Device for obtaining and administering unilamellar liposomes

Details Device for obtaining and administering unilamellar liposomes Device for obtaining and administering unilamellar liposomes

1985-06-06

1988-12-20

Lovering, Richard D.

Surgery

Means for introducing or removing material from body for...

Treating material introduced into or removed from body...

604190, 264 46, 424450, 4284022, 436829, A61M 500

Patent

active

047923318

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to an improved process for obtaining unilamella liposomes of high diameters and pharmacological application thereof for encapsulating an active principle for extemporaneous administration thereof.


BACKGROUND OF THE INVENTION

During the last few years, many studies have shown that liposomes can be used for encapsulation and transfer of an active substance into cells. To this end, phospholipid vesicles have been mainly prepared by physical techniques such as ultra-sounds, the so called "French press" technique or chemical procedures by using organic solvents or detergents followed by elimination of the latter.
However, the liposomes obtained nowadays present either structures or dimensions which do not always permit either encapsulation of certain macromolecules of a cumbersome nature or encapsulation of a relatively high quantity of active substance, with encapsulated quantities remaining until now limited to certain values.


SUMMARY OF THE INVENTION

Consequently, the purpose to reached by the Applicants is to find out a process for preparation of big unilamellar liposomes capable of encapsulating macromolecules of high dimensions (proteins or nucleic acids), on the one hand, and on the other hand, of encapsulating higher quantities of an active substance than was possible until now. Furthermore, such process should be sufficiently moderate and more especially should avoid contact with a an organic solvent, denaturing proteins or high energies such as ultra-sonic emission or the application of pressures capable of breaking up molecules of nucleic acids. The procedure having recourse to detergent appears to be the best method of preserving the structure and activity of such macromolecules.
Elimination of the detergent by dialysis or the filtration method on gel provides unilamellar liposomes. The internal volume of such liposomes varies with the nature of the detergent, the molecular ratio of the detergent to phospholipids, the lipid composition of the vesicles and the dialysis rate. The vesicle dimensions appear to be different in size but in the same order of magnitude whether it be after dialysis or after filtration on gel. However, scarceness and/or costs of the molecules to be encapsulated (antibodies, enzymes, purified or ARN carrier genes) require very high encapsulation rate and exclude procedures requiring large quantities of substance to be encapsulated as well as elimination of the detergent through filtration on gel.
The process according to the invention is based on the procedure of elimination of the detergent by dialysis and is an improvement thereof. As a matter of fact, one of the main disadvantages encountered upon formation of liposomes by the procedure utilizing detergents resides in that elimination of the detergents is never total; very often a significant quantity of detergent remains even after exhaustive dialysis. Thus, with the usually employed detergents, in particular, sodium cholate or desoxycholate and "Triton X-100", the residual quantity can be from a few % to 10%.
The Applicants however found in an entirely unexpected manner that if the detergent used for lipid solubilization for the formation of a liposome is a neutral detergent presenting a high critical micellar concentration under certain dialysis conditions, its quick and almost total elimination can be obtained thereby to permit realization of liposomes encapsulating an active principle such as a medicament practically free of detergent.
Furthermore, unilamella liposomes of high diameter from 200 to 1000 nm are obtained, which are therefore capable of encapsulating either large molecules in suitable quantities or high quantities of less cumbersome molecules thereby to remedy the disadvantages inherent to the known liposomes.
The Applicants have noted that for the particular application as the vehicle of a medicament practically free of detergent there can be cited as a detergent responding to the above definition, suitable according to the invention, octylglucoside whi

REFERENCES:
Rhoden et al.: "Formation of Unilamellar Lipid Vesicles of Controllable Dimensions by Detergent Dialysis", Biochemistry, vol. 18, No. 19, 1979, pp. 4173-4178.
Rosevear et al.: "Alkyl Glycoside Detergents: A Simpler Synthesis and Their Effects on Kinetic and Physical Properties of Cytochromec Oxidase", Biochemistry, vol. 19, No. 17, 1980, pp. 4108-4115.
Mimms et al.: "Phospholipid Vesicle Formation and Transmembrane Protein Incorporation Using Octyl Glycoside", Biochemistry, vol. 20, No. 4, 1981, pp. 833-840.
Chem. Abstr., vol. 95, No. 9, Aug. 31, 1981, p. 393, 76456k.
Chem Abstr., vol. 97, No. 5, Aug. 2, 1982, p. 216, 35020m.
Chem. Abstr., vol. 98, No. 1, Jan. 3, 1983, p. 227, 2483g.
Chem. Abstr., vol. 99, No. 25, Dec. 19, 1983, p. 354, 209201e.
Fry et al., "Rapid Separation of Low Molecular Weight Solutes from Liposomes without Dilution", Analytical Biochemistry, 90, 809-815 (1978).
Szoka, Jr. et al.: "Procedure for Preparation of Liposomes with Large Internal Aqueous Space . . . ", Proc. Natl. Acad. Sci. U.S.A., vol. 75, No. 9, pp. 4194-4198, Sep. 1978.

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