Pharmaceutical compositions for the treatment of solid tumors and uses.

Description

1 / 26 “PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF SOLID TUMORS AND USES”

[01] This technology is an addendum certificate to application BR102015031860-0, filed on 12 / 18 / 2015, entitled “Synthetic peptides, pharmaceutical compositions and uses”. This certificate describes pharmaceutical compositions comprising the synthetic peptides LyeTxImnK-Ac and LyeTxImnK (SEQ ID No. 2, acetylated or not, respectively), derived from the peptide LyeTxI-b (SEQ ID No. 1, of BR 102015031860-0), with the sequence minimized to 16 amino acids, containing an addition of a lysine at position 5, and their use for the production of drugs for the treatment of metastatic breast cancer.The use of pharmaceutical compositions containing the peptides resulted in a reduction in the metastatic activity of triple-negative breast cancer cells and, compared to the LyeTxI-b peptide, showed greater activity in increasing cell adhesion to extracellular matrix components, increasing focal adhesion, greater inhibition of MMP-2 secretion, and greater inhibition of cell invasion at non-cytotoxic concentrations.

[02] Cancer has been a leading cause of death in both developed and developing countries, decreasing life expectancy worldwide in the 21st century. In 2020, the number of new cancer cases was estimated at 19.3 million, and nearly 10 million deaths were caused by cancer worldwide, with female breast cancer being the most commonly diagnosed (11.7%), accounting for 2.3 million new cases. The triple-negative (TN) breast cancer subtype is the most aggressive and has been associated with a poor prognosis and resistance to existing treatments, with a lack of molecular targets for therapy. TN breast cancer is molecularly classified by the loss of expression of estrogen / progesterone hormone receptors. Petition 870240105262, dated 10 / 12 / 2024, page 12 / 46 Breast cancer is caused by the 2 / 26 (ER / PR) and human epidermal growth factor receptor 2 (HER2) genes. It accounts for 15-20% of all breast cancers and has the shortest survival rate. It is a highly invasive type of cancer, causing distant metastasis, mainly to the brain and viscera. Treatment strategies include surgery and radiotherapy, performed on the breast, lymph nodes, and adjacent areas. However, removal of the primary tumor in most solid breast cancers does not eliminate metastasis, which is one of the biggest obstacles to achieving a cure.

[03] The tumor microenvironment (TME) contains metastatic and non-metastatic tumor cells, stromal cells, including fibroblasts, immune cells, neuronal cells, and vascular endothelial cells. All these cells are embedded in the extracellular matrix (ECM) and are nourished by blood vessels resulting from angiogenesis. In cancer, fibroblasts frequently induce tumor progression by stimulating the proliferation and invasive phenotype of cancer cells, which increases their metastatic potential.

[04] Metastatic cells can escape the primary tumor, enter vascular systems, invade and colonize secondary organs, spreading throughout the body. In the invasion process, cancer cells move through the ECM, interacting with distinct proteins and supramolecular arrangements, penetrating epithelial and endothelial cell layers and invading various ECM structures.

[05] Cells interact with the ECM through their cell membrane adhesion receptors, such as integrins and transmembrane proteoglycans. Integrins are composed of two subunits, α and β, and the N-terminal domains of both subunits together form the ECM binding site. 18 α and 8 β subunits, with 24 heterodimer combinations, have been described and classified so far. Petition 870240105262, dated 10 / 12 / 2024, page 13 / 46 3 / 26 in 3 distinct groups: laminin-binding integrins (α1β1, α2β1, α3β1, α6β1, α7β1 and α6β4), collagen-binding integrins (α1β1, α2β1, α3β1, α10β1 and α11β1), leukocyte integrins (αLe2, αΜβ2, αΧβ2 and αDe2) and fibronectin-binding integrins (α5β1, ανβ1, ανβ3, ανβ5, ανβ6, αβ8 and αβ3). Integrins linked to the ECM component cluster and recruit adaptor molecules and signal transducer molecules, such as focal adhesion kinase (FAK) and members of the Src family, which serve as intracellular anchoring sites for the cytoskeleton and transmit signals into the cell (Eble JA, Niland S. The extracellular matrix in tumor progression and metastasis. Clin Exp Metastasis. 2019 Jun;36(3):171-198.

[06] Integrins are used as prognostic indicators in breast cancer and their interaction with extracellular matrix proteins triggers integrin clustering and the formation, disassembly, and reorganization of actin filaments, stress fibers, and focal adhesion complexes, regulating cell shape and cellular processes that require cell remodeling, such as cell adhesion, cell migration, and cell division. Inhibition of integrin functionality can alter adhesion properties, affecting cell contact with extracellular matrix components or endothelial cells, disrupting signal transduction cascades that support migration, or both.

[07] Matrix metalloproteinases (MMPs) are a family of calcium-dependent, zinc-containing endopeptidases capable of cleaving ECM components. Some MMPs are secreted and some are membrane-bound. They are primarily regulated by tissue inhibitors of metalloproteinases (TIMPs), which can be secreted or located on the cell surface and are responsible for controlling the degradation of ECM proteins by MMPs. One Petition 870240105262, dated 10 / 12 / 2024, page 14 / 46 4 / 26 Dysregulation of the balance between MMPs and TIMPs leads to cancer progression, among other pathological conditions. There are 23 different types of human MMPs, classified into six different groups: (1) collagenases (MMP-1, MMP-8 and MMP-13); (2) gelatinases (MMP-2 and MMP-9); (3) stromelysins (MMP-3 and MMP-10); (4) matrilysins (MMP-7 and MMP-26); (5) membrane metalloproteinases (MT-MMP): MMP-14, MMP-15, MMP-16, MMP-17, MMP-24 and MMP-25, which can digest a range of ECM proteins, such as gelatin, fibronectin and laminin, and can also activate pro-MMP-2; and the last group (6), with more heterogeneous structure and substrate specificity (MMP-12, MMP-19, MMP-20, MMP-21, MMP23, MMP-27 and MMP-28).

[08] Matrix metalloproteinases (MMPs) can degrade and remodel the ECM and have been associated with breast cancer progression and metastasis. Inhibition of some collagenase- and gelatinase-type MMPs, such as MMP-9 or MMP-14, using inhibitory monoclonal antibodies, has demonstrated promising antitumor effects in preclinical breast cancer models (Kwon MJ. Matrix metalloproteinases as therapeutic targets in breast cancer. Front Oncol. 2023 Jan 19;12:1108695. doi: 10.3389 / fonc.2022.1108695. PMID: 36741729; PMCID: PMC9897057).

[09] MMP-9 has been linked to cancer pathology, mainly by enabling cell invasion, metastasis, and angiogenesis. For this reason, MMP-9 has been evaluated as a biomarker for cancer (Huang H. Matrix Metalloproteinase-9 (MMP-9) as a Cancer Biomarker and MMP-9 Biosensors: Recent Advances. Sensors (Basel). 2018 Sep 27;18(10):3249. doi: 10.3390 / s18103249. PMID: 30262739; PMCID: PMC6211011). MMP-9 is expressed as pro-MMP-9 and is activated by a zymogen activation cascade initiated by MMP-14 in the cell membrane, which activates MMP-2 and MMP-13. Pro-MMP-9 is then Petition 870240105262, dated 10 / 12 / 2024, page 15 / 46 5 / 26 activated by MMP-2 and MMP-13, as well as MMP-3. MMP-3 is activated by plasmin via uPAr and uPA on the cell surface.

[10] Solid tumor therapy should be complemented with drugs that inhibit the ability of cancer cells to invade the ECM and develop secondary tumors, and should also target non-tumor cells in the TME that support cancer cell progression. Remodeling the immunosuppressive microenvironment in tumors is another important strategy to inhibit tumorigenesis and tumor progression. In this context, efforts in the field of anticancer drug discovery involve strategies to inhibit tumor progression and mechanisms related to motility, migration and / or invasion and metastasis.

[11] Among the drugs with antimetastatic activity available in the clinic are those that act on cell adhesion loss, the tumor microenvironment, the invasion cascade, cell migration, or interact with TME. Recently, the use of low-dose metronomic chemotherapy has shown effects in reducing metastasis by interfering with TME. Clinical trials are underway, such as the “Effect of Low Dose Metronomic Chemotherapy in Metastatic Breast Cancer” (clinicalTrials.gov, identifier NCT04350021). Several antimetastatic compounds are being studied in the laboratory, such as ruthenium compounds (NAMI-A). Animal studies with NAMI-A have shown a reduction in metastases at low-toxicity doses.These compounds exhibit antimetastatic activity at non-cytotoxic concentrations, leading to a reduction in migration rates, invasion, secretion of proteolytic enzymes such as MMP-2, in addition to modulating TME, increasing cell adhesion and the number of focal adhesion complexes, contributing to a reduction in metastasis (Gurgul I, Janczy-Cempa E, Mazuryk O, Lekka M, Lomzik M, Suzenet F, Gros PC, Brindell M. Petition 870240105262, dated 10 / 12 / 2024, p. 16 / 46 6 / 26 Inhibition of Metastasis by Polypyridyl Ru(II) Complexes via Modification of Cancer Cell Adhesion - In Vitro Functional and Molecular Studies. J Med Chem. August 11, 2022;65(15):10459-10470. doi: 10.1021 / acs.jmedchem.2c00580. Epub 2022 Jul 27).

[12] In recent decades, the field of anticancer drug discovery has introduced many antitumor agents from different natural sources, such as microorganisms, plants, animals and, especially, animal venoms. Peptide-based cancer treatment has gained much attention due to its advantages, such as affinity for binding to receptors, selectivity and specificity for tumor cells, as well as exhibiting low toxicity in normal tissues.

[13] Antimicrobial peptides (AMPs) have been studied as antitumor agents. The synthetic peptide of 24 amino acid residues LyeTxI-b (MW 2737.3 Da), corresponding to SEQ ID No. 1 of BR 102015031860-0, is an antimicrobial peptide derived from the native toxin LyetxI, from the spider Lycosa erythrognatha, and differs from this toxin by an acetylated N-terminus and a deletion of a His residue at position 16. The secondary structure of LyeTxI-b is a well-defined helical segment, from the second amino acid to the amidated C-terminus, without a clear partition between the hydrophobic and hydrophilic faces (Reis PVM, Boff D, Verly RM, Melo-Braga MN, Cortés ME, Santos DM, Pimenta AMC, Amaral FA, Resende JM, de Lima ME. LyeTxI-b, a Synthetic Peptide Derived From Lycosa erythrognatha Spider Venom, Shows Potent Antibiotic Activity in Vitro and in Vivo. PMC5897548).

[14] AMP LyeTxI-b showed in vivo activity in a septic arthritis model and, in addition to reducing the number of bacterial loads, also reduced immune cell migration and IL-Iβ cytokine levels and Petition 870240105262, dated 10 / 12 / 2024, p. 17 / 46 7 / 26 chemokine CXCL129. LyeTxI-b showed in vitro anticancer activity against solid tumor cells (HCT-116, MDA-MB-231 and MCF-7) and leukemic cells (HL60, THP-1). For the U87-MG glioblastoma cell line, the anticancer potential of LyeTxI-b was associated with a membranolytic mechanism (Abdel-Salam MAL, Carvalho-Tavares J, Gomes KS, Teixeira-Carvalho A, Kitten GT, Nyffeler J, Dias FF, Dos Reis PVM, Pimenta AMC, Leist M, de Lima ME, de Souza-Fagundes EM. The synthetic peptide LyeTxI-b derived from the venom of the spider Lycosa erythrognatha is cytotoxic to U-87 MG glioblastoma cells. 30449002).

[15] For human breast cancer cells (MCF-7, MDA-MB-231) and mouse breast cancer cells (4T1), LyeTxI-b showed IC50 values ​​of 5 to 7 μM and reduced the survival fraction of clonogenic MDAMB-231 cells, which are one of the cells responsible for tumor recurrence. In vivo studies using the 4T1 isogenic murine metastatic breast cancer model demonstrated that subcutaneous administration of the LyeTxIb peptide (5 mg / kg) significantly reduced tumor size, volume, and number of lung metastases compared to the control group. Quantification of cytokines and tumor growth factors by ELISA revealed that LyeTxI-b and carboplatin (5 mg / kg) showed a significant reduction in TGF-β expression in the tumor and spleen, a reduction in TNF-α in the brain and spleen, and suppression of IL-1β in the lung and tumor. Furthermore, LyeTxI-b induced an increase in the anti-inflammatory cytokine IL-10 in the tumor and reduced it in the spleen.Regarding IL-6, the peptide increased its levels in the tumor, spleen, and brain (Abdel-Salam MAL, Pinto B, Cassali G, Bueno L, Pêgas G, Oliveira F, Silva I, Klein A, Souza-Fagundes EM, de Lima ME, Carvalho-Tavares J. LyeTx Ib Peptide Attenuates Tumor Burden and. Petition 870240105262, dated 10 / 12 / 2024, p. 18 / 46 8 / 26 Metastasis in a 4T1 breast cancer model. Antibiotics (Basel). September 21, 2021;10(9):1136. doi: 10.3390 / antibiotics10091136).

[16] It is worth noting that IL-1 β activates focal adhesion kinase (FAK) and Src to induce the production of MMP-9, responsible for the invasion of breast cancer cells, while IL-10 inhibits tumorigenesis via downregulation of VEGF, IL-1 β, TNF-α, IL-6 and MMP-9. TGF-β and TNF-α also stimulate the production of MMP-9. Therefore, the effect of LyeTxI-b on cytokines and tumor growth factors demonstrated in vivo appears to be related to a lower invasive profile of breast cancer cells, and thus to the antimetastatic effect observed in vivo for this peptide.

[17] The state of the art presents the potential effect of the LyeTxI-b peptide for the treatment of metastatic breast cancer, as described below.

[18] The article by Avelar Júnior et al., entitled “LyeTxI-b, a Synthetic Peptide Derived From a Spider Venom, Is Highly Active in Triple-Negative Breast Cancer Cells and Acts Synergistically With Cisplatin”, published on April 3, 2022, in the journal Frontiers in Molecular Biosciences, demonstrated the cytotoxic effect of the LyeTx Ib peptide, alone and combined with cisplatin, against triple-negative cell lines (MDA-MD-231), with the peptide showing greater selectivity when administered alone and, in conjunction with the drug, showing a synergistic effect on cell death.

[19] The article by Abdel-Salam, et al., entitled “LyeTx Ib Peptide Attenuates Tumor Burden and Metastasis in a Mouse 4T1 Breast Cancer Model”, published on September 21, 2021 in the journal Antibiotics, indicates that the same synthetic peptide LyeTx Ib reduced tumor volume and metastatic nodules when administered subcutaneously or intratumorally in a murine model. Petition 870240105262, dated 10 / 12 / 2024, p. 19 / 46 9 / 26

[20] The present technology is a certificate of addition to application BR102015031860-0, filed on 12 / 18 / 2015, entitled “Synthetic peptides, pharmaceutical compositions and uses”. This certificate describes pharmaceutical compositions comprising the synthetic peptides LyeTxImnK-Ac and LyeTxImnK (SEQ ID No. 2, acetylated or not, respectively), derived from the peptide LyeTxI-b (SEQ ID No. 1 of BR 102015031860-0), with the sequence minimized to 16 amino acids, containing an addition of a lysine at position 5, and their use for the treatment of metastatic breast cancer. The use of pharmaceutical compositions containing the peptides resulted in reduced metastatic activity and, compared to application BR102015031860-0, resulted in greater activity in increasing cell adhesion to extracellular matrix components, increased focal adhesion, greater inhibition of MMP-2 secretion, and greater inhibition of cell invasion at non-cytotoxic concentrations.

[21] The 16 amino acid peptides were tested for their anticancer activity and showed an in vitro effect in increasing adhesion, decreasing migration and invasion, both for triple-negative breast cancer cells (MDA-MB-231) and for MCF-7 breast cancer cells, which demonstrates their potential as an antimetastatic agent for the treatment of breast cancer. The antimetastatic activity of the minimized peptides was verified at non-cytotoxic concentrations. The antimetastatic activity of the minimized peptides was evidenced by the reduction in MDAMB-231 cell invasion in type I collagen matrix: while the LyeTxI-b peptide (SEQ ID No. 1 of BR 102015031860-0) reduced cell invasion by 25%, the minimized peptides, here named LyeTxImnK-Ac (SEQ ID No. 2, acetylated) and LyeTxImnK (SEQ ID No. 2, non-acetylated), demonstrated a reduction of 40% and 30%, respectively. For the Petition 870240105262, dated 10 / 12 / 2024, page 20 / 46 10 / 26 MCF-7 cells, while LyeTxI-b showed a 15% reduction in invasion into type 1 collagen matrix, LyeTxImnK-Ac and LyeTxImnK reduced invasion by 40% and 35%, respectively. Furthermore, cells treated with the minimized peptides led to a reduction in the secretion of matrix metalloproteolytic enzymes (MMPs) of types 2 (MMP-2) and 9 (MMP-9), which are responsible for extracellular matrix degradation and, consequently, migration and invasion. This result reinforces the antimetastatic effect of the minimized peptides of the present invention. In MDA-MB-231 cells, LyeTxImnK (SEQ ID No2, non-acetylated) led to a greater than 70% reduction in MMP-2 secretion, which in turn led to a reduction of approximately 50% in MMP-9 activation (MMP2 is one of the enzymes that activates the proenzyme MMP-9).The LyeTxI-b peptide (SEQ ID No. 1 of BR 102015031860-0) showed a significant effect in reducing MMP-9 secretion, but did not alter MMP-2 secretion in MDA-MB-231 cells, which may be related to its more pronounced effect on inhibiting cell migration compared to the minimized peptides. For MCF-7, LyeTxImnK (SEQ ID No. 2, non-acetylated) reduced MMP-2 secretion by more than 90% and MMP-9 activation by more than 80%, compared to untreated cells. This effect on MCF-7 was also observed for LyeTxI-b (SEQ ID No. 1 of BR 102015031860-0) and for LyeTxImnK-Ac (SEQ ID No. 2, acetylated), but less pronounced (75% reduction for MMP-2 and 50-60% for active MMP-9). The minimized peptides increased breast cancer cell adhesion to type I collagen by 15 times and increased fibronectin adhesion by 2 times compared to untreated cells. The effect of LyeTxI-b (SEQ ID No. 1 of BR 102015031860-0) was less pronounced.Immunofluorescence analyses by confocal microscopy demonstrated that LyeTxImnK (SEQ ID No. 2, non-acetylated) increases the formation of... Petition 870240105262, dated 10 / 12 / 2024, page 21 / 46 11 / 26 focal adhesion complexes (FA). The effects demonstrated here in vitro for the minimized peptides demonstrate their antimetastatic properties, which can be exploited for drug development for the treatment of breast cancer, including triple-negative breast cancer.

[22] The synthetic peptide represented by SEQ ID No. 2 of the present technology was described in the article by Fuscaldi et al., entitled “Shortened derivatives from native antimicrobial peptide LyeTx I: In vitro and in vivo biological activity assessment” and published in the journal Experimental Biology and Medicine on November 11, 2020, but as a potential antimicrobial agent, not related to antitumor treatments. Therefore, there are no reports in the prior art on the antitumor activity of the synthetic peptides described in the present invention. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the increased adhesion of MDA-MB-231 cells to type I collagen and fibronectin at non-cytotoxic concentrations by the minimized peptides LyeTxI mnK-Ac and LyetxlmnK (SEQ ID No. 2, acetylated or not), when compared to LyeTxI (native toxin) and LyeTxI-b (SEQ ID No. 1 of BR 102015031860-0).

[23] Figure 2 indicates the increased adhesion of MCF-7 cells to type I collagen and fibronectin at non-cytotoxic concentrations, by the minimized peptides LyeTxI mnK-Ac and LyetxlmnK (SEQ ID No. 2, acetylated or not).

[24] Figure 3 demonstrates the reduction in invasion (A), cell migration (B) and secretion of MMP-2 and MMP-9 metalloproteinases (C) from MDA-MB-231 cells by the minimized peptides LyeTxI mnK-Ac and LyetxlmnK (SEQ ID No. 2, acetylated or not) compared with the peptide LyeTxIb (SEQ ID No. 1). Petition 870240105262, dated 10 / 12 / 2024, page 22 / 46 12 / 26

[25] Figure 4 demonstrates the reduction in invasion (A), cell migration (B) and secretion of MMP-2 and MMP-9 metalloproteinases (C) from MCF-7 cells by the minimized peptides LyeTxI mnK-Ac and LyetxlmnK (SEQ ID No. 2, acetylated or not), compared with the LyeTxI-b peptide (SEQ ID No. 1).

[26] Figure 5 shows the morphology analysis of MDA-MB-231 cells treated or not with LyeTxI-b (A) and the minimized peptide LyetxlmnK (B) (SEQ ID No. 2).

[27] Figure 6 shows the analysis of the presence of the complex and focus of adhesion in MDA-MB-231 cells treated or not with LyeTxI-b or the minimized peptide LyetxlmnK (SEQ ID No. 2).

[28] Figure 7 shows the interaction model between the LyeTxI-b peptide (SEQ ID No. 1 of BR 102015031860-0) in A, and the minimized peptide (SEQ ID No. 2) in B, with the transmembrane domain of integrin beta 1 (represented at the bottom of each figure). DETAILED DESCRIPTION OF THE TECHNOLOGY

[29] The present technology relates to pharmaceutical compositions comprising the synthetic peptides LyeTxImnK-Ac and LyeTxImnK (SEQ ID No. 2, acetylated or not, respectively), derived from the LyeTxI-b peptide (SEQ ID No. 1), with the sequence minimized to 16 amino acids, containing the addition of a lysine at position 5, and their use for the production of drugs for the treatment of metastatic breast cancer. The use of the pharmaceutical compositions containing the peptides resulted in a reduction in the metastatic activity of triple-negative breast cancer cells and, compared with the LyeTxI-b peptide, showed greater activity in increasing cell adhesion to extracellular matrix components, increased focal adhesion, greater inhibition of MMP-2 secretion and greater inhibition of cell invasion at non-cytotoxic concentrations. Petition 870240105262, dated 10 / 12 / 2024, page 23 / 46 13 / 26

[30] The LyeTxI-b peptide, which consists of the 24 amino acid sequence IWLTALKFLGKNLGKLAKQQLAKL (SEQ ID No. 1 of BR 102015031860-0), had its sequence minimized to 16 amino acids, containing the addition of a lysine at position 5, resulting in the synthetic peptides LyeTxImnK-Ac (Ac-IWLTKALKFLGKNLGK-NH2) and LyeTxImnK (H-IWLTKALKFLGKNLGK-NH2) (SEQ ID N° 2, acetylated or not, respectively).

[31] Pharmaceutical compositions for the treatment of solid tumors are characterized by comprising the synthetic peptide defined by SEQ ID No. 2 or its acetylated form, and / or pharmacologically acceptable salts thereof, and at least one pharmacologically acceptable excipient. The peptides in the compositions may be free and / or incorporated into carriers. Such carriers may be selected from the group comprising cyclodextrins, liposomes and / or other pharmacologically acceptable carriers. Such peptide and / or its isomers and / or its salts may be administered, together or separately, in combination or not with other antitumor agents that provide synergistic effects. The pharmaceutical compositions of the present invention may be administered intramuscularly, intravenously, nasally, sublingually, pulmonaryly, or topically.

[32] The pharmaceutical compositions of the present invention and the peptides of SEQ ID No. 2 can be used for the production of drugs for the treatment of metastatic breast cancer.

[33] The invention can be better understood by the following examples, which are not limiting.

[34] The following examples demonstrate experimental results indicating the in vitro antimetastatic effect of the minimized SEQ ID No2 peptides and the LyeTxI-b peptide (SEQ ID No1). Initially, the cytotoxicity of the two minimized peptides was determined. Petition 870240105262, dated 10 / 12 / 2024, page 24 / 46 14 / 26 LyeTxImnK-Ac and LyeTxImnK (SEQ ID No. 2, acetylated or non-acetylated, respectively) were compared with LyeTxI-b (SEQ ID No. 1 of BR 102015031860-0) and with the native toxin LyeTxI for MDA-MB231 and MCF-7 cells, in order to select non-cytotoxic concentrations. The non-cytotoxic concentrations were used to investigate the antimetastatic potential of the peptides through assays of: cell adhesion to extracellular matrix components such as collagen and fibronectin; analysis of focal adhesion complex formation, evaluation of cell migration and cell invasion; evaluation of MMP-2 and MMP-9 metalloproteinase secretion. EXAMPLE 1 - CYTOTOXICITY

[35] For the cytotoxicity assay, the following procedure was performed: 10,000 cells of the MDA-MB-231 and MCF-7 cell lines were seeded per well in a 96-well plate (90 pL / well) and treated with the peptides in a two-fold serial dilution (10 pL / well) for 48 ha at 37°C in a 5% CO2 atmosphere. Then, 2.5 mg / mL of MTT (Sigma® Co., St. Louis, MO, USA) was added. The plates were incubated for 4 ha at 37°C in a 5% CO2 atmosphere to obtain formazan crystals, the supernatant was removed by aspiration, and 100 pL of HCl-isopropanol (0.04 M) was added to the wells. After gentle agitation for 5 min at room temperature, the absorbance of the solubilized formazan product was measured at 560 nm.

[36] To assess the cytotoxicity of the peptides, the 50% inhibitory concentration of cell viability (IC50) was determined. The results are presented in Table 1, which shows the mean and confidence interval of four experiments performed in quadruplicate. Similar IC50 values ​​were observed between the larger peptides (LyeTxIb and native toxin LyeTxI), which showed IC50 <5 μM for the cell line. Petition 870240105262, dated 10 / 12 / 2024, page 25 / 46 15 / 26 MCF-7. The minimized peptides showed lower cytotoxicity, with IC50 values ​​around 17 μM for MCF-7. For the MDA-MB-231 cell line, a representative model of triple-negative breast cancer, an increase in IC50 values ​​was observed for the minimized peptides LyeTxImnK-Ac and LyeTxImnK (SEQ ID No. 2, acetylated or not) of 86 μM and 97.8 μM, respectively. The LyeTxl-b peptide (SEQ ID No. 1 of BR 102015031860-0) had the lowest IC50 value (4.5 μM), even when compared to the native toxin (LyeTxl = 13.6 pM). Table 1 - Cytotoxicity of peptides in breast cancer cell lines: comparison of IC50 values ​​(M) between the minimized peptides LyeTxImnk-Ac and LyeTximnK (SEQ ID No. 2, acetylated or not), LyeTxl-b (SEQ ID No. 1 of BR 102015031860-0) and the native toxin LyeTxI. Peptide MDA-MB-231 MCF-7 LyeTxI-b (SEQ ID NO. 1) 4.5 (3.5 to 5.9) 2.8 (2.2 to 3.6) LyeTxI (native toxin) 13.6 (11.1 to 16.6)* 4.1 (3.1 to 3.5) LyeTxI mnK (SEQ ID NO. 2) 97.8 (91.7 to 104.2) 16.0 (12.6 to 20.5) LyeTxImnK-Ac (SEQ ID NO. 2, acetylated) 86.1 (73.5 to 100.8) 17.0 (14.1 to 20.6)

[37] Having determined the IC50 for the four peptides, the analysis of their in vitro antimetastatic effect was carried out, using Petition 870240105262, dated 10 / 12 / 2024, page 26 / 46 16 / 26 non-cytotoxic concentrations of the peptide (concentrations below the IC50). EXAMPLE 2 - EFFECT OF PEPTIDES ON CELL ADHESION

[38] For cell adhesion assays, cell suspensions were prepared at the previously selected density for best visualization, with 50,000 cells for MDA-MB-231 and 100,000 cells for MCF-7, in medium without fetal bovine serum (FBS) and without antibiotics, and seeded into the wells. Immediately after seeding, the cells received treatment with the peptides: initially, a two-fold serial dilution of each peptide was performed, based on the previously calculated IC50 values ​​for each peptide. As a positive control for cell adhesion inhibition, cells were seeded in the presence of 10 mM EDTA. As a control for cell adhesion in the absence of treatment, cells were seeded with medium only. All treatments were performed in quadruplicate. The plates were incubated at 37°C, 5% CO2, for 2 to 3 hours for MDAMB-231 or 5 to 6 hours for MCF-7, for adhesion.After the cells became adherent and spread, the plates were washed once with PBS (100 pL / well), the adherent cells were fixed with 70% ethanol (100 pL / well, 10 minutes at room temperature) and stained with 0.2% crystal violet (50 pL / well, 30 minutes at room temperature). The plates were washed with water to remove the dye not absorbed by the cells. The crystal violet absorbed by the adherent cells was extracted by adding 0.2% Triton X-100 (100 pL / well) overnight under agitation. Absorbance was measured in a spectrophotometer at a wavelength of 560 nm. Data were expressed as the mean of the respective absolute values ​​and standard deviation.

[39] Loss of adhesion to the extracellular matrix by cancer cells, as well as loss of cell-cell adhesion, are key initial events in. Petition 870240105262, dated 10 / 12 / 2024, page 27 / 46 17 / 26 metastasis process and cancer progression. As already mentioned, in previous studies with the LyeTxI-b peptide (SEQ ID No. 1 of BR (102015031860-0) it was possible to observe a reduction in the number of metastatic nodules in the lungs of animals treated with this peptide, when studied in the isogenic model of triple-negative 4T1 breast cancer (Abdel-Salam et al, 2021). Having determined the IC50 for each peptide, the effect of these peptides on the adhesion of MDA-MB-231 and MCF-7 cells to two constituents of the extracellular matrix (type I collagen and fibronectin) was analyzed. Serial dilution of each peptide was performed at ten different concentrations, based on the respective IC50 value for each peptide. As shown in Figure 1, a significant increase in the adhesion of MDA-MB-231 cells to type I collagen and fibronectin by the minimized peptides was observed compared to the untreated control cells. The results presented in Figure 1 show that only the two minimized peptides had an effect on increasing the adhesion of MDA-MB-231 cells to type I collagen and fibronectin.It was observed that the minimized peptide LyeTxlmnK (SEQ ID No. 2) significantly increased cell adhesion at non-cytotoxic concentrations, from 25 to 3.12 μM in collagen and from 25 to 0.78 μM in fibronectin. The acetylated minimized peptide LyeTxlmnK-Ac (SEQ ID No. 2) showed a smaller effect than that observed for the LyeTxlmnK peptide. For LyeTxI-b (SEQ ID No. 1 of BR 102015031860-0) and the native toxin LyeTxI, no significant effect on increasing adhesion was observed.

[40] For MCF-7 cells, a significant increase in cell adhesion was also observed for both minimized peptides, at non-cytotoxic concentrations (8 to 1 μM), as shown in Figure 2. EXAMPLE 3 - EFFECT OF PEPTIDES ON CELL INVASION

[41] To evaluate the effect of peptides on cell invasion, MDA-MB-231 cells (2,000 / well) were centrifuged in the presence of 10 μg / mL Petition 870240105262, dated 10 / 12 / 2024, page 28 / 46 18 / 26 type I collagen cells were used to form spheroids in ultra-low adhesion plates; and MCF-7 cells were seeded directly into the well and incubated for 4 days to form spheroids. Once the spheroid was formed (d0), the cells were inserted into a type I collagen matrix (1 mg / mL) as previously described (Chong LH, Yip AK, Farm HJ, Mahmoud LN, Zeng Y and Chiam KH (2024), The role of cellmatrix adhesion and cell migration in breast tumor growth and progression. Front. Cell Dev. Biol. 12:1339251. doi: 10.3389 / fcell.2024.1339251) and incubated for 1 hour, followed by treatment with the peptides at a concentration of 1 / 8 of their respective IC50 values ​​determined in 2D and incubation for 72 hours. At the end of the 72 hours, the spheroids were photographed. Images were obtained before treatment (d0) and after 24 hours with 4x magnification. The area of ​​the spheroid was determined using ImageJ software (NIH, Rockville, USA).

[42] The local invasive capacity of tumor cells is considered an initial step in the complex process of metastasis. A three-dimensional (3D) spheroid culture model was used to determine the effect of peptides on tumor invasion. The peptides were studied at concentrations of their respective IC50 values ​​of 1 / 8, this being the value considered non-cytotoxic according to the literature (Gurgul I, JanczyCempa E, Mazuryk O, Lekka M, Lomzik M, Suzenet F, Gros PC, Brindell M. Inhibition of Metastasis by Polypyridyl Ru(II) Complexes through Modification of Cancer Cell Adhesion - In Vitro Functional and Molecular Studies. J Med Chem. 2022 Aug 11;65(15):10459-10470. doi: 10.1021 / acs.jmedchem.2c00580. Epub 2022 Jul 27. PMID: 35895090; PMCID: PMC9376949). Therefore, a concentration of 0.5 pM was used for the LyeTxI-b peptide, and 12.5 pM was used for the minimized peptides, LyeTxlmnK-Ac and LyeTxlmnK (SEQ ID No. 2, acetylated or not). Petition 870240105262, dated 10 / 12 / 2024, page 29 / 46 19 / 26

[43] In the 3D system, type I collagen was used, a fibrillar collagen that is the most abundant protein in the body and is found accumulated in the extracellular matrix (ECM) of connective tissues, such as tendons and skin.

[44] Type I collagen was obtained by acid extraction and used as an extracellular matrix substrate where the cells were embedded. This model mimics the main constituent of the interstitial matrix of solid tumors and has been used to test pro- or anti-invasive compounds. The results allow inferences about the effect of the tested compound on tumor growth in vivo, since the spheroids show progressive reduction of oxygen, nutrients, growth factors and limitations for drug penetration.

[45] The results showed that MDA-MB-231 cells, when cultured in a 3D system immersed in type I collagen, presented a spheroid of uniform morphology, rounded and with well-defined contours (Figure 3A, d0). After being inserted into the type I collagen matrix for three days (72h), the MDA-MB-231 cells began to invade the surrounding matrix, in a characteristic “starburst” pattern (Figure 3, A). However, these invasive protrusions were reduced in the presence of the three peptides tested. The invasion area, calculated using ImageJ software, showed a prominence for the minimized peptides (LyeTxlmnK-Ac and LyeTxlmnK, corresponding to SEQ ID No. 2, acetylated or not), which significantly inhibited the invasive capacity of MDA-MB-231 spheroids when compared to LyeTxI-b (SEQ ID No. 1 of BR 102015031860-0). This effect was also observed for the MCF-7 cell line (Figure 4, A).However, for this cell line, the spread of MCF-7 cells into the circulating matrix showed a different pattern, similar to a "cloud of cells," characteristic of MCF-7 cells, which exhibit a less invasive phenotype compared to MDA-MB-231. Petition 870240105262, dated 10 / 12 / 2024, pp. 30 / 46 20 / 26 EXAMPLE 4 - EFFECT OF PEPTIDES ON CELL MIGRATION

[46] Motility is an essential characteristic of metastatic cancer cells. The in vitro scratch wound healing assay was performed to determine whether peptides affect the migration of MDA-MB-231 and MCF-7 cells. For the migration assays, a 50,000 cell / ml suspension of MDA-MB-231 or MCF-7 cells was prepared. 70 μL of this suspension was pipetted into each chamber of the cell culture insert (ibidi, Munich, Germany; www.ibidi.de), followed by incubation for 16 hours in the presence of 10% FBS. The cell culture insert was removed after 16h, leaving a defined cell-free space of 500 μm. The wells were washed with PBS three times to remove debris and non-adherent cells. After obtaining a wound in the monolayer of the two cell lines, the cells were kept serum-free and then allowed to migrate in the presence or absence of peptides.Medium without FBS was added with or without the peptides at their respective IC50 values ​​and incubated for an additional 24 hours. Images were taken before treatment (d0) and after 24h, at 10x magnification, and cell-free space was determined using ImageJ software (NIH, Rockville, USA).

[47] Analysis of cell migration over the 24h period showed that the LyeTxI-b peptide was the only one that significantly reduced the migration of MDA-MB-231 cells, observed by the larger area of ​​the “wound” compared to the control after 24 hours of culture. The minimized peptides did not show a significant effect on the migration of MDA-MB231 cells (Figure 3 B) over the 24h period. Regarding MCF-7, there was no significant difference in the wound area measurement of the control culture, nor in the presence of the peptides during the 24 hours of the experiment (Figure 4, B), which can be explained by the less invasive phenotype of MCF-7. Petition 870240105262, dated 10 / 12 / 2024, page 31 / 46 21 / 26 EXAMPLE 5 - EFFECT OF PEPTIDES ON SECRETION OF Matrix metalloproteinases

[48] ​​Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that can hydrolyze extracellular matrix (ECM) proteins, including the basement membrane, which act as a barrier to the passage of tumor cells and are considered viable drug targets. Gelatinases, such as MMP-2 and MMP-9, are members of the MMP family that are capable of cleaving collagen, an important component of the basement membrane. Gelatinase expression is highly elevated in many malignant human tumors and is strongly correlated with the invasion and metastasis of cancer cells (Shin, S., Kim, C., Jung, Y. et al. The UPR inducer DPP23 inhibits the metastatic potential of MDA-MB-231 human breast cancer cells by targeting the Akt-IKK-NF-KB-MMP-9 axis. Sci Rep 6, 34134 (2016). https: / / doi.org / 10.1038 / srep34134).

[49] To examine whether the observed effects on the reduction of invasion of type I collagen-embedded spheroids by peptides were related to decreased MMP secretion, analysis of MMP-2 and MMP-9 secretion was performed with MDA-MB-231 and MCF-7 cell lines. A gel gelatinase activity assay was performed by zymography, a technique widely used to study enzymes that degrade the extracellular matrix, such as MMPs, from tissue extracts, cell cultures, among others. This simple and sensitive technique identifies MMPs by the degradation of their substrate and by their molecular weight and, therefore, helps to understand the role of MMPs in different contexts.

[50] To evaluate the effect of peptides on the secretion of matrix metalloproteinases (MMPs), cell culture medium was used in zymographic analyses, according to the following protocol, based on Toth and Fridman, 2001 (Toth M, Fridman R. Assessment of Petition 870240105262, dated 10 / 12 / 2024, pp. 32 / 46 22 / 26 Gelatinases (MMP-2 and MMP-9 by Gelatin Zymography. Methods Mol Med. 2001;57:163-74. doi: 10.1385 / 1-59259-136-1:163. PMID: 21340898; PMCID: PMC3845455). Cells were cultured to approximately 80% confluence in whole growth medium in 10 cm² flasks. The cell monolayer was washed twice with sterile PBS (5 mL) to completely remove serum and incubated with serum-free medium (2.0 mL) at 37 °C for 16 h ± 24 h. After this incubation, the cells were treated with the peptides and incubated for 24 h. The medium was collected and kept at 4 °C followed by centrifugation (400 g, 5 min) to remove cells and debris. For a better signal, the samples were concentrated 10 to 20 times with Vivaspin 2.0 (10 kDa). 20 to 30 μL of the sample were loaded onto a 10% SDS-PAGE gel copolymerized with gelatin.Medium with 10% serum was used as a positive control for MMP-9 (92 kDa and 84 kDa, pro- and active, respectively) and MMP-2 (72 kDa and 64 kDa, pro- and active, respectively) gelatinases. A molecular weight standard (200 to 50 kDa) was also applied. Electrophoresis was performed at constant voltage (130 V, up to 40 mA) for 90 to 100 min. The gel was placed in a plastic tray containing 50 mL of renaturation solution to remove SDS and renature the enzymes, and incubated for 30 min at room temperature with gentle stirring, then rinsed with 300 mL of dH2O and incubated at room temperature for 30 min in 50 mL of growth buffer with gentle stirring. The developing buffer was replaced with 100 mL of fresh developing buffer and the gel was incubated at 37°C for at least 16 h in a closed tray. The gel was stained with Coomassie blue for at least 1 h and decolorized until areas of gelatinolytic activity appeared as sharp, clear bands against the blue background.The images were obtained using ImageQuant LAS 4000 (GE) and analyzed using ImageJ software. The results... Petition 870240105262, dated 10 / 12 / 2024, pp. 33 / 46 23 / 26 obtained represent the average area under the curve of the bands corresponding to the MMPs (MMP-9: 92 kDa and 84 kDa, pro and active, respectively, and MMP-2: 72 kDa and 64 kDa, pro and active, respectively).

[51] Gelatin zymography showed that the gelatinase activity of MMP-2 and MMP-9 secreted by MDA-MB-231 in its inactive form was present in greater quantity in the control culture and was significantly suppressed in the presence of the three peptides (Figure 3, C). This same effect was also observed for MCF-7 cells, where a significant reduction in the secretion of active MMP-2 and MMP-9 was observed in relation to the control culture (Figure 4, C). EXAMPLE 6 - EFFECT OF PEPTIDES ON THE FORMATION OF FOCAL ADHESION COMPLEXES

[52] An initial and fundamental event for cancer cells to metastasize is the loss of cell adhesion between neighboring cells and with the extracellular matrix. Cell-extracellular matrix interactions are based on their focal adhesion (FA) and are essential for anchorage and migration. When cells spread or migrate on two-dimensional substrates, they form FAs. FA formation is initiated by the binding of integrins to their ligands on the substrate. Within 1 to 3 minutes of substrate exposure, adherent cells assemble clusters of about 50 bound integrins. In migrating cells, actin filaments push unbound integrins into small clusters at the leading edge of the cell. In both cases, the initiation of clustering requires actin polymerization but precedes the buildup of detectable strain. FAs transmit force from inside the cell to the substrate outside the cell and thus mediate cell-substrate adhesion and cell motility. Petition 870240105262, dated 10 / 12 / 2024, pp. 34 / 46 24 / 26

[53] Considering the effect on increased adhesion in MDAMB-231 cells, the analysis of the formation of focal adhesion complexes was performed with this cell line. The LyeTxI-b peptide (SEQ ID No. 1 of BR 102015031860-0), which showed antimetastatic activity in vivo (Abdel-Salam et al, 2021), and the minimized peptide with the best activity, LyeTxlmnK (SEQ ID No. 2 non-acetylated), which induced increased cell adhesion for type I collagen and fibronectin at more than one concentration (Figure 1), were selected. The peptides were studied at non-cytotoxic concentrations, at their respective 1 / 8 IC50 values. Fibronectin was the extracellular matrix component selected, considering the effect of LyeTxImnK (SEQ ID No. 2 non-acetylated) on cell adhesion, at a greater number of concentrations.

[54] Cells (3,000 cells / well) were seeded onto slides coated with fibronectin (0.2 ug / mL) in the presence or absence of peptides. After 3 hours, the cells were fixed with a solution containing 4% paraformaldehyde, 5% sucrose in PBS for 15 minutes. The cells were blocked with 2% BSA for 1 hour and the extracellular labeling antibody (integrin beta-1) was added for 24h. After extracellular labeling, intracellular labeling was performed. For this, the cells were permeabilized with a solution containing 0.2% BSA and 0.1% Triton-x100 for 1 hour. The cells were washed 3x with PBS and incubated with Paxillin antibody for 24h. The cells were washed again 3 times with PBS, followed by labeling with secondary antibodies, specific for the primary antibodies used labeled with fluorochromes, as well as phalloidin for actin labeling, for 2 hours.After washing (3 times with PBS), the nuclei were stained with a 5 pg / mL solution of Hoechst 3342, incubated for 5 minutes, and washed again with PBS. Images were acquired. Petition 870240105262, dated 10 / 12 / 2024, pp. 35 / 46 25 / 26 under a Zeiss confocal microscope, using 10x and 40x objectives. Two independent experiments were performed in triplicate.

[55] Studies of cell morphology and quantification of adhesion foci by immunofluorescence (Figure 5) show that MDAMB-231 cells from the control culture (after 3 hours of incubation) appear rounded in most fields, poorly spread and therefore weakly adhered to the fibronectin matrix. MDA-MB-231 cells treated with both peptides showed good adherence to fibronectin, with a "spread" morphology, in the vast majority of fields (Figure 5). It was possible to observe the characteristic morphological types of this cell line in its fusiform (mesenchymal), globular, and polygonal morphology. Plasma membrane protrusions, filopodia, and lamellopodia were observed, as previously described for this cell line (Tavianatou AG, Piperigkou Z, Barbera C, Beninatto R, Masola V, Caon I, Onisto M, Franchi M, Galesso D, Karamanos NK).Molecular size-dependent specificity of hyaluronan on functional properties, morphology and matrix composition of mammary cancer cells. Matrix Biol Plus. 2019 Jun 5;3:100008. doi:. 10.1016 / j.mbplus.2019.100008.). Antibody labeling to identify focal adhesion components such as beta-1 integrin (green), cytoskeleton (actin, red), and paxillin (yellow) showed that the untreated culture (control) exhibited diffuse labeling on the cell membrane. However, cells treated with LyeTxI-b (SEQ ID No. 1 of BR 102015031860-0) and LyeTxlmnK (SEQ ID No. 2 non-acetylated) peptides for 3 hours showed greater labeling intensity at the cell edges, demonstrating colocalization of beta-1 integrin, actin, and paxillin, where cell surface integrins cluster and make strong adhesive contact with extracellular matrix components (Figure 6). These results indicate Petition 870240105262, dated 10 / 12 / 2024, pp. 36 / 46 26 / 26 that LyeTxI-b (SEQ ID No. 1 of BR 102015031860-0) and LyeTxImnK (SEQ ID No. 2 non-acetylated) increase focal adhesion foci, which demonstrates the effect on increased fibronectin adhesion observed, which consequently leads to a reduction in cell migration and invasion, reducing the metastatic activity of the tested cells. EXAMPLE 7 - ANALYSIS OF THE POSSIBLE MECHANISM OF ACTION OF PEPTIDES

[56] The results presented in the six previous EXAMPLES demonstrate that the minimized peptides (SEQ ID No2), as well as LyeTxI-b (SEQ ID No. 1 of BR 102015031860-0), are able to deprive neoplastic cells of adhesion loss, contributing to the reduction of metastasis, by reducing their ability to migrate and / or invade.

[57] Analysis of the primary and secondary structure of the peptides allowed the creation of a model that can explain how peptides can act in this process. As demonstrated in Figure 7, both the LyeTxI-b peptide (SEQ ID No. 1 of BR 102015031860-0) and the LyeTxlmnK peptide (SEQ ID No. 2 non-acetylated) are able to interact with the transmembrane domain of the beta 1 subunit of the integrin, which prevents the beta 1 subunit from interacting with the transmembrane domain of the alpha subunit, maintaining the integrin in its active conformation, which promotes its greater adhesion to the components of the extracellular matrix. The interaction of the minimized peptide is more stable than that of the LyeTxI-b peptide (SEQ ID No. 1 of BR 1020150318600), which may explain the differentiated effects observed for this peptide. Petition 870240105262, dated 10 / 12 / 2024, pp. 37 / 46

Claims

1 / 1 CLAIMS 1. PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF SOLID TUMORS comprising a derivative of the synthetic peptide defined by SEQ ID No. 1, characterized by comprising the synthetic peptide defined by SEQ ID No. 2 or its acetylated form, and / or pharmacologically acceptable salts thereof, and at least one pharmacologically acceptable excipient.

2. PHARMACEUTICAL COMPOSITIONS, according to claim 1, characterized in that the peptides are free and / or incorporated into carriers.

3. PHARMACEUTICAL COMPOSITIONS, according to claim 2, characterized in that the carriers are selected from the group comprising cyclodextrins, liposomes and / or other pharmacologically acceptable carriers.

4. PHARMACEUTICAL COMPOSITIONS, according to claim 1, characterized in that the peptide and / or its isomers and / or its salts are administered, together or separately, in combination or not with other antitumor agents that provide synergistic effects.

5. PHARMACEUTICAL COMPOSITIONS, according to claim 1, characterized by being administered intramuscularly, intravenously, nasally, sublingually, pulmonaryly, orally or topically.

6. USE OF THE COMPOSITIONS, defined by claim 1, characterized by being for producing medicaments for the treatment of metastatic breast cancer.

7. USE OF THE PEPTIDE DEFINED BY SEQ ID No. 2, as defined in claim 1, characterized by being for the production of medicaments for the treatment of metastatic breast cancer. Petition 870240105262, dated 10 / 12 / 2024, pp. 38 / 46

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