KR20050024313A - Metalloproteinase inhibitors - Google Patents

Abstract

The present invention provides inhibitors of metalloproteinases that can be extracted from milk secretions such as milk and colostrum. This inhibitor is used to treat a range of diseases such as diseases of the gastrointestinal tract, cardiovascular conditions and wound healing. Also disclosed are methods of purifying inhibitors.

Description

Metalloproteinase inhibitors {METALLOPROTEINASE INHIBITORS}

The present invention relates to a composition derived from lactational secretion comprising milk and colostrum with anti-proteinase activity. More specifically, the composition is active against metalloproteinases and is useful for the treatment of wounds and disorders of the gastrointestinal tract.

No reference cited herein is to be construed as constituting the prior art. The discussion of references is intended to refer to what the author claims, and the applicant has the right to challenge the accuracy and adequacy of the cited references. Although a number of prior art publications are mentioned herein, it will be apparent that such references do not acknowledge that any of these documents forms part of the general technical knowledge common in Australia or any other country.

Proteinases are naturally occurring enzymes present in many tissues of the body. These enzymes normally work to break down proteins in specific ways. To prevent uncontrolled disruption of the target protein, the activity of the enzyme is regulated by the inhibitor substance. Thus, the combined and balanced action of proteinases and inhibitors regulates many important physiological processes by acting to regulate levels of biologically active or structurally important body proteins.

One important group of proteins is metalloproteinases. These enzymes are characterized by the presence of metal ions in order to catalyze proteolysis. About 17 different metalloproteinases that share important sequence homology have been identified and / or cloned. Metalloproteinases, depending on their structural and functional properties, include: (i) collagenase (metalloproteinase-1, 8 and 13); (ii) gelatinases A and B (metalloproteinase-2 and metalloproteinase-9); (iii) stromelysins 1 and 2 (metalloproteinases-3 and 10); (iv) matrilysin (MMP-7); Enamel lysine (MMP-20), macrophage metalloelasterase (MMP12), and MMP-19 (which forms a classical metalloproteinase) and (v) membrane-type metalloproteinases (MT-MMP- 1-4 and stromelysin-3, MMP-11) (W. Bode et al., Ann NY Acad Sci. 878, 73, 1999). These metalloproteinases share a common multi-domain structure but are glycosylated to different degrees at different sites. According to the sequence alignment, the assembly of these domains is early evolution and then diversified. Collectively, metalloproteinases can degrade all major components of the extracellular matrix (ECM).

Homeostasis of ECM is regulated by a fine balance between the synthesis of ECM proteins, the production of ECM-degrading extracellular matrix metalloproteinases (MMPs), and the presence of metalloproteinase inhibitors.

One class of metalloproteinases is tissue inhibitors (TIMPs) of metalloproteinases. The TIMP family has four conserved cysteine residues and includes four or more individual elements (TIMP-1-4) that express metalloproteinase inhibitory activity by forming a non-covalent complex with the metalloproteinases. It is done by Specifically, TIMPs bind 1: 1 stoichiometry to highly conserved active zinc-binding sites of metalloproteinases, but can also bind to other domains of metalloproteinase-2 and metalloproteinase-9. have. In addition to their inhibitory action, TIMPs appear to have other functions not thought to contribute directly to proteinase inhibition, including growth factor-like, anti-vessel-derived and anti-apoptitic activity. see.

TIMPs have been identified in a wide range of biological tissues such as bone, amniotic fluid, cartilage, aortic endothelial cells and skin fibroblasts. These tissues require substantial purification to isolate metalloproteinase inhibitors and are associated with biostability issues for human use. In addition, these sources may provide only a limited amount of TIMP.

One aspect of the present invention is to overcome or at least ameliorate one or more difficulties or deficiencies associated with many prior art by providing a rich source of metalloproteinase inhibitors and methods of purifying the inhibitors. Also disclosed are compositions comprising inhibitors and methods of treating various conditions and diseases using the compositions described herein.

1: Bovine Cheese Whey was subjected to a series of purification steps illustrated in Example 1A. This figure illustrates the elution profiles of protein and metalloproteinase inhibitors in EIA according to each chromatography step. Protein (-) was assessed by BCA assay (FIGS. 1A-C) or absorbance at 214 nm (FIG. 1D) (●). Metalloproteinase inhibitors of each fraction were measured by EIA as described in Example 1A.

2: SDS-PAGE analysis (left panel) and subsequent Western blot (right panel) analysis of purified metalloproteinase inhibitors using anti-TIMP-2 antibody. Samples of purified metalloproteinase inhibitors loaded in amounts of 2.8, 1.4 or 0.7 μg were allowed to flow under reducing conditions on a 10-20% Tris-tricin gel and stained using Coomas Brilliant Blue. A sample of purified metalloproteinase inhibitor loaded in an amount of 0.3 μg was also allowed to flow under reducing conditions on a 10-20% Tris-Triscine gel, but transferred to nitrocellulose to a monoclonal anti-TIMP-2 antibody (Chemicon ) And then HRP-conjugated both anti-mouse IgG (Silenus Labs). The HRP-labeled protein was then visualized with enhanced chemoluminescence (ECL). From this figure, it is confirmed that the metalloproteinase inhibitor is bovine TIMP-2.

Figure 3: Analytical RP-HPLC analysis of purified metalloproteinase inhibitors. Aliquots (10 μg) of purified metalloproteinase inhibitor formulations were analyzed on a mocrobore (2.1 × 100 mm, Brownlee Labs) on a C4 RP-HPLC column. Protein eluted with a linear gradient of 15-45% CH ₃ CN in 0.08% TFA over 2 minutes, then 45-80% CH ₃ CN over 5 minutes.

Figure 4: Mass spectrometric analysis of metalloproteinase inhibitors. Purity and molecular weight of the metalloproteinase inhibitors were measured using a VG Quattro triple quadrapole mass spectrometer according to the manufacturer's instructions. From this figure, it is confirmed that the metalloproteinase inhibitor is bovine TIMP-2.

5 shows a metalloproteinase activity assay used to test the activity of a metalloproteinase inhibitor. MMI here refers to a metalloproteinase inhibitor. This assay uses a biotinylated gelatinase substrate, which is degraded by active MMP-2 and MMP-9 (gelatinase) enzymes. Degradation of the biotinylated gelatinase substrate produces fragments with low biotin labels capable of binding to a proprietary 96-well plate specific to the substrate. The bound fragments are then detected (detectable at 405 nm) with streptavidin-enzyme complexes which produce a colored product upon addition of the enzyme substrate. Therefore, the greater the gelatinase activity, the lower the OD, while inhibiting the gelatinase activity increases the absorbance.

6: Metalloproteinase inhibitory activity of metalloproteinase inhibitors. Aliquots of pooled fractions from each step, and fractions 30 and 33 from the cellulose pin column, were assayed for metalloproteinase inhibitory activity using the Gelatinase Activity Assay Kit as described above. It was.

7: Rate of healing of normal and ischemic wounds at proximal (a), median (b) and distal (c) sites.

8: Western blotting of proteins from normal and ischemic wound sites. MMP-9 was upregulated in ischemic wounds at proximal (P) and median (M) sites but not at distal (c) sites.

Figure 9: Western blotting analysis of TIMP-2 of cellulopine fraction.

10: Ischemia and normal wound size (% of wound diameter at 0 o'clock in the median) after 10 days of treatment with saline or Composition A. Values are mean ± SE. ^* Indicates a significant difference by ANOVA for ischemic wounds treated with saline when P <0.05.

A first aspect of the invention provides a composition derived directly or indirectly from a mammalian lactation and comprising an inhibitor of a metalloproteinase. Applicants have found that milk and colostrum (particularly cow milk or bovine colostrum) contain an effective amount of a metalloproteinase inhibitor.

In a second aspect the present invention provides a method of treating, preventing or ameliorating a disorder associated with undesirable metalloproteinase activity, which method comprises a composition comprising a metalloproteinase inhibitor derived from lactation. Administering to the animal in need thereof an effective amount. This method is useful, for example, in the field of wound care, gastrointestinal disorders and cardiovascular disorders.

In a third aspect the present invention provides a method of at least partially purifying or enriching a metalloproteinase inhibitor, the method comprising providing a lactation or a derivative thereof, and

Milk secretions or derivatives thereof are subjected to centrifugation, microfiltration, ultrafiltration, ion-exchange chromatography, molecular sieve chromatography, affinity chromatography, reverse-phase high performance liquid chromatography and transient acidification. Applying to one or more processing steps selected from the group consisting of

A first aspect of the invention provides a composition derived directly or indirectly from a mammalian lactation and comprising an inhibitor of a metalloproteinase. Applicants have shown that milk and colostrum from ungulate animals are useful as a source of metalloproteinase inhibitors. The unexpected discovery of such inhibitors in milk and colostrum of ungulates provides a rich and continuous source of metalloproteinase inhibitors. As used herein, the term "metalloproteinase" includes proteases that degrade the components of the extracellular matrix by proteolysis. The term metalloproteinases includes (i) collagenase (metalloproteinase-1, 8 and 13); (ii) gelatinases A and B (metalloproteinase-2 and metalloproteinase-9); (iii) stromelysins 1 and 2 (metalloproteinases-3 and 10); (iv) matrilysin (MMP-7); Enamel lysine (MMP-20), macrophage metalloelasterase (MMP12), and MMP-19 (which forms a classical metalloproteinase) and (v) membrane-type metalloproteinases (MT-MMP- 1 to 4 and stromelysine-3, MMP-11).

In the present invention, the term "milk secretion" includes any secretion from the mammary gland of a mammal, including but not limited to milk and colostrum. Mammals can be humans, cattle, sheep, goats, camels or horses.

The present invention also encompasses the use of derivatives of lactation. Useful derivatives of the lactation include any of the altered proportions of fat and / or protein components thereof, including but not limited to cheese whey, skim milk, acid (casein) whey, dry milk powder, colostrum whey and skim colostrum. Product is included. Derivatives also include intermediates in the process such as chromatographic eluates, filtrates, retentates and the like.

Mammals are preferably ungulates. Cows are preferably cows. Compared to other ungulates, cows produce large amounts of milk on a regular basis that provides a relatively steady supply of metalloproteinase inhibitors. Cows that have recently begun produce large quantities of colostrum.

Preferably the metalloproteinase inhibitor is present in the composition at a concentration of about 0.01 μg / ml to about 100 mg / ml. More preferably, the metalloproteinase inhibitor is present at a concentration of about 0.1 μg / ml to about 1000 μg / ml. Even more preferably, the metalloproteinase inhibitor is present at a concentration of about 1 μg / ml to about 500 μg / ml. In a very preferred embodiment, the metalloproteinase inhibitor is present at a concentration of about 11 μg / ml, or about 45 μg / ml or 50 μg / ml when quantified with a fluorescence-quenching substrate assay as described in Example 3. do.

In a preferred form of the invention, the inhibitor is a tissue inhibitor of metalloproteinase (TIMP). As used herein, "tissue inhibitor of metalloproteinases" includes polypeptides that modulate the activity of metalloproteinases, including TIMP-1, TIMP-2, TIMP-3 and TIMP-4 It is not limited. The TIMP family has four conserved cysteine residues and includes four or more individual elements (TIMP-1-4) that express metalloproteinase inhibitory activity by forming a non-covalent complex with the metalloproteinases. It is done by Specifically, TIMPs bind 1: 1 stoichiometry to highly conserved active zinc-binding sites of metalloproteinases, but can also bind to other domains of metalloproteinase-2 and metalloproteinase-9. have. In addition to their inhibitory action, TIMPs appear to have other functions not thought to contribute directly to protease inhibition, including growth factor-like, anti-vascular derived and anti-apoptotic activity. TIMPs are expressed by various cell types and are present in most tissues. TIMPs have been reported in some body fluids, but there are no reports describing the presence of metalloprotease inhibitors in ungulate lactation.

TIMP-1 was originally isolated from the bones of rabbits and characterized as a collagenase inhibitor (A. Sellers and J. J. Reynolds. Biochem. J. 167, 353, 1977). Human TIMP-1 was then purified from amniotic fluid (G. Murphy et al., Biochem. J. 195, 167, 1981) and human fibroblasts and from a number of other sources (see J. Woessner for review). Methods in Mol. Biol. 151, 1, 2001). Human TIMP-1 consists of 184 amino acids comprising 12 cysteine residues forming six disulfide bonds and a characteristic six-loop structure. Human TIMP-1 may range from 30 to 34 kDa depending on the degree of glycosylation, but has a molecular weight of about 28.5 kDa and is strongly glycosylated.

Human TIMP-2 is initially the supernatant of human melanoma cells and fibroblasts (GI Goldberg et al., Proc. Natl. Acad. Sci. (USA) 86, 8207, 1989; W. Stetler-Stevenson et al., J. Biol. Chem. 264, 374, 1989) and crude media with 72 kDa progelatinase in controlled media of alveolar macrophages (SD Shapiro et al., J. Biol. Chem. 267, 1992). 21 kDa aglycosylated protein identified as protein. Human TIMP-2 contains six conserved six disulfide bond structures with amino acid levels 40% identical to human TIMP-1.

Bovine TIMP-2 was originally isolated from bovine cartilage and characterized as a collagenase inhibitor (J. Murray et al., J. Biol. Chem. 261, 4154, 1986). TIMP-2 was then subjected to a controlled medium of bovine aortic endothelial cells (Y. De Clerck et al., J. Biol. Chem. 264, 17445), which cloned and characterized bovine TIMP-2 cDNA sequences from these cells. , 1989) (T. Boone et al., Proc. Natl. Acad. Sci. (USA) 87, 2800, 1990). Bovine TIMP-2 cDNA encodes a leader sequence of 26 amino acids and a mature protein sequence of 194 amino acids. Bovine TIMP-2 has an amino acid level of 94% identical to human TIMP-2 and 41% and 42% homology with bovine TIMP-1 and TIMP-3, respectively. See Table 1 for the amino acid sequences of bovine TIMP-2 and human TIMP 1, 2, 3 and 4.

TIMP-3 was first identified and purified from chicken embryonic fibroblasts as 21 kDa aglycosylated protein (N. Pavloff et al., J. Biol. Chem. 267, 17321, 1992). Human homologues were then detected as serum-derived proteins in WI-38 fibroblasts (M. Wick et al., J. Biol. Chem. 269, 18953, 1994). Human TIMP-3 is 30% and 38% homologous with human TIMP-1 and human TIMP-2, respectively. More recently, the fourth element of the TIMP family, TIMP-4, has been identified through molecular cloning (J. Greene et al., J. Biol. Chem. 271, 30375, 1996). Human TIMP-4 has a predicted molecular weight of 22 kDa and the recombinant form of this protein has a molecular weight of 24 kDa.

In a more preferred form of the invention, the inhibitor is a bovine TIMP-2 polypeptide or a functional equivalent thereof. The term "functional equivalent" as used herein includes molecules having a capability substantially similar to TIMP-2 to inhibit the activity of metalloproteases. Functional equivalents are due to mutations in one or more of the nucleic acid sequences and thus include allelic variants of TIMP-2, which may alter the mRNA, and may or may not alter the structure or function of the polypeptide. do. Alleles of genes can arise as a result of natural deletions, substitutions, rearrangements or additions of nucleotides.

In the context of “allelic variants”, the term “variant” refers to an amino acid sequence modified by one or more amino acids. Variants may have conservative changes in which substituted amino acids have similar structural or chemical properties, eg, isoleucine is replaced by leucine. Variants may have non-conservative changes in which substituted amino acids have different structural or chemical properties, eg, alanine is substituted with glycine. The term “variant” also includes alterations in glycosylation and other variants, including allelic variations. Thus, altered proteins comprising these amino acid sequences are generally substantially equivalent to these proteins in function or structure and are thus defined as analogs. Preferably, the metalloproteinase inhibitor variants have 12 cystine residues of the original amino acid sequence that are conserved such that the polypeptide can form a non-covalent complex with the metalloproteinase. Even more preferably, the metalloproteinase inhibitor variant is TIMP-2 having 12 cystine residues of the original amino acid sequence that are conserved such that the polypeptide can form a non-covalent complex with the metalloproteinase.

Preferably, TIMP-2 has a molecular weight of 21,000 Da as measured by SDS-PAGE. In a more preferred form, TIMP-2 has an isoelectric point of about 7.0.

Preferably, TIMP-2 comprises the N terminal sequence NH2-CSCSPVHP. In a very preferred form, TIMP-2 has the following sequence.

Preferably, the inhibitor can inhibit metalloproteinase 2 and / or metalloproteinase 9. Metalloproteinase 2 is also known as gelatinase A. Metalloproteinase 2 is a proteolytic enzyme with a molecular weight of 72 kDa that catalyzes the degradation of collagen form IV by acting on peptide bonds. Metalloproteinase 9 is also known as gelatinase B. Metalloproteinase 2 is a proteolytic enzyme of 92 kDa molecular weight that catalyzes the degradation of collagen form IV by acting on peptide bonds.

Preferably, the inhibitor can inhibit the matrix metalloproteinases in membrane form, but not the tumor necrosis factor-alpha converting enzyme.

In view of the low sequence homology between TIMPs, it is not surprising that the four elements identified to date have individual biological functions. Unlike TIMP-1, TIMP-2 and TIMP-3 are effective inhibitors of membrane-form MMPs (MT-MMPs), but TIMP-3, but not TIMP-1, TIMP-2 or TIMP-4, is tumor necrosis factor-alpha It is important to inhibit the activity of the converting enzyme (TACE) (Amour et al., FEBS Lett. 435 39-44,1998).

Preferably, the inhibitor is not a growth factor and / or cannot stimulate the proliferation of L6 myoblasts.

In a more preferred form, the composition may comprise one or more cell growth stimulating factors having an approximately neutral to basic isoelectric point, and / or stimulate rat L6 myoblasts.

Preferably, the growth factor is transforming growth factor beta, insulin-like growth factor I, insulin-like growth factor II, betacellulin, any fibroblast growth factor element as known in the art, eg fiber Blast growth factor I or fibroblast growth factor II, insulin-like growth factor binding protein 1, 2, 3, 4, 5 or 6 and platelet-derived growth factor.

In a preferred form of the invention, lactation is first used in the composition after being treated with cheese whey.

Cheese whey is a by-product of the cheese industry, having essentially both fat and casein removed during cheese making. In the state of the art, cheese whey is intrinsically worthless and a potential contaminant, thus being the net cost of the industry. It is a low protein, high salt product available in large quantities. The major protein components in cheese whey are alpha lactalbumin (αLA) and beta lactoglobulin (βLG), which generally comprise at least 9% of the proteins present. High amounts of serum albumin, immunoglobulins and residual casein may also be present.

The composition may comprise one or more carriers and / or excipients. In a preferred form of the invention, the carrier and / or excipients are veterinary, nutriceutically, pharmaceutical or cosmetically acceptable. Pharmaceutical and cosmetic compositions according to the invention may be made suitable for administration by any suitable method. The composition can be made suitable for internal or topical administration. The composition may be in oral, infusion, topical or suppository form. Preferred delivery routes include skin, vaginal, intravenous, respiratory and gastrointestinal delivery. It is to be understood that the compositions described herein are not limited to human use, including any animal that can benefit from this composition.

Methods and pharmaceutical carriers for the manufacture of pharmaceutical compositions comprising compositions for topical administration are described in textbooks such as Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Easton, Pennsylvania, USA. It is well known in the art.

The composition of the present invention may be formulated to be suitable for oral administration. The compositions may be prepared in specific units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; As a powder or granules; As a solution or a suspension in an aqueous or non-aqueous liquid; It can be made into a mouth wash or into an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient can also be made into a mass, electuary or paste.

In addition to the components specifically described above, the compositions of the present invention may include other medicaments conventional in the art, containing in relation to the form of the composition, for example, compositions suitable for oral administration include sweeteners, thickeners and It may include additional agents such as flavoring agents.

In a preferred form of the invention, the compositions of the present invention comprise synthetic or biological polymers, glycosaminoglycans, or extracellular matrix molecules such as fibrin, collagen, gelatin, synthetic polymers, agarose, alginate, methylcellulose, Placed or impregnated (absorbent and non-absorbent) in hyaluronic acid, hydrocolloids, alginates, saline solutions, powders, ointments, plasters or irrigants or dressings, gauze, bandages, sutures, bandages, staples ), A carrier selected from the group consisting of transdermal patches or release dressings, toothpastes, chewing gums or resins, gargles or gels associated with prosthetic devices, screws or plates (biodegradable or non-biodegradable). do. Those skilled in the art will know appropriate carriers for use depending on the route or means of administration.

In another preferred form, the composition has one or more additional active ingredients selected from the group comprising antibiotics, anti-inflammatory agents, preservatives, other growth promoters, anesthetics. The compositions described herein may have a wound support associated with other molecules and / or adjuvants, carriers, solubilizers and growth factors as described above to aid in release, stability, solubility, activity. have. In addition, lactation or derivatives thereof or compositions of the invention may be used in combination with other compounds or molecules that work in concert with synergists, agonists and / or additives. The nature of these ingredients is not limited except that they must be pharmaceutically and physiologically acceptable for administration and should not reduce activity or render the active ingredient harmfully toxic.

The compositions of the present invention may be useful in combination with known therapeutic agents. When formulated at fixed doses, such combination products may employ inhibitors in the appropriate dosage range and other pharmaceutical actives within the approved dosage range. If the combination composition is inappropriate, the compositions can be used sequentially with known therapeutic agents.

The compositions of the present invention may also be useful for nutraceutical or cosmetic uses, and as such may be combined with other ingredients commonly used in such products in products. The term nutraceutical generally refers to any nutritional supplement which is designed for any particular clinical purpose as a general consumer food or used as a dietary supplement.

In a preferred form of the invention, the anti-inflammatory agent may at least partially inhibit the activity of tumor necrosis factor alpha. In a more preferred form, the anti-inflammatory agent is an antibody capable of binding to soluble tumor necrosis factor alpha such as REMICADE ^™ or soluble receptor such as ENBREL ^™ .

In a preferred embodiment, the composition comprises a number of basic cell growth stimulating factors having properties that stimulate the proliferation of rat L6 myoblasts. Even more preferably, the composition comprises transforming growth factor beta, insulin-like growth factor I, insulin-like growth factor II, betacellulin, fibroblast growth factor elements as known in the art, for example fibers Growth factor selected from the group comprising blast growth factor I or fibroblast growth factor II, insulin-like growth factor binding protein 1, 2, 3, 4, 5 or 6 and platelet-derived growth factor.

In a more preferred embodiment, the composition is fortified with metalloproteinase inhibitors. As used herein, the term "strengthening" means that the composition has a high proportion of a given component relative to the lactation from which the composition is derived.

Preferably, the TIMP-2 of the milk or colostrum or dairy product is present at a percentage purity of 0.01 to 10%. In a more preferred form, TIMP-2 in milk or colostrum or dairy is present at 10 to 30% percent purity. In an even more preferred form, the TIMP-2 in milk or colostrum or dairy is present at 30-70% percent purity. In an even more preferred form, TIMP-2 is present at 70 to 99.9% percent purity.

Preferably the composition has a neutral to basic pH.

In a preferred aspect of the invention, the composition has one or more of the following properties: reduction in the amount of alpha lactalbumin, beta lactoglobulin and casein compared to dairy products; Up to about 1% (w / w) salts of salts present in dairy products, casein up to 5% of dairy products, alpha lactalbumin, beta lactoglobulin, immunoglobulins and / or albumin.

In a second aspect, the present invention provides a method of treating, preventing or ameliorating a disorder associated with undesirable metalloproteinase activity, which method, in an animal in need thereof, comprises lactation as described herein. Administering an effective amount of a composition comprising a derived metalloproteinase inhibitor.

Preferably, this method is a method of treating wounds. The form of the curable surface wound is not limited and includes, but is not limited to, ulcers, conditions caused by surgery, therapeutically induced wounds, wounds associated with central nervous system disorders, exfoliative diseases of the skin, wounds associated with local or systemic infections, Congenital wounds, pathological wounds, traumatic and negligible wounds and burns. In a preferred form, the surface has undesirable metalloproteinase activity. In a preferred form, the skin of the wound is not complete.

In the method of treating a wound, the lactation or derivative or composition thereof of the present invention may be applied directly to the wound in a biologically acceptable carrier so as to be sustained release at a sufficient concentration in the wound environment. In the treatment of wounds, metalloproteinase inhibitors may be associated with wound support. As used herein, the term “wound aid” includes any means used to support or secure a wound and includes surgical securing means. The term includes bandages, dressings, sutures, staples, and the like. Supported wounds may be wounds caused by surgery or as a result of an accident or other injury. Lactose or derivatives or compositions thereof may be present on or impregnated on the surface of the wound support.

Preferably, the wound is an ulcer due to pressure, catheter disease, diabetes, autoimmune disease, sickle cell disease or hemophilia; Surgical outcomes; Therapeutic induction; Diseases of the central nervous system and deprived diseases of the skin; Local or systemic infections such as strawberry species, HIV, chickenpox or herpes infections; Congenital; Corneal damage to the eyes; Pathological wounds; Traumatic or fruiting wounds; Or an image.

In a preferred method, the concentration of metalloproteinase inhibitor is from about 0.1 ng / ml to about 10 μg / ml of fluid in the local environment of the wound site. Even more preferably, the concentration of metalloproteinase inhibitor is a fluid of about 1 ng / ml to about 1 μg / ml in the local environment of the wound site.

The invention also provides a method of preventing, ameliorating or treating a condition associated with gastrointestinal damage, disease or ulcer, which method comprises administering an effective amount of a composition described herein to an animal in need thereof. In a preferred method, the concentration of metalloproteinase inhibitor is from about 0.1 μg / ml to about 1000 μg / ml, even more preferably from about 1 μg / ml to about 500 μg / ml.

As used herein, the term "gastrointestinal damage, disease or ulcer" includes gastrointestinal damage or disease in the following forms:

(a) dental and oral wounds, including those associated with periodontal disease;

(b) peptic ulcer formation in the duodenum, stomach or esophagus due to radiation, non-steroidal anti-inflammatory drug (NSAID) treatment, Helicobacter pylori or chemotherapy;

(c) inflammatory bowel disease, such as ulcerative colitis or Crohn's disease;

(d) ulcers associated with stress conditions such as, for example, burns, trauma, sepsis, shock, intracranial surgery or head surgery;

(e) Mechloretamine, melphalan, busulfan, cytarabine, floxuridine, 5-fluorouracil, mercaptopurine, methotrexate, thioguanine, bleomycin, actinomycin-D Nutrient, including mucositis, according to radiotherapy and / or chemotherapy using agents such as daunorubicin, etoposide, mitomycin, vinblastine, vincristine, hydroxyurea or procarbazine damage to the inner layer of the alimentary tract;

(f) improper digestive tract function or digestive tract damage associated with premature birth, such as necrotic pre-enteritis or mild digestive motility;

(g) diarrhea conditions, such as those associated with bacterial, viral, fungal or protozoan infections, including AIDS;

(h) food intolerance, such as coeliac disease;

(i) cancers of the gastrointestinal tract, including the oral cavity, esophagus, stomach or intestines;

(j) surgically induced injuries, such as with some digestive tract resections, short digestive tract syndrome, hypertrophy, ileostomy, colon colitis;

(k) damage due to esophageal reflux;

(l) conditions associated with loss of barrier function, such as external burns, trauma, sepsis or shock;

(m) congenital conditions leading to improper gastrointestinal function or damage, such as volvulus and cystic fibrosis;

(n) Autoimmune diseases affecting the digestive tract, such as Sjogren's syndrome.

As used herein, the term “effective amount” means an amount sufficient to elicit a statistically significant response at a 95% confidence level (p <0.05 where the effect is due only to chance alone). Preferably, the effective amount is an amount that at least partially achieves the desired reaction of reducing metalloproteinase activity.

The composition may be administered in a therapeutically effective amount. A therapeutically effective amount may be used to achieve at least some desired effect, namely to alleviate or prevent symptoms of undesirable metalloproteinase activity, or to otherwise delay the onset of undesirable metalloproteinase activity, or By an amount necessary to inhibit its progression, to stop its onset or progression together, or to reduce metalloproteinase activity. Preferably, the term “therapeutically effective amount” as used herein means an amount sufficient to elicit a statistically significant response at a 95% confidence level (p <0.05 where the effect is due only to chance alone).

This amount is, of course, determined by the particular condition being treated, the severity of the condition, and the parameters of the individual patient, including age, physical condition, size, weight and other concurrent treatments, at the discretion of the attending physician. These factors are well known to those skilled in the art and can only be followed by routine experimentation. In general, it is desirable to determine the minimum effective amount in accordance with sound medical judgment. However, those skilled in the art will appreciate that high doses may be administered for medical, psychological or other reasons.

Symptom patients can be identified after a group of investigators carefully examine the symptoms in an injury, ulcer disease and metalloproteinase activity test.

The types of treatable gastrointestinal damage, disease or ulcer are not limited and include dental and oral wounds, peptic ulcers, inflammatory bowel disease, ulcers associated with stressful conditions, radiotherapy and / or chemotherapy damage, and inappropriate digestive tracts associated with premature birth Congenital conditions leading to impaired function or damage, diarrhea, food intolerance, cancer of the gastrointestinal tract, surgically induced damage, damage from esophageal reflux, loss of gut barrier function, inadequate gastrointestinal function or damage , And autoimmune diseases affecting the digestive tract and the digestive tract.

The composition may be administered at an appropriate time, including before, during or after gastrointestinal damage, disease or ulcer becomes evident.

In a preferred method, the condition is dental and oral wounds; Peptic ulcer of the duodenum, stomach or esophagus; Inflammatory bowel disease; Ulcers associated with stress conditions; Damage to the lining of the dietary tract; Improper digestive tract function or damage to the digestive tract associated with premature birth; Diarrhea status; Food intolerance; Cancer of the gastrointestinal tract; Surgically induced gut injury; Damage due to esophageal reflux; Conditions associated with loss of gut barrier function; Congenital conditions leading to inadequate gastrointestinal function or damage; Or autoimmune diseases affecting the digestive tract.

In a fifth aspect, the present invention provides a method for the prevention, amelioration and / or treatment of a disease associated with undesirable metalloproteinase activity, which method is directed to an animal in need thereof, Administering an effective amount of a derivative or composition thereof. The term "disorders associated with metalloproteinase activity" described herein includes the following diseases:

(i) undesired metalloproteinase activity may result in dilated cardiomyopathy, congestive heart failure, atherosclerosis, plaque rupture, reperfusion, injury, ischemia, and chronic obstructive pulmonary disease. Cardiovascular disorders that act on cardiovascular remodeling, including angioplastly restenosis and aortic aneurysms;

(ii) other tissues in which metalloproteinases are associated with irregular remodeling, including bone disorders such as osteosclerosis or osteoporosis, disorders of other tissues such as liver cirrhosis and fibrotic lung disease, disorders of nerve tissue such as multiple sclerosis Disorder of;

(iii) diseases associated with viral infections such as cytomegalovirus, retinitis, HIV and consequent AIDS syndrome, through which metalloproteinase activity is altered;

(iv) inflammatory bowel disease, Crohn's disease, ulcerative colitis, pancreatitis, diverticulitis, asthma or related lung disease, rheumatoid arthritis, gout, Reiter's Syndrome, lupus erthmatosis, ankylosing spondylitis ), Inflammation related disorders including the implications of metalloproteinases such as autoimmune keratitis, lung disease, bronchitis, emphysema, cystic fibrosis, acute respiratory distress syndrome;

(v) skin-related disorders, including application of metalloproteinases, including psoriasis, scleroderma, and atopic dermatitis, or disorders associated with ultraviolet damage to the skin causing aging and / or wrinkle formation of the skin .

Symptom patients are identified after a group of investigators have carefully examined the symptoms in affected tissues and tested for metalloproteinase activity.

In a preferred method, the condition is a cardiovascular disease, including but not limited to dilated cardiomyopathy, congestive heart failure, atherosclerosis, plaque rupture, reperfusion injury, ischemia, chronic blocked lung disease, angioplasty stenosis and aortic aneurysm ; Systemic diseases in which metalloproteinases are associated with irregular remodeling, including disorders of bone, liver, lung, and nervous tissue; Disorders associated with viral infections through which metalloproteinase activity is altered; Inflammation related disorders including the application of metalloproteinases; Psoriasis, scleroderma, and skin related disorders, including but not limited to the application of metalloproteinases, including but not limited to atopic dermatitis or disorders associated with ultraviolet damage to the skin causing aging and / or wrinkle formation of the skin. to be.

For all methods of treatment described herein, the daily dose may be routinely determined by the attending physician or veterinarian. Generally, the dose will vary depending on age, weight, individual patient's response, and the severity of the patient's symptoms. Suitable dosages of the inhibitors of the invention are generally from about 0.1 μg to about 100 mg per kg of body weight per day, preferably from about 1 μg to about 50 mg per kg of body weight per day. However, the dosage also depends on the composition used and the purity of the lactation or its derivatives.

In a third aspect, the present invention provides a method of at least partially purifying or enhancing a metalloproteinase inhibitor, the method comprising

Providing lactation or a derivative thereof, and

One or more treatments selected from the group consisting of centrifugation, microfiltration, ultrafiltration, ion-exchange chromatography, molecular sieve chromatography, affinity chromatography, reverse-phase high performance liquid chromatography and transient acidification Applying to the step.

The starting material can be a dairy filtrate that is substantially free of insoluble matter. Skim milk generally has higher protein and fat concentrations and lower salt concentrations than cheese whey. Skim milk also contains high concentrations of insoluble protein, especially particulate casein. To obtain a suitable composition from skim milk, if the selected cation-exchange resin does not have flow and adsorption properties that make it suitable for use with fat-containing and particulate-containing fluids, the skim milk is filtered prior to contacting it with the cation-exchange resin. To do this is usually necessary.

Lactose or derivatives thereof can be clarified by filtration or centrifugation, such as by filtration through a suitable sieve. Milk secretions or derivatives thereof can be filtered through hollow fiber cartridges of defined porosity.

Colostrum is the fluid produced during the first few days of lactation. Colostrum has a much higher protein concentration than skim milk or cheese whey. Due to the high protein concentration of colostrum, much less volume has to be applied to the cation-exchange resin, which facilitates the separation of metalloproteinase inhibitors. Colostrum, however, is high in fat and contains high concentrations of immunoglobulins in many mammalian species. Because of these two aspects, the preliminary filtration step of the process first needs to be particularly efficient, and then metalloproteinases, because immunoglobulins can be adsorbed to the resin, which will require proportionately more cation-exchange resins. Large scale separation of inhibitors is technically more difficult.

Multiple processing steps can be used to purify, enhance or activate metalloproteinase inhibitors. Suitability of the treatment step can be assessed by applying the lactation or its derivatives to a purification step advantageous for adsorption or separation and measuring the proportion of metalloproteinase inhibitors.

In a preferred embodiment, the treating step comprises ultrafiltration after cation exchange chromatography. The suitability of the cation exchange resin is such that the dairy product is passed through the test resin column at a neutral pH or other defined pH favoring adsorption or separation, and as a single band of about 21,000 Da according to gelatinase activity assay, SDS-PAGE. , N-terminal sequencing or mass spectrometry, or similar assays available in the prior art, can be used to determine the ratio of metalloproteinase inhibitors, and in particular TIMP-2. Preferably, the cation-exchange resin has a suitable pore size and suitable functional group for selectively adsorbing the metalloproteinase inhibitor. Sepharose-based cation exchange gels can be used. Sepharose is a trade name for agarose-based cation exchange resins.

The metalloproteinase inhibitor can be desorbed from the ion exchange resin to obtain a formulation with enhanced metalloproteinase inhibitory properties. The eluate can be concentrated and filtered using any suitable technique. The eluate can be concentrated, for example, by conventional ultrafiltration or additional chromatography or other procedures to obtain a composition comprising an inhibitor of metalloproteinases.

This method includes, for example, applying dairy products to a clarification step to remove insoluble matter therefrom; Adjusting the pH of the purification product to about 6.5 to 8.0; Contacting the clarified dairy product with a suitable cation-exchange resin; Eluting from the cation exchange resin at high ionic strength or high pH with a suitable buffer to provide the eluate; The eluate may be subjected to a concentration step and a diafiltration step to remove salts therefrom such that additional steps such as sequentially treating the lactation or derivatives thereof.

Eluates from cation-exchange resins are achieved at high ionic strength with which metalloproteinase inhibitors are recovered. For example, the metalloproteinase inhibitor adsorbed on the agarose-based resin can be eluted using 1M NaCl containing 0.25M NH ₄ OH.

The concentration step may comprise ultrafiltration. For example a 3000 Da exclusion film can be used. The diafiltration step used to remove the salt from the eluate may comprise diafiltration against 150 mM NaCl or a volatile salt, for example ammonium bicarbonate.

For example, a composition comprising an inhibitor of a metalloproteinase may be applied to a secretion or derivative in a filtration step to remove insoluble matter therefrom; Adjusting the pH of the filtrate to about 6.5 to 8.0; Contacting the filtrate with a suitable cation exchange resin to adsorb a metalloproteinase inhibitor having a basic to neutral isoelectric point thereto; Eluting the cation-exchange resin with a buffer to provide an eluate; By treating the eluate to remove salt therefrom, it may comprise sequentially treating the lactation or its derivatives.

Preferably, TIMP-2 having an isoelectric point 7.0 is a metallope of metalloproteinase 2 and metalloproteinase 9, as the activity of the metalloproteinase inhibitor is detected using a gelatinase activity assay. Adsorbed in an amount sufficient to be detected using a rotase inhibitory activity, the main protein having an acidic isoelectric point in the lactation or its derivatives is not absorbed; Eluting from cation exchange using a suitable buffer; The eluate is subjected to a concentration step and a diafiltration step to remove salts therefrom to obtain a composition comprising an inhibitor of a metalloproteinase.

Preferably, the metalloproteinase inhibitor is concentrated via an ultrafiltration step.

Preferably, the permeate or retentate comprising the metalloproteinase inhibitor obtained from the ultrafiltration step is excessively acidified.

The method may also further comprise an acidification step, for example. Preferably, the acidification is performed at a pH of about pH 3.0 or below. More preferably, the composition comprising the inhibitor of metalloproteinases is acidified to a pH of 2.0 to 3.0. Particularly preferred is an acidification pH of about 2.5.

Preferably the acidification is carried out using an inorganic acid, for example HCl. Acidification can be achieved by dissolving a composition comprising an inhibitor of a metalloproteinase in water, acidifying with a strong inorganic acid such as 5M HCl and drying.

The process of the present invention may further comprise removing the inert protein after acidification. Removal of inert proteins can be carried out using molecular sieve chromatography or ultrafiltration under acidic conditions. For example, a molecular sieve chromatography process can be used to obtain a composition comprising a metalloproteinase inhibitor with a molecular weight of 21,000 Da.

For example, a process for preparing a composition comprising an inhibitor of a metalloproteinase may include applying a dairy product to a filtration step to remove insoluble matter therefrom; Adjusting the pH of the filtrate to about 6.5 to 8.0; The filtrate is contacted with a suitable cation exchange resin such that the metalloproteinase inhibitor having a basic to neutral isoelectric point, and even more preferably TIMP-2 at isoelectric point 7.0, the activity of the metalloproteinase inhibitor is gelatinase As detected using an activity assay, it is adsorbed in an amount sufficient to be detected using the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9, and has an acid isoelectric point in dairy products. The main protein is not absorbed; Eluting from cation exchange using a suitable buffer; The eluate is subjected to a concentration step and a diafiltration step to remove salts therefrom; An acid source is provided; A composition comprising an inhibitor of a metalloproteinase may comprise sequentially processing dairy products that cause excessive acidification.

In a preferred form of the invention, the acidified permeate or retentate is subjected to gel filtration chromatography. Inactive proteins are removed using molecular sieve chromatography or ultrafiltration under acidic conditions. For example, a molecular sieve chromatography process is used to obtain a composition comprising a metalloproteinase inhibitor with a molecular weight of 21,000 Da. In a preferred form, the molecular sieve chromatography resin has a molecular weight of 10 kDa to 500 kDa. Even more preferably, the molecular sieve chromatography resin is a Cellufine 1000-M resin having a molecular weight of 10 kDa to 500 kDa.

The suitability of the molecular sieve chromatography resin is such that a milk secretion or derivative thereof is passed through the test resin column at neutral pH or other defined pH favoring separation and using gelatinase activity assays or similar assays available in the prior art. It can evaluate by measuring the ratio of a metalloproteinase inhibitor, especially TIMP-2.

Metalloproteinase inhibitors can be eluted from the column using a suitable eluate buffer as described in the prior art. Suitable eluate buffers include 150 mM NaCl, 10 mM HCl, pH 2.0.

In a very preferred form of the invention, first, a filtration step is used to remove insoluble matter therefrom; Adjusting the pH of the filtrate to about 6.5 to 8.0; The filtrate was contacted with a cation exchange resin, and the metalloproteinase inhibitor having a basic to neutral isoelectric point, and more preferably TIMP-2 at isoelectric point 7.0, the activity of the metalloproteinase inhibitor was gelatinase As detected using an activity assay, the adsorbate or derivative thereof is adsorbed in an amount sufficient to be detected using the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9. Processing sequentially. Main proteins with acidic isoelectric points in dairy products are not absorbed; Eluting from cation exchange using a suitable buffer; The eluate is subjected to a concentration step and a diafiltration step to remove salts therefrom; A source of acid is provided; Compositions comprising inhibitors of metalloproteinases are subjected to excessive acidification; The composition is contacted with a suitable molecular sieve chromatography resin such that the metalloproteinase inhibitor having a basic to neutral isoelectric point, and even more preferably TIMP-2 at isoelectric point 7.0, the activity of the metalloproteinase inhibitor is gelatinous. As detected using a tinase activity assay, the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9 to be enriched in an amount sufficient to be detected; Elution from the molecular sieve chromatography resin using a suitable buffer yields a composition further comprising an inhibitor of metalloproteinases.

In a preferred process, the fraction from the gel filtration chromatography step comprising a metalloproteinase inhibitor is subjected to anion exchange chromatography. Suitable anion exchange resins are metalloproteinase inhibitors, preferably metalloproteinase inhibitors having a basic to neutral isoelectric point, and even more preferably TIMP-2 at isoelectric point 7.0 is a metalloproteinase inhibitor. To be adsorbed in an amount sufficient to be detected using the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9 as detected using gelatinase activity assays. Will have the ability. Preferably, the anion exchange resin is a Q-Sepharose Fast Flow resin.

The suitability of the anion exchange resin is such that a lactate or derivative thereof is passed through a column of test resin at a neutral pH or other defined pH favoring adsorption or separation, and a gelatinase activity assay or similar assay available in the prior art. Can be evaluated by measuring the proportion of metalloproteinase inhibitors, in particular TIMP-2.

Compositions comprising inhibitors of metalloproteinases can be eluted from the resin using a suitable elution buffer. For example, a linear salt gradient of 0-0.5 M NaCl in 20 mM Tris-HCl can be used.

As a further example, a process for the preparation of a composition comprising an inhibitor of a metalloproteinase comprises first removing a insoluble material therefrom using a filtration step; Adjusting the pH of the filtrate to about 6.5 to 8.0; The filtrate was contacted with a cation exchange resin, and the metalloproteinase inhibitor having a basic to neutral isoelectric point, and more preferably TIMP-2 at isoelectric point 7.0, the activity of the metalloproteinase inhibitor was gelatinase As detected using an activity assay, it is adsorbed in an amount sufficient to be detected using the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9, and has an acid isoelectric point in dairy products. The main protein is not absorbed; Eluting from cation exchange using a suitable buffer; The eluate is subjected to a concentration step and a diafiltration step to remove salts therefrom; A source of acid is provided; Compositions comprising inhibitors of metalloproteinases are subjected to excessive acidification; The composition is contacted with a suitable molecular sieve chromatography resin such that a metalloproteinase inhibitor having a basic to neutral isoelectric point, and even more preferably TIMP-2 at isoelectric point 7.0, exhibits a reduced activity of the metalloproteinase inhibitor. As detected using a tinase activity assay, the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9 to be enriched in an amount sufficient to be detected; Eluting from the molecular sieve chromatography resin using a suitable buffer to obtain a composition further comprising an inhibitor of metalloproteinases; The composition is contacted with a suitable anion exchange chromatography resin so that a metalloproteinase inhibitor having a basic to neutral isoelectric point and, more preferably, TIMP-2 at isoelectric point 7.0, the activity of the metalloproteinase inhibitor is gelatinous. As detected using a tinase activity assay, the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9 is used to adsorb in an amount sufficient to be detected; Elution using an appropriate buffer from anion exchange chromatography resin to sequentially process the lactation or derivative thereof to obtain a composition comprising an inhibitor of metalloproteinases.

In a preferred embodiment, fractions from an anion exchange chromatography step comprising a metalloproteinase inhibitor are subjected to affinity chromatography. Preferably, the affinity chromatography column is a heparin-based affinity column.

Suitable affinity chromatography resins include metalloproteinases of metalloproteinase 2 and metalloproteinase 9, as the activity of the metalloproteinase inhibitor is detected using a gelatinase activity assay. In an amount sufficient to be detected using an inhibitory activity, a metalloproteinase inhibitor, preferably a metalloproteinase inhibitor having a basic to neutral isoelectric point, and even more preferably TIMP-2 at isoelectric point 7.0 Has the ability to adsorb. Preferably, the affinity chromatography resin is heparin affinity resin, and even more preferably the resin is Hi-trap heparin Sepharose resin (Amersham Pharmacia Biotech).

The suitability of affinity chromatography resins allows the dairy product to pass through a column of test resins at neutral pH or other defined pH favoring adsorption or separation, using gelatinase activity assays or similar assays available in the prior art. It can evaluate by measuring the ratio of a metalloproteinase inhibitor, especially TIMP-2.

Compositions comprising inhibitors of metalloproteinases can be eluted from the resin using a suitable elution buffer. For example, a linear salt gradient of 0.1-1.0 M NaCl in 20 mM Tris-HCl can be used. For example, a process for preparing a composition comprising an inhibitor of a metalloproteinase may include applying a dairy product to a filtration step to remove insoluble matter therefrom; Adjusting the pH of the filtrate to about 6.5 to 8.0; The filtrate was contacted with a cation exchange resin such that the metalloproteinase having a basic to neutral isoelectric point, and even more preferably TIMP-2 at isoelectric point 7.0, the activity of the metalloproteinase inhibitor was gelatinase activity. As detected using an assay, a main body having an acid isoelectric point in the dairy product adsorbed in an amount sufficient to be detected using the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9 Protein is not absorbed; Eluting from cation exchange using a suitable buffer; The eluate is subjected to a concentration step and a diafiltration step to remove salts therefrom; An acid source is provided; Compositions comprising inhibitors of metalloproteinases are excessively acidified; The composition is contacted with a suitable molecular sieve chromatography resin such that a metalloproteinase inhibitor having a basic to neutral isoelectric point, and even more preferably TIMP-2 at isoelectric point 7.0, exhibits a reduced activity of the metalloproteinase inhibitor. As detected using a tinase activity assay, the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9 to be enriched in an amount sufficient to be detected; Eluting from the molecular sieve chromatography resin using a suitable buffer to obtain a composition further comprising an inhibitor of metalloproteinases; The composition is contacted with a suitable anion exchange chromatography resin so that a metalloproteinase inhibitor having a basic to neutral isoelectric point and, more preferably, TIMP-2 at isoelectric point 7.0, the activity of the metalloproteinase inhibitor is gelatinous. As detected using a tinase activity assay, the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9 is used to adsorb in an amount sufficient to be detected; The anion exchange chromatography resin is eluted using a suitable buffer to obtain a composition comprising an inhibitor of a metalloproteinase; The composition is contacted with a suitable affinity chromatography resin such that a metalloproteinase inhibitor having a basic to neutral isoelectric point, and more preferably TIMP-2 at isoelectric point 7.0, exhibits a reduced activity of the metalloproteinase inhibitor. As detected using a tinase activity assay, the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9 is used to adsorb in an amount sufficient to be detected; Elution from an affinity chromatography resin using a suitable buffer may comprise sequentially processing dairy products to obtain compositions comprising inhibitors of metalloproteinases.

Preferably, the fraction from the affinity chromatography step is subjected to reverse phase high performance liquid chromatography. Suitable reversed phase high performance liquid chromatography resins are metalloproteinase inhibitors, preferably metalloproteinase inhibitors having basic to neutral isoelectric points, and even more preferably TIMP-2 at isoelectric point 7.0, As the activity of the inhibitor is detected using a gelatinase activity assay, adsorption in an amount sufficient to be detected using the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9 You can. Preferably, the reverse phase high performance liquid chromatography resin is selected from the group comprising a C4, C8 or C18 matrix. Even more preferably, the reverse phase high performance liquid chromatography resin is a C4 RP-HPLC column.

The suitability of reversed phase high performance liquid chromatography resins can be achieved by passing a dairy product through a column of test resin and using a gelatinase activity assay or a similar assay available in the prior art, to a metalloproteinase inhibitor, in particular TIMP-2. It can evaluate by measuring the ratio of.

Compositions comprising inhibitors of metalloproteinases can be eluted from the resin using a suitable elution buffer. For example, a 0-28% CH ₃ CN gradient can be used.

A process for preparing a composition comprising an inhibitor of a metalloproteinase comprises the steps of applying a dairy product to a filtration step to remove insoluble matter therefrom; Adjusting the pH of the filtrate to about 6.5 to 8.0; The filtrate was contacted with a cation exchange resin to obtain a metalloproteinase having a basic to neutral isoelectric point, and even more preferably TIMP-2 having an isoelectric point of 7.0, wherein the activity of the metalloproteinase inhibitor was gelatinase activity. As detected using an assay, the metalloproteinase 2 and metalloproteinase inhibitory activities of metalloproteinase 9 allow for adsorption in an amount sufficient to be detected, thereby having an acidic isoelectric point in the dairy product. The main protein is not absorbed; Eluting from cation exchange using a suitable buffer; The eluate is subjected to a concentration step and a diafiltration step to remove salts therefrom; A source of acid is provided; Compositions comprising inhibitors of metalloproteinases are subjected to excessive acidification; The composition is contacted with a suitable molecular sieve chromatography resin so that the metalloproteinase inhibitors having a basic to neutral isoelectric point, and even more preferably TIMP-2 at isoelectric point 7.0, have the activity of the metalloproteinase inhibitor. To be enriched in an amount sufficient to be detected using the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9, as detected using gelatinase activity assays; Eluting from the molecular sieve chromatography resin with a suitable buffer to obtain a composition comprising an inhibitor of metalloproteinases; The composition is contacted with a suitable anion exchange chromatography resin so that the basic to neutral isoelectric metalloproteinase inhibitors, and more preferably TIMP-2 at isoelectric point 7.0, the activity of the metalloproteinase inhibitors gelatin Adsorption in an amount sufficient to be detected using the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9 as detected using the first activity assay; The anion exchange chromatography resin is eluted using a suitable buffer to obtain a composition comprising an inhibitor of a metalloproteinase; The composition is contacted with a suitable affinity chromatography so that a metalloproteinase inhibitor having a basic to neutral isoelectric point and, more preferably, TIMP-2 at isoelectric point 7.0, the activity of the metalloproteinase inhibitor is gelatin Adsorbed in an amount sufficient to be detected using the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9 as detected using the first assay; The composition is contacted with a suitable reversed phase high performance liquid chromatography resin to provide a basic to neutral isoelectric metalloproteinase inhibitor and, more preferably, TIMP-2 at isoelectric point 7.0, wherein the activity of the metalloproteinase inhibitor is gelatin. As detected using a tinase activity assay, the metalloproteinase inhibitory activity of metalloproteinase 2 and metalloproteinase 9 is used to adsorb in an amount sufficient to be detected; Elution from a suitable reverse phase high performance liquid chromatography resin using a suitable buffer may comprise the continuous processing of the dairy product obtained by the composition further comprising an inhibitor of metalloproteinases.

Preferably the treatment step is ultrafiltration. Methods for preparing compositions comprising inhibitors of selective metalloproteinases include, for example, providing a source of dairy products, a suitable ultrafiltration membrane; Contacting the dairy product with a suitable ultrafiltration membrane to selectively enhance the filtrate's ability to inhibit metalloproteinases.

The inventors have surprisingly found that metalloproteinase inhibitors are present in dairy products with substantially enhanced TIMP-2 polypeptide activity. Based on the teachings herein, as biological and structural contributions of TIMP-2 are widely published in the scientific art, a composition comprising an inhibitor of a metalloproteinase using a suitable ultrafiltration membrane and using a suitable ultrafiltration Obtaining only requires only routine experimentation. Examples 1A, 1B, 2 and 3 demonstrate to the person skilled in the art the parameters of the physical properties of TIMP-2 and the behavioral properties of TIMP-2 according to various environmental conditions. Importantly, the teachings herein provide the solubility of TIMP-2, the charge and isoelectric point and protein size of TIMP-2 in various buffers and solvents. Using the teachings in the specification and a high level of knowledge in the art, those skilled in the art will purify TIMP-2 using a variety of combinations to easily select and use suitable ultrafiltration membranes with a high likelihood of success.

Suitable ultrafiltration membranes include dairy products as metalloproteinase inhibitors, preferably basic to neutral isoelectric metalloproteinase inhibitors, and even more preferably TIMP-2 at isoelectric point 7.0, as metalloproteinase inhibitors. As activity is detected using gelatinase activity assays, the ability to enhance the metalloproteinase 2 and metalloproteinase 9 activity to a sufficient amount to be detected using the metalloproteinase inhibitory activity. Have

The suitability of the ultrafiltration membrane allows the dairy product to pass through the ultrafiltration membrane at a defined pH, which is advantageous for separation, gelatinase activity assay, migration as a single band of about 21,000 Da according to SDS-PAGE, N-terminal sequence analysis or mass spectroscopy Assays or similar assays available in the prior art can be used to measure the proportion of metalloproteinase inhibitors, in particular TIMP-2. Preferably, the ultrafiltration membrane has a molecular weight range suitable for separating TIMP-2.

In a preferred embodiment, the treating step comprises purifying the lactation or derivative thereof through centrifugation and applying the obtained product indefinitely to a suitable affinity chromatography resin, such as a heparin affinity resin, even more preferably a resin. Is Hi-trap heparin Sepharose resin (Amersham Pharmacia Biotech). Compositions comprising inhibitors of metalloproteinases can be eluted from the resin using a suitable elution buffer. For example, a linear salt gradient of 0.1-1.0 M NaCl in 20 mM Tris-HCl can be used. The eluate can be subjected to a concentration step and a diafiltration step to remove salts therefrom, thereby obtaining a composition comprising an inhibitor of metalloproteinases.

As used herein, the term "comprising" means "including but not limited to" and the term "comprising" has a corresponding meaning.

The invention is now described in more detail with reference to the following examples. Examples 6 to 9 are prediction examples. However, the following detailed description is for illustrative purposes only and should be understood as never limiting the generality of the invention.

Example 1A

Method for producing a composition with enhanced metalloproteinase inhibitory activity

Step 1: Cation Exchange Chromatography

The pasteurized whey obtained as the final product of the cheese making was filtered through a 10 micron screen and a 0.2 micron Sartorou Microsart Sartocon II module to remove solids. The ultrafiltrate was adjusted to pH6.5 and applied to a column of S-Sepharose Fast Flow S cation exchange resin (Pharmacia) equilibrated with 50 mM sodium citrate buffer at pH6.5. After washing the column with the same buffer, the absorbed material was eluted using 1M NaCl solution containing 0.25M NH ₄ OH. This eluate was diafiltered against water until the conductivity reached 0 Tg and then concentrated by ultrafiltration; Both were treated using 3kDa-exclusion membranes. The resulting formulation was stored by lyophilization.

Step 2: transient acidification and gel filtration chromatography (molecular sieve chromatography)

A 1 L composition (total protein concentration = 40 mg.mL ⁻¹ ) with enhanced metalloproteinase inhibition properties prepared as described above was acidified to pH 2.0 overnight using HCl, filtered through a 1 μm membrane, Apply two 10 L Cellulpin 1000-M columns (molecular weight range of 10-500 kDa) (Amicon) connected in series at a flow rate of 60 mL.min ⁻¹ . Protein using 150mM NaCl, 10mM HCl, pH2.0, and eluted at a flow rate of 60mL.min ^-1. 450 mL fractions were collected and analyzed for metalloproteinase inhibitory activity using protein concentration (BCA assay) and enzyme immunoassay (EIA) (see below) (FIG. 1A). Aliquots of each fraction (20 μg) with enhanced metalloproteinase inhibition properties are also allowed to flow on a 4-20% reduced tris-glycine gel, transferred to nitrocellulose, and monoclonal anti-TIMP-2 The antibody was blotted for metalloproteinase inhibitory activity to confirm that bovine TIMP-2 was present at the correct molecular weight in the EIA-positive fraction (FIG. 1A, insertion).

Step 3: Anion Exchange Chromatography

The pooled cellulphine fraction was made to 50 mM NaCl, 20 mM Tris-HCl, pH8.0 using solid base Tris, adjusted to pH8.0 with 5M HCl, filtered through a 1 μm membrane, and FPLC system ( Pharmacia, Swede) is applied to a Q-Sepharose fast flow column (5 × 15 cm, Pharmacia, Sweden) at a flow rate of 5 mL · min ⁻¹ . The column was then washed with 20 mM Tris-HCl and the residual protein bound to the column (including metalloproteinase inhibitors) was washed with 15 mL min using a 2.1 L linear salt gradient of 0-0.5 M NaCl in 20 mM Tris-HCl. ^{Elute at} a flow rate of ^-1 . 30 mL fractions are collected and analyzed for protein concentration and metalloproteinase inhibitors (FIG. 1B). Subsequently, the fractions having metalloproteinase inhibitory activity are pooled.

Step 4: Affinity Chromatography

The pooled Q-Sepharose fast flow fraction is applied at a flow rate of 2 mL.min ^-1 to a Hi-trap heparin Sepharose column (series of 5 mL columns; Pharmacia, Sweden) attached to an FPLC system (Pharmacia, Sweden) .

The column is then washed with 0.1 M NaCl, 20 mM Tris-HCl, pH 8.0, and the residual protein bound to the column (including the metalloproteinase inhibitory fraction) is 75 mL linear of 0.1-1.0 M NaCl in 20 mM Tris-HCl. Elute at a flow rate of 15 mL.min ⁻¹ using a salt gradient. 1.0 mL fractions are collected and analyzed for protein concentration and metalloproteinase inhibitory activity (FIG. 1C). Subsequently, the fractions having metalloproteinase inhibitory activity are pooled.

Step 5: C4 RP-HPLC

The pooled Hi-trap Heparin Sepharose fraction was diluted 1: 2 using 0.1% TFA and delta-pack C4 RP-HPLC column (15 μm, 300 μs, 25 × 100 mm, Millipore-Waters) equilibrated with 0.1 TFA Waters), Lane Cove, New South Wales, Australia). After washing the column thoroughly with 0.1% TFA, the bound protein was 5 mL using a gradient of 0-28% CH ₃ CN over 25 minutes and a gradient of 28-36% CH ₃ CN in 0.08% TFA over 90 minutes. Eluate sequentially with a flow rate of .min ^-1 . 5 mL fractions are collected and assayed for metalloproteinase inhibitory activity (FIG. 1D). Fractions with metalloproteinase inhibitory activity are pooled and lyophilized.

The metalloproteinase inhibitory activity is present in the positive fraction from the final RP-HPLC step in the form of bovine TIMP-2 of 99% purity or less (including 99% purity) and has a molecular weight of about 21 kDa. These criteria were Coomassie stained reduced 10-20% gradient Tricine SDS-PAGE gel (see FIG. 2), analytical RP-HPLC (FIG. 3), mass spectrometry analysis (FIG. 4) and the N-terminus Inferred from sequence analysis.

TIMP-2 Enzyme Immunoassay

Individual wells of 96-well immunoplates (Nunc) are coated overnight at 4 ° C. using 90 μL of each sample (column fraction) in 15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH9.6. The plate is then washed four times with 100 μl of 0.05% Tween-20 in PBS and blocked for 1 hour at 37 ° C. in 1% BSA in PBS. Plates are washed four times with 0.05% Tween-20 in PBS and incubated at 37 ° C. for 1 hour using ¹ μg.mL ⁻¹ mouse anti-human TIMP-2 monoclonal IgG (Chemicon). Plates are washed again four times with 100 μl of 0.05% Tween-20 in PBS and incubated for 1 hour at 37 ° C. using both anti-mouse HRP conjugated IgG (1: 10000 dilution). Plates were then washed again four times with 0.05% Tween-20 in PBS and 200 μL of 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate per well. It develops at 37 degreeC for 1 hour, and absorbance is measured at 405 nm. If desired, EIA is developed using 200 μL of o-phenylenediamine dihydrochloride (OPD) and the absorbance is measured at 490 nm.

Example 1B

Selective preparation of compositions with enhanced metalloproteinase inhibitory activity

Centrifuge at 4C for 30 minutes at 41,000xg to determine the volume of reconstituted skim milk powder (reconstituted), or skim colostrum, which was originally burned in water. The identified feedstock is loaded at a flow rate of 5 mL · min ⁻¹ on a HiPrep 16/10 heparin FF column (16 × 100 mm, Pharmacia, Sweden) attached to an AKTA FPLC system (Pharmacia, Sweden). After washing the column with 10 mM sodium phosphate buffer, the residual protein bound to the column (including the metalloproteinase inhibitory fraction) was purified using 10 mM sodium phosphate buffer containing 2M NaCl, pH7.0, at 5 mL.min- ¹ . Eluate sequentially at flow rate. The eluted material is loaded on a HiPrep desalting column (Pharmacia, Sweden) to remove salt from the bound material and concentrated in Amicon ultrafiltration cells using a BioMax membrane (Millipore Corporation, Bedford, Mass.) With a 5,000 molecular weight. do. The composition is assayed for protein concentration and metalloproteinase inhibitory activity quantified by the method of Example 3 (FIG. 2 (b)).

Example 2

N-terminal Sequence Analysis of Metalloproteinase Inhibitors

N-terminal sequence analysis confirmed the identity of the amino acid sequence and thus the metalloproteinase inhibitor. 20 μg of purified metalloproteinase inhibitor was resuspended in 50 mM Tris-Cl pH8.5, 6M guanidine-HCl, 10 mM dithiothreitol (DTT) and incubated at 65 ° C. for 30 minutes. The denatured and reduced samples were then cooled to room temperature, alkylated with the addition of 40 mM iodoacetamide and incubated for 30 minutes. The sample is then acidified by addition of trifluoroacetic acid (TFA) and sequenced from the N-terminus by Edman degradation using a Hewlett-Packard G1000A protein sequencer according to the manufacturer's instructions. Check it. The N-terminal sequence (8 cycles) was as follows:

CSCSPVHP

Example 3

Metalloproteinase Inhibitory Activity of the Composition

The activity of metalloproteinase inhibitors was determined using a metalloproteinase gelatinase activity assay kit (Chemicon). This assay uses a biotinylated gelatinase substrate, which is cleaved using active MMP-2 and MMP-9 (gelatinase) enzymes. Degradation of the noninotinylated gelatinase substrate results in a fragment with a low biotin label capable of binding to a proprietary 96-well plate specific for the substrate. Bound fragments can then be detected using streptavidin-enzyme complex, which produces a colored product upon detection of the enzyme substrate (detectable at 405 nm). Therefore, the greater the gelatinase activity, the lower the OD, while the gelatinase activity is inhibited, the higher the absorbance (Fig. 5). P-aminophenylmercuric acetate (p-APMA) activated MMP-2 (6.7 ng) in TNC buffer (50 mM Tris-Cl pH7.5, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij-35) alone Or pre-incubate at 37 ° C. for 30 minutes with pooled fractions (10 μl) from each step of purification. A biotinylated gelatinase substrate is then added to each sample, incubated for 30 minutes at 37 ° C. and then MMP activity is measured according to the manufacturer's instructions. As shown in Figure 6, in the absence of MMP-2, the gelatinase substrate remains undigested (high absorbance), but in the presence of MMP-2 the substrate is degraded and low absorbance is observed. Pre-culture of MMP-2 with a metalloproteinase inhibitor-positive fraction from each step of purification clearly demonstrates the inhibitory effect of the metalloproteinase inhibitor on metalloproteinase activity. As expected, Fraction 33 from a Cellulpin column without a metalloproteinase inhibitor did not inhibit metalloproteinase activity (see FIG. 1), whereas Fraction 30 with a metalloproteinase inhibitor was shown. Was successful.

The metalloproteinase inhibitor activity of the milk extract compositions obtained from the various fraction pools of the purification methods summarized in Examples 1A and 1B was also determined using metalloproteinases (Peptide Institute, Osaka, Japan) using fluorescent-quenching substrates. Quantification Degradation of this substrate with metalloproteinases releases quencher particles from the proximal portion of the fluorescent tag, increasing fluorescence. Thus, increasing the amount of metalloproteinase inhibitor will reduce fluorescence. 1.5 μg / mL recombinant human MMP-2 was activated at 37 ° C. for 1 hour using 1 mM p-aminophenylmercuric acid (p-APMA). The pooled fractions assayed were generally diluted 1: 300 with Buffer E (0.1M Tris, 0.1M NaCl, 0.1M CaCl ₂ , 0.0% Brij-35), and 0 to 0 in buffer E 40 μl activated MMP-2. After a standard curve was obtained using 0.15 μg / mL recombinant human TIMP-2 (R & D Systems, Minneapolis, USA), 40 μl of each sample was plated into each well of a 96-well tray, which was buffered using 270 using Buffer E. Made to a total volume of μL / well. These samples were then incubated for 1 hour at RT, then 30 μl / well 16 μM fluorescent substrate was added and incubated at RT for 30-45 minutes. The reaction was stopped using 30 μl 200 mM EDTA and fluorescence was measured on a Wallac 1420 multilable counter (Perkin Elmer, Finland) (excitation / emission λ = 340-390 nm). The results from this experiment are shown in Table 2 below (“MMI” represents a metalloproteinase inhibitor and is subsequently identified as TIMP-2).

Example 4

Suitable pharmaceutical or veterinary compositions with enhanced metalloproteinase inhibitory activity

All units of components of the composition are measured in "parts."

The amount of milk product extract specified below is represented by "qs".

(i) saline solution

Active substance metalloproteinase inhibitor qs

Sodium chloride 0.9

Up to 100 deionized water

After the ingredients are thoroughly mixed, the pH of the composition is adjusted to 3.8-4.2 by addition of 10 mM HCl.

(ii) Setomacrogol cream

Active substance metalloproteinase inhibitor qs

Seto macrogol oil painting wax 15

Liquid Paraffin (Weight) 10

Chlorocresol 0.1

Propylene Glycol 5

Up to 100 distilled water

The cetomacrogol emulsifying wax dissolves with paraffin at about 70 ° C. Chlorocresol and propylene glycol are dissolved in about 50 parts of distilled water warmed to the same temperature. After mixing, the composition is weighted and stirred to cool. The milk product extract is then added to a suitable concentration and mixed thoroughly.

(iii) aqueous cream APF

Active substance metalloproteinase inhibitor qs

Oil Painting Ointment 30

Glycerol 5

Phenoxyethanol 1

Up to 100 distilled water

Emulsion ointment is dissolved at about 70 ° C. Phenoxyethanol is dissolved in distilled water warmed to the same temperature. The composition is mixed, weighted, stirred and cooled. The milk product extract is then added with thorough stirring.

(iv) buffered cream BPC 73

Active substance metalloproteinase inhibitor qs

Citric acid 5

Sodium Phosphate 25

Chlorocresol 1

Oil painting ointment 300

Distilled Water 669

After emulsifying ointment is dissolved with mild heat, sodium phosphate, citric acid and chlorocresol, which have been previously dissolved in distilled water at the same temperature, are added. The composition is stirred slowly until it cools down. The milk product extract is then added and mixed thoroughly.

(v) emulsifying ointment APF

Active substance metalloproteinase inhibitor qs

Oil wax 30

White Soft Paraffin 50

Liquid Paraffin (Weight) 20

The wax and paraffin are dissolved together and stirred until cooled. The milk product extract is then added to some of the bases to an appropriate concentration, gradually adding the rest, and then thoroughly mixed.

(vi) peptide ointment (as in neomycin and Bacitracin ointment BPC 73)

Active substance metalloproteinase inhibitor qs

Liquid Paraffin 10

Up to 100 white soft paraffin

Dissolve the white soft paraffin and add liquid paraffin. The mixture is stirred until it is cooled. The milk product extract is titrated with part of the base and added gradually to the rest of the base.

(vii) gel (when used in Lignocaine and Chlorhexidine gel APF)

Active substance metalloproteinase inhibitor qs

Tragacanth 2.5

Glycerol 25

Up to 100 distilled water

Tragacanth is mixed with most of glycerol and distilled water. After boiling, the mixture is cooled and lactate extract is added. The composition is weighted and mixed well. The finished product is protected from light.

(viii) spray (when used with adrenaline and atropine spray BPC 73)

Active substance metalloproteinase inhibitor qs

Sodium Metabisulfite 1

Chlorbutol 5

Propylene Glycol 50

Distilled water up to 1000

(ix) spray (when used in Indospray)

Active substance metalloproteinase inhibitor qs

Alcohol 95%

(x) lotion (when used with aminobenzoic acid lotion BPC73)

Active substance metalloproteinase inhibitor qs

Glycerol 20

Alcohol 95% 60

Up to 100 distilled water

(xi) Seto Macrogol Lotion APF

Active substance metalloproteinase inhibitor qs

Seto macrogol oil wax 3

Liquid Paraffin 10

Glycerol 10

Chlorhexidine Gluconate Solution 0.1

Up to 100 distilled water

The cetomacrogol emulsifying wax is dissolved at about 60 ° C. with liquid paraffin. To this mixture, a chlorhexidine solution previously diluted to 50 parts with distilled water at the same temperature is added with rapid stirring. After mixing, the composition is volume adjusted and stirred until cooled.

(xii) gargle

Active substance metalloproteinase inhibitor qs

Polyethylene glycol (7) -glycerol cocoenite 2

Glycerin (86%) 18

Peppermint Oil 10

Ethanol 55

Distilled Water 13

The active ingredient is dissolved in distilled water. Polyethylene glycol (7) -glycerol cocoate and glycerin are added and dissolved in solution. Dissolve the peppermint oil in ethanol and mix the two solutions with stirring. Dilute the solution to 1:10 before use.

(xiii) toothpaste

Active substance metalloproteinase inhibitor qs

Methyl Cellulose 0.8

Calcium Carbonate 30

Colloidal silica 3

Light liquid paraffin 2

Glycerin 20

Distilled water 100

The powder is moistened with methyl cellulose and active ingredient mixture, paraffin and glycerin in some distilled water. Mix with the remaining water and then homogenize the paste.

(xiv) parenteral solution components

Active substance metalloproteinase inhibitor qs

+/- human serum albumin 2

Arginine or Glycine 40

+/- Carbohydrate 40

Carbohydrates are glucose, mannose, dextrin, hydroxyethyl starch or mixtures thereof. pH is adjusted with phosphate, succinate, amino acids or mixtures thereof.

Example 5

Effectiveness as a treatment for chronic wounds of compositions derived from dairy products with enhanced properties that inhibit metalloproteinases

The present invention can be used to prevent and treat damage associated with surface wounds.

Metalloproteinases are generally not expressed in normal tissues but are upregulated in ulcerative and chronically inflamed tissues when tissue repair or remodeling is required. Chronic wounds are open wounds that include long term tissue loss. Burns are considered chronic wounds as skin ulceration due to pressure and problems with internal circulation leading to inadequate blood flow and tissue death. These wounds are characterized by excessive matrix metalloproteinases that prevent effective wound healing and closure. Metalloproteinase inhibitors, and in particular TIMP-2, inhibit these matrix metalloproteinases. In order to successfully test the effectiveness of TIMP-2 to aid wound healing, a suitable animal model is needed that reflects the characteristics of chronic wounds in human patients. Typical characteristics are as follows:

1) Chronic wounds have higher levels of MMP-9 and fewer ranges of MMP-2 than normal wounds, as determined by zymogram analysis (Trengrove et al., Wounde Repair Regen. 1999 Nov-Dec; 7 (6): 442-52);

2) MMP-1 (collagenase 1) and MMP-9 mRNA levels increase immediately after wound development, followed by an increase in MMP-2 mRNA expression (Soo et al. Plast Reconstr Surg. 2000 Feb; 105 (2) : 638-47);

3) MMP-2 and MMP-9 levels increase 5-10 fold in fluid from chronic leg ulcers (Wysocki et al, J Invest Dermatol. 1993 Jul; 101 (1): 64-8);

4) In chronic wounds, zymograms demonstrate an increase in gelatinase activity (MMP-2 and MMP-9), including low molecular weight proteinase species that may indicate activated or overactivated gelatinase fragments. Bullen et al, J Invest Dermatol. 1995, Feb; 104 (2): 236-40.

A rat model of chronic wounds developed by Chen et al (1999) was used to demonstrate the effectiveness of a dairy product with enhanced metalloproteinase inhibition properties as a treatment for chronic wounds. In this model, full thickness wounds were made on the back of a male rat (Sprague-Dawley rat) using a 6 mm skin biopsy punch in an electric drill (normal wound). Wound pairs were made at the proximal, median and distal. To make an ischemic wound, two parallel incisions were made along the wound side, the skin flap was lifted, repositioned and sutured. The healing of ischemic wounds in the central position was much slower than in normal wounds (FIG. 7B). In contrast, the healing rate in the proximal and distal is much faster than in the central position, and the ischemic effect is apparently lowest at the distal (FIGS. 7A & 7C). Distinguishing healing effects are thought to reflect well the total increase in MMP levels, which is mainly characterized by increased blood flow for different wound sites and increased in proMMP-9 and other unidentified gelatin degradation proteins.

Western immunoblot analysis was performed on pooled samples of normal (n = 3-5) and ischemic (N = 5-6) wounds at each wound site (FIG. 8). Levels of MMP-9 increased rapidly in ischemic wounds in the proximal and median and not in the distal region, reflecting the pattern seen in wound healing rates. Ischemic wounds demonstrated a much slower healing rate than normal wounds, and MMP-9 was upregulated in proximal and median ischemic wounds. This evidence suggests that rat ischemic wounds show typical features of chronic wounds seen in human patients.

Composition A with enhanced metalloproteinase inhibitory activity was obtained from whey to which steps 1 and 2 summarized in Example 1A were applied. The biologically active TIMP-2 component of Composition A was quantified using standard curves of purified TIMP-2 of fluorogen MMP inhibition summarized in Example 1A. Composition A included 10.82 μg / ml TIMP-2. Western immunoblot analysis of Composition A confirmed that TIMP-2 reactivity was a 22kDa form of TIMP-2 protein (FIG. 9).

Composition A was tested for the ability to promote ischemic wound healing in a rat chronic wound model. The test was conducted over 10 days with 8 rats per treatment group. On each day after surgery, wound size was measured by tracing around the wound on overhead transparency. The rats were then administered one dose (150 μl / wound) of pure composition A delivered in an alginate dressing. Ischemia regulating rats were administered sterile saline. After 10 days, Composition A treated rats had significantly improved healing compared to saline treated controls (FIG. 10).

Based on the results in Example 5 using a metalloproteinase inhibitor comprising TIMP-2 to improve the wound healing of chronic wounds, the inventors have found that the present invention used in this particular model is MMP-. It is expected to reduce MMP-9 activity, as measured by a Zymogram analysis of 9 (92 kDa) activity, to increase the rate of reduction of wounds and to reduce the time to functional recovery of the skin.

Example 6

Predictive Example Demonstrating a Methodology for Studying the Effectiveness of a Composition Enhancing the Inhibitory Properties of Metalloproteinases as a Treatment for Oral Mucositis

The present invention can be used to prevent and treat damage associated with mucositis. One skilled in the art can easily investigate the claimed invention for treating mucositis.

For example, investigations may be made of the effects of compositions enhanced on metalloproteinase inhibitory properties administered topically on chemotherapy-induced mucositis of Golden Syrian hamsters. In general, the test involves the continuous application of a composition with enhanced properties to inhibit metalloproteinases in the ball sacs of 10 hamsters treated with 5-fluorouracil.

The hamsters with similar mean weights are then divided into two groups of five. All hamsters are infused with 5-fluorouracil 90 mg / kg per day and 60 mg / kg intraperitoneally. On the 1st, 2nd and 3rd days, the ball patch is scraped six times in one direction with six strokes in the other orthogonal direction to obtain a uniform wound.

The group is treated with 0.3 ml of a commercial gargle as a vehicle or a composition with enhanced metalloproteinase inhibitory properties at a specified protein concentration. Firebag liquid treatment is applied daily for 1 minute, during which time the hamster is anesthetized with isofluran anesthesia. Ball bags are evaluated on days 5, 7, 8, 11, 13 and 15. Monitoring is based on a visual assessment (grading to size 1-10) of the ball bag taking into account the overall severity of the lesion, the degree of bruise, swelling and scarring. Body weight is reported as a percentage of the value on day 0.

Based on the results of Examples 1A, 1B, 2, 3 and especially the results of Example 5 showing the use of metalloproteinase inhibitors to treat wounds, the inventors have found that the invention used in this particular model The overall visual score, total ulcer area and weight loss are expected to reduce mucositis compared to vehicle treated groups when measured.

Example 7

Predictive Example Demonstrating Methodology for Studying the Effectiveness of Compositions with Enhanced Inhibitory Properties of Metalloproteinases to Reduce Bacterial Translocation of the Digestive Tract

The present invention is used to reduce bacterial potential in the digestive tract. One skilled in the art can readily investigate the claimed invention to reduce bacterial potential in the digestive tract.

The ability of the gut epithelium to provide a barrier to bacterial invasion provides an appropriate measure of mucosal repair and subsequent gut function.

For example, Sprague Dawley rats with high doses of chemotherapeutic agents are used as experimental models of damage to the lining of the digestive tract. Control rats were not treated with compositions with enhanced metalloproteinase inhibition properties, whereas experimental rats were treated with compositions with enhanced metalloproteinase inhibition properties for 5 days. Treated rats are fed a modified version of the composition with enhanced specific amount of metalloproteinase inhibitory properties in place of the same amount of casein. The treated rats were also administered the composition by gastric gavage on days 3, 4 and 5 of the experimental period. Control rats were fed unmodified and gavaged in the same protocol on the 3, 4 and 5 days of the same bovine serum albumin for the isisonitrogenous diet.

One group of control rats and treated rats are injected subcutaneously with 2.5 mg / kg methotrexate starting on days 1, 2 and 3. Additional controls are injected with simulated methotrexate and paired with methotrexate-injected controls.

Rats are kept in metabolism cages until blood is drawn on days 5, 8 and 12. The abdominal skin is soaked in 70% ethanol and then the intestines are removed under aseptic conditions. All visible mesenteric lymph nodes are placed in a sterile pre-weighed container and then the sample is weighed and the injection is added to a final concentration of 100 mg / ml. Tissues are homogenized in this solution using sterile glass-enhanced grinders. To determine the potential of Gram-negative bacteria to mesenteric lymphosomes, 40 or 60 mg of each tissue homogenate was placed on a MacConkey agar or blood agar plate and cultured aerobic bacteria for 48 hours at 35 ° C. The intestinal Gram negative bacterial colonies are then identified and counted using the API 20E strip. The frequency (% of animals showing detectable bacterial potential) and the average number of bacterial colonies per gram of tissue for each treatment group are calculated.

Based on the results of Examples 1A, 1B, 2 and 3, in particular the results of Example 5 showing the use of metalloproteinase inhibitors to treat wounds, the inventors have found that the present invention was used in this particular model. It is expected that the dislocation frequency will be lowered by this. We also expect that the number of colonies per gram of mesenteric lymph nodes in the treated group will be significantly lower.

Example 8

Predictive Example Demonstrating Methodology for Studying the Effectiveness of Compositions Enhancing the Inhibitory Properties of Metalloproteinases to Prevent Loss of Small Intestine Crypts and Villi of Rats Having Methotrexate-Induced Small Intestinal Mucositis

The present invention can be used to prevent the loss of small intestine crypts and villi of rats having methotrexate-induced small intestinal mucositis. One skilled in the art can investigate the claimed invention to prevent loss of small intestine crypts and villi of rats having methotrexate-induced small intestinal mucositis.

For example, high doses of chemotherapeutic agents and methotrexate are injected into Sprague Dawley rats as experimental models of the gut mucosa. In rats, methotrexate damages the small intestine but is not oral or colonic mucosa (Vanderhoof JA, Park JHY, Mohammapour H, Blackwood. Gastroenterology. 1990. 98: 1226-1231).

Rats weighed on average 140 g are kept in metabolic cages and fed a high carbohydrate diet. Control rats were not treated with a composition having a metalloproteinase activity, whereas treated rats were treated with a composition with a metalloproteinase inhibitor for 5 days. In addition, the treated rats were administered a composition having a metalloproteinase inhibitor by gastric gavage on the 3, 4 and 5 days of the experimental period. Control rats were fed unmodified and gavaged in the same protocol on the 3, 4 and 5 days of the same bovine serum albumin for the isisonitrogenous diet.

A group of control rats and test rats are injected subcutaneously with methotrexate 2.5 mg / kg starting on days 1, 2 and 3. Additional controls are injected with simulated methotrexate and paired with methotrexate-injected controls. Rats are kept in metabolic cages for five days and killed at this point to collect the gastrointestinal tract. Tissue samples are then collected from the proximal and distal small intestines. Tissue samples are fixed and stained for histological analysis using the method described in Read et al., J Endocrinol. 1992 133: 421-431, the entire disclosure of which is incorporated herein by reference.

Based on the results of Examples 1A, 1B, 2 and 3, in particular the results of Example 5 showing the use of metalloproteinase inhibitors to treat wounds, the inventors have found that the invention used in this particular model In detection of intact crypt area per unit area of the entire mucosa, we expect to reduce the loss of mucosal crypts in the proximal and distal small intestines compared to the control. The inventors also expect that the present invention used in this particular model will reduce the decrease in the length of villi compared to the control. In summary, the present invention was expected with reasonable expectation of success to prevent or promote the recovery of chemotherapy in the small intestine.

Example 9

Predictive Example Demonstrating Methodology for Studying the Effectiveness of a Composition Enhancing the Inhibitory Properties of Metalloproteinases as a Treatment for Gastric Ulcers

The present invention is used to prevent or treat damage associated with gastric ulcers induced by non-steroidal anti-inflammatory drug (NSAID) treatment. One skilled in the art can investigate the claimed invention to prevent or treat gastric ulcers induced by NSAID treatment.

For example, as an experimental model of NSAIDs-induced gastritis, Sprague Dawley rats with high doses of gavaged NSAIDs, indomethacin, are used. Control rats were not administered compositions with enhanced metalloproteinase inhibitory properties, whereas experimental rats received compositions with enhanced metalloproteinase inhibitory properties 72, 48, 24 hours or 30 minutes prior to indomethacin treatment. Prophylactic administration. Treated rats are supplied unmodified and are provided with a composition that enhances metalloproteinase inhibition properties in gastric gavage twice daily. Control rats were supplied unmodified and gavaged in the same volume of saline. Treated and control rats were fasted overnight and gavaged with 1 ml of indomethacin (100 mg / kg) to induce gastric ulcers. Both treated and control rats are lethal after 5 hours of indomethacin gavage to assess gastric ulcers.

The number, size, and area per unit mucosal surface area of gastric mucosal injury, induced by indomethacin treatment, and apoptosis or cell proliferation of the gastric epithelium are measured using techniques well known to those of skill in the art, between control and treatment groups In comparison, the reduction or prevention of gastric mucosal damage or the promotion of recovery is confirmed by the use of compositions with enhanced metalloproteinase inhibitory properties. Based on the results of Examples 1A, 1B, 2, 3 and 5, the inventors expect with reasonable expectation of success that the present invention reduces or prevents damage to the gastric mucosa or promotes recovery.

Finally, it should be understood that various modifications, changes and / or additions can be made without departing from the spirit of the invention as summarized herein.

<110> GROPEP LIMITED <120> METALLOPROTEINASE INHIBITORS <130> FPA07293 <150> AU PS 2820 <151> 2002-06-06 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 194 <212> PRT <213> Unknown <220> <223> BOVINE <400> 1 Cys Ser Cys Ser Pro Val His Pro Gln Gln Ala Phe Cys Asn Ala Asp 1 5 10 15 Ile Val Ile Arg Ala Lys Ala Val Asn Lys Lys Glu Val Asp Ser Gly 20 25 30 Asn Asp Ile Tyr Gly Asn Pro Ile Lys Arg Ile Gln Tyr Glu Ile Lys 35 40 45 Gln Ile Lys Met Phe Lys Gly Pro Asp Gln Asp Ile Glu Phe Ile Tyr 50 55 60 Thr Ala Pro Ala Ala Ala Val Cys Gly Val Ser Leu Asp Ile Gly Gly 65 70 75 80 Lys Lys Glu Tyr Leu Ile Ala Gly Lys Ala Glu Gly Asn Gly Asn Met 85 90 95 His Ile Thr Leu Cys Asp Phe Ile Val Pro Trp Asp Thr Leu Ser Ala 100 105 110 Thr Gln Lys Lys Ser Leu Asn His Arg Tyr Gln Met Gly Cys Glu Cys 115 120 125 Lys Ile Thr Arg Cys Pro Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp 130 135 140 Glu Cys Leu Trp Met Asp Trp Val Thr Glu Lys Asn Ile Asn Gly His 145 150 155 160 Gln Ala Lys Phe Phe Ala Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala 165 170 175 Trp Tyr Arg Gly Ala Ala Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu 180 185 190 Asp pro <210> 2 <211> 184 <212> PRT <213> Unknown <220> <223> HUMAN <400> 2 Cys Thr Cys Val Pro Pro His Pro Gln Thr Ala Phe Cys Asn Ser Asp 1 5 10 15 Leu Val Ile Arg Ala Lys Phe Val Gly Thr Pro Glu Val Asn Gln Thr 20 25 30 Thr Leu Tyr Gln Arg Tyr Glu Ile Lys Met Thr Lys Met Tyr Lys Gly 35 40 45 Phe Gln Ala Leu Gly Asp Ala Ala Asp Ile Arg Phe Val Tyr Thr Pro 50 55 60 Ala Met Glu Ser Val Cys Gly Tyr Phe His Arg Ser His Asn Arg Ser 65 70 75 80 Glu Glu Phe Leu Ile Ala Gly Lys Leu Gln Asp Gly Leu Leu His Ile 85 90 95 Thr Thr Cys Ser Phe Val Ala Pro Trp Asn Ser Leu Ser Leu Ala Gln 100 105 110 Arg Arg Gly Phe Thr Lys Thr Tyr Thr Val Gly Cys Glu Glu Cys Thr 115 120 125 Val Phe Pro Cys Leu Ser Ile Pro Cys Lys Leu Gln Ser Gly Thr His 130 135 140 Cys Leu Trp Thr Asp Gln Leu Leu Gln Gly Ser Glu Lys Gly Phe Gln 145 150 155 160 Ser Arg His Leu Ala Cys Leu Pro Arg Glu Pro Gly Leu Cys Thr Trp 165 170 175 Gln Ser Leu Arg Ser Gln Ile Ala 180 <210> 3 <211> 194 <212> PRT <213> Unknown <220> <223> HUMAN <400> 3 Cys Ser Cys Ser Pro Val His Pro Gln Gln Ala Phe Cys Asn Ala Asp 1 5 10 15 Val Val Ile Arg Ala Lys Ala Val Ser Glu Lys Glu Val Asp Ser Gly 20 25 30 Asn Asp Ile Tyr Gly Asn Pro Ile Lys Arg Ile Gln Tyr Glu Ile Lys 35 40 45 Gln Ile Lys Met Phe Lys Gly Pro Glu Lys Asp Ile Glu Phe Ile Tyr 50 55 60 Thr Ala Pro Ser Ser Ala Val Cys Gly Val Ser Leu Asp Val Gly Gly 65 70 75 80 Lys Lys Glu Tyr Leu Ile Ala Gly Lys Ala Glu Gly Asp Gly Lys Met 85 90 95 His Ile Thr Leu Cys Asp Phe Ile Val Pro Trp Asp Thr Leu Ser Thr 100 105 110 Thr Gln Lys Lys Ser Leu Asn His Arg Tyr Gln Met Gly Cys Glu Cys 115 120 125 Lys Ile Thr Arg Cys Pro Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp 130 135 140 Glu Cys Leu Trp Met Asp Trp Val Thr Glu Lys Asn Ile Asn Gly His 145 150 155 160 Gln Ala Lys Phe Phe Ala Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala 165 170 175 Trp Tyr Arg Gly Ala Ala Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu 180 185 190 Asp pro <210> 4 <211> 188 <212> PRT <213> Unknown <220> <223> HUMAN <400> 4 Cys Thr Cys Ser Pro Ser His Pro Gln Asp Ala Phe Cys Asn Ser Asp 1 5 10 15 Ile Val Ile Arg Ala Lys Val Val Gly Lys Lys Leu Val Lys Glu Gly 20 25 30 Pro Phe Gly Thr Leu Val Tyr Thr Ile Lys Gln Met Lys Met Tyr Arg 35 40 45 Gly Phe Thr Lys Met Pro His Val Gln Tyr Ile His Thr Glu Ala Ser 50 55 60 Glu Ser Leu Cys Gly Leu Lys Leu Glu Val Asn Lys Tyr Gln Tyr Leu 65 70 75 80 Leu Thr Gly Arg Val Tyr Asp Gly Lys Met Tyr Thr Gly Leu Cys Asn 85 90 95 Phe Val Glu Arg Trp Asp Gln Leu Thr Leu Ser Gln Arg Lys Gly Leu 100 105 110 Asn Tyr Arg Tyr His Leu Gly Cys Asn Cys Lys Ile Lys Ser Cys Tyr 115 120 125 Tyr Leu Pro Cys Phe Val Thr Ser Lys Asn Glu Cys Leu Trp Thr Asp 130 135 140 Met Leu Ser Asn Phe Gly Tyr Pro Gly Tyr Gln Ser Lys His Tyr Ala 145 150 155 160 Cys Ile Arg Gln Lys Gly Gly Tyr Cys Ser Trp Tyr Arg Gly Trp Ala 165 170 175 Pro Pro Asp Lys Ser Ile Ile Asn Ala Thr Asp Pro 180 185 <210> 5 <211> 195 <212> PRT <213> Unknown <220> <223> HUMAN <400> 5 Cys Ser Cys Ala Pro Ala His Pro Gln Gln His Ile Cys His Ser Ala 1 5 10 15 Leu Val Ile Arg Ala Lys Ile Ser Ser Glu Lys Val Val Pro Ala Ser 20 25 30 Ala Asp Pro Ala Asp Thr Glu Lys Met Leu Arg Tyr Glu Ile Lys Gln 35 40 45 Ile Lys Met Phe Lys Gly Phe Glu Lys Val Lys Asp Val Gln Tyr Ile 50 55 60 Tyr Thr Pro Phe Asp Ser Ser Leu Cys Gly Val Lys Leu Glu Ala Asn 65 70 75 80 Ser Gln Lys Gln Tyr Leu Leu Thr Gly Gln Val Leu Ser Asp Gly Lys 85 90 95 Val Phe Ile His Leu Cys Asn Tyr Ile Glu Pro Trp Glu Asp Leu Ser 100 105 110 Leu Val Gln Arg Glu Ser Leu Asn His His Tyr His Leu Asn Cys Gly 115 120 125 Cys Gln Ile Thr Thr Cys Tyr Thr Val Pro Cys Thr Ile Ser Ala Pro 130 135 140 Asn Glu Cys Leu Trp Thr Asp Trp Leu Leu Glu Arg Lys Leu Tyr Gly 145 150 155 160 Tyr Gln Ala Gln His Tyr Val Cys Met Lys His Val Asp Gly Thr Cys 165 170 175 Ser Trp Tyr Arg Gly His Leu Pro Leu Arg Lys Glu Phe Val Asp Ile 180 185 190 Val Gln Pro 195 <210> 6 <211> 8 <212> PRT <213> Unknown <220> <223> UNGULATE ANIMAL <400> 6 Cys Ser Cys Ser Pro Val His Pro 1 5 <210> 7 <211> 194 <212> PRT <213> Unknown <220> <223> UNGULATE ANIMAL <400> 7 Cys Ser Cys Ser Pro Val His Pro Gln Gln Ala Phe Cys Asn Ala Asp 1 5 10 15 Ile Val Ile Arg Ala Lys Ala Val Asn Lys Lys Glu Val Asp Ser Gly 20 25 30 Asn Asp Ile Tyr Gly Asn Pro Ile Lys Arg Ile Gln Tyr Glu Ile Lys 35 40 45 Gln Ile Lys Met Phe Lys Gly Pro Asp Gln Asp Ile Glu Phe Ile Tyr 50 55 60 Thr Ala Pro Ala Ala Ala Val Cys Gly Val Ser Leu Asp Ile Gly Gly 65 70 75 80 Lys Lys Glu Tyr Leu Ile Ala Gly Lys Ala Glu Gly Asn Gly Asn Met 85 90 95 His Ile Thr Leu Cys Asp Phe Ile Val Pro Trp Asp Thr Leu Ser Ala 100 105 110 Thr Gln Lys Lys Ser Leu Asn His Arg Tyr Gln Met Gly Cys Glu Cys 115 120 125 Lys Ile Thr Arg Cys Pro Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp 130 135 140 Glu Cys Leu Trp Met Asp Trp Val Thr Glu Lys Asn Ile Asn Gly His 145 150 155 160 Gln Ala Lys Phe Phe Ala Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala 165 170 175 Trp Tyr Arg Gly Ala Ala Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu 180 185 190 Asp pro

Claims (26)

A composition derived directly or indirectly from a mammalian lactation, comprising an inhibitor of a metalloproteinase. The method of claim 1, The metalloproteinases are (i) collagenase (metalloproteinase-1, 8 and 13); (ii) gelatinases A and B (metalloproteinase-2 and metalloproteinase-9); (iii) stromelysins 1 and 2 (metalloproteinases-3 and 10); (iv) matrilysin (MMP-7); Enamel lysine (MMP-20), macrophage metalloelasterase (MMP12), and MMP-19 and (v) membrane-type metalloproteinases (MT-MMP-1 to 4 and stromelysin-3, MMP -11) selected from the group consisting of. The method according to claim 1 or 2, The inhibitor is a composition, characterized in that the tissue inhibitor of metalloproteinases (TIMP). The method of claim 3, wherein Wherein said TIMP is a TIMP-2 polypeptide or a functional equivalent or fragment thereof. The method of claim 4, wherein Wherein said TIMP-2 has a molecular weight of about 21,000 Da and / or an isoelectric point of about 7.0 as measured by SDS-PAGE. The method of claim 5, The TIMP-2 comprises a N-terminal sequence of NH2-CSCSPVHP. The method of claim 6, The TIMP-2 has the following sequence: Or a functional equivalent or fragment thereof. The method according to any one of claims 1 to 7, The milk secretion is characterized in that milk or colostrum. The method according to any one of claims 1 to 8, Said mammal is selected from the group consisting of human, cow, sheep, goat, camel and horse. The method according to any one of claims 1 to 9, The mammal is a composition, characterized in that the emulsion. The method according to any one of claims 1 to 10, If the composition is indirectly derived from lactation, the intermediate product is selected from the group consisting of cheese whey, skim milk, acid (casein) whey, colostrum whey, skim colostrum and dry milk powder or combinations thereof. Composition. The method according to any one of claims 1 to 11, A composition comprising a pharmaceutically, veterinary, nutraceutical or cosmetically acceptable carrier and / or excipient. The method according to any one of claims 1 to 12, Wherein said inhibitor of metalloproteinase is present in a purity of about 70% to about 99% relative to the total protein content of the composition. A method of treating, preventing or ameliorating a disorder associated with undesired metalloproteinase activity, comprising administering to the animal in need thereof an effective amount of the composition according to claim 1. The method of claim 14, Such disorders include wounds caused by pressure, catheter disease, diabetes mellitus, autoimmune disease, sickle cell disease or hemophilia; Surgical outcomes; Therapeutic induction; Derived from disorders of the central nervous system and deprived diseases of the skin; Local or systemic infections such as strawberry species, HIV, chickenpox or herpes infections; Congenital; Corneal damage to the eyes; Pathological wounds; Traumatic or fruiting wounds; Or an image. The method of claim 14, Such disorders include dental and oral wounds; Peptic ulcer of the duodenum, stomach or esophagus; Inflammatory bowel disease; Ulcers associated with stress conditions; Damage to the lining of the dietary tract; Improper digestive tract function or damage to the digestive tract associated with premature birth; Diarrhea status; Food intolerance; Cancer of the gastrointestinal tract; Surgically induced gut injury; Damage due to esophageal reflux; Conditions associated with loss of gut barrier function; Congenital conditions leading to inadequate gastrointestinal function or damage; Or an autoimmune disease affecting the digestive tract. The method according to any one of claims 14 to 16, The composition is characterized in that the concentration of inhibitor is present at a concentration of about 0.1 ng / ml to about 10 μg / ml at the unwanted metalloproteinase active site. The method of claim 14, The disorders include cardiovascular disorders selected from the group including dilated myocardial disorders, congestive heart failure, atherosclerosis, plaque rupture, reperfusion injury, ischemia, chronic blocked pulmonary disease, angioplastic restenosis and aortic aneurysm; Tissue disorders in which metalloproteinases are associated with irregular remodeling, including disorders of bone, liver, lung, and nervous tissue; Disorders associated with viral infection by altering metalloproteinase activity; Inflammation related disorders including the application of metalloproteinases; Skin-related, including, but not limited to, psoriasis, scleroderma, and atopic dermatitis, or the application of metalloproteinases, including but not limited to diseases associated with ultraviolet damage to the skin causing aging and / or wrinkle formation of the skin Method characterized by the disabled. Providing lactation or a derivative thereof, and One or more treatments selected from the group consisting of centrifugation, microfiltration, ultrafiltration, ion-exchange chromatography, molecular sieve chromatography, affinity chromatography, reverse-phase high performance liquid chromatography and transient acidification Applying to the step, At least some purification or fortification of a metalloproteinase inhibitor. The method of claim 19, The method comprises ultrafiltration after cation exchange chromatography. The method of claim 20, The acidification step is followed by the ultrafiltration step. The method of claim 21, A gel filtration chromatography step following the excessive acidification step. The method of claim 22, And anion exchange chromatography followed by the gel filtration step. The method of claim 23, wherein And said affinity chromatography step is followed by said anion exchange chromatography step. The method of claim 24, Reverse phase high performance liquid chromatography following said affinity chromatography step. The method of claim 19, The method comprises the affinity chromatography step after the centrifugation step.