HUMAN LEUKOCYTE ELASTASE INHIBITORS PREPARED FROM PYOGENIC BACTERIA AND METHODS FOR THEIR PURIFICATION

Abstract: Proteinaceous materials extracted from pyogenic bacteria, streptococci, staphylococci and Klebsiella species, act as specific inhibitors of human luekocyte elastase and may be used to treat deseases which include elastolysis as part of their desease processes e.g., pulmonary emphysema, adult respiratory distress syndrome and bacterial pneumonites. The proteinaceous materials extracted from the pyogenic bacteria can be conveniently purified by providing an affinity column by various novel methods such as the following five step methods: (1) binding elastin to an affinity column support material (2) binding human leukocyte elastase to the elastin (3) binding the human leukocyte elastase inhibitors to the elastase (4) eluting the affinity column with a first elution medium capable of separating contaminating substances from the inhibitors and (5) separating the inhibitors from the elastase by use of a second elution medium having a raised salt concentration.

Description: HUMAN LEUKOCYTE ELASTASE INHIBITORS PREPARED FROM PYOGENIC BACTERIA AND METHODS FOR THEIR PURIFICATION TECHNICAL FIELD OF THE INVENTION This invention relates generally to substances for treating diseases which include elastin destruction as part of the disease process. More specifically this invention is concerned with proteinaceous substances which inhibit the elastolytic action of human leukocyte elastase and with methods for purifying human leukocyte elastase inhibitors. BACKGROUND ART Neutrophil proteases have been implicated in several human diseases since the turn of the century ( Me tchn ikof f E. _' Immun i te ' dans le Maladies Infectieuses/ Masson , Paris (1901)). A number of such proteases are known. This patent disclosure is particularly concerned with a class of protease found in normal/ circulating human polymorphonuclear leukocytes known for its elastolytic activity, see Janoff, A. Scherer, V. J. Exp. Med. 128:1137-1151 (1968). This class of protease is commonly referred to as human leukocyte elastase ( "HLE") / or alternatively, granulocyte elastase or neutrophil elastase, (for the purposes of this patent disclosure, the term HLE should be taken to include all three terms). HLE has been implicated in a wide variety of disease processes which produce elastolytic effects, e.g., pulmonary emphysema (Janoff, A., et al Am. Rev. Respir. Pis. 115:461-478, (1977) and Snider, G.L. Med. Clin . North Am.. 65:647-665, (1981)) , " rheumatoid arthritis and other arthritides involving acute inflammatory reactions (Janoff, A. et al J. Clin. Invest. 57:615-624 (1976) and Keiser, H. et al J. Cliri. Invest. 57:625-632 (1976)) and Adult Respiratory Distress Syndrome (Lee, C.T. et al N. Engl. J_. Med. 304:192-196 (1981) and McGuire, . . et al . Clin. Invest. 69:543-553 (1982)). The major tissue regulator of HLE in humans is alpha 1-proteinase inhibitor or alpha 1-antitrypsin. The alpha 1-proteinase inhibitor is often referred to as AlPi (Ohlsson, K.:Proteases and Biological Control (eds. E. Reich, D. Rifkin, E. Shaw) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 591-602 (1975)). When the normal AlPi/HLE balance is upset, elastin is destroyed. For example, it has been shown that a genetic deficiency of AlPi is associated with markedly increased risk of developing pulmonary emphysema (Eriksson, S. Ac ta . Med . Sea n d . 177 : ( suppl .432) 5-12 (1965) ) . Several other investigators have also suggested that oxidants in cigarette smoke can inactivate AlPi (Carp, H. and Janoff, A. Am. Rev. Respir. Pis. 118:617-621 (1978), Gadek, J.E. et al Science 206:1315-1316 (1979) and Beatty, K. et al J. Lab. Clin. Med. 100:186-192 (1982)) and that an "acquired" defect in lung AlPi is produced in cigarette smokers (Janoff, A. J_. Appl . P h y s i o 1 : Re s p i ra t . Env i ron . Exe rc i se Physical.55:285-293 (1983)). Consequently, various attempts have been made to develop inhibitors of HLE for potential use against those diseases wherein HLE has been implicated in a pathogenetic role. For example, investigators concerned with the treatment of pulmonary emphysema have tried several methods to improve the inhibitory screen of the lung. One such method is the infusion of pooled AlPi. This has been effective in replenishing the ntiproteinase screen in the lungs of patients with a genetic related AlPi deficiency, see for example, Gadek, J.E. et al . Clin . Invest. 68:1158-1165 (1981). However, this is an impractical approach since AlPi is cleared so rapidly from the circulation that frequent massive infusions are required. Other inv stigators are looking into the introduction of inhibitor-producing genetic materials into bacteria by recombinant DNA technologies. However, owing to the complexities of these technologies, progress in this area has been slow. To date no clinically effective elastase inhibitors have been reported. Furthermore, toxicity and antigenicity problems may be presented by materials produced by DNA technologies . Toxicity and antigenicity also have been the major problems associated with the use of those synthetic elastase inhibitors thus far produced. For example, certain peptide chloromethyl ketones (Powers, J.C. Am. Rev. Respir. Pis. 127:54-58 (1983)) have been shown to be effective i_n_ vivo (Janoff, A. and Rearing, R. Am. Rev. Respir. Pis. 121:1025-1029 (1980)), Kleinerman, J. et al Am. Rev. Respir. Pis. 121:381-387 (1980) and Ip, M.P.C. et al Am. Rev. Respir. Pis. 124:714-717 (1981)), but these agents have shown nephro toxicity ( Ranga , V. et al Am. Rev. Respir. Pis. 124:613-618 (1981)). More recently, mechanism-based irreversible inhibitors of HLE have been described, e.g., 3-chloro i socoumar in and 3,3-dichlorophthalide (Harper, J.W. et al J_. Am. Chem. Soc . 105:6518-6520 (1983) ) . But here again, nephrotoxity is an issue. Scientists following another investigatory path have found that HLE can be inhibited by secretory products of certain fungi. These products include "elasnin" , a highly alkylated 4-hydroxy-alpha-pyrone from Streptomyces noboritoensis (Nakagawa, A. et al J_. Org. Chem. A5:3268-3274 (1980)) and "elasta tinal" , a small peptidyl aldehyde from a different streptomyces strain (Umezawa, H., Aoyagi, T. , Okura , A., Morishima, H., Takeuchi, T. and Okawi , Y. . Antibiot. 26:787-789 (1973) and Okura, A. et al J. Antibiot. 28:337-339 (1975)). However, few efforts have been made to design substances based on structural knowledge of the reactive sites of naturally-occurring, endogenous elastase inhibitors even though the complete sequence of AlPi is now known (Carrell, R.W. et al Nature 298:329-334 (1982)). In the course of investigating the structure of the reactive si tes of naturally-occurring, endogenous elastase inhibitors. applicants have discovered a new class of naturally-occurring HLE inhibitors. They are found in pyogenic bacteria and can be purified by certain novel processes hereinafter described. DISCLOSURE OF THE INVENTION Applicants have discovered a novel class of HLE - in h i b i to r s in pyogenic bacteria, e.g. Streptococcus pneumoniae , Staphylococcus aureus and Klebsiella pneumoniae ( pneumococci ) . It should be noted that, for the purposes of this patent disclosure, the singular term "HLE inhibitor" and the plural term HLE inhibitors should be understood as being interchangeable since more than one HLE inhibitor may exist within the class. It should also be understood that, of these pyogenic bacteria, the pneumococcal inhibitors are the easiest to obtain in large yields. Therefore, they are the ones most extensively characterized. However, as will be indicated by some of the following portions of this patent disclosure, we have reason to believe they are prototypical of this newly discovered class of HLE-inhibitors. Their characterization can begin by noting that they are non-secretory, large molecular weight proteins. They are clearly different from both elasnin and elastatinal in their physical properties and kinetics of inhibition, as well as in their species of origin. Perhaps the most important single attribute of these inhibitors is the fact that they are specific to HLE. This is not true of any other known naturally occurring eukaryotic elastase inhibitor such as, for example, AlPi or bronchial- mucous proteinase inhibitor. Furthermore, since they are derived from bacteria which frequently live in the human body (e.g., pneumococci in nasopharynx, staphylococci on skin), often with no ill effects, they are less likely to be toxic or antigenic than those HLE inhibitors (1) designed on the basis of the known peptide-bond specifications of HLE such as the peptide chlorome thylke tones previously noted, (2) derived from genetically engineered bacteria, or (3) discovered through large-scale fungi screening programs. The HLE inhibitors herein disclosed can be purified according to known procedures; however they can be more effectively purified by a novel affinity column procedure hereinafter described. The first step in this novel process for purifying the HLE inhibitor materials is to bind elastin to an affinity column support material such as, for example, sepharose . Human leukocyte elastase is then bound to the elastin. Since this binding does not require the HLE molecules' active site, the bound HLE molecules have their active sites available for binding the HLE inhibitor molecules which are then introduced into the column as lysates of the pyogenic bacteria. After using a first elution medium to remove contaminants, the bindings between the HLE and the HLE inhibitor molecules are broken by use of a second elution medium having a raised salt concentration, i.e., a salt concentration higher than that of the first elution medium. This breaking of the HLE/HLE inhibitor binding takes place without breaking the binding between the elastase and the elastin, and thereby provides a novel and efficient process for purifying the sought after HLE inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the inhibition of human neutrophil elastase by extracts of S_. pneumoniae (III), after prolonged preincubation of enzyme with the bacterial protein at 37 °C. The extract was prepared by lysis of the bacteria in sodium deoxychola te , centrif uga tion and extensive dialysis of the supernate. Preincubation was carried out in o.OlM Tris at pH 8.0, plus 0.15M NaCl and enzyme assays were carried out in 0.2M Tris buffer, pH 8.0, containing 0.15M NaCl. Sodium deoxycholate alone had no effect on enzyme activity. The ( α ) line indicates a 30 minute preincubation; the ( ) line indicates a 60 minute preincub tion; and the ( o ) line shows the results of a 90 minute preincubation. The ( __, ) line indicates a 60 minute preincubation at 0°C. Figure 2 shows the chroma tography at 4°C of S_. pneumoniae (type III) extract against neutrophil elastase covalently bound to Sepharose. The running buffer was 0.01 M Tris at pH 8.0. Column dimensions were 1.0 x 10 cm. The solid line ( ) indicates absorbance at 280 nm (A280 nm) ; the shorter dash line ( — ) indicates inhibitory activity y_s neutrophil elastase. Clearly, two separate inhibitors of HLE are present in pneumococcal extracts (see I and II in figure 2). Subsequent experiments revealed that one of the agents was low molecular weight and was inactive in the presence of 1.15M NaCl while the other agent was high molecular weight and retained its activity in 0.15M NaCl. Figure 3 shows a Lineweaver-Burk analysis (plots o f re a c tion ve lo c i ty"! v s subs tra te concentra tion" 1 ) of sa lt-sensi tive and salt-resistant elastase inhibitors derived from pneumococci. Each assay mixture contained 36 ug/ l of pure human neutrophil elastase and 135 ug/ l of purified salt-sensitive inhibitor or of whole pneumococcal extract (crude salt-resistant inhibitor). The (O) line indicates enzyme alone; the ( A ) line indicates enzyme plus salt-sensitive inhibitor purified from type III pneumococci by preparative polyacrylamide gel electrophoresis; the ( _j ) line indicates enzyme plus crude salt-resistant inhibitor from type II pneumococci; and the ( • ) line indicates enzyme plus crude salt-resistant inhibitor from type III pneumococci. The reaction velocity V, indicates moles of substrate hydrolyzed per minute. he substrate concentration S is given in moles/liter. The graph indicates that the salt-sensitive agent inhibited competitively; the y-intercept (maximal velocity) is identical to that of the enzyme alone, whereas the x-intercept, affinity for substrate of (Km), is different from enzyme alone. By contrast, the salt-resistant agents from types II and II I pneumococci inhibi ted non-compe t i t i ve ly as can be observed from the different y-intercept and similar x-intercept values. Figure 4 shows the chroma tography of pneumococcal extract on Sephacryl S-300. The solid line ( ) indicates protein concentr tion expressed as absorbance at 280 nm. The short dashed line ( — ) indicates percentage inhibition of human neutrophil elastase in the presence of 0.15 NaCl. The long dash line ( ) indicates percentage inhibition of human neutrophil elastase in the absence of 0.15M NaCl. Figure 5 shows the results of SPS-gradient polyacrylamide g l electrophoresis of crude pneumococcal proteins and peak I protein obtained by molecular-sieve chroma tography (as in Figure 4). Peak I of Figure 4, which was enriched in specific activity of the salt-resistant inhibitor, also showed enrichment of one major band of high molecular weight protein, but contained trace amounts of lower molecular weight species as well (see Figure 5, panel A, lane 2). Identification of the enriched high MW band in Figure 5 (panel A, lane 2) as the high molecular-weight, salt-sensitive inhibitor was further supported by the observation that complexes of radio-iodinated neutrophil elastase and this inhibitor eluted in the high MW fraction during molecular-sieving through Sephacryl S-300 (See Figure 6). The molecular weight of this major band (putative salt-resistant inhibitor) was calculated to be 140,000 daltons, cf. arrow vs molecular weight markers in kilodaltons in Figure 5, panel B. Separate experiments revealed that this inhibitor was not active against trypsin (data not shown) or pancreatic elastase (see Table I). Therefore, a trypsin digestion experiment was performed to confirm that the inhibitor was at least partly protein in nature. Incubation of the partly purified inhibitor with TPCK-trypsin (defined in the Materials and Me thods-Pro teoly tic Enzymes portions of this patent disclosure) for 24 hours at ambient temperature resulted in loss of the major protein band and the appearance of smaller molecular weight fragments (see Figure 5, panel A, lane 3). After incubation with ImM di t hio threi tol and 3M urea, the trypsin-clea vage products were further dissociated into even lower molecular weight fragments. From these results, we have reason to believe that the high molecular weight inhibitor is susceptible to proteolysis and also that the molecule probably contains subunits which are stabilized by hydrophobic associations and/or disulfide bonds. Of considerable interest is the fact that the trypsin-cleavage products retained inhibitory activity against neutrophil elastase. Figure 6 shows the Sephacryl S-300 chroma tography of mixtures of isotope-labelled human neutrophil elastase and pneumococcal extract (type III pneu ococcus, reaction in 0.15 M NaCl). Curve 1, i.e. , the ( ) curve indicates 14 C-peptide chloromethyl ke tone - tr e a te elastase (100% inactivated) plus bacterial extract. Curve 2 ( ) is as above except treated elastase is 56% inactivated. Curve 3 ( • ) is as above except that the elastase is 10% inactivated. Elution volumes of 125ι-i a belled free elastase and 125j._ι a belled elastase complexed with the salt-resistant inhibitor are also marked in this Figure. V 0 indicates the Void Volume. Further explanation and interpretation are given in later portions of the patent specifications. Figure 7 indicates the double-immunodif fusion patterns given by Sephacryl S-300 eluted fractions containing neutrophil elastase complexed with pneumococcal inhibitor. The antiserum to neutrophil elastase is indicated in the central trough; the pure enzyme alone is indicated in the two wells labelled e; all other wells contained selected fractions from curve #3 of Figure 6 (every fifth fraction is numbered in the figure). Wells marked by "X" contained no sample. Gels were eluted to wash out unprecipi ta ted protein, dried and stained with Coomassie blue. Figure 8 plots the percent activity remaining (100 percent = fractional activity of 1) vs. molar ratio of inhibi tor /neutrophil elastase in reaction mixtures. Pure neutrophil elastase (Mr = 32,000) was incubated with partly-purified inhibitor (Sephacryl peak I), and the estimated Mr (i.e., molecular weight as determined by molecular radius) of the inhibitor was taken as about 140,000 (see the "Results" portion of the patent disclosure for further relevant discussions) . Extrapolation of the plot to zero percent remaining activity allowed estimation of the functional molar ratio of inhibitor /enzyme in the complex. Figure 9 depicts a proposed lysis mechanism of pneumococci cell wall material. Figure 10 shows chroma tographs of pneumococcal lysa te on Sepharose 2B and Sephadex G-100. Figure 11 is a gel-electrophoregram of smaller fragments of the inhibitor. Figure 12 shows the histologic appearance of mouse lung treated with various mixtures. Panel A represents a buffer control; panels B and C represent 50 ug neutrophil granule extract incubated for 10 minutes at 37"C with 200 ug nonspecific goat i mmunog 1 obu 1 in (protein control) and panel P represents 50 ug granule extract incubated as above with 200 ug pneumococcal inhibi tor-pe p t ide s . Significant hemorrhage can be noted in panels B and C, and a decrease in this manifesta ion of acute lung injury can be noted in panel P. Goat im unoglobulins alone did not produce acute injury. BEST MODES FOR CARRYING OUT THE INVENTION BACTERIA SOURCES ANP INITIAL EMBOPIMENTS Streptococcus pneumoniae type I (ATCC 9163), type II (ATCC 11733 ) , type III (ATCC 10813) and Staphylococcus aureas (ATCC 25923) were from the American Type Culture Collection (Rockville, Md). Other Streptococcus pneumoniae were obtained from blood cultures of patients with active pneumococcal pneumonia. The Klebsiella pneumoniae was isolated from clinical sources. Representative bacteria were grown in Todd-Hewitt (beef heart-infusion) broth at 37°C for 16-18 hours. Cells were then collected by cen trif uga tion at 1500 x g for 10 minutes, washed three times in cold phosphate-buffered saline, suspended in this same medium and lysed by various known techniques which were sometimes found to have certain specific advantages with regard to the kinds of cells being lysed. For example, sonication in the cold (Sonifer Cell Pisruptor, Model W140, Ultrasonics, Inc., Plainview, NY), or incubation for 1 hour at 37°C in the presence of 0.4 percent sodium deoxycholate were often used as the lysis technique. However, the latter procedure was employed only for S_. pneumoniae , which shows unique susceptibility to lysis in ionic detergents including bile salts. Another specific ' adaptation was found in the use of a French press (12,000 pounds per square inch) which easily lyses the Streptococcus pneu on iae but cannot lyse the Staphylococcus aureus. Alumina grinding worked well on Staphylococcus aureus but a slimy capsular material was formed when this technique was used on Strep to coccus pneumoniae . Freeze thaw techniques worked well on the Streptococcus pneumoniae but not on the S taphylococcus aureus. In any event, following lysis, cell debris was removed by high-speed cen tr if ugation . Sodium deoxycholate supernates were extensively dialysed against phosphate-buffered saline, and the extracts were frozen at about -85 °C until used. PREPARATION OF PROTEOLYTIC ENZYME The bovine trypsin utilized (freed of contaminating chymotrypsin activity by treatment with tosyl-phenylalanyl-chlorome thyl ke tone (TPCK)) was that supplied by Worthington Biochemical Corp. (Freehold, N J ) . Porcine pancreatic elastase was purchased from Elastin Products Co., Inc. (Pacific, MO) . Pure human neutrophil elastase and crude extracts of human neutrophil granules were prepared according to the methods of Feinstein and Janoff:A rapid method of purification of human granulocyte cationic neutral pro teases purification and further characterization of human granulocyte elastase. Biochem. Biophys. Acta 403:493-505 (1975). INHIBITION ASSAYS Inhibition of proteases by crude bacterial extracts or by purified bacterial inhibitors was expressed as a percent of control enzyme activity (without inhibitor) and normalized to the protein content of the extract or inhibitor fraction tested. Elastase activity (neutrophil and pancreatic enzymes) was measured using succinyl-L-alanyl-L-alanyl-L-alanyl -p-nitroanilide (Sigma Chemical Co., St. Louis, MO) as substrate, according to the method of Bieth, Speiss and Wermurth: The synthesis and analytical use of a highly sensitive and convenient substrate of elastase Biochem. Med. 11:350-357 (1974). Trypsin was assayed using N-benzoyl-P, L-arginine-p-nitroanilide (Sigma Chem. Co.) as the substrate. All enzyme activities were monitored kinetically in a Gilford Model 2400-S Recording Spec tropho tom ter (Gilford Instrument Laboratories Inc. , Oberlin, OH) , and rates of hydrolysis were calculated from the linear portions of the curves during the initial 5 to 10 minutes of incubation (22°C). Controls included substrates alone (substrate blanks) , substrates plus enzymes without inhibitors (enzyme controls), and substrates plus inhibitors without enzymes (inhibitor blanks). Sodium deoxycholate (0.4%) was demonstrated to be without effect on neutrophil elastase. Another test procedure was carried out as follows: Organisms: Streptococcus pneumoniae (Strep PN) was obtained from blood cultures of patients with active pneumococcal pneumonia from the University of Colorado Health Sciences Center, St. Joseph's Hospital, and the Penver V.A. Medical Center. The staphylococcus aureus utilized was a stock reference organism (ATCC #25923). All organisms were stored frozen at -70°C in trypticase soy broth made 1% in dextrose . Reagents: Tritiated bovine neck ligament elastin was prepared in one of our laboratories. Human neutrophil elastase and crude granule extracts were prepared from various normal subjects and from HL-60 cells (a continuous human cell line derived from a patient with a leukemia of neutrophil-like cells). Porcine pancreatic elastase was purchased from Elastin Products Co. (No. EC134). Bacterial Growth: Bacteria were grown in trypticase soy broth with 1% dextrose. The broth was innoculated and cultured at 37°C. Lysate preparation: The broth was removed from an incubator after the culture reached its last third of exponential growth curve as determined by optical density at 540nm and placed in a cold room to arrest growth. Broth was then centrifuged in cold at 20,000 x G for 10 minutes. The pellet was washed and centrifuged again. KNOWN PROCEPURE FOR INHIBITOR SPECIFICATION In some experiments, crude bacterial extracts were passed through a column of Sepharose to which pure human neutrophil elastase had first been covalently-linked. The latter was accomplished using cyanogen-bromide activated Sepharose according to the method of Cuatrecasas, P.:Protein purification by affinity chroma tograph . Perivations of agarose and polyacrylamide beads . Biol . Chem. 245:3059-3065 (1970). Chroma tography was carried out in 0.01M Tris-HCl buffer (pH 8.0) , and individual fractions were monitored for protein absorbance at 280nm and for inhibitory activity against neutrophil elastase. In other examinations, pneumococcal extracts were c hroma tographed on Sephacryl S-300, superfine, (Pharmacia Fine Chemicals, Uppsala, Sweden). The f ractiona tion range (Mr) of this gel is lxlO 4 - 1.5x10-5 f or globular proteins. Column dimensions were 3 cm x 75 cm and the buffer was 0.01M Tris-HCl, pH 8.0, containing 0.15M NaCl. Fractions were collected at a flow rate of 11 ml/hr. NOVEL PROCEPURE FOR INHIBITOR ENRICHMENT A novel method by which the initial material used for purification can be enriched in the inhibitor is to suspend the bacteria from which this inhibitor is to be purified in a nonionic, hypertonic medium such as about 0.6M sucrose. The bacterium are then subjected to lysozyme digestion. Treatment in this manner allows for solubilization of the cell wall constituents (of which this inhibitor appears to be a part) without disrupting the cell membranes. As a result, intracellular substances and cell membranes are not released into the solution and can be spun down leaving the inhibitor in the supernate. Extraneous proteins can then be segregated away, for example, by passing the supernate over a Sepharose 2B chromatography column (see for example Figure 5, column B) or further purified as described below. NOVEL PROCEPURE FOR INHIBITOR PURIFICATION Increased HLE inhibitor purification can be obtained by * a novel affinity column technique hereinafter described. This technique can be used as an adjunct to the novel lysozyme digestion procedure described above or it can be used on lysates of the pyogenic bacteria which have not undergone lysozyme digestion. The novel affinity column procedure begins with binding elastin to an insoluble solid support material such as, for example, sepharose. This binding can be accomplished by covalent binding, such as, for example, that achieved by use of a covalent ligand such as cyanogen bromide. HLE is then bound to the elastin by their normal interaction so that at least a portion of the active sites (enzymatic cleavage sites) of the HLE are still available for binding HLE inhibitors. Lysates of the pyogenic bacteria are then introduced to the affinity column so that the HLE inhibitors in the lysates of the pyogenic bacteria become bound to the active site of the neutrophil elastase. Preferably the lysates carrying the HLE inhibitor are introduced until the elastase molecules become saturated with inhibitor. The affinity column is then eluted with a first elution medium capable of separating contaminating substances from the HLE inhibitors. A low ionic strength neutral buffer (e.g., 0.1M Tris at pH 7.2) can be used for this purpose. Thereafter, the affinity column is eluted with a second elution medium capable of separating the HLE inhibitors from the HLE. A buffer (e.g., a salt medium such as a NaCl medium having a salt concentration of from about 0.01 to about 1 molar) having an ionic strength higher than that of the first elution medium can be used to break the binding between the HLE and the HLE inhibitor without breaking the binding between the HLE and the elastin and thereby providing an efficient method for purifying the HLE inhibitor. POLYACRYLAMIPE GEL ELECTROPHORESIS Polyacrylamide disc-gel electrophoresis was carried out at pH 8.6 in 12 percent acrylamide separation gels for two and one half hours at 3 mA per gel. Analytical gels were stained with Coomassie blue; preparative gels were manually sliced and selected slices- were extracted into 0.01M Tris-Cl buffer, pH 8.0, for 24hr at 37°. Gradient , so d i u m- d o d e c y 1 - s u 1 f a te (SPS ) polyacrylamide slab-gel electrophoresis was carried out according to the method of Laemmli : Cleavage of structural proteins during the assembly of the head of bac ter iophage T4, Nature 227:680-685 (1970) . Various purified proteins were used to calibrate the gels for estimation of molecular weight. After fixation, proteins were visualized by silver-staining as described by Wray, W. et al , Silver staining of proteins in polyacr lamide gels Anal. Bioche . 118:197-203 (1981). LABELLING PROCEPURE Radioactive labelling of pure neutrophil elastase was carried out according to the method of McFarlane A.S.:In vivo behavior of ll31-f ibrogen , J_. Clin . In e s . 42 : 356-361 (1963) , except that the concentration of unlabelled iodine was increased 10-fold. The catalytic site of the enzyme was protected during iodination by addition of 22mM N-s cc i 1 - ( L-a la n 1 ) 3 -p-n i troa n il ide to the reaction mixture. Under these conditions, catalytic activity of neutrophil elastase was unaffected by the labelling procedure. The 125 j. (as sodium iodide) was from Amersham (Arlington Heights, TL) and had a specific activity of 1.99 mCi/n mole. The final, labelled enzyme had a specific activity of 0.81 mCi/n mole . ENZYME INACTIVATION In selected experiments, neutrophil elastase was incubated under various conditions (see "Results" portion of this patent disclosure with Re¬ label led-N-ace tyl-L-alanyl-L-alanyl-L-prolyl-L-valyl chloromethyl ketone (specific ac ti i ty=2.78 mCi/m mole) to either partially or completely inactivate the enzyme. The preparation was then dialyzed extensively to remove any unreacted peptide chloromethyl ketone, and the inactivated enzyme mixed with pneumococcal extract and analysed for complex-formation. PROTEIN ASSAY Protein concentration of bacterial extracts were measured using the method of Lowry, i.e., Lowry, O.H., N.J. Roseborough, A.L. Farr and R.J. Randall : Protein measurement with the folin phenol reagent J_. Biol . Chem. 193:265-275 (1951). Bovine serum albumin was used as the reference. IMMUNOPIFFUSION TESTS Neutrophil elastase alone, or Sephacryl S-300 fractions containing complexes of elastase and pneumococcal inhibi tor were tested for immunorea c t i vi ty against a monospecific rabbit anti-human neutrophil elastase antiserum by double-immunodi f fusion in agarose gels. Gels were then washed free of soluble proteins, dried and stained with Coomassie blue to enhance visualization of precipitin lines. The rabbit antiserum was prepared by the methods described by Feinstein and Janoff in Bioche . Biophys. Acta 403:493-505 (1975). RESULTS Figure 1 shows that a second inhibitor of neutrophil elastase could, in fact, be detected in pneumococcal extracts, when the latter were incubated with enzyme in the presence of 0.15 M NaCl for prolonged times at 37 °C. Under these conditions, salt-sensitive inhibition was effectively prevented. This inhibition was found both in extracts prepared by sonication as well as by Na deoxychola te-induced lysis. Sodium deoxychola te alone had no inhibitory effect. The NaCl-resistant inhibitory activity could not be attributed to capsular polysaccharides since the concentration of these in the extracts was low and purified capsular polysacchar ides also failed to inhibit the enzyme in the presence of NaCl. In addition, "rough" strains of pneumococci (lacking capsules altogether) still show inhibitory activity (data not shown). Inhibition was not decreased when 0.15M NaCl was added to the assay buffer as well as to the preincubation buffer. Less inhibition was evident when preincubation was carried out for 1 hr at 0°C instead of 37°C (see Figure 1). MOPE OF INHIBITION COMPETITIVE VS. NONCOMPETITIVE BY EACH OF THE PNEUMOCOCCAL AGENTS: In order to further investigate the mode of inhibition by pneumococcal agents, the NaCl-sensi tive ( ionic-interaction-dependent) inhibitor was first isolated. Chroma tography of crude extract was carried out in weakly-ionizing buffer against neutrophil elastase bound covalently to Sepharose beads, as shown in Figure 2. The NaCl-resistant inhibitor appeared to elute in the run-through fractions (peak I) along with other proteins. This effect could have been caused by delayed complex formation between this inhibitor and immobilized enzyme at cold temperature. By contrast, faster ionic interactions between the NaCl-sensitive agent and bound enzyme caused passage of this inhibitor to be retarded and it was separately recovered peak-II (see Figure 2) . The latter fractions were concentrated by oncotic methods (Aquacide II-A, Calbiochem. La Jolla , CA) and subjected to analytical polyacrylamide disc-gel electrophoresis. The salt-sensitive inhibitor behaved as if it were highly acidic, migrating rapidly towards the anode at pH 8.6. Therefore, subsequent large scale separation of the salt-sensitive inhibitor was accomplished by extraction of extreme anodal slices of preparative polyacrylamide disc gels. INHIBITION METHOPS Each of the agents was then analysed for its mode of inhibition of neutrophil elastase by plotting reaction velocity-! against substrate concentration"! in the presence and absence of the agent. The NaCl-sensi tive agent ( inhibi tor-I ) , isolated by preparative electrophoresis as above, was tested against pure neutrophil elastase in weakly ionizing buffer. To analyse the second inhibitor, whole pneumococcal extract was preincubated with pure neutrophil elastase at 37 °C x 1 hour in the presence of 0.15M NaCl.. Results are given in Figure 3, where it can be seen that kinetics of inhibition by inhibitor-I corresponded to a competitive mode, consistent with inhibit'io.n by ionic interaction. Maximal velocity (y-intercept) at- high substrate concentrations was identical to that of enzyme without inhibitor, while affinity of enzyme for substrate (Km) or x-intercept differed significantly in the presence of this inhibitor. By contrast, inhibitor-II ( NaCl-re sis tan t ) decreased the maximal reaction velocity of the enzyme, even at saturating substrate concentration, but did not affect the affinity of the enzyme for its substrate (Km). This result is consistent with a non-competitive mode of inhibition for the salt-resistant agent, and suggests formation of stable complexes between neutrophil elastase and this inhibitor. SEPARATION OF THE INHIBITORS BY MOLECULAR-SIEVE CHROMATOGRAPHY: Figure 4 shows the chroma tographic pattern obtained when crude pneumococcal extract (prepared by lysis of S_. pneumoniae III in deoxychola te ) was passed through a column of Sephacryl S-300 as described under the "Methods" section of this patent application. The bulk of the salt-resistant inhibitory activity against neutrophil elastase ( inhibi tor-II ) appeared with protein eluting soon after the void volume (peak I in the figure) and therefore corresponded to material of high molecular weight. By contrast, when the salt-sensitive inhibitor (purified from polyacrylamide gels) was chroma tographed on the same Sephacryl S-300 column, a single-leak of inhibitory activity (in weakly ionizing buffer) appeared at the place indicated in the Figure, corresponding to material of much lower molecular weight. Thus, inhibitor-I is probably normally present in peak VI of the Sephacryl S-300 chroma togram. These data show that the two inhibitors are not only functionally disparate with regard to their mode of inhibition (see preceding section), but are also physically different molecules. The Inhibi tor-II was characterized in much more detail because ' of the greater likelihood that this agent might be capable of acting under physiological conditions (i.e., in the presence of physiological ion concentrations) . FURTHER CHARACTERIZATION OF INHIBITOR-II Figure 5 shows the results of SPS-gradient polyacr lami e g l el ctrophoresis of crude pneumococcal extract and of peak I protein obtained by mo le c u la r- s ie ve chroma tography (see preceding section). Peak I, which was enriched in specific activity of inhibitor-II , also showed enrichment of one major band of high molecular weight protein (panel A, lane 2) and contained, in addition, several fainter bands corresponding to lower molecular weight species. The molecular weight of the major band (putative inhibitor) was calculated to be about 140,000 daltons (see Figure 5, panel B) . Separate experiments revealed that inhibitor-II was not active against trypsin or pancreatic elastase. Therefore, a trypsin digestion experiment was performed to confirm that inhibitor-II was p oteinaceous in nature. Incubation of the partly-purified inhibitor-II with TPCK-trypsin (defined in the "Materials" section of this patent application) for 24 hours at ambient temperature resulted in disappearance of the major protein band at about 140,000 daltons. Under non-reducing conditions, elec trophorec tic analysis revealed smaller protein fragments after trypsin treatment (see Figure 5A, lane 3) . Under reducing conditions, however, the trypsin-cleavage products were converted into even lower molecular weight peptides. From these results, we conclude that inhibitor-II is susceptible to proteolysis and also that the molecule probably contains several polypeptide chains crossed-linked by disulfide bonds. PEMONSTRATION THAT INHIBITOR-II REQUIRES ACTIVE ENZYME TO FORM AN INHIBITORY GROWTH Many protein pr o t e i n a s e - i n h ib i t o r s act non-compe ti tively by posing as substrates for their target enzymes and then forming stable complexes with the latter, once peptide bond attack is initiated, see for example, Travis, J. and G.S. Salvesen :Human plasma proteinase inhibitors, Ann. Rev. Bioche . , 52:655-709 (1983). In these cases, an accessible catalytic-site in the enzyme is required for enzyme-inhibitor complex-formation to take place. To test whether inhibitor-II would similarly interact only with native proteinase, we treated human neutrophil elastase with a specific, irreversible, active-site-directed peptide chloromethyl ketone, see. Powers, J.C. et al : Specif icity of porcine pancreatic elastase, human leukocyte elastase and cathepsin G. Inhibition with peptide chloromethyl ketones Biochem. Biophys. Acta 4845:156-166 (1977). Conditions were varied to achieve different degrees of enzyme inactivation and then a determination was made of the extent of comp le x- f or ma t ion of the treated enzyme with pneumococcal inhibitor-II. Variable degrees of inactivation were achieved by employing high concentrations of n-succinyl-trialanyl-p-nitroanilide (a low molecular weight substrate of elastase) to compete with the chlorome th Ike tone inactivator for the enzyme's active-site. By varying reaction time as we l l as the concen tra tion ra tio of inactiva tor/substrate , levels of inactivation ranging from 10%-100% in three separate experiments were achieved. The peptide chlorome thy Ike tone was labelled with 1 4 C al lowing enzyme which con ta ined bound-inactivator to be detected during chroma tographic separation of complexes and free enzyme on Sephacryl S-300. Molecules of enzyme whose catalytic-sites had been protected by substrate and which had therefore remained active were, nevertheless, also labelled with the 14c- chlorome thylke tone . This could have occurred by virtue of nonspecific alkylation reactions between the highly reactive chloromethyl ketone and chemical groups at a distance from the enzyme's active-site (for example, the alpha amino group or tyrosine residues ) . These mixtures of ac tive , 14 C-labelled , neutrophil elastase and inactive, l 4 C-labelled enzyme (dialysed free of unbound inactivator, substrate, and p-ni troaniline were reacted with pneumococcal extract (in the presence of 0.15M NaCl and complexes of enzyme with pneumococcal inhibitor-II were then resolved by Sephacryl S-300 chroma tography. Results are given in Figure 6 where it can be seen that, as predicted, l 4 C-label eluted in two distinct peaks. One comes immediately after the void volume (enzyme-inhibitor complexes) followed by a retarded peak of enzyme alone. Again, as predicted, all the radioactivity eluted in this latter peak when 100% inactivated enzyme (confirmed by assay y_s succinyl- tr ialanyl-p-ni troanilide ) was reacted with the bacterial extract. For comparison, the same Sephacryl 300 column was also calibrated with 1 5 i-labe lied active neutrophil . elastase (free enzyme marker) and 125 i-labelled , active elastase plus pneumococcal extract (marker of enzyme-inhibitor II complexes). Elution volumes of these markers are also given in Figure 6. These results show that complexation of neutrophil elastase with pneumococcal inhibitor-II depended on an open binding-pocket in the enzyme . When fractions 60-80 in curve #3 of Figure 6 were subjected to double-immunodif fusion v monospecif ic, rabbit antiserum against human neutrophil elastase, the pattern shown in Figure 7 resulted. Since no enzyme activity could be detected in these fractions (only elastase that had been inhibited by complexation with pneumococcal inhibitor-II was present), the development of immunoprecipi tin lines showing identity with those given by free elastase suggested that at least some elastase antigenic sites remained exposed in the enzyme-inhibitor complex. To test whether the inhibitor binds covalently with serine-195 of HLE, elastase was reacted with 3 H-labelled diisopropyl f luorophospha te prior to incubation with the inhibitor. Under such conditions the elastase inhibitor complex is still formed. Moreover, complexes between active elastase and the inhibitor can be dissociated by mild denaturants, supporting the conclusion that they are not covalently bonded. SPECIFICITY ANP PISTRI3UTI0N OF THE TWO INHIBITORS: Table I shows that both pneumococcal inhibitors were specific for neutrophil elastase and neither inhibited porcine pancreatic elastase (or bovine trypsin). In addition. Table I shows that inhibitory activity against neutrophil elastase was also found in extracts of necrotizing organisms (K_. pneumoniae and S_. aureas ) , and that S_. pneumoniae types I and II ( non-necro tizing ) were not more inhibitory than S. pneumoniae type III, which is capable of causing tissue destruction. TABLE I Pistribution and Specificity of the Bacterial Inhibitors Assay Source Extract Test Per Cent Enzyme Buffer of Protein Enzyme Activity Extract (ug) Remaining S.pn. I 32 NE 58 S.pn. II 17 NE 76 Tris S.pn. III 17 NE 73 (o.2M,pH8) It 32 NE 62 II 40 NE 57 "* 40 T 100 "* 75 PPE 100 K.pn. 40 NE 55 II 40 PPE 58 S.aureas 40 NE 62 S.pn. II 70 NE 52 140 NE 29 Tris S. .pn. III 35 NE 14 (o.2M,rpH8 70 NE 10 plus 70 PPE 100 0.15M NaCl 140 PPE 100 K. .pn. 70 NE 92 140 NE 19 NE = neutrophil elastase (2 ug pure enzyme or 20 ug crude neutrophil granule protein) PPE = porcine pancreatic elastase (0.2 ug crystalline enzyme) T = bovine trypsin (1.0 ug crystalline enzyme) S.pn. = Streptococcus pneumoniae K.pn. = Klebsiella pneumoniae S. aureus = Staphylococcus aureus * = Three separate bacterial extracts were tested in these cases. CHARACTER OF INHIBITORS This discovery provides the first characterization of two, different neutrophil elastase inhibitors in pneumococci. Our data shows that inhibition by one of these agents (a low molecular weight, acidic substance that acts competitively and is dependent on ionic interactions to bind with the cationic enzyme) is suppressed by physiological concentration of NaCl. This inhibitor is therefore unlikely to play an important role in vivo . The second inhibitor however is, by contrast, active in the presence of 0.15M NaCl. This agent inhibits non-compe ti ively and requires an open binding-pocket but not an active catalytic site in its target enzyme to form inhibitory complexes with the latter. It appears to form a 1:1 molar complex with neutrophil elastase. Both pneumococcal inhibitors are specific for human neutrophil elastase; neither inhibits porcine pancreatic elastase. The second inhibitor's apparent molecular weight is about 140,000 daltons, yet it does not mask antigenic sites of the enzyme upon complexation with it. The high molecular weight inhibitor is sensitive to trypsin, and its tryptic degradation fragments are susceptible to further dissociation by reduction. Treatment of pneumococci with deoxycholic acid probably activates an autolytic enzyme in the cells which strips the peptide cross-links out of the cell wall peptidoglycan by cleaving bonds between alanine and muramic acid residues (see arrows. Fig 9). The pneumococci are lysed in the process, releasing free glycan strands (large molecular weight) , peptide bridges (low molecular weight) , a number of large molecular weight membrane-associated proteins and glycoproteins , as well as cytoplasmic constituents. When this lysate is chroma tographed on Sepharose 2B, the first peak after the void-volume contains a predominant large molecular weight protein or glycoprotein of 140,000 daltons which make a membrane protein (MP) similar to the high MW cell-surface glycoprotein antigens of other streptococci (see for example. Porn, et al . : Extracellular Antigens of Serotype III Group B Streptococci, Infect. I mun . 30:890-893, (1980) and OMP derived from gonococci. As would be expected, peak I of the Sepharose 2B chromatogram is also enriched in glycan fragments released during deoxychola te-induced lysis (Fig 9). However, most of the contaminating glycan can be removed from Sepharose peak I by treatment with lysozyme (see Fig 10, panel E). Inhibitory activity against human leukocyte elastase is associated with the large molecular weight pro te in or glycoprotein released during deoxychola te-induced lysis and recovered in Sepharose peak I (see panel A of Fig. 10). This protein (or glycoprotein) inhibitor is susceptible to degradation by beta glucuronidase (Fig. 10 panels B and C) or trypsin (Fig. 10 panel P). In either case, smaller fragments ranging in molecular weight between 22 and 45 kilodaltons can then be recovered from G-100 Sephadex columns (Fig. 10 panels C and P) and can be demonstrated on SPS-gradient polyacrylamide gels (Fig. 11). It is also of the utmost importance that these smaller fragments retain inhibitory activity against neutrophil elastase (Fig. 10 panels C and P). This fact should also facilitate the elucidation of the active site structure of the inhibitor. To summarize our overall characterization of the inhibitor we first note that it has a high molecular weight (140,000 daltons), but inhibitory activity is retained by smaller tryptic-peptides derived from the native agent. The inhibitor is specific for neutrophil elastase and it acts non-compe ti tively. Complex-formation between neutrophil elastase and the pneumococcal agent does not require covalent interaction of the inhibitor with serine-195 in the enzyme, since elastase inactivated by 3H-labeled diisopropyl f luorophospha te can still form such complexes. Moreover, complexes between active elastase and pneumococcal inhibitor can be dissociated by mild denaturants. On the other hand, enzyme whose substrate-binding pocket is blocked by a peptide chloromthy Ike tone does not react with pneumococcal inhibitor, suggesting that specific recognition-sites on the inhibitor, may interact non-covalently with the protease. Finally, ith respect to the potential physiological role of the major elastase inhibitor herein described, the following points are worth noting. First, solubilization of the inhibitor by deoxycholic acid-induced disruption of pneumococcal cell walls or by sonication of the bacteria may imply an intracellular ' localization of the agent and this, in turn, could raise questions regarding its ability to interact with extracellular enzyme. It should be remembered, however, that the same disintegration procedures also release molecules associated with cell wall peptidoglycan (see Figure 9). In this latter location, the inhibitor could have ready access to extracellular elastase. Furthermore, even if the agent were to be intracellular in location, following phagocytosis and lysis of pneumococci by neutrophils (or by antibody and complement in lung secretions). the inhibitor would be released and could then inactivate elastase. With respect to the rate of this inactivation, there is evidence of a slow association of the pneu ococcol with neutrophil elastase (see Figure 1). Effective _in vivo inhibition usually depends on fast association rates between inhibitors and their target enzymes. However, we will show in the next section of this patent application that the pneumococcal inhibitor can protect mouse lung _in_ vivo , against acute lung injury produced by neutrophil granule extracts, even when inhibitor and enzyme are administered immediately after mixing. IN VIVO PROTECTION PROVIPEP BY INHIBITOR Further studies were conducted to determine if the pneumococcal inhibitor could effectively block the activity of human neutrophil elastase _in_ vivo . If so, this might help account for the minimal lung destruction seen in most pneumococcal pneumonias. Moreover, it seemed important to test this question because non-covalent bonding of the inhibitor to the enzyme might permit the latter to dissociate under in vivo conditions. In this event, the physiological relevance of the inhibitor would be open to question. A model of acute lung injury was chosen to test the in_ vivo effectiveness of pneumococcal inhibitor. The sequestration of circulating 125 i-albumin in. the lungs, lung wet weight and lung hemorrhage caused by intratracheal protease instillation was measured. Both native pneumococcal inhibitor and its active tryptic peptides were tested for protection against these effects of the protease. Whole neutrophil granule extracts were employed as the source of leukocyte elastase in order to include other neutrophil granule constituents in the model. Because these are also secreted into pneumonic exudates, along with elastase, it was felt that whole granule extracts would more closely duplicate na tural ly-occur ing events. IN VIVO TEST METHOPS Human neutrophil granule extract was prepared according to the method of Feinstein, G. and A. Janoff : A rapid method of purification of human granulocyte cationic neutral proteases:purif ication and further characte ization of human granulocyte elastase Biochem. Biophys. Acta 403:493-505(1975). The pneumococcal inhibitor used was the large molecular weight fraction derived by Sepharose-2B chroma tography of bacterial extracts, the latter having been prepared by lysis of washed cells in deoxycholic acid. Gel-elec trophoresis data and previously discussed experiments had shown that this fraction contained the active 140,000 dalton elastase-inhibi tor . Other trials had shown that this pneumococcal fraction was not toxic to mice (data not shown), nor did it produce acute lung injury by itself (see Table II). In addition to the intact inhibitor, active tryp tic-fragments of the pneumococcal agent were also tested. These were prepared by 24 hour incubation of the large molecular weight fraction at room temperature with 2% (wt:wt) TPCK- trypsin , followed by inactivation of trypsin with N- tosy 1-lysy 1-chlorome thy 1 ketone and extensive dialysis to remove the unbound inactivator. Male, Swiss-Webster mice weighing between 25 and 30 g were anesthetized with intraperi toneal ketamine (Ketaset, Bristol Laboratories, Syracuse, NY). Each animal then received an intravenous injection of -*- 5ι_ι aDe ιι e cl bovine serum albumin (dose = 1.3 mg , specific activity = 0.314 uCi/ g). Immediately thereafter, the mice were treated with mixtures of granule extract and inhibitors (native or tryptic peptides). These mixtures were delivered to the lungs by a transoral technique, as described by Janoff, A. and R. Pea ing : Pre ven tion of ela s ta se -induced experimental emphysema by oral administration of a synthetic elastase inhibitor Am. Rev. Respir. Pis. 121:1025-1029 (1980). Control mice received granule extract mixed with TRIS buffer used to dissolve the inhibitor, or inhibitor mixed with PBS buffer used to dissolve granule proteins, or a mixture of the two buffers. Additional control groups were treated with granule extract mixed with goat immunoglobulins to serve as a source of nonspecific protein added at the same concentration as the active pneumococcal agent. In some experiments, the granule extract and inhibitor were premixed and incubated at 37° for 10 minutes before instillation, while in other trials the reagents were mixed at room temperature and instilled into mice immediately thereafter (0 min groups). Lungs were removed at time of death of the animal or after 1 hr in survivors, and acute lung injury was assessed using ' three methods: (a) total absorbance (at 412 nm) of hemoglobin in clarified supernates of 2.5% (wt:vol) lung homogenates prepared by grinding the tissues in distilled water; (b) cpm of 1 2 i _ a i b um i n in the lung, expressed as a percentage of injected dose and normalized either to lung dry weight or to body weight; and (c) lung wet wt/body wt ratios. Bovine serum albumin (Sigma Chem. Co., St. Louis, MO) was radiolabelled with 125 i using Bol ton-Hunter reagent (New England Nuclear, Boston, MA) according to the manufacturer's directions. Protein determinations were done according to the method of Lowry with bovine serum albumin as a standard. The statistical significance of difference between means for various experimental and treated groups was determined using Student's T-test. IN VIVO TEST RESULTS Four separate experiments involving over 100 mice were done, and the results are hereinafter summarized in Tables II and III. In all cases, 50 ug of granule extract protein and 200 ug of whole pneumococcal inhibitor protein or tryptic peptides were used. When goat immunoglobulin was used as a control protein, 200 ug were similarly employed. As can be seen from the data in Table II, the native pneumococcal inhibitor gave significant protection against acute lung injury caused by neutrophil granule extract, when the inhibitor and extract were preincubated for 10 minutes at 37°C before instilling the mixture into the mouse lungs. Protection was evident using all three parame ters of acute lung in jury_ ( lung wet w t , 125 j _ a ibumin sequestr tion, and hemoglobin absorbance of tissue extracts. TABLE II Experiments With Intact Pneumococcal Inhibitor Pre-Incubated With Granule Extract For 10 Minutes Treat¬ 12 5 I x -al ,.bum in Lung Total calculated ment (% dose re¬ wet wt absorbance: 2.5% (no. covered in ( mg ) /Body lung homogenate mice ) lung/g body wt (g) supernate at wt) 412 nm buffer 0.035 0.0058 nd control 0.010 0.0003 (5) granule 0.066 a 0.0081 a 29.4 extract 0.022 0.0014 0.7 alone (9) granule 0.045 b ' d 0.0066 b 17.5° extract 0.014 0.0009 0.9 + inhibitc >r (11) inhibitor 0.035 d 0.0059 d nd alone 0.007 0.0001 (5) (a) differs significantly from buffer control (pC 0.01) (b) differs significantly from granule extract alone (p ^ 0.05) (c) differs significantly from granule extract alone (p .0.01) (d) not significantly different from buffer control Table III summarizes the results of experiments using active tryptic peptides of the pneumococcal inhibitor. These were effective whether preincubation was for 10 minutes at 37° or for zero minutes at 22°, although greater protection was observed after longer preincubation intervals. Incubation of neutrophil granule extract with nonspecific goat immunoglogulins did not alter the ability of granule components to produce lung injury. TABLE III Experiments With Tryptic Peptides of Pneumococcal Inhibitor Pre-Incuba ted With Granule Extracts For 10 Minutes (in Panel A) or 0 minutes (in Panel B) 12 125 ∑ _ Treat- x T-albumin albumin Lung Wet A 412nm ment (% dose/ (% dose/ wt/body (no) body wt) lung dry wt) wt (A) buffer 0.030 28.8 0.0068 15.4 control + ■ 0.007 + 5.2 10.0006 + 3.2 (8) granule 0.11]? 77.8 0.009 30.7 a extract + 0.040 t 18.0 ± 0.0019 -8.1 (16) granule O.lOf 181.3 a o.oio 30.7 a extract + _t0.038 ± 60.4 t0.0017 6.8 goat glob¬ ulins (8) granule 0.04° 40.9 b 0.0080° 17. &' d extract + + 0.012 9.9 -0.0012 + 5.5 peDtides (16) (B. buffer 0.036 36.8 0.0055 22.7 control + 0.019 +• 12.1 ±0.0005 + 4.7 (8) granule 0.077 a 61.0 a 0.0090 a 37.0 a extract + 0.021 116.0 o.0010 + ■ 4.4 goat glob¬ ulins (8) granule 0.055 c 56.2 0.0075 c 30.0 C extract +0.018 + 16.1 ±0.0014 + 6.6 peptides (8)~ (a) differs significantly from buffer control ( p___ 0.01) (b) differs significantly from granule extract and/or granule extract + globulins (p ___ 0.001 (c) differs significantly from granule extract and/or granule extract + globulins (p_/_ 0.05) (d) not significantly different from buffer control Figure 12 shows the typical histologic appearance of buffer-treated mouse lung (panel A) vs_ lung tissue from animals receiving granule extract that had either been pre-incubated for 10 minutes at 37°C with goat immunoglobulins as a protein control (Panels B and C), or with inhibi tor-peptides (panel P). It is evident that the human neutrophil granule extract produced severe, acute lung injury in the mouse. It was primarily characterized by edema and hemorrhage. These responses were significantly reduced by the "pneumococcal agent. PISCUSSION OF IN VIVO TEST RESULTS The experiments herein described show that an inhibitor of neutrophil elastase obtained from pneumococci protected mouse lung against acute tissue injury produced by human neutrophil granule extracts. The degree of protection was related to the length of preincubation of granule extract with pneumococcal agent, and protection was enhanced when lower molecular weight tryptic-peptides, derived from the native inhibitor were used. Protection was not due to nonspecific effects of the added pneumococcal protection since equal amounts of goat immunoglobulins used in place of the pneumococcal inhibitor did not prevent injury. Although the pneumococcal agent specifically inhibits neutrophil elastase and is inactive against other proteases (trypsin, pancreatic elastase). unf ractionated neutrophil granule extracts were used rather than pure neutrophil elastase in the present s tudy . This seemed appropria te , since neutrophil-mediated acute lung injury may also involve other granule components such as: (1) cathepsin G, see Boudier , C. , P. Laurent and J.G. Bieth: Leukopro te inases and pulmonary emphysema : cathepsin G and other chymo trypsin-like proteinases enhance the elastolytic activity of elastase on lung elastin. Adv. Expt. Med. 167:313-317 (1980), (2) collagenase, (see, Lazarus, G. S. , J. R. Paniels, R. S. Brown, H. A. Bladen and H. M. Fullmer degradation of collagen by a human granulocyte collagenolytic system, J_. Clin . Invest. 47:2622-2628 (1968)), and (3) oxidants formed by the action of myeloperoxidases (another granule constituent), (see Clark, R. A.:Toxic effects of mye l ope ro x i da se an d H2O 2 se cre ted by neutrophils exposed to a soluble stimulus, Clin . Res . 27:209A (abstr) (1980) ; Carp, H. and A. Janoff: Potential mediator of inflammation. Phagocyte-derived oxidants suppress the elastase-inhibi tory- capacity of alpha 1-proteinase inhibitor in vi tro , J_. Clin . Invest* 66:987-995 (1980) and Greenwald, R. A. and W. W. Moy:Ξffect of oxygen-derived free radicals on hyaluronic acid. Ar thr i tis R he u m . 23:455-463 (1980)) . Under these conditions, the pneumococcal agent still gave significant protection. Previous chemical studies showed that the ' pneumococcal agent does not form a covalent complex with the active-site serine residue in neutrophil elastase. However, elastase-inhibi tor complexes appear to be stabilized by hydrophobic associations and inhibition is non-competitive. Perhaps for this reason complexes can form in_ vivo even in the presence of lung substrates for neutrophil elastase, and this could explain the partial protection observed when pre pa ra t ion s of gra nule e x tra c t and inhibi tory-peptides were instilled immediately after mixing. Thus, by virtue of their ability to specifically inhibit human leukocyte elastase, the above described inhibitors as well as their active site fragments can be used in treating a wide variety of diseases which produce elastolytic effects as part of their disease processes. Such diseases would include, but not be limited to, pulmonary emphysema, pneumonia (viral, bacterial, fungal, protozoan), abscesses, chronic bronchitis, hyaline membrane disease, adult respiratory distress syndrome (ARPS) , vasculitis, glomerulonephritis, bronchiectasis, interstitial lung disease, atherosclerosis, arthritis, psoriasis, cystic fibrosis, pemphigus, fasciitis and cellulitis. Obviously, treatment of such diseases by these inhibitors can be by any convenient delivery method, e.g. , inhalation, subcutaneous, intramuscular or intravenous injection, oral administration, salves, ointments or suppositories. It is also obvious that the inhibitors may be admixed with pharmaceutically acceptable carriers and/or other pharmaceutically compatible medicaments. Likewise, while the human leukocyte elastase inhibitors, the herein described novel methods for their enrichment and purificaiton as well as the herein described methods for their use have been described in great detail, it will be obvious to those skilled in this art that many modifications and/or extensions of these details can be made without departing from the scope and spirit of the teachings of this patent disclosure. Use of these inhibitors in the treatment of elastolytic diseases in humans would be an example of one obvious extension of these teachings.

Claims: WHAT IS CLAIMEP IS: 1. Proteinaceous human leukocyte elastase inhibitors derived from pyogenic bacteria and specific to human leukocyte elastase. 2. The proteinaceous human leukocyte elastase inhibitors of claim 1 wherein the pyogenic bacteria are sele-cted from the group consisting of S treptococcus pneumoniae , Klebsiella pneumoniae and S taphy lococcus aureus. 3. The proteinaceous human leukocyte elastase inhibitors of claim 1 wherein the pyogenic bacteria are Streptococcus pneumoniae. 4. Proteinaceous human leukocyte elastase inhibitors, derived from pyogenic bacteria, specific to human leukocyte elastase and further characterized as being non-secretory, high molecular weight proteins capable of remaining active in the presence of 0.15 M NaCl, acting in a non-competitive manner with human leukocyte elastase, and consisting of subunits stabilized by hydrophobic associations and/or disulfide bonds. 5. The human leukocyte elastase inhibitors of claim 4 wherein the inhibitors have an apparent molecular weight of about 140,000 daltons. 6. The proteinaceous human leukocyte elastase inhibitors of claim 4 wherein the pyogenic bacteria are pneumococci selected from the group consisting of S treptococcus pneumoniae , Klebsiella pneumoniae and S taph lococcus aureus. 7. The proteinaceous human leukocyte elastase inhibitors of claim 4 wherein the pyogenic bacteria are Streptococcus pneumoniae. 8. Proteinaceous human leukocyte elastase inhibitors which are fragments of human leukocyte elastase inhibitors derived from pyogenic bacteria, said fragments having active sites which are active against human leukocyte elastase and are further characterized as being derived from non-secretory, large molecular weight proteins capable of remaining active in the presence of 0.15 M NaCl, acting in a non-competitive manner with human leukocyte elastase, and consisting of subunits stabilized by hydrophobic associations and/or disulfide bonds. 9. The fragments of claim 8 wherein said fragments have an apparent molecular weight of less than about 140,000 daltons. 10. The proteinaceous human leukocyte elastase inhibitor fragments of claim 8 which are derived from the pyogenic bacteria selected from the group consisting of Streptococcus pneumon iae , Klebsiella pneumoniae and Staphylococcus aureus. 11. The proteinaceous human leukocyte elastase inhibitor fragments of claim 8 which are derived from S treptococcus pneumoniae. 12. A method of treating diseases which have elastolysis as part of their disease processes comprising administering an effective amount of human leukocyte elastase inhibitors which are derived from pyogenic bacteria. 13. The method of claim 12 wherein the human leukocyte elastase inhibitors are derived from pyogenic bacteria selected from the group consisting of Streptococcus pneumoniae , Klebsiella pneumoniae and Staphylococcus aureus. 14. The method of claim 12 wherein " the human leukocyte elastase inhibitors are derived from Streptococcus pneumoniae . 15. The method of claim 12 wherein the diseases having elastolysis as part of their disease processes are selected from the group consisting of pulmonary emphysema, pneumonia (viral, bacterial, fungal, protozoan), abscesses, chronic bronchitis, hyaline membrane disease, adult respiratory distress syndrome ( ARPS ) , vasculi tis , glomerulonephritis , b r o n c h i e c t a s i s , interstitial lung disease , atherosclerosis, arthritis, psoriasis, cystic fibrσsis, pemphigus, fasciitis and cellulitis. 16. A method of purifying human leukocyte elastase inhibitors derived from pyogenic bacteria comprising: (1) providing an affinity column by binding elastin to an insoluble support material; (2) binding human leukocyte elastase to the elastin so that a portion of the active sites of the human leukocyte elastase are available for binding human leukocyte elastase inhibitors; (3) introducing lysates of the pyogenic bacteria to the affinity column so that neutrophil elastase inhibitors in the lysates of the pyogenic bacteria become bound to the active site of the neutrophil elastase; (4) eluting the affinity column with a first elution medium which is capable of separating contaminating substances from the human leukocyte elastase inhibitors; and (5) eluting the affinity column with a second elution medium which is capable of separating the human leukocyte elastase inhibitors from the human leukocyte elastase. 17. The method of claim 16 wherein the insoluble support material is sepharose. 18. The method of claim 16 wherein the second elution medium has a salt concentration higher than that of the first elution medium. 19. A method of enriching human leukocyte elastase inhibitors derived from pyogenic bacteria comprising: (1) lysozyme digestion of pyogenic bacteria suspended in a nonionic, hypertonic medium such that bacteria cell wall components are solubilized without disrupting bacteria cell membranes and thereby minimizing release of intracellular substances; and (2) segregating extraneous proteins from the human leukocyte elastase inhibitors. 20. The method of claim 19 wherein the nonionic, hypertonic medium is sucrose having a concentration of about 0.6M. 21. The method of claim 19 wherein the segregating of extraneous proteins from the human leukocyte elastase inhibitor is by chroma tography of a cell supernate on Sepharose 2B.

Inventors: JANOFF AARON, SANDHAUS ROBERT A, VERED MICHA, JANOFF, AARON,, SANDHAUS, ROBERT, A., VERED, MICHA
Applicants: CORTECH INC, CORTECH, INC.
Application year: 1985
Main classification: C07K15
IPC codes: C07K14