Neuroprotection by levodopa and carbidopa in the setting of dopaminergic homeostasis after immature brain trauma

Abstract

This invention discloses the application of levodopa and carbidopa in neuroprotection of dopamine metabolic homeostasis after immature brain injury, relating to the field of pharmaceutical technology. Levodopa and carbidopa are used in a 4:1 mass ratio to treat progressive brain atrophy, Tau protein hyperphosphorylation, and cognitive impairment following immature brain traumatic brain injury (TBI). The ratio of levodopa to carbidopa is consistent with that of the clinical combination drug Sinemet®. This application works by reshaping the disordered dopamine metabolic homeostasis after TBI. This invention also discloses an LD / CD dosing regimen perfectly suited to the physiological characteristics of immature brains: using a 4:1 (w / w) ratio consistent with the clinical combination drug Sinemet®, carbidopa inhibits peripheral dopa decarboxylase activity, increasing the central bioavailability of levodopa by 4-5 times and significantly reducing peripheral nausea, vomiting, and other side effects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of levodopa and carbidopa in neuroprotection of dopamine metabolic homeostasis after immature brain injury. Background Technology

[0002] Traumatic brain injury is the leading cause of disability and death among children and adolescents worldwide. Unlike the adult brain, the immature brain is in a critical developmental window of synaptic pruning and myelination. When subjected to external force, it can not only induce acute brain parenchymal injury, but also trigger a secondary pathological cascade that lasts for months. This includes dopamine metabolism biphasic disorder, insufficient inhibitory phosphorylation of glycogen synthase kinase-3β leading to kinase overactivation, and hyperphosphorylation of microtubule-associated protein Tau at Thr231 and Ser404 sites, ultimately leading to progressive brain atrophy, impaired neural network construction, and long-term cognitive impairment.

[0003] Currently, there are no specific neuroprotective drugs for immature brain TBI. Existing treatment options can only treat complications such as increased intracranial pressure and epilepsy. Observation and rehabilitation training are the mainstream strategies. Children with TBI have a 3 to 5 times higher risk of developing intellectual disability and learning disabilities in the long term compared to ordinary children.

[0004] In the current technology, research on dopamine metabolism abnormalities after TBI is mostly limited to exploring basic mechanisms, and no clinically applicable pharmacological solutions have yet been developed. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of levodopa and carbidopa in neuroprotection of dopamine metabolic homeostasis after immature brain injury.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The application of levodopa and carbidopa in neuroprotection of dopamine metabolic homeostasis after immature brain injury involves using levodopa and carbidopa in the treatment of progressive brain atrophy, Tau protein hyperphosphorylation, and cognitive impairment following immature traumatic brain injury. The levodopa and carbidopa are mixed in a 4:1 mass ratio, consistent with the ratio of the clinical combination preparation Sinemet®. The dosage of levodopa is 30-60 mg / kg / day, and the dosage of carbidopa is 7.5-15 mg / kg / day. The first dose is initiated 1-2 hours after TBI, administered once daily via intraperitoneal injection for 7-28 days. This application works by reshaping the disordered dopamine metabolic homeostasis after TBI, rather than simply increasing the total amount of dopamine in the brain.

[0007] Preferably, the optimal dosage of levodopa is 50 mg / kg / day, and that of carbidopa is 12.5 mg / kg / day; this dosage is only the median lethal dose (LD50) of levodopa administered intraperitoneally to rats. 50 =800-1000mg / kg) 5%-6%, less than 1 / 10 LD 50 The safety threshold, and given the immature physiological characteristics of the blood-brain barrier, the dose is at a moderate to low level within the effective dose range, and there were no fatal events or serious organ toxicity during the observation period. The drug is administered after being dissolved in a solvent system consisting of 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% physiological saline.

[0008] Preferred method: This also includes a method for dynamically monitoring dopamine concentration in the brain using high-performance liquid chromatography, the method being implemented according to the following procedure: Sample pretreatment: 1, 3, 7, 14 and 28 days after TBI, the dermal tissue from the injured side was collected and added to pre-cooled 0.1M perchloric acid + 0.1% Na2EDTA + 0.1% Na2S2O5 extraction solution at a w / v ratio of 1:2.5. The tissue was homogenized at 60Hz for 90s × 2 times, sonicated in an ice-water bath for 10 min, and centrifuged at 12000rpm at 4℃ for 15 min. The supernatant was filtered through a 0.22μm needle filter before loading. Chromatographic conditions: EcoPak C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) + guard column of the same specifications was used. The column temperature was 30 ℃, the injection volume was 10 μL, the mobile phase was phosphoric acid aqueous solution (adjusted to pH=4.6 with ammonia): methanol = 90:10 (v / v) isocratic elution, the flow rate was 1.0 mL / min, the fluorescence detector excitation wavelength was 254 nm and the emission wavelength was 338 nm, and the run time was 15 min. Quantitative calculation: Plot a standard curve with dopamine standard concentration on the x-axis and peak area on the y-axis, and calculate using the following formula: Dopamine content (ng / g wet tissue) = (C×V) / M

[0009] Where C is the sample concentration, V is the final volume of 0.5 mL, and M is the amount of tissue weighed. Homeostasis assessment: If the brain dopamine concentration was significantly lower than that of the TBI group without TBI at at least 3, 7 and 28 days after injury after LD / CD intervention, and no unlimited accumulation was observed, then the dopamine metabolic homeostasis remodeling was deemed effective.

[0010] Preferably, the methodological validation of the HPLC method meets the following requirements: Specificity was confirmed to be free of impurities in the dopamine peak. The limit of detection (S / N=3:1) was ≤1.5 ng / mL, the limit of quantitation (S / N=10:1) was ≤5 ng / mL, the intra-day / inter-day precision RSD was ≤2%, the spiked recovery rate was 85%-115%, and the stability of the sample in an autosampler at 4℃ for 24 h was ≤3%.

[0011] Preferably, it also includes a step of assessing cognitive function recovery using behavioral tests, which includes: (1) New object recognition experiment to evaluate recognition memory: Adaptation period: For two consecutive days, rats were placed in a 50cm×50cm×40cm black open box and allowed to explore freely for 5 minutes each day to adapt to the experimental environment; Familiarization period: After the adaptation period, two identical objects (A+A) were placed in the box. The rats were placed with their backs to the objects and the exploration time of the two objects within 5 minutes was recorded (the distance between the nose tip and the object was <2cm or direct contact was counted as exploration). Samples with a total exploration time of <10s were excluded. Testing period: 1 hour after the familiarization period ends, one of the objects is replaced with a new object (A+B). The exploration time of the new / old object within 5 minutes is recorded. The recognition index = (exploration time of the new object / total exploration time) × 100% and the discrimination index = (exploration time of the new object - exploration time of the old object) / total exploration time are calculated to eliminate the interference of the difference in total exploration time between groups on the results. (2) Y-maze new arm experiment to assess spatial working memory: Training period: Randomly select one new arm of a closed Y maze (three arms, 30cm×8cm×15cm, with different visual cues at the arm ends), and allow rats to freely explore the two open arms for 10 minutes; Testing period: 1 hour after the end of the training period, the barriers were removed, all three arms were opened, and rats were placed in from the center. The number of times rats entered each arm and the dwell time within 5 minutes were recorded. The new arm exploration preference was calculated as (new arm dwell time / total exploration time) × 100%, excluding the interference of differences in the total number of entries between groups on motivation.

[0012] Preferred: The behavioral tests were conducted after the drug administration (1 month after injury). The identification index, discrimination index, and new arm exploration preference of the LD / CD intervention group were significantly higher than those of the TBI group without drug administration (p<0.05), and there was no significant difference in total exploration time and total number of entries between the two groups, confirming that cognitive function was restored rather than the exploration motivation was changed.

[0013] Preferred method: also includes molecular mechanism verification steps: the ratios of p-GSK-3β(Ser9) / total GSK-3β, p-Tau(Thr231) / total Tau, and p-Tau(Ser404) / total Tau were detected by Western Blot. After LD / CD intervention, the ratio of p-GSK-3β(Ser9) / total GSK-3β was significantly higher than that of the untreated TBI group (p<0.05), and the ratio of p-Tau / total Tau was significantly lower than that of the untreated TBI group. Moreover, the total Tau protein content was not lower than that of the TBI group, indicating that neuronal survival was preserved.

[0014] Preferred method: Includes a step of assessing brain injury volume using in vivo MRI: One month post-injury, a 9.4T small animal MRI scan is performed with T2WI sequence parameters TR=3500ms, TE=35ms, slice thickness 0.7mm, slice interval 0mm, FOV=35mm×35mm, and matrix 256×256. ITK-SNAP software is used to delineate T2WI high-signal areas and tissue defects layer by layer, based on a single voxel volume of 0.0131mm². 3 The lesion volume was calculated, and the lesion volume in the LD / CD intervention group was reduced by ≥80% compared with the untreated TBI group (p<0.0001).

[0015] Preferably, the immature brain is a mammalian larva 7-14 days after birth, preferably an SD rat 9-10 days after birth; the TBI model is a controlled cortical impaction (CCI) model, with impact parameters of 3mm probe diameter, 5.5m / s velocity, 1.5mm depth, and 50ms duration, and the sham surgery group only undergoes craniotomy without impact.

[0016] A pharmaceutical composition for achieving the aforementioned application comprises 30-60 mg / kg / day equivalent of levodopa, 7.5-15 mg / kg / day equivalent of carbidopa, and a pharmaceutically acceptable carrier, said carrier being the aforementioned 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline solvent system, or a clinically convertible liposome or cyclodextrin encapsulation formulation.

[0017] The application of the high-performance liquid chromatography method for dynamic monitoring of dopamine concentration in the brain in the preparation of a reagent for evaluating the efficacy of TBI in immature brain tissue.

[0018] The beneficial effects of this invention are as follows: 1. This invention discloses an LD / CD dosing regimen that is perfectly adapted to the physiological characteristics of immature brains: using a 4:1 (w / w) ratio consistent with the clinical compound preparation Sinemet®, carbidopa can inhibit peripheral dopa decarboxylase activity, thereby increasing the central bioavailability of levodopa by 4 to 5 times and significantly reducing peripheral nausea, vomiting and other side effects; the matching 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% physiological saline solvent system can effectively dissolve the drug and eliminate the interference of solvent stress on experimental results.

[0019] 2. This invention confirms through dynamic HPLC monitoring that after TBI, brain dopamine exhibits a biphasic imbalance characterized by an early abnormal surge followed by a sustained depletion. LD / CD intervention does not simply increase the total amount of brain dopamine, but rather activates the presynaptic D2 autoreceptor negative feedback through the dopamine produced by the conversion of exogenous L-DOPA, inhibiting the activity of endogenous tyrosine hydroxylase, reducing excessive dopamine synthesis and accelerating metabolic turnover, thus restoring the disordered dopamine metabolism to homeostasis. This fundamentally blocks the pathological cascade of dopamine excess → oxidative stress → GSK-3β deinhibition → Tau hyperphosphorylation.

[0020] 3. This invention uses commercially available clinical drugs, eliminating the need to develop brain-targeted gene delivery systems and avoiding the off-target risks of gene editing and the unknown long-term safety of the developing brain. The dosing regimen, detection methods, and evaluation paradigms can all be directly transferred to preclinical research and subsequent clinical trials, providing a feasible translational solution for drug therapy. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the controlled cortical impaction-induced TBI model in neonatal rats in this invention; Figure 2 This is the dopamine standard curve diagram used in this invention. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0023] Example 1: 1. Establishment of experimental animals and TBI model

[0024] SPF-grade male SD rats, 9-10 days old (PND) and weighing 19-25g, were selected and housed in an environment with a temperature of 22±2℃, humidity of 50±10%, and a 12h light-dark cycle. The mother rats had free access to food and water. The experimental protocol was approved by the Animal Ethics Committee of Chongqing Medical University.

[0025] like Figure 1As shown, a controlled cortical impaction (CCI) model of immature brain TBI was established using the following methods: Anesthesia was administered via intraperitoneal injection of 2.5% tribromoethanol (0.01 mL / g). A 1.5 cm incision was made along the midline of the skull to expose the skull. A 3 mm diameter bone window was created using a dental drill at a point 2 mm to the left of the sagittal line, between the coronal and lambdoid sutures (avoiding dura mater damage). The brain tissue was impacted with a probe of 3 mm diameter at a speed of 5.5 m / s, a depth of 1.5 mm, and a duration of 50 ms. After repositioning the bone fragments, the scalp was sutured. The mice were kept warm until awake and then returned to their mothers. The sham-operated group underwent only craniotomy without impaction.

[0026] 2. Dosage regimen and dosage selection

[0027] 2.1 Solvent and Drug Preparation

[0028] Solvent system formulation: 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% physiological saline (v / v), shake and mix until clear. Weigh levodopa (Sigma D9628) and carbidopa (Sigma C1338), dissolve them in the solvent to a final concentration of levodopa 10 mg / mL and carbidopa 2.5 mg / mL, i.e., LD:CD mass ratio of 4:1, which is completely consistent with the ratio of the clinical compound preparation Sinemet®. Prepare in the dark before use.

[0029] 2.2 Dosing regimen

[0030] The first intraperitoneal injection was initiated 1-2 hours after the TBI group rats were fully awakened. The dosage was levodopa 50 mg / kg / day + carbidopa 12.5 mg / kg / day, with an injection volume of 5 mL / kg. The administration was once daily at 9:00 AM for 28 consecutive days. The subgroup was observed for long-term observation until the drug was discontinued after 28 days of administration. MRI and behavioral assessments were completed one month after the injury.

[0031] The solvent control groups (Sham group and TBI group) were given an equal volume of blank solvent at the same time point to eliminate stress interference from solvent composition and drug administration.

[0032] 2.3 Dosage Selection

[0033] ① Rationality of the ratio: The 4:1 LD / CD ratio is the classic regimen for Parkinson's disease models. Carbidopa, as a peripheral DDC inhibitor, can block 70%-80% of levodopa from being prematurely converted into dopamine in the periphery, thereby increasing its central bioavailability by 4-5 times and reducing peripheral nausea, vomiting and other side effects. ② Dosage safety: LD50 of levodopa administered intraperitoneally in rats 50 The dosage is approximately 800-1000 mg / kg, and the 50 mg / kg dose in this regimen is only the LD50. 50 5%-6%, far lower than 1 / 10 LD50 The safety threshold; multiple studies have confirmed that no fatal events or serious organ toxicity occurred after continuous administration of this dose for 28 days; ③Immature brain adaptability: The blood-brain barrier and liver metabolic enzyme system of PND9-10 rats are not fully mature, and the central penetration rate of drugs is higher than that of adult animals. Therefore, a moderate to low level of the effective dose range (6-100mg / kg) is selected to further reduce safety risks while ensuring efficacy. ④ Observation period verification: Throughout the observation period of this experiment, there were no significant differences in the survival rate, weight gain curve, and general behavior of the LD / CD group rats compared with the solvent control group, confirming the safety of this protocol in developing rats.

[0034] 3. HPLC detection of dopamine concentration in brain tissue

[0035] 3.1 Sample Collection and Pretreatment

[0036] At 1, 3, 7, 14, and 28 days after TBI, patients underwent deep anesthesia via intraperitoneal injection of an excessive amount of tribromoethanol. The brain was then decapitated and the brain tissue was harvested. A 5mm × 5mm × 2mm section of tissue surrounding the lesion was dissected on ice, flash-frozen in liquid nitrogen, and stored at -80°C. Approximately 0.2g of brain tissue was accurately weighed and added to 0.5mL of pre-chilled extraction buffer (0.1M perchloric acid + 0.1% Na₂EDTA + 0.1% Na₂S₂O₅) at a ratio of 1:2.5 (w / v). The tissue was homogenized at 60Hz for 90s using a tissue homogenizer, repeated twice. The homogenate was sonicated in an ice-water bath for 10min, centrifuged at 12000rpm for 15min at 4°C, and the supernatant was carefully transferred to a new centrifuge tube. The volume was brought to 0.5mL with the extraction buffer, vortexed for 30s, filtered through a 0.22μm syringe filter, and transferred to a vial for storage at -80°C until analysis.

[0037] 3.2 Chromatographic conditions

[0038] An EcoPak C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used, with a tandem EcoPak C18 guard column of the same specifications (10 mm × 4.6 mm, 5 μm) at the front end; the column temperature was kept constant at 30 °C; the injection volume was 10 μL; the mobile phase was a mixture of phosphoric acid aqueous solution (pH precisely adjusted to 4.6 with ammonia) and methanol at a ratio of 90:10 (v / v), with isocratic elution; the flow rate was 1.0 mL / min; a fluorescence detector was used, with an excitation wavelength of 254 nm and an emission wavelength of 338 nm, and the run time was 15 min.

[0039] 3.3 Preparation of Standard Curve

[0040] like Figure 2As shown, dopamine standard (Yuanye Biotechnology) was precisely measured to prepare a series of standard working solutions with concentrations of 5, 10, 25, 50, 100, 250, and 500 ng / mL. 10 μL of each solution was injected under the above chromatographic conditions. Weighted least squares regression was performed with the standard concentration as the abscissa (X, ng / mL) and the peak area as the ordinate (Y) to obtain the standard curve equation. The correlation coefficient R0 was required. 2 ≥0.999.

[0041] 3.4 Content Calculation

[0042] Substitute the sample peak area into the standard curve equation to obtain the sample dopamine concentration C (ng / mL), and calculate the dopamine content in brain tissue using the formula: Dopamine content (ng / g wet tissue) = MC × V

[0043] Where V is the fixed volume of 0.5 mL and M is the tissue sample weight (g).

[0044] 3.5 Methodological Validation

[0045] ①Specificity: Compare the chromatograms of the standard, blank solvent, and brain tissue sample to confirm that there are no impurity peaks interfering with the position of the dopamine peak; ② Linear range: Standard curve R 2 ≥0.999, with a linear range covering 5-500 ng / mL; ③ Limit of detection and limit of quantitation: The limit of detection (LOD) is the concentration at a signal-to-noise ratio (S / N) of 3:1, which is approximately 1.5 ng / mL; the limit of quantitation (LOQ) is the concentration at an S / N of 10:1, which is approximately 5 ng / mL. ④ Precision: Using standards of high (400 ng / mL), medium (100 ng / mL), and low (10 ng / mL) concentrations, inject the samples 6 times consecutively on the same day. The intra-day precision RSD should be ≤1.5%; after 3 consecutive days of measurement, the inter-day precision RSD should be ≤2.0%. ⑤ Recovery rate: Brain tissue samples with known dopamine content were treated with high, medium and low concentrations of standards, and the samples were processed according to the pretreatment method. The recovery rate was 92%-107%. ⑥ Stability: The same brain tissue sample was placed in an autosampler at 4℃ for 0, 2, 4, 8, 12 and 24 hours before injection. The peak area RSD was ≤2.5%, confirming that the sample was stable within 24 hours.

[0046] 3.6 Test Results

[0047] The dopamine concentration in the brain of the TBI group showed a typical biphasic imbalance: it increased significantly 1 day after injury, reached its peak on 3 days, and then continued to decline, reaching a level significantly lower than the baseline level of the sham-operated group on 28 days. In contrast, the dopamine concentration in the LD / CD intervention group did not accumulate indefinitely, but was significantly lower than that in the TBI group at 3, 7 and 28 days (p<0.05). This confirms that exogenous levodopa inhibits the excessive synthesis of endogenous dopamine by activating the negative feedback of the presynaptic D2 autoreceptor, thereby reshaping the dopamine metabolic homeostasis.

[0048] 4. Behavioral tests

[0049] All behavioral tests employed an assessor-only single-blind design. Samples were coded before the test and unblinded after the test to eliminate subjective bias.

[0050] 4.1 New Object Recognition Experiment

[0051] The experimental setup consisted of a 50cm×50cm×40cm black open box, and the process was divided into three stages: ① Adaptation period: For two consecutive days, rats were placed in an empty experimental box and allowed to explore freely for 5 minutes each day to fully adapt to the experimental environment; ② Familiarization period: After the adaptation period, two identical objects (A+A) were placed in the box. The rats were placed with their backs to the objects and the exploration time of each object within 5 minutes was recorded (the distance from the nose tip to the object is <2cm or direct contact with the object is counted as exploration behavior). Samples with a total exploration time of <10s were excluded. ③ Testing period: One hour after the familiarization period ends, one of the objects is replaced with a new object (A+B). The exploration time for both the familiar and new objects is recorded within 5 minutes. Calculate: Recognition Index = (Time to explore new objects / (Time to explore new objects + Time to explore old objects)) × 100%

[0052] Discrimination Index = (Time to explore new objects - Time to explore old objects) / (Time to explore new objects + Time to explore old objects)

[0053] Results: The recognition index and discrimination index of the TBI group were significantly lower than those of the Sham group (p<0.001), while the two indices of the LD / CD group were significantly higher than those of the TBI group (p<0.01). There was no significant difference in the total exploration time among the three groups. This excluded the interference of differences in exploration motivation on the results and confirmed that LD / CD effectively reversed the recognition memory deficit caused by TBI.

[0054] 4.2Y Maze New Arm Experiment

[0055] The Y-maze consists of three arms with equal included angles (30cm × 8cm × 15cm), each with different visual cues at its end. The process is divided into two stages: ① Training period: Randomly select one arm as the new arm, close it with a baffle, and allow the rat to explore freely in the two open arms for 10 minutes; ② Testing period: One hour after the training period ended, remove the barriers, open all three arms, and place the rats into the maze from the center. Record the number of times the rats entered each arm and the dwell time within 5 minutes (complete entry of all four limbs into the arm is counted as one entry). Calculate: New player exploration preference = (New player dwell time / Total exploration time) × 100%

[0056] Results: The new arm exploration preference of the TBI group was significantly lower than that of the Sham group (p<0.05), while the new arm exploration preference of the LD / CD group was significantly higher than that of the TBI group (p<0.05). There was no significant difference in the total number of entries among the three groups. After excluding the interference of motor ability and exploration motivation, it was confirmed that LD / CD effectively restored the spatial working memory deficit caused by TBI.

[0057] Example 5: MRI Brain Injury Volume Assessment

[0058] One month post-injury, rats underwent in vivo imaging using a 9.4T small animal MRI scanner (Bruker BioSpec94 / 30USR). Rats were anesthetized with 2% isoflurane before scanning, and respiratory rate (60-80 breaths / min) and body temperature (37±0.5℃) were monitored. The scanning sequence was T2WI with the following parameters: TR=3500ms, TE=35ms, slice thickness=0.7mm, interslice spacing=0mm, FOV=35mm×35mm, matrix=256×256, NEX=3, covering the entire brain region from the prefrontal cortex to the cerebellum.

[0059] Damage volume analysis was performed using ITK-SNAP 3.8.0 software: regions of interest (ROIs) were delineated layer by layer in T2WI high signal and tissue defect areas, with a single voxel volume of 0.7mm × 0.137mm × 0.137mm ≈ 0.0131mm. 3 Damage volume = number of ROI voxels × 0.0131 mm 3 Simultaneously, the whole brain outline is drawn to calculate the whole brain volume, and the percentage of the damaged volume relative to the whole brain volume is calculated.

[0060] Results: No abnormal signals or tissue defects were observed in the Sham group; the lesion volume in the TBI group was 58.91±0.77 mm. 3 The lesion volume accounted for 6.47±0.08% of the total brain volume; the lesion volume in the LD / CD group decreased to 9.81±0.19 mm. 3 The brain volume was 1.168±0.023%, which was about 83% smaller than that of the TBI group (p<0.0001), confirming that LD / CD significantly reduced the progressive brain atrophy after TBI in immature brains at the macrostructural level.

[0061] 6. Molecular mechanism verification

[0062] Skin tissue was harvested from the injured side. Total protein was extracted using RIPA lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor. Protein concentration was determined by the BCA method. 30 μg of protein was loaded onto a 11% SDS-PAGE gel electrophoresis system. The gel was then wet-transferred to a PVDF membrane at 300 mA on ice for 120 min. After blocking with 5% BSA, the membrane was incubated overnight at 4°C with primary antibody: DDC (1:1000, Abcamab3905), p-GSK-3β (Ser9) (1:1000, CST#9323), and total GSK-3β. β (1:1000, CST#12456), p-Tau(Thr231) (1:1000, Abcamab151559), p-Tau(Ser404) (1:1000, Abcamab92676), total Tau (1:1000, Abcamab254256), GAPDH (1:5000, CST#5174), after washing with TBST, were incubated with HRP-labeled secondary antibody, and after ECL development, the gray values ​​were analyzed using ImageLab software.

[0063] result: ① DDC expression was significantly upregulated in the TBI group 1 day after injury, preceding Tau phosphorylation; DDC expression was further increased in the LD / CD group, suggesting a compensatory effect after suppression of endogenous synthesis; ②The ratio of p-GSK-3β(Ser9) / total GSK-3β in the TBI group was significantly lower than that in the Sham group, while the ratio in the LD / CD group was significantly higher than that in the TBI group (p<0.05), confirming that GSK-3β activity was inhibited; ③ The ratios of p-Tau(Thr231) / total Tau and p-Tau(Ser404) / total Tau in the TBI group were significantly higher than those in the Sham group, while both ratios in the LD / CD group were significantly lower than those in the TBI group (p<0.05), confirming that Tau hyperphosphorylation was blocked. ④ The total Tau protein content in the LD / CD group was significantly higher than that in the TBI group (p<0.05), suggesting that neuronal survival was preserved rather than non-specific degradation of Tau protein.

[0064] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of levodopa and carbidopa in the neuroprotection of dopamine metabolic homeostasis after immature brain injury, characterized in that, Levodopa and carbidopa are used in a drug to treat progressive brain atrophy, Tau protein hyperphosphorylation, and cognitive impairment following immature brain traumatic brain injury (TBI). The levodopa and carbidopa are mixed in a 4:1 mass ratio, consistent with the ratio of the clinical combination preparation Sinemet®. The application works by reshaping the disordered dopamine metabolic homeostasis after TBI.

2. The application of levodopa and carbidopa according to claim 1 in the neuroprotection of dopamine metabolic homeostasis after immature brain injury, characterized in that, The drug is administered after being dissolved in a solvent system consisting of 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% physiological saline.

3. The application of levodopa and carbidopa according to claim 2 in the neuroprotection of dopamine metabolic homeostasis after immature brain injury, characterized in that, This also includes a method for dynamically monitoring dopamine concentration in the brain using high-performance liquid chromatography, which is implemented according to the following procedure: Sample pretreatment: Take the dermal tissue from the injured side at 1d, 3d, 7d, 14d and 28d after TBI, add pre-cooled 0.1M perchloric acid + 0.1% Na2EDTA + 0.1% Na2S2O5 extraction solution at a w / v ratio of 1:2.5, homogenize with a tissue homogenizer, sonicate in an ice water bath, centrifuge, filter the supernatant through a needle filter and then load the sample; Chromatographic conditions: An EcoPak C18 reversed-phase column with a guard column of the same specifications was used, and the mobile phase was phosphoric acid aqueous solution: methanol = 90:10 isocratic elution. Quantitative calculation: Plot a standard curve with dopamine standard concentration on the x-axis and peak area on the y-axis, and calculate using the following formula: Dopamine content = (C × V) / M Where C is the sample concentration, V is the final volume of 0.5 mL, and M is the amount of tissue weighed. Homeostasis assessment: If the brain dopamine concentration was significantly lower than that of the TBI group without TBI at at least 3, 7 and 28 days after injury after LD / CD intervention, and no unlimited accumulation was observed, then the dopamine metabolic homeostasis remodeling was deemed effective.

4. The application of levodopa and carbidopa according to claim 3 in the neuroprotection of dopamine metabolic homeostasis after immature brain injury, characterized in that, The methodological validation of the HPLC method must meet the following requirements: Specificity was confirmed to be free of impurities in the dopamine peak, with a detection limit ≤1.5 ng / mL, a quantitation limit ≤5 ng / mL, an intra-day / inter-day precision RSD ≤2%, a spiked recovery rate of 85%-115%, and a sample stability RSD ≤3% in an autosampler at 4℃ for 24 hours.

5. The application of levodopa and carbidopa according to claim 1 in the neuroprotection of dopamine metabolic homeostasis after immature brain injury, characterized in that, It also includes a step of assessing cognitive function recovery using behavioral tests, which includes: (1) New object recognition experiment to evaluate recognition memory: Adaptation period: Rats were placed in a black open box to explore freely and adapt to the experimental environment; Familiarization period: After the adaptation period, two identical objects were placed in the box, and the rats were placed with their backs to the objects. The exploration time of the two objects was recorded. Testing period: After the familiarization period, one of the objects is replaced with a new object. The exploration time for the new / old object is recorded again. The recognition index is calculated as (exploration time of the new object / total exploration time) × 100%, and the discrimination index is calculated as (exploration time of the new object - exploration time of the old object) / total exploration time. The interference of the difference in total exploration time between groups on the results is eliminated. (2) Y-maze new arm experiment to assess spatial working memory: Training period: One new arm of the Y maze was randomly closed, allowing rats to explore freely in the two open arms; Testing period: After the training period, the barriers were removed, all three arms were opened, and rats were placed in from the center. The number of times rats entered each arm and the dwell time were recorded. The new arm exploration preference was calculated as (new arm dwell time / total exploration time) × 100%, excluding the interference of differences in the total number of entries between groups on motivation.

6. The application of levodopa and carbidopa according to claim 5 in the neuroprotection of dopamine metabolic homeostasis after immature brain injury, characterized in that, The behavioral tests were conducted after the administration of the drug. The identification index, discrimination index, and new arm exploration preference of the LD / CD intervention group were significantly higher than those of the TBI group without drug administration. The total exploration time and total number of entries were not significantly different from those of the TBI group, confirming that cognitive function was restored rather than the exploration motivation was changed.

7. The application of levodopa and carbidopa according to claim 1 in the neuroprotection of dopamine metabolic homeostasis after immature brain injury, characterized in that, It also includes molecular mechanism verification steps: the ratios of p-GSK-3β(Ser9) / total GSK-3β, p-Tau(Thr231) / total Tau, and p-Tau(Ser404) / total Tau were detected by Western Blot. After LD / CD intervention, the ratio of p-GSK-3β(Ser9) / total GSK-3β was significantly higher than that of the untreated TBI group, and the ratio of p-Tau / total Tau was significantly lower than that of the untreated TBI group. Moreover, the total Tau protein content was not lower than that of the TBI group, indicating that neuronal survival was preserved.

8. The application of levodopa and carbidopa according to claim 1 in the neuroprotection of dopamine metabolic homeostasis after immature brain injury, characterized in that, It also includes the steps of assessing brain injury volume with in vivo MRI: small animal MRI scans are performed after injury, and ITK-SNAP software is used to delineate T2WI high signal and tissue defect areas layer by layer to calculate the injury volume.

9. A pharmaceutical composition for achieving the application according to any one of claims 1-8, characterized in that, It contains 30-60 mg / kg / day equivalent of levodopa, 7.5-15 mg / kg / day equivalent of carbidopa, and a pharmaceutically acceptable carrier, wherein the carrier is the above-mentioned 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% physiological saline solvent system, or a clinically convertible liposome or cyclodextrin inclusion complex formulation.

10. The application of the method for dynamic monitoring of dopamine concentration in the brain by high performance liquid chromatography as described in claim 3 in the preparation of a reagent for evaluating the efficacy of TBI in immature brain tissue.

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