CS227010B2 - Suspensions of Drug- containing Liposomes - Google Patents

Abstract

Pharmaceutical suspensions of liposomes containing a drug (e.g. doxorubicin hydrochloride, aminosidine sulphate or 5- fluorouracil) are prepared by dissolution of lipidic components in a solvent, addition of an aqueous solution of the drug, and emulsification by insufflation of an inert gas until the solvent is completely removed by evaporation, and then the liposomes are separated from non-entrapped drug by shaking the suspension with a polymeric ion exchange resin or a polymeric absorbent resin to absorb the non- entrapped drug, then filtering off the resin. The ion exchange resin may be a strong or weak, anionic or cationic resin. The resultant liposomic suspensions may be lyophilised.

Description

The invention relates to a process for obtaining, extracting and stabilizing inhomogeneous systems, known as liposome suspensions, and stabilizing them by freeze-drying.

Liposomes are pharmaceutical kits in which the drug is present either dispersed or in other form as beads, consisting of aqueous concentric layers that adhere to the lipid (hydrophobic) layers: the drug may be present both in the aqueous layer and in the aqueous layer lipid layer (inside or outside) or in some way in a non-homogeneous system, usually known as a liposome suspension.

The hydrophobic layer is generally, but is not limited to, phospholipids such as castitin and sphyngomyelin, steroids such as cholesterol, more or less ionic surfactants such as dicetyl phosphate, stearylamine or phosphatidic acid, and / or other hydrophobic substances; the diameter of liposomes is usually in the range of 15 nm to 5 μιΐη.

The prior art manufacturing process usually comprises two main stages:

(a) production of a liposome suspension;

b) removing physically unbound drug from the liposome suspension,

In order to produce a lipcsome suspension, the lipid or lipophilic component is dissolved in a suitable solvent, which is then evaporated to dryness, usually under reduced pressure. The aqueous drug-containing layer is placed in a beaker containing the thin film evaporation residue and then subjected to mechanical shaking or ultrasonic shaking for a few seconds within a few hours. This results in an inhomogeneous layer, usually called a liposome suspension, which must be freed from the physically unbound drug by purification.

b) Removal of the physically unbound drug from the liposome suspension is generally accomplished by eluting the liposome suspension on the chromatography column using resins having exclusively molecular sieves such as Sephadex or Sepharose, 2B, 4B or 6B and others. This first results in liposomes, while the free drug is retained by the resin. This is followed by treatment in an ultra centrifuge at 100,000 g, after which the reaction mixture is washed with a buffered solution while still centrifuging in an ultra centrifuge.

Another method used to purify a liposome suspension is dialysis.

Gel filtration on resins such as Sepharose or Sephadex, according to the prior art, is a widespread brewing method used for the above-mentioned purpose, although it has the disadvantage of having to pre-saturate the resin bed in order to avoid a decrease in the yield of product in the eluate, which in addition is considerably diluted. Another disadvantage is that ultra-centrifuge processing cannot be used when the size of the liposomes is very small (for example in the range of 15 to 20 .mu.m) or when the composition or size of the bilayer is such that it would not withstand overloading 100,000 g in the centrifuge without leaking pharmaceuticals.

As far as dialysis is concerned, this method, in addition to requiring an extremely long operating time, is disadvantageous because of the undesirable adsorption of liposomes on the membranes, and also because it requires ultrafiltration, but which can only be used with very small dimensions and low molecular weight.

When using prior art resins, for example Sephadex or Sepharose, k. For this purpose, they are in each case gel resins acting as molecular sieves in which the product is trapped in their pores, which depends on the physical characteristics of its molecules, while the inventive method uses ion-exchange and absorption resins of organic or inorganic origin capturing the product by ionic chemical bonds, which makes it possible to dispense with subsequent chromatographic work-up; this is one of the advantages of the method according to the invention over the prior art; Another advantage is its very simple and easy operation as well as time savings.

Accordingly, the present invention provides a process for obtaining, extracting and stabilizing inhomogeneous systems, known as liposome suspensions, as well as purifying them from physically unbound medicaments, the method comprising treating the suspensions at a temperature in the range of 20-25 ° C. at atmospheric pressure for 10 to 120 minutes with liquid or solid polymers of synthetic or organic nature used as ion exchangers, or absorbent polymers, which polymers have chemical functionalities and are optionally cross-linked with a reactive chemical agent, such as -styrene , divinylbenzene, - acrylic acid and methacrylic acid, after which the purified suspension is freeze-dried.

Preferably, strong, medium or weak cation exchange resins such as phenol-formaldehyde resins, styrendivinylbenzene resins, acrylates, various hydrocarbons, and various condensation resins in liquid and solid form, having carboxyl, phosphonic and sulfone groups are used.

Strong anion exchange resins may also be used with trimethylammonium or dimethylethylammonium groups, or medium or weak anion exchange resins such as, for example, various resins having primary, secondary, tertiary amine or phosphine groups in the form of salts; other groups such as phenol-formaldehyde resins, styrendivinylbenzene resins, acrylates, methacrylates, various hydrocarbons and various condensation resins in liquid and solid form. These resins may be used in the form of salts or in activated form.

To carry out the process of the invention, a precisely weighed amount of at least one of the above resins is introduced directly into a beaker containing a liposome suspension to be subjected to purification, followed by shaking for 10 to 60 minutes. After filtration through a sintered-glass filter in which the ion exchange resin is retained with absorbed, physically unbound drug, a pure liposome suspension is obtained which can be freeze-dried.

A great advantage of this method of purifying the liposome suspension with ion exchange resins is that it is possible to maintain highly concentrated liposome suspensions (up to 5 mg / ml of Dissolve liposomes). It is not possible to maintain it on the column of molecular sieves (maximum 0.3 mg / ml). The liposome suspension thus obtained is very stable and does not tend to settle, such as torsion, in the suspensions obtained by centrifugation in an ultra centrifuge.

The same result is obtained when polymers and polymers without specific chemical function are used in place of the resin exchangers, which generally, but not exclusively, react on the basis of van der Waals forces and are generally known as adsorbent resins. These resins can be used to purify -suspensions of liposomes due to - polarity differences - between hydrophobic substances, which form the liposome envelope, and medicaments with a - more or less - hydrophilic nature. As adsorbents of inorganic origin, silica, alumina and the like can be used.

Final chemical stabilization - se. is achieved by drying the suspension - freeze-drying.

The invention is illustrated by the following examples. ,

Examplel

In the saponification beaker, the following lipid components are dissolved in chloroform and evaporated to dryness under reduced pressure: 1.5 g of egg lecithin, 0.4 g of cholesterol and 0.2 g of dicetyl phosphate. A 10 mg / ml solution of Doxorublubline hydride chloride in 0.007 N phosphate buffer was then added to the beaker and the mixture was sonicated for 1 minute. The suspension is then allowed to stand for 30 minutes under nitrogen at room temperature. Then, 2 g of methyl methacrylate polymer with divinyl-benzene, which is pre-activated to the - Na + -cycle, are added, with free carboxyl groups and a macroreticular structure which makes it possible to use it also in -hyd227010 organic solutions and is commercially available. available under the designation IRC-50 (manufactured by Rohm and Haas) (the amount is based on dry matter and corresponds to 5 ml of swollen resin).

The beaker is then shaken for about half an hour, then the suspension is filtered through a sintered glass filter.

The thus obtained liposomes of various sizes ranging from 0.5 to 2 μm and containing approximately 60% of the starting Doxorubicin are stabilized by freeze-drying.

* A p 1 a d 2

The procedure is as in Example 1, using the same lipid components and Doxorubicin. However, ultrasound shaking is performed for 10 minutes to obtain liposomes of less than 1 µm.

Since liposomes do not exhibit a completely homogeneous size, a non-granular resin is also used, which also acts as a sieve.

To this end, add 10 ml of a resin consisting of a transverse crosslinked styrene polymer divinylbenzene-Dowex 5Ό-o 4 with a particle size range of 74 to 149 μΐη, which has been previously activated in the Na ⁺ -cycle. After filtration, a suspension is obtained which contains liposomes having a size ranging from 0.12 to 0.8 gm and showing 75% of the starting Doxorubicin. Liposomes are stabilized by freeze-drying.

Example 3

A 10 mg / ml solution of 5-fluorouracil in 0.007 N phosphate buffer at pH 8 is poured into a saponification beaker containing the lipid phase obtained as described above. The resulting suspension is treated as described in Example 1 using a filter as the filter. 10 ml resin, consisting of a styrene polymer cross-linked with divinylbenzene-Amberlite IRA-400 (C1), which is pre-activated to the hydrochloride cycle. The liposomes thus obtained are stabilized by freeze-drying.

Example 4

The liposomes of 5-fluoro-racil are produced with the lipid components below: 1.5 g egg lecithin, 0.6 g cholesterol and 0.2 stearyl amine.

* 10 ml of a pre-activated resin consisting of a styrene polymer crosslinked with divinyl ⁹ benzene-Dowex 1 (particle size 149 to 300 .mu.m) was used as the cleaning system. The liposomes thus obtained are stabilized by freeze-drying.

Example 5

The procedure of Example 2 is repeated except that 15 grams of XAD 7 adsorbing resin (Rohm and Haas) is used instead of Dowex and the shaking time is extended to 40 minutes.

Filtration through a sintered glass filter G 1 gives a liposome suspension containing approximately 50% of the starting amount of Doxorubicin. Liposomes are stabilized by freeze-drying.

Example 6

1.5 g soya lecithin, 0.4 g cholesterol and 0.3 g diacetyl phosphate are dissolved in methylene chloride CH2Cl2 and a solution of aminosidine sulfate in 0.02 M phosphate buffer at pH 6.5 at 3 mg / ml is added to this solution.

Both phases are emulsified and the resulting emulsion is shaken, whereupon methylene chloride is completely ejected at room temperature by introducing nitrogen into the emulsion.

The suspension is stabilized for 4 hours at room temperature and the resin is poured into the beaker in an amount corresponding to 5 g of a dry resin consisting of a methacrylic acid polymer crosslinked with divinylbenzene-IRC-50 (Rohm and Haas).

After shaking for 1 hour, the liposome suspension is filtered through a sintered glass filter to remove the resin that retains the physically unbound drug. The liposome suspension is then stabilized by freeze-drying.

Example 1 a d 7

The procedure is as in Example 6, but 2.3 g of egg lecithin, 0.65 g of cholesterol and 0.15 g of octadecylamine are dissolved in 50 ml of methylene chloride CH2Cl2. The resulting solution was poured into a flask containing 250 mg of m-benzoylhydratropic acid in 150 mL of 0.02 M sodium potassium phosphate, pH 7.4.

While shaking, nitrogen is introduced into the flask until the organic solvent is completely removed and a liposome suspension is added to which 10 ml of anion exchange resin IRA 400 is added (ion cycle Cl, bulk density 0.71, true density 1.11, grain 380-450). After a 30 minute shaking, the resin is filtered off and the purified liposome suspension is freeze-dried.

Claims (10)

OBJECT OF THE INVENTION A method for obtaining, extracting and stabilizing non-homogeneous suspensions of 11posomes of known systems, as well as purifying them from physically unbound drugs, characterized in that the suspensions are treated at a temperature in the range of 20-25 ° C at atmospheric pressure for a time in the range 10 to 120 minutes with liquid or solid ion exchangers used by synthetic or organic polymers, or by adsorption polymers, which polymers have chemical functional groups and are optionally cross-linked with a reactive chemical agent such as styrene, divinylbenzene, acrylic acid and methacrylic acid then the purified suspension is freeze-dried. 2. A process according to claim 1, wherein strong, medium or weak cation exchange resins such as phenol-formaldehyde resins, styrendivinylbenzene resins, acrylates, methacrylates, various hydrocarbons and various condensation resins in liquid and solid form are used. having carboxyl, phosphone and sulfone groups. 3. The process according to claim 1, characterized in that strong anion exchange resins with trimethylammonium or dimethylethylammonium groups or medium or weak anion exchange resins, such as various resins having primary, secondary, tertiary amine or phosphine groups, are used. in the form of salts or other groups such as. are, for example, phenol-formaldehyde resins, styrendivinylbenzene resins, acrylates, methacrylates, various hydrocarbons and various condensation resins in liquid and solid form. 4. A process according to claim 1, wherein the resins are used in the form of salts or in activated form. 5. A method according to any one of claims 1 to 4, wherein the liposome suspension is completely or partially extracted after disrupting the lipid structure of the liposomes. 6. The method of claim 1, for purifying non-homogeneous systems, known as liposome suspensions, from physically unbound or liposome-bound drug by fully or partially extracting it, characterized in that it is a suspension. acts with organic adsorbents. polymers and / or. copolymers of divinylbenzene and styrene, or adsorbents of inorganic origin, for example silica or alumina. 7. The method of item 1, k. obtaining liposome suspensions, characterized in that the suspensions are obtained by injecting an inert gas such as nitrogen into a biphasic emulsion; by adding immiscible solvents. 8. The method of claim 1, wherein the purified liposome suspension is freeze-dried. 9. The process of claim 1 for obtaining liposome suspensions, characterized in that the suspensions are obtained by injecting an inert gas such as nitrogen and the like into a biphasic emulsion or by adding immiscible solvents. 10. The method of claim 1, wherein the purified liposome suspension is freeze-dried.