Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
2000-09-01
2004-12-28
Naff, David M. (Department: 1651)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C428S402200, C435S177000, C435S458000, C514S04400A, C536S022100
Reexamination Certificate
active
06835395
ABSTRACT:
BACKGROUND OF THE INVENTION
This application relates to oligodeoxynucleotide-containing small multilamellar lipid vesicles to methods of making such vesicles.
Oligodeoxynucleotides (ODN), ribozymes and plasmid DNA are recognized as having potential for many therapeutic applications. For example, sequence-specific antisense ODN may be used to inhibit expression of gene sequences. However, these large polyanionic macromolecules possess several inherent characteristics that restrict their pre-clinical and clinical utility for the therapy of chronic diseases. These include
(1) the cost and production of clinical grade materials (Prazeres et al.,
Trends Biotechnol.
17: 169-174(1999));
(2) degradation and inactivation by nucleases in plasma and cells (Akhtar et al.,
Life Sci.
49: 1793-801 (1991));
(3) poor intracellular delivery (Hope et al.,
Mol. Membr. Biol.
15: 1-14(1998), Rojanasukul, Y.,
Adv. Drug. Delivery Rev.
118: 115-131 (1996));
(4) rapid plasma elimination (Agrawal and Zhang,
Ciba Found. Symp.
209: 60-75 (1997), Crooke et al.,
J. Pharmacol. Exp. Ther.
227: 923-937 (1996)); and
(5) renal and dose-limiting hemodynamic toxicities (Henry et al.,
J. Pharmacol. Exp. Ther.
281: 810-816 (1997), Henry et al.,
Antisense Nucleic Acid Drug Dev.
7: 503-510 (1997), Galbraith et al.,
Antisense Res. Dev.
4: 201-206 (1994).
In an effort to overcome these problems, efforts have been made to formulate DNA and RNA-based therapeutics in lipid delivery systems. (Hope, et al., supra, Zelphati, et al.,
J. Liposome Res.
7: 31-49 (1997), Smyth-Templeton, et al.,
Nat. Biotechnol.
15: 647-652 (1997)). The development of such systems has been limited by two principal factors—low encapsulation efficiency (generally <10%) and low drug-to-lipid ratios (0.001-0.1%, w/w) when using neutral lipids. For example, utilization of cationic lipids has been shown to provide improved encapsulation efficiency. Thus, Gokhale et al. (
Gene Ther.
4: 1289-1299 (1997)) have described a lipid composition of PC:CH:DDAB (55:28:17 molar ratio) containing raf-1 ODN that sensitizes SQ-20B xenografted tumors to ionizing radiation. However, only 1% of the total administered dose remained in the circulation 5 minutes post irradiation.
In other efforts to improve encapsulation efficiencies, several approaches have been employed to entrap antisense ODN in lipid vesicles. For example, the minimum volume entrapment (MVE) procedure described by Thierry et al., (
Nucleic Acids Res.
20: 5691-5698 (1992),
Gene Regulation: Biology of Antisense RNA and DNA
(Erickson and Izant, eds.), pp. 147-161, Raven Press Ltd, New York (1992)) generates high encapsulation efficiencies and fairly high drug to lipid-ratios. However, the formulation employs 3-7% cardiolipin, which is a potent activator of rat and human complement, thus giving rise in increased hemodynamic toxicity. Furthermore, similar liposome formulations containing cardiolipin have been shown to exhibit very short circulation times. Thus, such compositions overcome some of the problems with free ODN therapeutics only at the expense of increasing other problems. The clinical application of this formulation of such compositions would therefore be expected to be limited. (Chonn, et al.,
J. Biol. Chem/
267: 18759-18765 (1992).
Another approach to improving encapsulation efficiency has been to chemically modify the ODN to make them more lipophilic. (Juliano and Akhtar,
Antisense Res. Dev.
2: 165-176 (1992), Tari et al,
Blood
84: 4601-4607 (1994). However, these molecules are insoluble in aqueous environments which has, at least until recently, placed limitations on their clinical development. (See Tari et al.,
J. Liposome Res.
8: 251-264 (1998)).
What these and similar studies clearly establish is that the interaction of anionic ODN and lipid carriers with the body is a very complicated one, which cannot be readily predicted. These and similar studies also show that there remains substantial room for improvement in such compositions in order to avoid the various drawbacks described above.
International Patent Publication No. WO 96/40964 discloses several methods for making lipid-nucleic acid particles One of these methods involves preparing a mixture of cationic and non-cationic lipids in an organic solvent, contacts and aqueous solution of nucleic acid with said mixture to provide a clear single phase, and removing the organic solvent. The list of organic solvent on Page 26 of this publication does not include ethanol, the solvent used in the present invention. Furthermore, electron microscopic observation of the lipid particles formed in this methodology shows that they are large unilamellar vesicles (See example 8 of WO 96/40964), and thus that they are structurally different from the compositions of the present invention.
SUMMARY OF THE INVENTION
It has now been determined that lipidic compositions with superior characteristics for in vivo delivery of oligodeoxynucleotides can easily and efficiently be made in the form of small multilamellar vesicles. Thus, the invention provides a composition comprising a population of nucleic acid-containing lipid vesicles in a liquid carrier, wherein at least a portion of the lipid vesicles are small:multilamellar vesicles. The small multilamellar vesicles comprise (a) a lipid component comprising 20-30 mol % of an ionizable amino lipid such as DODAP, and a steric barrier lipid such as PEG-CerC
14
; and (b) an oligodeoxynucleotide contained in the lumen or interlamellar spaces of the small multilamellar vesicles. The ODN and lipid components are preferably present in the small multilamellar vesicles in a mole ratio of from 0.15 to 0.25.
The compositions of the invention can be made by a method comprising the steps of:
(a) preparing a lipid mixture comprising 20-30 mol % of an ionizable amino lipid, a steric barrier lipid and additional lipid components selected from among neutral lipids and sterols in an ethanolic solvent;
(b) preparing a solution of oligodeoxynucleotide in an aqueous solvent having a pH at which the ionizable amino lipid is positively charged;
(c) adding the lipid mixture to the solution of oligodeoxynucleotide to form a mixture containing lipid vesicles;
(d) passing the mixture containing lipid vesicles through a filter to produce sized lipid vesicles in a solution containing ethanol;
(e) removing the ethanol from the sized lipid vesicles; and
(f) increasing the pH of the solution surrounding the sized lipid vesicles to in reduce the net positive charge on the exterior of the sized lipid vesicles. At least a portion of the A sized lipid vesicles are small multilamellar vesicles.
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Akhtar, S., et al., Liposome delivery of antisense oligonucleotides: adsortpion and efflux characteristics of phosphorothioate oligodeoxynucleotides; Journal of Controlled Release, 22 (1992) 47-56.
Bennett, C. Frank, Intracellular Delivery of Oligonucleotides with Cationic Liposomes, Chapter 14.
Bennett, C. Frank, et al., Pharmacokinetics in mice of a [3H]-labeled phosphorothioate oligonucleotide formulated in the presence and absence of a cationic lipid; Journal of Controlled Release, 41 (1996) 121-130.
Juliano, R.L. et al., Liposomes as a Drug Delivery System for Antisense Oligonucleotides; Antisense Research and Development 2:165-176 (1992).
Litzinger, David C., Limitations of Cationic Liposomes For Antisense Oligonucleotide Delivery in Vivo, Journal of Liposome Research, 7(1), 51-61 (
Ansell Steven M.
Cullis Pieter R
Dos Santos Nancy
Harasym Troy O.
Hope Michael J.
Naff David M.
Oppedahl & Larson LLP
The University of British Columbia
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