U.S. patent application number 17/196036 was filed with the patent office on 2021-08-19 for compositions and methods for drug delivery.
The applicant listed for this patent is BAXTER HEALTHCARE SA, BAXTER INTERNATIONAL INC.. Invention is credited to Shawn F. Bairstow, Mahesh V. Chaubal, Sarah Lee, Barrett Rabinow, Jane Werling.
Application Number | 20210251900 17/196036 |
Document ID | / |
Family ID | 1000005542465 |
Filed Date | 2021-08-19 |
United States Patent
Application |
20210251900 |
Kind Code |
A1 |
Rabinow; Barrett ; et
al. |
August 19, 2021 |
COMPOSITIONS AND METHODS FOR DRUG DELIVERY
Abstract
The present disclosure is directed to surface-modified particles
and to methods of making and using the same. The surface-modified
particles comprise a particle core and a coating associated with
the particle core, wherein the particle core comprises an active
agent, the coating comprises a surfactant having formula I, and the
surface-modified particle has an average size from about 1 nm to
about 2,000 nm: ##STR00001##
Inventors: |
Rabinow; Barrett; (Skokie,
IL) ; Bairstow; Shawn F.; (Gurnee, IL) ;
Chaubal; Mahesh V.; (Lake Zurich, IL) ; Lee;
Sarah; (Buffalo Grove, IL) ; Werling; Jane;
(Arlington Heights, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAXTER INTERNATIONAL INC.
BAXTER HEALTHCARE SA |
Deerfield
Glattpark (Opfikon) |
IL |
US
CH |
|
|
Family ID: |
1000005542465 |
Appl. No.: |
17/196036 |
Filed: |
March 9, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12467230 |
May 15, 2009 |
10952965 |
|
|
17196036 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/513 20130101;
A61K 9/145 20130101; A61K 31/7072 20130101; A61K 31/337 20130101;
A61K 31/365 20130101; A61K 31/538 20130101; A61K 31/496 20130101;
A61K 31/427 20130101; A61K 31/708 20130101; Y02A 50/30 20180101;
A61K 31/551 20130101; A61K 31/444 20130101; A61K 31/415 20130101;
A61K 31/4725 20130101; A61K 47/34 20130101; A61K 31/7068
20130101 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 31/496 20060101 A61K031/496; A61K 31/513 20060101
A61K031/513; A61K 31/415 20060101 A61K031/415; A61K 31/551 20060101
A61K031/551; A61K 31/538 20060101 A61K031/538; A61K 31/365 20060101
A61K031/365; A61K 31/4725 20060101 A61K031/4725; A61K 47/34
20060101 A61K047/34; A61K 31/337 20060101 A61K031/337; A61K 31/444
20060101 A61K031/444; A61K 31/7068 20060101 A61K031/7068; A61K
31/7072 20060101 A61K031/7072; A61K 31/427 20060101 A61K031/427;
A61K 31/708 20060101 A61K031/708 |
Claims
1. A surface-modified particle comprising a particle core and a
coating adsorbed to a surface of the particle core, wherein the
particle core comprises a small molecule active agents, a peptide
active agent or a protein active agent, the coating comprises a
surfactant having formula I, and the surface-modified particle has
a size from about 10 nm to about 1 .mu.m, does not comprise
polysaccharides, does not comprise colloidal silicon dioxide, and
does not comprise monoacylated monoglycerides: ##STR00004## wherein
n and m are 1; R.sup.1, R.sup.2, and R.sup.3 are methyl; and
R.sup.4 and R.sup.5 are independently selected from the group
consisting of cis-9-octadecenoyl and cis-9-octadecenyl.
2. The particle of claim 1, wherein R.sup.4 and R.sup.5 are
cis-9-octadecenoyl.
3. The particle of claim 1, wherein R.sup.4 and R.sup.5 are
cis-9-octadecenyl.
4. The particle of claim 1, wherein the coating further comprises a
second surfactant.
5. The particle of claim 4, wherein the second surfactant is
selected from the group consisting of anionic surfactants, cationic
surfactants, zwitterionic surfactants, nonionic surfactants,
surface active biological modifiers, and combinations thereof.
6. The particle of claim 4, wherein the second surfactant comprises
at least one of a poloxamer and a phospholipid.
7. The particle of claim 1, wherein the active agent is a
therapeutic agent.
8. The particle of claim 7, wherein the therapeutic agent is
selected from the group consisting of analgesics, anesthetics,
analeptics, adrenergic agents, adrenergic blocking agents,
adrenolytics, adrenocorticoids, adrenomimetics, anticholinergic
agents, anticholinesterases, anticonvulsants, alkylating agents,
alkaloids, allosteric inhibitors, anabolic steroids, anorexiants,
antacids, antidiarrheals, antidotes, antifolics, antipyretics,
antirheumatic agents, psychotherapeutic agents, neural blocking
agents, anti-inflammatory agents, antihelmintics, antibiotics,
anticoagulants, antidepressants, antiepileptics, antifungals,
antifibrotic agents, anti-infective agents, anti-parasitic agents,
antihistamines, antimuscarinic agents, antimycobacterial agents,
antineoplastic agents, antiprotozoal agents, antiviral agents,
anxiolytic sedatives, beta-adrenoceptor blocking agents,
corticosteroids, cough suppressants, dopaminergics, hemostatics,
hematological agents, hypnotics, immunological agents, muscarinics,
parasympathomimetics, prostaglandins, radio-pharmaceuticals,
sedatives, stimulants, sympathomimetics, vitamins, xanthines,
growth factors, hormones, antiprion agents, and combinations
thereof.
9. The particle of claim 1, wherein the active agent is an
antineoplastic agent selected from the group consisting of
paclitaxel, paclitaxel derivative compounds, alkaloids,
antimetabolites, enzyme inhibitors, alkylating agents, and
combinations thereof.
10. The particle of claim 1, wherein the active agent is
paclitaxel; and R4 and R5 are cis-9-octadecenoyl.
11. The particle of claim 1, wherein the active agent is
paclitaxel; and R.sup.4 and R.sup.5 are cis-9-octadecenyl.
12. The particle of claim 1, wherein the active agent is a protease
inhibitor.
13. The particle of claim 12, wherein the protease inhibitor is
selected from the group consisting of indinavir, ritonavir,
saquinavir, nelfinavir, and combinations thereof.
14. The particle of claim 1, wherein the active agent is a
nucleoside reverse transcriptase inhibitor.
15. The particle of claim 14, wherein the nucleoside reverse
transcriptase inhibitor is selected from the group consisting of
zidovudine, didanosine, stavudine, zalcitabine, lamivudine and
combinations thereof.
16. The particle of claim 1, wherein the active agent is a
non-nucleoside reverse transcriptase inhibitor.
17. The particle of claim 16, wherein the non-nucleoside reverse
transcriptase inhibitor is selected from the group consisting of
efavirenz, nevirapine, delaviradine, and combinations thereof.
18. The particle of claim 1, wherein the active agent is an
anti-inflammatory agent.
19. The particle of claim 18, wherein the anti-inflammatory agent
is selected from the group consisting of non-steroidal
anti-inflammatory drugs, nonselective cycloxygenase (COX)
inhibitors, COX-1 inhibitors, COX-2 inhibitors, lipoxygenase
inhibitors, corticosteroids, anti-oxidants, tumor necrosis factor
(TNF) inhibitors, and combinations thereof.
20. The particle of claim 1, wherein the active agent is selected
from the group consisting of celecoxib, rofecoxib, valdecoxib,
parecoxib, lumiracoxib, etoricoxib, and combinations thereof.
21. A pharmaceutical composition comprising a plurality of
particles of claim 1.
22. The particle of claim 1, wherein the particles are amorphous,
semicrystalline, crystalline, or a combination thereof.
23. The particle of claim 1, wherein the surface-modified particle
is capable of dissolution when taken up by cells or delivered to
tissue of a mammalian subject.
24. The particle of claim 1, wherein the surface-modified particle
includes at least 75% (w/w) active agent.
25. A surface-modified particle comprising a particle core and a
coating adsorbed to a surface of the particle core, wherein the
particle core consists of a peptide active agent, the coating
comprises a surfactant having formula I, and the surface-modified
particle has a size from about 10 nm to about 1 .mu.m, does not
comprise polysaccharides, does not comprise colloidal silicon
dioxide, and does not comprise monoacylated monoglycerides:
##STR00005## wherein n and m are 1; R.sup.1, R.sup.2, and R.sup.3
are methyl; and R.sup.4 and R.sup.5 are independently selected from
the group consisting of cis-9-octadecenoyl and
cis-9-octadecenyl.
26. A surface-modified particle comprising a particle core and a
coating adsorbed to a surface of the particle core, wherein the
particle core consists of a protein active agent, the coating
comprises a surfactant having formula I, and the surface-modified
particle has a size from about 10 nm to about 1 .mu.m, does not
comprise polysaccharides, does not comprise colloidal silicon
dioxide, and does not comprise monoacylated monoglycerides:
##STR00006## wherein n and m are 1; R.sup.1, R.sup.2, and R.sup.3
are methyl; and R.sup.4 and R.sup.5 are independently selected from
the group consisting of cis-9-octadecenoyl and
cis-9-octadecenyl.
27. A method of enhancing cellular uptake of an active agent, said
method comprising: contacting cells with surface-modified particles
under conditions sufficient to enhance cellular uptake of the
surface-modified particles, said particles comprising a particle
core and a coating adsorbed to a surface of the particle core,
wherein the particle core comprises a small molecule active agents,
a peptide active agent or a protein active agent, the coating
comprises a surfactant having formula I, and the surface-modified
particle has a size from about 10 nm to about 1 .mu.m, does not
comprise polysaccharides, does not comprise colloidal silicon
dioxide, and does not comprise monoacylated monoglycerides:
##STR00007## wherein n and m are 1; R.sup.1, R.sup.2, and R.sup.3
are methyl; and R.sup.4 and R.sup.5 are independently selected from
the group consisting of cis-9-octadecenoyl and
cis-9-octadecenyl.
28. The method of claim 27, wherein the cells are phagocytic
cells.
29. The method of claim 27, wherein said contacting is carried out
ex vivo.
30. The method of claim 27, wherein said contacting is carried out
in vivo.
31. The method of claim 27, wherein the cells are phagocytic cells
selected from the group consisting of macrophages, monocytes,
granulocytes, agranulocytes, neutrophils, and combinations
thereof.
32. The method of claim 27, wherein said contacting is effected by
administering to a subject an amount of said surface modified
particles effective to treat infectious diseases or disorders,
inflammatory diseases or disorders, neurodegenerative diseases or
disorders, or proliferative diseases or disorders.
33. The method of claim 32, wherein said administering is performed
intravenously, intraarterially, intramuscularly, subcutaneously,
intradermally, intraarticularly, intrathecally, epidurally,
intracerebrally, buccally, rectally, topically, transdermally,
orally, intranasally, via the pulmonary route, intraperitoneally,
intraophthalmically, or by a combination thereof.
34. The method of claim 32, wherein the subject has a
neurodegenerative disease or disorder selected from the group
consisting of Parkinson's disease, Alzheimer's disease, multiple
sclerosis, encephalomyelitis, encephalitis, Huntington's disease,
amyotrophic lateral sclerosis, frontotemporal dementia, prion
diseases, Creutzfeldt-Jakob disease, and adrenoleukodystrophy.
35. The method of claim 32, wherein the subject has an inflammatory
disease or disorder selected from the group consisting of
rheumatoid arthritis, Graves' disease, myasthenia gravis,
thyroiditis, diabetes, inflammatory bowel disease, autoimmune
oophoritis, systemic lupus erythematosus, and Sjogren's
syndrome.
36. The method of claim 32, wherein the subject has a proliferative
disease or disorder selected from the group consisting of colon
cancer, kidney cancer, non small cell lung cancer, small cell lung
cancer, head and neck cancer, cancers of the peritoneal cavity,
cervical cancer, breast cancer, prostate cancer, brain cancer,
sarcoma, melanoma, leukemia, acute lymphocytic leukemia, acute
myelogenous leukemia, chronic lymphocytic leukemia, chronic
myelogenous leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma,
myeloma, and glioblastoma.
37. The method of claim 32, wherein the subject has a proliferative
disease or disorder; and R.sup.4 and R.sup.5 are
cis-9-octadecenoyl.
38. The method of claim 32, wherein the subject has a proliferative
disease or disorder; and R.sup.4 and R.sup.5 are
cis-9-octadecenyl.
39. The method of claim 27, wherein the particle core consists of a
peptide active agent.
40. The method of claim 27, wherein the particle core consists of a
protein active agent.
41. The method of claim 27, wherein the surface-modified particle
includes at least 75% (w/w) active agent.
42. A method for treating a subject having an infectious disease or
disorder, an inflammatory disease or disorder, a neurodegenerative
disease or disorder, or a proliferative disease or disorder
comprising administering to said subject a plurality of
surface-modified particles into a body cavity having a site of
disease or inflammation, said surface-modified particles comprising
a particle core and a coating adsorbed to a surface of the particle
core, wherein the particle core comprises a small molecule active
agents, a peptide active agent or a protein active agent, the
coating comprises a surfactant of formula I, the surface-modified
particle has a size from about 10 nm to about 1 .mu.m, does not
comprise polysaccharides, does not comprise colloidal silicon
dioxide, and does not comprise monoacylated monoglycerides, and
said administration is effective in alleviating, treating, and/or
preventing symptoms or pathologies associated with said disease or
disorder: ##STR00008## wherein n and m are 1; R.sup.1, R.sup.2, and
R.sup.3 are methyl; and R.sup.4 and R.sup.5 are independently
selected from the group consisting of cis-9-octadecenoyl and
cis-9-octadecenyl.
43. The method of claim 42, wherein the body cavity is selected
from the group consisting of the peritoneal cavity, the bladder
cavity, the pulmonary cavity, the pleural cavity, the cardiac
cavity, the aqueous humor of the eye, and the vitreous humor of the
eye.
44. The method of claim 42, wherein the disease or disorder is
cancer and the active agent is an antineoplastic agent.
45. The method of claim 42, wherein the surface-modified particle
includes at least 75% (w/w) active agent.
46. The method of claim 42, wherein the particle core consists of a
peptide active agent.
47. The method of claim 42, wherein the particle core consists of a
protein active agent.
Description
BACKGROUND
Field of the Disclosure
[0001] The disclosure relates generally to compositions comprising
coated particles and to methods of making and using such
compositions for targeted drug delivery.
Brief Description of Related Technology
[0002] Nanoparticles (including nanospheres) and microparticles
(including microspheres) referred to herein collectively as
"particles," are solid or semi-solid particles having a diameter
from about 1 nm to about 10,000 nm (10 microns), preferably from
about 1 nm to about 2,000 nm (2 microns). Such particles can be
formed from a variety of materials, including proteins, synthetic
polymers, polysaccharides, nucleic acids, small molecules, and
combinations thereof, and have been used in many different
applications, primarily separations, diagnostics, and drug
delivery.
[0003] Compositions comprising such particles have been found to be
useful for drug delivery. For example, U.S. Patent Publication No.
2006/0073199 discloses that particles comprising an active agent
can be formulated as aqueous suspensions, and stabilized against
aggregation and particle growth by providing surfactant coatings on
or about the particles.
[0004] There is an on-going need for development of compositions
comprising particles and methods for making and using same,
particularly in delivering drugs of interest.
SUMMARY
[0005] One aspect of the invention is directed to a
surface-modified particle comprising a particle core and a coating
associated with the particle core. The particle core comprises an
active agent, such as a therapeutic agent or a diagnostic agent
(e.g., a small organic molecule or a biomacromolecule). The coating
comprises a surfactant having formula I:
##STR00002##
wherein n and m are independently selected from the group
consisting of 1, 2, 3, 4, 5, and 6; R.sup.1, R.sup.2, and R.sup.3
are independently selected from C.sub.1 to C.sub.8 alkyl; and
R.sup.4 and R.sup.5 are independently selected from the group
consisting of C.sub.6 to C.sub.40 alkyl, C.sub.6 to C.sub.40
alkenyl, C.sub.6 to C.sub.40 alkynyl, C(.dbd.O)(C.sub.5 to C.sub.39
alkyl), C(.dbd.O)(C.sub.5 to C.sub.39 alkenyl), and
C(.dbd.O)(C.sub.5 to C.sub.39 alkynyl). The surface-modified
particles according to the present invention generally have an
average size from about 1 nm to about 2,000 nm.
[0006] Another aspect of the invention is directed to a method of
enhancing cellular uptake of an active agent. The method comprises
contacting cells with surface-modified particles under conditions
sufficient to enhance cellular uptake of the surface-modified
particles. The particles comprise a particle core and a coating
associated with the particle core, wherein the particle core
comprises an active agent, the coating comprises a surfactant of
formula I, as defined herein, and the surface-modified particle has
an average size from about 1 nm to about 2,000 nm.
[0007] Another aspect of the invention is directed to a method for
treating a subject having an inflammatory disease or disorder
comprising administering to said subject a plurality of
surface-modified particles, said surface-modified particles
comprising a particle core and a coating associated with the
particle core, wherein the particle core comprises an active agent
(e.g., an anti-inflammatory agent), the coating comprises a
surfactant of formula I, as defined herein, the surface-modified
particle has an average size from about 1 nm to about 2,000 nm, and
said administration is effective in alleviating, treating, and/or
preventing symptoms or pathologies associated with said
inflammatory disease or disorder.
[0008] Another aspect of the invention is directed to a method for
treating a subject having a proliferative disease or disorder
comprising administering to said subject a plurality of
surface-modified particles, said surface-modified particles
comprising a particle core and a coating associated with the
particle core, wherein the particle core comprises an active agent
(e.g., an anti-proliferative such as an antineoplastic agent), the
coating comprises a surfactant of formula I, as defined herein, the
surface-modified particle has an average size from about 1 nm to
about 2,000 nm, and said administration is effective in
alleviating, treating, and/or preventing symptoms or pathologies
associated with said proliferative disease or disorder.
[0009] Another aspect of the invention is directed to a method for
treating a subject having an infectious disease or disorder
comprising administering to said subject a plurality of
surface-modified particles, said surface-modified particles
comprising a particle core and a coating associated with the
particle core, wherein the particle core comprises an active agent
(e.g., an anti-infective agent), the coating comprises a surfactant
of formula I, as defined herein, the surface-modified particle has
an average size from about 1 nm to about 2,000 nm, and said
administration is effective in alleviating, treating, and/or
preventing symptoms or pathologies associated with said infectious
disease or disorder.
[0010] In another aspect, the invention is directed to a method for
treating a subject having a neurodegenerative disease or disorder
comprising administering to said subject a plurality of
surface-modified particles, said surface-modified particles
comprising a particle core and a coating associated with the
particle core, wherein the particle core comprises an active agent
(e.g., an anti-neurodegenerative agent), the coating comprises a
surfactant of formula I, as defined herein, the surface-modified
particle has an average size from about 1 nm to about 2,000 nm, and
said administration is effective in alleviating, treating, and/or
preventing symptoms or pathologies associated with said
neurodegenerative disease or disorder.
[0011] Another aspect of the invention is directed to a method for
treating a subject having an infectious disease or disorder, an
inflammatory disease or disorder, a neurodegenerative disease or
disorder, or a proliferative disease or disorder comprising
administering to said subject a plurality of surface-modified
particles into a body cavity having a site of disease or
inflammation, said surface-modified particles comprising a particle
core and a coating associated with the particle core, wherein the
particle core comprises an active agent, the coating comprises a
surfactant of formula I, as defined herein, the surface-modified
particle has an average size from about 1 nm to about 2,000 nm, and
said administration is effective in alleviating, treating, and/or
preventing symptoms or pathologies associated with said disease or
disorder.
[0012] Each of the aforementioned methods for treating can be
effected by using cellular transport to deliver the
surface-modified particles to a target tissue of the subject, or by
localized administration of the surface-modified particles into a
body cavity having a site of disease (e.g., cancer, infection)
and/or inflammation in the subject such that the surface-modified
particles can be taken up by diseased or inflammatory cells located
within the body cavity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 (including FIG. 1A and FIG. 1B) provides graphs
showing uptake of DSPE-mPEG2000/poloxamer 188-coated paclitaxel
particles labeled with Oregon Green (No DOTAP) and DOTAP-coated
paclitaxel particles labeled with Oregon Green (DOTAP).
[0014] FIG. 2 (including FIG. 2A and FIG. 2B) provides graphs
showing uptake of DSPE-mPEG2000/poloxamer 188-coated paclitaxel
particles (DSPE-mPEG2000/poloxamer 188), DOTAP-coated paclitaxel
particles labeled with Oregon Green and stored for 3 months (DOTAP
Sample 1), freshly prepared DOTAP-coated paclitaxel particles
labeled with Oregon Green (DOTAP Sample 2), and protamine-coated
paclitaxel particles labeled with Oregon Green (Protamine).
[0015] FIG. 3 (including FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG.
3E and FIG. 3F) provides graphs showing uptake of
DSPE-mPEG2000/poloxamer 188-coated paclitaxel particles labeled
with Oregon Green (No DOTAP) and DOTAP-coated paclitaxel particles
labeled with Oregon Green (DOTAP). Cells were cultured for 1, 2, or
6 days prior to exposing the cells to the paclitaxel particles.
[0016] FIG. 4 (including FIG. 4A and FIG. 4B) provides graphs
showing uptake of DSPE-mPEG2000/poloxamer 188-coated paclitaxel
particles labeled with Oregon Green (DSPE-mPEG2000/poloxamer 188),
DOTAP-coated paclitaxel particles labeled with Oregon Green
(DOTAP/DSPE-mPEG2000/poloxamer 188), polylactic-co-glycolic
acid-coated paclitaxel particles labeled with Oregon Green
(PLGA/poloxamer 188), and phosphatidylserine-coated paclitaxel
particles labeled with Oregon Green (PS/DSPE-mPEG2000/poloxamer
188).
[0017] FIG. 5 (including FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D)
provides graphs showing uptake of DSPE-mPEG2000/poloxamer
188-coated paclitaxel particles labeled with Oregon Green
(DSPE-mPEG2000/poloxamer 188), DOTAP-coated paclitaxel particles
labeled with Oregon Green (DOTAP/DSPE-mPEG2000/poloxamer 188), and
cetyl trimethylammonium bromide-coated paclitaxel particles labeled
with Oregon Green (CTAB/DSPE-mPEG2000/poloxamer 188).
DETAILED DESCRIPTION
[0018] The claimed invention is susceptible of embodiments in many
different forms. Preferred embodiments, as disclosed herein, are to
be considered exemplary of the principles of the claimed invention
and thus not intended to limit the broad aspects of the claimed
invention to the embodiments illustrated.
[0019] One aspect of the invention provides a surface-modified
particle comprising a particle core and a coating associated with
the particle core. The particle core comprises an active agent
which is typically selected from the group consisting of small
molecules, peptides, and proteins, the coating comprises a
surfactant having formula I:
##STR00003##
wherein n and m are independently selected from the group
consisting of 1, 2, 3, 4, 5, and 6; R.sup.1, R.sup.2, and R.sup.3
are independently selected from C.sub.1 to C.sub.8 alkyl; and
R.sup.4 and R.sup.5 are independently selected from the group
consisting of C.sub.6 to C.sub.40 alkyl, C.sub.6 to C.sub.40
alkenyl, C.sub.6 to C.sub.40 alkynyl, C(.dbd.O)(C.sub.5 to C.sub.39
alkyl), C(.dbd.O)(C.sub.5 to C.sub.39 alkenyl), and
C(.dbd.O)(C.sub.5 to C.sub.39 alkynyl), and the surface-modified
particle has an average size from about 1 nm to about 2,000 nm.
[0020] As used herein, the term "alkyl" refers to straight chained
and branched saturated hydrocarbon groups, nonlimiting examples of
which include methyl, ethyl, and straight chain and branched propyl
and butyl groups. Alkyl groups optionally can be substituted, for
example, with one or more hydroxy (--OH), oxo (.dbd.O), halo (--F,
--Cl, --Br, or --I), and thio (--SH) groups or a combination
thereof.
[0021] As used herein, the term "alkenyl" refers to straight
chained and branched hydrocarbon groups containing at least one
carbon-carbon double bond, nonlimiting examples of which include
straight chain and branched hexadecenyl and octadecenyl groups.
Alkenyl groups optionally can be substituted, for example, with one
or more hydroxy (--OH), oxo (.dbd.O), halo (--F, --Cl, --Br, or
--I), and thio (--SH) groups or a combination thereof.
[0022] As used herein, the term "alkynyl" refers to straight
chained and branched hydrocarbon groups containing at least one
carbon-carbon triple bond, nonlimiting examples of which include
straight chain and branched hexadecynyl and octadecynyl groups.
Alkynyl groups optionally can be substituted, for example, with one
or more hydroxy (--OH), oxo (.dbd.O), halo (--F, --Cl, --Br, or
--I), and thio (--SH) groups or a combination thereof.
[0023] R.sup.1, R.sup.2, and R.sup.3 alkyl groups of formula I can
have, for example, from 1 to 8 carbon atoms, from 1 to 6 carbon
atoms, and/or from 1 to 4 carbon atoms. In some embodiments,
R.sup.1, R.sup.2, and R.sup.3 are independently selected from the
group consisting of methyl and ethyl.
[0024] R.sup.4 and R.sup.5 alkyl groups of formula I can have, for
example, from 6 to 40 carbon atoms, from 10 to 24 carbon atoms,
from 14 to 18 carbon atoms, from 5 to 39 carbon atoms, from 9 to 23
carbon atoms, and/or from 13 to 17 carbon atoms.
[0025] R.sup.4 and R.sup.5 alkenyl groups of formula I can have,
for example, 1, 2, 3, 4, 5, 6, or more double bonds. The R.sup.4
and R.sup.5 alkenyl groups can have, for example, from 6 to 40
carbon atoms, from 10 to 24 carbon atoms, from 14 to 18 carbon
atoms, from 5 to 39 carbon atoms, from 9 to 23 carbon atoms, and/or
from 13 to 17 carbon atoms.
[0026] R.sup.4 and R.sup.5 alkynyl groups of formula I can have,
for example, 1, 2, 3, 4, 5, 6, or more triple bonds. The R.sup.4
and R.sup.5 alkynyl groups can have, for example, from 6 to 40
carbon atoms, from 10 to 24 carbon atoms, from 14 to 18 carbon
atoms, from 5 to 39 carbon atoms, from 9 to 23 carbon atoms, and/or
from 13 to 17 carbon atoms.
[0027] In some embodiments, R.sup.4 and R.sup.5 are independently
selected from the group consisting of octyl, 2-ethylhexyl, nonyl,
decyl, dodecyl, tetradecyl, hexadecyl, cis-9-hexadecenyl,
octadecyl, 16-methylheptadecyl, trans-9-octadecenyl,
cis-9-octadecenyl, cis,cis-9,12-octadecadienyl,
trans,trans-9,12-octadecadienyl,
cis,cis,cis-9,12,15-octadecatrienyl,
trans,trans,trans-9,12,15-octadecatrienyl,
12-hydroxy-9-octadecenyl, eicosanyl, docosanyl, cis-13-docosenyl,
tetracosanyl, hexacosanyl, octacosanyl, triacontanyl,
tetratriacontanyl, octanoyl, decanoyl, dodecanoyl, tetradecanoyl,
hexadecanoyl, heptadecanoyl, octadecanoyl, eicosanoyl, docosanoyl,
tetracosanoyl, cis,cis,cis-9,12,15-octadecatrienoyl,
cis,cis,cis,cis-6,9,12,15-octadecatetraenoyl,
cis,cis,cis,cis,cis-5,8,11,14,17-eicosapentenoyl,
cis,cis,cis,cis,cis,cis-4,7,10,13,16,19-docosahexaenoyl,
cis,cis-9,12-octadecadienoyl, cis,cis,cis-6,9,12-octadecatrienoyl,
cis,cis,cis-8,11,14-eicosatrienoyl,
cis,cis,cis,cis-5,8,11,14-eicosatetraenoyl, cis-9-octadecenoyl,
trans-9-octadecenoyl, cis-13-docosenoyl, and
cis-15-tetracosenoyl.
[0028] In some embodiments, m and n are 1; R.sup.2, and R.sup.3 are
methyl; and R.sup.4 and R.sup.5 are cis-9-octadecenoyl, i.e., the
surfactant of formula I is
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP) or a
salt thereof. In other embodiments, m and n are 1; R.sup.1,
R.sup.2, and R.sup.3 are methyl; and R.sup.4 and R.sup.5 are
cis-9-octadecenyl, i.e., the surfactant of formula I is
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA), or a
salt thereof. In some embodiments, the active agent is paclitaxel;
m and n are 1; R.sup.1, R.sup.2, and R.sup.3 are methyl; and
R.sup.4 and R.sup.5 are cis-9-octadecenoyl, i.e., the surfactant of
formula I is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
(DOTAP) or a salt thereof. In other embodiments, the active agent
is paclitaxel; m and n are 1; R.sup.2, and R.sup.3 are methyl; and
R.sup.4 and R.sup.5 are cis-9-octadecenyl, i.e., the surfactant of
formula I is N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium
(DOTMA), or a salt thereof.
[0029] Another aspect of the present invention provides methods for
enhancing uptake of an active agent by phagocytic or non-phagocytic
cells by exposing the cells to a surface-modified particle
comprising a particle core and a coating associated with the
particle core, thereby forming cells loaded with the surface
modified particles. The particle core comprises an active agent
which is typically selected from the group consisting of small
molecules, peptides, and proteins, the coating comprises a
surfactant of formula I, as defined herein, and the
surface-modified particle has an average size from about 1 nm to
about 2,000 nm. Enhanced uptake by the cells of the active agent is
observed at least as compared to cells contacted with particles not
having a coating comprising a surfactant of formula I. Such methods
can be performed in vivo or ex vivo to form cells loaded with the
surface modified particles. In yet another aspect, the invention
also provides methods for delivery of a surface-modified particle
to a target tissue of a mammalian subject through cellular
transport using the aforementioned cells loaded with the surface
modified particles. It is contemplated that various methods of
administration, such as intravenous administration, intramuscular
administration, subcutaneous administration, and the like will
facilitate enhanced uptake of particles by cells that traffic to
the lymphatic system, the liver, and other tissue targets.
Subcutaneous administration, for example, is contemplated for
various diseases, including head and neck cancers which invade
locoregionally along the lymphatics.
[0030] As used herein, "target tissue" or "tissue target" refers to
the particular tissue of the subject to be treated. Examples of
such target tissues include, but are not limited to, the brain and
other portions of the central nervous system, the lymphatic system
(e.g., lymph nodes, bone marrow, spleen, thymus, etc.), the liver,
and any site of infection, inflammation, or tumor.
[0031] In addition to delivery by cellular transport, delivery to a
target tissue can be effected by localized administration of the
surface-modified particles into a body cavity having a site of
disease (e.g., cancer, infection) and/or inflammation in the
subject such that the surface-modified particles can be taken up by
diseased or inflammatory cells located within the body cavity so as
to deliver the active agent in close proximity to the diseased
tissue target. For example, cancers of the peritoneal cavity such
as ovarian cancer, peritoneal mesothelioma, peritoneal
carcinomatosis, and the like can be treated by intraperitoneally
administering the particles into the peritoneal cavity. Similarly,
bladder cancers, infections, and/or inflammation can be treated by
administering the particles into the bladder cavity; pulmonary
cancers, infections, and/or inflammation can be treated by
administering the particles into the pulmonary cavity (e.g, via
inhalation); cancers, infections, and/or inflammation of the
pleural cavity can be treated by administering particles into the
pleural cavity; cancers, infections, and/or inflammation of the
cardiac cavity can be treated by administering particles into the
cardiac cavity; and ophthalmic cancers, infections, and/or
inflammation can be treated by administering the particles into the
aqueous humor or vitreous humor of the eye. Advantageously, when
the surface-modified particles are administered proximate to and/or
adjacent to a site of disease or inflammation via administration to
a body cavity containing the site of disease or inflammation, the
surface-modified particles can be taken up by the diseased (e.g.,
cancerous, infected) or inflammatory cells located at the site of
disease or inflammation such that enhanced uptake by the diseased
or inflammatory cells of the surface modified particles according
to the invention is observed at least as compared to cells
contacted with particles not having a coating comprising a
surfactant of formula I.
[0032] As used herein, a "body cavity" refers to a relatively empty
space surrounded by a supporting tissue or a fluid-filled space
surrounded by a supporting tissue. As used herein, a body cavity
encompasses both the tissue surrounding (and defining) the cavity
and the complete interior of the cavity. Exemplary body cavities
include the peritoneal cavity, the bladder cavity, the pulmonary
cavity, the pleural cavity, the cardiac cavity, the aqueous humor
of the eye, and the vitreous humor of the eye.
[0033] In one aspect, the invention contemplates methods and
compositions for treating a subject having an inflammatory disease
or disorder comprising administering to said subject a plurality of
surface-modified particles, said surface-modified particles
comprising a particle core and a coating associated with the
particle core, wherein the particle core comprises an active agent
which is typically selected from the group consisting of small
molecules, peptides, and proteins, the coating comprises a
surfactant of formula I, as defined herein, the surface-modified
particle has an average size from about 1 nm to about 2,000 nm, and
said administration is effective in alleviating, treating, and/or
preventing symptoms or pathologies associated with said
inflammatory disease or disorder. In one aspect, the subject has an
inflammatory disease or disorder, and m and n are 1; R.sup.1,
R.sup.2, and R.sup.3 are methyl; and R.sup.4 and R.sup.5 are
cis-9-octadecenoyl. In one aspect, the active agent is an
anti-inflammatory agent. Delivery of the active agent can be
effected via cellular transport, as described herein, or by local
administration to the site of inflammation, as described
herein.
[0034] In another aspect, the invention contemplates methods and
compositions for treating a subject having a neurodegenerative
disease or disorder comprising administering to said subject a
plurality of surface-modified particles, said surface-modified
particles comprising a particle core and a coating associated with
the particle core, wherein the particle core comprises an active
agent which is typically selected from the group consisting of
small molecules, peptides, and proteins, the coating comprises a
surfactant of formula I, as defined herein, the surface-modified
particle has an average size from about 1 nm to about 2,000 nm, and
said administration is effective in alleviating, treating, and/or
preventing symptoms or pathologies associated with said
neurodegenerative disease or disorder. In one aspect, the subject
has a neurodegenerative disease or disorder, and m and n are 1;
R.sup.1, R.sup.2, and R.sup.3 are methyl; and R.sup.4 and R.sup.5
are cis-9-octadecenoyl. In one aspect, the active agent is an
anti-neurodegenerative agent. Delivery of the active agent can be
effected via cellular transport, as described herein.
[0035] In yet another aspect, the invention contemplates methods
and compositions for treating a subject having a proliferative
disease or disorder comprising administering to said subject a
plurality of surface-modified particles, said surface-modified
particles comprising a particle core and a coating associated with
the particle core, wherein the particle core comprises an active
agent which is typically selected from the group consisting of
small molecules, peptides, and proteins, the coating comprises a
surfactant of formula I, as defined herein, the surface-modified
particle has an average size from about 1 nm to about 2,000 nm, and
said administration is effective in alleviating, treating, and/or
preventing symptoms or pathologies associated with said
proliferative disease or disorder. In one aspect, the subject has a
proliferative disease or disorder, and m and n are 1; R.sup.1,
R.sup.2, and R.sup.3 are methyl; and R.sup.4 and R.sup.5 are
cis-9-octadecenoyl. In one aspect, the active agent is an
anti-proliferative agent such as an antineoplastic agent. Delivery
of the active agent can be effected via cellular transport, as
described herein, or by local administration to the site of
disease, as described herein.
[0036] In a still further aspect, the invention contemplates
methods and compositions for treating a subject having an
infectious disease or disorder comprising administering to said
subject a plurality of surface-modified particles, said
surface-modified particles comprising a particle core and a coating
associated with the particle core, wherein the particle core
comprises an active agent which is typically selected from the
group consisting of small molecules, peptides, and proteins, the
coating comprises a surfactant of formula I, as defined herein, the
surface-modified particle has an average size from about 1 nm to
about 2,000 nm, and said administration is effective in
alleviating, treating, and/or preventing symptoms or pathologies
associated with said infectious disease or disorder. In one aspect,
the subject has an infectious disease or disorder, and m and n are
1; R.sup.1, R.sup.2, and R.sup.3 are methyl; and R.sup.4 and
R.sup.5 are cis-9-octadecenoyl. In one aspect, the active agent is
an anti-infective agent such as an anti-fungal agent, an anti-viral
agent, an anti-bacterial agent, or an anti-parasitic agent.
Delivery of the active agent can be effected via cellular
transport, as described herein, or by local administration to the
site of disease, as described herein.
[0037] Thus, the methods of administration disclosed herein
contemplate administration of a therapeutically effective amount of
said surface modified particles. As used herein, the term
"therapeutically effective amount" refers to an amount of
surface-coated particles that is sufficient to alleviate,
ameliorate, clear, treat, and/or prevent symptoms or pathologies
associated with a disease or disorder contemplated for treatment in
accordance with the treatment methods disclosed herein.
Determination of therapeutically effective amounts is well within
the capability of those skilled in the art, especially in light of
the disclosure provided herein.
[0038] The following description of the surface-modified particle
applies to all embodiments disclosed herein. The active agent of
the surface-modified particle can be poorly water soluble or water
soluble. The active agent can be a therapeutic agent or a
diagnostic agent. The active agent can be a small molecule or a
biologic, such as a protein, a peptide, a carbohydrate, or a
complex, conjugate, or combination thereof. In one preferred
aspect, DNA, RNA, oligonucleotides, and polynucleotides are not
suitable active agents for use with the surface modified particles
of the invention. Active agents used in accordance
with the compositions and methods disclosed herein exhibit the
pharmaceutical activities normally associated with such active
agents notwithstanding that the active agents can be taken up and
subsequently delivered to target tissues by phagocytic or
non-phagocytic cells. As discussed above, active agents also can be
administered locally at a site of disease (e.g., cancer, infection)
and/or inflammation in a mammalian subject and taken up by diseased
cells (such as infected or cancerous cells), or inflammatory cells,
located at the site of disease and/or inflammation.
[0039] The active agent can be selected from a variety of known
pharmaceutical compounds such as, but not limited to: analgesics,
anesthetics, analeptics, adrenergic agents, adrenergic blocking
agents, adrenolytics, adrenocorticoids, adrenomimetics,
anticholinergic agents, anticholinesterases, anticonvulsants,
alkylating agents, alkaloids, allosteric inhibitors, anabolic
steroids, anorexiants, antacids, antidiarrheals, antidotes,
antifolics, antipyretics, antirheumatic agents, psychotherapeutic
agents, neural blocking agents, anti-inflammatory agents,
antihelmintics, anticoagulants, antidepressants, antiepileptics,
antifibrotic agents, anti-infective agents (e.g., antifungals,
antiviral agents such as antiretroviral agents, and antibiotics),
antihistamines, antimuscarinic agents, antimycobacterial agents,
antineoplastic agents, antiprotozoal agents, anxiolytic sedatives,
beta-adrenoceptor blocking agents, corticosteroids, cough
suppressants, dopaminergics, hemostatics, hematological agents,
hypnotics, immunological agents, muscarinics, parasympathomimetics,
prostaglandins, radio-pharmaceuticals, sedatives, stimulants,
sympathomimetics, vitamins, xanthines, growth factors, hormones,
and antiprion agents.
[0040] Examples of antineoplastic agents include, but are not
limited to, paclitaxel, paclitaxel derivative compounds, alkaloids,
antimetabolites, enzyme inhibitors, alkylating agents, and
combinations thereof.
[0041] The active agent also can be a protease inhibitor, such as
an HIV protease inhibitor. Examples of protease inhibitors include,
but are not limited to, indinavir, ritonavir, saquinavir,
nelfinavir, and combinations thereof.
[0042] The active agent can be a nucleoside reverse transcriptase
inhibitor. Examples of nucleoside reverse transcriptase inhibitors
include, but are not limited to, zidovudine, didanosine, stavudine,
zalcitabine, lamivudine, and combinations thereof.
[0043] The active agent can be a non-nucleoside reverse
transcriptase inhibitor. Examples of non-nucleoside reverse
transcriptase inhibitors include, but are not limited to,
efavirenz, nevirapine, delaviradine, and combinations thereof.
[0044] Examples of anti-inflammatory agents include, but are not
limited to, non-steroidal anti-inflammatory drugs, non-selective
cycloxygenase (COX) inhibitors, COX-1 inhibitors, COX-2 inhibitors,
lipoxygenase inhibitors, corticosteroids, anti-oxidants, tumor
necrosis factor (TNF) inhibitors, and combinations thereof.
Examples of COX-2 inhibitors include, but are not limited to,
celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib,
etoricoxib, and combinations thereof.
[0045] Diagnostic agents include x-ray imaging agents and contrast
media. Examples of x-ray imaging agents include WIN-8883 (ethyl
3,5-diacetamido-2,4,6-triiodobenzoate) also known as the ethyl
ester of diatrazoic acid (EEDA), WIN 67722, i.e.,
(6-ethoxy-6-oxohexyl-3,5-bis(acetamido)-2,4,6-triiodobenzoate;
ethyl-2-(3,5-bis(acetamido)-2,4,6-triiodo-benzoyloxy) butyrate (WIN
16318); ethyl diatrizoxyacetate (WIN 12901); ethyl
2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy)propionate (WIN
16923); N-ethyl 2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy
acetamide (WIN 65312); isopropyl
2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy) acetamide (WIN
12855); diethyl 2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy)
malonate (WIN 67721); ethyl
2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy) phenylacetate (WIN
67585); propanedioic acid,
[[3,5-bis(acetylamino)-2,4,5-triodobenzoyl]oxy]bis(1-methyl)ester
(WIN 68165); and benzoic acid,
3,5-bis(acetylamino)-2,4,6-triiodo-4-(ethyl-3-ethoxy-2-butenoate)
ester (WIN 68209). Contrast agents include those that are expected
to disintegrate relatively rapidly under physiological conditions,
thus minimizing any particle associated inflammatory response.
Disintegration can result from enzymatic hydrolysis, solubilization
of carboxylic acids at physiological pH, or other mechanisms. Thus,
poorly soluble iodinated carboxylic acids such as iodipamide,
diatrizoic acid, and metrizoic acid, along with hydrolytically
labile iodinated species such as WIN 67721, WIN 12901, WIN 68165,
and WIN 68209 are included.
[0046] Other contrast media include, but are not limited to,
particulate preparations of magnetic resonance imaging aids such as
gadolinium chelates, or other paramagnetic contrast agents.
Examples of such compounds are gadopentetate dimeglumine
(MAGNEVIST.RTM.) and gadoteridol (PROHANCE.RTM.).
[0047] A description of classes of therapeutic agents and
diagnostic agents and a listing of species within each class can be
found in Martindale, The Extra Pharmacopoeia, 31st Edition, The
Pharmaceutical Press, London, 1996 which is incorporated herein by
reference and made a part hereof. The listed therapeutic agents and
diagnostic agents are commercially available and/or can be prepared
by known techniques.
[0048] In a specific embodiment, the active agent is a poorly
water-soluble compound. What is meant by "poorly water soluble" is
a solubility of the compound in water of less than about 10 mg/mL,
and preferably less than about 1 mg/mL. These poorly water-soluble
compounds are particularly suitable for aqueous suspension
preparations since there are limited alternatives of formulating
these compounds in an aqueous medium. Advantageously, surfactants
of formula I, which provide the coatings in accordance with the
invention, can adsorb to the surface of particles comprising such
poorly water soluble active agents to form a substantially uniform
coating thereon. For example, the hydrophobic tail moieties of
surfactants of formula I can associate with hydrophobic regions on
the particle surface. In addition, surfactants of formula I are
positively charged, and thus electrostatic interactions between the
surfactant and negatively charged regions on the particle surface
can stabilize the coating comprising the surfactant of formula I.
In one preferred aspect, the poorly water soluble active agent
compound is an organic compound having a molecular weight less than
2500 grams/mol, less than 2000 grams/mol, and most typically less
than 1000 grams/mol, for example, between 200 grams/mol and 900
grams/mol. Such organic compounds are referred to herein as "small
molecules."
[0049] Alternatively, the invention can be practiced with
water-soluble compounds. To form aqueous suspensions of
water-soluble compounds the water soluble active compounds can be
entrapped in a solid carrier matrix (for example,
polylactate-polyglycolate copolymer, albumin, starch), or
encapsulated in a surrounding vesicle that is substantially
impermeable to the active agent. This encapsulating vesicle can be
a polymeric coating such as polyacrylate. Further, the small
particles prepared from these water soluble compounds can he
modified to improve chemical stability and control the
pharmacokinetic properties of the compounds by controlling the
release of the compounds from the particles. Examples of
water-soluble compounds include, but are not limited to, simple
organic compounds, proteins, peptides, nucleotides, and
carbohydrates.
[0050] The following description of particles also applies to all
embodiments disclosed herein. The particles can be amorphous,
semicrystalline, crystalline, or a combination thereof as
determined by suitable analytical methods such as differential
scanning calorimetry (DSC) or X-ray diffraction. Prior to
administration, the particles can be homogenized through a
homogenization process. The particles can also be homogenized
through a microprecipitation/homogenization process.
[0051] The coated particles generally have an average effective
particle size of generally from about 1 nm to about 2 .mu.m (or
2000 nanometers) as measured by dynamic light scattering methods
(e.g., photocorrelation spectroscopy, laser diffraction, low-angle
laser light scattering (LALLS), medium-angle laser light scattering
(MALLS)), light obscuration methods (Coulter method, for example),
rheology, or microscopy (light or electron). The preferred average
effective particle size depends on factors such as the intended
route of administration, formulation, solubility, toxicity and
bioavailability of the compound. Other suitable particle sizes
include, but are not limited to, about 10 nm to about 1 .mu.m,
about 50 nm to about 500 nm, and/or about 100 nm to about 250
nm.
[0052] In all embodiments, the coated particles are solid or
semi-solid particles comprising active agents. The coated particles
generally consist of at least 5% (w/w) active agent, for example,
at least 10% (w/w), at least 25% (w/w), at least 50% (w/w), and/or
at least 75% (w/w) or more active agent.
Preparation of the Particle Core
[0053] The processes for preparing the particles used in the
present invention can be accomplished through numerous techniques.
A representative, but non-exhaustive, discussion of techniques for
preparing particles follows.
I. Energy Addition Techniques for Forming Small Particle
Dispersions
[0054] In general, the method of preparing small particle
dispersions using energy addition techniques includes the step of
adding the active agent or pharmaceutically active compound, which
sometimes shall be referred to as a drug, in bulk form to a
suitable vehicle such as water or aqueous solution generally
containing one or more of the surfactants set forth below, or other
liquid in which the pharmaceutical compound is not appreciably
soluble, to form a first suspension, which shall be referred to as
a presuspension. Energy is added to the presuspension to form a
particle dispersion which is physically more stable than the
presuspension. Energy is added by mechanical grinding (e.g., pearl
milling, ball milling, hammer milling, fluid energy milling, jet
milling, or wet grinding). Such techniques are disclosed in U.S.
Pat. No. 5,145,684, which is incorporated herein by reference and
made a part hereof.
[0055] Energy addition techniques further include subjecting the
presuspension to high shear conditions including cavitation,
shearing or impact forces utilizing a microfluidizer. The present
invention further contemplates adding energy to the presuspension
using a piston gap homogenizer or counter current flow homogenizer
such as those disclosed in U.S. Pat. No. 5,091,188 which is
incorporated herein by reference and made a part hereof. Suitable
piston gap homogenizers are commercially available under the
product names EMULSIFLEX.TM. (Avestin) and FRENCH.RTM. Pressure
Cell (Thermo Spectronic). Suitable microfluidizers are available
from Microfluidics Corp.
[0056] The step of adding energy can also be accomplished using
sonication techniques. The step of sonicating can be carried out
with any suitable sonication device. Suitable devices include
Branson Model 5-450A and Cole-Parmer 500/750 Watt Model. Such
devices are well known in the industry. Typically the sonication
device has a sonication horn or probe that is inserted into the
presuspension to emit sonic energy into the solution. The
sonicating device, in a preferred form of the invention, is
operated at a frequency of from about 1 kHz to about 90 kHz and
more preferably from about 20 kHz to about 40 kHz or any range or
combination of ranges therein. The probe sizes can vary and
preferably are in distinct sizes such as 1/2 inch or 1/4 inch or
the like.
[0057] The dispersion of small particles can be sterilized prior to
administering. Sterilization can be accomplished by heat
sterilization, gamma irradiation, filtration (either directly as a
dispersion having particle sizes under 200 nm, or by sterile
filtration of the solutions used in the precipitation process,
prior to forming the solid dispersion), and by application of very
high pressure (greater than 2000 atmospheres), or by a combination
of high pressure and elevated temperature.
II. Precipitation Methods for Preparing Submicron Sized Particle
Dispersions
[0058] Small particle dispersions can also be prepared by
precipitation techniques. The following is a description of
examples of precipitation techniques.
[0059] Microprecipitation Methods. One example of a
microprecipitation method is disclosed in U.S. Pat. No. 5,780,062,
which is incorporated herein by reference and made a part hereof.
The '062 patent discloses an organic compound precipitation process
including: (i) dissolving the organic compound in a water-miscible
first solvent; (ii) preparing a solution of polymer and an
amphiphile in an aqueous second solvent and in which second solvent
the organic compound is substantially insoluble whereby a
polymer/amphiphile complex is formed; and (iii) mixing the
solutions from steps (i) and (ii) so as to cause precipitation of
an aggregate of the organic compound and the polymer/amphiphile
complex.
[0060] Other suitable precipitation processes are disclosed in U.S.
Pat. Nos. 6,607,784, 7,037,528, 6,869,617, 6,884,436, which are
incorporated herein by reference and made a part hereof. The
processes disclosed include the steps of: (1) dissolving an organic
compound in a water miscible first organic solvent to create a
first solution; (2) mixing the first solution with a second solvent
or water to precipitate the organic compound to create a
presuspension; and (3) adding energy to the presuspension in the
form of high-shear mixing or heat to provide a dispersion of small
particles. Optionally, the first organic solvent is removed from
the mixture by any suitable means such as centrifugation or
filtration methods. Moreover, the continuous phase of the
dispersion can be optionally replaced by another continuous phase
by removing the first continuous phase using methods such as
centrifugation and filtration, and adding a second continuous phase
and subsequently redispersing the solid material in the second
continuous phase. One or more optional surfactants set forth below
can be added to the first organic solvent, to the second aqueous
solution, or to both the first organic solvent and the second
aqueous solution.
[0061] Emulsion Precipitation Methods. One suitable emulsion
precipitation technique is disclosed in U.S. Patent Pub. No.
2005/0037083, which is incorporated herein by reference and is made
a part hereof. In this approach, the process includes the steps of:
(1) providing a multiphase system having an organic phase and an
aqueous phase, the organic phase having a pharmaceutically active
compound therein; and (2) sonicating the system to evaporate a
portion of the organic phase to cause precipitation of the compound
in the aqueous phase to form a dispersion of small particles. The
step of providing a multiphase system includes the steps of: (1)
mixing a water immiscible solvent with the pharmaceutically active
compound to define an organic solution, (2) preparing an aqueous
based solution with one or more surface active compounds, and (3)
mixing the organic solution with the aqueous solution to form the
multiphase system. The step of mixing the organic phase and the
aqueous phase can include the use of piston gap homogenizers,
colloidal mills, high speed stirring equipment, extrusion
equipment, manual agitation or shaking equipment, microfluidizer,
or other equipment or techniques for providing high shear
conditions. The crude emulsion will have oil droplets in the water
of a size of approximately less than 1 .mu.m in diameter. The crude
emulsion is sonicated to define a microemulsion and eventually to
provide a dispersion of small particles.
[0062] Another approach to preparing a dispersion of small
particles is disclosed U.S. Pat. No. 6,835,396, which is
incorporated herein by reference and made a part hereof. The
process includes the steps of: (1) providing a crude dispersion of
a multiphase system having an organic phase and an aqueous phase,
the organic phase having a pharmaceutical compound therein; (2)
providing energy to the crude dispersion to form a fine dispersion;
(3) freezing the fine dispersion; and (4) lyophilizing the fine
dispersion to obtain small particles of the pharmaceutical
compound. The small particles can be sterilized by the techniques
set forth below or the small particles can be reconstituted in an
aqueous medium and sterilized.
[0063] The step of providing a multiphase system includes the steps
of: (1) mixing a water immiscible solvent with the pharmaceutically
effective compound to define an organic solution; (2) preparing an
aqueous based solution with one or more surface active compounds;
and (3) mixing the organic solution with the aqueous solution to
form the multiphase system. The step of mixing the organic phase
and the aqueous phase includes the use of piston gap homogenizers,
colloidal mills, high speed stirring equipment, extrusion
equipment, manual agitation or shaking equipment, microfluidizer,
or other equipment or techniques for providing high shear
conditions.
[0064] Solvent-Antisolvent Precipitation. Small particle
dispersions can also be prepared using a solvent-antisolvent
precipitation technique disclosed by Fessi et al. in U.S. Pat. No.
5,118,528 and by Leclef et al. in U.S. Pat. No. 5,100,591 which are
incorporated herein by reference and made a part hereof. Both
processes include the steps of: (1) preparing a liquid phase of a
biologically active substance in a solvent or a mixture of solvents
to which may be added one or more surfactants; (2) preparing a
second liquid phase of a non-solvent or a mixture of non-solvents,
the non-solvent is miscible with the solvent or mixture of solvents
for the substance; (3) adding together the solutions of (1) and (2)
with stirring; and (4) removing of unwanted solvents to produce a
dispersion of small particles. These methods are distinguished from
those described under the above section, "Microprecipitation
Methods", in that they do not provide for a last step of adding
energy to the suspension in the form of high-shear mixing or
heat.
[0065] Phase Inversion Precipitation. Small particle dispersions
can be formed using phase inversion precipitation as disclosed in
U.S. Pat. Nos. 6,235,224, 6,143,211 and 6,616,869, each of which is
incorporated herein by reference and made a part hereof. Phase
inversion is a term used to describe the physical phenomena by
which a polymer dissolved in a continuous phase solvent system
inverts into a solid macromolecular network in which the polymer is
the continuous phase. One method to induce phase inversion is by
the addition of a nonsolvent to the continuous phase. The polymer
undergoes a transition from a single phase to an unstable two phase
mixture: polymer rich and polymer poor fractions. Micellar droplets
of nonsolvent in the polymer rich phase serve as nucleation sites
and become coated with polymer. The '224 patent discloses that
phase inversion of polymer solutions under certain conditions can
bring about spontaneous formation of discrete microparticles,
including nanoparticles. The '224 patent discloses dissolving or
dispersing a polymer in a solvent. A pharmaceutical agent is also
dissolved or dispersed in the solvent. For the crystal seeding step
to be effective in this process, it is desirable the agent is
dissolved in the solvent. The polymer, the agent and the solvent
together form a mixture having a continuous phase, wherein the
solvent is the continuous phase. The mixture is then introduced
into at least tenfold excess of a miscible nonsolvent to cause the
spontaneous formation of the microencapsulated microparticles of
the agent having an average particle size of between 10 nm and 10
.mu.m. The particle size is influenced by the solvent:nonsolvent
volume ratio, polymer concentration, the viscosity of the
polymer-solvent solution, the molecular weight of the polymer, and
the characteristics of the solvent-nonsolvent pair.
[0066] pH Shift Precipitation. Small particle dispersions can be
formed by pH shift precipitation techniques. Such techniques
typically include a step of dissolving a drug in a solution having
a pH where the drug is soluble, followed by the step of changing
the pH to a point where the drug is no longer soluble. The pH can
be acidic or basic, depending on the particular pharmaceutical
compound. The solution is then neutralized to form a dispersion of
small particles. One suitable pH shifting precipitation process is
disclosed in U.S. Pat. No. 5,665,331, which is incorporated herein
by reference and made a part hereof. The process includes the step
of dissolving of the pharmaceutical agent together with a crystal
growth modifier (CGM) in an alkaline solution and then neutralizing
the solution with an acid in the presence of suitable
surface-modifying surface-active agent or agents to form a small
particle dispersion of the pharmaceutical agent. The precipitation
step can be followed by steps of diafiltration clean-up of the
dispersion and then adjusting the concentration of the dispersion
to a desired level.
[0067] Other examples of pH shifting precipitation methods are
disclosed in U.S. Pat. Nos. 5,716,642; 5,662,883; 5,560,932; and
4,608,278, which are incorporated herein by reference and are made
a part hereof.
[0068] Infusion Precipitation Method. Suitable infusion
precipitation techniques to form small particle dispersions are
disclosed in U.S. Pat. Nos. 4,997,454 and 4,826,689, which are
incorporated herein by reference and made a part hereof. First, a
suitable solid compound is dissolved in a suitable organic solvent
to form a solvent mixture. Then, a precipitating nonsolvent
miscible with the organic solvent is infused into the solvent
mixture at a temperature between about -10.degree. C. and about
100.degree. C. and at an infusion rate of from about 0.01 ml per
minute to about 1000 ml per minute per volume of 50 ml to produce a
suspension of precipitated non-aggregated solid particles of the
compound with a substantially uniform mean diameter of less than 10
.mu.m. Agitation (e.g., by stirring) of the solution being infused
with the precipitating nonsolvent is preferred. The nonsolvent may
contain a surfactant to stabilize the particles against
aggregation. The particles are then separated from the solvent.
Depending on the solid compound and the desired particle size, the
parameters of temperature, ratio of nonsolvent to solvent, infusion
rate, stir rate, and volume can be varied according to the
invention. The particle size is proportional to the ratio of
nonsolvent:solvent volumes and the temperature of infusion and is
inversely proportional to the infusion rate and the stirring rate.
The precipitating nonsolvent may be aqueous or non-aqueous,
depending upon the relative solubility of the compound and the
desired suspending vehicle.
[0069] Temperature Shift Precipitation. Temperature shift
precipitation techniques may also be used to form small particle
dispersions. This technique is disclosed in U.S. Pat. No.
5,188,837, which is incorporated herein by reference and made a
part hereof. In an embodiment of the invention, lipospheres are
prepared by the steps of: (1) melting or dissolving a substance
such as a drug to be delivered in a molten vehicle to form a liquid
of the substance to be delivered; (2) adding a phospholipid along
with an aqueous medium to the melted substance or vehicle at a
temperature higher than the melting temperature of the substance or
vehicle; (3) mixing the suspension at a temperature above the
melting temperature of the vehicle until a homogenous fine
preparation is obtained; and then (4) rapidly cooling the
preparation to room temperature or below.
[0070] Solvent Evaporation Precipitation. Solvent evaporation
precipitation techniques are disclosed in U.S. Pat. No. 4,973,465
which is incorporated herein by reference and made a part hereof.
The '465 patent discloses methods for preparing microcrystals
including the steps of: (1) providing a solution of a
pharmaceutical composition and a phospholipid dissolved in a common
organic solvent or combination of solvents, (2) evaporating the
solvent or solvents and (3) suspending the film obtained by
evaporation of the solvent or solvents in an aqueous solution by
vigorous stirring to form a dispersion of small particles. The
solvent can be removed by evaporating a sufficient quantity of the
solvent to cause precipitation of the compound. The solvent can
also be removed by other well known techniques such as applying a
vacuum to the solution or blowing nitrogen over the solution.
[0071] Reaction Precipitation. Reaction precipitation includes the
steps of dissolving the pharmaceutical compound, and optionally
other excipients, into a suitable solvent to form a solution. The
compound may be added in an amount at or below the saturation point
of the compound in the solvent. The compound or any of the
excipients is precipitated from solution by reacting with a
chemical agent or by modification in response to adding energy such
as heat or UV light or the like such that the modified compound has
a lower solubility in the solvent and precipitates from the
solution to form a small particle dispersion. Precipitation of
excipient provides a solid matrix into which the drug is
sorbed.
[0072] Compressed Fluid Precipitation. A suitable technique for
precipitating by compressed fluid is disclosed in WO 97/14407 to
Johnston, which is incorporated herein by reference and made a part
hereof. The method includes the steps of dissolving a water
insoluble drug in a solvent to form a solution. The solution is
then sprayed into a compressed fluid, which can be a gas, liquid or
supercritical fluid. The addition of the compressed fluid to a
solution of a solute in a solvent causes the solute to attain or
approach supersaturated state and to precipitate out as fine
particles. In this case, the compressed fluid acts as an
antisolvent which lowers the cohesive energy density of the solvent
in which the drug is dissolved.
[0073] Alternatively, the drug can be dissolved in the compressed
fluid which is then sprayed into an aqueous phase. The rapid
expansion of the compressed fluid reduces the solvent power of the
fluid, which in turn causes the solute to precipitate out as small
particles in the aqueous phase. In this case, the compressed fluid
acts as a solvent.
[0074] In order to stabilize the particles against aggregation, a
surface modifier, such as a surfactant, is included in this
technique.
[0075] Spraying into Cryogenic Fluids. A suitable technique for
precipitating by compressed fluid is disclosed by Williams et al.
in U.S. Patent Pub. No. 2004/0022861, which is incorporated herein
by reference and made a part hereof. The method provides a system
and method for the production of small particles wherein the active
ingredient is mixed with water, one or more solvents, or a
combination thereof, and the resulting mixture sprayed at or below
the surface of a cryogenic fluid. Frozen particles are thereby
provided. Materials for encapsulating the solid particles may also
be added so that frozen particles are generated wherein the
encapsulating agent surrounds the active agent.
[0076] Protein Nanosphere/Microsphere Precipitation. Particles
utilized in this invention can also be produced from a process
involving mixing or dissolving macromolecules such as proteins with
a water soluble polymer. This process is disclosed in U.S. Pat.
Nos. 5,981,719, 6,090,925, 6,268,053, 6,458,387, and U.S. Patent
Pub. No. 2004/0043077, which are incorporated herein by reference
and made a part hereof. In an embodiment of the invention,
particles are prepared by mixing a macromolecule in solution with a
polymer or a mixture of polymers in solution at a pH near the
isoelectric point of the macromolecule. The mixture is incubated in
the presence of an energy source, such as heat, radiation, or
ionization, for a predetermined amount of time. The resulting
particles can be removed from any unincorporated components present
in the solution by physical separation methods.
[0077] There are numerous other suitable methodologies for
preparing small particle dispersions capable of use in accordance
with the invention.
III. Additional Methods for Preparing Particle Dispersions of
Pharmaceutical Compositions
[0078] The following additional processes for preparing particles
of pharmaceutical compositions (i.e. active agent or organic
compound) used in the present invention can be separated into four
general categories. Each of the categories of processes share the
steps of: (1) dissolving an organic compound in a water miscible
first solvent to create a first solution, (2) mixing the first
solution with a second solvent of water to precipitate the organic
compound to create a pre-suspension, and (3) adding energy to the
presuspension in the form of high-shear mixing or heat, or a
combination of both, to provide a stable form of the organic
compound having desired size ranges defined above. The mixing steps
and the adding energy step can be carried out in consecutive steps
or simultaneously.
[0079] The categories of processes are distinguished based upon the
physical properties of the organic compound as determined through
x-ray diffraction studies, differential scanning calorimetry (DSC)
studies, or other suitable study conducted prior to the
energy-addition step and after the energy-addition step. In the
first process category, prior to the energy-addition step the
organic compound in the presuspension takes an amorphous form, a
semi-crystalline form or a supercooled liquid form and has an
average effective particle size. After the energy-addition step the
organic compound is in a crystalline form having an average
effective particle size essentially the same or less than that of
the presuspension.
[0080] In the second process category, prior to the energy-addition
step the organic compound is in a crystalline form and has an
average effective particle size. After the energy-addition step,
the organic compound is in a crystalline form having essentially
the same average effective particle size as prior to the
energy-addition step but the crystals after the energy-addition
step are less likely to aggregate or form large crystals.
[0081] The lower tendency of the organic compound to aggregate or
form large crystals is observed by laser dynamic light scattering
and light microscopy.
[0082] In the third process category, prior to the energy-addition
step the organic compound is in a crystalline form that is friable
and has an average effective particle size. What is meant by the
term "friable" is that the particles are fragile and are more
easily broken down into smaller particles. After the
energy-addition step the organic compound is in a crystalline form
having an average effective particle size smaller than the crystals
of the pre-suspension. By taking the steps necessary to place the
organic compound in a crystalline form that is friable, the
subsequent energy-addition step can be carried out more quickly and
efficiently when compared to an organic compound in a less friable
crystalline morphology.
[0083] In the fourth process category, the first solution and
second solvent are simultaneously subjected to the energy-addition
step. Thus, the physical properties of the organic compound before
and after the energy addition step were not measured.
[0084] The energy-addition step can be carried out in any fashion
wherein the presuspension or the first solution and second solvent
are exposed to cavitation, shearing or impact forces. In one form,
the energy-addition step is an annealing step. Annealing is defined
in this invention as the process of converting matter that is
thermodynamically unstable into a more stable form by single or
repeated application of energy (direct heat or mechanical stress),
followed by thermal relaxation. This lowering of energy may be
achieved by conversion of the solid form from a less ordered to a
more ordered lattice structure. Alternatively, this stabilization
may occur by a reordering of the surfactant molecules at the
solid-liquid interface.
[0085] These four process categories are shown separately below. It
should be understood, however, that the process conditions such as
choice of surfactants or combination of surfactants, amount of
surfactant used, temperature of reaction, rate of mixing of
solutions, rate of precipitation and the like can be selected to
allow for any drug to be processed under any one of the categories
discussed next.
[0086] The first process category, as well as the second, third,
and fourth process categories, can be further divided into two
subcategories, Method A and B.
[0087] The first solvent according to the following processes is a
solvent or mixture of solvents in which the organic compound of
interest is relatively soluble and which is miscible with the
second solvent. Such solvents include, but are not limited to
water-miscible protic compounds, in which a hydrogen atom in the
molecule is bound to an electronegative atom such as oxygen,
nitrogen, or other Group VA, VIA and VII A in the Periodic Table of
elements. Examples of such solvents include, but are not limited
to, alcohols, amines (primary or secondary), oximes, hydroxamic
acids, carboxylic acids, sulfonic acids, phosphonic acids,
phosphoric acids, amides and ureas.
[0088] Other examples of the first solvent also include aprotic
organic solvents. Some of these aprotic solvents can form hydrogen
bonds with water, but can only act as proton acceptors because they
lack effective proton donating groups. One class of aprotic
solvents is a dipolar aprotic solvent, as defined by the
International Union of Pure and Applied Chemistry (IUPAC Compendium
of Chemical Terminology, 2nd Ed., 1997): a solvent with a
comparatively high relative permittivity (or dielectric constant),
greater than ca. 15, and a sizable permanent dipole moment, that
cannot donate suitably labile hydrogen atoms to form strong
hydrogen bonds, e.g. dimethyl sulfoxide.
[0089] Dipolar aprotic solvents can be selected from the group
consisting of: amides (fully substituted, with nitrogen lacking
attached hydrogen atoms), ureas (fully substituted, with no
hydrogen atoms attached to nitrogen), ethers, cyclic ethers,
nitriles, ketones, sulfones, sulfoxides, fully substituted
phosphates, phosphonate esters, phosphoramides, nitro compounds,
and the like. Dimethylsulfoxide (DMSO), N-methyl-2-pyrrolidinone
(NMP), 2-pyrrolidinone, 1,3-dimethylimidazolidinone (DMI),
dimethylacetamide (DMA), dimethylformamide (DMF), dioxane, acetone,
tetrahydrofuran (THF), tetramethylenesulfone (sulfolane),
acetonitrile, and hexamethylphosphoramide (HMPA), nitromethane,
among others, are members of this class.
[0090] Solvents may also be chosen that are generally
water-immiscible, but have sufficient water solubility at low
volumes (less than 10%) to act as a water-miscible first solvent at
these reduced volumes. Examples include aromatic hydrocarbons,
alkenes, alkanes, and halogenated aromatics, halogenated alkenes
and halogenated alkanes. Aromatics include, but are not limited to,
benzene (substituted or unsubstituted), and monocyclic or
polycyclic arenes. Examples of substituted benzenes include, but
are not limited to, xylenes (ortho, meta, or para), and toluene.
Examples of alkanes include but are not limited to hexane,
neopentane, heptane, isooctane, and cyclohexane. Examples of
halogenated aromatics include, but are not restricted to,
chlorobenzene, bromobenzene, and chlorotoluene. Examples of
halogenated alkanes and alkenes include, but are not restricted to,
trichloroethane, methylene chloride, ethylenedichloride (EDC), and
the like.
[0091] Other specific examples of solvents suitable for use as the
first solvent include, but are not limited to:
N-methyl-2-pyrrolidinone (also called N-methyl-2-pyrrolidone),
2-pyrrolidinone (also called 2-pyrrolidone),
1,3-dimethyl-2-imidazolidinone (DMI), dimethylsulfoxide,
dimethylacetamide, acetic acid, lactic acid, methanol, ethanol,
isopropanol, 3-pentanol, n-propanol, benzyl alcohol, glycerol,
butylene glycol (butanediol), ethylene glycol, propylene glycol,
monoacylated and diacylated monoglycerides (such as glyceryl
caprylate), dimethyl isosorbide, acetone, dimethylsulfone,
dimethylformamide, 1,4-dioxane, tetramethylenesulfone (sulfolane),
acetonitrile, nitromethane, tetramethylurea,
hexamethylphosphoramide (HMPA), tetrahydrofuran (THF), dioxane,
diethylether, tert-butylmethyl ether (TBME), aromatic hydrocarbons,
alkenes, alkanes, halogenated aromatics, halogenated alkenes,
halogenated alkanes, xylene, toluene, benzene, substituted benzene,
ethyl acetate, methyl acetate, butyl acetate, chlorobenzene,
bromobenzene, chlorotoluene, trichloroethane, methylene chloride,
ethylenedichloride (EDC), hexane, neopentane, heptane, isooctane,
cyclohexane, polyethylene glycol (PEG, for example, PEG-4, PEG-8,
PEG-9, PEG-12, PEG-14, PEG-16, PEG-120, PEG-75, PEG-150),
polyethylene glycol esters (examples such as PEG-4 dilaurate,
PEG-20 dilaurate, PEG-6 isostearate, PEG-8 palmitostearate, PEG-150
palmitostearate), polyethylene glycol sorbitans (such as PEG-20
sorbitan isostearate), polyethylene glycol monoalkyl ethers
(examples such as PEG-3 dimethyl ether, PEG-4 dimethyl ether),
polypropylene glycol (PPG), polypropylene alginate, PPG-10
butanediol, PPG-10 methyl glucose ether, PPG-20 methyl glucose
ether, PPG-15 stearyl ether, propylene glycol
dicaprylate/dicaprate, propylene glycol laurate, and glycofurol
(tetrahydrofurfuryl alcohol polyethylene glycol ether). A preferred
first solvent is N-methyl-2-pyrrolidinone. Another preferred first
solvent is lactic acid.
[0092] The second solvent is an aqueous solvent. This aqueous
solvent may be water by itself. This solvent may also contain
buffers, salts, surfactant(s), water-soluble polymers, and
combinations of these excipients.
[0093] Method A. In Method A, the organic compound ("active agent"
or "drug") is first dissolved in the first solvent to create a
first solution. The organic compound can be added from about 0.1%
(w/v) to about 50% (w/v) depending on the solubility of the organic
compound in the first solvent. Heating of the concentrate from
about 30.degree. C. to about 100.degree. C. may be necessary to
ensure total dissolution of the compound in the first solvent.
[0094] A second aqueous solvent is provided with one or more
optional surface modifiers such as an anionic surfactant, a
cationic surfactant, a zwitterionic surfactant, a nonionic
surfactant or a biologically surface active molecule added thereto.
Suitable anionic surfactants include but are not limited to alkyl
sulfonates, alkyl phosphates, alkyl phosphonates, potassium
laurate, triethanolamine stearate, sodium lauryl sulfate, sodium
dodecylsulfate, alkyl polyoxyethylene sulfates, sodium alginate,
dioctyl sodium sulfosuccinate, phosphatidyl glycerol, phosphatidyl
inosine, phosphatidylinositol, diphosphatidylglycerol,
phosphatidylserine, phosphatidic acid and their salts, sodium
carboxymethylcellulose, cholic acid and other bile acids (e.g.,
cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid,
glycodeoxycholic acid) and salts thereof (e.g., sodium
deoxycholate, etc.).
[0095] Zwitterionic surfactants are electrically neutral but
possess local positive and negative charges within the same
molecule. Suitable zwitterionic surfactants include but are not
limited to zwitterionic phospholipids. Suitable phospholipids
include phosphatidylcholine, phosphatidylethanolamine,
diacyl-glycero-phosphoethanolamine (such as
dimyristoyl-glycero-phosphoethanolamine (DMPE),
dipalmitoyl-glycero-phosphoethanolamine (DPPE),
distcaroyl-glycero-phosphoethanolamine (DSPE), and
dioleolyl-glycero-phosphoethanolamine (DOPE)). Mixtures of
phospholipids that include anionic and zwitterionic phospholipids
may be employed in this invention. Such mixtures include but are
not limited to lysophospholipids, egg or soybean phospholipid or
any combination thereof. The phospholipid, whether anionic,
zwitterionic or a mixture of phospholipids, may be salted or
desalted, hydrogenated or partially hydrogenated, or natural,
semisynthetic, or synthetic. The phospholipid may also be
conjugated with a water-soluble or hydrophilic polymer to
specifically target the delivery to macrophages in the present
invention. However, conjugated phospholipids may be used to target
other cells or tissue in other applications. A preferred polymer is
polyethylene glycol (PEG), which is also known as the monomethoxy
polyethyleneglycol (mPEG). The molecular weights of the PEG can
vary, for example, from 200 to 50,000. Some commonly used PEG's
that are commercially available include PEG 350, PEG 550, PEG 750,
PEG 1000, PEG 2000, PEG 3000, and PEG 5000. The phospholipid or the
PEG-phospholipid conjugate may also incorporate a functional group
which can covalently attach to a ligand including but not limited
to proteins, peptides, carbohydrates, glycoproteins, antibodies, or
pharmaceutically active agents. These functional groups may
conjugate with the ligands through, for example, amide bond
formation, disulfide or thioether formation, or biotin/streptavidin
binding. Examples of the ligand-binding functional groups include
but are not limited to hexanoylamine, dodecanylamine,
1,12-dodecanedicarboxylate, thioethanol,
4-(p-maleimidophenyl)butyramide (MPB),
4-(p-maleimidomethyl)cyclohexane-carboxamide (MCC),
3-(2-pyridyldithio)propionate (PDP), succinate, glutarate,
dodecanoate, and biotin.
[0096] Suitable cationic surfactants include but are not limited to
quaternary ammonium compounds, such as benzalkonium chloride,
cetyltrimethylammonium bromide, chitosans,
lauryldimethylbenzylammonium chloride, acyl carnitine
hydrochlorides, dimethyldioctadecylammomium bromide (DDAB),
dioleoyltrimethylammonium propane (DOTAP, also known as
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium),
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA),
dimyristoyltrimethylammonium propane (DMTAP),
dimethylaminoethanecarbamoyl cholesterol (DC-Chol),
1,2-diacylglycero-3-(O-alkyl)phosphocholine,
O-alkylphosphatidylcholine, alkyl pyridinium halides, or long-chain
alkyl amines such as, for example, n-octylamine and oleylamine.
Surfactants of formula I, as defined herein, also are suitable
cationic surfactants.
[0097] Suitable nonionic surfactants include: glyceryl esters,
polyoxyethylene fatty alcohol ethers (MACROGOL.TM. and BRIJ.TM.),
polyoxyethylene sorbitan fatty acid esters (polysorbates),
polyoxyethylene fatty acid esters (MYRJ.TM.), sorbitan esters
(SPAN.TM.), glycerol monostearate, polyethylene glycols,
polypropylene glycols, cetyl alcohol, cetostearyl alcohol, stearyl
alcohol, aryl alkyl polyether alcohols,
polyoxyethylene-polyoxypropylene copolymers (poloxamers),
poloxamines, methylcellulose, hydroxymethylcellulose,
hydroxypropylcellulose, hydroxypropylmethylcellulose,
noncrystalline cellulose, polysaccharides including starch and
starch derivatives such as hydroxyethylstarch (HES), polyvinyl
alcohol, and polyvinylpyrrolidone. In a preferred form, the
nonionic surfactant is a polyoxyethylene and polyoxypropylene
copolymer and preferably a block copolymer of propylene glycol and
ethylene glycol. Such polymers are sold under the trade name
POLOXAMER also sometimes referred to as PLURONIC.RTM., and sold by
several suppliers including Spectrum Chemical and Ruger. Among
polyoxyethylene fatty acid esters is included those having short
alkyl chains. One example of such a surfactant is SOLUTOL.RTM. HS
15, polyethylene-660-hydroxystearate, manufactured by BASF
Aktiengesellschaft.
[0098] Surface-active biological molecules include such molecules
as albumin, casein, hirudin or other appropriate proteins. For
example, proteins having hydrophilic and hydrophobic domains also
can be used. Polysaccharide surface active biologics are also
included, and consist of but are not limited to, starches,
heparins, and chitosans. Other suitable surfactants include any
amino acids such as leucine, alanine, valine, isoleucine, lysine,
aspartic acid, glutamic acid, methionine, phenylalanine, or any
derivatives of these amino acids such as, for example, amide or
ester derivatives and polypeptides formed from these amino
acids.
[0099] It may also be desirable to add a pH adjusting agent to the
second solvent. Suitable pH adjusting agents include, but are not
limited to, hydrochloric acid, sulfuric acid, phosphoric acid,
monocarboxylic acids (such as, for example, acetic acid and lactic
acid), dicarboxylic acids (such as, for example, succinic acid),
tricarboxylic acids (such as, for example, citric acid), THAM
(tris(hydroxymethyl)aminomethane), meglumine (N-methylglucosamine),
sodium hydroxide, and amino acids such as glycine, arginine,
lysine, alanine, histidine and leucine. The second solvent should
have a pH within the range of from about 3 to about 11. The aqueous
medium may additionally include an osmotic pressure adjusting
agent, such as but not limited to glycerin, a monosaccharide such
as dextrose, a disaccharide such as sucrose, a trisaccharide such
as raffinose, and sugar alcohols such as mannitol, xylitol and
sorbitol.
[0100] For oral dosage forms, one or more of the following
excipients may be utilized: gelatin, casein, lecithin
(phosphatides), gum acacia, cholesterol, tragacanth, stearic acid,
benzalkonium chloride, calcium stearate, glyceryl monostearate,
cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters,
polyoxyethylene alkyl ethers, e.g., macrogol ethers such as
cetomacrogol 1000, polyoxyethylene castor oil derivatives,
polyoxyethylene sorbitan fatty acid esters, e.g., the commercially
available TWEENS.TM., polyethylene glycols, polyoxyethylene
stearates, colloidal silicon dioxide, phosphates, sodium
dodecylsulfate, carboxymethylcellulose calcium,
carboxymethylcellulose sodium, methylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose phthalate, noncrystalline cellulose,
magnesium aluminum silicate, triethanolamine, polyvinyl alcohol
(PVA), and polyvinylpyrrolidone (PVP). Most of these excipients are
described in detail in the Handbook of Pharmaceutical Excipients,
published jointly by the American Pharmaceutical Association and
The Pharmaceutical Society of Great Britain, the Pharmaceutical
Press, 1986. The surface modifiers are commercially available
and/or can be prepared by techniques known in the art. Two or more
surface modifiers can be used in combination.
[0101] In a preferred form, the method for preparing small
particles of an organic compound includes the steps of adding the
first solution to the second solvent. The addition rate is
dependent on the batch size, and precipitation kinetics for the
organic compound. Typically, for a small-scale laboratory process
(preparation of 1 liter), the addition rate is from about 0.05 cc
per minute to about 10 cc per minute. During the addition, the
solutions should be under constant agitation. It has been observed
using light microscopy that amorphous particles, semi-crystalline
solids, or a supercooled liquid are formed to create a
pre-suspension. The method further includes the step of subjecting
the pre-suspension to an energy-addition step to convert the
amorphous particles, supercooled liquid or semicrystalline solid to
a more stable, crystalline solid state. The resulting particles
will have an average effective particles size as measured by
dynamic light scattering methods (e.g., photocorrelation
spectroscopy, laser diffraction, low-angle laser light scattering
(LALLS), medium-angle laser light scattering (MALLS)), light
obscuration methods (Coulter method, for example), rheology, or
microscopy (light or electron) within the ranges set forth above.
In process category four, the first solution and the second solvent
are combined while simultaneously conducting the energy-addition
step.
[0102] The energy-addition step involves adding energy through
sonication, homogenization, countercurrent flow homogenization,
microfluidization, or other methods of providing impact, shear or
cavitation forces. The sample may be cooled or heated during this
stage. In one form, the energy-addition step is effected by a
piston gap homogenizer such as the one sold by Avestin Inc. under
the product designation EmulsiFlex-C160. In another form, the
energy-addition step is accomplished by ultrasonication using an
ultrasonic processor such as the Vibra-Cell Ultrasonic Processor
(600 W), manufactured by Sonics and Materials, Inc. In yet another
form, the energy-addition step is accomplished by use of an
emulsification apparatus as described in U.S. Pat. No. 5,720,551,
which is incorporated herein by reference and made a part
hereof.
[0103] Depending upon the rate of energy addition, it may be
desirable to adjust the temperature of the processed sample to
within the range of from approximately -30.degree. C. to 30.degree.
C. Alternatively, in order to effect a desired phase change in the
processed solid, it may also be necessary to heat the
pre-suspension to a temperature within the range of from about
30.degree. C. to about 100.degree. C. during the energy-addition
step.
[0104] Method B. Method B differs from Method A in the following
respects. The first difference is a surfactant or combination of
surfactants is added to the first solution. The surfactants may be
selected from the groups of anionic, nonionic, cationic
surfactants, and surface-active biological modifiers set forth
above.
[0105] Comparative Example of Method A and Method B and U.S. Pat.
No. 5,780,062. U.S. Pat. No. 5,780,062 discloses a process for
preparing small particles of an organic compound by first
dissolving the compound in a suitable water-miscible first solvent.
A second solution is prepared by dissolving a polymer and an
amphiphile in aqueous solvent. The first solution is then added to
the second solution to form a precipitate that consists of the
organic compound and a polymer-amphiphile complex. The '062 patent
does not disclose utilizing the energy-addition step of this
process in Methods A and B. Lack of stability is typically
evidenced by rapid aggregation and particle growth. In some
instances, amorphous particles recrystallize as large crystals.
Adding energy to the pre-suspension in the manner disclosed above
typically affords particles that show decreased rates of particle
aggregation and growth, as well as the absence of recrystallization
upon product storage.
[0106] Methods A and B are further distinguished from the process
of the '062 patent by the absence of a step of forming a
polymer-amphiphile complex prior to precipitation. In Method A,
such a complex cannot be formed as no polymer is added to the
diluent (aqueous) phase. In Method B, the surfactant, which may
also act as an amphiphile, or polymer, is dissolved with the
organic compound in the first solvent. This precludes the formation
of any amphiphile-polymer complexes prior to precipitation. In the
'062 patent, successful precipitation of small particles relies
upon the formation of an amphiphile-polymer complex prior to
precipitation. The '062 patent discloses the amphiphile-polymer
complex forms aggregates in the aqueous second solution. The '062
patent explains the hydrophobic organic compound interacts with the
amphiphile-polymer complex, thereby reducing solubility of these
aggregates and causing precipitation. In the present process, it
has been demonstrated that the inclusion of the surfactant or
polymer in the first solvent (Method B) leads, upon subsequent
addition to second solvent, to formation of a more uniform, finer
particulate than is afforded by the process outlined by the '062
patent.
Coating of the Particles
[0107] The processes for coating the particles prepared by the
present invention can be accomplished through various techniques
known to those skilled in the art. The coating can be associated
with the particle through various associations, including covalent
and/or non-covalent associations (e.g., covalent bonding, ionic
interactions, electrostatic interactions, dipole-dipole
interactions, hydrogen bonding, van der Waal's forces,
hydrophobic/hydrophobic domain interactions, cross-linking, and/or
any other interactions).
[0108] Non-covalently bound coatings can be prepared, for example,
by the methods for preparing particle cores disclosed herein
provided that a surfactant according to formula I is used to
manufacture the particles, or by mixing a plurality of
pre-fabricated particles with a solution comprising a surfactant of
formula I, as defined herein, to form surface-modified particles
according to the invention. The solution can be mixed under
high-shear conditions using, for example, a microfluidizer, a
piston gap homogenizer, a counter-current flow homogenizer, or an
ultrasonic processor. To confirm the coating successfully adsorbs
to the particles, the surface electrical potential of the particles
can be determined by measuring the zeta potential before and after
the coating process. Other known methods for measuring the
adsorption of coatings also can be used, for example, the
surfactant of formula I can be modified with a fluorescent label
and absorption of the fluorescently-labeled surfactant of formula I
can be detected by fluorescence microscopy. Advantageously, the
coatings comprising a surfactant of formula I can associate with
the particle core, for example by adsorbing to the surface of
particles, which is an efficient method for associating coatings
comprising surfactants according to formula I to particle cores,
particularly particles comprising poorly water soluble active
agents, as explained above.
[0109] The coating can further include one or more additional
surfactants, including additional surfactants of formula I, by
adding the additional surfactants to the solution comprising the
surfactant of formula I, as defined herein, and then mixing the
pre-fabricated particles with said solution. Such additional
surfactant(s) can be selected from a variety of known anionic
surfactants, cationic surfactants, zwitterionic surfactants,
nonionic surfactants and surface active biological modifiers.
Suitable additional surfactants include the surfactants previously
set forth herein. Exemplary additional surfactants include, but are
not limited to, poloxamers, phospholipids, polyethylene
glycol-conjugated phospholipids, and polysorbates. Exemplary
combinations of additional surfactants include, but are not limited
to, poloxamers and phospholipids, poloxamers and polyethylene
glycol-conjugated phospholipids, and poloxamers and
polysorbates.
Cellular Uptake of Coated Particles
[0110] One embodiment of the present invention is directed to a
method of enhancing cellular uptake of an active agent, comprising
contacting cells with a plurality of surface-modified particles,
said particles comprising a particle core and a coating associated
with the particle core. The cells can be phagocytic cells, weakly
phagocytic cells, or non-phagocytic cells. The particle core
comprises an active agent which is typically selected from the
group consisting of small molecules, peptides, and proteins, the
coating comprises a surfactant of formula I, as defined herein, and
the surface-modified particle has an average size from about 1 nm
to about 2,000 nm. Uptake of the active agent by the cells is
thereby enhanced, at least relative to the uptake of active agent
when particles that do not comprise the aforementioned coating are
used.
[0111] Uptake by cells allows the active agent to be delivered to
target tissues in need of treatment because the various cell types
capable of enhanced uptake of the coated particles in accordance
with the disclosure also traffic to diseased (e.g., cancerous,
infected) or inflamed tissues. For example, neutrophils predominate
early in infection or inflammation, followed by monocyte-derived
phagocytes that leave the blood vasculature and enter infected
tissues, and such cells demonstrate enhanced uptake of the
surface-modified particles according to the invention at least
relative to particles not having a coating comprising a surfactant
according to formula I. Fixed macrophages (histiocytes) abound in
the liver, nervous system, lungs, lymph nodes, bone marrow, and
several other tissues, and such cells also demonstrate enhanced
uptake of the surface-modified particles according to the invention
at least relative to particles not having a coating comprising a
surfactant according to formula I. Tissues that are most affected
by bacterial, viral or fungal pathogens and which are inflamed can
be targeted by delivery of drug-loaded cells (granulocytes, for
example) having a propensity to be directed to these inflammation
sites by chemotaxis. Thus, by promoting uptake by the
aforementioned cells, the pharmaceutical agent is released from
these cells in a region where it is therapeutically most needed.
Thus, delivery of the agent to a target tissue for treatment of a
disease or disorder is facilitated by cells loaded with coated
particles according to the invention. Such diseases and disorders
include, but are not limited to, infectious diseases or disorders,
inflammatory diseases or disorders, neurodegenerative diseases or
disorders, and proliferative diseases or disorders.
[0112] There are numerous phagocytic cell types that are capable of
enhanced uptake of coated particles. These cells include, but are
not limited to, macrophages, monocytes, granulocytes, agranulocytes
and neutrophils. The present invention also encompasses weakly
phagocytic cells and non-phagocytic cells. Thus, other suitable
cell types include, but are not limited to, T-lymphocytes,
B-lymphocytes, null cells, natural killer cells, lymphocytes, red
blood cells, muscle cells, bone marrow cells, stem cells, bone
cells, vascular cells, organ tissue cells, neuronal cells,
basophils, eosinophils, dendritic cells, and endothelial cells.
Still other cells can be used to deliver the pharmaceutically
active compounds to a subject. Any cell type may be used in the
present invention so long as it is capable of uptake of the
particle. Uptake by the cells of the particles may include
phagocytosis, or other means of endocytosis, or
attachment/adsorption of the particle onto the surface of the
cells. Particles associated with the cell surface can also be taken
into the cells by pinocytosis, which is an invagination of the cell
membrane to form an intracellular capsule around the particle. In
pinocytosis ("cell drinking"), the engulfed particle is relatively
small (e.g., 20 nm) (Watts et al., Endocytosis: what goes in and
how?, Journal of Cell Science, 1992, volume 103(1), pages 1-8).
Pinocytosis occurs continuously in almost all eucaryotic cells.
Diseased cells, for example, cancerous cells, can also demonstrate
enhanced uptake of the surface modified particles according to the
invention at least as compared to cells contacted with particles
not having a coating comprising a surfactant of formula I.
[0113] As explained herein, the particles advantageously include a
coating which facilitates cellular uptake. In particular, the
coating can facilitate uptake by cells such as monocytes,
macrophages, and T-lymphocytes, which are capable of trafficking by
known mechanisms such as chemotaxis to a site of inflammation,
infection, and/or tumor and thereby deliver the particles to a
particular target tissue.
[0114] In one aspect of the invention, the contacting of the cells
to the surface-modified particle (to form cells loaded with the
active agent) is carried out ex vivo (i.e., outside of a mammalian
subject). Alternatively, or in addition, the contacting of the
cells to the surface-modified particle can be carried out in vivo
(i.e., inside a mammalian subject). An amount of the surface
modified particle that is effective to treat a disease or disorder
is used during the contacting step. One of ordinary skill
understands that a certain amount of the particles may be taken up
by a cell type that does not traffic to a target tissue of
interest, or is not released by the cell at the target tissue of
interest. Therefore, one of ordinary skill understands that the
amounts of particles administered may be optimized by routine
protocols, provided that such amounts are within established
administration protocols.
[0115] For ex-vivo administration, the cells can be isolated from a
mammalian subject using a cell separator or apheresis device. For
instance, the CS-3000.TM. cell separator (Fenwal Inc., Lake Zurich,
Ill.) or the ISOLEX.TM. cell separator (Baxter Healthcare Corp.,
Deerfield, Ill.) can be used to isolate various cells. Other
methods known to those skilled in the art of ex-vivo cell isolation
can be employed to obtain cells useful in the present invention.
Such methods include, but are not limited to, apheresis of
peripheral blood, mobilization of bone marrow cells through, e.g.,
G-CSF, M-CSF, or GM-CSF, or direct removal of marrow cells by
spinal, sternal, lumbar, or iliac crest puncture. The ex-vivo cells
can be maintained in a cell culture medium or other isolating
system known to those skilled in the art. Examples of such media
are Alserver's Solution, Ames' Medium, Eagle's Basal Medium, CHO
(Chinese Hamster Ovary) cell culture media, Click's Medium,
Dulbecco's Modified Eagle's Medium, phosphate-buffered saline,
phosphate-buffered dextrose or sucrose, Earle's Balanced Salt
Solution, Gene Therapy Medium-3, Gey's Balanced Salt Solution,
Glasgow Minimum Essential Medium, Hanks' Balanced Salt Solutions,
Hybridoma Media, Iscove's Modified Dulbecco's Medium,
Krebs-Henseleit Buffer with sugars, Leibovitz Media (L-15), M16
Medium, McCoy's Medium, MCDB, MDBK (Madin-Darby Bovine Kidney),
MDCK (Madin-Darby Canine Kidney), Medium 199, NCTC, Ham's Media
(e.g., Nutrient Mixture F 10), Coon's Modified Ham's Medium, RPMI,
and others such as those listed in Biochemicals & Reagents for
Life Science Research, Sigma-Aldrich Co. (St. Louis, Mo., USA). The
purpose of the culture so described may be for the purpose of
simple storage without loss of cells, or for cell proliferation or
expansion, by appropriate addition of growth factors, cytokines,
and nutrients, to encourage cell expansion. Such expansion would
minimize the number of times that a patient would have to be
prepared for removal of cellular samples.
[0116] Once isolated, the cells can be contacted with the coated
particles and incubated for a short period of time to allow for
cell uptake of the particles. The concentrations of particles used
in the ex-vivo procedure will vary due to several factors,
including, but not limited to, type of cells used, concentration of
cells, active agent employed, size of the small particle
dispersions, disease to be treated, and so on. Generally, however,
the cellular isolates are contacted with about 1 to about 300 mg/ml
of particles of the present invention. During contact of the
particles with the cells, the particles are at a concentration
higher than the thermodynamic saturation solubility, thereby
allowing the particles to remain in particulate form during uptake
and delivery to the mammalian subject. The cells can be incubated
with the particles for up to 24 hours or longer to permit
sufficient cell uptake of the drug particles.
[0117] Any method to effect uptake of particles of active agent by
ex vivo cells can be used with the requirement that the method does
not destroy or otherwise make the cells non-useful for
administration to a subject. For example, site-specific delivery of
the particle via a biorecognition molecule may be used. See, e.g.,
U.S. Patent Publication No. 2003/0092069, incorporated herein by
reference, which discloses the transferring of genes into specific
cells or tissues via a hollow nanoparticle. Other methods of
loading the ex-vivo cells include electroporation, sonoporation,
and other mechanical means that disrupt the cell membrane
(sonication, for example) and enable insertion of solid
particulates into the cells. Ultrasound was successfully used by
Zarnitsyn et al. (Zarnitsyn et al., Physical parameters influencing
optimization of ultrasound-mediated DNA transfection, Ultrasound
Med. Biol., 2004, volume 30(4), pages 527-538) to transiently
disrupt cell membranes and thereby facilitate the loading of DNA
into viable cells. Other mechanical procedures are well-known to
those experienced in the art, and are included as part of this
disclosure. Chemical methods of transiently destabilizing cell
membranes are also well known. Transfection reagents contain
surface active agents and include 293FECTIN.TM. Transfection
Reagent and LIPOFECTAMINE.TM., both products of Invitrogen
Corporation (Carlsbad, Calif.). Another example of a surfactant
used to transfer DNA into cells is the SAINT.TM. reagent from
Synvolux Therapeutics B. V. L. J. (Groningen, The Netherlands),
which is based on a pyridinium surfactant.
[0118] The following description of particles also applies to all
embodiments disclosed herein. For marginally soluble drugs, the
cell loading procedure can be utilized provided that the cells are
able to take up the coated active agent particles at a faster rate
than the competing dissolution process. The particles should be of
an appropriate size to allow for the cells to take up the coated
particles and deliver them to the target tissue before complete
dissolution of the particle. Because cells which are known to
traffic to the target tissue of interest are capable of taking up
the particles, the active agent is ultimately released from the
cells or otherwise delivered in the vicinity of the target tissue.
Furthermore, the concentration of the active agent composition
should be kept higher than the saturation solubility of the
composition so that the particle is able to remain in the
crystalline state during uptake.
[0119] The following description of particles also applies to all
embodiments disclosed herein. Administering of the surface-modified
particle can be performed by various techniques known in the art
for administering particles. Administering includes administering
the surface-modified particle to a mammalian subject. Suitable
methods for administering of the surface-modified particle include,
but are not limited to, administering the particle intravenously,
intraarterially, intramuscularly, subcutaneously, intradermally,
intraarticularly, intrathecally, epidurally, intracerebrally,
buccally, rectally, topically, transdermally, orally, intranasally,
via the pulmonary route, intraperitoneally, and/or
intraophthalmically. The step of administering can be by bolus
injection, by intermittent infusion, or by continuous infusion. The
amount of surface-modified particle and method of delivery can be
determined by skilled clinicians. Various factors will affect the
amount and method of delivery including, but not limited to, the
type of cells used (for ex vivo methods of administration), the
sex, weight and age of the subject to be treated, the type and
maturity of the disease or disorder to be treated, the active agent
to be administered, and so on. Generally, the active agent can be
provided in doses ranging from 1 pg compound/kg body weight to 1000
mg/kg, 0.1 mg/kg to 100 mg/kg, 0.1 mg/kg to 50 mg/kg, and 1 to 20
mg/kg, given in daily doses or in equivalent doses at longer or
shorter intervals, e.g., every other day, twice weekly, weekly, or
twice or three times daily.
[0120] Various diseases or disorders can be treated by the present
methods including, but not limited to, infectious diseases or
disorders, inflammatory diseases or disorders, neurodegenerative
diseases or disorders, and proliferative diseases or disorders. In
this regard, symptoms of such diseases or disorders can be
alleviated by the present methods.
[0121] "Infectious diseases or disorder" as used herein refers to a
condition caused by pathogenic microorganisms, such as bacteria,
viruses, parasites or fungi. Infectious diseases or disorders that
can benefit from the disclosed methods include, but are not limited
to, viral infections (including retroviral infections) such as
dengue, enterovirus infections, HIV, hepatitis B, hepatitis C, and
influenza; fungal infections; parasitic infections such as African
trypanosomiasis and malaria; and bacterial infections such as
cholera, meningitis, and tuberculosis.
[0122] "Inflammatory disease or disorder" as used herein refers to
a condition characterized by redness, heat, swelling, and pain
(i.e., inflammation) that typically involves tissue injury or
destruction. Inflammatory diseases or disorders are notably
associated with the influx of leukocytes and/or leukocyte
chemotaxis. Inflammatory conditions may result from infection with
pathogenic organisms or viruses and from noninfectious events
including but not limited to trauma or reperfusion following
myocardial infarction or stroke, immune responses to foreign
antigens, and autoimmune responses. Accordingly, inflammatory
conditions amenable to treatment with the methods and compounds of
the invention encompass conditions associated with reactions of the
specific defense system, conditions associated with reactions of
the non-specific defense system, and conditions associated with
inflammatory cell activation.
[0123] As used herein, the term "specific defense system" refers to
the component of the immune system that reacts to the presence of
specific antigens. Examples of inflammatory conditions resulting
from a response of the specific defense system include but are not
limited to the classical response to foreign antigens, autoimmune
diseases, and delayed type hypersensitivity response mediated by
B-cells and/or T-cells (i.e., B-lymphocytes and/or T-lymphocytes).
Chronic inflammatory diseases, the rejection of solid transplanted
tissue and organs including but not limited to kidney and bone
marrow transplants, and graft versus host disease (GVHD), are
further examples of inflammatory conditions resulting from a
response of the specific defense system.
[0124] The term "non-specific defense system" as used herein refers
to inflammatory conditions that are mediated by leukocytes that are
incapable of immunological memory (e.g., granulocytes including but
not limited to neutrophils, eosinophils, and basophils, mast cells,
monocytes, macrophages). Examples of inflammatory conditions that
result, at least in part, from a reaction of the non-specific
defense system include but are not limited to adult (acute)
respiratory distress syndrome (ARDS), multiple organ injury
syndromes, reperfusion injury, acute glomerulonephritis, reactive
arthritis, dermatitis with acute inflammatory components, acute
purulent meningitis, other central nervous system inflammatory
conditions including but not limited to stroke, thermal injury,
inflammatory bowel disease, granulocyte transfusion associated
syndromes, and cytokine-induced toxicity.
[0125] The therapeutic methods of the invention include methods for
the amelioration of conditions associated with inflammatory cell
activation. "Inflammatory cell activation" refers to the induction
by a stimulus (including but not limited to cytokines, antigens,
and auto-antibodies) of a proliferative cellular response, the
production of soluble mediators (including but not limited to
cytokines, oxygen radicals, enzymes, prostanoids, and vasoactive
amines), or cell surface expression of new or increased numbers of
mediators (including but not limited to major histocompatability
antigens and cell adhesion molecules) in inflammatory cells
(including but not limited to monocytes, macrophages, T
lymphocytes, B lymphocytes, granulocytes (polymorphonuclear
leukocytes including neutrophils, basophils, and eosinophils), mast
cells, dendritic cells, Langerhans cells, and endothelial cells).
It will be appreciated by persons skilled in the art that the
activation of one or a combination of these phenotypes in these
cells can contribute to the initiation, perpetuation, or
exacerbation of an inflammatory condition.
[0126] Other diseases or disorders which can be successfully
treated include diseases or disorders characterized by inflammation
or infection, including but not limited to, rheumatoid arthritis,
Graves' disease, myasthenia gravis, thyroiditis, diabetes,
inflammatory bowel disease, autoimmune oophoritis, systemic lupus
erythematosus, and Sjogren's syndrome.
[0127] Examples of neurodegenerative diseases or disorders which
can be successfully treated include, but are not limited to,
Parkinson's disease, Alzheimer's disease, multiple sclerosis,
encephalomyelitis, encephalitis (including HIV encephalitis),
Huntington's disease, amyotrophic lateral sclerosis (also known as
Lou Gehrig's disease), frontotemporal dementia, prion diseases,
Creutzfeldt-Jakob disease, and adrenoleukodystrophy. Other
neurodegenerative diseases or disorders which can be successfully
treated include Pick's disease, frontotemporal lobar degeneration,
progressive aphasia, and semantic dementia. Prion diseases, also
known as transmissible spongiform encephalopathies (TSEs), include
Creutzfeldt-Jakob disease, new variant Creutzfeldt-Jakob disease,
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,
and kuru. The neurodegenerative diseases or disorders also can be
Alexander disease, Alper's disease, ataxia telangiectasia, Batten
disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease),
Canavan disease, Cockayne syndrome, corticobasal degeneration,
HIV-associated dementia, Kennedy's disease, Krabbe disease, Lewy
body dementia, Machado-Joseph disease (spinocerebellar ataxia type
3), multiple system atrophy, neuroborreliosis, Pelizaeus-Merzbacher
disease, primary lateral sclerosis, Refsum's disease, Sandhoff
disease, Schilder's disease, schizophrenia, spinocerebellar ataxia,
spinal muscular atrophy, Steele-Richardson-Olszewski disease, and
tabes dorsalis.
[0128] 1001281 Proliferative diseases or disorders that can benefit
from the disclosed methods include, but are not limited to, colon
cancer, kidney cancer, non small cell lung cancer, small cell lung
cancer, head and neck cancer, cancers of the peritoneal cavity
(such as ovarian cancer), cervical cancer, breast cancer, prostate
cancer, brain cancer (including glioma), sarcoma, melanoma,
leukemia, acute lymphocytic leukemia, acute myelogenous leukemia,
chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin
lymphoma, non-Hodgkin lymphoma, myeloma, and glioblastoma.
Thyroiditis includes Hashimoto's thyroiditis, subacute thyroiditis
(also known as de Quervain's thyroiditis), silent thyroiditis (also
known as painless thyroiditis), post partum thyroiditis,
drug-induced thyroiditis, radiation-induced thyroiditis, and acute
suppurative thyroiditis.
[0129] The disclosure may be better understood by reference to the
following examples which are not intended to be limiting, but
rather only set forth exemplary embodiments in accordance with the
disclosure.
EXAMPLES
Example 1
Preparation of Paclitaxel Particles Having a DOTAP Coating
[0130] Paclitaxel particles were prepared using a
microprecipitation/homogenization procedure. Specifically,
paclitaxel (0.5 g) was dissolved in N-methyl pyrrolidone (NMP) (3
g) and then added, with rotor-stator mixing, to aqueous surfactant
solution A (25 mL). Solution A (pH .about.7.8 to 8.0) contained
sodium phosphate, dibasic, anhydrous (0.13 g), sodium phosphate,
monobasic, monohydrate (0.01 g), glycerin (2.2 g), DSPE-mPEG 2000
(0.2 g), and poloxamer 188 (0.5 g) in 100 mL water (Table 1).
TABLE-US-00001 TABLE 1 % (w/v) for % (w/v) Component Solution A for
Solution B Sodium phosphate, 0.127 0.127 dibasic, anhydrous Sodium
phosphate, 0.0144 0.0144 monobasic, monohydrate Glycerin 2.2 2.2
DSPE-mPEG 2000 0.2 0.2 Poloxamer 188 0.5 0.5 DOTAP 0.0 0.1 Water QS
to 100 mL QS to 100 mL
[0131] The resulting suspension was transferred to a homogenizer
(Avestin C5) and circulated at static pressure until the suspension
temperature reached at least 50.degree. C. The suspension was then
homogenized at a target pressure of 20,000.+-.2,000 psi and a
target temperature of 60.degree. C. for 60 minutes. The suspension
was collected and centrifuged for 30 minutes at 10,000 rpm. Upon
completion of the centrifuge cycle, the supernatant was decanted
and replaced with an equal volume of aqueous surfactant solution B.
Solution B (pH .about.7.8 to 8.0) contained the same components as
solution A and additionally contained
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate
(DOTAP, 0.1 g) (Table 1). The pellet was resuspended, and the
centrifugation was repeated twice more, using solution B as the
replacement surfactant each time. After the third resuspension, the
nanosuspension was homogenized for 30 minutes at a target pressure
of 20,000.+-.2,000 psi and a target temperature of 60.degree. C.
The final suspension contained particles having a size of
.about.160-170 nm.
[0132] Fluorescently-labeled paclitaxel particles were prepared
according to the procedure above by adding fluorescently-labeled
paclitaxel to the drug concentrate. Specifically, 400 .mu.g Oregon
Green-labeled paclitaxel (available from Invitrogen, Carlsbad,
Calif.) was added to the drug concentrate described above to yield
fluorescently-labeled paclitaxel particles with adequate
fluorescence intensity to be detected in flow cytometry and
fluorescent microscopy.
Example 2
Uptake by Human Mononuclear Cells of Paclitaxel Particles Having a
DOTAP Coating
[0133] The uptake of DOTAP-coated paclitaxel particles by human
mononuclear cells was compared to the uptake of protamine-coated
paclitaxel particles and DSPE-mPEG 2000/poloxamer 188-coated
paclitaxel particles. The protamine-coated paclitaxel particles
were prepared by adding 0.08 mL of a 25 mg/mL protamine solution to
0.01 mL of an Oregon Green-labeled paclitaxel suspension at 10
mg/mL.
[0134] Both DOTAP-coated particles and protamine-coated particles
are slightly positively charged under the conditions used for the
uptake experiments. Thus, the comparative experiment using
protamine-coated paclitaxel particles was designed to assess
whether enhanced uptake of paclitaxel particles could be solely
attributed to the positive charge of the coated particles.
[0135] Zeta potential measurements were performed on the paclitaxel
formulations used in the cell uptake experiments shown in FIG. 2
(and Table 2) by adding 30 .mu.L suspension to 10 mL of 10 mM HEPES
buffer pH 7.38. DOTAP- and protamine-coated paclitaxel
nanoparticles have slightly positive zeta potentials, while
DSPE-mPEG 2000/poloxamer 188-coated paclitaxel nanoparticles (which
lack DOTAP or protamine) have a negative zeta potential under the
tested conditions (data not shown).
[0136] Human mononuclear cells for use in the uptake experiments
were purified from the whole blood of human donors. These cells
were cultured in tissue culture treated 6-well plates (BD
Biosciences) for 5-7 days in Media A, with media exchanged every
2-3 days. Media A contained DMEM (Gibco BRL cat. no. 11960-051)
supplemented with the following to make 1 L: 1000U/ml recombinant
human macrophage-colony stimulating factor-1 (rhM-CSF-1)
(Chemicon), 100 mL heat-inactivated human serum, 10 mL 200 mM L
glutamine (Gibco BRL cat. no. 25030-081), 2 mL 50 mg/ml Gentamicin
(Sigma cat. no. G1397), and 400 .mu.L 25 mg/mL Ciprofloxacin (Bayer
code no. 89-001-1). The cells also were cultured on glass
coverslips for microscopy applications.
[0137] The adherent monocyte-derived macrophages were then treated
with paclitaxel formulations (paclitaxel particles having a DOTAP
coating, paclitaxel particles having a protamine coating, or
paclitaxel particles having a DSPE-mPEG 2000/poloxamer 188 coating)
at 37.degree. C. for various periods of time. The suspension
formulations contained paclitaxel (doped with Oregon Green-labeled
paclitaxel) at a final concentration of .about.10 .mu.M. After
incubation, the cells were washed at least three times with 2
mL/well phosphate-buffered saline (PBS). The cells were then
scraped in PBS and transferred to microfuge tubes (or fixed and
mounted if the cells were adherent to coverslips).
[0138] To assess uptake of the paclitaxel particles, the cells were
stained for CD14 expression and analyzed via flow cytometry. Gates
were established based on the dot plots for both the isotype
control (to establish the CD14+ selection gate) and the untreated
cells (to establish the Oregon Green selection gate). Paclitaxel
uptake was assessed by both the ratio of CD14+ cells
(monocyte-derived macrophages) positive for Oregon Green
fluorescence (% paclitaxel+/CD14+), i.e., the percentage of cells
that have internalized or adsorbed paclitaxel particles, and the
Mean Fluorescence Intensity (MFI). The MFI value directly
correlates with the concentration of paclitaxel contained within
the population of cells.
[0139] The uptake kinetics of the paclitaxel suspensions are shown
in FIGS. 1 and 2 (results are shown as both percentages of
paclitaxel positive cells after nanosuspension uptake and MFI of
cell-associated/internalized particles). In FIG. 1, cells were
exposed to the paclitaxel particles for 0, 3, 5 hours, while in
FIG. 2, cells were exposed to the paclitaxel particles for 0, 0.25,
0.5, and 1 hour. The DOTAP coating substantially improved the
uptake of paclitaxel particles as compared to DSPE-mPEG
2000/poloxamer 188-coated particles (FIGS. 1 and 2) and
protamine-coated particles (FIG. 2). These results suggest that
enhanced uptake of paclitaxel particles is not solely attributable
to the positive charge of the coated particles.
[0140] Additionally, FIG. 2 demonstrates the stability of the
paclitaxel formulations upon storage. DOTAP Sample 1 was stored for
approximately 3 months prior to the uptake experiments. DOTAP
Sample 2 was used in the uptake experiments shortly after
preparation. These results indicate that storage of DOTAP-coated
paclitaxel particles for several months does not significantly
affect the particle uptake kinetics.
[0141] Uptake of paclitaxel particles was quantified by reverse
phase HPLC. Paclitaxel uptake was measured after incubating the
cells with the paclitaxel suspensions for 15, 30, and 60 minutes.
Samples were prepared by adding acetonitrile (500 .mu.L) to a 500
.mu.L aliquot of each cell suspension and vortexing to mix. The
samples were then centrifuged at 10,000 rpm for 30 minutes at
25.degree. C. and the supernatants were analyzed by reverse phase
HPLC to determine the amount of paclitaxel in the sample (Table
2).
TABLE-US-00002 TABLE 2 Paclitaxel Levels in Cell Extracts (mg/mL)
15 minutes 30 minutes 60 minutes Untreated -- -- 0.00030 DOTAP
sample 1 0.00083 0.00126 0.00177 DOTAP sample 2 -- 0.00148 0.00203
Protamine -- 0.00046 0.00082 DSPE-mPEG 2000/poloxamer 188 -- --
0.00082
[0142] FIG. 3 shows uptake by monocyte-derived macrophages of
DOTAP-coated paclitaxel nanosuspensions after 1, 2, or 6 days of
culture. The cells were exposed to the paclitaxel particles for
various periods of time from 0 and 3.5 hours. The results indicate
that the longer the cells are cultured, the less responsive they
are to DOTAP-coated particles. It is theorized that the young cells
(cells which have been cultured in vitro for relatively short
periods of time) are capable of rapidly taking up DOTAP-coated
particles, while relatively older cells (cells which have been
cultured in vitro for longer periods of time) do not take up
DOTAP-coated particles as readily.
Example 3
Uptake by Human Mononuclear Cells of Paclitaxel Particles Having a
PLGA or a Phosphatidylserine Coating
[0143] The uptake of DOTAP-coated paclitaxel particles was compared
to the uptake of polylactic-co-glycolic acid (PLGA)-coated
paclitaxel particles and to the uptake of phosphatidylserine
(PS)-coated paclitaxel particles. PLGA-coated paclitaxel particles
and PS-coated paclitaxel particles were prepared in accordance with
the procedure described in Example 1, except that the PLGA
particles were sonicated, rather than homogenized, and were
formulated using a solution containing phosphate buffer, glycerin,
PLGA, and Poloxamer 188, and the PS particles were formulated using
a solution containing phosphate buffer, glycerin, DSPE-mPEG 2000,
Poloxamer 188, and phosphatidylserine.
[0144] The uptake kinetics of the paclitaxel suspensions are shown
in FIG. 4 (results are shown as both percentages of paclitaxel
positive cells after nanosuspension uptake and WI of cell
associated/internalized particles). The DOTAP coating substantially
improved the uptake of particles compared to PLGA-coated or
phosphatidylserine-coated particles. These results suggest that
enhanced uptake of paclitaxel particles is not solely attributable
to the presence of a polymer or surfactant coating.
Example 4
Uptake by Human Mononuclear Cells of Paclitaxel Particles Having a
CTAB Coating
[0145] The uptake of DOTAP-coated paclitaxel particles was compared
to the uptake of cetyl trimethylammonium bromide (CTAB)-coated
paclitaxel particles. CTAB-coated paclitaxel particles were
prepared in accordance with the procedure described in Example 1,
except that the CTAB particles were formulated using a solution
containing phosphate buffer, glycerin, DSPE-mPEG 2000, Poloxamer
188, and CTAB.
[0146] The uptake kinetics of the paclitaxel suspensions are shown
in FIG. 5 (results are shown as both percentages of paclitaxel
positive cells after nanosuspension uptake and WI of cell
associated/internalized particles). The DOTAP coating substantially
improved the uptake of particles compared to CTAB-coated particles.
These results suggest that enhanced uptake of paclitaxel particles
is not solely attributable to the presence of a coating having both
a positively charged group and a hydrophobic group.
Example 5
Uptake of Paclitaxel Particles Having a DOTAP Coating in Whole
Blood
[0147] Whole blood was drawn from a healthy human donor into EDTA
vacutainer (BD Biosciences). Paclitaxel nanosuspensions doped with
Oregon Green-labeled paclitaxel were incubated with the whole blood
(.about.10 .mu.M final concentration) for 1 hour at room
temperature in 1.7 mL microfuge tubes on a tube rotator. A fraction
of the whole blood was exposed to a hypotonic lysing solution (BD
Biosciences) to lyse the red blood cells. The lysed samples were
then stained for CD14 expression. Both the whole blood and stained
cells were analyzed via flow cytometry.
[0148] No apparent increase in Oregon Green fluorescence was
observed in either the red blood cell (RBC) or platelet
populations. A substantial increase in fluorescence was observed in
the CD14+ monocyte population in the lysed samples using the
DOTAP-formulated paclitaxel suspension. Paclitaxel formulations
having a DSPE-mPEG 2000/poloxamer 188 coating also showed some
uptake in the CD14+ monocyte population. There was no apparent
uptake in the other major cell populations as assessed by Oregon
Green fluorescence (data not shown). These results suggest that
DOTAP-coated paclitaxel particles are selectively taken up by
monocytes over red blood cells, platelets, and other cell types
present in blood.
Example 6
Uptake by Mouse Peritoneal Macrophages of Paclitaxel Particles
Having a DOTAP Coating
[0149] Peritoneal macrophages were isolated from mice and exposed
to paclitaxel particles having a DOTAP coating and to paclitaxel
particles without such a coating. Fluorescence images showed that
peritoneal macrophages exposed to the DOTAP-coated particles took
up greater amounts of paclitaxel than those exposed to DSPE-mPEG
2000/poloxamer 188-coated particles (data not shown). This example
supports that DOTAP enhances uptake of particles by peritoneal
macrophages.
Example 7
Uptake by Human OVCAR-3 Cells of Paclitaxel Particles Having a
DOTAP Coating
[0150] Human OVCAR-3 cells were transfected with Red Fluorescent
Protein (RFP) such that they fluoresced red. These cells were then
exposed to paclitaxel particles prepared using Oregon
Green-paclitaxel having a DOTAP coating and to paclitaxel particles
without a DOTAP coating. Fluorescence images showed that the
RFP-OVCAR-3 cells only took up particles when the particles were
coated with DOTAP (data not shown). There was no visible uptake of
the particles coated with DSPE-mPEG 2000/poloxamer 188. This
example supports that DOTAP enhances uptake of particles by human
ovarian cancer cells.
Example 8
Residence Time in Mice of Paclitaxel Particles Having a DOTAP
Coating
[0151] Oregon Green-labeled paclitaxel particles having a DOTAP
coating were injected subcutaneously into a mouse. Fluorescence
images were captured over time to demonstrate particle residence
time. The persistence of green fluorescence at 30 days indicated
that paclitaxel particles remained for at least 30 days when
injected subcutaneously (data not shown).
[0152] In a separate experiment, Oregon Green-labeled paclitaxel
particles having a coating containing DOTAP and a rhodamine-labeled
surfactant (Lissamine rhodamine B
1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine,
triethylammonium salt (rDHPE); Invitrogen, Carlsbad, Calif.) were
injected intraperitoneally (IP) into a healthy mouse. Fluorescence
images were captured over time to demonstrate particle residence
time. The data indicated that paclitaxel nanoparticles were cleared
rapidly (within about 24 hours) from the peritoneal space in a
healthy mouse (data not shown).
[0153] A mouse model was established wherein test mice were
implanted with RFP-OVCAR-3 cells and tumors were allowed to grow.
The tumors expressed RFP and had red fluorescence. Oregon
Green-labeled paclitaxel particles having a DOTAP coating were
administered by intraperitoneal injection to mice having
RFP-expressing tumors. The presence and location of the
DOTAP-coated paclitaxel particles were detected relative to the
tumors using fluorescence.
[0154] Both tumors and paclitaxel particles were observed by
fluorescence microscopy (red fluorescence for tumors, green
fluorescence for particles). Unlike healthy mice, in which
particles were rapidly cleared from the peritoneal cavity, the
DOTAP-coated paclitaxel particles were present in the tumor-bearing
mice up to 30 days post-injection, indicating that the tumors
present in the peritoneal cavity of the mouse were partially
responsible for the increased residence time. Moreover, the
DOTAP-coated paclitaxel particles frequently co-localized with
tumors. Thus, this example supports that the DOTAP-coated
paclitaxel particles target tumor sites as opposed to healthy
tissues, and are able to persist within the targeted tumor sites
for significant periods of time such that they can effectively
deliver a sustained release of the therapeutic drug.
[0155] Additionally, when DOTAP-coated paclitaxel particles were
present, the red fluorescence intensity diminished over time,
consistent with tumor cell death. Conversely, in the absence of
paclitaxel particles, the red fluorescence intensified over time.
Thus, this example further demonstrates that administration of
DOTAP-coated paclitaxel particles effectively treated cancer in
vivo.
[0156] While specific embodiments have been illustrated and
described, numerous modifications come to mind without departing
from the spirit of the invention and the scope of protection is
only limited by the scope of the accompanying claims.
* * * * *