Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Matrices
Reexamination Certificate
2001-04-09
2004-01-27
Page, Thurman K. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Matrices
C424S485000, C424S486000, C424S422000, C424S423000, C424S424000, C514S002600, C514S021800
Reexamination Certificate
active
06682760
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to cross-linked collagen matrices and preparations and more particularly to a novel method for cross linking collagen using reducing sugars and to cross linked collagen matrices and preparations formed by using this method.
BACKGROUND OF THE INVENTION
Collagens are key molecules of the animal kingdom accounting for approximately 25-30% of all proteins of mammalian organisms. Collagens are natural biopolymers that are organized as fibrillar networks and other forms of superstructures. The fibrillar collagens and particularly type I collagen have the highest incidence accounting for 80% of connective tissues proteins. The high incidence of the fibrillar collagens, their availability from animal sources and the ability to extract and prepare monomeric solutions of purified collagen which can be polymerized into three-dimensional fibrillar matrices make these collagens ideal candidates for natural biomaterials. In addition, the fibrillar collagens exhibit a high degree of conservation and are therefore weak antigens. The main antigenic sites of the fibrillar collagen molecules reside within the non-helical telopeptides which flank the helical portion of the molecule.
In vivo, the polymeric structure of the fibrillar collagens is stabilized by intermolecular cross-links, which are formed by an enzymatic process. Because of the staggered assembly of the collagen molecules, most of these cross-links bridge between the telopeptide domain of one molecule and the helical domain of an adjacent molecule. Additional cross-link formation by the process of glycation takes place as part of the collagen and connective tissues aging.
Glycation of proteins, including collagen, takes place as a physiological process of aging over the life course consequent to the exposure of proteins to glucose. It was found that glycated fibrillar collagens exhibit an increased level of cross-linking and therefore they are more resistant to degradation by collagenases, the specific enzymes which degrade collagen.
The process of glycation by glucose is slow because its physiological concentration in serum is relatively low and only a small proportion of it is found in the acyclic aldehyde form which is the reactive one. It was found that D(−)Ribose is 1000 folds more reactive than glucose in inducing glycation and cross-linking of collagen molecules in fibrillar collagens. For example, incubation of native fibrillar collagen in 0.2 M D(−)ribose for 5 days is equivalent to exposure to physiological concentration of glucose for 20 years. The cross-links produced by glycation bridge form mainly between the triple-helical domains of adjacent molecules.
The performance of collagen-based bioproducts depends on the one hand on controlling their functional longevity within the host and on the other hand on the preservation of the biological properties of the native collagen component. The functional longevity of the collagen component depends on its capacity to resist specific enzymatic degradation by collagenases (metaloproteinases). This capacity is directly related to the number of intramolecular and intermolecular cross-links within the collagen polymer. The higher the number of cross-links the higher the resistance to collagenase degradation.
Exemplary cross-linking agents of choice known in the art have been glutaraldehyde and other related non-physiological agents. These cross-linking agents react with amino acid residues of the collagen molecule to form intermolecular cross-links. However, these harsh agents may have negative effects on the biocompatibility and biological activity of cross-linked collagen-based bioproducts that are caused by alterations in the conformation of the collagen molecule and leaching out of the cross-linking agents. Thus, collagen products cross-linked by non-physiological agents are poorly accepted by and integrated within the host tissues. Furthermore, localized inflammation and more complex systemic reactions are disadvantageous side effects of glutaraldehyde cross-linked collagen products.
U.S. Pat. No. 4,971,954 to Brodsky et al. discloses the use of D(−)Ribose or other reducing physiological sugars as physiological agents for cross-linking collagen matrices by the process of glycation. However, the method disclosed by Brodsky et al. is efficient when the collagenous substrate consists of native collagen fibers, but is only partially effective for collagen matrices produced from reconstituted fibrillar collagen, particularly when the collagen is atelopeptide collagen. Atelopeptide collagen is produced by pepsin-solubilization of native collagen. Since pepsin cuts off the telopeptides of the collagen molecule which are antigenic, pepsin-solubilized collagen is the most utilized form of collagen in the biomedical industry.
In the method disclosed by Brodsky et al in U.S. Pat. No. 4,971,954, the cross-linking occurs by a process of glycation. In this process the acyclic form of D(−)Ribose condenses spontaneously with the &egr;-amino groups of lysyl and hydroxylysyl residues located in the triple helical domain of the collagen molecule. The condensation product is a Schiff base that undergoes Amadori rearrangement to form a ketoamine adduct. Ketoamines located on adjacent collagen molecules condense to form covalent cross-links, the exact nature of which has yet not been determined, even though fluorescent heterocyclic structures and others type have been recently proposed.
Brodsky et al. disclose the process of glycation for native fibrillar type I collagen, such as for example the native fibrillar type I collagen from rat tendon. However, cross-linking by the glycation method of Brodsky et al. is reversible. For example, in an article entitled “ISOLATION AND PARTIAL CHARACTERIZATION OF COLLAGEN CHAINS DIMERIZED BY SUGAR-DERIVED CROSS-LINKS”, published in
The Journal of Biological Chemistry
Vol. 263(33), pp. 17650-17657, 1988, Tanaka et al. show that rat tendon collagen cross-linked with D(−)Ribose for 1 day, is in the range of 50% reversibility at the end of a period of 5 days.
U.S. Pat. No. 5,955,438 to Pitaru et al. discloses, inter alia, a method for preparation of collagen matrices and membranes made from atelopeptide reconstituted collagen fibrils formed into a membrane and then cross-linked by a reducing sugar such as D(−)Ribose. The membrane or the implants made thereof are then subjected to critical point drying for drying and sterilization while preserving the three dimensional shape of the implants. The critical point drying procedure improves the resistance of the collagen matrix to collagenase degradation.
The cross-linking of native collagen with D(−)Ribose renders the native collagen fibers resistant to collagenase degradation. However, cross-linking of atelopeptide reconstituted collagen fibrils by D(−)Ribose is only negligibly effective in increasing their resistance to collagenase degradation. The reason for this is not clear. Since work by Tanaka et al. (see reference list) indicates that ribose-induced cross-links between native collagen molecules occur mainly between the triple-helical portions of adjacent collagen molecules, the removal of the telopeptides should not affect the degree of cross-linking of atelopeptide collagen. One possible explanation is that the packing of the atelopeptide collagen molecules in reconstituted collagen fibrils differs from the packing in native collagen fibrils (as discussed in Ref. 16 of the reference list). This difference in packing, in turn, may result in a change in the intermolecular distance or alignment which may cause a decrease in the strength or number of the covalent cross-links formed by D(−)Ribose.
SUMMARY OF THE INVENTION
There is therefore provided, in accordance with a preferred embodiment of the present invention, a method for preparing cross-linked collagen. The method includes the step of incubating collagen in a solution including water, at least one polar solvent, and at least one sugar, to
Noff Matitiau
Pitaru Shahar
Colbar R&D Ltd.
Di Nola-Baron Liliana
Eitan, Pearl, Latzer & Cohen Zedek LLP
Page Thurman K.
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