CA2233135A1 - Stabilised phospholipid compositions - Google Patents

Abstract

The stability of phospholipid compositions is enhanced by the inclusion of a buffer system comprising ammonia or a water soluble amine having a pH at 15 ~C
of less than or equal to 9.5.

Description

CA 0223313~ 1998-03-26 Stabilised Phospholipid Compositions This invention relates to stabilised aqueous phospholipid compositions.

Phospholipid compositions are used in a variety of diagnostic, therapeutic and cosmetic applications. For example, lipid compositions, in particular liposomes, are used to incorporate diagnostic and therapeutic agents, as vehicles for transfer of genetic material, as immunological adjuvants, in preparation of vaccines and in cancer detection. Clearly, the stability of the phospholipid is important for the protection of any entrapped substance from degradation reactions and also ~or optimum performance of the phospholipid itsel~.

One particular area of interest is in the field o~
diagnostic imaging. Contrast agents are employed to e~fect imaging enhancement in a variety of diagnostic techniques, the most important o~ these being X-ray imaging, magnetic resonance imaging (MRI), ultrasound imaging and nuclear medicine imaging. There is a continuing need ~or contrast agents which combine good storage stability and stability in vivo. Another area of particular interest is the development o~ stable phospholipid compositions for use in techniques involving autoclavation.

Studies have shown the use o~ trometamol and related buffer compounds to have a specific e~fect on the hydrolysis of phospholipids. Many of these studies show general acid/base catalysis by the trometamol buffer with increased hydrolysis of the phospholipids with increasing concentration o~ bu~fer species (see ~or = example Journal o~ Pharmaceutical Sciences 82: 362-366 CA 02233l3~ l998-03-26 (1993)).

However, inhibition of the hydrolysis o~ phospholipid compositions due to an interaction between trometamol and phospholipase contained within the compositions has also been reported (see for example Biochemical and Biophysical Res. Comm. 84: 238-247 (1978); Biochem. J.
~Q~: 799-801 (1982); Archives of Insect Biochemistry and Physiology 14: 1-12 (1990); and Journal of Bacteriology 175: 4298-4306 (1993)).

In Chemistry and Physics of Lipids 60: 93-99 (1991), the stability of phospholipids in liposomal aqueous suspension against oxidative degradation in air was investigated. It was demonstrated that lecithin was more resistant to hydrolysis in trometamol buffer than in pure water. This was indicated to relate specifically to the naturally occurring phospholipid having polyunsaturated fatty acid chains which are readily susceptible to peroxidation by a free-radical mechanism. As indicated in the disclosure, it is well-known that ultrasonic irradiation o~ water promotes the production of hydroxyl free radicals and hydrogen peroxide and that these active oxygen species are involved in oxidative degradation of phospholipids. The trometamol buffer appeared to provide resistance to hydrolysis and the reference indicates that the reduced oxidation/ hydrolysis observed is due to the fact that trometamol acts as an ef~icient scavenger of hydroxyl free radicals. A similar conclusion is reached in J.Pharm. Pharmacol. 45: 490-495 (1993), where a protective effect of buf~ers such as trometamol against lipid peroxidation is reported.

In W0-95/26205 (Nycomed/Daiichi) there are described diagnostic compositions containing multilamellar liposomes containing at least one imaging agent and CA 02233l3~ l998-03-26 being suspended in an aqueous medium containing said imaging agent, wherein the liposomes comprise a neutral phospholipid and a charged phospholipid, the average particle diameter of the liposomes is 50-3000 nm and the concentration of imaging agent in any aqueous phase filling the interior of the liposomes is substantially the same as that in the aqueous medium in which the liposomes are suspended.

It has now, surprisingly, been found that substantially saturated phospholipid compounds can be stabilised by buffers, the buffers providing a reduced degree of aqueous hydrolysis of the phospholipids.

Thus, viewed from one aspect the present invention provides an aqueous lipid composition, preferably a liposomal composition and preferably a composition in physiologically tolerable form, comprising one or more substantially saturated phospholipids in combination with a buffer system comprising ammonia or a water soluble amine having a pH at 15~C of less than or equal to 9.5, with the proviso that where said phospholipids comprise a combination of charged and neutral phospholipids and said composition is a liposomal composition containing a nonionic multiply hydroxylated X-ray contrast agent then said agent is not present within the liposomes and within the surrounding aqueous medium at substantially the same concentration.

The diagnostic compositions disclosed in WO-95/26205 thus are specifically disclaimed.

~ According to a further aspect of the present invention we provide a method for stabilising a substantially ~ saturated phospholipid composition, which method comprises including in a substantially saturated phospholipid composition a buffer system comprising CA 0223313~ 1998-03-26 ammonia or a water soluble amine having a pH at 15~C of less than or equal to 9.5, other than by adding a said buffer system to a liposomal composition containing a nonionic multiply hydroxylated X-ray contrast agent having said agent present within the liposomes and within the surrounding aqueous medium at substantially the same concentration.

For liposomal compositions, the bu~fer may be added before or after liposome generation.

Viewed from a further aspect the invention also provides a method o~ contrast enhanced imaging in which a contrast medium is administered to a subject (eg. a human or non-human animal, preferably a mAmmAl) and an image o~ the subject is generated, characterised in that as said contrast medium is used a composition according to the invention containing a contrast effective material. If desired, the contrast medium may be administered after activation o~ the contrast effective material, eg. by hyperpolarization.

Viewed from a still further aspect the invention provides a method o~ treatment in which a therapeutic or prophylactic agent is administered to a subject (eg. a human or non-human animal, preferably a mAmmAl), characterised in that there is administered a composition according to the invention containing a said therapeutic or prophylactic agent.

Viewed ~rom a yet ~urther aspect the invention provides a method of cosmetic treatment in which a cosmetic agent is administered to a subject (eg. a human or non-human animal, preferably a mAmm~l) characterised in that there is administered a composition according to the invention containing a said cosmetic agent.

CA 0223313~ 1998-03-26 In these methods, the compositions administered should contain an effective amount of the active agent (the contrast effective material, the therapeutic or prophylactic agent or the cosmetic agent), namely an amount sufficient to achieve contrast enhancement or to ~' achieve the desired therapeutic, prophylactic or cosmetic effect.

The phospholipids used in the compositions and methods of the invention may be charged or neutral (ie. carry no net charge). The use of neutral phospholipids however is particularly preferred as their protection against hydrolysis by the buffer system is particularly pronounced. Especially preferably the phospholipids in the compositions of the invention are entirely or substantially entirely neutral phospholipids.

The buffer systems for use in the methods or compositions of the present invention preferably have a pH of 6.0 to 9.5 at room temperature (15DC), more preferably 6.5 to 8.0, particularly preferably 6.8 to 7.8.

The compositions of the present invention show a reduced degree of hydrolysis of the phospholipid(s) when compared with formulations not including the speci~ied buffer system. Preferred compositions according to the present invention show a greater than 5% reduction in the extent of hydrolysis over a given time (eg. a normal shel~ life, for example 30 days or more) than occurs with formulations not including the buffer; more preferred compositions show a greater than 10~ and most ~ preferably greater than 25% reduction.

- The stabilisation achieved is of especial advantage during storage, during processing and during exposure of the phospholipid compositions to temperature, including CA 0223313~ 1998-03-26 during autoclaving.

In a pre~erred embodiment o~ the present invention the phospholipid compositions are stable at temperatures in the range ~rom 4 to 30~C; in a more pre~erred embodiment the compositions are stable ~or temperatures in the range ~rom 4 to 50~C; in another more preferred embodiment the compositions are stable for temperatures in the range o~ 4 to 125~C (which includes autoclaving) The phospholipid compositions are pre~erably stable under storage for a period of up to 2 years, more pre~erably up to 3 years, particularly pre~erably up to 5 years. "Stable" in this context means that at least 75%, pre~erably at least 80%, more pre~erably at least 90%, o~ undegraded phospholipid is present in the composition a~ter the speci~ied storage period.
-As indicated above, one particular advantage o~ thestabilization method of the invention is that the resulting phospholipid compositions have the ability to withstand a wide temperature range ~or a short period.
It is pre~erred, there~ore, that ~or stabilising phospholipid compositions to be autoclaved the bu~er system is added prior to autoclaving.

Bu~ers which may be employed in the methods or compositions o~ the present invention are pre~erably those o~ formula (I) NRlR2R3 (I) wherein Rl, R2 and R3, which may be the same or di~erent, each represents a hydrogen atom, a sugar residue, an alkyl group with 1 to 6 carbon atoms (which may carry one or more hydroxy, mercapto, carboxyl, sulphonic acid, carboxamido, imidazolyl, indolyl or hydroxy substituted phenyl groups), an alkylthio group with 1 to 6 carbon atoms and/or a group o~ the i~ormula -CA 0223313~ 1998-03-26 NR4R5 (in which R4 and R~, which may be the same or dif~erent, each represents a hydrogen atom, a carboxamido or -C(=NH)NH2 ~group or an alkyl group with 1 ~ to 6 carbon atoms); or any two of R1,R2 and R3 may, together with the intervening nitrogen atom, represent a pyrrolidine, morpholine or piperidine ring which may carry hydroxy, carboxyl, sulphonic acid or carboxamido groups.

Thus, for example, water soluble amines which may be employed as buffers include amino alcohols and amino sugars. More preferred ~m; ne~ include trometamol (tris(hydroxymethyl)methylamine, also denoted TRIS), N,N-bis(2-hydroxyethyl)-tris(hydroxymethyl)methylamine (denoted BIS-TRIS), 2-amino-2-methylpropane-1,3-diol (denoted AMPD), TES, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (denoted HEPES), diethanolamine, meglumine, triethanolamine and ammonia.

Especially preferred amines for use according to the invention are TRIS, BIS-TRIS, TES and meglumine in view of their advantageous physiological acceptability and/or advantageous pH values at room temperature.

The phospholipids for inclusion in the compositions of the present invention are; as indicated above, comprised of substantially saturated phospholipids. The term "substantially saturated" means that the fatty acid residues of the phospholipids are fully saturated (i.e contain no C-C double bonds) or that the extent of their unsaturation is very low,-e.g. as shown by an iodine value o~ no more than 10, pre~erably no more than 5. A
small proportion o~ unsaturated phospholipids giving an analogous overall extent~of unsaturation may also be - present in the compositions of the present invention.
The phospholipids may be ch~ged or neutral and may be of natural, synthetic or semi-synthetic origin CA 0223313~ 1998-03-26 (including chemically modified substantially saturated phospholipids). As mentioned above the use of neutral phospholipids is preferred.

The number of carbon atoms in the fatty acid residues is usually at least 14, pre~erably at least 16. The number oi~ carbon atoms in the fatty acid residue i8 also preferably 26 or less, eg. 25 or less, pre~erably 24 or less.

Neutral phospholipids use~ul in the present invention include, for example, neutral glycerophospholipids, for example a ~ully hydrogenated naturally occurring (e.g.
soybean- or egg yolk-derived) or synthetic phosphatidylcholine, particularly semisynthetic dipalmitoyl phosphatidylcholine (DPPC) or distearoyl phosphatidylcholine (DSPC), phosphatidylethanolamine (PE) or phosphatidylethanolamine-polyethyleneglycol (PE-PEG). More than one neutral phospholipid may be used.

Charged phospholipids useful in the present invention include, ~or example, positively or negatively charged glycerophospholipids. Negatively charged phospholipids include, ~or example, phosphatidylserine, ~or example a fully hydrogenated naturally occurring (e.g. soybean- or egg yolk-derived) or semi-synthetic phosphatidylserine, particularly semi-synthetic dipalmitoyl phosphatidylserine (DPPS) or distearoyl phosphatidylserine (DSPS); phosphatidylglycerol (PG), for example fully hydrogenated naturally occurring (e.g.
soybean- or egg yolk-derived) or semi-synthetic phosphatidylglycerol, particularly semi-~ynthetic or synthetic dipalmitoyl phosphatidylglycerol (DPPG); or distearoyl phosphatidylglycerol (DSPG);
phosphatidylinositol, for example a ~ully hydrogenated naturally occurring (e.g. soybean- or egg yolk-derived) or semi-synthetic phosphatidylinositol, particularly g semi-synthetic or synthetic dipalmitoyl phosphatidylinositol (DPPI) or distearoyl phosphatidylinositol (DSPI); phosphatidic acid, ~or example a ~ully hydrogenated naturally occurring (e.g.
soybean- or egg yolk-derived) or semi-synthetic phosphatidic acid, particularly semi-synthetic or synthetic dipalmitoyl phosphatidic acid (DPPA) or distearoyl phosphatidic acid (DSPA). Positively charged phospholipids include, for example, an ester o~
phosphatidic acid with an aminoalcohol, such as an ester o~ dipalmitoyl phosphatidic acid or distearoyl phosphatidic acid with hydroxyethylenediamine. Although such charged phospholipids are commonly used alone, more than one charged phospholipid may be used.

The concentration o~ the bu~er ~or use in the method o~
the present invention or in the compositions o~ the pre~ent invention is pre~erably in the range 2 mM to 200 mM, more preferably 2 mM to 100 mM and particularly pre~erably 2 mM to 20 mM.

The molar ratio o~ bu~er:lipid in the compositions of the present invention is pre~erably in the range 1:60 to 2000:1 (eg. 1:60 to 100:1), more pre~erably 1:60 to 1:0.02, particularly pre~erably 1:60 to 1:0.1. Another pre~erred range o~ molar ratio o~ bu~er:lipid ~or some diagnostic and other medical applications is 1:50 to 1:0.1, more pre~erably 1:20 to 1:0.5, particularly preferably 1:5 to 1:1.

The concentration o~ phospholipids in the compositions of the present invention ~or imaging and medical ~ applications is pre~erably in the range o.01 mM to 120 mM, eg. 1 mM to 120 mM.
-The phospholipid compositions o~ the present inventionmay be in any o~ the ~ormulation types generally CA 0223313~ 1998-03-26 encountered, for example liposomes, emulsions, micelles, microemulsions, lipid particles, lipid solutions and microbubbles. They can be produced by conventional procedures for each particular formulation type.

The method and the phospholipid compositions of the present invention are suitable for use in a variety of applications and in particular those where increased stability is of especial importance. The stabilised compositions can be used in a variety of diagnostic, therapeutic and cosmetic applications and particular mention can be made of phospholipid compositions for use with contrast media (X-ray, MRI, US and scintigraphy), and for use in cancer therapy, chemotherapy, therapy for fungal infections and treatment of psoriasis.

For use as diagnostic imaging contrast media, the liposomal compositions of the invention will include a contrast-effective material, eg. in the inner cavity of the liposomes, attached to the inner or outer wall of the liposome membrane or contained within the membrane, or in the liquid medium in which the liposomes are dispersed. By contrast effective it is meant that the material is capable of enhancing contrast in the imaging modality of interest. For conventional imaging modalities, eg. X-ray, MR, ultrasound, magnetotomography, electrical impedance tomography, scintigraphy, SPECT, PET, etc. the nature of appropriate contrast effective materials is well known, for example gases (eg. air, xenon, fluorinated compounds, etc.), radiation emitters, paramagnetic, superparamagnetic, ferrimagnetic and ferromagnetic materials (eg.
paramagnetic chelates o~ transition metals or lanthanides), heavy atom (eg. atomic number 47 or higher) compounds, eg. iodinated compounds (eg.
triiodophenyl compounds). Where the contrast effective material is gaseous (at ambient or body temperature), CA 0223313~ 1998-03-26 eg. air, xenon, helium, argon, hydrogen, nitrous oxide, oxygen, nitrogen, carbon dioxide, sulphur hexa~luoride, methane, acetylene, ~luorinated low molecular weight (eg. C1 to C7 ) hydrocarbons (pre~erably per~luorocarbons such as C2F6, C3F8, C4F1o and C5F12), 19F-containing gases, etc. it is conveniently contained within the liposome membrane. Where the contrast ef~ective material is water soluble (eg. a soluble triiodophenyl compound or a paramagnetic metal chelate) it is pre~erably in solution in the liposome core and especially pre~erably also in solution in the suspension medium.

Therapeutic or cosmetic agents may be similarly dispersed within the liposomal core, in or on the liposome membrane and/or in the suspension medium.
Conventional therapeutic or cosmetic agents capable o~
liposomal delivery may be used.
-For diagnostic compositions, e.g ~or X-ray and MRI/nuclear medicine, the concentration o~ total lipid is generally 5 mg/ml to 100 mg/ml (eg. 20 to 100 mg/ml, conveniently at least 40 or 50 mg/ml), pre~erably 10 mg/ml to 90 mg/ml, and more pre~erably 10 mg/ml to 80 mg/ml, in order to enhance encapsulation o~ contrast agent in the lipid. However, ~or ultrasound diagnostic compositions a pre~e~red range ~or the concentration o~
total lipid is generally 0.01 mg/ml to 20 mg/ml, pre~erably 0.01 mg/ml to 10 mg/ml (eg. 0.5 mg/ml to 10 mg/ml).

Where agents are encapsulated in the phospholipid (particularly in liposomes) this is pre~erably in the ~orm o~ an isotonic solution or suspension (relative to physiological osmotic pressure in the body). To obtain an isotonic solution or suspension, the agent is generally dissolved or suspended in a medium at a concentration which provides an isotonic solution. In CA 0223313~ 1998-03-26 the case where the agent alone cannot provide an isotonic solution because, ~or example, the solubility o~ the agent is insu~icient, other conventional tonicity adjusters (e.g non-toxic water soluble substances) may be added to the medium so that an isotonic solution is formed. Examples o~ such substances include: salts such as sodium chloride;
sugars such as mannitol, glucose, sucrose, mannose, galactose, sorbitol or the like; and polyhydric alcohols such as propylene glycol, glycerine and the like. I~
sorbitol is employed this is pre~erably at a concentration o~ 1 to 500 g/l, more pre~erably 0.1 to 20 g/lOOml. I~ glycerine is employed this is preferably at a concentration o~ 0.05 to 10 g/100 ml. I~ the phospholipid compositions are liposomal compositions, the amount of salts used is pre~erably as small as possible to facilitate stability of the liposomes during storage and autoclaving.

Isotonic solutions provided by means o~ the substances mentioned above are also pre~erably included in those phospholipid compositions according to the present invention which do not incorporate diagnostic, therapeutic or cosmetic agents.

The present phospholipid compositions may also contain various optional components in addition to the above-mentioned components. For example, vitamin E (~-tocopherol) and/or vitamin E acetate ester as an antioxidant may be added in an amount o~ 0.01 to 2 molar %, pre~erably 0.1 to 1 molar % relative to total amount o~ lipids.

- Diagnostic, therapeutic and cosmetic agents re~erred to above may be incorporated into the phospholipid compositions o~ the present invention by techniques well known in the art.

CA 02233l3~ l998-03-26 As indicated above, the prior art describes the inhibition of phospholipid hydrolysis to be by an indirect mechanism involving inhibition of a phospholipasei such a mechanism clearly does not apply in the present invention since the compositions concerned do not contain phospholipase. Similarly, the reduced oxidation/hydrolysis observed in the prior art using unsaturated phospholipids cannot be important in the method of the present invention involving saturated phospholipids (not withstanding that traces of unsaturated phospholipids can be present). The method of the present invention appears to demonstrate a dif~erent inhibition mechanism involving general inhibition of the acid/base catalysed hydrolysis of phospholipid esters.

The precise mechanism involved in the method of the present invention is not ~ully understood, particularly in terms of the improved stability on storage. However, it is well known (see for example Journal of Pharmaceutical Sciences 82: 362-366 (1993) and Phospholipid Handbook (Marcel Dekker Inc., 1993), pages 323-324) that phospholipids can be hydrolysed to form free fatty acids and lysophospholipids, which can be further hydrolysed to the corresponding glycerophospho compounds and free fatty acids; the final hydrolysis step gives glycerophosphoric acid by hydrolysis of the phosphate head group. Hydrolysis of the ester bond between glycerol and phosphoric acid seems to be di~ficult since no free phosphoric acid and glycerol is detected. Evidently the use of a buffer such as trometamol inhibits the acid/base catalysed hydrolysis o~ the fatty ester groups, as evidenced by the reduced liberation of free fatty acids; the final hydrolysis ~ step may also be inhibited- However, other hydrolysis mechanisms may also be involved.

The ~ollowing non-limiting Examples serve to ~urther illustrate the methods and compositions of the present invention.

CA 0223313~ 1998-03-26 Example 1 Composition: 1 ml containing:

Hydrogenated egg phosphatidylcholine 32 mg 1,2-Dipalmitoyl-sn-glycerO-3-phosphoethanolamine 4 mg 1,2-Dipalmitoyl-sn-glycero-3-phosphatidic acid sodium 4 mg Sorbitol 50 mg (Trometamol 1 mg) Water ~or injection ad 1 ml The composition was prepared by mixing the lipids with a mixture o~ chloro~orm, methanol and water (volume ratio 80:20:0.05). The mixture was heated on a water bath (at 50~C) to dissolve the lipids and the solvents were then removed by heating the solution in a rotary evaporator (at 50~C). Liposomes were then prepared by homogenisation and extrusion using standard techniques (including the addition o~ sorbitol). The dispersion was then split into two parts and a buf~er o~
trometamol/HCl having a pH of 7.4 was added to one o~
the two parts. The resulting composition was then filled into vials and autoclaved. Samples were stored at each of~ 30~, 40~ and 50~C for one month. The content o~ ~ree ~atty acids was measured before autoclaving, after autoclaving and after storage.

The presence o~ ~ree ~atty acids (FFA) in the phospholipid compositions was examined by elution on a thin layer chromatographic (TLC) plate coated with an O.25 mm thick layer oi~ silica gel 60 using a mixture of methanol, chloroform and ammonia (120:70: 8 by volume) as - the mobile phase. The sample was diluted 1:10 with methanol:dichloromethane (2:1 by volume) and an aliquot of the diluted sample was applied to the chromatographic plate. The amount of FFA was semi-quantified by CA 0223313~ 1998-03-26 comparison o~ the intensity of the spot o~tained from the sample and spots from palmitic acid standards corresponding to 1.25 mg/ml to 25 mg/ml concentrations.
The spots were developed with cupric sulphate spray reagent for about one hour at 170~C.

Degradation of phospholipids, measured as free fatty acids (mg/ml):

Wlthout trometamol With trometamol 1 month at 30~C s 5.0 s 2.5 1 month at 40~C 5 12.5 s 5.0 E~ple 2 Composition: 1 ml containing:

Hydrogenated egg phosphatidylcholine 36 mg 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol sodium 4 mg Sorbitol 50 mg (Trometamol 1 mg) Water for in~ection ad 1 ml Prepared as in Example 1.

Degradation of phospholipids, measured as free fatty acids (mg/ml):

Without trometamol With trometamol 1 month at 30~C ~ 1.25 s 1.25 1 month at 40~C s 2.5 s 1.25 1 month at 50~C s 12.5 5 2.5 The res~lts demonstrate that the samples containing trometamol show less degradation of phospholipids, CA 0223313~ 1998-03-26 measured as ~ree ~atty acids, compared to the samples without trometamol.

Further Examples o~ compositions according to the present invention are prepared as in Example 1 as ~ollows:

~xample 3 Phosphatidylcholine 20 mg Phosphatidylethanolamine 20 mg Glucose 50 mg Trometamol 0.5 mg Water ~or injection ad 1 ml Example 4 Phosphatidylglycerol 5mg Sucrose lOOmg Trometamol lmg Water ~o~ injection ad 1 ml Example 5 PEG-phosphatidylethanolamine 40mg Sorbitol 50mg Trometamol lOmg Water for injection ad 1 ml Example 6 Phosphatidylcholine 15mg Soya oleum 50mg Glycerol 24mg Trometamol lmg Water ~or injection ~ 1 ml CA 0223313~ 1998-03-26 ~xample 7 As a further Example demonstrating the method of the present invention the following composition (as disclosed in W0-95/26205) was also tested as in Example 1.

Composition: 1 ml containing:

Hydrogenated egg phosphatidylcholine (H-EPC) 51mg Hydrogenated egg phosphatidyl serine sodium (H-EPSNa) 5 mg Io~x~nol 400 mg Sorbitol 17 mg (Trometamol 1 mg) Water for injection ~ 1 ml The composition was prepared as in Example l but additionally adding an isotonic solution of iodixanol and sorbitol prior to the ~ormation of the liposome.

3egradation of phospholipids, measured as ~ree fatty acids (mg/ml):

Without trometamol With trometamol 1 month at 30~C ~ 2.5 s 1.25 1 month at 40~C s 5.0 ~ 2.5 1 month at 50~C s 25 0 ~ 12.5 The results o~ this Example demonstrate the use of the method of the present invention in stabilising a phospholipid composition additionally containing a contrast agent.

Examples 8 to 13 below'disclose the preparation of stabilized liposome suspensions suitable for use as CA 0223313~ 1998-03-26 contrast media in ultrasound (Examples 8 to 10) and magnetic resonance imaging (Examples 11 to 13) investigations. Ratios and percentages are by volume unless otherwise stated, except lipid ratios which are by weight. I~ l9F labelled fluorocarbons are used in Examples 8 to 10 these compositions could be used as MR
contrast media.

Example 8 Hydrogenated egg phosphatidylcholine (HEPC) and dipalmitoylphosphate (10:1) are dissolved in chloroform-methanol (2:1), and the solvent is then removed in a rotary evaporator. The lipids are then dispersed in purified water, and the dispersion is introduced in a gas tight glass reactor equipped with a high speed emulsifier. The gas in the reactor is air with 10%
C5Fl2 After preparation of the microbubbles, HEPES 5 mM
is added.

~xample 9 Distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidic acid (DPPA) and polyethyleneglycol (PEG 4000) in the ratio 25:1:2 are dissolved in tert-butanol, and the solvent is then removed in a rotary evaporator. The lipids are then dispersed in purified water, and the dispersion is introduced in a gas tight glass reactor equipped with a high speed emulsi~ier. The gas in the reactor is C3F8.
After preparation of the microbubbles, trometamol 8mM is added.

Exampl e 10 Dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidic acid (DPPA) and CA 0223313~ 1998-03-26 dipalmitoylphosphatidylethanolamine (DPPE) (8:1:1) are dissolved in chloro~orm-methanol-water (10:20:0.5), and the solvent is then removed in a rotary evaporator. The lipids are then dispersed in puri~ied water, and the dispersion is introduced in a gas tight glass reactor equipped with a high speed emulsi~ier. The gas in the reactor is C4Hlo. A~ter preparation o~ the microbubbles, TES 10 mM is added.

Example 11 Hydrogenated egg phosphatidylcholine (HEPC) and methoxy(PEG)-distearoylphosphatidylethanolamine (MPEG-DSPE) (9:1) are dry blended and dispersed in gadodiamide-caldiamide 0.5 M solution. Liposomes are then prepared by homogenisation and extrusion. HEPES 8 mM is added and the product is sterilised by ~ autoclaving.

Example 12 Hydrogenated egg phosphatidylcholine (HEPC) and dipalmitoylphosphatidylglycerol (DPPG) (9:1) are dry blended and dispersed in dimegluminegadopentetate-meglumine diethylenetriaminepentetate-meglumine 0.5 M
solution. Liposomes are then prepared by homogenisation and extrusion. TRIS 50 mM is added and the product is sterilised by autoclaving.

E~m~le 13 Hydrogenated egg phosphatidylcholine (HEPC) is dispersed in gadodiamide 0. 5 M solution. Liposomes are then prepared by homogenisation and extrusion. TES 50 mM is added and the product is sterilised by autoclaving.

Exam~le 14 Composition:

~ DSPA** lOmg DSPG++ lOmg Iohexol 390mg Bu~er q.s.
Water ~or injection ~ 1 mL

** distearoylphosphatidic acid f+ distearoylphosphatidyl glycerol (Both charged phospholipids) The compositions were prepared, as in Example 1, using TRIS, HEPES or TES bu~ers.

TES is 1- [tris(hydroxymethyl)methyl]-2-aminoethane sulphonic acid.

The concentrations o~ these bu~ers were chosen to give identical ionic strengths in the products. The pH and degradation data are ~et out in ~ull in Tables 1 and 2.

Table 1 Free fatty acids (mg/mL) Buffer On preparation On pre- After 1 After 3 Not autoclaved paration month 40~C months 40~C
Autoclaved Autoclaved Autoclaved TRIS 200 mM NMT 0.5 NMT 1.0 NMT 5.0 NMT 12.5 target pH 7.4 HEPES 168 mM NMT 1.0 NMT 1.0 NMT 5.0 NMT 12.5 target pH 7.4 TES 172 mM NMT 0.5 NMT 1.0 NMT 5.0 NMT 12.5 target pH 7.4 (NMT = not more than) CA 02233l35 l998-03-26 Table 2 pH
Bu~er On preparation On pre- A~ter l After 1 Not autoclaved paration month 40~C month 50~C
Autoclaved Autoclaved Autoclaved TRIS 200 mM 7.49 7.45 7.36 7.25 target pH 7.4 HEPES 168 mM 6.91 6.90 6.84 6.79 target pH 7.4 TES 172 mM 7.22 7.21 7.15 7.10 target pH 7.4 Example 15 Composition:

H-EPC* 6Omg Iodixanol 20Omg Sorbitol 37mg Bu~er q.s.
Water ~or injection ad 1 mL

* Hydrogenated egg phosphatidylcholine (a neutral phospholipid).

Three sets of compositions were prepared ~or comparison In one the bu~fer was TRIS, HEPES or TES, in a second no bu~er was used with pH being adjusted with NaOH/HCl, and in the third phosphate bu~fer or phosphate/citrate bu~er was used. The compositions were prepared as in Example 1.

Degradation o~ the phospholipid, measured as free ~atty acids (mg/mL) after autoclaving and a~ter 3 months storage at 40~C was as ~ollows:

CA 0223313~ 1998-03-26 Buffer After autoclaving 3 months, 40~C
TRIS 200 rrlM, pH 7.4 s 0.25 s 2.5 Phosphate buffer 75 mM
pH 7.4 s 1.0 s 5.0 (The concentrations of these buffers were chosen to give identical ionic strengths in the products).

The pH and degradation data are set out in full in Tables 3 and 4.

Table 3 Free fatty acids (mg/mL) Buffer On preparation On pre- 1 month 3 months Not autoclaved paration 40~C 40~C
~ Autoclaved Autoclaved Autoclaved TRIS 2 mM NMT 0.25 NMT 0.25 NMT 1.0 NMT 2.5 target pH 7.4 TRIS 8 mM NMT 0.25 NMT 0.25 NMT 1.O NMT 2.5 target pH 7.4 TRIS 50 mM NMT 0.25 NMT 0.25 NMT 0.5 NMT 2.5 target pH 7.4 TRIS 200 mM NMT 0.25 NMT 0.25 NMT 1.O NMT 2.5 target pH 7.4 TRIS 8 mM NMT 0.25 NMT 0.25 NMT 0.5 NMT 2.5 target pH 8.3 NaOH/HCl NMT 0.25 NMT 0.50 NMT 5.0 NMT 25 target pH (~ 12.5) 7.4 NaOH/HCl NMT 0.25 NMT 0.25 NMT 2.5 NMT 5 target pH 8.3 HEPES 7 mM NMT 0.25 NMT 0.25 NMT 1.O NMT 2.5 ~ target pH 7.4 TES 7 mM NMT 0.25 NMT 0.25 NMT 1.O NMT 5 target pH 7.4 Phosphate NMT 0.25 NMT 0.50 NMT 1.O NMT 5 buffer 3mM
target pH 7.4 CA 02233l3~ l998-03-26 Phosphate NMT 0.25 NMT 1.O NMT 5.O NMT 5 buffer 75 mM
target pH 7.4 Phosphate/ NMT 0.25 NMT 0.25 NMT 0.5 NMT 5 ,-citrate buffer 2.6 mM
target pH 7.4 Table 4 ~H
Buffer On preparation On pre- After 1 After 3 Not autoclaved paration month 40~C months 40~C
Autoclaved Autoclaved Autoclaved TRIS 2 mM 7.36 7.01 7.25 7.05 target pH 7.4 TRIS 8 ~M 7.34 7.30 7.28 7.22 target pH 7.4 TRIS 50 mM 7.44 7.43 7.44 7.41 target pH 7.4 TRIS 200 mM 7.48 7.50 7.50 7.50 target pH 7.4 TRIS 8 mM 8.14 8.07 8.08 7.99 target pH 8.3 NaOH/HC1 6.42 6.24 5.81 5.51 target pH 7.4 NaOH/HCl 7.18 7.19 6.04 6.85 target pH 8.3 HEPES 7 mM 6.99 6.89 6.83 6.80 target pH 7.4 TES 7 mM 7.28 7.21 7.20 7.11 target pH 7.4 Phosphate 7.46 7.75 6.71 6.64 buffer 3mM
target pH 7.4 Phosphate 7.35 7.17 7.07 6.99 buffer 75 mM
target pH 7.4 Phosphate/ 7.66 7.74 6.77 6.65 citrate buffer 2.6 mM
target pH 7.4 CA 0223313~ 1998-03-26 Thus a~ter 3 months storage, samples with TRIS and TRIS-like buffers (exemplified by HEPES and TES) show less degradation of the phospholipid as seen by the reduced ' level of free fatty acids compared with other buffers (phosphate buffer and phosphate/citrate buffer) and solutions without buffer (pH adjusted by NaOH/HCl).
Moreover, the reduction in pH observed after autoclaving and storage was less pronounced in the samples with TRIS, HEPES and TES (a reduction of less than 0.30 pH
units) compared with the other samples (a reduction of 0.30-1.00 pH units).

E~ le 16 Composition:

H-EPC 36mg MPEG-DSPE* 4mg Iodixanol 370mg Buffer q.s.
Water for injection ad 1 mL

* Methoxy(polyethyleneglycol)distearoylphosphatidyl-ethanolamine (a neutral phospholipid).

Three sets of compositions were prepared for comparison.
In one the buffer was TRIS, HEPES or TES, in a second no buffer was used with pH being adjusted with NaOH/HCl, and in the third phosphate buffer or phosphate/citrate buffer was used. The compositions were prepared as in Example 1.

Degradation of the phospholipid, measured as free fatty ~ acids (mg/mL) after autoclaving and after 1 month's storage at 40~C and 50~C was as follows:

CA 02233l35 l998-03-26 Buffer After autoclaving 1 month 40nc 1 month 50~C
T~S 172 mM, pH 7.4 ~ 0.25 - 0.5 - 1.0 Phosphate buffer 74 mM, pH 7.4 ~ 1.0 s 2.5 s 5.0 Phosphate/citrate 67 mM, pH 7.4 s 2.5 s 2.5 s 5.0 The concentrations o~ these bu~ers were chosen to give identical strengths in the products.

The pH and degradation data are set out in ~ull in Tables 5 and 6.

Table 5 Free ~atty acids (mg/mL) Buffer On preparation On pre- After 1 After 1 Not autoclaved paration month 40~C month 50'C
Autoclaved Autoclaved Autoclaved TRIS 200 mM NMT 0.5 NMT 0.5 -0.5 -1.0 target pH 7.4 NaOH/HCl NMT 0.5 NMT O.25 NMT 1.25 NMT 12.5 target pH 7.4 HEPES 168 mM NMT 0.25 NMT 0.25 ~0.5 NMT 2.5 target pH 7.4 TES 172 mM NMT 0.25 NMT 0.25 ~0.5 -1.0 target pH 7.4 Phosphate NMT 0.5 NMT 1.O NMT 2.5 NMT 5.0 buf~er 74 mM
target pH 7.4 Pho~phate/ NMT 0.25 NMT 2.5 NMT 2.5 NMT 5.0 ~ citrate buffer 67 mM
target pH 7.4 CA 0223313~ 1998-03-26 Table 6 pH
Buffer On preparation On pre- After 1 After 1 Not autoclaved paration month 40nC month 50~C
Autoclaved Autoclaved Autoclaved TRIS 200 mM 7.43 7.44 7.42 7.40 target pH 7.4 NaOH/HCl 6.52 5.84 5.54 4.98 target pH 7.4 HEPES 168 mM 6.91 6.89 6.89 6.91 target pH 7.4 TES 172 mM 7.18 7.16 7.18 7.19 target pH 7.4 Phosphate 7.37 7.20 7.23 7.22 buffer 74 mM
target pH 7.4 Phosphate/ 7.52 7.04 7.25 7.24 citrate buffer 67 mM
target pH 7.4

Claims (19)

Claims:

1. An aqueous lipid composition comprising one or more substantially saturated phospholipids in combination with a buffer system comprising ammonia or a water soluble amine having a pH at 15°C of less than or equal to 9.5, said composition having been autoclaved while containing said buffer system, with the proviso that where said phospholipids comprise a combination of charged and neutral phospholipids and said composition is a liposomal composition containing a nonionic multiply hydroxylated X-ray contrast agent then said agent is not present within the liposomes and within the surrounding aqueous medium at substantially the same concentration.

2. A composition as claimed in claim 1 containing liposomes.

3. A composition as claimed in either of claims 1 and 2 containing a contrast effective material.

4. A composition as claimed in claim 3 wherein said contrast effective material is an echogenic material.

5. A composition as claimed in claim 3 wherein said contrast effective material is a paramagnetic material.

6. A composition as claimed in claim 3 wherein said contrast effective material is a radiation emitting material.

7. A composition as claimed in claim 3 wherein said contrast effective material is an iodinated organic compound.

8. A composition as claimed in any one of claims 3 to 7 wherein said contrast effective material is present within liposomes in said composition.

9. A composition as claimed in either of claims 1 and 2 containing a therapeutic or prophylactic agent.

10. A composition as claimed in either of claims 1 and 2 containing a cosmetic agent.

11. A composition as claimed in any one of claims 1 to 10 wherein said buffer system comprises TRIS, BIS-TRIS, AMPD, HEPES or TES.

12. A composition as claimed in any one of claims 1 to 11 wherein said phospholipids are neutral phospholipids.

13. A method for stabilising a substantially saturated phospholipid composition, which method comprises including in a substantially saturated phospholipid composition a buffer system comprising ammonia or a water soluble amine having a pH at 15°C of less than or equal to 9.5 and autoclaving the buffer system containing composition, the inclusion of said buffer system being other than by adding a said buffer system to a liposomal composition containing a nonionic multiply hydroxylated X-ray contrast agent having said agent present within the liposomes and within the surrounding aqueous medium at substantially the same concentration.

14. A method as claimed in claim 13 wherein said buffer system is added to a pre-formed liposomal composition.

15. A method as claimed in claim 13 wherein said buffer system is admixed with a phospholipid composition and liposomes are generated in the resulting phospholipid and buffer containing mixture.

16. A method of contrast enhanced imaging in which a contrast medium is administered to a subject and an image of the subject is generated, characterised in that as said contrast medium is used a composition as claimed in any one of claims 3 to 9.

17. A method as claimed in claim 16 wherein image generation is effected by X-ray, ultrasound or MR
imaging.

18. A method of cosmetic treatment in which a cosmetic agent is administered to a subject, characterised in that there is administered a composition as claimed in claim 10.

19. A method of treatment in which a therapeutic or prophylactic agent is administered to a subject, characterised in that there is administered a composition as claimed in claim 9.