Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
2001-01-04
2004-06-15
Nguyen, Dave T. (Department: 1632)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C435S320100, C435S455000, C435S458000, C514S04400A
Reexamination Certificate
active
06749863
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to the systemic delivery of a therapeutic molecule via a liposome complex that is targeted to a pre-selected cell type. More specifically, the invention provides compositions and methods for cell-targeted gene transfer and gene therapy for human cancers whereby a therapeutic molecule is delivered to the targeted cancer cell via a ligand/liposome complex. Treatment of cell proliferative disease (e.g. cancer) results in substantial improvement of the efficacy of radiation and chemotherapeutic interventions.
2. Description of Related Art
The ideal therapeutic for cancer would be one that selectively targets a cellular pathway responsible for the tumor phenotype and would be nontoxic to normal cells. To date, the ideal therapeutic remains just that—an ideal. While cancer treatments involving gene therapy and anti-sense molecules have substantial promise, there are many issues that need to be addressed before this promise can be realized. Perhaps foremost among the issues associated with macromolecular treatments for cancer and other diseases is the efficient delivery of the therapeutic molecule(s) to the site(s) in the body where they are needed.
A variety of nucleic acid delivery systems (“vectors”) to treat cancer have been evaluated by others, including viruses and liposomes. The ideal vector for human cancer gene therapy would be one that could be systemically administered and then specifically and efficiently target tumor cells wherever they occur in the body. Viral vector-directed methods show high gene transfer efficiency but are deficient in several areas. The limitations of a viral approach are related to their lack of targeting and to the presence of residual viral elements that can be immunogenic, cytopathic, or recombinogenic.
A major deficiency of viral vectors is the lack of cancer cell specificity. Absent tumor targeting capability, viral vectors are limited in use to direct, local delivery that does not have the capability to reach metastatic disease—the ultimate cause of death for the majority of cancer patients.
The high titers achievable and the cell tropism that makes viruses attractive as gene therapy and gene transfer delivery vectors present some of their greatest deficiencies. Although the preparation of novel viruses with new targets for infection has been described in the literature, these vectors are problematic due to the need for growing virus to high titer. Consequently, a substantial amount of attention has been directed to non-viral vectors for the delivery of molecular therapeutics, including use in gene transfer and gene therapy.
Progress has been made toward developing non-viral, pharmaceutical formulations of genes for in vivo human therapy, particularly cationic liposome-mediated gene transfer systems. Cationic liposomes are composed of positively charged lipid bilayers and can be complexed to negatively charged, naked DNA by simple mixing of lipids and DNA such that the resulting complex has a net positive charge. The complex is easily bound and taken up by cells, with a relatively high transfection efficiency. Features of cationic liposomes that make them versatile and attractive for DNA delivery include: simplicity of preparation, the ability to complex large amounts of DNA, versatility in use with any type and size of DNA or RNA, the ability to transfect many different types of cells (including non-dividing cells) and lack of immunogenicity or biohazardous activity. The liposome approach offers a number of advantages over viral methodologies for gene delivery. Most significantly, since liposomes are not infectious agents capable of self-replication, they pose no risk of evolving into new classes of infectious human pathogens. Further, cationic liposomes have been shown to be safe and somewhat efficient for in vivo gene delivery. Since liposomes are not infectious agents, they can be formulated by simple mixing. Further, cationic liposomes have been shown to be safe and somewhat efficient for in vivo gene delivery. Clinical trials are now underway using cationic liposomes for gene delivery, and liposomes for delivery of small molecule therapeutics (e.g., chemotherapeutic and antifungal agents) are already on the market.
One disadvantage of cationic liposomes is that they lack tumor specificity and have relatively low transfection efficiencies as compared to viral vectors. However, targeting cancer cells via liposomes can be achieved by modifying the liposomes so that they bear a ligand recognized by a cell surface receptor. Receptor-mediated endocytosis represents a highly efficient internalization pathway in eukaryotic cells. The presence of a ligand on a liposome facilitates the entry of DNA into cells through initial binding of ligand by its receptor on the cell surface followed by internalization of the bound complex. Once internalized, sufficient DNA can escape the endocytic pathway to be expressed in the cell nucleus.
There now exists a substantial knowledge base regarding the molecules that reside on the exterior surfaces of cancer cells. Surface molecules can be used to selectively target liposomes to tumor cells, because the molecules that are found upon the exterior of tumor cells differ from those on normal cells. For example, if a liposome has the protein transferrin (Tf) on its surface, it can target cancer cells that have high levels of the transferrin receptor.
A variety of ligands have been examined for their liposome-targeting ability, including folic acid (folate), a vitamin necessary for DNA synthesis, and transferrin. Both the folate receptor and transferrin receptor levels are found to be elevated in various types of cancer cells including ovarian, oral, breast, prostate and colon. The presence of such receptors can correlate with the aggressive or proliferative status of tumor cells. The folate receptor has also been shown to recycle during the internalization of folate in rapidly dividing cells such as cancer cells. Moreover, the transferrin and folate-conjugated macromolecules and liposomes have been shown to be taken up specifically by receptor-bearing tumor cells by receptor mediated endocytosis. Thus the folate and transferrin receptors are considered to be useful as prognostic tumor markers for cancer and as potential targets for drug delivery in the therapy of malignant cell growth.
Failure to respond to radiotherapy and chemotherapy represents an unmet medical need in the treatment of many types of cancer. Often, when cancer recurs, the tumors have acquired increased resistance to radiation or to chemotherapeutic agents. The incorporation into cancer therapies of a new component which results in sensitization to these therapies would have immense clinical relevance. One way in which such chemo/radio sensitization could be achieved is via targeted gene therapy.
An important role for p53 in the control of cellular proliferation by the regulation of cell cycle events and induction of programmed cell death (apoptosis) has been established. Since it appears that most anti-cancer agents work by inducing apoptosis, inhibition of, or changes in, this pathway may lead to failure of therapeutic regimens. A direct link, therefore, has been suggested between abnormalities in p53 and resistance to cytotoxic cancer treatments (both chemo- and radiotherapy). It has also been suggested that the loss of p53 function may contribute to the cross-resistance to anti-cancer agents observed in some tumor cells. Various groups have established a positive correlation between the presence of mutant p53 and chemoresistance in mouse fibrosarcomas and in primary tumor cultures from breast carcinomas, human gastric and esophageal carcinomas, as well as B-cell chronic lymphoblastic leukemia. In addition, chemosensitivity via apoptosis reportedly was restored by expression of wtp53 in non-small cell lung carcinoma mouse xenografts carrying mutant p53.
A role for the tumor suppressor gene p53 in many critical cellular pathways, particularly in the cellular r
Chang Esther H.
Pirollo Kathleen
Xu Liang
Georgetown University
Nguyen Dave T.
Rothwell Figg Ernst & Manbeck
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