Nanostructured formulations containing sesquiterpene lactones and their use in the production of medicines for the treatment of tumors, hyperuricemia, inflammatory diseases, and gout.

Description

1 / 44 Nanostructured formulations containing sesquiterpene lactones and their use in the production of medicines for the treatment of tumors, hyperuricemia, inflammatory diseases, and gout. FIELD OF THE INVENTION

[001] Hyperuricemia leads to an increase in the formation of urate crystals, which can deposit in the joints and other tissues, causing the development of acute gouty arthritis, which has a prevalence of 1 to 2% in the adult population of developed countries. The number of medications used in the treatment of gout is limited. Furthermore, they have adverse effects and have proven ineffective in treating a considerable portion of patients. In this context, the study of Brazilian biodiversity is promising, since the abundance of natural reserves in Brazil represents the possibility of discovering new therapies. Eremantolide C / eremantolide C (EREC) and goiazensolide / goiazensolide (GOIA) have demonstrated promising pharmacological activities for the treatment of gout in previous studies. However, these sesquiterpene lactones showed low stability in the gastrointestinal tract and toxicity.

[002] In the present work, EREC and GOIA were formulated in liposomes as a strategic alternative to minimize toxicity, improve the stability of these substances, and simultaneously preserve their pharmacological activities. The liposomal formulations containing EREC and GOIA were characterized, evaluated through encapsulation efficiency, stability over 12 months in simulated physiological environments, and their antihyperuricemic, uricosuric, and hepatic xanthine oxidase inhibition activities were evaluated in Wistar rats. Anti-inflammatory activity was also evaluated in C57BL / 6 mice. The formulations exhibited nanometric size, narrow size distribution, and negative zeta potential, indicating their Petition 870260058359, dated 06 / 15 / 2026, page 4 / 49 2 / 44 Stability and uniformity. The efficient encapsulation of EREC and GOIA within liposomes emphasizes their potential for sustained release and therapeutic efficacy. Stability assessment revealed a small decrease in EREC concentration and notable stability in GOIA concentration. In vivo assays demonstrated that liposomal formulations reduced serum uric acid levels in hyperuricemic Wistar rats by increasing uric acid excretion and had an anti-arthritis effect by inhibiting neutrophil migration and the production of pro-inflammatory cytokines in mice induced with gout. Liposomal formulations with eremantolide C and goiazensolide effectively reduced the toxicity associated with these substances while maintaining their therapeutic effects. Liposomal formulations containing EREC and GOIA showed promise for use in the production of drugs for the treatment of neoplasms, hyperuricemia, gout, and inflammatory diseases. STATE OF THE ART

[003] The study carried out by De Souza et al. (https: / / doi.Org / 10.1016 / i.iep.2012.06.012) aimed to investigate whether the ethanolic extract of L. trichocarpha, the ethyl acetate fraction of the ethanolic extract and its main bioactive compounds, including eremantolideC / eremantolide C, can be useful in the treatment of gouty arthritis by combating hyperuricemia and inflammation. The ethanolic extract of L. trichocarpha, the ethyl acetate fraction of the ethanolic extract, and isolated compounds, including the sesquiterpene lactones eremantolide C / eremantolide C (EREC) and licnofolide / licnofolide (LIC), were evaluated for antihyperuricemic activity, reduction of hepatic xanthine oxidase, and anti-inflammatory activity (paw edema induced by monosodium urate crystals) in Swiss mice. LIC and EREC showed an antihyperuricemic effect at a dose of 25 mg / kg. However, they did not show an effect on the inhibition of hepatic xanthine oxidase. Petition 870260058359, dated 06 / 15 / 2026, page 5 / 49 3 / 44 The mechanism of anti-hyperuricemic activity for these sesquiterpene lactones is being elucidated. LIC and EREC reduced carrageenan-induced paw edema in Swiss mice, demonstrating anti-inflammatory activity in this animal model.

[004] Ferrari et al. (https: / / doi.org / 10.1002 / ptr.4736) evaluated the in vitro and in vivo anti-inflammatory activity of sesquiterpene lactones, LIC and EREC. Topical treatment with ointments containing LIC and EREC significantly reduced carrageenan-induced paw edema in Swiss mice. In vitro assays demonstrated that LIC inhibited nitric oxide (NO) production in lipopolysaccharide-stimulated J774A.1 macrophages and increased the production of the anti-inflammatory cytokine IL-10. The reduction in tumor necrosis factor-α (TNF-α) production by EREC was accompanied by a concentration-dependent increase in IL-10 production in J774A.1 macrophages. The anti-inflammatory effect of LIC appears to involve the inhibition of NO production and the increase in IL-10 production in this experimental model. The mechanism of EREC's effect in reducing carrageenan-induced paw edema can be attributed to the inhibition of TNF-α production and the stimulation of IL-10 production.

[005] Saúde-Guimarães et al. (https: / / doi.org / 10·1590 / S151605722014000200017), showed the in vitro antitumor activity of EREC. A sample of EREC was sent to the National Cancer Institute, USA (NCI, USA) where it was evaluated against a panel of 52 tumor cell lines from major human tumors derived from nine types of cancer. EREC completely inhibited the growth of thirty cell lines from eight cancer types, showing activity against all human leukemia (22.40 < IC100 < 50.10 pM) and colon (21.90 < IC100 < 46.80 μM) tumor cell lines, as well as activity against human lung (HOP92, IC100 = 41.70 pM) and central nervous system (U-251, IC100 = 37.10 pM) tumor cells, and five types of melanoma (LOX-IMVI, M14, SK-MEL-2, SK-MEL-5, UACC-62). Petition 870260058359, dated 06 / 15 / 2026, page 6 / 49 4 / 44 21.40 < IC100 < 53.70 μM), two types of ovarian tumor cells (OVCAR-3, OVCAR-5, 34.70 < IC100 < 38.90 μM), four types of renal tumor cells (786-0, RXF-393, SN12-C, TK-10, 28.80 < IC100 < 37.10 μM) and four types of breast tumor cells (MDA-MB-231 / ATCC, MDA-N, BT-549, T-47D, 30.90 < IC100 < 53.70 μM).

[006] All germacranolides with antitumor activity, except eremantolides, possess the α-methylene-γ-lactone group, responsible for the cytotoxicity of the molecule (https: / / doi.orq / 10.1021 / io00262a024). Eremantolides, despite the absence of α-methylene-γ-lactone in their chemical structure, possess antitumor activity. This activity is lower compared to sesquiterpene lactones possessing this group; however, eremantolides are less cytotoxic. Thus, sesquiterpene lactones of the GOIA type exhibit more pronounced antitumor activity than eremantolides (Saúde-Guimarães et al., 2014).This more pronounced activity is related to the presence of the α-methylene-Y-lactone group present in goiazensolides and absent in eremantolides, in addition to the presence of two more electrophilic centers: the angelate group at C-8 and carbon 5 (a ketonic carbonyl conjugated with the two double bonds D2, D4). Eremantolides, in turn, only have carbon 5 as a Michael acceptor center. According to Smith et al., carbon 5 is the electrophilic center responsible for the activity of eremantolides (https: / / doi.org / 10·1021 / ja00396a034).

[007] Ugoline et al. (https: / / doi.Org / 10.1016 / i.iep.2017.01.017) evaluated the anti-hyperuricemic, hepatic xanthine oxidase inhibition, and carrageenan-induced paw edema activities of GOIA in Swiss mice. GOIA (10 mg / kg) was able to reduce serum uric acid levels by inhibiting hepatic xanthine oxidase. Animals treated with GOIA (5.0 mg / kg) had the Petition 870260058359, dated 06 / 15 / 2026, page 7 / 49 5 / 44 Carrageenan-induced paw edema in Swiss mice reduced from the second hour of the experiment.

[008] Bernardes et al. (https: / / doi.Org / 10,1016 / i.bjp.2O18.12,008) demonstrated that EREC and GOIA exhibited anti-hyperuricemic activity at doses of 5 and 10 mg / kg, respectively. GOIA (5 mg / kg) increased urinary uric acid excretion and inhibited uric acid production by inhibiting hepatic xanthine oxidase at a dose of 10 mg / kg. EREC (5 and 10 mg / kg) showed a uricosuric effect and an effect on the inhibition of hepatic xanthine oxidase activity. Thus, these sesquiterpene lactones proved to be promising hypouricemic agents for treating hyperuricemia and gout.

[009] Bernardes et al. (https: / / doi.Org / 10.1016 / i.iep.2020.113738) showed the anti-hyperuricemic, uricosuric and hepatic xanthine oxidase inhibition action of EREC and GOIA in Wistar rats, the anti-inflammatory, antinociceptive and antioxidant action (effects on superoxide dismutase and catalase enzymes) in C57BL / 6 mice.

[0010] EREC and GOIA demonstrated, in previous studies described above, promising pharmacological activities for the treatment of gout. However, these sesquiterpene lactones showed low stability in the gastrointestinal tract and toxicity.

[0011] The predicted in silico physicochemical characteristics indicated that EREC has low solubility and high permeability. In vitro data for eremantholide C showed low solubility, with dose / solubility ratio values ​​(ml) of 9448.82, 10389.61, and 15000.00 for acetate buffer (pH 4.5), enzyme-free simulated intestinal fluid (pH 6.8), and phosphate buffer (pH 7.4), respectively. Furthermore, it showed high permeability, with an effective permeability of 30.4 × 10⁶ cm / s, a higher result compared to propranolol hydrochloride (9.23 × 10⁶ cm / s). Thus, the high permeability combined with its solubility, pharmacological activity, and low toxicity demonstrate the Petition 870260058359, dated 06 / 15 / 2026, page 8 / 49 6 / 44 Importance of EREC C as a potential drug candidate (https: / / doi.org / 10·1007 / s40199-021-00397-6)·

[0012] The polymorphic and physicochemical characterization of EREC, and the influence of these characteristics on its biopharmaceutical classification, were evaluated in two samples. EREC 1 showed melting at 234.7-241.6°C, while EREC 2 showed melting at 238.6-243.7°C. No polymorphic transition was observed during the intrinsic dissolution experiment. A single sharp endothermic peak was obtained for the EREC samples. X-ray diffraction showed no crystallographic differences between the EREC samples. EREC 1 and EREC 2 showed birefringence under polarized light and undefined morphology; however, the crystal shape was common to both samples. Thus, EREC does not exhibit classical or morphological polymorphism; Therefore, there is no influence of crystalline transitions on solubility and consequently on its biopharmaceutical classification and oral absorption process (https: / / doi.org / 10.1111 / jphp·13080).

[0013] Goiazensolide showed thermal stability close to 221.0°C and 210.0°C under a nitrogen and oxygen atmosphere, respectively. In silico data indicated that goiazensolide has high solubility and low permeability. The biopharmaceutical characteristics of goiazensolide are similar to those of commercially available drugs and attest to the feasibility of initiating the development process of a formulation containing this substance (https: / / doi.orq / 10.1093 / jpp / rqac020).

[0014] Liposomes stand out from other drug delivery nanosystems due to their high biocompatibility, versatility in composition and physicochemical characteristics, and ability to accommodate both water-soluble and lipid-soluble active substances. The benefits of incorporating active ingredients into liposomes include increased solubility for poorly water-soluble substances, modulation of their aggregation state, and modulation of their pharmacokinetics, potentially resulting in Petition 870260058359, dated 06 / 15 / 2026, page 9 / 49 7 / 44 Increased bioavailability via non-invasive routes (topical, nasal, pulmonary, and oral), reduced toxicity, and increased therapeutic activity. They can be composed of any lipid mixtures, provided they are capable of generating vesicular structures. Phospholipids are often preferred due to their ability to form bilayers and vesicles and their high biocompatibility. These phospholipids are well described in the state of the art (https: / / doi.org / 10.1002 / ejlt.201400219). Not limited to, liposomes can be conventional, typically formed by phosphatidylcholine, but may also contain cholesterol, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), phosphatidylglycerol, or other ionizable lipids. These liposomes may also include antioxidant lipids such as alpha-tocopherol.Liposomes can also be PEGylated, meaning that, in addition to the lipids used in conventional liposomes, they incorporate a PEGylated lipid in a molar proportion of 3 to 10% relative to the total lipids. Obtaining stable liposomal formulations is one of the challenges in the use of this type of nanosystem. The stability condition depends heavily on the lipid composition and the physicochemical characteristics of the drug used, and generally cannot be predicted a priori.

[0015] In the present invention, eremantolide C / eremantolide C (EREC) and goiazensolide / goiazensolide (GOIA) were formulated in liposomes as a strategic alternative to minimize toxicity, improve the stability of these substances, and simultaneously preserve their pharmacological activities. The liposomal formulations containing EREC and GOIA were characterized, evaluated through encapsulation efficiency, and assessed stability over 12 months in simulated physiological environments. Furthermore, the antihyperuricemic, uricosuric, and hepatic xanthine oxidase inhibition activities were evaluated in hyperuricemic Wistar rats, and the anti-inflammatory activity (inhibition of neutrophil migration and production of the cytokines IL1β, IL-6, and TNF-α) was evaluated in C57BL / 6 mice induced to arthritis by injection of a suspension of monosodium urate crystals into the joint. Petition 870260058359, dated 06 / 15 / 2026, page 10 / 49 8 / 44 thiofemoral. The formulations exhibited nanometric size, narrow size distribution, and negative zeta potential, indicating their stability and uniformity. The efficient encapsulation of EREC and GOIA within liposomes emphasizes their potential for sustained release and therapeutic efficacy. Stability assessment revealed a small decrease in EREC concentration and notable stability in GOIA concentration. In vivo assays demonstrated that the liposomal formulations reduced serum uric acid levels in Wistar rats by increasing uric acid excretion, and the anti-inflammatory effect in the monosodium urate crystal-induced arthritis model in the thiofemoral joint of C57BL / 6 mice occurred due to inhibition of neutrophil migration and reduction of pro-inflammatory cytokine production in the periarticular tissue of gout-induced mice.This study represents an advance in the treatment of gout, hyperuricemia, and inflammatory pain. Currently in Brazil, there are only two medications available for the control of hyperuricemia: allopurinol and lesinurade. However, not all patients respond to these treatments, making it necessary to search for new therapeutic options. Approximately 20% of hyperuricemic patients do not respond to treatment with allopurinol (http: / / dx.doi.org / 10.1016Zj.rbr.2016.06.009), the most widely used drug currently for the control of hyperuricemia. Liposomal formulations with EREC and GOIA effectively reduced the toxicity associated with these substances, while maintaining their therapeutic effects, making these products promising for use in the treatment of hyperuricemia, arthritis / inflammation, inflammatory pain, and gout.These formulations have an effect on reducing both hyperuricemia and the inflammation and inflammatory pain of gout, representing a difference compared to currently available medications for the treatment of hyperuricemia and gout, which do not have all the actions that these formulations have shown. Furthermore, the pharmaceutical formulations containing EREC and GOIA showed the described effects at much lower doses compared to other medications. Petition 870260058359, dated 06 / 15 / 2026, page 11 / 49 9 / 44 current medications and could expand treatment options for these pathologies. The search did not find nanostructured formulations containing the sesquiterpene lactones EREC and GOIA for the treatment of hyperuricemia, inflammatory diseases, gout, and neoplasms. BRIEF DESCRIPTION OF THE FIGURES

[0016] 1 Figure 1: Shows the TLC chromatographic profiles of some of the FAcOEt fractions containing EREC, revealed with anisaldehyde (A) and ceric sulfate (B).

[0017] Figure 2: Shows the chromatographic profiles of some of the FFJb and FFJc fractions and standard EREC, in TLC eluted with Hex:AcOEt (60:40) and revealed with ceric sulfate.

[0018] Figure 3: Shows the chromatographic profiles of some of the FFJ1b and EREC fractions, in TLC eluted with Hex:AcOEt (60:40) and revealed with ceric sulfate.

[0019] Figure 4: Shows standard FE and EREC chromatographic profiles on TLC eluted with Hex:AcOEt (40:60) and revealed with ceric sulfate, without heating.

[0020] Figure 5: Illustrates the scheme for the isolation of eremantolide C from aerial parts of Lychnophora trichocarpha.

[0021] Figure 6: A presents the chromatographic profiles by TLC of the FHex, FAcOEt and FMeOH fractions of the ECL extract of Lychnophora passerina and standard GOIA. Observation under UV light.

[0022] Figure 7: Shows the chromatographic profiles of some of the fractions obtained from the fractionations of FCCC, FDDD, FEEE and FGGG and GOIA, in TLC eluted with Hex:AcOEt (40:60) and revealed with ceric sulfate.

[0023] Figure 8: Shows the chromatographic profiles of some of the fractions obtained from the chromatographic fractionation of FCG and standard GOIA, in TLC eluted with Hex:AcOEt (40:60) and revealed with ceric sulfate. Petition 870260058359, dated 06 / 15 / 2026, page 12 / 49 10 / 44

[0024] Figure 9: Shows the chromatographic profiles of FGb and GOIA in TLC, eluted with Hex:AcOEt (40:60) and revealed with ceric sulfate.

[0025] Figure 10: Illustrates the scheme for the isolation of goiazensolide from aerial parts of Lychnophora passerina.

[0026] Figure 11: Shows the standard curve of eremantolide C (EREC) in a spectrophotometer at 254 nm.

[0027] Figure 12: Shows the standard curve of goiazensolide (GOIA) in a spectrophotometer at 254 nm.

[0028] Figure 13: Illustrates goiazensolide (A) and eremantolide C (B) in liposomes.

[0029] Figure 14: Shows the stability of the liposomal formulations LIPO + EREC (A) and LIPO + GOIA (B) during 0, 1, 2, 3, 6 and 12 months after their development. LIPO + EREC = liposomal formulation containing eremantolide C; LIPO + GOIA = liposomal formulation containing goiazensolide. Values ​​are expressed as mean ± SEM. One-Way ANOVA was used, followed by Tukey's test for statistical significance. *P<0.05 for multiple comparisons. The results show that the developed liposomal formulations are stable for up to 12 months.

[0030] Figure 15: Shows the stability of liposomal formulations containing the sesquiterpene lactones eremantolide C (A) and goiazensolide (B) by HPLC in simulated physiological environments: enzyme-free gastric fluid (pH= 1.2), acetate buffer (pH= 4.5) and simulated enzyme-free intestinal fluid (pH= 6.8), between 0h, 30 min, 1h and 7h. Two-Way ANOVA was used, followed by Tukey's multiple comparisons test for statistical significance. The results show that the stability of the liposomal formulations is influenced by pH.

[0031] Figure 16: Shows serum uric acid levels (A) and uric acid excretion (B) after treatment of hyperuricemic rats with clinically used drugs and liposomal formulations containing sesquiterpene lactones. Petition 870260058359, dated 06 / 15 / 2026, page 13 / 49 11 / 44 LIPO + EREC = liposomal formulation containing eremantolide C; LIPO + GOIA = liposomal formulation containing goiazensolide. Values ​​were expressed as mean ± SEM. One-Way ANOVA was used, followed by Dunnett's test for statistical significance. ***P<0.001 compared to the negative control (hyperuricemic group); ****P<0.0001 compared to the negative control (hyperuricemic group). The results show that the developed formulations are capable of reducing serum uric acid levels in animals induced to hyperuricemia.

[0032] Figure 17: Shows the effects of liposomal formulations containing sesquiterpene lactones on inflammation parameters: neutrophil migration (A) and quantification of IL-1β (B), IL-6 (C) and TNF-α (D) cytokines in the femorotibial periarticular tissue of mice induced to arthritis by an injection of MSU crystals. Negative control = DMSO: Tween 80: distilled water (10:10:80); LIPO = empty liposome; LIPO + EREC = liposomal formulation containing eremantolide C; LIPO + GOIA = liposomal formulation containing goiazensolide. Values ​​were expressed as mean ± SEM. One-Way ANOVA was used, followed by Dunnett's test for statistical significance. *P<0.05; **P<0.01; ***P<0.001 and ****P<0.0001 compared to the negative control. #P<0.05 compared to the negative control (One-Way ANOVA followed by Tukey's test). The results show that sesquiterpene lactones maintained their anti-inflammatory activity in liposomal formulations.

[0033] Figure 18: Shows the effects of liposomal formulations containing sesquiterpene lactones on the activity of the antioxidant enzymes superoxide dismutase (SOD) (A) and catalase (CAT) (B) in the periarticular femorotibial tissue of mice induced to arthritis by an injection of MSU crystals. Negative control = DMSO: Tween 80: distilled water (10:10:80); LIPO = empty liposome; LIPO + EREC = liposomal formulation containing eremantolide C; LIPO + GOIA = liposomal formulation containing goiazensolide. The values Petition 870260058359, dated 06 / 15 / 2026, p. 14 / 49 12 / 44 were expressed as mean ± SEM. One-way ANOVA was used, followed by Dunnett's test for statistical significance. *P<0.05; **P<0.01; ***P<0.001 and ****P<0.0001 compared to the negative control. DESCRIPTION OF THE INVENTION

[0034] This section will show in detail all the processes used in the preparation of nanostructured formulations containing sesquiterpene lactones for the treatment of tumors, hyperuricemia, inflammatory diseases and gout, aiming to describe clearly and sufficiently all the steps in the development of the formulations, supporting the descriptive sufficiency of this invention.

[0035] For the development of nanostructured formulations containing eremantolide C (EREC) and goiazensolide (GOIA), the following steps 1 to 7 must be considered: Step 1 - Processes for obtaining plant extracts

[0036] Process for the extraction of Lychnophora trichocarpha Spreng. The aerial parts of L. trichocarpha (0.945 kg) should be collected in Ouro Preto, Minas Gerais, in July. The dried aerial parts of the plant (0.779 kg) should be pulverized and extracted with 3 L of PA chloroform in a Soxhlet extractor for six hours.

[0037] Process for the extraction of Lychonophora passerina (Mart ex DC.) Gardn. The aerial parts of L. passerina should be collected in Serra do Cipó, Santana do Riacho, Minas Gerais, in June. The aerial parts of the plant species (751.0 g) should be dried in a forced-air oven at 37°C, pulverized in a knife mill (584.45 g) and exhaustively extracted with 7 L of PA chloroform by percolation for 15 days. The solvent should be evaporated under reduced pressure in a rotary evaporator, and the chloroform extract... Petition 870260058359, dated 06 / 15 / 2026, page 15 / 49 13 / 44 (ECL) obtained by keeping in a forced-air oven at 37°C or in a vacuum desiccator until constant weight (approximately 71.951 g). Step 2 - Processes for purifying sesquiterpene lactones

[0038] Process for the purification of EREC from the chloroform extract (ECL1) of L. trichocarpha. The dried chloroform extract (ECL1) should be fractionated by filtration column chromatography (CC) (glass column with 6 cm diameter x 60 cm height; silica gel 60 Merck®, 324.16 g, particles of 70-95 µm). Collect 33 fractions of 250 mL each. The mobile phase should consist of hexane (Hex), ethyl acetate (AcOEt) and methanol (MeOH). The solvents of the obtained fractions should be evaporated in a rotary evaporator at a temperature equal to or below 40°C. The hexane (FHex), ethyl acetate (FAcOEt), and methanolic (FMeOH) fractions must be completely dried in a forced-air oven at 37°C or in a vacuum desiccator until constant weight. Table 1 illustrates the chromatographic fractionation of the chloroform extract of L. trichocarpha. Table 1: Chromatographic fractionation of the chloroform extract of Lychnophora trichocarpha Code _ Dry residue weight Eluent Fractions . , (g) Fhex FAcOEt FMeOH Hex 1-10 6.25 AcOEt 11-28 29.751 MeOH 29-33 4.044

[0039] All fractions must be analyzed by Thin Layer Chromatography [TLC, stationary phase: silica gel 60G Merck, 0.25 mm thick plates, mobile phase: Hex:AcOEt (60:40), developers: ultraviolet (UV) light 365 nm and ceric sulfate]. EREC standard should be used for comparison. TLC analysis of the FAcOEt fraction, after spraying with ceric sulfate, without heating, should show white spots characteristic of LS. After heating the TLC to 100°C, a pink spot with a retention factor (Rf) of 0.7 should be observed, corresponding to EREC. FAcOEt (29.751 g) should be fractionated by CC (glass column with 4 cm diameter and 42 cm height; stationary phase: Petition 870260058359, dated 06 / 15 / 2026, page 16 / 49 14 / 44 silica gel 60 Merck; 110.451 g, particles of 70-95 µm; fractions of 50 mL each). Elution should be performed as indicated in Table 2. Table 2: Chromatographic fractionation of FAcOEt of the chloroform extract of Lychnophora trichocarpha Mobile Phase Collected Fractions Hex Hex:AcOEt (90:10) Hex:AcOEt (80:20) Hex:AcOEt (70:30) Hex:AcOEt (60:40) Hex:AcOEt (50:50) Hex:AcOEt (40:60) Hex:AcOEt (30:70) Hex:AcOEt (20:80) AcOEt MeOH 1-5 6-11 12-91 92-140 141-153 154-162 163-167 168-172 173-177 178-182 183-187

[0040] The fractions obtained from the chromatographic fractionation of FAcOEt should be evaporated under reduced pressure in a rotary evaporator at a temperature equal to or lower than 40°C and evaluated by TLC [Merck® aluminum plates with 0.2 mm thickness, stationary phase: silica gel 60 G Merck®; mobile phase: Hex:AcOEt (60:40); developers: ceric sulfate and anisaldehyde]. The fractions that show spots (bands) and Rfs similar to the EREC standard in TLC (Figure 1) should be pooled, weighed, and coded as shown in Table 3. Table 3: Data from the FAcOEt chromatographic fractionation of the chloroform extract of Lychnophora trichocarpha Combined Fractions Code Presence of LIC Presence of EREC Dry residue weight (g) 1-12 FAA No No - 12-32 FBB Yes No 1.058 33-50 FCC Yes No 1.088 51-57 FDD Yes No 1.251 58-62 FEE Yes No 0.798 63-67 FFF No Yes 1.538 68-77 FGG No Yes 9.792 78-82 FHH No Yes 2.159 83-87 FII No Yes 0.294 88-92 FJJ No Yes 0.267 93-97 FKK No No - 98-108 FLL No No - 109-138 FMM No No - 139-151 FNN No No - 152-187 FOO No No - Petition 870260058359, dated 06 / 15 / 2026, page 17 / 49 15 / 44

[0041] The pooled fraction groups containing EREC (FFF to FJJ) should be pooled, coded as for example FFJ (14.05 g) and fractionated in CC [glass column with 1.2 cm diameter and 58 cm height, stationary phase: silica gel 60 Merck, 14.312 g, particles of 70-95 µm, mobile phases: Hex, AcOEt and MeOH in increasing polarities; collection of 20 mL fractions each]. Elution should be performed as indicated in Table 4. Table 4: Chromatographic fractionation of the FFJ group Eluent Fractions Hex 1-10 Hex:AcOEt (90:10) 11-18 Hex:AcOEt (80:20) 19-34 Hex:AcOEt (75:25) 35-45 Hex:AcOEt (70:30) 46-60 Hex:AcOEt (65:35) 61-72 Hex:AcOEt (60:40) 73-80 Hex:AcOEt (55:45) 81-85 Hex:AcOEt (50:50) 86-90 Hex:AcOEt (40:60) 91-95 MeOH 95-100

[0042] Analysis of fractions 23 to 50 (FFJb and FFJc) by TLC [Merck® aluminum plates with 0.2 mm thickness, stationary phase: 60 G Merck® silica gel; mobile phase: Hex:AcOEt (60:40); developers: ceric sulfate and anisaldehyde], should show the presence of a stain (band) and Rf similar to that of the EREC standard, in addition to other substances (Table 5, Figure 2). These fractions should be pooled, weighed (6.018 g) and coded, for example FFJ1. FFJ1 should be fractionated in CC (stationary phase: Merck silica gel, 14.225 g; 70-95 µm particles; 1.2 cm diameter and 58 cm high glass column; mobile phases: Hex and AcOEt in increasing polarities; 20 mL fractions each) and eluted as shown in Table 6. Table 5: Data from the pooled fractions of the chromatographic fractionation of FFJ Combined fractions Code Presence of EREC Dry residue weight (g) 1-22 FFJa 23-38 FFJb No - Yes 5,125 Petition 870260058359, dated 06 / 15 / 2026, page 18 / 49 16 / 44 39-50 FFJc Yes 0.893 51-62 FFJd No - 63-80 FFJe No - 82-90 FFJf No - 91-100 FFJg No - Table 6: Chromatographic fractionation of FFJ1 Eluent Fractions Hex 1-5 Hex:AcOEt (95:5) 6-10 Hex:AcOEt (90:10) 11-15 Hex:AcOEt (80:20) 16-25 Hex:AcOEt (70:30) 26-40 Hex:AcOEt (65:35) 41-68 Hex:AcOEt (60:40) 69-85 Hex:AcOEt (55:45) 86-96 Hex:AcOEt (50:50) 97-100 MeOH 101-105

[0043] The fractions obtained must be evaporated at a temperature equal to or lower than 40°C. The chromatographic process must be monitored by TLC [Merck® aluminum plates with 0.2 mm thickness, stationary phase: 60 G Merck® silica gel; mobile phase: Hex:AcOEt (60:40); developers: ceric sulfate and anisaldehyde]. Fractions 33 to 63 must show the presence of a spot (band) and Rf similar to the EREC standard (Figure 3). These fractions must be combined, dried, weighed, and coded as FFJ1b as shown in Table 7. The solid present in FFJ1b must be added to 2 mL of ethyl ether, subsequently separated, filtered, dried, weighed (1.523 g), and coded as FE. FE should be compared by TLC with the standard EREC [Merck® aluminum plates with 0.2 mm thickness, stationary phase: silica gel 60 G Merck®; mobile phase: Hex:AcOEt (60:40); developers: ceric sulfate and anisaldehyde].FE should exhibit a chromatographic profile and Rf similar to that of standard EREC, indicating no presence of impurities (Figure 4). The complete scheme for isolating EREC from L. trichocarpha is illustrated in Figure 5. Table 7: Data on the fractions and coded groups from the chromatographic fractionation of FFJ1 Combined Fractions Code Presence of EREC Dry residue weight (g) 1-32 FFJ1a No - 33-63 FFJ1b Yes 2.658 64-85 FFJ1c No - Petition 870260058359, dated 06 / 15 / 2026, page 19 / 49 17 / 44 86-105 FFJ1d No

[0044] Process for the purification of GOIA from the chloroform extract (ECL2) of L. passerina. The dry ECL2 (71.951g) should be fractionated in a filtration column (glass column with 6cm diameter and 60cm height; stationary phase: silica gel 60 Merck, 345.81, particles of 70-95 µm; mobile phases: Hex, AcOEt and MeOH, fractions of 250mL each), as shown in Table 8. The solvents should be evaporated and the fractions kept in a forced-air oven at 37°C or in a vacuum desiccator until constant weight, generating the dry hexane (FHex), ethyl acetate (FAcOEt) and methanolic (FMeOH) fractions. Analysis of the FAcOEt fraction by TLC (Figure 6) should show the presence of GOIA, in addition to other substances. FAcOEt should be fractionated in CC (glass column with 4 cm diameter and 42 cm height; stationary phase: silica gel 60 Merck, 100,524, particles of 70-95 µm, 50 mL fractions), and eluted as indicated in Table 9. Table 8: Chromatographic fractionation of the chloroform extract of Lychnophora passerina Code Mobile Phase Collected Fractions Dry Residue Weight (g) FHex Hex 1-10 1.796 FAcOEt AcOEt 11-46 40.735 FMeOH MeOH 47-51 3.161 Table 9: Chromatographic fractionation of FAcOEt from Lychnophora passerina Mobile Phase | Fractions Collected | Hex | 1-13 | Hex:AcOEt (90:10) | 14-24 | Hex:AcOEt (80:20) | 25-50 | Hex:AcOEt (70:30) | 51-60 | Hex:AcOEt (60:40) | 61-80 | Hex:AcOEt (50:50) | 81-101 | Hex:AcOEt (40:60) | 102-109 | Hex:AcOEt (30:70) | 110-114 | Hex:AcOEt (20:80) | 115-119 | AcOEt | 120-124 | MeOH | 125-129 Petition 870260058359, dated 06 / 15 / 2026, page 20 / 49 18 / 44

[0045] The fractions obtained should be evaporated in a rotary evaporator, at a temperature equal to or lower than 40°C, and analyzed by TLC [silica gel 60 G, mobile phase Hex:AcOEt (40:60), developers ceric sulfate and anisaldehyde], together with the GOIA standard. Fractions 21 to 57 (FCCC to FGGG) should present chromatographic profiles and Rf similar to the GOIA standard (Figure 7). These should be pooled, weighed (15.208g) and coded as FCG, as shown in Table 10. FCG should be fractionated in CC [glass column 2.5cm in diameter and 76cm high; Stationary phase: Merck flash silica gel, 17.588 g, particles of 35-70 µm, mobile phase: dichloromethane:hexane (60:40), elution was isocratic, column pressure, 2 mL / minute, collect 189 fractions]. The fractions should be evaporated at a temperature equal to or below 40°C and analyzed by TLC [Merck 60 g silica gel, eluted with Hex:AcOEt (40:60), developers: exposure under UV light, followed by spraying with ceric sulfate and heating to 100°C].Fractions 84 to 97 should show spots and Rf values ​​similar to those presented by the GOIA standard (Figure 8). These fractions should be combined, weighed (3.819 g) and designated FGa. FGa should appear as a solid with a melting point range of 165.8-168.3°C, indicating the presence of GOIA and impurities. 5 mL of ethyl ether should be added to the solid, which should then be filtered, dried, weighed (1.225 g) and coded as FGb. Samples of FGb and standard GOIA should be applied to TLC [silica gel 60 G Merck, eluted with Hex:AcOEt (40:60) and revealed under UV light exposure, followed by spraying with ceric sulfate or anisaldehyde]. FGb should show a chromatographic profile and Rf value similar to the GOIA standard, without the presence of impurities (Figure 9). Figure 10 shows the scheme for the isolation of goiazensolid from aerial parts of L. passerina. Table 10: Data from the pooled fractions and coded groups of the chromatographic fractionation of FAcOEt from Lychnophora passerina Combined fractions Code Presence of GOIA Dry residue weight (g) 1-12 FAAA No - Petition 870260058359, dated 06 / 15 / 2026, page 21 / 49 19 / 44 15-20 FBBB No - 21-27 FCCC Yes 2.159 33-41 FDDD Yes 12.111 42-50 FEEE Yes 0.470 51-57 FGGG Yes 0.468 58-63 FHHH No - 64-77 FIII No - 78-93 FJJJ No - 94-129 FKKK No - Step 3 - Process for the chemical characterization of sesquiterpene lactones.

[0046] Procedure for characterizing the chemical structure of EREC and GOIA. FE and FGb should be solubilized in deuterated chloroform (CDCl3) and filtered in NMR tubes. The NMR spectra of FE and FGb should be obtained on a 400 MHz NMR spectrometer. TMS should be used as an internal reference standard. The coupling constants (J) should be calculated in Hertz (Hz), and the chemical shifts (δ) calculated in parts per million (ppm). The data from the obtained spectra should be compared with literature data for eremantolide C and goiazensolide, respectively. Analysis of the melting point of FE should show a melting range close to 215-215.3°C, in accordance with that of eremantolide C from the literature (214-215°C). FE should exhibit 1H and 13C NMR chemical shifts, COSY1H-1H, HSQC and HMBC (Tables 11 to 14) similar to those found in the literature for eremantolide C (Saúde et al., 1998; Saúde-Guimarães et al., 2007; Saúde-Guimarães et al., 2014).Analysis of the melting point of FGb should show a melting range (165.2-166.1°C) close to the range described for GOIA in the literature (168.7-169.5°C). FGb should exhibit 1H and 13C NMR chemical shifts, COSY WH, HSQC and HMBC (Tables 15 to 18) in accordance with literature data for goiazensolide (Soares et al., 2012). Table 11: 1H NMR data of FE (400 MHz) and eremantolid C (300 MHz; SaúdeGuimarães et al., 2007) in CDCLHERemantolide C __H(Saúde-Guimarães et al., 2007) _________________ ____________ Petition 870260058359, dated 06 / 15 / 2026, p. 22 / 49 20 / 44 δ (ppm) J (Hz) Multiplicity δ (ppm) J (Hz) Multiplicity material H-2 5.63 - s 5.63 - s H-5 6.04 - m 6.03 - m H-6 5.00 - m 5.00 - m H-7 2.82 4.3; 7.1 dd 2.83 4.0; 7.2 dd H-8 4.09 2.5; 4.3; 12 ddd 4.10 2.8; 4.0; 12.0 ddd H-9a 2.48 2.25; 13.5 dd 2.48 2.8; 13.6 dd H-9b 2.00 12; 13.5 dd 2.00 1.6; 7.2 dd H-13 1.18 - s 1.18 - s H-14 1.45 - s 1.46 - s H-15 2.05 2.1 t 2.06 2.0 t H-2'a 5.31 - s 5.31 - sl H-2'b 5.07 1.8 t 5.07 1.6 tl H-3' 1.91-s 1.91-s OH 3.79-s 3.54-s Table 12: 13C NMR data of FE (100 MHz) and eremantolide C (75 MHz; SaúdeGuimarães et al., 2007) at CDCI3 C Eremantholide C (Saúde-Guimarães et al., 2007) FE δ (ppm) δ (ppm) C1 205.89 205.92 C2 104.54 104.54 C3 187.27 187.33 C4 130.00 130.23 C5 134.77 134.72 C6 81.46 81.47 C7 62.53 62.48 C8 78.37 78.40 C9 43.46 43.50 C10 90.24 90.37 C11 59.88 60.05 C12 175.72 175.87 C13 21.94 21.93 C14 20.48 20.51 C15 20.30 20.35 C16 106.09 106.30 C1' 144.22 142.33 C2' 115.80 115.92 C3' 19.00 19.01 Table 13: COSY 1H-1H data of FE (CDCl3, 400 MHz) H δ 1H δ H COSY 1H-1H FE FE H-2 5.63 s - H-5 6.03 m H-6 (δ 5.00) H-15 (δ 2.06) H-5 (δ 6.03) H-6 5.00 m H-7 (δ 2.83) H-15 (δ 2.06) H-7 2.83 dd H-6 (δ 5.00) H-8 (δ 4.10) H-8 4.10 ddd H-7 (δ 2.82) Petition 870260058359, de 15 / 06 / 2026, pág. 23 / 49 21 / 44 H-9a (d 2.47) H-9b (d 2.00) H-9a 2.48 dd H-8 (d 4.10) H-9b (d 2.00) H-9b 2.00 dd H-8 (d 4.10) H-9a (d 2.47) H-13 1.18 s - H-14 1.46 s - H-15 2.06 t H-5 (d 6.03) H-6 (d 5.00) H-2'a 5.31 sl H-2'b (d 5.07) H-3' (d 1.91) H-2'b H-3' 5.07 tl 1.91 s H-2'a (d 5.31) H-3' (d 1.91) H-2'a (d 5.31) H-2'b (δ 5.07) OH 3.54 s - Tabela 14: Dados de HSQC e HMBC de FE (CDCh, 400 MHz) C δ 13C δ H HSQC δ H HMBC C1 205.92 - H-9b (δ 2.00); H-14 (δ 1.46) C2 104.54 H-2 (δ 5.63 s) - C3 187.33 - H-2 (δ 5.63); H-15 (d 2.06) C4 130.23 - H-15 (d 2.06) C5 134.72 H-5 (d 6.03 m) H-7 (d 2.82); H-15 (δ 2.06) C6 81.47 H-6 (δ 5.00 m) H-7 (δ 2.83) C7 62.48 H-7 (δ 2.83 dd) H-13 (δ 1.18) C8 78.40 H-8 (δ 4.10 ddd) H-9a (δ 2.48); H-9b (δ 2.00) C9 43.50 H-9a (δ 2.48 dd) H-9b (δ 2.00 dd) H-14 (δ 1.46) C10 90.37 - H-2 (δ 5.63); H-9b (δ 2.00) H-14 (δ 1.46) C11 60.05 - H-7 (δ 2.83); H-13 (δ 1.18) OH (δ 3.54) C12 175.87 - H-13 (δ 1.18) C13 21.93 H-13 (δ 1.18 s) H-7 (δ 2.83) C14 20.51 H-14 (δ 1.46 s) - C15 20.35 H-15 (δ 2.06 t) - H-7 (δ 2.82); H-13 (δ 1.18) C16 106.30 - H-2'a (δ 5.31); H-2'b (δ 5.07) H-3' (δ 1.91); OH (d 3.54) C1' 142.33 - H-3' (d 1.91) C2' 115.92 H-2'a (d 5.31) H-2'b (d 5.07) H-3' (d 1.91) C3' 19.01 H-3' (d 1.91) H-2'a (d 5.31) / H-2'b (d 5.07) Table 15: 1H NMR data of FGb (400 MHz) and goiazensolide (500 MHz; Soares et al., in CDCl3. H Goiazensolida (500 MHz, Soares et al., 2012) FGb δ (ppm) J (Hz) Multiplicidade δ (ppm) J (Hz) Multiplici dade H-2 5.83 - s 5.83 - s H-5 6.28 2.9; 1.5 dt 6.28 - m Petition 870260058359, of 15 / 06 / 2026, p. 24 / 49 22 / 44 H-6 5.34 2.9; 4.8; 2.1; 0.6 dddt 5.34 - m H-7 3.80 4.8; 2.6; 2.6; 0.8 dddt 3.80 - m H-8 4.54 2.6; 11.7; 2.1; 0.6 dddd 4.53 - m H- 9a 2.51 11.7; 13.8 dd 2.53 12.0; 14.0; dd H- 9b 2.32 2.1; 13.8; 0.8 ddd 2.33 1.6; 14.0 dd H13a 6.21 3.1; 0.7 dd 6.23 3.2 d H- 13b 5.48 2.6; 0.7 dd 5.49 2.4 d H- 14 1.54 - s 1.54 - s H- 15a H- 4.36 2.1; 1.5; 14.5 ddd 4.39 - sl 15b 4.40 2.1; 1.5; 14.5 ddd H- 3'a 6.02 0.8; 1.5 min 6.02 - sl H- 3'b 5.56 1.2; 1.5 dq 5.56 1.2 tl H- 4' 1.84 0.8; 1.2 dd 1.84 - sl Table 16: De13C NMR data of FGb (100 MHz) and goiazensolide (125 MHz, Soares et al., 2012) in CDCI3.' C Goiazensolida (125 MHz, Soares et al., 2012) FGb δ (ppm) δ (ppm) C1 204.7 204.7 C2 106.7 106.7 C3 184.4 184.5 C4 134.5 134.5 C5 135.3 135.1 C6 81.6 81.7 C7 50.9 50.9 C8 73.3 73.3 C9 43.9 43.9 C10 89.8 89.8 C11 133.2 133.1 C12 168.8 168.9 C13 124.7 124.8 C14 20.7 20.7 C15 63.2 63.1 C1' 166.9 166.9 C2' 135.4 135.3 C3' 126.6 126.7 C4' 17.9 18.0 Table 17: Data of COSY WH from FGb (CDCl3, 400 MHz) H δ 1H δ H COSY 1H-1H FGb FGb H-2 5.83 s - H-5 6.28 m H-6 (δ 5.34) H-5 (δ 6.28) H-6 5.34 m H-7 (δ 3.80) H-15a (δ 4.39) Petition 870260058359, 15 / 06 / 2026, pág. 25 / 49 23 / 44 H-7 3,80 m H-15b (δ 4,39) H-6 (δ 5,34) H-8 (δ 4,53) H-13a (δ 6,22) H-13b (δ 5,48) H-7 (δ 3,80) H-8 4,53 m H-9a (δ 2,53) H-9b (δ 2,33) H-9a 2,53 dd H-8 (δ 4,53) H-9b (δ 2,33) H-9b 2,33 dd H-8 (δ 4,53) H-9a (δ 2,53) H-13a 6,22 d H-7 (δ 3,80) H-13b 5,48 sl H-7 (δ 3,80) H-14 1,54 s - H-15a H-15b 4,39 sl H-6 (δ 5,34) H-3’a H-3’b 6,02 sl 5,56 tl H-3’b (δ 5,56) H-4’ (δ 1,77) H-3’a (δ 6,02) H-4’ (δ 1,77) H-4’ 1,84 s H-3’a (δ 6,02) H-3’b (δ 5,56) Table 18: HSQC and HMBC data from FGb (CDCh, 400 MHz) C δ 13C δ H HSQC δ H HMBC C1 204.7 - - C2 106.7 H-2 (δ 5.83) - C3 184.5 - - C4 134.5 - - C5 135.1 H-5 (δ 6.28) - C6 81.7 H-6 (δ 5.34) - C7 50.9 H-7 (δ 3.80) - C8 73.3 H-8 (δ 4.53) - C9 43.9 H-9a (δ 2.53) H-9b (δ 2.33) - C10 89.8 - - C11 133.1 - - C12 168.9 - - C13 124.8 H-13a (δ 6.23) H-13b (δ 5.49) - C14 20.7 H-14 (δ 1.84) - C15 63.1 H-15a (δ 4.39) H-15b (δ 4.39) - C1' 166.9 - - C2' 135.3 - - C3' 126.7 H-3'a (δ 6.02) H-3'b (δ 5.56) H-13a (δ 6.22) H-13b (δ 5.49) H-4' (δ 1.84) C4' 18.0 H-4' (δ 1.84) H-3'a (δ 6.02) H-3'b (δ 5.56) Step 4 - I prepare the nano-structured formulas based on sesquiterpenic lactones Petition 870260058359, 15 / 06 / 2026, pág. 26 / 49 24 / 44

[0047] Two liposomal formulations were prepared: LIPO + EREC (liposomal formulation containing soy phosphatidylcholine and EREC) and LIPO + GOIA (liposomal formulation containing soy phosphatidylcholine and GOIA), both formulations with a lipid:substance ratio of 10:0.5 to 40:2, preferably 20:1. For each of the two formulations, 73 mg of soy phosphatidylcholine (PC) (Phospholipon® 90G, Lipoid) were weighed into eppendorfs numbered 1 and 2. This amount of soy PC was dissolved in 75 µL of ethanol (EtOH). The solution was then gently stirred on a magnetic stirrer under heating at 40°C until completely dissolved. In parallel, 3.5 mg of EREC and 3.6 mg of GOIA were weighed. Next, these substances were added to Eppendorf tubes 1 and 2, respectively, containing the lipid. Thus, in each of the Eppendorf tubes, a ratio of 1:20 of LS:lipid was obtained. The resulting solution was incubated at 40°C until complete dissolution.PBS 1X was prepared by diluting 100 mL of PBS 10X (82 g sodium chloride; 10.5 g disodium phosphate and 3.55 g monobasic sodium phosphate in 1 L of Milli-Q water) in 900 mL of Milli-Q water (pH adjusted to 7.4). The ethanolic lipid solution was then rapidly injected into 750 µL of PBS 1X solution (pH = 7.4) using a 1 mL syringe, and the resulting suspension was maintained for 15 minutes at room temperature under magnetic stirring. Liposome size was calibrated by repeated extrusions (10 times) (Avanti® Mini-Extruder) onto polycarbonate membranes with 200 and 100 nm pores (Whatman® Nuclepore™). The liposome suspension was dialyzed against 1X PBS using a membrane (Spectra / Por® 6 Dialysis) with a molecular weight cutoff of 15 kDa (4h; 4°C) for EtOH removal. Empty liposomes (without substance) (LIPO) were also prepared using the same protocol, but without the addition of any substance. The prepared formulations were stored at 4°C. Step 5 - Characterization of the formulations Petition 870260058359, dated 06 / 15 / 2026, page 27 / 49 25 / 44

[0048] The average hydrodynamic diameter (z-mean), polydispersity index (PDI), and zeta potential (ζ) of vesicles in suspensions were determined by dynamic light scattering using a nanoparticle size analyzer (Zetasizer S90; Malvern, UK) after dilution of liposome suspensions in 1X PBS (1:100 v / v). These analyses were performed in triplicate.

[0049] LIPO + EREC and LIPO + GOIA were characterized in relation to the average hydrodynamic particle diameter (z-mean), PDI and zeta potential (ζ). These data are shown in Table 19. In both formulations developed, unimodal and narrow particle size distributions were evidenced by polydispersity index (PDI) values ​​of 0.03 to 0.15. LIPO + EREC and LIPO + GOIA presented nanometric size with a diameter between 106 and 113 nm, preferably between 108 and 112 nm. The size calibration process used 100 nm polycarbonate membranes. There was no significant difference between the size or PDI of the liposomal formulations containing the LS (LIPO + EREC and LIPO + GOIA) compared to the empty liposome (LIPO). Slightly negative zeta potentials were found in LIPO + EREC, LIPO + GOIA, and also in LIPO, with no significant difference between them.Sesquiterpene lactones were quantified in the initial (before dialysis) and final (after dialysis) liposome suspensions after redissolving the liposomal formulation in EtOH (1:20 v / v dilution), by measuring the absorbance at 254 nm (Cary® 50 UV-Vis spectrophotometer) and subsequently comparing it with standard curves constructed from pure EREC and GOIA solubilized in EtOH at different concentrations. This analysis was performed in triplicate.

[0050] The drug encapsulation efficiency (EE) was determined as: v Sesquiterpene lactone concentration in the final suspension EE (%) - 100 X-------------------------------;--------------------------;--Concentration of sesquiterpene lactone in the suspension before dialysis Petition 870260058359, dated 06 / 15 / 2026, page 28 / 49 26 / 44

[0051] The quantification of EREC and GOIA in liposomal formulations and the determination of encapsulation efficiency (EE) were performed using standard curves prepared with these pure substances. These standard curves provided linear equations, in which the absorbance exhibited by the sesquiterpene lactones in the spectrophotometer reading at 254 nm was a function of their concentration. The standard curves obtained are shown in Figures 11 and 12. From the linear equations obtained from the standard curves, the concentrations of EREC and GOIA in each formulation and the EE (%) were determined, which are expressed in Table 21. In both formulations developed, the unimodal and narrow particle size distributions were evidenced by PDI values ​​lower than 0.1. All formulations presented nanometric size with a diameter around 110 nm.There was no significant difference in size or PDI between the LIPO + EREC and LIPO + GOIA formulations compared to the empty liposome (LIPO). Slightly negative zeta potentials were found in both liposomal formulations LIPO + EREC and LIPO + GOIA, and also in LIPO, with no significant difference between them. LIPO + EREC showed an EREC concentration of 27.5 ± 6.5 pg / mL and EE close to 70%. LIPO + GOIA showed a GOIA concentration of 42.0 ± 5.0 pg / mL and EE of almost 80%. Table 19: Characterization of liposomal formulations containing sesquiterpene lactones (LS) Liposomal formulation z-average (nm ± SEM) PDI (value ± SEM) ζ (mV ± SEM) LS concentration (pg / mL ± SEM) EE (% ± SEM) LIPO * 110.9 ± 6.3 0.086 ± 0.031 -5.1 ± 1.6 - - LIPO + EREC * 108.5 ± 7.9 0.053 ± 0.026 -4.4 ± 0.1 27.5 ± 6.5 69.79 ± 3.550 LIPO + GOIA * 111.5 ± 2.3 0.049 ± 0.014 -4.7 ± 0.7 42.00 ± 5.0 79.62 ± 6.415 * LIPO = empty liposome (without substance); LIPO + EREC = liposomal formulation containing eremantolide C; LIPO + GOIA = liposomal formulation containing goiazensolide. EE - Encapsulation efficiency. Petition 870260058359, dated 06 / 15 / 2026, page 29 / 49 27 / 44

[0052] Figure 13 shows EREC and GOIA represented in liposomes. Step 6 - Evaluation of the stability of liposomal formulations for 12 months.

[0053] The LIPO + EREC and LIPO + GOIA formulations were evaluated for their stability after storage at 4°C, in relation to particle size distribution and sesquiterpene lactone concentration. Analyses were performed in triplicate after 1 week (i.e., 0 months) and 1, 2, 3, 6, and 12 months. Initially, the formulations were diluted in EtOH (1:20 v / v) and then read at 254 nm using a spectrophotometer (Cary® 50 UV-Vis). The results were calculated by projecting the absorbances onto previously constructed standard curves. The results are presented in Figure 14. At baseline (month 0), LIPO + EREC showed an EREC concentration of 27.21 ± 0.81 pg / mL, and subsequent months demonstrated a stable trend, with no statistically significant difference between them.When comparing each time point to the initial moment, only a small reduction in EREC concentration was observed (9.11%; 9.96%; 10.58% and 9.74% at 1, 2, 3 and 12 months, respectively). LIPO + GOIA was able to maintain the initial GOIA concentration for up to 12 months (Figure 14B). There was no significant difference in GOIA concentration during the evaluated time points. The concentration values ​​were 43.11 ± 0.23 pg / mL; 43.66 ± 0.41 pg / mL; 46.34 ± 1.0 pg / mL; 46.02 ± 1.0 pg / mL; 44.06 ± 1.1 pg / mL and 44.20 ± 0.66 pg / mL during 1 week and 1, 2, 3, 6, and 12 months after formulation development, respectively. This stability exhibited by the GOIA formulation can be attributed to the presence of the α-methyleneγ-lactone group in its structure, which stabilizes the molecule by resonance. Step 7 - Evaluation of the stability of liposomal formulations by HPLC in simulated physiological environments. Petition 870260058359, dated 06 / 15 / 2026, pp. 30 / 49 28 / 44

[0054] The solutions used were prepared as follows: i. Simulated gastric fluid without enzymes (SGF) (pH= 1.2): 2 g sodium chloride + 7 mL hydrochloric acid; volume made up to 1 L with Milli-Q water followed by pH adjustment. ii. Acetate buffer (pH = 4.5) = 2.99g sodium acetate trihydrate + 14mL 2M acetic acid; volume made up to 1 L with Milli-Q water followed by pH adjustment (Digimed DM20 pH meter) (United States Pharmacopeia, 2014; Caldeira et al., 2017). iii. Simulated intestinal fluid without enzymes (FISSE) (pH= 6.8) = 250 mL of a 0.2 M monobasic potassium phosphate solution + 112 mL of a 0.2 M sodium hydroxide solution; volume made up to 1 L with Milli-Q water followed by pH adjustment (Digimed DM20 pH meter) (United States Pharmacopeia, 2014; Caldeira et al., 2017). iv. EREC and GOIA that were efficiently encapsulated with liposomes should be quantified using HPLC (Waters Alliance® e2695) coupled to a UV-Vis detector (Waters 2489). The chromatographic conditions are described in Table 20. HPLC methods for the quantification and stability assessment of LIPO + EREC and LIPO + GOIA formulations in FGSSE (pH= 1.2), acetate buffer (pH= 4.5) and FISSE (pH= 6.8) were proposed based on an adaptation of the method developed and validated by Caldeira et al. (2017) for EREC; and by Ugoline et al. (2017) and Tana et al. (2022) for GOIA. The analytical curves were obtained by integrating the peak areas of EREC and GOIA quantified by HPLC at 267 nm. Variations in the peak areas of EREC and GOIA were analyzed at time zero, 30 minutes, 60 minutes, and 7 hours. Additionally, peak purity was evaluated using a diode array detector. Petition 870260058359, dated 06 / 15 / 2026, page 31 / 49 29 / 44 to attest to the absence of co-elution of other interfering substances with the chromatographic signal of sesquiterpene lactones. The results obtained were subjected to analysis of variance (ANOVA) using GraphPad Prism 8.0 software, followed by appropriate post-tests, and were expressed as mean ± SEM. A 95% confidence interval was adopted, with differences considered significant when the P-value was less than 0.05 (P < 0.05). The results are shown in Figure 15. Table 20: Chromatographic conditions for quantification of eremantolide C and goiazensolide by HPLC Chromatographic conditions Eremantolide C (EREC) (Caldeira et al., 2017) Goiazensolida (GOIA) (Ugoline et al., 2017; Tana et al., 2022). UV-Vis Detector UV-Vis Wavelength (nm) 267 267 Acidified water (acetic acid) 0.01%: acetonitrile (60:40) Acetonitrile: water (50:50) C18 (150 x 4.6 mm; 5 µm) C18 column (150 x 4.6 mm; 3 µm) Agilent Zorbax Eclipse XDB Temperature (°C) 30 30 Injection volume (pL) 25 20 Flow rate (mL / min) 1 1 Concentration range 10.0- 50.0 pg / mL 10.0- 50.0 pg / mL Retention time (min) 6.5 3.5

[0055] The stability of LIPO + EREC and LIPO + GOIA formulations in simulated physiological environments including FGSSE (pH= 1.2), acetate buffer (pH= 4.5) and FISSE (pH= 6.8) was evaluated by HPLC. As shown in Figure 15A, the initial concentration (time 0h) of EREC in LIPO + EREC was 11.23 pg / mL, 14.38 pg / mL and 15.15 pg / mL at pHs = 1.2, 4.5 and 6.8, respectively. After 30 minutes, a significant decrease in the EREC concentration to 8.87 pg / mL (21.02%) was observed at pH= 1.2, which may suggest the beginning of some degradation or instability in the acidic gastric environment. This result was expected, since, as described by Caldeira et al. (2017), EREC has a partition coefficient. Petition 870260058359, dated 06 / 15 / 2026, pp. 32 / 49 The 30 / 44 octanol-water (logP) ratio is approximately 2.0, thus resulting in greater permeability. The lower stability of EREC at acidic pHs is due to hydrolysis of the lactones in acidic environments. Furthermore, the absence of the α-methyleneγ-lactone group in the EREC structure prevents its resonance stabilization, as occurs with GOIA (Caldeira et al., 2017). At pH = 4.5, a slight decrease in concentration to 14.32 pg / mL (0.42%) was observed. At pH = 6.8, an increase in concentration to 15.35 pg / mL (1.32%) was observed, indicating a possible response to the less acidic environment. After 1 h, at pH = 1.2, the EREC concentration remained low (8.9 pg / mL), suggesting a sustained impact of gastric conditions. At pH = 4.5, the concentration increased by 12.57% (16.12 pg / mL). At pH = 6.8, a decrease of 8.08% (14.11 pg / mL) was observed, indicating a slight change in stability.After 7h, at pH= 1.2, a decrease in EREC concentration to 8.01 pg / mL (10.0%) was observed, again indicating the impact of gastric conditions. At pH= 4.5, a significant increase to 17.67 pg / mL (9.62%) was observed. At pH= 6.8, the EREC concentration decreased to 11.55 pg / mL (18.14%). As shown in Figure 15B, the initial concentration (time 0h) of GOIA in LIPO + GOIA was 15.99 pg / mL, 23.19 pg / mL, and 18.53 pg / mL at pHs = 1.2, 4.5, and 6.8, respectively. After 30 minutes, a significant increase in GOIA concentration to 17.17 pg / mL (7.38%) was observed at pH=1.2, which may suggest some potential alterations in the liposomal formulation in the gastric environment. At pH=4.5, an increase in concentration to 25.12 pg / mL (8.32%) was observed, which may indicate a possible release or alteration of GOIA in the acetate buffer.At pH= 6.8, an increase in concentration to 25.12 pg / mL (35.56%) was observed, indicating a possible response to the less acidic environment. This result was expected because, as demonstrated by Tana et al. (2022), GOIA has a logP of 1.3-1.4, thus favoring its intestinal absorption. After 1 h, at pH= 1.2, the GOIA concentration increased to 17.71 pg / mL (3.15%). At pH= 4.5, a. Petition 870260058359, dated 06 / 15 / 2026, pp. 33 / 49 31 / 44 concentration decreased to 24.46 pg / mL (2.63%). At pH= 6.8, a decrease of 3.57% (17.84 pg / mL) was observed, indicating a slight change in stability. After 7 h, at pH= 1.2, the GOIA concentration continued to increase to 18.26 pg / mL (3.11%), indicating stabilization under gastric conditions. At pH= 4.5, a significant increase to 25.84 pg / mL (5.64%) was observed. At pH= 6.8, the GOIA concentration increased to 18.73 pg / mL (4.99%). As demonstrated by Tana et al. (2022), GOIA has a pKa of 15.02, indicating a weakly acidic character, which causes it to remain in its non-ionized form throughout the physiological pH range. Due to the degree of ionization of GOIA in buffered media, it is possible to assume that there is no interference in the extent of its absorption in the GI tract. Step 8 - In vivo evaluation of anti-hyperuricemic activity

[0056] The anti-hyperuricemic activity assays of liposomal formulations containing sesquiterpene lactones were performed on male Wistar rats weighing between 180 and 280g, provided by the Animal Science Center of the Federal University of Ouro Preto. The rats were housed in groups of four, kept in an environment with controlled temperature (20-24°C), 12h / 12h light / dark cycle, with water and food ad libitum (pelleted and irradiated feed for mice, rats and hamsters, Nuvilab® brand).The experimental protocol received prior approval from the Ethics Committee on the Use of Animals (CEUA) of UFOP (number: 4079140421) and was in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication, revised in 1985) and with the Manual of Care and Procedures with Laboratory Animals of the Production and Experimentation Laboratory of the Faculty of Pharmaceutical Sciences and Institute of Chemistry of the University of São Paulo.

[0057] The following drugs and reagents were used to carry out the experiments: polysorbate 80 (Sigma-Aldrich); dimethyl sulfoxide (DMSO; Petition 870260058359, dated 06 / 15 / 2026, pp. 34 / 49 32 / 44 Synth®); xanthine (> 99%, Sigma-Aldrich); indomethacin (> 99%, Sigma-Aldrich); uric acid (> 98%, Sigma-Aldrich); benzbromarone (> 99%, Sigma-Aldrich); probenecid (> 98%, Sigma-Aldrich); potassium oxonate (> 99%, Sigma-Aldrich); allopurinol (> 98%, Sigma-Aldrich); lapachol (> 98%, Sigma-Aldrich); ketamine (Syntec) and xylazine (Syntec).

[0058] The animals were randomly assigned to seven experimental groups (n = 8). The night before the experiment, the animals were deprived of food and water. On the day of the experiment, to induce hyperuricemia, rats in groups 2 to 7 received an intraperitoneal (ip) injection of potassium oxonate (0.5 mL; 200 mg / kg) and a gavage of uric acid suspension (1 mL; 1 g / kg). 30 minutes after the induction of hyperuricemia, 0.2 mL of the treatments were administered according to Table 21. Rats in group 1 were not induced to hyperuricemia. After the treatments, the rats were individually placed in metabolic cages and given 100 mL of water. After 5 h, the animals' urine was collected in graduated tubes and stored at -20°C for later uric acid measurement. The animals' water intake was measured.The animals were then euthanized with a combination of ketamine (240 mg / kg) / xylazine (60 mg / kg), and blood samples were collected from the abdominal aorta and kept at 35°C until coagulation. The samples were then centrifuged at 3000 x g for 15 minutes (4°C). The supernatant (serum) was collected and stored at -20°C for subsequent quantification of uric acid. The results are shown in Figure 16A. Liver samples were also collected and stored at -80°C for subsequent evaluation of hepatic xanthine oxidase activity. Table 21: Treatments administered to Wistar rats via intraperitoneal injection - 0.2 mL Experimental Group Designation Treatment Received Dose 1 Normal Control Vehicle consisting of DMSO: Tween 80: distilled water (10:10:80) 0.2 mL Petition 870260058359, dated 06 / 15 / 2026, pp. 35 / 49 33 / 44 2. Hyperuricemic / Negative Control Vehicle consisting of DMSO: Tween 80: distilled water (10:10:80) 0.2 mL Positive control for 3. serum uric acid dosage Positive control for benzbromarone 10 mg / kg 4. serum uric acid dosage probenecid 50 mg / kg Positive control 5. evaluation of xanthine oxidase activity allopurinol 10 mg / kg hepatic 6. Liposomal formulation evaluated LIPO + EREC 25 pg / mL 7. Liposomal formulation evaluated LIPO + GOIA 40 pg / mL Step 9 - In vivo evaluation of uricosuric activity

[0059] Uric acid quantification: uric acid levels in blood and urine samples were quantified using a colorimetric technique with a diagnostic kit from Bioclin (Brazil), following the manufacturer's instructions. Initially, the pH of the urine samples was adjusted to 7.0-9.0 with a 5% aqueous NaOH solution. Then, the urine was diluted (1:10) in Milli-Q water. Subsequently, the following were added to three test tubes: i. Tube 1, labeled “Blank”: 1 mL of reagent 1 (Enzymatic Reagent: DHBS < 10 mmol / L, 4-aminoantipyrine < 10 mmol / L, peroxidase < 10.0 KU / L, uricase < 10.0 KU / L, buffer, surfactant, stabilizers and preservative); ii. Tube 2, labeled “Standard”: 25 μL of reagent 2 (Standard: uric acid 6 mg / dL, buffer, stabilizer and preservative) and 1 mL of reagent 1; iii. Tube 3, labeled “Sample”: 25 μL of serum or urine sample and 1 mL of reagent 1. The tubes were homogenized and placed in a water bath at 37°C for 5 minutes. Then, the absorbance was read in a spectrophotometer (Cary 50 Bio Varian), at 505 nm, calibrating the zero with the “Blank”. The quantification of acid Petition 870260058359, dated 06 / 15 / 2026, pp. 36 / 49 The 34 / 44 uric acid content was determined using the formulas given below. The results are shown in Figure 16B. Sample Abs X Standard Concentration X 10 Uric acid series (m&dL) = --------------------------------------------, e ' C7VVW7All-? Standard Absence ^X Urinary Volume (mL) Urine uric acid (me;24h) = ---------------------------100 Step 10 - Evaluation of xanthine oxidase activity

[0060] Reagents and solutions: 10X phosphate-buffered saline (PBS) (pH = 7.4), prepared by weighing: 82 g of NaCl; 10.5 g of Na2HPO4 and 3.55 g of NaH2PO4.H2O. These quantities were added to a volumetric flask and the volume was completed with Milli-Q water to 1 L. The pH of the solution was adjusted to 7.4. 1X PBS buffer was obtained by diluting 100 mL of 10X PBS in 900 mL of Milli-Q water.

[0061] Liver samples were homogenized in 1X PBS (5 mL; pH = 7.4) and centrifuged at 3000 χ² g (10 min; 4°C). The lipid layer was discarded, and the supernatant was centrifuged at 10000 χ² g for 60 minutes at 4°C. After that, 100 µL of liver homogenate were added to test tubes containing 5.4 mL of 1 mM potassium oxonate to prevent the conversion reaction of uric acid to allantoin. The mixture was pre-incubated for 15 minutes (37°C), and a xanthine solution (1.2 mL; 250 mM) was added to it to initiate the reaction. At times 0 and 30 minutes, the reaction was stopped by the addition of HCl (0.5 mL; 0.6 M). The samples were centrifuged at 3000 χ g (5 min). The supernatant was used for spectrophotometer reading (Varian 50 Bio UV / Vis) at 295 nm. The spectrophotometer was zeroed with a 1 mM potassium oxonate solution.The quantification of uric acid formed was done by the difference between the absorbance values ​​at times 0 and 30 minutes, which was compared with the calibration curve (Equation of the line: y = 0.9323x - 0.0915). Petition 870260058359, dated 06 / 15 / 2026, pp. 37 / 49 35 / 44 (R2 = 0.9973). Total protein content was determined by the Lowry method (1951), by spectrophotometry, using bovine albumin solution (BSA) as a standard. The assays were performed in triplicate. Enzyme activity was expressed as nmoles of uric acid produced per minute per 1 mg of protein. The results are presented in Table 22. Table 22: Effects of liposomal formulations with eremantolide C (EREC) and goiazensolide (GOIA) and clinically used medications on serum uric acid levels and xanthine oxidase activity in rats induced with hyperuricemia. Activity of Group Dose Water intake (mL ± SEM) Urine volume (mL ± SEM) Uric acid excretion (mg / dL.5h ± SEM) Serum uric acid (mg / dL ± SEM) Xanthine oxidase (nmol / min / mg Inhibition (%) protein ± WITHOUT) Normal Control - 6.25 ± 0.49 3.15 ± 0.60d 0.14 ± 0.03 3.95 ± 0.74d 6.28 ± 1.11d 74.98d Negative Control 6.01 ± 1.2 6.33 ± 0.92 0.44 ± 0.05 34.31 ± 3.38 25.10 ± (Hyperuricemic) 1.75 Positive Control (Benzbromarone) 10 mg / kg 10.51 ± 1.51d 9.43 ± 1.02d 3.55 ± 0.55d 10.13 ± 1.81d - - Positive Control 50 8.00 ± 2.16c 8.35 ± 2.15c 5.95 ± 0.39d 8.36 ± 1.27d (Probenecid) mg / kg Positive Control 10 5.75 ± 1.67 6.99 ± 1.08 0.36 ± 0.09 6.62 ± 0.87d 8.66 ± 65.51d (Allopurinol) mg / kg 1.34d LIPO + EREC 25 pg / mL 5.19 ± 0.31 7.09 ± 0.38b 3.34 ± 0.65d 5.02 ± 0.93d 25.06 ± 2.14 - LIPO + GOIA 40 pg / mL 6.44 ± 0.64 7.79 ± 0.34b 3.19 ± 0.78d 10.80 ± 0.85d 23.68 ± 3.34 - * Values ​​were expressed as mean ± SEM of eight animals. One-Way ANOVA was used, followed by Dunnett's test for statistical significance. aP< 0.05 compared to the hyperuricemic control; bP< 0.01 compared to the hyperuricemic control; cP< 0.001 compared to the hyperuricemic control; dP< 0.0001 compared to the hyperuricemic control. In vivo evaluation of anti-inflammatory / anti-arthritis activity.

[0062] In vivo assays to evaluate how liposomal formulations with EREC and GOIA modulate the inflammatory process were performed in male C57BL / 6 mice weighing between 20 and 25g, from the Animal Science Center of UFOP. The mice were maintained in a 12h / 12h light / dark cycle, with water and food ad libitum (pelleted and irradiated feed for mice, rats and hamsters, Nuvilab® brand). The experimental protocol received prior approval from the Ethics Committee on the Use of Animals (CEUA) of UFOP - protocol number 4079140421, and was in accordance with the Guide for the Care and Use of Laboratory Animals, Petition 870260058359, dated 06 / 15 / 2026, pp. 38 / 49 36 / 44 published by the US National Institutes of Health (NIH Publication, revised in 1985), and in accordance with the Manual of Care and Procedures for Laboratory Animals of the Production and Experimentation Laboratory of the Faculty of Pharmaceutical Sciences and Institute of Chemistry of the University of São Paulo.

[0063] The animals were randomly distributed into eleven (11) experimental groups (n = 6). The night before the experiment, the animals were deprived of food and water. On the day of the experiment, the animals initially received an oral administration of the following treatments (0.2 mL) shown in Table 23. One hour after the treatments, the mice were anesthetized by an ip injection of ketamine (150 mg / kg) / xylazine (10 mg / kg). After achieving sufficient depth of anesthesia, an intra-articular injection (10 μL) of MSU crystals suspended in 1X PBS was administered to groups 2 to 11 to induce gouty arthritis in the tibiofemoral joint of the mice. Group 1 received an intra-articular injection (10 μL) of 1X PBS. Six hours after the induction of gouty arthritis, the animals were euthanized by a lethal dose (ip) of ketamine / xylazine (450 / 30 mg / kg).The intra-articular cavity of each animal was washed twice with 5 μL of a 1X PBS:3% BSA solution, which was added to Eppendorf tubes containing 88 μL of the 1X PBS:3% BSA solution and 2 μL of EDTA, totaling 100 μL of intra-articular lavage. The lavage was subjected to flow cytometry analysis for neutrophil quantification. Periarticular tissue was collected and frozen at -80°C for subsequent analyses (quantification of pro-inflammatory cytokines). Table 23: Treatments administered to C57BL / 6 mice by gavage - 0.2 mL Experimental Group Designation Treatment Received Dose Vehicle consisting of 1 Normal control DMSO: Tween 80: distilled water (10:10:80) Vehicle consisting of 0.2 mL 2 Negative control DMSO: Tween 80: distilled water (10:10:80) 0.2 mL 3 Positive control Indomethacin 3 mg / kg 4 Liposomal formulation evaluated LIPO 250 ng / mL Petition 870260058359, dated 06 / 15 / 2026, pp. 39 / 49 37 / 44 5. Liposomal formulation evaluated: LIPO 500 ng / mL 6. Liposomal formulation evaluated: LIPO + EREC 100 ng / mL 7. Liposomal formulation evaluated: LIPO + EREC 250 ng / mL 8. Liposomal formulation evaluated: LIPO + EREC 500 ng / mL 9. Liposomal formulation evaluated: LIPO + GOIA 100 ng / mL 10. Liposomal formulation evaluated: LIPO + GOIA 250 ng / mL 11. Liposomal formulation evaluated: LIPO + GOIA 500 ng / mL Step 11 - Assessment of neutrophil migration

[0064] For this evaluation, initially, the eppendorf tubes containing the intra-articular lavage fluids were homogenized using a vortex mixer and transferred to flow cytometry tubes. 1 μL of the neutrophil marker (anti-mouse Ly6G PE (BD Biosciences)) was diluted in 499 μL of 1X PBS with 10% FBS and 10% rat serum. Then, 10 μL of the diluted marker were added to each tube containing the lavage fluids. The solutions in the tubes were vortexed and incubated for 20 minutes in the dark at room temperature. After that, each test tube received 1 mL of 1X PBS solution and was centrifuged at 3000 rpm for 15 minutes. The supernatants were discarded, and the samples were analyzed using a flow cytometer (FACS Calibur (BD Biosciences)) to quantify the neutrophils in each sample. The results are presented in Figure 17A. Step 12 - Quantification of pro-inflammatory cytokines

[0065] The periarticular tissue of the animals was removed from the -80°C freezer and homogenized with 1 mL of 1X PBS. The concentrations of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were determined using the standard murine ABTS ELISA kits for IL-1β, IL-6, and TNF-α (Peprotech®), following the manufacturer's recommendations.

[0066] Reagents, solutions and kits: Petition 870260058359, dated 06 / 15 / 2026, pages 40 / 49 38 / 44 i. Washing solution: 0.05% Tween 20 in 1X PBS; pH=7.2-7.4; ii. Diluent solution: 0.05% Tween 20 + 0.1% BSA in 1X PBS; filtered with a 0.2 μM pore membrane; iii. Blocking solution: 1% BSA in 1X PBS; filtered with a 0.2 μM pore membrane; iv. Pro-inflammatory cytokine levels are measured according to the kits, following the manufacturer's instructions; v. Murine TNF-α ABTS Elisa Development Kit - Standard Kit - code 900-K54 Peprotech; vi. Murine IL-1β ABTS Elisa Development Kit - Standard Kit - code 900-K47 - Peprotech; vii. Murine IL-6 ABTS Elisa Development Kit - Standard Kit - code 900-K50 - Peprotech; viii. Capture antibody (200 μg of rabbit antimurine IL-Ιβ and 2.5mg D- mannitol); ix. Detection antibody (50 μg of biotinylated rabbit antimurine IL-Ιβ and 2.5mg of D- mannitol); x. Murine IL-1 β standard (1 μg of recombinant murine IL-1 β, 2.2mg of BSA and 11mg of D- mannitol); xi. Capture antibody (101 μg of rabbit antimurine IL-6 and 2.5mg of D- mannitol); xii. Detection antibody (101 μg of biotinylated rabbit antimurine IL-6 and 20.2mg of BSA); xiii. Murine IL-6 standard (1 μg of recombinant murine IL-6, 2.2mg of BSA and 11mg of D- mannitol); xiv. Capture antibody (21 μg of rabbit antimurine TNF-α and 0.5mg of D- mannitol); xv. Detection antibody (11 μg of biotinylated rabbit antimurine TNF-α and 0.5 mg of D-mannitol); Petition 870260058359, dated 06 / 15 / 2026, pp. 41 / 49 39 / 44 xvi. Murine TNF-α standard (1 pg of recombinant murine TNF-α, 2.2 mg of BSA and 11 mg of D-mannitol).

[0067] Initially, the capture antibody was diluted in 1X PBS (1:10000) to achieve concentrations of 2.0 μg / mL (IL-1β assay) and 1.0 μg / mL (IL-6 and TNF-α assay), and 100 μL were added to each well of 96-well plates, which were incubated for 18 h (25°C). After this time, the plates were washed three times with washing solution. Then, 300 μL of the blocking solution were added to each well, and the plates were incubated for 1 h 30 (25°C). After this time, the wells were washed with washing solution (three times). After washing the plates, 100 μL of the standards and samples (periarticular tissue homogenates), previously vortex homogenates, were added in duplicate. The murine standards of IL-1β, IL-6, and TNF-α were diluted in the diluent solution, reaching concentrations of 4000 pg / mL to zero (for IL-1β), 4000 pg / mL to zero (for IL-6), and 1000 pg / mL to zero (for TNF-α), for the construction of standard curves.The plates were incubated for 2 hours (25°C) and then washed with washing solution (three times). The Avidin-HRP conjugate was diluted in 11 mL of diluent solution, and 100 μL were added to each well. The plates were incubated for 30 minutes (25°C), and then the wells were washed with washing solution (three times). Finally, 100 μL of ABTS solution were added to each well to initiate the colorimetric reaction, with the final reading taken after 30 minutes on an ELISA reader at 405 nm, with wavelength correction adjusted to 650 nm. The results are presented in Figure 17B-D. In vivo assessment of antioxidant activity

[0068] In vivo assays to evaluate how liposomal formulations with EREC and GOIA modulate oxidative stress were performed in male C57BL / 6 mice weighing between 20 and 25g, from the Animal Science Center of UFOP. The mice were kept in a Petition 870260058359, dated 06 / 15 / 2026, pages 42 / 49 40 / 44 light / dark cycle of 12h / 12h, with water and food ad libitum (pelleted and irradiated feed for mice, rats and hamsters, Nuvilab® brand). The experimental protocol received prior approval from the Ethics Committee on the Use of Animals (CEUA) of UFOP - protocol number 4079140421, and was in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication, revised in 1985), and in accordance with the Manual of Care and Procedures with Laboratory Animals of the Production and Experimentation Laboratory of the Faculty of Pharmaceutical Sciences and Institute of Chemistry of the University of São Paulo.

[0069] The animals were randomly distributed into eight (8) experimental groups (n=6). The night before the experiment, the animals were deprived of food and water. On the day of the experiment, the animals initially received an oral administration of the following treatments (0.2 mL) shown in Table 24. 1 h after the treatments, the mice were anesthetized by an ip injection of ketamine (150 mg / kg) / xylazine (10 mg / kg). After reaching a sufficient depth of anesthesia, an intra-articular injection (10 μL) of MSU crystals suspended in 1X PBS was administered to groups 2 to 8 to induce gouty arthritis in the tibiofemoral joint of the mice. Group 1 received an intra-articular injection (10 μL) of 1X PBS.

[0070] Twelve hours after the induction of gouty arthritis, the animals were euthanized by a lethal dose (ip) of ketamine / xylazine (450 / 30 mg / kg). Periarticular tissue was collected and frozen at -80°C for subsequent analyses (measurement of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT)). Table 24: Treatments administered to C57BL / 6 mice by gavage - d 0.2 mL Experimental Group Designation Treatment received Dose 1 Normal control Vehicle consisting of DMSO: Tween 80: distilled water 0.2 mL 2 Negative control (10:10:80) Vehicle consisting of DMSO: Tween 80: distilled water 0.2 mL (10:10:80) Petition 870260058359, dated 06 / 15 / 2026, pp. 43 / 49 41 / 44 3. Liposomal formulation evaluated: LIPO + EREC 100 ng / mL 4. Liposomal formulation evaluated: LIPO + EREC 250 ng / mL 5. Liposomal formulation evaluated: LIPO + EREC 500 ng / mL 6. Liposomal formulation evaluated: LIPO + GOIA 100 ng / mL 7. Liposomal formulation evaluated: LIPO + GOIA 250 ng / mL 8. Liposomal formulation evaluated: LIPO + GOIA 500 ng / mL

[0071] 20 μL of each dilution were pipetted onto an ELISA plate, and then 200 μL of the ABC solution were added to each well. The plate was left to stand for 10 minutes. Subsequently, 20 μL of Folin-Ciocalteu reagent (Sigma-Aldrich) were added and left to stand for 10 minutes. The plate reading was performed at 700 nm on the ELISA reader. Analyses were performed in the dark and in triplicate. Femorotibial periarticular tissue samples were prepared from a 25 μL dilution of each homogenate in 475 μL of Milli-Q water. The solutions were homogenized in a vortex mixer and placed in an ice bath. Then, 20 μL of each dilution were pipetted onto the ELISA plate. To each well, 200 μg of ABC solution were added and left to stand for 10 minutes. Then, 20 μg of Folin's reagent were added and left to stand for 10 minutes. The plate reading was taken at 700 nm using an ELISA reader.The analyses were performed in the dark and in triplicate. Step 13 - Evaluation of superoxide dismutase (SOD) activity

[0072] SOD activity was determined according to the method proposed by Dieterich et al. (2000), with adaptations. Initially, 22 mg of MTT were diluted in 4 mL of 1X PBS (pH=7.2), and 2 mg of pyrogallol in 160 mL of 1X PBS (pH=7.2). As the reagents are photosensitive, the vials containing the solutions were covered with aluminum foil. In an ELISA plate, 30 μg of the femorotibial periarticular tissue homogenates were pipetted. For the blank, 45 μg of 1X PBS buffer were pipetted; and, for the standard, 30 μg of Petition 870260058359, dated 06 / 15 / 2026, pages 44 / 49 42 / 44 PBS buffer. Then, 99 μL of 1X PBS and 6 μL of MTT solution were added to all wells. Subsequently, 15 μL of pyrogallol were added to all wells (except the blank). The plate was incubated for 5 minutes in an oven at 37°C. After this time, 150 μL of dimethyl sulfoxide (DMSO) were added to all wells to stop the reaction. Finally, the reading was taken on the spectrophotometer at 570 nm. The results are presented in Figure 18A. Step 14 - Evaluation of catalase (CAT) activity

[0073] CAT activity was determined according to the method proposed by Aebi (1984), with adaptations. Initially, to prepare the H2O2 solution, 40 μL of 30% H2O2 were diluted in 25 mL of 1X PBS buffer (pH = 7.2). The spectrophotometer was zeroed with distilled water. The negative control (blank) was performed by pipetting 10 μL of the sample into 500 μL of pure buffer in a cuvette. For sample analysis, 10 μL of each sample were added to 500 μL of the H2O2 solution in a cuvette, followed by homogenization. Readings were taken in a spectrophotometer at 240 nm. Absorbance readings were taken at time zero (T0), after 20 seconds (T20), and after 40 seconds (T40). All procedures were performed in the dark. The results are shown in Figure 18B. Step 15 - Statistical analysis of the data

[0074] The results obtained in the animal experiments were subjected to analysis of variance (ANOVA) using GraphPad Prism 6.0 software, followed by appropriate post-tests, and were expressed as mean ± SEM. A 95% confidence interval was adopted, with differences considered significant when the P-value was less than 0.05 (P< 0.05). Stage 14 - In vitro antitumor activity of eremantolide C Petition 870260058359, dated 06 / 15 / 2026, pages 45 / 49 43 / 44

[0075] EREC was evaluated by the National Cancer Institute (NCI, Bethesda, Maryland, USA) where its activity was assessed in 8 types of cancer and 60 human tumor cell lines that determined the antitumor effect, expressed in IC50 (concentration that inhibits 50% of tumor cell growth relative to control, in pg / mL), IC100 (concentration that inhibits 100% of tumor cell growth relative to control, in pg / mL) and LC50 (lethal concentration for 50% of tumor cells relative to control, in pg / mL). The NCI considered for EREC, LC50 values ​​less than or equal to 30.1 pg / mL, IC100 values ​​less than or equal to 17.0 pg / mL and IC50 values ​​less than or equal to 6.26 pg / mL as the most significant concentrations.Compound (2) showed LC50 values ​​between 18.1 and 29.3 pg / mL against eighteen tumor cell lines, CIC100 values ​​between 7.34 and 14.36 pg / mL against 26 cell lines and CIC50 values ​​between 1.65 and 5.47 pg / mL against 22 cell lines (Table 25). Table 25: In vitro antitumor activity study for eremantolide C (2). Concentration (pg / mL) Cancer Type / Cell Line CIC50 CIC100 CL50 Leukemia CCRF-CEM 1.83 9.1 HL-60 (TB) 1.65 7.72 30.7 RPMI-8226 3.60 11.69 SR 2.76 14.36 K-562 2.34 MOLT-4 5.46 Non-small cell lung cancer HOP-92 14.29 28.8 NCI-H2226 NCI-H23 31.5 Colon Cancer COLO 205 HCC-2998 4.29 8.82 18.1 HCT-116 4.60 9.27 18.6 HCT-15 3.94 9.52 22.9 KM12 6.64 12.84 24.7 SW-620 2.19 7.65 25.2 HT-29 15.43 Melanoma LOX IMVI M14 1.95 7.34 21.0 SK-MEL-2 4.22 10.97 28.3 SK-MEL-5 4.77 11.24 26.5 UACC-62 5.02 13.60 Petition 870260058359, dated 06 / 15 / 2026, pages 46 / 49 44 / 44 Ovarian Cancer IGROV 1 OVCAR-3 OVCAR-4 5.47 12.08 26.6 OVCAR-5 OVCAR-8 13.49 26.7 Renal Cancer 786-0 CAKI-1 5.40 10.03 18.6 RXF-393 3.67 10.38 29.3 SN12C 5.12 10.59 21.7 TK-10 UO-31 12.91 23.5 Prostate Cancer PC-3 Breast Cancer MCF7 11.63 20.9 MDA-MB 231 / ATCC 4.84 11.18 25.8 MDA-N 3.60 14.12 BT-549 MDA-MB-435 4.67 10.73 24.7 USC U251 12.98 Petition 870260058359, dated 06 / 15 / 2026, pp. 47 / 49

Claims

1 / 10 CLAIMS 1. NANOSTRUCTURED FORMULATIONS CONTAINING SESQUITERPENE LACTONES FOR THE TREATMENT OF NEOPLASMS, HYPERURICEMIA, INFLAMMATORY DISEASES AND GOUT characterized by comprising liposomes with an average diameter of less than 1000 nm incorporating furanoheliangolide type sesquiterpene lactones.

2. NANOSTRUCTURED FORMULATIONS, according to claim 1, characterized in that the active sesquiterpene lactones incorporated into the liposomal formulations are selected from the group of chemical entities with W or Z chemical structures, where each or any of R1, R2, R3, R4, R5 and R6 is (1) a substituent selected from groups consisting of hydrogen (-H), halogen (e.g., fluorine, chlorine or bromine), hydroxyl (-OH), alkoxyl (-OR'), acyl (-COR'), carboxyl (-CO2H), carboxylic esters (-CO2R'), amide ((CONR2'), amine (-NR2'), nitro (-NO2), nitroso (-NO), azo (N=N-), diazonium (-N2+), azide (-N3), hydrazine (-NR'-NR'2), cyano (-CN), isocyanate (CN-), cyanate (NCO-), isocyanate (OCN-), thioether (-SR'), thiol (-SH), sulfoxide (SOR'), sulfone (S(O)2R'), sulfonic acid (HO3S-), sulfonyl esters (RO3S-), sulfinic acid (HO2S-), sulfinyl esters (RO2S-), sulfenic acid (HOS-), sulfenyl esters (ROS-), where R' is an alkyl group,(1) an alkenyl or alkynyl group of 1 to 5 carbons; (2) an unsaturated, aliphatic, saturated, alicyclic or aromatic hydrocarbon radical having from 1 to 50 carbon atoms, preferably from 1 to 25 carbon atoms, and more preferably from 1 to 20 carbon atoms, which may be substituted with one or more of the substituents in (1) above; (3) a heterocyclic group having around 1 to 13 carbon atoms; (4) a glycoside residue; or (5) a peptide residue. Two or more of the R1, R2, R3, R4, R5 and R6 groups may be combined with any form of the listed cyclic parts listed above (I and II), wherein the substituents may be arranged in cyclic or acyclic form, so long as the furanone and α-methylene-Y-lactone pharmacophores retain the activities described in this patent, the atom or group represented by “X” may be oxygen (O), sulfur (S), nitrogen (N), imines (NR') or hydrazones (NR'-NR').where R' is an alkyl, alkenyl, or alkynyl group of 1-5 carbons.

3. Nanostructured formulations, according to claim 1, characterized in that the active substances incorporated in the liposomal formulations are sesquiterpene lactones defined by the following structural formula:

4. NANOSTRUCTURED FORMULATIONS, according to claim 1, characterized in that the active agents comprise active groups α-methylene-Y-lactone, furanone and conjugated esters and are preferably substances containing the chemical structures of furanoeliangolides X and Y, represented below: wherein, for chemical structure X, Ri can be any of the esters whose structures are given below: Ang O Tig OV MeAcr O A- JO MeBu O OH O ^^^OH^ Sar O OAc AcSar O )H OAc AcDHiBu OA OH OH DHiBu iVal ^o^^ iButi O OH V HMB O | Sen O ^^^O^ EpTig O Ά EpAng O Ά- EpMA O Ac . OAc oV o O^^CsHii o OxX“'X'·-'^ OO^y^ OH , OI o I II O^^ 1 oc O^^ OAc ) Ac , OA O [ 0''^^ AcO c OI A1 OAC .OA O [ O'^^^^^^ c O 1 O^^^^^^ OOH JA / OO yo H £ o O' / ^'x OAc ) H o O'^'^ OH 1 ΐώ e for the chemical structure Y, Ri can be any of the substituents whose chemical structures are given below: Petition 870240084167, dated 02 / 10 / 2024, page. 63 / 91 4 / 10 Where Ri can also be H or CH2CH3 or CH3 or CH3CH2CH2CH2CH3 or CH3CH2CH2CH2CH2CH2CH2CO (CeHisO, caprylic acid ester) or CH3CH2CH2CH2CH2CH2CH2CH2CH2CO (C10H19O, capric acid ester) or CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CO (C12H23O, lauric acid ester).or CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CO (C14H27O, myristic acid ester) or CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CO (C16H31O, palmitic acid ester) or CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CO (C18H35O, stearic acid ester) or CH3CH2CH2CH2CH2CH2CH2CH2CH=CHCH2CH2CH2CH2CH2CH2CO (C18H33O, oleic acid ester) or CH3CH2CH2CH2CH2CH2CH2CH2CH=CHCH2CH=CHCH2CH2CH2CH2CO (C18H31O, ester of linoleic acid)ou CH3CH2CH2CH2CH2CH=CHCH2CH=CHCH2CH=CHCH2CH2CH2CH2CO Petition 870240084167, dated 02 / 10 / 2024, p. 64 / 91 5 / 10 (C18H29O, ester of D-linoleic acid) or CH3CH2CH2CH2CH2CH2CH2CH2CH=CHCH2CHOHCH2CH2CH2CH2CH2CO (C18H32O2, ester of ricinoleic acid) or CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2C O (C20H39O, ester of arachidic acid) or CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2C H2CH2CO (C22H43O, ester of behenic acid).

5. NANOSTRUCTURED FORMULATIONS, according to claim 1, characterized in that the active agents comprise sesquiterpene lactones with any of the specific chemical structures and substituents represented in the following formulas: Petition 870240084167, dated 02 / 10 / 2024, p. 65 / 91 6 / 10 Petition 870240084167, dated 02 / 10 / 2024, p. 66 / 91 7 / 10 Petition 870240084167, dated 02 / 10 / 2024, p. 67 / 91 8 / 10 Petition 870240084167, dated 02 / 10 / 2024, p. 68 / 91 9 / 10 6. Nanostructured formulations containing sesquiterpene lactones according to claim 1, characterized in that the liposomes are composed of phosphatidylcholine. Petition 870240084167, dated 02 / 10 / 2024, pp. 69 / 91 10 / 10 7. Nanostructured formulations according to any one of claims 1 to 6, characterized by being administered via oral, intramuscular, subcutaneous, intraperitoneal, intranasal, inhalation, intra-articular, topical and / or intravenous routes.

8. USE OF THE NANOSTRUCTURED FORMULATIONS defined in claim 1, characterized by being for the production of a medicament for human and animal use for the treatment of hyperuricemia, inflammatory diseases, gout, and neoplasms in mammals, especially prostate cancer, breast cancer, cervical cancer, renal cancer, ovarian cancer, melanoma, colon cancer, leukemias, non-small cell lung cancer, among other multiple types. Petition 870240084167, dated 02 / 10 / 2024, pp. 70 / 91

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