Method and apparatus for liposome production

Chemical apparatus and process disinfecting – deodorizing – preser – Shock or sound wave – Including supersonic or ultrasonic energy generation means

Reexamination Certificate

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C264S004300, C425S005000, C428S402200, C424S450000

Reexamination Certificate

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06534018

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to a formulation for the delivery of a variety of beneficial and/or therapeutic compounds by encapsulation within liposomes, and a machine of unique design for the controlled production of same. Specifically the invention relates to a precisely controlled metering system for the mixing of the two or more components of the liposomal preparations so that the various factors affecting the consistency, reproducibility and efficacy of the product may be monitored and controlled. The present invention also relates to a method and apparatus for the production of liposomal suspensions, emulsions, ointments and creams.
2. Background
Liposomes are lipid vesicles made of membrane-like lipid bilayers separated by aqueous layers. Liposomes have been widely used to encapsulate biologically active agents for use as drug carriers since water- or lipid-soluble substances may be entrapped within the aqueous layers or within the bilayers themselves. There are numerous variables that can be adjusted to optimize this drug delivery system. These include, the number of lipid layers, size, surface charge, lipid composition and the methods of preparation.
Liposomes have been utilized in numerous pharmaceutical applications, including injectable, inhalation, oral and topical formulations, and provide advantages such as controlled or sustained release, enhanced drug delivery, and reduced systemic side effects as a result of delivery localization.
Materials and procedures for forming liposomes are well-known to those skilled in the art and will only be briefly described herein. Upon dispersion in an appropriate medium, a wide variety of phospholipids swell, hydrate and form multilamellar concentric bilayer vesicles with layers of aqueous media separating the lipid bilayers. These systems are referred to as multilamellar liposomes or multilamellar lipid vesicles (“MLVs”) and have diameters within the range of 10 nm to 100 &mgr;m. These MLVs were first described by Bangham, et al.,
J. Mol. Biol
. 13:238-252 (1965). In general, lipids or lipophilic substances are dissolved in an organic solvent. When the solvent is removed, such as under vacuum by rotary evaporation, the lipid residue forms a film on the wall of the container. An aqueous solution that typically contains electrolytes or hydrophilic biologically active materials is then added to the film. Large MLVs are produced upon agitation. When smaller MLVs are desired, the larger vesicles are subjected to sonication, sequential filtration through filters with decreasing pore size or reduced by other forms of mechanical shearing. There are also techniques by which MLVs can be reduced both in size and in number of lamellae, for example, by pressurized extrusion (Barenholz, et al.,
FEBS Lett.
99:210-214 (1979)).
Liposomes can also take the form of unilamellar vesicles, which are prepared by more extensive sonication of MLVs, and consist of a single spherical lipid bilayer surrounding an aqueous solution. Unilamellar vesicles (“ULVs”) can be small, having diameters within the range of 20 to 200 nm, while larger ULVs can have diameters within the range of 200 nm to 2 &mgr;m. There are several well-known techniques for making unilamellar vesicles. In Papahadjopoulos, et al.,
Biochim et Biophys Acta
135:624-238 (1968), sonication of an aqueous dispersion of phospholipids produces small ULVs having a lipid bilayer surrounding an aqueous solution. Schneider, U.S. Pat. No. 4,089,801 describes the formation of liposome precursors by ultrasonication, followed by the addition of an aqueous medium containing amphiphilic compounds and centrifugation to form a biomolecular lipid layer system.
Small ULVs can also be prepared by the ethanol injection technique described by Batzri, et al.,
Biochim et Biophys Acta
298:1015-1019 (1973) and the ether injection technique of Deamer, et al.,
Biochim et Biophys Acta
443:629-634 (1976). These methods involve the rapid injection of an organic solution of lipids into a buffer solution, which results in the rapid formation of unilamellar liposomes. Another technique for making ULVs is taught by Weder, et al. in “Liposome Technology”, ed. G. Gregoriadis, CRC Press Inc., Boca Raton, Fla., Vol. I, Chapter 7, pg. 79-107 (1984). This detergent removal method involves solubilizing the lipids and additives with detergents by agitation or sonication to produce the desired vesicles.
Papahadjopoulos, et al., U.S. Pat. No. 4,235,871, describes the preparation of large ULVs by a reverse phase evaporation technique that involves the formation of a water-in-oil emulsion of lipids in an organic solvent and the drug to be encapsulated in an aqueous buffer solution. The organic solvent is removed under pressure to yield a mixture which, upon agitation or dispersion in an aqueous media, is converted to large ULVs. Suzuki et al., U.S. Pat. No. 4,016,100, describes another method of encapsulating agents in unilamellar vesicles by freezing/thawing an aqueous phospholipid dispersion of the agent and lipids.
In addition to the MLVs and ULVs, liposomes can also be multivesicular. Described in Kim, et al.,
Biochim et Biophys Acta
728:339-348 (1983), these multivesicular liposomes are spherical and contain internal granular structures. The outer membrane is a lipid bilayer and the internal region contains small compartments separated by bilayer septum. Still yet another type of liposomes are oligolamellar vesicles (“OLVs”), which have a large center compartment surrounded by several peripheral lipid layers. These vesicles, having a diameter of 2-15 &mgr;m, are described in Callo, et al., Cryobiology 22(
3
):251-267 (1985).
Mezei, et al., U.S. Pat. Nos. 4,485,054 and 4,761,288 also describe methods of preparing lipid vesicles. More recently, Hsu, U.S. Pat. No. 5,653,996 describes a method of preparing liposomes utilizing aerosolization and Yiournas, et al., U.S. Pat. No. 5,013,497 describes a method for preparing liposomes utilizing a high velocity-shear mixing chamber. Methods are also described that use specific starting materials to produce ULVs (Wallach, et al., U.S. Pat. No. 4,853,228) or OLVs (Wallach, U.S. Pat. Nos. 5,474,848 and 5,628,936).
A comprehensive review of all the aforementioned lipid vesicles and methods for their preparation are described in “Liposome Technology”, ed. G. Gregoriadis, CRC Press Inc., Boca Raton, Fla., Vol. I, II & III (1984). This and the aforementioned references describing various lipid vesicles suitable for use in the invention are incorporated herein by reference.
Current methods of manufacturing liposomes are typically batch processes. Attempts at large scale or continuous manufacturing have largely been unsuccessful, primarily due to the problems associated with mixing an aqueous liquid phase with the lipid phase and the need to maintain the lipid phase at a relatively constant temperature.
Accordingly, there is a need for an improved method for the production of liposomes, preferably one that can produce liposomes in a continuous fashion rather than by batch methods, without the variations and uncontrolled differences which make large scale production of liposomal preparations problematic. In addition, there is a need for an improved method and apparatus for producing other liquid compositions, including but not limited to emulsions, ointments and creams. Those needs are met by the instant invention.
SUMMARY OF THE INVENTION
The present invention relates to a method for the continuous production of a composition of matter, such as lipid vesicles, by in-line mixing, said method comprising: (a) preparing a first phase, such as a lipid phase, and storing the lipid phase in a first storage means that is maintained at a set temperature; (b) preparing a second phase, such as an aqueous phase, and storing the aqueous phase in a second storage means that is maintained at a set temperature; (c) combining the lipid and aqueous phases by means of a mixing device having first and second metering systems, a pre-mixing system and a mixer, such

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