Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-03-19
2001-07-10
Russel, Jeffrey E. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S015800, C525S050000, C525S403000, C530S327000, C530S345000, C530S408000
Reexamination Certificate
active
06258774
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to chemical compounds for delivering therapeutic agents to tissues of a mammal, such as a human. More particularly, the invention involves a carrier for delivery of a therapeutic agent in which a biodegradable disulfide bond conjugates the therapeutic agent to a carrier of the present invention. The disulfide bond can be reduced in an appropriate environment in vivo so that a greater amount of the therapeutic agent is neither degraded nor excreted, but can be delivered to tissues, thus increasing its therapeutic effectiveness.
BACKGROUND OF THE INVENTION
It has been proposed that compounds, such as peptides, peptide mimetics, and oligonucleotides, or analogs or derivatives thereof, can be used as potential therapeutic agents. However, problems have been encountered in administering such compounds to a subject. For example, proteases and endonucleases present throughout the body digest such compounds, severely decreasing their biological activity. Other problems involve the elicitation of an immune response against the compound resulting in the degradation and inactivation of such compounds, and rapid renal clearance, particularly if the therapeutic agent has a low molecular weight. Hence, in order'to be effective, such therapeutic agents must be administered frequently, and parenterally rather than orally. An example of such a therapeutic agent is insulin, which is typically injected several times daily by diabetics.
In efforts to overcome these problems, researchers have attempted to modify chemically such therapeutic agents in order to manipulate their pharmacologic properties
1
, and perhaps enable them to survive longer in vivo before being degraded and removed from the blood stream. For example, one method of chemically modifying therapeutics is to append water-soluble polymer chains, such as polyethylene glycol (PEG), to the therapeutic agent
2
. Researchers have designed a PEG-lysine copolymer having multiple attachment sites
3
, and have conjugated the copolymer to low molecular weight therapeutic agents. However, such modifications have inherent limitations. For example, they frequently interfere with the bioavailability of the therapeutic. Consequently, if the target for a therapeutic agent is intracellular, and the modification of the therapeutic prevents its crossing of the cell membrane, then the bioavailability of the therapeutic agent is reduced due to the chemical modification.
Another limitation to attaching a water-soluble polymer to a therapeutic agent involves modulating the biological activity of the therapeutic agent in a deleterious manner. For example, if the modification of the therapeutic agent alters its three dimensional structure, then its ability to bind a receptor site it was designed to bind can be decreased, resulting in a decrease of activity.
Hence, what is needed is a carrier of a therapeutic agent that reduces the chance of an elicitation of an immune response against the therapeutic agent.
Moreover, what is needed is a carrier that protects therapeutic agents from protease/peptidase
uclease degradation in vivo, thereby eliminating the need for repetitive administration of the therapeutic agent.
In addition, what is needed is a carrier of a therapeutic agent that enhances cellular transmembrane delivery of the therapeutic agent.
Also, what is needed is a carrier of a therapeutic agent that does not release the therapeutic agent until the carrier has crossed the cell membrane, and once inside the cell, the carrier can release the therapeutic agent in a biologically active state.
What is also needed is a carrier of a therapeutic agent that does not interfere with the bioavailability of the therapeutic agent.
SUMMARY OF THE INVENTION
There is provided, in accordance with the present invention, a carrier for in vivo delivery of therapeutic agents that does not possess the shortcomings of other drug delivery carriers as described above, and offers the advantages of not interfering with the bioavailability of a therapeutic agent, protecting the therapeutic agent from proteolytic
ucleolytic degradation, from eliciting an immune response, and from rapid renal clearance, to name only a few.
Broadly, the present invention provides a carrier for in vivo delivery of a therapeutic agent comprising a thiol group, wherein the carrier comprises a polymer, and at least one thiol compound conjugated to the polymer, such that the thiol group of the thiol compound and the thiol group of the therapeutic agent form a disulfide bond. The disulfide bond can be broken in a physiologically relevant reducing environment, such as that found in the cytosol of a cell. Hence, Applicants have discovered a way to modify a therapeutic agent to minimize degradation in vivo, and yet not limit its bioavailability. In addition, the present invention is particularly well suited to deliver therapeutic agents to the cytosol of cells. Glutathione, a naturally occurring reducing agent is found predominantly in cell cytosol. Hence, glutathione can reduce a disulfide bond, and release a therapeutic agent from a carrier of the present invention within the cells of the target tissue. In addition, since more than one thiol compound can be conjugated to the polymer, the carrier of the present invention can deliver more than one molecule of therapeutic agent to a target cell or tissue.
Numerous therapeutic agents comprise thiol groups, and can be used to conjugate the therapeutic agent to a carrier of the present invention with a disulfide bond. For example, therapeutic agents which are peptides comprising a cysteine residue can be delivered in vivo with a carrier of the present invention. In addition, analogs or derivatives of peptides which serve as therapeutic agents can be made to comprise a thiol group so that they can be delivered in vivo with a carrier of the present invention. Even nucleotides and analogs or derivatives thereof, used in antisense therapy for example, can be easily modified to comprise a thiol group in order to be carried via a carrier of the present invention.
Another example of a therapeutic agent comprising a thiol group, which can be conjugated to a a carrier of the present invention for in vivo delivery, is a therapeutic agent which inhibits HIV-1 replication. More specifically, it has been determined that the HIV Tat protein strongly activates HIV transcription through its interactions with the TAR RNA region. The TAR RNA domain consists of the first 57 nucleotides of all virally encoded RNAs. The predicted TAR RNA secondary structure is a double-stranded stem with a 3-base bulge and a 6-base loop. HIV-1 Tat is a small nuclear protein containing 86-102 amino acids, and is encoded by multiply spliced mRNA. The 3-base bulge in TAR RNA and several other flanking nucleotides are essential for Tat-TAR interaction.
Tat protein apparently acts to promote transcription by binding through its basic domain to the 3-base bulge of TAR. This is accompanied by recruitment of host cellular factors, including Tat and TAR binding proteins, to the TAR RNA stem and 6-base loop, as well as to the complex of template DNA, transcription factors and RNA polymerase. Initiation of proviral gene expression appears to occur by activation of an NF-&kgr;B and/or Sp1-dependent promoter, resulting in production of viral transcripts at a sufficient level to provide synthesis of Tat protein, which then interacts with TAR to allow enhanced production of elongated HIV transcripts.
Efforts have been made to develop a therapeutic agent which binds TAR, and blocks Tat-TAR binding. For example, a 10-residue Tat peptide with an appended 4-mer antisense oligonucleotide can specifically bind to TAR RNA, as shown by its ability to stimulate RNase H-mediated cleavage at the site of oligonucleotide annealing to the 6-base single-stranded loop. In another example, a biotinylated peptide has also been shown to inhibit Tat binding to TAR (please see Choudhury, I., Wang, J., Rabson, A. B., Stein, S., Pooyan, S., Stein, S. and Leibowitz, M. J. (1998) Inhibi
Leibowitz Michael J.
Sinko Patrick J.
Stein Stanley
Klauber & Jackson
Russel Jeffrey E.
University of Medicine and Dentistry of New Jersey
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