Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Polymers from only ethylenic monomers or processes of...
Reexamination Certificate
1998-05-21
2001-10-23
Borin, Michael (Department: 1631)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
Polymers from only ethylenic monomers or processes of...
C514S002600, C514S449000, C514S549000
Reexamination Certificate
active
06306993
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to methods and compositions that are effective to enhance transport of biologically active agents, such as organic compounds, polypeptides, oligosaccharides, nucleic acids, and metal ions, across biological membranes.
References
Barsoum et al., PCT Pub. No. WO 94/04686 (1994).
Bonifaci, N., et al.,
Aids
9:995-1000.
Brugidou, J., et al.
Biochem. Biophys. Res. Comm
. 214(2):685-93 (1995).
Derossi, D., et al.,
J. Biol. Chem
. 269:10444-50 (1994).
Egholm, M. O., et al.,
Nature
365:566-568 (1993).
Eberle and Nuninger,
J. Org. Chem
. 57:2689 (1992).
Fawell, S., et al.,
Proc. Natl. Acad. Sci. USA
91:664-668 (1994).
Fletcher, M. D., et al.,
Chem. Rev
. 98:763 (1998).
Frankel et al., PCT Pub. No. WO 91/09958 (1991).
Gennaro, A. R., Ed.,
REMINGTON'S PHAMACEUTICAL SCIENCES
, 18
TH ED
., Mack Publishing Co., Easton, Pa. (1990).
Giannis, A., et al.,
Advances Drug Res
. 29:1 (1997).
Kessler, H.,
Angew. Chem. Int. Ed. Engl
. 32:543 (1993).
Lam, K. S., et al.,
Chem. Rev
. 97:411 (1997).
Langston, S.,
DDT
2:255 (1997).
Rivas, A., et al.,
J. Immunol
. 154:4423-33 (1995).
Ruegg, C., et al.,
J. Immunol
. 154:4434-43 (1995).
Ryser, H. J. P., PCT Pub. No. WO 79/00515 (1979).
Simon et al.,
Proc. Natl. Acad. Sci
. 89:9367 (1992).
Suffness, M., Ed.,
Taxol: Science and Applications
, CRC Press, New York, N.Y., pp. 237-239 (1995).
Shaheen et al.,
J. Virology
70:3392 (1996).
Tavladoraki et al.,
Nature
366:469 (1993).
Thompson, L. A., and Ellman, J. A.,
Chem. Rev
. 96:555 (1996).
Wong, S. S., Ed.,
Chemistry of Protein Conjugation and Cross-Linking
, CRC Press, Inc., Boca Raton, Fla. (1991).
Zuckermann, R. N.,
Chemtracts-Macromol. Chem
. 4:80 (1993).
All references cited within this application are incorporated herein by reference.
BACKGROUND OF THE INVENTION
The plasma membranes of cells present a barrier to passage of many useful therapeutic agents. In general, a drug must be freely soluble in both the aqueous compartments of the body and the lipid layers through which it must pass, in order to enter cells. Highly charged molecules in particular experience difficulty in passing across membranes. Many therapeutic macromolecules such as peptides and oligonucleotides are also particularly intractable to transmembrane transport. Thus, while biotechnology has made available a greater number of potentially valuable therapeutics, bioavailability considerations often hinder their medicinal utility. There is therefore a need for reliable means of transporting drugs, and particularly macromolecules, into cells.
Heretofore, a number of transporter molecules have been proposed to escort molecules across biological membranes. Ryser et al. (1979) teaches the use of high molecular weight polymers of lysine for increasing transport of various molecules across cellular membranes, with very high molecular weights being preferred. Although the authors contemplated polymers of other positively charged residues such as ornithine and arginine, operativity of such polymers was not shown.
Frankel et al. (1991) reported that conjugating selected molecules to the tat protein of HIV can increase cellular uptake of those molecules. However, use of the tat protein has certain disadvantages, including unfavorable aggregation and insolubility properties.
Barsoum et al. (1994) and Fawell et al. (1994) proposed using shorter fragments of the tat protein containing the tat basic region (residues 49-57 having the sequence RKKRRQRRR. Barsoum et al. noted that moderately long polyarginine polymers (MW 5000-15000 daltons) failed to enable transport of &bgr;-galactosidase across cell membranes (e.g., Barsoum on page 3), contrary to the suggestion of Ryser et al. (supra).
Other studies have shown that a 16 amino acid peptide-cholesterol conjugate derived from the Antennapedia homeodomain is rapidly internalized by cultured neurons (Brugidou et al., 1995). However, slightly shorter versions of this peptide (15 residues) are not effectively taken up by cells (Derossi et al., 1994).
The present invention is based in part on the applicants' discovery that conjugation of certain polymers composed of contiguous, highly basic subunits, particularly subunits containing guanidyl or amidinyl moieties, to small molecules or macromolecules is effective to significantly enhance transport of the attached molecule across biological membranes. Moreover, transport occurs at a rate significantly greater than the transport rate provided by a basic HIV tat peptide consisting of residues 49-57.
SUMMARY OF THE INVENTION
The present invention includes, in one aspect, a method for enhancing transport of a selected compound across a biological membrane. In the method, a biological membrane is contacted with a conjugate containing a biologically active agent that is covalently attached to at least one transport polymer. The conjugate is effective to promote transport of the agent across the biological membrane at a rate that is greater than the trans-membrane transport rate of the biological agent in non-conjugated form.
In one embodiment, the polymer consists of from 6 to 25 subunits, at least 50% of which contain a guanidino or amidino sidechain moiety, wherein the polymer contains at least 6, and more preferably, at least 7 guanidino or amidino sidechain moieties. In another embodiment, the polymer consists of from 6 to 20, 7 to 20, or 7 to 15 subunits. More preferably, at least 70% of the subunits in the polymer contain guanidino or amidino sidechain moiety, and more preferably still, 90%. Preferably, no guanidino or amidino sidechain moiety is separated from another such moiety by more than one non-guanidino or non-amidino subunit. In a more specific embodiment, the polymer contains at least 6 contiguous subunits each containing either a guanidino or amidino group, and preferably at least 6 or 7 contiguous guanidino sidechain moieties.
In another embodiment, the transport polymer contains from 6 to 25 contiguous subunits, from 7 to 25, from 6 to 20, or preferably from 7 to 20 contiguous subunits, each of which contains a guanidino or amidino sidechain moiety, and with the optional proviso that one of the contiguous subunits can contain a non-arginine residue to which the agent is attached.
In one embodiment, each contiguous subunit contains a guanidino moiety, as exemplified by a polymer containing at least six contiguous arginine residues.
Preferably, each transport polymer is linear. In a preferred embodiment, the agent is attached to a terminal end of the transport polymer.
In another specific embodiment, the conjugate contains a single transport polymer.
The transport-enhancing polymers are exemplified, in a preferred embodiment, by peptides in which arginine residues constitute the subunits. Such a polyarginine peptide may be composed of either all D-, all L- or mixed D- and L-arginines, and may include additional amino acids. More preferably, at least one, and preferably all of the subunits are D-arginine residues, to enhance biological stability of the polymer during transit of the conjugate to its biological target.
The method may be used to enhance transport of selected therapeutic agents across any of a number of biological membranes including, but not limited to, eukaryotic cell membranes, prokaryotic cell membranes, and cell walls. Exemplary prokaryotic cell membranes include bacterial membranes. Exemplary eukaryotic cell membranes of interest include, but are not limited to membranes of dendritic cells, epithelial cells, endothelial cells, keratinocytes, muscle cells, fungal cells, bacterial cells, plant cells, and the like.
According to a preferred embodiment of the invention, the transport polymer of the invention has an apparent affinity (Km) that is at least 10-fold greater, and preferably at least 100-fold greater, than the affinity measured for tat(49-75) peptide by the procedure of Example 6 when measured at room temperature (23° C.) or 37° C.
Biologically active agents (which encompass therapeutic agents) include, but are not limited to metal ions, w
Rothbard Jonathan B.
Wender Paul A.
Borin Michael
Reed & Associates
The Board of Trustees of the Leland Stanford Jr. University
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