WO2003082326A1

WO2003082326A1 - Use of aprotinin for treating wounds and inflammation

Info

Publication number: WO2003082326A1
Application number: PCT/EP2002/003579
Authority: WO
Inventors: Lars Bjorck; Heiko Herwald; Artur Schmidtchen
Original assignee: Hansa Medical Research Aktiebolag
Priority date: 2002-03-28
Filing date: 2002-03-28
Publication date: 2003-10-09
Also published as: AU2002257740A1

Abstract

Aprotinin or a variant, derivative, analogue or fragment thereof is provided for use in the treatment of a disease or condition associated with secretion of neutrophil derived substances. In particular, the conditions to be treated are ulcers or wounds such as chronic ulcers or wounds.

Description

USE OF APROTININ FOR TREATING WOUNDS AND INFLAMMATION

Field of the Invention

This invention relates to the treatment of diseases or conditions associated with secretion of neutrophil derived substances.

Background of the Invention

Polymorphonuclear leukocytes (PMN) are the most abundant leukocytes. They comprise about two-thirds of peripheral blood leukocytes, and have an important role in early host defense against invading microorganisms as they are the first cells encountered in the inflamed tissue area. The recruitment of PMNs from the bloodstream to a site of infection or inflammation involves their recognition of inflammatory mediators, their binding to adhesion molecules of the vascular endothelium, and their migration across the endothelial barrier. These processes involve a re-organization of PMNs including the up-regulation of adhesion receptors on the plasma membrane, the fusion of intracellular compartments/vesicles, and the secretion of bactericidal substances. The regulation of such events is dependent on a sophisticated procedure that includes the mobilization of neutrophilic proteins stored in different vesicles and granules. Chronic ulcers are a major health problem and an increasing burden to health care providers. At the molecular level, various alterations have been described in these ulcers. Initial pathogenetic steps involve activation of endothelial cells by up- regulation of various cell adhesion molecules (such as I-CAM) on endothelial cells, and their corresponding ligands on leukocytes, thus leading to extravasation of inflammatory cells into the wound area. The high influx of leukocytes and subsequent release of proteases, such as neutrophil elastase and gelatinases leads to high proteolyis in the surroundings of chronic ulcers. The resulting degradation of matrix molecules found in granulation tissue and in the basal lamina, such as fibronectin, laminin, and various collagens, hinders the formation of proper contacts between integrins of skin epithelial cells (keratinocytes) and the underlying matrices in these ulcers. Furthermore, various growth factors, such as plateled derived growth factor, are degraded, thus retarding cell-proliferation. These findings, together with the observation that keratinocytes display an altered, non-migratory, phenotype in venous ulcers, leads to a failure of re-epithelialization.

It is known that all chronic ulcers are constantly colonized or infected by various bacteria such as P. aeruginosa, E. faecalis and P. mirabilis. During recent years, clinical and experimental data support the view that these, and other pathogens, may contribute to the non-healing state of chronic ulcers. During infection, these pathogens, which are frequently found in chronic ulcers (P. aeruginosa; 20-30%>, E. faecalis 80%, P. mirabilis 15%), use a combination of virulence strategies. For instance, release of various proteinases, may modulate host responses involving kallikreins, coagulation factors, complement, cytokines, and antiproteinases.

Summary of the Invention According to the present invention, there is provided the use of aprotinin or a variant, derivative, analogue or fragment thereof in the manufacture of a medicament for use in the treatment of a disease or condition associated with secretion of neutrophil derived substances.

The invention also provides: - a method for the treatment of a patient who has suffered or is suffering from a disease or condition associated with secretion of neutrophil derived substances, which method comprises the step of administering to the patient a therapeutically effective amount of aprotinin or a variant, derivative, analogue or fragment thereof; - an agent for the treatment of a disease or condition associated with secretion of neutrophil derived substances comprising aprotinin or a variant, derivative, analogue or fragment thereof; and aprotinin or a variant, derivative, analogue or fragment thereof for use in the treatment of a disease or condition associated with secretion of neutrophil derived substances. Description of the figures

Figure 1 shows kinetics of changes in EC monolayer permeability in response to stimulation with rHBP. The permeability-increasing activity of HBP mani-fested as a dose-dependent decrease in transendothelial electrical resistance; rHBP 25 (O), 50 (©) and 75 (•) μg/ml, measured on left γ-axis. It also mamfested as an increase in protein flux across EC monolayer (albumin clearance) as illustrated for rHBP 75 μg/ml (A), measured on right γ-axis. Data are mean ± s.d,; «=6-8.

Figure 2 shows antibodies against HBP and dextran sulfate (DxSO₄) inhibit HPB- and PMN-evoked increase in EC permeability. Maximum change in transendothelial electrical resistance in response to stimulation with rHBP (H)₅ CD 18 cross-linking-induced PMN secretion E2 or chemoattractant (LTB₄)-induced PMN activation (D) in the absence (control) and the presence of anti-HPB or dextran sulphate. Similar neutralizing capacity of anti-HBP and dextran sulphate was found when fMLP instead of LTP₄ was used as a chemoattractant (data not shown). Data are mean ± s.d,; n=6 in each group.

Figure 3 involvement of neutrophil-derived granule proteins in PMN-evoked increase in EC permeability. Maximum change in transendothelial electrical resistance was measured after stimulation with intact CD 18 cross-linking-induced PMN secretion and PMN secretion depleted of HBP, elastase, and cathepsin G, respectively. The permeability-increasing activity of the PMN-derived secretion is lost after removal of HPB, whereas the activity remains intact after removal of elastase and/or cathepsin G. Data are mean ± s.d,; n=6 in each group.

Detailed description of the Invention

The present invention provides methods for treating diseases or conditions associated with secretion of neutrophil derived substances. These diseases or conditions may be characterised by excessive release of neutrophil derived substances. The neutrophil derived substance may be a neutrophilic protein, preferably heparin binding protein (HBP), also termed azurocidin or CAP37. HBP belongs to a family of serine proteases with an overall structure homologous to the elastase fold. However, the protein lacks enzymatic activity due to exchange of two essential amino acids in the catalytic triad. The secretion of heparin binding protein from neutrophils is preferably associated with acute or chronic bacterial infection. The treatment may be for acute conditions such as cutaneous vasculitis or

Sweets syndrome, or for chronic diseases such as psoriasis or psoriasis pustulosa. Preferably, chronic conditions are treated. The condition to be treated is preferably a skin condition or disease, for example a skin ulcer or wound. In particular, the invention is directed to the treatment of chronic ulcers and chronic wounds, and in particular skin ulcers or wounds. Chronic ulcers have various underlying causes, such as venous or arterial insufficiency, diabetes, vasculitis, or excessive pressure. The conditions to be treated in accordance with the present invention include those associated with bacterial infections, in particular bacterially infected ulcers and wounds such as skin ulcers and wounds. Venous leg ulcers (the major group of ulcers) represent a condition characterised by unregulated and excessive inflammation. The treatment in accordance with the present invention may be to reduce inflammatory reactions or to promote wound healing or reepithelialization in these tissues.

According to the present invention, the diseases and conditions associated with secretion of a neutrophil derived substance may be treated by administration of an inhibitor of the neutrophil derived substance. The inhibitor is preferably a HBP inhibitor. The HBP inhibitor may be an antagonist of HBP which blocks or interferes with the action of HBP. Alternatively, the HBP inhibitor may prevent the release of HBP from neutrophils. In particular, the inhibitor may block bacterially induced release of HBP from neutrophils.

The HBP inhibitor may be a serine protease inhibitor, preferably a Kunitz type protease inhibitor. More preferably, the HBP inhibitor is aprotinin or a variant, derivative, analogue or fragment thereof which maintains the ability to inhibit HBP. Aprotinin is a basic 6,500 Da protein present in several bovine organs and tissues, such as the lymph nodes, pancreas, lungs, parotid gland, spleen and liver. It is a single-chain polypeptide of 58 amino acid residues with the following amino acid sequence:

Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys Lys Ala Arg He

He Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gin Thr Phe Nal Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala Glu Asp Cys Met Arg

Thr Cys Gly Gly Ala

Certain aprotinin variants, derivatives, analogues or fragment thereof are known, for example aprotinin analogues and derivatives wherein Lys(15) is replaced with Gly, Ala, Nal, Leu, He, Met, Arg, L-a-butyric acid, L-norvaline, L-norleucine, dehydroalanine or L-homoserine. Lys(l 5) may also be replaced with Arg, Val, He, Leu, Phe, Gly, Ser, Trp, Tyr or Ala. Met(52) may be replaced with Glu, Nal, Leu, Thr or Ser. Alternatively, one or more of the amino acids in position 15, 16, 17, 18, 34, 39 and 52 may be replaced with another amino acid residue. Aprotinin or aprotinin analogues may be produced in yeast, in particular analogues lacking one or two amino acid residues at the Ν-terminal end, or in which Lys(41) and/or Arg(42) is replaced with another amino acid residue, in particular Ser. One or more of the amino acids in position 1, 2, 12-19, 38, 41 and 42 may be deleted or replaced with another amino acid residue. The amino acid substitutions are mainly found in the protease-binding region of the aprotinin molecule with a view to changing the protease-inhibition profile of aprotinin except for the substitution of Met(52) and the substitution of Lys(41) and/or Arg(42) which are carried out to facilitate the production of aprotinin in E. coli and yeast, respectively. Other inhibitors include anti HBP antibodies, dextran sulphate and other anionic compounds.

The invention also may utilise agents which inhibit HBP, and in particular which inhibit HBP release from neutrophils when in contact with endothelial cells or bacteria. The present invention also relates to the finding that release of HBP from endothelial cells leads to changes in the endothelial cell permeability. This activity may also be used as a basis for identifying novel HBP antagonists.

The present invention also provides assays for identifying HBP inhibitors. A preferred assay comprises:

(a) providing a sample of neutrophils; (b) contacting the neutrophils with a confluent monolayer of endothelial cells or with bacteria in the presence of a test substance; and

(c) monitoring for release and/or the effect of HBP.

The invention also provides an assay for identifying an antagonist of HBP, which comprises:

(a) providing a sample comprising a confluent monolayer of endothelial cells;

(b) contacting the sample with HBP and a test agent;

(c) measuring the change, if any, in transendothelial electrical resistance in the sample in response to HBP and the test agent; and comparing the change in electrical resistance in the test sample to the change in a control sample without a test agent, wherein a reduction in the percentage change in electrical resistance indicates that the test agent is a HBP antagonist.

In accordance with the assays of the present invention, the presence of a test substance on the ability of endothelial cells or bacteria to stimulate release of HBP from neutrophils may be monitored. This may be carried out, for example, by measuring levels of HBP in the presence and absence of a test substance when neutrophils are contacted with endothelial cells or with bacteria. Alternatively, the methods may involve monitoring for downstream effects of HBP, such as its effects on endothelial cell monolayers.

In another aspect of the present invention, the assay of the present invention is used to identify antagonists of HBP in which confluent layers of endothelial cells are provided and contacted with HBP in the presence of a test substance. Alterations in the endothelial cell layer are then monitored to determine the effect of the test substance on HBP activity. Such assays may include monitoring for changes in the endothelial cell monolayer structure that lead to increased permeability, for example, by directly visualising the endothelial cell monolayer or alternatively by monitoring for changes such as electrical resistance across the endothelial cell monolayer.

Inhibitors and antagonists identified in accordance with the present invention may then be used in the methods of treatment as described above in more detail for aprotinin. In preferred aspects of the invention, agents which inhibit release of HBP from neutrophils when in the presence of bacteria may be used to treat chronic bacterial infections such as chronic ulcers and wounds.

Aprotinin and other inhibitors of the invention may be administered to humans in various manners such as orally, intracranially, intravenously, intramuscularly, intraperitoneally, intra-nasally, intradermally, and subcutaneously. The preferred manner of administration is topical application. The particular mode of administration and dosage regimen will be selected by the attending physician, taking into account a number of factors including the age, weight and condition of the patient. The pharmaceutical compositions that contain aprotinin and other inhibitors of the invention as an active principal will normally be formulated with an appropriate pharmaceutically acceptable carrier or diluent depending upon the particular mode of administration being used. For instance, parenteral formulations are usually injectable fluids that use pharmaceutically and physiologically acceptable fluids such as physiological saline, balanced salt solutions, or the like as a vehicle. Oral formulations, on the other hand, may be solids, e.g. tablets or capsules, or liquid solutions or suspensions. The preferred formulation will be suitable for application to the skin, preferably suitable for application to skin ulcers and wounds. Preferably, the formulation is a cream or gel or other suitable formulation for topical application to the skin.

The amount of aprotinin and other inhibitors of the invention that is given to a patient will depend upon a variety of factors including the condition being treated, the nature of the patient under treatment and the severity of the condition under treatment. The timing of administration should be determined by medical personnel. The inhibitors of the invention may be impregnated in dressings or bandages to be applied to the skin. In particularly preferred aspects of the invention aprotinin or functional analogues thereof are provided in a topical formulation for the treatment of ulcers or wounds, in particular chronic ulcers or wounds such as those having bacterial infection. EXAMPLES Methods

EC monolayers

Bovine aorta ECs were isolated and cultured as previously described. The cells were seeded onto 3-μm pore size polycarbonate filters (Tissue Culture inserts, 10 mm; NUNC, Roskilde, Denmark) at a density of 2 x 10⁵ cells/filter and grown to confluent monolayers. The filter insert was placed in a two-compartment diffusion chamber with the monolayer separating an upper (luminal) and a lower (abluminal) fluid-filled well. Altered transendothelial electrical resistance and efflux of Evans blue dye-conjugated albumin were used as indices of stimulus-induced changes in EC barrier capacity.

CD 18 cross-linking-induced PMN activation and secretion

PMN activation and secretion was induced through antibody cross-linking of the common β chain (CD 18) of β integrins. Human PMN, isolated from leukocyte- rich plasma, were incubated with monoclonal antibody IB4 against CD 18 (3 μg per 1 x 10⁶ PMN) for 30 min, washed to remove unbound antibody, and subjected to CD 18 cross-linking through addition of goat F(ab')2 against mouse IgG (diluted 1:20; Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania). The PMN were sedimented by centrifugation at 300g for 15 min and the post-secretory cell-free supernatant was analyzed for its content of HBP through western-blot analysis. Proteins were separated by SDS-PAGE, and then transferred to nitrocellulose and incubated with rabbit antibody against human HBP or control rabbit IgG (Sigma) diluted in blocking buffer. Immunoreactivity was detected using horseradish peroxidase-conjugated goat antibody against rabbit IgG (Bio-Rad) and visualized by chemiluminescence (ECL, Bio-Rad).

The PMN post-secretory supernatant was incubated under gentle rotation for 15 min with each of anti-HBP, anti-cathepsin G (Athens Research and Technology, Athens, Georgia) and anti-elastase (The Binding Site, Birmingham, UK) antibodies (400 μg IgG/ml supernatant) coupled to Protein A Sepharose (CL-4B, Amersham Pharmacia Biotech, Uppsala, Sweden) for selective removal of each respective protein. The Sepharose beads were spun down, and the efficacy of immunoadsorption was verified with sandwich ELISA.

Stimulation of EC monolayers ECs were stimulated with recombinant human HBP or the PMN post- secretory supernatant. Alternatively, PMN (2 x 10⁶ ) were added to the luminal compartment (PMN to EC ratio = 10:1) and allowed to sediment for 10 min onto the EC monolayer. Activation of PMN was induced either with LTB4 (1 x 10^"8 M) or fMLP (1 x 10^"7 M) added to the abluminal compartment. Changes in transendothelial electrical resistance and albumin efflux were measured as described. EC monolayers were in some experiments pre-treated with the cell-permeant Ca⁺ chelator BAPTA/AM (5 μM; Molecular Probes Europe BV, Leiden, the Netherlands) for 30 min at 37 °C and washed twice. Antibody against HBP (50 μg/ml) or control rabbit IgG (50 μg/ml), or dextran sulfate (1 mg/ml; molecular weight: 500,000; Pharmacia, Uppsala, Sweden) was added to the luminal compartment prior to stimulation in some experiments. In a different set of experiments, rHBP or the mutant variant [G175QJHBP (75 μg/ml) was preincubated with the serine protease inhibitor aprotinin (20 μg/ml, Novo Nordisk, Bagsvaerd, Denmark) for 5 min prior to use.

Intracellular [Ca ] and F-actin distribution

Confluent EC monolayers were incubated for 30 min at 37 °C with the Ca⁺⁺ sensitive fluorescent probe fluo-3/AM (Molecular Probes) according to manufacturer's instructions. Changes in EC [Ca^""^"], following stimulation with rHBP (75 mg/ml) were measured through continuous registration of fluorescence intensity using a laser-scanning confocal imagmg system (Insight Plus; Meridian Instruments, Okemon, Michigan).

Confluent EC monolayers grown on Biomatrix-coated (Biomedical Technologies, Stoughton, Massachusetts) coverslips and incubated with rHBP or vehicle alone for 15 min at 37 °C were used for analysis of EC F-actin distribution. After fixation, the cells were permeabilized and stained for F-actin filaments with FITC-conjugated phalloidin (Sigma). In vivo experiments

The left cheek pouch of anesthetized Syrian golden hamsters was everted and prepared for microscopic observation as described previously (Raud J and Lindbom L, The Handbook of Immunopharmacology (ed. Brain, S.) 127-170, Academic, London, 1994). FITC-conjugated dextran (Mw: 150,000; Sigma) injected i.v. (250 mg/kg body weight) was used as plasma tracer to permit visualization in fluorescent light of changes in vascular macromolecular permeability. HBP (100 μg/ml) in bicarbonate buffer at 37 °C was topically applied to the cheek pouch and responses were recorded on video.

Dipole moment of HBP

The surface electrostatic potentials and dipole moment of HBP and elastase were calculated as described previously (Iversen et al., Nature Structural Biology 4, 265-268, 1997). All calculations were made using GRASP. Charges and atom radii were assigned by use of the full.crg and default.siz files within the GRASP package.

Statistical analysis.

Data are presented as mean ± s.d. Statistical significances were calculated using Mann-Whitney Rank Sum test for independent samples.

Results

Engagement of neutrophil β₂ integrins triggers HBP secretion

We activated human PMN in suspension through antibody-induced cross- linking of the common β₂ integrin subunit CD18. This mode of stimulation mimics adhesion-dependent engagement of β₂ integrins and leads to the secretion of proteins in the molecular range of 25-30 kD, which seem to be involved in PMN-induced alteration in EC permeability. Here, we demonstrate, through western-blot analysis, the presence of HBP in the cell-free postsecretory supernatant obtained after CD 18 cross-linking in suspended PMN. Incubation with control rabbit IgG did not recognise the protein. Recombinant HBP served as a positive control. The release of HBP was confirmed with ELISA (data not shown). Neutrophil-derived HBP increases endothelial permeability

In order to establish the role of HBP in PMN-induced derangement of the endothelial barrier we first examined the ability of HBP to provoke permeability changes in cultured bovine aorta EC monolayers. Administration of recombinant human HBP (rHBP) (25-75 μg/ml) to the luminal side of the EC monolayer elicited a prompt and dose-dependent decrease in transendothelial electrical resistance, reaching lowest value within 15 min after stimulation (30 ± 3%» of control at 75 μg/ml) and then leveling off (Fig. 1). Concomitant with the decrease in electrical resistance was an increase in macromolecular permeability of the monolayer. The activity profile for rHBP on EC permeability was similar to that previously reported for stimulation with cell-free supernatant from PMN activated through CD 18 cross- linking, and verified also in experiments using EC monolayers of human origin (HUVEC; data not shown).

We confirmed the peπneability-increasing activity of rHBP in vivo through intravital microscopy of microcirculation in the hamster cheek pouch. Topical application of rHBP (100 μg/ml) to the exposed tissue provoked prompt macromolecular leakage from postcapillary and small venules. Leaky spots were seen within two minutes and a maximum occurred after 7-10 minutes. After the tissue was washed with buffer, the leakage slowly subsided and disappeared completely within 30 min.

HBP induces Ca -dependent rearrangement of EC cytoskeleton

We used laser-scanning confocal microscopy to determine if HBP alters EC barrier function by inducing Ca^-dependent reorganization of the EC cytoskeleton. Administration of rHBP (75 μg/ml) to confluent EC monolayers (loaded with the Ca -sensitive fluorophore fluo-3/AM) provoked a rapid increase in fluore-scence intensity, indicating a rise in intracellular free [Ca^"1-1"], which peaked within 1-2 min and then gradually declined toward baseline levels. The Ca ^4" mobilization stimulated rearrangement of the EC cytoskeleton as indicated by the formation of actin-stress fibers spanning the cells. The coupling between the Ca⁺⁺ -dependent structural change in EC and impaired EC barrier function was evident because pretreatment with the cell-permeant calcium-chelator BAPTA AM abolished the HBP-evoked EC responses and increase in EC permeability (data not shown).

Anti-HBP attenuates PMN-evoked increases in EC permeability We administered purified antibodies against HBP or non-specific rabbit IgG

(50 μg/ml) to the EC monolayer before stimulation with rHBP (75 μg/ml). Anti- HBP, but not control rabbit IgG, attenuated the HBP-induced decline in electrical resistance (Fig. 2) as well as the increase in macromolecular permeability (net clearance volume was reduced by ~60% from 20.4 ± 3.4 μl in control to 8.6 ± 1.7 μl over a 60-min period).

In order to examine whether activated PMN exert effects on EC barrier function via the release of HBP, we sought to antagonize the cell-mediated response with anti-HBP antibodies. Stimulation of ECs either with cell-free postsecretory supernatant obtained after CD 18 cross-linking in suspended PMN or through transendothelial chemotactic activation of PMN layered on the EC monolayer (1 x 10^"8 M LTB4 or 1 x 10^"7 M fMLP) both evoked changes in EC permeability similar to that seen after challenge with rHBP (75 μg/ml) (Fig. 2). When stimulation was induced in the presence of antibodies against HBP, the increase in permeability was markedly suppressed (Fig. 2). In fact, the ability of anti-HBP to antagonize PMN- induced permeability changes exactly matched their potency in inhibiting the rHBP- induced increase in permeability.

Though human HBP is devoid of catalytic function, it is still capable of binding Kunitz type protease inhibitors (for example, aprotinin) with high affinity. We therefore investigated whether aprotinin could interfere with the activity of HBP. Preincubation of rHBP (75 μg/ml) with aprotinin (20 μg/ml) almost negated the HBP-evoked decrease in EC electrical resistance; in the presence of aprotinin the decline was only 10 ± 2% as compared with 70 + 3% after stimulation with HBP alone (P< 0.01; n = 6). We further demonstrated that aprotinin antagonized the effect of HBP by binding specifically to the inactive 'catalytic' site of the HBP molecule. A mutant variant of HBP ([G175QJHBP), which does not bind aprotinin because of the introduction of a bulky amino acid (replacement of glycine by glutamine) in the dead catalytic cleft, possessed permeability increasing activity similar to that of native HBP. However, aprotinin was unable to reverse its effect (67 ± 6% and 64 ± 5% decrease in electrical resistance with aprotinin absent and present, respectively; n =

6).

HBP mediates PMN-evoked alterations in EC permeability

The fact that EC hyperpermeability evoked both by PMN activation and rHBP stimulation was inhibited to the same extent by anti-HBP clearly indicates that secreted HBP is involved in PMN-induced alteration in vascular permeability. However, in the PMN secretion, in addition to HBP, we identified the structurally related granule proteins elastase and cathepsin G, previously implicated in inducing EC hyperpermeability. In order to find out to what extent these proteins contributed to the permeability increasing activity of the PMN-derived secretion we sought to deplete the postsecretory supernatant of HBP, elastase and cathepsin G, respectively. Specific immunoadsorption could completely and selectively remove each of these three proteins from the supernatant, as verified by sandwich ELISA (data not shown). Removal of HBP rendered the postsecretory supernatant completely inactive with regard to its capacity to induce EC permeability changes, whereas supernatant depleted of elastase and/or cathepsin G was found to induce a fall in electrical resistance of the same magnitude as that evoked by stimulation with native supernatant (Fig. 3). Clearly, these data suggest an exclusive role of HBP in mediating PMN-evoked alteration in vascular permeability.

All of the serprocidins display a positive surface charge. In HBP, a large patch of basic amino acid residues is concentrated on one side of the protein, giving the molecule a strong polarity. The surface electrostatic potential and dipole moment in HBP was calculated and rendered using GRASP, and compared with that in elastase. The centering and direction of the monopole vector in HBP and in elastase were found to be nearly identical. However, the dipole moment in HBP was three times larger (1089 Debye units) than that in elastase (358 Debye units) despite the great sequence homology between the molecules. Anionic compounds may interfere with the action of cationic proteins via charge interactions. We therefore examined whether the effect of rHBP on endothelial permeability could be neutralized by the polyanion dextran sulfate, similarly to what we previously have found for the EC response to stimulation with the PMN-derived post-secretory supernatant. Indeed, the permeability change in response to HBP stimulation was completely prevented by simultaneous administration of dextran sulfate (1 mg/ml) (Fig. 3). Again, the effectiveness by which the EC response to HBP stimulation was antagonized was strikingly similar to that found for the PMN-induced permeability increase.

Bacteria release HBP from human neutrophils Bacteria, originally derived from chronic ulcers, were grown overnight in

Todd Hewitt broth. After centrifugation, the supernatants (450 μl) were added to human blood (450 μl) and the release of HBP from neutrophils was measured by ELISA. Purified P. aeruginosa exotoxin (20 ng/ml), or P. aeruginosa elastase (3 U/ml) or LPS (50 ng/ml) did not release HBP. Indicated below are the bacterial strains that were tested (Table 1). Blood was lysed and the extent of release considered as 100%. The HBP releasing potential of the bacteria were graded as: -, less than 10% increase in HBP; +, 10-50 increase in HBP; and + +, 50-100% increase in HBP.

Bacteria Relea se of HBP

E. faecalis 231 +

P. mirabilis +

P. aeruginosa strainl3:l ++

10:5 ++

9:1 +

3:1 ++

15159 ++

42:3 ++

23:1 ++ Table 1. Bacterially induced release of HBP from neutrophils.

These results show that various ulcer-derived bacteria, such as those listed above, have the unique capacity to release HBP from human neutrophils. Furthermore, measurements of HBP in wound fluid from acute wounds and chronic ulcers indicate that HBP is significantly (10-30 fold) increased.

Claims

1. Use of aprotinin or a variant, derivative, analogue or fragment thereof in the manufacture of a medicament for use in the treatment of a disease or condition associated with secretion of neutrophil derived substances.

2. Use according to claim 1, wherein the disease or condition is characterised by excessive neufrophilic activation.

3. Use according to claim 2, wherein the disease or condition is a chronic ulcer or chronic wound.

4. Use according to any of the preceding claims, wherein the treatment is to reduce inflammatory reactions or to promote wound healing or reepithelialization.

5. Use according to any of the preceding claims, wherein the secretion of neufrophilic substances from neutrophils is associated with acute or chronic bacterial infection.

6. Use according to claim 2, wherein the disease or condition is cutaneous vasculitis, Sweets syndrome, psoriasis or psoriasis pustulosa.

7. A method for the treatment of a patient who has suffered or is suffering from a disease or condition associated with secretion of neutrophil derived substances, which method comprises the step of administering to the patient a therapeutically effective amount of aprotinin or a variant, derivative, analogue or fragment thereof.

8. An agent for the treatment of a disease or condition associated with secretion of neutrophil derived substances comprising aprotinin or a variant, derivative, analogue or fragment thereof.

9. Aprotinin or a variant, derivative, analogue or fragment thereof for use in the treatment of a disease or condition associated with secretion of neutrophil derived substances.