ALAMANDINE-(1-5) PEPTIDE, PHARMACEUTICAL COMPOSITION AND USES

Description

1 / 25 “ALAMANDINE-(1-5) PEPTIDE, PHARMACEUTICAL COMPOSITION AND USES”

[01] The present technology relates to the Alamandine-(1-5) peptide and its use for drug production. The peptide acts on the renin-angiotensin system as a triple agonist of MAS, MrgD and AT2 receptors.

[02] The renin-angiotensin system (RAS) is a biochemical cascade that degrades angiotensinogen into various bioactive peptides, which can activate different receptors promoting various specific effects.

[03] It is well known that the renin-angiotensin system plays a critical role in cardiovascular control and hydroelectrolytic homeostasis. The biologically active end products of this system are formed by a limited proteolysis process beginning with the hydrolysis of angiotensinogen by renin. Angiotensin (Ang) II and Ang (1-7) are key players in the RAS, acting as mediators of the classical and non-canonical axes, respectively. Both peptides can undergo decarboxylation of the N-terminal amino acid aspartate to alanine, forming Angiotensin A (Ang A) and Alamandine, respectively. While Ang A exhibits actions similar to those of Ang II, acting on AT1 and AT2 receptors, Alamandine differs from Ang-(1-7) by acting on MrgD receptors instead of Mas.

[04] Alamandine may have selective actions, as already reported for the insular cortex (Marins, FR et al. Alamandine but not angiotensin-(1-7) produces cardiovascular effects at the rostral insular cortex. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 321, R513-R521 (2021)). The hydrolysis of Angiotensin-(1-7) by Angiotensin-Converting Enzyme (ACE) may form the pentapeptide Angiotensin-(1-5), which has been described as an agonist Petition 870250077383, dated 08 / 30 / 2025, page 5 / 29 2 / 25 AT2R or Mas depending on the cell type and organ (Park, BM, Li, W. & Kim, SH Cardioprotective effects of angiotensin-(1-5) via Mas receptor in rats against ischemic-perfusion injury. Peptides 139, 170516 (2021); Yu, L., Yuan, K., Phuong, HTA, Park, BM & Kim, SH Angiotensin-(1-5), an active mediator of the renin-angiotensin system, stimulates ANP secretion via Mas receptor. https: / / doi.org / 10.1101 / 2024.04.05.588367 (2024)).

[05] There is no state-of-the-art data addressing the formation and actions of Alamandine-(1-5), a product of Alamandine, via hydrolysis by ECA.

[06] Patent document PI0800585-0, whose priority date is 13 / 02 / 2008, entitled “Pharmaceutical compositions of the Des[Asp1]-[Ala1]-angiotensin-(1-7) peptide and use of the Des-[Asp1]-[Ala1]-angiotensin-(1-7) peptide”, describes the use of the Des-[Asp1]-[Ala1]-angiotensin-(1-7) peptide for the treatment of vascular and cardiac diseases. The technology differs from the present technology in that it does not include the Alamandine-(1-5) peptide.

[07] Patent document BR1320120336056, whose priority date is 12 / 28 / 2012, entitled “Use of a pharmaceutical composition containing Alamandine for the treatment of hypertension and cardiac fibrosis”, describes the use of Alamandine for the treatment of hypertension and cardiac fibrosis through signaling via MrgD receptors. The document differs from the present technology in that it deals with Alamandine, and not Alamandine-(1-5).

[08] In the prior art, the peptide Alamandine(1-5) and its use for drug production have not been found. The present technology describes the formation of Alamandine-(1-5) from Alamandine by Petition 870250077383, dated 08 / 30 / 2025, page 6 / 29 3 / 25 ACE, present in human blood. This new angiotensin possesses unique activities, including a marked effect on cardiac inotropism. Alamandine-(1-5) (SEQ ID No. 1) described in the present technology is derived from Alamandine, formed by the hydrolysis of Alamandine by ACE. Unlike Alamandine, the results presented in the present technology, the subject of this patent application, demonstrate that Alamandine-(1-5) constricts aortic rings in mice, has a potent modulating effect on cardiac activity, reducing inotropism, and does not produce the same effects as Alamandine in the insular cortex. While Alamandine injected into this area increases renal sympathetic nerve activity, increases heart rate and blood pressure, Alamandine-(1-5) only increases blood pressure. Furthermore, Alamandine-(1-5) is able to bind to all three receptors of the protective axis of the renin-angiotensin system (But, MrgD is AT2) whereas Alamandine only binds to MrgD. BRIEF DESCRIPTION OF THE FIGURES

[09] Figure 1 shows the vascular effects of Alamandine-(1-5) [Ala 15] (SEQ ID No1) and Alamandine [Ala] in isolated vessels. (A) Cumulative concentration of Alamandine or Alamandine-(1-5) in aortic rings. (B) Effects of Alamandine-(1-5) with intact and denuded endothelium in aortic rings. (C) Effects of Alamandine-(1-5) in microvessels. (D) Representative image of vascular reactivity in response to Alamandine-(1-5) in a microvessel. Data were expressed as mean ± SEM and analyzed by Two-Way ANOVA followed by Fisher's multiple comparisons test. *p < 0.05.

[10] Figure 2 shows that Alamandine-(1-5) (SEQ ID No. 1) reduces the area of ​​ventricular myocyte shortening. (A) Concentration-dependent effect of Alamandine-(1-5) on isolated ventricular shortening. (B) Mean velocity tracing obtained from a single cycle of Petition 870250077383, dated 08 / 30 / 2025, p. 7 / 29 4 / 25 contraction-relaxation, in a control experiment and under the effect of Alamandine-(1-5). (C) Cardiomyocyte displacement velocity displayed in vector and magnitude fields, with visual and numerical intensity scale detected during maximum contraction velocity (MCS) and maximum relaxation velocity (MRS) in response to Alamandine-(1-5). (DF) A marked decrease in cardiomyocyte shortening and velocity parameters was observed in response to treatment with Alamandine-(1-5). (GH) Alamandine-(1-5) induced a reduction in the amplitude of Ca2+ transients, increasing the decay kinetics of the Ca2+ transient (T50). In the violin representations, solid and dashed lines indicate the medians and the 25th and 75th percentiles, respectively. The colored shapes show data density estimates. In (A) *p < 0.05. In (DH) *p < 0.05 Student's t-test when compared to the untreated control.

[11] Figure 3 depicts the effects of Alamandine-(1-5) on isolated rat hearts. After a stabilization period (20-30 min), the hearts were continuously perfused with Alamandine-(1-5) at doses of 20 or 200 pmol / L for 15 min. The graphs show the changes in (A) left ventricular end-systolic pressure (LVESP), (B) left ventricular end-diastolic pressure (LVEDP), (C) maximum rate of rise of left ventricular pressure (dP / dtmax), (D) maximum rate of fall of left ventricular pressure (dP / dtmin), (E) perfusion pressure, (F) heart rate (HR). Arrows indicate the start of perfusion with Alamandine-(1-5). #p < 0.05 (20 pmol / L) and *p < 0.05 (200 pmol / L) compared to the baseline of the respective group. Statistical significance was calculated using Two-Way ANOVA followed by Dunnett's multiple comparisons test. Each point represents the mean ± SEM. Petition 870250077383, dated 08 / 30 / 2025, page 8 / 29 5 / 25

[12] Figure 4 shows that Alamandine-(1-5) (SEQ ID No1) improves the baroreflex in SHR rats. Bradycardia baroreflex sensitivity index (ΔPI / ΔMAP, ms / mmHg) calculated by the ratio between baroreflex-mediated changes in heart rate (converted to pulse interval, in ms) by changes in mean arterial pressure (ΔMAP, in mmHg) produced by intravenous bolus injections of phenylephrine (1, 2.5, 5, 10 and 20 μg / ml), before and 90 minutes after ICV infusion of saline or Alamandine-(1-5) [Ala-(1-5), 0.04 pg / ml] in Wistar (A) and SHR (B) rats. Values ​​are mean ± SEM *p > 0.05 versus before infusion (paired t-test).

[13] Figure 5 depicts the effect of Alamandine-(1-5) (SEQ ID No1) on the insular cortex. Representative tracings showing the effects on baseline cardiovascular and autonomic parameters (RSNA, renal sympathetic nerve activity, %; iRSNA, peak renal sympathetic nerve activity, arbitrary units; blood pressure, mmHg; MAP, mean arterial pressure, mmHg; HR, heart rate, beats / min) elicited by microinjection of Alamandine (n = 7), Alamandine-(1-5) (n = 7) and saline (n = 4) at the rostral level of the posterior IC (A). (B) Changes in mean arterial pressure (MAP), (C) heart rate (HR), (D) renal sympathetic nerve activity (RSNA) elicited by microinjection of Alamandine (black bars), Alamandine-(1-5) (white bars) and saline (gray bars) at the rostral level of the posterior IC, coordinates +1.5 mm. Statistical analysis: one-way ANOVA, p < 0.05, * vs. Alamandine-(1-5) and Saline # vs. Alamandine and Saline.

[14] Figure 6 depicts the antihypertensive effect of Alamandine-(1-5) (SEQ ID No. 1) in SHR rats. Mean arterial pressure (MAP, mmHg; panel A), Heart rate (HR, beats / min; panel B), Systolic blood pressure (SAP, mmHg; panel C) before (baseline), 30 min and 6 h after intravenous bolus injection of Ala-(1-5) (30 pg / kg) in Wistar rats. Petition 870250077383, dated 08 / 30 / 2025, page 9 / 29 6 / 25 and SHR. Changes in SAP (mmHg; panel D) 6h after intravenous injection compared to baseline values ​​of saline and Ala-(1-5) in the Wistar or SHR groups. Values ​​are mean ± SEM. *p < 0.05 vs baseline (panels A and C) or saline (panel D) and #p < 0.05 vs Ala-(1-5) injection in Wistar rats (Two-way ANOVA, followed by Tukey's post-hoc test).

[15] Figure 7 represents the comparison of the involvement of the protective receptors of the RAS arm in the Alamandine-(1-5) (SEQ ID No1) response in aortic rings. Effects of Alamandine-(1-5) on aortic rings of MrgD KO (A), AT2 KO (B) and MAS KO (C). Data were expressed as mean ± SEM and analyzed by Two Way ANOVA followed by Fisher's test for multiple comparisons. p<0.05.

[16] Figure 8 shows that Alamandine-(1-5) (SEQ ID No1) activates Mas, MrgD, and AT2 receptors. (A) Representative images of nitric oxide production in EA.hy926, CHO-MAS, CHO-MrgD, and CHOAT2 cells loaded with DAF-FM and stimulated with Alamandine-(1-5) compared to unstimulated control. (B) Alamandine-(1-5) can significantly increase NO production in all cell types, indicating that it binds to and activates Mas, MrgD, and AT2 receptors (p<0.0001; n = 3; at least 90 cells per condition).

[17] Figure 9 (A) presents evidence that ACE is able to metabolize Alamandine forming Alamandine-(1-5) (SEQ ID No1), with the MS spectrum range in plasma of 610-900 m / z.

[18] Figure 9 (B) represents the MS spectral range in the plasma from 610 to 650 m / z.

[19] Figure 9 (C) shows the de novo sequencing of Synthesized alamandine (top panel) in aqueous solution and plasma-enriched alamandine (bottom panel).

[20] Figure 9 (D) shows the de novo sequencing of Alamandine-(1-5) synthesized (top panel) in aqueous solution and Petition 870250077383, dated 08 / 30 / 2025, p. 10 / 29 7 / 25 Alamandine-(1-5) formed in plasma (lower panel). The formation of Alamandine-(1-5) from plasma from angiotensinogen knockout mice (TLM mice) was suppressed in the presence of the ACE inhibitor captopril (Panels A and B).

[21] Figure 10 shows that Alamandine-(1-5) is circulating in mice and in human plasma. (A) Quantification of Alamandine and Alamandine-(1-5) in mouse plasma (B) and in humans. Mean ± SE of circulating alatensins in healthy individuals. Data obtained by UPLC-MS / MS peptide analyses as described previously. The t-test was used for comparison between male and female mice. **** = p < 0.0001. DETAILED DESCRIPTION OF THE TECHNOLOGY

[22] The present technology relates to the Alamandine-(1-5) peptide and its use for drug production. The peptide acts on the renin-angiotensin system as a triple agonist of MAS, MrgD and AT2 receptors.

[23] More specifically, the Alamandina-(1-5) peptide is characterized by being defined by the amino acid sequence SEQ ID No1.

[24] The SEQ ID No1 peptide is a triple agonist of MAS, MrgD and AT2 receptors.

[25] The pharmaceutical composition is characterized by containing the Alamandine-(1-5) peptide, defined by SEQ ID No. 1, in the presence of pharmaceutically and physiologically acceptable carriers and / or excipients.

[26] The pharmaceutical composition can be administered orally, intramuscularly, intravenously, subcutaneously, topically, transdermally or with devices that can be implanted or injected.

[27] The Alamandine-(1-5) peptide can be used for the production of medicines. Petition 870250077383, dated 08 / 30 / 2025, page 11 / 29 8 / 25

[28] The pharmaceutical composition can be used for the production of medicines.

[29] The present invention can be better understood through the following examples, which are not limiting. EXAMPLE 1 - VASOCONSTRICTION INDUCED BY ALAMANDINE(1-5) IN AORTIC RINGS OF MICE

[30] For in vivo and ex vivo analyses, male C57BL / 6J, MrgD KO, AT2R KO and Mas KO mice (10-14 weeks old) or SHR and Wistar rats were used, obtained from the animal facility of the Federal University of Minas Gerais, with experimental protocols approved by CEUA-UFMG (189 / 2023; 253 / 2022; 5 / 2018).

[31] Isolated aortic rings from the descending thoracic aorta were placed in an organ bath with Krebs-Henseleit solution (110.8 mmol.L-1NaCl, 5.9 mmol.L-1KCl, 25 mmol.L-1NaHCO3, 1.07 mmol.L-1MgSO4, 2.49 mmol.L-1CaCl2, 2.33 mmol.L-1NaH2PO4, 11.51 mmol.L-1glucose; pH 7.4) containing 95% O2 and 5% CO2 at 37°C, under a tension of 0.5 g (mice) or 1.5 g (rats) for 60 minutes for stabilization. After stabilization, the rings were pre-constricted with phenylephrine (100 nmol.L-1) and received a cumulative concentration (10-11 to 10-6M) of Alamandine-(1-5) (SEQ ID No1) to evaluate the vascular effect of this peptide. Some segments of mouse vessels were incubated with L-NAME (N-nitro-L-arginine methyl ester 100 μmol.L-1, Sigma-Aldrich).

[32] Mechanical activity was recorded isometrically using a force transducer and data acquisition system (DATAQ Instruments, USA). Second-order mesenteric resistance arteries (internal diameter <300 µm) were dissected and mounted on a multichannel wire myograph (Model 620M, DMT A / S, Aarhus NA, Denmark) containing heated (37 °C) and aerated (95% O2 and 5% CO2) physiological solution containing, in mmol.L-1: 118 NaCl, 4.7 KCl, 25 NaHCO3, Petition 870250077383, dated 08 / 30 / 2025, page 12 / 29 9 / 25 2.5 mg CaCl2.2H2O, 1.2 mg KH2PO4, 1.2 mg MgSO4.7H2O, 11 mg glucose, and 0.01 mg EDTA. After a 30-minute equilibration period, the arteries were exposed to 60 mM KCl to determine the maximum tension.

[33] After washing and 30 min of equilibration, 1 μmol.L-1 of phenylephrine (Sigma-Aldrich) was administered to induce contraction corresponding to half the maximum contraction of KCl, and then concentration-response curves for Alamandine-(1-5) (10-12 to 10-6 mol.L-1) were determined. The pentapeptide produced a significant increase in aortic ring tension (Figure 1, letter A). Contraction was already evident from a concentration of 10-11 mol.L-1. The contractile effect was only slightly increased in endothelium-deprived rings (Figure 1, letter B). In contrast, Alamandine-(1-5) produced a concentration-dependent relaxation in mouse mesenteric artery and in aortic rings of spontaneously hypertensive rats (SHR) and Wistar rats (Figure 1, letters C and D). EXAMPLE 2 - DECREASE IN CONTRACTILITY OF ISOLATED VENTRICULAR MYOCYTES FROM MICE INDUCED BY ALA-(1-5)

[34] Mouse hearts were rapidly collected, conditioned in cold Tyrode solution containing, in mmol.L-1: 140 NaCl, 5 KCl, 1 MgCl2, 0.33 NaH2PO4, 1.8 CaCl2, 10 glucose and 10 HEPES; pH 7.4 adjusted with NaOH, while the ascending aorta was cannulated and properly sealed. Ventricular myocytes were then isolated by the Langendorff method, which consists of an initial enzymatic dissociation, followed by mechanical dissociation, both at 37°C. The newly isolated ventricular myocytes were stored in Tyrode solution. Subsequently, cardiomyocytes were incubated for 10 min with Alamandine-(1-5) (10-7-10-9 mol.L-1), and intracellular Ca2+[Ca2+]i and cell contractility were evaluated. The Ca2+[Ca2+]i measurements Petition 870250077383, dated 08 / 30 / 2025, page 13 / 29 10 / 25 intracellular assays were performed on cardiomyocytes loaded with Fluo-4 AM dye (5 μmol.L-1, ThermoFisher). After a 30-minute period, the cells were washed once with Tyrode at room temperature to remove excess dye. Subsequently, the cells were treated with Alamandine-(1-5) (SEQ ID No. 1) and stimulated to contract at 1 Hz at room temperature. Images for intracellular Ca2+ concentration ([Ca2+]i) were acquired using a Zeiss 880 confocal microscope.

[35] For contractility measurements, ventricular myocytes were maintained in Tyrode's solution, subjected to electrical stimulation using platinum electrodes, with a pulse frequency of 1 Hz and a voltage of 40 V, each pulse lasting 5 ms. To capture cellular images (resolution of 640 x 480 pixels, presenting a pixel size of 0.25 μm / pixel and a depth of 8 bits), a high-speed digital CMOS camera (SILICON VIDEO 642 M, EPIX, Inc) was used, operating at a frame rate of 200 fps. After recording the contraction-relaxation cycles of untreated cells (control), the myocytes were exposed to Alamandine-(1-5) and the contractility measurements were recorded again. Image processing and analysis were conducted using CONTRACTIONWAVE software, employing the dense optical flow method. The cells were incubated with a range of Alamandine-(1-5) from 10 nmol.L-1 to 1 μmol.L-1 for 10 min.As shown in Figure 2, letter A, Alamandine-(1-5) induced a concentration-dependent decrease in the area of ​​shortening of ventricular myocytes, with more pronounced effects observed in cells exposed to 100 nmol.L-1 (Figure 2, letter A). Illustrative images of motion vectors acquired from control cells or cardiomyocytes exposed to Alamandine-(1-5) 100 nmol.L-1 are presented in Figure 2, letters B and C. Petition 870250077383, dated 08 / 30 / 2025, page 14 / 29 11 / 25

[36] As shown in Figure 2, letters D and F, Alamandine-(1-5) induced a significant decrease in area and shortening velocity parameters. To assess whether the reduction in contractility is due to changes in Ca2+ release, we recorded the Ca2+ transient amplitude by confocal microscopy in cells loaded with Fluo-4 AM. Consistent with the contractility data, cardiomyocytes exposed to Alamandine-(1-5) showed reduced and slower Ca2+ transients (Figure 2, letters G and H). EXAMPLE 3 - REDUCTION OF CARDIAC CONTRACTILITY IN Isolated rat hearts by ALA-(1-5)

[37] The Langendorff technique was used to evaluate the effect of Alamandine-(1-5) (SEQ ID No1) on left ventricular contractility and coronary arteries. Wistar rats were anesthetized with ketamine (100 mg / kg) and xylazine (10 mg / kg), then decapitated 10-15 min after intraperitoneal injection of 200 IU of heparin.

[38] Briefly, the thorax was opened and the heart was carefully dissected and perfused through an aortic stump with Krebs-Ringer (KRS) solution containing, in mmol.L-1: 118.4 NaCl, 1.2 KH2PO4, 4.7 KCl, 1.2 MgSO4-7H2O, 1.26 CaCb2H2O, 26.5 NaHCO3 and 11.7 glucose at 37 °C with constant oxygenation (5% CO2 and 95% O2). A water-filled balloon was inserted into the left ventricle through the left atrium to record the isovolumetric ventricular pressure and the volume was adjusted to maintain the left ventricular end-diastolic pressure between 8 and 12 mmHg. The maximum rate of increase in left ventricular pressure (dP / dtmax), the maximum rate of decline in left ventricular pressure (dP / dtmin), and the heart rate (HR) were calculated from the intraventricular pressure waveform.

[39] Coronary perfusion pressure was acquired using a pressure transducer connected to the aortic cannula and coupled to a Petition 870250077383, dated 08 / 30 / 2025, page 15 / 2912 / 25 data acquisition system (DATAQ Instruments, USA). During the stabilization period (20 min), the perfusion flow was adjusted to maintain the perfusion pressure between 70 and 100 mmHg. After that, the perfusion flow was kept constant and the hearts were perfused for another 15 min with KRS containing Alamandine-(1-5) 20 pmol.L-1 or 200 pmol.L-1. All experimental protocols were approved and carried out following the CEUA-UFG (protocol number 013 / 21). As shown in Figure 3, letter A, Alamandine gradually reduced LEVSP at a dose of 20 pmol.L-1. This effect was transient at a concentration of 200 pmol.L-1 (Figure 3, letter A). LEVDP was increased by Alamandine-(1-5) 200 pmol.L-1. No significant effect on LEVDP was observed at a concentration of 20 pmol.L-1 (Figure 3, letter B). Interestingly, at a concentration of 200 pmol.L-1o pentapeptide evoked a biphasic effect on dP / dtmax (Figure 3, letter C) and dP / dtmin (Figure 3, letter D), that is, a rapid and transient increase followed by a significant decrease in these parameters.

[40] At a concentration of 20 pmol.L-1, Alamandine-(1-5) produced only a gradual reduction in these parameters, and at a concentration of 200 pmol.L-1, it induced a reduction in perfusion pressure at the beginning of the perfusion period, indicating coronary vasodilation, which returned to baseline values ​​at the end of the analyzed period (Figure 3, letter E). In contrast, at a concentration of 20 pmol.L-1, Alamandine-(1-5) promoted a slight, but not significant, initial reduction in perfusion pressure followed by a progressive increase in this parameter. No effect was observed on HR (Figure 3, letter F). EXAMPLE 4 - CARDIOVASCULAR EFFECTS AND REFLEXES OF ACUTE ALAMANDINE-(1-5) ICV INFUSION

[41] Rats (SHR and Wistar, 12-14 weeks old) were anesthetized with a mixture of ketamine (50 mg / kg) and xylazine (10 Petition 870250077383, dated 08 / 30 / 2025, p. 16 / 29 Rats were subjected to stereotaxic implantation of a 25-gauge metallic cannula into the lateral ventricle and cemented to the skull with three anchoring screws. The cannula tip was placed 1.0 mm caudal to the bregma, 1.5 mm lateral to the midline, and 4.5 mm ventral to the dorsal surface of the skull, using the Paxinos & Watson atlas15 as a reference. The ICV cannula was connected to a polyethylene tube (PE-10), filled with sterile isotonic saline solution, and fixed to the interscapular region. After surgery, the rats received a polyantibiotic (20U; Pentabiotico®, Fort Dodge, Brazil) and flunixin meglumine (1 mg / kg, sc; Banamine®, Schering Plough, Brazil) for postoperative analgesia. The infusion site was verified post-mortem by the presence of Alcian blue dye (5%), injected through the IVC cannula (2 μL), only in the ventricular system.

[42] Cardiovascular parameters were recorded 5 to 7 days after stereotaxic surgery. One day before the experiments, under anesthesia with ketamine (50 mg / kg) and xylazine (10 mg / kg), the animals underwent implantation of polyethylene cannulas in the femoral artery and vein for recording cardiovascular parameters and baroreflex control of the HR test. Twenty-four hours later, a one-hour stabilization period of cardiovascular parameters was given, and baseline recording of MAP and HR and the baroreceptor reflex test were performed. The baroreflex was assessed by measuring reflex changes in HR (converted to pulse interval, PI) in response to transient increases in MAP (mmHg) induced by bolus injections of increasing doses of phenylephrine (PHE, 0.1 ml of 1 - 10 pg / ml, iv) as previously described.16 The baroreflex sensitivity index of each rat was calculated by the mean ratio (ΔPI / ΔMAP; ms / mmHg) of the maximum changes in PI and MAP with each dose of PHE. ICV infusion (4 pg / 12 μL / h, for 90 min) of Alamandine-(1-5) or saline solution (NaCl. Petition 870250077383, dated 08 / 30 / 2025, p. 17 / 29 14 / 25 The infusion (0.9%; control) was performed using a Hamilton® syringe connected to an infusion pump. Rats were randomly assigned to an Alamandine-(1-5) or control group. At the end of the ICV infusion, baroreflex sensitivity was assessed again. All experimental protocols were approved and performed following the CEUA-UFMG (protocol 113 / 2023). In conscious, freely moving rats, 90 minutes of Alamandine-(1-5) ICV infusion did not significantly alter baseline MAP or HR values ​​in normotensive rats (MAP: 103 ± 8 mmHg before and 105 ± 11 mmHg after 90 min infusion; HR: 312 ± 13 bpm before and 331 ± 10 bpm after 90 min infusion) or in hypertensive rats (MAP: 159 ± 8 mmHg before and 161 ± 10 mmHg after 90 min infusion; HR: 296 ± 7 bpm before and 310 ± 2 bpm after 90 min infusion). As expected, SHR animals showed a marked attenuation of baroreflex control of heart rate (0.3 ± 0.1 ms / mmHg) compared to Wistar rats (1.5 ± 0.5 ms / mmHg).

[43] As shown in Figure 4, letters A and B, SHR animals infused with Alamandine-(1-5) showed a significant improvement in baroreflex bradycardia sensitivity (0.5 ± 0.1 ms / mmHg) compared to the same animals before Alamandine-(1-5) infusion (0.3 ± 0.1 ms / mmHg). In contrast, Alamandine-(1-5) ICV infusion did not significantly alter the baroreflex control of HR in Wistar rats (1.4 ± 0.3 ms / mmHg). Cardiac autonomic balance was assessed by pharmacological blockade of cardiac muscarinic and β-adrenergic receptors. Interestingly, Alamandine-(1-5) did not produce significant changes in cardiac autonomic balance in either normotensive or hypertensive strains. EXAMPLE 5 - EFFECTS OF ALAMANDINE-(1-5) ON THE CORTEX INSULAR Petition 870250077383, dated 08 / 30 / 2025, page 18 / 29 15 / 25

[44] We recently showed that the insular cortex (IC) is a brain region where Alamandine has unique effects (increased blood pressure, heart rate and renal nerve activity) contrasting with the essentially absent effects of Ang-(1-7). Thus, our next aim was to determine the effect of Alamandine-(1-5) (SEQ ID No1) in this region.

[45] Under urethane anesthesia (1.4 g / kg, ip), the trachea was cannulated to maintain airway patency and the femoral artery and vein were cannulated (PE10 connected to PE50) to record PAP and drug injection or to supplement anesthesia, respectively. During the protocol, body temperature was maintained in the range of 36.5 to 37.0°C with a heating pad (Physitemp TCAT-2DF Controller). Blood pressure was recorded using a pressure transducer (MLT 0699, AD Instruments) and an electrocardiogram (ECG) was performed using the Sgoifo et al. protocol17 for heart rate (HR) measurement.

[46] Using a retroperitoneal approach, the left renal nerve was isolated and covered with mineral oil. The nerve was placed on a silver bipolar electrode to record renal sympathetic nerve activity (RSNA). The signal was amplified at 10K, filtered (100-1000 Hz), displayed on an oscilloscope (Tektronix 546B), and monitored by an audio amplifier. The filtered nerve activity signal was rectified, integrated (reset every second), and displayed. The noise level of the RSNA recording system was determined post-mortem and subtracted from the RSNA values ​​obtained during the experiment. The signals – ECG, MAP, and RSNA – were collected continuously and displayed on a computer connected to Powerlab 4 / 20 - LabChart 7.3.2 software (ADInstruments, Sydney, Australia). The animals were positioned in a stereotaxic frame, and a small unilateral craniectomy was performed to allow insertion. Petition 870250077383, dated 08 / 30 / 2025, page 19 / 29 16 / 25 of a glass pipette (Sigma-Aldrich) was injected into the insular cortex with the following coordinates: anteroposterior +1.5 mm, laterolateral 5.4 mm, and dorsoventral -7.0 mm. After all surgical procedures, a minimum period of 20 minutes was expected for stabilization of cardiovascular parameters. The duration of the microinjection of Alamandine(1-5) (400 pmol / 100 nL) into the IC was less than 10 seconds. As Alamandine (40 pmol / 100 nL, n=7) evoked a cardiovascular response only in the rostral IC, all experiments were performed only in this region. We evaluated changes in HR (beats / min), MAP (mmHg), and ANS (% baseline) evoked by unilateral microinjection of saline solution (0.9% NaCl, n = 4) and Alamandine-(1-5) (400 pmol / 100 nL, n = 7) in the rostral IC.

[47] All experiments were performed on separate groups of animals and the protocol was approved and carried out following the CEUA-UFMG (protocol number 11412 / 2012). At baseline, there was no difference in cardiovascular parameters between the groups (vehicle: 85 ± 2 mmHg and 388 ± 5 beats / min, Alamandine: 83 ± 9 mmHg and 389 ± 10 beats / min or Ala-(1-5): 91 ± 6 mmHg and 386 ± 7 beats / min). Microinjection of vehicle (saline solution, n=4) was performed as a control for non-specific autonomic and cardiovascular effects produced by volume microinjection and / or tissue damage. As shown in Figure 5, saline microinjection did not cause changes in the evaluated parameters (ΔMAP: 2±1 mmHg, ΔHR: 4±2 beats / min, ΔRSNA: 3±1%, Figure 5). Alamandine microinjection (n=7) into the rostral IC produced a sustained increase (18± 3 min) in MAP (Δ= 14 ±2 mmHg), and this effect was associated with a significant increase in HR (Δ= 32 ±4 beats / min) and RSNA (Δ= 36 ± 6%).These responses were obtained immediately after microinjection of Alamandine. When compared to the vehicle, and very different from what was observed with Alamandine alone... Petition 870250077383, dated 08 / 30 / 2025, page 20 / 29 17 / 25 microinjection of Alamandine-(1-5) (n=7) caused a significant increase in blood pressure without altering renal nerve activity or heart rate (ΔMAP: 22 ±2 mmHg, ΔHR: -1±3 beats / min, ΔRSNA: 1±3%; Figure 5). Unlike the response evoked by Alamandine, the effect of Alamandine-(1-5) had a shorter duration (5.88 ±1.9 min) without delay in the onset of effect. EXAMPLE 6 - IN VIVO CARDIOVASCULAR EFFECTS OF ALAMANDINE-(1-5) IN SHR RATS

[48] ​​One day before the experiments, the animals (SHR and Wistar, 12-14 weeks old, n= 6 each) were anesthetized with a mixture of Ketamine (50 mg / kg) and Xylazine (10 mg / kg). Polyethylene cannulas made with PE10 (4 cm for the artery and 3 cm for the vein) heat-polymerized with PE50 (approx. 17 cm) and filled with sterile 0.9% saline solution containing heparin (5000 IU / ml) were inserted into the abdominal aorta through the femoral artery (BP recordings) and into the femoral vein (drug injections). Catheters were externalized through the subcutaneous tissue to the interscapular region of the animal and fixed with surgical suture thread. Twenty-four hours later, cardiovascular parameters were recorded in conscious animals. The arterial catheter was connected to a pressure transducer, and pulsatile arterial pressure (PAP) was continuously recorded by an A / D data acquisition system (MP100; Biopac Systems, Inc., Santa Barbara, CA, USA).Mean arterial pressure (MAP) and heart rate (HR) were simultaneously derived from arterial pulse waves using software (AcqKnowledge5; Biopac Systems). After a one-hour stabilization period of cardiovascular parameters, the peptide Alamandine(1-5) was injected intravenously at a dose of 30 ug / kg. The injection volume was 0.1 mL / 100g. A similar volume of sterile saline solution was injected into SHR and Wistar rats. Petition 870250077383, dated 08 / 30 / 2025, page 21 / 29 18 / 25

[49] After injection, cardiovascular parameters were monitored for 6 hours. All experimental protocols were approved and performed following the CEUA-UFMG (protocol 113 / 2023). As shown in Figure 6, IV administration of Alamandine-(1-5) produced a progressive decrease in systolic blood pressure in free-moving SHR, reaching -33.3±3.0 mmHg, 6 hours after administration (systolic blood pressure: 208±7.6 mmHg before and 175±10.5 mmHg, 6 hours after injection). This marked effect on blood pressure was not accompanied by significant changes in HR (baseline: 348 ± 24.2 beats / min). The administration of Alamandine-(1-5) in Wistar rats produced only small changes in blood pressure (99 ± 11.5 mmHg, 6 hours after injection vs 109 ± 0.1 mmHg, before) without significant changes in heart rate (303 ± 12.3 beats / min, 6 hours after injection vs 328 ± 20.5 beats / min, before). Saline injection did not alter blood pressure or heart rate in Wistar or SHR rats. EXAMPLE 7 - INDUCTION OF NITRIC OXIDE PRODUCTION BY ACTIVATION OF MAS, MrgD AND AT2 RECEPTORS BY THE ACTION OF ALAMANDINE-(1-5)

[50] We evaluated NO measurements to test the activation of Alamandine-(1-5) receptors transfected into CHO cells and EA.hy926 cells. Cells were cultured overnight on glass slides and loaded with 10⁻⁶ mol.L⁻¹⁴,5-diaminofluorescein diacetate (DAF-2DA; Molecular Probes) for 30 minutes. After washing with PBS to remove excess probe, cells were stimulated for 30 min with Ala-(1-5) at concentrations ranging from 10⁻⁶ to 10⁻⁸ mol.L⁻¹ or the receptor agonist, leaving untreated cells as controls. Cells were then rinsed in PBS, fixed in 4% (w / v) paraformaldehyde solution for 15 min, and then Petition 870250077383, dated 08 / 30 / 2025, page 22 / 29 19 / 25 mounted in anti-fade mounting medium. Images were obtained on a Nikon Eclipse Ti confocal microscope (Nikon, Melville, NY, USA) with λex = 495 nm and λem = 505 nm. Fluorescence of the confocal images was quantified using ImageJ 14. Subsequently, we used pharmacological and genetic tools to gain insights into the mechanism of action of Alamandine-(1-5) using aortic rings from mice and cell culture. As shown in Figure 7, letter A, pretreatment of aortic rings with L-NAME only slightly increased the contractile effect of Alamandine-(1-5). The contractile effect of Alamandine-(1-5) was essentially abolished in aortic rings taken from MrgD-deficient mice and markedly attenuated in aortic rings taken from AT2-KO mice. In aortic rings of Mas-KO mice, the contractile effect of Alamandine-(1-5) was not significantly altered (Figure 7).Similarly, pretreatment with losartan did not alter the contractile effect of alamandine-(1-5) in this preparation.

[51] To investigate which of the classical RAS receptors Alamandine-(1-5) is acting on, we used EA.hy926, an endothelial cell line, and CHO cells transfected with Mas, MrgD, or AT2 receptors to evaluate nitric oxide production after stimulation with the peptide compared to unstimulated control cells (Figure 8, letters A and B). NO production was activated by Alamandine-(1-5) in EA.hy926 cells by 37% above the unstimulated control (p<0.0001). Alamandine-(1-5) also activated CHO cells transfected with Mas, MrgD, and AT2 receptors, increasing NO production by 77%, 85%, and 67%, respectively (p<0.0001). Together, these data showed that Alamandine-(1-5) can activate Mas, MrgD, and AT2 receptors. Petition 870250077383, dated 08 / 30 / 2025, pages 23 / 29 20 / 25 EXAMPLE 8 - EVIDENCE THAT THE ECA PLAYS A ROLE IMPORTANT ROLE IN THE FORMATION OF ALAMANDINE-(1-5) FROM ALAMANDINE IN PLASMA

[52] To explore the potential involvement of ACE in the conversion of Alamandine to Alamandine-(1-5) (SEQ ID No1), plasma samples were collected from angiotensinogen knockout (TLM) mice, which do not produce angiotensins. We conducted incubation experiments with Alamandine using TLM plasma in the presence and absence of the ACE inhibitor captopril. Mass spectrometry analysis (MALDI-TOF / TOF) was then employed to detect any potential formation of a fragment corresponding to the mass of Alamandine-(1-5) at 621 Da. As expected, TLM plasma (TLM_CT) did not show a peak corresponding to the mass of angiotensin. However, after 1 min (TLM-T1') and 3 min (TLM-T3') of TLM plasma incubated with Alamandine, the formation of a 621 Da peak was observed, corresponding to the mass of Alamandine-(1-5).In contrast, in the presence of captopril (TLM-T1'-CAP and TLM-T3'-CAP) no formation of a 621 Da peak was detected, highlighting the critical role of ACE activity in the formation of this pentapeptide, Figure 9 (A). The same mass spectrum was extended to the 610-650 m / z range to better visualize the formation of the 621 Da peak in the groups, Figure 9 (B). To confirm the identity of the enriched Alamandine, the pure synthetic peptide and the synthetic peptide in plasma were subjected to MS / MS fragmentation and sequenced using the de novo sequencing method, Figure 9 (C). To confirm the identity of Alamandine-(1-5), the pure synthetic peptide and the 621 Da peak in plasma were subjected to fragmentation and MS / MS sequencing, Figure 9 (D). Petition 870250077383, dated 08 / 30 / 2025, pages 24 / 29 21 / 25

[53] Overall, these results suggest that ACE is responsible for the rapid formation of Alamandine-(1-5) from Alamandine. In fact, Alamandine inhibits ACE activity in TLM mouse plasma in vitro with an EC50 of approximately 1.2 μmol.L-1. In contrast, Alamandine-(1-5) demonstrated an EC50 greater than 10 μmol.L-1 under the same conditions. These results are consistent with the MALDI-TOF / TOF findings and suggest that Alamandine acts as a competitive inhibitor of ACE. EXAMPLE 9 - ALAMANDINE CIRCULATES IN THE BLOOD OF MICE AND HUMANS

[54] Next, we used LC-MS / MS to assess whether Alamandine-(15) (SEQ ID No. 1) is present in human and mouse plasma samples. Blood samples from adult mice were collected from male and female animals (n=7 for each gender) by decapitation into tubes containing protease inhibitors (Roche Life Sciences, Basel, Switzerland) and 0.5% EDTA (v / v). Blood samples from young volunteers of both sexes (28±6 years and 67±15 kg) were collected by venipuncture into vacuum tubes containing EDTA and protease inhibitors on the same day as the clinical evaluation (between 8-11h). The blood was immediately centrifuged at 3000 rpm for 10 min, 4°C. Plasma samples were obtained and stored at -70°C until analyzed.

[55] Five milliliters of peripheral blood samples from healthy children (n=11) and pediatric patients with nephrotic syndrome (n=11) were collected by venipuncture into vacuum tubes containing EDTA on the same day as the clinical evaluation (between 8-11h). The blood was immediately centrifuged at 3000 rpm for 10 min, 4°C. Plasma samples were obtained and stored at -70°C until analyzed.

[56] The protocols were approved by CEP-UFMG (protocol number: CAAE-0547.0.203.000-11). The legal representatives of all Petition 870250077383, dated 08 / 30 / 2025, pages 25 / 29 22 / 25 participants (patients and healthy individuals) provided written informed consent prior to admission to the study. The study protocol did not interfere with any medical procedures. Follow-up and treatment were guaranteed even in case of refusal to participate in the study.

[57] Sample preparation for plasma angiotensin quantification. Samples were centrifuged (800g x 4°C for 10 minutes) and the plasma was stored at -80°C until extraction. Solid Phase Extraction (SPE) was performed using the Sep-Pak C183cc Vac RC cartridge, 500 mg sorbent (Waters, Milford, MA, USA). The C18 resin was activated by 2 sequential washes with 10 mL of 99% acetonitrile (ACN) / 0.1% formic acid (FA) (v / v), followed by 10 mL of 0.1% FA (v / v). Next, the resin was conditioned with 3 mL of 0.1% FA / 0.1% BSA (v / w), 10 mL of 10% acetonitrile / 0.1% FA (v / v), and 3 mL of 0.1% FA (v / v). The samples were then loaded and washed with 20 mL of 0.1% FA (v / v). For elution, 3 mL of 60% ACN / 0.1% FA (v / v) were loaded and collected in low-binding polypropylene tubes (Eppendorf, Hamburg, Germany). The samples were dried using a SpeedVac (Eppendorf, Hamburg, Germany).The dried samples were resuspended in 100 µL of 0.1% (v / v) formic acid (sample concentration factor = 10 times).

[58] Multiple Reaction Monitoring (MRM) Analysis. Chromatography was performed on an ACQUITY I-Class UPLC system (Waters, Milford, MA, USA) coupled to electrospray ionization (ESI) tandem mass spectrometry (LC-ESI-MS / MS Xevo TQ-S (Waters, Milford, MA, USA)). Chromatographic separation was performed on a C18 column (ACQUITY UPLC BEH C18 column, 130 Å, 1.7 μm, 2.1 mm x 100 mm, Waters, Milford, MA, USA) for 5.5 min per injection (10 pL volume per sample). Solvent A was made from 0.1% formic acid in Petition 870250077383, dated 08 / 30 / 2025, pages 26 / 29 23 / 25 H2O (v / v) and solvent B of 0.1% formic acid in ACN (v / v). The chromatographic gradient was defined as previously described 18 at a flow rate of 300 pL / min as follows (expressed as % of solvent B): i) 3-40% in 3.5 min, ii) 40-99% in 0.01 min, iii) 99% for 0.99 min, iv) 99-3% in 0.01 min and v) 3% for 0.99 min. Regarding the MS analysis, the main parameters were as follows: i) capillary = 3.5 kV; ii) cone = 20 V; iii) desolvation gas temperature (hydrogen) = 550 °C. The collision energy (CE) (argon gas) was adjusted for each target peptide ranging from 10 to 20 CE. The following transitions were monitored by MS in MRM mode. Alamandine: 286.1 < 136.1 and 286.1 < 327.2; Ang A: 335.1 < 231.8 and 335.1 < 513.2; Alamandine-(1-5): 621 < 210.94 and 621 < 508.1 m / z. The calibration curve was obtained using a stock solution containing a mixture of synthetic peptides.The applied calibration curve model (y = ax + b) proved to be accurate in the concentration range of 10 to 1000 pg^mL-1 (r=0.997). Data acquisition was performed using MassLynx MS software (Waters, Milford, MA, USA) and raw data evaluation using its TargetLynx™ application.

[59] MALDI TOF / TOF analysis. To evaluate whether Alamandine is metabolized from ACE to form Alamandine-(1-5), we took advantage of the global availability of angiotensinogen-deficient (TLM) mice.19 Blood samples were collected from TLM mice in heparin tubes and centrifuged (5000 RPM, 10 min, 4°C) for plasma separation. In tubes containing 470 pL of Tris-HCl buffer pH = 7.4, we added 20 pL of plasma from TLM mice and 10 pL of Alamandine solution at 500 pg.mL-1 (final concentration of 10 pg.mL-1). Samples were incubated at 37°C for 0, 1, and 3 minutes, with or without 1.2 μmol.L-1 of the ACE inhibitor, Captopril (Sigma-Aldrich). The reaction was stopped by the addition of a protease inhibitor cocktail (Roche Petition 870250077383, dated 08 / 30 / 2025, pages 27 / 29 24 / 25 Alamandine solution was added to the sample using a 0.5% (v / w) solution of Alamandine solution immediately at the appropriate time. Samples without Alamandine solution were used as controls. Prior to mass spectrometry analysis on an Autoflex III Smartbeam MALDI TOF / TOF spectrometer (Bruker Daltonics, Billerica, USA), we desalinated the sample using C18 P10 ZipTip® pipette tips (Millipore, Billerica, MA) following the manufacturer's instructions. Samples were eluted directly onto a polished MTP 384 steel target plate (Bruker Daltonics, Billerica, USA) and diluted (1:1, v / v) with an α-cyano-4-hydroxycinnamic acid matrix (SigmaAldrich, Saint Louis, USA). Sample-matrix cocrystallization was performed using the dry drop method.20 External calibration was performed by homogenizing Peptide Calibration Standard II (Bruker Daltonics, Billerica, USA) in a 1:1 ratio in an α-cyano-4-hydroxycinnamic acid matrix directly on the same target plate.The MS spectrum was obtained in positive reflected mode with 400 laser shots and an acceleration voltage maintained at 9.71-21.01 kV.

[60] As shown in Figure 10, letters A and B, Alamandine-(1-5) was detected in blood samples from healthy human volunteers and C57BL6 / J mice. The peptide was present in low concentrations in humans ranging from 0.2 to 8.7 pg / mL (mean 2.4 ± 1.3 pg / mL). In these samples, the concentration of the Alamandine-(1-5) precursor, Alamandine, ranged from 0.1 to 18.6 pg / mL (mean 3.7 ± 2.5 pg / mL). Alamandine-(1-5) was also detected in extracted mouse plasma samples (Figure 10, letter B).

[61] We previously reported that in pediatric patients with end-stage renal disease there was a marked increase in plasma angiotensin-(1-7). Here, we evaluated the concentration of Alamandine and Alamandine-(1-5) in pediatric patients with primary nephrotic syndrome (n=11) and compared them with measurements taken in Petition 870250077383, dated 08 / 30 / 2025, pages 28 / 29 25 / 25 healthy children matched for sex and age (n=11). In terms of disease response, 5 patients were in partial remission, 5 patients in complete remission, and only 1 patient was in disease relapse. All patients had normal renal function. Plasma levels of Alamandine-(1-5) were significantly higher in patients compared to controls (28.03±7.54 pg.mL-1 vs. 10.68±3.02 pg.mL-1 in controls, Figure 10, letter C). Despite a slight decrease in controls, plasma levels of Alamandine did not differ between the groups (19.54±6.33 pg.mL-1 vs. 26.09±6.12 pg.mL-1 in controls).

[62] No correlations were found between RAS molecules and proteinuria and with medications in use. However, Alamandine(1-5) was negatively correlated with plasma albumin levels (r= -0.695; p=0.038). In addition, Alamandine correlated negatively with total cholesterol (r= -0.700; p= 0.036), LDL (r= 0.800; p= 0.004) and HDL (r= -0.762; p= 0.038). Petition 870250077383, dated 08 / 30 / 2025, p. 29 / 29

Claims

1 / 1 CLAIMS 1. ALAMANDINE-(1-5) PEPTIDE, characterized in that it is defined by the amino acid sequence SEQ ID No.

1.

2. PHARMACEUTICAL COMPOSITION, comprising the peptide defined in claim 1, characterized by containing the Alamandine-(1-5) peptide, defined by SEQ ID No. 1, in the presence of pharmaceutically and physiologically acceptable carriers and / or excipients.

3. PHARMACEUTICAL COMPOSITION, according to claim 2, characterized by being administered orally, intramuscularly, intravenously, subcutaneously, topically, transdermally or with devices that can be implanted or injected.

4. USE of the peptide defined in claim 1, characterized by being in the production of medicines.

5. USE of the pharmaceutical composition, defined in claim 2, characterized by being in the production of medicines. Petition 870240108582, dated 12 / 19 / 2024, pp. 42 / 81

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