Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1995-05-16
2003-07-22
Kishore, Gollamudi S. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C428S402200, C264S004100, C264S004300
Reexamination Certificate
active
06596305
ABSTRACT:
FIELD OF THE INVENTION
The present invention is generally directed to a method of producing liposomes and particularly to a method in which the particle size of the liposome population is controlled by the amount of organic solvent.
BACKGROUND OF THE INVENTION
Methods of forming liposome vesicles for the association of a bioactive agent are well known. As used herein the term “association” shall mean bioactive agent which is encapsulated within the liposome and bioactive agent which, while not encapsulated, remains with the liposome and is not readily separated therefrom.
Some methods of forming liposomes employ an organic solvent to dissolve a lipid alone or the lipid and a bioactive agent such as a drug. For example, in Bally et al., U.S. Pat. No. 5,077,056, lipids are dissolved in an organic solvent and combined with an aqueous medium to form liposomes. Then a bioactive agent such as a drug is loaded into the preformed liposomes using a transmembrane concentration gradient. On the other hand, in Lenk et al., U.S. Pat. No. 5,082,664, a lipid and a bioactive agent are dissolved together in an organic solvent, and combined with an aqueous medium to form liposomes associated with the bioactive agent. In particular, the lipid and the bioactive agent (e.g. lipophilic drugs such as the prostaglandins) are co-dissolved in an aqueous-miscible organic solvent such as ethanol, then added slowly to an aqueous solution, which may additionally contain a drying protectant and/or a buffer, as discussed in the Lenk et al. patent. Both of these patents are hereby incorporated by reference into the present disclosure.
Another method for forming liposomes employs ethanol injection and is discussed in Batzri et al.,
Biochem. Biophys. Acta
. 298:1015 (1973). The ethanol injection method has been used to form liposomes having associated therewith a lipophilic or hydrophilic bioactive agent. When forming liposomes containing a lipophilic bioactive agent (e.g. prostaglandin), an optional preservative and the bioactive agent are added to the ethanol containing lipid. The resulting mixture is then slowly added to an aqueous medium. This process forms liposomes entrapping the aqueous medium. Ethanol injection processes, as well as other liposome formation processes, using a desalted charged lipid are disclosed in Popescu et al., U.S. Pat. No. 5,154,930, incorporated by reference into the present specification. A method of controlling size distribution of resultant liposomes in an ethanol infusion process is discussed in Aitcheson et al., U.S. Pat. No. 4,994,213.
For the formation of liposomes having a hydrophilic bioactive agent associated therewith (e.g. aminoglycoside antibiotics, such as gentamicin), the bioactive agent is added to the aqueous phase. The lipid and ethanol are combined to form a solution which is added to the aqueous phase and the resulting mixture is processed to form liposomes. The aqueous phase may be a solution of one or more drying protectants with or without a preservative.
The liposome preparations prepared by such methods typically contain liposomes having a wide variety of particle sizes. It is often desirable to reduce the size of the larger liposomes to obtain a single-modal population distribution encompassing a desired mean particle size. The term “single-modal population distribution” as used herein shall mean that most of the liposomes have a particle size within a continuous range of particle sizes encompassing the mean particle size. The term “mean particle size” shall mean the sum of the diameters of each liposome of the population divided by the total number of liposomes.
Size reduction to obtain a single-modal population distribution can be achieved by a number of methods such as by extrusion through a filter, as described in Pieter Cullis et al., U.S. Pat. No. 5,008,050, incorporated herein by reference.
A method of sizing liposomes by filtration through a 200 nm Unipore™ polycarbonate filter is discussed in Szoka,
Proc. Natl. Acad. Sci. U.S.A
. 75:4194-8 (1978). A size-processing method based on liposome extrusion through a series of uniform straight-pore type polycarbonate membranes is described in Hunt et al., U.S. Pat. No. 4,529,561.
U.S. Pat. No. 4,737,323, describes a method for sizing liposomes by extrusion through an asymmetric ceramic filter. Such filters are designed for operation at relatively high pressure, and can be backflushed to prevent clogging. U.S. Pat. No. 4,927,637, describes a method of sizing liposomes by passing them through a polymer filter having a web-like “tortuous-path” construction.
An alternative type of filter medium is described in Furneaux et al., U.S. Pat. No. 4,687,551. This patent discloses a filter sheet comprising an anodic aluminum oxide film having branched pores extending from one surface of the film to the other. The film is unique in that it includes a system of larger pores extending in from one face and a system of smaller pores extending in from the other face. The system of larger pores interconnects with the system of smaller pores such that the inner ends of one or more smaller pores are joined to the inner end of a larger pore and there are substantially no larger pores that terminate within the film.
The application of an aluminum oxide porous film to the size reduction of liposomes is disclosed in Royden M. Coe et al., U.S. Ser. No. 771,267 filed on Oct. 4, 1991.
Homogenization is another method for size reducing liposomes. In a simple homogenization method, a suspension of liposomes is repeatedly pumped under high pressure through a small orifice or reaction chamber until a desired size distribution is achieved.
The size reduction procedures described above for controlling the size of the final liposome product are time consuming and add significantly to the cost of producing liposomes. It would, therefore, be a significant advance in the art to provide a process for preparing liposomes in which the initial liposome preparation has a population of liposomes with a more uniform size distribution than that obtained with conventional liposome forming methods. As a consequence, the costly and time consuming post-production sizing procedures can be reduced or even eliminated.
It would be a further advance in the art to provide a process of making liposomes that can result in a single-modal population distribution of liposomes encompassing a preselected desired mean particle size.
SUMMARY OF THE INVENTION
The present invention is generally directed to a process of making liposomes by dissolving a lipid in an organic solvent. The concentration of the organic solvent is selected in accordance with a desired mean particle size. The resulting liposome population has a more uniform particle size distribution than prior processes where the concentration of organic solvent is not selected as required in the present invention.
In accordance with the present invention, there is provided a process for producing a population of liposomes having a desired mean particle size, the process comprising forming a mixture of vesicle-forming lipids in a single-phase solvent system containing a water-miscible organic solvent and water, the improvement comprising controlling the mean particle size of the population of liposomes by adjusting the initial concentration of solvent in said solvent system.
The present process can reduce or eliminate the need for costly and time consuming size reduction procedures to obtain a single-modal population distribution of liposomes encompassing a desired mean particle size. In accordance with one aspect of the invention, a desired mean particle size is chosen and the concentration of organic solvent selected which will produce a single-modal population distribution of liposomes encompassing the desired mean particle size, without having to rely on extensive post-production filtering procedures. I
In a particular embodiment of the present invention the step of adjusting the initial concentration of solvent comprises:
(a) forming a first test sample of a population of liposomes from a solvent system
Burns Doane Swecker & Mathis L.L.P.
Elan Pharmaceuticals Inc.
Kishore Gollamudi S.
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