Use of the compound SCH 23390 for the preparation of a medicament for the treatment of lead poisoning

Abstract

The application discloses application of a compound SCH23390 in preparation of a medicine for treating lead poisoning, and relates to the technical field of medicines. The application studies the efficiency of the compound SCH23390 in treatment of lead poisoning, first discloses the treatment effect of the compound SCH23390 on lead poisoning, breaks through the limitation of an existing drug target point, directly corrects abnormal nerve signals by inhibiting excessive activation of D1 neurons, avoids side effects of central excessive excitation, and has no significant toxic side effects under a treatment dose. In particular, the compound SCH23390 can significantly relieve specific abnormal behavior phenotypes caused by low-dose lead poisoning, and exhibits better neuroprotective characteristics than traditional chelating agents. The application breaks through the limitation of a single chelating antidote in the traditional field of lead poisoning treatment, and provides a new direction for development of anti-lead toxicity medicines with multiple target points and multiple mechanisms.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, and belongs to the new function of a compound, in particular, the present application relates to the application of a compound SCH23390 in the preparation of a drug for treating lead poisoning. BACKGROUND

[0002] Lead poisoning is a systemic toxic disease caused by excessive accumulation of lead ions (Pb 2+ ) and widely exists in the use of industrial pollution, lead-containing paint and traditional lead-containing products. Lead can accumulate in the central nervous system through the blood-brain barrier, causing irreversible damage to nerve function, manifested as cognitive impairment, abnormal behavior and multi-organ toxicity. It is worth noting that low concentration of lead exposure can cause abnormal increase of exploration behavior and increase of impulsivity. At present, the clinical treatment of lead poisoning mainly depends on chelating agents such as EDTA, DMSA, etc., but it can only partially remove the lead load in the body, and lacks targeted intervention for the abnormal behavior caused by lead exposure, and long-term use may be accompanied by side effects such as kidney damage and electrolyte disturbance, so it is urgent to develop new treatment strategies with detoxification and nerve function repair functions.

[0003] Compound SCH23390 is a dopamine D1 receptor antagonist, and is a small molecule compound with a clear chemical structure. Previous studies have focused on its potential value in central nervous system disease models, but its application in lead poisoning treatment has not been reported.

[0004] Based on this, the present application applies compound SCH23390 as an active ingredient in the study of lead poisoning mouse models, providing a new direction for the study of compound SCH23390 as a drug for treating lead poisoning. SUMMARY

[0005] Therefore, in order to overcome the shortcomings of the prior art, the present application provides the application of a compound SCH23390 in the preparation of a drug for treating lead poisoning, which uses compound SCH23390 as an active ingredient to alleviate the abnormal behavior phenotype caused by low-dose lead poisoning.

[0006] In order to achieve the above purpose, the present application provides the application of a compound SCH23390 in the preparation of a drug for treating lead poisoning; wherein the CAS number of compound SCH23390 is 87075-17-0, and the structural formula is:

[0007]

[0008] Preferably, the compound SCH23390 reverses the lead-induced D1-type neuron signal imbalance by inhibiting the over-activation of NAc D1-type neurons, alleviating the specific abnormal behavior phenotypes caused by low-dose lead poisoning, such as increased abnormal exploration behavior, impulsive behavior, etc.

[0009] Preferably, in the medicine, the dose of the compound SCH23390 is 15 μg / kg per day.

[0010] Illustratively, the present application studies the specific abnormal behavior phenotypes of lead-poisoned mice by compound SCH23390 through the construction of a lead-poisoned mouse model, including behavioral experiments, electrophysiological experiments, real-time recording of changes in neuronal activity during mouse activity by injecting adeno-associated viruses in brain regions, and studying the effects of compound SCH23390 on the behavior of lead-poisoned mice using the open field behavior paradigm.

[0011] Specifically, the construction of the lead-poisoned mouse model includes oral intake of lead ions by pregnant female mice and postnatal offspring, with an intake amount of 50 ppm / day.

[0012] Further, the intervention period includes intraperitoneal injection of compound SCH23390 at a dose of 15 μg / kg after the mice are 7 weeks old.

[0013] As one of the purposes of the application, the present application also provides a medicine for treating lead poisoning, which contains at least the active ingredient of compound SCH23390.

[0014] Preferably, the compound SCH23390 is the only active ingredient of the medicine.

[0015] Preferably, in the medicine, the active ingredient can also be a pharmaceutically acceptable salt, solvate or prodrug of compound SCH23390.

[0016] Preferably, the medicine is an oral preparation, an injection or a transdermal patch.

[0017] Preferably, the medicine further includes any one or a combination of several of the pharmaceutically acceptable auxiliary ingredients, distilled water or physiological saline, etc.

[0018] The beneficial technical effects obtained by the present application are:

[0019] 1. By using the technical solution of the present application, the compound SCH23390 can significantly alleviate the specific abnormal behavior phenotypes (such as abnormal exploration behavior and increased impulsivity) caused by low-dose lead poisoning, and exhibits better neuroprotective properties than traditional chelating agents. This discovery breaks through the limitations of relying on single chelation detoxification in the field of lead poisoning treatment, and provides a new direction for the development of multi-target, multi-mechanism anti-lead toxicity drugs.

[0020] 2. The application discloses a compound SCH23390, and the compound SCH23390 can effectively relieve abnormal behaviors caused by lead poisoning, and provides a reliable method and feasibility reference for a lead poisoning prevention and treatment scheme.

[0021] 3. The application discloses a compound SCH23390, and the compound SCH23390 can effectively relieve abnormal behaviors caused by lead poisoning, and provides a reliable method and feasibility reference for a lead poisoning prevention and treatment scheme. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 FIG. 2 is a comparison diagram of total movement distances of lead exposure group mice and control group mice in an open field in a typical embodiment of the application.

[0023] Figure 2 FIG. 3 is a comparison diagram of movement speeds of lead exposure group mice and control group mice in an open field in a typical embodiment of the application.

[0024] Figure 3 FIG. 4 is a comparison diagram of immobile times of lead exposure group mice and control group mice in an open field in a typical embodiment of the application.

[0025] Figure 4 FIG. 5 is a schematic diagram of mouse virus injection and optical fiber embedding in a typical embodiment of the application.

[0026] Figure 5 FIG. 6 is a schematic diagram of mouse optical fiber sites in a typical embodiment of the application.

[0027] Figure 6 FIG. 7 is a diagram of NAcD1-MSN average activity changes of lead exposure group mice and control group mice in an activity stage in an open field in a typical embodiment of the application.

[0028] Figure 7 FIG. 8 is a diagram of NAcD1-MSN activity average peak values of lead exposure group mice and control group mice in an activity stage in an open field in a typical embodiment of the application.

[0029] Figure 8 FIG. 9 is a diagram of NAcD1-MSN activity peak reaching times from activity starting of lead exposure group mice and control group mice in an open field in a typical embodiment of the application.

[0030] Figure 9 FIG. 10 is a diagram of neuron activity heat maps of lead exposure group mice and control group mice in a movement stage in an open field in a typical embodiment of the application.

[0031] Figure 10 Representative graph of AP of lead-exposed mice and control mice in a typical embodiment of the present application when 150 pA current was injected

[0032] Figure 11 Average AP frequency of lead-exposed mice and control mice in a typical embodiment of the present application when current was injected in sequence from 50 pA to 150 pA in increments of 50 pA.

[0033] Figure 12 Comparison graph of total movement distance in open field of lead + SCH23390 group, lead-exposed group of mice, and control group of mice in a typical embodiment of the present application.

[0034] Figure 13 Comparison graph of movement speed in open field of lead + SCH23390 group, lead-exposed group of mice, and control group of mice in a typical embodiment of the present application.

[0035] Figure 14 Comparison graph of immobility time in open field of lead + SCH23390 group, lead-exposed group of mice, and control group of mice in a typical embodiment of the present application. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the embodiments of the present application more clear, the technical scheme in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some embodiments of the present application, but not all the embodiments of the present application.

[0037] The application of a compound SCH23390 in the preparation of a drug for treating lead poisoning comprises the following steps: firstly, constructing a lead poisoning animal model, then performing behavior experiment, electrophysiological experiment and the like on the animal model, and finally performing intervention experiment by the compound SCH23390, and comparing the intervention experiment with the lead-exposed group without intervention. The total movement distance and movement speed of the mice injected with the compound SCH23390 in the open field are significantly reduced, and the immobility time is obviously increased, which indicates that the injection of the compound SCH23390 relieves the abnormal behavior caused by lead poisoning.

[0038] Specifically, the detection method for the research on the abnormal behavior of lead poisoning mice caused by lead poisoning by constructing a lead poisoning animal model and performing intervention experiment by the compound SCH23390 comprises the following steps:

[0039] (1) Constructing a lead poisoning animal model

[0040] The mother mouse is given lead exposure of 50 ppm from the gestation period until the weaning of the young mice, and then the young mice are continuously given lead exposure of the same concentration. The control group of mice ingests distilled water, and the ingestion mode is oral ingestion.

[0041] At 7 weeks of age, mice were used in an open field behavior paradigm to study the effects of lead exposure on abnormally increased exploratory behavior and impulsive-like behavior.

[0042] (2) Behavioral experiments

[0043] The open field test creates a low-stress, open space to observe the behavior, movement, and exploratory behavior of mice under low stress. The experiment is conducted within a cube-shaped space with dimensions of 50 cm x 50 cm x 50 cm x 50 cm. During the experiment, mice are placed in any corner of the space and allowed to move freely for 10 minutes, while their movement distance, speed, and duration of stillness are recorded.

[0044] (3) Electrophysiological experiments

[0045] At 6 weeks of age, adeno-associated virus rAAV-D1-GFP (injection site: AP: +1.42 mm, ML: ±0.80 mm, DV: -4.50 mm; injection dose: 150 nL) was injected into the brain region of mice with the nucleus accumbens (NAc). After three weeks of full viral expression, electrophysiological experiments were performed on brain slices of lead-exposed mice to record changes in neuronal action potentials (AP).

[0046] (4) Fiber Optic Recording

[0047] Mice were exposed to lead for 6 weeks, and then adeno-associated virus was injected into their NAc brain region.

[0048] rAAV-D1-DIO-GCaMP6m-WPRE-hGH polyA (injection site: AP: +1.42 mm, ML: ±0.80 mm, DV: -4.50 mm; injection dose: 150 nL), followed by fiber optic implantation (implantation site: AP: +1.42 mm, ML: ±0.80 mm, DV: -4.45 mm). Three weeks after full viral expression, behavioral experiments were performed on mice.

[0049] Seven or fourteen days after viral expression, a ceramic fiber optic cannula (200 μm in diameter, 0.37 NA, InperTechnology Co., Ltd.) was implanted at the same site above the NAc. Two weeks later, behavioral tests were performed to record real-time changes in neuronal activity during the mice's activities.

[0050] (5) SCH23390 behavioral intervention

[0051] After mice were exposed to lead for 7 weeks, they were randomly divided into three groups: control group, lead exposure group (Pb group or lead group) and lead + SCH23390 group.

[0052] The effect of compound SCH23390 on the behavior of lead poisoning mice is studied by using the open field behavior paradigm.

[0053] The research results of the above experiments show that the compound SCH23390 can significantly improve the abnormal behavior of lead poisoning mice in the open field, and has a significant improvement ability for the increased exploration behavior and impulsive behavior caused by lead poisoning.

[0054] Further, the compound SCH23390 can reverse the D1-type neuron signal imbalance induced by lead by inhibiting the overactivation of NAc D1-MSN.

[0055] The technical solutions of the present application are further described in detail below through specific examples.

[0056] Example 1

[0057] This example provides an intervention experiment of a compound SCH23390 on an animal model of lead poisoning, and the results show that the compound SCH23390 can significantly improve the abnormal behavior of lead poisoning mice in the open field, and has a significant improvement ability for the increased exploration behavior and impulsive behavior caused by lead poisoning.

[0058] Specifically, the steps of this example include:

[0059] 1. Construction of lead poisoning animal model and intervention of compound SCH23390

[0060] The c57 mother mice are individually raised after pregnancy, and the pregnant mother mice are randomly divided into a control group (ddH2O) and a lead group (50ppm lead acetate). The offspring are exposed to the same concentration after birth, and the behavior test is performed after the offspring are 7 weeks old. The exposure method is oral ingestion. More specifically, the c57 mother mice are exposed to lead from the first day of pregnancy to the offspring being 7 weeks old, and the exposure is through drinking water.

[0061] Before the behavior test, the lead exposure group mice are randomly divided into two groups, one group is injected with compound SCH23390 (physiological saline as solvent, injection volume is 0.25mL / one, concentration is 1.5 μg / mL) 30 min before the behavior test, injection amount is 15 μg / kg body weight, and the control group and the other lead exposure group are injected with the same volume of physiological saline.

[0062] 2. Stereotactic injection and optical fiber embedding

[0063] In order to monitor the calcium signal dynamics of NAc D1-type medium spiny neurons (D1-MSNs) of lead exposure mice in real time, the present application uses a gene coding calcium indicator virus system (rAAV-D1-DIO-GCaMP6m-WPRE-hGH polyA) for neuron-specific labeling.

[0064] The specific steps are as follows: select mice at 35 days after birth, intraperitoneally inject pentobarbital sodium (solvent is normal saline, dose is 60 mg / kg) to achieve deep anesthesia, and then fix on a brain stereotaxic instrument, and ensure the head stability through a tooth bar and an ear bar. The skin is cut along the midline of the skull (using sterilized surgical instruments), and the anterior fontanel and posterior fontanel are fully exposed, and the anterior fontanel is taken as the origin of the three-dimensional coordinate system (AP: 0 mm, ML: 0 mm, DV: 0 mm), and a stereomicroscope is used to assist fine leveling to ensure that the sagittal plane and the coronal plane of the skull are in a horizontal state. According to the target brain region coordinates (NAc core region: AP +1.42 mm, ML ±0.80 mm, DV -4.50 mm), a micro skull drill (diameter 0.9 mm) is used to gently drill a hole to avoid damaging the dura mater and blood vessels; a 33G microsyringe is connected to a nanosyringe pump, and the virus suspension is sucked at a speed of 160 nL / s, and then the virus is slowly injected at a speed of 50 nL / min (total dose 160 nL), and after the injection is completed, it is left for 10 minutes, and the needle is withdrawn after the virus particles are fully diffused; then the tip of the ceramic optical fiber (diameter 200 μm) is positioned 0.1 mm above the injection site (DV -4.45 mm), and the optical fiber clamp is slowly implanted, and the dental cement and stainless steel skull nail are used for double fixation to ensure the stability of the optical fiber-brain tissue interface.

[0065] Referring to Figure 4 , a schematic diagram of virus injection and optical fiber embedding in mice is shown; Figure 5 , a schematic diagram of the optical fiber site brain slice is shown, and it can be seen from the diagram that the virus injection site is correct. After suturing the skin incision, the mouse is placed in a constant temperature recovery cabin (37°C) until the spontaneous activity is restored. The virus expression period is set to 3 weeks, and then the calcium signal dynamics in the open field experiment are recorded synchronously through the optical fiber photometric system (470 nm excitation light, sampling rate 20 Hz).

[0066] Referring to Figures 6-8 , the average activity change, average peak value of activity, and time to reach the peak value of activity of NAc D1-MSN of the lead exposure group mice and the control group mice in the activity stage in the open field are shown, Figure 9 , a neuron activity heat map of the lead exposure group mice and the control group mice in the movement stage in the open field is shown, and it can be seen from the diagram that lead exposure causes the average activity intensity of NAc D1-MSN of mice to increase, the average peak value of activity to enhance, and the time to reach the peak value of activity to be unchanged.

[0067] 3. Preparation of electrophysiological brain slice and recording of D1-MSN action potential (AP)

[0068] Prepare a high concentration of glucose slice solution (formula: 210 mM sucrose, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 0.5 mM CaCl2, 7 mM MgCl2) mixed with crushed ice to form ice slurry, continuously pass 95% O2 / 5% CO2mixed gas for 20 minutes to obtain pre-cooled oxygenated slice solution ice slurry.

[0069] Pre-warm the artificial cerebrospinal fluid ACSF (126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose, 2 mM CaCl2, 1 mM MgCl2) to 34℃ and oxygen saturation to obtain oxygen-saturated ACSF.

[0070] After isoflurane anesthesia of the mice, quickly decapitate and take out the whole brain on an ice tray within 20 s, immerse in pre-cooled oxygenated slice solution ice slurry for 1 min to reduce metabolic activity; after trimming the brain tissue, fix it on the stage of the slicer using cyanoacrylate glue, and use a vibrating slicer (Leica VT1200S) to cut 300 μm thick NAc coronal sections; then transfer the sections to 34℃ oxygen-saturated ACSF for recovery for 30 minutes, and then incubate in 27℃ low flow rate ACSF for ≥1 hour to stabilize the physiological state of the neurons.

[0071] Using rAAV-D1-GFP labeled NAc slices, D1-MSNs (green fluorescent positive) are identified by fluorescence microscopy (excitation wavelength 488 nm). The Multiclamp 700B amplifier is used in conjunction with microelectrodes (impedance 4-6 MΩ, internal filling solution: 135 mM K-gluconate, 5 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 2 mM Mg-ATP) to record AP in current clamp mode.

[0072] Inject a step current (0-250 pA, step size 25 pA, duration 500 ms) to record AP frequency, threshold and half-width.

[0073] Reference Figure 10 For the 150 pA current injection in this example, representative diagrams of APs of lead-exposed mice and control mice are shown; reference Figure 11 For the current injection in increments of 50 pA from 50 pA to 150 pA in this example, the average AP frequency of lead-exposed mice and control mice is shown. As can be seen from the figure, lead exposure leads to an increase in the frequency of D-MSN APs of mice, indicating an increase in neuronal activity.

[0074] 4. Behavioral verification experiment

[0075] (1) Open field test quantifies lead poisoning behavior

[0076] Experimental device: 50x50x50 cm black polyethylene box, top-mounted infrared camera system (ANY-maze V6.0).

[0077] At the beginning of the experiment, the mouse was placed in any corner of the open field, and the mouse was allowed to freely explore in it for 10 minutes, and the total movement distance, movement speed and immobile time of the mouse were recorded. After the experiment, the bottom and inner wall of the open field were wiped with 75% alcohol to remove the odor of the previous mouse, so as to avoid affecting the activity of the next mouse in the open field.

[0078] Reference Figures 1-3 For comparison of the total movement distance, movement speed and immobile time of the control group (Ctrl group) and the lead exposure group (Pb group) in the open field, the mice in the control group were compared. The total movement distance of the mice in the lead exposure group (Pb group) in the open field was significantly increased, and the immobile time was significantly reduced, indicating that the mice showed increased exploration behavior; the movement speed of the lead exposure mice in the open field was increased, indicating that the mice showed impulsive behavior.

[0079] Reference Figures 12-14 For comparison of the total movement distance, movement speed and immobile time of the lead exposure group (lead group) and the lead+SCH23390 group (Pb+SCH23390) in the open field, it can be seen from the figure that compared with the lead exposure group, the total movement distance and movement speed of the mice injected with compound SCH23390 (lead+SCH23390 group) in the open field were significantly reduced, and the immobile time was significantly increased, indicating that injection of SCH23390 restored the abnormal behavior caused by lead poisoning.

[0080] In addition, the above experimental results show that the above-mentioned specific abnormal behavior phenotype caused by lead has no gender (Male and Female) difference in the mouse model.

[0081] In summary, the technical scheme of the present application detects the efficacy of compound SCH23390 in the treatment of lead poisoning, and provides that compound SCH23390 can significantly alleviate the specific abnormal behavior phenotype (such as abnormal exploration behavior, increased impulsivity, etc.) caused by low-dose lead poisoning, and exhibits better neuroprotective properties than traditional chelating agents. The technical scheme provides a reliable method and feasibility reference for the prevention and treatment of lead poisoning, and further breaks through the limitation of relying on single chelation detoxification in the field of lead poisoning treatment, and provides a new direction for the development of multi-target, multi-mechanism anti-lead toxicity drugs.

[0082] The above merely describes the preferred embodiments of the present application, and is not intended to limit the protection scope of the present application. The present application can have various changes and modifications for those skilled in the art. Any changes, modifications, replacements, integrations and parameter changes made to the embodiments within the spirit and principle of the present application, by conventional substitutions or capable of realizing the same functions without departing from the principles and spirit of the present application, all fall within the protection scope of the present application.

Claims

1. Use of a compound SCH23390 in the preparation of a medicament for treating lead poisoning; The compound SCH23390 is intervened in a lead poisoning mouse model; The lead poisoning mouse model is constructed by feeding pregnant female mice to postnatal offspring with lead ions, and the intake amount is 50 ppm / day; The intervention behavior of the lead poisoning mouse model includes intraperitoneal injection of the compound SCH23390 after the mice are 7 weeks old; The compound SCH23390 reverses lead-induced D1-type neuron signal imbalance by inhibiting the overactivation of NAc D1-type neurons, and alleviates the specific abnormal behavior phenotype caused by low-dose lead poisoning.

2. Use according to claim 1, characterized in that, In the medicament, the administration dose of the compound SCH23390 is 15 μg / kg per day.

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