WO2009043128A1

WO2009043128A1 - Use of protease inhibitors isolated from bauhinia sp. for the treatment of microbial infections and pharmaceutical composition

Info

Publication number: WO2009043128A1
Application number: PCT/BR2008/000298
Authority: WO
Inventors: Maria Luiza Vilela Oliva; Misako Uemura Sampaio; Fernanda Peruzzo De Caroli
Original assignee: Universidade Federal De São Paulo - Unifesp; Fundação De Amparo À Pesquisa Do Estado De São Paulo - Fapesp
Priority date: 2007-10-02
Filing date: 2008-10-02
Publication date: 2009-04-09
Also published as: BRPI0703559B8; BRPI0703559B1; BRPI0703559A2

Abstract

The present invention refers to the use of protease inhibitors isolated from Bauhinia sp. and/or synthetic peptides related to its primary structure with antimicrobial activity, as well as to a pharmaceutical composition comprising the same for the treatment of microbial infections.

Description

"USE OF PROTEASE INHIBITORS ISOLATED FROM BAOHINZA SP. FOR THE TREATMENT OF MICROBIAL INFECTIONS AND PHARMACEUTICAL COMPOSITION" .

FIELD OF THE INVENTION The present invention refers to the use of protease inhibitors isolated from Bauhinia sp. and synthetic peptides related to its primary structure with antimicrobial activity, as well as to a pharmaceutical composition comprising the same for the treatment of microbial infections.

BACKGROUND OF THE INVENTION

The antimicrobials are essential for preventing and treating infections which are more and more difficult to be treated, thus requiring more expensive and toxic drugs. Such requirement is due to the fact that many microorganisms, such as bacteria and fungi, are resistant to antimicrobials due to its own metabolic native properties or due to the resistance mechanism developed for protection. Among the most used antimicrobials are the beta- Iactamic compounds, more specifically the penicillins.

Penicillins, as all β-lactamic compounds, inhibit the development of bacteria by interfering in a specific stage of the cell wall synthesis. They covalently link to Penicillin-Binding Proteins (PBPs) , which are responsible for the transpeptidation catalysis in the cell wall synthesis. Thus, the transpeptidation is inhibited, a blockage in the cell wall peptidoglycan synthesis occurs and the bacteria die. Nevertheless, penicillins are also among the most used antibiotics in an inadequate and excessive way, thus generating resistant strains. The resistance to penicillins and other β-lactamic is generally developed by the following mechanisms: antibiotic inactivation by the production of β-lactamase enzymes; modification of penicillin binder proteins (PBPs) ; impairment of drug penetration to the PBPs; and presence of an efflux bomb; being the β-lactamase production the most common resistance mechanism.

Moreover, there are occurrences of hypersensitivity reactions in some people during the administration of penicillins and other β-lactamics. The allergenic reactions may consist of anaphylactic shock; serum disease type reactions, such as urticaria, fever, articular edema, angioneurotic edema, intense itching and respiratory difficulties, further than cutaneous eruptions.

Penicillins share chemical properties, action mechanisms, pharmacological and clinical effects with cephalosporins, monobactams, carbapenems and β-lactamase inhibitors. Therefore, patients with anaphylaxis history to penicillins also should not be medicated with other β- lactamics .

In the case of sepsis or endocarditis caused by methicillin-resistant staphylococci (MRSA) , parenteral vancomycin is commonly indicated as the main therapeutics. vancomycin is an antibiotic, glycopeptide, produced by Streptococcus orientalis. It is active only against Gram- positive bacteria, particularly staphylococci. It acts inhibiting the liberation of the cellular wall construction unit (disaccharide + peptide) of the lipidic carrier of the bacterial cellular membrane, impairing peptidoglycan polymerization. In the past, enterococci strains exhibited a uniform sensibility to vancomycin. Nevertheless, resistant strains already exist, mainly Enterococcus faecium. The determinants of the resistance to vancomycin in E. faecium and E. faecalis are located in a transposon present in a conjugative plasmid, allowing its fast transference between enterococci and potentially to other Gram-positive bacteria. Moreover, in 2002 a S. aureus strain was isolated with a high degree of resistance to vancomycin, where there was a conjugative plasmid in which Van A transposon was integrated as a horizontal genie transference sequence between E. faecalis species to a methicillin resistant S. aureus strain.

Vancomycin resistance in enterococci is due to a change at the D-Ala-D-Ala binding site of the peptidoglycan formation unit of the cell wall, wherein terminal D-AIa is replaced by D-lactate. Hence, there is critical loss of one hydrogen bond forming a high affinity linkage between vancomycin and its target, with loss of activity. This mechanism is also observed in S. aureus strains resistant to vancomycin which have obtained the enterococci resistance determinants.

Moreover, vancomycin administration presents the inconvenience of being done by parenteral route as it is poorly absorbed by the gastrointestinal tract and must be infused over a period of at least 60 minutes to avoid adverse reactions related to infusion, as erythematous or urticarial reactions, redness, tachycardia and hypotension. Among the most common reactions, the "red neck" or "red man" syndrome is pointed out. This redness related to infusion is not an allergic reaction, but a direct effect of vancomycin on mastocytes, causing histamine release. Ototoxicity and nephrotoxicity associated with high concentrations of the drug or interactions with aminoglycosides can also occur. Aminoglycosides constitute a group of bactericidal antibiotics originally obtained from various Streptomyces species. They are broadly used against Gram-negative enteric bacteria, mainly in bacteremia and sepsis, associated with vancomycin or with a penicillin in endocarditis and tuberculosis treatment. Aminoglycosides bind to specific ribosomal proteins of subunit 3OS and act inhibiting protein synthesis and reducing the accuracy of mRNA translation in ribosome. They have a fast bactericidal effect that seems to be strongly related to the capacity of inducing an incorrect reading.

The microorganisms can develop resistance to aminoglycosides through the following mechanisms: production of a transferase enzyme or enzymes which inactivate the aminoglycoside by adenylation, acetylation, or phosphorylation; interference in the egress of the aminoglycoside into the cell through carrier proteins; and deletion or modification of the receiving protein present at the ribosome subunit 3OS. Moreover, all aminoglycosides are ototoxic and nephrotoxic. And, in very high doses, they may cause an effect similar to curare with neuromuscular blockage resulting in respiratory paralysis.

Another class of antibiotics is constituted by tetracyclines, which are bacteriostatic antibiotics with a broad spectrum. They act as they bind to the bacterial ribosome subunit 3OS, thus blocking aminoacyl-tRNA binding to the acceptor site in the mRNA-ribosome complex. This blockage prevents the addition of amino acids to the growing peptide. There are also some resistance mechanisms to tetracyclines such as influx reduction or efflux increase of tetracyclines through an active transport proteic pump; ribosomal protection through protein production that interferes in the tetracycline binding to the ribosome; and enzymatic inactivation of tetracyclines.

Bacteria and other pathogenic agents, including fungi, develop virulence factors, such as adhesion molecules, liposaccharides, hemolysines, proteases which assist in the host invasion and colonization, thus facilitating dissemination and tissue destruction during the infection process (Holt & Bramanti, 1991; Grenier & Mayrand, 2000) .

The contact between bacterial cells is mediated by adhesion molecules named aggregation substance, AS. All genes that encode AS contain two Arg-Gly-Asp sites, RGD, which indicates a great potential of interaction with eukaryotic cells. Therefore, protein AS is responsible for bacterium-bacterium interaction and also for bacterium- eukaryotic cells interaction (Chow et al., 1993; Olmsted et al., 1994) . Bacterial adherence to the host cells is the first event for pathology development, and consequently for an infection.

Additionally, the complement factor activation is the first host response for an invasion caused by pathogenic agents. This process is mediated by proteases and proteolytic enzymes derived from pathogenic agents which could modify this system through two mechanisms: generic, such as Pseudomonas aeruginosas, or specific, such as S. pyogenes which degrades C5a; other mechanism would be the development of an alternative system in which pathogenic agents release proteases which would activate the complement factor itself, mainly C5 (Maeda et al., 1999) .

Furthermore, bradykinin mediators are induced by bacterial proteases which in need of nutrients directly or indirectly activate the kallikrein-kinin system; the activation of such system occurs due to bradykinin release from kininogen. Bradykinin is a powerful mediator of the inflammatory response which is able to generate vasodilatation, increase on vascular permeability, pain and edema. It is formed by high and low molecular weight kininogen. When is metabolized, bradykinin reduces circulating kininogen which amplifies human plasmatic kallikrein action (Travis & Potempa, 2000; Schmaier 2004) .

Thus, bradykinin mediators and nitric oxide are involved in infectious and inflammatory processes and also in cancer, being able to generate either a bacterial or tumor cell dissemination due to an increase in vascular permeability far from the main focus of the pathology.

(Maeda et al., 1999; Travis & Potempa, 2000) . Thus, it is important to point out that due to the fact that various stages of bacterial invasion and colonization in hosts are in part dependent on protease actuation. Protease inhibitors isolated from Bauhinia sp. could have an important role in the containment of such bacterial and fungicidal dissemination, either by the inhibitory effect of the proteases of these organisms or by the generation of fragments with activity against these agents .

Consequently, since microorganisms have already developed resistance mechanisms against the great majority of antibiotics used in therapeutics, including those used in hospital-acquired infections caused by MRSA, such as vancomycin, the development of a pharmaceutical composition with antimicrobial properties which acts through a distinct mechanism from those in which resistance has already been developed is required as an alternative treatment option for infections caused by resistant microorganisms.

Thus, taking into consideration that preliminary results indicated that the human plasmatic Bauhinia bauhinioides kallikrein inhibitor (BbKI) presented action over Staphylococcus aureus (ATCC 25923/29213) Gram-positive bacteria analyzed by microdilution and disc diffusion method, it is particularly interesting the development of a pharmaceutical composition of protease inhibitors isolated from Bauhinia sp. for the treatment of infections caused by pathogenic microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are part of the present specification and are included herein to illustrate certain aspects of the invention. The present invention can be better understood with reference to one or more of such figures in combination with the detailed description of the preferred embodiment presented herein.

Figure 1 shows the primary sequence of synthetic peptides derived from the reactive site region of BbKI. Figure 2 shows BbKI action over pathogenic bacteria in different concentrations: PSA - Pseudomonas aeruginosa

(ATCC 27853); E. coli - Escherichia coli (ATCC 25922); E. faecalis - Enterococcus faecalis (ATCC 29212) ; S. aureus -

Staphylococcus aureus (ATCC 25923) . 20,6 μM vancomycin is used as positive control for inhibition.

Figure 3 shows the quantification of BbKi inhibitory activity through the inhibition area. (A) - Staphylococcus aureus (ATCC 25923) in the presence of 5 μM BbKI, with formation of a 7,5 mm inhibition area; (B) - Staphylococcus aureus (ATCC 25923) in the presence of 20,6 μM vancomycin, with formation of a 18 mm inhibition area.

Figure 4 shows the action of the peptide containing the BbKi reactive site, RPGLPVRFESPLRINIIKENH₂, in different concentrations: PSA - Pseudomonas aeruginosa (ATCC 27853); E. coli - Escherichia coli (ATCC 25922); E. faecalis - Enterococcus faecalis (ATCC 29212) ; S. aureus - Staphylococcus aureus (ATCC 25923) . 20,6 μM vancomycin is used as positive control for inhibition.

Figure 5 shows the action of the peptide containing the BbKi reactive site, RPGLPVRFESPLNH₂, in different concentrations: PSA - Pseudomonas aeruginosa (ATCC 27853); E. coli - Escherichia coli (ATCC 25922); E. faecalis - Enterococcus faecalis (ATCC 29212); S. aureus Staphylococcus aureus (ATCC 25923). 20,6 μM vancomycin is used as positive control for inhibition. Figure 6 shows the action of the peptide containing the BbKi reactive site, GLPVRFESNH₂, in different concentrations: PSA - Pseudomonas aeruginosa (ATCC 27853); E. coli - Escherichia coli (ATCC 25922); E. faecalis - Enterococcus faecalis (ATCC 29212); S. aureus Staphylococcus aureus (ATCC 25923) . 20,6 μM vancomycin is used as positive control for inhibition.

Figure 7 shows the action of the peptide containing the BbKi reactive site, ESPLRINIIKESYNH₂, in different concentrations: PSA - Pseudomonas aeruginosa (ATCC 27853); E. coli - Escherichia coli (ATCC 25922); E. faecalis - Enterococcus faecalis (ATCC 29212); S. aureus Staphylococcus aureus (ATCC 25923) . 20,6 μM vancomycin is used as positive control for inhibition.

Figure 8 shows BbKI action over Staphylococcus aureus by the microdilution method. Staphylococcus aureus (ATCC 29213) is put in the presence of 0,25; 0,5; 1,0 and 2,0 nM

BbKI. As a positive control for inhibition, vancomycin was used in 34, 68, 136 and 272 nM concentrations.

Figure 9 shows graphically the BbKI action over Staphylococcus aureus by the microdilution method and CFU counts. Staphylococcus aureus (ATCC 29213) is put in the presence of 0,17; 0,34; 0,68; 1,0; 1,36; 1,70 and 2,0 nM BbKI. As a positive control for inhibition, vancomycin was used in 50 nM concentration.

Figure 10 shows, through the culture medium, BbKI action over Staphylococcus aureus by the microdilution method and CFU counts. Staphylococcus aureus (ATCC 29213) is put in the presence of 0,17; 0,34; 0,68; 1,0; 1,36; 1,70 and 2,0 nM BbKI. As a positive control for inhibition, vancomycin was used in 50 nM concentration. DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to the use of protease inhibitors isolated from Bauhinia sp. and/or synthetic peptides related to its primary structure with antimicrobial activity for the treatment of diseases such as, for example, hospital-acquired infections, sepsis, skin diseases such as folliculitis, furunculosis, carbuncle and impetigo, and deep infections such as osteomyelitis, bacteremia (frequently associated with metastatic abscesses), endocarditis, pneumonia and bacterial arthritis caused by pathogenic microorganisms, specially bacterial infectious agents. According to one aspect of the present invention, a pharmaceutical composition comprising an effective amount of at least one Bauhinia sp. protease inhibitor and/or at least one synthetic peptide related to its primary structure is provided comprising the BbKI reactive site with antimicrobial properties. Examples of such inhibitors include but are not limited to BbKI, RPGLPVRFESPLRINIIKENH₂, RPGLPVRFESPLNH₂, GLPVRFESNH₂, ESPLRINIIKESYNH₂, Bauhinia bauhinioides cruzipain inhibitor (BbCI), LAIAIITESFFL-NH₂ and peptides whose synthesis variations could improve the activity such as, for example, the peptides could have alternatives such as terminal carboxyl and so on instead of the terminal amine.

Typically, the pharmaceutical compositions of the present invention comprise at least one inhibitor in accordance with the present invention and at least one pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers include but are not limited to aqueous solutions, non-toxic excipients including salts, preservatives, buffers, more specifically saline solution, sodium phosphate buffer, sodium acetate, HEPES. The pH and the exact concentration of various composition constituents can be adjusted in accordance with the current practice.

Preferably, the pharmaceutical composition of the present invention comprises at least one protease inhibitor isolated from Bauhinia sp. and/or at least one synthetic peptide related to its primary structure in accordance with the present invention, the concentration being in the range of 5 - 500 mg, more preferably from 50 to 100 mg for the treatment of microbial infections.

Next, the present invention will be explained in a more detailed manner with the support of its preferred embodiment description.

The evaluation of BbKI antimicrobial action and peptides related to its primary structure was done in comparison with the following bacteria species: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and

Enterococcus faecalis.

Pseudomonas aeruginosa is an opportunistic Gram- negative bacillus responsible for hospital-acquired infections, chronic lung infection in patients with cystic fibrosis, urinary tract infections and ulcerative keratitis

(Bodey et al., 1983; Pier, 2000) .

Escherichia coli is a Gram-negative bacterium highly resistant to antibiotics. It is present in animal intestinal flora and also contaminating water, fruits, greens, vegetables, among other foods (Buchanan & Doyle,

1997) .

Its virulence is attributed to intestinal surface colonization and to enterotoxin production which induces the secretion of electrolytes and water, thus resulting in diarrhea (Guth, 2000) .

Staphylococcus aureus is a Gram-positive bacterium which is present in the skin flora, nasal mucous and oral cavity. This bacterium has the ability of multiplying and invading tissues. Therefore, it is related to secondary infections such as endocarditis, osteomyelitis, pneumonia, skin infections, meningitis, hospital-acquired infections, and septicemia, among others (Lowy, 2000; Tarkowski &

Wagner, 1998; Sakiniene, 1999). Staphylococcus aureus pathogenicity depends on the expression of the molecules secreted in the bacterial cell wall which will allow colonization and invasion in host cells, activating the proinflammatory cascade and stimulating an immune response (Dinges et al., 2000; Tarkowski et al., 2001) . Host cells invasion by Staphylococcus aureus can occur by four mechanisms: direct binding to epithelial cells; by cellular invasion sites where matrix proteins are exposed; through capillary migration and/or by binding to the host cell through cellular proteins with the production of various extracellular substances such as exotoxins, enterotoxins and leukocidin, cell-bacteria adhesion. Its adherence to the host is made by means of adhesion molecules such as collagen binding, fibronectin binding, among others (Tarkowski & Wagner, 1998; Lowy, 2000) . Enterococcus faecalis is an intestinal Gram-positive bacterium present in human and animal microflora which is responsible for various diseases such as: intestinal infections, endocarditis, meningitis, intra-abdominal and urinary tract infections, among others (Murray, 1998; Trick et al. , 1999) .

It is important to point out that although the invention has been executed with the above-mentioned pathogenic agents, the present invention is not limited to such agents and it can comprise other agents such as Candida albicans and others.

Purification and characterization of human plasmatic kallikrein inhibitor from Baύh±nia bauh±n±oides seed (BbKI) .

The Bauhinia seed inhibitors used to accomplish this work were isolated and characterized according to a previous established methodology described in the art. The purification stages used in BbKi isolation had the extraction with saline solution and its concentration through ketonic precipitation as an initial methodological phase. The chromatographic steps in Con A Sepharose 4B, DEAE-Sephadex A-50 and HiTrap Q, and trypsin-sepharose affinity showed to be effective in obtaining the inhibitor in a homogeneous form verified by the elution of a single peak in reverse phase chromatography of C₁₈ column. The N- terminal sequencing indicated kallikrein inhibitory activity. The process yield was 12% and the purification was of 6224,3 times.

BbKI presence in a single polypeptidic chain was confirmed by the detection of a single N-terminal sequence of the protein purified by ion-exchange chromatography. The sequence was also defined using other preparations. Moreover, the native protein molecular weight determined by mass spectrometry was 18,034, and is very similar to that obtained by the sequence.

The BbKI CD spectrum was registered from 200 to 240 nm (Araύjo et al., 2005). The content of BbKI secondary structure was estimated, with the following results: 44% α-helix, 29% β-sheet, 7% β-loop and 18% disordered. These results are compared with SBTI structure which presents 2% α-helix, 38% β-sheet, 23% β-loop and 37% disordered (Tetenbaum & Miller, 2001) . Nevertheless, the small elevation in the spectrum baseline at 230 nm is attributed to a disulfide bridge which could be explained by the fact that a single cysteine residue located at the C-terminal portion of the BbKI molecule, residue 154, may be involved in the formation of a dimeric molecule by the formation of an intermolecular disulfide bridge when the inhibitor is kept in solution for a long time, as occurs with the inhibitor isolated from Canavalia lineata, which presents a free cysteine (Terada et al., 1994).

The dissociation constant was measured by the determination of the residual enzymatic activity after the pre-incubation of BbKI and different enzymes. The results obtained with trypsin, K₁ = 20 nM, and HuPK, K₁ = 4,7 nM. The stoichiometry of the inhibition reaction shows that one mol of inhibitor, BbKI, reacts with one mol of trypsin to form the 1:1 inhibitor-enzyme complex.

The BbKI reactive site for plasmatic kallikrein was defined and various peptides were synthesized from Pi₃ to P₆' (Figure 1) . The stability of these complexes was periodically analyzed by the invariability of retention time in the reverse-phase column.

The dissociation constants values were calculated using an equation described by Morrison, 1969, assuming a "slow-tight binding" mechanism type. The low Ki value demonstrates the high affinity of the inhibitor with the enzyme, acting as a pseudo-substrate. The amino acid residue in Pi is the main responsible for the inhibitor interaction with the enzyme active site. The residues adjacent to Pi contribute for the inhibitor selectivity because the individual characteristics of each amino acid allow the occurrence of another interactions, such as hydrogen bridges, saline bridges or stereo hindrance, which are essential in the determination of protein inhibitory function (Bode & Huber, 1993, 2000; Wenzel & Tschesche, 1995; Travis & Potempa, 2000) . Comparing BbKI sequence with kininogen sequence in the region that contains Lys-BK, we can verify that BbKI 00298

presents the sequence homologous to this peptide in the reactive site region. In the inhibitor molecule, the HHRP sequence corresponds to kininogen MKRP (Figure 1) . The histidine presence with a positive character is favorable to the inhibitor interaction with the enzyme subsite Si, which is increased by the presence of arginine in P₂' .

Nevertheless, proline in P₃' does not favor enzyme cleavage. This similarity could explain the strong BbKI interaction with kallikreins. Study of the antimicrobial action of BbKI and peptides related to its primary structure

Disc diffusion method

The action of BbKI inhibitor and synthetic peptides related to the reactive site primary structure were evaluated through the inhibition zone (mm) over in vitro

ATCC (American Type Collection Culture) pathogenic bacteria.

The tests made followed NCCLS (National Committee for

Clinical Laboratory Standards) standards and in collaboration with the Laboratόrio Especial de

Microbiologia Clinica - Disciplina de Doenςas Infecciosas e

Parasitarias da Universidade Federal de Sao Paulo - UNIFESP

(LEMC/DIPA) . All the tests were made in triplicate.

The following bacterial strains were used: Pseudomonas aeruginosa (ATCC 27853) ; Staphylococcus aureus (ATCC

25923) ; Enterococcus faecallis (ATCC 29212) ; Escherichia coli (ATCC 25922) .

The inoculum was prepared by the Standard Method - log phase, where 3 to 8 colonies isolated in blood agar medium for 18 to 24 hours were transferred to a tube containing 5 mL of Muller Hinton Broth (Oxoid^®) and incubated at 35°C 000298

for 2 to 6 hours for inoculum standardization. Such standardization was made by the comparison between the inoculum turbidity with the 0,5 MacFarland scale tube (l,5xlθ⁸ CFU/mL) . Plate inoculation

The Mϋller-Hinto Agar plate (Oxoid^®) (150 x 15 x 4 mm) was inoculated (1 mL of inoculum/plate) in three directions with the preparation previously done with the aid of sterile "swabs" and kept for a rest period of 5 to 15 minutes. After this period, the sterile discs were placed following the NCCLS standards (National Committee for Clinical Laboratory Standards) . BbKI and the peptides related to its structure were added to these 6,0 mm diameter discs in various concentrations, according to Table 1. The plate was incubated at 36°C for 18 - 24 hours. The reading of the results was performed after such incubation through the inhibition areas (mm) around the discs .

The bacterial inoculum control in the presence of antibiotics was performed for all previously cited ATCC (American Type Collection Culture) strains.

Table 1 - Concentration of inhibitor and peptides used in the disc diffusion method.

The antimicrobial action of BbKI inhibitor and synthetic peptides related to the reactive site primary structure was evaluated through the inhibition area (mm) over in vitro ATCC (American Type Collection Culture) pathogenic bacteria.

The BbKI inhibitor demonstrated antibacterial activity only over Staphylococcus aureus (ATCC 25923) (Figures 2 and 3) with an inhibition area of 7,5 mm. Peptides with sequence of the BbKI reactive site region were also analyzed: RPGLPVRFESPLRINIIKENH₂ (Figure 4); RPGLPVRFESPLNH₂ (Figure 5); GLPVRFESNH₂ (Figure 6) ; ESPLRINIIKESYNH₂ (Figure 7) .

The most effective action was observed with the GLPVRFESNH₂ peptide which presented an inhibition area of 8 mm in a 88 μM concentration.

The results were compared with 20,6 μM vancomycin antibiotic (Oxoid©) used as a positive inhibition control.

BbKI, as well as the peptides in the respective concentrations, were not effective in these concentrations for Pseudomonas aeruginosa (ATCC 27853) , Enterococcus faecalis (ATCC 29212) and Escherichia coli (ATCC 25922) inhibition. Microdilution method and dose-dependence ratio

The action of BbKI and synthetic peptides related to the reactive site primary structure was evaluated over in vitro ATCC (American Type Collection Culture) pathogenic bacteria.

The tests were performed in collaboration with the Laboratόrio Especial de Microbiologia Clinica - Disciplina de Doenςas Infecciosas e Parasitarias da UNIFESP (LEMC/DIPA) . All the tests were made in triplicate. The bacterial strain used was Staphylococcus aureus

(ATCC 29213) and the inoculum preparation was made by the

Standard Method - log phase, 3 to 8 colonies isolated in blood agar medium for 18 to 24 hours were transferred to a tube containing 10 mL of 0,85% saline solution. This standardization was made by the comparison between inoculum turbidity with the 0,5 MacFarland scale tube (1,5 x 10⁸

CFU/mL) .

50 μL of 0,85% saline solution were inserted in an Elisa plate (96 wells) together with 50 μL of bacterial inoculum and crescent molar concentrations of the inhibitor, in accordance with the values shown in Table 2.

The respective positive and negative controls were done and vancomycin (Oxoid®) in different concentrations was used for the bacterial inoculum control with antibiotic (Table 2) .

Readings were made after a 24 hours incubation period using spectrophotometry (Spectronic Genesys 5) in a 550 nm absorbance . Table 2 - Inhibitor concentrations used in the test.

The BbKI action was evaluated over in vitro Staphylococcus aureus (ATCC 29213) , using the microdilution method.

0,5 nM BbKI demonstrated antibacterial activity over Staphylococcus aureus (ATCC 25923) (Figure 8) .

The results were compared with vancomycin antibiotic in the following concentrations: 34; 68; 136 and 272 nM (Oxoid^®) used as an inhibition positive control. Investigation of BbKI over Staphylococcus aureus using the microdilution method and CFU counting (colony-forming units) The action of inhibitors was evaluated using the microdilution method and colony-forming units counting over in vitro Staphylococcus aureus, ATCC 29213 (American Type Colection Culture) .

The tests made followed NCCLS (National Committee for Clinical Laboratory Standards) standards in cooperation with the Laboratόrio Especial de Microbiologia Clinica -

Disciplina de Doencas Infecciosas e Parasitarias da UNIFESP

(LEMC/DIPA) . All the tests were made in triplicate.

The inoculum preparation was made using the Standard Method - log phase, where 3 to 8 colonies isolated in blood agar medium for 18 to 24 hours were transferred to a tube containing 10 mL of 0,85% saline solution. Such standardization was made by the comparison between inoculum turbidity with the 0,5 MacFarland scale tube (l,5xlθ⁸ CFU/mL) .

Preparation of microdilution plates

In 1 mL tube containing 0,85% saline solution 50 μL of the bacterium inoculum were added together with crescent concentrations of BbKI, in accordance with Table 3. The respective positive controls were made in the absence of the protein and the negative controls were made in the presence of vancomycin (50 nM) . Plate preparation for CFU counting

After 30 minutes of incubation at 36°C, 10 μL aliquots were removed from the tubes and then diluted in 990 μL of 0,85% saline solution. Afterwards, 10 μL were transferred to a Petri plate containing blood agar medium. The plates were incubated at 36°C and CFU quantification was made after 24 hours of incubation. Table 3 - Inhibitor concentration used in the test.

BbKI action was evaluated over in vitro Staphylococcus aureus (ATCC 29213), using the microdilution method.

0,34 nM BbKI demonstrated antibacterial activity over Staphylococcus aureus (ATCC 25923) and demonstrated bactericidal activity at concentrations above 0,68 nM (Figures 9 and 10) .

The results were compared to vancomycin antibiotic at a concentration of 50 nM (Oxoid®) used as an inhibition positive control.

Thus, in brief, the antimicrobial activity was evaluated by two procedures: disc diffusion method and microdilution. BbKI and the peptides

RPGLPVRFESPLRINIIKENH₂, RPGLPVRFESPLNH₂, ESPLRINIIKESYNH₂ and R2008/000298

GLPVRFES-NH₂, whose sequence comprises P13 to Pε' residues of the structure, only presented action against Staphylococcus aureus (ATCC 25923) Gram-positive bacterium, and no effect over Escherichia coli (ATCC 25922) , Pseudomonas aeruginosa (ATCC 27853) and Enterococcus faecalis (ATCC 29212) was observed.

The native inhibitor, 5 μM BbKI (0,1 mg) , presented a 7,5 mm inhibition area while the synthetic peptides related to the reactive site primary structure, 45 μM RPGLPVRFESPLRINIIKENH2 (0,1 mg) , 73 μM RPGLPVRFESPLNH₂ (0,1 mg) , 58 μM ESPLRINIIKESYNH₂ (0,1 mg) and 88 μM GLPVRFESNH₂ (0,1 mg) , respectively presented an area of 7,0, 8,0, 7,0 and 8,0 mm (Figures 2 to 7).

The peptides and BbKI were more effective than the extracts of Mexican medicinal plants Hyptis pectinata and

Hyptis sualveololens, which in a 20 mg concentration present a 6,0 mm inhibition area over Staphylococcus aureus

(Rojas et al . , 1992). The results obtained with the alcoholic extract of Ononis spinosa L. and Bryonia syriaca Boiss, traditional Jordanian plants, in a 4,0 mg/disc concentration and an area of 12 mm (Mahasneh, et al., 1999) were not better than the one obtained with BbKI and RPGLPVRFESPLRINIIKENH₂, RPGLPVRFESPLNH₂, ESPLRINIIKESYNH₂ and GLPVRFESNH₂ peptides. Guava extract, 40 mg, presented an inhibition area between 8 - 10 mm (Gnan et al, 1999) . These results indicate that BbKI and the other related peptides present an important and specific antibacterial action for Staphylococcus aureus bacteria.

The cationic characteristics of the peptides derived from the BbKI structure suggest a strong interaction with the bacterium cellular membrane, causing permeabilization. Nevertheless, the hypothesis of interaction with membrane receptors cannot be eliminated since the structural analysis of the protein database shows that the peptides RPGLPVRFESPLRINIIKENH2, RPGLPVRFESPLNH₂, ESPLRINIIKESYNH₂ and GLPVRFES-NH₂ have a degree of similarity with adhesion and migration proteins. Thus, BbKI and the peptides derived from its structure can also interfere in protein/receptor interaction of the bacterium cellular membrane, preventing the adhesion process and, consequently, its replication. BbKI proved to be more efficient than the peptides derived from its reactive site by the disc diffusion method. Thus, dose-dependence relationship was established by the microdilution method, where BbKI (0,5 - 1,00 nM) inhibits Staphylococcus aureus development (Figure 8) . The results obtained by microdilution and colony- forming units counting method showed BbKI bactericidal activity over Staphylococcus aureus bacterium in concentrations above 0,68 nM (Figures 9 and 10).

Thus, taking into consideration that Staphylococus aureus is the bacteria most frequently responsible for hospital-acquired infections and sepsis, and that such infections are more and more difficult to be treated due to the development of resistance mechanisms, the need for new antimicrobial agents to be used in the treatment of such infections is clear.

Therefore, due the fact that protease inhibitors isolated from Bauhinia sp. and synthetic peptides related to its primary structure presented an important and specific antimicrobial activity for pathogenic agents, especially for Staphylococcus aureus bacteria, as is described above, it is interesting to use such inhibitors and synthetic peptides according to the present invention in the preparation of a pharmaceutical composition for the treatment of microbial infections.

Many modifications and variations of the present invention are apparent to a person skilled in the art without departing from the scope and spirit of the invention. Although the invention was described in relation to a specific preferred embodiment, it should be understood that the invention as claimed should not be improperly limited to such specific embodiment. Certainly, various modifications of the described embodiment to perform the invention which are obvious to a person skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.

Claims

1. Use of protease inhibitors isolated from Bauhinia sp. characterized in that it is in the preparation of a medicine comprising at least one of the referred inhibitors and/or at least one synthetic peptide related to its primary structure for the treatment of microbial infections .

2. Use, according to claim 1, characterized in that the referred at least one inhibitor and/or synthetic peptide is selected from the group consisting of BbKI, RPGLPVRFESPLRINIIKENH₂, RPGLPVRFESPLNH₂, GLPVRFESNH₂, ESPLRINIIKESYNH₂ and variants thereof.

3. Use, according to claim 1 or 2, characterized in that the referred infections include but are not limited to hospital-acquired infections, sepsis, skin diseases such as folliculitis, furunculosis, carbuncle and impetigo, and deep infections such as osteomyelitis, bacteremia, endocarditis, pneumonia and bacterial arthritis.

4. A pharmaceutical composition comprising protease inhibitors isolated from Bauhinia sp. and/or synthetic peptides related to its primary structure for the treatment of microbial infections characterized in that it comprises an effective amount of at least one of the referred inhibitors and/or at least one synthetic peptide related to its primary structure and at least one pharmaceutically acceptable carrier.

5. A pharmaceutical composition, according to claim 4, characterized in that the referred at least one inhibitor and/or synthetic peptide is selected from the group consisting of BbKI, RPGLPVRFESPLRINIIKENH₂, RPGLPVRFESPLNH₂, GLPVRFESNH₂ and ESPLRINIIKESYNH₂ and variants thereof.

6. A pharmaceutical composition, according to claim 4 or 5, characterized in that the referred at least one pharmaceutically acceptable carrier is selected from a group consisting of but not limited to aqueous solutions, non-toxic excipients such as saline solution, sodium phosphate buffer, sodium acetate and HEPES.

7. A pharmaceutical composition, according to claims 4 to

6, characterized in that the referred infections include but are not limited to hospital-acquired infections, sepsis, skin diseases such as folliculitis, furunculosis, carbuncle and impetigo, and deep infections such as osteomyelitis, bacteremia, endocarditis, pneumonia and bacterial arthritis.

8. A pharmaceutical composition, according to claims 4 to

7, characterized in that the referred at least one inhibitor and/or synthetic peptide is within the concentration range of 5 - 500 mg.

9. A pharmaceutical composition, according to claim 8, characterized in that the referred at least one inhibitor and/or synthetic peptide is within the concentration range of 50 - 100 mg.