Method for screening medicine for relieving morphine tolerance

Abstract

The invention discloses a method for screening a drug for relieving morphine tolerance, which adopts a TRPC5 protein target as a screening condition to screen to obtain a TRPC5 agonist as a drug for relieving morphine tolerance. According to the application disclosed by the invention, the direct association between the TRPC5 protein target spot and the opioid drug tolerance is confirmed, the morphine tolerance is effectively relieved by utilizing the TRPC5 protein agonist, a new target spot for opioid drug tolerance treatment is created, and the strong association between the TRPC5 protein target spot and the morphine tolerance also provides convenience for screening the drug for relieving the morphine tolerance. The combined administration of the TRPC5 protein agonist and morphine can relieve the tolerance of morphine, which prompts that the TRPC5 protein agonist and the opioid drugs can be prepared together to form the analgesic composition, so that the tolerance problem of the traditional opioid analgesic drugs is solved.

Description

technical field

[0001] The invention relates to the technical field of drug screening, in particular to a method for screening drugs for relieving morphine tolerance. Background technique

[0002] Morphine, the representative of opioids, has powerful analgesic effects and is the main drug for the treatment of severe, perioperative and chronic pain (1,2). With the prevalence of pain, the use of morphine in pain management has increased significantly over the past few decades. However, the large and frequent use of morphine will bring serious side effects, such as tolerance, that is, with the repeated use of the drug, the efficacy of the drug decreases, and it is necessary to increase the dosage or shorten the dosing interval to maintain the original efficacy. Morphine tolerance is a challenging clinical problem. There are numerous reports on the mechanisms of morphine tolerance, including down-regulation of opioid receptor numbers (3), changes in inhibitory neurons (4), and...

Examples

Embodiment 1

[0043] Example 1: Determination of the expression level of TRPC5 protein in the formation of morphine tolerance (the western blot experiments in the present invention are all carried out with reference to the "Guidelines for Molecular Biology Experiments"):

[0044] In vivo experiment: ICR mice were randomly divided into groups of 10. Each group was intrathecally injected with 10 μg (administration volume) / 10 μl (administration volume) morphine every day for 7 consecutive days. -L5), the protein expression of TRPC5 was detected by western blot. The experimental results showed that morphine could significantly down-regulate the protein level of TRPC5 (n=6), **p figure 2 shown).

[0045] In vitro experiments: In neuronal cell line (SH-SY5Y), after stimulation with 200 μM final concentration of morphine for 14 h, the protein expression of TRPC5 was detected by western blot. The experimental results showed that morphine could significantly down-regulate neuronal cells Protein leve...

Embodiment 2

[0046] Example 2: TRPC5 protein was used as the target to detect the improvement effect of TRPC5 agonists (Riluzole hydrochoride) on morphine tolerance. By opening the TRPC5 channel, it promotes the influx of calcium ions, thereby enhancing the efflux of GABA, and improving the analgesic effect of morphine.

[0047] Riluzole hydrochloride powder was purchased from MCE with a solubility of 100 mg / ml in DMSO (dimethyl sulfoxide), 50 mg of the powder was dissolved in 500 μl DMSO to give a 100 mg / ml stock solution. The 10 mg / ml morphine stock solution was diluted 10-fold with physiological saline to obtain 10 mg / 10 ml morphine (solution 1). The groups were set as control group (normal saline, saline), morphine group, morphine and TRPC5 agonist (Riluzole hydrochoride) co-administration group, and TRPC5 agonist (Riluzole hydrochoride) group. Wherein, 100 mg / ml riluzole hydrochloride stock solution was dissolved in 10 mg / 10 ml morphine to obtain a solution (solution 2) containing 2 ...

Embodiment 3

[0049]Example 3: Using TRPC5 protein as a target to detect TRPC5 agonist (BTD, 6-methyl-N-[3-(tricyclo[3.3.1.13,7]decyloxy)propyl]-4H-1, Ameliorative effect of 2,4-benzothiadiazine-3-propionamide-1,1-nitrogen dioxide) on morphine tolerance. BTD powder was purchased from TOCRIS, and 10 mg of the powder was dissolved in 217.58 μl DMSO to prepare a BTD stock solution with a concentration of 100 mM, ie, 46 μg / μl. The groups were set as control group (normal saline, saline), morphine group, morphine and TRPC5 agonist (BTD) co-administration group, and TRPC5 agonist (BTD) group. Wherein, 46 μg / μl BTD stock solution was dissolved in 10 mg / 10 ml morphine, to obtain a solution (solution 3) containing 2 μg / 10 μl TRPC5 agonist (BTD) in 10 μg / 10 μl morphine.

[0050] ICR mice were randomly divided into groups of 10. Each group was injected intrathecally with 10 μl of normal saline, 10 μl of solution 1, 10 μl of solution 3, and 10 μl of TRPC5 agonist (2 μg / 10 μl, BTD) for 30 min every day...

technical field

[0001] The invention relates to the technical field of drug screening, in particular to a method for screening drugs for relieving morphine tolerance. Background technique

[0002] Morphine, the representative of opioids, has powerful analgesic effects and is the main drug for the treatment of severe, perioperative and chronic pain (1,2). With the prevalence of pain, the use of morphine in pain management has increased significantly over the past few decades. However, the large and frequent use of morphine will bring serious side effects, such as tolerance, that is, with the repeated use of the drug, the efficacy of the drug decreases, and it is necessary to increase the dosage or shorten the dosing interval to maintain the original efficacy. Morphine tolerance is a challenging clinical problem. There are numerous reports on the mechanisms of morphine tolerance, including down-regulation of opioid receptor numbers (3), changes in inhibitory neurons (4), and...

Examples

Embodiment 1

[0043] Example 1: Determination of the expression level of TRPC5 protein in the formation of morphine tolerance (the western blot experiments in the present invention are all carried out with reference to the "Guidelines for Molecular Biology Experiments"):

[0044] In vivo experiment: ICR mice were randomly divided into groups of 10. Each group was intrathecally injected with 10 μg (administration volume) / 10 μl (administration volume) morphine every day for 7 consecutive days. -L5), the protein expression of TRPC5 was detected by western blot. The experimental results showed that morphine could significantly down-regulate the protein level of TRPC5 (n=6), **p figure 2 shown).

[0045] In vitro experiments: In neuronal cell line (SH-SY5Y), after stimulation with 200 μM final concentration of morphine for 14 h, the protein expression of TRPC5 was detected by western blot. The experimental results showed that morphine could significantly down-regulate neuronal cells Protein leve...

Embodiment 2

[0046] Example 2: TRPC5 protein was used as the target to detect the improvement effect of TRPC5 agonists (Riluzole hydrochoride) on morphine tolerance. By opening the TRPC5 channel, it promotes the influx of calcium ions, thereby enhancing the efflux of GABA, and improving the analgesic effect of morphine.

[0047] Riluzole hydrochloride powder was purchased from MCE with a solubility of 100 mg / ml in DMSO (dimethyl sulfoxide), 50 mg of the powder was dissolved in 500 μl DMSO to give a 100 mg / ml stock solution. The 10 mg / ml morphine stock solution was diluted 10-fold with physiological saline to obtain 10 mg / 10 ml morphine (solution 1). The groups were set as control group (normal saline, saline), morphine group, morphine and TRPC5 agonist (Riluzole hydrochoride) co-administration group, and TRPC5 agonist (Riluzole hydrochoride) group. Wherein, 100 mg / ml riluzole hydrochloride stock solution was dissolved in 10 mg / 10 ml morphine to obtain a solution (solution 2) containing 2 ...

Embodiment 3

[0049]Example 3: Using TRPC5 protein as a target to detect TRPC5 agonist (BTD, 6-methyl-N-[3-(tricyclo[3.3.1.13,7]decyloxy)propyl]-4H-1, Ameliorative effect of 2,4-benzothiadiazine-3-propionamide-1,1-nitrogen dioxide) on morphine tolerance. BTD powder was purchased from TOCRIS, and 10 mg of the powder was dissolved in 217.58 μl DMSO to prepare a BTD stock solution with a concentration of 100 mM, ie, 46 μg / μl. The groups were set as control group (normal saline, saline), morphine group, morphine and TRPC5 agonist (BTD) co-administration group, and TRPC5 agonist (BTD) group. Wherein, 46 μg / μl BTD stock solution was dissolved in 10 mg / 10 ml morphine, to obtain a solution (solution 3) containing 2 μg / 10 μl TRPC5 agonist (BTD) in 10 μg / 10 μl morphine.

[0050] ICR mice were randomly divided into groups of 10. Each group was injected intrathecally with 10 μl of normal saline, 10 μl of solution 1, 10 μl of solution 3, and 10 μl of TRPC5 agonist (2 μg / 10 μl, BTD) for 30 min every day...

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