Use of arginase inhibitors for the preparation of a medicament for the prevention and treatment of side effects of benzodiazepines

Abstract

The present application relates to the field of biological medicine technology, and particularly relates to application of arginase inhibitor in preparation of medicine for preventing and treating side effects of benzodiazepine drugs. The present application first breaks through the cognitive limitation of traditional "neuron receptor center theory", and clarifies that arginase synthetase 1 dependent pathological polarization of microglia is the core mechanism of benzodiazepine drug induced cognitive impairment. By targeting inhibiting abnormal up-regulation of arginase synthetase 1, the selective marker (C3) of inhibitory synapse and subsequent microglial phagocytosis can be effectively blocked, so that the density of inhibitory synapse and the hippocampal excitatory / inhibitory balance can be maintained in structure. The technical scheme of the present application can significantly reduce or even eliminate learning and memory impairment, drug tolerance and withdrawal hypersensitivity caused by long-term use of benzodiazepine drugs without weakening the sedative, anxiolytic and anticonvulsant effects of benzodiazepine drugs, and overcomes the technical defects of traditional intervention means and immune off-target of broad-spectrum anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of arginase inhibitors in the preparation of drugs for preventing and treating the side effects of benzodiazepines. Background Technology

[0002] Benzodiazepines (BZDs) are a class of drugs that regulate the gamma-aminobutyric acid type A receptor (GABA) through positive allosteric regulation. A Benzodiazepines (benzodiazepines) are drugs that enhance inhibitory conduction in the central nervous system and are widely used to treat mental illnesses such as anxiety, insomnia, epilepsy, and muscle spasms. Although their short-term efficacy is certain, clinical evidence shows that long-term use of benzodiazepines can lead to significant cognitive impairment, drug tolerance, and withdrawal syndrome. Epidemiological studies show that long-term users commonly experience decreased learning and memory abilities, impaired executive function, and a significantly increased risk of developing dementia later in life.

[0003] Currently, there is a lack of intervention programs based on molecular mechanisms for cognitive impairment, drug tolerance, and withdrawal syndrome caused by long-term use of benzodiazepines. Existing research is mostly limited to symptom relief or non-specific interventions, which has the following significant limitations: 1. Limitations in understanding the mechanism: Over-reliance on the "neuron-centrism" while neglecting immune remodeling; current technologies generally attribute the lateral effects of benzodiazepines to GABA. A Desensitization or subtype switching of receptors tends to be explained by physiological changes in the postsynaptic membrane of neurons. However, this understanding overlooks the immune polarization of microglia, which act as "synaptic pruners" in the central nervous system, under drug exposure. Current technology has not yet elucidated the complete pathological cascade of "benzodiazepine exposure—abnormal microglia polarization—pathological loss of inhibitory synapses," preventing research from addressing the essence of synaptic structural damage.

[0004] 2. The crudeness of intervention methods: "Off-target immune effects" caused by broad-spectrum anti-inflammatory regimens. Current interventions for neuroinflammation mostly use nonsteroidal anti-inflammatory drugs (NSAIDs) or broad-spectrum glial cell inhibitors (such as minocycline). These regimens are "indiscriminate inhibition," failing to identify and block attacks targeting specific synaptic types, and interfering with the normal physiological monitoring and neurotrophic support functions of microglia. This imprecise "one-size-fits-all" strategy often leads to severe immune adverse effects and makes it difficult to rebuild damaged neural circuit homeostasis.

[0005] 3. Lagging Clinical Strategies: The lack of reversible mechanisms for "compensatory neural network remodeling" means that the drug resistance and withdrawal symptoms of "hyperexcitability" caused by long-term benzodiazepine use are essentially structural manifestations of the breakdown of the excitation-inhibition balance in key brain regions such as the hippocampus after pathological pruning of inhibitory synapses. Existing clinical strategies (such as slow dose reduction and neurotransmitter regulation) only make minor adjustments to the damaged circuits, which is a "symptomatic treatment, not a cure." Currently, there is no technology that can prevent circuit remodeling in advance by blocking the physical disappearance of postsynapses, meaning that once cognitive impairment occurs, it is often irreversible.

[0006] 4. Misconceptions about key regulatory factors: The "pathogenic phenotype" of arginase synthase 1 under specific pharmacological conditions has been overlooked. This is the biggest bottleneck in current technology. In traditional inflammation models, arginase synthase 1 is often regarded as a marker of "anti-inflammatory" or "repair," and this preconceived scientific bias has led researchers to rarely explore its negative role in driving synaptic loss. Current technology has not revealed the synergistic coupling between arginase synthase 1 upregulation and complement C3 recognition of inhibitory synapses, nor has it realized that the arginase synthase 1 / C3 axis can serve as a precise target for blocking the selective loss of inhibitory synapses.

[0007] 5. Gaps in the Risk Assessment System: There is a lack of prospective molecular early warning indicators. Preclinical assessments of cognitive risks associated with benzodiazepines primarily rely on scale scores, which have a significant lag (by the time symptoms appear, synaptic loss has often reached the physiological compensation limit). Due to the lack of detection methods based on early molecular indicators such as arginase synthase 1 expression kinetics and complement C3 synaptic enrichment, clinicians are unable to identify risks in the early stages of circuit remodeling, greatly limiting the optimal window for preventative intervention.

[0008] Recent studies have revealed that microglial-mediated synaptic pruning plays a crucial role in the development of various neuropsychiatric disorders. The complement C3 / C3aR-CR3 signaling axis is widely considered a key molecular mechanism guiding microglia to recognize and engulf specific synapses. However, whether microglia are specifically activated under long-term benzodiazepine exposure, and how they participate in the selective loss of inhibitory synapses, has not yet been systematically elucidated. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide the application of arginase inhibitors in the preparation of drugs for preventing and treating the side effects of benzodiazepines. The present invention reveals the pathological mechanism of long-term benzodiazepine exposure leading to an imbalance of excitation / inhibition in the hippocampal dentate gyrus, and provides a technical solution that specifically inhibits the abnormal phagocytosis of inhibitory synapses by microglia through pathological synaptic elimination mediated by downregulation of Arg1 activity. The method of the present invention can be used to prepare drugs for the prevention and treatment of cognitive impairment, drug tolerance, and withdrawal reactions caused by benzodiazepines, and can also be used for high-throughput screening of related neuroprotective candidate drugs and for early injury risk assessment. The present invention has the advantages of high target specificity and high clinical translational value.

[0010] This invention provides the use of arginase inhibitors in the preparation of medicaments for the prevention and / or treatment of the side effects of benzodiazepine drugs.

[0011] In some embodiments, the side effects of the diazoxide drug are at least one of cognitive impairment, drug tolerance, and withdrawal symptoms.

[0012] In some embodiments, the cognitive impairment is impaired spatial working memory.

[0013] In some embodiments, the arginase inhibitor maintains the excitation / inhibition balance of the hippocampal neural circuit by inhibiting the abnormal upregulation of arginase in microglia and blocking the pathological phagocytosis of inhibitory synapses by microglia.

[0014] In some embodiments, the arginase inhibitor is selected from one or more of the following: small molecule compounds, nucleic acid interference molecules, antibodies, peptide inhibitors, and natural product extracts.

[0015] In some embodiments, the arginase inhibitor is selected from at least one of nor-NOHA, S-(2-boronethyl)-L-cysteine, 2-amino-6-boronocanoic acid, Numidargistat, OATD-02, and AZD0011.

[0016] In some embodiments, the arginase inhibitor is nor-NOHA.

[0017] In some embodiments, the dosage of nor-NOHA is 10-100 mg / kg per day.

[0018] In some embodiments, the dosage of nor-NOHA is 30-70 mg / kg per day.

[0019] In some embodiments, the arginase inhibitor and the benzodiazepine are administered simultaneously or sequentially.

[0020] In some embodiments, the sequential administration involves administering an arginase inhibitor before a benzodiazepine.

[0021] In some embodiments, the arginase inhibitor is used in combination with benzodiazepines to reduce or eliminate structural damage to inhibitory synapses while exerting the sedative, anxiolytic, and / or anticonvulsant effects of benzodiazepines.

[0022] In some embodiments, the medicament includes an arginase inhibitor and pharmaceutically acceptable excipients.

[0023] In some embodiments, the dosage form of the drug is an injection or an oral formulation.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention breaks through the limitations of the traditional "neuronal receptor-centric theory" by elucidating that arginase synthase 1-dependent microglial pathological polarization is the core mechanism of benzodiazepine-induced cognitive impairment. By targeting and inhibiting the abnormal upregulation of arginase synthase 1, the selective labeling (C3) of inhibitory synapses and subsequent microglial phagocytosis can be effectively blocked, structurally maintaining the density of inhibitory synapses and the hippocampal excitation / inhibition balance. The technical solution of this invention significantly reduces or even eliminates learning and memory impairment, drug tolerance, and withdrawal hypersensitivity caused by long-term use of benzodiazepines without weakening their sedative, anti-anxiety, and anticonvulsant effects, overcoming the technical defects of traditional intervention methods and the immune off-target effects of broad-spectrum anti-inflammatory drugs.

[0025] 2. This invention further establishes arginase synthase 1 as a key molecular target for the prevention and control of benzodiazepine side effects, supporting the development of combination therapies, fixed-dose compound preparations, and sustained-release drug delivery systems. Simultaneously, by detecting the expression kinetics and activity changes of arginase synthase 1, a quantifiable early warning system for cognitive decline risk is constructed, and a high-throughput drug screening platform is established. This provides systematic technical support for the safety assessment of benzodiazepines, the discovery of neuroprotective candidate drugs, and personalized precision medicine, possessing significant clinical translational value and promising industrial application prospects. Attached Figure Description

[0026] Figure 1 Long-term diazepam administration leads to GABA deficiency. DG The neuronal firing frequency decreased. Figure A shows a schematic diagram of multichannel electrode recording; Figure B shows a grid plot and typical trajectories with specified groups; Figure C shows GABA. DG Statistical analysis of spontaneous neuronal firing, n=3 mice per group; Figure 2This study showed that mice given long-term benzodiazepines exhibited cognitive impairment and microglial activation. Figure A illustrates the experimental design process; Figure B shows representative movement trajectories of the three groups of mice in the open field; Figure C is a statistical analysis of the total distance traveled by the three groups of mice in the open field (n=11-12 mice); Figure D is a schematic diagram of the new object recognition experiment; and Figure E is a statistical analysis of the time spent by the three groups of mice exploring new and old objects in the new object recognition experiment (n=11-12 mice). Figure F shows the statistical analysis of the total number of mice entering each arm in the Y maze in the three groups (n=14-15 mice). Figure G shows a representative heatmap of the three groups of mice in the Y maze. Figure H shows the statistical analysis of the percentage of mice freely alternating in the Y maze in the three groups (n=14-15 mice). Figure I shows a representative image (left) of Iba1 immunofluorescence staining (green) in the dentate gyrus of the hippocampus in the three groups of mice, a reconstructed immunofluorescence staining view of microglia (middle), and a three-dimensional reconstruction example of microglia (right). Scale bar: 20 μm (overview image), 10 μm (reconstructed microglia staining view and reconstruction example image), J figure is a statistical analysis of the total length of microglia fibers in the three groups of mice, K figure is a statistical analysis of the total area of ​​microglia fibers in the three groups of mice, L figure is a statistical analysis of the total volume of microglia fibers in the three groups of mice, M figure is a statistical analysis of the number of dendritic branches of microglia fibers in the three groups of mice, N figure is the trend of the number of intersections of microglia fiber branches in the three groups of mice, JN: n=6 mice, all data are expressed as mean ± standard deviation (Mean + SD). This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001; P < 0.0001; ns indicates no statistical difference; Figure 3 This study demonstrates that the inhibition of arginase synthase 1 in mice after administration of Nor-NOHA (an arginase inhibitor) improved cognitive function. Figure A shows the experimental design flowchart; Figure B shows representative movement trajectories of the four groups of mice in the open field; Figure C shows the statistical analysis of the total distance traveled by the four groups of mice in the open field (n=7-8 mice); Figure D shows the flowchart of the new object recognition experiment; Figure E shows the statistical analysis of the time spent exploring new and old objects in the new object recognition experiment for the four groups of mice (n=6-7 mice); Figure F shows the statistical analysis of the total number of arms entered by the four groups of mice in the Y maze (n=7 mice); Figure G shows representative heatmaps of the four groups of mice in the Y maze; and Figure H shows the statistical analysis of the percentage of free alternation in the Y maze for the four groups of mice (n=7 mice). All data are expressed as mean ± standard deviation (Mean + SD). This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001; P < 0.0001; ns indicates no statistical difference. Detailed Implementation

[0027] This invention provides the application of arginase inhibitors in the preparation of drugs for preventing and treating the side effects of benzodiazepines. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0028] To address the shortcomings of existing benzodiazepine lateral effect interventions, such as lack of specificity, unclear mechanisms, and difficulty in reversing circuit damage, this invention aims to provide a precise intervention scheme based on the "arginase synthase 1-C3-inhibitory synaptic phagocytosis" axis. Specific objectives include: 1. Elucidating and blocking the structural basis of benzodiazepine-related cognitive impairment. This invention aims to break away from the traditional "neuronal receptor-centric theory" and, for the first time, provide a technical approach to prevent cognitive impairment by intervening in arginase synthase 1-dependent pathological polarization of microglia. Its core objective is to reveal the molecular mechanism by which long-term benzodiazepine exposure induces selective labeling of inhibitory synapses (C3) and drives microglia to engage in depletion, providing theoretical support and technical means for diagnosing and treating related diseases from the perspective of "synaptic structure protection." 2. Achieving precise immune regulation with "synaptic subclass specificity." Addressing the deficiency of broad-spectrum anti-inflammatory therapy leading to immune imbalance, this invention aims to provide a non-inhibitory precise regulation strategy. By specifically targeting arginase synthase 1 or its downstream C3 loading process, this invention blocks only the erroneous cleavage of "inhibitory synapses" while preserving the normal anti-infection and neural support functions of microglia, thereby significantly improving the selectivity and safety of treatment. 3. Remodeling and Maintaining the Excitation / Inhibition Homeostasis of Neural Circuits. Another objective of this invention is to provide a method for effectively reversing the "hyperexcitation" remodeling of neural networks induced by benzodiazepines. By physically protecting the density of inhibitory synapses, this invention aims to block the development of drug resistance and post-withdrawal hypersensitivity at the source, providing a kinetic protection strategy superior to "slow tapering" for long-term patients, ensuring the functional integrity of neural circuits. 4. Developing Novel Drug-Enhancing Combination Formulations and Intervention Targets. This invention aims to establish arginase synthase 1 inhibitors and complement pathway modulators as novel drug targets for adjuvant therapy with benzodiazepines. By developing "two-in-one" combinations or fixed-dose compound formulations for use with benzodiazepines, this invention aims to automatically offset the synaptic toxicity of benzodiazepines while maintaining their sedative and anxiolytic effects, providing core technological reserves for the updating and iteration of sedative-hypnotic drugs. 5. Constructing a multi-dimensional risk warning and high-throughput drug screening and evaluation system. The purpose of this invention is to establish a comprehensive detection platform covering genes, proteins, and cell phenotypes. By detecting the expression kinetics of arginase synthase 1 and the deposition level of C3 at the synaptic site, this invention achieves: early screening of the risk of cognitive decline in long-term benzodiazepine users; high-sensitivity screening of candidate drugs aimed at reducing synaptic toxicity; and quantitative assessment of the neurological damage risk of existing clinical protocols, thereby promoting the precise and personalized application of psychotropic drugs.

[0029] In summary, this invention, through its original and in-depth elucidation of the pathological mechanism of "arginine synthase 1-mediated selective phagocytosis of inhibitory synapses by microglia," has successfully overcome the dual technical bottlenecks of "target deficiency" and "off-target efficacy" in the intervention of benzodiazepine-related cognitive impairment. The core value of this invention is reflected in the following dimensions: It pioneers a new paradigm of precise neuroimmune regulation: Unlike the traditional anti-inflammatory strategy of "broad-spectrum inhibition and systemic mobilization," this invention achieves precise "point-level" protection of inhibitory synapses by targeting the specific synaptic cleavage signal axis of arginine synthase 1 / C3. This approach reverses cognitive impairment while minimizing interference with normal neuroimmune homeostasis, establishing a high safety standard for the intervention of neuropsychiatric drug side effects. It constructs a circuit-level structural repair guarantee: This invention is the first to propose maintaining the excitation / inhibition balance in key brain regions such as the hippocampus by blocking physical synaptic loss, thereby fundamentally counteracting the drug resistance and withdrawal-related circuit overexcitation caused by long-term use of benzodiazepines. This marks a significant technological leap in clinical intervention, shifting from "receptor regulation" to "circuit remodeling and protection." It has driven technological innovation across the entire industry chain: This invention not only points the way for the development of next-generation "reduced toxicity and enhanced efficacy" benzodiazepine combination formulations, but also fills the technological gap in precise early warning of long-term benzodiazepine risks by establishing a multidimensional assessment system based on arginase synthase 1 expression and C3 deposition levels. In summary, the technical solutions covered by this patent not only possess significant inventiveness in pharmacological mechanisms, but also demonstrate broad application prospects in clinical translation, risk control, and the construction of novel drug screening platforms, providing a substantial theoretical foundation and technical support for precision medicine of neuroimmunological diseases.

[0030] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0031] Example 1

[0032] 1. Animal modeling

[0033] All experiments used mice aged 4–5 months. The animals were housed in an environment maintaining a 12-hour light-dark diurnal rhythm (light period 08:00–20:00), with room temperature controlled at 22±1 °C and relative humidity maintained at 40%–60%. Diazepam was dissolved in 0.5% sodium carboxymethyl cellulose aqueous solution and administered orally by gavage; flumazenil was dissolved in physiological saline containing 0.5% Tween 80 and administered intraperitoneally. Unless otherwise specified, all drug solutions were administered at a dose of 10 mL / kg body weight, once daily for 7 consecutive days. The experimental drug dosages were: diazepam (benzodiazepine) 5 mg / kg; flumazenil intraperitoneal injection dose of 15 mg / kg, administered 15 mins before diazepam gavage. Given that flumazenil's duration of action is shorter than that of diazepam, to maintain a stable antagonistic effect, mice were supplemented with flumazenil (10 mg / kg / day) via drinking water after the first injection. Under these conditions, flumazenil was dissolved in a solution containing 0.5% ethylene glycol to prepare drinking water for the mice to ingest freely. Arginase inhibitor nor-NOHA (purchased from MCE, catalog number: HY-112885B, CAS number: 2250019-93-1) was administered intraperitoneally daily (50 mg / kg / day).

[0034] 2. Multichannel in vivo electrophysiological recording

[0035] Mice to be implanted with a multichannel electrode array were housed individually. After three days of acclimatization, the customized multichannel electrode array was implanted into the dentate gyrus of the hippocampus. Mice were anesthetized with sodium pentobarbital (80 mg / kg) via intraperitoneal injection prior to surgery. All instruments were sterilized with 75% ethanol, and the entire procedure was performed under sterile conditions. A 1 cm × 1 cm cranial window was created by incising the scalp, leveling the electrode, and positioning it above the brain region. The electrode was then advanced downwards to the dentate gyrus at a speed of 0.1 mm / min. Two cranial screws were then implanted into the cerebellum, and the electrode ground wires were wrapped around the screws. Dental cement was then used for fixation. Signal acquisition was performed after at least a two-week recovery period, using the multichannel electrodes to simultaneously record signals from multiple neurons. Mice were allowed to acclimatize to the leads connecting the electrodes to their heads before recording. The experimental mice were placed in a cylindrical box encased in copper mesh, allowing free movement and undisturbed operation, during which multichannel electrical signals were continuously recorded. Daily recordings were performed for 7 consecutive days, 45 minutes each day. Specifically, changes in mouse neuronal activity were recorded before and after drug administration. Changes in neuronal activity before administration were recorded and recorded as day 0. Subsequently, mice were administered 5 mg / kg diazepam daily for 7 consecutive days, recorded as day 1, day 2, and so on up to day 7. Recordings were performed daily before administration, each session lasting 3 minutes, with intervals of 15 minutes between each recording. Neuronal signals were amplified, filtered with a bandwidth of 300-5000 Hz, and stored using NeuronStudio software. Multichannel in vivo recording typically uses a high-speed sampling frequency of 40 kHz for acquiring neuronal action potential signals. Threshold-triggered sampling techniques are generally used to reduce irrelevant data. For multichannel in vivo recording technology, distinguishing the firing activity of different neurons mainly relies on the differences in the waveforms of the recorded action potentials. Using multi-channel in vivo recording technology, we combined the in vivo firing patterns of mouse hippocampal DG neurons and processed them with offlinesortert software to obtain the firing time series of single neurons. Then, we used neuronexplorer to perform firing interval analysis and autocorrelation analysis. Based on the parameters of firing frequency and firing waveform, we were able to distinguish the firing activities of pyramidal neurons and interneurons in the hippocampal DG.

[0036] 3. Behavioral experiments

[0037] All behavioral experiments were conducted within the 08:00–20:00 light cycle. To avoid interference from olfactory residue, all behavioral apparatus was thoroughly cleaned between each trial. Unless otherwise specified, behavioral data collection and analysis were performed under double-blind conditions whenever possible. One hour before the behavioral tests, the mice were placed in the behavioral testing area to acclimatize.

[0038] Open field test: The open field test was used to evaluate the effects of different drug treatments on the spontaneous motor activity of mice. The apparatus used in the open field test was a 50 cm × 50 cm × 50 cm smooth white box. The central region was defined as a 25 cm × 25 cm area in the center of the open field. During the experiment, mice were gently placed in the center of the open field and allowed to explore freely for 10 minutes, with their movement trajectory recorded in real time by a video tracking system. The time spent in the central region, the number of times the mice entered the central region, and the total distance traveled were analyzed using EthoVision XT 17 software. The open field test serves as a baseline indicator for assessing differences in motor function among individuals. Before cognitive behavioral tests, the open field test is used to detect the spontaneous activity and exploratory behavior of mice, and to assess whether there are differences in their spontaneous motor abilities.

[0039] Novel Object Recognition Experiment: This experiment assesses the effects of different drug treatments on recognition memory in mice. During the experiment, mice were gently placed in the center of an open field apparatus for a 10-minute habituation phase, allowing them to freely explore the apparatus. After 24 hours of habituation, a familiarization phase began, where the mice were gently placed in the center of the apparatus and presented with two objects of identical shape and appearance, allowing them to freely explore for 10 minutes. After another 24 hours, a testing phase commenced, replacing one of the familiar objects with a novel object of different shape and color. The mouse was then gently placed back in the center of the apparatus and subjected to a 6-minute test. The entire process was recorded by a camera above the apparatus. The exploration time for the novel and familiar objects was recorded, and the ratio of the exploration time for the novel object to that for the familiar object was calculated as the recognition memory index. The novel object recognition experiment serves to assess inter-individual differences in cognitive function; a lower recognition memory index indicates poorer cognitive function in the mouse.

[0040] Y-maze: The Y-maze spontaneous alternation experiment was used to evaluate the effects of different drug treatments on spatial working memory in mice. The Y-maze apparatus consisted of three identical arms (30 cm × 6 cm × 15 cm). During the experiment, mice were gently placed in the center of the Y-maze and allowed to explore freely for 6 minutes, with the entire process recorded by a camera above the apparatus. The total number of times the mouse entered each arm was recorded, and the total number of entries in a triplet was defined as the total number of entries minus 2. A valid alternation was defined as consecutive entries into three different arms forming an overlapping triplet. The spontaneous alternation rate was calculated as the ratio of the number of alternations to the total number of triplet entries. The Y-maze is used to assess inter-individual differences in cognitive function; a lower spontaneous alternation rate indicates poorer cognitive function in the mouse.

[0041] 4. Three-dimensional reconstruction of mouse microglia

[0042] 40-micrometer-thick mouse brain slices were stained with Iba1 immunofluorescence. All images were acquired using a rotating confocal microscope (equipped with a 60× or 100× objective lens) to obtain stereoscopic images. To ensure the comparability of data between different groups, imaging parameters (including laser intensity, gain, and offset) were kept consistent throughout all experiments. Z-stack scanning was performed along the Z-axis with a step size of 1 micrometer to acquire images with a resolution of 512×512 pixels. Image data were analyzed using IMARIS 9.3.1 software (Bitplane). The "Filaments" module in IMARIS software was used to quantify the total length, total area, total volume, and number of branch points of microglia. Sholl analysis was used to analyze the morphological complexity of microglia. Each mouse was counted as an independent sample. Two tissue samples were randomly selected from each mouse for imaging and quantitative analysis, with a total sample size of 5 or 6 mice per group. The final morphological analysis of microglia was based on the average values ​​of each animal. Microglia phagocytosis was analyzed by comparing the total number of cells per animal. When microglia are activated, the total fiber length, total fiber area, total fiber volume, number of fiber branch points decrease, and morphological complexity is reduced.

[0043] 5. Experimental Results

[0044] The activity of GABAergic neurons in the DG region of free-moving mice before and after drug administration was detected. Figure 1 (Figure A). Based on the recorded pulse waveform characteristics, GABAergic neurons in the DG region were identified and classified. The results showed that, compared with before administration, the spontaneous pulse firing frequency of GABAergic neurons in the DG brain region was significantly reduced after diazepam administration. These results indicate that long-term diazepam administration leads to a decrease in the activity of GABADG neurons in mice. Figure 1 (Chinese BC).

[0045] To assess whether long-term diazepam administration leads to cognitive impairment in mice, the novel object recognition test and Y-maze test were used to evaluate cognitive function 24 hours after model establishment. As a baseline indicator for assessing inter-individual differences in motor function, the open field test was used to assess the mice's motor ability before cognitive behavioral tests. The open field test results showed no significant differences in motor behavioral indicators among the experimental groups, and no significant differences in motor ability among the groups, thus ruling out the interference of differences in motor ability on the subsequent cognitive test results. Figure 2 (British BC). In the new object recognition experiment, compared with the control group, the proportion of time mice in the diazepam group and the diazepam combined with flumazenil group to explore new objects was significantly reduced, indicating that cognitive ability was impaired. Figure 2(DE). Meanwhile, in the Y-maze test, the spontaneous alternation rate of mice in the diazepam group and the diazepam combined with flumazenil group was also significantly reduced, indicating a declining trend in their spatial working memory ability. The above results combined suggest that long-term diazepam administration leads to cognitive impairment in mice. Figure 2 (FH). Microglia were labeled using ionized calcium-binding aptamer 1 immunofluorescence staining. Figure 2 (I). Since the morphological changes of microglia can reflect their activation state, a semi-automated three-dimensional morphometric analysis was further performed. The results showed that, compared with the control group, after 7 days of treatment with diazepam or diazepam combined with flumazenil, the total fiber length of microglia in the DG region ( Figure 2 (J), surface area ( Figure 2 (middle K) and volume ( Figure 2 The number of dendritic branching points was also significantly reduced (L) and the number of dendritic branching points was also significantly reduced. Figure 2 Sholl analysis further confirmed that the number of radial cross-points around the cell bodies of microglia in the control group was significantly greater than that in the treatment group, suggesting that microglia transformed from a resting branching morphology to an activated state after diazepam treatment. Figure 2 (N).

[0046] To eliminate the interference of individual differences in baseline behavior on the results, an open field test was first used to assess the motor function of the mice. Specific groupings included: Control group: 0.5% sodium carboxymethyl cellulose solution; Arginase inhibitor + control group: 0.5% sodium carboxymethyl cellulose solution + arginase inhibitor, 50 mg / kg; Compared with the arginase inhibitor + diazepam group: arginase inhibitor, 50 mg / kg + diazepam, 5 mg / kg; Diazepam group: Diazepam 5 mg / kg.

[0047] Diazepam is dissolved in 0.5% sodium carboxymethyl cellulose solution. Arginine oxidase inhibitor is dissolved in physiological saline. Diazepam is administered by gavage, while arginine oxidase inhibitor is administered by intraperitoneal injection.

[0048] The results showed no significant differences in the motor ability indicators of the mice in the open field test among the groups, indicating that there was no difference in the motor ability among the groups. Figure 3 (B, C). In the novel object recognition experiment, compared with the control group, arginase inhibitor + control group, and arginase inhibitor + diazepam group, the diazepam group showed a significantly reduced proportion of exploration time for novel objects, indicating that long-term diazepam treatment impaired the recognition and memory abilities of mice, and this impairment was significantly improved after inhibiting arginase synthase 1. Figure 3(D, E). The Y-maze test results showed that the spontaneous change rate in the diazepam group was significantly decreased, indicating impaired spatial working memory. This impairment was significantly improved after inhibiting arginase synthase 1. However, compared with the control group, diazepam group, arginase inhibitor + control group, and arginase inhibitor + diazepam group, there was no significant difference in the number of times mice entered each arm, further indicating that the impairment was significantly improved after inhibiting arginase synthase 1 and that the above behavioral changes were not caused by differences in motor ability. Figure 3 (FH). The above results indicate that inhibiting arginase synthase 1 can significantly improve long-term diazepam-induced cognitive impairment in mice.

[0049] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of arginase inhibitors in the preparation of drugs for the prevention and / or treatment of the side effects of benzodiazepines.

2. The application according to claim 1, characterized in that, The side effects of the diazoxide drugs include at least one of cognitive impairment, drug tolerance, and withdrawal symptoms.

3. The application according to claim 2, characterized in that, The cognitive impairment is impaired spatial working memory.

4. The application according to claim 1, characterized in that, The arginase inhibitor is selected from at least one of nor-NOHA, S-(2-boronethyl)-L-cysteine, 2-amino-6-boronocanoic acid, Numidargistat, OATD-02, and AZD0011.

5. The application according to claim 4, characterized in that, The arginase inhibitor is nor-NOHA, and the dosage of nor-NOHA is 10-100 mg / kg per day.

6. The application according to claim 5, characterized in that, The dosage of nor-NOHA is 30-70 mg / kg per day.

7. The application according to claim 1, characterized in that, The arginase inhibitor and the benzodiazepine drug are administered simultaneously or sequentially.

8. The application according to claim 7, characterized in that, The sequential administration refers to administering an arginase inhibitor before a benzodiazepine.

9. The application according to claim 1, characterized in that, The drug includes arginase inhibitors and pharmaceutically acceptable excipients.

10. The application according to claim 9, characterized in that, The drug is available in either injectable or oral formulation.

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