Canadian Patents Database / Patent 2040237 Summary

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(12) Patent Application: (11) CA 2040237
(54) English Title: LONG-ACTING LIPOSOME PEPTIDE PHARMACEUTICAL PRODUCTS AND PROCESSES FOR THE PREPARATION THEREOF
(54) French Title: PRODUITS PHARMACEUTIQUES A BASE DE PEPTIDE LIPOSOMIQUE A ACTION PROLONGEE, ET LEURS PROCEDES DE PREPARATION
(52) Canadian Patent Classification (CPC):
  • 167/163
  • 167/170
(51) International Patent Classification (IPC):
  • A61K 9/127 (2006.01)
  • A61K 38/09 (2006.01)
  • A61K 38/58 (2006.01)
(72) Inventors :
  • KIBAT, PAUL-GERHARD (Germany)
  • SANDOW, JURGEN K. (Germany)
(73) Owners :
  • KIBAT, PAUL-GERHARD (Country Unknown)
  • SANDOW, JURGEN K. (Country Unknown)
  • HOECHST AKTIENGESELLSCHAFT (Germany)
(71) Applicants :
(74) Agent: BERESKIN & PARR LLP/S.E.N.C.R.L.,S.R.L.
(45) Issued:
(22) Filed Date: 1991-04-11
(41) Open to Public Inspection: 1991-10-13
(30) Availability of licence: N/A
(30) Language of filing: English

(30) Application Priority Data:
Application No. Country/Territory Date
P 40 11 864.9 Germany 1990-04-12

English Abstract


- 1 -
Abstract of the disclosure: HOE 90/F 112

Long-acting liposome peptide pharmaceutical products and
processes for the preparation thereof

Liposome products for peptides with extended peptide
release are described. The peptides have a molecular
weight between 500 and 10000, and the phospholipid
component of the liposome membrane has a phase-transition
temperature of at least 20°C and contains mainly
saturated fatty acids. The activity persists for more
than 14 days after s.c. or i.m. injection. Processes for
preparing these liposome products are also described.


Note: Claims are shown in the official language in which they were submitted.

- 18 - HOE 90/F 112

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A liposome product for peptides with extended peptide
release, wherein the peptides have a molecular weight
between about 500 and 10000, the phospholipid component
of the liposome membrane has a phase-transition temper-
ature of at least 20°C and mainly contains saturated
fatty acids, and the activity persists for more than
14 days after s.c. or i.m. injection.

2. A liposome product as claimed in claim 1, wherein one or
more of the following conditions are met

a) the peptide is an LHRH analog, a bradykinin anta-
gonist, HOE 427 or a hirudin derivative,
b) the phase-transition temperature of the phospholipid
component is above 30°C,
c) the mainly saturated fatty acids of the phospholipid
component have a chain length of at least 14 carbon
atoms,
d) the liposome membrane contains an added steroid,
e) the liposomes have an average volume-related parti-
cle size of at least 600 to 10000 nanometers,
f) the activity persists for at least 20 days.

3. A liposome product as claimed in claim 1, wherein one or
more of the following conditions are met

a) the peptide is an LHRH analog, HOE 140, HOE 427 or
HBW 023,
b) the phase-transition temperature of the phospholipid
component is at least 37°C,
c) the mainly saturated fatty acids of the phospholipid
component have a chain length of at least 14 carbon
atoms,
d) the liposome membrane contains an added steroid,
e) the liposomes have an average volume-related parti-
cle size of at least 600 to 1000 nanometers,
f) the activity persists for at least 30 days.

- 19 -
4. A liposome product as claimed in claim 1, wherein one or
more of the following conditions are met

a) the peptide is buserelin acetate or HOE 013,
b) the phase-transition temperature is at least 37°C,
c) the phospholipid component is dipalmitoyl-phosphati-
dyl-choline (DPPC) or hydrogenated lecithin from
natural sources,
d) the membrane contains added cholesterol,
d) the liposomes have an average volume-related parti-
cle size of at least 600 to 10000 nanometers,
f) the activity persists for at least 30 days.

5. A liposome product as claimed in claim 1, wherein
additional charge carriers are contained in the membrane
material.

6. A liposome product as claimed in claim 1, which contains
antioxidants and other auxiliaries with stabilizing or
release-influencing properties.

7. The use of a liposome product as claimed in claim 1 for
s.c. or i.m. injection.

8. A process for preparing a liposome product as claimed in
claim 1, which comprises

a) .alpha.) dissolving the phospholipid component and, where
appropriate, lipophilic additives in a suitable
organic solvent, removing the solvent and detach-
ing the resulting lipid matrix after adding an
aqueous solution of the peptide to form lipo-
somes, where the detachment takes place above the
phase-transition temperature of the phospholipid
component, or

.beta.) dissolving the phospholipid component and, where
appropriate, lipophilic additives, and the
peptide in a suitable organic solvent, removing

- 20 -
the solvent and detaching the resulting lipid
matrix using an aqueous medium, where the detach-
ment takes place above the phase-transition
temperature of the phospholipid component, or

.gamma.) dissolving the phospholipid component and, where
appropriate, lipophilic additives in a volatile
organic solvent and adding an aqueous peptide
solution which is immiscible with the organic
phase, converting the resulting two-phase system
by homogenization above the phase-transition
temperature of the phospholipid component into a
stable emulsion, and removing the organic solvent
with the formation of liposomes
and adjusting the liposome dispersions which have
been obtained by methods .alpha. to .gamma., where appropriate
after homogenization and equilibration, to the
required peptide content, and bottling and, where
appropriate, freeze-drying, or

b) preparing a lyophilisate of peptide-free liposomes
by methods .alpha., .beta. or .gamma., and dispensing an aqueous
peptide solution into a suitable vessel, where the
lyophilisate and peptide solution are combined
before administration.

- 21 -
9. The liposome product as claimed in claim 1 and
substantially as described herein.

Note: Descriptions are shown in the official language in which they were submitted.

2~23~

HOECHST AKTIENGESELLSCHAFT ~OE 90/F 112 Dr.D/pe

Description

;Long-acting liposome peptide pharmaceutical products and
processes for the preparation thereof

The invention relates to long-acting liposome peptide
pharmaceutical products for parenteral administration.
The preparations according to the invention are
administered subcutaneously (s.c.) or intramus-
cularly (i.m.~ and have a duration of action of more than
14 days. The invention furthermore relates also to
processes for preparing these products.

Liposomes are submicroscopic particles in the form of
hollow spheres. They possess a double membrane which i~
composed of amphiphilic molecules, usually phospholipids,
and surrounds an aqueous interior. They are composed of
body-like material, can act as carriers of a wide variety
of 6ubstances and can be spacif~cally adapted to meet
specific requirement~. In this connection, hydrophilic
pharmaceuticals are predominantly enclosed in the aqueous
interior volume, while lipophilic substances are mostly
bound in the membrane.

Liposomes are proposed as carrier ~ystems for a large
number of pharmaceuticals such as, for example, for
cytostatics, antiinfective agents and immunomodulators
(for example Yatvin, M.B. and Lelkes, P.I., Med. Phys. 9,
(1982)). Liposomal pharmaceutical products are mainly
administered parenterally, and often intravenous adminis-
tration is desired. The aim i8 usually to make use of a
depot effect, to reduce side effects or to increase
activity. After i.v. in~ection, liposomes, like all
colloidal sy~tems, are taken up by the cells of the
reticuloendothelial system (RES), eliminated with a half-
life not exceeding 2 days, and accumulate preferentially
ln the liver and spleen (Senior, J.H., CRC, Critical




.
- ,

~.

2 0 ~ 7
-- 2 --
Reviews in Therapeutic Drug Carrier Systems 3, 123
(1987)). Longer active levels are obtained after 8 .C . or
i.m. injection than after i.v. injections. The duration
of action of liposome products depends on the release of
~ubstance from the vesicles and on the transport thereof
from the injection 8ite, and on the breakdown of the
vesicles. Release of substance and breakdown are deter-
mined, in particular, by the composition of the liposome
membrane, while the tranport away depends on the particle
size, i.e. increases with decreasing particle size
(Arrowsmith et al., Int. J. Pharm. 20, 347-362 ~1984)~.
An additional factor is the lipid concentration in the
product (Jackson, A.J., Res. Comm. Chem. Pathol.
Pharmacol. 27, 293 (1980)).

The investigations, described in the publications indi-
cated above, on the i.m. or s.c. administration of
liposomal pharmaceutical carriers in no case showed
pharmaceutical release or retention of the product at the
site of administration for more than 14 days. On the
contrary, the pharmacokinetic investigations on these
liposome preparations of diverse vesicle composition and
with various pharmaceuticals also showed that either the
release of active substance was complete in the period of
14 days, or that the liposome had been broken down within
this time.

Liposome products for s.c. or i.m. in~ection for peptides
have already been described. A liposome formulation for
the long-term release of insulin is described, for
example, in GB-B 2,050,287. The international patent
application with the publication no. ~0 87/04592
describes a liposome release system for membrane-
impermeable molecules - calcitonin for example - which is
composed of a mixture of small S W (unilamellar lipo-
somes, particle size about 30-100 nm) containing active
substance with large MLV (multilamellar vesicles,
particle size about 200-10000 nm). Fukunaga et al.
(Endocrinology 115, 757 (1984)) describe an extended




- . , , :'' :'

_ 3 _ 2~2~7

hypocalcemic effect of calcitonin after liposomal encap-
sulation of the protein. According to the examples in
these publications, in no ca~e was it possible to find
an activity over more than 14 days.

For peptides, such as, for example, for LHRH analogs,
long-acting formulations based on biodegradable polymers
are described (compare, for example, EP-B 0 052 510 and
EP-B 0 145 240 for microcapsules, EP-~ 0 058 481 for
other controlled release systems). EP-A 0 299 402
describes long-acting formulations of LHRH analogs with
antagonistic activity.

Whereas liposome formulations are not mentioned in the
4 abovementioned publications~ GB-B 2 050 287 describes
an LHRH-containing liposome composition which, however,
contains, in contrast to the compositions of the present
invention, release modulators and has an elimination
half-life of about 4 days after ~.c. injection.

Liposomes as carrier systems for LHRH have also been
described by Schafer et al. (Pharmazie 42, 674 (1987) and
Pharmazie 42, 689 (1987)). They prepared MLV from mix-
tures of egg lecithin and phosphatidic acid and investi-
gated the pharmacokinetics after i.m. administration to
rabbits or pigs. The half-life for elimination from the
injection site did not exceed 20 hours. It was no longer
possible to measure LHRH blood levels after this time.

Surprisingly, it emerges with the special liposome
formulations characterized hereinafter that, in some
cases, they are still detectable after 35 days at the
in~ection site and they lead to significant blood levels
and pharmacological effect~.

The invention therefore relates to liposome products for
peptides with extended release of peptide, wherein the
peptides have a molecular weight between about 500 and
10000, the phospholipid component of the liposome




~.
.
.

_ 4 ~ 32S~7
membrane has a phase-transi~ion temperature of at lea~t
20~ and mainly contains saturated fatty acids, and the
activity persists for more than 14 days aftex s.c. or
i.m. injection.

The liposome preparations according to the invention
ensure, because of their special composition, an activity
over a period of more than 14 days. This means that the
products, because of their specific composition, both
remain for more th~n 14 days at the site o~ administra-
tion without being broken down, and release over thisperiod the enclosed peptide active substances in an
amount sufficient for the required activity.

The activity preferably persists for at least 20 days, in
particular 30 days and more.

The average volume-equivalent particle size of the
vesicles (liposomes) is preferably between 600 nm and
10000 nm, in particular above 800 nanometers, in order to
minimize the rate of transport away from the in~ection
site. The phospholipid component of the lipo~ome membrane
preferably has a phase-transition temperature of above
30~C, in particular at least 37C. It mainly contains
saturated fatty acid~ with a chain length of at least
14 carbon atoms.

Examples of suitable phospholipids are dimyristeyl-PC
(DMPC), distearoyl-PC ~DSPC), dipalmitoyl-PC (PC = phos-
phatidylcholine) or hydrogenated or partially hydro-
genated lecithins from natural sources. Suitable for
stabilization of the membrane are, for example, lipo-
philic additives of steroid6, such as cholesterol.

~he peptides encapsulated in liposomes (al80 in the form
of their physiologically tolerated salts) are of natural,
synthetic or semisynthetic origin and have specific
effects in the body. Thus, in the statements made herein-
before and hereinafter, peptides mean within the scope of



.

,
. . , , ' : . ' :,'

- 5 - ~ 7
the invention both free compounds and the physiologically
tolerated salts of the peptides characterized above. They
have a molecular weight of about 500 to 10000. Examples
of suitable peptides are LHRH analogs, bradykinin antago-
nists, insulin, vasopressin, oxytocin, calcitonin,heparin, hirudin and their synthetic or semisynthetic
analogs. Preferably encapsulated are LHRH analogs such
as, for example, buserelin, HOE 013 (Ac-D-Nal-p-Cl-D-Phe-
D-Trp-Ser-Tyr-D-Ser (~-L-Rha)-Leu-Arg-Pro-Aza~ly-NH2,
compare EP-A O 263 521, corresponding to US Patent
Application No. 390477). However, also suitable are, for
example, hirudins such as HBW 023 (R-DNA-hirudin di~-
closed in EP-A O 324 712, corresponding to ~S Patent
Application No. 295 422), HOE 427 (= ebiratide, ~4-
methionine dioxide, 8-D-lysine, 9-phenylamine]-~-MSH-
(4-9) (8-amino-octyl)amide triacetate, compare
EP-A O 179 332 corresponding to US Patents No. 4,623,715
and No. 4,696,913) and HOE 140 (= H-D-Arg-Arg-Pro-Hyp-
Gly-Thi-Ser-D-Tic-Oic-Arg-OH. 6CH3COOH, compare
EP-A O 370 453 corresponding to US Patent Application
No. 374 162).

It is known that the peptides ~uitable as active sub-
stances are active for only very short times after
administration in the living body (Banga et al., Int. J.
2~ Pharm. 45, 15-50 (1988)). They are inactivated by enzymes
or else chemical reactions and eliminated very rapidly.
The encapsulation of these peptides to give the liposome
products according to the invention makes it possible to
protect the substances from rapid metabolic inactivation
in the body and to ensure long-lasting continuous release
of unchanged active substance over lengthy periods.

The liposomes are either of the unilamellar or the
multilamellar type. The peptides can be located both in
the aqueous interior a~ solution and in the liposome
membrane. The release of active substance is controlled,
in particular, via the membrane, i.e. its nature and
possibly the content of active substance in the membrane



.
; .
'

2~2~7
-- 6 --
influence the duration of release of active substance.
Encapsulation of peptides in large, for example multi-
lamellar, liposomes increases, for example, the duration
of action owing to binding of the active substance to the
S carrier system to, for example, 20 days and more. With
the liposome products according to the invention,
liposomes are still to be found at the in~ection site
even after 30 days, for example. Moreover, an activity is
still detectable after this period.

The release of active substance can additionally be
controlled by additions of negatively or positively
charged charge carriers such hS, for example, dipalmi-
toyl-phosphatidyl-glycerol or s~earylamine in the
membrane portion, antioxidants or other auxiliaries with
stabilizing or release-influencing properties.

The liposomes can be prepared in principle by all methods
known from the literature, for example (Lichtenberg, D.,
Methods of Biochemical Analysis 33, 337 (1988)). Par-
ticularly suitable are preparation technologies which
provide larger liposomes.

The processe~ for preparing the liposome products accord-
ing to the invention comprise

a) ~) dissolving the phospholipid component andl where
appropriate, lipophilic additives in a suitable
organic solvent, removing the solvent and
detaching the resulting lipid matrix after adding
an aqueous solution of the peptide to form lipo-
somes, where the detachment takes place above the
phase-transition temperature of the phospholipid
component, or

~) dissolving the phospholipid component and, where
appropriate, lipophilic additives, and the
peptide in a suitable organic solvent, removing
the solvent and detaching the resulting lipid


- . . . . .
, ., , - ~.

. . . .

_ 7 ~ 2 3 ~
matrix usin~ an aqueous medium, where the
detachment takes place above the phase-transition
temperaturQ of the phospholipid component, or

7) dissolving the phospholipid component and, where
appropriate, lipophilic additives in a volatile
organic solvent and adding an aqueous peptide
solution which is immi~cible with the or~anic
phase, converting the resulting two-phase ~ystem
by homogenization above the phase-transition
temperature of the phospholipid component into a
~table emulsion, and removing the organic solvent
with the formation of liposomes

and adjustinq the liposome dispersions which have
been obtained by methods ~ to ~, where appropriate
after homogenization and equilibration, to the
required peptide content, and bottling and, where
appropriate, freeze-drying, or

b) preparing a lyophilisate of peptide free lipo~omes
by methods ~, ~ or 7, and di~pensing an aqueous
peptide solution into a ~uitable vessel, where the
lyophilisate and peptide solution are combined
before administration.

The lyophilisate~ obtained by the processes according to
the invention are converted by conventional methods, such
Z5 as, for example, addition of water ~or in~ec~ions/ into
forms suitable for i.m. or 8 . C . admini~tration.

The aq~ m~um used in the proces~ according to the
invention i8 composed of water or 8 mixture of w~ter and
an organic solvent such ns, $or example, methanol or
ethanol. It may additionally contain additive~ such as
sodium chloride or buffers, for example pho~phate buffer.
The aqueous peptide solutions can also have ~uch
additives.
The processes are expediently carried out as follows.




,

~ ' ' - , . ' . .:

Method a) - & -
20~2~7
u) Pho~pholipids and, where appropriate, lipophilic
additives (for example cholesterol) are dissolved in an
organic solvent such a~, for example, ethanol, methanol,
dichloromethane, chloroform or tert. butanol. The solvent
is removed by methods which permit no ob~ectionable
solvent residues and yield a lipid matri~ of maximum
surface area. Particularly sui~able for thi~ purp~se are
evaporation with rotary evaporators and lyophilization or
combinations of the methods.

To form liposomes, the li~id
matrix is detached after the addition of an aqueous
solution, which is buffered if neces~ary, of the peptide
pharmaceutical. This process must be carried out with the
mixture at temperatures above the phase-transition
temperature of the phospholipid component and, of course,
below a critical decompo6ition temperature of the
peptide. It is assisted by agitation of the vessel and by
the use of aids to increase the rate (for axample gla~s
beads or scrapers). The liposome disper6ion can subse-
quently also be 6ub~ected to a homogenization step, for
example with an Ultraturrax, high-pressure homogenizers
and comparable processes. The formed liposome~ are
equilibrated at elevated temperature until they have
reached a 6table ~tate and optimal swelling. The homo-
geneity of the dispersion is improved by removing coarse
fractions by filtering it, for example, through membrane
or glass filters of 1-20 ~m pore diameter.
If encap~ulation of the pharmaceutical is not guantlta-
tive, in many cases there i6 a need to remove the
unencapsulated fraction. Cross-flow filtration provides
particular advantages in the separation of bound and free
pharmaceutical and, on suitable choice of the membranes,
also allows removal of the fine liposome fr~ction
(smaller than about 400 nm). It is al60 possible to use
centrifugation proce~ses, chromatographic proces~s (gel,
ion exchange or absorption chromatography) or removal of
the free peptide by methods of adsorption or digestion.




-


- 9 - 2~2~
The finished liposome dispersion is e~amined for the
pharmaceutical concentration by suitable methods hnd is
diluted to the re~uired content. It i~ dispensed into
2~mpoules or vials and ~tored under suitable conditions.
All the process steps in the preparation of pharma-
ceutical preparations are carried out under aseptic
conditions.

~) Preparation i8 carried out in analogy to method ~ ~ut
the peptide i8 dissolved together with the lipoph~lic
constituents in the organic ~olvent. This process ~
particularly suitable for peptides with lipophilic
characteristics; 6uitable solvents are ethanol, methanol
and tert. butanol.

7 ) Phospholipids and lipophilic additives (for example
cholesterol) are dissolved in a volatile organic solvent
such as, for example, diethyl ether, diisopropyl ether or
a mixture thereof with dichloromethane or chloroform. To
this solution is added an aqueous peptide solution ~hich
is immiscible with the organic pha~e. The two-phase
system is converted into a stable emulsion by 8uitab1e
homogenization processes (Ultraturrax, ultrasound, high-
pressure homogenizer) at temperature6 above the phase-
transition temperature of the phospholipid component and
below a critical decomposition temperature of the
peptide. After this, the organic solvent is removed in
vacuo at the necessary temperature. The liposomes are
formed via a metastable, usually gel-like intermediate
stage and are substantially freed of impurities by
further removal of solvent.

The liposomes are further processed, purif1ed and
bottled as described for method o.
~ipo~omes which are prepared by method ~-~ and contain in
the a~ueou~ solution addition~ of cryoprotective
substances or to which cryoprotective~ have been added
after the preparation can be freeze-dried. The cho8en
additives and freeze-drying processes are mutually

lo ~ 2~7
appropriate so that the liposomes are easy to
reconstitute before administration and contain a large
proportion of the peptide pharmaceutical in ~ound form.
Examples of suitable cryoprotectives are mannitol,
xylitol, sorbitol, trehalo~e, dextrans, polyvinyl-
pyrrolidone, albumin, hydroxyethylstarch and modified
gelatin types.

Method b)

Liposomes containing no active substance are prepared in
accordance with method a) ~, ~ or ~ and lyophilized as
described above. To prepare the liposome dispersion,
sterile aqueou~ peptide solution is added to the lyophi-
lisate. This liposome dispersion can then be
administered.

Liposomes which contain no active substance and are
obtained by method a) ~, ~ or ~ are, where appropriate,
converted by suitable homogenization processes into
di~persions of small vesicles. A particularly suitable
process is high-pressure homogenization, for example
using a microfluidizer, but it is also possible besides
this to carry out a treatment with ultrasound or
Ultraturrax. The small lipo~omes produced by this can
subsequently be sub~ected to sterilization by filtration
before they are lyophilized as described above. These
liposomes are combined with the peptide solution before
administration. The dispersion obtained in this way
predominantly has large ve6icles.

~he liposome products according to the invention display
a long-lasting continuous release of active substance.
~0 They are furthermore di~tinguished by their great
stability on storage. Thus, as described in Example 9,
more than 99 ~ of the peptide is still liposome-bound
after storage for 12 months, and the particle size is
unchanged.

2 ~ 7
11
Example 1

200 mg of LHRH antagonist (HOE 013), 1348 mg of
hydrogenated egg lecithin (phase-transition temperature
about 53C) and 652 mg of cholesterol are dissolved in
50 ml of methanol at SOC. The solution i8 sterilized by
filtration through 0.2 ~m membrane filters and converted
into liposomes under aseptic conditions. For this, the
solvent is removed in a rotary evaporator at 55C until
a thin lipid matrix (film) is formed. 20 ml of sterile
sodium chloride solution are added to the lipid film
while passing in nitrogen, and the film i8 detached from
the vessel wall within 2 hours at 55C and shaken at 50C
overnight. The resulting liposome dispersion is filtered
through S ~m membrane filters and made up to 100 ml with
sodium chloride solution. The resulting dispersion is
transferred into polycarbonate centrifuge tubes and
centrifuged at 20,000 x g and 5C for 5 minutes. The
supernatant containing dissolved, unencapsulated LHRH
antagonist is removed. After addition of fresh sodium
chloride solution, the liposomes are redispersed and the
centrifugation i~ repeated 5 times; finally, the lipo-
somes are made up to 20 ml. After the active substance
content has been determined by HPLC, the liposome
dispersion is diluted with sodium chloride solution to
the final concentration of 1.6 mg/ml HOE 013 and
dispensed into sterile vials. The volume-related particle
size is, on average, 2300 nanometers, and the encapsu-
lation efficiency is 20 ~.

Example 2

2 x 1 ml of the liposomes ~corresponding to a single dose
of 3200 ~g of HOE 013) from Example 1 are in~ected
subcutaneously into female rats of about 200 g body
weight. The control comprises an identical test group of
animals which receiYes only solvent (sodium chloride
solution) (placebo) and a group of animals treated with
daily doses of LHRH antagonist solution (60 ~g) (in 5 %

- 12 - 2~237
strength mannitol solution). The suppression of estrus in
the animals is checked each week by estrus smear. On
clay 35, the concentration of HOE 013 in the urine is
measured and the 24 h excretion is calculated.

The re~lts (see Table 1) show that the animals in the
liposome group are still clearly suppressed after
35 days, in contrast to the two control groups. The
excretion rate on day 35 (4.5 ~g) demonstrates a
significant excretion of the antagonist in the case of
the liposome preparation. This excretion rate is closely
correlated with the plasma concentration.

Table l: Cycle suppression of female rats after sub-
cutaneous injection of LHRH antagonist HOE 013
or placebo
` .
Group Treatment Rats with cycle
No. ~dose) suppression/rats per group
Day of vaginal cytology
1 7 14 21 28 35
- - -

1 Control 0/11 0/11 0/11 0/11 0/11 0/11
(placebo)

2 Control
daily
injection 0/11 0/11 0/11 0/ll 0/11 0/11
(60 ~g
HOE 013
8.C.)

3 Liposomes
(single dose 0/8 8/8 8/8 8/8 8/8 8/8
of 3200 ~g
of HOE 013
s.c.)




.

- 13 ~ t
Example 3

40 mg of LHRH antagonist HOE 013, 262 mg of dipalmitoyl-
phosphatidyl-choline (DPPC)(phase-transitiontemperature
about 41C) and 138 mg of choleEterol are dissolved in
15 ml of methanol. The liposomes are prepared in analogy
to Example 1, but the volume of the aqueous phase for
film detachment and making up the liposome pelletæ is
4 ml.

Example 4

40 mg of LHRH antagonist HOE 013, 158.7 mg of dimyris-
toyl-phosphatidyl-choline (DMPC) (phase-transition
temperature about 23C) and 41.3 mg of cholesterol are
converted into liposomes as described in Example 3.

Example 5

The liposomes from Examples 1, 3 and 4 are tested for
their release in vitro. For this, 1 ml of the dispersion
is enclo~ed in dialysis tubes, placed in a vessel with
10 ml of buffer (tris-HCl 0.1 M, pH 7.4, isotonicized
with NaCl) a~d incubated at 37C with shaking. The buffer
solution is changed each day and analyzed for the content
of HOE 013. The results (see Table 2) show a marked
dependence of the release on the composition of the
liposome membrane.




- , .

- 14 - 2~237
Table 2

Day Peptide release in %
hydr. egg
lecithin/CH DPPC/CH DMPC/CH
~Example 1) (Example 3~ (Example 4)
O O O O
0.125 12.5 20.5 41.53
1 21.6 34.3 65.7
2 30.3 47.9 77.4
4 38.2 59.3 83.5
7 46.5 69.5 89.3
67.3 79.9 93.8
~4 74.4 89.9 97.5
21 92.8 96.5 10~.0
28 97.~ 98.7
98.1 99.8

Example 6

In place of HOE 013, 200 mg of buserelin acetate are
converted into liposomes as described in Example 1. The
volume-related particle size i8, on average, 1800 nm, and
the encapsulation efficiency is 10.6 %.

Example 7

250 mg of hydrogenated soybean lecithin are dissolved in
33.3 ml of diisopropyl ether and 16.7 ml of dichloro-
methane at 40C. 4 ml of a solution of 500 mg of hirudin
(HBW 023) in 10 mM phosphate buffer pH 7.4 are added. The
mixture is homogenized in an ultrasound bath for 1 min-
ute. The organic solvent is removed in a rotary evapor-
ator at 55C. The formed liposomes are equilibrated for
1 hour and then filtered through 5 ~m membrane filters.
After removal of the unencap~ulated fraction by centrifu-
gation at 8000 x g 3 times, the liposome pellet8 are made
up to 10 ml. The encapsulation ef$iciency is 11.5 %.



.. , . . . , ~



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.
.
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- 15 - 2~2~
Example 8

135 mg of hydrogena~ed egg lecithin are dissolved in 8 ml
of diisopropyl ether and 4 ml of dichloromethane at 40C.
4 ml of a solution of 20 mg of ebiratide (HOE 427) in
LO mM acetate buffer pH 3.5 are added. The mixture i8
llomogenized in an ultrasound bath for 1 minute. The
organic solvent i8 removed in a rotary evaporator at
55C. The formed lipo~omes are equilibrated for 1 hour
and then filtered through 5 ~m membrane filters. After
removal of the unencap~ulated fraction by centrifugation
at 16000 x g 3 times, the liposome pellets are made up to
10 ml. The encapsulation efficiency is 15 %.

Example 9

Liposomes from Example 1 are stored at 4C for 12 months
and then investigated for their storage stability. The
peptide fraction released into the dispersant (water)
from the liposomes after storage i8 removed by centrifu-
gation at 16000 rpm and determined by HPLC. After storage
for 12 months, 0.75 % of the encapsulated active sub-
stance has been released, and 99.25 % HOE 013 is still
bound in the liposomes. The average volume-related
particle size, determined by photon correlation spectro-
scopy, is 2300 nm, unchanged from the initial value.

Example 10

2000 mg of an equimolar mixture of dipalmitoyl-phospha-
tidyl-choline (DPPC), hydrogenated egg lecithin or egg
lecithin and cholesterol (CH) are dissolved in methanol.
The solvent is evaporated off in vacuo in a rotary
evaporator at 55C. The lipid matrix is detached with
20.0 ml of a solution of 200 mg of HOE 013 in 5.4 %
strength aqueous mannitol solution at 55C and equili-
brated in a shaking bath at 50C overnight. The formed
liposome dispersion is filtered through a 5 ~m filter and
then cooled to about 20C. The unencapsulated fraction is




. ,~ ' ., .
. . : . ~ ,

2~2~7
- 16 -
removed by centrifugation at 1000 rpm for 10 min. The
centrifugation is repeated twice after addition of 0.9 %
strength sodium chloride solution and redispersion.

T~e purified liposome fraction is diluted to the required
HOE 013 concentration and dispensed into sterile vials.
T~e encapsulation efficiency is
61.6 % for liposomes composed of DPPC/cholesterol
(50:50 mol %)
78.9 % for liposomes composed of hydrogenated egg leci-
thin (HPC)/cholesterol (50:50 mol %)
74.3 % for liposomes composed of egg lecithin
(PC)/cholesterol (50:50 mol %).

Example 11

Liposomes from Example 10 are in~ected s.c. into female
rats with an average body weight of 190-200 g. The dose
is 7.2 mg of HOE 013 per animal. The inhibition of the
cycles compared with a control group it3 determined by
vaginal cytology at fixed time points. The interval of
the average estrus suppression is
14 days for PC/CH liposomes (group 2)
34 days for HPC/CH liposomes (group 3)
48 days for DPPC/CH liposomes (group 4)

Table 3: Cycle suppression in female rats after 8.C.
in~ection of 7.2 mg of HOE 013 per an~mal

Group Rat~ with cycle ~uppre~31en/rat~ per group
Day after the ln~ectlon
O 1 2 3 6 7 8 9 12 14 16 21 23 28 31 34 37 41 44 48
1 (control) OJ8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8
2 0/8 0/8 0/8 1/8 8/8 6/8 8/8 8/8 8/8 8/8 8/8 7/8 7/8 7/8 6/8 6/8 3/8 2/8 2/8 0/8
3 0/7 0/7 0/7 1/7 6/7 5/7 7/7 6/7 7t7 7/7 7/7 7/7 6/7 S/7 S/7 5/7 4/7 5/7 3/7 3/7
4 1/8 1/8 2/8 S/8 7/8 8/8 7/8 6/8 7/8 4/8 3/8 2/8 1/8 2/8 3/8 4/8 0/8 0/8 0/8 0/8




,,
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- 20~02~
- 17 _
Example 12

3.37 g of hydrogenated egg lecithin and 1.63 g of choles-
terol are dissolved in 100 ml of methanol and evaporated
in a rotary evaporator at 60C for 30 minutes to give a
lipid film. After addition of glass bead6, 100 ml of
mannitol solution (5.4 %) equilibrated at 60C are added
and the film is detached by rotating the flask on a
rotary evaporator at 60C for 60 minutes.

The liposome dispersion is treated in a Nano~et (supplied
by Verstallen) at slit width 10 and a temperature of 60C
for 15 minutes. The small liposomes which are formed are
filtered through 0.2 ~m membrane filters and, after
cooling, dispensed into vials and then lyophilized.

To reconstitute the lyophilisates, a solution of 1 mg of
HOE 013 per ml of water for in~ections is added, and the
mixture is shaken at 60C.

The unencapsulated fraction i8 removed as in Example 1 by
repeated centrifugation. The lipo~ome-bound fraction is
28.9 % of the active substance.




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, . .
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,

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Title Date
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(22) Filed 1991-04-11
(41) Open to Public Inspection 1991-10-13
Dead Application 1993-10-11

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Filing $0.00 1991-04-11
Registration of Documents $0.00 1991-10-16
Current owners on record shown in alphabetical order.
Current Owners on Record
KIBAT, PAUL-GERHARD
SANDOW, JURGEN K.
HOECHST AKTIENGESELLSCHAFT
Past owners on record shown in alphabetical order.
Past Owners on Record
None
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.

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