FI63857C - FREQUENCY REQUIREMENT FOR FREQUENCY LIPOSOM PREPARATION - Google Patents

Description

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Patents and registration (44) Date of transfer to patent and registration. - ης n.

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England-England (GB) 19510/77 (71) Imperial Chemical Industries Limited, Imperial Chemical House, Millbank, London SW1P 3JF, England-England (GB) (72) John Raymond Evans, Macclesfield, Cheshire, Francis James Thomas Fildes, Macclesfield , Cheshire, Jean Elizabeth Oliver, Macclesfield, Cheshire, England-England (GB) (7U) Oy Kolster Ab (5U) Method for the preparation of lyophilized liposome preparations - Förfarande för framställning av frystorkade liposom-preparat (62) Divided by application 7Ö1012 63856) -Avdelad frän ansökan 781012 (utläggningsskrift 63856)

Liposomes have been described quite extensively in the literature and their structure is well known. They usually have an onion-like multilayer structure comprising a series of phospholithic bilayers separated by an aqueous substance. Another known type of liposome comprises a simple phospholipid bilayer enclosing an aqueous substance; these monolayer forms are sometimes referred to as "vesicles." In recent years, there has been a growing interest in using liposomes as carriers for compounds of interest due to one or more biological properties, e.g., drugs, proteins, enzymes, hormones, vitamins, and markers, etc. This broad group of biologically interesting compounds but is not limited to, are referred to herein as "biologically active compounds".

Encapsulation of a biologically active compound in liposomes can be accomplished by a variety of methods. The most commonly used method involves forming a phospholipid film (without or with a charged lipid) by evaporation from a solution in an organic solvent, e.g. chloroform, and then dispersing the film in a suitable aqueous medium. In the case of lipid-soluble biologically active compounds, i.e. those which combine with the lipid layers rather than the aqueous phase of the liposomes, the compound is usually molded into a film together with a phospholipid using a common organic solvent. In the case of water-soluble biologically active compounds, the compound is usually encapsulated in liposomes by dispersing a molded phospholipid film with an aqueous solution of the compound. The encapsulated compound is then separated from the free compound by centrifugation, chromatography or any other suitable procedure. In the case of biologically active compounds which associate with the lipid phase of liposomes, provided that they are present in an amount below or below their maximum lipid solubility, the liposomes prepared by the above method usually contain the majority of the lipid. from a compound bound to bilayers, in which case the separation of the free compound is not as crucial as in the case of water-soluble biologically active compounds which do not bind to lipid.

The above method is not suitable for large scale use. In addition, aqueous liposome dispersions have only limited stability and therefore have a limited shelf life. In addition, liposomes may accumulate or precipitate. Although such precipitates can usually be redispersed, the structure and size distribution of the original dispersion may change. Accumulation and precipitation can be reduced by combining charged lipids with liposomes, but this does not guarantee a satisfactory storage time. The loss of a biologically active compound from liposomes to an external aqueous medium is another factor that limits the use of these preparations as practical dosage forms. This is especially true for low molecular weight water-soluble compounds, but lipid-soluble compounds may also migrate into an external aqueous medium until equilibrium is reached. If the concentration of the compound is low and / or the volume of the external aqueous medium is large, this loss may represent a significant proportion of the total concentration of the biologically active compound in the liposomes.

All of these factors limit the use of liposomes as practical carriers for biologically active compounds, especially in drug therapy. In one embodiment, a film of lipid / biologically active compound is prepared and stored and then dispersed to form liposomes as needed just prior to use. However, the manufacture and storage of a unit dose film presents great practical difficulties and therefore this idea does not offer a practical solution to the problems presented above. The present invention relates to a method for providing a practical solution. Briefly, the method comprises preparing an aqueous liposome preparation in a manner known per se and then freeze-drying said aqueous preparation. The resulting lyophilized mixture can be stored and, if desired, dispersed in a suitable aqueous medium to obtain an aqueous liposome preparation.

Any conventional freeze-drying method can be used in carrying out the method of the invention. For the sake of brevity, the term "lyophilized potential liposome-containing compositions" is then used to refer to the lyophilized compositions (liposome precursors) available in accordance with this invention. When dispersed in a suitable aqueous medium, liposome preparations are obtained. When the lyophilized mixture containing the potential liposome is redispersed in a suitable aqueous medium, liposomes similar to those prepared by the known membrane dispersion method are unexpectedly formed. In the case of a lipid-soluble or lipid-bound biologically active compound, the compound is associated with liposomes in a large amount. On the other hand, the method of the invention is less suitable for water-soluble biologically active compounds that do not bind to lipid. The lyophilized 6 3 8 5 7 4 mixtures readily disperse when shaken with an aqueous medium and result in liposome formulations with a narrower size distribution than the corresponding preparation obtained by dispersing the molded film.

According to the invention, there is provided a process for preparing a lyophilized potential liposome-containing mixture, which process comprises preparing, in a manner known per se, an aqueous liposome preparation containing water, at least one liposome-forming amphipathic lipid, at least one lipid-soluble or lipid-binding biologically active compound and optionally at least one , which is a negative charge agent or a positive charge agent or cholesterol, which method is characterized in that said aqueous liposome preparation is lyophilized to obtain a lyophilized mixture containing a potential liposome.

Said aqueous liposome preparation can be prepared, for example, by the above-mentioned membrane dispersion method.

Any amphipathic lipid known to be suitable for the preparation of liposomes by known methods can be used in the method of this invention. Thus, a wide variety of lipids can be used in accordance with the invention, but those that are non-immunogenic and biodegradable are preferred. Examples of suitable lipids are phospholipids, for example natural lecithins, for example egg lecithin or soybean lecithin, or synthetic lecithins, e.g. saturated synthetic lecithins, e.g. choline or dilinoleyl-phosphatidylcholine. Either a single phospholipid or a mixture of phospholipids can be used.

As indicated above, the biologically active compound can be any lipid-soluble or lipid-binding compound that has a biologically interesting property. Thus, the compound may be a lipid-soluble or lipid-binding drug, protein, enzyme, hormone, vitamin or label. It is to be understood that lipid-soluble or lipid-binding biologically active compounds include certain water-soluble compounds, e.g., certain proteins.

Possible excipients include a substance which gives a negative charge, e.g. egg phosphatidic acid, dipalmitoyl-phosphatidic acid, disethyl phosphate or bovine brain ganglioside, or a substance which gives a positive charge, e.g. stearylamine or stearyl ester, cholesterol acetate or to the physical properties of the bilayers in liposomes as desired.

According to a further aspect of the invention there is provided a process for the preparation of an aqueous liposome preparation comprising water, at least one liposome-forming amphipathic lipid, at least one lipid-soluble or lipid-binding biologically active compound and optionally at least one excipient which is a negative charge agent or a positive charge agent or cholesterol. , characterized in that the lyophilized potential liposome-containing mixture prepared by the method described above is dispersed in an aqueous medium. A suitable aqueous medium is, for example, distilled water, isotonic saline or sterile or non-sterile buffer solution.

The invention is illustrated by the following examples.

Example 1

Egg lecithin (16.1 mg), egg phosphatidic acid (2 mg) and 3 3 H-cortisol-21-palmitate (hereinafter referred to as "H-CP", 1.66 mg) were dissolved in chloroform (5 ml) and the solution was poured In a 250 ml round-bottomed flask. The solvent was removed at room temperature by rotating the flask and blowing a stream of dry nitrogen into it. The lipid film thus obtained was then dispersed at room temperature in water (5 ml) to obtain a liposome preparation. Duplicate samples were taken 6,63857 (50 μΐ) for scintillation counting. The remainder of the liposome preparation was diluted to 25 ml with distilled water in an ultracentrifuge tube and ultracentrifuged at 120,000 g for 30 minutes. The supernatant was removed from the liposome stopper and the stopper was dissolved in distilled water (5 ml). Duplicate samples (50 μΐ) were taken from this dispersion and steroid binding was measured by scintillation counting. The remainder of the liposome dispersion was cooled using a mixture of methanol and solid carbon dioxide and the solvent was removed using a commercial freeze dryer. Thus, a lyophilized mixture containing a potential liposome was obtained.

The lyophilized mixture was stored for 5 days and then 0.9 w / v was added. brine (5 mL). Liposomes were gently formed by shaking the mixture in a flask at room temperature. Microscopic examination confirmed the presence of liposomes. Two days later, the liposomes were washed twice with 0.9 w / v.

saline by the method described above, except that saline was used instead of water. The steroid content of liposomes was determined by scintillation counting. Comparison of the radioactivity of the dispersions before and after lyophilization showed that 72% of the steroid present in the pre-freeze-dried preparation remained in the washed liposomes formed after lyophilization.

Example 2

Dipalmitoyl-phosphatidylcholine (hereinafter referred to as "DPPC" 29.8 mg) and 1 H-CP (3.32 mg) were dissolved in chloroform (5 ml) and cast into a thin film on the walls of a 250 ml round bottom flask by evaporating the solvent at room temperature using a dry stream of nitrogen. Distilled water (10 ml) was then added to the flask and the mixture was heated to 70 ° C with a water bath. Liposomes were formed by stirring the hot mixture with a bench shaker. Duplicate samples (50) were taken from the resulting dispersion for scintillation counting. The remainder of the dispersion was washed twice by diluting to 25 ml with distilled water and ultracentrifuging at 120,000 g for 30 min. The washed liposome stopper was redispersed in distilled water (10 mL) and duplicate samples (50 μl) were taken for scintillation counting. This dispersion (5 ml) was cooled in a cooling mixture of methanol and solid carbon dioxide and the solvent was removed in vacuo using a commercial freeze dryer.

7 65857 Thus, a lyophilized mixture containing a potential liposome was obtained and stored until needed.

Distilled water (5 ml) was added to the lyophilized mixture, and the resulting mixture was heated to 70 ° C in a water bath and gently shaken. Microscopic examination of the resulting milky suspension showed that it comprised a liposome suspension with a narrow size distribution. This suspension was ultracentrifuged at 120,000 g for 30 minutes and the liposome stopper was redispersed in distilled water (5 ml). Duplicate samples (50 μl) were taken from the resulting suspension to determine the final steroid content of the liposomes. Scintillation counting showed that 78% of the steroid combined with the initial washed dispersion was present in the final washed liposome preparation formed from the lyophilized mixture.

Claims (7)

8 63857 A process for preparing a lyophilized, potentially liposome-containing mixture which is physically and chemically stable and storage-stable, which process comprises preparing, in a manner known per se, an aqueous liposome preparation containing water, at least one liposome-forming amphipathic lipid, at least one lipid-soluble or a binding biologically active compound and optionally at least one excipient which is a negative charge agent or a positive charge agent or cholesterol, characterized in that said aqueous liposome preparation is lyophilized to obtain a lyophilized mixture containing a potential liposome. A method according to claim 1, characterized in that the aqueous liposome preparation is prepared by a membrane dispersion method. Process according to Claim 1 or 2, characterized in that the lipid is a phospholipid. Process according to Claim 3, characterized in that the phospholipid is natural or synthetic lecithin. Process according to one of Claims 1 to 4, characterized in that the biologically active compound is a medicament. A process for the preparation of an aqueous liposome preparation comprising water, at least one liposome-forming amphipathic lipid, at least one lipid-soluble or lipid-binding biologically active compound and optionally at least one excipient which is a negative charge agent or a positive charge agent or cholesterol, characterized in , dispersing the lyophilized mixture containing a potential liposome prepared by the method of any one of claims 1 to 5 in an aqueous medium. Process according to Claim 6, characterized in that the aqueous medium is distilled water, isotonic saline solution or sterile or non-sterile buffer solution.