Application of TRPV1 inhibitor in preparation of medicine for preventing and / or treating pain

Abstract

The invention discloses an application of a TRPV1 (transient receptor potential vanilloid 1) inhibitor in preparation of a medicine for preventing and / or treating pain. According to the technical scheme provided by the invention, the TRPV1 inhibitor is applied to the preparation of the medicine for preventing and / or treating the pain, and side effects causing body temperature change cannot be caused. The TRPV1 inhibitor PSFL2874 can directly act on a TRPV1 channel, has an inhibition effect on the TRPV1 channel, has no side effects such as tolerance addiction and the like, has different degrees of inhibition effects on a TRPV1 receptor at 30 nM to 10 mu M, and is small in dosage and remarkable in analgesic effect when being used for preventing or treating pain; the thermal pain response of a mouse suffering from inflammatory pain can be remarkably relieved through subcutaneous injection of the PSFL2874, it is indicated that the TRPV1 inhibitor provided by the invention has a remarkable analgesic effect, and the analgesic effect of the PSFL2874 is exerted through targeted action on a TRPV1 channel.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to an application of a TRPV1 inhibitor in the preparation of a drug for preventing and / or treating pain. Background Art

[0002] Transient receptor potential vanilloid 1 (TRPV1) is a polymodally activated cation channel widely expressed in nociceptive sensory neurons. It is a non-selective cation channel that can be polymodally activated by acid, noxious heat, and ligands. It is mainly expressed in a subset of small-diameter unmyelinated (C) and medium-diameter myelinated (Aδ) nociceptors. As one of the key polymodal receptors for detecting noxious stimuli, TRPV1 not only causes acute pain, but also persistent pain, especially pain associated with inflammation, and is therefore a potential analgesic target.

[0003] As an agonist of TRPV1 channels, capsaicin can bind to the corresponding sites on the TRPV1 channels to open the channels. However, high-dose or repeated doses of the agonist (capsaicin) can cause the TRPV1 channels to enter a persistent and reversible "desensitization state" that cannot be reactivated by capsaicin or other stimuli, causing the body to desensitize pain. Therefore, it is used to relieve and treat peripheral inflammatory pain and burning pain. The highly efficient TRPV1 agonist resiniferatoxin (RTX) effectively causes desensitization of neurons expressing TRPV1 in the dorsal root and trigeminal ganglia by inducing calcium overload in these neurons. However, this analgesic method is irreversible, so its application is currently limited to intractable cancer pain. However, the injection or consumption of capsaicin can drive a series of thermoregulatory processes, including vasodilation, sweating, and panting, all of which can promote the reduction of core body temperature, so they are usually not administered systemically in large doses.

[0004] Blocking the dysfunctional TRPV1 channels under pathological conditions with antagonists has also been shown to be a feasible way to relieve pathological pain, and several small molecules are currently in clinical trials. However, as a therapeutic target for various pains, TRPV1 agonists and antagonists have caused serious side effects of harmful temperature changes in various animal models and human clinical trials, hindering the development of innovative drugs targeting TRPV1. Summary of the invention

[0005] The main purpose of the present invention is to propose an application of a TRPV1 inhibitor in the preparation of a drug for preventing and / or treating pain, aiming to provide a TRPV1 inhibitor that can avoid the side effect of causing body temperature changes.

[0006] To achieve the above object, the present invention proposes the use of a TRPV1 inhibitor in the preparation of a drug for preventing and / or treating pain, and the structural formula of the TRPV1 inhibitor is as follows:

[0007]

[0008] Optionally, the TRPV1 inhibitor is able to avoid the side effect of causing changes in body temperature.

[0009] Optionally, the TRPV1 inhibitor has an inhibitory effect on the activation of TRPV1 receptors.

[0010] Optionally, the pain is pain caused by activation of TRPV1 channels.

[0011] Optionally, the pain is physiological or pathological pain caused by stimulation of TRPV1 channels.

[0012] Optionally, the drug comprises an active ingredient and a pharmaceutically acceptable excipient, and the active ingredient is a TRPV1 inhibitor.

[0013] The beneficial effects of the present invention are:

[0014] (1) In the technical solution provided by the present invention, the TRPV1 inhibitor is used in the preparation of a drug for preventing and / or treating pain without causing the side effect of changing body temperature.

[0015] (2) The TRPV1 inhibitor PSFL2874 can directly act on the TRPV1 channel and has an inhibitory effect on the TRPV1 channel. The inhibitor has no side effects such as tolerance and addiction. It has different degrees of inhibitory effects on the TRPV1 receptor at 30nM–10μM. When used for the prevention or treatment of pain, the dosage is small and the analgesic effect is significant.

[0016] (3) Subcutaneous injection of PSFL2874 can significantly alleviate the thermal pain response of mice with inflammatory pain, indicating that the TRPV1 inhibitor provided by the present invention has a significant analgesic effect, and the analgesic effect of PSFL2874 is exerted by targeting the TRPV1 channel. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is a graph showing the inhibitory effect of the compound PSFL2874 obtained in Example 2 of the present invention on TRPV1 channels;

[0019] Figure 2This is a graph showing the effect of PSFL2874 obtained in Example 3 of the present invention on the latency of thermal avoidance reaction in C57 mice;

[0020] Figure 3 This is a graph showing the effect of PSFL2874 obtained in Example 4 of the present invention on the latency of thermal avoidance reaction in C57 mice with TRPV1 gene knockout;

[0021] Figure 4 This is a graph showing the effects of PSFL2874 and AMG9810 on the body temperature of C57 mice obtained in Example 5 of the present invention.

[0022] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0023] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0024] In order to prevent or treat pain without causing the side effect of body temperature change, the present invention proposes the use of a TRPV1 inhibitor in the preparation of a drug for preventing and / or treating pain. The structural formula of the TRPV1 inhibitor is as follows:

[0025]

[0026] In the technical solution provided by the present invention, the TRPV1 inhibitor is used in the preparation of drugs for preventing and / or treating pain, without causing side effects of changes in body temperature; the TRPV1 inhibitor PSFL2874 can directly act on the TRPV1 channel and has an inhibitory effect on the TRPV1 channel, and the inhibitor has no side effects such as tolerance and addiction, and has different degrees of inhibitory effect on the TRPV1 receptor at 30nM-10μM. When used for preventing or treating pain, the dosage is small and the analgesic effect is significant.

[0027] Furthermore, the TRPV1 inhibitor can avoid the side effect of causing temperature changes. Experiments have shown that after the administration of the TRPV1 inhibitor PSFL2874, the body temperature of mice did not change during the continuous temperature detection process, indicating that the TRPV1 inhibitor does not affect the body temperature of experimental animals. Therefore, when the TRPV1 inhibitor is used in the preparation of drugs for the prevention and / or treatment of pain, it will not cause side effects of temperature changes such as fever.

[0028] Preferably, the TRPV1 inhibitor has an inhibitory effect on the activation of the TRPV1 receptor. The TRPV1 inhibitor PSFL2874 has different degrees of inhibitory effect on the TRPV1 receptor at 30nM-10μM.

[0029] In one embodiment of the present invention, the pain is caused by activation of TRPV1 channels. TRPV1 channels can be activated in multiple modes such as acid, noxious heat and ligands, and then the channel receptor proteins are widely expressed in the body's peripheral sensory nervous system and central brain nuclei, causing a feeling of pain.

[0030] Preferably, the pain is physiological or pathological pain caused by stimulation of the TRPV1 channel. Acidic substances released by the inflammatory response can stimulate and activate the TRPV1 channel, causing the receptor to produce a physiological pain response.

[0031] In another embodiment of the present invention, the drug includes an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is a TRPV1 inhibitor, and the excipient may be lactose, corn starch, magnesium stearate, etc., which play a role in seasoning, etc. The TRPV1 inhibitor, as an active ingredient, has a specific inhibitory effect on the activation of TRPV1 channels, thereby playing a role in treating pain.

[0032] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0033] Example 1 Synthesis of Compound PSFL2874

[0034] Weigh 1.9383g (10.3mmol) of naphthoic acid and add 50mL of ethanol. Stir at room temperature to obtain a bright yellow solution with a small amount of undissolved solid at the bottom. Add 4mL of 80% hydrazine hydrate, place in an oil bath at 80℃ and reflux for 12 hours, cool, and stand. The upper layer of the reaction bottle is a yellow solution and the lower part is turbid. Centrifuge, wash the separated off-white solid with ethanol, and dry to obtain 1.0980g of hydrazide in the form of a gray-green powder. Weigh 0.2299g (1.14mmol) of hydrazide, add 50mL of methanol, stir at room temperature, and it becomes slightly turbid. Add 4 drops of acetic acid and stir at room temperature. Weigh 0.5580g (3.19mmol) of 2,3-dichlorobenzaldehyde and add it to the above reaction system. The whole solution immediately becomes clear, and then quickly becomes turbid again. Continue stirring at room temperature for 12 hours, stand, centrifuge, wash the solid with methanol and dry, and obtain a white solid, which is PSFL2874.

[0035] Example 2 Inhibitory effect of compound PSFL2874 on TRPV1 receptor

[0036] 1. Experimental Materials

[0037] Plasmid: mTRPV1-pEGFP-N1;

[0038] Cell line: HEK293 (human embryonic kidney cells, from ATCC);

[0039] Transfection reagent: Calcium transfection reagent and common chemical reagents required were purchased from Sigma-Aldrich.

[0040] 2. Experimental equipment

[0041] The patch clamp digital-to-analog converter Digidata1440A, amplifier Axon 200B, membrane current recording software pCLAMP10.6, and analysis software Clampfit10.6 were purchased from Axon; the inverted microscope IX51 and fluorescent light source U-RFL-T were both purchased from 0lympus; the micromanipulator MC1000e was purchased from Siskiyou; and the electrode puller and polisher were from Narishige.

[0042] 3. Experimental steps

[0043] The whole-cell recording mode (clamp voltage is -60mV) was used for recording. The experiment was performed 24 hours after transfection of the corresponding plasmid of mTRPV1 in HEK293 cells. All recordings were performed at 23℃±2℃. The specific steps are as follows:

[0044] (1) First, the electrode is drawn. A borosilicate glass electrode with an outer diameter of 1.5 mm is drawn by a two-step method using a PC-10 electrode drawing instrument. The first step drawing temperature determines the shape of the electrode tip, and the second step drawing temperature determines the water resistance of the electrode (3-5 MΩ is better).

[0045] (2) After the drawn electrode is filled with about 1 / 3 of the Cs internal solution (note that the Cs internal solution should fill the tip of the electrode without bubbles), put the glass electrode containing the internal solution on the Ag / AgCl electrode wire. Be careful not to touch the electrode wire with your fingers to prevent the amplifier from being burned out by excessive current.

[0046] (3) Place the “Y”-shaped tube at the left edge of the low-power microscope field of view to ensure that the flow rate during drug administration is moderate;

[0047] (4) Find a single fluorescently labeled cell in good condition under a microscope, then use a micromanipulator to move the glass electrode so that its tip is lightly pressed against the cell membrane (the resistance increases by about 0.3 MΩ), and apply a negative pressure to the electrode to make the cell membrane sink into the glass electrode port and form an "Ω"-shaped seal with the inner wall of the electrode. At this time, the resistance between the inside of the electrode and the extracellular fluid is above GΩ. Gently apply negative pressure again to break the cell membrane to form a whole-cell mode. Record the current in the gap free mode. The sampling frequency of the cell is 10 kHz and the filter is 2 kHz.

[0048] (5) The drug administration method is through a "Y" shaped tube, relying on gravity to complete the switching between the external solution and the drug, with an interval of 1-2 minutes between two doses.

[0049] During the electrophysiological recording process, capsaicin was first administered through the Y tube to stimulate the TRPV1 channel, and the current value (Imax) was recorded; then capsaicin and different concentrations of PSFL2874 were administered simultaneously, and the current value (I0) was recorded to obtain Figure 1 .

[0050] As can be seen from the figure, I0 / Imax < 1, indicating that at the same concentration, the current stimulated by co-administering the inhibitor PSFL2874 and capsaicin is smaller than the current stimulated by capsaicin alone. The results show that PSFL2874 has an inhibitory effect on TRPV1 channels. And PSFL2874 has different degrees of inhibitory effect on TRPV1 receptors at 30nM-10μM.

[0051] Example 3 Effect of compound PSFL2874 on inflammatory pain in C57BL mice

[0052] 1. Animals: C57BL / 6 (18-22 g) were purchased from Jiangsu Huachuang Company, China. All animals were placed in a controlled environment (23±2°C, 50±10% humidity, 12 h light-dark cycle) with free access to food and water. All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals.

[0053] 2. Experimental equipment: electronic balance, cage ventilation system, micro-syringe and polyethylene experimental cage

[0054] 3. Experimental reagents: PSFL2874, complete Freund's adjuvant (CFA, Sigma-Aldrich, USA)

[0055] 4. Experimental methods:

[0056] The inflammatory pain model of C57BL / 6 mice was induced by injecting 20 μL of complete Freund's adjuvant (CFA, Sigma-Aldrich, USA) into the soles of the mice using a microsyringe; another group of mice was injected with 20 μL of normal saline into the soles of the feet as a blank control group (Vehicle). 72 hours after modeling, the model mice were divided into a model control group (control) and a group receiving different doses of PSFL2874. Blank solvent or different doses (0.0036, 0.036, 0.36 mg / kg) of PSFL2874 were respectively administered by sole injection. At different time points after administration, a photothermal analyzer (37370, Ugo Basile, Italy) was used to measure the latency (paw withdrawal latency, PWL) of the mice's avoidance response to thermal stimulation. The lower the PWL value, the more obvious the mouse's pain perception. The radiant heat was set to 35 and the cutoff time was 20s. The PWL test was repeated 3 times at each time point, and the average of these values ​​was taken for further analysis to obtain Figure 2 .

[0057] The experimental results showed that compared with the model control group, subcutaneous injection of different doses of PSFL2874 significantly prolonged the PWL value of mice with inflammatory pain modeling, and PSFL2874 could alleviate the thermal pain response of mice with inflammatory pain ( Figure 2 ), and as time goes by, within a certain concentration range, the higher the concentration of PSFL2874, the more obvious the effect of relieving the thermal pain response of mice. This shows that PSFL2874 has a significant analgesic effect.

[0058] Example 4 Effect of Compound PSFL2874 on Trpv1 Gene Knockout (Trpv1 - / - ) Effects of inflammatory pain in mice

[0059] Trpv1 was created using the commercial CRISPR / Cas9 method in experimental animals - / -Mice were purchased from Jicui Yaokang. The experimental equipment, reagents and methods were the same as those in Example 2. The thermal avoidance response of mice was recorded. Figure 3 .

[0060] The results showed that in Trpv1 - / - In mice, subcutaneous injection of different doses of PSFL2874 did not change the PWL value of mice with inflammatory pain model ( Figure 3 ), that is, PSFL2874 had no significant effect on inflammatory pain in Trpv1 knockout mice, indicating that the analgesic effect of PSFL2874 is exerted by targeting TRPV1 channels.

[0061] Example 5 Effect of compound PSFL2874 on body temperature of C57BL mice

[0062] 1. Animals: C57BL / 6 (18-22 g) were purchased from Jiangsu Huachuang Company, China. All animals were placed in a controlled environment (23±2°C, 50±10% humidity, 12 h light-dark cycle) with free access to food and water. All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals.

[0063] 2. Experimental equipment: non-contact handheld infrared thermometer (SMART SENSOR, ST9450, China), electronic balance, cage ventilation system, syringe and polyethylene experimental cage.

[0064] 3. Experimental reagents: PSFL2874, AMG9810 (MCE company).

[0065] 4. Experimental methods:

[0066] C57BL / 6 (18-22g) mice with shaved forehead hair were divided into three groups: blank control group (Vehicle), AMG9810 group and PSFL2874 group. They were given normal saline, AMG9810 (4mg / kg) and PSFL2874 (20mg / kg) by intraperitoneal injection (ip), respectively. At different time points after administration, the forehead body temperature was measured using a non-contact handheld infrared thermometer (SMART SENSOR, ST9450, China). Figure 4 .

[0067] The results showed that the positive control drug AMG9810 (TRPV1 channel inhibitor) had a significant effect on the body temperature of mice at a dose of 4 mg / kg, ip; while at a high dose of 20 mg / kg, ip, PSFL2874 did not cause changes in the body temperature of mice during the 60-minute temperature detection process ( Figure 4 ). This indicates that PSFL2874 does not affect the body temperature of experimental animals.

[0068] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. Use of a TRPV1 inhibitor in the preparation of a drug for preventing and / or treating pain, characterized in that: The structural formula of the TRPV1 inhibitor is shown below:

2. The use according to claim 1, characterized in that The TRPV1 inhibitor can avoid the side effect of causing changes in body temperature.

3. The use according to claim 1, characterized in that The TRPV1 inhibitor has an inhibitory effect on the activation of TRPV1 receptors.

4. The use according to claim 1, characterized in that The pain is pain caused by activation of TRPV1 channels.

5. The use according to claim 1, characterized in that The pain is physiological or pathological pain caused by stimulation of the TRPV1 channel.

6. The use according to claim 1, characterized in that The medicine comprises an active ingredient and pharmaceutically acceptable excipients, and the active ingredient is a TRPV1 inhibitor.

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