WO2021112662A1 - Peptide with anticancer activity against breast cancer - Google Patents

Peptide with anticancer activity against breast cancer Download PDF

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Publication number
WO2021112662A1
WO2021112662A1 PCT/MX2020/050045 MX2020050045W WO2021112662A1 WO 2021112662 A1 WO2021112662 A1 WO 2021112662A1 MX 2020050045 W MX2020050045 W MX 2020050045W WO 2021112662 A1 WO2021112662 A1 WO 2021112662A1
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peptide
breast cancer
peptides
cells
fskwll
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PCT/MX2020/050045
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Spanish (es)
French (fr)
Inventor
Catalina SORIANO CORREA
Carolina BARRIENTOS SALCEDO
Maria Isabel SOTO CRUZ
Martha LEGORRETA HERRERA
Mercedes BERMÚDEZ CORTÉS
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Universidad Nacional Autónoma de México
Universidad Veracruzana
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Publication of WO2021112662A1 publication Critical patent/WO2021112662A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the present invention is related to the field of medicine, particularly with the techniques and principles used in therapy for cancer treatment, specifically it refers to a peptide that has application for therapeutic purposes, especially against cells from cancer tumors.
  • breast MCF-7 and MBA-MB-231.
  • Peptides are chains of amino acids whose length can vary from two to less than one hundred amino acids, covalently linked by peptide bonds. They are present in nature and fulfill functions as vasoactive agents (Rhomberg, 2018), hormones (McLaughlin, 2019), neurotransmitters (Tringali, 2019), antioxidants
  • Primary structure refers to the number and sequence of amino acids present: by convention, it is written from the end that has a free N-initial amino group (located on the right) to the C-terminal end that contains the group a- free carboxyl (at the end of the peptide chain).
  • secondary structure the ordered arrangement of amino acids, the hydrogen bonds that form between the atoms and the single bonds of the peptide bond, allow the polypeptide chain to adopt lower free energy and therefore more stable conformations.
  • membranolytic peptides are not toxic to recipient cells and do not activate or suppress metabolic pathways. This is why membrane lysing peptides are good candidates for use in new therapeutic approaches.
  • a peptide Synthetic from natural peptides allows to modify their chemical and physicochemical properties, for example, to improve their solubility, pH, hydrophobicity and permeability in the target tissues, or to vary their structure by adding sentinel molecules without modifying their properties, which gives them therapeutic benefits.
  • computational chemistry and molecular modeling are potentially useful tools for the design of biopharmaceuticals, which aims to integrate applications aimed at rational design, use or obtaining chemical structures, to determine protein interaction maps ( protein-protein interactions) or to identify new active sites (molecular coupling and virtual screening), from collections of compounds (computational chemical libraries).
  • molecular simulation molecular dynamics allows to recreate macromolecules in their native environment.
  • molecules peptides
  • molecules can be redesigned in order to improve their chemical-biological properties, as well as, to increase their affinity, their specificity, or both (Georgoulia, 2019).
  • Solid phase synthesis consists of numerous steps of deprotection, activation and coupling, that is, instead of protecting the C-terminal with a chemical group, the C-terminal of the first amino acid is coupled to an activated solid support, such as polystyrene or polyacrylamide.
  • an activated solid support such as polystyrene or polyacrylamide.
  • This type of strategy has a dual function: the resin acts as the C-terminal protecting group and provides a quick method to separate the peptide from the different reaction mixtures during synthesis.
  • This synthesis is commonly performed in automated peptide synthesizers with high purity peptide production and good yield (Máde, 2014). This is crucial to continue the trials of the clinical stages.
  • Cancer is a public health problem in Mexico and worldwide, not only because of its clinical manifestations and high mortality, but also because of the great variety of individual and environmental risk factors with which it is associated (WHO, 2019 ; Máde, 2014). Cancer is an alteration of various metabolic pathways, which lead to uncontrolled cell proliferation and inhibition of apoptosis, as well as a lack of cell and tissue differentiation (Metzcar, 2019). Surgery, radiation, and chemotherapy are widely used therapeutic procedures to treat various types of cancer, however, these treatments are invasive and have serious side effects on the body. Currently, new gene therapies are in development that take advantage of the selective induction of apoptosis or necrosis.
  • the peptides with anticancer action have been divided into: those that induce necrosis (memebranolytics), those that activate the immune system (immunomodulators), those that lead to the activation of apoptotic pathways and those that block specific functions in neoplastic cells.
  • necrosis memebranolytics
  • immunomodulators those that lead to the activation of apoptotic pathways
  • the latter include those that interact with receptors, those that bind to cell adhesion proteins, and protein kinase inhibitors (Hilchie, 2019).
  • Benchie protein kinase inhibitors
  • Huang's group in 2000 proposed the Bcl-2 protein family as a target for the design of antitumor drugs, since they include anti and pro-apoptotic proteins with opposite biological functions.
  • peptides that block Bcl-2 or Bcl-xL derived from BH3-only proteins have been generated (Chipuk, 2008; Huang, 2000), and more recently also Lytic peptides have been developed from BH3 proteins named by the authors as ABH3 (Liu, 2016).
  • VmCT1 analogues FLGALWNVAKSVF
  • the FDA in the USA has registered and authorized the peptides: Trastuzumab, Bevacizumab, Pertuzumab, Denosumab and Buserelin.
  • Patent applications and patents that describe peptides are the United States patent US 7,632,814 B2 entitled “HYD1 peptides anti-cancer agents", the patent application KR20190073338A entitled “Pharmaceutical composition for inhibiting expression of CCND3 or PAK2 gene” , Australian patent application AU2019203377A1 entitled “Environmentally sensitive compositions”, patent application EP3228632 A1 entitled “Seiective anticancer chimeric peptide”, US patent application US2017042963 A1 entitled “Anticancer peptide for inhibiting proliferation of cancer stem cells and use”, the international patent application WO2016139684A2 entitled “A modified peptides as an anticancer agent”, the application of International patent WO2016099188 A1 entitled “Peptide having eight amino acid sequences derived from cage and retaining anticancer activity”
  • the peptide of the present invention was designed from theoretical studies of the electronic structure, physicochemical properties and chemical reactivity, determined through chemical-quantum descriptors at ab initio level.
  • the synthetic peptide of the present invention has a short action time, which is from 10 minutes at a concentration of 100 ng per 10 million cells, which represents an advantage as it is a low dose. and in a reduced exposure time.
  • the peptide of the present invention is highly selective for breast cancer cells as evidenced by assays on MCF-7 and MDA-MB-231 cell lines.
  • the peptide of the present invention is feasible to synthesize and has a chemical structure that gives it high stability, that is, it is stable up to 305 ° C.
  • the present invention is related to the design of a new peptide of pharmaceutical interest and with application in antitumor therapy in breast cancer cells.
  • the proposed peptide has a hexapeptide structure that corresponds to a sequence of six amino acids repeated 5 times (FSKWLL) 5 .
  • FSKWLL hexapeptide structure that corresponds to a sequence of six amino acids repeated 5 times
  • the peptide has membranolytic functions, which was confirmed indirectly through tests of cDNA expression microarrays. They showed that it is related to the suppression / activation of cell signaling pathways, which involve some members of the ABC transporter family, as well as apolipoprotein H, all of them involved in various cellular functions, such as metabolite flux. and signaling molecules, which in transformed cells leads to resistance to chemotherapeutic agents.
  • the purpose of this invention is to form part of the therapeutic schemes used in breast cancer. Particularly those tumors that do not show expression of the extracellular domain of the EGFR protein that is the target of the drug Herceptin. It may be useful after resection and before chemotherapy is started.
  • the proposed peptide was designed from theoretical studies at the atomic level, and its action time is from 10 minutes at a concentration of 100 ng per 10 million cells, which represents an advantage as it is a low dose and in reduced exposure time. It is highly selective for breast cancer cells as evidenced by assays on MCF-7 and MDA-MB-231 cell lines. In addition, it has a chemical structure that gives it high stability; that is, it is stable up to 305.0 ° C.
  • a peptide that aims to provide a peptide-type molecule, which has been called anti-5-peptide -Breast cancer (P-5ACM), which has high selectivity to cancer cells derived from breast adenocarcinoma, MCF-7 and MDA-MB-231. It is a further object of the present invention to provide a peptide that has advantages over those already existing in tumors that show resistance to chemotherapeutic agents or those for which there is no drug or a specific target.
  • P-5ACM anti-5-peptide -Breast cancer
  • An additional object of the present invention is to provide a non-toxic peptide (P-5ACM) for healthy cells in culture (MCF-10A), not transformed from mammary tissue or for human peripheral blood lymphocytes from healthy donors, both in cultures individual as in co-culture, that is, the peptide does not generate surrounding cellular toxicity by rapidly degrading after its effect.
  • P-5ACM non-toxic peptide
  • MCF-10A healthy cells in culture
  • P-5ACM a peptide
  • Figure 1A shows the three-dimensional chemical structure of the FSKWLL peptide, which corresponds to the minimum energy structure optimized at the HF / 6-31 G (d, p) level.
  • Figure 1B shows the three-dimensional chemical structure that corresponds to the structure of the hexapeptide, that is, it is the structure repeated five times of the peptide (FSKWLL) 5.
  • Figure 2 shows an infrared (IR) spectroscopy spectrogram of the peptide (FSKWLL).
  • IR infrared
  • Figure 3 is a graph showing a spectrogram of spectroscopy
  • Figure 4 is a graph showing a powder X-ray diffractogram of the hexapeptide. In the X-ray powder diffractogram, an amorphous behavior of the peptide can be observed.
  • Figure 5A is a graph showing a thermogravimetric analysis (TGA) of the hexapeptide
  • Figure 5B shows a graph showing a differential scanning calorimetry (DSC) analysis of the hexapeptide.
  • TGA thermogravimetric analysis
  • DSC differential scanning calorimetry
  • Figures 6A and 6B show images of cultured cells of the MCF-7 breast cancer cell line treated with the hexapeptide.
  • the cytotoxic effect of the peptide is observed, the cells are reduced in size and the nuclear membranes are damaged, without completely damaging the plasma membranes.
  • the images were obtained with confocal microscopy at 100X, with a numerical aperture of 0.9.
  • Figures 7A and 7B show a culture of MCF-7 cells derived from untreated breast cancer. MCF-7 cells in culture are seen to be extended, they form cell groups. Damage to cell membranes is not observed.
  • SVM Support Vector Machine
  • sequences of the proposed peptide family were sent for synthesis with the North American American Peptide Company, using the solid phase method. This company was requested because all products meet an interval of purity from 90 to 99%, and they are obtained in a lyophilized form.
  • HEXAPEPTIDE with amino acid sequence FSKWLL repeated 5 times (Figure 1 B) was found to have selective cytotoxic properties against cancer cells of the breast cancer cell lines MCF-7 and MDA-MB -231, both from mammary adenocarcinoma, starting 10 minutes after treatment.
  • the effective concentration of 100 ng / 10 6 cells was determined. Subsequently, an analysis of gene expression was made; the results showed the alteration of some proteins of the ABC transporter family, as well as apolipoprotein H, all of them involved in various cellular functions.
  • the peptide of the present invention comprises a six amino acid sequence FSKWLL repeated 5 times and is a hexapeptide having a purity range of 90 to 99% and is in lyophilized form.
  • the advantages of the proposed peptide are that: its cytotoxic effect occurs at a low concentration and the time required to achieve the effect of said peptide is short, which reduces the possibility of developing resistance, toxicity and presenting side effects. In addition, it enables a lower treatment cost, and is very stable at high temperatures.
  • the peptide of the present invention with a sequence of six amino acids FSKWLL repeated 5 times is useful in the preparation of a pharmaceutical composition for the treatment of breast cancer, in combination with an excipient or pharmaceutically acceptable diluent.
  • the peptide was synthesized by American Peptide, using the solid phase method. This company was used because all the products have a purity range of 90 to 99%, and it was obtained in a lyophilized form.
  • Peptide # 340572 synthesized by American Peptide was used, with a purity percentage of 95% and molecular weight of 3892.82 g / mol calculated by electrospray mass spectroscopy and isoelectric point (pl) of 10.6.
  • Figures 7A and 7B show images of a culture of MCF-7 cells derived from breast cancer without treatment. MCF-7 cells in culture are seen to be spread out and form cell clumps. In addition to the above, no damage to cell membranes is observed.
  • Thermo Scientific TM Nicolet TM iS TM 50 Infrared Spectrophotometer was used for the determination of the IR spectrum of the sample. An environmental white spectrum was first determined to avoid interference in the sample. Subsequently, a minimum amount of sample was placed, and the spectroscopic determination of the sample was carried out. The result was visualized with the OMNIC Spectra equipment software.
  • the graph in Figure 2 represents an infrared spectroscopy (IR) spectrogram of the peptide (FSKWLL). In the infrared spectrum it shows a band near 1650 cnr 1 that corresponds to the band of the amide I.
  • the bands near 1540 cnr 1 and 1200 cnr 1 correspond to the band of amide II, and of the band of amide III, respectively .
  • the band around 1400 cnr 1 corresponds to the bending of the COO- group.
  • the band corresponding to amide I, which is close to 1650 cnr 1 is characteristic of a molecular disorder so it does not form b-sheets, since they absorb about 1620 cnr 1 , however, it can form alpha helices .
  • a Raman microscopy equipment was used coupled to a Raman spectrophotometer adapted with a 630 nm laser of the Alpha 300M + model from the company Witec, Focus Innovation, and the Project Four and Control Four programs included in the package were used. of the team.
  • a minimum amount of sample was placed on a slide, and the sample was focused at 5x and 50x; and subsequently the laser radiation at 630 nm was impinged on the sample to obtain the Raman spectrum.
  • the graph in Fig. 3 shows the graph representing a Raman spectroscopy spectrogram of the peptide (FSKWLL).
  • thermogravimetric analysis thermogram a TA Instruments model Q50 equipment and the TA Universal Analysis package were used.
  • the sample holder was first tarred to zero the value of the mass measurement, later the sample was added.
  • the weight of the sample was 0.008 mg, and the temperature was varied in the computer program of 20 e C / min; the temperature range was from 30 ° C to 400 ° C.
  • thermogram a TA Instruments model Q200 equipment and the TA Universal Analysis package were used.
  • the sample was loaded into an aluminum capsule with a capacity between 10-50 ml.
  • the capsule was sealed by means of the sealing press, with an aluminum cap to prevent that, due to expansion or decomposition problems of the sample, it was projected out of the capsule and contaminating the equipment.
  • a reference capsule was used that was placed next to the capsule containing the sample. The sample was run for 20 minutes, at an initial temperature of 20 ° C and up to 400 ° C.
  • Figure 5a graphs a thermogravimetric analysis (TGA) of the hexapeptide
  • Figure 5B graphs the differential scanning calorimetry (DSC) analysis of the hexapeptide.

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Abstract

The present invention is related to the design of a new peptide of pharmaceutical interest and with application in antitumour therapy in breast cancer cells. The proposed peptide has a hexapeptide structure corresponding to a sequence of six amino acids repeated five times (FSKWLL)5. The purpose of this invention is to form part of the therapeutic regimes used in breast cancer, particularly in tumours without expression of the extracellular domain of the EGFR protein, which is targeted by the drug Herceptin. The peptide of the invention can be used following resection and prior to commencing administration of chemotherapy.

Description

PÉPTIDO CON ACTIVIDAD ANTICANCERÍGENA FRENTE AL CÁNCER DE MAMA PEPTIDE WITH ANTI-CANCER ACTIVITY AGAINST BREAST CANCER
CAMPO DE LA INVENCIÓN La presente invención se relaciona con el campo de la medicina particularmente con las técnicas y principios utilizados en terapia para tratamiento del cáncer, específicamente se refiere a un péptido que tiene aplicación con fines terapéuticos, especialmente contra células provenientes de tumores de cáncer de mama: MCF-7 y MBA-MB-231. FIELD OF THE INVENTION The present invention is related to the field of medicine, particularly with the techniques and principles used in therapy for cancer treatment, specifically it refers to a peptide that has application for therapeutic purposes, especially against cells from cancer tumors. breast: MCF-7 and MBA-MB-231.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
Los péptidos son cadenas de aminoácidos cuya longitud puede variar de dos a menos de cien aminoácidos, unidos covalentemente por enlaces peptídicos. Están presentes en la naturaleza y cumplen funciones como agentes vasoactivos (Rhomberg, 2018), hormonas (McLaughlin, 2019), neurotransmisores (Tringali, 2019), antioxidantesPeptides are chains of amino acids whose length can vary from two to less than one hundred amino acids, covalently linked by peptide bonds. They are present in nature and fulfill functions as vasoactive agents (Rhomberg, 2018), hormones (McLaughlin, 2019), neurotransmitters (Tringali, 2019), antioxidants
(Nwachukwu, 2019), antibióticos (Lewies, 2019), etc. La estructura de los péptidos y proteínas se aborda en cuatro niveles de complejidad: estructura primaria, secundaria, terciaria y cuaternaria. La estructura primaria se refiere al número y a la secuencia de los aminoácidos presentes: por convención, se escribe desde el extremo que tiene un grupo amino N-inicial libre (localizado a la derecha) hacia el extremo C-terminal que contiene al grupo a-carboxilo libre (al final de la cadena peptídica). En la estructura secundaria, el arreglo ordenado de aminoácidos, los puentes de hidrógeno que se forman entre los átomos y los enlaces sencillos del enlace peptídico, permiten que la cadena polipeptídica adopte conformaciones de menor energía libre y por lo tanto más estables. Esas conformaciones dependen de la secuencia primaria de los aminoácidos: Hélice a, lámina b, y otras como las hélices 3-10, hélices p; etc., y como estructuras supersecundarias se encuentran las horquillas b, hélice a- horquilla y motivos b-a-b (Tramontano, 2006). Existen varias funciones terapéuticas de los péptidos relacionadas con el tratamiento del cáncer, entre ellas destacan los péptidos que forman poros en la membrana celular y los que la desestabilizan, este tipo de péptidos forman parte del sistema inmune innato de varias especies, por ejemplo: las cecropinas en insectos, magaininas y dermaseptinas en anfibios, y las defensinas en los mamíferos. Poseen un relativo bajo peso molecular (menos de 5000 Da), baja antigenicidad y exhiben una estructura predominantemente catiónica antipática, lo que favorece su interacción con membranas celulares aniónicas (Ganz, 2003). El mecanismo de acción de estos péptidos consiste en la unión y la interrupción de la integridad estructural de la membrana a través de poros, agujeros o canales iónicos. Sin embargo, el mecanismo molecular exacto depende de la conformación específica de la molécula utilizada (Avci, 2018). (Nwachukwu, 2019), antibiotics (Lewies, 2019), etc. The structure of peptides and proteins is addressed at four levels of complexity: primary, secondary, tertiary, and quaternary structure. Primary structure refers to the number and sequence of amino acids present: by convention, it is written from the end that has a free N-initial amino group (located on the right) to the C-terminal end that contains the group a- free carboxyl (at the end of the peptide chain). In the secondary structure, the ordered arrangement of amino acids, the hydrogen bonds that form between the atoms and the single bonds of the peptide bond, allow the polypeptide chain to adopt lower free energy and therefore more stable conformations. These conformations depend on the primary sequence of the amino acids: Helix a, lamina b, and others such as helices 3-10, helices p; etc., and as supersecondary structures are hairpins b, helix a-hairpin and bab motifs (Tramontano, 2006). There are several therapeutic functions of peptides related to cancer treatment, among them the peptides that form pores in the cell membrane and those that destabilize it, this type of peptides are part of the innate immune system of several species, for example: cecropins in insects, magainins and dermaseptins in amphibians, and defensins in mammals. They have a relatively low molecular weight (less than 5000 Da), low antigenicity and exhibit a predominantly cationic antipathic structure, which favors their interaction with anionic cell membranes (Ganz, 2003). The mechanism of action of these peptides consists in the union and the interruption of the structural integrity of the membrane through pores, holes or ion channels. However, the exact molecular mechanism depends on the specific conformation of the molecule used (Avci, 2018).
La ventaja de los péptidos membranolíticos es que, no son tóxicos para las células receptoras y no activan o suprimen vías metabólicas. Es por ello por lo que, los péptidos que lisan membranas son buenos candidatos para utilizarse en nuevos enfoques terapéuticos. The advantage of membranolytic peptides is that they are not toxic to recipient cells and do not activate or suppress metabolic pathways. This is why membrane lysing peptides are good candidates for use in new therapeutic approaches.
En las últimas décadas, el desarrollo de fármacos se ha centrado principalmente en moléculas pequeñas y en biofármacos, no obstante, se estima que más del 90% de las moléculas estudiadas no logran llegar al mercado, debido a la toxicidad y a los efectos secundarios, más que a la eficacia, tolerabilidad y toxicidad. Los péptidos se degradan fácilmente en el interior del cuerpo humano, esta clase de biomoléculas por mucho tiempo se mantuvieron inelegibles para el desarrollo de fármacos. Sin embargo, gracias a los avances tecnológicos se ha despertado un gran interés en su uso, tanto como agentes de diagnóstico, como de agentes terapéuticos (Lau, 2018). Las tecnologías actuales tienen el potencial de generar un amplio espectro de fármacos peptídicos eficaces y seguros. Los péptidos con fines terapéuticos pueden ser de origen natural o completamente sintéticos, diseñados por métodos bioinformáticos y computacionales. El diseño de un péptido sintético a partir de péptidos naturales permite modificar sus propiedades químicas y fisicoquímicas, por ejemplo, mejorar su solubilidad, pH, hidrofobicidad y permeabilidad en los tejidos blanco, o bien variar su estructura añadiendo moléculas centinelas sin que se modifiquen sus propiedades, lo que les confiere ventajas terapéuticas. In recent decades, drug development has focused mainly on small molecules and biopharmaceuticals, however, it is estimated that more than 90% of the molecules studied fail to reach the market, due to toxicity and side effects, more than to efficacy, tolerability, and toxicity. Peptides are easily degraded inside the human body, this class of biomolecules for a long time remained ineligible for drug development. However, thanks to technological advances, great interest has been aroused in their use, both as diagnostic agents and as therapeutic agents (Lau, 2018). Current technologies have the potential to generate a broad spectrum of safe and effective peptide drugs. Peptides for therapeutic purposes can be of natural origin or completely synthetic, designed by bioinformatic and computational methods. The design of a peptide Synthetic from natural peptides allows to modify their chemical and physicochemical properties, for example, to improve their solubility, pH, hydrophobicity and permeability in the target tissues, or to vary their structure by adding sentinel molecules without modifying their properties, which gives them therapeutic benefits.
Por lo anterior, la química computacional y el modelado molecular son herramientas potencialmente útiles para el diseño de biofármacos, que tiene como objetivo la integración de aplicaciones encaminadas al diseño racional, al uso o a la obtención de estructuras químicas, para determinar mapas de interacción proteica (interacciones proteína-proteína) o para identificar nuevos sitios activos (acoplamiento molecular y cribado virtual), a partir de colecciones de compuestos (librerías químicas computacionales). Asimismo, la simulación molecular (dinámica molecular) permite recrear a las macromoléculas en su entorno nativo. Así, mediante técnicas computacionales, se pueden rediseñar moléculas (péptidos) con la finalidad de mejorar sus propiedades químico-biológicas, así como, para aumentar su afinidad, su especificidad, o ambas (Georgoulia, 2019). Therefore, computational chemistry and molecular modeling are potentially useful tools for the design of biopharmaceuticals, which aims to integrate applications aimed at rational design, use or obtaining chemical structures, to determine protein interaction maps ( protein-protein interactions) or to identify new active sites (molecular coupling and virtual screening), from collections of compounds (computational chemical libraries). Likewise, molecular simulation (molecular dynamics) allows to recreate macromolecules in their native environment. Thus, through computational techniques, molecules (peptides) can be redesigned in order to improve their chemical-biological properties, as well as, to increase their affinity, their specificity, or both (Georgoulia, 2019).
Actualmente, la síntesis experimental de péptidos en fase sólida es el método más común. La síntesis en fase sólida consiste en numerosos pasos de desprotección, activación y acoplamiento, es decir, en lugar de proteger al C-terminal con un grupo químico, el C-terminal del primer aminoácido se acopla a un soporte sólido activado, como el poliestireno o poliacrilamida. Este tipo de estrategia tiene una función doble: la resina actúa como el grupo protector C-terminal y proporciona un método rápido para separar el péptido de las diferentes mezclas de reacción durante la síntesis. Esta síntesis comúnmente se realiza en sintetizadores de péptidos automatizados con una producción de péptidos con alta pureza y un buen rendimiento (Máde, 2014). Esto es crucial para continuar con los ensayos de las etapas clínicas. El cáncer de mama es un problema de salud pública en México y a nivel mundial, no solo por sus manifestaciones clínicas y su alta mortalidad, sino también por la gran variedad de factores de riesgo individuales y ambientales con los que se le asocia (WHO, 2019; Máde, 2014). El cáncer es una alteración de diversas rutas metabólicas, que conducen a la proliferación celular incontrolada e inhibición de la apoptosis, así como a la falta de la diferenciación celular y tisular (Metzcar, 2019). La cirugía, radiación y la quimioterapia son procedimientos terapéuticos utilizados ampliamente para tratar los diversos tipos de cáncer, sin embargo, estos tratamientos resultan invasivos y tienen efectos colaterales graves sobre el organismo. Actualmente, se encuentran en desarrollo nuevas terapias génicas que aprovechan la inducción selectiva de apoptosis o necrosis. Los péptidos con acción anticancerígena se han dividido en: aquellos que inducen necrosis (memebranolíticos), los que activan al sistema inmune (inmunomoduladores), los que conducen a la activación de las rutas apoptóticas y los que bloquean funciones específicas en las células neoplásicas. En estos últimos se encuentran los que interactúan con receptores, los que se unen a proteínas de adhesión celular y los inhibidores de las proteínas cinasas (Hilchie, 2019). En 2007, Barrientos-Salcedo y colaboradores realizaron un análisis detallado de la estructura electrónica y de las propiedades fisicoquímicas de los aminoácidos del dominio de transactivación de p53: PPLSQETFSDLWKLL, lo que permitió generar una familia de péptidos sintéticos con características químicas teóricas específicas, los cuales cumplieron con la regla de Lipinski (Lipinski, 2001) para su síntesis. Los estudios experimentales realizados de dichos péptidos mostraron que, uno de los péptidos presentó efecto citotóxico contra células de cáncer de mama. Currently, experimental solid phase peptide synthesis is the most common method. Solid phase synthesis consists of numerous steps of deprotection, activation and coupling, that is, instead of protecting the C-terminal with a chemical group, the C-terminal of the first amino acid is coupled to an activated solid support, such as polystyrene or polyacrylamide. This type of strategy has a dual function: the resin acts as the C-terminal protecting group and provides a quick method to separate the peptide from the different reaction mixtures during synthesis. This synthesis is commonly performed in automated peptide synthesizers with high purity peptide production and good yield (Máde, 2014). This is crucial to continue the trials of the clinical stages. Breast cancer is a public health problem in Mexico and worldwide, not only because of its clinical manifestations and high mortality, but also because of the great variety of individual and environmental risk factors with which it is associated (WHO, 2019 ; Máde, 2014). Cancer is an alteration of various metabolic pathways, which lead to uncontrolled cell proliferation and inhibition of apoptosis, as well as a lack of cell and tissue differentiation (Metzcar, 2019). Surgery, radiation, and chemotherapy are widely used therapeutic procedures to treat various types of cancer, however, these treatments are invasive and have serious side effects on the body. Currently, new gene therapies are in development that take advantage of the selective induction of apoptosis or necrosis. The peptides with anticancer action have been divided into: those that induce necrosis (memebranolytics), those that activate the immune system (immunomodulators), those that lead to the activation of apoptotic pathways and those that block specific functions in neoplastic cells. The latter include those that interact with receptors, those that bind to cell adhesion proteins, and protein kinase inhibitors (Hilchie, 2019). In 2007, Barrientos-Salcedo et al. Carried out a detailed analysis of the electronic structure and the physicochemical properties of the amino acids of the transactivation domain of p53: PPLSQETFSDLWKLL, which allowed the generation of a family of synthetic peptides with specific theoretical chemical characteristics, which they complied with the Lipinski rule (Lipinski, 2001) for their synthesis. The experimental studies carried out on these peptides showed that one of the peptides had a cytotoxic effect against breast cancer cells.
El grupo de Huang en 2000 propuso la familia de proteínas Bcl-2 como blanco para el diseño de drogas antitumorales, ya que incluyen proteínas anti y proapoptóticas con funciones biológicas opuestas. En este sentido, se han generado péptidos que bloquean a Bcl-2 o Bcl-xL derivados de proteínas BH3-only (por ejemplo, EDIIRNIARHLAQVGDSMDR) (Chipuk, 2008; Huang, 2000), y más recientemente también se han desarrollado péptidos líticos a partir de proteínas BH3 nombrados por los autores como ABH3 (Liu, 2016). Huang's group in 2000 proposed the Bcl-2 protein family as a target for the design of antitumor drugs, since they include anti and pro-apoptotic proteins with opposite biological functions. In this sense, peptides that block Bcl-2 or Bcl-xL derived from BH3-only proteins (for example, EDIIRNIARHLAQVGDSMDR) have been generated (Chipuk, 2008; Huang, 2000), and more recently also Lytic peptides have been developed from BH3 proteins named by the authors as ABH3 (Liu, 2016).
Es bien conocido que varios péptidos con actividad antimicrobiana también presentan actividad antitumoral, con base en que tienen cargas positivas que les permiten interaccionar con membranas o moléculas blanco por medio de interacciones electrostáticas, hidrofobicidad o lipofilicidad tal que les permite entrar en contacto con las membranas celulares. Se ha estudiado mucho al respecto y recientemente, por ejemplo, se describió la actividad antitumoral de análogos de VmCT1 (FLGALWNVAKSVF) en células MCF-7 derivadas de cáncer de mama (Pedron, 2018; Felício, 2017; Gaspar, 2013). It is well known that several peptides with antimicrobial activity also have antitumor activity, based on the fact that they have positive charges that allow them to interact with membranes or target molecules through electrostatic interactions, hydrophobicity or lipophilicity such that they allow them to come into contact with cell membranes. . Much has been studied in this regard and recently, for example, the antitumor activity of VmCT1 analogues (FLGALWNVAKSVF) in MCF-7 cells derived from breast cancer was described (Pedron, 2018; Felício, 2017; Gaspar, 2013).
En los últimos años, los péptidos sintéticos para el tratamiento del cáncer se emplean debido a su efecto terapéutico, especificidad y toxicidad baja (Barron, 2019; Wilke, 2018; Gómez-Rivas, 2019; Garrison, 2019; Taieb, 2019). In recent years, synthetic peptides for the treatment of cancer are used due to their therapeutic effect, specificity and low toxicity (Barron, 2019; Wilke, 2018; Gómez-Rivas, 2019; Garrison, 2019; Taieb, 2019).
Para el desarrollo e innovación de péptidos sintéticos, se puede hacer uso de la química computacional, particularmente de los métodos químico-cuánticos, debido a que permiten la descripción detallada de la estructura electrónica, las propiedades fisicoquímicas y la estructura molecular en 3D, lo que facilita el diseño y la modificación racional y guiada de nuevas moléculas con características terapéuticas específicas. Como se describió anteriormente, en la literatura científica se cuenta con diversos reportes de péptidos antitumorales, muy promisorios, pero que, debido a su naturaleza bioquímica, sufren degradación por proteasas en la célula o en los tejidos, por lo que con base en el conocimiento detallado de su estructura química-molecular, se han propuesto modificaciones que impiden que se degraden y permiten que se ejerza su efecto, como es el caso de un grupo de péptidos monoméricos con actividad conocidaFor the development and innovation of synthetic peptides, use can be made of computational chemistry, particularly quantum-chemical methods, since they allow the detailed description of the electronic structure, the physicochemical properties and the molecular structure in 3D, which facilitates the design and rational and guided modification of new molecules with specific therapeutic characteristics. As described above, in the scientific literature there are various reports of antitumor peptides, very promising, but which, due to their biochemical nature, suffer degradation by proteases in the cell or in the tissues, so based on the knowledge Detailed description of its chemical-molecular structure, modifications have been proposed that prevent it from degrading and allow its effect to be exerted, as is the case of a group of monomeric peptides with known activity
[HRYYESSLEPWYPD (p6.7), FRYYESSLEPWDD (pDD), FRYYES- SLEPWDDD (pDDD), YGGFM (Met-enkephalin, M-enk), YGGFL (Leu- enkephalin, L-enk), ELYENKPRRPYIL (neurotensin, NT), RRPYIL (NT 8-13), yFGGFTGARKSARKLANQ (nociceptin, NC)], que se multimerizaron como dendrímeros (Bracci, 2003). En este sentido, también algunos péptidos ya se encuentran en fase clínica I como es el caso de LTX-35-Oncopore™ y su análogo LTX-302 (WKKW-DipKKWK-NH2) que se han empleado exitosamente como inmunoactivadores y oncolíticos de melanoma tipo B16 (Berge, 2010). Lo anterior gracias al conocimiento de la geometría molecular y las propiedades fisicoquímicas de dichos péptidos. La solicitud de patente P A/a/2004/000561 titulada “Péptidos eficaces en el tratamiento de tumores y otras condiciones que requieren la remoción o destrucción de células" con fecha del 16 de enero de 2004 y la solicitud de patente MX/a/2017/007338 titulada “Péptidos cíclicos derivados de cd44v6 para el tratamiento de enfermedades relacionadas con cánceres y angiogénesis” con fecha de 5 de junio de 2017, ninguna de las solicitudes antes indicadas describe un péptido como el que se reclama en la presente invención. Por otra parte, la FDA en USA tiene registrados y autorizados los péptidos: Trastuzumab, Bevacizumab, Pertuzumab, Denosumab y Buserelin. (https://webs.iiitd.edu.in/raqhava/thpdb/submitkev.php?ran=6327). Otras solicitudes de patente y patentes que describen péptidos son la patente norteamericana US 7,632,814 B2 titulada “HYD1 peptides anti -cáncer agents”, la solicitud de patente KR20190073338A titulada “Pharmaceutical composition for inhibiting expression of CCND3 or PAK2 gene”, la solicitud de patente australiana AU2019203377A1 titulada “Environmentally sensitive compositions”, la solicitud de patente EP3228632 A1 titulada “Seiective anticancer chimeric peptide”, la solicitud de patente norteamericana US2017042963 A1 titulada “Anticancer peptide for inhibiting proliferation of cáncer stem cells and use”, la solicitud de patente internacional WO2016139684A2 titulada “A modified peptides as an anticancer agent”, la solicitud de patente internacional WO2016099188 A1 titulada “Peptide having eight amino acid sequences derived from cage and retaining anticancer activity” y la patente US 9 067 970 B2 titulada “Anticancer agents comprising peptides with cancer-specific toxicity”, cabe destacar que ninguno de los documentos antes citados describe un péptido tal y como se reclama en la presente invención. [HRYYESSLEPWYPD (p6.7), FRYYESSLEPWDD (pDD), FRYYES- SLEPWDDD (pDDD), YGGFM (Met-enkephalin, M-enk), YGGFL (Leu- enkephalin, L-enk), ELYENKPRRPYIL (neurotensin, NT), RRPYIL (NT 8-13), and FGGFTGARKSARKLANQ (nociceptin, NC)], which multimerized as dendrimers (Bracci, 2003). In this sense, also some peptides are already in clinical phase I, such as LTX-35-Oncopore ™ and its analog LTX-302 (WKKW-DipKKWK-NH2) that have been used successfully as immunoactivators and oncolytics of melanoma type B16 (Berge, 2010). This is thanks to the knowledge of the molecular geometry and the physicochemical properties of said peptides. Patent application PA / a / 2004/000561 entitled "Effective peptides in the treatment of tumors and other conditions that require the removal or destruction of cells" dated January 16, 2004 and patent application MX / a / 2017 / 007338 entitled "Cyclic peptides derived from cd44v6 for the treatment of diseases related to cancers and angiogenesis" dated June 5, 2017, none of the aforementioned applications describes a peptide as claimed in the present invention. On the other hand, the FDA in the USA has registered and authorized the peptides: Trastuzumab, Bevacizumab, Pertuzumab, Denosumab and Buserelin. (https://webs.iiitd.edu.in/raqhava/thpdb/submitkev.php?ran=6327). Patent applications and patents that describe peptides are the United States patent US 7,632,814 B2 entitled "HYD1 peptides anti-cancer agents", the patent application KR20190073338A entitled "Pharmaceutical composition for inhibiting expression of CCND3 or PAK2 gene" , Australian patent application AU2019203377A1 entitled "Environmentally sensitive compositions", patent application EP3228632 A1 entitled "Seiective anticancer chimeric peptide", US patent application US2017042963 A1 entitled "Anticancer peptide for inhibiting proliferation of cancer stem cells and use", the international patent application WO2016139684A2 entitled "A modified peptides as an anticancer agent", the application of International patent WO2016099188 A1 entitled "Peptide having eight amino acid sequences derived from cage and retaining anticancer activity" and US patent 9 067 970 B2 entitled "Anticancer agents comprising peptides with cancer-specific toxicity", it should be noted that none of the aforementioned documents describes a peptide as claimed in the present invention.
El péptido de la presente invención se diseñó a partir de estudios teóricos de la estructura electrónica, propiedades fisicoquímicas y de reactivad química, determinadas a través de descriptores químico-cuánticos a nivel ab initio. The peptide of the present invention was designed from theoretical studies of the electronic structure, physicochemical properties and chemical reactivity, determined through chemical-quantum descriptors at ab initio level.
Los estudios experimentales mostraron que el péptido sintético de la presente invención tiene un tiempo de acción corto, que es a partir de los 10 minutos en una concentración de 100 ng por cada 10 millones de células, lo que representa una ventaja al ser una dosis baja y en un tiempo de exposición reducido. Experimental studies showed that the synthetic peptide of the present invention has a short action time, which is from 10 minutes at a concentration of 100 ng per 10 million cells, which represents an advantage as it is a low dose. and in a reduced exposure time.
El péptido de la presente invención es altamente selectivo a células cancerosas de mama como lo demuestran los ensayos en las líneas celulares MCF-7 y MDA-MB-231 . The peptide of the present invention is highly selective for breast cancer cells as evidenced by assays on MCF-7 and MDA-MB-231 cell lines.
El péptido de la presente invención es factible de sintetizar y posee una estructura química que le confiere una alta estabilidad, es decir, es estable hasta los 305 °C. The peptide of the present invention is feasible to synthesize and has a chemical structure that gives it high stability, that is, it is stable up to 305 ° C.
BREVE DESCRIPCIÓN DE LA INVENCIÓN BRIEF DESCRIPTION OF THE INVENTION
La presente invención se relaciona con el diseño de un nuevo péptido de interés farmacéutico y con aplicación en terapia antitumoral en células de cáncer de mama. El péptido propuesto posee una estructura hexapeptídica que corresponde a una secuencia de seis aminoácidos repetida 5 veces (FSKWLL)5. De acuerdo con las características químicas, estructurales y a sus propiedades fisicoquímicas, el péptido posee funciones membranolíticas, lo que se confirmó de manera indirecta a través de ensayos de microarreglos de expresión de cDNA. Mismos que demostraron que se relaciona con supresión/activación de vías de señalización celular, que involucran a algunos miembros de la familia de los transportadores ABC, así como a la apolipoproteína H, todos ellos, involucrados en diversas funciones celulares, tales como flujo de metabolitos y moléculas señalizadoras, que en células transformadas conlleva a la resistencia a los agentes quimioterapéuticos. The present invention is related to the design of a new peptide of pharmaceutical interest and with application in antitumor therapy in breast cancer cells. The proposed peptide has a hexapeptide structure that corresponds to a sequence of six amino acids repeated 5 times (FSKWLL) 5 . According to the chemical and structural characteristics and its physicochemical properties, the peptide has membranolytic functions, which was confirmed indirectly through tests of cDNA expression microarrays. They showed that it is related to the suppression / activation of cell signaling pathways, which involve some members of the ABC transporter family, as well as apolipoprotein H, all of them involved in various cellular functions, such as metabolite flux. and signaling molecules, which in transformed cells leads to resistance to chemotherapeutic agents.
Esta invención tiene como propósito formar parte de los esquemas terapéuticos empleados en el cáncer de mama. Particularmente aquellos tumores que no presentan expresión del dominio extracelular de la proteína EGFR que es el blanco del fármaco Herceptin. Puede ser útil después de la resección y antes de iniciar la administración quimioterapéutica. The purpose of this invention is to form part of the therapeutic schemes used in breast cancer. Particularly those tumors that do not show expression of the extracellular domain of the EGFR protein that is the target of the drug Herceptin. It may be useful after resection and before chemotherapy is started.
El péptido propuesto se diseñó a partir de estudios teóricos a nivel atómico, y su tiempo de acción es a partir de los 10 minutos en una concentración de 100 ng por cada 10 millones de células, lo que representa una ventaja al ser una dosis baja y en tiempo de exposición reducido. Es altamente selectivo a células cancerosas de mama como lo demuestran los ensayos en las líneas celulares MCF-7 y MDA-MB- 231 . Además, posee una estructura química que le confiere una alta estabilidad; es decir, es estable hasta los 305.0 °C. The proposed peptide was designed from theoretical studies at the atomic level, and its action time is from 10 minutes at a concentration of 100 ng per 10 million cells, which represents an advantage as it is a low dose and in reduced exposure time. It is highly selective for breast cancer cells as evidenced by assays on MCF-7 and MDA-MB-231 cell lines. In addition, it has a chemical structure that gives it high stability; that is, it is stable up to 305.0 ° C.
OBJETOS DE LA INVENCIÓN OBJECTS OF THE INVENTION
Teniendo en cuenta las limitaciones y desventajas de los tratamientos y fármacos descritos en el estado de la técnica, es un objeto de la presente invención proveer un péptido que tiene como objetivo proporcionar una molécula de tipo peptídico, al que se ha denominado péptido-5 anti-cáncer de mama (P-5ACM), el cual posee alta selectividad a células cancerosas derivadas de adenocarcinoma de mama, MCF-7 y MDA-MB-231 . Es un objeto más de la presente invención proveer un péptido que tiene ventajas sobre los ya existentes en tumores que presentan resistencia a agentes quimioterapéuticos o aquellos para los que no existe fármaco o un blanco específico. Taking into account the limitations and disadvantages of the treatments and drugs described in the state of the art, it is an object of the present invention to provide a peptide that aims to provide a peptide-type molecule, which has been called anti-5-peptide -Breast cancer (P-5ACM), which has high selectivity to cancer cells derived from breast adenocarcinoma, MCF-7 and MDA-MB-231. It is a further object of the present invention to provide a peptide that has advantages over those already existing in tumors that show resistance to chemotherapeutic agents or those for which there is no drug or a specific target.
Un objeto adicional de la presente invención es proveer un péptido (P-5ACM) no tóxico para las células sanas en cultivo (MCF-10A), no transformadas de tejido mamario ni para los linfocitos humanos de sangre periférica de donadores sanos, tanto en cultivos individuales como en co-cultivo, es decir, el péptido no genera toxicidad celular circundante al degradarse rápidamente después de su efecto. An additional object of the present invention is to provide a non-toxic peptide (P-5ACM) for healthy cells in culture (MCF-10A), not transformed from mammary tissue or for human peripheral blood lymphocytes from healthy donors, both in cultures individual as in co-culture, that is, the peptide does not generate surrounding cellular toxicity by rapidly degrading after its effect.
Es todavía más un objeto adicional de la presente invención proveer un péptido que posee una estructura tridimensional que le confiere una alta estabilidad a temperaturas elevadas (305 °C), de acuerdo con los resultados del análisis por calorimetría diferencial de barrido y en el análisis termogravimétrico DSC. It is still a further object of the present invention to provide a peptide that has a three-dimensional structure that confers it high stability at high temperatures (305 ° C), according to the results of the analysis by differential scanning calorimetry and in the thermogravimetric analysis. DSC.
Es aún otro objeto de la presente invención proveer un péptido (P-5ACM) para ser incluido en los esquemas terapéuticos empleados en el cáncer de mama. Particularmente, que se aplique en aquellos tumores que no presentan expresión del dominio extracelular de la proteína EGFR que es el blanco del fármaco Herceptin; después de la resección y antes de iniciar la administración quimioterapéutica. It is still another object of the present invention to provide a peptide (P-5ACM) to be included in the therapeutic schemes used in breast cancer. Particularly, that it be applied in those tumors that do not show expression of the extracellular domain of the EGFR protein that is the target of the Herceptin drug; after resection and before initiating chemotherapeutic administration.
Estos y otros objetos, particularidades y ventajas del péptido (P-5ACM) de la presente solicitud de patente, serán evidentes para un técnico en la materia a partir de la descripción detallada de ciertas modalidades, de las figuras que se acompañan, así como de las reivindicaciones anexas. BREVE DESCRIPCIÓN DE LAS FIGURAS DE LA INVENCIÓN These and other objects, particularities and advantages of the peptide (P-5ACM) of the present patent application will be apparent to a person skilled in the art from the detailed description of certain modalities, from the accompanying figures, as well as from the appended claims. BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION
Los aspectos novedosos que se consideran característicos de la presente invención se establecerán con particularidad en las reivindicaciones anexas. Sin embargo, la invención misma, tanto por su organización, así como por su método de operación, conjuntamente con otros objetos y ventajas de la misma, se comprenderán mejor en la siguiente descripción detallada de las modalidades de la presente invención, cuando se lea en relación con las figuras que se acompañan, en las cuales: The novel aspects that are considered characteristic of the present invention will be set forth with particularity in the appended claims. However, the invention itself, both for its organization, as well as for its method of operation, together with other objects and advantages thereof, will be better understood in the following detailed description of the modalities of the present invention, when read in relationship with the accompanying figures, in which:
La Figura 1A muestra la estructura química tridimensional del péptido FSKWLL, que corresponde a la estructura de mínima energía optimizada a nivel HF/6-31 G (d,p). Figure 1A shows the three-dimensional chemical structure of the FSKWLL peptide, which corresponds to the minimum energy structure optimized at the HF / 6-31 G (d, p) level.
La Figura 1 B muestra la estructura química tridimensional que corresponde a la estructura del hexapéptido, es decir, es la estructura repetida cinco veces del péptido (FSKWLL)5. Figure 1B shows the three-dimensional chemical structure that corresponds to the structure of the hexapeptide, that is, it is the structure repeated five times of the peptide (FSKWLL) 5.
La Figura 2 muestra un espectrograma de espectroscopia infrarroja (IR) del péptido (FSKWLL). En el espectro infrarrojo muestra una banda cerca de 1650 cnr1 que corresponde a la banda de la amida I. Las bandas cerca de 1540 cnr1 y 1200 cnr1 corresponden a la banda de amida II, y de la banda de amida III, respectivamente. La banda cerca de 1400 cnr1 corresponde a la flexión del grupo COO-. La banda correspondiente a la amida I, que se encuentra cerca de 1650 cnr1, es característica de un desorden molecular por lo que no forma b-láminas, ya que estas absorben cerca de 1620 cnr1, sin embargo, si puede formar hélices alfa. La Figura 3 es un gráfico que muestra un espectrograma de la espectroscopiaFigure 2 shows an infrared (IR) spectroscopy spectrogram of the peptide (FSKWLL). In the infrared spectrum it shows a band near 1650 cnr 1 that corresponds to the band of the amide I. The bands near 1540 cnr 1 and 1200 cnr 1 correspond to the band of amide II, and of the band of amide III, respectively . The band around 1400 cnr 1 corresponds to the bending of the COO- group. The band corresponding to amide I, which is close to 1650 cnr 1 , is characteristic of a molecular disorder so it does not form b-sheets, since they absorb about 1620 cnr 1 , however, it can form alpha helices . Figure 3 is a graph showing a spectrogram of spectroscopy
Raman del péptido (FSKWLL). En el espectrograma Raman se observa la banda cerca de 1650 cm-1, que corresponde a la amida I. Al igual que en el espectro de IR el resultado del estiramiento del C = O en el enlace peptídico, a diferencia del IR, en Raman la banda se observa en menor intensidad. Peptide Raman (FSKWLL). In the Raman spectrogram, the band near 1650 cm- 1 is observed, which corresponds to amide I. As in the IR spectrum, the result of the stretching of C = O in the peptide bond, unlike IR, in Raman the band is Observe in less intensity.
La Figura 4 es un gráfico que muestra un Difractograma de Rayos X en polvos del hexapéptido. En el difractograma de rayos-X en polvo, se puede observar un comportamiento amorfo del péptido. Figure 4 is a graph showing a powder X-ray diffractogram of the hexapeptide. In the X-ray powder diffractogram, an amorphous behavior of the peptide can be observed.
La Figura 5A es un gráfico que muestra un análisis termogravimétrico (TGA) del hexapéptido, mientras que la Figura 5B muestra un gráfico que expone un análisis por calorimetría diferencial de barrido (DSC) del hexapéptido. Se observa la pérdida de masa del péptido en función de la temperatura. En el análisis realizado por DSC a 90.43°C se detectó una ganancia de masa como consecuencia de la reacción de la muestra con la atmósfera que la rodea. Existe una pérdida de masa del 2.556% indicativo de una solvatación a 215.23 °C. Finalmente sufre descomposición a 306.18 °C, por lo que se concluye que es estable para su síntesis. Figure 5A is a graph showing a thermogravimetric analysis (TGA) of the hexapeptide, while Figure 5B shows a graph showing a differential scanning calorimetry (DSC) analysis of the hexapeptide. The mass loss of the peptide is observed as a function of temperature. In the analysis carried out by DSC at 90.43 ° C, a mass gain was detected as a consequence of the reaction of the sample with the surrounding atmosphere. There is a mass loss of 2.556% indicative of a solvation at 215.23 ° C. Finally it undergoes decomposition at 306.18 ° C, so it is concluded that it is stable for its synthesis.
Las Figura 6A y 6B muestran imágenes de células en cultivo de la línea celular de cáncer de mama MCF-7 tratadas con el hexapéptido. Se observa el efecto citotóxico del péptido, las células se reducen en tamaño y se dañan las membranas nucleares, sin que se dañen por completo las membranas plasmáticas. Las imágenes fueron obtenidas con microscopía confocal a 100X, con apertura numérica de 0.9. Figures 6A and 6B show images of cultured cells of the MCF-7 breast cancer cell line treated with the hexapeptide. The cytotoxic effect of the peptide is observed, the cells are reduced in size and the nuclear membranes are damaged, without completely damaging the plasma membranes. The images were obtained with confocal microscopy at 100X, with a numerical aperture of 0.9.
Las Figuras 7A y 7B muestran un cultivo de células MCF-7 derivadas de cáncer de mama sin tratamiento. Las células MCF-7 en cultivo se observan extendidas, forman grupos celulares. No se observa daño en las membranas celulares. Figures 7A and 7B show a culture of MCF-7 cells derived from untreated breast cancer. MCF-7 cells in culture are seen to be extended, they form cell groups. Damage to cell membranes is not observed.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
Ante el aumento de casos de muerte por cáncer y con la finalidad de proponer nuevas moléculas para el tratamiento de dicha enfermedad, se inició en el año 2007 la investigación de la presente invención. De acuerdo con una revisión bibliográfica exhaustiva se optó por emplear algoritmos Support Vector Machine (SVM), para generar secuencias peptídicas con características deseables para un péptido que interactúa con las membranas de las células cancerosas, pero se consideró que dichas secuencias no estuviesen reportadas en las bases de datos o como parte de proteínas naturales. Given the increase in cases of death from cancer and in order to propose new molecules for the treatment of this disease, the research of the present invention. According to an exhaustive bibliographic review, it was decided to use Support Vector Machine (SVM) algorithms to generate peptide sequences with desirable characteristics for a peptide that interacts with the membranes of cancer cells, but it was considered that said sequences were not reported in the databases or as part of natural proteins.
Una vez seleccionados los péptidos, se eligieron solo los cinco péptidos con el mayor puntaje de requerimientos en sus propiedades fisicoquímicas de acuerdo a las reglas de Lipinski, así como de un estudio detallado de la estructura electrónica, de sus propiedades fisicoquímicas y de reactividad química, a través de cálculos teóricos para determinar diversos descriptores químico-cuánticos, con lo que se estableció un modelo teórico con características químicas y estructurales específicas, mediante los métodos teóricos de Hartree-Fock (HF) y de la Teoría de Funcionales de la Densidad (DFT) con el funcional híbrido B3LYP, la metodología teórica está dada en Barrientos-Salcedo C, Arenas-Aranda D, Salamanca-Gómez F, Ortiz-Muñiz R, Soriano-Correa C. Cabe mencionar que, el péptido propuesto no se publicó en ese artículo. Once the peptides were selected, only the five peptides with the highest requirement score in their physicochemical properties were chosen according to Lipinski's rules, as well as a detailed study of the electronic structure, their physicochemical properties and chemical reactivity. through theoretical calculations to determine various chemical-quantum descriptors, thereby establishing a theoretical model with specific chemical and structural characteristics, using the theoretical methods of Hartree-Fock (HF) and the Density Functional Theory (DFT ) with the functional hybrid B3LYP, the theoretical methodology is given in Barrientos-Salcedo C, Arenas-Aranda D, Salamanca-Gómez F, Ortiz-Muñiz R, Soriano-Correa C. It should be mentioned that the proposed peptide was not published in that Article.
Los resultados de los cálculos teóricos permitieron generar una serie de secuencias peptídicas que tenían como características en común: una carga neta positiva y parámetros geométricos que le confieren una estructura tridimensional potencial para formar hélices alfa o para plegarse como láminas beta, de un tamaño entre 5 y 30 aminoácidos, con una vida media corta (no mayor a 24 horas), baja toxicidad, solubilidad, anfipaticidad y lipofilicidad con valores que se predice pueden traspasar las membranas con alta fluidez, por ejemplo, como las células cancerosas. The results of the theoretical calculations made it possible to generate a series of peptide sequences that had the following characteristics in common: a net positive charge and geometric parameters that give it a potential three-dimensional structure to form alpha helices or to fold as beta sheets, with a size between 5 and 30 amino acids, with a short half-life (no longer than 24 hours), low toxicity, solubility, amphipathicity and lipophilicity with values predicted to be able to cross membranes with high fluidity, for example, like cancer cells.
Las secuencias de la familia de péptidos propuestos se enviaron a sintetizar con la Compañía Norteamericana American Peptide, utilizando el método en fase sólida. Se solicitó a dicha compañía debido a que todos los productos cumplen un intervalo de pureza de 90 a 99%, y se obtienen de forma liofilizada. The sequences of the proposed peptide family were sent for synthesis with the North American American Peptide Company, using the solid phase method. This company was requested because all products meet an interval of purity from 90 to 99%, and they are obtained in a lyophilized form.
De acuerdo con los ensayos experimentales in vitro, se encontró que un HEXAPÉPTIDO con secuencia de aminoácidos FSKWLL repetida 5 veces (Figura 1 B) tuvo propiedades citotóxicas selectivas contra las células cancerosas de las líneas celulares de cáncer de mama MCF-7 y MDA-MB-231 , ambas procedentes de adenocarcinoma mamario, a partir de los 10 minutos después del tratamiento. According to in vitro experimental tests, a HEXAPEPTIDE with amino acid sequence FSKWLL repeated 5 times (Figure 1 B) was found to have selective cytotoxic properties against cancer cells of the breast cancer cell lines MCF-7 and MDA-MB -231, both from mammary adenocarcinoma, starting 10 minutes after treatment.
Se determinó la concentración efectiva de 100 ng/106 células. Posteriormente, se hizo un análisis de la expresión génica; los resultados mostraron la alteración de algunas proteínas de la familia de los transportadores ABC, así como la apolipoproteína H, todas ellas, involucradas en diversas funciones celulares. The effective concentration of 100 ng / 10 6 cells was determined. Subsequently, an analysis of gene expression was made; the results showed the alteration of some proteins of the ABC transporter family, as well as apolipoprotein H, all of them involved in various cellular functions.
Resumiendo, el péptido de la presente invención comprende una secuencia de seis aminoácidos FSKWLL repetida 5 veces y es un hexapéptido que tiene un intervalo de pureza de 90 a 99% y se encuentra en forma liofilizada. Las ventajas del péptido propuesto son que: su efecto citotóxico se presenta a una concentración baja y el tiempo que se requiere para alcanzar el efecto de dicho péptido es corto, lo cual reduce la posibilidad de desarrollar resistencia, toxicidad y de presentar efectos secundarios. Además, posibilita un menor costo de tratamiento, y es muy estable a altas temperaturas. In summary, the peptide of the present invention comprises a six amino acid sequence FSKWLL repeated 5 times and is a hexapeptide having a purity range of 90 to 99% and is in lyophilized form. The advantages of the proposed peptide are that: its cytotoxic effect occurs at a low concentration and the time required to achieve the effect of said peptide is short, which reduces the possibility of developing resistance, toxicity and presenting side effects. In addition, it enables a lower treatment cost, and is very stable at high temperatures.
Por lo anterior, y lo expuesto más adelante en ejemplos, el péptido de la presente invención con una secuencia de seis aminoácidos FSKWLL repetida 5 veces es útil en la preparación de composición farmacéutica para el tratamiento de cáncer de mamá, en combinación con un excipiente o diluyente farmacéuticamente aceptable. Due to the above, and what is explained later in examples, the peptide of the present invention with a sequence of six amino acids FSKWLL repeated 5 times is useful in the preparation of a pharmaceutical composition for the treatment of breast cancer, in combination with an excipient or pharmaceutically acceptable diluent.
La presente invención será mejor entendida a partir de los siguientes ejemplos, los cuales se presentan únicamente con fines ilustrativos para permitir la comprensión cabal de las modalidades de la presente invención, sin que ello implique que no existan otras modalidades no ilustradas en la presente solicitud de patente y que puedan llevarse a la práctica con base en la descripción detallada previamente realizada. Se describen los siguientes datos y resultados experimentales, esto con la finalidad de aportar los elementos necesarios para llevar a cabo la invención, mas no son limitativos del alcance de la misma. The present invention will be better understood from the following examples, which are presented for illustrative purposes only to allow a thorough understanding. of the modalities of the present invention, without implying that there are no other modalities not illustrated in the present patent application and that can be put into practice based on the detailed description previously made. The following data and experimental results are described, this in order to provide the necessary elements to carry out the invention, but they are not limiting of its scope.
EJEMPLOSEXAMPLES
RESULTADOS EXPERIMENTALES DETALLADOS DETAILED EXPERIMENTAL RESULTS
SÍNTESIS DEL PÉPTIDO PEPTIDE SYNTHESIS
El péptido fue sintetizado por American Peptide, utilizando el método en fase sólida. Se recurrió a esta compañía debido a que todos los productos tienen un intervalo de pureza de 90 a 99%, y se obtuvo de forma liofilizada. The peptide was synthesized by American Peptide, using the solid phase method. This company was used because all the products have a purity range of 90 to 99%, and it was obtained in a lyophilized form.
CARACTERÍSTICAS FISICOQUÍMICAS DEL HEXAPÉPTIDO PHYSICOCHEMICAL CHARACTERISTICS OF THE HEXAPEPTIDE
Se utilizó el péptido # 340572 sintetizado por American Peptide, con un porcentaje de pureza del 95% y peso molecular de 3892.82 g/mol calculado por espectroscopia de masas por electrospray y punto isoeléctrico (pl) de 10.6. Peptide # 340572 synthesized by American Peptide was used, with a purity percentage of 95% and molecular weight of 3892.82 g / mol calculated by electrospray mass spectroscopy and isoelectric point (pl) of 10.6.
ENSAYO DE CITOTOXICIDAD CYTOTOXICITY TEST
En un cultivo celular se determinó y sembró la concentración efectiva de 100 ng/106 células cancerosas de las líneas celulares de cáncer de mama MCF-7 y MDA-MB- 231 , ambas procedentes de adenocarcinoma mamario. Posteriormente, se hizo un análisis de la expresión génica; los resultados mostraron la alteración de algunas proteínas de la familia de los transportadores ABC, así como la apolipoproteína H, todas ellas, involucradas en diversas funciones celulares. De acuerdo con las pruebas experimentales in vitro, se encontró que un HEXAPÉPTIDO con secuencia de aminoácidos FSKWLL repetida 5 veces (Figura 1 B), tuvo propiedades citotóxicas selectivas contra las células cancerosas de las líneas celulares de cáncer de mama MCF- 7 y MDA-MB-231 , a partir de los 10 minutos después del tratamiento. Lo anterior se observa como se observa en las imágenes de células en cultivo de la línea celular de cáncer de mama MCF-7 tratadas con el hexapéptido mostradas en las figuras 6A y 6B. Dichas imágenes muestran efecto citotóxico del péptido, las células se reducen en tamaño y se dañan las membranas nucleares, sin que se dañen por completo las membranas plasmáticas. Las imágenes fueron obtenidas con microscopía confocal a 100X, con apertura numérica de 0.9. In a cell culture, the effective concentration of 100 ng / 10 6 cancer cells of the breast cancer cell lines MCF-7 and MDA-MB-231, both from mammary adenocarcinoma, was determined and seeded. Subsequently, an analysis of gene expression was made; the results showed the alteration of some proteins of the ABC transporter family, as well as apolipoprotein H, all of them involved in various cellular functions. According to in vitro experimental tests, it was found that a HEXAPEPTIDE with sequence of amino acids FSKWLL repeated 5 times (Figure 1 B), had selective cytotoxic properties against cancer cells of the breast cancer cell lines MCF-7 and MDA-MB-231, starting 10 minutes after treatment. This is observed as observed in the images of cells in culture of the MCF-7 breast cancer cell line treated with the hexapeptide shown in Figures 6A and 6B. These images show the cytotoxic effect of the peptide, the cells are reduced in size and the nuclear membranes are damaged, without completely damaging the plasma membranes. The images were obtained with confocal microscopy at 100X, with a numerical aperture of 0.9.
Por otro lado, las figuras 7A y 7B muestran imágenes de un cultivo de células MCF-7 derivadas de cáncer de mama sin tratamiento. Las células MCF-7 en cultivo se observan extendidas y forman grupos celulares. Además de lo anterior, no se observa daño en las membranas celulares. On the other hand, Figures 7A and 7B show images of a culture of MCF-7 cells derived from breast cancer without treatment. MCF-7 cells in culture are seen to be spread out and form cell clumps. In addition to the above, no damage to cell membranes is observed.
DIFRACCIÓN DE RAYOS-X EN POLVOS X-RAY DIFFRACTION IN POWDERS
Se utilizó un Difractómetro de rayos-X Bruker D2 PHASER con radiación de cobre Ka y SSD160 ojo de lince, y se utilizó la paquetería del software DIFFRAC SUITE™. Para la lectura de la muestra, ésta se colocó en un portamuestra de vidrio con la ayuda de una espátula, y se colocó en la plataforma. El patrón de difracción se obtuvo a temperatura ambiente y en ángulos de incidencia theta de 5 a 60 por un tiempo de 10 minutos y al finalizar, la muestra se recuperó totalmente. ESPECTROSCOPIA INFRARROJA A Bruker D2 PHASER X-ray diffractometer with Ka copper radiation and Lynx eye SSD160 was used, and the DIFFRAC SUITE ™ software package was used. For reading the sample, it was placed in a glass sample holder with the help of a spatula, and placed on the platform. The diffraction pattern was obtained at room temperature and at angles of incidence theta from 5 to 60 for a time of 10 minutes and at the end, the sample was fully recovered. INFRARED SPECTROSCOPY
Para la determinación del espectro de IR de la muestra se utilizó un Espectrofotómetro de Infrarrojo Nicolet™ iS™50 de Thermo Scientific™. Primero se determinó un espectro blanco del medio ambiente para evitar interferencias en la muestra. Posteriormente, se colocó una cantidad mínima de muestra, y se realizó la determinación espectroscópica de la muestra. El resultado se visualizó con el software del equipo OMNIC Spectra. El gráfico de la Figura 2, representa un espectrograma de espectroscopia infrarroja (IR) del péptido (FSKWLL). En el espectro infrarrojo muestra una banda cerca de 1650 cnr1 que corresponde a la banda de la amida I. Las bandas cerca de 1540 cnr1 y 1200 cnr1 corresponden a la banda de amida II, y de la banda de amida III, respectivamente. La banda cerca de 1400 cnr1 corresponde a la flexión del grupo COO-. La banda correspondiente a la amida I, que se encuentra cerca de 1650 cnr1, es característica de un desorden molecular por lo que no forma b-láminas, ya que estas absorben cerca de 1620 cnr1, sin embargo, si puede formar hélices alfa. For the determination of the IR spectrum of the sample, a Thermo Scientific ™ Nicolet ™ iS ™ 50 Infrared Spectrophotometer was used. An environmental white spectrum was first determined to avoid interference in the sample. Subsequently, a minimum amount of sample was placed, and the spectroscopic determination of the sample was carried out. The result was visualized with the OMNIC Spectra equipment software. The graph in Figure 2 represents an infrared spectroscopy (IR) spectrogram of the peptide (FSKWLL). In the infrared spectrum it shows a band near 1650 cnr 1 that corresponds to the band of the amide I. The bands near 1540 cnr 1 and 1200 cnr 1 correspond to the band of amide II, and of the band of amide III, respectively . The band around 1400 cnr 1 corresponds to the bending of the COO- group. The band corresponding to amide I, which is close to 1650 cnr 1 , is characteristic of a molecular disorder so it does not form b-sheets, since they absorb about 1620 cnr 1 , however, it can form alpha helices .
ESPECTROSCOPIA RAMAN RAMAN SPECTROSCOPY
Para la determinación del espectro Raman se utilizó un equipo de microscopía Raman acoplado a un espectrofotómetro Raman adaptado con un láser de 630 nm de modelo Alpha 300M+ de la compañía Witec, Focus Innovation y se utilizó el programa Project Four y Control Four incluidos en la paquetería del equipo. En el equipo (microscopio) se colocó una cantidad mínima de muestra en un portaobjetos, y se enfocó la muestra a 5x y 50x; y posteriormente se hizo incidir la radiación láser a 630 nm sobre la muestra para obtener el espectro Raman. El gráfico de la figura 3 muestra el gráfico que representa un espectrograma de la espectroscopia Raman del péptido (FSKWLL). En el espectrograma Raman se observa la banda cerca de 1650 cm-1, que corresponde a la amida I. Al igual que en el espectro de IR el resultado del estiramiento del C = O en el enlace peptídico, a diferencia del IR, en Raman la banda se observa en menor intensidad. TERMOGRAVIMETRÍA For the determination of the Raman spectrum, a Raman microscopy equipment was used coupled to a Raman spectrophotometer adapted with a 630 nm laser of the Alpha 300M + model from the company Witec, Focus Innovation, and the Project Four and Control Four programs included in the package were used. of the team. In the equipment (microscope) a minimum amount of sample was placed on a slide, and the sample was focused at 5x and 50x; and subsequently the laser radiation at 630 nm was impinged on the sample to obtain the Raman spectrum. The graph in Fig. 3 shows the graph representing a Raman spectroscopy spectrogram of the peptide (FSKWLL). In the Raman spectrogram, the band near 1650 cm- 1 is observed, which corresponds to amide I. As in the IR spectrum, the result of the stretching of C = O in the peptide bond, unlike IR, in Raman the band is observed in less intensity. THERMOGRAVIMETRY
Para la obtención del termograma de análisis termogravimétrico se utilizó un equipo de TA Instruments modelo Q50 y la paquetería de TA Universal Analysis. Para la determinación termogravimétrica primero se taró el porta muestras para ajustar a cero el valor de la medida de masa, posteriormente se adicionó la muestra. El peso de la muestra fue de 0.008 mg, y se varió la temperatura en el programa del ordenador de 20eC / min; el intervalo de temperatura fue de 30°C a 400 °C. To obtain the thermogravimetric analysis thermogram, a TA Instruments model Q50 equipment and the TA Universal Analysis package were used. For thermogravimetric determination, the sample holder was first tarred to zero the value of the mass measurement, later the sample was added. The weight of the sample was 0.008 mg, and the temperature was varied in the computer program of 20 e C / min; the temperature range was from 30 ° C to 400 ° C.
CALORIMETRÍA DIFERENCIAL DE BARRIDO SWEEP DIFFERENTIAL CALORIMETRY
Para la obtención del termograma de Calorimetría Diferencial de Barrido se utilizó un equipo de TA Instruments modelo Q200 y la paquetería de TA Universal Analysis. Para la determinación del termograma, la muestra se cargó en una cápsula de aluminio con una capacidad entre 10 - 50 mI. La cápsula se selló mediante la prensa selladora, con una tapa de aluminio para impedir que, por problemas de dilatación o descomposición de la muestra, ésta se proyectara fuera de la cápsula y contaminara el equipo. Además, se utilizó una capsula de referencia que se colocó a la par de la capsula que contenía la muestra. La muestra se corrió durante 20 minutos, a una temperatura inicial de 20°C y hasta 400°C. To obtain the Differential Scanning Calorimetry thermogram, a TA Instruments model Q200 equipment and the TA Universal Analysis package were used. For the determination of the thermogram, the sample was loaded into an aluminum capsule with a capacity between 10-50 ml. The capsule was sealed by means of the sealing press, with an aluminum cap to prevent that, due to expansion or decomposition problems of the sample, it was projected out of the capsule and contaminating the equipment. In addition, a reference capsule was used that was placed next to the capsule containing the sample. The sample was run for 20 minutes, at an initial temperature of 20 ° C and up to 400 ° C.
La Figura 5a expone en un gráfico un análisis termogravimétrico (TGA) del hexapéptido, mientras que el gráfico de la Figura 5B expone el análisis por calorimetría diferencial de barrido (DSC) del hexapéptido. Figure 5a graphs a thermogravimetric analysis (TGA) of the hexapeptide, while Figure 5B graphs the differential scanning calorimetry (DSC) analysis of the hexapeptide.
A través de ambos gráficos se observa la pérdida de masa del péptido en función de la temperatura. En el análisis realizado por DSC a 90.43°C se detectó una ganancia de masa como consecuencia de la reacción de la muestra con la atmósfera que la rodea. Existe una pérdida de masa del 2.556% indicativo de una solvatación a 215.23 °C. Finalmente sufre descomposición a 306.18 °C, por lo que se concluye que es estable para su síntesis. Through both graphs, the loss of mass of the peptide as a function of temperature is observed. In the analysis carried out by DSC at 90.43 ° C, a mass gain was detected as a consequence of the reaction of the sample with the surrounding atmosphere. There is a mass loss of 2.556% indicative of a solvation at 215.23 ° C. Finally it undergoes decomposition at 306.18 ° C, so it is concluded that it is stable for its synthesis.
La presente invención tiene usos importantes en el tratamiento contra la proliferación de células cancerosas de mama, como lo demuestra la actividad membranolítica y alta selectividad que posee el péptido propuesto en contra de las células de cáncer de mama. No obstante, su efecto depende de la dosis, pero esta es muy baja en comparación con las empleadas en otros péptidos con efectos semejantes. El efecto citotóxico que posee el péptido propuesto (P-5ACM), es muy útil y conveniente para que forme parte del esquema terapéutico contra células de cáncer de mama resistente a quimioterapia. La vía de administración debe ser por inyección intraductal a la glándula mamaria y puede ser directa debido a que no es tóxico y a su corto tiempo de vida media. The present invention has important uses in treating breast cancer cell proliferation, as evidenced by the activity membranolytic and high selectivity that the proposed peptide possesses against breast cancer cells. However, its effect depends on the dose, but this is very low compared to those used in other peptides with similar effects. The cytotoxic effect of the proposed peptide (P-5ACM) is very useful and convenient for it to form part of the therapeutic scheme against chemotherapy-resistant breast cancer cells. The route of administration should be by intraductal injection to the mammary gland and can be direct due to its non-toxicity and its short half-life.
Se hace constar que con relación a esta fecha el mejor método conocido por el solicitante para llevar a cabo la citada invención es el que resulta claro de la presente descripción de la invención. Aun cuando en la anterior descripción se ha hecho referencia a ciertas modalidades de la presente invención, debe hacerse hincapié en que son posibles numerosas modificaciones a dichas modalidades, pero sin apartarse del verdadero alcance de la invención, de tal modo que las características de la invención descritas en las modalidades de la misma, mostradas en las figuras y reclamadas en las reivindicaciones, pueden utilizarse individualmente o en cualquier combinación arbitraria para la realización de la presente invención, así como de diferentes modalidades que no hayan sido aquí descritas. Por consiguiente, debe entenderse que las modalidades de la presente invención son únicamente ilustrativas y no pretenden limitar el alcance de la presente invención, excepto por lo establecido tanto en el estado de la técnica como en las reivindicaciones anexas. It is noted that in relation to this date the best method known to the applicant to carry out the aforementioned invention is the one that is clear from the present description of the invention. Although reference has been made to certain embodiments of the present invention in the foregoing description, it should be emphasized that numerous modifications to such embodiments are possible, but without departing from the true scope of the invention, such that the characteristics of the invention described in the modalities thereof, shown in the figures and claimed in the claims, can be used individually or in any arbitrary combination for the realization of the present invention, as well as in different modalities that have not been described here. Accordingly, it should be understood that the embodiments of the present invention are illustrative only and are not intended to limit the scope of the present invention, except as set forth both in the state of the art and in the appended claims.

Claims

NOVEDAD DE LA INVENCIÓN NOVELTY OF THE INVENTION
REIVINDICACIONES
1 Un péptido caracterizado porque comprende una secuencia de seis aminoácidos FSKWLL repetida 5 veces 1 A peptide characterized by comprising a six amino acid sequence FSKWLL repeated 5 times
2.- El péptido de conformidad con la reivindicación 1 , caracterizado porque el péptido es un hexapéptido. 2. The peptide according to claim 1, characterized in that the peptide is a hexapeptide.
3.- El péptido de conformidad con la reivindicación 1 , caracterizado porque el péptido tiene un intervalo de pureza de 90 a 99% y se encuentra en forma liofilizada. 3. The peptide according to claim 1, characterized in that the peptide has a purity range of 90 to 99% and is in lyophilized form.
4.- El péptido de conformidad cualquiera de las reivindicaciones anteriores para usarse en el tratamiento de cáncer de mama. 4. The peptide according to any of the preceding claims for use in the treatment of breast cancer.
5.- Uso de un péptido de conformidad con cualquiera de las reivindicaciones 1 a 3 para preparar una composición farmacéutica para el tratamiento de cáncer de mamá. 5.- Use of a peptide according to any of claims 1 to 3 to prepare a pharmaceutical composition for the treatment of breast cancer.
6.- El uso de conformidad con la reivindicación 5, en donde el cáncer de mamá es un cáncer de mama como lo son las células MCF-7 o MDA-MB-231 . 6. The use according to claim 5, wherein the breast cancer is a breast cancer such as MCF-7 or MDA-MB-231 cells.
7.- Una composición farmacéutica que comprende como principio activo el péptido con una secuencia de seis aminoácidos FSKWLL repetida 5 veces y un excipiente o diluyente farmacéuticamente aceptable. 7.- A pharmaceutical composition that comprises as an active principle the peptide with a six amino acid sequence FSKWLL repeated 5 times and a pharmaceutically acceptable excipient or diluent.
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