Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1999-11-03
2001-11-20
Kishore, Gollamudi S. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C424S001210, C424S009321, C424S009510, C424S417000, C264S004100, C264S004300, C514S002600, C514S008100, C514S021800
Reexamination Certificate
active
06319517
ABSTRACT:
The invention relates to a pharmaceutical preparation comprising lyophilized liposomes encapsulating a biologically-active principle which is highly insoluble in water, and a process for preparing the 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 having a solubility in water ≦0.01% (w/v).
It is known that the use of liposomes in medical treatments has been held back by difficulties encountered in obtaining pharmaceutical preparations which are sufficiently stable both during lyophilization and over time. The said difficulties consist above all in the preparation of liposomes which neither burst nor pack together. In other words, the liposomes should remain whole and separate from one another.
The structural integrity of the liposomes is also particularly important in the case where the active principle is highly insoluble in water. In point of fact, the bursting of the liposomes during lyophilization and/or during preservation does not prevent the water-soluble active principles from going into 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 bursting of liposomes comprising active principles which are highly insoluble in water, reconstitution of the lyophilisate yields a solution comprising less active principle than required. The greater the quantity of burst liposomes, the greater the difference between the theoretical and the actual quantity of active principle in solution.
A lyophilization method of liposomes comprising a water-soluble biologically-active principle is described by U.S. Pat. No. 4,857,319. This document describes the lyophilization of liposomes preferably of an average size of about 50-100 nm, with the addition, as a preserving agent, of a disaccharide on the inside only (with the liposome content encapsulated), or on the outside only, or on both the inside and the outside. The disaccharide/lipid weight ratio ranges between 0.1:1 and 4:1. Preferably, the disaccharide is trehalose. The freezing phase is carried out at the temperature of liquid nitrogen (−195.8° C.).
In the aforementioned patent, stability characteristics during lyophilization were evaluated by means of measurements of retention of the active principle encapsulated in the liposomes after reconstitution of the lyophilisate via rehydration.
Table 2 of the aforementioned patent shows that retention is high (99-100%) only when the trehalose/lipid ratio is greater than 1.76 and the trehalose is present both on the inside and the outside of the liposomes. When the said ratio is equal to 0.11 and to 0.19, retention is equal to 22% and 49%, respectively, even if the trehalose is present both on the inside and the outside of the liposomes. By contrast, when the trehalose is present only on the outside, the amount of active principle retained is drastically reduced, even for very large quantities of trehalose. In point of fact, with a trehalose/lipid ratio of 3.9, the amount retained is only 26%.
Nevertheless, the aforementioned results are not reproducible when the biologically-active principle is highly insoluble in water. In fact, when the trehalose is added during preparation of the liposomes in order to encapsulate it within the lipid vesicles, a non-homogeneous, non-extrudible 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 remain substantially whole during lyophilization when the trehalose is added in small quantities to the liposomes before lyophilization only, and the said iyophilization is effected by carrying out the freezing phase at a temperature of between −5° and −70° C.
It is an object of the present invention to provide a 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.
After reconstitution by rehydration, the said composition retains in solution more than 95% of the biologically-active principle which is highly insoluble in water (Examples 1, 2 and 3).
Typical 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 their mixtures. Preferably, the lipids are phospholipids. The trehalose/lipid weight ratio, in turn, preferably lies between 1:2 and 1:1.
The average size of the liposomes may be between 50 and 250 nm. Preferably, it is between 50 and 100 nm.
A second object of the invention is constituted by a lyophilization process, characterized in that:
1) from 0.2 to 1.5 parts in weight of trehalose are added for each part in weight of lipids of an aqueous liposome composition in which the average size of the liposomes lies between 50 and 250 nm, and the said liposomes comprise a biologically-active principle which is highly insoluble in water;
2) the said composition is chilled via the lyophilizer chilling-plate to a temperature between −5° and −70° C., at a chilling rate of between 0.5° and 2° C./min.;
3) once the predetermined freezing temperature is reached, the said composition is kept at the said temperature for a period of between 2 and 5 hours;
4) a vacuum of between 5×10
−1
and 8×10
−2
millibar is applied, leaving the temperature of the chilling plate at the chilling temperature defined in point 2) for a period lasting between 2 and 5 hours;
5) the temperature of the chilling plate is brought to −1 50° C., and kept there until the water is completely removed.
The preferred operating conditions are as follows:
Phase 2
freezing temperature: −20° to −30° C.
chilling rate: 0.77° C./min.
Phase 3
time: 3 hours
Phase 4
vacuum: 6×10
−2
millibar
Phase 5
a) When the freezing temperature (Phase 2) is below −15° C., the temperature of the chilling plate is increased to −15° C. at a rate of between 0.5° and 2° C. and lyophilization takes place for 20 hours; then the temperature of the chilling plate is brought to −10° C., and after an hour to +5° C., and lyophilization occurs for 16 hours.
b) When the freezing temperature (Phase 2) is greater than or equal to −15° C., lyophilization is continued for 20 hours, after which the temperature of the chilling plate is taken to +5° C. and lyophilization 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
phosphatidyl ethanolamine : 0.1
triglycerides : 1
free fatty acids : 0.75
DL-&agr;-tocopherol : 0.15
Typically, the aqueous pharmaceutical liposome composition 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 the said dispersion in an aqueous phase;
c) letting the said suspension stand at ambient temperature for a period of between 0 and 48 hours;
d) heating to between 30° and 75° C. for 10-40 minutes;
e) freezing to between −150° and −2000° C.;
f) repeating p
Cavallo Giovanni
Marchitto Leonardo
Aziende Chimiche Riunite Angelini Francesco A.C.R.A.F. S.p.A.
Kishore Gollamudi S.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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