MXPA99007684A - Pharmaceutical preparation comprising lyophilized liposomes encapsulating an active principle which is highly insoluble in water, and the process for preparing thesaid preparation - Google Patents

Abstract

Lyophilized composition comprising trehalose and lipid liposomes in which a biologically-active principle has been incorporated, characterized in that the biologically-active principle is highly insoluble in water, the trehalose/lipid weight ratio is=1.5, and all of the trehalose was added to the outside of the liposomes already formed before lyophilization.

Description

Pharmaceutical preparation comprising lyophilized liposomes encapsulating an active ingredient, which is highly insoluble in water, and the process for preparing said preparation This invention relates to a pharmaceutical preparation comprising lyophilized liposomes which encapsulate a biologically active principle, which is highly insoluble in water, and a process for preparing said preparation. More particularly, the invention relates to a pharmaceutical preparation comprising lyophilized liposomes encapsulating a biologically active principle, which is highly insoluble in water and stable over time. In the course of the present description and the claims, the term "highly insoluble in water" is used to describe all those compounds which have a solubility in water < 0.01% (p / v). It is known that the use of liposomes in medical treatment has been retained by difficulties encountered in obtaining pharmaceutical preparations, which are sufficiently stable both during lyophilization and over time. The above-mentioned difficulties consist mainly in the preparation of liposomes, which neither explode nor pile up with each other. In other words, the liposomes must remain whole and separated from each other. The structural integrity of the liposomes is also particularly important in the case where the active principle is highly insoluble in water. In fact, the explosion of the liposomes during lyophilization and / or during storage does not prevent the water soluble active ingredients from going into the solution when the aqueous liposome solution is reconstituted before administration to the patient, by the addition of physiological solution. On the other hand, in the case of the explosion of liposomes comprising active principles, which are highly insoluble in water, the reconstitution of the lyophilisate produces a solution comprising less active ingredient than required. The greater the amount of liposomes that explode, the greater the difference between the theoretical amount and the actual amount of the active ingredient in the solution. A lyophilization method of liposomes comprising a water-soluble biologically active principle is described in US-A-4 857 31 9. This document describes the lyophilization of liposomes, preferably of an average size of about 50-1,00 nm, with the addition, as a preservative, of a disaccharide inside alone (with the encapsulated liposome content), or only on the outside, or both inside and outside. The weight ratio of disaccharide / lipid varies between 0.1: 1 and 4: 1. Preferably, the disaccharide is trehalose. The freezing phase is carried out at the temperature of liquid nitrogen (-1 95.8 ° C). In the aforementioned patent, the stability characteristics during lyophilization were evaluated by means of retention measurements of the active principle encapsulated in the liposomes after the reconstitution of the lyophilisate via rehydration. Table 2 of the aforementioned patent shows that retention is high (99-1 00%) only when the trehalose / lipid ratio is greater than 1.76 and trehalose is present both inside and outside of the liposomes. When the mentioned proportion is equal to 0.1 1 and to 0.1 9, the retention is equal to 22% and 49%, respectively, even if the trehalose is present both inside and outside of the liposomes. In contrast, when trehalose is only present on the outside, the amount of active substance retained is drastically reduced, even for very large amounts of trehalose. In fact, with a trehalose / lipid ratio of 3.09, the retained amount is only 26%. However, the aforementioned results are not reproducible when the biologically active principle is highly insoluble in water. In fact, when trehalose is added during the preparation of the liposomes in order to encapsulate it within the lipid vesicles, a non-extrudable, non-homogeneous suspension is obtained (Preparations for Comparison 1 and 2). Surprisingly, it has been discovered that liposomes comprising a biologically active principle, which is highly insoluble in water, remains substantially complete during lyophilization when trehalose is added in small amounts to the liposomes before lyophilization only, and said lyophilization is effected upon completion of the lyophilization. freezing phase at a temperature between -5 ° C and -70 ° C. An object of the present invention is to provide a lyophilized composition comprising liposomes of lipids and trehalose, in which a biologically active principle has been incorporated, characterized in that the active biologically active principle is highly insoluble in water, the weight ratio of trehalose / lpid is <; 1.5, and all of the trehalose was added to the outside of the liposomes already formed prior to lyophilization. After reconstitution by rehydration, said composition retains in solution more than 95% of the biologically active principle, which is highly insoluble in water (Examples 1, 2 and 3). The normal examples of biologically active principles, which are highly insoluble in water are: lonidamine, melatonin, cyclosporin A and bindarit. The lipids of the liposome composition to be subjected to the lyophilization process according to the invention are preferably chosen from the group comprising phosphoglycerides, glycerides, diglycerides, triglycerides, phospholipids, galactosyl and glucosyl lipids, cholesterol and its derivatives, sphingolipids. and its mixtures. Preferably, the lipids are phospholipids. The weight ratio of trehalose / lipid, in turn, preferably falls between 1: 2 and 1: 1. The average size of the liposomes can be between 50 and 250 nm. Preferably, it is between 50 and 1 00 nm. A second objective of the invention is constituted by a lyophilization process, characterized in that: 1) from 0.2 to 1.5 parts by weight of allose are added for each part by weight of lipids of an aqueous composition of liposomes, in the which the average size of the liposomes falls between 50 and 250 nm, and said liposomes comprise a biologically active principle, which is highly insoluble in water; 2) the aforementioned composition is cooled via the cooling plate of the Mofilizer at a temperature between -5 ° C and -70 ° C, at a cooling rate between 0.5 and 2 ° C / min; 3) once the predetermined freezing temperature is reached, said composition is maintained at said temperature for a period of between 2 and 5 hours; 4) a vacuum of between 50 and 8 Pa is applied, leaving the temperature of the cooling plate at the cooling temperature defined in point 2) during a period that lasts between 2 and 5 hours; 5) the temperature of the cooling plate is brought to -1 5 ° C, and it is kept there until the water is completely removed.

Preferred operating conditions are as follows: Phase 2 Freezing temperature: -20 to -30 ° C Cooling speed: 0.77 ° C / min. Phase 3 Time: 3 hours Phase 4 Vacuum: 6 Pa Phase 5 a) When the cooling temperature (Phase 2) is below -1 5 ° C, the temperature of the cooling plate is increased to -1 5 ° C at a speed between 0.5 and 2 ° C and lyophilization takes place for 20 hours; then the temperature of the cooling plate is brought to -10 ° C, and after one hour to + 5 ° C, and lyophilization occurs for 16 hours. b) When the freezing temperature (Phase 2) is greater than or equal to -1 5 ° C, lyophilization is continued for 20 hours, after which the temperature of the cooling plate is taken at + 5 ° C and Freeze drying occurs for 16 hours. A particularly preferred liposome composition according to the invention comprises: Component% (w / w) Phosphatidylcholine 94 Lysophosphatidylcholine 3 N-acyl-ethanolamine 1 Phosphatidyethanolamine 0.1 Triglycerides 1 Free fatty acids 0.75 DL-α-tocopherol 0.1 5 Normally, the aqueous pharmaceutical composition of liposomes of the invention is prepared by: a) dispersing a biologically active principle, which is highly insoluble in water in lipids at a temperature of between 20 and 30 ° C; b) suspending said dispersion in an aqueous phase; c) allowing said suspension to remain at room temperature for a period between 0 and 48 hours; d) heating to between 30 and 75 ° C for 1 0 - 40 minutes; e) Freeze between -150 and -2Ó0 ° C; f) repeat phases d) and e) at least twice, and no more than 8 times; g) filter through a filtration membrane with pores of 500-1000 nm in diameter; h) extrude through a membrane with pores of 50-400 nm in diameter; and simultaneous i) elimination of the active ingredient not trapped. The duration of phase c) depends on the amount of active ingredient highly insoluble in water, which is desired to trap in liposomes. The person skilled in the art will thus not have difficulty determining, by means of a few simple routine experiments, the appropriate time for each type of liposome composition and active ingredient. Preferably, the aqueous phase consists of a 0.05% aqueous solution) - 0.9% (w / v) sodium chloride. Normally, the amount of lipids used is 0.01 - 0.4 parts by weight for each part by weight of aqueous solution. In turn, the amount of active ingredient generally falls between 0.01 and 0.3 parts by weight for each part by weight of lipids. Generally, the extrusion is carried out using as an extrusion gas either compressed air or an inert gas chosen from the group comprising nitrogen, helium and argon. Preferably, helium is the interte gas. The pressure in the extrusion phase is preferably between 500 and 5500 kPa, and the temperature preferably falls between 20 and 75 ° C, even more preferably between 40 and 65 ° C. Typical examples of suitable extruders are the Lipex Biomembranes Thermobarrel Extruder type, or the Emulsiflex CC Avestin with CostarMR polycarbonate membrane with pores between 50 and 600 nm in diameter. Proceeding as described above, the aqueous compositions of liposomes are obtained comprising about 8 mg / ml melatonin, 3.8 mg / ml lonidamine, 1 mg / ml of cyclosporin A and 4 mg / ml of bindarit against a solubility in water of 3 x 1 0"3 mg / ml (lonidamine), 1 x 1 0 '1 mg / ml (bindarit) and, practically, the absolute insolubility of melatonin (GS Shida et al., "J. Pineal Res." 1994, 16, 198-201) and cyclosporin A ["Insoluble in Water", a monograph of cyclosporin A in "Analytical Profiles of Drug Substances" "(Analytical Profiles of Substances of Medicines), 1_6, 163, (1887)] The following examples should serve to illustrate the present invention without, however, limiting it.

PREPARATION 1 A liposome composition comprising a biologically active principle, which is highly insoluble in water, was prepared as described below. 1000 mg of melatonin were dispersed in 1 g of phospholipids at 30 ° C for 10 minutes by means of an Ultraturrax ™ type homogenizer. Immediately afterwards, said dispersion was suspended in 10 ml of 0.9% (w / v) aqueous solution of sodium chloride by means of said homogenizer and then heated in a waterbath at 55 ° C for 20 minutes.

The suspension thus obtained was subjected to the following cooling and heating cycle: cooling in liquid nitrogen for 1 minute, heating at 55 ° C until the complete fluidization of the phospholipids.

The mentioned cycle was repeated 6 times. The suspension was passed twice through a 0.6 μm filter using the Lipex Biomembrane device. In this way, a suspension of "Large Multilamellar Vesicle" (MLV) was obtained and subjected to 6 continuous extrusion cycles using a Lipex extruder Thermobarrel Biomembrane of 1 0 ml with Costar R 0. 1 μm to 55 polycarbonate filters. ° C, using helium as an extrusion gas at a pressure between 1000 and 4800 kPa.

PREPARATION I I We proceeded as described for Preparation I, using 2 g of phospholipids and 50 mg of lonidamine instead of 1 g of phospholipids and 1000 mg of melatonin.

PREPARATION I I I The procedure was as described for Preparation I, using 2 g of phospholipids and 200 mg of melatonin instead of 1 g of phospholipids and 1000 mg of melatonin.

PREPARATION IV Proceed as described for Preparation I I, except that the extrusion was done through a polycarbonate membrane of 0.2 μm, instead of 0.1 μm.

PREPARATION V 30 mg of cyclosporin A were dispersed in 2 g of phospholipids at 30 ° C for 10 minutes using a standard homogenizer Ultraturrax MR Immediately after, the aforementioned dispersion was suspended in a 0.9% (w / v) aqueous solution of sodium chloride using said homogenizer, and allowed to remain at room temperature for 24 hours. Following this, the suspension obtained was heated in a water bath at 65 ° C for 20 minutes. The suspension thus obtained was subjected to the following cooling and heating cycle: cooling in liquid nitrogen for 1 minute, heating at 65 ° C until the complete fluidization of the phospholipids.

Said cycle was repeated 6 times. The suspension was passed twice through a 0.6 μm filter using the Lipex Biomembranes device. In this way a suspension of "Ves ícula Grande Mutialamelar" (MLV) was obtained and subjected to 6 continuous extrusion cycles using a Lipex Biomembranes Thermobarrel extruder of 1 0 ml with CostarM R polycarbonate filters of 0.1 μm to 65 ° C, using helium as an extrusion gas at a pressure between 1000 and 4800 kPa.

PREPARATION VI The procedure was as described for Preparation I, using 2 g of phospholipids and 50 mg of bindarit, instead of 1 g of phospholipids and 100 mg of melatonin.

PREPARATION FOR COMPARISON 1 Preparation 1 A 100 mg of melatonin and 1 g of trehalose were dispersed in 1 g of phospholipids at 30 ° C for 10 minutes by means of a homogenizer of the UltraturraxM R. I type immediately afterwards, said dispersion being suspended in 1 ml. 0 ml of 0.9% (w / v) aqueous solution of sodium chloride using said homogenizer, and then it was heated in a water bath at 55 ° C for 20 minutes. The suspension thus obtained was subjected to the following cooling and heating cycle: cooling in liquid nitrogen for 1 minute, heating to 55 ° C until complete fluidization of the phospholipids.

This cycle was repeated 6 times. The suspension was passed twice through a 0.6 μm filter using the Lipex Biomem branes device. In this way, a very dense mass was obtained. The attempt to extrude by means of an extruder of type Lipex Biomem Thermobarrel braces of 1 0 mi with CostarM R polycarbonate filters of 0.1 μm at 55 ° C, using helium as an extrusion gas at a pressure between 1000 and 4800 kPa, It was not successful.

Preparation 1 B The procedure was as described for Preparation 1 A above, except that melatonin was omitted. A suspension of "Large Multilamellar Vesicle" (MLV) was obtained, which in turn turned out to be perfectly extrudable by means of a Lipex Biomembranes Thermobarrel 10 ml extruder with Costar R polycarbonate filters of 0.1 μm at 55 ° C, using helium as an extrusion gas at a pressure between 1000 and 4800 kPa.

PREPARATION FOR COMPARISON 2 Preparation 2A The procedure was as described for the purposes of Preparation for Comparison 1 A, except that 2 g of phospholipids were used instead of 1. In this case, a very dense, non-extrudable mass was also obtained.

Preparation 2B The procedure was as described for the preceding Preparation by Comparison 1B, except that 2 g of phospholipids were used instead of 1. In this case, a perfectly extrudable MLV suspension was also obtained.

EXAMPLE 1 Preparation I I was divided into 1 ml counts, and trehalose was added to each at the rate according to the weight ratio of trehalose / lipid given in Table 1/1. The lyophilization was carried out in a plate lyophilizer, as follows: 1) cooling to -25 ° C at the speed of 0.77 ° C / min; 2) maintenance of said temperature (-25 ° C) for 3 hours, 3) application of vacuum (6 Pa) and maintenance at said temperature (-25 ° C) for 2 hours; 4) heating at -1 5 ° C for 20 hours under a vacuum of 6 Pa; 5) heating at -1 0 ° C for 2 hours under a vacuum of 6 Pa; 6) heating to + 5 ° C for 20 hours under a vacuum of 6 Pa; 7) vacuum closure; 8) introduction of air.

The lyophilisate (1 ml) was rehydrated with 1 ml of distilled water and kept at room temperature for 30 minutes, in order to allow efficient reconstruction of the liposomes. 0.5 ml of said solution was further diluted with 10 ml of physiological solution in order to measure the average size of the liposomes with the N ICOM P 370 device. Table 1/1 shows the results obtained.

Table 1/1 Average size of liposomes before and after lyophilization From Table 1 / it can be seen that the absence of trehalose carries a certain degree of fusion of the liposomes, evidenced by the increase in its average size. Surprisingly, the increase in the amount of trehalose (trehalose / lipids 2: 1) also causes a certain degree of fusion with a consequent increase in the average size. Similar results have also been obtained with Preparation IV.

The average size of the liposomes and the amount of lon idamine were determined from a number of samples, freshly prepared as described above. Subsequently, the samples were replaced in the refrigerator at 5 ° C and sampled at given intervals, rehydrated to determine the amount of the active ingredient and the average size of the liposomes. The results thus obtained are given in Table 1/2.

TABLE 1/2 Proportion trehalose / lipids 1: 2 EXAMPLE 2 Preparation III was lyophilized as described in Example 1 above, and the average size of the liposomes before and after lyophilization (Table 2/1), as well as the average size of the liposomes and the amount of melatonin in the fresh preparations and those kept at 5 ° C were determined as described in the above-mentioned example (Table 2/2).

TABLE 2/1 Average size of the liposomes before and after lyophilization Similar results were also obtained with Preparation 1.

TABLE 2/2 Proportion of trehalose / lipids (w / w) 1: 1 EXAMPLE 3 Preparation VI was lyophilized as described in Example 1 above, and the average size of the liposomes before and after lyophilization (Table 3/1), as well as the average size of the liposomes and the amount of the bindarit in fresh preparations and those maintained at 5 ° C, were determined as described in the aforementioned Example (Table 3/2).

TABLE 3/1 Average size of liposomes before and after lyophilization TABLE 3/2 Proportion of trehalose / lipids (w / w) 1: 1 EXAMPLE FOR COMPARISON 1 Preparation I I was divided into 1 ml aliquots and trehalose was added to each aliquot according to the weight ratio of trehalose / lipids given in Comparison Table 1. The Preparation was then frozen at liquid nitrogen temperature (-1 95.8 ° C) and lyophilized for 20 hours, without any external temperature control. The lyophilisate (1 ml) was rehydrated with 1 ml of distilled water and kept at room temperature for 2 hours 0.5 ml of said solution was further diluted with 10 ml of physiological solution in order to measure the average size of the liposomes with the NI COMP 370 device. The results obtained are illustrated in Comparison Table 1 below.

COMPARISON TABLE 1 Average size of the liposomes before and after lyophilization From Comparison Table 1 we can observe that when lyophilization is carried out at the temperature of liquid nitrogen, either in the absence of trehalose or in the presence of trehalose in the proportions given in the Table above, a certain degree of of fusion of liposomes, evidenced by the increase in average size. In addition, the above data indicate that when lyophilization is carried out at the temperature of liquid nitrogen, the course of lyophilization by itself (for preparations of the same composition) does not always reproduce the same results.

Claims (10)

REIVI NDICAT

1 . The lyophilized compositcomprising lipid and trehalose liposomes in which a biologically active principle has been incorporated, characterized in that the biologically active principle is highly insoluble in water, the weight ratio of trehalose / lipids is < 1.5, and all of the trehalose was added to the outside of the liposomes already formed prior to lyophilizat

2. The lyophilized compositaccording to claim 1, characterized in that the biologically active principle, which is highly insoluble in water, is chosen from the group comprising lonidamine, melatonin, cyclosporin A and bindarit.

3. The lyophilized compositaccording to claim 1 or 2, characterized in that the lipids are chosen from the group comprising phosphoglycerides, glycerides, diglycerides, triglycerides, phospholipids, galactosyl and glucosyl lipids, cholesterol and its derivatives, sphingolipids and their mixtures.

4. The lyophilized compositaccording to claim 3, characterized in that the lipids are phospholipids.

5. The lyophilized compositaccording to any of claims 1 to 4, characterized in that the weight ratio of trehalose / lipid is between 1: 2 and 1: 1.

6. The lyophilized compositaccording to any of claims 1 to 5, characterized in that the average size of the liposomes is between 50 and 250 nm.

7. The lyophilized compositaccording to claim 6, characterized in that the average size of the liposomes is between 50 and 1 00 nm.

8. The process for lyophilizing a compositcomprising lipid and trehalose liposomes, in which a biologically active principle has been incorporated, characterized in that: a) from 0.2 to 1.5 parts by weight of trehalose are added for each part by weight of lipids of an aqueous compositof liposomes, in which the average size of the liposomes is between 50 and 250 nm, said liposomes comprising a biologically active principle, which is highly insoluble in water; b) said compositis cooled by means of the cooling plate of the lyophilizer at a temperature between -5 ° C and -70 ° C, at a cooling rate of between 0.5 and 2 ° C / min; c) once the predetermined freezing temperature has been reached, said compositis maintained at said temperature for between 2 and 5 hours; d) a vacuum of between 50 and 8 Pa is applied, leaving the temperature of the cooling plate at the cooling temperature defined in point b) for a period of between 2 and 5 hours; e) the temperature of the cooling plate is brought to -1 5 ° C, and it was kept there until the water was completely removed.

9. The lyophilizatprocess according to claim 8, characterized in that, in phase b), the freezing temperature is between -20 ° C and -30 ° C.

10. The lyophilizatprocess according to claim 8 or 9, characterized in that, in phase b), the cooling rate is 0.77 ° C / min. eleven . The lyophilizatprocess according to any of claims 8 to 10, characterized in that, in Step c), the time is 3 hours. The freeze-drying process according to any of claims 8 to 11, characterized in that, in Phase d), the vacuum is 6 Pa. SUMMARY A lyophilized compositcomprising lipid and trehalose liposomes, in which a biologically active principle has been incorporated, characterized in that the biologically active principle is highly soluble in water, the weight ratio of trehalose / lipid is < 1.5, and all of the trehalose was added to the outside of the liposomes already formed prior to lyophilizat