Process for the preparation of dispersible colloidal systems of amphiphilic lipids in the form of submicronic liposomes

0001The present invention relates to a process for the preparation of dispersible colloidal systems of amphiphilic lipids, in the form of submicron oligolamellar liposomes.

0002Numerous documents are known which describe the preparation and use of liposomes, in particular as vehicles for biologically active substances such as drugs, proteins, enzymes, diagnostic agents or cosmetic products. Thus, water-soluble substances can be encapsulated in the aqueous spaces of the liposome, or lipophilic substances can be incorporated into the lipid wall.

0003A process for the preparation of oligolamellar vesicular systems has already been described by Bangham et al. (J. Mol. Biol. G, 238-252; 1965). According to this process, the lipids and lipophilic substances are dissolved in an organic solvent and treated with an aqueous phase with vigorous stirring. However, this process, like most of the known processes which derive therefrom, does not make it possible to directly obtain liposomes of particle size less than a micrometer, which would allow much greater stability of the particles and their dispersions.

0004EP-A-0 130 577 describes a process for the preparation of liposomes by mixing the membrane components with a non-volatile solvent and dispersing the resulting mixture in an aqueous medium at high temperature.

0005The invention provides a simple and widely applicable process for the preparation of submicroscopic size liposomes.

0006The invention therefore relates to a process for the preparation of dispersible colloidal systems of amphiphilic lipids, in the form of submicron oligolamellar liposomes, the wall of which consists of said lipids and optionally of substance A and the core of which consists of water or an aqueous solution, and optionally containing a substance B, characterized in that:<ul id="ul0001" list-style="none"><li>(1) a liquid phase is prepared essentially consisting of a solution of the amphiphilic lipids and optionally of substance A in a volatile solvent or in a mixture of volatile solvents, and which may contain substance B in solution,</li><li>(2) a second liquid phase consisting essentially of water or an aqueous solution of substance B is prepared,</li><li>(3) the first phase is added to the second phase, with moderate stirring, so as to obtain, almost instantaneously, a colloidal suspension of liposomes;</li><li>(4) if desired, all or part of the solvent or mixture of solvents and water is removed, so as to obtain a colloidal suspension of desired concentration of liposomes.</li></ul>

0007Substance A, lipophilic in nature, is intended to modify the physical (electrical charge, rigidity) or chemical characteristics of the wall. It can be cholesterol, stearylamine, phosphatidic acid, alpha-tocopherol, a nonionic surfactant, etc.

0008Substance B is a biologically active substance, in particular a medicinal active principle or a medicinal precursor, a biological reagent or a cosmetic product. Substance B is introduced into phase (1) if it is lipophilic and into phase (2) if it is hydrophilic.

0009The amphiphilic lipids can be glycolipids, phospho-aminolipids, and in particular phospholipids, for example lecithins (egg, soy, etc.).

0010The volatile solvent is preferably an alcohol miscible with water in all proportions, in particular ethanol.

0011The concentration of lipids in the solvent can be 0.1 to 10% by weight, preferably 1 to 5% by weight.

0012It is advantageous that the volume of solvent used for phase (1) is between 5 and 100%, for example around 50%, of the volume of water in phase (2), in order to obtain small liposomes (in particular from 100 to 300 nm).

0013The term “moderate agitation” is understood to mean agitation such as magnetic agitation, from 10 to 500 rpm, for example around 100 rpm.

0014Thus the invention makes it possible to obtain drugs, in particular in injectable form, and cosmetic products which are very stable.

0015The following examples illustrate the invention.

Example 1: Preparation of liposomes.

0016<tables id="tabl0001" num="0001"><img file="EP0349429B1_D0001.tif" /></tables>

0017Phase 1 is added with magnetic stirring to phase 2. The medium immediately becomes opalescent by formation of liposomes. The average size of the liposomes, measured immediately after preparation, in a laser beam diffractometer (Nanosizer^RCoultronics), is 180 nm, with an average dispersion index of 0.5.

0018The alcohol is removed under reduced pressure, and the liposome suspension is filtered through a sintered glass (pores 9-15 nm).

0019The size of the liposomes, again measured in the filtrate, remains unchanged.

0020Transmission microscopy examination shows homogeneously sized oligolamellar liposomes.

Example 2: Preparation of liposomes containing cholesterol (variant of Example 1).

0021The procedure is as in Example 1, but adding 0.30 g of cholesterol to the alcoholic phase. The liposomes obtained having the same characteristics as in Example 1.

Example 3: Variant of Example 1.

0022The procedure is as in Example 1, but replacing the soy lecithin with egg lecithin. The liposomes obtained have the same characteristics as in Example 1.

Example 4: Variant of Example 2.

0023The procedure is as in Example 2, but replacing the soy lecithin with egg lecithin. The liposomes obtained have the same characteristics as in Example 1.

Example 5: Preparation of liposomes containing a hydrophilic active principle.

0024The procedure is as in Example 2, but adding 0.20 g of ampicillin (sodium salt) to the aqueous phase.

0025The incorporation rate of ampicillin in the liposomes, measured after separation of the liposomes from the aqueous phase by chromatography on Sephadex gel, is 10%.

Example 6: Preparation of liposomes containing a lipophilic active principle.

0026The procedure is as in Example 1, but adding 66.7 mg of muramyl-tripeptide-cholesterol to the organic phase. The rate of incorporation of the active ingredient is 100%.