EP0182906A1 - Aus pyogenen bakterien hergestellte menschliche leukozyt-elastase-inhibitoren und deren reinigungsverfahren - Google Patents

Aus pyogenen bakterien hergestellte menschliche leukozyt-elastase-inhibitoren und deren reinigungsverfahren

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Publication number
EP0182906A1
EP0182906A1 EP19850903486 EP85903486A EP0182906A1 EP 0182906 A1 EP0182906 A1 EP 0182906A1 EP 19850903486 EP19850903486 EP 19850903486 EP 85903486 A EP85903486 A EP 85903486A EP 0182906 A1 EP0182906 A1 EP 0182906A1
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Prior art keywords
human leukocyte
leukocyte elastase
inhibitor
inhibitors
elastase
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EP19850903486
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English (en)
French (fr)
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Aaron Janoff
Robert A. Sandhaus
Micha Vered
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Cortech Inc
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Cortech Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors

Definitions

  • 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.
  • Neutrophil proteases have been implicated in several human diseases since the turn of the century (Metchnikoff, E. L' Immunite' dans le Maladies Infectmaschines, 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).
  • HLE human leukocyte elastase
  • 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. Dis. 115:461-478, (1977) and Snider, G.L. Med. Clin. North Am.
  • the major tissue regulator of HLE in humans is alpha l-proteinase inhibitor or alpha l-antitrypsin.
  • the alpha l-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)).
  • AlPi Cholsson, K.:Proteases and Biological Control
  • elastin is destroyed.
  • a genetic deficiency of AlPi is associated with markedly increased risk of developing pulmonary emphysema (Eriksson, S. Acta. Med. Seand.
  • 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. J. Antibiot. 26:787-789 (1973) and Okura, A. et al J. Antibiot. 28:337-339 (1975)).
  • HLE-inhibitors in pyogenic bacteria, e.g. Streptococcus pneumoniae, Staphylococcus aureus and Klebsiella pneumoniae (pneumococci).
  • 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.
  • the pneumococcal inhibitors are the easiest to obtain in large yields. Therefore, they are the ones most extensively characterized.
  • 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 bronchialmucous proteinase inhibitor.
  • HLE inhibitors (1) designed on the basis of the known peptide-bond specifications of HLE such as the peptide chloromethylketones 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.
  • 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.
  • 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 deoxycholate, centrifugation and extensive dialysis of the supernate. Preincubation was carried out in 0.01M 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 ( ⁇ ) line indicates a 30 minute preincubation; the ( ⁇ ) line indicates a 60 minute preincubation; 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 chromatography 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 vs neutrophil elastase.
  • 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 - 1 vs 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/ml of pure human neutrophil elastase and 135 ug/ml of purified salt-sensitive inhibitor or of whole pneumococcal extract (crude salt-resistant inhibitor).
  • the (O) line indicates enzyme alone; the ( ⁇ ) line indicates enzyme plus salt-sensitive inhibitor purified from type III pneumococci by preparative polyacrylamide gel electrophoresis; the ( ⁇ ) 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.
  • the 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.
  • the salt-resistant agents from types II and II I pneumococci inhibi ted non-competitively as can be observed from the different y-intercept and similar x-intercept values.
  • Figure 4 shows the chromatography of pneumococcal extract on Sephacryl S-300.
  • the solid line (-------- ) indicates protein concentration 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 gel electrophoresis of crude pneumococcal proteins and peak I protein obtained by molecular-sieve chromatography (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).
  • Figure 7 indicates the double-immunodiffusion 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 unprecipitated protein, dried and stained with Coomassie blue.
  • Figure 9 depicts a proposed lysis mechanism of pneumococci cell wall material.
  • Figure 10 shows chromatographs of pneumococcal lysate 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 37oC with 200 ug nonspecific goat immunoglobulin (protein control) and
  • panel D represents 50 ug granule extract incubated as above with 200 ug pneumococcal inhibitor-peptides.
  • Significant hemorrhage can be noted in panels B and C, and a decrease in this manifestation of acute lung injury can be noted in panel D.
  • Goat immunoglobulins alone did not produce acute injury.
  • Streptococcus pneumoniae type I ATCC 9163
  • type II ATCC 11733
  • type III ATCC 10813)
  • Staphylococcus aureas ATCC 25923
  • 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.
  • bovine trypsin utilized (freed of contaminating chymotrypsin activity by treatment with tosyl-phenylalanyl-chloromethyl ketone (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 proteases:purification and further characterization of human granulocyte elastase. Biochem. Biophys. Acta 403:493-505 (1975).
  • Streptococcus pneumoniae (Strep PN) was obtained from blood cultures of patients with active pneumococcal pneumonia from the University of
  • 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.
  • pneumococcal extracts were chromatographed on Sephacryl S-300, superfine, (Pharmacia Fine Chemicals, Uppsala, Sweden).
  • the fractionation range (Mr) of this gel is 1x10 4 - 1.5x10 6 for 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.
  • 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.
  • Extraneous proteins can then be segregated away, for example, by passing the supernate over a Sepharose 2B chroma tography column (see for example Figure 5, column B) or further purified as described below.
  • 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.
  • 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
  • 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
  • a salt medium such as a NaCl medium having a salt concentration of from about 0.01 to about 1 molar
  • a buffer 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.
  • 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°.
  • Radioactive labelling of pure neutrophil elastase was carried out according to the method of McFarlane A.S.:In vivo behavior of I 131 -fibrogen, J. Clin. Inves . 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-succinyl-(L-alanyl)3-p-nitroanilide to the reaction mixture. Under these conditions, catalytic activity of neutrophil elastase was unaffected by the labelling procedure.
  • the 125 I (as sodium iodide) was from Amersham (Arlington Heights, IL) 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.
  • 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 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.
  • Neutrophil elastase alone, or Sephacryl S-300 fractions containing complexes of elastase and pneumococcal inhibi tor were tested for immunoreactivity against a monospecific rabbit anti-human neutrophil elastase antiserum by double-immunodiffusion 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 Biochem. Biophys. Acta 403:493-505 (1975).
  • 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 deoxycholate-induced lysis. Sodium deoxycholate 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.
  • the NaCl-sensitive ( ionic-interaction-dependent) inhibitor was first isolated. Chromatography 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.
  • Figure 4 shows the chroma tographic pattern obtained when crude pneumococcal extract (prepared by lysis of S. pneumoniae III in deoxycholate) 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 (inhibitor-II) appeared with protein eluting soon after the void volume (peak I in the figure) and therefore corresponded to material of high molecular weight.
  • the Inhibitor-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).
  • Figure 5 shows the results of SDS-gradient polyacrylamide gel electrophoresis of crude pneumococcal extract and of peak I protein obtained by molecular-sieve chromatography (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).
  • inhibitor-II was not active against trypsin or pancreatic elastase. Therefore, a trypsin digestion experiment was performed to confirm that inhibitor-II was proteinaceous 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, electrophorectic 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.
  • the peptide chloromethyIketone was labelled with 14 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 14 C- chloromethylketone. 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 ) .
  • 14 C-labelled, neutrophil elastase and inactive, 14 C-labelled enzyme dialysed free of unbound inactivator, substrate, and p-nitroaniline 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.
  • elastase was reacted with 3 H-labelled diisopropyl fluorophosphate 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.
  • 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-necrotizing) were not more inhibitory than S. pneumoniae type III, which is capable of causing tissue destruction.
  • 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)
  • K. pn. Klebsiella pneumoniae
  • S. aureus Staphylococcus aureus
  • 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.
  • this lysate is chroma tographed on Sepharose
  • 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. Immun. 30:890-893, (1980) and OMP derived from gonococci.
  • MP membrane protein
  • peak I of the Sepharose 2B chromatogram is also enriched in glycan fragments released during deoxycholate-induced lysis (Fig 9).
  • 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 naturally-occuring events.
  • 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:purification and further characterization 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 chromatography of bacterial extracts, the latter having been prepared by lysis of washed cells in deoxycholic acid. Gel-electrophoresis data and previously discussed experiments had shown that this fraction contained the active 140,000 dalton elastase-inhibitor.
  • 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.
  • 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 125 I -aibumin 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 Bolton-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.
  • 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.
  • 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 thepneumococcal agent.
  • 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.
  • 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.
  • 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.
  • 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.
  • pulmonary emphysema viral, bacterial, fungal, protozoan
  • abscesses chronic bronchitis
  • hyaline membrane disease hyaline membrane disease
  • ARPS adult respiratory distress syndrome
  • vasculitis vasculitis
  • 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.
  • 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.

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EP19850903486 1984-06-14 1985-06-12 Aus pyogenen bakterien hergestellte menschliche leukozyt-elastase-inhibitoren und deren reinigungsverfahren Withdrawn EP0182906A1 (de)

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IE911697A1 (en) * 1990-05-24 1991-12-04 Zeneca Ltd Therapeutic agent
US5162348A (en) * 1990-05-24 1992-11-10 Imperial Chemical Industries Plc Treatment of cystic fibrosis
ES2129401T3 (es) * 1990-05-24 1999-06-16 Zeneca Ltd Uso de un compuesto terapeutico para el tratamiento de bronquitis.
GB9403819D0 (en) * 1994-02-28 1994-04-20 Univ Leeds Control of parasites
FI963753L (fi) * 1994-03-23 1996-09-20 Tokyo Tanabe Co Uusi lääke hengitysteiden virustaudin hoitamiseksi
CA2387199A1 (en) * 1999-10-12 2001-04-19 Fujisawa Pharmaceutical Co., Ltd. Remedies for intractable wound

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