USOO57004.82A United States Patent 19 11 Patent Number: 5,700,482 Frederiksen et al. 45) Date of Patent: Dec. 23, 1997

54 PROCESS FOR THE PREPARATION OFA 5,123,414 6/1992 Unger ...... 128,654 LPOSOME DSPERSON UNDER 5,270,053 12/1993 Schneider et al...... 424/450 ELEWATED PRESSURE CONTENTS FOREIGN PATENT DOCUMENTS (75) Inventors: Lene Frederiksen; Klaus Anton, both 2145107 3/1985 United Kingdom. of Basel; Peter van Hoogevest, Riehen, 8801864 3.1988 WIPO . all of Switzerland 89.09593. 10/1989 WIPO Primary Examiner-Gollamudi S. Kishore 73) Assignee: Ciba-Geigy Corporation, Summit, N.J. Attorney, Agent, or Firm-Gregory D. Ferraro; Karen G. Kaiser; Irving M. Fishman 21 Appl. No.: 483,912 57 ABSTRACT 22 Fied: Jun. 7, 1995 The invention relates to a novel, advantageous process for Related U.S. Application Data the preparation of liposomes for the inclusion of water soluble or hydrophilic substances or mixtures of substances, 63 Continuation of Ser. No. 216,760, Mar, 23, 1994, aban which process provides the surprising advantage, in com doned. parison with known processes, that the proportion of sub stances or mixtures of substances actually included is 30 Foreign Application Priority Data increased and which, when used pharmaceutically, provides Mar. 24, 1993 CH Switzerland ...... 891f93 the advantage of sterile working conditions. In this process. a mixture consisting of at least one phospholipid and cus (51 int.C...... A61K 9/12 tomary lipophilic excipients is subjected to a mobile carrier 52 U.S. Cl...... 424/450; 264/4.1; 264/4.3 phase consisting of carbon dioxide and a polar organic 58 Field of Search ...... 424/450; 264/4.1, solvent (modifier) under supercritical pressure and tempera 264/4.3, 4.6 ture conditions, the compressed mixed phase is reduced to normal pressure and transferred to an aqueous phase com 56) References Cited prising a substance having water-soluble or hydrophilic U.S. PATENT DOCUMENTS properties for encapsulation in liposomes. 4,971,802 11/1990 Tarcsay et al...... 424/450 14 Claims, 1 Drawing Sheet

31 (D " a is a sm E F G H lso

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3. U.S. Patent Dec. 23, 1997 5,700,482

} 5,700.482 1 2 PROCESS FOR THE PREPARATION OFA The problem underlying the present invention is to LPOSOME DSPERSON UNDER provide a novel, improved process for the preparation of ELEWATED PRESSURE CONTENTS liposomes for the inclusion of water-soluble or hydrophilic substances or mixtures of substances, which process has the This is a CONTINUATION of Ser. No. 08/216,760, filed 5 surprising advantage in comparison with known processes Mar. 23, 1994 now abandoned. that the proportion of substances or mixtures of substances The present invention relates to a novel, advantageous actually included is increased and which, when used process for the preparation of a liposome dispersion. pharmaceutically, provides the advantage of working con Liposome dispersions comprising various inclusion com ditions that are as sterile as possible. pounds and phospholipids, such as lecithin, have been This problem is solved by the present invention, which described in numerous publications and have already been 10 relates to an advantageous process for the preparation of a tested clinically. In order to illustrate the prior art, European liposome dispersion. The liposome dispersion comprises: Patent Application (hereinafter referred to as EP-A) 178624 a) a substance for encapsulation in liposomes or a mixture is mentioned, in which there is described a liposome dis of substances for encapsulation having water-soluble or persion comprising synthetic, purified sodium 1,2-di-cis hydrophilic properties; octadecenoyl)-3-sn-phosphatidyl S-serine and 1-n- 15 hexadecanoyl-2-(9-cis-octadecenoyl)-3-sn-phosphatidyl b) at least one phospholipid of formula choline as phospholipids and lipophilic N-acetyl-D- muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2- 2.fH-O-R, dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)- O ethylamide or hydrophilic doxorubicin as encapsulated 20 R-o-h3 active ingredients. Those dispersions may be administered CH-O-P-O-R3 intravenously inter alia. Liposome dispersions comprising inclusion compounds OH without actual pharmacological properties, such as zinc wherein phthalocyanine, radioactive labelling compounds or fluores 25 R is Co-oacyl, cent compounds, are also known. EP-A-451 103 is men R2 is hydrogen or Co-oacyl, and tioned by way of illustration, in which there is described a R is hydrogen, 2-trimethylamino-1-ethyl, 2-amino-1- liposome dispersion comprising zinc-phthalocyanine which, ethyl, Calkyl, C-alkyl substituted by carboxy, following intravenous administration, may be used in Calkyl substituted by hydroxy, Calkyl substi so-called photodynamic chemotherapy only when stimu 30 tuted by carboxy and by hydroxy, or C-alkyl sub lated with focused light (LASER). stituted by carboxy and by amino, or salts of those Numerous processes for the preparation of liposome compounds; dispersions are described in the literature, for example c) water in the purity required for the intended applica treatment of an aqueous phospholipid dispersion with ultra tion; and, where appropriate, sonic waves; dispersion of phospholipids with surfactants in 35 d) additional excipients customary for the intended appli aqueous phase and removal of the surfactants by dialysis; cation. dissolution of phospholipids in organic solvents, removal of The process according to the present invention comprises the solvent by lyophilisation and dispersion of the residue in subjecting a mixture consisting of at least one phospholipid aqueous phase; infusion methods or reverse phase evapora b) and, where appropriate, lipophilic excipients d) custom tion. ary for the intended application, to a mobile carrier phase Many known preparation processes are disadvantageous consisting of carbon dioxide and a polar organic solvent since only a fraction of the amount of phospholipids used (modifier) under pressure and temperature conditions which forms liposomes, and those liposomes likewise comprise are higher than the critical pressure and the critical tempera only a fraction of inclusion compound. In addition, mixed ture of a pure carbon dioxide phase, reducing the com micelles, gel structures and double-layer aggregates of inde 45 pressed mixed phase that is obtainable to normal pressure finable size may also be formed. Also known are stability and transferring it to an aqueous phase comprising a sub problems, greatly varying liposome size distribution, a lack stance for encapsulation in liposomes or a mixture of of reproducibility of the processes themselves, high residual substances for encapsulation a) having water-soluble or amounts of organic solvents, residual amounts of hydrophilic properties and, where appropriate, water-soluble surfactants, etc. 50 excipients d) customary for the intended application, and, Furthermore, a common feature of all the preparation where appropriate, removing the organic solvent and/or processes hitherto known is that only a small proportion of separating of a fraction of liposomes having a desired the substance or mixture of substances to be encapsulated is diameter range and/or converting the liposome dispersion actually encapsulated in the double-layer membrane or in into a form suitable for the intended application. the interior space of the liposomes. The amount of active 55 In an especially preferred process variant, the phospho ingredient that is encapsulated can be increased by selecting lipid 1-n-hexadecanoyl-2-(9-cis-octadecenoyl)-3-sn a lipophilic substance or mixture of substances. However, if phosphatidyl choline (POPC) with the lipophilic excipient water-soluble or hydrophilic substances are to be cholesterol is subjected to a mobile carrier phase consisting encapsulated, the proportion of encapsulated substances of carbon dioxide with approximately from 5 to 7% ethanol always remains low in comparison with the total amount as modifier, above the critical pressure and critical tempera used. Water-soluble or hydrophilic substances are less prone ture of a pure CO phase (272 bar, 232 C.). After the to be enriched from the aqueous phase in a lipid phase. compressed mixed phase has been reduced to normal Moreover, lipid membranes have poor stability. When they pressure, an aqueous component comprising a water-soluble are leaking, the aqueous contents of the interior space of the active ingredient, such as EDATREXATE (10-EDAM), is liposomes are replaced by the aqueous phase surrounding 65 added thereto, whereupon liposomes comprising a high the liposomes, so that the degree of enrichment of a water proportion of that water-soluble inclusion compound form soluble active ingredient in the liposomes is being lowered. spontaneously. 5,700,482 3 4 Within the context of the description of the invention, the antibiotics, for example amplicillin, amoxycillin, cephalexin, terms mentioned hereinbefore and hereinafter are defined as cephradine, cefroxadin, cefaclor, erythromycin, follows: bacampicillin, minocycline or chloramphenicol, The expression: A substance for encapsulation in lipo antispasmodics, for example propantheline, atropine or somes or a mixture of substances for encapsulation having scopolamine, antitussives and antiasthmatics, for example water-soluble or hydrophilic properties-defines hydro theophylline, aminophylline, methylephedrine, procatechol, trimethoquinol, codeine, clofedanolol or dextromethorphan, philic or water-soluble substances and mixtures of sub diuretics, for example furosemide or acetazolamide, muscle stances of the prior art which are known to be capable of relaxants, for example chlorphenesin carbamate, tolperison, inclusion in liposomes having phospholipid double layers. eperison or baclofen, mild tranquilisers, for example Liposomes have been described in the literature in numer O oxazolam, diazepam, clotiazepam, medazepam, temazepam ous publications. Their construction and their use are the or fludiazepam, potent tranquilisers, for example sulpiride, subject of many studies. A distinction is made between clocapramine or Zotepin, beta-blockers, for example unilamellar liposomes having one double layer and multi pindolol, propranolol, carteolol, oxprenolol, metoprolol or lamellar liposomes having several double layers of phos labetalol, antiarrhythmics, for example procainamide, pholipids arranged in the manner of an onion skin. The size 5 disopyramide, ajimalin or quinidine, antigout agents, such as of the liposomes varies from approximately 1.0x10 to allopurinol, anticoagulants, such as ticlopidine, approximately 1.0x10 m. antiepileptics, for example phenytoin or valproat, The therapeutic use of liposomes as carriers especially of antihistamines, for example chlorpheniramine, clemastine, lipophilic pharmaceutical active ingredients is known. Lipo mequitazine, alimemazine, cyproheptadine, agents for treat somes have also been proposed as carriers of other lipophilic ing nausea and dizziness, for example diphenidol. substances having biological activity, such as proteins, for methochlopromide, domperidone or betahistine, example antibodies or enzymes, hormones, vitamins or antihypertensives, for example reserpine, rescinnamine, genes, or, for analytical purposes, as carriers of labelled methyldopa, prazosin, clonidine or budralazin, compounds. sympathomimetics, for example dihydroergotamine, isopro Liposomes and their preparation are described in the 25 terenol or etillefrin, expectorants, for example bromhexine, synoptical work by Gregoriadis G. (ed.) Liposome carbocisteine, L-ethylcysteine or L-methylcysteine, oral Technology, Vol. II, Incorporation of Drugs. Proteins and antidiabetics, for example glibenclamide or tolbutamide, Genetic Material, CRC Press 1984, cardiovascular agents, for example ubidecarenon or adenos In the case of the substance or mixture of substances for encapsulation in liposomes, a distinction is made between its 30 Preferred active ingredients are immunosuppressants, hydrophilic properties and its water-soluble properties. The such as cyclosporin, cytostatics, such as EDATREXATE hydrophilic property of a substance or mixture of substances (10-EDAM), doxorubicin, cytarabine, trifosamide, is understood as meaning its tendency to build up in the cyclophosphamide, fluorouracil or methotrexate, as well as phase interface of water, which is also known in the case of water-soluble sulfo derivatives of phthalocyanine, for surfactants. This requires the presence of so-called hydro 35 example tetrasulfophthalocyanine, which can be used in philic groups in the molecular structure of the substance or photodynamic chemotherapy. mixture of substances in question, which groups are able to Instead of a pharmaceutical active ingredient or an active interact with water in the sense of attraction. ingredient combination, the liposome dispersion may also A substance or mixture of substances is defined as water comprise other substances for encapsulation, such as radio soluble when part or all of its weighed amount has dissolved active labelling compounds or fluorescent compounds. in the aqueous phase and part or all of the substance or The nomenclature of the phospholipids (I) and the num mixture of substances in question is present in the solvent bering of the carbon atoms is in accordance with the water in molecularly disperse distribution. For pharmaceu recommendations made in Eur. J. of Biochem. 79, 11-21 tical purposes, the minimum concentration of dissolved (1977) “Nomenclature of Lipids” by the IUPAC-IUB Com active ingredient in water that is required for the efficacy is 45 mission on Biochemical Nomenclature (CBN) (sn sufficient, where appropriate in colloidally disperse distri nomenclature, stereospecific numbering). bution; no sedimentation of substances should occur. R and R as Coacyl are preferably straight-chained Active ingredients that are not readily soluble can be Coalkanoyl having an even number of carbon atoms and converted into water-soluble pharmaceutically acceptable straight-chained Coalkenoyl having a double bond and salts, for example into a hydrobromide, hydrochloride, 50 an even number of carbon atoms. mesylate, acetate, succinate, lactate, tatrate, fumarate, R and R2 as straight-chained Coalkanoyl having an sulfate, maleate, etc., and rendered usable for the process. even number of carbon atoms are, for example. Active ingredients that are not readily soluble may also be n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl or rendered water-soluble by conversion into water-soluble n-octadecanoyl. derivatives or by addition of the solubilisers mentioned 55 R and R as straight-chained Coalkenoyl having a below. double bond and an even number of carbon atoms are, for Suitable pharmaceutical active ingredients are the follow example, 6-cis-, 6-trans-, 9-cis- or 9-trans-dodecenoyl, ing active ingredients in water-soluble form, for example in -tetradecenoyl, -hexadecenoyl, -octadecenoyl or -icosenoyl, the form of water-soluble salts, or which have been rendered especially 9-cis-octadecenoyl (oleoyl). water-soluble by the addition of solubilisers: antiinflamma A phospholipid (I) wherein R is 2-trimethylamino-1- tory agents, for example dexamethasone, sodium dexam ethyl is referred to by the common name lecithin, and a ethasone sulfate, hydrocortisone or prednisolone, coronary phospholipid (I) wherein R is 2-amino-1-ethyl is referred to dilators, for example nifedipine, isosorbitol dinitrate, by the common name cephalin. There are suitable, for nitroglycerine, diltiazem, trapidil, dipyridamole or dilazep, example, naturally occurring cephalin or lecithin, for prostaglandins, for example prostaglandin E E or F. 65 example cephalin or lecithin from soybeans or chicken eggs, peripheral vasodilators, for example ifenprodil, cinepazet having different or identical acyl groups R and R or maleate, cyclandelate, cinnarizine or pentoxyphylline, mixtures thereof. 5,700.482 5 6 Preference is given to synthetic, substantially pure phos Component c)-water in the required purity for the pholipids (I) having different or identical acyl groups R and intended application-is present in the liposome dispersion R2. in the degree of purity prescribed for the particular use, the The term "synthetic" phospholipid (I) defines phospho water having been rendered germ- and pyrogen-free, for lipids which have a defined structure as regards R and R2. 5 example, in accordance with the provisions of the national Such synthetic phospholipids are preferably the lecithins and pharmacopoeias. For example, water for injection purposes cephalins defined above, the acyl groups RandR of which or sterilised water for injection purposes is used. have a specifically defined structure and are derived from a In addition, the liposome dispersion may comprise further defined fatty acid having a degree of purity greater than excipients d) that are necessary, for example, for the estab approximately 95%. R. and R may be the same or different 10 lishment of isotonic conditions, for example ionic additives, and unsaturated or saturated. Preferably, R is saturated, for such as sodium chloride, or non-ionic additives (structure example n-hexadecanoyl (=palmitoyl), and R is formers), such as sorbitol, mannitol or glucose, or water unsaturated, for example 9-cis-octadecenoyl (=oleoyl). soluble stabilisers for the liposome dispersion, such as The expression "naturally occurring" phospholipids (I) lactose, fructose or sucrose. In particular, the liposome defines phospholipids which do not have a defined structure 15 dispersion comprises those additives, for example sodium as regards R and R. Such natural phospholipids are like chloride or mannitol, in the prescribed amounts necessary wise lecithins and cephalins, the acyl groups R and R2 of for the establishment of isotonic conditions in the injection which are structurally indefinable and are derived from solutions. In an especially preferred embodiment of the naturally occurring fatty acid mixtures. process, the liposome dispersion is prepared with the lipo The requirement "substantially pure" phospholipid 20 philic excipient cholesterol. That excipient is added with the defines a degree of purity of more than 95% (by weight) of mentioned phospholipids to the mobile carrier phase con the phospholipid (I), which can be demonstrated by suitable sisting of CO, and the modifier ethanol. When that mixed methods of determination, for example by paper chroma phase is reduced to normal pressure, liposomes form in the tography. aqueous phase and wherein the excipient cholesterol is Special preference is given to synthetic, substantially pure 25 incorporated in the double layers consisting of phospholip phospholipids (I) wherein R is straight-chained Co ids. Liposomes having cholesterol in the double layer are 20alkanoyl having an even number of carbon atoms and R2 distinguished by increased stability. is straight-chained Coalkenoyl having a double bond and In addition to the water-soluble excipients, the liposome an even number of carbon atoms. dispersion may comprise further excipients that can be used In an especially preferred phospholipid (I), R is for liquid pharmaceutical formulations, which excipients n-dodecanoyl. n-tetradecanoyl, n-hexadecanoyl or increase the water-solubility of the mentioned active n-octadecanoyl and R is 9-cis-dodecenoyl, 9-cis ingredients, for example emulsifiers, wetting agents or tetradecenoyl, 9-cis-hexadecenoyl, 9-cis-octadecenoyl or surfactants, especially emulsifiers such as oleic acid, non 9-cis-icosenoyl. ionic surfacants of the fatty acid polyhydroxy alcohol ester In such phospholipid (I), R as Calkyl is, for example, 35 type, such as sorbitan monolaurate, monooleate, monostear methyl or ethyl. ate or monopalmitate, sorbitan tristearate or trioleate, poly R as C-alkyl substituted by carboxy, C-salkyl substi oxyethylene adducts of fatty acid polyhydroxy alcohol tuted by hydroxy or C-alkyl substituted by carboxy or by esters, such as polyoxyethylene sorbitan monolaurate, hydroxy is, for example, 2-hydroxyethyl. 2.3-dihydroxy-n- monooleate, monostearate, monopalmitate, tristearate or propyl, carboxymethyl, 1- or 2-carboxyethyl, trioleate, polyethylene glycol fatty acid esters, such as dicarboxymethyl 2-carboxy-2-hydroxyethyl or 3-carboxy polyoxyethylstearate, polyethyleneglycol400 stearate, poly 2,3-dihydroxy-n-propyl. ethylene glycol 2000 stearate, especially ethylene oxided R as C2-alkyl substituted by carboxy and by amino is, propylene oxide block polymers of the Pluronics type for example, 3-amino-3-carboxy-n-propy or 2-amino-2- (Wyandotte Chem. Corp.) or the Symperonice type (ICI). carboxy-n-propyl, preferably 2-amino-2-carboxyethyl. 45 The advantage of the process is that a large amount of a Phospholipids (I) comprising those groups may be in salt water-soluble substance, especially of a water-soluble phar form, for example in the form of the sodium or potassium maceutical active ingredient, such as EDATREXATE (10 salt. EDAM), doxorubicin, cytarabine or trifosamide, can be A very especially preferred phospholipid (I) is synthetic encapsulated in liposomes. The preparation processes hith 1-n-hexadecanoyl-2-(9-cis- octade cenoyl)-3-sn 50 erto known are disadvantageous for water-soluble inclusion phosphatidylcholine (POPC) having a purity of over 95%. compounds since only small amounts are encapsulated in The names given in parenthesis are also customary for the liposomes, while most of the compound remains in solution acyl radicals in the phospholipids (I): 9-cis-dodecenoyl in the aqueous phase. (lauroleoyl), 9-cis-tetradecenoyl (myristoleoyl), 9-cis There are used as phospholipid preferably the above hexadecenoyl (palmitoleoyl), 6-cis-octadecenoyl 55 mentioned natural or synthetic, substantially pure deriva (petroseloyl), 6-trans-octadecenoyl (petroselaldoyl), 9-cis tives of lecithin, especially 1-n-hexadecanoyl-2-(9-cis octadecenoyl (oleoyl), 9-trans-octadecenoyl (elaldoyl), octadecenoyl)-3-sn-phosphatidyl choline (POPC). 11-cis-octadecenoyl (vaccenoyl). 9-cis-icos enoyl Cholesterol is preferably used as the customary lipophilic (gadoleoyl), n-dodecanoyl (lauroyl), n-tetradecanoyl excipient. The incorporation of that excipient, which is also (myristoyl), n-hexadecanoyl (palmitoyl), n-octadecanoyl 60 present in stable, natural membranes, yields liposomes hav (stearoyl), n-icosanoyl (arachidoyl). ing an especially stable structure, which remain stable to A salt of the phospholipid (I) is preferably pharmaceuti storage for up to several months. cally acceptable. Salts are defined by the existence of The expression “mobile carrier phase consisting of carbon salt-forming groups in the substituent R and by the free dioxide and a polar organic solvent (modifier)" defines a hydroxy group at the phosphorus atom. The formation of 65 mixed phase consisting of carbon dioxide and polar solvent internal salts is also possible. Alkali metal salts, especially under pressure and temperature conditions which are in the sodium salts, are preferred. range of or above the critical point of that mixed phase. The 5,700,482 7 8 pressure and temperature conditions to be applied are higher gation for several hours, for example about 3 hours, in that than the critical pressure and the critical temperature of a gravitational field, liposomes are deposited, whereas small pure carbon dioxide phase (72 bar and 32°C.). There is used liposomes remain in dispersion and can be decanted. as polar organic solvent (modifier) preferably ethanol, but Repeated centrifugation results in complete separation of the also methanol, tert-butanol, isopropanol, n-propanol, methyl large liposomes from the small liposomes. isobutyl ketone, acetone, etc. Gel filtration especially can be used to separate off all the The pressure in the mobile carrier phase consisting of liposomes present in the aqueous phase having a diameter of carbon dioxide and the modifier, preferably ethanol, is more than about 6.0x10 m and also non-encapsulated increased to a value of at least 72 bar in a closed apparatus. components and excess, dispersed lipids that are present in see FIG. 1. Preferred are values in the supercritical range of O a pure CO phase of up to approximately 1000 bar, prefer high molecular weight aggregates and thus to produce an ably up to approximately 400 bar, especially from 200 to 300 aqueous dispersion having a fraction of liposomes of rela bar. Preferred working temperatures are temperatures from tively uniform size. above room temperature to approximately 100° C., prefer The completed formation of liposomes and their size ably from 50° to 60° C. The minimum supercritical condi 15 distribution in the aqueous phase can be demonstrated in a tions for a pure CO phase are approximately 72 bar and 32° manner known per se by various physical measuring C. (slightly varying figures are given in physicochemical methods, for example with freeze fracture samples and thin tables on account of differing measuring methods). sections under an electron microscope or by X-ray The expression "compressed mixed phase" defines a diffraction, by dynamic light scattering, by mass determi homogeneous mixture under supercritical or close to critical nation of the filtrate in an analytical ultracentrifuge and pressure and temperature conditions and is consisting of the especially by spectroscopy, for example in the nuclear mobile carrier phase, the phospholipid b) and, where magnetic resonance spectrum (H, C and 'P). appropriate, lipophilic excipients d) customary for the The liposome dispersion may be administered directly intended application, especially cholesterol. after removal of organic solvents, or it may be converted by The size of the liposomes formed in the aqueous phase is 25 freeze-drying into a lyophilisate, which is reconstituted dependent upon various conditions, for example the com immediately before administration by the addition of water position of the mobile carrier phase, the amount of active in the required injection volume. ingredient and the lipid components, the mixingratio thereof FIG. I depicts the closed apparatus in which the instant and the concentration in the aqueous dispersion, selection of invention may be carried out. pressure and temperature conditions, the rate of flow or 30 The process itself is carried out in a closed apparatus, variation in mixer types or capillary geometry, for example which is illustrated in greater detail in FIG. 1. The compo the length and diameter of the depressurisation capillary in nents of the apparatus are as follows, the numbering of the the apparatus used for the process. components corresponding to the reference numerals in the The removal of the organic solvent (modifier), which may drawing: be necessary for pharmaceutical applications, may be car 35 ried out by various methods, for example evaporation, 1. CO, reservoir Pure (99.9%) CO, from tank with dialysis or gel filtration/gel chromatography. Ethanol may be immersed pipe (Carbagas CH-Basel). removed from the aqueous solution by gel filtration 2 CO2 pump Gilson 306 (Symedic AG, CH-Zirich) with MGW Lauda RM6 refrigerating unit, (Sephadex8) G 50). Ethanol is preferably removed with CO IG instrumentengesellschaft AG using the counterflow principle with the application of 40 CH-Zirich. supercritical pressure and temperature conditions in accor 3 Pump pressure Gilson 805S (Symmedic). dance with the process described in U.S. Pat. No. 4492.808. safety device 4 Pulse damper Reduces vibrations of the pump. Aqueous dispersions having an acidic reaction are pref Approx. 2 m 118 capillary in diameter erably buffered to pH 7.0 to 7.8, preferably 7.2 to 7.4. (Supelico SA, CH-Gland). Pharmaceutically acceptable buffer solutions are preferably 45 5 Waste vessel For solvent residues. used for that purpose, the preparation of which is described 6 Modifier reservoir (Modifier) with ethanol. 7 Modifier pump Gilson 305 (Symmedic). in various national pharmacopoeias, for example the with European, U.S., German or British Pharmacopoeia. The 8 Pressure safety incorporated in Gilson 305. dispersion may be neutralised also by the addition of a device for pump pharmaceutically acceptable, dilute aqueous base, for SO 9 Static mixer Lee Wisco Mixer, Lee TCMA 252011.3T 648430, 2 units, Lee example dilute aqueous sodium hydroxide solution. Neu Hydraulische Miniaturkomponenten tralisation is customarily carried out with simultaneous pH D-Frankfurt. monitoring. Where necessary, the dispersion is made up with 1O Dynamic mixer Gilson 811B (Symmedic). sterile, germ-free and pyrogen-free water. 1. Filter SSI 05-0150 (Supelco). 55 12 Injector Gilson 231 and Gilson Dilutor It is possible to obtain an especially uniform size distri 401 (Symmedic), bution of the liposomes by after-treatment of the liposome 13 Check wave Spectra-Physics check wave dispersion, for example by treatment with ultrasonic waves with sleeve (Ciba-Geigy, CH-Basle). or extrusion through straight-pored filters (e.g. (Spectra Physics, CH-Allschwil). Nucleopore(s). 14 Pressure safety No.20631 25, 2500-25000 Psig wave (Haskel Inc. Burbank Ca, USA). The separation and isolation of a fraction of large lipo 15 Cross piece Walco (Supelico SA, rebored to somes from a fraction containing small liposomes, insofar as approx. 0.5 mm diameter). it is at all necessary, is likewise effected by means of 16 Manometer 0-600 bar (EKA, D-Staufen) for recycling system I. conventional separation methods, for example gel filtration 17 Recycling pump Gilson 303 (Symmedic, or ultrafiltration, for example with Sepharose( 4B or heating sleeve, Ciba-Geigy). Sephacryls (Pharmacia SE) as carrier, or by sedimentation 65 18 UW detector Linear UWIS 200 with SFC cell of the liposomes in an ultracentrifuge, for example with a and heating tube, gravitational field of 160,000xg. For example, after centrifu 5,700,482 9 10 -continued lipophilic constituents (phospholipid and, where appropriate, cholesterol) previously introduced into the 1.4 ul cell volume, 2 mm path length (Henngeler, extraction cell 19 are dissolved. The UV detector 18 displays CH-Riehen). the degree of homogenisation of the compressed lipid 9 Extraction cell Steel tube with threaded containing mixed phase with the lipophilic constituents connectors. introduced into the extraction cell 19. The detector signal is 2O Static mixer 1-3 mixing elements (SMXE DN3.2) recorded on a plotter. The extraction cell 19 is a pressure (Sulzer Chemtech, CH-Winterthur) stable steel tube with threaded connectors and filters at the Steel tube with threaded connector (Ciba-Geigy). inlet and outlet. Emptied chromatographic columns may 21 Pressure sensor Piezoresis five series 15 type PA-15 O also be used for that purpose. The pressure sensor 21 (Keller AG, CH-Winterthur) measures the pressure downstream of the recycling circuit I. Connection with minimum The pressure regulator 22 comprises a piezo crystal, which dead volume (Ciba-Geigy), 22 Pressure Piezo pressure regulator with piezo is controlled by the piezo driver 23 and thus establishes the regulator crystal and heating element required pressure conditions. The piezo driver 23 automati (EP-A-0 427 671). 5 cally establishes the required pressure independently of the 23 Piezo driver P-864 and PZT-control E-808. flow conditions. In the arrangement 24a,b-30a,b-31a,b- (Polyscience AG, CH-Cham). 24a/24b T-piece SS 01-0165 (Supelco), 24b preferred 32a,b water soluble or hydrophilic substance may be added rebored to 0.5 mm diameter. to the system which are to be encapsulated in liposomes. The 25 Static mixer 1 rod with 5 pieces (SMV-2 DN 10, arrangement 24b-30b-31b-32b in the low pressure range is Sulzer Chemtech, CH-Winterthur preferred. The addition in arrangement 24a-30a-31a-32a is Steel tube with threaded connector (Ciba-Geigy). also possible. The static mixer 25 serves as a homogeniser 26 Collecting vessel Three-necked round flask (50 ml) in the formation of liposomes. The recycling pump 27 27 Recycling pump Tube constriction pump conveys the aqueous phase from the collecting vessel 26 Kontron Analytic LC-Pump through the static mixer 33 into the recycling circuit II, Kontron inst. AG, CH-Zirich 28 Water bath with heating unit 25 which is defined by the arrangement 29-34-26-27-29. In that 29 Tpiece as 24 circuit, uncontrolled foam formation on depressurisation of 30a/30b Check valve as 13, 30b preferred the mixed phase is prevented and homogeneity of the 31a/31b Metering pump for water-soluble substance depressurised mixed phase is established. The water bath 28 as 7,31b preferred 32a32b Wessel Supply of water-soluble ensures that temperature conditions in the recycling circuit substance, 32b preferred 30 I, in the pump head of the recycling pump 17 and in the 33 Static mixer as 20 detection cell 18 are constant. The three-way taps A.C.D.F. ACDFGLJLN Three-way taps SSI O2-0124 G.I.J.L.Nallow the currents of the compressed phase to pass (Supelco). B.E.H.K.MO Two-way taps SSI O2-0120 through (one outer always open) or distribute them in two (Supelco). directions (two outlets open). The two-way taps B.E.H.K. M.O allow the currents to flow through or prevent them from 35 passing. The pumps 2 and 7 convey the CO, and the modifier from the reservoirs 1 and 6 into the apparatus. The CO pump is The process that can be carried out in the closed apparatus preferably cooled to -10° C., the CO having a density of according to FIG. 1 and the product currents can be approximately 1 g/ml and being easy to pump. The pump described using a representative phospholipid and a repre pressure safety device 3 displays the pressure of the pumps sentative lipophilic excipient (cholesterol) by means of the 2 and 7. The pulse damper 4 damps the pressure pulses of the following partial steps: pumps 2 and 7, which occur when the pump piston is oc) The closed apparatus described above and in FIG. 1 is retracted. The pump control 8 controls the flow and the charged with a mixed phase of CO/ethanol (=mobile phase mixing ratio of the two pumps 2 and 7. The static carrier phase) (pressure regulator: 250 bar, pressure mixers 9, 20, 25, 33 have no movable parts. Mixing occurs 45 regulator temperature: 90° C., water bath temperature: as a result of currents in a steel tube in which several mixing 60° C., adjustment at components 21 and 28). Taps elements (current breakers) are incorporated, which splits D.F. J.L and N are closed. Taps A,B,C,E,G,H.I.K.M and and collects the current lines. The dynamic mixer 10 has a O are open. The mobile phase flows through the movable part. The movement produces a turbulent current, apparatus in direction A.B,C,D,E,F,G,H.I.21,G.18, 17, which mixes the phases introduced. The filter 11 retains 50 15C or, in the alternative, A.B,C,D,15.17,18.G.H.I.21, undesired foreign particles in the mobile carrier phase and through the bypass AI, but not through the extrac consisting of CO and modifier. The injector 12 is provided tion cell 19. The pressure is adjusted to 250 bar using for further additions of modifier. The check valve 13 allows the pressure regulator 22. The flow rate of the metering the mobile carrier phase to pass only in the direction A-)B. pump 31 is dependent from the concentration of lipids Should the pressure in direction B fall and become less than 55 in the direpressurised mixed phase. the pressure in direction A, compressed phase is introduced B) The phospholipid, for example POPC, and the choles until stable, equal pressure conditions prevail. The adjust terol are weighed out into an extraction cell (molar ratio able pressure safety valve 14 opens in the case of undesired 7:3). The extraction cell is inserted at 19 and all overpressure. The cross piece 15 is open in all directions, components downstream of A and upstream before 21 The manometer 16 displays the pressure in the recycling are kept at a constant temperature of 54°C. The taps are circuit I, which is defined by the arrangement C-D-19-20 set in the same directions as in process step oc). F-G-18-17-15-C, or, preferably, C-15-17-18-G-F-20-D-C. y) Measurement of the homogeneity of the mobile carrier In the recycling circuit I, the compressed homogeneous phase at the detector 18. The UV absorption curve is mixed phase is under homogeneous conditions. The recy recorded using a plotter. cling pump 17 conveys the mobile carrier phase consisting 65 8) Taps D and F are opened and taps E and H are closed. of CO and modifier through the extraction cell 19 and the All other taps remain in the same positions as in process static mixer 20 in the recycling circuit I, whereupon the step o). The recycling circuit I: C.D. 19.20.F.G.18, 17, 5,700.482 11 12 15C or, in the alternative, C.15.17.18.G.F.20.19.D.C., are not being denatured during the liposome formation is thus closed. The check valve 13 maintains continu process. The following mounts were found in the liposome ous pressure in the recycling circuit I. Pressure equali dispersion that is obtainable: sation with pumps 2 and 7 via bypass A-I and pressure regulator 22. POPC e) The phospholipid weighed out with cholesterol in the 29.5 mg (55.8% yield) extraction cell 19 dissolves until equilibrium is reached Cholesterol in the compressed mixed phase. 6.2 mg (69.4% yield) ) When stable equilibrium has been reached (UV absorp molar ratio POPC:cholesterol-3:1. tion stable 18), taps A.I.D and F are closed. Taps E and Hare opened and the pump 31 is working. The remain EXAMPLE 2 ing taps are set in the same directions as in process step O). This example test is carried out according to steps O)-l) m). The compressed mixed phase comprising phospholip and the apparatus shown in FIG. 1, with the inclusion of the ids and cholesterol is decompressed out of the recycling 5 recycling circuit II and the addition of lipophilic or water circuit I through the pressure regulator 22. The mixed soluble substance. The components mentioned correspond phase is released from 24b into the aqueous phase to the reference numerals in FIG. 1, which are described containing the hydrophilic or water soluble substance, above: whereupon liposomes form spontaneously. Weighed amount: v) The aqueous liposome dispersion is diluted and 12.20 mg of cholesterol USPXX homogenised in the recycling circuit II consisting of the 59.40 mg of POPC arrangement 29-34-26-27-29. molar ratio of POPC to cholesterol: 2.5:1 l) When the UV absorption has reached the original level 0.425 mg/ml of zinc-phthalocyanine tetrasulfonate of the mobile carrier phase (no noticeable absorption 25 for phospholipids) and corresponds to the absorption in ZnPc(SOH) in dest, water process step y), the experiment is concluded and the CO, flow: liposome suspension is analysed qualitatively and 1.86 ml/min. at pump 2 quantitatively by HPLC. A portion of the suspension is 0.14 ml/min. at pump 7 examined under a light-optical microscope. 30 Flow rate: at metering pump 31 EXAMPLE 1. 0.005.59 min. Placebo test with the phospholipid 1-n-hexadecanoyl-2- 12.0 ul/min. (9-cis-octadecenoyl)-3-sn-phosphatidyl choline (POPC) 6.00-10.59 min. and cholesterol. The test is carried out according to steps 35 o)-L) and the apparatus shown in FIG. 1, but without using 4.5 l/min the recycling circuit II. The components mentioned above 11.00-17.00 min. correspond to the reference numerals in FIG. 1, which are 1.5 l/min. described above. The process is carried out analogously to Example 1 under Weighed amount: the conditions described therein. Analysis by means of 52.86 mg of POPC HPLC shows no decomposition products in the liposome 8.97 mg of cholesterol. dispersion. POPC, cholesterol and zinc-phthalocyanine tet Mobile carrier phase: rasulfonate are not denatured during the liposome formation CO and ethanol (96% viv). process. Before gel filtration, the following amounts were Detection: 45 found in the liposome dispersion that is obtainable: UV absorption at 210 nm. POPC: CO, flow: 45.70 mg (77% of the weighed amount) 0.465 ml/min, at pump 2. Cholesterol: Modifier 0.035 ml/min. at pump 7. 50 8.14 mg (67.0% of the weighed amount) Pressure: molar ratio POPC:cholesterol-3:1. Upstream of extraction cell at components 8 and 16: 1.0 ml of tiposome dispersion (1.03 mg of cholesterol and approx. 300 bar. 5.78 mg of POPC) are gel-filtered in order to remove Downstream of extraction cell at component 22: ZnPcCSOH) that has not been included. The following approx. 250 bar 55 Pressure variations of approx. 10 bar occur (after mounts are found in the fractions with liposomes: retraction of the pump piston) owing to the use of a POPC: recycling pump (piston pump-comp. 17). 4.84 mg (84% of the weighed amount) Temperature: Cholesterol: Water bath: 54° C. (comp. 28). 0.90 mg (87% of the weighed amount) Detection cell: approx. 54° C. (comp. 18). ZnPc(SOH), Recycling pump head; approx. 54 C. (comp. 17). 3.6 pug, of which 2.6 g in liposomes. Pressure regulator heated to 90° C. 72% active ingredient included with an inclusion vol Extraction cel: ume of approx. 0.7 l/mol lipid. Valco type (precolumn) with a volume of 350 pil. 65 What is claimed is: Analysis by means of HPLC shows no decomposition 1. A process for the preparation of a liposome dispersion products in the liposome suspension. POPC and cholesterol comprising 5,700.482 13 14 subjecting at least one phospholipid of the formula 3. A process according to claim 2, wherein hydrophilic or water-soluble pharmaceutical active ingredients are used as (H-O-R, the non-proteinaceous substance a) for encapsulation. R-O-CH O 4. A process according to claim 2, wherein zinc 2 phthalocyanine tetrasulfonate Znpc(SOH) is used as the CH-0--0-R non-proteinaceous substance a) for encapsulation. OH 5. A process according to claim 1, wherein synthetic, substantially pure 1-n-hexa- decanoyl-2-(9-cis wherein 10 octadecenoyl)-3-sn-phosphatidyl choline is used as the R1 is Co-oacyl, phospholipid b). R2 is hydrogen or Co-oacyl, and 6. A process according to claim 1, whereinsterilized water Ra is hydrogen, 2-trimethylamino-1-ethyl, 2-amino-1- ethyl, C-alkyl, C-alkyl substituted by carboxy, for injection purposes is used as component c). C-salkyl substituted by hydroxy, C-alkyl substituted 7. A process according to claim 1, wherein the liposome by carboxy and by hydroxy, or C2-alkyl substituted by 15 composition further comprises cholesterol. carboxy and by amino, or a salt of such phospholipid to 8. A process according to claim 1, wherein the liposome a mobile carrier phase consisting of carbon dioxide and composition further comprises at least one water-soluble a polar organic solvent under pressure and temperature excipient for the establishment of isotonic conditions. conditions which are higher than the critical pressure 9. A process according to claim 1, wherein the polar and the critical temperature of a pure carbon dioxide 20 phase, reducing the compressed mixed phase that is organic solvent is ethanol. obtainable to normal pressure and transferring it to an 10. A process according to claim 1, wherein a pressure aqueous phase comprising water in the purity required range of approximately from 72 to 400 bar is used. for the intended application and a non-proteinaceous 11. A process according to claim 10, wherein a pressure substance or a mixture of non-proteinaceous substances 25 range of approximately from 200 to 300 bar is used. having water-soluble or hydrophilic properties for 12. A process according to claim 1, wherein an elevated encapsulation in liposomes and removing the organic temperature of approximately from 32° to 100° C. is used. solvent and/or separating off a fraction of liposomes 13. A process according to claim 12, wherein an elevated having a desired diameter range and/or converting the temperature of approximately from 50° to 60° C. is used. liposome dispersion into a form suitable for the 30 intended application. 14. A process according to claim 1, wherein the liposome 2. A process according to claim 1, where the non dispersion that is obtainable is convened into a lyophilisate proteinaceous substance or mixture of substances for encap which is then reconstituted by the addition of water in the sulation is at least one compound selected from the group required injection volume. consisting of a radioactive labelling compound, and a phar maceutical active ingredient. sk xk k xk sk