Application of statin compound in preparation of alpha+beta4 type BK channel agonist

Abstract

The invention discloses application of a statin compound in preparation of an alpha + beta 4 type BK channel agonist, and belongs to the technical field of biological medicine. Specifically, by researching the influence of the statin compound on an ion channel, it is considered that the statin compound possibly has high activation activity on a BK alpha beta 4 channel. Through comparison of activation effects of the compounds on different BK channel subtypes, high selectivity of the compounds on BK alpha beta 4 channels is found, and compared with channels such as BK alpha and BK alpha beta 1, the activation effect of the compounds on the BK alpha beta 4 channels is improved by several times or even hundreds of times. Therefore, an alpha + beta 4 type BK channel agonist, namely a statin drug, is obtained, so that a new strategy for activating a specific BK channel is provided, and a new basis is provided for adjustment of a current statin medication scheme.

Description

TECHNICAL FIELD

[0001] The application relates to application of a statin in preparation of an alpha+beta4 type BK channel agonist and belongs to the technical field of biological medicines. BACKGROUND

[0002] Large conductance voltage-and calcium-activated potassium channel (BK) is encoded by KCNMA1 gene, can be opened or closed in response to changes in cell membrane potential and calcium ion concentration, also known as MaxiK, KCa1.1, Slo1. BK channel is widely distributed in vivo, participates in the regulation of neurotransmitter release, smooth muscle relaxation, cochlear hearing and other physiological processes; its dysfunction is related to diseases such as seizures and epilepsy, movement disorders, autism and mental retardation, tumors, hypertension, obesity and diabetes, and is a very important drug target. In different tissues and cells, BK channel structure subunit alpha (BK alpha) is often combined with different auxiliary subunits to form different channel subtypes to exert different functions. The currently found BK channel auxiliary subunits include beta subunit, gamma subunit and LINGO subunit. Among them, the beta subunit has four subtypes beta1-beta4, and the four subtypes have similar topological structures: two transmembrane regions (TM1, TM2), extracellular loop region and intracellular NH2 end and COOH end. Beta1 subunit is mainly expressed in smooth muscle and hair cells. It affects the gating characteristics of BK channel, increases the sensitivity of the channel to Ca 2+ , and also changes the pharmacological properties of BK channel. Beta2 has high homology with beta1 sequence, is mainly expressed in chromaffin cells and hippocampal neurons. Its NH2 end has a deactivation ball that can deactivate BK channel. Beta3 subunit is highly expressed in testis, pancreas and spleen, has high homology with beta2, and its physiological properties are also closer to beta2 subunit. Beta4 subunit is highly expressed in nerve cells, trabecular meshwork cells (TM) and is the only BK auxiliary subunit in Schlemm's canal cells (SC), in addition, it is also expressed in detrusor smooth muscle, intercalated cells of distal renal units. Unlike beta1-beta3, the combination of beta4 and alpha subunit reduces the Ca 2+ sensitivity of BK channel. The deletion or down-regulation of beta4 subunit is related to various diseases, and drugs that selectively activate BK alpha beta4 channel can be used for the treatment of related diseases.

[0003] At present, many modulators (including inhibitors and agonists) for BK channels have been developed, but there is no drug or compound that can selectively activate BKaP4 channels. The current BK channel agonists include synthetic benzimidazole compounds NS004 and NS1619, biaryl urea NS1608, fluoroindole BMS-204352, and diarylamine flufenamic acid and mefenamic acid, and some endogenous activators such as palmitoleic acid, eicosapentaenoic acid and eicosatetraenoic acid. However, most of these drugs have low activity, are not selective for BK channels and have poor drug properties. For example, the diarylamine opener has poor selectivity and has a great influence on other ion channels, so it has great limitations in application. Therefore, it is still necessary to find a highly selective activator for BKaP4 channels. SUMMARY

[0004] To solve the above problems, the present application has found, through a large number of screenings, that a compound, statin, has a significant activating effect on BKaP4 channels and has high selectivity, thereby realizing the function of regulating specific BK channels.

[0005] The first object of the present application is to provide the use of statin or its salt, isomer, derivative in the preparation of a BK channel agonist of a+P4 type.

[0006] Further, the statin is a liposoluble statin and / or a water-soluble statin.

[0007] Further, the statin is preferably a liposoluble statin, and the liposoluble statin includes one or more of simvastatin, atorvastatin, fluvastatin and lovastatin.

[0008] Further, the statin derivative includes an acid formed by ring opening (ester bond) of the statin.

[0009] The second object of the present application is to provide a BK channel agonist of a+P4 type, which contains statin or its salt, isomer, derivative.

[0010] Further, the statin is a liposoluble statin or a water-soluble statin.

[0011] Further, the statin is preferably a liposoluble statin, and the liposoluble statin includes one or more of simvastatin, atorvastatin, fluvastatin and lovastatin.

[0012] Further, the statin derivative includes an acid formed by ring opening (ester bond) of the statin.

[0013] Further, the α+β4 type BK channel agonist is a solid preparation or a liquid preparation.

[0014] Further, when the α+β4 type BK channel agonist is a liquid preparation, the effective concentration of the statin compound or its salt, isomer, derivative is in the nanomolar level. Preferably, the concentration is 0.1-100 nM. Of course, those skilled in the art can understand that the "effective concentration" herein is divided into in-vivo effective concentration and in-vitro effective concentration, which refers to the optimal concentration required to play a role at the target site in-vitro or in-vivo. Specifically, for in-vitro, the effective concentration can be equal to the administration concentration; for in-vivo, the effective concentration required to reach the in-vivo is in the nanomolar level, but considering the reasons such as metabolism, the administration concentration is usually greater than the effective concentration, and the skilled person can set it according to different administration methods.

[0015] A third object of the present application is to provide a pharmaceutical composition containing the α+β4 type BK channel agonist.

[0016] Further, the pharmaceutical composition can be prepared into oral preparations, capsules, tablets, powders or injections.

[0017] Further, the pharmaceutical composition further contains pharmaceutically acceptable excipients.

[0018] Further, the excipients include one or more of fillers, excipients, stabilizers, diluents, binders, disintegrants, lubricants, glidants, wetting agents, effervescent agents, coloring agents, sweeteners, fragrances, preservatives, dispersants, film formers, plasticizers, pore formers, opacifiers, retarders, solvents.

[0019] Further, the composition is composed of 0.1-100% of the statin compound and 99.9-0% of the excipients.

[0020] In the present application, the above definitions are only examples and are generally applicable in applications, pharmaceutical compositions and pharmaceutical products.

[0021] Advantages of the present application:

[0022] At present, there is no clear and effective drug for selective activation of BKaB4 channel in clinic, and through a large number of preliminary studies, it is found that statins can significantly activate BKaB4 channel, and show high selectivity relative to other ion channels. The statin provided by the application has low preparation cost, new use of old drugs, small side effects, and can be quickly put into clinical application, which not only provides a clue for subsequent development of specific activation drugs of the channel, but also expands the clinical indications of statins, and provides a new basis for adjusting the existing statin drug regimen, and provides application and theoretical basis for developing more specific and effective drugs in the future. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is the basic structure of statins.

[0024] Figure 2 It is the structural formula of different statin compounds.

[0025] Figure 3 It is the effect of simvastatin on BKaB4, BKa and BKaB1 channel currents under zero calcium conditions. A, BKaB4 channel current curves recorded before and after different concentrations of simvastatin were given. The upper insert indicates the applied voltage stimulation scheme. B, Time curves of 0, 0.1, 1.1, 6.1 and 16.1 nM simvastatin on BKaB4 current activation, the horizontal line on the top of the graph indicates the concentration and time period of drug addition, and Baseline indicates the condition before drug addition, i.e. the drug concentration is 0. C, Concentration-dependent curves of simvastatin activating BKaB4 (solid circle, n=7), BKa (hollow triangle, n=10) and BKaB1 (solid square, n=9) currents.

[0026] Figure 4 It is the effect of different concentrations of simvastatin on BKaB4, BKa and BKaB1 channel currents under 1 muM calcium conditions, and the vertical axis is the percentage of drug agonistic or inhibitory current (n=1-5).

[0027] Figure 5 It is the agonistic effect of simvastatin on BKaB4 and BKa channel currents under 1 muM calcium conditions. A, BKaB4 channel current curves recorded before and after different concentrations of simvastatin were given. B, Time curves of 0, 1, 6, 16, 116 nM simvastatin activating channel currents, the horizontal line on the top of the graph indicates the concentration and time period of drug addition, and Baseline indicates the condition before drug addition, i.e. the drug concentration is 0. C, Concentration-dependent curves of simvastatin activating BKaB4 (solid circle, n=5) and BKa (hollow triangle, n=5) currents.

[0028] Figure 6Effects of different statins on BKaP4 and BKa channels. A, the percentage of BKaP4 channel current activated by different statins (10 nM) in zero calcium condition (n = 5-7) and the percentage of BKa channel current activated by different statins (10 mM) in 1 mM calcium condition (n = 5-6). B, the percentage of BKaP4 channel current activated by different statins (10 nM) in 1 mM calcium condition (n = 5-6) and the percentage of BKa channel current inhibited or activated by different statins (10 mM) in 1 mM calcium condition (n = 5-7). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. DETAILED DESCRIPTION

[0029] The present application is further described in the following examples and with reference to the accompanying drawings and specific examples, which are provided to give a better understanding of the application and enable a worker of ordinary skill in the art to better utilize the application. The examples are not intended to limit the application as defined in the appended claims.

[0030] The present application relates to the following scheme:

[0031] At present, ion channel modulators are a common product form, such as the potassium ion channel positive modulator NS309 which has been issued in the market, and is a calcium ion activated potassium ion channel modulator. BKaP4 channels are associated with a variety of diseases, and there is no report on selective activation of BKaP4 channels at present, so it is imperative to develop a modulator product for BKaP4 channels.

[0032] Statins are a class of drugs for regulating blood lipids which are widely used in clinical, and they play a role in reducing lipids by inhibiting 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase. This class of drugs contains two basic pharmacophores: a mother nucleus (decahydronaphthalene ring or azacycle) and dihydroxyheptanoic acid (or is a lactone ring or is an open ring hydroxy acid) Figure 1Dihydroxyheptanoic acid is an essential group for inhibiting HMG-CoA reductase and requires ring-opening to be active. Statins can be divided into two main categories based on solubility: lipophilic and hydrophilic. Lipophilic statins include atorvastatin, fluvastatin, lovastatin, and simvastatin, while hydrophilic statins include pravastatin and rosuvastatin. Simvastatin's parent nucleus is a decahydronaphthalene ring, which is metabolized in vivo into ring-opened simvastatin acid to exert its lipid-lowering effect. Lovastatin shares the same parent nucleus as simvastatin, the difference being that lovastatin lacks a methyl group on the decahydronaphthalene ring side chain; while pravastatin is obtained by ring-opening lovastatin through a lactone ring. Atorvastatin and fluvastatin have nitrogen-containing heterocyclic parent nuclei, unlike simvastatin. Figure 2 In addition to their lipid-lowering effects, statins also possess pleiotropic effects independent of cholesterol, such as anti-inflammatory, anti-oxidative stress, anti-cancer, and anti-psychiatric effects. It is generally believed that statins exert these pleiotropic effects by inhibiting the synthesis of downstream products in the mevalonate pathway, thereby inhibiting the isoprenelation of small GTPases (guanosine triphosphatase).

[0033] In this invention, a cell model was constructed in which the structural subunit α of a BK channel binds to the auxiliary subunits β1 and β4. The regulatory effects of statins on different BK channel subtypes were studied in conjunction with their response to electrical stimulation. The results showed that the regulatory effects of statins on different BK channels, such as BKα, BKαβ1, and BKαβ4, varied significantly. At different concentrations, all statins exhibited selective activation of the BKαβ4 channel, which can be used to prepare products activating the BKαβ4 channel. It should be noted that, as is known in the art, the BKαβ1, BKαβ2, and BKαβ3 channels have similar sensitivity to calcium ions. However, the mechanistic study in this invention indicates that the binding of statins is affected by calcium ions. Therefore, this invention only needs to compare the differences in the activation effects of statins on BKαβ1 and BKαβ4 channels to obtain specific results.

[0034] In this invention, the BKαβ4 channel refers to the channel subtype formed by the combination of the BK channel structural subunit α (BKα) and the auxiliary subunit β4, and has the same meaning as descriptions such as "α+β4 type BK channel" and "high conductivity potassium channel subtype containing auxiliary subunit β4".

[0035] Pharmaceutical Composition

[0036] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing a statin compound, and may also contain other active ingredients, as well as a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, and the pH value may vary depending on the properties of the substances being formulated, which can be adjusted as needed by a skilled technician. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): oral, intraperitoneal, intravenous, or local administration.

[0037] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of a statin compound and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): fillers, excipients, stabilizers, diluents, binders, lubricants, surfactants, or combinations thereof.

[0038] The above pharmaceutical formulations should be matched with the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. They can also be prepared into other suitable forms to improve bioavailability, etc. Pharmaceutical compositions such as injections and solutions should preferably be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount.

[0039] When using a drug combination, a safe and effective amount of the drug is administered to an individual. The specific dosage should also take into account factors such as the route of administration and the patient's health condition, all of which are within the scope of a skilled physician's expertise.

[0040] Example: Selective activation of BKαβ4 channel by statins

[0041] This invention utilizes the transfection reagent polyethyleneimine (PEI) to transfect BKα (GenBank) TM accession numberL16912), BKβ4 (GenBank TM accession numberAF207992) and BKβ1 (GenBank TM The plasmid (accession number U25138.1) was transfected and expressed in a heterologous expression system (human embryonic kidney cells HEK293T). The effects of different statin drugs on BK currents were then detected using single-cell patch-clamp technology. The complete technical protocol involved is as follows:

[0042] 1. Drug preparation

[0043] (1) Simvastatin solution: Weigh 1.89 mg of simvastatin and dissolve it in 45.1 μL of DMSO to prepare a 100 mM stock solution. Aliquot the stock solution into 10 μL tubes and store them in a -20°C freezer for later use. Before performing patch-clamp experiments, dilute the stock solution with ddH2O to the required concentration (both DMSO and ddH2O were filtered through a 0.22 μm microporous membrane before preparation).

[0044] (2) Other statins: Lipid-soluble statins (lovastatin, atorvastatin, fluvastatin) are prepared similarly to simvastatin. Before the experiment, they are prepared into a 10 mM stock solution using DMSO, and then diluted to the required concentration using ddH2O during the experiment. Water-soluble statins (simvastatin acid, pravastatin) are prepared by weighing a certain amount of powder, dissolving it in sterile ddH2O to prepare a 10 mM stock solution, and storing it at -20°C.

[0045] 2. Preparation of electrophysiological solutions

[0046] (1) Preparation of electrode internal solution

[0047]

[0048] Note: Dissolve in ddH2O, adjust pH to 7.2 with MeSO3H, bring the solution to a final volume of 500mL, filter through a 0.22μm microporous membrane, and store at room temperature.

[0049] (2) Preparation of intracellular fluid

[0050] 0μM Ca 2+ Solution formulation

[0051]

[0052] Note: Dissolve in ddH2O, adjust pH to 7.2 with MeSO3H, bring the solution to a final volume of 1000mL, filter through a 0.22μm microporous membrane, and store at room temperature.

[0053] 1μM Ca 2+ Solution formulation

[0054]

[0055] Note: Dissolve in ddH2O, adjust pH to 7.2 with MeSO3H, bring the solution to a final volume of 1000mL, filter through a 0.22μm microporous membrane, and store at room temperature.

[0056] 100μM Ca 2+ Solution formulation

[0057]

[0058] Note: Dissolve in ddH2O, adjust pH to 7.2 with MeSO3H, bring the solution to a final volume of 1000mL, filter through a 0.22μm microporous membrane, and store at room temperature.

[0059] 3. Cell Culture

[0060] HEK293T cells were cultured in high-glucose DMEM medium containing 1% penicillin, streptomycin, and 10% FBS. During culture, the cells were placed in a 37°C incubator with 5% CO2 and 100% saturated humidity. To ensure normal cell growth and proliferation, passage was necessary when the cell density reached 90%, typically every 2-3 days.

[0061] 4. Cell transfection

[0062] Seed cells into 12-well plates one day in advance. On the second day, when the cell density reaches 60%-70%, plasmid transfection can be performed. The specific steps for transfection are as follows (taking a 12-well plate as an example):

[0063] 1) Prepare several 1.5mL EP tubes, label the plasmid to be transfected on the EP tube caps, and add 100μL of serum-free and antibiotic-free DMEM medium to each tube.

[0064] 2) Calculate the required plasmid volume according to the plasmid concentration, add 1 μg of plasmid and 1.6 μL of transfection reagent PEI to the corresponding EP tubes for dilution, and let stand for 5 min.

[0065] 3) After the static period, add the culture medium containing the plasmid to the culture medium containing PEI and mix well. Incubate together for 15 minutes.

[0066] 4) Remove the cells to be transfected (12-well plate) from the incubator in advance, replace with fresh serum-free and antibiotic-free culture medium, add the incubated liposome complex to the 12-well plate, mix gently, and put it back into the cell culture incubator for culture.

[0067] 5) 6-8 hours after transfection, replace the culture medium in the 12-well plate with fresh serum-containing and antibiotic-containing medium, and continue culturing for 12-16 hours before proceeding with subsequent experiments.

[0068] 5. Cell drops

[0069] The purpose of cell dropping is to transfer transfected cells from a 12-well plate onto a coverslip for subsequent patch-clamp experiments. The specific steps for cell dropping are as follows:

[0070] 1) Before the experiment, use a glass cutter to cut the 20*20mm coverslip into several small pieces evenly and soak them in anhydrous ethanol.

[0071] 2) Use pointed tweezers to pick up the glass slide in the operating table, sterilize the glass slide over an alcohol lamp, and then place it in a plastic culture dish (usually a cell culture dish with a diameter of 35mm).

[0072] 3) Drop the PDL solution onto the glass slide, ensuring the solution covers the entire slide, and let it stand for 15 minutes.

[0073] 4) After absorbing the PDL solution, add sterile ddH2O to the slide for cleaning, absorb the ddH2O, and then let the slide air dry naturally.

[0074] 5) Remove the 12-well plate from the incubator, aspirate the culture medium, wash twice with 1 mL of PBS and discard the PBS, add 300 μL of 0.25% trypsin to digest for 30 seconds, add 1 mL of high-glucose DMEM culture medium, and gently blow the cells off.

[0075] 6) Collect the cells in a 1.5 mL EP tube and centrifuge at 1100 rpm for 3 min.

[0076] 7) Discard the supernatant, gently tap the cell pellet to disperse it, dilute the cells according to the amount collected in the EP tube, and then drop the diluted solution onto a glass slide. The slide must be completely dry at this point; if there is moisture on the slide, the cell suspension will flow into the dish, resulting in no cells on the slide. Cover the dish and let it stand for 15-20 minutes.

[0077] 8) Then gently add 2 mL of fresh culture medium along the inner wall of the dish, and slowly place the cells back into the incubator. Perform patch-clamp experiments within 4-24 hours. If the time is too long, the cell condition on the slide will deteriorate, affecting the experimental procedure.

[0078] 6. Patch clamp experiment

[0079] This invention uses an inside-out mode to record the macroscopic current of the BK channel. The experimental room temperature needs to be maintained at 23-25℃. The specific operating steps are as follows:

[0080] 1) First, the glass electrode is drawn. The glass electrode used in this invention has a length of 100 mm, an outer diameter of 1.50 mm, an inner diameter of 0.86 mm, and contains a liquid-conducting wire (SUTTER INSTRUMENT, BF150-86-10). The shape and resistance of the electrode are controlled by adjusting the parameters of the drawing instrument. The electrode tip diameter used in the experiment is 1-2 μm, and the water resistance is approximately 3-6 MΩ.

[0081] 2) Add cell bath solution to the culture dish, place the prepared HEK293T cell slide in the culture dish, place the culture dish in the center of the stage, and observe the cells in real time using a CCD imaging system. Select cells with smooth surfaces and clear edges for the experiment.

[0082] 3) Fill the electrode with charged electrode internal solution, remove any air bubbles from the tip, and install it onto the electrode holder, ensuring the silver wire is submerged below the liquid surface. Micro-manipulate the electrode electrically to immerse it in the bath solution, compensating for the liquid junction potential. Then, bring the electrode into contact with the cell; the electrode resistance will increase. Apply a negative pressure to the cell to rapidly increase the sealing resistance until a gigohm seal is achieved. Afterward, quickly move the electrode upward or to the side to remove it from the cell, establishing an inside-out mode. Apply the appropriate voltage stimulation to the membrane and record changes in the macroscopic current at a filtered frequency of 2.9 kHz. During recording, add the drug directly to the bath solution and mix with a pipette.

[0083] 4) All voltage stimulation schemes provided in this invention are square wave stimulations, including a 20ms pre-stimulation at -80mV, followed by a 20ms stimulation at different test voltages (-100mV to 260mV, with different voltage stimulations used depending on the channel subtype and calcium concentration; specific values ​​are shown in the corresponding results section), and finally a 10ms stimulation at -80mV to record the tail current. When recording the conductance-voltage (GV) curve, voltage stimulation is applied every 2 seconds. When recording the concentration-time curve, voltage stimulation is applied every 5 seconds.

[0084] 7. Patch-clamp data analysis

[0085] When analyzing the channel voltage activation characteristics, the conductance-voltage (GV) curve is obtained by measuring the steady-state current value and fitted using the Boltzmann function. The equation is as follows: G / Gmax=1 / (1+exp((VV)) 50 ) / κ)). Among them, V 50 This represents the half-activation voltage, which is the stimulation voltage corresponding to when the channel conductance reaches half of its maximum conductance. The smaller this value, the more sensitive the channel is to voltage stimulation; while κ is the slope factor.

[0086] When analyzing the excitation or inhibition effects of drugs on channel currents, 10-20 baseline current curves are first recorded, and the steady-state current values ​​are measured and averaged as the initial current value I0 before drug administration. With the baseline stable, the drug is added, and voltage stimulation is applied every 5 seconds, recording the corresponding current curves. After the drug effect stabilizes, five more current curves are recorded, and the steady-state current values ​​of these five curves are measured and averaged as I0. drug To calculate the percentage of activation or inhibition of the electric current by the drug (ΔI%). The calculation formula is as follows: ΔI% = (I drug -I0) / I0*100%. The drug concentration-dependent curve is fitted with the Hill equation to obtain the half-maximal effective concentration (MCC) and the Hill coefficient. Hill equation: ΔI% = ΔI% max / (1+(EC50 / [drug]) n ), where ΔI% refers to the percentage of drug activation current, ΔI% max EC refers to the maximum percentage of drug activation current. 50 The half-maximal effective concentration (WMC) of a drug refers to the drug concentration, while n is the Hill coefficient, which reflects the number of drug-channel proteins that bind to it.

[0087] 8. Experimental Results

[0088] (1) Simvastatin selectively activates the BKαβ4 channel

[0089] Since the gating and pharmacological properties of BK channels are affected by calcium ions, the effects of simvastatin on BKαβ4, BKα, and BKαβ1 channels were compared under zero calcium and calcium (1 μM calcium) conditions.

[0090] Under zero calcium conditions and 150 mV voltage stimulation, simvastatin exhibited concentration-dependent activation of the currents of BKαβ4, BKα, and BKαβ1 channels; however, the drug's effect on the BKαβ4 channel was hundreds of times stronger than its effect on the BKα and BKαβ1 channels. Figure 3 As shown, 1.1 nM simvastatin significantly activated the BKαβ4 current, increasing it by 45.21 ± 3.75% (n = 9). The same drug concentration had no effect on the BKα and BKαβ1 channel currents. The concentration-response curve of the drug was fitted using the Hill equation, yielding the EC50 of simvastatin activating the BKαβ4 channel at zero calcium. 50 The value is 10.92 nM, and the Hill coefficient is 0.35. Figure 3 C). Under the same conditions, simvastatin activates the EC50 of BKα and BKαβ1 channels. 50 The concentrations were 4.27 μM and 43.5 μM, respectively, with Hill coefficients of 0.42 and 0.35. Figure 3 C).

[0091] At 1μM Ca 2+ Under these conditions, 0.1–11.1 μM simvastatin still exhibits agonistic activity towards BKαβ4 channels, although this effect diminishes with increasing concentration. However, it shows weak agonistic activity (29.27 ± 5.20%, n = 5) at low concentrations (0.1 μM) and inhibitory activity (-16.10 ± 1.97% and -31.38 ± 5.78%, n = 5) at high concentrations (1.1 μM and 11.1 μM). Its effects on BKαβ1 channels are similar to those on BKα channels. Figure 4As shown, 100 nM simvastatin can increase the BKαβ4 current by 101.71 ± 11.77%, which is 3.5 times the effect on the BKα current and 10.7 times the effect on the BKαβ1 current.

[0092] Further comparisons were made of the effects of simvastatin on BKαβ4 channels and BKα channel currents within a low concentration range. The concentration-response curve of the drug was fitted using the Hill equation to obtain the EC50 of simvastatin activating BKαβ4 channels. 50 The value is 1.45nM, and the Hill coefficient is 0.90 (n=5). Figure 5 C). Simvastatin at 1 μM Ca 2+ EC activation of BKα channel under certain conditions 50 The value is 0.73nM, and the Hill coefficient is 0.51 (n=5). Figure 5 C). Although simvastatin activates the EC50 of BKαβ4 and BKα channels under these conditions. 50 The effects are similar, but the activation effect on the BKαβ4 channel is significantly stronger than that on the BKα channel.

[0093] In summary, compared to BKα and BKαβ1 channels, simvastatin activates the BKαβ4 channel current regardless of the presence of calcium ions, and its activation effect on the BKαβ4 channel current is significantly stronger than that of BKα and BKαβ1 channels, as evidenced by an EC value hundreds of times lower at zero calcium levels. 50 And when calcium is present, in similar EC values 50 It has an activation effect several times higher than that of time.

[0094] (2) Comparison of the effects of other statins on BKαβ4 and BKα channels

[0095] The effects of several statins (simvastatin, simvastatin acid, lovastatin, pravastatin, atorvastatin, and fluvastatin) at a concentration of 10 nM on BKαβ4 channel current and the effects of these statins at a concentration of 10 μM on BKα channel current were compared under 0 and 1 μM calcium conditions. The results are as follows: Figure 6As shown: Lovastatin's effect on BKαβ4 channels is similar to that of simvastatin, but its effect on BKα channels is weaker. Atorvastatin activates both BKα and BKαβ4 channels at both zero and 1 μM calcium levels; its activation of BKαβ4 channels at zero calcium is more than twice that of simvastatin, while its effect at 1 μM calcium is slightly weaker. Fluvastatin's effect on both BKαβ4 and BKα channels at zero calcium is similar to that of simvastatin, but its effect at 1 μM calcium is weaker than that of simvastatin. Simvastatin acid and pravastatin have very weak effects on both BKαβ4 and BKα channels.

[0096] Overall, at zero calcium, 1 / 1000 concentrations of simvastatin and fluvastatin (10 nM) activated the BKαβ4 channel current to the same level as the activation effect of 10 μM concentration, while 10 nM lovastatin and atorvastatin had 2.2 and 4.2 times the activation effect of 10 μM, respectively. At 1 μM calcium, simvastatin, lovastatin, and fluvastatin activated the BKαβ4 channel current but inhibited it; 10 nM atorvastatin had twice the activation effect of 10 μM. These results indicate that simvastatin, lovastatin, atorvastatin, and fluvastatin selectively activate BKαβ4 channels within the nanomolar concentration range.

[0097] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Use of a statin compound or a salt, isomer, derivative thereof in the preparation of an α+β4 type BK channel agonist.

2. Use according to claim 1, characterized in that, The statin compound is a liposoluble statin compound and / or a water-soluble statin compound.

3. Use according to claim 2, characterized in that, The liposoluble statin compound includes one or more of simvastatin, atorvastatin, fluvastatin, and lovastatin.

4. Use according to claim 1, characterized in that, The statin compound derivative includes an acid formed by ring opening of the statin compound.

5. An alpha + beta type 4 BK channel agonist, characterized in that, The α+β4 type BK channel agonist contains the statin compound or a salt, isomer, derivative thereof.

6. The alpha+beta class 4 BK channel agonist of claim 5, wherein, The α+β4 type BK channel agonist is a solid preparation or a liquid preparation.

7. The alpha+beta class 4 BK channel agonist of claim 6, wherein, When the α+β4 type BK channel agonist is a liquid preparation, the effective concentration of the statin compound or a salt, isomer, derivative thereof is in the nanomolar level.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition contains the α+β4 type BK channel agonist of any one of claims 5-7.

9. The pharmaceutical composition of claim 8, wherein, The pharmaceutical composition further contains a pharmaceutically acceptable excipient.

10. The pharmaceutical composition of claim 9, wherein, The excipient includes one or more of a filler, an excipient, a stabilizer, a diluent, a binder, a disintegrant, a lubricant, a glidant, a wetting agent, an effervescent agent, a coloring agent, a sweetener, an aromatic agent, a preservative, a dispersant, a film former, a plasticizer, a pore former, an opacifier, a retarder, a solvent.

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