Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-10-14
2003-07-01
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S021800, C514S002600, C514S008100, C514S012200, C530S350000, C530S356000
Reexamination Certificate
active
06586406
ABSTRACT:
BACKGROUND OF THE INVENTION
The limited capacity of articular cartilage to regenerate represents a major obstacle in the management of degenerative and traumatic joint injuries. The maintenance of a functional joint surface requires that articular chondrocytes respond to extracellular signals that are generated from growth and differentiation factors, mechanical stimuli, and interactions with specific components of the extracellular matrix. The invention is directed to an extracellular matrix of type I collagen, type II collagen, type I collagen plus hyaluronate, or type II collagen plus hyaluronate, and differentiation factor-5 (GDF-5), a member of the bone morphogenetic protein (BMP) family that is involved in joint development on the chondrogenic activity of growth.
Coordinated function of many cell types is regulated by integration of extracellular signal derived from soluble factors inducing growth factors and insoluble molecules such as extracellular matrix (ECM). The skeletal elements of the vertebrate limb are derived during embryonic development from mesenchymal cells, which condense and initiate a differentiation program that result in cartilage and bone. Bone morphogenetic proteins may play a crucial role in mesenchymal condensations in skeletal patterning, including the process of joint formation. This is based upon in situ hybridization and immunostaining showing that GDF-5 is predominantly found at the stage of precartilaginous mesenchymal condensation and throughout the cartilaginous cores of the developing long bone; and null mutation in GDF-5 (frameshift mutation at the mouse brachypodism locus) resulting in disruption of the formation of approximately 30% of the joints in the limb. This includes the complete absence of joint development between the proximal and medial phalanges in the forefeet and hindfeet. Further evidence of the role of GDF-5 in regulating the cellular condensation required for chondrogenesis and joint formation comes from null mutation of noggin gene which is a known antagonist of bone morphogenetic protein function. While, in mice lacking noggin, cartilage condensation initiated, the process of joint formation failed as judged by the absence of GDF-5 expression.
Despite the importance of joint formation in skeletal patterning and human disease, relatively little is known about the molecular mechanisms that control where and when a joint will form. In the limb, joints typically arise by the splitting of larger skeletal precursors, rather than by collision or apposition of separate elements. This process takes place through a series of steps including: 1) initial formation of specialized regions of high density that extend in transverse stripes across developing cartilage element; 2) programmed cell death and changes in matrix production in the center of the interzone, creating a three layer structure; 3) differentiation of articular cartilage at the two edges of the interzone; and 4) accumulation of fluid-filled spaces that coalesce to make a gap between opposing skeletal elements. Expression of GDF-5 is initiated in the region of joint development 24-36 hours before the morphological appearance of the interzone. The expression continues for at least 2-3 days at a particular site, and is still evident at the three-layered interzone stage of joint development. The expression level of GDF-5 then decreases at later stages of joint formation. In vitro biological and biochemical analyses of recombinant hGDF-5 suggest that the primary physiological role of GDF-5 may be restricted to early stages of chondrogenesis of mesenchymal progenitor cells. This is based on a showing that: 1) GDF-5 stimulates mesenchymal aggregation and chondrogenesis in rat limb bud cells; 2) GDF-5 fails to stimulate alkaline phosphatase activity measured utilizing well differentiated osteoblastic cell type MC3T3-E1 cells; 3) GDF-5 stimulates alkaline phosphatase activity in rat osteoprogenitor cells ROB-C26 which is more primitive and less differentiated; 4) GDF-5 binds to distinct heterodimer of receptor for BMPs which is expressed more prevalently in less differentiated cells of mesenchymal origin.
SUMMARY OF THE INVENTION
This invention is directed to a method and composition for inducing or enhancing chondrogenesis in cells with an extracellular matrix containing GDF-5. The extracellular matrix consists of type I collagen, type II collagen, type I collagen plus hyaluronate or type II collagen plus hyaluronate, and contains growth and differentiation factor-5, GDF-5. An effective amount of GDF-5 to induce or enhance chondrogenesis is about 1 ng to 10 mg/ml matrix protein. A matrix is a solid porous composition having a relatively fixed three-dimensional structure.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Chondrogenesis is induced by an extracellular matrix composition of type I collagen, type II collagen, type I collagen plus hyaluronate, or type II collagen plus hyaluronate containing GDF-5. Type I and II collagen represent the most abundant ECM protein in bone and cartilage, respectively.
Collagen may be obtained from bone, tendons, skin, or the like. The collagen source may be any convenient animal source, mammalian or avian, including bovine, porcine, equine, or the like, or chicken, turkey or other domestic source of collagen.
Hyaluronic acid is a naturally-occuring polysaccharide containing alternating N~acetyl~D~glucosamine and D~glucuronic acid monosaccharide units linked with beta 1-4 bonds and disaccharide units linked with beta 1-3 glycoside bonds. It occurs usually as the sodium salt and has a molecular weight range of about 50,000 to 8×10
6
.
The collagen or collagen-hyalurate mixture is provided as a matrix, typically by lyophilization. The collagen-hyaluronate is formed by treating collagen with an active formyl aldehyde hyaluronate, formed as described in U.S. Pat. No. 5,866,165, incorporated by reference herein. The collagen hyaluronate composition is also provided as a matrix by lyophilization.
The matrix is preferably implanted with an effective amount of GDF-5, which is about 1 mg to 10 mg/ml of matrix protein.
To show in vitro application, fetal rat calvarial cells (FRC's) were plated on various purified extracellular matrix proteins in the presence of recombinant human GDF-5 (100 ng/ml) for 3 weeks and scored for differentiation at the level of morphology, overall proteoglycan synthesis and deposition, and aggrecan and type II collagen expression. Results show that GDF-5 stimulated chondrogenic nodule formation of FRC's plated only on type I or type II collagen. Chondrogenic nodules stained heavily with alcian blue and were positive for type II collagen and aggrecan-expression, as judged by immunohistochemical and transcriptional analyses. Cells in monolayer that surround the nodules were negative for the chondrogenic markers. In sharp contrast, GDF-5 failed to stimulate chondrogenesis in FRC's plated on fibronectin, type IV collagen or tissue culture plastic.
Plastic plates were first coated with different ECM proteins including type I and II collagen, type IV collagen, or fibronectin. The results show that GDF-5 stimulated the formation of chondrogenic cell aggregate that bind heavily to the alcian blue stain. Under these conditions GDF-5 fails to stimulate the formation of characteristic nodules in FRC cultured in the presence of vehicle alone, type IV collagen, or fibronectin. Plastic culture 12 well (Costar, Cambridge, Mass.) were coated with 0.01% (w/v) of the indicated extracellular matrix proteins for 2 hours at 37C°. After removal of nonadsorbant protein, fetal rat calvarial cells were plated at a density of 2×10
5
cells/well in DMEM containing 10% FBS. Culture plates were then maintained for 21 days in culture media supplemented with or without GDF-5 (100 ng/ml). Plates were then stained overnight with alcian blue stain (0.5% w/v in 3% acetic acid), washed and photographed. For quantitation of alcian blue, cells were solubilized in 8M urea, and the amount of stain was quantitated using spectrophotometer (Mole
Daverman Robin
Heidaran Mohammad A.
Liu Lin-Shu
Spiro Robert C.
Beyer Weaver & Thomas
Depuy Acromed, Inc.
Low Christopher S. F.
Robinson Hope A.
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