HK1077236A - Compositions comprising collagen and metalloprotease inhibitors - Google Patents

Description

Compositions and methods of using COLLAJOLIE

Background

Technical Field

The present invention relates generally to pharmaceutical compositions and methods, and more particularly to compositions and methods for increasing the duration of activity of a grafted collagen material.

Background

Collagen is one of the most abundant proteins in mammals and represents up to 30% of the dry weight of the human body (see l.c. junqueira and j.carreiro, Basic Histology, 4 th edition, lange medical Publications, Los Altos, calif., 1983, pages 89-119). Collagen provides strength and flexibility to the skin, hair and nails, and is also a major and essential component of muscles, tendons, cartilage, ligaments, joints and blood vessels.

Collagen can be found in at least five different naturally occurring forms produced by several different cell types. Type I collagen is the most abundant form of collagen and can be found throughout the body. It is produced by fibroblasts, osteoblasts, odontoblasts, and chondroblasts and can be found in bone, dentin, dermis, and fibrocartilage. Type II collagen is produced by chondroblasts and can be found primarily in cartilage. Type III collagen is produced by smooth muscle fibroblasts, reticulocytes, neural membrane cells, and liver cells. Its primary function is to maintain organ architecture and can be found in smooth muscle, endoneurial, arterial, uterine, liver, spleen, kidney, and lung tissues.

Type IV collagen is primarily thought to be involved in support and filtration and may be found in the epithelial and endothelial basement layers and basement membranes. Type V collagen is found in the fetal membranes, blood vessels, and placental basement membrane.

Collagen has been proposed for use in the treatment of various medical applications, including, for example, cosmetic surgery, arthritis, skin regeneration, transplantation, organ replacement, and the treatment of wounds and burns (see, for example, U.S. Pat. nos. 6,309,670, 5,925,736, 5,856,446, 5,843,445, 5,800,811, 5,783,188, 5,720,955, 5,383,930, 5,106,949, 5,104,660, 5,081,106, 4,837,379, 4,604,346, 4,485,097, 4,546,500, 4,539,716, and 4,409,332).

However, collagen has presented several problems associated with medical applications. For example, in transplantation, collagen preparations containing impurities are effective immunogens that can stimulate inflammatory responses. Similarly, non-human forms of collagen such as bovine collagen have been associated with chronic cellular inflammatory responses that can lead to scar tissue and adhesion formation, and transient low grade fever. In addition, the duration of implantable collagen is limited, requiring repeated manipulations on a regular basis.

The present invention discloses novel compositions, devices and methods for prolonging the activity of collagen-based implants, and additionally provides other related advantages.

Summary of The Invention

Briefly, the present invention provides compositions and methods for prolonging the activity of collagen-based implants. Collagen-based biomaterials are used to provide structure and support in a variety of medical procedures including skin injections for cosmetic purposes (wrinkles, scars, cosmetic defects), periurethral bulking agents to treat incontinence, and vascular "plugs" to create hemostasis following vascular puncture procedures. Although extremely effective, collagen implants have short duration of in vivo activity because the material is rapidly broken down by degradative enzymes (mainly collagenase and other matrix metalloproteinases) released by leukocytes and connective tissue cells (fibroblasts) adjacent to the implant. The result is that the collagen implantation procedure must be repeated at frequent intervals to maintain functional activity.

Various naturally occurring and synthetically produced molecules have been developed for other purposes of inhibiting collagenase activity (e.g., in the treatment of malignancies, arthritis, and other diseases) (collectively referred to as "matrix metalloproteinase inhibitors" or "collagenase inhibitors"). The present invention describes compositions that combine collagen and a compound that inhibits collagenase activity to produce a collagen-based implant ("Collajoline") with enhanced in vivo durability. Particularly preferred compounds or factors inhibit MMP-1, MMP-8, MMP-13, and/or NEAP-14 activity. Representative examples of MMPI suitable for use in the present invention include TIMP-1, tetracycline, doxycycline, minocycline, batimastat *, marimastat *, RO-1130830, CGS 27023A, BMS-275291, CMT-3, solistat, ilomastat, CP-544439, prinomastat, PNU-1427690, SU-5402 and tokacat.

Herein, in one aspect of the invention, a composition comprising collagen and MMPI is provided. In certain embodiments MMPI is a tissue inhibitor of matrix metalloprotease (e.g., TIMP-1, TIMP-2, TIMP-3, or TIMP-4). In other embodiments, the MMPI is tetracycline, or an analog or derivative thereof (e.g., minocycline or doxycycline); hydroxamates (e.g., batimastat, marimastat, or tocacate); or RO-1130830, CGS 27023A, CMT-3, solirestat, ilomastat, CP-544439, prinomastat, PNU-1427690, SU-5402, or BMS-275291. In other embodiments the collagen is type I or type II collagen. In still other embodiments the compositions provided herein can comprise other compounds or compositions, including, for example, thrombin and/or dyes. In further embodiments, the composition may be sterilized in a manner suitable for human administration.

In certain aspects of the invention, methods of making the compositions described herein are provided, comprising the step of mixing collagen and one or more MMPI described herein. In related embodiments, these methods may additionally comprise the step of admixing a dye or thrombin. In further embodiments, the composition may be sterilized.

In other aspects of the invention, the above-described compositions may be used for the treatment and/or prevention of various medical conditions. For example, in one aspect of the invention, a method for repairing and/or augmenting skin or tissue is provided that includes injecting into the skin or tissue a composition as described above. In various embodiments, the composition may be injected into the lips in order to correct or enhance the lips, or into the skin (e.g., in the face in order to correct scars or eliminate wrinkles).

In other aspects of the invention, there are provided methods for treating or preventing urinary incontinence comprising administering to a patient a composition as described above. In certain embodiments, the composition may be administered periurethrally or transurethrally.

In still other aspects of the present invention, there is provided a method for sealing a surgical site comprising the step of administering to a patient a composition as described above. Representative examples of such surgical sites include vascular access sites (e.g., sealants may be used as vascular sealants).

In another aspect, the present invention provides a method of preparing collajolie, comprising admixing collagen and at least one MMPI. In a particular embodiment, collajolie is prepared containing at least two MMPI. In certain embodiments, the collagen is type I or type II collagen. In other embodiments, the MMPI is a tissue inhibitor of matrix metalloproteinase (TIMP), such as TIMP-1, TIMP-2, TIMP-3, or TIMP-4. In still other embodiments, the MMPI is tetracycline, or an analog or derivative thereof, such as minocycline or doxycycline. In yet another embodiment, the MMPI is a hydroxamate such as batimastat, marimastat, or tokacat. In other embodiments, the MMPI is RO-1130830, CGS-27023A, or BMS-275291. In additional embodiments, the MMPI is a polypeptide inhibitor, such as an inhibitor of metalloprotease maturase. In further embodiments, the MMPI is a thiol-based compound. In other embodiments, the MMPI is a bisphosphonate with structure (I):

wherein R' and R "are independently hydrogen, halogen such as chlorine, hydroxyl, optionally substituted amino, optionally substituted thio, or optionally substituted alkyl, alkanyl, alkenyl, alkynyl, alkanediyl, alkyldiyl, alkyleno, heteroalkyl, heteroalkanyl, heteroalkenyl, heteroalkynl, heteroalkanyl, heteroalkanediyl, heteroalkeno, aryl, aralkyl, heteroaryl, heteroaralkyl. In other embodiments, the MMPI is first blended with at least one polymer prior to blending with the collagen. In related embodiments, the polymer is biodegradable, such as albumin, gelatin, starch, cellulose, dextran, polysaccharides, fibrinogen, poly (esters), poly (D, L lactide), poly (D, L-lactide) -co-glycolide, poly (glycolide), poly (e-caprolactone), poly (hydroxybutyrate), poly (alkyl carbonate), poly (anhydride), or poly (orthoester), and copolymers and mixtures thereof. In another embodiment, any of the methods of making collajolie described above further comprises the step of sterilizing the mixture.

These and other aspects of the invention will become apparent upon reference to the following detailed description and attached drawings. In addition, various references are recited herein which describe in more detail certain methods or compositions (e.g., compounds, proteins, vectors, and their production, etc.), and are hereby incorporated by reference in their entirety. It is also understood when referring to the PCT application that the basic or cited U.S. applications are also incorporated herein by reference in their entirety.

Detailed Description

Before setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms that will be used hereinafter.

By "collagen" is meant herein all forms of collagen described or referenced herein, including those that have been processed or modified. Representative examples include type I and type II collagen. Collagen may be prepared from human or animal sources, or may be produced using recombinant techniques.

"matrix metalloproteinase inhibitor" or "MMPI" refers to a compound, agent or composition that inhibits matrix metalloproteinase activity. Representative examples of MMP inhibitors ("MMPI") include Tissue Inhibitors of Metalloproteinases (TIMPs) (e.g., TIMP-1, TIMP-2, TIMP-3, or TIMP-4), alpha₂Macroglobulin, tetracyclines (e.g., tetracycline, minocycline, and doxycycline), hydroxamates (e.g., batimastat, marimastat, and tocatet), chelating agents (e.g., EDTA, cysteine, acetylcysteine, D-penicillamine, and gold salts), synthetic MMP fragments, succinyl mercaptopurine, phosphonamides (phosphonamide), and hydroxamic acid.

Unless otherwise indicated, any concentration or percentage range recited herein should be understood to include any integer within the range and fractions thereof, such as concentrations in the tenth and hundredth of an integer. Herein, "about" or "substantially comprising" means the mean ± 15%.

I. Collagen protein

Collagen is the major component of skin, cartilage, bone, and connective tissue, and exists in several different types or forms, with types I, II, III, and IV being most common. Collagen is typically isolated from natural sources such as bovine bone, cartilage or rawhide. Bone is typically defatted, crushed, dried and demineralized to extract collagen. In contrast, bovine cartilage or rawhide is typically chopped and digested with enzymes other than collagenase (to remove contaminating proteins). Collagen may also be produced from human tissue (patient's own or donor tissue) or by recombinant methods.

In certain embodiments of the invention, it is preferred that the collagen is prepared as an immunologically inactive sterile composition. They may be soluble (e.g. the commercially available Vitrogen * 100 collagen solution) or recombinant fibrillar atelopeptide collagen, such as Zydem * collagen implant (ZCI).

Representative examples of patents that disclose collagen-containing compositions, devices, and/or delivery of such compositions and devices include U.S. Pat. nos. 4,164,559, 4,424,208, 4,140,537, 4,563,350, 4,582,640, 4,642,117, 4,743,229, 4,776,890, 4,795,467, 4,888,366, 5,035,715, 5,162,430, 5,304,595, 5,324,775, 5,328,955, 5,413,791, 5,428,428, 5,446,091, 5,475,052, 5,523,348, 5,527,856, 5,543,441, 5,550,187, 5,565,671, 5,580,541, 5,614,587, 5,616,689, 5,464, 5,693,341, 5,545,744,678, 5,744,519,35,744,744,671, 5,744,744,756, 5,744,35,936, 5,976,115,936, 5,936, 5,115,936, 5,936, 5,102,936, 5,102,102,102,102,102,102,102,102,102,102,102,102,102,102,102,102,102,102,102,102,102,102,102,102, 6,6,102,67,102,102,102,67,102,67,67,102,67,67, 6,6,150,67,67,67,67,102,102,.

II. Matrix Metalloproteinase (MMP) inhibitors

Metalloproteinases (MMPs) are a group of naturally occurring zinc-dependent enzymes that are involved in the breakdown and turnover of macromolecules of the extracellular matrix. More than 23 metalloproteases have been identified to date, and have been broadly classified into the enzyme families of collagenase, stromelysin, gelatinase, elastase and matrilysin (matrilysin). Metalloproteases are derived from a variety of cell types, including neutrophils, monocytes, macrophages and fibroblasts.

MMPs are the major enzymes involved in collagen breakdown and normal turnover in vivo. Although many MMPs are capable of breaking down several connective tissue components including collagen, the enzymes with the highest specificity for collagen come from the collagenase family (e.g., MMP-1, MMP-8, MMP-13, and MMP-14). Metalloprotease activity is naturally inhibited in vivo by a family of inhibitors known as "tissue inhibitors of metalloproteinases" or "TIMPs" which bind to the active region of metalloproteinases rendering it inactive. The natural balance between enzymatic activity and inhibition regulates the rate of metabolism of the extracellular matrix under physiological conditions.

Assays for measuring MMP inhibition are readily known in the art and include, for example, the following: cawston T.E., Barrett A.J., "A Rapid and reproducible assay for collagenase using [14C]acetylated collagen, "anal. biochem.35: 1961-1965 (1963); cawston T.E., Murphy G, "Mammalian collagenases," Methods in Enzymology 80: 711 (1981); koshy p.t.j., Rowan a.d., Life p.f., Cawston t.e., "96-well plate assays for measuring collagen activity using (3) H-acetylated collagen," anal. biochem.99: 340-; stack m.s., Gray r.d, "company of vertabrate collagenase and gelatinase using a newfluorogenic substrate," j.biol.chem.264: 4277-4281 (1989); and Knight C.G., Willencrock F., Murphy G, "A novel subunit-layered peptide gene senThe positive continuous assays of the matrix metalloproteinases, "FEBS Lett 296: 263-266(1992). In the context of the present invention, MMPI preferably has a concentration of mM to nM (10-3 to 10)^-9) Inhibitory Concentration (IC).

When collagen is implanted as part of a therapeutic regimen, it is also gradually metabolized by enzymes from the MMP family until it is completely resorbed. This gradual loss of structural integrity due to enzymatic degradation of the collagen implant leads to a loss of functional activity, which leads to implant failure and ultimately the need for subsequent re-intervention. Efforts to prolong the activity of collagen implants have focused on cross-linking collagen implants in order to slow down enzymatic degradation. The present invention describes the incorporation of an agent or agents capable of inhibiting MMP activity into a collagen implant so as to tilt the physiological equilibrium to favor collagen preservation. The present invention is compatible with, and can be used in conjunction with, collagen crosslinking strategies such as those designed to increase the retention time of collagen implants.

Because pathological production of MMPs has been associated with the progression of a variety of clinically important disease processes such as tumor metastasis and chronic inflammatory diseases such as osteoarthritis and rheumatoid arthritis, a number of naturally occurring and synthetic agents have been developed to inhibit MMP activity. Not surprisingly, modulation of MMP activity is an important and highly regulated process in vivo. As a result, the existence of many sites in the pathway leading to MMP production allows the development of molecules capable of inhibiting MMP synthesis or activity. The types of agents capable of inhibiting MMP activity are described in more detail below.

Briefly, various cytokines (e.g., TNF- α, IL-1, FGF and others) are capable of stimulating pathways leading to MMP production. Inhibitors of these cytokines, which inhibit their cellular receptors, have been shown to inhibit MMP synthesis in certain circumstances and would be suitable for use in the present invention. Upon binding to its cellular receptor, stimulation of MMP production triggers the production of a variety of second messengers and cellular signaling molecules such as jun kinase, JKK, etc. -inhibition of these molecules can also reduce MMP production. A variety of transcription factors (e.g., c-fos, c-jun, NF-. kappa.B, c-myc) are involved in the transcription of MMP genes, inhibitors of these transcription factors and their products (e.g., AP-1 protein) can also reduce the amount of MMP transcribed and can be used for the purposes of the present invention. Similarly, inhibition of the MMP gene itself (e.g., gene knock-out) or MMP RNA (e.g., antisense, ribozyme, tetracycline, doxycycline, minocycline) can be used in the present invention to reduce the amount of active MMP enzyme in the region surrounding the collagen implant.

In addition, the function and activity of metalloproteases can be inhibited after they have been secreted from the cell. Because MMPs are secreted from cells as inactive precursor proteins (called pre-MMPs) which are subsequently converted to active enzymes by highly specific enzymatic cleavage (catalyzed by enzymes such as plasmin, mast cell proteases, cathepsin G, plasma kallikrein and others), the transition of a MMP from its inactive to active state can be inhibited (thereby maintaining it in an inactive form). Inhibitors of enzymes responsible for the transition of an MMP from its inactive to active state may also be used in the present invention. Finally, the function of an activated MMP can be directly inhibited by several mechanisms such as chelating its zinc metal active center (e.g., EDTA, cysteine, acetylcysteine, D-penicillamine, and gold salts; hydroxamates such as batimastat, marimastat, tocatet, actinonin, Matylystatins; phosphonic acid inhibitors; phosphonates; thiols and thiodiimines (sulfoximines) which form monodentate ligands with catalytic zinc; carboxylates which complex catalytic zinc to form bidentate ligands; succinylmercaptoketones and mercaptoalcohols). These compounds are very effective in inhibiting MMP activity and will be particularly useful for the purposes of the present invention. An important class of MMPI exert their effects by binding to MMPs, leading to the formation of inactive complexes. These compounds, known as Tissue Inhibitors of Metalloproteinases (TIMPs) such as TIMP-1, TIMP-2, TIMP-3, and TIMP-4, are capable of inhibiting the activity of almost all MMPs. Although any TIMP is suitable for the purposes of the present invention, TIMP-1 (to a lesser extent TIMP-2) is particularly preferred because of its highest specificity for inhibiting collagenase. It should also be noted that any compound that increases TIMP production will tilt the equilibrium in favor of collagen preservation and may be used in the present invention. Still other inhibitors act by inhibiting binding of MMPs to their substrates (e.g., synthetic MMP fragments, synthetic collagen fragments), and may also be used for the purposes of the present invention, alone or in combination with other MMPIs. It will be clear to those skilled in the art that any substance capable of inhibiting the production, activation or enzymatic function of MMP enzymes will be a desirable substance for the purposes of the present invention, regardless of the particular mechanism of inhibition.

Representative examples of MMPI include actinoamidin (3- [ [1- [ [2- (hydroxymethyl) -1-pyrrolidinyl)]Carbamoyl radical]-octanoyl-hydroxamic acid](ii) a Bromocyclic adenosine monophosphate; n-chlorotaurine; batimastat, also known as BB-94; CT1166, also known as N1{ N- [2- (morpholinosulfonylamino) -ethyl ]]-3-cyclohexyl-2- (S) -propionamido } -N4-hydroxy-2- (R) - [3- (4-methylphenyl) propyl]Succinamide (biochem. J.308: 167-175 (1995)); estramustine (estradiol-3-bis (2-chloroethyl) carbamate); eicosapentaenoic acid; marimastat (BB-2516); matlystatin-B; peptidyl hydroxamic acids such as pNH₂-Bz-Gly-Pro-D-Leu-D-Ala-NHOH (Biophys. biochem. Res. Comm.199: 1442-1446 (1994)); n-phosphonoalkyl dipeptides such as N- [ N- ((R) -1-phosphonopropyl) - (S) -leucinyl]- (S) -phenylalanine-N-carboxamide (J Med chem.37: 158-169 (1994)); protocatechualdehyde (3, 4-dihydroxybenzaldehyde); ro-31-7467, also known as 2- [ (5-bromo-2, 3-dihydro-6-hydroxy-1, 3-dioxo-1H-phenyl [ de ]]Isoquinoline 2-yl) methyl](hydroxy) - [ phosphinyl group]-N- (2-oxo-3-azacyclotridecyl) -4-methylpentanamide; tetracyclines such as (4- (dimethylamino) -1, 4, 4a, 5, 5a, 6,11, 12 a-octahydro-3, 6,10, 12, 12 a-pentahydroxy-6-methyl-1, 11-dioxo-2-tetracarboxamide), doxycycline (α -6-deoxy-5-hydroxy-tetracycline) minocycline (7-dimethylamino-6-dimethyl-6-deoxytetracycline), and methacycline (6-methyleneoxytetracycline); trifluoroacetate (J.Med chem.36: 4030-4039 (1993)); and based on 1, 10-phenanthroline (o-phenanthroline [4- (N-hydroxyamino) -2R-isobutyl-3S- (thiopentan-2-ylthiomethyl) -succinyl]Compounds of (e.g. L-phenylalanine-N-carboxyalkylamino-formamides, e.g. N- [1- (R) -carboxy-3- (1, 3-dihydro-2H-benzo [ f)]Isoindol-2-yl) propyl]-N', N "-dimethyl-L-leucinamide.

Other representative MMPI's include, for example, chelating agents (EDTA, cysteine, acetylcysteine, D-penicillamine, and gold salts), bis (dioxopiperazines) (see U.S. Pat. No. 5,866,570, Liang et al), neovistat (inhibits the gelatin and elastin breakdown activities of MMP-9, and MMP-12, see U.S. Pat. No. 6,168,807 Aeterna laboratory); KB-R7785(Akzo Nobel); ilomastat (Glycomed/Ligand; U.S. patent 5,892,112); RPR-122818(Aventis) solimastat (British Biotech, WO 99/25693), BB-1101, BB-2983, BB-3644(British Biotech); BMS-275291 (see Rizvi et al, Proceedings of the1999 AACR NCI EORTC International Conference "# 726," A Phase I, safety and pharmaceutical scientific trial of BMS-275291, a Matrix Metalloproteinaseeinhibitor (MMPI), in Patients with advanced or statistical cancer "), D-1927, D-5410, CH-5902, CH-138 (Celltech); CMT-3, dermostatt (CollaGenex-U.S. Pat. No. 5,837,696); DAC-MMPI (Conjuchem); RS-1130830 and RS-113-080(Hoffmann-La Roche); GM-1339 (Ligand); GI-155704A (GlaxoSmith Kline); ONO-4817 (ONO); AG-3433, AG-3088, prinomastat (Agouron; U.S. Pat. No. 5,753,653), CP-544439 (Pfizer; U.S. Pat. No. 6,156,798); POL-641 (Polifarma); SC-964, SD-2590, PNU-142769 (Pharmacia; WO 97/32846), SU-5402 (Pharmacia; WO 98/50356); PGE-2946979, pGE-4304887(Procter & Gamble); cellulase (fibrise) -conjugate (Schering-AG); EF-13 (Scotia-pharmaceuticals); s-3304 (Shionogi); CGS-25015 and CGS-27023A (Novartis), XR-168(Xenova), and RO1130830 (Fisher et al, 219 American Chemical Society National Meeting, San Francisco, CA, 3 months 26-30 days, "ORGN 830" Synthesis of RO1130830, a Matrix Metalloprotein Inhibitor: Evolution of a research scheme to Plant Production "). Other MMPI are disclosed in us patent 4,235,885; 4,263,293, respectively; 4,276,284, respectively; 4,297,275, respectively; 4,367,233, respectively; 4,371,465, respectively; 4,371,466, respectively; 4,374,765, respectively; 4,382,081, respectively; 4,558,034, respectively; 4,704,383, respectively; 4,950,755, respectively; 5,270,447, 6,294,694, and 6,329,550.

Representative examples of classes of MMPI, discussed in more detail below, include (1) tissue inhibitors of matrix metalloproteinases (TIMPs); (2) tetracycline, (3) hydroxamates, (4) synthetic MMP fragments (e.g., peptide inhibitors), (5) thiol-based compounds, and (6) bisphosphonates.

1. Tissue inhibitors of matrix metalloproteinases

Tissue inhibitors of matrix metalloproteinases (TIMPs) are classified based on their ability to inhibit metalloproteinases, structural similarity to each other, 12 cysteines forming important disulfide bonds in secondary structure, and the presence of a VIRAF motif that interacts with the metal ion of metalloproteinases. The nucleic acid and amino acid sequences of TIMPs have been described: TIMP-1 (Doherty AJP et al (1985) Nature 318: 66-69), TIMP-2(Boone TC et al (1990) Proc Natl Acad Sci 87: 2800-; (see also, Boone T.C., et al, "cDNA cloning and expression of a metallic inhibitor related to a viral inhibitor of metallic inhibitors," Proc. Natl.Acad. Sci.USA, 87: 2800-.

TIMP-1 is a 30kD protein and is the most commonly expressed TIMP molecule. It contains two asparagine residues as sugar binding sites, one in Loop 1 and one in Loop 2 (Murphy and Dochery, supra). In addition, a truncated form of TIMP-1, which contains only the first three loops of the molecule, is capable of inhibiting MMP. Although TIMP-1 is a better interstitial collagenase inhibitor than TIMP-2 (Howard E W et al (1991) J Biol Chem 266: 13070-75), the 23kDTIMP-2 molecule is the most effective inhibitor of gelatinases A and B. TIMP-3 is a 21kD protein that inhibits collagenase 1, stromelysin, and gelatinases A and B (Apte S.S. et al (1995) J biol chem 270: 14313-18) and can be induced with mitotic agents (Wick et al (1994).

As noted above, any of the four TIMP molecules is capable of inhibiting the activity of almost all MMPs identified thus far and is suitable for the purposes of the present invention. However, TIMP-1, which has the highest specificity for collagenase, will be particularly preferred for incorporation into collagen implants.

2. Tetracyclines

Tetracyclines are a class of analogs and derivative compounds originally known for their use as antibiotics. Many tetracyclines, including tetracycline, doxycycline, minocycline, and others, have been shown to inhibit the production and activity of MMPs. While the exact mechanism is not fully understood, MMP inhibition can occur by down-regulating MMP expression and/or post-translationally by chelating the zinc metal active site. Given their broad application and low toxicity, these compounds would be particularly effective for incorporation into collagen implants.

The parent compounds of the tetracycline family have the following general structure:

the polycyclic cores may be numbered as follows:

tetracyclines, as well as 5-OH (oxytetracycline) and 7-Cl (chlorotetracycline) derivatives, occur in nature and are well known antibiotics. Other tetracyclines include, for example, aprepitetracycline, tetracycline, hydroxymethyltetracycline, demeclocycline, doxycycline, ethambutracycline, guanpemetrycline, lymecycline, meglumine, mepycycline, minocycline, methacycline, cyclomycin, picycycline, pirimicycline, and demeclocycline.

Tetracyclines may also be modified so that they retain their structural relationship to the antibiotic tetracycline, but their antibiotic activity is substantially or completely reduced by chemical modification. Representative examples of Chemically Modified Tetracyclines (CMT) include, for example, CMT-1 (4-des (dimethylamino) -tetracycline), CMT-2 (tetracyclinecarbonitrile), CMT-3 (6-desmethyl-6-deoxy-4-des (dimethylamino) tetracycline), CMT-4 (7-chloro-4-des (dimethylamino) tetracycline), CMT-5 (tetracycline pyrazole), CMT-6 (4-hydroxy-4-des (dimethylamino) tetracycline), CMT-7 (4-des (dimethylamino) -12 α -deoxytetracyclines), CMT-8 (6-deoxy-5 α -hydroxy-4-des (dimethylamino) tetracycline), CMT-9 (4-des (dimethylamino) -12 a-deoxyanhydro-tetracycline), and CMT-10 (4-des- (dimethylamino) minocycline).

Representative examples of tetracyclines (including tetracycline derivatives) are described in: U.S. Pat. No. 4,3,622,627, Blackwood et al; 3,846,486, Marcus; 3,862,225, Conover, etc.; 3,895,033, Murakami et al; 3,901,942, Bernard, etc.; 3,914,299, Muxfeldt; 3,925,432, Gillchrest; 3,927,094, Villax; 3,932,490, Fernandez; 3,951,962, Murakami et al; 3,983,173, Hartung et al; 3,991,111, Murakami et al; 3,993,694, Martin, etc.; 4,060,605, Cotti; 4,066,694, Blackwood, etc.; 4,081,528, Armstrong; 4,086,332, Armstrong; 4,126,680, Armstrong; 4,853,375, Krupin, etc.; 4,918,208, Hasegawa, etc.; and 5,538,954, Koch et al. (see generally, Mitscher, L.A., The Chemistry of tetracyline Antibiotics, ch.6, Marcell Dekker, New York, 1978).

Further examples of tetracycline derivatives are described in: U.S. Pat. No. 4,666,897, Golub et al, 4,704,383, McNamara et al, 4,904,647, Kulcsar et al, 4,935,412, McNamara et al, 5,223,248, McNamara et al, 5,248,797, Sum et al, 5,281,628, Hlavka et al, 5,326,759, Hlavka et al, 5,258,371, Golub et al, 5,308,839, Golub et al, 5,326,759, 5,401,863, Hlavka et al, 5,308,839, Golub et al, 5,308,839, Hlvaka et al, 5,308,839, Backer et al, 5,308,839, Backer et al, Zheng et al, 5,308,839, Heggie et al, 5,308,839, WO Vhe et al, WO 5,308,839, and WO 5,308,839.

3. Hydroxamic acid esters

Another class of MMP inhibiting compounds are hydroxamates (or hydroxamic acids). Although the exact mechanism of MMP inhibition is not clearly known, it is believed that these compounds exert their effect primarily through interaction with the zinc metal active site in the enzyme (e.g., by coordinating with catalytic zinc in a bidentate fashion to assume a trigonal bipyramidal geometry). A variety of hydroxamates have been synthesized and tested in several disease states with mixed clinical outcomes. However, given their selective activity against MMPs and their excellent safety and tolerability, these agents would be particularly preferred for incorporation into collagen implants to enhance the durability of the implants.

Hydroxamates (or hydroxamic acids) have the general structure shown below:

wherein A is HN (OH) -CO-or HCO-N (OH) -; r¹Is C₂-C₅An alkyl group; r²Is a characteristic group of a natural alpha amino acid, which may be protected, with the proviso that R²Is not H or methyl; r³Is H, NH₂，OH，SH，C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Alkylamino radical, C₁-C₆Alkylthio, aryl (C)₁-C₆Alkyl), or amino (C)₁-C₆Alkyl), hydroxy (C)₁-C₆Alkyl), mercapto (C)₁-C₆Alkyl) or carboxyl (C)₁-C₆Alkyl), wherein amino, hydroxyl, mercapto or carboxyl may be protected, amino may be acylated or carboxyl may be amidated; r⁴Is H or methyl; r⁵Is H, C₁-C₆Alkyl radical, C₁-C₆Alkoxy (C)₁-C₆Alkyl), di (C)₁-C₆Alkoxy) methylene, carboxyl, (C)₁-C₆Alkyl) carbonyl (C)₁-C₆Alkoxy) carbonyl, arylmethoxycarbonyl, (C)₁-C₆Alkyl) aminocarbonyl or arylaminocarbonyl; and R is⁶Is H or methyl; or R²And R⁴Together form a radical (CH)₂)_nWherein n is an integer from 4 to 11; or R⁴And R⁵Together form cyclopropane, and pharmaceutically acceptable salts of these hydroxamate compounds, which are acidic or basic. In this regard, see, for example, EP-A-0236872.

Wherein R is¹Is C₁-C₆An alkyl group; r²Is C₁-C₆Alkyl, benzyl, hydroxybenzyl, benzyloxybenzyl, (C)₁-C₆Alkoxy) benzyl or benzyloxy (C)₁-C₆Alkyl groups); a is (CHR)³-CHR⁴) Or (CR)³＝CR⁴) A group; r³Is hydrogen, C₁-C₆Alkyl, phenyl or phenyl (C)₁-C₆Alkyl groups); and R is⁴Is H or C₁-C₆Alkyl, phenyl (C)₁-C₆Alkyl), cycloalkyl or cycloalkyl (C)₁-C₆Alkyl groups). In this regard, see, for example, EP-A-0214639.

Wherein R is¹Is hydrogen or hydroxy, R²Is hydrogen or alkyl, R³Is C₃-C₆Alkyl radical, R⁴Is hydrogen, alkyl, -CH₂Z, wherein Z is optionally substituted phenyl or heteroaryl, or R⁴Is a group C (HOR)⁸)R⁹Wherein R is⁸Is hydrogen, CH₂Alkyl of Ph, wherein Ph is optionally substituted phenyl, and R⁹Is hydrogen or alkyl; and R is⁵Is hydrogen or alkyl. In this regard, see, for example, EP-A-320118.

Wherein R is¹Is hydrogen, alkyl or optionally substituted aryl, R²Is hydrogen or aryl such as CO alkyl or COZ, wherein Z is optionally substituted aryl; r³Is C_3-6Alkyl radical, R₄Is hydrogen, alkyl, -CH₂R₁₀Wherein R is₁Is optionally substituted phenyl or heteroaryl, or R₄Is a group C (HOR)¹¹)R¹²Wherein R is¹¹Is hydrogen, alkyl or CH₂Ph, wherein Ph is optionally substituted phenyl, and R¹²Is hydrogen or alkyl; and R is⁵Is hydrogen, alkyl or a group C (HR)¹³)COR¹⁴Wherein R is¹³Is hydrogen, or alkyl, and R¹⁴Is hydroxy, alkoxy, or-NR⁶R⁷Wherein R is⁶Or R⁷Each of which is hydrogen or alkyl, or R⁶And R⁷Together with the nitrogen atom to which they are bound, form a 5-, 6-or 7-membered ring containing an optional oxygen or sulfur atom or another optional nitrogen atom in the ring, which is optionally substituted by alkyl. In this regard, see, for example, EP-A-0322184.

Wherein R is¹And R²Independent of each otherIs H, alkyl, alkoxy, halogen or CF₃，R³Is H, an acyl group, such as a cocoalkyl group or COZ, wherein Z is an optionally substituted aryl group, or a group RS, wherein R is an organic residue such that RS provides an in vivo cleavable disulfide bond; r⁴Is C₃-C₆Alkyl radical, R⁵Is H, alkyl, -CH₂R¹⁰Wherein R is¹⁰Is optionally substituted phenyl or heteroaryl, or a radical C (HOR)¹¹)R¹²Wherein R is¹¹Is hydrogen, alkyl or CH₂Ph, wherein Ph is optionally substituted phenyl, and R¹²Is hydrogen or alkyl; r⁶Is hydrogen, alkyl or C (HR)¹³)COR¹⁴Wherein R is¹³Is hydrogen, or alkyl, and R¹⁴Is hydroxy, alkoxy, or-NR⁷R⁸Wherein R is⁷Or R⁸Each of which is hydrogen or alkyl, or R⁷And R⁸Together with the nitrogen atom to which they are bound, form a 5-, 6-or 7-membered ring containing an optionally oxygen, sulfur or optionally substituted nitrogen atom in the ring; x is (CH)₂)_nWherein n is 0,1, or 2; and Y is CH₂. In this regard, see, for example, EP-A-358305.

Wherein R is hydrogen, C₁-C₆Alkyl or optionally substituted benzyl, R¹Is hydrogen or C₁-C₆Alkyl radical, R²Is C₃-C₆Alkyl radical, R³Is hydrogen, alkyl, -CH₂Z, wherein Z is optionally substituted phenyl or heteroaryl, or R³Is a group C (HOR)⁷)R⁸Wherein R is⁷Is hydrogen, alkyl or CH₂Ph, wherein Ph is optionally substituted phenyl, and R⁸Is hydrogen or alkyl; r⁴is-CH₂-(CH₂)_nOR⁵，-CH₂-(CH₂)_nOCOR⁶or-CH (R)⁹)COR¹⁰Wherein n is an integer from 1 to 6; r⁵，R⁶And R⁹Is hydrogen or C₁-C₆An alkyl group; and R is¹Is hydroxy or O (C)₁-C₆Alkyl) or NR⁵R⁶Wherein R is⁵And R⁶May be linked to form a heterocyclic ring; or R³And R⁴Are linked together to (CH)₂)_mWherein m is an integer of 4 to 12. In this regard, see, for example, EP-A-0401963.

Wherein R is¹Is H, C₁-C₆Alkyl, phenyl, thienyl, substituted phenyl, phenyl (C)₁-C₆) Alkyl, heterocyclic radical, (C)₁-C₆) Alkylcarbonyl, phenacyl or substituted phenacyl; or, when n is 0, R¹Represents SR^xWherein R is^xRepresents a group of the formula:

R²is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl, phenyl (C)₁-C₆) Alkyl, cycloalkyl (C)₁-C₆) Alkyl or cycloalkenyl (C)₁-C₆) An alkyl group; r³Is an amino acid side chain or C₁-C₆Alkyl, benzyl, (C)₁-C₆Alkoxy) benzyl, benzyloxy (C)₁-C₆Alkyl) or benzyloxybenzyl; r⁴Is H or C₁-C₆An alkyl group; r⁵Is H or methyl; n is 0,1 or 2; and A represents C₁-C₆A hydrocarbon chain optionally substituted by one or more C₁-C₆Alkyl, phenyl or substituted phenyl; and their salts and N-oxides. In this regard, see, for example, PCT International publication No. WO 90/05719.

Wherein R is¹Is H, C₁-C₆Alkyl radical, C₂-C₆Alkenyl, phenyl (C)₁-C₆) Alkyl radical, C₁-C₆Alkylthiomethyl, phenylthiomethyl, substituted phenylthiomethyl, phenyl (C)₁-C₆) Alkylthiomethyl, or heterocyclic thiomethyl or R¹denotes-SR^xWherein R is^xRepresents the following groups:

R²represents a hydrogen atom, or C₁-C₆Alkyl radical, C₁-C₆Alkenyl, phenyl (C)₁-C₆) Alkyl, cycloalkyl (C)₁-C₆) Alkyl, or cycloalkenyl (C)₁-C₆) An alkyl group; r³Represents an amino acid side chain or C₁-C₆Alkyl, benzyl, (C)₁-C₆) Alkoxybenzyl, benzyloxy (C)₁-C₆) Alkyl, or benzyloxybenzyl; r⁴Represents a hydrogen atom, or a methyl group; n is an integer from 1 to 6; and A represents a group-NH₂Substituted acyclic amines or heterocyclic bases; or a salt and/or N-oxide and/or (when the compound is a thio compound) a sulfoxide or sulfone thereof. In this regard, see, for example, PCT International publication WO 09/05716.

Wherein R is¹Is H, C₁-C₆Alkyl radical, C₁-C₆Alkenyl, phenyl (C)₁-C₆) Alkyl radical, C₁-C₆Alkylthiomethyl, phenylthiomethyl, substituted phenylthiomethyl, phenyl (C)₁-C₆) Alkylthiomethyl or heterocyclic thioA methyl group; or R¹represents-S-R^xWherein R is^xRepresents the following groups:

R²represents a hydrogen atom, or C₁-C₆Alkyl radical, C₁-C₆Alkenyl, phenyl (C)₁-C₆) Alkyl, cycloalkyl (C)₁-C₆) Alkyl, or cycloalkenyl (C)₁-C₆) An alkyl group; r₃Represents an amino acid side chain or C₁-C₆Alkyl, benzyl, (C)₁-C₆) Alkoxybenzyl, benzyloxy (C)₁-C₆) Alkyl or benzyloxybenzyl; r⁴Represents a hydrogen atom or a methyl group; r⁵Represents a group (CH)₂) A; or R⁴And R⁵Together represent the following groups:

q represents CH₂Or CO; m is an integer of 1 to 3; n is an integer from 1 to 6; a represents a hydroxyl group, (C)₁-C₆) Alkoxy group, (C)₂-C₇) Acyloxy, (C)₁-C₆) Alkylthio, phenylthio, (C)₂-C₇) An amido or N-pyrrolidone group; or a salt and/or N-oxide and/or (when the compound is a thio compound) a sulfoxide or sulfone thereof. In this regard, see, for example, PCT International publication No. WO 91/02716.

Wherein R is¹Is H, C₁-C₆Alkyl, phenyl, substituted phenyl, phenyl (C)₁-C₆Alkyl), or a heterocyclic group; or R¹Is ASO_nR⁷Wherein A represents C₁-C₆A hydrocarbon chain optionally substituted by one or more C₁-C₆Alkyl, phenyl or substituted phenyl, n is 0,1, or 2, and R⁷Is C₁-C₆Alkyl, phenyl, substituted phenyl, phenyl (C)₁-C₆Alkyl), heterocyclic group, (C)₁-C₆Alkyl) aroyl, thienyl or phenacyl; r²Is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Alkenyl, phenyl (C)₁-C₆Alkyl) or cycloalkyl (C)₁-C₆Alkyl groups); r³And R⁴Selected from hydrogen, halogen, cyanoamino, amino (C)₁-C₆) Alkyl, aminodi (C)₁-C₆) Alkyl, amino (C)₁-C₆) Alkanoyl, aminobenzoylmethyl, amino (substituted) phenyl, amino acids or derivatives thereof, hydroxy, oxy (C)₁-C₆) Alkyl, oxyacyl, formyl, carboxylic acid, carbamoyl, carboxy (C)₁-C₆) Alkylamides, carboxyphenylamides, carboxy (C)₁-C₆) Alkyl, hydroxy (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkoxy (C)₁-C₆) Alkyl or acyloxy (C)₁-C₆) Alkyl radical (C)₁-C₆) An alkyl carboxylic acid, or (C)₁-C₆) Alkylcarboxy (C)₁-C₆) An alkyl group; or R³Is OCH₂COR⁸And R⁴Is hydrogen, wherein R⁸Is hydroxy, C₁-C₆Oxyalkyl radical, C₁-C₆Oxyalkylphenyl, amino, C₁-C₆Aminoalkyl radical, C₁-C₆Aminodialkyl radical, C₁-C₆Aminoalkylphenyl, amino acids or derivatives thereof; or R³Is OCH₂CH₂OR⁹And R is⁴Is hydrogen, wherein R⁹Is C₁-C₆Alkyl radical, C₁-C₆Alkylphenyl, phenyl, substituted phenyl, (C)₁-C₆Alkyl) acyl, or phenacyl; or R³Is OCH₂CN and R⁴Is hydrogen; r⁵Is hydrogen or C₁-C₆Alkyl, or (C)₁-C₆) An alkyl phenyl group; r⁶Is hydrogen or methyl; or a salt thereof. In this regard, see, e.g., PCT International application PCT/GB 92/00230.

Two preferred compounds for use in the present invention are mentioned in us patent 5,872,152: [4- (N-hydroxyamino) -2R-isobutyl-3S-thienylthiomethyl ] succinyl ] -L-phenylalanine-N-carboxamide having the following structure:

and [4- (N-hydroxyamino) -2R-isobutyl-3S-phenylthiomethyl ] succinyl ] -L-phenylalanine-N-carboxamide having the following structure

As used herein to describe MMP inhibitors containing hydroxamic acid moieties, the following terms have the indicated meanings. The term "C₁-C₆Alkyl "refers to a straight or branched chain hydrocarbon group containing 1 to 6 carbon atoms, wherein exemplified alkyl is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl and hexyl. The term "C₁-C₆Alkenyl "means a straight or branched chain hydrocarbon radical containing 1 to 6 carbon atoms and containing at least one or more double bonds, each of which is E or Z stereochemistry where applicable, where the term includes, for example, α, β -unsaturated methylene, ethenyl, 1-propenyl, 1-and 2-butenyl and 2-methyl-2-propenyl, and where in preferred embodiments C is₁-C₆Alkenyl is C₂-C₆An alkenyl group. The term "C₃-C₆Cycloalkyl "refers to an alicyclic group containing 3 to 6 carbon atoms, with cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl being exemplary cycloalkyl groups. The term "C₄-C₆Cycloalkenyl radicals "Refers to cycloaliphatic radicals having from 4 to 6 carbon atoms and additionally containing one or more double bonds, of which cycloalkenyl radicals are exemplified by cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl. The term "halogen" refers to fluorine, chlorine, bromine or iodine. The term "amino acid side chain" refers to the linkage to-CH (NH) in the following R or S amino acid₂) Characteristic side chains attached to the (COOH) moiety: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, histidine, arginine, glutamic acid, and aspartic acid.

Representative examples of hydroxamates and methods of synthesizing hydroxamates are described in detail in the following: U.S. Pat. nos. 4,599,361, 4,720,486, 4,743,587, 4,996,358, 5,183,900, 5,189,178, 5,239,078, 5,240,958, 5,256,657, 5,300,674, 5,304,604, 5,310,763, 5,412,145, 5,442,110, 5,473,100, 5,514,677, 5,530,161, 5,643,964, 5,652,262, 5,691,382, 5,696,082, 5,700,838, 5,747,5145,006, 5,763,621, 5,821,262, 5,840,939, 5,849,951, 5,859,253, 5,861, 5,866,717, 5,872,152, 5,791, 5,840,939, 5,695,695, 5,090,304,107,304,123,123,436, 5,124, 5,201,304,123,123,123,123,123,123,127,46,9, 5,695,695, 5,107,124, 5,123,123,123,123,123,107,123,123,123,123,123,123,123,107,123,123,124, 6,123,123,107,107,123,123,123,123,123,123,124, 6,123,123,123,123,123,107,123,123,123,123,123,123,123,123. Representative foreign and international applications and publications include EP-A-0231081, EP-A-0236872, EP-A-0274453, EP-A-0489577, EP-A-0489579, EP-A-0497192, EP-A-0574758, and EP-A-0575844, as well as WO90/05716, WO90/05719, WO91/02716, WO 92/09563, WO 92/17460, WO92/13831, WO 92/22523, WO 93/09090, WO 93/09097, WO 93/20047, WO93/24449, WO 93/24475, WO 94/02446, WO 94/02447, WO 94/21612, WO94/21625, WO 94/24140, WO 94/25434, WO 94/25435, and WO 99/06361. Many hydroxamates are also readily available from various commercial sources.

4. Polypeptide inhibitors

In other aspects of the invention polypeptide (including polypeptide derivatives) inhibitors of matrix metalloproteinases may be used to extend the duration and effectiveness of collagen. Representative examples of polypeptide inhibitors include those disclosed in U.S. Pat. nos. 5,300,501, 5,530,128, 5,569,665, 5,714,491, and 5,889,058.

5. Mercapto-based compounds

Mercapto-based compounds may also be used as MMPI. Representative examples include mercaptoketones and mercaptoalcohols such as those described in U.S. Pat. Nos. 5,831,004, 5,840,698, and 5,929,278; mercapto sulfides such as those described in us patent 5,455,262.

6. Diphosphonic acid esters

Bisphosphonates are compounds which are related to inorganic pyrophosphoric acid (see generally H.Fleisch, Endocr.Rev., 19 (1): 80-100 (1998); also, H.Fleisch, Bisphosphates in BoneDisease: From The Laboratory to The Patient (1997, 3 rd edition.) The Parthenon publishing Group, New York and London). Generally diphosphonates have the following structure

Wherein the substituents R' and R "independently represent a hydrogen or halogen atom, a hydroxyl group, an optionally substituted amino group or an optionally substituted thio group or an optionally substituted hydrocarbon residue. In one aspect, one of R 'and R' is hydroxy, hydrogen or chloro.

Representative examples of bisphosphonates include, for example, alendronate ((4-amino-1-hydroxybutylidene) diphosphate); clodronic acid (dichloromethane diphosphonic acid); etidronate ((1-hydroxyethylidene) diphosphonic acid); (ii) aminohydroxy diphosphonic acid ((3-amino-1-hydroxypropyl) diphosphonic acid); risedronate ([ -hydroxy-2- (3-pyridyl) methylene ] diphosphonic acid); tiludronate (([ (4-chlorophenyl) thio ] -methylene) diphosphonic acid); zolendronate; [ 1-hydroxy-3- (methyl-pentylamino) -propylidene ] diphosphonate (BM 21.0955); [ (cycloheptylamino) methylene ] diphosphonate (YM 175); 1-hydroxy-3- (1-pyrrolidinyl) -propylidene ] diphosphonate (EB-1053); [ 1-hydroxy-2- (1H-imidazole) -1-yl ] ethylene ] diphosphonate (CGP 42' 446) and (1-hydroxy-2-imidazole- [1, 2-a ] pyridin-3-yl-ethylene) diphosphonate (YM 529).

Representative examples of bisphosphonates are described in us patents 5,652,227 and 5,998,390.

7. Combinations of MMPs

In certain embodiments of the invention, more than one MMPI may be used (e.g., two or more MMPIs may be usedCombination ofUse). Synergistic MMPI include, for example, tetracycline and bisphosphonates (see, e.g., U.S. patents 5,998,390 and 6,114,316). Other MMPI implementations may be used as wellGroup of Combination of Chinese herbsIncluding, for example, MMPI (e.g., hydroxamates and tetracyclines) that inhibit MMPs at various stages.

III. Preparation

As mentioned above, collagen is a fibrous protein, which may be obtained from a natural source or recombinantly produced. Representative examples of U.S. patents that describe collagen-based compositions and methods of making the compositions include U.S. patents 6,166,130, 6,051,648, 5,874,500, 5,705,488, 5,550,187, 5,527,856, 5,523,291, 4,582,640, 4,424,208, and 3,949,073.

The MMPI compositions of the present invention can be prepared in a variety of ways. For example, MMPI can be dissolved directly in the collagen solution. If the MMPI is stable in the collagen solution, a composition containing collagen and MMPI can be prepared in a single application device. If the MMPI is not stable in the collagen solution for a significant period of time, the composition may be prepared as a two-component system in which the components are mixed just prior to use.

The MMPI compositions of the invention can also be produced by placing the MMPI factor in a carrier. Representative examples of carriers can include polymeric and non-polymeric carriers (e.g., liposomes or vitamin-based carriers, and can be biodegradable or non-biodegradable. examples of biodegradable compositions include albumin, gelatin, starch, cellulose, dextran, polysaccharides, fibrinogen, poly (esters), [ such as poly (D, L lactide), poly (D, L-lactide-co-glycolide), poly (e-caprolactone), and copolymers and mixtures thereof ] poly (hydroxybutyrate), poly (alkyl carbonate), poly (anhydride), or poly (orthoester), (see generally Illum, L., Davids, S.S. (eds.) "Polymers in Controlled Drug Delivery," Wright, StoBril, 1987; Arshalady, J., rolled Release 17: 1-22 (1991); Pitt, int. J. Pharm Phard 59: 173 (1990); Holland et al, controlled Release 4: 155-0180(1986)). Representative examples of non-biodegradable polymers include copolymers of ethylene oxide and propylene oxide [ Pluronic polymer-BASF ], EVA copolymers, silicone rubber, poly (methacrylate) based polymers and poly (acrylate) based polymers. Particularly preferred polymeric carriers include poly (D, L-lactic acid) oligomers and polymers, poly (glycolic acid), copolymers of lactic acid and glycolic acid, poly (caprolactone), poly (valerolactone), polyanhydrides, caprolactone and/or lactic acid, and/or copolymers of glycolic acid with polyethylene glycol or methoxypolyethylene glycol, and mixtures thereof.

Polymer carriers may be formulated in a variety of forms including, for example, rod-like devices, pellets, tablets or capsules (see, for example, Goodell et al, am. J. Hosp. phase.43: 1454-. The MMPI factor may be attached by encapsulation in a polymer matrix, or bound by covalent bonding, or encapsulated in microcapsules. In certain preferred embodiments of the invention, the MMPI compositions are provided in non-encapsulated formulations such as microspheres (ranging in size from a few nanometers to a few micrometers), pastes, threads of varying sizes, films and sprays.

Preferably, the MMPI compositions of the invention (which in certain embodiments comprise one or more MMPI factors, and a polymeric carrier) are formulated in a manner suitable for the intended use. In certain aspects of the invention, the MMPI compositions should be biocompatible and release one or more MMPI factors over a period of days to months. For example, an "immediate release" or "burst" MMPI composition is provided that releases greater than 10%, 20%, or 25% of the MMPI factor (e.g., tetracycline) over a 7-10 day period. In certain embodiments the "immediate release" composition should be capable of releasing chemotherapeutic levels (where applicable) of the desired MMPI factor. In other embodiments, "low release" MMPI compositions are provided that release less than 5% (w/v) of MMPI factor over a period of 7-10 days. In addition, the MMPI compositions of the present invention should preferably be stable for several months and capable of being produced and maintained under sterile conditions.

In certain aspects of the invention, the MMPI compositions may be formulated to any size between about 0.050nm to about 500 μm, depending on the particular application. For example, when used for cosmetic tissue augmentation purposes (as discussed below), it is generally preferred that the MMPI composition be formulated as microspheres of about 0.1 to about 100 μm, preferably about 0.5 to about 50 μm, and most preferably about 1 to about 25 μm. Alternatively the composition may also be applied as a solution, wherein the MMPI is dissolved in the micelles. The micellar composition may be polymeric in nature. The most preferred polymeric composition for use as polymeric micelles is a copolymer of MePEG and poly (D, L-lactide). Alternatively the composition may also be administered as a solution, wherein the MMPI is encapsulated in liposomes (see above). Alternatively the composition may also be applied as a solution wherein the MMPI is encapsulated in the oil phase of an emulsion or microemulsion.

The MMPI compositions of the invention may also be prepared in various "cream" or gel forms. For example, in one embodiment of the present invention, MMPI compositions are provided that are liquid at one temperature (e.g., a temperature greater than 37 ℃, such as 40 ℃,45 ℃,50 ℃,55 ℃,60 ℃) and solid or semi-solid at another temperature (e.g., ambient body temperature, or any temperature below 37 ℃). The "thermal pastes" disclosed herein can be readily prepared.

Representative examples of MMPI factors that incorporate those described above are described in more detail in the examples below.

In another aspect of the invention, a polymeric carrier is provided which is adapted to contain and release a hydrophobic compound, the carrier comprising a hydrophobic compound bound to a carbohydrate, protein or polypeptide. In certain embodiments, the polymeric carrier contains or comprises a region, pocket, or particle of one or more hydrophobic compounds. For example, in one embodiment of the present invention, the hydrophobic compound may be incorporated into a matrix comprising the hydrophobic compound, followed by incorporation of the matrix into a polymeric carrier. A variety of matrices may be used in this regard, including for example carbohydrates and polysaccharides such as starch, cellulose, dextran, methylcellulose, and hyaluronic acid, proteins or polypeptides such as albumin, collagen and gelatin. In an alternative embodiment, the hydrophobic compound may be comprised in a hydrophobic core comprised in a hydrophilic shell. For example, paclitaxel may be incorporated into a hydrophobic core (e.g., poly D, L lactic acid-PEG or MePEG aggregates) having a hydrophilic shell, as described in the examples below.

1. collagen-MMP prodrugs

In certain aspects of the invention, MMPI compositions can be formulated in such a way as to covalently attach MMPI to collagen used in a particular application. MMPI can be linked to collagen directly or through a linker molecule (e.g., polyethylene glycol). Once the collagen-MMP prodrug system is introduced and/or administered to the desired site, MMPI can inhibit MMPs while still attached to collagen, or it can inhibit MMPs after it has been cleaved (hydrolytically and/or enzymatically) from collagen.

For TIMP, a heterobifunctional crosslinking agent (e.g., thio-EMCS [ Pierce ]) may be used to covalently bind TIMP to collagen. More specifically, the TOMP can react with the thio-EMCS, whereby the maleimide group reacts with the-SH group of the cysteine contained in the TIMP sequence. The activated TIMP may then be reacted with the collagen solution. The collagen-TIMP conjugate may then be used for tissue augmentation applications.

2. Additional compositions

In certain embodiments of the present invention, the collagen/MMPI compositions provided herein may be further modified to enhance their utility. For example, in one embodiment a dye or other coloring agent may be added to enhance the color development of the collagen/MMPI composition. The dye or colorant may be permanent or transient (e.g., methylene blue). In other embodiments, compounds or factors that aid in coagulation (e.g., thrombin) may be added to the compositions described herein.

IV. Clinical application

1. Skin injection

Various injectable collagen products have been developed for soft tissue augmentation to correct facial scarring, reduce facial lines and augment lips. Specifically, the implant is shown to be used to treat various topographical defects, including (but not limited to) correcting acne scars, atrophy from disease or trauma, glabellar wrinkles, nasolabial folds, or defects following rhinoplasty, skin grafting, or other surgical procedures, and other soft tissue defects.

Several commercially available products were used for this purpose, including Zyderm I * (3.5% bovine collagen saline with 0.3% lidocaine), Zyderm II * (6.5% bovine collagen), Zyplast * (mcghan medical Corporation; 3.5% bovine skin collagen cross-linked with glutaraldehyde, dispersed in phosphate buffered saline with 0.3% lidocaine) and fibre (serno-gelatin, a combination of epsilon-amino-hexanoic acid and saline, combined with the patient's plasma in a 1: 1 ratio prior to injection). Other collagen-based injectable products, including those derived from non-bovine or human sources, may also be used in this embodiment.

Unfortunately, repeated "trimming" operations are often required because the implant is colonized by host connective tissue cells and inflammatory cells (conize), which produce metalloproteinases, such as collagenase that degrades collagen implants over time. Injectable collagen containing a metalloproteinase inhibitor (MMPI), alone or in a sustained release formulation (as described above), will result in an implant with increased durability and reduced number of subsequent repeated injections.

Although any of the above-described metalloprotease inhibitors may be suitably added to the skin collagen injection, the following are particularly preferred: TIMP-1, tetracycline, doxycycline, minocycline, batimastat *; marimastat *; ro-1130830, CGS 27023A, BMS-275291, CMT-3, solistat, ilomastat, CP-544439, prinomastat, PNU-1427690, SU-5402, and tocatet.

Regardless of the formulation used, administration of MMPI-loaded collagen injections will be performed in the following manner. Before applying the material, the patient should have completed two skin tests (performed in 2 weeks) to detect allergy. If these tests are negative, an MMPI loaded injection may be administered to the patient. A frozen pre-loaded syringe is used, with a fine gauge needle (30 or 32 gauge), containing no more than 309cc of implant material. The patient is placed in a sitting position with the table top tilted slightly backwards. Topical lidocaine and/or prilocaine can be used for anesthesia. The needle is inserted and advanced into the epidermal tissue at an angle to the skin. Sufficient implant material is extruded to repair the soft tissue topographical defects. In the case of Zyderm * loaded with MMPI, overcorrection is required (injecting more material than is ultimately needed) because a significant portion of the injected material dissipates within hours after injection. Zyplast * loaded with MMPI is typically used to correct deeper streaks and is injected deeper into the skin. Because the material is more rigid, no overcorrection is necessary.

As noted above, subsequent trim injections may be required to maintain maximum correction. However, a collagen injection loaded with a metalloprotease inhibitor will last longer than its unloaded counterpart, will provide long-term correction and reduce the need for repeat injections.

The total amount of injected material depends on the location of the corrected topographical defect; however, the total amount of injected material should not exceed 33cc for collagen-based products. The following MMPI loaded compositions will be described on a per cc dose basis.

a. Collagen skin injection loaded with marimastat *

A preferred composition is a collagen/saline suspension of 0.001% -30% marimastat * per cc (i.e. 1. mu.g-30 mg marimastat by weight). A particularly preferred dose is 0.01-1.5% marimastat * (i.e. 10. mu.g to 1.5mg) per cc of collagen/saline suspension. Thus, the total dose delivered in 30cc therapy will not exceed 45mg (or less than the 50mg daily single dose determined to be well tolerated). In one embodiment, 0.001-30% marimastat is loaded in PLGA microspheres or other polymer based microspheres, which are reloaded in collagen, in order to produce a sustained release of the material over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

b. Collagen injection loaded with batimastat *

A preferred composition is 0.001% to 30% batimastat * (i.e. 1 μ g to 30mg batimastat by weight) per cc of collagen/saline suspension. A particularly preferred dose is 0.01-5% (i.e., 10. mu.g to 5mg by weight) per cc of collagen/saline suspension. Thus, the total dose delivered in 30cc therapy will not exceed 150mg of batimastat * (or be less than 300mg/m with established good tolerability)²A single dose). In one embodiment, the height is adjustedInsoluble batimastat * was loaded in PLGA microspheres or other polymer-based microspheres, which were reloaded in collagen in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

c. Collagen skin injection loaded with doxycycline

Preferred compositions are 0.001% -30% doxycycline (1 μ g-30mg by weight doxycycline) per cc of injectable collagen/saline suspension. A particularly preferred dose is 0.01-3% (10. mu.g to 3mg doxycycline by weight) per cc of collagen/saline suspension. Thus, the total dose administered in 30cc of treatment will not exceed 90mg (or less than a well-tolerated daily dose of 100 mg). In one embodiment, from 0.001% to 30% of doxycycline is loaded into PLGA microspheres or other polymer-based microspheres, which are then loaded into collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

d. Skin collagen injection loaded with tetracycline

Preferred compositions are 0.001% -30% tetracycline (1 μ g-30mg tetracycline by weight) per cc of injectable collagen/saline suspension. A particularly preferred dose is 0.01-30% tetracycline (10. mu.g to 30mg tetracycline by weight) per cc of collagen/saline suspension. Thus, the total dose administered in 30cc of treatment will not exceed 900mg (or less than a well-tolerated daily dose of 1 g). In one embodiment, 0.001% -30% tetracycline is loaded into PLGA microspheres or other polymer-based microspheres, which are reloaded into collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

e. Epidermal collagen injection loaded with minocycline

Preferred compositions are 0.001% -30% minocycline (1 μ g-30mg minocycline by weight) per cc of injectable collagen/saline suspension. A particularly preferred dose is 0.01-6% minocycline (10 μ g to 6mg by weight minocycline) per cc of collagen/saline suspension. Thus, the total dose administered in 30cc of treatment will not exceed 180mg (or less than a well-tolerated daily dose of 200 mg). In one embodiment, 0.001% -30% minocycline is loaded into PLGA or other polymer-based microspheres, which are reloaded into collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

f. Tocatel-loaded epidermal collagen injection

Preferred compositions are 0.001% -30% tokacart (1. mu.g-30 mg of tokacart by weight) per cc of injectable collagen/saline suspension. A particularly preferred dose is 0.01-5% tokkat (10. mu.g to 5mg of tokkat by weight) per cc of collagen/saline suspension. Thus, the total dose administered in 30cc of treatment will not exceed 150 mg. In one embodiment, 0.001% -30% of tocatel is loaded into PLGA microspheres or other polymer-based microspheres, which are then loaded into collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

2. Urinary incontinence

Injectable collagen is often used to treat urinary incontinence. The following embodiments detail the composition of metalloprotease inhibitor-loaded collagen products and their methods of use in treating this common medical condition.

Briefly, incontinence or involuntary loss of urine is a common medical condition affecting 20% of women and 1-2% of men at some time during their life. The most common form of urinary incontinence is stress incontinence, or inadvertent urine leakage in response to activity that causes an increase in intra-abdominal pressure, such as sneezing, coughing or straining (straining). This occurs when the pressure within the bladder (pressure within the bladder) exceeds the pressure in the urethra, forcing urine from the bladder into the urethra without detrusor (bladder muscle) contractions. Several diseases are believed to lead to stress urinary incontinence, including:

(1) the bladder neck descends and the internal urethral spinocter exits the abdomen.

(2) Internal urethral spinocter disorder due to trauma, surgery, childbirth, or malignancy.

Corrective measures are primarily intended to support the urethra and bladder neck of the abdominal cavity by surgical or non-surgical methods. The second approach involves the use of urethral bulking agents (including collagen) which are designed to increase urethral pressure and reduce urinary incontinence.

Although periurethral and transurethral collagen injections have been used with great success in managing stress urinary incontinence, most cases require more than one treatment due to the limited durability of collagen implants. The use of MMPI-based collagen injections can maintain the activity of the implant and reduce the need and frequency of subsequent periurethral and transurethral injections.

Several commercially available collagen-based products are available for the treatment of stress urinary incontinence. Contigen * (35 mg/ml purified bovine glutaraldehyde crosslinked collagen dispersed in phosphate buffered saline obtained by CR Bard *) was widely used as a bulking agent. Other collagen-based injectable products, including those derived from non-bovine, human or recombinant sources, may also be used in this embodiment. Using Contigen *, crosslinked collagen starts to degrade within about 12 weeks and completely degrades within 10-19 months. Although the percentage of improvement in their incontinence initially showed after treatment was 58-100%, collagen resorption caused the need for repeated procedures in the above time intervals in most patients. In the present invention, MMPI is added to an injectable based on a sustained release form of collagen to reduce the rate of degradation of the implant and prolong its in vivo activity beyond that seen with collagen alone (e.g., consistently greater than 1 year in most patients and greater than 2 years in a substantial proportion of other patients).

Transurethral technique：

Regardless of the formulation used, the MMPI loaded collagen was administered via urethral injection in the following manner. Before applying the material, the patient should have completed two skin tests (performed 2 weeks apart) to detect allergy. If these tests are negative, an MMPI-loaded collagen injection can be administered to the patient. A frozen single use pre-loaded syringe with a fine gauge needle (23 gauge transurethral needle, with a stabilizing cannula) containing 2.5ml of implant material was used. The patient was placed at the site of cystectomy and 10ml of 2% lidocaine was injected into the urethra for anesthesia. In women, the bladder neck is viewed with a cystoscope. The needle was inserted through the injection port of the cystoscope at an acute angle at 4 o' clock into the plane directly below the bladder mucosa at a distance of 1-1.5cm from the bladder neck. The needle was then advanced with the cystoscope parallel to the long axis of the urethra until it was just below the bladder neck mucosa. MMPI loaded collagen was slowly injected into the site. The operation is then repeated at the 8 o' clock position. Methylene blue or other non-toxic coloring agents may be added to the implant to aid in the development of the injectable formulation.

Periurethral injection

Periurethral injection of MMPI loaded collagen can also be used to treat incontinence. As mentioned above, the patient should have completed two skin tests (performed 2 weeks apart) to detect allergy prior to application of the material. If these tests are negative, an MMPI-loaded collagen injection can be administered to the patient. A frozen single use pre-loaded syringe with a fine gauge needle (periurethral needle) containing 2.5ml of implant material was used. The patient was placed at the site of cystectomy and 10ml of 2% lidocaine was injected into the urethra for anesthesia, and the bladder neck was observed with cystoscopy (the urethra can also be observed by suprapubic cystoscopy in males). The needle is inserted transvaginally or suprapubically into the area immediately adjacent and lateral to the urethra. When it reaches the appropriate location near the bladder neck (as viewed with a cystoscope and as described above), MMPI loaded collagen is slowly injected into the site. Methylene blue or other non-toxic coloring agents may be added to the implant to aid in the development of the injectable formulation.

Although any MMPI-loaded collagen injection that is possible may be suitable for transurethral or periurethral treatment of incontinence, MMPI such as TIMP-1, tetracycline, doxycycline, minocycline, batimastat *, marimastat *, Ro-1130830, CGS 27023A, BMS-275291, CMT-3, solistat, ilomastat, CP-544439, prinomastat, PNU-1427690, SU-5402, and tokacart are particularly preferred. The following compositions are ideally suited for use as urethral bulking agents:

a. collagen periurethral/transurethral injection loaded with marimastat *

A preferred composition is a collagen/saline suspension of 0.001% -30% marimastat * per cc (i.e. 1. mu.g-30 mg marimastat by weight). A particularly preferred dose is 0.01-1.5% marimastat * (i.e. 10. mu.g to 1.5mg) per mL of collagen/saline suspension. Thus, the total dose delivered in 2.5ml treatment will not exceed 45mg (or less than the 50mg daily single dose determined to be well tolerated). In one embodiment, 0.001-30% marimastat is loaded in PLGA microspheres or other polymer based microspheres, which are reloaded in collagen, in order to produce a sustained release of the material over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

b. Collagen periurethral/transurethral injection loaded with batimastat *

A preferred composition is 0.001% to 30% batimastat * (i.e. 1 μ g to 30mg batimastat by weight) per cc of collagen/saline suspension. A particularly preferred dose is 0.01-30% (10. mu.g to 30mg by weight) per mL of collagen/saline suspension. Thus, the total dose delivered in 2.5cc therapy will not exceed 75mg of batimastat * (or be less than 300mg/m with established good tolerability)²A single dose). In one embodiment, 0.001-30% batimastat * is loaded in PLGA microspheres or other polymer based microspheres, which are reloaded in collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

c. Collagen periurethral/transurethral injection loaded with doxycycline

Preferred compositions are 0.001% -30% doxycycline (1 μ g-30mg by weight doxycycline) per mL injectable collagen/saline suspension. A particularly preferred dose is 0.01-3% doxycycline (10. mu.g to 3mg doxycycline by weight) per mL of collagen/saline suspension. Thus, the total dose administered in 2.5mL treatment will not exceed 75mg (or less than a well-tolerated daily dose of 100 mg). In one embodiment, from 0.001% to 30% of doxycycline is loaded into PLGA microspheres or other polymer-based microspheres, which are then loaded into collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

d. Collagen periurethral/transurethral injection loaded with tetracycline

A preferred composition is 0.001% -30% tetracycline (1. mu.g-30 mg tetracycline by weight) per mL of injectable collagen/saline suspension. A particularly preferred dose is 0.01-30% tetracycline (10. mu.g to 30mg tetracycline by weight) per mL of collagen/saline suspension. Thus, the total dose administered in 2.5mL treatment will not exceed 75mg (or less than a well-tolerated daily dose of 1 g). In one embodiment, 0.001% -30% tetracycline is loaded into PLGA microspheres or other polymer-based microspheres, which are reloaded into collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

e. Collagen periurethral/transurethral injection loaded with minocycline

Preferred compositions are 0.001% -30% minocycline (1 μ g-30mg minocycline by weight) per cc of injectable collagen/saline suspension. A particularly preferred dose is 0.01-6% minocycline (10 μ g to 6mg by weight minocycline) per cc of collagen/saline suspension. Thus, the total dose administered in 30cc of treatment will not exceed 180mg (or less than a well-tolerated daily dose of 200 mg). In one embodiment, 0.001% -30% minocycline is loaded into PLGA or other polymer-based microspheres, which are reloaded into collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

f. Tukatide-loaded periurethral/transurethral injection containing epidermal collagen

Preferred compositions are 0.001% -30% tokacart (1. mu.g-30 mg of tokacart by weight) per mL of injectable collagen/saline suspension. A particularly preferred dose is 0.01-5% tokkat (10. mu.g to 5mg of tokkat by weight) per mL of collagen/saline suspension. Thus, the total dose administered in 2.5mL treatment will not exceed 75 mg. In one embodiment, 0.001% -30% of tocatel is loaded into PLGA microspheres or other polymer-based microspheres, which are then loaded into collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

3. Surgical sealant

Collagen has been widely used as a surgical sealant; particularly as a vascular sealant to stop bleeding after a femoral puncture into a blood vessel and to stop bleeding during surgery.

Cannulation of the femoral artery is the initial step in obtaining access to the vascular system as part of many conventional medical procedures such as, for example, modular coronary angiography using a stent graft, cerebrovascular angiography, coronary angioplasty, coronary stenting, cerebrovascular aneurysm repair, abdominal aneurysm repair, and several other procedures. For many of these indications, larger devices must be introduced into the femoral artery, requiring an "off-site" approach on the artery. Once the intervention has been completed and the catheter hub removed, it is often difficult to pack bleeding from the bony spur site (particularly because many patients are on anticoagulant therapy). Collagen-based vascular sealants have been developed for application at puncture sites to "seal" wounds and initiate healing of arteriotomies. This can allow the patient to walk more quickly and prevent serious complications such as hematoma formation, or in severe cases bleeding and massive blood loss. Hemostatic collagen sealants are also used during surgery to seal the adventitia (external) or cutting surface of blood vessels, bones and tissues as an aid to hemostasis when controlling bleeding by a bandage is ineffective or impossible. These products are used in cardiovascular, systemic, hepatic and orthopedic surgical procedures.

Several collagen-based blocking agents are commercially available, including Vasoseal^TM(produced by Datascope *) and CoStasis^TM(manufactured by Cohesion Technologies *). The production of MMPI loaded collagen-based sealant will prolong the activity of the collagen implant and allow for a complete healing process prior to resorption of the implant. This may be particularly useful in controlling surgical bleeding, where the vascular repair site may not be readily accessible post-operatively.

Vasoseal^TMIs an example of a collagen "embolization" (plug) kit for femoral artery puncture repair. Briefly, an arteriotomy-locator is inserted into the cannula using an introducer prior to removal of the vascular access cannula. Once the arteriotomy positioner is properly moved to the site, the operating cannula and introducer are removed with the artery compressed. Advancing a tissue dilator over the positioner and advancing a cannula over the dilator such that the cannula is positioned over the outer surface of the site of the arteriotomy; the positioner and dilator are then removed. A collagen cartridge (containing 80-100mg of purified bovine collagen plug) was inserted into the cannula and the collagen plug was injected over the arteriopuncture wound (2 injections may be required). The MMPI loaded femoral artery sealant was used in exactly the same way, but still remained longer than the site allowing complete healing to occur, thus reducing the risk of later rebleeding. Examples of embolic formulations of MMPI loaded collagen are provided below.

CoStasis^TMIs a sprayable liquid, is an example of a collagen-based surgical sealant for hemostasis that would benefit from the addition of MMPI (see, e.g., U.S. patent nos. 5,290,552, 5,614,587, 5,744,545, 5,786,421, 5,936,035, 6,096,309, and 6,280,727). To use the system, the patient's own plasma is collected and drawn into a syringe connected to the connection device. The collagen suspension (in 40mM CaCl)₂20mg/mL bovine collagen in buffer and at least 300U/mL bovine thrombin) syringe was attached to the other portion of the connector. The coupling device mixes the contents of the collagen/thrombin syringe with the contents of the patient plasma syringe. Conversion of autologous fibrinogen to fibrin egg by bovine thrombinLeukocytes, which form a collagen/fibrin gel matrix in the presence of collagen, adhere to the bleeding site. The mixture was then sprayed by syringe at the bleeding site. MMPI was added as a component of the collagen/thrombin suspension as described above. Collagen sealants loaded with MMPI will have greatest utility in surgical procedures where long-term hemostasis may be required until tissue healing occurs.

a. Marimastat * -loaded collagen surgical sealant

A preferred composition is 0.001% -30% marimastat * per mL (i.e., 1. mu.g-30 mg marimastat by weight) of collagen/thrombin suspension (in 40mM CaCl)₂20mg/mL bovine collagen in buffer and at least 300U/mL bovine thrombin). A particularly preferred dose is 0.01-10% marimastat * (i.e. 10. mu.g to 10mg) per mL of collagen/thrombin suspension. Thus, the total dose delivered in 5.0ml treatment will not exceed 50mg (or equal to a single daily dose of 50mg for established good tolerance). In one embodiment, 0.001-10% marimastat is loaded in PLGA microspheres or other polymer-based microspheres, which are loaded in a collagen/thrombin suspension in order to produce a sustained release of the material over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

b. Palmastat * -loaded collagen surgical sealant

A preferred composition is 0.001% -30% batimastat * (i.e., 1. mu.g-30 mg batimastat * by weight) per mL of collagen/thrombin suspension (in 40mM CaCl)₂20mg/mL bovine collagen in buffer and at least 300U/mL bovine thrombin). A particularly preferred dose is 0.01-30% (10. mu.g to 30mg by weight) per mL of collagen/thrombin suspension. Thus, the total dose delivered in 5mL treatment will not exceed 150mg of batimastat * (or be less than 300mg/m with established good tolerability)²A single dose).In one embodiment, 0.001-30% batimastat * is loaded in PLGA microspheres or other polymer-based microspheres, which are loaded in a collagen/thrombin suspension in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

c. Doxycycline-loaded collagen surgical sealant

A preferred composition is 0.001% -20% doxycycline (1. mu.g-30 mg by weight doxycycline) per mL injectable collagen/thrombin suspension (in 40mM CaCl)₂20mg/mL bovine collagen in buffer and at least 300U/mL bovine thrombin). A particularly preferred dose is 0.01-20% doxycycline (10. mu.g to 20mg by weight doxycycline) per mL of collagen/thrombin suspension. Thus, the total dose administered in 5mL treatment will not exceed 100mg (or equal to a well tolerated daily dose of 100 mg). In one embodiment, from 0.001% to 20% of doxycycline is loaded into PLGA microspheres or other polymer-based microspheres, which are then loaded into a collagen/thrombin suspension in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

d. Tetracycline loaded collagen surgical sealant

A preferred composition is 0.001% -30% tetracycline (1. mu.g-30 mg tetracycline by weight) per mL injectable collagen/thrombin suspension (in 40mM CaCl)₂20mg/mL bovine collagen in buffer and at least 300U/mL bovine thrombin). A particularly preferred dose is 0.01-30% tetracycline (10. mu.g to 30mg tetracycline by weight) per mL of collagen/thrombin suspension. Thus, the total dose administered in 5mL treatment will not exceed 150mg (or less than a well-tolerated daily dose of 1 g). In one implementationIn a protocol, 0.001% -30% tetracycline is loaded into PLGA microspheres or other polymer-based microspheres, which are then loaded into a collagen/thrombin suspension to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

e. Minocycline loaded collagen surgical sealant

A preferred composition is 0.001% -30% minocycline (1. mu.g-30 mg by weight minocycline) per mL injectable collagen/thrombin suspension (in 40mM CaCl)₂20mg/mL bovine collagen in buffer and at least 300U/mL bovine thrombin). A particularly preferred dose is 0.01-20% minocycline (10. mu.g to 20mg minocycline by weight) per mL of collagen/thrombin suspension. Thus, the total dose administered in 5mL treatment will not exceed 100mg (or be less than a well-tolerated daily dose of 200 mg). In one embodiment, 0.001% -30% minocycline is loaded into PLGA microspheres or other polymer-based microspheres, which are then loaded into a collagen/thrombin suspension to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

f. Tocatel-loaded epidermal collagen surgical sealant

A preferred composition is 0.001% -30% tokkat (1. mu.g-30 mg tokkat by weight) per mL injectable collagen/thrombin suspension (in 40mM CaCl)₂20mg/mL bovine collagen in buffer and at least 300U/mL bovine thrombin). A particularly preferred dose is 0.01-10% tocatel (10. mu.g to 10mg of tocatel by weight) per mL of collagen/thrombin suspension. Thus, the total dose administered in 5mL treatment will not exceed 50 mg. In one embodiment, 0.001 will be used% -30% of tocatel loaded in PLGA microspheres or other polymer-based microspheres, which are loaded in a collagen/thrombin suspension in order to produce a sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

g. Marimastat * -loaded collagen strand puncture sealant

A preferred composition is 0.001% -10% marimastat * (i.e. 1. mu.g-30 mg marimastat by weight) per dose of collagen (80-100mg collagen plug). A particularly preferred dose is 0.01-10% marimastat * (i.e. 10. mu.g to 10mg) per collagen plug. In one embodiment, 0.001-10% marimastat is loaded in PLGA microspheres or other polymer based microspheres, which are reloaded in collagen, in order to produce a sustained release of the material over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

h. Collagen femoral puncture sealant loaded with batimastat *

A preferred composition is 0.001% -30% batimastat * (i.e. 1. mu.g-30 mg batimastat * by weight) per dose of collagen (80-100mg collagen plug). A particularly preferred dose is 0.01-30% (10. mu.g to 30mg by weight) per collagen plug. In one embodiment, 0.001-30% batimastat * is loaded in PLGA microspheres or other polymer based microspheres, which are reloaded in collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

i. Collagen femoral puncture closure loaded with doxycyclineAgent for treating cancer

A preferred composition is 0.001% -20% doxycycline (1. mu.g-30 mg by weight doxycycline) per dose of collagen (80-100mg collagen plugs). A particularly preferred dose is 0.01-20% doxycycline (10. mu.g to 20mg by weight doxycycline) per collagen plug. In one embodiment, from 0.001% to 20% of doxycycline is loaded into PLGA microspheres or other polymer-based microspheres, which are then loaded into collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

j. Collagen femoral puncture sealant loaded with tetracycline

A preferred composition is 0.001% -30% tetracycline (1. mu.g-30 mg tetracycline by weight) per dose of collagen (80-100mg collagen plug). A particularly preferred dose is 0.01-30% tetracycline (10. mu.g to 30mg tetracycline by weight) per collagen plug. In one embodiment, 0.001% -30% tetracycline is loaded into PLGA microspheres or other polymer-based microspheres, which are reloaded into collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

k. Collagen femoral puncture sealant loaded with minocycline

A preferred composition is 0.001% -30% minocycline (1. mu.g-30 mg minocycline by weight) per dose of collagen (80-100mg collagen plug). A particularly preferred dose is 0.01-20% minocycline (10 μ g to 20mg minocycline by weight) per collagen plug. In one embodiment, 0.001% -30% minocycline is loaded into PLGA or other polymer-based microspheres, which are reloaded into collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

l. Tocate-loaded epidermal collagen protein femoral puncture sealant

Preferred compositions are 0.001% -30% tokkat (1. mu.g-30 mg tokkat by weight) per dose of collagen (80-100mg collagen plugs). A particularly preferred dose is 0.01-10% tokkat (10. mu.g to 10mg of tokkat by weight) per collagen plug. In one embodiment, 0.001% -30% of tocatel is loaded into PLGA microspheres or other polymer-based microspheres, which are then loaded into collagen, in order to produce sustained release of the drug over a period of days to months. Injectable collagen of any origin (e.g., bovine, human, or recombinant; crosslinked or uncrosslinked) will be suitable for combination with the above to produce the desired end product.

It should be readily apparent to one skilled in the art that any of the above MMPI, or derivatives or analogs thereof, can be used to produce variations of the above compositions without departing from the spirit and scope of the invention.

Examples

Example 1

Preparation of collagen

Collagen source

Peeling from freshly killed rabbits. Shaving the removed skin, defatting by sharp peeling and cutting into 2cm²And (4) a square shape. Skin squares were freeze dried at ambient temperature for 24 hours and then in solid CO₂Ground in a mill with the aid of a mill to produce a powder.

Dissolution

A suspension of powdered skin was prepared by adding the powdered material to a 0.5M acetic acid solution to give a skin concentration of 5g dry weight skin/liter. The suspension was cooled to 10 ℃. Freshly prepared pepsin solution (0.5 g in 10ml 0.01N HCl) was added to the skin suspension and the mixture was incubated at 10 ℃ for 5 days with occasional stirring.

Pepsin removal

After the enzymatic treatment, the remaining pepsin in the mixture was denatured by adding 5ml Tris base and adjusting the pH to 7.0 with 3N NaOH at 4 ℃.30 g NaCl was stirred into the mixture to maintain the collagen in solution. After 4 hours, the mixture was centrifuged at 30,000g for 30 minutes to remove precipitated pepsin.

Purification of

The enzymatically treated collagen was precipitated from the supernatant by adding an additional 140g of NaCl. The solution was stirred and allowed to stand at 4 ℃ for 4 hours. The precipitated collagen was centrifuged at 30,000g for 30 minutes. The collagen precipitate obtained was resuspended in 200ml of deionized water. 0.5N acetic acid was added to make the final volume one liter. The collagen was precipitated from the solution by adding 50g of NaCl, allowing the solution to stand at 4 ℃ for 5 hours and centrifuging at 30,000g for 30 minutes.

Sterilization

The collagen pellet was resuspended in 200ml distilled water, transferred to a sterilized dialysis tube and dialyzed against 50 volumes of 1N acetic acid for 72 hours. Collagen was then dialyzed against 50 volumes of 0.001N acetic acid for 24 hours, during which time the solution was changed 3 times. The dialysis solution was then concentrated by placing the dialysis tube on a sterile absorbent towel in a laminar flow bacterial barrier until a concentration of 12-15mg collagen/ml solution was reached. The concentrated solution was then dialyzed against 50 volumes of 0.001N acetic acid for 24 hours. The collagen solution was then stored in sterile vials at 4 ℃.

Adding a polymerization accelerator to the concentrate

A buffered saline solution (NaCl 2.5mM/l, NaHPO) was added at 4 ℃ just prior to use₄0.1mM/l, pH7.4) was added to the collagen solution in a volume to volume ratio of 10: 1 (collagen: buffer) and the buffered concentrate was transferred to a cooled (4 ℃ C.) syringe. For certain applications (e.g., cosmetic tissue augmentation), the buffered saline solution may also contain 0.3-1% (w/v) local anesthetic (e.g., lidocaine).

Example 2

Preparation of TIMP-1 loaded microspheres using the W/O/W method

Specifically, 100mg of 50/50PLGA polymer (IV ═ 0.15) was added to 12mL of dichloromethane. To this was added 800. mu.L of Phosphate Buffered Saline (PBS) solution or TIMP-1 in PBS (concentration typically 1-10 mg/mL). The mixture was then homogenized (20 seconds at 6,000 rpm). Once formed, the solution was dispersed into 100mL of 1.0% aqueous polyvinyl alcohol (PVA) solution and immediately homogenized (40 seconds at 8,000 rpm) to form an aqueous-in-oil-in-water multiple emulsion. Polydisperse particles (mostly less than 10 microns in size) are formed under these conditions. The solvent was then slowly removed by evaporation and the microspheres were collected by centrifugation. The particles were washed (5 times) with deionized water and then frozen in a dry ice/acetone bath, freeze-dried overnight to yield a white free-flowing powder of microspheres.

Microspheres with longer degradation curves were prepared using the method described above using 85/15 PLGA (IV ═ 0.68).

The above method may also be used to prepare microspheres comprising TIMP-2, TIMP-3 and TIMP-4.

Example 3

Preparation of tetracycline loaded microspheres using the W/O/W method

Tetracycline loaded microspheres were prepared in a manner similar to that described in the examples above, except that tetracycline hydrochloride was used.

Example 4

Preparation of doxycycline-loaded microspheres using the W/O/W method

Microspheres loaded with doxycycline were prepared in a manner similar to that described in the examples above, except that doxycycline hydrochloride was used.

Example 5

Preparation of minocycline-loaded microspheres using a W/O/W process

Minocycline-loaded microspheres were prepared in a manner similar to that described in the examples above, except that minocycline hydrochloride was used.

Example 6

Preparation of batimastat loaded microspheres using an oil in water process

Preparation of PVA solution

In a 1000ml beaker, 1000ml of distilled water and 100g of PVA (Aldrich 13-23K, 98% hydrolysis) were added. Place a 2 "stir bar in the beaker. The suspension was heated to 75-80 ℃ while stirring. Once the PVA was completely dissolved (a clear solution formed), the PVA solution (w/v) was cooled to room temperature and filtered through a syringe inline filter.

Preparation of PLGA solution with batimastat

100mg of batimastat and 900mg of PLGA (50/50, IV ═ 0.15) were weighed and transferred to a 20ml scintillation vial. 10mL of HPLC grade Dichloromethane (DCM) was added to the vial to dissolve the PLGA and batimastat. The sample was placed on an orbital shaker (device 4) until the polymer and batimastat dissolved.

Preparation of microspheres with a diameter of less than 25 μm

100ml of 10% PVA solution was transferred to a 400ml beaker. The beaker was held upright using double-sided tape. A 3-blade stir bar was placed in the beaker and adjusted to a height of about 0.5cm above the bottom of the beaker. The stirrer motor (Dyna-Mix from Fisher Scientific) was started to 2.5. 10ml of PLGA/batimastat solution was poured into the PVA solution during stirring. The stirring speed was gradually increased to a fixed value of 5. Stirring is continued for 2.5-3.0 hours. The resulting microspheres were filtered through 2 metal sieves (53 μm (top) and 25 μm (bottom)) into a 100ml beaker to remove any large size material. The microspheres were washed with distilled water while filtering. The microspheres collected in the filtrate were centrifuged (1000rpm, 10min.) to precipitate the microspheres. The supernatant was removed using a pasteur pipette and the pellet resuspended with 100ml of distilled water. This process was repeated 2 additional times.

The washed microspheres were transferred to a glass container. The transfer was accomplished by rinsing the beaker with a small amount of distilled water (20-30 ml). The container was sealed with Parafilm and placed in a freezer at-20 ℃ overnight. The frozen solution of microspheres was then freeze-dried using a freeze-dryer for about 3 days. The dried microspheres were transferred to a 20ml scintillation vial and stored at-20 ℃. The microspheres are then terminally sterilized by irradiation with at least 2.5Mrad cobalt-60 (Co-60) x-rays.

Example 7

Preparation of Marimastat-loaded microspheres using an oil-in-water process

Marimastat-loaded microspheres were prepared in a similar manner to that described in the above examples, except that marimastat was used instead of batimastat.

Example 8

Preparation of tocatel-loaded microspheres using an oil-in-water process

The preparation of tolcatide-loaded microspheres was carried out in a similar manner to that described in the above examples, except that tolcatide was used instead of batimastat.

Example 9

Preparation of collagen solution containing micellar batimastat

Preparation of polymers

The polymer was synthesized by main (bulk) ring opening polymerization using DL-lactide and methoxy poly (ethylene glycol) [ MePEG 2000] in the presence of 0.5% w/w stannous octoate.

Briefly, the reaction glassware was washed and rinsed with sterile water from Irrigation USP, dried at 37 deg.C, and then depyrogenated at 250 deg.C for at least 1 hour. MePEG 2000 and DL-lactide (240 g and 160g, respectively) were weighed and transferred to a round bottom flask using a stainless steel funnel. A2-inch Teflon * -coated magnetic stir bar was added to the flask. A glass stopper was used to seal the flask, which was then immersed in a preheated oil bath up to the neck. The oil bath was maintained at 140 ℃ using a temperature controlled electric furnace. After the MePEG and DL-lactide had melted and reached 140 ℃, 2mL of 95% stannous octoate (catalyst) was added to the flask. The flask was shaken vigorously immediately after addition to ensure rapid mixing, and then returned to the oil bath. The reaction was allowed to proceed for 6 hours with heating and stirring. The liquid polymer was then poured into a stainless steel tray, covered and left in the fume hood overnight (about 16 hours). The polymer solidifies in the pan. The plate top was sealed using Parafilm *. The sealed polymer-containing trays were placed in a freezer at-20 ℃. + -. 5 ℃ for 0.5 hour.

The polymer was then removed from the refrigerator and transferred to glass storage bottles and stored at 2-8 ℃.

Preparation of micellar batimastat (batimastat/polymer matrix)

The reaction glassware was washed and rinsed with sterile water from Irrigation USP, dried at 37 deg.C, and then depyrogenated at 250 deg.C for at least 1 hour. First, a phosphate buffer, 0.08M, pH7.6 was prepared. The buffer was dispersed in a volume of 1mL per vial. The vial was heated at 90 ℃ for 2 hours to dry the buffer. The temperature was then raised to 160 ℃ and the vial was dried for an additional 3 hours.

The polymer was dissolved in THF at a concentration of 10% w/v with stirring and heating. The polymer solution was then centrifuged at 3000rpm for 30 minutes. The supernatant was decanted and allowed to stand. Additional THF was added to precipitate and centrifuged a second time at 3000rpm for 30 minutes. The second supernatant and the first supernatant were combined. Batimastat was weighed and then the supernatants were added and combined. The solution was brought to the desired final concentration with THF to prepare a 9.9% polymer solution containing 1.1% batimastat.

To prepare a batch visualization of the final product vials, micellar batimastat was dispersed in vials containing dry phosphate buffer at a volume of 1mL per vial. The vial was placed in a 50 ℃ vacuum oven. The vacuum was maintained at-80 kPa and the vial was held in the oven overnight (15-24 hours). The vials were stoppered with Teflon-backed gray butyl stoppers and sealed with aluminum seals. The batimastat/polymer matrix was sterilized using 2.5Mrad gamma-irradiation. Each vial contained about 11mg of batimastat, 99mg of polymer, and 11mg of phosphate. Vials were stored at 2 ℃ to 8 ℃ until set up.

Preparation of micellar batimastat/collagen gel

In a sterile biosafety cabinet, 2ml of sterile saline was added to a vial (prepared as above) containing about 11mg of batimastat, 99mg of polymer and 11mg of phosphate. The contents of the vial were dissolved in 2mL of sterile saline by placing the vial in a 37 ℃ water bath for about 30 minutes with intermittent stirring. Using a sterile 1mL syringe, an aliquot of 1mL micellar batimastat solution was aspirated from the vial and injected into 29mL collagen gel. The samples were mixed to produce a homogeneous solution of micellar batimastat in the collagen gel. The samples were then loaded into 1mL syringes for in vivo experiments.

Example 10

Preparation of 2-component micelle kit

Preparation of freeze-dried micelle batimastat

A solid composition capable of forming micelles upon constitution with an aqueous medium containing collagen was prepared as follows:

briefly, 41.29g MePEG (MW 2,000g/mol) was mixed with 412.84g 60: 40 MePEG: the poly (DL-lactide) diblock copolymer (see examples provided above) was combined, heated to 75 ℃ in a mineral oil bath and stirred by overhead stirring blades. Once a clear liquid was obtained, the mixture was cooled to 55 ℃. To the mixture was added 45.87g of a 200ml solution of batimastat in tetrahydrofuran. The solvent was added at about 40ml/min and the mixture was stirred at 55 ℃ for 4 hours. After stirring for this time, the liquid composition was transferred to a stainless steel pan and placed in a forced air oven at 50 ℃ for about 48 hours to remove residual solvent. The composition was then cooled to room temperature and allowed to solidify to form a batimastat-polymer matrix.

Phosphate buffer was prepared by combining 237.8g of disodium phosphate heptahydrate, 15.18g of sodium dihydrogen phosphate monohydrate in 1600ml of water. To the phosphate buffer, 327g of batimastat-polymer matrix was added and stirred for 2 hours to dissolve the solid. After a clear solution was obtained, the volume was adjusted to 2000ml with additional water. Vials were filled with 15ml aliquots of this solution and freeze dried by cooling to-34 ℃ for 5 hours, heating to-16 ℃ while reducing the pressure to less than 0.2mm Hg for 68 hours, heating to 30 ℃ while maintaining the reduced pressure, followed by another 20 hours. The result is a freeze-dried matrix that can be built up to form a clear micellar solution.

Preparation of 2-component kit

Weigh 40mg of the freeze-dried micellar batimastat material into a capped 1mL syringe. The plunger was replaced and the syringe sealed in a plastic bag using a heat sealer. The samples were sterilized using 2.5Mrad gamma irradiation. Immediately prior to application, the plastic bag containing the sterile lyophilized material was opened and connected to a two-up syringe connector (supplier, cat #). A syringe containing 2mL of 3.5% bovine collagen (95% type I and 5% type III) was attached to the remaining end of the duplex syringe connector. The plunger of the syringe containing the collagen material is pushed in to transfer the collagen material to the syringe containing the micelle material. The material was transferred from one syringe to another until a homogeneous solution was obtained. The material was then transferred to a syringe that originally contained collagen. The syringe was disconnected from the connector and a 30 gauge needle was connected to the syringe. The material is now ready for use.

Example 11

Preparation of 2-component micelle kit

Weigh 40mg of the freeze-dried micellar batimastat material into a capped 1mL syringe. The plunger was replaced and the syringe sealed in a plastic bag using a heat sealer. The samples were sterilized using 2.5Mrad gamma irradiation. Immediately prior to use, the plastic bag containing the sterile lyophilized material was opened and connected to the duplex syringe connector. A syringe containing 2mL of 3.5% bovine collagen (95% type I and 5% type III) was attached to the remaining end of the duplex syringe connector. The plunger of the syringe containing the collagen material is pushed in to transfer the collagen material to the syringe containing the micelle material. The material was transferred from one syringe to another until a homogeneous solution was obtained. The material was then transferred to a syringe that originally contained collagen. The syringe was disconnected from the connector and a 30 gauge needle was connected to the syringe. The material is now ready for use.

Example 12

Liposome formulations

MLV liposomes

In a 50mL round bottom flask, a total of 100mg egg phosphatidylcholine (Avanti Polar Lipids) and cholesterol (Sigma) [ 5: 1 molar ratio ] was added to 5mL dichloromethane. Once dissolved, 3mg of batimastat was added to the solution. Solvent was removed using rotavap under low vacuum. The lipid-drug mixture is dried overnight under vacuum. 5mL of 0.9% NaCl solution was added to the dry lipid-drug mixture. The solution was slowly rotated for 1 hour using a rotavap and 37 ℃ water bath apparatus. When 5% maltose was added to the 0.9% NaCl makeup solution, the samples were frozen and freeze-dried in acetone dry ice to produce a solid product.

An amount of the freeze-dried material of microspheroidal batistat (prepared as described above) was weighed into a capped 1mL syringe according to the specific dosage required. The plunger was replaced and the syringe sealed in a plastic bag using a heat sealer. The samples were sterilized using 2.5Mrad gamma irradiation. Immediately prior to use, the plastic bag containing the sterile lyophilized material was opened and connected to the duplex syringe connector. Syringes containing 3.5% bovine collagen (95% type I and 5% type III) were attached to the remaining end of the duplex syringe connector. The plunger of the syringe containing the collagen material is pushed in to transfer the collagen material to the syringe containing the micelle material. The material was transferred from one syringe to another until a homogeneous solution was obtained. The material was then transferred to a syringe that originally contained collagen. The syringe was disconnected from the connector and a 30 gauge needle was connected to the syringe. The material is now ready for use.

SUV liposomes

The liposomes prepared above were reduced in size by placing the samples in an ultrasonic bath (45 ℃) for 10 minutes. The solution was changed from an opaque milky solution to a transparent blue solution. The solution is used as or freeze-dried to produce a solid product. The solid product can be used to prepare a collagen solution in a manner similar to that described above.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims (62)

1. A composition comprising collagen and at least one metalloproteinase inhibitor (MMPI).

2. The composition according to claim 1 wherein the MMPI is a tissue inhibitor of matrix metalloproteinase (TIMP).

3. The composition according to claim 2, wherein the TIMP is TIMP-1 or TIMP-2.

4. The composition according to claim 2, wherein said TIMP is TIMP-3 or TIMP-4.

5. A composition according to claim 1 wherein the MMPI is tetracycline, or an analogue or derivative thereof.

6. A composition according to claim 5 wherein the MMPI is tetracycline.

7. A composition according to claim 6, wherein the tetracycline is minocycline or doxycycline.

8. A composition according to claim 1 wherein the MMPI is a hydroxamate.

9. The composition according to claim 8 wherein the hydroxamate is batimastat, marimastat, or tocacatte.

10. The composition according to claim 1 wherein said MMPI is RO-1130830, CGS-27023A or BMS-275291.

11. The composition according to claim 1 wherein said MMPI is a polypeptide inhibitor.

12. The composition according to claim 11, wherein the polypeptide inhibitor is an inhibitor of metalloprotease maturase.

13. A composition according to claim 1 wherein the MMPI is a mercapto-based compound.

14. The composition according to claim 1 wherein the MMPI is a bisphosphonate with structure (I):

wherein R' and R "are independently hydrogen, halogen, hydroxy, optionally substituted amino, optionally substituted thio, or optionally substituted alkyl, alkanyl, alkenyl, alkynyl, alkadiyl, alkyleno, heteroalkyl, heteroalkanyl, heteroalkenyl, heteroalkynyl, heteroalkanediyl, heteroalkalkeno, aryl, aralkyl, heteroaryl, heteroaralkyl.

15. The composition according to claim 14 wherein said MMPI is a bisphosphonate with R' and R "being hydroxyl, hydrogen, or chlorine.

16. A composition according to claim 1 comprising at least two MMPI.

17. The composition according to claim 16 wherein said at least two MMPI's comprise tetracycline, or an analog or derivative thereof, and a bisphosphonate.

18. The composition according to claim 16 wherein said at least two MMPI's comprise tetracycline, or an analog or derivative thereof, and a hydroxymate.

19. A composition comprising collagen, at least one metalloproteinase inhibitor (MMPI), and at least one polymer.

20. The composition of claim 19, wherein the polymer is biodegradable.

21. The composition of claim 19, wherein the biodegradable polymer is selected from the group consisting of albumin, gelatin, starch, cellulose, dextran, polysaccharides, fibrinogen, poly (esters), poly (D, L lactide), poly (D, L-lactide-co-glycolide), poly (e-caprolactone), poly (hydroxybutyrate), poly (alkyl carbonate), poly (anhydride), or poly (orthoester), and copolymers and mixtures thereof.

22. The composition of claim 19, wherein the polymer is non-biodegradable, wherein the non-biodegradable polymer is selected from the group consisting of copolymers of ethylene oxide and propylene oxide, ethylene vinyl acetate copolymers, silicone rubber, poly (methacrylate) based polymers, and poly (acrylate) based polymers.

22. The composition of claim 20, wherein the polymer is in the form of a stick, pellet, tablet or capsule.

23. The composition of claim 20, wherein the polymer is in the form of a microsphere, a paste, a thermal paste, a thread, a film or a spray.

24. The composition of claim 1 wherein the MMPI is associated with the polymer by being enclosed in a polymer matrix, by covalent bonding, or by encapsulation.

25. The composition of claim 1 wherein the MMPI is covalently linked to collagen, either directly or through a linker.

26. The composition of claim 25 wherein said MMPI linked to collagen is released by chemical or enzymatic cleavage of a covalent bond.

27. The composition of claim 19, further comprising a matrix, wherein the MMPI is incorporated into the matrix, the matrix selected from the group consisting of carbohydrates, polysaccharides, starch, cellulose, dextran, methylcellulose, hyaluronic acid, polypeptides, albumin, collagen, and gelatin.

28. A composition according to claim 1, wherein the collagen is type I or type II.

29. A composition according to claim 19, wherein the collagen is type I or type II.

30. A composition according to any one of claims 1 to 29, wherein the composition is sterile.

31. A composition according to any one of claims 1 to 29, additionally comprising thrombin or a dye.

32. A composition according to any one of claims 1 to 29 which additionally comprises a pharmaceutically acceptable diluent, carrier or excipient.

33. A method for repairing or augmenting skin or tissue, comprising injecting into the skin or tissue a composition according to claim 30.

34. The method according to claim 33, wherein said injection is into the lips.

35. A method according to claim 33, wherein the injection is into the skin on the face.

36. A method for treating or preventing urinary incontinence, comprising administering to a patient a composition according to claim 30, such that said urinary incontinence is treated or prevented.

37. A method according to claim 36 wherein the composition is administered periurethrally.

38. The method according to claim 37, wherein the composition is administered transurethrally.

39. A method of sealing a surgical site comprising administering to a patient a composition according to claim 30.

40. A method according to claim 39 wherein said site is an area proximal to a blood vessel.

41. A method of making collajolie comprising admixing collagen and at least one MMPI.

42. A method according to claim 41 wherein the collagen is type I or type II collagen.

43. A method according to claim 41 wherein the MMPI is a tissue inhibitor of matrix metalloproteinase (TIMP).

44. The method according to claim 43, wherein said TIMP is TIMP-1 or TIMP-2.

45. The method according to claim 43, wherein said TIMP is TIMP-3 or TIMP-4.

46. A method according to claim 41 wherein the MMPI is tetracycline, or an analogue or derivative thereof.

47. A method according to claim 46 wherein the MMPI is tetracycline.

48. A method according to claim 47, wherein the tetracycline is minocycline or doxycycline.

49. The method according to claim 41 wherein said MMPI is a hydroxamate.

50. The method according to claim 49 wherein the hydroxamate is batimastat, marimastat, or tokacat.

51. The method of claim 41 wherein said MMPI is RO-1130830, CGS-27023A or BMS-275291.

52. The method according to claim 41, wherein said MMPI is a polypeptide inhibitor.

53. A method according to claim 52 wherein the polypeptide inhibitor is an inhibitor of the metalloprotease maturase.

54. The method according to claim 41, wherein said MMPI is a thiol-based compound.

55. The method according to claim 41 wherein said MMPI is a bisphosphonate having structure (I):

wherein R' and R "are independently hydrogen, halogen, hydroxy, optionally substituted amino, optionally substituted thio, or optionally substituted alkyl, alkanyl, alkenyl, alkynyl, alkadiyl, alkyleno, heteroalkyl, heteroalkanyl, heteroalkenyl, heteroalkynyl, heteroalkanediyl, heteroalkalkeno, aryl, aralkyl, heteroaryl, heteroaralkyl.

56. The method according to claim 55 wherein said MMPI is a bisphosphonate with R' and R "being hydroxy, hydrogen, or chlorine.

57. A method according to claim 41 comprising at least two MMPI.

58. A method according to claim 41 wherein the MMPI is first blended with at least one polymer prior to blending with the collagen.

59. The method of claim 58, wherein the polymer is biodegradable.

60. The method of claim 59 wherein the biodegradable polymer is selected from the group consisting of albumin, gelatin, starch, cellulose, dextran, polysaccharides, fibrinogen, poly (esters), poly (D, L lactide), poly (D, L-lactide-co-glycolide), poly (e-caprolactone), poly (hydroxybutyrate), poly (alkyl carbonate), poly (anhydride), or poly (orthoester), and copolymers and mixtures thereof.

61. The method of claim 41, further comprising the step of sterilizing the mixture.