CA2047023A1 - Process for the production of an injectable liposome dispersion - Google Patents
Process for the production of an injectable liposome dispersionInfo
- Publication number
- CA2047023A1 CA2047023A1 CA002047023A CA2047023A CA2047023A1 CA 2047023 A1 CA2047023 A1 CA 2047023A1 CA 002047023 A CA002047023 A CA 002047023A CA 2047023 A CA2047023 A CA 2047023A CA 2047023 A1 CA2047023 A1 CA 2047023A1
- Authority
- CA
- Canada
- Prior art keywords
- cis
- octadecenoyl
- synthetic
- dispersion
- carrier liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
Abstract
Process for the production of an injectable 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.
Description
` -- 2~7023 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 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 l-n-hexa-decanoyl-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 ean be administered, inter alia, intravenously.
The injectable liposome dispersions, however, are disadvantageous since, as the preliminary step, they require the produetion of a dry preparation, for example Iyophilisate 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~wàrming and dispersing~the organie solution in water. Beeause of the~high eontent of organie solvents and possible thermal degradation products, these solutions are physiologieally unaeeeptable for injeetion solutions, in partieular for intravenous àdministration.
The~objeet of the present invention is to produce an injectable liposome dispersion by mixing~the eomponents, while avoiding the preliminary step of the dry preparation, by choosing~a pharmaceutically~suitable, non-toxic solvent. This object is achieved by the present lnvention, whlch relates to a novel, advantageous process for the production of an .
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injectable liposome dispersion.
The invcntion relates to a process for the production of an injectable liposome dispersion comprising a) a phospholipid of the formula CH2_ O--R1 R2--o--CH o 3CH2--o--p--O--(CnH2n)--N--Rb (I), o~3 Rc in which R1 is C10 20acyl, R2 is hydrogen or C1O 20acyl~ Ra~ Rb and Rc are hydrogen or Cl4alkyl and n is an integer from two to four, optionally combined with an additional b) phospholipid of the formula CH2_ 0--R3 R4--o--2CH O
I ll (II), 3CH2----P----Rs 0~
in which R3 is Cl~20acyl, R4 is hydrogen or Cl0 20acyl and R5 is hydrogen, Cl4alkyl, Cl salkyl substituted by carboxy, C2 5alkyl substituted by hydroxy, C2 5allcyl substituted ;by carboxy and hydroxy or C2 salkyl substituted by carboxy and amino, ~ , c)~the :active ingredient to be injected or an acdve ingredient combination and ~d) a~pharmaceutically acceptable carrier liquid and, optionally, further adjuncts suitable for injeFuon~preparations.
The process is characterised in that a solution or suspension of the components a) and c~ or a3, b~ and c) in concentrated acetic acid is dispersed in the carrier liquid c) and the .
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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 lipoxomes 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.
The process in particular has the advantage that the organic solvent acetic acid can be 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 (I) and if appropriate (II) having an inner space and a spherical shape ("onion skin-like" construclion of the double layers or membranes - multilamellar). The order of size of the liposomes is variable between about 1.0 x lo-8 up to about 1.0 x 10-5 m.
The therapeutic use of liposomes as carriers of active ingredients of different types is known. Liposomes have furtherrnore been proposed as carriers of proteins, for example antibodies or enzymes, hormones, vitamins or genes, or for analytical purposes as carriers ' ~' ' ' ' '- . .
`- 2047023 of labelled compounds.
Injectable liposome dispersions are described in the review by Gregoriadis G. (editor) Liposome Tcchnology, Vol. II, Incorporation of Drugs, Proteins and Genetic Material, CRC Press 1984. In the review by Knight, C.G. (editor), Liposomes: From PhysicalStructure 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 l 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 formu1a I, Rl and R2 with the meanings C~0 20acyl are preferably straight-chain Cl0.20alkanoyl having an even number of C atoms and straight-chain Cl0 20alkenoyl having a double bond and an even number of C atoms.
Straight-chain Cl0 20alkanoyl Rl and R2 having an even number of C atoms are, for ~example, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl or n-octadecanoy L
Straight-chain Cl0 20alkenoyl Rl and R2 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 -(CnH2n)- is unbranched or branched alkylene, for example, l,l-ethylene, 1,1-, 1,2- or 1,3-propylene or 1,2-, 1,3- or 1,4-butylene. 1,2-ethylene (n = 2) is preferred.
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Phospholipids of the formula I are, for example, naturally occurring cephalins in which R
Rb and Rc are hydrogen, or naturally occurring lecithins in which Ra~ E~b and Rc are 20~7023 methyl, for example cephalin or lecithin from soya beans, bovine brain, bovine liver or egg yolk having different or identical acyl groups R~ and R2 or mixtures thereof.
Synthetic, essentially pure phospholipids of the formula I having different or identical acyl groups Rl and R2 are preferred.
The term "synthetic" phospholipid of the formula 1 defines phospholipids which have a uniform composition with respect to Rl and R2. Such synthetic phospholipids are preferably the lecithins and cephalins defined above, whose acyl groups Rl and R2 have a defined structure and are derived from a defined fatty acid having a degree of purity higher than about 95 %. R~ and R2 can be identical or different and unsaturated or saturated. Rl is preferably saturated, for example n-hexadecanoyl, and R2 unsaturated, for example 9-cis-octadecenoyl (= oleoyl).
The term "naturally occurring" phospholipids of the formula I defines phospholipids which with respect to Rl and R2 do not have a unifonn composition. Such natural phospholipids are likewise lecithins and cephalins whose acyl groups Rl and R2 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 (I), 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 RI has the meaning straight-chain Cl0.20alkanoyl having an even number of C
atoms and R2 has the meaning straight-chain Cl0 20alkenoyl having a double bond and an even number of C atoms. Ra~ Rb and Rc are methyl and n is two.
.
In a particularly preferred phospholipid of the formula I, R~ is n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl or n-octadecanoyl and R2 is 9-cis-dodecenoy1, 9-cis-tetradecenoyl, 9-cis-hexadecenoyl, 9-cis-octadecenoyl or 9-cis-icosenoyl. R2 is tetradeceyl, 9-cis-hexadecenoyl, 9-cis-octadecenoyl or 9-cis-icosenoyl. Ra~ Rb and Rc are methyl and n is tWO.
A very pardcularly preferred phospholipid of the fonnula I is synthetic l-n-hexa-decanoyl-2-(9-cis-octadecenoyl)-3-sn-phosphatidylcholine having a purity of more than 95 ~, ~
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20~7023 %.
In a phospholipid of the formula II, R3 and R4 are defined with the same meaningCl0 20acyl as for Rl and R2 in a phospholipid of the formula I.
The acyl groups R3 and R4 can be different or identical.
Rs having the meaning C~4alkyl is, for example, methyl or ethyl.
Rs having the meanings Cl salkyl substituted by carboxyl, C2 salkyl substituted by hydroxyl or C2 salkyl substituted by carboxy or hydroxy are, for example, 2-hydroxyethyl, 2,3-dihydroxy-n-propyl, carboxymethyl, 1- or 2-carboxyethy], dicarboxymethyl, 2-carboxy-2-hydroxyethyl or 3-carboxy-2,3-dihydroxy-n-propyl.
R5 having the meaning C2 salkyl 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 II containing these groups can be present in salt form, for example as the sodium or potassium salt.
Synthetic, essentially pure phospholipids of the formula II having different or identical acyl groups R3 and R4 are preferred.
The def:nitions stipulated for phospholipids of the formula I mentioned above apply with respect to the definition "synthetic" and the requirement "essentially pure".
In a particularly preferred phospholipid of the forrnula II, R3 and R4 have the meaning straight-chain Cl0 20alkenoyl having a double bond and an even number of C atoms. R5 is 2-amino-2-carboxyethyl.
~, I n the preferred phospholipid of the formula II, R3 and R4 having the meaning "Cl0 20alkenoyl having a double bond and an even number of C atoms" are preferably 9-cis-dodecenoyl 9-cis-tetradecenoyl, 9-cis-hexadecenoyl, 6-cis-octadecenoyl, 6-trans-octadecenoyl, 9-cis-octadecenoyl, 9-trans-octadecenoyl, 1 l-cis-octadecenoyl or 9-cis-icosenoyl.
In~ a particularly preferred phospholipid of the formula lI, R3 and R4 have identical '~' :
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meanings, for exan1ple 9-cis-dodecenoyl, 9-cis-~e~radecenoyl, 9-cis-hexadecenoyl, 9-cis-octadecenoyl or 9-cis-icosenoyl.
A very particularly preferred phospholipid of the forrnula II is synthetic sodium 1,2-di-(9-cis-octadecenoyl)-3-sn-phosphatidyl-S-serine having a purity of more than 95 %.
The names given in brackets are also customary for the acyl radicals in the phospholipids of the formulae I and II:
9-cis-dodecenoyl (lauroleoyl), 9-cis-tetradecenoyl (myristoleoyl), 9-cis-hexadecenoyl (palmitoleoyl), 6-cis-octadecenoyl (petroseloyl), 6-trans-octadecenoyl ~petroselaidoyl), 9-cis-octadecenoyl (oleoyl), 9-trans-octadecenoyl (elaidoyl), 1 l-cis-octadecenoyl (vaccenoyl), 9-cis-icosenoyl (gadoleoyl), n-dodecanoyl (lauroyl), n-tetradecanoyl (myristoyl), n-hexadecanoyl (palmitoyl), n-octadecanoyl (stearoyl), n-icosanoyl (arachidoyl).
Water-soluble, but also sparingly soluble, if appropriate crystalline, lipophilic active ingredients which can be administered 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,pharmaceutically acceptable salts, for example as the hydrobromide, hydrochloride, mesylate, acetate, succinate, lactate, tartrate, fumarate, sulfate, maleate, etc.
Suitable pharrnaceutical active ingredients are, for example, antiinflammatory agents, for example indomethacin, acetylsalicylic acid, 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 examp1e prostaglandin El, E2 or F
peripheral vasodilators, for example if enprodil, cinepazet maleate, cyclandelate, cinnarizine or pentoxyphylline, antibiotics, for example ampicillin, amoxycillin, cephalexin, cefradin, cefroxadin, cefactor, erythromycin, bacampicillin, minocyclin or chlorarnphenicol, antispasmodic agents, for example propantheline, atropine or scopolamine, antitussives and antiasthmatics, for example theophylline, aminophylline, methylephedrine, procatechol, trimethoquinol, codeine, clofedanol or dextromethorphan, diuretics, for example furosemide or acetazolamide, muscle-relaxing agents, for example chlorphenesin carbamate, tolperisone, eperisone or baclofen, weak tranquillisers, for ..... .
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20~7023 example oxazolam, diazepam, clotiazepam, medazepam, temazepam or fludiazepam, strong tranquillisers, for example sulpiride, clocapramine or zotepine, beta-blockers, for example pindolol, propranolol, carteolol, oxprenolol, metoprolol or labetalol, antiarrhythmics, for example procainamide, disopyramide, ajimalin or quinidine, antiarthritic agents such as allopurinol, anticoagulants such as ticlopidine, antiepileptics, for example phenyloin, valproate or carbamazepine, antihistaminics, for example chlorpheniramine, clemastine, mequitazine, alimemazine, cyproheptadine, agents against nausea and vertigo, for example diphenidol, metoclopramide, domperidone or betahistine, hypotensive agents, for example reserpine, rescinnamine, methyldopa, prazosine, clonidine or budralazine, sympathomimetics, for example dihydroergotamine, isoproterenol or etilefrine, expectorants, for example bromhexine, carbocysteine, L-ethylcysteine or L-methylcysteine, oral antidiabetics, for example glibenclamide or tolbutamide, and cardiovascular agents, for example ubidecarenone or adenosine.
Preferred antihypercalcaemics are those from the calcitonin series, for example 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-isoglutaminyl-L-a1anine-2-( 1 ,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryl-oxy)ethylamide, disodium N-acetyl-D-muramyl-L-alanyl-D-glutamic acid (C~-L-alanine--2-(1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamide, sodium N-acetyl--D-muramyl-L-alanyl-D-isoglutamine, sodium N-acetyldesmethyl-muramyl--L-alanyl-D-isoglutamine, N-acetyl-D-muramyl-L-alanyl-D-glutamine-o~-n-butyl ester, Na-(N-acetyl-D-muramyl-L-alanyl-D-isoglutaminyl)-N~-stearoyl-L-lysine or 6-0-stearoyl-N-acetylD-muramyl-L-alanyl-D-isoglutamine.
The pharmaceutically acceptable carrier liquid d) is water which has been processed microorganism- and pyrogen-free according to the directions of the national pharrnacopoeias.
The components a), b) and c) or a) and c) are contained in the carrier liquid d) as liposomes in such a 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 intravenously.
g Non-toxic adjuncts which can be used for injection preparations can be present in the carrier liquid d), for example water-soluble adjuncts which are necessary for the production of isotonic conditions, for example ionic additives such as sodium chloride or non-ionic additives (structure-forming agen~) such as sorbitol, mannitol or glucose 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-ionic surfactants of the fatty acid polyhydroxyalcohol ester type such as sorbitan monolaurate, oleate, stearate or palmitate, sorbitan tristearate or trioleate, polyoxyethylene adducts of fatty acid polyhydroxy alcohol esters such as polyoxyethylene sorbitan monolaurate, oleate, stearate, palmitate, tristearate or trioleate, polyethylene glycol fatty acid esters such as polyoxyethyl stearate, polyethylene glycol 40û stearate, polyethylene glycol 2000 stearate, in palticular 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.
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Depending on the solubility of the components a) and c) or a), b) and c) in the concentrated acetic acid, a solution is formed. This is then dispersed in the carrier liquid d), to which, if desired, suitable adjuncts for injection preparations have been added.
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The dispersion itself is carried out, for example, by shaking (for example vortex mixer) or stirring the carrier liquid. The formation of liposomesj 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), preferabIy 2 to 20 per cent by weight, of the acetic acid soluaon or dispersion can be dispersed in the aqueous phase.
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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 36C, 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 months).
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 reducin~ the concentration of the individual lipid components.
The size and structure (multilamellar/unilamellar) 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 having 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 ultrasonic waves gives a high content of unilamellar liposomes in the dispersion.
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 pharmacopoeias, for example the European, U.S., German or British pharmacopoeia. The dispersion can also be neutralised byaddition of a pharmaceutically acceptable, dilute aqueous base, for example dilute sodium hydroxide soludon. The dispersion is customarily neutralised with simultaneous pH
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, for 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 ultrasound or extrusion through even-pore filters (for example Nucleopore~)).
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-` 2047023 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 gel filtration or ultrafiltration, for example using Sepharose~) 4B or Sephacryl(g) (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 cen~rifugation in this gravitational field, while the small liposomes remain dispersed and can be decanted. A complete separation of the large liposomes from the small liposomes is achieved after centrifugation several times.
All liposomes in the aqueous phase having a diameter greater than about 6.0 x lo-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 cxample using freeze-fracture samples and thin sections in the electron microscope or by X-ray diffrac~ion, by dynamic light scattering, by mass determination of the filtrate in the analy~ical ultracentrifuge and primarily by spectroscopy, for example in the nuclear magnetic resonance specuum (IH, 13C and 31p).
The liposome dispersion can be administered directly, but by freeze-drying can also be converted into a Iyophilisate which is reconstituted with the intended injection volume by addition of water immediately before administration.
The invention relates primarily to a process for the production of an intravenously administrable liposome dispersion comprising a) synthetic, essentially pure 1-n-hexadecanoyl-2-(9-cis-octadecenoyl)-3-sn-phosphatidylcholine (I), 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 pharmaceutlcally acceptable carrier liquid and, if appropriate, adjuncts suitable for .
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, .~ ' ' ' ' ~047023 injec~ion 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-hexadecanoyl-2-(9-cis-octadecenoyl)-3-sn-phosphatidylcholine (I), if appropriate combined with b) synthetic, essentially pure sodium 1,2-di-(9-cis-octadecenoyl)-3-sn-phospha-tidyl-S-serine (II), c) carbamazepine, synthetic human calcitonin or N-acetyl-D-muramyl-L-alanyl-D-iso-glutaminyl-I,-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-hexa-decanoyl-2-(9-cis-octadecenoyl~-3-sn-phosphatidylcholine and at least 95 % pure 1,2-di-(9-cis-octadecenoyl)-3-sn-phosphatidyl-S-serine in a weight ratio of 7:3 and 1 mg of N-acetyl-D-muramyl-L-alanyl-D-isoglutaminyl-~alanine-2-(1,2-dipalmitoyl-sn-glycero3-hydroxyphosphoryloxy)ethylamide are dissolved in 0.25 ml of glacial acetic acid in a round-bottomed flask. The equimolar amount of 2N NaOH
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 = 7.2). The buffer solution is chosen such that a pH of the dispersion of about 7.2 to 7.4 is obtained ~; ~ 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 1-n-hexadecanoyl-2-lyso-3-sn-phosphatidyl-choline, 40 mg of at least 95 % pure 1,2-di(9-cis-octadecenoyl)-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 P acetic acid with stirring.~ An equimolar amount of 2N NaO~I solution (4.1 5 ml) is added with stirring. A liposome dispersion is formed. The other steps are carried out analogously to Example 1.
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` - 2047023 Example 3: 250 mg of the phospholipid mixture according to Example 1 are dissolved in 250 111 of glacial acetic acid containing 310 I,lg 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 forrned is diluted to a volume of 20 ml by addition of microorganism-free, pyrogen-free water for injection. This dispersion is suitable for intramuscular administration .
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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 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 l-n-hexa-decanoyl-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 ean be administered, inter alia, intravenously.
The injectable liposome dispersions, however, are disadvantageous since, as the preliminary step, they require the produetion of a dry preparation, for example Iyophilisate 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~wàrming and dispersing~the organie solution in water. Beeause of the~high eontent of organie solvents and possible thermal degradation products, these solutions are physiologieally unaeeeptable for injeetion solutions, in partieular for intravenous àdministration.
The~objeet of the present invention is to produce an injectable liposome dispersion by mixing~the eomponents, while avoiding the preliminary step of the dry preparation, by choosing~a pharmaceutically~suitable, non-toxic solvent. This object is achieved by the present lnvention, whlch relates to a novel, advantageous process for the production of an .
. .
;, . . . . . .
, -, .
injectable liposome dispersion.
The invcntion relates to a process for the production of an injectable liposome dispersion comprising a) a phospholipid of the formula CH2_ O--R1 R2--o--CH o 3CH2--o--p--O--(CnH2n)--N--Rb (I), o~3 Rc in which R1 is C10 20acyl, R2 is hydrogen or C1O 20acyl~ Ra~ Rb and Rc are hydrogen or Cl4alkyl and n is an integer from two to four, optionally combined with an additional b) phospholipid of the formula CH2_ 0--R3 R4--o--2CH O
I ll (II), 3CH2----P----Rs 0~
in which R3 is Cl~20acyl, R4 is hydrogen or Cl0 20acyl and R5 is hydrogen, Cl4alkyl, Cl salkyl substituted by carboxy, C2 5alkyl substituted by hydroxy, C2 5allcyl substituted ;by carboxy and hydroxy or C2 salkyl substituted by carboxy and amino, ~ , c)~the :active ingredient to be injected or an acdve ingredient combination and ~d) a~pharmaceutically acceptable carrier liquid and, optionally, further adjuncts suitable for injeFuon~preparations.
The process is characterised in that a solution or suspension of the components a) and c~ or a3, b~ and c) in concentrated acetic acid is dispersed in the carrier liquid c) and the .
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--- . .
:: . - ; . :
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 lipoxomes 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.
The process in particular has the advantage that the organic solvent acetic acid can be 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 (I) and if appropriate (II) having an inner space and a spherical shape ("onion skin-like" construclion of the double layers or membranes - multilamellar). The order of size of the liposomes is variable between about 1.0 x lo-8 up to about 1.0 x 10-5 m.
The therapeutic use of liposomes as carriers of active ingredients of different types is known. Liposomes have furtherrnore been proposed as carriers of proteins, for example antibodies or enzymes, hormones, vitamins or genes, or for analytical purposes as carriers ' ~' ' ' ' '- . .
`- 2047023 of labelled compounds.
Injectable liposome dispersions are described in the review by Gregoriadis G. (editor) Liposome Tcchnology, Vol. II, Incorporation of Drugs, Proteins and Genetic Material, CRC Press 1984. In the review by Knight, C.G. (editor), Liposomes: From PhysicalStructure 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 l 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 formu1a I, Rl and R2 with the meanings C~0 20acyl are preferably straight-chain Cl0.20alkanoyl having an even number of C atoms and straight-chain Cl0 20alkenoyl having a double bond and an even number of C atoms.
Straight-chain Cl0 20alkanoyl Rl and R2 having an even number of C atoms are, for ~example, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl or n-octadecanoy L
Straight-chain Cl0 20alkenoyl Rl and R2 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 -(CnH2n)- is unbranched or branched alkylene, for example, l,l-ethylene, 1,1-, 1,2- or 1,3-propylene or 1,2-, 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 R
Rb and Rc are hydrogen, or naturally occurring lecithins in which Ra~ E~b and Rc are 20~7023 methyl, for example cephalin or lecithin from soya beans, bovine brain, bovine liver or egg yolk having different or identical acyl groups R~ and R2 or mixtures thereof.
Synthetic, essentially pure phospholipids of the formula I having different or identical acyl groups Rl and R2 are preferred.
The term "synthetic" phospholipid of the formula 1 defines phospholipids which have a uniform composition with respect to Rl and R2. Such synthetic phospholipids are preferably the lecithins and cephalins defined above, whose acyl groups Rl and R2 have a defined structure and are derived from a defined fatty acid having a degree of purity higher than about 95 %. R~ and R2 can be identical or different and unsaturated or saturated. Rl is preferably saturated, for example n-hexadecanoyl, and R2 unsaturated, for example 9-cis-octadecenoyl (= oleoyl).
The term "naturally occurring" phospholipids of the formula I defines phospholipids which with respect to Rl and R2 do not have a unifonn composition. Such natural phospholipids are likewise lecithins and cephalins whose acyl groups Rl and R2 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 (I), 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 RI has the meaning straight-chain Cl0.20alkanoyl having an even number of C
atoms and R2 has the meaning straight-chain Cl0 20alkenoyl having a double bond and an even number of C atoms. Ra~ Rb and Rc are methyl and n is two.
.
In a particularly preferred phospholipid of the formula I, R~ is n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl or n-octadecanoyl and R2 is 9-cis-dodecenoy1, 9-cis-tetradecenoyl, 9-cis-hexadecenoyl, 9-cis-octadecenoyl or 9-cis-icosenoyl. R2 is tetradeceyl, 9-cis-hexadecenoyl, 9-cis-octadecenoyl or 9-cis-icosenoyl. Ra~ Rb and Rc are methyl and n is tWO.
A very pardcularly preferred phospholipid of the fonnula I is synthetic l-n-hexa-decanoyl-2-(9-cis-octadecenoyl)-3-sn-phosphatidylcholine having a purity of more than 95 ~, ~
- ,: .
~;
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20~7023 %.
In a phospholipid of the formula II, R3 and R4 are defined with the same meaningCl0 20acyl as for Rl and R2 in a phospholipid of the formula I.
The acyl groups R3 and R4 can be different or identical.
Rs having the meaning C~4alkyl is, for example, methyl or ethyl.
Rs having the meanings Cl salkyl substituted by carboxyl, C2 salkyl substituted by hydroxyl or C2 salkyl substituted by carboxy or hydroxy are, for example, 2-hydroxyethyl, 2,3-dihydroxy-n-propyl, carboxymethyl, 1- or 2-carboxyethy], dicarboxymethyl, 2-carboxy-2-hydroxyethyl or 3-carboxy-2,3-dihydroxy-n-propyl.
R5 having the meaning C2 salkyl 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 II containing these groups can be present in salt form, for example as the sodium or potassium salt.
Synthetic, essentially pure phospholipids of the formula II having different or identical acyl groups R3 and R4 are preferred.
The def:nitions stipulated for phospholipids of the formula I mentioned above apply with respect to the definition "synthetic" and the requirement "essentially pure".
In a particularly preferred phospholipid of the forrnula II, R3 and R4 have the meaning straight-chain Cl0 20alkenoyl having a double bond and an even number of C atoms. R5 is 2-amino-2-carboxyethyl.
~, I n the preferred phospholipid of the formula II, R3 and R4 having the meaning "Cl0 20alkenoyl having a double bond and an even number of C atoms" are preferably 9-cis-dodecenoyl 9-cis-tetradecenoyl, 9-cis-hexadecenoyl, 6-cis-octadecenoyl, 6-trans-octadecenoyl, 9-cis-octadecenoyl, 9-trans-octadecenoyl, 1 l-cis-octadecenoyl or 9-cis-icosenoyl.
In~ a particularly preferred phospholipid of the formula lI, R3 and R4 have identical '~' :
.
~- .
.
, . :
:. ., .
~ ' , .
meanings, for exan1ple 9-cis-dodecenoyl, 9-cis-~e~radecenoyl, 9-cis-hexadecenoyl, 9-cis-octadecenoyl or 9-cis-icosenoyl.
A very particularly preferred phospholipid of the forrnula II is synthetic sodium 1,2-di-(9-cis-octadecenoyl)-3-sn-phosphatidyl-S-serine having a purity of more than 95 %.
The names given in brackets are also customary for the acyl radicals in the phospholipids of the formulae I and II:
9-cis-dodecenoyl (lauroleoyl), 9-cis-tetradecenoyl (myristoleoyl), 9-cis-hexadecenoyl (palmitoleoyl), 6-cis-octadecenoyl (petroseloyl), 6-trans-octadecenoyl ~petroselaidoyl), 9-cis-octadecenoyl (oleoyl), 9-trans-octadecenoyl (elaidoyl), 1 l-cis-octadecenoyl (vaccenoyl), 9-cis-icosenoyl (gadoleoyl), n-dodecanoyl (lauroyl), n-tetradecanoyl (myristoyl), n-hexadecanoyl (palmitoyl), n-octadecanoyl (stearoyl), n-icosanoyl (arachidoyl).
Water-soluble, but also sparingly soluble, if appropriate crystalline, lipophilic active ingredients which can be administered 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,pharmaceutically acceptable salts, for example as the hydrobromide, hydrochloride, mesylate, acetate, succinate, lactate, tartrate, fumarate, sulfate, maleate, etc.
Suitable pharrnaceutical active ingredients are, for example, antiinflammatory agents, for example indomethacin, acetylsalicylic acid, 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 examp1e prostaglandin El, E2 or F
peripheral vasodilators, for example if enprodil, cinepazet maleate, cyclandelate, cinnarizine or pentoxyphylline, antibiotics, for example ampicillin, amoxycillin, cephalexin, cefradin, cefroxadin, cefactor, erythromycin, bacampicillin, minocyclin or chlorarnphenicol, antispasmodic agents, for example propantheline, atropine or scopolamine, antitussives and antiasthmatics, for example theophylline, aminophylline, methylephedrine, procatechol, trimethoquinol, codeine, clofedanol or dextromethorphan, diuretics, for example furosemide or acetazolamide, muscle-relaxing agents, for example chlorphenesin carbamate, tolperisone, eperisone or baclofen, weak tranquillisers, for ..... .
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20~7023 example oxazolam, diazepam, clotiazepam, medazepam, temazepam or fludiazepam, strong tranquillisers, for example sulpiride, clocapramine or zotepine, beta-blockers, for example pindolol, propranolol, carteolol, oxprenolol, metoprolol or labetalol, antiarrhythmics, for example procainamide, disopyramide, ajimalin or quinidine, antiarthritic agents such as allopurinol, anticoagulants such as ticlopidine, antiepileptics, for example phenyloin, valproate or carbamazepine, antihistaminics, for example chlorpheniramine, clemastine, mequitazine, alimemazine, cyproheptadine, agents against nausea and vertigo, for example diphenidol, metoclopramide, domperidone or betahistine, hypotensive agents, for example reserpine, rescinnamine, methyldopa, prazosine, clonidine or budralazine, sympathomimetics, for example dihydroergotamine, isoproterenol or etilefrine, expectorants, for example bromhexine, carbocysteine, L-ethylcysteine or L-methylcysteine, oral antidiabetics, for example glibenclamide or tolbutamide, and cardiovascular agents, for example ubidecarenone or adenosine.
Preferred antihypercalcaemics are those from the calcitonin series, for example 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-isoglutaminyl-L-a1anine-2-( 1 ,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryl-oxy)ethylamide, disodium N-acetyl-D-muramyl-L-alanyl-D-glutamic acid (C~-L-alanine--2-(1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamide, sodium N-acetyl--D-muramyl-L-alanyl-D-isoglutamine, sodium N-acetyldesmethyl-muramyl--L-alanyl-D-isoglutamine, N-acetyl-D-muramyl-L-alanyl-D-glutamine-o~-n-butyl ester, Na-(N-acetyl-D-muramyl-L-alanyl-D-isoglutaminyl)-N~-stearoyl-L-lysine or 6-0-stearoyl-N-acetylD-muramyl-L-alanyl-D-isoglutamine.
The pharmaceutically acceptable carrier liquid d) is water which has been processed microorganism- and pyrogen-free according to the directions of the national pharrnacopoeias.
The components a), b) and c) or a) and c) are contained in the carrier liquid d) as liposomes in such a 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 intravenously.
g Non-toxic adjuncts which can be used for injection preparations can be present in the carrier liquid d), for example water-soluble adjuncts which are necessary for the production of isotonic conditions, for example ionic additives such as sodium chloride or non-ionic additives (structure-forming agen~) such as sorbitol, mannitol or glucose 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-ionic surfactants of the fatty acid polyhydroxyalcohol ester type such as sorbitan monolaurate, oleate, stearate or palmitate, sorbitan tristearate or trioleate, polyoxyethylene adducts of fatty acid polyhydroxy alcohol esters such as polyoxyethylene sorbitan monolaurate, oleate, stearate, palmitate, tristearate or trioleate, polyethylene glycol fatty acid esters such as polyoxyethyl stearate, polyethylene glycol 40û stearate, polyethylene glycol 2000 stearate, in palticular 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 a), b) and c) in the concentrated acetic acid, a solution is formed. This is then dispersed in the carrier liquid d), 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 liposomesj 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), preferabIy 2 to 20 per cent by weight, of the acetic acid soluaon or dispersion can be dispersed in the aqueous phase.
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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 36C, 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 months).
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 reducin~ the concentration of the individual lipid components.
The size and structure (multilamellar/unilamellar) 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 having 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 ultrasonic waves gives a high content of unilamellar liposomes in the dispersion.
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 pharmacopoeias, for example the European, U.S., German or British pharmacopoeia. The dispersion can also be neutralised byaddition of a pharmaceutically acceptable, dilute aqueous base, for example dilute sodium hydroxide soludon. The dispersion is customarily neutralised with simultaneous pH
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, for 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 ultrasound or extrusion through even-pore filters (for example Nucleopore~)).
~ . .. : ' ' ~
-` 2047023 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 gel filtration or ultrafiltration, for example using Sepharose~) 4B or Sephacryl(g) (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 cen~rifugation in this gravitational field, while the small liposomes remain dispersed and can be decanted. A complete separation of the large liposomes from the small liposomes is achieved after centrifugation several times.
All liposomes in the aqueous phase having a diameter greater than about 6.0 x lo-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 cxample using freeze-fracture samples and thin sections in the electron microscope or by X-ray diffrac~ion, by dynamic light scattering, by mass determination of the filtrate in the analy~ical ultracentrifuge and primarily by spectroscopy, for example in the nuclear magnetic resonance specuum (IH, 13C and 31p).
The liposome dispersion can be administered directly, but by freeze-drying can also be converted into a Iyophilisate which is reconstituted with the intended injection volume by addition of water immediately before administration.
The invention relates primarily to a process for the production of an intravenously administrable liposome dispersion comprising a) synthetic, essentially pure 1-n-hexadecanoyl-2-(9-cis-octadecenoyl)-3-sn-phosphatidylcholine (I), 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 pharmaceutlcally acceptable carrier liquid and, if appropriate, adjuncts suitable for .
.
, .~ ' ' ' ' ~047023 injec~ion 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-hexadecanoyl-2-(9-cis-octadecenoyl)-3-sn-phosphatidylcholine (I), if appropriate combined with b) synthetic, essentially pure sodium 1,2-di-(9-cis-octadecenoyl)-3-sn-phospha-tidyl-S-serine (II), c) carbamazepine, synthetic human calcitonin or N-acetyl-D-muramyl-L-alanyl-D-iso-glutaminyl-I,-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-hexa-decanoyl-2-(9-cis-octadecenoyl~-3-sn-phosphatidylcholine and at least 95 % pure 1,2-di-(9-cis-octadecenoyl)-3-sn-phosphatidyl-S-serine in a weight ratio of 7:3 and 1 mg of N-acetyl-D-muramyl-L-alanyl-D-isoglutaminyl-~alanine-2-(1,2-dipalmitoyl-sn-glycero3-hydroxyphosphoryloxy)ethylamide are dissolved in 0.25 ml of glacial acetic acid in a round-bottomed flask. The equimolar amount of 2N NaOH
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 = 7.2). The buffer solution is chosen such that a pH of the dispersion of about 7.2 to 7.4 is obtained ~; ~ 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 1-n-hexadecanoyl-2-lyso-3-sn-phosphatidyl-choline, 40 mg of at least 95 % pure 1,2-di(9-cis-octadecenoyl)-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 P acetic acid with stirring.~ An equimolar amount of 2N NaO~I solution (4.1 5 ml) is added with stirring. A liposome dispersion is formed. The other steps are carried out analogously to Example 1.
::
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` - 2047023 Example 3: 250 mg of the phospholipid mixture according to Example 1 are dissolved in 250 111 of glacial acetic acid containing 310 I,lg 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 forrned is diluted to a volume of 20 ml by addition of microorganism-free, pyrogen-free water for injection. This dispersion is suitable for intramuscular administration .
::
.
`:
.
Claims (5)
1. A process for the production of an injectable liposome dispersion comprising a) a phospholipid of the formula (I), in which R1 is Cl0-20acyl, R2 is hydrogen or C10-20acyl,Ra,Rb and Rc are hydrogen or C1-4alkyl and n is an integer from one to four, optionally combined with an additional b) phospholipid of the formula (II), in which R3 is C10-20acyl, R4 is hydrogen or C10-20acyl and R5 is hydrogen,C1-4alkyl, C1-5alkyl substituted by carboxy, C2-5alkyl substituted by hydroxy, C2-5alkyl 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, if appropriate, further adjuncts suitable for injection preparations, characterised in that a solution or suspension of the components a) and c) or a), 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-octadecenoyl)-
3-sn-phosphatidylcholine (I), 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.
3. 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-octadecenoyl)-3-sn-phosphatidylcholine (I), if appropriate combined with b) synthetic, essentially pure sodium 1,2-di-(9-cis-octadecenoyl)-3-sn-phospha-tidyl-S-serine (II), 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 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 out.
3. 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-octadecenoyl)-3-sn-phosphatidylcholine (I), if appropriate combined with b) synthetic, essentially pure sodium 1,2-di-(9-cis-octadecenoyl)-3-sn-phospha-tidyl-S-serine (II), 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 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 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 a), b) 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.
5. The liposome dispersion obtainable by the process according to claim 1.
FO 7.4/RS/lb*
FO 7.4/RS/lb*
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH237190 | 1990-07-17 | ||
| CH2371/90-8 | 1990-07-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2047023A1 true CA2047023A1 (en) | 1992-01-18 |
Family
ID=4232208
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002047023A Abandoned CA2047023A1 (en) | 1990-07-17 | 1991-07-15 | Process for the production of an injectable liposome dispersion |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0467838A3 (en) |
| JP (1) | JPH0570342A (en) |
| KR (1) | KR920002166A (en) |
| AU (1) | AU639812B2 (en) |
| CA (1) | CA2047023A1 (en) |
| IE (1) | IE912485A1 (en) |
| IL (1) | IL98783A0 (en) |
| NZ (1) | NZ238966A (en) |
| PT (1) | PT98321A (en) |
| ZA (1) | ZA915545B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9224502D0 (en) * | 1992-11-23 | 1993-01-13 | New Roger R C | Method of preparing a lipid-containing formulation |
| ATE226210T1 (en) | 1997-08-18 | 2002-11-15 | Max Planck Gesellschaft | PHOSPHOLIPIDE ANALOGOUS COMPOUNDS |
| JP2020069470A (en) * | 2018-10-29 | 2020-05-07 | 株式会社げんてん本店 | Method for manufacturing liposome and method for manufacturing liposome containing liquid |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USRE30748E (en) * | 1976-10-12 | 1981-09-22 | Phosphatidyl quaternary ammonium compounds | |
| EP0088046B1 (en) * | 1982-02-17 | 1987-12-09 | Ciba-Geigy Ag | Lipids in the aqueous phase |
| JPS607932A (en) * | 1983-06-29 | 1985-01-16 | Dai Ichi Seiyaku Co Ltd | Preparation of liposome |
| CA1260393A (en) * | 1984-10-16 | 1989-09-26 | Lajos Tarcsay | Liposomes of synthetic lipids |
-
1991
- 1991-07-09 EP EP19910810542 patent/EP0467838A3/en not_active Ceased
- 1991-07-10 IL IL98783A patent/IL98783A0/en unknown
- 1991-07-12 JP JP3172639A patent/JPH0570342A/en active Pending
- 1991-07-15 CA CA002047023A patent/CA2047023A1/en not_active Abandoned
- 1991-07-15 PT PT98321A patent/PT98321A/en not_active Application Discontinuation
- 1991-07-15 NZ NZ238966A patent/NZ238966A/en unknown
- 1991-07-16 IE IE248591A patent/IE912485A1/en unknown
- 1991-07-16 AU AU80464/91A patent/AU639812B2/en not_active Ceased
- 1991-07-16 ZA ZA915545A patent/ZA915545B/en unknown
- 1991-07-16 KR KR1019910012151A patent/KR920002166A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| AU639812B2 (en) | 1993-08-05 |
| ZA915545B (en) | 1992-03-25 |
| AU8046491A (en) | 1992-01-23 |
| IE912485A1 (en) | 1992-01-29 |
| EP0467838A3 (en) | 1992-09-02 |
| PT98321A (en) | 1992-05-29 |
| EP0467838A2 (en) | 1992-01-22 |
| JPH0570342A (en) | 1993-03-23 |
| IL98783A0 (en) | 1992-07-15 |
| KR920002166A (en) | 1992-02-28 |
| NZ238966A (en) | 1993-03-26 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |