JPH0133446B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides pharmaceutical compositions (lipidophilic liposomes),
Concerning methods for their production and purification.
More particularly, the present invention relates to a new method for purifying liposomal suspensions obtained by known methods and stabilizing them by lyophilization.

Liposomes are pharmaceutical compositions containing a drug dispersed or present in various forms in microspheres consisting of aqueous concentric layers adhered to a lipid layer (hydrophobic). The drug can be present in both the aqueous and lipid layers (inside or outside), or either way, in a heterogeneous system commonly known as a liposomal suspension.

Generally, but not absolutely, the hydrophobic layer is composed of phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, such as dicetyl phosphate, stearylamine or phosphatidium phosphate. Includes more or less ionic surfactant materials and/or other hydrophobic materials. The diameter of liposomes is generally 15 nm.
~5μ range. The manufacturing process, commonly carried out by known techniques, consists of two major steps: preparation of liposomes and purification of liposomes from unencapsulated drug.

(1) Preparation of liposomes The lipid or parent lipid component is generally dissolved in a suitable solvent which is then evaporated to dryness under vacuum.

The drug-containing aqueous layer is added as a thin layer to the flask containing the residue and the whole is subjected to mechanical or ultrasonic vibration for a period of 10 seconds to several hours. The heterogeneous layer thus obtained, generally referred to as a liposome suspension, must be purified from unencapsulated drug.

(2) Separation of liposomes from non-encapsulated drug This operation is usually carried out using resins with only molecular sieving function, such as Sepharose 2B, 4B or 6B.
It is carried out by elution on a chromatographic column using other methods.

Liposomes are first collected while free drug is retained in the resin.

Other methods used are ultracentrifugation at 100,000 g followed by washing by ultracentrifugation, always in a buffer solution. Dialysis can also be used as an alternative method.

The present invention relates to a generally synthetic or organic liquid resin having a chemical function that can be used as an ion exchanger, such as styrene, divinylbenzene, acrylic acid or methacrylic acid, commonly known as ion exchange resins. The present invention relates to a new method for purifying heterogeneous layers known as liposome suspensions with both polymers in solid form.

A precise amount of one or more of the resins mentioned above is introduced directly into the flask containing the liposome suspension to be purified, and this is
Shake for ~60 min. After passing through a sintered glass filter capable of retaining the ion exchange resin adsorbed with unencapsulated drug, a pure suspension of liposomes is obtained. This can be subjected to lyophilization. This method of purifying liposome suspensions using ion-exchange resins produces high concentrations of liposome suspensions (5 mg/ml for doxorubicin HCl) that cannot be maintained by molecular sieve column chromatography (up to 0.3 mg/ml). to)
shows the great advantage of retaining The liposome suspensions obtained in this way are very stable and, in contrast to those obtained by ultracentrifugation, do not tend to sediment.

Instead of ion exchange resins, use polymers and copolymers without specific chemical functionalities, commonly known as adsorbent resins, which usually, but not necessarily, react by Van der Waals forces. It is also achieved by These resins can be used to purify liposome suspensions due to the difference in polarity between the hydrophobic material and the somewhat hydrophilic drug that forms the liposome shell. Final chemical stabilization is achieved by lyophilization of the liposome suspension.

Reference Example 1 In a saponification flask, the following amount of lipids, i.e.
1.5 g egg lecithin, 0.4 g cholesterol and 0.2 g dicetyl phosphate are dissolved in chloroform and evaporated to dryness under vacuum. A solution of doxorubicin hydrochloride (10 mg/ml concentration) in 0.007N phosphate buffer is added to the flask and the suspension is subjected to ultrasonic vibration for 1 minute. Leave the suspension at room temperature under nitrogen for 30 min.

``IRC-'' previously activated in the sodium form.
50” resin (Rohm & Haas product) 2g
(Weight refers to dry weight and corresponds to 5 ml of swollen resin). This resin is obtained by polymerization of methyl methacrylate properly cross-linked with specific chemical reagents such as divinylbenzole, has carboxyl functionality and a macronetwork structure, and is suitable for use in hydrophobic solutions. It is possible.

The flask was shaken for approximately 30 minutes and the suspension
Pass through G1 porous sheet.

The liposomes obtained in this way, having various sizes from 0.5 to 2 μ and containing approximately 60% of the starting amount of doxorubicin, are stabilized by lyophilization.

Reference example 2 In order to obtain liposomes with a size of 1μ or less,
Operated as above, and using the same amounts of lipid and doxorubicin, the ultrasonic shaking time was extended to 10 minutes.

Since the liposomes did not give a completely uniform size, a non-granular resin was used that also had a sieving function. Therefore, the type of resin known on the market as "Dowex" 50-X-4 (100-200 mesh) pre-activated in the sodium form.
Add 10ml.

After filtration, a liposome-containing suspension is obtained having various sizes from 0.2 to 0.8 μ and containing 75% of the starting doxorubicin. This liposome is stabilized by lyophilization.

Reference Example 3 A solution of 5-fluorouracil at a concentration of 10 mg/ml in a 0.007N phosphate buffer with a pH of 8 is poured into a saponification flask containing the lipid phase prepared as described above. This suspension was previously activated as the hydrochloride salt as described in Example 1.
10 ml of a resin of the type known on the market as "Amberlite" IRA-400 (Cl) was used as overuse resin for processing.

The liposomes thus obtained are stabilized by lyophilization.

Reference Example 4 By operating as described above, the following amount of lipids, i.e.
Liposomes of 5-fluorouracil are prepared using 1.5 g of egg lecithin, 0.4 g of cholesterol, and 0.2 g of stearylamine.

"Dowex" 1 (50-100
10 ml of a resin of the type known on the market as methane) are used as the purification system. The liposomes thus obtained are stabilized by lyophilization.

Reference example 5 Reference example 2 is repeated, but with “Dowex”
ROHM instead of 50W-X (100-200 meters)
15 g of adsorbent resin XAD7 from And Haas is used and the shaking time is increased to 40 minutes. After passing through a sintered glass filter G1, a suspension of liposomes containing about 50% of the starting amount of doxorubicin is obtained. This liposome is stabilized by lyophilization.

Example 1 1.5g soybean lecithin, 0.4g cholesterol and 0.3g dicetyl phosphate in appropriate amounts
Dissolved in CH ₂ Cl ₂ and to this solution, PH6.5
Aminocidine sulfate 3 mg/in 0.02M phosphoric acid buffer
Add 70ml of the ml solution.

The two phases are emulsified and shaken, and nitrogen is bubbled through at nitrogen temperature until completely free of methylene chloride.

The suspension is allowed to stabilize for 4 hours at room temperature and then an amount of "Amberlite" IRC-50 resin corresponding to 5 g of dry resin is poured into the flask. After shaking for 1 hour, the liposome suspension is passed through a sintered glass filter to remove resin retaining unencapsulated drug. This liposome suspension is stabilized by vacuum lyophilization.

Example 2 According to the method described in Example 1, 2.3 g egg lecithin, 0.65 g cholesterol and 0.15 g octadecylamine were dissolved in 50 ml CH ₂ Cl ₂ and
The solution was then poured into a flask containing 150 ml of Na,K-phosphate buffer (0.02M, PH 7.4) containing 250 mg of 5-fluorouracil. Inert gas (N ₂ ) was bubbled through the flask under shaking to completely remove the organic solvent and form liposomes. To this solution was added 10 ml of anion exchange resin IRA400 (Cl) manufactured by Rohm and Haas.

After shaking for 30 minutes, the resin was filtered off and the purified liposome suspension was lyophilized.

Example 3 3.22g lecithin, 1.13g cholesterol,
0.956 g dicetyl phosphate and 16 mg vitamin E acetate were dissolved in a small amount of CH ₂ Cl ₂ .

On the other hand, 448 mg of doxorubicin hydrochloride and
7.77g of lactose, sodium phosphate
0.066M, NaCl0.0388M, EDTA disodium salt
It was dissolved in a buffer solution (PH5.6) consisting of 0.1mM.

The above organic solvent solution and aqueous solution were mixed and emulsified by shaking. Next, nitrogen was blown in at room temperature to completely remove methylene chloride. The suspension was treated with ultrasound. Then add loam to this flask.
80 g of an ion exchange resin known under the trade name "IRC-50" manufactured by And Haas or adsorption resin XAD-7 manufactured by Rohm and Haas was poured. After 1 hour of shaking, the resin was filtered off and the liposome suspension was filtered through a membrane filter with a porosity of 3 mcm. The liposome suspension was placed in a vial and placed in a freeze dryer for freeze drying.

Claims (1)

[Claims] 1. In preparing a liposome suspension containing doxorubicin hydrochloride, aminocidine sulfate or 5-fluorouracil, (a) a lipid component is dissolved in a solvent, and an aqueous solution of the drug is added thereto. A liposome suspension is then prepared by blowing inert gas to emulsify it until the solvent is completely removed by evaporation, and (b) the suspension is mixed with a polymeric ion exchange resin or polymer. A process for the preparation of liposomal suspensions, characterized in that the liposomal suspensions are purified from uncollected drugs by treatment with a sorbent resin and subjected to freeze-drying for stabilization. 2. The ion exchange resin is generally, but not necessarily limited to, carboxylic acids of different matrices, such as phenol formaldehyde, styrene-divinylbenzene, acrylates, methacrylates, various hydrocarbons in liquid and solid form, and various condensation resins; 2. The method according to claim 1, characterized in that it is of various weak, average or strong strength cation types defined by the phosphonic acid and sulfonic acid functional groups. 3. The ion exchange resins generally contain different matrices, such as, but not necessarily limited to, phenol formaldehyde, styrene-divinylbenzene, acrylates, methacrylates, various hydrocarbons in liquid and fixed form, and various condensation resins. Various salts formed with quaternary ammonium, primary, secondary and tertiary amine functionality or other functionalities defined by various weak, average or strong strength anion types. The method according to item 1, characterized in that: 4. Process according to claim 1, characterized in that the resin can be used in salt-forming or active form. 5. In purifying a heterogeneous system known as a liposomal suspension from non-encapsulated drugs, said suspension is free of any specific chemical functionality and is usually, but not necessarily, 2. Process according to claim 1, characterized in that it is treated with polymers and/or copolymers, generally known as adsorbents, which react by means of Waals forces. 6. Process according to item 5 above, characterized in that, in addition to its aliphatic and aromatic nature, the adsorbent may also be of inorganic origin. 7. A method according to item 1, characterized in that the suspension is obtained by blowing an inert gas (for example nitrogen or the like) into a two-phase emulsion type.