U.S. patent application number 09/969374 was filed with the patent office on 2002-08-01 for small particle liposome aerosols for delivery of anti-cancer drugs.
This patent application is currently assigned to Research Development Foundation. Invention is credited to Gilbert, Brian, Giovanella, Beppino C., Knight, J. Vernon, Koshkina, Nadezhda, Waldrep, J. Clifford, Wellen, Clyde W..
Application Number | 20020102296 09/969374 |
Document ID | / |
Family ID | 25463629 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020102296 |
Kind Code |
A1 |
Giovanella, Beppino C. ; et
al. |
August 1, 2002 |
Small particle liposome aerosols for delivery of anti-cancer
drugs
Abstract
The small particle liposome or lipid complex aerosol compounds
and methods of treatment of the present invention involve lipid- or
water soluble anti-cancer drugs incorporated into liposomes or
other lipid complexes. The liposomes and complexes are administered
in aqueous dispersions from a jet nebulizer to the respiratory
tract of an individual. Various anti-cancer drugs may be used,
including 20-S-Camptothecin, 9-Nitro-camptothecin,
9-Amino-camptothecin, 10,11 -methylenedioxy-camptot- hecin and
taxol or its derivatives. Administration of these drugs by
inhalation provides faster and more efficient absorption of the
anticancer drug than does intramuscular administration or oral
administration.
Inventors: |
Giovanella, Beppino C.;
(Houston, TX) ; Knight, J. Vernon; (Houston,
TX) ; Waldrep, J. Clifford; (The Woodlands, TX)
; Koshkina, Nadezhda; (Houston, TX) ; Gilbert,
Brian; (Houston, TX) ; Wellen, Clyde W.;
(Houston, TX) |
Correspondence
Address: |
Dr. Benjamin Adler
Adler & Associates
8011 Candle Lane
Houston
TX
77071
US
|
Assignee: |
Research Development
Foundation
|
Family ID: |
25463629 |
Appl. No.: |
09/969374 |
Filed: |
October 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09969374 |
Oct 1, 2001 |
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09353496 |
Jul 15, 1999 |
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09353496 |
Jul 15, 1999 |
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08933254 |
Sep 23, 1997 |
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Current U.S.
Class: |
424/450 ; 424/45;
424/85.5; 514/251; 514/263.3; 514/27; 514/283; 514/449 |
Current CPC
Class: |
A61K 31/513 20130101;
A61K 31/7048 20130101; A61K 31/03 20130101; A61K 31/4745 20130101;
A61P 35/00 20180101; Y10S 514/938 20130101; A61K 31/175 20130101;
A61K 31/519 20130101; A61K 31/52 20130101; A61K 9/0078 20130101;
A61K 31/337 20130101; A61K 38/21 20130101; A61K 9/127 20130101;
A61P 31/00 20180101 |
Class at
Publication: |
424/450 ; 424/45;
514/27; 514/283; 514/449; 514/263.3; 424/85.5; 514/251 |
International
Class: |
A61K 038/21; A61L
009/04; A61K 031/525; A61K 031/522; A61K 031/4745; A61K 031/337;
A61K 031/7048 |
Claims
What is claimed is:
1. A method for treating cancer, comprising the step of delivering,
via small particle aerosol, aqueous dispersions of anti-cancer
drugs in liposomes or lipid complexes to the respiratory tract of
an individual in need of such treatment.
2. The method of claim 1, wherein said anti-cancer drug is selected
from the group consisting of 20-S-camptothecin,
9-nitro-camptothecin, 9-amino-camptothecin,
10,11-methylenedioxy-camptothecin, taxol, taxol-A, mitotane,
methotrexate, mercaptopurine, lomustine, interferon, 5-fluorouracil
and etopiside.
3. The method of claim 2, wherein said anti-cancer drug is selected
from the group consisting of 20-S-camptothecin,
9-nitro-camptothecin, 9-amino-camptothecin,
10,11-methylenedioxy-camptothecin and taxol.
4. The method of claim 1, wherein said delivering step is performed
by a jet nebulizer.
5. The method of claim 4, wherein said liposomes are sheared to a
diameter of less than 500 nm by said jet nebulizer.
6. The method of claim 1, wherein a final concentration of said
anticancer drug in said liposomes or lipid complexes is no greater
than 5.0 mg/ml.
7. The method of claim 6, wherein a final concentration of said
anticancer drug in said liposomes or lipid complexes is no greater
than 1.0 mg/ml.
8. A liposome or lipid complex for delivery of anticancer drugs via
small particle aerosol, wherein a ratio of anticancer drug to lipid
is about 1:1 to about 1:200 wt:wt.
9. The liposome or lipid complex of claim 8, wherein a ratio of
anticancer drug to lipid is about 1:10 to about 1:100 wt:wt.
10. The liposome or lipid complex of claim 9, wherein a ratio of
anticancer drug to lipid is about 1:10 to about 1:50 wt:wt.
11. The liposome or lipid complex of claim 8, wherein said
anticancer drug is camptothecin or a derivative of camptothecin,
said lipid is dilauroylphosphatidylcholine, and said ratio is about
1:10 to about 1:50.
12. The liposome or lipid complex of claim 8, wherein said
anticancer drug is taxol or its derivatives, said lipid is
dilauroylphosphatidylcholine and said ratio is about 1:25 to about
1:40.
13. A liposome produced by the following steps: dissolving a
lipid-soluble anticancer drug in an appropriate solvent to produce
dissolved anticancer drug; dissolving a lipid suitable for the
formulation and delivery of anticancer drugs by aerosol in an
appropriate solvent to produce dissolved lipid; combining said
dissolved anticancer drug and said dissolved lipid to produce a
solution, wherein said dissolved anticancer drug is at a
concentration not exceeding about 10% of the total volume of said
solution and a ratio of said anticancer drug to said suitable lipid
is in a range of 1:1 to 1:200 wt:wt of said solution; and
evaporating said solvents from said solution to produce a
powder.
14. The liposome of claim 13, further comprising the step of
dissolving said powder in sterile water to produce a suspension,
wherein a concentration of said anticancer drug in said sterile
water is no more than about 5.0 mg/ml.
15. The liposome of claim 13 wherein said suitable lipid for
formulating and delivering drugs by aerosol is
dilauroylphosphatidylcholine.
16. The liposome of claim 13, wherein said anti-cancer drug is
selected from the group consisting of 20-S-camptothecin,
9-nitro-camptothecin, 9-amino-camptothecin,
10,11-methylenedioxy-camptothecin, taxol, taxol-A, mitotane,
methotrexate, mercaptopurine, lomustine, interferon, 5-fluorouracil
and etopiside.
17. The liposome of claim 13, wherein said anti-cancer drug is
selected from the group consisting of 20-S-Camptothecin,
9-Nitro-camptothecin, 9-Amino-camptothecin and
10,11-methylenedioxy-camptothecin, and taxol.
18. The liposome of claim 13, wherein a ratio of anticancer drug to
lipid is about 1:10 to about 1:100 wt:wt.
19. The liposome of claim 18, wherein a ratio of anticancer drug to
lipid is about 1:10 to about 1:50 wt:wt.
20. A liposome produced by the following steps: dissolving a
lipid-soluble anticancer drug selected from the group of
20-S-camptothecin (CPT), 9-nitrocamptothecin (9-NC) and other lipid
soluble camptothecin derivatives in a volume of DMSO to produce
dissolved anticancer drug; mixing said dissolved anticancer drug
with an appropriate solvent; dissolving a lipid suitable for the
formulation and delivery of drugs by aerosol in an appropriate
solvent to produce a dissolved lipid; combining said dissolved
anticancer drug and said dissolved lipid to produce a solution,
wherein said DMSO is at a concentration not exceeding about 10% of
the total volume of said solution and a ratio of said anticancer
drug to said suitable lipid is in a range of 1:1 to 1:200 of said
solution; and evaporating said solvents from said solution to
produce a powder.
21. The liposome of claim 20, wherein said lipid is
dilauroylphosphatidylcholine.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates generally to the field of
pharmacology and cancer treatment. Specifically, the present
invention provides formulations and methods for small particle
aerosol delivery by inhalation of aqueous dispersions of liposomes
carrying anti-cancer drugs to the respiratory tract.
[0003] 2. Description of the Related Art
[0004] 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. 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 (Hallman, M., et al. Inositol
Supplementation in Premature Infants with Respiratory Distress
Syndrome, N. Eng. J. Med. 1992 326:1233-1239).
[0005] 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.
Additionally, lipid complexes of various sizes can be used.
[0006] Nebulization shears liposomes and other lipid complexes to
sizes readily discharged from the nozzle of the nebulizer.
Liposomes and other lipid complexes 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 or complexes
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. 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.
[0007] The prior art is deficient in formulations and methods for
small particle aerosol delivery of aqueous dispersions of liposomes
or lipid complexes containing anti-cancer drugs. The present
invention fulfills this long-standing need and desire in the
art.
SUMMARY OF THE INVENTION
[0008] The small particle liposome or lipid complex aerosol
compounds and methods of treatment of the present invention involve
lipid-soluble or water-soluble anti-cancer drugs incorporated into
liposomes or other lipid complexes. These drug-carrying lipids then
are administered in aqueous dispersions from a jet nebulizer. The
present invention demonstrates that speedier and more efficient
systemic absorption of drug is actualized after pulmonary
administration by aerosol than is actualized by intramuscular or
oral administration.
[0009] One object of the present invention is to provide a method
for treating cancer, comprising the step of delivering, via small
particle aerosol, aqueous dispersions of anti-cancer drugs to the
respiratory tract of an individual in need of such treatment.
Examples of anticancer drugs available for use in this embodiment
of the invention include, but are not limited to,
20-S-camptothecin, 9-nitro-camptothecin, 9-amino-camptothecin,
10,11-methylenedioxy-camptothecin, taxol, taxol-A, mitotane,
methotrexate, mercaptopurine, lomustine, interferon, 5-fluorouracil
and etopiside. In a more preferred embodiment of this object, the
anti-cancer drug is selected from the group consisting of
20-S-camptothecin, 9-nitro-camptothecin, 9-amino-camptothecin,
10,11-methylenedioxy-camptothecin and taxol. Additionally, in a
preferred embodiment of the present objective, the delivery of the
anticancer drug is performed by a jet nebulizer.
[0010] In another object of the present invention, there is
provided a lipid complex or liposome for delivery of anticancer
drugs via small particle aerosols comprising an anticancer drug and
a lipid, wherein the anticancer drug is at a concentration not
exceeding about 10% of the total volume of the preparation and a
ratio of the anticancer drug to the suitable solvent is in the
range of about 1:1 to about 1:200, preferably in a range of about
1:10 to about 1:100, and most preferably in a range of about 1:10
to about 1:50 (wt:wt) of the preparation. One specific embodiment
of this object includes 9-nitro-camptothecin and
dilauroylphosphatidylcholine in a ratio of about 1:fo to 1:50
wt:wt; with a particularly preferred embodiment having a
9-nitro-camptothecin and dilauroylphosphatidylcholine of about 1:50
wt:wt. In another embodiment, there is provided a liposome for
delivery of anticancer drugs via small particle aerosols comprising
Taxol and dilauroylphosphatidylcholine in a ratio of about 1:30
wt;wt.
[0011] In yet another embodiment of the present invention, there is
provided a liposome produced by the following steps: dissolving a
lipid-soluble anticancer drug in a solvent suitable for dissolving
the anticancer drug to produce dissolved anticancer drug; adding
the dissolved anticancer drug to a dissolved lipid suitable for
formulation and delivery of drugs by aerosol to produce a solution,
wherein the dissolved anticancer drug is at a concentration not
exceeding about 10% of the total volume of the solution and a ratio
of the anticancer drug to the lipid is in the range of about 1:1 to
about 1:200, preferably in a range of about 1:10 to about 1:100,
and most preferably in a range of about 1:10 to about 1:50 (wt:wt)
of the solution; and freezing and lyophilizing the solution. At
this point, the solution may be stored frozen for later use or
dissolved in sterile water for use, producing a suspension, wherein
the concentration of the anticancer drug in the sterile water in
the suspension is no more than about 5.0 mg/ml.
[0012] A preferred embodiment of the above object provides
liposomal preparations of 20-S-camptothecin (CPT),
9-nitrocamptothecin (9-NC) and other lipid soluble camptothecin
derivatives, produced by the following steps: preparing
concentrated stock solutions of said 20-S-camptothecin (CPT),
9-nitrocamptothecin (9-NC) or other-lipid soluble camptothecin
derivatives and lipids in compatible solvents; adding appropriate
volumes of the 20-S-camptothecin (CPT), 9-nitrocamptothecin (9-NC)
or other-lipid soluble camptothecin derivative and lipid
concentrated stock solutions to a volume of t-butanol to form a
second solution, wherein a concentration of said 20-S-camptothecin
(CPT), 9-nitrocamptothecin (9-NC) and other lipid soluble
camptothecin derivatives does not exceed 10% of said second
solution and wherein a ratio of drug to lipid is in the range of
about 1:1 to about 1:200, preferably in a range of about 1:10 to
about 1:100, and most preferably in a range of about 1:10 to about
1:50 (wt:wt) in said second solution; freezing said second
solution; and lyophilizing said second solution to produce a powder
preparation. At this point, the powder preparation may be stored
frozen for later use or dissolved in sterile water producing a
suspension, wherein a concentration of said anticancer drug in said
suspension is no more than about 5 mg/ml.
[0013] A more particular embodiment provides liposomes produced by
the following steps: preparing a concentrated stock solutions of
anticancer drug, for example 100 mg CPT in 1 ml t-butanol or 100 mg
9-NC in DMSO, preparing a stock solution of lipid, for example, 100
mg DLPC in 1 ml butanol; adding appropriate volumes of said
concentrated stock solutions to a volume of t-butanol to form a
second solution wherein a final volume is about 10 ml, a volume of
DMSO, if any, does not exceed 10% (vol:vol) of said final volume, a
concentration of anticancer drug does not exceed 10% (wt:wt) of the
total volume, and wherein a ratio of drug to lipid is in a range of
about 1:1 to about 1:200, preferably in a range of about 1:10 to
about 1:100, and most preferably in a range of about 1:10 to about
1:50 (wt:wt); freezing said second solution; and lyophilizing said
frozen solution to produce a powder preparation. Tthe powder
preparation may then be stored frozen for later use or dissolved in
sterile water producing a suspension. Generally, the concentration
of the anticancer drug in the suspension is no more than about 5
mg/ml.
[0014] Another preferred embodiment of the object above provides a
liposome produced by the following steps: mixing taxol with
synthetic alpha lecithin: dilauroylphosphatidylcholine; dissolving
the taxol-DLPC in t-butanol to produce a preparation; and freezing
and lyophilizing the preparation. Liposomes are produced by adding
sterile, pure water at a temperature above 25.degree. C., wherein
the final concentration of taxol to dilauroylphosphatidylcholine is
about 1:1 to about 1:200, preferably in a range of about 1:10 to
about 1:100, and most preferably in a range of about 1:25 to about
1:40 (wt:wt). In addition to alpha lecithin, other natural or
synthetic lecithins may be used, including but not limited to egg
yolk phosphatidylcholine, hydrogenated soybean phosphatidylcholine,
dimyristophosphatidylcholine,
diolyeolyl-dipalmitoyleolylphosphatidylchol- ine and dipalmitoyl
phosphatidylcholine.
[0015] The efficiency of incorporation of 9-NC and other
camptothecin derivatives and anticancer drugs into liposomes can be
tested by layering an aqueous dispersion of lyophilized
drug-liposome preparation over a Percoll.TM. gradient and
centrifuging. Unincorporated drug collects at the bottom of the
tube, but drug incorporated into liposomes collects at the
interface between the Percoll gradient and the water phase. One
qualitative test of incorporation efficiency is the observation of
drug crystals when the dispersion of drug-liposomes are examined by
microscopy under polarized light. Other methods are also available,
for example, analytical HPLC methods can be used to quantitatively
assess non-encapsulated, crystalized drug.
[0016] 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. These
embodiments are given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The appended drawings have been included herein so that the
above-recited features, advantages and objects of the invention
will become clear and can be understood in detail. 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 should not be considered to limit the scope of the
invention.
[0018] FIG. 1: shows the effect of treatment with 9-NC-DLPC
liposome aerosol on xenografted human breast cancer in nude
mice.
[0019] FIG. 2: shows the effect of further treatment with 9-NC-DLPC
liposome aerosol in mice selected from FIG. 1.
[0020] FIG. 3: shows the effect of treatment with 9-NC-DLPC
liposome aerosol on xenografted human colon cancer (Squires) in
nude mice.
[0021] FIG. 4: shows the effect of treatment with 9-NC-DLPC by
liposome aerosol or by oral administration on the growth of human
lung cancer xenografts (Spark) in nude mice as measured by tumor
volume.
[0022] FIG. 5: shows the output of Taxol-DLPC liposomes (1:30; 1
mg/ml) with the Aerotech II nebulizer.
[0023] FIG. 6: shows the recovery of taxol from the preparation
described in FIG. 5; taxol-DLPC dosage at 15 bpm, 500 ml TV.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The following definitions are provided. Terms not
specifically defined are meant to interpreted as is customary in
the art.
[0025] 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 (See Knight, V., Viral and Mycoplasmal Infections of the
Respiratory Tract. 1973, Lea and Febiger, Phila. Pa., pp. 2).
"Liposome aerosols" consist of 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 man or animals (Knight, V. and Waldrep, J. C.
Liposome Aerosol for Delivery of Asthma Medications; see also In
Kay, B., Allergy and Allergic Diseases, 1997, Blackwell
Publications, Oxford, England, Vol. I pp. 730-741). The size of the
aerosol droplets defined for this application are those described
in U.S. patent 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 MMAD may be less than 1 .mu.m,
such as 0.8 .mu.m. Based on the studies disclosed by the present
invention, the liposomes m a y constitute substantially all of the
volume of the droplet when it has equilibrated to ambient relative
humidity.
[0026] 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.
[0027] As used herein, the terms "20-S-camptothecin" or "CPT"
refers to a plant alkaloid with anti-cancer properties.
[0028] As used herein, the terms "9-nitro-camptothecin" or "9-NC",
"9-amino-camptothecin" or "9-AC," and
"10,11-methylenedioxy-camptothecin" or "MDC" refer to active
anti-cancer drugs derived from 20-S-camptothecin that are insoluble
in water but are soluble in certain lipid solvents.
[0029] As used herein, the terms "dilauroylphosphatidylcholine" or
"DLPC" is a lipid used to formulate liposomes.
[0030] The present invention is directed to a method for treating
cancer, comprising the step of delivering, via small particle
aerosol, aqueous dispersions of anti-cancer drugs to the
respiratory tract of an individual in need of such treatment. In a
preferred embodiment of this object, the anti-cancer drug is
selected from the group consisting of 20-S-camptothecin,
9-nitro-camptothecin, 9-amino-camptothecin and 10, 1
1-methylenedioxy-camptothecin. Additionally, in a preferred
embodiment of the present objective, the delivery of the anticancer
drug is performed by a jet nebulizer.
[0031] Additionally, there is a liposome for delivery of anticancer
drugs via small particle aerosols comprising an anticancer drug and
a lipid, wherein the anticancer drug is at a concentration not
exceeding about 10% of the total volume of the preparation and a
ratio of the anticancer drug to the suitable solvent is in the
range of about 1:1 to about 1:200, preferably in a range of about
1:10 to about 1:100, and most preferably in a range of about 1:10
to about 1:50 (wt:wt) of the preparation. One specific embodiment
of this object includes 9-nitro-camptothecin and
dilauroylphosphatidylcholine in a ratio of about 1:10 to 1:50
wt:wt; with a particularly preferred embodiment having a
9-nitro-camptothecin and dilauroylphosphatidylcholine of about 1:50
wt:wt.
[0032] Taxol is another anti-cancer drug that is lipid soluble and
is incorporated easily into a liposome formulation. The optimal
ratio for taxol and dilauroylphosphatidylcholine is a ratio of
about 1:1 to about 1:200, preferably in a range of about 1:10 to
about 1:100, and more preferably in a range of about 1:25 to about
1:40 (wt:wt). A most preferred embodiment provides a taxol to DLPC
ratio of about 1:30. Taxol is directly dissolved in t-butanol
without use of DMSO as is used for some camptothecins. The Taxol
liposomal preparation is otherwise similar to that of the
camptothecins.
[0033] Further, the present invention is directed to liposomes for
delivery of anticancer drugs via small particle aerosols produced
by the following steps: dissolving a lipid-soluble anticancer drug
in a solvent suitable for dissolving the anticancer drug to produce
dissolved anticancer drug; adding the dissolved anticancer drug to
a dissolved lipid suitable for formulation and delivery of drugs by
aerosol to produce a solution, wherein the dissolved anticancer
drug is at a concentration not exceeding about 10% of the total
volume of the solution and a ratio of the anticancer drug to the
suitable solvent is in the range of about 1:1 to about 1:200 of the
solution; and freezing and lyophilizing the solution. At this
point, the solution may be stored frozen for later use or dissolved
in sterile water to produce a suspension, wherein the concentration
of the anticancer drug in the sterile water in the suspension is no
more than about 5.0 mg/ml. A particular embodiment of the present
invention provides a liposome produced by the following steps:
dissolving a lipid-soluble anticancer drug selected from the group
of 20-S-camptothecin, 9-nitro-camptothecin, 9-amino-camptothecin
and 10,11-methylenedioxy-camptothecin in 100% DMSO to produce
dissolved anticancer drug; and adding said dissolved anticancer
drug to dilauroylphosphatidylcholine dissolved in t-butanol to
produce a solution, wherein the dissolved anticancer drug is at a
concentration not exceeding about 5% of the total volume of the
solution and the ratio of anticancer drug to
dilauroylphosphatidylcholine is about 1:50 in the solution. The
solution is frozen and lyophilized overnight. For use, the
lyophilized solution is suspended in appropriate volumes of
sterile, distilled water. In addition, other methods of liposome
preparation known in the art may be utilized, for example, rotary
evaporation can be used instead of lyophilization.
[0034] 9-NC-DLPC aerosol is prepared by first dissolving the drug
in DMSO; to do so, heating to 50-60.degree. C. may be required.
This solution is added to a larger volume of t-butanol, such that
the DMSO solution does not exceed 5-10% of the total t-butanol and
DMSO volume combined. The organic solvents DMSO and t-butanol are
evaporated from the solution on liquid nitrogen resulting in a
slightly yellow powder. For use, distilled sterile water is added
to the vials containing the drug at the appropriate concentration
and added to the reservoir of the nebulizer. The Aerotech II.TM.
nebulizer CIS-USA, Inc., Bedford Mass. is currently employed, but
other nebulizers with similar aerosol-generating properties may be
used.
[0035] A particular embodiment of the present invention is directed
to a liposome produced by the following steps: mixing taxol with
synthetic alpha lecithin: dilauroylphosphatidylcholine; dissolving
the taxol-DLPC in t-butanol to produce a solution; and freezing and
lyophilizing the solution. Liposomes are produced by adding
sterile, pure water at a temperature above 25.degree. C., wherein
the final concentration of taxol to dilauroylphosphatidylcholine is
about 1:1 to about 1:200, preferably in a range of about 1:10 to
about 1:100, and more preferably in a range of about 1:25 to about
1:40 (wt:wt).
[0036] It is contemplated specifically that the pharmaceutical
compositions of the present invention be used for aerosol delivery
of aqueous dispersions of liposomes carrying anti-cancer drugs to
the respiratory tract. A person having ordinary skill in this art
would readily be able to determine, without undue experimentation,
the appropriate dosages of these aerosol formulations. When used in
vivo for therapy, the aerosol formulations of the present invention
are administered to the patient in therapeutically effective
amounts; i.e., amounts that eliminate or reduce the tumor burden.
As with all pharmaceuticals, the dose and dosage regimen will
depend upon the nature of the cancer (primary or metastatic), the
characteristics of the particular drug (e.g., its therapeutic
index), the patient, the patient's history and other factors. The
amount of aerosol formulation administered will typically be in the
range of about 8 .mu.g/kg of patient weight/day to about 100
.mu.g/kg of patient weight/day for 9-NC. Again, dose and dosage
regimen will vary depending on a number of factors known to those
skilled in the art. See Remington's Pharmaceutical Science, 17th
Ed. (1990) Mark Publishing Co., Easton, Pa.; and Goodman and
Gilman's: The Pharmacological Basis of Therapeutics 8th Ed (1990)
Pergamon Press.
[0037] The small particle liposome aerosol compounds and methods of
treatment of the present invention involve lipid- or water-soluble
anti-cancer drugs incorporated into liposomes. The liposomes are
administered in aqueous dispersions from a jet nebulizer. Various
anti-cancer drugs may be used, including 20-S-camptothecin,
9-nitro-camptothecin, 9-amino-camptothecin,
10,11-methylenedioxy-camptoth- ecin and taxol.
[0038] 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
[0039] Anti-Cancer Drugs CPT and its 9-NC Derivative
[0040] Representative of the anti-cancer drugs of the present
invention are 20-S-camptothecin and its 9-NC derivative. Other
camptothecins such as 9-AC and 10,11-MDC, as well as other
anticancer formulations known in the art, also possess properties
suitable for use. All these anti-cancer drugs may be used in
liposome formulations.
[0041] Table 1 shows a comparison of blood or plasma concentrations
of 9-NC and the time of peak concentrations following oral
administration to humans, dogs and mice. (Hinz, H. R., et. al.
Pharmacokinetics of the in vivo and in vitro conversion of 9-Nitro
20-(S)-camptothecin to 9-Amino-20-(S)-camptothecin in Humans, Dogs
and Mice. Cancer Research 1994 54:3096-3100). Single oral doses
ranged from 0.1 to 1.0 mg/kg for humans and dogs and were 4.1 mg/kg
for mice. There may be some differences in pharmacokinetics between
CPT and 9-NC, but it is a reasonable possibility that the foregoing
differences are predictive of the properties of both agents.
1TABLE 1 Comparison of 9-NC concentrations in blood or plasma
following single oral, intranasal or aerosol dosage Blood or plasma
Time of maximum concentration concentration Oral dose (Single
doses) Human 0.1 mg/kg 483 ng/ml 3.4 h (7 mg/dose) (T1/2 = 2.5 h)
1.0 mg/kg 1247 ng/ml 5.3 h (70 mg/dose) (T1/2 = 4.9 h) Dog 1.0
mg/kg 19.1 ng/ml 0.7 h (10 mg/dose) (T1/2 = 6.4 h) Mouse 4.1 mg/kg
732 ng/ml 6 min (0.124 mg/dose) (T1/2 = 10 h) *Intranasal Mouse 233
.mu.g/kg 213 ng/ml end of instillation (7 .mu.g/mouse) *Inhaled in
liposome aersol (30 min) Mouse 16.2 .mu.g/kg 13.9 ng/ml end of (486
ng/mouse) aerosolization *Intransal and aerosol results from Baylor
Aerosol Laboratory, the others from Hinz, H. R. (see text for
reference)
[0042] Table 2 describes the method of calculating aerosol dosage
in the mouse, and this is the basis for determining dosages cited
in Table 1. Much of the inhaled drug is deposited in the
nasopharynx of the mouse because of the complex nose structure of
rodents. Similarly, nasal instillation leads to high nasopharyngeal
deposition. Material deposited here is promptly transported to the
esophageal orifice and swallowed. About 10-15 percent of the
inhaled aerosol dose, however, will penetrate to the peripheral
lungs.
[0043] In humans with mouth breathing, only small amounts of the
aerosol particles will deposit in the mouth and virtually none will
deposit in the nasopharynx. Material deposited in the central
airways is returned to the pharynx by muco-ciliary action where it
is swallowed. In the case of primary lung tumors which often occur
at bronchial bifurcations, drug will deposit on tumor surfaces and
be adsorbed directly into the tumor mass. Material deposited beyond
the 16th Weibel generation, which is beyond the ciliated
epithelium, will not be moved upward; thus, in tumors within the
peripheral lung parenchyma, the drug will deposit in adjacent areas
and be absorbed directly into the tumor mass. In addition, a major
advantage of the present invention is that drug deposited beyond
the ciliated epithelium is picked up by capillaries in the
interstitial space and the lymphatics of the lungs and will enter
the circulation.
2TABLE 2 Calculation of deposited doses of 9-NC in different
species when administered by aerosol NEBULIZER: AeroTech II; 10
L/min; Measured 1.7% efficiency RESERVOIR: 9-Nitrocamptothecin
(9NC)-DLPC liposomes: 100, 500 or 1,000 .mu.g 9NC/mL and 1:50 (w/w)
mg DLPC/mL Dose Calculation Total Weibel 9NC in 9NC in Drug
Generations: Resv Aerosol Body Wt Rx Time Drug Dose (.mu.g/ Resp
Secr 0-16 17-23 Species Drug MMAD (.mu.g/mL) (.mu.g/L) (kg)
(min/day) K* (.mu.g/kg/min) kg/day) (.mu.g/day) (.mu.g/mL)**
(.mu.g/mL)*** Human [adult] 9NC 0.8 100 1.8 70 15 0.076 0.14 2.0
142 0.68 1.4 5.8 Human [adult] 9NC 0.8 100 1.8 70 30 0.076 0.14 4.1
284 1.35 2.7 11.7 Human [adult] 9NC 1.2 500 8.5 70 15 0.076 0.64
9.6 675 3.21 6.5 27.7 Human [adult] 9NC 1.2 500 8.5 70 30 0.076
0.64 19.3 1353 6.44 13.0 55.5 Human [adult] 9NC 1.5 1,000 15.9 70
15 0.076 1.20 18.0 1259 6.00 12.1 51.6 Human [adult] 9NC 1.5 1,000
15.9 70 30 0.076 1.20 36.0 2518 11.99 24.2 103.3 Dog 9NC 1.2 500
8.5 30 15 0.100 0.85 12.8 383 4.26 8.6 36.7 Dog 9NC 1.2 500 8.5 30
30 0.100 0.85 25.6 767 8.52 17.2 73.4 Cotton Rat 9NC 1.2 500 8.5
0.075 30 0.350 2.98 89.5 6.71 29.8 Cotton Rat 9NC 1.2 500 8.5 0.075
60 0.350 2.98 178.9 13.42 59.6 Cotton Rat 9NC 1.2 500 8.5 0.068 30
0.350 2.98 89.5 6.08 29.8 Mouse 9NC 0.8 100 1.8 0.030 15 0.300 0.54
8.1 0.24 2.7 Mouse 9NC 0.8 100 1.8 0.030 30 0.300 0.54 16.1 0.48
5.4 Mouse 9NC 1.2 500 8.5 0.030 15 0.300 2.55 38.3 1.15 12.8 Mouse
9NC 1.2 500 8.5 0.030 30 0.300 2.56 76.7 2.30 25.6 Mouse 9NC 1.2
500 8.5 0.030 60 0.300 2.56 153.4 4.60 51.1 Mouse 9NC 1.2 500 8.5
0.030 120 0.300 2.56 306.7 9.20 102.2 Mouse 9NC 1.5 1,000 15.9
0.030 15 0.300 4.76 71.4 2.14 23.8 Mouse 9NC 1.5 1,000 15.9 0.030
30 0.300 4.77 143.1 4.29 47.7 *K (human/adult): 0.108 L-min/kg
.times. 0.7; Assuming Nose and mouth breathing, Mouth-only = 1/2 K
(cotton rat): 0.7 L-min/kg .times. 0.5 K (mouse): 1 L-min/kg
.times. 0.3 K (dog, golden): 0.2 L-min/kg .times. 0.5 **Estimated
Peak after each treatment: secretion volume = 1 mL/kg ***Based on
data from Patton (3570) and Philadelphia info; Mouth-only breathing
in man
[0044] Table 3 shows the tissue distribution of CPT following 15
minutes of nebulization in DLPC liposome aerosol. The deposited
dose was calculated to be 486 ng per mouse. The mean concentrations
in lungs and liver were similar with smaller concentrations in the
other sites examined. Table 4 shows tissue distributions over a
period of one hour following intranasal instillation of 7 .mu.g per
mouse (233 .mu.g/kg). Drug was cleared promptly from the lungs so
that by 15 minutes after stopping nebulization only negligible
amounts of drug were present in the lungs. Liver, kidney and spleen
had substantial, amounts of drug initially which gradually
diminished through the one hour of study. Interestingly, blood
concentrations were the least throughout the study. These studies
indicate substantial immediate deposition of drug in the lungs with
rapid clearance to the viscera. The amount of drug contributed by
absorption from swallowed drug is uncertain.
3TABLE 3 Tissue distribution of CPT following 15 minutes inhalation
of CPT liposome aerosol Animal Organ CPT (ng/gm) tissue 1 Lung 52.0
Liver 44.3 Spleen 12.0 Kidney 29.3 Blood 7.1 2 Lung 48.0 Liver 44.3
Spleen 16.4 Kidney 21.8 Blood 8.3 3 Lung 27.0 Liver 21.9 Spleen
11.4 Kidney 18.0 Blood 22.6 4 Lung 77.5 Liver 178.0 Spleen 25.0
Kidney 50.0 Blood 17.7 MEAN (.+-.SD) Lung 51.1 .+-. 20.7 Liver 72.1
.+-. 71.4 Spleen 16.2 .+-. 6.3 Kidney 29.8 .+-. 14.3 Blood 13.9
.+-. 7.5 The CPT concentration in the liposomal preparation in the
nebulizer was 0.2 mg/ml aerosol was generated with an Aerotech II
nebulizer operating at a flow rate of 10 L/min.
[0045]
4TABLE 4 Time dependent organ distribution of CPT after intranasal
administration Time (minutes) 0 15 30 60 Organ ng/gm of tissue Lung
1287 .+-. 657 19 .+-. 3 36 .+-. 23 7 .+-. 3 Liver 651 .+-. 418 255
.+-. 101 66 .+-. 17 34 .+-. 7 Kidney 542 .+-. 174 190 .+-. 57 49
.+-. 13 24 .+-. 21 Spleen 351 .+-. 137 84 .+-. 32 21 .+-. 8 7 .+-.
2 Blood 213 .+-. 19 53 .+-. 20 8 .+-. 3 4 .+-. 2 Remarks: CPT was
administered in liposomal formulation prepared with DLPC with
initial drug concentration 0.2 mg/ml. 35 .mu.L of suspension was
installed to each animal (group of 3 animals was treated for each
time point).
[0046] Table 5 shows the distribution of drug in blood and viscera
following intramuscular injection of CPT. Drug disappeared very
slowly from the site of intramuscular injection in the first 12
hours, with only very small concentrations detected in the liver an
d virtually no drug present at other sites. Concentrations in the
blood were negligible throughout the study. The dose administered
was the same as that given by intranasal instillation. These
findings indicate a speedier and more efficient systemic absorption
of drug after pulmonary administration of drug than by the
intramuscular route. It is likely that deposition in organs and
vascular spaces will increase the opportunity for exposure to
albumin molecules and degradation to the carboxyl form of the
drug.
5TABLE 5 Time dependent organ distribution of CPT after
intramuscular administration Time (minutes) 0 30 60 1200 Organ
ng/gm tissue Lung 2 .+-. 1 4 .+-. 2 3 .+-. 3 4 .+-. 3 Liver 3 .+-.
1 87 .+-. 74 136 .+-. 107 126 .+-. 116 Spleen 2 .+-. 1 18 .+-. 9 11
.+-. 5 5 .+-. 1 Kidney 2 .+-. 0 40 .+-. 14 26 .+-. 7 15 .+-. 5
Blood 2 .+-. 1 12 .+-. 5 8 .+-. 1 4 .+-. 1 Site of inj. 6918 .+-.
265 4309 .+-. 1548 4609 .+-. 1412 1544 .+-. 751 Remarks: CPT
initial stock 5 mg/ml in DMSO was suspended in saline (1.4 .mu.L
stock + 48.6 .mu.L saline) and total 50 .mu.L of suspension was
injected i.m. in each mice. Group of 3 animals was treated for each
time point.
EXAMPLE 2
[0047] Stability of Liposomes Consisting of DLPC and 9-AC
[0048] Table 6 shows the stability of liposomes with fixed weight
ratio of 9-NC and DLPC of 1:50 (w/w) but with increasing
concentrations of constituents from 0.1 mg/ml to 1.0 mg/ml of drug.
The samples were tested under various conditions after vortexing,
but before start of nebulization, after nebulization for 1.5 to 2
minutes (sample taken from the fluid in the reservoir of the
nebulizer) and from the aerosol that was collected in an all-glass
impinger (All-Glass Impinger, Ace Glass Co., Vineland N.J.).
6TABLE 6 Liposome particle size and drug crystal formation in
preparations of 9-NC DLPC liposome formulations Concentration
(mg/ml) Liposome Crystals presence 9NC DLPC Sample particle size,
nm (visual estimation) 0.1 5.0 1 8006 - 2 798 + 3 332 - 0.2 10.0 1
6201 - 2 434 + 3 812 - 1.0 50.0 1 5448 ++ 2 718 ++ 3 816 + 1 sample
was tested after vortexing 2 samples was taken from nebulizer
reservoir after 1.5 to 2 minutes of aerosolization 3 sample was
from aerosol collected in an AGI containing water
[0049] The most stable preparation was the one with the lowest
concentration of constituents. A few crystals appeared in the
reservoir following nebulization. Nebulization caused a ten-fold
reduction in the diameter of the liposome particles, due to the
shear forces associated with nebulization. There was further
reduction in the diameter of liposome particles recovered from the
aerosol. This finding is consistent with selection of smaller
particles for discharge in aerosol. The lack of crystals suggests
that crystals may not nebulize as readily as liposomes. With larger
dosages of liposomes, size reduction following nebulization
occurred, but particles recovered from aerosol were not reduced in
size compared to particles that had been cycled in the reservoir of
the nebulizer.
EXAMPLE 3
[0050] Kinetics of Lactone Ring Opening
[0051] The anti-tumor activity of several of the camptothecins are
diminished following dissolution in aqueous media. This is due to a
hydrolyzable alpha-hydroxy lactone ring (ring E). The change
results from acyl cleavage yielding the biologically inactive
carboxylate form of the molecule. The lactone ring form of the drug
is sheltered in liposomes (Burke, T. G., Biochemistry 1993
32:5352-5364), but the carboxyl form of the drug has high affinity
for human serum albumin. This leads to rapid conversion of lactone
to carboxylate in the presence of human serum albumin, and thus to
loss of anti-cancer activity. Deposition within the lungs on
alveolar surfaces where there is little albumin and/or interaction
with constituents of the liposomes is clearly a factor in
preserving the anti-cancer effect of 9-NC.
EXAMPLE 4
[0052] Effect of 9-NC on Growth of Human Breast Cancer Explants in
Mice
[0053] FIG. 1 shows growth in the area of subcutaneous breast
cancer xenografts during treatment with 9-NC-DLPC liposome aerosol.
There were six 9-NC treated and 5 control mice. Treatments were
given 15 minutes daily, five days per week. The dose was 8.1
.mu.g/kg per day. The deposited dose in the respiratory tract of
each mouse was estimated to be 234 ng per day. The data on tumor
size was normalized and the divergence of tumor size (% initial
tumor growth) in the two groups was highly significant by day 17 of
treatment (P<0.011). After this time, control mice were
sacrificed because of the presence of large necrotic tumor masses.
FIG. 2 shows the course of events with two treated mice which were
subsequently followed with higher doses of drug, following a period
of 16 days without treatment. A few days following restart of
treatment with a five-fold increase in the dose of 9-NC liposome
aerosol, the size of tumors in the treated animals diminished
rapidly, and were no longer visible by the 85th day after start of
treatment.
EXAMPLE 5
[0054] Effect of 9-NC on Human ColoRectal Cancer Xenografts in Nude
Mice
[0055] A similar study was performed in nude mice with human colon
carcinoma xenografts and is shown in FIG. 3. There were 15 treated
and 20 control mice. Ten controls received empty DLPC liposomes and
10 received no treatment. Control animals who received no treatment
or DLPC only showed a consistent and rapid increase in tumor size
until they were sacrificed on day 36. The overall rate of tumor
growth was 7 to 11 times greater in control than in 9-NC-treated
mice. The treated animals were divided into two groups of 10 each.
One group received 77 .mu.g/kg/daily, five days per week throughout
the entire experiment. The other received 77 .mu.g/kg per day five
days per week until day 35 when the dose was increased to 153
.mu.g/kg per day five days per week until day 46 when it was
increased to 307 .mu.g/kg on the same schedule until day 61. There
was slightly less increase in tumor size in the group receiving the
higher dose, but the differences were not statistically
significant, and the data are combined in the figure. Four mice in
the DLPC treatment group were sacrificed because of large tumors or
tumor necrosis before day 61, and six mice in the no treatment
group were sacrificed for the same reasons before day 61. In the
treatment group five mice were sacrificed because of tumor necrosis
or emaciation before day 61. The emaciated mice were in the high
dose group, suggesting drug toxicity. One additional treated mouse
was sacrificed because of rectal prolapse. Based on these findings
of reduced rate of tumor growth, day 28, (P<0.007, Student t
test, 2 tailed) and reduced mortality there is an unequivocal
therapeutic effect of 9-NC treatment (P<0.002.about.chi square
test).
EXAMPLE 6
[0056] Effect of 9-NC on Human Lung Carcinoma Xenografts in Nude
Mice
[0057] Additionally, studies were performed on the effect of
treatment with 9-NC-DLPC via liposome aerosol or via oral
administration on the growth of human lung cancer xenografts
(Spark) in nude mice as measured by tumor volume. Treatment was
initiated about two weeks after tumor implantation. Control animals
showed a rapid increase in volume of tumors. Animals who received
oral dosage with the liposome drug aqueous suspension in doses of
100 .mu.g/kg/day--more than twice the aerosol dosage--did not
respond to treatment. See FIG. 4.
[0058] Both aerosol and oral doses were doubled on day 13. The
increased dosage was followed by decrease in the size of tumors
treated with aerosol, but there was no decrease in size of tumors
in mice given oral treatment. Thus, despite the fact that half or
more of aerosol dosage administered to mice is deposited in the
nose, head, trachea and upper bronchi and is promptly carried by
the mucociliary system to the esophagus where it is swallowed, the
fraction of inhaled drug that is deposited in the lung is
principally responsible for the effect on tumor growth.
[0059] The most likely explanation of the clear efficiency of
aerosol delivery is the rapid entry of the drug to the circulation
where it is returned to the left heart, and then to the aorta an d
peripheral circulation. Thus, the drug would reach the tumors o n
"first pass" without having passed through the liver, which would
remove large amounts of drug from blood.
EXAMPLE 7
[0060] Animal Models
[0061] Nude Mice: Swiss immunodeficient nude mice of the NIH-1 high
fertility strain, bred and housed at the Stehlin Institute were
used for these experiments (Giovella, B. C., et al., Complete
Growth Inhibition of Human Cancer Xenografts in Nude Mice by
Treatment with 20-(S)-Camptothecin, Cancer Research 1991
51:3052-3055).
[0062] Human Cancer Xenografts: Human heterotransplants were
established in nude mice. For an implant, approximately 50 mg of
wet weight of finely minced tumor in 0.5 ml of Eagles minimum
essential medium was injected under the skin over the right dorsal
chest region. The animals were started on treatment with the
experimental drug about 10 days after implantation of tumors.
Tumors of breast cancers were measured in two dimensions (i.e.
area) with calipers, while colon cancers were measured in three
dimensions (i.e. volume) with calipers.
EXAMPLE 8
[0063] Camptothecin Liposome Aerosol Formulation and
Administration
[0064] CPT and 9-nitrocamptothecin were provided by Dr. Beppino
Giovanella of the Stehlin Institute, Houston, Tex. DLPC was
obtained from Avanti Polar Lipids, Pelham, Ala. Aerotech II
nebulizers were obtained from Cis-USA, Inc., Bedford, Mass.
[0065] For formulation of liposomes, 9-NC (100 mg/ml) or CPI (10
mg/ml) was dissolved in 100% DMSO, and added to DLPC dissolved in
tertiary butanol (40.degree. C.) so the final DMSO concentration
did not exceed 5 percent of the total volume and the ratio of drug
to lipid was 1:50 (w/w). The final suspension was clear. If
precipitation occurred, it was reheated to 50-60.degree. C. The
preparation was frozen in liquid nitrogen and lyophilized
overnight. For use the material was dissolved in sterile water to
the appropriate drug concentration, not exceeding 1.0 mg/ml for
either drug. The efficiency of incorporation of drug in the
liposomes was examined qualitatively by microscopic examination
under polarized light. Unincorporated drug was seen as
bi-refringent crystals. The efficiency of incorporation was
examined by centrifugation of aqueous suspensions of liposomes on
Percoll.TM. gradients. One-tenth ml of suspension was layered over
2 ml of gradient and centrifuged at 2000 rpm for 25-30 minutes.
Liposomes layer at the water-Percoll interface, while
unincorporated drug was deposited at the bottom of the tube. Many
other lipids may be substituted for DLPC in the formulation and use
of liposomes for delivery of drugs by aerosol (Sugarman, S. M., et.
al. Lipid-complexed campothecin: formulation and initial
biodistribution and anti-tumor activity studies. Cancer
Chemotherapy Pharmacol. 1996 37:531-538).
EXAMPLE 9
[0066] HPLC Analysis
[0067] The Waters (Milford, Mass.) 710B Wisp automatic injector and
Waters Nova-Pak C18 column at room temperature was used to
quantitate CPT and 9-NC. The mobile phase was 30% acetonitrile and
70% of 0.1% glacial acetic acid. CPT was detected using the Waters
470 scanning fluorescence detector set to an excitation wavelength
of 370 nm and an emission wavelength of 440 nm. 9-NC was detected
using the Waters 440 absorbence detector and monitoring at 254 nm.
The data were analyzed with the Waters Millenium software.
EXAMPLE 10
[0068] Aerosol Droplet Measurement
[0069] The size of aerosol droplets was measured with the Andersen
ACFM non-viable ambient particle sizing sampler (Andersen
Instruments, Inc., Atlanta, Ga.) by methods previously described
(Waldrep, J. C. et. al., J Aerosol Med. 1994 7:135-145). Mass
median aerodynamic diameters and geometric standard deviations were
determined using KaleidaGraph 2.0 (Synergy Software, Reading Pa.).
The aerosol droplets consisted of an aqueous suspension of
liposomes. Liposome diameters were measured in aqueous suspension
with the Model 3300 NICOMP Laser Particle Sizer.
EXAMPLE 11
[0070] Preparation and Efficiency of Taxol-DLPC Liposomes
Formulated by Aerosol Delivery
[0071] Taxol is another anti-cancer drug that is lipid soluble and
is incorporated easily into liposomal formulation. Taxol is
dissolved directly in t-butanol without use of DMSO similar to the
camptothecins. The taxol liposomal preparation is otherwise like
that of the camptothecins.
[0072] The optimal taxol to DLPC ratio was found to be about 1:30
wt:wt. Formulations compatible with nebulization and aerosol
delivery formulations were produced at 1 mg Taxol and with 30 m g
DLPC per ml. For optimized Taxol-DLPC liposomes, 5 mg of Taxol was
mixed with 150 mg of synthetic alpha-lecithin:
dilauroylphosphatidylcholine (DLPC). Working at 37.degree. C., the
drug/DLPC was mixed in 20 mls of tertiary butanol with stirring.
After mixing, the drug/lipid preparation was pipetted into glass
vials, frozen rapidly, and lyophilized overnight to remove the
t-butanol leaving a powder. Multi-lamellar liposomes were produced
by adding 5 mls of ultra pure water above the DLPC phase transition
temperature (Tc) at 25.degree. C. to deliver a final standard drug
concentration of 1 mg Taxol: 30 mg DLPC per ml. The mixture was
incubated for 30 minutes at room temperature with intermittent
mixing to produce multilamellar vesicular (MLV) liposomes. Aliquots
were removed for determination of drug concentration by HPLC.
[0073] FIG. 5 shows the particle size distribution of taxol-DLPC
liposome aerosol with a MMAD of 1.4 .mu.m and a geometric standard
deviation of 2.0 FIG. 6 shows the recovery of taxol from the
preparation described in FIG. 5. In the lung model (Harvard
Respirator) used to measure the output of taxol from the nebulizer
(Aerotech II), 87.75 liters of aerosol were sampled yielding 3000
.mu.g of taxol. There was thus an aerosol concentration of 34.2
.mu.g/l. From this information, the dose of aerosol deposited in
the respiratory tract following inhalation can be calculated.
[0074] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. Further, these patents and publications are
incorporated by reference herein to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
[0075] One skilled in the art will appreciate readily that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those objects,
ends and advantages inherent herein. 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.
* * * * *