U.S. patent application number 10/517122 was filed with the patent office on 2006-06-08 for hazard-free microencapsulation for structurally delicate agents, an application of stable aqueous-aqueous emulsion.
Invention is credited to Tuo Jin, Hua Zhu, Jiahao Zhu.
Application Number | 20060121121 10/517122 |
Document ID | / |
Family ID | 27404043 |
Filed Date | 2006-06-08 |
United States Patent
Application |
20060121121 |
Kind Code |
A1 |
Jin; Tuo ; et al. |
June 8, 2006 |
Hazard-free microencapsulation for structurally delicate agents, an
application of stable aqueous-aqueous emulsion
Abstract
This invention provides method for sustained release delivery of
structurally delicate agents such as proteins and peptides. Using
unique emulsion system (Stable polymer aqueous-aqueous emulsion),
proteins and peptides can be microencapsulated in polysacchride
glassy particles under a condition free of any chemical or physical
hazard such as organic solvents, strong interfacial tension, strong
shears, elevated temperature, large amount of surfactants, and
cross-linking agents. Proteins loaded in these glassy particles
showed strong resistance to organic solvents, prolonged activity in
hydrated state, and an excellent sustained release profile with
minimal burst and incomplete release when being further loaded in
degradable polymer microspheres. This invention provides a simple
yet effective approach to address all the technical challenges
raised in sustained release delivery of proteins.
Inventors: |
Jin; Tuo; (Tianjin, CN)
; Zhu; Hua; (Plainboro, NJ) ; Zhu; Jiahao;
(Brooklyn, NY) |
Correspondence
Address: |
Albert Wai-Kit Chan;Law Offices of Albert Wai-Kit Chan
World Plaza, Suite 604,
141-07 20th Avenue
Whitestone
NY
11357
US
|
Family ID: |
27404043 |
Appl. No.: |
10/517122 |
Filed: |
June 3, 2003 |
PCT Filed: |
June 3, 2003 |
PCT NO: |
PCT/CN03/00431 |
371 Date: |
January 26, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60418100 |
Oct 11, 2002 |
|
|
|
Current U.S.
Class: |
424/490 ;
264/4 |
Current CPC
Class: |
A61K 9/0043 20130101;
A61K 9/113 20130101; A61K 9/19 20130101; A61K 9/5031 20130101; A61K
9/1075 20130101; A61K 9/1652 20130101; A61K 9/5073 20130101; A61K
9/0073 20130101; A61K 9/0024 20130101; A61K 9/5036 20130101; A61K
38/00 20130101 |
Class at
Publication: |
424/490 ;
264/004 |
International
Class: |
A61K 9/16 20060101
A61K009/16; B29C 39/10 20060101 B29C039/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 3, 2002 |
US |
60384971 |
Nov 8, 2002 |
US |
10291327 |
Claims
1. A method for encapsulating agents into particles through stable
aqueous-aqueous emulsification comprising: a. selecting
polysaccharides as the dispersed phase for aqueous-aqueous
emulsification, selecting aqueous polymers as the continuous phase,
and selecting an stabilizing agent and its concentration for
aqueous-aqueous emulsification, to provide a stable polymer
aqueous-aqueous emulsion which is capable of encapsulating an agent
into the polysaccharide dispersed phase; b. providing at least one
agent; c. controlling the size and shape of the agent-loaded
polysaccharide particles into appropriate size range; d. drying the
emulsion; and e. removing the continuous phase after drying by
washing the sample with solvent(s) which do not penetrate into the
dried dispersed phase nor affect the loaded delicate agent(s).
2. A composition used in the method of claim 1, including an
aqueous dispersed phase, an aqueous continuous phase and an aqueous
surface modifier, capable to form a stable aqueous-aqueous
emulsion.
3. The composition of claim 2, comprising sufficient amount of
polysaccharides or derivatives thereof capable of forming the
dispersed phase of the aqueous-aqueous emulsion and protecting
agents encapsulated.
4. The composition of claim 3, wherein the polysaccharide is
selected from the group consisting of dextran, starch, cellulose
and its derivatives, and agarose and all type of poly- or
oligo-sugars, which possess similar structure.
5. The composition of claim 4, wherein the average molecular weight
of the polysaccharides is ranged from 2,000 to 2,000,000.
6. The composition of claim 3, wherein the agent is a biologically
active agent.
7. The composition of claim 6, wherein the agent is selected from
the group consisting of proteins, peptides, DNA/RNA, liposomes, and
live viruses.
8. The composition of claim 7, wherein the protein or peptide is
selected from the group consisting of erythropoietin (EPO),
granulocyte colony stimulating factor (G-CSF), granulocyte
macrophage colony stimulating factor (GM-CSF), interferon and
.beta., growth hormone, calcitonin, tissue-type plasminogen
activator (TPA), factor VIII, factor IX, hirudin, dornabe, and
other therapeutic proteins or peptides.
9. The composition of claim 3, further comprising a small molecular
sugar as complimentary agents for better protection of agents
encapsulated in the polysaccharide dispersed phase during
successive steps.
10. The composition of claim 9, wherein the small molecular sugar
is selected from trehalose, manitol, sucrose, lactose or
glycerin.
11. The composition of claim 2, comprising an aqueous polymer,
which is immiscible with the polysaccharides, to form the
continuous phase of the aqueous-aqueous emulsion.
12. The composition of claim 11, wherein the aqueous polymer in the
continuous phase is polyethylene glycol (PEG), polyethylene oxide
(PEO), polyvinyl pyrrolidone (PVP), or polyvinyl alcohol (PVA).
13. The composition of claim 12, wherein the average molecular
weight of the polymer is ranged from 2,000 to 2,000,000.
14. The composition of claim 2, comprising an aqueous polymer as
the surface modifier of the dispersed phase.
15. The composition of claim 14, wherein the polymeric surface
modifier is selected from sodium alginate, hyaluronate,
carboxymethyl cellulose, carboxymethyl dextran, dextran sulfate,
and other dextran or starch devertives, or other polymers that
possess negatively charged backbone and positively charged counter
ions.
16. The method of claim 1, wherein the emulsion is dried through
lyophilization, spray drying or a conventional drying process to
solidify the agent-encapsulated polysaccharide dispersed phase.
17. Dried polysaccharide dispersed phase prepared by the method of
claim 16, possessing an average diameter of 1-5 .mu.m for
inhalation and for double microencapsulation, and of 1-50 .mu.m for
other applications.
18. A method of encapsulating dried polysaccharide dispersed phase
into biodegradable polymer microspheres for controlled release of
bioactive agent(s) comprising: a) utilizing a solid-in-oil-in-water
(S-O-W) emulsification process or a solid-in-oil-in-oil process
with the dried polysaccharide dispersed phase as the solid phase;
b) selecting a biodegradable polymer, dissolving the polymer in an
organic solvent and suspending the dried polysaccharide dispersed
phase in the polymer solution; c) selecting polymeric surfactant(s)
for dispersing the solution of the biodegradable polymers in a
water solution of a small molecular salt; d) the concentration of
the slat solution ranges from 0.5% to 50%; e) removing the organic
solvent by extraction or evaporation.
19. The method of claim 18, wherein the biodegradable polymer is
PLGA, poly-pseudo CBZ-serine or other polymers.
20. Particulates of degradable polymers prepared using the method
of claim 18, wherein dried polysaccharide dispersed phase is
distributed in the matrix.
21. Particulates of claim 20, wherein the ratio of dried
polysaccharide dispersed phase to the degradable polymer is within
the range of 1:2 to 1:40.
22. A composition of any one of claims 2-15 for or acceptable for
pharmaceutical applications.
Description
CROSS REFERENCE OF RELATED APPLICATION
[0001] This application claims priority of U.S. Ser. No.
60/384,971, filed Jun. 3, 2002, and U.S. Ser. No. 60/418,100, filed
Oct. 11, 2002, the contents of which are incorporated by reference
here into this application.
[0002] Throughout this application, various references are referred
to. Disclosures of these publications in their entireties are
hereby incorporated by reference into this application to more
fully describe the state of the art to which this invention
pertains.
FIELD OF THE INVENTION
[0003] The present invention demonstrates a novel method for
preparing a novel particulate glassy system which effectively
preserve structure/activity of proteins peptides, DNA, liposomes
and live viruses during formulation process, storage, and
application.
BACKGROUND OF THE INVENTION
[0004] Due to the impermeability and short half-life, most of
protein therapeutics require frequent injection. To reduce
injection frequency, development of sustained release dosage forms
has been a long-standing research focus since 1970s (1). In spite
of extensive research efforts (2), up to now, sustained release
formulation technology has succeeded in only one protein drug,
recombinant human growth hormone (rhGH). The major roadblocks are
invariably the protein instability in formulation process and at
the site of release (3, 4) as well initial burst and incomplete
release.
[0005] Various strategies to improve protein stability in
microencapsulation have been reported in last decades (3, 5, 6).
Many of these approaches, however, only address one or some issues,
leaving others unsolved or even creating new problems. Some methods
are feasible for only specific proteins, and some reports are
contradictory to each other due to different focal points of
researchers. For example, for the only commercially available
long-acting protein, sustained release rhGH, the protein was
stabilized by forming complex with zinc ions (7) based on that
natural hGH forms complex with zinc in secretory granules (8). When
zinc was co-encapsulated with another protein, erythropoietin (EPO)
for example, up to 40% of released proteins was aggregated (9),
which could result in unwanted immunogenisity. In order to protect
proteins from organic solvents used in microencapsulation, sugars,
inorganic salts or other conceivable excipients are used to
preformulate proteins into solid particles prior to
microencapsulating them into degradable polymer microspheres
through a solid-in-oil-water (S-O-W) emulsification process (7, 9,
10). These excipients often resulted in considerable burst release
due to strong osmotic pressure created by their high solubility
(11) and rapid dissolution (12). When highly soluble ammonium
sulfate was used to stabilize EPO in microencapsulation, burst
release accounted up to 55% of total drug (9).
[0006] Cleland and Jones studied the effects of various excipients
on protection of rhGH and interferon (IFN-) in
water-in-oil-in-water (W-O-W) and S-O-W encapsulation processes,
and found that mannitol or trehalose were the best in preventing
proteins from aggregation during microencapsulation process were
prevented (6). Sanchez et al. examined the protection effects of
similar excipients for another protein, tetanus toxoid, and found
dextran, that was ineffective for recovering rhGH and IFN-.gamma.
in Cleland and Jones report, showed best protection for the protein
(based on ELISA) at the release phase under a hydrated condition
(10). It seems that small sugars offer better protection in
dehydration steps (drying), while polysaccharides are more
effective in a hydrated step (release) (13). A burst release of 60%
of total loading was observed from dextran included PLGA
microspheres prepared by Sanchez et al. This burst release may be
attributed to the particle size of the co-lyophilized
protein-excipient particles (14, 15).
[0007] The size of pre-formed protein particles plays an important
role in a S-O-W process. Morita et al. demonstrated that when the
mean diameter of solid protein particles increased from 5 to 20
.mu.m, the initial release almost doubled, and encapsulation
efficiency dropped from 80% to 20% (15). Cleland et al. discussed
different approaches for reducing protein particle size for a S-O-W
process (6). Homogenizing a lyophilized protein-excipients powder
in organic solvents can only result particles above 10 .mu.m in
diameter, while milling the powders to smaller size may cause
protein denature due to the shears and heat generated (6). Spray
drying may produce protein particles to desired size, but shear and
heat at atomization as well as the presence of air-liquid interface
may cause denaturation (6, 16). Moreover, surfactants must be used
in spray drying and spray freeze-drying that facilitate contact and
interaction between proteins and dichloromethane (the solvent most
frequently used in microencapsulation) (6). Maa et al. reported
that complexation of rhGH with zinc prior to spray drying can
effectively prevent aggregation of the protein (16). Again, zinc
complexation can denatrue proteins other than rhGH (9). Morita et
al. prepared fine protein particles by a freezing-induced
precipitation with a co-solution of proteins and PEG (15, 17). But
the protein particles still have to be exposed to organic solvents
directly during microencapsulation. Direct contact of unprotected
proteins with PLGA will cause incomplete release by strong
adsorption of the proteins on the internal surface of the polymer
matrix (18). To avoid the hydrophilic-hydrophobic interface,
aqueous two-phase systems were used for preparing polysaccharide
particles (19, 20). However, the dispersed phases need to be
stabilized by covalent or ionic cross-linking, another potential
cause for protein denaturation.
[0008] For sustained release of delicate proteins, an approach that
can address all these important issues is highly desired. Due to
the long-standing difficulties discussed above, it is unlikely that
this task can be accomplished with the existing approaches.
Microencapsulation strategies based on new scientific concepts are
required.
[0009] In one of our previous patent application, we have reported
(as the first time according to best of our knowledge) a unique
microencapsulation system, stable polymer aqueous-aqueous emulsion
(24). This system differs from conventional emulsions in that both
the dispersed and continuous phases are aqueous. The system is also
different from so-called polymer aqueous two-phase systems that
form two block phases in absence of continuous mixing. This
emulsion is stable for up a week without any (covalent or ionic)
cross-linking treatment. Due to these unique characteristics,
delicate therapeutics such as proteins, liposomes or live viruses
can be loaded into the droplets of this emulsion under a condition
free of chemical or physical hazards such as organic solvents,
concentrated salts, extreme pH, crosslink agents, high shear
stress, high interfacial tension and high temperature. By
freeze-drying or other drying methods, dispersed phase of the
emulsion can form glassy particles of defined shape and uniform
size for various delivery purposes (inhalation or sustained
release). Our previous work has established the proof-of-principle
that all the stability problems raised in protein
microencapsulation, such as the processes of protein loading,
drying, storage and release (3), can be addressed using this unique
system. In addition, all the ingredients used are those proven for
injection into human.
[0010] This present application further demonstrates applications
of this stable aqueous-aqueous emulsion system in delivery of
protein drugs. Proteins can be loaded into the dispersed phase of
the aqueous-aqueous emulsion system and form glassy particles by
freeze-drying thereafter. The entire preparation process is free of
any chemical physical hazards. Protein activity can be well
preserved during this preparation process. Proteins loaded in the
glassy particles made via the emulsion system (called AqueSphere(s)
hereafter) showed strong resistance to organic solvents, prolonged
activity in hydrated state at 37.degree. C., as well as linear
release profile with minimal burst and incomplete release when
being further loaded in degradable polymer microsphere.
SUMMARY OF THE INVENTION
[0011] It is an object of this invention to provide a method to
prepare polymeric microspheres for sustained release of protein
therapeutics. The method is an application of material system,
stable polymer aqueous-aqueous emulsion and AqueSphres
(polysaccharide glassy particles made by solidification of the
emulsion system), which were described in our earlier patent
application (24). The method comprises 1) loading proteins in the
droplets of the stable aqueous-aqueous emulsion system; 2)
preparation of AqueSpheres with diameter ranging between 1-5
microns for inhalation protein delivery; 3) encapsulation of
AqueSpheres into PLGA and other degradable polymer microspheres and
injectable implants; 4) preparation of AqueSpheres loaded with
structurally delicate substances other than proteins (such as
liposomes and live viruses) for inhalation, nasal spray and other
therapeutic uses.
[0012] A major difficulty that delayed development of sustained
release or non-invasive protein formulations is that proteins are
denatured during the formulation process. To prevent protein
denature, a formulation process must be free of (or proteins must
be protected from) the chemical physical hazards discussed above.
In achieving this objective, however, properties and functions of
the final product such as particle size and shape, release profile,
loading efficiency, prolonged activity at the site of release and
so forth should not be compromised. It is also preferred that the
manufacture process can be easy, reproducible and environmentally
friendly.
[0013] The present invention has demonstrated a simple solution for
all these objectives above.
[0014] First, fragile biological agents such as proteins can be
loaded into the dispersed phase of the stable polymer
aqueous-aqueous emulsion system (24) under a condition free of any
chemical or physical hazard. A uniform size distribution of the
droplets can be achieved by a conventional emulsification process
under appropriate shear stress and low interfacial tension (due to
the aqueous-aqueous nature). Then the system can be freeze-dried to
dry powder in which the polymer droplets converted to glassy
particles of uniform sizes (1-5 um in diameter). Once the glassy
particles are formed, the structure of the loaded are preserved and
protected. Due to its hydrophilicity and high glassy transition
temperature, the system offers strong resistance to organic
solvents as well as resistance to ambient temperature and moisture
(in terms of protein activity retention). The bio-agents-loaded
AqueSpheres can therefore be used for inhalation drug delivery
(based on their size range) or subjected to further formulation
process with biodegradable hydrophobic polymers for sustained
release.
[0015] For preparation of sustained release microspheres,
AqueSpheres can be loaded into PLGA (or other degradable polymers)
microspheres by conventional solid-in-oil-in-water (S-O-W) or
solid-in-oil-in-oil (S-O-O) emulsification methods. A recovery
experiment from PLGA microspheres indicated that the AqueSpheres
remain intact inside of the microspheres (Example 4).
[0016] Bioactivity of the proteins loaded in AqueSpheres was
retained after contacted with organic solvents and after
microencapsulation process as assayed in cell proliferation
(Example 5, 6, and 7), indicating that conformation of proteins
were well protected in the glassy matrix of polysaccharide. In
addition, the activity retention of proteins after miroencapsuleted
in PLGA microspheres (Example 7) suggests high encapsulation
efficiency.
[0017] The most challenging task in developing sustained release
protein dosage forms is to ensure protein activity in a hydrated
state at physiological temperature (21). Hydration and temperature
elevation will increase the mobility of proteins and lower the
energy barrier for protein hydrolysis, aggregation and conformation
change. With the present technology, proteins loaded in AqueSpheres
showed prolonged activity in a hydrated state at 37.degree. C.
(Example 8). Recombinant human erythropoietin (rhEPO) which has in
vivo half life of 8.5 hrs and in vitro half life of a day showed a
half life of a week under a hydrated condition when loaded in
AqueSpheres (Example 8). The AqueSphere matrix formed a viscous
phase surrounding the proteins so that limited protein mobility and
the chance for proteins to contact with each other and other
species (the degradable polymer and enzymes).
[0018] Burst effect, defined as rapid release of considerable
amount of loadings in the initial period of administration, is
another common problem in developing sustained release dosage forms
of protein drugs. Burst effect is found for both injectable
implants and microsphere formulations, although the causes may be
different. Accompanying with burst effect is incomplete release
that part of the proteins loaded strongly interact with the polymer
matrix and are not able to release in the required period. Having
proteins pre-encapsulated in AqueSpheres prior to loading into
degradable polymers can effectively prevent burst effect, and at
the same time, reduce the portion of incomplete release (Example
9).
[0019] Moreover, AqueSpheres helps to reduce local acidity
generated by polymer degradation. Local acidity is another cause
believed for protein denaturation during release period.
AqueSpheres form inter-connected channels when being hydrated in
degradable polymer matrix that their viscous nature limits
diffusion of macromolecular proteins but permeable to small
molecular buffers. This nature allow the local acidity be buffered
in the sustained release process. In addition, the surface modifier
(sodium alginate) itself possesses significant buffer effect.
[0020] This invention provides a simple yet effective solution for
all the long-standing technical difficulties in developing
sustained release protein microspheres (3-5).
DETAILED DESCRIPTION OF THE FIGURES
[0021] FIG. 1. Stable polymer aqueous-aqueous emulsion loaded with
myoglobin in the dispersed phase. The picture was taken one week
after the samples were prepared.
[0022] (1) Dispersed phase: 1 ml, containing 5 w/w % myoglobin and
20 w/w % dextran; Continuous phase: 5 ml, containing 1 w/w % sodium
alginate and 20 w/w % PEG.
[0023] (2) Dispersed phase: 1 ml, containing 5 w/w % myoglobin and
20 w/w % dextran; Continuous phase: 10 ml, containing 1 w/w %
sodium alginate and 20 w/w % PEG.
[0024] (3) Dispersed phase: 0.5 ml, containing 5 w/w % myoglobin
and 20 w/w % dextran; Continuous phase: 10 ml, containing 1 w/w %
sodium alginate and 20 w/w % PEG.
[0025] (4) Dispersed phase: 1 ml, containing 5 w/w % myoglobin and
20 w/w % dextran; Continuous phase: 5 ml, containing 20 w/w %
PEG.
[0026] (5) Dispersed phase: 1 ml, containing 5 w/w % myoglobin and
20 w/w % dextran; Continuous phase: 5 ml, containing 1 w/w % sodium
alginate, 20 w/w % PEG and 10 mM NaCl.
[0027] (6) Dispersed phase: 1 ml, containing 5 w/w % myoglobin and
20 w/w % dextran; Continuous phase: 5 ml, containing 1 w/w % sodium
alginate, 20 w/w % PEG and 100 mM NaCl.
[0028] The brown dispersed phase (myoglobin/dextran) in samples (4)
and (6) started to fuse right after preparation and formed a block
phases at the bottom of over night. Those in sample (1), (2), (3)
and (5) were unchanged in a week as observed using a
microscope.
[0029] FIG. 2. Microscopic images of stable aqueous-aqueous
emulsion and polysacchride particles.
[0030] (2A) Microscopic image of the stable aqueous-aqueous
emulsion shown in FIG. 1-1; (2B) microscopic image after (2A) was
freeze-dried and washed with dichloromethane (to remove the dried
PEG phase
[0031] FIG. 3. Preparation of polylactic-glycolic acid (PLGA)
microspheres by a S-O-W double emulsification
[0032] 3A) Microscopic image of a S-O-W double emulsion for which
AqueSpheres are evenly suspended in the organic PLGA phase.
[0033] 3B) Solidified PLGA microspheres in which AqueSpheres are
encapsulated.
[0034] FIG. 4. Microscopic image of AqueSpheres recoved from PLGA
microspheres (as shown in FIG. 3B). The size and shape of recovered
AqueSpheres are identical to that before encapsulated in PLGA
microspheres (FIG. 2B).
[0035] FIG. 5. Comparation of catalytic activity of
.beta.-galactosidase assayed at each step of microencapsulation
using AqueSphere technology.
[0036] Compared with .beta.-galactosidase loaded in a fresh
aqueous-aqueous emulsion, its activity only slightly reduced in
subsequent steps.
[0037] FIG. 6. Bioactivity of rhEPO assayed by proliferation of TF1
cells after each preparation step.
[0038] Equivalent amounts of rhEPO were reconsitituted and
incubated with TF1 cells after emulsification, freeze-drying, and
washing with dichloromethane, respectively. Cells proliferated were
counted under a microscope. Numbers of cells per well were averaged
from three wells.
[0039] FIG. 7. Bioactivity of recombinant human granulocyte
macrophage colony stimulating factor (rhGM-CSF) assayed by
proliferation of TF1 cells after each preparation step.
[0040] Equivalent amounts of rhGM-CSF were reconsitituted and
incubated with TF1 cells after emulsification, freeze-drying,
washing with dichloromethane, and recovery from PLGA microspheres
in which the protein was encapsulated, respectively. Cells
proliferated were counted under a microscope. Numbers of cells per
well were averaged from three wells.
[0041] FIG. 8. Bioactivity of rhEPO assayed by proliferation of TF1
cells after incubation in a hydrated form at 37.degree. C. Activity
after incubattion in a hydrated state at physiological temperature:
The protein loaded in AqueSpheres was added with water twice of
their mass and incubated 37.degree. C. for different days prior to
cell culture. The activity was indicated by the average number of
cells grew in each well. For control, equivalent amount of rhEPO
was incubated in a PBS buffer and assayed under identical
conditions.
[0042] FIG. 9. Bioactivity of rhGM-CSF assayed by proliferation of
TF1 cells after incubation in a hydrated form at 37.degree. C.
Activity after incubattion in a hydrated state at physiological
temperature: The protein loaded in AqueSpheres was added with water
twice of their mass and incubated 37.degree. C. for different days
prior to cell culture. The activity was indicated by the average
number of cells grew in each well. For control, equivalent amount
of rhGM-CSF was incubated in a PBS buffer and assayed under
identical conditions.
[0043] FIG. 10. Catalytic activity of AqueSphere-loaded
.beta.-galactisidase as a function of incubation time in a hydrated
state at 37.degree. C. The activity was compared with that
incubated in a trehalose solution. Concentration of sugars (or
polysaccharide) was 30 w/w % in both hydrated AqueSpheres and
trehalose.
[0044] FIG. 11. Release profile of myoglobin from PLGA
microspheres. The release study was carried out by suspending 50 mg
microspheres in 2 ml of 0.1 M BPS buffer at 37.degree. C. Amount of
myoglobin released was assayed using a BCA method. .diamond-solid.:
Pure myoglobin particles directly encapsulated in microspheres made
of ester-end PLGA with lactide/glycolide ratio of 50/50 and
molecular weight of 6K; .diamond.: Myoglobin-dextran particles
encapsulated in microspheres made of the same PLGA as above.
[0045] FIG. 12. Release profiles of myoglobin microencapsulated in
PLGA microspheres as AqueSpheres. .largecircle.: from microspheres
of PLGA with lactide/glycolide ratio (L/G) of 50/50 and molecular
weight (MW) of 12K; .quadrature.: from microspheres of PLGA with
L/G of 65/35 and MW of 12K; .DELTA.: from microspheres of PLGA with
L/G of 75/25 and MW of 12K; .box-solid.: from microspheres of PLGA
with L/G of 65/35 and MW of 20K.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The present invention provides a method to use polymer
aqueous-aqueous emulsion system [24] to deliver proteins and other
biological agents in a sustained release dosage forms. When
biological agents are loaded in a polysaccharide solution, followed
by emulsification and freeze-dry, their structure is "fixed" in a
hydrophilic glassy matrix. Such glassy particles (AqueSpheres)
offer a series advantages that cannot be all achieved by any other
reported method.
[0047] Small and uniform particle sizes of pre-protected proteins
play an important role in control of the burst release and
improving encapsulation efficiency in a S-O-W or a S-O-O
micronecapsulation process (6, 13). This invention provides a
method to prepare protein-loaded polysaccharide glassy particles of
defined shape and uniform size (1-3 um, Examples 1 and 2) under a
condition free of organic solvents, strong interfacial tension,
strong shears, elevated temperature, large amount of surfactants,
and (covalent or ionic) cross-linking agents. These factors are
known to denature proteins in one or several steps of
microencapsulation process (3,6,21). As discussed above, however,
no a method known to date (W/O emulsion, spray drying, spray
freeze-drying, freeze-drying, milling, and in situ cross-linking)
can be used for preparing protein particles without compromising
with the hazards above.
[0048] In addition, spray drying and spray freeze-drying can only
be used to prepare particles with low molecular weight sugars or
salts as the protein stabilizers because polysaccharide solutions
are too viscous to spray. The present stabilized emulsification
method allows viscous aqueous solution be easily dispersed. As
discussed later, polysaccharide stabilizers possesses a number of
advantages for both protein stabilization and release kinetics.
[0049] Once loaded in the polysaccharide particles, delicate
proteins can be protected from contact with organic solvents during
microencapsulation processes. .beta.-galactosidase, recombinant
human ethrypoietin (rhEPO) and recombinant human granulocyte
macrophage colony stimulating factor (rhGM-CSF) were loaded in
AqueSpheres and washed with dichloromethane (DCM) and/or
encapsulated in PLGA microspheres with DCM as the solvent. The
bioactivity of these proteins can be well retained as determined
with activity assay after the preparation treatments (Examples 5,
6, and 7). Contact with organic solvents is believed as the major
chemical hazards in microencapsulation processes using degradable
polymers (3).
[0050] In addition to resistance to organic solvents, AqueSpheres
can protect proteins from aggregation and conformation change in a
hydrated state at physiological temperature. Protecting delicate
protein under such a condition is regarded as the most challenging
technical hurdle in sustained protein release (21). We incubated
hydrated-AqueSphres, loaded with rhEPO, rhGM-CSF and
.beta.-galactosidase respectively, at 37.degree. C., and found that
protein activity were well retained (Examples 8). For rhEPO, its
half-life in hydrated AqueSpheres was 7 times longer than that in a
BPS buffer (FIG. 8 and Example 8). For rhGM-CSF, there was no
significant declining in bioactivity after incubation for 9 days
(FIG. 9, and Example 8). For .beta.-galactosidase, a comparison was
made between AqueSphres and trehalose (a well recommended protein
stabilizer (6) matrix under the same incubation condition. Proteins
protected by AqueSpheres were 5 times active as that protected by
trehalose after incubation for a week (FIG. 10 and Example 8).
[0051] AqueSpheres, when encapsulated in degradable polymer
microspheres, offer an ideal release profile with extended linear
kinetics and free of burst. Polylactic-glycolic acid (PLGA)
microspheres are know to release loaded macromolecules in three
phases (22): an initial burst due to rapid diffusion of the
molecules located in the surface region (25) or internal
water-filled pores (14) of the microspheres, a lag phase after the
initial burst, and an accelerated release due to bulky degradation
of the polymer. A burst effect, for which more than 50% loading may
be released in the first day after administration, may be dangerous
for many therapeutic agents. Due to the small and uniform size,
particles prepared by this method dispersed evenly in the matrix of
degradable polymers (Example 3) that there is no a surface-rich
protein distribution. In addition, unlike small molecular weight
protein stabilizers that readily dissolve (cause high osmotic
pressure (11)) and rapidly diffuse out of the polymer matrix,
AqueSpheres form a viscous phase that fills the diffusion channels
when hydrated. Since the molecules of polysaccharide themselves
diffuse gradually from the polymer matrix (23), protein burst can
be suppressed (Example 9) by the viscous stabilizers. Moreover, the
diffusion process may be extended so that it overlaps with the
degradation process to give a single phase release kinetics
(Example 9).
[0052] Interaction between proteins loaded and the degradable
polymers is another problem that causes incomplete release and
insoluble protein aggregation (18). In the present method, the
protein molecules are surrounded by the viscous polysaccharides in
side of a hydrated microsphere during the entire release period
(23) so that the chance for protein-polymer contact is reduced.
Release profiles of myoglobin encapsulated in PLGA microspheres
directly and the encapsulated through AqueSpheres are compared in
FIG. 11 (Example 9). For direct microencapsulation, less than 20%
of the loaded proteins were release over 45 days. While for
encapsulation through AqueSpheres, 70% of the loadings were
released for the same period.
[0053] Local acidity in the PLGA matrix is another cause for
protein denature (3). When the polymer degrades, the degradation
products (lactic acid and glycolic acid as well as their oligomers)
may be trapped inside of the polymer matrix and cause the local pH
to decrease. In our system, AqueSpheres form an interconnected
viscous phase when hydrated. These viscous channels, although less
permeable to macromolecular proteins, are permeable to small
molecular buffers so that the acidity degradation fragments may be
buffered. In addition, alginate used as the surface modifier for
the aqueous-aqueous emulsion (example 1) possesses a buffer effect.
In a titration test, the pH was stabilized around 5 when 100 .mu.l
of 0.1 N HCl was added to 0.9 ml 150 mM (based on the monomer)
alginate solution. For same amount of water, 10 .mu.l of the same
acid caused pH to drop to 1.
[0054] The present invention provides, as the first time, a simple
yet inclusive solution by which all the technical challenges in
sustained release protein delivery can be addressed. With this
method, delicate proteins can be protected in steps of both
formulation and administration, and release approximately
constantly with minimal burst and incomplete release. The system
demonstrated is expected to have a wide variety of applications for
delivery of delicate therapeutics.
[0055] The invention will be better understood by reference to the
Examples which follow, but those skilled in the art will readily
appreciate that the specific examples are only illustrative and are
not meant to limit the invention as described herein, which is
defined by the claims which follow thereafter.
EXAMPLES
Example 1
Stability of Polymer Aqueous-aqueous Emulsion
[0056] Stability of polymer aqueous-aqueous emulsion was examined
by observation of the fusion (the size change) of the dispersed
phase under a microscope and by observation of formation of block
phases of the colored dispersed phase directly by eyes as a
function of time. The dispersed phase was formed by a dextran
solution. Three concentrations of the dextran solution, 5, 20 and
40 w/w %, were used in the experiments without significant
difference in the results, i.e. for either of the concentration,
stable aqueous-aqueous emulsion was formed. For the average
molecular weight of dextran, <M>.sub.W=10,000, 67,000 and
500,000 were tested without significant difference in results. The
continuous phase contained PEG with concentration 5, 20, and 40 w/w
% in different tests, for all of which, stable emulsion was formed.
Average molecular weight of PEG used were 8000 and 22,000. As
emulsion stabilizers, sodium alginate, carboxymethyl dextran,
carboxymethyl cellulose were tested. All these stabilizing agents
showed effectiveness in stabilizing the aqueous-aqueous emulsion.
Sodium alginate (<M>.sub.W was represented by low, medium or
high viscosity) was used in most of experiments for its abundant
sources. The concentration of the emulsion stabilizers, 0.2, 1, 5
w/w %, were used in experiments, respectively. The emulsion
stabilizers were co-dissolved with the dispersed phase and the
continuous phase, respectively. No significant difference in
emulsion stability was observed. For direct observeation, colored
molecules, blue dextran (<M>.sub.W=50,000 and 1,000,000) or
myoglobin was added into the dispersed phase as an indicator.
[0057] Emulsions with various concentrations of sodium chloride
were prepared by adding the dextran solution to the PEG solution,
followed by homogenizing with a mechanic homogenizer. Dextran to
PEG ratio was 1:5 to 1:20. After the emulsions were prepared, a
drop of the sample was subjected to a microscope for microscopic
image taking. Then the samples were left in bottles for continuous
observation.
[0058] FIG. 1 shows a picture of a polymer aqueous-aqueous emulsion
after mixing was stopped and the emulsion stored at room
temperature for a week. Myoglobin was used as a model protein that
was loaded in the dispersed phase, showing the rusty color. Among
the six samples, sample 4 was prepared without sodium alginate.
Sample 6 was the same as samples 1 except that sodium chloride was
added (to reach 100 mM). For these two samples, fusion of the
dispersed phase occurred right after stirring was stopped, which
led to formation of two block phases in a few hours. For the other
four samples in FIG. 1, the droplet diameter remained in the range
of 3-7 .mu.m (FIG. 2 A) during the week. This result supported our
hypothesis that charged polymer molecules adsorbed at the droplet
surface and created a diffuse double layer. Increasing the
concentration of sodium ions, the counter ions of alginate, shelled
the surface charge, reduced the magnitude of the zeta potential,
and thus caused droplets to coalescence. Reducing the dextran/PEG
ratio to 1:15 lead to an emulsion stable for two weeks.
[0059] In this experiment, the partition coefficient of myoglobin
between the continuous phase and the dispersed phase was 1:50, as
determined by absorbance at 410 nm, indicating that the majority of
myoglobin was in the dextran phase. In addition to myoglobin,
recombinant human granulocyte macrophage colony stimulating factor
(rhGM-CSF) and liposomes carrying amphotericin B (AmB) were also
loaded in the system and formed glassy particles similar to those
in FIG. 2B. About 93% of rhGM-CSF and 95% of AmB/liposomes were
partitioned in the dispersed phase as detected by activity assay
(See later discussion) and UV absorbance at 408 nm,
respectively.
[0060] FIG. 2A shows a microscopic image of a polymer
aqueous-aqueous emulsion prepared with the sodium chloride free
solutions described above. Emulsion droplets with a uniform size
distribution between 3-8 microns in diameter were obtained.
Example 2
Preparation of AqueSpheres
[0061] AqueSpheres were prepared simply by freeze-drying the stable
emulsions of above. After freeze-drying, the dextran droplets
converted to solid particles. However, the most of dextran
particles were dispersed in a solid matrix formed by the continuous
phase, PEG. The PEG can be removed by washing the lyophilized
powder with methylene chloride or acetonitrile. These solvents did
neither dissolve nor swell the dried dextran phase. FIGS. 2A and 2B
showed the microscopic images of the dispersed phase at different
preparation stages: after emulsification, after freeze-drying
followed by rinsing with dichloromethane (to remove PEG), and after
recovery from PLGA coating, respectively. After freeze-drying, the
diameter of the dispersed phase remained uniform but dropped from
3-7 .mu.m to 1-3 .mu.m, a reasonable size reduction from loss of
water (See FIG. 2B). These images indicated that no droplet fusion
occurred during lyophilization. This size range of the dried
particles (1-3 .mu.m) is ideal for inhalation delivery of
therapeutics and is also suitable for preparation of degradable
polymer coated microspheres via double encapsulation (S-O-W) (5,
13).
Example 3
Microencapsulation of AqueSpheres into PLGA Microspheres
[0062] AqueSpheres can be further microencapsulated into the matrix
of PLGA and other biodegradable polymer microspheres through a
"solid-in-oil-in-water" emulsification process. In the present
study, PLGA with lactic:glycolic ratio of 50:50 and 75:25 were
used. AqueSpheres prepared as in Example 2 were first suspended in
a PLGA/dichloromethane solution (10-20%) at the AqueSphere/PLGA
ratio of 1:2 to 1:20, then added into a water solution containing
0.1-10% sodium chloride and 0.1-4% polyvinyl alcohol (PVA) or PEG
or polyvinyl parralidone (PVP) under stirring. The volume ratio of
the two solutions was 1:2 to 1:10. After an emulsion was formed,
the organic solvent was extracted by pouring the system into large
volume of cold water (10 times of the emulsion) under stirring.
FIGS. 3A and 3B show the microscopic images of the PLGA droplets
before solvent extraction and PLGA particles after solvent
extraction, respectively. Before solvent extraction, the PLGA
droplets were transparent within which the encapsulated AqueSpheres
were evenly distributed. After hardening by solvents removing, the
PLGA particles lost transparency.
Example 4
Recovery of AqueSpheres from PLGA Particles
[0063] AqueSpheres can be recovered from the PLGA microspheres
prepared as in Example 3. AqueSpheres loaded PLGA particles were
re-dissolved in dichloromethane or acetonitrile, followed by
centrifugation. This procedure was repeated 4 to 6 times. FIG. 4
shows the AqueSpheres recovered from PLGA microspheres by the above
mentioned procedure. The particle size and shape of AqueSpheres
remain the same as before being encapsulated in PLGA microspheres.
The result suggests that hydration of AqueSpheres during the
microencapsulation process is not significant.
[0064] A weight measurement was carried out to examine
encapsulation efficiency of AqueSpheres by the PLGA microspheres. A
relatively constant weight ratio of dextran to PLGA was obtained
before (1:19) and after (1.06:19) microencapsulation, suggesting
high encapsulation efficiency. This conclusion consists with our
result on protein activity assay before and after encapsulation
(See Example 7).
Example 5
Protection of .beta.-galactosidase by AqueSpheres Against Organic
Solvents
[0065] To examine the effectiveness of AqueSpheres in protecting
delicate proteins against organic solvents, .beta.-galactosidase,
an enzyme with quadral structure and molecular weight of 434 KD,
was loaded into AqueSpheres. The protein was dissolved in a dextran
solution (MW=10-500 KD, 5-25% in concentration) at the ratio of
10-100 units/ml and emulsified into a PEG solution as in Example 1.
After freeze-dying, the PEG phase was removed by washing with
dichloromethane (a popular solvent used in preparation of PLGA
microspheres) several times as in Example 4. Then, the obtained
protein-loaded AqueSpheres were re-dissolved in a buffer and
assayed by hydrolysis of o-nitrophenyl-.beta.-D-galactopyranoside
(ONPG). As indicated in FIG. 5, the catalytic activity of the
enzyme only decreased less than 10% after the procedure from
Example 1 through Example 2 (included emulsification, freeze-drying
and washing with dichloromethane). The result was reproducible by
three runs. This 10% activity loss includes loss of the proteins by
partition between the dextran and PEG phases in the emulsification
process and by the washing process, as well as those denatured in
freeze-drying and in the washing process and lost during the
washing process. This result indicates that delicate proteins
inside of AqueSperes can be well protected against organic solvents
during microencapsulation process.
Example 6
Partition of rhEPO and rhGM-CSF in the Dispersed and the Continuous
Phases of the Aqueous-aqueous Emulsion
[0066] A partition experiment was carried out to determine the
efficiency of proteins being loaded in the dispersed phase of the
emulsion system. The aqueous-aqueous emulsion containing
recombinant human erythropoietin (rhEPO) or recombinant human
granulocyte-macrophage colony stimulating factor (rhGM-CSF) was
centrifuged, followed by a cell proliferation assay using a TF1
cell line. Protein activity was measured by counting the numbers of
cells per well under a microscope. About 94% of rhEPO and 93% of
rhGM-CSf were found in the dextran phase by the partition
experiment.
Example 7
Protection of rhEPO and rhGM-CSF by AqueSpheres Against Organic
Solvents
[0067] Protein-protection by AqueSpheres was further examined with
the two proteins rhEPO and rhGM-CSF. The proteins were loaded in
AqueSpheres and treated according the procedure identical to that
in Example 5. Bioactivity of the proteins was assayed by the same
cell proliferation method as for partition (Example 6). The
proteins before encapsulation and recovered from AqueSpheres (after
washing with dichloromethane) were added into same cell
suspensions, respectively. The result for rhEPO is shown in FIG. 6.
After freeze-drying, the activity retention for rhEPO was 85% as
indicated by the drop of cell count from 27800 to 23700 per well.
Washing the lyophilized powder (so the Peg phase was removed)
resulted a further drop of the cell count to 22600, indicating that
the activity retention was 95%. Because only 94% of proteins were
remained in the dextran phase after washing with organic solvent
(Example 6), the activity retention was 100% after contact with the
organic solvent.
[0068] FIG. 7 shows the result of activity assay for rhGM-CSf after
each preparation step. Freeze-drying the protein-loaded emulsion to
a dry powder caused the average number of cells per well slightly
reduced from 130900 to 122600, indicating roughly 94% of activity
retention. After washing the freeze-dried powder with
dichloromethane to remove residual PEG, the cell count decreased to
111100 per well, a 9% further reduction. Much of this 9% reduction,
however, was caused by rhGM-CSF partitioned in the continuous phase
(about 7% of total rhGM-CSF, Example 6) that was washed away along
with PEG. Encapsulating the protein-loaded dextran particles into
PLGA microspheres did not cause further activity decrease as
indicated by an average cell count of 118900 per well. The high
activity retention also indicated high encapsulation efficiency
that was indicate by a weight measurement (Example 4).
Example 8
Activity Retention of rhEPO, rhGM-CSF and .beta.-galactosidase by
AqueSpheres in Hydrated State at Physiological Temperature
[0069] It has been widely believed that the most challenging task
in developing sustained release dosage forms of protein drugs is to
ensure protein activity in a hydrated state at physiological
temperature (18). During sustained release, the degradable polymer
microspheres will absorb water and swell, and the encapsulated
protein molecules will be exposed to a hydrated condition at body
temperature. Hydration and temperature elevation will increase the
mobility of protein molecules that increases the chance for
chemical or physical changes of protein (19). To examine protein
stability under physiological conditions, water was added to the
dextran particles loaded with rhEPO or rhGM-CSF (to formed a
viscous 30 w/w % dextran solution) and incubated at 37.degree. C.
Protein activity in FT1 cell proliferation was shown in FIGS. 8 and
9 as a function of incubation time.
[0070] For rhEPO, activity of those protected by AqueSpheres
gradually declined to about 50% in a week (FIG. 80). For
unprotected rhEPO, however, the same amount of activity declining
took only one day. Half-life of rhEPO is 8.5 hrs in vivo due to
enzymatic catalysis in the body. Clearly the viscous polysaccharide
phase, formed by hydration of AqueSphere, can extend the protein
activity at physiological condition for significant period of
time.
[0071] Similar result was obtained for rhGM-CSF (FIG. 9). For
protected rhGM-CSF, activity retention was 85% after 10 days of
incubation. That of unprotected VhGM-CSF was 56% for the same
incubation period.
[0072] The protection effect of polysaccharide stabilizers for
.beta.-galactosidase in hydrated state was compared with that of
trehalose. The activity assessment was carried out same as in
Example 5. After 7 days of incubaion at 37.degree. C., the activity
for the protein stabilized by polysaccharide declined to 89% while
that stabilized by trehalose declined to 17%. Extending the
incubation time to two weeks resulted in a further activity
reduction to 48% for hydrated AqueSpheres but 0% for that incubated
in trehalose solution.
Example 9
Protein Release Profile with Mimimal Burst and Incomplete Release
from PLGA Microspheres
[0073] Burst effect and incomplete release are another common
problem in development of sustained release dosage form of protein
drugs. Due to burst effect, 30-70% of proteins loaded maybe release
immediately after administration. Incomplete release referes to
that 20-40% of the loadings remained as insoluble residues. This
undesired release can be prevented by pre-loading proteins in
AqueSphere. The protein was loaded into AqueSpheres (0.1-20%)
through the aqueous-aqueous emulsification process first. Then the
protein-loaded AqueSpheres were encapsulated in PLGA microspheres
(1-20%) using a S-O-W technique. Loading capacity of myoglobin in
PLGA was 0.25 to 5%. PVA, PEG and PVP were dissolved in the water
phase (0.1-5%) as surfactants. FIG. 11 shows release profiles of
myoglobin encapsulated to PLGA (with the end group blocked)
microspheres with and without protection of AqueSpheres. When
myoglobin was encapsulated as pure protein particles into
microspheres made of ester-end PLGA, only 17% of the loaded protein
was released over 45 days. For myoglobins encapsulated after
pre-loaded in AqueSpheres, up to 75% of the loaded protein was
released linearly over 45 days without a burst release in the
beginning. Such a burst-free linear release was also achieved when
the myoglobin-dextran particles were encapsulated in microspheres
of a relatively hydrophilic acid-ended PLGA (FIG. 12).
[0074] FIG. 12. shows the myoglobin release profiles from
microspheres made of acid-end PLGA (molecular weight=12K) with
lactide:glycolide ratio of 50:50, 65:35 and 75:25, respectively.
For all these samples, myoglobin were pre-formulated to AqueSpheres
prior to encapsulation into PLGA microspheres. About 7 to 12%
loadings were released in the first day, followed by a linear
kinetics. From microspheres made of PLGA with L/G of 50/50 and
65/35 and MW of 12K, protein release was over 90% in 50 days,
almost complete. Increase in the L/G ratio from 65/35 to 75/25
resulted in slightly a decreased release rate as that 80% of
loadings was released in the same time period. Release rate also
declined by increase of molecular weight (MW) from 12K to 20K. For
the PLGA with L/G ratio of 65/35, 65% of myoglobin encapsulated was
released during 50 days. In either of the cases, the release
profile were almost linear. Encapsulation efficiency of myoglobin
into PLGA microspheres by this methods was about 90% based on
analysis of the protein content in the supernatant of after the
preparation process.
Example 10
Bioactivity of GM-CSF Released from PLGA Microsphers
[0075] The protein, rhGM-CSF was loaded into PLGA microspheres
through AqueSpheres as the methods described in Example 1, 2 and 3.
The protein to dextran ratio was 1:500 and the AqueSphere to PLGA
ratio was 1:5. The rhGM-CSF loaded PLGA microspheres were suspended
in a buffer solution and incubated at 37.degree. C. The supernatant
was collected each day and replaced by fresh buffer. The collected
supernatant was diluted by 20 times and assayed as in Example 7.
The activities measured are plotted against the sampling dates in
FIG. 13. The activity was roughly constant up to day 24 after
incubation, then dropped to the level of negative control at day
32.
[0076] It has been widely recognized that local acidity generated
inside of the PLGA microsphres due to the polymer degradation is
one of the major cause for protein denature during the release
period {26}. To examine the effect of scidity on the activity of
rhGM-CSF, the protein was incubated in dextran solutions at pH of
1, 2, 3, 4, 5 and 6, respectively, for one day prior to activity
assay. Compared with the sample incubated at pH 6, the activity
reduced by 75% at pH 4, and reduced to 45% when the pH was below 2.
This pH dependent activity declining was not observed for the
protein released from the PLGA microspheres (FIG. 13). This result
suggests that local acidity was not accumulated in the matrix of
the PLGA microspheres. Probably AqueSpheres formed viscouse
channels upon hydration which is, although less permeable to
macromolecular agents, highly permeable to small molecular buffer
so that the acidic group generated by PLGA degradation were
buffered during the protein release period.
REFERENCES
[0077] 1. R. Langer, Folkman, J., Nature 263, 793-800 (1976).
[0078] 2. CAS, "Search on Chemical Abstracts resulted in 962
research articles and patents on the subject of sustauned release
of proteins based on degradable polymers." (2002). [0079] 3. M. V.
Weert, Hennink, W. E., Jiskoot, W., Pharm. Res. 17, 11591167
(2000). [0080] 4. R. T. Bartus, Tracy, M. A., Emerich, D. F., Zale,
S. E., Science 281, 1161-1162 (1998). [0081] 5. P. A. Burke,
Handbook of pharmaceutical controlled release technology Marcel
Dekker, 661-692 (2000). [0082] 6. J. L. Cleland, Jones J. S.,
Pharm. Res. 13, 1464-1475 (1996). [0083] 7. O. L. Johson,
Pharmaceutical Research 14, 730-735, (1997). [0084] 8. B. C.
Cunningham, Mulkerrin, M. G., Wells, J. A., Science 253, 545-548
(1991). [0085] 9. S. E. Zale, Burke, P. A., Berstein, H., Brickner,
A., in U.S. Pat. No. 5,716,644. (USA, 1998). [0086] 10. A. Sanchez,
Villamayor, B., Guo, Y., Mclver, J., Alonso, M. J., Intern. J.
Pharm. 185, 255-266 (1999). [0087] 11. S. P. Schwendeman, Tobio,
M., Jaworowicz, M., Alonso, M. J., Langer, R., J.
Microencapsulation 15, 299-318 (1998). [0088] 12. M. Morlock, Koll,
H., Winter, G., Kissel, T., European Journal of pharmaceutics and
biopharmaceutics 43, 29-36 (1997). [0089] 13. S. Yoshioka, Aso, Y.,
Kojima, S., Pharmaceutical Research 14, 736-741 (1997). [0090] 14.
M. v. d. Weert2, Hof, R. V., Weerd, J. v. d., Heeren, M. A.,
Posthuma, G., Hennink, W. E., Crommelin D. J. A., J. Controlled
Release 68, 31-40 (2000). [0091] 15. T. Morita, Horikiri, Y.,
Suzuki, T., Yoshino, H., International Journal of Pharmaceutics
219, 127-137 (2001). [0092] 16. Y.-F. Maa, Nguyen, P-A., Hsu, S.
W., J. Pharm. Sci., 87, 152-159 (1997). [0093] 17. T. Morita,
Horikiri, Y., Yamahara, H., Suzuki, T., Yoshino, H., Pharm. Res.
17, 1367-1373 (2000). [0094] 18. T. G. Park, Lee, H. Y., Nam, Y.
S., J. Controlled Release 55, 181-191 (1998). [0095] 19. O.
Franssen, W. E. Hennink, Intern. J. Pharm., 168, 1-7 (1998). [0096]
20. F. Lamberti, in WO96/40071 (1996).; [0097] 21. S. P.
Schwendeman, Cardamone, M., Brandon, M. R., Klibanov, A., Langer,
R., Stability of proteins and their delivery from biodegradable
polymer microspheres. S. C. H. Bernstein, Ed., Microparticulate
Systems for the Delivery of Proteins and Vaccines (Mercel Dekker,
New York, 1996), vol. 77. [0098] 22. W. R. Liu, Langer, R.,
Klibanov, A. M., Biotech. Bioeng. 37, 177-184 (1991). [0099] 23. B.
Bittner, Morlock, M., Koll, H., Winter, G., Kissel, T., Eur. J.
Pharm. Biopharm. 45, 295-305 (1998). [0100] 24. T. Jin, L. Chen, H.
Zhu, U.S. patent application Ser. No. 09/886,555 (2001). [0101] 25.
H. Takahata, Lavelle, E. C., Coombes, A. G. A., Davis, S. S., J.
Controlled Release 50, 237-246 (1998). [0102] 26. G. Zhu, S. R.
Mallery, S. P. Schwendeman, Nature Biotech., 18, 52-57 (2000)
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