Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-09-15
2001-06-26
Nolan, Patrick J. (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S324000, C435S007100
Reexamination Certificate
active
06252045
ABSTRACT:
TECHNICAL FIELD
This invention relates primarily to the enhancement of drug absorption across epithelial and endothelial barriers using occludin inhibitors.
BACKGROUND OF THE INVENTION
In mammalian cells, intercellular junctions are typically categorized into four types, based on early electron microscope studies: adherens junctions, desmosomes, gap junctions, and tight junctions. Recent research interest has focused on the molecular organization and functions of these junctions, to not only explain cell-cell interactions and communication within multi-cellular organisms, but also to regulate paracellular permeability for therapeutic purposes.
Drug absorption across epithelial and endothelial tissue is limited in several stages. One important barrier is created by intercellular tight junctions which limit movement of substances between cells (Anderson, J. M., and Van Itallie, C. M.,
Am. J. Physiol
. (GI and Liver) 269:G467-G475 (1995)). The tight junction barrier appears to be created by extracellular contacts of a transmembrane protein called occludin. The protein was originally cloned from the chicken (Furuse, M., et al.,
J. Cell Biol
. 123: 1777-1788 (1993)). Occludin has subsequently been cloned and sequenced from human, mouse, dog and rat kangaroo (Ando-Akatsuka, Y., et al.,
J Cell Biology
133: 43-47 (1996). Human occludin has also been cloned and sequenced by applicants (Genbank Accession U53823; see SEQ ID NOs 1 and 2 and FIG.
1
).
Tight junctions create a regulated paracellular barrier to the movement of water, solutes, macromolecules, immune cells, and the like between and among both epithelial and endothelial cells. New evidence has elucidated information about proteins involved in this dynamic regulation.
It would be beneficial to utilize this information to alter paracellular permeability for specific medical purposes.
SUMMARY OF THE INVENTION
It is an object of the invention to provide the sequence of cloned human occludin.
It is another object of the invention to provide a method for the selective enhancement of transmucosal or transvascular drug delivery. It has been demonstrated that peptides corresponding to the extracellular fragments of human occludin are capable of inhibiting cell to cell adhesion (see the examples that follow). Further it has been shown that peptides corresponding to extracellular sequences of occludin can interrupt the transmonolayer barrier properties of cultured epithelial cells (Wong and Gumbiner, B. (1997)
J. Cell Biology
136:399-409. Also see FIG.
2
.
These and other objects are accomplished by the present invention, which provides cloned human occludin and methods for altering occludin's barrier properties. The sequence of occludin provides occludin-based screening assays for occludin inhibitors such as binding assays, assays that measure adhesive properties, and the like, particularly those involving the extracellular loop sequences given below. In some embodiments, fibroblast adhesion measurements, e.g., those employing electrical resistance or transmonolayer flux measurements are employed.
In other embodiments, the invention provides a method for enhancing drug delivery by disrupting the intercellular seal provided by occludin. In accordance with this embodiment of the invention, effective amounts of occludin inhibitors and/or mimics such as peptide fragments of occludin and the like compounds identified in screens are administered to a patient, typically in combination with another drug or a mixture of drugs. Mimics include, but are not limited to, peptides analogous to sequences disclosed herein that have sequence alterations that enhance solubility or other properties desirable for achieving desirable pharmacological effects described hereafter. Administration can be local or systemic; local administration is preferred in some embodiments.
REFERENCES:
Ngo et al. The Protein Folding Problem and Tertiary Structure Prediction pp. 433 & 492 & 495, 1994.
Anderson James M.
Van Itallie Christina M.
Krinsky Mary M.
Nolan Patrick J.
Yale University
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