Note: Descriptions are shown in the official language in which they were submitted.
~0~~8~3
HOECHST A~CTTENf~ESE~2~SCHAFT HOE 91/F 092 Dr. D/PI
Description
Long--eating biodegradable microparticles and a process
for their preparation
The invention relates 'to long-acting, biologically
degradable microparticles based on poly(lactide-co
glycolide) (~PLGA) which contain therapeutically active
peptides or their physiologically tolerable salts as the
active substance, and to a process for the preparation of
such microparticles.
For pharmaceuticals which are enzymatically broken down
in the gastrotestinal tract, such as, for example,
proteins or peptides, in addition to nasal and dermal
administration - often with only a low absorption rate
parenteral pharmaceutical forms are particularly
important. Long-term treatment of certain diseases makes
it appear desirable to develop parenteral depot pharma-
ceutical forms which release the pharmaceutical
continuously over the course of several weeks. In the
literature, in addition to implants (compare, fox example
EP-E-0,058,481) microcapsules/microspheres are also
described for these purposes.
In order to avoid operative removal o~ these injection
forms, biodegradable polymers are used. In addition to
polyamides, polyanhydrides, poly(ortho)est-ers and
polyacetals, polyesters are in particular employed.
Usually, polyesters based on the monomers lactic acid and
glycolic acid or their copolymers are described as
suitable polymers.
For the preparation of mierocapsules/microspheres,
various processes, such as, far example phase separation
(EP-H-0,052,510), solvent extraction (EP-B-0,145,240) or
spray drying (EP-A-0,315,875 ~ South African Patent
- 2 -
88/8396, CH 666,06 A5, compare Derwent Ref. No.
88-228036/33) are suitable. Compared to the other
processes, spray drying has the advantage that the
encapsulation efficiency is as a rule higher 'than in the
other processes mentioned and that no other auxiliaries
are needed for encapsulation, which auxiliaries would
then lead to possible contamination in the product. It is
disadvantageous that the spray drying has to be carried
out at relatively high temperatures, i.e. as a rule far
above the boiling point of the respective solvents. If
suspensions, emulsions or solwtions of copolymers of
lactic and glycolic acid (PLCA) are subjected to spray
drying under custamary conditions, no microparticles are
obtained, but thread-like structures which are not
suitable for an injectable depot form (EP-A-0,315,875).
The microcapsules obtained by spray drying at 59°C
described in CH 666,406 A5 do not contain PLGA as the
biologically degradable support material but D,L-oligo-
lactoyl-N-(L)-phenylalanine.
Surprisingly, it has now been found that DLGA polymer
solutions and apprapriate aqueous suspensions and emul-
sions can be sprayed to give microparticles if the
spraying temperature is less than 6'0°C and the spray flow
rate is above 500 NL/h. These microparticles are addi-
tionally distinguished, compared to microparticles which
are prepared by the other processes mentioned, by a lower
residual solvent content (organic solvent, water), which
is below 1~. The latter is of great importance, in
particular in the case of injectables, since high demands
are made here on the purity of a pharmaceutical.
The invention therefore relates to microparticles which
contain a peptide as the active substance and
poly(lactide-co-glycolide) (DLGA) as the carrier
material, which are obtained by spray drying at
temperatures below 60°C at a spray flow rate of above
500 NL/h.
(1 NL = 22.415 liters).
3
In the embodiments above and below, microparticles are
also understood as meaning mic:cocapsules and micro-
spheres, i.e. both particles in which the active sub-
stance is completely or partially endorsed by polymer,
and particles in which the active substance is dispersed
in a PLGA matrix.
Peptides are understood as meaning natural and synthetic
peptides and also their physiologically tolerable salts.
The invention furthermore relates to a process for the
preparation of microparticles which contain a peptide as
the active substance and PLGA as the carrier material,
which compr:Lses suspending or dissolving the peptide in
a solution of the carrier material or emulsifying an
aqueous solution of the peptide in a solution of the
carrier material and spraying the suspension, solution or
emulsion at a temperature below 60°C at a spray flow rate
of greater than 500 NL/h.
The microparticles according to the invention preferably
contain water-soluble peptides. The molecular weight of
the peptides is preferably over 800. Particularly suit-
able peptides are LHRH agonists anti antagonists such as,
for example buserelin, HOE 013 (Ac-D-Nal-p-Cl-D-Phe-D-
Trp-Ser-Tyr-D-Ser-(a-L-Rha)-Leu-Arg-Pro-Azagly-NH2,
campare EP-A-0,263,521), nafarelin, triptorelin,
leuprorelin and goserelin. Peptides of other types are
also suitable such as, for example, TRH, vasopressin,
calcitonin, insulin or HOE 427 {= ebiratide, [4
methionine dioxide, 8-D-lysine, 9-phenylamine]-a-MSIi
{4-9)-(8-aminooctyl)amide triacetate, compare EP-A
0,179,332).
The biologically degradable support ma'~erial is PLGA. The
release of active substance from the microparticles is
not only affected by the physicochemical properties of
the active substance but also by the properties of the
polymer or of the polymer mixtuxes such as molecular
weight, molar composition and sequence of the lactic acid
and glycolic acid units in the polymer. The carrier
material may comprise, for example, a mixture of 50:50
PZ,GA and 40:60 PT~G~1. The higher the molecular weight or
the inherent viscosity, the longer the release of active
substance lasts. The inherent viscosity (20°C,
chloroform, 0.1~) is preferably 0.1 to 0.8 dl/g. The
molar ratio of lactide to glycolide units is in the
range, for example, from 85:15 to 40:60; preferably 50:50
poly(d,l-lactide-co-glycolide) is used which has a
viscosity of 0.1 to 0.7 dl/g, in particular of 0.1 to
0.5 dl/g.
Depending on the properties .of the polymer, the micro-
particles prepared by the process according to the
invention release the active substance over a period of
preferably 2 weeks to 3 months. The degree of loading
also has an influence, even if minor, on the duration of
active compound release. In the process according to the
invention, microparticles having a degree of peptide
loading of less than 20~, preferably of less than 12~,
are preferably obtained.
In the microparticles prepared by the process according
to the invention, the particle size, which also affects
the release, is less than 200 yam, preferably less than
100 ~sm. In particular, the particle size is less than 50
~sm.
The microparticles are prepared by spraying of a
solution, emulsion or suspension of the active substance
in the polymer solution. Solvents which can be employed,
for example, are chloroform, methylene chloride, Dr3~',
acetone, ethyl acetate, glacial acetic acid and water or
mixtures thereof. Preferably, methylene chloride, glacial
acetic acid and water are used.
In the process according to the invention, the peptide is
preferably dissolved in water and added to the solution
~~~z~D~~~
-- 5
of PLGA in, for example, methylene chloride. The emulsion
formed in this way is preferably sprayed. Spraying can
also be carried out after addition of, for example,
methanol, a solution then being formed.
The spraying temperature is preferably 50° to 30°C, in
particular 40° to 30°C. The spray flow rate must not be
too low, as otherwise thread formation can occur. Tt is
preferably about 800 ~L/h, as better drying properties
are associated with a higher spray flow rate.
In the case of the use of a laboratory sprayer (for
exempla Biichi Mini Spray dryer 190), the pressure of the
spray medium, for example air or nitrogen, should be in
the range 3-8 bar. The pump capacity of the sprayer must
be suited to the conditions (3-20 ml/min). Hy appropriate
adjustment of the aspirator, for example 18 scale
divisions, it is ensured that all the air or nitrogen in
the spray medium is removed.
The PLGA polymer should be dissolved in as little solvent
as possible, polymers with a high content of lactide
requiring a larger amount of solvent. Tn principle, the
concentration of the polymer in the solution, emulsian or
suspension to be sprayed should be selected such that
spraying is possible under the selected conditions.
Preferably, the PLGA concentration in methylene chloride
is less than 15~ by weight, calculated relative to
methylene chloride.
The invention is illustrated by the following examples.
The viscosity given in the examples for PLGA is the
inherent viscosity of a 0>1~ strength salution in
chloroform at 20°C determined by generally customary
methods.
~'he spray parameters a ) aspirator and b ) pump capacity
are adjusted in all examples to 18 (for aspirator) or
~(~6~88~
s --
(for pump capacity) scale divisions.
Example 1e Spray medium m glacial acetic acid
a) 0.064 g of buserelin acetate is dissolved in 2.0 g of
water. The aqueous solution is mixed with a solution of
1.936 g of 50:50 PLGA (IV = 0.4 dl/g) in 40 g of glacial
acetic acid and sprayed in a laboratory spray dryer.
The spray parameters are adjusted as followss
1. Inlet temperature 50°C
2. Spray flow rate 800 NL/h
The microparticles are obtained as a white, pourable
powder. Yields 1.2 g = 60~ of theory.
Active substance content
(degree of loading): 3~
Residual solvent contents
glacial acetic acid 0>8~
b) In the same manner, microparticles are prepared using
0.192 g of buserelin acetate and I.SOS g of 50x50 PLGA
(IV = 0.4 dl/g). Yield: 1.2 g = 60~ of theory.
Active substance contents
Residual solvent contents
Glacial acetic acid: O.S~
For 20 mg of microparticles, the in vitro release rates
were determined as follows:
20 mg of microparticles are introduced into 2.0 ml of a
pH '7.4 phosphate buffer solution and eluted for 24 h at
+37°C. After separation of the elcaate from the micro
particles, the elcaate is tested for buserelin content
using an HPLC method. The remaining microparticles axe
once more added to 2.0 ml of phosphate buffer and again
eluted for 24 h at 37°C. This process is continued
analogously over 56 days.
The amounts of buserelin acetate determined from the
7 _
eluates are compiled in extract form in the following
table:
Active Substance Active Substance
Content Content
3~ 9~
Day 2 5.8 ~g 23.2 psg
Day 9 a 0,7 ~g 57.7 ~g
Day 15 13.4 ~g 51.9 ug
Day 23 20.3 ~g 16.7 ~g
Day 30 13.2 ~g 8.4 ~g
Day 37 4.1 ~g 3.4 ,ug
Day 44 2.5 ~g 1.5 ~g
Day 51 0.7 ~g 0.7 ~g
Day 5s 0.7 ~g 0.7 ~g
Example 2: Spray medium ~ chloroform
a) 0.108 g of buserelin acetate is dissolved in 2.0 g of
water. Using a rotor~stator homogenizer, the agueous
solution is emulsified in a solution of 1.892 g of 50:50
PLCA (IV = 0.4 d1/g) in 92 g of chloroform and sprayed in
a laboratory spray dryer.
The spray parameters are adjusted as follows:
1. Inlet temperature 30°C
2. Spray flow rate 800 NL/h
The microparticles axe obtained as a white, pourable
powder. Yield: 0.9 g = 45~ of theory.
Active substance content: 5~
b) In the same manner, microparticles are prepared using
0.152 g of buserelin acetate and 1.848 g of 50:50 PLEA
(IV = 0.4 dl/g). Yield: 1 g = 50~ of theory.
Active substance content: 7~
For 20 mg of microparticles, the in vitro release rates
were determined analogously to Example 1 over 35 days:
g
The amounts of buserelin acetate determined from the
eluates are compiled in extract :Form in the following
table:
Active Substance Active Substance
Content Content
5 ~ 7 ~
Day 2 49.7 ~g 56.2 ~sg
Day 9 7.3 fag 10.1 ~g
Day 16 30.3 ~g 37.8 ~g
Day 23 30.0 Esg 38.6 ~g
Day 30 10.9 ~g 13.6 ug
Day 35 11.5 ~cg 19:.5 ~g
Example 3t Spray medium - methylene chloride/water/-
methanol
a) 0.128 g of buserelin acetate is dissolved in 2.0 g of
water. The aqueous solution is mixed with a solution of
1.872 g of 50:50 PLGA (IV - 0e~4 dl/g) a.n ~6 g of
methylene chloride. 9 g of methanol are added to the
mixture. A clear solution is formed which is sprayed in
a laboratory spray dryer.
The spray parameters are adjusted as follows:
1. Inlet temperature 30°C
2. Spray flow rate 800 I3z/h
The microparticles are obtained as a white, pourable
powder. 'Yields 1.2 g = 60~ of theory.
Active substance content: 6~
Residual solvent contents Water 0.9~
Methylene chloride 0.06
Methanol ~ 0.01
~'or 20 mg of microparticles, the in vitro release rates
were determined analogously to Example 1 over 28 days:
The amounts of buserelin acetate determined from the
eluates are compiled in extract form in the following
table:
Active Substance Content
6~
Day 2 93.7 ~g
Day 9 5.1 ~sg
Day is 7.4 ~g
Day 23 12.1 ~~g
Day 28 12, 2 fag
Example 4: Spray medium - methylene chloride/water/m
methanol
a) 0.084 g of buserelin acetate is dissolved in 4.0 g of
water. The aqueous solution is mixed with a solutian of
1.916 g of 50:50 PT~GA (IV ~ 0.42 d1/g) i~ 65 ml of
methylene chloride. 25 g of methanol are added to the
mixture. A clear solution is formed which is sprayed in
a laboratory spray dryer.
The spray parameters are adjusted as follows:
1. Tnlet temperature 3!)°C
2. Spray flow rate 800 ~1Z/h
The micraparticles are obtained as a white, pourable
powder. Yields 0.8 g = 40& of theory..
Active substance contents 4~
b) In the same manner, microparticles are prepared using
0.168 g of buserelin acetate and 1.832 g of 50:50 P~GA
(IV ~ 0.42 dl/g). Yield: 0.8 g = 40~ of theory.
Active substance content: 8~
For 20 mg of microparticles, the in vitro release rates
3U were determined as follows:
20 mg of microparticles are introduced into 2.0 ml of a
~~~~88~
_ 10 _
pH 7.4 phosphate buffer solution and eluted for 24 h at
+37°C. After separation of the elua~te from the micro-
particles, the eluate is tested for buserelin content
using a radioimmunoassay (~tIAp The remaining micro-
s particles are added to 2.0 m1 of phosphate buffer once
again and again eluted for 24 h at 3?°C.
This process is analogously continued over 63 days,
The amounts of buserelin acetate determined from the
eluates are compiled in extract form in the following
30 table:
Active Substance Active Substance
Content Content
4~ 8~
Day 2 129.3 ~sg 280.1 ~sg
15 Day 9 1.7 pig 4.7 ~g
Day 16 1.9 ~g 5.7 ug
Day 23 6.3 ug 3.0 gag
Day 30 6.4 ~sg 8.1 ug
day 37 3.1 fag 5.6 peg
20 Day 44 1.6 ~.g 3.9 ~sg
Day 51 3.1 ~g 2.4 ~,g
Day 58 0.7 ~cg 1.4 ~g
Day 63 0.7 gag 1.4 ~sg
Example 5: Spray medium -~ chloroform/water/dimethyl-
25 formamide
a) 0:13 g of buserelin acetate is dissolved in 2.0 g of
water. Using a rotor-stator homogenizer, the aqueous
solution is emulsified in a solution of 1.87 g of 50150
PLGA (T~ = 0.4 dl/g) in 41.4 g of chloroform and 4.6 g of
30 dimethylformamide and sprayed in a laboratory spray
dryer.
The spray parameters are adjusted as follows:
1. Inlet temperature 30°C
2. Spray flow rate 800 NL/h
- 11 -
The microparticles are obtained as a white, pourable
powder. Yields 1.2 g = 60~ of theory.
Active substance content: 6~
For 20 mg of microparticles the in vitro release rates
were determined analogously 'to Example 4 over 63 days:
The amounts of buserelin acetate determined from the
eluates are compiled in extract form in the following
table:
Active substance content
6~
Day 2 32.1 ~g
Day 9 16.8 ~g
Day 16 49.1 ~sg
Day 23 39.1 ~g
Day 30 17.5 fag
Day 37 5.7 ~sg
Day 44 2.5 ~g
Day 51 2.5 ~g
Day 58 1.4 ~g
Day 63 1.4 gag
Example 6: Spray medium - methylene: chloride
a) 0.085 g of buserelin acetate is dissolved in 2.0 g of
water. t7sing a rotor~stator homogenizer, the acyueous
solution is emulsified in a solution of 1.915 g of 50:50
FLGA (IV = 0.4 dl/g) in 46 g of methylene chloride and
sprayed in a laboratory spray dryer.
The spray parameters are adjusted as follows:
1. Inlet temperature 30°C
2. Spray flow rate 800 I~L/h
The microparticles axe obtained as a white, pourable
powder. Yield: 0.6 g = 30~ of theory.
2~6~~83
- 12
Active substance content: 4~
b) Tn the same manner, microparticles are prepared using
0.128 g of buserelin acetate and 1.872 g of 50:50 FLGA
(IV = 0.4 dl/g). "Yield: 0.7 g = 35~ of theory.
Active substance contewt: 6~
c) and also using 0.170 g of buserelin acetate and
1.872 g of 50:50 FLGA (IV = 0.4 dl/g).
Yield: 0.4 g = 20$ of theory.
Active substance content: 8~S
For 20 mg of the microparticles, the in vitro release
rates were determined analogously to Example 4 over 63
days:
"the amounts of buserelin acetate determined from the
eluates are compiled in extract form in the following
table:
Active Active Active
substance substance substance
content content conter_t
4~ 6~ 8~
Day 2 39.4 ~cg 49.8 gag 88.0 ~cg
Day 9 1.1 ~g 2.7 psg 4.7 ~g
Day 16 5.9 ~cg 34.3 ~g 21.7 fag
Day 23 17.1 ~sg 51.5 ~g 28.0 ~sg
Day 30 4.2 ~sg 18.4 ~sg 3.2 ~g
Day 37 1.5 ~g 5.8 gag 2.2 ~g
Day 44 1.7 ~g 2.5 gag 2.8 ~g
Day 51 0.4 ~sg 0.8 ~g 2.4 ~sg
Day 58 0.3 ~g 0.? ~g 2.5 ~sg
Day 63 0.3 ~g 0.3 ~ug 2.5 ~g
Example 7: Spray medium - methylene chloride/water
0.032 g of buserelin acetate is dissolved in 1.0 g of
crater. Using a rotor-stator homogenizer, the aqueous
- 13
solution is emulsified in a solution of 0.968 g of 85:15
PLGA in 23 g of methylene chloride and sprayed in a
laboratory spray dryer.
The spray parameters are adjusted as follows:
1. Inlet temperature 40°C
2. Spray flow rate 800 NL/h
The microparticles are obtained as a white, pourable
powder. Yield: 0.7 g = 70~ of theory.
Active substance content: 3~
Example 8: Spray medium - methylene chlaride/water
0.064 g of buserelin acetate is dissolved in 2.0 g of
water. Using a rotor-stator homogenizer, the aqueous
solution is emulsified in a solution of 1.936 g of 50:50
PLGA (IV = 0.7 dl/g) in 92 g of methylene chloride and
sprayed in a laboratory spray dryer.
The spray parameters are adjusted as follows:
1. Inlet temperature 40°C
2. Spray flow rate 8(IO NL/h
The microparticles are obtained as a white, pourable
powder, yield: 0.9 g = 45~ of theory.
Active substance content: 3~
Release of active substance determined analogously to
Example 1
Active substance content
3~
Day 2 48.1 beg
Day 23 3.9 ,fig
Day 37 7.1 ~g
Day 44 3.9 ~g
Day 51 1.0 ~cg
~~~~88~
.. 14 _
Example 9: Spray medium ~ methylene chloride/wate:r
0.106 g of buserelin acetate is dissolved in 1.0 g of
water. Using a rotor-°stator homagenizer, the aqueous
solution is emulsified in a solution of 0.894 g of 50:50
PLGA (IV = 0.1 dl/g) in 23 g of methylene chloride and
sprayed in a laboratory spray dryer.
The spray parameters are adjusted as follows:
1. Inlet temperature 40'°C
2. Spray flow rate 800 Ny/g
The microparticles are obtained as a white, pourable
powder. Yield: 0.3 g = 30~ of theory.
Active substance content: 10~
Release of the active substance determined analogously to
Example 1
Active substance content
10$
Day 2 32.7 ~g
day 9 19.6 ~g
Day 16 20.u' ~g
Day 23 15.4 fag
Day 28 15.4 ~sg
Example 10: Spray medium ~ methylene chloride/water
0.13 g of buserelin acetate is dissolved in 2.0 g of
water. Using a rotor-stator hamogenizer, the aqueous
solution is emulsified in a solution of 1.87 g of 75x25
PLGA (IV = 0.5 dl/g) in 46 g of methylene chloride and
sprayed in a laboratory spray dryer.
The spray parameters are adjusted as follows:
1. Inlet temperature 30~C
2. Spray flow rate 800 NZ/h
15 - 2~~~~~~
The microparticles are obtained as a white, pourable
powder. Field: 1.2 g = 60~ of theory.
Active substance content: 6$
Example 11: Spray medium - methylene chloride/water
0.128 g of buserelin acetate is dissolved in 2.0 g of
water. Using a rotor-stator homogenizes, the aqueous
solution is emulsified in a solution of 1.872 g of 75:25
PLGA (IV = 0.8 dl/g) in 138 g of methylene chloride and
sprayed in a laboratory spray dryer.
The spray parameters ass adjusted as follows:
1. Inlet temperature: 30°C
2. Spray flaw rate 800 NL/h
The microparticles are obtained as a white, pourable
powder. Yield: 0.9 g = 45~ of theory.
Active substance content: 6~
Example 12: Spray medium - methylene chloride/water
0.13 g of buserelin acetate is dissolved in 2.0 g of
water. Using a rotor~stator homogenizes, the aqueous
solution is emulsified in a solution of 1.87 g of 47:53
PLGA (IV = 0.3 dl/g) in 46 g of methylene chloride and
sprayed in a laboratory spray dryer.
The spray parameters are adjusted as follows:
1. Inlet temperature 30°C
2. Spray flow rate 800 NL/h
The microparticles are obtained as a white, pourable
powder, yield: 1.3 g = 65~k of theory.
Active substance content: 6~
Example 13: Spray medium ~- methylene chloride
0.13 g of buserelin acetate is suspended in a solution of
~0~~~~3
- 16 -
1.87 g of 50:50 PLGA (IV - 0,4 dl/g) and 46 g of
methylene chloride using a rotor-stator homogeni~er. The
suspension is sprayed in a laboratory spray dryer.
The spray parameters are adjusted as follows:
1. Inlet temperature 40°C
2. Spray flow rate 800 NL/h
The microparticles are obtained as a white, pourable
pawder. Yield: 0.5 g ~ 25~ of theory.
Active substance content: 6~
Example 14: Spray medium - methylene chlaride/water
a) 0.2 g of Hoe 427 is dissolved in 1.0 g of water. Using
a rotor-stator homogeni~er, the aqueous solution is
emulsified in a solution of 3.8 g of 50:50 PLGA (IV =
0.4 dl/g) in 92 g of methylene chloride and sprayed in a
laboratory spray dryer.
The spray parameters are adjusted as follows:
1. Inlet temperature 40°~
2. Spray flow rate 800 ~1L/h
The microparticles are obtained as a white, pourable
powder. Yield: 1.9 g = 48~ of theory.
Active substance content: 5~
b) In the same manner, microparticles are prepared using
0.6 g of Hoe 427 and 3.4 g of 50:50 PLGA (IV = 0.4 dl/g),
Yield: 2.8 g = 70~ of theory.
Active substance content: 15~
c) and also using 0.4 g of Hoe 427 and 3.6 g of 50:50
PLGA (IV = 0,4 d1/g).
Yields 2 g = 50~ of theory.
Active substance content: 10~s
- 17 -
Example 15s Spray medium - methylene chloride/water
0.08 g of Hoe 013 is dissolved in 1.0 g of water. Using
a rotar-stator homogenizer, the aqueous solution is
emulsified in a solution of 0.92 g of 50:50 PLGA (IV
0.4 dl/g) in 23 g of methylene chloride and sprayed in a
laboratory spray dryer.
The spray parameters are adjusted as followss
1. Inlet temperature 30°U
2. spray flow rate 800 NL/h
20 The microparticles are obtained as a white, pourable
powder. Fields 0.5 g = 50~ of theory.
Active substance content: 6~
Release of active substance determined analogously to
Example 1
Active substance content
6~
Day 2 13.9 ~sg
Day 9 5.9 pig
Day lfi 3.1 ~g
Day 23 3.7 ug
Day 28 2.2 ~g
