BR102019024710A2 - COMPOUND, USE, PHARMACEUTICAL COMPOSITION, METHOD OF DETECTION AND METHOD OF LOADING AND/OR INTERNALIZING A COMPOUND INTO EUKARYOTIC CELLS - Google Patents

Abstract

compound, use, pharmaceutical composition, detection method, and method of carrying and/or internalizing a compound into eukaryotic cells. the invention belongs to the technical fields of pharmaceutical sciences, immunology, diagnosis and/or cancer treatment. specifically, the invention relates to compounds for targeting or internalizing molecular entities into the intracellular domain of eukaryotic or neoplastic cells, to their use to modify other pharmaceutically active ingredients, to a pharmaceutical composition comprising said compound, and to a method for diagnosis, prognosis, and /or treatment for cancer or other cell-altering conditions.

Description

COMPOUND, USE, PHARMACEUTICAL COMPOSITION, METHOD OF DETECTION AND METHOD OF LOADING AND/OR INTERNALIZING A COMPOUND INTO EUKARYOTIC CELLS Field of Invention

[0001] The invention belongs to the technical fields of Pharmaceutical Sciences, Immunology, Diagnosis and/or Treatment for Cancer. Specifically, the invention relates to compounds for targeting molecular entities to the intracellular domain of neoplastic/eukaryotic cells, to their use to modify other pharmaceutically active ingredients, to a pharmaceutical composition comprising said compound and to a method for diagnosis, prognosis and/or treatment of cancer or other cell-altering conditions.

Description of the State of the Technique

[0002] Targeting active pharmaceutical ingredients (API) to tumor cells is a highly desirable feature for combating cancer and related cellular abnormalities. Even more desirable is to target APIs selectively at tumor cells.

[0003] Cell penetration peptides (CPP) are well known in the art. In general, CPPs typically do not exceed 30 residues in length and generally carry a positive charge, which facilitates electrostatic interactions with the negatively charged cell surface. The compound of the invention is also peptide in nature, but does not have conventional characteristics of CPPs: their origin, sequence, chemical charge, type of bond and general physicochemical properties are very different.

[0004] The compound of the invention derives from the study of Amblyomin-X, which is a Kunitz-like protein homolog, identified in the transcriptome of the salivary glands of the adult tick Amblyomma sculptum, which was studied by the present inventors. The recombinant protein form of Amblyomin-X showed antitumor activity via apoptosis induction and proteasome inhibition. In addition, this molecule showed pro-apoptotic effects on tumor cells1-3 and decreased tumor growth and metastases in vivo2,3. Amblyomin-X's mechanism of action involves proteasome inhibition, which occurs preferentially through the proteasome's trypsin-like (T-L) activity2. Some of the present inventors have shown that although the primary target of Amblyomin-X appears to be the proteasome, this macromolecule also inhibits autophagy through a suggested mechanism involving the activation of mTOR, which is transported by cytoplasmic dynein4. Furthermore, some of the inventors have reported the proapoptotic effect of Amblyomin-X on these human tumor cells associated with inhibition of proteasome function, ER stress (increased expression of UPR markers), [Ca2+] mobilization, mitochondrial dysfunction, PARP cleavage and activation of caspase3. Interestingly, none of these changes were observed in normal human fibroblast cells5

• 1. Akagi EM, Junior PL, Simons SM, Bellini MH, Barreto SA, ChudzinskiTavassi AM. Pro-apoptotic effects of Amblyomin-X in murine renal cell carcinoma "in vitro". Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 2012; 66:64-9.
• 2. Chudzinski-Tavassi AM, De-Sa-Junior PL, Simons SM, Maria DA, de Souza Ventura J, Batista IF, et al. A new tick Kunitz type inhibitor, Amblyomin-X, induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system. Toxicon: official journal of the International Society on Toxinology 2010; 56:1145-54.
• 3. Ventura JS, Faria F, Batista IF, Simons SM, Oliveira DG, Morais KL, et al. A Kunitz-type FXa inhibitor affects tumor progression, hypercoagulable state and triggers apoptosis. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 2013; 67:192-6.
• 4. Pacheco MT, Berra CM, Morais KL, Sciani JM, Branco VG, Bosch RV, et al. Dynein function and protein clearance changes in tumor cells induced by a Kunitz-type molecule, Amblyomin-X. PloS one 2014; 9:e111907.
• 5. Morais KL, Pacheco MT, Berra CM, Bosch RV, Sciani JM, Chammas R, et al. Amblyomin-X induces ER stress, mitochondrial dysfunction, and caspase activation in human melanoma and pancreatic tumor cell. Molecular and cellular biochemistry 2016; 415:119-31.
• 6. de Souza JG, Morais KL, Angles-Cano E, Boufleur P, de Mello ES, Maria DA, et al. Promising pharmacological profile of a Kunitz-type inhibitor in murine renal cell carcinoma model. Oncotarget 2016; 7:62255-66.
• 7. Pacheco MT, Morais KL, Berra CM, Demasi M, Sciani JM, Branco VG, et al. Specific role of cytoplasmic dynein in the mechanism of action of an antitumor molecule, Amblyomin-X. Experimental cell research 2016; 340:248-58.
• 8. Benavent Acero FR, Perera Negrin Y, Alonso DF, Perea SE, Gomez DE, Farina HG. Mechanisms of Cellular Uptake, Intracellular Transportation, and Degradation of CIGB-300, a Tat-Conjugated Peptide, in Tumor Cell Lines. Molecular pharmaceutics 2014; 11:1798-807.
• 9. Kristensen M, Birch D, Morck Nielsen H. Applications and Challenges for Use of Cell-Penetrating Peptides as Delivery Vectors for Peptide and Protein Cargos. International journal of molecular sciences 2016; 17.
• 10. Batista IF, Ramos OH, Ventura JS, Junqueira-de-Azevedo IL, Ho PL, Chudzinski-Tavassi AM. A new Factor Xa inhibitor from Amblyomma cajennense with a unique domain composition. Archives of biochemistry and biophysics 2010; 493:151-6.
• 11. Benedetti G, Morais KL, Guerreiro JR, de Oliveira EF, Hoshida MS, Oliveira L, et al. Bothrops jararaca peptide with anti-hypertensive action normalizes endothelium dysfunction involved in physiopathology of preeclampsia. PloS one 2011; 6:e23680.
• 12. Riedl S, Rinner B, Asslaber M, Schaider H, Walzer S, Novak A, et al. In search of a novel target - phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy. Biochimica et biophysica acta 2011; 1808:2638-45.
• 13. Bohm G, Muhr R, Jaenicke R. Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein engineering 1992; 5:191- 5.
• 14. Deleage G, Geourjon C. An interactive graphic program for calculating the secondary structure content of proteins from circular dichroism spectrum. Computer applications in the biosciences: CABIOS 1993; 9:197-9.
• 15- North American Patent No.: US 8,883,740 entitled Peptides, Compositions and Uses Thereof.
• 16 – Canadian patent application CA2712807 entitled Peptides, Compositions and Uses Thereof.
• 17- Chudzinski-Tavassi AM, Carrijo-Carvalho LC, Waismam K, Farsky SH, Ramos OH, Reis CV. A lipocalin sequence signature modulates cell survival. FEBS Lett. 2010 Jul 2;584(13):2896-900.
• 18- Carrijo-Carvalho LC, Maria DA, Ventura JS, Morais KL, Melo RL, Rodrigues CJ, Chudzinski-Tavassi AM. A lipocalin-derived Peptide modulating fibroblasts and extracellular matrix proteins. J Toxicol. 2012;2012:325250.
• 19- Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973 Nov;52(11):2745-56.
• 20- Alvarez Flores MP, Fritzen M, Reis CV, Chudzinski-Tavassi AM. Losac, a factor X activator from Lonomia obliqua bristle extract: its role in the pathophysiological mechanisms and cell survival. Biochem Biophys Res Commun. 2006 May 19;343(4):1216-23.
• 21- Perea SE, Reyes O, Puchades Y, Mendoza O, Vispo NS, Torrens I, Santos A, Silva R, Acevedo B, López E, Falcón V, Alonso DF. Antitumor effect of a novel proapoptotic peptide that impairs the phosphorylation by the protein kinase 2 (casein kinase 2). Cancer Res. 2004 Oct 1;64(19):7127-9.

[0005] Despite years of intensive study of Amblyomin-X by the inventors, they are so far not aware of any previous reports describing or suggesting the compound of the invention, let alone its surprising and relevant technical effects. The closest scientific works are recited below.

• 1. Akagi EM, Junior PL, Simons SM, Bellini MH, Barreto SA, Chudzinski Tavassi AM. Pro-apoptotic effects of Amblyomin-X in murine renal cell carcinoma "in vitro". Biomedicine & pharmacotherapy = Biomedicine & pharmacotherapie 2012; 66:64-9.
• 2. Chudzinski-Tavassi AM, De-Sa-Junior PL, Simons SM, Maria DA, de Souza Ventura J, Batista IF, et al. A new tick Kunitz type inhibitor, Amblyomin-X, induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system. Toxicon: official journal of the International Society on Toxinology 2010; 56:1145-54.
• 3. Ventura JS, Faria F, Batista IF, Simons SM, Oliveira DG, Morais KL, et al. Kunitz-type FXa inhibitor affects tumor progression, hypercoagulable state and triggers apoptosis. Biomedicine & pharmacotherapy = Biomedicine & pharmacotherapie 2013; 67:192-6.
• 4. Pacheco MT, Berra CM, Morais KL, Sciani JM, Branco VG, Bosch RV, et al. Dynein function and protein clearance changes in tumor cells induced by a Kunitz-type molecule, Amblyomin-X. PloSone 2014; 9:e111907.
• 5. Morais KL, Pacheco MT, Berra CM, Bosch RV, Sciani JM, Chammas R, et al. Amblyomin-X induces ER stress, mitochondrial dysfunction, and caspase activation in human melanoma and pancreatic tumor cell. Molecular and cellular biochemistry 2016; 415:119-31.
• 6. de Souza JG, Morais KL, Angles-Cano E, Boufleur P, de Mello ES, Maria DA, et al. Promising pharmacological profile of a Kunitz-type inhibitor in murine renal cell carcinoma model. Oncotarget 2016; 7:62255-66.
• 7. Pacheco MT, Morais KL, Berra CM, Demasi M, Sciani JM, Branco VG, et al. Specific role of cytoplasmic dynein in the mechanism of action of an antitumor molecule, Amblyomin-X. Experimental cell research 2016; 340:248-58.
• 8. Benavent Acero FR, Perera Negrin Y, Alonso DF, Perea SE, Gomez DE, Farina HG. Mechanisms of Cellular Uptake, Intracellular Transportation, and Degradation of CIGB-300, a Tat-Conjugated Peptide, in Tumor Cell Lines. Molecular Pharmaceuticals 2014; 11:1798-807.
• 9. Kristensen M, Birch D, Morck Nielsen H. Applications and Challenges for Use of Cell-Penetrating Peptides as Delivery Vectors for Peptide and Protein Cargo. International journal of molecular sciences 2016; 17.
• 10. Batista IF, Ramos OH, Ventura JS, Junqueira-de-Azevedo IL, Ho PL, Chudzinski-Tavassi AM. A new Factor Xa inhibitor from Amblyomma cajennense with a unique domain composition. Archives of biochemistry and biophysics 2010; 493:151-6.
• 11. Benedetti G, Morais KL, Guerreiro JR, de Oliveira EF, Hoshida MS, Oliveira L, et al. Bothrops jararaca peptide with antihypertensive action normalizes endothelium dysfunction involved in physiopathology of preeclampsia. PloSone 2011; 6:e23680.
• 12. Riedl S, Rinner B, Asslaber M, Schaider H, Walzer S, Novak A, et al. In search of a novel target - phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy. Biochimica et biophysica acta 2011; 1808:2638-45.
• 13. Bohm G, Muhr R, Jaenicke R. Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein engineering 1992; 5:191-5.
• 14. Deleage G, Geourjon C. An interactive graphic program for calculating the secondary structure content of proteins from circular dichroism spectrum. Computer applications in the biosciences: CABIOS 1993; 9:197-9.
• 15- North American Patent No.: US 8,883,740 entitled Peptides, Compositions and Uses Thereof.
• 16 – Canadian patent application CA2712807 entitled Peptides, Compositions and Uses Thereof.
• 17- Chudzinski-Tavassi AM, Carrijo-Carvalho LC, Waismam K, Farsky SH, Ramos OH, Reis CV. A lipocalin sequence signature modulates cell survival. FEBS Lett. 2010 Jul 2;584(13):2896-900.
• 18- Carrijo-Carvalho LC, Maria DA, Ventura JS, Morais KL, Melo RL, Rodrigues CJ, Chudzinski-Tavassi AM. A lipocalin-derived Peptide modulating fibroblasts and extracellular matrix proteins. J Toxicol. 2012;2012:325250.
• 19- Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973 Nov;52(11):2745-56.
• 20- Alvarez Flores MP, Fritzen M, Reis CV, Chudzinski-Tavassi AM. Losac, a factor X activator from Lonomia obliqua bristle extract: its role in the pathophysiological mechanisms and cell survival. Biochem Biophys Res Commun. 2006 May 19;343(4):1216-23.
• 21- Perea SE, Reyes O, Puchades Y, Mendoza O, Vispo NS, Torrens I, Santos A, Silva R, Acevedo B, López E, Falcón V, Alonso DF. Antitumor effect of a novel proapoptotic peptide that impairs the phosphorylation by the protein kinase 2 (casein kinase 2). Cancer Res. 2004 Oct 1;64(19):7127-9.

[0006] To the best knowledge of the inventors, no document in the art discloses or even suggests the teachings of the present invention.

Summary of the Invention

[0007] The present invention provides compounds for directing/internalizing molecular entities to the intracellular domain of neoplastic or cancerous eukaryotic cells. The invention also provides the use of said compound to modify other active pharmaceutical ingredients such that said modified active ingredients are selectively targeted to eukaryotic, neoplastic or cancerous cells. The invention also provides a pharmaceutical composition comprising said compounds and a method for diagnosing, prognosticating and/or treating cancer or other altered cellular conditions.

[0008] The inventive concept underlying the objects of the invention is a polypeptide compound having at least 80% identity to the polypeptide of SEQ ID No.1, SEQ ID No.6 or SEQ ID No.7. Embodiments of the compound of the invention include: a polypeptide of SEQ ID No.1, SEQ ID No.6 or SEQ ID No.7; its fragments; and/or cyclic modified forms thereof, including those modified with amide, alkyl-, alkoxy, halogen, hydroxy- or PEG groups, as well as those modified with other functional groups or amino acids/peptides, including unnatural amino acids such as d-forms of amino acids, salts of any of the above entities; and/or combinations thereof.

[0009] In one embodiment, the compound of the invention is used to modify small molecules so that the resulting complex is enabled with the ability to penetrate tumor cells.

[0010] In another embodiment, the compound of the invention is used to modify other peptide molecules so that the resulting complex is enabled with the ability to penetrate tumor cells.

[0011] These and other objects of the invention will be more readily appreciated by those skilled in the art from the evidence supporting the detailed description of the invention, as well as the appended claims.

Brief Description of Figures

[0012] Figure 1 reveals the non-cytotoxicity induced by an embodiment of compound of the invention (termed C-ter), and with reference only to the Kunitz domain. After 48 h, the cell viability of tumor cells (SKMEL-28 and MIA PaCa-2) was measured by MTT using increasing concentrations (micromolar) of C-ter and Kunitz. Positive controls: 20 µM complete amblyomin-X, 5 µM staurosporine, 80 nM bortezomib (BTZ80) and 5 µM MG132.

[0013] Figure 2 shows the effects of an embodiment of the compound of the invention (C-ter) on the cell viability of tumor and non-tumor cells, compared to the Kunitz domain of Amblyomin-X. Cells were incubated with increasing concentrations of C-ter or Kunitz for 48 h at 37°C. MG-132 (5) was used for 48 h, a known proteasome inhibitor, as a positive control. Then, cell viability was measured by MTT reduction.

[0014] Figure 3 shows the internalization of an embodiment of a compound of the invention by tumor. (A) Cells were incubated with C-ter488 (20) and, for comparison, Kunitz-488 or Amblyomin-X-488 (20), for 4h. Green fluorescence was monitored with confocal laser scanning microscopy. The core was stained with 5 µM Syto59. (B) Microinjection of Kunitz-488 and Amblyomin-X-488 triggers cell death in human melanoma cells. Cells were microinjected with 20 µM Kunitz or Amblyomin-X and incubated for 4 h, when cell death was scored, based on green fluorescence and PI staining. (C) Percentage of positive cells for nuclear translocation of C-ter and Amblyomin-X in SKMEL-28 and MIA PaCa-2 cells. Both vesicles (C-ter and Amblyomin-X) or nuclear translocation were analyzed using MetaXpress software (Molecular Devices, CA).

[0015] Figure 4 shows that the compound of the invention is taken up by the tumor and, to a lesser extent, by non-tumor cells (HUVEC cells, HDFa). Cells were incubated with Amblyomin-X-488 (1 µM), Cter-488 (10 µM), Kunitz-488 (10 µM) or Alexa Fluor 488 (0.5 nM) for 4 h. Green fluorescence was monitored with confocal laser scanning microscopy.

[0016] Figure 5 shows the compound of the invention used to modify a pharmaceutical ingredient used for diagnostic purposes. The incorporation of C-ter-488 was initiated by lipid rafts. Tumor cell lines were treated with C-ter-488 at the indicated times; cells were then incubated with transfer of Alexa Fluor 555 (100 mg/mL), Alexa Fluor 555 cholera toxin B subunit (50 mg/mL) or Alexa Fluor 555 dextran (1 mg/mL) in ligation medium. at 37°C for 20 min. Next, green and red fluorescence were monitored by a Molecular Devices ImageXpress Micro Confocal plate scanning microscope. The nucleus was stained with Hoechst 332. The overlap between C-ter and endocytosis markers was analyzed using MetaXpress software (Molecular Devices, Sunnyvale, CA).

[0017] Figure 6 shows that the compound of the invention and/or its cargo is/are targeted to the caveosome and/or lysosomes as an intracellular destination. Tumor cell lines were treated with 488-Cter at the indicated concentration. Then, cells were incubated with a primary antibody against caveolin-1 or LAMP-2, followed by a secondary antibody conjugated to Alexa Fluor 555. Further, after treatment, cells were incubated with Lyso Tracker Red DND 99 (100 nM ) in binding medium at 37°C for 20 min. Then, the green and red fluorescences were monitored by a Micro Devices plate scanning microscope from Molecular Devices ImageXpress. The nucleus stained with Hoechst 332. The overlap between C-ter and intracellular markers was analyzed using MetaXpress software (Molecular Devices, Sunnyvale, CA).

[0018] Figure 7 shows selective uptake of Amblyomin-X via PS (phosphatidylserine) recognition. (A) The amount of PS exposed in the cells was analyzed by FITC-Annexin V staining, followed by FACS analysis or confocal microscopy. (B) SK-MEL-28 cells were pretreated with Na3VO4 (sodium orthovanadate - an inhibitor of PS synthesis) for 2h. Then, the cells were incubated with Amblyomin-X-488 (1), P1-488 (10) for 4h. Green fluorescence was monitored with confocal laser scanning microscopy.

[0019] Figure 8 presents the circular dichroism analysis of the inventive compound C-ter and Kunitz for comparison. Spectra were measured at a peptide concentration of 20 µM. The Amblyomin-X result is from Pasqualoto et. al., 2014 - Protein & Peptide Letters.

[0020] Figure 9 shows the charge delivery capacity of the compound of the invention. C-ter-Alexa 488 was efficiently absorbed in HUVECs after 4 hours, with 20 µM being the best uptake concentration. The P4-Alexa 488 peptide was barely detected after 4h at 10 µM, while said compound modified with the compound of the invention, that is, P4-Cter-Alexa488 was efficiently absorbed at the concentration of 10 µM already after 1 h. Green fluorescence was monitored by High Content Screening (HCS).

[0021] Figure 10 shows that the compound of the invention, itself or chemically modified (Cter and P4-Cter), is absorbed by HUVECs. Representative images of HCS for uptake of C-ter (4h, 20 µM) and P4 (4h, 10 µM). Green fluorescence was monitored by High Content Screening (HCS). CTRL Alexa 488 is a control of Alexa Fluor 488 dye alone.

[0022] Figure 11 schematically shows the compound of the invention (C-ter) chemically linked to a peptide known as P15 (Seq ID No 5).

[0023] Figure 12 shows that the compound of the invention provides for the uptake by HUVECs of yet another peptide known as P15. A) presents representative images of HCS for incorporation of Control (CTRL) C-ter (4h, 10 µM), P15 (4h, 10 µM) and C-ter-P15 (4h, 10 µM); B) shows a graph with cell quantification: vesicle count (transfluor, 9 sites/well) of test compounds vs. control.

[0024] Figure 13 shows a schematic of the Amblyomin-X sequence. The upper panel shows the entire sequence of Amblyomin-X and delimitation of the Kunitz domain and carboxy-terminal end. In the lower panel, it shows the specification of each sequence derived from the carboxy-terminal end for synthetic peptide synthesis with or without labeling.

[0025] Figure 14 shows the standardization of the concentration of labeled synthetic peptides in SK-Mel-28 cells during 6 h of treatment by HCS. SK-Mel-28 cells were incubated with the different labeled synthetic peptides for 6 h and evaluated by HCS. The peptides F1CFITC and F2C-FITC had the highest fluorescence intensity at concentrations of 10 μM. The difference between the F3C-FITC peptide that did not fluoresce even at the highest concentration. In the lower panel, images of the negative (unmarked) and positive (full C-ter) controls. Nuclei were stained in blue by DAPI (500 nM).

[0026] Figure 15 shows quantification of SK-Mel-28 cells incubated with F1C-FITC and F2C-FITC for 1h, 2h, 4h and 6h and evaluated by HCS. The quantification of the relative fluorescence resulting from the internalization of the F1C-FITC and F2C-FITC peptides in even shorter times at a concentration of 5 μM was performed, evidencing greater ease of internalization of the F2CFITC peptide. This result is supported by the similar number of cells between treatments.

Detailed Description of the Invention and some embodiments

[0027] The present invention provides a compound for targeting molecular entities to the intracellular domain of neoplastic/cancerous cells. The compound of the invention is peptide and has at least 80% identity to the polypeptide of SEQ ID No.1, SEQ ID No.6 or SEQ ID No.7. Embodiments of the compound of the invention include: a peptide of SEQ ID No. 1, SEQ ID No.6 or SEQ ID No.7; a fragment thereof; and/or modified forms thereof, including modified cyclic, amide, alkyl-, alkoxy, halogeno, hydroxy- or PEG, as well as modified with other functional groups or amino acids/peptides, including unnatural amino acids such as D-forms of amino acids, salts of any of the above entities; and/or combinations thereof.

[0028] In one embodiment the compound of the invention is peptide and has at least 90% identity to the polypeptide of SEQ ID No.1, SEQ ID No.6 or SEQ ID No.7.

[0029] In one embodiment the compound of the invention is peptide and has at least 95% identity to the polypeptide of SEQ ID No.1, SEQ ID No.6 or SEQ ID No.7.

[0030] The compound of the invention is synthetic. Said compound is useful for the preparation of a product of pharmaceutical interest selected from diagnostics, prognostics and/or therapeutic use in vertebrates, preferably a mammal.

[0031] The invention also provides the use of said compound to modify other active pharmaceutical ingredients such that said active ingredients are selectively targeted to neoplastic/cancerous cells. The invention also provides a pharmaceutical composition comprising said compound and a method for diagnosing, prognosticating and/or treating cancer or other altered cellular conditions.

[0032] In one embodiment, the compound of the invention is used to modify small molecules, so that the resulting complex is enabled with the ability to penetrate tumor cells. In another embodiment, the compound of the invention is used to modify other peptide molecules so that the resulting complex is capable of penetrating tumor cells. Detailed examples embodied in the present invention include the modification of two different chemical entities as shown in Table 1 below.

[0033] Both chemical entities shown in the table above have little or no cellular penetrating ability, but when bound to the compound of the invention have cellular penetrating ability. Furthermore, said chemical entities, when linked to the compound of the invention, also have the ability to selectively penetrate tumor cells.

[0034] For purposes of the present invention the following definitions are used:

[0035] Product of pharmaceutical interest

[0036] In the context of the present patent application, "compound of pharmaceutical interest" should be understood as any molecular entity that comprises the compound described as an inventive concept common to the present patent application, also including molecular entities obtained by chemical modification/derivatization of the same, with the inclusion of other functional groups, linear or branched side chains, alteration of hydrophilicity or hydrophobicity, among others, provided that they comprise SEQ ID No. 1 as a nucleus or a fragment thereof as defined above, with the exception of natural entities and already known.

[0037] Pharmaceutical composition

[0038] In the context of the present patent application, "pharmaceutical composition" should be understood as any composition that contains an active principle, with prophylactic, palliative and/or curative purposes, acting in order to maintain and/or restore the homeostasis, and can be administered orally, topically, parenterally, enterally and/or intrathecally.

[0039] Pharmaceutically acceptable formulation

[0040] In the context of the present patent application, "pharmaceutically acceptable formulation" is to be understood as a formulation containing pharmaceutically acceptable excipients and carriers well known to those skilled in the art, such as the development of convenient doses and treatments for use in particular compositions that may be described in a number of treatment regimens, including oral, parenteral, intravenous, intranasal, intravitreal and intramuscular, intracerebral, intracerebroventricular and intraocular and their administration and/or formulation.

[0041] Modified peptide

[0042] In the context of the present patent application, "modified peptide" should be understood as an unnatural peptide, artificially modified or obtained by synthesis, including halides, cyclized, amidated, alkylated, alkoxylated, hydroxylated, PEGylated, others functional groups on any amino acid, or its salt forms, as well as with an amino acid or peptide, including unnatural ones such as the d-amino acid forms. The peptide compound can be pegylated using techniques known to those skilled in the art, such as, for example, pegylation with reagents containing the succinimidyl group, which react preferentially with primary amines present in the N-terminal region of the peptide. The peptide compound of the invention can be alkylated to any amino acid using techniques known to those skilled in the art, including, for example, the Mitsunobu reaction described in the article by Reichwein & Liskamp (Reichwein JF & Liskamp RMJ. Site-specific N-alkylation of peptides on the solid phase. Tetrahedron Letters, Volume 39, Issue 10, 5 March 1998, Pages 1243-1246). Said article describes the introduction of any alkyl group into a specific amide function of a peptide. The peptide compound of the invention may be alkoxylated, substituted with halogens, hydroxy or other functional groups at any amino acid using techniques known to those skilled in the art, including, for example, those described in the book/publication Special Periodic Reports, Amino Acids, Peptides and Proteins : Volume 42, Royal Society of Chemistry, 2013. The peptide compound of the invention can be modified with other molecular species useful in diagnostic and/or therapeutic applications, such as Biotin, using techniques known to those skilled in the art.

[0043] Cyclic or circular peptide

[0044] In the context of the present patent application, "cyclic, cyclized or circular peptide" is to be understood as a peptide that has had a covalent bond between the two ends of a linear peptide molecule by any method known in the art, particularly by enzyme activity. The cyclic peptide can be used as a replacement for the linear peptide due to the fact that it is more difficult to degrade, since its ends or attack zones by hydrolyzing enzymes are not as exposed as in a linear peptide.

[0045] In the context of the present patent application, the term "small molecule" is to be understood to mean conventional non-peptide/biological pharmaceutical entities, both for diagnostic use and for therapeutic use.

Example 1 - Process to obtain the compound of the invention

[0046] In this embodiment, the compound of the invention is the polypeptide of SEQ ID No.1 (C-ter) and was prepared by chemical synthesis by CEA. The compound was solubilized in H2O.

Example 2 - The use of the compound of the invention to deliver cargo to different cell types

[0047] Cell lines and culture conditions

[0048] Human melanomas (SK-MEL-28), pancreatic adenocarcinomas (MIA-PaCa-2) and murine renal adenocarcinoma cells (RENCA) were obtained and cultured according to the instructions of the American Type Culture Collection (ATCC, Manassas, GO). Human dermal fibroblast cells (HDFa) were obtained from Invitrogen. Endothelial cells were obtained from human umbilical veins (HUVECs), as previously described11. The study received prior approval from the Research Ethics Committee and the free and informed consent form was obtained from female donors. The HUVECs were seeded in T75 flasks previously covered with 2% gelatin and cultured in RPMI medium, supplemented with 10% fetal bovine serum, Lglutamine (2 mM), streptomycin sulfate (100 mg/ml), penicillin (100 U/ml). ml) sodium pyruvate (100 mM), 2-mercaptoethanol (10 mM), ECGF (10 mg/ml) and heparin (45 µg/ml), pH 7.4. In all experiments, HUVECs were used in the second or third pass. All cell lines were routinely cultured in a humidified 5% CO2 incubator at 37°C.

Cell Viability Test

[0049] Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, after 48 hours of treatment with indicated concentrations of each compound, 20 µl of 5 mg/ml MTT was added to the cells and the plates were incubated for 3 h at 37°C. Subsequently, the medium was discarded, the dark blue formazan crystalline product was dissolved in 100 mL of dimethyl sulfoxide (DMSO), and the absorbance was measured in a Spectra MAX 190 microplate reader (Molecular Devices, Sunnyvale, USA) at 540 nm.

Cell uptake studies

[0050] The compound of the invention (C-ter) was chemically bonded with the amine reactive dye (tetrafluorophenyl ester of alexa fluor 488 tetrafluorophenyl acid (TFP)) using the Alexa Fluor® 488 Microscale Protein Labeling Kit (Molecular Probes) , following the manufacturer's instructions. Other entities (Amblyomin-X and its fragment called Kunitz domain) were also modified as described above, for comparative analysis.

[0051] To investigate the role of PS in C-ter uptake, cells were pretreated with Annexin V (5 μg/mL) or Na3VO4 (25 μM) and then incubated with fluorochrome-conjugated molecules for 4 hours .

[0052] The dye-conjugated protein was named 488-Amblyomin-X. Briefly, cells (10 4 cells/well) were seeded in 96-well plates. Once stabilized, the culture medium was replaced and the cells were incubated with 488-Amblyomin-X (20 µM) at 37 °C for 2h, 4h and 24h. Cells were then incubated with Alexa Fluor 555 transferrin (100 µg/mL), Alexa Fluor 555 cholera toxin B subunit (50 µg/mL) or Alexa Fluor 555 dextran (1 mg/mL) in binding medium. 37°C for 20 min. The nuclei were stained with Hoechst 332. Then, the green and red fluorescences were monitored by a Molecular Devices ImageXpress Micro Confocal plate scanning microscope (Molecular Devices, Sunnyvale, CA) or by the LSM 510 Meta confocal microscope (Zeiss, Jena, Germany) followed by evaluation of the overlap between 488-Amblyomin-X markers and endocytosis using MetaXpress software (Molecular Devices, Sunnyvale, CA).

Microinjection Assay

[0053] SK-MEL-28 cells were plated on cellocate glass coverslips for 24 h prior to injection. The microinjection solution was prepared with: 20 µM Amblyomin-X-488 or 10 µM Kunitz-647. Staurosporine (10 µM) was added directly to the medium for 6 hours and used as a control for PI staining. Control cells were maintained in culture medium. The solution was injected into the cytoplasm of the cells (compensation pressure: 30 hPa; injection pressure: 100 hPa; injection time: 0.3 s). Immediately after injection, cells were replaced with fresh medium in the 37°C incubator. After 4 h, cells were stained with PI (1μg/mL - contain 100 ng/mL RNAse A) for 15 min at room temperature. Then, the cells were washed twice with 1X PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. Finally, the cells were washed with 1X PBS and a drop of Vectashield® antifade mounting solution (Vecta Labs) was applied to the slide with the coverslip containing the cells facing down and then sealed. The analysis was performed using a Zeiss 510 LSMS confocal microscope (Zeiss, Germany).

Intracellular trafficking by indirect immunofluorescence.

[0054] Tumor cells were grown in 96-well plates (104 cells/well) and treated with 488-Amblyomin-X (20 µM) to indicate timings. Cells were washed twice with PHEM-glycine buffer (2 mM HEPES, 10 mM EGTA, 2 mM MgCl2, 60 mM Pipes, 100 mM Glycine, pH 6.9) and fixed with 4% paraformaldehyde for 3 h at room temperature. . The washing step was repeated and then the cells were incubated with a cell permeabilization solution (0.1% Tween in PHEM) for 5 minutes at room temperature. Samples were washed and incubated with 1% BSA blocking solution for 30 minutes at room temperature. Next, the primary antibody was incubated overnight at 4°C: (i) mouse anti-human caveolin-1 1:100 (Santa Cruz Biotechnology, Inc., USA); (ii) 1:50 rabbit anti-human LAMP-2 (Abcam). A washing procedure was performed and the samples were incubated with Alexa Fluor® 555 anti-rabbit goat (InvitrogenTM Life Technologies Inc., USA) and anti-rabbit Alexa Fluor® 647 (InvitrogenTM Life Technologies Inc., USA) secondary antibodies. ), both at 1:200 dilution for 1 h at room temperature in the dark. Core stained with Hoechst 332. Next, green and red fluorescences were monitored by a Molecular Devices ImageXpress Micro Confocal plate scanning microscope (Molecular Devices, Sunnyvale, CA) followed by evaluation of overlap between 488-Amblyomin-X markers and endocytosis. using MetaXpress software (Molecular Devices, Sunnyvale, CA) or vesicle interactions by Imaris software.

Membrane detection containing OS

[0055] To measure endogenous PS exposure on the outer leaflet of the membrane, cultured cells were harvested, washed with phosphate-buffered saline (PBS) and resuspended in 1X Annexin binding buffer (Invitrogen™, Life Technologies Inc. ) containing 2.5 µl of FITCAnnexin V, followed by incubation for 15 min in the dark at room temperature. Analysis was performed by FACS-Calibur or Aria flow cytometer (Becton Dickinson, St. Louis, MO). Data were evaluated using FlowJo software (Tree Star, Inc, Ashland, OR).

[0056] In a second approach, similar to that described by Riedl et. al., 2011, cells (1 × 105 ) were seeded in 35 mm dishes. Then the medium was removed and the same annexin binding protocol was applied as mentioned above. Then, unfixed cells were washed twice in appropriate culture medium and immediately observed under a confocal microscope LSM 510 Meta (Zeiss, Jena, Germany).

Circular Dichroism (CD)

[0057] The CD spectra were recorded on a JASCO J-810 spectropolarimeter equipped with a thermoelectric sample temperature controller (Peltiersystem).

[0058] C-ter domain samples (20 μM) were diluted in phosphate buffered saline (PBS), pH 7.4, or water using a total volume of 550 μL. Milli-Q water and protein-free buffer were used to calibrate the instrument. Scans were collected from 190 to 260 nm at 21 °C using a light-length quartz cell (Helma) of 1.0 mm (200 μL). The data were corrected and adjusted to the input buffer and the mean residual ellipticity of the molars was calculated based on the molecular mass of each compound. The secondary structure estimation was performed using the CDNN CD13 spectrum deconvolution software and the DicroProt14 program.

Statistical analysis

[0059] Comparisons were performed using Two-way ANOVA analysis followed by Tukey's Post Hoc test or tTest, employing GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA). Criteria for statistical significance were established as * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001.

Cargo delivery capability of the compound of the invention

[0060] Endothelial cell culture was obtained from umbilical cords by digesting umbilical cord veins with collagenase as described previously (19) with some modifications (20). Endothelial cells between the first and third passage were used for all experiments. Cells were seeded in 96-well culture plates (Advance, Greiner), special for immunofluorescence, at a density of 8x103 cells/well pre-coated with 2% (w/v) gelatin in EGM®-2 BulletKit® medium for HUVECs (Lonza, CC-3162) allowed to seed at 37°C in a humidified atmosphere of 5% CO 2 . Treated cells were incubated in RPMI medium containing 1% fetal bovine serum (FBS) and Alexa-Fluor 488-labeled peptides (C-ter/Seq ID No 1, P4/Seq ID No 2, or P4-Cter/Seq ID No. No 3) at different concentrations and times. After treatment, cells were fixed with 4% paraformaldehyde and analyzed in ImageXpress Micro Confocal High-Content Imaging (HCS, Molecular Devices) in Widefield mode and the number of vesicles containing the peptides in cells was quantified using MetaXpress High-Content Image Acquisition and Analysis Software (Molecular Devices).

Example 3 - Use of the compound of the invention in a pharmaceutical product Effect of the compound of the invention (Cter) on tumor and non-tumor cells

[0061] The effect of the compound of the invention alone was investigated on the viability of human tumor cells, including melanoma (SK-MEL-28) and pancreatic adenocarcinoma (Mia-PaCa-2) cells using MTT assays. After 48 hours, no change was observed. In contrast, AmblyominX and positive controls decreased the cell viability of both tumor cells (Figure 1A)

[0062] In addition, preliminary results were obtained with murine renal adenocarcinoma cells (RENCA), human dermal fibroblasts (HDFa) and human umbilical vein endothelial cells (HUVEC). Except for decreasing the viability of RENCA by 1 μM, again no change was observed (Figure 2).

Only the C-ter domain is taken up by tumor and non-tumor cells

[0063] The Cter domain was complexed with the cell impermeable Alexa Fluor 488. After 4 hours, the Cter domain (20) was observed to be taken up by SK-MEL-28 (tumor cells). However, Kunitz (20 µM for 4h) was not detected inside the cells (Figure 3A), suggesting that the C-ter is responsible for the uptake of Amblyomin-X. Furthermore, after microinjection of Kunitz or complete Amblyomin-X (both 20 µM), propidium iodide (PI) staining was detected in SK-MEL-28 (Figure 3B), suggesting a decrease in cell viability. Microinjection cells were not used as a control. This result indicates that the Kunitz domain may be responsible for cytotoxicity and its internalization is crucial for this effect.

[0064] In addition, we quantified the uptake of C-ter and Amblyomin-X and observed that the accumulation of C-ter within cells is faster than Amblyomin-X (Table 2), and few cells were positive for nuclear translocation (Figure 3C).

[0065] Similar results were obtained with HUVEC and HDFa in preliminary experiments (Figure 4), that is, C-ter uptake by these cells. We investigated whether higher concentrations of Kunitz (100 µM) would affect uptake studies and, after 4 hours of treatment, vesicles ten times smaller than Amblyomin-X were detected (Table 3).

C-ter incorporation by lipid rafts and caveosomes and lysosomes as intracellular fate

[0066] We investigated the endosomal pathway used by C-ter by colocalization assay using AlexaFluor 555-labeled dextran, cholera toxin B subunit (CTxB) or transferrin. As seen in Figure 5, overlapping points were found between C-ter and all markers, but the highest were with CTxB, specifically at the shortest treatment time, suggesting that C-ter uptake occurs by caveolin-mediated endocytosis. . Thus, co-localization with cav-1 (Figure 6) occurred mainly after 4 hours of treatment. Furthermore, C-ter was found in colocalization with late endosomal markers, LysoTracker and LAMP-2, especially in MIA PaCa-2.

[0067] The compound of the invention retains its ability to penetrate tumor cells even after pretreatment with Na3VO4 or pretreatment with Annexin V (a PS ligand).

[0068] Tumor cells have more exposure of anionic phospholipids (phosphatidylserine - PS) on their cell surfaces. As can be seen in Figure 6, tumor cell lines (SK-MEL-28) are more positive for Annexin-5 labeling, suggesting more PS exposure compared to non-tumorigenic cells (HDFa and HUVEC), regardless of activation of apoptosis. Furthermore, when human melanoma cells were pretreated with Na3VO4 (sodium orthovanadate - an inhibitor of PS synthesis), or, in other words, when PS exposure was decreased, C-ter uptake was not affected. by pretreatment with Na3VO4. Annexin V (a PS ligand). Amblyomin-X, in contrast, was only slightly detected within these cells.

Example 4 - Structural analysis of the compound of the invention C-ter is a compound of random structure

[0069] The analysis of circular dichroism shows that the compound of the invention is mostly of random structure (Figure 8).

Cargo delivery capability by conjugation with a cell membrane permeable molecule

[0070] To further demonstrate the ability of C-ter to be used as a chemical cargo delivery entity, the compound of the invention (Seq ID No 1, 50 amino acids) was chemically linked to a peptide having 10 amino acids belonging to the cytoprotective peptide (SEQ ID No. 2, sequence YAIGYSSKDYK-OH, called peptide P4, References 15-18), thus forming a hybrid peptide. P4 is known for its low penetration ability. The hybrid peptide in this embodiment of the invention thus comprises 60 amino acids with the following sequence (SEQ ID No. 3):

[0071] YAIGYSSKDYKGGGGEEQTHFHFESPKLISFKVQDYWILNDIMKKNLT GISLKSEEEDADSGEID

[0072] Cter, P4 and P4-C-ter peptides were labeled with cell impermeable Alexa Fluor 488. The ability of P4 and P4-Cter peptides bound to Alexa 488 was analyzed by HUVECs at 1 h, 2 h and 4 h using different concentrations: 0.5 µM, 1 µM, 10 µM and 20 µM (Figure 9). This was compared to the uptake of Alexa 488 bound C-ter peptide (0.5 µM, 1 µM, 10 µM and 20 µM concentration) after 4 hours, as shown in previous experiments for SK-MEL-28 (Figure 3). The results showed that P4-Alexa 488 was not detected inside the cells after 4h (Figure 9A and Figure 10), in contrast to P4-C-ter-Alexa 488, which was captured and quantified (Figure 9B and Figure 10) inside of the cells. This result shows that C-ter provided the internalization of the P4 peptide into the cells.

[0073] In view of the results presented above, it is possible to conclude that the compound of the invention has the ability to deliver cargo within tumor cells, this cargo being a peptide or a small molecular entity. Furthermore, when comparing functions, the C-terminal domain is responsible for the uptake of Amblyomin-X by tumor cells, while the Kunitz-like domain of Amblyomin-X is associated with its cytotoxicity to tumor cells. Also, Cterminal domain internalization and intracellular traffic are similar to Amblyomin-X

[0074] First, the C-ter charge delivery capability indicates that this molecule is similar in function to a cell-penetrating peptide (CPP). However, C-ter does not have conventional characteristics of CPPs, since its origin, sequence, chemical charge, bond type and general physicochemical properties are very different. In general, CPPs typically do not exceed 30 residues in length and generally carry a positive charge, which facilitates electrostatic interactions with the negatively charged cell surface. In contrast, the C-ter has 50 residues and a negative formal charge8, 9. Furthermore, if an endocytic mechanism is uplifted by a CPP for intracellular delivery of its charge, a successful outcome relates to the endosomal escape potential. before being directed to lysosomes for degradation or recycling of endosomes for transport back to the plasma membrane and subsequent extracellular release9. In this regard, the compound of the invention showed promising results. Even with co-localization in late endosome markers, it was found in the other subcellular structure (caveosomes).

Example 5 - The compound of the invention also provides for the internalization of the P15 peptide

[0075] In this embodiment, yet another hybrid peptide was synthesized in order to validate the ability of the compound of the invention (Cter) as a cargo delivery peptide by conjugating the Cter sequence (50 amino acids) to a molecule poorly permeable to the cell membrane. called P15 (Seq ID No. 4, 7128.99 Da, Perea et al., 2004). Figure 11 schematically shows the compound of the invention chemically linked to a peptide known as P15, forming the complex of Seq ID No 5.

[0076] The compound of the invention (Cter, 5856 Da), the P15 peptide (1290.54 Da) and Cter-P15 (7128.99 Da) were labeled with the Alexa Fluor 488 TFP dye (Life Technologies # A30006) according to the instructions of the manufacturer.

[0077] To show the ability to internalize the compound of the invention, SK-MEL-28 cells were seeded in a 96-well plate (Advanced, Greiner # 655-986) at the density of 7x103 cells/well for 24h in DMEM High Glucose , medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Sigma). The compounds Cter-Alexa 488, P15-Alexa 488 and Cter-P15-Alexa 488 at a concentration of 10 µM were added to the cells and incubated for 4 h. Cells were submitted to fixation steps for further analysis by High Content Screening (HCS). For this, the cell monolayers were washed three times with PHEM buffer (2 mM HEPES, 10 mM EGTA, 2 mM MgCl2, 60 mM PIPES - pH 6.9) and fixed for 1 h with cold 4% paraformaldehyde. Nuclei were stained with Hoechst (5 µM) for 5 min. The samples were subjected to high-content image analysis on ImageCpress Micro Confocal High-Content Imaging (HCS, Molecular Devices) in confocal mode and the number of vesicles containing the peptides in the cells was quantified using the MetaXpress HighContent Image Acquisition & Analysis software. . (Molecular Devices).

[0078] Figure 12 shows the charge delivery capacity of the compound of the invention. A) Representative images of HCS for uptake of Cter-Alexa 488, P15-Alexa 488 and Cter-P15-Alexa 488. B) Quantification of the number of vesicles by green fluorescence by High Content Screening (HCS). The results show that Cter-Alexa 488 (10 µM) was efficiently absorbed into SK-MEL-28 after 4 h while the P15-Alexa 488 peptide was poorly detected. Compound P-15 modified with the compound of the invention, i.e. Cter-P15-Alexa 488 was efficiently absorbed at the concentration of 10 µM after 4h.

[0079] Example 6 - Additional fragments of the CTer fraction of Amblyomin-X and their internalization

[0080] Three synthetic peptides derived from the carboxy-terminal end of Amblyomin-X were synthesized. These peptides were named: F1C (AA1 – AA20) SEQ ID No.6, F2C (AA16 – AA35) SEQ ID No.7 and F3C (AA35 – AA50) SEQ ID No.8, as well as the respective isothiocyanate-tagged version of fluorescein-FITC (F1C-FITC, F2C-FITC, F3C-FITC). After the concentration standardization and quantification of the synthetic peptides labeled with the fluorophore, the internalization assays were started in SK-Mel-28 cells in short incubation times using HCS. For this, initially two concentrations of each probe (1μM and 10μM) were used to perform incubations of 1h and 6h. Internalization of F1C-FITC and F2C-FITC was observed at both concentrations, in a dose-dependent manner. F3C-FITC did not show internalization even at higher concentrations used. The images were analyzed by confocal microscopy and the internalization of F1C-FITC and F2C-FITC peptides was observed, showing a similar pattern of subcellular localization. The amount of F2C-FITC within SK-Mel-28 cells was higher compared to the amount of F1C-FITC, suggesting that F2C-FITC is more easily taken up by cells. This difference in fluorescence emission can be guaranteed by the close number of cells between treatments, which supports the better result of F2CFITC. With this set of results, it can be suggested that the F2C fragment derived from the carboxy-terminal portion of Amblyomin-X may be involved in the molecular internalization and could be considered a CPP.

[0081] Methodology. Approximately 1x10 3 SKMel-28 cells were seeded per well of 96-well plates (Greiner bio-one, Kremsmunster, Austria), which were maintained under the same conditions described by Pacheco et al (2014). For treatment with different synthetic peptides labeled with fluorescein isothiocyanate (FITC) concentrations of 1, 5 and 10 μM were used. As a control, cells were treated with the FITC-labeled carboxy-terminal end (C-Ter-FITC). At 1, 2, 4 and 6 h after treatment, cells were washed three times with warm 1X PBS and then fixed in 4% paraformaldehyde for 30 min. After the fixation time, cells were incubated with a solution of DAPI (4',6-diamidino-2-phenylindole) in PHEM-Triton X-100 buffer for 15 min for nucleation labeling. After staining the nucleus, the solution was removed and the cells washed three times with 1x warm PBS for fluorescence analysis.

[0082] Results. Three synthetic peptides derived from the carboxy-terminal end of Amblyomin-X were synthesized. These peptides were named: F1C (AA1 – AA20), F2C (AA16 – AA35) and F3C (AA35 – AA50), as well as the respective fluorescein isothiocyanate-FITC labeled version (F1C-FITC, F2C-FITC and F3C-FITC). ) (Figure 13).

[0083] After the standardization of concentration and quantification of the synthetic peptides labeled with fluorophore, the internalization assays were started in SK-Mel-28 cells in short incubation times using HCS. For this, initially two concentrations of each probe (1 and 10 μM) were used to perform incubations of 1 and 6 h (Figure 14). Higher fluorescence intensity of F1C-FITC and F2C-FITC was observed at both concentrations and as expected, in a dependent manner. F3C-FITC did not show fluorescence even at the highest concentrations used (Figure 14). As a positive control, the entire end of the carboxy-terminal labeled with FITC (C-Term-FITC) was used, already well known for its internalization capacity, but the probe aliquot used did not work as expected (Figure 14).

[0084] Given the fluorescence emission of each labeled peptide, it was decided to work with an intermediate concentration (5 μM) for further internalization assays. In this way, quantitative analysis of the fluorescence of F1C-FITC and F2C-FITC peptides in SK-Mel-28 cells was performed, demonstrating greater ease of internalization of F2C-FITC than F1CFITC during 1, 2, 4 and 6 h of incubation. (Figure 15). This difference in fluorescence emission can be guaranteed by the close number of cells between the different treatments (Figure 15), which supports the better result resulting from the internalization of the F2C-FITC peptide (Figure 14).

[0085] Those skilled in the art will value the knowledge presented herein and may reproduce the invention in the modalities presented and in other variants and alternatives, covered by the scope of the claims below.

Claims (9)

A compound characterized in that it comprises at least 80% identity with the compound defined in SEQ ID NO: 1, SEQ ID NO: 6 or SEQ ID NO: 7. Compound according to claim 1, characterized in that it is in cyclic, amidated, alkylated, alkoxylated, halogenated, hydroxylated or PEGylated form, or modified with other functional groups and/or with unnatural amino acids, such as d-forms of amino acids; their salts; or combinations thereof. Compound according to claim 1 or 2, characterized in that it is chemically bonded or fused to an active pharmaceutical ingredient. Compound according to any one of the preceding claims, characterized in that the pharmaceutically active ingredient is selected from: a compound for diagnostic use; a fluorophore; a small molecule; a polypeptide; an antineoplastic agent; or combinations thereof. A compound according to any one of the preceding claims, characterized in that it is as defined in SEQ ID NO: 3 or in SEQ ID NO: 5. Use of a compound as defined in any one of the preceding claims for the preparation of a medicament to treat cancer or related diseases. Pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound as defined in any one of claims 1 to 5. Method of detection of cellular and/or biochemical alterations characterized by comprising the steps of:

• (i) selecting one or more eukaryotic cells in vitro;
• (ii) contacting said cells with a compound as defined in any one of claims 1 to 5, or a molecular entity comprising said compound, such that said compound penetrates said cell; and
• (iii) detect/assess the status of the cellular/biochemical change.

Method of carrying and/or internalizing a compound into eukaryotic cells characterized in that it comprises:

• - chemically linking or fusing one or more compounds of carrier or internalization interest with a compound as defined in claim 1 or 2 generating a chemically linked or fused compound;
• - producing a pharmaceutical composition comprising said chemically bound or fused compound;
• - administering said pharmaceutical composition to a eukaryotic cell.

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