The present invention, in some embodiments thereof, relates to compositions which comprise procollagen and uses of same in promoting wound healing, treating fibrosis and promoting angiogenesis.
The rapid response of the mammalian body to initiate the healing response to prevent life threatening bleeding and infection has evolved to ensure survival, often at the expense of efficient regeneration of the damaged tissue. The wound healing process entails different stages, some being sequential, while others concomitant. However, all stages are carefully orchestrated at the damaged tissue site to regenerate a tissue with normal functionality. The sequence of events involves clotting, inflammation, tissue deposition (migration and proliferation) and finally tissue remodeling.
At the time of tissue injury, blood is released from damaged vessels leading to the formation of a fibrin fiber mesh with platelets entrapped within. The mesh functions as a scaffold for recruited cells to migrate towards and throughout. The activated platelets degranulate and release chemotactic agents including cytokines and growth factors such as transforming growth factor-β1 (TGF-β1), resulting in recruitment of fibroblasts and keratinocytes. Several days after injury the fibroblasts begin to replace the damaged tissue by depositing new collagen matrices. Collagen fibers gradually increase in thickness and align along the stress line of the wound. In normal scar formation, collagen fibers typically align in parallel to the epidermis. This newly formed granulation tissue is eventually organized and contracted into a more dense structure by myofibroblasts.
Scars usually form as a result of the normal progression of the wound healing response and are composed of connective tissue deposited during the healing process. Most scars exhibit a certain degree of both abnormal organization (as seen in scars of the skin) and amounts of connective tissue (as seen in scars of the central nervous system). However, alterations in the normal tissue production cascade result in less than optimal wound healing with excessive deposit of scarring tissue resulting in keloid and hypertrophic scar formation, also termed fibrosis. Hypertrophic scars are characterized by excessive collagen deposition, altered collagen remodeling and contraction and differ from keloid scars in that they are defined within the boundaries of the wound site.
Transforming growth factor-β1 (TGF-β1) plays an important role in these healing processes and has been reported to mediate the transition of fibroblasts into myofibroblasts. This fibroblast subtype is characterized by α-smooth muscle actin (α-SMA) expression and is involved in wound contraction. TGF-β1 induces collagen deposition by upregulation of both mRNA stability and expression of procollagen. In addition, it reduces collagen degradation rates by inhibiting the expression of matrix metalloproteinases (MMPs) while inducing the expression of tissue inhibitors of metalloproteinases (TIMPs).
Aside from the MMP/TIPMP balance, the accessibility of a collagen molecule to such enzymatic activity is also a central factor in determining collagen degradation rates. This accessibility is primarily determined by the organizational state of the collagen (helical monomers versus monomers organized into fibrils) and the extent of crosslinking between collagen triple helices.
Types I and III collagen are fibril-forming collagens, which constitute the bulk of the dermal extracellular matrix. Collagen is synthesized as a procollagen precursor, in which three collagen polypeptides coil into each other, forming the triple helix. These helices are subsequently linked together at the final step of collagen fibril biosynthesis. Type I procollagen consists of two alpha 1 collagen chains and a single alpha 2 chain. Type III is composed of three alpha 1 chains.
In all of the fibrillar collagen molecules, the three polypeptide chains are constructed from a repeating Gly-X-Y triplet, where X and Y can be any amino acid but are frequently the imino acids proline and hydroxyproline. An important feature of fibril forming collagens is that they are synthesized as precursor procollagens containing globular N- and C-terminal extension propeptides.
Each procollagen molecule assembles within the rough endoplasmic reticulum from its three constituent polypeptide chains. As the polypeptide chain is co-translationally translocated across the membrane of the endoplasmic reticulum, hydroxylation of proline and lysine residues occurs within the Gly-X-Y repeat region. Once the polypeptide chain is fully translocated into the lumen of the endoplasmic reticulum, the three pro-alpha chains associate via their C-propeptides to form a trimeric molecule allowing the Gly-X-Y repeat region to form a nucleation point at its C-terminal end, ensuring correct alignment of the chains. The Gly-X-Y region then folds in a C-to-N direction to form a triple helix.
The C-propeptides, and to a lesser extent the N-propeptides, maintain procollagen solubility during its passage out of the cell [Bulleid et al., Biochem Soc Trans. 2000; 28(4):350-3]. Following or during secretion of procollagen molecules into the extracellular matrix, propeptides are typically cleaved by procollagen N- and C-proteinases, thereby triggering spontaneous self-assembly of collagen molecules into fibrils [Hulmes, 2002 J Struct Biol. 137(1-2):2-10].
Removal of the propeptides by procollagen N- and C-proteinases dramatically lowers the solubility of procollagen and is necessary to initiate the self-assembly of collagen into fibers at 37° C. Crucial to this assembly process are the short non triple-helical peptides called telopeptides which are the remnants of the N- and C-terminal propeptides following digestion with N/C proteinases. These peptides act to ensure correct covalent registration of the collagen molecules within the fibril structure via their crosslinkable aldehydes by lowering the critical concentration necessary for self-assembly (Bulleid et al., 2000, supra).
To date, animal-derived collagen is the major source of collagen for medical applications. Animal-purified collagen is fully processed containing crosslinked telopeptides which render it highly insoluble. Solubilization of animal-purified collagen is typically effected using an extraction method which involves proteolytic removal of the telopeptide region with proteloytic enzymes such as trypsin, yielding atelocollagen which can be solubilized (see U.S. Pat. Nos. 3,934,852; 3,121,049; 3,131,130; 3,314,861; 3,530,037; 3,949,073; 4,233,360 and 4,488,911 for general methods for preparing purified soluble collagen). Atelocollagen undergoes fibrillogenesis under physiological conditions, to form fibers. Such fibers are relatively stable structures, resistant to proteolysis by MMPs. However, these fibers lack the molecular domains found in procollagen, essential to natural wound healing processes and to the natural formation of collagen structures.
As mentioned, alterations in the normal tissue production cascade during the process of wound healing may lead to excessive deposition of scarring tissue resulting in fibrosis.
U.S. Pat. No. 6,448,278 and references therein describe specific procollagen C-proteinase (PCP) inhibitors for the treatment of various medical conditions associated with unregulated production of collagen, including pathological fibrosis or scarring.
Zhang Y et al., 1999, 13(1):51-4 teach direct stimulation of procollagen I (alpha 1) gene expression by administration of platelet-derived wound healing factor (PDWHF).
Saggers, et al., [Wounds 13(2):66-71, 2001] reported that acid-soluble collagen isolated from rat tail tendons inhibits types I and III procollagen mRNA expression in human dermal fibroblasts grown on collagen-coated dishes. The anabolic steroid, oxandrolone, antagonized such collagen substrate inhibition of procollagen mRNA expression. These findings suggest that oxandrolone may directly enhance wound healing by increasing the expression of procollagen mRNA in fibroblasts associated with a collagen matrix analogous to the healing wound.
U.S. Patent App. Nos. 20030199441 and 20050282737 teach medicaments for treating or preventing fibrotic diseases. They describe application of a (poly) peptide with antifibrotic activity, comprising at least one N-terminal procollagen (III) propeptide and a C-terminal procollagen (III) propeptide, or a fragment of the (poly) peptide.