Note: Descriptions are shown in the official language in which they were submitted.
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SURFACE-MODIFIED MICROPARTICLES AND
METHODS OF FORMING AND USING THE SAME
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent
Application
Serial No. 60/675,372 filed April 27, 2005 and U.S. Provisional Patent
Application Serial
No. 60/750,903 filed December 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
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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 charge. In this example, the method
furtlier 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
rnicroparticle.
[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.
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[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 conipound
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 Cma,, and tax that is different from the Cma, and
t,,,a,; of the core
microparticle.
[00010] 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
[00011] Fig. 1 is a flow chart showing an exemplary method set forth in the
present
disclosure;
[00012] Fig. 2 is a schematic illustration of the fabrication of monolayers of
charged
compounds on a preformed microparticle;
[00013] Fig. 3 is a graph that shows the change in zeta potential of insulin
microparticles with alternatingly charged monolayers (Examples 1A & 1B);
[00014] 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);
[00015] 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);
[00016] Fig. 6 is a laser scanning confocal (LSC) micrograph of insulin
microparticles with one FITC-labeled protamine monolayer (Example 2B);
[00017] 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);
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[00018] 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);
[00019] 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);
[00020] Fig. 10 is a graph showing changes in zeta-potential of insulin
microparticles
following subsequent depositions of a cliondroitin sulfate monolayer and a
gelatin A
monolayer (Example 3B);
[00021] 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);
[00022] 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);
[00023] 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);
[00024] Fig. 14 is a graph showing the zeta-potential of insulin
microparticles
following deposition of each monolayer as described in Exanzple 5;
[00025] 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);
[00026] 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);
[00027] Fig. 17 is a graph showing release profiles of insulin from insulin
microparticles coated with three monolayers (Example 7);
[00028] 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);
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[00029] Fig. 18B is a graph showing the serum glucose depression versus time
profiles of rats treated with single subcutaneous injection of uncoated
insulin
inicroparticles, or protamine-coated insulin microparticles Example 8);
[00030] 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);
[00031] 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);
[00032] 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);
[00033] 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);
[00034] 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);
[00035] 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);
[00036] 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);
[00037] 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);
[00038] Fig. 24 is a flow chart showing another exemplary method of preparing
microparticles set forth in the present disclosure;
[00039] 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);
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[00040] Fig. 26 is a graph showing the effect of reaction pH on the in vitr=o
1-hour
percentage of cumulative insulin release of insulin microparticles surface-
modified with
one monolayer of protarnine sulfate, poly-l-lysine, or poly-l-arginine
(Example 14);
[00041] 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);
[00042] Fig. 28 is a graph showing the in vitro release profiles of PLL-
insulin
microparticles thermally treated at different temperatures; and
[00043] 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 C, and PLA-
modified
insulin microparticles treated at 4 C (Example 18); and
[00044] 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 C,
and PLA-
modified insulin microparticles treated at 4 C (Example 18).
DETAILED DESCRIPTION
[00045] 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.
[00046] "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 viti o
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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
enviromnent 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 ffizrther
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 2kD or less (such as 1.5kD or less, or
1kD or less),
macromolecules (such as those having a molecular weight of 2kD or greater,
preferably
5kD 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 7Ø
[00047] "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
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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.
[00048] "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 defmed 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.
[00049] "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 (e.g.,
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-l-antitrypsin
(AAT), DNase,
superoxide dismutase (SOD), lysozyme, ribonuclease, hyaluronidase,
collagenase, human
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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), LHRH 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.
[00050] "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 (e.g., 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 more thereof.
[00051] "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). Carbohydrates 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.
[00052] "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
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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.
[00053] "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.
[00054] "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, W, 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 (e.g., 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.
[00055] "Macromolecule" refers to a material capable of providing a three-
dimensional (e.g., tertiary and/or quatemary) structure, and includes carriers
and certain
active agents of the present disclosure. Non-limiting macromolecules used to
form the
microparticles include, itzter alia, polymers, copolymers, proteins (e.g.,
enzymes,
recombinant proteins, albumins like human serum albumin), peptides, lipids,
carbohydrates, polysaccharides, nucleic acids, vectors (e.g., virus, viral
particles),
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complexes and conjugates thereof (e.g., 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.,
[00056] "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.
[00057] "Monodisperse size distribution" refers to a preferred microparticle
size
distribution in which the ratio of the volume diameter of the 90th percentile
(i.e., the
average particle size of the largest 10% of the microparticles) to the volume
diameter of
the 10th 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 gaps 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
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used to indicate a monodisperse size distribution. Calculation of GSD is known
and
understood to one skilled in the art.
[00058] "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 microparticles and non-crystalline
microparticles as understood by one skilled in the art.
[00059] "Solid" refers to a state that includes at least substantially solid
and/or semi-
solid, but excludes gel, liquid, and gas.
[00060] "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).
[00061] "Carrier" refers to a compound, typically a macromolecule, having a
primary
function to provide a three-dimensional structure (including tertiary and/or
quatemary
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.
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[00062] "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.
[00063] "Suspension" or "dispersion" refers to a mixture, preferably fmely
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).
[00064] "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.
[00065] "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 the 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 monolayers)
of interest,
preferably when subjected to certain conditions (such as in solution or
suspension).
[00066] "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.,
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conjugates, aggregates, and/or coniplexes thereof), each of which
independently has
and/or is capable of having a net charge of the same sign.
[00067] "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, etc.),
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.
[00068] "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.
[00069] "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 (e.g., solvent, pH).
[00070] "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).
[00071] "Ambient temperature" refers to a temperature of around room
temperature,
typically in a range of about 20 C to about 40 C.
[00072] "Subject" or "patient" refers to animals, including vertebrates like
mammals,
preferably humans.
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[00073] "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 lcidney), 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.
[00074] "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."
[00075] "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 (e.g., monosaccharides,
polysaccharides,
carbohydrates), vitamins, steroids, steroid analogs, cofactors, active agents,
nucleic acids,
viruses, bacteria, toxins, antigens, other ligands, precursors thereof, and
derivatives
thereof.
[00076] "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
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include maleimide groups, disulfide groups (e.g., ortho-pyridyl disulfide),
vinylsulfone
groups, azide groups, and a-iodo acetyl groups.
[00077] "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 non-toxic 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,
fumaric, toluensulfonic, methanesulfonic, ethane dislfonic, oxalic,
isethionic).
[00078] "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.
[00079] "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.
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[00080] "Metabolite" refers to a form of a compound obtained in a subject body
by
action of the body on the administered form of the compound. 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.
[00081] "Diagnostic agent" refers to any material or substance useful in
connection
with methods for perceptually observing (e.g., imaging) a norrnal 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.
[00082] "Cross-link," "cross-linked" and "cross-linking" generally refer to
the
linking 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 free 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
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materials/substances, but otherwise is non-reactive to the
materials/substances), and/or
energy sources (e.g., heating; cooling; high-energy radiations like
electromagnetic, e-
beam, and nuclear; acoustic radiations like ultrasonic; etc.).
[00083] "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.
[00084] "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.
[00085] "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.
[00086] "Dipole-dipole interaction" refers an intermolecular attraction
between two
or more polar molecules, such as a first molecule having an uncharged, partial
positive end
S+ (e.g., electropositive head group like the choline head group of
phosphatidylcholine)
and a second molecule having an uncharged, partial negative end S- (e.g., an
electronegative atom like heteroatom 0, 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.
[000871 "Hydrogen bond" refers to an attractive force or bridge between a
hydrogen
atom covalently bonded to a first electronegative atom (e.g., 0, 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).
[00088] "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
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generally associated with momentary dipole moments induced by neighboring
molecules
undergoing changes in electron distribution.
[00089] "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.
[00090] "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.
[00091] "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).
[00092] "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-
limitin.g
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
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vesicle or an inner and/or outer surface thereof; interspersion, conjugations,
and/or
complexation between different materials).
[00093] "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).
[00094] "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.
[00095] "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).
[00096] "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
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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.
[00097] "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.
[00098] 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 Mõ or weight
average
molecular weight Mw), 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.
[00099] 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.
[000100] "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.
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[000101] 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.
[000102] 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 m or less,
and may
have a monodisperse or polydisperse size distribution.
[000103] Methods of pre-forming the preformed microparticle are not
particularly
limiting, and include those disclosed in U.S. Patent No. 6,458,387 and U.S.
Patent
Publication No. 2005/0142206, which are incorporated herein by reference in
their
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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).
[000104] 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,
PEG-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),
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polypropylene glycol (PPG), 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.
[000105] 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.
[000106] 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
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).
[000107] 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
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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
C/minute to
50 C/minute, preferably 0.2 C/minute to 30 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.
[000108] 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., M 2+ 2+ 2' 2+ 3+ 2" 2
g., , g, Ca, Cu, Fe, Fe), anions (e.g., C03 , S04 ), 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.
[000109] 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,
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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)
NaC1.
[000110] 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 COZ, ethane, and xenon. Non-limiting examples of co-solvents include
acetonitrile,
dichloromethane, ethanol, methanol, water, and 2-propanol.
[000111] 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.
[000112] 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-
inflaixmlatory
agents, antimalarials, antimetabolites, antimuscarinic agents,
antimycobacterial agents,
antineoplastic agents, antiosteoporosis agents, antipathogen agents,
antiprotozoal agents,
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adhesion molecules, antipyretics, antirheumatic agents, antiseptics,
antithyroid agents,
antiulcer agents, antiviral agents, anxiolytic sedatives, astringents, beta-
adrenoceptor
blocking agents, biocides, blood clotting factors, calcitonin, cardiotonics,
chemotherapeutics, cholesterol lowering agents, cofactors, corticosteroids,
cough
suppressants, cytokines, diuretics, dopaminergics, estrogen receptor
modulators, enzymes
and cofactors thereof, enzyme inhibitors, growth differentiation factors,
growth factors,
hematological agents, hematopoietics, hemoglobin modifiers, hemostatics,
hormones and
hormone analogs, hypnotics, hypotensive diuretics, immunological agents,
irnmunostimulants, immunosuppressants, inhibitors, ligands, lipid regulating
agents,
lymphokines, muscarinics, muscle relaxants, neural blocking agents,
neurotropic agents,
paclitaxel and derivative compounds, parasympathomimetics, parathyroid
hormone,
promotors, prostaglandins, psychotherapeutic agents, psychotropic agents,
radio-
pharmaceuticals, receptors, sedatives, sex hormones, sterilants, stimulants,
thrombopoietics, trophic factors, sympathomimetics, 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.
[000113] 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.
[000114] 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,
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D, E, K, and the like), and herbal extracts. The nutritional supplements may
be
commercially available and/or prepared by known techniques.
[000115] As discussed above, the active agent may be a compound having a
molecular
weight of 2kD 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.
[000116] 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 ageints, 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, banlbuterol, tulobuterol, and
salts thereof.
Non-limiting examples of anti-fungal agents iinclude itraconazole,
fluconazole,
amphotericin B, and salts thereof.
[000117] 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)-
2,4,6-triiodobenzoyloxy)propionate (WIN 16923); N-ethyl 2-(3,5-bis(acetamido)-
2,4,6-
triiodobenzoyloxy-acetamide (WIN 65312); isopropyl 2-(3,5-bis(acetamido)-2,4,6-
triiodobenzoyloxy)acetamide (WIN 12855); diethyl 2-(3,5-bis(acetamido)-2,4,6-
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
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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.
[000118] 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.
[000119] 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 naicroparticles may have an overall density
of 0.5
g/cm3 or greater, preferably 0.75 g/cm3 or greater, more preferably 0.85 g/cm3
or greater.
The density may be generally up to about 2 g/cm3, preferably 1.75 g/cm3 or
less, more
preferably 1.5 g/cm3 or less.
[000120] 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 bulking 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%.
[000121] Surface modification of the preformed microparticles is achieved,
without
limitation, by forming, in a controlled manner, at least one monolayer
containing at least
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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.
[000122] 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 microparticle 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.
[000123] The surface-modified microparticle may further contain one or more
additional altematingly 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.
[000124] 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
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and/or swelling, and have a polydisperse or, preferably, monodisperse, size
distribution.
The preformed microparticle, may be free of lipids and/or encapsulation.
[000125] 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
to 7.5, such as a physiological pH of 7 to 7.4 or endosomal pH of 5 to 6.5.
[000126] 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 formation. 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.
[000127] 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
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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 exaniple, 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 tvent, 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.
[000128] 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
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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 posiiive 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).
[000129] 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 compouri.ds, 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 30%. A non-limiting example of the solution contains about 16% (w/v)
polyethylene glycol and 0.7% (w/v) NaCI. 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 C
to 5 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.
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[000130] 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 ZnC12.
[000131] Thus, in one example, deposition of the formed monolayer may take
place in
a solution that includes buffered saline (that is, 0.7% NaCI buffer) and 8% or
more by
weight or volume of a SRAIVIA 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 SRAlVIA 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 work in solutions containing 16% or less SRAlVIA.
[000132] 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 Ahat 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 C
and 15 C, preferably between 1 C and 10 C, more preferably between 2 C and 5
C, most
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preferably less than 5 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
[000133] 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.
[000134] 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 altematingly 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.
[000135] Thus, turning briefly to Fig. 2, co-incubation of the preformed
microparticles
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
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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 tempera.ture during the incubation
and the
optional washing is optimized based on the solubility of the active agent and
the charged
compound 20.
[000136] 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 intermedi.ate 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 altematingly 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.
[000137] 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
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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.
[000138] 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.
[000139] In a furtlier 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.
[000140] 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 C to 10 C, such as 4 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 I
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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 C and 40 C, more preferably between 10 C and 30 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 C or lower, most
preferably 10 C or
lower, such as 4 C. The resulting mixture may fiuth.er undergo optional
washings as
described herein to yield additionally treated, surface-modified,
microparticles.
[000141] 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 exam.ples of suitable
suspensions
include microparticles (such as insulin microspheres) in a buffer, such as a
PEG buffer
containing 16% PEG, 0.7% NaCI, 67mM 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/mi 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 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
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as described above. 'Optional washings may be carried out on the suspension
prior to the
additional treatment.
[000142] The additional treatments may be carried out immediately after the
fornzation
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
monolaye'r on the
preforrn.ed microparticles, the monolaye'r 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, fift.h). In a fiuther 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.
[000143] As described in Exaiuple 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 (%CRIh) 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
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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 (%CR24h) that
is greater
than %CRII,. 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% NaC1, pH 7.4, free of divalent cation) at 37 C, may be
capable of
displaying a %CRIh of 50% or less and/or a ratio of %CR24h to %CRIh of greater
than 1:1.
The OoCRIh may preferably be 40% or less, more preferably 30% or less,
further
preferably 20% or less, most preferably 10% or less. The ratio of %CR24h to
%CR1h 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.
[000144] Without being bound to any particular theory, it is believed that the
additional treatment following the monolayer fomiation 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.
[000145] Deposition of additional alternatingly charged monolayers of charged
compounds beyond the formed monolayer may fiu-ther 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
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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.
[000146] 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 interaction. 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 more 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 polyelectrolytes 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-
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phosphoethanolamine-N-(carboxyfluorescein) (FITC-EA), 1,2-distaeroyl-sn-
glycero-
3[phospho-rac-O(1-glycerol)](sodium salt) (DSPG), 1,2-dipalmitoyl-sn-glycero-3-
phosphate (monosodium salt) (DPPA) 1,2-dioleoyl-3-dimethylammonium-propane (D
[0001471 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.
[000148] 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
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used as a food additive, for prevention and/or treatment of a condition or
disorder in a
subject.
[000149] 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
[000150] 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 (USI QCM System, Model 260303, Sanwa Tsusho
Co.,
Ltd, Japan). To form each monolayer, the resonators were incubated in 0.25 M
NaCI
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.
[000151] 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 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%
NaC1, pH
5.8; b) 16% PEG-0.7% NaC1 pH 7.0; c)0.16% acetic acid-0.026% ZnC12.
Concentrations
of PEG, NaCI and ZnC12 in the buffers for assembly may be varied for
optimization.
[000152] The monolayers, after forming, were dried in a stream of nitrogen
gas. The
frequency change of the resonators following forrnation 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.
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Microparticle Net Surface Charge Measurements
[000153] For microparticle net surface charge (zeta potential) measurements a
Zeta
Potential Analyzer (Model ZetaPALS, Brookhaven Instruments Corp., Holtsville,
NY)
was used. A 40 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 C to minimize microparticle disintegration.
In Vitro Release (IVR)
[000154] 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% NaCI, 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 C. At designated time intervals 400
L of the
IVR medium was transferred into a microfuge tube and centrifuged for 2 minutes
at 13k
rpm. A 300 L aliquot of the supernatant was removed and stored at -80 C until
analyzed
by Bicinchoninic Acid (BCA) assay as understood by one skilled in the art. A
300 L
aliquot of fresh releasing buffer was added to the microfuge tube to
reconstitute the pellet.
The 400 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 dimethyl sulfoxide
(DMSO) and a
surfactant and pH neutralization.
EXAMPLES
Example lA: Microspheres with Polystyrene Sulfonate Monolayer
[000155] 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 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).
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Example 1B: Microspheres with Multiple Alternating Monolayers
of Polystyrene Sulfonate and Polyallylamine Hydrochloride
[000156] The PSS-modified inicroparticles 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 lA, 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
[000157] The procedures of Example lA were used to form a polyanion monolayer
on
insulin microspheres at pH 4.8 (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.
[000158] 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.
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Example 3A: Microspheres with Multiple Monolayers of Oppositely
Charged Polyions
[000159] The resulting microspheres of Examples 1A & 2A were used as
intermediate
microspheres, re-suspended in the aqueous solution (16% PEG, 0.7% NaCI, pH
4.8) in the
presence of 0.3 mg/mL polycation PLL, and incubated for 1 hr at 2 C to fornl 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.
[000160] 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% NaCI. 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
[000161] 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 C. Fomlation of the condroitin sulfate
monolayer, according to the procedures of Exainple lA, 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 forination of each monolayer is shown in FIG. 10.
Example 4A: Monolayer Formation in the Presence of Polyvalent
Cation and PEG
[000162] The procedures of Example 1 B were used to form monolayers of
protamine
and condroitin sulfate about preformed insulin microspheres, with the
exception that the
aqueous solution had a pH of 6.4 and contained 16% PEG, 0.7% NaCI, 0.16% (w/v)
acetic
acid, and 0.026% (w/v) ZnC12. Comparative examples were formed using a Zn-free
&
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acetate-free 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
[000163] 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 1o NaC1,
0.16% acetic
acid, and 0.026% ZnC12. Zeta-potentials of the resulting microspheres are
shown in FIG.
12.
Example 5: Microspheres Surface-modified with Liposomes
[0001641 Liposomes containing 60% cationic lipid 1,2-dioleoyl-3-
dimethylanunonium-propane (DAP) and 20% zwitterionic 1,2-dioleoyl-sn-glycero-3-
phosphocholine (DOPC), suspended in an aqueous solution of 16% PEG, 0.7% NaC1,
0.16% acetic acid and 0.026% ZnC12 at pH 7.0 were used to co-incubate with
preformed
insulin microspheres (pre-washed with the same aqueous solution) at 2 C for 1
hr. The
procedures of Example 4B were applied to form a subsequent monolayer of
chondroitin
sulfate.
[000165] 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
[000166] Protamine-modified insulin microspheres were formed using the
procedures
of Example 2B 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
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[0001671 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
[0001681 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 7Ø 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 (Cm~,,) and the time to achieve Cma, (t,Y,a,Y), 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 Cm,, greater in the
surface
modified microparticles as compared to Cma~, and ta" of the unmodified,
preformed
microparticles. As demonstrated by this Example, the C. of the surface
modified
microparticle was 2.5 fold greater than the CIõa,, of the preformed
microparticle. In other
examples, the Cma,, 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. of the
preformed microparticle.
Table 1.
Parameter Preformed Microparticles Modified Microparticles
AUCo-m 203.3 46.5 780.9 81.3
MRTQ-7h 1.7 0.2 2.9 0.2
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Cmax 103.5 27.3 259.0 52.9
tmax 0.55 0.41 2.60 0.55
Example 9: Surface Modification in Presence of Various Solubility Reducing
Agents
[000169] Aqueous media of protamine sulfate (0.15 mg/mL) used to incubate the
preformed insulin microparticles contained one of PLURONIC 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 lA 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
[000170] Procedures of Example lA 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 protaniine concentration in the reaction
mediumcorrelated
with reduction of insulin release, with an observed maximum effective
concentration of
about 0.3 mg/mL.
[000171] 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
[000172] 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 protamine
sulfate and chondroitin sulfate, respectively, at 2 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
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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
[000173] Preformed intravenous immunoglobulin (IVIG) microspheres were
modified with alternating monolayers of chondroitin sulfate and protanline
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 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
[000174] In order to determine surface charge characteristics of preformed
irisulin
microspheres in solubility reducing media containing 16% PEG, pH of the 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
[000175] At 4 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) NaCI, and 67 mM sodium acetate] at one of the following pH values:
5.7, 5.9,
6.5, and 7Ø 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/mi 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 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.
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The surface-modified microspheres were then subjected to in vitro release
following the
protocol disclosed herein.
[000176] 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 unxnodified insulin microspheres (insulin SNPcore,
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 SNPeore. 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.
[000177] The zeta potential of the PLA-modified insulin microspheres was
higher
(above 20 mV) at reaction pH values close to insulin SNP~ore, and lower (below
20 mV) at
reaction pH values away from insulin SNP.re. 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 SNPcore, and greater (about 40 mV) at reaction pH
values away
from insulin SNPeOre. 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 SNPcore,
and greater (about 40 mV or greater) at reaction pH values away from insulin
SNPcore.
[000178] As shown in FIG. 26, the in vitro 1-hour percentage of cumulative
release
(%CRII,) 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 %CRlh was generally greater at reaction pH values close to insulin
SNPcore than
that at reaction pH values away from insulin SNP,.ore, 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
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%CRIh, 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.
[000179] In cases where it may be desired to have a %CRIh 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
microsplleres) may be surface-modified at reaction pH away from SNPcore using
certain
charged compounds (e.g., protamine sulfate, PLA). In cases where it may be
desired to
have a 1oCR1h 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 SNPcOTe using certain charged compounds (such as PLL).
Example 15: Surface-modified Nucleic Acid Microspheres
[000180] 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 micro
spheres 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 1 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 C for 45 minutes to form the surface-modified microspheres,
which were
then centrifugally washed with nuclease-free 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
[000181] 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) NaC1, and 67 mM sodium acetate at pH 7.0
and
4 C. At a concentration of 6 mg/ml, a 1.5 ml aqueous solution of a polycation
(ProtS,
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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 C with
continuous
agitation for 30 minutes to form the surface-modified insulin microspheres
having the
respective polycation monolayer. Next, the reaction mixtures were fizrther
incubated for
another 30 minutes, at a temperature of 4 C, 15 C, 28 C, or 37 C. Then the
thermally-
treated insulin microspheres were centrifugally (3000 rpm at 4 C for 10
minutes) collected
and washed three times with fresh aliquots of the buffer at pH 7.0 and 4 C.
Zeta potential
data and in vitro release profiles of the thermally-treated, surface-modified
insulin
microspheres were generated as described herein above.
[000182] It was found, unexpectedly, that certain thermal treatments as
described
above (such as incubation at a temperature of 15 C, 28 C, or 37 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 loCRlh being lower following incubation at 28 C than
that
following incubation at 15 C), which are consistently less than that following
incubation
at non-elevated temperature (i.e., 4 C).
[000183] As exemplified in Table 2, the initial release or "burst" phase in
the in vitro
release profiles, as represented by %CR1h, 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 C). The amount of reduction in %CRIh 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 2. Percentage of Reduction in %CRIh of PLL-modified Insulin Microspheres
Sample (2n Incubation %CRIh % Reduction (in %CRIh as compared to
Temp) Control)
Control (4 C) 57.4
Thermal 1 (15 C) 48.3 16
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Thermal 2 (28 C) 33.1 42
Thermal3 (37 C) 34.1 40
The reduction effect of the thermal treatment (at 28 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 3. Effect of Thermal Treatment (at 28 C) on %CRIh of Surface-modified
Insulin Microspheres
Polycation %CRIh without %CR1h with Thermal % Reduction in
Thermal Treatment Treatment (Example %CRIh
(Example 14) 16)
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
[000184] 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, SoCRIh of the surface-modified hGH microspheres
following
incubation at 28 C was reduced as compared to that of the surface-modified hGH
microspheres following incubation at 4 C.
Table 4. Percentage of Reduction in %CRIh of PLA-modified hGH Microspheres
Sample (2n Incubation %CRIh % Reduction (in %CRIh as compared to
Temp) Control)
Control (4 C) 24.9
Thermal 1 (15 C 10.8 57
Thermal2 (28 C) 9.6 61
Thermal 3 (37 C) 13.0 48
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Example 18: Effect of Thermally-treated, Surface-modified Microparticles In
Vivo
[000185] 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 C),
and the
other portion incubated at 4 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) NaCI, 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, CRIh was likewise reduced following heat treatment.
[000186] PLA-modified insulin microspheres treated at 4 C provided comparable
serum insulin concentration profile, serum glucose depression profile, Cma,
and tm.. In
comparison, PLA-modified insulin microspheres treated at 28 C provided
flattened and
right-shifted serum insulin concentration profile, right-shifted serum glucose
depression
profile, depressed Cm,,,, and prolonged tm,~..
Table 5. In Vivo Insulin Release from Different Insulin Microspheres
Parameter Unmodified PLA-modified, 4 C PLA-modified, 28 C
Cmax 479.1 147.0 463.5 136.9 256.8 95.8
tmax 1.1 0.5 1.1 0.5 2.0 0.7
[000187] Fig. 29 A 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 C, or PLA-
modified
insulin microspheres treated at 4 C (Example 18);
[000188] 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 C, or PLA-
modified
insulin microspheres treated at 4 C (Example 18);
[000189] 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.
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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.
