Field of the Invention The present invention relates to the treatment of inflammatory disorders by the administration of leukocyte elastase inhibitors.

Background of the Invention A number of inflammatory diseases, including cystic fibrosis, glomerulo-nephritis, rheumatoid arthritis, chronic obstructive pulmonary disease and emphysema, register a progressive modification of tissue architecture that eventually impairs organ function. In these cases, both serine-proteinases and metallo-proteinases have been demonstrated as instrumental in extracellular matrix alteration. Among the serine-proteinases, leukocyte elastase (LE), mainly released upon stimulation by neutrophil leukocytes at a site of inflammation, has the potential to preferentially disrupt the elastic network. In addition, LE can activate a number of matrix metalloproteinases (MMPs), and inactivate their tissue inhibitors (TIMPs).

Leukocyte elastase is physiologically counterbalanced by endogenous serine-proteinase inhibitors, such as al-proteinase inhibitor (a1- PI, also known as al-antitrypsin), a2-macroglobulin, and secretory leukoproteinase inhibitor. Any enzyme/inhibitor imbalance may lead directly to increased lysis of extracellular matrix macromolecules and increased risk of tissue injury in the immediate vicinity of activated neutrophils. In particular, a1- PI deficiency is the most prevalent potentially fatal hereditary disease in Caucasian individuals, and is an important risk factor for pulmonary emphysema. An increase of leukocyte elastase may also occur as a result of increased recruitment of leukocytes to the lung sustained by viral or bacterial pathogens that are encouraged by environmental conditions or life habits.

Additionally, a functional deficiency of inhibitors can occur due to inhibitor inactivation in the lung by oxidation from cigarette smoke or oxygen radicals released from inflammatory leukocytes.

In treating an imbalance between leukocyte elastase and serine- proteinase inhibitors, exogenous elastase inhibitors are known drugs. Also, direct a1-PI replacement is a potential therapeutic approach currently under investigation. To date, no genetically engineered a1-PI is yet available for this purpose, but a number of heterocyclic inhibitors quite specific for LE have been developed, with Ki values in the range of 10-5-10-7 M. Peptide chloromethyl ketones are also effective inhibitors in animal models of emphysema, and have served as standard of comparison for newly developed inhibitors, but present side effects that make them unsuitable for human therapeutic use.

Epigallocatechin-3-gallate (EGCG) is the major polyphenol or catechin from green tea. Several beneficial effects of EGCG are known, as seen from the following examples. U. S. Pat. Nos. 5,318,986 and 5,670,154 both granted to Hara and Honda (1994,1997) disclose that tea polyphenols including EGCG inhibit the enzyme activity of alpha amylase and tyrosinase.

U. S. Pat. No. 5,605,929 to Liao and Liang (1997) discloses catechins including EGCG inhibit the enzyme activity of 5 alpha reductase. U. S. Pat. No.

5,391,568 to Chung (1995) discloses that EGCG inhibits lung cancer in a mammal. However, the effect of catechins, particularly EGCG, on the level of enzyme activity of leukocyte elastase is unknown.

It is the object of the present invention to provide a method for the treatment of inflammatory disorders. More specifically, it is an object of the present invention to provide a method for the treatment of inflammatory inhibitors wherein an agent that inhibits leukocyte elastase is administered.

These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

Brief Summary of the Invention The present invention is directed to a method for treating inflammatory diseases by inhibiting the activity of leukocytic elastase. The

method includes administering to a host in need a therapeutical effective amount of catechin. Preferably, the catechin for inhibilitating the activity of leukocytic elastase is selected from epigallocatechin-3-gallate and epigallocatechin. Most preferably, the catechin is epigallocatechin-3-gallate.

The catechin can be suitable for oral, rectal, nasal, topical, vaginal, parenteral, inhalation, or insuffalation administration.

The present invention is further directed to a method for reducing leukocyte elastase activity in mammals. The method includes administering an effective leukocyte activity reducing amount of at least one catechin.

Preferably, the catechin is selected from epigallocatechin-3-gallate and epigallocatechin.

Brief Description of the Drawings FIGURE 1A shows a graph comparing the inhibition of leukocytic elastase by EGCG and EGC with increasing concentrations of synthetic substrate.

FIGURE 1B shows a linear inverse graph of FIGURE 1A comparing the inhibition of leukocytic elastase by EGCG and EGC with increasing concentrations of synthetic substrate.

FIGURE 2 shows a graph comparing the enzymatic activity of leukocytic elastase, cathepsin G, and thrombin on synthetic substrate with increasing amounts of epigallocatechin-3-gallate.

FIGURE 3A shows a graph comparing the percentage of substrate degradation inhibitor over time at different catechin concentrations.

FIGURE 3B shows a graph disclosing the inhibition of elastolytic activity by certain inhibitors expressed as a percentage of that exerted by EGCG.

FIGURE 3C shows a graph comparing the inhibition of substrate degradation of standard class-specific proteinase inhibitors with EGCG.

FIGURES 4A, 4B, and 4C each show a gelatin-zymography of EGCG interference of leukocyte elastase activation of MMP-2 and MMP-9.

Detailed Description of the Preferred Embodiments This invention is directed to a pharmacologically acceptable composition for inhibiting leukocyte elastase (LE) in a mammal. The composition includes a catechin and a pharmaceutically acceptable carrier with the catechin present in the composition in an effective amount to inhibit LE in the mammal. The invention is also directed to a method of inhibiting LE in a mammal, which includes the step of administering to the mammal a catechin in pure form or in a pharmaceutical acceptable carrier.

As used herein, catechins includes tea catechins and catechin derivatives. Suitable catechins for use in the composition or method can be isolated from natural sources such as green tea. Particularly, green tea catechins including, but not limited to epigallocatechin-3-gallate (EGCG) and epigallocatechin (EGC), are inhibitors of leukocyte elastase (LE). Catechins in pure form or in a pharmacologically acceptable carrier will find benefit in treating conditions and disorders where there is an advantage in inhibiting the LE enzyme. Catechins, particularly EGCG and EGC, may be useful to inhibit the activity LE in patients suffering from inflammatory conditions in a dose dependant, noncompetitive manner.

There is also evidence that the LE enzyme may be directly or indirectly involved in the pathophysiology of a number of inflammatory diseases including cystic fibrosis, glomerulo-nephritis, rheumatoid arthritis, chronic obstructive pulmonary disease, and emphysema. Catechins can prove helpful in treating these disorders.

In addition to inhibiting LE activity and inflammatory disorders associated with LE, catechins have been found to interfere with LE activation of matrix metalloproteins (MMPs). LE, in addition to altering extracellular matricies, can activate a number of MMPs and inactivate their tissue inhibitors.

Catechins, particularly EGCG, can interfere with LE activation of MMPs where activity is instrumental to endothelial and tumor cells in cutting through extracellular matrix barriers during angiogenetic and inovasive/metastatic process and pulmonary emphysema.

Pharmaceutical formulations for catechins may include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual),

vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration, or for administration by inhalation or insufflation. The formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the steps of bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules ; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid ; or as an oil-in-water liquid emulsion; or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.

Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use. Extemporaneous

injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or polyethylene glycol.

Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavored basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose and acacia.

For administration by inhalation, the active ingredient is conveniently delivered from an insufflator, nebulizer pressurized packs or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, the active ingredient may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form, in for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.

The active ingredient may also be used in combination with other therapeutic agents, for example, anti-inflammatory agents, particularly non- steroidal anti-inflammatory drugs (NSAIDs), and vasodilator prostaglandins.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

Having generally described the invention, the following examples are included for purposes of illustration so that the invention may be more readily understood and are in no way intended to limit the scope of the invention unless otherwise specifically indicated.

EXAMPLES METHODS AND MATERIALS REAGENTS Elastase and cathepsin G from human leukocytes, porcine pancreatic elastase, elastase substrate N-methoxysuccinyl-ala-ala-pro-val p- nitroanilide, EGCG, EGC, all the inhibitors, cytochalasin B, and formyl-Met- Leu-Phe (fMLP) were purchased from Sigma Chemical Co. (St. Louis, MO).

Thrombin from human plasma, cathepsin G substrate suc-ala-ala-pro-phe- pNA, and thrombin substrate H-sar-pro-arg-pNA were obtained from Calbiochem-Novabiochem (Nottingham, UK). Human k-elastin was obtained as already described.

SUBSTRATE DEGRADATION BY SERINE-PROTEINASES Leukocyte and pancreatic elastases, cathepsin G, and thrombin were solubilized (250 mU/ml) in Hepes buffer [0.1 M Hepes, 0.5 M NaCI, 10% dimethyl sulfoxide (DMSO)] at pH 8.0 (elastase and thrombin) and 7.5 (cathepsin G). All the inhibitors were freshly prepared 5X in the same buffers, except phenylmethylsulfonyl fluoride (PMSF; 10X), for which 10% ethanol was added. Elastase substrate was prepared (20X) in 100% ethanol, and cathepsin substrate was prepared (20X) in 100% DMSO. Dilutions of the inhibitor were premixed with the enzymes in micrometer wells and maintained for 15 min at 4°C ; 5 pI specific substrate at various concentrations (10-500 uM) was then added to 100 pi final volume, and the mixture was incubated at 37°C. At 20-30-min intervals, the intensity of the color developed by digested substrate was measured at 405 nm with a Titertek Multiskan (Flow Laboratories, McLean, VA), and the control background was subtracted.

Logarithmic plot and double-reciprocal plot of the results allowed the IC50s and the type and Ki of inhibition exerted on LE by EGCG to be deduced.

Elastin-zymography and gelatin-zymography were also used to confirm the inhibition of LE and thrombin by EGCG. Without heating, 10 mU LE and 100 mU thrombin were electrophoresed in 0.15% k-elastin-and 0.1% gelatin-containing 10% polyacrylamide, respectively. The gel was then washed twice for 15 min with 2.5% Triton X-100; cut into slices corresponding

to the lanes and then put in different tanks containing the stated concentrations of EGCG; incubated for 72 h at 37°C in Hepes-buffer (as above); stained for 30 min with 30% methanol/10% acetic acid containing 0.5% Coomassie brilliant blue R-250; and destained in the same solution without dye. Clear bands represent areas of gelatinolysis on the blue background.

ELASTASE-MEDIATED GELATINASE ACTIVATION Serum-less HT1080 human fibrosarcoma cell-conditioned medium was used as a source of gelatinases, being pro-MMP-9 the prevalent type. LE and EGCG were diluted in Hepes buffer to 1 mU/ui and 25 uM, respectively, and then 5 pi LE was mixed with 18 NI flavanol solution at 4°C.

After 15 min, 150 pI medium was added, and the volumes adjusted to 180 ul.

Following 4 h incubation at 37°C, 60 pi sodium dodecyl sulfate (SDS)- electrophoresis buffer 4X was added, and after mixing, 1/10 of the volume (24 NI) was processed for gelatin-zymography (6% polyacrylamide), which was developed overnight in Tris-buffer (50 mM Tris-HCI, 200 mM NaCI, 10 mM Cal2, pH 7.4) and stained as above.

NEUTROPHIL ISOLATION AND ELASTOLYTIC ACTIVITY Neutrophils were isolated under endotoxin-free conditions from buffy-coats of healthy donors according to a single-step separation procedure.

The resulting cell population contained 96-98% neutrophils with traces of eosinophils and mononuclear cells.

The cells were suspended in phenol red-less DME/F12 (Sigma- Aldrich, St. Louis, MO), mixed 1: 1 with Hepes buffer containing uM concentration of inhibitor, directly seeded (2.5X105 cells/200, ul) onto plastic microwells and incubated at 37°C in 5% CO2 in air. After 15 min, 5 ug/ml cytochalasin B was added, followed after 5 min by 100 nM fMLP. The elastase substrate was then added at 0.5 mM final concentration, and the absorbance was measured at 405 nm, as above, at 20-30-min intervals up to 2 h.

The inhibition of elastolytic activity was also tested in medium conditioned 6 h by the same number of neutrophils, incubated with 0.5 mM

substrate 5 uM inhibitor. The lysis of the substrate was measured as above, and the inhibition referred as percent of control after 2 h.

WESTERN BLOTTING Samples of medium conditioned by an equal number of neutrophils 5 uM EGCG were electrophoresed in 10% polyacrylamide gel in SDS and electroblotted to a Hybond-C Extra nitrocellulose membrane (Amersham Pharmacia Biotech, Little Chalfont, UK). The Western blotting was developed by standard procedure using rabbit anti-human neutrophil elastase or cathepsin G antibody 1: 200 (Inalco, Milano, Italy) and horseradish peroxidase-labeled anti-rabbit immunoglobulin G (IgG) 1: 1500 (Sigma Chemical Co.). Antigen detection was achieved by incubating the membrane for 1 min at room temperature with 0.125 ml/cm2 enhanced chemiluminescence (ECL)-detection solution and exposing it to Hyperfilm MP (both from Amersham Pharmacia Biotech).

NEUTROPHIL-PROMOTED PRO-MMP-9 ACTIVATION Neutrophils (5X105 in 200 pi Dulbecco's modified Eagle's medium) were seeded onto plastic microwells, activated by cytochalasin-fMLP as above, and incubated at 37°C in 5% CO2 in air with or without 5 uM EGCG.

After 6 h, 30 ul aliquots of the conditioned medium were clarified and processed directly for gelatin-zymography.

Example 1 EGCG INHIBITS DEGRADATION OF SYNTHETIC SUBSTRATE BY HUMAN LE To assay the inhibition exerted on human LE by EGCG and EGC, 5 mU purified LE was incubated for up to 2 h with the two flavanols in the presence of increasing concentrations of its synthetic substrate (N- methoxysuccinyl-ala-ala-pro-val p-ni-troanilide). Within the 0.1-0.5 mM range of substrate, LE activity is strongly inhibited by 1 uM EGCG but affected very little by 1 uM EGC, and no synergistic effect was registered. FIGURE 1A shows the inhibition of enzymatic activity (5mU) by 1 uM EGCG and EGC

compared to the control or increasing synthetic substrate concentration. The inhibition exerted by EGCG is dose-dependent and noncompetitive, as determined by double-reciprocal plotting of the results obtained at different flavanol concentrations in FIGURE 1B ; the plots share a common-1/Michaelis constant (Km) on the abscissa, and the calculated Kg is 0.34 pM. The S in FIGURE 1 B denotes substrates.

When 5 mU LE was incubated with elastase substrate in the presence of 1 uM inhibitor, the degradation was reduced 65% by EGCG and completely blocked by a1-PI, whereas ovomucoid, aprotinin, and PMSF lacked any effect. The inhibition by EGCG was maintained with a constant slope throughout the 2 h of measurement, and the deduced IC50 was 0.4 uM. This concentration is approximately 50-fold lower than that shown for MMP-2 and MMP-9 and 10,000 that shown for uPA as shown in FIGURE 2. Under the same conditions, we deduced a 30-fold lower ICso for a1-PI (13 nM) but much higher values for aprotinin (0.2 mM), PMSF (0.4 mM), and ovomucoid (4 mM).

The inhibition of LE was also verified by k-elastinzymography, developed in the presence of increasing concentrations of EGCG; dose-dependent inhibition of in situ degradation of k-elastin by 50 mU LE was indeed evident within 0.1-2 pM flavanol.

In contrast, when human thrombin (50 mU) and cathepsin G (5 mU) were incubated with their specific synthetic substrate (500 uM and 100 uM) in the presence of increasing amounts of EGCG, the deduced ICso was 60 uM and 1 mM, respectively, as shown in FIGURE 2. Furthermore, no inhibition of porcine pancreatic elastase was obtained using EGCG up to 1 mM (unpublished results). The inhibition of thrombin activity was also verified by gelatin-zymography developed in the presence of increasing concentrations of EGCG; dose-dependent inhibition of in situ degradation of gelatin by 100 mU thrombin was indeed evident within 10-103 pM flavanol.

Readily achievable plasma concentrations of the major flavanol of green tea, EGCG, inhibit the activity of human LE in a dose-dependent, noncompetitive manner, with Ksi below 350 nM. As determined from the IC50s in the experimental conditions used, this phyto-component is only 30X less potent than a1-PI, the endogenous inhibitor most involved in balancing

elastolytic burst in inflamed lungs. Conversely, EGCG exerts an inhibition superior to a variety of natural and synthetic inhibitors: its IC50 is 1/40 that shown for the microbial elastase-like proteinase inhibitor elastinal and between 1/50 and 1/200 that of some substituted cephalosporines,-lactams and trifluoro-methyl ketones-the latter ones recently suggested for the treatment of diseases characterized by neutrophil and LE involvement. The inhibition is much superior to that exerted by some standard class-specific, serine- proteinase inhibitors ovomucoid, aprotinin, and PMSF which also exert moderate activity on LE, and is maintained by EGCG over the 2-h period of measurement with a constant slope, suggesting a durable EGCG effect at body temperature.

Example 2 EGCG INHIBITS DEGRADATION OF LE SUBSTRATE BY NEUTROPHILS When freshly isolated human peripheral blood neutrophils (2.5X105), shortly preincubated with increasing concentrations of EGCG (0.3-9 , uM), were activated with cytochalasin B-fMLP, the degradation of elastase substrate (500 uM) was inhibited in a dose-dependent manner, as shown in FIGURE 3A. This was revealed by the increased absorbance at 405 nm as a result of the release of colored digestion products. Almost 30% inhibition was measured within the first 45 min with 9 uM EGCG, after which the effect decreased progressively during the following 60 min at all concentrations.

To directly compare in culture the effect of EGCG and other cell- compatible proteinase inhibitors, FIGURE 3B shows that 2.5 X 105 activated neutrophils were also incubated up to 1 h with elastase substrate in the presence of the same concentration (5 uM) of EGCG, aprotinin, 1-10 phenanthroline, or pepstatin A. Aprotinin developed approximately 30% of the inhibition exerted by EGCG, 1-10 phenanthroline <5%, and pepstatin A <10%, as confirmed in duplicate experiments.

The inhibition by other standard class-specific proteinase inhibitors was also tested in the cell-free system, on culture medium conditioned for 6 h by freshly isolated neutrophils and incubated up to 2 h with elastase substrate. FIGURE 3C shows that while 5 uM EGCG reduced up to

67% of the substrate degradation measured in the control, this was almost completely suppressed by the same concentration of a1-PI but only <15% and <5% by 10 mM ethylenediaminetetraacetate (EDTA) and N-ethylmaleimide (NEM), respectively, in duplicate experiments.

EGCG is also effective on neutrophil culture. In fact, non- cytotoxic concentrations of flavanol restrain the elastolytic activity of freshly isolated neutrophils in a dose-dependent manner, although less efficiently compared with the biochemical assays. At any concentration of flavanol, the inhibition of neutrophil elastolytic activity declines progressively over the first 2 h of incubation, although we verified that LE is released into the medium only during the initial phase following activation. The constant slope of the time- course inhibition registered with purified LE should rule out the possibility that this decline may be attributable to the thermo-instability of the flavanol. Most likely, the loss of EGCG potential may be attributable to flavanol oxidation by the hydrogen peroxide secreted after a lag period during a prolonged respiratory burst induced on neutrophils by cytochalasin-fMLP treatment.

Under the same conditions, aprotinin is 70% less effective in comparison with the same concentration of flavanol ; this moderate effect is not in conflict with the complete lack of inhibition of LE in the test tube, because it may be a result of a block of other serine-proteinases active on the substrate.

Certainly, most of the elastolytic activity released by the neutrophils is attributable to serine-proteinases as inferred from the complete inhibition of substrate degradation in the presence of a1-PI and the marginal inhibition in the presence of metallo-proteinase (1.10 phenanthroline, EDTA) and cysteineproteinase inhibitors (pepstatin A, NEM). Furthermore, be-cause of the two serine-proteinases released mostly by neutrophils, cathepsin G is, in comparison, largely insensitive to EGCG, the prevalent serine-proteinase inhibited by uM flavanol should be LE. Indeed, the presence of LE into the medium was proved by specific antibodies, which revealed unmodified levels of the proteinase in the presence of the flavanol. Conversely, the abundant MMP-9 secreted by neutrophils may be, in part, responsible for the elastolytic metalloproteinase activity.

Example 3 EGCG INHIBITS MMP-9 ACTIVATION BY PURIFIED LE AND ISOLATED NEUTROPHILS The possibility that EGCG may interfere with LE activation of MMPs, in particular with gelatinases MMP-2 and MMP-9, was also analyzed.

When culture medium containing mostly pro-MMP-9 was incubated for 4 h with 0.5 mU LE before assaying in gelatin-zymography, the conversion of the zymogen to the activated form of MMP-9 was restrained substantially by the presence of 2.5 uM EGCG, as shown in FIGURE 4A, but not EGC. Under the same conditions, LE alone did not produce substantial increments of the MMP- 2 activated form.

Parallel results were obtained when freshly isolated neutrophils were incubated for 6 h in the presence of 5 uM flavanol. Gelatin-zymography of the culture medium shows abundant MMP-9, as already demonstrated, most in the zymogen form, plus an unidentified gelatinolytic band of lower relative mobility (Mr). FIGURE 4B shows that although already less represented in the control, the activated form of MMP-9 was diminished substantially in the presence of EGCG. The level of total secreted MMP-9 registered no substantial difference. EGC had no effect.

Regardless of the respective contribution in elastin degradation, MMP-9 and LE are extracellularly blocked by the flavanol, which thus has the potential to contain the degradative neutrophil activity in an in vivo context in the case of endogenous inhibitor failure. In fact, EGCG has already been shown to inhibit two gelatinases, MMP-2 and MMP-9, whose activity is instrumental to endothelial and tumor cells in cutting through extracellular matrix barriers during angiogenetic and invasive/metastatic processes and pulmonary emphysema. We showed that the IC50 of EGCG for these MMPs lies in the range of 10-30 pM, and the second-most abundant flavanol in green tea, EGC, shows much lower efficacy (1/30). Biochemical assays now reveal that EGCG exerts an even stronger inhibition of LE (50X). This inhibition occurs at concentrations approximately 25-fold and two orders of magnitude lower than the cytotoxic threshold already demonstrated on transformed and normal endothelial cells, respectively, and similar to those in the plasma of

moderate green tea drinkers (0.1-0.3 uM). Again, EGC exerts very little inhibition.

When purified LE is incubated with pro-MMP-9 in a cell-free system, the conversion of this zymogen into activated form is restrained substantially in the presence of EGCG. In addition, the activation of pro-MMP- 9 secreted by freshly isolated neutrophils is somewhat limited when they are cultured briefly in the presence of the flavanol. This reduced activation may play an important role in airway inflammation and tumor disease; it would contribute to the down-regulation of local proinflammatory interleukin (IL)-1ß activity, restraint of transforming growth factor-p (TGF- (3) induction of tumor- cell invasion and angiogenesis, preservation of the most potent inhibitor of LE, restraint of neutrophil recruitment by chemoattractant fragments of a1-PI, preservation of the underlying elastin structure of the lung, and containment of degradation of the basement membrane molecular scaffold, a prerequisite for angiogenesis and tumor-cell invasion.

Regarding angiogenesis, elastase has been shown to convert plasminogen into angiostatin, a potent inhibitor of angiogenesis, and elastase activity inhibition by EGCG could inhibit production of angiostatin, thus promoting inflammation. This paradox may be only apparent; in fact, angiostatin is only one of the endogenous inhibitors of angiogenesis (e. g., endostatin and thrombospondin), and inhibition of LE and inflammation reduces the expression of a number of proangiogenic factors, i. e., fibroblast growth factor (FGF)-ß, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and cytokines.

Regarding tumor-cell invasion, the inhibition of direct lytic activity of LE on collagen IV must also be taken into account as a potential contributor to the documented reduction of tumor-invasive processes. Conversely, the level of MMP-9 expression by neutrophils is not affected substantially by EGCG. Thus, their potential ability to traverse basement membrane barriers and their recruitment at the site of inflammation for immune-surveillance are not likely to be impaired upon treatment with flavanol concentration effective on LE (10'7 M). Indeed, neutrophil motility-proteolysis necessary to transmigrate

in vitro through endothelial cell monolayers is reduced by EGCG with an IC50 of approximately 10-5 M.

In addition, thrombin efficiently activates one of the invasion- related gelatinases, MMP-2, as registered in a microvascular endothelial cell model. Thrombin is here shown to be inhibited by EGCG at concentrations higher than those effective on MMP-2; thus, degradation of basement membrane by sprouting endothelial cells during angiogenesis would be restrained primarily by direct inhibition of MMP-2. Conversely, the prevention of cardiovascular pathologies ascribed to green tea consumption and the demonstrated antithrombotic activities of catechins may not be attributable to direct inhibition of thrombin by EGCG, whose plasma level in green tea drinkers reaches 1/200 the ICso for the proteinase. Even so, the potential use of flavanols as thrombin inhibitors merits deeper investigation.

Furthermore, an IC50 of 8 X 10-8 M has been measured for EGCG on the invasive behavior of tumor cells in vitro], over 100-fold lower than that mentioned above for neutrophil transmigration. Whatever lies behind these differences, they offer the possibility of inhibiting disruption of elastic scaffold by LE and tumor-cell aggressiveness, without substantially impairing the physiological traffic of neutrophils, and should certainly facilitate future pharmacological applications of this natural inhibitor.

Example 4 EGCG DOES NOT REDUCE ELASTASE AND CATHEPSIN G SECRETION BY NEUTROPHILS To verify whether EGCG restrains LE and cathepsin G secretion, 106 freshly isolated neutrophils were preincubated in serum-less medium with and without 5 uM flavanol and activated with cytochalasin A-fMLP as above; then the conditioned medium was analyzed directly at 1,2, and 4 h by Western blotting for the two serine-proteinases. FIGURE 4C shows that the presence of the flavanol had no measurable effect on the level of LE released into the culture medium, which remained constant from 1 h onward. Also, the presence of the flavonal had no measurable effect on cathyssin G levels.

Cathepsin G, stored in the azurophilic granules of neutrophils and monocytes and released upon cell stimulation or lysis, can degrade a wide array of matrix and humoral proteins, although less potently than LE, and activate MMP-9. While cathepsin G release is not affected by uM concentrations of EGCG, the inhibition exerted by the flavanol on its activity is markedly weaker than that on LE (1/2500) but also less pronounced than that on MMP-2 (1/50): the IC50 on cathepsin G is in the mM range, close to that on uPA, and may contribute to the residual MMP-9 activation at EGCG concentrations over one order of magnitude of the IC50 for LE. The differential effectiveness of EGCG on the activity of LE and cathepsin G may prove to be useful in clinical application of the flavanol, considering the need to preserve a number of cathepsin G-mediated reactions, i. e., coagulation, immune response, and wound debridement.

While certain representative embodiments and details of the present invention have been shown for the purposes of illustrating the invention, it will be apparent to those skilled in the art that various changes and modifications may be made therein without departing from the spirit or scope of the invention.