U.S. patent application number 09/888781 was filed with the patent office on 2001-10-25 for porous particles for deep lung delivery.
This patent application is currently assigned to The Penn Research Foundation, Inc.. Invention is credited to Ben-Jebria, Abdellaziz, Caponetti, Giovanni, Edwards, David A., Hanes, Justin, Hrkach, Jeffrey S., Langer, Robert S., Lotan, Noah.
Application Number | 20010033829 09/888781 |
Document ID | / |
Family ID | 24629425 |
Filed Date | 2001-10-25 |
United States Patent
Application |
20010033829 |
Kind Code |
A1 |
Edwards, David A. ; et
al. |
October 25, 2001 |
Porous particles for deep lung delivery
Abstract
Improved porous particles for drug delivery to the pulmonary
system, and methods for their synthesis and administration are
provided. In a preferred embodiment, the porous particles are made
of a biodegradable material and have a mass density less than 0.4
g/cm.sup.3/. The particles may be formed of biodegradable materials
such as biodegradable polymers. For example, the particles may be
formed of a functionalized polyester graft copolymer consisting of
a linear .alpha.-hydroxy-acid polyester backbone having at least
one amino acid group incorporated therein and at least one
poly(amino acid) side chain extending from an amino acid group in
the polyester backbone. In one embodiment, porous particles having
a relatively large mean diameter, for example greater than 5 .mu.m,
can be used for enhanced delivery of a therapeutic agent to the
alveolar region of the lung. The porous particles incorporating a
therapeutic agent may be effectively aerosolized for administration
to the respiratory tract to permit systemic or local delivery of
wide variety of therapeutic agents.
Inventors: |
Edwards, David A.; (Boston,
MA) ; Caponetti, Giovanni; (Somerville, MA) ;
Hrkach, Jeffrey S.; (Cambridge, MA) ; Lotan,
Noah; (Haifa, IL) ; Hanes, Justin; (Baltimore,
MD) ; Langer, Robert S.; (Newton, MA) ;
Ben-Jebria, Abdellaziz; (State College, PA) |
Correspondence
Address: |
Anabela Cristina Taylor, Esq.
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
Two Militia Drive
Lexington
MA
02421-4799
US
|
Assignee: |
The Penn Research Foundation,
Inc.
University Park
PA
|
Family ID: |
24629425 |
Appl. No.: |
09/888781 |
Filed: |
June 25, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09888781 |
Jun 25, 2001 |
|
|
|
09569153 |
May 11, 2000 |
|
|
|
6254854 |
|
|
|
|
09569153 |
May 11, 2000 |
|
|
|
08655570 |
May 24, 1996 |
|
|
|
Current U.S.
Class: |
424/43 ; 514/1.7;
514/1.8; 514/11.3; 514/13.7; 514/17.7; 514/2.4; 514/3.7; 514/44A;
514/5.9; 514/54; 514/9.7 |
Current CPC
Class: |
A61K 9/0075 20130101;
Y10S 514/826 20130101; A61K 31/137 20130101; A61K 9/1647 20130101;
Y10S 514/851 20130101 |
Class at
Publication: |
424/43 ; 514/3;
514/44; 514/54 |
International
Class: |
A61K 038/28; A61K
009/00; A61K 031/715; A61K 048/00 |
Claims
What is claimed is:
1. An essentially dry composition comprising: amorphous
biodegradable particles for delivering a therapeutic, prophylactic
or diagnostic agent to the deep lung, wherein the particles have a
mass density less than 0.4 g/cm.sup.3, a mass mean diameter between
5 .mu.m and 30 .mu.m and an aerodynamic diameter less than the mass
mean diameter and between 1 and 5 .mu.m, and comprise a
therapeutic, prophylactic or diagnostic agent.
2. The composition of claim 1 wherein the particles further
comprise a pharmaceutically acceptable excipient.
3. The composition of claim 2 wherein the pharmaceutically
acceptable excipient is selected from the group consisting of
organic materials, inorganic materials, surfactants, polymers and
any combinations thereof.
4. The composition of claim 3 wherein the pharmaceutically
acceptable excipient is a non-polymeric organic material.
5. The composition of claim 4 wherein the agent is selected from
the group consisting of proteins, peptides, polysaccharides,
lipids, nucleic acids and combinations thereof.
6. The composition of claim 5 wherein the agent is a protein.
7. The composition of claim 4 wherein the agent is insulin.
8. The composition of claim 4 wherein the agent is selected from
the group consisting of antibodies, antigens, antibiotics,
antivirals, hormones, vasoactive agents, neuroactive agents,
anticoagulants, immunomodulating agents, cytotoxic agents,
antisense agents and genes.
9. The composition of claim 4 wherein the agent is an agent for the
treatment of asthma, emphysema, or cystic fibrosis.
10. The composition of claim 4 wherein a targeting molecule is
attached to the particle.
11. The composition of claim 4 further comprising a
pharmaceutically acceptable carrier for administration of the
lungs.
12. The composition of claim 3 wherein the pharmaceutically
acceptable excipient is a surfactant.
13. The composition of claim 12 wherein the agent is selected from
the group consisting of proteins, peptides, polysaccharides,
lipids, nucleic acids and combinations thereof.
14. The composition of claim 13 wherein the agent is a protein.
15. The composition of claim 12 wherein the agent is insulin.
16. The composition of claim 12 wherein the agent is selected from
the group consisting of antibodies, antigens, antibiotics,
antivirals, hormones, vasoactive agents, neuroactive agents,
anticoagulants, immunomodulating agents, cytotoxic agents,
antisense agents and genes.
17. The composition of claim 12 wherein the agent is an agent for
the treatment of asthma, emphysema, or cystic fibrosis.
18. The composition of claim 12 wherein a targeting molecule is
attached to the particle.
19. The composition of claim 12 further comprising a
pharmaceutically acceptable carrier for administration of the
lungs.
20. The composition of claim 3 wherein the pharmaceutically
acceptable excipient is a polymeric material.
21. The composition of claim 3 wherein the pharmaceutically
acceptable excipient includes a functionalized polyester graft
copolymer comprising: a linear .alpha.-hydroxy-acid polyester
backbone having at least one amino acid group incorporated therein;
and at least one poly(amino acid) side chain extending from an
amino acid group in the polyester backbone.
22. The composition of claim 21 wherein the polyester is a polymer
of an .alpha.-hydroxy acid selected from the group consisting of
lactic acid, glycolic acid, hydroxybutyric acid and valeric acid,
and copolymers thereof.
23. The composition of claim 21 wherein the linear
.alpha.-hydroxy-acid polyester backbone comprises a poly(lactic
acid) polymer having at least one lysine group incorporated
therein; and wherein the poly(amino acid) extends from at least one
lysine group in the polyester backbone.
24. The composition of claim 23 wherein the poly(amino acid) is a
polymer of an amino acid selected from the group consisting of
aspartic acid, lysine, alanine, and any combination thereof.
25. The composition of claim 23 wherein the polymer is selected
from the group consisting of poly(lactic
acid-co-lysine-graft-lysine) and poly(lactic
acid-co-lysine-graft-alanine-lysine).
26. The composition of claim 21 wherein the particles comprise a
blend of a polyester and the functionalized polyester graft
copolymer.
27. The composition of claim 26 wherein the particles comprise a
blend of poly(lactic acid-co-lysine-graft-lysine) and poly(lactic
acid-co-glycolic acid-block-ethylene oxide).
28. The composition of claim 21 wherein the poly(amino acid) side
chain includes from about 10 to about 100 amino acids.
29. The composition of claim 21 wherein the polymer has an amino
acid content of about 7-72%.
30. The composition of claim 3 wherein the pharmaceutically
acceptable excipient is selected from the group consisting of
polyamides, polycarbonates, polyalkylenes, poly(alkylene glycol),
poly(alkylene oxide), poly(ethylene terephthalate), polyvinyl
alsohols, polyvinyl ehters, polyvinyl esters, polymers or acrylic
acid, polymers of methacrylic acid, celluloses, polysacchardies,
peptides, proteins, blends and copolymers tehreof.
31. A method for delivery to the deep lung of the pulmonary system
comprising: administering to the respiratory tract of a patient in
need of treatment, prophylaxis or diagnosis an effective amount of
an essentially dry composition comprising amorphous biodegradable
particles for delivering a therapeutic, prophylactic or diagnostic
agent; wherein the particles have a mass density less than 0.4
g/cm.sup.3, a mass mean diameter between 5 .mu.m and 30 .mu.m and
an aerodynamic diameter less than the mass mean diameter and
between 1 .mu.m and 5 .mu.m, and comprise a therapeutic,
prophylactic or diagnostic agent.
32. The method of claim 31 wherein the particles further comprise a
pharmaceutically acceptable excipient.
33. The method of claim 31 wherein the pharmaceutically acceptable
excipient is selected from the group consisting of organic
materials, inorganic materials, surfactants, polymers and any
combinations thereof.
34. The method of claim 33 wherein the pharmaceutically acceptable
excipient is a non-polymeric organic material.
35. The method of claim 33 wherein the agent is selected from the
group consisting of proteins, peptides, polysaccharides, lipids,
nucleic acids and combinations thereof.
36. The method of claim 35 wherein the agent is a protein.
37. The method of claim 33 wherein the agent is insulin.
38. The method of claim 33 wherein the agent is selected from the
group consisting of antibodies, antigens, antibiotics, antivirals,
hormones, vasoactive agents, neuroactive agents, anticoagulants,
immunomodulating agents, cytotoxic agents, antisense agents and
genes.
39. The method of claim 33 wherein the agent is an agent for the
treatment of asthma, emphysema, or cystic fibrosis.
40. The method of claim 33 wherein a targeting molecule is attached
to the particle.
41. The method of claim 33 wherein the composition is administered
together with a pharmaceutically acceptable carrier for
administration to the lungs.
42. The method of claim 41 wherein the carrier is a liquid or a
powder.
43. The method of claim 33 wherein delivery is by dry powder
inhaler.
44. The method of claim 33 wherein the pharmaceutically acceptable
excipient is a surfactant.
45. The method of claim 44 wherein the agent is selected from the
group consisting of proteins, peptides, polysaccharides, lipids,
nucleic acids and combinations thereof.
46. The method of claim 45 wherein the agent is a protein.
47. The method of claim 44 wherein the agent is insulin.
48. The method of claim 44 wherein the agent is selected from the
group consisting of antibodies, antigens, antibiotics, antivirals,
hormones, vasoactive agents, neuroactive agents, anticoagulants,
immunomodulating agents, cytotoxic agents, antisense agents and
genes.
49. The method of claim 44 wherein the agent is an agent for the
treatment of asthma, emphysema, or cystic fibrosis.
50. The method of claim 44 wherein a targeting molecule is attached
to the particle.
51. The method of claim 44 wherein the composition is administered
together with a pharmaceutically acceptable carrier for
administration of the lungs.
52. The method of claim 51 wherein the carrier is a liquid or a
powder.
53. The method of claim 44 wherein delivery is by dry powder
inhaler.
54. The method of claim 33 wherein the pharmaceutically acceptable
excipient is a polymeric material.
55. The method of claim 33 wherein the pharmaceutically acceptable
excipient includes a functionalized polyester graft copolymer
comprising: a linear .alpha.-hydroxy-acid polyester backbone having
at least one amino acid group incorporated therein; and at least
one poly(amino acid) side chain extending from an amino acid group
in the polyester backbone.
56. The method of claim 55 wherein the polyester is a polymer of an
.alpha.-hydroxy acid selected from the group consisting of lactic
acid, glycolic acid, hydroxybutyric acid and valeric acid, and
copolymers thereof.
57. The method of claim 55 wherein the linear .alpha.-hydroxy-acid
polyester backbone comprises a poly(lactic acid) polymer having at
least one lysine group incorporated therein; and wherein the
poly(amino acid) extends from at least one lysine group in the
polyester backbone.
58. The method of claim 57 wherein the poly(amino acid) is a
polymer of an amino acid selected from the group consisting of
aspartic acid, lysine, alanine, and any combination thereof.
59. The method of claim 57 wherein the polymer is selected from the
group consisting of poly(lactic acid-co-lysine-graft-lysine) and
poly(lactic acid-co-lysine-graft-alanine-lysine).
60. The method of claim 55 wherein the particles comprise a blend
of a polyester and the functionalized polyester graft
copolymer.
61. The method of claim 60 wherein the particles comprise a blend
of poly(lactic acid-co-lysine-graft-lysine) and poly(lactic
acid-co-glycolic acid-block-ethylene oxide).
62. The method of claim 55 wherein the poly(amino acid) side chain
includes from about 10 to about 100 amino acids.
63. The method of claim 55 wherein the polymer has an amino acid
content of about 7-72%.
64. The method of claim 33 wherein the pharmaceutically acceptable
excipient is selected from the group consisting of polyamides,
polycarbonates, polyalkylenes, poly(alkylene glycol), poly(alkylene
oxide), poly(ethylene terephthalate), polyvinyl alsohols, polyvinyl
ehters, polyvinyl esters, polymers or acrylic acid, polymers of
methacrylic acid, celluloses, polysacchardies, peptides, proteins,
blends and copolymers tehreof.
Description
RELATED APPLICATION(S)
[0001] This application is a Continuation of U.S. application Ser.
No. 09/569,153 filed on May 11, 2000 which is a Continuation of
U.S. application Ser. No. 08/655,570, filed May 24, 1996; the
entire teachings of both are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to porous polymeric
particles for drug delivery to the pulmonary system.
[0003] Biodegradable polymeric particles have been developed for
the controlled-release and delivery of protein and peptides drugs.
Langer, R., Science, 249: 1527-1533 (1990). Examples include the
use of biodegradable particles for gene therapy (Mulligan, R. C.
Science, 260: 926-932 (1993)) and "single-shot" vaccine delivery
(Eldridge et al., Mol. Immunol., 28: 287-294 (1991)) for
immunization. Protein and peptide delivery via degradable particles
is restricted due to low bioavailability in the blood stream, since
macromolecules and/or microparticles tend to poorly permeate
organ-blood barriers of the human body, particularly when delivered
either orally or invasively.
[0004] Aerosols for the delivery of therapeutic agents to the
respiratory tract have been developed. The respiratory tract
includes the upper airways, including the oropharynx and larynx,
followed by the lower airways, which include the trachea followed
by bifurcations into the bronchi and bronchioli. The upper and
lower airways are called the conductive airways. The terminal
bronchioli then divide into respiratory bronchioli which then lead
to the ultimate respiratory zone, the alveoli, or deep lung. Gonda,
I. "Aerosols for delivery of therapeutic and diagnostic agents to
the respiratory tract," in Critical Reviews in Therapeutic Drug
Carrier Systems, 6: 273-313, (1990). The deep lung, or alveoli, are
the primary target of inhaled therapeutic aerosols for systemic
delivery.
[0005] Inhaled aerosols have been used for the treatment of local
lung disorders including asthma and cystic fibrosis (Anderson, et
al., Am. Rev. Rev. Respir. Dis., 140: 1317-1324 (1989)) and have
potential for the systemic delivery of peptides and proteins as
well (Patton and Platz, Advanced Drug Delivery Reviews, 8: 179-196
(1992)). However, pulmonary drug delivery strategies present many
difficulties for the delivery of macromolecules; these include
protein denaturation during aerosolization, excessive loss of
inhaled drug in the oropharyngeal cavity (typically exceeding 80%),
poor control over the site of deposition, irreproducibility of
therapeutic results owing to variations in breathing patterns, the
quick absorption of drug potentially resulting in local toxic
effects, and phagocytosis by lung macrophages.
[0006] Local and systemic inhalation therapies can often benefit
from a relative slow controlled release of the therapeutic agent.
Gonda, I., "Physico-chemical principles in aerosol delivery," in:
Topics in Pharmaceutical Science 1991, D. J. A. Crommelin and K. K.
Midha, Eds., Stuttgart: Medpharm Scientific Publishers, pp. 95-117,
1992. Slow release from a therapeutic aerosol can prolong the
residence of an administered drug in the airways or acini, and
diminish the rate of drug appearance in the blood stream. Also,
patient compliance is increased by reducing the frequency of
dosing. Langer, R., Science, 249: 1527-1533 (1990); and Gonda, I.,
"Aerosols for delivery of therapeutic and diagnostic agents to the
respiratory tract," in Critical Reviews in Therapeutic Drug Carrier
Systems, 6: 273-313, 1990.
[0007] The human lungs can remove or rapidly degrade hydrolytically
cleavable deposited aerosols over periods ranging from minutes to
hours. In the upper airways, ciliated epithelia contribute to the
"mucociliary excalator" by which particles are swept from the
airways toward the mouth. Pavia, D., "LungMucociliary Clearance,"
in Aerosols and the Lung: Clinical and Experimental Aspects,
Clarke, S. W. and Pavia, D., Eds., Butterworths, London, 1984. In
the deep lungs, alveolar macrophages are capable of phagocytosing
particles soon after their deposition. Warheit, M. B. and Hartsky,
M. A., Microscopy Res. Tech., 26: 412-422 (1993); and Brain, J. D.,
"Physiology and Pathophysiology of Pulmonary Macrophages," in The
Reticuloendothelial System, S. M. Reichard and J. Filkins, Eds.,
Plenum, New York, pp. 315-327, 1985. As the diameter of particles
exceeds 3 .mu.m, there is increasingly less phagocytosis by
macrophages. However, increasing the particle size also minimizes
the probability of particles (possessing standard mass density)
entering the airways and acini due to excessive deposition in the
oropharyngeal or nasal regions. Heyder, J., et al., J. Aerosol
Aci., 17: 811-825 (1986). An effective slow-release inhalation
therapy requires a means of avoiding or suspending the lung's
natural clearance mechanisms until drugs have been effectively
delivered.
[0008] Therapeutic dry-powder aerosols have been made as solid
(macroscopically nonporous) particles, with mean diameters less
than approximately 5 .mu.m to avoid excessive oropharyngeal
deposition. Ganderton, D., J. Biopharmaceutical Sciences 3: 101-105
(1992); and Gonda, I., "Physico-Chemical Principles in Aerosol
Delivery," in Topics in Pharmaceutical Sciences 1991, Commelin, D.
J. and K. K. Midha, Eds., Medpharm Scientific Publishers,
Stuttgart, pp. 95-115, 1992.
[0009] There is a need for improved inhaled aerosols for pulmonary
delivery of therapeutic agents. There is a need for the development
of drug carriers which are capable of delivering the drug in an
effective amount into the airways or the alveolar zone of the lung.
There further is a need for the development of drug carriers for
use as inhaled aerosols which are biodegradable and are capable of
controlled release within the airways or in the alveolar zone of
the lung.
[0010] It is therefore an object of the present invention to
provide improved carriers for the pulmonary delivery of therapeutic
agents. It is a further object of the invention to provide inhaled
aerosols which are effective carriers for delivery of therapeutic
agents to the deep lung. It is another object of the invention to
provide carriers for pulmonary delivery which avoid phagocytosis in
the deep lung. It is a further object of the invention to provide
carriers for pulmonary drug delivery which are capable of
biodegrading and releasing the drug at a controlled rate.
SUMMARY OF THE INVENTION
[0011] Improved porous particles for drug delivery to the pulmonary
system, and methods for their synthesis and administration are
provided. In a preferred embodiment, the porous particles are made
of a biodegradable material and have a mass density less than 0.4
g/cm.sup.3. The particles may be formed of biodegradable materials
such as biodegradable polymers. For example, the particles may be
formed of a functionalized polyester graft copolymer consisting of
a linear .alpha.-hydroxy-acid polyester backbone having at least
one amino acid group incorporated therein and at least one
poly(amino acid) side chain extending from an amino acid group in
the polyester backbone. In one embodiment, porous particles having
a relatively large mean diameter, for example, greater than 5
.mu.m, can be used for enhanced delivery of a therapeutic agent to
the airways or the alveolar region of the lung. The porous
particles incorporating a therapeutic agent may be effectively
aerosolized for administration to the respiratory tract to permit
systemic or local delivery of a wide variety of therapeutic
agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a graph comparing total particle mass of porous
and non-porous particles deposited on the nonrespirable and
respirable stages of a cascade impactor following
aerosolization.
[0013] FIG. 2 is a graph comparing total particle mass deposited in
the trachea and after the carina (lungs) in rat lungs and upper
airways following intratracheal aerosolization during forced
ventilation of porous PLAL-Lys particles and non-porous PLAL
particles.
[0014] FIG. 3 is a graph comparing total particle recovery of
porous PLAL-Lys particles and non-porous PLAL particles in rat
lungs and following broncho alveolar lavage.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Biodegradable particles for improved delivery of therapeutic
agents to the respiratory tract are provided. The particles can be
used in one embodiment for controlled systemic or local drug
delivery to the respiratory tract via aerosolization. In one
embodiment, the particles are porous particles having a mass
density less than 1.0 g/cm.sup.3, preferably less than about 0.4
g/cm.sup.3. The porous structure permits deep lung delivery of
relatively large diameter therapeutic aerosols, for example greater
than 5 .mu.m in mean diameter. The particles also may include a
rough surface texture which can reduce particle agglomeration and
provide a highly flowable powder, which is ideal for aerosolization
via dry powder inhaler devices, leading to lower deposition in the
mouth and throat.
Mass Density and Diameter of Porous Particles
[0016] As used herein the term "porous particles" refers to
particles having a total mass density less than about 0.4
g/cm.sup.3. The mean diameter of the particles can range, for
example, from about 100 nm to 15 .mu.m, or larger depending on
factors such as particle composition, and the targeted site of the
respiratory tract for deposition of the particle.
Particle Size
[0017] In one embodiment, particles which are macroscopically
porous, and incorporate a therapeutic drug, and having a mass
density less than about 0.4 g/cm.sup.3, can be made with mean
diameters greater than 5 .mu.m, such that they are capable of
escaping inertial and gravitational deposition in the oropharyngeal
region, and are targeted to the airways or the deep lung. The use
of larger porous particles is advantageous since they are able to
aerosolize more efficiently than smaller, non-porous aerosols such
as those currently used for inhalation therapies.
[0018] The large (>5 .mu.m) porous particles are also
advantageous in that they can more successfully avoid phagocytic
engulfment by alveolar macrophages and clearance from the lungs, in
comparison to smaller non-porous particles, due to size exclusion
of the particles from the phagocytes' cytosolic space. Phagocytosis
of particles by alveolar macrophages diminishes precipitously as
particle diameter increases beyond 3 .mu.m. Kawaguchi, H. et al.,
Biomaterials 7: 61-66 (1986); Krenis, L. J. and Strauss, B., Proc.
Soc. Exp. Med., 107: 748-750 (1961); and Rudt, S. and Muller, R.
H., J. Contr. Rel., 22: 263-272 (1992). The porous particles thus
are capable of a longer term release of a therapeutic agent.
Following inhalation, porous degradable particles can deposit in
the lungs (due to their relatively low mass density), and
subsequently undergo slow degradation and drug release, without the
majority of the particles being phagocytosed by alveolar
macrophages. The drug can be delivered relatively slowly into the
alveolar fluid, and at a controlled rate into the blood stream,
minimizing possible toxic responses of exposed cells to an
excessively high concentration of the drug. The porous polymeric
particles thus are highly suitable for inhalation therapies,
particularly in controlled release applications. The preferred
diameter for porous particles for inhalation therapy is greater
than 5 .mu.m, for example between about 5-15 .mu.m.
[0019] The particles also may be fashioned with the appropriate
material, diameter and mass density for localized delivery to other
regions of the repiratory tract such as the upper airways. For
example higher density or larger particles may be used for upper
airway delivery.
Particle Deposition
[0020] Inertial impaction and gravitational settling of aerosols
are predominant deposition mechanisms in the airways and acini of
the lungs during normal breathing conditions. Edwards, D. A., J.
Aerosol Sci. 26: 293-317 (1995). Both deposition mechanisms
increase in proportion to the mass of aerosols and not to particle
volume. Since the site of aerosol deposition in the lungs is
determined by the intrinsic mass of the aerosol (at least for
particles of mean aerodynamic diameter greater than approximately 1
.mu.m), diminishing particle mass density by increasing particle
porosity permits the delivery of larger particles into the lungs,
all other physical parameters being equal.
[0021] The low mass porous particles have a small aerodynamic
diameter in comparison to the actual sphere diameter. The
aerodynamic diameter, d.sub.aer, is related to the actual sphere
diameter, d (Gonda, I., "Physico-chemical principles in aerosol
delivery," in Topics in Pharmaceutical Sciences 1991 (eds. D. J. A.
Crommelin and K. K. Midha), pp. 95-117, Stuttgart: Medpharm
Scientific Publishers, 1992) by the formula:
d.sub.aer=d{square root}.rho.
[0022] where the particle mass density .rho. is in units of
g/cm.sup.3. Maximal deposition of monodisperse aerosol particles in
the alveolar region of the human lung (.about.60%) occurs for an
aerodynamic diameter of approximately d.sub.aer=3 .mu.m. Heyder, J.
et al., J. Aerosol Sci., 17: 811-825 (1986). Due to their small
mass density, the actual diamter d of porous particles comprising a
mondisperse inhaled powder that will exhibit maximum deep-lung
deposition is:
d=3/{square root}.rho. .mu.m (where .rho.<1);
[0023] where d is always greater than 3 .mu.m. For example, porous
particles that display a mass density, .rho.=0.1 g/cm.sup.3, will
exhibit a maximum deposition for particles having actual diameters
as large as 9.5 .mu.m. The increased particle size diminishes
interparticle adhesion forces. Visser, J., Powder Technology,
58:1-10. Thus, large particle size increases efficiency of
aerosolization to the deep lung for particles of low mass
density.
Particle Materials
[0024] The porous particles preferably are biodegradable and
biocompatible, and optionally are capable of biodegrading at a
controlled rate for delivery of a drug. The porous particles can be
made of any material which is capable of forming a porous particle
having a mass density less than about 0.4 g/cm.sup.3. Both
inorganic and organic materials can be used. For example, ceramics
may be used. Other non-polymeric materials may be used which are
capable of forming porous particles as defined herein.
Polymeric Particles
[0025] The particles may be formed from any biocompatible, and
preferably biodegradable polymer, copolymer, or blend, which is
capable of forming porous particles having a density less than
about 0.4 g/cm.sup.3.
[0026] Surface eroding polymers such as polyanhydrides may be used
to form the porous particles. For example, polyanhydrides such as
poly[(p-carboxyphenoxy)hexane anhydride] ("PCPH") may be used.
Biodegradable polyanhydrides are described, for example, in U.S.
Pat. No. 4,857,311, the disclosure of which is incorporated herein
by reference.
[0027] In another embodiment, bulk eroding polymers such as those
based on polyesters including poly(hydroxy acids) can be used. For
example, polyglycolic acid ("PGA") or polylactic acid ("PLA") or
copolymers thereof may be used to form the porous particles,
wherein the polyester has incorporated therein a charged or
functionalizable group such as an amino acid as described
below.
[0028] Other polymers include polyamides, polycarbonates,
polyalkylenes such as polyethylene, polypropylene, poly(ethylene
glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly
vinyl compounds such as polyvinyl alcohols, polyvinyl ethers, and
polyvinyl esters, polymers of acrylic and methacrylic acids,
celluloses, polysaccharides, and peptides or proteins, or
copolymers or blends thereof which are capable of forming porous
particles with a mass density less than about 0.4 g/cm.sup.3.
Polymers may be selected with or modified to have the appropriate
stability and degradation rates in vivo for different controlled
drug delivery applications.
Polyester Graft Copolymers
[0029] In one preferred embodiment, the porous particles are fomed
from functionalized polyester graft coppolymers, as described in
Hrkach et al., Macromolecules, 28:4736-4739 (1995); and Hrkach et
al., "Poly(L-Lactic acid-co-amino acid) Graft Copolymers: A Class
of Functional Biodegradable Biomaterials" in Hydrogel and
Biodegradable Polymers for Bioapplications, ACS Symposium Series
No. 627, Raphael M. Ottenbrite et al., Eds., American Chemical
Society, Chapter 8, pp. 93-101, 1996, the disclosures of which are
incorporated herein by reference. The functionalized graft
copolymers are copolymers of polyesters, such as poly(glycolic
acid) or poly(lactic acid), and another polymer including
functionalizable or ionizable groups, such as a poly(amino acid).
In a preferred embodiment, comb-like graft copolymers are used
which include a linear polyester backbone having amino acids
incorporated therein, and poly(amino acid) side chains which extend
from the amino acid groups in the polyester backbone. The
polyesters may be polymers of a .alpha.-hydroxy acids such as
lactic acid, glycolic acid, hydroxybutyric acid and valeric acid,
or derivatives or combinations thereof. The inclusion of ionizable
side chains, such as polylysine, in the polymer has been found to
enable the formation of more highly porous particles, using
techniques for making microparticles known in the art, such as
solvent evaporation. Other ionizable groups, such as amino or
carboxyl groups, may be incorporated, covalently or noncovalently,
into the polymer to enhance porosity. For example, polyaniline
could be incorporated into the polymer.
[0030] An exemplary polyester graft coppolymer, which may be used
to form porous polymeric particles is the graft copolymer,
poly(lactic acid-co-lysine-graft-lysine) ("PLAL-Lys"), which has a
polyester backbone consisting of poly(L-lactic acid-co-Z-L-lysine)
(PLAL), and grafted lysine chains. PLAL-Lys is a comb-like graft
copolymer having a backbone composition, for example, of 98 mol %
lactic acid and 2 mol % lysine and poly(lysine) side chains
extending from the lysine sites of the backbone.
[0031] PLAL-Lys may be synthesized as follows. First, the PLAL
copolymer consisting of L-lactic acid units and approximately 1-2%
N.epsilon.-carbobenzoxy-L-lysine (Z-L-lysine) units is synthesized
as described in Barrera et al., J. Am. Chem. Soc., 115:11010
(1993). Removal of the Z protecting groups of the randomly
incorporated lysine groups in the polymer chain of PLAL yields the
free .epsilon.-amine which can undergo further chemical
modification. The use of the poly(lactic acid) copolymer is
advantageous since it biodegrades into lactic acid and lysine,
which can be processed by the body. The existing backbone lysine
groups are used as initiating sites for the growth of poly(amino
acid) side chains.
[0032] The lysine .epsilon.-amine groups of linear poly(L-lactic
acid-co-L-lysine) copolymers initiate the ring opening
polymerization of an amino acid N-carboxyanhydride (NCA) to produce
poly(L-lactic acid-co-amino acid) comb-like graft copolynmers. In a
preferred embodiment, NCAs are synthesized by reacting the
appropriate amino acid with triphosgene. Daly et al., Tetrahedron
Lett., 29:5859 (1988). The advantage of using triphosgene over
phosgene gas is that it is a solid material, and therefore, safer
and easier to handle. It also is soluble in THF and hexane so any
excess is efficiently separated from the NCAs.
[0033] The ring opening polymerization of amino acid
N-carboxyanhydrides (NCAs) is initiated by nucleophilic initiators
such as amines, alcohols, and water. The primary amino initiated
ring opening polymerization of NCAs allows good control over the
degree of polymerization when the monomer to initiator ratio (M/I)
is less than 150. Kricheldorf, H. R. in Models of Biopolymers by
Ring-Opening Polymerization, Penczek, S., Ed., CRC Press, Boca
Raton, 1990, chapter 1; Kricheldorf, H. R.,
.alpha.-Aminoacid-N-Carboxy-Anhydrides and Related Heterocycles,
Springer-Verlag, Berlin, 1987; and Imanishi, Y. in Ring-Opening
Polymerization, Ivin, K. J. and Saegusa, T., Eds., Elsevier,
London, 1984, Volume 2, chapter 8. Methods for using lysine
.epsilon.-amine groups as polymeric initiators for NCA
polymerizations are described in the art. Sela, M. et al., J. Am.
Chem. Soc., 78:746 (1956).
[0034] In the reaction of an amino acid NCA with PLAL, the
nucleophilic primary .epsilon.-amine of the lysine side chain
attacks C-5 of the NCA leading to ring opening and formation of the
amino acid amide along with the evolution of CO.sub.2. Propagation
takes place via further attack of the amine group of the amino acid
amides on subsequent NCA molecules. The degree of polymerization of
the poly(amino acid) side chains, the corresponding amino acid
content in the graft copolymers and their resulting physical and
chemical characteristics can be controlled by changing the M/I
ratio for the NCA polymerization--that is, changing the ratio of
NCA to lysine .epsilon.-amine groups of pLAL. Thus, in the
synthesis, the length of the poly(amino acid), such as
poly(lysine), side chains and the total amino acid content in the
polymer may be designed and synthesized for a particular
application.
[0035] The poly(amino acid) side chains grafted onto or
incorporated into the polyester backbone can include any amino
acid, such as aspartic acid, alanine or lysine, or mixtures
thereof. The functional groups present in the amino acid side
chains, which can be chemically modified, include amino, carboxylic
acid, sulfide, guanidino, imidazole and hydroxyl groups. As used
herein, the term "amino acid" includes natural and synthetic amino
acids and derivatives thereof. The polymers can be prepared with a
range of amino acid side chain lenghts, for example, about 10-100
or more amino acids, and with an overall amino acid content of, for
example, 7-72% or more depending on the reaction conditions. The
grafting of poly(amino acids) from the pLAL backbone may be
conducted in a solvent such as dioxane, DMF, or CH.sub.2Cl.sub.2 or
mixtures thereof. In a preferred embodiment, the reaction is
conducted at room temperature for about 2-4 days in dioxane.
[0036] Alternatively, the porous particles for pulmonary drug
delivery may be formed from polymers or blends of polymers with
different polyester/amino acid backbones and grafted amino acid
side chains. For example, poly(lactic
acid-co-lysine-graft-alanine-lysine) ("PLAL-Ala-Lys"), or a blend
of PLAL-Lys with poly(lactic acid-co-glycolic acid-block-ethylene
oxide) ("PLGA-PEG") ("PLAL-Lys-PLGA-PEG") may be used.
[0037] In the synthesis, the graft copolymers may be tailored to
optimize different characteristic of the porous particle including:
i) interactions between the agent to be delivered and the copolymer
to provide stabilization of the agent and retention of activity
upon delivery; ii) rate of polymer degradation and, thereby, rate
of drug release profiles; iii) surface characteristics and
targeting capabilities via chemical modification; and iv) particle
porosity.
Formation of Porous Polymeric Particles
[0038] Porous polymeric particles may be prepared using single and
double emulsion solvent evaporation, spray drying, solvent
extraction and other methods well known to those of ordinary skill
in the art. Methods developed for making microspheres for drug
delivery are described in the literature, for example, as described
by Mathiowitz and Langer, J. Controlled Release Vol. 5:13-22
(1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987);
and Mathiowitz, et al., J. Appl. Polymer Sci.35: 755-774 (1988),
the teachings of which are incorporated herein. The selection of
the method depends on the polymer selection, the size, external
morphology, and crystallinity that is desired, as described, for
example, by Mathiowitz, et al., Scanning Microscopy 4:329-340
(1990); Mathiowitz, et al., J. Appl. Polymer Sci. 45:125-134
(1992); and Benita, et al., J. Pharm. Sci. 73:1721-1724 (1984), the
teachings of which are incorporated herein.
[0039] In solvent evaporation, described for example, in
Mathiowitz, et al., (1990), Benita, and U.S. Pat. No. 4,272,398 to
Jaffe, the polymer is dissolved in a volatile organic solvent, such
as methylene chloride. Several different polymer concentrations can
be used, for example, between 0.05 and 0.20 g/ml. The drug, either
in soluble form or dispersed as fine particles, is added to the
polymer solution, and the mixture is suspended in an aqueous phase
that contains a surface active agent such as poly(vinyl alcohol).
The aqueous phase may be, for example, a concentration of 1%
poly(vinyl alcohol) w/v in distilled water. The resulting emulsion
is stirred until most of the organic solvent evaporates, leaving
solid microspheres, which may be washed with water and dried
overnight in a lyophilizer.
[0040] Microspheres with different sizes (1-1000 microns) and
morphologies can obtained by this method which is useful for
relatively stable polymers such as polyesters and polystryrene.
However, labile polymers such as polyanhydrides may degrade due to
exposure to water. For these polymers, solvent removal may be
preferred.
[0041] Solvent removal was primarily designed for use with
polyanhydrides. In this method, the drug is dispersed or dissolved
in a solution of a selected polymer in a volatile organic solvent
like methylene chloride. The mixture is then suspended in oil, such
as silicon oil, by stirring, to form an emulsion. Within 24 hours,
the solvent diffuses into the oil phase and the emulsion droplets
harden into solid polymer microspheres. Unlike solvent evaporation,
this method can be used to make microspheres from polymers with
high melting points and a wide range of molecular weights.
Microspheres having a diameter for example between one and 300
microns can be obtained with this procedure.
Targeting of Particles
[0042] Targeting molecules can be attached to the porous particles
via reactive functional groups on the particles. For example,
targeting molecules can be attached to the amino acid groups of
functionalized polyester graft copolymer particles, such as
PLAL-Lys particles. Targeting molecules permit binding interaction
of the particle with specific receptor sites, such as those within
the lungs. The particles can be targeted by attachment of ligands
which specifically or non-specifically bind to particular targets.
Exemplary targeting molecules include antibodies and fragments
thereof including the variable regions, lectins, and hormones or
other organic molecules capable of specific binding for example to
receptors on the surfaces of the target cells.
Therapeutic Agents
[0043] Any of a variety of therapeutic, prophylactic or diagnostic
agents can be delivered. Examples include synthetic inorganic and
organic compounds, proteins and peptides, polysaccharides and other
sugars, lipids, and nucleic acid sequences having therapeutic,
prophylactic or diagnostic activities. Nucleic acid sequences
include genes, antisense molecules which bind to complementary DNA
to inhibit transcription, and ribozymes. The agents to be
incorporated can have a variety of biological activities, such as
vasoactive agents, neuroactive agents, hormones, anticoaguulants,
immunomodulating agents, cytotoxic agents, antibiotics, antivirals,
antisense, antigens, and antibodies. In some instances, the
proteins may be antibodies or antigens which otherwise would have
to be administered by injection to elicit an appropriate response.
Compounds with a wide range of molecular weight can be
encapsulated, for example, between 100 and 500,000 grams per
mole.
[0044] Proteins are defined as consisting of 100 amino acid
residues or more; peptides are less than 100 amino acid residues.
Unless otherwise stated, the term protein refers to both proteins
and peptides. Examples include insulin and other hormones.
Polysaccharides, such as heparin, can also be administered.
[0045] The porous polymeric aerosols are useful as carriers for a
variety of inhalation therapies. They can be used to encapsulate
small and large drugs, release encapsulated drugs over time periods
ranging from hours to months, and withstand extreme conditions
during aerosolization or following deposition in the lungs that
might otherwise harm the encapsulated therapeutic.
[0046] The porous particles may include a therapeutic agent for
local delivery within the lung, such as agents for the treatment of
asthma, emphysema, or cystic fibrosis, or for systemic treatment.
For example, genes for the treatment of diseases such as cystic
fibrosis can be administered.
Administration
[0047] The particles including a therapeutic agent may be
administered alone or in any appropriate pharmaceutical carrier,
such as a liquid, for example saline, or a powder, for
administration to the respiratory system.
[0048] Aerosol dosage, formulations and delivery systems may be
selected for a particular therapeutic application, as described,
for example in Gonda, I. "Aerosols for delivery of therapeutic and
diagnostic agents to the respiratory tract," in Critical Reviews in
Therapeutic Drug Carrier Systems, 6:273-313 (1990), and in Moren,
"Aerosol dosage forms and formulations," in Aerosols in Medicine.
Principles, Diagnosis and Therapy, Moren, et al., Eds., Esevier,
Amsterdam, 1985, the disclosures of which are incorporated herein
by reference.
[0049] The greater efficiency of aerosolization by porous particles
of relatively large size permits more drug to be delivered than is
possible with the same mass of nonporous aerosols. The relative
large size of porous aerosols depositing in the deep lungs also
minimizes potential drug losses caused by particle phagocytosis.
The use of porous polymeric aerosols as therapeutic carriers
provides the benefits of biodegradable polymers for controlled
released in the lungs and long-time local action or systemic
bioavailability. Denaturation of macromolecular drugs can be
minimized during aerosolization since macromolecules are contained
and protected within a polymeric shell. Coencapsulation of peptides
with peptidase-inhibitors can minimize peptide enzymatic
degradation.
[0050] The present invention will be further understood by
reference to the following non-limiting examples.
EXAMPLE 1
Synthesis of Porous Poly[(p-carboxyphenoxy)-hexane Anhydride]
("PCPH") Particles
[0051] Porous poly[(p-carboxyphenoxy)-hexane anhydride] ("PCPH")
particles were synthesized as follows. 100 mg PCPH (MW -25,000) was
dissolved in 3.0 mL methylene chloride. To this clear solution was
added 5.0 mL 1% w/v aqueous polyvinyl alcohol (PVA, MW
.about.25,000, 88 mole % hydrolyzed) saturated with methylene
chloride, and the mixture was vortexed (Vortex Genie 2, Fisher
Scientific) at maximum speed for one minute. The resulting
milky-white emulsion was poured into a beaker containing 95 mL 1%
PVA and homogenized (Silverson Homogenizers) at 6000 RPM for one
minute using a 0.75 inch tip. After homogenization, the mixture was
stirred with a magnetic stirring bar and the methylene chloride
quickly extracted from the polymer particles by adding 2 mL
isopropyl alcohol. The mixture was continued to stir for 35 minutes
to allow complete hardening of the microparticles. The hardened
particles were collected by centrifugation and washed several times
with double distilled water. The particles were freeze dried to
obtain a free-flowing powder void of clumps. Yield, 85-90%.
[0052] The mean diameter of this batch was 6.0 .mu.m, however,
particles with mean diameters ranging from a few hundred nanometers
to several millimeters may be made with only slight modifications.
Scanning electron micrograph photos of a typical barch of PCPH
particles showed the particles to be highly porous. The particles
have a mass density less that 1 g/cm.sup.3 as indicated by the fact
that the particles float when dispersed in an organic solvent.
EXAMPLE 2
Synthesis of PLAL-Lys and PLAL-Lys-Ala Polymeric and Copolymeric
Particles
Porous PLAL-Lys Particles
[0053] PLAL-Lya particles were prepared by dissolving 50 mg of the
graft copolymer in 0.5 ml dimethylsulfoxide, then adding 1.5 ml
dichloromethane dropwise. The polymer solution is emulsified in 100
ml of 5% w/v polyvinyl alcohol solution (average molecular weight
25 KDa, 88% hydrolyzed) using a homogenizer (Silverson) at a speed
of approximately 7500 rpm. The resulting dispersion is stirred
using a magnetic stirrer for 1 hour. Following this period, the pH
is brought to 7.0-7.2 by addition of 0.1 N NaOH solution. Stirring
is continued for an additional 2 hours until the methylene chloride
is completely evaporated and the particles hardened. The particles
are then isolated by centrifugation at 4000 rpm (1600 g) for 10
minutes (Sorvall RX-5B). The supernatant is discarded and the
precipitate washed three times with distilled water, the dispersion
frozen in liquid nitrogen, and lyophilized (Labconco freeze dryer
8) for at least 48 hours. Particle sizing is performed using a
Coulter counter. Average particle mean diameters ranged from 100 nm
to 14 .mu.m, depending upon processing parameters such as
homogenization speed and time. All particles exhibit high porosity
(net mass density less than 0.4 g/cm.sup.3). Scanning electron
micrograph photos of the particles showed them to be highly
porous.
Porous PLAL-Ala-Lys Particles
[0054] 100 mg of PLAL-Ala-Lys is completely dissolved in 0.4 ml
trifluoroethanol, then 1.0 ml methylene chloride is added dropwise.
The polymer solution is emulsified in 100 ml of 1% w/v polyvinyl
alcohol solution (average molecular weight 25 KDa, 80% hydrolyzed)
using a sonicator (Sonic & Materal VC-250) for 15 seconds at an
output of 40 W. 2 ml of 1% PVA solution is added to the mixture and
it is vortexed at the highest speed for 30 seconds. The mixture is
quickly poured into a beaker containing 100 ml 0.3% PVA solution,
and stirred for three hours allowing evaporation of the methylene
chloride. Scanning electron micrograph photos of the particles
showed them to be highly porous.
Porous Copolymer Particles
[0055] Polymeric porous particles consisting of a blend of PLAL-Lys
ad PLGA-PEG were made. 50 mg of the PLGA-PEG polymer (molecular
weight of PEG: 20 KDa, 1:2 weight ratio of PEG:PLGA, 75:25
lactide:glycolide) was completely dissolved in 1 ml
dichloromethane. 3 mg of poly(lactide-co-lysine)-polylysine graft
copolymer is dissolved in 0.1 ml dimethylsulfoxide and mixed with
the first polymer solution. 0.2 ml TE buffer, pH 7.6, is emulsified
in the polymer solution by probe sonication (Sonic & Materal
VC-250) for 10 seconds at an output of 40W. To this first emulsion,
2 ml of distilled water is added and mixed using a vortex mixer at
4000 rpm for 60 seconds. The resulting dispersion is agitated by
using a magnetic stirrer for 3 hours until methylene chloride is
completely evaporated and micropheres formed. The spheres are then
isolated by centrifugation at 5000 rpm for 30 min. The supernatant
is discarded, the precipitate washed three times with distilled
water and resuspended in 5 ml of water. The dispersion is frozen in
liquid nitrogen and lyophilized for 48 hours.
[0056] Variables which may be manipulated to alter the size
distribution of the particles include: polymer concentration,
polymer molecular weight, surfactant type (e.g., PVA, PEG, etc.),
surfactant concentration, and mixing intensity. Variables which may
be manipulated to alter the porosity of the particles include:
polymer concentration, polymer molecular weight, rate of methylene
chloride extraction by isopropyl alcohol (or another miscible
solvent), volume of isopropyl alcohol added, inclusion of an inner
water phase, volume of inner water phase, inclusion of salts or
other highly water-soluble molecules in the inner water phase which
leak out of the hardening sphere by osmotic pressure, causing the
formation of channels, or pores, in proportion to their
concentration, and surfactant type and concentration.
[0057] By scanning electron microscopy (SEM), the PLAL-Lys-PLGA-PEG
particles were highly porous. The particles had a mean particle
diameter of 7 .mu.m.+-.3.8 .mu.m. The blend of PLAL-Lys with
poly(lactic acid) (PLA) and/or PLGA-PEG copolymers can be adjusted
to adjust particle porosity and size. Additionally, processing
parameters such as homogenization speed and time can be adjusted.
Neither PLAL, PLA nor PLGA-PEG alone yields a porous structure when
prepared by these techniques.
EXAMPLE 3
Rhodamine Isothiocyanate Labeling of PLAL and PLAL-Lys
Particles
[0058] Lysine amine groups on the surface or porous (PLAL-Lys) and
nonporous (PLAL) microparticles with similar mean diameters (6-7
.mu.m) and size distibutions (standard deviations 3-4 .mu.m) were
labeled with Rhodamine isothiaocyanate. The mass density of the
porous PLAL-Lys particles was 0.1 g/cm.sup.3 and that of the
nonporous PLAL particles was 0.8 g/cm.sup.3.
[0059] The rhodamine-labeled particles were characterized by
confocal microscopy. A limited number of lysine functionalities on
the surface of the solid particle were able to react with rhodamine
isothiocyanate, as evidenced by the fluorescent image. In the
porous particle, the higher lysine content in the graft copolymer
and the porous particle structure result in a higher level of
rhodamine attachment, with rhodamine attachment dispersed
throughout the interstices of the porous structure. This also
demonstrates that targeting molecules can be attached to the porous
particles for interaction with specific receptor sites within the
lungs via chemical attachment of appropriate targeting agents to
the particle surface.
EXAMPLE 4
Aerosolization of PLAL and PLAL-Lys Particles
[0060] To determine whether large porous particles can escape
(mouth, throat, and inhaler) deposition and more efficiently enter
the airways and acini than nonporous particles of similar size,
aerosolization and deposition of porous PLAL-Lys (mean diameter 6.3
.mu.m.+-.3.3 .mu.m) or nonporous PLAL (mean diameter 6.9
.mu.m.+-.3.6 .mu.m) particles were examined in vitro using a
cascade impactor system.
[0061] 20 mg of the porous or nonporous microparticles were placed
in gelatin capsules (Eli Lilly), the capsules loaded into a
Spinhaler dry powder inhaler (DPI) (Fisons), and the DPI activated.
Particles were aerosolized into a Mark I Andersen Impactor
(Anderson Samplers, GA) from the DPI for 30 seconds at 28.3 l/min
flow rate. Each plate of the Andersen Impactor was previously
coated with Tween 80 by immersing the plates in an acetone solution
(5% w/vol) and subsequently evaporating the acetone in a oven at
60.degree. for 5 min. After aerosolization and deposition,
particles were collected from each stage of the impactor system in
separate volumetric flasks by rinsing each stage with NaOH solution
(0.2 N) in order to completely degrade the polymers. After
incubation at 37.degree. C. for 12 h, the fluorescence of each
solution was measured (wavelengths of 554 nm excitation, 574 nm
emission).
[0062] Particles were determines as nonrespirable (mean aerodynamic
diameter exceeding 4.7 .mu.m: impactor estimate) if they deposited
on the first three stages of the impactor, and respirable (mean
aerodynamic diameter 4.7 .mu.m or less) if they deposited on
subsequent stages. FIG. 1 shows that less than 10% of the nonporous
(PLAL) particles that exit the DPI are respirable. This is
consistent with the large size of the microparticles and their
standard mass density. On the other hand, greater the 55% of the
porous (PLAL-Lys) particles are respirable, even though the
geometrical dimensions of the two particle types are almost
identical. The lower mass density of the porous (PLAL-Lys)
microparticles is responsible for this improvement in particle
penetration, as discussed further below.
[0063] The nonporous (PLAL) particles also inefficiently aerosolize
from the DPI; typically, less than 40% of the nonporous particles
exited the Spinhaler DPI for the protocol used. The porous
(PLAL-Lys) particles exhibited much more efficient aerosolization
(approximately 80% if the porous microparticles typically exited
the DPI during aerosolization).
[0064] The combined effects of efficient aerosolization and high
respirable fraction of aerosolized particle mass means that a far
greater fraction of a porous particle powder is likely to deposit
in the lungs than of a nonporous particle powder.
EXAMPLE 5
In Vivo Aerosolization of PLAL and PLAL-Lys Particles
[0065] The penetration of porous and non-porous polymeric PLAL and
PLAL-Lys microparticles into the lungs was evaluated in and in vivo
experiment involving the aerosolization of the microparticles into
the airways of live rats.
[0066] Male Sprague Dawley rats (150-200 g) were anesthetized using
ketamine (90 mg/kg)/xylazine (10 mg/kg). The anesthetized rat was
placed ventral side up on a surgical table provided with a
temperature controlled pad to maintain physiological temperature.
The animal was cannulated about the carina with an endotracheal
tube connected to a Harvard ventilator. The animal was force
ventilated for 20 minutes and 300 ml/min. 50 mg of porous
(PLAL-Lys) or nonporous (PLA) microparticles were introduced into
the endotracheal tube.
[0067] Following the period of forced ventilation, the animal was
euthanized and the lungs and trachea were separately washed using
bronchoalveolar lavage. A tracheal cannula was inserted, tied into
place, and the airways were washed with 10 ml aliquots of HBSS. The
lavage procedure was repeated until a total volume of 30 ml was
collected. The lavage fluid was centrifuged (400 g) and the pellets
collectted and resuspended in 2 ml of phenol red-free Hanks
balanced salt solution (Gibco, Grand Island, N.Y.) without
Ca.sup.2+ and Mg.sup.2+ (HBSS). 100 ml were removed for particle
counting using a hemacytometer. The remaining solution was mixed
with 10 ml of 0.4 N NaOH. After incubation at 37.degree. C. for 12
h, the fluorescence of each solution was measured (wavelengths of
554 nm excitation, 574 nm emission).
[0068] FIG. 2 is a bar graph showing total particle mass deposited
in the trachea and after the carina (lungs) in rat lungs and upper
airways following intratracheal aerosolization during forced
ventilation. The PLAL-Lys porous particles had a mean diameter 6.9
.mu.m.+-.4.2 .mu.m. The nonporouse particles PLAL particles had a
mean diameter of 6.7 82 m.+-.3.2 .mu.m. Percent tracheal porous
particle deposition was 54.54.+-.0.77, and nonporous deposition was
76.98.+-.1.95. Percent porous particle deposition in the lungs was
46.75.+-.0.77, and nonporous deposition ws 23.02.+-.1.95.
[0069] The nonporous (PLAL) particles deposited primarily in the
trachea (approximately 79% of all particle mass that entered the
trachea). This result is similar to the in vitro perfomance of the
nonporous microparticles and is consistent with the relatively
large size of the nonporous particles. Approximately 54% if the
porous (PLAL-Lys) particle mass deposited in the trachea.
Therefore, about half of the porous particle mass that enters the
trachea traverses through the trachea and into the airways and
acini of the rat lungs, demonstrating the effective penetration of
the porous particles into the lungs.
[0070] Following bronchoalveolar lavage, particles remaining in the
rat lungs were obtained by careful dissection of the individual
lobes of the lungs. The lobes were placed in separate petri dishes
containing 5 ml of HBSS. Each lobe was teased through 60 mesh
screen to dissociate the tissue and was then filtered through
cotton gauze to remove tissue debris and connective tissue. The
petri dish and gauze were washed with an additional 15 ml of HBSS
to maximize microparticle collection. Each tissue preparation was
centrifuged and resuspended in 2 ml of HBSS and the number of
particles counted in a hemacytometer. The particle numbers
remaining in the lungs following the bronchoalveolar lavage are
shown in FIG. 3. Lobe numbers correspond to: 1) left lung, 2)
anterior, 3) median, 4) posterior, 5) postcaval. A considerably
greater number of porous PLAL-Lys particles enters every lobe of
the lungs than the nonporous PLAL particles, even though the
geometrical dimensions of the two types of particles are
essentially the same. These results reflect both the efficiency of
porous particle aerosolization and the propensity of the porous
particles to escape deposition prior to the carina or first
bifurcation.
[0071] Modifications and variations of the present invention will
be obvious to those skilled in the art from the foregoing detailed
description. Such modifications and variations are intended to come
within the scope of the following claims.
* * * * *