Pharmaceutical composition comprising chloroquine for inhibiting k-atp channel-mediated vasodilation

Abstract

The present invention relates to a pharmaceutical composition comprising chloroquine or a pharmaceutically acceptable salt thereof, thereby exhibiting a vasodilation inhibition effect.

Description

Pharmaceutical composition for inhibiting K ATP channel-mediated vasodilation comprising chloroquine

[0001] The present invention is K ATP It relates to a pharmaceutical composition for inhibiting channel-mediated vasodilation.

[0002]

[0003] Adenosine triphosphate (ATP)-sensitive potassium (K) receptors regulate oxygen and nutrient supply to vascular smooth muscle ATP ) channels are involved in regulating tissue perfusion by inducing vasodilation by endogenous vasodilators in pathophysiological conditions such as acidosis, hypoxia, and ischemia. Chloroquine and hydroxychloroquine have similar pharmacological properties such as lipid solubility, high oral absorption, and large volume of distribution, and are used in the treatment of rheumatoid arthritis, malaria, and systemic lupus erythematosus. In addition, after chloroquine and hydroxychloroquine were reported to inhibit severe acute respiratory syndrome coronavirus 2 (SARS coronavirus 2), they were used as alternative drugs for the treatment of COVID-19. However, cardiac toxicity such as ventricular tachycardia and QT prolongation were reported in subsequent clinical trials. Chloroquine has high toxicity, and it is more toxic than hydroxychloroquine because the plasma concentration ratio indicating a toxic effect (chloroquine to hydroxychloroquine) is less than approximately 0.39. Furthermore, chloroquine has a K ATP It increases insulin secretion through channel inhibition, causing the side effect of hypoglycemia. Although K in vascular smooth muscle and pancreas ATP Although the channels share some properties, their actions are induced by modulators such as ATP, sulfonylurea compounds, and potassium channel-opening drugs. ATP There are differences in the properties of the channels.

[0004] The production of oxygen compounds, including reactive oxygen species (ROS), is caused by K ATPIt has been reported to inhibit vasodilation by channels. Chloroquine has been shown to induce hearing loss and myocardial toxicity in rats with pressure overload cardiomegaly through the production of oxygen compounds and subsequent oxidative stress. However, chloroquine has been shown to inhibit K ATP The effect on channel-mediated vasodilation is unknown.

[0005]

[0006] The present invention aims to provide a pharmaceutical composition for inhibiting vasodilation.

[0007]

[0008] The present invention relates to a pharmaceutical composition for inhibiting vasodilation comprising chloroquine.

[0009] In the present invention, the vasodilation may be vasodilation due to drug treatment.

[0010] In the present invention, the vasodilation is K ATP It may be vasodilation due to channel agonists.

[0011]

[0012] The pharmaceutical composition of the present invention exhibits an inhibitory effect on vasodilation.

[0013]

[0014] Figure 1. Effects of chloroquine (CQ, A) and hydroxychloroquine (HCQ, B) on levchromakalim-induced vasodilation in deendothelialized rat aorta. n = 13, 12, 7, and 6 are control, 10, and 10, respectively. -5 M CQ, 3×10 -5 M CQ and 3×10 -6 M CQ. n = 12, 12, 5 and 6 are control group, 10 respectively. -5 M HCQ, 3×10 -5 M HCQ and 3×10 -6For M HCQ. Data are presented as mean ± standard deviation and expressed as a percentage of phenylephrine-induced contraction. n represents the number of rats. Log ED50 of levchromakalim-induced vasodilation: **p< 0.01, ***p< 0.001 vs. control. Maximal levchromakalim-induced vasodilation: § p< 0.05, #p< 0.01, †p< 0.001 vs. control. Log ED50 is the log of levchromakalim that produces half (50%) of the maximal levchromakalim-induced vasodilation.

[0015] Figure 2. Effects of N-acetyl-L-cysteine ​​(NAC, A), glibenclamide (B), and chloroquine (CQ), alone or in combination, on levchromakalim-induced vasodilation in deendothelialized rat aorta. Data (n = 6) are presented as mean ± standard deviation and expressed as a percentage of phenylephrine-induced contraction. n represents the number of rats. A: *p < 0.05, ***p < 0.001 vs. CQ alone. B: ***p < 0.001 vs. control.

[0016] Figure 3. Effects of chloroquine (CQ) alone and in combination with lipid emulsion (LE) on levchromakalim-induced vasodilation in deendothelialized rat aorta. Data (n = 6) are presented as mean ± standard deviation and expressed as a percentage of phenylephrine-induced contraction. n represents the number of rats. ***p < 0.001 vs. control.

[0017] Figure 4. Effect of chloroquine (CQ) on diltiazem (A) and sodium nitroprusside (SNP, B)-induced vasodilation in deendothelialized rat aorta. Data (n = 5) are presented as mean ± standard deviation and expressed as a percentage of phenylephrine-induced contraction. n represents the number of rats. ***p < 0.001 vs. control.

[0018] Figure 5. Restoration of levchromakalim (LMK)-induced membrane hyperpolarization in vascular smooth muscle cells by high concentrations of chloroquine (CQ) and hydroxychloroquine (HCQ). Membrane potentials were recorded within 1 min after chemical treatment in current-clamp mode (I = 0). Each bar represents the mean ± standard deviation (n = 7). Values ​​are from three independent experiments. ***p < 0.001 vs. control, ††p < 0.01, and †††p < 0.001 vs. LMK alone.

[0019] Figure 6. Effects of chloroquine (CQ) and N-acetyl-L-cysteine ​​(NAC) alone or in combination (A) and of CQ and hydroxychloroquine (HCQ) (B) on reactive oxygen species (ROS) in rat aortic vascular smooth muscle cells. Intracellular ROS levels were measured using fluorescence microscopy after staining with the fluorescent dye 2,7-dichlorofluorescein diacetate (H2DCFDA). Scale bar: 100 μm. Data (n = 3 and 4 for A and B, respectively) are presented as median ± interquartile range (25 to 75%) and are expressed relative to the control value, where n represents the number of independent experiments. A: *p < 0.05 and ***p < 0.001, versus control. †††p < 0.001, versus CQ alone. B: ***p < 0.001, versus control. #p< 0.001, 6×10 -5 M HCQ vs. FI, fluorescence intensity.

[0020]

[0021] The present invention is described in detail below.

[0022]

[0023] The present invention relates to a pharmaceutical composition for inhibiting vasodilation comprising chloroquine or a pharmaceutically acceptable salt thereof.

[0024] Chloroquine is a compound having the structure of chemical formula 1 below.

[0025] [Chemical Formula 1]

[0026]

[0027] The term pharmaceutically acceptable salt means any organic or inorganic addition salt of acetylgentistin at a concentration that is relatively non-toxic and harmless to the patient and that does not diminish the beneficial effects of acetylgentistin due to side effects attributable to this salt.

[0028] The salt may be an acid addition salt or a metal salt obtained using a base. Any acid or base known in the art may be used without limitation.

[0029] Vasodilation can be, for example, vasodilation due to drug treatment. Drugs include, for example, K ATP It may be a channel agonist. K ATP The channel agonist may be, for example, revchromakalim.

[0030] The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods. Carriers, excipients, and diluents that can be contained in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, they are usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the above compound with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0031] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, and weight, but may be administered once or several times daily at a dosage of 0.001 to 100 mg / kg, preferably 0.01 to 10 mg / kg. Furthermore, the dosage may be increased or decreased depending on the route of administration, severity of the disease, sex, body weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any way.

[0032]

[0033] The present invention will be described in more detail with reference to the following examples.

[0034]

[0035] Materials and Methods

[0036] The experimental protocol (GNU-210429-R0036) was approved by the Institutional Animal Care and Use Committee of Gyeongsang National University. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals established by the National Institutes of Health.

[0037]

[0038] Preparation and isometric tension measurements of isolated rat aortas.

[0039] Male Sprague-Dawley rats (weight: 230-280 g, Coretech, Pyeongtaek, Gyeonggi-do) were housed in a small hole in a rat cage with 100% CO 2The rats were euthanized by feeding them with . The thorax was opened, and the descending thoracic aorta was excised from the thoracic cavity and immersed in Krebs solution containing sodium chloride (118 mM), glucose (11 mM), sodium bicarbonate (25 mM), calcium chloride (2.4 mM), potassium chloride (4.7 mM), monopotassium phosphate (1.2 mM), and magnesium sulfate (1.2 mM). Fat and connective tissue surrounding the isolated rat aorta immersed in Krebs solution were removed under a microscope. The isolated rat aorta was then cut into 2.5 mm-long segments. The endothelium of all isolated rat aortas was stripped by rolling back and forth two 25-gauge needles inserted into the lumen of the isolated rat aorta. The isolated thoracic aorta was suspended in a Grass isometric transducer (FT-03; Grass Instrument, Quincy, MA, USA) mounted in an organ bath maintained at 37°C. At the same time, the existing Krebs solution was replaced with a fresh solution every 30 minutes. The Krebs solution was maintained at pH 7.4 by supplying gas containing 5% carbon dioxide and 95% oxygen. Endothelial removal was confirmed using the following method: Phenylephrine (10 -7 After addition of acetylcholine (10 M), stable and sustained contractions were induced, followed by phenylephrine-induced contractions in a long-term bath with aortas. -5 M) was added. Aortas with less than 15% acetylcholine-induced relaxation were considered as endothelial-free aortas. Fresh Krebs solution was then added several times to the organ bath to wash the aortas, which showed acetylcholine-induced relaxation in phenylephrine-induced contraction and eventually restored baseline resting tension. The following experimental protocol was then performed. Because levchromakalim-induced vasodilation is partly mediated by endothelial nitric oxide, all endothelialized rat aortas used in this experiment were treated with nitric oxide synthase inhibitor N to prevent endothelial nitric oxide release due to the presence of residual endothelium. ω-nitro-L-arginine methyl ester(L-NAME; 10 -4 M) was preprocessed.

[0040]

[0041] Experimental protocol

[0042] First, toxic concentrations of chloroquine (10 -5 , 3Х10 -5 , and 6Х10 -5 M) and hydroxychloroquine (10 -5 , 3Х10 -5 , and 6Х10 -5 K) in the deendothelialized rat aorta of M ATP The effects of the channel agonist levchromakalim on vasodilation were analyzed. Endothelialized rat aortas were pretreated with chloroquine or hydroxychloroquine for 20 min. Phenylephrine (10 -6 After inducing sustained and stable contractions in isolated endothelial rat aortas, levchromakalim (10 -8 to 10 -5 M) was added to produce revchromakalim-induced vasodilation regardless of the presence or absence of endothelium.

[0043] Second, ROS and K ATP To confirm the role of channel inhibitors, the effect of chloroquine on inhibition of levchromakalim-induced vasodilation was investigated. The ROS scavenger N-acetyl-L-cysteine ​​(NAC, 5X10 -3 M) and K ATP Channel inhibitor glibenclamide (5Х10 -6 M) to investigate the effect of chloroquine (6Х10 -5 The effect of chloroquine alone on the inhibition of levchromakalim-induced vasodilation was investigated. The deendothelialized rat aortas were treated with chloroquine alone for 20 minutes, NAC and glibenclamide for 35 minutes each, or pretreated with NAC or glibenclamide for 15 minutes and then treated with chloroquine for 20 minutes. Phenylephrine (10-6 After M) induced stable sustained contraction, revchromakalim (10 -8 to 10 -5 M) was added to produce levchromakalim-induced vasodilation under treatment with chloroquine, NAC, and glibenclamide alone or in combination with NAC plus chloroquine or glibenclamide plus chloroquine, regardless of the presence or absence of endothelium.

[0044] Thirdly, the effect of lipid emulsion (Intralipid; 1%) on the inhibition of levchromakalim-induced vasodilation by lipophilic chloroquine was investigated. The deendothelialized rat aortas were pretreated with lipid emulsion (1%) for 15 min and then treated with chloroquine (6Х10 -5 M) for 20 minutes or treated with chloroquine (6Х10 -5 M) was treated for 20 minutes. Then, phenylephrine (10 -6 After M) induced stable sustained contraction, revchromakalim (10 -8 to 10 -5 M) was added to produce levchromakalim-induced vasodilation with or without endothelium, either alone or in combination with chloroquine and lipid emulsion.

[0045] Fourth, to determine whether the inhibition of levchromakalim-induced vasodilation by chloroquine was specific, chloroquine (6Х10 -5 M) on vasodilation by the calcium channel inhibitor diltiazem and the nitric oxide donor sodium nitroprusside. The aorta was treated with chloroquine (6Х10 -5 M) was pretreated for 20 minutes. Phenylephrine (10 -6 After M) induced sustained and stable contractions, diltiazem (3Х10 -8 to 3Х10 -4 M) or sodium nitroprusside (10 -10 to 10 -7M) was treated in combination or to induce vasodilation in the presence or absence of chloroquine.

[0046]

[0047] Vascular smooth muscle cell culture

[0048] Vascular smooth muscle cells (VSMCs) were isolated from the descending dural aorta and cultured in Dulbecco's modified Eagle medium supplemented with 100 mg / ml streptomycin, 100 U / ml penicillin, and 10% heat-treated fetal bovine serum. Cells at passages 3–5 were used and incubated in a humidified atmosphere containing 5% carbon dioxide at 37°C.

[0049]

[0050] Measurement of resting membrane potential of vascular smooth muscle cells

[0051] The membrane potential of vascular smooth muscle cells (VSMCs) was measured in current-clamp mode (I = 0) using the whole-cell patch-clamp technique. The membrane potential was amplified using a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Union City, CA, USA). The bath solution (pH 7.4) consisted of 135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The pipette solution (pH 7.3) consisted of 150 mM KCl, 1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES. The pipette tip resistance was 4–6 MΩ. Compounds were dissolved in the bath solution at the desired concentrations. Experimental data were analyzed using Clampfit (pCLAMP, version 9.2, Axon Instruments).

[0052]

[0053] Measurement of intracellular reactive oxygen species

[0054] Chloroquine-induced ROS production was measured in vascular smooth muscle cells (VSMCs) using 2,7-dichlorofluorescein diacetate (H2DCFDA). Cells seeded on glass coverslips (2X10 in 6-well culture plates) 5 (at a density of 10 cells / well) were cultured overnight in a carbon dioxide incubator at 37°C, and then transferred to serum-free medium. Cells were treated with chloroquine (6Х10 -5 M) or hydroxychloroquine (6Х10 -5 M) for only 10 minutes or NAC (5Х10 -3 M) for only 70 minutes, or NAC (5Х10 -3 M) for 1 hour and then chloroquine (6Х10 -5M) was treated for 10 minutes. Afterwards, the cells were washed with phosphate-buffered solution (PBS). The cells were incubated for 30 minutes at 37°C for 10 -5 M H2DCFDA was treated and washed twice with PBS. ROS production was captured using a fluorescence microscope (Nikon Eclipse Ti2; Nikon Co., Tokyo, Japan), and the captured cell images were analyzed.

[0055]

[0056] compound

[0057] The highest purity compounds were used. Chloroquine, hydroxychloroquine, L-NAME, glibenclamide, H2DCFDA, diltiazem, sodium nitroprusside, NAC, phenylephrine, and acetylcholine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Intralipid (20%) was purchased from Fresenius Kabi AB (Upsala, Sweden). DMEM medium (Dulbecco's modified Eagle medium), fetal bovine serum, and penicillin-streptomycin were purchased from Gibco (Life Technologies, Grand Island, NY, USA). Revchromakalim was purchased from Tocris Bioscience (Bristol, United Kingdom). Revchromakalim was dissolved in ethanol (final concentration: 0.19%). All other drugs were dissolved in distilled water.

[0058]

[0059] Statistical analysis

[0060] The primary outcome was the effect of chloroquine, hydroxychloroquine, NAC, glibenclamide, and lipid emulsion on levchromakalim-induced vasodilation. The Kolmogorov-Smirnov test was used to test for normality. Generalized linear mixed-effects models (Stata version 14.1, Stat Corp LP, Lakeway Drive, College Station, TX, USA) were used to analyze the effects of chloroquine, hydroxychloroquine, NAC, glibenclamide, and lipid emulsion or their combination on levchromakalim, diltiazem, and sodium nitroprusside-induced vasodilation. To calculate the log of revchromakalim to produce half (50%) of the amount of revchromakalim in revchromakalim-induced vasodilation in the presence or absence of chloroquine or hydroxychloroquine, nonlinear regression was performed by fitting the revchromakalim dose-response curve to a sigmoidal curve in Prism 5.0 (Graphad Software Inc, San Diego, CA, USA). The log ED50 and maximum revchromakalim-induced vasodilation were compared to determine the effect of chloroquine or hydroxychloroquine on revchromakalim-induced vasodilation. The log ED50 effect of chloroquine and hydroxychloroquine on revchromakalim-induced vasodilation was analyzed using the Kruskal-Wallis test and Dunn's multiple comparison test, respectively, and the log ED50 ratio was used to compare the inhibitory efficacy of chloroquine and hydroxychloroquine on revchromakalim-induced vasodilation. An unpaired Student's t-test was used to compare the Log ED50 ratios between the chloroquine and hydroxychloroquine groups. The effects of chloroquine and hydroxychloroquine on levchromakalim-induced membrane potential were analyzed using a one-way analysis of variance, followed by a Bonferroni multiple comparison test.The effects of chloroquine, hydroxychloroquine, and NAC, or their combination, on ROS production were analyzed using the Kruskal-Wallis test and Dunn's multiple comparison test.

[0061]

[0062] result

[0063] Chloroquine (10 -5 , 3Х10 -5 and 6Х10 -5 M) inhibited vasodilation induced by revchromakalim (Log ED50: p < 0.01, 10 -5 M chloroquine vs. control; p< 0.001, 3Х10 -5 and 6Х10 -5 M chloroquine vs. control; Fig. 1A). Also, hydroxychloroquine (10 -5 , 3Х10 -5 and 6Х10 -5 M) also inhibited revchromakalim-induced vasodilation (Log ED50: 10 -5 , 3Х10 -5 and 6Х10 -5 M hydroxychloroquine; p < 0.001 vs. control; Fig. 1B). However, 10 -5 The Log ED50 ratio of levchromakalim-induced vasodilation in the M chloroquine-treated group was 10 -5 M was greater than that in the hydroxychloroquine treatment group (p = 0.001; Log ED50 ratio: 10 -5 M chloroquine = 1.72 ± 0.32 vs. 10 -5 M hydroxychloroquine = 1.29 ± 0.22), suggesting that chloroquine inhibits levchromakalim-induced vasodilation more than hydroxychloroquine. On the other hand, in the chloroquine-treated group, high concentrations of levchromakalim (10 -5 Maximal vasodilation induced by M) was increased compared to the control group (6Х10 -5 M chloroquine: p< 0.001 vs. control; 3Х10-5 M chloroquine: p< 0.01 vs. control; Fig. 1A). Also, at high concentrations (6Х10 -5 Hydroxychloroquine at high concentrations (10 -5 M) slightly increased the maximal vasodilation induced by revchromakalim (p<0.05 vs. control; Fig. 1B). NAC (5X10 -3 M) and chloroquine (6Х10 -5 M) combined treatment with chloroquine (6Х10 -5 M) Increased vasodilation by revchromakalim compared to single dose (10 -6 and 3Х10 -6 p< 0.001 in M ​​Revchromakalim; 10 -5 M Revchromakalim (p<0.05; Fig. 2A). Glibenclamide (5X10 -6 M) and chloroquine (6Х10 -5 M) or glibenclamide (5Х10 -6 M) alone eliminated vasodilation caused by revchromakalim (5Х10 -6 M glibenclamide + 6Х10 -5 M chloroquine or 5Х10 -6 M Glibenclamide alone: ​​10 -7 10 in -5 p<0.001 vs. control in M ​​Revchromakalim; Fig. 2B). Lipid emulsion (1%) and chloroquine (6Х10 -5 Combination therapy with chloroquine (M) did not significantly alter vasodilation induced by levchromakalim compared with chloroquine alone (Fig. 3). Chloroquine (6X10 -5 M) increased diltiazem-induced vasodilation (10 -6 10 in -4 M diltiazem (p<0.001 vs. control; Fig. 4A). However, chloroquine (6Х10 -5 M) did not affect vasodilation induced by sodium nitroprusside (Fig. 4B). Revchromakalim (10 -5M) induced membrane hyperpolarization in rat aortic vascular smooth muscle cells (p<0.001 vs. control; Fig. 5). Chloroquine (3Х10 -5 M) and hydroxychloroquine (3Х10 -5 M) is Revchromakalim (10 -5 M) inhibited membrane hyperpolarization induced by (p< 0.01: 3Х10 -5 M chloroquine + levchromakalim vs levchromakalim alone; p<0.001: 3Х10 -5 M hydroxychloroquine + levchromakalim vs levchromakalim alone; Figure 5). Chloroquine (6X10 -5 M) increased ROS production in rat aortic vascular smooth muscle cells (p<0.001 vs. control; Fig. 6A). However, NAC (5Х10 -3 Pretreatment with M) is chloroquine (6Х10 -5 M) inhibited ROS production induced by hydroxychloroquine (p<0.001 compared to chloroquine alone; Fig. 6A). Additionally, hydroxychloroquine (6Х10 -5 M) also increased ROS production (p<0.001 vs. control; Fig. 6B). Chloroquine (6Х10 -5 M) is hydroxychloroquine (6Х10 -5 M) produced more ROS than the control group (p< 0.001; Fig. 6B).

Claims

1. A pharmaceutical composition for inhibiting vasodilation comprising chloroquine or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition for inhibiting vasodilation according to claim 1, wherein the vasodilation is vasodilation caused by drug treatment.

3. In claim 1, the vasodilation is K ATP A pharmaceutical composition for inhibiting vasodilation, which is vasodilation by a channel agonist.

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