BRPI0703175B1 - ANTI-INFLAMMATORY AND ANTIALERGIC CYCLIC PEPTIDES - Google Patents

Abstract

anti-inflammatory and anti-allergic cyclic peptides. The present invention relates to synthetic cyclic peptides comprising a sequence of 13 l-amino acids in their primary structure, which exhibit anti-inflammatory and antiallergic activities useful for the treatment of acute and chronic inflammation and / or allergies and are particularly useful for treatment of acute or chronic allergic asthma. The invention also discloses a pharmaceutical composition containing such peptides, use and method for treating or preventing acute and / or chronic inflammatory and / or allergic disorders.

Description

ANTI-INFLAMMATORY AND ANTI-ALLERGIC CYCLIC PEPTIDES

Field of the Invention

The present invention ^Z refers to synthetic cyclic peptides comprising a sequence of 13 L-amino acids in their primary structure, which have anti-inflammatory and antiallergic activities, useful for the treatment of inflammations and / or allergies, acute or chronic, being particularly useful for the treatment of acute or chronic allergic asthma. The invention also describes a pharmaceutical composition containing such peptides, use and method for treating or preventing inflammatory and / or allergic, acute and / or chronic disorders.

Abbreviations

The following abbreviations will be used throughout the specification:

SeqOl refers to The Seq.ID.n ° 01; Seq02 refers to The Seq.ID.n ° 02; Seq03 refers to The Seq.ID.n ° 03; Seq04 refers to The Seq.ID.n ° 04; SeqOS refers to The Seq.ID.n ° 05; Seq06 refers to The Seq.ID.n ° 06; Seq07 refers to The Seq.ID.n ° 07; Seq08 refers to The Seq.ID.n ° 08; Seq09 refers to The Seq.ID.n ° 09.

Fundamentals of the Invention

Inflammation is a homeostatic, dynamic and protective response that the host has against the various aggressions to which it is subjected. The acute phase of inflammation is characterized by changes in vascular caliber that lead to increased blood flow and structural changes in the microcirculation, allowing plasma proteins and leukocytes to leave the circulation which is accompanied by a series of cellular and tissue events aimed at recovery of homeostasis.

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In relation to tissue events, in this phase the recruitment of leukocytes to the injury site is observed, in which the predominant influx is primarily of neutrophils followed by the recruitment of mononuclear cells, this influx being responsible for the resolution of the inflammatory process, removing the harmful agent and restoring tissue homeostasis.

Neutrophils are the first cells to reach the lesion site and they predominate numerically in the acute period of tissue injury. They play an important role in the defense of the organism, as they can phagocytize and destroy the harmful agent. In addition, they can be activated by cytokines produced by macrophages residing in tissue and endothelial cells.

Once the monocytes in the blood leave the circulation and enter the injury site, they differentiate into macrophages, which are the final effector cells of the inflammatory reaction. Macrophages phagocytize foreign particles and destroy them through the synthesis of reactive oxygen species such as nitric oxide. Sequentially, the activated macrophage acts by removing cell debris, inducing antigen elimination and resolving the inflammatory reaction, as well as initiating the adaptive immune response.

Regarding cellular events, leukocytes recruited to the lesion site initiate the process of secretion of pro-inflammatory or alert cytokines (IL1, TNF-α and IL-6) that trigger a series of reactions

humorous and cellular, both at the site of inflammation and

distance.

The activation of stromal cells by cytokines causes secretion of chemotactic chemokines for neutrophils and cells mononuclear cells. At the site of inflammation

initially vasodilation of post-capillary venules and changes in blood flow (deceleration), resulting in

3/24 leukocyte marginalization along the vascular endothelium, a process mediated by selectins and their ligands, rich in carbohydrates.

When leukocyte activation occurs, the bearing stops with firm adhesion to the endothelium, this event results from the binding of βΐ and β2 integrins expressed in leukocytes to several members of the immunoglobulin superfamily expressed in the endothelium (ICAM-1, ICAM-2 and

VCAM-1). Finally, leukocytes migrate between endothelial cells in the apical region to the basolateral surface (diapedesis) towards the extravascular space. Subsequent subendothelial migration through extravascular tissue is dependent on gradients of chemokines, chemotactic cytokines and adhesion interactions with the extracellular matrix. In the end, already in the inflammatory focus, the leukocytes expand their cytotoxic functions, releasing oxidants, proteases and other products such as growth factors and cytokines. Eosinophils unlike neutrophils can survive in tissues for long periods, sometimes weeks, depending on the cytokines of the microenvironment

Regarding the cytokines involved in this process, pro-inflammatory cytokine TNF-α, produced by monocytes, macrophages, activated NK cells and T lymphocytes, increases the expression of endothelial cell adhesion molecules; activates neutrophils; stimulates macrophages to produce IL-1, IL6, and IL-8; increases the expression of MHC class I; increases the synthesis of prostaglandins by cells of the hypothalamus; acts on hepatocytes in the production of serum amyloid protein; suppresses the cell division of the marrow; reduces tissue perfusion (by decreasing myocardial contractility); relaxes the tone of vascular smooth muscle and promotes intravascular thrombosis.

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The cytokine IL ~ 1, produced by macrophages, endothelial and epithelial cells, promotes the synthesis of IL-2 by T lymphocytes and the differentiation of B lymphocytes; promotes the synthesis of prostaglandins; increases the proliferation of T and B lymphocytes; acts on macrophages inducing the synthesis of IL-1, IL-6, TNF-α and IL-8; the synthesis of acute phase proteins; cachexia; increases the cell division of the marrow.

The cytokine IL-6, produced by macrophages, endothelial cells and T lymphocytes, acts on the hepatocyte in the production of fibrinogen; growth factor for activated B lymphocytes; increases the cell division of the marrow.

These cellular and tissue events cause effects characterized by pain, increased temperature, erythema, edema and loss of function at the injury site during inflammation. Failures in the regulation of the control of the inflammatory process happen and can cause the destruction of epithelial, endothelial or muscle cells and promote dysfunction of the cell or the affected organ.

Asthma, for example, is a chronic pulmonary inflammatory disease characterized by episodes of bronchoconstriction and pulmonary eosinophilic inflammation. Bronchoconstriction of the acute phase is caused by pharmacological mediators released by mast cells activated by the binding of the allergen to the allergen-specific IgE molecules on its surface and the bronchial reactivity of the late phase is associated with severe pulmonary eosinophilic inflammation. T lymphocytes, mainly of the Th2 subtype, orchestrate these responses through the production of cytokines such as IL-4, IL-5, IL-9 and IL13 that are genetically controlled.

The presentation of antigen through mature lung dendritic cells is the basis of the Th2 sensitization process that takes place in patients with allergies or in animals exposed to an aeroallergen.

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Evidence of inflammation in asthma was initially derived from findings of preferential accumulation of CD2 ⁺ T lymphocytes of the Th2 type, mast cells and macrophages in the lungs of patients with fatal asthma. Analyzes of bronchoalveolar lavage 5 in asthmatic patients during late phase reactions (LPR) also reveal the presence of a high number of CD4 ⁺ T lymphocytes and eosinophils.

Activated CD4 ⁺ T cells are the main source of cytokines (IL-4, IL-5, IL-9 and IL-13) which are responsible for the development of a pulmonary allergic inflammatory response, typically Th2, with a predominance of an eosinophilic infiltrate and IgE production. Eosinophils are attracted to the lung through the action of several factors such as cytokines and chemokines that promote chemotaxis, the increase in the expression of adhesion molecules in the vascular endothelium and on the cell surface, cell activation and transmigration.

Once activated, eosinophils release their granular content (main basic protein, neurotoxin derived from 20 eosinophils, eosinophil cationic protein and eosinophil peroxidase), which in addition to being toxic to helminths and bacteria, directly stimulates bronchial smooth muscle, causing contraction and promoting increased reactivity towards cholinergic mediators.

In an already established pulmonary inflammation, eosinophils are able to amplify the response, recruiting new cells, due to the localized production of pro-inflammatory cytokines. Chronic eosinophilic inflammation and IgE antibodies have been directly associated with asthma severity 30.

Chronic inflammation in asthma is accompanied by pulmonary hyperreactivity (increased response to bronchoconstriction

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4 * to nonspecific stimuli) and airway remodeling.

Many factors can trigger an asthma attack, including allergens, bacterial or viral infections, exercise, abrupt weather changes, or exposure to airway irritants, such as tobacco smoke. The main causes of asthma triggering in children under the age of age are viral infections caused by parainfluenza (PIV), respiratory syncytial virus (RSV) and picornavirus (rhinovirus and enterovirus).

Sensitive asthmatic individuals, when exposed to an allergen in aerosol form, manifest symptoms that begin within 5 to 10 minutes. During this immediate phase of asthma, bronchial smooth muscle contraction, local edema formation, increased mucus secretion by the bronchial epithelial glands and a small cellular infiltrate are observed. These characteristics are explained by the action of mediators such as histamine, prostaglandin D2 (PGD2), leukotriene C4 (LTC4) and platelet activating factor (PAF - platelet activating factor) and cytokines TNF-α and IL-1 released by mast cells activated by allergen-specific IgE antibodies.

According to estimates by the National Center for Health Statistics of the Center for Disease Control and Prevention (NCHS-CDC, 2005, Hyattsville, MD) approximately 22.2 million people suffer from asthma in the United States, including 6, 5 million are under the age of 18. In the United States alone, about 11 people die from asthma a day. In Brazil, estimates show that 10% of the population has asthma with 2500 deaths per year, with 4 being ^the cause of hospitalization in the Unified Health System. Among asthmatic patients, 60% are difficult to control.

Currently, treatments for inflammation and asthma are aimed at using medications divided into two

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7/24 categories: 1) drugs for the improvement of acute symptoms (60% of asthmatics) represented mainly by β ₂ agonists with fast onset, ipratropium bromide which is an anticholinergic agent quaternary ammonium and aminophylline which is a derivative of xanthine that causes muscle relaxation for maintenance, used smoothly of the bronchi; and 2) drugs to prevent asthma symptoms, mainly represented by inhaled and systemic corticosteroids.

However, the use of these drugs, especially in prolonged or repeated cases, has limitations determined by the non-specificity of action and the consequent unwanted side effects to the patient. In addition, current anti-inflammatory medications are not able to significantly prevent loss of lung function in asthma.

Drugs that aim to treat inflammation while potentially minimizing the risks of side effects and that are more specific are constantly being investigated and some examples can be mentioned.

WO 03/070194 presents a proposal to esterify corticosteroids with α-amino acids and can function as useful prodrugs for the treatment of rhinitis and asthma, particularly by inhalation, and for the treatment of inflammation, particularly by local or topical administration. However, the advantage presented by the proposed prodrugs is not clear, and the proposed treatment still depends on the actions of corticosteroids.

WO 2004/068928 relates to peptides isolated from the defensive secretion of the toad Bombina maxima that are bradykinin B ₂ agonists and can be used to treat and / or prevent disorders associated with bradykinin including disorders

8/24 cardiovascular diseases, inflammation, asthma, allergic rhinitis, pain, angiongenesis, among others.

In another document, US 2003/0152564, a peptide corresponding to positions 62-71 of the human C-reactive protein sequence is presented, which is capable of inhibiting, in vitro, the enzymatic activity of human leukocyte elastase and / or cathepsin Human G can be used for the treatment of chronic inflammatory conditions such as rheumatoid arthritis, pulmonary emphysema, cystic fibrosis, bronchitis, asthma and some acute syndromes of respiratory disorders.

US 2007/0123455 proposes a method and compositions that comprise human proteins S100A8 and / or S100A9 and are suitable for treating inflammatory disorders such as allergy, asthma, atherosclerosis, autoimmune diseases, infections, among others.

US patent 5,290,762 describes a method for the prophylaxis or treatment of inflammatory diseases in a patient, which comprises administering to the injury site an amount of at least one serine protease inhibitor. The document claims as a group of serine protease inhibitors, any inhibitor of leukocyte protease secretion, C-reactive protein, serum amyloid A protein, alpha-2-macroglobulin, alpha-2-antiplasmin.

The involvement of serine protease inhibition for the treatment of inflammation has been extensively investigated. For example, human tryptase is a serine protease, found in mast cells, similar to trypsin. Tryptase is a mediator of a number of allergenic and inflammatory pathologies, including rhinitis, conjunctivitis and asthma. Thus, inhibitors of this serine protease can be used successfully for the treatment of respiratory and allergic diseases.

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Among the serine protease inhibitors, the best studied are the peptides described by Bowman [Poc Soc Expd Med 1946 63: 547] and Birk et al [Bull Res Coucil Israel, Sec A 1962 11:48; Biochem Biophys Acta 1963 67: 326] (where the name BBI - Bowman Birk Inhibitors came from). BBIs are found abundantly in dicotyledonous and monocotyledonous plants, among which stands out the one called SFTI-1 (sunflower trypsin inhibitor-1) which is a bicyclic peptide, composed of 14 amino acids and, until today, is the smallest and more potent natural Bowman-Birk inhibitor. SFTI-1 has been used against pathogens and insects in transgenic plants, but it can also be used to prevent cancer, dengue and other inflammatory diseases and

In addition to peptides being obtained from substances produced for example, they are also targets that can be considered as nature capable of reaching allergies.

with pharmacological applications may be produced by plants, animals, as poisons by investigation. Animal toxins molecules developed by specific and selective targets, inhibiting or stimulating physiological reactions.

Particularly noteworthy are the proteins called natterins that are isolated from the poison of the fish Thalassophryne nattereri found in Brazil. These proteins form a family of toxins with molecular mass around 38 kDa and whose sequences have high homology with each other and are capable of inducing innumerable biological activities such as edema and nociception.

From the natterins, it was possible to observe sequences of amino acids that are not obtained per se through the purification of the natural poison, which meanwhile stood out due to the similarity with the structures of the Bowman-Birk type peptides. The attempt to isolate such amino acid sequences from natterins is frustrated, since r

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J * requires a long and laborious process, resulting in very small amounts of fragments of impure peptides, not exactly corresponding to the peptides of interest of this invention. Such difficulty prevents their industrial application.

Summary obtained from the present invention by peptide synthesis sequence showing 4 positions

L-amino acids have artificial cyclic peptides comprising one in its primary structure, the amino acid cysteine in those specifically in the peptide chain and the amino acid lysine at position 11 of the peptide chain, being characterized by containing a disulfide bridge formed through the thiol group of the residues of cysteine at positions 4 and 13, forming a cyclic peptide. Said synthetic cyclic peptides have anti-inflammatory and anti-allergic activity, particularly with potential application for the treatment of acute and / or chronic asthma.

Description of the Figures

Figure 1: Michaelis-Menten kinetics obtained for the hydrolysis of the substrate Abz-FRSSRQ-EDDnp by the catalytic activity of trypsin.

Figure 2: Effect of the different types of Seq on the leukocyte bearing induced in the microcirculation by LPS (lipopolysaccharide). * p <0.05 compared to the LPS group.

Figure 3: Effect of the change of essential amino acids in the sequence of Seq07 and Modi, ys / Aia ^{on the} leukocyte bearing induced in the microcirculation by LPS (lipopolysaccharide). * p <0.05 compared to the LPS group.

Figure 4: Seq07 prevention of LPS-induced peritonitis (lipopolysaccharide). Mice injected with LPS (10 gg / mL) were treated 30 minutes before with Seq07 t

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11/24 intraperitoneally (4 or 40 nmol). The figure represents the count of the total number of cells (A), neutrophils (B) and macrophages (C) of the peritoneal lavage, after twenty-four hours. The results are expressed as the mean ± SEM. * p <

0.05 compared to the LPS group.

Figure 5: Effect of Seq07 (4 or 40 nmol)

recruitment of leukocytes and eosinophils in bronchoalveolar lavage in acute asthma.

The bronchoalveolar lavage of mice was subjected to counting the total number of cells (A) and eosinophils (B). The results represent the mean ± SEM of 6 animals / group. * p <0.001 compared to the Asthma group.

Figure 6: Effect of Seq07 on leukocyte and eosinophil recruitment in bronchoalveolar lavage in chronic asthma. The bronchoalveolar lavage was subjected to counting the total number of cells (A) and eosinophils (B). The results represent the mean ± SEM of 6 animals / group. * p <0.001 compared to the Asthma group

Detailed Description of the Invention

The present invention relates to synthetic cyclic peptides comprising a sequence of 13 L-amino acids in their primary structure, preferably presenting protective chemical groups at one or both ends of the peptide, said peptides have anti-inflammatory and anti-allergic activity.

The peptides of the present invention have the amino acid cysteine at positions 4 and 13 of the peptide chain and the amino acid lysine at position 11 of the peptide chain.

In addition, the peptides included in the present invention preferably have the amino acids valine or leucine or isoleucine or another hydrophobic and neutral amino acid in position 1 of their basic sequence.

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In addition, the peptides included in the present invention preferably have the amino acids arginine or glutamine or another hydrophilic and polar amino acid, and may have a positive charge, in position 3 of its basic sequence.

In addition, the peptides included in the present invention preferably have the amino acids arginine or threonine or serine or aspartate or another amino acid

hydrophilic and polar in position 5 of its basic sequence.

In addition, the peptides included in the present invention preferably have the amino acids methionine or isoleucine or leucine or another hydrophilic and neutral amino acid at position 7 of its basic sequence.

In addition, the peptides included in the present invention preferably have the amino acids glycine or aspartate in position 9 of their basic sequence.

In particular, the peptides of the present invention are characterized by containing a disulfide bridge formed through the thiol group of the cysteine residues of positions 4 and 13, forming a cyclic peptide.

Chemical protection of the peptide molecule can be accomplished by adding protective chemical groups to the carboxy-terminal end of the peptide, or to the amino-terminal end of the peptide, or by protecting both ends of the molecule. Preferably, the protection of the molecule can occur by amidation of the carboxyl of the amino acid cysteine of position 13 of the peptides of the present invention, or by the acetylation of the amino acid residue of position 1 of the peptide chain, or even by the protection of both the carboxy-terminal end and the protection of the amino- terminal simultaneously.

In the main embodiment of the present invention, peptides are characterized by the amino acid sequences identified in this document by the abbreviations SeqOl, * 13/24

Seq02, Seq03, Seq04, Seq05, Seq06, Seq07, Seq08 and Seq09 and derivatives, homologues, analogs and / or mimetics thereof are also included in the scope of this invention.

Within the scope of the present invention are also the 5 amino acid substitutions, deletions or modifications that can be understood in an obvious manner, by a person skilled in the art, from the peptides taught here.

Examples of these modifications, but not limited to them, are: the individual or alternate or joint replacement of proline residues by hydroxylated proline (hydroxyproline); the individual or alternate or joint replacement of methionine residues by oxidized methionine; and the individual or alternate or joint replacement of L-amino acids by D-amino acids.

Also within the scope of the present invention are the larger peptides that include in their basic sequence the amino acid sequences described here. Similarly, peptides having deletions of positions 1, 2 and 3 as described herein, while maintaining the cyclic structure 20 that occurs between the amino acids of positions 4 to 13, are also included in the scope of the present invention.

Cyclic peptides included in the present invention, alone or in combination containing at least two of the peptides, have the ability to inhibit leukocyte adhesion and bearing in laboratory tested mammals. In addition to inhibition of leukocyte adhesion and rolling, there is a decrease and prevention of leukocyte recruitment, particularly neutrophils and macrophages, in the peritoneal cavity of laboratory guinea pigs 30 injected with the inflammatory agent LPS (lipopolysaccharide), with treatment with representatives of the peptides of the present invention.

In mammals immunized and challenged with ovalbumin, which causes the manifestation of acute murine asthma, f

14/24 cyclic peptides of the present invention alone or in combination containing at least two of the peptides, administered by the intramuscular, intranasal and oral routes, are also shown to inhibit the recruitment of the total number of leukocytes to the alveolar space, a decrease characterized particularly due to the absence of eosinophils in the bronchoalveolar lavage.

The cyclic peptides of the present invention alone or in combination with at least two of the peptides, are also capable of decreasing the total number of leukocytes recruited into the alveolar space, with a decrease in eosinophils, in a conventional experimental model for the development of chronic asthma in animals.

In view of the characteristics reported above, it is observed that the cyclic peptides of the present invention alone or in combination containing at least two of the peptides, are capable of inhibiting, in mammals, inflammatory manifestations, pathological allergic manifestations where the influx of eosinophils such as asthma, bronchitis, 20 rhinitis and dermatitis, as well as associated pathological manifestations such as arthritis, disorders of the immune system, lymphocyte dysfunction during the immune response, tumors, cell adhesion and / or manifestations of parasitic diseases. The results obtained for asthma were compared with the conventional treatment of asthma, using dexamethasone, in which the peptides of the present invention were equally efficient.

Additionally, it is observed that the cyclic peptides of the present invention are resistant to digestion with trypsin and can be administered orally. Other routes of administration that can be used for the peptides of the present invention, but not limited to them, are the intranasal, intramuscular and intravenous routes.

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In addition to being resistant to the action of trypsin, the peptides of the present invention are also capable of inhibiting the proteolytic action of the enzyme trypsin presenting Ki in the range of 20 to 63.6 nM. This property of the peptides of the present invention suggests that such peptides can be classified as inhibitors of serine protease, since trypsin is an important enzyme representing the

family of serine proteases. Knowing that human tryptase is a serine protease found in mast cells, being very similar to trypsin, and that its inhibition has been successful in the treatment of respiratory and allergic diseases, it can be suggested that the activity verified for peptides in present invention is related to the inhibition of serine proteases.

The peptides of the present invention have no cytotoxic effect on macrophages, as well as having no harmful effect on muscle fibers and on the microcirculation of the cremaster muscle of animals in the laboratory.

Furthermore, the peptides of the present invention alone or in combination have not been shown to be immunogenic, as they do not induce the synthesis of specific antibodies and, above all, do not induce lethality.

In one aspect of the present invention, the cyclic peptides SeqOl, Seq02, Seq03, Seq04, Seq05, Seq06, Seq07, Seq08 and Seq09 can be obtained by employing known peptide synthesis techniques, such as classic chemical synthesis in solution or in solid phase; enzymatic synthesis;

or through the recombinant DNA technique.

The chemical synthesis of the cyclic peptides of the present invention can preferably be carried out in solid phase, and, more preferably, with the use of the FMOC (9-Fluorenylmethoxycarbonyl) strategy to provide protection to the alpha-amino groups present in the reaction.

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Purification of synthetic peptides can be carried out by chromatography techniques known in the art. Preferably, liquid chromatography is used, the purities and identities of the peptides being confirmed by mass spectrometry.

After the purification of the peptides synthesized according to the above description, there is the stage of formation of the disulfide bridges for each peptide. Disulfide bridges occur between the cysteine residues of positions 4 and 13 in the same peptide chain by the air reduction method, for example, by dissolving the peptide in ammonia bicarbonate solution between 0.01 and 0.5M in a proportion of 0.05 to 15 mg / mL with stirring until complete disulfide bridge formation. Each peptide can be isolated by lyophilization and purified using a preparative liquid chromatography system.

Another object of the present invention relates to a pharmaceutical composition containing the peptides SeqOl, Seq02, Seq03, Seq04, SeqOS, Seq06, Seq07, Seq08 or Seq09, their derivatives, homologues, analogs and / or mimetics, which may be in the form of pharmaceutically acceptable salts thereof, alone or in combination containing at least two of the peptides, and one or more vehicles comprised within a diluent, excipient or pharmaceutically acceptable solvent.

Another object of the present invention is the method for treating or preventing inflammatory and / or allergic disorders, acute or chronic, such as, but not limited to, bronchial asthma, rhinitis, arthritis, atopic dermatitis, immune system disorders, lymphocyte dysfunction during immune response, tumors, cell adhesion and / or manifestations of parasitic diseases, through the administration, to a mammal, of the cyclic peptides SeqOl, Seq02, Seq03, Seq04, SeqOS, Seq06, Seq07, Seq08 or Seq09, alone or in combination, or in %

17/24 τί pharmaceutical compositions containing at least one of the peptides of the present invention.

Thus, another object of the present invention is the use of the cyclic peptides SeqOl, Seq02, Seq03, Seq04, Seq05, Seq06, Seq07, Seq08 and Seq09, alone or in combination containing at least two of the peptides, in the manufacture of drugs used in the prevention or treatment of inflammatory and / or allergic disorders, acute or chronic, such as, but not limited to, bronchial asthma, rhinitis,

arthritis, atopic dermatitis, immune system disorders, lymphocyte dysfunction during the immune response, tumors, cell adhesion and / or manifestations of parasitic diseases. According to this development, the synthetic cyclic peptides of the present invention, alone or in combination containing at least two of the peptides, can be used to obtain a prodrug or drug that inhibits the onset of inflammatory and / or allergic conditions in mammals.

The administration of the cyclic peptides of the present invention or pharmaceutical compositions containing them, can be carried out, but not limited, by the intramuscular, intranasal, intravenous and oral routes, the oral route being the preferred route of administration.

The examples cited below are merely illustrative, and should be used only for a better understanding of the developments contained in the present invention; however, they should not be used in order to limit the objects described.

EXAMPLE 1A: Trypsin Inhibition

Studies of inhibition of trypsin with the peptide SeqOl revealed a simple competitive inhibition, as shown in Figure 1 (simple intercept on the X axis) with the Ki value in the range of ΙΟμΜ - 0.1 nM.

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The substrate used was Abz-Phe-Arg-Ser-Ser-ArgGln-EDDnp, a fluorescent analogue of bradykinin purified using the HPLC (High Pressure Liquid Chromatography) technique until its purity level was greater than 95% (single peak).

EXAMPLE 1B: Tests:

a) Enzymatic reactions for the determination of K _m ek _ca t

The concentration of the substrate solution was determined by the fluorometric measurement of the Abz product formed after total trypsin hydrolysis.

The hydrolysis reactions were carried out on a Hitachi F-2000 spectrofluorimeter, adjusted with excitation and emission slits of 10 and 20 nm, excitation and emission wavelengths 320 and 420 nm, respectively. The reaction temperature in the cuvette was maintained at 37 ° C in a thermostabilized compartment.

The enzymatic activity of trypsin (0.5 - 5 nM) was determined in 50 mM Tris-HCl buffer, pH 8.0, containing 20 mM NaCI at 37 ° C, with the addition of approximately 0.1 - 6.0 μΜ fluorescent substrates. The increase in fluorescence was monitored continuously for 5 min on the spectrofluorimeter. When necessary, the correction of the fluorescence reading was made using the empirical equation described (Araújo et al., Biochemistry 25; 8519, 2000). The kinetic data were determined from the initial hydrolysis speeds (substrate consumption less than 10%) and the calculations involving the kinetic parameters (K _ra , V _max and k _cat ) were performed using the equation described by MichaelisMenten in the GRAFIT program for Windows 3.0.

b) Determination of inhibition constants (Ki)

For the realization of trypsin inhibition kinetics with the peptides of the present invention, three substrate concentrations were used: a) corresponding to half of the

19/24 K _m value (0.8 μΜ); b) corresponding to the full amount of

K _m (1.6 μΜ); c) corresponding to little more than twice the μΜ). The enzyme was used at a concentration of 3.0 nM and nM; and inhibitors (SeqOl a

100

Seq09) in nM and ΙμΜ), in addition to the control, for each substrate concentration, with the substrate consumption being less than

10% of the total concentration. The incubation was

carried out at 37 ° C in Tris-HCl buffer, pH 8.0, containing 20 mM

NaCl at 37 ° C. In the characterization of inhibition, the Lineweaver-Burk plot (1 / V x 1 / S) was used, and the Ki value was determined through the relationship between the apparent inhibition constant (Ki _(app) ) and the K _ra value. substrate: Ki _(a p _P ) = [I] / [(v ₀ / ví) -1] and Ki = K _{i (app)} / [(K _m / [S]) + l] where;

[I] = inhibitor concentration; (vq / vj.) = relationship between hydrolysis rates in the absence and presence of inhibitor, respectively. K _m / [S] = relationship between the substrate's K _m and its concentration.

The results obtained are summarized in Table 1 below:

Table 1

Identifying string Ki (nM) SeqOl 22.5 Seq02 24.0 Seq03 63, 6 Seq04 42.0 Seq05 44.0 Seq06 24.2 Seq07 20, 0 Seq08 24.8 Seq09 21.8

EXAMPLE 2: Inhibition of Leukocyte Adhesion and Rolling in vivo:

a) Leukocyte bearing for the peptides of the present invention:

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Ο assay to check the inhibition of leukocyte adhesion and rolling was performed in mice (n = 3 / per group), which were anesthetized with sodium pentobarbital (Hypnol®, 50 mg / kg) and kept on a plate with controlled temperature (37 ' C), then underwent surgical manipulation in the scrotum to expose the

cremaster muscle, which was fixed around a transparent area of the plate. This was positioned on the optical microscope dolly to allow visualization in vivo of the local microcirculation. The preparations were kept moist and heated by irrigation with PBS (phosphate buffered solution) (0.15 M). The inflammatory agent LPS (lipopolysaccharide), at a concentration of 1 pg / mL diluted in 20 pL, was applied topically to the cremaster muscle. The appearance of the post-capillary venule was recorded before and after the application of LPS (lipopolysaccharide). The SeqOl to Seq09 peptides (1 μΜ) were applied topically 15 min after LPS application. Events in the pre- and post-capillary venules, arterioles and muscle fibers were observed for up to 30 minutes. Leukocyte rolling, firm adherence and leakage in post-capillary venules of 20-40 pm in diameter were recorded and quantified every 1 minute for 10 minutes. Transmigration was defined as the number of leukocytes in extravascular tissue in a 100 pm segment. The cremaster microcirculation was analyzed using the epi-illumination technique using an intravital fluorescence microscope (Axio Imager Al, Carl Zeiss, Germany) as described by Sperandio et al (J. Exp. Med., 19; 197 (10): 1355 -1363, 2003).

The tested peptides induced a decrease in the leukocyte bearing in the post-capillary venules of the mouse cremaster, in the order of 50 to 90%, as shown in figure 2.

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b) Leukocyte rolling with modified peptide: Deletion of amino acids from positions 1, 2 and 3

The above assay was repeated with a modified peptide (Mod-3) in which the removal of amino acids from positions 1, 2 and 3 was promoted as shown below:

SeqOl Ile 1-Ρ1Ό2- Arg ₃ -Cys ₄ -Ar g ₅ -Ly S6-Met ₇ -Proe-Gly 9- Val 10- Ly s 1 ₍ -Met 12-Cy s 13 Mod (-3) 1 1 Cys ₄ -Arg ₅ -Lys6-Met7-Pro8-Gly9-Val 1 ₀ -Lys] 1 -Met 12-Cys 13

This modification was intended to verify the essentiality for the anti-inflammatory activity of the cyclic structure resulting from the disulfide bridge between the thiol groups of the cysteine residues of positions 4 and 13 of the amino acids of the present invention. The peptide from which the amino acids were taken maintained leukocyte-bearing inhibitory activity compared to the peptides described in the present invention, as shown in Figure 2.

c) Effect of Lys substitution at position 11 on the activity of the peptides of the present invention

The leukocyte rolling assay was repeated, according to the description of example 2, for a modified peptide in which the lysine of (ModLyg / Alg) position 11 of one of the peptides representing this invention was replaced.

invention by an alanine (amino acid with properties contrary to gives lysine), in order to if check The essentiality gives Lysn for the activity From peptides gives

present invention, as shown below:

SeqOl 1 1 Ile [-Pro2-Arg3-Cys ₄ -Arg5-Lys6-Met ₇ -Pro8-Gly9-V to 1 o-Ly si ₍ -Met 12-Cys 13 ModLys / Wing 1 i Ile 1 -Pro2-Arg3-Cys ₄ -Arg5-LySü-Met ₇ -Pro8-Gly9-V al i o-Alaj [-Met 12-Cys 13

The modified peptide (Mod _Lys / Aia) was not efficient in inhibiting the leukocyte rolling, as shown in figure 3, in comparison with the Seq07 peptide,

22/24 showing the importance of the presence of Lysu for the maintenance of this pharmacological activity.

EXAMPLE 3: Prevention of LPS-induced peritonitis

Mice were injected intraperitoneally with 4 nmol or 40 nmol of the peptide identified by Seq07 dissolved in 500 gL of sterile saline, 30 minutes before

perform an intraperitoneal injection of LPS (055: BR 20 pg / mL, Sigma) diluted in 500 gL of sterile saline. Animals injected only with sterile saline were considered as controls (Control). 24h after LPS injection, the animals were sacrificed with a high dose of chloral hydrate to obtain the washing of the peritoneal cavity for counting the number of cells (A), neutrophils (B) and macrophages (C). The material removed was centrifuged for 10 minutes at 1500 rpm at 4 ° C, the supernatant was separated and frozen at -20 ° C for future analysis and the remaining material containing the cells, resuspended in PBS 0.1% BSA. The total count was performed in a Neubauer chamber and for the differential count, an aliquot of the cell suspension was placed on slides and subjected to centrifugation in a Citospin centrifuge, stained with Hema 3 · and examined under an optical microscope, counting a total of 300 cells. The absolute number of each cell population was obtained by multiplying the percentages by the total number of cells found in the sample volume.

Both concentrations of the tested peptide (4 nmol and 40 nmol) prevented the recruitment of leukocytes in the peritoneal cavity of mice injected with LPS by about 50% (compared to the control). It can also be seen through figure 4 that the peptide prevented the recruitment of neutrophils and macrophages.

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EXAMPLE 4: Inhibition of Acute and Chronic Pulmonary Allergic Inflammatory Response

The induction of acute pulmonary asthma was observed in male BALB / c animals, 7 weeks old, which were immunized with 1% ovalbumin (OVA) (grade V 10 qg, Sigma) adsorbed in 1.6 mg of aluminum hydroxide in days 0 and 7. From the 14th day, the animals were submitted to 3 challenges per week with 1% aerosol OVA. Animals immunized and only challenged with PBS (phosphate buffered solution) were considered control (Control). To induce chronic asthma, the animals were challenged 3 times a week for 3 consecutive weeks.

For the aerosol challenge, the animals were placed in a closed plastic box, adapted to an ultrasonic nebulizer (US-800, ICEL) and exposed to the inhalation of 2 mL of 1% ovalbumin solution (grade V, Sigma) in saline during 20 minutes. 24h after the last aerosol challenge, the animals were sacrificed with a high dose of chloral hydrate to obtain peripheral blood, bronchoalveolar lavage (BAL) and lung tissue to count the total number of leukocytes and count the number of eosinophils.

Groups of animals received treatment with 4 nmol or 40 nmol of the peptide identified by Seq07 intranasally and intraperitoneally 30 minutes before each challenge and orally 1 hour before each challenge. The group of animals with chronic asthma was treated only with 4 nmol or 40 nmol of the peptide identified by Seq07 intraperitoneally 30 minutes before each challenge.

To obtain the BAL, the animals of all groups (OVA, treated or Control) were sacrificed with 10% chloral hydrate, exanquinated, and the cannula trachea and air space were washed with 3 1 mL aliquots of HBSS (Hanks Balanced Salt Solution) + EDTA

24/24 (ethylenediaminetetraacetic acid). After collection, the BAL was immediately centrifuged at 800 rpm for 10 minutes, the supernatant discarded and the cell button resuspended in 1 mL of HBSS + 0.1% BSA (bovine serum albumin) for cell counting.

Total BAL cells were counted in the Neubauer chamber and aliquots containing 5 x

10 ⁵ cells were centrifuged on glass slides using a

cytocentrifuge, for 5 minutes, at 600 rpm. To determine the percentage of different cell populations, the slides were stained with Hema 3 and 300 cells were counted in randomly selected fields. To obtain the absolute number of each cell population in the BAL, the percentages were multiplied by the total number of cells found in the volume.

treatment of animals with acute asthma, with 4 nmol or 40 nmol of the peptide tested in all routes of administration caused a decrease in the total number of leukocytes recruited into the alveolar space, and this decrease was characterized by the absence of eosinophils, as can be seen in figure 5. The effect of the tested peptide on the recruitment of cells to the lung was similar to the effect induced by dexamethasone at a dose of 0.3 mg / kg, orally.

The intraperitoneal treatment of animals presenting chronic asthma, with 4 nmol or 40 nmol of the tested peptide, caused a decrease in the total number of leukocytes recruited into the alveolar space, with a decrease in eosinophils, as can be seen in figure 6. The effect of peptide tested in the recruitment of cells to the lung was similar to the effect induced by dexamethasone at a dose of 0.3 mg / kg, orally.

Claims (3)

1. Synthetic cyclic peptides characterized by consisting of 13 amino acids, presenting protective chemical groups at one or both of its ends and specifically presenting the amino acid cysteine at positions 4 and 13 and the amino acid lysine at position 11 of the peptide chain and a disulfide bridge between the thiol group of the cysteine residue 4 and the thiol group of the cysteine residue 13 having anti-inflammatory and / or antiallergic activity, in which the said peptides consist of consensus sequences selected from the group consisting of: Ile ₁ -Pro ₂ -Arg ₃ -Cys ₄ -Arg ₅ -Lys ₆ -Met ₇ -Pro _8- Gly ₉ -Val ₁₀ -Lys ₁₁ Met12-Cys ₂ 3 (Seq. ID. N ° 01) Val1-Glu2-Gln3-Cys4-Thr5-Ile6-Ile7-Gly8-Asp9-Glu10-Lys11Asp12-Cys13 (Seq. ID. N ° 02) Val1-Glu2-Gln3-Cys4-Thr5-Ile6-Ile7-Gly8-Asp9-Ala10-Lys11Asp12-Cys13 (Seq. ID. N ° 03) Val ₁ -Gln ₂ -Gln ₃ -Cys ₄ -Be ₅ -Glu ₆ -Ile ₇ -Ala ₈ -Gly ₉ -Ala ₁₀ -Lys ₁₁ Pro12-Cys13 (Seq.ID. n ° 04) Leu1-His2-Arg3-Cys4-Asp5-Lys6-Ile7-Ala8-Asp9-Ala10-Lys11Pro12-Cys13 (Seq. ID. N ° 05) He1-Pro2-Arg3-Cys4-Arg5-Ala6-Met7-Pro8-Gly9-Val10-Lys11Met12-Cys13 (Seq. ID. N ° 06) Ile1-Pro2-Arg3-Cys4-Args-Lys6-Met7-Pro8-Gly9-Val10-Lys11Met ₁₂ -Cys ₁₃ -NH ₂ (Seq. ID. N ° 07) Ac-Ile1-Pro2-Arg3-Cys4-Args-Lys6-Met7-Pro8-Gly9-Val10Lys11-Met12-Cys13 (Seq. ID. N ° 08) Ac-Ile1-Pro2-Arg3-Cys4-Arg5-Lys6-Met7-Pro8-Gly9-Val10Lys11-Met12-Cys13-NH2 (Seq. ID. No. 09). Petition 870180142336, of 10/18/2018, p. 11/13 2/3 2. Peptides, in wake up with claim 1, characterized by fact that amino acids are L- amino acids. 3. Peptides in wake up with claim 1 characterized by fact that proline residues are replaced individually, alternately or together with hydroxy-proline residues. Peptides according to claim 1 characterized by the fact that the methionine residues are replaced individually, alternately or together with oxidized methionine residues. 5. Peptides according to claim 1, characterized by the fact that the L-amino acids are substituted individually, alternately or together by D-amino acids. 6. Peptides according to claim 1, characterized by being anti-inflammatory and / or antiallergic. 7. Peptides, according to claim 1, characterized by being anti-asthmatic. 8. Peptides, according to claim 1, characterized by being trypsin inhibitors. 9. Pharmaceutical composition characterized by comprising the peptides as defined in the claim 1 alone or in combination containing two or more of said peptides, or their pharmaceutically acceptable salts and one or more pharmaceutically acceptable excipients, where the peptide is present in an effective dose to treat or Petition 870180142336, of 10/18/2018, p. 12/13 3/3 prevent acute and / or chronic inflammatory and / or allergic disorders. Pharmaceutical composition according to claim 9, characterized in that it is administered by the oral, intranasal, intramuscular or intravenous routes. 11. Use of one or more of the peptides as defined in claim 1, characterized for being for the manufacture of a medicine for the treatment or prevention, in a mammal, of acute and / or chronic inflammatory and / or allergic diseases, asthma, rhinitis , arthritis, atopic dermatitis, immune system disorders, lymphocyte dysfunction during the immune response, tumors, cell adhesion and / or parasitic diseases.