WO2016051000A1 - Peptide isolated in a lupin hydrolysate and use thereof in the treatment of inflammatory diseases - Google Patents

Peptide isolated in a lupin hydrolysate and use thereof in the treatment of inflammatory diseases Download PDF

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Publication number
WO2016051000A1
WO2016051000A1 PCT/ES2015/070705 ES2015070705W WO2016051000A1 WO 2016051000 A1 WO2016051000 A1 WO 2016051000A1 ES 2015070705 W ES2015070705 W ES 2015070705W WO 2016051000 A1 WO2016051000 A1 WO 2016051000A1
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Prior art keywords
peptide
expression
nucleotide sequence
protein
inflammatory
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PCT/ES2015/070705
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Spanish (es)
French (fr)
Inventor
Francisco MILLÁN RODRÍGUEZ
Justo Javier PEDROCHE JIMÉNEZ
María del Mar YUST ESCOBAR
María del Carmen MILLÁN LINARES
Álvaro VILLANUEVA LAZO
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Consejo Superior De Investigaciones Científicas (Csic)
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Publication of WO2016051000A1 publication Critical patent/WO2016051000A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

  • the present invention relates to a peptide isolated from a protein hydrolyzate, wherein said peptide exhibits anti-inflammatory and / or immunomodulatory activity, which makes its use possible in the treatment of inflammatory diseases. Therefore, the present invention is directed to the field of the treatment of inflammatory diseases.
  • Inflammation is characterized by increased blood flow to the tissue causing temperature increase, redness, swelling and pain.
  • Short-term or acute inflammation is an attempt of protection by the organism to eliminate harmful stimuli and to start the healing process.
  • Long term or chronic inflammation leads to various inflammatory disorders and chronic diseases.
  • the acute phase is characterized by its short duration, the exudation of liquid and plasma proteins and the migration of leukocytes, predominantly neutrophils.
  • the chronic phase is characterized by a longer duration, the presence of lymphocytes and macrophages, blood vessel proliferation, fibrosis and necrosis.
  • Inflammation is involved in the onset and development of numerous diseases, including cancer, neurological diseases, cardiovascular diseases, hypertension, blood pressure, atherosclerosis, diabetes, obesity, respiratory disorders, multiskeletal disorders, gastrointestinal disorders and autoimmune disorders. .
  • the immune system is responsible for responding to any type of aggression in the body. It has its origin in the lymphoid organs (lymph nodes, spleen, thymus, and bone marrow) and a large number of cell subtypes (immunocompetent cells) are involved, including monocytes and neutrophils.
  • Macrophages are one of the most studied cells of the immune system. They are usually in all tissues. In them, macrophages acquire their unique and different morphological and functional properties (for example, Kupffer cells in the liver and alveolar macrophages in the lungs) (Locati, M., Mantovani, A., & Sica, A. ( 2013). Advances in Immunology, 120, 163-184).
  • a macrophage has a half-life of 2-4 months.
  • stimuli that induce macrophage activation for example opsonins, cytokines and growth factors
  • mononuclear phagocytes polarize and express different specialized functional properties.
  • Classically activated macrophages (M1) induced by interferon (IFN) and / or tumor necrosis factor (TNF) show a Th1 type phenotype, are proinflammatory effectors and have bactericidal functions, while alternately activated macrophages (M2) exhibit a phenotype Th2 type and are involved in the resolution of inflammation and tissue healing.
  • Cytokines are the regulatory molecules of host responses to infection, immune response, inflammation and trauma. There are two types of cytokines: proinflammatory and anti-inflammatory. Therefore, proinflammatory cytokine inhibitors have been considered as potential candidates for anti-inflammatory drugs.
  • Feeding is one of the main factors that affects the modulation of the inflammatory response that occurs in the body. Through various effects on cells and their metabolic pathways, food causes changes in the normal functioning of the different systems involved in the inflammatory process, as food can behave as cellular metabolites and can act as procellular or anti-inflammatory intracellular messengers.
  • curcumin a Polyphenol present in turmeric binds to a variety of biomachromolecules that indicate that curcumin is highly pleiotropic with anti-inflammatory, hypoglycemic, antioxidant, wound healing, and anti-microbial activity (Gupta, SC, et al. (2011). Natural Product Reports, 28, 1937-1955).
  • curcumin a Polyphenol present in turmeric binds to a variety of biomachromolecules that indicate that curcumin is highly pleiotropic with anti-inflammatory, hypoglycemic, antioxidant, wound healing, and anti-microbial activity.
  • milk proteins are considered the most important source of bioactive peptides (Wada, Y., & Lonnerdal, B. (2014). The Journal of Nutritional Biochemistry, 25, 503-514) and protein hydrolysates have been obtained with anti-inflammatory properties from milk casein (Mao, XY., Cheng, X., Wang, X., & Wu, SJ. (2011). Food Chemistry, 126, 484-490).
  • milk casein Moo, XY., Cheng, X., Wang, X., & Wu, SJ. (2011). Food Chemistry, 126, 484-490.
  • the present invention is directed to a peptide with anti-inflammatory and / or immunomodulatory activity, which makes its use possible in the treatment of inflammatory diseases.
  • the inventors of the present invention started from a lupine protein hydrolyzate and, after several purification and isolation processes, obtained a peptide that had the ability to inhibit the expression of proinflammatory cytokines, such as TNF and I L-1 ⁇ or promote the expression of anti-inflammatory cytokines such as IL-10, which indicated its anti-inflammatory and immunomodulatory activity, and its potential use in the treatment of diseases that occur with inflammation. Therefore, based on this discovery, a series of inventive aspects have been developed that will be described in detail below.
  • the peptide of the invention with anti-inflammatory and / or immunomodulatory activity comprises the sequence Gly-Pro-Glu-Thr-Ala-Phe-Leu-Arg (SEQ ID NO: 1) .
  • the present invention relates to a peptide comprising the amino acid sequence SEQ ID NO: 1, or a variant thereof with a sequence identity of 75, 80, 85, 90, 95, 96 , 97, 98 or 99% with SEQ ID NO: 1, hereinafter "peptide of the invention", wherein said peptide does not comprise the amino acid sequence consisting of the sequence SEQ ID NO: 2.
  • peptide is understood as the linear chain of amino acids linked together by chemical bonds of the amide type.
  • the number of amino acids that can be part of the peptide of the invention ranges, for example, from 8 to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140 or 150 amino acids .
  • identity or “sequence identity” is understood as the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences. Depending on the number of common residues between the aligned sequences, a degree of identity expressed as a percentage will be obtained.
  • the degree of identity between two sequences of Amino acids can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as BLAST [AltschuI SF et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215 (3): 403-10].
  • the BLAST programs for example, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are in the public domain on the website of The National Center for Biotechonology Information (NCBI).
  • a peptide has anti-inflammatory activity when it is capable of inducing the expression of IL-10, a potent anti-inflammatory cytokine, as well as suppressing the expression of TNF and / or I L-1 ⁇ , proinflammatory cytokines , in macrophages derived from THP-1 cells.
  • a peptide exhibits immunomodulatory activity, when it has the capacity to modulate the response of human immune system cells such as macrophages, these being, in turn, capable of modulating the function of other immune cells such as T lymphocytes.
  • Examples of tests on how to evaluate whether a peptide has anti-inflammatory and / or immunomodulatory activity are, for example, the tests described in the illustrative Examples of the present invention that accompany the description.
  • One of the characteristics of the peptide of the invention is that it does not comprise the amino acid sequence Gly-Pro-Glu-Thr-Ala-Phe-Leu-Arg-Leu (SEQ ID NO: 2).
  • This peptide is located in the National Center for Biotechnology Information or NCBI databases with accession number B3A0G0, version B3A0G0 Gl: 460425300.
  • the peptide of the invention can be attached to one or both of its ends other types of molecules, such as a marker, a signal peptide, etc., and said molecules can be directly linked to the carboxyl or amino terminal ends of the peptide or linked through a spacer or linker.
  • the peptide of the invention comprises a marker attached to one of its ends.
  • marker means that molecule that allows the identification of the peptide of the invention.
  • the label can be a "marker peptide” for the identification and / or location of the peptide of the invention since said markers correspond to binding sites to certain molecules or atoms, such as the histidine chain, the GST, the avidin, the streptavidin, etc., or because they are easily detectable by immunochemical techniques, such as hemagglutinin, VSV-G, HSVtk, FLAG, V5, myc, etc., or because they are easily observable, such as fluorescent proteins.
  • the VSV-G marker peptide belongs to the vesicular stomatitis vesicle glycoprotein.
  • the HSVtk marker peptide belongs to the thymidine kinase of the herpes simplex virus 1.
  • the FLAG peptide is an 8 amino acid epitope designed specifically as a recombinant protein marker.
  • V5 is a small epitope present in the P and V proteins of simian virus 5 paramyxovirus (SV5).
  • the myc epitope has 10 amino acids and is part of the human c-myc transcription factor sequence.
  • the marker is selected from the group consisting of c-myc, FLAG, HA, histidine chain, GST, biotin, VSV-G, HSVtk, V5, biotin, avidin, streptavidin, protein binding to maltose and a fluorescent protein.
  • the peptide of the invention comprises attached to one of its ends a signal peptide.
  • signal peptide is understood as the sequence of between 3 and 60 amino acids that directs the transport of the peptide to a specific subcellular location, such as the endoplasmic reticulum, the mitochondria or the nucleus. He Signal peptide can also direct the transport of the protein out of the cell, which could be equivalent to its secretion or its transport to the cell periplasm, in the case of cells such as Escherichia coli. Examples of signal peptide to direct the transport of a peptide include, but are not limited to, pelB, stll, ecotin, lamB, herpes GD, 1 pp, alkaline phosphatase, invertase, alpha factor and the leader sequence of protein A.
  • the signal peptide is selected from the group consisting of pelB, stll, ecotin, lamB, herpes GD, 1 pp, alkaline phosphatase, invertase, alpha factor and the protein A leader sequence.
  • the peptide of the invention carries both the label and the signal peptide simultaneously, either at different ends of the peptide of the invention or at the same end.
  • junctions [signal peptide] - [marker] - [peptide of the invention] and [marker] - [signal peptide] - [peptide of the invention] are also contemplated, where the [marker] binding - [signal peptide] can go to the amino or carboxyl terminal of the peptide of the invention. Likewise, if it is considered convenient, more than one marker can also be used.
  • both the label and the signal peptide can be linked to the peptide of the invention directly or through a spacer or linker.
  • spacer or “linker” or “linker” is understood as the molecule that serves as a link between two domains or two molecules.
  • the domains or molecules would be the peptide of the invention, the marker and the signal peptide.
  • nucleotide sequence of the invention which encodes the peptide of the invention.
  • nucleotide sequence is understood as the nucleotide chain linked to each other through phosphate groups.
  • nucleic acid can be formed by deoxyribonucleotides, ribonucleotides, modified nucleotides or any substrate that can be incorporated into a DNA or RNA polymer by a polymerase or by a synthetic reaction.
  • encodes refers to the genetic code that determines how a nucleotide sequence is translated into a polypeptide or a protein.
  • the order of nucleotides in a sequence determines the order of amino acids along a peptide, a polypeptide or a protein.
  • the nucleotide sequence of the invention can be inserted into a vector, for example, an expression vector. Therefore, in another aspect, the invention relates to a vector, hereinafter "vector of the invention", comprising the nucleotide sequence of the invention.
  • vector refers to a nucleic acid molecule that can be used to transport or transfer a second nucleic acid molecule into a cell.
  • said second nucleic acid molecule is the nucleotide sequence of the invention.
  • a vector may contain different functional elements that include, but are not limited to, transcription control elements, such as promoters or operators, regions or enhancers of transcription factor binding, and control elements to initiate and terminate transcription.
  • Vectors include, but are not limited to: plasmids, cosmids, viruses, phages, recombinant expression cassettes and transposons. Some vectors are capable of replicating or dividing autonomously after being introduced into the host cell, such as bacterial vectors with an origin of bacterial replication or mammalian episomal vectors. Others Vectors can be integrated into the genome of the host cell and thus replicated together with the cell genome.
  • an expression vector comprises, in addition to the nucleotide sequence encoding the peptide of the invention, a promoter that directs its transcription (eg, pT7, plac, ptrc, ptac, pBAD, ret, etc.), to which is operatively linked, and other necessary or appropriate sequences that control and regulate said transcription and, where appropriate, the translation of the product of interest, for example, transcription initiation and termination signals (tlt2, etc.), polyadenylation signal , origin of replication, ribosome binding sequences (RBS), coding sequences of transcriptional regulators, (enhancers), transcriptional silencers (silencers), repressors, etc.
  • a promoter that directs its transcription eg, pT7, plac, ptrc, ptac, pBAD, ret, etc.
  • a promoter that directs its transcription eg, pT7, plac, ptrc,
  • expression vectors can be selected according to the conditions and needs of each specific case among expression plasmids, viral vectors (DNA or RNA), cosmids, artificial chromosomes, etc. which may also contain markers that can be used to select cells transfected or transformed with the gene or genes of interest.
  • the choice of the vector will depend on the host cell and the type of use to be performed.
  • said vector can be a viral vector, such as adenovirus, adenovirus associated viruses as well as retroviruses and, in particular, lentiviruses, etc., or non-viral such as pcDNA3, pHCMV / Zeo, pCR3.1, pEFI / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAXI, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d, pTDTI, etc.
  • viral vector such as adenovirus, adenovirus associated viruses as well as retroviruses and, in particular, lentiviruses, etc.
  • non-viral such as pcDNA3, pHCMV / Zeo, pCR3.1, pEFI / His, pIND /
  • said vectors can be used to transform, transfect or infect cells, both prokaryotic and eukaryotic.
  • the obtaining of said vector can be carried out by conventional methods known to those skilled in the art, as well as for the transformation of microorganisms and eukaryotic cells different widely known methods can be used - chemical transformation, transfection, electroporation, microinjection, etc. - described in various manuals widely known to those skilled in the art.
  • the invention in another aspect, relates to a cell, hereinafter "cell of the invention” comprising the nucleotide sequence or the vector of the invention described above.
  • Suitable host cells in the context of the present invention include, but are not limited to, prokaryotic cells, such as bacterial cells, for example E. coli, and eukaryotic cells, such as yeast cells, for example, S. cerevisiae, animal cells , preferably from mammalian, fungal cells, for example, Aspergillus sp. or insect cells, for example, Spodoptera frugiperda.
  • the invention relates to a peptide extract, hereinafter "peptide extract of the invention", which comprises the peptide of the invention.
  • peptide extract is understood as the solution obtained by centrifugation from a chemical, physical and / or enzymatic modification made in a starting material of a plant, animal, fungal and / or microbial nature. , and in which a set of peptides can be found as the main component among which it comprises the peptide of the invention.
  • composition of the invention which comprises the peptide, the nucleotide sequence, the vector, the cell and / or the peptide extract of the invention.
  • composition comprises combinations of the peptide, the nucleotide sequence, the vector, the cell or the peptide extract of the invention.
  • the peptide, the nucleotide sequence, the vector, the cell and / or the peptide extract of the invention must be present in a therapeutically effective amount in the composition so that they can exert the anti-inflammatory effect. and / or immunomodulator.
  • therapeutically effective amount is understood as an amount of the peptide of the invention that, administered in doses and for the necessary period of time to a subject, is effective in achieving the desired prophylactic or therapeutic result.
  • a “therapeutically effective amount” of the peptide or pharmaceutical composition of the invention may vary with the disease stage, age, sex and weight of the individual, and refers to an amount that It has no adverse effects or toxicity and is capable of achieving the desired prophylactic or therapeutic effect.
  • the pharmaceutical composition of the invention can be administered by any appropriate route (for example, oral, sublingual, perioral, intranasal, parenteral, transdermal, topical, etc.), for which the pharmaceutically acceptable excipients and vehicles necessary for the formulation of the pharmaceutical form of administration chosen.
  • any appropriate route for example, oral, sublingual, perioral, intranasal, parenteral, transdermal, topical, etc.
  • the pharmaceutically acceptable excipients and vehicles necessary for the formulation of the pharmaceutical form of administration chosen for which the pharmaceutically acceptable excipients and vehicles necessary for the formulation of the pharmaceutical form of administration chosen.
  • the different pharmaceutical forms of drug administration and their preparation are general knowledge for the person skilled in the art.
  • the form adapted to oral administration refers to a physical state that can allow oral administration.
  • Said form adapted to oral administration is selected from the list comprising, but not limited to, drops, syrup, herbal tea, elixir, suspension, extemporaneous suspension, drinkable vial, tablet, capsule, granulate, seal, pill, tablet, tablet, tablet and lyophilized.
  • parenteral administration refers to a physical state that can allow its injectable administration, that is, preferably in a liquid state.
  • Parenteral administration can be carried out via intramuscular, intraarterial, intravenous, intradermal, subcutaneous or intraosseous administration but not limited to these types of parenteral administration routes.
  • composition is present in a form adapted to sublingual, nasal, intrathecal, bronchial, lymphatic, rectal, transdermal or inhaled administration.
  • composition of the invention may comprise other components, such as diluents, excipients, salts, etc.
  • the composition of the invention further comprises an excipient.
  • excipient is understood as an inactive substance used to incorporate the active ingredient (peptide of the invention). It can also be used to help the process by which the composition is manufactured.
  • excipients include, but are not limited to, binders (keep the ingredients of a tablet together, such as hydroxypropyl, cellulose, xylitol, sorbitol, mannitol, etc.), diluents (fill in the contents of a tablet or capsule, such as vegetable cellulose, dibasic calcium phosphate, safflower flower, etc.), disintegrators (compounds that expand and dissolve when they get wet causing the tablet to break when it reaches the digestive tract and release the active substances for absorption), lubricants (prevent the ingredients from being grouped into lumps, such as talcum, silica, etc.), coatings (protect the tablet's ingredients from external agents, such as cellulose , synthetic polymers, polysaccharides, etc.), sweeteners, flavorings
  • the composition may be in the form of a solid, for example powders, granules, tablets, gelatin capsules, liposomes or suppositories.
  • Suitable solid supports may be, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine and wax.
  • Other pharmaceutically acceptable excipients and / or carriers suitable for administration to animals will be known to those skilled in the art.
  • compositions according to the present invention can also be presented in liquid form, for example, solutions, emulsions, suspensions or syrups.
  • suitable liquid supports may be, for example, water, organic solvents, such as glycerol or glycols, as well as mixtures thereof, in varying proportions, in water.
  • composition may also comprise salts, principally, pharmaceutically acceptable salts, including, but not limited to, the addition salts of inorganic acids, such as hydrochloride, hydrobromide, iodhydrate, sulfate, phosphate, diphosphate and nitrate, or organic acids , such as acetate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, palmoate and stearate.
  • salts formed from bases such as sodium or potassium hydroxide.
  • the composition of the invention is a pharmaceutical composition or a nutritional composition.
  • pharmaceutical composition means the composition that can be administered to a subject, for example, a human, orally, parenterally, topically, by inhalation spray, intranasally or by rectal route, in unit dosage formulations containing non-toxic carriers, diluents, adjuvants, vehicles and the like.
  • pharmaceutical composition includes compositions for use in human or animal health (veterinary compositions).
  • nutritional composition means that composition ingested by living beings by which the body receives, transforms and uses chemical substances contained in food.
  • the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier.
  • the “carrier” or carrier is preferably an inert substance in order to facilitate the incorporation of other compounds, allow better dosing and administration or give consistency and form to the pharmaceutical composition. Therefore, the carrier is a substance that is used in the medicament to dilute any of the components of the pharmaceutical composition of the present invention to a certain volume or weight; or that even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and form to the medicine.
  • the pharmaceutically acceptable carrier is the diluent.
  • pharmaceutically acceptable refers to the excipient or vehicle being allowed and evaluated so as not to cause harm to the subject to which it is administered. Furthermore, the excipient and the vehicle must be pharmaceutically suitable, that is, they must allow the activity of the compounds of the pharmaceutical composition and be compatible with said components.
  • the peptide, the nucleotide sequence, the vector and / or the cell of the invention as well as the pharmaceutical compositions containing them can be used together with other additional drugs to provide a combination therapy.
  • additional drugs may be part of the same pharmaceutical composition or, alternatively, they may be provided in the form of a separate composition for simultaneous or non-simultaneous administration to the pharmaceutical composition comprising the peptide, the nucleotide sequence, the vector and / or the cell of the invention.
  • composition of the invention further comprises an anti-inflammatory compound.
  • anti-inflammatory compounds include, but are not limited to, steroidal compounds, non-steroidal compounds (also called NSAIDs) and the group of disease-modifying anti-rheumatoid drugs or DMARDs.
  • Anti-steroidal anti-inflammatory drugs include, without limitation, corticosteroids, such as dexamethasone, prednisone, etc.
  • non-steroidal anti-inflammatories include, but are not limited to, COX-2 inhibitors, acetylsalicylic acid, derivatives of propyl acid (such as ibuprofen and naproxen), derivatives of acetic acid (such as indomethacin), enolic acids (such as piroxicam ) and paracetamol.
  • DMARDs include, but are not limited to, metrotexate, penicillamine, chloroquine, sulfasalazine and gold salts.
  • the peptide of the invention has anti-inflammatory and / or immunomodulatory activity, which allows its use in the treatment of diseases or disorders that occur with inflammation.
  • the invention relates to the use of the peptide, the nucleotide sequence, the vector, the cell and / or the peptide extract of the invention in the manufacture of a medicament.
  • the possibility of using combinations of the peptide, the nucleotide sequence, the vector, the cell or the peptide extract of the invention in the preparation of a medicament is contemplated.
  • the term "medicament” and “pharmaceutical composition” are considered to be equivalent and, therefore, the invention also relates to the peptide, nucleotide sequence, vector and / or cell of the invention. in the elaboration of a pharmaceutical composition as described in previous paragraphs.
  • the invention relates to the use of the peptide, the nucleotide sequence, the vector, the cell and / or the peptide extract of the invention in the preparation of a medicament with anti-inflammatory and / or immunomodulatory activity.
  • a medicament exhibits anti-inflammatory activity when it has the capacity to induce the expression of IL-10, a potent anti-inflammatory cytokine, as well as to suppress the expression of TNF and / or I L-1 ⁇ , cytokines of nature proinflammatory, in macrophages derived from THP-1 cells.
  • a medicament has immunomodulatory activity, when it has the capacity to modulate the response of human immune system cells such as macrophages, these being, in turn, capable of modulating the function of other immune cells such as T lymphocytes.
  • the invention relates to the use of the peptide, the nucleotide sequence, the vector, the cell and / or the peptide extract of the invention, in the preparation of a medicament for the treatment of an inflammatory disease.
  • the possibility of the use of combinations of the peptide, the nucleotide sequence, the vector, the cell or the peptide extract of the invention in the preparation of a medicament for the treatment of an inflammatory disease is not limited to the peptide, the nucleotide sequence, the vector, the cell and / or the peptide extract of the invention.
  • treatment means the set of means used in a subject to alleviate or cure a disease, or to eliminate or decrease the symptoms of a disease.
  • subject means a member of an animal species, preferably a mammal, and includes, but is not limited to, a domestic animal, a primate and a human; In the context of the present invention, the individual is preferably a human, male or female, of any race or age.
  • inflammatory disease is understood to be that disease that causes inflammation and causes high levels of TNFa in the individual suffering from it.
  • the inflammatory disease is selected from the group consisting of an autoimmune disease, cancer, atherosclerosis, osteoarthritis, arthritis, diabetes, obesity, metabolic syndrome, asthma, Crohn's disease, hypertension and dermatitis.
  • autoimmune disease is understood as that disease in which the immune system of an organism attacks its own cells. Examples of autoimmune diseases include, but are not limited to, acquired immunodeficiency syndrome (AIDS), autoimmune lymphoproliferative syndrome, hemolytic anemia, inflammatory diseases, and thrombocytopenia, acute or chronic immune diseases associated with organ transplantation and allergies.
  • AIDS acquired immunodeficiency syndrome
  • autoimmune lymphoproliferative syndrome hemolytic anemia
  • inflammatory diseases and thrombocytopenia
  • acute or chronic immune diseases associated with organ transplantation and allergies acute or chronic immune diseases associated with organ transplantation and allergies.
  • cancer is understood as that disease in which abnormal cells divide without control and can invade other tissues, causing a metastasis.
  • cancer and “tumor” are considered equivalent.
  • cancer include, but are not limited to, lymphomas, leukemia, breast cancer, colon cancer, lung cancer, colorectal cancer, esophageal cancer, skin cancer, prostate cancer and stomach cancer.
  • the invention relates to a method, hereafter "Method of the invention", to obtain the peptide of the invention, comprising: a) Hydrolyzing a protein extract from a legume with an endoprotease to obtain a protein hydrolyzate, b) subject the protein hydrolyzate obtained in step a) to tangential ultracentrifugation by using a membrane with a pore size of at least 1 kDa, c) perform a reverse phase chromatography of the permeate obtained in the ultracentrifugation of stage b ) and analyze the ability of the collected fractions to inhibit the expression of proinflammatory cytokines by cellular assays, and d) subject to purification by gel chromatography to the fraction obtained in step c) capable of decreasing or inhibiting the expression of proinflammatory cytokines Regarding a control.
  • Method of the invention to obtain the peptide of the invention, comprising: a) Hydrolyzing a protein extract from a legume with an endoprotease to obtain
  • the method of the invention comprises hydrolyzing a protein extract from a legume with an endoprotease to obtain a protein hydrolyzate.
  • endoproteases Any of the endoproteases that exist in the state of the art can be used to practice step a) of the method of the invention.
  • the endoproteases are broad spectrum and hydrolyse amide bonds within the protein chain.
  • Examples of endoproteases include, but are not limited to, serine proteases, Neutrase®, Flavourzyme® and Protamex®.
  • the endoprotease is Alcalase® or subtilisin.
  • Protein hydrolysis is normally carried out in a reactor, with stirring control, pH, temperature and process time.
  • the substrate is dissolved or resuspended in water until the pH and temperature stabilize;
  • the endoprotease is then added, starting the hydrolysis.
  • a decrease in pH occurs due to the breakage of the peptide bonds.
  • the pH should be maintained at the optimum of the enzyme by adding diluted base.
  • the enzyme can be inactivated with heat, by a decrease in pH or with a combination of both of them. Or it can also be removed from the medium by filtration and finally precipitated protein.
  • the substrate of protein hydrolysis can be any legume plant.
  • Leguminous plants are woody or herbaceous plants with vegetable type fruit, capable of fixing atmospheric nitrogen by their symbiosis are the bacterial genus Rhizobium.
  • Examples of legumes include, but are not limited to, beans (Phaseolus vulgaris), soybeans (G ⁇ ycine max), beans (Vicia faba), enteja (Lens cuiinaris), chickpeas (Cicer ar ⁇ etinum), peas (Pisum sativum), carob (Ceratonia siiiqua ), lupine (Lupinus aibus), peanut (Arachis hypogaea), alfalfa (Medicago sativa), clover (Trifolium pratense) and veza (Vicia sativa).
  • the legume is a lupine plant.
  • the protein hydrolyzate also called peptide extract
  • it is subjected to tangential ultracentrifugation by using a membrane with a pore size of at least 1 kDa [step b) of the method of the invention].
  • ultracentrifugation employed in a tangential ultracentrifugation.
  • Ultracentrifugation is a widely known process in the state of the art used to separate the components of a suspension based on the size and density of its components by centrifugal force.
  • ultracentrifugation is used to filter the peptide hydrolyzate and separate the peptides with a molecular weight greater than 1 kDa from the peptides with a weight molecular less than or equal to 1 kDa.
  • a permeate and a retained are obtained.
  • membranes with other pore sizes can be employed, for example, at least 3, at least 5, at least 10, at least 30, at least 50 or at least 100 kDa.
  • the membrane employed in the method of the invention is at least 10 kDa.
  • Reverse Phase Chromatography is a technique that allows molecules to be separated based on their polarity, in which the stationary phase is chemically modified silica particles with saturated, unsaturated or aromatic hydrocarbons of different types. This makes the stationary phase an apolar matrix. Therefore, mixtures of polar solvents, such as water, acetonitrile, ethyl acetate, acetone, aliphatic alcohols, etc., are used for this type of chromatography. In the separation of amino acids and peptides by reverse phase the most commonly used resin has a linear chain of 18 carbons and is called Ci 8 [
  • the sample in this invention the permeate
  • the sample is applied to the column in acidified aqueous solution, for example, with trifluoroacetic acid, a volatile organic acid that is very pure.
  • acidified aqueous solution for example, with trifluoroacetic acid, a volatile organic acid that is very pure.
  • trifluoroacetic acid a volatile organic acid that is very pure.
  • inflammatory cytokines include, but are not limited to, TNF, I L-1 ⁇ , IL6, CCR2, and CCL2.
  • Trials on how to analyze the expression of proinflammatory cytokines include, but are not limited to, RNA isolation and quantification by quantitative PCR of the cytokines produced by the cells, determination of the production of cytokines produced by the cells by ELISA, etc. as well as the tests described in the examples illustrating the invention. Tests and techniques on how to analyze the expression of pro-inflammatory cytokines are widely known in the state of the art and their use is routine practice for the person skilled in the art.
  • step d) of the method of the invention the fraction obtained in step c) capable of decreasing or inhibiting the expression of pro-inflammatory cytokines with respect to a control is subjected to purification by gel chromatography.
  • Gel chromatography is a technique used to separate molecules based on their size. The separating capacity lies primarily in the gel, whose matrix consists of a large number of microscopic porous spheres. These spheres consist of long chains of polymers joined together by chemical bonds to form a three-dimensional network.
  • the gels normally used for the separation of proteins and peptides are of three types: dextran, agarose and polyacrylamide.
  • the sample in this invention the fraction selected after reverse phase chromatography
  • Elution is performed in a socratic manner with an aqueous solution that may include, but is not limited to, formic acid, hydrochloric acid, ammonium acetate, phosphate buffered saline, ammonium bicarbonate, trichloroacetic acid.
  • an aqueous solution that may include, but is not limited to, formic acid, hydrochloric acid, ammonium acetate, phosphate buffered saline, ammonium bicarbonate, trichloroacetic acid.
  • control is understood as the level of proinflammatory cytokine expression obtained in a cellular assay to which the fractions collected after the reverse phase chromatography of step c) have not been added.
  • the fraction that is capable of inhibiting or decreasing the expression of proinflammatory cytokines with respect to a control will correspond to the peptide of the invention.
  • Techniques on how to isolate peptides from a fraction are widely known in the state of the art and any of them can be used.
  • FIG. 1 mRNA expression of TNF (A), IL-1 ⁇ (B), IL-6 (C), CCR2 (D), CCL2 (E), and CCL18 (F) in THP-1 derived macrophages after 6 hours of treatment with PM 1 (> 10 kDa) and PM2 ( ⁇ 10 kDa). Values marked with different letters are significantly different (P ⁇ 0.05).
  • FIG. 1 mRNA expression of IL-6 (A), CCR2 (B), CCL2 (C), and CCL18 (D) in THP-1 derived macrophages after 6 h of treatment with F3 and F7 obtained by RPC. Values marked with different letters are significantly different (P ⁇ 0.05).
  • Figure 4 Elution profile of F3 obtained in a Superdex peptide 10/300 gel filtration column.
  • Figure 5. mRNA expression of IL-6 (A), CCR2 (B), and CCL2 (C), in THP-1 derived macrophages after 6 h of treatment with F3-5 obtained in a Superdex gel filtration column 10/300 peptide. Values marked with different letters are significantly different (P ⁇ 0.05).
  • Figure 6. Expression of the mRNA of CCL18 (A) and IL-10 (B) in macrophages derived from THP-1 after 6 h of treatment with fraction F3-5. Values marked with different letters are significantly different (P ⁇ 0.05).
  • FIG. 7 Expression of the mRNA of TNF (A), I L-1 ⁇ (B), and IL-6 (C) in THP-1 derived macrophages after 6 h of treatment with the peptide synthesized at 100 ⁇ g / mL (P1) and at 500 ⁇ g / mL (P2). Values marked with different letters are significantly different (P ⁇ 0.05).
  • FIG. 8 Expression of the mRNA of CCL2 (A) and CCR2 (B) in THP-1 derived macrophages after 6 h of treatment with the peptide at the concentration of 100 ⁇ g / mL (P1) and 500 ⁇ / ⁇ . ( ⁇ 2). Values marked with different letters are significantly different (P ⁇ 0.05).
  • FIG. 9 Expression of the mRNA of CCL18 (A) and IL-10 (B) in THP-1 derived macrophages after 6 h of treatment with the peptide at the concentration of 100 ⁇ g / mL (P1) and 500 ⁇ g / mL (P2). Values marked with different letters are significantly different (P ⁇ 0.05).
  • FIG 10. Effect on the production of TNF (A), IL-1 ⁇ (B), and IL-10 (C) of macrophages derived from THP-1 cells after 6 hours (1) and 48 hours (2) of treatment with two concentrations of the synthesized peptide, P1 (100 ⁇ g / mL) and P2 (500 ⁇ g / mL). Values marked with different letters are significantly different (P ⁇ 0.05).
  • Figure 11. Percentage production of nitric oxide (NO) in the supernatant of macrophages derived from THP-1 after 48 hours of treatment with the peptide at 100 ⁇ g / mL (P1) and 500 ⁇ g / mL (P2).
  • NO nitric oxide
  • Example 1 Isolation of the peptide of the invention.
  • Ultrafiltration processes have been used to purify bioactive peptides from protein hydrolysates (Vo, TS, Ryu, BM, & Kim, S.-K. (2013). Purification of novel anti-inflammatory peptides from enzymatic hydrolysate of the edible microalgal Spirulina maximum Journal of Functional Foods, 5, 1336-1346).
  • the enzyme was inactivated by heating at 85 ° C for 15 minutes, and the mixture was centrifuged at 8500 rpm for 20 minutes. The centrifugal supernatant was used as substrate for the purification of anti-inflammatory peptides by tangential ultrafiltration with a membrane with a molecular weight cut-off size of 10 kDa.
  • the protein recovery performance analysis in the retained (PM1) and permeate (PM2) as well as the protein content of the fractions obtained by ultrafiltration were evaluated, with PM2 being the one with the highest activity (Table 1).
  • TNF TNF, IL-1 ⁇ , IL6, CCR2, CCL2 (pro-inflammatory cytokines) and CCL18 (anti-inflammatory cytokine)
  • the expression of TNF ( Figure 1A) and CCL2 ( Figure 1 E) was significantly reduced by the two fractions obtained by ultrafiltration, while the rest of proinflammatory cytokines, except CCR2, were also inhibited although no significant differences were observed.
  • both fractions appear to induce the expression of CCL18 but this induction was not significant according to the statistical studies performed.
  • TNF and I L-1 ⁇ were evaluated by quantitative PCR, being positive (+) in cases where the expression of cytokines was repressed and negative (-) in the case that it was induced or similar to the control sample ( Figure 4).
  • the F3-5 fraction showed a positive result for TNF expression, therefore this fraction was chosen for the rest of the analysis. No significant differences were observed in the case of IL6 expression ( Figure 5A), however, a drastic reduction in CCR2 and CCL2 expression was observed for the F3-5 fraction (-59.54%) and (-74, 45%), respectively ( Figure 5B and 5C).
  • Example 2 Anti-inflammatory activity of the peptide of the invention.
  • macrophages derived from THP-1 were treated with the same at two different concentrations. The concentration of less than 100 ⁇ g / mL was named as P1 and the highest concentration of 500 ⁇ g / mL as P2.
  • the concentration of TNF, I L-1 ⁇ and IL-10 in the cell culture supernatants was determined after the treatments at the two concentrations described above and at different hours (6 and 48 hours) for Observe the possible fluctuations in protein levels since each one presents different renewal rates.
  • the production of TNF and I L-1 ⁇ was decreased after 6 hours by the peptide at the lowest concentration or P1 (100 ⁇ g / mL) significantly with respect to the control, being also different from that obtained with the peptide at the highest concentration [Figure 10A (1) and 10B (2)].

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Abstract

The invention relates to the peptide of SEQ ID n° 1, isolated in a lupin protein hydrolysate (Lupinus Angustifolius L.), to the method for the production thereof by means of hydrolysis with alcalase, and to the use there of in the treatment of inflammatory diseases.

Description

PÉPTIDO AISLADO EN UN HIDROLIZADO DE ALTRAMUZ Y SU USO EN EL TRATAMIENTO DE ENFERMEDADES INFLAMATORIAS  PEPTIDE ISOLATED IN A LIQUID HYDROLYZE AND ITS USE IN THE TREATMENT OF INFLAMMATORY DISEASES
DESCRIPCIÓN DESCRIPTION
La presente invención se refiere a un péptido aislado de un hidrolizado proteico, en donde dicho péptido presenta actividad antiinflamatoria y/o inmunomoduladora, lo que hace posible su uso en el tratamiento de enfermedades inflamatorias. Por lo tanto, la presente invención va dirigida al campo del tratamiento de enfermedades inflamatorias. The present invention relates to a peptide isolated from a protein hydrolyzate, wherein said peptide exhibits anti-inflammatory and / or immunomodulatory activity, which makes its use possible in the treatment of inflammatory diseases. Therefore, the present invention is directed to the field of the treatment of inflammatory diseases.
ESTADO DE LA TÉCNICA STATE OF THE TECHNIQUE
La inflamación se caracteriza por el aumento del flujo sanguíneo al tejido provocando el aumento de temperatura, enrojecimiento, hinchazón y dolor. La inflamación a corto plazo o aguda es un intento de protección por el organismo para eliminar los estímulos nocivos y para iniciar el proceso de curación. A largo plazo o inflamación crónica, sin embargo, conduce a varios trastornos inflamatorios y enfermedades crónicas. La fase aguda se caracteriza por su breve duración, la exudación de líquido y de proteínas plasmáticas y la migración de leucocitos, predominantemente neutrófilos. La fase crónica se caracteriza por una duración mayor, la presencia de linfocitos y macrófagos, la proliferación de vasos sanguíneos, la fibrosis y la necrosis. Inflammation is characterized by increased blood flow to the tissue causing temperature increase, redness, swelling and pain. Short-term or acute inflammation is an attempt of protection by the organism to eliminate harmful stimuli and to start the healing process. Long term or chronic inflammation, however, leads to various inflammatory disorders and chronic diseases. The acute phase is characterized by its short duration, the exudation of liquid and plasma proteins and the migration of leukocytes, predominantly neutrophils. The chronic phase is characterized by a longer duration, the presence of lymphocytes and macrophages, blood vessel proliferation, fibrosis and necrosis.
La inflamación está involucrada en el inicio y desarrollo de numerosas enfermedades, incluyendo el cáncer, enfermedades neurológicas, enfermedades cardiovasculares, la hipertensión, la presión arterial, la aterosclerosis, la diabetes, la obesidad, trastornos respiratorios, trastornos multiesqueléticos, trastornos gastrointestinales y trastornos autoinmunes. El sistema inmune es el encargado de responder ante cualquier tipo de agresión en el organismo. Tiene su origen en los órganos linfoides (ganglios linfáticos, bazo, timo, y médula ósea) y en su acción participan una gran cantidad de subtipos celulares (células inmunocompetentes) entre las que se encuentran monocitos y neutrófilos. Después de 24-72 horas de permanecer en el torrente sanguíneo, los monocitos lo abandonan y atraviesan el endotelio de los capilares hacia el tejido conectivo, donde se diferencian a macrófagos o células dendríticas y desempeñan sus funciones relacionadas con la fagocitosis, inflamación, presentación de antígenos y hemostasia. Los macrófagos son una de las células más estudiadas del sistema inmune. Están generalmente en todos los tejidos. En ellos, los macrófagos adquieren sus propiedades morfológicas y funcionales, únicas y diferentes (por ejemplo, células de Kupffer en el hígado y los macrófagos alveolares en los pulmones) (Locati, M., Mantovani, A., & Sica, A. (2013). Advances in Immunology, 120, 163-184). Un macrófago tiene una vida media de 2-4 meses. En los tejidos existen estímulos que inducen la activación del macrófago (por ejemplo opsoninas, citoquinas y factores de crecimiento), incrementando rápidamente su metabolismo, motilidad y actividad fagocítica. En respuesta a las citoquinas y productos microbianos, los fagocitos mononucleares se polarizan y expresan distintas propiedades funcionales especializadas. Los macrófagos activados clásicamente (M1) inducidos por interferón (IFN) y/o factor de necrosis tumoral (TNF) muestran un fenotipo tipo Th1 , son efectores proinflamatorios y tienen funciones bactericidas, mientras que los macrófagos activados alternativamente (M2), exhiben un fenotipo tipo Th2 y están involucrados en la resolución de la inflamación y la curación del tejido. Las citoquinas son las moléculas reguladoras de las respuestas del huésped a la infección, la respuesta inmune, la inflamación y trauma. Hay dos tipos de citoquinas: proinflamatorias y antiinflamatorias. Por lo tanto, los inhibidores de las citoquinas proinflamatorias se han considerado como posibles candidatos de fármacos antiinflamatorios. Inflammation is involved in the onset and development of numerous diseases, including cancer, neurological diseases, cardiovascular diseases, hypertension, blood pressure, atherosclerosis, diabetes, obesity, respiratory disorders, multiskeletal disorders, gastrointestinal disorders and autoimmune disorders. . The immune system is responsible for responding to any type of aggression in the body. It has its origin in the lymphoid organs (lymph nodes, spleen, thymus, and bone marrow) and a large number of cell subtypes (immunocompetent cells) are involved, including monocytes and neutrophils. After 24-72 hours of remaining in the bloodstream, the monocytes leave it and pass through the endothelium of the capillaries into the connective tissue, where they differentiate into macrophages or dendritic cells and perform their functions related to phagocytosis, inflammation, antigen presentation and hemostasis. Macrophages are one of the most studied cells of the immune system. They are usually in all tissues. In them, macrophages acquire their unique and different morphological and functional properties (for example, Kupffer cells in the liver and alveolar macrophages in the lungs) (Locati, M., Mantovani, A., & Sica, A. ( 2013). Advances in Immunology, 120, 163-184). A macrophage has a half-life of 2-4 months. In the tissues there are stimuli that induce macrophage activation (for example opsonins, cytokines and growth factors), rapidly increasing their metabolism, motility and phagocytic activity. In response to cytokines and microbial products, mononuclear phagocytes polarize and express different specialized functional properties. Classically activated macrophages (M1) induced by interferon (IFN) and / or tumor necrosis factor (TNF) show a Th1 type phenotype, are proinflammatory effectors and have bactericidal functions, while alternately activated macrophages (M2) exhibit a phenotype Th2 type and are involved in the resolution of inflammation and tissue healing. Cytokines are the regulatory molecules of host responses to infection, immune response, inflammation and trauma. There are two types of cytokines: proinflammatory and anti-inflammatory. Therefore, proinflammatory cytokine inhibitors have been considered as potential candidates for anti-inflammatory drugs.
Aunque la ciencia moderna ha desarrollado varios medicamentos para el tratamiento de las enfermedades crónicas, la mayoría de estos fármacos son enormemente caros y están asociados con efectos secundarios graves y el aumento de la morbilidad. La alimentación es uno de los principales factores que incide en la modulación de la respuesta inflamatoria que se da en el organismo. A través de diversos efectos sobre las células y sus rutas metabólicas, la alimentación provoca cambios en el normal funcionamiento de los diferentes sistemas involucrados en el proceso inflamatorio, pues los alimentos pueden comportarse como metabolitos celulares pudiendo actuar como mensajeros intracelulares pro o antiinflamatorios. Although modern science has developed several medications for the treatment of chronic diseases, most of these drugs are enormously expensive and are associated with serious side effects and increased morbidity. Feeding is one of the main factors that affects the modulation of the inflammatory response that occurs in the body. Through various effects on cells and their metabolic pathways, food causes changes in the normal functioning of the different systems involved in the inflammatory process, as food can behave as cellular metabolites and can act as procellular or anti-inflammatory intracellular messengers.
En concreto, algunos agentes de la dieta han mostrado potencial para inactivar las moléculas inflamatorias mediante la unión directa. Por ejemplo, la curcumina, un polifenol presente en la cúrcuma, se une a una variedad de biomacromoléculas que indican que la curcumina es altamente pleiotrópica con actividad antiinflamatoria, hipoglucémica, antioxidante, cicatrización de heridas, y anti-microbiana (Gupta, S.C., et al. (2011). Natural Product Reports, 28, 1937-1955). Existen diversos estudios sobre péptidos antiinflamatorios derivados de la síntesis química (Selvatici, R., Siniscalchi, A., & Spisani, S. (2013). Mini-Reviews in Medicinal Chemistry, 13, 553-564). Hasta ahora, las proteínas de la leche se consideran la fuente más importante de péptidos bioactivos (Wada, Y., & Lonnerdal, B. (2014). The Journal of Nutritional Biochemistry, 25, 503-514) y se han obtenido hidrolizados proteicos con propiedades antiinflamatorias a partir de caseína de leche (Mao, X-Y., Cheng, X., Wang, X. , & Wu, S-J. (2011). Food Chemistry, 126, 484-490). Sin embargo, existen pocos estudios sobre el efecto de los hidrolizados de proteínas vegetales en los marcadores de inflamación. Specifically, some diet agents have shown potential to inactivate inflammatory molecules through direct binding. For example, curcumin, a Polyphenol present in turmeric binds to a variety of biomachromolecules that indicate that curcumin is highly pleiotropic with anti-inflammatory, hypoglycemic, antioxidant, wound healing, and anti-microbial activity (Gupta, SC, et al. (2011). Natural Product Reports, 28, 1937-1955). There are several studies on anti-inflammatory peptides derived from chemical synthesis (Selvatici, R., Siniscalchi, A., & Spisani, S. (2013). Mini-Reviews in Medicinal Chemistry, 13, 553-564). Until now, milk proteins are considered the most important source of bioactive peptides (Wada, Y., & Lonnerdal, B. (2014). The Journal of Nutritional Biochemistry, 25, 503-514) and protein hydrolysates have been obtained with anti-inflammatory properties from milk casein (Mao, XY., Cheng, X., Wang, X., & Wu, SJ. (2011). Food Chemistry, 126, 484-490). However, there are few studies on the effect of plant protein hydrolysates on inflammation markers.
En este sentido, se ha visto que la hidrólisis de la proteína de soja reduce la expresión de algunos de ellos (Vernaza, M.G., et al. (2012). Food Chemistry, 134, 2217-2225). Por otro lado, se ha visto que las semillas con un alto contenido en proteínas como el altramuz, disminuye las concentraciones de glucosa e insulina en sangre en los animales y los seres humanos (Baldeón, M.E., Castro, J., Villacrés, E., Narváez, L, & Fornasini, M. (2012). Nutrición Hospitalaria, 27, 1261-1266). Asimismo, los alimentos a base de proteínas de altramuz dulce están ganando atención por parte de la industria y el consumidor debido a su posible papel en la prevención de la enfermedad cardiovascular (Belski, R. (2012). Advances in Food and Nutrition Research, 66, 147- 215), donde la mayoría de los marcadores de riesgo tienen un componente pro- inflamatorio. In this sense, it has been seen that the hydrolysis of soy protein reduces the expression of some of them (Vernaza, M.G., et al. (2012). Food Chemistry, 134, 2217-2225). On the other hand, it has been seen that seeds with a high protein content such as lupine, lower blood glucose and insulin concentrations in animals and humans (Baldeón, ME, Castro, J., Villacrés, E. , Narváez, L, & Fornasini, M. (2012). Hospital Nutrition, 27, 1261-1266). Likewise, sweet lupine protein-based foods are gaining attention from the industry and the consumer due to their possible role in the prevention of cardiovascular disease (Belski, R. (2012). Advances in Food and Nutrition Research, 66, 147-215), where most of the risk markers have a pro-inflammatory component.
Por lo tanto existe en el estado de la técnica la necesidad de proporcionar nuevos compuestos "naturales" que puedan emplearse en el tratamiento de enfermedades antiinflamatorias, alternativos a los que existen en el estado de la técnica de origen sintético en la mayoría de los casos, que no estén asociados con efectos secundarios graves y morbilidad. Therefore, there is a need in the state of the art to provide new "natural" compounds that can be used in the treatment of anti-inflammatory diseases, alternative to those that exist in the state of the art of synthetic origin in most cases, that are not associated with serious side effects and morbidity.
DESCRIPCIÓN DE LA INVENCIÓN La presente invención va dirigida a un péptido con actividad antiinflamatoria y/o inmunomoduladora, lo que hace posible su uso en el tratamiento de enfermedades inflamatorias. Para la identificación de dicho péptido, los inventores de la presente invención partieron de un hidrolizado proteico de altramuz y, tras varios procesos de purificación y aislamiento, obtuvieron un péptido que presentaba la capacidad de inhibir la expresión de citoquinas proinflamatorias, tales como TNF e I L-1 β o promover la expresión de citoquinas antiinflamatorias como IL-10, lo que indicaba su actividad antiinflamatoria e inmunomoduladora, y su uso potencial en el tratamiento de enfermedades que cursan con inflamación. Por lo tanto, en base a este descubrimiento, se han desarrollado una serie de aspectos inventivos que serán descritos en detalle a continuación. DESCRIPTION OF THE INVENTION The present invention is directed to a peptide with anti-inflammatory and / or immunomodulatory activity, which makes its use possible in the treatment of inflammatory diseases. For the identification of said peptide, the inventors of the present invention started from a lupine protein hydrolyzate and, after several purification and isolation processes, obtained a peptide that had the ability to inhibit the expression of proinflammatory cytokines, such as TNF and I L-1β or promote the expression of anti-inflammatory cytokines such as IL-10, which indicated its anti-inflammatory and immunomodulatory activity, and its potential use in the treatment of diseases that occur with inflammation. Therefore, based on this discovery, a series of inventive aspects have been developed that will be described in detail below.
Péptido de la invención Mediante espectrometría de masas, los inventores averiguaron que el péptido de la invención con actividad antiinflamatoria y/o inmunomoduladora comprende la secuencia Gly-Pro-Glu-Thr-Ala-Phe-Leu-Arg (SEQ ID NO: 1). Peptide of the Invention By mass spectrometry, the inventors found that the peptide of the invention with anti-inflammatory and / or immunomodulatory activity comprises the sequence Gly-Pro-Glu-Thr-Ala-Phe-Leu-Arg (SEQ ID NO: 1) .
Por lo tanto, en un aspecto, la presente invención se relaciona con un péptido que comprende la secuencia de aminoácidos SEQ ID NO: 1 , o una variante del mismo con una identidad de secuencia del 75, 80, 85, 90, 95, 96, 97, 98 o 99% con la SEQ ID NO: 1 , de aquí en adelante "péptido de la invención", en el que dicho péptido no comprende la secuencia de aminoácidos que consiste en la secuencia SEQ ID NO: 2. En el contexto de la presente invención, se entiende por "péptido" a la cadena lineal de aminoácidos unidos entre sí mediante enlaces químicos de tipo amídico. El número de aminoácidos que pueden formar parte del péptido de la invención va, por ejemplo, desde 8 hasta 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140 o 150 aminoácidos. Therefore, in one aspect, the present invention relates to a peptide comprising the amino acid sequence SEQ ID NO: 1, or a variant thereof with a sequence identity of 75, 80, 85, 90, 95, 96 , 97, 98 or 99% with SEQ ID NO: 1, hereinafter "peptide of the invention", wherein said peptide does not comprise the amino acid sequence consisting of the sequence SEQ ID NO: 2. In the In the context of the present invention, "peptide" is understood as the linear chain of amino acids linked together by chemical bonds of the amide type. The number of amino acids that can be part of the peptide of the invention ranges, for example, from 8 to 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140 or 150 amino acids .
En la presente invención se entiende por "identidad" o "identidad de secuencia" al grado de similitud entre dos secuencias de nucleótidos o aminoácidos obtenido mediante el alineamiento de las dos secuencias. Dependiendo del número de residuos comunes entre las secuencias alineadas, se obtendrá un grado de identidad expresado en tanto por ciento. El grado de identidad entre dos secuencias de aminoácidos puede determinarse por métodos convencionales, por ejemplo, mediante algoritmos estándar de alineamiento de secuencias conocidos en el estado de la técnica, como por ejemplo BLAST [AltschuI S.F. et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215(3):403-10]. Los programas BLAST, por ejemplo, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, son de dominio público en la página web de The National Center for Biotechonology Information (NCBI). In the present invention, "identity" or "sequence identity" is understood as the degree of similarity between two nucleotide or amino acid sequences obtained by aligning the two sequences. Depending on the number of common residues between the aligned sequences, a degree of identity expressed as a percentage will be obtained. The degree of identity between two sequences of Amino acids can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as BLAST [AltschuI SF et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215 (3): 403-10]. The BLAST programs, for example, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are in the public domain on the website of The National Center for Biotechonology Information (NCBI).
El experto en la materia entiende que las mutaciones en la secuencia de nucleótidos de los genes que dan lugar a sustituciones conservativas de aminoácidos en posiciones no críticas para la funcionalidad de la proteína son mutaciones evolutivamente neutras que no afectan a su estructura global ni a su funcionalidad. Dichas variantes caen dentro del ámbito de la presente invención. Están incluidas también dentro del ámbito de la invención aquellas variantes de la proteína SEQ ID NO: 1 que presenten inserciones, deleciones o modificaciones de uno o más aminoácidos respecto a dicha SEQ ID NO: 1 , y conserven, además, las mismas funciones que dicha proteína SEQ ID NO: 1 , es decir, actividad antiinflamatoria y/o inmunomoduladora. The person skilled in the art understands that mutations in the nucleotide sequence of genes that give rise to conservative amino acid substitutions at positions not critical to protein functionality are evolutionarily neutral mutations that do not affect their overall structure or functionality. . Such variants fall within the scope of the present invention. Also included within the scope of the invention are those variants of the protein SEQ ID NO: 1 that have insertions, deletions or modifications of one or more amino acids with respect to said SEQ ID NO: 1, and also retain the same functions as said protein SEQ ID NO: 1, that is, anti-inflammatory and / or immunomodulatory activity.
En la presente invención se entiende que un péptido tiene actividad antiinflamatoria cuando tiene capacidad de inducir la expresión de IL-10, una potente citoquina antiinflamatoria, así como de reprimir la expresión de TNF y/o I L- 1 β , citoquinas de naturaleza proinflamatoria, en macrófagos derivados de células THP-1. In the present invention it is understood that a peptide has anti-inflammatory activity when it is capable of inducing the expression of IL-10, a potent anti-inflammatory cytokine, as well as suppressing the expression of TNF and / or I L-1β, proinflammatory cytokines , in macrophages derived from THP-1 cells.
En la presente invención, se entiende que un péptido presenta actividad inmunomoduladora, cuando presenta capacidad de modular la respuesta de células del sistema inmune humano como los macrófagos, siendo éstos, a su vez, capaces de modular la función de otras células inmunes como son los linfocitos T. In the present invention, it is understood that a peptide exhibits immunomodulatory activity, when it has the capacity to modulate the response of human immune system cells such as macrophages, these being, in turn, capable of modulating the function of other immune cells such as T lymphocytes.
Ejemplos de ensayos sobre como evaluar si un péptido tiene actividad antiinflamatoria y/o inmunomoduladora son, por ejemplo, los ensayos descritos en los Ejmeplos ilustrativos de la presente invención que acompañan a la descripción. Examples of tests on how to evaluate whether a peptide has anti-inflammatory and / or immunomodulatory activity are, for example, the tests described in the illustrative Examples of the present invention that accompany the description.
Una de las características del péptido de la invención es que no comprende la secuencia de aminoácidos Gly-Pro-Glu-Thr-Ala-Phe-Leu-Arg-Leu (SEQ ID NO: 2). Este péptido se localiza en las bases de datos del National Center for Biotechnology Information o NCBI con numero de acceso B3A0G0, versión B3A0G0 Gl:460425300. One of the characteristics of the peptide of the invention is that it does not comprise the amino acid sequence Gly-Pro-Glu-Thr-Ala-Phe-Leu-Arg-Leu (SEQ ID NO: 2). This peptide is located in the National Center for Biotechnology Information or NCBI databases with accession number B3A0G0, version B3A0G0 Gl: 460425300.
El péptido de la invención puede llevar unido a uno o ambos de sus extremos otros tipos de moléculas, tales como un marcador, un péptido señal, etc., y dichas moléculas pueden ir unidas directamente a los extremos carboxilo o amino terminal del péptido o unidas a través de un espaciador o linker. The peptide of the invention can be attached to one or both of its ends other types of molecules, such as a marker, a signal peptide, etc., and said molecules can be directly linked to the carboxyl or amino terminal ends of the peptide or linked through a spacer or linker.
Así, en una realización particular, el péptido de la invención comprende unido a uno de sus extremos un marcador. Thus, in a particular embodiment, the peptide of the invention comprises a marker attached to one of its ends.
En el contexto de la presente invención, se entiende por "marcador" a aquella molécula que permite la identificación del péptido de la invención. El marcador puede ser un "péptido marcador" para la identificación y/o la localización del péptido de la invención ya que dichos marcadores corresponden a sitios de unión a determinadas moléculas o átomos, como la cadena de histidinas, la GST, la avidina, la estreptavidina, etc., o porque son fácilmente detectables por técnicas inmunoquímicas, como hemaglutinina, VSV-G, HSVtk, FLAG, V5, myc, etc., o porque son fácilmente observables, como las proteínas fluorescentes. El péptido marcador VSV-G pertenece a la glicoproteína del virus vesicular de la estomatitis. El péptido marcador HSVtk pertenece a la timidina quinasa del virus del herpes simple 1. El péptido FLAG es un epítopo de 8 aminoácidos diseñado específicamente como marcador de proteínas recombinantes. V5 es un pequeño epítopo presente en las proteínas P y V del paramixovirus del virus de simio 5 (SV5). El epítopo myc tiene 10 aminoácidos y es parte de la secuencia del factor de transcripción c-myc humano. Por lo tanto, en otra realización más particular, el marcador se selecciona del grupo que consiste en c-myc, FLAG, HA, cadena de histidinas, GST, biotina, VSV-G, HSVtk, V5, biotina, avidina, estreptavidina, proteína de unión a maltosa y una proteína fluorescente. En otra realización particular, el péptido de la invención comprende unido a uno de sus extremos un péptido señal. In the context of the present invention, "marker" means that molecule that allows the identification of the peptide of the invention. The label can be a "marker peptide" for the identification and / or location of the peptide of the invention since said markers correspond to binding sites to certain molecules or atoms, such as the histidine chain, the GST, the avidin, the streptavidin, etc., or because they are easily detectable by immunochemical techniques, such as hemagglutinin, VSV-G, HSVtk, FLAG, V5, myc, etc., or because they are easily observable, such as fluorescent proteins. The VSV-G marker peptide belongs to the vesicular stomatitis vesicle glycoprotein. The HSVtk marker peptide belongs to the thymidine kinase of the herpes simplex virus 1. The FLAG peptide is an 8 amino acid epitope designed specifically as a recombinant protein marker. V5 is a small epitope present in the P and V proteins of simian virus 5 paramyxovirus (SV5). The myc epitope has 10 amino acids and is part of the human c-myc transcription factor sequence. Therefore, in another more particular embodiment, the marker is selected from the group consisting of c-myc, FLAG, HA, histidine chain, GST, biotin, VSV-G, HSVtk, V5, biotin, avidin, streptavidin, protein binding to maltose and a fluorescent protein. In another particular embodiment, the peptide of the invention comprises attached to one of its ends a signal peptide.
En la presente invención se entiende por "péptido señal" a la secuencia de entre 3 y 60 aminoácidos que dirige el transporte del péptido hacia una localización subcelular determinada, como puede ser el retículo endoplasmático, la mitocondria o el núcleo. El péptido señal puede dirigir también el transporte de la proteína fuera de la célula, lo que podría equivaler a su secreción o a su transporte al periplasma celular, en el caso de células como Escherichia coli. Ejemplos de péptido señal para dirigir el transporte de un péptido incluyen, pero no se limitan a, pelB, stll, ecotina, lamB, herpes GD, 1 pp, fosfatasa alcalina, invertasa, factor alfa y la secuencia líder de la proteína A. Así, en otra realización más particular, el péptido señal se selecciona del grupo que consiste en pelB, stll, ecotina, lamB, herpes GD, 1 pp, fosfatasa alcalina, invertasa, factor alfa y la secuencia líder de la proteína A. Como entiende el experto en la materia, también cabe la posibilidad de que el péptido de la invención lleve unidos simultáneamente tanto el marcador como el péptido señal, bien en diferentes extremos del péptido de la invención o en el mismo extremo. Así, dentro de la presente invención también se contemplan las uniones [péptido señal]- [marcador]-[péptido de la invención] y [marcador]-[péptido señal]-[péptido de la invención], donde la unión [marcador]-[péptido señal] pueden ir en el extremo amino o carboxilo terminal del péptido de la invención. Asimismo, si se cree conveniente, también pueden emplearse más de un marcador. In the present invention, "signal peptide" is understood as the sequence of between 3 and 60 amino acids that directs the transport of the peptide to a specific subcellular location, such as the endoplasmic reticulum, the mitochondria or the nucleus. He Signal peptide can also direct the transport of the protein out of the cell, which could be equivalent to its secretion or its transport to the cell periplasm, in the case of cells such as Escherichia coli. Examples of signal peptide to direct the transport of a peptide include, but are not limited to, pelB, stll, ecotin, lamB, herpes GD, 1 pp, alkaline phosphatase, invertase, alpha factor and the leader sequence of protein A. Thus In another more particular embodiment, the signal peptide is selected from the group consisting of pelB, stll, ecotin, lamB, herpes GD, 1 pp, alkaline phosphatase, invertase, alpha factor and the protein A leader sequence. skilled in the art, it is also possible that the peptide of the invention carries both the label and the signal peptide simultaneously, either at different ends of the peptide of the invention or at the same end. Thus, within the present invention, the junctions [signal peptide] - [marker] - [peptide of the invention] and [marker] - [signal peptide] - [peptide of the invention] are also contemplated, where the [marker] binding - [signal peptide] can go to the amino or carboxyl terminal of the peptide of the invention. Likewise, if it is considered convenient, more than one marker can also be used.
Finalmente, tanto el marcador como el péptido señal pueden ir unidos al péptido de la invención directamente o a través de un espaciador o linker. Finally, both the label and the signal peptide can be linked to the peptide of the invention directly or through a spacer or linker.
En la presente invención, se entiende por "unido directamente" a la unión covalente entre dos grupos diferentes que no tiene ningún átomo intermedio entre los dos grupos que están uniéndose. In the present invention, "directly bound" is understood as the covalent bond between two different groups that has no intermediate atom between the two groups that are joining.
En la presente invención se entiende por "espaciador" o "ligador" o "linker" a la molécula que sirve de nexo de unión entre dos dominios o dos moléculas. En el contexto de la presente invención, los dominios o moléculas serían el péptido de la invención, el marcador y el péptido señal. In the present invention, "spacer" or "linker" or "linker" is understood as the molecule that serves as a link between two domains or two molecules. In the context of the present invention, the domains or molecules would be the peptide of the invention, the marker and the signal peptide.
Métodos para producir el péptido de la invención son ampliamente conocidos en el estado de la técnica. El péptido de la invención, y cualquiera de sus realizaciones particulares, puede producirse por síntesis en un laboratorio, o por expresión en una célula de la secuencia de nucleótidos que codifica el péptido de la invención. Así, otro aspecto, la invención se relación con una secuencia de nucleótidos, de aquí en adelante "secuencia de nucleótidos de la invención", que codifica el péptido de la invención. En la presente invención se entiende por "secuencia de nucleótidos" a la cadena de nucleótidos unidos entre sí a través de grupos fosfato. La secuencia de nucleótidos, denominada comúnmente ácido nucleico, puede estar formada por desoxirribonucleótidos, ribonucleótidos, nucleótidos modificados o cualquier sustrato que pueda ser incorporado en un polímero de ADN o ARN por una polimerasa o por una reacción sintética. Methods for producing the peptide of the invention are widely known in the state of the art. The peptide of the invention, and any of its particular embodiments, can be produced by synthesis in a laboratory, or by expression in a cell of the nucleotide sequence encoding the peptide of the invention. Thus, another aspect, the invention relates to a nucleotide sequence, hereinafter "nucleotide sequence of the invention", which encodes the peptide of the invention. In the present invention, "nucleotide sequence" is understood as the nucleotide chain linked to each other through phosphate groups. The nucleotide sequence, commonly referred to as nucleic acid, can be formed by deoxyribonucleotides, ribonucleotides, modified nucleotides or any substrate that can be incorporated into a DNA or RNA polymer by a polymerase or by a synthetic reaction.
El término "codifica" se refiere al código genético que determina cómo una secuencia de nucleótidos se traduce en un polipéptido o una proteína. El orden de los nucleótidos de una secuencia determina el orden de los aminoácidos a lo largo de un péptido, un polipéptido o una proteína. The term "encodes" refers to the genetic code that determines how a nucleotide sequence is translated into a polypeptide or a protein. The order of nucleotides in a sequence determines the order of amino acids along a peptide, a polypeptide or a protein.
La secuencia de nucleótidos de la invención puede insertarse en un vector, por ejemplo, un vector de expresión. Por lo tanto, en otro aspecto, la invención se relaciona con un vector, de aquí en adelante "vector de la invención", que comprende la secuencia de nucleótidos de la invención. The nucleotide sequence of the invention can be inserted into a vector, for example, an expression vector. Therefore, in another aspect, the invention relates to a vector, hereinafter "vector of the invention", comprising the nucleotide sequence of the invention.
En la presente descripción, el término "vector" se refiere a una molécula de ácido nucleico que puede usarse para transportar o transferir una segunda molécula de ácido nucleico al interior de una célula. En la presente invención, dicha segunda molécula de ácido nucleico es la secuencia de nucleótidos de la invención. In the present description, the term "vector" refers to a nucleic acid molecule that can be used to transport or transfer a second nucleic acid molecule into a cell. In the present invention, said second nucleic acid molecule is the nucleotide sequence of the invention.
Un vector puede contener diferentes elementos funcionales que incluyen, pero no se limitan a, elementos de control de la transcripción, como promotores u operadores, regiones o potenciadores de la unión a factores de transcripción, y elementos de control para iniciar y terminar la transcripción. Los vectores incluyen, pero no se limitan a: plásmidos, cósmidos, virus, fagos, casetes de expresión recombinantes y transposones. Algunos vectores son capaces de replicarse o dividirse autónomamente tras ser introducidos en la célula huésped, como los vectores bacterianos con un origen de replicación bacteriano o los vectores episomales de mamíferos. Otros vectores pueden integrarse en el genoma de la célula huésped y replicarse así junto con el genoma celular. A vector may contain different functional elements that include, but are not limited to, transcription control elements, such as promoters or operators, regions or enhancers of transcription factor binding, and control elements to initiate and terminate transcription. Vectors include, but are not limited to: plasmids, cosmids, viruses, phages, recombinant expression cassettes and transposons. Some vectors are capable of replicating or dividing autonomously after being introduced into the host cell, such as bacterial vectors with an origin of bacterial replication or mammalian episomal vectors. Others Vectors can be integrated into the genome of the host cell and thus replicated together with the cell genome.
En general, un vector de expresión comprende, además de la secuencia de nucleótidos que codifica el péptido de la invención, un promotor que dirige su transcripción (por ejemplo, pT7, plac, ptrc, ptac, pBAD, ret, etc.), al que está operativamente enlazado, y otras secuencias necesarias o apropiadas que controlan y regulan dicha transcripción y, en su caso, la traducción del producto de interés, por ejemplo, señales de inicio y terminación de transcripción (tlt2, etc.), señal de poliadenilación, origen de replicación, secuencias de unión a ribosomas (RBS), secuencias codificantes de reguladores transcripcionales, (enhancers), silenciadores transcripcionales (silencers), represores, etc. Ejemplos de vectores de expresión apropiados pueden seleccionarse de acuerdo con las condiciones y necesidades de cada caso concreto entre plásmidos de expresión, vectores virales (ADN o ARN), cósmidos, cromosomas artificiales, etc. que pueden contener, además, marcadores utilizables para seleccionar las células transfectadas o transformadas con el gen o genes de interés. La elección del vector dependerá de la célula huésped y del tipo de uso que se quiera realizar. Por ejemplo, dicho vector puede ser un vector viral, tales como adenovirus, virus asociados a los adenovirus así como retrovirus y, en particular, lentivirus, etc., o no viral tales como pcDNA3, pHCMV/Zeo, pCR3.1 , pEFI/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXI, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1 , pML2d, pTDTI, etc. Dichos vectores pueden ser administrados directamente al sujeto por métodos convencionales. Alternativamente, dichos vectores pueden ser utilizados para transformar, transfectar o infectar células, tanto procariotas como eucariotas. La obtención de dicho vector puede realizarse por métodos convencionales conocidos por los técnicos en la materia al igual que para la transformación de microorganismos y células eucariotas se pueden utilizar diferentes métodos ampliamente conocidas - transformación química, transfección, electroporación, microinyección, etc. - descritos en diversos manuales ampliamente conocidos por el experto en la materia. In general, an expression vector comprises, in addition to the nucleotide sequence encoding the peptide of the invention, a promoter that directs its transcription (eg, pT7, plac, ptrc, ptac, pBAD, ret, etc.), to which is operatively linked, and other necessary or appropriate sequences that control and regulate said transcription and, where appropriate, the translation of the product of interest, for example, transcription initiation and termination signals (tlt2, etc.), polyadenylation signal , origin of replication, ribosome binding sequences (RBS), coding sequences of transcriptional regulators, (enhancers), transcriptional silencers (silencers), repressors, etc. Examples of appropriate expression vectors can be selected according to the conditions and needs of each specific case among expression plasmids, viral vectors (DNA or RNA), cosmids, artificial chromosomes, etc. which may also contain markers that can be used to select cells transfected or transformed with the gene or genes of interest. The choice of the vector will depend on the host cell and the type of use to be performed. For example, said vector can be a viral vector, such as adenovirus, adenovirus associated viruses as well as retroviruses and, in particular, lentiviruses, etc., or non-viral such as pcDNA3, pHCMV / Zeo, pCR3.1, pEFI / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAXI, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d, pTDTI, etc. Such vectors can be administered directly to the subject by conventional methods. Alternatively, said vectors can be used to transform, transfect or infect cells, both prokaryotic and eukaryotic. The obtaining of said vector can be carried out by conventional methods known to those skilled in the art, as well as for the transformation of microorganisms and eukaryotic cells different widely known methods can be used - chemical transformation, transfection, electroporation, microinjection, etc. - described in various manuals widely known to those skilled in the art.
En otro aspecto, la invención se relaciona con una célula, de aquí en adelante "célula de la invención" que comprende la secuencia de nucleótidos o el vector de la invención descritos anteriormente. Las células huéspedes adecuadas en el contexto de la presente invención incluyen, sin limitar a, células procariotas, tales como células bacterianas, por ejemplo E. coli, y células eucariotas, tales como células de levaduras, por ejemplo, S. cerevisiae, células animales, preferiblemente de mamífero, células fúngicas, por ejemplo, Aspergillus sp. o células de insectos, por ejemplo, Spodoptera frugiperda. In another aspect, the invention relates to a cell, hereinafter "cell of the invention" comprising the nucleotide sequence or the vector of the invention described above. Suitable host cells in the context of the present invention include, but are not limited to, prokaryotic cells, such as bacterial cells, for example E. coli, and eukaryotic cells, such as yeast cells, for example, S. cerevisiae, animal cells , preferably from mammalian, fungal cells, for example, Aspergillus sp. or insect cells, for example, Spodoptera frugiperda.
En otro aspecto, la invención se relaciona con un extracto peptídico, de aquí en adelante "extracto peptídico de la invención", que comprende el péptido de la invención. In another aspect, the invention relates to a peptide extract, hereinafter "peptide extract of the invention", which comprises the peptide of the invention.
En el contexto de la presente invención, se entiende por "extracto peptídico" a la solución obtenida por centrifugación a partir de una modificación química, física y/o enzimática realizada en un material de partida de naturaleza vegetal, animal, fúngica y/o microbiana, y en el cual puede encontrarse como componente principal un conjunto de péptidos entre los cuales comprende el péptido de la invención. In the context of the present invention, "peptide extract" is understood as the solution obtained by centrifugation from a chemical, physical and / or enzymatic modification made in a starting material of a plant, animal, fungal and / or microbial nature. , and in which a set of peptides can be found as the main component among which it comprises the peptide of the invention.
En otro aspecto, la invención se relaciona con una composición, de aquí en adelante "composición de la invención", que comprende el péptido, la secuencia de nucleótidos, el vector, la célula y/o el extracto peptídico de la invención. Dentro del presente aspecto inventivo se contempla la posibilidad de que la composición comprenda combinaciones del péptido, la secuencia de nucleótidos, el vector, la célula o el extracto peptídico de la invención. In another aspect, the invention relates to a composition, hereinafter "composition of the invention", which comprises the peptide, the nucleotide sequence, the vector, the cell and / or the peptide extract of the invention. Within the present inventive aspect the possibility is contemplated that the composition comprises combinations of the peptide, the nucleotide sequence, the vector, the cell or the peptide extract of the invention.
Como entiende el experto en la materia, el péptido, la secuencia de nucleótidos, el vector, la célula y/o el extracto peptídico de la invención tienen que estar presentes en una cantidad terapéuticamente eficaz en la composición para que estos puedan ejercer el efecto antiinflamatorio y/o inmunomodulador. As the person skilled in the art understands, the peptide, the nucleotide sequence, the vector, the cell and / or the peptide extract of the invention must be present in a therapeutically effective amount in the composition so that they can exert the anti-inflammatory effect. and / or immunomodulator.
En la presente invención se entiende por "cantidad terapéuticamente efectiva" a una cantidad del péptido de la invención que, administrada en dosis y durante el período de tiempo necesario a un sujeto, sea efectiva a la hora de conseguir el resultado profiláctico o terapéutico deseado. Una "cantidad terapéuticamente efectiva" del péptido o composición farmacéutica de la invención puede variar con el estadio de la enfermedad, la edad, el sexo y el peso del individuo, y se refiere a una cantidad que no presenta efectos adversos ni toxicidad y es capaz de alcanzar el efecto profiláctico o terapéutico deseado. In the present invention, "therapeutically effective amount" is understood as an amount of the peptide of the invention that, administered in doses and for the necessary period of time to a subject, is effective in achieving the desired prophylactic or therapeutic result. A "therapeutically effective amount" of the peptide or pharmaceutical composition of the invention may vary with the disease stage, age, sex and weight of the individual, and refers to an amount that It has no adverse effects or toxicity and is capable of achieving the desired prophylactic or therapeutic effect.
La composición farmacéutica de la invención puede administrarse por cualquier vía apropiada (por ejemplo, oral, sublingual, perioral, intranasal, parenteral, transdérmica, tópica, etc.), para lo cual se utilizarán los excipientes y vehículos farmacéuticamente aceptables necesarios para la formulación de la forma farmacéutica de administración elegida. Las distintas formas farmacéuticas de administración de fármacos y de su preparación son conocimiento general para el experto en la materia. The pharmaceutical composition of the invention can be administered by any appropriate route (for example, oral, sublingual, perioral, intranasal, parenteral, transdermal, topical, etc.), for which the pharmaceutically acceptable excipients and vehicles necessary for the formulation of the pharmaceutical form of administration chosen. The different pharmaceutical forms of drug administration and their preparation are general knowledge for the person skilled in the art.
La forma adaptada a la administración oral se refiere a un estado físico que pueda permitir su administración oral. Dicha forma adaptada a la administración oral se selecciona de la lista que comprende, pero sin limitarse, gotas, jarabe, tisana, elixir, suspensión, suspensión extemporánea, vial bebible, comprimido, cápsula, granulado, sello, pildora, tableta, pastilla, trocisco y liofilizado. The form adapted to oral administration refers to a physical state that can allow oral administration. Said form adapted to oral administration is selected from the list comprising, but not limited to, drops, syrup, herbal tea, elixir, suspension, extemporaneous suspension, drinkable vial, tablet, capsule, granulate, seal, pill, tablet, tablet, tablet and lyophilized.
La forma adaptada a la administración parenteral se refiere a un estado físico que pueda permitir su administración inyectable, es decir, preferiblemente en estado líquido. La administración parenteral se puede llevar a cabo por vía de administración intramuscular, intraarterial, intravenosa, intradérmica, subcutánea o intraósea pero sin limitarse únicamente a estos tipos de vías de administración parenteral The form adapted to parenteral administration refers to a physical state that can allow its injectable administration, that is, preferably in a liquid state. Parenteral administration can be carried out via intramuscular, intraarterial, intravenous, intradermal, subcutaneous or intraosseous administration but not limited to these types of parenteral administration routes.
Otra posibilidad es que la composición farmacéutica se presente en una forma adaptada a la administración sublingual, nasal, intratecal, bronquial, linfática, rectal, transdérmica o inhalada. Another possibility is that the pharmaceutical composition is present in a form adapted to sublingual, nasal, intrathecal, bronchial, lymphatic, rectal, transdermal or inhaled administration.
La composición de la invención puede comprender otros componentes, tales como diluyentes, excipientes, sales, etc. Así, en una realización particular, la composición de la invención comprende, adicionalmente, un excipiente. The composition of the invention may comprise other components, such as diluents, excipients, salts, etc. Thus, in a particular embodiment, the composition of the invention further comprises an excipient.
En la presente invención se entiende por "excipiente" a una sustancia inactiva usada para incorporar el principio activo (péptido de la invención). Además puede ser usado para ayudar al proceso mediante el cual la composición es manufacturada. Ejemplos de excipientes incluyen, sin limitar a, aglutinantes (mantienen los ingredientes de una tableta unidos, tales como hidroxipropil, celulosa, xilitol, sorbitol, manitol, etc.), diluyentes (rellenan el contenido de una pastilla o cápsula, tales como, celulosa vegetal, fosfato cálcico dibásico, flor de cártamo, etc.), desintegradores (compuestos que se expanden y disuelven cuando se les moja causando que la tableta se rompa cuando llegue al tracto digestivo y libere los principio activos para su absorción), lubrificantes (previenen que los ingredientes se agrupen en terrones, tales como talco, sílica, etc.), recubridores (protegen a los ingredientes de la tableta de los agentes externos, tales como celulosa, polímeros sintéticos, polisacáridos, etc.), edulcorantes, saborizantes y colorantes. La composición puede estar en forma de un sólido, por ejemplo polvos, gránulos, comprimidos, cápsulas de gelatina, liposomas o supositorios. Los soportes sólidos apropiados pueden ser, por ejemplo, fosfato de calcio, estearato de magnesio, talco, azúcares, lactosa, dextrina, almidón, gelatina, celulosa, metilcelulosa, carboximetilcelulosa de sodio, polivinilpirrolidina y cera. Otros excipientes y/o portadores farmacéuticamente aceptables apropiados su administración a animales serán conocidos por los expertos en la materia. In the present invention, "excipient" is understood as an inactive substance used to incorporate the active ingredient (peptide of the invention). It can also be used to help the process by which the composition is manufactured. Examples of excipients include, but are not limited to, binders (keep the ingredients of a tablet together, such as hydroxypropyl, cellulose, xylitol, sorbitol, mannitol, etc.), diluents (fill in the contents of a tablet or capsule, such as vegetable cellulose, dibasic calcium phosphate, safflower flower, etc.), disintegrators (compounds that expand and dissolve when they get wet causing the tablet to break when it reaches the digestive tract and release the active substances for absorption), lubricants (prevent the ingredients from being grouped into lumps, such as talcum, silica, etc.), coatings (protect the tablet's ingredients from external agents, such as cellulose , synthetic polymers, polysaccharides, etc.), sweeteners, flavorings and dyes. The composition may be in the form of a solid, for example powders, granules, tablets, gelatin capsules, liposomes or suppositories. Suitable solid supports may be, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine and wax. Other pharmaceutically acceptable excipients and / or carriers suitable for administration to animals will be known to those skilled in the art.
Las composiciones farmacéuticas según la presente invención también se pueden presentar en forma líquida, por ejemplo, soluciones, emulsiones, suspensiones o jarabes. Los soportes líquidos apropiados pueden ser, por ejemplo, agua, disolventes orgánicos, tales como glicerol o glicoles, así como sus mezclas, en proporciones variables, en agua. The pharmaceutical compositions according to the present invention can also be presented in liquid form, for example, solutions, emulsions, suspensions or syrups. Suitable liquid supports may be, for example, water, organic solvents, such as glycerol or glycols, as well as mixtures thereof, in varying proportions, in water.
La composición también puede comprender sales, principalmente, sales farmacéuticamente aceptables entre las que se incluyen, sin limitar a, las sales de adición de ácidos inorgánicos, tales como clorhidrato, bromhidrato, yodhidrato, sulfato, fosfato, difosfato y nitrato, o de ácidos orgánicos, tales como acetato, maleato, fumarato, tartrato, succinato, citrato, lactato, metanosulfonato, p-toluenosulfonato, palmoato y estearato. También dentro del alcance de la presente invención, cuando se pueden utilizar, se encuentran las sales formadas a partir de bases, tales como el hidróxido de sodio o de potasio. The composition may also comprise salts, principally, pharmaceutically acceptable salts, including, but not limited to, the addition salts of inorganic acids, such as hydrochloride, hydrobromide, iodhydrate, sulfate, phosphate, diphosphate and nitrate, or organic acids , such as acetate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, palmoate and stearate. Also within the scope of the present invention, when they can be used, are salts formed from bases, such as sodium or potassium hydroxide.
En una realización particular, la composición de la invención es una composición farmacéutica o una composición nutricional. En la presente invención, se entiende por "composición farmacéutica" a la composición que puede administrarse a un sujeto, por ejemplo, un humano, por vía oral, por vía parenteral, por vía tópica, mediante pulverización de inhalación, por vía intranasal o por vía rectal, en formulaciones de dosificación unitaria que contienen portadores, diluyentes, adyuvantes, vehículos y similares no tóxicos. El término composición farmacéutica incluye composiciones para su uso en sanidad humana o animal (composiciones veterinarias). In a particular embodiment, the composition of the invention is a pharmaceutical composition or a nutritional composition. In the present invention, "pharmaceutical composition" means the composition that can be administered to a subject, for example, a human, orally, parenterally, topically, by inhalation spray, intranasally or by rectal route, in unit dosage formulations containing non-toxic carriers, diluents, adjuvants, vehicles and the like. The term pharmaceutical composition includes compositions for use in human or animal health (veterinary compositions).
En la presente invención, se entiende por "composición nutricional" a aquella composición ingerida por los seres vivos por el cual el organismo recibe, transforma y utiliza sustancias químicas contenidas en los alimentos. In the present invention, "nutritional composition" means that composition ingested by living beings by which the body receives, transforms and uses chemical substances contained in food.
En una realización particular, la composición farmacéutica de la invención comprende, además, un vehículo farmacéuticamente aceptable. In a particular embodiment, the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier.
El "vehículo" o portador, es preferiblemente una sustancia inerte con la finalidad de facilitar la incorporación de otros compuestos, permitir una mejor dosificación y administración o dar consistencia y forma a la composición farmacéutica. Por tanto, el vehículo es una sustancia que se emplea en el medicamento para diluir cualquiera de los componentes de la composición farmacéutica de la presente invención hasta un volumen o peso determinado; o bien que aún sin diluir dichos componentes es capaz de permitir una mejor dosificación y administración o dar consistencia y forma al medicamento. Cuando la forma de presentación es líquida, el vehículo farmacéuticamente aceptable es el diluyente. The "carrier" or carrier, is preferably an inert substance in order to facilitate the incorporation of other compounds, allow better dosing and administration or give consistency and form to the pharmaceutical composition. Therefore, the carrier is a substance that is used in the medicament to dilute any of the components of the pharmaceutical composition of the present invention to a certain volume or weight; or that even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and form to the medicine. When the form of presentation is liquid, the pharmaceutically acceptable carrier is the diluent.
La expresión "farmacológicamente aceptable" hace referencia a que el excipiente o el vehículo está permitido y evaluado de modo que no cause daño al sujeto al que se le administra. Además, el excipiente y el vehículo deben ser farmacéuticamente adecuados, es decir, deben permitir la actividad de los compuestos de la composición farmacéutica y ser compatible con dichos componentes. The term "pharmacologically acceptable" refers to the excipient or vehicle being allowed and evaluated so as not to cause harm to the subject to which it is administered. Furthermore, the excipient and the vehicle must be pharmaceutically suitable, that is, they must allow the activity of the compounds of the pharmaceutical composition and be compatible with said components.
El péptido, la secuencia de nucleótidos, el vector y/o la célula de la invención así como las composiciones farmacéuticas que los contienen pueden ser utilizados junto con otros fármacos adicionales para proporcionar una terapia de combinación. Dichos fármacos adicionales pueden formar parte de la misma composición farmacéutica o, alternativamente, pueden ser proporcionados en forma de una composición separada para su administración simultánea o no a la de la composición farmacéutica que comprende el péptido, la secuencia de nucleótidos, el vector y/o la célula de la invención. The peptide, the nucleotide sequence, the vector and / or the cell of the invention as well as the pharmaceutical compositions containing them can be used together with other additional drugs to provide a combination therapy. Such additional drugs may be part of the same pharmaceutical composition or, alternatively, they may be provided in the form of a separate composition for simultaneous or non-simultaneous administration to the pharmaceutical composition comprising the peptide, the nucleotide sequence, the vector and / or the cell of the invention.
Así, en una realización particular, la composición de la invención comprende, adicionalmente, un compuesto antiinflamatorio. Thus, in a particular embodiment, the composition of the invention further comprises an anti-inflammatory compound.
Ejemplos de compuestos antiinflamatorios incluyen, sin limitar a, compuestos esteroideos, compuestos no esteroideos (también denominados AINES) y el grupo de fármacos antirreumoatoides modificadores de la enfermedad o FARME. Entre los anti inflamatorios esteroideos se incluyen, sin limitar a, corticoides, tales como la dexametasona, la prednisona, etc. Ejemplos de antiinflamatorios no esteroideos incluyen, sin limitar a, inhibidores de la COX-2, ácido acetil salicílico, derivados del ácido propílico (como el ibuprofeno y naproxen), derivados del ácido acético (como la indometacina), ácidos enólicos (como el piroxicam) y paracetamol. Ejemplos de FARME incluyen, sin limitar a, el metrotexato, la penicilamina, la cloroquina, la sulfasalazina y las sales de oro. Usos del péptido de la invención Examples of anti-inflammatory compounds include, but are not limited to, steroidal compounds, non-steroidal compounds (also called NSAIDs) and the group of disease-modifying anti-rheumatoid drugs or DMARDs. Anti-steroidal anti-inflammatory drugs include, without limitation, corticosteroids, such as dexamethasone, prednisone, etc. Examples of non-steroidal anti-inflammatories include, but are not limited to, COX-2 inhibitors, acetylsalicylic acid, derivatives of propyl acid (such as ibuprofen and naproxen), derivatives of acetic acid (such as indomethacin), enolic acids (such as piroxicam ) and paracetamol. Examples of DMARDs include, but are not limited to, metrotexate, penicillamine, chloroquine, sulfasalazine and gold salts. Uses of the peptide of the invention
Como se ha explicado al comienzo de la presente descripción, el péptido de la invención tiene actividad antiinflamatoria y/o inmunomoduladora, lo que permite su uso en el tratamiento de enfermedades o trastornos que cursan con inflamación. As explained at the beginning of the present description, the peptide of the invention has anti-inflammatory and / or immunomodulatory activity, which allows its use in the treatment of diseases or disorders that occur with inflammation.
Por lo tanto, en otro aspecto, la invención se relaciona con el uso del péptido, la secuencia de nucleótidos, el vector, la célula y/o el extracto peptídico de la invención en la elaboración de un medicamento. Dentro del presente aspecto inventivo se contempla la posibilidad del uso de combinaciones del péptido, la secuencia de nucleótidos, el vector, la célula o el extracto peptídico de la invención en la elaboración de un medicamento. Therefore, in another aspect, the invention relates to the use of the peptide, the nucleotide sequence, the vector, the cell and / or the peptide extract of the invention in the manufacture of a medicament. Within the present inventive aspect, the possibility of using combinations of the peptide, the nucleotide sequence, the vector, the cell or the peptide extract of the invention in the preparation of a medicament is contemplated.
En la presente invención, se considera que el término "medicamento" y "composición farmacéutica" son equivalentes y, por lo tanto, la invención también se relaciona con el péptido, la secuencia de nucleótidos, el vector y/o la célula de la invención en la elaboración de una composición farmacéutica tal como la descrita en párrafos anteriores. In the present invention, the term "medicament" and "pharmaceutical composition" are considered to be equivalent and, therefore, the invention also relates to the peptide, nucleotide sequence, vector and / or cell of the invention. in the elaboration of a pharmaceutical composition as described in previous paragraphs.
En otro aspecto, la invención se relaciona con el uso del péptido, la secuencia de nucleótidos, el vector, la célula y/o el extracto peptídico de la invención en la elaboración de un medicamento con actividad antiinflamatoria y/o inmunomoduladora. In another aspect, the invention relates to the use of the peptide, the nucleotide sequence, the vector, the cell and / or the peptide extract of the invention in the preparation of a medicament with anti-inflammatory and / or immunomodulatory activity.
En la presente invención, se entiende que un medicamento presenta actividad antiinflamatoria cuando tiene capacidad de inducir la expresión de IL-10, una potente citoquina antiinflamatoria, así como de reprimir la expresión de TNF y/o I L- 1 β , citoquinas de naturaleza proinflamatoria, en macrofagos derivados de células THP-1. In the present invention, it is understood that a medicament exhibits anti-inflammatory activity when it has the capacity to induce the expression of IL-10, a potent anti-inflammatory cytokine, as well as to suppress the expression of TNF and / or I L-1β, cytokines of nature proinflammatory, in macrophages derived from THP-1 cells.
En la presente invención, se entiende que un medicamento presenta actividad inmunomoduladora, cuando presenta capacidad de modular la respuesta de células del sistema inmune humano como los macrofagos, siendo éstos, a su vez, capaces de modular la función de otras células inmunes como son los linfocitos T. In the present invention, it is understood that a medicament has immunomodulatory activity, when it has the capacity to modulate the response of human immune system cells such as macrophages, these being, in turn, capable of modulating the function of other immune cells such as T lymphocytes.
En otra realización particular, la invención se relaciona con el uso del péptido, la secuencia de nucleótidos, el vector, la célula y/o el extracto peptídico de la invención, en la elaboración de un medicamento para el tratamiento de una enfermedad inflamatoria. Tal como se ha expresado anteriormente, dentro del presente aspecto inventivo se contempla la posibilidad del uso de combinaciones del péptido, la secuencia de nucleótidos, el vector, la célula o el extracto peptídico de la invención en la elaboración de un medicamento para el tratamiento de una enfermedad inflamatoria. In another particular embodiment, the invention relates to the use of the peptide, the nucleotide sequence, the vector, the cell and / or the peptide extract of the invention, in the preparation of a medicament for the treatment of an inflammatory disease. As stated above, within the present inventive aspect the possibility of the use of combinations of the peptide, the nucleotide sequence, the vector, the cell or the peptide extract of the invention in the preparation of a medicament for the treatment of an inflammatory disease
En la presente invención se entiende por "tratamiento" o "terapia" al conjunto de medios que se emplean en un sujeto para aliviar o curar una enfermedad, o para eliminar o disminuir los síntomas de una enfermedad. En la presente invención se entiende por "individuo" o "sujeto" a un miembro de una especie de animal, preferentemente mamífero e incluye, pero no se limita a, un animal doméstico, un primate y un humano; en el contexto de la presente invención, el individuo es preferiblemente un ser humano, masculino o femenino, de cualquier raza o edad. En la presente invención se entiende por "enfermedad inflamatoria" a aquella enfermedad que cursa con inflamación y que provoca unos niveles elevados de TNFa en el individuo que la padece. En una realización particular, la enfermedad inflamatoria se selecciona del grupo que consiste en una enfermedad autoinmune, cáncer, ateroesclerosis, artrosis, artritis, diabetes, obesidad, síndrome metabolico, asma, enfermedad de Crohn, hipertensión y dermatitis. En la presente invención se entiende por "enfermedad autoinmune" a aquella enfermedad en la que el sistema inmunitario de un organismo ataca a sus propias células. Ejemplos de enfermedades autoinmunes incluyen, sin limitar a, síndrome de inmunodeficiencia adquirida (SIDA), síndrome linfoproliferativo autoinmune, anemia hemolítica, enfermedades inflamatorias, y trombocitopenia, enfermedades inmunes agudas o crónicas asociadas con el trasplante de órganos y alergias. In the present invention, "treatment" or "therapy" means the set of means used in a subject to alleviate or cure a disease, or to eliminate or decrease the symptoms of a disease. In the present invention, "individual" or "subject" means a member of an animal species, preferably a mammal, and includes, but is not limited to, a domestic animal, a primate and a human; In the context of the present invention, the individual is preferably a human, male or female, of any race or age. In the present invention, "inflammatory disease" is understood to be that disease that causes inflammation and causes high levels of TNFa in the individual suffering from it. In a particular embodiment, the inflammatory disease is selected from the group consisting of an autoimmune disease, cancer, atherosclerosis, osteoarthritis, arthritis, diabetes, obesity, metabolic syndrome, asthma, Crohn's disease, hypertension and dermatitis. In the present invention, "autoimmune disease" is understood as that disease in which the immune system of an organism attacks its own cells. Examples of autoimmune diseases include, but are not limited to, acquired immunodeficiency syndrome (AIDS), autoimmune lymphoproliferative syndrome, hemolytic anemia, inflammatory diseases, and thrombocytopenia, acute or chronic immune diseases associated with organ transplantation and allergies.
En la presente invención se entiende por "cáncer" a aquella enfermedad en la que células anormales se dividen sin control y pueden invadir otros tejidos, provocando una metástasis. En la presente invención, los términos "cáncer"y "tumor" se consideran equivalentes. Ejemplos de cáncer, incluyen, sin limitar a, linfomas, leucemias, cáncer de mama, cáncer de colon, cáncer de pulmón, cáncer colorrectal, cáncer de esófago, cáncer de piel, cáncer de próstata y cáncer de estómago. In the present invention "cancer" is understood as that disease in which abnormal cells divide without control and can invade other tissues, causing a metastasis. In the present invention, the terms "cancer" and "tumor" are considered equivalent. Examples of cancer include, but are not limited to, lymphomas, leukemia, breast cancer, colon cancer, lung cancer, colorectal cancer, esophageal cancer, skin cancer, prostate cancer and stomach cancer.
Realizaciones particulares del medicamento o la composición farmacéutica han sido descritas en detalles en párrafos anteriores, tales como las formas de presentación del medicamento, los excipientes, cantidades terapéuticas, formas de administración, etc. y son aplicables al presente aspecto inventivo. Particular embodiments of the medicament or pharmaceutical composition have been described in detail in previous paragraphs, such as the forms of presentation of the medicament, the excipients, therapeutic amounts, forms of administration, etc. and are applicable to the present inventive aspect.
Método de obtención del péptido de la invención Method of obtaining the peptide of the invention
En otro aspecto, la invención se relaciona con un método, de aquí en adelante "Método de la invención", para obtener el péptido de la invención, que comprende: a) Hidrolizar un extracto proteico procedente de una leguminosa con una endoproteasa para obtener un hidrolizado proteico, b) someter el hidrolizado proteico obtenido en la etapa a) a una ultracentrifugación tangencial mediante el empleo de una membrana con un tamaño de poro al menos 1 kDa, c) realizar una cromatografía en fase reversa del permeado obtenido en la ultracentrifugación de la etapa b) y analizar la capacidad de las fracciones recogidas de inhibir la expresión de citoquinas proinflamatorias mediante ensayos celulares, y d) someter a una purificación mediante cromatografía de gel a la fracción obtenida en la etapa c) capaz de disminuir o inhibir la expresión de las citoquinas proinflamatorias con respecto a un control. In another aspect, the invention relates to a method, hereafter "Method of the invention", to obtain the peptide of the invention, comprising: a) Hydrolyzing a protein extract from a legume with an endoprotease to obtain a protein hydrolyzate, b) subject the protein hydrolyzate obtained in step a) to tangential ultracentrifugation by using a membrane with a pore size of at least 1 kDa, c) perform a reverse phase chromatography of the permeate obtained in the ultracentrifugation of stage b ) and analyze the ability of the collected fractions to inhibit the expression of proinflammatory cytokines by cellular assays, and d) subject to purification by gel chromatography to the fraction obtained in step c) capable of decreasing or inhibiting the expression of proinflammatory cytokines Regarding a control.
En una primera etapa [etapa a)] el método de la invención comprende hidrolizar un extracto proteico procedente de una leguminosa con una endoproteasa para obtener un hidrolizado proteico. In a first stage [step a)] the method of the invention comprises hydrolyzing a protein extract from a legume with an endoprotease to obtain a protein hydrolyzate.
Métodos para obtener extractos proteicos a partir de vegetales son ampliamente conocidos en el estado de la técnica y su aplicación es práctica de rutina para el experto en la materia. Una vez obtenido el extracto proteico, se procede a la hidrólisis del mismo mediante una endoproteasa. Cualquiera de las endoproteasas que existen en el estado de la técnica puede usarse para poner en práctica la etapa a) del método de la invención. En una realización particular, las endoproteasas son de amplio espectro e hidrolizan enlaces amídicos dentro de la cadena de la proteína. Ejemplos de endoproteasas incluyen, sin limitar a, serinproteasas, Neutrase®, Flavourzyme® y Protamex®. En una realización particular, la endoproteasa es Alcalase® o subtilisina. Methods for obtaining protein extracts from vegetables are widely known in the state of the art and their application is routine practice for those skilled in the art. Once the protein extract is obtained, hydrolysis is carried out by means of an endoprotease. Any of the endoproteases that exist in the state of the art can be used to practice step a) of the method of the invention. In a particular embodiment, the endoproteases are broad spectrum and hydrolyse amide bonds within the protein chain. Examples of endoproteases include, but are not limited to, serine proteases, Neutrase®, Flavourzyme® and Protamex®. In a particular embodiment, the endoprotease is Alcalase® or subtilisin.
La hidrólisis proteica se realiza normalmente en un reactor, con control de agitación, pH, temperatura y tiempo del proceso. El sustrato se disuelve o resuspende en agua hasta que el pH y la temperatura se estabilizan; a continuación se agrega la endoproteasa dando inicio a la hidrólisis. A medida que ésta progresa se produce una disminución del pH debido a la rotura de los enlaces peptídicos. En los casos de hidrólisis enzimática el pH debe ser mantenido en el óptimo de la enzima mediante la adición de base diluida. Para finalizar la hidrólisis proteica la enzima puede ser inactivada con calor, mediante una disminución del pH o con una combinación de ambos. O también puede ser retirada del medio mediante filtración y la proteína finalmente precipitada. Protein hydrolysis is normally carried out in a reactor, with stirring control, pH, temperature and process time. The substrate is dissolved or resuspended in water until the pH and temperature stabilize; The endoprotease is then added, starting the hydrolysis. As it progresses a decrease in pH occurs due to the breakage of the peptide bonds. In cases of enzymatic hydrolysis the pH should be maintained at the optimum of the enzyme by adding diluted base. To end protein hydrolysis the enzyme can be inactivated with heat, by a decrease in pH or with a combination of both of them. Or it can also be removed from the medium by filtration and finally precipitated protein.
El sustrato de la hidrólisis proteica puede ser cualquier planta leguminosa. Las plantas leguminosas son plantas leñosas o herbáceas con fruto tipo legumbre, capaces de fijar nitrógeno atmosférico por su simbiosis son el género bacteriano Rhizobium. Ejemplos de leguminosas incluyen, sin limitar a, judía (Phaseolus vulgaris), soja (Gíycine max), haba (Vicia faba), ¡enteja (Lens cuiinaris), garbanzo (Cicer aríetinum), guisante (Pisum sativum), algarroba (Ceratonia siiiqua), altramuz (Lupinus aibus), cacahuete (Arachis hypogaea), alfalfa (Medicago sativa), trébol (Trifolium pratense) y veza (Vicia sativa). No obstante, en una realización particular, la leguminosa es una planta de altramuz. The substrate of protein hydrolysis can be any legume plant. Leguminous plants are woody or herbaceous plants with vegetable type fruit, capable of fixing atmospheric nitrogen by their symbiosis are the bacterial genus Rhizobium. Examples of legumes include, but are not limited to, beans (Phaseolus vulgaris), soybeans (Gíycine max), beans (Vicia faba), enteja (Lens cuiinaris), chickpeas (Cicer aríetinum), peas (Pisum sativum), carob (Ceratonia siiiqua ), lupine (Lupinus aibus), peanut (Arachis hypogaea), alfalfa (Medicago sativa), clover (Trifolium pratense) and veza (Vicia sativa). However, in a particular embodiment, the legume is a lupine plant.
Una vez obtenido el hidrolizado proteico (también denominado extracto peptídico), éste es sometido a una ultracentrifugación tangencial mediante el empleo de una membrana con un tamaño de poro de al menos 1 kDa [etapa b) del método de la invención]. Once the protein hydrolyzate (also called peptide extract) is obtained, it is subjected to tangential ultracentrifugation by using a membrane with a pore size of at least 1 kDa [step b) of the method of the invention].
En el contexto de la presente invención, la ultracentrifugación empleada en una ultracentrifugación tangencial. La ultracentrifugación es un procedimiento ampliamente conocido en el estado de la técnica usado para separar los componentes de una suspensión en base al tamaño y la densidad de sus componentes por acción de la fuerza centrífuga. En la presente invención, mediante el empleo de una membrana de tamaño de poro de, al menos, 1 kDa, la ultracentrifugación se usa para filtrar el hidrolizado peptídico y separar los péptidos con un peso molecular mayor de 1 kDa de los péptidos con un peso molecular menor o igual de 1 kDa. Así, tras finalizar la ultracentrifugación tangencial, se obtiene un permeado y un retenido. En el retenido estarán los péptidos con una masa molecular mayor de 1 kDa, y en el permeado estarán los péptidos con un peso molecular menor o igual de 1 kDa. No obstante, en el contexto de la presente invención, pueden emplearse membranas con otros tamaños de poro, por ejemplo, al menos 3, al menos 5, al menos 10, al menos 30, al menos 50 o al menos 100 kDa. En una realización particular, la membrana empleada en el método de la invención es de, al menos, 10 kDa. A continuación, se realiza una cromatografía en fase reversa del permeado y se analiza la expresión de la citoquinas proinflamatorias en las fracciones recogidas [etapa c) del método de la invención]. La cromatografía en fase reversa (Reverse Phase Cromatography o RPC) es una técnica que permite separar moléculas en base a su polaridad, en la que la fase estacionaria es de partículas de sílica químicamente modificadas con hidrocarburos saturados, insaturados o aromáticos de diferentes tipos. Esto convierte a la fase estacionaria en una matriz apolar. Por lo tanto, para este tipo de cromatografías se emplean mezclas de solventes polares, tales como agua, acetonitrilo, acetato de etilo, acetona, alcoholes alifáticos, etc. En la separación de aminoácidos y péptidos mediante fase reversa la resina más comúnmente empleada posee una cadena lineal de 18 carbonos y se denomina como Ci8 [|SiO?|-(CH?)i7-CHs1. La muestra (en esta invención el permeado) se aplica a la columna en solución acuosa acidificada, por ejemplo, con ácido trifluoroacético, un ácido orgánico volátil y que se requiere muy puro. A fin de eluír de la columna los aminoácidos más apolares es necesario aumentar la concentración de acetonitrilo en el sistema. In the context of the present invention, the ultracentrifugation employed in a tangential ultracentrifugation. Ultracentrifugation is a widely known process in the state of the art used to separate the components of a suspension based on the size and density of its components by centrifugal force. In the present invention, by using a pore size membrane of at least 1 kDa, ultracentrifugation is used to filter the peptide hydrolyzate and separate the peptides with a molecular weight greater than 1 kDa from the peptides with a weight molecular less than or equal to 1 kDa. Thus, after completing the tangential ultracentrifugation, a permeate and a retained are obtained. In the retentate there will be peptides with a molecular mass greater than 1 kDa, and in the permeate there will be peptides with a molecular weight less than or equal to 1 kDa. However, in the context of the present invention, membranes with other pore sizes can be employed, for example, at least 3, at least 5, at least 10, at least 30, at least 50 or at least 100 kDa. In a particular embodiment, the membrane employed in the method of the invention is at least 10 kDa. Next, a reverse phase permeate chromatography is performed and the expression of proinflammatory cytokines in the fractions collected [step c) of the method of the invention] is analyzed. Reverse Phase Chromatography (RPC) is a technique that allows molecules to be separated based on their polarity, in which the stationary phase is chemically modified silica particles with saturated, unsaturated or aromatic hydrocarbons of different types. This makes the stationary phase an apolar matrix. Therefore, mixtures of polar solvents, such as water, acetonitrile, ethyl acetate, acetone, aliphatic alcohols, etc., are used for this type of chromatography. In the separation of amino acids and peptides by reverse phase the most commonly used resin has a linear chain of 18 carbons and is called Ci 8 [| SiO? | - (CH ? ) And 7 -CHs1. The sample (in this invention the permeate) is applied to the column in acidified aqueous solution, for example, with trifluoroacetic acid, a volatile organic acid that is very pure. In order to elute the most apolar amino acids from the column, it is necessary to increase the concentration of acetonitrile in the system.
Una vez eluída la columna y recogidas las distintas fracciones, se procede a analizar la capacidad de las fracciones recogidas para inducir la expresión de citoquinas proinflamatorias mediante ensayos celulares. Ejemplos de citoquinas inflamatorias incluyen, sin limitar a, TNF, I L- 1 β , IL6, CCR2, y CCL2 . Ensayos sobre como analizar la expresión de citoquinas proinflamatorias, incluyen, pero no se limitan a, aislamiento de ARN y cuantificacion mediante PCR cuantitativa de las citoquinas producidas por las células, determinación de la producción de citoquinas producidas por las células mediante ELISA, etc. así como los ensayos descritos en los ejemplos que ilustran la invención. Ensayos y técnicas sobre como analizar la expresión de citoquinas proinflamatorias son ampliamente conocidas en el estado de técnica y su empleo es práctica de rutina para el experto en la materia. Once the column is eluted and the different fractions collected, the capacity of the collected fractions to induce the expression of proinflammatory cytokines is analyzed by cellular assays. Examples of inflammatory cytokines include, but are not limited to, TNF, I L-1β, IL6, CCR2, and CCL2. Trials on how to analyze the expression of proinflammatory cytokines, include, but are not limited to, RNA isolation and quantification by quantitative PCR of the cytokines produced by the cells, determination of the production of cytokines produced by the cells by ELISA, etc. as well as the tests described in the examples illustrating the invention. Tests and techniques on how to analyze the expression of pro-inflammatory cytokines are widely known in the state of the art and their use is routine practice for the person skilled in the art.
Finalmente, en la etapa d) del método de la invención, se somete a una purificación mediante cromatografía de gel a la fracción obtenida en la etapa c) capaz de disminuir o inhibir la expresión de las citoquinas proinflamatorias con respecto a un control. La cromatografía de gel es una técnica empleada para separar moléculas en función de su tamaño. La capacidad separadora reside fundamentalmente en el gel, cuya matriz consta de un gran número de esferas porosas microscópicas. Estas esferas están constituidas por largas cadenas de polímeros unidas entre sí por enlaces químicos para formar una red tridimensional. Los geles normalmente utilizados para la separación de proteínas y péptidos son de tres tipos: dextrano, agarosa y poliacrilamida. La muestra (en esta invención la fracción seleccionada tras la cromatografía de fase reversa) se aplica a la columna en solución acuosa. La elución se realiza de modo ¡socrático con una solución acuosa que puede incluir, pero no se limita a, ácido fórmico, ácido clorhídrico, acetato amónico, tampón fosfato salino, bicarbonato amónico, ácido tricloroacético. Una vez eluída la columna y recogidas las distintas fracciones, se procede a analizar la capacidad de las mismas de inducir la expresión de citoquinas proinflamatorias mediante los ensayos celulares descritos anteriormente. Ejemplos de citoquinas proinflamatorias incluyen, sin limitar a, TNF, IL- 1 β, IL6, CCR2, y CCL2. Finally, in step d) of the method of the invention, the fraction obtained in step c) capable of decreasing or inhibiting the expression of pro-inflammatory cytokines with respect to a control is subjected to purification by gel chromatography. Gel chromatography is a technique used to separate molecules based on their size. The separating capacity lies primarily in the gel, whose matrix consists of a large number of microscopic porous spheres. These spheres consist of long chains of polymers joined together by chemical bonds to form a three-dimensional network. The gels normally used for the separation of proteins and peptides are of three types: dextran, agarose and polyacrylamide. The sample (in this invention the fraction selected after reverse phase chromatography) is applied to the column in aqueous solution. Elution is performed in a socratic manner with an aqueous solution that may include, but is not limited to, formic acid, hydrochloric acid, ammonium acetate, phosphate buffered saline, ammonium bicarbonate, trichloroacetic acid. Once the column has been eluted and the different fractions have been collected, their ability to induce proinflammatory cytokine expression is analyzed using the cell assays described above. Examples of proinflammatory cytokines include, but are not limited to, TNF, IL-1β, IL6, CCR2, and CCL2.
En la presente invención, se entiende por "control" al nivel de expresión de citoquinas proinflamatorias obtenido en un ensayo celular al que no se le han añadido las fracciones recogidas tras la cromatografía en fase reversa de la etapa c). In the present invention, "control" is understood as the level of proinflammatory cytokine expression obtained in a cellular assay to which the fractions collected after the reverse phase chromatography of step c) have not been added.
La fracción que sea capaz de inhibir o disminuir la expresión de citoquinas proinflamatorias con respecto a un control, corresponderá al péptido de la invención. Técnicas sobre como aislar los péptidos de una fracción son ampliamente conocidos en el estado de la técnica y puede emplearse cualquier de ellos. The fraction that is capable of inhibiting or decreasing the expression of proinflammatory cytokines with respect to a control will correspond to the peptide of the invention. Techniques on how to isolate peptides from a fraction are widely known in the state of the art and any of them can be used.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y figuras se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. BREVE DESCRIPCIÓN DE LAS FIGURAS Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention. BRIEF DESCRIPTION OF THE FIGURES
Figura 1. Expresión ARNm de TNF (A), IL-1 β (B), IL-6 (C), CCR2 (D), CCL2 (E), y CCL18 (F) en macrófagos derivados de THP-1 después de 6 horas de tratamiento con PM 1 (>10 kDa) y PM2 (<10 kDa). Los valores marcados con letras distintas son significativamente diferentes (P<0,05). Figure 1. mRNA expression of TNF (A), IL-1β (B), IL-6 (C), CCR2 (D), CCL2 (E), and CCL18 (F) in THP-1 derived macrophages after 6 hours of treatment with PM 1 (> 10 kDa) and PM2 (<10 kDa). Values marked with different letters are significantly different (P <0.05).
Figura 2. Perfil de elución de la fracción PM2 (<10 kDa) usando cromatografía de fase reversa por FPLC. Se recogieron 17 fracciones y se midió la expresión del ARNm de TNF e I L-1 β en macrófagos derivados de THP-1. Figure 2. Elution profile of the PM2 fraction (<10 kDa) using reverse phase chromatography by FPLC. 17 fractions were collected and the expression of TNF and I L-1β mRNA in macrophages derived from THP-1 was measured.
Figura 3. Expresión ARNm de IL-6 (A), CCR2 (B), CCL2 (C), y CCL18 (D) en macrófagos derivados de THP-1 después de 6 h de tratamiento con F3 y F7 obtenidas por RPC. Los valores marcados con letras distintas son significativamente diferentes (P<0,05). Figure 3. mRNA expression of IL-6 (A), CCR2 (B), CCL2 (C), and CCL18 (D) in THP-1 derived macrophages after 6 h of treatment with F3 and F7 obtained by RPC. Values marked with different letters are significantly different (P <0.05).
Figura 4. Perfil de elución de F3 obtenida en una columna de filtración en gel Superdex peptide 10/300. Figura 5. Expresión ARNm de IL-6 (A), CCR2 (B), y CCL2 (C), en macrófagos derivados de THP-1 después de 6 h de tratamiento con F3-5 obtenida en una columna de filtración en gel Superdex peptide 10/300. Los valores marcados con letras distintas son significativamente diferentes (P<0,05). Figura 6. Expresión del ARNm de CCL18 (A) e IL-10 (B) en macrófagos derivados de THP-1 después de 6 h de tratamiento con la fracción F3-5. Los valores marcados con letras distintas son significativamente diferentes (P<0,05). Figure 4. Elution profile of F3 obtained in a Superdex peptide 10/300 gel filtration column. Figure 5. mRNA expression of IL-6 (A), CCR2 (B), and CCL2 (C), in THP-1 derived macrophages after 6 h of treatment with F3-5 obtained in a Superdex gel filtration column 10/300 peptide. Values marked with different letters are significantly different (P <0.05). Figure 6. Expression of the mRNA of CCL18 (A) and IL-10 (B) in macrophages derived from THP-1 after 6 h of treatment with fraction F3-5. Values marked with different letters are significantly different (P <0.05).
Figura 7. Expresión del ARNm de TNF (A), I L-1 β (B), e IL-6 (C) en macrófagos derivados de THP-1 después de 6 h de tratamiento con el péptido sintetizado a 100 μg/mL (P1) y a 500 μg/mL (P2). Los valores marcados con letras distintas son significativamente diferentes (P<0,05). Figure 7. Expression of the mRNA of TNF (A), I L-1β (B), and IL-6 (C) in THP-1 derived macrophages after 6 h of treatment with the peptide synthesized at 100 μg / mL (P1) and at 500 μg / mL (P2). Values marked with different letters are significantly different (P <0.05).
Figura 8. Expresión del ARNm de CCL2 (A) y CCR2 (B) en macrófagos derivados de THP-1 después de 6 h de tratamiento con el péptido a la concentración de 100 μg/mL (P1) y de 500 μς/Γηί. (Ρ2). Los valores marcados con letras distintas son significativamente diferentes (P<0,05). Figure 8. Expression of the mRNA of CCL2 (A) and CCR2 (B) in THP-1 derived macrophages after 6 h of treatment with the peptide at the concentration of 100 μg / mL (P1) and 500 μς / Γηί. (Ρ2). Values marked with different letters are significantly different (P <0.05).
Figura 9. Expresión del ARNm de CCL18 (A) e IL-10 (B) en macrófagos derivados de THP-1 después de 6 h de tratamiento con el péptido a la concentración de 100 μg/mL (P1) y de 500 μg/mL (P2). Los valores marcados con letras distintas son significativamente diferentes (P<0,05). Figure 9. Expression of the mRNA of CCL18 (A) and IL-10 (B) in THP-1 derived macrophages after 6 h of treatment with the peptide at the concentration of 100 μg / mL (P1) and 500 μg / mL (P2). Values marked with different letters are significantly different (P <0.05).
Figura 10. Efecto sobre la producción de TNF (A), IL-1 β (B), e IL-10 (C) de los macrófagos derivados de células THP-1 después de 6 horas (1) y 48 horas (2) de tratamiento con dos concentraciones del péptido sintetizado, P1 (100 μg/mL) y P2 (500 μg/mL). Los valores marcados con letras distintas son significativamente diferentes (P<0,05). Figura 11. Porcentaje de producción de óxido nítrico (NO) en el sobrenadante de los macrófagos derivados de THP-1 después de 48 horas de tratamiento con el péptido a 100 μg/mL (P1) y 500 μg/mL (P2). Figure 10. Effect on the production of TNF (A), IL-1β (B), and IL-10 (C) of macrophages derived from THP-1 cells after 6 hours (1) and 48 hours (2) of treatment with two concentrations of the synthesized peptide, P1 (100 μg / mL) and P2 (500 μg / mL). Values marked with different letters are significantly different (P <0.05). Figure 11. Percentage production of nitric oxide (NO) in the supernatant of macrophages derived from THP-1 after 48 hours of treatment with the peptide at 100 μg / mL (P1) and 500 μg / mL (P2).
EJEMPLOS EXAMPLES
A continuación se ilustrará la invención mediante unos ensayos realizados por los inventores, que pone de manifiesto la efectividad del producto de la invención. The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.
Ejemplo 1 - Aislamiento del péptido de la invención. Example 1 - Isolation of the peptide of the invention.
Purificación de péptidos por ultrafiltración Purification of peptides by ultrafiltration
Procesos de ultrafiltración se han utilizado para purificar péptidos bioactivos de hidrolizados de proteínas (Vo, T.S., Ryu, B. M., & Kim, S.-K. (2013). Purification of novel anti-inflammatory peptides from enzymatic hydrolysate of the edible microalgal Spirulina máxima. Journal of Functional Foods, 5, 1336-1346). LPH (Lupin Protein Hydrosylates) obtenidos mediante hidrólisis enzimática de aislado proteico de altramuz con Alcalase®, en un reactor con control de pH y temperatura, empleándose las siguientes condiciones: pH 8, 50° C, E/S=03 UA/g proteína, 15 minutos. La enzima fue inactivada por calentamiento a 85° C durante 15 minutos, y la mezcla fue centrifugada a 8500 rpm durante 20 minutos. El sobrenadante de centrifugación fue utilizado como substrato para la purificación de péptidos antiinflamatorios mediante ultrafiltración tangencial con una membrana con un tamaño de corte de peso molecular de 10 kDa. El análisis de rendimiento de recuperación proteica en el retenido (PM1) y permeado (PM2) así como el contenido proteínico de las fracciones obtenidas por ultrafiltración fueron evaluados, siendo PM2 el que presentó la mayor actividad (Tabla 1). Ultrafiltration processes have been used to purify bioactive peptides from protein hydrolysates (Vo, TS, Ryu, BM, & Kim, S.-K. (2013). Purification of novel anti-inflammatory peptides from enzymatic hydrolysate of the edible microalgal Spirulina maximum Journal of Functional Foods, 5, 1336-1346). LPH (Lupine Protein Hydrosylates) obtained by enzymatic hydrolysis of lupine protein isolate with Alcalase®, in a reactor with pH and temperature control, using the following conditions: pH 8, 50 ° C, I / O = 03 UA / g protein , 15 minutes. The enzyme was inactivated by heating at 85 ° C for 15 minutes, and the mixture was centrifuged at 8500 rpm for 20 minutes. The centrifugal supernatant was used as substrate for the purification of anti-inflammatory peptides by tangential ultrafiltration with a membrane with a molecular weight cut-off size of 10 kDa. The protein recovery performance analysis in the retained (PM1) and permeate (PM2) as well as the protein content of the fractions obtained by ultrafiltration were evaluated, with PM2 being the one with the highest activity (Table 1).
Tabla 1. Valores de rendimiento en peso y riqueza proteica de las fracciones del hidrolizado proteico de altramuz (HPA) obtenidas por ultrafiltración. Table 1. Performance values by weight and protein richness of fractions of lupine protein hydrolyzate (HPA) obtained by ultrafiltration.
Figure imgf000024_0001
Por otra parte, la expresión de TNF, IL-1 β, IL6, CCR2, CCL2 (citoquinas proinflamatorias) y CCL18 (citoquina antiinflamatoria), fue evaluada mediante PCR cuantitativa, en relación con el control sin tratamiento. La expresión de TNF (Figura 1A) y CCL2 (Figura 1 E) fue reducida significativamente por las dos fracciones obtenidas por ultrafiltración, mientras que el resto de citoquinas proinflamatorias, excepto CCR2, también fueron inhibidos aunque no se observaron diferencias significativas. Por otro lado, ambas fracciones parecen inducir la expresión de CCL18 pero esta inducción no fue significativa a tenor de los estudios estadísticos realizados. Estos resultados junto con el mayor rendimiento en peso y el contenido proteico obtenidos en la fracción PM2 (84,28% y 81 ,96%, respectivamente) y el hecho de que péptidos con una masa molecular relativamente baja presenten una mayor absorción intestinal y biodisponibilidad y que por consiguiente, puedan mantener su bioactividad hasta los objetivos específicos, condujo a elegir la fracción PM2 (<10 kDa) para el siguiente paso de purificación. Purificación por cromatografía de fase reversa a partir de PM2 de altramuz.
Figure imgf000024_0001
On the other hand, the expression of TNF, IL-1β, IL6, CCR2, CCL2 (pro-inflammatory cytokines) and CCL18 (anti-inflammatory cytokine), was evaluated by quantitative PCR, in relation to the control without treatment. The expression of TNF (Figure 1A) and CCL2 (Figure 1 E) was significantly reduced by the two fractions obtained by ultrafiltration, while the rest of proinflammatory cytokines, except CCR2, were also inhibited although no significant differences were observed. On the other hand, both fractions appear to induce the expression of CCL18 but this induction was not significant according to the statistical studies performed. These results together with the greater yield in weight and the protein content obtained in the PM2 fraction (84.28% and 81.96%, respectively) and the fact that peptides with a relatively low molecular mass have a greater intestinal absorption and bioavailability and that therefore, they can maintain their bioactivity up to the specific objectives, led to choosing the PM2 fraction (<10 kDa) for the next purification step. Purification by reverse phase chromatography from lupine PM2.
PM2 fue utilizado para posterior purificación de péptidos antiinflamatorios por cromatografía de fase reversa empleándose una columna Resource 15RPC 3 ml_, donde diecisiete fracciones (1-17) fueron recolectadas, evaluándose los valores de rendimiento de recuperación en relación a la recuperación total realizada en el anterior paso de purificación (Tabla 2). La expresión de TNF y de I L- β, ambas citoquinas proinflamatorias, fue evaluada por PCR cuantitativa tras incubación en macrófagos THP-1 durante 6 horas, siendo positivo (+) en los casos en que la expresión de citoquinas fuera reprimida y negativo (-) para el caso que fuera inducida o similar a la muestra control (Figura 2). El gráfico muestra que sólo las fracciones F3 y F7 redujeron la expresión de ambas citoquinas. Posteriormente, la expresión de las citoquinas IL6, CCR2, CCL2 y CCL18 fue evaluada para comprobar cuál de las dos fracciones presentaba mayor potencial antiinflamatorio. El análisis de la expresión génica mostró una reducción significativa de la expresión de CCR2 para F3 y F7, citoquina que no había sido reprimida por ninguna de las fracciones obtenidas por ultrafiltración (Figura 3B). La expresión de su ligando, CCL2 fue también reducida de manera significativa por ambas fracciones, observándose una mayor represión del gen por F7 (Figuras 3C). Además, la expresión de IL6 fue reducida por F3 y F7, no encontrándose diferencias significativas entre ambas fracciones (Figura 3A). Por otro lado, la expresión del CCL18 fue inducida aumentando los valores frente al control (Figura 3D) por las dos fracciones. Así, debido a los resultados de represión de la expresión de citoquinas y la evaluación del rendimiento en peso, la fracción F3, obtenida tras purificación por RPC, fue seleccionada por tener un rendimiento proteico casi dos veces mayor que el obtenido con la fracción F7 (Tabla 2). Tabla 2. Valores de rendimiento en peso después de la purificación por cromatografía de fase reversa por FPLC. PM2 was used for subsequent purification of anti-inflammatory peptides by reverse phase chromatography using a Resource 15RPC 3 ml_ column, where seventeen fractions (1-17) were collected, evaluating recovery performance values in relation to the total recovery performed in the previous purification step (Table 2). The expression of TNF and I L- β, both cytokines proinflammatory, was evaluated by quantitative PCR after incubation in THP-1 macrophages for 6 hours, being positive (+) in cases where the expression of cytokines was repressed and negative (-) for the case that was induced or similar to the sample control (Figure 2). The graph shows that only fractions F3 and F7 reduced the expression of both cytokines. Subsequently, the expression of the cytokines IL6, CCR2, CCL2 and CCL18 was evaluated to check which of the two fractions had the greatest anti-inflammatory potential. The analysis of gene expression showed a significant reduction in the expression of CCR2 for F3 and F7, cytokine that had not been repressed by any of the fractions obtained by ultrafiltration (Figure 3B). The expression of its ligand, CCL2 was also significantly reduced by both fractions, with a greater repression of the gene by F7 (Figures 3C). In addition, the expression of IL6 was reduced by F3 and F7, with no significant differences found between both fractions (Figure 3A). On the other hand, the expression of CCL18 was induced by increasing the values against the control (Figure 3D) by the two fractions. Thus, due to the repression results of the cytokine expression and the evaluation of the weight performance, the F3 fraction, obtained after RPC purification, was selected as having a protein yield almost twice as high as that obtained with the F7 fraction ( Table 2). Table 2. Performance values by weight after purification by reverse phase chromatography by FPLC.
Fracción Rendimiento en peso (%)  Fraction Yield by weight (%)
Fl 7,13  Fl 7.13
F2 1,89  F2 1.89
F3 10,04  F3 10.04
F4 15,42  F4 15.42
F5 16,45  F5 16.45
F6 10,04  F6 10.04
F7 5,38  F7 5.38
F8 2,47  F8 2.47
F9 4,80  F9 4.80
FIO 6,98  FIO 6.98
Fll 2,76 F12 6,11 Fll 2,76 F12 6.11
F13 2,33  F13 2.33
F14 2,33  F14 2.33
F15 1,60  F15 1.60
F16 1,75  F16 1.75
F17 2,32  F17 2.32
Purificación mediante cromatografía de gel filtración de la fracción F3 de altramuz.Purification by gel chromatography filtration of the F3 fraction of lupine.
La cromatografía de filtración en gel se ha utilizado en numerosos estudios para la purificación e identificación de péptidos bioactivos de hidrolizados de proteínas de fuentes tales como soja (Hyun, J.K., In Young, B., Chang-Won, A., Suyong, L, & Hyeon, G.L. (2007). Peptides, 28, 2098-2103). La fracción F3 fue purificada en una columna de Superdex-peptide con un rango óptimo de separación entre 7- 0.1 kDa. El volumen eluído se dividió en cinco fracciones nombradas como F3-1 , F3-2, F3-3, F3-4 y F3-5 (Figura 4). Cada fracción se liofilizó, evaluándose posteriormente su actividad antiinflamatoria. La expresión de TNF y I L-1 β fue evaluada por PCR cuantitativa, siendo positivo (+) en los casos en que la expresión de citoquinas fuera reprimida y negativo (-) para el caso que fuera inducida o similar a la muestra control (Figura 4). La fracción F3-5 mostró un resultado positivo para la expresión de TNF, por lo tanto esta fracción fue elegida para el resto del análisis. No se observaron diferencias significativas en el caso de la expresión de IL6 (Figura 5A), sin embargo, se observó una reducción drástica en la expresión CCR2 y CCL2 por la fracción F3-5 (-59,54%) y (-74,45%), respectivamente (Figura 5B y 5C). Estos resultados indican que F3-5 tiende a disminuir la capacidad proinflamatoria de los macrófagos activados tipo Th1 disminuyendo la expresión de citoquinas implicadas en procesos proinflamatorios. Gel filtration chromatography has been used in numerous studies for the purification and identification of bioactive peptides from protein hydrolysates from sources such as soybean (Hyun, JK, In Young, B., Chang-Won, A., Suyong, L , & Hyeon, GL (2007). Peptides, 28, 2098-2103). The F3 fraction was purified on a Superdex-peptide column with an optimum separation range between 7- 0.1 kDa. The eluted volume was divided into five fractions named F3-1, F3-2, F3-3, F3-4 and F3-5 (Figure 4). Each fraction was lyophilized, subsequently evaluating its anti-inflammatory activity. The expression of TNF and I L-1β was evaluated by quantitative PCR, being positive (+) in cases where the expression of cytokines was repressed and negative (-) in the case that it was induced or similar to the control sample ( Figure 4). The F3-5 fraction showed a positive result for TNF expression, therefore this fraction was chosen for the rest of the analysis. No significant differences were observed in the case of IL6 expression (Figure 5A), however, a drastic reduction in CCR2 and CCL2 expression was observed for the F3-5 fraction (-59.54%) and (-74, 45%), respectively (Figure 5B and 5C). These results indicate that F3-5 tends to decrease the proinflammatory capacity of activated Th1 type macrophages by decreasing the expression of cytokines involved in proinflammatory processes.
Con el objetivo de corroborar esta afirmación, se evaluó la capacidad de F3-5 para modular los niveles de CCL18 e interleuquina-10 (IL-10). Ambas citoquinas antiinflamatorias juegan un papel crucial en la prevención de las patologías inflamatorias y autoinmunes. Tanto los niveles de expresión de CCL18 como de IL-10 fueron inducidos de forma significativa con respecto al control sin tratamiento por F3-5 (Figura 6) confirmándose la hipótesis de que la fracción F3-5 presenta actividad antiinflamatoria. Posteriormente, en la fracción F3-5 fue identificado el péptido Gly-Pro-Glu-Thr-Ala- Phe-Leu-Arg por espectrometría de masas (PM: 889.60 Da) y sintetizado para continuar los estudios en la producción y la expresión génica de citoquinas por ELISA y PCR, respectivamente. Tras la comparación de esta secuencia de aminoácidos con las bases de datos del estado de la técnica, se vio que formaba parte de la familia de las leguminosas. In order to corroborate this statement, the ability of F3-5 to modulate the levels of CCL18 and interleukin-10 (IL-10) was evaluated. Both anti-inflammatory cytokines play a crucial role in the prevention of inflammatory and autoimmune pathologies. Both the expression levels of CCL18 and IL-10 were significantly induced with respect to the control without treatment by F3-5 (Figure 6) confirming the hypothesis that fraction F3-5 exhibits anti-inflammatory activity. Subsequently, in the F3-5 fraction, the Gly-Pro-Glu-Thr-Ala-Phe-Leu-Arg peptide was identified by mass spectrometry (PM: 889.60 Da) and synthesized to continue studies in gene production and expression of cytokines by ELISA and PCR, respectively. After comparing this amino acid sequence with the prior art databases, it was found that it was part of the legume family.
Ejemplo 2 - Actividad antiinflamatoria del péptido de la invención. Una vez sintetizado el péptido, los macrófagos derivados de THP-1 fueron tratados con el mismo a dos concentraciones diferentes. A la concentración menor de 100 μg/mL se le nombró como P1 y a la más elevada de 500 μg/mL como P2. Example 2 - Anti-inflammatory activity of the peptide of the invention. Once the peptide was synthesized, macrophages derived from THP-1 were treated with the same at two different concentrations. The concentration of less than 100 μg / mL was named as P1 and the highest concentration of 500 μg / mL as P2.
Como se puede observar en la Figura 7 la expresión de TNF (Figura 7A) e I L- β (Figura 7B) se reprimió significativamente con respecto al control, y no se observaron diferencias entre los tratamientos. Por otro lado P1 parecía inducir la expresión de la IL-6 mientras que P2 parecía reducir dicha expresión. De todos modos, con ninguno de los dos tratamientos se obtuvieron diferencias significativas con respecto a la expresión control de la IL-6. Este primer screening nos mostraba una vez más que el péptido, anteriormente purificado a partir del hidrolizado y ahora, sintetizado a partir de su secuencia, mostraba una potencial actividad antiinflamatoria por reducir la expresión de las citoquinas proinflamatorias TNF e I L-1 β a las concentraciones anteriormente usadas e incluso a una dosis 5 veces inferior a la usada en los ensayos anteriores. As can be seen in Figure 7, the expression of TNF (Figure 7A) and I L-β (Figure 7B) was significantly suppressed with respect to the control, and no differences were observed between treatments. On the other hand P1 seemed to induce the expression of IL-6 while P2 seemed to reduce said expression. In any case, no significant differences were obtained with respect to the control expression of IL-6 with either treatment. This first screening showed us once again that the peptide, previously purified from the hydrolyzate and now, synthesized from its sequence, showed a potential anti-inflammatory activity by reducing the expression of TNF and I L-1 β proinflammatory cytokines at previously used concentrations and even at a dose 5 times lower than that used in the previous tests.
En cuanto al efecto del péptido sobre la capacidad quimiotáctica de los macrófagos derivados de THP-1 humanos se observó que contribuyó a una reducción significativa de la expresión del ligando CCL2 (Figura 8A). Su receptor CCR2, sin embargo, sólo mostró diferencias con respecto al control después del tratamiento con P1 (Figura 8B). Regarding the effect of the peptide on the chemotactic capacity of human THP-1 derived macrophages, it was observed that it contributed to a significant reduction in CCL2 ligand expression (Figure 8A). Its CCR2 receptor, however, only showed differences with respect to the control after treatment with P1 (Figure 8B).
Por otro lado, el análisis de la expresión de las citoquinas antiinflamatorias (Figura 9A y 9B) mostró una pequeña diferencia con la fracción F3-5, ya que para ésta, se obtuvieron valores de expresión de CCL18 mucho más altos que el control y en este caso, con el péptido a cualquiera de las concentraciones usadas, no se observaron diferencias significativas con respecto al control (Figura 9A). Esto no fue así para la expresión de la IL-10 que fue inducida de manera significativa por el péptido a ambas concentraciones (Figura 9B). Estas pequeñas diferencias sobre la expresión de las citoquinas pro y antiinflamatorias entre la fracción F3-5 y el péptido sintetizado pueden deberse a la interacción de las células con alguna secuencia aminoacídica minoritaria diferente a la sintetizada presente en la fracción seleccionada. On the other hand, the analysis of the expression of the anti-inflammatory cytokines (Figure 9A and 9B) showed a small difference with the F3-5 fraction, since for this, CCL18 expression values were obtained much higher than the control and in In this case, with the peptide at any of the concentrations used, no significant differences were observed with respect to the control (Figure 9A). This was not the case for the IL-10 expression that was significantly induced by the peptide at both concentrations (Figure 9B). These small differences on the expression of pro and anti-inflammatory cytokines between the F3-5 fraction and the synthesized peptide may be due to the interaction of the cells with some minor amino acid sequence different from the one synthesized present in the selected fraction.
Para demostrar el efecto antiinflamatorio del péptido se determinó la concentración de TNF, I L-1 β e IL-10 en los sobrenadantes de los cultivos celulares después de los tratamientos a las dos concentraciones anteriormente descritas y a distintas horas (6 y 48 horas) para observar las posibles fluctuaciones en los niveles de proteínas ya que cada una de ellas presenta diferentes tasas de renovación. La producción de TNF y de I L-1 β fue disminuida después de 6 horas por el péptido a la concentración más baja o P1 (100 μg/mL) de forma significativa con respecto al control, siendo además, diferente a la obtenida con el péptido a la concentración mayor [Figura 10A(1) y 10B(2)]. Estos datos coinciden en parte con los obtenidos por PCR cuantitativa, en los que la adición de ambas concentraciones del péptido al medio de reacción provocó una reducción significativa de la expresión de ambas citoquinas proinflamatorias. Para la IL-10, hay que esperar a las 48 horas para observar el aumento en la producción de la citoquina [Figura 10C(2)], siendo significativamente diferente con respecto al control en el caso de P1. Por lo tanto, es la dosis más baja del péptido o P1 (100 μg/mL) la que actúa tanto a nivel transcripcional como postranscripcional, ejerciendo un efecto antiinflamatorio a corto plazo (6 horas) mediante la inhibición de la secreción de TNF e I L-1 β y a más largo plazo (48 horas) por su capacidad de inducir la secreción de la citoquina antiinflamatoria IL-10. To demonstrate the anti-inflammatory effect of the peptide, the concentration of TNF, I L-1β and IL-10 in the cell culture supernatants was determined after the treatments at the two concentrations described above and at different hours (6 and 48 hours) for Observe the possible fluctuations in protein levels since each one presents different renewal rates. The production of TNF and I L-1β was decreased after 6 hours by the peptide at the lowest concentration or P1 (100 μg / mL) significantly with respect to the control, being also different from that obtained with the peptide at the highest concentration [Figure 10A (1) and 10B (2)]. These data coincide in part with those obtained by quantitative PCR, in which the addition of both concentrations of the peptide to the reaction medium caused a significant reduction in the expression of both proinflammatory cytokines. For IL-10, wait 48 hours to observe the increase in cytokine production [Figure 10C (2)], being significantly different from the control in the case of P1. Therefore, it is the lowest dose of the peptide or P1 (100 μg / mL) that acts both transcriptionally and post-transcriptionally, exerting an anti-inflammatory effect in the short term (6 hours) by inhibiting the secretion of TNF and I L-1 β and longer term (48 hours) for its ability to induce the secretion of the anti-inflammatory cytokine IL-10.
Por último, y para corroborar una vez más que el péptido sintetizado presenta un potencial antiinflamatorio, se evaluó el efecto del péptido a diferentes concentraciones (P1 y P2) sobre la liberación del NO (Figura 1 1). La reducción significativa del porcentaje de NO producido por los macrófagos después de la exposición durante 48 horas con el péptido a 100 μg/mL y 500 μg/mL, demuestra la actividad antiinflamatoria del péptido sintetizado por su capacidad de inhibir este mediador inflamatorio. Finalmente, podemos establecer que el péptido identificado como Gly-Pro-Glu-Thr-Ala- Phe-Leu-Arg presenta actividad antiinflamatoria in vitro a través del estudio de la expresión de diversas citoquinas por PCR cuantitativa, y el análisis de los niveles de estas proteínas por ELISA y NO involucrados en procesos inflamatorios. Finally, and to confirm once again that the synthesized peptide has an anti-inflammatory potential, the effect of the peptide at different concentrations (P1 and P2) on NO release was evaluated (Figure 1 1). The significant reduction in the percentage of NO produced by macrophages after exposure for 48 hours with the peptide at 100 μg / mL and 500 μg / mL demonstrates the anti-inflammatory activity of the peptide synthesized by its ability to inhibit this inflammatory mediator. Finally, we can establish that the peptide identified as Gly-Pro-Glu-Thr-Ala-Phe-Leu-Arg presents anti-inflammatory activity in vitro through the study of the expression of various cytokines by quantitative PCR, and the analysis of the levels of these proteins by ELISA and NOT involved in inflammatory processes.

Claims

REIVINDICACIONES
1. Un péptido que comprende la secuencia de aminoácidos SEQ ID NO: 1 , o una variante del mismo con una identidad del 75, 80, 85, 90, 95, 96, 97, 98 o 99% con la SEQ ID NO: 1 , en el que dicho péptido no comprende la secuencia de aminoácidos que consiste en la secuencia SEQ ID NO: 2. 1. A peptide comprising the amino acid sequence SEQ ID NO: 1, or a variant thereof with an identity of 75, 80, 85, 90, 95, 96, 97, 98 or 99% with SEQ ID NO: 1 , wherein said peptide does not comprise the amino acid sequence consisting of the sequence SEQ ID NO: 2.
2. Péptido según la reivindicación 1 , caracterizado porque comprende unido a uno de sus extremos un marcador. 2. Peptide according to claim 1, characterized in that it comprises a marker attached to one of its ends.
3. Péptido según la reivindicación 2, en el que el marcador se selecciona del grupo que consiste en c-myc, FLAG, HA, cadena de histidinas, GST, biotina, VSV-G, HSVtk, V5, biotina, avidina, estreptavidina, proteína de unión a maltosa y una proteína fluorescente. 3. Peptide according to claim 2, wherein the marker is selected from the group consisting of c-myc, FLAG, HA, histidine chain, GST, biotin, VSV-G, HSVtk, V5, biotin, avidin, streptavidin, maltose binding protein and a fluorescent protein.
4. Péptido según cualquiera de las reivindicaciones 1 a 3, caracterizado por que comprende unido a uno de sus extremos un péptido señal. 4. Peptide according to any one of claims 1 to 3, characterized in that it comprises a signal peptide attached to one of its ends.
5. Una secuencia de nucleótidos que codifica un péptido según cualquiera de las reivindicaciones 1 a 4. 5. A nucleotide sequence encoding a peptide according to any one of claims 1 to 4.
6. Un vector que comprende una secuencia de nucleótidos según la reivindicación 5. 6. A vector comprising a nucleotide sequence according to claim 5.
7. Una célula que comprende una secuencia de nucleótidos según la reivindicación 5, o un vector según la reivindicación 6. 7. A cell comprising a nucleotide sequence according to claim 5, or a vector according to claim 6.
8. Un extracto peptídico que comprende un péptido según cualquiera de las reivindicaciones 1 a 4. 8. A peptide extract comprising a peptide according to any one of claims 1 to 4.
9. Una composición que comprende un péptido según cualquiera de las reivindicaciones 1 a 4, una secuencia de nucleótidos según la reivindicación 5, un vector según la reivindicación 6, una célula según la reivindicación 7 y/o un extracto peptídico según la reivindicación 8. 9. A composition comprising a peptide according to any one of claims 1 to 4, a nucleotide sequence according to claim 5, a vector according to claim 6, a cell according to claim 7 and / or a peptide extract according to claim 8.
10. Composición según la reivindicación 9, en el que la composición es una composición farmacéutica o una composición nutricional. 10. Composition according to claim 9, wherein the composition is a pharmaceutical composition or a nutritional composition.
1 1. Uso de un péptido según cualquiera de las reivindicaciones 1 a 4, una secuencia de nucleótidos según la reivindicación 5, un vector según la reivindicación 6, una célula según la reivindicación 7 y/o un extracto peptídico según la reivindicación 8 en la elaboración de un medicamento. Use of a peptide according to any one of claims 1 to 4, a nucleotide sequence according to claim 5, a vector according to claim 6, a cell according to claim 7 and / or a peptide extract according to claim 8 in the Preparation of a medicine.
12. Uso de un péptido según cualquiera de las reivindicaciones 1 a 4, una secuencia de nucleótidos según la reivindicación 5, un vector según la reivindicación 6, una célula según la reivindicación 7 y/o un extracto peptídico según la reivindicación 8, en la elaboración de un medicamento para el tratamiento de un enfermedad inflamatoria. 12. Use of a peptide according to any one of claims 1 to 4, a nucleotide sequence according to claim 5, a vector according to claim 6, a cell according to claim 7 and / or a peptide extract according to claim 8, in the development of a medication for the treatment of an inflammatory disease.
13. Método para obtener un péptido según cualquiera de las reivindicaciones 1 a 4, que comprende: 13. Method for obtaining a peptide according to any of claims 1 to 4, comprising:
a) Hidrolizar un extracto proteico procedente de una leguminosa con una endoproteasa para obtener un hidrolizado proteico, a) Hydrolyze a protein extract from a legume with an endoprotease to obtain a protein hydrolyzate,
b) someter el hidrolizado proteico obtenido en la etapa a) a una ultracentrifugación tangencial mediante el empleo de una membrana con un tamaño de poro al menos 1 kDa, b) subject the protein hydrolyzate obtained in step a) to tangential ultracentrifugation by using a membrane with a pore size of at least 1 kDa,
c) realizar una cromatografía en fase reversa del permeado obtenido en la ultracentrifugación de la etapa b) y analizar la capacidad de las fracciones recogidas de inducir la expresión de citoquinas proinflamatorias mediante ensayos celulares, y d) someter a una purificación mediante cromatografía de gel a la fracción obtenida en la etapa c) capaz de disminuir o inhibir la expresión de las citoquinas proinflamatorias con respecto a un control. c) perform a reverse phase chromatography of the permeate obtained in the ultracentrifugation of stage b) and analyze the ability of the collected fractions to induce the expression of proinflammatory cytokines by cellular assays, and d) subject to purification by gel chromatography at fraction obtained in step c) capable of decreasing or inhibiting the expression of proinflammatory cytokines with respect to a control.
14. Método según la reivindicación 13, en la que la leguminosa es una planta de altramuz. 14. Method according to claim 13, wherein the legume is a lupine plant.
15. Método según la reivindicación 13 o 14, en el que la endoproteasa es una endoproteasa de amplio espectro, preferiblemente, subtilisina. 15. A method according to claim 13 or 14, wherein the endoprotease is a broad spectrum endoprotease, preferably subtilisin.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020192865A1 (en) 2019-03-22 2020-10-01 Symrise Ag Plant peptides and their applications

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000062789A1 (en) * 1999-04-19 2000-10-26 Laboratoires Pharmascience Peptide extract of lupine and pharmaceutical or cosmetic or nutritional composition comprising same

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000062789A1 (en) * 1999-04-19 2000-10-26 Laboratoires Pharmascience Peptide extract of lupine and pharmaceutical or cosmetic or nutritional composition comprising same

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MILLÁN-LINARES, M.C. ET AL.: "Anti-inflammatory activity of lupine (Lupinus angustifolius L.) protein hydrolysates in THP-1-derived macrophages''.", JOURNAL OF FUNCTIONAL FOODS., vol. 8, May 2014 (2014-05-01), pages 224 - 233 *
MILLÁN-LINARES, M.C. ET AL.: "Lupine protein hydrolysates inhibit enzymes involved in the inflammatory pathway''.", FOOD CHEMISTRY., vol. 151, 15 May 2014 (2014-05-15), pages 141 - 147, XP028668485, DOI: doi:10.1016/j.foodchem.2013.11.053 *
PREDEL, R. ET AL.: "Peptidomics-based phylogeny and biogeography of Mantophasmatodea (Hexapoda).", SYSTEMATIC BIOLOGY, vol. 61, no. 4, 16 April 2012 (2012-04-16), pages 609 - 629 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020192865A1 (en) 2019-03-22 2020-10-01 Symrise Ag Plant peptides and their applications

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