Metalloproteinase inhibitors
The present invention provides inhibitors of metalloprotineases that may be extracted from lactational secretions such as milk and colostrum. The inhibitors have use in the treatment of a range of diseases such as disorders of the gastrointestinal tract, cardiovascular conditions and wound healing. Methods for purifiying the inhibitors are also disclosed.
1 . A composition derived directly or indirectly from a lactational secretion of an ungulate animal, the composition comprising an inhibitor of a metalloproteinase, wherein the inhibitor of metalloproteinase is enriched as compared with the lactational secretion from which the composition is derived.
2 . A composition according to claim 1 wherein the metalloproteinase is selected from the group consisting of (i) the collagenases (metalloproteinases-1, 8 and 13); (ii) the gelatinases A and B (metalloproteinase-2 and metalloproteinase-9); (iii) the stromelysins 1 and 2 (metalloproteinases-3 and 10 ); (iv) matrilysin (MMP-7); enamelysin (MMP-20), macrophage metalloelastase (MMP12), and MMP-19 and (v) the membrane-type metalloproteinases (MT-MMP-1 to 4 and stromelysin-3, MMP-11).
3 . A composition according to claim 1 wherein the inhibitor is a tissue inhibitor of a metalloproteinase (TIMP).
4 . A composition according to claim 3 wherein the TIMP is a TIMP-2 polypeptide or a functional equivalent or fragment thereof.
5 . A composition according to claim 4 wherein the TIMP-2 has a molecular weight of about 21,000 Da as determined by SDS-PAGE and/or has an isoelectric point of about 7.0.
6 . A composition according to claim 5 wherein the TIMP-2 comprises the following N-terminal sequence: NH2-CSCSPVHP.
7 . A composition according to claim 6 wherein the TIMP-2 comprises the following sequence:
NH2_CSCSPVHPQQAFCNADIVIRAKAVNKKEVDSGNDIYGNPIKRIQYEIKQIK MFKGPDQDIEFIYTAPAAAVCGVSLDIGGKKEYLIAGKAEGNGNMHITLCDFIVP WDTLSATQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKNIN GHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP_COOH,
or a functional equivalent or fragment thereof.
8 . A composition according to claim 1 wherein the lactational secretion is milk or colostrum.
9 . A composition according to claim 1 wherein the ungulate animal is selected from the group consisting of a cow, a sheep, a goat, a camel and a horse.
10 . A composition according to claim 1 wherein if the composition is derived indirectly from a lactational secretion, an intermediate product is selected from the group consisting of cheese whey, skim milk, acid (casein) whey, colostral whey, defatted colostrum and dried milk powder or a combination thereof.
11 . A composition according to claim 1 comprising a pharmaceutically, veterinarily, nutriceutically or cosmetically acceptable carrier and/or excipient.
12 . A composition according to claim 1 wherein the inhibitor of a metalloproteinase is present at a purity of from about 70% to about 99% with respect to the total protein content of the composition.
13 . A method for treating, preventing or ameliorating a disorder associated with undesirable metalloproteinase activity, the method including administering to an animal in need thereof an effective amount of a composition according to claim 1 .
14 . A method according to claim 13 wherein the disorder is a wound caused by pressure, vascular disease, diabetes, autoimmune disease, sickle cell diseases or hemophilia; a result of surgery; therapeutically induced; associated with disorders of the central nervous system, and resulting from any exfoliative disease of the skin; associated with either local or systemic infection such as yaws, HIV, chicken pox or herpes infection; congenital; a corneal injury to the eye; a pathological wound; a traumatic or accidental wound; or a burn.
15 . A method according to claim 13 wherein the disorder is a dental or oral wound; peptic ulceration of the duodenum, stomach or esophagus; inflammatory bowel disease; an ulcer associated with stress conditions; damage to the lining of the alimentary tract; inadequate gut function or damage to the gut associated with prematurity; a diarrheal condition; a food intolerance; a cancer of the gastrointestinal tract; surgically induced damage to the gut; damage due to esophageal reflux; a condition associated with loss of gut barrier function; a congenital condition resulting in inadequate gastrointestinal function or damage; or an autoimmune disease that affects the gut.
16 . A method according to claim 13 wherein the composition is administered so that the concentration of the inhibitor is present in a concentration of from about 0.1 ng/ml to about 10 μg/ml at the site of undesirable metalloproteinase activity.
17 . A method according to claim 13 wherein the disorder is a disorder of the cardiovascular system selected from the group including dilated cardiomyopathy, congestive heart failure, atherosclerosis, plaque rupture, reperfusion injury, ischemia, chronic obstructive pulmonary disease, angioplastly restenosis, aortic aneurism; a disorder of a tissue where a metalloproteinase is involved in the irregular remodeling including disorders of bone, liver, lung and nervous tissues; a disorder relating to viral infection whereby metalloproteinase activity is altered; a disorder relating to inflammation involving the implication of metalloproteinases; a disorder relating to skin involving the implication of a metalloproteinase, including but not limited to psoriasis, scleroderma and atopic dermatitis or disorders relating to ultraviolet damage of skin which results in the skin having an aged and/or wrinkled appearance.
18 . A method for at least partially purifying or enriching a metalloproteinase inhibitor, the method including the steps of;
providing a lactational secretion of an ungulate animal or a derivative of the lactational secretion, and
subjecting the lactational secretion or derivative to one or more treatment steps selected from the group consisting of centrifugation, micro-filtration, ultra-filtration, ion-exchange chromatography, molecular sieve chromatography, affinity chromatography, reverse-phase high performance liquid chromatography and transient acidification.
19 . A method according to claim 18 , the method comprising the steps of cation exchange chromatography followed by ultrafiltration.
20 . A method according to claim 19 wherein the ultrafiltration step is followed by an acidification step.
21 . A method according to claim 20 wherein the transient acidification step is followed by a gel filtration chromatography step.
22 . A method according to claim 21 wherein the gel filtration step is followed by an anion exchange chromatography step.
23 . A method according to claim 22 wherein the anion exchange chromatography step is followed by an affinity chromatography step.
24 . A method according to claim 23 wherein the affinity chromatography step is followed by a reverse phase high performance liquid chromatography step.
25 . A method according to claim 18 , the method comprising a centrifugation step followed by an affinity chromatography step.