Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1997-02-20
2002-06-04
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
06399094
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a liposomal preparation, its production and use. Areas of application are pharmacy and medicine, particularly its use as carrier systems for drug substances, vaccines, diagnostics and vectors.
2. Brief Description of the Background of the Invention Including Prior Art
Liposomes have gained increasing importance in the past years as carriers or encapsulation means for various substances, particularly in medical applications (see Liposomes from Physics to Applications, D. D. Lasic, Elsevier, Amsterdam 1993). Among the various types of liposomes mostly those are regarded favorable, which have a small to intermediate vesicle size such as from about 10 to 300 nm with good size uniformity and which have one single membrane shell and provide a unilamellar vesicle. Moreover, the active substances should be entrapped or incorporated in the liposomes in an efficient manner.
For the preparation of liposomes, numerous methods have been described. These methods, however, fulfill only insufficiently at least one of the above mentioned main requirements—small to intermediate liposome size or high encapsulation efficiency. This is mainly the case when the active substance is water soluble and thus should be encapsulated and entrapped in the aqueous interior of the liposomes. The so-called DRV-method and the so-called REV-method both yield good encapsulation efficiencies but at the same time yield populations of inhomogeneous, mostly large liposomes. In contrast, other previously reported preparation methods based on ultrasonic treatment, high-pressure homogenization or detergent removal and dialysis can achieve in case of homogeneous, small to intermediate sized liposomes only a poor encapsulation efficiency, particularly with water soluble substances (Liposome Technology 2nd edition, volume I-III, G. Gregoriadis (Ed.), CRC Press Inc., Boca Raton, Flo., 1993, 37-48; D. Bachmann et al. Preparation of Liposomes using a Mini-Lab 8.30 H high-pressure homogenizer, Int. J. Pharm. 91, 1/93, 69-74).
Liposomes, after three decades of research, are still gaining increasing interest with special emphasis more recently on their use as drug carrier systems. For therapeutic purposes, they must be loaded with active substances. This is more easily achieved with lipophilic or amphiphilic molecules as they have a tendency to be incorporated in the liposomal membrane. In contrast, hydrophilic molecules must be encapsulated in the aqueous interior which, in general, cannot easily be performed in an efficient manner.
A variety of liposome preparation techniques have been developed over the past three decades, none of them, however, perfectly fulfilling the two basic requirements homogeneous and not too large liposome sizes and efficient encapsulation of hydrophilic molecules at the same time. Whereas the dehydration-rehydration vesicles DRV techniques (C. Kirby and G. Gregoriadis, 1984, Dehydration-rehydration vesicles: a simple method for high yield drug entrapment in liposomes, Biotechnology, pp. 979-984), and reverse-space evaporation techniques REV (F. Szoka and D. Papahadjopoulos, 1978, Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation, Proc. Natl. Acad. Sci., USA 75, 4194-4198), achieve high encapsulation efficiencies but only relatively large and heterogeneous liposome sizes, it is just the opposite with high-pressure homogenization (M. Brandl, D. Bachmann, M. Drechsler, and K. H. Bauer, 1990, Liposome preparation by a new high-pressure homogenizer Gavlin Macron Lab 40 Drug Dev. Ind. Pharm. 16, 2167-2192; M. Brandl, D. Bachmann, M. Drechsler, and K. H. Bauer, 1993, Liposome Preparation using High-pressure Homogenizers, in G. Gregoriadis (Ed.) Liposome Technology 2nd edition, Vol. 1, pp. 49-65, CRC, Boca Raton), or detergent depletion techniques (J. Brunner, P. Skrabal, and H. Hauser, 1976, Single bilayer vesicles prepared without sonication, physico-chemical properties, Biochim. Biophys. Acta 455, 322-331). The concept at the beginning of the invention was fairly straightforward: for low-molecular weight hydrophilic molecules, which represent the majority of classical drug substances, the degree of entrapment into liposomes can be regarded a result of partition of the aqueous drug solution at the moment of liposome formation into a compartment inside the liposomes and a compartment in-between the liposomes.
The ratio of volume inside the liposomes compared to the total aqueous volume of the preparation is defined as encapsulation efficiency. The ratio can theoretically be increased by two alterations within the system: Firstly, when at constant lipid concentration, only a few large liposomes are formed instead of many small liposomes, the entrapped aqueous volume is increased. For technical, and in the case of i.v. injection, also for pharmacokinetic reasons, the enlargement of liposome size is confined to narrow limits. In the case of i.v. injection liposomes sizes between 70 and not more than 200 (D. Liu, A. Mori, and L. Huang, 1992, Role of liposome size and res. blockade in controlling biodistribution and tumor uptake of gml-containing liposomes, Biochem. Biophys. Acta 1104, 95-101), and sufficient size homogeneity (D. Liu and L. Huang, 1992, Size homogeneity of a liposome preparation is crucial for liposome biodistribution in vivo, J. Liposome Res. 2, 57-66), are regarded as favorable for most applications. Secondly, with increasing lipid concentration, more liposomes per unit volume of the preparation are formed. This again should lead to an increase in the ratio of aqueous space inside compared to in-between the liposomes as long as liposome shape and lamellarity are unchanged. Phospholipid, however, when dispersed in aqueous medium at or above lipid contents of 200 to 300 mM result in highly viscous dispersions up to semisolid consistency. Although it was expected that these viscous to semisolid preparations would no longer be liposome dispersions, it was of interest to prepare such “pastes” preferentially with homogeneous physicochemical characteristics. Based on previous experience with liposome preparation by the one-step technique (M. Brandl, D. Bachmann, M. Drechsler, and K. R. Bauer, 1990, Liposome preparation by a new high-pressure homogenizer Gavlin Macron Lab 40 Drug Dev. Ind. Pharm. 16, 2167-2192, M. Brandl, D. Bachmann, M. Drechsler, and K. H. Bauer, 1993, Liposome Preparation using High-pressure Homogenizers, in G. Gregoriadis (Ed.) Liposome Technology 2nd edition, Vol. 1, pp. 49-65, CRC, Boca Raton), high-pressure homogenization for “forced hydration” of lipids was employed.
In order to study the inner structure of the pastes in terms of homogeneity, freeze fracture electron microscopical visualization was used. It was noticed that these pastes may retain hydrophilic markers and thus potentially serve as depot formulations for controlled release of drugs. The drug release behavior of the pastes was further analyzed in a standard in-vitro continuous flow through apparatus as normally employed for release tests of ointments. Preliminary reports of this project have been published recently, M. Brandl and R. Reszka, 1995, Preparation and characterization of phospholipid membrane gels as depot formulations for potential use as implants, Proc. Intern. Symp. Control. Rel. Bioac. Mater. 22, 472-473; M. Brandl, C. Tardi, M. Menzel, and R. Reszka, 1995, Highly concentrated phospholipid dispersions: preparation by high pressure homogenization and analysis of drug release. Proceedings 4th Liposome Res. Days. Freiburg PSI (not completely legible in publication); M. Brandl, D. Bachmann, R. Reszka, and M. Drechsler, 1996, Unilamellar Liposomal Preparations with High Active Substance Content.
SUMMARY OF THE INVENTION
1. Purposes of the Invention
The goal of the invention is to make a liposomal preparation with good encapsulation efficiency for active substances, based on homogeneous liposomes of small to intermediate size.
These and other objects
Bachmann Dieter
Brandl Martin
Drechsler Markus
Reszka Regine
Brandl Martin
Kishore Gollamudi S.
Norris & McLaughlin & Marcus
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