Biochemistry and Pharmacology of ICI 200,880, a Synthetic Peptide Inhibitor of Human Neutrophil Elastase JOSEPH C. WILLIAMS, ROSS L. STEIN," RALPH E. GILES, AND ROBERT D. KRELL Pulmonary Pharmacology Section Department of Pharmacology ICI Pharmaceuticals Group ICI Americas Inc. Wilmington, Delaware 19897

A large body of circumstantial evidence indicating that a protease-antiprotease imbalance contributes to the pathogenesis of emphysema has been accumulated. Several lines of evidence suggest that the elastase contained within polymorphonuclear leukocytes (PMN) may represent the most important protease limb of this hyp0thesis.l Even though the evidence for human neutrophil elastase (HNE) involvement in human emphysema is circumstantial, this hypothesis does suggest a rational approach for the design of chemicals with therapeutic potential. Many protease inhibitors have been synthesized and biologically evaluated.? These inhibitors include both irreversible inhibitors such as the peptide chloromethyl ketone^,^ reversible inhibitors such as the peptide boronic acids,4 cephalosporin inhibitor^,^ and peptide aldehydes.6 Moreover, several biosynthetically derived natural inhibitors including alphal-proteinase inhibitor,' Eglin C,B and secretory leukoproteinase inhibitor (SLPI)9.'ohave been described both biochemically and pharmacologically. Variously, these compounds have inhibited HNE both in vitro and in vivo with varying degrees of success. This communication describes the biologic profile of ICI 200,880 (FIG. l ) , a selective, potent, peptide trifluoromethylketone inhibitor of HNE that prevents the acute effects and halts the progression of elastase-induced destructive lung lesions in experimental animals. METHODS

Determination of Kinetics of Inhibition of Human Neutrophil Elastase Substrate, Methoxysuccinyl-Ala-Ala-Pro-Val-pNA, was hydrolyzed by HNE releasing p-nitroanalide which was continuously measured spectrophotometrically by monitoring absorbance changes at 410 nm. Both substrate and inhibitor were dissolved in DMSO. Fifty microliters of both substrate and inhibitor or DMSO were added to a cuvette containing 2.895 ml of buffer I (10 mM Na phosphate, 500 mM NaCI, pH 7.6). The cuvette was placed in a thermostatically


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WILLIAMS ef al.: 1CI 200,880


controlled, water-jacketed holder in the cell compartment of a Cary 210 spectrophotometer (Varian Techtron, PTX, Ltd, Victoria, Australia) and allowed to reach thermal equilibrium. The temperature was maintained at 25 +. 0.1"C. The reaction was initiated by the addition of 5 pI of 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. Triplicate determinations were conducted for each inhibitor concentration. Determination of Protease Selectivity

The protease selectivity of ICI 200,880 was determined by using minor modifications of the foregoing protocol. All substrates were dissolved in DMSO and combined with inhibitor (dissolved in the same solvent). Equal volumes (50 p l ) of inhibitor and substrate were added to 2.895 ml of buffer I in a cuvette which was placed in a spectrophotometer for continuous monitoring of absorbance changes. When the reaction mixtures had reached thermal equilibrium (25'0, the reactions


FIGURE 1. Structure of ICI 200,880. The chemical name is [4-(4-chlorophenyl sulfonycarl-(RS)-(l-trifluoroacetyl-2-methylprolyl)amide]. bamoyl) benzoyl-L-valyl-L-prolyl-

were initiated by the addition of 5 p1 of enzyme and absorbance was monitored at either 410 or 348 nm, depending on the substrate leaving group. Assay conditions for the various enzymes were as follows: Porcine pancreatic elastase: Substrate; succinyl-Ala-Ala-Ala-pNA: buffer I:

enzyme concentration; 24 nM: wavelength; 410 nm. Bovine pancreatic chymotrypsin:

Substrate; succinyl-Ala-Ala-Pro-Phe-pNA: buffer I: enzyme concentration; 3.3 nM: wavelength; 410 nm. Human plasma thrombin:

Substrate; BZ-Phe-Val-Ala-pNA: buffer I: enzyme concentration; 5 units/ml: wavelength; 410 nm. Acetylcholinesterase:

Substrate ; acetylcholine : buffer 1: enzyme concentration; 14 nM: wavelength; 420 nm.



Human leukocyte cathepsin G: Substrate; succinyl-Ala-Ala-Pro-Phe-pNA: 0. I M Tris, 0.7 mM NaN,, pH 8.3. enzyme concentration; 25 nM: wavelength; 410 nm. Angiotensin-converting enzyme: Substrate; FA-Phe-Gly-Gly: 50 mM HEPES, 300 mM NaCI, pH 7.5: enzyme concentration; 10 nM: wavelength; 348 nm. Trypsin: Substrate; Bz-Phe-Val-Arg-pNA: buffer I: enzyme concentration; 13 nM: wavelength; 410 nm. Papa in : Substrate; N-CBZ-Gly-pNPE: 0.2 M NaP04, 0.005 M EDTA, 1.65% DMSO, 0.8% CH3CN, pH 6.0: enzyme concentration; 87 nM: wavelength; 410 nm. Pepsin: Substrate; Leu-Ser-(NO&Phe-NIeu-Ala-Leu-OCH3: 0. I M , Na Citrate, 3.3% DMSO, pH 3.0. enzyme concentration; 9.5 nM: wavelength; 410 nm.

Pharmacokinetic Evaluation of ICI 200,880 This assay utilizes the HNE inhibitory activity of the inhibitors to assess their presence in biologic fluids. In the species evaluated, the endogenous elastase inhibitors found in biologic fluids are also trypsin inhibitors. By complexing the endogenous inhibitors with exogenous trypsin, the levels of ICI 200,880 in various biologic fluids could be quantified. The pharmacokinetic characterization of the compound was determined as follows: before and at varying times after intravenous administration, blood samples (0.2 ml) were obtained via cardiac puncture under light anesthesia. The blood was expressed into 2-ml centrifuge tubes, allowed to clot for 30 minutes, and centrifuged in a Beckman microfuge. Fifty microliters of serum were then combined with 50 pl of trypsin (5 mg/ml) and allowed to interact for 5 minutes. The trypsin-treated serum (10 pI) was then added to a 0.52-ml cuvette containing buffer with 20 nM HNE. After an additional 30 minutes of incubation, the reaction was started with the addition of substrate (350 pI of 1.6 mM Methoxysuccinyl-Ala-Ala-Pro-Val-pNA) and the reaction monitored spectrophotometrically at 410 nm in a Baxter-Rotochem centrifugal analyzer. Percentage of inhibition of HNE by trypsinized serum was calculated. The actual serum concentrations of the inhibitors were determined by comparing the percentage of inhibition of the test sample with the percentage of inhibition determined for a standard curve for ICI 200,880 prepared in control blood. Log serum drug concentration was plotted versus time after drug administration, and an approximate tV2 elimination time was determined from the resultant curve. The retention of ICI 200,880 in the lungs of hamsters after inhalation exposure was determined by exposing animals, in a muzzle only exposure system, to an aerosol of ICI 200,880. Compound was dissolved in phosphate-buffered saline solution (PBS) at a concentration of 10 mg/ml and aerosols were generated using a Reteca nebulizer. The animals were exposed to the resultant atmosphere for 60 minutes. At various times after the end of the exposure, the animals were killed, and the lungs were removed and lavaged with PBS. Lavage samples were processed and analyzed for ICI 200,880 as just described.

WILLIAMS et al.: ICI 200,880


Induction of Acute Lung Injury with HNE

Male, Syrian hamsters (90to 110 g) were lightly anesthetized with Brevital sodium (30 mg/kg ip) and the trachea was surgically exposed. A dose of 400 pg of HNE in 0.3 mlO.01 M PBS was injected into the exposed trachea via a %-inch, 23gauge needle. The incision was closed with stainless steel surgical staples and the animals were allowed to recover. Twenty-four hours after the injection of HNE, the animals were killed with an overdose of pentobarbital sodium. The lungs and heart were resected and the lungs and trachea carefully cleaned of extraneous material. After 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 was recorded. Total red and white blood cell counts were determined using a Coulter counter. The data are expressed as lung weight/ 100 g body weight and total cells recovered (cells/ml x volume recovered). Therapeutic Effect of ICI 200,880 in PPE- and HNE-Induced Emphysema in Hamsters PPE Therapeutic Trial

On day 0, male, Syrian hamsters (90-100 g) were lightly anesthetized with Brevital sodium and received intratracheal injections of vehicle or 100 or 150 pg/ animal PPE in a total volume of 0.3 ml PBS. The animals were allowed to recover and placed in cages with food and water allowed ad libitum. Twenty-four hours after administration of enzyme, the animals were divided into equal groups and received twice daily subcutaneous injections of either 50 or 100 pmol/kg ICI 200,880 or vehicle for either 14 or 28 days after which they were killed and the lungs prepared for morphometric analysis as will be described. Two groups of animals received an intratracheal injection of PBS followed by twice daily administration of ICI 200,880 ( I 0 0 pmol/kg sc) for either 14 or 28 days after which the lungs were removed and processed for morphometric analysis as will be described.

H N E Therapeutic Trial On day 0, animals received intratracheal injections of either vehicle or 400 p g HNE/animal as described in the previous paragraph. Twenty-four hours later the animals were divided into equal groups and were treated twice daily with subcutaneous injections of either 50 or 100 pmollkg ICI 200,880 or vehicle. After 56 days of drug or vehicle treatment, all animals were killed and lungs were prepared as will be described. A separate group of animals received an intratracheal injection of PBS followed by subcutaneous administration of 100 pmol/kg of ICI 200,880 for the duration of the study. Morphometric Analysis

At the indicated times after enzyme administration, the animals were killed with an injection of pentobarbital sodium, the thorax was opened, and the lungs and heart were removed. The heart and other extraneous material were carefully



dissected away, and the lungs were washed in saline solution, blotted dry, and weighed. The lungs were then inflated and fixed with 10% phosphate-buffered formalin (pH 7.0) at 25 cm formalin pressure. Five-micron hematoxylin and eosin sections were prepared by JDJ Laboratories (Elkton, Maryland) and mean linear intercept determined using an Optomax Image Analyzer (Hollis, New Hampshire). Statistical Analysis

Data were evaluated using Student's two-tailed t test with p

Biochemistry and pharmacology of ICI 200,880, a synthetic peptide inhibitor of human neutrophil elastase.

ICI 200,880 is representative of a new chemical class of inhibitors of human neutrophil elastase (HNE). The compound demonstrated competitive kinetics...
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