Note: Descriptions are shown in the official language in which they were submitted.
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
1
DISPERSION OF POLOXAMER-PROTEIN PARTICLES, METHODS OF
MANUFACTURING AND USES THEREOF
The present invention relates to a method for preparing poloxamer-protein
particles.
It also relates to poloxamer-protein particles obtainable by this method,
dispersion thereof, and their use in methods of encapsulation, in particular
of
microencapsulation.
Protein delivery from microparticles made of biodegradable polymers such
as poly(D,L-lactide-co-glycolide) (PLGA) is very interesting to avoid protein
proteolysis and complete its sustained release. So, this formulation has been
extensively studied. Nevertheless, protein successful delivery from PLGA
microparticles is not still achieved. The most important hurdles are related
to
protein stability issues during the formulation process in one hand and during
the
release period in the other hand (Schwendeman et al., 1996; van de Weert et
al.,
2000; Bilati et al., 2005; Tamber et al., 2005; Wang et al., 2005). While
protein
stabilization during the formulation process is being reached thanks to the
use of
additives (Pean et al., 1998; Pean et al., 1999), protein release according to
a
zero-order profile and without denaturation was scarcely reported (Kim et al.,
2006; Park et al., 2006; Lee et al., 2007).
The release of small hydrophobic molecules from PLGA microspheres is
governed by drug diffusion through aqueous pores in the initial phase and by
polymer degradation at later stages. For proteins, an initial massive release
(burst
effect) followed by an incomplete release was frequently observed due to
instability problems (Crotts et al., 1998; Aubert-Pouessel et al., 2004).
Moisture-
induced aggregation and ionic interactions were supposed to occur in the
initial
phase of the microsphere hydration. Later, during polymer erosion, non-
specific
protein adsorption onto the degrading surface area and covalent/noncovalent
aggregation due to the formation of acidic PLGA degradation products were
reported as factors responsible for this incomplete release (Park et al.,
1998)
The use of stabilizing additives was the most widely employed strategy to
minimize protein degradation associated with the direct environment of
degrading
PLGA (Morlock et al., 1997). However, stabilizers only influenced the first
day
release because of their rapid diffusion from the microparticles (Sanchez et
al.,
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
2
1999). Alternatively, complex methods were engineered to allow the complete
and
sustained release of proteins, i.e. protein chemical modification (Castellanos
et al.,
2005), preparation of heterogeneously structured microspheres (Jiang et al.,
2003)
and formation of porous microspheres (Kim et al., 2006).
It has now been developed a new method for preparing microencapsulated
protein, which allows to obtain a complete and continuous release of the
protein in
a biologically active form. Advantageously, this method can be easily applied
to a
wide range of proteins.
More specifically, it has been discovered that when the protein was
precipitated in the presence of a poloxamer, particles of protein/poloxamer
were
formed. Now, these poloxamer-protein particles are particularly advantageous
for
preparing microspheres loaded with protein, notably by the s/o/w method or by
prilling. It has indeed been observed that the solid state protein allows to
stabilize
the protein during the release step so that the use of stabilizing agents such
as
albumin or trehalose is not required. Further, precipitation of the protein
with
poloxamer allows to improve the release profile of the protein from
microspheres,
notably to obtain a continuous and sustained release of the protein in a
biologically
active form, notably for at least 20 days. Thus, advantageously, the presence
of
poloxamer does not induce a burst effect. As poloxamer is a surfactant, it is
assumed that its presence limits the interactions between the protein and the
polymer which encapsulates the protein, thus enabling to improve the release
of
the protein.
Poloxamer-protein particles
Thus, in one aspect, the invention is directed to a method for preparing a
dispersion of poloxamer-protein particles, said method comprising the steps
of:
i) preparing an aqueous solution comprising a protein and a poloxamer;
ii) contacting the obtained solution with a water-miscible protein non solvent
in a sufficient amount to form a dispersion of poloxamer-protein particles;
and optionally
iii) recovering the obtained poloxamer-protein particles.
This method is particularly advantageous for preparing non denaturated solid
state protein as small poloxamer-protein particles which are particularly
useful for
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
3
subsequent encapsulation. Advantageously, this method can be applied to a wide
range of protein with high yields.
As used herein, the term "particles" refers to an aggregated physical unit of
solid material.
As used herein, the expression "poloxamer-protein particle" refers to a
particle comprising a precipitated protein in combination with a poloxamer,
notably
one or more molecules of precipitated proteins combined with one or more
molecules of poloxamer.
The particles according to the invention are preferably nanoparticles.
Nanoparticles are understood as particles having a median diameter d50
inferior to 1 m.
Preferably, the median diameter of the poloxamer-protein particles of the
invention ranges from 50 to 200 nm and is notably of about 150 nm.
As used herein, the terms "median diameter d50" refers to the particle
diameter so that 50% of the volume of the particles population have a smaller
diameter.
The median diameter d50 according to the invention is determined by virtue of
a particle size measurement performed on the suspensions according to the
method based on light diffraction.
As used herein, the term "poloxamer" refers to a nonionic block copolymer
comprising a hydrophobic chain of polyoxypropylene and a hydrophilic chain of
polyoxyethylene. Such poloxamers may be linear or branched, and include
notably
tri-blocks or tetra-blocks copolymers. They notably include poloxamines such
as
Tetronic 1107 (BASF). Especially preferred poloxamers are those having a
hydrophile-lipophile balance (HLB) not less than 10, preferably not less than
18,
and most preferably not less than 24. Most preferred poloxamers are ones that
are
pharmaceutically acceptable for the intended route of administration of the
protein
particles.
Preferred poloxamers are composed of a central hydrophobic chain of
polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene.
Preferably, the poloxamer is selected from the group consisting of poloxamer
188, 407, 338 and 237.
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
4
The molar ratio (mol/mol) of poloxamer/protein is not critical for the
precipitation of protein and may vary in a wide range.
Preferably, the protein is a therapeutic protein. Examples of suitable
proteins
include notably enzymes, growth factors, cytokines, hormones, antibodies,
fragments of antibodies or coagulation factors. As growth factors, mention may
be
made of Nerve Growth Factor, Brain Derived Neurotrophic Factor, Neurotrophin
3,
the Transforming Growth Factor beta family, the Glial Cell Line-Derived
Neurotrophic Factor family, the Fibroblast Growth Factors, Endothelium Growth
Factor, Platelet-Derived Growth Factor. Cytokines such as interferon a, 13, y,
hormones such as Human Growth Hormone, ErythropoIetine, interleukins such as
IL-1, IL-2, chemokines, antigens and mixture thereof are other examples. The
protein could also be a diagnostic agent, a cosmetic or a nutritional
supplement.
It has been observed that the precipitation yield of protein according to the
invention generally increases with the molecular weight of protein. Thus, the
method according to the invention may be applied to a wide range of protein
molecular weights.
Preferably, the molecular weight of protein is not less than 8 KDa, more
preferably not less than 10 KDa.
As an example, the protein molecular weight may range from 10 to 950 KDa,
and notably from 10 to 200 KDa.
The water-miscible protein non solvent is used in a sufficient amount to
precipitate the protein as small particles.
A volume ratio of water miscible solvent/aqueous solution ranging from 5 to
100 is generally sufficient to induce the precipitation of the protein in the
presence
of poloxamer.
The formation of protein-poloxamer particles may occur in a wide range of
temperature. Thus, preferably, the water-miscible protein non solvent is
contacted
with said solution comprising the protein and the poloxamer at a temperature
ranging from 1 to 25`C. More preferably, it is cont acted at a temperature
ranging
from 2 to 10 eC and most preferably of about zleC. I ndeed, it has been
observed
that the formation of protein-poloxamer particles is more reproducible at low
temperatures. This may be particularly advantageous when such poloxamer-
protein particles are intended to be encapsulated, insofar as poloxamer-
protein
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
particles allow to improve the release profile of protein from microspheres or
microparticles.
Preferably, the water-miscible protein non solvent is also a non-solvent of
the
poloxamer.
5 As
used herein, "protein non solvent" or "poloxamer non solvent" means a
solvent wherein the protein, the poloxamer respectively precipitates.
The water-miscible protein non solvent is preferably biocompatible.
As used herein, "biocompatible" refers to those solvents which are, within the
scope of sound medical judgment, suitable for contact with the tissues of
human
beings and animals without excessive toxicity, irritation, allergic response,
or other
problem complications commensurate with a reasonable benefit/risk ratio.
Preferably, the water-miscible non solvent is glycofurol also called
tetraglycol
or tetrahydrofurfurylpolyethylene-glycol or tetrahydrofurfuryl alcohol, or
also
polyethylene-glycol ether (CAS: 9004-76-6).
The volume of glycofurol may represent 80 % to 99 % of the volume of the
aqueous solution.
Preferably, the aqueous solution further contains a salt.
Advantageously, the use of a salt in combination with a water-miscible non
solvent of the protein promotes and/or enhances the precipitation of the
protein. It
notably allows to reach better yields of precipitation. Further, protein
activity is
preserved after precipitation and redissolution which is scarcely observed
with
salt-induced precipitation.
The salt concentration of the aqueous solution may vary in a wide range. It is
generally predetermined according to the nature of the protein.
For a given amount of one protein, at a fixed solution pH, a fixed temperature
and a fixed volume ratio of water/water-miscible non solvent, the person
ordinary
skilled in the art may determine a minimal suitable salt concentration by
routine
work, typically by adding increasing amounts of salt up to observing the
precipitation of the protein. In this respect, it can be noted that the
presence of
poloxamer does not affect the formation of protein precipitates.
Preferably, the concentration of salt ranges from 0.01M to 3M.
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
6
Preferably, the salt is a water soluble electrolyte. Tris[hydroxymethyI]-
aminomethane, NaCI, KCI, (NH4)2SO4 or a mixture thereof may be used. Among
these, NaCI is particularly preferred.
The precipitation yield of the protein may be further optimized by adjusting
three parameters: the ratio between the volumes of the aqueous phase and of
the
water- miscible protein non solvent, the ionic strength and the mass of
protein.
As an example, by using optimized conditions, a precipitation yield superior
to 95% may be obtained for lysozyme.
The particles may be recovered by using any conventional methods, notably
centrifugation.
In a particular aspect of the invention, the water-miscible protein non
solvent
further contains a dissolved wall-forming polymer. In this respect, the water-
miscible protein non solvent is preferably glycofurol.
As used herein, the wording "wall-forming polymer" refers to polymers
capable of forming the structural entity of a matrix individually or in
combination.
Biodegradable and biocompatible wall-forming polymers are preferred,
especially
for injectable applications. Examples of such polymers include notably poly(a-
hydroxyacides) such as polylactides (PLA), poly(lactide-co-glycolide)
copolymers
(PLGA's), polyethylene glycol conjugated with a copolymer of lactic acid and
glyocolic acid (PLGA'-PEG's) or with a polymer of lactic acid, polyesters such
as
poly-c-caprolactones, poly(orthoesters) and triglycerides and mixtures
thereof.
Preferably, the molar ratio (mol/mol) of poloxamer/polymer ranges from 1 to
30, more preferably from 2 to 10 and is most preferably of about 5.
This embodiment is particularly advantageous as the obtained dispersion
may be directly implemented in an encapsulation method such as prilling,
without
need of recovering the formed poloxamer-protein particles.
In view of encapsulating poloxamer-protein particles and notably of improving
the release profile of protein from microspheres or microcapsules, it is
particularly
preferred to use a molar ratio (mol/mol) of poloxamer/protein in the range of
1 to
30, more preferably of 2 to 20, and most preferably of about 17.
In a further aspect, the invention is directed to a dispersion of poloxamer-
protein particles obtainable by the method of the invention.
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
7
In a still further aspect, the invention is directed to poloxamer-protein
particles obtainable by the method of the invention.
Encapsulated poloxamer-protein particles
The poloxamer-protein particles of the present invention can further be
encapsulated within matrices of wall-forming polymers to form encapsulated
particles. The encapsulation may be accomplished by any process known in the
art such as the emulsification/solvent extraction process or the prilling
method. In
respect of the emulsification/solvent extraction process, reference could be
made
notably to Pean et al., 1998.
In a preferred aspect, (micro)encapsulation of the particles according to the
invention is accomplished by an emulsification/solvent extraction process.
Thus, the invention is directed to a method for encapsulating poloxamer-
protein particles comprising :
i) preparing a s/o/w emulsion containing :
- as a continuous phase, an aqueous phase, and
- as a discontinuous phase, an organic solvent containing dispersed
poloxamer-protein particles as defined above and a wall-forming polymer, said
wall-forming polymer being soluble in said organic solvent, and insoluble in
said
continuous phase,
ii) solidifying said discontinuous phase, thereby forming encapsulated
poloxamer-protein particles ; and optionally
iii) recovering the obtained encapsulated poloxamer-protein particles.
As used herein, the term "emulsion" refers to a heterogenous system of one
immiscible liquid (discontinuous phase) dispersed in another liquid
(continuous
phase) in the form of droplets. The size of the droplets of the emulsion may
range
from 1 pm to several hundred pm, for example to 100 pm.
As used herein, "s/o/w emulsion" means a solid-in-oil-in-water emulsion and
refers to an emulsion wherein poloxamer-protein particles are dispersed in the
organic solvent forming the discontinuous phase.
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
8
The emulsion may be prepared by any conventional methods which may
include notably shearing, high pressure homogenization, static mixing,
sonication,
phase inversion induced by temperature or/and pressure.
Preferably, the s/o/w emulsion is formed by pouring the discontinous phase in
the continuous phase under stirring.
As an example, the organic solvent forming the discontinuous phase may be
selected from the group consisting of dichloromethane, acetone, ethyl acetate
and
chloroform or a mixture thereof.
Preferably, the wall-forming polymer is a polymer of lactic acid, a copolymer
of lactic acid and glycolic acid, in particular poly(D,L-lactide-co-glycolide)
(PLGA)
or polyethylene glycol conjugated PLGA's (PLGA'-PEG's) such as the triblock
copolymer PLGA-PEG-PLGA.
Preferably, the continuous phase further comprises a surfactant such as
poly(vinyl alcohol).
In a preferred aspect, the solidification of the discontinuous phase is
performed by extracting the organic solvent from the discontinuous phase, thus
desolvating the wall-forming polymer and solidifying the discontinuous phase.
Preferably, the solvent is extracted by adding a second solvent, said second
solvent being miscible with the solvent of the discontinuous phase and not a
solvent of the wall-forming polymer contained in the discontinuous phase,
thereby
forming a mixture of solvents which is miscible in the continuous phase.
As an example, the second solvent may be water, ethanol, propylene glycol
or polyethylene glycols.
The obtained encapsulated particles may be recovered by using any
conventional methods such as filtration or centrifugation and can be
lyophilized
after their washing.
In a further preferred aspect, (micro)encapsulation of the particles according
to the invention is accomplished by the technique known as laminar jet break-
up or
"prilling" which is particularly advantageous because it is industrially
feasible and
induces the formation of monodispersed microparticles (Serp et al., 2000).
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
9
Thus, the invention is directed to a method for encapsulating poloxamer-
protein particles comprising:
i) providing a dispersion of poloxamer-protein particles containing a wall-
forming polymer as defined above or dispersing poloxamer-protein particles as
defined above in a solution of a wall-forming polymer dissolved in a solvent;
ii) forming droplets of the dispersion of step i);
iii) solidifying said droplets;
and optionally
iv) recovering the obtained encapsulated poloxamer-protein particles.
Preferably, the droplets are solidified by extracting the solvent of the
dispersion.
The solvent of the dispersion may notably be extracted with a second
solvent, said second solvent being miscible with the solvent of the dispersion
and
being a non-solvent of said wall-forming polymer, thereby forming encapsulated
poloxamer-protein particles.
Encapsulated poloxamer-protein particles may notably be prepared
according to this method, by breaking apart a laminar jet of a dispersion as
defined
in step i), into monosized droplets by means of a vibrating nozzle device. The
obtained droplets may then be solidified by falling into a solidification
bath, notably
a bath of a second solvent which is a non solvent of the wall-forming polymer
and
which is miscible with the solvent of the dispersion.
Preferably, the solvent of the dispersion is glycofurol and the wall-forming
polymer is PLGA. In this context, the droplets of the dispersion may be
solidified
notably into a PGLA non-solvent bath of water, ethanol, glycerol, polyethylene
glycol, propan-2-ol, propylene glycol or a mixture thereof. This embodiment is
particularly advantageous as no toxic solvent is used.
In a still further aspect, the invention is also directed to the encapsulated
poloxamer-protein particles obtainable according to the invention.
Encapsulated particles are notably microencapsulated particles, and may be
microcapsules or microspheres. As used herein, "microspheres" are matrix
systems in which the poloxamer-protein combination is dispersed.
"Microcapsules"
CA 02701377 2015-04-21
are composed of a nucleus of poloxamer-protein combination coated with a layer
of polymer.
Microcapsules may be obtained with the prilling method, while microspheres
may be obtained with the s/o/w emulsification solvent extraction process or by
5 prilling.
Advantageously, the poloxamer-protein combination allows to obtain
microspheres in which the protein is homogeneously dispersed in the matrix of
wall-forming polymer and thus to improve the release profile of the
encapsulated
protein.
10 Preferably, the encapsulated poloxamer-protein particles have a median
diameter ranging from Ito 1000 pm.
Advantageously, the encapsulated poloxamer-protein particles display an
improved release profile of the protein as compared to encapsulated protein
particles wherein the protein has not been precipitated with a poloxamer. More
specifically, it has been observed that encapsulated poloxamer-protein
particles
according to the invention allow a continuous release of the protein without
any
burst effect over 20 days.
The invention is also directed to a pharmaceutical composition comprising
encapsulated poloxamer-protein particles according to the invention.
FIGURES
Figure 1: Optimization of the lysozyme precipitation: spatial representation
of the
experimental design results.
Numbers in the circles refer to the percentage of lysozyme recovered under a
biologically active form, after precipitation and dissolution, for each
experiment.
Figure 2: Evolution of lysozyme zeta potential when adding poloxamer 188 in
solution.
Figure 3: In vitro release profile of lysozyme (mean SD) from PLGA
microspheres
(MS) without poloxamer 188 (2 batches twice) and with poloxamer 188 (example
CA 02701377 2015-04-21
11
7) (3 batches) and from PLGA-PEG-PLGA microspheres containing poloxamer
188 (example 9) (7 batches).
Figure 4 : In vitro release profile of TGF beta 3 with poloxamer 188 from PLGA
microspheres assessed by ELISA and biological activity of released TGF beta 3
assessed by a bioassay.
EXAMPLES
Materials
Lysozyme (chicken egg white) and its substrate: Micrococcus lysodeikticus,
glycofurol (tetraglycol or a-[(tetrahydro-2-furanyl) methyl]-w-hydroxy-
poly(oxy-1,2-
ethanediy1) and buffer compounds were obtained from Sigma-Aldrich (Saint
Quentin Fa'Javier, France). TGF beta 3 was purchased from Abcys (Paris,
France). TGF beta 3 ELISA kit was from R&D systems (Lille, France). Poloxamer
was kindly supplied by BASF (Levallois-Perret, France). Capped 75/25 PLGA,
provided by Phusis (Saint-lsmier, France), had a mean molecular weight of
27,000
Da (Polydispersity index, 1=1.9) as determined by size exclusion
chromatography
(standard: polystyrene). PLGA50:50-PEG-PLGA50:50 (RGP t 50106, 10% PEG
with 6,000Da, i.v. 0.75) was purchased from Boehringer-lngelheim (lngelheim,
Germany). Polyvinyl alcohol (Mowiole 4-88) was from Kuraray Specialities
Europe
(Frankfurt, Germany).
Method for preparing protein particles
Firstly, the protein was dissolved in a non-buffered saline solution and then
mixed with glycofurol at room temperature. 30 minutes later, the protein
particles
were recovered by centrifugation (10,000 g, 30 min, 4 C). Mixing and
centrifugation times of 30 min were selected in order to optimize the
precipitation
yield.
Method for preparing protein-poloxamer particles
Firstly, protein and poloxamer 188 were codissolved in a saline solution. This
protein-poloxamer solution was then mixed with glycofurol to prepare a protein-
poloxamer dispersion. 30 minutes later, the protein-poloxamer particles were
CA 02701377 2015-04-21
12
recovered by centrifugation (10,000 g, 30 min, 4 C). Mixing and centrifugation
times of 30 min were selected in order to optimize the precipitation yield.
Optimization of the precipitation yield of a protein widely available such as
lysozyme
To define the optimum conditions of precipitation of lysozyme, an
experimental design was used. Preliminary studies (not shown) using the
technique described revealed that three parameters influence protein
precipitation
yield: the ratio between the volumes of aqueous phase and of glycofurol, the
ionic
strength and the mass of protein. The presence of poloxamer does not affect
the
protein precipitation.
To study these three variables, a Doehlert matrix was chosen. Fifteen
experiments were carried out. Each experiment was repeated three times. The
experimental domain for each factor is described as follows:
- ionic strength of the aqueous phase (U1): 0.01 to 0.59 M (5 levels),
- volume of the aqueous phase (U2): 25 to 155 pl (7 levels)
- protein quantity (U3): 0.1 to 0.9 mg (3 levels).
The volume of glycofurol was the complement for 1m1 of suspension.
Experimental design is reported in Table 1.
CA 02701377 2015-04-21
12a)
IsrExp. Ionic strenght Aqueous phase volume Protein quantity
U1 U2 U3
(M) (pl) (mg)
1 0.59 90.0 0.50
2 0.01 90.0 0.50
3 0.44 155.0 0.50
4 0.16 25.0 0.50
0.44 25.0 0.50
6 0.16 155.0 0.50
7 0.44 111.7 0.90
8 0.16 68.3 0.10
9 0.44 68.3 0.10
0.30 133.4 0.10
11 0.16 111.7 0.90
12 0.30 46.6 0.90
13 0.30 90.0 0.50
14 0.30 90.0 0.50
0.30 90.0 0.50
Table 1 represents the values of each factor (U1, U2, U3) for the fifteen
experiments.
5
The measured response was the percentage of reversible lysozyme particles
collected (precipitation yield) (Figure 1). For its determination, the
dispersion of
protein particles in glycofurol was centrifuged, the supernatant eliminated
and the
pellet of protein particles dissolved in TRIS-HCI 0.01M buffer, pH 7.4 in
order to
10 determine its active mass. Biological activity of lysozyme was
determined by
measuring the turbidity change in a Micrococcus lysodeikticus bacterial cell
suspension.
Nemrod W software (2000, LPRAI, Marseille) was used for generation and
exploitation of the statistical experimental design.
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
13
Optimization of the precipitation yield of a protein scarcely available (TGF-
63)
To define protein behaviour in the presence of salt and glycofurol, a rapid
salt
screening may be employed. An aqueous protein solution (50 pl) containing the
protein quantity wanted to be precipitated (from 20 to 1000 pg) is deposited
in the
well of a 96-well plate. Then, saline solutions (50 pl) with growing salt
concentrations (from 0 to 3M) are added in each well. Finally, glycofurol (300
pl) is
adjoined and the absorbance is measured at 350 nm. An increase in the
absorbance is related to the formation protein particles. The presence of
poloxamer does not affect the formation of protein particles.
Size of the lysozyme-poloxamer 188 particles
The size of the lysozyme-poloxamer 188 particles dispersed in glycofurol was
determined by light diffraction (Mastersizer 2000, Malvern Instruments,
Worcestershire, UK).
Zeta potential
To monitor the formation of a combination between lysozyme and poloxamer
188 in solution, lysozyme solution in acetic acid 0.1M containing increasing
amount of poloxamer 188 were prepared. The protein concentration was
maintained at 10mg/ml. The corresponding zeta potential was measured as a
function of protein to poloxamer ratio, using a Zetasizer Nano-ZS (Malvern
Instruments, Worcestershire, UK).
FTIR
The secondary structure of lysozyme with or without poloxamer 188 inside
the microspheres was determined by FTIR spectroscopy. Microspheres loaded
with 5% w/w of protein (with respect to the amount of PLGA) were studied in
order
to detect the protein. FTIR studies were conducted with a Bucker IFS 28
equipped
with a DTGS detector. 500 scans (4000-400cm-1) at 2 cm-1 resolution were
averaged to obtain each spectrum. Lyophilized microspheres were measured as
KBr pellets (4-5 mg of microspheres per 200 mg of KBr). All spectra were
analyzed in the amide I region (1700-1600cm-1) using the program OPUS version
2Ø In all cases, a linear baseline between 2000-1800 cm-1 was subtracted.
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
14
Infrared band position and the number of bands in the amide I region were
calculated by Levenberg-Marquardt algorithm using the program OPUS. The
secondary structure contents were calculated from the area of the individual
assigned bands and their fraction of the total area in the amide I region.
BSA-FITC distribution in microsphere
Microspheres were loaded with BSA-FITC or with BSA-FITC/poloxamer 188
as described above. A laser scanning confocal imaging system (Olympus light
microscope Fluoview FU300, Paris, France) was employed to observe BSA-FITC
distribution in microspheres. Dry microspheres were dispersed on a glass
slide;
fluorescence images of cross-sections were taken by an optical sectioning. All
the
images were obtained using a single resolution.
Preparation of the microencapsuled lysozyme-poloxamer 188 particles
PLGA microspheres loaded with lysozyme-poloxamer 188 particles were
prepared using a s/o/w emulsion solvent extraction-evaporation process adapted
from Pean et al. (Pean et al., 1998). Briefly, 0.9 mg of protein-poloxamer 188
particles (0.6% w/w with respect to the amount of PLGA) were prepared in the
optimum conditions of precipitation and collected as described above. Then,
they
were carefully dispersed in an organic solution (2 ml; 3:1 methylene
chloride:acetone) containing 150 mg of PLGA. The resulting organic suspension
was then emulsified in a poly(vinyl alcohol) aqueous solution (90 ml, 4% w/v)
maintained at 1`C and mechanically stirred at 550 rpm for 1 min (heidolph RZR
2041, Merck Eurolab, Paris, France). After addition of 100 ml of deionized
water
and stirring for 10 min, the resulting s/o/w emulsion was added to deionized
water
(500 ml) and stirred further for 20 min to extract the organic solvent.
Finally, the
formed microparticles were filtered on a 5 pm filter (HVLP type, Millipore SA,
Guyancourt, France), washed five times with 100 ml of deionized water and
freeze-dried. The average volume diameter and the size distribution of the
resulting microspheres were evaluated using a MultisizerTM 3 Coulter Counter
(Beckman Coulter, Roissy CDG, France).
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
Lysozyme encapsulation efficiency
Protein encapsulation yield was determined considering the biologically-
active entrapped protein. Lysozyme PLGA microspheres (10 mg, 3 batches) were
dissolved in 0.9 ml DMSO in silanized glass tube. After 1 hour, 3 ml of 0.01M
HCI
5 was added. The solution was left to stand for one more hour, and then
incubated
with Micrococcus lysodeikticus suspension for lysozyme activity determination.
In vitro release profile of lysozyme from microspheres
The in vitro release profile of lysozyme from PLGA microspheres was determined
10 by adding 500 pL of TRIS-HCI 0.01M buffer, pH 7.4, containing 0.1(Y0 w/v
BSA and
0.09% w/v NaCI to 10 mg of microspheres into centrifugation tubes. The tubes
were closed, incubated in a water bath at 37cC and agitated at 125 rpm. At
determined time intervals, the tubes were centrifuged for 5min at 3000rpm. The
500 pl of the supernatant were collected for analysis and replaced by fresh
buffer.
15 The percentage of biologically-active released lysozyme was measured by
enzymatic assay.
Results
Example 1. Preparation of 900 uci of lysozyme particles coupled
with poloxamer 188
45 pl of a solution containing 900pg of lysozyme and 9 mg of poloxamer 188 in
NaCI 0.3 M are added to glycofurol to form a 1 ml suspension at room
temperature. The complex particles are recovered by centrifugation (10,000g,
min, 4`C) and elimination of the supernatant.
Example 2. Preparation of 300 pci of lysozyme particles
coupled with poloxamer 188
10 pl of a solution containing 300 pg of lysozyme and 3 mg of poloxamer 188 in
NaCI 0.3 M are added to glycofurol to form a 1m1 suspension. The poloxamer-
protein particles are recovered by centrifugation (10,000 g, 30 min, 4cC) and
elimination of the supernatant.
CA 02701377 2015-04-21
16
Example 3. Preparation of 9mg of lysozyme particles coupled with poloxamer 188
450 pl of a solution containing 9 mg of lysozyme and 90 mg of poloxamer 188 in
NaCl 0.3 M are added to glycofurol to form a 10 ml suspension. The complex
particles are recovered by centrifugation (10,000 g, 30 min, 4 C) and
elimination of
the supernatant.
Example 4. Activity of lysozyme particles coupled with
poloxamer 188 after dissolution
The pellet of poloxamer-protein particles described in Example 1 and 2 is
dissolved in an appropriate solvent in order to determine its active mass
(TRIS-
HCI 0.01M buffer, pH 7.4). Biological activity of lysozyme is determined by
measuring the turbidity change in a Micrococcus lysodeikticus bacterial cell
suspension. More than 80% of the protein is recovered in an active form.
Example 5. Size of the lysozyme/poloxamer 188 particles
The size of the poloxamer-protein particles suspended in glycofurol (described
in
example 1) was determined by light diffraction. The average particle size is
about
100nm with a narrow distribution.
Example 6. Evolution of lysozyme zeta potential in presence of poloxamer 188
The interaction of lysozyme with increasing amount of poloxamer 188 in aqueous
solution was controlled by zeta potential measurements. When poloxamer 188
was added, lysozyme zeta potential shifts from 15 to 5 mV (Figure 2). This
decrease of the net surface charge on lysozyme may be considered as the result
of molecular combination of lysozyme with poloxamer 188.
Example 7. Microencapsulation of the lysozyme/poloxamer 188 particles
in PLGA microspheres
Preparation of the PLGA microspheres loaded with lysozyme/poloxamer 188
particles
Lysozyme-loaded PLGA microspheres were prepared using a solid-in-oil-in-water
(s/o/w) emulsion solvent extraction-evaporation process. Briefly, 900 pg of
protein
CA 02701377 2015-04-21
17
particles coupled with poloxamer 188 (0.6% protein w/w with respect to the
amount of PLGA) were prepared as described in example 1 and collected as
described above. Then, they were carefully dispersed in an organic solution (2
ml;
3:1 methylene chloride:acetone) containing 150 mg of uncapped 75/25 PLGA
(mean molecular weight of 27,000 Da, polydispersity index of 1.9). The
resulting
organic suspension was then emulsified in a poly(vinyl alcohol) aqueous
solution
(90 ml, 4% w/v) maintained at 1 C and mechanically stirred at 550 rpm for 1
min.
After addition of 100m1 of deionized water and stirring for 10 min, the
resulting
s/o/w emulsion was added to deionized water (500 ml) and stirred further for
20
min to extract the organic solvent. Finally, the formed microparticles were
filtered
on a 5 pm filter washed with 500 ml of deionized water and freeze-dried. The
resulting microspheres had an average volume diameter of about 60 pm.
In vitro release study
The in vitro release profile of lysozyme from PLGA microspheres was determined
by adding 500 pL of TRIS-HCI 0.01M buffer, pH 7.4, containing 0.1% w/v BSA and
0.09% w/v NaCl to 10mg of microspheres into eppendorf tubes, incubated in a
water bath at 37 C, agitated at 125 rpm. At determined time intervals, the
tubes
were centrifuged for 5 min at 3000 rpm. The 500 pl of the supernatant were
collected for analysis and replaced by fresh buffer. The percentage of
released
biologically-active lysozyme was measured by enzymatic assay. The effect of
poloxamer 188 on the in vitro release profile is shown in Figure 3.
Example 8
To characterize the encapsulated protein-poloxamer particles of example 7, the
secondary structure of encapsulated lysozyme was characterized by Fourier
transform infrared (FTIR) spectroscopy (Table 2). The protein amide I IR
infrared
spectra were analyzed for the secondary structure composition and the
secondary
structure was quantified. Firstly, FTIR spectroscopy demonstrated that few
protein
structural perturbations were induced by the encapsulation and by the presence
of
poloxamer. Only minor spectral changes occurred in the amide I band (1700-1600
cm-1), which is sensitive to protein structure. Analysis of the spectra by
Gaussian
curve-fitting revealed few change in the a-helical and in 13-sheet content;
the
. CA 02701377 2015-04-21
18
secondary structure was within the error the same as for the powder prior to
encapsulation.
Conditions a-helix p-sheet
Native lysozyme (powder) 24.5 37.9
Lysozyme in PLGA microspheres 24.9 34.9
Lysozyme + poloxamer188 in PLGA 23.9 20.2
microspheres
Table 2 Secondary structure of lysozyme under various conditions
(as determined by FTIR spectroscopy)
Example 9. Microencapsulation of the lysozyme/poloxamer 188 particles in PLGA-
PEG-PLGA microspheres (150 mg microsphere batch)
PLGA-PEG-PLGA (10% PEG 6 000Da) was employed. The same procedure as
example 7 was followed to prepare the lysozyme-loaded PLGA-PEG-PLGA
microspheres except that the mechanically stirring was adjusted to 850 rpm to
obtain 60 pm microspheres. The effect of the polymer type on the in vitro
release
profile is shown in figure 3.
Example 10. BSA-FITC/poloxamer 188 distribution in PLGA-microspheres
Preparation of the BSA-FITC/poloxamer 188 particles
45 pl of a solution containing 900 pg of BSA-FITC and 9mg of poloxamer 188 in
NaCI 0.3 M are added to glycofurol to form a 1 ml suspension at room
temperature. The complex particles are recovered by centrifugation (10,000 g,
min, 4 C) and elimination of the supernatant.
Preparation of the PLGA microspheres loaded with lysozyme/poloxamer 188
25 particles
BSA-FITC loaded PLGA microspheres were prepared according to the procedure
described in example 7. Briefly, 900 pg of protein particles coupled with
poloxamer
188 (0.6% protein w/w with respect to the amount of PLGA) were carefully
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
19
dispersed in an organic solution (2 ml; 3:1 methylene chloride:acetone)
containing
150 mg of PLGA. The resulting microspheres had an average volume diameter of
about 60 pm.
Con focal analysis
A confocal image system was employed to observe BSA-FITC distribution in
microspheres. Dry microspheres were dispersed on a glass slide; fluorescence
images of cross-sections were taken by an optical sectioning. The presence of
poloxamer revealed a better protein distribution inside the microspheres.
Example 11. Preparation of 125 nq of TGF-133 (Transforminq Growth Factor-beta
3) particles coupled with poloxamer 188
100 pl of a solution containing 125 ng of TGF-(33 and 1.25 mg of poloxamer 188
in
10 mM phosphate buffer (pH7, NaCI 2M) were added to 740 mg glycofurol. The
complex particles was recovered by centrifugation (10,000 g, 30 min) and
elimination of the supernatant. The presence of poloxamer 188 did not affect
the
precipitation yield as determined by ELISA quantification.
Example 12 : Preparation of 50 uq of TGF 133 particles coupled with poloxamer
188
1.220 ml of solution containing 50 pg of TGF beta 3 and 3 mg of poloxamer 188
in
10 mM phosphate buffer (pH 7.4, NaCI 2M) are added to 8.68 g of glycofurol;
After
min, the particles are recovered by centrifugation (10,000g, 30 min, 4C) and
elimination of the supernatant.
Example 13 : Preparation of 50 mq PLGA microspheres batch loaded with
protein/poloxamer 188 particles
Protein particles coupled with poloxamer 188 were carefully dispersed in an
organic solution (670 pl; 3:1 methylene chloride:acetone) containing 50 mg of
capped 75/25 PLGA (mean molecular weight of 27,000 Da, polydispersity index of
1.9). The resulting organic suspension was then emulsified in a poly(vinyl
alcohol)
aqueous solution (30 ml, 4% w/v) maintained at 1cC and mechanically stirred at
550 rpm for 1 min. After addition of 33m1 of deionized water and stirring for
10 min,
CA 02701377 2015-04-21
the resulting s/o/w emulsion was added to deionized water (167 ml) and stirred
further for 20 min to extract the organic solvent. Finally, the formed
microparticles
were filtered on a 5 pm filter washed with 500 ml of deionized water and
freeze-
dried. The resulting microspheres had an average volume diameter of about
5 60 pm.
Example 14: Microencapsulation of TGF 133
TGF beta 3 was precipitated as mentioned in example 12.
In parallel, HAS (Human Albumin Serum) particles was prepared : 10 pl of NaCI
10 0.3 M containing 250 pg HAS are added to 1.077 g of glycofurol. After 30
min, the
HAS particles were recovered by centrifugation (10 000 g, 30 min, 4 C) and
elimination of the supernatant.
The HAS particles were dispersed in 3x100 pl polymer organic solution
(composition described in example 13), particles of poloxamer-TGF beta 3 were
15 dispersed in the same conditions. Both suspensions were mixed and used
to
prepare PLGA microspheres as described in example 13.
Example 15: TGF-I33 (Transforming Growth Factor-beta3) profile release
TGF beta 3 was encapsulated as mentioned in example 14.
20 The in vitro release profile of TGF beta 3 from PLGA microspheres was
determined by 500 pl of PBS buffer, pH , containing 1% w/v BSA to 10 mg of
microspheres into eppendorf tubes, incubated in a bath at 37 C, agitated at
125
rpm. The 500 pl of the supernatant were collected for analysis and replaced by
fresh buffer. The percentage of released TGF beta3 was determined by ELISA
and the biological activity of released TGF beta 3 was evaluated by a bioassay
(Figure 4).
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
21
REFERENCES
Aubert-Pouessel, A. Venier-Julienne, M.G. Clavreul, A. Sergent, M. Jollivet,
C.
Montero-Menei, C.N. Garcion, E. Bibby, D.C. Menei, P. and Benoit, J.P., 2004.
In
vitro study of GDNF release from biodegradable PLGA microspheres, J Control
Release, 95, 3, 463-75.
Bilati, U. Allemann, E. and Doelker, E., 2005. Strategic approaches for
overcoming
peptide and protein instability within biodegradable nano- and microparticles,
Eur J
Pharm Biopharm, 59, 3, 375-88.
Castellanos, I.J. Al-Azzam, W. and Griebenow, K., 2005. Effect of the covalent
modification with poly(ethylene glycol) on alpha-chymotrypsin stability upon
encapsulation in poly(lactic-co-glycolic) microspheres, J Pharm Sci, 94, 2,
327-40.
Crotts, G. and Park, T.G., 1998. Protein delivery from poly(lactic-co-glycolic
acid)
biodegradable microspheres: release kinetics and stability issues, J
Microencapsul, 15, 6, 699-713.
Jiang, G.Qiu, W. and DeLuca, P.P., 2003. Preparation and in vitro/in vivo
evaluation of insulin-loaded poly(acryloyl-hydroxyethyl starch)-PLGA composite
microspheres, Pharm Res, 20, 3, 452-9.
Kim, H.K.Chung, H.J. and Park, T.G., 2006. Biodegradable polymeric
microspheres with "open/closed" pores for sustained release of human growth
hormone, Journal of Controlled Release, 112,2, 167-174.
Lee, E.S. Kwon, M.J. Lee, H. and Kim, J.J., 2007. Stabilization of protein
encapsulated in poly(lactide-co-glycolide) microspheres by novel viscous
S/VV/O/VV method, Int J Pharm, 331, 1, 27-37.
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
22
Morlock, M. Koll, H. Winter, G. and Kissel, T., 1997. Microencapsulation of rh-
erythropoietin, using biodegradable poly(D,L-lactide-co-glycolide): protein
stability
and the effects of stabilizing excpients, Eur J Pharm Bioph, 43, 29-36.
Park, T.G.Yong Lee, H. and Sung Nam, Y., 1998. A new preparation method for
protein loaded poly(D, L-lactic-co-glycolic acid) microspheres and protein
release
mechanism study, J Control Release, 55, 2-3, 181-91.
Park, J.H. Ye, M. Yeo, Y. Lee, W.K. Paul, C. and Park, K., 2006. Reservoir-
type
microcapsules prepared by the solvent exchange method: effect of formulation
parameters on microencapsulation of lysozyme, Mol Pharm, 3, 2, 135-43.
Pean, J.M. Boury, F. Venier-Julienne, M.G. Menei, P. Proust, J.E. and Benoit,
J.P.,
1999. Why does PEG 400 co-encapsulation improve NGF stability and release
from PLGA biodegradable microspheres, Pharm Res, 16, 8, 1294-9.
Pean, J.M. Venier-Julienne, M.G. Boury, F. Menei, P.Denizot, B. and Benoit,
J.P.,
1998. NGF release from poly(D,L-lactide-co-glycolide) microspheres. Effect of
some formulation parameters on encapsulated NGF stability, J Control Release,
56, 1-3, 175-87.
Sanchez, A. Villamayor, B. Guo, Y. McIver, J. and Alonso, M.J., 1999.
Formulation
strategies for the stabilization of tetanus toxoid in poly(lactide-co-
glycolide)
microspheres, Int J Pharm, 185, 2, 255-66.
Schwendeman, S.P. Cardamone, M. Klibanov, A. Langer, R. and Brandon, M.R.,
1996. Stability of proteins and their delivery from biodegradable polymer
microspheres. In: Cohen, S. and Bernstein, H. (Ed.), Microparticulate systems
for
the delivery of proteins and vaccines, Marcel Dekker, New York, pp. 1-49.
Serp, D. Cantana, E. Heinzen, C. Von Stockar, U. and Marison, I.W., 2000.
Characterization of an encapsulation device for the production of monodisperse
alginate beads for cell immobilization, Biotechnol Bioeng, 70, 1, 41-53.
CA 02701377 2010-03-31
WO 2009/043874 PCT/EP2008/063147
23
Tamber, H. Johansen, P. Merkle, H.P. and Gander, B., 2005. Formulation aspects
of biodegradable polymeric microspheres for antigen delivery, Advanced Drug
Delivery Reviews, 57, 3 SPEC. ISS., 357-376.
Van de Weert, M. Hennink, W.E. and Jiskoot, W., 2000. Protein instability in
poly(lactic-co-glycolic acid) microparticles, Pharm Res, 17, 10, 1159-67.
Wang, W., 2005. Protein aggregation and its inhibition in biopharmaceutics,
Int J
Pharm, 289, 1-2, 1-30.
