claim 11, wherein said conventional liposome is
formed from a lipid comprising a phosphatidylcholine or a
poly(ethylene glycol) modified phospholipid.
13. The method of claim 12, wherein said phosphatidylcholine is
dilauroylphosphatidylcholine.
14. The method of claim 11, wherein said sterically stabilized
liposome is formed from modified phospholipids.
15. The method of claim 14, wherein said modified phospholipid is
dimyristylphosphoethanolamine poly(ethylene glycol) 2000.
16. The method of claim 10, wherein said liposome carries a
lipophilic drug in a liposomal formulation.
17. The method of claim 16, wherein said lipophilic drug is
selected from the group consisting of amphotericin B, nystatin,
glucocorticoids, an immunosuppressive and an anti-cancer drug.
18. The method of claim 17, wherein said anti-cancer drug is
selected from the group consisting of camptothecin, camptothecin
derivatives and paclitaxel.
19. The method of claim 1, wherein said drug is selected from the
group consisting of therapeutic proteins, therapeutic peptides, DNA
genes, sense oligonucleotides, anti-sense oligonucleotides and
viral vectors.
20. The method of claim 19, wherein said DNA gene is
chloramphenicol acetyl transferase or p53.
21. The method of claim 19, wherein said DNA gene is delivered via
a polycationic polymer carrier or a cationic liposome.
22. The method of claim 21, wherein said polycationic polymer is
polyethylenimine.
23. The method of claim 22, wherein said polyethylenimine has a
nitrogen:phosphate ratio of about 10:1 to about 20:1.
24. The method of claim 23, wherein said polyethylenimine has a
nitrogen:phosphate ratio of 10:1.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This patent application claims benefit of priority of
provisional application, U.S. Serial No. 60/169,038, filed Dec. 4,
1999, now abandoned.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the fields of
pharmacology and drug delivery. More specifically, the present
invention relates to a method of using carbon dioxide gas to
increase pulmonary deposition of an aerosolized drug during
inhalation therapy.
[0004] 2. Description of the Related Art
[0005] Small particle liposome aerosol treatment consists of
lipid-soluble or water-soluble anti-cancer drugs incorporated into
liposomes, which are administered from aqueous dispersions in a jet
nebulizer (see U.S. Pat. No. 5,049,388). Aerosols of 1-3 .mu.m mass
median aerodynamic diameter, generated upon nebulization, enable
targeted delivery onto surfaces of the respiratory tract. The
deposited liposomes subsequently release drug locally within the
lung or into the blood circulation with delivery to extra-pulmonary
tissue.
[0006] If the drug is lipid soluble, it will associate with the
lipid molecules in a manner specific to the lipid employed, the
anti-cancer drug employed and possibly it may be modified further
by various soluble constituents which may be included in the
suspending aqueous medium. Such soluble constituents may include
buffering salts and possibly inositol to enhance the synthesis and
secretion of surfactant phospholipid in lung tissue and to minimize
respiratory distress already present or that which might result
from the aerosol treatment (7).
[0007] If the drug is water soluble, it may be incorporated by
appropriate procedures in aqueous vesicles that exist in concentric
spaces between lipid bilayers (lamellae) of the multilamellar
liposome. Unilamellar liposomes may be prepared; however, their
capacity to entrap either lipid-soluble or water-soluble drugs is
diminished since entrapment is restricted to one central vesicle.
Aerosol water droplets may contain one or more drug-liposomes.
Moreover, it is also possible to incorporate more than one drug in
a aerosol liposome treatment, either by mixing different
drug-containing liposomes, or by using liposomes wherein the drugs
have been combined and incorporated together into liposomes.
[0008] Nebulization shears liposomes to sizes readily discharged
from the nozzle of the nebulizer. Liposomes up to several microns
in diameter are typically sheared to diameters of less than 500 nm,
and may be considerably smaller than that depending on the
operating characteristics of the nebulizer and other variables.
Shearing of water-soluble drugs contained in liposomes will release
appreciable amounts of the water soluble compound, perhaps 50
percent. This is not a contraindication to their use, but it means
that two forms of the drug preparation is administered, and the
effect includes the therapeutic effect that would be produced by
both forms if either form had been given alone. Many other details
of liposome aerosol treatment are described in U.S. Pat. No.
5,049,388.
[0009] In general, the underlying objective of inhalation therapy
is the topical delivery of aerosolized particles of pharmaceutical
drugs into the central airways and to peripheral regions of the
respiratory tract. However, the deposition fraction of the inhaled
particles even for the optimal size range of 1-2 .mu.m mass median
aerodynamic diameter is only approximately 20%. Pulmonary
deposition of inhaled aerosols is influenced significantly by
particle size, hygroscopic properties and airway geometry (1,2).
The breathing pattern is also an important variable that determines
the deposition pattern of inhaled particles (1,2).
[0010] Specifically, breath holding markedly increases pulmonary
deposition due to increased residence time of particles within the
lung. This allows a longer period for gravity sedimentation to
occur especially in the small peripheral airways and to ensure that
the aqueous particles can equilibrate fully in the near 100%
humidity and reach their maximum size, which further enhances their
deposition (1,2). Computer simulations demonstrate that a
thirty-second breath holding maneuver in humans can increase the
deposition fraction 3.2 times. The physiological principle of this
effect is due to increased particle intake upon deep inspiration in
which the inhaled volume may be as much as 8-fold higher than the
amount inhaled with basal tidal breathing. This larger volume of
tidal breathing leads to penetration of particles to the furthest
recesses of the lung where airway diameters are smallest, and thus
deposition due to gravity and maximum particle size occurs with
greatest efficiency.
[0011] By extension of this physiological property, direct
utilization of factors which could increase the volume of inspired
air (containing aerosol particles) would subsequently markedly
increase the deposited fraction in the central airways and to an
even greater extent in the peripheral lung. Carbon dioxide
(CO.sub.2) is the most important natural regulator of respiration.
Carbon dioxide diffuses freely from the tissues into the blood
according to the existing pressure gradient. Increased levels of
carbon dioxide in the blood readily diffuse into the cerebrospinal
fluid where there is conversion into HCO.sub.3.sup.- and H.sup.+.
Central chemoreceptors on the ventral surface of the medulla
respond to increased H.sup.+ in the CSF and cause a compensatory
increase in ventilation (rate and tidal volume).
[0012] Investigators have utilized carbon dioxide inhalation to
manipulate ventilation in experimental animals and humans.
Inhalation of 5% carbon dioxide causes as much as 192% increase in
tidal volume (3). This increase is rapid and reaches a sustained
plateau throughout the duration of exposure (4). Once the carbon
dioxide exposure ceases, the changes in ventilation reverse within
minutes to basal level (4). Similarly, inhalation of 5% carbon
dioxide by humans results in a 3-fold increase in the minute volume
(5). Inhalation of 5% or 7.5% of carbon dioxide by normal humans
for two minutes resulted in increases in frequency of breathing by
6.7% and 19%, respectively, and increases in tidal volumes by 31%
and 52%, respectively, so that minute volumes were increased by 34%
and 75%, respectively (6). Longer exposures to these concentrations
would have produced even greater responses (5).
[0013] Camptothecin analogues and taxanes are chemical agents
currently being developed as chemotherapeutic agents (21,26). The
anticancer drugs, paclitaxel (PTX) and different camptothecin (CPT)
derivatives are clinically active in the treatment of a variety of
human tumors, including lung cancer. These drugs show beneficial
results in clinical trials when used as single agents or in
combination with other drugs (21). These drugs are given
systemically by oral or intravenous routes of administration; the
most effective route for paclitaxel is continuous intravenous
infusion (22,24) whereas lipophilic congeners of camptothecin
administered orally prove most effective.
[0014] The development of toxic side effects is often a major
limitation in such therapeutic regimens. Several subcutaneous human
cancer xenografts in nude mice (23) and in experimental murine
pulmonary metastasis (6) have been successfully treated using
liposomal formulations of camptothecin and 9-nitrocamptothecin
(9NC) administered by the aerosol route as an alternative method of
therapy. Pharmacokinetic studies in mice with camptothecin showed
that inhalation of liposomal camptothecin produced substantial drug
levels in the lungs and other organs, which cleared rapidly after
cessation of aerosol delivery (17). In spite of these levels,
aerosol delivery systems are generally only 15-20% efficient in
drug deposition (29, 30); thus increasing pulmonary deposition
would be advantageous.
[0015] Using these systemic routes of drug delivery, a certain
amount of drug egresses from the blood stream and localizes in the
respiratory tissue, but lungs are not the main organs for drug
deposition. The utilization of conventional liposomes as carriers
for these drugs does not improve the pulmonary deposition of drugs
administered by commonly used systemic routes (11,27). Nebulization
is a very effective route for target drug delivery to the
respiratory tract (17); e.g., camptothecin. Dogs with spontaneously
arising primary and metastatic lung tumors have been successfully
treated when new formulations of doxorubicin and PTX are delivered
via aerosolization (16). However in these instances, aerosols were
generated using normal air.
[0016] Gene delivery to different tissues has been accomplished
using both viral and nonviral vectors. Although the use of nonviral
vectors avoids the immunogenic response associated with viral
vectors, nonviral vectors, such as cationic lipids and polycationic
polymers, have not been associated generally with the high levels
of gene expression characteristic of viral vectors. However,
polyethyleneimine (PEI), a cationic polymer, is effective both in
tissue culture and in vivo (36). The protonable nitrogen on every
third nitrogen provides polyethyleneimine with a huge buffering
capacity. Polyethyleneimine can effectively traffic DNA to the
nucleus (37) and protect DNA against DNAse degradation (36). Both
linear and branched forms of polyethyleneimine have been shown to
produce high levels of transgene expression in various tissues such
as lung, brain, and kidney (39-41). Polyethyleneimine has also been
used to efficiently deliver DNA to tumors in vivo (42).
[0017] Aerosol delivery is a noninvasive way to deliver genes of
interest to the lungs and could potentially be used to treat
diseases such as lung cancer and cystic fibrosis. However, the
levels of transgene expression have not been very high due, in some
cases, to loss of DNA viability during nebulization (43). PEI can
protect the DNA during nebulization (44) and can result in higher
levels of transfection in the lung than most of the other cationic
lipids tested (44,45). PEI-mediated transfection is also resistant
to inhibition by lung surfactants (46).
[0018] Increased efficiency of drug deposition to the respiratory
tract by the inhalation route is achieved by several ways: 1)
changing the concentration of drug in the formulation used for
aerosolization (31); 2) using more efficient types of nebulizers
(32); 3) increasing the duration of treatment; or 4) changing the
breathing patterns (4). As previously stated, carbon dioxide is a
natural modulator of respiration. The inhalation of air containing
low concentrations of CO.sub.2 (from about 3-7%) caused similar
changes in breathing patterns and was tolerated well (13, 6). No
difference in breathing patterns was observed between inhalation of
5%CO.sub.2-in-air and moderate physical exercise in man (32).
Similar effects of 5%CO.sub.2-in-air may be obtained in man using
aerosol treatment. Thus utilization of CO.sub.2-enriched air for
nebulization as a modulator of inhalation therapy can result in
more effective pulmonary delivery of chemotherapeutic agents.
[0019] The prior art is deficient in the lack of a means of
enhancing the pulmonary deposition of an aerosolized drug during
inhalation therapy. The present invention fulfills this
longstanding need and desire in the art.
SUMMARY OF THE INVENTION
[0020] The present invention provides a method of increasing the
deposition of aerosolized drug in the respiratory tract of an
individual or animal, comprising the step of administering said
aerosolized drug in an air mixture containing up to about 10%
carbon dioxide gas. 2.5%, 5%, and 7.5% carbon dioxide
concentrations have been used herein. The aerosol may be
administered for 1 to 30 minutes or even longer. The administered
drug may be a soluble drug, an insoluble drug or a therapeutic
composition, e.g., oligonucleotide, gene, peptide, or protein, that
may be dissolved in solution and directly aerosolized with a jet
nebulizer or incorporated into a carrier such as liposomes, slow
release polymers or polycationic polymers prior to
aerosolization.
[0021] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention given for
the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which
will become clear, are attained and can be understood in detail,
more particular descriptions of the invention briefly summarized
above may be had by reference to certain embodiments thereof which
are illustrated in the appended drawings. These drawings form a
part of the specification. It is to be noted, however, that the
appended drawings illustrate preferred embodiments of the invention
and therefore are not to be considered limiting in their scope.
[0023] FIG. 1 shows the tissue distribution of camptothecin after a
30 min exposure to liposome aerosol generated with normal air
(solid) or with 5%CO.sub.2-enriched air (hatched). At the end of
treatment (30 min) organs from three mice per group are resected
and the drug content determined by HPLC. Mean values with SD are
calculated. P values for 5%CO.sub.2-air compared to normal air are
0.02, 0.13, 0.04, 0.04, 0.03, and 0/01 for lungs, liver, spleen,
kidney, blood and brain, respectively (Student's t-test,
two-tailed).
[0024] FIG. 2 shows the pulmonary concentration-time curve for
CPT-liposomes administered for 30 min. by aerosol generated with
normal air (O) or with 5%CO.sub.2-enriched air (.circle-solid.).
For each time point lungs from three mice are resected and the drug
content determined by HPLC. Mean values with SD are calculated.
[0025] FIG. 3 shows the pulmonary concentration-time curve for
PTX-liposomes administered for 30 min. by aerosol generated with
normal air (O) or with 5%CO.sub.2-enriched air (.circle-solid.).
For each time point lungs from three mice are combined and the drug
content determined by HPLC. Each experiment is repeated three times
and mean values with SD are calculated.
[0026] FIG. 4 shows the comparison of tissue paclitaxel levels in
the lungs of mice exposed to aerosols containing different
liposomal formulations. Equivalent levels of exposure to paclitaxel
are achieved in a 5% CO.sub.2-in-air aerosol of sterically
stabilized paclitaxel-liposomes prepared from
dimyristylphosphoethanolamine poly (ethylene glycol) 2000 as when
DLPC is utilized.
[0027] FIG. 5 shows the comparison between CAT expression in lung
by PEI-DNA aerosol generated using air or air containing 5%
CO.sub.2. One milligram of CAT plasmid was complexed with PEI at an
N:P ratio of 10:1 and the resulting complex aerosolized to mice for
30 min. The lungs are harvested after 24 h and the CAT assay is
performed as described. Values are means .+-.SD (n=6 mice per
group, P=0.001).
[0028] FIG. 6 shows the effect of percent CO2 on the efficiency of
PEI-DNA transfer to the lung by aerosol. Different percentages of
CO2-in-air are used with a fixed amount of CAT plasmid. The
complexes were aerosolized using 0%, 2.5%, 5%, 10% carbon dioxide
and control. Mice are sacrificed, the lungs harvested, and the CAT
assay was performed. Values are expressed as means .+-.SD.
[0029] FIG. 7 shows that the gene expression in lung by PEI-DNA
aerosol was dose dependent. Increasing doses of CAT plasmid were
aerosolized using 5% CO.sub.2-in-air at a fixed N:P ratio of 10:1.
There is an increase in both the total amount of DNA delivered and
the concentration of PEI-DNAdelivered. Mice were sacrificed after
24 h, the lungs are harvested, and the Cat protein is assayed.
Values are means .+-.SD (n=5 mice per group).
[0030] FIG. 8 shows the effect of N:P ratios on the efficiency of
PEI-DNA transfer to the lung by aerosol. Different PEI-DNA (N:P)
ratios are used with a fixed amount CAT plasmid (2 mg). The complex
is aerosolized using 5% CO.sub.2-in-air. Mice are sacrificed after
24 h, the lungs are harvested, and the CAT assay is performed.
Values are means .+-.SD (n=5 mice per group).
[0031] FIG. 9 shows the effect of N:P ratios on luciferase gene
expression in the lung. A fixed amount of luciferase plasmid (2 mg)
is delivered at different N:P ratios. The complexes are aerosolized
using 5% CO.sub.2-in-air. Mice are sacrificed 24 h after aerosol
delivery, lungs are harvested, and luciferase activity is
determined. Values are means .+-.SD (n=5 mice per group).
[0032] FIG. 10 shows the time course of transgene expression after
single PEI-DNA aerosol exposure.
[0033] In FIG. 10A mice are delivered an aerosol containing 2 mg of
CAT plasmid at a N:P ratio of 15:1 using 5% CO.sub.2-in-air. Mice
are sacrificed at different time points and the lungs are harvested
and immediately frozen. The CAT assay is performed after the last
time point. Values are means .+-.SD (n=5 mice per time point).
[0034] FIG. 10B shows the persistence of CAT expression using two
different N:P ratios. Both groups of mice (n=5 mice each per time
point per group) are delivered 2 mg of CAT plasmid at a 15:1 or
10:1 NPP ratio using 5% CO.sub.2-in-air. The time points for the
10:1 ratio are 1, 2, 3, and 6 days post aerosol exposure and for
the 15:1 ratio are 1, 3, 7, and 10 days post aerosol exposure.
[0035] FIG. 11 shows tissue distribution of transgene after single
PEI-DNA aerosol exposure. The same groups of mice are used as in
FIG. 9 (from the 10:1 group). Different tissues are harvested and
immediately frozen. The CAT protein is assayed after the last time
point. Values are means .+-.SD (n=5 mice per time point). Levels of
CAT in non-lung tissues in the aerosol-exposed group are not
different from the control tissues (P>0.1).
[0036] FIG. 12 shows the histological analysis of PEI-DNA
aerosol-treated lungs. Two milligrams of CAT plasmid is complexed
with PEI at a N:P ratio of 15:1 and the complex was aerosolized ot
five mice for 30 min using 5% CO.sub.2-in-air. Mice are sacrificed
24 h later and lungs are harvested and fixed in formalin. Thin
sections are stained with hemtoxylin and eosin (H&E).
[0037] FIG. 12A: bronchiole (control);
[0038] FIG. 12B: bronchiole (treated). Magnification
100.times..
[0039] FIG. 13 shows the inhibition of B16-F10 lung metastasis by
PEI-p53 aerosol delivery.
[0040] FIG. 13A: Tumor index was claculated by the formula: Tumor
index=lung weights.times.average grade for the group. Values are
means .+-.SD (n=10 mice per group).
[0041] FIG. 13B: Representative lungs from control, PEI-Lucand
PEI-p53 treated mice are presented (n=10 mice per group). Lungs
from PEI-treated group (not shown) are similar in shape, size, and
number of tumor foci to those shown for control and PEI-Luc-treated
groups. Data are representative of two separate experiments.
[0042] FIG. 13C: Lung weights of mice from different groups. Values
are means .+-.SD (n=10 mice per group).
DETAILED DESCRIPTION OF THE INVENTION
[0043] The present invention provides a method of increasing the
deposition of aerosolized drug in the respiratory tract of an
individual or animal, comprising the step of administering said
aerosolized drug in an air mixture containing up to about 10%
carbon dioxide gas. Preferred concentrations include 2.5%, 5% and
7.5% carbon dioxide gas. The aerosol may be administered for 1 to
30 minutes or even longer.
[0044] The instant invention is directed to the aerosol delivery of
a water soluble drug. Such a drug may be directly prepared as a
water solution or a buffered solution and directly aerosolized.
Representative water soluble drugs include antibiotics like
tobramycin and pentamidine; muclolytics like acetyl cytsteine;
bronchodilators like albuterol; parasympathetic agents like
ipratropium bromide; enzymes like DNase; and anti-virals like
ribavirin.
[0045] Alternatively, the instant invention may be used to deliver
an insoluble drug that is associated with a carrier prior to
aerosol delivery. Possible carriers include liposomes, slow release
polymers and polycationic polymers. Lipsomes are an especially
useful carrier for lipophilic drugs such as amphotericin B;
nystatin; glucocorticoids; immunosuppressives like CsA, FK506,
rapamycin or mycophenolate; and anti-cancer drugs like
camptothecin, camptothecin derivatives, and paclitaxel. The
liposomes may be formed from such lipids as the phospholipid
dilauroylphosphatidylcholine (DLPC) or they may be sterically
stabilized liposomes formulated with modified phospholipids such as
dimyristylphosphoethanolamine poly(ethylene glycol) 2000. Slow
release polymers, such as poly(lactic acid-co-glycolic acid)
(PLGA), or polycationic polymers, such as polyethyleneimine (PEI),
may be utilized.
[0046] The instant invention may also be applied to the delivery of
therapeutic proteins, therapeutic peptides, DNA genes, sense
oligonucleotides, anti-sense oligonucleotides, and viral vectors.
Representative examples of DNA genes are the chloramphenical acetyl
transferase gene (CAT) or the p53 gene. Preferably, these genes are
delivered via a polycationic polymer carrier such as
polyethylenimine. Cationic liposomes also may be utilized as
carriers. The polyethylenimine may have a nitrogen:phosphate ratio
from about 10:1 to about 20:1. In a preferred embodiment, the PEI
nitrogen:phosphate ratio is about 10:1.
[0047] The following definitions are provided. Terms not
specifically defined are meant to be interpreted as is customary in
the art.
[0048] As used herein, the term "aerosols" refers to dispersions in
air of solid or liquid particles, of fine enough particle size and
consequent low settling velocities to have relative airborne
stability (8).
[0049] As used herein, the term "liposome aerosols" refers to
aqueous droplets within which are dispersed one or more particles
of liposomes or liposomes containing one or more medications
intended for delivery to the respiratory tract of humans or animals
(9).
[0050] As used herein, the size of the aerosol droplets defined for
this application are those described in U.S. Pat. No. 5,049,338,
namely mass median aerodynamic diameter (MMAD) of 1-3 .mu.m with a
geometric standard deviation of about 1.8-2.2. However, with low
concentrations of 9-NC and possibly other camptothecin derivatives,
the mass median aerodynamic diameter may be less than 1 .mu.m, such
as 0.8 .mu.m. Based on the studies disclosed by the present
invention, the liposomes may constitute substantially all of the
volume of the droplet when it has equilibrated to ambient relative
humidity.
[0051] As used herein, the "Weibel Lung Model" refers to a
classification of the structure of the human lungs that recognizes
23 consecutive branchings of the airways of humans. The trachea is
labeled 0, bronchi and bronchioles extend through branches 16.
These portions of the airways contain ciliated epithelium and mucus
glands. Together they constitute the mucociliary blanket.
Branchings 17-23 compose the alveolar portion of the lung and do
not have a mucociliary blanket. Thus, particles deposited here are
not carried up the airway to be swallowed.
[0052] It is postulated herein that under controlled experimental
conditions of hypercapnia, deposition of inhaled drug particles
would greatly increase over levels observed during basal tidal
breathing conditions. The use of carbon dioxide gas/air mixtures to
drive continuous flow jet nebulizers could greatly increase the
efficiency of the drug dose delivered to the peripheral lung
(Weibel's generations 17-23). By analogy, this system could be
effectively utilized to increase the biological efficiency of
inhaled drugs. This concept could be theoretically employed with
any drug, gene, oligonucleotide, or protein/peptide formulation
(soluble, liposomal, crystalline, or polymer-based carrier such as
polyethylenimine) and any gas or air driven jet nebulizer
[0053] The current invention is primarily directed toward the use
of carbon dioxide gas to increase the depth and frequency of
breathing during inhalation therapy with as aerosolized drug to
result in increased minute volumes. The increased tidal lung volume
results in enhanced pulmonary deposition of the inhaled drug
particles, particularly in the lung periphery which may not be
fully ventilated at low levels of breathing. The increased minute
volume resulting from increased frequency and greater depth of
breathing both contribute to the increased minute volume.
[0054] Administering an aerosolized drug in an air mixture
containing up to about 10% carbon dioxide gas results in increased
deposition of the drug in the respiratory system, measurably
improving efficiency and therapeutic efficacy of the aerosol drug
delivery. Preferred concentrations include 2.5%, 5% and 7.5% carbon
dioxide gas. The aerosol may be administered for 1 to 30 minutes or
even longer. The enhancing effect of the carbon dioxide is evident
within 30 seconds. The respiratory effects of carbon dioxide are
transient and can be employed repeatedly.
[0055] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion.
EXAMPLE 1
[0056] Materials
[0057] PIX was obtained from Xechem (New Brunswick, N.J.). CPT was
obtained from Sigma (St. Louis, Mo.) and 9NC from ChemWerth
(Woodbridge, Conn.). Dilauroylphosphatidylcholine (DLPC) was
purchased from Avanti Polar Lipids (Alabaster, Ala.). DMSO was
purchased from Sigma (St. Louis, Mo.) and HPLC grade other organic
solvents were obtained from Fisher Scientific. Sterile water for
irrigation came from Baxter Healthcare Corporation (Deerfield,
Ill.).
[0058] ICR mice (7-8 weeks old) were obtained from Harlan-Sprague
Dawley (Indianapolis, Ind.) and housed in standard cages with food
and water provided ad libitum. Female C57BL/6 mice (8-9 weeks old)
and female Balb/C mice (5-7 weeks old) were obtained from
Harlan-Sprague Dawley (Houston, Tex.). All animal care was in
accordance with Baylor College of Medicine Institutional Animal
Care and Use Committee.
[0059] The bacterial chloramphenicol acetyl transferase gene (CAT,
p4119, Ref. 15) is primarily used as the reporter gene for
measuring transgene expression. The CAT gene is under the control
of human cytomegalovirus (CMV) early promoter/enhancer element. The
luciferase plasmid (pGL3, Promega, Madison, Wis.) modified by
insertion of the CMV promoter/enhancer element and the human growth
hormone polyadenylation sequence was a gift from Dr. Michael Barry
(Center for Cell and Gene Therapy, Baylor). All plasmids are
purified on Qiagen columns (Qiagen, Valencia, Calif.) and are
endotoxin free. The plasmids are quantitated by UV absorbance at
260 nm. Agarose gel analysis revealed th plasmids to be a mixture
of primarily supercoiled plasmid with a small amount of nicked
plasmid.
[0060] The plasmid containing the p53 gene was obtained from Dr. Y.
K. Fung (Children's Hospital, Los Angeles, Calif.). The p53 gene is
under the control of human cytomegalovirus (CMV) promoter/enhancer
element. The plasmid used as a control contains the firefly
luciferase (Luc) gene and was obtained from Dr. Michael Barry
(Baylor College of medicine). The plasmids were purified
commercially by Bayou Biolabs (Harahan, La.), were endotoxin free
and were quantitated using UV absorbance. Agarose gel analysis
revealed the plasmids to be primarily in the supercoiled form with
a small amount of nicked plasmid.
[0061] B16-F10 melanoma cell line was obtained from Division of
Cancer treatment and Diagnosis Center (DCTDC, NCE, Frederick, MD)
and cultivated in DMEM supplemented with 10% fetal calf serum. The
cell line has been shown to form tumors in the lung (15).
Twenty-five thousand B16-F10 cells in 200 .mu.l of media are
injected per mice via the tail vein of C57BL/6 mice. Lung
metastases are visually detected within 2 weeks after inoculation
of cells. The cells were used at passages 3-12.
EXAMPLE 2
[0062] Statistics
[0063] After performing one-way analysis of variance (ANOVA) to
compare the means, a two-tailed unpaired Student's t test was done.
A difference was considered significant if P.ltoreq.0.05.
EXAMPLE 3
[0064] Preparation of Liposomes
[0065] Stock solutions of DLPC, PTX and camptothecin are prepared
in t-butanol at 100, 10 and 1 mg/ml, respectively, using previously
described methods (17). Aliquots of paclitaxel and DLPC are mixed
at a weight ratio of 1:10. The camptothecin to DLPC weight ratio is
1:50. The drug-phospholipid mixture is then frozen in liquid
nitrogen and lyphilized overnight to a dry powder. The formulations
are stored sealed at -20.degree. C. Before use the mixture is
reconstituted with sterile water for irrigation and vortexed until
a homogeneous multi-lamellar liposomal suspension is obtained. The
initial concentrations of camptothecin and paclitaxel in suspension
prior to nebulization are 0.5 mg/ml and 10 mg/ml, respectively. The
size of liposomes before and after nebulization is determined using
Nicomp Submicron Particle Sizer Model 370 9NICOMP, Santa Barbara,
Calif.).
EXAMPLE 4
[0066] Aerosol Particle Size Characteristics
[0067] The characteristics of aerosol particles containing
liposomal encapsulated drugs are estimated using an Andersen/AFCM
nonviable ambient particle sizing sampler (Andersen Instruments,
Atlanta, Ga.) as described (31). The concentration of drug in
aerosols produced by air or gas mixtures flowing at 10 L/min
through AERO-MIST nebulizer is also measured by collecting samples
for 3 min starting one minute after aerosolization initiation. The
mass median aerodynamic diameter (MMAD) and geometric standard
deviation (GSD) are calculated as described (30, 31) using
KaleidaGraph 2.0 software (synergy Software, Reading, Pa.).
EXAMPLE 5
[0068] Aerosol Delivery of Paclitaxel and Camptotbecin
[0069] The treatment of mice with aerosol is performed as
previously described (16-18). Briefly, an AERO-MIST jet nebulizer
(CIS-USA, Bedford, Mass.) is used to generate aerosol particles at
the air flow rate of 10 L/min. Mice are placed in sealed plastic
cage (23.times.18.times.13 cm) and exposed to aerosol for 30 min.
The aerosol is generated with normal or 5%CO.sub.2-enriched air
obtained by mixing normal air and CO.sub.2 with a blender (Bird 3m,
Palm Springs, Calif.) and the CO.sub.2 concentrations are
calibrated with a Fluid Fyrite (Bacharach Inc., Pittsburgh, Pa.).
At each time point 3 mice are removed from the cage and sacrificed
by exposure to Isoflurane, USP (Abbott Laboratories, Chicago, Ill.)
and exsanguination. Organs are resected, weighed and kept frozen at
-70.degree. C. until extraction.
EXAMPLE 6
[0070] Extraction of Drug From Tissues
[0071] Before extraction, samples are thawed and immediately cut in
small pieces with scissors. To extract paclitaxel from tissues, 3
ml of ethylacetate is added to each sample and homogenized in a
mini-beadbeater (Wig-L-Bug, Model 3110B, Crescent Dental MFR. Co.,
Lyons, Ill.) for 2 min. Homogenates are tranferred to 10 ml conical
glass centrifuge tubes and centrifuged at 1,000.times. g for 10
min. The supernatant fraction is separated and organic solvent is
evaporated with air. The residue is reconstituted in 0.2 ml of
methanol:acetonitrile (2:1, v/v), sonicated in a water-bath
sonicator and centrifuged at 1,000.times. g for 10 min. Supernatant
fractions are warmed at 37.degree. C. for 30 min and analyzed by
HPLC.
[0072] The extraction procedure for camptothecin and 9NC is as
previously described (17). Briefly, after thawing tissue, 20 .mu.g
of 9NC in 20 .mu.l is added to organs as an internal standard to
determine the extraction efficiency. The samples are cut in small
pieces and 1 ml of 0.1% aqueous acetic acid solution, pH 3.2 is
added to each sample. After the homogenization in a
mini-beadbeater, the homogenates are centrifuged at 1,000.times. g
for 5 min. The supernatant fractions are re-extracted with 8 ml of
methylene chloride. The organic fraction is separated and dried
under air at room temperature. The dried samples are reconstituted
in 0.2 ml of acetronitrile.
EXAMPLE 7
[0073] HPLC analysis
[0074] Paclitaxel is quantified by reverse-phase HPIC monitoring on
a Waters 486 UV absorbance detector at 227 nm (Waters, Milford,
Mass.). All measurements are made at room temperature on Waters
Nova-Pak C18 column (3.9.times.150 cm). The mobile phase is
composed of 49% acetonitrile and 51% water. The flow rate ia 1.5
ml/min. A 25 .mu.l aliquot of each sample is injected and data is
analyzed with Waters millennium Software. For PTX extraction
efficiency determination, identical procedures are performed when a
known amount of paclitaxel is added to each tissue and compared to
the extracted amount of paclitaxel. The extraction efficiency (%)
is calculated as ((amount of paclitaxel after extraction)/(amount
of added paclitaxel)).times.100. For all tested tissues the average
extraction efficiency is 89.+-.4% (data not shown) and this index
is used to calculate the final concentrations of drug in the
tissues.
[0075] HPLC analysis of camptothecin is performed using a Waters
NovaPak C18 column (3.9.times.150 cm) (17). Chromatograms for
camptothecin are monitored on Waters 470 scanning fluorescent
detector (.lambda.ex=360 nm, .lambda.em=455 nm) while 9NC is
detected using Waters 440 UV absorbance detector monitoring at 254
nm. The mobile phase is composed of 30% acetonitrile and 70% of
0.1% acetic acid solution in water, pH 3.5 at a flow rate 1.2
ml/min (16,17).
EXAMPLE 8
[0076] Aerosol Characteristics of Liposome Formulations
[0077] The properties of CPT-DLPC and PIX-DLPC liposomes and their
aerosol characteristics are summarized in Table 1. The utilization
of 5%CO.sub.2-air did not change the concentration of either drug
in the aerosol or their MMAD and GSD (P>0.1; Student's t-test,
two-tailed). The nebulization procedure reduces the size of
liposome particles in solution from micron- to nano-particles for
both drug formulations. The size of liposomes of CPT-DLPC decreased
from 2.54.+-.0.91 .mu.m before nebulization to 0.49.+-.0.07 .mu.m
after nebulization using the 5% CO.sub.2-air mixture. For the
PTX-DLPC formulation these values are 13.14.+-.12.15 .mu.m and
0.23.+-.0.17 .mu.m, respectively. The aerosol particle size before
or after nebulization is not different for either PTX-DLPC or
CPT-DLPC administered by aerosol using normal or 5% CO.sub.2-air
(P>0.5; Student's t-test, two-tailed).
1TABLE 1 Aerosol and liposome characteristics for PTX-DLPC and
CPT-DLPC formulations using 5% CO2-air versus normal air Drug
Concentration Aerosol droplets Liposome particle size, .mu.m Drug
Air in Aerosol, MMAD Before After Formulation Composition .mu.g/L
.mu.m GSD Nebulization Nebulization CPT-DLPC, Normal 9.0 .+-. 1.3
1.6 .+-. 0.3 2.1 .+-. 0.1 3.72 .+-. 1.10 0.34 .+-. 0.11 0.5 mg
CPT/ml 5% CO2 9.2 .+-. 1.9 1.7 .+-. 0.5 2.3 .+-. 0.2 2.54 .+-. 0.91
0.49 .+-. 0.07 PTX-DLPC, Normal 153.0 .+-. 27 2.0 .+-. 0.2 1.8 .+-.
0.03 12.49 .+-. 8.06 0.13 .+-. 0.18 10 mg PTX/ml 5% CO2 175.0 .+-.
9 2.2 .+-. 0.2 1.9 .+-. 0.1 13.14 .+-. 12.15 0.23 .+-. 0.17 Values
are means .+-. SD (n = 3 for each value). MMAD, mass median
aerodynamic diameter; GSD, geometric standard deviation
EXAMPLE 9
[0078] Tissue Distribution and Pharmacokinetics of CPT-DLPC After
Delivery by Aerosol Generated With Normal or 5%CO2-Enriched Air
[0079] ICR mice are divided into two groups: the first group (n=4)
received CPT-DLPC formulation via aerosol generated with normal air
for 30 min, so their breathing parameters are not changed during
treatment; the second group (n=6) inhaled the same formulation but
in the atmosphere of 5%CO.sub.2-enriched air.
[0080] Inhalation of aerosols generated with 5%CO.sub.2-air caused
a significant increase in deposition of camptothecin into the lungs
(2.1-3.5-fold) (FIG. 1). CPT is detected at 134.+-.123 and
476.+-.216 ng/g of lung tissue of mice from the first and second
groups, respectively. The use of 5%CO.sub.2-in-air did not change
tissue distribution patterns. The concentrations of drug in the
liver, spleen, kidney, blood and brain after inhalation of CPT-DLPC
aerosol generated with 5%CO.sub.2-air are also increased.
[0081] The pharmacokinetic deposition of camptothecin in lungs
during and after 30 mins exposure to aerosols of CPT-DLPC using
normal or 5%CO.sub.2-air is determined (FIG. 2). The pulmonary
concentrations of camptothecin increased during the treatment with
the maximum concentration (Cmax) at the end of aerosol treatment
(30 min.) and subsequently lung concentrations started to decline.
The peak respiratory levels are 232.+-.158 and 486.+-.78 ng/g of
the tissue for normal and 5%CO.sub.2-air, respectively. During the
15 min after the aerosol has been stopped, the concentrations of
the drug decrease exponentially. Clearance half-lives (T1/2) for
both treatments are 12-15 min. The profiles of the pharmacokinetic
curves are very similar for both types of treatment. Only trace
amounts of drug are detected in the lungs 90 min. after the end of
aerosolization (120 min. time point) with either air source.
EXAMPLE 10
[0082] Tissue Distribution and Pharmacokinetics of PTX Drug After
Treatment With Aerosol PTX-DLPC Generated by Normal or
5%CO.sub.2-Enriched Air
[0083] Due to the limitations of the detection method, a liposomal
formulation of paclitaxel at 10 mg of PTX/ml suspension is used.
Mice are sacrificed halfway through exposure (15 min), at the end
of treatment (30 min), and at several time points following the end
of treatment. Mice are exposed to PIX-DLPC aerosol generated with
either normal air or air containing 5%CO.sub.2.
[0084] Pulmonary paclitaxel Cmax values are achieved at the end of
treatment (30 min) with either air source (FIG. 3). In the 5%
CO.sub.2-enriched air group Cmax is 4.2-fold higher than in the
ambient air group (23.1.+-.4.3 and 5.5.+-.0.2 .mu.g/g,
respectively). This carbon dioxide induced enhancement is unrelated
to the liposomal formulation (FIG. 4). Sterically stabilized
paclitaxel liposomes prepared using dismyristylphosphoethanolamine
poly (ethylene glycol) 2000 and dilauroylphosphatidylcholine are
deposited in the lung at equivalent levels when 5% CO.sub.2-in-air
is utilized.
[0085] Treatment with 5%CO.sub.2 produced 5.7-fold higher area
under the lung-concentration-time curve compared to normal air
(33.7 and 5.9 .mu.g-hr/g, respectively). In both cases PTX
concentrations started to decrease from the pulmonary tissue after
the treatment ended. T1/2.alpha. and T1/2.beta. values for
paclitaxel in the lungs are 0.3 and 1.6 hr, respectively, when
normal air is used for aerosol generation. T1/2.alpha. is 0.7 hr
and T1/2.beta. is 5.1 hr for paclitaxel administered by liposome
aerosol produced with 5%CO.sub.2-air. Comparative analysis for the
other organs, such as liver, spleen, kidney and blood was
performed; however, the levels of paclitaxel in these tissues using
normal air for aerosolization are below detectable levels.
[0086] The tissue distribution of paclitaxel after liposome aerosol
delivery using 5%CO.sub.2-air is presented in Table 2. The highest
concentrations of the drug are detected in the lungs. Lower
concentrations are found in the other organs. Analysis of the area
under the concentration-time curve (AUC) over a 3 hr. period for
different organisms using the trapezoidal rule shoes the following
AUC values for lungs, liver, kidney, blood and brain: 34.+-.2,
9.8.+-.1.9, 2.4.+-.1.4, 2.8.+-.1.5, 0.13.+-.0.10, 0.23.+-.0.2 .mu.g
PTS-hr/g of tissue, respectively.
2TABLE 2 PTX deposition in tissues during and after 30 min exposure
to aerosol PTX-DLPC* generated with 5% CO2-air Time PTX
concentration (.mu.g/g of tissue) (hr) Lungs Liver Spleen Kidney
Blood Brain 0.25 20.3 .+-. 7.8 1.5 .+-. 0.8 0.6 .+-. 0.3 1.4 .+-.
0.0 0.25 .+-. 0.03 0.14 .+-. 0.16 0.5 23.1 .+-. 4.3 5.7 .+-. 3.0
1.4 .+-. 0.9 1.6 .+-. 0.1 0.18 .+-. 0.08 0.16 .+-. 0.02 0.75 18.0
.+-. 3.6 5.5 .+-. 1.8 0.5 .+-. 0.4 1.4 .+-. 0.1 0.08 .+-. 0.09 0.11
.+-. 0.03 1.0 14.8 .+-. 9.5 4.8 .+-. 3.9 2.6 .+-. 2.7 1.2 .+-. 0.7
0.07 .+-. 0.07 0.11 .+-. 0.03 1.5 8.7 .+-. 2.8 2.8 .+-. 0.8 1.0
.+-. 1.6 0.7 .+-. 0.3 0.03 .+-. 0.06 0.09 .+-. 0.08 2.0 6.5 .+-.
2.9 3.1 .+-. 0.7 0.6 .+-. 0.4 0.4 .+-. 0.3 0.01 .+-. 0.02 0.04 .+-.
0.04 3.0 7.1 .+-. 2.8 2.3 .+-. 0.6 0.5 .+-. 0.2 0.4 .+-. 0.1 0.01
.+-. 0.02 0.05 .+-. 0.05 Values are means .+-. SD of three
experiments (organs from 3 mice were combined and processed in each
experiment
EXAMPLE 11
[0087] Effect of Carbon Dioxide Induced Respiratory Patterns on
Drug Deposition
[0088] The increased pulmonary drug concentrations found in the
lungs after inhalation of 5%CO.sub.2-in-air could be explained by
changed respiratory patterns. Breathing patterns of mice in the
atmosphere of 5%CO.sub.2-enriched air are visually observed to
become deeper and slower and to return to normal almost immediately
after the end of treatment. Histological analysis did not reveal
any changes in pulmonary tissue. Plethysmograph studies that have
been performed by other researchers have demonstrated that
inhalation of 5%CO.sub.2-enriched air increased ventilation in
mammalians primarily because of the increase in tidal volume
(approximately 170-180%) (18,19). The average pulmonary deposition
of camptothecin and paclitaxel increased approximately 2-4-fold.
This disproportion with the increase of tidal volume may be due to
some other physiological changes in breathing parameters, e.g.,
breathing frequency, respiratory duration of inspiratory and
expiratory cycles, and minute ventilation (13). By deep and
complete expiration with breath holding the retention of the
aerosol increased almost twice in comparison with normal breathing
(15).
EXAMPLE 12
[0089] Preparation of PEI-DNA Complexes
[0090] PEI (25 kDa, branched) was purchased from Aldrich Chemical
(Milwaukee, Wis.). A PEI stock solution was prepared at a
concentration of 4.3 mg/ml (0.1 M in nitrogen) in PBS, pH 7-7.5.
PEI and DNA are mixed separately in 5 ml water at the required
concentrations. The PEI solution is slowly vortexed and the DNA
solution is added to it to make a final volume of 10 ml. The
mixture is alloed to stand at room temperature for about 15-20 min.
before nebulization. The resulting charge ratio is expressed as PEI
nitrogen:DNA phosphate (N:P), which can be calculated by taking
into account that DNA has 3 nmol of phosphate per microgram and 1
.mu.l of 0.1 M PEI solution has 100 nmol of amine nitrogen. A 10:1
N:P ratio corresponds to a 1:29:1 PEI:DNA weight ratio.
EXAMPLE 13
[0091] Aerosol Delivery of PEI:DNA Complexes
[0092] Mice are placed in plastic cages that are sealed with tape
before aerosol delivery (48). This is an unrestrained, whole-body
aerosol exposure system. PEI-DNA complexes are aerosolized using an
Aero-Mist nebulizer (CIS-US, Inc., Bedford, Mass.) at 10 liters/min
flow rate using air or air containing 5% CO2. Aero-Mist is a
high-output, efficient nebulizer demonstrated to produce aerosols
in the optimal range of 1-2 .mu.m MMAD with a geometric standard
deviation (GSD) of 2.9 using an Andersen cascade impactor (Andersen
Instruments, Atlanta, Ga.) by a method previously described (50). A
source of dry air (Aridyne 3500, Timeter, Lancaster, Pa.) is
delivered to a Bird 3M gas blender (Palm Springs, Calif.) attached
to an air compressor and a CO.sub.2 tank. The resulting mixture of
air and CO.sub.2 is delivered to the nebulizer. The final
concentration of 5% CO.sub.2 in air is determined using a Fyrite
solution (Bacharach, Pittsburgh, Pa.). Nebulization of 10 ml
solution took approximately 30 min.
EXAMPLE 14
[0093] CAT Assay
[0094] Mice are anesthetized and sacrificed after each time point
and the lungs and other tissues are harvested, weighed, and
immediately frozen. A CAT ELISA kit (Boehringer Mannheim GmbH,
Mannheim, Germany) is used for measureing in vivo expression. The
tissues are homogenized in 700 .mu.l CAT assay lysis buffer using a
Wig-L-Bug bead homogenizer (Crescent Dental Mfg., Lyons, Ill.).
After centrifuging the homogenates, 200 .mu.l of the extract is
used for the CAT ELISA performed in a 96-well plate format. The
abosorbance is read using a microtiter plate reader (Molecular
Devices, Sunnyvale, Calif.). Nave mice are used as controls. The
CAT activity is expressed as ng of CAT/g of tissue using a standard
curve prepared with purified CAT enzyme. The sensitivity of the
assay is further enhanced according to suggestions from the
manufacturer so that it can detect levels of CAT protein as low as
0.1-0.3 pg/well.
EXAMPLE 15
[0095] Luciferase Assay
[0096] Mice are anesthetized and sacrificed and the lungs are
harvested. A luciferase assay kit (Promega) is used to measure
luciferase expression. The lungs are homogenized in 1 ml of
luciferase assay lysis buffer using a Wig-L-Bug bead homogenizer.
After centrifuging the homogenates, 10 .mu.l of the extract is
added to 50 .mu.l of luciferase substrate and the luminescence read
for 10 s in a 96-well plate on a luminometer (Microlumat LB 96 P,
EG & G Berthold, Germany). Naive mice are used as controls. The
luciferase activity is expressed as RLU/10s/g of tissue. In this
system, 10.sup.7 RLU corresponds to 1 ng of luciferase using
purified luciferase from Promega.
EXAMPLE 16
[0097] Histological Analysis of Tissue Sections
[0098] Mice are anesthetized with isoflurane and sacrificed by
exsanguination via the abdominal aorta. Lungs are isolated,
cannulated, and fixed by inflation with 10% neutral buffered
formalin, embedded in paraffin, and processed for histological
analysis. Thin sections are cut at 4 .mu.m and observed under the
microscope for any signs of inflammation or toxicity using the
hematoxylin and eosin stain.
EXAMPLE 17
[0099] Myeloperoxidase (MPO) Assay
[0100] Twenty-four hours after aerosol exposure, mice are
anesthetized with isoflurane and sacrificed by exsanguination via
the abdominal aorta. The lungs are harvested after perfusion
through the heart with saline. The tissue is homogenized in
hexadecyltrimethylammonium bromide (0.5% HTAB in 50 mM phosphate
buffer, pH 6.0; 5 ml HTAB/g of tissue) as previously described
(51). After centrifugation, the MPO activity in the supernatant is
determined using o-diasinidine dihydrochloride (0.167 mg/ml) plus
0.0005% hydrogen peroxide. The absorbance is measured at 460 nm
using a microtiter plate reader (Molecular Devices). The absolute
values after 15 min are recorded. Naive mice are used as
controls.
EXAMPLE 18
[0101] Nebulization of PEI-DNA Complexes with 5% CO.sub.2 Enhances
the Transgene Expression in Lung Compared to Normal Air
[0102] Breathing 5% CO.sub.2 in air has been associated with an
increase in the tidal volume and breathing frequency in mice and
humans (52-54). When 5% CO.sub.2-in-air is utilized to deliver the
PEI-DNA aerosol, the mice can be visually observed to be breathing
deeper and more rapidly. Inhalation of aerosols containing 5%
CO.sub.2 could lead to greater inhalation of aerosol particles and
correspondingly higher transgene expression compared to that
achieved with aerosol delivered by air due to increased tidal
volume and breathing frequency.
[0103] PEI-DNA complexes are delivered to Balb/C mice by aerosol
using either normal air or air containing 5% CO.sub.2. A fixed
amount of CAT plasmid (1 mg/10 ml of solution), at a N:P ratio of
10:1, is aerosolized for 30 min as indicated above. The lungs are
harvested after 24 h and CAT assay is performed to determine the
degree of transfection. Five percent CO.sub.2-in-air lead to a
three-fold increase (P=0.001) in the levels of CAT detected
compared to aerosol nebulized with air alone (FIG. 5). Also, 5%
CO.sub.2 does not change the particle size of the resulting
drug-liposome aerosol particles.
[0104] Enhancement of PEI-DNA transfer to the lung by aerosol using
different percentages of CO.sub.2-in-air with a fixed amount of CAT
plasmid is also examined. The complexes are aerosolized using 0%,
2.5%, 5%, 10% and control amounts of carbon dioxide in air. The CAT
activity assayed indicates using either 2.5% or 10% provides as
good a level of transfection as using 5% CO.sub.2-in air (FIG.
6).
[0105] It is possible that enhanced CO.sub.2 has an effect on the
transfection efficiency of PEI-DNA complexes by changing some other
physiological parameters. However, CO.sub.2 does not significantly
alter the pH of the PEI-DNA solution nor does the particle size of
the resulting aerosol droplets, as compared to those of air,
significantly change. The increase in transgene expression in the
lungs is most likely due to increased deposition of aerosol
particles. Five percent CO.sub.2-in-air also could help to optimize
the aerosol delivery of other polymer-DNA or cationic lipid-DNA
complexes (45). This percentage of CO.sub.2 has been well tolerated
by humans and has been shown to increase the minute volume (54,55),
so this strategy could be efficacious against pulmonary diseases in
humans provided that the size, geometry and physiology of the human
pulmonary system is taken into consideration.
EXAMPLE 19
[0106] DNA Transfer by PET is Dose Dependent
[0107] To further optimize the transgene expression, the N:P ratio
is kept constant at 10:1 and the amount of DNA is varied from 250
.mu.g to 4 mg per 10 ml of the aerosolized solution. This leads to
an increase in the reservoir concentration as well as the amount of
total DNA nebulized in the aerosol output.
[0108] The nebulized output from the Aerotech II nebulizer was
calculated to be approximately 80%. About 72% of the reservoir DNA
was delivered to the inhalation chamber as estimated using an
all-glass impinger (AGI) (50). The remainder was trapped in the
T-connector and tubing. Based on murine obligate nasal breathing,
pulmonary physiology (minute volume and deposition fraction) (50),
and the output concentration of aerosol (4.8 ug/liter), the amount
of DNA deposited in the lungs of a mouse is estimated to be
approximately 4-5 .mu.g during 30 min of aerosol exposure (for a
starting reservoir concentration of 2 mg DNA/10 ml solution). These
calculations are based on normal air breathing; the deposition
could be higher in the presence of 5% CO.sub.2 due to the increased
tidal volume and breathing frequency (53).
[0109] The complexes are aerosolized using 5% CO.sub.2-in-air with
2 mg DNA giving the highest level of CAT expression in the lung
(FIG. 7). The levels of CAT measured with 250 .mu.g DNA are not
statistically different from control lungs (P=0.34). Also, when 4
mg of DNA is dissolved in 10 ml at a N:P ratio of 10:1, it leads to
some visual precipitation of the DNA, which may account for no
further increase in the level of CAT detected in the lungs compared
to 2 mg (P=0.51).
[0110] It should be noted that there is an increase in both the
concentration and the amount of DNA delivered. However, it may be
possible to further increase the expression in the lung by
increasing the exposure time of aerosol at the optimal
concentrations. These expression levels in the lung are comparable
to those using other delivery systems (34).
EXAMPLE 20
[0111] Optimization of PEI-DNA Ratios
[0112] Although PEI can protect the DNA during nebulization and
also result in higher transgene expression in the lungs after
aerosol delivery when compared to most other cationic lipids,
determination of optimal parameters for gene delivery is
beneficial. The charge interaction between any cationic vehicle and
the negatively charged DNA is an important factor determining the
efficiency of the transfection of the complex. Previous studies
have examined the optimum PEI-DNA (N:P) ratio for transfection in
the lung (38, 56). However, these studies involved an intravenous
mode of PEI-DNA delivery. Gene delivery by aerosol could require
different conditions.
[0113] To determine the charge ratio that would be ideal for in
vivo aerosol delivery, different PEI-DNA (N:P) ratios for their
ability to transfect the lung are evaluated. The amount of DNA is
kept constant at 2 mg and the PEI concentration is varied to obtain
ratios of 5:1, 10:1, 12.5:1, 15:1, 17.5:1, and 20:1. These ratios
are chosen based on previous in vitro and in vivo (by instillation)
studies (43). The complexes are aerosolized using 5%
CO.sub.2-in-air. A N:P ratio of 15:1 gave the highest level of CAT
expression in lung, whereas 5:1 resulted in a very low level of CAT
expression (FIG. 8). There is statistically no difference between
10:1, 12.5:1, 15:1, 17.5:1, and 20:1 ratios (P>0.1), but a
significant difference between 15:1 and 20:1 (P=0.05) and between
10:1 and 15:1 (P=0.014).
[0114] To determine the optimal ratio for a plasmid other than CAT,
different N:P ratios for the expression of the luciferase gene in
the lung are tested. The ratios evaluated are 5:1, 10:1, 15:1,
20:1, 30:1, and 40:1. The optimum curve for luciferase shifted to
the right compared to CAT, with the highest expression at 20:1
(P<0.05 compared to other ratios) (FIG. 8). This suggests that
different plasmids might require different N:P ratios; the
different size of luciferase plasmid leads to a structurally
different complex with PEI compared to that of the CAT plasmid. It
could also be due to a difference in plasmid purity and the
proportion of supercoiled structure. Still there is a considerable
overlap in the optimum N:P ratios of these two plasmids. The
optimum ratios for different plasmids may be different. Considering
experimental variablility, a ratio between 10:1 and 20:1 should
work suitably. A ratio lower than 10:1 did not give very high
transfection in the lung. These results are in agreement with those
obtained using branched 25K PEL although the mode of delivery was
intravenous (56).
EXAMPLE 21
[0115] Time Course of CAT Expression in Lung Following Single
Aerosol Delivery
[0116] CAT expression was also used to monitor the time course of
gene expression. The analysis of the persistence of CAT expression
following a single aerosol delivery provides important information
for planning a treatment regime for therapeutic studies. Two
milligrams of CAT plasmid is aerosolized, using 5% CO.sub.2-in-air,
to the mice at two different N:P ratios, 15:1 and 10:1. Different
time points examined for the 10:1 group are 1, 2, 3, and 6 days
post aerosol exposure. Lungs and other tissues are harvested at
different time points and frozen immediately. All tissues are
assayed simultaneously after the last time point (day 6). For the
15:1 group the mice are sacrificed 1, 3, 7, and 10 days after
aerosol treatment. The lungs are harvested, weighed, and frozen
after each time point and the CAT protein is assayed after the last
time point (day 10).
[0117] For both N:P ratios examined, the CAT expression is highest
at 24 h and remains constant (statistically no difference between
day 1 and day 3, P=0.4 for the 15:1 ratio and P=0.12 for the 10:1
ratio) for over three days (FIGS. 10A and 10B). The CAT level falls
to about 50% of peak levels after a week and significant levels are
detected even after 10 days (P=0.001 compared to control).
[0118] This suggests that the delivery may be more than adequate
for a variety of clinical applications. The persistence of gene
expression up to day 10 is similar to or greater than that of other
cationic lipids used for instillation or aerosol delivery of genes
(34,58).
EXAMPLE 22
[0119] Tissue Distribution of Transgene
[0120] Intravenous or intraperitoneal delivery of DNA vectors
generally results in expression in a variety of tissues. In order
to determine if aerosol delivery of PEI-DNA results in systemic
gene delivery, different tissues are harvested from the same group
of mice as the above experiment (from the 10:1) group) and the CAT
assay is performed after the last time point. The tissues examined
are lung, liver, spleen, kidney, thymus, brain, and blood. The
level of CAT detected in non-lung tissues was very low and not
significantly different (P>0.1 for all the tissues) from the
control tissues (FIG. 11).
[0121] The tissue distribution data show that gene expression
following aerosol delivery in this system is confined to the lung,
indicating minimal systemic delivery. In contrast to the lung,
tissues such as liver, spleen, and kidney, which normally exhibit
detectable levels of expression when genes are delivered via
intravenous or intraperitoneal administration, exhibited
insignificant or no detectable CAT expression when delivered by
PEI-DNA aerosol. This is important if the expression of the gene of
interest is to be restricted to the lungs. In other studies, the
intratracheal mode of gene delivery has been used to localize the
gene to the lungs (58). However, this is a rather invasive
technique compared to aerosol and generally results in less uniform
deposition to the peripheral regions of the lung. Aerosol delivery
helps to distribute the particles noninvasively and uniformly
through out the lungs (49).
EXAMPLE 23
[0122] Histological Analysis Shows no Signs of Inflammation
[0123] In order to determine if aerosol delivery of PEI-DNA
complexes leads to any kind of toxicity or acute inflammation in
this system, two milligrams of CAT plasmid is complexed with PEI at
a N:P ratio of 15:1 and the mice are exposed to aerosol for 30 min
using 5% CO.sub.2-in-air. The mice are sacrificed after 24 h and
the lungs are fixed in formalin and stained with hematoxylin and
eosin. The lungs did not show any evidence of histological
abnormality, e.g., inflammatory cell infiltration or damage to the
lungs when thin sections are examined (FIG. 12). Use of 5%
CO.sub.2-in-air to optimize pulmonary gene delivery by PEI-DNA
aerosol seems to be safe and highly specific for the lung.
[0124] Although high levels of expression are detected in this
system even a week after single aerosol exposure, some therapies
may require repeated and frequent delivery of genes. The effects of
prolonged PEI-DNA aerosol exposure on lungs and other tissues needs
to be determined.
EXAMPLE 24
[0125] Myeloperoxidase Assay Does not Reveal any Inflammation
[0126] Acute pulmonary inflammation is mediated in part by
polymorphonuclear leukocyte (PMN) sequestration to the peripheral
tissues. A biochemical marker for polymorphonuclear leukocyte is
myeloperoxidase (MPO), which is a heme-containing enzyme found in
the azurophilic granules and its often utilized as an inflammation
marker in the lungs (18). To assess neutrophil infiltration into
the lungs, 2 mg of CAT plasmid is complexed with PEI at a N:P raio
of 15:1 and the mice are exposed to aerosol for 30 min using 5%
CO.sub.2-in-air. The mice are sacrificed after 24 h, the lungs were
harvested, and the myeloperoxidase assay is performed (Table
3).
[0127] The myeloperoxidase contents in the control and
aerosol-exposed lungs were not significantly different (P=0.92).
The myeloperoxidase assay did not reveal any difference between the
control and aerosol-exposed lungs, i.e., there is no difference in
the absolute absorbance values (OD) between control and
aerosol-exposed lungs, even 15 min after incubation of the reaction
(OD of 0.078.+-.0.009 for control and 0.084.+-.0.004 for
aerosol-exposed lungs, P>0.5).
3TABLE 3 Myeloperoxidase (MPO) Assay for Evaluation of Neutrophil
Infiltration into the Lungs Group Control Aerosol Lung MPO activity
0.0398 .+-. 0.01 0.0404 .+-. 0.008 (.delta.A/min/g tissue) Note.
Two milligrams of CAT plasmid was complexed with PEI at a N:P ratio
of 15:1 and the complex was aerosolized to five mice for 30 min
using 5% CO2-in-air. Mice were sacrificed 24 h later, lungs were
harvested, and the MPO assay was performed. Values are means .+-.
SD (n = 5 mice per group).
EXAMPLE 25
[0128] P53 Assay
[0129] P53 expression was examined using an ELISA kit (Roche
Diagnostics, Indianapolis, Ind.). For in vitro expression, B16-F10
cells grown in tissue culture plates (20,000 cells/well in a
48-well plate) were transfected with PEI:DNA complexes for 24 h.
The cultures were then washed and cells lysed using cell lysis
buffer. After centrifugation, 100 .mu.l of the lysate was used for
p53 ELISA. The p53 levels were normalized to the total protein
content measured by the BCA protein assay (Pierce, Rockford, Ill.).
For in vivo expression, mice were exposed to PEI:p53 aerosol,
sacrificed 24 h later and the lungs harvested and weighed. The
lungs were homogenized in 1 ml of ice cold cell lysis buffer (20 mM
Tris, 0.5 mM EDTA, 1% Nonidet P40, 0.05% SDS, 1 mM PMSE, 1 .mu.g/ml
pepstatin, 2 .mu.g/ml leupeptin) using a Wig-L-Bug bead homogenizer
(Crescent, Lyons, Ill.). After centrifugation at 4.degree. C., 100
.mu.l of the supernatant was used for p53 FLISA performed in a
96-well plate. The absorbance (450 nm) was read in triplicate using
a Molecular Devices (Sunnyvale, Calif.) microtiter plate reader.
The amount of p53 was determined using a standard curve prepared
with purified 053. The assay can detect p53 levels as low as 10
pg/ml and the linear measuring range of the assay is 50-1000 pg/ml.
The total protein content in the lungs was determined using the BCA
protein assay.
EXAMPLE 26
[0130] P53 Expression in Mouse Lung Following Aerosol Delivery of
PEI-p53 Complexes
[0131] PEI-p53 complexes are prepared as done for PEI:DNA complexes
described above. Two milligrams of p53 plasmid is complexed with
polyethyleneimine at a PEI:DNA (N:P) ratio of 10:1 and aerosolized
to the C57BL/6 mice using 5% CO.sub.2-in-air. Mice were placed in
plastic cages that were sealed with tape before aerosol delivery.
This is an unrestrained, whole body aerosol exposure system.
PEI-p53 complexes were aerosolized using an Aero-Mist nebulizer in
the presence of 5%CO.sub.2 as described for aerosolization of
polyethyleneimine:CAT complexes previously herein.
[0132] P53 expression in lung was analyzed by ELISA 24 h after
aerosol delivery of the PEI-p53 complexes to the mice. Aerosol
delivery of complexes lead to about a four fold increase in the
levels of p53 detected in the lung tissue compared to that detected
in the lings of nave mice. The level of p53 in the control mice is
0.0398.+-.0.01 pg/mg protein and the level in the aerosolized mice
is 0.0404.+-.0.008 pg/mg protein (values are means .+-.SD) (59).
Exposure to PEI-Luc did not result in any increase in the p53
levels (data not shown).
EXAMPLE 27
[0133] Inhibition of B16-F10 Lung Metastasis by Aerosol Delivery of
PEI-p53
[0134] C57BL/6 mice were injected intravenously with 25,000 B16-F10
cells on day 0. The mice were treated with polyethyleneimine-p53
aerosol complexes generated using 5% CO.sub.2 twice a week starting
the day after inoculation of the cancer cells into the mice (on
days 1, 4, 8, 11, 15, 18, and 22) with the last treatment on day 22
postinjection (a total of seven aerosol exposures). Control groups
included untreated mice, mice treated with polyethyleneimine or
with polyethyleneimine-Luc aerosol complexes. The control animals
start dying around day 24 post tumor cell inoculation, which is
when the therapy was stopped and the experiment terminated. The
dosage of treatment was 2 mg plasmid/10 ml of aerosolized solution
at a polyethyleneimine:DNA (N:P) ratio of 10:1. This is the total
amount of DNA aerosolized to the mice. The amount of DNA delivered
per mouse is estimated to be about 4-5 .mu.g in the presence of
normal air and is increased in the presence of 5% CO.sub.2 due to
the increase in tidal and minute volumes.
[0135] On day 24 post tumor inoculation, the mice were sacrificed
and the lungs fixed and tumor index was calculated. The mice
treated with PEI-p53 had a very low tumor index (P<0.001
compared to all other groups) whereas all the control groups had
large number of tumor nodules (FIGS. 13A, 13B). A majority of
untreated mice and mice treated with either polyethyleneimine alone
or with polyethyleneimine-Luc had numerous uncountable tumor
nodules with concomitant invasion into the chest wall and had
metastases in extrapulmonary tissue such as the neck and abdominal
lymph nodes. However, all of the mice treated with
polyethyleneimine-p53 complexes had very small and distinct tumor
foci with no invasion into the chest wall and no extrapulmonary
metastatic tumors. There was no effect of 5% CO.sub.2 alone on the
growth of tumors compared to untreated mice (data not shown). The
lung weights also showed a significant difference (P<0.01)
between PEI-p53 treated group and all the control groups (FIG.
13C).
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[0198] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. These patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0199] One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The present examples along with the methods, procedures,
treatments, molecules, and specific compounds described herein are
presently representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention as
defined by the scope of the claims.
* * * * *