Biologic Characterization of ICI 200,880 and ICI 200,355, Novel Inhibitors of Human Neutrophil Elastase1 , 2
JOSEPH c. WILLIAMS, RONALD c. FALCONE, CATHY KNEE, ROSS L. STEIN, ANNE M. STRIMPLER, BARBARA REAVES, RALPH E. GILES, and ROBERT D. KRELL Introduction
A large body of circumstantial evidence has been accum ulated indicating that a protease-antiprotease imbalance contributes to the pathogenesis of emphysema. The evidence favoring the involvement of PMN elastase include the presence of abnormally high numbers of neutrophils in the bronchoalveolar lavage of emphysematous patients (1) and in the interstitium of lungs of smokers, a group predisposed to the development of emphysema (2). Additionally, and debatably, human neutrophil elastase (HNE) has been immunolocalized in emphysematous regions of resected human lung (3, 4). Even though the evidence for HNE involvement in human emphysema is unproved, this hypothesis does suggest a rational approach for the design of chemicals with therapeutic potential. Many HNE inhibitors have been synthesized and biologically evaluated (5). These inhibitors include both irreversible inhibitors such as the peptide chloromethylketones (6), reversibleinhibitors such as the peptide boronic acids (7), cephalosporin inhibitors (8), and peptide aldehydes (9). Moreover, several biosynthetically derived natural inhibitors, including alpha-proteinase inhibitor (10), eglin C (11), and secretory leukoproteinase inhibitor (SLPI) (12, 13)have been described both biochemically and pharmacologically. These compounds have inhibited HNE both in vitro and in vivo with varying degrees of success. This communication describes the biologic profiles of ICI 200,880 and ICI 200,355 (figure 1), selective, potent, peptide trifluoromethyl ketone inhibitors of HNE that prevent the acute effects and halt the progression of elastase-induced destructive lung lesions in experimental animals. Methods Determination of Kinetics of Inhibition of Human and Hamster Neutrophil Elastase Substrate, methoxysuccinyl-ala-ala-pro-val-p-
SUMMARY ICI 200,880 and its close structural analog, ICI 200,355, are representatives of a new chemical class of inhibitors of human neutrophil elastase (HNE). Both compounds are substituted tripeptide ketones, which demonstrated competitive kinetics versus HNE, with identical KI velues of 5.0 x 10-10 M. The selectivity of ICI 200,880 for HNE versus a variety of enzymes ranged from 150·fold [relative to porcine pancreatic elastase (PPE)] to greater than 360,000-fold in favor of HNE. The compound effectively Inhibited HNE-hydrolysls of bovine ligamentum nuchae elastin. In pharmacoklnetlc stUdies, ICI200,880 and IC1200,355displayed long retention times when administered directly to the lung and were rapidly eliminated after Intravenous administration. Pretreatment of hamsters with either Inhibitor before Intratracheal administration of HNE produced dose- and timedependent Inhibition of enzyme-Induced Increases In lung weight, total lavegeable red cells, and totallavageable white cells. Aerosol administration of ICI200,880 produced similar results. Subcutaneous administration of either 50 or 100 Ilmollkg (tWice/day)of IC1200,880for 14or 28 days prevented the time-dependent Increase in alveolar diameter produced by a single Intratracheal dose of PPE when compound dosing was initiated 24 h after the enzyme. Treatment of hamsters with the same protocol and doses of ICI200,880 for 8 wk prevented the destructive lesion Induced by a single Intratracheal dose of HNE. It Is concluded that IC1200,880and IC1200,355 have biochemical, pharmacoklnetlc, and pharmacologic profiles that make them useful therapeutic agents for understandIng the role of HNE in various diseases. ICI 200,880 Is presently being evaluated in humans. AM REV RESPIR DIS 1991; 144:875-883
nitroanilide, was hydrolyzed by HNE releasing p-nitroanilide, which was continuously measured spectrophotometrica1lyby monitoring absorbance changes at 410nm. 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 buffer I (10 mM Na phosphate, 500 mM NaCI at pH 7.6). The cuvette was placed in a thermostatically 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 ± 0.10 C. The reaction was initiated by the addition of 5 III 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.
Inhibition of Isoforms of Human Neutrophil Elastase HNE, purified by the method of Viscarello and coworkers (14), was applied to a preparative Mono-S cation exchange column of a fast protein liquid chromatography (FPLC) (pharmacia, Piscataway, NJ) system and separated into three major peaks. "Center cuts" from each peak were separately applied to a sec-
ond analytic Mono-S cation exchangecolumn of a FPLC and rechromatographed to apparent homogeneity as evidenced by the elution of a single protein peak. Each isoform was examined kinetically against the substrate and inhibitors as described above.
Determination of Protease Selectivity The protease selectivity of ICI 200,880 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 (50Ill) of inhibitor and substrate were added to 2.895 ml of buffer 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 C), the reactions were initiated by the addition of 5 IIIenzyme, and absorbance was monitored at either 410 or 348 nm, depending on the substrate leaving (Receivedin originalform September 24, 1990and in revised form April 16, 1991) 1 From the Pulmonary Pharmacology Section, Department of Pharmacology, ICI Pharmaceuticals Group, ICI Americas Inc., Wilmington, Delaware. 2 Correspondence and requests for reprints should be addressed to Joseph C. Williams, Pulmonary Pharmacology Section, Department of Pharmacology, ICI Pharmaceuticals Group, ICI Americas Inc., Wilmington, DE 19897.
875
WILLIAMS, FALCONE, KNEE, STEIN, STRIMPLER, REAVES, GILES, AND KRELL
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group. Assay conditions for the various enzymes were as follows: Porcinepancreaticelastase. Substrate, suecinyl-ala-ala-ala-pNA; buffer, I; enzyme concentration, 24 nM; wavelength, 410 nm. Bovinepancreaticchymotrypsin. Substrate, succinyl-ala-ala-pro-phe-pNA; buffer, I; enzyme concentration, 3.3 nM; wavelength, 410 nm. Human plasma thrombin. Substrate, BZphe-val-ala-pNA; buffer, I; enzyme concentration, 5 units/ml; wavelength, 410 nm. Acetylcholinesterase. Substrate, acetylcholine; buffer, I; enzyme concentration, 14nM; wavelength, 420 nm. Human leukocyte cathepsin G. Substrate, succinyl-ala-ala-pro-phe-pNA; buffer, 0.1 M TRIS and 0.7 mM NaN3 at pH 8.3; enzyme concentration, 25 nM; wavelength, 410 nm. Angiotensin-converting enzyme. Substrate, FA-phe-gly-gly; buffer, 50 mM HE PES and 300 mM NaCI at 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. Papain. Substrate, N-CBZ-gly-pNPE; buffer, 0.2 M NaPO., 0.005 M EDTA, 1.65070 DMSO, and 0.8010 MeCN at pH 6.0; enzyme concentration, 87 nM; wavelength, 347.5 nm. Pepsin. Substrate, Leu-ser-(N02)-phe-Nleuala-leu-OMe; buffer, 0.1 M Na citrate and 3.3010 DMSO at pH 3.0; enzyme concentration, 9.5 nM; wavelength, 410 nm. Thermolysin. Substrate, Leu-ser-(N0 2)phe-NLeu-ala-leu-OMe; buffer, I; enzyme concentration, 2 nM; wavelength, 310 nm.
Inhibition of HNE-Catalyzed Hydrolysis of Insoluble Elastin Bovine neck elastin was suspended at a concentration of 20 mg/ml in 0.1 M tricine, 0.75 M NaCl (pH, 8.0) buffer containing I III Triton X-loo/m!. The suspension was stirred at room temperature for 18h, washed on a glassfritted 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 10mg/ml, Aliquots of substrate (8 ml) were added to 25-ml flasks, and HNE was added to a final concentration of 0.2 IlM. The flasks were stirred continuouslyat 25° C. Ten minutes after the initiation of the reaction, inhibitor was added to final concentrations ranging from 0.2 to 0.8 IlM. At various times 0.75-ml samples were 'emoved from the reaction flasks and added LO centrifuge tubes containing 0.75 ml 0.1 M acetic acid, 4.0 M NaCI at pH 4.7. After cen-
=
Fig. 1. Structures of IC1200,880 and ICI 200,355. Thechemical namesare: 4-(4chloro (ICI 200,880) or bromo (iCI 200,355) phenyl sulfonycarbamoyl) benzoyl-L-valyl-L-prol ine-1 (RS)-(l-trifluoroacetyl-2-methylpropyl)amide.
Sr
trifugation, the absorbance of the supernatant at 276 nm was determined.
Pharmacokinetic Evaluation of ICI 200,880 and ICI 200,355 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 and ICI 200,355 in various biologic fluids could be quantified. The pharmacokinetic characterizations of the compounds were determined as follows: before and at varying times after intravenous, subcutaneous, or oral administration of compounds to either hamsters, monkeys, or dogs, blood samples (0.2 ml) were obtained via cardiac puncture under light anesthesia (hamsters), withdrawn from the subcubital vein (dogs), or from the pelvic venous sinus (monkeys). The blood was expressed into 2-ml centrifuge tubes, allowed to clot for 30 min, and centrifuged in a Beckman microfuge (Beckman Instruments, Fullerton, CA). Fifty microliters of serum were then combined with 50 III trypsin (5 mg/ml) and allowed to interact for 5 min. The trypsintreated serum (10Ill)was then added to a 0.52ml cuvette containing buffer with 20 nM HNE. After an additional 30-min incubation, the reaction was started with the addition of substrate (350 IIIof 1.6mM methoxysuccinylala-ala-pro-val-pNA), and the reaction was monitored spectrophotometrically at 410 nm in a Baxter Rotochem centrifugal analyzer. Percent inhibition of HNE by trypsinized serum was calculated. The actual serum concentrations ofthe inhibitors were determined by comparing percent inhibition of the test sample with the percent inhibitions determined for a standard curve for ICI 200,880 or ICI 200,355prepared in control blood. Log serum drug concentration was plotted versus time postdrug administration and an approximate elimination half-time was determined from the resultant curve. To determine persistence in the lung after intratracheal administration of ICI 200,880 and ICI 200,355, male Syrian hamsters were prepared for intratracheal administration of compound as describedbelow. At various times after compound administration, the animals were killed, and the lungs were removed and lavaged with phosphate-buffered saline (PBS). As the inhibition of HNE was negligible in control hamster lung lavage, the inhibition
of HNE by drug-treated animals was determined without addition of trypsin. Lavage concentrations were determined by comparing test results with results of a standard curve prepared in control lavage fluid. Log lavage drug concentration was plotted versus time postdrug administration and an approximate retention half-time was determined. The retention of ICI 200,880 in the lungs of hamsters after inhalation exposure was determined by exposing animals, in a nose-andmuzzle-only exposure system, to an aerosol of ICI 200,880. Compound was dissolved in PBS at a concentration of 10 mg/ml, and aerosols weregenerated using a Retec nebulizer. The animals were exposed to the resultant atmosphere for 60 min. 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 described above.
Induction of Acute Lung Injury with HNE Male Syrian hamsters weighing 90 to llO g were anesthetized intraperitoneally with Brevital sodium (30 mg/kg), and the trachea was surgically exposed. A dose of 400 ug HNE, in 0.3 ml 0.01 M PBS, was injected into the exposed trachea via a half-inch, 23-gauge 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 were carefully cleaned of extraneous material. After measurement of wet lung weight, the tracheas were cannulated and lavaged three times with 2 ml PBS. The recovered lavages were pooled for each animal, and the volume was recorded. Total red and white cells were determined using a Coulter counter (Coulter Electronics, Hialeah, FL). The data are expressed as lung weight/Its) g body weight and total cells recovered (cells ml x volume recovered). Percent inhibition of the resultant lesions by inhibitors was calculated as (a - b/a) x 100 where a is the response in the absence of inhibitors, and b is the response in the presence of inhibitors. Both a and b were corrected for baseline. Therapeutic Effect of ICI 200,355 in PPE- and HNE-induced Emphysema in Hamsters PPE therapeutic trial. On Day 0, male Syrian hamsters weighing90 to 100g wereanesthetized with Brevital sodium and received intratracheal injections of vehicle or 100or 150 ug/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 receivedtwice daily subcutaneous injections of either 50 or 100 umol/kg ICI 200,880 or vehicle for either 14
BIOLOGIC CHARACTERIZATON OF ICI 200,880 AND ICI 200,355
or 28 days, after which they were killed and the lungs were prepared for morphometric analysis as described below. Two groups of animals received intratracheal injections of PBS followedby twice daily subcutaneous administration of ICI 200,880 (100 umol/kg) for either 14or 28 days, after which the lungs were removed and processed for morphometric analysis as described below. HNE therapeutic trial. On Day 0, animals received intratracheal injections of either vehicle or 400 I1g HNE/animal as described above. Twenty-four hours later the animals weredivided into equal groups and weretreated twice daily with subcutaneous injections of either 50 or 100 umol/kg ICI 200,880 or vehicle.After 56 days of drug or vehicletreatment, all animals were killed, and the lungs were prepared as described below. A separate group of animals receivedan intratracheal injection of PBS followed by subcutaneous administration of 100 umol/kg of ICI 200,880 for the duration of the study. Morphometric analysis. At the indicated times after enzyme administration, the animals werekilledwith 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, blotted dry, and weighed. The lungs were then inflated and fixed with 10% phosphatebuffered formalin (pH, 7.0) at 25-cm formalin pressure. Sections 5 11m thick, stained with hematoxylin-eosin, were prepared by JDJ Laboratories (Elkton, MD), and mean linear intercepts weredetermined using an Optomax image analyzer (Optomax, Hollis, NH). Statistical analysis. Data were evaluated using either Student's two-tailed t test or oneway analysis of variance and Duncan's multiple range test, with p < 0.05 considered statistically significant. Source of materials. Bovine pancreatic chymotrypsin, human plasma thrombin, acetyl cholinesterase, angiotensin-converting enzyme, bovine pancreatic trypsin, pepsin, and all synthetic substrates were purchased from Sigma Chemical Co. (St. Louis, MO). Porcine pancreatic elastase was purchased from Worthington Biochemicals (Freehold, NJ), human neutrophil elastase, cathespin G, and insoluble bovine legamentum nuchae elastin werepurchased from Elastin Products (Owensville, MO). Hamster neutrophil elastase was prepared from hamster neutrophils by the method ofViscarello and coworkers (14).All other reagents were of the highest grade commercially available.Male syrian hamsters were purchased from Charles River Laboratories (Wilmington, MA), Beagle dogs from LREHazelton (Kalamazoo, MI), and Macaca fasicularis monkeys from Charles River Primates (Long Island, NY).
Results
Biochemical Characterization of ICI 200,880 Potency and selectivity of ICI 200,880
877 0.08
Fig. 2. Kinetic analysis of the hydrolysis of methoxysuccinyl ala-ala-pro-valylpNA by HNE (1 x 10-' M) alone [IJ = oand in the presence of 0.3and 1.2 iJM ICI200,880.The ordinate reflectsthe formation of p-nitroanilide by HNE versus time on the abscissa. Note the curvature in the reaction progress curve, which indicates the "slow-binding" nature of the inhibition of HNE by ICI 200,880.
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against several hydrolases. ICI 200,880 was evaluated as an inhibitor of several hydrolases, including hamster and human neutrophil elastase. Similar data for ICI 200,355 have been reported previously (15) and are virtually identical to ICI 200,880. As illustrated in Figure 2, ICI 200,880 is a potent, slow-binding inhibitor of human neutrophil elastase with a K, value of (5.0 ± 1.0) x 10-10 M and a Ks« of (8 ± 1) x 10-4 M- 1 s:'. Human neutrophil elastase exists as a mixture of isoforms (15). The three major isoforms were isolated and purified, and the ability of ICI 200,880 to inhibit those isoforms was evaluated. As illustrated in table 1, ICI 200,880 was an equally effective inhibitor of all three isozymes. Likewise, the major endogenous inhibitor of HNE, alpha-l-proteinase inhibitor, also equally inhibited all three isoforms. In contrast, the compound was a very weak inhibitor of a variety of other proteases that are representative of the four classes (table 2), the exception being porcine pancreatic elastase where a 150-fold selectivity ratio was observed. Inhibition ofHNE catalyzed hydrolysis ofinsoluble elastin. The ability ofICI 200,880 to inhibit HNE-hydrolysis of insoluble elastin is illustrated in figure 3. In these experiments, the reaction was initiated by combining HNE with insolu-
TABLE 1 KINETIC PARAMETERS FOR THE ISOFORMS OF HUMAN LEUKOCYTE ELASTASE (HLE) Isoforms
MeOSuc-ala-ala-pro-val-CH 2CI 10-3 KilKi, M-' S-1 Alpha-l-protease inhibitor 10" Kon, M-' So, IC1200,880 10" Kon, M-' S-l 10~o~M
HLE
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3.3 3.1 2.5
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ble bovine ligament elastin followed 10 min later by the addition of ICI 200,880. The compound produced a concentrationrelated inhibition of the hydrolysis of insoluble elastin by HNE. A Henderson plot (16) of these data yielded a K, of 1.0 ± 0.2 nM (mean ± SEM, n = 3), a value in good agreement with the K; of 5 x 10-10 M obtained versus the synthetic substrate. ICI 200,355 provided virtually identical data (15).
Pharmacologic Characterization of ICI200,880 and tct 200,355 Pharmacokinetic evaluation. When administered as an intravenous bolus to hamsters, ICI 200,880 (20 umol/kg) and ICI 200,355 (10umol/kg) are rapidly distributed and eliminated from the bloodstream with estimated half-times of 62 and 52 min, respectively (figure 4). However, when administered by intratracheal instillation (figure 5) the compounds are retained in the lung for a longer period of time; half-time retention of 179 min
TABLE 2 HYDROLASE SELECTIVITY OF ICI 200,880
Hydrolase Serine HNE PPE Hamster neutrophil elastase Cathespin G Chymotrypsin Thrombin Trypsin Acetylcholine esterase Cysteine Papain Metallo Thermolysin ACE Acid Pepsin
Ki' (nM) 0.5 74 7.3 180,000 Nit 65,000 NI NI NI NI NI 45,000
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Fig. 6. Pharmacokinetics for ICI200,880 and methoxysuccinyl-ala-ala-pro-valchloromethylketone (CMK) administered by aerosol to hamsters. A 10 mglml solution of ICI 200,880 or CMK was aerosolized for 60 min with a Retec nebulizer. At various times after administration the animals were killed, and the lungs were resected and lavaged, and the amount of compound in lavage fluid was determined as described in METHODS. The symbols (closed circles, open squares, closed squares) represent three separate experiments with ICI 200,880; 0 represent the CMK data. Symbols with vertical lines represent the mean ± SEM of six to 10 animals per point.
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creases of lung weight/body weight, total cells, and total white cells (figure 10). Forty-eight hours after exposure, significant (p < 0.05) inhibition of the HNEinduced increase of total cells, but not lung weight/body weight or total white cells, was apparent (data not shown). The protective effect of a single subcutaneously administered bolus dose of ICI 200,880 (86 umol/kg) 1 h prior to intratracheal administration of 100 ug/animal HNE is shown in figure 11. As is apparent, the compound significantly (p < 0.05) inhibited the HNEinduced increases of lung weight/body weightand totallavageable cells.A reduction of totallavageable total white cells was also apparent, but statistical significance (p < 0.05) was not achieved. The effect of leI 200,880 on the
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progressionofpulmonary "emphysemaDose (urnoleranlrnal, i.t.) like" lesions induced by PPE or HNE. The ability of ICI 200,880 administered Fig. 7. Dose- and time-response relationship for inhibisubcutaneously to inhibit the progression tion of HNE-induced acute lung injury by ICI 200,880. of an emphysema-like lesion induced by Hamsters received various doses of ICI 200,880 inPPE was evaluated. Increases in mean tratracheally 2 to 24 h prior to administration of 400 I1g HNE, also administered intratracheally; 24 h after HNE linear intercept (MLI) were produced by . the animals were killed, the lungs were resected, intratracheal administration of either 100 weighed, and lavaged. lung weights (A), total lavageor 150 IJ,g of PPE. As is apparent in fig- able cells (B), and tolallavageable white cells (C) were ure 12A, 2 wk after PPE administration, determined. Symbols represent the mean (n = 9 to 20). increases (P < 0.05) in MLI were ob- Asterisks indicate p < 0.05compared with control values. served. In those animals that had received ICI 200,880 subcutaneously (50 or 100 umol/kg), the resultant PPE-induced le- ed in PPE-induced lesions that were sigsion was significantly (p < 0.05) reduced nificantly (P < 0.05)lessseverethan those regardless of the dose of PPE ad- observed after vehicle. Subcutaneous administered. In those animals that were ministration of ICI 200,880 at doses of evaluated after 4 wk of therapy, similar 50 and 100 umol/kg twice/day after inresults were obtained (figure 12B). That tratracheal treatment with PBS had no is, administration of ICI 200,880 result- effect on MLI.
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WILLIAMS, FALCONE, KNEE, STEIN, STRIMPLER, REAVES, GILES, AND KRELL
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JOSEPH c. WILLIAMS, RONALD c. FALCONE, CATHY KNEE, ROSS L. STEIN, ANNE M. STRIMPLER, BARBARA REAVES, RALPH E. GILES, and ROBERT D. KRELL Introduction
A large body of circumstantial evidence has been accum ulated indicating that a protease-antiprotease imbalance contributes to the pathogenesis of emphysema. The evidence favoring the involvement of PMN elastase include the presence of abnormally high numbers of neutrophils in the bronchoalveolar lavage of emphysematous patients (1) and in the interstitium of lungs of smokers, a group predisposed to the development of emphysema (2). Additionally, and debatably, human neutrophil elastase (HNE) has been immunolocalized in emphysematous regions of resected human lung (3, 4). Even though the evidence for HNE involvement in human emphysema is unproved, this hypothesis does suggest a rational approach for the design of chemicals with therapeutic potential. Many HNE inhibitors have been synthesized and biologically evaluated (5). These inhibitors include both irreversible inhibitors such as the peptide chloromethylketones (6), reversibleinhibitors such as the peptide boronic acids (7), cephalosporin inhibitors (8), and peptide aldehydes (9). Moreover, several biosynthetically derived natural inhibitors, including alpha-proteinase inhibitor (10), eglin C (11), and secretory leukoproteinase inhibitor (SLPI) (12, 13)have been described both biochemically and pharmacologically. These compounds have inhibited HNE both in vitro and in vivo with varying degrees of success. This communication describes the biologic profiles of ICI 200,880 and ICI 200,355 (figure 1), selective, potent, peptide trifluoromethyl ketone inhibitors of HNE that prevent the acute effects and halt the progression of elastase-induced destructive lung lesions in experimental animals. Methods Determination of Kinetics of Inhibition of Human and Hamster Neutrophil Elastase Substrate, methoxysuccinyl-ala-ala-pro-val-p-
SUMMARY ICI 200,880 and its close structural analog, ICI 200,355, are representatives of a new chemical class of inhibitors of human neutrophil elastase (HNE). Both compounds are substituted tripeptide ketones, which demonstrated competitive kinetics versus HNE, with identical KI velues of 5.0 x 10-10 M. The selectivity of ICI 200,880 for HNE versus a variety of enzymes ranged from 150·fold [relative to porcine pancreatic elastase (PPE)] to greater than 360,000-fold in favor of HNE. The compound effectively Inhibited HNE-hydrolysls of bovine ligamentum nuchae elastin. In pharmacoklnetlc stUdies, ICI200,880 and IC1200,355displayed long retention times when administered directly to the lung and were rapidly eliminated after Intravenous administration. Pretreatment of hamsters with either Inhibitor before Intratracheal administration of HNE produced dose- and timedependent Inhibition of enzyme-Induced Increases In lung weight, total lavegeable red cells, and totallavageable white cells. Aerosol administration of ICI200,880 produced similar results. Subcutaneous administration of either 50 or 100 Ilmollkg (tWice/day)of IC1200,880for 14or 28 days prevented the time-dependent Increase in alveolar diameter produced by a single Intratracheal dose of PPE when compound dosing was initiated 24 h after the enzyme. Treatment of hamsters with the same protocol and doses of ICI200,880 for 8 wk prevented the destructive lesion Induced by a single Intratracheal dose of HNE. It Is concluded that IC1200,880and IC1200,355 have biochemical, pharmacoklnetlc, and pharmacologic profiles that make them useful therapeutic agents for understandIng the role of HNE in various diseases. ICI 200,880 Is presently being evaluated in humans. AM REV RESPIR DIS 1991; 144:875-883
nitroanilide, was hydrolyzed by HNE releasing p-nitroanilide, which was continuously measured spectrophotometrica1lyby monitoring absorbance changes at 410nm. 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 buffer I (10 mM Na phosphate, 500 mM NaCI at pH 7.6). The cuvette was placed in a thermostatically 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 ± 0.10 C. The reaction was initiated by the addition of 5 III 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.
Inhibition of Isoforms of Human Neutrophil Elastase HNE, purified by the method of Viscarello and coworkers (14), was applied to a preparative Mono-S cation exchange column of a fast protein liquid chromatography (FPLC) (pharmacia, Piscataway, NJ) system and separated into three major peaks. "Center cuts" from each peak were separately applied to a sec-
ond analytic Mono-S cation exchangecolumn of a FPLC and rechromatographed to apparent homogeneity as evidenced by the elution of a single protein peak. Each isoform was examined kinetically against the substrate and inhibitors as described above.
Determination of Protease Selectivity The protease selectivity of ICI 200,880 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 (50Ill) of inhibitor and substrate were added to 2.895 ml of buffer 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 C), the reactions were initiated by the addition of 5 IIIenzyme, and absorbance was monitored at either 410 or 348 nm, depending on the substrate leaving (Receivedin originalform September 24, 1990and in revised form April 16, 1991) 1 From the Pulmonary Pharmacology Section, Department of Pharmacology, ICI Pharmaceuticals Group, ICI Americas Inc., Wilmington, Delaware. 2 Correspondence and requests for reprints should be addressed to Joseph C. Williams, Pulmonary Pharmacology Section, Department of Pharmacology, ICI Pharmaceuticals Group, ICI Americas Inc., Wilmington, DE 19897.
875
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CI
ICI 200,355 R
group. Assay conditions for the various enzymes were as follows: Porcinepancreaticelastase. Substrate, suecinyl-ala-ala-ala-pNA; buffer, I; enzyme concentration, 24 nM; wavelength, 410 nm. Bovinepancreaticchymotrypsin. Substrate, succinyl-ala-ala-pro-phe-pNA; buffer, I; enzyme concentration, 3.3 nM; wavelength, 410 nm. Human plasma thrombin. Substrate, BZphe-val-ala-pNA; buffer, I; enzyme concentration, 5 units/ml; wavelength, 410 nm. Acetylcholinesterase. Substrate, acetylcholine; buffer, I; enzyme concentration, 14nM; wavelength, 420 nm. Human leukocyte cathepsin G. Substrate, succinyl-ala-ala-pro-phe-pNA; buffer, 0.1 M TRIS and 0.7 mM NaN3 at pH 8.3; enzyme concentration, 25 nM; wavelength, 410 nm. Angiotensin-converting enzyme. Substrate, FA-phe-gly-gly; buffer, 50 mM HE PES and 300 mM NaCI at 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. Papain. Substrate, N-CBZ-gly-pNPE; buffer, 0.2 M NaPO., 0.005 M EDTA, 1.65070 DMSO, and 0.8010 MeCN at pH 6.0; enzyme concentration, 87 nM; wavelength, 347.5 nm. Pepsin. Substrate, Leu-ser-(N02)-phe-Nleuala-leu-OMe; buffer, 0.1 M Na citrate and 3.3010 DMSO at pH 3.0; enzyme concentration, 9.5 nM; wavelength, 410 nm. Thermolysin. Substrate, Leu-ser-(N0 2)phe-NLeu-ala-leu-OMe; buffer, I; enzyme concentration, 2 nM; wavelength, 310 nm.
Inhibition of HNE-Catalyzed Hydrolysis of Insoluble Elastin Bovine neck elastin was suspended at a concentration of 20 mg/ml in 0.1 M tricine, 0.75 M NaCl (pH, 8.0) buffer containing I III Triton X-loo/m!. The suspension was stirred at room temperature for 18h, washed on a glassfritted 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 10mg/ml, Aliquots of substrate (8 ml) were added to 25-ml flasks, and HNE was added to a final concentration of 0.2 IlM. The flasks were stirred continuouslyat 25° C. Ten minutes after the initiation of the reaction, inhibitor was added to final concentrations ranging from 0.2 to 0.8 IlM. At various times 0.75-ml samples were 'emoved from the reaction flasks and added LO centrifuge tubes containing 0.75 ml 0.1 M acetic acid, 4.0 M NaCI at pH 4.7. After cen-
=
Fig. 1. Structures of IC1200,880 and ICI 200,355. Thechemical namesare: 4-(4chloro (ICI 200,880) or bromo (iCI 200,355) phenyl sulfonycarbamoyl) benzoyl-L-valyl-L-prol ine-1 (RS)-(l-trifluoroacetyl-2-methylpropyl)amide.
Sr
trifugation, the absorbance of the supernatant at 276 nm was determined.
Pharmacokinetic Evaluation of ICI 200,880 and ICI 200,355 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 and ICI 200,355 in various biologic fluids could be quantified. The pharmacokinetic characterizations of the compounds were determined as follows: before and at varying times after intravenous, subcutaneous, or oral administration of compounds to either hamsters, monkeys, or dogs, blood samples (0.2 ml) were obtained via cardiac puncture under light anesthesia (hamsters), withdrawn from the subcubital vein (dogs), or from the pelvic venous sinus (monkeys). The blood was expressed into 2-ml centrifuge tubes, allowed to clot for 30 min, and centrifuged in a Beckman microfuge (Beckman Instruments, Fullerton, CA). Fifty microliters of serum were then combined with 50 III trypsin (5 mg/ml) and allowed to interact for 5 min. The trypsintreated serum (10Ill)was then added to a 0.52ml cuvette containing buffer with 20 nM HNE. After an additional 30-min incubation, the reaction was started with the addition of substrate (350 IIIof 1.6mM methoxysuccinylala-ala-pro-val-pNA), and the reaction was monitored spectrophotometrically at 410 nm in a Baxter Rotochem centrifugal analyzer. Percent inhibition of HNE by trypsinized serum was calculated. The actual serum concentrations ofthe inhibitors were determined by comparing percent inhibition of the test sample with the percent inhibitions determined for a standard curve for ICI 200,880 or ICI 200,355prepared in control blood. Log serum drug concentration was plotted versus time postdrug administration and an approximate elimination half-time was determined from the resultant curve. To determine persistence in the lung after intratracheal administration of ICI 200,880 and ICI 200,355, male Syrian hamsters were prepared for intratracheal administration of compound as describedbelow. At various times after compound administration, the animals were killed, and the lungs were removed and lavaged with phosphate-buffered saline (PBS). As the inhibition of HNE was negligible in control hamster lung lavage, the inhibition
of HNE by drug-treated animals was determined without addition of trypsin. Lavage concentrations were determined by comparing test results with results of a standard curve prepared in control lavage fluid. Log lavage drug concentration was plotted versus time postdrug administration and an approximate retention half-time was determined. The retention of ICI 200,880 in the lungs of hamsters after inhalation exposure was determined by exposing animals, in a nose-andmuzzle-only exposure system, to an aerosol of ICI 200,880. Compound was dissolved in PBS at a concentration of 10 mg/ml, and aerosols weregenerated using a Retec nebulizer. The animals were exposed to the resultant atmosphere for 60 min. 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 described above.
Induction of Acute Lung Injury with HNE Male Syrian hamsters weighing 90 to llO g were anesthetized intraperitoneally with Brevital sodium (30 mg/kg), and the trachea was surgically exposed. A dose of 400 ug HNE, in 0.3 ml 0.01 M PBS, was injected into the exposed trachea via a half-inch, 23-gauge 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 were carefully cleaned of extraneous material. After measurement of wet lung weight, the tracheas were cannulated and lavaged three times with 2 ml PBS. The recovered lavages were pooled for each animal, and the volume was recorded. Total red and white cells were determined using a Coulter counter (Coulter Electronics, Hialeah, FL). The data are expressed as lung weight/Its) g body weight and total cells recovered (cells ml x volume recovered). Percent inhibition of the resultant lesions by inhibitors was calculated as (a - b/a) x 100 where a is the response in the absence of inhibitors, and b is the response in the presence of inhibitors. Both a and b were corrected for baseline. Therapeutic Effect of ICI 200,355 in PPE- and HNE-induced Emphysema in Hamsters PPE therapeutic trial. On Day 0, male Syrian hamsters weighing90 to 100g wereanesthetized with Brevital sodium and received intratracheal injections of vehicle or 100or 150 ug/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 receivedtwice daily subcutaneous injections of either 50 or 100 umol/kg ICI 200,880 or vehicle for either 14
BIOLOGIC CHARACTERIZATON OF ICI 200,880 AND ICI 200,355
or 28 days, after which they were killed and the lungs were prepared for morphometric analysis as described below. Two groups of animals received intratracheal injections of PBS followedby twice daily subcutaneous administration of ICI 200,880 (100 umol/kg) for either 14or 28 days, after which the lungs were removed and processed for morphometric analysis as described below. HNE therapeutic trial. On Day 0, animals received intratracheal injections of either vehicle or 400 I1g HNE/animal as described above. Twenty-four hours later the animals weredivided into equal groups and weretreated twice daily with subcutaneous injections of either 50 or 100 umol/kg ICI 200,880 or vehicle.After 56 days of drug or vehicletreatment, all animals were killed, and the lungs were prepared as described below. A separate group of animals receivedan intratracheal injection of PBS followed by subcutaneous administration of 100 umol/kg of ICI 200,880 for the duration of the study. Morphometric analysis. At the indicated times after enzyme administration, the animals werekilledwith 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, blotted dry, and weighed. The lungs were then inflated and fixed with 10% phosphatebuffered formalin (pH, 7.0) at 25-cm formalin pressure. Sections 5 11m thick, stained with hematoxylin-eosin, were prepared by JDJ Laboratories (Elkton, MD), and mean linear intercepts weredetermined using an Optomax image analyzer (Optomax, Hollis, NH). Statistical analysis. Data were evaluated using either Student's two-tailed t test or oneway analysis of variance and Duncan's multiple range test, with p < 0.05 considered statistically significant. Source of materials. Bovine pancreatic chymotrypsin, human plasma thrombin, acetyl cholinesterase, angiotensin-converting enzyme, bovine pancreatic trypsin, pepsin, and all synthetic substrates were purchased from Sigma Chemical Co. (St. Louis, MO). Porcine pancreatic elastase was purchased from Worthington Biochemicals (Freehold, NJ), human neutrophil elastase, cathespin G, and insoluble bovine legamentum nuchae elastin werepurchased from Elastin Products (Owensville, MO). Hamster neutrophil elastase was prepared from hamster neutrophils by the method ofViscarello and coworkers (14).All other reagents were of the highest grade commercially available.Male syrian hamsters were purchased from Charles River Laboratories (Wilmington, MA), Beagle dogs from LREHazelton (Kalamazoo, MI), and Macaca fasicularis monkeys from Charles River Primates (Long Island, NY).
Results
Biochemical Characterization of ICI 200,880 Potency and selectivity of ICI 200,880
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Fig. 2. Kinetic analysis of the hydrolysis of methoxysuccinyl ala-ala-pro-valylpNA by HNE (1 x 10-' M) alone [IJ = oand in the presence of 0.3and 1.2 iJM ICI200,880.The ordinate reflectsthe formation of p-nitroanilide by HNE versus time on the abscissa. Note the curvature in the reaction progress curve, which indicates the "slow-binding" nature of the inhibition of HNE by ICI 200,880.
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against several hydrolases. ICI 200,880 was evaluated as an inhibitor of several hydrolases, including hamster and human neutrophil elastase. Similar data for ICI 200,355 have been reported previously (15) and are virtually identical to ICI 200,880. As illustrated in Figure 2, ICI 200,880 is a potent, slow-binding inhibitor of human neutrophil elastase with a K, value of (5.0 ± 1.0) x 10-10 M and a Ks« of (8 ± 1) x 10-4 M- 1 s:'. Human neutrophil elastase exists as a mixture of isoforms (15). The three major isoforms were isolated and purified, and the ability of ICI 200,880 to inhibit those isoforms was evaluated. As illustrated in table 1, ICI 200,880 was an equally effective inhibitor of all three isozymes. Likewise, the major endogenous inhibitor of HNE, alpha-l-proteinase inhibitor, also equally inhibited all three isoforms. In contrast, the compound was a very weak inhibitor of a variety of other proteases that are representative of the four classes (table 2), the exception being porcine pancreatic elastase where a 150-fold selectivity ratio was observed. Inhibition ofHNE catalyzed hydrolysis ofinsoluble elastin. The ability ofICI 200,880 to inhibit HNE-hydrolysis of insoluble elastin is illustrated in figure 3. In these experiments, the reaction was initiated by combining HNE with insolu-
TABLE 1 KINETIC PARAMETERS FOR THE ISOFORMS OF HUMAN LEUKOCYTE ELASTASE (HLE) Isoforms
MeOSuc-ala-ala-pro-val-CH 2CI 10-3 KilKi, M-' S-1 Alpha-l-protease inhibitor 10" Kon, M-' So, IC1200,880 10" Kon, M-' S-l 10~o~M
HLE
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9 4
11 6
7 3
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ble bovine ligament elastin followed 10 min later by the addition of ICI 200,880. The compound produced a concentrationrelated inhibition of the hydrolysis of insoluble elastin by HNE. A Henderson plot (16) of these data yielded a K, of 1.0 ± 0.2 nM (mean ± SEM, n = 3), a value in good agreement with the K; of 5 x 10-10 M obtained versus the synthetic substrate. ICI 200,355 provided virtually identical data (15).
Pharmacologic Characterization of ICI200,880 and tct 200,355 Pharmacokinetic evaluation. When administered as an intravenous bolus to hamsters, ICI 200,880 (20 umol/kg) and ICI 200,355 (10umol/kg) are rapidly distributed and eliminated from the bloodstream with estimated half-times of 62 and 52 min, respectively (figure 4). However, when administered by intratracheal instillation (figure 5) the compounds are retained in the lung for a longer period of time; half-time retention of 179 min
TABLE 2 HYDROLASE SELECTIVITY OF ICI 200,880
Hydrolase Serine HNE PPE Hamster neutrophil elastase Cathespin G Chymotrypsin Thrombin Trypsin Acetylcholine esterase Cysteine Papain Metallo Thermolysin ACE Acid Pepsin
Ki' (nM) 0.5 74 7.3 180,000 Nit 65,000 NI NI NI NI NI 45,000
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creases of lung weight/body weight, total cells, and total white cells (figure 10). Forty-eight hours after exposure, significant (p < 0.05) inhibition of the HNEinduced increase of total cells, but not lung weight/body weight or total white cells, was apparent (data not shown). The protective effect of a single subcutaneously administered bolus dose of ICI 200,880 (86 umol/kg) 1 h prior to intratracheal administration of 100 ug/animal HNE is shown in figure 11. As is apparent, the compound significantly (p < 0.05) inhibited the HNEinduced increases of lung weight/body weightand totallavageable cells.A reduction of totallavageable total white cells was also apparent, but statistical significance (p < 0.05) was not achieved. The effect of leI 200,880 on the
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880
WILLIAMS, FALCONE, KNEE, STEIN, STRIMPLER, REAVES, GILES, AND KRELL
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