AU639812B2 - Process for the production of an injectable liposome dispersion - Google Patents

Description

Our Ref: 367732 t 98 12 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STAND)ARD PATENT 9 4.

9 S *99* *9S~ @499 *@e9 9 9999 9 4 *9 V999 see Applicant(s) Address for Service: Cibai-Geigy AG Klybeckstrasse 141 4002 BASLE

SWITZERLAND

ARTHUR S. CAVE CO.

Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Process for the production of an injectable liposome dispersion 9 4 9**9 4 Invention Title: The following statement is a full description of this invontion, including the best method of performing it known to me:- 5020 -1- Process for the production of an injectable liposome dispersion The present invention relates to a novel, advantageous process for the production of an injectable liposome dispersion. This can primarily be used for intravenous administration.

Injectable liposome dispersions containing various active ingredients and phospholipids such as lecithin and phosphatidylserine as adjuncts are described in numerous publications and have already been clinically tested. To illustrate the prior art, European Patent se* Application 178 624 is mentioned, in which a liposome dispersion containing synthetic, purified sodium 1,2-di-(9-cis-octadecenoyl)-3-sn-phosphatidyl-S-serine and 1-n-hexadecanoyl-2-(9-cis-octadecenoyl)-3-sn-phosphatidylcholine as phospholipids and N-acetyl-D-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2- dipalmitoyl-sn-glycero- -3-hydroxyphosphoryloxy)ethylamide as the encapsulated active ingredient is described.

This dispersion can be administered, inter alia, intravenously.

The injectable liposome dispersions, however, are disadvantageous since, as the preliminary step, they require the production of a dry preparation, for example lyophilisate or film residue, which is dispersed in water to form liposomes in situ before administration.

According to U.S. Patent Specification 4 687 661, liposome dispersions can also be produced without the preliminary step of the dry preparation by dissolving the lipid components in selected, non-volatile organic solvents such as glycerol or ethylene glycol and warming and dispersing the organic solution in water. Because of the high content of organic solvents and possible thermal degradation products, these solutions are physiologically unacceptable for injection solutions, in particular for intravenous administration.

The object of the present invention is to produce an injectable liposome dispersion by mixing the components, while avoiding the preliminary step of the dry preparation, by choosing a pharmaceutically suitable, non-toxic solvent. This object is achieved by the present invention, which relates to a novel, advantageous process for the production of an -2injectable liposome dispersion.

The invention relates to a process for the production of an injectable liposome dispersion comprising a) a phospholipid of the formula 1

CH

2 R 21

R

2 -O CH

O

I I Ra .o 3 CH2- p-O (CnH 2 n) -N-Rb \c in which R, is C 1 0 20 acyl, R 2 is hydrogen or Co.

20 acyl, Ra, Rb and R are hydrogen or C-4alkyl and n is an integer from two to four, optionally combined with an additional b) phospholipid of the formula

CH

2

R

0* 9 2

R

4 H

O

31 (II), 3CH 2

R

S in which R 3 is C 10 2 0 acyl, R 4 is hydrogen or Clo 20 acyl and R 5 is hydrogen, Claalkyl,

C.C

1 alkyl substituted by carboxy, C2-5alkyl substituted by hydroxy, C2-salkyl substituted by carboxy and hydroxy or C2-5alkyl substituted by carboxy and amino, c) the active ingredient to be injected or an active ingredient combination and d) a pharmaceutically acceptable carrier liquid and, optionally, further adjuncts suitable for injection preparations.

The process is characterised in that a solution or suspension of the components a) and c) or b) and c) in concentrated acetic acid is dispersed in the carrier liquid c) and the dispersion thus obtained is brought to a physiologically acceptable pH-level and, optionally, adjuncts suitable for injection preparations are added and, optionally, a fraction of liposomes having a desired diameter range is separated.

The advantageous liposome dispersion which can be produced by this process is free from solid particles and relatively large lipid aggregates, stable at room temperature for at least several hours, reproducible in respect of the amount proportion of the components, toxicologically acceptable and therefore particularly suitable for intravenous administration to humans.

S The process in particular has the advantage that the organic solvent acetic acid can be S, converted into the physiologically acceptable acetate salt after dispersion by addition of a dilute aqueous base, such as dilute sodium hydroxide solution. In the case of complete neutralisation, therefore, the injection solution is free from organic solvents and not a problem for intravenous administration. The complicated step of removing the organic solvent from the dispersion becomes unnecessary.

The terms mentioned above and in the following are preferably defined as follows in the context of the description of the invention: The injectable liposome dispersion can be administered parenterally, preferably intravenously, but also intramuscularly, for example for the formation of a depot, or subcutaneously, for example for the administration of local anaesthetics.

The injectable liposome dispersion contains liposomes in the form of unilamellar, preferably multilamellar, large and small liposomes comprising a double layer arrangement of the phospholipids of the formula I and if appropriate of the formula II having an internal space and a spherical shape (unilamellar) or comprising several concentric double layer arrangements of the phospholipids and if appropriate (II) having an inner space and a spherical shape ("onion skin-like" construction of the double layers or membranes multilamellar). The order of size of the liposomes is variable between about 1.0 x 10- 8 up to about 1.0 x 10-" m.

The therapeutic use of liposomes as carriers of active ingredients of different types is known. Liposomes have furthermore been proposed as carriers of proteins, for example antibodies or enzymes, hormones, vitamins or genes, or for analytical purposes as carriers of labelled compounds.

Injectable liposome dispersions are described in the review by Gregoriadis G. (editor) Liposome Technology, Vol. II, Incorporation of Drugs, Proteins and Genetic Material, CRC Press 1984. In the review by Knight, C.G. (editor), Liposomes: From Physical Structure to Therapeutic Applications, Elsevier 1981, the advantages of such an administration form based on liposomes are summarised in chapter 16 on p. 166.

The expression "lower" used in connection with organic radicals, for example lower alkyl, lower alkylene, lower alkoxy, lower alkanoyl etc. means that such organic radicals, if not expressly defined otherwise, contain up to and including 7 and preferably up to and including 4 carbon atoms.

The nomenclature of the phospholipids of the formulae I and II and the numbering of the C atoms follows from the recommendations (sn nomenclature, stereospecific numbering) given in Eur. J. of Biochem. 79, 11-21 (1977) "Nomenclature of Lipids" by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN).

In a phospholipid of the formula I, R 1 and R 2 with the meanings Cl 0 20 acyl are preferably straight-chain Clo 0 20 alkanoyl having an even number of C atoms and straight-chain

C

10 20 alkenoyl having a double bond and an even number of C atoms.

Straight-chain C 0 20 alkanoyl R, and R 2 having an even number of C atoms are, for O example, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl or n-octadecanoyl.

Straight-chain Clo 0 20 alkenoyl R. and R 2 having a double bond and an even number of C atoms are, for example, 6-cis-, 6-trans-, 9-cis- or 9-trans-dodecenoyl, -tetradecenoyl, -hexadecenoyl, -octadecenoyl or -icosenoyl, in particular 9-cis-octadecenoyl (oleoyl).

In a phospholipid of the formula I, n is an integer from two to four, preferably two. The group of the formula -(CnH 2 is unbranched or branched alkylene, for example, 1,1-ethylene, 1,2- or 1,3-propylene or 1,3- or 1,4-butylene. 1,2-ethylene (n 2) is preferred.

Phospholipids of the formula I are, for example, naturally occurring cephalins in which Ra, Rb and R c are hydrogen, or naturally occurring lecithins in which Ra, Rb and R e are methyl, for example cephalin or lecithin from soya beans, bovine brain, bovine liver or egg yolk having different or identical acyl groups R 1 and R 2 or mixtures thereof.

Synthetic, essentially pure phospholipids of the formula I having different or identical acyl groups R 1 and R 2 are preferred.

The term "synthetic" phospholipid of the formula I defines phospholipids which have a uniform composition with respect to R 1 and R 2 Such synthetic phospholipids are preferably the lecithins and cephalins defined above, whose acyl groups R, and R 2 have a defined structure and are derived from a defined fatty acid having a degree of purity r ~higher than about 95 R 1 and R 2 can be identical or different and unsaturated or saturated. Ri is preferably saturated, for example n-hexadecanoyl, and R 2 unsaturated, for example 9-cis-octadecenoyl oleoyl).

The term "naturally occurring" phospholipids of the formula I defines phospholipids which with respect to R 1 and R 2 do not have a uniform composition. Such natural phospholipids are likewise lecithins and cephalins whose acyl groups R 1 and R 2 are structurally undefinable and derived from naturally occurring fatty acid mixtures.

The requirement "essentially pure" phospholipid defines a degree of purity of more than 95 (by weight) of the phospholipid which can be proved by suitable analytical methods, for example by paper chromatography.

Particularly preferred synthetic, essentially pure phospholipids of the formula I are those in which R 1 has the meaning straight-chain C 10 o 20 alkanvl having an even number of C atoms and R 2 has the meaning straight-chain C 1 0 2 0 alkenoyl having a double bond and an S even number of C atoms. R a Rb and R c are methyl and n is two.

In a particularly preferred phospholipid of the formula I, R 1 is n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl or n-octadecanoyl and R 2 is 9-cis-dodecenoyl, 9-cis-tetradecenoyl, 9-cis-hexadecenoyi, 9-cis-octadecenoyl or 9-cis-icosenoyl. R 2 is tetradecenoyl, 9-cis-hexadecenoyl, 9-cis-octadecenoyl or 9-cis-icosenoyl. Ra, Rb and Rc are methyl and n is two.

A very particularly preferred phospholipid of the formula I is synthetic 1-n-hexadecanoyl-2-(9-cis-octadecenoyl)-3-sn-phosphatidylcholine having a purity of more than -6- In a phospholipid of the formula 11, R 3 and R 4 are defined with the Same meaning

CIO-

20 acy1 as for R, and R 2 in a phospholipid of the formula 1.

The acyl groups R 3 and R 4 can be different or identical.

R

5 having the meaning CI-4alkyl is, for example, methylt or ethyl.

R

5 having the meanings C 1 -5alkyl substituted by carboxyl, C 2 5 alkyl substituted by hydroxyl or C 2 5 alkyl substituted by ca-rboxy or hydroxy are, for example, 2-hydroxyothlyl, 2,3-dihydroxy-n-propyl, carboxyrnethyl, I- or 2-carboxyethyl, dicarboxymethyl, 2-carboxy-2-hiydroxyethyl or 3-carboxy-2,3-dihydroxy-n-propyl.

R

5 having the meaning C 2 5 alkyl substituted by carboxy and amino is, for example, 3-amino-3-carboxy-n-propyl or 2-amino-2-carboxy-n-propyl, preferably 2-amino-2-carboxyethyl. Phospholipids of the formula 11 containing these groups can be present in salt form, for example as the sodium or potassium salt.

Synthetic, essentially pure phospholipids of the formula 11 having different or identical so: 9 acyl groups R 3 and R 4 are preferred.

The definitions stipulated for phospholipids of the formula I mentioned above apply with 0 respect to the dcfinitil-n "synthetic" and the reqluirement "essentially pure".

:*In a particularly preferred phospholipid of the formula I1, R 3 and R 4 h1ave the meaning sees*: straighit-chain CIO- 20 alkenoyl having a dotuble bond and an even number of C atoms. R 5 is 2-amino-2-carboxyethyl.

In the preferred phospholipid of the formula 11, R 3 and R 4 having the meaning

"CIO-

20 alkenoyl having a double bond and an even number of C atoms" are preferably 9-cis-dodecenoyl 9-cis- tetradlecenoyl, 9-cis-hiexadecenoyl, 6-cis-octadecenoyl, 6-trans-octadecenoyl, 9-cis-octadecenoyl, 9-trans-octadecenoyl, Ii -cis-octadecenoyl or 9-cis-icosenoyl.

In a particularly preferred phospholipid of the formula IL. R 3 and R 4 have identical

I-

mcanings, for example 9-cis-dodlccnoyl, 9-cis-tctradeconoyl, 9-cis-hexadeccnoyl, 9-cis-octadcccnoyl or 9-cis-icosenoyl.

A very particularly preferred phospholipld of the formula II is synthetic sodium 1,2-di-(9-cis-octadecenoyl)-3-sn-phosphiatidyl-S-ser-ine having a purtity of more than 95 The names given in brackets are also customary for the acyl radicals in the phospholipids of the formulae I and 11: 9-cis-dodecenoyl (laurolcoyl), 9-cis-tetradecenoNVl (myristolcoyl), 9-cis-hlexad'.cunoyl (palmitolcoyl), 6-cis-octadecenoyl (petroseloyl), 6-trans-octadecenoyl (petroselaidoyl), 9. 9-cis-octadecenoyl (olcoyl), 9-trans-octadecenoyl (claidoyl), 1 1-cis-octadecenoyl (vaccenoyl), 9-cis-icosenoyl (gadolcoyl), n-dodecanoyl (lauroyl), n-tctradecanoyl (myristoyl), n-hexadecanoyl (palmitoyl), n-octadccanoyl (stearoyl), n-icosanoyl (arachidoyl).

War-soluible, but also sparingly soluble, if appropriate crystalline, lipophlilic active ingredients which can be admiinistered by means of injection Solutions are primarily suitable as the injectable active ingredient or active ingredient combination.

Sparingly soluble active ingredients are present, for example, as water-soluble, s:9" pharmaceutically acceptable salts, for example as thle hydrobromide, hydrochloride, mesylate, acetate, succinate, lactate, tartrate, fumarate, sulfate, maleate, etc, Suitable pharmaceutical active ingredients are, for example, antiinflammaitory a~gents, for example indomethacin, acetylsalicylic i-cid, ketoprofen, ibuprofen, mefenamic acid, dexamethasone, sodium dexamethasone sulfate, hydrocortisone or prednisolone, coronary .:dilators, for example nifedipine, isosorbide dinitrate, nitroglycerin, diltiazem, trapidil, dipyridamole or dilazep, prostaglandins, for example prostagiandin EI, E 2 or 2X peripheral vasodilators, for example ifenprodil, cinepazet maleate, cyclandelate, cinnarizine or pentoxyphylline, antibiotics, for examnple ampicillin, amnoxy,,illin, cephalexin, cefradin, cefroxadin, cefactor, erythrom-ycin, bacampicillin, minocyclin or chioramphenicol, antispasmodic agents, for example propantheline, atropine or scopolamiine, antitussives and antiasthmatics, for example tiieophylline, amiinophylline, methylephedrine, procatechol, trimiethoquinol, codeine, clofedanol or dextromethorphan, diuretics, for example furosemiide or acetazolamide, muscle-relaxing agents, for example chlorphenesin carbamate, tolperisone, eperisone or baclofen, weak tranqluillisers, for 8examplc oxazolam, diazepam, clotiazepam, medazcpam, temazepami or fludiazepam, strong tranquillisers, for example sulpiride, clocapramine or zotepinc, beta-blockers, for example pindolol, propranolol, carteolol, oxprenolol, metoprolol or labetalol, antiarrhythmics, for example procainamide, disopyramide, ajimalin or quinidine, antiarthritic agents such as allopurinoi, anticoagulants such as ticlopidine, antiepileptics, for example phenytoin, vaiproate or carbamazepine, anti histaminics, for example chiorpheniramine, clernastine, mnequitazine, alimemazine, cyproheptadine, agents against nausea and vertigo, for example diphenidol, metoclopramide, clomperidone or betahistine, hypotensive agents, for example reserpine, rescinnamine, methyldopa, prazosine, clonidine or budralazine, sympathomimectics, for example dihydroergotamnine, isoproterenol or ctilefrine, expectorants, for example bromhe%-xine, cabocysteine, L-othylcysteine or L-methylcysteine, oral antidiabetics, for example glibenclamnide or ',itutamide, and cardiovascular agents, for example uibidecarenone or adenosine, Preferred antihiypercalcaemics are those from the calcitonin series, for example 0* synthetically preparable salmon, human and porcine calcitonin and the eel calcitonin preparation, for example 1,7-Asu-eel calcitonin (elcatonin), or lipophilic or hydrophilic immunomodulators from the muramyl peptide series, for example N-acetyl-D-muramyl- -L-alanyl-D-isoglutaminy1-L-alanine-2-( 1,2-dipalmitoyl-sn-glycero-3-hiydroxyphlosphoryloxy)ethiylamide, disodiumn N-acetyl-D-mur~amyl-L-alanyl-D-glu tam ic acid (CY-L-alanine- ,2-d ipalm itoyl-s n-g-lycero-3 -hy(!'roxyph osph oryloxy)e thylami de, sodium N-acetyl- -D-mu ram yl-L-alanyl-D-iso ohiutamine, sodium N-acetyldesmethyl-muramyl- -L-alanyl-D-isoglutamnine, N-acetyl- D-muram yl-L- alan yl- D-gluLItamnine-c(x-n-bu tyl ester, N(X-(N-acetyl-D-muranyl-L-alanyl-D-isoluItaminyl)-N?-stearoyl-L-lysine or 6-O-stearoyl-N-acetylD-muramvl- L-alanyl-D-isogiLutanmine, The pharmaceutically acceptable carrier liquid d) is water which has been processed microorganism and pyrogen-free according to the directions of the national pharmacopoeias.

The components b) and c) or a) and c) tae contained in the carrier liquid d) as liposomes in such at way that no solids or solid aggregates such as micelles reform for several days to weeks and the clear or possibly slightly opalescent liquid containing the components mentioned, if appropriate after filtration, can be administered as an injection solution, preferably intravenotusly.

-9- Non-toxic adjuncts which can be used for injection preparations can be present in the carrier liquid for example water-soluble adjuncts which are necessary for the production of isotonic cc ,ditions, for example ionic additives such as sodium chloride or non-ionic additives (structure-forming agents) such as sorbitol, mannitol or glucos.: or water-soluble stabilisers, for the liposome dispersion such as lactose, fructose or sucrose.

These additives, for example sodium chloride or mannitol, are in particular present in the amounts described above which are necessary for the production of isotonic conditions in the injection solutions.

In addition to the water-soluble adjuncts, emulsifiers, wetting agents or surfactants, which can be used for liquid pharmaceutical formulations, can be present in the carrier liquid, in particular emulsifiers such as oleic acid, non-lonic surfactants of the fatty acid polyhydroxyalcohol ester type such as sorbitan monoiaurate, oleate, stearate or palmilate, sorbitan tristearate or trioleate, polyoxyethylene adducts of fatty acid polyhydroxy alcohol esters such as polyoxyethylene sorbitan monolaurate, olcate, stearate, palmitate, tristearate S or trioleate, polyethylene glycol fatty acid esters such as polyoxyethyl stearate, polyethylene glycol 400 stearate, polyethylene glycol 2000 stearate, in particular ethylene oxide/propylene oxide block polymers of the Pluronic® (Wyandotte Chem. Corp.) or Synperonic® (ICI) type.

Concentrated acetic acid has a content of more than 90 (by weight), the remaining content by weight being water. Concentrated purified acetic acid having a content of more than 95 in particular more than 99 is preferred, and is known under the trivial name glacial acetic acid.

Depending on the solubility of the components a) and c) or b) and c) in the concentrated acetic acid, a solution is formed. This is then dispersed in the carrier liquid to which, if desired, suitable adjuncts for injection preparations have been added.

The dispersion itself is carried out, for example, by shaking (for example vortex mixer) or stirring the carrier liquid. The formation of liposomes, which can be large, small, unilamellar or multilamellar, takes place spontaneously, i.e. without additional supply of energy from outside and at a high rate. 0.1 to 50 per cent by weight (relative to the total weight of the aqueous dispersion), preferably 2 to 20 per cent by weight, of the acetic acid solution or dispersion can be dispersed in the aqueous phase.

The components mentioned can also be dispersed by using a high pressure homogeniser, for example as described in Pharm. Ind. 52, No. 3 (1990) on pages 343-347, and as a result a particularly uniform liposome dispersion can be produced.

Dispersion is carried out at temperatures below about 36 0 C, preferably at room temperature. If appropriate, the process is carried out with cooling and/or under an inert gas atmosphere, for example a nitrogen or argon atmosphere. The liposomes obtainable are stable in the aqueous phase for a very long time (up to several weeks or monrths).

The size of the liposomes formed depends, inter alia, on the amount of active ingredient and lipid components, and their mixing ratio and concentration in the aqueous dispersion.

Thus, aqueous phases having a high content of small or large liposomes can be produced by increasing or reducing the concentration of the individual lipid components, 9* S' The size and structure (multilamellar/unilainellar) of the liposomes formed is also dependent on the choice of the process in question. On shaking or stirring, for example with conventional stirrers fitted with a propeller or blade or with a magnetic stirrer, dispersions are obtained ha; ng a high content of large multilamellar liposomes. An increase in the stirring frequency or changing to phase mixers having high shear forces causes an increase in the content of small, multilamellar liposomes. Treatment with S ultrasonic waves gives a high content of unilamellar liposomes in the dispersion.

S Acid-reacting aqueous dispersions are preferably buffered to pH 7.0 to 7.8, preferably 7.2 to 7.4. Pharmaceutically acceptable buffer solutions can preferably be used for this, whose production is described in various national pharmacopocias, for example the European, German or British pharmacopoeia. The dispersion can also be neutralised by addition of a pharmaceutically acceptable, dilute aqueous base, for example dilute sodium hydroxide solution. The dispersion is customarily neutralised with simultaneous oH monitoring. If appropriate, the dispersion is made up to the necessary injection volume with sterile, microorganism-free and pyrogen-free water. The injection preparation can be administered directly, foi example subcutaneously, preferably intravenously.

A particularly uniform size distribution of the liposomes can be obtained by post-treatment of the liposome dispersion, for example by acoustic irradiation with ulh tsound or extrusion through even-pore filters (for example Nucleopore®).

11 The separation and isolation of a fraction of large liposomes from a fraction containing small liposomes, if necessary at all, is carried out by means of conventional separation methods, for example g, 1 filtration or ultrafiltration, for example using Sepharose® 4B or Sephacryl® (Pharmacia SE) as carriers, or by sedimentation of the liposomes in the ultracentrifuge, for example using a gravitational field at 160,000 x g. Liposomes sediment, for example, after several hours, for example about three hours of centrifugation in this gravitational field, while the small liposomes remain dispersed and can be decanted. A complete separation of the large liposomes from the smn:1 liposomes is achieved after centrifugation several times.

All liposomes in the aqueous phase having a diameter greater than about 6.0 x 10- 8 m and non-encapsulated components and excess, dispersed lipids which are present in high molecular weight aggregates can be separated, in particular by gel filtration, and an aqueous dispersion containing a fraction of liposomes having relatively uniform size can thus be prepared, The resultant formation of liposomes and their content in the aqueous phase can be analyzed in a manner known per se by applying various physical analytical methods, for example using freeze-fracture samples and thin sections in the electron microscope or by X-ray diffraction, by dynamic light scattering, by mass determination of the filtrate in the analytical ultracentrifuge and primarily by spectroscopy, for example in the nuclear magnetic resonance spectrum (1H, 'C and 31 p).

The liposome dispersion can be administered directly, but by freeze-drying can also be converted into a lyophilisate which is reconstituted with tie intended injection volume by addition of water immediately before administration.

The invention relates primarily to a process for the production of an intravenously administrable lipoome dispersion comprising a) synthetic, essentially pu, 1-n-hexadecanoyl-2-(9-cis-octadecenoyl)- 3-sn-phosphatidylcholine if appropriate combined with b) synthetic, essentially pure sodium 1,2-di-(9-cis-octadecenoyl)-3-sn-phosphatidyl-S-serine (II), c) the active ingredient to be administered intravenously and d) a pharmaceutically acceptable carrier liquid and, if appropriate, adjuncts suitable for 12injection preparations.

The invention relates in particular to a process for the production of an intravenously administerable liposome dispersion comprising a) synthetic, essentially pure 1-n-hex d.ecanoyl-2-(9-cis-octadecenoyl)- 3-sn-phosphatidylcholine (1 appropriate combined with b) synthetic, essentially pure sodium 1,2-di-(9-cis-octadecenoyl-3-sn-phosphatidyl-S-serine (II), c) carbamazepine, synthetic human calcitonin or N-acetyl-D-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 ,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamide and d) a pharmaceutically acceptable carrier liquid and, if appropriate, adjuncts suitable for injection preparations.

The following examples illustrate the invention.

Example 1: 250 mg of a phospholipid mixture containing at least 95 pure 1-n-hexadccanoyl-2-(9-cis-octadecenoyl)-3-sn-phosohatidylcholine and at least 95 pure 1,2-di-(9-cis-octadecnoyl)-3-sn-phosphatidyl-S-serine in a weight ratio of 7:3 and 1 mg of N-acetyl-D-muramyl-L-alanyl-D-isoaglutaminyl-L-alanine- 2-(1,2-dipalmitoyl-sn-glycero3-hydro.yphosphoryloxy)ethylamide are dissolved in 0.25 ml of glacial acetic acid in a round-bottomed flask. The equimolar amount of 2N NaOH 9 solution 2.075 ml) is added with stirring. A liposome dispersion is formed, which is made up to the desired volume (50 ml) with a suitable buffer solution (pH The buffer solution is chosen such that a pH of the dispersion of about 7.2 to 7.4 is obtained S and an osmolarity of about 290 mosmol. The dispersion can be made microorganism-free in an autoclave before administration.

Example 2: 80 mg of at least 95 pure l-n-hexadecanoyl-2-lyso-3-sn-phosphatidylcholine, 40 mg of at least 95 pure 1 ,2-di(9-cis-octadccenoy)- 3-sn-phosphatidylethanolamine and 80 mg of oleic acid together with 15 mg of carbamazepine are weighed into a glass vial. This mixture is dissolved in 0.5 ml of glacial acetic acid with stirring. An equimolar amount of 2N NaOH solution (4.15 ml) is added with stirring. A liposome dispersion is formed. The other steps are carried out analogously to Example 1.

13- Example 3: 250 mg of the phospholipid mixture according to Example 1 are dissolved in 250 il of glacial acetic acid containing 310 .gg of synthetic human calcitonin in a round-bottomed flask. This solution is treated with 2.08 ml of sterile-filtered 2N NaOH solution with stirring and homogenised at setting 10 using a vortex device. The liposome dispersion formed is diluted to a volume of 20 ml by addition of microorganism-free, pyrogen-free water for injection. This dispersion is suitable for I tramuscular administration.

0e*

A

a ft a f, 4

I

Claims (5)

1. A process for the production of an injectable liposome dispersion comprising a) a phospholipid of the formula 1 CH 2 R 1 R-O-2CH I II e/Ra 3 CH2-O- p- (CnH 2 -N-Rb 0 \\R *se. A. S. S. eAS. in which R, is C 1 o_ 20 acyl, R 2 is hydrogen or Co 10 20 acyl, Ra, Rb and R e are hydrogen or Ci-4alkyl and n is an integer from one to four, optionally combined with an additional b) phospholipid of the formula 'CH 2 R3 2,, v 14 J I II 3CH2- P-0- Rs 0 in which R 3 is C 1 o-2 0 acyl, R 4 is hydrogen or Clo. 20 acyl and R 5 is hydrogen, Clalkyl, C 1 s-alkyl substituted by carboxy, C 2 5 alkyl substituted by hydroxy, C2. 5 alkyl substituted by carboxy and hydroxy or C 2 .5alkyl substituted by carboxy and amino, c) the active ingredient to be injected or an active ingredient combination and d) a pharmaccutically acceptable carrier liquid and, if appropriate, further adjuncts suitable for injection preparations, characterised in that a solution or suspension of the components a) and c) or b) and c) in concentrated acetic acid is dispersed in the carrier liquid d) and the dispersion obtainable is brought to a physiologically acceptable pH level and, optionally, adjuncts suitable for injection preparations are added and, optionally, a fraction of liposomes having a desired diameter range is separated.

2. A process according to claim 1 for the production of an intravenously administerable liposome dispersion comprising a) synthetic, essentially pure 1-n-hexadecanoyl-2-(9-cis-octadeccnoyl)-

3-sn-phosphatidylcholine if appropriate combined with b) synthetic, essentially pure sodium 1,2-di-(9-cis-octadecenoyl)-3-sn-phospha- tidyl-S-serine (II), c) the active ingredient to be administered intravenously and d) a pharmaceutically acceptable carrier liquid and, if appropriate, adjuncts suitable for injection preparations, characterised in that the measures mentioned in claim 1 are carried out. Ob 0 3. A process according to claim 1 for the production of an intravenously administerable liposome dispersion comprising Io a) synthetic, essentially pure 1-n-hexadecanoyl-2-(9-cis-octadecenoyl)- 3-sn-phosphatidylcholine if appropriate combined with b) synthetic, essentially pure sodium 1,2-di-(9-cis-octadecenoyl)-3-sn-phospha- tidyl-S-serine (II), 4 0 c) carbamazepine, synthetic human calcitonin or N-acetyl-D-muramyl-L-alanyl-D-iso- glutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamide and Ip d) a pharmaceutically acceptable carrier liquid and, if appropriate, adjuncts suitable for injection preparations, characterised in that the process according to claim 1 are carried S. out.

4. A process according to any one of claims 1 to 3, wherein a solution or suspension of the components a) and c) or 1b' and c) in glacial acetic acid is dispersed in the carrier liquid d) and the dispersion obtainable is brought to a pH level of 7.2 to 7.4. The liposome dispersion obtainable by the process according to claim 1. 16

6. Any novel injectable liposome dispersion substantially as herein described with reference to any one of the Examples. DATED this 16th day of July, 1991. CIBA-GEIGY AG By Its Patent Attorneys ARTHUR S. CAVE CO. 0 0090 *'e 06 0 *006 OeeO 0 bee S I. 0e S. .0e 0 0050 0* 00 S 0S S 00i rw C C 0000 a 4~e.e.c U Process for the production of an iniectable liposome dispersion Abstract The present invention relates to a novel, advantageous process for the production of an injectable liposome dispersion. The phospholipid components and the active ingredient to be administered are dissolved in concentrated acetic acid, the solution is neutralised and the injection solution, which can be administered directly, is dispersed. 6 0t4 4099 6 a4