Application of compound SCH23390 in preparation of medicine for treating lead poisoning

Abstract

The invention discloses application of a compound SCH23390 in preparation of a medicine for treating lead poisoning, and relates to the technical field of medicines. The invention studies the efficacy of the compound SCH23390 in the treatment of lead poisoning, reveals the treatment effect of the compound SCH23390 on lead poisoning for the first time, breaks through the limitation of existing drug targets, directly corrects abnormal neural signals by inhibiting excessive activation of D1 neurons and avoids the side effect of central hyperexcitation, and has no obvious toxic or side effect under the treatment dosage, especially, the compound SCH23390 can be used for treating lead poisoning. The compound SCH23390 can obviously relieve specific abnormal behavior phenotypes caused by low-dose lead poisoning, and shows the nerve protection characteristic superior to that of a traditional chelating agent. The limitation that the traditional lead poisoning treatment field depends on a single chelating antidote is broken through, and a brand new direction is provided for developing multi-target and multi-mechanism anti-lead-toxicity drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and pertains to novel functional uses of compounds. In particular, the present invention relates to use of a compound SCH23390 in preparing a drug for treating lead poisoning. Background Art

[0002] Lead poisoning is a disease caused by lead ions (Pb 2+ ) is a systemic toxic disease caused by excessive accumulation, which is widely present in industrial pollution, lead-containing paints and the use of traditional lead-containing products. Lead can accumulate in the central nervous system through the blood-brain barrier, leading to irreversible neurological damage, manifested as cognitive impairment, behavioral abnormalities and multi-organ toxicity. It is worth noting that low-concentration lead exposure can lead to an abnormal increase in exploratory behavior and increased impulsivity. At present, the clinical treatment of lead poisoning mainly relies on chelating agents, such as EDTA, DMSA, etc., but they can only partially eliminate the lead load in the body, lack targeted intervention for neurobehavioral abnormalities caused by lead exposure, and long-term use may be accompanied by side effects such as kidney damage and electrolyte imbalance. There is an urgent need to develop new treatment strategies that have both detoxification and neurological function repair effects.

[0003] The compound SCH23390 is a dopamine D1 receptor antagonist and 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 the treatment of lead poisoning has not been reported.

[0004] Based on this, the present invention applies compound SCH23390 as an active ingredient to the study of lead poisoning mouse model, providing a new direction for the research of compound SCH23390 as a drug for treating lead poisoning. Summary of the Invention

[0005] In view of this, in order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a use of a compound SCH23390 in the preparation of a drug for treating lead poisoning, wherein the compound SCH23390 is used as an active ingredient to alleviate the specific abnormal behavioral phenotype caused by low-dose lead poisoning.

[0006] In order to achieve the above object, the present invention provides a use of a compound SCH23390 in the preparation of a medicament for treating lead poisoning; wherein the compound SCH23390 has a CAS number of 87075-17-0 and a structural formula of:

[0007] Preferably, compound SCH23390 reverses lead-induced D1 neuron signal imbalance by inhibiting excessive activation of NAc D1 neurons, thereby alleviating specific abnormal behavioral phenotypes caused by low-dose lead poisoning, such as abnormal increase in exploratory behavior and impulsive behavior.

[0008] Preferably, in the drug, the dosage of compound SCH23390 is 15 μg / kg per day.

[0009] Illustratively, the present invention constructs a lead poisoning mouse model to study the effects of compound SCH23390 on specific abnormal behavioral phenotypes of lead-poisoned mice, including behavioral experiments and electrophysiological experiments. By injecting adeno-associated virus into the brain area, changes in neuronal activity during mouse activities are recorded in real time, and an open field behavioral paradigm is used to study the effects of compound SCH23390 on the behavior of lead-poisoned mice.

[0010] Specifically, the construction of the lead poisoning mouse model includes orally ingesting lead ions from pregnant mice to offspring after birth, with the intake amount being 50 ppm / day.

[0011] Furthermore, the intervention period includes intraperitoneal injection of compound SCH23390 at a dose of 15 μg / kg after the mice are 7 weeks old.

[0012] As one of the objects of the invention, the present invention also provides a drug for treating lead poisoning, the active ingredient of which at least includes compound SCH23390.

[0013] Preferably, compound SCH23390 is the sole active ingredient of the medicament.

[0014] Preferably, in the drug, the active ingredient may also be a pharmaceutically acceptable salt, solvate or prodrug of compound SCH23390.

[0015] Preferably, the drug is an oral preparation, an injection or a transdermal patch.

[0016] Preferably, the drug further comprises any one or a combination of pharmaceutically acceptable auxiliary ingredients, distilled water or physiological saline.

[0017] The beneficial technical effects obtained by the present invention are: 1. Using the technical solutions of this invention, the compound SCH23390 significantly alleviates specific abnormal behavioral phenotypes (such as abnormal exploratory behavior and increased impulsivity) caused by low-dose lead poisoning and exhibits neuroprotective properties superior to traditional chelators. This discovery overcomes the limitation of single-agent chelation detoxification in the treatment of lead poisoning and provides a new direction for the development of multi-target, multi-mechanism anti-lead toxicity drugs.

[0018] 2. Using the technical solution of the present invention, the efficacy of compound SCH23390 in the treatment of lead poisoning was tested. The results showed that the compound can effectively alleviate abnormal behaviors caused by lead poisoning, providing a reliable method and feasibility reference for the prevention and treatment of lead poisoning.

[0019] 3. This invention reveals for the first time the therapeutic effect of compound SCH23390 on lead poisoning, breaking through the limitations of existing drug targets. By inhibiting the overactivation of D1 neurons, it directly corrects abnormal neural signaling, avoiding the side effects of central nervous system overexcitation, and has no significant toxic side effects at therapeutic doses. Based on the experimental results in a lead poisoning mouse model, it can be inferred that drugs containing the active substance compound SCH23390 may be suitable for children and patients with long-term medication needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison of the total movement distance of lead-exposed mice and control mice in an open field in a typical embodiment of the present invention.

[0021] Figure 2 This is a comparison of the movement speeds of lead-exposed mice and control mice in an open field in a typical embodiment of the present invention.

[0022] Figure 3 This is a comparison of the immobility time of lead-exposed mice and control mice in an open field in a typical embodiment of the present invention.

[0023] Figure 4 Schematic diagram of mouse virus injection and optical fiber embedding in a typical embodiment of the present invention.

[0024] Figure 5 Schematic diagram of the optical fiber location of mice in a typical embodiment of the present invention.

[0025] Figure 6 This figure shows the average activity changes of NAcD1-MSNs in the lead-exposed group and the control group during the activity phase in the open field in a typical embodiment of the present invention.

[0026] Figure 7 This is the average peak value of NAcD1-MSN activity during the activity phase in the open field in the lead-exposed group mice and the control group mice in a typical embodiment of the present invention.

[0027] Figure 8 It is the time from the start of activity to the peak of NAc D1-MSN activity in the open field of lead-exposed mice and control mice in a typical embodiment of the present invention.

[0028] Figure 9 This is a heat map of neuronal activity during the movement phase of lead-exposed mice and control mice in an open field in a typical embodiment of the present invention.

[0029] Figure 10 Representative schematic diagram of AP in lead-exposed mice and control mice when 150 pA current was injected in a typical embodiment of the present invention Figure 11 The average AP frequency of lead-exposed mice and control mice when currents ranging from 50 pA to 150 pA were injected in increments of 50 pA in a typical embodiment of the present invention.

[0030] Figure 12 This is a comparison chart of the total movement distance of mice in the lead + SCH23390 group, the lead exposure group, and the control group in an open field in a typical embodiment of the present invention.

[0031] Figure 13 This is a comparison of the movement speeds of mice in the lead + SCH23390 group, the lead exposure group, and the control group in an open field in a typical embodiment of the present invention.

[0032] Figure 14 This is a comparison of the immobility time of mice in the lead + SCH23390 group, the lead exposure group, and the control group in an open field in a typical embodiment of the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments.

[0034] A method for preparing a drug for treating lead poisoning comprises: first constructing an animal model of lead poisoning, then conducting behavioral experiments and electrophysiological experiments on the animal model, and finally intervening in the experiment with the compound SCH23390. The results are compared with those of an uninterventional lead-exposed group, where the total movement distance and movement speed of mice injected with the compound SCH23390 in an open field are significantly reduced, while the immobility time is significantly increased, indicating that the injection of the compound SCH23390 alleviates abnormal behavior caused by lead poisoning.

[0035] Specifically, the lead poisoning animal model construction and the intervention experiment of compound SCH23390 to study the abnormal behavior caused by lead poisoning in lead-poisoned mice include the following detection methods: (1) Construction of lead poisoning animal model The mother mice were exposed to 50 ppm lead from the beginning of pregnancy until the pups were weaned, and then the pups continued to be exposed to the same concentration of lead. The mice in the control group consumed distilled water orally.

[0036] When the mice were 7 weeks old, an open field behavioral paradigm was used to study the effects of lead exposure on abnormal increases in exploratory behavior and impulsive-like behavior.

[0037] (2) Behavioral experiments The open field experiment creates a low-stress, open space for observing the behavior, movement, and exploratory performance of mice under low-stress conditions. The experiment is conducted in a 50 cm long, wide, and high cube. During the experiment, mice are placed in any corner of the arena and allowed to move freely within it for 10 minutes. The distance moved, speed, and duration of rest are recorded.

[0038] (3) Electrophysiological experiments When mice were 6 weeks old, adeno-associated virus rAAV-D1-GFP was injected into the nucleus accumbens (NAc) of the mice (injection site: AP: +1.42 mm, ML: ±0.80 mm, DV: -4.50 mm; injection dose: 150 nL). Three weeks after the virus was fully expressed, electrophysiological experiments were performed on brain slices of lead-exposed mice to record changes in neuronal action potentials (APs).

[0039] (4) Fiber optic recording Six weeks after exposure to lead, mice were injected with adeno-associated virus into the NAc region of the brain. rAAV-D1-DIO-GCaMP6m-WPRE-hGH polyA (injection site: AP: +1.42 mm, ML: ±0.80 mm, DV: -4.50 mm; injection volume: 150 nL) was injected, and an optical fiber was implanted (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 the mice.

[0040] Seven or 14 days after viral expression, a ceramic fiber optic cannula (200 μm diameter, 0.37 NA, InperTechnology Co., Ltd) was implanted in the same location above the NAc. Two weeks later, behavioral testing was performed to record real-time changes in neuronal activity during mouse activities.

[0041] (5) SCH23390 Behavioral Intervention 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.

[0042] The open field behavioral paradigm was used to study the effect of compound SCH23390 on the behavior of lead-poisoned mice.

[0043] The results of the above experiments show that compound SCH23390 can significantly improve the abnormal behavioral performance of lead-poisoned mice in the open field, showing a significant ability to improve the increased exploratory behavior and impulsive behavior caused by lead poisoning.

[0044] Furthermore, compound SCH23390 can reverse lead-induced D1-type neuronal signaling imbalance by inhibiting NAc D1-MSN overactivation.

[0045] The technical solution of the present invention is further described in detail below through specific embodiments.

[0046] Example 1

[0047] This example provides an intervention experiment of compound SCH23390 on an animal model of lead poisoning. The results show that compound SCH23390 can significantly improve the abnormal behavioral performance of lead-poisoned mice in the open field, demonstrating a significant improvement in the increased exploratory behavior and impulsive behavior caused by lead poisoning.

[0048] Specifically, the steps of this embodiment include: 1. Construction of lead poisoning animal model and intervention with compound SCH23390 C57 mother mice were housed individually after pregnancy, and the pregnant mother mice were randomly divided into a control group (ddH2O) and a lead group (50 ppm lead acetate). The offspring were exposed to the same concentration after birth until behavioral tests were conducted when the offspring were 7 weeks old. The exposure method was oral intake; more specifically, C57 mother mice were exposed to lead from the first day of conception until the offspring were 7 weeks old, and the lead was ingested through drinking water.

[0049] Before the behavioral test, the lead-exposed mice were randomly divided into two groups. One group was intraperitoneally injected with compound SCH23390 (normal saline as solvent, injection volume of 0.25 mL / mouse, concentration of 1.5 μg / mL) at a dose of 15 μg / kg body weight 30 minutes before the behavioral test. The control group and the other group of lead-exposed mice were injected with an equal volume of normal saline.

[0050] 2. Stereotaxic Injection and Fiber Optic Placement To monitor the calcium signal dynamics of D1 medium spiny neurons (D1-MSNs) in NAc of lead-exposed mice in real time, the present invention used a genetically encoded calcium indicator virus system (rAAV-D1-DIO-GCaMP6m-WPRE-hGH polyA) for neuron-specific labeling.

[0051] The specific procedures are as follows: 35-day-old mice were selected and deeply anesthetized via intraperitoneal injection of sodium pentobarbital (60 mg / kg in saline). The mice were then secured in a stereotaxic apparatus, with the head stabilized by tooth bars and ear bars. The skin was incised along the midline of the skull (using sterile surgical instruments) to fully expose the anterior and posterior bregmas. The anterior bregma was designated as the origin of the three-dimensional coordinate system (AP: 0 mm, ML: 0 mm, DV: 0 mm). A stereomicroscope was used to finely level the skull, ensuring that both the sagittal and coronal planes were level. According to the coordinates of the target brain region (NAc core area: AP +1.42 mm, ML ±0.80 mm, DV -4.50 mm), a micro-skull drill (0.9 mm diameter) was used to gently drill a hole to avoid damaging the dura mater and blood vessels; a 33G microinjection needle was connected to a nano-injection pump, and the virus suspension was aspirated at a rate of 160 nL / s. Then, the virus was slowly injected at a rate of 50 nL / min (total dose 160 nL). After the injection, the needle was allowed to stand for 10 minutes until the virus particles were fully diffused and then the needle was withdrawn; the tip of the ceramic optical fiber (200 μm diameter) was then positioned 0.1 mm above the injection site (DV -4.45 mm), slowly implanted using an optical fiber clamp, and double fixed with dental cement and stainless steel skull nails to ensure the stability of the optical fiber-brain tissue interface.

[0052] See Figure 4 , is a schematic diagram of virus injection and optical fiber embedding in mice; Figure 5 A schematic diagram of the fiber optic site in the brain slice shows the correct site for viral injection. After suturing the skin incision, the mouse was placed in a constant-temperature resuscitation chamber (37°C) until spontaneous activity resumed. The viral expression period was set for 3 weeks, followed by simultaneous recording of calcium signal dynamics during an open-field experiment using a fiber optic photometry system (470 nm excitation light, 20 Hz sampling rate).

[0053] See Figure 6-Figure 8 , respectively, the changes in the average activity of NAcD1-MSNs, the average peak value of the activity, and the time when the activity reaches the peak value in the open field during the activity phase of the lead-exposed group mice and the control group mice in this embodiment, Figure 9 This is a heat map of neuronal activity in the lead-exposed group and the control group during the movement phase in the open field. It can be seen from the figure that lead exposure causes the average activity intensity of NAc D1-MSN in mice to increase and the average peak value of activity to increase, while the time for the activity to reach the peak remains unchanged.

[0054] 3. Electrophysiological Brain Slice Preparation and D1-MSN Action Potential (AP) Recording Prepare a high-concentration glucose slicing solution (formula: 210 mM sucrose, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 0.5 mM CaCl2, 7 mM MgCl2) and mix it with crushed ice to form an ice slurry. Continuously pass 95% O2 / 5% CO2 mixed gas for 20 minutes to obtain pre-cooled oxygenated slicing solution ice slurry.

[0055] 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) was preheated to 34°C and saturated with oxygen to obtain oxygen-saturated ACSF.

[0056] Mice were anesthetized with isoflurane and rapidly decapitated. The whole brain was removed within 20 seconds on an ice tray and immersed in a precooled oxygenated slicing solution slurry for 1 minute to reduce metabolic activity. After trimming, the brain tissue was fixed to the microtome stage using cyanoacrylate glue, and 300-μm-thick NAc coronal sections were cut using a vibrating microtome (Leica VT1200S). The slices were then transferred to 34°C oxygen-saturated aCSF for 30 minutes to recover and then incubated in 27°C low-flow aCSF for ≥1 hour to stabilize neuronal physiological state.

[0057] Using rAAV-D1-GFP-labeled NAc slices, D1-MSNs (green fluorescence) were identified by fluorescence microscopy (excitation wavelength 488 nm). APs were recorded in current-clamp mode using a patch-clamp amplifier (Multiclamp 700B) coupled with microelectrodes (impedance 4-6 MΩ, refill solution: 135 mM K-gluconate, 5 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 2 mM Mg-ATP).

[0058] A step current (0-250 pA, 25 pA step, 500 ms duration) was injected, and the AP frequency, threshold, and half-amplitude width were recorded.

[0059] See Figure 10 This is a representative schematic diagram of APs in lead-exposed mice and control mice when 150 pA current was injected in this example; see Figure 11 This figure shows the average AP frequency of lead-exposed mice and control mice when currents ranging from 50 pA to 150 pA were injected in increments of 50 pA in this example. As can be seen from the figure, lead exposure causes an increase in the frequency of D-MSN APs in mice, indicating enhanced neuronal activity.

[0060] 4. Behavioral Verification Experiment (1) Open field test to quantify lead poisoning behavior Experimental setup: 50 × 50 × 50 cm black polyethylene box with an infrared camera system (ANY-maze V6.0) installed on the top.

[0061] At the beginning of the experiment, mice were placed in any corner of the open field and allowed to explore freely for 10 minutes. The total distance traveled, movement speed, and time spent immobile were recorded. After the experiment, the floor and inner walls of the open field were wiped with 75% alcohol to remove any residual odor from the previous mouse and to prevent it from affecting the next mouse's movement within the field.

[0062] See Figure 1-Figure 3 , is a comparison chart of the total movement distance, movement speed and immobility time of the control group (Ctrl group) and the lead exposure group (Pb group) in the open field. Compared with the mice in the control group, the total movement distance of the mice in the lead exposure group (Pb group) in the open field was significantly increased, while the immobility time was significantly reduced, indicating that the mice had increased exploratory behavior; the movement speed of the lead-exposed mice in the open field increased, indicating that the mice had impulsive behavior.

[0063] See Figure 12-14 , is a comparison of the total movement distance, movement speed and immobility 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 mice injected with compound SCH23390 (lead + SCH23390 group) in the open field were significantly reduced, while the immobility time was significantly increased, indicating that the injection of SCH23390 restored the abnormal behavior caused by lead poisoning.

[0064] In addition, the above experimental results showed that there was no gender (male and female) difference in the above-mentioned specific abnormal behavioral phenotypes caused by lead in the mouse model.

[0065] In summary, the present invention's technical solution, through testing the efficacy of compound SCH23390 in the treatment of lead poisoning, demonstrates that compound SCH23390 can significantly alleviate specific abnormal behavioral phenotypes (such as abnormal exploratory behavior and increased impulsivity) caused by low-dose lead poisoning, and exhibits neuroprotective properties superior to traditional chelators. This technical solution provides a reliable method and feasibility reference for the prevention and treatment of lead poisoning. Furthermore, this invention further overcomes the limitation of the field of lead poisoning treatment that relies on a single chelation detoxification method, providing a new direction for the development of multi-target, multi-mechanism anti-lead toxicity drugs.

[0066] 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 drug for treating lead poisoning.

2. The use according to claim 1, characterized in that The compound SCH23390 inhibits the overactivation of NAc D1 neurons, reverses the lead-induced imbalance in D1 neuron signaling, and alleviates the specific abnormal behavioral phenotypes caused by low-dose lead poisoning.

3. The use according to claim 2, characterized in that The compound SCH23390 intervened in the lead poisoning mouse model; The construction of the lead poisoning mouse model includes orally ingesting lead ions from pregnant mothers to offspring after birth, with the intake amount being 50 ppm / day.

4. The use according to claim 3, characterized in that The intervention period of the lead poisoning mouse model included intraperitoneal injection of compound SCH23390 after the mice were 7 weeks old.

5. The use according to claim 1, characterized in that Among the drugs, the dosage of compound SCH23390 is 15 μg / kg per day.

6. A medicine for treating lead poisoning, characterized in that: The active ingredient thereof includes at least compound SCH23390.

7. The drug according to claim 6, characterized in that Compound SCH23390 is the only active ingredient of the drug.

8. The drug according to claim 6, characterized in that In the medicine, the active ingredient may also be a pharmaceutically acceptable salt, solvate or prodrug of compound SCH23390.

9. The drug according to claim 6, characterized in that The medicine is an oral preparation, an injection or a transdermal patch.

10. The drug according to any one of claims 6 to 9, characterized in that The medicine further comprises pharmaceutically acceptable auxiliary components, physiological saline or distilled water.

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