WO1996016648A1

WO1996016648A1 - Method for regulating metalloproteinase activity

Info

Publication number: WO1996016648A1
Application number: PCT/US1995/015529
Authority: WO
Inventors: George Anthony Calvert Murrell
Original assignee: New York Society For The Ruptured And Crippled Maintaining The Hospital For Special Surgery
Priority date: 1994-11-29
Filing date: 1995-11-21
Publication date: 1996-06-06
Also published as: AU4247996A

Abstract

Disclosed is a method for regulating the activity of matrix metalloproteinases in a biological tissue. The method comprises adjusting the concentration of nitric oxide to which said metalloproteinases are exposed. The method may be used to treat a variety of disease states, such as in tumor metastasis, otitis media, pulmonary emphysema, and schleroderma. The invention may also be used to prevent the loosening of surgical implants.

Description

METHOD FOR REGULATING METALLOP OTEINASE ACTIVITY

Field of the Invention

This invention pertains to methods and compositions for regulating the activity of metalloproteinases in a tissue by regulating the concentration of nitric oxide within, or in the vicinity of, the tissue. The present invention is particularly applicable to tissues affected by a pathological process that is caused by, or substantially affected by, inappropriate expression or activity of metalloproteinases. The methods of the invention may be used to treat lung tissue that has been damaged by emphysema, to inhibit tumor metastasis, to reduce implant loosening, and to treat other conditions caused or affected by expression of metalloproteinases.

Background of the Invention

Matrix metalloproteinases (MMPs) are neutral proteases that degrade components of the extracellular matrix

(Grant et al. , (1992), Matrix Supp . , 1:217-223). MMPs are zinc-dependent, are secreted as zymogens, are activated in vitro by organomercurial reagents, are autocatalytically processed following activation, and are inhibited by a class of proteins designated tissue inhibitors of metalloproteinases

(TIMPS) (Stetler-Stevenson et al. , (1993), Ann. Rev. Cell Biol . , 9_:541-573) . There are three currently recognized classes of MMPs: interstitial collagenases, stromelysins, and gelatinases (type IV collagenases) .

Interstitial collagenases degrade the triple helical domains of the fibrillar collagens (types I, II, III, and X) . The stromelysins include stromelysin l, 2, and 3, as well as matrilysin, and have a broad range of substrates including proteoglycans. The gelatinases degrade denatured collagen, as well as native collagen containing local helical disruptions. Importantly, MMPs have been implicated in a number of different physiological processes, including pathological conditions, that involve digestion of the extracellular matrix. For example, the metastatic potential of different types of tumor cells shows a direct correlation with the level and/or activity of MMPs, and an inverse correlation with the level and/or activity of TIMPs. (Stetler-Stevenson et al. , (1993), Ann . Rev. Cell Bid . , 2:541-573.)

MMPs are also thought to play a critical role in causing the tissue damage that is characteristic of arthritis. In articular cartilage, the major identified MMPs are collagenase and stromelysin. Both of these enzymes are secreted by chondrocytes and synovial cells in response to inflammatory mediators such as endotoxin, interleukin 1-β and tumor necrosis factor-α (Mitchell et al., (1993), Biochem. Biophys . Res . Comm. , 19JS:1133-1142; Williams et al. , (1991), J. Orthop . Res . , .9:258-265) . Their activity is also regulated by endogenous TIMPS (Gavrilovic et al. , (1987), J. Cell Sci . , 87:357) . Importantly, the activity of these enzymes is greatly enhanced during articular catabolism as in e.g. septic arthritis, osteoarthritis, and rheumatoid arthritis.

Other pathological conditions in which inappropriate activity of MMPs is thought to cause or exacerbate the condition include: otitis media (Juhn et al. , Ann. Otol . Rhinol . Layrngol . Suppl . 163 : 43 -5, 1994) ; periodontal disease (Rikfin et al. , J. Periodontol . 64 : 819 , 1993); pulmonary emphysema (D'Armiento et al. Cell 71 :955, 1992); and adult respiratory distress syndrome (ARDS) (Elsasser et al., Schweiz Med. Wochsenschr. 121 :1530, 1991) . In systemic schleroderma, a reverse correlation has been shown between disease indicators and the level of collagen-degrading activity in blood cells (Solov'eva et al. , Ter.Arkh . 63 : 88, 1991).

Compounds that have been suggested for use as MMP regulators include: endogenous protease inhibitors such as TIMPs (Alvarez et al. , (1990), J. Natl . Cancer Inst . , 82:589) ; microbial protease inhibitors such as leupeptin and antipain; synthetic peptides and peptide derivatives; and hydroxamic acid derivatives (Dodwell et al., (1993), Cane. Treat . Rev. , 19.:283) . The applicability of these compounds is, however, limited by: the difficulty in synthesizing sufficiently large quantities (e.g. TIMPs) , instability in biological environments (e.g. antipain) , and uncertain potency (e.g. synthetic peptides) . The present invention is based on the finding that nitric oxide plays a key role in the activation of MMPs.

Nitric oxide (NO*) is synthesized from the amino acid L- arginine by a family of enzymes, the nitric oxide synthases

(NOSs) . Its size (one of the ten smallest molecules) and its unpaired electron (denoted • ) , make it a highly reactive and locally diffusible free radical.

NOSs comprise inducible and constitutive forms. The constitutive forms of NOS release NO* at low levels at relatively stable concentrations. Examples of constitutive forms include endothelial cell and brain NOS. NO* synthesized by vascular endothelium is responsible for the regulation of blood pressure, while in the central nervous system NO* is a neurotransmitter involved in memory and motor function.

Inducible forms of NOS are found in phagocytic cells, hepatocytes, and in cartilage, and release high levels of NO* in response to the E. coli lipopolysaccliaride ( PS or endotoxin) and to inflammatory mediators such as interleukin-lβ (IL-lβ) , tumor necrosis factor-o; (TNF-c.) and interferon-β. In the circulatory system, the inducible forms of NOS are thought to play a role during host defense and immunological reactions.

McCartney-Francis et al. ( J. Exp.Med. 178:749. 1993) discloses the use of N-monomethyl arginine, an NOS inhibitor, to reduce the symptoms of arthritis in an animal model. The

McCartney-Francis study, however, does not address the relationship between NO* levels and MMP activity. Thus, until the present invention, it was not known that local concentrations of NO* could be manipulated as a way of regulating MMPs. It has now been unexpectedly discovered that NO* is responsible for the inflammatory induction of MMPs in certain cells and tissues including articular chondrocytes. It has also been found that the underlying pathological state caused by the activation of MMPs in the affected tissues can be alleviated or inhibited by reducing the exposure of such tissues to NO. It is therefore an object of the present invention to provide methods and compositions for regulating MMPs by altering the level of NO in or near the cells that synthesize metalloproteinases. A further object of the invention is to provide a method of treating or alleviating otitis media, pulmonary emphysema, and diseases of collagen metabolism such as systemic scleroderma, and to reduce bone inflammation caused by degeneration of surgical implants. A further object of the invention is to provide a method of preventing or slowing tumor metastasis in mammals.

Summary of the Invention

It has now been discovered that pathological conditions mediated by the action of matrix metalloproteinases

(MMPs) can be treated by adjusting the concentration of nitric oxide to which the MMPs are exposed within, or in the vicinity of, the affected tissue. These pathological conditions include metastasis of malignant tumors, otitis media, pulmonary emphysema, adult respiratory distress syndrome (ARDS) , loosening of surgical implants, and systemic scleroderma. When it is desired to reduce the activity of MMPs, an agent is administered that reduces the concentration of nitric oxide. Conversely, when it is desired to increase the activity of MMPs, an agent is administered that provides nitric oxide locally in the vicinity of the MMPs. Also encompassed by the invention are pharmaceutical compositions for treating tumor metastasis, otitis media, pulmonary emphysema, ARDS, and scleroderma.

Brief Description of the Drawings

Figure 1(a) shows a time response of Nitrite (N0₂ ^") release in bovine articular chondrocytes, when stimulated with 1 μg/ml endotoxin. Figure 1(b) shows a dose response of N0₂ ^~ release by bovine articular chondrocytes to endotoxin. N = 6 for each group; means ± SEM, *** = p < 0.001 as compared with t₀ using paired two-way Student's t tests (a), or with control using un-paired two-way Student's t tests (b) . Cell viability was tested utilizing mitochondrial- ependent reduction of 3-

(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide

(MTT) . No treatment inhibited mitochondrial MTT conversion to formazan. Figure 2 (a) shows nitrite (N0₂ ^") release by fresh explants of human cartilage. Figure 2(b) shows nitrite (N0₂ ^") release by three month old explants of bovine occipital cartilage (b) . N0₂ ^' production is stimulated by endotoxin (1 μg/ml) , and this stimulation is inhibited by 1 mg/ml of the nitric oxide synthase inhibitor, Nw-nitro-L-arginine methyl ester (L-NAME) . N = 6 for each group; mean ± SEM, *** = p < 0.0001 as compared with control using un-paired two-way Student's t test.

Figure 3 (a) shows nitrite (N0₂') release in cultured bovine chondrocytes. Figure 3(b) shows the measurement of viability of the cells in Figure 3(a) utilizing mitochondrial- dependent reduction of 3- (4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide (MTT) to formazan. Figure 3(c) shows collagenolytic activity in cell/media of cultures in Figure 3(a) over 24 hrs. N0₂ ^" production and collagenolytic activity was stimulated by IL-1 (50 ng/ml interleukin-lβ) and this stimulation was inhibited by 1 mg/ml of the nitric oxide synthase inhibitor, Nw-nitro-L-arginine methyl ester (L-NAME) or 20 μg/ml of the protein synthesis inhibitor, cycloheximide (cyclohex) , without affecting cell viability. N = 6 for each group; mean ± SEM. Mean collagenolytic activity in control group = 1.2 μg collagen/10⁶ cells/24 hrs. *** = p < 0.001 as compared with control, + = p 0.05, + + = p < 0.01 as compared with IL-1 alone using un-paired two-way Student's t test. Figure 4 (a) shows nitrite release in cultured bovine cartilage explants. Figure 4(b) shows caseinolytic (stromelysin) activity of the media of explants as in Figure 4 (a) , removed after 24 hrs incubation with 10 μg/ml endotoxin and 1 mg/ml of the nitric oxide synthase inhibitor, Nw-nitro-L- arginine methyl ester (L-NAME) . Figure 4(c) shows nitrite (N0₂ ^" ) release, and Figure 4(d) shows stromelysin activity, respectively, of explants removed after 72 hrs incubation with 100 ng/ml interleukin-lβ (IL-1) and L-NAME. N0₂ ^" production and stromelysin activity were stimulated by endotoxin and IL-1, and this stimulation was inhibited by the nitric oxide synthase inhibitor, L-NAME. N = 6 for each group; mean ± SEM. Mean stromelysin activity in control group in (b) = 8.6 μg casein/g w/w cartilage/24 hrs., in (d) = 25 μg casein/g w/w cartilage/24 hrs. * = p < 0.05, ** = p < 0.01, *** = p 0.001 as compared with control, + = p < 0.05, +++ = p < 0.001 as compared with endotoxin alone using un-paired two-way Student's t test.

Figure 5 shows a correlation between stromelysin activity and nitrite of the media of cultured bovine cartilage explants. The explants were incubated for 24 hrs. in media alone (o) , l mg/ml Nw-nitro-L-arginine methyl ester, L-NAME

(+) , 10 μg/ml endotoxin (A) , or endotoxin and L-NAME Φ) .

Correlation coefficient (D = 0.8, p < 0.001. Figure 6 shows the effects of exogenously generated nitric oxide (NO*) on stromelysin activity of cultured chondrocytes. NO* was generated by the NO* donor S-nitroso-N- acetyl-D,L-penicillamine (SNAP) . Mean stromelysin activity of the control group = 2.4 μg casein/10⁶ cells/24 hrs. *** = p < 0.001 as compared with control using un-paired two-way Students's t test. No treatment inhibited mitochondrial- dependent reduction of 3- (4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide (MTT) to formazan.

Detailed Description of the Invention

All patent applications, patents, and literature references cited in this specification are hereby incorporated by reference in their entirety. In case of inconsistencies, the present description, including definitions, will control. Defin ti ons :

1. "Matrix metalloproteinase" (MMP) is defined herein as a neutral protease that is metal-dependent, is secreted as a zy ogen, and, after activation by proteolytic processing, acts to degrade extracellular matrix components. Examples of MMPs include interstitial collagenases, stromelysins, and gelatinases. (Stetler-Stevenson et al., (1993), Ann . Rev. Cell Biol . , 2:541-573) . 2. "NO* -sensitive MMP" is defined herein as a metalloproteinase whose expression and/or activity in a given tissue is affected by the concentration of NO* in or near that tissue.

3. A "NONOate" is a compound that, when present in a physiological environment, provides NO* in that environment.

4. An "NO* scavenger" is a compound that binds and neutralizes NO* .

5. An "NOS inhibitor" is a compound that inhibits the production of NO by nitric oxide synthases (NOSs) . 6. A surgical "implant" is used herein to denote a non-absorbable device constructed of non-organic material (e.g. metal and/or plastic) that is implanted in the body. Examples include artificial joints such as total knee or hip replacements; heart valves; stents that keep hollow structures open as in e.g. urethral or vascular stents; dental implants; and screws, plates, and rods used for fixation of fractures or for bony reconstructive procedures.

The present invention is directed to regulating the activity of matrix metalloproteinases (MMPs) in a tissue by modulating the levels of nitric oxide (NO*) in that tissue. It has now been found that NO* plays a key regulatory role in the activation of MMPs. Inflammatory mediators such as interleukin 1-/3, tumor necrosis factor-α and endotoxin increase the activity of MMPs by increasing the level of NO* (see Example 1 below) . Furthermore, this effect is mimicked by NO* donor compounds that provide NO* locally (NONOates) . Agents that inhibit nitric oxide synthase (NOS) , as well as agents that scavenge NO*, are useful in inhibiting MMPs and in treating pathological conditions mediated by MMPs. Thus, it is contemplated that pathological conditions such as otitis media, periodontal disease, tumor metastasis, pulmonary emphysema, adult respiratory distress syndrome, and loosening of surgical implants can be treated by reducing the exposure of MMPs in the affected tissues to NO* . In addition, diseases in which MMP activity is lower than normal, as in e.g. scleroderma, may be treated by increasing the concentration of NO* in the vicinity of the relevant MMPs. In one embodiment, the method of the invention is carried out by reducing or increasing the concentration of NO* in the vicinity of the affected tissue. By "vicinity" is meant tissues, extracellular fluids, or environments that are in direct contact with the affected tissue. An example of a tissue that is in direct contact with an affected tissue is the fibrous layer of tissue (pseudocapsule) surrounding a tumor. Examples of extracellular fluids that are in direct contact with affected tissues are saliva, which contacts the gums, or synovial fluid, which contacts chondrocytes. An example of an extracellular environment is the extracellular matrix of a tissue. It will be understood that the maximal distance at which an NO* -inhibiting or NO* -stimulating treatment will be effective in regulating MMP activity will vary from tissue to tissue and between different pathological situations. In order to be effective in inhibiting MMPs, the concentration or level of NO* to which the affected tissue is exposed should be decreased by at least 50% and up to 100% of the amount of NO* to which the tissue is exposed in the absence of treatment. Preferably, NO* is decreased by at least 75%, and most preferably, by at least 90%. The duration of time during which NO* levels are decreased will vary, depending upon the particular pathological condition being treated.

Conversely, in order to be effective in activating MMPs, the concentration or level of NO* to which the tissue is exposed should be increased by at least about 150% and up to about 10,000% (i.e. 100-fold). Preferably, the amount of N0» to which the tissue is exposed as a result of the treatment is between about 150% and about 1000% higher as compared with the amount of N0^# to which the tissue is exposed in the absence of such treatment. The most preferred range is between about 400% and about 1000% the amount of NO» to which the tissue is normally exposed. The types of tissue that may be treated using the methods of the present invention include any tissue in which a pathological condition is caused or exacerbated by the action of NO* -sensitive MMPs, or a tissue in which, conversely, activation of MMPs provides therapeutic benefit. Non-limiting examples include a tissue comprising a malignant tumor or cells derived therefrom, where the pathological condition comprises metastasis of tumor cells from the site of the tumor to other sites in the body; the epithelium and underlying soft tissue, cartilage, and bone comprising the middle ear, which are inflamed in otitis media; gums and underlying bone and ligament, which are inflamed in gingivitis and periodontitis; lung tissue, including bronchoalveolar tissue, which is destroyed in emphysema and is inflamed in adult respiratory distress syndrome (ARDS) ; bone and fibrous tissue, which are subject to inflammatory destruction as a result of surgical implants; and, skin and other tissues that are affected by systemic scleroderma, a disease of collagen metabolism.

The invention can be employed to regulate NO-- sensitive MMPs that are found to be involved in pathological conditions. One of ordinary skill in the art can establish whether or not a particular pathological condition is mediated by NO* -sensitive MMPs using routine experimentation and commercially available reagents. To make this determination, in vi tro explants of the tissue under consideration are prepared using methods known in the art for the particular tissue. The cultured tissue is then tested for the presence of NOS isoforms that are inducible by inflammatory mediators, and for enhancement of MMP activity by the same mediators. For example, individual cultures are exposed to IL-l/3 at a range of concentrations between about l and about 200 ng/ml, tumor necrosis factor (TNF) -on at a range of concentrations between about 1 and about 100 ng/ml, or E. coli endotoxin at a range concentrations between about 1 and about 200 μg/ml, for a period of time ranging between 6 hours and 48 hours. NOS activity is then measured as a function of production of N0₂ ^" (a stable product of NO*), and MMP activity is measured by any appropriate proteolysis assay (see Example l for collagenolytic and caseinolytic assay conditions.)

Generally, an increase in N0₂ ^" production of more than 30% in the inflammatory mediator-treated tissue relative to the control (untreated) tissue indicates the presence of one or more inducible forms of NOS. Furthermore, an inflammatory mediator-caused enhancement of MMP activity of more than 30% over that observed in the control tissue indicates an N0- regulated MMP. To confirm the presence of an NO-regulated MMP in the tissue under investigation, the induction of MMP activity by inflammatory mediators is measured in the presence of an NOS inhibitor or an NO scavenger. Conversely, the effect of a NONOate on MMP activity is also tested.

In a target tissue, i.e. one containing N0- regulatable MMPs, an NOS inhibitor or an NO scavenger will prevent the induction of MMP activity by 50% or more, and incubation with a NONOate alone will enhance MMP activity by 100% or more. If the criteria described above are met, the tissue under study is a proper target for the methods and compositions of the present invention.

The present invention encompasses the therapeutic use of agents that inhibit NO* in clinical practice. These agents are administered to patients afflicted with malignant tumors, pulmonary emphysema, adult respiratory distress syndrome, otitis media, or periodontal disease, or recipients of surgical implants. The objective of such administration is to reduce the concentration of NO within, or in the vicinity of, the tissue affected by the pathological condition, thus reducing the activity of MMPs in or near that tissue. Compounds that may be employed to reduce the concentration of NO* for the purpose of regulating MMPs include without limitation N-w-nitro-L- arginine methyl ester (L-NAME) , monomethyl arginine (NMMA) , aminoguanidine or its derivatives, and N-w-nitro-L-arginine (L- NMA) . The preferred compound to use to reduce NO* is NMMA (Alexis Corp. , San Diego, CA) . In addition, NO* scavengers such asCPTIO (2- (4-carboxyphenyl) -4,4,5,5, -tetramethylimidazoline- 1-oxyl-3-oxide, Alexis Corp.) may also be used. In certain pathological conditions such as scleroderma, it is desirable to increase the activity of MMPs by providing NO* in the vicinity of the affected tissue. The compounds that may be employed to increase the N0*» concentration, which are collectively known as NONOates, include without limitation sodium nitroprusside; N- (Ethoxycarbonyl) -3- (4-morpholinyl)sydnoneimine (Molsidomine) ; 3-morpholinosydnonimine (SIN-1) ; 1,2,3,4-Oxatriazolium, 5- amino-3- (3,4-di-chlorophneyl) -chloride (GEA 3162); 1,2,3,4- Oxatriazolium,5-amino-3- (30chloro-2-methyl-phenyl)chloride (GEA

5024) ; 1,2,3,4-Oxatriazolium,3- (3-chloro-2-methylphenyl) -5-

[ [ [cyanomethylamino] carbonyl]amino] -hydroxide inner salt (GEA

5583); S-nitroso-N-acetyl-D,L-penicillamine (SNAP); l-[{4',5'-

Bis(carboxymethoxy) -2' -nitrophenyl)methoxy] -2-oxo-3,3,diethyl- l-triazene dipotassium salt (CNO-4) ; and [l-(4',5'- Bis(carboymethoxy) -2' -nitrophenyl)methoxy] -2-oxo-3,3-diethyl-1- triazine diacetoxymethyl ester (CNO-5) , all of which are available from Alexis Corp. (San Diego, CA) . Additional compounds include nitroglycerin, diethylamine-NO (DEA/NO) , IPA/NO, sper ine-NO (SPER/NO) , sulfite-NO (SULFI/NO) , OXI/NO, and DETA/NO, the synthesis of which is described in Drago, R.S., in Free Radicals in Organi c Chemistry (Advances in Chemistry Series), Number 36, pages 143-149, 1962; and in Maragos et al. , J. Med. Chem. 34 : 3242, 1991. Briefly, these compounds are prepared by reaction of nitric oxide with a nucleophile. The preferred NO**-generating compound is DETA/NO, which has a half-life of about 1 day and is thus particularly suited for sustained-released formulations described below (Hrabie et al., J. Org. Chem. 58 : 1472 , 1993). In addition to the compounds identified above, it is possible to use other NO* -inhibiting or NO* -providing compounds, provided they are biologically acceptable i.e. non- toxic under the conditions of treatment employed. These compounds may be identified using the methods described in co- pending U.S. patent application titled "Regulation of Wound Healing by Nitric Oxide", filed September 29, 1994, which is incorporated by reference. In practicing the present invention, NOS inhibitor or NO* scavenger compounds, or NONOates, can be administered in conjunction with any pharmaceutically acceptable carrier known in the art, e.g. in sterile isotonic saline; creams or ointments such as those containing waxes, fatty acids, and propylene glycol; sprays; subcutaneous pumps; or slow-release formulations such as those containing polylactic acid- polyglycolic acid (PLAGA) . The active compounds are formulated at concentrations ranging from about 0.1 mg/ml to about 5 mg/ml, preferably about 0.4 mg/ml. The pharmaceutical formulations of the present invention need not in themselves contain the entire amount of the agent that is effective in inhibiting or activating MMPS, as such effective amounts can be reached by administration of a single application or dose, or a plurality of applications or doses of such pharmaceutical formulations.

In addition to the active compound, the formulation may comprise analgesics (e.g. lidocaine) , antioxidants (e.g. superoxide dismutase, vitamin C, vitamin E) inhibitors of xanthine oxidase (e.g. allopurinol) , corticosteroids, or combinations thereof.

NOS inhibitors, NO* scavengers, or NONOates may be administered topically or internally, or applied during surgery. The only limitation on the route of administration is that the compounds must be available to decrease or increase the concentration of NO* in the vicinity of the affected tissue for a sufficient period of time to affect MMP activity. The NO* -inhibiting compounds may also be injected directly into the affected tissue, or may be included in irrigation, lavage, or other fluids used during surgery.

The preferred mode of administration for each condition is one that will bring the active compound as close as possible to the affected tissue. In the preferred embodiment, an NOS inhibitor or NO* scavenger is brought into direct contact with the affected tissue. Thus, a preferred method of inhibiting tumor metastasis is to bring the NOS inhibitor or NO* scavenger into direct contact with the tumor mass (or to the area in which the tumor was situated, after removal of the tumor) . This can be accomplished by direct injection into the tumor, and/or by topical application of a cream or ointment (containing the NOS inhibitor or NO* scavenger) to the tumor or lesion. Similarly, a preferred method for treating otitis media is to administer an NOS inhibitor or NO scavenger in the form of ear drops, and a preferred method for treating periodontal disease is to apply an NOS inhibitor or NO* scavenger directly to the gums in the form of a spray or ointment. Because NO* affects many physiological processes, for optimal use in practicing the present invention an agent should act locally i.e. within or in the immediate vicinity of the affected tissue. In some cases, the anatomy of the affected tissue (e.g. a joint, or an area of skin) naturally restricts the activity of the agent to the tissue or its immediate environment. In other cases, this restriction is achieved by employing a formulation (such as a cream, ointment, or slow- release polymer) that limits the diffusion of the agent, or by employing an agent with a relatively brief duration of activity.

It will be understood that, in practicing the present invention, treatment of a given pathological condition signifies lessening or amelioration of at least one recognized symptom of the disease. In addition, the ability to refrain from administration of conventional drugs that are currently used to treat the condition (e.g. cytotoxic drugs or anti- inflammatory agents) is also significant. Furthermore, a patient suffering from a progressive disease is deemed to have received a significant benefit if the disease fails to progress and the patient is maintained in a stable condition.

The present invention also encompasses the in vitro use of NO* -mediated regulation of MMPs in diagnosis and drug development. For example, tissue samples derived from biopsy can be tested for their content of NO* -sensitive MMPs as part of a comprehensive diagnostic battery. Furthermore, MMPs in crude, partially purified, or purified form can be activated in vitro using the methods of the present invention (i.e. by increasing the concentration of NO* to which they are exposed) , and the activated MMPs can then serve as excellent targets for systematic testing of other potentially therapeutic MMP- inhibiting compounds.

In one embodiment of the present invention, NMMA or L-NAME is formulated in sterile saline at a concentration of 1 mg/ml and administered by injection into a tumor mass. In another embodiment, NMMA or L-NAME is formulated at a concentration of 1 mg/ml in a cream or ointment containing petrolatum, propylene glycol, and waxes, which is applied to skin in the area of a malignant melanoma.

In another embodiment, an NOS inhibitor or NO scavenger compound is chemically linked, using methods known in the art, to a compound that specifically recognizes a tumor e.g. a tumor-specific antibody. The hybrid compound is administered intravenously, in a manner that ensures that effective amounts reach the tumor cells.

In yet another embodiment, NMMA is formulated in a spray or ointment suitable for use in the mouth, which is applied to gums to treat or prevent periodontal disease. In yet another embodiment, NMMA is formulated in sterile saline at a concentration of 1 mg/ml, which is administered as ear drops to treat or prevent recurring otitis media.

In yet another embodiment, NMMA is formulated at a concentration of 1 mg/ml in an inhalable spray, which is used to treat or prevent pulmonary emphysema.

In yet another embodiment, an NO* -inhibiting agent or NO* scavenger compound is sprayed onto the surface of a surgical prosthesis, incorporated into the gelatin or other material coating the prosthesis, or added to polymethyl methyacrylate or other bone cement used to stabilize the implant. Wear particles that are released from the surface of the treated prosthesis thus fail to induce MMP-activating levels of NO* in the vicinity of the prosthesis. The N0-- inhibiting agent or NO* scavenger may also be formulated in a controlled-release polymer and injected around a prosthesis or into an artificial joint cavity. In yet another embodiment, DETA/NO is formulated at a concentration of 1 mg/ml in a dermatological cream, which is applied to skin lesions caused by scleroderma.

The following working examples are intended to serve as non-limiting illustrations of the present invention.

Example 1; Nitric Oxide Activates Metalloproteinases in Articular Cartilage

This study was carried out to determine whether induction of NO is responsible for the inflammatory activation of MMPs. The procedure was as follows: A. Materials and Methods Materials

Dulbecco's modification of Eagle's medium (DMEM) , Ca²⁺ and Mg²⁺ free Dulbecco's phosphate buffered saline (PBS) and Hank's solution, antibiotic-antimycotic solution (#600--

5240; 10,000 U/ml penicillin G sodium; 10,000 μg/ml streptomycin sulfate; 25 μg/ml amphotericin β) , HEPES solution

(238.3 g/1) , trypsin solution (0.25% (w/v) trypsin in Hank's solution) and fetal calf serum (FCS) were purchased from Gibco

Laboratories Ltd, New York, NY. Tissue culture plates were from Falcon^®, Becton Dickinson & Co, New York, NY. MADV S65

(0.65μm pore size) multiscreen plates and multiscreen punches were purchased from Millipore Corporation, Philadelphia, PA. Picopros^® scintillation vials and UltimaGold^® scintillation fluid were purchased from Packard Technologies, Downers Grove, IL. ³H-thymidine and ³⁵S-sulphate were purchased from Amersham Corp, Chicago, IL. Tumor necrosis factor-α (TNF-c.) was a gift from Grace Wong, Genentech Corp, South San Francisco, CA. ³H- type I collagen (1.0 mCi/mg) was purchased from New England Nuclear, Boston, MA. NG- monomethyl-L-arginine monoester was from Calbiochem, La Jolla, CA; and S-nitroso-N-acetyl-D,L- penicillamine (SNAP) was from Alexis Corporation, San Diego, CA. All other chemicals and biochemicals were purchased from Sigma Chemical Co., St Louis, MO.

Sample collection and culture conditions

Human mesici were obtained from four patients at arthroscopic surgery for large meniscal tears. Shoulder capsule and synovium were obtained from a 21 year old female undergoing a forequarter amputation for chondrosarcoma. Human cartilage explants were obtained from a 7 year old female undergoing a knee fusion for proximal femoral focal deficiency. Bovine tissue was obtained from the hooves of freshly slaughtered calves. Bovine cartilage explants were obtained from occipital articular cartilage. Canine tissues were harvested from adult mongrel dogs euthanized for other research purposes. Each sterile tissue sample was immediately placed in cold (4°C) tissue culture medium containing 90% (w/v) , 1% (v/v) antibiotic-antimycotic solution, 0.22% (w/v) NaHC0₃ with 10% (v/v) fetal calf serum, pH 7.35, and dissected into 1 mm cubes. For cell culture from explants, six cubes were placed into each well of a six well tissue culture plate and cultured at 37°C. Media were changed every three days throughout the culture period. When confluent, the explants and cell layers were trypsinized with 0.25% trypsin solution. Cells from passage one or two were used for all assays. Chondrocytes were obtained by collagenase digestion of slices of bovine articular occipital cartilage in 0.025% (w/v) collagenase, 1% (v/v) antibiotic-antimycotic solution, 2% (v/v) HEPES solution with vigorous agitation at 37°C for six hours. The cell suspension was then spun at 30,000 rpm for 10 minutes and the supernatant discarded. The remaining cells were then resuspended in fresh media and plated in 75 ml culture flasks. Morphological analysis

The morphological characteristics of cultured cell lines were evaluated directly and from photographs utilizing an Olympus BHS inverted microscope and C35AD4 camera (Olympus Corp, Tokyo, Japan) . Objective morphological analyses (cell length, diameter, form, area) were performed from photographs utilizing a digitizing tablet and Sigma Scan for Windows^® software program (Jandel Scientific, San Rafael, CA) . Ultrastructural characteristics were assessed by transmission electron microscopy. Nitrite release

Nitrite (N0₂.) , a stable end-product of nitric oxide, was measured in the media of cultured cells and explants utilizing the spectrophotometric method based on the Greiss reaction. The absorbance was measured at 550/650 run with a 340 ATTC microplate photometer (Tecan US Inc, Research Triangle Park, NC) . Cell viability

Cell respiration, an indicator of cell viability, was assessed by the mitochondrial-dependent reduction of 3- (4,5- dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromide (MTT) to formazan. Cells in 96-well plates were incubated with MTT (0.4 mg/ml for 60 min) . Culture medium was removed by aspiration and the cells were solubilized in dimethyl sulfoxide (200 μl) . The extent of reduction of MTT to formazan within cells was quantified by measurement of absorbance at 550nm/650nm. Formazan production was expressed as a percentage of that observed in cells treated with media alone. Thymid ne Incorporation

Each well of a 96-well MADV tissue culture plate was seeded with 5 x 104 cells and cultured for 24 hours. The medium was then replaced with 250 μl medium containing 4.0 μCi ³H-thymidine and carrier thymidine to a final concentration of 6.0 μM thymidine and the various agents to be tested. After 24 hours of incubation at 37°C, the cell layer was washed twice with PBS, lysed, the DNA allowed to precipitate with 95% ethanol (2 x 10 min) , and the radioactive filters punched with a multiscreen punch into 4 ml scintillation vials. 0.4 ml of 0.45% (w/v) sodium hypochlorite was then added to each vial. The vials were mixed for 39 minutes, 0.3 ml of scintillant was added to each vial, and the specimens were counted using a Beckman LS 2800 Scintillation Spectrophotometer (Beckman Instruments, Fullerton, CA) . Collagenolytic Assay

The assay for radiolabeled collagen degradation by conditioned media of explants of bovine occipital cartilage was that of Williams et al. (J. Orthop. Res . 9: 258, 1991). For direct measurement of collagenolytic activity of cultured chondrocytes, this method was adapted to a multiwell system. In brief, 10⁵ cells/well were seeded in DMEM with 10% (v/v) FCS in 96 well-multiwell tissue culture plates. The following morning, the media were removed and the cells washed with 200 μl PBS (x2) . Fresh media (DMEM without FCS) containing 0.256 μCi/ml ³H-collagen along with the reagents to be tested were added to each well. The final volume for each well was 250 μl. After incubation for 24 hrs at 37°C, 100 μl of media were transferred for nitrite estimation. 100 μl of media was transferred to 1 ml multiwell tubes for collagenolytic activity, mixed with 100 μl precipitation solution (4:1:10 (v/v); 0.2 M EDTA:0.1% (w/v) type I collagen:75% (v/v) saturated (NH₄)₂S0₄) and allowed to precipitate at 4°C overnight. The tubes were then centrifuged at 30,000 rpm for 15 minutes and 100 μl of each supernatant transferred to scintillation vials, mixed with 3 ml of scintillation fluid, and counted in a liquid scintillation spectrophotometer. Maximal substrate degradation was estimated using bacterial collagenase (1,000 ng) and blank reactions were executed with media alone. The inhibitory effects of 1,10-phenanthroline (1 mM) and EDTA (10 mM) on IL-1 and LPS induced collagenolytic activity were quantified by adding these reagents to the reaction mixture.

Caseinolytic Activity

The assay for radiolabeled casein degradation of conditioned media of explants of bovine occipital cartilage was that of Williams et al. For direct caseinolytic activity of cultured chondrocytes, this method was adapted to a multiwell system. In brief, the cells were seeded as for collagenolytic activity. The following, morning, the media was removed and the cells washed with 200 μl PBS (x2) . Fresh media (usually DMEM without FCS) containing 0.08 μCi/ml ¹⁴C-casein along with the reagents to be tested were added to each well. The final volume for each well was 250 μl. After incubation for 24 hrs at 37°C, 100 μl of media was transferred for nitrite estimation. 100 μl of media was transferred to 1 ml multiwell tubes for caseinolytic activity, mixed with 150 μl ice cold 20% (v/v) trichloracetic acid and 50 μl unlabeled casein (3 mg/ml) , and allowed to precipitate at 4°C overnight. The tubes were then centrifuged at 30,000 rpm for 15 min and 100 μl of each supernatant transferred to scintillation vials, mixed with 3 ml of scintillation fluid, and counted in a liquid scintillation spectrophotometer. Maximal substrate degradation was estimated using bacterial trypsin (0.025% w/v final concentration) and blank reactions were executed with media alone. The inhibitory effects of 1, 10-phenanthroline (I mill) and EDTA (10 mM) on IL-1 and LPS induced collagenolytic activity were quantified by adding these reagents to the reaction mixture. Statistical Analysis

Statistical Analysis was performed using two-tailed Student's t tests.

B. RESULTS

Articular^* chondrocytes

All primary and secondary cultures of bovine occipital articular chondrocytes had characteristic cobblestone morphological appearance. This appearance was preserved when the cell lines were maintained at high cell density. Nitric oxide synthase activity in chondrocytes and cartilage Figures 1-3 show the stimulation of NO production by inflammatory mediators. ILl-β (1-100 ng/ml), TNF-α (1-10 μg/ml) and endotoxin (1-100 μg/ml) induced N0₂. release in a time and dose-dependent fashion in both human and bovine cartilage explants and in bovine chondrocytes (Figures 1A-2B) . Non-stimulated cells did not release measurable N0₂. (Fig IB) . The release of N0₂. was inhibited by co-incubation with the nitric oxide synthase inhibitors Nw-nitro-L-arginine methyl ester (L-NAME) , N^monomethyl-L-arginine monoester or aminoguanidine, and with the protein synthesis inhibitor, cycloheximide. These agents alone did not affect viability (n = 8, data not shown) .

Metal loproteinase activity in chondrocytes and cartilage

ILl-β (50 ng/ml) stimulated a 3-fold increase in collagenolytic activity of cultured bovine chondrocytes. This activity was completely inhibited with 1 mg/ml of the nitric oxide synthase inhibitor, L-NAME or with 20 μg/ml of the protein synthesis inhibitor, cycloheximide (Fig 3C) . Endotoxin

(10 μg/ml) and IL-lβ (100 ng/ml) also induced stromelysin activity in the media of cultured bovine cartilage by 50-100% (Figure 9A,B) . In each case the increase in metalloprotease activity was accompanied by an increase in nitric oxide synthase activity and was inhibited by the nitric oxide synthase inhibitor, L-NANE (Figure 4, A-D) . Metal loproteinase activity versus nitric oxide synthase activity

Unstimulated cartilage explants released basal amounts of stromelysin activity. When stimulated by endotoxin, this activity increased in a near linear fashion, There was an excellent correlation between metalloprotease activity and N0₂. levels in the media of explants of articular cartilage was established (r = 0.8, p < 0.001; Fig 5).

Effects of exogenous NO' on me tall opro einase activity

NO* exogenous generated with 10⁴-10³ M of the N0-- donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) increased caseinolytic activity of cultured bovine chondrocytes in a dose-dependent fashion (Fig 6) .

C. DISCUSSION This study is the first to show that induction of chondrocyte metalloproteinase activity is dependent upon, and mediated by, NO* . The induction of NOS activity in chondrocytes was a time-dependent process that involved protein synthesis. Once induced, NOS activity correlated with increasedmetalloproteinase activity in chondrocyte culture and in conditioned media from cartilage explants. Exogenous NO* also induced metalloproteinase activity.

Example 2: Suppression of Tumor Metastasis Using NOS Inhibitors or NO* Scavengers

The following examples illustrate the use of the present invention in clinical practice to inhibit the metastatic spread of malignant tumors. a) Topical : A patient presents with a skin lesion, which is biopsied and confirmed to be a malignant melanoma. A cream is formulated containing the following components: white petrolatum (70% v/v) ; emulsifying waxNF (5% v/v) ; propylene glycol (10% v/v) ; propylene carbonate (5% v/v) ; glyceryl monostearate (5% v/v) ; white wax (5% v/v) ; and an NOS inhibitor or NO* scavenger (0.4 mg/ml of ointment) . The cream is rubbed into the site of the original lesion before and after surgical excision of the tumor, and the application is continued daily for six months. b) Internal: A patient presents with a mass in the right thigh. After pre-operative evaluation to determine the extent of invasion and the presence of any metastases, the tumor is biopsied and confirmed to be a soft tissue sarcoma. A wide excision of the tumor is then performed i.e. a margin of at least 2 cm of "normal" tissue is removed along with the tumor. In the remaining cavity, polylactic acid-polyglycolic acid (PLAGA) beads containing an NOS inhibitor (formulated as described in Example 8 below) are placed throughout the wound. The formulation will release the NOS inhibitor over several weeks.

Ec>.τn i t- 3; Prevention of Implant Failure Using NOS Inhibitors or NO Scavengers One of the causes of failure of surgical implants is the mechanical generation of wear particles of 1-100 μm in diameter, which are shed from the surface of the implant. These particles are targets for phagocytosis by phagocytes and fibroblasts, and stimulate the secretion of inflammatory cytokines, e.g. TNF-c- and IL-lβ, by these cells. The cytokines induce NOS, which produces NO, which in turn activates MMPs in the vicinity of the implant. The MMPs digest the tissue surrounding the implant, resulting eventually in the loosening of the implant from bone or soft tissue. Examples of implants in which the present invention is applied are: total hip replacement (including femoral and/or acetabular prostheses) ; and total knee replacement (including femoral and tibial components) . The prostheses may comprise titanium and other alloys and/or plastics such as polyethylene. A solution is prepared containing NMMA (1 mg/ml) and gelatin (10%) , which is sprayed on the surface of the implant to form a 2mm-thick gelatin coating. Alternatively, in a total knee replacement, a lmg/ml solution of NMMA is placed in a pump such as that used to deliver insulin, which is implanted subcutaneously in the thigh, with its delivery site in the joint space. The pump is programmed to deliver the NMMA solution into the joint space at a rate of 1 μl/hour for about 30 days.

^*eτr»τn ^*i 4; T-QΛt-irmmt of Periodontal Disease Using an NOS

Inhibitor or NO Scavenger This experiment is carried out to illustrate that the present invention can be used to treat inflammatory gum diseases that involve the action of MMPs on gum and underlying tissues.

Gingivitis and periodontitis are characterized by bleeding, redness, and pain in the gums and at the base of the teeth.

The following formulation is prepared, containing, by weight percent: poloaxmer 407, 10% (CCA Industries, E.

Rutherford, NJ) ; glycerin, 5%; sodium saccharin, 0.1%; aminoguanidine, 1 mM.

The solution is applied with the tip of a cotton swab applicator at least twice a day for 10 days, or until abatement of the symptoms described above.

Example 5; Treatment of Otitis Media Using an NOS Inhibitor or NO Scavenger

This experiment is carried out to illustrate the use of the present invention to treat ear inflammations such as otitis media and otitis externa. A patient presents with a painful ear. Otoscopic examination reveals a swollen red eustachian tube, injected tympanic membrane, and loss of the normal light reflex on the tympanic membrane.

An ear drop solution is formulated as follows: polymyxin B sulfate, 1,000 units/ml; neomycin sulfate, 3.5 mg/ml; hydrocortisone, 10 mg/ml; thimerosal, 0.01%; and aminoguanidine, lmM. The ear drops are administered 3 drops every four hours for four days, or until the symptoms described above are reduced or disappear.

τgτrjnτn ^*ιe 6_t -~-t-fflcmt of Pulmonary Emphysema Using yn NOS Inhibitor or NO Scavenger

Emphysema is a progressive deterioration of lung tissue that involves progressive loss of the structural architecture of the alveoli and bronchial tree. A typical patient presents with symptoms of persistent fruity cough and shortness of breath, and a definitive diagnosis is made using X-ray and/or bronchoscopy.

NMMA is formulated at a concentration of 1 mg/ml in normal saline, for use in an inhalable spray.

The spray is used in conjunction with commercially available inhalers such as Vertolin Inhalation Aerosol (Albuterol, USP; Allen and Harburys Division of Glaxo, Research Park Triangle, NC) , which allow pre-determined amounts to be administered and facilitate the penetration of the spray into the lungs. The spray is administered in an amount of three puffs every four hours. A decrease in the symptoms described above, and a more normal appearance of the lungs in X-ray, indicate that the treatment has been efficacious.

Example 7: Treatment of Scleroderma Using a NONOate

Scleroderma is a disease involving deposition of excessive amounts of collagen, as well as a reduced secretion of collagenase-type MMPs in the affected tissue. In the skin, a thin epidermis overlies compact bundles of collagen which lie parallel to the epidermis. As a consequence, the skin becomes firm, thickened, and leathery in appearance. More progressive pathological changes in the skin result in flexion contractures of the fingers, as well as dark and uneven pigmentation.

DETA/NO is formulated in an ointment having the following composition: DETA/NO (1 mg/ml) ; white petrolatum (70% v/v) ; emulsifying waxNF (5% v/v) ; propylene glycol (10% v/v) ; propylene carbonate (5% v/v) ; glyceryl monostearate (5% v/v) ; white wax (5% v/v) . The ointment is applied three times daily to affected areas of skin.

-^gfam ia fl; Sustained Release Formulations A sustained-release formulation of an NOS inhibitor is formulated as follows: 20g of an NOS inhibitor such as L- NAME and 80 g polylactic acid-polyglycolic acid (PLAGA) are dissolved in 100 ml methyl chloride. 0.5 g polyvinyl alcohol is added. The mixture is stirred for 24 hours, until the solvent has evaporated. The resulting microspheres are washed in distilled water and freeze-dried, after which they are stored under nitrogen at -20°C.

Claims

What is claimed is: 1. A method for regulating the activity of matrix metalloproteinases in a biological tissue, which comprises adjusting the concentration of nitric oxide to which said metalloproteinases are exposed.

2. The method of claim 1, wherein said ajusting comprises increasing said concentration.

3. The method of claim 1, wherein said adjusting comprises decreasing said concentration.

4. The method of claim 1, which comprises administering an agent that decreases said concentration of nitric oxide.

5. The method of claim 4 which comprises decreasing the concentration of nitric oxide by about 50% to about 100% below the concentration of nitric oxide that is present in the absence of said agent.

6. The method of claim 4 which comprises administering said agent in a manner that places said agent in contact with said tissue.

7. The method of claim 4 which comprises administering monomethyl arginine.

8. A method for inhibiting metastasis of malignant tumor cells, which comprises decreasing the concentration of nitric oxide in the vicinity of said cells.

9. A pharmaceutical composition for inhibiting metastasis of a malignant tumor, which comprises an agent that decreases the local concentration of nitric oxide in said tumor and a pharmaceutically acceptable carrier.

10. The composition of claim 9 wherein the concentration of said agent is between about 0.1 mg/ml and about 5 mg/ml.

11. A method for treating otitis media in a patient in need of such treatment, which comprises administering effective amounts for treating otitis media of an agent that reduces the concentration of nitric oxide in the middle ear sinuses of said patient.

12. Apharmaceutical composition for treating otitis media, which comprises an agent that decreases the local concentration of nitric oxide and a pharmaceutically acceptable carrier.

13. The composition of claim 12 wherein the concentration of said agent is between about 0.1 mg/ml and about 5 mg/ml.

14. A method for treating pulmonary emphysema in a patient in need of such treatment, which comprises administering by inhalation effective amounts for treating said emphysema of an agent that decreases the local concentration of nitric oxide in the pulmonary tissue of said patient.

15. A pharmaceutical inhalable composition for treating pulmonary emphysema, which comprises an agent that decreases the local concentration of nitric oxide in pulmonary tissue after inhalation, and a pharmaceutically acceptable carrier.

16. The composition of claim 15 wherein the concentration of said agent is between about 0.1 mg/ml and about 5 mg/ml.

17. A method for treating scleroderma in a patient in need of such treatment, which comprises administering effective amounts for treating scleroderma of an agent that increases the concentration of nitric oxide in the vicinity of skin affected by said scleroderma.

18. A pharmaceutical composition for treating scleroderma, which comprises an agent that increases the concentration of nitric oxide in the vicinity of skin affected by said scleroderma, and a pharmaceutically aceeptable carrier.

19. The composition of claim 18 wherein the concentration of said agent is between about 0.1 mg/ml and about 5 mg/ml.

20. A method for preventing loosening of surgical implants, which comprises administering effective amounts for preventing said loosening of an agent that decreases the concentration of nitric oxide in the vicinity of said implant.