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    • 专利摘要:
    PEPTIDE AS A MEDICINE, IN PARTICULAR FOR THE TREATMENT OF CANCER. The present invention relates to a SEQ 1 sequence peptide: INWLKIAKKVAGML-NH2, and one or more of its chosen variants, among the SEQ sequences. 2 to SEQ. 25, or one of its mixtures. In particular, these peptides are used as a medicine, notably for the treatment of cancer. The present invention also relates to a pharmaceutical composition, comprising these peptides.
    公开号:BR112012029975B1
    申请号:R112012029975-1
    申请日:2011-05-20
    公开日:2020-10-27
    发明作者:Esther Suzy Arlette Fellous；Jorge Kalil；Mario Palma
    申请人:Esther Suzy Arlete Fellours；Jorge Kalil；Mario Palma；
    IPC主号:
    专利说明:

    [001] The present invention relates to a pharmaceutical composition, comprising at least one particular peptide for its application as a medicine in the treatment of cancer.
    [002] To treat cancer, there are conventionally three types of therapies: surgery to remove the tumor, radiation therapy to destroy the tumor by lightning, and medical treatments (chemotherapy). Surgery and radiation therapy are heavy treatments that are difficult for the patient to endure. In addition, these three treatments should be more often associated, as they are complementary. They must be combined within a programmed strategy.
    [003] With regard to medical treatments, they aim to treat not only the affected organ, but also the entire organism by administering anti-cancer drugs. Thus, unlike surgery or radiotherapy that treat the tumor only locally, only medical treatments have a general action that allows to treat a tumor that has already spread or is in the process of doing so. There are several types of medical treatments: cytotoxic chemotherapy and hormone therapy are the most classic; immunotherapy, gene therapy and non-cytotoxic therapies are still in the field of research.
    [004] The document WO00 / 02581 refers to the use of a peptide for the manufacture of a medicine intended for the treatment or prophylaxis of cancer, the peptide being constituted notably by the sequences EARPALLTSRLRFIPK, DGLRPIVNMDYVVGAR, GVPEYGCWNLRKTWNF, ILAKFLHVYV or ELAKFLHVYV or ELL.
    [005] The publication “Monitoring the positioning of short polycationic peptides in model lipid bilayers by combining hydrogen / deuterium exchange and electrospray ionization mass spectrometry”, from Biochemistry and Biophysics Acta, 2008, pages 2797 to 2805, describes the use of a spectrometer electrospray ionization mass analysis to analyze the hydrogen / deuterium exchange properties of the Apoica-MP INWLKIAKKVAGML-NH2 peptide.
    [006] Despite extensive research in this field, there is currently no drug that is both effective in the treatment of cancers and that preserves the healthy cells of a human or animal organism. Today's chemotherapy always leads to more or less serious side effects.
    [007] The purpose of the present invention is to propose a new medicament peptide and a new pharmaceutical composition that avoid all or part of these side effects.
    [008] For this purpose, the invention has as its object a peptide comprising at least one sequence chosen from: the sequences SEQ ID NO: 2 to SEQ ID NO: 25.
    [009] The invention also relates to a peptide comprising at least one sequence chosen from: the sequences SEQ ID NO: 1 to SEQ ID NO: 25 for use as a medicine.
    [0010] Preferably, the peptides of sequences SEQ ID NO: 1 to SEQ ID NO: 25 are used in the treatment of cancer. In particular, the peptide sequences acting as an active ingredient to treat cancer are as follows (Table 1): Table I




    [0011] The applicant's work on these peptides from SEQ ID NO: 1 to SEQ ID NO: 25 has allowed us to put, surprisingly, evidence that these peptides have a cytotoxic activity against cancer cells, not being toxic or very slightly toxic , for the host cells to be treated. The active peptides, according to the invention, specifically alter the microtubules of cancer cells.
    [0012] Constituents of one of the three filament networks of the cytostructure, the microtubules are complex and unstable polymers, whose base unit is the dimer of tubulins α and β. They are at the origin of multiple cellular movements, axonal transport, or motility, for example, and, above all, they provide the base material for the constitution of the mitotic spindle. Microtubules are hollow tubes of variable length and undergoing perpetual reform. Thus, by disrupting the dynamics of cancer cell microtubules, the peptides, according to the invention, also act as antimitotics, preventing the proliferation of cancer cells (reducing or stopping the mitosis of cancer cells).
    [0013] The active peptides, according to the invention, would also specifically alter the plasma membranes of cancer cells and would not induce these same changes in the healthy cells of the host organism. This could be explained by the fact that the active peptides, because of their polycationic character, would act on the plasma membranes of cancer cells rich in negative charges. Therefore, the active peptides, according to the invention, have an action on two different cellular compartments: the intracellular cyto-structure and the plasma membrane of cancer cells.
    [0014] Active peptides, which comprise only 10 to 14 amino acids, also have the advantage of being very easy to synthesize.
    [0015] Preferably, the peptide (s) mentioned in table I, applied for use as a medicine for the treatment of cancer, are notably effective for the treatment of breast cancer, glioblastoma, breast cancer brain, cervix, colorectal, cutaneous, endometrium, stomach, liver, gastrointestinal stroma, malignant haemopathies (leukemia, multiple myeloma, lymphomas (Hodgkin's disease, non-hodgkin's lymphoma), hepatocellular carcinoma, Kaposi's sarcoma cancer of the larynx, mesothelioma, cancer of the esophagus, eye, osteo-sarcoma, cancer of the ovary, pancreas, skin (notably mouth), cancer of the lung, prostate, rhabdomyosarcoma, cancer of the kidney, breast, testis, thyroid, soft tissue, bladder carcinoma, myeloma (bone cancer) and plasmacytoma.
    [0016] According to another characteristic of the invention, peptides of sequences SEQ ID NO: 1 to SEQ ID NO: 25 are produced by chemical synthesis.
    [0017] Another purpose of the present invention relates to a pharmaceutical composition, comprising one of the peptides, as defined above.
    [0018] Advantageously, the composition is in the form of a solution, an aqueous suspension, or in the dry state in the form of uncoated or coated tablets, such as pills, jellies, capsules or powders.
    [0019] If the pharmaceutical composition is in the dry state, it can be mixed with one or more inert diluents, such as starch, cellulose, sucrose, lactose or silica, etc. It can also comprise other substances in the dry state such as one or more lubricants, such as magnesium stearate, or talc, a dye, a coating (dragees) or a varnish.
    [0020] If the pharmaceutical composition according to the invention is in liquid form, it may comprise pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents, such as water, ethanol, glycerol, vegetable oils or paraffin oil. The pharmaceutical composition can comprise other liquid substances than diluents, for example, moistening, sweetening, thickeners, flavoring or stabilizing products.
    [0021] Preferably, the composition, possibly associated with a pharmaceutically acceptable non-toxic inert carrier excipient, is characterized by the fact that it is administered topically in the form of cutaneous, transcutaneous or percutaneous application; oral; parenteral; nasal or bronchial.
    [0022] The pharmaceutical composition for parenteral administration may preferably be an aqueous or non-aqueous solution, a suspension or an emulsion. As a solvent or vehicle, water, polypropylene glycol, vegetable oils, in particular olive oil or sesame oil, injectable organic esters, for example, ethyl oleate or other suitable organic solvents, will be mentioned. The composition rhabdomyosarcoma, cancer of the kidneys, sinus, testis, thyroid, soft tissue, bladder carcinoma, myeloma (bone cancer) and plasmacytoma.
    [0023] According to another characteristic of the invention, peptides of sequences SEQ ID NO: 1 to SEQ ID NO: 25 are products by chemical synthesis.
    [0024] Another purpose of the present invention relates to a pharmaceutical composition, comprising one of the peptides, as defined above.
    [0025] Advantageously, the composition is in the form of a solution, an aqueous suspension, or in the dry state in the form of uncoated or coated tablets, such as pills, jellies, capsules or powders.
    [0026] If the pharmaceutical composition is in the dry state, it can be mixed with one or more inert diluents, such as starch, cellulose, sucrose, lactose or silica, etc. It can also comprise other substances in the dry state such as one or more lubricants, such as magnesium stearate or talc, a dye, a coating (dragees) or a varnish.
    [0027] If the pharmaceutical composition according to the invention is in liquid form, it may comprise pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents, such as water, ethanol, glycerol, vegetable oils or paraffin oil. The pharmaceutical composition can comprise other liquid substances than diluents, for example, moistening, sweetening, thickeners, flavoring or stabilizing products.
    [0028] Preferably, the composition, possibly associated with a non-toxic pharmaceutically acceptable excipient or inert vehicle, is characterized by the fact that it is administered topically in the form of cutaneous, transcutaneous or percutaneous application; oral; parenteral; nasal or bronchial.
    [0029] The pharmaceutical composition for parenteral administration may preferably be an aqueous or non-aqueous solution, a suspension or an emulsion. As a solvent or vehicle, water, propylene glycol, vegetable oils, in particular olive oil or sesame oil, injectable organic esters, for example, ethyl oleate or other suitable organic solvents, will be mentioned. The pharmaceutical composition can also contain adjuvants, in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents.
    [0030] For topical administration, the drugs will be advantageously administered in the form of ointments, creams, gels or patches.
    [0031] In particular, the pharmaceutical composition is sterile.
    [0032] Sterilization can be done in several ways, for example by aseptic filtering, incorporating into the composition of the sterilizing agents, by irradiation or by heating. Medicines can also be prepared in the form of sterile solid compositions that can be dissolved at the time of use in sterile water or any other sterile injectable medium.
    [0033] The pharmaceutical composition may, in addition, be used alone or in combination, in a specific treatment with other drugs, or in combination with radiotherapy or surgery.
    [0034] The invention will be better understood and other purposes, details, characteristics and advantages of it will appear more clearly during the following description of a particular embodiment of the invention, given only by way of illustration and not limitation, with Reference: to the attached drawings . Examples of pharmaceutical tests are, moreover, proposed to illustrate, but in no case would they know how to be interpreted as limiting the scope of the invention.
    [0035] In these drawings:
    [0036] - figure 1 represents the effect of the SEQ ID NO: 1 peptide on the microtubule polymerization of a healthy rat brain at doses of 23.3 pM, 46.6 pM, 70 pM and 93.3 pM, as well as the effect of a sample proves not to have been subjected to the peptide of SEQ ID NO: 1; in particular, figure 1 represents the evolution of in vitro polymerization of rat brain microtubules, quantified by measurements of optical density at 345 nm of wavelength, as a function of time in minutes and in the presence or not of a SEQ ID peptide. NO: 1;
    [0037] - Figure 2 represents the peptide effect of SEQ ID NO: 1 at doses of 46.6 pm, 70 pm, 93.3 pM and 116 pM on the stability of healthy rat brain microtubules, assessed by the percentage microtubular depolymerization induced by a 30 min treatment at 4 ° C of the microtubules previously connected at 37 ° C for 15 min;
    [0038] - figure 3 represents photos of the cell line A549 (lung cancer) treated with the peptide of SEQ ID NO: 1 to 15 pM and not treated with this peptide (control at 0 pM), of the cell line NMT- 1 treated with the peptide of SEQ ID NO: 1 to 15 pM and not treated with that peptide (control) and of the cell line PANC-1 (pancreatic cancer) treated with the peptide of SEQ ID NO: 1 to 15 pM and not treated with that peptide (control);
    [0039] - figure 4 represents photos of non-tumor cell lines, FIBRO 1 and FIBRO 2 (fibroplastic cells) treated with the peptide of SEQ ID NO: 1 to 15 pM and not treated with this peptide (control);
    [0040] - figure 5 represents four graphs that represent the reliability of cells of different strains: T (Cos kidney lineage); G (lung line A549); C (HepG2 liver strain); R (pancreatic PANC1 lineage); S (pancreatic lineage MiaPaCa); X (adrenal line H2 95 R); Y (melanoma strain MNT-1); leukemic lymphocyte lines L1 (HL 60); L3 (U 937); L4 (NB 4); L5 (MenoMacβ), and a lineage of normal L6 lymphocytes, depending on the peptide concentration of SEQ ID NO: 1;
    [0041] - figure 6 represents 4 phase contrast images of cancer cells A 549 (lung cancer), showing their morphology in the absence of the SEQ ID NO: 1 peptide (evidence images A and B) and the alterations of its morphology induced by 15 pM of the peptide of SEQ ID NO: 1 (image C taken 4 hours after injection of the peptide of SEQ ID NO: 1 and image D taken 2 hours after injection of the peptide of SEQ ID NO: 1) ; and
    [0042] - Figure 7 represents a graph, showing the effect of the peptide of SEQ ID NO: 1 (concentration of 15 pM) on the loss of adhesion to the substrate of cancer cells A 549 as a function of time. MATERIALS AND METHODS A1) Synthesis of peptides SEQ ID NO: 1 to SEQ ID NO: 25 and purification
    [0043] The peptides are prepared by synthesis on solid phase fractionated manually, using N-9-fluoro phenyl methoxy-carbonyl with a TGS Novasyn resin. The side chains protect the groups, including t-butyl for serine and t-butoxy carbonyl for lysine. To prepare the peptides that have an N-terminal acetylated residue, after coupling the last amino acid residue, the anhydrous acid was added to the resin-peptide complexes and kept under stirring for 1 hour to promote acetylation.
    [0044] The cleavage of the resin-peptide complexes was carried out by treatment with trifluoro acetic acid / 1,2-ethane dithiol / anisol / phenol / water (82.5; 2.5; 5; 5; 5 by volume) at a concentration of 10 ml.g-1 at room temperature for 2 hours. Filtration was then carried out to remove the resin. Then, ethyl ether at 4 ° C was added, causing the crude peptides to precipitate, which were then collected after centrifugation at 1000 g for 15 minutes at room temperature. The crude peptides were then solubilized in water and chromatographed under RP-HPLC, using a semi-preparatory column (250 mm * 10.0 mm; 5 pm) C-18 from Shiseidido under an isocratic purification with 40% (v / v) of aceto-nitrile, containing 0.1% (v / v) TFA at a rate of 2 ml / min. Purification was set at 215 nm with a shimadzu UV-DAD detector (ultraviolet detector with diode bars), model SPD-M10A) and each purified fraction was manually collected in a 2 ml glass flask. The sequences of the synthesized peptides (acetylated and non-acetylated) were determined by HPLC and ESI-MS analyzes. A2) Peptides of SEQ ID NO: 1 to SEQ ID NO: 25
    [0045] So, after the peptides were synthesized, purified and examined, tests were done to determine their biological activities. Most of them were also shown to have antibacterial activity.
    [0046] Furthermore, it appeared, surprisingly and unexpectedly, that the peptides, according to the invention, had properties that alter the microtubules of cancer cells, and notably the peptide SEQ ID NO: 1 for which the results are described below. This peptide is a short peptide of 14 amino acids. This peptide has the following amino acid sequence: INWLKIA KKVA GML -NH2 (SEQ ID NO: 1).
    The sequence peptides SEQ ID NO: 2 to SEQ ID NO: 25 are variants or homologues of the peptide of SEQ ID NO: 1. These variants differ from the peptide SEQ ID NO: 1 by one to three amino acids (absence or substitution of one or more amino acids) and / or the absence of the -NH2 group at the N-terminal end. These variants have been shown to be as effective as the peptide of SEQ ID NO: 1, or have shown slightly less efficacy than the peptide of SEQ ID NO: 1, but are generally more active on types of cancers that respond a little worse to peptide SEQ ID NO: 1. EXPERIMENTAL TESTS 81) Effect of the SEQ ID NO: 1 sequence peptide on the properties of the purified microtubules (figure 1)
    [0048] Purified rat brain microtubules were prepared by an in vitro process dependent on the binding temperature at 37 ° C and shutdown at 4 ° C in the presence of GTP (guanosine triphosphate) described by FELLOUS et al. (1977) "microtubule assembly in vitro, purification of assembly-promoting factors", Eur J. Biochem, August 15, 19787, 15; 78 (1): 167-174.
    [0049] The in vitro polymerization of the microtubules was followed by the measurement of turbidity changes at 345 nm every 30 seconds during an incubation period of 15 minutes. The optical density measurements were made with a UVICON thermostat spectrophotometer at 37 ° C and equipped with an automatic charger that can accommodate 6 vats.
    [0050] As shown in figure 1, the peptide of SEQ ID NO: 1 induces a significant increase in turbidity during the process of microtubule polymerization in relation to the test sample not comprising the peptide of SEQ ID NO: 1 and serving as a control polymerization of microtubules. The increase in absorbance observed in the presence of the SEQ ID NO: 1 peptide goes with the increase in its concentration and can reach a very high level, which can only be explained by the only increase in normal polymerized microtubules. These data thus imply the formation of abnormal microtubule structures or unorganized tubulin aggregates in the presence of the SEQ ID NO: 1 peptide. Therefore, figure 1 demonstrates that the SEQ ID NO: 1 peptide has a direct action on microtubules that the intracellular microtubules should therefore be a target of the peptide. In addition, this polymerization of abnormal microtubules is similar to the polymerization of microtubules that occurs in the presence of a high rate of an already known anticancer: Taxol. 82) Action of the peptide SEQ ID NO: 1: similar to the action of Taxol (figure 2).
    [0051] Another argument that strongly suggests that the peptide of SEQ ID NO: 1 and Taxol show a certain similarity in their mechanism of action is shown in figure 2.
    [0052] The same type of experiments using the same technology as for B1) was performed to assess the stability of microtubules formed in the presence of increasing concentrations of the peptide of SEQ ID NO: 1. This stability is indicated by the inability of these microtubules to be depolymerize after treatment at 4 ° C for 30 minutes.
    [0053] Indeed, a decrease in the capacity of the microtubules to be depolymerized is observed in the presence of SEQ ID NO: 1, when they are subjected to the temperature of 4 ° C for 30 minutes. This decrease is all the more important the higher the concentration of SEQ ID NO: 1. It becomes total if the concentration of SEQ ID NO: 1 is very high. The microtubules formed in the presence of very high concentrations of the peptide of SEQ ID NO: 1 actually lose their ability to depolymerize when they are subjected for 30 minutes to a temperature of 4 ° C.
    [0054] The fact that the peptide of SEQ ID NO: 1 has a mechanism of action very close to that of Taxol is a certain advantage of the present invention. It should also be added that the applicant's observations, demonstrating the reversible effect of the SEQ ID NO: 1 peptide on microtubular properties, give it a greater advantage over other anti-microtubular peptides such as dolostatins, whose effect on microtubules is reversible .
    [0055] With tests at the cellular level, it is confirmed that the peptide of SEQ ID NO: 1 has a mechanism of action similar to that of Taxol. These tests show, in fact, that the peptide of SEQ ID NO: 1 abnormally and irreversibly increases the intracellular concentration of polymerized and / or aggregated tubulin, thus inducing a blockage in the mitosis process.
    However, the peptide of SEQ ID NO: 1 does not appear to bind on the same tubulin site as Taxol, as several strains obtained from taxol-resistant breast tumors and which maintained this resistance property in culture. These in vitro cells show a high sensitivity to the peptide of SEQ ID NO: 1 and some analogs of that peptide (cf. paragraph B9).
    [0057] This observation is greater, as it will be possible to consider an effective therapy, thanks to the peptide SEQ ID NO: 1 to 25 in patients who do not respond or no longer respond to a treatment by Taxol. 83) Effect of the peptide of SEQ ID NO: 1 on the alteration of the organization of the microtubule network of cancer cells via immunostained fluorescence labeling of the attached tubulins (figures 3).
    [0058] To verify the hypothesis that the peptide of SEQ ID NO: 1 could prevent the proliferation of cancer cells, altering the microtubule network (that is, having a mechanism of action close to that of the "taxol-like mechanism"), Immunofluorescence labeling experiments on the microtubule structures of cells treated or not by the peptide of SEQ ID NO: 1 were carried out, using the process described below.
    [0059] Cancer cells of different strains (A549): lung cancer, NMT1: melanoma, Panel: pancreatic cancer) were incubated on glass slides with different concentrations of the peptide of SEQ ID NO: 1 for 4 hours at 37 ° C . Then, the cells were fixed with PFA, washed with PBS and incubated in a PBS buffer (phosphate buffered saline) containing 1% BSA, 0.1% Triton X 100 for 30 minutes at 37 ° C. After washing with PBS, the cells were incubated with an anti-beta tubulin monoclonal antibody overnight in a cold chamber. Then, the glass slides were washed with PBS and the cells were incubated with a goat anti-mouse antibody coupled with fluorescein isothiocyanate (FITC) for 60 minutes at room temperature. After washing with PBS, the labeled cells were visualized with a fluorescence microscope. The dapi staining technique was applied to mark the nuclei in blue.
    [0060] As shown in figure 3, when lung cancer A 549 cells are treated by the peptide of SEQ ID NO: 1, and this even during a short period of time, there is formation of very dense polymers of tubulin which are a mixture of microtubules and aggregates. When these cells are not treated with the peptide of SEQ ID NO: 1, the microtubule network is very organized and undisturbed. Figure 3 also shows the peptide effect of SEQ ID NO: 1 on two other varieties of cancer cells, melanoma cells MNT-1 and pancreatic PANC 1 cells. When cells are treated with peptide SEQ ID NO: 1, its microtubular network is also altered as shown by the significant increase in fluorescence in relation to the fluorescence of untreated cells. However, the microtubular change seen in treated MNT-1 and PANC 1 cells is less important than in A549 cells. This can be explained by the fact that the treatment period was not enough or by the fact that the tubulins of the MNT-1 and PANC 1 strains show differences in their molecular structure in relation to the tubulins of A549 cells and would thus be able to induce a reduction in their affinity for the peptide SEQ ID NO: 1. 84) Activity of the peptide of SEQ ID NO: 1 on healthy cells (figure 4)
    [0061] The specific activity of the peptide of SEQ ID NO: 1 on cancer cells and not on healthy cells was confirmed by the experiments shown in figure 4. Two varieties of non-tumorigenic fibroblastic cells: FIBRO 1 and FIBRO 3 were brought into contact with the peptide of SEQ ID NO: 1, in order to see if the healthy cell microtubule network was also disturbed by the peptide. Now, figure 4 demonstrates that human fibroblasts that represent a variety of normal cells do not fully respond to the peptide of SEQ ID NO: 1 (no density modification of the polymerized tubulin), at least with concentrations in peptide of SEQ ID NO: 1 used by cancer cells (15 pM). This observation represents an important advance of the present invention. Very few antitumor agents have a very large difference in cytotoxicity to cancer cells versus healthy cells in the host organism (ie, very toxic to cancer cells and slightly toxic to healthy host cells).
    [0062] This last point has two important consequences: it is, in fact, very likely that few side effects will appear, when the peptide of SEQ ID NO: 1 and its analogs will be used as a therapeutic agent in the mammal, particularly in Man .
    [0063] In addition, it will be possible to significantly increase the effectiveness of the peptide of SEQ ID NO: 1 and its analogs, increasing both its dosage and its treatment time. 85) measure of cellular proliferation and reliability (figure 5)
    [0064] The following cell lines (table II) were analyzed to determine and quantify the peptide activity of SEQ ID NO: 1 in table II below, the fibroblasts M and N are not tumor, the L.1 lymphocytes, L.3, L.4 and L.5 are leukemic, while L.6 lymphocytes are normal lymphocytes.
    [0065] Approximately 10,000 cells were cultured in a volume of 0.2 ml_ per well in 96-well microplates for 24 hours before treatment by the peptide of SEQ ID NO: 1. The cells were incubated for 48 to 72 hours at 37 ° C in a CO2 incubator in the presence or not of the peptide of SEQ ID NO: 1 in different concentrations.
    [0066] Following this incubation, the MTT reagent or 3- (4,5 dimethyl -2-yl) -2,5 diphenyl tetrazolium bromide (0.5 mg / ml in a volume of 0.1 ml) added to each well and incubated for 2 hours at 37 ° C, Table II


    [0067] The tetrazolium ring containing MTT is then reduced by the succinate dehydrogenase of the living cells in formazan. Then the float was removed and 0.1 ml of lysis buffer (isopropanol containing 10% Triton X 100 and 10% 1N HCI) was added to dissolve the formazan blue-violet precipitate formed. After a few minutes, the absorbance was determined at room temperature, using a microplate reading with a test wavelength of 562 nm. Wells that do not comprise SEQ ID NO: 1 peptide were used in order to control the reliability of control cells. IC 50 values (peptide concentration of SEQ ID NO: 1, causing 50% cytotoxicity on these cell lines) for each variety of cells were evaluated by the ratio of the absorbance of the treated cells to the absorbance of the control cells.
    [0068] Another method of measuring cell reliability was used. This method consists of making a direct visual count of the number of cells, assisting a Malassez cell, under different treatment conditions. This method allows a better evaluation of the steps that precede cell death (better visibility under the microscope by direct vision of cells) than by the MTT test.
    [0069] As shown in figure 5 and table III, the peptide of SEQ ID NO: 11 prevents the proliferation of tumor cells. The more the dose of the SEQ ID NO: 1 peptide is increased, the more the proliferation of cancer cells will be prevented. It has been observed that doses of SEQ ID NO: 1 peptide toxic to cancer cells are not toxic to normal cells like normal lymphocytes or fibroblasts. From the applicant, the peptide SEQ ID NO: 1 decreases the proliferation of cancer cells, probably altering the formation of microtubules of the mitotic spindle.
    [0070] It has also been observed that the sensitivity of different varieties of tumor cells varies. Table III below proves that the IC 50 value is very low for very sensitive tumor cells, such as the A549 cell variety (lung cancer). The IC 50 value is higher for less sensitive cell varieties, such as pancreatic cells (PANC 1 cell range) or MNT 1 human melanoma cell varieties (DU 145 cell range) of glioblastoma cell varieties (U87 - M6). In the latter case, it substantially depends on the exposure time of the glioblastoma cells with the peptide of SEQ ID NO: 1. This observation suggests that the peptide of SEQ ID NO: 1 could have a different action and efficacy, depending on the type of cells carcinogenic. A modulation of the dose or length of treatment can then be considered to act effectively on certain types of cancers. The set of results in Table III shows that the peptide of SEQ ID NO: 1 has a very broad antitumor activity whereas the peptide of SEQ ID NO: 1 has a broad antitumor activity that could be more important than that of taxol to combat different types of cancers.
    [0071] Table III also shows the very low toxicity of the peptide SEQ ID NO: 1 in different types of normal cells such as lymphocytes or fibroblasts.
    [0072] This table shows that the peptide of SEQ ID NO: 1 shows an activity in a large panel of cancer cells (a measure of cell reliability). Table III

    B6) Effects of the peptide from and on the structure of the plasma membrane (Figures 6 and 7)
    [0073] The effects of SEQ ID NO: 1 on the structure and properties of the plasma membrane have been highlighted by direct viewing experiments on cells treated with a phase-contrast microscope and by counting experiments on cells that adhere and those that have lost its ability to adhere to the substrate.
    [0074] Figure 6 shows that, in the presence of the SEQ ID NO: 1 peptide, the number of A549 tumor cells has decreased significantly (after 2 and 4 hours of treatment) and that, in addition, their cell contour is greatly altered in consequence of changes in the plasma membrane. This adds to the changes that affect the microtubular cyto structure.
    [0075] Figure 7 shows in effect that the number of A 549 cells, which retains their adhesive properties to the substrate, decreases as a result of the incubation time and that, on the contrary, the number of A 549 cells that float in what fluctuates by cause of a loss of adhesion to the substrate, increases with the time of culture.
    [0076] The peptide of SEQ ID NO: 1, due to its polycationic character, modifies the plasma membrane properties of canker-rich cells rich in negatively charged phospholipids, destabilizing the structure of the double lipid layer of these cells. This membrane destabilization causes cell deformations either by inducing cytoplasmic extensions or by deformations of the cell contour as shown in the experiment illustrated in figure 6, in which an abnormal cell contour with increased cell volume is seen in the treated cells. The destabilization of the membranes also causes functional changes such as the loss of adhesion to the culture substrate as shown in figure 7.
    [0077] These membrane changes are responsible for major cell deaths. Therefore, the peptide of SEQ ID NO: 1 and certain variants have a double effect, cytostatic (inhibition of mitosis) and cytotoxic (cell death). This double effect amplifies the antitumor action of these peptides.
    [0078] The peptide of SEQ ID NO: 1 and certain analogs may not only alter the membranes of cancer cells. They can alter the membranes of other cell types, such as bacterial or fungal cells. 87) Effects of SEQ ID NO: 1 peptide analogs.
    [0079] The analogs or variants of the peptide SEQ ID NO: 1 were tested on the A549 strain that corresponds to a lung cancer that is still very difficult to take care of. The tested variants are as follows: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 2.
    [0080] The evaluation of the activity of different tested variants can be summarized as follows:

    [0081] In conclusion of the analysis of the activity of the peptide variants of SEQ ID NO: 1, it is possible to say that at least four peptides that have a modified sequence in relation to that of SEQ ID NO: 1, namely peptides of SEQ ID NO: 3, SEQ ID NO: 4. SEQ ID NO: 7 and SEQ ID NO: 9, have a cytotoxic activity fully equivalent to the peptide SEQ ID NO: 1 against A 549 cells.
    [0082] Another result refers to the peptide of SEQ ID NO: 7. This peptide has been shown not only to be active against MNT-1 melanoma cells with an IC 50q eu between 10 and 15 pM and also against cells of T47 sinus cancer with an IC50 between 10 and 15 pM.
    [0083] In addition, by increasing the dosage or increasing the treatment in time of the peptides, it will be possible to improve the effectiveness of the treatment against cancer. For example, by increasing the dosage of SEQ ID NO: 1, it will be possible to obtain better results for treating moderately sensitive tumors such as certain pancreatic PANC-1 tumors or glioblastoma. 88) Resistance of cancer cells to peptides SEQ ID NO: 1 to 25.
    [0084] Finally, according to the indications of the preliminary experiments, the peptide of SEQ ID NO: 1 and its analogs would have the advantage of not inducing a fast process of resistance. 89) Sensitivity of taxol-resistant cells to SEQ ID NO: 1 and SEQ ID NO: 7 sequence peptides.
    [0085] The work was carried out on 2 cell lines TX1 and TX 2 derived from breast tumors that no longer respond to Taxol. The two strains showed a very good sensitivity to at least two peptides namely the peptide of SEQ ID NO: 1 and the peptide of SEQ 7. More precisely, the results of growth inhibition already found significant after 24 H of culture in the presence of these peptides.
    [0086] After 48 hours of culture in the presence of peptides, the growth inhibition assessments are as follows:

    [0087] For the two types of resistant cells TX1 and TX2, the peptides SEQ ID NO: 1 of SEQ ID NO: 7 have a very good sensitivity, since the IC50 index evaluated was between 3 and 7 pM.
    权利要求:
    Claims (5)
    [0001]
    1. Peptide, characterized by the fact that it comprises at least one sequence chosen from the sequences of SEQ ID NOs: 2, 3, 4, 7, 9, 18 and 19.
    [0002]
    2. Peptide according to claim 1, characterized by the fact that it is produced by synthesis.
    [0003]
    3. Pharmaceutical composition, characterized by the fact that it comprises a peptide or a mixture of peptides as defined in claim 1.
    [0004]
    4. Pharmaceutical composition according to claim 3, characterized in that it is presented in the form of a solution, an aqueous suspension, or in the dry state in the form of uncoated or coated tablets, such as pills, jellies, capsules, or powders.
    [0005]
    5. Composition according to claim 3 or 4, possibly associated with a pharmaceutically acceptable excipient or inert non-toxic vehicle, characterized by the fact that it is topically administered in the form of cutaneous, transcutaneous application. nea or percutaneous; oral; parenteral; nasal or bronchial.
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    同族专利:
    公开号 | 公开日
    EP2576596A1|2013-04-10|
    US20130230591A1|2013-09-05|
    WO2011148083A4|2012-02-02|
    EP2576596B1|2016-01-20|
    BR112012029975A2|2017-02-21|
    CN102971336B|2015-11-25|
    FR2960542A1|2011-12-02|
    WO2011148083A1|2011-12-01|
    FR2960542B1|2012-08-17|
    CN102971336A|2013-03-13|
    US9133242B2|2015-09-15|
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    US7030211B1|1998-07-08|2006-04-18|Gemvax As|Antigenic peptides derived from telomerase|
    CN101225106B|2007-01-15|2010-10-20|李�昊|Short peptide suppressing growth of cancer cell and uses thereof|
    CN101497652B|2008-02-01|2011-04-06|李�昊|Peptide for inhibiting solid tumor and leukaemia cancer cell growth and use thereof|
    CN101235079A|2008-03-04|2008-08-06|福州大学|Short peptide for inhibiting growing of liver cancer cell and application thereof|US9907837B2|2012-05-11|2018-03-06|Gemvax & Kael Co., Ltd.|Composition for preventing or treating cachexia|
    ES2691070T3|2012-05-11|2018-11-23|Kael-Gemvax Co.,Ltd|Anti-inflammatory peptides and composition comprising the same|
    CN104822698B|2012-07-11|2018-08-10|珍白斯凯尔有限公司|Cell-penetrating peptides and the conjugate comprising the peptide and composition|
    CN105263508B|2013-04-19|2019-10-25|珍白斯凯尔有限公司|Treat and prevent the composition of ischemia injury|
    RU2725744C2|2013-06-07|2020-07-03|Джемвакс Энд Каэл Ко., Лтд.|Biological markers which can be used in cancer immunotherapy|
    EP3011967B1|2013-06-21|2020-06-17|Gemvax & Kael Co., Ltd.|Hormone secretion regulator, composition containing same, and method for controlling hormone secretion using same|
    KR102359396B1|2013-11-22|2022-02-08|주식회사 젬백스앤카엘|Peptide having angiogenesis inhibitory activity and composition containing same|
    EP3085380B1|2013-12-17|2020-06-17|Gemvax & Kael Co., Ltd.|Composition for treating prostate cancer|
    CN106456697B|2014-04-11|2019-12-17|珍白斯凯尔有限公司|Peptide having fibrosis-inhibiting activity and composition containing the same|
    JP6466971B2|2014-04-30|2019-02-06|ジェムバックス アンド カエル カンパニー，リミティド|Organ, tissue or cell transplant composition, kit and transplant method|
    CN104031120B|2014-05-29|2016-08-24|王凤羽|A kind of peptide variant treating cancer and application thereof|
    KR20160076911A|2014-12-23|2016-07-01|주식회사 젬백스앤카엘|Peptides for treating ophthalmopathy and the Composition Comprising the Same|
    JP6751097B2|2015-02-27|2020-09-02|ジェムバックス アンド カエル カンパニー，リミティド|Peptide for preventing hearing damage and composition containing the same|
    EP3318265B1|2015-07-02|2021-08-18|Gemvax & Kael Co., Ltd.|Peptide having anti-viral effect and composition containing same|
    JP2019513750A|2016-04-07|2019-05-30|ジェムバックス アンド カエル カンパニー，リミティド|Peptide having efficacy of increasing telomerase activity and extension of telomeres, and composition containing the same|
    CN106167513A|2016-07-09|2016-11-30|青岛大学|There is peptide and the application thereof of anticancer growth activity|
    CN105936643A|2016-07-09|2016-09-14|青岛大学|Peptide with activity on inhibiting growth of cancer cells and applications thereof|
    CN106084012A|2016-07-09|2016-11-09|青岛大学|There is peptide and the application thereof of anticancer growth activity|
    CN105936642A|2016-07-09|2016-09-14|青岛大学|Peptide with activity on inhibiting growth of cancer cells and applications thereof|
    CN106146620A|2016-07-09|2016-11-23|青岛大学|There is peptide and the application thereof of anticancer growth activity|
    MA52363A|2018-04-26|2021-03-03|Agenus Inc|THERMAL SHOCK PROTEINPEPTIDIC COMPOSITIONS AND THEIR METHODS OF USE|
    法律状态:
    2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law|
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2019-07-02| B07E| Notice of approval relating to section 229 industrial property law|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |
    2019-09-03| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|
    2020-04-14| B09A| Decision: intention to grant|
    2020-10-27| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 20/05/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1054106A|FR2960542B1|2010-05-27|2010-05-27|PEPTIDE AS A DRUG, ESPECIALLY FOR THE TREATMENT OF CANCER|
    FR1054106|2010-05-27|
    PCT/FR2011/051150|WO2011148083A1|2010-05-27|2011-05-20|Peptide for use as a medicament, in particular for the treatment of cancer|
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