Method for diagnosing fragile X syndrome by using positron emission tomography probe targeting Sigma-1 receptor

Abstract

The invention discloses a method for specifically targeting a positron emission tomography (PET) probe (+)-[18F] YL1 of a Sigma-1 receptor (Sig-1R) and application of the positron emission tomography (PET) probe (+)-[18F] YL1 in fragile X syndrome (FXS). The method comprises the following steps: 1, synthesizing and characterizing the (+)-[18F] YL1; 2, testing cognitive impairment and engraving plate compulsive behaviors of the FXS mouse model; 3, (+)-[< 18 > F] YL1 positron emission tomography (PET) of targeted Sig-1R; and 4, analyzing the correlation between the PET quantitative result of each brain region and the cognitive impairment and the severity of the inscribing forcing. According to the method, (+)-[18F] YL1 PET quantification is mainly carried out on each brain region, correlation analysis is carried out on each brain region and cognitive impairment and engraving plate forcing severity of different dimensions, so that Sig-1R expression change of each brain region in FXS is measured, and understanding of a potential relationship between a Sig-1R-related brain function network and external behavior performance is deepened. The invention provides a new thought for the field, and is expected to provide a new target insight and an effective iconography means for diagnosis and treatment evaluation of FXS. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of medical imaging, and particularly relates to a method for using a positron emission tomography probe targeting a Sigma-1 receptor for diagnosing fragile X syndrome. Background Art

[0002] FXS is the leading genetic cause of intellectual disability and autism spectrum disorders (ASD). Its main pathogenesis is due to the silencing of the Fmr1 gene on chromosome X, which leads to the loss of FMRP, which is essential for brain development. Although many hypotheses for the diagnosis or treatment of FXS have been proposed, these hypotheses are far from being exhaustive and satisfactory, so further efforts are needed to decipher the complexity of FXS pathophysiology and advance the development of therapeutic drugs.

[0003] FMRP deficiency is associated with endoplasmic reticulum stress or dysfunction of many ion channels, especially abnormal calcium ion flow, leading to impaired synaptic function and structure. In FXS animal models, changes in synaptic function are considered to be the basis of this neurodevelopmental disorder. Geng et al. found that FMRP interacts with VDAC to regulate the formation and function of endoplasmic reticulum-mitochondrial contact sites (ER-mitochondrial contact sites, ERMCS), and named them MAM. This structure is sensitive to mitochondrial calcium (mito-Ca 2+ ) homeostasis. fFMRP-deficient cells exhibit excessive ERMCS formation and ER-mitochondrial Ca 2+ Transfer, and promote mitochondrial ROS accumulation and dysfunction. Therefore, regulating MAM-related signaling pathways may help rescue FMRP function. The various regulatory mechanisms of Sig-1R, which is mainly enriched in MAM, are closely related to the pathophysiology of FXS and are considered to be one of the potential diagnostic and therapeutic targets for FXS.

[0004] PET is an imaging technology that can display biological molecule metabolism and receptor binding in vivo. PET imaging with Sig-1R as the target can track the expression changes of Sig-1R in the disease state in vivo. The present invention has developed a PET molecular probe (+)-[ 18 F]YL1, and performed PET / CT-MRI imaging on FXS model mice to explore the changes in Sig-1R expression in the brain of FXS model mice, and through correlation analysis between PET quantitative results and behavioral phenotypes, to explore the application prospects of this probe in FXS. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defect of the prior art that there is a lack of effective imaging means for diagnosing fragile X syndrome, and to provide a method for diagnosing fragile X syndrome by using a positron emission tomography probe targeting Sigma-1 receptors.

[0006] The present invention mainly solves the above technical problems through the following technical solutions. One of the technical solutions of the present invention is: an imaging method for auxiliary diagnosis of fragile X syndrome, comprising: 1. (+)-[ 18 F]YL1 synthesis and characterization; 2. Cognitive impairment and stereotyped compulsive behavior testing in FXS mouse model; 3. (+)-[ 18 F]YL1 positron emission tomography (PET); 4. Correlation analysis between the PET quantitative results of each brain region and the severity of cognitive impairment and stereotypy.

[0007] When the mouse changes as follows, it indicates that the (+)-[ 18 F]YL1 can assess the disease:

[0008] (1)(+)-[ 18 F]YL1 PET imaging showed increased uptake in the brain of FXS mice.

[0009] (2)(+)-[ 18 The correlation analysis between the quantitative results of F]YL1 PET and various brain regions related to spatial cognition, social cognition and stereotyped repetition showed a strong correlation.

[0010] The positive and progressive effects of the present invention are:

[0011] The present invention develops a probe (+)-[ 18 F]YL1 and its synthesis method, and use it to non-invasively evaluate the expression level of Sig-1R in the brain of Fmr1 gene knockout mice at the living level, and explore the correlation between Sig-1R expression level and behavior in FXS, thereby deepening the understanding of the relationship between Sig-1R-related brain functional networks and external behavioral manifestations, and providing a basis for Sig-1R as a target for diagnosis and drug treatment of FXS. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The structure of a representative tracer targeting the Sigma-1 receptor;

[0013] Figure 2 Design a flow chart for the experiment;

[0014] Figure 3 for (+)-[ 18 F] Radiosynthetic route and HPLC quality control results of YL1;

[0015] Figure 4 for (+)-[ 18 F] Metabolic stability results of YL1 in rat brain;

[0016] Figure 5 Schematic diagram of mouse behavioral testing and its test results;

[0017] Figure 6 for (+)-[ 18 F]YL1 PET-MRI images and quantification and ex vivo autoradiography images and quantification results;

[0018] Figure 7 Staining of the cortex and hippocampus of WT and FXS mice;

[0019] Figure 8 Different dimensions of cognitive impairment and severity of stereotypy and compulsion are associated with different brain regions (+)-[ 18 F] Correlation analysis of YL1 uptake. DETAILED DESCRIPTION

[0020] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] 1. Experimental design

[0022] First, the Fmr1 gene was knocked out using Crispr / Cas9 technology to simulate the gene methylation transcriptional silencing mechanism in humans. Then, the gene was identified and verified by PCR, and a mouse model that can be used for experiments was successfully constructed. Subsequently, mouse behavioral tests, (+)-[ 18 F]YL1 PET-MRI scanning, autoradiography, and immunofluorescence staining were performed for (+)-[ 18 The feasibility of F]YL1 for the diagnosis and treatment of FXS was evaluated. Figure 2 .

[0023] 2. (+)-[ 18 Radiosynthesis and HPLC procedures of F]YL1

[0024] Experimental method: (+)-[ 18 F]YL1 was synthesized by direct nucleophilic substitution labeling (see Figure 3 A), and the radiochemical purity of the product was checked by HPLC and co-injection.

[0025] Experimental results: The radioactive peak of the final product is similar to that of the standard (+)-[ 19The UV absorption peaks of F]TZ3108 were 14.7 min and 13.9 min respectively, and the retention times were basically the same (see Figure 3 BC). The average radiochemical purity of the final product was 98.9±2.0%.

[0026] 3. Biodistribution experiment

[0027] Experimental method: 20 SD rats weighing about 200 g were randomly divided into 5 groups (5-min group, 30-min group, 30-min blocking group, 60-min group and 120-min group), with 4 rats in each group. 2.2-3.7 MBq (+)-[ 18 F]YL1 probe 5, 30, 60, and 120 minutes later (the 30-minute blocking group required a tail vein injection of 1 mg / kg SA4503 5 minutes before probe injection), the SD rats were killed by cervical dislocation and the organs of interest (brain, blood, heart, liver, kidney, lung, spleen, pancreas, muscle, bone, intestine) were quickly dissected. The brain tissue was peeled off and quickly placed on ice for brain region segmentation, which was divided into cortex, striatum, hippocampus, thalamus, cerebellum, brainstem and other brain tissues. After gently wiping the blood on the surface of the organ with paper, put it into the bottom of the corresponding γ counter matching flow tube and weigh it. The weight of the tissue is the difference between the weight before and after the sample is loaded into the γ counter matching flow tube. The γ counter is used to count the weight of each organ (+)-[ 18 F] Radioactivity of YL1. All measurements were background subtracted and decay corrected to the time of injection and then averaged. Data are expressed as percentage of injected dose per gram of tissue (%ID / g).

[0028] Experimental results: As shown in Table 1, the expression of the probe (+)-[ 18 F] Biodistribution of YL1, and uptake was calculated as %ID / g. Specific uptake of the probe was assessed at 30 min after pretreatment with the blocker SA4503 (1 mg / kg) 5 min before tracer injection. As a recognized, highly potent and selective Sig-1R agonist, SA4503 has been shown to have neuroprotective effects.

[0029] (+)-[ 18 F]YL1 rapidly penetrates the blood-brain barrier and accumulates in the rat brain. 18 The brain uptake (%ID / g) of the probe (+)-[ 18 F]YL1 has good uptake capacity and stability in the rat brain. 18Compared with the brain uptake of F]TZ3108 (1.586±0.505 and 1.321±0.082 at 5 and 60 minutes, respectively, the K of Sig-1R i =0.48 nM), (+)-[ 18 F]YL1 has a higher potency (K for Sig-1R i =0.14 nM), which may help the probe (+)-[ 18 F]YL1 binds stably to Sig-1R and enhances the imaging effect; (+)-[ 18 The higher brain uptake of F]YL1 may be due to variability in biodistribution experiments or (+)-[ 18 F]YL1 has a higher binding affinity to Sig-1R. In addition, we found that at 5 minutes, multiple brain regions (including cerebellum, brainstem, cortex, thalamus, and hippocampus) showed a strong affinity for (+)-[ 18 The uptake of F]YL1 was relatively high, with the highest uptake in the cerebellum (2.557±0.251) and the lowest uptake in the striatum (1.905±0.204). 18 F]YL1 was gradually washed out in the brain, but still maintained relatively high levels in all brain regions at 120 min; at 120 min, the regional brain uptake in the cortex, striatum, hippocampus, thalamus, cerebellum, and brainstem were 1.702±0.233, 1.435±0.180, 1.615±0.172, 1.572±0.138, 1.607±0.215, and 2.174±0.281, respectively. In the blocking study, compared with the 30-min control group without blocking compounds, the total brain uptake of rats pretreated with 1 mg / kg SA4503 increased from 1.627±0.097 to 1.659±0.063, among which the uptake in the cortex, hippocampus, striatum and thalamus increased, especially the uptake in the cortex increased significantly from 1.723±0.168 to 2.149±0.094 (p<0.01), and the uptake in the hippocampus increased significantly from 1.551±0.137 to 1.741±0.069 (p<0.05). This may be due to the fact that SA4503 affects the internalization process of Sig-1R, increases the availability of Sig-1R in these brain regions, and binds to Sig-1R subtypes in a competitive binding manner, while the affinity of the probe to Sig-1R (K i(+)-TZ3108-Sig-1R =0.14nM) is superior to SA4503 (K iSA4503-Sig-1R=4.6 nM); however, the uptake in the cerebellum and brainstem decreased, with the uptake in the cerebellum significantly decreasing from 1.736 ± 0.099 to 1.389 ± 0.109 (p < 0.01), and the uptake in the brainstem significantly decreasing from 1.900 ± 0.233 to 1.415 ± 0.081 (p < 0.01). This may be due to the wider distribution of Sig-1R in the cerebellum and brainstem, suggesting that the probe binds more specifically to Sig-1R in these brain regions.

[0030] Consistent with the distribution of Sig-1R in peripheral tissues, the lungs showed a strong affinity for (+)-[ 18 The initial uptake of F]YL1 was the highest, i.e., 16.620±1.778 at 5 minutes after injection; by 30 minutes, the activity in the lungs had dropped to 5.971±1.671, and at 120 minutes, the activity in the lungs had dropped to 2.165±0.347. The activity in blood was very low (0.042 ± 0.008 after 5 min, decreasing to 0.018 ± 0.004 after 60 min and 0.016 ± 0.007 after 120 min); the activity in liver increased from 1.778 ± 0.374 after 5 min to 3.501 ± 0.335 after 120 min, indicating that the probe was mainly metabolized by the liver. No in vivo defluorination was observed: the bone resorption rate was 0.690 ± 0.177 at 5 min and 0.551 ± 0.076 at 120 min; the total bone resorption was low, with negligible changes from 5 to 120 min. Liver defluorination was observed at all time points , lungs, kidneys, spleen, and pancreas. In the blocking study, the uptake of the probe in the pancreas, kidneys, and lungs of rats pretreated with 1 mg / kg SA4503 was reduced compared to the 30-minute control group without blocking compounds, especially the uptake in the pancreas, which was significantly reduced from 2.002±0.196 to 1.030±0.125 (p<0.001). This is consistent with the distribution of Sig-1R in these peripheral organs. The increased uptake observed in the liver (from 2.145±0.660 to 3.225±0.232) may reflect the increased clearance when target tissue uptake is blocked.

[0031] Observed (+)-[ 18 The distribution of (+)-[ 18 F]YL1 can penetrate the blood-brain barrier (BBB) ​​and accumulate in brain regions where Sig-1R expression levels are known to be high. 18 F]YL1 is rapidly cleared from the blood and shows specific uptake in Sig-1R-enriched tissues without obvious defluorination in vivo. Table 1 is (+)-[18 F] Results of biodistribution experiment of YL1 in rats:

[0032] 4. Metabolic stability experiment

[0033] Experimental methods:

[0034] (1) In vitro metabolic stability test:

[0035] Take 1-5 μL of tracer (+)-[ 18 F]YL1, after being diluted 1000 times with the mobile phase, was allowed to stand at room temperature for 60 minutes, 50 μL was injected into the analytical HPLC column (mobile phase was phase A: deionized H2O containing 0.1% trifluoroacetic acid, phase B: acetonitrile, 25% acetonitrile + 75% H2O; flow rate was 1 mL / min; UV wavelength: 254 nm), and 15 minutes of HPLC-grade flow-through was collected at a time interval of 1 minute / tube. Each tube was read for 30 seconds using a γ counter. The counts (Counts) of each tube were decay-corrected to the injection time and connected to form a curve. The ratio of the peak area of ​​the product peak and the metabolic peak to the total area under the curve was calculated by the software Origin 2022 (OriginLab, Northampton, Massachusetts, USA). This group of data is the in vitro control group (In vitro control group) injected for 60 minutes for in vivo metabolic stability.

[0036] (2) In vivo metabolic stability experiment:

[0037] ① Treatment of experimental mice: Prepare an adult male SD rat weighing about 200 g and inject (+)-[ 18 F] YL1 probe (30-37 MBq), SD rats were killed by cervical dislocation under deep anesthesia with 2-3% isoflurane after 5, 30 and 60 minutes, and then the brain was quickly removed by opening the chest.

[0038] ② Brain sample processing: weigh, cut into pieces, put into a mortar, add an appropriate amount of liquid nitrogen to grind, add 1-2mL of phosphate buffer saline (PBS) containing protease inhibitors to continue grinding, take the homogenate and put it into a 2mL centrifuge tube, place it in a 4℃ refrigerated centrifuge for 5 minutes (10000g / min). After centrifugation, separate the supernatant and precipitate. Take the supernatant and cold acetonitrile at a volume ratio of 2:3 and shake and mix well, then centrifuge at high speed for 5 minutes (10000g / min). Take 100μL of the supernatant after centrifugation and mix it with 100μL of the mobile phase at a ratio of 1:1, filter it with a 0.22μm filter membrane, and take 50μL for HPLC.

[0039] ③On-machine reading: After the supernatant was injected into the analytical HPLC column (mobile phase A: deionized H2O containing 0.1% trifluoroacetic acid, phase B: acetonitrile, V (acetonitrile): V (H2O) = 25:75; flow rate is 1 mL / min; UV wavelength: 254 nm), 15 minutes of HPLC-grade flow-through was collected at a time interval of 1 minute / tube. Each tube was read for 30 seconds using a γ counter. The counts (Counts) of each tube were decay-corrected to the injection time and connected to form a curve. The ratio of the peak area of ​​the product peak and the metabolic peak to the total area under the curve was calculated by the software Origin 2022 (OriginLab, Northampton, Massachusetts, USA).

[0040] Experimental results: The metabolic stability of the probe is shown in Figure 4 . Tracer (+)-[ 18 The in vitro stability results of F]YL1 suggest that its properties are still quite stable even after 60 minutes of incubation in the mobile phase. According to the previous quality control analysis results of this probe, the peak that appears at 9 minutes is the product peak, i.e., (+)-[ 18 F]YL1, and the peaks that appeared at other times were metabolite peaks. 18 After 15, 30 and 60 minutes of treatment with F]YL, the percentages of the product peak were 94.23%, 90.09% and 90.74%, respectively, showing a slowly decreasing trend, which indicates that the probe is relatively stable in in vivo imaging.

[0041] 5. Behavioral assessment

[0042] Since the symptoms of Fragile X syndrome are diverse, we studied the manifestations of Fragile X syndrome from three main aspects: cognition, social interaction, and stereotyped repetition.

[0043] (1) Open field test

[0044] Experimental methods: To evaluate the spontaneous activity, anxiety level, and spatial cognition of WT and Fmr1 KO mice, we performed an open field test in a black, light-proof cube box (50 cm × 50 cm × 40 cm) made of acrylic resin. The mice were placed in the center of the open field, and a series of related behaviors were automatically tracked by VisuTrack software for 10 minutes. The total movement distance, immobility time, and the proportion of the time spent in the center area to the total time spent were measured. After each experiment, the surface of the open field was wiped with 75% ethanol to prevent odor crosstalk between two adjacent experiments.

[0045] Results: Several specific behaviors of mice in the open field were evaluated, including the total distance moved, the average speed of movement, the time spent immobile during the test, and the proportion of the time spent in the central area of ​​the open field to the total test time. Compared with WT mice in the same litter, the total distance moved by Fmr1 KO mice was significantly shorter during the same test time (WT: 5483.34±344.95cm; Fmr1 KO: 4390.21±202.76cm; p<0.05)( Figure 5 A), the immobility time was longer (WT: 123.13±11.30s; Fmr1 KO: 180.21±15.14s; p<0.01) ( Figure 5 B), which indicates that Fmr1 KO mice have reduced exploratory behavior in the open field and are less anxious about being exposed to an open environment. In addition, based on the fact that mice will naturally approach the edge of the field in a novel environment, this trait is called tactility, which protects mice from invasion by outsiders. Therefore, the time spent in the central area is a reflection of the spatial cognitive ability of mice. Normal mice will avoid the open environment, quickly leave the central area, and move around the periphery. In this experiment, Fmr1 KO mice spent more time in the central area of ​​the open field than WT mice (WT: 7.82±0.78%; Fmr1 KO: 12.58±1.31%; p<0.01)( Figure 5 C), which indicates that Fmr1 KO mice have poor cognitive ability in novel environments and may have spatial cognition impairment.

[0046] (2) New object recognition experiment

[0047] Experimental methods: The memory and learning ability of WT and Fmr1 KO mice were evaluated by novel object recognition test. One day before the experiment (habituation phase), mice were placed in a 35 cm × 35 cm × 25 cm object-free test box to explore freely for 30 minutes to familiarize themselves with the environment. On the first training day (familiarization phase), two identical blocks (referred to as blocks FamiliarA and FamiliarA′) were placed on one side of the test box. The mice were placed in the test box from the other side with their backs to the blocks and allowed to explore freely for 10 minutes. After 24 hours, the recognition test phase began. Block B was replaced with another new block that had never been touched (referred to as block Novel B). The mice were placed in the test box again in the same way and at the same location to explore for 10 minutes. VisuTrack software was used to record and analyze the sniffing observation time of the mice on the two blocks during the familiarization phase and the test phase.

[0048] Calculate the preference ratio of mice to different blocks:

[0049] Novelty preference = sniffing time on new blocks ÷ sum of sniffing time on new blocks and familiar blocks × 100

[0050] Familiarity preference = sniffing time on familiar blocks ÷ sum of sniffing time on new blocks and familiar blocks × 100

[0051] Calculate the discrimination ratio of mice to different blocks:

[0052] Discrimination index = (sniffing time for new blocks - sniffing time for familiar blocks) ÷ sum of sniffing time for new blocks and familiar blocks × 100

[0053] Results: To further explore the cognitive abilities of Fmr1 KO mice, we performed a novel object recognition test based on the spontaneous tendency of mice to spend more time exploring novel objects rather than familiar objects. During the familiarization phase, neither Fmr1 KO mice nor their littermates WT mice showed a clear preference for the blocks placed in the test box (WT ObjectA preference : 56.35±2.35; KO objectApreference :57.20±3.29; p=0.8383)( Figure 5 D). However, as shown in the figure, in the test phase, when one of the blocks was replaced by a new block, the discrimination index of the Fmr1 KO mouse group was significantly lower than that of the WT group (WT Novelpreference :77.77±2.68;KO Novelpreference : 61.07±3.71; p<0.05; WT Familiarpreference : 22.23±2.68; KO Novelpreference : 38.93±3.71; p<0.05; WT discrimination index: 55.54±5.35%; KO discrimination index: 22.14±7.41%, p<0.05)( Figure 5 EF), indicating that Fmr1 KO mice have impaired discrimination between familiar and novel objects compared with WT mice

[0054] (3) Three-Box Social Experiment

[0055] Experimental methods: To study the social behavior and cognitive ability of WT and Fmr1 KO mice, we conducted a three-box social experiment in a rectangular test box (60 cm × 45 cm × 22 cm). To eliminate the influence of visual orientation, two black cylindrical social cages with longitudinal transparent plastic strips were placed diagonally on both sides of the chamber.

[0056] The experiment was conducted in three stages. In the habituation stage (10 minutes), the test mice were placed in the central chamber with the cage door open and allowed to freely explore the three-chamber apparatus in an empty cage. Then the sociability test stage (10 minutes) was carried out, in which a stimulus mouse (StrangerA) of the same strain as the test mouse was placed in one of the social cages, and there was no previous interaction, while the other cage remained empty. In this stage of the experiment, the test mouse was placed in the central chamber with the cage door open, and was allowed to move freely and interact with the stimulus mouse. Finally, the social novelty preference test (10 minutes) was carried out, and another new stimulus mouse (Stranger B) was placed in the empty social cage in the previous stage. The rest of the operations were the same as the sociability test stage. After each stage, the test box and social cage were wiped clean with 75% ethanol. The number and duration of mutual contact between the test mouse and the empty cage, StrangerA or B were measured using VisuTrack software, and the contact range was defined as 4 cm around the social cage.

[0057] Calculate the social preference of mice during the sociability test phase:

[0058] Sociability index = the time the mouse explores the stimulus mouse or the empty cage / the total time the mouse explores the stimulus mouse and the empty cage × 100

[0059] Calculate the social novelty preference of mice in the social novelty preference test phase:

[0060] Social novelty preference index = the exploration time of mice towards new stimulus mice or familiar stimulus mice / the total exploration time of mice towards new and familiar stimulus mice × 100

[0061] Experimental results: To test the social interest and social memory of WT and Fmr1 KO mice, we conducted a three-box social experiment. In the sociability test phase, by comparing the exploration time of experimental mice with unfamiliar mice and empty cages, we found that Fmr1 KO mice, like WT mice, preferred to interact with unfamiliar mice rather than empty cages ( Figure 5 GH). Subsequently, in the social novelty preference test phase, the exploration time of the experimental mice with the familiar unfamiliar mice in the previous phase and the new unfamiliar mice were compared. The results showed that WT mice preferred to explore the new unfamiliar mice, while Fmr1 KO mice explored the new unfamiliar mice significantly less than the familiar mice (p<0.05) ( Figure 5 IJ). The above results indicate that Fmr1 KO mice have social cognitive impairment.

[0062] (4) Buried beads experiment

[0063] Experimental methods: We conducted a bead burying experiment to investigate whether WT and Fmr1 KO mice have stereotyped and compulsive behaviors. A 6-cm-thick corncob bedding was laid in a new cage, and 12 glass marbles with a diameter of 20 mm were evenly arranged in a "3×4" pattern on the flattened bedding, with each marble separated by 5 cm. Mice were placed in the cage one by one, and the number of glass marbles buried by the mouse within 30 minutes was recorded. If 2 / 3 or more of the volume of the glass marble was covered by the bedding, it was considered buried.

[0064] Experimental results: To explore the stereotyped and compulsive behaviors of Fmr1 KO mice, we performed a bead burying test. The cage was padded with 6 cm deep corn cob bedding, on which 12 glass marbles were placed at equal distances, allowing the mice to freely explore the new cage without any interference. After 30 minutes, the number of marbles buried by WT mice and Fmr1 KO mice was significantly different by unpaired t-test (WT: 5.00±0.89; KO: 8.16±0.8; p<0.05) ( Figure 5 KL), indicating that Fmr1 KO mice exhibit repetitive and stereotyped behaviors.

[0065] 6. Micro MRI Scan

[0066] Mice were subjected to magnetic resonance imaging to determine the anatomical location of brain regions in PET images. MRI procedures were performed on a 9.4T miniature MRI scanner (BioSpec 94 / 30USER, Bruker BioSpin MRI Gmbh, Germany) and Paravision 5.1 software (www.bruker.com). Structural imaging data were acquired using a two-dimensional T2-weighted (T2W) TurboRapid Acquisition with Refocused Echoes (TurboRARE) sequence with the following parameters: matrix size = 150 × 150, field of view (FOV) = 15 × 15 mm, number of consecutive slices = 55, slice thickness = 0.3 mm; effective spatial resolution = 0.1 × 0.1 × 0.3 mm, repetition time (TR) = 6200 ms, echo time (TE) = 22.5 ms, average = 6, RARE factor = 8. The resulting MRI images are used to re-integrate with the PET / CT images to assist in positioning, calibration, and VOI delineation.

[0067] 7. PET / CT imaging acquisition

[0068] PET / CT images were acquired by a small animal microPET / CT imaging system (nanoScan PET / CT 82s). First, the pre-imaging mice were weighed and anesthetized with isoflurane (2% induction, 1%-1.5% maintenance); a homemade indwelling needle was implanted in the tail vein, and the mice were placed in a prone position on the scanning bed. During the entire imaging process, the important physiological parameters of the animals were continuously monitored and recorded by a physiological monitoring system. The temperature of the scanning room was controlled at 22-24°C. After acquiring a CT scouting image to assist in planning the scanning area for PET / CT acquisition, a radioactive tracer (+)-[ 18 F]YL1 (10-15μCi / g), and 30min dynamic PET / CT images were immediately acquired. Then CT spiral scanning was performed for image fusion and attenuation correction. Finally, the 3DOSEM (3-dimensional ordered subsets expectation maximization) algorithm was used to reconstruct the acquired PET raw data into 27 frames (3×10s; 4×60s; 10×150s; 10×3s). The main parameters are as follows:

[0069] ①CT positioning image scanning parameters: tube voltage 35kVp, tube current 980uA, exposure time 170ms;

[0070] ②CT spiral scanning parameters: tube voltage 50 kVp, tube current 980 uA, exposure time 300 ms, pitch 1.0, projection number 20, Binning 1:4;

[0071] ③CT real-time reconstruction parameters: small voxel thin layer reconstruction, filter type butter worth, filter cutoff 100;

[0072] ④PET scanning parameters: 1FOV, field of view size 98.5mm, consistent with mode 1-5;

[0073] ⑤PET reconstruction parameters: conforming to modes 1-5, voxel size 0.4, reconstruction method Tera-Tomo 3D (i.e., 3DOSEM), 4 iterations, 4 subsets, random, scatter, and attenuation corrections.

[0074] 8. PET-MRI imaging data analysis

[0075] Analysis methods: PMOD software (version 4.203) was used to analyze dynamic PET / CT data. The volume of interest (VOIs) was delineated to obtain the original time activity curve (TAC) and semi-quantitative standardized uptake value (SUV). The image derived input function (IDIF) of the aortic arch was used as the blood input function, and the built-in Kinetic module of PMOD software was used to analyze the (+)-[ 18 The dynamic data of F]YL1 were fitted with LoganAIF model to obtain the total distribution volume (V T ).

[0076] Experimental results: The brain regions of interest were delineated and semi-quantitatively analyzed by PMOD software. The preliminary TAC of different brain regions was compared and it was found that almost all brain regions showed differential uptake ( Figure 6 AB), indicating that Fmr1 gene silencing induced (+)-[ 18 F]YL1 uptake increased. Combined with different brain regions (+)-[ 18 The TAC curves and AUC of F]YL1 also showed that the brain regions of Fmr1 KO mice were more sensitive to (+)-[ 18 F]YL1 radioactivity uptake increased ( Figure 6 C).

[0077] SUV is only a semi-quantitative method, so we further used the fully quantitative non-compartmental model of the reversible receptor system, the LoganAIF model, to analyze the (+)-[ 18 The raw TAC data of aortic arch TAC was used as the input function, and the raw (+)-[ 18 LoganAIF graphical analysis of F]YL1 TAC data showed that the VT value of the brain region of Fmr1 KO mice was significantly higher than that of WT mice ( Figure 6 D), which further indicates that Sig-1R expression in the brain increases when Fmr1 gene is silenced and FMRP is deficient.

[0078] 9. (+)-[ 18 In vitro autoradiography of F]YL1

[0079] Experimental method: Under 2% isoflurane anesthesia, 3.3-3.7×10 4 MBq tracer (+)-[ 18F]YL1, WT mice and Fmr1 KO mice were returned to their original cages. After waiting for 30 minutes, the test mice were killed by cervical dislocation and the brains were removed. After embedding with OCT, the mouse brain tissue was sectioned coronally using a freezing microtome, with a distance of 30 μm between the anterior and posterior slices. Subsequently, the brain slices were exposed to the Beaver micropatterned gas detection system (Ai4R Inc, France) for 3 hours for in vitro autoradiography. The autoradiography images were quantified by BeaQuant 3.3 software (Ai4R, France). The radioactivity in the cortex (CTX), striatum (STR), hippocampus (HIP) and thalamus (THA) was quantified (cp / min / mm 2 At least eight regions of interest (ROIs) were drawn for each brain region. Data from technical replicates were statistically analyzed.

[0080] Experimental results: (+)-[ 18 F]YL1 autoradiography results are shown in Figure 6 Compared with WT mice, Fmr1 KO mice showed a significant decrease in the expression of (+)-[ 18 F]YL1 uptake is greater ( Figure 4-8 ), which is consistent with the results of PET / CT, further proving that Sig-1R expression is upregulated in FXS at the microscopic level.

[0081] 10. Immunofluorescence

[0082] Experimental methods: Mice were deeply anesthetized with a 3.0% isoflurane / oxygen mixture and transcardially perfused with saline and 4% cold paraformaldehyde. The brain was removed and dehydrated by sucrose gradient and embedded in OCT, and then cut into 5 μm size. The mouse brain was fluorescently stained using the primary antibody targeting Sig-1R (rabbit anti-SIGMAR1 polyclonal antibody, 1:300, 15168-1-AP, Proteintech). The slides were placed at 4°C to incubate the primary antibody overnight. After the primary antibody incubation, the slides were washed three times with phosphate buffered saline (PBS). The primary antibody was labeled with the corresponding secondary antibody (AlexaFluor 488-labeled goat anti-rabbit IgG, 1:400, GB25303, Servicebio) at room temperature for 1 hour. After the secondary antibody incubation, the slides were rinsed three times with PBS and incubated with DAPI (G1012, Wuhan Saiweier Biotechnology Co., Ltd.). Images were collected using a Nikon Eclipse C1 microscope and an upright fluorescence microscope with multiple laser diode modules, with DAPI excitation wavelengths of 330-380 nm and emission wavelengths of 420 nm; 488 excitation wavelengths of 465-495 nm and emission wavelengths of 515-555 nm. All images were taken under the same fluorescence settings, and the brightness and contrast were subsequently adjusted identically to ensure accurate pathological quantification. At the same time, the slides were scanned and stored using a 3DHISTECH Pannoramic MIDI scanner. These images were browsed, analyzed, and screenshotted by CaseViewer (version 2.4).

[0083] Experimental results: From the obtained immunofluorescence images ( Figure 7 ) showed that the expression of Sig-1R in the cortex and hippocampus of Fmr1 KO mice was higher than that in the control WT mice of the same age. This result is consistent with the previous (+)-[ 18 The results of in vivo PET / CT scanning of F]YL1 were consistent with those of autoradiography, which further supported the conclusion that Sig-1R expression was upregulated when Fmr1 gene silencing led to FMRP deficiency.

[0084] 11. Correlation analysis between PET dynamics quantification and different behavioral phenotype results

[0085] Experimental method: In order to explore the relationship between the quantitative results of PET and the phenotypes reflected by different behavioral experiments, we performed pharmacokinetic fitting results (V T ) were subjected to Pearson correlation analysis with the open field test (percentage of time spent in the central area), the novel object recognition test (discrimination index), the three-box social experiment (social novelty preference index) and the buried bead experiment (number of buried beads).

[0086] Experimental results:

[0087] (1) Correlation analysis between PET dynamics quantification and open field test results:

[0088] Among all 14 brain regions, V represents the expression of Sig-1R in the brain region. T There is a strong linear positive correlation between the value and the parameter representing the proportion of time spent in the central area of ​​spatial cognition ability ( Figure 8 A), the correlation coefficient r was between 0.56-0.65; except for the cerebellum (p=0.0601), this correlation was statistically significant in other brain regions (p<0.05), which may suggest that the spatial cognitive impairment of FXS may be related to the expression of Sig-1R in the whole brain.

[0089] (2) Correlation analysis between PET dynamics quantification and three-box social experiment results

[0090] In order to explore the relationship between PET quantitative results and social cognitive impairment, we analyzed the pharmacokinetic results of V in different brain regions of the two groups of mice. T Pearson correlation analysis was performed with the results of the three-box social experiment (social novelty preference). The results showed that among all 14 brain regions, V, which represents the expression of Sig-1R in the brain region, T There is a strong linear negative correlation between the value and the social novelty preference representing social cognitive ability ( Figure 8 B), the correlation coefficient r was between 0.64 and 0.74, and this correlation was statistically significant (p<0.05), which may suggest that the social cognitive impairment of FXS may be related to the expression of Sig-1R in the whole brain.

[0091] (3) Correlation analysis between PET kinetic quantification and embedded bead experiment results

[0092] In order to explore the relationship between PET quantitative results and repetitive stereotypic behaviors, we analyzed the pharmacokinetic results of V in different brain regions of the two groups of mice. T Pearson correlation analysis was performed with the results of the bead burying experiment (the number of marbles buried). The results showed that among all 14 brain regions, V, which represents the expression of Sig-1R in the brain region, T There is a strong linear positive correlation between the value and the number of marbles buried, which represents repetitive stereotyped behavior ( Figure 8 C), the correlation coefficient was between 0.64-0.71, and this correlation was statistically significant (p<0.05), which may suggest that the repetitive stereotyped behavior of FXS may be related to the expression of Sig-1R in the whole brain.

[0093] (4) Correlation analysis between PET dynamics quantification and novel object recognition experimental results

[0094] Among all 14 brain regions, V represents the expression of Sig-1R in the brain region. T The correlation between the values ​​and the discrimination index representing the cognitive ability of object recognition is very weak ( Figure 8 D), the correlation coefficient r was between -0.14 and -0.43, and this correlation was not statistically significant (p>0.05), which may suggest that the object recognition cognitive impairment of FXS may not be significantly correlated with the expression of Sig-1R in the whole brain.

[0095] 12. Statistical processing

[0096] All data were analyzed using unpaired two-tailed Student's t-test and are presented as mean ± standard error. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad, La Jolla, CA). To assess correlation, Pearson's correlation coefficient r was calculated. The statistical significance level was set at p < 0.05.

Claims

1. A fluorine-18 labeled Sigma-1 receptor targeting compound, characterized in that: Its structural formula is: ； in, 18 F is a radioactive signal group.

2. The fluorine-18 labeled Sigma-1 receptor targeting compound labeled precursor according to claim 1, characterized in that: Its structural formula is: ； Where R is nitro; the compound is chiral and has an optical rotation of [α] D 20 =+7.3° (1.1 mg / mL, MeOH).

3. The method for preparing a fluorine-18 labeled Sigma-1 receptor targeting compound according to claim 1, comprising the following steps: (1) Use 1 ml (acetonitrile: water = 9:1) of potassium carbonate (5 mg) and K 222 (15 mg) of the mixed solution will 18 F - Wash from the QMA column into a vial, transfer the fluoride solution to dryness with nitrogen at 110°C (3 min), and add 1 ml of anhydrous acetonitrile twice for azeotropic drying; (2) Dissolve 2.5 mg of the precursor in 200 μl of anhydrous acetonitrile and blow dry with nitrogen at 60 °C. Dissolve the dried precursor in 1 ml of DMSO for later use; (3) The reconstituted precursor was pumped into the reaction bottle, sealed with a cap, and heated at 130 °C for 15 min. The reaction solution was passed through an activated C18 column to remove 18 F - ; (4) A mixed solution of 40% H2O (containing 0.1% TFA) and 60% ethanol was used as the mobile phase for semi-preparative separation of the labeled product at a flow rate of 1.5 ml / min. The flow-through was collected for 18 to 26 min and then passed through a C18 column to capture the final product.

4. Use of a fluorine-18 labeled Sigma-1 receptor targeting compound as claimed in claim 1 or 2 in the preparation of a reagent for positron emission tomography.

5. As claimed in claim 4, the fluorine-18 labeled Sigma-1 receptor targeting compound ((+)-[ 18 F]YL1) combined with PET brain area quantification in the auxiliary diagnosis of FXS, characterized by: The Sig-1R changes in each brain region include: The brain of FXS mice is divided into 14 brain regions, including cortex, striatum, hippocampus, thalamus, cerebellum, basal forebrain, hypothalamus, amygdala, brainstem, central gray matter, superior colliculus, olfactory bulb, midbrain, hypocolithium, (+)-[ 18 F]YL1 PET increased quantitatively in various brain regions, indicating that Sig-1R expression was upregulated.

6. As claimed in claim 4, the Sigma-1 receptor targeting compound labeled with fluorine-18 ((+)-[ 18 F]YL1) combined with PET brain area quantification in the auxiliary diagnosis of FXS, characterized by: The correlation between the Sig-1R expression level in each brain region and the severity of cognitive impairment and stereotyped repetition includes: (1) Spatial cognitive impairment and (+)-[ 18 F] Correlation analysis of YL1 PET quantification showed that there was a positive correlation in 13 brain regions except the cerebellum, and the more severe the spatial cognition, the higher the PET quantification; (2) Social cognition and (+)-[ 18 F] Correlation analysis of YL1 PET quantification showed that there was a negative correlation in all 14 brain regions, and the better the social cognitive performance, the lower the PET quantification; (3) Severity of stereotyped repetition and (+)-[ 18 F] Correlation analysis of YL1 PET quantification showed that there was a positive correlation in all 14 brain regions, and the more severe the stereotyped repetition, the higher the PET quantification; (4) Object recognition cognitive impairment and (+)-[ 18 Correlation analysis of F]YL1 PET quantification showed that there was no correlation in any of the 14 brain regions.

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