IE46572B1 - Method for the manufacture of liposome compositions - Google Patents

Description

This invention relates to an improved method for the manufacture of liposomes.

Liposomes are quite widely described in the literature, and their structure is well known. Usually they have'an onion-like multilamellar structure comprising a plurality of phospholipid bilayers spaced one from another by aqueous material. Another type of liposome which is.known consists of a single phospholipid bilayer enclosing aqueous material; these unilamellar forms are' sometimes referred to as vesicles.

In recent years there has been increasing interest in the use of liposomes as carriers of compounds which are of interest because of one or other biological property, for example medicaments, proteins, enzymes, hormones, vitamins and marker compounds. It is to be understood that this broad group of biologically interesting compounds, which includes medicaments (human and veterinary) but is not restricted thereto, will be referred to in this specification as biologically active compounds.

The encapsulation of a biologically active compound in liposomes can be achieved by a variety of methods.

The method most commonly used involves casting a film of phospholipid (in the absence or presence of a charged lipid) by evaporation from a solution in an 48572 - 3 organic solvent, for example chloroform, and then dispersing the film in a suitable aqueous medium. In the case of lipid-soluble biologically active compounds, that is, those which associate with the lipid layers rather than the aqueous phase of the liposomes, the compound is usually cast as a film together with a phospholipid, using an organic solvent for both ingredients. In the case of water-soluble biologically active compounds the compound is usually encapsulated in liposomes by dispersing a cast phospholipid film in an aqueous solution of the compound. The unencapsulated compound is then removed from the encapsulated compound by centrifugation, chromatography or some other suitable procedure. In the case of biologically active compounds which associate with the lipid phase of liposomes, provided they are present in an amount below their maximum lipid solubility or below the maximum amount that can be bound by the lipid, liposomes prepared by the above method usually contain most of the compound bound in the lipid bilayers, and removal of the unencapsulated compound from the liposomes is not so critical as in the case of water-soluble biologically active compounds which do not bind to lipid.

The above-mentioned method does not lend itself to large scale usage. In addition, aqueous liposome dispersions only have limited stability and therefore their storage life is limited. Moreover, the liposomes can aggregate and precipitate as a sediment. Although such sediments can usually be re-dispersed, the structure and size distribution of the original dispersion may be changed. Aggregation and sedimentation can be reduced by the incorporation of charged lipids into the liposomes, but this does not guarantee a satisfactory storage life. The loss of the biologically active compound from the liposomes into the external aqueous medium is another factor which restricts the potential of these preparations as practicable dosage forms. This - 4 is particularly severe for low molecular weight, watersoluble compounds, but lipid-soluble compounds too can partition into the external aqueous medium until equilibrium is reached. If the content of compound is small, and/or the volume of the external aqueous medium is large, this loss can represent a significant proportion of the total content of the biologically active compound in the liposomes.

All of these factors restrict the use of liposomes as practicable carriers of biologically active compounds, particularly in medicament therapy. One solution might be to prepare and store the lipid/biologically active compound film, and then disperse the film to form liposomes as needed just before use. However, unit dose film preparation and storage presents serious practical difficulties, and therefore this idea does not provide a practical solution to the problems outlined above.

The present invention is concerned with a method which does provide a practical solution. In brief, the method comprises preparing an aqueous liposome preparation by any known method and then freeze-drying the preparation, whereafter the resulting freeze-dried mixture is stored' and, when desired, dispersed in an aqueous medium so as to give an aqueous liposome preparation. Any conventional freeze-drying procedure can be used in carrying out the freeze-drying method of this invention. For brevity hereinafter, the expression freeze-dried, potential liposome, mixtures will be used for the freeze-dried mixtures obtained according to this invention which, upon dispersion in a suitable aqueous medium, afford the desired aqueous liposome preparations. Unexpectedly, when a freeze-dried, potential liposome, mixture of this invention is redispersed in a suitable aqueous medium, for example isotonic saline, liposomes are formed which are similar to those prepared by the known film dispersion 6 5 7 2 - 5 method. In the case of a lipid-soluble or lipid-bound biologically active compound, the compound is re-incorporated into the liposomes to a large extent.

On the other hand, as explained below the method of the invention is not so suitable for those watersoluble biologically active compounds which do not bind to lipid. The freeze-dried mixtures disperse easily when shaken with an aqueous medium, and it appears that they lead to liposome preparations having a narrower size distribution than a corresponding preparation obtained by dispersing a cast film. This narrower size distribution might be advantageous as regards the reproducibility of the effect of the former liposome preparations over the latter.

According to the invention there is provided a method for the manufacture of a freeze-dried, potential liposome, mixture which comprises preparing by any known method an aqueous liposome composition comprising in the liposomes at least one biologically active compound and optionally at least one adjuvant as hereinafter defined, and then freeze-drying the aqueous liposome composition to produce a freeze-dried, potential liposome, mixture.

Any amphipathic lipid which is known to be suitable for preparing liposomes by known methods can be used in the method of this invention. Thus a wide variety of lipids may be used according to this invention, but those which are non-immunogenic and bio-degradable are preferred. Example of suitable lipids are the phospholipids, for example the natural lecithins, for example egg lecithin or soya bean lecithin, or synthetic lecithins, for example saturated synthetic lecithins, for example dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoyl-phosphatidylcholine, or unsaturated synthetic lecithins, for example dioleyl46572 - 6 phosphatidylcholine or dilinoleyl-phosphatidylcholine. Either a single phospholipid or a mixture of phospholipids may be used.

As indicated above, the biologically active compound may be any compound having a property of biological Interest. Thus, for example, the compound may be a medicament, protein, enzyme, hormone, vitamin or marker compound. It is to be understood that the method of this invention is particularly useful in the case of lipid-soluble or lipid-bound biologically active compounds (which include some water-soluble compounds, for example some proteins). The same methods are not so suitable for water-soluble, non-lipid-bound, biologically active compounds because in those cases only a relatively small fraction of the compound is re-incorporated into the liposomes upon dispersion of the freeze-dried mixture. Nevertheless, this drawback is acceptable provided that a suitable excess of the water-soluble biologically active compound is incor20 porated into the freeze-dried mixture. Also, when liposomes are prepared from such a mixture, if the presence of unencapsulated biologically active compound in the external aqueous medium is disadvantageous, the unencapsulated compound must be removed by one of the above-mentioned methods. Thus, the suitability of the method of the invention in the case of a water-soluble, non-lipid-bound, biologically active compound depends upon all of the relevant facts, including (1) the nature of the compound's activity, (2) the compound's potency, (3) the amount of the compound incorporated in the liposome preparation produced according to this invention and (4) the desirability or not of the compound being present in the external aqueous medium.

The said adjuvants consist of: (a) substances which are known in this art to provide a negative 6 5 7 2 - Ί charge, for example egg phosphatidic acid, dipalmitolylphosphatidic acid, dicetyl phosphate or beef brain ganglioside; (b) substances which are known in this art to provide a positive charge, for example stearylamine or stearylamine acetate? and (c) substances which are known in this art to effect the physical properties of the lipid bilayers in the liposomes in some desirable way, for example rendering them more fluid or more rigid, as required, for example cholesterol.

According to a further feature of the invention there is provided the freeze-dried, potential liposome, mixture which is obtained by the method described immediately above.

According to a further feature of the invention there is provided a method for the manufacture of an aqueous liposome preparation containing in the liposomes at least one biologically active compound and optionally at least one adjuvant as defined hereinbefore, which comprises dispersing a freeze-dried, potential liposome, mixture obtained by the method described immediately above, in an aqueous medium.

As a suitable aqueous medium there may be mentioned for example, distilled water, isotonic saline, or a sterile or non-sterile buffer solution.

The invention is illustrated by tho rollowlny Examples:Example 1 Egg leceithin (16.1 mg.), egg phosphatidic acid o (2 mg.) and H-cortisol 21-palmitate (hereinafter ο H-CP; 1.66 mg.) were dissolved in chloroform (5 ml.), and the solution was poured into a 250 ml. roundbottomed flask. The solvent was removed at room temperature by rotating the flask and blowing a stream of dry nitrogen into it. The lipid film thereby - 8 obtained was then dispersed at room temperature in water (5 ml.), giving a liposome preparation. Duplicate samples (50 μΐ.) were removed for scintillation counting. The remainder of the liposome preparation was diluted to 25 ml. with distilled water in an ultracentrifuge tube, and ultra-centrifuged at 120,000 g for 30 minutes. The supernatant liquid was removed from the liposome plug, and the plug was dispersed in distilled water (5 ml.). Duplicate samples (50 μΐ.) of this dispersion were taken, and the steroid incorporation was measured by scintillation counting. The remainder of the liposome dispersion was frozen, using a methanolsolid carbon dioxide mixture, and the solvent removed using a commercial freeze-dryer. There was thus obtained a freeze-dried, potential liposome, mixture.

The freeze-dried mixture was stored for five days, and to it was then added 0.9% w/v saline (5 ml.). Liposomes were formed by gently 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 the liposomes was determined by scintillation counting.

Comparison of the radioactivity of the dispersions before and after freeze-drying showed that 72% of the steroid present in the washed preparation before freezedrying was retained in the washed liposomes formed after freeze-drying.

Example 2 Dipalmitoyl-phosphatidylcholine (hereinafter DPPC; 29.8 mg.) and ^H-CP (3.32 mg.) were dissolved in chloroform (5 ml.) and cast as a thin film on the wall of a 250 ml. round-bottomed flask by evaporating the solvent at room temperature using a stream of dry - 9 nitrogen. Distilled water (10 ml.) was then added to the flask, and the mixture was heated to 70°C. on a water bath. Liposomes were formed by agitating the hot mixture on a bench vibromixer. Duplicate samples (50 μΐ.) of the resulting dispersion were removed for scintillation counting. The remainder of the dispersion was washed twice hy diluting to 25 ml. with distilled water and ultracentrifugation at 120,000g for 30 minutes. The washed liposome plug was re-dispersed in distilled water (10 ml.), and duplicated samples (50 μΐ.) were taken for scintillation counting. This dispersion (5 ml.) was frozen in a freezing mixture consisting of methanol and solid carbon dioxide, and the solvent was removed under vacuum using a commercial freeze-dryer. There was thus obtained a freeze-dried, potential liposome, mixture which was stored until reguired.

Distilled water (5 ml.) was added to the freezedried mixture, and the resulting mixture was heated to 70°C. on a water bath, and gently shaken. Microscopic examination of the resulting milky suspension showed it to consist of a suspension of liposomes, with a narrow size distribution. This suspension was ultracentrifuged at 120,000g for 30 minutes, and the liposome plug was then redispersed in distilled water (5ml). Duplicate samples (50 μΐ.) of the resulting suspension were taken for assaying the final steroid content of the liposomes. Scintillation counting showed that 78% of the steroid incorporated in the original washed dispersion was present in the final washed liposome preparation formed from the freeze-dried mixture.

Duplicate samples (6mg.) of dried liposome plugs prepared from (a) the original film and (b) the freezedried mixture were then weighed into sample holders for differential scanning calorimetry (hereinafter DSC). The DSC spectra of the mixtures between 0°C, and 5O°C. were recorded on a Perkin Elmer differential , - 10 scanning calorimeter. Control samples for DSC were also 3 prepared by mixing the same weights of DPPC and H-CP as in the original mixture, and then mixing them with 50% by weight of water. These served as non-liposome control mixtures. The DSC spectra of these control mixtures were measured as described above.

The DSC spectrum of DPPC alone consists of a main transition endotherm at 41°C. and a pre-transition endotherm at 35°C. The half-peak line width of the main endotherm is approximately 3°C. The experiments described above showed that, in the non-liposome control mixtures, both peaks were observed in the DSC spectra of the mixtures, and the line width remained at about 3°C. This is believed to show that in simple mixtures (i.e. not liposomes) the steroid does not change the DSC spectrum of the lipid. The spectra of the duplicate liposome preparations showed one transition only (the main endotherm), and the average line width of those preparations was 5.8°C., a considerable broadening compared with the control mixtures. This broadening results from the molecular interaction of the lipid and steroid in the liposomes prepared by the above method. Therefore, there can be no doubt that the liposome preparations prepared both from the original film and from the freeze-dried mixture contained the steroid in the liposomes.

Example 3 Egg lecithin (15 mg.), cholesterol (2.09 mg.) and dicetyl phosphate (1.55 mg.) were dissolved in chloro30 form (5 ml.), and cast as a thin film on the wall of a test tube. ^gl-Angiotensln 11 (0.1 mg.) in 3.3mM phosphate buffer (pH 7.4; 1 ml.) was added to the tube.

The lipid was dispersed in the aqueous medium with the aid of a bench vibromixer to form lipoaomea. The lipo35 some dispersion was washed twice by diluting to 26 ml. 48573 - 11 with 3.3 mM phosphate buffer (pH 7.4), followed by ultracentrifugation at 120,000g for 1 hour. The washed liposome plug was redispersed in 3.3 mM phosphate buffer (pH 7.4; 5ml.) and duplicate samples (0.25 ml.) were removed for scintillation counting, 4ml. of the remaining suspension were placed in a test tube, frozen (methanol-solid carbon dioxide), and freeze-dried. The resulting freeze-dried, potential liposome, mixture was resuspended in 3.3mM phosphate buffer (pH 7.4; 2ml.) and washed twice as before. The washed liposome plug was resuspended in 3.3mM phosphate buffer (pH. 7.4; 4ml.). Duplicate samples (0.25 μΐ.) were removed for scinitillation counting. 26% of the initial amount of angiotensin IX was retained in the liposomes after the first liposome preparation and washing. 28% of this 26%, that is 7% of the initial amount of angiotensin II, was retained in the liposomes after freeze-drying and reconstitution.

The 3.3mM phosphate buffer (pH 7.4) used in this Example and Example 4 was prepared by dissolving potassium dihydrogen phosphate (O.895g.) and disodium hydrogen phosphate dihydrate (4.765g.) in distilled water, and making the solution up to 1 litre with distilled water.

Example 4 Egg lecithin (15 mg.), cholesterol (2.09 mg.) and dicetyl phosphate (1.55 mg.) were dissolved in chloroform (5 ml.), and cast as a thin film on the wall of a test tube, 3H-Inulin (5 mg.) in 3.3mM phosphate buffer (pH 7.4; 1 ml.) was added to the tube. The lipid was dispersed in the inulin solution with the aid of bench vibromixer to form liposomes. The liposome dispersion was washed twice by diluting to 26ml. with 3.3mM phosphate buffer (pH 7.4), followed by ultracentrifugation at 120,000g for 1 hour. The washed liposome plug was redispersed in 3.3mM phosphate buffer (pH 7.4; 2.1 - 12 ml.) and duplicate samples (1 μΐ.) were removed for scintillation counting. The remaining suspension was frozen (methanol-solid carbon dioxide mixture) and freeze-dried in a test tube. The resulting freeze5 dried, potential liposome, mixture was resuspended in 3.3mM phosphate buffer (pH 7.4; 1 ml.) to form liposomes and washed twice as before. The washed liposome plug was resuspended in 3.3mM phosphate buffer (pH 7.4, 2 ml.), and duplicate samples (1 μΐ.) were taken for scintillation counting. 21% of the initial amount of inulin was retained in liposomes after the first liposome preparation and washing. 17% of this 21%, that is 4% of the initial amount of inulin, was retained in the liposomes after freeze-drying and reconstitution.

Claims (17)

1. CLAIMS:1. A method for the manufacture of a freeze-dried, potential liposome, mixture which comprises preparing by any known method an aqueous liposome composition comprising in the liposomes at least one biologically active compound and optionally at least one adjuvant as defined hereinbefore, and the freeze-drying of the aqueous liposome composition to produce a freeze-dried, potential liposome, mixture.

2. A method as claimed in claim 1 in which the liposomes contain at least one phospholipid.

3. A method as claimed in claim 2 in which the phospholipid is a natural or synthetic lecithin.

4. A method as claimed in claim 3 in which the lecithin is egg lecithin or dipalmitoyl-phosphatidylcholine.

5. A method as claimed in any one of claims 1 to 4 in which the biologically active compound is a lipidsoluble or lipid-bound compound.

6. A method as claimed In any one of claims 1 fo 4 in which the biologically active compound is a watersoluble, non-lipid-bound compound.

7. A method as claimed in any one of claims 1 to 6 in which the biologically active compound is a medicament.

8. A method as claimed in claim 1 in which the adjuvant is egg phosphatidic acid, dipalmitoylphosphatidic acid, dicetyl phosphate or beef brain ganglioside. 46372 - 14

9. A method as claimed in claim 1 in which the adjuvant is stearylamine or stearylamine acetate.

10. A method as claimed in claim 1 in which the adjuvant is cholesterol.

11. A freeze-dried, potential liposome, mixture which is obtained by the method claimed in any one of claims 1 to 10.

12. A method for the manufacture of an aqueous liposome preparation containing in the liposomes at least one biologically active compound and optionally at least one adjuvant as defined hereinbefore, which comprises dispersing a freeze-dried, potential liposome, mixture as claimed in claim 11, in an aqueous medium.

13. A method as claimed in claim 12 in which the aqueous medium is distilled water, isotonic saline, or a sterile or non-sterile buffer solution.

14. An aqueous liposome preparation containing in the liposomes at least one biologically active compound and optionally at least one adjuvant as defined hereinbefore, whenever obtained by a method claimed in claim 12 or 13.

15. A method as claimed in claim 1, substantially as described in any of the Examples.

16. A freeze-dried, potential liposome, mixture as claimed in claim 11, substantially as described in any of the Examples.

17. A method as claimed in claim 12, substantially as described in any of the Examples.