WO2023019333A1 - Macrocyclic mimetic antagonists with biological activity, uses thereof and compositions - Google Patents

Macrocyclic mimetic antagonists with biological activity, uses thereof and compositions Download PDF

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WO2023019333A1
WO2023019333A1 PCT/BR2022/050318 BR2022050318W WO2023019333A1 WO 2023019333 A1 WO2023019333 A1 WO 2023019333A1 BR 2022050318 W BR2022050318 W BR 2022050318W WO 2023019333 A1 WO2023019333 A1 WO 2023019333A1
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seq
3mpr
peptides
harg
acid
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Portuguese (pt)
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Joari Marques DE MIRANDA
Russolina Benedeta ZINGALI
Eleonora KURTENBACH
Hamilton SILVA JUNIOR
Rafael Soares de AQUINO
Jenifer NOWATZKI
Nailton Monteiro Do NASCIMENTO JÚNIOR
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Novageia Biotecnologia Ltda
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/58—Reptiles
    • A61K35/583—Snakes; Lizards, e.g. chameleons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • 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.
  • one of the objectives of the invention is to provide proteins and peptides and macrocycles with an anti-inflammatory effect. It is a further object of the present invention to provide compositions containing the proteins and peptides with anti-inflammatory effect with fewer side effects and lower production cost.
  • RNAseq RNAseq
  • RNAseq 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 ).
  • the disintegrin domain was expressed in a heterologous way, 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.
  • r-Bothrojatina this recombinant protein, called r-Bothrojatina
  • r-Bothrojatina 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.
  • SBFS solid phase peptide synthesis
  • the present invention relates to macrocyclic synthetic peptides with anti-inflammatory activity.
  • the present invention also relates to medicinal preparations containing said synthetic peptides.
  • RNAseq transcriptome analysis of the venom-producing gland of B. jararaca snakes
  • 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.
  • 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.
  • ECM extracellular matrix
  • peptides characterized by comprising Formula (I) are provided:
  • X1 is 3mpr, Cys or 2mac, preferably 3mpr;
  • X2 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gin, 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, Gin, 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, Gin, Arg, Ser, Thr, Val, Trp, Tyr, Harg or Cha, preferably Tyr; It is
  • X6 is Pro or abz2, preferably Pro; on what
  • Hglu is homoglutamic acid
  • 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,
  • SEQ ID NO:7 Preferably consisting of 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.
  • a vector 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).
  • a pharmaceutical composition comprising one or more peptides of the invention and a pharmaceutically acceptable carrier.
  • a composition which comprises the protein of SEQ ID NO:1 (Bothrojantin) in addition to the peptides of the invention.
  • the pharmaceutical composition is presented in the form of a transdermal patch, solution, suspension, emulsion, tablets or capsules.
  • the pharmaceutical composition is an injectable solution, in the form of microparticles or nanoparticles.
  • 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.
  • Figure 1 depicts the predicted sequence and alignment of the new disintegrin sequence (Bothrojatina) with other similar proteins containing the KGD/RGD domain.
  • 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.
  • (B) shows the three-dimensional model of the Bothrojatin protein structure based on the SWISS-MODEL homology system. .
  • 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 ).
  • Figure 2 represents the steps of expression and purification of the new recombinant disintegrin r-Bothrojatina.
  • A the amino acid sequence of the recombinant protein r-Bothrojatin expressed in pET-15b vector is shown, fused to the N-terminal 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.
  • B a representative affinity chromatography profile of the purification steps of the r-Bothrojatin protein is shown.
  • FIG. 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 it is measured the percentage of adhesion of cells to their fibrinogen substrate using CHO-K1 cells previously transiently transfected with plasmids encoding a2b[33 integrin (present on the surface of platelets and inhibited by Eptifibatide), activated with MnCl2, and subjected to treatment with different concentrations of Bothrojatine and Eptifibatide (control: cells transfected with empty vector, not activated with MnCl2 or using bovine serum albumin as substrate); and C) or blood clotting assay in the presence of Bothrojatin or heparin (positive control for inhibition
  • FIG 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).
  • Figure 5 shows the effects of the cyclic peptides of the invention on cell adhesion, migration and invasion, reproducing the biological effects of r-Bothrojatine.
  • 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 through assays with transwell inserts as described for Figure 4 for cell migration (E) and cell invasion (F).
  • FIG. 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 integrin inhibitor peptide a2b[33, Eptifibatide (results presented as percent 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).
  • 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.
  • 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.
  • 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.
  • Bothrojatin disintegrin and cyclic peptides inhibited the migration of isolated leukocytes 24 and 48 h after sodium thioglycolate stimulation; Less significant effects were seen in isolated cells after 3 h and 72 h.
  • B An analysis of the integrins present in leukocytes isolated from the peritoneum indicated a stable expression pattern of a3, a4 subunits and upregulation of (31 specifically at 24 and 48 h after thioglycolate treatment.
  • Figure 9 shows the effect of 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.
  • 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.
  • B-C The joint treatment of antagonistic antibodies anti-a4 and (31 with Bothrojatine showed no cumulative effect.
  • 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 peptide S900050 promotes a decrease in the process of weight loss induced by TNBS (A), a decrease in the process of colon contraction induced by a inflammatory process (B) and a decrease in the rate of migration of inflammatory cells to the intestinal wall of mice. Statistical significance was determined between groups that received (solid square) and did not receive (solid circle) S900050 treatment by ANOVA (*P ⁇ 0.05, **P ⁇ 0.01).
  • 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 average size (dl 0.5) around 52 pm; 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.
  • the light scattering peak classifies the sample at the average size (dl 0.5) around 103 qm;
  • On the left is a scanning electron microscopy photograph illustrating the spherical structure of the nanoparticles that make up the LAR or Depot formulation.
  • 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 (n° 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 RNAIaterTM solution (Qiagen) and frozen at -70 °C until use.
  • RNAeasy kit Qiagen
  • 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.
  • the samples were purified by the SPRI method (solid phase reversible immobilization).
  • 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).
  • a secondary assembly was performed using an assembler more suitable for larger sequences, the 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.
  • the iAssembler http://bioinfo.bti.cornell.edu/tool/iAssembler
  • 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).
  • Bothrojatina The sequence of the new identified protein, called Bothrojatina, was then cloned into commercial plasmids (pGEM-T, pET-15b and pSELECT-His) for the production of recombinant protein r- Bothrojatina. 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.
  • 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
  • phase reverse phase reverse (column C18- Allcrom) in Akta Avant (GE) system.
  • buffer changes were made according to the specifications of each column.
  • 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.
  • snake venom is a rich source of biologically active compounds containing proteins with high affinity and selectivity for specific components in cells.
  • proteins with biological activity of snake venoms the inventors highlight the metalloproteases, which are enzymes that play an important role in envenomation-related pathogenesis.
  • the P-ll class metalloproteases contain a disintegrin domain with potent integrin binding and inhibition activity.
  • 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 the KGD pharmacophoric nucleus as its center. Table 1 shows the sequences obtained in this process.
  • Table 1 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).
  • 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).
  • This domain may have greater specificity of binding 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.
  • one of the objects of the invention is also the macrocycles (cyclic structures) through disulfide bridges, peptide bonds, or any other chemical reactions capable of joining the ends of the linear structures of molecules, regardless of the number of amino acid residues or structural subunits.
  • THP-1 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.
  • Bothrojatine inhibited the migration of THP-1, a cell of human monocytic lineage, in a concentration-dependent manner ( Figure 4E).
  • ECM extracellular matrix
  • 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.
  • autoimmune diseases including rheumatoid arthritis, reactive arthritis, multiple sclerosis, scleroderma, diabetes mellitus, atopic dermatitis, Hashimoto's thyroiditis (Graves' disease), lupus erythemat
  • 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 (cell line from human umbilical vein endothelial cells, 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.
  • fibroblasts hamster kidney cell line, BHK
  • epithelial cells murine melanoma cell line, B16F10
  • primary vascular endothelial cells cell line from human umbilical vein endothelial cells, HUVEC
  • monocytic cells human monocytic cell line, THP-1
  • blocking antibodies were used as a tool to study the specific inhibition of integrins by the compounds tested and the specificity of the inhibitory peptides on the integrins studied.
  • Blocking antibodies against a3, a4 and (31) integrins were used on murine peritoneal leukocytes isolated after 24 hours of intraperitoneal injection of sodium thioglycolate.
  • the antibody interference assay confirmed the role of a4 and (31) integrins, but not a3 integrin on murine leukocyte migration in vitro ( Figure 10A).
  • Bothrojatin had a comparable inhibitory effect as their factors used independently, suggesting that they may be the target of the same active site on the cell surface ( Figure 10B - C)
  • Figure 10B - C Taken together, these results suggest that at least one of the ways that Bothrojatine, or its derivative peptides, have to inhibit inflammation is through the inhibition of leukocyte invasion mediated by a4 integrin(31).
  • 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.
  • 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.
  • Non-limiting examples of pharmaceutical formulations of the invention are tablets, capsules, dragees, pills, solutions, suspensions, emulsions, ovules.
  • 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%.
  • osmotic regulators such as carboxymethylcellulose, mannitol, among others
  • 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 pm.
  • 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 apolar 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 in an aqueous solution in a proportion of 1 to 50%, preferably 10%, which may be: water, phosphate buffer, acetate, borate, pamoate, sodium chloride, carbonates, polyvinyl alcohol, among others.
  • the deposit formulation can have micro or nano dimensions, with a dispersion peak between 10 qm to 100 pm, preferably 45 pm for micro and 20 qm 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 that can be: mechanical with rotor-stator, angled blades, microfluid, pressurizer, ultrasound or a combination thereof.
  • agitation 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.
  • 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/cm 2 , releasing between 6 and 100 mcg/h of the active ingredient.

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Abstract

The present invention relates to macrocyclic synthetic peptides with anti-inflammatory activity. The present invention also relates to medicinal preparations containing these synthetic peptides. More specifically, the invention relates to synthetic peptides with an R/KGDX pharmacophoric nucleus, with a linear or cyclic structure, produced by chemical Fmoc synthesis with cell adhesion modulation activity and an anti-inflammatory effect, for example. These compounds have an activity similar to that of disintegrins (integrin antagonists), are produced from disintegrins isolated from the venom of snakes of the Brazilian biodiversity, and are composed of natural and synthetic amino acids. The invention also relates to compositions containing these peptides and to the use thereof to prepare medicinal drugs.

Description

ANTAGONISTAS MIMÉTICOS MACROCÍCLICOS COM ATIVIDADE BIOLÓGICA, SEUS USOS E COMPOSIÇÕES Campo técnico MACROCYCLIC MIMETIC ANTAGONISTS WITH BIOLOGICAL ACTIVITY, THEIR USES AND COMPOSITIONS Technical field
[01 ] A presente invenção se refere a novos peptídeos sintéticos macrocíclicos com atividade anti-inflamatória. A presente invenção também se refere a preparações medicinais contendo os referidos peptídeos sintéticos. [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.
Antecedentes da invenção Background of the invention
[02] Tendo em vista as limitações relativas ao uso terapêutico de produtos biológicos, a indústria farmacêutica busca prospectar novas moléculas farmoquímicas. Uma das estratégias mais utilizadas para a descoberta de novas entidades químicas é a triagem a partir de compostos naturais. O desenvolvimento de novos antagonistas de integrinas, por exemplo, utiliza frequentemente de proteínas isoladas da peçonha de serpentes como estruturas moleculares líderes (lead structures) para o desenvolvimento de fármacos. Várias proteínas presentes na peçonha de serpentes têm sido identificadas possuindo grande afinidade e seletividade para diferentes alvos, especialmente proteínas de membrana tais como canais iônicos, receptores e transportadores. Essas características as tornam atrativas como modelos para o desenvolvimento de novos fármacos. [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] As substâncias naturais há muito servem como fontes de produtos terapêuticos aplicados no tratamento das mais diversas enfermidades. Atualmente há no mercado farmacêutico um verdadeiro arsenal de fármacos derivados da medicina tradicional oriundos de plantas, fungos, organismos unicelulares e animais (Pure Appl. Chem., 1999, Vol. 71 , No. 9, pp. 1655-1661). O processo de descoberta de novos fármacos vem sofrendo mudanças, através da incorporação de tecnologias como a química combinatória, a triagem de compostos em larga escala (bibliotecas de compostos), síntese de novo e, sobretudo, o uso de técnicas genômicas e proteômicas de triagem em larga escala (high throughput screening). Por outro lado, compostos oriundos da triagem de produtos naturais (extratos, peçonhas, purificados parciais etc.) raramente são adequados para comercialização, devido à problemas como baixa biodisponibilidade, meia-vida, toxicidade, especificidade, pureza etc. Frequentemente, os produtos naturais servem como estruturas líderes, que são pontos de partida para que modificações moleculares possam ser inseridas (ou miniaturizadas no caso de macromoléculas), criando compostos com as características farmacológicas almejadas. [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). For another 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] Entre as possíveis origens dos produtos naturais, o uso de animais peçonhentos e suas toxinas frequentemente fornecem alvos moleculares para o desenvolvimento de novas entidades químicas (NEQs) e medicações inovadoras. Há hoje no mercado farmacêutico diferentes exemplos de produtos que devem seu desenvolvimento à pesquisa científica sobre peçonha animais (Nature Reviews Drug Discovery 2003, 2:790-802; Toxins 2019; 11 (5):303). [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] Várias publicações de patentes têm descrito proteínas de peçonhas de serpentes, e fragmentos peptídicos derivados dos mesmos, com atividade biológica e ação no tratamento de diversas enfermidades. [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] O documento US5344783 revela um ensaio para rastrear a peçonha de serpente quanto à presença ou ausência de inibidores da agregação plaquetária (PAIs - do inglês platelet aggregation inhibitors) com base na ligação específica ao receptor. Usando este ensaio, a identificação e caracterização do PAI em uma ampla gama de amostras de peçonha de serpente foram realizadas. O PAI purificado de várias dessas peçonhas de serpente ativas é descrito. Além disso, os PAIs sem a sequência de adesão Arg-Gly-Asp, mas contendo Lys-Gly-Asp, são preparados e inibem especificamente a ligação do fibrinogênio ou do fator de von Willebrand ao GP llb-llla. [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] O documento US5686570, por sua vez, revela um ensaio para rastrear a peçonha de serpente quanto à presença ou ausência de inibidores da agregação plaquetária (PAIs) com base na ligação específica ao receptor. Usando este ensaio, foi realizada a identificação e caracterização de PAIs em uma ampla faixa de amostras de peçonha de serpente. O PAI isolado e purificado de vários dessas peçonhas de serpente ativas é descrito e caracterizado. Além disso, PAIs sem a sequência de adesão Arg-Gly-Asp (RGD), mas contendo um fragmento específico, são revelados e mostraram inibir especificamente a ligação de fibrinogênio ou Fator de von Willebrand para GP llb-llla. [07] Document US5686570, in turn, reveals a assay to screen snake venom for the presence or absence of platelet aggregation inhibitors (PAIs) based on specific receptor binding. 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 the binding of fibrinogen or von Willebrand Factor to GP llb-llla.
[08] Já o documento US5851839 revela um grupo de peptídeos que são, ou estão relacionados a, inibidores da agregação plaquetária isolados e purificados de várias peçonhas de serpentes. Estes peptídeos são úteis como agentes terapêuticos para o tratamento e prevenção de distúrbios isquêmicos associados a plaquetas. Mais especificamente, este documento revela peptídeos que bloqueiam receptores específicos para proteínas adesivas envolvidas na aderência e agregação plaquetária. [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] Como pode-se perceber, o estado da técnica apresenta diversos compostos derivados de peçonha de serpente com atividade antiagregante plaquetária e antitrom bótica. Entretanto, ao usar metodologia semelhante com um espécime da biodiversidade brasileira, a saber, Bothrops jararaca, os inventores verificaram que uma proteína isolada não apresentou o mesmo efeito que o macrociclo Eptifibatide, cuja estrutura é derivada da desintegrina barbourina isolada de Sistrurus m. barbouri (Journal of Biological Chemistry, 1991 ; 266: 9359-62). Ao contrário, a nova proteína apresentou pouco ou nenhum efeito na agregação plaquetária. Assim, os inventores verificaram que a nova proteína isolada apresentou, de maneira surpreendente, efeito anti-inflamatório. [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] Dessa maneira, um dos objetivos da invenção é fornecer proteínas e peptídeos e macrociclos com efeito anti-inflamatório. É ainda objetivo da presente invenção fornecer composições contendo as proteínas e peptideos com efeito anti-inflamatório com menos efeitos colaterais e com menor custo de produção. [010] Thus, one of the objectives of the invention is to provide proteins and peptides and macrocycles with an anti-inflammatory effect. It is a further object of the present invention to provide compositions containing the proteins and peptides with anti-inflammatory effect with fewer side effects and lower production cost.
[011] Na presente invenção, os inventores empregaram técnicas de sequenciamento de alto desempenho (high throughput sequencing) direcionado para a identificação de novas toxinas diretamente sobre o mRNA das glândulas de peçonha. Esta abordagem permitiu a descoberta de novas estruturas líderes, a partir de proteínas (ou mRNA) minoritárias na composição da peçonha, que não seriam identificadas com o emprego de técnicas tradicionais de venômica (fracionamento cromatográfico e proteômica). O alto poder analítico do método aplicado (RNAseq) permitiu a identificação de sequências codificantes de proteínas inéditas, como é o caso da sequência descrita na presente invenção. Essa nova sequência foi clonada e expressa em sistema heterólogo sendo sua forma recombinante denominada r-Bothrojatina, de acordo com o comitê para a padronização da nomenclatura das desintegrinas (J. Thromb Haemost 2007 5:1971 ). [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] Na presente invenção, foram utilizados métodos de sequenciamento genético de alto desempenho utilizando a técnica de RNAseq para identificar novos alvos moleculares de natureza peptídica que não seriam identificados por técnicas mais tradicionais de venômica (Curr Pharm Des, 2007;13(28):2927-34). Através do sequenciamento do DNA complementar das glândulas de peçonha de serpentes da espécie B. jararaca foi identificada a sequência codificante parcial de uma metaloproteinase desconhecida pelo estado da arte. O domínio desintegrina foi expresso de maneira heteróloga, com a adição de alguns aminoácidos na sua porção N-terminal, e essa proteína recombinante, denominada r-Bothrojatina, apresentou propriedades anti- adesivas e anti-inflamatórias inéditas para o domínio presente na desintegrina, conforme demonstrado em ensaios funcionais in vitro e in vivo. Com base na caracterização funcional e na sequência da Bothrojatina, foi sintetizada e caracterizada uma família de moléculas farmoquímicas por meio de síntese de peptídeos de fase sólida (SPFS). Breve descrição da invenção [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 way, 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] A presente invenção se refere a peptídeos sintéticos macrocíclicos com atividade anti-inflamatória. A presente invenção também se refere a preparações medicinais contendo os referidos peptídeos sintéticos. [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] Foi realizada uma análise de transcriptoma da glândula produtora de peçonha de serpentes B. jararaca (RNAseq) para prospecção de novas sequências de proteínas (e estruturas líder) com alto potencial biológico. [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] A presente invenção propicia o desenvolvimento e caracterização de uma nova família de moléculas que apresenta atividades surpreendentes e inesperadas para o estado da arte, uma vez que são moléculas derivadas de um domínio de desintegrina KGD e desta origem havia conhecimento somente de efeitos sobre a cascata de coagulação/hemostasia. [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] Esta nova família de moléculas possui atividade e especificidade distintas das descritas até então, afetando a interação de células imunes com elementos da matriz extracelular (MEC), mas não a cascata de coagulação ou o processo de agregação plaquetária. [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] Em uma modalidade da presente invenção, são fornecidos peptídeos caracterizados por compreender a Fórmula (I): [017] In one embodiment of the present invention, peptides characterized by comprising Formula (I) are provided:
X1-X2-Gly-X4-X5-X6-Cys Fórmula (I) X1-X2-Gly-X4-X5-X6-Cys Formula (I)
Onde Where
X1 é 3mpr, Cys ou 2mac, preferencialmente 3mpr; X1 is 3mpr, Cys or 2mac, preferably 3mpr;
X2 é Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Vai, Trp, Tyr, Harg, Orn ou Dab, preferencialmente Harg; X2 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr, Harg, Orn or Dab, preferably Harg;
X4 é Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Vai, Trp, Tyr, Harg, Gla ou Hglu, preferencialmente Glu; X4 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr, Harg, Gla or Hglu, preferably Glu;
X5 é Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Vai, Trp, Tyr, Harg ou Cha, preferencial mente Tyr; eX5 is Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr, Harg or Cha, preferably Tyr; It is
X6 é Pro ou abz2, preferencialmente Pro; em que X6 is Pro or abz2, preferably Pro; on what
Harg é homoarginina Harg is homoarginine
Hglu é ácido homoglutâmico Hglu is homoglutamic acid
Orn é ornitina orn is ornithine
Dab é ácido alfa gama diaminobutírico Dab is alpha gamma diaminobutyric acid
Gla é ácido gama carboxiglutâmico 3mpr é 3-ácido mercaptopropiônico 2mac é ácido 2-mercaptoacético abz2 é ácido 2-aminobenzóico Cha é ciclohexil alanina, Gla is gamma carboxyglutamic acid 3mpr is 3-mercaptopropionic acid 2mac is 2-mercaptoacetic acid abz2 is 2-aminobenzoic acid Cha is cyclohexyl alanine,
Em que consiste, preferencialmente, nas sequências SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10 e SEQ ID NO:11 , seus ciclos, enantiômeros e fragmentos dos mesmos. Preferably consisting of 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] Em uma modalidade, é fornecido um vetor que compreende uma construção de DNA consistindo na sequência definida pela SEQ ID NO: 1 (Bothrojatina), operacionalmente ligada às sequências promotora e terminadora heterólogas e capaz de expressar a proteína da SEQ ID NO:1 (Bothrojatina). [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] Em uma modalidade da invenção, é fornecido uma composição farmacêutica que compreende um ou mais peptídeos da invenção e um veículo farmaceuticamente aceitável. Em outra modalidade da invenção, é fornecido uma composição que compreende, a proteína da SEQ ID NO:1 (Bothrojantin) em adição aos peptídeos da invenção. [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] Em uma modalidade da invenção, a composição farmacêutica é apresentada na forma de um adesivo transdérmico, solução, suspensão, emulsão, comprimidos ou cápsulas. Em uma modalidade da invenção, a composição farmacêutica é solução injetável, na forma de micropartículas ou nanopartículas. [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] Em uma modalidade da invenção, os peptídeos e as composições da invenção são utilizados para a preparação de um medicamento para uso em doenças inflamatórias, doenças autoimunes, doenças hematológicas e doenças neurológicas. [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.
Breve descrição das figuras Brief description of figures
[022] A Figura 1 descreve a sequência predita e o alinhamento da nova sequência de desintegrina (Bothrojatina) com outras proteínas semelhantes contendo o domínio KGD/RGD. Em (A), é apresentada a sequência parcial do gene da metaloproteinase da peçonha de serpente (SVMP, do inglês snake venom metaloproteinase) encontrada a partir da análise de dados de RNA-Seq de glândula de peçonha de B. jararaca, a sequência recombinante de aminoácidos do domínio desintegrina com 76 aminoácidos (em destaque cinza claro), sendo 13 resíduos de cisteínas (sublinhados) e um domínio KGD (em destaque cinza escuro) encontram-se em destaque. Em (B) é apresentado o modelo tridimensional da estrutura da proteína Bothrojatina baseado no sistema de homologia SWISS-MODEL Para esse modelo foi utilizado como molde a estrutura da desintegrina Bitistatina B (PDB: 2MP5) que apresenta 72% de identidade com a nova desintegrina. Em (C) é apresentada a análise de alinhamento múltiplo da Bothrojatina e várias outras desintegrinas conhecidas de peçonhas de serpentes. O domínio de ligação à integrinas está destacado em azul e os aminoácidos adjacentes importantes para ligação e seletividade das moléculas estão sublinhados. Os resíduos de cisteína, importantes para o enovelamento das desintegrinas, são conservados entre as moléculas e estão em destaque cinza escuro. O domínio NCQ está destacado em cinza claro. A identidade (% de identidade) da Bothrojatina com cada desintegrina está anotado à direita. Os números de acesso no GenBanK para cada desintegrina conhecida são: 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] A figura 2 representa as etapas de expressão e purificação da nova desintegrina recombinante r-Bothrojatina. Em (A), é apresentada a sequência de aminoácidos da proteína recombinante r- Bothrojatina expressa em vetor pET-15b fusionada ao epítopo de histidina N- terminal e sítio de clivagem por trombina (local da clivagem em destaque com o triângulo preto), gerando uma proteína resultante com 4 aminoácidos no domínio N-terminal (destacados em negrito) em comparação com o domínio nativo. Em (B) é apresentado um perfil de cromatografia de afinidade representativo das etapas da purificação da proteína r- Bothrojatina. A linha azul representa o valor de absorção de proteínas da amostra em 280 qm por volume de eluição, enquanto a linha verde representa 0 % de tampão B de eluição por volume de eluição. Em (C) é apresentada uma imagem representativa de um gel SDS-PAGE das amostras obtidas nas etapas de purificação da Bothrojatina recombinante expressa em E. coli., onde: 1- padrão de peso molecular; 2- fração das proteínas solúveis totais; 3- fração após primeira etapa de purificação cromatográfica; 4- fração após segunda etapa de polimento mostrando a proteína pura. Em (D) é apresentado uma imagem representativa de Western Blotting utilizando anticorpo capaz de reconhecer o epítopo de histidinas fusionado a proteína recombinante, onde: 1- r-Bothrojatina purificada; 2- r-Bothrojatina purificada após a remoção do epítopo de histidinas. Em (E) é apresentado um espectro representativo de análise de dicroísmo circular da r-Bothrojatina., demonstrando que a proteína recombinante resultante do processo de purificação possui estrutura secundária. [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. (B) shows the three-dimensional model of the Bothrojatin protein structure based on the SWISS-MODEL homology system. . In (C) the multiple alignment analysis of Bothrojatin and several other known disintegrins 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 r-Bothrojatin expressed in pET-15b vector is shown, fused to the N-terminal 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 µm 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] A Figura 3 mostra que a desintegrina Bothrojatina não apresenta efeito antitrombótico quando comparado com conhecidos compostos antiplaquetários e anticoagulantes, conforme mostrado pela falta de atividade observada em: A) ensaio de agregação de plaquetas (em comparação com o composto antiplaquetário Eptifibatide), onde a agregação plaquetária é induzida por adição de ADP; B) ensaio de adesão celular, onde é medido o percentual de adesão das células ao seu substrato fibrinogênio utilizando células CHO-K1 previamente transfectadas transientemente com plasmídeos codificantes da integrina a2b[33 (presente na superfície de plaquetas e inibidas pelo Eptifibatide), ativadas com MnCl2, e submetidas a tratamento com diferentes concentrações de Bothrojatina e Eptifibatide (controle: células transfectadas com vetor vazio, não ativadas com MnCl2 ou utilizando albumina de soro bovino como substrato); e C) ou ensaio de coagulação sanguínea na presença de Bothrojatina ou heparina (controle positivo para inibição), medido por tempo de tromboplastina parcialmente ativada (aPTT), induzido por adição de ADP. Para A, B e C os valores são representativos da média de três experimentos ± SE. * p <0,05, ** p <0,01 (one way ANOVA). [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 it is measured the percentage of adhesion of cells to their fibrinogen substrate using CHO-K1 cells previously transiently transfected with plasmids encoding a2b[33 integrin (present on the surface of platelets and inhibited by Eptifibatide), activated with MnCl2, and subjected to treatment with different concentrations of Bothrojatine and Eptifibatide (control: cells transfected with empty vector, not activated with MnCl2 or using bovine serum albumin as substrate); and C) or blood clotting assay in the presence of Bothrojatin or heparin (positive control for inhibition), measured by partially activated thromboplastin time (aPTT), induced by 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] A Figura 4 apresenta os efeitos da desintegrina Bothrojatina sobre a adesão, migração e invasão celular. Um ensaio de adesão celular à laminina foi utilizado para testar o efeito de concentrações crescentes de desintegrina Bothrojatina na adesão à laminina em diferentes tipos ou linhagens celulares: (A) B16F10; (B) BHK; (C) HUVEC; e (D) THP-1. A migração de células THP-1 foi monitorada pela passagem das células por entre a membrana porosa das câmaras do inserto transwell (E) e a invasão celular foi monitorada pela capacidade das células atravessarem a membrana porosa do inserto transwell e a superfície de matrigel (F). Os resultados representam a média de pelo menos três experimentos independentes. A significância estatística foi determinada por ANOVA (* p <0,05, ** p <0,01 ). [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] A Figura 5 apresenta os efeitos dos peptídeos cíclicos da invenção sobre a adesão, migração e invasão celular, reproduzindo os efeitos biológicos da r-Bothrojatina. Um ensaio de adesão celular à laminina foi utilizado para testar o efeito de concentrações crescentes dos peptídeos cíclicos S900050, S900066 e S900064 às células (A) B16F10; (B) BHK; (C) HUVEC; e (D) THP-1. A migração e invasão celular de células THP-1 foi verificada através de ensaios com insertos de transwell, conforme descrito para a Figura 4 para migração celular (E) e invasão celular (F). [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-Bothrojatine. 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 through assays with transwell inserts as described for Figure 4 for cell migration (E) and cell invasion (F).
[027]A Figura 6 apresenta o limitado efeito antitrombótico dos peptídeos cíclicos da invenção. Os peptídeos cíclicos S900050, S900066 e S900064 tiveram um limitado efeito antiplaquetário e anticoagulante, conforme mostrado pelos resultados observados em: (A) ensaio de agregação de plaquetas induzido por adição de ADP e utilizando como controle positivo o peptídeo inibidor de integrina a2b[33, Eptifibatide (resultados apresentados como percentual de inibição de agregação plaquetária); e (B) ensaio de coagulação sanguínea por quantificação do tempo de tromboplastina parcialmente ativada (aPTT), em comparação com os efeitos de heparina e Bothrojatina. A significância estatística foi determinada por ANOVA (* P <0,05, ** P <0,01 ). [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 integrin inhibitor peptide a2b[33, Eptifibatide (results presented as percent 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] A Figura 7 apresenta o efeito inibitório sobre a migração celular de leucócitos peritoneais murinos in vivo pela desintegrina Bothrojatina e dos peptídeos cíclicos da invenção S900050, S900066 e S900064. A atividade anti-inflamatória da desintegrina Bothrojatina e dos peptídeos cíclicos foi testada utilizando um modelo animal de peritonite induzida por tioglicolato de sódio. (A) A Bothrojatina inibiu a migração de leucócitos de forma dependente da dose. (B) Os peptídeos S900050, S900066 e S900064 também inibiram a migração de leucócitos para o peritônio. A desintegrina Bothrojatina e os peptídeos cíclicos foram administrados de forma intravenosa na veia lateral da cauda 1 h antes da injeção intraperitoneal de tioglicolato. A concentração de leucócitos foi determinada no lavado peritoneal após 3 h do estímulo anti-inflamatório. [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] A Figura 8 apresenta os efeitos inibitórios da desintegrina Bothrojatina e dos peptídeos cíclicos da invenção sobre a migração de leucócitos peritoneais 24 e 48 h após indução com tioglicolato de sódio. Os leucócitos peritoneais foram coletados em diferentes tempos após estimulação por tioglicolato de sódio e sua capacidade de migrar em um modelo utilizando inserções transwell foi avaliada. (A) A desintegrina Bothrojatina e os peptídeos cíclicos inibiram a migração de leucócitos isolados 24 e 48 h após o estímulo de tioglicolato de sódio; efeitos menos significativos foram observados em células isoladas após 3 h e 72 h. (B) Uma análise das integrinas presentes nos leucócitos isolados do peritônio indicaram um padrão de expressão estável das subunidades a3, a4 e supra-regulação de (31 especificamente às 24 e 48 h após o tratamento de tioglicolato. [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 stimulation; Less significant effects were seen in isolated cells after 3 h and 72 h. (B) An analysis of the integrins present in leukocytes isolated from the peritoneum indicated a stable expression pattern of a3, a4 subunits and upregulation of (31 specifically at 24 and 48 h after thioglycolate treatment.
[030] A Figura 9 apresenta o efeito do peptídeo S90050 sobre a migração de neutrófilos para o peritônio durante a inflamação induzida por tioglicolato de sódio. 48 h após o estímulo por tioglicolato de sódio, foi monitorado por citometria de fluxo a migração de leucócitos marcados com anticorpos anti-GR-1-APC (neutrófilos), anti-F4/80-ALEXA Flúor 488 (macrófagos) e anti-CD3-PE (células T). A Figura mostra resultados representativos onde as células não foram estimuladas (controle), e estimuladas e tratadas com PBS (PBS) ou tratadas com S90050. [030] Figure 9 shows the effect of 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] A Figura 10 apresenta a análise do papel de diferentes integrinas na migração de leucócitos para o peritônio de camundongos após a indução de inflamação por tioglicolato de sódio, utilizando um modelo de inativação com anticorpos. Células do peritônio de camundongos foram isoladas 24 após estímulo com tioglicolato de sódio e a sua migração foi analisada utilizando insertos de transwell. A) a migração dos leucócitos peritoneais foi inibida pelo bloqueio de anticorpos contra a4 e (31 , mas não contra a3. B-C) O tratamento conjunto dos anticorpos antagonistas anti- a4 e (31 com a Bothrojatina apresentou efeito não cumulativo. [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) The migration of peritoneal leukocytes was inhibited by blocking antibodies against a4 and (31 , but not against a3. B-C) The joint treatment of antagonistic antibodies anti-a4 and (31 with Bothrojatine showed no cumulative effect.
[032] A Figura 11 apresenta o efeito anti-inflamatório do peptídeo S900050 em um modelo animal de colite química induzida pela adição de Ácido 2,4,6-trinitrobenzeno sulfônico (TNBS) em camundongos Balb/c. É observado que o tratamento intravenoso com o peptídeo S900050 promove uma diminuição no processo de perda de peso induzido por TNBS (A), uma diminuição no processo de contração do cólon induzida por um processo inflamatório (B) e uma diminuição da taxa de migração de células inflamatórias para a parede intestinal dos camundongos. A significância estatística foi determinada entre grupos que receberam (quadrado sólido) e não receberam (círculo sólido) o tratamento com S900050 por ANOVA (* P <0,05, ** P <0,01 ). [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 peptide S900050 promotes a decrease in the process of weight loss induced by TNBS (A), a decrease in the process of colon contraction induced by a inflammatory process (B) and a decrease in the rate of migration of inflammatory cells to the intestinal wall of mice. Statistical significance was determined between groups that received (solid square) and did not receive (solid circle) S900050 treatment by ANOVA (*P<0.05, **P<0.01).
[033] A Figura 12 apresenta a análise de formulações micro- e nanoestruturadas compostas do polímero biodegradável ácido poli lático co-glicólico (PLGA). (A) Cromatograma de identificação de um dos peptídeos da invenção após a purificação, que é caracterizado dentro do gradiente da fase móvel com um pico de intensidade máxima no meio do ciclo cromatográfico (pico em 15063). (B) Gráfico de espalhamento dinâmico de luz (do inglês DLS - diffraction light scattering) das micropartículas da formulação. O pico do espalhamento de luz classifica a amostra no tamanho médio (dl 0,5) em tomo de 52 pm; à esquerda é apresentada uma fotografia de microscopia eletrônica de varredura ilustrando a estruturação esférica das micropartículas componentes da formulação LAR (do inglês long-acting release - liberação de ação prolongada) ou Depot (depósito). (C) Gráfico de espalhamento dinâmico de luz das nanopartículas da formulação. O pico do espalhamento de luz classifica a amostra no tamanho médio (dl 0,5) em torno de 103 qm; à esquerda é apresentada uma fotografia de microscopia eletrônica de varredura ilustrando a estruturação esférica das nanopartículas componentes da formulação LAR ou Depot. [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 average size (dl 0.5) around 52 pm; 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 qm; On the left is a scanning electron microscopy photograph illustrating the spherical structure of the nanoparticles that make up the LAR or Depot formulation.
Descrição detalhada da invenção Detailed description of the invention
[034] Os inventores realizaram análise do transcriptoma da glândula produtora de peçonha de serpentes da espécie B. jararaca (por RNAseq) para prospecção de novas sequências de proteínas (e estruturas líder) com alto potencial biológico. O manejo dos animais foi realizado de acordo com os Princípios de Ética em Pesquisa Animal e aprovado pela Comissão de Ética para Experimentação Animal do Centro de Ciências da Saúde da Universidade Federal do Rio de Janeiro (n° 077/10). Espécimes de B. jararaca foram coletadas e as glândulas de peçonha foram dissecadas após 4 dias da coleta manual da peçonha, quando a taxa de transcrição é máxima. As glândulas de peçonha foram imediatamente colocadas em solução RNAIater™ (Qiagen) e congeladas à -70 °C até seu uso. [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 (n° 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 RNAIater™ solution (Qiagen) and frozen at -70 °C until use.
[035] O tecido das glândulas de peçonha foi homogeneizado através de um rotor-extrator e o RNA total foi extraído através do kit RNAeasy (Qiagen) conforme instruções do fabricante. O mRNA foi extraído (através do kit Dynabeads® mRNA Purification kit - Invitrogen), purificado e fragmentado com ZnCl2 de maneira que os fragmentos gerados apresentem tamanho médio de aproximadamente 700 pb. O mRNA fragmentado foi convertido em cDNA (kit cDNA Synthesis System - Roche). As extremidades dos fragmentos foram polidas e reparadas (com as enzimas T4 DNA polimerase e T4 polinucleotídeo quinase) através da incubação em um termociclador por 20 min a temperaturas de 10 a 15 °C, 20 min a 25 °C e por 20 min a 75 °C. Posteriormente, foi feita a ligação de adaptadores para sequenciamento com a enzima T4 DNA. [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.
[033] Para a remoção de fragmentos pequenos de polinucleotídeos, as amostras foram purificadas pelo método SPRI (solid phase reversible immobilization). Nesse procedimento, as partículas magnéticas são incubadas com o DNA e, durante essa incubação, as partículas magnéticas são ligadas ao DNA mas não à fragmentos pequenos de DNA (por exemplo, primers, nucleotídeos livres etc.). Após a retenção das partículas magnéticas por uma placa imantada, essas são lavadas para remoção dos fragmentos pequenos não ligados e o DNA remanescente ligado às partículas é eluido para obtenção de uma biblioteca de cDNA. O cDNA assim obtido foi ligado às partículas (beads) específicas para o protocolo de sequenciamento através da plataforma 454 de piro-sequenciamento (Roche). Do processo de sequenciamento foram obtidas 487018 sequências (comumente denominadas de reads). Em seguida, foi realizada a montagem das sequências de novo utilizando Newbler 2.5 (454, Life Sciences), que é um montador (assembler) especializado em NGS (next generation sequencing), com as opções -cNDA, - urt e -mi98. A partir dos resultados do Newbler foi executada uma montagem secundária utilizando um montador mais adequado a sequências maiores, o iAssembler (http://bioinfo.bti.cornell.edu/tool/iAssembler), para reduzir a redundância e unir mais sequências que não foram unidas pelo Newbler usando um script que utiliza o MIRA como montador primário e o CAP3 como secundário, além de aplicar correções pós-montagem. Como resultado, foram obtidas montagens de dois transcriptomas, obtendo-se para cada transcriptoma um conjunto de genes únicos, comumente denominados unigenes. [033] 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. Then, the sequences were assembled again using Newbler 2.5 (454, Life Sciences), which is an assembler. specialized in NGS (next generation sequencing), with the -cNDA, -urt and -mi98 options. Based on the Newbler results, a secondary assembly was performed using an assembler more suitable for larger sequences, the 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.
[034] Os inventores da presente invenção compararam os unigenes obtidos nas montagens da invenção com sequências de mRNA de B. jararaca obtidas em bancos próprios e de colaboradores, para verificar se as sequências originais do mRNA da presente invenção não haviam sido previamente identificadas por outros grupos e estavam presentes nos bancos de dados. Considerando apenas os unigenes que tiveram similaridade maior de 90% e cobrindo pelo menos 90% de seu tamanho, para uma amostra apenas 12,4% dos reads já haviam sido descritos e para outra amostra apenas 40,85% dos reads já haviam sido descritos. Esses dados comprovam a eficiência da metodologia utilizada para a identificação de novas proteínas com alto potencial biológico. A análise dos transcriptoma obtidos identificou uma sequência parcial de metaloproteinase de peçonha de serpente contendo o domínio desintegrina (domínio de ligação à integrina KGD) (Figura 1A e 1 B). [034] 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).
[035] Para a anotação funcional, em cada uma das duas montagens, foi realizada busca por similaridade utilizando o BLASTX contra o banco de dados de proteínas NCBI nr, com e-value 10"5, para identificar possíveis proteínas homólogas, e, também, BLASTN contra o banco de nucleotídeos, NCBI nt, para tentar identificar supostas proteínas quando não houve hit com o banco nr. Os resultados do BLASTX foram então importados no software Blast2G0, onde foi possível anotar parte dos unigenes com termos de Gene Ontology (GO), códigos da Enzyme Commission (EC), e vias metabólicas, através do KEGG (Kyoto Encyclopedia of Genes and Genomes). A Figura 1C apresenta esse resultado comparativo entre a Bothrojatina e outras desintegrinas conhecidas. [035] 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 NCBI nt bank of nucleotides, to try to identify supposed proteins when there was no hit with the nr bank. The BLASTX results were then imported into the Blast2G0 software, where it was possible to annotate part of the unigenes with Gene Ontology terms ), Enzyme Commission (EC) codes, and metabolism, through KEGG (Kyoto Encyclopedia of Genes and Genomes). Figure 1C presents this comparative result between Bothrojatine and other known disintegrins.
[036] A sequência da nova proteína identificada, denominada Bothrojatina, foi então clonada em plasm ídeos comerciais (pGEM- T, pET-15b e pSELECT-His) para a produção da proteína recombinante r- Bothrojatina. De acordo com a estratégia de purificação utilizada, a proteína é expressa no mesmo sentido de leitura com uma cauda de Histidina na porção N-terminal. A proteína recombinante foi purificada em sistemas de cromatografia de afinidade (coluna HisTrap HP- GE), troca iônica (coluna Mono Q 5/50 GL - GE), gel filtração (coluna Sephacryl S-100 HR ou Superdex 200- GE) ou fase reversa (coluna C18- Allcrom) em sistema Akta Avant (GE). Para cada sistema, foram feitas as trocas de tampão de acordo com as especificações de cada coluna. Após a purificação, a pureza da proteína foi verificada por SDS-PAGE e confirmada por técnica de Western Blotting utilizando anticorpo capaz de reconhecer o epítopo de histidinas fusionado à proteína recombinante. [036] The sequence of the new identified protein, called Bothrojatina, was then cloned into commercial plasmids (pGEM-T, pET-15b and pSELECT-His) for the production of recombinant protein r- Bothrojatina. 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.
[037] O epítopo de histidinas foi retirado da Bothrojatina recombinante pela clivagem com trombina (5-20 U/mg de proteína) em reação durante 12-16 horas a temperatura ambiente. As amostras foram então aplicadas em membranas de filtração tangencial com tamanhos variando entre 100 e 3 kDa, preferencialmente 30 e 3 KDa, para remoção da trombina contam inante e o fragmento proteico da cauda de poli-His. Ao final dessa etapa a Bothrojatina recombinante purificada foi estocada a -20 °C até o uso. A figura 2 representa as etapas de expressão e purificação da nova desintegrina recombinante Bothrojatina. [037] 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.
[038] A peçonha de serpentes é uma rica fonte de compostos biologicamente ativos contendo proteínas com alta afinidade e seletividade para componentes específicos nas células. Dentre as proteínas com atividade biológica de peçonhas de serpente, os inventores destacam as metaloproteases, que são enzimas que tem um papel importante na patogênese relacionada ao envenenamento. Entre as diferentes metaloproteinases encontradas, as metaloproteases da classe P-ll contêm um domínio desintegrina com potente atividade de ligação e inibição das integrinas. [038] 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 of snake venoms, the inventors highlight the metalloproteases, which are enzymes that play an important role in envenomation-related pathogenesis. Among the different metalloproteinases found, the P-ll class metalloproteases contain a disintegrin domain with potent integrin binding and inhibition activity.
[039] No trabalho publicado (J Proteomics, 2016, 135, 73- 89), os inventores descreveram que usaram dados de proteômica para confirmar sequências encontradas no transcriptoma da presente invenção. A caracterização funcional e estrutural da Bothrojatina serviu de estrutura líder para a síntese química de moléculas macrocíclicas contendo aminoácidos naturais e análogos não naturais, reproduzindo os efeitos anti-inflamatórios observados com a desintegrina recombinante. A invenção parte de sequências truncadas da sequência da Bothrojatina, tendo como centro o núcleo farmacofórico KGD. A tabela 1 mostra as sequências obtidas nesse processo. Estão incluídas na tabela 1 as sequências obtidas a partir de simulações de ancoragem molecular (molecular docking) in sílico, no formato de SEQ ID NO: X (as SEQ ID NO:4-6 da tabela 1 correspondem às mesmas sequências SEQ ID NO:4-6 do arquivo de listagem de sequências; as SEQ ID NO: 7-11 correspondem aos peptídeos utilizados nos Exemplos). [039] 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 the KGD pharmacophoric nucleus as its center. 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).
Tabela 1. Listagem de sequências da invenção
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Table 1. Sequence listing of the invention
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
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). 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).
[040] Três sequências (S900064-13-mer e S900066-11- mer e S009349-8-mer) obtidas destas construções mostraram-se promissoras. Ainda neste grupo, alterações foram produzidas, como a redução do número de resíduos e alteração de grupos funcionais, e mais duas sequências foram incluídas: Mpa-KGDMDC (S000015) e 7-mer Mpa-HarGEYPC (S900050). A triagem experimental destes compostos confirmou que a estratégia de miniaturização e mimetização da estrutura da Bothrojatina foi capaz de manter as atividades anti-inflamatórias observadas com a proteína recombinante. Baseado nos resultados obtidos, foram realizadas modificações à estrutura original da Bothrojatina para criar uma biblioteca virtual de compostos, para ser empregada em estudos de ancoragem molecular (molecular docking) in silico, triagem in vitro e in vivo. [040] 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.
[041] Diferentes modificações foram incorporadas às estruturas descritas acima, como a redução de grupos carboxílicos na sequência (remoção de aminoácido negativos ou substituição por polares ou apoiares), a remoção do N-terminal (eliminação dos aminoácidos anteriores ao núcleo farmacofórico KGD, e o remodelamento do C-terminal (resíduos posteriores ao resíduo polar ácido do núcleo farmacofórico) formando um novo núcleo farmacofórico R/KGDX, sendo X um resíduo apoiar ou polar não carregado capaz de aumentar a especificidade deste núcleo, preferencialmente resíduos que conferem alguma rigidez estrutural (tensão angular ou impedimento esférico) com estruturas homo- ou heterocíclicas integradas à estrutura básica sem no entanto conferir carga efetiva. Este domínio pode apresentar maior especificidade de ligação se há a formação de molécula cíclica (macrocíclico), conferindo uma estruturação diferente do peptídeo linear (J. Biol. Chem. 1997, 272-21341-21348; J. Biol. Chem., 2000, 275-21785- 21788). Todas essas sequências encontram-se representadas na Tabela 1. Dessa forma, além das estruturas acíclicas, também é um dos objetos da invenção os macrociclos (estruturas cíclicas) através de ponte dissulfeto, ligação peptídica ou por meio de quaisquer outras reações químicas capazes de unir as extremidades das estruturas lineares das moléculas, independentemente do número de resíduos de aminoácidos ou subunidades estruturais. [041] Different modifications were incorporated into the structures described above, such as the reduction of carboxylic groups in the sequence (removal of negative amino acids or replacement by polar or apolar), removal of the N-terminus (removal of amino acids prior to the pharmacophoric core KGD, and remodeling of the C-terminus (residues subsequent to the polar acidic residue of the pharmacophoric core) forming a new pharmacophoric core R/KGDX, with X being a residue non-charged polar or non-polar capable of increasing the specificity of this nucleus, preferably residues that confer some structural rigidity (angular tension or spherical impediment) with homo- or heterocyclic structures integrated into the basic structure without, however, conferring effective charge. This domain may have greater specificity of binding 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 bridges, peptide bonds, or any other chemical reactions capable of joining the ends of the linear structures of molecules, regardless of the number of amino acid residues or structural subunits.
Exemplos Exemplo 1 [042] Os ensaios experimentais demonstram que a desintegrina recombinante Bothrojatina não apresenta efeito antiplaquetário e anticoagulante quando comparado com compostos conhecidos (Figura 3). O gráfico apresentado na Figura 3 mostra o percentual de células aderentes ao fibrinogênio de células transfectadas e ativadas (definido como 100% de adesão). Examples Example 1 [042] Experimental assays demonstrate that the recombinant disintegrin Bothrojatine does not show 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).
[043] A capacidade de migração de THP-1 em direção a um estímulo inflamatório foi aferida num ensaio de migração utilizando uma câmara de Boyden modificada (inserto transwell), que cria duas câmaras de cultivo separadas por uma membrana porosa. A adição da Bothrojatina inibiu a migração de THP-1 , uma célula de linhagem monocítica humana, de uma maneira dependente da concentração (Figura 4E). Fisiologicamente, é essencial que os leucócitos possam aderir e migrar por entre as células endoteliais, para posteriormente degradar a matriz extracelular (MEC) dos vasos sanguíneos, de forma a alcançar os locais da inflamação, tanto em condições fisiológicas como em condições patológicas. Foram simuladas as propriedades invasoras de leucócitos in vitro através da recuperação da superfície do inserto com uma camada de gel à base de matriz de colágeno e laminina (geltrex® - ensaio de invasão de matriz de gel). Mais uma vez, a desintegrina inibiu a invasão da matriz por THP-1 em uma maneira dependente da dose (Figura 4F). Esses resultados indicam que Bothrojatina tem um potencial anti-inflamatório, servindo como molécula líder para o desenvolvimento de NEQs antagonistas de integrinas. [043] 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 cell of human monocytic lineage, 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.
Exemplo 2 Example 2
[044] A capacidade de desintegrinas em modular a interação entre as integrinas e seus respectivos substratos está relacionada com diferentes mecanismos patológicos. Especificamente, a inibição da resposta inflamatória através da regulação da atividade de integrinas está relacionada com o tratamento de doenças autoimunes, incluindo atrite reumatoide, artrite reativa, esclerose múltipla, esclerodermia, diabetes mellitus, dermatite atópica, tireoidite de Hashimoto (doença de graves), lúpus eritematoso, síndrome de Sjogrem, vitiligo, doença de Crohn e célica, retocolite ulcerativa, miocardites e vasculites autoimunes, psoríase, anemia falciforme, para citar algumas. [044] 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.
[045] Para testar seu potencial anti-inflamatório foi testado o efeito da Bothrojatina e os respectivos macrociclos derivados do mesmo sobre a interação integrina e seu substrato (Figura 4A a 4D). Inicialmente foram, então, utilizados ensaios de adesão celular em placa para avaliar o efeito da Bothrojatina sobre a interação com diferentes proteínas constituintes da MEC. Uma triagem do efeito inibitório da Bothrojatina utilizando diferentes substratos (fibrinogênio, fibronectina, colágeno V, vitronectina e laminina) e células, como por exemplo fibroblastos (linhagem celular de rim de hamster, BHK), células epiteliais (linhagem celular de melanoma murino, B16F10), células primárias de endotélio vascular (linhagem de células endoteliais da veia umbilical humana, HUVEC) e células monocíticas (linhagem celular monocítica humana, THP-1 ) apresentou efeito inibitório na ligação celular à laminina. Efeitos marginais foram observados com outros substratos testados. [045] 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 (cell line from human umbilical vein endothelial cells, 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.
[046] Para demonstrar que peptídeos cíclicos derivados da Bothrojatina reproduzem os efeitos biológicos observados anteriormente, novos ensaios de adesão, migração e invasão celular in vitro foram realizados. Em concordância com o efeito biológico promissor exibido pela Bothrojatina, os compostos macrocíclicos derivados da sua estrutura tridimensional também foram capazes de inibir a adesão, migração e invasão celular (Figura 5). Além disso, semelhante à Bothrojatina, os peptídeos sintéticos testados tiveram efeito limitado sobre a cascata de coagulação ou ligação do fibrinogênio- integrina a2b[33 presentes na agregação plaquetária (Figura 6). [046] 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 limited effect on the coagulation cascade or fibrinogen-a2b[33 integrin binding present in platelet aggregation (Figure 6).
[047] Os resultados in vivo comprovam o potencial anti- inflamatório da Bothrojatina e seus peptídeos cognatos. A utilização de um modelo animal com inflamação aguda de peritonite química (injeção intraperitoneal de tioglicolato) confirmou o efeito inibitório sob a migração de células inflamatórias (Figura 7). [047] 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).
Exemplo 3 Example 3
[048] O potencial terapêutico de desintegrinas está associado à sua capacidade de agir especificamente sob diferentes integrinas. No caso, foi analisado o efeito da Bothrojatina e seus compostos derivados sobre a migração de leucócitos peritoneais. Leucócitos peritoneais de camundongos foram coletados às 3, 24, 48 e 72 horas após o estímulo pró- inflamatório com tioglicolato, e o seu perfil de migração in vitro foi avaliado na presença de Bothrojatina ou peptídeos derivados. Os peptídeos e a desintegrina inibiram a migração in vitro de populações de leucócitos isolados às 24 e 48 h após a instilação do tioglicolato (Figura 8A). Análise do perfil de expressão de integrina utilizando anticorpos monoclonais específicos revelaram expressão estável das integrinas a3 e a4 e a super-regulação específica de (31 em ambos os tempos de 24 e 48 horas após o tratamento de tioglicolato (Figura 8B). [048] 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 pro-inflammatory stimulation with thioglycolate, and their in vitro migration profile was evaluated in the presence of Bothrojatine or derived peptides. Peptides and disintegrin inhibited 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 the specific up-regulation of (31) at both the 24 and 48 hour time points after thioglycolate treatment ( Figure 8B ).
[049] Foi testado ainda o efeito do peptídeo S90050 sobre a migração de neutrófilos para o peritônio durante a inflamação induzida por tioglicolato de sódio 48 h após o estímulo por tioglicolato de sódio. Os resultados monitorados por citometria de fluxo mostram migração de macrófagos, neutrófilos e células T de células não estimuladas (controle), e estimuladas e tratadas com PBS (PBS) ou tratadas com S90050 (Figura 9). [049] 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).
[050] Em seguida, foram utilizados anticorpos bloqueadores como ferramenta para estudar a inibição específica de integrinas pelos compostos testados e a especificidade dos peptídeos inibitórios sobre as integrinas estudadas. Foram utilizados anticorpos de bloqueio contra as integrinas a3, a4 e (31 , em leucócitos peritoneais murinos isolados após 24 horas de injeção intraperitoneal de tioglicolato de sódio. O ensaio de interferência de anticorpos confirmou o papel da integrinas a4 e (31 , mas não da integrina a3 sobre a migração de leucócitos murinos in vitro (Figura 10A). A combinação da Bothrojatina com anticorpos inibitórios teve efeito inibidor comparável como seus fatores utilizados independentemente, sugerindo que eles podem ser o alvo do mesmo sítio ativo na superfície da célula (Figura 10B- C). Considerados em conjunto, estes resultados sugerem, que ao menos uma das formas que a Bothrojatina, ou seus peptídeos derivados, têm para inibir a inflamação é através da inibição da invasão de leucócitos mediada pela integrina a4(31 . [050] Then, blocking antibodies were used as a tool to study the specific inhibition of integrins by the compounds tested and the specificity of the inhibitory peptides on the integrins studied. Blocking antibodies against a3, a4 and (31) integrins were used on murine peritoneal leukocytes isolated after 24 hours of intraperitoneal injection of sodium thioglycolate. The antibody interference assay confirmed the role of a4 and (31) integrins, but not a3 integrin on murine leukocyte migration in vitro (Figure 10A). The combination of Bothrojatin with inhibitory antibodies had a comparable inhibitory effect as their factors used independently, suggesting that they may be the target of the same active site on the cell surface (Figure 10B - C) Taken together, these results suggest that at least one of the ways that Bothrojatine, or its derivative peptides, have to inhibit inflammation is through the inhibition of leukocyte invasion mediated by a4 integrin(31).
Exemplo 4 Example 4
[051] A especificidade de ligação das desintegrinas é determinante para a sua aplicação terapêutica. Baseado nos resultados anteriores que indicam uma especificidade da Bothrojatina ou seu peptídeo cíclico S900050 para inibição de integrina do tipo a4|31 , foi testada a sua capacidade terapêutica para o tratamento de doenças autoimunes. Mas especificamente, baseado na sua capacidade de inibir a migração leucocitária foi utilizado um modelo animal para o estudo de doenças inflamatórias intestinais. [051] The binding specificity of disintegrins is crucial for their therapeutic application. Based on previous results indicating a specificity of Bothrojatine or its peptide cyclic S900050 for inhibition of α4|31-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.
[052] O modelo animal de colite química mimetiza características histológicas e imunológicas de doenças inflamatórias que acometem o sistema gastrointestinal. A utilização do modelo de colite induzidas por instilação retal com ácido 2,4,6-trinitrobenzenosulfonico (TNBS) induz um mecanismo de colite com migração transmural de células inflamatórias, quadro semelhante ao observado em casos como doença de Crohn e retocolite ulcerativa. A utilização deste modelo de colite é vastamente utilizada para o estudo de doenças inflamatórias do sistema gastrointestinal analisando as respostas imunológicas associadas a inflamação da mucosa intestinal, sendo o efeito do tratamento acompanhado de forma prática por sintomas como: (a) perda de peso, (b) contração do cólon e (c) aumento no número de células inflamatórias. [052] 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.
[053] Um quadro de colite foi induzido em camundongos Balb/c por instilação retal com TNBS (TNBS 4% em 50% etanol), seguido ou não da administração intravenosa do peptídeo cíclico S900050 em um período de 24 hs após a indução da colite, a instilação com PBS foi utilizada como controle. Foi realizada uma análise contínua da perda de peso, e 7 dias após o começo do procedimento foi realizada a eutanásia dos animais e medida a extensão do cólon e do número de células inflamatórias na membrana basal intestinal. Os resultados demonstraram que o peptídeo S900050 tem um efeito benéfico para o tratamento de doenças inflamatórias intestinais (Figura 11 ). Administração de S900050 apresentou diferenças significativas quanto a diminuição da perda de peso e na contração do cólon, o que sugere uma menor presença de células inflamatórias intestinais. Esses resultados demonstram um potencial terapêutico de S900050 para o tratamento de doenças autoimunes. [053] 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.
Exemplo 5 Example 5
[054] Exemplos não limitativos de formulações farmacêuticas da invenção são, comprimidos, cápsulas, drágeas, pílulas, soluções, suspensões, emulsões, óvulos. [054] Non-limiting examples of pharmaceutical formulations of the invention are tablets, capsules, dragees, pills, solutions, suspensions, emulsions, ovules.
[055] Formulou-se composições farmacêuticas de Bothrojatina, ou peptídeos miméticos, na concentração entre 0,01 mg/mL e 1 g/mL, preferencialmente 0,1 mg/mL, em formulação de solução injetável com água para injeções e reguladores osmóticos, como por exemplo, carboximetilcelulose, manitol, dentre outros, na proporção entre 0,5% e 50%, preferencialmente entre 5% e 10%. Foram utilizados surfactantes não iônicos, como: álcool alifático etoxilado, polioxietilenos, ésteres carbônicos, ésteres de polietileno glicol, sorbitóis, monoalcanolaminas, dentre outros, na proporção entre 0,01 % e 2%, preferencialmente poloxamer 188 na proporção entre 0,1 % e 1 %. A mistura é homogeneizada por agitação mecânica com velocidade entre 100 e 2000 rpm, preferencialmente entre 600 e 1000 rpm. A força iônica do meio pode ser corrigida para pH fisiológico ou levemente ácido, entre 4 e 8, preferencialmente 5,2 ou 7,4. Quando pronta a solução é filtrada em ponto de bolha 0,1 ou 0,22 pm. [055] 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 pm.
Exemplo 6 Example 6
[056] Foram formuladas composições farmacêuticas de Bothrojatina, ou peptídeos miméticos, na concentração de 0,01 mg/mL a 1 mg/mL, preferencialmente 0,5 mg/mL, em solventes oleosos, polares ou apoiares. Os solventes são tais como: cloreto de metileno, metanol, álcool benzílico, acetato de etila, éter etílico, dimeticona, dentre outros. Obteve-se a mistura da proteína ou peptídeo com polímero biodegradável, biocompatível (podendo ser como exemplo, mas não restrito a, PLGA nas suas opções comerciais 85:15, 75:25, 65:35, 50:50 ou 45:55), na concentração de 10 a 1000 mg/mL em solvente orgânico compatível, mas preferencialmente em torno de 150 mg/mL, como: cloreto de metileno ou acetato de etila. A forma final foi obtida emulsificando-se em solução aquosa na proporção de 1 a 50%, preferencialmente 10%, podendo ser: água, tampão fosfato, acetato, borato, pamoato, cloreto de sódio, carbonatos, álcool polivinílico, dentre outros. [056] 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 apolar 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 in an aqueous solution in a proportion of 1 to 50%, preferably 10%, which may be: water, phosphate buffer, acetate, borate, pamoate, sodium chloride, carbonates, polyvinyl alcohol, among others.
[057]A formulação de depósito pode ter dimensões micro ou nano, com pico de dispersão entre 10 qm a 100 pm, preferencialmente 45 pm para micro e 20 qm para nano, definidas pela presença de surfactantes na concentração de 0,01 % a 2%, preferencialmente, mas não restrito a 0,1 % do brometo de didodecildimetilamônio, sob efeito da agitação que pode ser: mecânica com rotor-estator, pás anguladas, microfluído, pressurizador, ultrassom ou a combinação deles. Um exemplo não limitativo de formulação em nanopartículas pode ser encontrado na Figura 12. [057] The deposit formulation can have micro or nano dimensions, with a dispersion peak between 10 qm to 100 pm, preferably 45 pm for micro and 20 qm 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 that 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.
Exemplo 7 Example 7
[058] Foram também formuladas composições da Bothrojatina, ou peptídeos miméticos, na concentração de 0,01 mg/mL a 1 mg/mL, preferencialmente 0,5 mg/mL na forma de adesivos transdérmicos compostos por adesivos metacrilatos: dextroalfatocoferol, polibutilmetacrilato, metilmetacrilato, copolímero acrílico, óleo de silicone e películas de poliestireno com polivinis. As dimensões do adesivo podem ser customizadas para o uso, contendo entre 1 e 10 mg de NCE/cm2, liberando entre 6 e 100 mcg/h do ativo. [058] 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/cm 2 , releasing between 6 and 100 mcg/h of the active ingredient.
[059] Todos os documentos citados neste relatório descritivo são aqui incorporados por referência. [059] All documents cited in this specification are hereby incorporated by reference.
[060]Tendo sido revelados alguns exemplos de execução preferenciais e alternativos à presente invenção, resta claro que nenhum dos exemplos prestados é limitativo ao escopo de proteção da presente invenção, sendo este definido e limitado tão somente pelo quadro reivindicatório apenso e pelas reivindicações nele contidas. [060] 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 solely by the attached set of claims and the claims contained therein .

Claims

28 REIVINDICAÇÕES 28 CLAIMS
1 . Peptídeo caracterizado por compreender a Fórmula (I): 1 . Peptide characterized by comprising Formula (I):
X1 -X2-Gly-X4-X5-X6-Cys Fórmula (I)X1 -X2-Gly-X4-X5-X6-Cys Formula (I)
Onde Where
X1 é 3mpr, Cys ou 2mac, preferencialmente 3mpr; X1 is 3mpr, Cys or 2mac, preferably 3mpr;
X2 é Ala, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Vai, Trp, Tyr, Harg, Orn ou Dab, preferencialmente Harg; X2 is Ala, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr, Harg, Orn or Dab, preferably Harg;
X4 é Ala, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Vai, Trp, Tyr, Harg, Gla ou Hglu, preferencialmente Glu; X4 is Ala, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr, Harg, Gla or Hglu, preferably Glu;
X5 é Ala, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Vai, Trp, Tyr, Harg, DOPA ou Cha, preferencialmente Tyr; e X5 is Ala, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr, Harg, DOPA or Cha, preferably Tyr; It is
X6 é Pro ou ABZ2, preferencialmente Pro; em que X6 is Pro or ABZ2, preferably Pro; on what
Harg é homoarginina Harg is homoarginine
Hglu é ácido homoglutâmico Hglu is homoglutamic acid
Orn é ornitina orn is ornithine
Dab é ácido alfa gama diaminobutírico Dab is alpha gamma diaminobutyric acid
Gla é ácido gama carboxiglutâmico Gla is gamma carboxyglutamic acid
3mpr é 3-ácido mercaptopropiônico 2mac é ácido 2-mercaptoacético ABZ2 é Ácido 2 aminobenzóico 3mpr is 3-mercaptopropionic acid 2mac is 2-mercaptoacetic acid ABZ2 is 2-aminobenzoic acid
Cha é ciclohexil alanina, Cha is cyclohexyl alanine,
Em que consiste, preferencialmente, nas sequências SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 e SEQ ID NO:1 1 (que correspondem, respectivamente, a CRIPKGDDMDDC, 3mpr-RIPKGDDMDC, 3mpr-KGDDMDC, 3mpr-KGDMDC e 3mpr-Harg-GEYPC), seus ciclos, enantiômeros e fragmentos dos mesmos.Preferably consisting of the sequences SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11 (corresponding respectively to CRIPKGDDMDDC, 3mpr-RIPKGDDMDC , 3mpr-KGDDMDC, 3mpr-KGDMDC and 3mpr-Harg-GEYPC), their cycles, enantiomers and fragments thereof.
2. Vetor caracterizado por compreender uma construção de DNA consistindo na sequência definida pela SEQ ID NO: 1 (Bothrojatina), operacionalmente ligada às sequências promotora e terminadora heterólogas e capaz de expressar a proteína da SEQ ID NO:1 . 2. Vector characterized by comprising a construction of DNA consisting of the sequence defined by SEQ ID NO:1 (Bothrojatine), operably linked to heterologous promoter and terminator sequences and capable of expressing the protein of SEQ ID NO:1.
3. Composição farmacêutica caracterizada por compreender um ou mais peptídeos, conforme definido na reivindicação 1 , e um veículo farmaceuticamente aceitável. 3. Pharmaceutical composition, characterized in that it comprises one or more peptides, as defined in claim 1, and a pharmaceutically acceptable vehicle.
4. Composição, de acordo com a reivindicação 3, caracterizada por compreender, adicionalmente, a proteína Bothrojantin, definida na SEQ ID NO: 1. 4. Composition according to claim 3, characterized in that it additionally comprises the protein Bothrojantin, defined in SEQ ID NO: 1.
5. Composição farmacêutica, de acordo com a reivindicação 3 ou 4, caracterizada por ter a forma de um adesivo transdérmico, solução, suspensão, emulsão, comprimidos ou cápsulas. 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.
6. Composição farmacêutica, de acordo com a reivindicação 5, caracterizada pelo fato de ser uma solução injetável, na forma de micropartículas ou nanopartículas. 6. Pharmaceutical composition, according to claim 5, characterized in that it is an injectable solution, in the form of microparticles or nanoparticles.
7. Uso dos peptídeos conforme definidos na reivindicação 1 e das composições conforme definidas nas reivindicações 3, 4, 5 ou 6 caracterizado por ser para a preparação de um medicamento para uso em doenças inflamatórias, doenças autoimunes, doenças hematológicas e doenças neurológicas. 7. 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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Citations (6)

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US5747447A (en) * 1992-04-30 1998-05-05 Cor Therapeutics Stable polypeptide composition
WO2008088548A2 (en) * 2006-12-26 2008-07-24 National Taiwan University Disintegrin variants and pharmaceutical uses thereof
WO2010093976A1 (en) * 2009-02-12 2010-08-19 University Of Southern California Bioadhesive patch for sutureless closure of soft tissue
WO2020148528A1 (en) * 2019-01-15 2020-07-23 Bicyclerd Limited BICYCLIC PEPTIDE LIGANDS SPECIFIC FOR INTEGRIN αVβ3

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US5686570A (en) * 1989-06-16 1997-11-11 Cor Therapeutics, Inc. Platelet aggregation inhibitors
US5721210A (en) * 1990-07-09 1998-02-24 Tanabe Seiyaku Co., Ltd. Cyclic cell adhesion modulation compounds
US5747447A (en) * 1992-04-30 1998-05-05 Cor Therapeutics Stable polypeptide composition
WO2008088548A2 (en) * 2006-12-26 2008-07-24 National Taiwan University Disintegrin variants and pharmaceutical uses thereof
WO2010093976A1 (en) * 2009-02-12 2010-08-19 University Of Southern California Bioadhesive patch for sutureless closure of soft tissue
WO2020148528A1 (en) * 2019-01-15 2020-07-23 Bicyclerd Limited BICYCLIC PEPTIDE LIGANDS SPECIFIC FOR INTEGRIN αVβ3

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