EFFECT OF NEROL COMPLEXED WITH β-CYCLODEXTRIN IN THE TREATMENT OF ACUTE MYOCARDIAL INFARCTION IN RATS

Description

/ 11 EFFECT OF NEROL COMPLEXED WITH β-CYCLODEXTRIN IN THE TREATMENT OF ACUTE MYOCARDIAL INFARCTION IN RATS

[001] This patent application relates to a pharmaceutical formulation of the inclusion complex of nerol ((2Z)-3,7-dimethylocta-2,6-dien-1-ol) with β-cyclodextrin (β-CD) with cardioprotective action in a rat model of acute myocardial infarction. It is an intraperitoneal application product for the treatment of acute myocardial infarction injury in rats. The inclusion complex containing nerol + β-CD proved effective in reducing myocardial injury by decreasing the infarct area, creatine phosphokinase MB (CK-MB) levels, and improving the mechanical function of the heart, as well as electrocardiographic changes.

[002] This work was designed seeking a technologically innovative product focused on developing a formulation that is economically viable and has fewer adverse effects for the treatment of injury resulting from acute myocardial infarction. The complexation of nerol with β-CD is important because it improves the chemical stability of nerol, its solubility, and its biological effect.

[003] Cardiovascular diseases (CVDs) have high morbidity and mortality rates. The increase in the number of hospitalizations related to CVDs occurs due to several factors and should be treated immediately after the onset of the first symptoms. In this sense, data show that the impact of deaths from these pathologies was 17.9 million individuals in 2019, equivalent to 32% of global deaths. Furthermore, among 20 million people who suffered from CVDs, approximately 12 million were fatal due to acute myocardial infarction (MI) (WHO, 2021). In Petition 870250048864, dated 11 / 06 / 2025, page 4 / 15 / 11 In low- and middle-income countries, such as Brazil, more than three-quarters of deaths are due to CVDs. Furthermore, of the 17 million premature deaths (under 70 years old) due to non-communicable diseases in 2019, 38% were caused by CVDs (Ministry of Health, 2021).

[004] From a pathological point of view, MI is defined as the death of cardiomyocytes caused by an ischemic insult. The diagnosis of MI depends on the sensitivity and specificity of clinical criteria, electrocardiographic findings, imaging studies, and biomarkers used to detect cardiomyocyte death (Heusch, 2020). Thus, MI is a cardiac dysfunction in which reperfusion of coronary blood flow is necessary to minimize the size of the myocardial infarction and cardiac dysfunction; however, this process also causes additional cardiac damage. However, when myocardial demand is increased, flow restriction prevents increased oxygen supply, resulting in ischemia and causing angina pectoris, characterized by chest pain due to lack of blood. In the vast majority of cases, MI results from atherosclerotic coronary artery disease complicated by superimposed thrombosis (Frangogiannis, 2015).Among the treatments for ischemic heart injury, pharmacological treatment, ischemic preconditioning, medical gases, or vitamin therapy stand out (Naito et al., 2020). However, a universally accepted treatment is still lacking. Therefore, studies are needed to discover new therapeutic agents that can be applied before, during, or after reperfusion to protect the heart against damage from ischemic heart injury, to reduce cardiac death, and to prevent the development of heart failure (Gunata & Parlakpinar, 2020). Petition 870250048864, dated 11 / 06 / 2025, page 5 / 15 / 11

[005] The development of a new natural product capable of reducing the damage caused by MI is of great importance to medicine. Among natural products, the class of terpenes stands out. Among terpenes, the monoterpene nerol (C10H18O) stands out, present in several essential oils (Chen & Viljoen, 2010). Nerol was primarily isolated from neroli oil, with a scent similar to bergamot oil, resulting in orange blossom bergamot, which is widely used in perfume production (Gonza et al., 2016). Nerol exhibits antioxidant activity with superoxide radical scavenging action (Mileva et al., 2014) with promising antioxidant activity in alveolar macrophages of rats stressed with hydroperoxides, inhibiting the generation of reactive oxygen species (ROS) (Tiwari & Kakkar, 2009).Studies have shown that nerol was able to reduce atrial contractility and L-type calcium current, exhibiting antiarrhythmic properties in guinea pigs (Menezes-Filho, 2019). According to Cheng et al. (2021), nerol showed an effective cardioprotective effect by activating the PI3K / AKT pathway, reducing cell apoptosis in H9c2 cells subjected to hypoxia / reoxygenation (Cheng et al., 2021).

[006] However, the cardioprotective effects of nerol against isoproterenol-induced acute myocardial infarction are still undefined. Nerol has a hydrophobic nature, conferring low affinity in aqueous media, limiting its use in in vivo systems. Drugs that are poorly soluble in water have low bioavailability, with dissolution being the limiting factor for their absorption. Among the techniques used to increase dissolution and decrease toxicity, the complexation of drugs with cyclodextrins stands out (Grillo et al., 2008). In order to suppress this chemical characteristic, Petition 870250048864, dated 11 / 06 / 2025, page 6 / 15 / 11, encapsulation is used in drug delivery systems, among which the technological use of complexation with cyclodextrins (CDs) stands out, mitigating this limitation.

[007] CDs are cyclic oligosaccharides composed of six (α-CD), seven (β-CD), or eight (γ-CD) glucopyranose units linked by α-1,4-glycosidic bonds (Stella; He, 2008), allowing them to form inclusion complexes with other substances. β-CD has been widely used to increase the solubility, bioavailability, and reduce the side effects of drugs (Silva, 2014). Thus, complexing nerol with β-CD will result in an easily handled powder that may exhibit greater bioavailability, better cardioprotective effect, and greater potency. Furthermore, β-CD has low cost and low toxicity (Oliveira-Filho et al., 2018). Therefore, obtaining a formulation of nerol complexed with β-CD is important to evaluate its cardioprotective effect in the acute myocardial infarction model in rat hearts.This is a formulation already patented by our research group (BR1020230141226) entitled “PHARMACEUTICAL FORMULATION CONTAINING NEROL AND β-CYCLODEXTRIN: METHOD OF OBTAINING, CHARACTERIZATION AND CARDIOPROTECTIVE EFFECT IN CARDIAC ISCHEMIA AND REPERFUSION INJURY”. This formulation was tested in another pathological model of acute myocardial infarction induced by the administration of a high dose of isoproterenol. The present invention is not limited to the methods and components described. The invention is capable of other embodiments and of being practiced in various ways. It should be understood that the description employed here is for descriptive purposes only and should not be considered limiting. Petition 870250048864, dated 11 / 06 / 2025, page 7 / 15 / 11

[008] Preparation of the nerol inclusion complex

[009] The nerol + β-CD inclusion complex was obtained by lyophilization, which consisted of adding 20 mL of distilled water to a mortar and adding nerol and β-CD (molar ratio of 1:1). The mixture was kept under magnetic stirring (36 h, at 150 rpm). The mixture was maintained at a temperature of 4 °C. Then, the frozen solution was subjected to lyophilization (-50 °C) to remove all moisture. The nerol + β-CD was stored in hermetically sealed glass containers.

[0010] Acute myocardial infarction model in rats

[0011] IM was induced by administering isoproterenol (ISO) (100 mg / kg; ip) on two consecutive days, with a 24-hour interval between applications (Mesquita, 2017). Thirty minutes after each ISO administration, the animals were treated with nerol + eCD (5, 10, or 25 mg / kg; ip). ISO and nerol + eCD were diluted in saline solution (0.9% NaCl). Pure nerol was diluted in DMSO (1%). The animals were divided into 5 groups: 1) Control: animals received eCD administration and were not subjected to IM; 2) Vehicle + IM: animals received isoproterenol (100 mg / kg, ip) and saline + eCD administration; 3) Nerol + IM: animals received isoproterenol (100 mg / kg ip) and nerol (10 mg / kg); 4) Nerol + eCD: animals received isoproterenol (100 mg / kg, ip) and nerol + eCD administration (5, 10 and 25 mg / kg).

[0012] Twenty-four hours after the last ISO administration, the animals were weighed on a precision scale and anesthetized with ketamine and xylazine (80 mg / kg and 10 mg / kg, respectively). Then, an incision was made at the level of the xiphoid process to expose the heart, which was removed through the aortic arch. The heart was cleaned and Petition 870250048864, dated 11 / 06 / 2025, page 8 / 15 / 11 weighed on a precision scale. In addition, the tibia of each rat was measured using a caliper. The data were presented as the ratio of heart mass to body mass and as the ratio of heart mass to tibia size (Souza et al., 2020).

[0013] Left ventricular developed pressure (LVDP) was determined using a latex balloon that was inserted into the left ventricle. Signals were captured by a pressure transducer (MTL0699 / A®), amplified (BRIDGE Amp FE221, ADInstrument®), digitized (PowerLab 4 / 35 ADInstrument®), and stored on a computer. From the left ventricular pressure recording, the following were determined: LVDP, maximum (+dP / dt) and minimum (-dP / dt) ventricular pressure derivatives, end-diastolic pressure (EDP), and heart rate.

[0014] The infarct area was determined by staining with triphenyltetrazolium chloride (TTC) according to Fishbein et al. (1981). On the third day after infarction (2 days), the hearts underwent three cross-sections. For measuring the infarct area, the middle third of the heart, 0.5 cm thick, was used. The heart segment was kept at 37°C in a 1% TCC solution (diluted in Krebs solution) for 20 min, and subsequently fixed in buffered formalin for 30 min. Viable cells acquired a red color, while non-viable cells appeared pale. After this procedure, the images of the sections were scanned using a printer (HP® J4660) and subsequently analyzed using ImageJ® 1.38x software. The results were presented as the percentage of the left ventricular area occupied by the infarct, used to determine the extent of myocardial injury. Petition 870250048864, dated 11 / 06 / 2025, page 9 / 15 / 11

[0015] Twenty-four hours after the last ISO administration, the animals were sacrificed, and blood was collected and stored in a collection tube without anticoagulant. Subsequently, it was centrifuged at 4,500 rpm for 15 minutes at room temperature. After obtaining the serum, the standard operating procedure for assays was performed according to the manufacturer's protocol. Assays were performed in triplicate for the enzyme creatine phosphokinase MB fraction (CPK-MB). A commercial assay kit from Labtest® was used, and the sample analyses were performed on the LABMAX 240 PREMIUM analyzer.

[0016] For electrocardiographic evaluation, rats were anesthetized with ketamine / xylazine (80 mg / kg and 10 mg / kg, respectively, i.p.) and maintained in the supine position with spontaneous breathing. For surface ECG recording, three stainless steel electrodes were implanted subcutaneously. ECG signals were amplified (HP7754A, HP7754B, Hewlett-Packard, Chicago, USA), digitized (DI-710, WindaqPro, Dataq, Ohio, USA), and stored on a computer for offline processing. In all experimental groups, heart rate (HR), QT interval (QTi), QRS complex duration, and intrinsic deflection (ID) were measured over 10 consecutive beats. QTi was corrected for HR using Bazett's equation. The ECG was recorded for 15 min (Gondim et al., 2017; Souza et al., 2019).

[0017] The results obtained in the various experiments were expressed as mean ± SEM and the differences were evaluated by means of one-way or two-way analysis of variance (ANOVA), followed by Tukey's test.

[0018] Figure 1A shows representative images of the infarct area of ​​the experimental groups evaluated. Figure 1B shows that isoproterenol significantly increased the infarct area compared to the control group. However, Petition 870250048864, dated 11 / 06 / 2025, p. 10 / 15 / 11: The hearts of animals treated with pure nerol (N10) and nerol complexed with eCD at doses of 5 (NC5), 10 (NC10), and 25 mg / kg (NC25) showed a reduction in the extent of the infarcted area, being more pronounced in the NC10 and NC25 groups. However, there was no significant difference between NC10 and NC25. It is worth noting that NC10 was more effective in reducing the infarcted area when compared to N10. Regarding myocardial contractility, Figure 1C shows a significant reduction in LVDP in the MI group compared to the control. On the other hand, treatment of animals with N10, NC5, NC10, and NC25 showed improvement in LVDP, with NC10 and NC25 being more efficient than N10 and NC5. Regarding the biochemical marker of myocardial injury, Figure 1D shows the elevation of CK-MB levels in the IM group, which was reduced with the N10, NC5, NC10, and NC25 treatments.These results showed that complexed nerol proved to be more effective than pure nerol at the same dose, i.e., 10 mg / kg. Among the 3 tested doses of complexed nerol (5, 10, and 25 mg / kg), the 10 mg / kg dose proved to be more effective in cardioprotection compared to the 5 mg / kg dose and was similar to the 25 mg / kg dose. Therefore, the chosen dose of nerol+e-CD in this study was 10 mg / kg.

[0019] Regarding morphometric parameters, Figure 2A shows representative images of the heart sizes of the evaluated experimental groups, showing that isoproterenol significantly increased heart size compared to the control group. However, the hearts of animals treated with NC10 showed a reduction in heart size. Figure 2B shows that there was a decrease in body weight in the animals of the infarcted group compared to the control and NC10-treated groups. We can observe in Figures 2C and 2D that the hearts of the IM group Petition 870250048864, dated 11 / 06 / 2025, page 11 / 15 / 11 showed a significant increase in the heart weight / tibia size and heart weight / body weight ratios, respectively, which were reduced with the treatment of animals with NC10, indicating effective protection against cardiac hypertrophy caused by MI.

[0020] Figure 3A shows representative images of the left ventricular developed pressure (LVDP) of the evaluated experimental groups. Figure 3B indicates that isoproterenol-induced MI promoted a marked decrease in LVDP, which was increased in the NC10 group, demonstrating recovery of the heart's contractile function. Regarding heart rate (HR), Figure 3C shows that the heart rate of the NC10 group was significantly lower than in the MI group. The other parameter used to evaluate contractile function was end-diastolic pressure (EDP), shown in Figure 3D, which measures the left ventricle's ability to relax and adapt to variations in blood volume, activating the Frank-Starling mechanism and shaping its contractility. In this result, we observed that the MI group had a reduction in EDP compared to the control group, and that it was recovered in the animals of the NC10 group.Figure 3E shows, respectively, the maximum derivatives of contraction (+dP / dt) and relaxation (-dP / dt), which decreased in the IM group and increased with the treatment of animals with NC10.

[0021] Regarding electrocardiographic parameters, Figure 4A shows representative ECG images, indicating with the ST elevation arrow and increased HR in the IM group. Figure 4B shows that there was a decrease in PRi (ms) in the IM group and no change in the NC10 group with no significant difference from the control. In Figure 4C it was seen that, in the IM group, there was an increase in Petition 870250048864, dated 11 / 06 / 2025, page 12 / 15 / 11 duration of the QRS complex and that treatment with NC10 was able to reverse this increase. Figure 4D shows that the QTc interval was decreased in the group treated with NC10 when compared to the IM group and there was no significant difference in relation to the control group.

[0022] Another parameter evaluated was heart rate variability, where Figures 5A and B show that infarction induction increased autonomic sympathetic modulation, verified by the increase in LF and decrease in HF in the IM group. The LF / HF ratio (Figure 5C) was also increased in the IM group. On the other hand, note that in animals treated with NC10, the LF, HF, and LF / HF values ​​returned to the control situation.

[0023] Thus, it was possible to demonstrate with the results obtained and described above that there was myocardial injury and mechanical and electrical dysfunction of the heart induced by MI and that these were mitigated by treating the animals with NC10. LIST OF FIGURES Figure 1: Area of ​​infarction in cardiac tissues of the different experimental groups. (A) Representative images of infarction areas in the hearts, (B) Percentage of damaged area in each group and (C) Left ventricular pressure (LVDP), (D) Total Creatine Phosphokinase - Fraction (CPKMB). CTR (control), MI (acute myocardial infarction), N10 (pure nerol 10mg / Kg), NC5 (complexed nerol 5mg / Kg), NC10 (complexed nerol 10mg / Kg) and NC25 (complexed nerol 25mg / Kg). Data are expressed as mean ± SEM. One-way ANOVA followed by Tukey's post-hoc test was used. *p < 0.05 vs CTR; #p < 0.05 vs MI; φρ < 0.05 vs N10 and δp < 0.05 vs NC5. Figure 2: Evaluation of morphometric parameters in the different experimental groups. (A) Representative images of the hearts, (B) Body weight (C) Heart weight of the animals normalized by tibia size, (D) Heart weight of the animals normalized by body mass. CTR (control), MI (acute myocardial infarction) and NC10 (nerol complex 10mg / Kg). Data were expressed as mean ± SEM. One-way ANOVA followed by Tukey's post-hoc test was used. *p < 0.05 vs CTR and #p < 0.05 vs MI. Figure 3: Effect of treatment with complexed nerol on ventricular contractility in animals undergoing myocardial infarction (MI). (A) Images with tracings representative of left ventricular strength, (B) LVEDP (left ventricular pressure), (C) heart rate (bpm: beats per minute), (D) EDP (end-diastolic pressure) and (E) Max dP / dt (maximum derivative of left ventricular contraction) Min dP / dt (minimum derivative of left ventricular relaxation). CTR (control), MI (acute myocardial infarction) and NC10 (complexed nerol 10mg / Kg). Petition 870250048864, dated 11 / 06 / 2025, page 13 / 15 / 11 data were expressed as mean ± SEM. A one-way ANOVA test followed by Tukey's post-hoc test was used. *p < 0.05 vs CTR and #p < 0.05 vs IM. Figure 4: Effect of complexed nerol treatment on electrocardiographic changes in myocardial infarction (MI). (A) Representative images of electrocardiographic recordings, (B) PRi interval, (C) QRS complex and (D) corrected QT interval. CTR (control), MI (acute myocardial infarction) and NC10 (complexed nerol 10mg / Kg). Data are expressed as mean ± SEM. One-way ANOVA followed by Tukey's post-hoc test was used. *p < 0.05 vs CTR and #p < 0.05 vs MI. Figure 5: Effect of resistance training on changes in heart rate variability in myocardial infarction (MI). (A) LF, low frequency band, (B) HF, high frequency band, (C) LF / HF ratio. CTR (control), MI (acute myocardial infarction) and NC10 (nerol complex 10mg / Kg). Data are expressed as mean ± SEM. One-way ANOVA followed by Tukey's post-hoc test was used. *p < 0.05 vs CTR and #p < 0.05 vs MI. Petition 870250048864, dated 11 / 06 / 2025, p. 14 / 15

Claims

CLAIMS 1- Nerol complexed with β-cyclodextrin, characterized by being preferentially included in β-cyclodextrin. 2- Nerol complexed with β-cyclodextrin, characterized by being obtained preferably by the lyophilization method. 3- Nerol complexed with β-cyclodextrin, characterized by its effectiveness in the treatment of acute myocardial infarction induced by isoproterenol. 4- Nerol complexed with β-cyclodextrin, characterized by being more effective in the treatment of acute myocardial infarction caused by isoproterenol compared to pure nerol. 5- Nerol complexed with β-cyclodextrin, characterized by being preferentially useful in the treatment of injury induced by acute myocardial infarction due to isoproterenol. Petition 870240109881, dated 12 / 23 / 2024, page 17 / 23

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