A calcium ion channel Ca V 3.2 Application of inhibitors in the preparation of pain medications

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a calcium ion channel Ca0. V 3.2 Application of inhibitors in the preparation of pain medications. The compound AM-760 or a pharmaceutically acceptable salt thereof described in this invention can target calcium ion channels Ca2+. V 3.2; A mouse pain model was induced by plantar injection of 4% formalin solution. Intraperitoneal injection of 10 mg / kg AM-760 significantly improved formalin-induced acute and persistent pain in mice. This compound is similar to existing Ca... V 3. Compared with the channel inhibitor TTA-A2, it has a stronger analgesic effect and is of great significance in pain treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a calcium ion channel Ca0. V 3.2 Application of inhibitors in the preparation of pain medications. Background Technology

[0002] Pain is ubiquitous in humans and many animals, with complex mechanisms and widespread impact. Chronic pain severely affects normal work and life, increases the economic burden on patients, and has become a major public health problem. Currently, medications for mild pain mainly consist of nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and acetaminophen, which act on nerve endings. However, these drugs have insufficient analgesic effects and cause side effects on the digestive system. Medications for moderate or severe pain mainly consist of opioid analgesics that act on the central nervous system, such as morphine. However, these drugs pose serious risks of abuse, overdose addiction, and side effects such as nausea, constipation, and vomiting. Therefore, developing novel analgesics that act on other targets, have good efficacy, and have fewer side effects is of great significance.

[0003] In recent years, ion channels have demonstrated significant analgesic advantages. T-type calcium ion channels possess unique electrophysiological characteristics, such as low-voltage activation, voltage-dependent rapid inactivation, and slow deactivation after channel closure. Therefore, they play a crucial regulatory role in the excitability of neurons in both the peripheral and central nervous systems. Studies have shown that changes in the expression and function of T-type calcium ion channels are closely related to the occurrence and duration of pain, and can serve as targets for pain treatment. T-type calcium ion channels include three subtypes: Ca2+, Ca2+, and Ca2+. V 3.1, Ca V 3.2 and Ca V 3.3, where Ca V 3.2 Its role in pain is unanimously recognized by researchers. Ca V 3.2 Encoded by the CACNA1H gene, it is widely expressed in the peripheral and central nervous systems, regulates neuronal excitability, participates in pain sensation, and plays an important role in somatic pain, visceral pain, and neuropathic pain.

[0004] Therefore, inhibitors targeting the CaV3.2 ion channel have the potential to become novel analgesics. Summary of the Invention

[0005] The purpose of this invention is to provide a compound with analgesic effects and its applications. The compound in this invention affects calcium channels (Ca). V 3.2 It has a significant inhibitory effect and a marked analgesic effect, and has the potential to be developed into analgesic drugs.

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

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

[0008]

[0009] It has analgesic effects; the compound with the above formula is 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol, and its molecular formula is C. 21 H 27 NO, with a molecular weight of 309.45 g / mol, belongs to the piperidine alcohol class of compounds. This compound targets the calcium ion channel Ca2+. V 3.2.

[0010] Preferably, the compound is:

[0011]

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

[0013] Furthermore, the method of administration is oral 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 includes water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oil, and polyalkylene glycol.

[0016] The beneficial effects of this invention:

[0017] This invention provides a calcium ion channel Ca V 3.2 Application of inhibitors in the preparation of pain medications: Compound 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol is a calcium channel inhibitor that inhibits calcium channel Ca2+. V 3.2 It has a significant inhibitory effect. This compound can effectively alleviate pain in a mouse formalin model and can be used as an analgesic. This compound is similar to existing Ca... V 3. Compared with the channel inhibitor TTA-A2, it has a stronger analgesic effect and is of great significance in pain treatment drugs. Attached Figure Description

[0018] Figure 1 This indicates that the model compound described in this invention is effective against TTA-A2 and Ca. V 3.2 Inhibition diagram of the channel.

[0019] Figure 2 This refers to the effect of the 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol compound of the present invention on Ca. V 3.2 Inhibition diagram of the channel.

[0020] Figure 3 The diagram shows the analgesic effect of the 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol compound described in this invention on a formalin-induced mouse pain model. Detailed Implementation

[0021] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0022] The 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol (ID: AM-760 / 12201013) described in this invention is Figure 1 The compounds shown are not particularly limited, and those skilled in the art can prepare them based on common knowledge in the field, or obtain the compounds described in this invention through commercial sales.

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

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

[0025] In the following examples, the compound reacts with Ca V The function of channel 3.2 is detected using the FLIPR-TERA instrument (Molecular Devices). FLIPR is a homogeneous, dynamic, cellular 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 reflects the effects of the drug on acute and persistent pain.

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

[0028] The model compound TTA-A2 affects the calcium ion channel Ca V The role of 3.2

[0029] Stable transfection of Ca V 3.2 HEK-293 cells (Cav3.2-HEK293) were seeded at 20,000 cells / well into 384-well black-bottomed permeable culture plates, with 20 μl of culture medium added to each well. The plates were cultured at 37°C and 5% CO2 for 24 h. When the cell confluence reached 90%, the culture medium was removed, and 20 μl of FLIPR calcium assay dye was added to each well. The plates were then incubated at 37°C in the dark for 1 h.

[0030] TTTA-A2 is a reported specific inhibitor of the Cav3.2 channel. In this embodiment, it was used as a positive control to evaluate the pharmacological function of Cav3.2-HEK293 cells. Freshly prepared serially diluted TTA-A2 was added to the wells of the compound plate (maximum concentration 25 μM, serially diluted 2-fold, for a total of 12 concentrations, with 5 replicates for each concentration). Different concentrations of TTA-A2 were added to the 384-well cell plate using a FLIPR automated sample loading system and incubated for 20 min. Then, 20 mM of the stimulant CaCl2 was added. Fluorescence was read according to the parameter settings: excitation wavelength 470–495 nm, emission wavelength 515–575 nm, exposure time 0.05 s, data collected once per second for 120 s. The changes in fluorescence signal over time caused by different concentrations of TTA-A2 were detected using real-time sample loading and recording.

[0031] according to Figure 1 At 20mM Ca 2+ Activate Ca V In the case of channel 3.2, the fluorescence signal changes induced by different concentrations of TTA-A2 inhibiting Cav3.2 channel exhibited an inverted S-shaped increase / decrease curve, with a clear dose-response relationship. The IC50 of the inhibitory effect of TTA-A2 on Cav3.2 channel was [not specified]. 50 The value was 0.23 μM. These results indicate that the Cav3.2-HEK293 cell model can effectively reflect the drug's effect on Ca2+. V 3.2 The function of the channel, applicable to drug-induced calcium absorption. V 3.2 In vitro evaluation of the channel.

[0032] Example 1

[0033] 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol (ID: AM-760 / 12201013) inhibits calcium ion channels Ca2+. V The role of 3.2

[0034] Stable transfection of Ca V 3.2 HEK-293 cells (Cav3.2-HEK293) were seeded at 20,000 cells / well into 384-well black-bottomed permeable culture plates, with 20 μl of culture medium added to each well. The plates were cultured at 37°C and 5% CO2 for 24 h. When the cell confluence reached 90%, the culture medium was removed, and 20 μl of FLIPR calcium assay dye was added to each well. The plates were then incubated at 37°C in the dark for 1 h.

[0035] Freshly prepared serially diluted AM-760 was added to the well plates of the compound (maximum concentration 25 μM, serially diluted 2-fold, for a total of 12 concentrations, with 5 replicates for each concentration). Using a FLIPR automated sample loading system, different concentrations of AM-760 were added to 384-well cell culture plates and incubated for 20 min. Then, 20 mM of the stimulant CaCl2 was added. Fluorescence was read according to the parameters set: excitation wavelength 470–495 nm, emission wavelength 515–575 nm, exposure time 0.05 s, data collected once per second for 120 s. Real-time sample loading and recording were used to detect the changes in fluorescence signal over time caused by different concentrations of TTA-A2.

[0036] according to Figure 2 At 20mM Ca 2+ Activate Ca V In the case of channel 3.2, the fluorescence signal changes induced by different concentrations of AM-760 inhibiting the Cav3.2 channel exhibited an inverted S-shaped increase / decrease curve, with a clear dose-response relationship. The IC50 of AM-760's inhibitory effect on the Cav3.2 channel was [not specified]. 50 The value is 0.15 μM.

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

[0038] Example 2

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

[0040] Formalin injection induces two phases of pain: the first phase is immediate pain after injection, lasting approximately 10 minutes; the second phase begins about 15-20 minutes after injection and lasts for more than 60 minutes. TTA-A2 is an inhibitor targeting the Cav3.2 channel, and studies have shown that this compound has analgesic activity; it was used as a positive control in this example. Mice were randomly assigned to 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 was intraperitoneal injection at a dose of 10 mg / kg, while the control group received an equal volume of saline. Animals were placed in clean containers and allowed to acclimatize for 15-30 minutes. Formalin solution was drawn using a microsyringe; 20 μl was drawn using a 50 μl microsyringe with a 30G needle. The control solution or experimental drug was injected, and 20 minutes were allowed for the drug to take effect in the body. Gently grasp the animal, stretch its hind paw, and inject a 4% formalin solution (inject into the central skin of the paw). Immediately after injecting the formalin, return the animal to the container and start timing to record the duration of pain.

[0041] The results are as follows Figure 3 As shown, in phase I, the time for mice in the saline control group (Con), TTA-A2 group, and AM-760 group to lick / bite their right hind paw was (59.40±12.79) s, (55.80±16.21) s, and (49.53±13.37) s, respectively. The AM-760 group showed a significant difference in phase I pain scores induced by formalin compared to the saline group (P<0.05). In phase II, the time for mice in the Con, TTA-A2, and AM-760 groups to lick / bite their right hind paw was (246.73±56.92) s, (198.13±43.08) s, and (158.40±41.85) s, respectively. The TTA-A2 and AM-760 groups showed significant differences in phase II pain scores induced by formalin compared to the saline group (P<0.05). The pain response scores of the two time phases were summed up. The time spent licking / biting the right hind paw of mice in the TTA-A2 group and AM-760 group was significantly shorter than that in the Con group. Compared with the TTA-A2 group, the time spent licking / biting the right hind paw of mice in the AM-760 group was significantly shorter (P<0.05).

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

[0043] As can be seen from the results of the above embodiments, the 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol (ID: AM-760 / 12201013) compound of the present invention has a positive effect on Ca2+. V Channel 3.2 exhibits significant inhibitory activity and can significantly improve formalin-induced acute and persistent pain in mice. The 3-isopropyl-4-methyl-2,6-diphenyl-4-piperidinol (ID: AM-760 / 12201013) compound described in this invention can be used in the preparation of drugs for the prevention or treatment of pain.

Claims

1. A calcium ion channel Ca V 3.2 The use of inhibitors in the preparation of pain medications, characterized in that, The inhibitor is a compound of the following formula or a pharmaceutically acceptable salt thereof: 。

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