Liposomes containing particulate materials

Details Liposomes containing particulate materials Liposomes containing particulate materials

2001-09-27

2002-09-17

Kishore, Gollamudi S. (Department: 1615)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C424S204100, C424S234100, C424S240100, C424S246100, C424S258100, C424S265100, C424S269100, C264S004100, C264S004300, C264S004600

Reexamination Certificate

active

06451338

ABSTRACT:

The present invention relates to liposome preparations capable of use in administration of organic solvent labile materials, such as whole live or attenuated cells, to human or animal bodies. Such preparations have utility in delivery of labile bioactive materials whereby a slow release is provided which may b e targeted to specific body areas. A method for the manufacture of such preparations is also provided.
The use of liposomes in the administration of vaccine agents is well known, and their adjuvant activity has been demonstrated by numerous studies into immunopotentiation of a large variety of bacterial, viral, protozoan, protein and peptide vaccines; see reviews by Gregoriandis G (1990) Immunol Today, 11, 89-97 and Alving C R (1991) J Immunol Meth, 140, p1-13.
These studies have all been carried out using liposomes produced by techniques which generate vesicles of submicron average diameter (see Gregoriadis G (ed) (1993) Liposome Technology, 2nd Edition, Volumes I-III CRC Press, Boca Raton, 1993) which are capable of accomodating peptides and proteins, but not capable of efficiently carrying larger vaccines. Such larger vaccines include a number of attenuated or killed viruses and bacteria such as measles, polio virus,
Bordetella pertussis
, Bacille Calmette-Guerin and
Salmonella typhi
(see Mimms C A et al (1993) Medical Microbiology, Chapter 36, Mosby).
Although most of these vaccines are highly immunogenic, there are circumstances where their administration in sufficiently large liposomes may be a preferred alternative. For instance, in the case of multiple vaccines consisting of a mixture of soluble and particulate (eg. microbial) antigens or vaccine formulations also containing cytokines, simultaneous presentation of all materials to immunocompetant cells via a common liposome carrier may be advantageous in terms of improving the immunogenicity to antigens.
Furthermore, liposomes incorporating antigenic material in their aqueous phase are known to prevent interaction of the antigen with its antibodies in pre-immunized animals and ensuing allergic reactions or antigen neutralisation (Gregoriadis and Allison (1974) FEBS Lett., 45, 71-74. It can thus be seen that liposomes could be beneficial if employed as carriers for administration of vaccines to infants for prophylaxis against agents for which maternal antibodies were present, eg, such as measles, or to individuals with hypersensitivity to vaccine contaminants.
It is known to incorporate particulate materials into large liposomes having average diameter up to 9.2 &mgr;m by methods wherein solvents such as chloroform are formed into spherules containing smaller water droplets (see Kim and Martin (1981) Biochimica et Biophysica Acta, 646, 1-9). Using this technique materials such as Collagen, DNA and bacteria (
Streptococcus salivarius
) were entrapped, but it was noted that labile globular proteins such as serum albumen and haemoglobin did not allow liposome formation, presumably due to surface denaturation, and that protein denaturation occurred. Such method is unsuitable for the encapsulation of labile materials due to the damaging and cytotoxic effects of the organic solvent, and certainly unsuitable for the encapsulation of whole (live) or attenuated bacteria, protozoa, viruses or multicellular animal or plant cells.
Methods for entrapping soluble materials in liposomes without use of organic solvents in the encapsulation step have been known for several years (see Kirby and Gregoriadis (1984) Liposome Technology, Vol I, Gregoriadis G (ed), CRC Press, Inc Boca Raton, Fla., pp 19-28; Deamer and Uster (1983) Liposomes, Ostro M J (ed) Marcel Dekker, Inc, NY. pp27-51; Deamer and Barchfield (1982) J Mol Evol 18, 203-206), and are based upon a method which dehydrates preformed liposomes then rehydrates them in the presence of the soluble materials. In these methods the soluble materials enter with water as the liposomes fuse together resulting in material being entrapped in multilamella liposomes. The liposomes used were 40 to 80 nm in diameter before freeze drying and the multilamellar product vesicle volume resulting was still smaller. Such volume and structure are unsuitable for encapsulating micrometer size and/or living materials, and entrapment levels for soluble drugs are not as high as for unilamella liposomes due to relatively low surface area for entry into the vesicles. The same technique has also been applied to small unilammela liposomes for the purpose of encapsulating aqueous solutions (see EP 0171710).
The aforesaid process is relatively mild and has been used to successfully encapsulate labile solutes such as factor VIII (see Kirby and Gregoriadis (1984) Biotechnology, 2, 979-984) and tetanus toxoid (Gregoriadis et al (1987) Vaccine, Vol 5, p145-151). It relies upon solute entering the liposomes as they form while rehydration water enters. Despite such work on solutes, there has still not been provided a method for the encapsulation of whole (live) or attenuated organisms, cells or other insoluble structures bearing labile entities, without damaging them; whether bacterial, protozoan, viral or otherwise.
Furthermore, no method has yet been provided for encapsulating water labile soluble materials in larger liposomes, whether unilamellar or multilamella, that would allow targeting at specific tissues with still higher quantities of material.
The present inventors have now surprisingly found that dehydration/rehydration is capable of successful encapsulation of insoluble particulates such as whole live or attenuated organisms, cells, or microscopic water insoluble structures having organic solvent labile activity, whereby organisms are not killed and activity is retained. The invention allows micrometer sized unilamella and multilamella liposomes to be produced, (ie. 0.1-50 &mgr;m diameter liposomes) which in contrast with the liposomes of the prior art, are capable of entrapping micrometer size and/or living material, and have inner vesicles of relatively high capacity, being similar in size to their outer diameter in the case of the unilamella giant liposomes.
It is particularly surprisingly that (i) when micrometer sized liposomes are dehydrated then rehydrated in this manner, unilamella liposome structure is retained which offers improved capacity for soluble material as well as the ability to retain particulates described above and (ii) where conditions are used such that multilamella liposomes are formed containing insoluble or undissolved material they are of micron size rather than the previously obtained 40 to 80 nm in diameter.
Thus in a first aspect of the invention there is provided a method for forming liposomes of greater than 0.1 &mgr;m diameter, preferably greater than 1 &mgr;m diameter, containing undissolved or insoluble particulate biologically, chemically or physically active material comprising (a) forming unilamellar liposomes (b) freeze drying the liposomes so formed and then (c) rehydrating them in intimate admixture with the undissolved or insoluble material to be contained therein.
Where unilamella liposomes are to be produced step (a) forms liposomes of greater than 0.1 &mgr;m in diameter and uses these in step (b). Where multilamella liposomes are to be produced the size of the liposomes need not be fixed in step (a), but determined by the undissolved or insoluble material with which they are preferably freeze dried with in step (b) prior to rehydration in step (c).
The freeze drying step is, in the case of both unilamella and multilamella liposomes, preferably carried out on a mixture of the liposomes and material to be entrapped and may be carried out by known methods for freeze drying liposomes. The rehydration step is preferably controlled such that the number of liposomes destroyed by osmotic pressures induced by solute concentrations generated by water entering the vesicles is minimised.
In a second aspect the present invention further provides liposomes produced by the method of the first aspect of the invention, and particularly provides liposomes characterised in

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