WO1993000926A1

WO1993000926A1 - Treatment of periodontal disease with protease inhibitors

Info

Publication number: WO1993000926A1
Application number: PCT/US1992/005549
Authority: WO
Inventors: Philip A. Pemberton; Michael A. Curtis
Original assignee: Children's Medical Center Corporation
Priority date: 1991-07-02
Filing date: 1992-07-01
Publication date: 1993-01-21
Also published as: AU2299592A

Abstract

Periodontal diseases can be treated by administering to an individual a pharmaceutical composition comprising an effective therapeutic amount of a serine protease inhibitor or a portion thereof, having a reactive site containing a basic residue or a peptide corresponding to the reactive site, and a physiologically acceptable carrier. Preferably, the serine protease inhibitor specifically inhibits the trypsin-like activity of Porphyromonas gingivalis and has an active site containing an arginine or lysine residue.

Description

TREATMENT OF PERIODONTAL DISEASE WITH PROTEASE INHIBITORS

Background of the Invention

The technical field of this invention is treatment of bacterial diseases and, in particular, treatment of periodontal diseases.

The anaerobic gram-negative rod, Porphyromonas αinαivalis. is strongly implicated in the initiation and progression of certain forms of periodontitis (a group of inflammatory diseases of the periodontium) . The organism expresses an array of hydrolytic enzyme activities including aryl sulphatase, heparinase, collagenase, alkaline phosphatase and chymotrypsin-like and trypsin-like protease activities. Mayrand, D. and S.C. Holt, Microbiol. Rev. 52: 134-152 (1988). The concerted action of these enzymes contributes to the soft and hard tissue destruction typical of advanced adult periodontal disease primarily by perturbation of the host protease:antiprotease homeostasis and also, to some degree, by direct action on the host's tissue components. The trypsin-like enzyme activity of P. αinαivalis has been identified as a major virulence factor. See e.g., Sorsa, T. e_£ al. J. Periodont. Res. 22: 375-380 (1987).

Though the enzyme activity is referred to as trypsin-like, based on its preferential hydrolysis of lysine and arginine containing peptide bonds, it is still unclear whether the enzyme should be classified as a serine protease or a cysteine protease. As no amino acid sequence data are currently available, it has only been possible to define the nature of the catalytic site of the enzyme on the basis of inhibitor studies. Such studies have produced conflicting conclusions, largely because of the lack of truly class-specific inhibitors. The P. σinσivalis trypsin-like enzyme activity has been classified as a cysteine protease based on inhibition by thiol blocking compounds. However, it has been suggested that the enzyme behaves more like a serine protease. Sorsa, T. e_fc al. J. Periodont. Res. 22: 375-380 (1987); and Tsutsui, H. e_t al. Infect. Im un. 5_5_: 420-427 (1987) .

Periodontal diseases are a group of inflammatory conditions affecting the supporting tissues of the teeth. The most common form, adult periodontitis, produces moderate to severe tissue destruction in up to 15-20% of dentate individuals in most populations of the world. Adult periodontitis is a major cause of morbidity and tooth loss, and provides a potentially dangerous focus of infection with systemic and metastatic implications in susceptible patients. The management of periodontal diseases constitutes a major health burden, with the United States alone spending more than one billion dollars each year. Despite public and private expenditures, the majority of patients remain untreated because patients do not present or practitioners do not recognize the need. More effective diagnostic, preventative and disease stabilization treatments are urgently required. Summarv of the Invention

Pharmaceutical compositions are disclosed for use in treating peridontal diseases. The compositions comprise natural or recombinant serine protease inhibitors having a reactive site containing a basic residue, or a portion thereof comprising the reactive site, and a physiologically acceptable carrier. These serine protease inhibitors specifically inhibit the trypsin-like activity of Porphyro onas oinαivalis. Typically, the reactive site of the inhibitor contains an arginine or lysine residue.

The pharmaceutical composition can be administered to an individual afflicted by a periodontal disease. For example, in an individual infected by £_,. σinσivalis. an effective therapeutic amount of a serine protease inhibitor having a reactive site containing a lysine or an arginine residue can be administered to arrest the proteolysis mediated by this organism. The composition can be administered in the form of a toothpaste, outhwash, irrigation, or slow release compositions (e.g., microcapsules or inhibitor-impregnated matrices) applied directly to the tooth pocket or otherwise applied topically to inflamed surfaces.

The invention will next be described in connection with certain examples and experimental results; however, it should be clear that various changes, additions and subtractions can be made by those skilled in the art without departing from the spirit or scope of the invention. Brief Description of The Figures

FIGS. 1A and IB show the inhibition of the trypsin-like enzyme activity of P_^. qingivalis W83 culture supernatant by preincubation with human serine protease inhibitors antithrombin III and α2-antiplasmin respectively.

FIG. 2 shows the effect of preincubation with serine protease inhibitors human Cl-inhibitor, antithrombin III and α2-antiplasmin on _s_ gingivalis W83 culture supernatant trypsin-like enzyme activity.

FIG. 3 shows the effect of αl-antitrypsin and αl-antitrypsin (Pittsburgh) on trypsin-like enzyme activity in culture supernatant ([]), cell sonicates (o) and IEF enzyme preparation (x) of P_s_ gingivalis W83.

FIG. 4 shows a Western blot using the anti-serpin antiserum of the reaction products between antithrombin III and either thrombin (Lane 1) or cell sonicate of IL. gingivalis W83 (Lane 3). Lane 2 shows antithrombin III alone.

FIG. 5 shows a Western blot using the anti-serpin antiserum of the reaction products between αl-antitrypsin (Pittsburgh) and either thrombin (Lane 1) or cell sonicate of Pj. gingivalis W83 (Lane 3). Lane 2 shows αl-antitrypsin (Pittsburgh) alone.

SUBSTITUTE SHEET Detailed Description of The Invention

A serine protease inhibitor or a portion thereof, having a reactive site containing a basic residue is used in a pharmaceutical composition for the treatment of a periodontal disease. Preferably, an inhibitor which specifically inhibits the trypsin-like activity of Porphyromonas gingivalis, or a peptide having an amino acid sequence consisting essentially of the reactive site of the inhibitor, and a pharmaceutically acceptable carrier are administered to an individual afflicted by a periodontal disease related to infection by this organism. Typically, the reactive site of the inhibitor contains an arginine or lysine residue.

Serine protease inhibitors useful in pharmaceutical compositions or diagnostic reagents include naturally occuring proteins, such as serpins isolated from human serum, recombinant protein produced by standard techniques, portions of natural or recombinant proteins consisting essentially of the reactive site of a serpin, or synthetic peptides having amino acid sequences corresponding to the reactive site of a serpin. Isolated human plasma serpins are glycoproteins comprising single polypeptide lengths of Mr 40-70 kD with a variable number of attached oligosaccharide side chains. They possess a single reactive center which is carboxy-terminally placed in the primary structure. Examples of serpins useful in compositions include α2-antiplasmin, antithrombin III, αl-antitrypsin (Pittsburgh) ,plasminogen activator inhibitor, protein C inhibitor, protease nexin I or mouse αl-antichymotrypsin. The pharmaceutical composition or diagnostic reagent can include a portion of a natural or recombinant serpin, such as a synthetic peptide having an amino acid sequence corresponding to the reactive site of the serpin. A useful peptide comprises tyrosine-alanine-lysine-arginine (SEQ ID NO: 1) (amino to carboxyl terminal) . Peptides having amino acid sequences corresponding to the reactive site of a serpin can be isolated by hydrolysis of human plasma serpins or synthesized by standard solid-phase techniques.

In addition to the inhibitor, the pharmaceutical composition can include an effector or co-factor, such as heparin which is known to bind to and significantly enhance the rate of inhibition produced by certain serine protease inhibitors against their target enzyme (e.g., the antithrombin and thrombin interaction) .

Typically, serpins present a reactive site to cognate or target serine protease(s) as substrates. Upon interaction between a protease and a serpin inhibition or catalysis results. The primary sequence of the reactive site determines the specificity of the inhibitor. The target serine protease specifically recognizes the P2-P1 residues. The PI' site is almost invariably Ser or Thr and the P2' site is usually hydrophobic. Thus, inhibitors with PI Lys or Arg tend to inhibit trypsin-like proteases. The reactive site of an inhibitory serpin interacts with a target protease in a 1:1 stoichiometry. The serine protease inhibitors useful in pharmaceutical compositions can be genetically engineered to be free of sites of inactivation for proteases in the oral environment. For example, plasmids containing the wild-type αl-antitrypsin or antithrombin III genes can be used for site-directed mutagenesis of the active site. George P.M., et al. Blood 73(2): 490-496 (1989). The mutant αl-antitrypsin genes can be expressed in, and purified from, the yeast Saccharomyces cerevisiaie with the mutant sequences attached to the ubiquitin gene to provide higher yields of the expressed protein. Rosenberg S., e_t al. Nature 312: 77-80 (1984). The translational product is truncated to liberate native αl-antitrypsin which is then purified by a combination of ion exchange, affinity, and size exclusion chromotography and tested for inhibition of P. gingivalis trypsin-like enzyme activity.

Reactive sites can also be engineered into the antithrombin III gene to exploit the observed protease:inhibitor complex stability conferred by ligand occupancy of antithrombin Ill's heparin-binding domain. These mutants can be expressed in a baculovirus expression system

(Maxbac™ instruction manual (version 1.4) pp. 1-41 (1990) Invitrogen Corp.) rather than the yeast system, as it has been shown that the final product is incorrectly glycosylated in the yeast system (Christey P.B., Ph.D. Thesis Cambridge University U.K. (1990)) whereas the baculoviral system can partially glycosylate translation products (Miller L.K., Ann. Rev. Microbiol. 42: 177-199 (1988) and Miller L.K., Bioessavs 11(4) : 91-95 (1989)) and also produce a high yeild. The mutated gene can be ligated into the vector pVL941 and co-transfected with wild-type baculovirus into the host cell line Spodoptera fruoiperda (Sf9) . The appropriate recombinants can be identified by the absence of occlusion bodies and checked to ensure the sequence is correct and in the appropriate orientation. The recombinants are then used to infect larger volumes of cells from which the recombinant proteins can be isolated by ion-exchange and affinity chromatography. Travis J., e_£. al. J. Biol. Chem. 260: 4384 (1985); McCay E.J., Thromb. Res. 21: 375-382 (1981). Purified recombinant proteins can be tested for their ability to inhibit the trypsin-like enzyme activity of P_i_ gingivalis.

The method of this invention can be used to treat a periodontal disease in an individual, particularly a disease related to infection by the organism P_j_ gingivalis. According to the method, a pharmaceutical composition comprising an effective therapeutic amount of a serine protease inhibitor or a portion thereof, having a reactive site containing a basic residue, or a peptide having an amino acid sequence corresponding to the reactive site is administered to the individual. An effective therapeutic amount of the inhibitor is an amount sufficient to arrest the proteolysis mediated by P. gingivalis.

Typically, the pharmaceutical composition includes a pharmaceutically-acceptable carrier such as sterile water, ethanol, polyol or suitable mixtures thereof. ^'As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial, and antifungal agents, isbtonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the inhibitor, it's use in the pharmaceutical compositions and diagnostic reagents is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The pharmaceutical composition can be administered in the form of a toothpaste, mouthwash, irrigation, or slow release compositions (e.g., microcapsules or inhibitor-impregnated matrices) applied directly to the tooth pocket or otherwise applied topically to inflamed areas.

The invention can also be used for diagnostic purposes to detect and/or quantify the extent of periodontal infection by P_,_ gingivalis or related organisms. In one approach, a peptidyl fragment of the inhibitors of the present invention can be converted into a diagnostic substrate with an appropriate indicator composition. By applying a scraping of a suspect infection site (e.g., a plaque from the tooth pocket or gingival crevice) when bacterial infection is present, the serine proteases will cleave the diagnostic substrate to produce reaction products which are indicative of P. gingivalis presence. Indicator compositions useful in such diagnostic substrates include chromophores such as nitroanilides, napthalamides, and coumarins. This invention is illustrated further by the following Examples.

Examples

P. gingivalis W83, a highly proteolytic strain, was innoculated from a 48 hour horse blood agar plate into dialysed BM broth, a peptone/yeast medium supplemented with 5 mg haemin/1. Shah, H.N., e£ al. J. APPI. Bact. 41: 473-492 (1976). The innoculated broths were incubated anaerobically (80% N2, 10% H2 10% CO2) for 6 days at 37°C.

Culture supernatant fluid was collected after centrifugation (18,000 x g, 30 min, 4°C) and then filter sterilised (0.22 μm filter). Vesicles were precipitated by 40% saturation with ammonium sulphate and discarded. Soluble proteins were precipitated by 70% saturation with ammonium sulphate and then dissolved in and dialysed against tris(hydroxymethyl)methylamine (50 mM, pH 7.2). The volume of the dialysate was then brought to 1/lOth the original culture volume, filter sterilised (0.22 μm pore size) and stored at -70°C. Aliquots of the dialysate were isoelectrically focussed in preparative flatbeds (pH 3.5 - 10; Application Note 198, LKB Instruments, Bromma, Sweden). Trypsin-like enzyme activity free from any other contaminating enzyme activity was found in fractions of pi 7.5 and pi 4.6. Harper, F.H., et al. Clinical and Molecular Aspects of Anaerobes. Borriello S.P., ed., Wrightson Biomedical, Petersfield, England p. 175 (1990). The pi 4.6 material, which is in agreement with previous published estimates of the pi of this enzyme (Fujimara, S. and T. Nakamura Infect. Immun. 55: 716-720 (1987); Ono, M. , e_t ZI_Λ. Oral Microbiol. Immunol. 2: 77-81 (1987)), was then dialysed overnight against Tris buffer to remove ampholytes and stored at -70°C until required. Where indicated, non-fractionated culture supernatant was also used.

Bacterial cells were pelleted by centrifugation and washed once in Tris buffer. Following resuspension to 1/5 v/v in HEPES buffer (N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic acid, 10 mM, pH 7.2), they were sonicated on ice for 1 minute intervals (xlO) with intermittent cooling at maximum power using a Soniprep 150 (MSE Scientific Instruments, Crawley, England) . The sonicate was then centrifuged (40,000g, 60min, 40°C) to remove unbroken cells and cell membranes and then stored at -70°C until required.

Antithrombin III, αl-antitrypsin and Cl-inhibitor were purified from human plasma by various chromatographic procedures. Alpha 1-antitrypsin was purified by thiol exchange chromatography on glutathione sepharose (Laurell, C.B., et al. Eur. J. Biochem. 57: 107-113 (1975)) followed by ion-exchange chromatography on Q-sepharose. Antithrombin III was purified by heparin-agarose chromatography (McKay, E.J. Thromb. Res. 21: 375-382 (1981)) and contaminating heparin removed by ion exchange chromatography on Q-sepharose. Cl-inhibitor was purified by salt fractionation, affinity chromatography, ion exchange chromatography and size exclusion chromatography according to Harrison, R.A. Biochem. 22: 5001-5007 (1983) . Human α-2 antiplasmin was purchased from Calbiochem (La Jolla, CA, USA). Alpha 1-antitrypsin (Pitts) was a gift from Professor RW Carrell, University Department of Haematology, Hills Rd, Cambridge, U.K.

Diisopropyl fluorophosphate (DFP) was obtained from Aldrich Chemical Company (Gillingham, England) . Human plasma low molecular weight kininogen was from Novabiochem (Nottingham, England) and p-amidinophenyl-methylsulphonylfluoride (p-APMSF) hydrochloride from Calbiochem (La Jolla, CA, USA). Transepoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64) and egg white cystatin were both purchased from Sigma (Poole, Dorset, England).

Trypsin-like enzyme activity was assessed by two procedures. First, by measuring the rate of cleavage of the fluorimetric substrate

N-benzoyl-L-arginine 7-amido-4-methylcoumarin hydrochloride (BAAMC - Sigma Chemical Co. Ltd, Poole, England) . Assays were routinely performed in 3 ml Tris HC1 (50 mM, pH 7.2, 10 mM CaCl₂, 1 mM cysteine HC1) containing 3.7 mM BAAMC at room temperature. Following addition of 'the sample, the rate of increase in relative fluorescence (i.e., rate of hydrolysis of BAAMC) was measured (excitation:380 nm; emission:460 nm; SFM-25 fluorimeter, Kontron Instruments, Zurich, Switzerland) . Enzyme activity was expressed in units of relative fluorescence released/minute and percentage inhibition calculated relative to a control cuvette. Additionally, trypsin-like activity was determined spectrophotometrically by measuring the rate of hydrolysis of Nα-benzpyl-DL-arginine p-nitroanilide (BAPNA) hydrochloride (0.46 mM in 50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 1 mM cysteine-HCl) at 405 nm.

Inhibitors which are generally presumed to be useful discriminators between cysteine and serine proteases were examined for their ability to inhibit the trypsin-like enzyme activity of Pj_ gingivalis in a BAPNA assay. 10 μg whole cell sonicate protein was used/ml assay. Because of the presence of multiple molecular weight forms of the enzyme we are unable to give a precise molarity to any of the enzyme preparations used in this investigation. However if it is generously assumed that the whole of the protein present in the sonicate preparation corresponded to enzyme protein and an arbitrary minimum molecular weight of 10,000 is chosen, then the maximum amount of enzyme present in 10 μg sonicate protein would be 1 nmole. The absolute maximum concentration of trypsin-like enzyme in an assay volume of 1 ml would be 1 μM. Hence, inhibitors whose mechanism of action involve a 1:1 stoichiometry with the enzyme were used in the assay at concentrations equal to or greater than 1 μM in order to ensure that they would be* in excess.

For inhibition experiments the enzyme and inhibitor were pre-incubated in Tris buffer containing calcium and cysteine for 10 minutes prior to determination of enzyme activity. All experiments were performed in duplicate on at least two separate occasions and the results are presented as mean data. The standard deviations of the means did not exceed ±5% in each case. Egg white cystatin and human low molecular weight kiningen, both protein inhibitors of cysteine proteases, were ineffective inhibitors of the trypsin-like activity of P_j_ gingivalis.

Trans-epoxysuccinyl compound, E64, an irreversible inhibitor of cysteine proteases that does not affect cysteine residues in other enzymes or low molecular weight thiols, caused a decrease in activity of the enzyme but only when incorporated into the assay at 50-100 fold higher concentration than the theoretical maximum enzyme molarity. pAPMSF, a specific inhibitor of trypsin-like serine proteases caused a reduction in enzyme activity, but only at high concentrations. DFP, a broad spectrum serine protease inhibitor which is considered diagnostic for serine proteases was completely ineffective (Table 1) .

The IEF enzyme activity was not inhibited by phenylmethanesulphonyl fluoride but was completely abolished by tosyllysylchloromethylketone, tosylphenylalanylchloromethylketone, leupe tin, chymostatin, antipain (all effective against both serine and cysteine proteases) and by the chelating agents ethylenediaminetetra-acetic acid and ethyleneglycol-bis-tetra-acetic acid. Other inhibitors, including peptides containing the reactive site residue Arg or Lys modified at the C-terminus with either a boronic acid, chloromethyl ketone, or aldehyde moiety, can be useful in the present invention. TABLE 1

Concentration μM Activity (%)

Cystatin 2.5 110

99 96 97

92 77 63

96 100

84

19

Human plasma serpins with an arginine residue in the Pi position of the reactive site were examined for the ability to inhibit the trypsin-like activity of p_,_ gingivalis in BAAMC assays. The amino acid configurations at the reactive site of αl-antitrypsin, αl-antitrypsin (Pitts), antithrombin III, α2-antiplasmin and Cl-inhibitor are shown in Table 2. TABLE 2

Amino Acid Configuration at the Reactive Site of Human Plasma Arginine Serpins

Target Reactive Site Protease (Pi - Pi')

αl-Antitrypsin Elastase Met-Ser

αl-Antitrypsin Thrombin Arg-Ser (Pitts)

Antithrombin III Thrombin Arg-Ser

α2-Antiplasmin Plasmin Arg-Met

Cl-inhibitor Cl Arg-Thr

In the case of antithrombin III and α2-antiplasmin, a dose dependent inhibition, to greater than 90% and 80% respectively, was observed using culture supernatant (8 μl) as the enzyme preparation (FIGS. 1A and IB). Cl-inhibitor was ineffective over the same concentration range (FIG. 2) . Inhibition of enzyme activity by αl-antitrypsin (Pitts) was examined using the culture supernatant, sonicate preparation, and IEF purified enzyme adjusted to the same specific activity. In each case, the mutant serpin inhibited enzyme activity to greater than 85% whereas normal αl-antitrypsin showed no effect over the same concentration range (FIG. 3).

The complete, or near complete, inhibition of all the trypsin-like enzyme activity in unfractionated culture supernatant and whole cell sonicates of P_,_ gingivalis by αl-antitrypsin (Pitts), antithrombin III and α2-antiplasmin suggests that the multiple molecular weight forms of the thiol dependent enzyme activity (Fujimara, S. and T.

Nakamura Oral Microbiol. Immunol. 4.: 227-229 (1989); Grenier, D., e_£ al. Infect. Immun. 57: 95-99 (1989)) share a similar catalytic mechanism and may represent active degradative products of the same enzyme.

Although Cl-inhibitor has arginine as the reactive site Pi residue, it did not inhibit P. gingivalis trypsin-like enzyme activity. This may be explicable for a number of reasons: 1) It is the largest serpin characterised to date comprising a unique heavily glycosylated N-terminal domain which may recognise specific elements on its physiological target(s) (Bock, S.C., e al. Biochem. 25: 4292-4301 (1986); 2) It is the only serpin which has a threonine in the Pi' position; 3) Hypervariability within the region surrounding the reactive site. Other residues partly contribute to the specificity requirements (e.g., P2) • It is also true that the arginine directed target proteases Clr and Cls are controlled by by Cl-inhibitor exclusively indicating interactions in addition to the Pi specificity to be important. Currently, there is no evidence for serpin inhibition of any class of protease other than the serine proteases. Thus, the inhibition of the trypsin-like enzyme by antithrombin III, αl-antitrypsin (Pitts) and α2-antiplasmin strongly suggests that the enzyme is a serine protease. By implication the inhibition of activity by thiol-reactive agents may thus be a consequence of the blockage of a free cysteine residue(s) not directly involved in the catalytic mechanism.

The reaction products following incubation of antithrombin III and αl-antitrypsin with either bovine thrombin (Sigma) or cell sonicates of P. gingivalis were examined by SDS/PAGE and immunoblotting using anti-serpin antisera (see below) . 2 μg antithrombin III or αl-antitrypsin were incubated at room temperature with either 20 units thrombin or 20 μl whole cell sonicate (3.3 μg total protein) for 10 minutes in Tris buffer (pH 7.2, 50 mM, 10 mM calcium chloride, 1 mM cysteine) . The reaction was stopped by the addition of SDS/PAGE sample reducing buffer and EDTA to a final concentration of 10 mM.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS/PAGE) was performed essentially according to Laemmli, U.K. Nature 227: 680-685 (1970) on 10% acrylamide/bisacrylamide separating gels incorporating a 4% stacking gel. Western blotting was performed by electroelution for 2 hours at 400 mA onto nitrocellulose in a carbonate transfer buffer, pH 9.9. Dunn, S.D. Anal. Biochem. 157: 144-153 (1986). Bovine serum albumin (BSA) in phosphate buffered saline (5%) was the blocking agent and immunochemical detection performed using the following antisera and dilutions: rabbit anti-αl-antitrypsin (Unipath, Bedford, U.K.), 1:40,000; rabbit anti-antithrombin III, 1:1000; horseradish peroxidase-conjugated swine anti-rabbit immunoglobulins (Dakopats, Glostrup, Denmark), 1:500. Color development was performed using diaminobenzidine.

As shown in FIG. 4, antithrombin III was cleaved by the cell sonicate enzyme preparation with the formation of a modified inhibitor with molecular weight approximately 5000 lower than the intact serpin. In contrast to the incubation of inhibitor with its physiological target enzyme, thrombin, no higher molecular weight species reactive with antiserpin antiserum could be detected.

Antithrombin III (40 μg) was incubated with the IEF enzyme preparation (40 μg) in an imidazole buffer (60 mM, pH 7.3) containing 5 mM L-cysteine, 10 mM calcium chloride, 2.5 mM 2-mercaptoethanol and 4.0% (v/v) dimethyl sulphoxide in a final volume of 200 μl. The incubation was allowed to react at room temperature for 10 minutes and the reaction stopped by addition of electrophoresis buffer (SDS/PAGE, non-reducing) and heating at 90°C for 5 minutes.

A 5-20% SDS/PAGE mini-gel was prepared (Hoeffer Scientific Equipment, California, USA) and 5 μg (80 pmol) samples of native and proteolytically modified antithrombin III were electrophoresed under non-reducing conditions. The proteins were transblotted (Matsudaira, P. J. Biol. Chem. 262: 10035-10038 (1987)) onto "Problot" (Applied Biosystems Incorporated, California, USA) using 10 mM ^KH2P°4 0.05% SDS, pH 6.8 as the continuous buffer system. Protein bands were detected by staining with Coomassie blue R-250, excised and directly sequenced on an Applied Biosystems model 477A protein sequencer.

The results of the cleavage site analysis of antithrombin III are shown in Table 3. These data indicate inactivation by cleavage between arg(394)-ser(395) , the reactive centre of the molecule, as well as some (approximately 45%) N-terminal modification between residues lys(39)-ile(40) . Walsh, K.A. Proteases and

Biological Control, p 1-11. Reich, E., Rifkin, D.B. and E. Shaw, (ed) Cold Spring Harbor Lab., Cold Spring Harbor, New York (1975). Both sites are in keeping with the reported specificity of the protease.

Table 3

pmol

Native ATIII H G S P V D I 7 Q A 48 (SEQ ID NO: 2)

P_i. gingivalis modified ATIII 1. H G S P V D I 7 Q A 40 (SEQ ID NO: 2)

2. S L N P N R V T F K 38 (SEQ ID NO: 3)

3. I P E A T N R R V ? 20 (SEQ ID NO: 4)

Incubation of αl-antitrypsin (Pitts) with cell sonicate resulted in cleavage of the inhibitor and formation of two major lower molecular weight species (approximate M_r -= 40,000). Similar, lower molecular weight products of αl-antitrypsin (Pitts) were also produced following incubation of the serpin with thrombin (FIG. 5; lanes 1 and 3) suggesting these components represent reactive centre cleavage products of native or N-terminally cleaved protein. In the latter incubation, higher molecular weight bands immunoreactive with anti αl-antitrypsin antiserum were also generated, indicative of stable enzyme/serpin complex formation. Attempts to demonstrate complex formation between the P. gingivalis trypsin-like enzyme in cell sonicates and αl-antitrypsin (Pitts) on immunoblots were unsuccessful.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Pemberton, Philip S Curtis, Michael A.

(ii) TITLE OF INVENTION: Treatment of Periodontal

Disease with Protease Inhibitors

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Lahive & Cockfield

(B) STREET: 60 State Street, Suite 510

(C) CITY: Boston

(D) STATE: MA

(E) COUNTRY: USA

(F) ZIP: 02109

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: ASCII TEXT

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT

(B) FILING DATE: 01-JUL-1992 (C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/724,823

(B) FILING DATE: 02-JUL-1991 (C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Engellenner, Thomas J.

(B) REGISTRATION NUMBER: 28,711 (C) REFERENCE/DOCKET NUMBER: CME-026

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 227-7400

(B) TELEFAX: (617) 227-5941 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Tyr Ala Lys Arg

1

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

His Gly Ser Pro Val Asp lie Xaa Gin Ala 1 5 10 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Ser Leu Asn Pro Asn Arg Val Thr Phe Lys 1 5 10

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ. ID NO:4

lie Pro Glu Ala Thr Asn Arg Arg Val 1 5 Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

1. A pharmaceutical composition for treating a periodontal disease, comprising an effective therapeutic amount of a serine protease inhibitor having a reactive site containing a basic residue, and a physiologically acceptable carrier,

2. A pharmaceutical composition of claim 1, wherein the basic residue comprises arginine or lysine.

3. A pharmaceutical composition of claim 2, wherein the serine protease inhibitor is selected from the group consisting of α2-antiplasmin, antithrombin III, αl-antitrypsin (Pittsburgh), plasminogen activator inhibitor, protein C inhibitor, protease nexin I or mouse αl-antichymotrypsin.

4. A pharmaceutical composition of claim 1, wherein the serine protease inhibitor specifically inhibits the trypsin-like activity of Porphyromonas gingivalis.

5. A pharmaceutical composition of claim 1 further comprising an amount of heparin sufficient to enhance the rate of inhibition of the trypsin-like activity of Phorphyromonas gingivalis by the serine protease inhibitor or portion thereof.

6. A pharmaceutical composition of claim 1, wherein the serine protease inhibitor is a synthetic peptide which comprises

5 tyrosine-alanine-lysine-arginine (SEQ ID NO: 1).

7. A method for treating a periodontal disease in an individual, comprising administering to the individual a pharmaceutical composition

10 comprising an effective therapeutic amount of a serine protease inhibitor having a reactive site containing a basic residue, and a physiologically acceptable carrier.

15 8. A method of claim 7, wherein the basic residue is arginine or lysine.

9. A method of claim 8, wherein the serine protease inhibitor is selected from the group consisting 20 of α2-antiplasmin, antithrombin III, αl-antitrypsin (Pittsburgh) , plasminogen activator inhibitor, protein C inhibitor, protease nexin I or mouse αl-antichymotrypsin.

25 10. A method of claim 7, wherein the serine protease inhibitor specifically inhibits the trypsin-like activity of Porphyromonas gingivalis.

11. A diagnostic assay for periodontal infection in an individual by Porphyromonas gingivalis or a related organism, comprising the steps of: (a) providing a sample of suspect infection from the individual and a diagnostic substrate comprising a serine protease inhibitor and an indicator composition; and

(b) contacting the sample with the diagnostic substrate such that enzymes present in the sample bind with the serine protease inhibitor and modify the indicator composition.

12. A diagnostic assay of claim 11, wherein the serine protease inhibitor is a synthetic peptide having a reactive site containing a basic residue.

13. A diagnostic assay of claim 12, wherein the basic residue comprises arginine or lysine.

14. A diagnostic assay of claim 11, wherein the indicator composition is a chromphoric indicator.

15. A diagnostic assay of claim 14, wherein the indicator composition is selected from a group consisting of nitroanilides, coumarins, and napthalamides.

16. A reagent for detecting the presence of Porphyromonas gingivalis infection in an individual, comprising a serine protease inhibitor and an indicator composition, the inhibitor having a binding affinity for an enzyme released by P_j_ gingivalis organisms, such that binding of the serine protease inhibitor modifies the state of the indicator composition.

10

17. A reagent of claim 16, wherein the serine protease inhibitor is a synthetic peptide having a reactive site containing a basic residue.

15 18. A reagent of claim 17, wherein the basic residue comprises arginine or lysine.

19. A reagent of claim 16, wherein the indicator composition is a chromphoric indicator. 0

20. A reagent of claim 19, wherein the indicator composition is a nitroanilide.