BRPI0703559B1 - use of protease inhibitors isolated from bauhinia sp. for the treatment of microbial infections and pharmaceutical composition - Google Patents

Abstract

use of protease inhibitors isolated from bauhinia sp. for the treatment of microbial infections and pharmaceutical composition. The present invention relates to the use of protease inhibitors isolated from bauhinia sp. and / or synthetic peptides related to their primary antimicrobial activity structure, as well as a pharmaceutical composition comprising them for the treatment of microbial infections.

Description

(54) Title: USE OF ISOLATED PROTEASE INHIBITORS FROM BAUHINIA SP. FOR THE TREATMENT OF MICROBIAL INFECTIONS AND PHARMACEUTICAL COMPOSITION (51) lnt.CI .: A61K 38/04; A61K 38/03; A61K 38/10; A61K 38/08; A61P 31/10; A61P 31/04 (73) Holder (s): UNIVERSIDADE FEDERAL DE SÃO PAULO - UNIFESP. SÃO PAULO STATE RESEARCH FOUNDATION (72) Inventor (s): MARIA LUIZA VILELA OLIVA; MISAKO UEMURA SAMPAIO; FERNANDA PERUZZO DE CAROLI (85) National Phase Start Date: 10/02/2007

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Invention Patent Descriptive Report for: USE OF DOS

PROTEASE INHIBITORS ISOLATED FROM BAUHINIA SP. TO

TREATMENT OF MICROBIAL INFECTIONS AND PHARMACEUTICAL COMPOSITION.

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

Antimicrobials are essential for the prevention and treatment of infections that are increasingly difficult to be treated, thus requiring more expensive and more toxic drugs. This need is due to the fact that several microorganisms, such as bacteria and fungi, are resistant to antimicrobials due to their own innate metabolic characteristics or due to the resistance mechanism developed by protection.

In between antibiotics more used are the compounds beta-lactams, more specifically at penicillins. Penicillins , a example of all the antibiotics β-lactams, inhibit growth of

r

2/35 bacteria when interfering in a specific stage of cell wall synthesis. They bind covalently to Penicillin-Binding Proteins (PLP), which are responsible for catalyzing transpeptidation in cell wall synthesis. Thus, transpeptidation is inhibited, the peptidoglycan synthesis of the cell wall is blocked and the bacteria dies.

However, penicillins are also among the most used antibiotics inappropriately and in excess, consequently generating resistant strains. Resistance to penicillins and other β-lactams is generally developed by the following mechanisms: inactivation of the antibiotic by the production of βlactamases enzymes; modification of the Binding Proteins of

Penicillin (PLP); impairment of drug penetration to PLP; and presence of an efflux pump. The production of β-lactamases is the most common mechanism of resistance.

Besides that, there is occurrences of reactions in hypersensitivity in some people during The administration of penicillins and other β-lactams. At allergic reactions can consist in shock anaphylactic; disease-like reactions serum like urticaria, fever, edema articular, edema angioneurotic, pruritus intense, and

3/35 difficulty breathing, in addition to several skin rashes.

Penicillins share chemical characteristics, mechanisms of action, pharmacological and clinical effects with cephalosporins, monobactamines, carbapenens and β-lactamase inhibitors, which are also βlactam compounds. Therefore, patients with a history of penicillin anaphylaxis should also not be treated with other β-lactams.

For cases of sepsis or endocarditis caused by methicillin-resistant staphylococci (MRSA), parenteral Vancomycin is usually indicated as the main therapy. Vancomycin is an antibiotic, glycopeptide, produced by Streptococcus orientalis. It is active only against Gram-positive bacteria, particularly staphylococci. It acts by inhibiting the release of the cell wall construction unit (disaccharide + peptide) from the lipid transporter of the bacterial cell membrane, compromising the polymerization of the peptidoglycan.

In the past, strains of enterococci exhibited uniform sensitivity to Vancomycin. However, resistant strains already exist, mainly Enterococcus faecium. The determinants of Vancomycin resistance in E. faecium and E. faecalis are located in a transposon present in a

4/35 conjugative plasmid, allowing its rapid transfer between enterococci and, potentially, to other Gram-positive bacteria. In addition, in 2002 a strain of S. aureus with high resistance to Vancomycin was isolated, where there was a conjugative plasmid in which transposon Van A was integrated as a horizontal gene transfer sequence between E. species.

faecalis for a methicillin-resistant strain of S. aureus.

Vancomycin resistance in enterococci results from a modification in the D-Ala-D-Ala binding site of the cell wall peptidoglycan formation unit, in which the

Terminal D-wing is replaced by D-lactate. As a result, there is a critical loss of a hydrogen bond, which forms a high affinity bond between Vancomycin and its target, with loss of activity. This mechanism is also observed in strains of S. aureus resistant to Vancomycin that have acquired the determinants of resistance of enterococci.

In addition, Vancomycin has the drawback of being made parenterally, as it is poorly absorbed by the gastrointestinal tract, and should be infused over a period of at least 60 minutes to avoid infusion-related adverse reactions, such as erythematous or urticaria, flushing, tachycardia

5/35 and hypotension. Among the most common reactions, the so-called red neck syndrome or red man syndrome stands out. This blushing related to the infusion is not an allergic reaction, but a direct effect of

Vancomycin on mast cells, causing histamine release. Ototoxicity and nephrotoxicity associated with high drug concentrations or interactions with aminoglycosides may also occur.

Aminoglycosides are a group of 10 bactericidal antibiotics originally obtained from various species of Streptomyces. They are most widely used against Gram-negative enteric bacteria, especially in bacteremia and sepsis, in combination with Vancomycin or a penicillin in endocarditis and for the treatment of tuberculosis. Aminoglycosides bind to specific ribosomal proteins of the 3 OS subunit, and act by inhibiting protein synthesis and reducing the translation fidelity of mRNA in the ribosome. They have a rapid bactericidal effect that seems to be strongly correlated with the ability to induce incorrect reading.

Microorganisms can develop resistance to aminoglycosides through the following mechanisms: production of a transferase enzyme or enzymes that inactivate the

6/35 aminoglycoside by adenylation, acetylation or phosphorylation; interference with the entry of aminoglycoside in the cell, through transport proteins; and deletion or alteration of the receptor protein existing in the VOS 3 OS subunit?

ribosome. In addition, all aminoglycosides are ototoxic and nephrotoxic. And, in very high doses, they can have an effect similar to curare, with neuromuscular block resulting in respiratory paralysis.

Another class of antibiotics is tetracyclines, which are broad-spectrum bacteriostatic antibiotics. They act by binding to the 3OS subunit of the bacterial ribosome, blocking the aminoaciltRNA binding to the acceptor site in the mRNA-ribosome complex. This block prevents the addition of amino acids to the growing peptide. There are also some mechanisms of resistance to tetracyclines such as decreasing the influx or increasing the efflux of tetracyclines through an active transport protein pump; ribosomal protection, through the production of proteins that interfere in the binding of tetracyclines to the ribosome; and enzymatic inactivation of tetracyclines.

Bacteria and other pathogens, including fungi, develop virulence factors, such as adhesion molecules, liposaccharides, hemolysins, proteases, which

Ί / 3Β assist in host invasion and colonization, thus facilitating tissue dissemination and destruction during the infection process (Holt & Bramanti, 1991;

Grenier & Mayrand, 2000).

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

Additionally, activation of the complement factor is the host's first response to invasion by pathogens. This process is mediated by proteases and proteolytic enzymes, derived from pathogenic agents, which can alter this system through two mechanisms: generic, such as Pseudomonas aeruglnosas, or specific, such as S. pyogenes that degrades C5a; another mechanism would be the development of an alternative system in which pathogens release proteases that will

8/35 activate the complement factor itself, mainly C5 (Maeda et al., 1999).

In addition, bradykinin mediators are induced by bacterial proteases that, in need of nutrients, directly or indirectly activate the kallikrein-kinin system; the activation of this system occurs due to a release of bradykinin from the kininogen. Bradykinin is a potent inflammatory response mediator capable of generating vasodilation, increased vascular permeability, pain and edema. It is formed by high and low molecular mass kininogens. When metabolized, bradykinin reduces circulating kininogen which amplifies the action of human plasma kallikrein (Travis & Potempa,

2000; Schmaier 2004).

Thus, the mediators of bradykinin and nitric oxide are involved in inflammatory, infectious processes and also in cancer, which can generate a spread, be it bacterial or tumor cells, due to increased vascular permeability, far from the main focus of the pathology. (Maeda et al., 1999; Travis &

Potempa, 2000).

Thus, it is important to emphasize that, due to the various stages of the invasion and colonization of the host by bacteria depend in part on the performance of proteases, the

9/35 protease inhibitors isolated from Bauhínia sp. they could play an important role in containing this bacterial and fungicidal spread, either by the inhibitory effect of proteases from these organisms, or by the generation of fragments with activity against these agents.

Therefore, bearing in mind that microorganisms have already developed mechanisms of resistance to the vast majority of antibiotics used in therapy, including those used in cases of hospital infections caused by

MRSA, like Vancomycin, shows the need to develop a pharmaceutical composition with antimicrobial properties that acts through a mechanism different from those in which resistance has already been developed, as an alternative treatment option for infections caused by resistant microorganisms.

Thus, taking into account that preliminary results indicated that the human plasma kallikrein inhibitor from Bauhínia bauhinioides (BbKI), had an action on the Gram-positive bacteria

Staphylococcus aureus (ATCC 25923/29213) analyzed by the diffusion and microdilution disc method, the development of a pharmaceutical composition from the protease inhibitors isolated from Bauhínia sp. for the treatment of

10/35 infections caused by pathogenic microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are part of this report and are included here in order to illustrate certain aspects of the invention. The present invention can be better understood with reference to one or more of these figures, in combination with the detailed description of the preferred embodiment presented here.

Figure 1 shows the primary sequence of synthetic peptides derived from the region of the reactive BbKI site.

Figure 2 shows the action of BbKI on 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). Vancomycin 20.6 μΜ is used as a positive inhibition control.

Figure 3 shows the quantification of the inhibitory affinity of BbKI across the area of inhibition. (THE)

Staphylococcus aureus (ATCC 25923) in the presence of BbKI 5 μΜ, with the formation of an inhibition area of 7.5 mm; (B) Staphylococcus aureus (ATCC 25923) in the presence of Vancomycin 20.6 μΜ, with an 18 mm inhibition area.

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Figure 4 shows the action of the peptide containing the reactive BbKI site, RPGLPVRFESPLRINIIKENH ₂ , in different concentrations: PSA - Pseudomonas aeruginosa (ATCC 27853);

E. coli - Escherichia coli (ATCC 25922); E. faecalis 5 Enterococcus faecalis (ATCC 29212); S. aureus

Staphylococcus aureus (ATCC 25923). Vancomycin 20.6 μΜ is used as a positive inhibition control.

Figure 5 shows the action of the peptide containing the reactive BbKI site, RPGLPVRFESPLNH ₂ , 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). Vancomycin 20.6 μΜ is used as a positive inhibition control.

Figure 6 shows the action of the peptide containing the reactive BbKI site, GLPVRFESNH ₂ , 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). Vancomycin 20.6 μΜ is used as a positive inhibition control.

Figure 7 shows the action of the peptide containing the reactive BbKI site, ESPLRINIIKESYNH ₂ , in different concentrations: PSA - Pseudomonas aeruginosa (ATCC 27853);

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E. coli - Escherichia coli (ATCC 25922); E. faecalis Enterococcus faecalis (ATCC 29212); 5. aureus

Staphylococcus aureus (ATCC 25923). Vancomycin 20.6 μΜ is used as a positive inhibition control.

Figure 8 shows the action of BbKI on the

Staphylococcus aureus by the microdilution method. Staphylococcus aureus (ATCC 29213) is placed in the presence of BbKI 0.25; 0.5; 1.0 and 2.0 nM. As a positive control of inhibition, Vancomycin was used in the concentrations of

34, 68, 136 and 272 nM.

Figure 9 shows graphically the action of BbKI on Staphylococcus aureus by the microdilution and CFU counting method. Staphylococcus aureus (ATCC 29213) is placed in the presence of BbKI 0.17; 0.34; 0.68; 1.0; 1.36;

1.70 and 2.0 nM. As a positive inhibition control, Vancomycin at a concentration of 50 nM was used.

Figure 10 shows, through the culture media, the action of BbKI on Staphylococcus aureus by the method of microdilution and CFU counting. Staphylococcus aureus (ATCC 29213) is placed in the presence of BbKI 0.17; 0.34; 0.68; 1.0; 1.36; 1.70 and 2.0 nM. As a positive inhibition control, Vancomycin at a concentration of 50 nM was used.

DETAILED DESCRIPTION OF THE INVENTION

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The present invention relates 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 hospital infections, sepsis, skin diseases such as folliculitis, furunculosis, carbuncle and impetigo, and deep infections such as osteomyelitis, bacteremia (often associated with metastatic abscesses), bacterial endocarditis, pneumonia and arthritis caused by pathogenic microorganisms, especially bacterial infectious agents.

In accordance with an aspect of the present invention, a pharmaceutical composition is provided comprising an effective amount of at least one protease inhibitor of Bauhinia sp. and / or at least one synthetic peptide related to its primary structure, comprising the reactive BbKI site, with antimicrobial properties. Examples of such inhibitors include, but are not limited to BbKI, rpglpvrfesplriniikenh ₂ , RPGLPVRFESPLNH ₂ , GLPVRFESNH ₂ ,

ESPLRINIIKESYNH2, Bauhinia bauhinioides cruzipain inhibitor (BbCI), LAIAIITESFFL-NH ₂ , and peptides whose variations in synthesis can improve activity, for example, instead of the terminal amine, the peptides may have alternatives such as terminal carboxyl and so on

14/35 on.

Typically, the pharmaceutical compositions of the present invention comprise at least one inhibitor according to 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, sodium phosphate buffer, sodium acetate, HEPES. The pH and the exact concentration of various components of the composition can be adjusted according to standard practice.

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

In the following, the present invention will be explained in more detail with the help of the description of its preferred modality.

The evaluation of the antimicrobial action of BbKI and peptides related to its primary structure was

15/35 performed against the following bacterial species: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Enterococcus faecalis.

Pseudomonas aeruginosa is a gram-negative, opportunistic bacillus responsible for nosocomial infection, 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 contaminates water, fruits, vegetables, vegetables and other foods (Buchanan &

Doyle, 1997).

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

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

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Tarkowski & Wagner, 1998; Sakiniene, 1999).

The pathogenicity of Staphyiococcus aureus depends on the expression of molecules secreted in the bacterial cell wall that will allow colonization and invasion to host cells, activating the pro-inflammatory cascade and stimulating an immune response (Dinges et al., 2000;

Tarkowski et al., 2001).

Invasion of host cells by Staphylococcus aureus can occur through four mechanisms: direct attachment to epithelial cells; by cell invasion sites where matrix proteins are exposed; through capillarity migration and / or by binding to the host cell via cellular proteins, such as the production of various extracellular substances, such as exotoxins, enterotoxins and leukocidin, cell-bacterial adhesion. Its adhesion to the host is made by means of adhesion molecules, such as binding to collagen, binding to fibronectin, among others (Tarkowski & Wagner, 1998; Lowy, 2000).

Enterococcus faecalis is a Gram-positive bacterium of intestinal origin, present in human and animal microflora and responsible for several diseases, such as: intestinal infections, endocarditis, meningitis, intra-abdominal infections and urinary tract, among others (Murray, 1998;

Trick et al., 1999).

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It is important to note that although the invention has been carried out with the aforementioned pathogens, the present invention is not limited to such agents, and may comprise other agents such as candida albicans and others.

Purification and characterization of the human plasma kallikrein inhibitor from the seed of Bauhinia bauhinioides (BbKI)

The Bauhinia seed inhibitors used for this work were isolated and characterized according to a previously established methodology and described in the technique.

The purification steps used in the isolation of BbKI, had as their initial methodological phase, extraction with saline solution and concentration of it through ketone precipitation. The chromatographic steps in Con A Sepharose 4B, in DEAE-Sephadex A-50 and HiTrap Q, and trypsin-Sepharose affinity proved to be effective for obtaining the inhibitor homogeneously verified by eluting a single peak in the reverse phase ermatography of the Ci ₈ column. N-terminal sequencing indicated kallikrein inhibitory activity. The process yield was 12% and the purification 6224.3 times.

BbKI the presence of a single polypeptide chain was

18/35 confirmed by the detection of a single N-terminal sequence of the purified protein by ion exchange chromatography. The sequence was also defined using other preparations. In addition, the molecular weight of the native protein determined by mass spectrometry was Mr. 18.034 and is very similar to that obtained by the sequence.

The BbKI CD spectrum was recorded from 200 to 240 nm (Araújo et al., 2005). The content in structure secondary in BbKI was estimated, with the next results: 44% a-helix, 29% β-leaf, 7% β-turn

and 18% disorderly. These results are compared with the structure of the SBTI, which presents 2% of a-helix, 38% of β-leaf, 23% of β-loop and 37% of disorderly (Tetenbaum & Miller, 2001). However, the small rise in the spectrum baseline at 230 nm is attributed to the disulfide bridge that could be explained by the fact that a single cysteine residue located in the C-terminal portion of the BbKI molecule, residue 154, may be involved in the formation of a dimeric molecule through the formation of an intermolecular disulfide bridge when the inhibitor is kept in solution for a long time, as occurs with the isolated inhibitor of Canavalía lineata, which presents a free cysteine (Terada et al., 1994).

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The dissociation constant was measured by determining the residual enzymatic activity after pre-incubation BbKI and different enzymes. The results obtained with trypsin, Ki = 20 nM, and HuPK, Ki = 4.7 nM. Stoichiometry of the inhibition reaction shows that one mole of inhibitor, BbKI, reacts with one mole of trypsin to form the 1: 1 inhibitor-enzyme complex.

The reactive site of BbKI for plasma kallikrein was defined and several peptides were synthesized, from P ₁₃ to P ₆ ', Figure 1. The stability of these complexes was analyzed periodically by the invariability of the retention time in the reverse phase column.

The values of the dissociation constants were calculated using the equation described by Morrison,

1969, assuming a slow-tight binding mechanism.

The low Ki value demonstrates the inhibitor's high affinity for the enzyme, acting as a pseudo-substrate. The amino acid residue in P _x is primarily responsible for the interaction of the inhibitor with the active site of the enzyme. The residues adjacent to Ρχ contribute to the selectivity of the inhibitor, since the individual characteristics of each amino acid allow other interactions to occur, such as hydrogen bonds, saline bonds or stereo impedance, which are fundamental in determining the inhibitory function of

20/35 protein (Bode & Huber, 1993, 2000; Wenzel & Tschesche,

1995; Travis & Potempa, 2000).

Comparing the sequence of BbKI with the sequence of kininogen in the region containing Lys-BK, we can verify that BbKI presents the sequence homologous to this peptide in the region of the reactive site. In the inhibitor molecule, the HHRP sequence corresponds to the kininogen MKRP (Figure 1). The presence of histidine with a positive character is favorable to the interaction of the inhibitor with the Si subsite of the enzyme, which is increased by the presence of arginine in P ₂ '. However, P ₃ 'proline does not favor the enzyme divage. This similarity could explain the strong interaction of BbKI with kallikreins.

And all of the antimicrobial action of BbKI and peptides related to its primary structure

Disc Diffusion Method

The action of the BbKI inhibitor and the synthetic peptides related to the primary structure of the reactive site were evaluated, through the inhibition zone (mm), on pathogenic bacteria ATCC (American Type Collection

Culture), in vitro.

The tests carried out followed the standards standardized by the NCCLS (National Committee for Clinical Laboratory Standards) and in collaboration with the Special Laboratory of

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Clinical Microbiology - Infectious Diseases Discipline and

Parasitic diseases of the Federal University of São Paulo - UNIFESP (LEMC / DIPA). All tests were performed 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 using the Standard Method 10 Log phase, where 3 to 8 colonies isolated in blood Agar medium, from 18 to 24 hours, were transferred to a tube containing 5 mL of Müller Hinton Broth (Oxoid®) and incubated at 35 ° C from 2 to 6 hours for standardization of the inoculum. This standardization was carried out by comparing the turbidity of the inoculum with the 0.5 tube on the MacFarland scale (1.5 x ^{10 8} UFC / mL).

Inoculation on the plate

The Müller-Hinto Agar (Oxoid®) plate (150 x 15 x 4 mm) was inoculated (1 mL of inoculum / plate) in three directions, with the preparation carried out previously, with the aid of sterile swabs and maintained for 5 to 15 minutes at rest. After that period the sterile discs were placed in compliance with the NCCLS (National Committee for Clinical Laboratory Standards) standards. In these 6.0 mm discs

22/35 diameter BbKI and the peptides related to its structure in varying concentrations were added, according to the

Table 1. The plate was incubated at 36 ° C for 18-24 hours. The results were read after this incubation through the inhibition areas (mm) around the discs.

Control of the bacterial inoculum in the presence of antibiotics was performed for all the ATCC (American Type Collection Culture) strains previously mentioned. Table 1 - Concentration of inhibitor and peptides used in the diffusion disc method.

INHIBITOR CONCENTRATION (μΜ) BbKI 0.4 0.8 2.5 5.0 rpglpvrfesplriniikenh ₂ 2.7 4.5 27 45 rpglpvrfesplnh ₂ 4.4 7.3 44 73 glpvrfesnh ₂ 6.6 11 55 88 esplriniikesynh ₂ 3.5 5.8 35 58 Vancomycin 30 pg (Oxoid) 20.6

The antimicrobial action of the BbKI inhibitor and synthetic peptides related to the primary structure of the reactive site were evaluated, through the inhibition area (mm), on pathogenic bacteria ATCC (American Type Collection

Culture), in vitro.

The BbKI inhibitor demonstrated antibacterial activity

23/35 only on Staphylococcus aureus (ATCC 25923), Figures 2 and 3, with an inhibition area of 7.5 mm. Peptides with the sequence of the region of the reactive BbKI site were also analyzed: RPGLPVRFESPLRINIIKENH ₂ , Figure 4;

RPGLPVRFESPLNH ₂₁ Figure 5; GLPVRFESNH _{2 f} Figure 6;

ESPLRINIIKESYNH _{2 /} Figure 7. ·

The most effective action was observed with the GLPVRFESNH ₂ peptide, which presented an 8 mm inhibition area at a concentration of 88 μΜ.

¹⁰ The results were compared with the antibiotic

Vancomycin 20.6 μΜ (Oxoid) used as a positive inhibition control.

BbKI as the peptides, in the respective concentrations, were not effective in those concentrations, in the inhibition of

Pseudomonas aerugínosa (ATCC 27853), Enterococcus faecalis (ATCC 29212) and Escherichia coli (ATCC 25922).

Microdilution method and dose-dependency relationship

The action of BbKI and synthetic peptides related to the primary structure of the reactive site were evaluated on pathogenic bacteria ATCC (American Type Collection

Culture), in vitro.

The tests carried out followed in collaboration with the Special Laboratory of Clinical Microbiology - Discipline

24/35 of Infectious and Parasitic Diseases of UNIFESP (LEMC / DIPA). All tests were performed in triplicate.

The bacterial strain used was Staphylococcus aureus 5 (ATCC 29213) and the inoculum was prepared using the Standard Method - Log phase, 3 to 8 colonies isolated in blood Agar medium, from 18 to 24 hours, were transferred to a tube containing 10 mL of 0.85% saline solution. This standardization was carried out by comparing the turbidity of the inoculum with the 0.5 tube on the MacFarland scale (1.5 x 10 ⁸ CFU / mL).

In an Elisa plate (96 wells), 50 µl of 0.85% saline solution were inserted together with 50 µl of bacterial inoculum and increasing molar concentrations of the inhibitor, according to values shown in Table 2.

The respective positive and negative controls were performed, and Vancomycin (Oxoid®), in different concentrations, was used to control the bacterial inoculum with antibiotics, Table 2.

Through spectrophotometry (Spectronic Genesys 5) at 550 nm absorbance the readings were taken after 24 hours of incubation.

Table 2 - Concentrations of the inhibitor used in the test.

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INHIBITOR CONCENTRATION (nM) BbKI 0.25 BbKI 0.50 BbKI 1.00 BbKI 2.00 Vancomycin (Oxoid * ¹ ) 34 Vancomycin (Oxoid *) 68 Vancomycin (Oxoid ^) 136 Vancomycin (Oxord ^) 272

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

BbKI, at a concentration of 0.5 nM, demonstrated antibacterial activity on Staphylococcus aureus (ATCC 25923),

Figure 8.

The results were compared with the antibiotic

Vancomycin in concentrations: 34; 68; 136 and 272 nM (Oxoid ^) used as a positive inhibition control.

Investigation of BbKI on Staphylococcus aureus using the microdilution and UFC counting method (Units

Colony Forming Machines)

The action of the inhibitors was evaluated using the microdilution method and counting the units forming

26/35 colonies on Staphylococcus aureus, ATCC 29213 (American Type Collection Culture), in vitro.

The tests carried out followed the standards standardized by the NCCLS (National Committee for Clinicai Laboratory

Standards) and in collaboration with the Special Laboratory of

Clinical Microbiology - Infectious Diseases Discipline and

UNIFESP parasites (LEMC / DIPA). All tests were performed in triplicate.

The inoculum was prepared using the Standard Method 10 Log phase, where 3 to 8 colonies isolated in blood Agar medium, from 18 to 24 hours, were transferred to a tube containing mL of 0.85% saline. This standardization was performed by comparing the turbidity of the inoculum with the 0.5 tube of the MacFarland scale (1.5 x ^{10 8)}

CFU / mL).

Preparation of microdilution plates

In 1 mL tubes containing 0.85% saline, 50 // L of the bacterial inoculum were added together with increasing concentrations of BbKI, as shown in Table 3. The due positive controls were performed in the absence of the protein and the negative controls were performed in the presence of Vancomycin (50 nM).

Preparation of UFC count plates

After 30 minutes of incubation at 36 ° C,

27/35 10 μΐ aliquots of the tubes and then diluted in 990 μΐ of 0.85% saline. Then, 10 μΐ were transferred to a Petri dish containing blood Agar medium. The plates were incubated at 36 ° C and the CFU quantification was performed after 24 hours of incubation.

Table 3 - Concentration of the inhibitor used in the test.

INHIBITOR CONCENTRATION (nM) BbKI 0.17 BbKI 0.34 BbKI 0.68 BbKI 1.00 BbKI 1.36 BbKI 1.70 BbKI 2.00 Vancomycin (Oxoid'j 50

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

BbKI, at a concentration of 0.34 nM, demonstrated antibacterial activity on Staphylococcus aureus (ATCC 25923), and at concentrations above 0.68 nM it demonstrated bactericidal activity, Figures 9 and 10.

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The results were compared with the antibiotic Vancomycin, at a concentration of 50 nM (Oxoid), used as a positive inhibition control.

Thus, in summary, the antimicrobial action was evaluated by two procedures: disk diffusion method and microdilution. BbKI, and peptides

RPGLPVRFESPLRINIIKENH ₂ , RPGLPVRFESPLNH ₂ , ESPLRINIIKESYNH2 and

GLPVRFES “NH ₂ , whose sequence comprises the P ₁₃ to Pg 'residues of the structure, showed action only against Gram-positive bacteria Staphylococcus aureus (ATCC 25923), with no effect on Escherichia coli (ATCC 25922), Pseudomonas aeruginosa ( ATCC 27853) and

Enterococcus faecalis (ATCC 29212).

The native inhibitor, BbKI at 5 μΜ (0.1 mg), showed an area of inhibition of 7.5 mm, while synthetic peptides related to the primary structure of the reactive site, 45 μΜ (0.1 mg) of RPGLPVRFESPLRINIIKENH ₂ , 73 μΜ (0.1 mg) of RPGLPVRFESPLNH ₂ , 58 μΜ (0.1 mg)

ESPLRINIIKESYNH2 and 88 μΜ (0.1 mg) GLPVRFESNH ₂ had an area of 7.0, 8.0, 7.0 and 8.0 mm respectively (Figures 7).

Peptides and BbKI were more effective than extracts from Mexican medicinal plants Hyptis pectinata and Hyptis sualveololens, which at a concentration of 20 mg,

29/35 shows an area of inhibition of 6.0 mm over Staphylococcus ãureus (Rojas et al., 1992). The results obtained with alcoholic extract of Ononís spínosa L. and Bryonia syriaca Boiss, traditional plants of Jordan, concentration of 4.0 mg / disc and 12 mm of area (Mahasneh, et al., 1999), were not better than those obtained with BbKI and peptides RPGLPVRFESPLR1NIIKENH ₂ , RPGLPVRFESPLNH ₂ ,

ESPLRINIIKESYNH ₂ and GLPVRFESNH ₂ . The guava extract, 40 mg, showed an area of inhibition between 8-10 mm (Gnan et al, 1999). These results indicate that BbKI and related peptides have an important and specific antibacterial action for the bacterium Staphylococcus aureus.

The cationic characteristics of peptides derived from the BbKI structure suggest a strong interaction with the bacterial cell membrane causing permeabilization. However, the hypothesis of interaction with membrane receptors cannot be eliminated since the structural analysis in the protein database shows that the peptides

RPGLPVRFESPLRINIIKENH2, RPGLPVRFESPLNH ₂ , ESPLRINIIKESYNH ₂ and

GLPVRFES-NH ₂ have similarity with adhesion and migration proteins. Thus, BbKI and the peptides derived from its structure may also be interfering with the protein / receptor interaction of the bacterial cell membrane,

30/35 preventing the adhesion process and consequently its replication.

BbKI, through the diffusion disc method, was more efficient than peptides derived from its reactive site. Then, using the microdilution method, a dose-dependency relationship was established, where BbKI (0.5 - 1.00 nM) inhibits the development of

Staphylococcus aureus (Figure 8).

The results obtained through the microdilution method and counting the colony-forming units, showed the bactericidal action of BbKI on the Staphylococcus aureus bacteria, in concentrations above 0.68 nM (Figures 9 and 10).

Thus, taking into account that Staphylococus aureus is the most frequent bacterium responsible for nosocomial infection and sepsis, and that such infections are increasingly difficult to be treated due to the development of resistance mechanisms, the need for new antimicrobial agents for used to treat these infections.

Therefore, considering that protease inhibitors isolated from Bauhinia sp. and the synthetic peptides related to its primary structure showed an important antimicrobial action and

31/35 specific for pathogens, especially for the bacterium Staphylococcus aureus, as described above, it is interesting to use such inhibitors and synthetic peptides, according to the present invention, for the elaboration of a pharmaceutical composition for the treatment of microbial infections.

Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to the specific preferred embodiment, it should be understood that the invention as claimed should not be improperly limited to that specific embodiment. Certainly, various modifications of the described modality to carry out the invention that are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.

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Claims (2)

1. Pharmaceutical composition for the treatment of microbial infections characterized by containing: - inhibitors and / or synthetic peptides selected from the group consisting of: Bauhinia bauhinioides kallikrein inhibitor (BbKI), SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 and variants. - a pharmaceutically acceptable carrier, selected from the group of: non-toxic excipients, such as saline, sodium phosphate buffer, sodium acetate, HEPES. 2. Pharmaceutical composition, according to claim 1, characterized in that said inhibitors and / or peptide are in a concentration of 5 to 500mg, being preferably from 50 to 100mg. 3. Use of the composition as defined in claim 1, characterized in that it is directed at bacterial infections such as hospital infections, sepsis, skin diseases such as folliculitis, furunculosis, carbuncle and impetigo, and deep infections such as osteomelitis, bacteramy, endocarditis, pneumonia and bacterial arthritis. Petition 870180062478, of 7/19/2018, p. 8/10 r / Ί -Ρ- ¹ '·. V? BbKI HHRP-G FESPLRINI v 7 RP-G FESPLRINI v r i 1 RP · GFESPL .p- ¹ '·. I ϊ G F E S ESPLRINIIKESY FIGURE 1 2/7 Inhibition area (mm) FIGURE 3 $ 3/7 rpglpvrfesplriniikenh ₂ (μΜ) FIGURE 4 RPGLPVRFESPLNH (μΜ) FIGURE 5 * 4/7 <? GLPVRFESNHí (μΜ) FIGURE 6 zone ESPLRINIIKESYNH (pM) FIGURE 7 * 5/7 Α550 _Λ 2 Ί FIGURE 8 * * F '6/7 FIGURE 9 FIGURE 10