Biosynthetic Limonene Derivatives for the Treatment of Anxiety and Seizures in Zebrafish

Description

/ 4 Biosynthetic Limonene Derivatives for the Treatment of Anxiety and Seizures in Zebrafish Field of invention

[001] The present invention relates to the field of pharmacology and medicinal chemistry of bioactive compounds, specifically to the use of biosynthetic derivatives of limonene with anxiolytic and anticonvulsant properties.

[002] The invention involves the use of these derivatives in an aquatic model, such as zebrafish (Danio rerio), to evaluate their effects on specific neural receptors, including GABA and 5-HT receptors.

[003] The approach adopted combines structural and functional analyses to better understand the molecular interactions of limonene derivatives with these receptors, aiming to elucidate the underlying neuropharmacological mechanisms and contribute to the development of new therapies for anxiety disorders and epilepsy.

[004] The results obtained from this invention are promising, as they demonstrate, through an experimental model, the biological activities with anxiolytic and anticonvulsant action. Background of the invention

[005] Anxiety disorders and epilepsy are common and debilitating neurological conditions that significantly impact the quality of life of affected individuals. Conventional treatments for anxiety and seizures generally involve the use of benzodiazepines, antidepressants, and anticonvulsants. However, these therapies have limitations, including the development of tolerance, unwanted side effects, and long-term dependence. Therefore, there is a growing demand for therapeutic alternatives that are more effective and have fewer adverse effects. Petition 870250004978, dated 22 / 01 / 2025, page 33 / 36 / 4

[006] Natural substances, especially those with neuropharmacological activities, have received attention due to their therapeutic potential and more favorable safety profile. Limonene, a monoterpene found in citrus fruits, has demonstrated potential in several pharmacological applications, including antioxidant, anti-inflammatory, and neuroprotective properties. Preliminary studies indicate that limonene derivatives may modulate neuronal activities, suggesting promising anxiolytic and anticonvulsant effects. However, the understanding of the molecular mechanisms and the specificity of its interactions with central nervous system receptors, such as GABA and 5-HT, is still limited.

[007] The zebrafish (Danio rerio) model has stood out in neuroscientific research as an effective and translational system for evaluating substances with anxiolytic and anticonvulsant activities, due to its genetic and functional similarity to the mammalian nervous system. The use of zebrafish allows for the rapid and efficient analysis of bioactive compounds, including the identification of potential molecular targets and the elucidation of neuropharmacological mechanisms.

[008] Thus, the present invention seeks to explore limonene derivatives as anxiolytic and anticonvulsant agents, using the zebrafish model. Furthermore, it proposes the study of the interactions of these compounds with GABA and 5-HT receptors for a deeper understanding of the mechanisms involved, aiming to develop effective therapeutic alternatives with a lower risk of side effects.

[009] Currently, several medications are available for the treatment of anxiety and seizures, including benzodiazepines, anticonvulsants, and selective serotonin reuptake inhibitors (SSRIs). However, these treatments often have undesirable side effects, such as excessive sedation, dependence, and tolerance, which limits their long-term effectiveness. Therefore, there is a continuing need to develop new therapeutic agents that offer efficacy in treating these conditions with an improved safety profile. Brief description of the drawings Figure 1 shows biosynthetic derivatives of limonene. Petition 870250004978, dated 22 / 01 / 2025, page 34 / 36 / 4 Figure 2 shows the effect of (S)- (-)LIM (A), (R)- (+)LIM, PERY (C), PA (D) and PAC (E) on anxiety behavior in adult zebrafish in light / dark conditions (0-5 min). Values ​​represent the mean ± standard error of the mean for 6 animals / group; ANOVA followed by Tukey's test (*p<0.05; **p<0.01; ****p<0.0001 vs. control; #p<0.05; # # p<0.01 vs. DZP; # # # # p<0.0001 vs. DZP). Figure 3 shows the effect of (S)- (-)LIM, (R)- (+)LIM, PERY, PA and PAC against serotonergic antagonists on anxiolytic behavior. Dzp - Diazepam (4 mg / kg; 20 μL; ip); Control - 3% DMSO (20 μL; ip). Values ​​represent the mean ± standard error of the mean (SEM) for 6 animals / group. ANOVA followed by Tukey (* p < 0.05, ** p < 0.01, **** p < 0.0001 vs. control; # # p < 0.01; # # # #p < 0.0001 vs. Dzp). Figure 4 shows the effect of (S)-(-)LIM and (R)-(+)LIM against PTZ-induced seizures. Dzp - Diazepam (4 mg / kg; 20 μL; ip); Control - DMSO 3% (20 μL; ip). Values ​​represent the mean ± standard error of the mean (SEM) for 6 animals / group. ANOVA followed by Tukey; (#p < 0.05, # #p < 0.01, # # #p < 0.001; # # # #p < 0.0001 vs. Dzp). Figure 5 shows the effect of PERY against PTZ-induced seizures (8A-8C). Anticonvulsant mechanism via GABA antagonist FMZ (9A-9C). Dzp - Diazepam (4 mg / kg; 20 μL; i.p.); Control - DMSO 3% (20 μL; i.p.). Values ​​represent the mean ± standard error of the mean (SEM) for 6 animals / group. ANOVA followed by Tukey; (* p < 0.05, ** p < 0.01, *** p < 0.0001, **** p < 0.0001 vs. control; # # p < 0.01; # # # #p < 0.0001 vs. Dzp, ( # # # #p < 0.0001 vs. FMZ + PERY). Figure 6 shows the effect of PAC against PTZ-induced seizures (10A-10C). Anticonvulsant mechanism via GABA antagonist FMZ (11A-11C). Dzp - Diazepam (4 mg / kg; 20 μL; i.p.); Control - 3% DMSO (20 μL; i.p.). Values ​​represent the mean ± standard error of the mean (SEM) for 6 animals / group. ANOVA followed by Tukey; (** p < 0.01, **** p < 0.0001 vs. control; # p < 0.05; # # # p < 0.001, # # # # p < 0.0001 vs. Dzp, (# # # # p < 0.0001 vs. FMZ + PAC). Figure 7 shows the effect of PA against PTZ-induced seizures (12A-12C). Anticonvulsant mechanism via GABA antagonist FMZ (13A-13C). Dzp - Diazepam (4 mg / kg; 20 μL; ipsi); Control - 3% DMSO (20 μL; ipsi). The values ​​represent the Petition 870250004978, dated 01 / 22 / 2025, page 35 / 36 / 4 mean ± standard error of the mean (SEM) for 6 animals / group. ANOVA followed by Tukey; (** p < 0.01, *** p < 0.0001, **** p < 0.0001 vs. control; # # #p < 0.001, # # # #p < 0.0001 vs. Dzp, (# # #p < 0.0001, # # # #p < 0.0001 vs. FMZ +PA). Verification of Results

[0010] The toxicity test of limonene enantiomers and their oxidized derivatives was carried out over 96 hours.

[0011] No significant toxicity or deaths were observed, even at the highest dose, allowing the determination of an LD50 greater than 40 mg / kg.

[0012] Anxiolytic activity was assessed using a light and dark area preference test.

[0013] Both enantiomers, (S)-(-)-LIM and (R)-(+)-LIM, exhibited anxiolytic effects.

[0014] (S)-(-)-LIM demonstrated efficacy at all doses tested.

[0015] The anxiolytic efficacy was influenced by serotonergic antagonists, suggesting involvement of the serotonergic system.

[0016] In the anticonvulsant test, oxidized derivatives, especially perilic acid (PAC), significantly delayed PTZ-induced seizures.

[0017] This anticonvulsant effect was blocked by the antagonist flumazenil (FMZ), indicating the involvement of the GABAergic system.

[0018] PAC, as the most oxidized derivative, was the most effective in reducing anxiety and slowing the progression of seizures.

[0019] The results suggest that the oxidation of limonene compounds may increase their therapeutic efficacy, both in anxiolytic and anticonvulsant activity. Petition 870250004978, dated 01 / 22 / 2025, page 36 / 36

Claims

CLAIMS 1. Pharmaceutical composition comprising the limonene enantiomers, (R)-(+)limonene and (S)-(-)-limonene, and / or their oxidized derivatives, for use in the treatment of anxiety disorders and seizures, characterized by not presenting significant toxicity up to 96 hours at doses up to 40 mg / kg, indicating a promising safety profile for therapeutic applications.

2. Pharmaceutical composition according to Claim 1, characterized by including (R)-(+)limonene, whose anxiolytic activity is mediated by modulation of serotonin (5HT) receptors.

3. Pharmaceutical composition according to Claim 1, characterized by including (S)-(-)limonene, whose anxiolytic activity is not reversed by antagonists of the tested receptors, indicating a mechanism of action independent of these receptors.

4. Pharmaceutical composition according to Claim 1, characterized by including perilic acid (PAC), the most oxidized derivative of limonene, which exhibits anxiolytic effects at all doses tested and significantly delays the progression of seizures to the most severe stage (stage III).

5. Pharmaceutical composition according to Claim 4, characterized by perilic acid (PAC) acting to delay PTZ-induced seizures through a molecular structure similar to diazepam (DZP), suggesting a shared mechanism of anxiolytic and anticonvulsant action.

6. Method of using a composition according to Claim 1 for modulating anxiety and seizure responses, characterized by the use of limonene enantiomers and their oxidized derivatives as anxiolytic and anticonvulsant agents, due to the influence of oxidation effects on therapeutic potency.

7. Pharmaceutical composition according to Claim 1, characterized by the inclusion of molecular structures similar to diazepam (DZP) in oxidized limonene derivatives, allowing anxiolytic and anticonvulsant effects by modulating similar pathways. Petition 870250004978, dated 01 / 22 / 2025, pp. 32 / 36

Images