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 .
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2 0 ~ 7 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 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 - . . . . .
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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.
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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.
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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 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 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 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.
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- 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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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 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 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 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 ,, , , ::
- 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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