Ca 2+ Medicament for preventing and treating addiction of morphine by formononetin based on CaMK-CREB pathway and application thereof

Abstract

The present application relates to a kind of based on-CaMK-CREB path's formononetin anti morphine addiction drug monomer and its application.Formononetin (Formononetin) as single active ingredient, by inhibiting end flow, down-regulation CaMKII expression and the phosphorylation level of downstream signal molecules (such as PKA, ERK, CREB) regulation, significantly reduce morphine-induced addictive behavior and molecular adaptation.The pharmaceutical composition provided in the present application is standardized, the purity is detected by HPLC≥98%, ensure the batch quality consistency.Combined with behavior (CPP, spontaneous movement test) and molecular biology (qPCR, Western Blot, ELISA) experiment, the cascade effect path that formononetin is revealed by "end flow inhibition→CaMKII phosphorylation block→CREB dependent gene down-regulation", provide new strategy for the development of single-component anti-addiction drug.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of formononetin as a single active ingredient in the preparation of antimorphine addiction drugs, which regulates Ca2+... 2+ The CaMK-CREB signaling pathway inhibits the formation of opioid dependence. Background Technology

[0002] Opioids are commonly used analgesics in clinical practice. They bind to opioid receptors in the brain, blocking pain signals and triggering the release of large amounts of dopamine. Opioid addiction (such as morphine) is a chronic, relapsing brain disorder, the core mechanism of which involves abnormal activation of the mesolimbic dopamine system and alterations in neuroplasticity. Currently, the U.S. Food and Drug Administration has approved three drugs for treating opioid use disorder: methadone, buprenorphine, and extended-release naltrexone. However, these drugs all have the problems of requiring long-term use, high addictiveness, and relapse upon discontinuation. Therefore, the development of novel, safe, and effective anti-addiction drugs is of significant clinical importance.

[0003] Formononetin is a major isoflavone component of traditional Chinese medicines such as Astragalus membranaceus and Rhodiola rosea. Studies have shown that it possesses neuroprotective, anti-inflammatory, and synaptic plasticity-regulating effects. Based on previous research on compound 511 and considering formononetin's high lipid solubility, small molecular weight, and easy membrane transduction, it is speculated that formononetin, the main component of Astragalus membranaceus, plays a crucial role in combating morphine addiction. However, the mechanism of action of formononetin in opioid addiction remains unclear, particularly whether it intervenes in the classic morphine addiction pathway through Ca2+. 2+ There has been no systematic research on the function of signaling pathways regulating addiction-related brain regions such as the ventral tegmental area (VTA) of the midbrain.

[0004] This invention is the first to discover that strychnosine specifically inhibits Ca2+. 2+ The CaMK-CREB signaling pathway significantly reduces morphine-induced addictive behavior and molecular adaptation, providing a new strategy for developing single-component anti-addiction drugs. Summary of the Invention

[0005] This invention provides a drug with gentianin as the sole active ingredient, which exerts its anti-morphine addiction effect through the following mechanism:

[0006] (I) Inhibition of Ca 2+ Influx: Strigolactone can reverse morphine-induced increase in intracellular calcium ion concentration in dopaminergic neurons in the VTA brain region;

[0007] (ii) Downregulation of CaMKII expression: Western Blot results showed that gentianin significantly reduced the upregulation of CaMKII protein expression caused by chronic morphine exposure;

[0008] (III) Regulation of downstream signaling molecules: Stupatin inhibits the phosphorylation levels of PKA, ERK, and CREB, and blocks the transcription of morphine addiction-related genes.

[0009] The pharmaceutical composition comprises the following components:

[0010] Active ingredient: Strigolide, purity ≥98% (HPLC detection)

[0011] Pharmaceutical excipient: Sodium carboxymethyl cellulose (CMC-Na) was selected.

[0012] Preferred dosage: 20-60 mg / kg / day

[0013] Preparation method: Weigh 0.5g of sodium carboxymethyl cellulose and add 100mL of pure water. Stir at 75℃ and speed setting 2 until the solution becomes clear to obtain a 0.5% sodium carboxymethyl cellulose solution. Weigh 25mg, 50mg, and 75mg of gentianin into 50mL EP tubes and label them as 10mg / kg, 20mg / kg, and 30mg / kg FNT, respectively. Add 25mL of 0.5% sodium carboxymethyl cellulose solution to each tube and vortex until homogeneous, forming a milky white turbid liquid.

[0014] Beneficial effects:

[0015] (i) Standardization of ingredients: As the only active ingredient, argentin has a purity of ≥98% as detected by HPLC, which avoids the uncontrollability of the interaction between multiple components in compound drugs and ensures the consistency of quality between batches.

[0016] (II) Multi-dimensional mechanism verification: Combining behavioral (CPP, spontaneous movement test) and molecular biology (qPCR, Western Blot, ELISA) experiments, the mechanism is fully revealed through "Ca 2+ The cascade pathway of "influenza inhibition → CaMKII phosphorylation blockade → CREB-dependent gene downregulation" is followed. Attached Figure Description

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The advantages and / or other aspects will become clearer.

[0018] Figure 1 Changes in conditional position preference scores in morphine addiction were investigated using strychnine as an intervention.

[0019] Figure 2The effect of styracin on the expression levels of different CaMKII isoform mRNAs in the VTA, NAc, and mPFC brain regions of morphine-addicted mice.

[0020] Figure 3 The effect of styracilin on calcium ion signaling in the VTA brain region of morphine-addicted mice.

[0021] Figure 4 The effect of styracin on cAMP expression levels in the VTA brain region of morphine-addicted mice.

[0022] Figure 5 The effect of styracifoliin on the expression of CAMKII and P-CREB.

[0023] Figure 6 Ca in the VTA brain region of the stylosin mouse 2+ The impact on the expression levels of downstream related proteins. Detailed Implementation

[0024] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0025] In this embodiment, the antimorphine addiction drug formulation is: argentin, purity ≥98% (HPLC detection), supplemented with sodium carboxymethyl cellulose as a pharmaceutical excipient.

[0026] The preparation method is as follows: Take 0.5g of sodium carboxymethyl cellulose, add 100mL of pure water, stir at 75℃ until clear, and obtain a 0.5% solution. Weigh 25mg, 50mg, and 75mg of gentianin respectively, place them in 50mL EP tubes, and label them as 10mg / kg, 20mg / kg, and 30mg / kg FNT. Add 25mL of 0.5% sodium carboxymethyl cellulose solution to each, vortex to mix, and form a milky white suspension.

[0027] A mouse model of morphine addiction was established: A mouse morphine conditional place preference (CPP) model was used, and the responses of mice in the black-and-white box experiment were jointly detected to clarify the pharmacodynamic effects of gentianin and observe its influence on anti-morphine addiction in mice, evaluating the improvement of anti-morphine addiction by gentianin. Six-week-old healthy male C57BL / 6J mice, weighing 18-22g, were selected. First, the baseline of conditioned place preference in mice was determined, and then the mouse morphine conditional place preference model was established. Mice with a natural preference for the black box were randomly divided into 6 groups: Control group (intraperitoneal injection of physiological saline 10 mg / kg + oral administration of carboxymethyl cellulose sodium 40 mg / kg), Model group (intraperitoneal injection of morphine 10 mg / kg + oral administration of carboxymethyl cellulose sodium 40 mg / kg), FNT (argentin) 20 group (intraperitoneal injection of morphine 10 mg / kg + oral administration of FNT 20 mg / kg), FNT40 group (intraperitoneal injection of morphine 10 mg / kg + oral administration of FNT 40 mg / kg), FNT60 group (intraperitoneal injection of morphine 10 mg / kg + oral administration of FNT 60 mg / kg), and NLX (naloxone) group (intraperitoneal injection of morphine 10 mg / kg + intraperitoneal injection of NLX 0.1 mg / kg). After 5 days of conditioned place preference training, the mice were tested.

[0028] The non-natural preference box (white box) served as the morphine-supplemented box, while the natural preference box (black box) served as the saline-pairing box. During training, the gates between the boxes were closed, ensuring the animals could only remain in their designated boxes. Administered morphine twice daily, 6 hours apart (morning and afternoon), according to body weight. Both the model group and the treated group received intraperitoneal injections of morphine when placed in the white box, and intraperitoneal injections of saline when placed in the black box. The training period was 30 minutes. During testing, the gates between the boxes were first closed, and each mouse was placed in a neutral compartment for 5 minutes. Then, the gates were opened, allowing the mice to freely move within the conditioned place preference box for 30 minutes. The time spent in the conditioned place preference box was recorded, and the CPP score was calculated. CPP score = Time (white box time) - Time (black box time). Figure 1 result.

[0029] like Figure 1 As shown, where, #### There was a significant difference in CPP scores between the Mod group and the Sal group (P < 0.0001); *** There was a significant difference in CPP scores between the FNT40 group and the Mod group (P < 0.001). **** There was a significant difference in CPP scores between the FNT60 group and the MOD group (P < 0.0001). This indicates that gentianin can effectively intervene in the increase in CPP scores caused by morphine addiction, and there was no significant difference in treatment efficacy between the medium and high dose groups.

[0030] like Figure 2 qPCR test results showed that ### Compared with the control group (Sal), the mRNA expression level of CaMK2A gene in the VTA brain region of the model group (Mod) mice was significantly upregulated (P<0.001), indicating that chronic morphine exposure significantly activated CaMK2A gene transcription in the VTA brain region. *** Compared with the model group, the gentianin intervention group (FNT) showed a significant decrease in the mRNA expression level of the CaMK2A gene (P < 0.001), suggesting that gentianin can effectively inhibit the upregulation of CaMK2A gene expression induced by chronic morphine. Further analysis showed no significant difference in the mRNA expression level of the CaMK2A gene in the NAc and mPFC brain regions (P > 0.05). Furthermore, the mRNA expression levels of CaMK2B, CaMK2D, and CaMK2G genes in the VTA, NAc, and mPFC brain regions were not statistically significant (P > 0.05). These results indicate that the intervention effect of gentianin on chronic morphine addiction is mainly concentrated in the VTA brain region and specifically regulates the expression of the CaMK2A gene.

[0031] like Figure 3 As shown in the figure, after 5 days of conditioned position preference (CPP) training, real-time calcium signal changes in dopaminergic neurons in the VTA brain region of experimental mice were collected during the test period. Compared with the control group (Sal), the model group (Mod) showed significantly upregulated calcium signals in the VTA brain region of mice in the drug-accompanied box, indicating that chronic morphine exposure significantly enhanced calcium ion activity in the VTA brain region of dopaminergic neurons. In the non-drug-accompanied box, the calcium signal in the model group of mice was slightly downregulated compared with the control group, but there was no significant difference. In the gentianone intervention group (FNT), the calcium signal in the VTA brain region of mice was significantly downregulated compared with the model group in the drug-accompanied box, and the calcium signal in the non-drug-accompanied box returned to the control group level. This result is consistent with the behavioral performance in the conditioned position preference experiment. These results indicate that gentianone can significantly inhibit the increase in calcium ion signal levels in the VTA brain region induced by chronic morphine, further confirming its molecular mechanism of intervening in morphine addiction by regulating the calcium signaling pathway.

[0032] like Figure 4 As shown, the cAMP expression level in the VTA region of morphine-addicted mice was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that... ## Compared with the control group (Sal), the cAMP expression level of the model group (Mod) mice was significantly increased (P<0.01), indicating that chronic morphine exposure significantly activated the cAMP signaling pathway in the VTA brain region. *Compared with the model group, the cAMP expression level in the gentianin intervention group (FNT) was significantly reduced (P < 0.05), suggesting that gentianin can effectively inhibit the upregulation of the cAMP signaling pathway induced by chronic morphine. These results indicate that gentianin intervenes in the molecular adaptation process related to morphine addiction by regulating the cAMP signaling pathway, further supporting the molecular mechanism of its anti-addiction effect.

[0033] like Figure 5 Immunohistochemical results showed that ## Compared with the control group (Sal), the number of CaMKII positive cells in the VTA region of the model group (Mod) mice was significantly increased (P<0.01), indicating that chronic morphine exposure significantly upregulated the expression of CaMKII protein in the VTA brain region. ** Compared with the model group, the number of CaMKII-positive cells in the gentianin intervention group (FNT) was significantly reduced (P < 0.01), suggesting that gentianin can effectively inhibit the upregulation of CaMKII protein expression induced by chronic morphine. Immunofluorescence assay results showed that... #### Compared with the control group (Sal), the number of p-CREB immunopositive cells in the VTA region of the model group (Mod) mice was significantly increased (P<0.0001), indicating that chronic morphine exposure significantly activated the CREB signaling pathway in the VTA brain region. **** Compared with the model group, the number of p-CREB immunopositive cells was significantly reduced in the gentianin intervention group (FNT) (P < 0.0001), suggesting that gentianin can effectively inhibit the upregulation of p-CREB protein expression induced by chronic morphine. These results indicate that gentianin intervenes in the molecular adaptation process related to morphine addiction by inhibiting the expression of CaMKII and p-CREB proteins, further supporting the molecular mechanism of its anti-addiction effect.

[0034] like Figure 6 As shown, Western blotting analysis of protein expression levels in the VTA region of morphine-addicted mice revealed that... # Compared with the control group (Sal), the expression levels of CaMKII and p-CREB proteins in the VTA region of the model group (Mod) mice were significantly upregulated (P<0.05); ## Compared with the control group (Sal), the expression level of p-ERK protein in the VTA region of the model group (Mod) mice was significantly increased (P<0.01); ### Compared with the control group (Sal), the expression level of p-PKA protein in the VTA region of the model group (Mod) mice was significantly increased (P<0.001); ** Compared with the model group, the expression levels of p-PKA and p-CREB proteins in the strychnosine intervention group (FNT) were significantly reduced (P < 0.01).* Compared with the model group, the expression levels of CaMKII and p-ERK proteins were significantly reduced in the genistein intervention group (FNT) (P < 0.05). This suggests that genistein inhibits the expression of CaMKII and p-ERK proteins. 2+ Activation of the CaMKII-CREB signaling pathway effectively reversed the molecular adaptation process induced by chronic morphine. These results indicate that gentianin regulates CaMKII-CREB signaling pathways. 2+ The CaMKII-CREB signaling pathway intervenes in molecular adaptation processes related to morphine addiction, further supporting the molecular mechanism of its anti-addiction effect.

Claims

1. A drug monomer for treating morphine addiction, characterized in that, It contains strychnosine as the sole active ingredient, with a purity of ≥98% (HPLC detection).

2. The pharmaceutical composition according to claim 1, characterized in that, The strychnosin inhibits Ca... 2 + Influx, downregulation of CaMKII expression and regulation of phosphorylation levels of PKA, ERK, and CREB, reducing morphine-induced addictive behavior and molecular adaptation.

3. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition can be combined with a pharmaceutically acceptable carrier to form a solution.

4. The pharmaceutical composition according to claim 1, characterized in that, The preferred dosage of the pharmaceutical composition is 20-60 mg / kg / day.

5. The method for preparing the pharmaceutical composition according to claim 1, characterized in that, Includes the following steps: (a) Weigh 0.5g of sodium carboxymethyl cellulose, add 100mL of pure water, stir at 75℃ until clear, and obtain a 0.5% sodium carboxymethyl cellulose solution; (ii) Weigh 25mg, 50mg and 75mg of gentianin respectively, add 25mL of 0.5% sodium carboxymethyl cellulose solution, vortex and mix well to form a milky white suspension.

6. The pharmaceutical composition according to claim 5, characterized in that, It is used to treat opioid addiction, especially morphine addiction.

Images