Application of calcium channel CaV3.2 inhibitor in preparation of medicine for treating pain

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of a calcium channel CaV3.2 inhibitor in preparation of a medicine for treating pain. According to the present invention, the compound AM-760 or the pharmaceutically acceptable salt thereof can target the calcium ion channel CaV3.2; a mouse pain model is induced by injecting a 4% formalin solution to the pelma of a mouse, acute pain and persistent pain of the mouse induced by formalin can be remarkably improved by intraperitoneal injection of 10 mg / kg AM-760, and compared with an existing CaV3. Channel inhibitor TTA-A2, the compound has a stronger analgesic effect and has important significance in pain treatment drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and particularly relates to a calcium ion channel Ca V 3.2 Application of inhibitors in the preparation of drugs for treating pain. Background Art

[0002] Pain is common in humans and many animals. Its mechanism is relatively complex and has a wide range of impacts. Chronic pain seriously affects normal work and life, increases the economic burden on patients, and has become a major public health problem. At present, the main drugs used to treat mild pain are non-steroidal anti-inflammatory drugs such as aspirin and acetaminophen that act on nerve endings, but these drugs have insufficient analgesic effects and have side effects on the digestive system; the main drugs used to treat moderate or severe pain are opioid analgesics that act on the central nervous system, such as morphine. However, these drugs have serious risks of abuse and overdose addiction, and also have side effects such as nausea, constipation, and vomiting. Therefore, it is of great significance to develop a new analgesic that acts on other targets, has good efficacy, and has few side effects.

[0003] In recent years, ion channels have shown good analgesic advantages. T-type calcium channels have some unique electrophysiological characteristics such as low voltage activation, voltage-dependent fast inactivation, and slow inactivation after channel closure. Therefore, they play an important regulatory role in the excitability of neurons in both the peripheral and central nervous systems. Studies have shown that the expression and functional changes of T-type calcium channels are closely related to the occurrence and persistence of pain, and can be used as a target for pain treatment. T-type calcium channels include three subtypes, namely Ca V 3.1 Ca V 3.2 and Ca V 3.3, where Ca V 3.2 The role of Ca in pain is unanimously recognized by researchers. V 3.2 is encoded by the CACNA1H gene and is widely expressed in the peripheral nervous system and central nervous system. It regulates neuronal excitability and participates in pain sensation. It plays an important role in somatic pain, visceral pain and neuropathic pain.

[0004] Therefore, inhibitors targeting CaV3.2 ion channels have the potential to become new analgesics. Summary of the invention

[0005] The present invention aims to provide a compound having analgesic effect and its application. The compound of the present invention has an analgesic effect on calcium channel Ca V 3.2 It has obvious inhibitory effect and analgesic effect, and has the potential to be developed into analgesic drugs.

[0006] The present invention is achieved through the following technical solutions.

[0007] In a first aspect, the present invention provides a calcium ion channel Ca V 3.2 Use of an inhibitor in the preparation of a drug for treating pain, wherein the inhibitor is a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0008]

[0009] It has analgesic effect. The name of the above compound is 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol, with molecular formula C 21 H 27 NO, with a molecular weight of 309.45 g / mol, belongs to the piperidinol class of compounds. The compound targets the calcium ion channel Ca V 3.2.

[0010] Preferably, the compound is:

[0011]

[0012] Preferably, the active ingredient is administered in a dose of 50 ng / kg to 50 mg / kg.

[0013] Furthermore, the administration method is oral administration or injection.

[0014] In a second aspect, the present invention provides a pharmaceutical composition comprising the compound according to the first aspect or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0015] Preferably, the pharmaceutically acceptable carrier, diluent or excipient comprises water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oil, polyalkylene glycol.

[0016] Beneficial effects of the present invention:

[0017] The present invention provides a calcium ion channel Ca V 3.2 Application of inhibitors in the preparation of drugs for the treatment of pain. The compound 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol is a calcium channel inhibitor. V 3.2 has a significant inhibitory effect. The compound can effectively reduce the pain of the mouse formalin model and can be used as an analgesic drug. V 3. Compared with the channel inhibitor TTA-A2, it has a stronger analgesic effect and is of great significance in pain treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results show that the model compound of the present invention has an effect on TTA-A2 and Ca V 3.2 Diagram of the inhibitory effect of the channel.

[0019] Figure 2 The 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol compound of the present invention is shown to be effective for Ca V 3.2 Diagram of the inhibitory effect of the channel.

[0020] Figure 3 The figure shows the analgesic effect of the 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol compound of the present invention on the formalin-induced pain model in mice. DETAILED DESCRIPTION

[0021] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0022] The 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol (ID No.: AM-760 / 12201013) described in the present invention is Figure 1 The compounds shown are not particularly limited, and those skilled in the art can prepare them according to common knowledge in the art, or obtain the compounds described in the present invention from commercial sources.

[0023] The present invention will be described in detail below through examples.

[0024] In the following examples, unless otherwise specified, all reagents and materials used are commercially available.

[0025] In the following examples, the compounds were V The effect of channel 3.2 was detected by the FLIPR-TERA instrument (Molecular Devices). FLIPR is a homogeneous, dynamic, cellular level fluorescence detection method that detects changes in intracellular calcium ion concentration in real time based on changes in intracellular fluorescence signals in the presence of calcium-sensitive fluorescent dyes.

[0026] In the following examples, a formalin-induced mouse pain model was used, which can reflect the effects of drugs on acute and persistent pain.

[0027] In the following examples, the experimental ICR mice used were purchased from Beijing Jinmuyang Experimental Animal Breeding Company, male, weighing 18-22 g, and 8 weeks old.

[0028] Effect of model compound TTA-A2 on calcium channel Ca V 3.2 Function

[0029] Stably transfected Ca V 3.2 HEK-293 cells (Cav3.2-HEK293) were seeded into 384-well black bottom transparent culture plates at 20,000 cells / well, 20 μl of culture medium was added to each well, and the plates were incubated at 37°C with 5% CO 2 When the cell confluence in the well reaches 90%, remove the culture medium in the well, add 20 μl of FLIPR calcium assay dye to each well, and incubate at 37°C in the dark for 1 hour.

[0030] TTTA-A2 is a reported specific inhibitor of Cav3.2 channels. In this example, it was used as a positive control to evaluate the pharmacological function of Cav3.2-HEK293 cells in this example. Freshly prepared gradient dilutions of TTA-A2 were added to the compound plate (maximum concentration 25 μM, gradient dilutions were performed in 2-fold equal proportions, a total of 12 concentrations were diluted, and 5 replicates were performed for each concentration); different concentrations of TTA-A2 were added to the 384-well cell plate using the FLIPR automatic sample loading system and incubated for 20 minutes, and then 20 mM of the stimulator CaCl was added. 2 Fluorescence reading was performed according to the parameter settings, with an excitation wavelength of 470-495 nm, an emission wavelength of 515-575 nm, an exposure time of 0.05 s, and data collected once per second for 120 s. Real-time sampling and recording were used to detect the changes in fluorescence signals caused by different concentrations of TTA-A2 over time.

[0031] according to Figure 1 , in 20 mM Ca 2+ Activated Ca V In the case of Cav3.2 channels, the changes in fluorescence signals caused by different concentrations of TTA-A2 inhibiting Cav3.2 channels showed an "inverted S"-shaped increase and decrease curve, and there was an obvious dose-effect relationship. The IC value of the inhibitory effect of TTA-A2 on Cav3.2 channels 50 The value was 0.23 μM. The above results show that the Cav3.2-HEK293 cell model can well reflect the effect of drugs on Ca V 3.2 Channel function, suitable for drug-induced Ca V 3.2 In vitro evaluation of the channel.

[0032] Example 1

[0033] 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol (ID number: AM-760 / 12201013) has an inhibitory effect on calcium channels Ca V 3.2 Function

[0034] Stably transfected Ca V 3.2 HEK-293 cells (Cav3.2-HEK293) were seeded into 384-well black bottom transparent culture plates at 20,000 cells / well, 20 μl of culture medium was added to each well, and the plates were incubated at 37°C with 5% CO 2 When the cell confluence in the well reaches 90%, remove the culture medium in the well, add 20 μl of FLIPR calcium assay dye to each well, and incubate at 37°C in the dark for 1 hour.

[0035] Freshly prepared gradient dilutions of AM-760 were added to the compound plate (maximum concentration 25 μM, gradient dilutions were performed in 2-fold equal proportions, a total of 12 dilutions, and 5 replicates for each concentration); different concentrations of AM-760 were added to the 384-well cell plate using the FLIPR automatic sample loading system and incubated for 20 minutes, and then 20 mM of the stimulator CaCl was added 2 Fluorescence reading was performed according to the parameter settings, with an excitation wavelength of 470-495 nm, an emission wavelength of 515-575 nm, an exposure time of 0.05 s, and data collected once per second for 120 s. Real-time sampling and recording were used to detect the changes in fluorescence signals caused by different concentrations of TTA-A2 over time.

[0036] according to Figure 2 , in 20 mM Ca 2+ Activated Ca V In the case of Cav3.2 channel, the change of fluorescence signal caused by different concentrations of AM-760 inhibiting Cav3.2 channel showed an "inverted S" increase and decrease curve, and there was an obvious dose-effect relationship. IC 50 The value is 0.15 μM.

[0037] The above results indicate that AM-760 is an inhibitor of Cav3.2 channels, and its inhibitory activity on Cav3.2 channels is better than that of the model compound TTA-A2.

[0038] Example 2

[0039] Analgesic activity of 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol (ID number: AM-760 / 12201013) in vivo

[0040] Formalin injection can cause two phases of pain sensation, the first phase is the pain immediately after injection, which lasts about 10 minutes; the second phase starts about 15-20 minutes after injection and lasts for more than 60 minutes. TTA-A2 is an inhibitor targeting Cav3.2 channels. Studies have shown that the compound has analgesic activity and is used as a positive control in this embodiment. During the experiment, mice were randomly divided into a saline control group (Con group), a positive control group (TTA-A2 group) and an experimental compound group (AM-760 group), with 12 mice in each group. The administration method was intraperitoneal injection at a dose of 10 mg / kg, and the control group was injected with an equal volume of saline. The animals were placed in a clean container box and allowed to adapt to the environment for 15-30 minutes. Use a microsyringe to extract the formalin solution, and use a 50μl microsyringe with a 30G needle to extract 20μl. Inject the control solution or experimental drug, and wait for 20 minutes after injection to allow the drug to take effect in the body. The animal was gently grasped, its hind paw stretched, and injected with 4% formalin solution (injected into the central skin of the plantar paw). Immediately after the formalin injection, the animal was returned to the container and a timer was started to record the pain time.

[0041] The results are as follows Figure 3 As shown in the figure, in the first phase, the time for mice in the saline control group (Con), TTA-A2 group and AM-760 group to lick / bite the right hind paw was (59.40±12.79) s, (55.80±16.21) s and (49.53±13.37) s, respectively. The pain scores of mice in the AM-760 group for formalin-induced phase I were significantly different from those in the saline group (P<0.05). In the second phase, the time for mice in the Con group, TTA-A2 group and AM-760 group to lick / bite the right hind paw was (246.73.40±56.92) s, (198.13±43.08) s and (158.40±41.85) s, respectively. The pain scores of mice in the TTA-A2 and AM-760 groups for formalin-induced phase II were significantly different from those in the saline group (P<0.05). The pain response scores of the two phases were added up. The time that mice in the TTA-A2 and AM-760 groups licked / bite their right hind paws was significantly lower than that in the Con group. Compared with the TTA-A2 group, the time that mice in the AM-760 group licked / bite their right hind paws was significantly lower (P<0.05).

[0042] In summary, in the formalin-induced pain model, the 10 mg / kg TTA-A2 group and the AM-760 group had significant analgesic activity, and AM-760 had a rapid onset of action and better analgesic activity than TTA-A2.

[0043] From the results of the above examples, it can be seen that the 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol (ID No.: AM-760 / 12201013) compound of the present invention has an effect on Ca V 3.2 channel has obvious inhibitory effect, and can significantly improve formalin-induced acute pain and persistent pain in mice. The 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol (ID No.: AM-760 / 12201013) compound of the present invention can be used in the preparation of drugs for preventing or treating pain.

Claims

1. A calcium channel Ca V 3.2 Use of an inhibitor in the preparation of a drug for treating pain, characterized in that: The inhibitor is a compound of the following formula or a pharmaceutically acceptable salt thereof:

2. The calcium ion channel Ca according to claim 1 V 3.2 Use of an inhibitor in the preparation of a drug for treating pain, characterized in that: The compound is:

3. The calcium ion channel Ca according to claim 1 or 2 V 3.2 Use of an inhibitor in the preparation of a drug for treating pain, characterized in that: The active ingredient is administered in doses ranging from 50 ng / kg to 50 mg / kg.

4. The calcium ion channel Ca according to claim 3 V 3.2 Use of an inhibitor in the preparation of a drug for treating pain, characterized in that: The administration method is oral administration or injection.

5. A pharmaceutical composition comprising the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutically acceptable carriers, diluents or excipients include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils and polyalkylene glycols.

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