MXPA99007683A - Aqueous pharmaceutical composition comprising an active ingredient which is highly insoluble in water - Google Patents

Abstract

Aqueous pharmaceutical composition comprising an active ingredient which is highly insoluble in water, dispersed in liposomes and method of preparation thereof.

Description

Aqueous pharmaceutical composition comprising an active ingredient, which is highly insoluble in water This invention relates to an aqueous pharmaceutical composition comprising an active ingredient, which is highly insoluble in water. In particular, it relates to a pharmaceutical composition, in which the active ingredient is dispersed in liposomes. A large amount of research has been done to find new liposomal preparations in the pharmaceutical field. However, many difficulties have arisen, particularly in relation to active ingredients which are highly insoluble in water. In particular, those with a solubility in water < 0.01% (p / v). In fact, the technique currently used to produce liposomes comprising active ingredients of low water solubility comprises: a) solubilization of the active ingredient and the preselected phospholipids in a suitable organic solvent, for example, chloroform; b) evaporation of this solvent under reduced pressure to give a film active ingredient / phospholipid; c) addition of a second organic solvent, for example, terbutyl alcohol; d) freezing of the obtained solution at the temperature of liquid nitrogen; e) lyophilization of the frozen solution; f) hydration of the lyophilized solution with a buffer solution to give a suspension of multilamellar liposomes (MLV); and g) treating this suspension with ultrasound to give a suspension of smaller liposomes (SUV). An example of this method is described by A. Sharma et al. "Pharmaceutical Research", 2 (6), 889-896 (1 994). However, this technique has the disadvantage of being very laborious and leads to the presence of traces of organic solvents in the liposomes.

However, the mentioned authors refer to having investigated several techniques for the preparation of MLV liposomes, such as, the hydration of the dry lipid films (manual agitation), freezing thawing and various techniques, such as, extrusion and ultrasound treatment for then reduce (post-processing) the size of the liposomes (MLV-SUV) and conclude that the method described in detail above, and comprising the phases a) ag) mentioned, proved to be the most acceptable (loc.cit. page 890 , right column, lines 51 -57). Without. However, the aforementioned authors do not indicate how said first technique was combined for the preparation of MLV liposomes and said second technique, which reduces the size of the liposomes. WO-A-96 40664, EP-A-0 578 629, DE-A-4 038 075 and DE-A-4 430 593 describe pharmaceutical compositions wherein a water-insoluble active ingredient is dispersed in liposomes. Such an active ingredient is cyclosporin A, melatonin and, respectively, taxol. However, none of the aforementioned documents describes a method for preparing an aqueous liposome composition, which combines the freeze-thaw technique with extrusion.

Surprisingly, it has now been found that the technique of freezing and thawing combined with extrusion allows the preparation of aqueous compositions of liposomes of active ingredients with a solubility in water < 0.01% (w / v) without the use of any organic solvent. In this description, and the claims that follow, the active ingredients with a solubility in water < 0.01% are defined as "highly insoluble in water". Accordingly, it is a first objective of this invention to provide a pharmaceutical composition according to claim 1. The following examples are typical of active ingredients, which are highly insoluble in water: lonidamine (solubility: 3x1 0"6 g / ml), melatonin [" practically insoluble ", G. S. Shida et al.," J. Pineal Res. ", 16, 1 98-201, (1994)], cyclosporin-A [" water-soluble ", mono- logy on cyclosporin-A in" Analytical Profiles of Drug Substances "(Analytical Profiles of Drug Substances) 1_6, 1 63, (1 987) 3 and bindarit (solubility: 1 x 1 0"4 g / ml) The liposomes of the compositions according to this invention are preferably made of a component selected from the group comprising phosphoglycerides, glycerides, diglycerides, triglycerides, phospholipids, glucosyl and galactosyl lipids, cholesterol and its derivatives, sphingolipids and mixtures thereof More preferably, they are made of phospholipids, A typical example of the liposome composition according to this invention comprises phosphatidylcholine, lysophosphatidylcholine , N-acyl phosphatidylcholine, phosphatidyl ethanolamine, phosphatidylserine, sphingomyelin, non-polar lipids, triglycerides, free fatty acids, DL-α-tocopherol A preferred liposome composition according to this invention comprises: Component% (w / w) Phosphatidylcholine 85-97 Lisophosphatidylcholine 0-5 N-acyl-ethanolamine 0-4 Phosphatidyl ethanolamine 0-1 0 Triglycerides 0-4 Free fatty acids 0-3 DL-α-tocopherol 0-1 A particularly preferred composition of iiposomes according to this invention comprises: Component% (w / w) Phosphatidylcholine 94 Lisophosphatidylcholine 3 N-acyl-ethanolamine 1 Phosphatidyl ethanolamine 0.1 Triglycerides 1 Free fatty acids 0.75 DL-α-tocopherol 0. 1 5 Typically, the size of the liposomes according to this invention is less than 500 nm. Preferably, it is from 50-250 nm. A second objective of this invention is to provide a method for the preparation of an aqueous pharmaceutical composition according to claim 6. The duration of phase c) depends on the amount of highly insoluble active ingredient in water to be trapped in the liposomes. The person skilled in the art therefore finds no difficulty since a few simple routine experiments will determine the correct time for each type of liposome composition and active ingredient. The aqueous phase should preferably be made from an aqueous solution of sodium chloride at 0.05% -0.9% (w / v). Normally, the amount of lipid used is between 0.01 -0.4 parts by weight for each part by weight of aqueous solution. In turn, the amount of active ingredient is usually between 0.01 and 0.3 parts by weight per part by weight of lipid. Normally, the disperser is a homogenizer of the Ultraturrax ™ type. Normally, the extrusion is carried out using compressed air or an inert gas, chosen from the group comprising nitrogen, helium and argon, as the extrusion gas. The preferred inert gas is helium. In the extrusion phase, the pressure will preferably be between 500 and 5500 kPa and the temperature will preferably be between 20 and 75 ° C, and even more preferably between 40 ° C and 65 ° C. Normal examples of suitable extruders are those of the Lipex Biomembranes Thermobarrel type or of the Emulsiflex CC Avestin type with filters with CostarMR membranes of polycarbonate with pores between 50 and 600 nm. Normally, phase h) is repeated at least twice and no more than 8 times. Preferably 6 times. The following examples illustrate this invention without limiting it in any way.

EXAMPLE 1 100 mg of meiatonin was dispersed in 1 g of phospholipid at 30 ° C for 10 minutes using an Ultraturrax ™ type homogenizer. Immediately afterwards, this dispersion was suspended in 10 ml of 0.9% aqueous sodium chloride solution (w / v) using the aforementioned homogenizer, and then heated in a waterbath at 55 ° C for 20 minutes. The suspension obtained in this form was subjected to the following cooling and heating cycle: cooling in liquid nitrogen for 1 minute, heating at 55 ° C until the phospholipids are completely fluid. This cycle was repeated 6 times. The suspension was passed twice through a 0.6 μm filter with the Lipex Biomembrane apparatus. Thus, a suspension of "Large Vesicles" was obtained Multilamellar "(MLV), which was subjected to 6 cycles of continuous extrusion using a 1 0 ml extruder of the Lipex Biomembrane Extruder Thermobarrel type with CostarMR polycarbonate filters of 0.1 μm at 55 ° C, using helium, as the extrusion gas , at a pressure between 1 000 and 4800 kPa .. Operating as described above, three batches of the product were prepared (LM / 1 86, LM / 188 and LM / 190) .The following tests were performed on the batches: * amount of melatonin in the aqueous composition of liposomes (HPLC analysis); * size of liposome; * amount of melatonin trapped in liposomes.The following table shows the parameters measured and their importance: The data obtained are given in Table 1, which shows: - the concentration of melatonin obtained in the aqueous formulation of liposomes was expressed as an average value of the three batches, 8.05x1 0"3 g / ml; the liposomes for the three lots was 93 nm; the trapped quantity, expressed as an average value for the three lots, was 80.5 μg / mg; the formulations did not show any phenomenon of liposome aggregation.

TABLE 1 (*) expressed as μg of medicine per mg of phospholipids used.

The following procedure was used for the analysis of H PLC: - fixed phase: reverse phase column PKB-1 00 (250 x 4.6 mm, 5 μm Supelco); - mobile phase: water: acetonitrile 80: 20 (v / v); - detection: UV 254 nm. Two pieces of apparatus were used for the analysis of the average size of the liposomes: 1) DELSA 440 Coulter, 2) NI COMP Submicron Model 370 Particle Dimension. The procedure was as follows: a) for tests performed with the apparatus 1) , 1 ml of liposome suspension was diluted with 10 ml of 0.9% aqueous sodium chloride solution (w / v); b) for the tests carried out with the apparatus 2), 0.5 ml of solution a) was diluted in 10 ml of 0.9% aqueous sodium chloride solution (w / v).

EXAMPLE 2 Proceed as described in Example 1 above, using 2 g of phospholipid and 50 mg of lonidine in place of 1 g of phospholipid and 100 mg of melatonin. In this way three batches of the product are prepared (LM / 195, GN / 1 and GN / 2L). The data obtained is given in Table 2, which shows: the concentration of lonidamine in the aqueous composition was from the initial solubility value of 3 x 10"6 g / ml to an average value for the three lots of 3.83 x 1 0"3 g / ml; the average size of the liposomes for the three lots was 79.6 nm; the trapped quantity, expressed as an average value for the three lots, was 19.2 μg / mg; the formulations did not show any phenomenon of liposome aggregation.

TABLE 2 (*) expressed as μg of medicine per mg of phospholipids used.

EXAMPLE 3 Proceed as described in Example 1 above, using 2 g of phospholipid and 200 mg of melatonin instead of 1 g of phospholipid and 100 mg of melatonin. In this way, three batches of the product were prepared (GN / 1 M, GN / 2M and G N / 3M). The data obtained are given in Table 3, which shows: - the concentration of melatonin in the aqueous formulation of liposomes, expressed as an average value for the three lots, was 13.5 x 10 0 3 g / ml, the average size of the liposomes for the three batches was 92.6 nm - the trapped amount, expressed as an average value of the three batches, was 67.6 μg / mg, the formulations did not show any liposome aggregation phenomenon.

TABLE 3 (*) expressed as μg of medicine per mg of phospholipids used.

EXAM PLO 4 Proceed as described in Example 2 above, except that the extrusion was done through a polycarbonate membrane of 0.2 μm instead of 0.1 μm. In this way three batches of the product were prepared (GN / 3L, GN / 4L and GN / 5L). The data obtained is given in Table 4, which shows that by increasing the lonidamine from 20 mg to 50 mg, the amount of phosphoiipid for 1 to 2 g and extruding with a 0.2 μm membrane instead of a 0.1 μm membrane, a significant increase in the concentration of lonidamine in the aqueous composition is obtained in example 2. In fact, an average value of 4.47 x 10-3 g / ml was obtained for the concentration of lonidamine.

TABLE 4 (*) expressed as μg of medicine per mg of phospholipids used.

EXAMPLE 5 20 mg of cyclosporin-A were dispersed in 1 g of phospholipid at 30 ° C for 10 minutes using an Ultraturrax ™ type homogenizer. Immediately thereafter, the dispersion was suspended in an aqueous solution of sodium chloride at 0.9% (w / v) using the aforementioned homogenizer, and then heated in a waterbath at 65 ° C for 20 minutes. The suspension obtained in this manner was subjected to the following cooling and heating cycle: cooling in liquid nitrogen for 1 minute, heating to 65 ° C until the phospholipids are completely fluid. This cycle was repeated 6 times. The suspension was passed twice through a 0.6 μg filter with the Lipex Biomembrane apparatus. Thus, a suspension of "Large Multilamellar Vesicles" (MLV) was obtained, which was subjected to 6 cycles of continuous extrusion using a 1 0 ml extruder of the Lipex Biomembrane Extruder Thermobarrel type with CostarMR polycarbonate filters from 0.1 μm to 65 ° C, using helium as the extrusion gas at a pressure between 1000 and 4800 kPa. In this way, three batches of the product were prepared (LM / 416A, LM / 416B and LM / 41 6C). The data obtained are given in Table 5, which shows: - the concentration of cyclosporin-A in the aqueous formulation of liposomes, expressed as an average value for the three batches, was 0.96 x 10-3 g / ml; - the average size of the liposomes for the three batches was 1 03 nm; the trapped quantity, expressed as an average value for the three lots, was 9.6 μg / mg; the formulations did not show any phenomenon of liposome aggregation.

TABLE 5 (*) expressed as μg of medicine per mg of phospholipids used.

EXAMPLE 6 Proceed as described in Example 1 above, using 2 g of phospholipids and 50 mg of bindarit instead of 1 g of phospholipids and 100 mg of meiatonin. In this way three batches of the product were prepared (LM / 356, LM / 357 and LM / 358). The data obtained are given in Table 6, which shows: the concentration of bindarit in the aqueous composition of liposomes was from the initial solubility value of 1 x 1 0-4 g / ml to an average value for the three batches of 4 mg / ml; the average size of the liposomes for the three lots was 108.3 nm; the trapped quantity, expressed as an average value for the three lots, was 20.2 μg / mg; the formulations did not show any phenomenon of liposome aggregation.

TABLE 6 (*) expressed as μg of medicine per mg of phospholipids used.

EXAMPLE 7 30 mg of cyclosporin-A were dispersed in 2 g of phospholipid at 30 ° C for 10 minutes using a U-type homogenizer ltraturrax ™. Immediately afterwards, this dispersion was suspended in an aqueous solution of sodium chloride at 0.9% (w / v) using the aforementioned homogenizer and allowed to stand at room temperature for 24 hours. Then the suspension obtained was heated in a waterbath at 65 ° C for 20 minutes. The suspension obtained in this manner was subjected to the following cooling and heating cycle: cooling in liquid nitrogen for 1 minute, heating at 65 ° C until the phospholipids are completely fluid. This cycle was repeated 6 times. This suspension was passed twice through a 0.6 μm filter with the Lipex Biomembrane apparatus. Thus, a suspension of "Large Vesicles" was obtained Multilamellar "(MLV), which was subjected to 6 continuous extrusion cycles using an extruder of the Lipex Biomembrane Extruder Thermobarrel 10 ml type with CostarMR polycarbonate filters of 0. 1 μm at 65 ° C, using helium as the extrusion gas At a pressure of between 1000 and 4800 kPa, three batches of the product were prepared (LM / 422a, LM / 422b and LM / 422c) The data obtained are given in Table 7, which shows: the concentration of cyclosporin-A in the aqueous liposome formulation, expressed as an average value for the three batches, was 3 mg / ml, the average size of the liposomes for the three batches was 1 19.5 nm, the trapped amount, expressed as a value average for the three batches was 1 5 μg / mg, the formulations did not show any liposome aggregation phenomenon.

TABLE 7 (*) expressed as μg of medicine per mg of phospholipids used.

Claims (13)

1. REIVI NDI CATIONS 1 . An aqueous pharmaceutical composition, wherein an active ingredient having a solubility in water not greater than 0.01% (w / v) is dispersed in liposomes.
2. 2. A composition according to claim 1, characterized in that the liposomes are composed of a component selected from the group comprising phosphoglycerides, glycerides, diglycerides, triglycerides, phospholipids, galactosyl and glucosyl lipids, cholesterol and its derivatives, sphingolipids and mixtures thereof. same.
3. 3. A composition according to claim 1 or 2, characterized in that the liposomes are constituted by a composition comprising phosphatidylcholine, lysophosphatidylcholine, N-acyl-phosphatidylcholine, phosphatidyl ethanolamine, phosphatidylserine, sphingomyelin, non-polar lipids, triglycerides, free fatty acids, DL-a-tocopherol.
4. 4. A composition according to any of claims 1 to 3, characterized in that the amount of lipids is between 0.01 and 0.4 parts by weight for each part by weight of water.
5. 5. A composition according to any of claims 1 to 4, characterized in that the amount of the active ingredient is between 0.01 and

   0. 3 parts by weight for each part by weight of lipids.
6. 6. A method for the preparation of an aqueous pharmaceutical composition with an active ingredient having a solubility in water not greater than 0.01% (w / v) dispersed in liposomes, characterized in that it comprises the following phases: a) dispersion of this active ingredient in lipids at a temperature between 20 and 30 ° C; b) suspension of this dispersion in an aqueous phase; c) rest of this suspension at room temperature for a period between 0 and 48 hours; d) heating at 30-75 ° C for 1 0-40 minutes; e) freezing at -150 ° C / -200 ° C; f) repetition of phases d) and e) at least twice and no more than 8 times; g) filtration through a filtration membrane with pores of 500-1000 nM in diameter; h) extrusion through a membrane with pores of 50-400 nM diameter; and at the same time i) removal of any active ingredient, which is not trapped.
7. 7. A method according to claim 6, characterized in that the aqueous phase is constituted by the aqueous solution of sodium chloride a

   0. 05% -0.9% (p / v).
8. 8. A method according to claim 6 or 7, characterized in that the amount of lipids used is between 0.01 and 0.4 parts by weight for each part by weight of water.
9. 9. A method according to any of claims 6 to 8, characterized in that the amount of the active ingredient used is between 0.01 and 0.3 parts by weight for each part by weight of lipids. 1 0.
10. A method according to any of claims 6 to 9 above, characterized by the use in phase h) of an extrusion gas chosen from the group comprising air, nitrogen, helium and argon. eleven .
11. A method according to claim 10, characterized in that the extrusion gas has a pressure between 500 and 5500 kPa.
12. 12. A method according to any of the preceding claims 6 to 11, characterized in that phase h) is carried out at a temperature between 20 and 75 ° C.
13. 13. A method according to claim 1 2, characterized in that the temperature is between 40 and 65 ° C. A method according to any one of claims 6 to 12 above, characterized in that phase h) is repeated at least twice and no more than 8 times.