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INFLAMMATION AND IMMUNOPHARMACOLOGY

Inhibition of Leukocyte Elastase, Polymorphonuclear Chemoinvasion, and Inflammation-Triggered Pulmonary Fibrosis by a 4-Alkyliden--lactam with a Galloyl Moiety

Department of Experimental Biomedical Sciences, Medical School of Padova, Padova, Italy (I.D.A., L.S., S.G.); Department of Chemistry `G. Ciamician', University of Bologna, Bologna, Italy (P.G., D.G., A.Q.); Department of Pathology, Medical School of Padova, Padova, Italy (F.C., C.G.); and Department of Clinical Medicine I, Medical School of Padova, Padova, Italy (E.B., F.P., C.A.)

Received September 27, 2005; accepted October 24, 2005.

	Abstract

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-Lactams, a well known class of antibiotics, have been investigated as inhibitors of the disruptive protease released by inflammatory cells, leukocyte elastase (LE). We have synthesized a new -lactam with an N-linked galloyl moiety, the latter identified as strategic in conferring anti-LE properties to some flavonols. This N-galloyl-derivative -lactam inhibits the LE activity with a K_i of 0.7 µM, whereas it exerts weak activity against cathepsin G and protease-3 (IC₅₀ > 100 µM), and matrix metalloproteinase (MMP)-2 and MMP-9. Without affecting chemotactic response and viability of polymorphonuclear (PMN) leukocytes, the compound efficiently restrains their chemoinvasion (IC₅₀ of 1-2 µM) blocking the LE-triggered activation of pro-MMP-9, instrumental to extravasation. Daily i.p. injection of compound enhances resolution in a pulmonary inflammation model, significantly reducing consequent fibrosis. These results indicate that the new -lactam is a potent anti-inflammatory compound with therapeutic potential.

Human tissues are protected from excessive LE activity by endogenous inhibitors, such as 1-protease-ihibitor (1-PI), 2-macroglobulin (Lee and Downey, 2001), and secretory leukoprotease inhibitor (Rice and Weiss, 1990), but any enzyme/inhibitor imbalance may lead to increased lysis of ECM macromolecules and increased risk of tissue injury in areas infiltrated by activated PMN leukocytes (Lee and Downey, 2001). Furthermore, given the LE ability to degrade multiple cytokines, receptors, and complement components, a negative modulation of the inflammatory response may favor antigen persistence, leading to chronic inflammation.

As for the possibility of using exogenous LE inhibitors for therapeutic purposes, most of the inhibitors so far developed cause side effects that make them unsuitable for human use (Teshima et al., 1982). Recently, a number of -lactams, compounds widely used as antimicrobial drugs, have been identified as inhibitors of serine enzymes, in particular LE (Konaklieva, 2002). A core structure of four-membered cyclic amide (-lactam or azetidin-2-one) is the common feature of these molecules; the first LE inhibitor -lactams were naturally occurring bicyclic compounds, such as clavams and cephalosporins (Knight et al., 1992), but more recently synthetic monocyclic -lactams have been developed. Because the latter perform with extremely good safety profiles and infrequent side effects (Kuhn et al., 2004), they could represent a good model base for designing powerful drugs able to inhibit LE and restore the altered protease/antiprotease ratio at the inflammatory sites.

View larger version (25K): [in this window] [in a new window]   Fig. 1. A, scheme of synthesis of compounds 1 and 2 (4-alkyliden--lactam). B, structure of EGCG with encircled galloyl moiety. C, scheme of synthesis of compound 3, here named N-galloyl-4-alkyliden--lactam.

Some catechins (vegetable secondary metabolites of the flavonoid family), and in particular those with a galloyl group (Fig. 1B), have been shown to exert a very powerful inhibition of LE activity (Garbisa et al., 2000; Sartor et al., 2002a,b), but their absorption, bioavailability, and metabolic fate await full clarification. We have thus synthesized and tested a number of monocyclic -lactam derivatives with a galloyl moiety-like group in different positions (Cainelli et al., 2005); some of these, such as the {3-[1-(tert-butyl-dimethylsilanyloxy)-ethyl]-4-oxo-1-(3,4,5-tris-benzyloxy-benzoyl)-azetidin-2-ylidene}-acetic acid ethyl ester (Fig. 1C), exert indeed an improved anti-LE activity. Here, we show that this latter compound wields the most potent inhibition of LE so far reported for its category and restrains PMN leukocyte chemoinvasion, protecting against inflammatory events taking place in the bleomycin-triggered animal model of lung fibrosis.

	Materials and Methods

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Materials. Elastase (LE) and proteinase-3 (PR-3) from human leukocytes were obtained from Elastin Products Company Inc. (Owensville, MO); cathepsin G, elastase substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide, cytochalasin B, and formyl-Met-Leu-Phe (fMLP) were from Sigma-Aldrich (St. Louis, MO); PR-3 substrate methoxysuccinyl-Lys-(2-picolinoyl)-Ala-Pro-Val-p-nitro-anilide was from Bachem (Bubendorf, Switzerland); cathepsin G substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide was from Calbiochem-Nova-biochem (Nottingham, UK); fetal calf serum (FCS) was from Biochrom (Berlin, Germany); basement membrane matrix (Matrigel) was from BD Biosciences Discovery Labware (Bedford, MA); polyvinylpyrrolidone-free polycarbonate filters were from Millipore (Billerica, MA); and Ficoll-Paque Plus (<0.12 enzyme unit/ml) was from GE Healthcare (Little Chalfont, Buckinghamshire, UK). All of the other reagents, if not specified, were purchased from Sigma-Aldrich.

Synthesis and Characterization of -Lactams. The {3-[1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-4-oxo-azetidin-2-ylidene}-acetic acid ethyl ester (hereafter named 4-alkyliden--lactam; Fig. 1A, compound 2) and its derivative {3-[1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-4-oxo-1-(3,4,5-tris-benzyloxy-benzoyl)-azetidin-2-ylidene}-acetic acid ethyl ester (hereafter named N-galloyl-4-alkyliden--lactam; Fig. 1C, compound 3), obtained by inserting a galloyl moiety with O-benzyl protections at the nitrogen atom, were prepared according to the procedure reported previously (Cainelli et al., 2005) and is outlined here.

Commercial reagents were used as received without additional purification. Anhydrous solvents were obtained commercially and used without further drying. ¹H and ¹³C NMR values were recorded on a Varian Mercury 400, Inova 300, or Gemini 200 instrument using a 5-µm probe. All chemical shifts have been quoted relative to deuterated solvent signals, in ppm, J in Hz. Fourier transform infrared: Nicolet 205 measured as films or Nujol mull between NaCl plates and reported in centimeter^-1. Thin-layer chromatography: Merck 60 F₂₅₄. Column chromatography: Merck Silica Gel 200-300 mesh. HPLC-mass spectrometry: HPLC: HP1100 (Agilent Technologies, Palo Alto, CA) column ZOBRAX-Eclipse XDB-C8 (Agilent Technologies). The products were eluted with CH₃CN/H₂O, gradient: from 30 to 80% of CH₃CN. Mass spectrometry: MSD1100 single-quadrupole mass spectrometer (Agilent Technologies), full-scan mode from m/z 50 to m/z 2600, scan time 0.1 s in positive ion mode, electrospray ionization spray voltage 4500 V, nitrogen gas 35 psi, drying gas flow 11.5 ml/min, fragmentor voltage 20 V. The []²⁵_D values were determined with a PerkinElmer 343 polarimeter (PerkinElmer Life and Analytical Sciences, Boston, MA). Compounds 1 and 2 (Fig. 1A) were prepared according to the procedure reported previously (Cainelli et al., 2005).

Compound 3 [3-[1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-4-oxo-1-(3,4,5-tris-benzyloxy-benzoyl)-azetidin-2-ylidene]-acetic acid ethyl ester was prepared as follows (Fig. 1C): compound 2 (0.381 g; 1.017 mmol) and 3,4,5-tribenzyloxybenzoyl chloride (0.513 g; 1.119 mmol) were dissolved in anhydrous acetone (10 ml); K₂CO₃ (0.141 g; 1.017 mmol) was added, and the reaction mixture was stirred at room temperature for 4 h. Then, K₂CO₃ was filtered, the solvent was removed, and the crude oily residue was immediately purified by flash chromatography (cyclohexane/ethyl acetate = 95/5) to give compound 3, yield 44%, pale yellow oil. ¹H NMR (CDCl₃, 300 MHz) 0.17 (s, 3H), 0.19 (s, 3H), 0.96 (s, 9H), 1.26 (d, J = 6.3 Hz, 3H), 1.35 (t, J = 7.2 Hz, 3H), 4.25 (q, J = 7.2 Hz, 2H), 4.34 (dd, J = 4.8 Hz, J = 1.2 Hz, 1H), 4.82 (m, 1H), 5.20 (s, 6H), 6.66 (, J = 1.2 Hz, 1H), 7.29-7.48 (m, 17H). ¹³C NMR (CDCl₃, 75.5 MHz) -4.9, -4.6, 14.3, 17.9, 19.8, 25.7, 60.3, 64.1, 65.4, 71.4, 75.2, 100.5, 109.9, 125.8, 127.4, 127.5, 128.0, 128.2, 128.4, 128.5, 136.6, 137.3, 143.3, 149.3, 152.4, 164.1, 164.6, 166.2. []²⁵_D =+47.3 (CHCl₃,c = 4.16). Infrared (film): 2930, 1841, 1718, 1683, 1328, 1197 cm^-1. HPLC: R_t = 9.64 min. The in vitro antioxidant activity of the N-galloyl-4-alkyliden--lactam was evaluated by the Trolox equivalent antioxidant capacity method (Miller et al., 1993), a spectrophotometric measurement of antioxidant-triggered discoloration of a green solution containing a pre-formed organic cation radical.

Neutrophil Isolation. Neutrophils were isolated under endotoxin-free conditions from buffy coats of healthy donors, according to a separation procedure described previously in detail (Dri et al., 1999). In brief, freshly prepared buffy coats were diluted 1:3 in PBS, and centrifuged at 180g for 10 min three times to reduce contamination by platelets. After further rinsing in PBS, cells were layered on Ficoll density gradient and centrifuged at 280g for 30 min. Peripheral blood mononuclear cells were collected at the interface, and PBS and residual Ficoll were removed. The pellet, which contained erythrocytes and PMN leukocytes, was diluted with 3 volumes of ammonium chloride lysis solution (155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA, pH 7.4) and incubated at 4°C for 10 min. After centrifugation (180g for 10 min at 4°C), the lysis was repeated to eliminate residual erythrocytes. The final pellet, assessed by direct observation of nuclear morphology to consist of >96% PMN leukocytes, was immediately used for the assays, after dilution in the proper buffer solution.

Inhibition of Proteolytic Activities. Concentrated stock solutions of LE and cathepsin G were prepared in HEPES buffer (0.1 M HEPES, 0.5 M NaCl, and 10% DMSO), and PR-3 in glycine buffer (0.1 M glycine and 0.1 M NaCl), at pH 7.8, 7.5, and 3.2, respectively, as described previously (Sartor et al., 2002b; Pezzato et al., 2003). N-Galloyl-4-alkyliden--lactam and substrates were freshly prepared 20x in DMSO, and dilutions of the -lactam were premixed with the enzyme in 96-well plates and maintained 30 min at 4°C. Then, 5 µl of substrate (8 mM for LE, 10 mM for cathepsin G, and 4 mM for PR-3) was added to 100 µl of final volume, and the mixture was incubated at 37°C. At 20-min intervals, the intensity of the color developed by the digested substrate was measured at 405 nm using a Titertek Mutiskan (Flow Laboratories, Irvine, Scotland, UK), and the control background was subtracted (in triplicate experiments). The reactions developed linearly for as long as 120 min; data from 60 min were used for plotting the colorimetric reactions. Double-reciprocal plot of the results allowed the type and K_i of inhibition exerted over LE to be deduced.

MMP-9 Activation by LE and PMN Leukocytes. Serum-less medium (Dulbecco's modified Eagle's medium, DMEM) conditioned 90 min by PMN leukocytes under gentle shaking was used as a source of gelatinases, with pro-MMP-9 being the prevalent type. LE and N-galloyl-4-alkyliden--lactam were diluted in DMEM to 1 mU/ml and 2 mM, respectively (the pH of the medium was preadjusted to 7.8, to optimize the LE activity and to preserve the -lactam stability); 5 µl of LE was mixed with -lactam solution at 4°C, and after 15 min the medium was added. After 1-h incubation at 37°C, SDS-electrophoresis buffer (4x) was added, and the samples were processed for 6% polyacrylamide gelatin-zymography (see below).

Gelatin-Zymography. Without heating the samples, zymography was performed by electrophoresing the samples in 0.1% gelatin containing 6% polyacrylamide, in the presence of SDS. After electrophoresis, the gels were washed twice for 30 min with 2.5% Triton X-100 and incubated overnight at 37°C in Tris buffer (50 mM Tris-HCl, 200 mM NaCl, and 10 mM CaCl₂, pH 7.4). For gelatinase inhibition assays, the N-galloyl-4-alkyliden--lactam was freshly solubilized in DMSO and diluted (1, 10, and 100 µM) in the Tris buffer used for developing separate slices of gelatinase zymograms, which were incubated as described above.

The gels were then 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 on the blue background represent areas of gelatinolysis. Digestion bands were quantitated using an image analyzer system with Gel-Doc 2000 and QuantityOne software (Bio-Rad, Hercules, CA).

PMN Chemotaxis, Chemoinvasion, and Viability. The chemotaxis and chemoinvasion of PMN leukocytes in response to fMLP was tested using the modified Boyden chamber assay, as reported previously (Benelli et al., 2000). Gelatin (nonbarrier for chemotaxis) and Matrigel (barrier for chemoinvasion) were used as matrix substrates for cell migration toward a chemoattractant represented by 10^-7 M solution of fMLP in DMEM without phenol red (control experiments were performed in absence of chemoattractant). Polyvinylpyrrolidone-free polycarbonate filters (5-µm pore size) were all precoated by immersion in gelatin solution (0.1%) and part over-coated with 50 µl of 0.66 mg/ml Matrigel. After seeding 2 x 10⁶ cells onto the filters, and 45-min (chemotaxis) or 2-h (chemoinvasion) incubation in serum-free medium with or without the N-galloyl-4-alkyliden--lactam, nonmigrated cells were removed from the upper surface of the filters, and those that actively migrated to the lower chamber were harvested and directly counted using a hemocytometer. The results of quintuplicate experiments were averaged after background subtraction (control experiments).

The effect of the N-galloyl-4-alkyliden--lactam on short-term PMN leukocyte viability was also verified. Freshly isolated PMN leukocytes were seeded (5 x 10⁵) onto 96-well plates and incubated in 5% CO₂ air at 37°C in 90 µl of DMEM without phenol red, supplemented with or without 10% FCS, and with or without the -lactam at increasing concentrations. After 4 and 20 h, the cell viability was determined by CellTiter 96 assay (Promega, Madison, WI).

Bleomycin-Induced Pulmonary Fibrosis. Ten- to 12-week-old C57BL6/N mice (weighing 22-25 g) from Charles River (Lecco, Italy) were maintained 14 days in a germ-free environment and allowed free access to food and water before use. Then, pulmonary fibrosis was induced in 32 animals by intratracheal instillation of bleomycin as described previously (Keane et al., 1999). Here, bleomycin was dissolved to a concentration of 1.25 U/ml in sterile PBS and vortexed extensively before each 100-µl aliquot (5 U/kg) was used. The mice were anesthetized and the trachea was exposed and entered with a 29-gauge needle. Intratracheal instillation was then performed slowly, and the skin wound was sealed and treated with Betadine solution. The experiment group of mice (20 animals) was given i.p. injection of 1 mM N-galloyl-4-alkyliden--lactam in 40 µl of PBS, at 9:00 AM and 6:00 PM every day, excluded Sunday, starting 3 days before (10 mice) or the same day of (10 mice) instillation; the control group was left untreated. All experiments conformed to the regulatory standards and were approved by the Medical School Committee of Padova.

Preparation of the Tissues. Four weeks after intratracheal instillation, the mice were weighed, anesthetized, heparinized, and exsanguinated via the femoral artery. The heart and lungs were removed en bloc; the lungs were dissected away from the external vasculature and bronchi and sectioned parasagittally, superior to inferior. Liver and kidneys were also explanted, and all the specimens were fixed in buffered 4% paraformaldehyde for morphological studies.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Inhibition of PMN leukocyte proteases by the N-galloyl-4-alkyliden--lactam. A, LE, PR-3, and cathepsin G (cat.G) were preincubated 15 min with increasing concentration of the -lactam and then the specific chromogenic substrates were added, and the mixture was incubated 60 min at 37°C. The absorbance (405 nm) of triplicate digestions was averaged (S.D. always <10%) and expressed as percentage of controls. In the same conditions, the precursor molecule (2 in Fig. 1A) at 100 µM inhibits only 66% LE activity (Cainelli et al., 2003a). B, the double-reciprocal plot demonstrates noncompetitive inhibition of LE by increasing concentration of compound (mean values of triplicate samples, with S.D. <10%). S, substrate.

Determination of Hydroxyproline. The hydroxyproline content of mouse lung was determined by standard methods (Moore et al., 2001), with slight modifications. After rinsing with PBS, the upper left lung lobe was defatted, dried, weighed, and hydrolyzed for 22 h at 110°C in 6 N HCl. Aliquots were then assayed by adding chloramine T solution for 20 min, 3.15 M perchloric acid for 5 min, and Erlich's reagent at 60°C for 20 min. Absorbance was measured at 561 nm, and the amount of hydroxyproline was determined against a standard curve.

Statistics. Data are expressed as the means of three determinations for biochemical and four determinations for migration and invasion assays (four PMN leukocyte donors separately). Morphological examination was performed, analyzing at least two sections from each lung per animal, and the data were averaged both as mean score (MS) and mean percentage (MP), separately for the two experimental groups (with and without 3-day pretreatment). Comparisons were conducted by one-way analysis of variance, with significance set at p < 0.05.

	Results

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Synthesis of the N-Galloyl-4-alkyliden--lactam. The 4-alkyliden-azetidin-2-ones represent a relatively new class of -lactam compounds: few examples were studied in the 1980s (Greengrass and Hoople, 1981; Prasad et al., 1982), with no further development. These compounds have been recently obtained using an original and well established protocol starting from 4-acetoxy-azetidinones and diazoesters (Cainelli et al., 2003b). We demonstrated that the unsaturated system on the alkylidene side chain activates these monocyclic -lactams toward serine proteases such as human LE (Cainelli et al., 2003a). N-Galloyl-4-alkyliden--lactam was prepared with a two-step synthesis (Fig. 1): AlCl₃-mediated addition of ethyl diazoacetate to 3-(2-tert-butyldimethylsilyloxyethyl)-4-acetoxy-azetidin-2-one and subsequent N- acylation reaction with tri-O-benzyl-galloyl chloride (Cainelli et al., 2005). ¹H and ¹³C NMR analyses confirmed the chemical structure presented in Fig. 1C for the target compound.

The oxidant property of the new compound was tested with the Trolox equivalent antioxidant capacity analysis, which registered an extremely low Trolox equivalent value (17.3 µM in 6 min, from 2.12 mM N-galloyl-4-alkyliden--lactam in ethanol), indicating that the compound has no relevant antioxidant activity. The pH value of all buffers containing this compound as described in this section was always found unmodified.

Inhibition of Proteolytic Activities. When the three PMN leukocyte proteases (LE, cathepsin G, and PR-3) were preincubated with N-galloyl-4-alkyliden--lactam and then mixed with their specific synthetic substrates, a dose-dependent inhibition was registered, which maintained a constant slope throughout the 2 h of measurement. The 60-min plot shows that the IC₅₀ for LE is very low (below micromolar concentration), whereas that of cathepsin G and PR-3 are more than 100 µM (Fig. 2A). The double-reciprocal plotting of the results obtained at different -lactam concentrations for LE—sharing a common-1/Michaelis constant (K_m) on the abscissa—reveals that this inhibition is noncompetitive, with a K_i = 0.7 µM (Fig. 2B).

The effect of the N-galloyl-4-alkyliden--lactam was also studied on the MMP-2 and MMP-9 activities contained in a culture medium conditioned by tumor cells (Garbisa et al., 1999). When the -lactam was present (0-100 µM) in the buffer during the development of gelatin-zymography of the medium, the MMP-9-corresponding digestion bands were not inhibited, and those of MMP-2 were inhibited only by 25% at the highest concentration (not shown).

Effects on Chemotaxis and Chemoinvasion. The effect of the N-galloyl-4-alkyliden--lactam on PMN leukocyte migration toward fMLP was measured by using a modified Boyden chamber assay. On gelatin-coated filters (chemotaxis), PMN leukocytes showed a 2-fold increase in migration to fMLP over unstimulated controls; in this case, 15-min pretreatment with N-galloyl-4-alkyliden--lactam—and its presence during the assay—did not affect PMN chemotaxis, within the tested range of concentrations (0-6 µM) (Fig. 3). Controls with the -lactam in the lower chamber excluded that the compound itself has chemotactic activity (not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 3. Chemoinvasion, but not chemotaxis, of PMN leukocytes is inhibited by N-galloyl-4-alkyliden--lactam. The modified Boyden chamber assay shows that PMN chemotaxis through gelatin (open circles), toward fMLP in the bottom chamber, is not restrained by the -lactam present in the top chamber; whereas a parallel experiment shows that PMN chemoinvasion through Matrigel (full circles) is restrained in a dose-dependent manner, with an IC50 of 1 to 2 µM. Examples of quadruplicate experiments (45 min, c. taxis; 2 h, c. invas.); average of quadruplicate ± S.D.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Inhibition of LE-triggered activation of pro-MMP-9 by N-galloyl-4-alkyliden--lactam. Gelatin-zymography of equal volumes of MMP-9 gelatinase-containing culture medium, preincubated 2 h at 37°C with 0.5 mU of commercial LE, and increasing concentrations of -lactam. Numbers in white are percentage of inhibition, as from densitometry of the activated form of MMP-9.

Effects on MMP-9 Activation by LE. Usually, the medium conditioned 1 to3hby freshly isolated PMN leukocytes contains a prevalent gelatinolytic activity that is recognized as the zymogen form of MMP-9, and a limited proportion of this enzyme is present in the activated form (varying from 9:1 to 8:2 ratio in different preparations). Preliminary data indicated that within the range of concentrations used, the new -lactam does not inhibit LE release from PMN leukocytes (data not shown); but, to boost the LE-triggered MMP-9 activation, aliquots of culture medium obtained from PMN leukocytes were incubated at 37°C for 2 h with 5 mU of purified LE; in the presence of µM N-galloyl-4-alkyliden--lactam, the conversion of the zymogen to the activated form of MMP-9 was restrained in a dose-dependent manner, with >40% decrease at 0.23 µM, and >80% at 6.3 µM, as verified by gelatin-zymography and densitometry (Fig. 4).

Effects on Induced Pulmonary Fibrosis. Pulmonary fibrosis was induced in mice by intratracheal instillation of bleomycin, and the experiment group of mice was given i.p. injection of N-galloyl-4-alkyliden--lactam every day for 4 weeks, whereas the control group was left untreated (as described in detail under Materials and Methods).

At the end of the experiment, the weight of mice treated i.p. with -lactam [(+)--lactam] showed no significant differences compared with controls [(-)-bleomycin]; and from the start of the experiment (T₀), mice registered a gain 2.36 times higher than that of the (-)--lactam mice (from 17.0 to 20.3 and 18.4 g, respectively; p < 0.01) (Fig. 5). No side effects were noted throughout the experiment. Intraperitoneal administration of the compound twice daily for 31 days, starting 3 days before bleomycin treatment, registered some nonmarginal effects even before animal sacrifice: all 10 (+)--lactam mice seemed healthy during the experiment, whereas of the 12 (-)--lactam controls 2 died after 8 to 10 days, and six mice presented signs of dyspnea, ataxia, and characteristic fur changes the second week after treatment. A similar effect was registered without 3-day pretreatment (not shown).

View larger version (88K): [in this window] [in a new window]   Fig. 5. Daily i.p. N-galloyl-4-alkyliden--lactam protects mice from inflammation-triggered pulmonary fibrosis. Foci of inflammatory infiltration (arrows in A; H&E), cuboidalization of epithelial cells (arrows in B), and peribronchial and interstitial fibrosis (blue-green in C; Heidenhaim trichrome) are clearly visible in example of lungs of bleomycin-treated mice, whereas reduced or normal parenchyma is seen in bleomycin + -lactam-treated animals after 4 weeks (D and E, H&E; F, Heidenhaim trichrome). All histologies are at 25x original magnification. Image analysis quantitation confirms the reduction of bleomycin-induced inflammation (histogram 1), cuboidalization (histogram 2), and fibrosis (histogram 3) in bleomycin + -lactam-treated mice. Eventually, the weight gain (from T0) in bleomycin + -lactam-treated mice was significantly higher than in those treated with bleomycin alone (histogram 4).

To quantitatively determine the extent of fibrosis, hydroxyproline content in the lung was measured as a surrogate for lung collagen deposition. Whereas in untreated animals the value was 12.3 µg/lobe, in those treated with bleomycin the values measured 4 weeks after instillation were 15.8 and 14.2 µg/lobe with p < 0.01, in (-)--lactam and (+)--lactam, respectively. No degenerative changes were observed in heart, liver, and kidney tissues, thus excluding drug toxicity.

	Discussion

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Here, we describe the activity of a new N-galloyl-4-alkyliden--lactam exhibiting the most potent inhibition of LE so far reported for this group, and only 1 order of magnitude lower than the endogenous 1-PI; this primary effect leads to restraint of MMP-9 activation, containment of PMN leukocyte recruitment, and eventually marked down-grading of inflammation-triggered pulmonary fibrosis.

Chemistry and Biochemistry. The addition of a galloyl group, with O-benzyl protection of all three hydroxy groups, to the nitrogen atom of 4-alkyliden--lactam markedly increases the inhibition exerted on LE, in comparison with the precursor molecule: the LE activity is inhibited >95% by the new compound at 10 µM, but only 66% by 100 µM precursor--lactam (Cainelli et al., 2003a, 2005). Although the new compound inhibits LE activity in a dose-dependent noncompetitive manner, with K_i in the micromolar range (0.7 µM), the precursor showed a nonconsistent slope of inhibition, precluding calculation of K_i. As for the majority of the 4-alkylidene-azetidin-2-ones tested, the registered inhibition was maintained with a constant slope over the 2-h period of measurement, suggesting a stable effect at body temperature.

The new N-galloyl-4-alkyliden--lactam is thus the most potent LE inhibitor of its class, and the presence of the polyphenolic substituent in this -lactam was a good choice for boosting this activity. This new compound is as good as (-)-epigallocatechin-3-gallate (EGCG), the most powerful exogenous inhibitor of LE so far described (IC₅₀ = 0.4 µM) (Sartor et al., 2002a); and, like EGCG, it is not as active against proteinase-3 and cathepsin G (IC₅₀ > 100 µM), also released by PMN leukocytes. In contrast, the inhibition of MMP-2 and MMP-9, two gelatinases instrumental in cell invasion, is markedly lower (IC₅₀ > 100 µM versus 10-30 µM) (Garbisa et al., 1999), widening the span between the efficacy on gelatinases and LE, and thus rendering the -lactam even more specific for this potent proteolytic weapon. In addition, and in line with the effects obtained with other LE inhibitors (Clemente et al., 2001), the new -lactam hinders the LE-triggered activation of the zymogen form of MMP-9, the major protease involved in PMN leukocyte migration across basement membrane (BM) during extravasation (see below).

We have previously shown that the galloyl group is essential for conferring to the catechins their anti-LE properties (Garbisa et al., 2000; Sartor et al., 2002a); and in the attempt to boost this character on -lactams, we thus designed a new series of compounds with a phenolic group added to the nitrogen atom of the -lactam ring (Cainelli et al., 2005). The excellent anti-LE property exerted by the N-galloyl-4-alkyliden--lactam, in which the three hydroxyls are protected by benzyl residues, now confirms that although the galloyl framework is crucial for the inhibitory activity toward LE, the free hydroxyls are not. The latter were indeed shown to be mostly important in conferring antigelatinolytic properties (Sartor et al., 2002a).

As a preliminary approach, the possibility has been excluded that this anti-LE property may descend from antioxidant characteristics (well known, in contrast, for the catechin group, from which the galloyl residue has been adopted). Some authors reported that the mechanism of LE inhibition by -lactams involves a reaction with the nucleophilic serine-195 at the active site of elastase, which opens the -lactam ring to form a stable acyl-enzyme intermediate (Knight et al., 1992); but recently, other mechanisms have been defined (Taylor et al., 1999; Gerard et al., 2004). Although the non-competitive inhibition registered for the new compound excludes the binding of this -lactam to the LE active site and should run off the former mechanism, no hints are yet available to suggest the latter or any other mechanism. Regard-less, the new -lactam exerts an inhibition much superior than other natural and synthetic inhibitors of LE, in particular some standard class-specific serine-proteinase inhibitors such as phenylmethylsulfonyl fluoride and aprotinin (Sartor et al., 2002a), is only 1 order of magnitude less potent than the endogenous inhibitor 1-PI (Campbell et al., 2000), and exerts this property at noncytotoxic concentration.

PMN Leukocytes in Vitro. Leukocyte extravasation and trafficking into the tissues are crucial events in the host defense response, but overactivation and migration of PMN leukocytes to the site of inflammation, with their disruptive proteolytic load, can negatively contribute to inflammatory tissue injury and lead to eventual pathological tissue alterations. We now show that PMN leukocyte capacity of invading in vitro a reconstituted BM barrier (Matrigel) is markedly restrained by micromolar concentrations of the new -lactam (IC₅₀ < 2 µM). Although these concentrations do not affect PMN leukocyte viability, our biochemical results suggest that a key role is played in this down-modulation (directly) by the inhibition of degradation of BM components by LE activity and (indirectly) by the consequent impairment of LE-triggered activation of MMP-9.

Here, it is worth mentioning that micromolar EGCG (a green tea phytofactor in which a pivotal role of the galloyl moiety in LE inhibition was first demonstrated; Sartor et al., 2002a) efficiently inhibits PMN chemotaxis and inflammatory recruitment (Donà et al., 2003). In contrast, the new compound, obtained by adding the galloyl moiety to the lactam ring, while restraining chemoinvasion, is completely ineffective on PMN leukocyte chemotaxis: this suggests that it does not affect the cytoskeleton, but instead impairs mainly the proteolytic machinery. Preliminary data, indicating that it does not inhibit LE release from PMN leukocytes, seem also to exclude an effect on exocytosis processes.

The same dissected effects have been reported for PMN leukocytes treated with tissue inhibitors of metalloproteinases-1, the preferential MMP-9 inhibitor. In addition, in this case, trans-BM migration was inhibited without affecting chemotaxis or degranulation (Delclaux et al., 1996). Together, these results strongly suggest that MMP-9 is a major factor on PMN migration across BM and that LE is instrumental to this process by activating pro-MMP-9. This hypothesis is now reinforced by the clear dose-dependent inhibition of both chemoinvasion (Fig. 3) and LE-triggered MMP-9 activation in vitro by micromolar N-galloyl-4-alkyliden--lactam (Fig. 4).

Despite that information on absorption, metabolism, and bioavailability of this new -lactam are not yet available, we verified whether this effect, registered in vitro, is efficaciously translated in vivo in the restrain of inflammatory cell recruitment and consequent tissue damage (see below).

Pulmonary Fibrosis. Chronic inflammation of the alveolar space is thought to mediate the development of pulmonary fibrosis, a disorder with an aggressive course (5-year survival of 50%) that is characterized by fibroblast proliferation and extracellular matrix remodeling, resulting in irreversible distortion of the lung's architecture (Selman et al., 2001). Intratracheal bleomycin challenge is a model of acute lymphocyte-dependent lung injury resulting in areas of patchy and chronic inflammation, with release of proteases, and production of cytokines, chemokines, and growth factors that mediate the eventual subpleural, peribronchiolar, and perivascular deposition of extracellular matrix and scar tissue formation that are characteristic of pulmonary fibrosis (Christensen et al., 1999; Moore et al., 2001). Protection from induced pulmonary fibrosis in chemoattractant chemokine receptor knockout (CCR2^-/-) mice, in comparison with wild type, was shown to be independent from differential recruitment of inflammatory cells, and no statistical differences were registered in the PMN leukocyte populations (Corbel et al., 2002).

Here, histological staining and semiquantitative analysis clearly showed that in animals given i.p. N-Galloyl-4-alkyliden--lactam both the inflammatory cell infiltration and the patchy fibrosis were significantly reduced (by 78 and 81% as mean percentage, respectively) (Fig. 5), and quantitative determination of hydroxyproline (a marker of collagenous proteins) confirmed a significant reduction (-46%) of inflammation-triggered extracellular matrix collagen fiber deposition. A contributory factor to the antifibrotic effect might well be the inhibition exerted by N-galloyl-4-alkyliden--lactam on LE-triggered activation of MMP-9, the gelatinase involved in the damage to lung extracellular matrix (Lazo and Hoyt, 1990; Egeblad and Werb, 2002), and whose level significantly increases in induced pulmonary fibrosis concomitantly with collagen deposition (Selman et al., 2001), as illustrated above.

Cuboidalization, a histological marker of epithelial reactive changes, was also less evident in the treated animals: the lower epithelial damage parallels indeed the dramatically reduced inflammatory infiltration in the lungs of treated animals. Although the principal target and the mechanism of bleomycin-triggered lung injury are not well identified (van Acker et al., 1995), the possibility of a direct protective effect of this -lactam on alveolar epithelial cells may not be excluded and deserves further investigation.

Conclusions. To what extent the antifibrosis effect exerted by the N-galloyl-4-alkyliden--lactam is mainly the result of the restraint in the PMN default activity and turnover, and how much of the down-modulation of other cells remains to be determined. Certainly, this new -lactam is a potent inhibitor of one of the most aggressive PMN leukocyte proteases (LE) and may be effective in preventive and therapeutic treatment of individuals exposed to risk of excessive or chronic inflammation.

Recurrent inflammation is considered a potential causative step of tumoral transformation in some types of tissue, and inflammatory infiltration of the primary mass is a common occurrence (Balckwill and Mantovani, 2001). Whether the inhibition exerted by the new compound on the LE-triggered activation of MMP-9, which is instrumental also in cancer invasion (Lazo and Hoyt, 1990), successfully hinders the metastatic aggressiveness of tumor cells is under investigation.

	Acknowledgements

 
We are grateful to Prof. R. Cervellati (Department of Chemistry "G. Ciamician", University of Bologna) for measures of antioxidant activity, Dr. G. De Silvestro (Immuno-transfusion Service, Padova Hospital, Padova, Italy) for supplying buffy coats, and Dr. S. Biggin for revision of the English manuscript.

	Footnotes

 
This research was supported by grants from the Associazione Italiana per la Ricerca sul Cancro, the Ministero dell'Istruzione, dell'Università e della Ricerca Italian Government (PRIN 2004), and the Universities of Padova and Bologna (Italy).

doi:10.1124/jpet.105.096248.

ABBREVIATIONS: LE, leukocyte elastase; PMN, polymorphonuclear; ECM, extracellular matrix; MMP, matrix metalloproteinase; 1-PI, 1-protease-inhibitor; PR-3, proteinase-3; fMLP, N-formyl-Met-Leu-Phe; FCS, fetal calf serum; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; MS, mean score; MP, mean percentage.

¹ Both authors contributed equally to this work.

Address correspondence to: Dr. Spiridione Garbisa, Department of Experimental Biomedical Sciences, Viale G. Colombo 3, 35121 Padova, Italy. E-mail: garbisa{at}unipd.it

	References

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