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Biochemical and Pharmacological Characterization of ICI 186,756: A Novel, Potent, and Selective Inhibitor of Human Neutrophil Elastase J. C. Williams, R. L. Stein, A. M. Strimpler, B. Reaves, and R. D. Krell

ABSTRACT: ICI 186,756 is a representative of a new chemical class of synthetic inhibitors of human neutrophil elastase W E ) . This compound demonstrated competitive inhibition of HNE with a Ki of 3.6 f 0.8 x mol/L. The selectivity of ICI 186,756for HNE versus a variety of enzymes ranged from a minimum of 870fold to > 640,000. The compound efectively inhibited hydrolysis of bovine ligamentum nuchae elastin by HNE. Pretreatment of hamsters with ICI 186,756 up to 2 h before intratracheal administration of HNE inhibited enzyme-induced increases in lung weight, total lavageable red cells, and total lavageable white cells measured 24 h a f t . . HNE administration. In contrast, similar lung efects produced by intratracheal administration of porcine pancreatic elastase (WE) were not inhibited by ICI 186,756. Treatment of hamsters with 43 pmol/kg ficj of ICI 186,756 for 14 or 28 days modulated the increases in alveolar diameter produced by both PPE and HNE, respectively. It is concluded that ICI 186,756 is a potent, competitive, and selective inhibitor of HNE that may be useful in understanding the role of this enzyme in animal models of various diseases. Furthermore, the maintenance or progression of emphysema-like lesions induced in hamsters by PPE do not appear to be due to the persistence of that enzyme within the lung.

From the Pulmonay Section, Department of Pbarmacolo~ICI Pharmaceuticals Group, ICI Americas, Inc., Wilmington, Delaware; Merck, Sharp & Dobme Research Laboratories, Rahway, New Jersey; and the Department of Pbamacology, Yale University School of Medicine, New Haven, Connecticut. Address all correspondence to Dv. Joseph C. Williums, ICI Phanaceuticals Group2ICI Americas, Inc., Wilmington, DE 19897. Received 12 February 1990; accepted 28 August 1990.

Experimental Lung Research 17:725-741 (1991) Copyright 0 1991 by Hemisphere Publishing Corporation

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INTRODUCTION A large body of circumstantial evidence has accumulated indicating that a protease-antiprotease imbalance may contribute to the pathogenesis of emphysema. Several lines of evidence suggest that the elastase of polymorphonuclear leukocytes may be the most important protease. These include: (1) the presence of abnormal numbers of neutrophils in bronchoalveolar lavage fluid obtained from smokers, a group predisposed to the development of emphysema [I]; (2) the increased numbers of neutrophils in the interstitium of the lungs of smokers, with and without emphysema [2]; and (3) the immunolocalization of human neutrophil elastase (HNE) in the emphysematous regions of resected human lungs [3], an observation that has been questioned by Fox et al. [4].Although the evidence of HNE involvement in human emphysema is circumstantial, this hypothesis is directly testable through the use of potent, selective inhibitors of HNE. Many protease inhibitors have been synthesized and evaluated both in vitro and in vivo [S]. They include both irreversible inhibitors, such as peptide chloromethylketones originally described by Powers et al. [6] and reversible inhibitors such as peptide boronic acids [7] and peptide aldehydes IS]. Moreover, there exist several biosynthetically derived natural inhibitors including a,-proteinase inhibitor [9], Eglin C [lo], and secretory leukoproteinase inhibitor [11]. These compounds have inhibited H N E both in vitro and in vivo with varying degrees of success. This paper describes the biologic characteristics of ICI 186,756 (Fig. l), a potent, selective, low-molecular-weight peptide aldehyde inhibitor of H N E that prevents the acute effects and modulates elastase-induced destructive lung lesions in experimental animals.

Y

.H

( I \ ) N H

0

(R,S)-N2-(3-carboxy-l-oxopropyl)-N6-[(phenylmethoxy)carbonyll-

L-lysyl-L-valyl-N-(l-formyl-2-methylpropyl)-L-Prolinamide Figure 1 Srructure and chemical name of ICI 186,756.

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727

METHODS

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Determination of Kinetics of Inhibition of Human Neutrophil Elastase The substrate methoxysuccinyl-ala-ala-pro-Val-pnitroanalide was hydrolyzed by HNE releasing pnitroanalide (PNA), which was continuously measured spectrophotometrically by monitoring absorbance changes at 410 nm. Both substrate and inhibitor were dissolved in dimethyl sulfoxide (DMSO). Fifty pl of both the substrate and inhibitor or DMSO were added t o a cuvette containing 2.895 ml buffer I (10 mm Na phosphate, 500 mmol/L NaCl, p H 7.6). The cuvette was placed in a thermostatically controlled, water-jacketed holder in the cell compartment of a Cary B210 spectrophotometer and allowed to reach thermal equilibrium. The temperature was maintained at 25 k 0.1OC. The reaction was initiated by the addition of 5 pl enzyme solution (0.14 mg/ml). Absorbance was continuously monitored and stored in a DEC PDP 11/32 minicomputer. Initial and steady-state velocities were calculated by a fit of the experimental data to a linear dependence on time by linear least-squares analysis and Ki's determined by Dixon analysis [ 121. Triplicate determinations were conducted for each inhibitor concentration.

Determination of Protease Selectivity The protease selectivity of ICI 187,656 was determined by using minor modifications of the protocol above. All substrates were dissolved in DMSO and combined with inhibitor (dissolved in the same solvent). Equal volumes (50 p1) of inhibitor and substrate were added to 2.895 ml of buffer I in a cuvette, and the cuvettes were placed in the spectrophotometer described above. When the reaction mixtures had reached thermal equilibrium (25OC) the reactions were initiated by the addition of 5 pl of enzyme, and absorbance was continuously monitored at the wavelength indicated below. Assay conditions for the various enzymes were as foliows: Porcine pancreatic elastase Substrate: succinyl-ala-ala-ala-pNA Buffer: Buffer I Enzyme concentration: 24 nmol/L Wavelength: 410 nm Bovine pancreatic chymotrypsin Substrate: succinyl-ala-ala-pro-phe-pNA Buffer: Buffer I Enzyme concentration: 3.3 nmol/L Wavelength: 410 nm

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al.

Human plasma thrombtn Substrate: Benzoyl-phe-Val-ala-pNA Buffer: Buffer I Enzyme concentration: 5 units/ml Wavelength: 410 nm Bovine liver cathepsin B Substrate: Benzyloxycarbonyl-gly-o-nitrophenol Buffer: 0.2 mol/L Na phosphate, 0.01 mol/L ethylenediaminetetraacetic acid (EDTA), 0.02mol/L cysteine, pH 5 Enzyme concentration: 33 p.g/rnl Wavelength: 380 nm Bovine pancreatic carboxpeptidase A Substrate: Hippuryl-phe Buffer: Buffer I Enzyme concentration: 3.3 nmol/L Wavelength: 348 nm A cetylcholinesterase Substrate: Acetylcholine Buffer: Buffer I Enzyme concentration: 14 nmol/L Wavelength: 420 nm Human leukocyte cathepsin G Substrate: succinyl-ala-ala-pro-phe-pNA Buffer: 0.1 mol/L Tris, 0.7 mmol/L NaN,, pH 8.3 Enzyme concentration: 25 nmol/L Wavelength: 410 nm Angiotensin converting enzyme Substrate: 3-(2-furylacryloyl)-phe-gly-gly Buffer: 50 mmol/L HEPES, 300 mmol/L NaCl, pH 7.5 Enzyme concentration: 10 nmol/L Wavelength: 348 nm

Inhibition of HNE-Catalyzed Hydrolysis of Insoluble Elastin Bovine neck elastin was suspended at a concentration of 20 mg/ml in 0.1 mol/L Tricine, 0.5 mol/L NaCl, p H 8 buffer containing 250 pl triton xl00 per 250 ml. The suspension was stirred at room temperature for 18 h, washed on a glass-fritted funnel with buffer and resuspended in fresh buffer. The elastin solution was diluted with an equal volume of buffer to give a final concentration of 10 mg/ml. Aliquots of substrate (8 ml) were added to 25-ml flasks and H N E was added to produce a final concentration of 1.2 pmol/L. The flasks were stirred continuously at 25°C. Ten minutes after the initiation of the reaction, inhibitor was added to final concentrations of either 1.2 or 12 pmol/L. At various times, 0.75 ml samples were removed from the reaction flasks and added to centrifuge tubes containing 0.75 mlO.1 mol/L acetic acid, 4 mol/L NaC1, p H 4.7. After centrifugation, the absor-

Biology of ICI 186,756

729

bance of the supernatant at 276 nm was determined on a Cary B210 spectrophotometer.

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Induction of Acute Lung Injury with HNE Male Syrian hamsters were lightly anesthetized with metho-hexital sodium (30 mg/kg, ip) and their tracheae were surgically exposed. A dose of 400 pg of HNE or 100 pg porcine pancreatic elastase (PPE), in 0.3 ml 0.01 mol/L phosphate buffered saline (PBS) was injected into the exposed trachea through a Y2-in23-gauge needle. The incision was closed with stainless steel surgical staples, and the animals were allowed to recover. The animals were killed with an overdose of pentobarbital sodium 24 h after the injection of HNE. The lungs and heart were resected and the lungs and trachea carefully cleaned of extraneous material. Following measurement of wet lung weight, the tracheas were cannulated and lavaged three times with 2 ml of PBS. The recovered lavages were pooled for each animal and the volume recorded. Total red and white blood cell counts were determined using a Coulter counter (Model D2N). The data are expressed as lung weight/100 g body weight and total cells recovered (cells/ml times volume recovered).

Prophylactic Effect of ICI 186,756 in PPE- and HNE-Induced Acute Lung Injury

Dose-response relationship. Male Syrian hamsters were lightly anesthetized with metho-hexital sodium and the tracheae exposed as described above. Various doses of ICI 186,756, dissolved in 0.3 ml of PBS, were injected into the trachea by a %-in 23-gauge needle. At 30 min after compound administration, the animals received intratracheal injections of either H N E or PPE. At 24 h after enzyme administration, the extent of the lesions were evaluated as described above.

Duration of action of ICI 186,7>6. Hamsters were lightly anesthetized and prepared for intratracheal injection of 1.2 pmol ICI 186,756 as described above. At the times indicated in the results section, the animals received intratracheal injections of 400 p g HNE per animal and the extent of the lesion was evaluated as described above. Therapeutic Effect of ICI 186,756 in PPE- and HNE-Induced Emphysema in Hamsters PPE therapeutic trial. On day 0, male Syrian hamsters (weighing 90 to 100 g) were lightly anesthetized with metho-hexital sodium and received intratracheal injections of vehicle or 100 or 150 pg of PPE per animal in a total volume of 0.3 ml PBS. The animals were allowed to recover and then placed

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in cages with food and water ad libitum. On day 14, 10 animals of each group were killed and the lungs prepared as described in the following section on morphometric analysis. The remaining animals were divided into equal groups and received twice daily sc injections of either 43 pmol/kg ICI 186,756 or vehicle for an additional 14 days, after which they were killed and the lungs prepared for morphometric analysis as described below. Previous studies in our laboratory have indicated that this protocol produces a stable or slowly progressive lung lesion when evaluated by measurement of mean linear intercept.

HNE therapeutic trial. O n day 0, animals received intratracheal injections of either vehicle or 300, 400, or 500 pg of HNE per animal as described under the preceding section. On day 28, 10 animals of each group were killed and the lungs prepared for morphometric analysis. The remaining animals were divided into equal groups and were treated twice daily with sc injections of either 43 pmol/kg ICI 186,756 or vehicle. After 28 days of drug or vehicle treatment, all animals were killed and the lungs prepared as described below. As with PPE, preliminary studies were performed with HNE t o determine the experimental protocol necessary to produce a chronic lung lesion that is slowly progressive or sustained for 56 days in the absence of intervention.

Morphometric analysis. At the indicated times after enzyme administration, the animals were killed with a lethal injection of pentobarbital sodium; in each the thorax was opened and the lungs and heart removed. The heart and other extraneous material were carefully dissected away, and the lungs were washed in saline, blotted dry, and weighed. The lungs were then inflated and fixed with 10% phosphate buffered formalin at 25 cm formalin pressure. Next, 5 pm hematoxylin and eosin sections were prepared ('JDJ Laboratories, Elkton, MD) and mean linear intercepts were determined using an Optomax Image Analyzer (Optomax, Inc., Hollis,

NH).

Source of materials. Bovine pancreatic chymotrypsin, human plasma thrombin, acetylcholinesterase, angiotensin converting enzyme, bovine pancreatic trypsin, pepsin, and all synthetic substrates were purchased from Sigma Chemical Co. (St. Louis, MO). PPE was purchased from Worthington Biochemicals (Freehold, NJ), and HNE, bovine neck elastin, and cathespin G were purchased from Elastin Products (Owensville, MO). Hamster neutrophil elastase was purified from hamster polymorphonuclear leukocytes according to the method of Viscarello et al. [13]. All other reagents were of the highest grade commercially available. Male Syrian hamsters were purchased from Charles River Laboratories (Wilmington, MA).

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Statistics. Data were evaluated using either Student’s t test or one-way analysis of variance and Duncan’s multiple range test with P < .05 considered statistically significant.

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RESULTS

Biochemical Characterization of ICI 186,756 ICI 186,756 was evaluated as an inhibitor of several hydrolases including hamster neutrophil elastase and HNE. As illustrated in Table 1, ICI 186,756 was a potent inhibitor of both hamster neutrophil elastase and H N E with Ki values of 10 and 3.6 nmol/L (n = 15), respectively. Lineweaver-Burk analysis demonstrated that ICI was a competitive inhibitor of HNE (Fig. 2). In contrast, the compound was a weak inhibitor of a variety of other proteases, with the exception of papain (Table 1).Notably, ICI 186,756 was over 2000 times more potent as an inhibitor of HNE than PPE. The ability of ICI 186,756 to inhibit HNE-hydrolysis of insoluble elastin is illustrated in Fig. 3. In these experiments, the reaction was initiated by combining H N E with insoluble bovine ligament elastin followed 10 min later by the addition of ICI 186,756. When inhibitor concentration was equal to enzyme concentration (1.2 pmol/L) approximately 50% inhibition of elastinolysis was observed. When the inhibitor was in tenfold excess, inhibition was approximately 90%.

Pharmacologic Characterization of ICI 186,756 Inhibition of HNE-induced acute lung injury by ICI 186,756. The ability of ICI 186,756 to inhibit HNE-induced acute lung injury was evaluated. The compound was administered at doses of 0.12, 0.6, or 1.2 pmol per animal intratracheally, 30 min before intratracheal administration of 400 pg of HNE. The compound produced a significant ( P < .05)dose-dependent inhibition of the HNE-induced increases of lung weight, total lavageable red blood cells, and total lavageable white blood cells (Fig. 4a-c). It should also be noted that administration of ICI 186,756, alone, produced slight but significant ( P < .05)increases of all three parameters. An indication of the duration of action of ICI 186,756 on the HNEinduced acute lung injury is illustrated in Fig. 5. The compound was administered at a dose of 1.2 pmol per animal by intratracheal injection 15, 30, 60, 120, or 240 min before administration of 400 pg of HNE. ICI 186,756 produced significant (P < .05) inhibition of the HNE-induced increases of lung weight for at least 4 h. Likewise, the increase of total lavageable red blood cells was inhibited ( P < .05) for 4 h. In contrast, the ability of ICI 186,756 to inhibit HNE-induced accumulation of white

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blood cells was lost by 4 h. Additionally, ICI 186,756, alone, produced a slight but significant (P < .05) accumulation of white blood cells within the lung; however, in contrast to previous studies, it produced no significant effect on lung weight and body weight or on lavageable red blood cells ( P > .05).

Inhibition of PPE-induced acute lung injury by ICI 186,756. The ability of ICI 186,756 to inhibit PPE-induced acute lung injury was also evaluated. The intratracheal administration of 1.2 pmol per animal of ICI 186,756 30 min before the intratracheal administration of 100 pg per animal of PPE failed to inhibit the effects of PPE on any of the measured parameters (Table 2). Once again, ICI 186,756, alone, produced a small but significant ( P < .05)elevation in lavageable white blood cells. Effect of ICI 186,756 on the development of pulmonary emphysema-like lesions induced by PPE or HNE. The ability of ICI 186,756, administered subcutaneously, to modulate emphysema-like lesions induced by PPE was evaluated. Increases in mean linear intercept (MLI) were produced by intratracheal administration of either 100 or 150 pg of PPE. As illustrated in Fig. 6, 2 weeks after PPE administration, dose-dependent increases (P < .05)in MLI were observed. At this time, the 100 and 150 pg of PPE groups were divided into two subgroups. One group received twice daily subcutaneous injections of 43 pmol/kg of ICI 186,756, the other served as vehicle controls. As illustrated in Fig. 6, during weeks 2 t o 4, the lesion did not progress in either PPE-dose group. ICI 186,756 produced an apparently Table 1 Evaluation of the Hydrolase Selectivity of ICI 186,756 Hydrolase Serine proteases Human neutrophil elastase Hamster neutrophil elastase Porcine pancreatic elastase Human plasma thrombin Bovine pancreatic chymotrypsin Cysteine proteases Papaya laytex papain Bovine liver cathepsin B Metalloprot ease Bovine pancreatic carboxypeptidase A Error estimates are less than o r equal to 20Y0 of the mean.

"K, values determined from Dixon plots.

Ki (nmol/L)" 3.6 10.0 7,000.0 39,000.0 > 1,000,000.0 50.0

> 50,000.0 > 1,000,000.0

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Biology of ICI 186,756

I

I

I

I

I

0

5

60 -

I

I

I

I

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55 50 45 -

.-

E

\

5 Ti>

40-

35302520 15-

-15 -10 -5

10 15 20 25

Figure 2 Lineweaver-Burk analysis of the inhibition of human neutrophil elastase hydrolysis of the synthetic substrate methoxysuccinyl Val-pro-Val-pnitroanaline by ICI 186,756. The compound appears to be a competitive inhibitor with a Ki = 3.6 nmol/L.

complete regression (P < .05) of the lesion in the low-dose group and significant regression ( P < .05) in the high-dose group. The ability of ICI 186,756 to inhibit the maintenance of an emphysemalike lesion induced by HNE was likewise evaluated. Increases in MLI were apparent 4 weeks after intratracheal administration of all three different doses of HNE. However, the extent of the lesion was not dependent on the dose of H N E (Fig. 7). As with the PPE study, at 4 weeks the animals within each HNE-dose group were subdivided into two groups: one group received twice daily subcutaneous injections of 43 pmol/kg of ICI 186,756, and the other received vehicle. Between weeks 4 and 8 the initial lesion induced by low-dose (300 pg per animal) H N E regressed spontaneously and did not differ from vehicle control (P < .05). Consequently, ICI 186,756 did not demonstrate an effect. In contrast, both the 400 and 500 pg per animal HNE groups demonstrated modest, non-dose-related increases in MLI above the 4-week lesion level. Under these conditions, ICI 186,756 appeared to produce a modest but insignificant (P > .05) regression of the lesion induced by 400 pg of HNE, whereas in the 500 pg dose group,

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progression between weeks 4 t o 8 was significantly inhibited by ICI 186,756 (P < .05).

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DISCUSSION ICI 186,756 has been demonstrated t o be a potent, competitive, and selective inhibitor of HNE, both in vitro and in vivo. The Ki, 3.6 nmol/L, compares favorably with that of the substituted 0-lactam reported by Bonney et 31. [14] and with that of the peptide aldehyde inhibitors reported by Kennedy et al. [8] and Hassal et al. [15]. Although ICI 186,756 is a potent inhibitor of HNE, as well as of hamster neutrophil elastase, it is a much weaker inhibitor of PPE and papain and has little or no affinity for the other hydrolases evaluated. The compound is also capable of halting HNEcatalyzed hydrolysis of insoluble elastin even when added after initiation of the reaction. The ability of ICI 186,756 to prevent acute HNE-induced lung lesions was based on the observation that intratracheal administration of HNE produces a marked hemorrhagic and inflammatory response that is appar-

I .o

0.9 0.8 0.7 u)

2 0.6 n

0 0.5 c(1

a,

n

0.4

0.3 0.2 0.1

0.0 60

180 240 300 Incubation Time (rnin) 120

360

O i Addition of ICI 186,756

Figure 3 Inhibition of HNE-elastinolysis by ICI 186,756. Bovine ligamentum nuchae elastin (20 mg/ml) was digested with H N E (1.2 pmol/L) in the presence or absence of two concentrations of ICI 186,756. Inhibitor or vehicle was added 10 min after addition of HNE. AOD 275 was used to determine the rate of hydrolysis.

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Wet LwlBw

Total Lavagable RBC's 1.4 1.2

-

.-f 0.80.60.4 0.0 m

1.0

-*

B.

A

*

-

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# Total Lavagable WBC's

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14-

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-.0

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#

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4

0 2

3 3

9

2 8 ? w* u I=

1.2

HNE

.6

.3 vmole

+ lC1186,756

Figure 4 Dose-response relationship for ICI 186,756 inhibition of HNE-induced acute lung injury in hamsters. PBS, ICI 186,756 and/or HNE (400 pg per animal) were administered intratracheally and the animals sacrificed 24 h later. Lungs were removed and lavaged and (u) lung weighdbody weight, (b) total lavageable red blood cells, or (c) total lavageable white blood cells quantitated. When ICI 186,756 was used in combination with HNE, the compound was administered 30 min before and the animals sacrificed 24 h after the enzyme. Bars with vertical lines represent mean f SEM of n = animals (number of bars). Significantly 'I( > .05) from * PBS or # HNE controls.

ent in the first 24 h after enzyme administration [16, 171. Not unexpectedly, PPE also produced an acute lung injury. The doses of PPE and H N E used in these studies were chosen to provide a rigorous system in which to evaluate an elastase inhibitor. Although the doses of enzymes used were at or slightly above the maxima of the dose-response curves for the production of acute lung injury (unreported observations), they were within the range of doses reported to produce an emphysema-like lesion in hamsters

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I

0

I

I

60

120 180 Dose Interval (min)

1

240

Figure 5 Duration of action of ICI 186,756 administered intratracheally (1.2 pmol per animal) in the HNE-induced acute lung injury model. The time between intratracheal administration of ICI 186,756

-

and H N E was varied and the animals sacrificed 24 h after enzyme. Symbols with vertical lines represent total lavageable red cells, and A = total the mean f SEM for n = animals, 0 = LW/BW, 0 lavageable white cells.

[8, 17-19, 22; unpublished observations]. ICI 186,756 produced a dosedependent inhibition of both hemorrhage and inflammation, as indicated

by lavageable red blood cells, wet lung weight, and lavageable white blood cells, when administered intratracheally 30 min before HNE administration. The doses used ranged from approximately 60 to 600 pg of comTable 2 Lack of Effect of ICI 186,756 on Porcine Pancreatic Elastase-Induced Acute Lung Injury in Hamsters" Treatment

LW/BW

PBS ICI 186,756 PPE PPE + ICI 186,756

0.61 0.64 0.89 0.82

f 0.01 (12) f 0.01 (11) f 0.05 (16) 2 0.03 (13)

WBC x lo6

RBC x lo6

1.45 2.90 8.69 11.60

19.4 25.8 152.0 117.0

f 0.48

f 0.50b f 1.21 f 1.93

f 4.80

f 4.80 k 16.10 f 20.10

"1.2 pmol of ICI 186,756 per animal was administered intratracheally followed after 30 min by intratracheal administration of 100 pg of PPE per animal; 24 h later, the animals were sacrificed and parameters measured as described in the text. Values are mean f SEM. Numbers in parentheses are the number of animals. PBS indicates phosphate buffered saline (10 mmol/L, p H 7.4); LW/BW, lung weighdbody weight; WBC, white blood cells; RBC, red blood cells. 'P < .05 relative to PBS.

737

Biology of ICI 186,756

4 weeks post PPE

+ drug UI

p < .05 cf saline, I# cf matched PPE = 18 to 20 *

n v)

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0

.-ti E

W

f 30 20

Dose of PPE (it) Figure 6 Inhibition of PPE-induced emphysema by ICI 186,756. PPE (100 or 150 fig per animal) or saline was administered intratracheally and the insult allowed to progress for 2 weeks. At the end of the 2-week period, one group of animals from each dose group was sacrificed to evaluate the extent of the lesions. The remaining animals were divided into two groups: one received twice daily sc administrationsof ICI 186,756 (43 pmol/kg) and the other receiving vehicle. Two weeks later, all animals were sacrificed and mean linear intercept (Lm) determined. Bars with vertical lines represent the mean SEM of n animals. Note that between the 2- and +week period the lesion did not progress in the 100 fig dose group.

*

-

pound per animal and resulted in 50% to 90% inhibition of hemorrhage, 47% to 100% inhibition of edema, and 48% to 80% inhibition of accumulation of white blood cells in the lung. The ability of the compound to inhibit HNE-induced edema and hemorrhage persisted for at least 4 h, whereas the accumulation of white blood cells in the lavage was significantly inhibited for only 2 h. Hassel et al. [15] reported a similar inhibition of HNE-induced hemorrhage using another peptide-based aldehyde at doses of 10 to 1000 pg per animal. Bonney et al. [14] reported that 10 and 100 pg of L-659,286 produced 18% and 90% inhibition of the hemorrhagic response induced by 50 pg of HNE, respectively. ICI 186,756 was also demonstrated to be capable of inhibiting an ongoing destructive lung lesion initiated by either H N E or PPE. Permitting lesions to develop before intervention with ICI 186,756 offered two advantages: (1) The vast majority of the instigating enzyme would be either inactivated or cleared from the lung before inhibitor administration [2O], and (2) presumably, this model more faithfully reflects the human disease. As previously reported [17], the lesion induced by PPE was both more rapid in onset and more severe than that induced by HNE. We also observed that the lowest doses of PPE and H N E did not progress with time

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80

p < .05 * cf saline, /f cf matched HNt weeks post HNE 0 N = 10/group weeks post HNE * 8 weeks post HNE drug Ell

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Z 70C g 60:* W

E

5040-

30-

+*

.A

*

1 aline

T

500 pg Dose of HNE (it)

Figure 7 Inhibition of HNE-induced emphysema by ICI 186,756. HNE (300,400, or 500 p g per animal) or saline was administered intratracheally and the insult allowed to progress for 4 weeks. At the end of the 4-week period, one group of animals from each dose group were sacrificed to evaluate the extent of the lesion by measuring mean linear intercept (Lm). The remaining animals from each HNE-dose group were divided into two subgroups: one received ICI 186,756 (43 pmol/kg) twice daily sc, the other received vehicle for 4 weeks. All animals were sacrificed and Lm determined as described in the text. Bars with vertical lines represent means SEM for n animals.

*

-

and, in fact, appeared to regress. It has been our experience, particularly with PPE, that low doses frequently produce early destructive lesions that, based solely on MLI values, regress with time [unpublished observations]. In contrast, the high doses of both enzymes produced lesions that were maintained for the duration of the study. The parenteral administration of ICI 186,756 between weeks 2 and 4 in the PPE study and between weeks 4 and 8 in the H N E study appeared to facilitate the return to control values in the low-dose groups of both enzymes (significantly so for the PPE group) and prevented the progression in the high-dose groups. The report by Giles et al. [21] that progesterone administered after the establishment of papain-induced emphysema-like lesions in rats resulted in significant reversal of both MLI and functional residual capacity is the only other report of therapeutic efficacy of any treatment regime. The increased number of white blood cells in lung lavage following administration of ICI 186,756 is enigmatic. The compound alone was not chemoattractant for PMNs [unpublished observations], suggesting that endotoxin contamination was not a factor. Nor were increased white blood cells a consistent feature of inhibitors from the same chemical class. The possibility that the compound indirectly released chemoattractants cannot be discounted.

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Several hypotheses have been offered to explain the progressive nature of the lung injury following a single insult with PPE [18]. One suggests that the persistence of extremely low levels of the enzyme in the lung may account for the progressive nature of the lesion. The experiments with ICI 186,756 demonstrating inhibition of the maintenance of the PPE-induced chronic lesion, despite low affinity for PPE in vitro confirmed by a lack of in vivo activity in the acute lung injury studies, would seem to discount this possibility. More consistent with these data is the notion that the initial insult sets in motion an endogenous inflammatory process that perpetuates the acute lesion. Additional support for this hypothesis is found in the observation of Snider et al. [18] that the numbers of polymorphonuclear leukocytes in hamster lung lavage are severalfold greater than in control animals for up to 60 days following elastase treatment. The observation that ICI 186,756 inhibited the maintenance of the PPE-induced lesion, coupled with the demonstrated ability of the compound to inhibit hamster neutrophil elastase, creates speculation that autologous neutrophil elastase may be involved. In a similar vein, Lucey et al. [22] have suggested that, following intratracheal HNE administration in hamsters, both growth and tissue destruction take place at the same time and counterbalance one another. This may account for the lack of progression of the HNE-induced emphysema-like lesions. Martorana et al. [23] made similar observations when documenting the lack of progression of PPE-induced emphysemalike lesions in young hamsters. If the tissue destruction was due to endogenous elastase supplied by the hamsters’ own neutrophils, the addition of exogenous elastase inhibitors such as ICI 186,756 should allow growth and repair in the absence of destruction and a subsequent decrease in average alveolar diameter. The demonstrated ability of ICI 186,756 to prevent the maintenance of the HNE-induced lesions in these studies support the hypothesis set forth by Lucey et al. [23]. The properties of ICI 186,756 described above suggest that this and similar compounds may be of value in examining the role of HNE in animal models of various disease processes. The authors express their appreciation to Dr. Bruce Gomes for his assistance with the elastinolysis experiments and to Sharon Bailor for her contributions to this manuscript’s preparation.

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20. Stone PJ, Pereira W, Biles D, Snider GL, Kagan HM, Franzblau C: Studies on the fate of pancreatic elastase in the hamster lung: 14C-guanidatedelastase. Am Rev Respir Dis 116:49-56, 1977. 21. Giles RE, Finkel MP, Williams JC: The therapeutic effect of progesterone in papain-induced emphysema. Proc SOCExp Biol Med 147:489-493, 1974. 22. Lucey EC, Stone PJ, Christensen TG, Breuer R, Snider GL: An 18-month study of the effects on hamster lungs of intratracheally administered human neutrophil elastase. Exp Lung Res 14:671-686, 1988. 23. Maratorana PA, van Evan P, Schaper J: The effect of lung growth on the evolution of elastase-induced emphysema in the hamster. Lung 160:19-27, 1982.

Biochemical and pharmacological characterization of ICI 186,756: a novel, potent, and selective inhibitor of human neutrophil elastase.

ICI 186,756 is a representative of a new chemical class of synthetic inhibitors of human neutrophil elastase (HNE). This compound demonstrated competi...
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