GB1575344A - Method for the manufacture of liposome composition - Google Patents

Description

(54) METHOD FOR THE MANUFACTURE OF LIPOSOME COMPOSITIONS (71) We, IMPERIAL CHEMICAL IN DUSTRIES LIMITED, Imperial Chemical House, Millbank, London SW1P 3JF a British.'Company do hereby declare the invention, for which we pray that a patent may be.granted to us, and the method by which it is to be performed, to be particular ly described in and by the following state ment: 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 mul.tilamellar structure comprising a plural ity 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 in terest in the use of liposomes as carriers of compounds which are of interest because of one or other biological property. for exam ple medicaments. proteins, enzymes, hor -mones, vitamins and marker compounds. It is to be understood that this broad group of biologically interesting compounds, which includes medicaments (human and veterin ary) but is not restricted thereto, will be referred to in this specification as "biologic- ally active cqmpqunds".

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 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 is particularly severe for low molecular weight. water-soluble compounds, but lipidsoluble 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 linosomes.

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 mix ture is stored and, when desired, dispersed in an aqueous medium so as to give an aqueous liposome preparation. Any con veritional 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 prepa rations. Unexpectedly, when a freeze-dried, potential liposome, mixture of this inven tion is re-dispersed in a suitable aqueous medium, for example isotonic saline, lip somes are formed which are similar to those prepared by the known film dispersion 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 water soluble 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 corres ponding preparation obtained by dispersing a cast film. This narrower size distribution might be advantageous as regards the repro ducibility of the effect of the former lipo some preparations over the latter.

According to the invention there is pro vided 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 freeie- 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 dioleylphosphatidylcholine 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 watersoluble compounds, for example some proteins). The said 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 incorporated 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 suita- bility 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) subst ances which are known in this art to provide a negative charge, for example egg phospha 'tidic acid, dipalmitolyl-phosphatidic 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 affect 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 immedi ately above.

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

As 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 the follow ing Examples: Example 1 Egg leceithin (16.1mg.), egg phosphatidic acid (2mg.) and JH-cortisol 21-palmitate (hereinafter "jH-CP"; 1.66 mg.) were dis solved in chloroform (5ml.), and the solu tion was poured into 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 thereby obtained was then dis persed at room temperature in water (5ml.), giving a liposome preparation. Duplicate samples (50 EL1.) were removed for scintilla 'tion counting. The remainder of the lipo some preparation was diluted to 25ml. with distilled water in an ultracentrifuge tube, and ultra-centrifuged at 120,000g for 30 minutes. The supernatant liquid was re 'moved from the liposome plug, and the plug was dispersed in distilled water (5ml.).

Duplicate samples (50 ul.) of this dispersion were taken, and the steroid incorporation was measured by scintillation counting. The remainder of -the liposome dispersion was frozen, using a methanol-solid carbon diox id 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 0.9loo w/v saline (Sml.). 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 freeze drying was retained in the washed liposomes formed after freeze-drying.

Example 2 Dip,almitoyl-phosph'atidylcholine (hereinafter "DPPC"; 29.8mg.) and 3H-CP (3.32mg.) were dissolved in chloroform (5ml.) and cast as a thin film on the wall of a 250ml. round-bottomed flask by evaporating the solvent at room temperature using a stream of dry nitrogen. Distilled water (10ml.) 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 (501l1.) of the resulting dispersion were removed for scintillation counting. The rernainder of the dispersion was washed twice by diluting to 25ml. with distilled water and ultracentrifugation at 120,000g for 30 minutes. The washed liposome plug was re-dispersed in distilled water (10ml.), and duplicated samples (50F1.) were taken for scintillation counting. This dispersion (5ml.) 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 required.

Distilled water (5ml.) was added to the freeze-dried 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 (5(jyl.) 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 lipo some plugs prepared from (a) the original film and (b) the freeze-dried mixture were then weighed into sample holders for differential scanning calorimetry (hereinafter -"DSC"). The DSC spectra of the mixtures between 0 C. and 50 C. were recorded on a Perkin Elmer differential scanning calorimeter. Control samples for DSC were also 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 de scribed above The DSC spectrum of- DPPC alone consists of a main transistion endotherm at 41 C and a pre-transistion 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 bout ut 3 C. This is believed to show that in simple mixtures (i.e. not liposomes) the steroid does not change @e the DSC spectrum of the lipid. The spectra of the duplicate liposome preparations showed one transi tion only (the main endotherm), and the average line width of those preparations was 5.8 C. a considerable broadening com pared with the control mixtures. This broadening results from the molecular in teraction of the lipid and steroid in the liposomes prepared by the above method.

Therefore, there can be no doubt that the liposome preparations prepared hoth from the original film and from the freeze-dried mixture contained the steroid in the liposomes.

Example 3 Egg lecithin ( 15mg.), cholesterol (2.09mg.) and diectyl phosphate (1.55mg,) were dissolved in chloroform (5ml.), and cast as a thin film on the wall of a test tube.

@25I-Angiotensin @@ (0.1mg.) in 3.3mM phos phate buffer (pH 7.4: 1ml.) was added to the tube. The lipid was dispersed in the aqueous medium with the aid of a bench vibromixer to form liposomes. The lipo sonic dispersion was washed twice by dilut ing to 26mL with 3.3mM phosphate butter (pH 7.4). followed by ultracentrifugation at 120,000g for I hour. The washed liposome plug was redispersed ill 3.3mM phosphate buffer (pH 7.4; 5ml.) and duplicate samples (0.25ml.) were removed for scintillation counting, 4mL of the remaining suspension were placed in a test tube. frozen (metha- nol-solid carbon dioxide), and freeze-dried.

The resulting freeze-dried, potential lipo some, 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 buf- fer (pH. 7.4; 4ml.). Duplicate samples (0.25 l.) were removed for scintillation counting. 266 of the initial amount of angiotension II 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 Il. 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 (0.895g.) 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 (15mg.), cholesterol (2.09 mg.) and dicetyl phosphate (1.55mg.) were dissolved in chloroform (5ml.), and cast as a thin film on the wall of a test tubc. 3l-l-lnulin (5mg.) in 3.3mM phosphate buffer (pH 7.4; 1ml.) 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 bv 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, 2.1ml,) and duplicate samples (1 l.) were removed for scintillation counting. The remaining sus pension was frozen (methanol-solid carbon dioxide mixture) and freeze-dried in a test tube. The resulting freeze-dried, potential liposome mixture was resuspended in 3.3mM phosphate buffer (pH 7.4; 1ml.) to form liposomes and washed twice as before.

The washed liposome plug was resuspended in 3.3mM phosphate buffer (pH 7.4. 2ml,). and duplicate samples (1111. ) were taken for scintillation counting. 21% of the initial amount of inulin was retained iii liposomes after the first liposome preparation and washing. 17% of this 21%. that is 4% of the initial amount of iiiulin. 'v-is retained iii the liposomes after freeze-drying and reconstitution.

Claims (17)

WHAT WE CLAIM IS:

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

2. A method as @@ claimed iii claino 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 lipid-soluble or lipid-bound compound.

6. A method as claimed in any one of claims 1 to 4 in which the biologically active compound is a water-soluble, non-lipidbound 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, dipalmitoyl-phosphatidic acid, dicetyl phosphate or beef brain ganglioside.

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.