Application of O-GlcNAc glycosyl hydrolase inhibitor in preparation of medicine for treating depression caused by opioid drug withdrawal

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an O-GlcNAc glycosyl hydrolase inhibitor in preparation of a medicine for treating depression caused by opioid withdrawal. Experimental research shows that after being used for a long time, the opioids are abstinged, so that the glycosylation modification of dopaminergic neurons O-GlcNAc in a covered region on the abdominal side of a mouse is reduced, and the phenotypes of pleasant sensation loss and despair-like depression are caused. Intracranial sustained release and intraperitoneal injection of the O-GlcNAc glycosyl hydrolase inhibitor MK-8719 can recover the glycosylation modification level of dopaminergic neurons O-GlcNAc, can significantly improve pleasant sensation loss and despair symptoms caused by withdrawal of opioid analgesic drugs, and has an anti-depression curative effect. Therefore, the O-GlcNAc glycosyl hydrolase inhibitor MK-8719 can effectively reduce pleasant sensation loss and despair-like behaviors, the inhibition effect is clear in target and remarkable in effect, and the O-GlcNAc glycosyl hydrolase inhibitor MK-8719 can be applied to preparation of drugs for treating depression caused by withdrawal of opioid analgesic drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of O-GlcNAc glycosyl hydrolase inhibitors in the preparation of drugs for treating opioid withdrawal-induced depression. Background Technology

[0002] Opioid use disorder (OPD) and its comorbidity with depression pose a serious global public health challenge. Statistics show that approximately 60 million people worldwide use opioids annually, a significant proportion of whom experience depressive symptoms and negative emotions after withdrawal. Opioid use disorder is highly correlated with the development of depression-related affective disorders, and currently, there are no effective treatments. The prevalence of major depressive disorder among Opioid use disorder patients is 40-60%, and patients with major depressive disorder have a 2-3 times increased risk of developing Opioid use disorder. Addicts have a 3-4 times higher risk of developing severe depressive disorder than the general population. These two conditions mutually reinforce each other, significantly increasing relapse rates, suicide risk, and treatment resistance after detoxification. Dysfunction of dopaminergic neurons in the ventral tegmental area (VTA) of the midbrain is closely related to the pathogenesis of drug addiction and mood disorders such as depression. Dopaminergic neurons are involved in regulating reward, motivation, and emotional behavior; reduced activity can lead to core symptoms of depression such as anhedonia and hopelessness-like behavior, and promote relapse after opioid withdrawal. Therefore, exploring the molecular mechanisms for restoring VTA dopamine neuronal homeostasis is of great significance for developing effective treatment strategies for opioid withdrawal-induced depression.

[0003] O-GlcNAc glycosylation has been identified as a key post-translational modification regulating neuronal function. This modification involves the covalent linking of N-acetylglucosamine (GlcNAc) to intracellular protein serine / threonine residues catalyzed by O-GlcNAc glycosyltransferase (OGT), while O-GlcNAc glycosidase (OGA) is responsible for its hydrolytic removal, forming a dynamic and reversible regulatory system. O-GlcNAc glycosylation is most abundant in the mammalian brain, widely involved in energy metabolism, synaptic plasticity, and neural circuit function regulation. Decreased neuronal O-GlcNAc levels can lead to dysfunction of cell membrane ion channels and inhibit neuronal excitability; conversely, upregulation of O-GlcNAc modification in hippocampal neurons can improve cognitive function in aged mice and alleviate memory impairment in an Alzheimer's disease model. Importantly, specific knockout of OGA in substantia nigra dopamine neurons enhances O-GlcNAc modification levels, promotes striatal dopamine release, and significantly improves synaptic function and motor impairment in a Parkinson's disease model. The use of pharmacological methods (such as OGA inhibitors) to enhance O-GlcNAc glycosylation and restore neuronal energy homeostasis mediated by O-GlcNAc glycosylation in dopaminergic neurons disrupted by addictive drugs could provide an application strategy for the treatment of addiction and depression. However, there are currently no reports on the use of O-GlcNAc glycosylhydrolase inhibitors in the treatment of opioid withdrawal-induced depression. Summary of the Invention

[0004] The purpose of this invention is to provide the use of an O-GlcNAc glycosyl hydrolase inhibitor in the preparation of a drug for treating opioid withdrawal-induced depression and a drug for treating opioid addiction.

[0005] This invention has found that O-GlcNAc glycosyl hydrolase inhibitors can effectively alleviate anhedonia and hopelessness-like behaviors, and that this therapeutic effect has a clear target and significant efficacy. Based on this, this invention proposes the use of O-GlcNAc glycosyl hydrolase inhibitors in the preparation of drugs for treating opioid withdrawal-induced depression.

[0006] In this invention, the O-GlcNAc glycosyl hydrolase inhibitor has the activity of MK-8719, and the molecular formula of MK-8719 is C9H. 14 F2N2O3S, with a molecular weight of 268.28, has the following structure:

[0007] ;

[0008] The English name of MK-8719 is:

[0009] (3aR,5S,6S,7R,7aR)-5-(difluoromethyl)-2-(ethylamino)-3a,6,7,7a-tetrahydro-5Hpyrano[3,2-d]thiazole-6,7-diol.

[0010] Furthermore, opioids include opioids containing morphine, fentanyl, oxycodone, etc., and include various dosage forms.

[0011] Furthermore, the depression caused by opioid withdrawal includes depression caused by various dosage forms of opioids, as well as depression comorbid with these drug addictions.

[0012] Furthermore, the medication for treating depression, including the O-GlcNAc glycosyl hydrolase inhibitor MK-8719, also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the dosage concentration of the aforementioned MK-8719 is 20-100 mg / kg, preferably 30-70 mg / kg.

[0014] Furthermore, the dosage forms of the medication for treating depression include sustained-release formulations, injections, tablets, pills, capsules, granules, or suspensions.

[0015] Furthermore, the administration methods include oral administration and intravenous injection.

[0016] The present invention has the following technical effects:

[0017] Opioid withdrawal after long-term use leads to decreased O-GlcNAc glycosyltransferase (OGT) and O-GlcNAc glycosylation modification in dopamine neurons of the ventral tegmental region of mice, causing anhedonia and hopelessness-like depression phenotypes. Intracranial sustained-release and intraperitoneal injection of the O-GlcNAc glycosylhydrolase inhibitor MK-8719 can restore the O-GlcNAc glycosylation modification level of dopamine neurons, significantly improving anhedonia and hopelessness-like symptoms induced by opioid withdrawal, and exhibiting antidepressant efficacy. This invention's O-GlcNAc glycosylhydrolase inhibitor MK-8719 effectively reduces anhedonia and hopelessness-like behaviors, with a clearly defined and significant inhibitory target, making it suitable for the preparation of drugs to treat opioid withdrawal-induced depression, and possessing significant translational application value. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the depressive-like behavior in mice induced by long-term withdrawal from morphine administration at escalating doses.

[0019] Figure 2This diagram illustrates the result of long-term morphine withdrawal leading to a decrease in the levels of O-GlcNAc glycosyltransferase OGT and O-GlcNAc glycosylation modification in VTA dopamine neurons in the ventral tegmental area.

[0020] Figure 3 This is a schematic diagram illustrating the correlation analysis results between the scores of anhedonia and despair-like behavior in mice and O-GlcNAc glycosylation modification.

[0021] Figure 4 This is a schematic diagram showing the results of intracranial MK-8719 sustained-release administration significantly alleviating morphine withdrawal-induced depressive-like behavior in mice.

[0022] Figure 5 This is a schematic diagram showing the results of intracranial MK-8719 sustained-release administration significantly increasing the O O-GlcNAc glycosylation modification level of VTA dopamine neurons in the ventral tegmental area.

[0023] Figure 6 This is a schematic diagram showing the results of intraperitoneal administration of MK-8719 significantly alleviating morphine withdrawal-induced depressive-like behavior in mice. Detailed Implementation

[0024] The present invention will be further described below through embodiments. Obviously, various modifications and variations can be made to the present invention without departing from its essence, and these modifications and variations are also within the scope of protection claimed in this application.

[0025] Unless otherwise specified, the reagents and instruments used in the examples section are commercially available, conventional products.

[0026] Experimental Materials, Methods and Statistical Analysis

[0027] 1. Reagents

[0028] Morphine Hydrochloride Injection (Shenyang No. 1 Pharmaceutical Co., Ltd., 10 mg / ml, batch number: 161109-1)

[0029] Compound MK-8719 (selleck, lot number: S8890), purity 99.61%

[0030] OGT Polyclonal Antibody (Thermo Scientific, Lot No.: PA5-22071)

[0031] Antibody (O-GlcNAc) Monoclonal Antibody (RL2) (Thermo Fisher, Lot No.: MA1-072)

[0032] Antibody anti-TH (Merck, batch number: MAB318).

[0033] 2. Laboratory animals and grouping

[0034] Clean-grade adult male C57 / BL6 mice (wild-type mice) were purchased from Shanghai Silex Laboratory Animal Co., Ltd., aged 8-12 weeks and weighing 25-28 g. Before surgery, the animals were housed in our laboratory animal room for 7 days with free access to food, a room temperature of 20-22℃, humidity of 55±5%, and a 12-hour day-night cycle. Experiments began one week after the animals acclimatized to the environment. Mice were randomly divided into groups of 10-15.

[0035] 3. Administration method

[0036] Establishment of a morphine-induced depressive-like behavior model: Mice were given morphine via intraperitoneal injection twice daily (at 10:00 AM and 10:00 PM) for 6 consecutive days in cages: 20 mg / kg on day 1, 40 mg / kg on day 2, 60 mg / kg on day 3, 80 mg / kg on day 4, 100 mg / kg on day 5, and 100 mg / kg on day 6, followed by a 3-week withdrawal period.

[0037] For sustained-release administration to the brain: MK-8719 is dissolved in filtered sterile standard artificial cerebrospinal fluid (ACSF) with the following composition: (124 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 24 mM NaHCO3, 5 mM HEPES, 12.5 mM glucose, 2.4 mM CaCl2, and 1.2 mM MgCl2), pH 7.3-7.4; osmotic pressure 290-310 mOsm. It is diluted to the required concentration for micro-osmosis in the brain region before perfusion of the sustained-release capsules.

[0038] Control group (Vehicle) mice were administered an equal volume of physiological saline via intraperitoneal injection. MK-8719 group mice were administered MK-8719 (dissolved in physiological saline) via intraperitoneal injection. Intraperitoneal administration was once daily for 21 consecutive days.

[0039] 4. Micro-osmotic pump implantation scheme

[0040] The sustained-release pump was filled with a solution containing MK-8719 using a microsyringe, and then incubated in 0.9% medical saline at 37°C for approximately 60 hours. A sustained-release pump capsule (2004 W type, injection rate: 0.25 μl / h, duration of action: 4 weeks, Reward, China) containing 200 μl of MK-8719 solution (2 μM; Selleck, catalog number: S8890) or an equal volume of artificial cerebrospinal fluid was then fixed subcutaneously in the back. A plastic catheter was inserted into the ventral tegmental area (VTA) of the target brain region, with the cannula pre-embedded above the VTA. The VTA brain region coordinates were: AP: +3.2 mm, ML: ±0.5 mm, DV: -4.0 mm. AP and ML were referenced to the anterior fontanelle, and DV was referenced to the skull surface. The catheter was then connected to the osmotic pump. Behavioral data were collected 3 weeks after the recovery period.

[0041] 5. Detection and assessment of depressive-like behaviors

[0042] Sucrose water preference test: Mice were acclimatized to the room, water bottles, and sucrose solution before the sucrose water preference test. They were deprived of water (but not food) for 12 hours before the experiment, and the results were measured for 2 hours. The experiment was conducted entirely in single cages, with mice given a choice between 2% sucrose water and drinking water throughout the test (the two water bottles were switched midway). The amount of water consumed by each mouse was weighed, and the evaluation index was: sucrose water preference score = sucrose water consumption / (sucrose water consumption + pure water consumption). A decrease in sucrose water preference can be used as an indicator of anhedonia, a core symptom of depression in mice.

[0043] Tail suspension test: A mouse was suspended upside down by attaching medical tape to the tip of its tail 1 cm from the end, with the other end attached to the top of a box. The height between the tip of the tail and the ground was approximately 30 cm. A 6-minute video recording was made to document the animal's behavior and the duration of stillness. After recording, the medical tape was removed, the animal was carefully removed, the data was recorded, and the animal was returned to its original cage. Motionlessness in mice can be considered a manifestation of learned helplessness and subsequent abandonment of struggle. The longer the mouse remained still, the more severe its depressive, hopeless-like behavior. The duration of stillness was recorded using VisuTrack animal behavior trajectory analysis software (Shanghai Xinruan Information Technology Co., Ltd.).

[0044] Forced swimming experiment: Mice were acclimatized to the laboratory environment for at least one week prior to the experiment (12-hour light / dark cycle, free access to food and water). The forced swimming experiment was conducted in a transparent acrylic cylindrical tank with a diameter of 20 cm and a height of 40 cm. The water depth was ≥ 15 cm, ensuring that the mice could not touch the bottom or use their tails for support, and that their noses could remain above the water surface. The water temperature for breathing was 23 ± 2℃. The swimming time for mice was 6 minutes. After 2 minutes of acclimatization, the time the mice remained motionless (in seconds) was recorded as an indicator of despair behavior. After the experiment, the mice were quickly removed, dried with a towel, and returned to their cages for warming and recovery. The time the mice remained motionless (maintaining balance or floating quietly on the water surface) was recorded using VisuTrack animal behavior trajectory analysis software (Shanghai Xinruan Information Technology Co., Ltd.).

[0045] 6. Immunofluorescence staining and analysis

[0046] Mice were deeply anesthetized and their thoracic cavities were opened. The hearts of mice were perfused with 30 mL of physiological saline at a constant flow rate of 6 mL / min using a constant flow pump. The brains were then perfused with 30 mL of 4% paraformaldehyde for 7 minutes, followed by whole-brain harvesting and fixation with 4% paraformaldehyde. The brains were dehydrated using a gradient of 20% and 30% sucrose solutions. Frozen sections (30 μm) were then placed in brain slice preservation solution and stored at -20°C. Ventral tegmental area (VTA) brain slices were washed three times with 1× PBS solution for 10 minutes each time. Primary antibodies (OGT: 1:200; O-GlcNAc: 1:200; anti-TH: 1:500) were diluted with antibody diluent, incubated on a shaker at room temperature for 45 minutes, and then incubated overnight at 4°C. During elution, the brains were washed three times with 1× PBS solution for 10 minutes each time. Fluorescein-labeled secondary antibodies (Cy3 or Alexa 647, 1:500 or 1:1000) were diluted with 1× PBS and incubated at room temperature for 1–2 hours. The slides were rinsed three times with 1× PBS for 10 minutes each time. The slides were then mounted with anti-fluorescence quenching mounting medium. Images were acquired using a Nikon A1 confocal microscope with a 20× objective lens. Indices of fluorescence intensity (IOD) were analyzed using Image-Pro Plus 6.0.

[0047] 7. Data Processing and Statistical Analysis

[0048] Data analysis and processing were performed using GraphPad Prism 8 software (GraphPad Software), and results are expressed as mean ± standard error. The expression is denoted by ). For two sets of data that conform to a normal distribution and homogeneity of variance, the Unpaired t-test is used. For two sets of measurements from the same mouse, the Paired t-test is used if the data conforms to a normal distribution and homogeneity of variance. For two sets of data that do not conform to a normal distribution, the Mann-Whitney U test is performed. Correlation analysis is performed using linear regression. P < 0.05 is considered significant.

[0049] Example 1: Long-term withdrawal from morphine with increasing doses induced depressive-like behavior in mice.

[0050] Mice were randomly divided into two groups (saline and morphine withdrawal groups), with 12-15 mice in each group. A model of depressive-like behavior induced by escalating morphine administration was established: mice were placed in cages and administered escalating intraperitoneal injections of morphine twice daily (at 10:00 AM and 10:00 PM) for 6 consecutive days: Day 1: 20 mg / kg; Day 2: 40 mg / kg; Day 3: 60 mg / kg; Day 4: 80 mg / kg; Day 5: 100 mg / kg; Day 6: 100 mg / kg, followed by a 3-week withdrawal period. The saline group received an equal volume of saline solution intraperitoneally daily.

[0051] Behavioral results such as Figure 1 As shown, after 3 weeks of morphine withdrawal at escalating doses, mice exhibited depressive-like behaviors, including anhedonia and despair-like behaviors. In the sucrose preference test, mice in the morphine withdrawal group showed significant anhedonia behavior, specifically a significant decrease in their sucrose preference score. Figure 1 A); In the tail suspension test, morphine-withdrawing mice showed a significant increase in immobility time. Figure 1 B), in the forced swimming experiment, morphine-withdrawn mice also showed a significant increase in immobility time. Figure 1 C).

[0052] Depend on Figure 1 It is evident that, compared with the saline control group, the morphine escalation dose withdrawal for 3 weeks successfully established the phenotype of anhedonia and hopeless behavior, the core symptoms of depression.

[0053] Example 2: Long-term morphine withdrawal caused a decrease in O-GlcNAc glycosyltransferase OGT and O-GlcNAc glycosylation modification in VTA dopamine neurons in the ventral tegmental area.

[0054] Brains were harvested from mice in the saline and morphine withdrawal groups after perfusion, fixed, dehydrated, sectioned, and subjected to immunofluorescence staining. The degree of OGT and O-GlcNAc modification in dopaminergic neurons of the ventral tegmental area (VTA) of the mouse midbrain was imaged using laser confocal microscopy. The immunofluorescence staining results are shown below. Figure 2 As shown, the fluorescence intensity of both OGT and O-GlcNAc was significantly reduced in dopamine neurons. This suggests that O-GlcNAc glycosyltransferases OGT and O-GlcNAc glycosylation in VTA dopamine neurons may be involved in withdrawal-induced depressive behavior.

[0055] Example 3: The scores of anhedonia and despair-like behavior in mice were positively correlated with O-GlcNAc glycosylation modification.

[0056] Further through correlation analysis, such as Figure 3 As shown, it was found that the sucrose preference score in mice was positively correlated with the O-GlcNAc modification level of VTA dopamine neurons. The lower the sucrose preference score, the more severe the anhedonia, and the lower the O-GlcNAc modification level of dopamine neurons. Figure 3 A). The duration of immobility during tail suspension and forced swimming was negatively correlated with the O-GlcNAc modification level of VTA dopamine neurons. The longer the immobility, the more severe the despair behavior and the higher the O-GlcNAc modification level of dopamine neurons. (Figure 3B-C).

[0057] These results indicate that downregulation of O-GlcNAc modification levels in dopamine neurons in the ventral tegmental area (VTA) is positively correlated with the severity of withdrawal-induced depressive-like behaviors in mice, and that reduced O-GlcNAc modification levels in dopamine neurons are involved in the occurrence of morphine withdrawal-induced depressive behaviors. This suggests that selectively enhancing O-GlcNAc glycosylation in dopamine neurons can alleviate morphine withdrawal-induced depressive behaviors, thereby playing a role in treating addiction and acting as an antidepressant.

[0058] Example 4: Intracranial MK-8719 sustained-release administration significantly alleviated morphine withdrawal-induced depressive-like behavior in mice.

[0059] To investigate the effects of intracranial sustained-release MK-8719 on morphine withdrawal-induced depressive behavior in mice, wild-type mice were immediately randomly divided into an MK-8719 group (MK-8719) and a control group (Vehicle) after escalating morphine administration, and microosmotic pumps were implanted. The MK-8719 group received microinfusion of MK-8719 (2 μM, injection rate: 0.25 μl / h) into the ventral tegmental area (VTA), while the Vehicle group received microinfusion of sterile artificial cerebrospinal fluid. Three weeks later, sucrose preference test, tail suspension test, and forced swimming test were performed sequentially.

[0060] Test results are as follows Figure 4As shown, compared with the control group, the MK-8719 group significantly increased the score in the sucrose preference test and reduced the time spent stationary in the tail suspension and forced swimming tests. This suggests that the intracranial sustained-release O-GlcNAc glycosylhydrolase OGA inhibitor MK-8719 can significantly alleviate morphine withdrawal-induced depressive-like behavior in mice.

[0061] Mice in the morphine withdrawal group exhibited significant anhedonia behavior, specifically a significant decrease in sucrose preference scores (Chow: 79.52 ± 2.08; HFD: 55.19 ± 4.17, p < 0.05). Figure 2 (As shown in D); In the tail suspension test, morphine-withdrawing mice showed a significant increase in immobility time, and in the forced swimming test, morphine-withdrawing mice also showed a significant increase in immobility time.

[0062] Example 5: Intracranial MK-8719 sustained-release administration significantly increased the O-GlcNAc glycosylation modification level of VTA dopamine neurons in the ventral tegmental area.

[0063] Brains were harvested from mice in the MK-8719 group and the control group after perfusion, fixed, dehydrated, and sectioned for immunofluorescence staining. The degree of O-GlcNAc modification in dopaminergic neurons of the ventral tegmental area (VTA) of mice was imaged using a laser confocal microscope. The immunofluorescence staining results are shown below. Figure 5 As shown, the fluorescence intensity of O-GlcNAc in dopaminergic neurons was significantly enhanced. These results suggest that the O-GlcNAc glycosylhydrolase inhibitor MK-8719 alleviates morphine-induced depressive-like behavior in mice by increasing the level of O-GlcNAc modification in dopaminergic neurons in the ventral tegmental area (VTA).

[0064] Example 6: Intraperitoneal administration of MK-8719 significantly alleviated morphine withdrawal-induced depressive-like behavior in mice.

[0065] To enhance the translational value of the research, the effects of intraperitoneal administration of MK-8719 on morphine withdrawal-induced depressive behavior in mice were further investigated. After administering escalating doses of morphine, wild-type mice were immediately randomly divided into an MK-8719 group (MK-8719) and a control group (Vehicle). Mice in the MK-8719 group received intraperitoneal injection of MK-8719 (dissolved in physiological saline). The intraperitoneal administration was once daily for 21 consecutive days. Mice in the control group (Vehicle) received an equal volume of physiological saline as a carrier via intraperitoneal injection. Three weeks later, sucrose preference, tail suspension, and forced swimming tests were performed sequentially.

[0066] Test results are as follows Figure 6As shown, compared with the control group, the MK-8719 group significantly increased the score in the sucrose preference test and reduced the time spent stationary in the tail suspension and forced swimming tests. This suggests that intraperitoneal administration of MK-8719 (20 mg / kg) can also significantly alleviate morphine withdrawal-induced depressive-like behavior in mice.

Claims

1. The application of O-GlcNAc glycosyl hydrolase inhibitors in the preparation of drugs for treating opioid withdrawal-induced depression, among which, The O-GlcNAc glycosyl hydrolase inhibitor, whose activity is MK-8719, has the molecular formula C9H. 14 F2N2O3S, with a molecular weight of 268.28, has the following structure: 。 2. The application according to claim 1, characterized in that, The opioid drugs mentioned include opioids containing morphine, fentanyl, oxycodone, etc., and include various dosage forms.

3. The application according to claim 1 or 2, characterized in that, The depression caused by opioid withdrawal includes depression caused by various dosage forms of opioids, as well as depression comorbid with these drug addictions.

4. The application according to claim 3, characterized in that, The medication for treating depression includes the O-GlcNAc glycosyl hydrolase inhibitor MK-8719, and pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The dosage of MK-8719 is 20-100 mg / kg.

6. The application according to claim 4, characterized in that, The dosage forms of the medication for treating depression include sustained-release formulations, injections, tablets, pills, capsules, granules, or suspensions.

7. The application according to claim 4, characterized in that, The administration methods include oral administration and intravenous injection.

8. A pharmaceutical composition for treating opioid withdrawal-induced depression, characterized in that, Including O-GlcNAc glycosyl hydrolase inhibitors, whose activity is known as MK-8719.

9. The pharmaceutical composition according to claim 8, characterized in that, It also includes pharmaceutically acceptable excipients.

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