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WO2024179420A1 - Utilisation de lys01 ou d'un sel de celui-ci dans la préparation d'un inhibiteur de canal potassique kir4.1 - Google Patents

Utilisation de lys01 ou d'un sel de celui-ci dans la préparation d'un inhibiteur de canal potassique kir4.1 Download PDF

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WO2024179420A1
WO2024179420A1 PCT/CN2024/078649 CN2024078649W WO2024179420A1 WO 2024179420 A1 WO2024179420 A1 WO 2024179420A1 CN 2024078649 W CN2024078649 W CN 2024078649W WO 2024179420 A1 WO2024179420 A1 WO 2024179420A1
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lys05
mice
lys01
potassium channel
salt
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Chinese (zh)
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高召兵
周晓宇
郑月明
许海燕
汪佩
詹丽
顾跃玲
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Shanghai Institute of Materia Medica of CAS
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Shanghai Institute of Materia Medica of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics

Definitions

  • the present invention belongs to the field of medicine. Specifically, the present invention relates to the use of Lys01 or a salt thereof in the preparation of a Kir4.1 inhibitor. In addition, the present invention also provides the use of Lys01 or a salt thereof in the preparation of a drug for treating and/or preventing a disease with the Kir4.1 potassium channel as a therapeutic target, and the use of Lys01 or a salt thereof in the preparation of a diuretic.
  • Lys01 N'-(7-chloroquinolin-4-yl)-N-[2-[(7-chloroquinolin-4-yl)amino]ethyl]-N-methylethane-1,2-diamine.
  • Lys05 is the trihydrochloride form of Lys01.
  • Depression is a neurological disorder that seriously affects human health and quality of life, shortens patients' life expectancy, and increases socioeconomic burden.
  • the global incidence of depression is about 16%, and the incidence of depressive disorders in China is about 3.02% (Malhi GS et al., Lancet. 2018, 392 (10161): 2299-2312; Smith K., Nature. 2014, 515 (7526): 181.).
  • Traditional antidepressants such as monoamine reuptake inhibitors, are widely used in clinical practice, but have obvious limitations, mainly including: 1. Delayed onset, often requiring 3 to 4 months of continuous treatment to improve patients' depression. 2. Low effectiveness, there are still 30% of refractory depression patients in clinical practice (Trevino K.
  • BDNF brain-derived neurotrophic factor
  • Kir4.1 hyperfunction genetic mutations are also related to neurological diseases such as autism and autism-epilepsy comorbidity (Nwaobi SE et al., Acta Neuropathol.2016,132(1):1-21.).
  • one object of the present invention is to provide the use of Lys01 or a salt thereof in the preparation of Kir4.1 potassium channel inhibitors.
  • the Kir4.1 potassium channel inhibitor may be a heterologous Kir4.1 potassium channel inhibitor or an endogenous Kir4.1 potassium channel inhibitor.
  • the diseases that use Kir4.1 potassium channel as a therapeutic target include neurological diseases such as depression, autism, epilepsy-autism comorbidity, Huntington's disease, and kidney diseases such as urinary retention.
  • the present invention also provides the use of Lys01 or a salt thereof in the preparation of a diuretic.
  • the salt of Lys01 can be an acid addition salt, and the acid forming the salt can be hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-Toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxy
  • Lys01 or its salt can be administered alone or in any convenient pharmaceutical form (e.g., in the form of a composition or preparation with a pharmaceutically acceptable carrier).
  • Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices may also be included.
  • suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.
  • suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.
  • compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • Lys01 or its salt can be administered alone or in combination with other therapeutic drugs (such as other antidepressants).
  • a safe and effective amount of the compound of the present invention is administered to an individual (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 20 to 500 mg.
  • the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill range of skilled physicians.
  • the present invention shows that Lys05, the trihydrochloride of Lys01, inhibits the function of Kir4.1 channel, which can inhibit endogenous Kir4.1 channel current and regulate the resting membrane potential of glial cells; animal experiments show that Lys05 can quickly relieve the depressive-like behavior of animals. Therefore, the present invention shows that Lys05 can be used as a heterologous Kir4.1 channel inhibitor and an endogenous Kir4.1 channel current inhibitor, and is used to prepare drugs for preventing and/or treating neuropsychiatric diseases and kidney diseases with Kir4.1 channel as a therapeutic target.
  • Figure 1 shows the effect of Lys05 on Kir4.1 current in Example 1 of the present invention, wherein: A is a representative current diagram of Kir4.1 channel before and after administration of 10 ⁇ M Lys05; B is the dose-effect curve of Lys05 of Kir4.1 channel.
  • Figure 2 shows the effect of Lys05 on the endogenous Kir4.1 current of astrocytes in Example 2 of the present invention, wherein: A is a representative current graph of the endogenous Kir4.1 current of the normal astrocyte group (top) and the Kir4.1 siRNA-knockdown group (bottom) under a step voltage stimulation scheme before and after the administration of 10 ⁇ M Lys05; B is a representative current graph of the endogenous Kir4.1 current of the normal astrocyte group (left) and the Kir4.1 siRNA-knockdown group (right) under a ramp voltage stimulation scheme before and after the administration of 10 ⁇ M Lys05; C is a statistical graph of the inhibition rate of endogenous Kir4.1 current of the normal astrocyte group (left) and the Kir4.1 siRNA-knockdown group (right) under a ramp voltage stimulation scheme after the administration of 10 ⁇ M Lys05.
  • Figure 3 shows the effect of Lys05 on the resting membrane potential level of astrocytes in Example 2 of the present invention, wherein: A is a representative graph of the changes in the resting membrane potential level of the normal astrocyte group and the Kir4.1 siRNA-knockdown group before and after the administration of 10 ⁇ M Lys05; B is a statistical graph of the changes in the resting membrane potential level of the normal astrocyte group and the Kir4.1 siRNA-knockdown group before and after the administration of 10 ⁇ M Lys05; C is a representative graph of the changes in the resting membrane potential level of the normal astrocyte group and the Kir4.1 siRNA-knockdown group before and after the administration of 100 ⁇ M BaCl 2 (non-selective Kir4.1 channel inhibitor); D is a statistical graph of the changes in the resting membrane potential level of the normal astrocyte group and the Kir4.1 siRNA-knockdown group before and after the administration of 100 ⁇ M BaCl 2 .
  • Figure 4 shows the effect of Lys05 on the depressive behavior induced by Kir4.1 overexpressing mice in Example 3 of the present invention, wherein: A is a schematic diagram of the Kir4.1 overexpressing adeno-associated virus vector; B is a schematic diagram of the modeling and detection scheme of the depressive behavior of mice induced by overexpressing Kir4.1 in the lateral habenula tissue; C is a representative fluorescence schematic diagram of the lateral habenula tissue of mice after expressing the Kir4.1 adeno-associated virus; D is a statistical graph of the effect of intraperitoneal administration of different doses of Lys05 (5 and 10 mg/kg) on the immobility time (left) and the number of immobility times (right) of normal mice and Kir4.1 overexpressing mice in the forced swimming test; E is a statistical graph of the effect of intraperitoneal administration of different doses of Lys05 (5 and 10 mg/kg) on the sucrose preference of normal mice and Kir4.1 overexpressing mice in the sucrose preference experiment.
  • Figure 5 shows the effect of Lys05 on the acute forced swimming test in mice in Example 4 of the present invention, wherein: A is a schematic diagram of the forced swimming drug administration and detection scheme; B is a statistical chart of the effects of intraperitoneal administration of different doses of Lys05 (45, 60 and 70 mg/kg) and the positive control drug imipramine (30 mg/kg) on the immobility time of mice.
  • Figure 6 shows the effect of Lys05 on corticosterone-induced depressive behavior in mice in Example 4 of the present invention, wherein: A is a schematic diagram of the modeling and detection scheme of corticosterone-induced depressive behavior in mice; B is a statistical diagram of the effect of intraperitoneal administration of different doses of Lys05 (5 and 10 mg/kg) and the positive control drug S-ketamine (10 mg/kg) on the total movement distance (left) and central area movement time (right) in the open field test of mice; C is a statistical diagram of the effect of intraperitoneal administration of different doses of Lys05 (5 and 10 mg/kg) and the positive control drug S-ketamine (10 mg/kg) on the immobility time in the tail suspension test of mice; D is a statistical diagram of the effect of intraperitoneal administration of different doses of Lys05 (5 and 10 mg/kg) and the positive control drug S-ketamine (10 mg/kg) on the immobility time in the tail suspension test of mice.
  • A is a schematic diagram of the modeling
  • Figure 7 shows the effect of Lys05 on the depressive behavior of mice in the novelty-suppressed feeding model in Example 4 of the present invention, wherein: A is the effect of a single intraperitoneal administration of Lys05 (5 and 10 mg/kg) and the positive control drug S-ketamine (5 and 10 mg/kg) on the feeding latency of mice; B is the effect of multiple intraperitoneal administration of Lys05 (3, 5 and 10 mg/kg) on the feeding latency of mice.
  • Figure 8 shows the effect of Lys05 on the depressive behavior of mice induced by olfactory bulbectomy in Example 5 of the present invention, wherein: A is a schematic diagram of the modeling and detection scheme (single administration) of depressive behavior in mice induced by olfactory bulbectomy; B is a statistical graph of the effect of a single intraperitoneal administration of different doses of Lys05 (10 and 30 mg/kg) and the positive control drug imipramine (30 mg/kg) on the total movement distance in the open field test of mice; C is a statistical graph of the effect of a single intraperitoneal administration of different doses of Lys05 (10 and 30 mg/kg) and the positive control drug S-ketamine (10 mg/kg) on the total movement distance in the open field test of mice; D is a schematic diagram of the modeling and detection scheme (multiple administration) of depressive behavior in mice induced by olfactory bulbectomy; E is a statistical graph of the effect of intraperitoneal administration of different doses of Lys05 (10 and 20 mg/kg
  • Example 1 Lys05 inhibits Kir4.1 potassium channel current
  • CHO Chinese Hamster Ovary cell (CHO) (Cell Bank, Chinese Academy of Sciences) culture medium formula: 50/50 DMEM/F-12 (Gibco), added with 10% fetal bovine serum (Fetal bovine serum) (Gibco, Australia), 2mM L-glutamine (Invitrogen).
  • the plasmid carrying the Kir4.1 gene was expressed in CHO cells using the liposome transfection method. Specifically: the plasmid encoding the Kir4.1 gene was synthesized by Beijing Liuhe BGI Gene Co., Ltd. and the number published in the NCBI database is NM_002241.5. 24 hours before transfection, the CHO cells were digested with trypsin (Sigma) and plated on a dish with a diameter of 35 mm. The transfection used Lipofectamine2000 TM reagent (Invitrogen) and was performed according to the operating steps provided.
  • the extracellular solution was continuously perfused by a BPS perfusion system (ALA Scientific Instruments).
  • the electrical signals were filtered at 2kHz and converted to digital signals using DigiData 1322A in pClamp 9.2 software (Molecular Devices). Series resistance compensation was 60-80%, and a ramp stimulation voltage protocol of -120 mV to +50 mV was used during the recording of electrophysiological studies.
  • the step voltage recording scheme of Kir4.1 potassium channel is: clamp voltage -80mV, a series of 500ms stimulation voltage (from -120mV to +40mV, 20mV interval) to induce current.
  • the ramp voltage recording scheme of Kir4.1 potassium channel current is: clamp voltage -80mV, first give -120mV, 100ms super voltage stimulation to induce Inward potassium current, a ramp voltage stimulation of -120mV to +50mV for 500ms was given, and an outward current was induced at +50mV stimulation voltage for 100ms.
  • Electrophysiological data were analyzed using Clampfit 10.2 (Molecular Devices), followed by GraphPad Prim 8 (GraphPad Software), the dose-effect curve was fitted using the Hill equation, and image processing was performed using Adobe Illustrator CS6 (Adobe) software.
  • Inhibition rate % (1-I/I 0 ) ⁇ 100%, where I/I 0 is the inhibition intensity, I 0 is the current value before administration, and I is the current value after administration. All experimental data are expressed as mean ⁇ standard error (mean ⁇ SEM).
  • Example 2 Lys05 inhibits endogenous Kir4.1 potassium current and depolarized membrane potential levels in mouse cortical astrocytes
  • Kir4.1 is specifically expressed in glial cells in the brain, highly expressed in astrocytes, and mediates the "K + space buffer" function. This process depends on the hyperpolarized resting membrane potential of astrocytes, and reabsorbs potassium ions released to the extracellular space by neural activity into the cell. As the main potassium channel in astrocytes, Kir4.1 channels play a key role in mediating endogenous potassium currents in astrocytes and regulating resting membrane potential levels. Therefore, inhibiting, knocking out or knocking down Kir4.1 potassium channels will significantly affect the above processes.
  • a mixed system of transfection reagent and small interfering RNA (siRNA) (targeting Kir4.1 gene, siRNA positive chain sequence: GGAGCACAUUGCUGACAAATT; antisense chain sequence: UUUGUCAGCAAUGUGCUCCTT) was prepared, and the mixture was incubated at room temperature for 10 minutes to obtain the transfection reagent-siRNA complex. During this period, the cultured astrocytes were digested and inoculated into the well plate, and the transfection reagent-siRNA complex was added before the cells adhered to the wall. The electrophysiological experiment was recorded 72 hours after the astrocytes were transfected with siRNA.
  • siRNA small interfering RNA
  • the endogenous potassium current of cultured primary astrocytes or astrocytes knocked down Kir4.1 was recorded by whole-cell voltage clamp using an Axonpatch-700B amplifier (Molecular Devices).
  • the endogenous potassium current recording was performed using a BPS perfusion system to give a 10 ⁇ M Lys05 detection solution or a 100 ⁇ M BaCl 2 solution, and the protocol was the same as in Example 1.
  • the extracellular solution containing only 1 ⁇ DMSO (volume percentage) was perfused to elute the drug effect until a stable state was reached.
  • the resting membrane potential level was recorded by whole-cell current clamp.
  • the recording scheme was as follows: the cell was clamped at 0pA without any current stimulation, and the membrane potential amplitude was continuously recorded in the "Gap-free" mode.
  • the electrode inlet resistance was 2-3M ⁇
  • the series resistance compensation was 60-80%
  • the recording temperature was 20-22°C.
  • BaCl 2 non-selective inhibitor of Kir4.1 channel, Sigma, analytical grade
  • Electrophysiological data analysis was performed using Clampfit 10.2, GraphPad Prim 8 and Adobe Illustrator CS6 software for data analysis and image processing software analysis.
  • Inhibition rate % (1-I/I 0 ) ⁇ 100%, where I/I 0 is the inhibition intensity, I 0 is the current value before administration, and I is the current value after administration. All experimental data are expressed as mean ⁇ standard error (mean ⁇ SEM). The unpaired t-test method was used to analyze the data for significance, where ns (no significance) indicates that there is no statistical difference between the two groups, **P ⁇ 0.01, ****P ⁇ 0.0001 indicates that the difference between the two groups is statistically significant.
  • Lys05 inhibits endogenous Kir4.1 potassium currents and depolarizes resting membrane potential levels in primary astrocytes.
  • Example 3 Lys05 alleviates depressive-like behavior induced by overexpression of Kir4.1 channels in the lateral habenular nucleus of mice
  • the lateral habenula is an important brain region for regulating depression. Activating the glutamatergic neurons in the lateral habenula can directly or indirectly act on the ventral tegmental area, the "reward center", inhibiting the activity of dopaminergic neurons and leading to reduced dopamine release in the brain.
  • Overexpression of Kir4.1 in the lateral habenula astrocytes by in situ virus injection can induce clustered discharges of lateral habenula neurons and drive mice to produce a depressive-like phenotype. Knocking down the Kir4.1 protein in the lateral habenula astrocytes or expressing a dominant negative mutant channel can alleviate depressive symptoms. Therefore, the effect of Lys05 on the depressive-like behavior induced by overexpression of Kir4.1 in the lateral habenula was further investigated.
  • Kir4.1 overexpressing adeno-associated virus (AAV2/5-gfaABC1D-eGFP-Kir4.1, titer: 1.94 ⁇ 10 13 vg/mL) and control virus (AAV2/5-gfaABC1D-eGFP, titer: 1.79 ⁇ 10 13 vg/mL) were synthesized by TeraTu Biotech, and the viral vectors are shown in FIG4A .
  • mice were anesthetized, and the middle hair on the top of the head of the mouse was removed with a hair trimmer.
  • the mouse was fixed in a stereotaxic instrument (Stoelting). The scalp was gently cut, the periosteum was peeled off, the skull was exposed, and the anterior fontanelle was determined. According to the atlas, the position of the bilateral lateral habenula was determined (lateral habenula position coordinates: AP, -1.32mm, ML, ⁇ 0.42mm, DV, -2.8mm), and a small hole was drilled in each.
  • the microinjector needle connected to the automatic microinjection pump was inserted into the drilled holes on both sides in turn, and the adeno-associated virus overexpressing Kir4.1 (0.2 ⁇ L, 1:5 dilution) was injected at a rate of 0.1 ⁇ L/min. After the injection, the needle was left to stand for 10 minutes and then withdrawn. The scalp was sutured and the wound was wiped with iodine disinfectant.
  • the control group of mice was injected with a virus expressing only green fluorescent protein. After surgery, the mice were placed in a warm environment until they woke up, and behavioral experiments were performed 21 days later. After the experiment, the mouse brain was dissected, and the injection position was confirmed by preparing slices to observe the autofluorescence of the virus.
  • mice were housed in single cages 3 days in advance for adaptation training.
  • two drinking bottles containing 1% sucrose solution and drinking water were placed in the mice's cages. After 24 hours, the positions of the two drinking bottles were swapped to avoid the mice from developing positional preferences, and they were kept there for another 24 hours. The mice were then prohibited from drinking water for 24 hours.
  • the formal testing phase two new drinking bottles containing 1% sucrose solution and drinking water were prepared and placed in the cages for the mice to drink freely. The test lasted for a total of 24 hours. At the 12th hour, the positions of the two drinking bottles were swapped to eliminate the influence of position. The two drinking bottles were weighed before and after the experiment to calculate the solution consumption.
  • the sucrose preference of mice is defined as the percentage of sucrose solution consumption to total consumption.
  • the experimental results showed that 21 days after the lateral habenula was microinjected with Kir4.1 overexpressing virus, spontaneous green fluorescence produced by viral expression was observed in the lateral habenula brain region of mice (B, C in Figure 4).
  • the forced swimming model is a fast and effective in vivo model evaluation method for antidepressant drugs.
  • the performance of the test mice in the forced swimming model was first evaluated. Compared with the EGFP control group mice, the immobility time and number of immobility of mice overexpressing Kir4.1 increased significantly (D in Figure 4), indicating that typical depressive-like behavior was induced.
  • Lys05 can alleviate depressive-like behaviors, suggesting that it may also have a therapeutic effect on other classic depression models.
  • mice were made to chronically ingest corticosterone by drinking corticosterone solution daily. Specifically, the mice in the corticosterone group were provided with 0.45% 2-hydroxypropyl- ⁇ -cyclodextrin solution (0.45% HP- ⁇ -CD) containing corticosterone (35 ⁇ g/mL, Sigma) as daily drinking water, which was freely available, and the mice in the control group were provided with 0.45% HP- ⁇ -CD solution. This was provided for 14 consecutive days, and then the corticosterone content was halved every two days, and continued for 6 days. The corticosterone solution was replaced every two days to avoid degradation. Behavioral evaluation was performed from the 21st day.
  • mice were placed in a new cage one day in advance and fasted for 24 hours.
  • the test environment was a 50 ⁇ 50 ⁇ 20 cm white plastic box with a light intensity of 1100-1200 lux.
  • a single feed pellet was placed in the center of the test box, and the mice were placed in the corner of the test box.
  • the timer was started from the time the mice were placed in the box to detect the delay time for the mice to eat the first bite of food. After the mice ingested the food, they were immediately placed in a cage with pre-weighed food in the cage. The food intake of the mice in the original cage within 5 minutes was tested as a control.
  • Lys05 dosing solution weigh 22.5, 30, 35 and 1.5 mg Lys05 respectively and dissolve in 500 ⁇ L DMSO. Dissolve completely, add 9.5 mL 1% Tween-80 (take 1 mL Tween-80 and dissolve in 99 mL normal saline) to obtain 2.25, 3, 3.5 and 0.15 mg/mL Lys05 injection solution. One hour before the evaluation of depressive behavior (acute forced swimming model is 30 min), inject Lys05 injection solution (final 5% DMSO + 95% 1% Tween-80) into the right abdominal cavity of the test mouse at 0.2 mL/10 g body weight to obtain 45, 60, 70 or 3 mg/kg Lys05 dosing amount. The preparation method of 5 and 10 mg/kg Lys05 solution is the same as that of Example 3.2.
  • imipramine dosing solution Weigh 15 mg of imipramine and dissolve it in 10 mL of normal saline to obtain 1.5 mg/mL imipramine injection. One hour before the evaluation of depressive behavior (30 minutes for the acute forced swimming model), inject imipramine injection into the right abdominal cavity of the test mouse at 0.2 mL/10 g body weight to obtain an imipramine dosage of 30 mg/kg.
  • S-ketamine dosing solution Take 0.2 mL of 25 mg/mL S-ketamine injection and dissolve it in 9.8 mL of raw The mice were injected with 0.2 mL/10 g body weight S-ketamine injection solution into the right abdominal cavity 1 hour before the depressive behavior evaluation to obtain a 10 mg/kg S-ketamine dosage.
  • Open field test Place the mouse in the center of the open field test box, and the mouse behavior analysis and processing system automatically records the mouse's movement status within 5 minutes. After each mouse test, clean up the excrement and wipe the open field with alcohol to remove residual odor.
  • Novelty suppressed feeding test see 4.3 for the method
  • the corticosterone-induced mouse depression model is one of the widely used classic depression models because it effectively simulates the cognitive dysfunction and affective disorders caused by elevated glucocorticoid levels in clinical patients with depression.
  • the inventors found that after C57 mice were continuously fed with drinking water containing corticosterone for 21 days, they showed typical depressive-like behaviors in both the tail suspension test and the sucrose preference test (Figure 6).
  • S-ketamine is a rapid antidepressant drug used in clinical practice.
  • the novelty-suppressed feeding model reflects the conflict between anxiety and seeking food rewards in mice when dealing with a new environment.
  • the ability of sudden change is strong or weak, which is a classic depression/anxiety behavior evaluation model.
  • this model has the advantage of predicting the onset time of antidepressant drugs.
  • the traditional antidepressant fluoxetine needs to be taken effect after chronic administration, while the rapid antidepressant ketamine can exert its efficacy after 1 hour of administration.
  • 3 5 and 10mg/kg Lys05 can further shorten the feeding latency of mice, showing better antidepressant effects (B in Figure 7).
  • Example 5 Lys05 rapidly relieves depressive behavior in mice induced by olfactory bulbectomy
  • mice were anesthetized, and the hair in the middle of the top of the head of the mice was removed with a hair trimmer.
  • the mice were fixed in a stereotaxic instrument (Stoelting), the scalp was gently cut, the periosteum was peeled off, the skull was exposed, and the anterior fontanelle was determined.
  • the position of the olfactory bulb was determined according to the atlas, and a small hole was drilled on each side.
  • the needle of the Morton syringe connected to a micro vacuum pump was inserted into the drill hole, and the olfactory bulb was slowly sucked out. Avoid damaging the prefrontal cortex during the suction process.
  • mice were operated the same except that the olfactory bulbs were not sucked out. After the operation, the mice were placed in a warm environment until they woke up, and then they were kept in a single cage to reduce other factors that caused the aggressive reaction of the mice. After the experiment, the mouse brain was dissected to observe whether the olfactory bulb was completely removed. The data of animals with damaged prefrontal cortex and more than 30% of olfactory bulbs remaining during the operation were excluded.
  • Lys05 dosing solution weigh 10 mg Lys05 and dissolve it in 500 ⁇ L DMSO. After complete dissolution, add 9.5 mL 1% Tween-80 (take 1 mL Tween-80 and dissolve it in 99 mL saline) to obtain 1 mg/mL Lys05 injection solution. One hour before the evaluation of depressive behavior, inject 0.2 mL/10 g body weight of Lys05 injection solution into the right abdominal cavity of the test mouse (final 5% DMSO + 95% 1% Tween-80) to obtain a Lys05 dosing dose of 20 mg/kg.
  • the preparation method of 10 and 30 mg/kg Lys05 dosing solutions is the same as in Examples 3 and 4.
  • S-ketamine dosing solution Take 60 ⁇ L of 25 mg/mL S-ketamine injection and dissolve it in 9.94 mL of raw The mixture was diluted with saline to obtain a 0.15 mg/mL S-ketamine injection solution, and the S-ketamine injection solution was injected into the right abdominal cavity of the test mice at a rate of 0.2 mL/10 g body weight 1 hour before the depressive behavior evaluation to obtain a 3 mg/kg S-ketamine dosage.
  • the preparation method of the 10 mg/kg S-ketamine dosing solution was the same as in Example 4.
  • mice with olfactory bulbectomy showed extremely active behavior and spontaneous anxiety
  • the total movement distance of mice and the percentage of central movement distance to total movement distance in the open field test were the main evaluation indicators of the olfactory bulbectomy model. Consistent with literature reports, the inventors found that the total movement distance of mice in the olfactory bulbectomy group was significantly increased compared with that of mice in the sham operation group (A, B in Figure 8).
  • the antidepressant effect of intraperitoneal injection of Lys05 on olfactory bulbectomy mice was evaluated 1 hour after administration. After administration of 30 mg/kg Lys05, the total movement distance of mice in the administration group was significantly decreased compared with that of mice in the control group, and it was dose-dependent (B in Figure 8).
  • the traditional antidepressant imipramine which is slow-acting, is ineffective after a single administration, and even shows a sedative effect in mice in the sham operation group (B in Figure 8).
  • a single administration of the fast-acting antidepressant S-ketamine produced a rapid antidepressant effect similar to Lys05 that shortened the movement distance of mice (C in Figure 8).
  • the olfactory bulb resection model is one of the most accurate models for predicting the onset time of traditional antidepressants. Continuous administration for 5 days can make imipramine exert an antidepressant effect.
  • Lys05 can exert a stronger antidepressant effect in the olfactory bulb resection model, and the traditional antidepressant imipramine also produces a drug effect after chronic administration (D, E in Figure 8).
  • the above results show that Lys05 can produce a rapid onset of antidepressant effects.

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Abstract

La présente invention concerne une utilisation de Lys01 ou d'un sel de celui-ci dans la préparation d'un inhibiteur de canal potassique Kir4.1. Spécifiquement, la présente invention concerne une utilisation de Lys01 ou d'un sel de celui-ci dans la préparation d'un inhibiteur de Kir4.1, une utilisation de Lys01 ou d'un sel de celui-ci dans la préparation d'un médicament pour le traitement et/ou la prévention d'une maladie à l'aide d'un canal potassique Kir4.1 en tant que cible thérapeutique, et une utilisation de Lys01 ou d'un sel de celui-ci dans la préparation d'un diurétique. Le trihydrochlorure Lys05 de Lys01 présenté dans la présente invention a une forte activité inhibitrice du canal potassique Kir4.1, peut supprimer un courant endogène du canal potassique Kir4.1, peut dépolariser le potentiel de la membrane de repos de l'astrocyte et peut atténuer les comportements de type dépressif induits par la surexpression du canal potassique Kir4.1 dans l'habénula latérale de la souris. Le Lys05 exerce un effet antidépresseur rapide sur une série de modèles de dépression chez la souris, tels que le modèle de nage forcée aiguë, le modèle d'alimentation supprimée par la nouveauté, le modèle de dépression induite par la corticostérone et le modèle de bulbectomie olfactive.
PCT/CN2024/078649 2023-03-01 2024-02-27 Utilisation de lys01 ou d'un sel de celui-ci dans la préparation d'un inhibiteur de canal potassique kir4.1 Pending WO2024179420A1 (fr)

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CN202310187958.5A CN118576596A (zh) 2023-03-01 2023-03-01 Lys01或其盐在制备Kir4.1钾离子通道抑制剂中的应用

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103687853A (zh) * 2011-04-29 2014-03-26 宾夕法尼亚大学托管会 新型双氨基喹啉化合物及其制备的药物组合物和它们的用途
CN108853505A (zh) * 2017-05-09 2018-11-23 浙江大学 钾离子通道抑制剂治疗抑郁症的用途和药物组合物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103687853A (zh) * 2011-04-29 2014-03-26 宾夕法尼亚大学托管会 新型双氨基喹啉化合物及其制备的药物组合物和它们的用途
CN108853505A (zh) * 2017-05-09 2018-11-23 浙江大学 钾离子通道抑制剂治疗抑郁症的用途和药物组合物

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MARMOLEJO-MURILLO LETICIA G.; ARéCHIGA-FIGUEROA IVáN A.; MORENO-GALINDO ELOY G.; NAVARRO-POLANCO RICARDO A.; RODRíG: "Chloroquine blocks the Kir4.1 channels by an open-pore blocking mechanism", EUROPEAN JOURNAL OF PHARMACOLOGY, ELSEVIER SCIENCE, NL, vol. 800, 20 February 2017 (2017-02-20), NL , pages 40 - 47, XP029939020, ISSN: 0014-2999, DOI: 10.1016/j.ejphar.2017.02.024 *
ZHOU XIAOYU, ZHAO CHENG, XU HAIYAN, XU YIXIANG, ZHAN LI, WANG PEI, HE JINGYI, LU TAOTAO, GU YUELING, YANG YAN, XU CHANJUAN, CHEN Y: "Pharmacological inhibition of Kir4.1 evokes rapid-onset antidepressant responses", NATURE CHEMICAL BIOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 20, no. 7, 1 July 2024 (2024-07-01), New York, pages 857 - 866, XP093204534, ISSN: 1552-4450, DOI: 10.1038/s41589-024-01555-y *

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