Introduction

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Human leukocyte elastase (HLE) belongs to the chymotrypsin family of serine proteinases. The optimum pH of this enzyme is close to neutrality, and the catalytic site is composed of three hydrogen-bonded amino acid residues: His57, Asp102, and Ser195 (in chymotrypsin numbering), which form the so-called catalytic triad. The enzyme is composed of a single peptide chain of 218 amino acid residues and four disulfide bridges. It shows 30 to 40% sequence identity with other elastinolytic or nonelastinolytic serine proteinases. HLE preferentially cleaves the oxidized insulin B chain with Val at the P1 position, but it also hydrolyzes bonds with Ala, Ser, or Cys in the P1 position (Skiles and Jeng, 1999).

HLE is located in the azurophilic granules of polymorphonuclear leukocytes (PMNLs), where the HLE concentration is rather high (3 µg of enzyme/10⁶ cells) (Liou and Campbell, 1995). The major physiological function is to digest bacteria and immune complexes and to take part in the host defense process (Travis et al., 1991). HLE aids in the migration of neutrophils from blood to various tissues such as the airways in response to chemotactic factors (Banda et al., 1988). HLE also takes part in wound healing, tissue repair, and in the apoptosis of PMNLs (Trevani et al., 1996).

In addition to elastin (highly flexible and highly hydrophobic component of lung connective tissue, arteries, skin, and ligaments), elastase cleaves many proteins with important biological functions, including different types of collagens (Kittelberger et al., 1992), membrane proteins, and cartilage proteoglycans (Janusz and Doherty, 1991). HLE also indirectly favors the breakdown of extracellular matrix proteins by activating procollagenase, prostromelysin, and progelatinase (Rice and Banda, 1995). HLE inactivates a number of endogenous proteinase inhibitors such as ₂-antiplasmin, ₁-antichymotrypsin, antithrombin, and tissue inhibitor of metalloproteinases (Higushi et al., 1992).

Extracellular elastase activity is tightly controlled in the pulmonary system by ₁-protease inhibitor (₁PI), responsible for protection of the lower airways from elastolytic damage, whereas the secretory leukocyte proteinase inhibitor protects mainly the upper airways (Vogelmeier et al., 1991). In a number of pulmonary pathophysiological states, e.g., pulmonary emphysema (Fujita et al., 1990), chronic bronchitis (Fujita et al., 1990), and cystic fibrosis (Griese et al., 2001), endogenous elastase inhibitors are inefficient in regulating HLE activity.

HLE is considered to be the primary source of tissue damage associated with inflammatory diseases such as pulmonary emphysema (Groutas, 1987), adult respiratory distress syndrome (ARDS) (McGuire et al., 1982), chronic bronchitis (Llewellyn-Jones et al., 1996), chronic obstructive pulmonary disease (COPD) (Piccioni et al., 1992), pulmonary hypertension (Cowan et al., 2000), and other inflammatory diseases (Adeyemi et al., 1985) as well as bronchopulmonary dysplasia in premature neonates (Stiskal et al., 1998).

HLE is involved in the pathogenesis of increased and abnormal airway secretions commonly associated with airway inflammatory diseases (Fujimoto et al., 1995). Thus, bronchoalveolar lavage (BAL) fluid from patients with chronic bronchitis and cystic fibrosis had increased HLE activity. Furthermore, excessive elastase has been proposed to contribute not only to these chronic inflammatory diseases but also to acute inflammatory diseases such as ARDS and septic shock. These findings stimulated interest in the search for agents with elastase inhibitory activity, and many synthetic inhibitors of HLE have been described and reviewed previously (Metz and Peet, 1999; Skiles and Jeng, 1999; Leung et al., 2000).

Recently, we have synthesized a novel elastase inhibitor with low molecular weight, 2-(9-(2-piperidinoethoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yloxymethyl)-4-(1-methylethyl)-6-methoxy-1,2-benzisothiazol-3(2H)-one-1,1-dioxide, C₂₇H₃₂N₄O₇S (SSR-69071), for the treatment of COPD, ARDS, cystic fibrosis, asthma, and other inflammatory diseases. SSR69071 is a saccharine derivative with a molecular mass of 565.64 Da (Fig. 1A). In the present study, we report on the biochemical and pharmacological properties of SSR69071. For comparison, (S)-1-[(S)-2-(methoxycarbonylamino)-3-methylbutyryl]-N-[(S)-2-methyl-1-(trifluoroacetyl)propyl]pyrrolidine-2-carboxamide (ZD8321), a selective and orally active elastase inhibitor (Veale et al., 1997), was synthesized and used as a reference in the biochemical and pharmacological studies (Fig. 1B).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   A, chemical structure of SSR69071. B, chemical structure of ZD8321.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals

Male NMRI mice (approximately 6 weeks old and weighing between 18 and 20 g) and CDBR male rats (weighing 98 to 204 g) were obtained from Charles River Hungary Kft (Budapest, Hungary). Male New Zealand White rabbits (3.5-5 kg) were obtained from LabNyul Kft (Hungary). All experimental and housing procedures were approved by the Institutional Animal Care and Use Committee of Sanofi-Synthelabo Research Budapest and by the Hungarian Animal Ethic Committee. Animals were acclimatized to housing conditions for at least 1 week before the experiments.

Reagents and Drugs

Human sputum elastase (875 U/mg) and elastin Congo red were obtained from Elastin Product Co. Inc.(Owensville, MO). Brij-35, casein, glycerolformal, dimethicon, carboxymethylcellulose, Tween 80, cremophor EL, Meo-Suc-Ala-Ala-Pro-Val-pNa, porcine pancreatic elastase (5 U/mg), and carrageenan were purchased from Sigma-Aldrich Kft (Budapest, Hungary).

SSR69071 and ZD8321 were synthesized in the chemistry laboratory of Sanofi-Synthelabo Research (Budapest, Hungary). Their purity, determined by high-performance liquid chromatography and thin layer chromatography, was >95%.

In Vitro Studies

Elastase Assay. Human leukocyte elastase. Human sputum elastase was used as human leukocyte elastase (Green et al., 1991). The enzyme was dissolved in assay buffer (50 mM HEPES/NaOH, pH 7.8, containing 0.5 M NaCl and 0.1 mg/ml bovine serum albumin) at a 656 U/ml (30 µM) concentration. This solution was diluted in assay buffer 10 times and was stored in aliquots (80 µl) at 80°C.

Preparation of murine and rat neutrophil extracts. Peritoneal neutrophils were obtained from mice and rats pretreated for 16 h with a 1% casein (100 ml/kg) solution i.p. After peritoneal lavage the neutrophils were centrifuged (2000g for 10 min) and resuspended in Tris-HCl buffer (0.1 M, pH 7.5) containing 0.1% Brij-35 and 1 M MgCl₂ and lysed by sonication. The lysate was used as the source of neutrophil elastase (Kawabata et al., 1991).

Preparation of rabbit neutrophil extracts. Rabbit PMNLs were obtained from peripheral blood of New Zealand White rabbits after removal of erythrocytes by sedimentation at unit gravity through dextran (Boyum, 1976). After brief ultrasonic homogenization and alternate freezing and thawing, the suspension was centrifuged (12,000g, 10 min) and the supernatant was used as enzyme source.

Determination of elastase inhibitory activity. The elastase activity was monitored using the specific chromogenic substrate Meo-Suc-Ala-Ala-Pro-Val-pNa. The assay mixture contained 130 µl (8.5 U) of elastase, 100 µl of substrate (final concentration, 400 µM), 20 µl of inhibitor or dimethyl sulfoxide, and 50 µl of assay buffer. Final volume of the assay was 300 µl. The assay was performed in microtiter plates placed in a kinetic plate reader (V_max kinetic plate reader; Molecular Devices Corp., Sunnyvale, CA). The assay was started by addition of elastase, and the change in absorbance at 410 nm was continuously monitored at 25°C. Because both SSR69071 and ZD8321 are slow, tight-binding inhibitors of human leukocyte elastase, the assay reaction was monitored for 8.5 h (read interval, 90 s; number of readings, 340) (Williams et al., 1991).

Determination and calculation of kinetic inhibitory constants for human leukocyte elastase inhibitors. Slow, tight-binding competitive inhibitors exhibited time-dependent inhibition (Cha, 1975; Williams and Morrison, 1979). Three kinetic constants describe this type of inhibition: K_i, inhibition constant; k_on, the second-order kinetic constant describing the inactivation process; and k_off, the rate constant for the dissociation of enzyme inhibitor complex. The progress curve for the enzyme reaction in the presence of a slow tight-binding inhibitor does not display a simple linear product versus time relationship (Williams and Morrison, 1979; Morrison and Walsh, 1988); as a result of slow onset of inhibition, product formation over time will be a curvilinear function.

(2)

Determination of IC₅₀ values for human leukocyte elastase-catalyzed hydrolysis of insoluble elastin. The ability of SSR69071 to inhibit the hydrolysis of insoluble elastin by human leukocyte elastase was determined spectrophotometrically using elastin Congo red as a substrate according to the method of Naughton and Sanger (1961) with minor modifications. Elastin Congo red (final concentration, 10 mg/ml) and human elastase (final concentration, 20 nM) were incubated with various concentration of inhibitors in 1.2 ml of 0.1 M HEPES buffer, pH 7.8, containing 0.2 M NaCl and 0.1 mg/ml bovine serum albumin at 37°C for 20 h. After the incubation, the reaction was stopped by centrifugation at 3000 rpm for 15 min at room temperature. Finally, absorbance of the supernatant at 495 nm was measured with spectrophotometer (Biochrom 4030; LKB, Uppsala, Sweden).

Ex Vivo Experiments

Ex Vivo Inhibition of Human Leukocyte Elastase Activity in Mouse Bronchoalveolar Lavage Fluid after Oral Administration. SSR69071 was suspended in cremophor LE/distilled water [1:3 (v/v)] and administered in a volume of 10 ml/kg. Animals in the control group received the vehicle alone. For dose-dependence studies, the animals were treated orally with SSR69071 (3-, 6-, 10-, and 20-mg/kg doses) or ZD8321 (10-, 20-, 50-, or 100-mg/kg doses) 1 h before BAL fluid collection. In time-dependence studies, animals were treated orally with SSR69071 (20 mg/kg) or ZD8321 (100 mg/kg) 10, 30, 60, 120, 240, 480, or 1440 min before BAL collection. After oral treatment, the animals were euthanized, and the trachea was exposed and a small incision was made for insertion of a polyethylene cannula. A needle attached to a 1.0-ml syringe was inserted into the cannula, and 0.5 ml of air was withdrawn from the airways. One-milliliter sterile physiological saline was then instilled into the airways, and the chest was briefly and gently massaged. Finally, the syringe was removed from the cannula and the BAL fluid was collected.

In Vivo Experiments

HLE-Induced Lung Hemorrhage in Mice. To test the effectiveness of HLE inhibitors, overnight fasted NMRI mice were treated orally via gastric tube. Investigated compounds were suspended in cremophor EL/distilled water [1:3 (v/v)], and animals were treated orally in a volume of 10 ml/kg with SSR69071 (0.3-, 1-, 3-, or 10-mg/kg doses) or with ZD8321 (1-, 3-, 10-, or 30-mg/kg doses) 30 min before the intratracheal instillation of HLE. Animals were anesthetized, and the trachea was exposed by a 5-mm incision of the neck. Animals received 10 IU of HLE dissolved in 0.025 ml of ice-cold sterile physiological saline. The solution was very slowly injected into the trachea. Three hours after HLE instillation, animals were euthanized with an overdose of urethane. BAL fluid was collected as described above. This procedure was repeated three times and the total volume of BAL was recorded. Triton X-100 was then added to the collected BAL fluid [final concentration, 0.2% (v/v)] to ensure cell disruption. The optical density of the supernatant was determined by spectrophotometer at 540 nm, and it was correlated with hemoglobin content (Corteling et al., 2002). Values are given as mean ± S.E.M. The ID₅₀ (and 95% confidence intervals) was determined from the dose-response relationship using linear regression.

Paw Edema Models on Rats. To test the effectiveness of HLE inhibitors, overnight fasted rats were treated orally via the gastric tube or intravenously through the jugular vein. Animals were treated intravenously 10 min before or orally 120 min before intraplantar injection of HLE or carrageenan. SSR69071 was suspended in 0.2 ml of Tween 80, 0.2 g of carboxymethylcellulose, and 0.01 ml of dimethicon in 100 ml of distilled water, and the animals received either drug or vehicle in a volume of 1 ml/kg. For intravenous administration, SSR69071 was dissolved in glycerol-formal in a volume of 1 ml/kg. The paw edema was evoked by the intraplantar injection of 0.9% saline solution of HLE (50 U/0.1 ml/right hind paw) or intraplantar injection of 0.9% saline solution of carrageenan (0.1 ml/right hind paw), both in control and drug-treated groups. The paw volume was measured immediately (control value) and at 0.5, 1, 2, 3, 4, and 5 h after HLE or carrageenan injection by a plethysmograph (type 7150; Ugo Basile, Comerio, Italy).

Statistical Analysis

In vitro IC₅₀ values were calculated with an unweighted method for least-square fit of data (Grafit version 4.0; Erithacus Software Ltd., Staines, UK). Statistical analysis was performed using SAS/STAT (SAS Institute, Inc., Cary, NC) and RS/1 software packages (Domain Solutions Corp., Cambridge, MA). The significance of differences were obtained using the Kruskal-Wallis test and Student's t test. A value of *p < 0.05, **p < 0.01, or ***p < 0.001 was considered as statistically significant (SAS, version 6.12).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

In Vitro Studies

The elastase activity was measured with the help of a synthetic chromogenic substrate. Biphasic reaction progress curves were obtained during the inhibition of HLE by SSR69071 and ZD8321 as illustrated in Fig. 2. The progress curve for the enzyme reaction in the presence of a slow tight-binding inhibitor does not show a simple linear product versus time relationship. Product formation over time will be a curvilinear function because of the slow onset of inhibition for these compounds. As shown in Fig. 2, SSR69071 is a more potent elastase inhibitor than ZD8321. Inhibition constant (K_i) and the constant for inactivation process (k_on) were 16.8 ± 1.4 pM and 0.183 ± 0.013 ×10⁶/mol s for SSR69071. The dissociation rate constant of enzyme-inhibitor complex (k_off) of SSR69071, calculated as described under Materials and Methods, was 3.11 ± 0.37 × 10⁶/s. In the same experimental conditions the K_i value for ZD8321 was 5.57 ± 0.18 nM (Table 1).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Progress curves for the inhibition of HLE by SSR69071 and ZD8321, regarding hydrolysis of the synthetic substrate methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide. SSR69071 and ZD8321 were shown to be slow tight-binding inhibitors of HLE.

Based on these results, SR69071 is a potent, competitive, and slow tight-binding type inhibitor of HLE. Both k_on and k_off values suggested that the enzyme-inhibitor complex has a fast association and a slow dissociation rate.

The effects of SSR69071, ZD8321, and ₁-antitrypsin were compared on the basis of their IC₅₀ values. HLE (5 nM) was incubated for 10 min with different concentrations of inhibitors, and the remaining HLE activity was determined on the basis of hydrolysis of synthetic substrate. Inhibitory activities were determined and the IC₅₀ values were calculated. SSR69071, ZD8321, and ₁-antitrypsin inhibited HLE dose dependently with IC₅₀ values of 3.9, 99, and 10.9 nM, respectively.

The ability of SSR69071 and ZD8321 to inhibit HLE hydrolysis of insoluble elastin was also evaluated. The IC₅₀ values of SSR69071 and ZD8321 to inhibit insoluble elastin degradation were 13 and 350 nM, respectively.

To determine species specificity of SSR69071, its elastase inhibitory effect was investigated on leukocyte elastase isolated from different species. SSR69071 showed high species specificity displaying a 2 log lower K_i value for the human compared with the rat and mouse enzyme and 3 log lower for the rabbit enzyme. K_i values for mice, rat, and rabbit elastase enzyme were 1.7, 3, and 58 nM, respectively (Table 2).

SSR69071 is highly specific inhibitor of elastase type endopeptidases because it had no effect on the various receptors and enzymes [71 receptors and 27 enzymes tested by CEREP (Paris, France) and MDS Pharma Services (Taipei, Taiwan)] up to 10 µM in vitro (data not shown). The enzyme-selectivity profile includes the inhibition of other proteolytic enzymes, e.g., serine proteinases (cathepsin G, tryptase, and thrombin), cysteine peptidases (cathepsin B), and metallopeptidases (endothelin-converting enzyme, angiotensin-converting enzyme, collagenase IV, neutral endopeptidase, MMP-2, MMP-3, MMP-7, and MMP-9).

Ex Vivo Results

The activity of SSR69071 was determined in the BAL fluid after oral administration in mice. The elastase inhibitory potency of BAL was compared before and after oral treatment with SSR69071. We determined the dose dependence and the time dependence of this activity. Diluted BAL from vehicle-treated mice did not significantly inhibit HLE (average HLE activity, 10.65 ± 0.46 mOD/min). SSR69071, administered orally, dose dependently inhibited the human leukocyte elastase in the BAL fluid from 3 to 20 mg/kg (Fig. 3). This effect was not significant at the dose of 3 mg/kg (6 ± 5%) but reached a statistical level of significance from 6 mg/kg (27 ± 6%, p < 0.05). The maximal ex vivo enzyme activity inhibition was observed at the dose of 20 mg/kg (87 ± 3%, enzyme activity = 1.1 ± 0.26 mOD/min, p < 0.05). The calculated ID₅₀ was 10.5 mg/kg p.o. In the same experimental conditions, ZD8321 showed little ex vivo inhibitory potency because the highest dose tested (100 mg/kg) was only effective with a 44 ± 7% inhibition of the elastase activity (Fig. 3).

View larger version (8K): [in this window] [in a new window]   Fig. 3.   Dose dependence of the ex vivo activity of SSR69071 and ZD8321 in mice. Animals were treated orally 1 h before the BAL collection. HLE was added exogenously into the BAL, and elastase activity was determined against a synthetic substrate (as indicated under Materials and Methods) and the elastase inhibitory activity was calculated. Values are means ± S.E.M. for 10 to 15 mice. Student's t test, *, p < 0.05; **, p < 0.01; ***, p < 0.001.

SSR69071 was orally administered (20 mg/kg) at different pretreatment times (10, 30, and 60 min and 2, 4, 6, and 24 h). Data obtained showed that HLE activity was significantly inhibited up to 6 h with a maximum inhibitory activity observed at 30 min (90 ± 3%, 1.2 ± 0.36 mOD/min) with even some significant inhibition seen after 10 min. Furthermore, HLE activity was still decreased by 42 ± 9% (p < 0.05) after a 24-h pretreatment time (Fig. 4).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Time dependence of the ex vivo activity of SSR69071 and ZD8321 in mice. Animals were treated orally at the specified time before the BAL collection. HLE was added exogenously into the BAL, and elastase activity was determined with synthetic substrate (as indicated under Materials and Methods) and the elastase inhibitory activity was calculated. Values are means ± S.E.M. for 10 to 15 mice. Student's t test, *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Conversely, ZD8321, administered at 100 mg/kg, displayed a moderate efficacy with a maximum HLE activity inhibition observed at 30 min (44 ± 9%). This activity decreased, but still remained statistically significant after a 2-h pretreatment time (26 ± 12%, p < 0.05) (Fig. 4.).

In Vivo Activity

Lung Hemorrhage in Mice. The ability of SR69071 to protect animals from HLE-induced lung hemorrhage was evaluated in mice. Intratracheal instillation of HLE (10 U, 10-15 µg) caused a severe lung hemorrhage in mice.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Effect of ZD8321 and SSR69071 on HLE-induced lung hemorrhage in mice. SSR69071 was administered orally 30 min before HLE (10 U/animal) instillation. Values are means ± S.E.M. for 9 to 12 mice. Kruskal-Wallis test, *, p < 0.05; **, p < 0.01; ***, p < 0.001.

HLE-Induced Paw Edema in Rats. The paw edema of this model is thought to reflect an increase in the permeability of the peripheral capillaries caused by elastase. Both SSR69071 and ZD8321 dose dependently inhibited the HLE-induced paw edema after intravenous administration, with SSR69071 being more effective than ZD8321. The greatest inhibition was observed at 30 mg/kg (74.7%, p  0.001 and 53%, p  0.001 for SSR69071 and ZD8321, respectively). The calculated ID₃₀ values were 0.03 mg/kg i.v. for SSR69071 and 0.57 mg/kg i.v. for ZD8321 (Fig. 6A).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   A, inhibitory effect of ZD8321 and SSR69071 on HLE-induced paw edema in rats. Compounds were administered intravenously 10 min before 50 U/paw of HLE injection. The ID30 value is 0.03 mg/kg i.v. for SSR69071 and 0.57 mg/kg i.v. for ZD8321. Values are means for five rats. Student's t test, n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, inhibitory effect of ZD8321 and SSR69071 on HLE-induced paw edema in rats. Compounds were administered orally 2 h before 50 U/paw of HLE injection. The ID30 value is 2.7 mg/kg p.o. for SSR69071 and 4.2 mg/kg p.o. for ZD8321. Values are means for five rats. Student's t test, n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, time course effect of ZD8321 and SSR69071 on HLE-induced paw edema in rats. Compounds in a dose of 3 mg/kg were administered orally at given times before 50 U/paw of HLE injection. Values are means for five rats. Student's t test, n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Carrageenan-Induced Paw Edema in Rats. Both SSR69071 and ZD8321 dose dependently inhibited the carrageenan-induced paw edema after intravenous administration. SSR69071 was more effective than ZD8321, maximal edema inhibition being 59% (p  0.001) and 47.4% (p  0.001), respectively. The calculated ID₃₀ values of SSR69071 and ZD8321 were 1.0 and 2.5 mg/kg, respectively (Fig. 7A).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   A, inhibitory effect of ZD8321 and SSR69071 on carrageenan-induced paw edema in rats. Compounds were administered intravenously 10 min before 0.9% solution of carrageenan injection (0.1 ml/paw). The ID30 value is 1.0 mg/kg i.v. for SSR69071 and 2.5 mg/kg i.v. for ZD8321. Values are means for five rats. Student's t test, n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, inhibitory effect of ZD8321 and SSR69071 on carrageenan-induced paw edema in rats. Compounds were administered orally 2 h before 0.9% solution of carrageenan injection (0.1 ml/paw). The ID30 value is 2.2 mg/kg p.o. for SSR69071 and 3.1 mg/kg p.o. for ZD8321. Values are means for five rats. Student's t test, n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, time course effect of ZD8321 and SSR69071 on carrageenan-induced paw edema in rats. Compounds were administered orally in 3 mg/kg p.o. doses at given times before 0.9% solution of carrageenan injection (0.1 ml/paw). Values are means for five rats. Student's t test, n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

SSR69071 is a potent, competitive, and slow tight-binding inhibitor of HLE in vitro with a K_i value of 16.8 pM (Table 1). SSR69071 is more effective than FK706 (Shinguh et al., 1997), ONO-5046 (Kawabata et al., 1991), and GW-311616A (Norman, 1998), according to the K_i values. SSR69071 has a fast association rate and slow dissociation rate, resulting in a stable, slowly reversible HLE-inhibitor complex. The low value of k_off suggests that the SSR69071-enzyme complex is barely dissociated, resulting in the slow reversibility of inhibition. These properties of SSR69071 suggest an extremely high activity and long duration of action in humans.

The activity of SSR69071 was compared with that of ₁PI, because it is responsible for protection of the lower airways from elastolytic damage (Vogelmeier et al., 1991). SSR69071, using methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide as a substrate, was approximately 3 times more potent than ₁-proteinase inhibitor, with IC₅₀ values being 3.9 and 10.9 nM, respectively. Moreover, a more than 1000-fold higher concentration of ₁PI on a weight basis was needed to inhibit enzyme activity because of its very high molecular mass (SSR69071: molecular mass = 0.56 kDa, IC₅₀ = 0.0022 µg/ml; ₁PI: molecular mass = 54 kDa, IC₅₀ = 5.9 µg/ml). In this respect, low molecular mass inhibitors, which are therapeutically effective at low doses, would have an advantage over high molecular mass inhibitors such as ₁PI.

We examined the ability of SSR69071 to inhibit elastases from different sources by using a synthetic substrate. SSR69071 inhibited all types of elastase in a dose-dependent manner, but at a much higher concentration compared with HLE. The K_i values of SSR69071 for elastase-type endopeptidases such as rat, mouse, and rabbit neutrophil elastase and porcine pancreatic elastases were 3, 1.7, 58, and 100 nM, respectively. Thus, SSR69071 has a weak activity on porcine pancreatic type elastase and showed high species specificity, displaying a 100- to 1000-fold lower K_i for human than for the rat, mouse, and rabbit enzyme. The strong species selectivity of SSR69071 (human versus rodent elastase) should be kept in mind when analyzing the effects of the compound on pharmacological animal models. The results in rats and mice may underestimate the expected in vivo potency of SSR69071 in humans.

The inhibitory activity of SSR69071 was not confined to synthetic peptide substrates but extended to the degradation by HLE of macromolecular substrates such as elastin. The elastin Congo red derivative was used because of easy detection. Because it was insoluble, it is not suitable for kinetic studies. When using insoluble elastin as the substrate of HLE, much longer incubation times (20 h) were required than those used in the steady-state experiments, to gather sufficient reaction products for quantitative analysis. Similar to the study with a synthetic substrate, SSR69071, produced a dose-dependent inhibition of HLE-mediated elastolysis, which yielded an IC₅₀ value of 13 nM.

The activity of SSR69071 was investigated in various in vivo animal models. The acute hemorrhagic assay conducted in the hamster is a widely used model for the assessment of in vivo activity of HLE inhibitors (Fletcher et al., 1990; Williams et al., 1991; Veale et al., 1997). This model is thought to be predictive for efficacy in emphysema (Fletcher et al., 1990). Because the pharmacokinetic properties of SSR69071 were not compatible with studies in hamster, we set up and validated animal models in mice for the assessment of in vivo activity of SSR69071.

The activity of SSR69071 was determined after oral administration in mice, in the BAL fluid. SSR69071 showed a dose-dependent efficacy (ED₅₀ = 10.5 mg/kg p.o. after 1-h pretreatment time). SSR69071 occurred in the BAL after oral treatment with an apparent fast absorption rate, because 10 min after the oral treatment significant HLE inhibitory activity (73%) was observed (Fig. 4). This activity demonstrated a very good penetration in the lungs from the systemic circulation. A maximum inhibitory activity was observed at 30 min (90%), the activity being still decreased by 42 ± 9% (p < 0.05) after 24-h pretreatment time, which indicated a long duration of action.

The ability of SSR69071 to protect animals from HLE-induced lung hemorrhage was evaluated in mice. Intratracheal instillation of HLE (10 IU) caused a severe lung hemorrhage. Oral administration of SSR69071 dose dependently and potently inhibited the lung hemorrhage with an ID₅₀ value of 2.8 mg/kg.

The good oral activity of SSR69071 is very important because only a limited number of published elastase inhibitors show oral activity (Herbert et al., 1992; Metz and Peet, 1999; Skiles and Jeng, 1999; Leung et al., 2000) and their active doses are quite high, between 10 and 50 mg/kg (Herbert et al., 1992; Veale et al., 1997; Metz and Peet, 1999; Skiles and Jeng, 1999; Leung et al., 2000).

HLE is considered to play a crucial role in many inflammatory conditions where leukocytes infiltrate the site of inflammation and are activated by various stimuli (Fujie et al., 1999). Shinguh et al. (1997) showed that selective elastase inhibitors could prevent edema formation induced by elastase in an experimental model in mice. Human neutrophil elastase also elicited paw edema as did other irritants such as zymosan, carrageenan, and bradykinin. The paw edema in this model is thought to reflect increases in permeability of the peripheral capillaries (Shinguh et al., 1997). Nakagawa et al. (1986) showed that a selective elastase inhibitor attenuated the vascular permeability increase, leukocyte cell migration, and development of granulated tissue induced by carrageenan.

Intravenous and oral treatments with SSR69071 were effective on HLE- and carrageenan-induced paw edema in rats in a dose- and time-dependent manner.

The maximal edema inhibition was obtained 2 h after oral SSR69071 administration. Furthermore, a significant inhibitory effect was observed up to 5 h after the drug administration. SSR69071 dose dependently inhibited HLE- and carrageenan-induced paw edema in rats. The calculated ID₃₀ value of SSR69071 was 2.7 mg/kg on HLE-induced and 2.2 mg/kg on carrageenan-induced paw edema in rats.

The oral activity of SSR69071 was demonstrated in mice and rats, in two different animal models, two HLE-dependent models, and in an inflammatory model in rats (carrageenan-induced edema formation).

In view of possible clinical development, we have performed a preliminary safety study in rats administered SSR69071 at a dose of 50 mg/kg/day orally for 14 days (~20-fold the pharmacological dose in this species). After a 2-week period, we could observe neither mortality, clinical signs, changes in hematology or biochemical parameters, nor histological alteration, including liver (data not shown).

In conclusion, SSR69071 has been shown to be a potent and selective inhibitor of HLE, exhibiting good oral activity in various rodent models (despite its lower potency on rat and mouse elastase) with a potential in the treatment of inflammatory bronchopulmonary diseases such as COPD and chronic bronchitis.