Application of medicinal preparation in preparation of medicine for preventing formation of tobacco addiction and / or improving tobacco addiction

Abstract

The invention provides an application of a medicinal preparation in preparation of a medicine for preventing formation of tobacco addiction and / or improving tobacco addiction. The medicinal preparation comprises the following components: 0.1-100 mg / mL of tetrahydroberberine, 0-0.05 g / mL of an acidity regulator, 0-0.5 g / mL of a sweetening agent and 0-0.1 g / mL of an organic solvent. The pharmaceutical preparation can prevent the formation of the tobacco addiction, effectively relieve the tobacco addiction and help smoking cessation, and the pharmaceutical preparation has clear action mechanisms for preventing the formation of the tobacco addiction and improving the tobacco addiction and does not have obvious side effects.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of smoking cessation, and particularly relates to application of a pharmaceutical preparation in preparation of a medicine for preventing the formation of a smoking habit and / or improving a smoking habit. BACKGROUND

[0002] Nicotine dependence (smoking habit) is a chronic and recurrent brain disease, and its core mechanism lies in that nicotine continuously activates the brain reward pathway by acting on the nicotinic acetylcholine receptor (nAChR) in the central nervous system, especially the alpha4beta2-nAChR subtype rich in the ventral tegmental area and the nucleus accumbens, thereby leading to strong psychological dependence and physical dependence. At present, the main smoking cessation intervention methods in the world mainly include nicotine replacement therapy (NRT), non-nicotine prescription drugs (such as varenicline and bupropion) and behavior cognitive therapy. However, these methods have limitations: NRT can only alleviate withdrawal symptoms, and the relapse rate is high; although varenicline and the like are partial agonists of alpha4beta2-nAChR, they have clear side effects such as nausea, abnormal dreams and neuropsychiatric symptoms, which limit their long-term and extensive application.

[0003] Moreover, most of the existing intervention strategies start after an individual has formed a regular smoking habit or even developed dependence, which belongs to "after-the-fact remedy". For high-risk groups (such as adolescents and individuals with a family history) or individuals in the early stage of smoking who have not yet formed a stable neural dependence, there is a lack of safe, effective and easily accepted early preventive intervention methods. The remodeling effect of nicotine on the brain reward system starts rapidly, and once a stable neural adaptation is formed, it is extremely difficult to reverse.

[0004] Therefore, developing an anti-nicotine dependence drug with high efficiency, safety and dual effects of prevention and treatment is still a key problem to be broken through in the current public health field. SUMMARY

[0005] To solve the problems and deficiencies in the prior art, the application provides application of a pharmaceutical preparation in preparation of a medicine for preventing the formation of a smoking habit and / or improving a smoking habit, which can prevent the formation of a smoking habit, effectively alleviate a smoking habit and help quit smoking, and the mechanism of preventing the formation of a smoking habit and improving a smoking habit is clear, and there is no obvious side effect.

[0006] According to a first aspect of the application, application of a pharmaceutical preparation in preparation of a medicine for preventing the formation of a smoking habit and / or improving a smoking habit is provided, and the pharmaceutical preparation comprises the following components: tetrahydroberberine 0.1-100 mg / mL, acidity regulator 0-0.05 g / mL, sweetener 0-0.5 g / mL and organic solvent 0-0.1 g / mL.

[0007] Tetrahydroberberine (THB) belongs to tetrahydroisoquinoline alkaloids, which is derived from the rhizome of the plant Corydalis tuber, and can also be synthesized by hydrogenation of berberine. Compared with berberine, THB also has a variety of significant biological activities. Current research has found that THB has the effects of anti-hypertension, anti-arrhythmia, anti-fibrillation, anti-acute myocardial infarction, and treatment and protection of ischemia-reperfusion injury. It has also been reported that THB has the effects of anti-oxidation and regulating gastrointestinal function. However, there is no related report about the effect of tetrahydroberberine in preventing the formation of smoking addiction and improving smoking addiction in the prior art.

[0008] The pharmaceutical preparation provided by the present application uses tetrahydroberberine as the active ingredient, and the use amount of the active ingredient is clear, and the reasonable use amount of the acidity regulator, sweetener, organic solvent and the like is further limited. Therefore, compared with conventional smoking cessation traditional Chinese medicine, the present application directly uses tetrahydroberberine in traditional Chinese medicine plants as the active ingredient, and the use amount is clear, realizing the qualitative change from the traditional Chinese medicine "mixture" to the "clear active substance", which fundamentally distinguishes the mechanism of the present pharmaceutical preparation from the traditional Chinese medicine compound with complex components and unclear target. At the same time, it can significantly improve the drug experience and improve the long-term medication compliance of patients (especially smokers) by adjusting the taste (sweetener) and stability (acidity regulator) of the auxiliary materials. The organic solvent can also better dissolve the raw materials and improve the use effect of the tetrahydroberberine active ingredient.

[0009] Further experiments have proved that the pharmaceutical preparation provided by the present application can specifically act on the key target point of α4β2-nAChR (i.e. effectively inhibit the nicotine receptor α4β2-nAChR), and it is a non-competitive inhibitor with high subtype selectivity, and does not affect α7-nAChR and α1-nAChR. The above shows that the mechanism of the pharmaceutical preparation provided by the present application in preventing the formation of smoking addiction and improving smoking addiction is clear, so as a drug, it will not produce unpredictable nervous system or muscle side effects due to affecting α7-nAChR (related to learning and memory, neuroinflammation) or α1-nAChR (related to muscle contraction), that is, the overall side effects are smaller.

[0010] Secondly, the pharmaceutical preparation provided by the present application can inhibit the excitation of VTA region of nicotine caused by the dopaminergic neurons, and the injection of the pharmaceutical preparation alone can cause the increase of the discharge activity of the dopaminergic neurons. That is, under the whole condition, the pharmaceutical preparation provided by the present application shows two effects, on the one hand, it can eliminate the excitation effect of nicotine; on the other hand, it can increase the discharge activity of the dopaminergic neurons to a certain extent; it conforms to the double target theory of smoking cessation. Specifically, in the presence of nicotine, the pharmaceutical preparation acts as a strong antagonist to block the excessive and abnormal strong activation of nicotine to the dopamine system, which eliminates the core motivation of smoking, that is, the instant pleasure brought by nicotine, so that smoking becomes "no feeling", thereby helping the smoker to break the vicious cycle of "smoking-pleasure-reinforcement". And in the absence of nicotine, the pharmaceutical preparation itself can act as a mild agonist / modulator to slightly increase the baseline activity of the dopamine system, which can alleviate the withdrawal symptoms during the smoking cessation process, such as depression, irritability, inattention, lack of pleasure, etc. It provides a smooth and safe "compensatory support" for the brain, helping the smoker to smoothly pass through the most difficult physiological and psychological withdrawal period without relying on tobacco.

[0011] Thirdly, the pharmaceutical preparation provided by the present application can obviously prevent the formation of smoking addiction, specifically, the pre-use of the pharmaceutical preparation can greatly slow down the formation of position preference, indicating that the pharmaceutical preparation actively intervenes and prevents the consolidation process of nicotine reward memory. Without such intervention, the preference will be normally formed, and the pre-administration prevents this process.

[0012] In summary, the pharmaceutical preparation provided by the present application, which takes tetrahydroberberine as the main active ingredient, has clear application mechanism in smoking cessation, specific action, and small side effects, and can achieve the dual effects of preventing the formation of smoking addiction and improving smoking addiction, which has important practical significance for the prevention and treatment of smoking addiction.

[0013] In the application of the above-mentioned pharmaceutical preparation in the preparation of a drug for preventing the formation of smoking addiction and / or improving smoking addiction, the acidity regulator includes at least one of citric acid, malic acid, tartaric acid, acetic acid, lactic acid, gluconic acid and sorbic acid; the sweetening agent includes at least one of white granulated sugar, glucose, fructose and sucrose; and the organic solvent includes at least one of dimethyl sulfoxide and ethanol. The selection of the above-mentioned acidity regulator, sweetening agent and organic solvent as auxiliary materials can not only fully exert the functions of these substances themselves, such as the stability of the acidity regulator, the inherent bitter taste of the sweetening agent, the sufficient dissolution of the organic solvent to improve the bioavailability and ensure the efficacy, but also can synergistically promote the maximization of the efficacy and reduce the side effects of the drug.

[0014] In the use of the above-mentioned pharmaceutical preparation in the preparation of a drug for preventing the formation of a smoking habit and / or improving a smoking habit, the pharmaceutical preparation comprises the following components: tetrahydroberberine 0.1-100 mg / mL, an acidity regulator 0.01-0.05 g / mL, a sweetener 0.05-0.2 g / mL, and an organic solvent 0.01-0.1 g / mL. In the above-mentioned concentrations of each material, the concentration range (0.1-100 mg / mL) of tetrahydroberberine covers the entire demand window from low-dose prevention to high-dose treatment, ensuring that it can reach an effective blood drug concentration in the body, while avoiding ineffective or excessively high concentrations that cause unintended reactions. The concentration range of the acidity regulator is 0.01-0.05 g / mL, which maintains the stability of the system pH while avoiding excessive acidity that stimulates the mucosa. The concentration range of the sweetener is 0.05-0.2 g / mL, which can effectively mask the bitter taste and improve palatability, and the amount is controlled to be not higher than 0.2 g / mL, which can avoid causing a sharp fluctuation in blood sugar or the risk of tooth decay. The concentration range of the organic solvent is 0.01-0.1 g / mL, which can minimize the potential irritancy and safety risks of the solvent while ensuring the sufficient dissolution of the active ingredient.

[0015] In the use of the above-mentioned pharmaceutical preparation in the preparation of a drug for preventing the formation of a smoking habit and / or improving a smoking habit, the acidity regulator comprises citric acid and sorbic acid; the sweetener comprises fructose and sucrose; and the organic solvent is dimethyl sulfoxide. The use of the above-mentioned acidity regulator and organic solvent can be more conducive to the stability and bioavailability of the active ingredient, and thus can be more conducive to the prevention and treatment of a smoking habit by the pharmaceutical preparation.

[0016] In the use of the above-mentioned pharmaceutical preparation in the preparation of a drug for preventing the formation of a smoking habit and / or improving a smoking habit, the pharmaceutical preparation comprises the following components: tetrahydroberberine 1 mg / mL, citric acid 0.04 g / mL, sorbic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, and dimethyl sulfoxide 0.1 g / mL. In the above-mentioned materials and specific concentrations, each material can better exert a synergistic effect, which is conducive to increasing the efficacy and reducing side effects.

[0017] In the use of the above-mentioned pharmaceutical preparation in the preparation of a drug for preventing the formation of a smoking habit and / or improving a smoking habit, the drug is a mouth spray type drug. The drug of the present application is a mouth spray type drug, which can be used immediately when a patient experiences a smoking habit craving, and can be administered instantly in any situation, greatly eliminating the use obstacles of traditional oral or chewable preparations, and is particularly suitable for use in work, social situations, etc., making the patient more willing to adhere to the standard use for a long time and ensuring the prevention and treatment effect.

[0018] In the use of the above-mentioned pharmaceutical preparation in the preparation of a drug for preventing the formation of a smoking habit and / or improving a smoking habit, the pharmaceutical preparation prevents the formation of a smoking habit and / or improves a smoking habit by inhibiting the expression of α4β2-nAChR.

[0019] In the use of the above pharmaceutical preparation in the preparation of a drug for preventing the formation of a smoking habit and / or improving a smoking habit, the pharmaceutical preparation does not affect the expression of α7-nAChR and α1-nAChR.

[0020] In the use of the above pharmaceutical preparation in the preparation of a drug for preventing the formation of a smoking habit and / or improving a smoking habit, the pharmaceutical preparation prevents the formation of a smoking habit and / or improves a smoking habit by inhibiting the excitatory effect of nicotine on the VTA area of dopaminergic neurons; and / or, the pharmaceutical preparation prevents the formation of a smoking habit and / or improves a smoking habit by affecting the place preference behavior of nicotine.

[0021] In the use of the above pharmaceutical preparation in the preparation of a drug for preventing the formation of a smoking habit and / or improving a smoking habit, the pharmaceutical preparation is used to prevent the formation of a nicotine addiction, and / or the pharmaceutical preparation is used to improve a nicotine addiction.

[0022] In summary, the specific pharmaceutical preparation provided by the present application with tetrahydroberberine as the core active ingredient can effectively prevent the formation of a smoking habit and / or improve (treat) a smoking habit, has a clear mechanism, good drug efficacy, and small side effects, is a new application of a smoking cessation drug, and has very high clinical conversion value and market prospects. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 1 is the electrophysiological experimental results of the inhibitory effect of the pharmaceutical preparation in Example 1 on nicotine; wherein A is the real-time inhibitory trajectory of the pharmaceutical preparation in this example on the nicotine-induced current, and B is the quantitative concentration-effect relationship of the inhibitory effect of the pharmaceutical preparation in this example.

[0024] Figure 2 Figure 2 is the electrophysiological experimental results of the increase in the firing frequency of VTA area dopaminergic neurons by nicotine in Example 1; wherein A is a plot of the instantaneous firing frequency as a function of time, i.e., a time-frequency curve, and B is the normalized firing rate of different groups.

[0025] Figure 3 Figure 3 is the effect of the pharmaceutical preparation on the firing frequency of VTA area dopaminergic neurons caused by nicotine; wherein A is the measured result after treatment with the pharmaceutical preparation, and B is the measured result of prevention by the pharmaceutical preparation.

[0026] Figure 4 Figure 4 is the place preference behavior of nicotine, the pharmaceutical preparation, and the pharmaceutical preparation on nicotine; the sample size N = 10.

[0027] Figure 5For comparison of the situation before and after using the oral spray of the pharmaceutical preparation, A is the comparison of the number of cigarettes smoked per day before and after using the oral spray of the pharmaceutical preparation, B is the average number of cigarettes smoked per day within 31 days before and after using the oral spray of the pharmaceutical preparation, C is the interval between two smoking times before and after using the oral spray of the pharmaceutical preparation, and D is the average interval between two smoking times within 31 days before and after using the oral spray of the pharmaceutical preparation. DETAILED DESCRIPTION

[0028] In order for those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments.

[0029] Embodiment 1 The pharmaceutical preparation used in this embodiment includes the following components: tetrahydroberberine 1 mg / mL, citric acid 0.04 g / mL, sorbic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, and dimethyl sulfoxide 0.1 g / mL.

[0030] The following experiments were carried out using the above pharmaceutical preparation: Experiment 1: Transfection of human nicotine α4β2-nAChR receptors into epithelial cell line (SH-EP1) and patch-clamp whole-cell recording experiment Figure 1 1. The steps of the experiment of transfecting the epithelial cell line (SH-EP1) with human nicotine α4β2-nAChR receptor are as follows: (1) Preparation before transfection Plasmid construction: An expression vector (pcDNA3.1) suitable for mammalian cells is selected, and the coding genes of the target nAChR subunits (α4β2-nAChR) are cloned respectively. Each subunit plasmid should carry a different resistance screening marker (neomycin and hygromycin). The transfection molar ratio of the subunit plasmids is usually set to 1:1.

[0031] Cell culture: SH-EP1 cells are inoculated in a 6-well culture plate coated with 0.01% polylysine, and DMEM high-glucose culture medium containing 10% fetal bovine serum is used. Culture at 37°C, 5% CO2, and transfect when the cell confluence reaches 70-80%.

[0032] (2) Transfection procedure (using Lipofectamine 3000 reagent) Solution preparation: (a) Dilute DNA: Mix 0.8 μg of α4 subunit plasmid and 0.8 μg of β2 subunit plasmid in 125 μL of Opti-MEM serum-free medium. (b) Dilute transfection reagent: Add 3.75 μL of Lipofectamine 3000 reagent to 125 μL of Opti-MEM serum-free medium, and let it stand at room temperature for 5 minutes.

[0033] Complex formation: Diluted DNA solution was mixed with diluted transfection reagent solution, mixed well by pipetting, and incubated at room temperature for 15 minutes to form DNA-liposome complex.

[0034] Transfection: 250 μL of DNA-liposome complex was added dropwise into one well of a 6-well plate containing 1.5 mL of complete medium. The plate was gently shaken horizontally and then returned to the 37°C incubator.

[0035] (3) Post-transfection treatment Culture and expression: Six hours after transfection, the medium was replaced with 2 mL of fresh complete medium. nAChR protein expression was detected 24 hours after transfection, and functional membrane localization expression peaked 48-72 hours after transfection.

[0036] Enhanced expression strategy: When the medium was replaced, a sodium butyrate solution at pH 7.4 was added to a final concentration of 10 mM to promote multi-subunit correct assembly and membrane localization.

[0037] 2. Patch-clamp whole-cell recording experiment steps (to verify α4β2-nAChR function): (1) Preparation before experiment 1) Preparation of recording electrodes: Borosilicate glass capillary with an outer diameter of 1.5 mm and an inner diameter of 0.86 mm was used to pull recording electrodes in two steps on a P-97 puller. The first step parameters were set as: Heat = 510, Pull = 60, Velocity = 80; the second step parameters were: Heat = 510, Pull = 120, Velocity = 80. The diameter of the tip of the pulled electrode was about 1-2 μm.

[0038] 2) Electrode filling with internal solution: The internal solution formula was: potassium chloride 140 mM, ethylene glycol tetraacetic acid 10 mM, HEPES 10 mM, magnesium chloride 2 mM, and the pH was adjusted to 7.3 with potassium hydroxide, and the osmotic pressure was adjusted to 290-300 mOsm / L. After filling, the electrode resistance was controlled within the range of 3-5 MΩ. The electrode was placed in the electrode holder before use, and the tip was tapped to remove air bubbles.

[0039] 3) Cell treatment and perfusion system preparation a. SH-EP1 cells 48 hours after transfection were removed from the incubator, and the medium was discarded.

[0040] b. Add 1 mL of preheated membrane enzyme (0.25%) and digest for 1 minute, then add 1 mL of serum-containing medium to terminate digestion.

[0041] c. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells in 1 mL extracellular solution. The extracellular solution formula is (unit mM): sodium chloride 140 mM, potassium chloride 5 mM, calcium chloride 2 mM, magnesium chloride 1 mM, glucose 10 mM, HEPES 10 mM, and adjust the pH to 7.4 with sodium hydroxide, and the osmotic pressure to 310-320 mOsm / L.

[0042] d. Drop the cell suspension in the center of the perfusion chamber with a diameter of 35 mm, and let the cells adhere for 15 minutes.

[0043] e. Install the chamber on the inverted microscope stage and continuously perfuse the extracellular solution at a flow rate of 2 mL / min through the gravity perfusion system.

[0044] (2) Whole-cell recording mode (i.e. the following whole-cell voltage clamp mode) is established a. Find a single cell (target cell) with good adhesion, full shape, and green fluorescence (indicating successful transfection) under a 40x objective lens.

[0045] b. Manipulate the three-dimensional micro-manipulator to vertically and slowly approach the target cell with the recording electrode filled with internal solution. After the electrode is filled, a +5 mV test voltage pulse is applied on the patch clamp amplifier (such as Multiclamp 700B) to monitor the electrode impedance. The compensation liquid junction potential.

[0046] c. When the electrode tip lightly touches the cell membrane, immediately apply a negative pressure suction of 20-30 cm H2O to form a gigohm (GΩ) seal between the membrane and the electrode tip. The seal resistance reaches more than 1 GΩ to form a cell attachment mode.

[0047] d. Continue to apply a short and strong negative pressure suction or a membrane breakage electric shock (-1 V, 5 ms pulse) to connect the internal solution of the electrode with the intracellular solution, forming a whole-cell recording mode. At this time, the membrane capacitance and series resistance are automatically compensated. The series resistance compensation is up to 80%, and the change of series resistance during recording is less than 20%.

[0048] (3) Drug application and data recording a. In the whole-cell voltage clamp mode, set the cell clamp potential to -40 mV. Use pClamp 11.0 software to collect signals through Digidata 1550B digital-to-analog converter, set the sampling frequency to 10 kHz, and set the filter frequency to 2 kHz.

[0049] b. Preparation and application of nicotine solution: Prepare 3 μΜ nicotine working solution and gradient diluted drug preparation solution (using the drug preparation prepared in this example as solute, prepare drug preparation solution with concentration of 3 μg / mL, 10 μg / mL, 30 μg / mL and 100 μg / mL respectively) with extracellular fluid. Store in the dark at 4°C before use.

[0050] c. Apply drug (diluted drug preparation solution) through an eight-channel fast switching perfusion system (such as Warner Instruments VC-8). Set one of the pipes as extracellular fluid containing 3 μΜ nicotine, and the rest as normal extracellular fluid.

[0051] d. Start the perfusion system to make the cell continuously immersed in normal extracellular fluid (extracellular fluid). After recording a 60-second baseline current, control the valve through software to quickly switch the perfusion fluid to nicotine solution, and the stimulation lasts for 5 seconds. Then switch back to normal extracellular fluid until the current returns to baseline.

[0052] e. Repeat the application of nicotine stimulation 2-3 times for each cell, with an interval of 3 minutes between each stimulation to ensure that the receptor recovers from desensitization.

[0053] (4) Data analysis a. Analyze the recorded current curve using Clampfit 11.0 software.

[0054] b. Measure the amplitude of the inward peak current induced by nicotine, recorded in pA. Calculate the average of 2-3 responses in the same cell.

[0055] c. Calculate the proportion of functional current (peak current > 50 pA) that can be recorded in transfected cells. In this experiment, the α4β2-nAChR subtype is used, and the proportion should be higher than 60%.

[0056] d. Divide the original current amplitude by the membrane capacitance of the cell (unit pF) to get the current density (pA / pF) for standardizing the recording results of different size cells.

[0057] The experimental results are shown in Figure 1 , wherein, Figure 1A is the real-time inhibition trajectory of different drug concentrations on nicotine-induced current in this embodiment. Under a constant clamping voltage (-40 mV), the application of 3 μM nicotine (NIC) induces a stable and continuous inward current (downward deflection, amplitude about 200 pA). In the presence of nicotine, the drug preparation is added in turn at concentrations of 3 μg / mL, 10 μg / mL, 30 μg / mL, and 100 μg / mL. It can be clearly observed that the nicotine current is gradually and reversibly inhibited, and the current amplitude decreases with increasing drug concentration. This directly proves that the drug preparation has a direct inhibitory effect on the function of α4β2-nAChR and has a rapid and concentration-dependent characteristic. The drug preparation inhibits α4β2-nAChR-mediated current, which is a non-competitive inhibition. That is, the drug preparation inhibits α4β2-nAChR with specificity, because the same concentration of the drug preparation does not affect the expression of α7-nAChR and α1-nAChR.

[0058] Figure 1 B is the quantitative concentration-effect relationship of the inhibitory effect of the drug preparation in this embodiment. The horizontal coordinate is the concentration of the drug preparation (μg / mL), which is a nonlinear horizontal coordinate axis measured in log2 units. The vertical coordinate is the normalized current amplitude (%), which is 100% when nicotine acts alone. From Experiment 2: Recording of the firing activity of dopaminergic neurons in the reward area (VTA) of the brain of anesthetized mice using the single-cell extracellular recording method B, it can be seen that as the concentration of the drug preparation increases from 1 μg / mL to 3 mg / mL (3000 μg / mL), the amplitude of the nicotine-induced current is inhibited from 100% to nearly 0%, and the curve presents a typical "S" type (or inverse "S" type) concentration-effect relationship, which is consistent with the characteristics of receptor antagonists in pharmacology. The IC 50 value of the curve is 66.77 μg / mL, which is basically at the middle point of the gradient concentration, indicating that the drug concentration curve is reasonably set and the measurement is complete, providing a more scientific reference range for subsequent dose design (such as the determination of the effective concentration of the drug preparation). It should be noted that IC 50 is a key parameter for measuring the potency of inhibitors, which means that 50% of the maximum current of nicotine is required to inhibit the drug preparation. This value indicates that the drug preparation can effectively inhibit the function of the receptor in the micromolar concentration range.

[0059] The above results show that the drug preparation of this embodiment can effectively inhibit the function of the nicotine receptor subtype α4β2-nAChR. And for the first time, it is found that the drug preparation acts on the α4β2-nAChR.

[0060] Figure 2 Figure 3 The specific method of this experiment is as follows: 1. Surgical preparation and animal fixation a. Select 8-12 weeks old C57BL / 6J male mice, weighing 22-28 grams. Anesthetize the mice by intraperitoneal injection of urethane solution (20% concentration, 1.2 g / kg body weight). Confirm the depth of anesthesia by the loss of toe pinch reflex and stable respiration (about 60-80 times per minute).

[0061] b. Fix the anesthetized mouse on a stereotaxic instrument. Use ear bars to fix the head, ensuring that the bregma and lambda are at the same level. Adjust the temperature-controlled pad to maintain the rectal temperature of the mouse at 37.0 ± 0.5 °C.

[0062] c. Make a 1.5 cm incision along the midline of the skull, bluntly separate the periosteum, and expose the bregma and lambda. Clean the skull surface with 3% hydrogen peroxide solution to clearly show the coronal and lambdoid sutures.

[0063] 2. Craniotomy and electrode positioning a. According to the mouse brain stereotaxic atlas, determine the coordinates of the ventral tegmental area: 3.1 mm posterior to the bregma, 0.5 mm lateral to the midline.

[0064] b. Use a microskull drill to drill a circular bone window with a diameter of about 2 mm at the target coordinates. Continuously drop room temperature normal saline to cool down during operation. Carefully peel off the dura mater with fine forceps to expose the cerebral cortex surface.

[0065] c. Fix the tungsten microelectrode (impedance 2-5 MΩ) on the microelectrode pusher. Slowly lower the electrode vertically at a speed of 5 microns per second, and record the depth as zero when it penetrates the cortex surface.

[0066] 3. Neuron recording and identification a. When the electrode tip reaches the estimated depth of the VTA (4.2-4.8 mm below the skull surface), start the cell search.

[0067] b. Use a bioelectric signal acquisition system (such as Plexon OmniPlex) to set the sampling frequency to 40 kHz and the high-pass filter to 300 Hz. The amplification factor is 10,000, and the noise level is controlled within 15 μV.

[0068] c. Identify dopamine neurons using the following criteria: record spontaneous action potentials, waveform width greater than 1.1 ms (from trough to peak); discharge frequency of 1-5 Hz, irregular or clustered discharge pattern; no excitatory response to noxious stimuli (such as tail clamping).

[0069] 4. Data collection and analysis a. Record stable neuronal firing for 10 minutes as baseline activity. Single neuron signals were isolated from background noise by online sorting, setting threshold at 3 times the standard deviation.

[0070] b. Analyze firing properties using Spike2 software: calculate mean firing rate (unit: Hz); generate inter-spike histogram to analyze firing pattern; record the rising phase time, falling phase time and amplitude of action potential waveform; apply electrophoresis (-10 μA, 10 minutes) to deposit Chicago blue dye at recording site. After the experiment, perfuse and fix the brain, make 50 μm thick coronal sections, and verify the recording site in VTA region by Nissl staining.

[0071] 5. Quality control standards The signal-to-noise ratio of each successfully recorded dopaminergic neuron should be greater than 4:1.

[0072] The stable recording time of a neuron should be no less than 5 minutes, and the coefficient of variation of waveform parameters during this period should be less than 15%.

[0073] A maximum of 3 VTA sites should be recorded from each mouse, and the recording depths should be spaced at least 100 μm apart.

[0074] The experimental results are shown in Figure 2 , Figure 3 As shown in Figure 2 , Figure 2 , pre-injection of the drug preparation can inhibit the excitatory effect of nicotine on dopaminergic neurons in the VTA region, and injection of the drug preparation alone can itself cause an increase in the firing activity of dopaminergic neurons.

[0075] Among them, Figure 3 A is the instantaneous firing rate graph as a function of time, and the time-frequency curve graph shows the continuous process of neuronal firing activity as a function of time. The horizontal axis represents time, and the vertical axis represents the instantaneous firing rate (defined as 100% of the average frequency during the baseline period before injection, to visually display the relative amplitude of the change), which indicates that before the injection marker, the firing rate of the neuron remains at a relatively stable, low baseline level (baseline period), and after the injection marker, the curve representing the firing rate can be observed to rise rapidly and steeply. DA neurons (n=5) indicates that the curve is the average of the recording data of 5 dopaminergic neurons, proving the universality and repeatability of the effect. The above shows that intravenous injection of nicotine can rapidly and strongly activate dopaminergic neurons in the VTA region, maintaining their firing rate at a much higher level than the baseline for a long time.

[0076] Figure 2B is the standardized discharge rate of different groups, used to compare the statistical differences of multiple electrophysiological parameters before and after administration. Among them, the abscissa represents four different electrophysiological parameters for quantifying the characteristics of neuronal discharge activity, which describes the excitability of neurons from different aspects. "Rate" represents the discharge frequency, which refers to the number of action potentials generated by neurons per unit time. "Bursting" represents burst discharge, which refers to the discharge mode of neurons suddenly emitting a series of high-frequency action potentials during the resting period. "SO power" represents slow oscillation power, which specifically refers to the slow oscillation frequency band of local field potential signals. SO is sleep, especially slow wave sleep, and a decrease in this indicator indicates that sleep is disturbed. "CV" represents the coefficient of variation, which is used to quantify the regularity or variability of neuronal discharge intervals. The figure shows that for the Rate group and the Bursting group, the gray column is significantly higher than the white column, which quantitatively proves that nicotine not only increases the overall discharge frequency, but also specifically induces reward-related burst discharge patterns. In the SO power record, the indicator of the nicotine group decreases, indicating that nicotine interferes with the sleep waves of the mouse brain. There is no significant change in the CV group, indicating that nicotine does not change the regularity of neuronal discharge intervals, but only increases the excitability of neurons.

[0077] Figure 3 A is the measured result after treatment with the drug (N = 5). As can be seen from the figure, under no treatment, the baseline data value of all samples is 100. Without using the drug preparation, simple nicotine treatment makes the average value of Rate increase to 132.9, the average value of Bursting increase to 593.0, and the average value of SO decrease to 39.7, which is consistent with the nicotine treatment measurement of B. After treatment with the drug preparation, compared with the nicotine treatment group, the average value of Rate decreases to 102.2, the average value of Bursting decreases to 118.6, and the average value of SO increases to 99.2, basically returning to the baseline data level, indicating that after smoking, using this drug preparation for treatment helps to quit smoking. Experiment 3: Effect of nicotine on the place preference value of mice

[0078] Figure 4 B is the measured result of pre-treatment with the drug (N = 5). As can be seen from the figure, under no treatment, the baseline data value of all samples is 100. First, after using the drug preparation, the values of the four indicators of Rate, Bursting, SO, and CV are basically unchanged, indicating that the drug preparation basically does not affect the sample itself. After pre-treatment with the drug preparation and then nicotine treatment, the above four indicators do not show statistically significant changes compared to the baseline data. It is indicated that after pre-treatment with the drug preparation, the effect of nicotine on the neurons of the sample is inhibited, and this drug preparation helps to prevent smoking addiction.

[0079] ​The above results show that under the whole condition, the pharmaceutical preparation provided by the application shows two effects, on the one hand, it can eliminate the stimulating effect of nicotine, and on the other hand, it itself can prevent the firing activity of dopaminergic neurons caused by nicotine. It conforms to the double-target point smoking cessation theory.

[0080] Figure 4 The specific practice of the experiment is as follows: 1. Experimental device and pretreatment Preparation of conditional position preference box: a standard three-box CPP device (60 cm long x 30 cm wide x 30 cm high) is used, and a liftable partition is provided in the middle of the box.

[0081] The two preferred side boxes are distinguished by completely different sensory clues: A box: the bottom plate is a stainless steel grid (grid spacing 1 cm), and the side wall is a vertical black and white stripe (stripe width 2 cm).

[0082] B box: the bottom plate is a smooth transparent acrylic plate, and the side wall is a horizontal black and white stripe (stripe width 2 cm).

[0083] An infrared camera (sampling frequency 25 Hz) is installed above the box and connected to the ANY-maze behavior tracking system.

[0084] Before the experiment, clean the box thoroughly with 75% ethanol solution to remove odor residues.

[0085] 2. Experimental procedure (1) Pretreatment stage (day 1): Place the mice (C57BL / 6J male, 8 weeks old) in the central area of the CPP box, remove the partition, and allow them to freely explore the entire box for 15 minutes.

[0086] Record the initial residence time of the mice in the two boxes. Eliminate individuals with significant congenital preference for either side (single residence time > 80% of total time).

[0087] According to the initial preference, randomly pair the mice into nicotine control group and other nicotine + pharmaceutical preparation pairing groups, ensuring that there is no statistical difference in the initial preference of all groups.

[0088] (2) Conditioning training stage (days 2-4, once in the morning and once in the afternoon each day): Morning training: restrict the mice to the non-preferred side box for 30 minutes. All mice are injected intraperitoneally with normal saline (0.9% NaCl, injection volume 10 mL / kg body weight).

[0089] Afternoon training: nicotine control group was injected with nicotine hydrogen tartrate solution (dose 0.5 mg / kg, calculated as free base, dissolved in normal saline) intraperitoneally; nicotine + drug formulation pair group was injected with equal volume of nicotine solution and drug formulation solution.

[0090] Immediately after injection, mice were confined to their initial preferred side chamber for 30 minutes.

[0091] Each training interval was at least 4 hours apart, and all training was completed in a fixed time period (9:00-12:00; 14:00-17:00).

[0092] (3) Test phase (day 5): Without drug, mice were placed in the center zone, the partition was removed, and they were allowed to freely explore the entire chamber for 15 minutes.

[0093] ANY-maze system automatically recorded: a. residence time in each chamber; b. shuttle times; c. total distance of movement.

[0094] The test environment maintained a light intensity of 20 lux, and background white noise was controlled at 50 dB.

[0095] 3. Data analysis and judgment criteria CPP score calculation: CPP score = (residence time in the paired side in the test phase - residence time in the paired side in the pre-treatment phase) (unit: seconds).

[0096] Judgment criteria: the CPP score of the nicotine control group was significantly greater than 0, and there was a statistically significant difference compared with the control group, that is, it was determined that nicotine-induced place preference was formed.

[0097] Motor control analysis: compare the total movement distance of the two groups in the test phase to exclude the non-specific movement activation effect of nicotine. Analyze the shuttle times to evaluate the degree of exploration activity of the animals.

[0098] Data processing criteria: exclude abnormal data with total movement distance less than 200 cm or freezing time more than 50% of the total time during the test. Use two-factor repeated measures ANOVA to compare the differences between groups, with a significance level set at p<0.05. The effective sample size of each group was not less than 10 mice.

[0099] The experimental results are shown in Figure 4 , and Experiment 4: Testing the effect of smoking cessation using a mouth spray prepared from the above pharmaceutical preparationIt is known that nicotine can significantly increase the position preference value of mice (135, n=10). But if the drug preparation solution (100 μg / mL, i.e. 1 mg / kg) is added before each injection of nicotine, the formation of nicotine position preference can be greatly slowed down. If the nicotine injection is waited for 4 days, the dependence has already been formed (135, n=10), the drug preparation can remove the position preference behavior of the formed nicotine. The drug preparation has no effect on the position aversion behavior caused by high concentration of nicotine. Here it should be noted that, Figure 5 In the above, NIC represents nicotine, and Drug represents the drug preparation.

[0100] The above results show that in the free-moving mice, low concentration of nicotine can cause position preference behavior, and high concentration of nicotine can cause position aversion behavior. The drug preparation can eliminate the position preference of nicotine but has no effect on the position aversion behavior.

[0101] Figure 5 The specific practice of the experiment is as follows: 30 male subjects, with an average age of 45.1±1.8 years (Mean±SE) and an average smoking age of 20.3±1.7 years. The above drug preparation is used to prepare an oral spray, and the 30 male subjects use the oral spray for 31 days.

[0102] The experimental results are shown in Figure 5 A, 5B, 5C, 5D, wherein, Figure 5 A is a comparison of the number of cigarettes smoked per day before and after using the oral spray; Figure 5 B is the average number of cigarettes smoked per day before and after using the oral spray; ​ C is the interval between two smoking times before and after using the oral spray; ​ D is the average interval between two smoking times after using the oral spray.

[0103] It can be seen that the average number of cigarettes smoked per day before using the oral spray is 15.9±8.2. After using the oral spray, the average number of cigarettes smoked per day is reduced to 7.6±4.9 (p<0.05), and the subjects can endure not to smoke for 2.3±1.4 hours under normal circumstances, but after using the oral spray, the time can be extended to 7.5±4.8 hours (p<0.05).

[0104] The above results show that the drug preparation oral spray can effectively alleviate the craving for cigarettes and help quit smoking.

[0105] Example 2 The difference between this example and Example 1 is that the drug preparation used includes the following components: tetrahydroberberine 1 mg / mL, citric acid 0.04 g / mL, sorbic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, and ethanol 0.1 g / mL.

[0106] Example 3 This example differs from Example 1 in that the pharmaceutical preparation employed includes the following ingredients: tetrahydroberberine 1 mg / mL, malic acid 0.04 g / mL, sorbic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, dimethyl sulfoxide 0.1 g / mL.

[0107] Example 4 This example differs from Example 1 in that the pharmaceutical preparation employed includes the following ingredients: tetrahydroberberine 1 mg / mL, tartaric acid 0.04 g / mL, acetic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, dimethyl sulfoxide 0.1 g / mL.

[0108] Example 5 This example differs from Example 1 in that the pharmaceutical preparation employed includes the following ingredients: tetrahydroberberine 1 mg / mL, lactic acid 0.04 g / mL, gluconic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, dimethyl sulfoxide 0.1 g / mL.

[0109] Example 6 This example differs from Example 1 in that the pharmaceutical preparation employed includes the following ingredients: tetrahydroberberine 1 mg / mL, citric acid 0.04 g / mL, sorbic acid 0.03 g / mL, white sugar 0.1 g / mL, glucose 0.1 g / mL, dimethyl sulfoxide 0.1 g / mL.

[0110] Example 7 This example differs from Example 1 in that the pharmaceutical preparation employed includes the following ingredients: tetrahydroberberine 0.01 mg / mL, citric acid 0.04 g / mL, sorbic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, dimethyl sulfoxide 0.1 g / mL.

[0111] Example 8 This example differs from Example 1 in that the pharmaceutical preparation employed includes the following ingredients: tetrahydroberberine 10 mg / mL, citric acid 0.04 g / mL, sorbic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, dimethyl sulfoxide 0.1 g / mL.

[0112] Example 9 This example differs from Example 1 in that the pharmaceutical preparation employed includes the following ingredients: tetrahydroberberine 50 mg / mL, citric acid 0.04 g / mL, sorbic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, dimethyl sulfoxide 0.1 g / mL.

[0113] Example 10 The present example differs from Example 1 in that the pharmaceutical preparation used comprises the following components: tetrahydroberberine 100 mg / mL, citric acid 0.04 g / mL, sorbic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, dimethyl sulfoxide 0.1 g / mL.

[0114] Comparative Example 1 The present comparative example differs from Example 1 in that the pharmaceutical preparation used comprises the following components: tetrahydroberberine 0.05 mg / mL, citric acid 0.04 g / mL, sorbic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, dimethyl sulfoxide 0.1 g / mL.

[0115] Comparative Example 2 The present comparative example differs from Example 1 in that the pharmaceutical preparation used comprises the following components: tetrahydroberberine 120 mg / mL, citric acid 0.04 g / mL, sorbic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, dimethyl sulfoxide 0.1 g / mL.

[0116] Test Analysis The pharmaceutical preparations of Examples 2-10 and Comparative Examples 1-2 were used to prepare mouth sprays, in a similar manner to that of Experiment 4 in Example 1, and 35 male subjects were asked to use the mouth sprays, and the average number of cigarettes smoked per day after using the mouth sprays was recorded. The results are shown in Table 1.

[0117] Table 1 Average number of cigarettes smoked per day after using the mouth sprays of Examples 1-10 and Comparative Examples 1-2

[0118] As can be seen from Table 1, the average number of cigarettes smoked per day after using the mouth sprays showed a clear tendency to decrease compared to before using the mouth sprays, and too low or too high a concentration of tetrahydroberberine can affect the use effect; too low a concentration can result in insufficient active ingredients, and the effect can decrease; too high a concentration can result in insufficient dissolution of tetrahydroberberine, and the drug absorption effect can be poor, thereby weakening the use effect. Also, when the components of the mouth sprays are changed, the use effect can also be affected, which shows that when the pharmaceutical preparation of the mouth spray comprises "tetrahydroberberine 1 mg / mL, citric acid 0.04 g / mL, sorbic acid 0.03 g / mL, fructose 0.1 g / mL, sucrose 0.1 g / mL, dimethyl sulfoxide 0.1 g / mL", the smoking cessation effect for the subjects is better.

[0119] The above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application is described in detail with reference to the above examples, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently, and these modifications or replacements are within the protection scope of the present application.

Claims

1. The use of a pharmaceutical preparation in the preparation of a drug for preventing and / or improving nicotine addiction, characterized in that, The pharmaceutical preparation comprises the following components: tetrahydroberberine 0.1~100mg / mL, acidity regulator 0~0.05g / mL, sweetener 0~0.5g / mL, and organic solvent 0~0.1g / mL.

2. The use of the pharmaceutical preparation as described in claim 1 in the preparation of a medicament for preventing and / or improving nicotine addiction, characterized in that: The acidity regulator includes at least one of citric acid, malic acid, tartaric acid, acetic acid, lactic acid, gluconic acid, and sorbic acid. The sweetener includes at least one of white sugar, glucose, fructose, and sucrose; The organic solvent includes at least one of dimethyl sulfoxide and ethanol.

3. The use of the pharmaceutical preparation as described in claim 1 in the preparation of a medicament for preventing and / or improving nicotine addiction, characterized in that, The pharmaceutical preparation comprises the following components: 0.1-100 mg / mL of tetrahydroberberine, 0.01-0.05 g / mL of acidity regulator, 0.05-0.2 g / mL of sweetener, and 0.01-0.1 g / mL of organic solvent.

4. The use of the pharmaceutical preparation as described in claim 3 in the preparation of a medicament for preventing and / or improving nicotine addiction, characterized in that: The acidity regulators include citric acid and sorbic acid; The sweeteners include fructose and sucrose; The organic solvent is dimethyl sulfoxide.

5. The use of the pharmaceutical formulation as described in claim 4 in the preparation of a medicament for preventing and / or improving nicotine addiction, characterized in that, The pharmaceutical preparation comprises the following components: 1 mg / mL tetrahydroberberine, 0.04 g / mL citric acid, 0.03 g / mL sorbic acid, 0.1 g / mL fructose, 0.1 g / mL sucrose, and 0.1 g / mL dimethyl sulfoxide.

6. The use of the pharmaceutical preparation as described in claim 1 in the preparation of a medicament for preventing and / or improving nicotine addiction, characterized in that: The drug is a mouth spray.

7. The use of the pharmaceutical preparation according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or improving nicotine addiction, characterized in that: The pharmaceutical preparation prevents nicotine addiction and / or improves nicotine addiction by inhibiting α4β2-nAChR expression.

8. The use of the pharmaceutical preparation according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or improving nicotine addiction, characterized in that: The drug formulation does not affect the expression of α7-nAChR and α1-nAChR.

9. The use of the pharmaceutical preparation according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or improving nicotine addiction, characterized in that: The pharmaceutical preparation prevents and / or improves nicotine addiction by inhibiting the excitatory effect of nicotine on dopaminergic neurons in the VTA region; And / or, the pharmaceutical formulation prevents nicotine addiction and / or improves nicotine addiction by influencing nicotine position preference behavior.

10. The use of the pharmaceutical preparation according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or improving nicotine addiction, characterized in that: The pharmaceutical preparation is used to prevent the development of nicotine addiction, and / or the pharmaceutical preparation is used to improve nicotine addiction.

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