Drug – bio-affecting and body treating compositions – Extract – body fluid – or cellular material of undetermined... – Digestive system
Reexamination Certificate
2000-02-02
2001-12-18
Witz, Jean C. (Department: 1651)
Drug, bio-affecting and body treating compositions
Extract, body fluid, or cellular material of undetermined...
Digestive system
Reexamination Certificate
active
06331319
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to enzymatically treated submucosal tissue and methods for its preparation and use. More particularly, the present invention is directed to submucosal tissue that is at least partially digested with galactosidase.
BACKGROUND OF THE INVENTION
It is known that compositions comprising the tunica submucosa of the intestine of warm-blooded vertebrates can be used advantageously as tissue graft materials. See U.S. Pat. Nos. 4,902,508 and 5,281,422, the disclosures of which are expressly incorporated herein by reference. The tissue graft compositions described in those patents are characterized by excellent mechanical properties, including high compliance, a high burst pressure point, and an effective porosity index which allows such compositions to be used beneficially for vascular graft and connective tissue graft constructs. When used in such applications the graft constructs appear not only to serve as a matrix for the regrowth of the tissues replaced by the graft constructs, but, indeed, to promote or induce such regrowth of endogenous tissue. Common events to this remodeling process include widespread and rapid neovascularization, proliferation of granulation mesenchymal cells, biodegradation/resorption of implanted intestinal submucosal tissue material, and lack of immune rejection.
It is also known that intestinal submucosa can be fluidized by comminuting and/or enzymatic digestion, without loss of its apparent biotropic properties, for use in less invasive methods of administration (e.g., by injection or topical application) to host tissues in need of repair. See U.S. Pat. No. 5,275,826, the disclosure of which is expressly incorporated herein by reference.
Submucosal tissue grafts have been successfully used as a xenograft in vascular, dura mater, urinary bladder, and orthopedic applications, and as dermal grafts. The remodeled tissue resembles the native tissue, both grossly and histologically, such that the original submucosal tissue graft is generally unidentifiable when remodeling is complete. Despite its xenogeneic nature, vertebrate submucosal tissue has not induced a clinical rejection response in the animal systems in which it has been tested, including rats, mice, dogs, cats, rabbits, and sheep. Thus, submucosal tissue is a potentially useful xenogeneic graft material for use in humans.
Galactosyl-&agr;(1,3)galactose (referred to as the Gal epitope) is a glycosyl modification of cell surface components and some serum proteins in all mammals, except humans and Old World apes. The epitope comprises a terminal galactose moiety linked to another galactose moiety through an &agr;1-3 linkage. It has been shown that human serum contains naturally occurring IgG and IgM antibodies directed against this epitope. It is estimated that 1% of all circulating IgG in humans is anti-Gal. This high level of anti- Gal epitope antibodies is thought to be produced in response to endogenous bacteria in the gastrointestinal system; the lipopolysaccharides of those bacteria contain the Gal epitope. Xenogeneic transplantation of organ tissue into a human host results in IgG and IgM antibodies binding to the Gal epitope (especially for those epitopes located on endothelial cells), the initiation of an inflammatory reaction, and vascular thrombosis and hyperacute xenograft rejection of the transplant. Accordingly, a major obstacle to successful xenotransplantation of porcine and other non-Old World ape vertebrate species organs into humans is the presence of Gal epitopes on the tissues of those organs.
As disclosed herein, the Gal epitope has also been found in porcine submucosal tissue prepared in accordance with the procedures disclosed in U.S. Pat. Nos. 4,902,508 and 5,281,422. It is not known whether the Gal epitope exists as a naturally occurring component of the submucosal tissue or whether the epitope is a remnant of cell lysis, and remains attached to the submucosal tissue during processing of the submucosal tissue.
There have been no reports of submucosal tissue graft constructs inducing an immune response after implantation in the animal systems in which it has been tested, including rats, mice, dogs, cats, rabbits, and sheep. However, due to the association of the Gal epitope with hyperacute xenograft whole organ transplant rejection in humans, a preferred submucosal tissue graft construct would comprise submucosal tissue substantially free of the Gal epitope.
SUMMARY OF THE INVENTION
The present invention is directed to submucosal tissue that has been treated with galactosidase to produce submucosal tissue substantially free of detectable amounts of the Gal epitope. Such “Gal free” submucosal tissue is used to form tissue graft constructs for the replacement and repair of damaged or diseased endogenous tissues.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions:
The term “Gal epitope” refers to a glycosyl modification [galactosyl-&agr;(1,3)galactose] of cellular compounds present on the cell surface or on serum proteins of all mammals except humans and Old World apes.
The term “glycosidase” as use herein refers to an enzyme that cleaves/destroys the terminal a-linked galactose present on most vertebrate cellular components. For example one glycosidase useful in accordance with the present is &agr;-galactosidase.
The term “glycosaminoglycanase” (GAGase) as use herein refers to an enzyme that hydrolyzes glycosaminoglycans (GAG) including, for example chondroitin sulfate, hyaluronic acid heparin and heparin sulfate.
The term “Gal free submucosal tissue” refers to submucosal tissue that is substantially free of all detectable amounts of the Gal epitope as determined by the antibody and lectin assays described in detail in Example 1.
The present invention is directed to warm blooded vertebrate submucosal tissue that is substantially free of the Gal epitope, and methods for its preparation and use. More particularly, the present invention is directed to submucosal tissue that has been at least partially digested with an enzyme, such as &agr;-galactosidase, to diminish the level of the Gal epitope present in the submucosal tissue. The galactosidase treated submucosal tissue is used in accordance with the present invention as a non-immunogenic tissue graft composition and as an in vitro cell culture substrate.
The galactosidase treated submucosal tissue of the present invention is derived from vertebrate submucosa and comprises naturally associated extracellular matrix proteins, glycoproteins and other factors. Preferably the submucosal tissue comprises intestinal submucosa of a warm-blooded vertebrate, and one particularly preferred source of the submucosal tissue is the small intestine of warm-blooded vertebrates. Suitable submucosal tissue comprises the tunica submucosa delaminated from the tunica muscularis and at least the luminal portion of the tunica mucosa. In one preferred embodiment of the present invention the submucosal tissue is intestinal submucosa comprising the tunica submucosa and basilar portions of the tunica mucosa including the larnina muscularis mucosa and the stratum compactum which layers are known to vary in thickness and in definition dependent on the source vertebrate species. Submucosal tissue can also be prepared from other organs of vertebrate species, for example, from the urogenital system, including the urinary bladder (see U.S. Pat. No. 5,554,389), and other portions of the digestive tract including the stomach. The disclosures of U.S. Pat. No. 5,554,389 is expressly incorporated herein.
The preparation of submucosal tissue for use in accordance with this invention is described in U.S. Pat. Nos. 4,902,508 and 5,554,389. To sumrnmarize, submucosal tissue is prepared from vertebrate intestine (or other organ source), preferably harvested from porcine, ovine or bovine species, but not excluding other species, by subjecting the intestinal tissue to abrasion using a longitudinal wiping motion to remove the outer layers, comprising smooth muscle tissues, and the innermost layer, i.e., a
Badylak Stephen F.
McPherson Timothy B.
Record Rae Denese
Barnes & Thornburg
Purdue Research Foundation
Witz Jean C.
LandOfFree
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