Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02238352 1998-OS-21
Process for the Production of Morphologically Uniform
Microcapsules and Microcapsules that are Produced
According to this Process
The invention relates to a process for the production of
morphologically uniform microcapsules that contain peptides,
proteins, or other water-soluble biologically active substances
as active ingredients, and microcapsules that are produced
according to this process.
As is generally known, peptides and proteins represent
active ingredients with sizeable pharmacodynamics, which,
however, are broken down upon oral administration because of
their hydrolysis sensitivity in the acidic environment of the
stomach, as well as enzymatic degradation, and thus are partially
inactivated in such a way that their action in the
gastrointestinal tract is considerably reduced.
Rapid inactivation of proteins and peptides can be observed,
however, even after parenteral administration and especially
after intravenous administration because of the half-life, which
is very often very short. This means that despite sizeable
pharmacodynamics and theoretically lower therapeutic dosages,
multiple administrations of higher dosages may be necessary,
which mean a large burden on the patients.
Suitable formulations that avoid the above-mentioned
drawbacks are depot systems in the form of polymer microcapsules
or polymer nanocapsules, which are also known extensively for
peptides and are described in the literature.
, CA 02238352 1998-OS-21
2
They have the advantages that
-- Peptides and proteins are protected against rapid
inactivation,
-- lower dosages are pharmacologically effective,
-- multiple administration can be reduced,
-- controlled release of peptides and proteins is possible
in principle,
-- the encapsulated active ingredients are transported in
a directed manner, and
-- undesirable side-effects can be reduced.
The known processes for microencapsulation or
nanoencapsulation of water-soluble substances can be divided as
follows:
-- Coacervation or emulsion phase separation
-- encapsulation by spray drying
-- solvent-evaporation in an organic or aqueous phase.
All processes include the embedding of active ingredients
into a biodegradable polymer matrix or copolymer matrix.
Polymers that are known from the literature for this purpose
are polyamides, polyanhydrides, polyesters, polyorthoesters,
polyacetates, polylactones, polyorthocarbonates, i.a. To date,
polylactide-co-glycolide polymers have mainly been used.
Thus, pharmaceutical compositions of water-soluble peptides
and proteins in capsule form, which were produced based on
coacervation or emulsion phase separation, are known from US
4,675,189 (Syntex Inc.), US 4,835,139 (Debiopharm S.A.) and EP
302 582 B1 (Southern Research Inst.).
CA 02238352 1998-OS-21
3
According to this disclosure, processes are described in
which the copolymer that is used, preferably poly-(lactide-co-
glycolide)-polymer, is dissolved in a halogenated organic
solvent, preferably dichloromethane, and an aqueous peptide
solution is dispersed in this solution. Then, a so-called
coacervation agent is added. The coacervation agent is soluble
in the organic solvent, but the polymer is insoluble in the
coacervation agent, causing precipitation of the polymer with the
inclusion of the dispersed polypeptides. As a coacervation
agent, usually silicone oil is used for phase separation. After
the silicone oil is added, a large amount of heptane, which
ensures the setting of the microcapsules, must be added as well.
The encapsulation efficiency of this method is approximately
70$ (US 4,835,136). The microcapsules that are produced have a
diameter of 1 to 500 ~.cm, preferably 10 to 50 ~Cm according to the
examples.
In addition to the use of toxicologically problematic agents
such as dichloromethane, heptane, and silicone oil, the drawbacks
of this process also include the need to use large amounts of
solvent, which results from the encapsulation using coacervation
agents, such as silicone oil.
A process that is described in EP-A 315875 (Hoechst AG) for
the production of biodegradable microcapsules of water-soluble
peptides and proteins is based on the spray-drying process, in
which an aqueous peptide or protein solution is emulsified in an
organic polymer solution, and this emulsion is spray-dried.
CA 02238352 1998-OS-21
4
As a biodegradable polymer, a mixture of polyhydroxybutyric
acid and poly (lactide-co-glycolide) polymer is used in a mixing
ratio of between 99:1 to 20:80.
The peptide or protein is present in micronized form or in
an aqueous solution. As a solvent, chloroform, dichloromethane,
DMF or a solvent mixture that consists of
water/ethanol/chloroform are considered. According to the
examples, chloroform is used. The spray drying is carried out at
temperatures of between preferably 45 and 95°C.
Disadvantageous in this process is the potential risk of
explosion when a non-halogenated solvent is used and high
temperatures are used simultaneously during the drying process.
Moreover, the use of non-flammable solvents such as
dichloroethane results in toxicologically harmful residual
solvent contamination in the end product. In addition, spray-
dried microcapsules basically show a strong tendency to
agglomerate; agglomerates of about 100 E.cm in size are produced.
Microparticles that are produced according to the "solvent-
evaporation-process'° are described in two Canadian Patent
Applications CA 2,100,925 (Rhone-Merieux) and CA 2,099,941
(Tanabe Seiyaku Co.).
Usually, with this method, the aqueous peptide or protein
solution is dispersed into an organic polymer solution, or active
ingredient crystals are suspended in the polymer solution. After
a second aqueous phase is added with a surfactant, the polymer
solvent is evaporated.
CA 02238352 1998-OS-21
This method is highly variable, and normally W/O- or complex
W/O/W-emulsions are produced.
According to CA 2,099,941, water-soluble active ingredients
and biodegradable polymers are first dissolved in a solvent or a
solvent mixture in which they are both soluble. Then, this
solvent is removed, and the solid dispersion that is produced is
dissolved in an organic solvent that is not water-miscible. The
resulting solution (oil phase) is emulsified in an aqueous phase,
so that a W/O-emulsion is produced.
Finally, the organic solvent of the oil phase of this
emulsion is evaporated.
Concrete examples of the patent relate to poly (lactide-co-
glycolide) polymers (PLGA) as a matrix and a hormone (TRH) that
releases thyreotropin or its derivatives as an active ingredient,
which are first dissolved in a mixture that consists of
acetonitrile/ethanol and optionally water, or only acetonitrile,
or that consists of acetonitrile and aqueous gelatin, or of
dichloromethane and ethanol.
As an organic solvent in the solution of the solid
dispersion, dichloromethane or chloroform is used. An aqueous
polyvinyl alcohol solution represents the aqueous phase.
The size of the microcapsules is approximately a diameter of
1 to 100 Vim, according to the concrete examples about 50 um to
< 100 Vim.
According to CA 2,100,925, microcapsules of LHRH hormone and
analogs are produced by prior dispersion of the LHRH hormone in
powder form in two organic solvents, whereby one solvent (above-
CA 02238352 1998-OS-21
6
mentioned dispersion solvent) makes it possible to produce a
homogeneous suspension of the pulverized hormone by simple
stirring. The second solvent is readily water-miscible and thus
makes the microdispersion of the organic phase in aqueous phase
possible.
As a second solvent, dichloromethane or, alternatively,
chloroform is used. The capsules have a diameter of between 1
and 250 E.cm. Preferably, the capsules are larger than 50-60 E.tm.
The morphology of the microcapsules that are thus produced
is also very different. As already explained above, the
halogenated solvents that are used are toxicologically harmful.
In addition, this process also requires sizeable amounts of
surfactants.
The object of the invention was to develop a simple and
gentle process for the production of morphologically uniform,
non-agglomerating microcapsules using toxicologically harmless
solvents, which have an encapsulation efficiency of at least 85~,
preferably over 90~, and is to yield microcapsules in a size
range of 200 nm to 500 ~Cm with a high degree of concentration.
In addition, the process is to make "scaling-up" possible.
The object of the invention is achieved surprisingly simply
using the "Induced Phase Transition" method, which is implemented
by virtue of the fact that a polymer that is commonly used for
microcapsule production, such as a polyester that consists of
hydroxycarboxylic acids or a block polymer that consists of
hydroxycarboxylic acids and polyethylene glycol (PEG), is
dissolved in a halogen-free, solvent or solvent mixture that is
CA 02238352 1998-OS-21
7
not water-miscible or is partially water-miscible and the
buffered active ingredient solution, which has a pH of between
6.0 - 8.0, is dispersed in this solution. Homogenization
produces a stable W/O-emulsion to which an aqueous solution that
contains a surfactant or a mixture of surfactants is added as an
outer phase while being stirred, in such a way that a three-phase
W/O/W emulsion is obtained. Then, the solvent or solvent mixture
.is removed with commonly used methods, preferably in a vacuum
and/or air/nitrogen stream. The microcapsules are concentrated
and optionally freeze-dried.
In this case, the particle size is controlled by the
stirring speed, whereby smaller particles (< 8 Vim) -- such as are
required if the product is intended for intravenous
administration -- are obtained at higher stirring speeds.
Optionally, after the solvent is removed, the microcapsules
are additionally subjected to "cross-flow" filtration, by which
residual surfactant and residual solvent portions are removed.
As a result, it is possible to reduce or to avoid the "initial
burst," i.e., a large release of active ingredients immediately
after administration (because of active ingredients that adhere
to the particle surface).
For freeze-drying, cryoprotectors such as sugar, sugar
alcohols, or polyvinylpyrrolidone derivatives are optionally
added.
Preferred polyesters of hydroxycarboxylic acids that can be
used in the process according to the invention are:
. CA 02238352 1998-OS-21
8
Polyglycolides (PGA) and copolymers of glycolides, such as
glycolide/lactide copolymers (PGA/PLLA) or glycolide/trimethylene
carbonate copolymers (PGA/TMC); L-polylactides (PLA) and
stereocopolymers of polylactides such as poly-L-lactide (PLLA),
poly-DL-lactide copolymers and L-lactide/DL-lactide copolymers;
copolymers of PLA such as lactide/tetramethylglycolide
copolymers, lactide/S-valerolactone copolymer and lactide/~-
caprolactone copolymer; poly-f3-hydroxybutyrate (PHBA), PHBA/!3-
hydroxyvalerate copolymers (PHBA/HVA), poly-f3-hydroxypropionate
(PHPA), poly-p-dioxanone (PDS), poly-6-valerolactone,
hydrophobized polysaccharides, - hyaluronic acid, dextrans or
hydrophobized amylopectin and poly-~-caprolactone.
As block copolymers of polyesters of hydroxycarboxylic acids
and linear or star-polyethylene glycol (PEG), the substances
named below can be used in the process according to the
invention:
AB-Block copolymers that consist of PLA and PEG, ABA-
triblock copolymers that consist of PLA-PEG-PLA, S(3)-PEG-PLA
block copolymers and S(4)-PEG-PLA block copolymers.
The polymer Resomer~R~ 505, especially Resomer~R~ RG-756 or
Resomer~R~ RG-858, is preferred according to the invention.
Resomer~R~ is a trademark of the Bohringer Ingelheim Company.
In this case, this is a (DL-lactide-co-glycolide)-polymer.
Halogen-free solvents or solvent mixtures that are preferred
according to the invention are acetone, ethanol, alkyl acetates
such as methyl, ethyl, propyl, isopropyl or butyl acetate, alkyl
formates such as methyl-, ethyl-, propyl-, isopropyl- or butyl
CA 02238352 1998-OS-21
9
formate, triacetin, triethyl citrate and/or C~-C4 alkyl lactates,
e.g., methyl or ethyl lactate.
Ethyl acetate, isopropyl acetate, and propyl formate are
especially preferably used.
For the purposes of this invention, buffered solutions are
aqueous solutions of peptides, proteins or their physiologically
compatible salts or of other water-soluble biologically active
substances, which are preferably adjusted with a tris
(hydroxymethyl) aminomethane solution or a phosphate buffer
solution to a pH of between 6.0 and 8.0, preferably a pH of 6.5
to 7.4.
Another buffer that can be used according to the invention
is the citrate buffer, whereby the buffer concentrations are
generally in the range of 5 mmol/1 to 300 mmol/1.
Any water-soluble peptides or proteins can be encapsulated
with the process according to the invention. The process
according to the invention is especially suitable for
encapsulating human serum albumin, insulin, interferon, and LHRH
antagonists or their analogs.
Morphologically uniform microcapsules of human serum
albumin, insulin, interferons, and the peptides that are
mentioned below can quite especially advantageously be produced
with the process according to the invention:
a) DesA(2)Nal-beta-Ala-DCpa-Deal-Ser-Tyr-DCit-Leu-Arg-Pro-
DAla-NH2 ,
b) DesA(2)Nal-Gly-DCpa-DPal-Ser-Tyr-DCit-Leu-Arg-Pro-DAla-
NHZ ,
CA 02238352 1998-OS-21
c) Ac-DNal-DCpa-DPal-Ser-Tyr-DLys(Mor)-Leu-Lys(Mor)Pro-
DAla-NHZ .
The meanings of DesA(2)Nal and D-Cit and the chemical
structures of peptides a) to c) are presented in Figure 1 or 2.
For the purposes of the invention, preferred as surfactants
are substances from the Poloxamere~R~ group, polyethylene glycol
alkyl ethers, polysorbates (Tween~R~, Span~R~), saccharose esters
(Sisterna~R~, The Netherlands), saccharose esters (Ryoto sugar
esters, Tokyo), gelatin, polyvinylpyrrolidone, fatty alcohol
polyglycoside, charps, charpso, decyl-f3-D-glycopyranoside, decyl-
f3-D-maltopyranoside, dodecyl-f3-D-maltopyranoside, sodium-oleate,
Poloxamine~R~ group, polyethylene glycol, polyvinyl alcohol,
polyoxyethylated fatty acid ether (Brij~R~) Triton X 100 or
mixtures thereof.
Polyvinyl alcohol, Brij~R~, Poloxamere~R~ Poloxamine~R~ and
Tween~R~ are preferably used.
The subject of the invention is also morphologically uniform
microcapsules that are produced according to the above-mentioned
process and have a diameter of 200 nm to 500 Vim, preferably
between 0.2 to 8 /..cm.
Because of the advantageous conformation of polymer and
solvent, no formation of agglomerates of the microcapsules occurs
in the process according to the invention.
Thus, Figures 3 and 4 show light-microscopic pictures of the
microcapsules according to the invention that are produced
according to Example 10 (Fig. 3) or according to Example 15 (Fig.
4). A millimeter in the imaging corresponds to 1 ~.Cm in reality.
CA 02238352 1998-OS-21
11
The pictures clearly show the uniform morphology; particle
agglomerates are not present.
The encapsulation efficiency of the process is at least 85~;
preferably encapsulation efficiencies of between 90 and 95~ are
achieved. The mass of the encapsulated active ingredient
100/mass of the active ingredient that is used is defined as
encapsulation efficiency. The degree of concentration of the
microcapsules that are produced is between 3 to 30~ (degree of
concentration = mass of active ingredient - 100/mass of active
ingredient + mass of polymer).
Then, the invention is to be explained in more detail in the
embodiments, without limiting the latter to it.
, CA 02238352 1998-OS-21
' 12
Example 1
1.7 g of the polymer Resomer~R~ RG-756 is dissolved in 29 ml
of ethyl acetate and moved into a steel vessel (height 11.5 cm,
inside diameter 8 cm). Then, 3 ml of an aqueous 5 mmol
tris(hydroxymethyl)aminomethane solution (pH 7.4) that contains
200 mg of human albumin is dispersed with the aid of a mechanical
stirrer (Dispermat-FT, VMA-Getzmann GmbH, 5 cm dissolver disk)
into the polymer solution for 6 minutes at 10,000 rpm below room
temperature. 45 ml of an aqueous solution, consisting of a 2~
polyvinyl alcohol solution (molecular weight 9,000 - 10,000,
Aldrich) is added to the W/O-emulsion that is produced while
being stirred (8,000 rpm). After a dispersion time of 10
seconds, the W/O/W-emulsion is moved into a 500 ml three-necked
flask and stirred using a KPG stirrer. The solvent ethyl acetate
is then removed at 20°C by applying a vacuum (900 mbar),
nitrogen, or air feed. After 5 hours, the suspension is washed
with 5 1 of water or an aqueous solution and concentrated by
evaporation in a desired suspension volume. "Cross-flow"
filtration is carried out using a Sartocon Mini~R~ (Sartorius AG,
Gottingen) Systems. The solvent-free and almost emulsifier-free
suspension is mixed with a cryoprotector (for example with a
sugar, sugar alcohol, or polyvinyl pyrrolidone derivative),
frozen as quickly as possible, for example with liquid nitrogen,
and freeze-dried.
The lyophilizate that is resuspended with water or with an
aqueous solution contains microcapsules with a human albumin
content of 9~ (human albumin mass - 100/human albumin mass +
CA 02238352 1998-OS-21
13
polymer mass = degree of concentration), and they have a diameter
of from 0.2 to 8 ~,m. The encapsulation efficiency is 86~.
Example 2
The procedure is the same as in Example 1, whereby 1.7 g of
Resomer~R~ RG-756 is not dissolved in 29 ml of ethyl acetate, but
rather in 40 ml of methyl acetate.
Example 3
The procedure is the same as in Example 1, whereby instead
of 1.7 g of the polymer Resomer~R~ RG-756, 1.1 g of the polymer
Resomer~R~ RG-858 is used.
Example 4
The procedure is the same as in Example 1, whereby instead
of 1.7 g of the polymer resomer~R~ RG-756, 3.0 g of the polymer
Resomer~R~ RG-858 is used.
Example 5
The procedure is the same as in Example 1, whereby instead
of a 2~ PVA solution, a 2~ Brij~R~ 35 solution is used.
Example 6
The procedure is the same as in Example 1, whereby instead
of a 2~ PVA solution, a 2~ Brij~R~ 96 solution is used.
CA 02238352 1998-OS-21
14
Example 7
The procedure is the same as in Example 1, whereby instead
of a 2~ PVA solution, a 2~ Tween~R~ 20 solution is used.
Example 8
1.1 g of the polymer Resomer~R~ RG-858 is dissolved in 29 ml
of ethyl acetate and moved into a steel vessel (height 11.5 cm,
inside diameter 8 cm).
Then, 7 ml of a 5 mmol tris(hydroxymethyl)aminomethane
solution (pH 7.4) that contains 50 mg of the peptide DesA(2)Nal-
beta-Ala-DCpa-deal-Ser-Tyr-DCit-Leu-Arg-Pro-DAla-NHZ (peptide a)
and 2 ml of ethanol is dispersed with the aid of a mechanical
stirrer (Dispermat-FT, VMA-Getzmann GmbH, 5 cm dissolver disk)
into the polymer solution for 6 minutes at 10,000 rpm below room
temperature. 45 ml of an aqueous solution, consisting of a 2~
polyvinyl alcohol solution (molecular weight 9,000 - 10,000,
Aldrich) is added to the W/O-emulsion that is produced while
being stirred (8,000 rpm). After a dispersion time of 10
seconds, the W/O/W-emulsion is moved into a 500 ml three-necked
flask and stirred using a KPG stirrer. The solvent ethyl acetate
is then removed at 20°C by applying a vacuum (900 mbar), nitrogen
or air feed. After 5 hours, the suspension is washed with 5 1 of
water or an aqueous solution and concentrated by evaporation in a
desired suspension volume. A "cross-flow" filtration is carried
out using a Sartocon Mini~R~ (Sartorius AG, Gottingen) System with
a polyolefin membrane (cutoff 0.2 Vim). The solvent-free and
almost emulsifier-free suspension is mixed with a cryoprotector
CA 02238352 1998-OS-21
(for example with a sugar, sugar alcohol or polyvinyl pyrrolidone
derivative), frozen as quickly as possible, for example with
liquid nitrogen, and freeze-dried.
The lyophilizate that is resuspended with water or with an
aqueous solution contains microcapsules with an active ingredient
content of 4~. The microcapsules have a diameter of from 0.2 to
8 Vim. The encapsulation efficiency is 93~.
Example 9
1.1 g of the polymer Resomer~R~ RG-858 is dissolved in 29 ml
of ethyl acetate and moved into a steel vessel (height 11.5 cm,
inside diameter 8 cm). Then, 5 ml of an aqueous 5 mmol
tris(hydroxymethyl)aminomethane solution (pH 7.4) that contains
48 mg of the peptide DesA(2)Nal-Gly-DCpa-DPal-Ser-Tyr-DCit-Leu-
Arg-Pro-DAla-NHZ (peptide b) is dispersed with the aid of a
mechanical stirrer (Dispermat-FT, VMA-Getzmann GmbH, 5 cm
dissolver disk) into the polymer solution for 6 minutes at 10,000
rpm below room temperature. 45 ml of an aqueous solution,
consisting of a 2~ polyvinyl alcohol solution (molecular weight
9,000 - 10,000, Aldrich) is added to the W/O-emulsion that is
produced while being stirred (8,000 rpm). After a dispersion
time of 10 seconds, the W/O/W-emulsion is moved into a 500 ml
three-necked flask and stirred using a KPG stirrer. The solvent
ethyl acetate is then removed at 20°C by applying a vacuum (900
mbar), nitrogen or air feed. After 5 hours, the suspension is
washed with 5 1 of water or an aqueous solution and concentrated
by evaporation in a desired suspension volume. The use of a
CA 02238352 1998-OS-21
16
"cross-flow°° filtration, for example with a Sartocon Mini~R~
(Sartorius AG, Gottingen) System with a polyolefin membrane
(cutoff 0.2 Vim), is advantageous. The solvent-free and almost
emulsifier-free suspension can be mixed with a cryoprotector (for
example with a sugar, sugar alcohol or polyvinyl pyrrolidone
derivative) and is frozen as quickly as possible, for example
with liquid nitrogen, and freeze-dried.
The lyophilizate that is resuspended with water or with an
aqueous solution contains microcapsules with an active ingredient
content of 4$. The microcapsules have a diameter of from 0.2 to
8 ~tcm. The encapsulation efficiency is 95.7.
Example 10
1.1 g of the polymer Resomer~R~ RG-858 is dissolved in 30 ml
of propyl formate and moved into a steel vessel (height 11.5 cm,
inside diameter 8 cm). Then, 5 ml of an aqueous 5 mmol
tris(hydroxymethyl)aminomethane solution (pH 7.0) that contains
50 mg of the LHRH antagonist Ac-DNal-DCpa-DPal-Ser-Tyr-DLys
(Mor)-Leu-Lys(Mor)Pro-DAla-NH2 (peptide c) is dispersed with the
aid of a mechanical stirrer (Dispermat-FT, VMA-Getzmann GmbH, 5
cm dissolver disk) into the polymer solution for 6 minutes at
10,000 rpm below room temperature. 45 ml of an aqueous solution,
consisting of a 2~ polyvinyl alcohol solution (molecular weight
x,000 - 10,000, Aldrich), is added to the W/O-emulsion that is
produced while being stirred (8,000 rpm). After a dispersion
time of 10 seconds, the W/O/W-emulsion is moved into a 500 ml
three-necked flask and stirred using a KPG stirrer. The solvent
CA 02238352 1998-OS-21
17
propyl formate is then removed at 20°C by applying a vacuum (900
mbar), nitrogen or air feed. After 5 hours, the suspension is
washed with 5 1 of water or an aqueous solution and concentrated
by evaporation in a desired suspension volume. A "cross-flow"
filtration is carried out with a Sartocon Mini~R~ (Sartorius AG,
Gottingen) System with a polyolefin membrane (cutoff 0.2 um).
The solvent-free and almost emulsifier-free suspension is frozen
as quickly as possible with liquid nitrogen and freeze-dried.
The lyophilizate that is resuspended with water or with an
aqueous solution contains microcapsules with an active ingredient
content of 3.9~, and the microcapsules have a diameter of from
0.2 to 8 Vim. The encapsulation efficiency is 90.7.
Example 11
1.1 g of the polymer Resomer~R~ RG-858 is dissolved in 30 ml
of isopropyl acetate and moved into a steel vessel (height 11.5
cm, inside diameter 8 cm). Then, 5 ml of an aqueous 5 mmol
tris(hydroxymethyl)aminomethane solution (pH 7.0) that contains
50 mg of the LHRH antagonist as in Example 10 is dispersed with
the aid of a mechanical stirrer (Dispermat-FT, VMA-Getzmann GmbH,
cm dissolver disk) into the polymer solution for 6 minutes at
10,000 rpm below room temperature.
45 ml of an aqueous solution, consisting of a 2~ polyvinyl
alcohol solution (molecular weight 9,000 - 10,000, Aldrich) is
added to the W/O-emulsion that is produced while being stirred
(8,000 rpm). After a dispersion time of 10 seconds, the W/O/W-
emulsion is moved into a 500 ml three-necked flask and stirred
CA 02238352 1998-OS-21
1 ti
using a KPG stirrer. The solvent isopropyl acetate is then
removed at 20°C by applying a vacuum (900 mbar), nitrogen or air
feed. After 5 hours, the suspension is washed with 5 1 of water
or an aqueous solution and concentrated by evaporation in a
desired suspension volume. A "cross-flow" filtration is carried
out with a Sartocon Mini~R~ (Sartorius AG, Gottingen) System with
a polyolefin membrane (cutoff 0.2 ~Cm), and the solvent-free and
almost emulsifier-free suspension is freeze-dried.
The lyophilizate that is resuspended with water or with an
aqueous solution contains microcapsules with an active ingredient
content of 2.9~, and the microcapsules have a diameter of from
0.2 to 8 ~.im. The encapsulation efficiency is 90.6.
Example 12
The procedure is the same as in Example 1, whereby the 5
mmol tris(hydroxymethyl)aminomethane solution (pH 7.0) is
replaced by a 5 mmol phosphate buffer solution (PBS, pH 7.2).
Example 13
The procedure is the same as in Example 1, whereby instead
of 200 mg of HSA that is dissolved in 3 ml of tris-buffer (pH =
7.4), 750 mg of HSA that is dissolved in 5 ml of tris-buffer (pH
- 7.4) is used.
The lyophilizate that is resuspended in water or aqueous
solutions contains microcapsules with an HSA content of 30~. The
encapsulation efficiency is 90.9.
CA 02238352 1998-OS-21
' 19
Example 14
The procedure is the same as in Example 13, whereby instead
of 2~ polyvinyl alcohol solution, a 2~ Poloxamer F 127 solution
is used.
Example 15
The procedure is the same as in Example 13, whereby instead
of 2~ polyvinyl alcohol solution, a 2~ Poloxamine T 707 solution
is used.
Example 16
The procedure is the same as in Example 13, whereby instead
of 2~ polyvinyl alcohol solution, a 2~ Poloxamine T 908 solution
is used.
Example 17
The procedure is the same as in Example l, whereby 200 mg of
HSA is replaced by 200 mg of insulin (human, recombinant (pfs),
Sigma Chemie [Sigma Chemistry) No. I 0259).
Example 18
The procedure is the same as in Example l, whereby 200 mg of
HSA is replaced by 200 mg of interferon (human leukocyte (pfs)
(a-IFH, Le), Sigma Chemie No. I 1008).
CA 02238352 1998-OS-21
~u
Example 19
The procedure is the same as in Example 1, whereby 200 mg of
HSA is replaced by 200 mg of insulin (human, gamma (pfs) (y-IFN),
Sigma Chemie No. I 6507).
Example 20
120 mg of insulin is dissolved in 0.8 ml of HC1 (O.iN) and
mixed with 2 ml of NaCl solution (0.9~). Then, the pH of the
solution is set at 6-7 with NaOH (0.1N). This solution is added
to a solution of 500 mg of polymer RG-858 in 10 ml of ethyl
acetate and then stirred with Ultraturax (at 10,000-15,000 rpm)
for 3-4 minutes. Then, 50 ml of a 2~ aqueous Polaxamine T707
solution is added while being stirred. After the addition has
been completed, the suspension is moved into a three-necked
flask, which is equipped with a stirrer. The solvent (ethyl
acetate) is removed by applying a vacuum while being stirred.
The remaining residue is washed in a cross-flow filtration
(Sartocon Mini~R~ Sartorius AG, Gottingen) with 5 liters of water.
The residue that contains nearly solvent-free and surfactant-free
microcapsules is, optionally with the addition of a
cryoprotector, quickly frozen and freeze-dried.
The lyophilizate that is resuspended with water or with an
aqueous solution contains microcapsules with a degree of
concentration of 15~ by weight.
CA 02238352 1998-OS-21
21
Example 21
The procedure is the same as in Example 20, whereby instead
of the 2~ Polaxamine T707 solution, a 2~ Polaxamer-407 (F127)
solution is used.
The degree of concentration of the microcapsules that are
obtained is 17~.
Example 22
The procedure is the same as in Example 20, whereby instead
of the 2$ Polaxamine T707 solution, a 2~ Polaxamer-188 (F68)
solution is used.
The degree of concentration of the microcapsules that are
obtained is 16~.
Example 23
The procedure is performed analogously to Example 20. Here,
however, 700 mg of polymer and 223 mg of insulin are used. The
lyophilizate that is resuspended in water or an aqueous solution
contains microcapsules with an insulin content of 20~. -
