IE911231A1 - Long-acting liposome peptide pharmaceutical products and¹processes for the preparation thereof - Google Patents

Abstract

Liposome compositions for peptides with prolonged release of peptides 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 DEG C and contains predominantly saturated fatty acids. The activity persists for more than 14 days after s.c. or i.m. injection. Also described are methods for preparing these liposome compositions.

Description

HOECHST AKTIENGESELLSCHAFT HOE 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 intramuscularly (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 is 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 substances and can be specifically adapted to meet specific requirements. 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 systems 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 administration is desired. The aim is usually to make use of a depot effect, to reduce side effects or to increase activity. After i.v. injection, liposomes, like all colloidal systems, are taken up by the cells of the reticuloendothelial system (RES), eliminated with a halflife not exceeding 2 days, and accumulate preferentially in the liver and spleen (Senior, J.H., CRC, Critical - 2 Reviews in Therapeutic Drug Carrier Systems 3, 123 (1987)). Longer active levels are obtained after s.c. or i.m. injection than after i.v. injections. The duration of action of liposome products depends on the release of substance from the vesicles and on the transport thereof from the injection site, and on the breakdown of the vesicles. Release of substance and breakdown are determined, 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 indicated 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. injection 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. WO 87/04592 describes a liposome release system for membraneimpermeable molecules - calcitonin for example - which is composed of a mixture of small SUV (unilamellar liposomes, 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 hypocalcemic effect of calcitonin after liposomal encapsulation of the protein. According to the examples in these publications, in no case 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-B 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 s.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 mixtures of egg lecithin and phosphatidic acid and investigated 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 injection site and they lead to significant blood levels and pharmacological effects.

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 membrane has a phase-transition temperature 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.

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 than 14 days at the site of administra10 tion without being broken down, and release over this period 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 injection site. The phospholipid component of the liposome membrane preferably has a phase-transition temperature of above 30°C, in particular at least 37’C. It mainly contains saturated fatty acids with a chain length of at least 14 carbon atoms .

Examples of suitable phospholipids are dimyristoyl-PC (DMPC), distearoyl-PC (DSPC), dipalmitoyl-PC (PC = phosphatidylcholine) or hydrogenated or partially hydrogenated lecithins from natural sources. Suitable for stabilization of the membrane are, for example, lipophilic additives of steroids, such as cholesterol.

The peptides encapsulated in liposomes (also 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 hereinbefore and hereinafter, peptides mean within the scope of - 5 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 antago5 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-PheD-Trp-Ser-Tyr-D-Ser (α-L-Rha)-Leu-Arg-Pro-Azagly-NH2, compare EP-A 0 263 521, corresponding to US Patent Application No. 390477). However, also suitable are, for example, hirudins such as HBW 023 (R-DNA-hirudin disclosed in EP-A 0 324 712, corresponding to US Patent Application No. 295 422), HOE 427 (= ebiratide, [415 methionine dioxide, 8-D-lysine, 9-phenylamine]-a-MSH(4-9) (8-amino-octyl)amide triacetate, compare EP-A 0 179 332 corresponding to US Patents No. 4,623,715 and No. 4,696,913) and HOE 140 (= H-D-Arg-Arg-Pro-HypGly-Thi-Ser-D-Tic-Oic-Arg-OH. 6CH3COOH, compare EP-A 0 370 453 corresponding to US Patent Application No. 374 162).

It is known that the peptides suitable as active substances are active for only very short times after administration in the living body (Banga et al., Int. J.

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 as 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 - 6 influence the duration of release of active substance. Encapsulation of peptides in large, for example multilamellar, liposomes increases, for example, the duration of action owing to binding of the active substance to the 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 injection 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 as, for example, dipalmitoyl-phosphatidyl-glycerol or stearylamine 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)). Particularly suitable are preparation technologies which provide larger liposomes.

The processes for preparing the liposome products according to the invention comprise a) a) dissolving the phospholipid component and, 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 liposomes, where the detachment takes place above the phase-transition temperature of the phospholipid component, or β) 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 matrix using an aqueous medium, where the detachment takes place above the phase-transition temperature of the phospholipid component, or 7) dissolving the phospholipid component and, where 5 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 a to 7, 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 α, β or 7, and dispensing an aqueous peptide solution into a suitable vessel, where the lyophilisate and peptide solution are combined before administration.

The lyophilisates obtained by the processes according to the invention are converted by conventional methods, such as, for example, addition of water for injections, into forms suitable for i.m. or s.c. administration.

The aoueous medium used in the process according to the invention is composed of water or a mixture of water and an organic solvent such as, for example, methanol or ethanol. It may additionally contain additives such as sodium chloride or buffers, for example phosphate buffer.

The aqueous peptide solutions can also have such additives.

The processes are expediently carried out as follows.

.-Aw a) θ a) Phospholipids and, where appropriate, lipophilic additives (for example cholesterol) are dissolved in an organic solvent such as, for example, ethanol, methanol, dichloromethane, chloroform or tert, butanol. The solvent is removed by methods which permit no objectionable solvent residues and yield a lipid matrix of maximum surface area. Particularly suitable for this purpose are evaporation with rotary evaporators and lyophilization or combinations of the methods.

To form liposomes, the lipid matrix is detached after the addition of an agueous solution, which is buffered if necessary, 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 decomposition temperature of the peptide. It is assisted by agitation of the vessel and by the use of aids to increase the rate (for example glass beads or scrapers). The liposome dispersion can subsequently also be subjected to a homogenization step, for example with an Ultraturrax, high-pressure homogenizers and comparable processes. The formed liposomes are equilibrated at elevated temperature until they have reached a stable state and optimal swelling. The homogeneity of the dispersion is improved by removing coarse fractions by filtering it, for example, through membrane or glass filters of 1-20 pm pore diameter.

If encapsulation of the pharmaceutical is not quantitative, in many cases there is a need to remove the unencapsulated fraction. Croes-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 fraction (smaller than about 400 nm). It is also possible to use centrifugation processes, chromatographic processes (gel, ion exchange or absorption chromatography) or removal of the free peptide by methods of adsorption or digestion. - 9 The finished liposome dispersion is examined for the pharmaceutical concentration by suitable methods and is diluted to the required content. It is dispensed into ampoules or vials and stored under suitable conditions.

All the process steps in the preparation of pharmaceutical preparations are carried out under aseptic conditions. β) Preparation is carried out in analogy to method a but the peptide is dissolved together with the lipophilic constituents in the organic solvent. This process is particularly suitable for peptides with lipophilic characteristics; suitable 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 which is immiscible with the organic phase. The two-phase system is converted into a stable emulsion by suitable homogenization processes (Ultraturrax, ultrasound, highpressure homogenizer) at temperatures above the phasetransition 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, purified and bottled as described for method a.

Liposomes which are prepared by method o-7 and contain in the aqueous solution additions of cryoprotective substances or to which cryoprotectives have been added after the preparation can be freeze-dried. The chosen additives and freeze-drying processes are mutually - 10 appropriate so that the liposomes are easy to reconstitute before administration and contain a large proportion of the peptide pharmaceutical in bound form. Examples of suitable cryoprotectives are mannitol, xylitol, sorbitol, trehalose, dextrans, polyvinylpyrrolidone, albumin, hydroxyethylstarch and modified gelatin types.

Method b) Liposomes containing no active substance are prepared in 10 accordance with method a) a, 0 or γ and lyophilized as described above. To prepare the liposome dispersion, sterile aqueous peptide solution is added to the lyophilisate. This liposome dispersion can then be administered.

Liposomes which contain no active substance and are obtained by method a) α, β or γ are, where appropriate, converted by suitable homogenization processes into dispersions 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 liposomes produced by this can subsequently be subjected 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 vesicles.

The liposome products according to the invention display a long-lasting continuous release of active substance.

They are furthermore distinguished 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. - 11 Example 1 200 mg of LHRH antagonist (HOE 013), 1348 mg of hydrogenated egg lecithin (phase-transition temperature about 53°C) and 652 mg of cholesterol are dissolved in 50 ml of methanol at 50eC. The solution is sterilized by filtration through 0.2 μτα membrane filters and converted into liposomes under aseptic conditions. For this, the solvent is removed in a rotary evaporator at 55*C 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 is detached from the vessel wall within 2 hours at 55°C and shaken at 50°C overnight. The resulting liposome dispersion is filtered through 5 pm 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 5°C 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 is repeated 5 times; finally, the liposomes 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 encapsulation efficiency is 20 %.

Example 2 2 x 1 ml of the liposomes (corresponding to a single dose of 3200 μg of HOE 013) from Example 1 are injected subcutaneously into female rats of about 200 g body weight. The control comprises an identical test group of animals which receives only solvent (sodium chloride solution) (placebo) and a group of animals treated with daily doses of LHRH antagonist solution (60 pg) (in 5 % - 12 strength mannitol solution). The suppression of estrus in the animals is checked each week by estrus smear. On day 35, the concentration of HOE 013 in the urine is measured and the 24 h excretion is calculated.

The results (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 yg) 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 1: Cycle suppression of female rats after subcutaneous injection of LHRH antagonist HOE 013 or placebo Group No. Treatment (dose) Rats with cycle 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) Control daily injection (60 μ? HOE 013 s.c. ) 0/11 0/11 0/11 0/11 0/11 0/11 Liposomes (single dose of 3200 μς of HOE 013 s.c.) 0/8 8/8 8/8 8/8 8/8 8/8 - 13 Example 3 mg of LHRH antagonist HOE 013, 262 mg of dipalmitoylphosphatidyl-choline (DPPC) (phase-transition temperature about 41 °C) and 138 mg of cholesterol are dissolved in 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 pellets is 4 ml.

Example 4 40 mg of LHRH antagonist HOE 013, 158.7 mg of dimyristoyl-phosphatidyl-choline (DMPC) (phase-transition temperature about 23°C) 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 enclosed in dialysis tubes, placed in a vessel with 10 ml of buffer (tris-HCl 0.1 M, pH 7.4, isotonicized with NaCl) and incubated at 37‘C 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.

Table 2 Day Peptide release in % hydr. egg lecithin/CH DPPC/CH DMPC/CH (Example 1) (Example 3) (Example 4) 0 0 0 0 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 10 67.3 79.9 93.8 14 74.4 89.9 97.5 21 92.8 96.5 100.0 28 97.2 98.7 35 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 is, 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 dichloro25 methane at 40°C. 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 minute. The organic solvent is removed in a rotary evaporator at 55°C. The formed liposomes are equilibrated for 1 hour and then filtered through 5 pm membrane filters.

After removal of the unencapsulated fraction by centrifugation at 8000 x g 3 times, the liposome pellets are made up to 10 ml. The encapsulation efficiency is 11.5 %. - 15 Example 8 135 mg of hydrogenated egg lecithin are dissolved in 8 ml of diisopropyl ether and 4 ml of dichloromethane at 40°C. 4 ml of a solution of 20 mg of ebiratide (HOE 427) in mM acetate buffer pH 3.5 are added. The mixture is homogenized in an ultrasound bath for 1 minute. The organic solvent is removed in a rotary evaporator at 55°C. The formed liposomes are equilibrated for 1 hour and then filtered through 5 pm membrane filters. After removal of the unencapsulated 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 4 °C for 12 months and then investigated for their storage stability. The peptide fraction released into the dispersant (water) from the liposomes after storage is removed by centrifugation at 16000 rpm and determined by HPLC. After storage for 12 months, 0.75 % of the encapsulated active sub20 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 spectroscopy, is 2300 nm, unchanged from the initial value.

Example 10 2000 mg of an equimolar mixture of dipalmitoyl-phosphatidyl-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 55°C. The lipid matrix is detached with .0 ml of a solution of 200 mg of HOE 013 in 5.4 % strength aqueous mannitol solution at 55 *C and equilibrated in a shaking bath at 50°C overnight. The formed liposome dispersion is filtered through a 5 pm filter and then cooled to about 20°C. The unencapsulated fraction is - 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.

The purified liposome fraction is diluted to the required 5 HOE 013 concentration and dispensed into sterile vials.

The encapsulation efficiency is 61.6 % for liposomes composed of DPPC/cholesterol (50:50 mol %) 78.9 % for liposomes composed of hydrogenated egg leci10 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 injected s.c. into female 15 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 is determined by vaginal cytology at fixed time points. The interval of the average estrus suppression is 20 14 days for PC/CH liposomes (group 2) days for HPC/CH liposomes (group 3) days for DPPC/CH liposomes (group 4) Table 3: Cycle suppression in female rats after s.c. injection of 7.2 mg of HOE 013 per animal Group Rats with cycle suppression/rats per group Day after the injection 0 1 2 3 6 7 8 9 12 14 16 21 23 28 31 34 37 41 44 48 1 (control) 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 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 7/7 7/7 7/7 7/7 6/7 5/7 5/7 5/7 4/7 5/7 3/7 3/7 4 1/8 1/8 2/8 5/8 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 - 17 Example 12 3.37 g of hydrogenated egg lecithin and 1.63 g of cholesterol are dissolved in 100 ml of methanol and evaporated in a rotary evaporator at 60 °C for 30 minutes to give a lipid film. After addition of glass beads, 100 ml of mannitol solution (5.4 %) equilibrated at 60°C are added and the film is detached by rotating the flask on a rotary evaporator at 60°C for 60 minutes.

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

To reconstitute the lyophilisates, a solution of 1 mg of 15 HOE 013 per ml of water for injections is added, and the mixture is shaken at 60°C.

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

Claims (10)

1. Patent claims

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 temperature 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 antagonist, 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 particle 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 37C, 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 particle size of at least 600 to 10000 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 e C, c) the phospholipid component is dipalmitoyl-phosphatidyl-choline (DPPC) or hydrogenated lecithin from natural sources, d) the membrane contains added cholesterol, d) the liposomes have an average volume-related particle 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) a) dissolving the phospholipid component and, 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 liposomes, where the detachment takes place above the phase-transition temperature of the phospholipid component, or β) 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 matrix using an aqueous medium, where the detachment takes place above the phase-transition temperature of the phospholipid component, or 5 γ) 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 10 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 15 been obtained by methods a to γ, 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 20 by methods a, β or γ, and dispensing an agueous peptide solution into a suitable vessel, where the lyophilisate and peptide solution are combined before administration.

9. 9.

10. 10. 12. 12. A liposome product according to claim 1, substantially as hereinbefore described and exemplified. Use according to claim 7, substantially as hereinbefore described. A process for preparing a liposome product according to claim 1, substantially as hereinbefore described and exemplified . A liposome product according to claim 1, whenever prepared by a process claimed in a preceding claim.