Formulation with flavonoids and potential to induce or modulate plasticity and neuroprotection
A flavonoid and phenolic acid formulation enhances neuroplasticity and reduces neural damage by promoting dendritic outgrowth and complexity, addressing the limitations of existing treatments for chemotherapeutic neurotoxicity.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- PONTIFICIA UNIV JAVERIANA
- Filing Date
- 2024-10-18
- Publication Date
- 2026-04-23
AI Technical Summary
Existing treatments for neurotoxic and cognitive effects from chemotherapeutic drugs fail to induce neuroplasticity and effectively reduce neural damage, while existing flavonoid formulations primarily focus on antioxidant properties without addressing structural plasticity.
An aqueous or alcoholic formulation with a high content of flavonoids and phenolic acids, including specific ratios of Chrysin-glucoside, vicenin, and other flavonoids, enhances dendritic outgrowth and complexity in cortical neurons, reducing cytotoxicity and promoting neuroplasticity.
The formulation improves neuroplasticity, enhances dendritic structure, and reduces neural damage, as evidenced by improved cognitive performance and structural plasticity in animal models, suggesting a therapeutic strategy for chemotherapeutic-induced neurotoxicity.
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Abstract
Description
[0001] Formulation with flavonoids and potential to induce or modulate plasticity and neuroprotection
[0002] FIELD OF THE INVENTION
[0003] [1] The present invention relates to an aqueous or alcoholic formulation characterized by a high content of flavonoids. This formulation exhibits the potential to induce or modulate structural plasticity and neuroprotection. More particularly, the formulation of the present invention can improve dendritic outgrowth and complexity in cortical neurons, reduce cytotoxicity of other treatments, and thus, effectively reduce neural damage related to chemotherapy and other medical treatments.
[0004] [2] Furthermore, the formulation of the present invention can be used as an alternative antiproliferative co-adjuvant in anti-cancer and other chemical treatments.
[0005] BACKGROUND OF THE INVENTION
[0006] [3] Treatment of neurotoxic and cognitive effects derived from chemotherapeutic drugs is one milestone for patients and survivors. Pharmacological interventions with other drugs are among the primary tools assessed to lessen the neurological damage of chemical treatments. [4] Prior art collectively demonstrates the existence of previous knowledge and innovation in the field compositions comprising alkaloids. They highlight the potential benefits and need for formulation strategies associated with such compositions.
[0007] [5] Particularly, the invention disclosed in patent document EP 2786758 reveals a pharmaceutical composition which can improve memory and learning ability, thus effectively preventing and treating disorders such as dementia and amnesia.
[0008] [6] In the same approach, patent document WO 2003 / 053336 discloses a flavonoid compound with antioxidant properties, optionally formulated in a pharmaceutically acceptable carrier. This compound or combination of compounds provides significant, effective relief of peripheral neural or vascular ailments symptoms.
[0009] [7] WO2021121443 relates to use of a flavonoid extract obtained from the species Talipariti datum sw., formulations and treatment method for improve different types of memory, both short and long term, associated with various pathologies and population ageing in humans.
[0010] [8] The document KR20160103729 reveals composition for prevention and treatment of cognitive disorder comprising extracts of mulberry. This composition is a food supplement. [9] Also, Ferraz C., et. al. in 2020 reviewed the therapeutic potential of flavonoids in pain and inflammation, focusing on how the development of formulations containing flavonoids, along with the understanding of their structure-activity relationship, can be harnessed to identify novel flavonoid-based therapies to treat pathological pain and inflammation.
[0011]
[0010] In this scenario, antioxidant formulations are promising due to their exceptional effect of blocking the chemotherapeutic neurotoxic pathway rather than correcting the final effects. More particularly, among these compounds, polyphenols like flavonoids have been demonstrated to foster neuroprotective effects.
[0012]
[0011] The objective of the invention is to provide a formulation that, in addition to neuroprotection, provides neuroplasticity in the neuronal network.
[0013] SUMMARY OF THE INVENTION
[0014]
[0012] The present invention relates to aqueous or alcoholic formulation with a high content of flavonoids comprising a combination of flavonoids for prevention and treatment of cognitive impairments due to medical treatments or other conditions. More particularly, the present invention relates to an aqueous or alcoholic formulation which can improve dendritic outgrowth, spinogenesis, and complexity in cortical neurons, reduce cytotoxicity of other treatments, and thus, effectively reduce neural damage related to chemotherapy and other medical treatments.
[0015]
[0013] The formulation of the present invention comprises a combination of flavonoids and phenolic acids for the prevention of pathologies that involve alterations in the maintenance and formation of dendrites; to prevent them from negatively impacting cognitive performance. Thus, the invention provides a formulation that improves neuroplasticity in the neural network.
[0016] BRIEF DESCRIPTION OF DRAWINGS
[0017]
[0014] Some embodiments of the present invention are illustrated as an example and are not limited by the figures of the accompanying drawings.
[0018]
[0015] Figure 1. Neuroprotective effect of the formulation of the invention. A) Aqueous formulation of the invention pg / mL-1; B) Alcoholic formulation of the invention pg / mL-1.
[0019]
[0016] Figure 2. Effect of the invention's formulation on cortical neurons morphology (DIV6). Scholl analysis that allows quantifying the number of intersections at different distances from the soma of a neuron. A.
[0020] Representative images of neurons labeled against the MAP2 protein. B. Quantification of the number of intersections to compare between concentrations.
[0021]
[0017] Figure 3. Determination of dendritic tree complexity. Effect on the number of branches (nBranch), branching complexity, and median overall process length in cortical neurons when using the formulation of the invention. A. Number of dendritic branches. B. Complexity of the arborization of the neuron.
[0022]
[0018] Figure 4. General design of the in vivo study.
[0023]
[0019] Figure 5. Parameters evaluated in the open field test in Balb mice C. A. Distance traveled in meters. B. Average speed (m / s). C. Time spent in the center of the arena. Stockings ± SEM, ANOVA *P<0.05.
[0024]
[0020] Figure 6. Parameters evaluated in the Y -maze test in Balb C mice. A: Total distance covered in meters. B. Total time in seconds. C. Time spent in the new arm. D. Discrimination index. Stockings ± SEM, ANOVA ***P<0.001.
[0025]
[0021] Figure 7. Parameters evaluated in the Novel Object Recognition test in Balb C mice. A: Total distance traveled in meters. B. Total time in seconds. C. Discrimination index. Stockings ± SEM. ANOVA *P<0.05.
[0026]
[0022] Figure 8. Representative images of the dendritic spines of Balb C mice. Golgi Cox stain. ScalelO mm. DETAILED DESCRIPTION OF THE INVENTION
[0027]
[0023] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well as the singular forms, unless the context clearly indicates otherwise.
[0028]
[0024] It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.
[0029]
[0025] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one having ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
[0026] In describing the invention, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefits and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques. Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion.
[0030]
[0027] Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.
[0031]
[0028] The present invention relates to an aqueous or alcoholic formulation with a high content of flavonoids comprising a combination of flavonoids for the prevention and treatment of cognitive impairments due to medical treatments or other conditions.
[0032]
[0029] In one embodiment, the formulation comprises a mixture of flavonoids and phenolic acids.
[0033]
[0030] In this particular embodiment, the formulation comprises a mixture made up of at least two flavonoids, accounting for at least 50% of the formulation, and phenolic acids corresponding to at least 40%, while other flavonoids in combination with polyphenols can be present in at least 10% of the formulation.
[0031] Particularly, the formulation disclosed herein comprises as a main compound a phenolic acid with content exceeding 25%, followed by Chrysin-glucoside with a percentage of at least 20%, vicenin or apigenin glycoside with more than 15 %, and a mixture of other flavonoids in combination with polyphenols with more than 10% (Table 1 and Table 2).
[0034]
[0032] The formulation of the present invention comprises a combination of flavonoids, containing a mixture of flavonoids accounting for at least 50% of the formulation and phenolic acids corresponding to at least 40%, plus 10% of other compounds.
[0035] Table 1. Flavonoids in pre-formulation and proportion of them
[0036] Table 2. Phenolic acids in pre-formulation and proportion of them
[0037] Phenolic acids % prepreparations formulation
[0038] 1 6.1
[0039] 2 3.35
[0040] 3 7.84
[0041] 4 28.26 Total 49,832481
[0033] In a particular embodiment, in the formulation of the present invention, the combination of flavonoids mainly comprises vitexin-2"-O- rhamnoside and luteolin-7-O-glucoside and a combination of other flavonoids with polyphenols. Preferably, the mixture of other flavonoids in combination with polyphenols comprises vicenin, apigenin, chrysin, glucoside, spinosyn and, cyclopasifloric acid glucoside, other isomers, and mixtures of these and related molecules.
[0042]
[0034] In the same embodiment of the invention, the ratio in the mixture is as follows: Phenolic acids 2: Chrysin-glucoside 1: vicenin 0.5: other flavonoids 0.5: (Mixture 2: 1: 0.5: 0.5).
[0043]
[0035] In another embodiment of the invention, it is expected that for each 100 mg of the mixture there will be more than 40 mg of phenolic acids, more than 20 mg of Chrysin-glucoside, more than 15 mg of vicenin and more than 10 mg of other flavonoids.
[0044]
[0036] Particularly, the formulation of the invention is in concentrations ranging from 0.01 pg / mL to 5 pg / mL. In this embodiment, these concentrations can be extrapolated to establish effective dosing strategies and explore other concentrations.
[0045]
[0037] From these concentrations, the optimal dosage range and concentration of the formulation as a whole and of flavonoids and other components can be established and can harness their neuroprotective and plasticity-inducing properties fully.
[0046]
[0038] This formulation can enhance neurogenesis, help promote dendritogenesis, and therefore improve neurological health and brain function.
[0047]
[0039] In another embodiment, it is also possible to obtain pharmaceutical forms of the formulation or dosage for treating, ameliorating, mitigating, alleviating, or reducing cognitive impairment associated with multiple conditions. These pharmaceutical forms can include capsules, emulsions, or syrups.
[0048]
[0040] In another embodiment, the formulation of the invention can be combined with other neurological enhancers and other additives or natural or artificial compounds.
[0049]
[0041] In contrast, the formulation of the invention can also be combined or co-administered with any proportion or dose of a cytotoxic agent.
[0050]
[0042] Examples
[0051]
[0043] These examples serve as illustrative embodiments, showcasing the invention's potential applications without intending to limit the scope of the claims. The examples provided herein are meant to inspire and guide those skilled in the art, but they should not be construed as exhaustive or exclusive representations of the invention.
[0052]
[0044] Example 1 : Preparation of the formulation of the invention
[0053]
[0045] The formulation of the invention was prepared by combination of a mixture of flavonoids and water for 10 minutes (1:10 P / V).
[0054] Subsequently, the product obtained was lyophilized, weighed, and stored in moisture-free conditions, without exposure to direct light, and away from possible contaminants. These conditions were maintained for at least three batches of the formulation.
[0055]
[0046] Example 2: In vitro activity evaluation of the formulation of the invention in neurons
[0056]
[0047] In in vitro assays in neurons cultured for 6 days (DIV 6), changes were observed in the quantified morphological parameters such as the number of ramifications and the complexity of the ramifications. Specifically, changes in dendritic complexity were observed when they were treated with the formulation to the concentrations of 0.01 and 0.03 mg per mL.
[0057]
[0048] Neuroprotective effect of the aqueous and alcoholic formulation of the invention on Doxorubicin-induced damage in cultured neurons
[0058] (DIV6). Cells were pre-treated for 24 h with the formulation of the invention followed by a 24-h exposure to Dox at IC20 (gray bars) or
[0059] IC40 (black bars) for 24 h. Dashed-line represents the viability of untreated cortical neurons. At selected formulation concentrations its effect on cortical neurons is minimal (open bars). Cell viability was determined after the formulation of the invention and drug treatment via MTT assay. At the assessed formulation of the invention concentrations, cell viability is calculated as the absorbance ratio of A (formulation of the invention + Dox) / A (formulation of the invention) > 100%. Data are presented as Mean ± Standard Error of Mean (SEM). *, p < 0.05; **, p < 0.01 (Figure 1).
[0060]
[0049] Figure 2 shows the correlation between the number of intersections at different distances from the soma of a neuron and the complexity of the dendritic tree. In a healthy neural this complexity is high, while in an unhealthy one, the number of intersections is lower.
[0061]
[0050] The effect of the formulation of the invention on the morphology of cultured neurons (DIV6) was assessed. Figures 2 and 3 show representative images of cortical neurons at different concentrations of the formulation of the invention that evidence significant morphology changes relative to untreated neurons. Horizontal bars cluster regions by treatment whose number of intersections share the same significant difference relative to the control group.
[0062]
[0051] Figure 3 shows other parameters associated with the complexity of the dendritic tree. Violin plots shown of the aqueous formulation of the invention represent the effect on the number of branches (nBranch), branching complexity in cortical neurons. *, p < 0.05, **, p < 0.01, ***, p < 0.005 (n > 60).
[0063]
[0052] Example 3: In vivo results of the formulation of the invention in neuroprotection and structural plasticity.
[0064]
[0053] The neuroprotective and synaptic structural plasticity effects of the formulation of the invention were identified in a mouse model of chemobrain induced with the chemotherapeutic Doxorubicin (Dox), by evaluating the effects of the formulation at the behavioral and structural level. Figure 4 shows the experimental strategy.
[0065]
[0054] Balb C mice were used and administered Doxorubicin (Dox) (2.5 mg / kg for three weeks) intraperitoneally and co-administered 6 doses of the formulation of the invention. As control groups, mice treated with (Dox) and saline solution were used. Subsequently, the effects of cotreatment with chemotherapy were evaluated and histopathological analyses were performed.
[0066]
[0055] In the final segment of the study, behavioral tests were performed. The open field test was used to quantitatively assess general locomotor activity, exploratory levels, and habituation to the environment. Figure 5A shows the total distance traveled, the average speed in Figure 5B, and the time spent in the center of the arena in Figure 5C. No significant differences were found in the first two parameters. However, significant differences were found between the control and Dox group in the time the mice spent in the center, as well as comparing Dox and the co-treatment with the formulation of the invention. These differences in the wandering of the rodents both, in the center and on the periphery may depend on the size of the open field and the variability of the data.
[0067]
[0056] The Y-maze allows the evaluation of spatial learning and working memory, Figure 6 shows the results of the performance of the groups in this test. No significant differences were found in total distance traveled (6A), and total time of exploration (6B). However, the discrimination index (6C) shows that the Dox group exhibits negative values that correlate with a decrease in working memory and spatial memory in the comparison by ANOVA in one way a significance of *P<0.05. Additionally, an improvement in performance in this test was observed for the group co-treated with Dox and the formulation of the invention (Dox- Formulation), which suggests a neuroprotective effect on cognitive function.
[0068]
[0057] This test allowed us to evaluate short-term memory by exposing the mice to two identical objects and the following exposures, evaluating the degree of exploration of the familiar object against a new one. Figure 7 shows the results of the group's performance in this test. No significant differences were found in the total distance traveled (7A), and the total exploration time (7B). However, the discrimination index shows that the
[0069] Dox group exhibits negative values that correlate with decreased working memory and spatial memory in the comparison by ANOVA in one way a significance of *P<0.05. Additionally, an improvement in performance in this test was observed for the group co-treated with Dox-Pass, which suggests a neuroprotective effect on cognitive function.
[0070]
[0058] The results showed that mice co-treated with the formulation of the invention and Dox performed similarly to controls in memory tests. Meanwhile, animals treated with Dox had a performance that evidenced cognitive impairment. These results suggest that co-treatment with the formulation of the invention improves performance on behavioral tests that assess short-term memory. Additionally, these results may indicate that the formulation helps preserve the structural plasticity at the cellular level, as evidenced in the performance of cognitive tests.
[0071]
[0059] To validate this hypothesis, the brains of control and treatment animals were evaluated by microscopy to determine changes in dendritic spines. For this purpose, 150 mm sections were made using a vibratome and were subjected to Golgi Cox staining. A Zeiss microscope with an 80x objective was used and images were acquired with an Axiocam 305 color. The results are shown below (Figure 8).
[0060] At the histopathological level, it was evidenced that the mice treated with the formulation of the invention (Formulation and Dox- Formulation) retained the structure and number of dendritic spines compared to the mice treated with Dox. In the quantification, a loss in the number of spines was observed for the Dox treatment group, which were recovered with co-treatment with the formulation (Dox-Formulation). Additionally, it is observed that there is an increase in the density of the thorns of the Formulation group when compared to the control. These results suggest that the improvement in the performance of short-term memory tests is correlated with changes in structural and synaptic plasticity induced by the components of the formulation of the invention. Particularly, in the Dox-Formulation group, which would allow us to propose that the formulation of the invention has not only a neuroplastic, dendritogenic effect, but also a spinogenic effect with the preservation of spines-like structures. These findings suggest a neuroprotective potential of the formulation, which revealed both functional and structural effects, which outlines the formulation as a useful therapeutic strategy to reduce the side effects of chemotherapy. Particularly, chemo brain in cancer patients. This makes it a promising formulation in neuroprotection in pathologies associated with loss of dendritic and synaptic structures.
Claims
CLAIMS1. Formulation with flavonoid content, comprising a mixture of flavonoids, phenolic acids, and other flavonoids in combination with polyphenols, and solvents selected from aqueous or alcoholic or a mixture of these.
2. The formulation of claim 1, wherein the mixture of flavonoids comprises at least two flavonoids, accounting for at least 50% of the formulation, phenolic acids correspond to at least 40% and other flavonoids can be present in at least 10% of the formulation.
3. The formulation of claim 1, wherein the mixture of flavonoids mainly comprises vitexin-2"-O-rhamnoside and luteolin-7-O-glucoside and a combination of other flavonoids with polyphenols.
4. The formulation of claim 1, wherein the mixture of other flavonoids also comprises vicenin, apigenin, chrysin, glucoside, spinosyn and cyclopasifloric acid glucoside, other isomers and mixtures of these and related molecules.
5. The formulation of claim 1, wherein the concentration of the formulation is 0.01 and 10 pg / mL.
6. The formulation of claim 5, wherein the concentration of the formulationis 0.01 and 0.03 pg / mL.
7. Use of formulation of claim 1 for neuroprotection, preservation of neural plasticity, and reduction of side effects of chemotherapy.
8. Use of formulation of claim 1 as antiproliferative co-adjuvant in anti-cancer treatment.