IE911350A1

IE911350A1 - Process for the production of aqueous liposome suspensions¹containing active ingredients

Info

Publication number: IE911350A1
Application number: IE135091A
Authority: IE
Original assignee: Schering Ag
Priority date: 1990-04-24
Filing date: 1991-04-23
Publication date: 1991-11-06
Also published as: FI103178B1; AU651969B2; HUT62457A; ZA913091B; EP0478727A1; EP0478727B1; FI103178B; DK0478727T3; NO915012L; CA2041075A1; FI916120A0; DE4013580A1; PT97468A; AU7593391A; ATE141503T1; WO1991016039A1; HU215960B; HU9200235D0; DE59108097D1; JPH04506814A

Abstract

Described is a process for the preparation of aqueous liposome suspensions containing active substances, the suspensions being prepared from a solution of a liposome-forming substance, or mixture of such substances, in a lower alcohol with a maximum of three carbon atoms and an aqueous phase, the active ingredients being dissolved or suspended in the lipid solution and/or the aqueous phase. The process is characterized in that the aqueous phase is incorporated, by mixing, in the alcoholic solution and the lower alcohol removed by vacuum distillation and, if necessary, the liposome suspension freeze-dried.

Description

Summary of the invention The invention relates to a process for the production 5 of aqueous liposome suspensions containing active ingredients from a solution of a substance or a mixture of substances (also designated below as lipid or lipid mixture), which forms liposomes in a lower alcohol with at most 3 carbon atoms and an aqueous phase, the active ingredient or the active ingredient mixture is dissolved or suspended in the lipid solution and/or the aqueous phase, characterized in that the aqueous phase is added to the alcoholic solution with homogenization, and the lower alcohol is removed by vacuum distillation.

As is known, liposomes are self-contained spherical or elliptical lipid vesicles, which enclose an aqueous phase. Depending on the number of lipid double layers present, large and small unilamellar liposomes (LUV and SUV) are distinguished from multilamellar liposomes (MLV).

The first liposomes were produced by dispersion of lipid films in aqueous phases. The MLV dispersions thus obtained can be converted to suv by using suitable homogenization processes, such as, e.g., ultrasonic irradiation or high-pressure homogenization. But MLV and SUV exhibit only a small inclusion volume (volume of i'll enclosed aqueous phase [liter/mol lipid]), so that they are not suitable for effective inclusion of hydrophilic substances.

In contrast, LUV exhibit a clearly increased inclusion 5 volume and thus an increased inclusion capacity (measured on the basis of the % of the total active ingredient that can possibly be contained in the inclusion volume).

The most common methods, up to this day, for production of LUV can be summarized under the generic term of solvent evaporation methods.

The method probably most known and best with respect to the inclusions obtained is the REV (reverse phase evaporation) method of Papahadjopoulos (U.S. patent no. 4,235,871). In this case, the lipid or lipid mixture is first dissolved in a solvent which is not, or only slightly, water-miscible (generally, diethyl ether or chloroform). After addition to the aqueous phase containing pharmaceutical substances, the mixture is then converted to a W/0 emulsion (water-in-oil emulsion) by ultrasonic irradiation. The solvent is then removed from the latter using a rotary evaporator, and a gel is first formed which is converted to a liposome suspension by increasing the vacuum or adding water.

In contrast to this, in the ether injection method introduced by Deamer and Banghara [Biochem. Biophys. Acta, 443 (1976) 629-634], the lipid, also dissolved in diethyl ether, is injected into a warm aqueous solution (50-60°C) under partial vacuum. The liposomes obtained after filtration through a 1.2 micron filter are heterogeneous and exhibit sizes between 150-250 nm.

The solvents used in these methods are to be viewed critically for toxicological and safety reasons. mrr\ —I’i ϋ.υχ .w'iui-l-h wniic iu . cl i iu · t 0+4 X kJ HkJf++ TkJ-4 - 3 But, if ethanol is used as a solvent in a process preceding the ether injection method, greatly diluted SUV dispersions result which are concentrated by ultrafiltration [Biochem. Biophys. Acta, 298 (1973) 1015-1019]. The average size of the liposomes thus obtained is clearly under 50 nm and the MLV portion is indicated as 6%.

An embodiment of this process is described in EP-A 0 253 619. In this process, for example, an ethanolic lipid and pharmaceutical agent solution, which constitutes a total of 10% of the total formulation, are injected under pressure of 1,000 to 3,000,000 h Pa (hecto Pascals) in an aqueous phase, which is agitated by a homogenizer.

According to this process, which is very expensive, small unilamellar liposomes result. This process is unsuitable for production of liposome suspensions which contain hydrophilic active ingredients.

Also worth mentioning is a method for the production of liposomes with water-soluble solvents, which includes the production of a monophase (WO 85/00751). This one-phase mixture is generally produced from 5 ml of the lipidcontaining solvent (e.g., ethanol) and 0.2 ml of the aqueous component. The solvent is then removed by introducing an inert gas during the simultaneous ultrasonic irradiation of the mixture, and a film results. The latter is then resuspended with an aqueous phase. In this case, so-called MPV (monophasic vesicles) result, which exhibit several lipid double layers. Relative to the standard MPV, the MPVs produced by the above-described process exhibit increased stability in buffers as well as increased inclusions.

Finally, the process described in EP-A 0 349 429, in which an ethanolic lipid solution optionally containing the active ingredient is introduced into an aqueous phase with - 4 light stirring, is also to be mentioned. As our tests show, considerably lower inclusion capacities are achieved in this previously known process than by the process according to the invention. Moreover, the process according to the invention has the advantage that with its help, even with lipid concentration of over 10% relative to the ethanolic phase, high inclusion capacities or even a further increase of the inclusion capacities can be achieved.

In comparison with these previously known methods, the 10 process according to the invention has the advantage that it is technically feasible on a large scale in a simple way.

The use of strongly toxic or very easily flammable solvents is avoided. The very good reproducibility of the process according to the invention is also to be emphasized relative to the obtained active ingredient inclusions and liposome sizes, as well as the very high active ingredient inclusion in the liposomes, up to over 50%, and the unusually high active ingredient concentrations, obtained by the process according to the invention, in the liposome suspensions (up to 800 mg/ml). Active ingredient inclusion means the percentage of the total active ingredient employed which is contained in the inclusion volume of the lipids. Further, it is worth mentioning that it is possible to produce liposome suspensions, which have only a very low residual solvent content and which have a good shelf life, with the help of the process according to the invention. The process according to the invention can also be performed problemfree under aseptic conditions.

To perform the process according to the invention, the same substances for forming liposomes can be used as in the previously known processes.

Substances forming suitable liposomes are usually amphiphatic lipids or lipid mixtures, such as, for example, - 5 phospholipids, sphingomyelins or ether phospholipids. These lipids can have straight-chain or branched, saturated or unsaturated, similar or different acyl side chains.

Suitable phospholipids are, for example, the phosphatidylcholines, the phosphatidylethanolamines, the phosphatidylserines, the phosphatidylinositols, the phosphatidylglycerols or the phosphatidic acids. Suitable ether phospholipids are, for example, the plasmalogens (Dr. Otto-Albert Neumueller: Roempps Chemie-Lexikon; Franck‘sche Verlagshandlung, Stuttgart (DE) 2665, 3159, 3920 and 4045).

For the production of liposomes, lipids or mixtures thereof and in particular also mixtures of these lipids with cholesterol, cholesterol hemisuccinate, alpha-tocopherol, alpha-tocopherol hemisuccinate and/or charge carriers, such as, for example, stearyl amine, stearic acid, diethyl phosphate, oleic acid, palmitic acid, bile acid, such as, for example, cholic acid, glycocholic acid, can be used. Suitable mixtures can contain about up to 60 mole percent of cholesterol and up to 20 mole percent of charge carrier. As solvent for the phospholipids or mixtures, preferably isopropanol or in particular ethanol is used.

In principle, the process according to the invention should also be feasible with water-soluble low-boiling solvents other than lower alcohols; for example, tetrahydrofuran is suitable. But, performance of this process embodiment is considerably more expensive.

To perform the process according to the invention, alcoholic solutions are preferably used which contain 0.2 g 0 to 30 g of liposome-forming substance or substance mixture per 100 ml of solvent. If necessary, these solutions are produced by heating the components. i υ · - 6 The process according to the invention is performed, as was already mentioned, in such a way that the aqueous phase is added to the alcoholic solution with mixing (such as, for example, vigorous stirring) and the lower alcohol is removed, for example, by vacuum distillation or by introducing nitrogen.

Optionally, the dispersion obtained after combining the aqueous phase and the alcoholic solution is mixed for some time (about 10 to 180 minutes) at a temperature of about 20 to 90°C, before the solvent is completely or partially separated.

The temperatures suitable for the process according to the invention are dependent on the solubility of the liposome-forming substances as well as their phase transition temperature (tc), the heat stability of the active ingredient or active ingredient mixture and the vapor pressure of the alcohol to be separated. The temperature is preferably between 20°C and 90°c and in particular between 40°C and 70°C. in general, it will be sufficient to operate with a vacuum of 10 h Pa to 100 h Pa.

The aqueous phase used for the process according to the invention can optionally contain buffer substances and/or isotonizing additives. Suitable additives are, for example, inorganic or organic salts or buffer substances, such as sodium chloride, Tris buffer, phosphate buffer, citrate buffer, glycine buffer, citrate-phosphate buffer, maleate buffer, etc. Mono- or di-saccharides, such as glucose, lactose, saccharose or trehalose, sugar alcohols, such as mannitol, sorbitol, xylitol or glycerine or water-soluble polymers, such as dextran or polyethlyene glycol.

Since the lipids and also several active ingredients are sensitive to oxidation, the aqueous phase can be mixed - 7 with antioxidants, such as sodium ascorbate, tocopherol or sodium bisulfite.

The aqueous liposome suspensions produced according to the process of the invention are used preferably for encapsulating water-soluble active ingredients.

Such water-soluble active ingredients are, for example, diagnostic agents, such as the X-ray contrast media iotrolan, iopromide, iohexol, iosimide, metrizamide, salts of amidoacetic acid, iotroxic acid, iopamidol, -hydroxyacetamido-2,4,6-triiodo-isophthalic acid-(2,3dihydroxy-N-methylpropyl)-(2-hydroxyethyl)-diamide (-ZK 119095) and 3-carbamoyl-5-[N-(2-hydroxyethyl)-acetamido]2,4,6-triiodo-benzoic acid-[(IRS,2SR)-2,3-dihydroxy-lhydroxymethylpropyl]-amide (-ZK 139129) or NMR contrast media, such as gadolinium DTPA, gadolinium DOTA and the gadolinium complex of 10-[l-hydroxymethyl-2,3dihydroxypropyl]-1,4,7-tris-[(carboxymethyl)-1,4,7,10tetraazacydodecane].

Suitable therapeutic active ingredients are, among others, antibiotic agents, such as gentamycin or kanamycin, cytostatic agents, such as doxorubicin hydrochloride or cyclophosphamide and virustatic agents, such as vidarabine or active ingredients, such as mitoxantrone hydrochloride.

These water-soluble active ingredients are dissolved in the aqueous phase before performance of the process according to the invention.

Further, the aqueous liposome suspensions can also be used to encapsulate slightly soluble active ingredients in water.

Such active ingredients are, for example, plant protecting agents, such as slightly soluble insecticides or herbicides and in particular slightly soluble pharmaceutical active ingredients. - 8 Slightly water-soluble or insoluble pharmaceutical active ingredients of the following active ingredient groups are suitable, for example, for production of aqueous suspensions according to the process of the invention.

Gestagenically active steroid hormones, such as, for example, 13-ethyl-17beta-hydroxy-l8,19-dinor-l7alpha-pregn4-en-20-yl-3-one (-levonorgestrel), 13-ethyl-17beta-hydroxy18,19-dinor-17alpha-pregna-4,15-dien-20-yn-3-one (-gestoden) or l3-ethyl-17beta-hydroxy-ll-methylene-l8,19-dinor-17alpha10 pregn-4-en-20-yn (=desogestrel).

Estrogenally active steroid hormones, such as 3hydroxy-l,3,5-(10)-estratrien-17-one (-estrone) or l,9-nor17alpha-pregna-l,3,5(10)-trien-20-yn-3,17beta-diol (ethinylestradiol).

Androgenically active steroid hormones, such as, for example, 17beta-hydroxy-4-androsten-3-one (-testosterone) and its ester or 17beta-hydroxy-lalpha-methyl-5alphaandrosten-3-one (=iuesterolone).

Antiandrogenically active steroid hormones, such as, for example, 17alpha-acetoxy-6-chloro-lbeta,2beta-dihydro3H-cyclopropa(1,2)-pregna-l,4,6-triene-3,20-dione (cypoteronacetate).

Corticoids, such as, for example, llbeta,17alpha,21trihydroxy-4-pregnene-3,20-dione (-hydrocortisone), llbeta,17alpha,21-trihydroxy-l,4-pregnadiene-3,20-dione (-prednisolone) , llbeta,17alpha,21-trihydroxy-6alpha-methyl1,4-pregnatriene-3,20-dione (-methylprednisolone) and 6alpha-fluoro-llbeta,21-dihydroxy-16alpha-methyl-l,4pregnadiene-3,20-dione (-diflucortolone) and their esters.

Ergolines, such as, for example, 3-(9,10-dihydro-6methyl-8alpha-ergolinyl)-l,l-diethylurea (-ergoline), 3-(2bromo-9,10-dihydro-6-methyl-8alpha-ergolinyl)-1,1IE 911350 - 9 diethylurea (-bromergoline) or 3-(6-methyl-8alphaergolinyl)-1,1-diethylurea (=terguride).

Antihypertensive agents, such as, for example, 7alphaacetylthio-17alpha-hydroxy-3-oxo-4-pregnene-21-carboxylic acid-gamma-lactone (=spironolactone) or 7alpha-acetylthio15beta,16beta-methylene-3-oxo-17alpha-pregna-l,4-diene21,17-carbolactone (=raespirenone).

Anticoagulants, such as, for example, 5-[hexahydro-5hydroxy-4-(3-hydroxy-4-methyl-l-octen-6-ynyl)-2(1H)10 pentalenylidene)]-pentanoic acid (=iloprost).

Psychopharmacological agents, such as, for example, 4-(3-eye1openty1oxy-4-methoxy-pheny1-2-pyrrο1idone (=rolipram) and 7-chloro-l,3-dihydro-l-methyl-5-phenyl-2H1,4-benzodiazepin-2-one (=diazepara). carotinoids, such as, for example, alpha-carotin and beta-carotin.

Fat-soluble vitamins, such as, for example, vitamins of the vitamin A, vitamin D, vitamin E and vitamin K group.

Beta-carbolines, as they are described, for example, in 20 European patent applications 234,173 and 239,667, are another group. As beta-carbolines, for example, there can be mentioned 6-benzoyloxy-4-methoxymethyl-beta-carboline-3carboxylic acid-isopropyl ester (=becarail) and 5-(4chlorophenoxy)-4-methoxymethy1-beta-carboline-3-carboxylic acid-isopropyl ester (=C1-PHOCIP).

Slightly soluble or insoluble contrast media are also worth mentioning, such as the X-ray contrast medium iodipamide ethyl ester or NMR contrast media, such as the iron or manganese porphyrin chelates or also the magnetites.

As suitable active ingredients, salicylic acid, retinoic acid and azalaic acid further can be mentioned. Antimycotins, such as the amphotericin B, iconacol or meconazol can also be used. - 10 Before performance of the process according to the invention, these active ingredients are dissolved or suspended in the alcoholic solution or in the aqueous phase.

In the process according to the invention, generally 5 0.05 to 10 g and preferably 1 to 3 g of active ingredient is used per g of substance (substance mixture) for forming liposomes. Sterile liposome suspensions can be produced in a simple way by the process according to the invention by both solutions being sterilized by filtration before performance of the process and all subsequent process steps being performed under aseptic conditions.

The inclusions which are achievable in the liposome suspensions are dependent, of course, on the type of active ingredient and the substance (substance mixture) for forming liposomes as well as their ratio to one another.

Optionally, the unencapsulated active ingredient can be removed, for example, by ultrafiltration, dialysis, microfiltration or centrifuging.

The liposome suspension obtained can be directly stored. Alternatively, the liposome suspension can be converted by freeze-drying to a stable form of storage. Before the freeze-drying, if necessary, among others, for example, additives, such as mannitol or sorbitol and/or electrolytes and viscosity-influencing agents such as sodium chloride can be added to the materials to be freeze-dried.

The lyophilizates can be resuspended with bidistilled water or aqueous phases, which can contain the same additives as the aqueous phases used for liposome production. 0 The amount of resuspension medium can in this case be 0.5 to 20, but preferably 1 to 6 ml per g of lyophilizate. The suspensions obtained can be used directly or after filtration through filters of suitable pore size.

The liposomes produced by the process according to the invention can, for example, be used if they contain contrast media for NMR or X-ray diagnosis in various diagnostic processes. These include, for example, the diagnosis of tumors in organs of the reticulo-endothelial system (for example, liver and spleen) or other intravascularly accessible tissues as well as the arthrography. The extravascular administration of such liposome suspensions (such as, for example, subcutaneous, intramuscular or intraperitoneal) can also be used for diagnostic purposes, such as, for example, the indirect and direct lymphography, or for therapeutic purposes (such as intramuscular depot preparations) . Of course, it is also possible to administer the preparations orally, and the preparations optionally can be filled in coated capsules resistant to gastric juices.

Further, the liposome suspensions containing active ingredients produced by the process according to the invention can be applied in the case of suitable lipid composition as blood pool agents or as target-directed pharmaceutical vehicles.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and unless otherwise indicated, all parts and percentages are by weight.

The entire disclosures of all applications, patents and publications, cited above and below, and of corresponding - 12 application Federal Republic of Germany P 40 13 580.2, filed April 24, 1990, are hereby incorporated by reference.

In the following examples, the following abbreviations are used: PC - Phosphatidylcholine S 100 of the Lipoid KG CH = company Powdered cholesterol (JP) of the Merck AG SA = company Very pure stearic acid of the Fluka AG 10 company DPPG = Dipalmitoylphosphatidylglycerol DPPG of the HSPC - Lipoid KG company Hydrogenated soyabean lecithin (Phospholipon 15 DCP = 9OH) of the Nattermann AG company Dicetyl phosphate of the Sigma company - 13 EXAMPLES Example l 9.25 g of lipid mixture (PC/CH/SA — 4:5:1 mol:mol:mol) is dissolved in 100 ml of ethanol and sterilized by filtration at 70°C. Then, the solution is transferred to a reaction vessel heated moderately to 55°C and it is mixed with stirring (300 rpm) with an active ingredient solution sterilized by filtration (containing 27.75 g of iopromide and 200 ml of aqueous 20 mmol tris-HCl buffer: pH 7.5).

Then, the alcohol is distilled off at 55°C in a vacuum of 5,000 Pa and the resulting liposome suspension is examined for its properties.

Then, the liposome suspension is bottled directly in portions of 20 g in 50 ml glass infusion bottles and it is freeze-dried. The lyophilizates obtained are resuspended so that an active ingredient concentration of about 200 mg/ml results and also examined.

Example 2 Performance of the test takes place as described in example 1, except that 18.5 g of iopromide is used.

Performance of the test takes place as described in example 2, except that 13.9 g of lipid mixture is used.

Example_4 Performance of the test takes place as described in example 2, except that 18.5 g of lipid mixture is used.

Example 5 Performance of the test took place as described in example 2, except that iotrolan is used instead of iopromide.

BKQpH.g Performance of the test takes place as described in example 2, except that iopamidol is used instead of iopromide.

Bacample 7 Performance of the test takes place as described in example 2, except that iohexal is used instead of iopromide Example 8 Performance of the test takes place as described in example 2, except that the nonionic contrast medium -hydroxyacetamide-2,4,6-triiodo-isophthalic acid-(2,3dihydroxy-N-methylpropyl)-(2-hydroxyethyl)-diamide is used instead of iopromide.

Example 9 Performance of the test took place as described in example 2, except that a lipid mixture of PC/CH/SA — 4:4:2 mol:mol:mol is used.

Example 10 Performance of the test takes place as described in example 2, except that a lipid mixture of PC/CH/PG of 4:5:1 mol:mol:mol is used.

,E 911350 Example 11 Performance of the test takes place as described in example 2, except that a lipid mixture of PC/CH/SA of 6:3:1 mol:mol:mol is used.

Example 1¾ Performance of the test takes place as described in example 2, except that a lipid mixture of PC/CH/DCP of 4:5:1 is used.

Example 13 Performance of the test takes place as described in example 2, except that a lipid mixture of PC/CH 1:1 is used.

Example 14 Performance of the test takes place as described in example 2, except that a lipid mixture of PC/HSPC/CH/SA of 2:2:5:1 is used.

Example 15 Performance of the test takes place as described in example 2, except that the batch size was increased tenfold.

Exaaais_n Performance of the test takes place as described in example 2, except that a one hundred and fiftyfold larger batch is used.

The following table shows the results reached in examples 1 to 16. Η cu Ο* (Φ - 17 Example 17 The residual ethanol content of the liposome suspensions, produced according to example 2, before and after freeze-drying is determined on 8 batches by gas chromatography. The ethanol contents are determined in the original liposome suspension with 0.59 ± 0.010% (m/V).

ExafflPlfiL 19 Liposome suspensions and lyophilizates produced according to example 2 are stored at 4, 25 and 40°C. To judge the stability, appearance, size of inclusion and pH — in the case of the lyophilizates after resuspension — are determined at the respective time.

After several months of storage, the resuspended lyophilizates showed no significant deviations relative to the examined sizes.

In pharmacological tests, the liposome suspensions thus produced show the following properties: A Resuspended lyophilizates produced according to example 20 2 are examined relative to their acute toxicity after a single administration to a mouse and rat.

The respective LD50 is determined to be: 2.8 g of I/Kg body weight mouse (total iodine) or 3.0 g of I/kg body weight rat.

Test B For resuspended lyophilizates produced according to example 2, the subacute toxicity is determined in rats (n = 6). - 18 After repeating the dose of 1 g of I/kg body weight (total iodine) 6 times, each at an interval of 3 days, no changes of histopathological or clinical parameters are determined.

Ttak C Resuspended lyophilizates obtained according to example 2 are examined relative to their organ connection after intravenous administration in rats. hour after the administration of 100 mg of I/kg body 10 weight (total iodine), 0.54 I/g of wet weight corresponding to 24% of the administered dose is detected in the liver.

Test D Resuspended lyophilizates obtained according to example 2 are administered intravenously in a dose of 100 mg of total iodine/kg body weight to rabbits with liver tumors.

The resulting difference in density of liver/tumor is 35 Hounsfied units (HU) after 1 hour.

Test B Resuspended lyophilizates obtained according to example 2 are examined on dogs relative to their suitability for indirect CT lymphography. hours after interdigital administration of 200 mg of total iodine (in 3 ml of liposome suspension), at most 200 HU of density increase is measured in CT in the popliteal and iliac lymph nodes.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

- Mrr-ru- bi ix:, .i_ Νϋ:'/ίοιί"·χ4ο—b4i0 - 19 From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims

1. WHAT IS CLAIMED IB;

1. A process for the production of an aqueous liposome suspension comprising: preparing a solution containing at least one liposomeforming substance in a lower alcohol solvent having 1-3 carbon atoms; preparing an aqueous phase; dissolving and/or suspending at least one active ingredient in said solution and/or said aqueous phase; introducing said aqueous phase into said solution with mixing; removing said lower alcohol by vacuum distillation to form a liposome suspension; and optionally freeze-drying said liposome suspension.

2. A process according to claim 1, wherein said active ingredient is water-soluble and is dissolved and/or suspended in said aqueous phase.

3. A process according to claim 1, wherein said active ingredient is a contrast medium.

4. A process according to claim 2, wherein said active ingredient is a contrast medium. —r - -U- · · I t u_4__i

5. I * I i_ .^r- 1 II ,I_J I ^_|_ I 1LJ · ( c_i_> C.'-t·-) O^XCJ Wfcj ί i “ tJ I - 21 5. A process according to claim 1, wherein the active ingredient inclusion in the liposomes is up to 50%.

6. A process according to claim 1, wherein the active ingredient concentration in said liposome suspension is up to 800 mg/ml.

7. A process according to claim 1, wherein said solution contains 0.2-30 g of said liposome-forming substance per 100 ml of said lower alcohol solvent.

8. A process according to claim 1, wherein, after said aqueous phase is introduced into said solution and prior to removal of said lower alcohol, the combination of aqueous phase and solution are mixed for 10-180 minutes at a temperature of 20-90°C.

9. A process according to claim 1, wherein said vacuum distillation is performed at a vacuum of 10 h Pa to 100 h Pa.

10. A process according to claim 1, wherein said liposome suspension is freeze-dried to form lyophilizates and said lyophilizates are subsequently resuspended in an aqueous resuspension medium, the amount of said resuspension medium being 0.5-20 ml per g of lyophilizate.

11. A process according to claim 1, wherein 0.05-10 g of active ingredient per g of said liposome-forming substance is used to prepare said liposome suspension. -22described with prepared by a 13 or 14.

12. A process according to claim 3, wherein said contrast medium is an X-ray contrast medium.

13. A process according to claim 4, wherein said active ingredient is an X-ray contrast medium.

14. A process substantially as hereinbefore reference to the Examples.

15. An aqueous liposome suspension whenever process as claimed in any of claims 1 to

16. In a method for the diagnosis of tumors, the improvement wherein a suspension according to any of claims 1 to 15 is employed as a diagnostic medium and said active ingredient is a tumor diagnostic agent.

17. In a method of indirect lipography the improvement wherein a suspension according to any of claims 1 to 15 is employed as a diagnostic medium and said active ingredient is a lipographic diagnostic agent.

18. A method of diagnosis substantially as hereinbefore described with reference to the Examples.

19. A method of indirect lipography substantially as hereinbefore described with reference to the Examples.