COMPLEXOS DE INCLUSÃO DE FILACANTONA EM SS-CICLODEXTRINA E EM SULFOBUTIL-ÉTER-SS-CICLODEXTRINA PARA USO COMO AGENTES CITOTÓXICOS

Description

1 / 14 “PHYLACANTHONE INCLUSION COMPLEXES IN β-CYCLODEXTRIN AND IN SULFOBUTYL ETHER-AND-CYCLODEXTRIN FOR USE AS CYTOTOXIC AGENTS” Field of invention

[001] The present invention relates to obtaining two potential cytotoxic agents containing phyllacanthones, a bis-nor-diterpene isolated from the stem bark of the species Cnidoscolus quercifolius Pohl, complexed in β-cyclodextrin and sulfobutyl-ether-cyclodextrin. The species Cnidoscolus quercifolius Pohl belongs to the Euphorbiaceae family, known as a source of various compounds with cytotoxic activity. Even though they represent great therapeutic potential, some of these compounds exhibit high volatility and low water solubility, which impairs their effectiveness and eventual incorporation into formulations. In this sense, cyclodextrins, macrocyclic and non-toxic oligosaccharides, are considered ideal for the formation of inclusion complexes which, in turn, can improve the physicochemical stability, reducing losses due to evaporation of the complexed compounds and possibly enhancing their therapeutic effects.In this way, the innovation may represent a pharmaceutical alternative with potential application as a cytotoxic agent, mainly against cutaneous melanoma.

[002] Currently, there is a growing number of publications and patent applications in the pharmaceutical field involving inclusion complexes with cyclodextrins. The favorable size of the internal cavity diameter of cyclodextrins allows the formation of inclusion complexes with most drugs, improving their physicochemical properties and enhancing their pharmacological effects - as exemplified by patents BR122018068797B1, BR0415338-3, BR200303884-A, BR200401621-A, US20030220293-A1, US20030212043-A1, US6464988, US6071964, GB2376231-A and WO9718245. To date, there are no patents relating to inclusion complexes with cyclodextrins involving the compound under study. Fundamentals of the invention

[003] Melanoma is a type of cancer that has one of the largest and fastest-growing progressions. Petition 870240108102, dated 12 / 18 / 2024, page 9 / 27 2 / 14 mutation rates. What is observed is that the genes carrying these mutations are already detectable in their benign stage, and this suggests that changes in gene expression are related to disease progression (AROZARENA; WELLBROCK, 2019). Risk factors include sun exposure, freckle density, skin color, eye color, hair color, family history, and a number of susceptibility genes, with sun exposure being the main environmental risk factor (VUONG et al., 2020).

[004] Conventional therapy includes agents such as hydroxyurea, which was the first approved treatment for metastatic malignant melanoma. However, it has low efficacy, and therefore, there has been a constant search for more promising treatments. Dacarbazine, in turn, is an alkylating agent that is administered intravenously, metabolized in the liver by cytochrome P450 and eliminated via the kidneys, presenting significant toxic effects, such as the development of hepatic veno-occlusive disease, nausea and vomiting (RAMOS, 2009).

[005] IFN-α was the first exogenous cytokine to show antitumor activity in advanced melanomas, exhibiting immunoregulatory, antiproliferative, differentiation-inducing, apoptotic, and antiangiogenic activity in various neoplasms. However, what is observed in clinical practice is that treatment with high doses of IFN-α is accompanied by multiple adverse effects, such as flu-like symptoms, anorexia, adverse skin reactions, hepatotoxicity, fatigue, and depressive symptoms, limiting its use (KORN et al., 2008).

[006] Given this, the need to innovate and develop new treatments is highlighted in order to increase the quality of life and survival rate of patients (AYDOGMU§-OZTÜRK et al., 2020). In this context, natural products present themselves as a potent source of new drugs, which can be applied in the treatment of melanoma, considered one of the most aggressive forms of skin cancer (OLIVEIRA-JÚNIOR, 2017).

[007] Admittedly, medicinal plants are the main source of highly effective traditional medicines in the treatment of many types of cancer, possessing significant effects. It is known that their use can also reduce the Petition 870240108102, dated 12 / 18 / 2024, page 10 / 27 3 / 14 adverse side effects related to these treatments (AYDOGMU§-OZTÜRK et al., 2020).

[008] Among the species with this potential, we can mention Cnidoscolus quercifolius Pohl, belonging to the Euphorbiaceae family. Popularly known as "Favela", it is a species with thick leaves, tuberous roots and small white flowers, widely disseminated throughout the Caatinga (SANTOS; SILVA; SILVA, 2020). According to Sobrinho (2011), the bark of this species contains cytotoxic substances, such as methyl-favelin, favelol and favelone. In addition to these, diterpenes such as phyllacanthones and triterpenes such as 3-β-O-cinnamoyl-lupeol have already been identified. This justifies the fact that extracts from the stem bark of this species have a high percentage of cell growth inhibition, being commonly used to treat tumors and inflammatory processes (SOBRINHO, 2011).

[009] The compounds present in these extracts exhibit significant cytotoxic activity in several human tumor cell lines (A2058 cell line), and are attributed with antitumor properties (TORRES, 2018). Furthermore, studies demonstrate that phylacantoin specifically showed a significant cytotoxic effect on human promyelocytic leukemia and human lung carcinoma cell lines (PAULA et al., 2016).

[010] Despite their significant therapeutic potential, oxygenated diterpenes, such as phylactone, are a class of compounds that often exhibit high volatility and low water solubility, which reduces their therapeutic efficacy and impairs their stability in therapeutic formulations. To overcome these problems, various carriers can be used to improve the physicochemical properties of these compounds, thus enhancing their effects (ABRIL-SANCHEZ et al., 2019). Among these carriers, cyclodextrins deserve special mention.

[011] Cyclodextrins (CDs) are non-toxic, macrocyclic oligosaccharides, represented as a truncated cone with a relatively hydrophilic surface and Petition 870240108102, dated 12 / 18 / 2024, page 11 / 27 4 / 14 a hydrophobic central cavity, being chemically and physically stable molecules and with a cavity capable of containing hydrophobic molecules in its internal portion. These properties make them ideal for solubilizing compounds in aqueous media due to the polarity of their surface, forming inclusion complexes that, in turn, can improve and increase the physicochemical stability, reducing losses due to evaporation of the encapsulated compounds (OLIVEIRA-FILHO, 2019).

[012] Thus, the present innovation stemmed from the objective of obtaining and characterizing the formation of two inclusion complexes containing the bis-nor-diterpene phyllacanthone, isolated from the stem bark of Cnidoscolus quercifolius Pohl, in β-cyclodextrin and in sulfobutyl-ether-cyclodextrin, evaluating the cytotoxic effect of these complexes on human melanoma cells. Due to the great potential of this diterpene, it is considered that complexation can significantly optimize its physicochemical properties and, consequently, its pharmacological effects.

[013] The invention can be better understood through the following detailed description: Description of the invention and embodiment of the invention

[014] Initially, the stem bark of Cnidoscolus quercifolius Pohl was collected in the municipality of Petrolina-PE, located on the premises of the Agricultural Sciences Campus of the Federal University of the São Francisco Valley (latitude 09°19'43''S, longitude 40°33'09''W). The species under study was registered on the platform of the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen), under registration number AB179D2. The material had been previously identified by a botanist and a comparison was made with the exsiccata of the species already deposited (voucher #19202) in the Vale do São Francisco Herbarium (HVASF) of the Federal University of the São Francisco Valley (UNIVASF). After this process, the plant material was subjected to drying in an oven with air circulation, at an average temperature of 40 °C, for 72 hours. After drying, the material was then pulverized in a mechanical mill, obtaining a total of 900 g of stem bark powder.

[015] The dried and pulverized plant material was subjected to exhaustive maceration, Petition 870240108102, dated 12 / 18 / 2024, page 12 / 27 5 / 14 in a stainless steel container, using 95% ethanol as solvent. Subsequently, the extractive solution was filtered and concentrated in a rotary evaporator under reduced pressure, at an average temperature of 50 °C. After obtaining the crude ethanolic extract of the stem bark of Cnidoscolus quercifolius Pohl (61 g), 30 g of this extract were subjected to vacuum liquid chromatography, using silica gel 60 as the stationary phase.

[016] The subfractions were subjected to preparative thin-layer chromatography (TLC) using silica gel 60 (2-25 μm), with a fluorescence indicator (Merck®).

[017] The isolated substances were subsequently analyzed by nuclear magnetic resonance (NMR), and the objective was to isolate bis-nor-diterpene phyllacanthone from the chloroform fraction, as previously reported in the literature. For this purpose, 3 g of the chloroform fraction (from which the diterpene had already been isolated, according to previous studies) was subjected to column chromatography on silica gel. The system used was an isocratic hexane / ethyl acetate (97:3) system, with the entire process monitored using an ultraviolet (UV) light source. At the end of the process, the column was washed with ethyl acetate, and 15 subfractions (C1-C15) were obtained.

[018] As expected, bis-nor-diterpene phyllactone was isolated from the stem bark of the species, and its chemical structure was confirmed by nuclear magnetic resonance. Paula et al. (2016) report that the isolation of compounds in Cnidoscolus species reinforces their importance as potential chemomarkers for the genus. Furthermore, the cytotoxic activity reported for this compound and other compounds isolated from the genus may aid in the development of new anticancer agents. Precisely for this reason, seeking techniques that can optimize and enhance their effects, such as the formation of inclusion complexes with cyclodextrins, becomes extremely relevant.

[019] Based on this, the next step was to obtain the inclusion complexes of philacantone in β-cyclodextrin (Sigma-Aldrich®) and in β-sulfobutyl ether. Petition 870240108102, dated 12 / 18 / 2024, page 13 / 27 6 / 14 cyclodextrin (CycloLab®). For this, a 1:1 molar ratio of terpene to cyclodextrin was used. The materials were weighed and placed in an Erlenmeyer flask. Thus, the complexes started from 100 mg of phylacantoin to 0.998 g of β-cyclodextrin and from 100 mg of phylacantoin to 1.970 g of sulfobutyl-ether β-cyclodextrin. To form the complexes, a solution of 75% water and 25% ethanol was added to the mixture, for a total of 250 mL of solution. The amount of phylacantoin used was added to the volume of ethanol used, and the mixture was subjected to ultrasound (10 minutes, at 25 °C). After that, solutions containing water and β-cyclodextrin or sulfobutyl ether-cyclodextrin were added to the mixtures, which were then left in a magnetic stirrer for 48 hours.The solution of the phyllactone / ε-cyclodextrin complex was lyophilized, and the solution of the phyllactone / sulfobutyl-ether-β-cyclodextrin complex was left in an oven (at 45 °C) in a glass container for 48 h until completely dry.

[020] As mentioned, the drying techniques for the obtained complexes were lyophilization and oven drying. Some studies demonstrate that the dissolution rate of isolated compounds and physical mixtures does not vary, while complexes can enhance this rate. After drying, dry, loose, and amorphous powders were obtained, and these characteristics may ensure greater stability, as well as greater guest-cyclodextrin interaction.

[021] For the characterization of the complexes, initially 1H nuclear magnetic resonance (1H NMR) experiments were acquired using D2O and conducted on a Bruker™ 400 MHz ASCEND III spectrometer, operating at 9.4 Tesla, observing the 1H nucleus at 400.13 MHz, equipped with a 5 mm direct detection probe and field gradient on the z-axis. The 1H NMR chemical shifts were expressed relative to the TMS signal in δ 0.00 (reference compound) and the coupling constants (J) were expressed in Hz.

[022] The formation of inclusion complexes by NMR technique was observed from the chemical shifts of the cyclodextrin protons that are inside the cavity, hydrogen 3 (H-3) and hydrogen 5 (H-5), which will undergo a greater alteration (compared to the protons positioned to the outside of the Petition 870240108102, dated 12 / 18 / 2024, page 14 / 27 7 / 14 cavity) once the visiting molecule is inserted within the cavity. Experiments for the complexes between cyclodextrins and phyllacantone demonstrated that hydrogens 3 and 5 were in a region of lower frequency when forming an inclusion complex with the terpenes, indicating that they were shielded in the presence of the visiting molecule.

[023] To complement the evidence of complex formation, the surfaces of philacantone, cyclodextrins, physical mixtures, and inclusion complexes were examined using a TescanVEGA3 scanning electron microscope. Samples were prepared by mounting the powders on carbon tape placed under an aluminum foil. Subsequently, the powders were coated with gold powder for 12 minutes and examined using SEM at 10 kV.

[024] Scanning electron microscopy revealed that cyclodextrins have a very characteristic and extremely evident morphology. Phylactone, in turn, is found in the form of crystals. The physical mixtures exhibit characteristics that can be attributed to the isolated carriers and, possibly, to phylactone (in both cases). Thus, in the physical mixtures, the terpene is not inserted into the cavity of the host molecule. The analysis of the obtained complexes, however, shows quite unique morphological changes, and the reduction in particle size and the resulting amorphous structure are notable. The literature reports that the formation of a complex is characterized by the acquisition of a finer morphology, which may resemble "fine shavings" covering the entire surface of the material.

[025] The materials were also characterized by Fourier transform infrared (IR) spectroscopy. The IR spectra of the analyzed samples were obtained using KBr pellets as a solid support. To prepare the pellet, approximately 1 mg of the sample was used for 100 mg of KBr, which were ground until a fine powder was obtained. The mixture was then subjected to a pressure of 78.5 kN, using a Shimadzu® hydraulic press for a period of ten minutes. After obtaining the KBr pellet, it was analyzed on a Shimadzu® IRTracer-100 spectrometer, in the region between 4000 and 400 Petition 870240108102, dated 12 / 18 / 2024, page 15 / 27 8 / 14 cm-1, with 45 scans and a resolution of 8 cm-1.

[026] Through analysis of the IR spectra, it is possible to observe that the spectra of the physical mixtures resemble the combination of the spectra of cyclodextrins with phylcantone, clearly showing characteristic bands of the two components. The analysis of the spectra of the two complexes, however, showed a strong similarity to the absorption pattern of cyclodextrins and showed a reduction in the intensity of the characteristic bands of the diterpene. Absorption bands are clearly noted in the region of 3000 cm-1 (OH stretching), 2900 cm-1 (CH stretching), at 1154 cm-1 (CO stretching) and 1026 cm-1 (C-OC stretching), all characteristic of cyclodextrins, with a slight shift in the OH stretching and CH stretching bands.

[027] This information helps to prove that the formation of the complexes was effective, since the inclusion of a molecule in the cyclodextrin cavity generates a greater conformational restriction, reducing the free movement of the included molecules and thus reducing the signal intensity. A computational molecular docking study was carried out to corroborate the results obtained, using the graphical interfaces of the Autodock Vina (TROTT; OLSON, 2010) and Autodock Tools (MORRIS et al., 2009) programs, obtaining an initial set of conformations for subsequent semi-empirical refinement.

[028] The structure of β-cyclodextrin was retrieved from the RCSB-PDB crystallographic database (www.rcsb.org) PDB ID:5MK9. The structure of sulfobutyl-ether-βcyclodextrin was drawn from the three-dimensional structure of 5MK9 in Chemsketch, following the sulfonation pattern of the inclusion complex used experimentally. The Chimera software (PETTERSEN et al., 2004) was used for hydrogen addition. The 3D structure of philacantone was obtained from the PubChem database (www.pubchem.ncbi.nlm.nih.gov), with hydrogens also added using the Chimera software.

[029] For Vina's calculations, the gridbox dimensions were 14 x 13 x 13 Å and 21 x 21 x 21 Å with a standard spacing of 1.0 Å between grid points, centered on β-cyclodextrin and sulfobutyl-ether-cyclodextrin, respectively. From the Petition 870240108102, dated 12 / 18 / 2024, page 16 / 27 9 / 14 of the best poses found in Vina, semi-empirical calculations were performed using PM6-DH2 with the MOPAC2016 package (STEWART, 2016).

[030] The final interaction energies (ΔE), after semi-empirical calculations considering the heat of formation (ΔH° f) of each system, were evaluated as: ΔΕ=ΔΗ f(complex)— [ΔΗ f(cyclodextrin) +ΔΗ f(phylacanthone)]

[031] Based on the results obtained, it was possible to observe that the best poses for the complex obtained after semi-empirical calculations by Molecular Docking demonstrate that the most stable form of complex formation between philacantone and β-cyclodextrin occurs when the guest inserts through the widest cavity of βCD (with an interaction energy of -89.8068 kcal / mol). The most stable pose for the complex between philacantone and sulfobutyl-ether-cyclodextrin presented an interaction energy of -87.4032 kcal / mol.

[032] The dissolution profile of philacantone and inclusion complexes was analyzed according to the methodology proposed by Oliveira et al. (2019), seeking to understand how complexation can optimize the solubility of methylfavelin. Initially, the maximum wavelength of philacantone was determined by scanning wavelengths of 200-400 nm in saline solution (0.15 M) acidified to pH 1.5 with chloride acid, at 37 °C using a UV-Vis spectrophotometer. For the determination of the calibration curve, philacantone solutions ranging from 2-30 pg.mL-1 (Y = 0.0033X + 0.0077, R2 = 0.9911) were used, where X is the philacantone concentration and Y is the absorbance at 260 nm at 37 °C. For the controlled release study, philacantone and the complex were placed in 50 mL of saline solution. The experiments were performed on a magnetic stirrer with controlled temperature and agitation (7 °C and 100 rpm, respectively) for a total time of 100 minutes.At periodic intervals, samples of the release medium were taken and analyzed by spectrophotometry at 260 nm to determine the amount of philacantone released. True triplicates were performed for all samples.

[033] The dissolution test revealed that, throughout the entire experiment (100 minutes), there is no dissolution of philacant in the medium. At the same time of Petition 870240108102, dated 12 / 18 / 2024, page 17 / 27 In the 10 / 14 experiment, it was observed that the dissolution of philacantone from the two inclusion complexes is extremely rapid (mainly in the first two minutes). Thus, the release profile of the complex is observed to be immediate, indicating that complexation can significantly increase the solubility of philacantone under the tested conditions. This observation is generally well associated with greater in vivo bioavailability of organic compounds. Optimized solubility, combined with the intrinsic permeation capacity of hydrophobic molecules, generally contributes to better bioavailability.

[034] Furthermore, the complexation efficiency of the methods used was evaluated. To determine the concentration of philacantone that was actually complexed, the methodology used was that proposed by Ferraz et al. (2020). First, the surface-adsorbed philacantone was removed from both complexes by washing 10 mg of sample with 10 mL of ethanol for 10 minutes with agitation. The resulting solution was centrifuged at 4500 g for 5 min at 25 °C to promote the removal of any cyclodextrin residue. Then, the supernatant containing the adsorbed philacantone was collected and filtered through a membrane filter (0.22 μm filter, Millipore™).

[035] An aliquot (1000 μL) of the filtrate was added to quartz cuvettes for absorbance reading in a UV-Vis spectrophotometer at 260 nm. True triplicates were made for all samples. A standard curve (30-60 μg / mL, R2= 0.9911) was also constructed under the same conditions to calculate the philacantone concentration. Finally, the complexation efficiency (EC%) was calculated according to the equation: EC% = (mass of phylacanthone recovered / mass of phylacanthone in the complex) x 100

[036] In the present study, the two complexes obtained showed a satisfactory complexation efficiency of 89.60% and 77.56% (for the philacantone complex in β-CD and in sulfobutyl-ether-cyclodextrin, respectively). Many factors can influence the interactions between guest molecules and cyclodextrins, including Petition 870240108102, dated 12 / 18 / 2024, page 18 / 27 11 / 14 the type of cyclodextrin (which differ in cavity size), the complexation method, and the hydrophobicity level of the guest molecule (FENYVESI et al., 2016). The high complexation efficiency values ​​observed indicate that a large amount of phyllacanthones was effectively complexed, corroborating the characterization data presented.

[037] The cytotoxic activity of the complexes and isolated philacantone was determined by the MTT method, described by Mossman (1983) and adapted by Oliveira-Júnior (2017). Samples were solubilized in DMSO (dimethyl sulfoxide) and diluted in culture medium, then deposited in a sterile 96-well microplate. After cell suspension and incubation, the MTT solution was added to each well. The absorbance of each well was read at 550 nm using the VERSAmax microplate reader (Molecular Devices®, France). The percentage of cell growth inhibition (IC%) was calculated using the following equation: IC% = 100 - [(Aa - Ab) / (Ac - Ab)] x 100 Considering that: Aa represents the absorbance of wells containing cells treated with the samples; Ab represents the absorbance of empty wells (blank) and Ac represents the absorbance of wells containing only cells cultured in culture medium + 1% DMSO, with the results obtained in IC%. For all samples, eight replicates were performed in independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. P-values ​​< 0.05 were considered statistically significant. All analyses were performed using GraphPad Prism® 5.0 software (GraphPad Prism Software, Inc., San Diego, CA, USA).

[038] The results indicated that phyllacanthon, at a concentration of 50 μM, exhibited a cell growth inhibition percentage of 35.50 ± 4.81%. The inclusion complexes of phyllacanthon in β-cyclodextrin and in sulfobutyl ether-β-cyclodextrin showed a growth inhibition percentage of 42.98 ± 3.70% and 39.95 ± 2.5%, respectively, both with statistically significant differences. Petition 870240108102, dated 12 / 18 / 2024, page 19 / 27 12 / 14 significant compared to the control group (p < 0.05).

[039] Micrographs for the cytotoxic activity of phylactone and inclusion complexes showed a reduction in cell population promoted by the treatments. These results are in agreement with previous reports by Oliveira Júnior et al. (2022), who described the moderate concentration-dependent cytotoxic potential and cell inhibition promoted by phylactone against A2058 melanoma cells. References

[040] ABRIL-SÁNCHEZ, C. et al. Evaluation of the properties of the essential oil citronellal nanoencapsulated by cyclodextrins. Chemistry and Physics of Lipids, v. 219, p. 72- 78, 2019.

[041] AROZARENA, I.; WELLBROCK, C. Phenotype plasticity as enabler of melanoma progression and therapy resistance. Nature Reviews Cancer, v. 19, n. 7, p. 377- 391, 2019.

[042] AYDOGMU§-OZTÜRK, F. et al. Preclinical study of the medicinal plants for the treatment of malignant melanoma. Molecular Biology Reports, v. 47, n. 8, p. 59755983, 2020.

[043] FENYVESI, É.; VIKMON, M.; SZENTE, L. Cyclodextrins in food technology and human nutrition: benefits and limitations. Critical reviews in food science and nutrition, v. 56, n. 12, p. 1981-2004, 2016.

[044] FERRAZ, C. A. A. et al. Complexation with β-cyclodextrin enhances apoptosismediated cytotoxic effect of harman in chemoresistant BRAF-mutated melanoma cells. European Journal of Pharmaceutical Sciences, v. 150, p. 105353, 2020

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[049] OLIVEIRA-FILHO, RD Development and characterization of molecular complexes of Alpinia zerumbet (Pers.) Burtt & Smith essential oil with sulfobutyl-ether-and-cyclodextrin. Doctoral thesis. Federal University of Ceará, 2019.

[050] OLIVEIRA-JUNIOR, RG et al. Bis-nor-diterpene from Cnidoscolus quercifolius (Euphorbiaceae) induces tubulin depolymerization-mediated apoptosis in BRAF mutant melanoma cells. Chemico-Biological Interactions, v. 355, p. 109849, 2022.

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Claims

1 / 3 CLAIMS 1. “PHILACANTHONE INCLUSION COMPLEX IN β-CYCLODEXTRIN FOR USE AS A CYTOTOXIC AGENT”, characterized by the lyophilized powder containing the bis-nor-diterpene phyllacanthon and the macrocyclic oligosaccharide β-cyclodextrin.

2. “PHILACANTHONE INCLUSION COMPLEX IN β-CYCLODEXTRIN FOR USE AS A CYTOTOXIC AGENT”, characterized by the lyophilized powder containing the bis-nor-diterpene phyllacanthon and the macrocyclic oligosaccharide β-cyclodextrin, according to claim 1, for the purpose of increasing the solubility of phyllacanthon and improving the physicochemical stability of phyllacanthon.

3. “PHILACANTHONE INCLUSION COMPLEX IN β-CYCLODEXTRIN FOR USE AS A CYTOTOXIC AGENT”, characterized by the lyophilized powder containing the bis-nor-diterpene philacanthone and the macrocyclic oligosaccharide β-cyclodextrin, according to claims 1 and 2, with cytotoxic activity against tumor cell lines.

4. "PHILACANTHONE INCLUSION COMPLEX IN β-CYCLODEXTRIN FOR USE AS A CYTOTOXIC AGENT", characterized by the lyophilized powder containing the bis-nor-diterpene phyllacanthon and the macrocyclic oligosaccharide β-cyclodextrin, according to claims 1, 2 and 3, as a cytotoxic agent with application in the treatment of cutaneous melanoma.

5. "PHILACANTHONE INCLUSION COMPLEX IN β-CYCLODEXTRIN FOR USE AS A CYTOTOXIC AGENT", characterized by the lyophilized powder containing the bis-nor-diterpene philacanthone and the macrocyclic oligosaccharide β-cyclodextrin, according to claims 1, 2, 3 and 4, used in pharmaceutical formulations with an antitumor purpose, associated or not with other active ingredients.

6. “PHYLACANTHONE INCLUSION COMPLEX IN SULFOBUTYL ETHER-βCYCLODEXTRIN FOR USE AS A CYTOTOXIC AGENT”, characterized by the lyophilized powder containing the bis-nor-diterpene phylacanthon and the macrocyclic oligosaccharide sulfobutyl ether-βcyclodextrin.

7. “PHYLACANTHONE INCLUSION COMPLEX IN SULFOBUTYL ETHER-βCYCLODEXTRIN FOR USE AS A CYTOTOXIC AGENT”, characterized by the lyophilized powder containing the bis-nor-diterpene phylacanthon and the macrocyclic oligosaccharide sulfobutyl ether-βcyclodextrin, according to claim 6, for the purpose of increasing the solubility of phylacanthon and improving the physicochemical stability of phylacanthon.

8. “PHYLACANTHONE INCLUSION COMPLEX IN SULFOBUTYL ETHER-βCYCLODEXTRIN FOR USE AS A CYTOTOXIC AGENT”, characterized by the lyophilized powder containing the bis-nor-diterpene phylacanthone and the macrocyclic oligosaccharide sulfobutyl ether-βcyclodextrin, according to claims 6 and 7, with cytotoxic activity against tumor cell lines.

9. "PHYLACANTHONE INCLUSION COMPLEX IN SULFOBUTYL ETHER-βCYCLODEXTRIN FOR USE AS A CYTOTOXIC AGENT", characterized by the lyophilized powder containing bis-nor-diterpene phylacanthone and the macrocyclic oligosaccharide sulfobutyl ether-βcyclodextrin, according to claims 6, 7 and 8, as a cytotoxic agent with application in the treatment of cutaneous melanoma.

10. "PHYLACANTHONE INCLUSION COMPLEX IN SULFOBUTYL ETHER-β-CYCLODEXTRIN FOR USE AS A CYTOTOXIC AGENT", characterized by the lyophilized powder containing the bis-nor-diterpene phylacanthone and the macrocyclic oligosaccharide sulfobutyl ether-β-cyclodextrin, according to claims 6, 7, 8 and 9, used in pharmaceutical formulations for antitumor purposes, associated or not with other active ingredients. Petition 870240108102, dated 12 / 18 / 2024, pages 24 / 27 3 / 3