BR102021016135A2 - MACROCYCLIC MIMETIC ANTAGONISTS WITH BIOLOGICAL ACTIVITY, THEIR USES AND COMPOSITIONS - Google Patents

Abstract

The present invention relates to macrocyclic synthetic peptides with anti-inflammatory activity. The present invention also relates to medicinal preparations containing said synthetic peptides. Specifically, the invention refers to synthetic peptides with a pharmacophoric nucleus R/KGDX, in a linear or cyclic structure, obtained by means of Fmoc chemical synthesis with activities in the modulation of cell adhesion and effect, for example, anti-inflammatory. Such compounds have disintegrin-like activity (integrin antagonists) and originate from disintegrins isolated from the venom of snakes from Brazilian biodiversity, being composed of natural and unnatural amino acids. The invention also relates to compositions containing these peptides and their use for the preparation of medicaments.

Description

MACROCYCLIC MIMETIC ANTAGONISTS WITH BIOLOGICAL ACTIVITY, THEIR USES AND COMPOSITIONS technical field

[01] The present invention relates to new macrocyclic synthetic peptides with anti-inflammatory activity. The present invention also relates to medicinal preparations containing said synthetic peptides.

Background of the invention

[02] In view of the limitations related to the therapeutic use of biological products, the pharmaceutical industry seeks to prospect new pharmochemical molecules. One of the most used strategies for the discovery of new chemical entities is the screening from natural compounds. The development of new integrin antagonists, for example, often uses proteins isolated from snake venom as lead molecular structures for drug development. Several proteins present in snake venom have been identified as having high affinity and selectivity for different targets, especially membrane proteins such as ion channels, receptors and transporters. These characteristics make them attractive as models for the development of new drugs.

[03] Natural substances have long served as sources of therapeutic products applied in the treatment of the most diverse diseases. Currently, the pharmaceutical market has a veritable arsenal of drugs derived from traditional medicine from plants, fungi, unicellular organisms and animals (Pure Appl. Chem., 1999, Vol. 71, No. 9, pp. 1655-1661). The process of discovering new drugs has undergone changes, through the incorporation of technologies such as combinatorial chemistry, screening of compounds on a large scale (compound libraries), de novo synthesis and, above all, the use of genomic and proteomic screening techniques on a large scale (high throughput screening). On the other hand, compounds derived from the screening of natural products (extracts, venoms, partial purification, etc.) are rarely suitable for commercialization, due to problems such as low bioavailability, half-life, toxicity, specificity, purity, etc. Often, natural products serve as leader structures, which are starting points for which molecular modifications can be inserted (or miniaturized in the case of macromolecules), creating compounds with the desired pharmacological characteristics.

[04] Among the possible origins of natural products, the use of venomous animals and their toxins often provide molecular targets for the development of new chemical entities (NEQs) and innovative medications. Today, there are different examples of products on the pharmaceutical market that owe their development to scientific research on animal venoms (Nature Reviews Drug Discovery 2003, 2:790-802; Toxins 2019; 11(5):303).

[05] Several patent publications have described snake venom proteins, and peptide fragments derived from them, with biological activity and action in the treatment of various diseases.

[06] Document US5344783 reveals an assay to screen snake venom for the presence or absence of platelet aggregation inhibitors (PAIs) based on specific binding to the receptor. Using this assay, the identification and characterization of PAI in a wide range of snake venom samples was performed. The PAI purified from several of these active serpent venoms is described. In addition, PAIs lacking the Arg-Gly-Asp adhesion sequence but containing Lys-Gly-Asp are prepared that specifically inhibit the binding of fibrinogen or von Willebrand factor to GP IIb-IIIa.

[07] Document US5686570, in turn, reveals an assay to screen snake venom for the presence or absence of platelet aggregation inhibitors (PAIs) based on specific binding to the receptor. Using this assay, the identification and characterization of PAIs was performed in a wide range of snake venom samples. PAI isolated and purified from several of these active snake venoms is described and characterized. Furthermore, PAIs lacking the Arg-Gly-Asp (RGD) adhesion sequence, but containing a specific fragment, are disclosed and shown to specifically inhibit binding of fibrinogen or von Willebrand Factor to GP IIb-IIIa.

[08] Document US5851839 reveals a group of peptides that are, or are related to, platelet aggregation inhibitors isolated and purified from various snake venoms. These peptides are useful as therapeutic agents for the treatment and prevention of platelet-associated ischemic disorders. More specifically, this document discloses peptides that block specific receptors for adhesive proteins involved in platelet adhesion and aggregation.

[09] As can be seen, the state of the art presents several compounds derived from snake venom with antiplatelet and antithrombotic activity. However, when using a similar methodology with a specimen of Brazilian biodiversity, namely Bothrops jararaca, the inventors verified that an isolated protein did not have the same effect as the Eptifibatide macrocycle, whose structure is derived from the barbourine disintegrin isolated from Sistrurus m. barbouri (Journal of Biological Chemistry, 1991; 266: 9359-62). In contrast, the new protein had little or no effect on platelet aggregation. Thus, the inventors found that the new isolated protein had, surprisingly, an anti-inflammatory effect.

[010] Thus, one of the objectives of the invention is to provide proteins and peptides and macrocycles with an anti-inflammatory effect. It is also an objective of the present invention to provide compositions containing proteins and peptides with anti-inflammatory effect with less side effects and lower production cost.

[011] In the present invention, the inventors employed high-throughput sequencing techniques aimed at identifying new toxins directly on the mRNA of venom glands. This approach allowed the discovery of new leader structures, based on minor proteins (or mRNA) in the composition of the venom, which would not be identified using traditional techniques of venomics (chromatographic fractionation and proteomics). The high analytical power of the applied method (RNAseq) allowed the identification of unpublished protein coding sequences, as is the case of the sequence described in the present invention. This new sequence was cloned and expressed in a heterologous system, its recombinant form being called r-Bothrojatine, according to the committee for the standardization of the nomenclature of disintegrins (J. Thromb Haemost 2007 5:1971).

[012] In the present invention, high-performance genetic sequencing methods were used using the RNAseq technique to identify new molecular targets of a peptide nature that would not be identified by more traditional techniques of venomics (Curr Pharm Des, 2007;13(28) :2927-34.). By sequencing the complementary DNA of the venom glands of snakes of the species B. jararaca, the partial coding sequence of a metalloproteinase unknown by the state of the art was identified. The disintegrin domain was expressed in a heterologous manner, with the addition of some amino acids in its N-terminal portion, and this recombinant protein, called r-Bothrojatina, showed unprecedented anti-adhesive and anti-inflammatory properties for the domain present in disintegrin, as demonstrated in in vitro and in vivo functional assays. Based on the functional characterization and sequence of Bothrojatine, a family of pharmacochemical molecules was synthesized and characterized by means of solid phase peptide synthesis (SPFS).

Brief description of the invention

[013] The present invention relates to macrocyclic synthetic peptides with anti-inflammatory activity. The present invention also relates to medicinal preparations containing said synthetic peptides.

[014] A transcriptome analysis of the venom-producing gland of B. jararaca snakes (RNAseq) was performed to prospect new protein sequences (and leader structures) with high biological potential

[015] The present invention provides the development and characterization of a new family of molecules that presents surprising and unexpected activities for the state of the art, since they are molecules derived from a KGD disintegrin domain and from this origin there was knowledge only of effects on the coagulation/hemostasis cascade.

[016] This new family of molecules has activity and specificity distinct from those described so far, affecting the interaction of immune cells with elements of the extracellular matrix (ECM), but not the coagulation cascade or the process of platelet aggregation.

[017] In one embodiment of the present invention, peptides characterized by comprising Formula (I) are provided: X1-X2-Gly-X4-X5-X6-Cys Formula (I) Where X1 is 3mpr, Cys or 2mac, preferably 3mpr ; X2 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, Harg, Orn or Dab, preferably Harg; X4 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, Harg, Gla or Hglu, preferably Glu; X5 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, Harg or Cha, preferably Tyr; and X6 is Pro or abz2, preferably Pro; where Harg is homoarginine Hglu is homoglutamic acid Orn is ornithine Dab is alpha gamma diaminobutyric acid Gla is gamma carboxyglutamic acid 3mpr is 3-mercaptopropionic acid 2mac is 2-mercaptoacetic acid abz2 is 2-aminobenzoic acid Cha is cyclohexyl alanine, What it consists of, preferably, in the sequences SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, their cycles, enantiomers and fragments thereof.

[018] In one embodiment, a vector is provided comprising a DNA construct consisting of the sequence defined by SEQ ID NO: 1 (Bothrojatine), operatively linked to the heterologous promoter and terminator sequences and capable of expressing the protein of SEQ ID NO: 1 (Bothrojatine).

[019] In one embodiment of the invention, there is provided a pharmaceutical composition comprising one or more peptides of the invention and a pharmaceutically acceptable carrier. In another embodiment of the invention, a composition is provided which comprises the protein of SEQ ID NO:1 (Bothrojantin) in addition to the peptides of the invention.

[020] In one embodiment of the invention, the pharmaceutical composition is presented in the form of a transdermal patch, solution, suspension, emulsion, tablets or capsules. In one embodiment of the invention, the pharmaceutical composition is an injectable solution, in the form of microparticles or nanoparticles.

[021] In one embodiment of the invention, the peptides and compositions of the invention are used for the preparation of a medicine for use in inflammatory diseases, autoimmune diseases, hematological diseases and neurological diseases.

Brief description of figures

[022] Figure 1 depicts the predicted sequence and alignment of the new disintegrin sequence (Bothrojatina) with other similar proteins containing the KGD/RGD domain. In (A), the partial sequence of the snake venom metalloproteinase gene (SVMP) found from the analysis of RNA-Seq data from the venom gland of B. jararaca, the recombinant sequence is shown. amino acid sequence of the disintegrin domain with 76 amino acids (light gray highlight), with 13 cysteine residues (underlined) and a KGD domain (dark gray highlight) are highlighted. In (B) the three-dimensional model of the Bothrojatin protein structure based on the SWISS-MODEL homology system is shown. For this model, the structure of the disintegrin Bitistatin B (PDB: 2MP5) was used as a template, which presents 72% of identity with the new disintegrin. In (C) the multiple alignment analysis of Bothrojatin and several other disintegrins known from snake venoms is presented. The integrin-binding domain is highlighted in blue and adjacent amino acids important for binding and selectivity of molecules are underlined. Cysteine residues, important for disintegrin folding, are conserved between molecules and are highlighted in dark gray. The NCQ domain is highlighted in light gray. The identity (% identity) of Bothrojatine with each disintegrin is noted on the right. The GenBanK accession numbers for each known disintegrin are: agkistin (Q8AWX7.1), jararin (DQ375440.1), jerdonitin (P83912.1), albolatin (P0C6B6.1), stejnitin (P0DM87.1), barbourin (P22827 .1), piscivostatin-beta (Q805F4.1), salmosin-2 (AAC42597.1), ussuristatin-2 (Q7LZT4.1), jarastatin (Q0NZX5.1), DisBA-01 (Q801Z4.2), saxatilin (Q9DGH6 .1), bothrostatin (AAK15542.1), EC3B (P81631.1), EO5A (CAJ40966.1), VLO5B (P0C6B0.1).

[023] Figure 2 represents the steps of expression and purification of the new recombinant disintegrin r-Bothrojatina. In (A), the amino acid sequence of the recombinant protein rBothrojatina expressed in pET-15b vector is shown, fused to the Nterminal histidine epitope and thrombin cleavage site (cleavage site highlighted with the black triangle), generating a resulting protein with 4 amino acids in the N-terminal domain (highlighted in bold) compared to the native domain. In (B) a representative affinity chromatography profile of the purification steps of the r-Bothrojatin protein is shown. The blue line represents the protein absorption value of the sample at 280 ηm per elution volume, while the green line represents 0% Elution Buffer B per elution volume. In (C) a representative image of an SDS-PAGE gel of the samples obtained in the steps of purification of the recombinant Bothrojatine expressed in E. coli. is presented, where: 1- standard of molecular weight; 2- fraction of total soluble proteins; 3- fraction after first chromatographic purification step; 4- Fraction after second polishing step showing pure protein. In (D) a representative Western Blotting image is shown using an antibody capable of recognizing the histidine epitope fused to a recombinant protein, where: purified 1-r-Bothrojatine; Purified 2-r-Bothrojatine after removal of histidine epitope. In (E) a representative spectrum of circular dichroism analysis of r-Bothrojatine is shown, demonstrating that the recombinant protein resulting from the purification process has a secondary structure.

[024] Figure 3 shows that Bothrojatine disintegrin has no antithrombotic effect when compared to known antiplatelet and anticoagulant compounds, as shown by the lack of activity observed in: A) platelet aggregation assay (compared to the antiplatelet compound Eptifibatide), where platelet aggregation is induced by addition of ADP; B) cell adhesion assay, where the percentage of cell adhesion to its fibrinogen substrate is measured using CHO-K1 cells previously transiently transfected with plasmids coding for the α2bβ3 integrin (present on the surface of platelets and inhibited by Eptifibatide), activated with MnCl2, and submitted to treatment with different concentrations of Bothrojatina and Eptifibatide (control: cells transfected with empty vector, not activated with MnCl2 or using bovine serum albumin as substrate); and C) or blood coagulation assay in the presence of Bothrojatin or heparin (positive control for inhibition), measured by partially activated thromboplastin time (aPTT), induced by the addition of ADP. For A, B and C the values are representative of the average of three experiments ± SE. *p<0.05, **p<0.01 (one way ANOVA).

[025] Figure 4 shows the effects of Bothrojatine disintegrin on cell adhesion, migration and invasion. A laminin cell adhesion assay was used to test the effect of increasing concentrations of Bothrojatine disintegrin on laminin adhesion in different cell types or cell lines: (A) B16F10; (B) BHK; (C) HUVEC; and (D) THP-1. Migration of THP-1 cells was monitored by the passage of cells through the porous membrane of the transwell insert chambers (E) and cell invasion was monitored by the ability of cells to cross the porous membrane of the transwell insert and the matrigel surface (F ). Results represent the average of at least three independent experiments. Statistical significance was determined by ANOVA (*p<0.05, **p<0.01).

[026] Figure 5 shows the effects of the cyclic peptides of the invention on cell adhesion, migration and invasion, reproducing the biological effects of r-Bothrojatina. A laminin cell adhesion assay was used to test the effect of increasing concentrations of the cyclic peptides S900050, S900066 and S900064 on (A) B16F10; (B) BHK; (C) HUVEC; and (D) THP-1. Cell migration and invasion of THP-1 cells was verified by transwell insert assays as described for Figure 4 for cell migration (E) and cell invasion (F).

[027] Figure 6 shows the limited antithrombotic effect of the cyclic peptides of the invention. The cyclic peptides S900050, S900066 and S900064 had a limited antiplatelet and anticoagulant effect, as shown by the results observed in: (A) assay of platelet aggregation induced by addition of ADP and using as positive control the α2bβ3 integrin inhibitor peptide, Eptifibatide ( results presented as percentage inhibition of platelet aggregation); and (B) blood clotting assay by quantification of partially activated thromboplastin time (aPTT), in comparison with the effects of heparin and Bothrojatin. Statistical significance was determined by ANOVA (*P<0.05, **P<0.01).

[028] Figure 7 shows the inhibitory effect on cell migration of murine peritoneal leukocytes in vivo by the disintegrin Bothrojatina and the cyclic peptides of the invention S900050, S900066 and S900064. The anti-inflammatory activity of Bothrojatin disintegrin and cyclic peptides was tested using an animal model of sodium thioglycolate-induced peritonitis. (A) Bothrojatine inhibited leukocyte migration in a dose-dependent manner. (B) Peptides S900050, S900066 and S900064 also inhibited the migration of leukocytes to the peritoneum. Bothrojatin disintegrin and cyclic peptides were administered intravenously in the lateral tail vein 1 h before the intraperitoneal injection of thioglycolate. The concentration of leukocytes was determined in the peritoneal lavage 3 h after the anti-inflammatory stimulus.

[029] Figure 8 shows the inhibitory effects of Bothrojatine disintegrin and cyclic peptides of the invention on the migration of peritoneal leukocytes 24 and 48 h after induction with sodium thioglycolate. Peritoneal leukocytes were collected at different times after stimulation by sodium thioglycolate and their ability to migrate in a model using transwell inserts was evaluated. (A) Bothrojatin disintegrin and cyclic peptides inhibited the migration of isolated leukocytes 24 and 48 h after sodium thioglycolate stimulus; Less significant effects were seen in isolated cells after 3 h and 72 h. (B) An analysis of integrins present in leukocytes isolated from the peritoneum indicated a stable expression pattern of α3, α4 subunits and β1 upregulation specifically at 24 and 48 h after thioglycolate treatment.

[030] Figure 9 shows the effect of the peptide S90050 on the migration of neutrophils to the peritoneum during inflammation induced by sodium thioglycolate. 48 h after stimulation with sodium thioglycolate, the migration of leukocytes labeled with anti-GR-1-APC (neutrophils), anti-F4/80-ALEXA Fluor 488 (macrophages) and anti-CD3 antibodies was monitored by flow cytometry -PE (T cells). The Figure shows representative results where cells were not stimulated (control), and stimulated and treated with PBS (PBS) or treated with S90050.

[031] Figure 10 presents the analysis of the role of different integrins in the migration of leukocytes to the peritoneum of mice after induction of inflammation by sodium thioglycolate, using an inactivation model with antibodies. Mouse peritoneum cells were isolated 24 hours after stimulation with sodium thioglycolate and their migration was analyzed using transwell inserts. A) peritoneal leukocyte migration was inhibited by blocking antibodies against α4 and β1, but not against α3. B-C) The joint treatment of anti-α4 and β1 antagonist antibodies with Bothrojatine showed a non-cumulative effect.

[032] Figure 11 shows the anti-inflammatory effect of the peptide S900050 in an animal model of chemical colitis induced by the addition of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in Balb/c mice. It is observed that intravenous treatment with the s900050 peptide promotes a decrease in the process of weight loss induced by TNBS (A), a decrease in the process of colon contraction induced by an inflammatory process (B) and a decrease in the migration rate of inflammatory cells to the intestinal wall of mice. Statistical significance was determined between groups receiving (solid square) and not receiving (solid circle) treatment with S900050 by ANOVA (*P<0.05, **P<0.01).

[033] Figure 12 shows the analysis of micro- and nanostructured formulations composed of the biodegradable polymer co-glycolic polylactic acid (PLGA). (A) Chromatogram identifying one of the peptides of the invention after purification, which is characterized within the mobile phase gradient with a peak of maximum intensity in the middle of the chromatographic cycle (peak at 15063). (B) Dynamic light scattering graph (DLS – diffraction light scattering) of the microparticles of the formulation. The light scattering peak classifies the sample at the average size (dl 0.5) around 52 μm; On the left is a scanning electron microscopy photograph illustrating the spherical structure of the microparticles that make up the LAR (long-acting release) or Depot formulation. (C) Dynamic light scattering graph of the nanoparticles in the formulation. The light scattering peak classifies the sample at the average size (dl 0.5) around 103 ηm; On the left is a scanning electron microscopy photograph illustrating the spherical structure of the nanoparticles that make up the LAR or Depot formulation.

Detailed description of the invention

[034] The inventors performed analysis of the transcriptome of the venom-producing gland of snakes of the species B. jararaca (by RNAseq) to prospect new protein sequences (and leader structures) with high biological potential. The animals were handled in accordance with the Principles of Ethics in Animal Research and approved by the Ethics Committee for Animal Experimentation of the Health Sciences Center of the Federal University of Rio de Janeiro (nº 077/10). Specimens of B. jararaca were collected and the venom glands were dissected after 4 days of manual venom collection, when the transcription rate is at its maximum. Venom glands were immediately placed in RNAlater™ solution (Qiagen) and frozen at -70°C until use.

[035] The tissue of the venom glands was homogenized using a rotor-extractor and the total RNA was extracted using the RNAeasy kit (Qiagen) according to the manufacturer's instructions. The mRNA was extracted (using the Dynabeads® mRNA Purification kit - Invitrogen), purified and fragmented with ZnCl2 so that the fragments generated had an average size of approximately 700 bp. The fragmented mRNA was converted into cDNA (cDNA Synthesis System kit - Roche). The ends of the fragments were polished and repaired (with T4 DNA polymerase and T4 polynucleotide kinase enzymes) by incubation in a thermocycler for 20 min at temperatures from 10 to 15 °C, 20 min at 25 °C and for 20 min at 75 °C. °C. Subsequently, adapters were ligated for sequencing with the T4 DNA enzyme.

[036] For the removal of small fragments of polynucleotides, the samples were purified by the SPRI method (solid phase reversible immobilization). In this procedure, the magnetic particles are incubated with the DNA and, during this incubation, the magnetic particles are bound to the DNA but not to small DNA fragments (eg primers, free nucleotides, etc.). After retaining the magnetic particles by a magnetic plate, they are washed to remove small unbound fragments and the remaining DNA bound to the particles is eluted to obtain a cDNA library. The cDNA thus obtained was linked to the specific particles (beads) for the sequencing protocol through the pyro-sequencing platform 454 (Roche). From the sequencing process, 487018 sequences (commonly called reads) were obtained. Next, the sequences were assembled again using Newbler 2.5 (454, Life Sciences), which is an assembler specialized in NGS (next generation sequencing), with the options -cNDA, -urt and -mi98. Based on the Newbler results, a secondary assembly was performed using an assembler more suitable for larger sequences, iAssembler (http://bioinfo.bti.cornell.edu/tool/iAssembler), to reduce redundancy and assemble more sequences than were joined together by Newbler using a script that uses MIRA as the primary assembler and CAP3 as the secondary assembler, in addition to applying post-assembly patches. As a result, assemblies of two transcriptomes were obtained, obtaining for each transcriptome a set of unique genes, commonly called unigenes.

[037] The inventors of the present invention compared the unigenes obtained in the assemblies of the invention with mRNA sequences of B. jararaca obtained in their own banks and collaborators, to verify that the original mRNA sequences of the present invention had not been previously identified by others groups and were present in the databases. Considering only the unigenes that had similarity greater than 90% and covering at least 90% of their size, for a sample only 12.4% of the reads had already been described and for another sample only 40.85% of the reads had already been described . These data prove the efficiency of the methodology used for the identification of new proteins with high biological potential. Analysis of the transcriptome obtained identified a partial sequence of snake venom metalloproteinase containing the disintegrin domain (KGD integrin binding domain) (Figure 1A and 1B).

[038] For functional annotation, in each of the two assemblies, a similarity search was performed using BLASTX against the NCBI nr protein database, with e-value 10−5 , to identify possible homologous proteins, and also , BLASTN against the nucleotide bank, NCBI nt, to try to identify putative proteins when there was no hit with the nr bank. The BLASTX results were then imported into the Blast2GO software, where it was possible to annotate part of the unigenes with Gene Ontology (GO) terms, Enzyme Commission (EC) codes, and metabolic pathways, through KEGG (Kyoto Encyclopedia of Genes and Genomes) . Figure 1C presents this comparative result between Bothrojatine and other known disintegrins.

[039] The sequence of the new identified protein, called Bothrojatina, was then cloned into commercial plasmids (pGEMT, pET-15b and pSELECT-His) for the production of the recombinant protein rBothrojatina. According to the purification strategy used, the protein is expressed in the same reading order with a Histidine tag in the N-terminal portion. The recombinant protein was purified using affinity chromatography (HisTrap HP-GE column), ion exchange (Mono Q 5/50 GL - GE column), gel filtration (Sephacryl S-100 HR or Superdex 200-GE column) or phase reverse (column C18- Allcrom) in Akta Avant (GE) system. For each system, buffer changes were made according to the specifications of each column. After purification, the purity of the protein was verified by SDS-PAGE and confirmed by the Western Blotting technique using an antibody capable of recognizing the histidine epitope fused to the recombinant protein.

[040] The histidine epitope was removed from recombinant Bothrojatina by cleavage with thrombin (5-20 U/mg of protein) in reaction for 12-16 hours at room temperature. Samples were then applied to tangential filtration membranes ranging in size from 100 to 3 kDa, preferably 30 to 3 kDa, to remove contaminating thrombin and the poly-His tail protein fragment. At the end of this step, purified recombinant Bothrojatine was stored at -20 °C until use. Figure 2 represents the steps of expression and purification of the new recombinant disintegrin Bothrojatine.

[041] Snake venom is a rich source of biologically active compounds containing proteins with high affinity and selectivity for specific components in cells. Among the proteins with biological activity from snake venoms, the inventors highlight the metalloproteases, which are enzymes that play an important role in the pathogenesis related to envenomation. Among the different metalloproteinases found, the P-II class metalloproteases contain a disintegrin domain with potent integrin binding and inhibition activity.

Onde Harg é homoarginina; Hglu é ácido homoglutâmico; Orn é ornitina; Dab é ácido alfa gama diaminobutírico; Gla é ácido gama carboxiglutâmico; 3mpr é 3- ácido mercaptopropiônico; 2mac é ácido 2-mercaptoacético; abz2 é ácido 2- aminobenzóico e Cha é ciclohexil alanina. Todos os peptídeos relacionados na tabela 1 podem ser lineares ou ciclos, preferencialmente ciclos (ciclo-ditio).[042] In the published work (J Proteomics, 2016, 135, 73-89), the inventors described that they used proteomics data to confirm sequences found in the transcriptome of the present invention. The functional and structural characterization of Bothrojatine served as a leading structure for the chemical synthesis of macrocyclic molecules containing natural amino acids and unnatural analogues, reproducing the anti-inflammatory effects observed with recombinant disintegrin. The invention starts from truncated sequences of the Bothrojatine sequence, having as its center the KGD pharmacophoric nucleus. Table 1 shows the sequences obtained in this process. Included in Table 1 are the sequences obtained from molecular docking simulations (molecular docking) in silico, in the format of SEQ ID NO: X (SEQ ID NO:4-6 in Table 1 correspond to the same sequences as SEQ ID NO: 4-6 of the sequence listing file; SEQ ID NO: 7-11 correspond to the peptides used in the Examples).

where Harg is homoarginine; Hglu is homoglutamic acid; Orn is ornithine; Dab is alpha gamma diaminobutyric acid; Gla is gamma carboxyglutamic acid; 3mpr is 3-mercaptopropionic acid; 2mac is 2-mercaptoacetic acid; abz2 is 2-aminobenzoic acid and Cha is cyclohexyl alanine. All peptides listed in table 1 can be linear or cycles, preferably cycles (cyclo-dithio).

[043] Three sequences (S900064-13-mer and S900066-11-mer and S009349-8-mer) obtained from these constructs showed promise. Also in this group, changes were produced, such as reducing the number of residues and changing functional groups, and two more sequences were included: Mpa-KGDMDC (S000015) and 7-mer Mpa-HarGEYPC (S900050). The experimental screening of these compounds confirmed that the strategy of miniaturization and mimicry of the Bothrojatine structure was able to maintain the anti-inflammatory activities observed with the recombinant protein. Based on the obtained results, modifications were made to the original structure of Bothrojatine to create a virtual library of compounds, to be used in molecular docking studies (molecular docking) in silico, in vitro and in vivo screening

[044] Different modifications were incorporated into the structures described above, such as the reduction of carboxyl groups in the sequence (removal of negative amino acids or replacement by polar or nonpolar), removal of the N-terminus (elimination of amino acids prior to the KGD pharmacophoric core, and remodeling of the C-terminus (residues after the acidic polar residue of the pharmacophoric nucleus) forming a new pharmacophoric nucleus R/KGDX, X being an uncharged polar or polar residue capable of increasing the specificity of this nucleus, preferably residues that confer some structural rigidity (angular strain or steric hindrance) with homo- or heterocyclic structures integrated into the basic structure without, however, conferring effective charge. This domain may have greater binding specificity if there is the formation of a cyclic molecule (macrocyclic), giving a different structure to the linear peptide (J. Biol. Chem. 1997, 272-21341-21348; J. Biol. Chem., 2000, 275-21785-21788). All these sequences are represented in Table 1. Thus, in addition to the acyclic structures, one of the objects of the invention is also the macrocycles (cyclic structures) through disulfide bridge, peptide bond or through any other chemical reactions capable of uniting the ends of the linear structures of the molecules, regardless of the number of amino acid residues or structural subunits.

Examples Example 1

[045] Experimental assays demonstrate that the recombinant disintegrin Bothrojatina does not have antiplatelet and anticoagulant effect when compared to known compounds (Figure 3). The graph presented in Figure 3 shows the percentage of cells adherent to the fibrinogen of transfected and activated cells (defined as 100% adhesion).

[046] The ability of THP-1 to migrate toward an inflammatory stimulus was measured in a migration assay using a modified Boyden chamber (transwell insert), which creates two culture chambers separated by a porous membrane. The addition of Bothrojatine inhibited the migration of THP-1, a human monocytic lineage cell, in a concentration dependent manner ( Figure 4E ). Physiologically, it is essential that leukocytes can adhere and migrate between endothelial cells, to subsequently degrade the extracellular matrix (ECM) of blood vessels, in order to reach the sites of inflammation, both in physiological and pathological conditions. In vitro leukocyte invasive properties were simulated by recovering the surface of the insert with a layer of gel based on a collagen matrix and laminin (geltrex® - gel matrix invasion assay). Again, disintegrin inhibited matrix invasion by THP-1 in a dose-dependent manner (Figure 4F). These results indicate that Bothrojatine has an anti-inflammatory potential, serving as a lead molecule for the development of integrin antagonist NEQs.

Example 2

[047] The ability of disintegrins to modulate the interaction between integrins and their respective substrates is related to different pathological mechanisms. Specifically, inhibition of the inflammatory response through regulation of integrin activity is related to the treatment of autoimmune diseases, including rheumatoid arthritis, reactive arthritis, multiple sclerosis, scleroderma, diabetes mellitus, atopic dermatitis, Hashimoto's thyroiditis (Graves' disease), lupus erythematosus, Sjogrem's syndrome, vitiligo, Crohn's and celiac disease, ulcerative colitis, autoimmune myocarditis and vasculitis, psoriasis, sickle cell anemia, to name a few

[048] To test its anti-inflammatory potential, the effect of Bothrojatine and the respective macrocycles derived from it on the interaction of integrin and its substrate was tested (Figure 4A to 4D). Initially, then, assays of cellular adhesion in plaque were used to evaluate the effect of Bothrojatine on the interaction with different constituent proteins of the ECM. A screening of the inhibitory effect of Bothrojatine using different substrates (fibrinogen, fibronectin, collagen V, vitronectin and laminin) and cells, such as fibroblasts (hamster kidney cell line, BHK), epithelial cells (murine melanoma cell line, B16F10 ), primary vascular endothelial cells (human umbilical vein endothelial cell line, HUVEC) and monocytic cells (human monocytic cell line, THP-1) showed an inhibitory effect on cell binding to laminin. Marginal effects were observed with other tested substrates.

[049] To demonstrate that cyclic peptides derived from Bothrojatina reproduce the biological effects previously observed, new in vitro adhesion, migration and cell invasion assays were performed. In line with the promising biological effect exhibited by Bothrojatine, the macrocyclic compounds derived from its three-dimensional structure were also able to inhibit cell adhesion, migration and invasion (Figure 5). Furthermore, similar to Bothrojatine, the synthetic peptides tested had a limited effect on the coagulation cascade or binding of fibrinogen and α2bβ3 integrin present in platelet aggregation (Figure 6).

[050] The in vivo results prove the anti-inflammatory potential of Bothrojatine and its cognate peptides. The use of an animal model with acute inflammation of chemical peritonitis (intraperitoneal injection of thioglycolate) confirmed the inhibitory effect on the migration of inflammatory cells (Figure 7).

Example 3

[051] The therapeutic potential of disintegrins is associated with their ability to act specifically on different integrins. In this case, the effect of Bothrojatine and its derivative compounds on the migration of peritoneal leukocytes was analyzed. Peritoneal leukocytes from mice were collected at 3, 24, 48 and 72 hours after proinflammatory stimulation with thioglycolate, and their in vitro migration profile was evaluated in the presence of Bothrojatine or derived peptides. Peptides and disintegrin inhibited the in vitro migration of isolated leukocyte populations at 24 and 48 h after thioglycolate instillation (Figure 8A). Analysis of the integrin expression profile using specific monoclonal antibodies revealed stable expression of α3 and α4 integrins and specific up-regulation of β1 at both 24 and 48 hour times after thioglycolate treatment ( Figure 8B ).

[052] We also tested the effect of peptide S90050 on the migration of neutrophils to the peritoneum during inflammation induced by sodium thioglycolate 48 h after stimulation by sodium thioglycolate. The results monitored by flow cytometry show migration of macrophages, neutrophils and T cells from unstimulated cells (control), and stimulated and treated with PBS (PBS) or treated with S90050 (Figure 9).

[053] Next, blocking antibodies were used as a tool to study the specific inhibition of integrins by the tested compounds and the specificity of the inhibitory peptides on the integrins studied. Blocking antibodies against α3, α4 and β1 integrins were used in murine peritoneal leukocytes isolated after 24 hours of intraperitoneal injection of sodium thioglycolate. Antibody interference assay confirmed the role of α4 and β1 integrins, but not α3 integrin on murine leukocyte migration in vitro ( Figure 10A ). The combination of Bothrojatine with inhibitory antibodies had comparable inhibitory effect as their factors used independently, suggesting that they may target the same active site on the cell surface (Figure 10BC). Taken together, these results suggest that at least one of the ways that Bothrojatine, or its derivative peptides, inhibit inflammation is through the inhibition of leukocyte invasion mediated by α4β1 integrin.

Example 4

[054] The binding specificity of disintegrins is crucial for their therapeutic application. Based on previous results that indicate a specificity of Bothrojatine or its cyclic peptide S900050 for inhibition of α4β1-type integrin, its therapeutic capacity for the treatment of autoimmune diseases was tested. More specifically, based on its ability to inhibit leukocyte migration, an animal model was used to study inflammatory bowel diseases.

[055] The animal model of chemical colitis mimics histological and immunological features of inflammatory diseases that affect the gastrointestinal system. The use of the colitis model induced by rectal instillation with 2,4,6-trinitrobenzenesulfonic acid (TNBS) induces a mechanism of colitis with transmural migration of inflammatory cells, a situation similar to that observed in cases such as Crohn's disease and ulcerative colitis. The use of this model of colitis is widely used for the study of inflammatory diseases of the gastrointestinal system by analyzing the immune responses associated with inflammation of the intestinal mucosa, the effect of the treatment being accompanied in a practical way by symptoms such as: (a) weight loss, ( b) contraction of the colon and (c) increase in the number of inflammatory cells

[056] A condition of colitis was induced in Balb/c mice by rectal instillation with TNBS (4% TNBS in 50% ethanol), followed or not by intravenous administration of the cyclic peptide S900050 within a period of 24 hours after colitis induction , instillation with PBS was used as a control. A continuous analysis of weight loss was performed, and 7 days after the start of the procedure, the animals were euthanized and the extent of the colon and the number of inflammatory cells in the intestinal basement membrane were measured. The results demonstrated that the S900050 peptide has a beneficial effect for the treatment of inflammatory bowel diseases (Figure 11). Administration of S900050 showed significant differences in terms of decreased weight loss and colonic contraction, which suggests a lower presence of intestinal inflammatory cells. These results demonstrate a therapeutic potential of S900050 for the treatment of autoimmune diseases.

Example 5

[057] Non-limiting examples of pharmaceutical formulations of the invention are tablets, capsules, dragees, pills, solutions, suspensions, emulsions, ovules

[058] Pharmaceutical compositions of Bothrojatine, or mimetic peptides, were formulated at a concentration between 0.01 mg/mL and 1 g/mL, preferably 0.1 mg/mL, in an injectable solution formulation with water for injections and osmotic regulators , such as carboxymethylcellulose, mannitol, among others, in the proportion between 0.5% and 50%, preferably between 5% and 10%. Non-ionic surfactants were used, such as: ethoxylated aliphatic alcohol, polyoxyethylenes, carbonic esters, polyethylene glycol esters, sorbitols, monoalkanolamines, among others, in a proportion between 0.01% and 2%, preferably poloxamer 188 in a proportion between 0.1% and 1%. The mixture is homogenized by mechanical stirring at a speed between 100 and 2000 rpm, preferably between 600 and 1000 rpm. The ionic strength of the medium can be corrected to physiological or slightly acidic pH, between 4 and 8, preferably 5.2 or 7.4. When ready, the solution is filtered at a bubble point of 0.1 or 0.22 µm.

Example 6

[059] Pharmaceutical compositions of Bothrojatine, or mimetic peptides, were formulated at a concentration of 0.01 mg/mL to 1 mg/mL, preferably 0.5 mg/mL, in oily, polar or non-polar solvents. Solvents are such as: methylene chloride, methanol, benzyl alcohol, ethyl acetate, ethyl ether, dimethicone, among others. A mixture of the protein or peptide with a biodegradable, biocompatible polymer was obtained (which may be, for example, but not restricted to, PLGA in its commercial options 85:15, 75:25, 65:35, 50:50 or 45:55) , at a concentration of 10 to 1000 mg/mL in a compatible organic solvent, but preferably around 150 mg/mL, such as: methylene chloride or ethyl acetate. The final form was obtained by emulsifying it in an aqueous solution in a proportion of 1 to 50%, preferably 10%, which could be: water, phosphate buffer, acetate, borate, pamoate, sodium chloride, carbonates, polyvinyl alcohol, among others.

[060] The deposit formulation can have micro or nano dimensions, with a dispersion peak between 10 ηm to 100 μm, preferably 45 μm for micro and 20 ηm for nano, defined by the presence of surfactants in a concentration of 0.01% to 2 %, preferably, but not restricted to 0.1% of didodecyldimethylammonium bromide, under the effect of agitation which can be: mechanical with rotor-stator, angled blades, microfluid, pressurizer, ultrasound or a combination thereof. A non-limiting example of nanoparticle formulation can be found in Figure 12.

Example 7

[061] Compositions of Bothrojatine, or mimetic peptides, were also formulated at a concentration of 0.01 mg/mL to 1 mg/mL, preferably 0.5 mg/mL in the form of transdermal patches composed of methacrylate patches: dextroalphatocopherol, polybutylmethacrylate, methylmethacrylate, acrylic copolymer, silicone oil and polyvinyl polystyrene films. The dimensions of the patch can be customized for use, containing between 1 and 10 mg of NCE/cm2, releasing between 6 and 100 mcg/h of the active.

[062] All documents cited in this specification are hereby incorporated by reference.

[063] Having revealed some preferred and alternative examples of execution of the present invention, it is clear that none of the examples provided is limiting the scope of protection of the present invention, which is defined and limited only by the attached set of claims and the claims contained therein .

Claims (7)

Peptide characterized by comprising Formula (I): X1-X2-Gly-X4-X5-X6-Cys Formula (I) Where X1 is 3mpr, Cys or 2mac, preferably 3mpr; X2 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, Harg, Orn or Dab, preferably Harg; X4 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, Harg, Gla or Hglu, preferably Glu; X5 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, Harg, DOPA or Cha, preferably Tyr; and X6 is Pro or ABZ2, preferably Pro; where Harg is homoarginine Hglu is homoglutamic acid Orn is ornithine Dab is alpha gamma diaminobutyric acid Gla is gamma carboxyglutamic acid 3mpr is 3-mercaptopropionic acid 2mac is 2-mercaptoacetic acid ABZ2 is 2-aminobenzoic acid Cha is cyclohexyl alanine, What it preferably consists of , in the sequences SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11 (which correspond, respectively, to CRIPKGDDMDDC, 3mpr-RIPKGDDMDC, 3mpr-KGDDMDC, 3mpr -KGDMDC and 3mpr-Harg-GEYPC), their cycles, enantiomers and fragments thereof. Vector characterized by comprising a DNA construct consisting of the sequence defined by SEQ ID NO: 1 (Bothrojatine), operatively linked to the heterologous promoter and terminator sequences and capable of expressing the protein of SEQ ID NO:1. Pharmaceutical composition, characterized in that it comprises one or more peptides, as defined in claim 1, and a pharmaceutically acceptable vehicle. Composition according to claim 3, characterized in that it additionally comprises the protein Bothrojantin, defined in SEQ ID NO: 1. Pharmaceutical composition according to claim 3 or 4, characterized in that it is in the form of a transdermal patch, solution, suspension, emulsion, tablets or capsules. Pharmaceutical composition, according to claim 5, characterized by the fact that it is an injectable solution, in the form of microparticles or nanoparticles. Use of the peptides as defined in claim 1 and of the compositions as defined in claims 3, 4, 5 or 6 characterized in that it is for the preparation of a medicine for use in inflammatory diseases, autoimmune diseases, hematological diseases and neurological diseases.

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