SPECIFICATIONLiposomic SuspensionsThe invention relates to the preparation ofliposomes. Liposomes are pharmaceutical compositions in which the drug is contained in corpuscles consisting of aqueous and lipidic concentric layers. The drug can be contained both in the aqueous and in the lipidic layer. The lipidic layer generally comprises a phospholipid, such as lecithin or sphyngomyelin, a steroid, for example cholesterol, and an ionic tensioactive substance, such as dicetylphosphate, stearylamine or phosphatidic acid. Other hydrophobic materials may be present. Diameters of liposomes generally range from 1 5 nm to 5,.
The procedure usually employed for preparing liposomes comprises two main steps, preparation and separation.
A conventional preparation step comprises dissolving the lipidic components in a suitable solvent, such as chloroform, which is then evaporated off, generally under reduced pressure.
To the flask containing the residue as a thin layer, an aqueous solution of the drug is added and the whole is emulsified by submission to ultrasonic shaking for a time ranging from 10 seconds to some hours. The liposomic suspension so obtained contains an important fraction of nonentrapped drug which must be separated from the liposomes.
A conventional separation step is carried out by column chromatography with a resinous molecular sieve, for example Sepharose 2B, 4B or 6B. Sepharose is a Trade Mark. The liposomes elute first, whereas the free drug is retained by the resin. An alternative separation step, also conventional, is the ultracentrifugation at100,000 g and subsequent washing, also by ultracentrifugation, with buffered solution. A third conventional separation step is dialysis.
British Patent Specification No. 7,838,495 (2004745A), in our former name of SocietaFarmaceutici Italia S.p.A., describes a separation step which comprises shaking the suspension with a polymeric ion exchange resin or a polymeric absorbent resin and filtering off the resin. Suitable polymeric ion exchange resins for use in this separation step include both solid and liquid synthetic organic polymers, usually but not necessarily crosslinked by functional moieties.
They may be of the weak, average or strong cationic type, cross-linked by carboxylic, phosphonic or sulphonic functions, for example.
Alternatively they may be of the strong anionic type, 1 st and 2nd type, or of weak or average anionic type, cross-linked by salified quaternary ammonium, primary, secondary or tertiary aminic or phosphinic functions, for example. Suitable matrices include phenolformaldehyde, styrenedivinylbenzol, acrylates, methacrylates, and various hydrocarbons and other condensation resins.
They may be used in salified or activated form.
The polymeric absorbent resins can be employed owing to the difference of polarity between thehydrophobic substances forming the liposomic shell and the drugs which are hydrophilic; aliphatic, aromatic and inorganic absorbent resins can be used. Shaking is preferably carried out for10 to 60 minutes. This purification procedure enables very concentrated liposomic suspensions to be obtained (for example, up to 5 mg/ml of doxorubicin hydrochloride). Such concentrations cannot be achieved by chromatography with a resinous molecular sieve (max 0.3 mg/ml). The liposomic suspension achieved is very stable and not inclined to sediment, unlke those obtained by ultracentrifugation.
The conventional use of ultrasonic shaking for emulsification in the preparation step, however is liable to cause titanium contamination of the liposomic suspension from the horns, tips or microtips of the sonic processor, and furthermore certain large molecules may be disrupted by the ultrasonic energy.
The invention provides a method for the preparation of a liposomic suspension, the method comprising (a) preparing the liposomic suspension by dissolution of lipidic components in a solvent, addition of an aqueous solution of a drug, and emulsification by insufflation of an inert gas until the solvent is completely removed by evaporation, and (b) the liposomic suspension by shaking the suspension with a polymeric ion exchange resin or a polymeric absorbent resin.
Definitive chemical stabilisation may be achieved by lyophilisation of the liposomic suspension.
Preferred drugs for use in the method according to the invention include doxorubicin hydrochloride, aminosidine sulphate and 5fluorouracil.
The resins used in step (b) may be as described above, specifically suitable ones being Rohm a Haas IRC-50, Dowex 50-X 4 100--200 mesh,Amberlite IRA-400 (Cl), Dowex I 50-100 mesh and Rohm a Haas XAD-7. These resins are specified by the Trade Marks applied to them.
The invention is illustrated by the followingExamples.
Example 11.5 g of soya-lecithin, 0.4 g of cholesterol and 0.3 g of dicetylphosphate were dissolved in dichloromethane. A solution of aminosidine sulphate in 0.02M buffer phosphate at pH 6.5 at the concentration of 3 mg/ml was added. The two phases were emulsified under shaking, nitrogen being bubbled through at room temperature until complete removal ofthe dichloromethane.
The suspension was stood at room temperature for 4 hours and then into the flask an amount equivalent to 5 g of dry resin of Rohm andHaas IRC-50 resin was poured. After 1 hour of shaking the liposomic suspension was filtered through a sintered glass filter to remove the resin which had retained the non-entrapped drug. The liposomic suspension was then stabilised by lyophilisation.
Example 2Operating as described in Example 1, 2.3 g of egg lecithin, 0.65 g of cholesterol and 0.15 g of octadecylamine were dissolved in 50 ml of dichloromethane and the solution was poured into a flask containing 250 mg of mbenzoylhydratropic acid (generic nameKetoprofen) in 1 50 ml of Na, K buffer phosphate 0.02 M at pH 7.4. Inert gas (nitrogen) was bubbled into the flask, under shaking, until complete removal of the organic solvent and resulting formation of the liposomic suspension.
10 ml of anion exchange resin IRa 400 (Cl-) manufactured by Rohm and Haas were added to the suspension. After 30 minutes of shaking, the resin was removed by filtration and the purified liposomic suspension was lyophilized.