Title:  Chitin hydrogels, methods of their production and use

United States Patent:  6,124,273

Inventors:  Drohan; William N. (Springfield, VA); MacPhee; Martin J. (Gaithersburg, MD); Miekka; Shirley I. (Gaithersburg, MD); Singh; Manish S. (Columbia, MD); Elson; Clive (Halifax, CA); Taylor, Jr.; John R. (New York, NY)

Appl. No.:  960555

This invention is directed to the preparation and utilization of supplemented chitin hydrogels, such as chitosan hydrogels. Further provided are biomaterials comprising same. The particular supplement delivered by the chitin hydrogel is selected as a function of its intended use. In one embodiment, this invention provides a composition of matter, comprising a chitin hydrogel or chitin-derived hydrogel, wherein the hydrogel does not inhibit full-thickness skin wound healing.

SUMMARY OF THE INVENTION

In one embodiment, this invention provides a composition of matter, comprising a chitin hydrogel or chitin-derived hydrogel, wherein the hydrogel does not inhibit full-thickness skin wound healing.

In another embodiment, this invention provides a composition of matter comprising a supplemented hydrogel comprising at least one growth factor.

In another embodiment, this invention provides a composition of matter comprising a supplemented hydrogel comprising at least one growth factor and/or a drug.

In another embodiment, this invention provides a composition of matter that promotes the directed migration of animal cells, comprising: a hydrogel; and an effective concentration of at least one growth factor, wherein the concentration of the growth factor is effective in promoting the directed migration of the animal cells.

In another embodiment, the present invention provides a composition of matter that promotes wound healing, comprising: a hydrogel; and an effective concentration of at least one growth factor, wherein the concentration is effective in promoting wound healing.

In another embodiment, the present invention provides a composition of matter that promotes the endothelialization of a vascular prosthesis, comprising: a hydrogel; and an effective concentration of at least one growth factor, wherein the concentration is effective in promoting the endothelialization of a vascular prosthesis.

In another embodiment, the present invention provides a composition of matter that promotes the proliferation and/or differentiation of animal cells, comprising: a hydrogel; and an effective concentration of at least one growth factor, wherein the concentration is effective in promoting proliferation and/or differentiation of animal cells.

In another embodiment, the present invention provides a composition of matter that promotes the localized delivery of at least one drug.

In another embodiment, the present invention provides a composition of matter that promotes the localized delivery of at least one growth factor.

In another embodiment, the present invention provides a process for promoting the healing of wounds, comprising applying to the wound, a composition that contains a supplemented hydrogel and an effective concentration of at least one growth factor, wherein the concentration is effective to promote wound healing.

In another embodiment, the present invention provides a process for promoting the endothelialization of a vascular prosthesis, comprising applying to the vascular prosthesis a composition that contains a chitin hydrogel and an effective concentration of at least one growth factor, wherein the concentration is effective to promote the endothelialization of a vascular prothesis.

In another embodiment, the present invention provides a process for promoting the proliferation and/or differentiation of animal cells, comprising placing the cells in sufficient proximity to a chitin hydrogel which contains an effective concentration of at least one growth factor, wherein the concentration is effective in promoting the proliferation and/or differentiation of the cells.

In a further embodiment, the present invention provides a process for the localized delivery of at least one drug to a tissue, comprising applying to the tissue a chitin hydrogel which contains at least one drug.

In another embodiment, the present invention provides a process for the localized delivery of at least one growth factor to a tissue, comprising applying to the tissue a chitin hydrogel which contains at least one growth factor.

In another embodiment, this invention provides a process for producing the directed migration of animal cells, comprising: placing in sufficient proximity to the cells, a chitin hydrogel which contains an effective concentration of at least one growth factor, wherein the concentration is effective to produce the desired directed migration of said cells.

In another embodiment, this invention provides a simple to use, fast acting, field-ready bandage for applying a hydrogel to wounded tissue in a patient, comprising an occlusive backing, affixed to which is a layer of dry materials comprising an effective amount of dry, purified chitin or chitosan to produce a tissue-sealing hydrogel matrix upon hydration. Further embodiments pertain to the use and preparation of the chitin bandage.

In yet another embodiment, this invention provides a simple to use, fast acting, field-ready dressing for treating wounded tissue in a patient, is formulated as an expandable foam comprising an effective amount of purified chitin or chitosan to produce a tissue-sealing hydrogel matrix upon hydration. Further embodiments pertain to the use and preparation of the chitin dressing.

In another embodiment, this invention provides a mixture of chitin hydrogel, DBM and/or purified BMP's. This mixture provides a matrix that allows the cellular components of the body to migrate into it and thus produce osteoinduction where needed. The matrix composition, enzymes (such as thrombin and plasmin), BMPs, growth factors and DBM and their concentrations are adequately formulated to optimize the longevity of this temporal scaffolding structure and the osteoinduction which needs to occur. All of the chitin hydrogel components are biodegradable, but during osteogenesis the mixture provides a non-collapsible scaffold that can determine the shape and location of the newly formed bone. Soft tissue collapse into the bony nonunion defect, which is a problem in bone reconstructive surgery, will thus be avoided. The use of hydrogel supplemented with growth factors such as CIF-A and CIF-B, infra, which promote cartilage development, will be useful in the reconstruction of lost, damaged or missing cartilage and/or bone.

In a preferred embodiment, an effective concentration of HBGF-1 is added to a chitin preparation to provide a growth factor-supplemented chitin hydrogel that possesses the ability to promote wound healing. In another preferred embodiment, an effective amount of a platelet-derived extract is added to the chitin hydrogel.

In other preferred embodiments, an effective concentration of a mixture of at least two growth factors are added to the chitin hydrogel and an effective amount of the growth factor-supplemented hydrogel is applied to the wounded tissue.

In addition to growth factors, drugs, polyclonal and monoclonal antibodies, oligonucleotides and other compounds, including, but not limited to, DBM, BMPs, osteogenic or cartilage inducing compositions may be added to the hydrogel. They accelerate wound healing, combat infection, neoplasia, and/or other disease processes, mediate or enhance the activity of the growth factor in the hydrogel, and/or interfere with hydrogel components which inhibit the activities of the growth factor in the hydrogel. These drugs may include, but are not limited to: antimicrobial compositions, including antibiotics, such as tetracycline, ciprofloxacin, and the like; antimycogenic compositions; antivirals, such as gangcyclovir, zidovudine, amantidine, vidarabine, ribaravin, trifluridine, acyclovir, dideoxyuridine, and the like, as well as antibodies to viral components or gene products; antifungals, such as diflucan, ketaconizole, nystatin, and the like; and antiparasitic agents, such as pentamidine, and the like. The drugs may further include anti-inflammatory agents, such as .alpha.-1-anti-trypsin, .alpha.-1-antichymotrypsin, and the like; cytokines and interferons, such as .alpha.- or .beta.- or .gamma.-interferon, .alpha.- or .beta.-tumor necrosis factor, and the like, and interleukins.

In an additional preferred embodiment, an effective concentration of cytotoxin or cell proliferation inhibiting composition is delivered by the chitin hydrogel. An effective concentration at least one cytotoxin or cell proliferation inhibiting composition is added to the chitin hydrogel.

In another embodiment, genetically altered cells and/or other cells may also be included in the hydrogels of this invention.

In additional embodiments, anything which does not destroy the matrix or the supplementary components added thereto can be added to the hydrogels of this invention.

In another embodiment, the supplemented hydrogel can be used in organoids and could contain, for example, growth factors such as FGF-1, FGF-2, FGF-4 and OP-1, or any recognized growth factor, including those listed above.

In another embodiment, this invention provides a composition that promotes the localized delivery of a poorly water soluble form of an antibiotic, such as the free base form of TET, and/or other drug.

In another embodiment, the present invention provides a method for cross-linking a chitin hydrogel.

In another embodiment, the present invention provides a system for the delivery of a supplement from a chitin hydrogel.

In another embodiment, the present invention provides a composition of matter for subcutaneous delivery that promotes the controlled, extended release of proteins for absorption into the blood stream.

In another embodiment, the present invention provides a composition of matter that promotes the delivery of coagulation proteins or factors or anticoagulant proteins or factors.

In another embodiment, the present invention provides a composition of matter that promotes the delivery of plasma proteins, comprising applying or injecting by subcutaneous, intradermal, intermuscular, intraperitoneal or intravenous injection a chitin hydrogel which contains at least one plasma protein, whereupon the concentration is effective to achieve therapeutic levels in situ or in the blood stream in individuals who have congenital or acquired deficiencies or defects of the protein.

In another embodiment, the present invention provides a composition of matter that promotes the delivery of Factor IX, comprising injecting or applying by subcutaneous, intradermal, intramuscular, intraperitoneal or intravenous injection of a chitin hydrogel which contains Factor IX, whereupon the concentration is effective to achieve therapeutic levels in the blood plasma in individuals with Hemophilia B.

In another embodiment, the present invention provides a composition of matter that promotes the delivery of Factor VIII, comprising injecting or applying by subcutaneous, intradermal, intramuscular, intraperitoneal or intravenous injection a chitin hydrogel which contains Factor VIII, whereupon the concentration is effective to achieve therapeutic levels in the blood plasma in individuals with Hemophilia A.

The present invention has several advantages over previously used compositions and methods. The first advantage is that the growth factor- and/or drug-supplemented chitin hydrogels of the present invention have many of the characteristics of an ideal biodegradable carrier, namely: they can be formulated to be free of non-mammalian proteins, thus eliminating or minimizing immunogenicity problems and foreign-body reactions; their administration is versatile; and their removal from the host's tissues is not required because the matrices are degraded by the host's own natural lytic system.

A second advantage is that the present invention provides a good way to effectively deliver growth factors, analgesics, antimicrobial compositions, anti-inflammatory compounds, antibodies, anticoagulants, antiproliferatives, cytokines, cytotoxins, chemotherapeutic drugs, interferons, hormones, hydroxyapatite, lipids, oligonucleotides, osteoinducers, polymers, polysaccharides, proteoglycans, polypeptides, protease inhibitors, proteins (including plasma proteins), steroids, vasoconstrictors, vasodilators, vitamins, minerals, stabilizers and the like, for a prolonged period of time to an internal or external wound. This is particularly advantageous since it appears that some growth factor receptors must be occupied for at least 12 hours to produce a maximal biological effect. Previously, there was no way to do this. The present invention allows for prolonged contact between the growth factor and its receptors to occur, and thus allows for the production of strong biological effects.

A third advantage of the present invention is that animal cells can migrate into and through, and grow in the chitin hydrogel of the present invention. This aids engraftment of the cells to neighboring tissues and prostheses, which cannot be achieved using commercially available European tissue sealants.

A fourth advantage is that because of its initial liquid nature, the hydrogel of the present invention can cover surfaces more thoroughly and completely than many previously available delivery systems. This is especially important for the use of the present invention in coating biomaterials and in promoting the endothelialization of a biomaterial, such as a vascular prostheses. The supplemented hydrogel will not only coat the interior and exterior of the vascular prosthesis, but also fill the pores contained therein, permitting the actual induction of migrating cells into the biomaterial. As a result, engraftment of autologous endothelial cells will occur along the whole length of the vascular prosthesis, thereby decreasing its thrombogenicity and antigenicity. Previously, engraftment started at the ends of the vascular prosthesis and proceeded, if at all, toward the interior of the same. To date, total engraftment of a biomaterial into a human is uncommon, primarily because delays allow thrombogenicity and antigenicity to develop. Moreover, previously used vascular prostheses have been primarily seeded with nonautologous cells, enhancing the possibility of rejection by the body. The cells were easily washed off by the shearing force of blood passing through a vascular prosthesis.

A fifth advantage is that the supplemented hydrogels of the present invention can be molded and thus can be custom made into almost any desired shape. For example, the chitin hydrogel can be supplemented with BMPs and/or DBM and can be custom made into the needed shape to most appropriately treat a bone wound. This cannot be done with DBM powder alone, because DBM powder will not maintain its shape. Moreover, the supplemented chitin hydrogel can be readily dried into thin sheets.

A sixth advantage is that the antibiotic-supplemented hydrogel of this invention, such as with TET, has been found to unexpectedly increased the longevity and stability of the chitin hydrogen, as compared to that of the unsupplemented hydrogel. This increased stability continues even after appreciable quantities of the antibiotic no longer remain in the matrix. For example, soaking a newly formed hydrogel in a saturated solution of TET produced from free base TET, or in a solution of CIP HCl, produces a matrix which is stable and preserved even after substantially all the TET or CIP has dissipated. As a result, one can expect an increased storage shelf life of the hydrogel, and possibly an increased persistence in vivo.

The seventh advantage of the present invention is a direct result of the prolonged longevity and stability of the chitin hydrogel. As a result of this unexpected increase in stability of the matrix, antibiotic or antiproliferative-supplemented hydrogel can be used to produce localized, long term delivery of a drug and/or a growth factor, and the like. This delivery will continue even after the stabilizing drug, such as TET or CIP, has substantially left the matrix. Inclusion of a solid form, preferably a poorly water soluble form of a drug, such as in free base form, into a matrix that has been stabilized by, for example, TET or CIP, permitting site-specific delivery of the supplement for an extended period of time. Some forms of drugs, such as free base TET, advantageously allow for both stabilization of the matrix and prolonged drug delivery, while other drugs may do one or the other, but not both. Such applications are not previously known in the art.

An eighth advantage of the present invention is that it allows site-directed angiogenesis to occur in vivo. While others have demonstrated localized non-specific angiogenesis, no one else has used a chitin hydrogel to promote site-directed angiogenesis.

A ninth advantage of the present invention is that because the components of the chitin hydrogel can be formulated into several forms of simple to use, fast-acting field dressings, it is now possible to control bleeding from hemorrhaging trauma wounds, thereby saving numerous lives that previously would have been lost. Although life-saving methods of treating such wounds are possible by trained medical personal or in fully-equipped clinics and hospitals, the present invention satisfies society's long-felt need for an easy-to-use, first-aid (or even self-applied) treatment that will, in emergency or disaster situations, allow an untrained individual to treat traumatic injuries to control hemorrhage until medical assistance is available.

A tenth advantage of the present invention is that, because the components of the chitin hydrogel can be formulated so as to release proteins and polypeptides at controlled rates for extended times, it is now possible to deliver proteins into peripheral sites by subcutaneous, intradermal, intramuscular, intraperitoneal or intravenous injection, in chitin hydrogel formulations which will release the protein at a rate suitable for absorption of the protein into the blood plasma at therapeutic and/or prophylactic levels.

Claim 1 of 32 Claims

What is claimed is:

1. A composition of matter that provides sustained release of proteins, said composition comprising a covalently cross-linked N,O-carboxymethyl chitosan hydrogel having a protein to be delivered incorporated therein in sufficient concentration to provide said sustained release.

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