Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Particulate form
Reexamination Certificate
2001-02-16
2003-11-25
Page, Thurman K. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Particulate form
C424S489000, C424S499000, C424S484000, C424S488000, C424S422000, C424S423000, C424S426000, C514S772100
Reexamination Certificate
active
06652886
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to delivery of a bioactive agent. More particularly, the invention relates to a composition and method for delivering bioactive agents, such as DNA, RNA, oligonucleotides, proteins, peptides, and drugs, by facilitating their transmembrane transport or by enhancing their adhesion to biological surfaces. It relates particularly to a biodegradable cationic copolymer of a poly(alkylenimine) (PAI) and a hydrophilic polymer wherein the PAI and the hydrophilic polymer are covalently linked by a biodegradable linkage. The cationic copolymers of the present invention can be used in drug delivery and are especially useful for delivery of nucleic acids or any anionic bioactive agents.
BACKGROUND OF THE INVENTION
Biodegradable polymers are gaining attention as drug delivery systems. R. Langer, New Methods of Drug delivery, 249 Science 1527-1533 (1990); B. Jeong et al., Biodegradable Block Copolymers as Injectable Drug-delivery Systems, 388 Nature 860-862 (1997). Delivering bioactive agents from a biodegradable delivery system is highly desirable because the need for a surgical procedure to remove the delivery system is avoided. Controlled release of bioactive agents can reduce the required frequency of administration by maintaining the concentration of the therapeutic agent at desired levels. One important means of maintaining the proper concentration is by controlling the degradation rate of the biodegradable drug delivery system.
Gene therapy is generally considered as a promising approach, not only for the treatment of diseases with genetic defects, but also in the development of strategies for treatment and prevention of chronic diseases such as cancer, cardiovascular disease and rheumatoid arthritis. However, nucleic acids, as well as other polyanionic substances, are rapidly degraded by nucleases and exhibit poor cellular uptake when delivered in aqueous solutions. Since early efforts to identify methods for delivery of nucleic acids in tissue culture cells in the mid 1950's, steady progress has been made towards improving delivery of functional DNA, RNA, and antisense oligonucleotides in vitro and in vivo.
The gene carriers used so far include viral systems (retroviruses, adenoviruses, adeno-associated viruses, or herpes simplex viruses) or nonviral systems (liposomes, polymers, peptides, calcium phosphate precipitation and electroporation). Viral vectors have been shown to have high transfection efficiency when compared to non-viral vectors, but due to several drawbacks, such as targeting only dividing cells, random DNA insertion, their low capacity for carrying large sized therapeutic genes, risk of replication, and possible host immune reaction, their use in vivo is severely limited.
An ideal transfection reagent should exhibit a high level of transfection activity without needing any mechanical or physical manipulation of the cells or tissues. The reagent should be non-toxic, or minimally toxic, at the effective dose. It should also be biodegradable in order to avoid any long-term adverse side-effects on the treated cells. When gene carriers are used for delivery of nucleic acids in vivo, it is essential that the gene carriers themselves be nontoxic and that they degrade into non-toxic products. To minimize the toxicity of the intact gene carrier and its degradation products, the design of gene carriers needs to be based on naturally occurring metabolites.
Because of their sub-cellular size, nanoparticles are hypothesized to enhance interfacial cellular uptake, thus achieving in a true sense a local pharmacological drug effect. It is also hypothesized that there would be enhanced cellular uptake of drugs contained in nanoparticles (due to endocytosis) compared to the uptake of the corresponding free drug. Nanoparticles have been investigated as drug carrier systems for tumor localization of therapeutic agents in cancer therapy, for intracellular targeting (antiviral or antibacterial agents), for targeting to the reticuloendothelial system (parasitic infections), as an immunological adjuvant (by oral and subcutaneous routes), for ocular delivery with sustained drug action, and for prolonged systemic drug therapy.
As compared to viral gene carriers, there are several advantages to the use of non-viral based gene therapies, including their relative safety and low cost of manufacture. Non-viral gene delivery systems such as cationic polymers or synthetic gene carriers, e.g. poly-L-lysine (PLL), are being widely sought as alternatives and investigated intensively to circumvent some of the problems encountered with use of viral vectors. J. Cheng et al., Effect of Size and Serum Proteins on Transfection Efficiency of Poly((2-dimethylamino)ethyl methacrylate)-plasmid nanoparticles, 13 Pharm. Res. 1038-1042 (1996). There are several polymeric materials currently being investigated for use as gene carriers, of which poly-L-lysine (PLL) is the most popular, but few of them are biodegradable. Biodegradable polymers, such as polylactic/glycolic acid(negatively charged), and polylactide/glycolide(neutral) have been used as gene carriers in the form of non-soluble particulates. Amarucyama et al, Nanoparticle DNA Carrier with PLL Grafted Polysallanide Copolymer and Polylactic Acid, 8 Bioconjugate, 735-739(1997). In general, cationic polymers are known to be toxic and the PLL backbone is barely degraded under physiological conditions. It remains in cells and tissues and causes an undesirably high toxicity. A. Segouras & R. Dunlan, Methods for Evaluation of Biocompatibility of Synthetic Polymers, 1 J.Mater.Sci in Medicine, 61-68(1990).
PAIs such as poly(ethylenimine) (PEI) and polyspermine have been known as efficient gene carriers with high cationic charge potentials. Branched PEI consists of approximately 25, 50 and 25% of primary, secondary and tertiary amines and is able to condense and deliver DNA in vitro and in vivo, W. T. Godbey et al., 51 J. Biomed. Mater. Res. 321 (2000); W. T. Godbey et al., 60 J. Contr. Rel. 149 (1999); D. D. Dunlap et al., 25 Nucleic Acids Research 3095 (1997); O. Boussif et al., 92 Proc. Nat l Acad. Sci. USA 7297 (1995). Primary amines of PEI are reported to participate in forming complexes with DNA by ionic interaction with phosphate groups, while the secondary and tertiary amines cause a substantial endosomal disruption after endocytosis due to their buffering effect which contributes to the high transfection efficiency of PEI. The high transfection efficiency of PEI, along with its cytotoxicity, strongly depends on its molecular weight. It is generally believed that PEI with a molecular weight higher than 25 K displays a high transfection efficiency and toxicity, while PEI with molecular weight less than 1.8 K shows almost no transfection, but is less toxic, S. Brunner et al., 7 Gene Ther. 401 (2000); D. Fischer et al., 16 Pharm. Res. 1273 (1999); W. T. Godbey et al., 45 J. Biomed. Mater. Res. 268 (1999). In addition, just like most cationic polymers, PEI has drawbacks since complexes of PEI and DNA are often poorly soluble under physiological conditions, A. V. Kabanov et al., 6 Bioconjugate Chem. 7 (1995).
Di-block and graft copolymers of PEI and PEG have been synthesized and investigated by several research groups, Y. Akiyama et al., 33 Macromolecules 5841 (2000); S. V. Vinogradov et al., 9 Bioconjugate Chem. 805 (1998). Although copolymers of high molecular weight PEI and PEG exhibit considerable transfection efficiency, with the employment of high molecular weight PEI, cytotoxicity still remains as a problem. In addition, none of the existing copolymers of PEI and PEG are biodegradable.
In view of the foregoing, development of a gene carrier for gene therapy and drug delivery that is non-toxic, biodegradable, and capable of forming nanoparticles, or transfection complexes will be appreciated and desired. The novel gene carrier of the present invention comprises a novel cationic copolymer of a poly(alkylenimine) (PAI) and a hydrophilic polymer, wherein the PAI and the hydrophilic polymer are covalently linke
Ahn Cheol Hee
Kim Sung Wan
Di Nola-Baron Liliana
Expression Genetics
Page Thurman K.
Thorpe North & Western LLP
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