Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Particulate form
Reexamination Certificate
2002-04-04
2004-11-09
Nguyen, Dave T. (Department: 1635)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Particulate form
C424S009322, C536S023100
Reexamination Certificate
active
06814980
ABSTRACT:
1. FIELD OF THE INVENTION
The subject invention is in the field of sustained drug delivery using micro-encapsulation of bioactive agents. In particular, the invention describes improved methods for incorporating polyanionic bioactive agents into polymeric microspheres and/or nanospheres through the use of a condensing agent, as well as microspheres and/or nanospheres prepared by the method.
2. BACKGROUND OF THE INVENTION
2.1. Gene Therapy
Gene therapy was originally conceived as a specific gene replacement therapy for correcting heritable defects by delivering functionally active therapeutic genes into targeted cells. Initial efforts toward somatic gene therapy have largely relied on indirect means of introducing genes into tissues, called ex vivo gene therapy. In ex vivo protocols, target cells are removed from the body, transfected or infected in vitro with vectors carrying recombinant genes, and re-implanted into the body (“autologous cell transfer”). A variety of transfection techniques are currently available and used to transfer DNA in vitro into cells, including calcium phosphate-DNA precipitation, DEAE-Dextran transfection, electroporation, liposome mediated DNA transfer, and transduction with recombinant viral vectors. Such ex vivo treatment protocols have been proposed to transfer DNA into a variety of different cell types including epithelial cells (Morgan et al., U.S. Pat. No. 4,868,116; Morgan & Mulligan, WO87/00201; Morgan et al., 1987,
Science
237:1476-1479; Morgan & Mulligan, U.S. Pat. No. 4,980,286), endothelial cells (WO89/05345), hepatocytes (Wilson & Mulligan, WO89/07136; Wolff et al., 1987,
Proc. Natl. Acad. Sci. USA
84:3344-3348; Ledley et al., 1987,
Proc. Natl. Acad. Sci. USA
84:5335-5339; Wilson et al., 1990,
Proc. Natl. Acad. Sci. USA
87:8437-8441), fibroblasts (Palmer et al., 1987,
Proc. Natl. Acad. Sci. USA
84:1055-1059; Anson et al., 1987,
Mol. Biol. Med.
4:11-20; Rosenberg et al., 1988,
Science
242:1575-1578; Naughton & Naughton, U.S. Pat. No. 4,963,489), lymphocytes (Anderson et al., U.S. Pat. No. 5,399,346; Blaese et al., 1995,
Science
270:475-480) and hematopoietic stem cells (Lim et al., 1989,
Proc. Natl. Acad. Sci. USA
86:8892-8896; Anderson et al., U.S. Pat. No. 5,399,346).
Direct in vivo gene transfer has recently been attempted with formulations of DNA trapped in liposomes (Ledley et al., 1987,
J. Pediatrics
110:1), in proteoliposomes that contain viral envelope receptor proteins (Nicolau et al., 1983,
Proc. Natl. Acad. Sci. USA
80:1068), and with DNA coupled to a polylysine-glycoprotein carrier complex. In addition, “gene guns” have been used for gene delivery into cells (Australian Patent No. 9068389). Some have even speculated that naked DNA, or DNA associated with liposomes, can be formulated in liquid carrier solutions suitable for injection into interstitial spaces for transfer of DNA into cells (Felgner, WO90/11092).
Perhaps one of the greatest problems associated with currently devised gene therapies, whether ex vivo or in vivo, is the inability to transfer DNA efficiently into a targeted cell population and to achieve high level expression of the gene product in vivo. Viral vectors are regarded as the most efficient system, and recombinant, replication-defective viral vectors have been used to transduce (via infection) cells both ex vivo and in vivo. Such vectors have included retroviral, adenoviral and adeno-associated, and herpes viral vectors. While highly efficient at gene transfer, the major disadvantages associated with the use of viral vectors include the inability of many viral vectors to infect non-dividing cells; problems associated with insertional mutagenesis; inflammatory reactions to the virus and potential helper virus production; antibody responses to the viral coats; and the potential for production and transmission of harmful virus to other human patients.
The efficiency of gene transfer into cells directly influences the resultant gene expression levels. In addition to the general low efficiency with which most cell types take up and express foreign DNA, many targeted cell populations are found in very low numbers in the body, so that the low efficiency of presentation of DNA to the specific targeted cell types further diminished the overall efficiency of gene transfer.
In many approaches aimed at increasing the efficiency of gene transfer into cells, the nucleic acid is typically complexed with carriers that facilitate the transfer of the DNA across the cell membrane for delivery to the nucleus. The carrier molecules bind and condense DNA into small particles which facilitate DNA entry into cells through endocytosis or pinocytosis. In addition, the carrier molecules act as scaffolds to which ligands may be attached in order to achieve site- or cell-specific targeting of DNA.
The most common DNA condensing agents used in the development of nonviral gene delivery systems include polylysine (Laemmli, 1975,
Proc. Natl. Acad. Sci. USA
72:4288-92; Wolfert & Seymour, 1996,
Gene Therapy
3:269-73) and low molecular weight glycopeptides (Wadhwa et al., 1995,
Bioconjugate Chemistry
6:283-291). Polylysine amino groups have been derivatized with transferrin, glycoconjugates, folate, lectins, antibodies, or other proteins to provide specificity in cell recognition, without compromising the polylysine's binding affinity for DNA.
Clearly, improved methods of gene delivery are needed. Such methods should be amenable to use with virtually any gene of interest and should permit the introduction of genetic material into a variety of cells and tissues.
2.2. Receptor-mediated Gene Delivery
Receptor-mediated gene delivery has emerged as a potentially useful approach for introduction of DNA into cells in vivo. An advantage of this gene delivery method is the ability to target DNA to specific tissue or cell types based on the recognition of ligands by unique receptors expressed on the cell surface (Wu et al., 1988,
J. Biol. Chem.
263:14621-14624; Christiano et al., 1993,
Proc. Natl. Acad. Sci. USA
90:2122-2126; Huckett et al., 1990,
Biochem. Pharmacol.
40:253-263; Perales et al., 1994,
Eur. J. Biochem.
226:255-266). In addition, this particular delivery system is not limited by the size of the DNA and the system does not involve the use of infectious agents.
Receptor-mediated gene transfer has considerable potential for use in human gene therapy if the method can be developed to a point where it is both a reliable and efficient approach for delivery in targeted host cells. The major shortcomings of currently available techniques are transient, variable and low level expression of the transferred DNA. Any method designed to increase the efficiency of transfer of DNA into the cell will facilitate the successful development of receptor-mediated gene delivery protocols.
2.3. Microspheres and Nanospheres
Oftentimes, it is desirable to deliver pharmaceutical or other bioactive agents intracellularly rather than, or in addition to, extracellularly. Such applications are particularly useful where, for example, the bioactive agent cannot easily penetrate or traverse the cellular membrane. Examples of such bioactive agents include oligonucleotides such as antisense DNA and RNA, ribozymes, DNA for gene therapy, transcription factors, growth factor binding proteins, signaling receptors and the like. Also desirable is sustaining this delivery of bioactive agents over an extended period of time.
Microspheres and/or nanospheres are a widely used vehicle for delivering drugs intracellularly, and for sustaining the delivery for an extended time. Generally, microspheres and/or nanospheres comprise a biocompatible biodegradable polymeric core having a bioactive agent incorporated therein. Microspheres are typically spherical and have an average diameter of about 1 to 900 &mgr;m, while nanospheres are typically spherical and have an average diameter of less than 1 &mgr;m, usually less than about 300 nm.
Advantages of microsphere and/or nanosphere (hereinafter collectively “microsphere”) bioactive formulations inclu
Cohen Hagit
Labhasetwar Vinod
Levy Robert J.
Dierker and Associates, P.C.
Nguyen Dave T.
Schnizer Richard
The Regents of the University of Michigan
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