U.S. patent application number 11/380556 was filed with the patent office on 2006-11-23 for surface-modified microparticles and methods of forming and using the same.
Invention is credited to Ramin Darvi, Yuri Lvov, Julia Rashba-Step, Terrence L. Scott, Tatsiana Shutava.
Application Number | 20060260777 11/380556 |
Document ID | / |
Family ID | 36928379 |
Filed Date | 2006-11-23 |
United States Patent
Application |
20060260777 |
Kind Code |
A1 |
Rashba-Step; Julia ; et
al. |
November 23, 2006 |
SURFACE-MODIFIED MICROPARTICLES AND METHODS OF FORMING AND USING
THE SAME
Abstract
Surface-modified microparticles and methods of making and using
such particles are disclosed. The surface modified microparticles
include a preformed or core microparticle that contains at least
one active agent. The outer surface of the preformed or core
microparticle carries a net surface charge. A monolayer is
associated with the outer surface of the preformed or core
microparticle. The monolayer includes at least one charged compound
that has a charge that is different from the net surface charge of
the preformed or core microparticle,
Inventors: |
Rashba-Step; Julia; (Newton,
MA) ; Scott; Terrence L.; (Winchester, MA) ;
Darvi; Ramin; (Lexington, MA) ; Lvov; Yuri;
(Ruston, LA) ; Shutava; Tatsiana; (Minsk,
BY) |
Correspondence
Address: |
BAXTER HEALTHCARE CORPORATION
ONE BAXTER PARKWAY
DF2-2E
DEERFIELD
IL
60015
US
|
Family ID: |
36928379 |
Appl. No.: |
11/380556 |
Filed: |
April 27, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60675372 |
Apr 27, 2005 |
|
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|
60750903 |
Dec 16, 2005 |
|
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Current U.S.
Class: |
162/181.1 |
Current CPC
Class: |
A61K 9/5057 20130101;
A61P 5/00 20180101; A61K 9/1658 20130101; A61K 9/5089 20130101;
A61K 9/5073 20130101; C07K 17/08 20130101; A61K 9/5026 20130101;
A61K 9/5036 20130101; A61K 9/1688 20130101; A61K 9/5052 20130101;
A61K 9/5047 20130101; A61K 9/0019 20130101; A61K 9/1635 20130101;
A61P 5/50 20180101; A61K 9/0024 20130101; A61K 9/16 20130101; A61K
9/1272 20130101; A61K 31/7105 20130101 |
Class at
Publication: |
162/181.1 |
International
Class: |
D21H 23/00 20060101
D21H023/00 |
Claims
1. A method for preparing a surface-modified microparticle,
comprising: providing a solid and amorphous preformed microparticle
comprising at least one active agent, said microparticle having an
outer surface carrying a net surface charge; exposing at least said
outer surface of said preformed microparticle to at least one
charged compound having a net charge that is opposite in sign to
said net surface charge; and forming a monolayer comprising said at
least one charged compound, whereby said formed monolayer is
associated with said outer surface,
2. The method of claim 1, wherein said formed monolayer has an
outer surface carrying a net surface charge that is different from
that of said preformed microparticle.
3. The method of claim 1, wherein said outer surface of said
preformed microparticle consists substantially of said at least one
active agent.
4. The method of claim 2, wherein said formed monolayer is a
saturated monolayer and said net surface charge is opposite in sign
to that of said preformed microparticle.
5. The method of claim 1, wherein said preformed microparticle
comprises, by weight, approximately 40% to less than 100% of said
at least one active agent distributed substantially homogeneously
throughout said preformed microparticle.
6. The method of claim 1 wherein said preformed microparticle
comprises, by weight, 80% or greater of said at least one active
agent distributed substantially homogenously throughout said
preformed microparticle.
7. The method of claim 1, wherein said exposing further comprises:
providing a solution comprising said at least one charged compound
and one or more of water, a buffer, and a water-miscible organic
solvent and one or more solubility reducing agents; and contacting
said preformed microparticle in said solution.
8. The method of claim 1, wherein said net surface charge is
contributed substantially by said at least one active agent.
9. The method of claim 7, wherein said one or more solubility
reducing agents comprise one or more of alcohols, carbohydrates,
non-ionic aqueous-miscible polymers, and inorganic ionic compounds
comprising polyvalent cations.
10. The method of claim 7, wherein said solution comprises, in
weight-to-volume percentage, 16% polyethylene glycol and 0.7%
sodium chloride and has a pH of between 4and 10.
11. The method of claim 7, wherein said solution has a pH that
differs from a surface-neutral point of said preformed
microparticle by 0 to less than 0.3.
12. The method of claim 7, wherein said solution has a pH that
differs from a surface-neutral point of said preformed
microparticle by 0.3 or greater.
13. The method of claim 1, wherein said exposing is carried out at
a temperature between 2.degree. C. and 5.degree. C.
14. The method of claim 1, further comprising carrying out one or
more treatments to the surface-modified microparticle, the
treatments comprising manipulations of temperature, pressure, pH or
a combination thereof.
15. The method of claim 14, wherein the treatment comprises heating
a suspension including the microparticle at an elevated
temperature.
16. The method of claim 15 further comprising allowing said
suspension to arrive at a depressed temperature that is lower than
said elevated temperature.
17. The method of claim 7, further comprising separating said
surface-modified microparticle from said solution.
18. The method of claim 1, wherein said at least one charged
compound comprises one or more of polyelectrolytes, charged
polyaminoacids, charged polysaccharides, polyionic polymers,
charged peptides, charged proteinaceous compounds, charged lipids
optionally in combination with uncharged lipids, charged lipid
structures, and derivatives thereof.
19. The method of claim 1, further comprising: exposing at least
said outer surface of a formed monolayer to at least one different
charged compound having a net charge that is opposite in sign to
said net surface charge of said formed monolayer; and forming a
subsequent monolayer comprising said at least one different charged
compound, wherein said subsequent monolayer is associated with said
formed monolayer
20. The method of claim 19, wherein said subsequent monolayer has
an outer surface carrying a net surface charge that is opposite in
sign to that of said formed monolayer.
21. The method of claim 20, further comprising forming one to five
additional alternatingly charged monolayers.
22. The method of claim 20, further comprising forming an odd
number of additional alternatingly charged monolayers.
23. The method of claim 1, further comprising forming said
surface-modified microparticle capable of controlled release of
said at least one active agent.
24. The method of claim 23, wherein said controlled release
comprises an initial burst and a substantially linear release
profile.
25. The method of claim 1, wherein said preformed microparticle is
spherical.
26. The microparticle of claim 1 wherein said at least one active
agent is a proteinaceous compound.
27. The method of claim 1, wherein said preformed microparticle is
free of covalent crosslinking and free of hydrogel.
28. The method of claim 1, wherein said preformed microparticle is
free of lipids and free of encapsulation.
29. A method for preparing a surface-modified microparticle,
comprising: providing a liquid continuous phase system comprising
at least one solvent, at least one active agent, at least one
phase-separation enhancing agent; inducing a phase change at
optionally a controlled rate in said system to cause a liquid-solid
phase separation; forming a solid phase that comprises an amorphous
and solid microparticle comprising said at least one active agent
and having an outer surface carrying a net surface charge, and a
liquid phase that comprises said solvent and said at least one
phase-separation enhancing agents; exposing at least said outer
surface of said formed microparticle to at least one charged
compound having a net charge that is opposite in sign to said net
surface charge; and forming a monolayer comprising said at least
one charged compound, wherein said formed monolayer is associated
with said outer surface of said formed microparticle.
30. The method of claim 29, wherein said method is free of washing
said formed microparticle prior to said exposing.
31. The method of claim 29, further comprising washing said formed
microparticle in the presence of at least one phase-separation
enhancing agents prior to said exposing.
32. The method of claim 29, wherein said exposing is carried out in
the presence of at least one phase separation enhancing agents.
33. The method of claim 29 wherein said preformed microparticle
comprises, by weight, 80% or greater of said at least one active
agent distributed substantially homogenously throughout said
preformed microparticle.
34. The method of claim 34, wherein said exposing comprises:
providing a solution that comprises said at least one charged
compound, optionally a polyvalent cation, and, in weight-to-volume
percentage, 16% polyethylene glycol and 0.7% sodium chloride, and
incubating said formed microparticle in said solution at a
temperature of 2.degree. C. to 5.degree. C. over a period of 1
second to 10 hours.
35. The method of claim 29, wherein said at least one charged
compound is provided in a solution having a pH that differs from a
surface-neutral point of said formed microparticle by 0 to less
than 0.3.
36. The method of claim 29, wherein said at least one charged
compound is provided in a solution having a pH that differs from a
surface-neutral point of said formed microparticle by 0.3 or
greater.
37. The method of claim 29, further comprising forming said
surface-modified microparticle having a monodisperse or
polydisperse size distribution.
38. A method for preparing a surface-modified microparticle,
comprising: providing an amorphous and solid preformed
microparticle comprising at least one active agent, said preformed
microparticle having an outer surface carrying a net surface
charge; exposing at least said outer surface of said preformed
microparticle to at least one charged compound having a net charge
that is opposite in sign to said net surface charge of said
preformed microparticle; forming an intermediate microparticle that
comprises said preformed microparticle and a formed monolayer
comprising said at least one charged compound, wherein said formed
monolayer is associated with said outer surface of said preformed
microparticle; exposing at least said formed monolayer to at least
one different charged compound; forming said surface-modified
microparticle that comprises said intermediate microparticle and a
subsequent monolayer comprising said at least one different charged
compound, wherein said surface-modified microparticle has a release
profile for release of said at least one active agent that is
different from the release profile of said intermediate
microparticle.
39. The method of claim 38 further comprising carrying out one or
more treatments to said intermediate microparticle, the treatment
comprising manipulation of temperature, pressure, pH or a
combination thereof.
40. A method of claim 39 wherein the treating comprises heating a
suspension including the microparticle at an elevated
temperature.
41. A microparticle comprising: a solid, amorphous core
microparticle comprising 80% or greater, by weight, of at least one
active agent, said core microparticle comprising an outer surface
carrying a net surface charge; and a monolayer comprising at least
one charged compound associated at least by electrostatic
interaction with said outer surface of said core microparticle,
said charged compound having a net charge that is sufficiently
different from said net surface charge of said core
microparticle,
42. The microparticle of claim 41 wherein said at least one active
agent is homogeneously distributed across said core
microparticle.
43. The microparticle of claim 41 wherein said core microparticle
is spherical.
44. The microparticle of claim 41 wherein said monolayer has a
thickness of less than 50 nm.
45. The microparticle of claim 41 wherein said charge of said
charged compound is opposite in sign to said net surface charge of
said microparticle outer surface.
46. The microparticle of claim 41 further comprising another
monolayer comprising at least one different charged compound, said
other monolayer carrying a net charge that is different from said
net charge of said monolayer associated with said core
microparticle.
47. The microparticle of claim 46 wherein said net surface charge
of said other monolayer is opposite in sign to that of said
monolayer associated with said core microparticle.
48. The microparticle of claim 41 wherein said active agent is
insulin or human growth hormone.
49. A microparticle comprising at least 80% by weight of at least
one active agent, the microparticle being solid, and an outer
surface of the microparticle comprising at least one charged
polymer associated with the active agent, wherein the microparticle
is capable of displaying a 1-hour percentage of cumulative release
of the active agent of 50% or less when subjected to in vitro
release in a release buffer at pH 7.4 and 37.degree. C., the
release buffer consisting 10 mM Tris, 005% (w/v) Brij 35, and 0.9%
(w/v) NaCl in water.
50. A microparticle for in vivo administration comprising a solid,
microparticle having at least 80%, by weight, of at least one
proteinaceous compound homogeneously distributed across said
microparticle, wherein the outer surface of said microparticle
comprises at least one charged compound associated with said outer
surface, wherein upon administration, said microparticle provides a
C.sub.max and t.sub.max that is than the C.sub.max and t.sub.max of
said core microparticle.
51. The microparticle of claim 50 wherein said monolayer associated
with said microparticle outer surface comprises a positively
charged compound selected from the group consisting essentially of
positively charged polyelectrolytes, positively charged
polyaminoacids and positively charged polysaccharides.
52. The microparticle of claim 50 wherein said monolayer associated
with said core comprises a negatively charged compound selected
from the group consisting essentially of negatively charged
polyelectrolytes, negatively charged polyaminoacids and negatively
charged polysaccharides.
53. The microparticle of claim 51 wherein said another monolayer
comprises a negatively charged compound selected from the group
consisting essentially of negatively charged polyelectrolytes,
negatively charged polyaminoacids and negatively charged
polysaccharides.
54. The microparticle of claim 52 wherein said another monolayer
comprises a positively charged compound selected from the group
consisting essentially of positively charged polyelectrolytes,
positively charged polyaminoacids and positively charged
polysaccharides.
55. The microparticle of claim 50 wherein said proteinaceous
compound is insulin or human growth hormone.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/675,372 filed Apr. 27, 2005 and U.S.
Provisional Patent Application Ser. No. 60/750,903 filed Dec. 16,
2005.
FIELD AND BACKGROUND
[0002] The present disclosure is generally directed to
microparticles containing one or more active agents and delivery
methods of such microparticles to subjects. More particularly, the
present disclosure is directed to surface modifications of the
microparticles such that they are capable of controlled release of
one or more active agents. The present disclosure is further
directed to methods for making and using such surface-modified
microparticles.
[0003] Microparticles have been used in many different
applications, including the controlled delivery and/or release of
active agents. Controlling or modifying the release profile of an
active agent can, if desired, prolong the levels (such as
therapeutic levels) of the active agent in the blood stream of the
recipient, improve pharmacokinetics and pharmacodynamics, and
result in greater convenience to the recipient.
SUMMARY
[0004] The present disclosure is generally directed to methods for
preparing a surface-modified microparticle. In one example, the
method includes providing an amorphous and solid preformed
microparticle including at least one active agent. The preformed
microparticle has an outer surface carrying a net surface charge.
The method further includes exposing at least the outer surface of
the preformed microparticle to at least one charged compound having
a net charge that is opposite in sign to the net surface charge of
the preformed microparticle outer surface. A monolayer of the
charged compound is formed whereby the monolayer is associated with
the preformed microparticle outer surface.
[0005] The present disclosure is also directed to a method for
preparing a surface-modified microparticle including providing a
liquid continuous phase system containing a solvent, at least one
active agent, and one or more phase-separation enhancing agents.
The method includes inducing a liquid-solid phase separation at,
optionally, a controlled rate to cause a liquid-solid separation,
and forming a solid phase that includes a solid and amorphous
microparticle containing the active agent, the microparticle having
an outer surface carrying a net surface charge, while the solvent
and the phase-separation enhancing agents remain in the liquid
phase. After forming the microparticle, at least the outer surface
of the formed microparticle is exposed to at least one charged
compound having a net charge that is opposite in sign to the net
surface charge of microparticle outer surface. The method further
includes forming a monolayer including at least one charged
compound on the formed microparticle whereby the formed monolayer
is associated with the microparticle outer surface.
[0006] The present disclosure is also directed to a method for
preparing a surface modified microparticle that includes providing
an amorphous and solid preformed microparticle including at least
one active agent, the preformed microparticle having an outer
surface carrying a net surface charges In this example, the method
further includes exposing at least the outer surface of the
preformed microparticle to at least one charged compound having a
net charge that is opposite in sign to the net surface charge of
the preformed microparticle. An intermediate microparticle is
formed that includes the preformed microparticle and a formed
monolayer including the at least one charged compound wherein the
formed monolayer is associated with the preformed microparticle
outer surface. The formed monolayer is then exposed to at least a
different charged compound to form a surface modified microparticle
that includes the intermediate microparticle and a subsequent
monolayer including at least the one different charged compound.
The surface modified microparticle has a release profile for
release of the at least one active agent that is different from the
release profile of the intermediate microparticle.
[0007] The present disclosure is also directed to a microparticle
that includes a solid, amorphous core microparticle that includes
between 80% or greater than by weight, of at least one active
agent. The outer surface of the core microparticle carries a
selected net surface charge. A monolayer of at least one charged
compound carrying a net surface charge that is sufficiently
different from the surface charge of the core microparticle outer
surface to allow for association therewith, is associated with the
core microparticle outer surface at least by, but not limited to,
electrostatic interaction with the outer surface.
[0008] A solid microparticle including at least 80%, by weight, of
at least one active agent where the outer surface of the
microparticle includes at least one charged compound associated
with the active agent may display a 1-hour percentage of cumulative
release of the active agent that is 50% or less when subjected to
in vitro release in a suitable buffer at selected pH and
temperature,
[0009] Furthermore, a solid, preformed microparticle having at
least 80%, by weight, of at least one proteinaceous compound where
the outer surface of the preformed microparticle has at least one
charged compound associated with the proteinaceous compound are
suitable for in vivo administration Upon such administration, the
microparticle provides a C.sub.max and t.sub.max that is different
from the C.sub.max and t.sub.max of the core microparticle.
[0010] Further details of the above-described microparticles,
methods of making microparticles and methods for controlling the
release of active agents from the microparticles are discussed
below.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 is a flow chart showing an exemplary method set forth
in the present disclosure;
[0012] FIG. 2 is a schematic illustration of the fabrication of
monolayers of charged compounds on a preformed microparticle;
[0013] FIG. 3 is a graph that shows the change in zeta potential of
insulin microparticles with alternatingly charged monolayers
(Examples 1A & 1B);
[0014] FIG. 4 is a graph that shows the change in zeta potential of
insulin microparticles, each, with one monolayer of different
polyanionic compounds (Example 2A);
[0015] FIG. 5 is a graph that shows the change in zeta potential of
insulin microparticles, each with one monolayer of different
polycationic compounds (Example 2B);
[0016] FIG. 6 is a laser scanning confocal (LSC) micrograph of
insulin microparticles with one FITC-labeled protamine monolayer
(Example 2B);
[0017] FIG. 7 is a graph showing surface electric charge alteration
of insulin microparticles, each with a first monolayer of different
polyanionic compounds and a second monolayer of poly-L-lysine
(Example 3A);
[0018] FIG. 8 is a LSC micrograph of insulin microparticles with a
first monolayer of PSS and a second monolayer of FITC-labeled PLL
(Example 3A);
[0019] FIG. 9 is a graph showing progressive film thicknesses after
each successive deposition of alternatingly charged polyelectrolyte
monolayers (e.g., PLL and chondroitin sulfate) on the QCM electrode
(Example 3A);
[0020] FIG. 10 is a graph showing changes in zeta-potential of
insulin microparticles following subsequent depositions of a
chondroitin sulfate monolayer and a gelatin A monolayer (Example
3B);
[0021] FIG. 11 is a graph comparing zeta-potential of insulin
microparticles with monolayers of protamine sulfate and chondroitin
sulfate in the presence and absence of zinc cations in the
PEG-containing reaction medium (Example 4A);
[0022] FIG. 12 is a graph showing zeta-potential of insulin
microparticles with monolayers of protamine sulfate and chondroitin
sulfate in presence of zinc cations in a PEG-free reaction medium
(Example 4B);
[0023] FIG. 13 is a graph showing LSC micrograph of insulin
microparticles with one monolayer of Rodamine B-labeled protamine
(top left) and one monolayer of FITC-labeled DAP (top right)
(Example 5);
[0024] FIG. 14 is a graph showing the zeta-potential of insulin
microparticles following deposition of each monolayer as described
in Example 5;
[0025] FIG. 15 is a graph showing insulin release profiles from
microparticles coated with a monolayer of protamine sulfate with
various concentrations of the polycation in the reaction medium
(Example 6);
[0026] FIG. 16 is a graph showing insulin release profiles from
microparticles with a first monolayer of protamine sulfate and a
second monolayer of carboxymethyl cellulose at various
concentrations of the polyanion in the reaction medium (Example
7);
[0027] FIG. 17 is a graph showing release profiles of insulin from
insulin microparticles coated with three monolayers (Example
7);
[0028] FIG. 18A is a graph showing the serum human insulin (hINS)
concentration versus time profiles in rats that have received a
single subcutaneous injection of uncoated insulin microparticles,
or protamine-coated insulin microparticles (Example 8);
[0029] FIG. 18B is a graph showing the serum glucose depression
versus time profiles of rats treated with single subcutaneous
injection of uncoated insulin microparticles, or protamine-coated
insulin microparticles Example 8);
[0030] FIG. 19 is a graph showing alteration in the surface charge
of insulin microparticle by deposition of one monolayer of
protamine in various solubility-reducing media at pH 7.0 (Example
9);
[0031] FIG. 20A is a graph showing the effect of protamine
concentration in the reaction medium on the surface charge of
insulin microparticles, and the extent of the dissolution after 48
h from initiation of the in vitro release (Example 10);
[0032] FIG. 20B is a graph showing the effect of CMC concentration
in the reaction medium on the surface charge of protaimne-coated
insulin microparticles, and the extent of their dissolution after
48 h from initiation of the in vitro release (Example 10);
[0033] FIG. 20C is a graph showing the effect of protamine
concentration in the reaction medium on the surface charge of
insulin microparticles coated with one monolayer of protamine and
one monolayer of CMC, and the extent of their dissolution after 48
h from initiation of the in vitro release (Example 10);
[0034] FIG. 21A is a graph showing the alteration of zeta-potential
of hGH microparticles following subsequent depositions of protamine
sulfate and chondroitin sulfate monolayers (Example 11);
[0035] FIG. 21B is a graph showing profiles of hGH release from
microparticles coated with one, two, or three monolayers of
alternating protamine and condroitin sulfate (Example 11);
[0036] FIG. 22 is a graph showing the alteration of zeta-potential
of intravenous immunoglobulin (IVIG) microparticles following
subsequent depositions of chondroitin sulfate and protamine sulfate
monolayers (Example 12);
[0037] FIG. 23 is a graph showing net surface charge
characteristics of insulin microspheres in 16% PEG solution across
a pH range of 4 to 7.5 (Example 13);
[0038] FIG. 24 is a flow chart showing another exemplary method of
preparing microparticles set forth in the present disclosure;
[0039] FIG. 25 is a graph showing the effect of reaction pH on the
zeta potential of insulin microparticles that have been
surface-modified with one monolayer of protamine sulfate,
poly-l-lysine, or poly-l-arginine (Example 14);
[0040] FIG. 26 is a graph showing the effect of reaction pH on the
in vitro 1-hour percentage of cumulative insulin release of insulin
microparticles surface-modified with one monolayer of protamine
sulfate, poly-l-lysine, or poly-l-arginine (Example 14);
[0041] FIG. 27 is a laser scanning confocal (LSC) micrograph of
nucleic acid microparticles surface-modified with one monolayer of
rodamine B-labeled poly-l-lysine (Example 15);
[0042] FIG. 28 is a graph showing the in vitro release profiles of
PLL-insulin microparticles thermally treated at different
temperatures; and
[0043] FIG. 29A is a graph showing the serum insulin concentration
versus time profiles in rats that have received a single
subcutaneous injection of uncoated insulin microparticles,
PLA-modified insulin microparticles treated at 28.degree. C., and
PLA-modified insulin microparticles treated at 4.degree. C.
(Example 18); and
[0044] FIG. 29B is a graph showing the serum glucose depression
concentration versus time profiles in rats that have received a
single subcutaneous injection of uncoated insulin microparticles,
PLA-modified insulin microparticles treated at 28.degree. C., and
PLA-modified insulin microparticles treated at 4.degree. C.
(Example 18).
DETAILED DESCRIPTION
[0045] Unless otherwise defined herein, scientific and technical
terminologies employed in the present disclosure shall have the
meanings that are commonly understood and used by one of ordinary
skill in the art. Unless otherwise required by context, it will be
understood that singular terms shall include plural forms of the
same and plural terms shall include the singular. Specifically, as
used herein and in the claims, the singular forms "a" and "an"
include the plural reference unless the context clearly indicates
otherwise. Thus, for example, the reference to a particular
microparticle is a reference to one such microparticle or a
plurality of such microparticles, including equivalents thereof
known to one skilled in the art. Also, as used herein and in the
claims, the terms "at least one" and "one or more" have the same
meaning and include one, two, three or more. The following terms,
unless otherwise indicated, shall be understood to have the
following meanings when used in the context of the present
disclosure.
[0046] "Active agent" refers to naturally occurring, synthetic, or
semi-synthetic materials (e.g., compounds, fermentates, extracts,
cellular structures) capable of eliciting, directly or indirectly,
one or more physical, chemical, and/or biological effects, in vitro
and/or in vivo. The active agent may be capable of preventing,
alleviating, treating, and/or curing abnormal and/or pathological
conditions of a living body, such as by destroying a parasitic
organism, or by limiting the effect of a disease or abnormality by
materially altering the physiology of the host or parasite. The
active agent may be capable of maintaining, increasing, decreasing,
limiting, or destroying a physiologic body function The active
agent may be capable of diagnosing a physiological condition or
state by an in vitro and/or in vivo test. The active agent may be
capable of controlling or protecting an environment or living body
by attracting, disabling, inhibiting, killing, modifying, repelling
and/or retarding an animal or microorganism. The active agent may
be capable of otherwise treating (such as deodorizing, protecting,
adorning, grooming) a body. Depending on the effect and/or its
application, the active agent may further be referred to as a
bioactive agent, a pharmaceutical agent (such as a prophylactic
agent, a therapeutic agent), a diagnostic agent, a nutritional
supplement, and/or a cosmetic agent, and includes, without
limitation, prodrugs, affinity molecules, synthetic organic
molecules, polymers, molecules with a molecular weight of 2 kD or
less (such as 1.5 kD or less, or 1 kD or less), macromolecules
(such as those having a molecular weight of 2 kD or greater,
preferably 5 kD or greater), proteinaceous compounds, peptides,
vitamins, steroids, steroid analogs, lipids, nucleic acids,
carbohydrates, precursors thereof and derivatives thereof. Active
agents may be ionic or non-ionic, may be neutral, positively
charged, negatively charged, or zwitterionic, and may be used
singly or in combination of two or more thereof. Active agents may
be water insoluble, but more preferably are water soluble. Active
agents may have an isoelectric point of 7.0 or greater, but
preferably less than 7.0.
[0047] "Microparticle" refers to a particulate that is solid
(including substantially solid or semi-solid, but excluding gel,
liquid and gas), having an average geometric particle size
(sometimes referred to as diameter) of less than 1 mm, preferably
200 microns or less, more preferably 100 microns or less, most
preferably 10 microns or less. In one example, the particle size
may be 0.01 microns or greater, preferably 0.1 microns or greater,
more preferably 0.5 microns or greater, and most preferably from
0.5 microns to 5 microns. Average geometric particle size may be
measured by dynamic light scattering methods (such as
photocorrelation spectroscopy, laser diffraction, low-angle laser
light scattering (LALLS), medium-angle laser light scattering
(MALLS)), light obscuration methods (such as Coulter analysis
method), or other methods (such as rheology, light or electron
microscopy). Particles for pulmonary delivery will have an
aerodynamic particle size determined by time of flight measurements
or Andersen Cascade Impactor measurements. Microparticles may have
a spherical shape (sometimes referred to as microspheres) and/or
may be encapsulated (sometimes referred to as microcapsules).
Certain microparticles may have one or more internal voids and/or
cavities. Other microparticles may be free of such voids or
cavities. Microparticles may be porous or, preferably non-porous.
Microparticles may be formed from, in part or in whole, one or more
non-limiting materials, such as the active agents, carriers,
polymers, stabilizing agents, and/or complexing agents disclosed
herein.
[0048] "Peptides" refer to natural, synthetic, or semi-synthetic
compounds formed at least in part from two or more of the same or
different amino acids and/or imino acids. Non-limiting examples of
peptides include oligopeptides (such as those having less than 50
amino/imino acid monomer units, including dipeptides and
tripeptides and the like), polypeptides, proteinaceous compounds as
defined herein, as well as precursors and derivatives thereof
(e.g., glycosylated, hyperglycosylated, PEGylated, FITC-labeled,
salts thereof). Peptides may be used singly, or in combination of
two or more thereof. Peptides may be neutral, positively charged,
negatively charged, or zwitterionic, and may be used singly or in
combination of two or, more thereof
[0049] "Proteinaceous compounds" refer to natural, synthetic,
semi-synthetic, or recombinant compounds of or related structurally
and/or functionally to proteins, such as those containing or
consisting essentially of a-amino acids covalently associated
through peptide linkages. Non-limiting proteinaceous compounds
include globular proteins (edge, albumins, globulins, histones),
fibrous proteins (e.g., collagens, elastins, keratins), compound
proteins (including those containing one or more non-peptide
component, e.g., glycoproteins, nucleoproteins, mucoproteins,
lipoproteins, metalloproteins), therapeutic proteins, fusion
proteins, receptors, antigens (such as synthetic or recombinant
antigens), viral surface proteins, hormones and hormone analogs,
antibodies (such as monoclonal or polyclonal antibodies), enzymes,
Fab fragments, cyclic peptides, linear- peptides, and the like.
Non-limiting therapeutic proteins include bone morphogenic
proteins, drug resistance proteins, toxoids, erythropoietins,
proteins of the blood clotting cascade (e.g., Factor VII, Factor
VIII, Factor IX, et al.), subtilisin, ovalbumin,
alpha-1-antitrypsin (AAT), DNase, superoxide dismutase (SOD),
lysozyme, ribonuclease, hyaluronidase, collagenase, human growth
hormone (hGH), erythropoietin, insulin and insulin-like growth
factors or their analogs, interferons, glatiramer,
granulocyte-macrophage colony-stimulating factor, granulocyte
colony-stimulating factor, desmopressin, leutinizing hormone
release hormone (LHRH) agonists (e.g., leuprolide, goserelin,
buserelin, gonadorelin, histrelin, nafarelin, deslorelin,
fertirelin, triptorelin), LHER antagonists, vasopressin,
cyclosporine, calcitonin, parathyroid hormone, parathyroid hormone
peptides, insulin, glucogen-like peptides, and analogs thereof
Proteinaceous compounds may be neutral, positively charged,
negatively charged, or zwitterionic, and may be used singly or in
combination of two or more thereof.
[0050] "Nucleic acids" refer to natural, synthetic, semi-synthetic,
or recombinant compounds formed at least in part from two or more
of the same or different nucleotides, and may be single-stranded or
double-stranded. Non-limiting examples of nucleic acids include
oligonucleotides (such as those having 20 or less base pairs, e.g.,
sense, anti-sense, or missense), aptamers, polynucleotides (e g.,
sense, anti-sense, or missense), DNA (e.g., sense, anti-sense, or
missense), RNA (erg., sense, anti-sense, or missense), siRNA,
nucleotide acid constructs, single-stranded or double-stranded
segments thereof, as well as precursors and derivatives thereof
(e.g., glycosylated, hyperglycosylated, PEGylated, FITC-labeled,
nucleosides, salts thereof). Nucleic acids may be neutral,
positively charged, negatively charged, or zwitterionic, and may be
used singly or in combination of two or mole thereof.
[0051] "Carbohydrates" refer to natural, synthetic, or
semi-synthetic compounds formed at least in part from monomeric
sugar units. Non-limiting carbohydrates include polysaccharides,
sugars, starches, and celluloses, such as carboxymethylcellulose,
dextrans, hetastarch, cyclodextrins, alginates, chitosans,
chondroitins, heparins, as well as precursors and derivatives
thereof (e.g., glycosylated, hyperglycosylated, PEGylated,
FITC-labeled, salts thereof). Carbodydrates may be ionic or
non-ionic, may be neutral, positively charged, negatively charged,
or zwitterionic, and may be used singly or in combination of two
or, more thereof.
[0052] "Lipids" refer to natural, synthetic, or semi-synthetic
compounds that are generally amphiphilic. The lipids typically
comprise a hydrophilic component and a hydrophobic component.
Non-limiting examples include fatty acids, neutral fats,
phosphatides, oils, glycolipids, surfactants, aliphatic alcohols,
waxes, terpenes and steroids. Lipids may be ionic or non-ionic, may
be neutral, positively charged, negatively charged, or
zwitterionic, and may be used singly or in combination of two or
more thereof
[0053] "Complexing agent" refers to a material capable of forming
one or more non-covalent associations with the active agent.
Through such associations, the complexing agent is capable of
facilitating the loading of one or more active agents into the
microparticle, retaining the active agent(s) within the
microparticle, and/or otherwise modifying the release of the active
agent(s) from the microparticle. Complexing agents may be ionic or
non-ionic, may be neutral, positively charged, negatively charged,
or zwitterionic, and may be used singly or in combination of two or
more thereof.
[0054] "Stabilizing," used especially in conjunction with an agent
(e.g., compound), a process, or a condition, refers to the
capability of such agent, process or condition to, at least in
part, form the microparticles (or a composition or formulation or
kit containing such microparticles), facilitate the formation
thereof, and/or enhance the stability thereof (e.g., the
maintenance of a relatively balanced condition, like increased
resistance against destruction, decomposition, degradation, and the
like). Non-limiting stabilizing processes or conditions include
thermal input/output (e.g., heating, cooling), electromagnetic
irradiation (e.g., gamma rays, X rays, UV, visible light, actinic,
infrared, microwaves, radio waves), high-energy particle
irradiation (e.g., electron beams, nuclear), and ultrasound
irradiation. Non-limiting stabilizing agents include lipids,
proteins, polymers, carbohydrates, surfactants, salts (erg.,
organic, inorganic, with cations that are monovalent or polyvalent,
metallic, organic, or organometallic, and anions that are
monovalent or polyvalent, organic, inorganic, or organometallic),
as well as certain of the carriers, the active agents, the
crosslinkers, the co-agents, and the complexing agents disclosed
herein. The stabilizing agents may be ionic or non-ionic, may be
neutral, positively charged, negatively charged, or zwitterionic,
and may be used singly or in combination of two or more
thereof.
[0055] "Macromolecule" refers to a material capable of providing a
three-dimensional (e.g., tertiary and/or quaternary) structure, and
includes carriers and certain active agents of the present
disclosure. Non-limiting macromolecules used to form the
microparticles include, inter alia, polymers, copolymers, proteins
(edge, enzymes, recombinant proteins, albumins like human serum
albumin), peptides, lipids, carbohydrates, polysaccharides, nucleic
acids, vectors (e.g., virus, viral particles), complexes and
conjugates thereof (egg, by covalent and/or non-covalent
associations, between two macromolecules like carbohydrate-protein
conjugates, between an active agent and a macromolecule like
hapten-protein conjugates, the active agent may or may not be
capable of having a tertiary and/or quaternary structure), and
mixtures of two or more thereof, preferably having a molecular
weight of 1,500 or greater. Macromolecules may be neutral,
positively charged, negatively charged, or zwitterionic, and may be
used singly or in combination of two or more thereof.
[0056] "Spherical" refers to a geometric shape that is at least
"substantially spherical." "Substantially spherical" means that the
ratio of the longest length (i.e., one between two points on the
perimeter and passes the geometric center of the shape) to the
shortest length on any cross-section that passes through the
geometric center is about 1.5 or less, preferably about 1.33 or
less, more preferably 1.25 or less. spherical does not require a
line of symmetry. Further, the microparticles may have surface
texturing (such as continuous or discrete lines, islands, lattice,
indentations, channel openings, protuberances that are small in
scale when compared to the overall size of the microparticles) and
still be spherical. Surface contact there between is minimized in
microparticles that are spherical, which minimizes the undesirable
agglomeration of the microparticles. In comparison, microparticles
that are crystals or flakes typically display significant
agglomeration through ionic and/or non-ionic interactions at
relatively large flat surfaces.
[0057] "Monodisperse size distribution" refers to a preferred
microparticle size distribution in which the ratio of the volume
diameter of the 90.sup.th percentile (i.e., the average particle
size of the largest 10% of the microparticles) to the volume
diameter of the 10.sup.th percentile (i.e., the average particle
size of the smallest 10% of the microparticles) is about 5 or less,
preferably about 3 or less, more preferably about 2 or less, most
preferably about 1.5 to 1. Consequently, "polydisperse size
distribution" refers to one where the diameter ratio described
above is greater than 5, preferably greater than 8, more preferably
greater than 10. In microparticles having a polydisperse size
distribution, smaller microparticles may fill in the caps between
larger microparticles, thus possibly displaying large contact
surfaces and significant agglomeration there between A Geometric
Standard Deviation (GSD) of 2.5 or less, preferably 1.8 or less,
may also be used to indicate a monodisperse size distribution.
Calculation of GSD is known and understood to one skilled in the
art.
[0058] "Amorphous" refers to materials and constructions that are
"substantially amorphous," such as microparticles having multiple
non-crystalline domains (or lacking crystallinity altogether) or
otherwise non-crystalline. Substantially amorphous microparticles
of the present disclosure are generally random solid particulates
in which crystalline lattices constitute less than 50% by volume
and/or weight of the microparticles, or are absent, and include
semi-crystalline micropaticles and non-crystalline microparticles
as understood by one skilled in the art.
[0059] "Solid" refers to a state that includes at least
substantially solid and/or semi-solid, but excludes gel, liquid,
and gas.
[0060] "Preformed microparticle" refers to a microparticle
fabricated using one or more non-limiting methods, such as those
known to one skilled in the art, without surface modification as
described herein, having or capable of having on its outer surface
a net surface electric charge that is positive, negative, or
neutral. A preformed microparticle is also referred to herein as
"core microparticle" or "core." The preformed or core microparticle
typically comprises one or more active agents and, optionally, one
or more carriers, which, independently, may be compartmentalized in
a portion of the preformed or core microparticle or preferably be
distributed substantially homogeneously throughout the preformed
microparticles. The net surface charge, preferably being non-zero,
may be contributed primarily or at least, substantially, by the
active agent(s) and/or the optional carrier(s).
[0061] "Carrier" refers to a compound, typically a macromolecule,
having a primary function to provide a three-dimensional structure
(including tertiary and/or quaternary structure) The carrier may be
unassociated or associated with the active agent (such as
conjugates or complexes thereof) in forming microparticles as
described above. The carrier may further provide other functions,
such as being an active agent, modify release profile of the active
agent from the microparticle, and/or impart one or more particular
properties to the microparticle (such as contribute at least in
part to the net surface charge). In one example, the carrier is a
protein (such as albumins, preferably human serum albumin) having a
molecular weight of 1500 Daltons or greater,
[0062] "Polymer" or "polymeric" refers to a natural, synthetic, or
semi-synthetic molecule having in a main chain or ring structure
two or more repeating monomer units. Polymers broadly include
dimers, trimers, tetramers, oligomers, higher molecular weight
polymer, adducts, homopolymers, random copolymers,
pseudo-copolymers, statistical copolymers, alternating copolymers,
periodic copolymer, bipolymers, terpolymers, quaterpolymers, other
forms of copolymers, substituted derivatives thereof, and mixtures
thereof, and narrowly refer to molecules having 10 or more
repeating monomer units. Polymers may be linear, branched, block,
graft, monodisperse, polydisperse, regular, irregular, tactic,
isotactic, syndiotactic, stereoregular, atactic, stereoblock,
single-strand, double-strand, star, comb, dendritic, and/or
ionomeric, may be ionic or non-ionic, may be neutral, positively
charged, negatively charged, or zwitterionic, and may be used
singly or in combination of two or more thereof
[0063] "Suspension" or "dispersion" refers to a mixture, preferably
finely divided, of two or more phases (e g., solid, liquid, gas),
such as solid in liquid, liquid in liquid, gas in liquid, solid in
solid, solid in gas, liquid in gas, and the like. The suspension or
dispersion may preferably remain stable for extended periods of
time (e.g., minutes, hours, days, weeks, months, years).
[0064] "Resuspending" refers to changing microparticles from a
non-flowable (e.g., solid) state to a flowable (e.g., liquid) state
by adding a flowable medium (e.g., a liquid), while retaining most
or all of the characteristics of the microparticles. The liquid may
be, for example, aqueous, aqueous miscible, or organic.
[0065] "Charged" and "electrically charged" refer interchangeably
to the capability of providing one, two, three, or more formal
units of electrical charges of the same or opposite sign and/or the
presence of such charges (i.e., "charged" refers to chargeable
and/or charged). The electrical charges may be provided by and/or
be present in tile form of one or more of the same or different
organic and/or organometallic moieties (e.g., ionic groups,
ionizable groups, precursors thereof) in the compound (e.g.,
polyelectrolytes, proteins) or the structure (e.g., the preformed
microparticle, the monolayer's) of interest, preferably when
subjected to certain conditions (such as in solution or
suspension).
[0066] "Charged compound" and "electrically charged compound" refer
interchangeably to a single compound that is charged as described
above, or a combination of two or more different compounds in
unassociated and/or associated forms (e.g., conjugates, aggregates,
and/or complexes thereof), each of which independently has and/or
is capable of having a net charge of the same sign.
[0067] "Monolayer" refers to a single layer formed over a
three-dimensional substrate, from a composition of one or more
compounds (such as a charged compound as described above). The
monolayer may be a continuous and nonporous monolayer, a continuous
and porous monolayer (such as a lattice network), a non-continuous
monolayer of a plurality of discrete elements (e.g., islands,
strips, clusters, etch), or a combination thereof. Typically, the
monolayer will be decomposable or degradable, such as
biodegradable, enzymatically or hydrolytically degradable and the
like, to allow for non-diffusional release of an active agent (from
the microparticle) over which the monolayer is deposited The
monolayer may have a thickness of 100 nm or less, preferably 50 nm
or less, more preferably 20 nm or less, most preferably 10 nm or
less. In one example, the monolayer is formed through self-assembly
of a charged compound.
[0068] "Saturated monolayer" refers to a monolayer as defined above
that is incapable of further incorporating, cumulatively, an excess
amount of the composition forming the monolayer when subjected to
the same set of conditions under which the monolayer is formed.
Saturated monolayers are preferred monolayers used in
surface-modified microparticles.
[0069] "Net charge" and "net electric charge" are used
interchangeably and refer to the sum of all formal units of
electric charge a charged compound is capable of having or has,
such as in a flowable medium under certain conditions (preferably
in a solution of certain pH). The net charge may be positive,
negative, or zero (such as in zwitterionic compounds), and is
condition-dependent (erg., solvent, pH).
[0070] "Net surface charge" and "net surface electric charge" are
used interchangeably and refer to an overall cumulative electric
charge on an outermost surface of a three-dimensional structure
(e.g., a microparticle, a monolayer). The net surface charge may be
positive, negative, or zero, and is condition-dependent (e.g.,
solvent, pH).
[0071] "Ambient temperature" refers to a temperature of around room
temperature, typically in a range of about 20.degree. C. to about
40.degree. C.
[0072] "Subject" or "patient" refers to animals, including
vertebrates like mammals, preferably humans.
[0073] "Region of a subject" refers to a localized internal or
external area or portion of the subject (e.g., an organ), or a
collection of areas or portions throughout the entire subject
(e.g., lymphocytes). Non-limiting examples of such regions include
pulmonary region (e.g., lung, alveoli, gastrointestinal region
(e.g., regions defined by esophagus, stomach, small large
intestines, and rectum), cardiovascular region (e.g., myocardial
tissue), renal region (e.g., the region defined by the kidney, the
abdominal aorta, and vasculature leading directly to and from the
kidney), vasculature (i.e., blood vessels, e.g., arteries, veins,
capillaries, and the like), circulatory system, healthy or diseased
tissues, benign or malignant (e.g., tumorous or cancerous) tissues,
lymphocytes, receptors, organs and the like, as well as regions to
be imaged with diagnostic imaging, regions to be administered
and/or treated with an active agent, regions to be targeted for the
delivery of an active agent, and regions of elevated
temperature,
[0074] "Therapeutic" refers to any pharmaceutic, drug, prophylactic
agent, contrast agent, or dye useful in the treatment (including
prevention, diagnosis, alleviation, suppression, remission, or
cure) of a malady, affliction, disease or injury in a subject.
Therapeutically useful peptides and nucleic acids may be included
within the meaning of the term "pharmaceutic" or "drug."
[0075] "Affinity molecule" refers to any material or substance
capable of promoting binding and/or targeting of regions in vivo
and/or tissues/receptors in vitro. Affinity molecules, including
receptors and targeting ligands, may be natural, synthetic, or
semi-synthetic, may be ionic or non-ionic, may be neutral,
positively charged, negatively charged, or zwitterionic, and may be
used singly or in combination of two or more thereof. Non-limiting
affinity molecules include proteinaceous compounds (e.g.,
antibodies, antibody fragments, hormones, hormone analogues,
glycoproteins and lectins), peptides, polypeptides, amino acids,
sugars, saccharides (egg monosaccharides, polysaccharides,
carbohydrates), vitamins, steroids, steroid analogs, cofactors,
active agents, nucleic acids, viruses, bacteria, toxins, antigens,
other ligands, precursors thereof, and derivatives thereof.
[0076] "Precursor" refers to any material or substance capable of
being converted to a desired material or substance, preferably
through a chemical and/or biochemical reaction or pathway, such as
anchoring a precursor to a material. Non-limiting precursor
moieties include maleimide groups, disulfide groups (e.g.,
ortho-pyridyl disulfide), vinylsulfone groups, azide groups, and
.alpha.-iodo acetyl groups
[0077] "Derivative" refers to any material or substance formed from
a parent material or substance, preferably through a chemical
and/or biochemical reaction or pathway considered routine by one of
ordinary skill in the art. Non-limiting examples of derivatives
include glycosylated, hyperglycosylated, PEGylated, FITC-labelled,
protected with protecting groups (e.g., benzyl for alcohol or
thiol, t-butoxycarbonyl for amine), as well as salts, esters,
amides, conjugates, complexes, manufacturing related compounds, and
metabolites thereof. Salts may be organic or inorganic, with
cations that are monovalent or polyvalent, metallic, organic, or
organometallic, and anions that are monovalent or polyvalent,
organic, inorganic, or organometallic. Preferred salts are
pharmaceutically acceptable, and include, without limitation,
mineral or organic acid salts of basic residues (e.g., amines),
alkali or organic salts of acidic residues (e.g., carboxylic
acids), and the like, such as conventional nontoxic salts or the
quaternary ammonium salts of the parent compound formed from
non-toxic inorganic acids (e.g., hydrochloric, hydrobromic,
sulfuric, sulfamic, phosphoric, nitric) and organic acids (e.g.,
acetic, propionic, succinic, glycolic, stearic, lactic, malic,
tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,
phenylacetic, glutamic, benzoic, salicylic, sulfanilic,
2-acetoxybenzoic, famaric, toluensulfonic, methanesulfonic, ethane
dislfonic, oxalic, isethionic).
[0078] "Analog" refers to a compound having a chemically modified
form of a principle compound or class thereof, which maintains the
pharmaceutical and/or pharmacological activities characteristic of
the principle compound or class
[0079] "Prodrug" refers to any covalently bonded carriers that
release an active agent in vivo when administered to a subject.
Prodrugs are known to enhance numerous desirable qualities (e.g.,
solubility, bioavailability, manufacturing) of the active agents
Prodrugs may be prepared by modifying functional groups (e.g.,
hydroxy, amino, carboxyl, and/or sulfhydryl groups) present in the
active agent in such a way that the modifications are reversed (e
g., modifier group cleaved), either in routine manipulation or in
vivo, to afford the original active agent. The transformation in
vivo may be, for example, as the result of some metabolic process,
such as chemical or enzymatic hydrolysis of a carboxylic,
phosphoric or sulphate ester, or reduction or oxidation of a
susceptible functionality.
[0080] "Metabolite" refers to a form of a compound obtained in a
subject body by action of the body on the administered form of the
compounds. For example, a demethylated metabolite may be obtained
in the body after administration of a methylated compound bearing a
methyl group. Metabolites may themselves have biological,
preferably therapeutic, activities.
[0081] "Diagnostic agent" refers to any material or substance
useful in connection with methods for perceptually observing (e.g.,
imaging) a normal or abnormal biological condition or state, or
detecting the presence or absence of a pathogen or a pathological
condition. Non-limiting diagnostic agents include contrast agents
and dyes for use in connection with radiography imaging (e.g.,
X-ray imaging), ultrasound imaging, magnetic resonance imaging,
computed tomography, positron emission tomography imaging, and the
like. Diagnostic agents further include any other agents useful in
facilitating diagnosis in vivo and/or in vitro, whether or not
imaging methodology is employed.
[0082] "Cross-link," "cross-linked" and "cross-linking" generally
refer to the lining of two or more materials and/or substances,
including any of those disclosed herein, through one or more
covalent and/or non-covalent (e.g., ionic) associations.
Cross-linking may be effected naturally (e.g., disulfide bonds of
cystine residues) or through synthetic or semi-synthetic routes,
for example, optionally in the presence of one or more
cross-linkers (i.e., a molecule X by itself capable of reacting
with two or more materials/substances Y and Z to form a cross-link
product Y--X-Z, where the associations of Y--X and X-Z are
independently covalent and/or non-covalent), initiators (i-e., a
molecule by itself capable of providing reactive species like flee
radicals for the cross-link reaction, e g., thermally decomposable
initiators like organic peroxides, azo initiators, and
carbon-carbon initiators, actinically decomposable initiators like
photoinitiators of various wavelengths), activators (i e., a
molecule A capable of reacting with a first material/substance Y to
form an activated intermediate [A-Y], which in turn reacts with a
second material/substance Z to form a cross-link product Y-Z, while
A is chemically altered or consumed during the process), catalysts
(i.e., a molecule capable of modifying the kinetics of the
cross-link reaction without being chemically modified during the
process), co-agents (i.e., a molecule that, when co-present with
one or more of the initiators, activators, and/or catalysts, is
capable of modifying the kinetics of the cross-link reaction and/or
being incorporated into the cross-link product of the two or more
materials/substances, but otherwise is non-reactive to the
materials/substances), and/or energy sources (edge, heating;
cooling; high-energy radiations like electromagnetic, e-beam, and
nuclear; acoustic radiations like ultrasonic; etc.).
[0083] "Covalent association" refers to an intermolecular
interaction (e.g., a bond) between two or more individual molecules
that involves the sharing of electrons in the bonding orbitals of
two atoms.
[0084] "Non-covalent association" refers to an intermolecular
interaction between two or more individual molecules without
involving a covalent bond. Intermolecular interaction depends on,
for example, polarity, electric charge, and/or other
characteristics of the individual molecules, and includes, without
limitation, electrostatic (e.g., ionic) interactions, dipole-dipole
interactions, van der Waal's forces, and combinations of two or
more thereof.
[0085] "Electrostatic interaction" refers to an intermolecular
interaction between two or more positively or negatively charged
moieties/groups, which may be attractive when two are oppositely
charged (i.e., one positive, another negative), repulsive when two
charges are of the same sign (i.e., two positive or two negative),
or a combination thereof.
[0086] "Dipole-dipole interaction" refers an intermolecular
attraction between two or more polar molecules, such as a first
molecule having an uncharged, partial positive end .delta..sup.+
(e.g., electropositive head group like the choline head group of
phosphatidylcholine) and a second molecule having an uncharged,
partial negative end .delta..sup.- (e.g., an electronegative atom
like heteroatom O, N, or S in a polysaccharide). Dipole-dipole
interaction also refers to intermolecular hydrogen bonding in which
a hydrogen atom serves as a bridge between electronegative atoms on
separate molecules and in which a hydrogen atom is held to a first
molecule by a covalent bond and to a second molecule by
electrostatic forces.
[0087] "Hydrogen bond" refers to an attractive force or bridge
between a hydrogen atom covalently bonded to a first
electronegative atom (e.g., O, N, S) and a second electronegative
atom, where the first and second electronegative atoms may be in
two different molecules (intermolecular hydrogen bonding) or in a
single molecule (intramolecular hydrogen bonding).
[0088] "Van der Waal's forces" refers to the attractive forces
between non-polar molecules that are accounted for by quantum
mechanics. Van der Waal's forces are generally associated with
momentary dipole moments induced by neighboring molecules
undergoing changes in electron distribution.
[0089] "Hydrophilic interaction" refers to an attraction toward
water molecules, where a material/compound or a portion thereof may
bind with, absorb, and/or dissolve in water. This may result in
swelling and/or the formation of reversible hydrogels.
[0090] "Hydrophobic interaction" refers to a repulsion against
water molecules, where a material/compound or a portion thereof do
not bind with, absorb, or dissolve in water.
[0091] "Biocompatible" refers to materials/substances that are
generally not injurious to biological functions and do not result
in unacceptable toxicity (e.g., allergenic responses or disease
states).
[0092] "In association with" and "associated with" refer in general
to the one or more interactions between, and/or incorporation of,
different materials (typically those that are part of the
microparticles), one or more of such materials and one or more
structures (or portions thereof) of the microparticles, and
different structures (or portions thereof) of the microparticles.
The materials of the microparticles include, without limitation,
ions such as monovalent and polyvalent ions disclosed herein, as
well as compounds such as active agents, stabilizing agents,
cross-link agents, charged or uncharged compounds, the various
polymers disclosed herein, and combinations of two or more thereof.
The structures of the microparticles and portions thereof include,
without limitation, core, core microparticle, preformed
microparticle, monolayer, intermediate microparticle,
surface-modified microparticle, portions of such structures (such
as outer surfaces, inner surfaces), domains between such structures
and portions thereof, and combinations of two or more thereof.
Various associations, being reversible or irreversible. Migratory
or non-migratory, may be present singly or in combination of two or
more thereof. Non-limiting associations include, without
limitation, covalent and/or non-covalent associations (e.g.,
covalent bonding, ionic interactions, electrostatic interactions,
dipole-dipole interactions, hydrogen bonding, van der Waal's
forces, cross-linking, and/or any other interactions),
encapsulation in layer/membrane, compartmentalization in center or
vesicles or between two layers/membranes, homogeneous integration
throughout the microparticle or in a portion thereof (e.g.,
containment in, adhesion to, and/or affixation to center or layer
or vesicle or an inner and/or outer surface thereof; interspersion,
conjugations, and/or complexation between different materials).
[0093] "Tissue" refers generally to an individual cell or a
plurality or aggregate of cells specialized and capable of
performing one or more particular functions. Non-limiting, tissue
examples include membranous tissues, (e.g., endothelium,
epithelium), blood, laminae, connective tissue (e.g., interstitial
tissue), organs (e.g., myocardial tissue, myocardial cells,
cardiomyocites), abnormal cell(s) (e.g., tumors).
[0094] "Receptor" refers to a molecular structure within a cell or
on its surface, generally characterized by its selective binding of
a specific substance, e.g., ligand. Non-limiting receptors include
cell-surface receptors for peptide hormones, neurotransmitters,
antigens, complement fragments, and immunoglobulins, and
cytoplasmic receptors for steroid hormones.
[0095] "Controlled release" refers to a predetermined in vivo
and/or in vitro release (e.g., dissolution) profile of an active
agent, as compared to the release profile of the active agent in
its native form. The active agent is preferably associated with a
microparticle or a composition or formulation containing such a
microparticle, as disclosed herein, such that one or more aspects
of its release kinetics (e.g., initial burst, quantity and/or rate
over a specified time period or phase, cumulative quantity over a
specific time period, length of time for total release, pattern
and/or profile, etc.) are increased, decreased, shortened,
prolonged, and/or otherwise modified as desired. Non-limiting
examples of controlled release include immediate/instant release
(i.e., initial burst or rapid release), extended release, sustained
release, prolonged release, delayed release, modified release,
and/or targeted release, occurring individually, in combination of
two or more thereof or in the absence of one or more thereof (e. g.
extended or sustained release in the absence of an initial
burst).
[0096] "Extended release" refers to the release of an active agent,
preferably in association with a microparticle or a composition or
formulation containing such a microparticle, as disclosed herein,
over a time period longer than the free aqueous diffusion period of
the active agent in its native form. The extended release period
may be hours (e.g., at least about 1, 2, 5, or 10 hours), days
(e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 30, 40,
45, 60, or 90 days), weeks (at least about 1, 2, 3, 4, 5, 6, 10,
15, 20, 30, 40, or 50 weeks), months (at least about 1, 2, 3, 4, 6,
9, or 12 months), about 1 or more years, or a range between any two
of the time periods. The pattern of an extended release may be
continuous, periodic, sporadic, or a combination thereof.
[0097] "Sustained release" refers to an extended release of an
active agent such that a functionally significant level of the
active agent (i.e., a level capable of bring about the desired
function of the active agent) is present at any time point of the
extended release period, preferably with a continuous and/or
uniform release pattern. Non-limiting examples of sustained release
profiles include those, when displayed in a plot of release time
(x-axis) versus cumulative release (y-axis), showing at least one
upward segment that is linear, step-wise, zig-zagging, curved,
and/or wavy, over a time period of 1 hour or longer.
[0098] Other than in the operating examples, or unless otherwise
expressly specified, all of the numerical ranges, amounts, values
and percentages such as those for quantities of materials, times,
temperatures, reaction conditions, ratios of amounts, values for
molecular weight (whether number average molecular weight M.sub.n
or weight average molecular weight M.sub.w), and others disclosed
herein should be understood as modified in all instances by the
term "about." Accordingly, unless indicated to the contrary, the
numerical parameters set forth in the present disclosure and
attached claims are approximations that may vary as desired. At the
very least, each numerical parameter should at least be construed
in light of the number of reported significant digits and by
applying ordinary rounding techniques.
[0099] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the disclosure are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Furthermore, when numerical ranges of varying scope are set forth
herein, it is contemplated that any combination of these values
inclusive of the recited values may be used.
[0100] "Formed from" and "formed of" denote open, language. As
such, it is intended that a composition "formed from" or "formed
of" a list of recited components be a composition comprising at
least these recited components, and can further include other
non-recited components during formulation of the composition
[0101] Examples provided herein, including those following "such
as" and "e.g.," are considered as illustrative only of various
aspects of the present disclosure and embodiments thereof, without
being specifically limited thereto. Any suitable equivalents,
alternatives, and modifications thereof (including materials,
substances, constructions, compositions, formulations, means,
methods, conditions, etc.) known and/or available to one skilled in
the art may be used or carried out in place of or in combination
with those disclosed herein, and are considered to fall within the
scope of the present disclosure,
[0102] In one example, each of the surface-modified microparticles
of the present disclosure preferably contains an amorphous (e.g.,
such as free of crystalline structures) and solid preformed
microparticle associated, at least at its outer surface, with at
least one monolayer containing at least one charged compound. The
preformed microparticle contains at least one active agent and/or
at least one macromolecule having a molecular weight of 4,500
Daltons or greater. The macromolecule may be the active agent or
may be different from the active agent. The macromolecule may be a
carrier, a stabilizing agent, or a complexing agent (e.g.,
proteinaceous compounds, polyelectrolytes). The active agent and/or
the macromolecule may constitute 40% to 100% or less, and typically
at least 80%, such as 90% or more or 95% or more, by weight of the
preformed microparticle. Preferably, the active agent and/or the
macromolecule is/are distributed homogeneously throughout the core
microparticle. An outer surface of the preformed microparticle
carries a net surface charge, which may be attributed, at least in
part, and more typically in large part, to the active agent and/or
the macromolecule, especially when the outer surface is formed of
the active agent and/or the macromolecule. The preformed
microparticle may be free of covalent crosslinking, hydrogel,
lipids, and/or encapsulation. Alternatively, the preformed
microparticle may contain one or more charged compounds, covalent
crosslinking, and/or encapsulation. The one or more charged
compounds in the preformed microparticle may be distributed
homogeneously throughout the preformed microparticle, or
compartmentalized in specific portions thereof, such as in a layer.
The preformed microparticle may preferably have a particle size of
10 .mu.m or less, and may have a monodisperse or polydisperse size
distribution.
[0103] Methods of pre-forming the preformed microparticle are not
particularly limiting, and include those disclosed in U.S. Pat. No.
6,458,387 and U.S. Patent Publication No. 2005/0142206, which are
incorporated herein by reference in their entirety. In one example,
a single flowable continuous phase system (such as liquid, gas, or
plasma, preferably a solution or suspension) is formulated to
contain one or more active agents, a medium, and one or more
phase-separation enhancing agents (PSEAs) The medium is preferably
a liquid solvent (e.g., hydrophilic or hydrophobic organic
solvents, water, buffers, aqueous-miscible organic solvents, and
combinations of two or more thereof), more preferably an aqueous or
aqueous-miscible solvent. Suitable organic solvents include,
without limitation, methylene chloride, chloroform, acetonitrile,
ethylacetate, methanol, ethanol, pentane, the likes thereof, and
combinations of two or more thereof (such as a 1:1 mixture of
methylene chloride and acetone). The active agent and the PSEA may
independently be dissolved, suspended, or otherwise homogeneously
distributed within the medium. When subjecting the flowable system
to certain conditions (such as a temperature below the phase
transition temperature of the active agent in the medium), the
active agent undergoes a liquid-solid phase separation and forms a
discontinuous, preferably solid, phase (such as a plurality of core
microparticles suspended in the medium), while the PSEA remains in
the continuous phase (such as being dissolved in the medium).
[0104] The medium can be organic, containing an organic solvent or
a mixture of two or more inter-miscible organic solvents, which may
independently be aqueous-miscible or aqueous-immiscible. The
solution can also be an aqueous-based solution containing an
aqueous medium or an aqueous-miscible organic solvent or a mixture
of aqueous-miscible organic solvents or combinations thereof. The
aqueous medium can be water, a buffer (e.g., normal saline,
buffered solutions, buffered saline), and the like. Suitable
aqueous-miscible organic solvents may be monomers or polymers, and
include, but are not limited to, N-methyl-2-pyrrolidinone
(N-methyl-2-pyrrolidone), 2-pyrrolidinone (2-pyrrolidone),
1,3-dimethyl-2-imidazolidinone (DMI), dimethylsulfoxide,
dimethylacetamide, acetic acid, lactic acid, acetone, methyl ethyl
ketone, acetonitrile, methanol, ethanol, n-propanol, isopropanol,
3-pentanol, benzyl alcohol, glycerol, tetrahydrofuran (THF),
polyethylene glycol (PEG, e.g., PEG-4, PEG-8, PEG-9, PEC-12,
PEG-14, PEG-16, PEG-120, PEG-75, PEG-150), PEG esters (e.g., PEG-4
dilaurate, PEG-20 dilaurate, PEG-6 isostearate, PEG-8
palmitostearate, PEG-150 palmitostearate), PEG sorbitans (such as
PEG-20 sorbitan isostearate), PEG ethers (such as monoalkyl and
dialkyl ethers, e.g., PEG-3 dimethyl ether, PEG-4 dimethyl ether,
and glycofurol), polypropylene glycol (PPS), PPG esters (such as
polypropylene glycol alginate (PGA), PPG dicaprylate, PPG
dicaprate, PPG laurate), alkoxylated linear alkyl diols (such as
PPG-10 butanediol), alkoxylated alkyl glucose ether (e.g., PPG-10
methyl glucose ether, PPG-20 methyl glucose ether), PPG alkyl
ethers (such as PPG-15 stearyl ether), alkanes (e.g., propane,
butane, pentane, hexane, heptane, octane, nonane, decane), and
combinations of two or more thereof.
[0105] In a preferred example, a solution of the PSEA in a first
solvent is provided, in which the PSEA is soluble in or miscible
with the first solvent. The active agent is mixed in, directly or
as a second solution in a second solvent, with the first solution.
The first and second solvent may be the same or at least miscible
with each other. Preferably the active agent is added at a
temperature equal to or lower than ambient temperature,
particularly when the active agent is a heat labile molecule such
as certain proteinaceous compounds. However, the system may be
heated to increase solubility of the active agent in the system, as
long as the activity of the active agent is not adversely
affected.
[0106] When the mixture is brought to phase separation conditions,
the PSEA, while remaining in the liquid continuous phase, enhances
and/or induces a liquid-solid phase separation of the active agent
from the solution (such as by reducing solubility of the active
agent), forming the core microparticles (the solid discontinuous
phase), which may preferably be microspheres. Suitable PSEA
compounds include, but are not limited to, natural and synthetic
polymers, linear polymers, branched polymers, cyclo-polymers,
copolymers (random, block, grafted, such as poloxamers,
particularly PLURONIC.RTM. F127 and F68), terpolymers, amphiphilic
polymers, carbohydrate-based polymers, polyaliphatic alcohols,
poly(vinyl)polymers, polyacrylic acids, polyorganic acids,
polyamino acids, polyethers, polyesters, polyimides, polyaldehydes,
polyvinylpyrrolidone (PVP), and surfactants. Suitable or exemplary
PSEAs include, without limitation, polymers acceptable as
pharmaceutical additives, such as PEGs (e.g., PEG 200, PEG 300, PEG
3350, PEG 8000, PEG 10000, PEG 20000, etc,), poloxamers, PVP,
hydroxyethylstarch, amphiphilic polymers, as well as non-polymers
(such as mixtures of propylene glycol and ethanol).
[0107] Conditions capable of enhancing, inducing, promoting,
controlling, suppressing, retarding, or otherwise affecting the
liquid-solid phase separation include, without limitation, changes
in temperature, pressure, pH, ionic strength and/or osmolality of
the solutions, concentrations of the active agent and/or the PSEA,
the likes thereof, as well as rates of such changes, and
combinations of two or more thereof. Such conditions may desirably
be applied before and up to the phase separation, or even during
the phase separation. In one example, the system is exposed to a
temperature below the phase transition temperature of the active
agent therein, alone or in combination with adjustments to the
concentrations of the active agent and/or the PSEA, as described in
U.S. Patent Application Publication 2005/0142206, the entire
disclosure of which is incorporated herein by reference. The rate
of temperature drop may be held constant or altered in any
controlled manner, as long as it is within a range of 0.2.degree.
C./minute to 50.degree. C./minute, preferably 0.2.degree. C./minute
to 30.degree. C./minute. Freezing point depressing agents (FPDAs),
used individually or in combination of two or more thereof, may be
mixed in the system directly or in solutions (such as aqueous
solutions) thereof, particularly for systems in which the freezing
point is higher than the phase transition temperature of the active
agent. Suitable FPDAs include, without limitation, propylene
glycol, sucrose, ethylene glycol, alcohols (e.g., ethanol,
methanol), and aqueous mixtures thereof.
[0108] In one example, the preformed microparticles may further
comprise one or more excipients that negligibly affect the phase
separation. The excipient may imbue the core microparticles and/or
the compounds therein (e.g., the active agent, the optional
carrier) with additional characteristics such as increased
stability, controlled release of the active agent from the
preformed microparticles, and/or modified permeation of the active
agent through biological tissues. Suitable excipients include, but
are not limited to, carbohydrates (e.g., trehalose, sucrose,
mannitol), polyvalent cations (preferably metal cations, e.g.,
Zn.sup.2+, Mg.sup.2+, Ca.sup.2+, Cu.sup.2+, Fe.sup.2+, Fe.sup.3+),
anions (e.g., CO.sub.3.sup.2-, SO.sub.4.sup.2-), amino acids (such
as glycine), lipids, phospholipids, fatty acids and esters thereof,
surfactants, triglycerides, bile acids and conjugates and salts
thereof (e.g., cholic acid, deoxycholic acid, glycocholate,
taurocholate, sodium cholate), and any polymers disclosed
herein.
[0109] The preformed microparticles may optionally be separated
from the solution and washed prior to the surface modification as
disclosed herein, or be surface-modified without separation or
washing. Separation means include, without limitation,
centrifugation, dialysis, sedimentation (creaming), phase
separation, chromatography, electrophoresis, precipitation,
extraction, affinity binding, filtration, and diafiltration. For
active agents with relatively low aqueous solubility, the washing
medium may be aqueous, optionally containing one or more solubility
reducing agents (SRAs) and/or excipients as disclosed herein
Preferred SRAs are capable of forming insoluble complexes with the
active agents and/or carriers in the microparticles, and include,
without limitation, compounds such as salts that comprise divalent
or polyvalent cations (such as those disclosed herein). For active
agents with relatively high aqueous solubility (such as
proteinaceous compounds), the washing medium may be organic, or
aqueous but containing at least one SRA or precipitating agent
(such as ammonium sulfate). In one example, the washing medium is
the same solution used in the phase separation reaction, such as an
aqueous solution including approximately 16% (w/v) PEG and 0.7%
(w/v)
[0110] It is preferred that the washing medium has a low boiling
point for easy removal by, for example, lyophilization,
evaporation, or drying The washing medium may be a supercritical
fluid or a fluid near its supercritical point, used alone or in
combination with a co-solvent. Supercritical fluids may be solvents
for the PSEAs, but not for the preformed microparticles.
Non-limiting examples of supercritical fluids include liquid
CO.sub.2, ethane, and xenon. Non-limiting examples of co-solvents
include acetonitrile, dichloromethane, ethanol, methanol, water,
and 2-propanol.
[0111] As indicated above, active agents with varying degrees of
solubility in water may be employed in the microparticles described
herein. While water insoluble active agents may be used, water
soluble active agents are preferred.
[0112] The active agent may be a pharmaceutical agent. Depending on
its effect and/or application, the pharmaceutical agent includes,
without limitation, adjuvants, adrenergic agents, adrenergic
blocking agents, adrenocorticoids, adrenolytics, adrenomimetics,
alkaloids, alkylating agents, allosteric inhibitors, anabolic
steroids, analeptics, analgesics, anesthetics, anorexiants,
antacids, anthelmintics, anti-allergic agents, antiangiogenesis
agents, anti-arrhythmic agents, anti-bacterial agents, antibiotics,
antibodies, anticancer agents. anticholinergic agents,
anticholinesterases, anticoagulants, anticonvulsants, antidementia
agents, antidepressants, antidiabetic agents, antidiarrheals,
antidotes, antiepileptics, antifolics, antifungals, antigens,
antihelmintics, antihistamines, antihyperlipidemics,
antihypertensive agents, anti-infective agents, anti-inflammatory
agents, antimalarials, antimetabolites, antimuscarinic agents,
antimycobacterial agents, antineoplastic agents, antiosteoporosis
agents, antipathogen agents, antiprotozoal agents, adhesion
molecules, antipyretics, antirheumatic agents, antiseptics,
antithyroid agents, antiulcer agents, antiviral agents, anxioltic
sedatives, astringents, beta-adrenoceptor blocking agents,
biocides, blood cloning factors, calcitonin, cardiotonics,
chemotherapeutics, cholesterol lowering agents, cofactors,
corticosteroids, cough suppressants, cytokines, diuretics,
dopaininergics, estrogen receptor modulators, enzymes and cofactors
thereof enzyme inhibitors, growth differentiation factors, growth
factors, hematological agents, hematopoietics, hemoglobin
modifiers, lemostatics, hormones and hormone analogs, hypnotics,
hypotensive diuretics, immunological agents, immunostimulants,
immunosuppressants, inhibitors, ligands, lipid regulating agents,
lymphokines, muscarinics, muscle relaxants, neural blocking agents,
neurotropic agents, paclitaxel and derivative compounds,
parasympathomimetics, parathyroid hormone, piromotors,
prostaglandins, psychotherapeutic agents, psychotropic agents,
radio-pharmaceuticals, receptors, sedatives, sex hormones,
sterilants, stimulants, thrombopoietics, trophic factors,
sympathonmimetics, thyroid agents, vaccines, vasodilators,
vitamins, xanthines, as well as conjugates, complexes, precursors,
and metabolites thereof. The active agent may be used individually
or in combinations of two or more thereof In one example, the
active agent is a prophylactic and/or therapeutic agent that
includes, but is not limited to, peptides, carbohydrates, nucleic
acids, other compounds, precursors and derivatives thereof, and
combinations of two or more thereof
[0113] As discussed above, the active agent may be a cosmetic
agent. Non-limiting cosmetic agents include inter-alia emollients,
humectants, free radical inhibitors, anti-inflammatories, vitamins,
depigmenting agents, anti-acne agents, antiseborrhoeics,
keratolytics, slimming agents, skin coloring agents and sunscreen
agents. Non-limiting compounds useful as cosmetic agents include
linoleic acid, retinol, retinoic acid, ascorbic acid alkyl esters,
polyunsaturated fatty acids, nicotinic esters, tocopherol
nicotinate, unsaponifiables of rice, soybean or shea, ceramides,
hydroxy acids such as glycolic acid, selenium derivatives,
antioxidants, beta-carotene, gamma-orizanol and stearyl glycerate.
The cosmetic agents may be commercially available and/or prepared
by known techniques.
[0114] As discussed above, the active agent may be a nutritional
supplement. Non-limiting nutritional supplements include proteins,
carbohydrates, water-soluble vitamins (e.g., vitamin C, B-complex
vitamins, and the like), fat-soluble vitamins (e.g., vitamins A, D,
E, K, and the like), and herbal extracts. The nutritional
supplements may be commercially available and/or prepared by known
techniques.
[0115] As discussed above, the active agent may be a compound
having a molecular weight of 2 kD or less. Non-limiting examples of
such compounds include steroids, beta-agonists, anti-microbials,
antifungals, taxanes (antimitotic and antimicrotubule agents),
amino acids, aliphatic compounds, aromatic compounds, and urea
compounds
[0116] In one example, the active agent may be a therapeutic agent
for prevention and/or treatment of pulmonary disorders.
Non-limiting examples of such agents include steroids,
beta-agonists, anti-fungals, anti-microbial compounds, bronchial
dialators, anti-asthmatic agents, non-steroidal anti-inflammatory
agents (NSAIDS), AAT, and agents to treat cystic fibrosis.
Non-limiting examples of steroids include beclomethasone (such as
beclomethasone dipropionate), fluticasone (such as fluticasone
propionate), budesonide, estradiol, fludrocortisone, flucinonide,
triamcinolone (such as triamcinolone acetonide), flunisolide, and
salts thereof. Non-limiting examples of beta-agonists include
salmeterol xinafoate, formoterol fumarate, levo-albuterol,
bambuterol, tulobuterol, and salts thereof. Non-limiting examples
of anti-fungal agents include itraconazole, fluconazole,
amphotericin B, and salts thereof.
[0117] As discussed above, the active agent may be a diagnostic
agent. Non-limiting diagnostic agents include x-ray imaging agents
and contrast media. Non-limiting examples of x-ray imaging agents
include ethyl 3,5-diacetamido-2,4,6-triiodobenzoate (WIN-8883,
ethyl ester of diatrazoic acid);
6-ethoxy-6-oxohexyl-3,5-bis(acetamido)-2,4,6-triiodobenzoate (WIN
67722);
ethyl-2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy)-butyrate (WIN
16318); ethyl diatrizoxyacetate (WIN 12901); ethyl
2-(3,5-bis(acetamido)-7,4,6-triiodobenzoyloxy)propionate (WIN
16923); N-ethyl
2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy-acetamide (WIN
65312); isopropyl
2-(3,5-bis(acetamide)-2,4,6-triiodobenzoyloxy)acetamide (WIN
12855); diethyl
2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxymalonate (WIN 67721);
ethyl 2-(3,5-bis(acetamido)-2,4,6-triiodobenzoyloxy)phenyl-acetate
(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-triodo-4-(ethyl-3-ethoxy-2-butenoate)ester
(WIN 68209). Preferred contrast agents desirably disintegrate
relatively rapidly under physiological conditions, thus minimizing
any particle associated inflammatory response. Disintegration may
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 or others may be preferred.
[0118] As discussed above, the active agents may be used in a
combination of two or more thereof. Non-limiting examples include a
steroid and a beta-agonist, e.g., fluticasone propionate and
salmeterol, budesonide and formeterol, etc
[0119] Preformed microparticles may be substantially free of
internal voids and/or cavities (such as being free of vesicles),
substantially free of encapsulation, substantially free of lipids,
substantially free of hydrogel or swelling, substantially and
non-porous, amorphous solid, and/or spherical as those terms are
defined herein. Preformed microparticles may have multiple surface
channel openings, the diameter of which are generally 100 nm or
less, preferably 10 nm or less, more preferably 5 nm or less, most
preferably 1 nm or less. Preformed microparticles may have an
overall density of 0.5 g/cm.sup.3 or greater, preferably 0.75
g/cm.sup.3 or greater, more preferably 0-85 g/cm.sup.3 or greater.
The density may be generally up to about 2 g/cm.sup.3, preferably
1.75 g/cm.sup.3 or less, more preferably 1.5 g/cm.sup.3 or
less.
[0120] Preformed microparticles may exhibit a high payload of the
at least one active agent. Depending on the formulation and the
physical/chemical nature of the compounds, there are typically at
least 1000 or more, such as a few million to hundreds of millions
of the active agent molecules in each of the preformed
microparticles. The weight percentage of the active agent in the
preformed microparticle may be any of the amount below or greater,
or any ranges there between, but less than 100%: 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98%, 99%. While incorporation of a significant amount of
hulking agents and/or other excipients is not required in the
preformed microparticles, one or more of such compounds may be
present therein. In any event, the desired integrity and/or
activity are retained for a majority (50% or greater, preferably
75% or greater, more preferably 90% or greater, most preferably 95%
or greater) of the active agent, if not 100%.
[0121] Surface modification of the preformed mictoparticles is
achieved, without limitation, by forming, in a controlled manner,
at least one monolayer containing at least one charged compound
about the preformed microparticle. When two or more such monolayers
are formed, each contains different charged compounds, and
preferably each carries on its outer surface a net surface charge
that is different in sign and/or value from that of the preceding
one and/or the subsequent one, if present. Deposition of such
monolayers one at a time allows for optimal control over various
properties of the resulting microparticles, allowing one to tailor
or "fine-tune" the microparticles to achieve a desired result.
[0122] Preferably, the monolayer immediately about the performed
microparticle ("formed monolayer") contains one or more charged
compounds, each independently having a net charge that is opposite
in sign to the net surface charge of the core microparticle. The
preformed micropartiele may at least, in part, be penetrable by the
charged compound in the formed monolayer An outer surface of the
formed monolayer may carry a net surface charge that is different
from, preferably opposite in sign to, that of the preformed
microparticle outer surface, especially when the formed monolayer
is a saturated monolayer as defined herein. The charged compounds
may include one or more of polyelectrolytes, charged
polyaminoacids, charged polysaccharides, polyionic polymers,
charged proteinaceous compounds, charged peptides, charged lipids
optionally in combination with uncharged lipids, charged lipid
structures, and derivatives thereof.
[0123] The surface-modified microparticle may further contain one
or more additional alternatingly charged monolayers, such that the
surface-modified microparticle has a desired release profile of the
active agent. This number is not particularly limited, but may
typically be between 1 to 7, such as 2, 3, 4, 5, or 6. Optionally,
one or more of such charged monolayers may independently have one
or more of the same or different active agents, such as an affinity
molecule, especially a targeting ligand, associated covalently
and/or non-covalently thereto, preferably on their respective outer
surfaces. Alternatively or in combination, the core microparticle
may have one or more portions, such as a center or an underlying
layer (a charged monolayer, for example), containing at least one
such active agent, preferably on the outer surface of the
portion.
[0124] The preformed microparticle, the surface-modified
microparticle, and any intermediates there between, if any, may be
and/or have one or more of the following characteristics: spherical
as defined herein, free of covalent crosslinking, free of hydrogel
and/or swelling, and have a polydisperse or, preferably,
monodisperse, size distribution. The preformed microparticle, may
be free of lipids and/or encapsulation.
[0125] Preferably, the surface-modified microparticle is capable of
controlled release, especially sustained release, of the active
agent, with a non-limiting release profile such as an initial burst
and a linear release profile, and may be provided as a suspension
or a dry powder in compositions or formulations for pharmaceutical,
therapeutic, diagnostic, cosmetic, and/or nutritional applications
As discussed above, the controlled release may occur within a
selected pH environment. In that regard, preferably the controlled
release may occur within a pH range of approximately 2 to 10, and
more preferably approximately 5 to 7.5, such as a physiological pH
of 7 to 7.4 or endosomal pH of 5 to 6.5.
[0126] Controlled deposition of the one or more monolayers may
further involve alteration of the net surface charge of the
microparticle (such as the preformed microparticle onto which one
or more of the monolayers have been deposited) through a controlled
manipulation of one or more conditions, such as changes in
temperature, pressure, pH, ionic strength and/or osmolality of the
reaction medium, concentrations of components within the reaction
medium, the likes thereof, as well as rates of such changes, and
combinations of two or more thereof. Such controlled manipulations
may desirably be applied before and up to the deposition of the one
or more monolayers, or even during the monolayer formations. In one
example, the net surface charge of the microparticle is capable of
being positive, neutral, and negative. The net surface change is
selected through, for example, a controlled change in one or more
of the conditions described above, such as a controlled change in
pH. In one example, the pH of the solution is selected such that
the net surface charge of the microparticle is negative, and the
difference between the pH of the solution and the surface-neutral
point of the microparticle is less than 0.3, alternatively equal to
or greater than 0.3, preferably 0.5 or greater, more preferably 0.8
or greater, most preferably 1 or greater.
[0127] FIG. 1 illustrates one exemplary method of providing
surface-modified microparticles in accordance with the present
disclosure. is A suspension of a plurality of preformed
microparticles used as three-dimensional substrates for the
deposition is first provided. Non-limiting methods of forming the
preformed microparticle include those disclosed herein and any
other methods known to those of skill in the art. One such method
is illustrated on the top portion of FIG. 24, which involves
providing a solution containing, the active agent and the
phase-separation enhancing agent, inducing a liquid-solid phase
separation through, for example, controlled cooling, and forming
the preformed microparticle. In one example, any one, two, or more,
or all of the compounds used to form the preformed microparticles
may preferably be distributed homogeneously throughout each
preformed microparticle (e.g., being present at similar
concentrations in the center, on the surface, and anywhere else
therein). It will be understood that methods of surface
modification as disclosed herein may be incorporated in whole or in
part into the underlying methods of fabricating the preformed
microparticles or made to be a continuation thereof, as illustrated
in FIG. 24. Between the pre-formation of the unmodified
microparticle and the surface modification, the preformed
microparticle may be separated from the liquid phase and,
optionally, washed, preferably in the presence of the
phase-separation enhancing agent. For example, the washing medium
may be the same solution used during phase separation, containing
the phase-separation enhancing agent. Alternatively, the preformed
microparticle is not separated from the liquid phase or washed. In
any event, as shown in FIGS. 1 and 24, the suspension or a
re-suspension of the preformed microparticle is combined and mixed
with a solution that includes at least one suitable charged
compound.
[0128] As described above, the preformed microparticles may have a
weight percent (wt. %) loading of the active agent of 40% or more,
preferably 60% or more, or 80% or more, or 90% or more, or 95% or
more, and less than 100%, typically 98% or less. The preformed
microparticles may further have, or are capable of being induced
(such as from a neutral state) to have, a net surface electric
charge. In one example, the net surface charge is contributed
primarily or essentially by the active agent and/or the carrier, if
any, present in the preformed microparticles; the compound(s) may
preferably be homogeneously distributed therein. Alternatively, the
active agent is compartmentalized in one or more portions of the
preformed microparticle, such as a center or an underlying layer (a
charged monolayer, for example), preferably distributed
substantially homogeneously within the portion or primarily on an
outer surface thereof. The preformed microparticles may be exposed
to (such as mixed with) at least one charged compound having or
capable of having a net electrical charge that is, preferably,
opposite in sign to the net surface charge of the preformed
microparticle, thereby forming the formed monolayer of the charged
compound about the preformed microparticle. The formed monolayer or
the surface modified microparticle has a net surface electric
charge that may be the same in sign as that of the preformed
microparticle, zero or, preferably, opposite in sign to that of the
preformed microparticle. In other words, if the outer surface of
the preformed microparticle has a negative net surface charge (such
as determined by zeta-potential measurements), then the formed
monolayer may preferably have on its outer surface a positive net
surface charge. Alternatively, if the preformed microparticle has a
positive net surface charge, then the formed monolayer may
preferably have a negative net surface charge. Deposition of the
monolayer can take place in an aqueous medium (e.g., water, buffer,
or aqueous solution containing some water miscible organic solvent
of the type previously described, or one that may be present in the
manufacture of the preformed microparticle).
[0129] To prepare the surface-modified microparticle, a
non-limiting method includes pre-forming or otherwise providing the
unmodified microparticle, exposing it to the one or more charged
compounds, which may be provided in a solution into which the
microparticle may be immersed, and forming the monolayer. The
solution may contain one or more of water, a buffer, and a
water-miscible organic solvent, and one or more solubility reducing
agents (e.g., alcohols, carbohydrates, non-ionic aqueous-miscible
polymers, and/or inorganic ionic compounds containing monovalent or
polyvalent cations), with a concentration in weight-to-volume
percentage of 5% to 50%, preferably 10% to 90%. A non-limiting
example of the solution contains about 16% (w/v) polyethylene
glycol and 0.7% (w/v) NaCl. The pH of the solution, typically in a
range of 4 to 10, may be adjusted to be same as or close to the
surface-neutral point of the core microparticle (such as with a
difference of 0 to less than 0.3), or away from that (such as with
a difference of 0.3 pH units or greater). The charged compound may
be present in the solution at a concentration of 0.05 mg/mL to 10
mg/mL. The preformed microparticle and the charged compound are
co-incubated in the solution, preferably at a temperature of
2.degree. C. to 5.degree. C. or up to ambient temperature over a
period of 1 second to 10 hours. The formation of the monolayer may
be carried out in a controlled manner. The resulting
surface-modified microparticle or an intermediate thereof may be
separated from the solution with optional washing. The washing
medium may be the same as the solution described above. The
procedure may be repeated using alternatingly charged compounds to
form the alternatingly charged monolayers, if desired
[0130] As indicated above, the reaction system can include one or
more solubility reducing and/or viscosity increasing agents
(SRA/VIA), as well as one or more PSEAs. Suitable SRA/VIAs and
PSEAs include, without limitation, those known to one skilled in
the art and those disclosed herein, such as alcohols (e.g.,
ethanol, glycerol), carbohydrates (such as sucrose), non-ionic
aqueous-miscible polymers (e.g., PEG, PVP, block copolymers of
polyoxyethylene and polyoxypropylene(poloxamers), hetastarch,
dextran, etc.), and inorganic ionizable compounds containing
polyvalent (e.g., divalent, trivalent) cations (e.g., metal and
organic cations such as those disclosed herein), such as
ZnCl.sub.2.
[0131] Thus, in one example, deposition of the formed monolayer may
take place in a solution that includes buffered saline (that is,
0.7% NaCl buffer) and 8% or more by weight or volume of a SRA/VIA
such as PEG, preferably 12% or more, more preferably 15% or more;
typically 30% or less, preferably 25% or less, more preferably 20%
or less, most preferably about 16% or more. The amount of SRA/VIA
required in the solution will depend, in part, on the stability of
the active agent, as well as the dissolution profile of the
monolayer(s). Certain charged compounds (such as the polycations
gelatin B and chitosan) may worlk in solutions containing 16% or
less SRA/VIA.
[0132] The pH of the solution at which the net surface charge of
the microparticle is zero is referred herein as the surface-neutral
point of the microparticle in the particular solution. In certain
examples, the pH of the solution may be adjusted to be at or near,
the surface-neutral point of the microparticle in the solution with
a difference there between of less than 0.3 (pH units), preferably
0.25 or less, more preferably 0.2 or less. In other examples, the
pH of the solution may preferably be adjusted away from the
surface-neutral point of the microparticle in the solution, with a
difference there between of 0.3 (pH units) or greater, preferably
0.5 or greater, more preferably 0.8 or greater, most preferably 1
or greater. It has been observed that in certain examples,
adjusting the solution pH away from the surface-neutral point of
the microparticle can affect dissolution kinetics of the active
agent therein. Incubation of the microparticles in the solution can
be performed at or, preferably, below ambient temperature, but
preferably above the freezing temperature of the solution, to
minimize disintegration of the microparticles. Incubation
temperature may even be lower than the freezing temperature of the
solution when one or more FPDAs disclosed herein are used. For
example, the incubation temperature may be between 0.degree. C. and
15.degree. C., preferably between 1.degree. C. and 10.degree. C.,
more preferably between 2.degree. C. and 5.degree. C., most
preferably less than 5.degree. C. In general, the concentration of
the charged compound in the solution for each monolayer fabrication
may be equal to, less than, and/or greater than one of the
following, or in a range between any two thereof: 0.05 mg/mL, 0.1
mg/mL, 0.5 mg/mL, 1 mg/mL, 10 mg/mL, 5 mg/mL, 3 mg/mL. When the
preformed microparticle is co-incubated with the charged compound
in the solution, a weight ratio of the preformed microparticle to
the charged compound may be 1:1 or greater, preferably 2:1 to 10:1,
more preferably 2.5:1 to 7:1
[0133] Incubation time may be adjusted to achieve the desired
charge modification (such as neutralization or charge reversal),
monolayer coverage, and/or monolayer thickness. Depending on the
particular reaction (such as ingredients and/or conditions), the
incubation time may be equal to, shorter than, and/or longer than
one of the following, or in a range between any two thereof: 10
hours, 5 hours, 3 hours, 10 minutes, 30 minutes, 100 minutes, 75
minutes, 60 minutes, 15 minutes, 5 minutes, 1 minute, 30 seconds,
10 seconds, 5 seconds, 1 second. Each monolayer may have a
thickness that is equal to, less than, and/or greater than one of
the following, or in a range between any two thereof: 100 nm, 50
nm, 20 nm, 5 nm, 1 nm, 0.5 nm, 0.1 nm, 2 nm, 10 nm. A typical
monolayer of the present disclosure is less than 100 nm in
thickness, preferably less than 10 nm.
[0134] Without wishing to be bound by any particular theory, it is
believed that a factor in controlling release of the active agent
from the microparticles may be the type and/or degree of
interaction and/or association(e.g., non-covalent association,
ionic complexation) that occurs at or near the outer surface of the
preformed microparticle (such as the interface with the formed
monolayer), which may involve the active agent, the charged
compound, and/or other components, if any. In some cases, a strong
interaction or association at this interface slows down, delays,
and/or otherwise hinders dissolution of the active agent, and is
believed to stabilize the surface-modified microparticle and
facilitate fabrication of additional alternatingly charged
monolayers, if desired. In addition, as described in greater detail
below, the interaction can be further affected by the subsequent
formation of additional alternatingly charged monolayers.
[0135] Thus, turning briefly to FIG. 2, co-incubation of the
preformed microparticles 10 and a charged compound 20, preferably
in a solution, results in intermediate microparticles 40 with a
single monolayer of the charged compound 20 formed on and
associated with at least the outer surface of the preformed
microparticle 10. Following incubation, the suspension of the
intermediate microparticles 40 may then be separated from the
solution through centrifugation, filtration, diafiltration, and/or
other separation methods. The intermediate microparticles 40 are
optionally washed with a washing solution (preferably an aqueous
medium, such as the SRA-containing buffer described above, as
generally shown in FIG. 1). The temperature during the incubation
and the optional washing is optimized based on the solubility of
the active agent and the charged compound 20.
[0136] If further surface modification is desired or required,
after the optional washing, the intermediate microparticle 40 may
be further exposed to (such as mixed with) a different charged
compound 30, preferably in a solution, to form a subsequent
monolayer of the charged compound 30 about and associated with the
formed monolayer of the intermediate microparticle 40. Charged
compound 30 preferably has a net electric charge opposite in sign
to that of the charged compound 20. The subsequent monolayer may be
formed immediately about the formed monolayer. The intermediate
microparticle 50 may have a net surface electric charge that is
same in sign as that of the intermediate microparticle 40, neutral
or, preferably, opposite in sign to that of the intermediate
microparticle 40. As shown in FIG. 1, the monolayer formation
procedure may be repeated as in the previous cycle, to form
microparticles 50 and 60 that have additional and, preferably but
not necessarily, adjacent alternatingly charged monolayers, each
associated with the preceding monolayer (FIG. 2). The total number
of the monolayers to be formed may be selected or predetermined
such that controlled release of the active agent with a desired
release profile is achievable in the surface-modified
microparticle. As set forth above, this number can be an integer of
1, 2, 3, 4, 5, 6, 7, or greater, preferably 100 or less, more
preferably 20 or less, and most preferably 10 or less,
[0137] In another example, one or more of the charged compounds
forming the monolayers may be active agent(s) identical to or
different from the one in the preformed microparticle. For example,
one or more of the odd numbered (e.g., first, third) monolayers may
independently be formed of the same or different charged active
agent(s), having net electric charge(s) opposite in sign to the net
surface charge of the preformed microparticle. Alternatively or in
combination, one or more of the even numbered (e.g., second, forth)
monolayers may independently be formed of the same or different
charged active agent(s), having net electric charge(s) same in sign
as the net surface charge of the preformed microparticle. With
reference to FIG. 2, charged compound 20 or 30 may be an active
agent that is the same as or different from the one in the
preformed microparticle 10, and charged compound 30 or 20,
respectively, may be an otherwise inert charged compound or a
charged active agent different from the one in the preformed
microparticle.
[0138] In another example, one or more active agents, charged
and/or uncharged, may be incorporated into one or more of the
monolayers through covalent and/or non-covalent associations. Such
monolayer-bound active agent(s) may be the same as that of the
preformed microparticle, or different therefrom. Such a
construction may allow controlled release (e.g., extended release,
sustained release) of the monolayer-bound active agent(s).
Alternatively or in combination, one of more of such
monolayer-bound active agent(s) may be affinity molecules, such as
targeting ligands, which may selectively bring the underlying
microparticle to a predetermined region to achieve targeted
delivery of the active agent within the core microparticle.
[0139] In a further example, the surface-modified microparticles
described above having one or more monolayers of charged compounds,
preferably in a suspension, may undergo one or more physical and/or
chemical treatments to further modify one or more characteristics
of the surface-modified microparticles, such as, but not limited
to, the release profile of the active agent therein. The treatments
may be carried out immediately after the formation of the
surface-modified microparticles and prior to any optional washing,
or immediately following the optional washings. The treatment may
involve manipulation of one or more parameters of the reaction
mixture, such as, without limitation, temperature, pH, and/or
pressure. Typically the one or more parameters may be adjusted
(such as increased or decreased) from an initial value to a second
value and held for a period of time, and then adjusted (such as
decreased or increased) to a third value or returned or allowed to
return to the initial value and held for another period of
time.
[0140] The thermal treatment, for example, may involve a heating
stage and a cooling stage. Prior to the additional treatment, the
suspension may be kept at a relatively low temperature below
ambient temperature to at least minimize dissolution of the
microparticles therein, preferably the temperature at which the
surface-modified microparticles are formed, more preferably
2.degree. C. to 10.degree. C., such as 4.degree. C. During the
heating stage, the suspension may be heated to a temperature and
incubated at this elevated temperature for an incubation period of
1 minute to 5 hours, preferably 15 minutes to 1 hour, such as 30
minutes. The elevated temperature may be higher than the relatively
low temperature at which the suspension was kept prior to the
additional treatment, and lower than a degradation temperature of
the surface-modified microparticles in the suspension, preferably
between 5.degree. C. and 40.degree. C., more preferably between
10.degree. C. and 30.degree. C. The heating stage may optionally be
immediately followed with a cooling stage, during which the
suspension may be chilled at a temperature, rapidly or gradually in
a controlled manner and optionally incubated at this depressed
temperature for an incubation period of 1 minute to 5 hours,
preferably 15 minutes to 1 hour, such as 30 minutes. In one
example, chilling is achieved by washing with a chilled washing
solution. Alternatively, the suspension may be allowed to return to
or close to its original temperature or to a selected temperature
below the temperature to which the suspension was heated. The
depressed temperature may be lower than the elevated temperature,
and higher than a freezing temperature of the suspension,
preferably at or below ambient temperature, optionally equal to or
different from the relatively low temperature at which the
suspension was kept prior to the additional treatment, more
preferably 15.degree. C. or lower, most preferably 10.degree. C. or
lower, such as 4.degree. C. The resulting mixture may further
undergo optional washings as described herein to yield additionally
treated, surface-modified, microparticles.
[0141] Surface-modified microparticles suitable for the additional
treatment described above include those formed from amorphous,
solid, and homogenous preformed microparticles having 40% to less
than 100%, or more typically 80% or greater, by weight, of an
active agent as described herein. Non-limiting examples of suitable
suspensions include microparticles (such as insulin microspheres)
in a buffer, such as a PEG buffer containing 16% PEG, 0.7% NaCl, 67
nM Na acetate, and having a pH in the range of 5 to 8 (e.g., 5.7,
5.9, 6.5, 7.0). The microparticles may have a concentration in the
buffer of 0.01 mg/ml to 50 mg/ml, preferably 0.1 mg/ml to 10 mg/ml,
such as 1 mg/ml. A charged compound or a mixture of two or more
thereof, such as protamine sulfate, poly-L-lysine, and/or
poly-L-arginine, may be mixed into the suspension to provide a
concentration of 0,01 mg/ml to 10 mg/ml, preferably 0.1 mg/ml to 1
mg/ml, such as 0.3 mg/ml. The mixture may be incubated at the
relatively low temperature, such as 4.degree. C., and under
agitation for an incubation period of 10 seconds to 5 hours, such
as 1 hour, to ensure the formation of a monolayer of the charged
compound on the outer surface of each of the preformed
microparticles. Then the suspension may be subjected to the thermal
treatment as described above. Optional washings may be carried out
on the suspension prior to the additional treatment.
[0142] The additional treatments may be carried out immediately
after the formation of any one or more of the monolayers as
disclosed herein. In one example, the additional treatment may be
carried out immediately after the formation of a single monolayer
on the preformed microparticles, the monolayer being formed of
positively charged compounds or negatively charged compounds. When
optionally one or more additional monolayers are formed on the
first monolayer, the additional treatment may or may not be carried
out immediately following the formation of such additional
monolayers. In another example, two or more monolayers may be
formed sequentially on the core microparticles, and the additional
treatment may be carried out only immediately after a single
predetermined monolayer (such as the last monolayer; the first
monolayer, or any other monolayer there between) is formed. In a
further example, two or more monolayers may be formed sequentially
on the core microparticles, and the additional treatment may be
carried out immediately after the formation of each and every
monolayer having one or more predetermined characteristics, such as
containing positively charged or negatively charged compounds, or
containing a particular compound (e.g., active agent, affinity
molecule, derivative) or moiety (e.g., functional group, label), or
being a particular monolayer from the core (e.g., first, second,
third, fourth, fifth). In a further example, the additional
treatment may be carried out immediately after the formation of
each monolayer of a predetermined set, which may be all of the
monolayers or a subset thereof
[0143] As described in Example 16 below, the surface-modified
microparticles following the additional treatment may display
modifications in net surface charge (zeta potential) and/or release
profile of the active agent therein. With certain charged compounds
(such as PLL and PLA, but not protamine sulfate), a change (such as
an increase) in the surface charge of the surface-modified
microparticles may be observed. When subjected to the in vitro
release protocol as disclosed herein, the additionally treated,
surface-modified microparticles are capable of displaying a
reduction in the 1-hour percentage of cumulative release (%
CR.sub.1 h) of the active agent therein, as compared to the
surface-modified microparticles without the additional treatment.
Inasmuch as it is believed that the initial burst of the active
agent release typically occurs within the first hour, the example
demonstrates that the initial burst of the active agent release may
be significantly reduced by the additional treatment. The same
additionally treated, surface-modified microparticles may be
capable of continued, preferably sustained, release beyond 1 hour,
preferably beyond 24 hours, more preferably beyond 48 hours, most
preferably beyond 7 days, having a 24-hour percentage of cumulative
release (% CR.sub.24 hr) that is greater than % CR.sub.1 h. As a
result of the additional treatment, the surface-modified
microparticles of the present disclosure, when subjected to in
vitro release in a release buffer (10 mM Tris, 0.05% Brij 35, 0.9%
NaCl, pH 7.4, free of divalent cation) at 37.degree. C., may be
capable of displaying a % CR.sub.1 h of 50% or less and/or a ratio
of % CR.sub.24 h to % CR.sub.1 h of greater than 1:1. The %
CR.sub.1 h may preferably be 40% or less, more preferably 30% or
less, further preferably 20% or less, most preferably 10% or less.
The ratio of % CR.sub.24 h, to % CR.sub.1 h may preferably be
1.05:1 or greater, more preferably 1.1:1 or, greater, but not more
than 10:1, preferably 5:1 or less, more preferably 2:1 or less,
most preferably 1.5:1 or less.
[0144] Without being bound to any particular theory, it is believed
that the additional treatment following the monolayer formation as
disclosed herein allows the charged compound in the monolayer and
the molecules (e.g., the active agent, the optional carrier
molecules in the preformed microparticle, the charged compound in
the preceding monolayer) that comprises the outer surface of the
substrate (e.g., the preformed microparticle, the preceding
monolayer) to rearrange and form an association that is much
stronger than the electrostatic interaction between the monolayer
and the outer surface of the substrate prior to the additional
treatment. It is believed that through the additional treatment a
modified shell is formed on the outer surface of the
surface-modified microparticle, the modified shell containing a
homogenous mixture of the charged compound and the molecules that
form the outer surface of the substrate.
[0145] Deposition of additional alternatingly charged monolayers of
charged compounds beyond the formed monolayer may further affect,
among other things, the release profile of the active agent in the
preformed microparticle. As previously described, depending on the
attractive forces at the interface between the preformed
microparticle and the formed monolayer; strong association between
the two may be observed. This may result in retarding the quantity
and/or rate of release of the active agent. The release profile may
be further modified by forming one or more additional alternatingly
charged monolayers about the formed monolayer. Without being
restricted to any particular theory, it is believed that addition
of a second oppositely charged monolayer may ease the association
between the formed monolayer and the preformed microparticle,
thereby enhancing the release of the active agent. Subsequent
application of the alternatingly charged monolayers, arranged
consecutively with optional interleaving layers of active agents,
if desired, can allow fine-tuning of active agent release from the
surface-modified microparticles, as shown in some of the examples
disclosed herein.
[0146] Suitable charged compounds that may be used in accordance
with the present invention may be charged compounds capable of
associating with any substrate, preferably by, but not limited to
non-covalent association and, more preferably, electrostatic
interactions. Thus, suitable charged compounds include positively
charged, negatively charged, or zwitterionic, and include, but are
not limited to, polyelectrolytes, charged polyaminoacids,
polysaccharides, polyionic polymers, ionomers, charged peptides,
charged proteinaceous compounds, charged lipids optionally in
combination with uncharged lipids, charged lipid structures such as
liposomes, precursors and derivatives thereof, and combinations of
two or mote thereof. Non-limiting examples include negatively
charged polyelectrolytes such as polystyrene sulfonate (PSS) and
polyacrylic acid (PAA), negatively charged polyaminoacids such as
polyaspartic acid, polyglutamic acid, and alginic acid, negatively
charged polysaccharides such as chondroitin sulfate and dextran
sulfate, positively charged polyclectrolytes such as polyallyl
amine hydrochloride (PAH) and poly(diallyldimethyl ammonium
chloride (PDDA), positively charged polyaminoacids such as
poly(L-lysine) hydrochloride, polyornithine hydrochloride, and
polyarginine hydrochloride, and positively charged polysaccharides
such as chitosan and chitosan sulfate. Also useful as charged
compounds in the present invention are, without limitation,
biocompatible polyionic polymers (e.g., ionomers, polycationic
polymers such as polycationic polyurethanes, polyethers,
polyesters, polyamides; polyanionic polymers such as polyanionic
polyurethanes, polyethers, polyesters, polyamides), charged
proteins (e.g., protamine, protamine sulfate, xanthan gum, human
serum albumin, zein, ubiquitins, and gelatins A & B), and
charged lipids (e.g., phosphatidyl choline, phosphatidyl serine).
Also included are derivatives (e.g., glycosylated,
hyperglycosylated, PEGylated, FITC-labeled, salts thereof),
conjugates, and complexes of the charged compound disclosed herein.
More specifically, suitable positively charged lipids (that is,
polyanionic lipids), negatively charged lipids (that is,
polycationic lipids), and zwitterionic lipids include
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluoresce-
in) (FITC-EA),
1,2-distaeroyl-sn-glycero-3[phospho-rac-O(1-glycerol)](sodium salt)
(DSPG), 1,2-dipalmitoyl-sn-glyceuo-3-phosphate (monosodium salt)
(DPPA) 1,2-dioleoyl-3-dimethylammonium-propane (D
[0147] Furthermore, lipid structures (such as liposomes) can be
used in alternate deposition with charged compounds. Uncharged
(such as non-ionic) lipids may be used in combination with
electrically charged lipids to form one or more of the monolayers,
and the molar ratio there between can be optimized to achieve
minimum permeability of the active agent through the monolayer.
[0148] The surface-modified microparticle disclosed herein,
typically containing a preformed microparticle and one or more
monolayers, preferably has a release profile of the active agent
that is different from that of the core microparticle. Non-limiting
examples of the differences in release profile include a reduction
in the initial burst, an extension of release time, a display of
linear/constant release over a time period, and/or a reduction in
rate of release over a prolonged time period. The surface-modified
microparticles may be present, preferably in a functionally (e.g.,
therapeutically, pharmaceutically, diagnostically) effective
amount, as a suspension or dry powder in a liquid or solid
composition or formulation, in the presence or absence of one or
more of preservatives, isotonicity agents, pharmaceutically
acceptable carriers, and stabilizing agents. Such compositions and
formulations may be administered in an effective amount to a
subject for prevention or treatment of a condition or state, or as
a nutritional supplement, or for the purpose of physical
enhancement or psychological well-being. Such compositions and
formulations may be incorporated into a diagnostic method, tool, or
kit for in vitro and/or in vivo detection of a substance,
condition, or disorder being present or absent, or a disposition
for such a condition or disorder. For example, the substance, upon
contact, may form an association (e.g., conjugate, complex) with
the surface-modified microparticle or a portion thereof (such as
the core microparticle), which is capable of providing one or more
signals for detection. The one or more signals may be one or more
moieties labeled on one or more portions of the association (e.g.,
the substance, the microparticles), or may be elicited upon the
formation of the association (e.g., emission of light, discharge of
another substance). Additionally, the surface-modified
microparticles may be incorporated into a nutritional and/or
dietary supplement or a food composition, or used as a food
additive, for prevention and/or treatment of a condition or
disorder in a subject.
[0149] Set forth below are analytical methods and several examples
of surface-modified microparticles fabricated in accordance with
the present disclosure. All of the charged monolayers formed in the
examples are believed to be saturated monolayers as described
herein. Readings and measurements reported were recorded using
instruments and methods described below.
Quartz Crystal Microbalance (QCM) Measurements
[0150] The QCM method was used to confirm the presence of
layer-by-layer assembly of electrically charged compounds in the
presence of an SRA-containing solution. Precursor films of multiple
(e.g., 2, 3, or 4) PAH/PSS bilayers (i.e., each bilayer includes a
PAH monolayer immediately adjacent to a PSS monolayer) were
deposited on 9 MHz silver resonators of a QCM (JJSI QCM System,
Model 260303, Sanwa Tsusho Co., Ltd, Japan). To form each
monolayer, the resonators were incubated in 0.25 M NaCl aqueous
buffer with a charged compound concentration of 1.5 mg/mL, at room
temperature for 15 minutes, washed three times with deionized (DI)
water, and dried. Instead of the aqueous buffer, the monolayers can
also be formed using DI water solution having a charged compound
concentration of 3 mg/mL.
[0151] To form each monolayer of the electrically charged compound
of interest on the precursor film, the coated resonators were
further incubated in aqueous buffers containing the respective
charged compounds at +2.degree. C. for about 1 hour, followed by
washings with DI water. For polyelectrolytes and charged proteins,
the concentration of the charged compound in the buffer was in a
range of 0.1 mg/mL to 3 mg/mL, preferably 1 mg/mL. For charged
lipids, the concentration of the charged compound in the buffer
(suspension) was 0.1 mg/mL to 3 mg/mL, preferably 0.25 mg/mL to 1
mg/mL. Different buffers were used, including (expressed in w/v
percentages): a) 16% PEG-0.7% NaCl, pH 5.8; b) 16% PEG-0.7% NaCl pH
7.0; c)0.16% acetic acid-0.026% ZnCl.sub.2. Concentrations of PEG,
NaCl and ZnCl.sub.2 in the buffers for assembly may be varied for
optimization.
[0152] The monolayers, after forming, were dried in a stream of
nitrogen gas. The frequency change of the resonators following
formation of each monolayer was monitored and converted into
thickness as understood by one skilled in the art, the results of
which are shown in FIG. 9,
Microparticle Net Surface Charge Measurements
[0153] For microparticle net surface charge (zeta potential)
measurements a Zeta Potential Analyzer (Model ZetaPALS, Brookhaven
Instruments Corp., Holtsville, N.Y.) was used A 40 .mu.L aliquot of
each sample under investigation was added to 1.5 mL of the
corresponding salt-free PEG solution, mixed, and the resulting
suspension was immediately used for the measurements. The
temperature of the suspension was equilibrated to 8.degree. C. to
minimize microparticle disintegration.
In Vitro Release (IVR)
[0154] To generate the IVR profile of the active agent (such as
insulin), a 10 mL aliquot of a releasing buffer (10 mM Tris, 0.05%
Brij 35, 0.9% NaCl, pH 7.4) was added into a glass vial containing
0.5 mL of the concentrated particle suspension (equivalent to 3 mg
of insulin), mixed, and incubated at 37.degree. C. At designated
time intervals 400 .mu.L of the IVR medium was transferred into a
microfuge tube and centrifuged for 2 minutes at 1.3 k rpm. A 300
.mu.L aliquot of the supernatant was removed and stored at
-80.degree. C. until analyzed by Bicinchoninic Acid (BCA) assay as
understood by one skilled in the art. A 300 .mu.L aliquot of flesh
releasing buffer was added to the microfuge tube to reconstitute
the pellet. The 400 .mu.L suspension was transferred back to the
IVR. Total active agent content of the microparticle was determined
by BCA assay after complete dissolution of the microparticle in an
aqueous alkaline solution containing dimetbyl sulfoxide (DMSO) and
a surfactant and pH neutralization.
EXAMPLES
Example 1A
Microspheres with Polystyrene Sulfonate Monolayer
[0155] Insulin microspheres formed using a phase separation method
disclosed herein (i.e., preformed microparticles) were incubated in
an aqueous solution of 16% (w/v) PEG and 0.7% (w/v) NaCl in the
presence of 0.3 mg/mL polyion PSS at 2.degree. C. and pH 4.8 for 1
hr. To remove the unassociated PSS centrifugal washing (at 3000 rpm
for 15 minutes) was applied twice, each using the initial volume of
the aqueous solution described above as the washing medium to
re-suspend the microspheres. Comparison of zeta-potential values of
the unmodified and modified microspheres confirms the formation of
the PSS monolayer (FIG. 3).
Example 1B
Microspheres with Multiple Alternating Monolayers of Polystyrene
Sulfonate and Polyallylamine Hydrochloride
[0156] The PSS-modified microparticles of Example 1A were used as
intermediate microparticles to form a subsequent monolayer of
polycation PAH, Similar formation and washing procedures were used
as described in Example 1A, except that PAH was substituted for PSS
at the same concentration. The procedures were repeated to form
desired number of alternating monolayers, FIG. 3 illustrates the
zeta-potential values of four consecutive depositions of the
PSS/PAH bilayer assembly after formation of each monolayer.
Example 2A
Microspheres Surface-Modified at a pH Below the Surface-Neutral
Point of the Microspheres
[0157] The procedures of Example 1A were used to form a polyanion
monolayer on insulin microspheres at pH 418 (below surface-neutral
point of the microspheres, which was observed to be about 5.6) FIG.
4 depicts zeta-potential values of insulin microspheres with a
monolayer of polyacrylic acid (model polyanion), dextran sufate,
polyaspartic acid, polyglutamic acid, and alginate. The
zeta-potential of the preformed insulin microspheres at pH of 4.8
showed a positive net surface charge. Following the formation of
the respective polyanion monolayers, the net surface charges of the
resulting surface-modified microspheres were negative.
Example 2B
Microspheres Surface-Modified at a pH Above the Surface-Neutral
Point of the Microspheres
[0158] The procedures of Examples 1A & 1B were used to form a
polycation monolayer on insulin microspheres at pH 7.0 (above
surface-neutral point of the microspheres). FIG. 5 depicts
zeta-potential values of insulin microspheres with a monolayer of
polydiallyldimethylammonium chloride (PDDA, model polycation),
protamine sulfate (ProtS), poly-l-arginine (PLA), and poly-l-lysine
(PLL). The zeta-potential of the preformed insulin microspheres at
pH of 7.0 showed a negative net surface charge. Following the
formation of the respective polycation monolayers, the net surface
charges of the resulting surface-modified microspheres were
positive LSC micrograph of insulin microspheres with FITC-labeled
protamine monolayer, as shown in FIG. 6, confirmed formation of the
polycation monolayer.
Example 3A
Microspheres with Multiple Monolayers of Oppositely Charged
Polyions
[0159] The resulting microspheres of Examples 1A & 2A were used
as intermediate microspheres, re-suspended in the aqueous solution
(16% PEG, 0.7% NaCl, pH 4.8) in the presence of 0.3 mg/mL.
polycation PLL, and incubated for 1 hr at 2.degree. C. to form a
subsequent monolayer of PLL over the formed polyanion monolayer.
Net surface charge reversal of the microspheres, shown in FIG. 7,
confirmed formation of the polyion monolayers. LSC micrograph of
insulin microspheres with a formed PSS monolayer and a subsequent
FITC-labeled PLL monolayer, shown in FIG. 8, demonstrated surface
deposition of the polycation PLL.
[0160] A QCM as described above was used to measure the thickness
of each polyion monolayer. The reaction medium contained PLL or
chondroitin sulfate at 1 mg/mL in 16% PEG, 0.7% NaCl The film
assembly was constructed by consecutive incubation of the QCM
resonators in the reaction media containing the polyions for 15
minutes each, followed intermediately with DI water washes and
drying with nitrogen stream. FIG. 9 illustrates the progressive
film thickness following formation of each monolayer. Depending on
the polyion, each monolayer was estimated to increase the total
thickness by about 1 nm or less, with an averaged increase of about
0.5 nm.
Example 3B
Microspheres with Multiple Monolayers of Oppositely Charged
Biocompatible Polyions
[0161] Condroitin sulfate and gelatin A were used to form multiple
monolayers of oppositely charged polyions about preformed insulin
microspheres, in an aqueous solution of 16% PEG & 0.7% NaCl, at
pH 4,8 and 2.degree. C. Formation of the condroitin sulfate
monolayer, according to the procedures of Example 1A, was followed
by subsequent formation of the gelatin A monolayer. The procedures
were repeated to form a total of 6 alternatively charged
monolayers. Reversal of net surface charge of the microspheres
following formation of each monolayer is shown in FIG. 10.
Example 4A
Monolayer Formation in the Presence of Polyvalent Cation and
PEG
[0162] The procedures of Example 1B were used to form monolayers of
protamine and condroitin sulfate about preformed insulin
microspheres, with the exception that the aqueous solution had a
pH1 of 6.4 and contained 16% PEG, 0.7% NaCl, 0.16% (w/v) acetic
acid, and 0.026% (w/v) ZnCl.sub.2. Comparative examples were formed
using a Zn-free & acetate-flee aqueous solution with a pH of
6.4 containing 16% PEG and 0 7% NaCl. Zeta-potentials of the
resulting microspheres are shown in FIG. 11
Example 4B
Monolayer Formation in the Presence of Polyvalent Cation and
Absence of PEG
[0163] The procedures of Example 1B were used to form monolayers of
protamine and condroitin sulfate about preformed insulin
microspheres, with the exception that the aqueous solution was
PEG-free, had a pH of 7.0, and contained 0.7% NaC], 0.16% acetic
acid, and 0.026% ZnCl12 Zeta-potentials of the resulting
microspheres are shown in FIG. 12.
Example 5
Microspheres Surface-modified with Liposomes
[0164] Liposomes containing 60% cationic lipid
1,2-dioleoyl-3-dimethylanimonium-piopane (DAP) and 20% zwitterionic
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), suspended in an
aqueous solution of 16% PEG, 0.7% NaCl, 0.16% acetic acid and
0,026% ZnCl.sub.2 at pH 7.0 were used to co-incubate with preformed
insulin microspheres (pre-washed with the same aqueous solution) at
2.degree. C. for 1 hr. The procedures of Example 41B were applied
to form a subsequent monolayer of chondroitin sulfate.
[0165] Alternatively, liposomes containing anionic
1,2-distearoyl-sn-glycero-3[phosphor-rac-(1-glycero)]) (DSPG,
sodium salt), DOPC and cholestrol were deposited on
protamine-modified insulin microspheres, using the procedures of
Example 4B. FIG. 13 illustrates LSC micrographs of microspheres
containing Rodamine B-labeled protamine monolayer (top right),
microspheres containing FITC-labeled DAP (top left), and
microspheres containing both (bottom left), Zeta-potential values
of the microspheres following each deposition are shown in FIG.
14.
Example 6
Sustained Release of Insulin from Protamine-Modified
Microspheres
[0166] Protamine-modified insulin microspheres were formed using
the procedures of Example 21B at various concentrations of the
polyion in the reaction medium The IVR profiles of resulting
protamine-modified insulin microspheres are shown in FIG. 15. An
increase in concentration of polyion reduced the initial burst and
subsequent release rate of insulin from the surface-modified
microspheres.
Example 7
Release Modification with Multiple Monolayers
[0167] The resulting microspheres of Example 2B were used as
intermediate microparticles, about which carboxymethyl cellulose
(CMC) was deposited at various concentrations in the reaction
medium. The IVR profiles shown in FIG. 16 demonstrated the
capability of the subsequent monolayer in further modifying the
release of active agents from the surface-modified microspheres.
Deposition of an additional protamine monolayer was able to
partially or fully restore the release profile (FIG. 17).
Example 8
In Vivo Release of Insulin from Protamine-Modified Insulin
Microspheres
[0168] In vivo release of insulin from protamine-modified insulin
microspheres was investigated in chemically induced Sprague-Dawley
rats. The surface-modified microspheres prepared according to
Example 2B were administered as a suspension in 16% PEG 3350, pH
7.0. The preformed insulin microspheres free of surface
modification were administered in the PEG solution, or in
phosphate-buffered saline, pH 7,4, as control. The animals received
an initial subcutaneous dose of 1 IU/kg of the microspheres. An
ELISA assay was used to determine the recombinant human insulin
(rhINS) serum levels in the collected samples. The results, as
illustrated in Table 1 and FIGS. 18A and 18B, demonstrate the
significant effect of the surface-modification on pharmacokinetics
of the administered dose. Specifically, the surface-modification
increased the maximum serum concentration of rhINS (C.sub.max) and
the time to achieve C.sub.max (t.sub.max), as well as the area
under the rhINS concentration-time curve (AUC) and the mean
residence time (MRT) of the protein. The serum glucose depression
(FIG. 18B) also was in agreement with the corresponding serum
rhINS. As shown below, the increase in C.sub.max greater in the
surface modified microparticles as compared to C.sub.max and
t.sub.max of the unmodified, preformed microparticles. As
demonstrated by this Example, the C.sub.max of the surface modified
microparticle was 2.5 fold greater than the C.sub.max of the
preformed microparticle. In other examples, the C.sub.max of the
surface modified microparticle may be increased 1.1 fold or
greater, 1.25 fold or greater, 1.5 fold or greater, 2.0 fold or
greater than the C.sub.max of the preformed microparticle.
TABLE-US-00001 TABLE 1 Parameter Preformed Microparticles Modified
Microparticles AUC.sub.0-7 h 203.3 .+-. 46.5 780.9 .+-. 81.3
MRT.sub.0-7 h 1.7 .+-. 0.2 2.9 .+-. 0.2 C.sub.max 103.5 .+-. 27.3
259.0 .+-. 52.9 t.sub.max 0.55 .+-. 0.41 2.60 .+-. 0.55
Example 9
Surface Modification in Presence of Various Solubility Reducing
Agents
[0169] Aqueous media of protamine sulfate (0.15 mg/mL) used to
incubate the preformed insulin microparticles contained one of
PLURONICOR F-68 or F-127 (10% or 16% w/v), glycerol (20%, 40%, or
60% v/v), and ethanol (10% v/v). Procedures as described in Example
1A were followed. Zeta-potential values of the microspheres before
and after surface modification, shown in FIG. 19, indicated
formation of protamine monolayer.
Example 10
Effect of Concentration of Charged Compound on Release Profile of
Surface-Modified Microspheres
[0170] Procedures of Example 1A were followed, in which the
concentration of protamine sulfate was varied in a range of 0.1
mg/mL to 1.5 mg/mL. FIG. 20A illustrates the relationship between
zeta-potential of the microspheres and the cumulative release of
insulin at 48 hrs. An increase in protamine concentration in the
reaction mediumeorrelated with reduction of insulin release, with
an observed maximum effective concentration of about 0.3 mg/mL.
[0171] The protamine-modified microspheres prepared at a protamine
concentration of 1.5 mg/mL were further modified with polyanion
carboxymethyl cellulose or chondroitin sulfate in the concentration
range of 0.05-1.2 mg/mL or 0.1-1.2 mg/mL, respectively Formation of
the subsequent monolayer significantly reversed the release
reduction effect of the protamine monolayer, as shown in FIGS. 20B
and 20C. The results suggested the ability of a few monolayers in
fine-adjusting the release profile of the microparticles in a
controlled manner.
Example 11
Surface Modification of hGH Microspheres
[0172] Preformed hGH microspheres were incubated, in alternating
sequence, in aqueous media (16% PEG 3350, 0.7% NaCl, pH 6.0)
containing 0.3 mg/mL prolamine sulfate and chondroitin sulfate,
respectively, at 2.degree. C. for 1 hr each, to form the
alternatingly charged monolayers. FIG. 21A depicts the
zeta-potential of the microspheres after deposition of each
monolayer. The IVR profiles of surface-modified hGH microspheres
with one, two, or three monolayers are compared with that of the
unmodified preformed hGH microspheres in FIG. 21B.
Example 12
Surface Modification of Microspheres of Intravenous
Immunoglobulin
[0173] Preformed intravenous immunoglobulin (IVIG) microspheres
were modified with alternating monolayers of chondroitin sulfate
and protamine sulfate. For each monolayer, the incubation was
carried out in a pH 7.0 aqueous medium containing 12.5% PEG 8000,
50 mM ammonium acetate, and 0.15 mg/mL of the respective polyion at
4.degree. C. for 1 hr. Centrifugal washing was used to remove
excess polyions. FIG. 22 depicts the zeta-potential of the
microspheres after deposition of each monolayer.
Example 13
Surface Charge Characteristics of Microspheres in Aqueous PEG
Media
[0174] In order to determine surface charge characteristics of
preformed insulin microspheres in solubility reducing media
containing 16% PEG, pH of tie media was adjusted in a range of
4-7.5. Zeta-potential of the microspheres were determined in each
medium, and plotted versus the corresponding pH, as shown in FIG.
23. The surface-neutral point for the preformed insulin
microspheres was estimated to be 5.6. As the pH of the medium
decreased below or increased above the surface-neutral point, the
net surface charge of the preformed insulin microspheres became
more and more positive or negative, respectively.
Example 14
Effect of Reaction pH on Zeta Potential and Release Profile of
Surface-Modified Microspheres
[0175] At 4.degree. C., insulin microspheres (20 mg) formed using a
phase separation method disclosed herein were suspended in 19 ml of
a buffer [containing 16% (w/v) PEG, 0.7% (w/v) NaCl, and 67 mM
sodium acetate] at one of the following pH values: 5.7, 5.9, 6.5,
and 7.0. Zeta potential of the unmodified microspheres in the
buffer of different pH was measured as described herein above.
Protamine sulfate, poly-l-lysine, or poly-l-arginine was added to
the suspension as 1 ml of a 6 mg/ml solution in the same buffer at
the same pH as that of the suspension. The resulting reaction
mixtures each had a microsphere concentration of 1 mg/ml and a
polycation concentration of 0.3 mg/ml. The reaction mixtures were
incubated at 4.degree. C. for one hour and then centrifugally
washed (3000 rpm for 15 minutes) three times with 20 ml fresh
aliquots of the buffer at the respective pH values of the reaction
mixtures. Zeta potentials of the resulting surface-modified
microspheres in the resuspensions of the last washing were measured
as described above. The surface-modified microspheres were then
subjected to in vitro release following the protocol disclosed
herein.
[0176] As shown in FIG. 25, the formation of the polycation
monolayer qualitatively reversed (from negative to positive) the
surface charge of insulin microspheres at the different reaction pH
values described above. The zeta potential of the surface-modified
microspheres and the magnitude of the charge reversal appeared to
depend at least in part on the reaction pH and/or the polycation.
Specifically, the zeta potential of the PLL-modified insulin
microspheres was higher (in a general range of 15 mV or greater,
such as about 20 mV) at reaction pH values (e.g., 5.7, 5.9) close
to the surface-neural point of the unmodified insulin microspheres
(insulin SNP.sub.core, about 5.6), and lower (in a general range of
less than 15 mV, such as about 8 mV) at reaction pH values (e.g.,
6.5, 7.0) away from insulin SNP.sub.core. The magnitude (about 30
mV) of the charge reversal following the formation of the PLL
monolayer was the same across the different reaction pH values
specified above.
[0177] The zeta potential of the PLA-modified insulin microspheres
was higher (above 20 mV) at reaction pH values close to insulin
SNP.sub.core, and lower (below 20 mV) at reaction pH values away
from insulin SNP.sub.core. The magnitude of the charge reversal
following the formation of the PLA monolayer was less (about 30 mV)
at reaction pH values close to insulin SNP.sub.core, and greater
(about 40 mV) at reaction pH values away from insulin SNP.sub.core.
The zeta potential (about 20 mV) of the ProtS-modified insulin
microspheres was the same across the different reaction pH values
specified above. The magnitude of the charge reversal following the
formation of the protamine sulfate monolayer was less (about 30 mV
or less) at reaction pH values close to insulin SNP.sub.core, and
greater (about 40 mV or greater) at reaction pH values away from
insulin SNP.sub.core.
[0178] As shown in FIG. 26, the in vitro 1-hour percentage of
cumulative release (% CR.sub.1 h) of insulin from the
surface-modified insulin microspheres was affected by the reaction
pH and/or the polycation used in the surface modification reaction.
Specifically, insulin % CR.sub.1 h was generally greater at
reaction pH values close to insulin SNP.sub.core, than that at
reaction pH values away from insulin SNP.sub.core, with the
difference there between ranging from greater than 5% to 10% to 20%
to less than 30%. At the same reaction pH, PLA-modified
microspheres and ProtS-modified microspheres had comparable insulin
% CR.sub.1 h, the level of which was less than that of PLL-modified
microspheres, with the differences there between ranging generally
from 20% to 30% or more.
[0179] In cases where it may be desired to have a % CR.sub.1 h, of
less than 50%, preferably 40% or less, more preferably 30% or less,
most preferably 20% or less, the preformed microparticles of the
present disclosure (such as the unmodified insulin microspheres)
may be surface-modified at reaction pH away from SNP.sub.core using
certain charged compounds (e.g., protamine sulfate, PLA) In cases
where it may be desired to have a % CR.sub.1 h, of 50% or greater,
preferably 60% or greater, more preferably 70% or greater, most
preferably 75% or greater, the preformed microparticles of the
present disclosure (such as the unmodified insulin microspheres)
may be surface-modified at reaction pH close to SNP.sub.core using
certain charged compounds (such as PLL).
Example 15
Surface-Modified Nucleic Acid Microspheres
[0180] Nucleic acid microspheres were formed according to the
disclosures of U.S. Patent Application Publication Nos.
2006-0018971 and 2006-0024240, the entirety of which are
incorporated herein by express reference thereto. Each of the
microspheres had a homogenous mixture containing at least 80% (such
as 85% to 90%) by weight of a CD40 siRNA and 15% or less (such as
6% to 10%) by weight of poly-l-lysine. The nucleic acid
microspheres were suspended in 100 .mu.l of nuclease-free deionized
water (pH 7.0) containing 1 mg/ml Rodamine B-labeled PLL (70 kD).
The suspension was incubated with agitation at 4.degree. C. for 45
minutes to form the surface-modified microspheres, which were then
centrifugally washed with nuclease-flee deionized water (pH 7.0).
The zeta-potentials of the unmodified microspheres and the
surface-modified microspheres were measured to be -24 mV and 34 mV,
respectively. Clearly, surface modification of the nucleic acid
microspheres through formation of a polycation monolayer is capable
of reversing their surface electric charge. Laser scanning confocal
micrograph of Rodamine B-labeled PLL is shown in FIG. 27,
confirming successful formation of the monolayer on the outer
surface of the nucleic acid microspheres.
Example 16
Thermal Treatment of Surface-Modified Microparticles
[0181] Preformed, unmodified insulin microspheres (12 mg), such as
those formed from controlled phase separation as disclosed herein,
were suspended in 1.5 ml of a buffer containing 16% (w/v) PEG, 0.7%
(w/v) NaCl, and 67 mM sodium acetate at pH 7.0 and 4.degree. C. At
a concentration of 6 mg/ml, a 1.5 ml aqueous solution of a
polycation (ProtS, PLL, or PLA) dissolved in the same buffer was
mixed with the suspension, resulting in reaction mixtures having a
microsphere concentration of 4 mg/ml and a polycation concentration
of 3 mg/ml. The reaction mixtures were incubated at 4.degree. C.
with continuous agitation for 30 minutes to form the
surface-modified insulin microspheres having the respective
polycation monolayer. Next, the reaction mixtures were further
incubated for another 30 minutes at a temperature of 4.degree. C.,
15.degree. C., 28.degree. C., or 37.degree. C. Then the
thermally-treated insulin microspheres were centrifugally (3000 rpm
at 4.degree. C. for 10 minutes) collected and washed three times
with fresh aliquots of the buffer at pH 7.0 and 4.degree. C. Zeta
potential data and in vitro release profiles of the
thermally-treated, surface-modified insulin microspheres were
generated as described herein above.
[0182] It was found, unexpectedly, that certain thermal treatments
as described above (such as incubation at a temperature of
15.degree. C., 28.degree. C., or 37.degree. C., but not limited
thereto) were capable of selectively and differentially altering
certain characteristics (such as zeta potential and release
profile) of the surface-modified microparticles without adversely
affecting other properties thereof (such as particle size, extended
release phase). As illustrated in FIG. 28, the incubation at
different elevated temperatures as described above results in
different levels of initial insulin release from the
polycation-modified insulin microspheres (insulin % CR.sub.1 h
being lower following incubation at 28.degree. C. than that
following incubation at 15.degree. C.), which are consistently less
than that following incubation at non-elevated temperature (i.e.,
4.degree. C.).
[0183] As exemplified in Table 2, the initial release or "burst"
phase in the in vitro release profiles, as represented by %
CR.sub.1 h, of the thermally-treated, surface-modified
microparticles were reduced as compared to that of the
surface-modified microparticles that did not undergo the thermal
treatment (such as those incubated at a second temperature of
4.degree. C.). The amount of reduction in % CR.sub.1 h, of the
active agent released in vitro from the thermally-treated,
surface-modified microparticles, with respect to the control, can
be 10% or greater, such as 15% or greater, 25% or greater, or 40%
or greater, TABLE-US-00002 TABLE 2 Percentage of Reduction in %
CR.sub.1 h of PLL-modified Insulin Microspheres Sample (2.sup.nd %
Reduction (in % CR.sub.1 h as compared to Incubation Temp) %
CR.sub.1 h Control) Control (4.degree. C.) 57.4 Thermal 1
(15.degree. C.) 48.3 16 Thermal 2 (28.degree. C.) 33.1 42 Thermal 3
(37.degree. C.) 34.1 40
[0184] The reduction effect of the thermal treatment (at 28.degree.
C.) on the initial release phase of the in vitro release of insulin
was consistently observed in the polycations tested above.
Unexpectedly, different polycations exert different levels of
reduction on the initial release of insulin. As illustrated in
FIGS. 26 and 28, and summarized in Table 3, the reduction effect
observed in PLA-modified insulin microspheres of the present
disclosure is greater than that in PLL-modified insulin
microspheres, while the reduction effect observed in ProtS-modified
insulin microspheres is less than that in PLL-modified insulin
microspheres, TABLE-US-00003 TABLE 3 Effect of Thermal Treatment
(at 28.degree. C.) on % CR.sub.1 h of Surface-modified Insulin
Microspheres % CR.sub.1 h without % CR.sub.1 h with Thermal Thermal
Treatment Treatment (Example % Reduction in Polycation (Example 14)
16) % CR.sub.1 h PLL 50.1 28.4 43 ProtS 28.9 15.1 48 PLA 19.5 5.1
74
Example 17
Thermal Treatment of Surface-Modified Microparticles
[0185] Preformed, unmodified hGH microspheres, such as those formed
from controlled phase separation as disclosed herein, underwent the
same thermal treatments as described in Example 16. Zeta potential
data and in vitro release profiles of the thermally-treated,
surface-modified hGH microspheres were generated as described
herein above. As exemplified in Table 4, % CR.sub.1 h, of the
surface-modified hOH microspheres following incubation at
28.degree. C. was reduced as compared to that of the
surface-modified hGH-microspheres following incubation at 4.degree.
C. TABLE-US-00004 TABLE 4 Percentage of Reduction in % CR.sub.1 h
of PLA-modified hGH Microspheres Sample (2.sup.nd % Reduction (in %
CR.sub.1 h as compared to Incubation Temp) % CR.sub.1 h Control)
Control (4.degree. C.) 24.9 Thermal 1 (15.degree. C.) 10.8 57
Thermal 2 (28.degree. C.) 9.6 61 Thermal 3 (37.degree. C.) 13.0
48
Example 18
Effect of Thermally-Treated, Surface-Modified Microparticles In
Vivo
[0186] Unmodified insulin microspheres were prepared using the
controlled phase separation method disclosed herein. Two portions
of the unmodified insulin microspheres were surface-modified with
PLA, with one portion thermally-treated (at 28.degree. C), and the
other portion incubated at 4.degree. C. as control, according to
the procedures described in Example 16. Injectable compositions
each containing one of the three different insulin microspheres
suspended in a buffer [16% (w/v) PEG, 0.7% (w/v) NaCl, at pH 7.0]
were prepared. The compositions were administered subcutaneously to
normal Spague-Dawley rats at a dose of 4 IU/kg. An ELISA assay was
used to determine the insulin serum levels in the collected
samples. The results are illustrated in Table 5 and FIGS. 29A and
29B. As shown in FIG. 29A, CR.sub.1 h was likewise reduced
following heat treatment.
[0187] PLA-modified insulin microspheres treated at 4.degree. C.
provided comparable serum insulin concentration profile, serum
glucose depression profile, C.sub.max, and to I.sub.max comparison,
PLA-modified insulin microspheres treated at 28.degree. C. provided
flattened and right-shifted serum insulin concentration profile,
right-shifted serum glucose depression profile, depressed
C.sub.max, and prolonged t.sub.max. TABLE-US-00005 TABLE 5 In Vivo
Insulin Release from Different Insulin Microspheres PLA- Parameter
Unmodified PLA-modified, 4.degree. C. modified, 28.degree. C.
C.sub.max 479.1 .+-. 147.0 463.5 .+-. 136.9 256.8 .+-. 95.8
t.sub.max 1.1 .+-. 0.5 1.1 .+-. 0.5 2.0 .+-. 0.7
[0188] FIG. 29A is a graph showing the serum insulin concentration
versus time profiles in rats that have received a single
subcutaneous injection of uncoated insulin microspheres,
PLA-modified insulin microspheres treated at 28.degree. C., or
PLA-modified insulin microspheres treated at 4.degree. C. (Example
18);
[0189] FIG. 29B is a graph showing the serum glucose depression
versus time profiles of rats treated with single subcutaneous
injection of uncoated insulin microspheres, PLA-modified insulin
microspheres treated at 28.degree. C., or PLA-modified insulin
microspheres treated at 4.degree. C. (Example 18);
[0190] It is to be understood that the embodiments disclosed herein
are merely exemplary of aspects of the disclosure, which may be
embodied in various different forms. Therefore, specific details
and preferred embodiments disclosed herein are not to be
interpreted as limiting, but merely as a basis for the claims and
as a representative basis for teaching one skilled in the art to
variously employ the subject matter disclosed herein in any
appropriate manner. The embodiments which have been described are
illustrative of some of the applications of the principles of the
present disclosure, and modifications may be made, including those
combinations of features that are individually disclosed or claimed
herein.
* * * * *