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

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

There are numerous documents describing the preparation and use of liposomes, such as vehicle-biologically active substances such as drugs, proteins, enzymes, diagnostic agents or cosmetics. Thus, water-soluble substances can be encapsulated in the aqueous spaces of the liposome, or lipophilic materials may be incorporated in the lipid wall.

Systems a process for preparing oligolamellar vesicle has already been described by Bangham et al. (J. Mol. Biol.<u>13</u>, 238-252; 1965). According to this method, 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 known methods derived therefrom, does not allow to directly obtain liposomes of submicron particle size, which would allow a much greater stability of the particles and their dispersions.

EP-A-0130577 describes a liposome preparation method by mixing membrane components with a non-volatile solvent and dispersing the resulting mixture in an aqueous medium at elevated temperature.

The invention provides a simple and widely applicable, liposome preparation of submicroscopic size.

The invention therefore relates to a system method for preparing colloidal dispersible amphiphilic lipids, in the form of oligolamellar liposomes submicron whose wall is constituted by the said lipids and optionally a substance A and of which the core consists Parde water or an aqueous solution, optionally containing a substance B, characterized in that:<ul><li>(1) preparing a liquid phase consisting essentially of a solution of 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, the concentration of lipids in the solvent volatile being from 0.1 to 10% by weight</li><li>(2) preparing a second liquid phase consisting essentially of water or an aqueous solution of substance B,</li><li>(3) is added under moderate agitation, without the use of the Hamilton syringe first phase to the second phase, so as to obtain almost instantaneously a colloidal suspension of liposomes;</li><li>(4) if desired, are removed all or part of the solvent or mixture of solvents and water, so as to obtain a colloidal suspension of desired liposome concentration.</li></ul>

The substance A, lipophilic, is intended to modify the physical characteristics (electrical load, stiffness) or chemical from the wall. It may be cholesterol, stearylamine, phosphatidic acid, alpha-tocopherol, a nonionic surfactant, etc ...

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

The amphiphilic lipids can be glycolipids, phospho-aminolipids, and in particular phospholipids, such as lecithins (egg, soybean, etc ...) ..

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

The lipid concentration in the solvent may be preferably 1 to 5% by weight.

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

The expression "moderate stirring" agitation such as magnetic stirring, 10 to 500 rpm, for example about 100 rpm.

Thus, the invention provides drugs, including injectable form, and cosmetics that are very stable.

The following examples illustrate the invention.

example 1

Preparation of liposomes.

<tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="2" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col2" align="left">organic phase 1</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">soy lecithin (Epikuron 170)</entry><entry namest="col2" nameend="col2" align="char" char=",">2.0 9</entry></row><row><entry namest="col1" nameend="col1" align="left">absolute ethanol</entry><entry namest="col2" nameend="col2" align="char" char=",">50.0 g</entry></row></tbody></tgroup><tgroup cols="2" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col2" align="left">aqueous phase 2</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="left">Water</entry><entry namest="col2" nameend="col2" align="char" char=",">100.0 g</entry></row></tbody></tgroup></table></tables>

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

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

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

The review by transmission microscopy shows oligolamellar liposomes of uniform size.

example 2

Preparation of liposomes containing cholesterol (Variant of Example 1).

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

example 3

: Variant of Example 1.

The procedure is as in Example 1, but replacing soybean lecithin of egg lecithin. The obtained liposomes have the same characteristics as in Example 1.

example 4

: Variant of Example 2.

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

example 5

Preparation of liposomes containing a hydrophilic active principle.

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

The 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.

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