Combination of spider venom peptide and chemotherapeutic agents, composition comprising spider venom peptide, related uses and kit

Abstract

The present invention relates to the field of cancer therapies. In particular, the present invention relates to the development of new therapies for the treatment of solid tumours. The present invention relates to a combination of a peptide derived from the venom of the spider species Phoneutria nigriventer, especially the LW-9 fraction, and classic chemotherapeutic agents, in particular doxorubicin, as an alternative for the treatment of solid tumours, including breast cancer. Additionally, the present invention describes a kit comprising an effective dosage form of peptide fractions of the venom of the spider species Phoneutria nigriventer, an effective dosage form of an anthracycline, and instructions for use. An additional objective of the present invention is to describe a composition comprising the peptide fraction LW-9 as well as the use thereof in the treatment of solid tumours.

Description

COMBINATION OF SPIDER VENOM PEPTIDE AND CHEMOTHERAPEUTIC AGENTS, COMPOSITION COMPRISING SPIDER VENOM PEPTIDE, RELATED USES AND KIT FIELD OF THE INVENTION

[0001] The present invention falls within the field of cancer therapies. In particular, the present invention relates to the development of novel therapies for the treatment of solid tumors.

[0002] The present invention relates to a combination of a peptide derived from the venom of the spider species Phoneutria nigri-venter, especially the LW-9 fraction, and classic chemotherapeutic agents, in particular doxorubicin, as an alternative for the treatment of solid tumors, including breast cancer. Additionally, the present invention describes a kit of parts comprising an effective dosage form of peptide fractions from the venom of the spider species Phoneutria nigri-venter, an effective dosage form of an anthracycline, and instructions for use. It is a further objective of the present invention to describe a composition comprising the LW-9 peptide fraction as well as its use in the treatment of solid tumors. FUNDAMENTALS OF THE INVENTION

[0003] The following paragraphs are intended to introduce the reader to a more detailed description, but without intending to limit the subject matter claimed in this disclosure.

[0004] Cancer treatment is a major challenge. Chemotherapy drugs routinely used to treat breast cancer have limited efficacy and serious side effects. Doxorubicin is a chemotherapy drug from the anthracycline class, which is very active. against breast cancer. However, the drug frequently causes fatigue, nausea, decreased appetite, mouth sores, diarrhea, among other adverse symptoms that significantly impair the patient's quality of life, even making the continuation of treatment unfeasible in some cases. Treatment using doxorubicin associated with a new peptide synthesized from a prototype identified in spider venom significantly reduced tumor size (murine experimental model) compared to treatment with doxorubicin alone, in addition to reducing adverse effects. Animals treated with the drug combination showed greater feed consumption, weight gain, and greater exploratory activity compared to the group treated with doxorubicin alone, and had no observable signs of pain and discomfort. Analyses of the mechanism of the test peptide demonstrated that it activates the immune system.Therefore, combining an anthracycline (which kills tumor cells by damaging DNA) with an immunomodulatory agent represents a promising strategy, since the therapy will utilize different mechanisms against the tumor, complementing each other.

[0005] After several decades remaining in the shadow of synthetic molecules, drug discovery from natural sources is undergoing a renaissance (Wainwright et al., 2022). These products of natural origin can be used as: a) direct sources of therapeutic agents; b) starting points for semi-synthetic routes for obtaining drugs; and c) prototypes for the synthesis of new drugs (Calixto, 2019; Mariottini & Grice, 2019). It is estimated that around 35% of the revenue from... The pharmaceutical industry focuses on drugs derived directly or indirectly from plants, microorganisms, or animals. Classic examples include drugs that revolutionized the history of medicine, such as morphine and penicillin (Thomford et al., 2018). Approximately one-third of the drugs approved in the last 20 years by the American Food and Drug Administration (EDA) were derived from natural sources, mainly plants. However, the use of molecules extracted from animals represents a challenge. Only 3% of the 1652 drugs approved by the FDA between 1983 and 2014 were of animal origin, which points to the need to expand studies in this field (Newman & Crag, 2016).

[0006] In the context of cancer therapy, the use of natural products is considered especially strategic (Amaral et al., 2019). Important chemotherapeutic agents such as Taxol (Taxus brevi folia) and Vinblastine (Catharanthus roseus) can be cited as examples. Additionally, toxins from animal venoms, mainly from bees, snakes, and arthropods, have shown promising results in cancer treatment in preclinical models through antiangiogenic effects, induction of apoptosis, inhibition of cell proliferation, reduction of tissue invasion and metastasis, as well as immunomodulatory effects (Orsolic, 2012; Calderon et al., 2014; Raposo et al., 2017; Jimenez et al., 2018; Li et al., 2018; Wu et al., 2019; Santos et al., 2019; Barreto et al., 2020; Bonfanti et al., 2020; Kisaki et al., 2021; Munhoz et al., 2021; Caballero et al., 2022). Mato et al., 2023). However, in addition to such studies not having progressed over the years, they were not The immunomodulatory effects of these natural products have been evaluated in the context of cancer, much less in breast cancer.

[0007] Many of the antitumor effects observed with animal venoms are mediated by peptides, a class of molecules predominant in most venoms. Venom peptides (VPs) exhibit high specificity and selectivity for tumor cells, and may be useful as prototypes for the design of more specific peptide drugs for targeted cancer therapies. Because VPs naturally target mammalian receptors, they show high selectivity for ion channels and defined receptors in the cell membrane (Mahadevappa et al. 2017). VPs offer many advantages compared to proteins or monoclonal antibodies in targeted therapies, as they are generally highly resistant to pH, temperature, and protease degradation.Furthermore, VPs are frequently naturally stabilized through post-translational modifications, such as C-terminal amidation, sulfation of tyrosine residues, bromination of tryptophans, and glycosylation of threonines, and can be further stabilized by chemical modifications, such as the incorporation or cyclization of D-amino acids (Mahadevappa et al., 2017) and conjugation with poly(ethylene glycol) (PEG), for example (Roberts et al., 2012). An important aspect is that advances in large-scale peptide synthesis techniques may make VP-derived drugs more accessible to patients.

[0008] Cancer is a multifactorial, complex, and highly prevalent disease of great global importance. There is a The constant search for treatments that are more effective and have fewer adverse effects, improving the quality of life and overall survival of patients, has led to a shift in therapeutic strategies considering the molecular profile of tumors. This has resulted in the detection of new targets, leading to two new approaches in cancer treatment: 1) individualized / personalized therapy (targeted drugs) based on the molecular signature of tumors; and 2) a focus on the tumor microenvironment, leading to the development of immunotherapies (Zugazagoitia et al., 2016). Another strategy gaining traction is the combination of drugs with different mechanisms, promoting the treatment of the disease from multiple angles.

[0009] Immunotherapy, which has been successfully used in the treatment of various types of tumors (Hedge & Chen, 2020; Khoury et al., 2019; Queirolo et al., 2019), is not yet a reality for the treatment of most solid tumors. Despite these limitations, some agents that are in clinical trials show positive responses. Clinical trials include dendritic cell (DC) vaccines, CAR T therapies, the use of PD-1 and P-I1 inhibitors, among others (McGranahan et al., 2019; Choi et al., 2019; McKelvey et al., 2020). However, although limited, the results obtained in clinical trials to date suggest that immunotherapies are promising in the treatment of solid tumors, pointing to the need to use strategies to improve their performance. Much research has focused on obtaining a specific response to tumor antigens by loading DCs with individual tumors or synthetic antigens obtained from tumors. Currently, However, researchers have focused on combination therapies (e.g., the NCT04201873 clinical trial, which uses DC vaccines combined with the anti-PD-1 monoclonal antibody, pembrolizumab). The use of adjuvants that increase the immunogenicity of the tumor microenvironment, therefore, presents great potential (Anguille et al., 2019; Sabado et al., 2017).

[0010] Spiders are considered one of the most diverse animals, with over 47,000 described species. It is estimated that there are more than 10 million biologically active peptides present in spider venoms, although only around 1,400 have been characterized to date (Peigneur et al., 2018). The venom of the spider *Phoneutria nigriventer* - PnV (found in South America and popularly known as "Armadeira") contains peptides with potential pharmacological and biotechnological applications (Raposo et al., 2017; Diniz et al., 2018).

[0011] PnV permeabilizes the blood-brain barrier (Raposo et al., 2007, 2012, 2014; Cruz-Höfling, Tavares, Raposo, 2016) and causes alterations in the morphology and cytoskeleton of astrocytes (Raposo et al., 2016). These results led to the hypothesis that the venom could contain molecules with an antitumor effect, for example, in gliomas, since these tumors are mainly derived from glial cells, especially astrocytes.

[0012] Immunomodulation in cancer treatment refers to the use of therapies that aim to modulate the patient's immune system to help the body recognize and fight cancer cells more effectively. The immune system plays a fundamental role in... Detection and destruction of abnormal cells, including cancerous cells. However, cancer can evade immune detection or suppress the immune response, allowing tumor cells to proliferate. Therefore, the use of a drug combined, simultaneously or not, with another mechanism represents an advance in this technical field of therapies for cancer treatment.

[0013] Thus, the objective of the present invention is to describe an innovative and surprising strategy for immunotherapy against cancer. STATE OF THE ART

[0014] Some prior art documents describe strategies for treating solid tumors. However, there is no evidence to date of a treatment involving the combination of classic chemotherapeutic agents, exemplified in the present application by doxorubicin, with the LW-9 peptide, with an amino acid sequence corresponding to QKKDRFLGLM (peptide of 1221.53 Da, SEQ ID NO.: 1), derived from the venom of the spider species Phoneutria nigriventer, for the treatment of solid tumors, particularly for the treatment of breast cancer.

[0015] Several groups around the world are researching new alternative therapies for cancer treatment. However, research specifically using drug combinations containing doxorubicin, aiming to improve expected effects and reduce adverse effects, is not yet available. Current protocols indicate cyclical therapies, aiming to reduce adverse effects and provide time for the body to recover. Furthermore, cyclical protocols utilize different chemotherapeutic agents. However, they all share the mechanism related to promoting damage to the viability of the tumor cell, which results in inevitable damage to healthy cells.

[0016] Some studies in the literature propose strategies for the treatment of breast cancer, which involve, for example, the development of new biopharmaceuticals for this purpose. However, none of the documents available in the state of the art describe the use of the LW-9 peptide as an immunomodulator in combination with classic chemotherapeutic agents.

[0017] The publication titled "Molecular characterization of malignant tumors in dogs and identification of neoplasms responsive to new biopharmaceuticals: a translational approach," published in 2021, describes the increase in malignant neoplasms in dogs, including breast cancer, as well as strategies for treating the disease. The document demonstrated that canine mammary tumor cells responded very well to treatment with peptides from the venom of the spider Phoneutria nigri venter. However, despite the document anticipating the potential use of peptides from the spider species Phoneutria nigri venter, it is silent regarding the LW-9 peptide, as well as the use of any possible combination of this peptide and conventional chemotherapeutic agents, such as doxorubicin, in the treatment of breast cancer.Furthermore, the document neither describes nor even suggests the use of peptides derived from the spider species Phoneutria nigri venter as an immunomodulator in cancer treatment, particularly in the treatment of breast cancer.

[0018] The publication titled "Does the Sequence of The 2020 study, "Anthracycline and Taxane Matter? The NeoSAMBA Trial," describes how adjuvant chemotherapy for breast cancer reduced mortality by almost half compared to the absence of adjuvant chemotherapy. The article describes how doxorubicin delivery followed by CMF (cyclophosphamide, methotrexate, and fluorouracil) significantly reduced the risk of disease recurrence and death compared to alternating doxorubicin and CMF regimens. However, the document does not mention the LW-9 peptide, derived from the venom of the spider species *Phoneutria nigriventer*, as an alternative for cancer treatment, much less its use in combination with doxorubicin for the treatment of solid tumors, especially breast tumors.

[0019] In turn, the publication "Metaboomic analysis of macrophages modulated with a molecule isolated from spider venom: potential application in the development of new cancer treatments", published in 2022, describes the venom of the spider Phoneutria nigriventer (PnV) as a mixture of bioactive compounds with pharmacological potential (PEIGNEUR et al., 2018). The document describes that studies have shown that PnV provided antitumor effects in vitro and in vivo (BARRETO et al., 2019; BARRETO et al., 2020; BONFANTI et al., 2020). However, despite discussing the advantages of peptides derived from the venom of the spider Phoneutria nigriventer, the document neither reveals nor even suggests the use of combining LW-9, for example, acting as an immunomodulator, with anthracyclines, the so-called classic chemotherapeutic agents, such as doxorubicin, in cancer treatment. mammary.

[0020] The 2022 publication titled "Bioactive peptides from venoms against glioma progression" describes the possibility of combination therapy in cancer treatment. Specifically, the document suggests the combined use of standard cancer therapies with bioactive peptides derived from spider venom. The document describes how peptides from the venom of the spider *Phoneutria nigriventer* act as immunomodulators. The publication further describes that PnV fractions increase the number of monocytes in the blood of xenogeneic mice and the number of tumor-infiltrating macrophages. The publication demonstrated that the LW-9 fraction of *Phoneutria nigriventer* venom increased the cytotoxic and phagocytic activity of macrophages through immunomodulation. Furthermore, it suggested that macrophages were re-educated into a non-TAMs phenotype, resulting in increased destruction of cancerous cells.However, despite the foregoing, the aforementioned publication differs from the present patent application in that: (i) the mechanism explored was direct cytotoxicity in tumor cells, and not immunomodulation, (ii) there is no reference to the combination of components of the venom of the spider species Phoneutria nigriventer with classic chemotherapeutic agents, such as doxorubicin, and (iii) the study is focused only on the benefits of using the venom in a specific class of tumor, glioma.

[0021] Regarding the publication entitled "Adjuvant systemic treatment in breast cancer," from 2001, he describes, in a rather simplified way, about adjuvant treatments in patients with breast cancer. However, besides the fact that the aforementioned publication neither describes nor even suggests the use of the specific combination of LW-9 and doxorubicin, the chemotherapeutic treatments in said publication are also not based on the patient's immunohistochemical profile. Therefore, despite the aforementioned publication sharing the same technical field as the present invention, there is no evidence regarding the use of the LW-9 peptide as an immunomodulator, associated with classic anthracyclines, especially doxorubicin, that would allow for more effective treatment in patients with breast cancer and, moreover, with substantially reduced side effects.

[0022] Therefore, the aforementioned prior art, even when combined with each other, in any combination thereof, would not motivate a person skilled in the art to deduce or obtain the teachings described in this patent application. SUMMARY OF THE INVENTION

[0023] The present invention relates to a combination of classic chemotherapeutic agents and peptides derived from the venom of the spider species Phoneutria nigrivente for the treatment of solid tumors. In particular, the present invention describes a combination comprising the LW-9 peptide, with an amino acid sequence corresponding to QKKDRFLGLM, which corresponds to a 1221.53 Da peptide, identified from screening using purified molecules from the venom of the spider Phoneutria nigrivente, for the treatment of breast cancer.

[0024] Based on the experiments performed (in vivo tests), treatment with LW-9 significantly slowed tumor progression, especially in combination with the classic chemotherapy drug used in clinical practice, namely doxorubicin, chosen as a positive control. In addition to interfering with tumor progression, the LW-9 peptide improved animal welfare, increasing feed intake and body weight throughout the treatment period, compared to the untreated control and the group that received only doxorubicin.

[0025] Furthermore, it was observed that the LW-9 peptide, at a dose of approximately 1 pg / kg, did not cause alterations in the evaluated hepatic and renal functional markers (AST, ALT, creatinine, and urea), unlike the dose of approximately 10 pg / kg, for which some toxic effects were observed. Finally, the results of flow cytometry analyses revealed that the peptide, mainly in association with the classic chemotherapeutic agent, doxorubicin, at a dose of approximately 5 mg / kg, significantly reduced the expression of PD-L-1 in the tumor microenvironment and also surprisingly increased the expression of CD3 and GITR, indicating activation of the immune response with alterations in markers that suggest a good prognosis.

[0026] PDL-1 is a transmembrane protein that plays a crucial role in regulating the immune system. The main function of PDL-1 is to suppress the body's immune response, playing an important role in regulating autoimmunity and preventing an excessive immune response that can damage healthy body tissues. However, this function can also be... exploited by cancer cells to avoid being destroyed by the immune system. Assessing PD-L-1 expression is an important part of evaluating cancer patients, especially those considering immunotherapy, a class of treatments aimed at strengthening the immune response against cancer.

[0027] Cancer is a multifactorial disease, and the combination of drugs with different mechanisms has been a promising treatment strategy. It is widely known that classic chemotherapeutic agents cause serious side effects, responsible for the low quality of life of patients. These adverse effects sometimes make the continuation of treatment unfeasible. The drug tested in this study proved to be effective, especially in combination with doxorubicin, in addition to reducing the adverse effects of classic therapy, with great potential for clinical use.

[0028] To that end, a first embodiment of the present invention describes a combination of classic chemotherapeutic agents, exemplified by doxorubicin, and the LW-9 peptide, derived from the venom of the spider species *Phoneutria nigriventer*. However, any cytotoxic chemotherapeutic agent that acts directly on tumor cells, either by damaging DNA or by altering mitosis, can be used in this combination. Among the classic chemotherapeutic agents mentioned are platinum-based chemotherapeutic agents (Carboplatin); other anthracyclines (Mitoxantrone in the case of cardiac patients who cannot receive doxorubicin) and oxazaphosphorines (Cyclophosphamide).

[0029] In a second embodiment of the present invention, the use of a combination of classic chemotherapeutic agents, exemplified by doxorubicin, and the LW-9 peptide, for the treatment of solid tumors is described. In particular, the present invention describes the use of a combination of the LW-9 fraction of the venom of the spider Phoneutria nigriventer and doxorubicin for the treatment of breast cancer.

[0030] In a third embodiment of the present invention, the use of the LW-9 peptide fraction from the venom of the spider species Phoneutria nigriventer, in isolation, is described in the manufacture of a medicament / composition for the treatment of solid tumors in an individual in need thereof.

[0031] In a preferred embodiment of the present invention, the LW-9 peptide fraction from the venom of the spider species Phoneutria nigriventer is used for the treatment of breast tumors.

[0032] In a fourth embodiment of the present invention, a kit of parts for the treatment of solid tumors in a subject in need thereof is described, comprising the following elements: a) an effective dosage form of peptide fractions of the venom of the spider species Phoneutria nigriventer, b) an effective dosage form of an anthracycline, and c) instructions for use.

[0033] In a preferred embodiment of the present invention, the kit of parts for the treatment of solid tumors comprises approximately 1 pg / kg of LW-9 and approximately 5 mg / kg of a classic chemotherapeutic agent. Specifically, the present invention provides a kit of parts for the treatment of solid tumors, comprising approximately 1 pg / kg of LW-9 and... also, approximately 5 mg / kg of doxorubicin.

[0034] Regarding the kit of parts for the treatment of solid tumors and its instructions for use, the LW-9 peptide from a) must be kept in the freezer at a temperature of -20 °C, and when used, it must be reconstituted in saline solution and kept refrigerated at a temperature of -20 °C until application, which must occur within a period of 5 hours. The application must be performed intravenously (IV).

[0035] In turn, the anthracycline in b) of the parts kit, particularly doxorubicin, should be kept refrigerated at a temperature between 2 and 8 °C. It should be administered intravenously (IV) at a dosage of approximately 5 mg / kg. The LW-9 peptide should be kept in a freezer at -20 °C, removed from refrigeration, and then allowed to stand at room temperature for 15 minutes, with homogenization before use.

[0036] In a fifth embodiment of the present invention, a composition is described comprising a therapeutically effective amount of an LW-9 peptide fraction from the venom of the spider species Phoneutria nigriventer and a pharmaceutically acceptable carrier.

[0037] A sixth embodiment of the present invention describes the use of a therapeutically effective amount of an LW-9 peptide fraction from the venom of the spider species Phoneutria nigriventer in the manufacture of a composition and / or medicament for the treatment of solid tumors. BRIEF DESCRIPTION OF THE FIGURES

[0038] The figures described are presented for a complete and thorough understanding of the purpose of this invention.

[0039] Figure 1 shows a microscopic image of the EO771 murine mammary adenocarcinoma cell line.

[0040] Figure 2A presents a graph related to the purification of the LW-9 peptide by HPLC and analysis by ESI-Q-ToF spectrometry (Xevo GS, Waters CO.), in which the molecular mass of said peptide was determined.

[0041] Figure 2B shows the ion selected and fragmented by the collision with argon gas to generate an MS / MS mass spectrometry profile.

[0042] Figure 2C shows the ion selected and fragmented by the collision with argon gas to generate an MS / MS mass spectrometry profile.

[0043] Figure 3 shows in A a 2D model of the LW-9 peptide, and in B and C they show models of the 3D structure of the LW-9 peptide.

[0044] Figure 4 presents a diagram showing the LD50 and toxicity class of the LW-9 peptide. In Figure 4, the left-hand panel shows the 2D structure of the peptide. The right-hand panel shows a summary of the molecule's structural characteristics.

[0045] Figure 5 presents representative images of the mammary neoplasm developed in a control animal at the end of the experimental period (approximately 30 days after tumor inoculation). The tumors were measured with calipers every 48 hours to monitor tumor progression.

[0046] Figure 6 presents graphs related to the average weight (A) and tumor volume (B) measurements. The figures show that treatment with LW-9 1 pg / Kg induced significant tumor regression, both when administered Alone, as well as in combination with doxorubicin. LW-9 10 mg / kg had a lower result than the 1 mg / kg dose. *p < 0.05, **p < 0.01, compared to the untreated control group. Doxo = doxorubicin.

[0047] Figure 7 presents representative images of the tumors in each experimental group.

[0048] Figure 8 presents serum dosage graphs of hepatic and renal toxicity markers. Graphs A and B show the dosages of AST and ALT, respectively. Treatment with LW-9 at a dose of 10 mg / kg induced an increase in AST compared to the untreated control group. *p < 0.05, **p < 0.01, compared to the untreated control group. Graphs C and D show the dosages of Creatine and Urea, respectively.

[0049] Figure 9 presents graphs of average feed consumption (A) and weight variation (B) of the animals over the period. *p < 0.05, ***p < 0.001, compared to the untreated control; ##p < 0.01, ###p < 0.001, compared to the group treated only with doxorubicin.

[0050] Figure 10 presents flow cytometry graphs for the detection of immune system activation markers. Graphs A to C show a count of PDL-1+ (A), CD3+ / GITR+ (B), and CD3+ / GITR- (C) cells in the tumor microenvironment. Graphs D and E show a count of CD3+ / GITR+ (D) and CD3+ / GITR- (E) cells in the spleen. *p < 0.05, ***p < 0.001, compared to the untreated control; ##p < 0.01, ###p < 0.001, compared to the group treated with doxorubicin only.

[0051] Figure 11 presents flow cytometry graphs for the detection of activation markers of the system. immunological. Graphs A to C show a count of PDL-1+ (A), CD3+ / GITR+ (B), CD3+ / GITR- (C) cells in the tumor microenvironment. Graphs D and E show a count of CD3+ / GITR+ (D) and CD3+ / GITR- (E) cells in the spleen. *p < 0.05, ***p < 0.001, compared to the untreated control; ##p < 0.01, ###p < 0.001, compared to the group treated with doxorubicin (doxo) only. DETAILED DESCRIPTION OF THE INVENTION

[0052] Unless otherwise specified, the terms used throughout this descriptive report have their common meanings in the art, within the context of the disclosure, and in the specific context in which each term is used. Certain terms that are used to describe the disclosure are discussed below, or elsewhere in this descriptive report, to provide additional guidance to the technician regarding the description of the disclosure. Publications cited herein are specifically incorporated by reference in their entirety.

[0053] It will be appreciated that the same thing can be said in different ways. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed here. No special significance should be placed on whether a term is elaborated or discussed here. Synonyms for certain terms are provided, but the exemplification of some synonyms does not preclude the potential use of others not listed here.

[0054] In general, the present invention relates to a synergistic combination of a spider venom peptide, fraction LW-9, and classic chemotherapeutic agents, exemplified by doxorubicin, for use in the treatment of tumors. Solid tumors, such as breast tumors, and related kits.

[0055] Doxorubicin is a widely used medication in the treatment of various types of cancer. It is a chemotherapeutic agent from the anthracycline group, which are compounds with antitumor properties. Doxorubicin is one of the most commonly prescribed anthracyclines and has been used clinically for decades due to its effectiveness in treating various forms of cancer.

[0056] Doxorubicin works by inhibiting DNA replication and RNA synthesis in cancer cells. It does this by intercalating itself between the nitrogenous bases of DNA, which prevents cell division and leads to the death of cancer cells. In addition, doxorubicin also generates oxygen free radicals that can damage the DNA of cancer cells, further contributing to its cytotoxic effect.

[0057] Like other chemotherapeutic agents, doxorubicin can cause a variety of side effects. These may include nausea, vomiting, hair loss (alopecia), bone marrow suppression (resulting in anemia, neutropenia, and thrombocytopenia), heart damage (cardiotoxicity), oral mucositis, and skin reactions, among others. Cardiotoxicity is a particular concern with doxorubicin and can be dose-limiting. For this reason, seeking a more effective response in the treatment of solid tumors, especially breast tumors, as well as reducing the toxic effects of doxorubicin, the invention proposes the combination of doxorubicins and the LW-9 peptide fraction, which proved effective in treating a subject with the disease and also showed improvement in the patient's condition. The patient benefits from the reduction or even elimination of side effects resulting from the use of anthracyclines.

[0058] Regarding peptide fractions from spider venom, it is noteworthy that certain peptides have been explored for their potential therapeutic uses. For example, the LW-9 peptide fraction, a peptide of 1221.53 Da, was identified, characterized, and synthesized from screening performed using purified molecules from the venom of Phoneutria nigri venter.

[0059] A combination is understood to be an association of two or more active ingredients in the form of a product. The combination may be contained in a single form or in separate forms for simultaneous administration. In this sense, the present invention relates to a combination of the LW-9 fraction of the venom of the spider species Phoneutria nigriventer and classic chemotherapeutic agents, such as anthracyclines, which are presented in separate forms, however, for simultaneous administration, as evidenced by the working examples described in the application.

[0060] In this sense, the present invention describes a protocol for administering the LW-9 peptide fraction to a subject every 48 hours, in combination with a single dose of doxorubicin. This is because, due to its toxicity, doxorubicin administration is only recommended every 21 days. Thus, in the protocol disclosed in the present invention, for the treatment of solid tumors, the first dose of LW-9 is administered simultaneously with doxorubicin, with subsequent doses of LW-9 administered separately. In summary, the protocol of the present invention provides for doses of doxorubicin administered simultaneously with the LW-9 peptide fraction (a every 21 days) with maintenance using only LW-9 every 48 hours.

[0061] This is because, to date, satisfactory results have not been obtained for a single formulation comprising the LW-9 fraction and anthracyclines. In this sense, considering the complex immunomodulatory activity of the peptide, which activated macrophages, NK cells, DCs, and lymphocytes, the present invention describes a therapeutic strategy combining the LW-9 peptide fraction and anthracyclines, in which the said LW-9 peptide fraction is administered by systemic injection, acting as an immunoadjuvant in the treatment. The administration of said LW-9 peptide fraction can also occur alone, at a dose of approximately 1 ug / kg intravenously every 48 hours for 15 days (7 applications), or even in combination with a doxorubicin-type anthracycline.This strategy is capable of providing a more complete activation of the innate and adaptive immune system, as well as the drug's action on the tumor cells themselves, in addition to allowing for combined use with classic chemotherapeutic agents, enabling a multi-target strategy.

[0062] A multi-target strategy is a therapeutic approach in which multiple molecular targets are simultaneously targeted to treat a specific disease or condition. This strategy is especially relevant in the context of therapy development, where different targets may be involved in a complex pathological process. Therefore, the multi-target strategy is extremely important for cancer treatment, given that it involves a network of biological processes and molecular mechanisms. interconnected.

[0063] Therefore, to identify and characterize the LW-9 peptide as potentially relevant in the immunoadjuvant treatment of solid tumors, several experimental tests were performed to confirm the viability of these new molecules in relation to their chemotherapeutic effect. Characterization and synthesis of the peptide

[0064] After identifying the LW-9 fraction of the venom of the spider Phoneutria nigriventer, the peptide was purified by HPLC and analyzed by ESI-Q-ToF spectrometry (Xevo GS, Waters Co.). The sample was inserted into the spectrometer using a columnless liquid chromatography system, with elution using 50% acetonitrile in water containing 0.1% formic acid. Computational analyses (in silico) Molecular modeling

[0065] The three-dimensional structure of the PnTkP-VI peptide from Phoneutria nigriventer, not available in the PDB, was constructed using protein homology techniques. For this, the amino acid sequence (QKKDRFLGLM) of the LW-9 peptide was acquired from the UniProtKB database (https: / / www.uniprot.org / ), in FASTA format, located by code P86303. Then, the I-TASSER server (https: / / zhanggroup.org / I-TASSER / ) was used to predict the three-dimensional structure of the peptide by homology.

[0066] The visualization of the three-dimensional structure of the PnTkP-VI peptide from Phoneutria nigriventer in the models generated in I-TASSER was performed using the Bíovía Discovery Studio Visualizer program, version 21.1.0.20298. The program generates images of three-dimensional and conformational structures of the peptide. It offers a wide variety of methods that can be used to demonstrate and visualize the spatial differences of each amino acid present in the structure. Evaluation of the physicochemical, pharmacokinetic, and toxicological properties of the LW-9 peptide.

[0067] The physicochemical parameters, according to Lipinski's rule of 5 (Lipinski, 2000), were analyzed using the MarvinSketch version 23.12 tool available at the link (https: / / chemaxon.com / marvin), which are LogP, number of hydrogen donors and acceptors, and molecular mass.

[0068] For the evaluation of pharmacokinetic and toxicological properties, the web-based program PreADMET, available at the link (https: / / preadmet.bmdrc.kr / ), was used, which allows for the assessment of the absorption, distribution, metabolism, excretion (ADME), and toxicity of the peptide. The pharmacokinetic parameters evaluated were BBB (blood-brain barrier penetration), Caco2 (in vitro cell permeability nm / s), CYP_2C19 (in vitro inhibition of CYP 2019), CYP_2C9 (in vitro inhibition of CYP 2C9), CYP_2D6 (in vitro inhibition of CYP 2D6), CYP_2D6_substrate (in vitro CYP 2D6 substrate), CYP_3A4 (in vitro inhibition of CYP_3A4), HIA (human intestinal absorption), MDCK (in vitro cell permeability of MDCK nm / s), Pgp (in vitro inhibition of P-glycoprotein), and Plasma Protein Binding.

[0069] The toxicological parameters analyzed were: Carcino mouse (Carcinogenicity in mice), Carcino rat (Carcinogenicity in rats), hERG inhibition (inhibition). hERG in vitro), TA100 10RLI (result of the in vitro Ames test on the TA100 strain), TA100_NA (result of the in vitro Ames test on the TA100 strain without metabolic activation), TA1535 lORLI (result of the in vitro Ames test on the TA1535 strain with metabolic activation by rat liver homogenate), TA1535 NA (result of the in vitro Ames test on the TA1535 strain without metabolic activation). Analysis of LD50

[0070] Estimates of the LD50 (median lethal dose) and the probability of the LW-9 peptide causing hepatotoxicity, immunotoxicity, and cytotoxicity were performed using the ProTox-2 platform available at the link (https: / / tox-new.charite.de / protox_II / ). Expansion and maintenance of the murine mammary carcinoma lineage (EO771)

[0071] Murine mammary adenocarcinoma cells (EO771) (Figure 1) were thawed and maintained in Iscove's Modified Dulbecco's Medium (IMDM) culture medium supplemented with 10% fetal bovine serum (FBS), 50 mg / mL of Penicillin and Streptomycin (Gibco Inc., Billings, MT, USA), in an incubator at 37 °C containing 5% CO2. Animals

[0072] The assays were performed on adult (8 weeks old) C57BL / 6 (C-Black) females obtained from the Multidisciplinary Center for Biological Research in the Area of ​​Laboratory Animal Science (CEMIB - UNICAMP) and maintained in a colony established in the Animal Bioterium of the Immunology Area, Department of Structural and Functional Biology, Institute of Biology / UNICAMP. The animals remained in microisolator-type cages, with water and Food was provided ad libitum and the light / dark cycle was 12 / 12 hours. The experimental protocol followed the 5 principles of the Brazilian College of Animal Experimentation (COBEA) and was approved by the Ethics Committee on the Use of Animals (CEUA / UNI CAMP; 6175 / 2023). Tumor cell inoculation

[0073] Murine mammary adenocarcinoma cells (EO771) were cultured and, after 90% confluence, were released using a cell scraper. The cells were washed 3 times with 1 M phosphate-buffered saline (PBS), counted, and diluted in the same solution at a concentration of 1.5 x 10⁵ in 50 µL, and then inoculated into the 4th o Left mammary gland (via intradermal injection). Examples of implementation Treatment and analysis of clinical parameters in animals with tumors.

[0074] The animals were monitored for tumor progression in response to treatments for 30 days. Primary tumors were measured every 48 hours using a caliper. The treatments described below were initiated after the appearance of a 0.2 mm diameter tumor mass (approximately 15 to 20 days after tumor inoculation), a protocol that most closely simulates the moment when breast cancer diagnosis and treatment initiation occur. Treatment was carried out for 15 days; therefore, all animals were euthanized approximately 30 days after tumor cell inoculation. The doses used for LW-9 were based on previous studies (an estimate of blood concentration was made based on concentrations previously used in experimental models). Doxorubicin was administered as a single dose (5 mg / kg) on ​​the first day of treatment (along with the first dose of LW-9). The dosage used was based on the use of the drug in veterinary patients with breast cancer (one dose of doxorubicin every 30 days), as well as in the literature (Ewens et al., 2006). All groups described below received inoculation of tumor cells (Table 1). Table 1 - Treatment groups

[0075] The following clinical parameters were evaluated (animal welfare and treatment efficacy): 1. Feed consumption (weighing the feed); 2. Weight gain or loss (weighing the animal); 3. Overall survival and disease-free survival; 4. Presence of pain (signs such as: hunched posture, little movement, half-closed eyes, tremors, goosebumps); 5. Tumor progression (measurement of the tumor with Caliper measurement and, at the end of the experiment, weighing and measurements of the dissected tumor; appearance of metastasis or ascites).

[0076] In turn, the following toxicity parameters were evaluated: 1. Weighing and macroscopic and microscopic evaluation of organs (liver, kidneys, lungs, and spleen); 2. Analysis of blood parameters (complete blood count and measurement of liver function markers (AST and ALT) and kidney function markers (creatinine and urea)). Laboratory analyses and histopathology

[0077] Thirty days after tumor inoculation, the animals were euthanized using an overdose of anesthetic (ketamine 300 mg / kg and xylazine 30 mg / kg), and biological material (blood and organs) was immediately collected for analysis. Tumor volume was calculated according to Weirich (Provenzi et al., 2015), using the ellipsoid formula (volume calculation for solid tumors): length x depth x thickness x 0.523. Blood was collected in heparinized tubes, which were sent for analysis to LABCARE - Veterinary Laboratory (Campinas, SP); complete blood count and measurements of AST and ALT (liver function), creatinine and urea (renal function) were performed.

[0078] Kidneys, liver, spleen, and mammary tumor were dissected, weighed, measured, and processed for paraffin embedding and histopathological analysis. The samples were placed in a 50 mL Falcon tube containing 10% formaldehyde, covering the sample. This container was labeled with the animal's identification and the date of collection. After 48 hours of fixation, the material was processed for paraffin embedding. Briefly: fragments of approximately 3 cm. The fragments were washed under running water and transferred to 70% ethanol for several hours. Then, the fragments were immersed in an increasing gradient of ethanol (70% to 100%) and subsequently cleared in xylene and impregnated with histological paraffin (Paraplast, Sigma-Aldrich) in an oven at 58°C. Using slightly heated forceps, the fragments were placed in the paraffin-filled embedding mold. The molds were then placed on a surface at room temperature and, after drying, were unblocked.

[0079] For histopathological analysis, 5 µm thick sections of the material were cut using a microtome, followed by hematoxylin-eosin staining and observation under a light microscope. Analysis of cell populations in the spleen and TME by flow cytometry.

[0080] For flow cytometry analyses, animals were euthanized by anesthetic overdose (300 mg / kg ketamine + 30 mg / kg xylazine, i.p.) and the primary tumor and spleen were dissected. To obtain a cell suspension, biological material was removed from the animals and transferred to Petri dishes containing serum-free medium, macerated using a steel sieve, and centrifuged in tubes with a 70 µm cell strainer. After collecting the suspension in the tube, red blood cells were lysed using cell lysis buffer - ACK (ammonium chloride-potassium). The contents were then centrifuged (1500 rpm, 5 min, 4°C) and the pellet was resuspended using 1 mL of IMDM medium supplemented with FBS and kept refrigerated. After viability detection, using the method of... Trypan blue was used to plate the cells in 2 x 10 5Cells per well were prepared in triplicate using a 96-well plate. Immunostaining was then performed with the following antibodies: anti-Ki67 (#SolA15), anti-CD3 (#145-2C11), anti-GITR (#DTA-1), and anti-PD-Ll (#CD274) (incubation for 30 min at 4°C in a dark chamber). All antibodies used were from eBioscience (USA) or Abeam (USA). The preparations (20,000 cells) were acquired using a flow cytometer (BD FACSVerse, Department of Genetics, Evolution and Bioagents, Institute of Biology, UNICAMP) and the data were analyzed using the FlowJo® VX program (Tree Star Inc., Ashland, OR). Statistical analysis

[0081] The significance level was analyzed using the one-way ANOVA or two-way ANOVA test, followed by Dunnett's or Tukey's multiple comparisons test. The t-test was used to compare each treatment with the control. The result was expressed as the mean ± standard error. Analyses were performed using GraphPad 8.0 (GraphPad Software Inc., San Diego, CA) and Stata 11.1 (StataCorp, College Station, TX, USA). Results

[0082] Biochemical analysis by mass spectrometry revealed that the peptide has the amino acid sequence: PyrKKDRFLGLM-CONH2 (Figure 2A). The molecular mass of the peptide was determined using MS mode (Figure 2A), and this ion was selected and fragmented by argon gas collision to generate an MS / MS profile (Figure 2B and Figure 2C). Analysis of the fragments allowed for sequencing of the... The peptide and its identification, SEQ ID NO:1, were determined by comparison with peptides isolated from venoms of the same spider species already described, which exhibit exactly the same fragmentation profile.

[0083] The synthesis was performed by the company Aminotech (Purity equal to or greater than 98% in HPLC).

[0084] Through computational analyses, 2D and 3D visualizations of the LW-9 peptide structure were assembled using molecular modeling techniques. It was possible to observe that the molecule has a strong polar characteristic present in the amino acids that compose it (Figure 3).

[0085] For a molecule to be administered orally, it must meet the parameters of Lipinski's rule or the rule of five (Santos et al., 2018). The molecule must consist of up to 5 hydrogen bond donors, 10 hydrogen bond acceptors, a maximum molecular mass of 500 g / mol, and an octanol-water partition coefficient (LogP) between 0 and 5. (Lipinski, 2000). According to the rules established by Lipinski, the LW-9 peptide violated the number of hydrogen donors, number of hydrogen acceptors and molar mass above 500 Da, the LogP is in accordance with the rule, however it presents a negative value, which shows a hydrophilic and polar profile of this structure (Table 2). Table 2 - Evaluation of the physicochemical parameters of the LW-9 peptide Hydrogen donors Hydrogen acceptors Spring mass 1221.53

[0086] The first stage of pharmacokinetics is drug absorption, a process that depends on the substance's ability to overcome biological barriers such as the gastrointestinal tract (Souza et al., 2007). Analysis of absorption revealed that the LW-9 peptide does not exhibit a good rate of gastrointestinal absorption, as well as low permeability through the blood-brain barrier (Table 3). Table 3 - Evaluation of the absorption, distribution and excretion parameters of the LW-9 peptide. BBB (permeability through the blood-brain barrier), CACo2 (in vitro cell permeability), HIA (human intestinal absorption), MDCK (in vitro cell permeability in canine kidney), PgP (in vitro inhibition of P-glycoprotein), PBP (plasma protein binding).

[0087] Furthermore, the permeability of the peptide in vitro in human colorectal carcinoma cells also demonstrated low potential. Regarding P-glycoprotein (P-gp) activity, the LW-9 peptide did not inhibit its activity. P-gp is an efflux transporter responsible for removing substances from cells (Huber et al., 2010). An important role of P-gp is to protect the central nervous system from xenobiotics (Szakács et al., 2008). The inability to inhibit P-gp is further evidence suggesting low oral absorption and poor passage through the blood-brain barrier. MDCK is another parameter that assesses the permeability of the molecule in canine kidney cells in vitro; Similarly, the LW-9 peptide demonstrated low permeability potential due to its polarity in water. Given the results suggesting low absorption of the peptide, the intraperitoneal route was chosen for administration in preclinical experiments.

[0088] PBP is a characteristic that, within pharmacokinetics, assesses the distribution and degree of binding to plasma proteins. The LW-9 peptide demonstrates a low rate of binding to plasma proteins, which suggests good distribution (high apparent volume of distribution).

[0089] Metabolism is a very important phase of the pharmacokinetic process; therefore, it is essential to understand the interactions of the LW-9 peptide with cytochrome P450 isoenzymes: CYP2C19 (I), CYP2C9 (I), CYP2D6 (I), CYP2D6 (S), CYP3A4 (I), and CYP3A4 (S). These enzymes are responsible for metabolizing chemical substances. By adding chemical groups to activate or inactivate drugs, the inhibition of these enzymes may cause drug interactions (Audi and Pussi, 2000). The LW-9 peptide has been shown to inhibit GYP 2C19 and CYP3A4, and may interact with the metabolism of other drugs (Table 4). Table 4 - Evaluation of metabolism parameters in the CYP450 isoenzymes of the LW-9 peptide. (-) Negative for inhibition of cytochrome P450 isoenzymes, (+) Positive for inhibition of cytochrome P450 isoenzymes GYP 2C19 (I) (in vitro inhibition of CYP 2C19), CYP 2C9 (I) (in vitro inhibition of CYP 2C9), CYP 2D6 (I) (in vitro inhibition of CYP 2D6), CYP 2D6 (S) (in vitro CYP 2D6 substrate), CYP 3A4 (I) (in vitro inhibition of CYP 3A4), CYP 3A4 (S) (in vitro CYP 3A4 substrate).

[0090] In general, carcinogenicity studies are conducted on organisms with metabolisms as similar as possible to that of humans, as recommended by the Brazilian National Health Surveillance Agency (ANVISA). Table 5 shows that the LW-9 peptide did not exhibit carcinogenicity in mice and rats. Since this is a non-clinical study, in silico analysis does not guarantee complete pharmacological and toxicokinetic safety of a drug, making it necessary to conduct further studies with the test substance. In vitro and in vivo carcinogenicity and mutagenicity studies will be performed later. However, regarding the in silico prediction data performed in PreADMET, the use of data from the National Toxicology Program (NTP) and the Food and Drug Administration (EDA), through the application of algorithms, provides greater reliability to the results.Furthermore, the application of the server enables the reduction of costs and analysis time (Jónsdóttir et al., 2005; Benfenati, 2016; Efinger et al., 2018). Table 5 - Evaluation of toxicological parameters of the Lw-9 peptide. Carcino mouse (negative for carcinogenicity in mice), Carcino rat (negative for carcinogenicity in rats), hERG inhibition (*) (low risk for hERG gene channel inhibition in vitro), (**) medium risk for hERG gene channel inhibition in vitro), (***) high risk for hERG gene channel inhibition in vitro.

[0091] Regarding the toxicological parameters presented in Table 5, the PreADMET server predicts hERG inhibition. This parameter allows for the evaluation of the influence of the drug under test on cardiac function, due to the ability of the hERG gene to block the voltage-gated potassium channel, which is essential for the action potential in the heart. Thus, by inhibiting the coding of the channel, the repolarization phase of the action potential is slowed down, resulting in a prolonged QT interval and potentially leading to cardiac problems such as heart failure and arrhythmias (Jing et al., 2015). The LW-9 peptide showed ambiguity in hERG inhibition, i.e., an undefined response to hERG. The aforementioned classification is unclear regarding the potential risk and cannot, on its own, exclude or confirm cardiac risk; it should be complemented with other tests.

[0092] The data presented in Table 6 show some results regarding the possibility of mutagenicity induced by the LW-9 peptide. The LW-9 peptide did not inhibit any strain tested in the in silico study, which represents an excellent safety margin in this respect. The Ames test is potentially relevant when considering the mutagenic characteristics of a given substance. This test is capable of detecting mutations in the genetic material involved in the synthesis of... The amino acid histidine (Kauffmann et al., 2020). For the test, Salmonella typhimurium strains are used to check for the possibility of mutagenicity, which is a widely accepted test (Zeiger, 2019). Table 6 - Analysis of the in silico Ames test of the LW-9 peptide. TA 100 (-S9) (negative for the in vitro Ames test in the TA100 strain without metabolic activation), TA 100 (+S9) (negative and positive for the in vitro Ames test in the TA100 strain with metabolic activation by rat liver homogenate), TA 1535 (-S9) (negative for the in vitro Ames test in the TA1535 strain without metabolic activation), TA 1535 (+S9) (positive for the in vitro Ames test in the TA1535 strain with metabolic activation by rat liver homogenate).

[0093] The Ames test is based on the use of indicator strains of S. typhimurium, sensitive to substances capable of inducing different types of mutation. In the presence of mutagenic agents, these strains revert their auxotrophic character to histidine synthesis and begin to form colonies in a medium devoid of this amino acid (Moreira et al., 2002). As can be seen in Table 6, the strains used in the Ames test performed in the PreADMET program (TA 100 and TA 1535) are in agreement with the genotoxicity strains, giving the test greater reliability. However, the guideline recommends the use of 5 distinct strains. Each strain tested in the Ames test suggests a different interpretation.

[0094] According to Grandis (2016), the TA100 and TA1535 strains are directly related to mutations in the guanine-cytosine pair. The first detects the presence of mutagenic agents capable of generating base pair substitutions in the genetic material. The second represents a specific substitution in the pair, resulting from a methylation reaction. In the test, the strains are represented according to the presence or absence of the S9 fraction, being identified, respectively, by (+S9) and (-S9). This fraction simulates metabolic conditions and is composed of microsomal and cytosolic hepatic fractions (Calixto, 2012). Thus, by performing the test in the presence of the S9 fraction, it is possible to test the mutagenicity of the metabolites produced by each substance submitted to the test, by simulating metabolic activation by the rat liver. It should be noted that, despite the sensitivity of the Ames test, complementary tests should be performed, and it cannot, alone, exclude or confirm genotoxicity.

[0095] ProTox-II is a virtual laboratory for predicting the toxicities of small molecules. Predicting compound toxicities is an important part of the drug design and development process. Computational toxicity estimates are not only faster than determining toxic doses in animals, but they can also help reduce the amount of animal experimentation.

[0096] ProTox-II incorporates molecular similarity, fragment propensities, most frequent features, and machine learning (cross-validation CLUSTER based on fragment similarity), based on a total of 33 Models for predicting various toxicity endpoints, such as acute toxicity, hepatotoxicity, cytotoxicity, carcinogenicity, mutagenicity, immunotoxicity, adverse outcome pathways (Tox21), and toxicity targets.

[0097] According to ProTox-II, the LW-9 peptide showed an excellent LD50 of 1500 mg / kg. Furthermore, its classification is class 4 with a good safety margin, allowing for testing of different doses of the molecule, as shown in Figure 4.

[0098] Animals inoculated with neoplastic cells developed a detectable tumor mass between 15 and 20 days after inoculation. The onset of development was homogeneous in all animals, which were randomly divided into treatment groups after the detection of a 0.2 mm tumor mass. Therefore, there was little variation in the timing of treatment initiation, as the day after cell inoculation was not considered, but rather the appearance of a tumor mass with the aforementioned dimensions. In this sense, Figure 5 shows representative images of the final tumor mass (around 30 days after cell inoculation) in the untreated control group.

[0099] At the end of the experimental period, the animals were euthanized and the tumor was dissected, weighed, and measured to calculate the volume. In this context, Figure 6 shows the comparison between the measurements (A - tumor weight; B - tumor volume). Treatment with the positive control, doxorubicin, did not significantly reduce tumor measurements. This may be due to the treatment regimen (single dose and euthanasia 15 days after administration). In veterinary tumors, three administrations of doxorubicin were performed, with an interval of 20 to 30 days between them. Treatment with LW-9 (dose of 1 pg / kg, i.p., every 48 hours) promoted significant tumor regression compared to the untreated control. The combination of doxorubicin and LW-9 1 pg / kg also induced a significant reduction, significantly greater than LW-9 alone in relation to tumor volume. Interestingly, LW-9 at a dose of 10 pg / kg had a lower result than the dose of 1 pg / kg, indicating that there may be saturation of the mechanism involved and / or induction of resistance mechanisms. In this sense, Figure 7 shows representative images of the tumors from each experimental group.

[0100] Analysis of liver and kidney toxicity markers (Figure 8) showed that the only treatment that induced toxicity was LW-9 10 pg / kg. There was an increase in AST and a reduction in urea compared to the untreated control. Even though this dose was 10 times higher than the dose considered therapeutic in the present study, this result points to the need for further investigations regarding liver and kidney damage caused by the peptide.

[0101] The animal welfare analysis included weighing the feed to assess average consumption during the experimental period, graph A (Figure 9), as well as the animals' weight throughout the period, graph B (Figure 9). The results demonstrated that animals treated with doxorubicin had a reduction in feed consumption compared to the untreated control group, which is expected due to the adverse effects of this drug on the gastrointestinal tract (Carvalho et al., 2009). Animals treated with LW-9 1 pg / Kg did not have altered consumption. Regarding the control group, feed consumption was significantly higher compared to the group treated with doxorubicin. The combination of the two treatments (LW-9 1 pg / Kg + doxorubicin) induced a reduction in feed consumption compared to the untreated control group; however, consumption was significantly higher compared to the group treated only with doxorubicin, indicating that the side effects were possibly attenuated by the combination of drugs. Interestingly, treatment with the 10 times higher dose of LW-9 (10 pg / Kg) also induced a significant reduction in average feed consumption compared to the untreated control group.

[0102] The animals' weight varied greatly throughout the experimental period in all groups, except for the group treated with the drug combination, which remained stable. At the end of the 30-day follow-up, the untreated control group, the group treated with doxorubicin, and the group treated with LW-9 10 pg / kg showed the greatest weight loss, while the group treated with LW-9 1 pg / kg showed the greatest weight gain (significantly greater than the control and doxorubicin groups). The group that received the combination (LW-9 1 pg / kg + doxorubicin) had the most stable weight throughout the treatment, showing neither loss nor gain.

[0103] Flow cytometry results, relating to the count of cells positive for the markers (Figure 10), demonstrated that treatment with LW-9 1 pg / Kg + doxorubicin induced a significant reduction in cells expressing PDL-1, graph A (Figure 9) in the tumor microenvironment, compared to the untreated control. PDL-1 present in tumor cells, when combined with its ligand, PD-1 in immune system cells, promotes the inhibition of... The immune response, self-tolerance, inhibits apoptosis of regulatory T cells (T-reg), and has been considered a limiting factor in the effectiveness of immunotherapies (Han et al, 2020). Therefore, the reduction of PD-L1 by the combined therapy is indicative of a good treatment prognosis and the efficiency of immune activation. Corroborating this finding, therapy with LW-9 1 pg / Kg + doxorubicin also increased the number of cells doubly expressing CD3 and GITR (10, B) and expressing only CD3 (10, C) in the tumor microenvironment. The increase in CD3 indicates greater lymphocyte activation, and GITR (Glucocorticoid-induced TNF receptor) has been associated with a good cancer prognosis. GITR is a type II transmembrane receptor, a member of the TNFR superfamily, constitutively expressed on Treg cells and induced on activated CD8+ and CD4+ T cells (Burugu et al., 2018). The binding of GITR to GITR-L (expressed on antigen-presenting cells) inhibits Treg activity (Coe et al.)., 2010; Ainda et al. , 2014) while stimulating effector T cells (Knee et al. , 2016), making GITR activation an attractive strategy for cancer immunotherapy. The reduction of PD-L1 along with the increase in GITR suggests that LW-9 may be a molecule with great immunomodulatory potential for cancer treatment.

[0104] Surprisingly, there was no increase in markers in the spleen, suggesting that there is specific immunoactivation in the tumor environment, a positive aspect for the effectiveness of the therapy, without inducing side effects resulting from an overactivation of the immune response, which can cause cytokine storm, inflammation, and autoimmunity.

[0105] Figure 11 shows the MFI (Intensity Mean Fluorescence) of the markers (i.e., the total expression of the markers in the sample, and not the number of cells expressing them). These results confirm the increased expression of the markers by the combined treatment (LW-9 1 pg / Kg + doxorubicin) in the tumor microenvironment, compared to the untreated control, graphs A to C (Figure 11). Furthermore, in the case of MFI, treatment with LW-9 1 pg / Kg alone also promoted an increase in CD3 and GITR in the tumor microenvironment, graphs A and B (Figure 11). Corroborating the positive cell count data, which indicate local activation in the tumor microenvironment (and not in circulating cells), in the spleen there was a reduction in MFI and CD3, graphs D and E (Figure 11), but there was an increase in GITR, graph F (Figure 11).

[0106] The present invention is defined herein in terms of its preferred embodiment. Nevertheless, a person skilled in the art is perfectly capable of observing that modifications may be made to the information described herein, such modifications still being covered by the same scope of the subject matter described and claimed.

Claims

CLAIMS 1. Combination characterized by comprising anthracyclines and peptide fractions from the venom of the spider species Phoneutria nigriventer.

2. Combination according to claim 1, characterized in that the peptide fraction is LW-9, with an amino acid sequence corresponding to sequence SEQ ID NO: 1, of 1221.53 Da.

3. Combination according to claims 1 and 2, characterized in that the anthracycline is doxorubicin.

4. Combination, according to any one of claims 1 to 3, characterized in that the LW-9 peptide is present in a concentration of about 1 ug / kg and doxorubicin is present in a concentration of about 5 mg / kg.

5. Combination, according to any one of claims 1 to 4, characterized by being in the treatment of solid tumors.

6. Combination, according to any one of claims 1 to 5, characterized by being used in the treatment of breast cancer.

7. Combination, according to any one of claims 1 to 6, characterized in that it is prepared for intraperitoneal administration.

8. Kit of parts for the treatment of solid tumors, characterized by comprising the following elements: (a) an effective dosage form of peptide fractions of the venom of the spider species Phoneutria nigríventer, (b) an effective dosage form of an anthracycline, (c) instructions for use.

9. Kit according to claim 8, characterized in that the peptide fraction of a) is the LW-9 fraction and the anthracycline of b) is doxorubicin.

10. Kit, according to claim 8 or 9, characterized in that it comprises about 1 ug / kg of LW-9.

11. Kit, according to claim 8 or 9, characterized in that it comprises approximately 5 mg / kg of doxorubicin.

12. Use of anthracyclines and peptide fractions from the venom of the spider species Phoneutria nigriventer, characterized by the fact that they are used in the manufacture of a combination and / or kit for the treatment of solid tumors.

13. Use according to claim 12, characterized in that the solid tumor is a breast tumor.

14. Use according to claim 12, characterized in that the combination and / or kit is prepared for intraperitoneal administration.

15. Composition characterized by comprising the LW-9 peptide fraction and a pharmaceutically acceptable carrier.

16. Use of LW-9 peptide fraction from spider venom of the species Phoneutria nigriventer, characterized by its use in the manufacture of a composition, as defined in claim 15, for the treatment of solid tumors.

17. Use according to claim 16, characterized in that the solid tumor is a tumor. mammary.

18. Use according to claim 16 or 17, characterized in that the composition is prepared for intraperitoneal administration.

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