Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-05-20
2003-11-11
Wilson, James O. (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S041000
Reexamination Certificate
active
06645947
ABSTRACT:
BACKGROUND OF THE INVENTION
The attachment of cells and proteins to substrates is a well-known problem that has presented itself in a number of contexts. For example, in cell cultures to produce antibodies, fibroblasts attach to extracellular matrix proteins bound to the tissue culture substrate. Similarly, in urinary catheters, bacterial cells attach to the walls of the catheter; in arterial catheters, platelets attach to the tip of the catheter; and in contact lenses, proteins coat the surfaces of the lenses.
Various bioadhesives are known in the art. U.S. Pat. No. 4,615,697, issued to Robinson et al., defines a bioadhesive as a material that requires a force of at least about 50 dynes/cm
2
to separate two adhered, freshly excised pieces of rabbit stomach, following the procedure disclosed therein. The bioadhesive disclosed in Robinson et al. is a water-swellable, but water insoluble, fibrous, cross-linked carboxy-functional polymer.
Various attempts to ameliorate the problem of attachment of cells and proteins to substrates have been employed, but none have been found to be satisfactory. It would be desirable to solve this problem using a biocompatible substance that is adherent to substrates and inhibits cellular and protein attachment.
Certain cells, such as macrophages and fibroblasts, are referred to as “substrate-dependent cells” because they are active and proliferate only when attached to a surface or substrate. The attachment occurs via a family of proteins (“attachment molecules or proteins”), such as vitronectin and fibrinectin, which are found in the extracellular matrix. A surface that is coated with a material that is strongly adhesive may inhibit the attachment of substrate dependent cells by blocking attachment of extracellular matrix proteins. Hence, adhesive materials, as described herein, are useful in compositions or can form devices that inhibit the attachment of certain proteins and certain types of cells.
SUMMARY OF THE INVENTION
The present invention features a method of inhibiting cellular attachment to substrates. The invention is based, in part, on the discovery of adherent coatings of N,O-carboxymethylchitosan (“NOCC”), and in particular that adherent coatings of NOCC may be applied to various substrates, such as mammalian tissue, so as to inhibit attachment of other cells, such as substrate dependent cells. Further, it has been discovered that these adherent coatings of NOCC may be used in other areas where inhibition of cell or protein attachment is desirable, such as in the preparation of cell populations, on medical devices, and with cell-based products. The invention also has application to the inhibition of the attachment of proteins to surfaces.
The present invention provides a composition that is adherent to a variety of synthetic materials and mammalian tissues. The present invention also provides a method of inhibiting cellular and protein attachment to a substrate by applying adherent coatings of NOCC to the substrate such that the attachment of cells and proteins is inhibited. The amount of adherent NOCC in the composition should be effective to inhibit the attachment of substrate-dependent cells, preferably in a concentration of 0.05-5%(w/v), most preferably in a concentration of 0.1-2.5%(w/v).
In one embodiment, the invention provides a composition and method of inhibiting attachment of substrate-dependent cells to a substrate by applying a composition containing adherent NOCC to a substrate such that attachment of substrate-dependent cells is inhibited. The method may be applied to inhibit substrate-dependent cell attachment to mammalian tissue, medical devices, fermentation units, bioreactors and solid supports. In preferred embodiments, the substrate-dependent cells which are inhibited include fibroblasts, macrophages, epithelial cells, and endothelial cells.
In another embodiment, the invention provides a composition and method of inhibiting attachment of proteins to a substrate by applying a composition containing adherent NOCC to a substrate such that attachment of proteinaceous material is inhibited. The method may be applied to inhibit protein attachment to contact lenses, medical devices, fermentation units, bioreactors and solid supports.
In another embodiment, the invention may be used in a method of obtaining a population of cells, e.g. mammalian cells, by supplementing culture media with adherent NOCC, growing the population of cells in the supplemented media, and allowing the cells to grow or differentiate, such that substrate-dependent cells do not proliferate within the cell population.
In another embodiment, the invention provides a method of obtaining cells suitable for use in protein or antibody production by supplementing culture media with adherent NOCC and growing the cells in the supplemented media, such that intercellular attachment (or clumping) within the cell population is inhibited and production of proteins or antibodies is enhanced.
In yet another embodiment, the invention provides a method of inhibiting attachment of inflammatory cells and platelets to a medical device by coating said device with a composition containing adherent NOCC, such that platelet or inflammatory cell attachment to the medical device is inhibited. In preferred embodiments, the internal medical device is either a stent or shunt. In other preferred embodiments, the inflammatory cell includes fibroblasts, macrophages, and monocytes.
In still another embodiment, the invention includes a method of inhibiting fibroblast attachment in a cell-based product in contact with a solid support by introducing adherent NOCC into the cell based product such that fibroblast attachment is inhibited.
In another embodiment, the invention provides a composition and method of delivering drugs, proteins, and other therapeutic agents from an adhesive device or composition that is adherent to soft (mucosal or non-mucosal) tissue or hard tissue. In preferred embodiments, the adherent delivery device can be used as a buccal, oral, vaginal, inhalant, or the like delivery system. The device can be in a variety of forms including solutions, creams, pellets, particles, beads, gels, and pastes.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the inhibition of cellular and protein attachment to various substrates. The method of the invention uses an adherent coating of N,O-carboxymethylchitosan (“NOCC”) which provides unexpected benefits in inhibiting cellular and protein attachment.
NOCC is a derivative of chitin, which is found in the shells of crustaceans and many insects. Chitin and its derivatives are normally biocompatible, naturally resorbed by the body, and have previously been used for sustained drug release, bone induction and hemostasis (Chandy and Sharma,
Biomat. Art. Cells
&
Immob. Biotech.
19:745-760 (1991); Klokkevold, P. et al.,
J. Oral Maxillofac. Sur.
50:41-45 (1992)). Due to its prevalence, chitin may be obtained relatively cheaply, largely from waste products. As disclosed in U.S. Pat. No. 4,619,995, issued to Hayes, NOCC has carboxymethyl substituents on some of both the amino and primary hydroxyl sites of the glucosamine units of the chitosan structure. NOCC may be used in an uncrosslinked form as a solution or may be cross-linked or complexed into a stable gel. Because of its advantageous physical properties, and its relative low cost, NOCC presents advantageous properties for use in inhibiting cellular and protein attachment.
Definitions
The term “inhibit,” or any form thereof, is defined in its broadest sense and includes minimize, prevent, repress, suppress, curb, constrain, restrict and the like.
The terms “adherent NOCC” or “an adherent coating of NOCC” mean a coating or composition of NOCC that exhibits an adhesion between freshly excised tissue of at least about 100 dynes/cm
2
, using the procedure described in Example 1.
The term “substrate” refers to any object to which cells can attach. Examples of substrates include, without limitation, mammalian tissue (including both hard tissue, such as bone, and
Elson Clive M.
Lee Timothy D. G.
Chitogenics, Inc.
Crane Lawrence E.
Lahive & Cockfield LLP
Loren, Esq. Ralph A.
Wilson James O.
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