Pharmaceutical compositions based on different fractions of heteroctenus junceus scorpion venom, method and uses

Abstract

The invention relates to a composition used as an anti-carcinogenic agent having low cardiotoxicity and high antimicrobial stability. The composition comprises at least one fraction of Heteroctenus junceus scorpion venom, wherein said fraction can be selected from the list comprising: F4, F5.3 and any recognisable and identifiable combination of the two, wherein both fractions have an anti-carcinogenic effect, which is mainly associated with the total or partial inhibition of tumour progression.

Description

[0001] PHARMACEUTICAL COMPOSITIONS BASED ON DIFFERENT FRACTIONS OF THE VENOM OF THE SCORPION HETEROCTENUS JUNCEUS, METHOD AND USES.

[0002] Field of invention

[0003] The present invention is generally related to the fields of biochemistry, molecular biology, pharmacy, pharmacology, and physiology. Specifically, this disclosure is a novel formulation derived from the venom of the scorpion Heteroctenus junceus (formerly known as Rhopalurus junceus) for the treatment of pathological conditions, particularly as an anticancer agent against tumors of epithelial origin, with reduced toxicity to human hNav1.5 ion channels, which are responsible for initiating the action potential in cardiomyocytes. The methods and techniques used to obtain and develop the formulation, as well as the in vitro experimental results, are described.

[0004] Background of the invention

[0005] The background to this invention includes prior knowledge of the existence of ion channels, which form pores in the cell membrane and allow the passive flow of ions in the direction of their electrochemical gradient, generating electrical currents. The relevant physiological role of ion channels lies in their participation in cellular processes such as membrane potential generation, signal transduction, neurotransmitter release, muscle contraction, hormone secretion, volume regulation, growth, motility, migration, proliferation, and apoptosis, among others. Given these characteristics, it is relevant that variations in the expression of some of these channels can lead to the development of pathological processes such as cancer.

[0006] Cancer represents one of humanity's major health problems, currently responsible for more than 9.7 million deaths each year. Genomic instability is a primary cause and a fundamental characteristic of human cancer.

[0007] One of the genes that increases genetic instability is that of ion channels, contributing to the transition to a more aggressive cancer phenotype. Numerous lines of evidence have accumulated showing that the overexpression of these ion channels plays a particularly prominent role in key cancer features such as unrestricted proliferation, evasion of apoptosis, neovascularization, tissue invasion, and metastasis (Prevarskaya N, Skryma R, Shuba Y. Ion Channels in Cancer: Are Cancer Hallmarks Oncochannelopathies? Physiol Rev. 2018; 98(2):559-621). Overexpression of the Kv1.1 channel has been identified in several cancer types and is associated with proliferation, migration, cell signaling, and other processes.This characteristic makes it possible to use it as a target to reduce or inhibit its activity and therefore the characteristics associated with the development of cancer (Ouadid-Ahidouch H, Chaussade F, Roudbaraki M, Slomianny C, Dewailly E, Delcourt P, Prevarskaya N. KV1.1 K(+) channels identification in human breast carcinoma cells: involvement in cell proliferation. Biochem Biophys Res Commun. 2000; 278(2):272-277).

[0008] All peptides from scorpion venoms that act on potassium channels, identified to date, are known to have between 30 and 40 amino acids with 3 to 4 disulfide bridges, allowing for a compact conformation. Studies conducted in different laboratories have identified these peptides as channel pore blockers. These peptides act by binding to the outer vestibule of the channel and blocking ion conduction by physically occluding the pore (Garcia ML, Gao Y, McManus OB, Kaczorowski GJ. Potassium channels: from scorpion venoms to high-resolution structure. Toxicon. 2001; 39(6):739-748).

[0009] H. junceus, similar to other scorpion venoms, contains a mixture of peptides with molecular weights between 3 kDa and 90 kDa, which constitute the major component, along with proteins, amino acids, and free amines. Under natural conditions, scorpion venom is an opalescent fluid with a pH of 7.12, which also contains mucus, lipids, carbohydrates, inorganic salts, and low molecular weight organic molecules. Peptides present in scorpion venoms have been shown to possess a wide variety of pharmacological properties, including particularly anticancer properties (Ding J, Chua PJ, Bay BH, Gopalakrishnakone P. Scorpion venoms as a potential source of novel cancer therapeutic compounds. Exp Biol Med (Maywood). 2014; 239(4):387-393; Raposo C. Scorpion and spider venoms in cancer treatment: state of the art, challenges, and perspectives. J Clin Transí Res. 2017; 3(2):233-249).

[0010] Several patents describe the anticancer properties of the venom of the scorpion H. junceus. The first patent appeared in the 1990s, describing the extraction of the venom directly from scorpions and the preparation of a solution of H. junceus venom diluted in water. Its antitumor activity was empirically demonstrated by administering this solution to domestic animals with spontaneous tumors, and a reduction in tumor size was observed (CU 22413 A1).

[0011] Additionally, in 2009 a patent was filed that uses the same solution of H. junceus scorpion venom described previously, the novelty of which lies in the fact that it was subjected to a polarization process and used in the treatment of people with cancer (US 2009 / 0123558). In the first two cases, although it maintained its natural state, there was no pharmaceutical development; the results were empirical, based on the same therapeutic properties known from centuries of traditional medicine use of this natural product.

[0012] In 2011, a third patent was described that scientifically demonstrated the anti-inflammatory, analgesic, and anticancer properties of the venom of the scorpion H. junceus. This patent differs substantially from previous patents by describing, through experimental evidence, the potential therapeutic effects of this scorpion venom in humans, using in vitro and in vivo experimental models (WO / 2012 / 041261). Furthermore, this patent, for the first time, describes the involvement of several peptides present in the venom as responsible for these effects. However, in all patents to date, the formulations remain a mixture of scorpion venom in water, obtained using artisanal methods, which could lead to potential contamination of the formulation.

[0013] On the other hand, the occurrence of cardiotoxic effects in scorpion venoms has been reported, due to the presence of toxins that interact with ion channels present in the heart (Aboumaád B, Tiger A, Khattabi A, Soulaymani R, Lahssaini M, Benhassain SM, Iba N. Cardiac involvement and its complications about three cases of severe scorpion envenomation. Toxicon. 2014; 78:78-82; In particular, toxins that act on the adult human sodium channel subtype 1.5 (hNav1.5), responsible for the initiation of the action potential in cardiomyocytes, have been described (Jiang et al., 2021; Saucedo et al., 2012).Toxins that interact with this channel are classified into two types: α-NaTx, which bind to domain IV of the sodium channel and delay the rapid inactivation of the channel current, and p-NaTx, which bind to domain II of the sodium channel and modify its activation, generally causing a reduction in the peak channel current (Quintero-Hernández V et al., 2013). The effect of toxins on the hNav1.5 channel (Jalali A et al., 2005; Saucedo AL et al., 2012) is partly responsible for the cardiotoxic effects induced by scorpion venoms. Damage to this hNav1.5 ion channel is associated with the development of pathologies such as long QT syndrome, Brugada syndrome, atrial fibrillation, and ventricular dysplasia, among others (Dong C et al., 2020). Therefore, it is evident from the previous state of the art that there are no prior studies that have identified the presence of these toxins in the venom of the scorpion R. junceus.The development of new pharmaceutical compositions, using scorpion venom as an active component and its use in humans, requires the identification and elimination of the presence of these toxins, which will improve pharmacological safety, reduce toxicological risks and increase the stability of the formulation, allowing the potential use of other routes of administration.

[0014] Summary of the Invention

[0015] The invention relates to a composition used as an anticancer agent with reduced cardiac toxicity and high antimicrobial stability, comprising at least one fraction of the venom of the scorpion Heteroctenus junceus, wherein said fraction may be selected from the list comprising: F4, F5.3, and any combination thereof, recognizable and identifiable, wherein both fractions exhibit an anticancer effect, primarily associated with the total or partial inhibition of tumor progression. Description of the Invention

[0016] The invention relates to a liquid formulation used as an anticancer agent with reduced cardiac toxicity and high antimicrobial stability, comprising: a) at least a fraction of the venom of the scorpion Heteroctenus junceus (0.5-2 mg / mL); b) preservatives represented by at least methylparaben (0.05%) and propylparaben (0.02%) w / w; c) distilled water; and d) optionally, other pharmaceutical excipients.

[0017] The invention comprises at least two recognizable and identifiable fractions (F4 and F5.3), where both fractions exhibit an anticancer effect, primarily associated with the total or partial inhibition of tumor progression.

[0018] In one embodiment of the invention, the first fraction or F4 comprises at least two identifiable peptides, wherein at least one of these peptides is the peptide code Uniprot A0A1 U9WSZ2, and wherein the second fraction comprises at least three identifiable peptides, wherein at least one of these peptides is the peptide code UNIPROT C0HJT0.

[0019] In one embodiment of the invention, it comprises a formulation containing at least the first fraction (F4), characterized in that it contains at least one I-listone 3a (as a fraction marker) and wherein said formulation is effective in the treatment of patients with cancers of epithelial origin, particularly non-small cell lung cancer.

[0020] In another embodiment of the invention, it comprises a formulation containing a mixture of fractions (F4 and F5.3) at different concentrations, characterized in that said mixture has an enhanced effect with anticancer activity in the treatment of patients with cancers of epithelial origin and who suffer from other chronic or acute diseases, including coronary diseases.

[0021] In another embodiment of the invention, it comprises a formulation containing at least a fraction of the list comprising: F4, F5.3 and the reconstituted poison, for the treatment of cancer patients and those with pre-existing chronic diseases, including coronary and respiratory diseases.

[0022] In one embodiment of the invention, the isolated F4 and F5.3 fractions have an acidic pH (~1.0 to 3.0), the isolated F8.3 and F9 fractions (fractions considered toxic, since they affect the hNav1.5 ion channel) have a physiological pH (~6.0 to 7.0), and additionally, the sum of the F4 and F5.3 fractions corresponds to only 1% of the composition of the complete venom; and wherein, furthermore, the residue or resulting composition of the venom (approximately the remaining 99%) reduces its pH from physiological pH (6-7) to an acidic pH (pH of ~1.0 to 3.0). Where the pH stabilization role would allow to ensure a biological functioning of the peptides and components present in the venom and / or in any of its fractions with antitumor activity, by at least one of the fractions isolated from the original venom, which can be selected from the list comprising: fraction F4, fraction F5.3 and a mixture of both, as part of the proposed invention.

[0023] In one embodiment of the invention, the components of the isolated F4 and F5.3 fractions maintain their structure (Figure 2), resulting in resistance / independent to environments with acidic pH and / or physiological pH, maintaining their antitumor action, activity, or effectiveness.

[0024] In another different embodiment of the invention, it comprises a formulation containing an extract of the venom of the scorpion Heteroctenus junceus without the fractions that have an effect on the hNav1.5 ion channel (fractions F8.3 and F9), responsible for the occurrence of chronic diseases, including coronary and cardiopulmonary diseases, further enriched with at least one of the fractions F4, F5.3, characterized by their effect on non-small cell lung cancer.

[0025] In another different embodiment of the invention, it comprises a formulation containing an extract of the venom of the scorpion Heteroctenus junceus without the fraction identified as toxic or dangerous for patients with chronic diseases, including coronary and cardiopulmonary diseases, further enriched with at least one of the fractions F4 or F5.3, characterized by their anticancer effect in non-small cell lung cancer.

[0026] Examples of realization of the invention

[0027] Example 1

[0028] Poison fractionation (Example of the implementation of the invention methodology)

[0029] The venom of the scorpion H. junceus was conveniently diluted in double-distilled water and fractionated by high-performance liquid chromatography (HPLC). A total of 6 mg was fractionated in each run, using a Shodex 218 TP C18 reversed-phase analytical column (250 mm x 10 mm, 4.6 pm, 300 Å pore size), previously equilibrated with a water / trifluoroacetic acid (TFA) mixture (0.12% v / v TFA) installed in the HPLC system (Shimadzu, Japan), equipped with a photodiode array detector. The fractions were eluted at a flow rate of 1 mL / min and a linear gradient of 0 to 60% using an acetonitrile / TFA mixture (0.1% v / v TFA in acetonitrile). Each run lasted 60 min and the detection signal of the chromatogram fractions was obtained at a wavelength of 230 nm.The fractions from each chromatographic run, eluted at the same retention times, were collected manually, pooled, dried in a concentrator (Labconco, Kansas, MO, USA), and stored at -20°C until use. HPLC-grade solvents and reagents (Sigma-Aldrich, St. Louis, Missouri, USA) were used. Figure 1 shows the chromatogram obtained from the injection of H. junceus scorpion venom. The fractions that exhibited the effect on the hNav1.5 sodium channel were removed from each venom run, and the remaining fractions were collected and reconstituted together (pooled). To verify the removal of these fractions, the reconstituted venom (VR) was run on the HPLC under the same chromatographic conditions used for the whole venom (VC). Figure 2 shows the chromatogram of the VR after the removal of the aforementioned fractions.

[0030] Retention time fraction 8.3: 32.58 min

[0031] Fraction 9 retention time: 35.77 min

[0032] Example 2

[0033] Electrophysiological recordings (Example of the safety and / or security of the formulation of the invention, with respect to the complete venom containing fractions with potential risk to a subgroup of users)

[0034] Adherent HEK-293 cells, maintained in DMEM-LG 5% fetal bovine serum (FBS), 1% Fungizone, and 1% Penicillin-streptomycin, were subcultured and plated onto 35 mm plates with 12 mm coverslips. Prior to 24 h post-subculture, the cells were transfected simultaneously with plasmid DNA containing the gene encoding the hNav1.5 channel and plasmid DNA containing the gene encoding the green fluorescent protein (GFP), using lipofectamine 3000. Finally, the cells were incubated for 48–72 h at 37°C with 5% CO2. Cells containing the transfected channel and exhibiting green fluorescence under a fluorescence microscope were used to obtain electrophysiological recordings.

[0035] Transfected cells cultured on coverslips were placed in a recording chamber (RC-25, Warner Instruments) mounted on an anti-vibration table under an inverted Olympus 1X71 microscope. A Patch Clamp L / M-EPC 7 amplifier was used to amplify the current signals, which were digitized with a Digidata 1550B and acquired using pClamp 10.7 software. Pipette displacement and position were controlled with a Scientifica Patchstar micromanipulator. Recording electrodes were fabricated from borosilicate capillaries using a horizontal drawr (P-87, Shutter Instruments), exhibiting an electrical resistance between 2 and 3 MΩ, and filled with internal solution.The patch-clamp technique in whole-cell mode was used to obtain current recordings using the following protocol: initial stimulation at -120 mV for 200 ms, followed by a pulse of -20 mV for 100 ms, and finally at -120 mV for 30 ms, under constant perfusion at 1 mL / min. Changes in current were recorded under different concentrations (pg / mL) (0, 50, 100, 150, 250, 400, 500, 750, 1000) of whole scorpion venom, reconstituted scorpion venom, and a pool of fractions removed from the whole scorpion venom (for the latter two, the concentration was calculated based on their proportion in the whole venom). The reconstituted venom and the pool of fractions represent 75.63% and 24.37%, respectively, of the whole venom. In all cases, they were added to the external solution prior to the protocol and adjusted to pH 7.4.The currents for each sample were recorded after 1 minute of current stabilization using only external solution. Each sample was then immediately applied, with 1.5 minute washes (external solution) between each concentration. The initial minute of recording was used as a control, and changes in current inactivation (considering the residual current 10 ms after the depolarizing pulse is applied (ohms / Lmax)) and changes in maximum current (L) were evaluated. peak ) in the different concentrations evaluated.

[0036] The composition of the internal solution in mM was as follows: 80 CsCl, 50 Aspartic Acid, 1 CaCl2, 1 MgCl2, 10 HEPES, 11 EGTA, 5 MgATP, 0.3 GTP, pH adjusted to 7.4 with CsOH and an osmolarity of approximately 300 mOsm. The composition of the external solution in mM was as follows: 140 NaCl, 5 KCl, 2 CaCl2, 1.2 MgCl2, 10 HEPES, 5 Glucose, pH adjusted to 7.2 with NaOH and an osmolarity of 300 mM.

[0037] The patch-clamp technique was used to determine the effect of scorpion venom and the pool of fractions on sodium currents originating from the hNav1.5 channel expressed in HEK-293 cells. Depolarizing pulses in cells expressing the hNav1.5 channel showed sodium currents that rapidly activated and inactivated within 5 ms (Figure 3). Perfusion of different concentrations of whole scorpion venom and the pool of fractions individually caused concentration-dependent inhibition of the current (Figures 3A and 3B). Perfusion of different concentrations of reconstituted venom showed less inhibition of the current than the other samples (Figure 3C).

[0038] Additionally, the effect of the different treatments on the rapid inactivation of the hNav1.5 sodium channel was measured 10 ms after the depolarizing pulse. Perfusion of various concentrations of whole scorpion venom and the pool of fractions individually resulted in a dose-dependent decrease in the rapid inactivation rate (Figures 3A and 3B). The application of different concentrations of reconstituted venom showed a smaller magnitude in the delay of the channel inactivation current (Figure 3C). The effect of the different samples on the activation and inactivation currents at the saturating concentration of 750 pg / ml is shown in Figure 3D. Whole scorpion venom and the pool of fractions showed maximum inhibition of the current and similar effects on channel inactivation, characterized in both cases by the persistence of an inactivation current (Figure 3D).In the reconstituted venom, these effects were of lesser magnitude compared to the other samples (Figure 3D). The magnitude of the effect of the whole venom and the pool of fractions showed values ​​greater than 0.7 relative to the maximum current for each case, which corresponds to less inactivation of the sodium channels (Figure 4A). In both cases, the effect on inactivation was different when compared to the reconstituted venom, which only reached 0.2 of the maximum current (Figure 4B). EC values. 50 The EC50 values ​​for the inactivated whole venom and the pool of fractions were 191.75 pg / mL and 57.33 pg / mL, respectively. The EC50 could not be determined for the reconstituted venom. 50 because maximum levels of effect on inactivation were not reached in the concentration range evaluated, which confirms the low impact of this condition on the inhibition of channel inactivation.

[0039] The mean inhibitory concentrations (EC) 50 The EC values ​​for each condition were obtained by plotting the normalized currents against each concentration. The whole venom and the pool of fractions inhibited the current at values ​​above 80% of the maximum current (Figure 4B). 50 The values ​​for the whole venom and the pool of fractions were 128.745 pg / mL and 74.70 pg / mL, respectively. The EC value 50 The effect of the reconstituted venom could not be determined within the evaluated concentration range, and the effect on current inhibition reached values ​​below 40% of the maximum current only at concentrations equivalent to 1000 pg / mL (Figure 4B). Comparison of the effect on current inhibition confirmed that there are differences between the whole venom and the pool of fractions when compared to the reconstituted venom (Figure 4D).

[0040] The nature of the effects observed in these experiments allows us to conclude that the complete venom of the scorpion H. junceus contains components that have alpha and beta effects on the activity of the hNav1.5 sodium channel. The fractions exhibiting these effects were isolated and then combined for study using electrophysiological experiments, which confirmed the presence of inhibitory effects on activation (beta effect) and on the prolongation of the inactivation process (alpha effect) of this channel. The concentration values, both for the scorpion venom and the pool of fractions, at which effects on this hNav1.5 channel were observed were much higher than those of the venoms of other scorpion species analyzed for this channel.These findings demonstrate that the components responsible for the alpha and beta effects are expressed in low proportions or may exhibit very low affinities for this channel compared to components from other species. The presence of protein fractions in scorpion venom, which interact with the hNav1.5 sodium channel, justifies their removal from the venom, thus improving pharmacological safety by reducing potential pathological effects linked to the modulation of the activity of this hNav1.5 sodium channel. The absence of these fractions in the reconstituted venom largely reduced the effects on this channel and, therefore, the potential toxic effects linked to the interaction with this ion channel, which is responsible for initiating the cardiac action potential. Example 3.

[0041] Cell lines

[0042] The A549 (ATCC, CCL-185™) and HT-29 (AddexBio, T0016002) cell lines were used for the experiments. A549 and HT-29 cells were maintained in 90% (w / v) modified Dulbecco medium (DMEM) with inactivated fetal bovine serum (FBS), 10% (v / v), penicillin (100 U / mL), streptomycin (100 pg / mL), and fungizone (100 pg / mL).

[0043] Cell Viability

[0044] Cell viability was assessed using the MTT assay. HT-29 and A549 cells were seeded in a 96-well cell culture plate at a density of 5x10 3Cells / well were added to various concentrations of whole (VC) and reconstituted (VR) scorpion venom (0, 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2 mg / mL). The 96-well plates were incubated for 72 h at 37°C with 5% CO2. Then, 10 pL of MTT solution (5 mg / mL) (Merck, USA) was added to the 96-well culture plates, and the cells were incubated for 3 h at 37°C with 5% CO2. The culture medium was decanted from the plate, and 150 pL of DMSO (100%) was added to each well, and A values ​​were obtained. 560nm with the Synergy™ HTX multimode microplate reader (Agilent BioTek, USA). The IC value 50 The results were obtained from the concentration-effect curves and calculated as % viability inhibition = (Abs. of negative control - Abs. of treated wells / Abs. of negative control) x 100, for each cell line. The experiments were repeated three times and five technical replicates were used.

[0045] Cells incubated for 72 h with different concentrations of whole scorpion venom and reconstituted venom showed similar cytotoxicity to tumor cells (Figure 5). MTT assays in both cell lines confirm that the observed changes are due to decreased cell viability. Both venoms induce a dose-dependent decrease in cell viability, as shown in Figure 5. IC50 values 50 For the whole venom, the values ​​were 0.77±0.09 mg / mL in A549 and 0.95±0.07 mg / mL in HT-29. While for the reconstituted venom, the IC50 values ​​were 50 were 1.04±0.15 mg / mL in A549 and 1.02±0.2 mg / mL in HT-29. Comparatively, the IC values 50The results were no different when both venoms were compared in each of the tumor cell lines. These data show that removing the fractions did not affect the anticancer effect of the reconstituted venom on tumor cells, compared to the whole venom.

[0046] Example 4

[0047] Cell cycle analysis (Example of the formulation's effectiveness against tumor cells) Cells were cultured in 60 mm plates at a density of 5 x 10 5 cells / plate and after incubation at 37°C overnight, were treated with the IC value 50Cells from whole scorpion venom and reconstituted venom were obtained by trypsinization, centrifuged at 1500 rpm for 5 min at 4°C, and washed twice with cold phosphate-buffered saline (PBS, pH 7.4). For cell cycle stabilization, cells were fixed in ice-cold methanol (100%) for 1 h at -20°C. The cells were washed twice with cold PBS, centrifuged at 1500 rpm for 5 min at 4°C, and then resuspended in a solution containing 100 pg / mL RNase A (Thermo Scientific, USA) and incubated at 37°C for 1 h. Finally, 50 pg / mL propidium iodide solution (Merck, USA) was added, and the cells were incubated at room temperature for 15 min in the dark. Cell cycle phases were determined by flow cytometry and analyzed using FlowJo software. Ten phases were recorded for each experiment.000 events, the trials were performed in duplicate and the experiments were repeated twice.

[0048] The effect of whole and reconstituted venom on the cell cycle was analyzed in A549 and HT-29 cells over 72 hours. The proportion of cells in each cell cycle stage was determined by incorporating propidium iodide into DNA and measured by flow cytometry. Treatment with both venoms showed similar behavior in modifying cell cycle distribution compared to control cells (Figure 6). In both venoms for each tumor cell line, the greatest cell accumulation was observed in the G2 / M phase, significantly different from control cells (Figures 6B and 6D). The similarity of the effect on the cell cycle in both cases demonstrates that reconstituted venom does not differ from whole venom in tumor cells.

[0049] Example 5

[0050] Cell death mechanism (Example of the effectiveness of the formulation for the control of tumor cells, even without the presence of fractions potentially risky for chronic patients)

[0051] The apoptosis event was detected by flow cytometry using the Annexin V-FITC / PI commercial kit (Cell Signaling Technologies, USA). Tumor cells were seeded in 60 mm (5 x 10) plates. 5 cells / plate) and were cultured at 37°C, 5% CO2 overnight. After this period, the cells were treated with the IC50 value. 50Whole scorpion venom and reconstituted venom were incubated at 37°C with 5% CO2 for 72 h. Plates cultured with untreated cells were used as controls. Cells from the different treatments were harvested individually by trypsinization, collected in 1.5 ml tubes, washed twice with cold PBS (4°C), and centrifuged at 1500 rpm for 5 min at 4°C. Tubes containing each treatment were resuspended in binding buffer (250 pL) and stained with annexin V-FITC / PI according to the manufacturer's instructions. Cells were incubated for 10 min at 4°C in the dark, and apoptosis was detected using flow cytometry and analyzed with FlowJo software. For each experiment, 10,000 events were recorded. Assays were performed in duplicate, and experiments were repeated twice.

[0052] The type of cell death induced in A549 and HT-29 tumor cells was studied by flow cytometry after treatment with whole and reconstituted venom. Cells single-stained with annexin V-FITC and those double-stained with annexin V-FITC / PI represented the total number of cells undergoing apoptosis; cells single-stained with Pl represented the total number of cells undergoing necrosis. As shown in Figure 7, the behavior was similar in both tumor cell lines for both formulations and significantly different from the untreated control. Treatment for 72 h induced similar increases in the proportion of cells killed by apoptosis for whole (47.05 ± 4.22%) and reconstituted (41.51 ± 5.39%) venom in A549 (Figures 7A and 7B). Likewise, whole and reconstituted scorpion venom increased the proportion of apoptotic cells equivalently in HT-29 with 51.45±4.48% and 49.52±4.30% respectively (Figures 7C and 7D). The control group showed only 5.65 ± 1.53% and 7.24 ± 1.28% of cells killed by apoptosis in A549 and HT-29, respectively. This evidence confirms apoptosis as the predominant mechanism of cell death induced by both the whole venom and the reconstituted venom. Removing the fractions that affect the hNav1.5 channel from the reconstituted venom does not affect its anticancer effect, and it maintains the same properties as the whole venom.

[0053] Example 6

[0054] Design and development of the oral liquid formulation using H. junceus scorpion venom as the active ingredient (Example of the formulation's stability, based on its preparation methodology)

[0055] Methylparaben (MP) and propylparaben (PP) were used together as preservatives. To obtain the mixture, 0.5 g of MP and 0.22 g of PP were weighed and stirred for 4 hours or until complete dissolution was achieved. The aqueous solution containing the parabens (MP:PP) remained in the final formulation at a proportion not exceeding 0.2% (Aulton ME. and Taylor K., 2013).

[0056] Methylcellulose (MC) was used as a thickening agent. 0.1g of methylcellulose was dissolved in 10 mL of the aqueous MP / PP mixture to obtain a 0.1% concentration (Remington JP., 2006; Rowe RC., 2020).

[0057] The reconstituted scorpion venom solution was found at 5 mg / mL and was used as the active ingredient in the formulation. To obtain the final formulation, the reconstituted scorpion venom was dissolved in the aqueous mixture containing the paraben blend and methylcellulose (MP / PP / MC), resulting in a final concentration of 0.8 mg / mL (Jouyban A., 2009; Molinero M. and García M., 2014). The pH of the final formulation was adjusted to a final pH between 6 and 7. Finally, the formulation was packaged in 60 mL amber PET bottles, commonly used for packaging oral liquid products in the pharmaceutical industry.

[0058] To verify the stability of the formulation and ensure that the excipients did not interfere with the active ingredient, high-performance liquid chromatography (HPLC) analyses were performed. A total of 10 pL of the final formulation was injected into a Kromasil C18 reversed-phase analytical column (250 mm x 10 mm, 4 pm, 300 Å pore size), previously equilibrated with a water / trifluoroacetic acid (TFA) mixture (0.12% v / v TFA) installed in the HPLC system (Shimadzu, Japan), equipped with a UV / VIS detector. The sample eluted at a flow rate of 1 mL / min with a linear gradient of 0 to 50% using an acetonitrile / TFA mixture (0.1% v / v TFA in acetonitrile). Each run lasted 65 min and the detection signal of the reconstituted venom was 230 nm while the detection wavelength of the excipients was 254 nm.

[0059] The major components of the reconstituted scorpion venom could be observed and differentiated from the excipients present in the formulation, as shown in Figure 8. In Figure 8A, at a wavelength of 230 nm, the major peaks of the reconstituted scorpion venom appear at retention times of 37.51 min, 39.56 min, and 40.94 min, respectively. In the same chromatogram, two prominent peaks are observed at 35.11 min and 49.88 min, corresponding to the preservatives methylparaben and propylparaben. Additionally, the 254 nm wavelength served to confirm only the presence of both preservatives in the formulation. The stability of the formulation was studied using the same protocol and wavelengths after 30 days of incubation at 4°C and 28°C. The formulation showed no variations in retention times or in the total area of ​​the formulation components when evaluated by HPLC chromatography (Figure 8).Only the area of ​​one of the scorpion venom spikes decreased by approximately 10% without altering other parameters. This confirms the compatibility of the various excipients used with the active ingredient and the stability of the formulation for at least 30 days.

[0060] Example 7

[0061] Microbiological analysis (Example of the viability and safety of using the formulation in other administration formats, including parenteral route)

[0062] Microbiological analysis was performed to determine the protective effect of the preservative mixture on the microbiological stability of formulations based on reconstituted scorpion venom. The aerobic plate count method, based on the Bacteriological Analytical Manual (Maturin L. and Peeler JT, 1998), was used. Samples of the formulation were prepared and stored at 4°C and 28°C for 30 days in 60 mL amber PET bottles, commonly used for liquid preparations in the pharmaceutical industry. The materials, supplies, and reagents used were not sterile to allow for the evaluation of the efficacy of the paraben mixture used as a preservative. The presence of mesophilic bacteria and pathogens, primarily E. coli and S. aureus (Feng P., et al., 1998), was determined.

[0063] For the enumeration of E. coli, 90 mm diameter Petri dishes containing brilliant green bile agar were prepared. The growth of the remaining mesophilic microorganisms, including S. aureus, was performed on 90 mm Petri dishes containing tryptone soy agar (TSA). In duplicate, 0.1 mL of each formulation was applied to the surface of each Petri dish and incubated at 35°C ± 1°C for 48 h. In all cases, the observation of colony growth on the Petri dish was considered positive for the presence of bacteria in the samples. On brilliant green bile agar, colonies that develop a red color with a pink halo and gas production are considered positive for E. coli.

[0064] Microbiological analyses to determine the efficacy of the methylparaben and propylparaben preservative mixture showed no bacterial growth in any of the Petri dishes where the formulation samples were applied, after a 30-day incubation period at two different temperatures. This evidence demonstrates that the preservative system was effective in protecting the product from microbial contamination when used in the formulation based on reconstituted scorpion venom.

[0065] Each of the individual advantages described for the pharmaceutical composition in the present invention is relevant to the current state of the art. However, the pharmaceutical composition itself solves a highly sensitive stability problem for current formulations obtained empirically and artisanally, considering the contamination they can and do suffer due to their method of preparation.It also considers the additional advantage of eliminating components that present a toxicological risk; this makes it possible to explore other routes of administration besides the oral route (the one used until now for previous formulations), such as the use of parenteral routes that allow the active ingredients to reach the bloodstream almost unchanged. This means higher concentrations of active components at the active site, as it avoids the degradative effect of the gastrointestinal tract on protein solutions and a shorter time to reach the anatomical sites that represent the therapeutic targets of the active ingredient contained in this pharmaceutical composition. Example 8.

[0066] Obtaining active fractions against non-small cell lung cancer

[0067] Reversed-phase chromatography

[0068] The venom of the scorpion *Heteroctenus junceus* was separated by reversed-phase high-performance liquid chromatography (HPLC) using a C18 column (Intersustain C18, 5 µm pore size, 4.6 mm ID x 250 mm column length, GL Science Inc., Japan). A 2 mL loop was used, and 6 mg of scorpion venom was injected into each chromatographic run. The mobile phases used were: phase A: HPLC-grade water with 0.1% TFA; phase B: acetonitrile with 0.1% TFA. Each chromatographic run was performed in a linear gradient from 0 to 45% acetonitrile for 45 min at 1 mL / min. The column temperature was 26°C, and the signal was detected at 230 nm. The fractions of interest were collected individually.At the end of the runs, all the collected fractions were concentrated overnight at 4°C in a concentrator (Centrivap concentrator system, Labconco, United States) and finally the concentrated fractions were stored at -20°C.

[0069] Results

[0070] Reversed-phase chromatography

[0071] To separate and obtain the different fractions of interest from the scorpion venom, several injections were performed on a reversed-phase C18 column coupled to an HPLC system. Figure 9 shows the standard chromatographic pattern of the venom, and the chromatographic peak corresponding to a retention time of 25.5 min is visible and was designated fraction 4 (F4). The characteristics of fraction F4 suggest that it may be of a high degree of purity. Additionally, fraction F5.3 was collected at a retention time of 27 min, and given the characteristics of this fraction, it was re-purified as it suggests the presence of more than one component.

[0072] Example 9 - Cell lines and cell viability

[0073] Cell lines

[0074] The A549 cell line (ATOO, CCL-185™) was used for the experiments. A549 cells were maintained in 90% (w / v) modified Dulbecco medium (DMEM) with inactivated fetal bovine serum (FBS), 10% (v / v), penicillin (100 U / mL), streptomycin (100 pg / mL), and fungizone (100 pg / mL). Cell viability

[0075] Cell viability was assessed using the MTT assay. A549 cells were seeded in a 96-well cell culture plate at a density of 5*10 3Cells / well were added to various concentrations of fractions F4 and F5.3 (15.1, 31.25, 62.5, 125, 250, 500, 1000 µg / mL). The 96-well plates were incubated for 72 h at 37°C with 5% CO2. Then, 10 pL of MTT solution (5 mg / mL) (Merck, USA) was added to the 96-well culture plates, and the cells were incubated for 3 h at 37°C with 5% CO2. The culture medium was decanted from the plate, and 150 pL of DMSO (100%) was added to each well, and A values ​​were obtained. 560nm with the Synergy™ HTX multimode microplate reader (Agilent BioTek, USA). The IC50 value 50 The results were obtained from the concentration-effect curves and calculated as % viability inhibition = (Abs. of negative control - Abs. of treated wells / Abs. of negative control) x 100, for the cell line. The experiments were repeated three times and three technical replicates were used.

[0076] Cells incubated for 72 h with different concentrations of each fraction showed similar behavior regarding the reduction of cell viability in A549 lung tumor cells (Figure 10). The MTT assay indicates that both fractions induce a dose-dependent decrease in cell viability, as shown in Figure 10. The IC50 values 50 For fraction F4 it was 378.2±68.4 pg / mL (0.38mg / mL), while for fraction F5.3 the CI value 50 were 372.6±47.1 pg / mL (0.37mg / mL). This experimental evidence confirms that both fractions are responsible for the anticancer effect observed in the whole venom (VC) and the reconstituted venom (VR).

[0077] Regarding the reproducibility of the invention, it is important to note that the technologies for obtaining the different fractions of the proposed invention are described and widely known in the prior art. However, in particular, the details of obtaining the different fractions of the proposed invention are preferably described in embodiments 1 and 9.

[0078] Example 10 Mass spectrometry and identification of the major component in fractions F4 and F5.3

[0079] The major components of fractions F4 and F5.3 were identified by mass spectrometry. Each sample was individually dissolved in a solution containing 8 M urea in 25 mM NaHCO3. The sample was reduced with 20 mM DTT in 25 mM NFLHCO3 for 1 h at room temperature. Additionally, the samples were alkylated in a solution of 20 mM ICH2CONH2 in 25 mM NFLHCO3 and incubated for 1 h in the dark at room temperature. Subsequently, the sample was diluted in 25 mM NaHCO3, cleaned using a Sep-Pak C18 column (Waters), and dried in a rotary concentrator at 1000 rpm at 10°C overnight. 200 ng of the peptide were injected into a nanoElute nanoUHPLC (Bruker Daltonics) coupled to a timsTOF Pro mass spectrometer (Bruker Daltonics) using an Aurora UHPLC column (25 cm x 75 pm ID, 1.6 pm C18, IonOpticks, Australia). Liquid chromatography employed a 90-min gradient from 2% to 35% buffer B (0.1% formic acid / acetonitrile).Data collection was performed using TimsControl 2.0 software (Bruker Daltonics) under 10 PASEF cycles, with a mass range of 100–1700 m / z, capillary ionization at 1.5 kV and 180°C, and a time-of-flight frequency of 10 kHz at a resolution of 50,000 FWHM. The data collected by the mass spectrometer were analyzed using PEAKS Studio X+ software (Bioinformatics Solutions) with the PEAKS IMS protein identification module. Mass tolerance parameters of 50 ppm were used, employing monoisotopic masses and 0.05 Da ionic fragments. Non-specific digestion mode was selected, with a maximum of two loss cleavages per peptide. The following were used as post-translational modifications (PTMs): Carbamidomethylation of cysteine ​​(fixed PTM), Methionine Oxidation (M), N-terminal Acetylation, Asparagine and Glutamine Deamination (NQ), and Lysine and N-terminal Carbamylation (K, X, N), as variable PTMs.All peptides were filtered using an ALC>80%. The ALO (Average Local Confidence) refers to the average confidence that an amino acid is present in the peptide de novo at a particular position, expressed as a percentage.

[0080] Result

[0081] The identification of the major component sequence in fractions F4 and F5.3 was performed using MS / MS mass spectrometry. The sequence with the highest number of unique peptides in fraction F4 matched a peptide in the Uniprot database with the sequence PGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVGLF EDTNLCAIHAKR, Uniprot code A0A1 U9WSZ2, corresponding to a histone H3a with a molecular weight of 8552 Da and 74 amino acids, present in the venom of the scorpion Heteroctenus junceus. On the other hand, the analysis of the MS / MS spectra of fraction F5.3 revealed a major component with a monoisotopic mass of 3945.55 Da. Sequencing of the F5.3 fraction and comparison with the Uniprot databases revealed a 100% match with the peptide code C0HJT0 present in the venom of the scorpion Heteroctenus junceus, with sequence

[0082] TVIDVKCTSPKQCVPACKAAMGTVRAKCMNGKCKCYI and 37 aa in length. The identification of these components and their correspondence with peptides already described in this species confirm the positive identification of these peptides and their major presence in the fractions where they were identified, making it possible for them to be considered as markers of these fractions.

[0083] Brief Description of the Figures

[0084] Figure 1: Chromatogram of the complete venom of the scorpion H. junceus (VC) obtained by injection of 6 mg of venom into an analytical C18 reversed-phase HPLC column, using a flow rate of 1 mL / min, and a mobile phase of acetonitrile / 0.1% TFA. Gray bands: Fractions of scorpion venom with an effect on the human adult sodium channel 1.5 (hNav1.5), with potential toxicity to cardiac cells (fractions: F8.3 and F9).

[0085] Figure 2: Chromatogram of reconstituted (VR) H. junceus scorpion venom, obtained by HPLC injection of 2 mg of venom into an analytical C18 reversed-phase column, using a flow rate of 1 mL / min, and a mobile phase of acetonitrile / 0.1% TFA. Chromatogram without the fractions with potential toxicity to cardiac cells (fractions: F8.3 and F9).

[0086] Figure 3: Current recordings of the effect of different samples on the adult sodium channel hNav1.5. A) whole venom (VC). B) pool of fractions (pool). C) reconstituted venom (without fraction with potential toxicity to cardiac cells) (VR). D) Representative recording of the effect of different samples on channel currents at the saturating concentration of 750 pg / mL.

[0087] Figure 4: (Toxicity) Effect on peak current and inactivation of the adult human hNav1.5 sodium channel, expressed in HEK-293 cells. Whole scorpion venom (red), reconstituted scorpion venom (green), pool of fractions (F8.3 + F9) isolated by HPLC (blue). A) Dose-response curves of channel inactivation inhibition (where greater inactivation inhibition implies greater toxicity). B) Bar chart of the magnitude of the effect on channel inactivation inhibition at the saturating concentration of 750 pg / ml. C) Dose-response curves of channel current inhibition. D) Bar chart of the magnitude of the effect on channel current inhibition at the saturating concentration of 750 pg / ml for whole venom. The pool of fractions and the reconstituted venom were analyzed based on their proportion of the whole venom. The mean effective concentration (EC) value 50The values ​​were obtained by plotting the normalized currents as a function of each concentration evaluated for each of the experimental conditions. The bars represent an average of 3 measurements. *p<0.05 compared to the reconstituted poison.

[0088] Figure 5: (Effectiveness: Inhibition of cell viability) Dose-response curves of tumor cells treated with scorpion venom, obtained by the MTT assay: A) A549 and B) HT-29. The IC50 values 50 These results were obtained by fitting the dose-response curves to the Hill equation. It can be observed that by eliminating the fractions with potential toxicity (IF8.3 + F9), the effectiveness of the formulation is maintained (i.e., including fractions F4 and F5.3).

[0089] Figure 6: (Effectiveness: Cell cycle arrest) Effect of whole (VC) and reconstituted (VR) H. junceus scorpion venom (including F4 and F5.3, but excluding F8.3 and F9) on the cell cycle in A549 and HT-29 tumor cells. A) Representative histogram of the cell cycle in the A549 cell line after 72 h of scorpion venom treatment. B) Bar chart representing the percentage of cells in G0 / G1, S, and G2 / M in A549 cells. C) Representative histogram of the cell cycle in the HT-29 cell line after 72 h of scorpion venom treatment. D) Bar chart representing the percentage of cells in G0 / G1, S, and G2 / M in HT-29 cells. Bars represent an average of 3 measurements. *p<0.05 compared to control.

[0090] Figure 7: (Effectiveness: Induction of cell death by apoptosis) Analysis of cell death events in A549 and HT-29 tumor cells treated with whole (VC) and reconstituted (VR) H. junceus scorpion venom. Scatter plots representing propidium iodide (y-axis) and Annexin-V (x-axis) as measures of apoptotic cell death in A: A549 and C: HT-29, respectively. B) Bar charts of the percentage of total apoptosis determined by flow cytometry in B: A549 and D: HT-29, respectively (n=4). *p<0.05 compared to the control.

[0091] Figure 8: Chromatogram of the formulation containing reconstituted H. junceus scorpion venom (VR) and preservatives (where the present chromatogram would not be distinguishable from a chromatogram performed on whole scorpion venom). Chromatographic standards were obtained by injection of 100 pL of the formulation into an analytical C18 reversed-phase HPLC column, using a flow rate of 1 mL / min, and an acetonitrile / 0.1% TFA mixture as the mobile phase. A) Chromatogram obtained at a wavelength of 230 nm. B) Chromatogram obtained at a wavelength of 254 nm.

[0092] Figure 9: Chromatogram of H junceus scorpion venom, identifying fractions F4 and F5.3. 6 mg of venom was injected into a reversed-phase C18 column coupled to an HPLC system. The run was performed for 45 min at 1 mL / min in a 0-45% ACN gradient. The dashed line corresponds to the percentage of acetonitrile during the run time.

[0093] Figure 10: (Effectiveness: Inhibition of cell viability) Dose-response curves of tumor cells treated with fractions F4 and F5.3, obtained by the MTT assay: The IC50 values 50 These values ​​were obtained by fitting the dose-response curves to the Hill equation. They exhibited similar behavior to VC and VR. VC: whole scorpion venom; VR: reconstituted scorpion venom

Claims

CLAIMS 1. A liquid composition used as an anticancer agent with reduced cardiac toxicity and high antimicrobial stability, CHARACTERIZED in that it comprises: a) at least a fraction of the venom of the scorpion Heteroctenus junceus (0.1-2 mg / mL), b) preservatives represented by at least methylparaben (0.05%) and propylparaben (0.02%) w / w, c) distilled water and, d) pharmaceutical excipients, wherein furthermore, the fraction in a) may be selected from the list comprising the recognizable and identifiable fractions: F4 and F5.3, wherein both fractions exhibit a potentiated anticancer effect, mainly associated with the inhibition of tumor progression.

2. The composition of claim 1, CHARACTERIZED in that the first fraction or F4 comprises at least two identifiable peptides, wherein at least one of these peptides corresponds to a peptide code UNIPROT A0A1U9WSZ2; and wherein the second fraction or F5.3 comprises at least three identifiable peptides, wherein at least one of these peptides corresponds to the peptide code UNIPROT COHJTO.

3. The composition of claim 1, CHARACTERIZED in that it comprises at least the first fraction (F4) containing at least the peptide A0A1U9WSZ2 (as a fraction marker) and wherein said composition is effective in the treatment of patients with cancers of epithelial origin, particularly non-small cell lung cancer.

4. The composition of claim 1, CHARACTERIZED in that it comprises at least the second fraction (F5.3), wherein said fraction in turn comprises a mixture of peptides, wherein said peptide mixture contains at least the peptide identified as COHJTO (as a marker of the fraction) and wherein said composition is effective in the treatment of patients with cancers of epithelial origin, particularly non-small cell lung cancer.

5. A composition for the treatment of tumor cells and / or tumors in human and animal patients, CHARACTERIZED in that it comprises a mixture of fractions (4 and 5.3) of the venom of the scorpion H. junceus at different concentrations of each fraction, wherein each fraction is maintained in a range of between 0.12 mg / mL and 1.2 mg / mL, wherein said mixture also has a potentiated effect with anticancer activity in the treatment of patients with cancers of epithelial origin and who suffer from other chronic or acute diseases, including coronary diseases.

6. A composition for the treatment of tumor cells and / or tumors in human and animal patients, CHARACTERIZED in that it comprises at least one fraction from the list comprising: F4, F5.3, and the venom reconstituted with at least one of said fractions, and wherein furthermore the concentration of the fractions present in the reconstituted venom is in the range of 0.12 mg / mL and 1.2 mg / mL, and wherein furthermore it has an enhanced therapeutic effect for the treatment of cancer patients and those with pre-existing chronic diseases, including coronary and respiratory diseases.

7. The composition of claims 3, 4, 5 and 6, CHARACTERIZED in that the components of the isolated fractions 4 and 5.3 maintain their structure, resulting in resistance / independentness to environments with acidic pH, while also maintaining their antitumor action, activity or effectiveness.

8. A composition used as an anticancer agent with reduced cardiac toxicity and high antimicrobial stability, CHARACTERIZED in that it comprises an extract of the venom of the scorpion H. junceus without the fractions that have an effect on the hNav1.5 ion channel corresponding to fractions F8.3 and F9, identified as potentially toxic or dangerous for patients with chronic diseases, including coronary and cardiopulmonary diseases, wherein said composition is further enriched with at least one of the fractions F4, F5.3, characterized by their potentiated antiproliferative effect in non-small cell lung cancer.

Images