Application of hydrogenated quinidine in preparation of medicine for treating migraine

Abstract

The invention provides an application of hydrogenated quinidine in preparation of a medicine for treating migraine. It is found for the first time that the hydrogenated quinidine can relieve pain behaviors (including reduction of a pain threshold value, reduction of head scratching behaviors and reduction of freezing time) and photophobia behaviors caused by migraine, and the hydrogenated quinidine has a good treatment or prevention effect on migraine diseases. Hydroquinidine has a great application prospect in preparation of medicines for treating migraine.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of hydroquinidine in preparing medicine for treating migraine. Background Art

[0002] Migraine is a common and disabling disease that affects the quality of life and has affected more than one billion people worldwide. It is characterized by moderate to severe headaches, often accompanied by nausea, vomiting, photophobia, phonophobia and other symptoms.

[0003] The pathogenesis of migraine is complex and not yet fully understood. At present, the main theories about the pathogenesis of migraine include vascular theory, neuron theory (cortical spreading depression theory, trigeminovascular theory), inflammatory mediator theory, etc. Among them, the trigeminovascular theory occupies a dominant position in the pathophysiological mechanism of migraine. This theory believes that the onset of migraine involves three mechanisms: dilation of intracranial and extracranial blood vessels supplying the meninges, release of vasoactive peptides by perivascular nerves causing neurogenic inflammation, and reduced inhibition of central pain transmission. Neurogenic inflammation is the key link, characterized by dilation of meningeal blood vessels, plasma extravasation, platelet activation, and mast cell degranulation.

[0004] Therefore, it is necessary to develop a drug for the treatment of migraine. Summary of the invention

[0005] The present invention aims to provide an application of hydroquinidine in the preparation of a medicament for treating migraine. The present invention firstly finds that hydroquinidine can alleviate the pain behavior and photophobia caused by migraine, and has a good therapeutic or preventive effect on migraine disease.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The invention provides application of hydroquinidine in preparing a medicine for treating migraine.

[0008] Furthermore, the medicine also includes pharmaceutically acceptable excipients and carriers.

[0009] Furthermore, the auxiliary material includes at least one of a filler, a disintegrant, a binder, an excipient, a lubricant, a sweetener or a colorant.

[0010] Furthermore, the carrier includes at least one of physiological saline, phosphate buffer solution, calcium gluconate solution, ethanol and water.

[0011] Furthermore, the dosage form of the drug includes at least one of granules, tablets, pills, capsules, and injections.

[0012] The pharmaceutical dosage form provided by the present invention can be prepared according to conventional methods in the art. For example, in order to prepare hydroquinidine into granules, tablets or pills, etc., various pharmaceutically acceptable excipients known in the art can be used, such as diluents, wetting agents, disintegrants, binders, lubricants, glidants, etc.; diluents can be sucrose, starch, dextrin, microcrystalline cellulose, calcium hydrogen phosphate, calcium carbonate lactose, mannitol, xylitol, etc.; wetting agents can be ethanol, water, isopropanol, etc.; binders can be syrup, starch slurry, acacia gum slurry, dextrin, sodium carboxymethyl cellulose, microcrystalline cellulose, gelatin slurry, methyl cellulose, ethyl cellulose, hydroxypropyl methylcellulose, acrylic resin, carbomer, etc.; disintegrants can be cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, etc.; lubricants and glidants can be stearates, tartaric acid, liquid paraffin, talc, silicon dioxide, polyethylene glycol, etc.

[0013] Furthermore, if necessary, preservatives, fragrances, colorants, flavoring agents or other additives may be added to the pharmaceutical preparations.

[0014] Furthermore, the dosage of the hydroquinidine is 2-5 mg / kg.

[0015] Furthermore, the hydroquinidine relieves migraine-like behaviors, improves migraine-related photosensitization behaviors, and reduces the levels of inflammatory factors associated with migraine.

[0016] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0017] 1. The use of hydroquinidine provided by the present invention in the preparation of a drug for treating migraine has discovered for the first time that hydroquinidine can alleviate pain behaviors (including lowering pain threshold, reducing head scratching behavior, and reducing freezing time) and photophobia caused by migraine, and has a good therapeutic or preventive effect on migraine diseases.

[0018] 2. The present invention has found through research that hydroquinidine regulates neuroinflammatory response by mediating the ELK1 / UBB signaling pathway, thereby achieving the effect of improving migraine. The ELK1 / UBB signaling pathway is used as the target of hydroquinidine in screening for the preparation of drugs for treating migraine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 Molecular docking and interaction diagram of hydroquinidine (HQ) and Ubb.

[0021] Figure 2 The results of evaluating the effect of hydroquinidine (HQ) on improving pain-related behaviors in CSD model mice, where (a) is the periorbital pain threshold, (b) is the number of facial scratching, and (c) is the freezing time.

[0022] Figure 3 Figure 3. Behavioral diagram to evaluate the effect of hydroquinidine (HQ) on improving the photophobia of CSD model mice, where (a) is the time spent in the light chamber, (b) is the movement trajectory of the mouse in the light / dark box, and (c) is a representative trajectory heat map.

[0023] Figure 4 The figure shows the WB method to evaluate the expression of Ub, p-Elk1, Elk1, IL-1β, IL-6 and TNF-α in the cortex of CSD model mice regulated by hydroquinidine (HQ), where (a) is the electrophoresis running result and (b) is the statistical result of protein expression.

[0024] Figure 5 The results of evaluating the effect of hydroquinidine (HQ) on improving pain-related behaviors in IS model mice, where (a) is the periorbital pain threshold, (b) is the number of facial scratching, and (c) is the freezing time.

[0025] Figure 6 To evaluate the effect of hydroquinidine (HQ) on improving the photophobia of IS model mice, (a) is the time spent in the light chamber, (b) is the movement trajectory of the mice in the light / dark box, and (c) is a representative trajectory heat.

[0026] Figure 7 The results of evaluating the effect of hydroquinidine (HQ) on improving pain-related behaviors in NTG model mice, where (a) is the periorbital pain threshold, (b) is the number of facial scratching, and (c) is the freezing time.

[0027] Figure 8 To evaluate the effect of hydroquinidine (HQ) on improving the photophobia of NTG model mice, (a) is the time spent in the light chamber, (b) is the movement trajectory of the mouse in the light / dark box, and (c) is a representative trajectory heat map.

[0028] Fig. 9 The figure shows the expression of Ub, p-Elk1, Elk1, IL-1β, IL-6 and TNF-α in the cortex of IS model mice and NTG model mice evaluated by WB method, where (a) is the electrophoresis running result, (b) and (c) are the statistical results of protein expression. DETAILED DESCRIPTION

[0029] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0030] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0031] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0032] The present application will be described in detail below with reference to embodiments and experimental data.

[0033] Example 1: Hydroquinidine (HQ) improves pain-related behaviors in CSD model mice

[0034] 1. Experimental materials:

[0035] 1.1 Experimental animals

[0036] This study included SPF female (C57BL / 6J) mice weighing 17-20 g. They were purchased from Charles River Laboratories. All animal experiments were conducted in the clean animal laboratory of Renmin Hospital of Wuhan University. During the experiment, mice were free to eat and drink, and the light-controlled laboratory alternated day and night, with a room temperature of 1826°C and a humidity of (55±2)%.

[0037] 1.2 Experimental drugs and reagents

[0038] 3% isoflurane (provided by the Animal Experiment Center of Renmin Hospital of Wuhan University).

[0039] 1.5% isoflurane (provided by the Animal Experiment Center of Renmin Hospital of Wuhan University).

[0040] Hydroquinidine was purchased from Aladdin Biochemical Technology Co., Ltd. with a purity of 98%. When used, it was prepared with phosphate buffered saline (PBS) to 1 mg / ml, and the mass ratio of the effective amount of hydroquinidine to the unit mass of the mouse was preferably 3 mg / kg.

[0041] Potassium chloride (KCl) was purchased from China National Pharmaceutical Group Co., Ltd. When used, it was prepared with phosphate buffered saline (PBS) to 1 mol / l.

[0042] Sodium chloride (NaCl) was purchased from China National Pharmaceutical Group Co., Ltd. (%, %). It was prepared with phosphate buffered saline (PBS) to 1 mol / l before use.

[0043] Phosphate buffered saline (PBS) was purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0044] Sumatriptan (SUMA) is a commonly used drug for the treatment of migraine in clinic, and was purchased from Anhui Jingke Biotechnology Co., Ltd. It was prepared into a 50 mg / ml stock solution with phosphate buffered saline (PBS), and the stock solution was diluted 500 times with PBS when used, and the dosage for mice was 6 ml / kg.

[0045] 1.3 Experimental instruments and equipment

[0046] Disposable syringes, heating pads, brain stereotaxic instrument (Shenzhen Ruiwode Technology Life Science Co., Ltd.), surgical instruments, dental drills and drill bits (Shenzhen Ruiwode Technology Life Science Co., Ltd.), 5 μL microsyringes, electrophysiological recorders, cotton balls, guide cannulas, dental cement, Von Frey fiber filaments (North Coast Medical, USA), light / dark box testing equipment, etc.

[0047] 2. Experimental methods:

[0048] 2.1 Experimental groups: There were 12 mice in each group, including sham-VEH group, CSD-VEH group, CSD-hydroquinidine (CSD-HQ) group, and CSD-sumatriptan (CSD-SUMA) group.

[0049] 2.2 Construction of CSD migraine model

[0050] Mice were anesthetized with 3% isoflurane, maintained with 1.5% isoflurane, and placed on a stereotaxic apparatus with a heating pad to maintain body temperature. A midline skin incision was made on the back of the head to expose the skull. In this model, two holes with a diameter of approximately 2 mm were drilled, and cotton balls soaked in 1 mol / l KCl were placed in the occipital foramen to induce CSD. 5 microliters of KCl solution were added every 15 minutes to keep it moist, and recording electrodes were placed in the prefrontal cortex. CSD signals were recorded using an electrophysiological recorder. 1 mol / l NaCl was used instead of KCl in the control group of mice.

[0051] 2.3 Behavioral testing

[0052] 2.3.1 Pain behavior test: The test was conducted 24 hours after the model was established. The entire assessment was performed by an experimenter who was unaware of the grouping, including spontaneous pain behavior and mechanical hyperalgesia.

[0053] Spontaneous pain-related behaviors include head scratching and freezing behavior. The number of head scratching was recorded as the mouse scratching the scalp and orbital skin area with its forepaws, which is generally considered to be a manifestation of aversive behavior caused by craniofacial pain. Freezing behavior is the most prominent behavior in the CSD model, defined as complete immobility except for breathing. Freezing behavior is expressed as the ratio (%) of freezing time to total recording time.

[0054] Mechanical hyperalgesia was assessed using the Von Frey test. A series of standardized Von Frey filaments, starting with a 0.008 g filament, were applied continuously to the orbital region with an interval of 1–2 min between consecutive stimulations. A positive response was considered if the mouse stroked the face with the ipsilateral forepaw, quickly retracted the head from the stimulation, or vocalized. If the response was negative, the mechanical stimulation was gradually increased until a positive response was obtained, and then the stimulation was reduced until a negative result was observed. The minimum force that produced a positive response was recorded as the threshold force. Each mouse was tested at least three times, and the average threshold was measured.

[0055] 2.3.2 Photophobia behavior test

[0056] Photophobia was assessed using the light / dark box test. The light / dark box consisted of a bright and a dark compartment with a passage between the two compartments (each compartment measured 30 × 30 × 20 cm). The box was placed in a dedicated room where mice could acclimate to the environment before testing. At the beginning of the experiment, mice were placed in the center of the box and allowed to explore freely for a total of 5 minutes. The movement trajectory of the mice was detected. The time the mice spent in the bright compartment was recorded to assess photophobia.

[0057] 2.4 Index detection

[0058] Western blot staining was used to detect the protein expressions of Ub, p-Elk1, Elk1, IL-1β, IL-6 and TNF-α in the mouse cortex, with GAPDH as the internal reference.

[0059] The brief steps of WB are as follows: Use appropriate lysis buffer to lyse tissue and extract protein. Mix the protein sample with protein loading buffer and heat denature it, then separate it by SDS-PAGE gel electrophoresis. Then transfer the protein in the gel to a PVDF membrane, block it with skim milk powder, and incubate it overnight with primary antibodies (Ub, 1:1000; p-Elk1, 1:1000; Elk1, 1:1000; IL-1β, 1:1000; IL-6, 1:1000; TNF-α, 1:1000; GAPDH, 1:5000). Incubate the secondary antibody at room temperature for 1 hour, and finally use chemiluminescence software to expose the protein bands.

[0060] 3. Results

[0061] 3.1 Mouse behavioral results

[0062] like Figure 2-3 As shown, compared with the control group (Sham-VEH), the periorbital pain threshold, facial scratching times and freezing time of CSD model mice were improved after hydroquinidine treatment, showing a pain-relieving effect.

[0063] 3.2 WB method to determine the protein expression in the cortical area of ​​each group of mice

[0064] like Figure 4 As shown, hydroquinidine reduced the expression levels of Elk1, p-Elk1, and inflammatory factors IL-1β, IL-6, and TNF-α induced by CSD, and reversed the decrease in Ub protein caused by CSD.

[0065] Example 2: Hydroquinidine (HQ) improves pain-related behaviors in IS and NTG model mice

[0066] 1. Experimental materials:

[0067] 1.1 Experimental animals

[0068] Same as Example 1.

[0069] 1.2 Experimental drugs and reagents

[0070] Inflammatory soup (containing 1 mM prostaglandin E2, 1 mM serotonin, 1 mM histamine and 1 mM bradykinin) was purchased from Solebao Biotechnology Co., Ltd.

[0071] Nitroglycerin (NTG) was purchased from Henan Runhong Pharmaceutical Co., Ltd.

[0072] The rest is the same as in Example 1.

[0073] 1.3 Experimental instruments and equipment

[0074] Same as Example 1.

[0075] 2. Experimental Methods

[0076] 2.1 Experimental Grouping

[0077] IS migraine mouse model grouping: 6 mice in each group, including sham operation-vehicle (Sham-VEH) group, inflammatory soup-vehicle (IS-VEH) group, IS-hydroquinidine (IS-HQ) group, and IS-sumatriptan (IS-SUMA) group.

[0078] NTG migraine mouse model grouping: 6 mice in each group, including control-vehicle (CON-VEH) group, nitroglycerin-vehicle (NTG-VEH) group, nitroglycerin-sumatriptan (NTG-SUMA) group, and nitroglycerin-hydroquinidine (NTG-HQ) group.

[0079] 2.2 Construction of IS migraine mouse model

[0080] Mice were anesthetized with 3% isoflurane, maintained with 1.5% isoflurane, and placed on a stereotaxic frame with a heating pad to maintain body temperature. A small hole (1 mm in diameter, 1 mm posterior, 1 mm lateral to Bregma) was drilled in the skull, penetrating the skull but leaving the dura intact, and a guide cannula was implanted in the hole and fixed with dental cement for IS delivery, and the wound was sutured postoperatively. 20 μL of IS (1 mM prostaglandin E2, 1 mM serotonin, 1 mM histamine, and 1 mM bradykinin) or saline was delivered into the cannula every day for 7 consecutive days.

[0081] 2.3 NTG-induced migraine model

[0082] The NTG migraine mouse model was established by intraperitoneal injection of NTG (10 mg / kg) once every other day for a total of 5 injections.

[0083] 2.3 Behavioral testing

[0084] Same as Example 1.

[0085] 2.4 Index detection

[0086] Same as Example 1

[0087] 3. Results

[0088] 3.1 Mouse behavioral results

[0089] like Figure 5-6 As shown, the pain-related behaviors (including periorbital pain threshold, facial scratching times, and freezing time) of IS model mice were significantly improved after hydroquinidine treatment.

[0090] like Figure 7-8 As shown, the pain-related behaviors (including periorbital pain threshold, facial scratching times, and freezing time) of NTG model mice were significantly improved after hydroquinidine treatment.

[0091] 3.2 WB method to determine the protein expression in the cortical area of ​​each group of mice

[0092] like Fig. 9 As shown in the results, the Ub protein level in the cortex of IS model mice decreased, and treatment with hydroquinidine and sumatriptan could reverse this phenomenon. In the IS model, hydroquinidine also showed a certain regulatory effect on the expression of Elk1 and inflammatory factors. Similar to the IS model, the decrease in Ub protein levels in the cortex of NTG model mice was also reversed after hydroquinidine treatment. Hydroquinidine also had a significant regulatory effect on the expression levels of Elk1 and inflammatory factors induced by NTG.

[0093] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0094] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of hydroquinidine in the preparation of a medicament for treating migraine.

2. The use according to claim 1, characterized in that: The medicine also includes pharmaceutically acceptable excipients and carriers.

3. The use according to claim 1, characterized in that: The auxiliary materials include at least one of a filler, a disintegrant, a binder, an excipient, a lubricant, a sweetener or a colorant.

4. The use according to claim 1, characterized in that: The carrier includes at least one of physiological saline, phosphate buffer solution, calcium gluconate solution, ethanol and water.

5. The use according to claim 1, characterized in that: The dosage form of the drug includes at least one of granules, tablets, pills, capsules and injections.

6. The use according to claim 1, characterized in that: The dosage of the hydroquinidine is 2-5 mg / kg.

7. The use according to claim 1, characterized in that: The hydroquinidine relieves migraine-like behavior.

8. The use according to claim 1, characterized in that: The hydroquinidine improves the behavior of migraine-related photosensitization.

9. The use according to claim 1, characterized in that: The hydroquinidine reduces the levels of inflammatory factors associated with migraine.

10. Application of ELK1 / UBB signaling pathway as the target of hydroquinidine in screening drugs for the treatment of migraine.

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