Application of Felodipine in the preparation of drugs to improve cognitive impairment

Abstract

This application belongs to the field of pharmaceutical application technology, specifically relating to the application of felodipine in the preparation of drugs for improving cognitive impairment. This application discloses a novel use of felodipine as an M-potassium channel inhibitor, which enhances hippocampal neuronal excitability and improves long-term potentiation by inhibiting Kv7.2 / Kv7.3 channel activity, thereby exerting a cognitive-improving effect and providing a new drug option for the treatment of cognitive impairment. This application also elucidates the molecular mechanism by which felodipine inhibits M-potassium channels, confirming that it exerts its effect by binding to K319 and H324 residues in the S6 region of the Kv7.2 channel, providing a theoretical basis for the structural optimization of felodipine and the development of novel M-potassium channel inhibitors.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical application technology, specifically relating to the application of felodipine in the preparation of drugs to improve cognitive dysfunction. Background Technology

[0002] Cognitive impairment is a group of neurological diseases characterized primarily by impaired cognitive functions such as memory, thinking, and learning. It mainly includes various types such as Alzheimer's disease, vascular dementia, and Lewy body dementia. With the accelerating aging of the population, the incidence of cognitive impairment is rising year by year, placing a heavy economic burden and care pressure on patients' families and society. Currently, commonly used cognitive-improving drugs mainly include cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists, but these drugs have limited efficacy, significant side effects, and insufficient targeting, making it difficult to meet clinical needs.

[0003] The Kv7 family consists of voltage-gated potassium channel subunits encoded by the KCNQ gene, comprising five members: Kv7.1 through Kv7.5. Kv7.2, Kv7.3, and Kv7.5 are primarily distributed in the central nervous system, and the low-threshold voltage-gated potassium channels formed by these subunits are known as "M-channels." M-channels play a crucial role in regulating neuronal excitability and synaptic plasticity, and their abnormal activity is closely related to cognitive impairment. Studies have shown that inhibiting M-channels can enhance neuronal firing frequency and improve long-term potentiation (LTP) function in the hippocampus, thereby improving cognitive function.

[0004] Felodipine is a dihydropyridine calcium channel antagonist primarily used clinically to treat hypertension. Its mechanism of action involves blocking voltage-gated calcium channels to dilate blood vessels and lower blood pressure. However, current technology has not yet elucidated the interaction between felodipine and neural M-potassium channels, nor its pharmacological activity in improving cognitive function. Therefore, exploring novel applications of felodipine in the treatment of cognitive impairment and clarifying its mechanism of action has significant clinical application value and innovative implications. Summary of the Invention

[0005] Based on this, one embodiment of this application provides the use of felodipine in the preparation of a drug for improving cognitive impairment.

[0006] This application provides, in one aspect, the use of felodipine in the preparation of a drug for improving cognitive impairment, wherein the drug exerts its effect by inhibiting M-potassium channels.

[0007] In some embodiments, the M-potassium channel is a Kv7.2 / Kv7.3 channel.

[0008] In some embodiments, the cognitive impairment is selected from one or more of Alzheimer's disease, vascular dementia, or scopolamine-induced cognitive impairment.

[0009] In some embodiments, the effective dose of felodipine in the drug is 0.1 mg / kg to 10 mg / kg.

[0010] In some embodiments, the drug is administered via intraperitoneal injection or oral administration.

[0011] In some of these embodiments, felodipine inhibits M-potassium channel activity by binding to K319 and H324 residues in the S6 region of the Kv7.2 channel.

[0012] In some embodiments, the drug also includes a pharmaceutically acceptable carrier or excipient.

[0013] In some embodiments, the dosage form of the drug is selected from tablets, capsules, injections, or oral solutions.

[0014] In some of these embodiments, the half-maximal inhibitory concentration (IC50) of felodipine for M-potassium channels is... 50 The range is 0.5μM-1.5μM.

[0015] In some embodiments, the subject of the drug is a mammal.

[0016] This application provides the use of felodipine in the preparation of drugs to improve cognitive impairment. Felodipine, as an M-potassium channel inhibitor, can inhibit both macroscopic and single-channel currents of M-potassium channels in a concentration-dependent manner. Whole-cell patch-clamp techniques confirmed that felodipine has a significant inhibitory effect on Kv7.2 / Kv7.3 channels, with an IC50 value of [missing value]. 50 The effective concentration was 1.1 ± 0.3 μM, comparable to the inhibitory effect of the positive control XE991. Single-channel patch-clamp experiments further confirmed that felodipine reduced the opening probability of M-potassium channels without altering single-channel conductance, indicating a direct action on channel gating mechanisms. By inhibiting M-potassium channels, felodipine effectively enhanced the excitability of hippocampal neurons, increased neuronal firing frequency, and protected long-term potentiation function in the hippocampus, thereby improving cognition. This mechanism of action is distinctly different from felodipine's traditional calcium channel antagonism, providing a novel theoretical basis for its application in the treatment of cognitive impairment. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The results of the concentration-dependent inhibitory effect of felodipine on Kv7.2 / Kv7.3 provided in one embodiment of this application; Figure 2 The results of the suppression of single-channel current of Kv7.2 / Kv7.3 by felodipine provided in an embodiment of this application; Figure 3 The concentration-dependent inhibition results of felodipine on other Kv7 subtypes provided in one embodiment of this application; Figure 4 The activation kinetics of felodipine on other Kv7 subtypes provided in one embodiment of this application; Figure 5 Identification results of key residues for felodipine-mediated Kv7.2 inhibition provided in an embodiment of this application; Figure 6 The results of the inhibition of natural M current and enhancement of neuronal firing by felodipine provided in an embodiment of this application; Figure 7 Felodipine, provided in one embodiment of this application, protects against scopolamine-induced long-term enhanced hippocampal dysfunction. Figure 8 The results of felodipine in improving scopolamine-induced cognitive impairment in mice, as provided in one embodiment of this application. Detailed Implementation

[0019] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0020] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings: The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0022] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0023] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0024] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0025] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0026] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0027] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0028] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0029] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0030] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0031] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0032] The term "M-type Potassium Channel" refers to a voltage-gated potassium ion channel assembled from members of the Kv7 family (primarily Kv7.2 and Kv7.3 subunits) encoded by the KCNQ gene. It is named "M-current" because its current is inhibited by muscarinic receptor agonists. This channel is widely expressed in the central nervous system, playing a crucial role, particularly in brain regions related to learning and memory, such as the hippocampus and cortex. M-channels influence synaptic plasticity and neural network excitability by regulating the resting membrane potential and firing frequency of neurons, making them key targets for cognitive function regulation.

[0033] The term "Kv7.2 / Kv7.3 channel" refers to Kv7.2 and Kv7.3, which are the main constituent isoforms of M-channels in the central nervous system, typically existing as heterotetramers and forming functional M-currents. Kv7.2 / Kv7.3 channels are characterized by low-threshold activation, slow activation, and non-inactivation, making them suitable for modulating neuronal excitability within the subthreshold membrane potential range. This application demonstrates through whole-cell patch-clamp experiments that felodipine has preferential inhibitory activity against Kv7.2 / Kv7.3 channels, and its IC50 inhibitory activity against single isoforms of Kv7.2 and Kv7.3 is [not specified]. 50 The values ​​were 3.1±2.5 μM and 11.2±2.5 μM, respectively, showing good subtype selectivity.

[0034] The term "cognitive dysfunction" refers to a clinical syndrome characterized by a decline in higher neurological functions such as memory, learning, attention, and executive function. It is commonly seen in neurodegenerative diseases such as Alzheimer's disease, vascular dementia, Lewy body dementia, and Parkinson's disease dementia. The types of cognitive dysfunction covered in this application include, but are not limited to, Alzheimer's disease, vascular dementia, and scopolamine-induced cognitive dysfunction models, encompassing common experimental models and clinical disease types.

[0035] The term "half-maximal inhibitory concentration" (IC50) 50 IC 50 IC is a commonly used indicator to measure the inhibitory efficacy of a drug on a target, representing the concentration of the drug required to reduce channel activity by 50%. 50 The smaller the value, the stronger the inhibitory effect of the drug on the target. In this application, felodipine has an IC50 value for the Kv7.2 / Kv7.3 channel. 50 The value was 1.1 ± 0.3 μM, indicating that it has significant inhibitory activity at the micromolar level, providing an important reference for its dosage design as a cognitive improvement drug.

[0036] To address the lack of highly effective and targeted drugs for the treatment of cognitive impairment in the existing technology, this application provides the use of felodipine in the preparation of drugs to improve cognitive function.

[0037] The first aspect of this application provides the use of felodipine in the preparation of a drug for improving cognitive impairment, said drug acting by inhibiting M-potassium channels.

[0038] Felodipine, as an M-potassium channel inhibitor, can inhibit both macroscopic and single-channel currents of M-potassium channels in a concentration-dependent manner. Whole-cell patch-clamp techniques confirmed that felodipine has a significant inhibitory effect on Kv7.2 / Kv7.3 channels, with an IC50 concentration of [missing value]. 50 The effective concentration was 1.1 ± 0.3 μM, comparable to the inhibitory effect of the positive control XE991. Single-channel patch-clamp experiments further confirmed that felodipine reduced the opening probability of M-potassium channels without altering single-channel conductance, indicating a direct action on channel gating mechanisms. By inhibiting M-potassium channels, felodipine effectively enhanced the excitability of hippocampal neurons, increased neuronal firing frequency, and protected long-term potentiation function in the hippocampus, thereby improving cognition. This mechanism of action is distinctly different from felodipine's traditional calcium channel antagonism, providing a novel theoretical basis for its application in the treatment of cognitive impairment.

[0039] In some embodiments, the M-potassium channel is a Kv7.2 / Kv7.3 channel. Kv7.2 / Kv7.3 channels are the main M-channel subtypes in the central nervous system, widely distributed in brain regions closely related to cognitive function, such as the hippocampus and cortex. Experimental data show that felodipine has preferential inhibitory activity against Kv7.2 / Kv7.3 channels, with an IC50 value of [missing information]. 50 The value was significantly lower than that of other Kv7 subtypes, demonstrating good target selectivity.

[0040] In some embodiments, the M-potassium channel is a Kv7.2 channel or a Kv7.3 channel. Felodipine has an inhibitory effect on both Kv7.2 and Kv7.3 channels, IC 50 The values ​​were 3.1±2.5μM and 11.2±2.5μM, respectively, providing multiple target selection options for clinical drug use.

[0041] In some embodiments, the cognitive impairment is selected from one or more of Alzheimer's disease, vascular dementia, or scopolamine-induced cognitive impairment. Animal experiments have shown that felodipine can significantly improve scopolamine-induced cognitive impairment in mice. In the new object recognition test, the discrimination index of the 1 mg / kg felodipine group reached 0.10 ± 0.02, which is comparable to that of the positive control drug donepezil, confirming its effectiveness in the treatment of cognitive impairment.

[0042] In some embodiments, the effective dose of felodipine in the drug is 0.1-10 mg / kg. Experimental results showed that the effect of felodipine in improving cognitive impairment was dose-dependent, with discrimination indices of 0.05±0.03, 0.10±0.02, and 0.24±0.02 for the 0.1 mg / kg, 1 mg / kg, and 10 mg / kg dosing groups, respectively, providing a reference for clinical dosage selection.

[0043] In some embodiments, the drug is administered via intraperitoneal injection or oral administration. Felodipine has good bioavailability and can exert its cognitive-improving effects through multiple routes of administration.

[0044] In some embodiments, felodipine inhibits M-potassium channel activity by binding to K319 and H324 residues in the S6 region of the Kv7.2 channel. Molecular docking and mutagenesis experiments confirmed that K319 and H324 are key amino acid residues for felodipine binding to the Kv7.2 channel, and the double mutant K319A / H324A reduces the IC50 of felodipine. 50 Increasing the value from the micromolar level to greater than 100 μM significantly reduced the channel's sensitivity to felodipine.

[0045] In some embodiments, the drug further includes a pharmaceutically acceptable carrier or excipient. Depending on the route of administration and dosage form requirements, suitable carriers and excipients can be selected to improve drug stability, bioavailability, and patient compliance.

[0046] In some embodiments, the dosage form of the drug is selected from tablets, capsules, injections, or oral liquids. Specifically, tablets may use microcrystalline cellulose, lactose, magnesium stearate, etc., as excipients; injections may use water for injection, sodium chloride, glucose, etc., as solvents or osmotic pressure regulators.

[0047] In some embodiments, the half-maximal inhibitory concentration (IC50) of felodipine for the Kv7.2 / Kv7.3 channels is... 50 The concentration range is 0.5-1.5 μM. This concentration range indicates that felodipine has high inhibitory activity against Kv7.2 / Kv7.3 channels, providing a reference for the effective dosage design of it as a cognitive-improving drug.

[0048] In some embodiments, the M-potassium channel may be selected from one or more of Kv7.1, Kv7.2, Kv7.3, Kv7.4, or Kv7.5. Specifically, felodipine has preferential inhibitory activity against Kv7.2 / Kv7.3 channels, IC50 50 The value is 1.1 ± 0.3 μM; for the Kv7.2 channel IC 50 The value is 3.1 ± 2.5 μM; for the Kv7.3 channel IC50 The value is 11.2 ± 2.5 μM; for the Kv7.4 channel IC 50 The value is 5.6 ± 0.9 μM; for the Kv7.1 channel IC 50 The value is 14.8 ± 3.4 μM; for the Kv7.5 channel IC 50 The value is 12.1 ± 2.4 μM.

[0049] In some embodiments, the cognitive impairment may be selected from one or more of Alzheimer's disease, vascular dementia, Lewy body dementia, Parkinson's disease dementia, frontotemporal dementia, scopolamine-induced cognitive impairment, or age-related cognitive impairment.

[0050] In some embodiments, the effective dose of felodipine can be selected from any value among 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 5 mg / kg, or 10 mg / kg. Specifically, for intraperitoneal injection, the preferred dose is 0.1-10 mg / kg; for oral administration, the preferred dose is 1-20 mg / kg.

[0051] In some embodiments, the inhibitory concentration of felodipine on the Kv7.2 / Kv7.3 channels can be selected from any value among 0.1 μM, 0.5 μM, 1 μM, 3 μM, 5 μM, or 10 μM. (The last part, "close to IC," appears to be an unrelated fragment and is omitted from the translation.) 50 At a concentration of (1.1±0.3μM), felodipine showed the best inhibitory effect on Kv7.2 / Kv7.3 channels.

[0052] In some embodiments, the binding site of felodipine to the Kv7.2 channel is located in the S6 region, with key amino acid residues including K319 and H324. Specifically, K319 and H324 residues interact with felodipine through hydrogen bonds and hydrophobic interactions, and the double mutation K319A / H324A can reduce the IC50 of felodipine. 50 The value increased to greater than 100 μM.

[0053] In some embodiments, the drug can be administered via a method selected from intraperitoneal injection, oral administration, intravenous injection, subcutaneous injection, or intramuscular injection. Specifically, intraperitoneal injection has a rapid onset of action and is suitable for the treatment of acute cognitive impairment; oral administration is convenient for long-term use and is suitable for the maintenance treatment of chronic cognitive impairment.

[0054] This application has the following beneficial effects: This application reveals for the first time a novel use of felodipine as an M-potassium channel inhibitor, which enhances hippocampal neuronal excitability and improves long-term potentiation by inhibiting Kv7.2 / Kv7.3 channel activity, thereby exerting a cognitive-improving effect and providing a new drug option for the treatment of cognitive impairment.

[0055] This application elucidates the molecular mechanism by which felodipine inhibits M-potassium channels, demonstrating that it exerts its effects by binding to K319 and H324 residues in the S6 region of the Kv7.2 channel, providing a theoretical basis for the structural optimization of felodipine and the development of novel M-potassium channel inhibitors.

[0056] This application systematically verified the cognitive-improving effects of felodipine at multiple levels, from molecular, cellular, tissue to whole animal, through electrophysiological patch-clamp technology, molecular docking technology, and animal behavioral experiments, providing sufficient experimental evidence for the clinical translation of felodipine as a new drug for the treatment of cognitive impairment.

[0057] Felodipine, as a marketed drug, has had its safety and pharmacokinetic characteristics fully verified. Its application in the treatment of cognitive impairment can greatly shorten the drug development cycle and reduce development costs, and has important clinical application value and social significance.

[0058] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0059] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0060] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0061] Example 1 Whole-cell patch-clamp assays confirmed that felodipine has a concentration-dependent inhibitory effect on Kv7.2 / Kv7.3. The effect of felodipine on the Kv7.2 / Kv7.3 current stably expressed in CHO cells was tested using perforated whole-cell patch-clamp recording technology. 3 μM felodipine resulted in a 73.6 ± 5.4% inhibition rate of the Kv7.2 / Kv7.3 current recorded at 0 mV. Figure 1 (A) As a positive control, 3 μM of the M channel-specific inhibitor XE991 showed an inhibition rate of 81.4 ± 1.5% on the Kv7.2 / Kv7.3 current. Figure 1(A) By perfusing different concentrations of felodipine ranging from 0.1 to 30 μM, a concentration-dependent inhibition of the Kv7.2 / Kv7.3 current was observed. 50 The value is 1.1 ± 0.3 μM ( Figure 1 (as shown in B and Table 1).

[0062] This application further investigates the effect of felodipine on the voltage-dependent activation of the Kv7.2 / Kv7.3 channels. (The last sentence appears to be incomplete and possibly refers to an IC designation.) 50 At a concentration of 1 μM, felodipine had no significant effect on the voltage-gated activation of the Kv7.2 / Kv7.3 current. 1 / 2 The value changed from -10.3±1.5mV to -9.1±0.7mV. Figure 1 (See C in Table 1). Furthermore, felodipine slowed the activation kinetics of the Kv7.2 / Kv7.3 channels, increasing the time constant from 415.6 ± 39.6 ms to 540.2 ± 62.4 ms, while the deactivation kinetics remained largely unchanged (from 23.8 ± 3.4 ms to 21.9 ± 2.9 ms). Figure 1 (D in Table 2).

[0063] Table 1: Felodipine vs. Kv7 Channel IC 50 and V 1 / 2 Function

[0064] All data are expressed as mean ± standard error (mean ± SEM). n = 5–8, *P < 0.05, paired t-tests were used for analysis.

[0065] Table 2: Effects of felodipine on the activation and deactivation time constants of the Kv7 channel

[0066] All data are expressed as mean ± standard error (mean ± SEM). n = 4–7, *P < 0.05, paired t-tests were used for analysis.

[0067] Example 2 Single-channel patch-clamp assays confirmed the direct inhibitory effect of felodipine on the Kv7.2 / Kv7.3 single-channel current. The effect of felodipine on Kv7.2 / Kv7.3 single channels was tested using cell-attached single-channel recording technology. 1 μM felodipine significantly reduced the opening probability of Kv7.2 / Kv7.3 single channels from 0.33±0.05 to 0.13±0.02, but did not change the single-channel conductance—the experimental group maintained 8.6±0.4 pS, which was not significantly different from the control group's 8.5±0.6 pS. Figure 2 ).

[0068] Example 3 Whole-cell patch-clamp assays confirmed the concentration-dependent inhibition of felodipine on other Kv7 isoforms. The inhibitory effect of felodipine on transient expression of Kv7.1-Kv7.5 isoforms in HEK293T cells was tested using whole-cell patch-clamp techniques. Figure 3 As shown, all Kv7 channel subtypes exhibit concentration dependence, but their potency differs significantly. IC 50 The values ​​were: 14.8±3.4 μM for Kv7.1, 3.1±2.5 μM for Kv7.2, 11.2±2.5 μM for Kv7.3, 5.6±0.9 μM for Kv7.4, and 12.1±2.4 μM for Kv7.5. These results indicate that felodipine exhibits preferential inhibition of neuronal Kv7.2 / Kv7.3 channels in other Kv7 subtypes.

[0069] Example 4 Whole-cell patch-clamp assays confirmed the voltage-dependent activation of felodipine on other Kv7 isoforms.

[0070] This application further investigates felodipine in its near-IC50 range. 50 The effect of time on the voltage-dependent activation characteristics of each subtype. For example... Figure 4 As shown in Figure A, felodipine has no significant effect on the voltage-dependent activation curve of Kv7.1. In contrast, 3 μM felodipine causes the activation curve of the Kv7.2 channel to shift to the right, and the half-activation voltage (V) increases. 1 / 2 The voltage changed from -23.1±1.3mV to -8.1±0.9mV. Figure 4 (B) Felodipine has no significant effect on the voltage-dependent activation of Kv7.3, Kv7.4, or Kv7.5 channels. Under controlled conditions, the V7.3 and V7.4 of Kv7.3 and Kv7.4 are... 1 / 2 The values ​​were -7.5±1.8 and -5.2±3.0 mV, respectively, compared to -3.9±1.6 and -6.3±1.5 mV when applied to filodipine; while the V of Kv7.5 1 / 2 The values ​​were -26.8±2.2mV and -22.3±3.2mV, respectively (see...). Figure 4 C in Figure 4 D and Figure 4 (E in the text).

[0071] Example 5 Pair docking confirms identification of key residues in felodipine-mediated Kv7.2 inhibition. To identify the residues crucial for felodipine-mediated Kv7.2 inhibition, molecular docking of felodipine with Kv7.2 (PDB number: 7CR0) was performed using the Glide model from MOE2020. The docking predicted a potential binding pocket with a docking score of -6.9. Figure 5 A in the example. Figure 8 As shown in section B, residues K319 and H324 in the S6 region were identified as crucial sites for felodipine binding. These residues interact through hydrogen bonds and hydrophobic interactions. To verify this finding, this application mutated K319 and H324 to alanine and evaluated their effect on felodipine inhibition of Kv7.2. These two mutations reduced the inhibitory effect of 10 μM felodipine from 76.6 ± 3.4% to 43.4 ± 2.4% and 27.7 ± 3.5%, respectively. Figure 5 In step B), the sensitivity of the channel to felodipine was reduced, but not significantly. To further verify K319 and H324, this application double-mutated them to obtain the K319A / H324A double mutant. After double mutation, the inhibition rate of felodipine was significantly reduced, and the IC50 was lower. 50 Greater than 100 μM. This further confirms that K319 and H324 are two relatively important amino acid residues that felodipine binds to.

[0072] The W236 residue in the S5 region is a key site for RTG-induced Kv7.2 activation and ML252-induced Kv7 inhibition. This application further investigated whether W236, F240, and L299 (residues adjacent to W236) play important roles in felodipine-mediated Kv7.2 inhibition. Figure 5 As shown in B, the alanine mutations of these residues did not alter the sensitivity of the Kv7.2 channel to 3 μM felodipine: compared to 76.6 ± 3.4% in the wild-type control group, the inhibition rate was 73.8 ± 6.8% for the W236A mutant, 76.9 ± 9.7% for the F240A mutant, and 69.4 ± 10.4% for the L299A mutant. These results indicate that residues K319 and H324 are crucial for felodipine-induced Kv7 inhibition.

[0073] Example 6 Whole-cell patch-clamp assays confirmed the inhibitory effect of felodipine on innate M-currents and the enhancement of neuronal firing in hippocampal neurons. Hippocampal tissue from C57 mice within 24 hours of birth was isolated, and primary hippocampal neurons were prepared using trypsin digestion. These neurons were seeded onto poly-L-lysine-coated coverslips and cultured at 37°C in a 5% CO2 incubator for 14–21 days. Whole-cell patch-clamp recording was used to record Muller's current (M-current): a depolarization protocol to -20 mV (lasting 500 ms) followed by repolarization to -60 mV induced M-currents. Perfusion with 3 μM, 10 μM felodipine, and 3 μM XE991 was performed, and the M-current inhibition rate was calculated. Resting membrane potential (RMP) was recorded: in current-clamp mode, membrane potentials were recorded without current injection to observe the effect of felodipine on RMP. Neuronal firing activity was recorded: in current-clamp mode, a 50 pA current injection was applied, and the firing frequency before and after felodipine treatment was recorded. Felodipine at concentrations of 3 μM and 10 μM inhibited M-current by 20.1 ± 0.1% and 38.8 ± 2.0%, respectively. As a positive control, 3 μM XE991 inhibited M-current by 18.2 ± 2.0%. 3 μM and 10 μM felodipine significantly depolarized the resting membrane potential (RMP) of hippocampal neurons, producing potential shifts of 6.5 ± 1.6 mV and 12.8 ± 2.3 mV, respectively, changing the RMP from -56.2 ± 7.9 mV to -50.6 ± 7.9 mV and from -54.2 ± 1.7 mV to -41.5 ± 7.8 mV. Similarly, 3 μM XE991 depolarized the RMP by 6.3 ± 3 mV. Figure 6 (B in the text). Furthermore, under current injection conditions, 3 μM and 10 μM felodipine significantly enhanced hippocampal neuronal firing activity (…). Figure 6 C in Figure 6 (D in the text). These results indicate that felodipine enhances the excitability of hippocampal neurons by inhibiting M-currents.

[0074] Example 7 Brain slice patch-clamp confirmed that felodipine protected against scopolamine-induced long-term hippocampal enhancement dysfunction. Mice were randomly divided into four groups: control group (saline), scopolamine treatment group (Scop+Veh), felodipine intervention group (Scop+Fel), and RTG reversal group (Scop+Fel+RTG). Mice were administered drugs intraperitoneally: the control group received saline; the Scop+Veh group received 1 mg / kg scopolamine, followed by saline 30 min later; the Scop+Fel group received 1 mg / kg scopolamine, followed by 10 mg / kg felodipine 30 min later; and the Scop+Fel+RTG group received 1 mg / kg scopolamine, followed by 10 mg / kg felodipine 30 min later, and then 10 mg / kg RTG 30 min later. One hour after drug administration, the mice were rapidly decapitated, and the hippocampus was harvested to prepare 300 μm thick slices, which were then incubated in ACSF for 1 hour (32°C, oxygenated). Brain slice patch-clamp recording technique was used to record field excitatory postsynaptic potentials (fEPSPs) in the CA1 region of the hippocampus. The stimulating electrode was placed on the Schaffer collateral branch, and the recording electrode was placed in the pyramidal cell layer of the CA1 region. The stimulation intensity was set to 30%-50% of the maximum fEPSP amplitude. After stabilizing for 15 min, high-frequency stimulation (HFS: 100 Hz, 1 s duration, 10 s interval, 3 groups) was applied to induce LTP, and recording continued for 45 min. Changes in the fEPSP slope were analyzed. In the control group, field excitatory postsynaptic potentials (fEPSPs) were significantly enhanced after HFS stimulation and lasted for 45 min. In the scopolamine-treated group (Scop+Veh), the long-term potentiation (LTP) was impaired, changing from 1.5±0.09 to 0.7±0.05. Figure 7 (B in the figure, p < 0.001). Notably, the combined use of felodipine reversed scopolamine-induced LTP damage, resulting in a fEPSP slope of 1.7 ± 0.7 (in the figure). Figure 7 In the case of B (p < 0.05), using RTG can reverse the effect of felodipine, and the slope of fEPSP becomes 1.0 ± 0.05 ( Figure 7 (B in the figure, p < 0.05). These findings suggest that felodipine can protect the hippocampus from scopolamine-induced LTP damage. This effect may stem from felodipine's inhibition of Kv7.2 / Kv7.3 channels, thereby enhancing its neuroprotective properties in hippocampal memory.

[0075] Example 8 Animal experiments have shown that felodipine improves scopolamine-induced cognitive impairment in mice. This application further used the Novel Object Recognition (NOR) test to evaluate the effect of felodipine on scopolamine-induced cognitive impairment in mice. After intraperitoneal injection of 1 mg / kg scopolamine, the novel object exploration time in mice was significantly reduced, and the discrimination index (DI) decreased to -0.24 ± 0.02, while the DI in the solvent control group was 0.12 ± 0.1, indicating that the scopolamine-induced cognitive impairment model was successfully established. Figure 8 In this experiment, different doses of D01 were administered, and their effect on the time to explore new objects was measured. The results showed that exploration time increased in a dose-dependent manner: the discrimination index (DI) was 0.05 ± 0.03 in the 0.1 mg / kg group, 0.10 ± 0.02 in the 1 mg / kg group, and 0.24 ± 0.02 in the 10 mg / kg group. As a positive control, the 1 mg / kg XE991 group also showed a significant increase in exploration time for new objects, with a DI of 0.08 ± 0.04. Donepezil at 10 mg / kg also significantly increased exploration time for new objects, with a DI of 0.13 ± 0.04. In contrast, concurrent administration of 1 mg / kg felodipine and 10 mg / kg RTG slightly reversed the improving effect of felodipine. These results suggest that felodipine can improve scopolamine-induced cognitive impairment by increasing neuronal excitability.

[0076] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. The application of felodipine in the preparation of drugs for improving cognitive impairment, characterized in that, The drug works by inhibiting M-potassium channels.

2. The application according to claim 1, characterized in that, The M-potassium channel is a Kv7.2 / Kv7.3 channel.

3. The application according to claim 1, characterized in that, The cognitive impairment is selected from one or more of Alzheimer's disease, vascular dementia, or scopolamine-induced cognitive impairment.

4. The application according to claim 1, characterized in that, The effective dosage of felodipine in the drug is 0.1 mg / kg to 10 mg / kg.

5. The application according to any one of claims 1 to 4, characterized in that, The drug can be administered via intraperitoneal injection or oral administration.

6. The application according to claim 3, characterized in that, Felodipine inhibits M-potassium channel activity by binding to K319 and H324 residues in the S6 region of the Kv7.2 channel.

7. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

8. The application according to claim 7, characterized in that, The dosage form of the drug is selected from one of tablets, capsules, injections, or oral liquids.

9. The application according to claim 1, characterized in that, The half-maximal inhibitory concentration (IC50) of felodipine on M-potassium channels 50 The range is 0.5μM-1.5μM.

10. The application according to claim 1, characterized in that, The subjects of the drug are mammals.

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