CA2091113A1 - Liposomal active compound formulations and process for their production - Google Patents

Abstract

Liposomal active compound formulations and process for their production ABSTRACT OF THE DISCLOSURE

The invention relates to a liposomal dihydropyridine formulation which can be administered parenterally and to a process for its production.

LeA28936

Description

2 ~ 3 The invention relates to a liposomal dihydropyridine formulation which can be administered parenterally and to a process for its production.

Dihydropyridines are, in general, poorly water-soluble.
For parenteral administration, therefore, use with water-miscible, tolerable organic solvents or use of particular injection or infusion formulations is relied upon. These include parenteral nanoemulsions, nanosuspensions (hydrosols) and liposomes.

Organic solvent systems as carriers for dihydropyridines have a number of disadvantages: on account of too low a tolerability in the peripheral vessels they must either be administered via a central catheter or diluted with a simultaneously administered co-infusion. Complications due to supersaturation can occur here owing to the solubility ratios. Furthermore, depending on the type oi dihydropyridine, considerable amounts of organic solvents and possibly other auxiliaries had to be added. In particular in th~ case of relatively long-term intravenous therapies, this would lead to an amount of undesired solvent which is no longer tolerable.

It is known that sparingly soluble active compounds can be administered parenterally in a liposomal admini-stration form (Liposome Technology, Vol I~III, ed.
Gregoriadis). It is also known that liposome dispersions Le A 28 986 - l -2 ~ 1 3 can be prepared by various techniques. ~xamples which may be mentioned are: high-pressure homogenisation (EP-A-0,101,007), ultrasonication, dialysis, extrusion, microfluidisation and dilution processes~ Under the known preparation techniques, processes which do not use organic solvents are also described which, however, only function under certain conditions and/or require a high industrial outlay.

In particular for nimodipine, it is described, for example, in DE 3,515,335 how this particularly sparingly soluble dihydropyridine can be formulated as liposomes using organic solvents.

It has surprisingly been found that with sparingly soluble active compounds and even with the e~tremely low solubility of nimodipine the use of organic solvents in the preparation of liposomes can be avoided. The liposomes according to the invention are prepared accord-ing to the following process: cryoprotector, pH
stabiliser, antioxidant, phospholipid and active compound are prehomogenised without the use of organic solvents in a water receiver into which nitrogen is passed. The predispersion is then subjected to high pressure homogenisation at elevated temperature until the desired liposome size is obtained.

It was not foreseeable that even with a dihydropyridine which is as sparingly soluble as nimodipine, stable liposomes can be formed without the active compound Le A 28 936 - 2 -recrystallising.

It is known that additions of polyalcohols ~O~fenlegungs-schrift DE 3,515 r 335 A1) can prevent the recrystal-lisation of active compounds such as dihydropyridines in liposome dispersions.

It has surprisingly been found that active compounds, in particular dihydropyridines, can be formulated keeping to active compound:phospholipid ratios of 1:20 to 1:60 without the presence of organic solvents and without the addition of polyalcohols to inhibit recrystallisation, which is disadvantageous from the medical point of view.
At the said active compound:phospholipid ratios, the active compound neither recrystallises during preparation in the dispersion nor during storage in the lyophilisate.
Using the active compound:phospholipid ratio according to the invention, a storage-stable liposome formulation is thus `also achieved without the addition of polyalcohols.
In particular, it was not foreseeable that the active compound does not recrystallise even if the liposome lyophilisates are reconstituted after 2 years' storage at 6C.

The invention relates to stable pharmaceutical prepara-tions which can be administered parenterally and are free o~ organic solvents, based on liposomes with a phospho-lipid membrane having an active compound:phospholipidratio between 1:20 and 1:100 and an average liposome diameter between 40 and 200 nm which after reconstitution Le A 28 936 - 3 -have a pH between 4.5 and 6.5.

Pharmaceutical preparations are particularly preferred whose liposome membrane contains between 20 and 60 parts by weight of phospholipid and contains 1 part of active compound, the preferred active compounds employed being dihydropyridines of the formula (I) ~ X (I) Y~Z

in which Ydenotes the group NO2, CN or -COORl, where 10R1 represents Cl-C4-alkyl, optionally substituted by C1-C3~alkoxy/
Z represents the group CO-NH-cycloalkyl (3-7 C atoms) or -COOR2, where R2 denotes C1-Cl~-alkyl, optionally substituted by 15C1-C3-alkoxy, trifluoromethyl, N-methyl-N-benzylamino or benzyl, R3 represents Cl-C4-alkyl, cyano or hydroxyalkyl (1-4 C
atoms) and X represents 1 or 2 identical or different sub-20stituents from the group nitro, halogen, Cl-C4-alkyl, Le A 28 936 - 4 -2~

Cl-C4-alkoxy, benzoyl and a 2,3-ring member consist-ing of =N-O-N=, 2-cycloalkylmethylthio, 2-(4-methyl-benzyloxy) or trifluoromethyl.

The invention also relates to a process for the production of the preparations according to the invention, characterised in that 1 part by weight of the active compound and 20-60 parts by weight of phospholipid - are predispersed for an adequate period together with an antioxidant, a cryoprotector and a pH stabiliser at temperatures between about 10 and B0C, preferably 20 to 80C, and a pH between 5 and 7.5, preferably 6 and 7, in water into which nitrogen has been passed using a stirrer and the liposomes are then homogenised to an average size between 40 and 200 nm in an abrasion-free high-pressure jet homogeniser at temperatures between 20C and 80C and a pressure between 400 and 1000 bar and the dispersion thus obtained is optionally then poured into suitable vials and rapidly frozen at temperatures between -50C
and -70C. It is particularly advantageous to employ glucose as the cryoprotector and to select a ratio between cryoprotector and phospholipid of between 1:1 and 4:1. If appropriate, the liposomes prepared in this way can additionally be sterilised after homogenisation, for example by filtration through a 0.2 ~m filter.

To achieve sho:rt drying times, it may be advantageous to employ the shell-freeze or the spin-freeze technique.

Suitable reconstitution media which may be mentioned are Le A 2~ 936 - 5 -isotonic glucose solution (5% strengtn) or water for injections. To improve the flocculation behaviour after reconstitution it is recommended where appropriate that the liposome membrane contains up to 10~ by weight of a charge inducer. Those which may be mentioned as preferred are DMPG-Na (dimyristoyl-phosphatidylglycerol sodium salt), DPPG-Na (dipalmitoyl-phosphatidylglycerol sodium salt) or DSPG-Na (distearoyl-phosphatidylglycerol sodium salt).

The pharmaceutical preparations according to the inven-tion are distinguished by excellent temperature and storage stability. They can be stored, for example, at temperatures below 30C for at least two years without loss of quality occurring after reconstitution. The reconstituted dispersions are stable for up to 24 h and show no flocculation or aggregation phenomena of the phospholipid.

The liposomes according to the invention are generally suitable for lipophilic active compounds, in particular for dihydropyridines such as nifedipine, nisoldipine, nimodipine, nitrendipine, felodipine or amlodipine. The technique is also particularly suitable for very sparingly soluble dihydropyridines such as nimodipine.

The preparation of the liposomes according to the invention comprises several sub-steps. First, after initial weighing of the substances used, a prehomogenate is prepared using conventional rotor-stator stirrers. The Le A 28 936 - 6 -predispersions here contain the phospholipid and nimodipine in the said ratio between 20~1 and 40:1. The predispersions furthermore contain known cryoprotectors such as sucrose, glucose or fructose, antioxidants such as ascorbic acid and pH stabilisers such as arginine.

Suitable phospholipids for the construction of the liposomes for parenteral administration are all phospho-lipids having the customary purity for this purpose. The phospholipids can in this case be either unsaturated or, to obtain other pharmacokinetic profiles, saturated. As natural phospholipids, soya or egg phospnolipids are just as suitable as synthetic phospholipids. To obtain par-ticularly translucent liposome dispersions and to improve the flocculation behaviour after reconstitution, the addition of up to 10% of charge inducers such as Na-DMPG, Na-DPPG or Na-DSPG is advantageous.

The prehomogenate is filtered through a 5 ~m filter before high-pressure homogenisation and then homogenised to the desired liposome size between 40 and 50 nm in a customary high-pressure jet homogeniser at temperatures between 20 and 80C, preferably between 40 and 80C, under an inert gas, such as nitrogen protection.

For the purpose of microorganism reduction, the homogenised liposome dispersion is filtered through a customary 0.2 ~m filter and then lyophilised. For reasons of optimum cryoprotector action, the phospholipid:cryo-protector ratio should be between 1:1 and 1:4, preferably Le A 28 936 - 7 -between 1:1.8 and 1:4.
For the purpose of lyophilisation, the di~persion is poured into vials or infusion bottles, the bottles are placed in the freeze-dryer and the product is frozen therein with the aid of the charging spaces pre-cooled to -50C. To achieve particularly rapid drying times, it is advantageous to freeze the bottles or vials while rotating by the spin- or shell-free~ing principle.

The liposomes prepared according to the invention are of between about 40 and 50 nm size even after homogenisation and have a narrow distribution around this average value.

The liposome lyophilisates prepared according to the invention can be reconstituted before administration using the customary parenteral solutions. Preferably, the reconstitution is carried out using 5% strength glucose sol~tion or water for injections. The lyophilisates can be reconstituted within one minute with repeated shaking.

The reconstituted liposome dispersions according to the invention are preferably administered at the desired rate using customary infusion pump systems such as Perfusor~, Imed~ or Ivac~. For safety reasons, the reconstituted liposome dispersion is filtered in-line through a com-mercially available filter before administration.

Le A 2B 936 - 8 -ExemPlarv embodiments Example 1 Nitrogen is passed into aoo g of water for injection for 10 min. l g of ascorbic acid, 150 g of glucose and 1 g of L-arginine are then dissolved in this amount of water.
1.667 g of nimodipine and 83.35 g o~ highly pure egg phospholipid (for example Lipoid E PC) are dispersed in this medium and it is made up to 1055.5 g with water into which nitrogen has been passed (active compound: phospho-lipid ratio = 1:50).

This dispersion is prehomogenised for 30 min at 75C and under nitrogen protection using a high-speed stirrer (for example Ultra-Turrax). The predispersion is filtered through a 5 ~m filter.

The predispersion is then homogenised for one hour at 75C; 800 bar and under nitrogen protection in an abrasion-free high-pressure jet homogeniser. Sterile filtration (0.2 ~m) is carried out after cooling to room temperature. The content is 100% of the theoretical content.

The dispersion is poured to 15.3 ml into 250 or 50 ml bottles and frozen to give a thin product cake with the aid of spin- or shell-freezing at -65C (dry ice-ethanol cooling mixture).

The frozen product is transferred to the charging spaces Le A 28 936 - 9 -~ ~ $ ~ 3 of a lyophiliser which is precooled to -50C and dried at -30C and 0.05 mbar for 14 h. Afterdrying at 30C and O.001 mbar lasts 7 h.

The lyophilisate is reconstituted with isotonic glucose solution to 250 ml or with water for injection to 50 ml.
The content is 100% of the theoretical content.

Liposome properties:
before after recon-lyophilisation stitution _ Average s~ze 45 nm 50 nm Dispersity index K2 0.350 0.500 Turbidity 170 units 40 units Osmolarity 1.2 Osmol 360 mOsmol pH 6.5 4.5 Particle load (HIAC) <10 particles after in-line filtration (> 2 ~m/ml) Example 2 Nitrogen is passed into 800 g of water for injection for 10 min. 1 g of ascorbic acid, 100 g of glucose and 1 g of L-arginine are then dissolved in this amount of water.
1.667 g of nimodipine and 50.00 g of highly pure soya phospholipid (for example Phospholipon 90) are dispersed in this medium and it is made up to 1055.5 g with water into which nitrogen has been passed (active compound:

Le A 28 936 - 10 -phospholipid ratio = 1:30).

This dispersion is prehomogenised for 30 minutes at 75C
and under nitrogen protection using a high-speed stirrer (for example Ultra-Turrax). The predispersion is filtered through a 5 ~m filter.

The predispersion is then homogenised for one hour at 75C, 800 bar and under nitrogen protection in an abrasion-free high-pressure jet homogeniser. Sterile filtration (0.2 ~m) is carried out after cooling to room temperature. ~he content is 100% of the theoretical content.

In contrast to Example 1, after pouring into appropriate bottles the dispersion is frozen at -65C without use of spin- or shell-freezing.

The product is dried at +30C and 0.05 mbar for 7 h (main drying) and afterdried at ~30C and 0.001 mbar. The liposome properties from Example 1 are retained.

Example 3 Nitrogen is passed into 800 g of water for injection purposes for 10 min. 1 g of ascorbic acid, 100 g of glucose and 1 g of L-arginine are then dissolved in this amount of water. 1.667 g of nimodipine, A5 g of highly pure sakurated phospholipid (for example Epikuron 200 SH) and 5 g of DMPG-Na are dispersed in this medium and it is Le A 28 936 2~ g ~ ~

made up to lOS5.5 g with water into which nitrogen has been passed (active compound: phospholipid ratio = 1:30).

~his dispersion i5 prehomogenised for 30 minutes at 75C
and under nitrogen protection using a high-speed stirrer (for example Ultra-Turrax). The predispersion is filtered through a 5 ~m filter.

The predispersion is then homogenised for one hour at 75C, 800 bar and under nitrogen protection in an abrasion-free high-pressure jet homogeniser. Sterile filtration (0.2 ~Lm) is carried out after cooling to room temperature. The content is 100% of the theoretical content.

The dispersion is poured to 15.3 ml into 250 ml or 50 ml bottles and frozen to give a thin product cake with the aid of spin- or shell-freezing at -65C (dry ice-ethanol cooli~g mixture).

The frozen product is transferred to the charging spaces of a lyophiliser which is precooled to -50~C and dried at -30C and 0.05 mbar for 14 h. Afterdrying lasts for 7 h at 30~C and 0.001 mbar.

The lyophilisate is reconstituted with isotonic glucose solution to 250 ml or with water for injections to 50 ml.
The content is 100~ of the theoretical content.

Le A 28 936 - 12 -Liposome properties:
before after lyo-lyophilisation philisation Average size 39 nm 58 nm Dispersity index K2 0.530 0.630 Turbidity 130 units 40 units Osmolarity 700 mOsmol 355 mOsmol pH 6.5 6.1 Example 4 Nitrogen is passed into 800 g of water for injections for 10 min. 1 g of ascorbic acid, 50 g of glucose and 1 g of L-arginine are then dissolved in this amount of water.
1.667 g of nimodipine and 25 g of egg phospholipid (for example Lipoid E 80) are dispersed in this medium and it is màde up to 1055.5 g with water into which nitrogen has been passed (active compound: phospholipid ratio 1:15).

This dispersion is prehomogenised for 30 min at 75C and under nitrogen protection using a high-speed stirrer (for example Vltra-Turrax). The predispersion is filtered through a 5 ~m filter.

The predispersion is then homogenised for one hour at 75C, 800 bar and under nitrogen protection in an abrasion-free high-pressure jet homogeniser. Sterile filtration (0.2 ~m~ is carried out after cooling to room Le A 28 936 - 13 ~

2~3~3 temperature. The active compound content ~t this active compound: phospholipid ratio (1:15) is 72~ of theory.

The dispersion is poured to 15.3 ml into 250 ml or 50 ml bottles and frozen to give a thin product cake with the aid of spin- or shell-freezing at -65C (dry ice-ethanol cooling mixture)~

The froæen product is ~ransferred to the charging spaces of a lyophiliser which is precooled to -50C and dried at -30C and 0.05 mbar for 14 h. Afterdrying at 30~C and 0.001 mbar lasts 7 h.

The lyophilisate is reconstituted with isotonic glucose solution to 250 ml or with water for injection to 50 ml.
The content of the reconstituted dispersion is 77% of the theoretical content.

ExamPle 5 Nitrogen is passed into 800 g of water for injection for 10 min. 1 g of ascorbic acid, 23.34 g of glucose and l g of L-arginine are then dissolved in this amount of water.
1.667 g of nimodipine and 11.68 g of egg phospholipid (for example Lipoid E 80) are dispersed in this medium and it is made up to 1055.5 g with water into which nitrogen has been passed (active compound: phospholipid ratio 1:7).

This dispersion i5 prehomogenised for 30 min at 75C and Le A 28 936 - 14 -under nitrogen protection using a high-speed stirrer (for example Ultra-Turrax). The predispersion is filtered through a 5 ~m filter. Active compound crystals can be detected on the filter.

The predispersion is then homogenised for one hour at 75C, 800 bar and under nitrogen protection in an abrasion-free high-pressure jet homogeniser. Sterile filtration (0.2 ~m) is carried out after cooling to room temperature. The content of the liposome dispersion is 18% of the theoretical content.

Le A 28 936 - 15 -

Claims (14)

1. Stable pharmaceutical preparation which can be administered parenterally and is free of organic solvents, based on liposomes having a phospholipid membrane, the active compound/phospholipid ratio being between 1:20 and 1:100 and the average liposome diameter being between 40 and 200 nm and which, after reconstitution, has a pH of between 4.5 and 6.5.

2. Pharmaceutical preparation according to Claim 1, the liposome membrane containing between 20 and 60 parts by weight of phospholipid and containing 1 part of active compound.

3. Pharmaceutical preparation according to Claim 1 which after reconstitution has an osmotic pressure between 270 and 370 mOsmol.

4. Pharmaceutical preparation according to Claim 1, containing as active compound a dihydropyridine of the formula (I) (I) Le A 28 936 in which Y denotes the group NO2, CN or -COOR1, where R1 represents C1-C4-alkyl, optionally substituted by C1-C3-alkoxy, Z represents the group CO-NH-cycloalkyl (3-7 C atoms) or -COOR2, where R2 denotes C1-C10-alkyl, optionally substituted by C1-C3-alkoxy, trifluoromethyl, N-methyl-N-benzylamino or benzyl, R3 represents C1-C4-alkyl, cyano or hydroxyalkyl (1-4 C atoms) and X represents 1 or 2 identical or different substituents from the group nitro, halogen, C1-C4-alkyl, C1-C4-alkoxy, benzoyl and a 2,3-ring member consisting of =N-O-N=, 2-cycloalkylmethylthio, 2-(4-methylbenzyloxy) or trifluoromethyl.

5. Pharmaceutical preparation according to claim 4 wherein the active compound is selected from the group consist-ing of nifedipine, nisoldipine, nimodipine, nitrendipine, felodipine and amlodipine.

6. Pharmaceutical preparation according to claim 5 wherein the active compound is nimodipine.

7. A liposome having a phospholipid membrane and free of organic solvents, said liposome containing an active compound wherein the active compound phospholipid ratio is from about 1:20 to about 1:100 wt/wt and the average liposome diameter is between 40 and about 200 nm.

8. A liposome according to claim 7 which has been lyophilized.

9. A pharmaceutical preparation prepared by reconstituting a lyophilized liposome according to claim 8 in a liquid vehicle suitable for parenteral administration, wherein said preparation has a pH from about 4.5 to about 6.5.

10. Process for the production of a pharmaceutical preparation according to any one of claims 1 to 6 which can be administered parenterally, characterised in that 1 part by weight of the active compound and 20 to 60 parts by weight of phospholipid are predispersed for an adequate period together with an antioxidant, a cryoprotector and a pH stabiliser at temperatures between about 10°C and 80°C and a pH between 5 and 7.5 in water into which nitrogen has been passed using a high-speed stirrer.

11. Process according to claim 10 characterised in that cryoprotector and phospholipid are employed in a ratio of between 1:1 and 4:1.

12. Process according to claim 10 characterised in that the cryoprotector employed is glucose.

13. Process according to claim 10 characterised in that after predispersion the liposomes are homogenised to an average size between 40 and 200 nm in a high-pressure jet homogeniser at temperatures between 20 and 80°C and a pressure between 400 and 1,000 bar, and the dispersion obtained is optionally rapidly frozen at -50°C to -70°C.

14. Process according to claim 13, characterised in that after homogenisation the dispersion is sterilised by filtration through a 0.2 µm filter.