• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Peptide and pharmaceutical compositions thereof for use as an antimicrobial and in the treatment of cancer. The present invention relates to a peptide derived from defensin 3 of the red beetle of the flour Tribolium castaneum and pharmaceutical compositions containing it and to its use as an antimicrobial in the treatment of infections caused by Gram + bacteria, Gram-bacteria and fungi. The present invention also relates to the use of said peptide and pharmaceutical compositions containing it, in the treatment of cancer, in particular, breast cancer. The described compound is a peptide derived from defensin 3 of the red beetle of T. castaneum flour. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2717685A1
    申请号:ES201731455
    申请日:2017-12-22
    公开日:2019-06-24
    发明作者:García María Dolores Real；Segarra Carolina Rausell；Robles Inmaculada García；Jardón María Benito；Fort Aída Robles
    申请人:Universitat de Valencia；
    IPC主号:
    专利说明:

    [0001]
    [0002] Peptide and pharmaceutical compositions thereof for use as an antimicrobial and in the treatment of cancer
    [0003]
    [0004] FIELD OF THE INVENTION
    [0005]
    [0006] The present invention relates to pharmaceutical compounds and compositions for use as an antimicrobial in the treatment of infections caused by Gram + bacteria, Gram- bacteria and fungi. The present invention also relates to pharmaceutical compounds and compositions for use in the treatment of cancer, in particular, breast cancer. The compound described is a peptide derived from defensin 3 of the red beetle of the Tríbolium castaneum flour .
    [0007]
    [0008] BACKGROUND OF THE INVENTION
    [0009]
    [0010] Antimicrobial peptides (AMPs ) are promising candidates as new antibiotic agents, since they have an alternative mechanism of action to conventional antibiotics. They are excellent agents to counteract the development of antibiotic resistance. The speed with which antimicrobial peptides promote cell death is an additional factor that helps reduce the likelihood of developing resistance. On the other hand, these peptides can also act synergistically with conventional antibiotics, enhancing and improving their therapeutic activity.
    [0011]
    [0012] Antimicrobial peptides are small in size, generally have positive charges and a relationship between hydrophilic and hydrophobic residues that provides them with amphipathic characteristics. The combination of hydrophobicity with the cationic character allows them to interact electrostatically with microbial membranes that typically have anionic and lipid-rich surfaces.
    [0013]
    [0014] These peptides are part of the innate immunity of all living organisms, from bacteria, to vertebrates, to fungi, insects and plants, although not at the same level. While in invertebrates they constitute the main weapon of defense of your immune system, in vertebrates, they also modulate adaptive immunity through activity. Chemotactic, proinflammatory and anti-inflammatory signaling and also help the healing process.
    [0015]
    [0016] Tríbolium castaneum, the red flour beetle, is an insect responsible for stored food pests, and a model organism for research in developmental biology, evolution, immunity, etc.
    [0017]
    [0018] The document Contreras et al., 2015 describes the antimicrobial effect of fragments of the T. castaneum (Tc) beetle defenses against Bacillus thuringiensis (Bt). Against the entomopathogenic toxins Cry3Aa and Cry3Ba of this bacterium, the larvae of the insect develop an immune response in which the expression of the 2 and 3 defenses of T. castaneum (TcDef2 and TcDef3) is induced. The document describes that TcDef3-pep, a 29 amino acid TcDef2 peptide fragment, has antimicrobial activity against Escherichia coli, Staphylococcus aureus and Candida albicans, especially highlighting the susceptibility of S. aureus. The TcDef3-pep peptide corresponds to the sequence identified by SEQ ID NO: 2 of the present invention.
    [0019]
    [0020] Rajamuthiah et al., 2015 describes antimicrobial peptides with activity against S. aureus. The authors describe a peptide obtained from T. castaneum (XM_968482, encodes peptide XP_973575.3) with antibacterial activity. Although the authors of this article refer to defensin 1, the amino acid sequence shown in Figure 1A corresponds to defensin 3 of T. castaneum.
    [0021]
    [0022] Tonk et al., 2015 describes the antimicrobial activity of T. castaneum defensins against Gram bacteria. The three defensins (Def1, Def2 and Def3) have activity against Micrococcus luteus and B. thuringiensis , and only Def1 and Def2 have activity against Staphylococcus epidermis. The authors test the activity of defensins against S. aureus, observing the absence of antimicrobial activity against this microorganism (Figure 2 of the cited document).
    [0023]
    [0024] The five types of cancer that cause the highest mortality are lung, liver, colorectal, gastric and breast. Specifically, breast cancer is the one with the highest incidence in Europe (458,718 cases, 13% of the total) and the second worldwide (1.7 million cases, 12% of the total), occupying the third and fifth place in terms of mortality, respectively.
    [0025] Breast carcinomas have neoplasms with great heterogeneity, which have led to the need to develop different classification systems: histological, molecular and functional markers. The most commonly used clinical classification is the molecular marker dependent, which includes estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2). According to the presence or absence of these markers, breast tumors are distributed in four basic subtypes: ER or PR positive and HER2 negative ([ER + | PR +] HER2-), ER or PR positive and HER 2 positive ([ER + | PR +] HER2 +), ER and PR negative and HER2 positive ([ER- | PR-] HER2 +), also called HER2 positive and finally, ER and PR negative and HER2 negative ([ER- | PR-] HER2-) also called triple negative (TNBC, the English Triple Negative Breast Cancer), being the most aggressive and the worst prognosis.
    [0026]
    [0027] The treatment of breast cancer requires a personalized multidisciplinary approach for each individual, which combines surgery, radiotherapy, chemotherapy and hormonal therapies. Triple negative breast cancer poses several additional problems, since it does not respond to hormonal therapies since it lacks receptors, it is associated with a risk of metastasis four times higher than other types of breast tumors, and, in addition, the risk peak Recurrence is observed between the first and third year, with the majority of deaths occurring in the first five years. Chemotherapy is the only treatment that currently improves the outcome of TNBC, but its high cytotoxicity also for healthy cells and the appearance of chemoresistance causes other treatment options, such as molecule-directed therapies, to be investigated. Unfortunately, most of these types of therapies involve the acquisition of resistance by the tumor cells that limit the effectiveness of the treatment. An interesting improvement would be the development of new drugs against triple negative breast cancer, that selectively kill tumor cells or reduce their proliferation and that are not affected by the known resistance mechanisms.
    [0028]
    [0029] DETAILED DESCRIPTION OF THE INVENTION
    [0030]
    [0031] In the context of products with antimicrobial activity, the problem of the prior art is to provide peptides derived from T. castaneum defensin 3 with improved antimicrobial activity with respect to peptides derived from T. castaneum defensin 3 described in the state of technique
    [0032] The present invention solves said problem by providing a peptide and a pharmaceutical composition thereof, which have not been described in the state of the art. This new peptide and pharmaceutical composition thereof have an unexpected effect and show a greater activity against S. aureus than other peptides derived from T. castaneum defensin 3 with antimicrobial activity described in the prior art.
    [0033]
    [0034] The sequence peptide SEQ ID NO: 1 has 31 amino acids, an additional amino acid at each end relative to the TcDef3-pep peptide, described in Contreras et al., 2015, this being the structural difference between the two peptides.
    [0035]
    [0036] The peptide described in Rajamuthiah et al., 2015, called Tca1, has activity against S. aureus, but its sequence has 44 amino acids, compared to 31 of SEQ ID NO: 1.
    [0037]
    [0038] In the context of products with activity against cancer, the problem of the prior art is to provide effective compounds for use in the treatment of cancer.
    [0039]
    [0040] The present invention solves said problem by providing a peptide comprising the sequence SEQ ID NO: 1 and a pharmaceutical composition thereof, effective for use in the treatment of cancer. This new use in the treatment of cancer has not been previously described in the state of the art.
    [0041]
    [0042] Here, the term "PaSK" refers to the peptide identified by the sequence SEQ ID NO: 1. Therefore, herein the terms and expressions "PaSK", "peptide consisting of the sequence SEQ ID NO: 1 "and" sequence SEQ ID NO: 1 ", are interchangeable.
    [0043]
    [0044] Here, the term "TcDef3-pep" refers to the peptide identified by the sequence SEQ ID NO: 2. Therefore, herein the terms and expressions "TcDef3-pep", "peptide consisting of the sequence SEQ ID NO: 2 ”and“ sequence SEQ ID NO: 2 ”, are interchangeable.
    [0045]
    [0046] Peptide and pharmaceutical composition comprising the peptide
    [0047]
    [0048] The present invention provides a peptide consisting of the sequence SEQ ID NO: 1.
    [0049] In one embodiment, the present invention provides a pharmaceutical composition comprising the peptide consisting of the sequence SEQ ID NO: 1 and at least one pharmaceutically acceptable carrier or excipient.
    [0050]
    [0051] In one embodiment, the carrier is selected from the group consisting of organic nanoparticles, selected from the group consisting of: lipids, nanoemulsions, polymeric micelles, SCK nanoparticles, liposomes, nanogels, hydrogels, lipoplexes, polylexes; polymers selected from the group consisting of: albumin, cellulose, chitosan, alginate, gelatin, poly-ε-caprolactone (PCL), hydroxyethyl starch (HES; MEA), polyglycolate (PGA), poly (lactic-co-glycolide) , polylactide (PLA), poly (d, 1-lactide-co-glycolide) (PLGA), polyethylene glycol (PEG), W- (2-Hydroxypropyl) methacrylamide (poly (HPMA); PHPMA) and dextran; dendrimers, selected from the group consisting of: polyether hydroxylamine (PEHAM), polyamidoamine (PAMAM), polyetheramine, polypropyleneimine and polyglycerol; nanofibers, selected from the group consisting of: carbon nanotubes, poly (d, 1-lactide-co-glycolide) nanofibers (PLGA), polyethylene glycol (PEG), chitosan, polyvinyl alcohol (PVA), polylactide (PLA), polyethylene oxide and polycaprolactone (PCL); and inorganic nanoparticles, selected from the group consisting of: gold nanoparticles, metal oxide nanoparticles, titanium oxide nanoparticles, platinum oxide nanoparticles, superparamagnetic iron oxide nanoparticles (SPIO-NPs), diamond-based nanoparticles and nanoparticles QD
    [0052]
    [0053] Here, the term "nanoemulsions" refers to heterogeneous mixtures of two immiscible liquids with an emulsifier that stabilizes the dispersed drops.
    [0054]
    [0055] Here, the term "polymeric micelles" refers to self-assembled amphiphilic copolymer chains in aqueous medium, which have a hydrophobic core surrounded by a hydrophilic crown. Polymeric micelles include polymeric micelles of the polyionic type (PIC) of the English term "polyion complex" and polymer-metal complex micelles.
    [0056]
    [0057] The acronym "SCK" comes from the English term "shell cmss-linked knedel-like." Here, the term "SCK nanoparticles" refers to self-assembled amphiphilic copolymer chains in polymeric micelles, in which selective crosslinking is performed in the outer layer.
    [0058] Here, the term "lipoplexes" refers to complexes formed by nucleic acids (DNA / RNA) and cationic lipids. They include PEGylated lipoplexes.
    [0059]
    [0060] Here, the term "polylexes" refers to complexes formed by nucleic acids (DNA / RNA) and polymers. They include PEGylated pollexlexes.
    [0061]
    [0062] Here, the term "carbon nanotubes" refers to single-wall or multi-wall cylindrical graphene sheets.
    [0063]
    [0064] The acronym "SPIO-NPs" comes from the English term "superparamagnetic iron oxides nanoparticles".
    [0065]
    [0066] The acronym "QD" comes from the English term "quantum dots." Here, the term "QD nanoparticles" refers to semiconductor inorganic nanoparticles, widely used as fluorophores in imaging techniques.
    [0067]
    [0068] In one embodiment, the pharmaceutical composition comprises an effective amount of the peptide consisting of the sequence SEQ ID NO: 1.
    [0069]
    [0070] The amount of the compound that provides an objectively identifiable improvement in the patient's condition, recognized by a qualified observer and where said patient is treated with a pharmaceutical composition comprising said amount of the compound is defined as effective amount, for the purposes of the present invention. .
    [0071]
    [0072] Inert pharmaceutically acceptable excipients, for the purposes of the present invention, are ingredients such as, but not limited to: cosolvents, surfactants, oils, humectants, emollients, preservatives, stabilizers and antioxidants. Said excipient or vehicle can be, for example, a diluent. The pharmaceutical composition may be in crystalline, powdered, granular, compacted solid, liquid, solution, suspension, elixir, syrup, emulsion, cream, gel, drop, mist, vapor or spray form. Conventional techniques can be used for the preparation of the pharmaceutical composition. For example, the pharmaceutical composition may be comprised in a capsule, tablet, pill, oblong tablet, ampoule, envelope, syringe, cartridge, nebulizer or other container.
    [0073]
    [0074] The pharmaceutical composition of the invention can be administered alone, or in combination with other active ingredients.
    [0075] The pharmaceutical composition of the invention can be administered to a subject in different ways, depending on whether the treatment is local or systemic, and depending on the area to be treated. Thus, for example, the pharmaceutical composition of the invention can be administered to a subject by ocular, vaginal, rectal, intranasal, oral, inhalation or parenteral route, either intradermally, subcutaneously, intramuscularly, intraperitoneally, intrarectally, intraarterially, intralymphatically. , intravenous, intrathecal, intraocular, intracranial and intratracheal. Parenteral administration, if used, is usually done by injection. Solutions for injection can be prepared in various ways, such as liquid solutions or suspensions, solid forms suitable for dissolving or suspended before injection, or as emulsions. Other forms of parenteral administration use slow or sustained release systems, so that a constant dose is achieved. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions, and may also contain diluents and other diluting buffers and additives. Examples of non-aqueous solvents are: propylene glycol, polyethylene glycol, vegetable oils such as olive oil and organic esters for injection such as ethyl oleate. Examples of aqueous solvents are: water, aqueous alcohol solutions, emulsions or suspensions, which include saline and buffer. Examples of parenteral vehicles are: sodium chloride solution, Ringer's dextrose, sodium chloride and dextrose, etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, etc. Formulations for topical administration may include creams, lotions, gels, drops, suppositories, aerosols, liquids and powders. Certain conventional pharmaceutical carriers, aqueous bases, oily or powder bases, thickeners, etc. They may also be necessary. Compositions for oral administration may include powders or granules, suspensions or solutions in water or non-aqueous medium, capsules or tablets. It may be desirable to include thickeners, flavorings, diluents, emulsifiers, dispersants, etc.
    [0076]
    [0077] The pharmaceutical composition of the invention can be administered in single or multiple doses.
    [0078]
    [0079] In one embodiment, the pharmaceutical composition further comprises an antibiotic agent.
    [0080]
    [0081] In one embodiment, said antibiotic agent is selected from the group consisting of fusidic acid, arsfenamine, clindamycin, chloramphenicol, ethambutol, fosfomycin, furazolidone, isoniazid, lincomycin, linezolid, metronidazole, mupirocin, nitrofurantoin, pyrazinamide, platensimicin, quinupristin, tinuphicin, quinupristine or an antibiotic of the selected class of: aminoglycosides, ansamycins, carbacefem, carbapenem, cephalosporins, glycopeptides, macrolides, monobactams, penicillins, polypeptides, quinolones, sulfonamides and tetracyclines.
    [0082]
    [0083] Peptide or pharmaceutical composition for use as a medicine and as an antimicrobial
    [0084]
    [0085] In one embodiment, the present invention provides the peptide consisting of the sequence SEQ ID NO: 1 or the pharmaceutical composition, for use as a medicament.
    [0086]
    [0087] In one embodiment, the present invention relates to the peptide or pharmaceutical composition for use as an antimicrobial medicament.
    [0088]
    [0089] In a preferred embodiment, the present invention relates to the peptide consisting of SEQ ID NO: 1 or the pharmaceutical composition comprising it, for use as an antimicrobial medicament in the treatment of infections caused by Gram + bacteria, Gram- bacteria and fungi.
    [0090]
    [0091] In a preferred embodiment, said Gram + bacterium is S. aureus.
    [0092]
    [0093] In a preferred embodiment, said Gram-bacterium is E. coli.
    [0094]
    [0095] In a preferred embodiment, said fungus is C. albicans.
    [0096]
    [0097] Peptide or pharmaceutical composition for use in the treatment of cancer
    [0098]
    [0099] In one embodiment, the present invention provides a peptide comprising the sequence SEQ ID NO: 1 or a pharmaceutical composition comprising said peptide and at least one pharmaceutically acceptable carrier or excipient, for use in the treatment of cancer.
    [0100]
    [0101] In one embodiment, the pharmaceutical composition comprises an effective amount of the peptide comprising the sequence SEQ ID NO: 1.
    [0102]
    [0103] They are pharmaceutically acceptable excipients, inert ingredients such as, but not limited to: cosolvents, surfactants, oils, humectants, emollients, preservatives, stabilizers and antioxidants. Said excipient or vehicle can be, for example, a diluent. The pharmaceutical composition may be in crystalline, powder, granular, solid compacted, liquid, in solution, suspension, elixir, syrup, emulsion, cream, gel, drop, mist, vapor or spray. Conventional techniques can be used for the preparation of the pharmaceutical composition. For example, the pharmaceutical composition may be comprised in: a capsule, tablet, pill, oblong tablet, ampoule, envelope, syringe, cartridge, nebulizer or other container.
    [0104]
    [0105] The pharmaceutical composition can be administered alone or in combination with other active ingredients.
    [0106]
    [0107] The pharmaceutical composition can be administered to a subject in different ways, depending on whether the treatment is local or systemic, and depending on the area to be treated. Thus, for example, the pharmaceutical composition can be administered to a subject by eye, vaginal, rectal, intranasal, oral, inhalation or parenteral route, whether intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralinatic, intravenous, intrathecal, intraocular, intracranial and intratracheal. Parenteral administration, if used, is usually done by injection. Solutions for injection can be prepared in various ways, such as liquid solutions or suspensions, solid forms suitable for dissolving or suspended before injection, or as emulsions. Other forms of parenteral administration use slow or sustained release systems, so that a constant dose is achieved. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions, and may also contain diluents and other diluting buffers and additives. Examples of non-aqueous solvents are: propylene glycol, polyethylene glycol, vegetable oils such as olive oil and organic esters for injection such as ethyl oleate. Examples of aqueous solvents are: water, aqueous alcohol solutions, emulsions or suspensions, which include saline and buffer. Examples of parenteral vehicles are: sodium chloride solution, Ringer's dextrose, sodium chloride and dextrose, etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, etc. Formulations for topical administration may include creams, lotions, gels, drops, suppositories, aerosols, liquids and powders. Certain conventional pharmaceutical carriers, aqueous bases, oily or powder bases, thickeners, etc. They may also be necessary. Compositions for oral administration may include powders or granules, suspensions or solutions in water or non-aqueous medium, capsules or tablets. It may be desirable to include thickeners, flavorings, diluents, emulsifiers, dispersants, etc.
    [0108] The pharmaceutical composition can be administered in single or multiple doses.
    [0109]
    [0110] In a particular embodiment, the present invention provides a peptide consisting of the sequence SEQ ID NO: 1 or a pharmaceutical composition comprising the peptide consisting of the sequence SEQ ID NO: 1 and at least one pharmaceutically acceptable carrier or excipient, for Use in cancer treatment.
    [0111]
    [0112] In one embodiment, the cancer is selected from the group consisting of breast cancer, breast cancer resistant to anti-HER2 therapy, breast carcinoma, breast adenocarcinoma, gastric carcinoma, gastric adenocarcinoma, colon carcinoma, colon adenocarcinoma, carcinoma of pancreas, pancreatic adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, ovarian adenocarcinoma, ovarian carcinoma, endometrial carcinoma, cervical carcinoma, lung carcinoma, pulmonary adenocarcinoma, non-small cell lung cancer small cell, thyroid carcinoma, metastatic papillary thyroid carcinoma, follicular thyroid carcinoma, bladder carcinoma, transitional cell carcinoma of the urinary bladder, prostate carcinoma, glial lineage cancer of the central nervous system (glioma), sarcomas, fibrosarcoma, fibrous histiocytoma malignant, human Edwing sarcoma, est sarcoma endometrial rome, osteosarcoma, rhabdomyosarcoma, melanoma, embryonic cancers, neuroblastoma, medulloblastoma, retinoblastoma, nephroblastoma, hepatoblastoma, hematological cancers, B or T cell leukemia, non-Hodgkin lymphoma, non-Hodgkin lymphoma of B or T cell, Burkoma lymphoma Hodgkin lymphoma, leukemia, B or T cell lymphoma, and multiple myeloma.
    [0113]
    [0114] In a preferred embodiment, the cancer is breast cancer.
    [0115]
    [0116] In a more preferred embodiment, breast cancer is triple negative breast cancer.
    [0117]
    [0118] The peptide of the present invention can efficiently penetrate heterogeneous tumor cells, which allows them to exert their intrinsic anticancer activity or in synergy with other therapeutic agents, and has a lower probability of developing resistance than other therapeutic agents.
    [0119]
    [0120] In one embodiment, the peptide or pharmaceutical composition is for use in the treatment of cancer in combination with a treatment with a chemotherapeutic agent, with a treatment with an immunotherapeutic agent, or with a radiotherapy treatment.
    [0121] In one embodiment, said chemotherapeutic agent is selected from the group consisting of: anastrozole, capecitabine, carboplatin, oxaliplatin, cyclophosphamide, cisplatin, docetaxel, doxorubicin, eribulin, fulvestrant, imiquimod, letrozole, paclitaxel, romidepsin, triciribin-5-fluorouracil, triciribin-5-fluorouracil and gemcitabine.
    [0122]
    [0123] In one embodiment, said immunotherapeutic agent is selected from the group consisting of: dovitinib, ipilimumab, lapatinib, margetuximab, neratinib, nivolumab, olaparib, palbociclib, pembrolizumab, pertuzumab, ruxolitinib, trastuzumab and veliparib.
    [0124]
    [0125] In an embodiment of the pharmaceutical composition for use in the treatment of cancer, said carrier is selected from the group consisting of organic nanoparticles, selected from the group consisting of: lipids, nanoemulsions, polymeric micelles, SCK nanoparticles, liposomes, nanogels, hydrogels, lipoplexes, pollexlexes; polymers selected from the group consisting of: albumin, cellulose, chitosan, alginate, gelatin, poly-caprolactone (PCL), starch hydroxyethyl (HES; MEA), polyglycolate (PGA), poly (lactic-co-glycolide) , polylactide (PLA), poly (d, 1-lactide-co-glycolide) (PLGA), polyethylene glycol (PEG), W- (2-Hydroxypropyl) methacrylamide (poly (HPMA); PHPMA) and dextran; dendrimers, selected from the group consisting of: polyether hydroxylamine (PEHAM), polyamidoamine (PAMAM), polyetheramine, polypropyleneimine and polyglycerol; nanofibers, selected from the group consisting of: carbon nanotubes, poly (d, 1-lactide-co-glycolide) nanofibers (PLGA), polyethylene glycol (PEG), chitosan, polyvinyl alcohol (PVA), polylactide (PLA), polyethylene oxide and polycaprolactone (PCL); and inorganic nanoparticles, selected from the group consisting of: gold nanoparticles, metal oxide nanoparticles, titanium oxide nanoparticles, platinum oxide nanoparticles, superparamagnetic iron oxide nanoparticles (SPIO-NPs), diamond-based nanoparticles and nanoparticles QD
    [0126]
    [0127] In one embodiment, the pharmaceutical composition for use in the treatment of cancer further comprises a chemotherapeutic agent or an immunotherapeutic agent.
    [0128]
    [0129] In one embodiment, said chemotherapeutic agent is selected from the group consisting of: anastrozole, capecitabine, carboplatin, oxaliplatin, cyclophosphamide, cisplatin, docetaxel, doxorubicin, eribulin, fulvestrant, imiquimod, letrozole, paclitaxel, romidepsin, triciribin-5-fluorouracil, triciribin-5-fluorouracil and gemcitabine.
    [0130] In one embodiment, said immunotherapeutic agent is selected from the group consisting of: dovitinib, ipilimumab, lapatinib, margetuximab, neratinib, nivolumab, olaparib, palbociclib, pembrolizumab, pertuzumab, ruxolitinib, trastuzumab and veliparib.
    [0131]
    [0132] The results of Examples 3 and 4 demonstrate that PaSK (peptide consisting of the sequence SEQ ID NO: 1) inhibits tumor growth and proliferation by regulating cell cycle progression, affecting the G1 / S transition in cancer cells of triple negative breast. The p53 tumor suppressor protein normally mediates cell cycle arrest and apoptosis in response to genotoxic stress. MDA-MB-231 cells have point mutations that make the p53 protein not functional in them. Thus, the inhibition of growth and proliferation of triple negative breast cancer cells by non-P53-dependent PaSK provides the PaSK peptide with great interest for therapeutic use in breast tumors in which p53 is often mutated.
    [0133]
    [0134] Definitions
    [0135]
    [0136] Here, the term "cancer" may encompass all types of oncogenic processes and / or cancerous growths. In embodiments, the cancer includes primary tumors, as well as metastatic tissues or malignant transformed cells, tissues or organs. In embodiments, the cancer encompasses all histopathologies and stages, for example, stages of invasiveness / severity, of a cancer. In embodiments, the cancer includes recurrent and / or resistant cancer. The terms "cancer" and "tumor" can be used interchangeably.
    [0137]
    [0138] Unless otherwise defined, all technical and scientific terms have the same meaning as those commonly understood by a person skilled in the art in the field of the invention. Similar and equivalent methods and materials to those described herein can be used in the practice of the present invention.
    [0139]
    [0140] Throughout the description and the claims, the term "comprises", "comprising" and its variants are not of a limiting nature and, therefore, are not intended to exclude other technical characteristics.
    [0141]
    [0142] Throughout the description and claims, the term "consisting" or "consisting of", when referring, in particular, to biological sequences, means that the compounds of the invention are precisely restricted to the fragment identified as such by the indicated sequence.
    [0143]
    [0144] BRIEF DESCRIPTION OF THE FIGURES
    [0145]
    [0146] Figure 1 Antimicrobial activity of hBD-3, TcDef3-pep and PaSK peptides against S. aureus. Graphs of the fluorescence obtained in the flow cytometer windows of the negative controls (A) and positive control hBD-3 (B). Graphs of the fluorescence obtained in the flow cytometer windows of the TcDef3-pep (C) and PaSK (D) peptides. In the ordinate axis, FITC refers to fluorescein and indicates the cells that have incorporated SYBR green. In the abscissa axis, PerCP-Cy5.5-A refers to peridinin-chiorophyll proteins and corresponds to the cells that have captured propidium iodide. The P2 window shows live cells and the P3 window shows dead cells.
    [0147]
    [0148] Figure 2 Comparison of cytotoxicity between hBD-3, TcDef3-pep and PaSK peptides against S. aureus. The activity was measured at a concentration of 25 pg / mL in 2 replicates. The activity for the negative control (control), positive control hBD-3, TcDef3-pep and PaSK is shown. The graph represents the average of the two replicas with their corresponding standard deviations.
    [0149]
    [0150] Figure 3 Antimicrobial activity of the TcDef3-pep and PaSK peptides against S. aureus. The points represent the means of two replicas with their corresponding standard deviations. TcDef3-pep peptide data acquired from Contreras et al., 2015.
    [0151]
    [0152] Figure 4 Images of the damage caused by peptides in S. aureus performed with scanning electron microscopy. Cells not treated with peptides (AB) and treated with hBD-3 (CD), TcDef3-pep (EF) and PaSK (GH) are shown at a concentration of 25 pg / mL for 1 hour. Continuous arrows indicate breaks and bulges in the membrane, dashed arrows indicate cytoplasmic remains. The number representing the scale is 3 pm (A), 2 pm (B), 4 pm (C), 2 pm (D), 4 pm (E), 500 nm (F), 2 pm (G) and 2 pm (H).
    [0153]
    [0154] Figure 5 Images obtained by scanning electron microscopy of the biofilm produced by S. aureus cells treated with TcDef3-pep (A) and PaSK (B), at a concentration of 25 pg / mL for 1 h. The number representing the scale is 1 pm (A, upper photo), 2 pm (A, central photo), 4 pm (A, lower photo), 4 pm (B, upper photo), 2 pm (central photo) and 3 pm (photo below).
    [0155] Figure 6 Images of the damage caused by peptides in S. aureus performed with transmission electron microscopy. Cells not treated with peptides (AB) and treated with hBD-3 (CD), TcDef3-pep (EF) and PaSK (GH) are shown at a concentration of 25 pg / mL for 1 h. Cytoplasmic and cell wall debris (6), malformations, detachments in the membrane and thinning of the peptidoglycan (4), cell lysis (5), cytoplasmic vacuolization (7), division septal malformation (8) and very structures are indicated electrodensates that only appear in cells treated with the PaSK peptide (9). The number representing the scale is 200 nm (A), 100 nm (B), 600 nm (C), 200 nm (D), 400 nm (E), 200 nm (F), 600 nm (G) and 200 nm (H).
    [0156]
    [0157] Figure 7 Images obtained by electron microscopy of biofilm transmission produced by S. aureus cells treated with the TcDef3-pep (A) and PaSK (B) peptides, at a concentration of 25 pg / mL for 1 h. The number representing the scale is 400 nm (A, left), 400 nm (A, right), 400 nm (B, left), 400 nm (B, right)
    [0158]
    [0159] Figure 8 Determination of the cytotoxic effect of the PaSK peptide on two triple negative breast cancer cell lines, one human (MDA-MB-231) and one mouse (4T1), and one normal mouse mammary epithelial cell line (HC-11) for an MTS essay. Results are shown for a bovine fetal serum concentration of 0.5% and 24 h of incubation. The PaSK peptide has an average IC 50 of approximately 200 pM (700 pg / mL) in all three cases.
    [0160]
    [0161] Figure 9 The PaSK peptide inhibits the proliferation of human triple negative breast cancer cells (MDA-MB-231). The cells were incubated with Oregon Green. Cell proliferation analysis was performed by flow cytometry of tumor cells labeled with Oregon Green fluorophore. The results of the non-proliferative control (peak with black area), cells not treated with the peptide (peak with white area) and cells treated with 50 pM (200 pg / mL) of PaSK peptide (peak with area are shown) in gray color).
    [0162]
    [0163] Figure 10 Cytotoxic activity of the PaSK peptide against tumor cells MDA-MB-231. Graphs of live and dead cells obtained in the flow cytometer, control (A) and treatment with PaSK (B). In the ordinate axis, SSC-A (Side Scatter) refers to the granularity of the cells of the selected population. In the abscissa axis, PerCP-Cy5.5-A refers to Peridinin-chiorophyll proteins and corresponds to the cells that have captured the propidium iodide. Activity was measured at a concentration of 200 pg / mL of PaSK in Two replicas Viability graph in which the means of two replicas are represented with their corresponding standard deviations (C).
    [0164]
    [0165] Figure 11 Antiproliferative activity of the PaSK peptide against tumor cells MDA-MB-231. Analysis of cell cycle progression using ModFit LT software, A) Control B) PaSK (100 pg / mL). The distribution of cells in the different phases of the cell cycle was detected in 2 replicates. C) Graph of the percentage of cells observed in each phase of the cell cycle, in which the means of two replicas are represented with their corresponding standard deviations. The asterisk indicates statistically significant differences between treatments (t-Student test, p <0.05). D) Scheme of the cell cycle showing the phase affected by the action of the PaSK peptide.
    [0166]
    [0167] Figure 12 Discriminant analysis of the quantitative measurements of proteins obtained in the control and treatment replicas of the differential proteomic analysis with Marker View 1.3 software. Eight samples (4 control replicates (C1-C4 samples) and 4 replicas of PaSK treatment (samples P1-P4)) were analyzed.
    [0168]
    [0169] Figure 13 Differential protein clusters by molecular function. Protein clusters with diminished (A) and increased (B) abundance in the treatment / control relationship, performed at Uniprot (http://www.uniprot.org).
    [0170]
    [0171] Figure 14 Images of the damage caused by PaSK in tumor cells MDA-MB-231 performed with scanning electron microscopy. Cells not treated with the PaSK (AB) peptide and treated with PaSK at a concentration of 100 pg / mL for 72 hours (DF) are shown. The membrane (1) and its expansions (5) are indicated in the cells without treatment. The membrane of the cells treated with PaSK (2) and invaginations and bulges (3) are indicated. The number representing the scale is 10 pm (A), 5 pm (B), 10 pm (C), 50 pm (D), 10 pm (E) and 10 pm (F).
    [0172]
    [0173] Figure 15 Images of the damage caused by PaSK in tumor cells MDA-MB-231 performed with transmission electron microscopy. Cells not treated with the PaSK (AB) peptide and treated with PaSK at a concentration of 100 pg / mL for 72 hours (DF) are shown. The membrane (1) and its expansions (5) are indicated in the cells without treatment. The membrane of the cells treated with PaSK (2), invaginations and bulges (3) and ruptures of the membrane and vesicles with cytoplasmic remains (4) are indicated. The number representing the scale is 2 pm (A), 2 pm (B), 2 pm (C), 2 pm (D), 1 pm (E), 2 pm (F).
    [0174]
    [0175] DESCRIPTION OF EMBODIMENTS
    [0176]
    [0177] Materials and methods
    [0178]
    [0179] Peptides used
    [0180] The synthetic peptides used in this study were: TcDef3-pep and PaSK, fragments of T. castaneum 3 defensin , and human hBD-3 (PeptaNova).
    [0181]
    [0182] Bacterial strain of Staphylococcus aureus subsp. aureus
    [0183] CECT 4013 strain of S. aureus subsp. aureus, strain accessible to the public deposited in the Spanish Type Culture Collection (CECT).
    [0184]
    [0185] MDA-MB-231 human mammary gland tumor cell line
    [0186] The human mammary gland tumor cell line MDA-MB-231, identified as ATCC® HTB-26 ™, a publicly accessible cell line deposited with the American Type Culture Collection (ATCC) deposit authority was used. The cells were grown in 75 cm2 bottles with DMEM medium, supplemented with 10% bovine fetal serum, 1% penicillin and streptomycin and 0.1% fungizone, and maintained at 37 ° C in an atmosphere containing 5% CO 2 .
    [0187]
    [0188] Preparation of sample
    [0189] MDA-MB-231 cells were cultured in 6-well plates with a density of 5x104 cells per well, to which 200 pL or 400 pL of PaSK (final concentrations 100 pg / mL and 200 pg / mL) were added or the same volume of medium (solvent with which the peptide dilution was made) in the corresponding controls. The cells were incubated at 37 ° C for 72 h.
    [0190]
    [0191] Flow cytometry of S. aureus cells
    [0192] From S. aureus cells grown overnight in LB liquid medium (1% peptone, 0.5% yeast extract and 1% NaCl) at 37 ° C and under stirring, aliquots were made in new liquid medium and left grow to obtain an optical density at 600 nm (OD600) of 0.5, optimal for detection in the flow cytometer. Aliquots with a concentration of 5x106 cfu / 100pL of S. aureus were prepared to which 10, 15, 20 and 25 pg / mL were added of PaSK, 25 pg / mL of TcDef3-pep and H 2 O (negative control), solvent with which the dilutions of the peptides were made. The mixtures were incubated at 37 ° C for 8 h and labeled with SYBR Green (Invitrogen) fluorochromes (25 pL of a solution of SYBR Green 25X in H 2 O) staining all cells and propidium iodide (Sigma) (10 pL of propidium iodide 1 mg / mL) staining dead cells. Finally, the cell death produced by the peptides was analyzed by flow cytometry with the BD Facs Verse (Becton Dickinson) equipment, from the cell culture and flow cytometry section of the Central Research Support Services of the University of Valencia . The experiments were performed in duplicate.
    [0193]
    [0194] The flow cytometer forces the cells to pass one by one through a needle creating a thin row of liquid and detects how the laser beam (argon laser emission at 488 nm, in the equipment used) interacts with the cells, depending on how incident light and fluorescence emitted by excited fluorochromes are deflected. The results are presented in the form of graphs obtained with the BD Facs suite analysis program v1.0.5.3841 (Becton Dickinson). First, a control graph was made with the parameters FSC (Forward Scatter) and SSC (Side Scatter) to search for all cellular events. These parameters provide cell size and granularity, respectively. Next, a second graph is made with the SSC and FITC parameters (fluorescence parameter indicating the cells that have SYBR Green), to differentiate the cells from the rest of the particles that may be in the sample. Finally, a third graph is made with the fluorescence parameters FITC and PerCPCy5.5 (indicates the cells stained with propidium iodide) to differentiate the cells that have only been stained with SYBR Green from those that have been stained with propidium iodide and that will be the ones that have died.
    [0195]
    [0196] Flow cytometry of human mammary gland tumor cells MDA-MB-231 To estimate cell viability, cells of the MDA-MB-231 cell line were incubated with 0.5 pL of a 1: 1000 dilution of the fluorochrome propidium iodide (IP ) 1 mg / mL (Sigma-Aldrich), with which the dead cells were stained.
    [0197]
    [0198] The CycleTEST ™ PLUS DNA Reagent Kit (Sigma-Aldrich) was used to analyze the cell cycle. Two washings were performed with citrate buffer (containing sodium citrate, sucrose and dimethyl sulfoxide (DMSO)), centrifuging the samples in each of them at 300xg for 5 min, and a final wash, in which the number of cells was adjusted at 5x105 and centrifuged at 400xg for 5 min. Next, 250 pL of solution A (containing trypsin in a detergent buffer with spermine tetrahydrochloride), 200 pL of solution B (containing a trypsin and ribonuclease A inhibitor, in a citrate stabilizing buffer with spermine tetrahydrochloride) and 200 pL of solution C (includes IP and citrate stabilizing buffer with spermine tetrahydrochloride), leaving between each solution an interval of 10 min at room temperature and performing the last one, before filtering, in Cold and dark chamber. The analysis of the cell death produced by the peptide and the phase of the cell cycle in which the cells were found were performed with the BD Facs Verse (Becton Dickinson) equipment. The cell cycle modeling was carried out using Modfit LT software, version 3.3.11. The experiments were performed in duplicate.
    [0199]
    [0200] MTS method
    [0201] Cell viability was determined using the MTS method. MDA-MB-231 cells cultured in 96-well plates at 37 ° C in a humidified atmosphere with 10% CO 2 up to a density of 5x103 cells / well, were incubated with different concentrations of the PaSK peptide for 24 h. Then, 20 pL of the MTS / PMS solution (3- (4,5-dimethylliazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium / phenacin methosulfate was added ) to each well and after incubating for 2 h in the dark, the absorbance reading was carried out at 490 nm in a plate reader. The percentage reduction in the number of cells was calculated by equation (1 - E / C) * 100, where E is the absorbance of the cells treated with the peptide and C the absorbance of the samples of the control cells.
    [0202]
    [0203] Scanning Electron Microscopy of S. aureus cells
    [0204] The cells were prepared in the same manner as for the flow cytometry technique and the same aliquots and treatments were performed. The samples were incubated at 37 ° C for 1 h. The bacteria were fixed with Karnosky (2.5% paraformaldehyde and 0.5 glutaraldehyde) for 2 h at 4 ° C. After washing (centrifugation and removal of the supernatant), they were fixed with 2% osmium tetraoxide for another 2 h, washed again and filtered using a 0.2 pm filter. Then, the cells were dehydrated in graduated series of ethanol (30 °, 50 °, 70 °, 90 °, 100 °) for 10 min, in each gradation. To perform the critical point drying the ethanol was replaced with liquid CO 2, the temperature increased to accelerate the evaporation and the pressure was lowered slowly to maintain the exact form of the bacteria. Finally, it was shaded with palladium gold for 2 min and the results were observed in the FEG-SEM HITACHI S4800 at 10 Kv.
    [0205]
    [0206] Scanning Electron Microscopy of human mammary gland tumor cells MDA-MB-231
    [0207] MDA-MB-231 cells, controlled or treated with 100 pg / mL of PaSK, were prepared as described in the "Sample preparation" section. The cells were fixed with Karnosky (paraformaldehyde 2.5% and glutaraldehyde 0.5%) for 2 h at 4 ° C. After washing (centrifugation and removal of the supernatant), they were fixed with 2% osmium tetraoxide for another 2 h, washed again and filtered using a 0.2 pm filter. Then, the cells were dehydrated in graduated series of ethanol (30 °, 50 °, 70 °, 90 °, 100 °) for 10 min, in each gradation. To perform the critical point drying the ethanol was replaced with liquid CO 2, the temperature increased to accelerate the evaporation and the pressure was lowered slowly to maintain the exact shape of the cells. Finally, it was shaded with palladium gold for 2 min and the results were observed in the FEG-SEM HITACHI S4800 at 10 Kv.
    [0208]
    [0209] Transmission Electron Microscopy of S. aureus cells and human mammary gland tumor cells MDA-MB-231
    [0210] The samples were prepared in the same way as for scanning electron microscopy, with the exception of the ethanol graduated series of dehydration in which only 90 ° gradation was reached. Next, the resin samples were included in 4 steps: 96 ° ethanol - 2: 1 LR-White resin (20 h), 100 ° ethanol - 2: 1 resin (20 h), 100 ° ethanol - 1: 2 resin (20 h) and 100% resin (24 h at 60 ° C). Once the samples were included in the resin, the block for ultra-thin 60 nm cuts was prepared in the Leica UC6 Ultracut Microtome. Finally, the cuts were contrasted with lead and the results were observed in the TEM JEOL-JEM1010 at 70kV.
    [0211]
    [0212] Example 1. Analysis of the antimicrobial activity of the PaSK peptide against S. aureus
    [0213]
    [0214] In this example, the antimicrobial activity of the PaSK peptide was compared with the antimicrobial activity of the TcDef3-pep peptide. The antimicrobial activity of the TcDef3-pep peptide against S. aureus has been described in Contreras et al., 2015.
    [0215]
    [0216] The antimicrobial activity of the PaSK peptide was determined by flow cytometry. This technique is based on the dispersion of light by cells and the use of fluorochromes to discriminate between living and dead cells. All cells are permeable to SYBR green fluorochrome, while only dead cells are for propidium iodide, since it requires that the bacterial membranes are damaged to penetrate inside. Therefore, the use of these two fluorochromes provides a method for determining viable quantifying the loss of cell viability from the increase in propidium iodide inside dead cells.
    [0217] After incubation of the cells of the CECT 4013 strain of S. aureus with 25 pg / mL of the TcDef3-pep and PaSK peptides, the percentage of dead cells was determined. As a positive control, at the same concentration, the human defensin hBD-3 was used, which has antimicrobial activity against different types of microorganisms, including S. aureus. Figures 1A-1D show the graphs corresponding to the fluorescence obtained in the flow cytometer windows. The P2 window shows live cells and the P3 window shows dead cells.
    [0218]
    [0219] The percentages of dead cells and the standard deviations (SD) of the two replicates analyzed for the treatments with each peptide and for the control cells are shown in Figure 2. The cytotoxicity of hBD-3 and TcDef3-pep was about 70% and 55%, respectively. However, the PaSK peptide has a percentage of almost 100% cell death. Mortality was statistically significant with the two-tailed t-Student test, p <0.05 in the 3 peptides with respect to the negative control and in the PaSK peptide also with respect to TcDef3-pep and hBD-3 (Figure 2).
    [0220]
    [0221] The antimicrobial activity of the PaSK peptide was also analyzed at lower concentrations, 10, 15 and 20 pg / mL, under the same conditions of the previous experiments. Figure 3 shows the percentage of cell death at these concentrations. It is observed that at the concentration of 10 pg / mL in which the TcDef3-pep peptide showed a mortality percentage of 9.2% (Contreras et al., 2015), the PaSK peptide already has a cytotoxicity close to 100%.
    [0222]
    [0223] Example 2. Effect of the TcDef3-pep and PaSK peptides on the morphology of S. aureus
    [0224]
    [0225] Scanning Electron Microscopy
    [0226] Scanning electron microscopy provides high resolution images and was used to visualize the damage exerted by the peptides on the bacteria membrane. S. aureus CECT 4013 cells were treated with the hBD-3, TcDef3-pep and PaSK peptides at a concentration of 25 pg / mL, for 1 h, and analyzed by scanning electron microscopy (Figure 4). In the untreated cells the typical circular and uniform conformation of S. aureus with the intact membrane was observed, while in the treated cells different types of morphological alterations are noticed. The images of the cells treated with hBD-3 show structural damage and lysis of the membrane (continuous arrows), in addition to a large leak of the cytoplasmic content (dashed arrows). In the cells treated with the TcDef3 peptide, bulges in the bacterial membrane are observed (arrows continuous) and some cytoplasmic remains (dashed arrows), although not as excessive as in the treatment with hBD-3. Finally, cells treated with the PaSK peptide show irregular membrane morphologies (continuous arrow) and complete cell decays with the consequent release of cytoplasmic residues (dashed arrow). With this treatment, small circular structures of smaller size than S. aureus cells can be seen .
    [0227]
    [0228] In the images obtained by scanning electron microscopy of the cells treated with the peptides, the formation of biofilm in the bacteria was observed (Figure 5). It was ruled out that exopolysaccharide formation was caused by lack of nutrients or other characteristics of the culture medium, since samples treated with hBD-3 (positive control) and those carrying H 2 O (negative control) grew with the same medium and did not form biofilm (Figure 4).
    [0229]
    [0230] Transmission Electron Microscopy
    [0231] Transmission electron microscopy provides high resolution images and was used to visualize the ultrastructural damage exerted by the peptides inside the bacteria. S. aureus CECT 4013 cells were treated with hBD-3, TcDef3-pep and PaSK peptides at a concentration of 25 pg / mL, for 1 hour and analyzed by transmission electron microscopy (Figure 6). In the untreated cells, the cytoplasmic membrane (3) and the intact peptidoglycan wall (2) were observed, typical of a Gram such as S. aureus, as well as the dividing septum (1) without malformations, while in the cells treated different types of alterations are noticed. The images of the cells treated with hBD-3 show malformations (4) and lysis of the membrane (5), in addition to a large leak of the cytoplasmic content, remnants of detached wall (6) and vacuolation of the cytoplasm (7). In the cells treated with the TcDef3-pep peptide, malformations and bulges are observed in the bacterial membrane (4), some cytoplasmic remains and detached wall remains (6). Finally, the cells treated with the PaSK peptide show detachments, malformations and thinning of the membrane (4), leakage of cytoplasmic remains (6) and malformations and inhibition of the septum of division (8). Small circular structures were observed (9). These structures show a different electrodensity from the cells and lack a cytoplasmic membrane and a peptidoglycan wall. The biofilm formed by the cells treated with the peptides was also observed in the images obtained by transmission electron microscopy (Figure 7) and was not observed in the cells treated with the human defensin hBD-3 (Figure 6).
    [0232] Example 3. Analysis of the cytotoxic and antiproliferative activity of the PaSK peptide
    [0233]
    [0234] The cytotoxic effect of the PaSK peptide on two triple negative breast cancer cell lines, one human (MDA-MB-231) and one mouse (4T1), and one normal mammary epithelial cell mouse line (HC-) eleven). For this, an MTS cell viability test was carried out based on the use of a tetrazolium-derived compound that is reduced in living cells to form a soluble colored product.
    [0235]
    [0236] Different concentrations of peptide (5 pg / mL at 700 pg / mL) were tested at different times (3 h, 24 h and 48 h) and in the presence of different concentrations of fetal bovine serum in the assay (0.5% and 10%), since it has been described that serum affects the activity of antimicrobial peptides. The results obtained showed that for a concentration of fetal bovine serum in the 0.5% assay and 24 h of incubation, the PaSK peptide exhibits dose-dependent cytotoxic activity in the two triple negative breast cancer cell lines under study and also in the normal mammary epithelial cell line, with an average IC 50 of approximately 200 pM (700 pg / mL) in all three cases (Figure 8). These data confirm that the PaSK peptide exhibits cytotoxic activity in mammalian cells at high concentrations. However, in a range of peptide concentrations around 100 pM (400 pg / mL), PaSK was significantly more cytotoxic for human triple negative breast cancer cells (MDA-MB-231).
    [0237]
    [0238] A cell proliferation analysis was carried out by flow cytometry of Oregon Green fluorophore labeled tumor cells subsequently treated with a concentration of 50 pM (200 pg / mL) of PaSK or H 2 O peptide as a control. This analysis allowed exploring the use of PaSK in a type of combined therapeutic strategy. The combined use of therapeutic agents aimed at inhibiting the growth of the tumor and its maintenance and agents capable of stimulating the patient's immune response in the treatment of cancer is an alternative therapy strategy to conventional strategies based solely on cytotoxic agents to reduce the impact. of the two main problems associated with the exclusive use of cytotoxic agents in cancer therapy: non-specific toxicity and the emergence of resistance.
    [0239]
    [0240] The Oregon Green reagent covalently binds to free amino groups of the cells giving them a homogeneous fluorescence, which is distributed between the daughter cells during each cell division. Since the fluorescence intensity of the cells is divided in half approximately after each cell division, the final fluorescence intensity after Treatment with the peptide and in the untreated control cells in relation to the initial fluorescence intensity of the cells provides information on how many cell division cycles have occurred in each case. The results obtained are shown in Figure 9. Cells treated with the PaSK peptide have a higher fluorescence intensity than untreated cells, indicating that the peptide inhibits the proliferation of human triple negative breast cancer cells.
    [0241]
    [0242] Example 4. Analysis of the cytotoxic and antiproliferative activity of the PaSK peptide
    [0243]
    [0244] In this example, the cytotoxic and antiproliferative activity of the PaSK peptide was analyzed by flow cytometry, performing a cell viability analysis and a cell cycle analysis. After the treatment of the MDA-MB-231 tumor cells with 200 pg / mL (cell viability experiments) and 100 pg / mL (cell cycle analysis) of PaSK the percentage of viable cells was determined, as well as the cycle phase cell in which they stopped proliferation.
    [0245]
    [0246] Figures 10A and 10B show the graphs corresponding to the cell viability analyzed from the fluorescence of propidium iodide (IP), represented in the graphs coming from the flow cytometer (the P3 region represents dead cells). In the control experiment (Figure 10A), a cell viability of 98% was obtained, while in the treatment with PaSK (Figure 10B) the cell viability was 93%.
    [0247]
    [0248] Figure 10C shows the percentage of living cells and the standard deviations (SD) of the two replicates analyzed for PaSK treatment and for control cells. The mortality percentage of the PaSK peptide at a concentration of 200 pg / mL against MDA-MB-231 cells was around 5%, statistically significant with respect to the control.
    [0249]
    [0250] Figures 11A and 11B show the graphs with the distribution of cells found in each phase of the cell cycle obtained by flow cytometry, detecting the fluorescence of propidium iodide. In the control cells and in the cells treated with PaSK the following distribution was observed: in phase G1 52.0% and 57.9%, in phase S 40.0% and 33.2% and in phase G28.0% and 9.0%, respectively.
    [0251]
    [0252] Figure 11C shows the percentage of cells that are in each phase of the cell cycle, G1, S and G2 and the standard deviations (SD) of the two replicates analyzed for the PaSK treatment and for control cells. It was compared if there were statistically significant differences in each phase of the cycle between the control cells and the cells treated with the peptide using the two-tailed t-Student test. Figure 11D is a schematic of the cell cycle showing the phase affected by the action of the PaSK peptide.
    [0253]
    [0254] The PaSK peptide shows antiproliferative activity, since the treated cells show a statistically significant decrease in the percentage of S-phase cells and an increase in G1 phase, although the latter lacks statistical significance (Figure 11C and 11D).
    [0255]
    [0256] Example 5. Differential proteomic analysis in triple negative breast cancer cells treated with the PaSK peptide
    [0257]
    [0258] A differential proteomic analysis was performed by SWATH to elucidate the molecular mechanisms by which the peptide exerts antiproliferative activity against triple negative breast cancer cells MDA-MB-231 without killing the cells.
    [0259]
    [0260] Eight samples were analyzed (4 replicates of the control and 4 replicates of the PaSK treatment) in which a total of 1,571 proteins (1% FDR) were quantified. From the quantitative data, a discriminant analysis was performed with the Marker View 1.3 software, obtaining two clearly differentiated groups, the control replicas and the treatment replicas (Figure 12).
    [0261]
    [0262] A t-Student test (p <0.05) was performed between both conditions (control and treatment) and of the 1,571 total quantified proteins (1% FDR), 31 were differentially expressed. Of these, 24 decreased their abundance and 7 increased it in the treatment with respect to the control (not shaded and shaded, respectively, in Table 1).
    [0263] Table 1 Differential protein expression of MDA-MB-231 cells treated and not treated with the PaSK peptide. In the treated samples versus control samples, the non-shaded and shaded proteins decrease and increase their abundance, respectively (t-Student test, p <0.05). T means treated samples, C means control samples.
    [0264]
    [0265]
    [0266]
    [0267]
    [0268]
    [0269] Additionally, the differentiation of differential proteins into functional groups was carried out in Uniprot, where the groupings were observed descriptively for both the proteins with diminished and increased abundances in the treatment / control relationship. Figure 13 shows the distribution by molecular function. Of the proteins with diminished abundance, the majority functions correspond to proteins of union (91.7%), those that present catalytic activity (33.3%), with structural molecular activity (16.7%), regulators of molecular function (12.5%) and finally, transcriptional co-activators, transporters and translation initiators (4.2%) (Figure 13A). On the other hand, of the proteins with increased abundance, the majority are also binding proteins (85.7%), which have catalytic activity (71.4%), with antioxidant activity and enzymatic regulation activity, each (28.6%) and finally , with electron transport activity (14.3%) (Figure 13B).
    [0270]
    [0271] The results of the differential proteomic analysis confirm that the PaSK peptide acts on intracellular targets. Of the 31 proteins with statistically significant differential expression, 24 decreased their expression and 7 increased it in MDA-MB-231 cells treated with the peptide (Table 1). These are proteins with oncogenic capacity, some of which have been described as being overexpressed in tumor cells of different types and are involved, for example, in the transport of vesicles, signal transduction and apoptosis, in the alteration of metabolic pathways. , in processes of DNA repair, transcription or post-transcriptional regulation and translation, in cell adhesion and motility related to metastases and in responses to oxidative stress.
    [0272]
    [0273] Of the 24 proteins that decrease their expression in cells treated with PaSK, in relation to cell cycle regulation, the eukaryotic translation initiation factor 3B (EIF3B), the ribosomal proteins S13 and L5 (RPS13 and RPSL5, respectively) stand out ) and Protein 1 related to La (LARP1). EIF3 is a protein complex that organizes a network of interactions between several eukaryotic translation initiation factors that are associated in the 40S subunit and participate in the different reactions involved in the translation. In addition, it has other regulatory functions such as restarting the translation of polycistronic mRNAs and acting as a receptor for protein kinases that control protein synthesis. It has been shown that negative regulation of EIF3B expression causes the accumulation of G0 / G1 phase cells, significantly reducing the number of S-phase tumor cells, suggesting that EIF3B may be associated with an inhibition in DNA replication. which leads to a reduction of the cell growth rate.
    [0274]
    [0275] The ribosomal proteins S13 and L5 are part of the 40S and 60S subunit of the ribosome, respectively. It has been previously described that overexpression of RPS13 in gastric cancer cells promoted the growth and transition from the G1 phase to the S phase of the cell cycle, while when RPS13 was negatively regulated in said cells, it increased the number of cells arrested in G1 phase It has been described that the loss of RPL5 cancels ribosome biogenesis and protein synthesis. Such loss does not induce a complete stop of the cell cycle, but strongly inhibits its progression. The decrease in both EIF3B and RPL5 would induce a cell cycle control point independent of p53. These results are consistent with those obtained in the analysis of the antiproliferative activity of the PaSK peptide of Example 4, where a significant decrease in MDA-MB-231 cells in S phase was observed (Figure 11C), which suggests an independent cell cycle control of p53, since MDA-MB-231 cells possess the mutated and non-functional p53 gene, as discussed above.
    [0276]
    [0277] Three of the proteins with increased abundance in cells treated with the PaSK peptide are glutathione peroxidase 1 (GPX1), thioredoxin reductase 1 (TXNRD1) and sulfur: quinone oxidoreductase (SQRDL). All three proteins are involved in the response to oxidative stress and are usually overexpressed in tumor cells. Because oxidation protection activates survival genes and inhibits apoptosis, overexpression of such proteins could be a response of MDA-MB-231 cells to the anti-cancer activity of the PaSK peptide.
    [0278]
    [0279] Example 6. Effect of the PaSK peptide on the morphology of triple negative breast cancer cells
    [0280]
    [0281] Both scanning electron microscopy and transmission electron microscopy provide high resolution images and were used to visualize the morphological and ultrastructural change produced by the PaSK peptide in the membrane and inside the tumor cells. MDA-MB-231 cells were treated with the PaSK peptide at a concentration of 100 pg / mL, for 72 h, and analyzed by scanning electron microscopy (Figure 14) and transmission electron microscopy (Figure 15). The images reveal that the PaSK peptide induced major morphological changes in the MDA-MB-231 tumor cells. Both in scanning electron microscopy and in transmission electron microscopy in untreated cells, a circular conformation was observed with the continuous and intact membrane and the typical hair cells of human cells (1). However, in the cells treated with PaSK an irregular membrane was observed in which the villi appear to be decomposing forming a network that is disintegrating (2). In addition, they presented very pronounced invaginations (3) and complete ruptures of the membrane, which in some cases were recirculated again forming vesicles with cytoplasmic remains (4). Although what appeared to be membrane expansions was also observed in untreated cells, said membrane is continuous (5) and was not interrupted as in the treated cells These results show that the PaSK peptide has a mechanism of membranolytic action.
    [0282]
    [0283] FREE TEXT OF THE LIST OF SEQUENCES
    [0284]
    [0285] SEQ ID NO: 1
    [0286] Defensin 3 of Tríbolium castaneum; PaSK fragment
    [0287]
    [0288] SEQ ID NO: 2
    [0289] Defensin 3 of Tríbolium castaneum; TcDef3-pep fragment
    [0290]
    [0291] REFERENCE LIST
    [0292]
    [0293] - Contreras, E., Benito-Jardón, M., López-Galiano, M. J., Real, M. D. and Rausell, C. (2015).
    [0294] Tríbolium castaneum immune defense genes are differentially expressed in response to Bacillus thuringiensis toxins sharing common receptor molecules and exhibiting disparate toxicity. Developmental and Comparative Immunology. 50, 139-145.
    [0295] - Rajamuthiah, R. et al. (2015). A defensin from the model beetle Tríbolium castaneum acts synergistically with telavacin and daptomycin against multidrug resistant Staphylococcus aureus. PLOS One. 10 (6): 1-14.
    [0296] - Tonk, M. et al. (2015). Tribolium castaneum defensins are primarily active against Grampositive bacteria. J Invertebrate Pathol. 132: 208-215.
    权利要求:
    Claims (22)
    [1]
    1. Peptide consisting of the sequence SEQ ID NO: 1.
    [2]
    2. A pharmaceutical composition comprising the peptide according to claim 1 and at least one pharmaceutically acceptable carrier or excipient.
    [3]
    3. The pharmaceutical composition according to claim 2, characterized in that said carrier is selected from the group consisting of organic nanoparticles, selected from the group consisting of: lipids, nanoemulsions, polymeric micelles, SCK nanoparticles, liposomes, nanogels, hydrogels, lipoplexes, polylexes; polymers selected from the group consisting of: albumin, cellulose, chitosan, alginate, gelatin, poly-caprolactone (PCL), starch hydroxyethyl (HES; MEA), polyglycolate (PGA), poly (lactic-co-glycolide) , polylactide (PLA), poly (d, 1-lactide-co-glycolide) (PLGA), polyethylene glycol (PEG), W- (2-Hydroxypropyl) methacrylamide (poly (HPMA); PHPMA) and dextran; dendrimers, selected from the group consisting of: polyether hydroxylamine (PEHAM), polyamidoamine (PAMAM), polyetheramine, polypropyleneimine and polyglycerol; nanofibers, selected from the group consisting of: carbon nanotubes, poly (d, 1-lactide-co-glycolide) nanofibers (PLGA), polyethylene glycol (PEG), chitosan, polyvinyl alcohol (PVA), polylactide (PLA), polyethylene oxide and polycaprolactone (PCL); and inorganic nanoparticles, selected from the group consisting of: gold nanoparticles, metal oxide nanoparticles, titanium oxide nanoparticles, platinum oxide nanoparticles, superparamagnetic iron oxide nanoparticles (SPIO-NPs), diamond-based nanoparticles and nanoparticles QD
    [4]
    4. The pharmaceutical composition according to claim 3, further comprising an antibiotic agent.
    [5]
    5. The pharmaceutical composition according to claim 4, characterized in that said antibiotic agent is selected from the group consisting of fusidic acid, arsfenamine, clindamycin, chloramphenicol, ethambutol, fosfomycin, furazolidone, isoniazid, lincomycin, linezolid, metronidazole, mupirocin, nitrofurantoin, pyrazinamide, platensimycin, quinupristin, rifampicin and tinidazole or an antibiotic of the selected class of: aminoglycosides, ansamycins, carbacefem, carbapenem, cephalosporins, glycopeptides, macrolides, monobactamics, penicillins, polypeptides, quinolones, sulfolidases, quinolones, quinolones, tetrabolones
    [6]
    6. The peptide according to claim 1 or the pharmaceutical composition according to any of claims 2 to 5, for use as a medicament.
    [7]
    7. The peptide or pharmaceutical composition for use according to claim 6, as an antimicrobial medicament.
    [8]
    8. The peptide or pharmaceutical composition for use according to claim 7, as an antimicrobial medicament in the treatment of infections caused by Gram + bacteria, Gram- bacteria and fungi.
    [9]
    9. The peptide or pharmaceutical composition for use according to claim 8, characterized in that said Gram + bacterium is Staphylococcus aureus.
    [10]
    10. The peptide or pharmaceutical composition for use according to claim 8, characterized in that said Gram-bacterium is Escherichia coli.
    [11]
    11. The peptide or pharmaceutical composition for use according to claim 8, characterized in that said fungus is Candida albicans.
    [12]
    12. A peptide comprising the sequence SEQ ID NO: 1 or a pharmaceutical composition comprising the peptide comprising the sequence SEQ ID NO: 1 and at least one pharmaceutically acceptable carrier or excipient, for use in the treatment of cancer.
    [13]
    13. The peptide or pharmaceutical composition for use according to claim 12, characterized in that the cancer is selected from the group consisting of breast cancer, breast cancer resistant to anti-HER2 therapy, breast carcinoma, breast adenocarcinoma, carcinoma gastric, gastric adenocarcinoma, colon carcinoma, colon adenocarcinoma, pancreatic carcinoma, pancreatic adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, ovarian carcinoma, ovarian adenocarcinoma, ovarian carcinoma, endometrial carcinoma, cervical carcinoma , lung carcinoma, pulmonary adenocarcinoma, non-small cell lung cancer, small cell lung cancer, thyroid carcinoma, metastatic papillary thyroid carcinoma, thyroid follicular carcinoma, bladder carcinoma, transitional cell carcinoma of the urinary bladder, prostate carcinoma, glial lineage cancer of the central nervous system (glioma), sarcomas , fibrosarcoma, malignant fibrous histiocytoma, human Edwing sarcoma, endometrial stromal sarcoma, osteosarcoma, rhabdomyosarcoma, melanoma, embryonic cancers, neuroblastoma, medulloblastoma, retinoblastoma, nephroblastoma, hepatoblastoma, hematologic cancers, B or T cell leukemia, non-Hodgkin lymphoma, non-Hodgkin B or T cell lymphoma, Burkitt lymphoma, Hodgkin lymphoma, leukemia, B or T cell lymphoma and multiple myeloma.
    [14]
    14. The peptide or pharmaceutical composition for use according to claim 13, characterized in that the cancer is breast cancer.
    [15]
    15. The peptide or pharmaceutical composition for use according to claim 14, characterized in that the breast cancer is triple negative breast cancer.
    [16]
    16. The peptide or pharmaceutical composition for use in the treatment of cancer, according to any of claims 12 to 15, in combination with a treatment with a chemotherapeutic agent, with a treatment with an immunotherapeutic agent, or with a radiotherapy treatment.
    [17]
    17. The peptide or pharmaceutical composition for use according to claim 16, characterized in that said chemotherapeutic agent is selected from the group consisting of anastrozole, capecitabine, carboplatin, oxaliplatin, cyclophosphamide, cisplatin, docetaxel, doxorubicin, eribulin, fulvestrant, imiquimod , letrozole, paclitaxel, romidepsin, triciribin, xemestane, 5-fluorouracil and gemcitabine.
    [18]
    18. The peptide or pharmaceutical composition for use according to claim 16, characterized in that said immunotherapeutic agent is selected from the group consisting of dovitinib, ipilimumab, lapatinib, margetuximab, neratinib, nivolumab, olaparib, palbociclib, pembrolizumab, pertuzumab, ruxolitinib trastuzumab and veliparib.
    [19]
    19. The pharmaceutical composition for use according to any of claims 12 to 18, characterized in that the carrier is selected from the group consisting of organic nanoparticles, selected from the group consisting of: lipids, nanoemulsions, polymeric micelles, SCK nanoparticles, liposomes, nanogels, hydrogels, lipoplexes, pollexlexes; polymers selected from the group consisting of: albumin, cellulose, chitosan, alginate, gelatin, poly-caprolactone (PCL), starch hydroxyethyl (HES; MEA), polyglycolate (PGA), poly (lactic-co-glycolide) , polylactide (PLA), poly (d, 1-lactide-co-glycolide) (PLGA), polyethylene glycol (PEG), W- (2-Hydroxypropyl) methacrylamide (poly (HPMA); PHPMA) and dextran; dendrimers, selected from the group consisting of: polyether hydroxylamine (PEHAM), polyamidoamine (PAMAM), polyetheramine, polypropyleneimine and polyglycerol; nanofibers, selected from the group consisting of: carbon nanotubes, poly (d, 1-lactide-co-glycolide) nanofibers (PLGA), polyethylene glycol (PEG), chitosan, polyvinyl alcohol (PVA), polylactide (PLA), polyethylene oxide and polycaprolactone (PCL); and inorganic nanoparticles, selected from the group consisting of: gold nanoparticles, metal oxide nanoparticles, titanium oxide nanoparticles, platinum oxide nanoparticles, superparamagnetic iron oxide nanoparticles (SPIO-NPs), diamond-based nanoparticles and nanoparticles QD
    [20]
    20. The pharmaceutical composition for use according to any of claims 12 to 19, further comprising a chemotherapeutic agent or an immunotherapeutic agent.
    [21]
    21. The pharmaceutical composition for use according to claim 20, characterized in that said chemotherapeutic agent is selected from the group consisting of anastrozole, capecitabine, carboplatin, oxaliplatin, cyclophosphamide, cisplatin, docetaxel, doxorubicin, eribulin, fulvestrant, imiquimod, letrozole, paclitaxel, romidepsin, triciribin, xemestane, 5-fluorouracil and gemcitabine.
    [22]
    22. The pharmaceutical composition for use according to claim 20, characterized in that said immunotherapeutic agent is selected from the group consisting of dovitinib, ipilimumab, lapatinib, margetuximab, neratinib, nivolumab, olaparib, palbociclib, pembrolizumab, pertuzumab, ruxolitinib, trastuzuma Veliparib
    类似技术:
    公开号 | 公开日 | 专利标题
    Wang et al.2017|Hyaluronic acid-coated chitosan nanoparticles induce ROS-mediated tumor cell apoptosis and enhance antitumor efficiency by targeted drug delivery via CD44
    Ma et al.2013|Modulation of drug-resistant membrane and apoptosis proteins of breast cancer stem cells by targeting berberine liposomes
    Yu et al.2012|Mitochondrial targeting topotecan-loaded liposomes for treating drug-resistant breast cancer and inhibiting invasive metastases of melanoma
    Wen et al.2014|Antagonism of tumoral prolactin receptor promotes autophagy-related cell death
    Nakayama et al.2016|Preclinical activity of selinexor, an inhibitor of XPO1, in sarcoma
    Hilchie et al.2016|Mastoparan is a membranolytic anti-cancer peptide that works synergistically with gemcitabine in a mouse model of mammary carcinoma
    Wen et al.2017|Micellar delivery of miR-34a modulator rubone and paclitaxel in resistant prostate cancer
    US8106024B2|2012-01-31|Method of treating cancer with an RPN2 gene expression inhibitor
    Xiao et al.2016|YAP induces cisplatin resistance through activation of autophagy in human ovarian carcinoma cells
    Guo et al.2010|Effects of stealth liposomal daunorubicin plus tamoxifen on the breast cancer and cancer stem cells
    US9770482B2|2017-09-26|Targeting the EGFR-SGLT1 interaction for cancer therapy
    Hossainzadeh et al.2019|Silibinin encapsulation in polymersome: A promising anticancer nanoparticle for inducing apoptosis and decreasing the expression level of miR‐125b/miR‐182 in human breast cancer cells
    Vallespi et al.2010|Identification of a novel antitumor peptide based on the screening of an Ala‐library derived from the LALF32–51 region
    Ling et al.2018|An ABCG2 non-substrate anticancer agent FL118 targets drug-resistant cancer stem-like cells and overcomes treatment resistance of human pancreatic cancer
    Ju et al.2018|PTD modified paclitaxel anti-resistant liposomes for treatment of drug-resistant non-small cell lung cancer
    Qi et al.2016|Targeting CD133+ laryngeal carcinoma cells with chemotherapeutic drugs and siRNA against ABCG2 mediated by thermo/pH-sensitive mesoporous silica nanoparticles
    Morán et al.2018|Dual responsive gelatin-based nanoparticles for enhanced 5-fluorouracil efficiency
    Hong et al.2019|Improving the anticancer effect of afatinib and microRNA by using lipid polymeric nanoparticles conjugated with dual pH-responsive and targeting peptides
    Michlewska et al.2019|Ruthenium dendrimers against acute promyelocytic leukemia: in vitro studies on HL-60 cells
    Wang et al.2004|Bimodal effect of hypoxia in cancer: role of hypoxia inducible factor in apoptosis
    ES2717685B2|2020-07-10|Peptide and pharmaceutical compositions thereof for use as an antimicrobial and in the treatment of cancer
    Zhang et al.2017|Big data analysis of global advances in pharmaceutics and drug delivery 1980-2014
    Wang et al.2016|Eradication of CD44-variant positive population in head and neck tumors through controlled intracellular navigation of cisplatin-loaded nanomedicines
    Chen et al.2006|The role of the VPS4A-exosome pathway in the intrinsic egress route of a DNA-binding anticancer drug
    Haque et al.2021|ROS-mediated necrosis by glycolipid biosurfactants on lung, breast, and skin melanoma cells
    同族专利:
    公开号 | 公开日
    ES2717685B2|2020-07-10|
    WO2019122489A1|2019-06-27|
    EP3730513A1|2020-10-28|
    CN111491947A|2020-08-04|
    CA3085408A1|2019-06-27|
    EP3730513A4|2021-10-27|
    JP2021509106A|2021-03-18|
    US20210002339A1|2021-01-07|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2019-06-24| BA2A| Patent application published|Ref document number: 2717685 Country of ref document: ES Kind code of ref document: A1 Effective date: 20190624 |
    2020-07-10| FG2A| Definitive protection|Ref document number: 2717685 Country of ref document: ES Kind code of ref document: B2 Effective date: 20200710 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201731455A|ES2717685B2|2017-12-22|2017-12-22|Peptide and pharmaceutical compositions thereof for use as an antimicrobial and in the treatment of cancer|ES201731455A| ES2717685B2|2017-12-22|2017-12-22|Peptide and pharmaceutical compositions thereof for use as an antimicrobial and in the treatment of cancer|
    PCT/ES2018/070824| WO2019122489A1|2017-12-22|2018-12-21|Peptide and pharmaceutical compositions of same for use as an antimicrobial and in cancer treatment|
    CA3085408A| CA3085408A1|2017-12-22|2018-12-21|Peptide and pharmaceutical compositions therof for use as an antimicrobial and in cancer treatment|
    EP18890389.2A| EP3730513A4|2017-12-22|2018-12-21|Peptide and pharmaceutical compositions of same for use as an antimicrobial and in cancer treatment|
    US16/955,370| US20210002339A1|2017-12-22|2018-12-21|Peptide and pharmaceutical compositions of same for use as an antimicrobial and in cancer treatment|
    CN201880082529.9A| CN111491947A|2017-12-22|2018-12-21|Peptides and pharmaceutical compositions thereof for use as antimicrobial agents and for the treatment of cancer|
    JP2020533058A| JP2021509106A|2017-12-22|2018-12-21|Peptides and their pharmaceutical compositions for use as antibacterial agents and in the treatment of cancer|
    [返回顶部]