CN112185518A - Developing method of TSPO translocator PET probe in neuroinflammation - Google Patents
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Abstract
The invention relates to a method for developing a TSPO translocator PET probe in neuroinflammation, belonging to the technical field of medical imaging. The method comprises the steps of establishing a cerebral ischemia rat model, and carrying out PET imaging treatment by using a small animal PET scanner; the binding specificity of 18F-VUIIS1009A and 18F-VUIIS1009B were evaluated separately; performing PET image co-registration and analysis, and performing imaging analysis by using an arterial plasma input function AIF (automated air imaging) to generate a Logan graph and a parameter image of a total distribution volume VT of each radioactive tracer; generating a parametric image for a binding capacity constant BPND by using a reference Logan model with a healthy brain as a reference region; and statistical analysis is performed. The invention can more accurately reflect the expression condition of TSPO in the nervous system diseases, thereby more accurately evaluating the progression degree of neuroinflammation in various nervous system diseases.
Description
Technical Field
The invention relates to a method for developing a TSPO translocator PET probe in neuroinflammation, belonging to the technical field of medical imaging.
Background
Neuroinflammation is currently a response to local injury to the Central Nervous System (CNS) or to a remote pathological event, and commonly occurs in a variety of neurological disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD), Multiple Sclerosis (MS), Huntington's Disease (HD), and Amyotrophic Lateral Sclerosis (ALS). Neuroinflammation often plays an important role in a variety of neuropathologies, and diagnosis and treatment of neuroinflammation is generally considered critical for monitoring of disease progression and evaluation of new therapies. In these experiments neuroimaging provides a non-invasive tool to characterize and monitor neuroinflammation in vivo. As one of the neuroimaging methods, Positron Emission Tomography (PET) can be used for nanomolar levels while visualizing quantitative molecular targets with high sensitivity and specificity.
Translocater protein (TSPO, 18kDa) is one of the most popular PET imaging biomarkers for neuroinflammation, and has been experimentally validated for over 20 years in neuroinflammation studies. TSPO, formally known as the peripheral benzodiazepine receptor, is a mitochondrial outer membrane protein located primarily in peripheral organs including the lung, heart, kidney and nasal epithelium. In healthy brain, the expression level of TSPO is extremely low, but when microglia are activated in neuroinflammation due to brain injury, the expression of TSPO is too high. Overexpression of TSPO is considered as a biomarker of neuroinflammation in many neurological diseases (including stroke, AD, PD, MS, HD and ALS). In previous experiments, TSPO imaging has been used as a powerful tool to elucidate neuroinflammatory effects and to evaluate therapeutic effects on brain diseases in clinical and preclinical experiments.
Over the past few decades, many TSPO-PET radiotracers have been developed to test neuroinflammation in a variety of diseases. Of these radiotracers, the most common one so far is 11C-PK11195, but it has several limitations including high non-specific binding, low brain penetration, low signal-to-noise ratio (SNR), low sensitivity, poor quantification capability. Thus, a number of experiments were performed to develop novel TSPO radiotracers, including phenoxyarylacetamide derivatives labelled with 11C (11C-DAA1106, 11C-PBR28) or 18F (18F-FEDAA1106, 18F-FEPPA, 18F-PBR06), imidazopyridine derivatives (11C-CLINME) and pyrazolopyrimidine derivatives (18F-DPA-714). Among them, 18F-DPA-714 is a pyrazolopyrimidine radiotracer with high TSPO binding affinity. 18F-DPA-714, labeled 18F, has a longer half-life (110 min) than 11C-PK11195(20 min). It is widely used in TSPO-PET imaging to dynamically monitor brain inflammatory responses and disease progression and exhibits higher bioavailability, lower non-specific binding and higher non-displaceable Binding Potential (BP)ND). Clinical trials using 18F-DPA-714 have been conducted in ALS, AD and post-stroke trials, showing great promise in neuroimaging trials.
Although TSPO PET imaging has been performed in a variety of neuroinflammatory diseases, there are some drawbacks, including relatively low neuroinflammatory uptake, low SNR and small differences in TSPO expression (semi-quantitative parameters (% ID/cc or SUV)T，BPNDAnd DVR-like macro-parameters, in order to fully characterize the pharmacokinetics of the radiotracer, TSPO expression levels, as well as to increase SNR and improve imaging vision. Indeed, radiotracers with higher binding specificities generally exhibit higher values of macroscopic parameters (e.g., BP)ND) Higher SNR, and therefore better visual impact in PET imaging, and higher sensitivity to TSPO density variations.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) the existing 11C-PK11195 radioactive tracer has high nonspecific binding, low brain penetration, low signal-to-noise ratio (SNR), low sensitivity and poor quantification capability.
(2) Currently, there is still relatively low neuroinflammatory uptake, low SNR and low sensitivity of detection of TSPO expression applying TSPO PET imaging to a variety of neuroinflammatory diseases.
The difficulty in solving the above problems and defects is:
(1) lack of TSPO-PET probe with high sensitivity, low signal-to-noise ratio and strong quantification capability.
(2) There is a lack of diagnostic and image evaluation and analysis methods for corresponding probes.
(3) Preclinical and clinical diagnosis and corresponding analytical data are lacking.
The significance of solving the problems and the defects is as follows: the expression condition of TSPO in the nervous system diseases can be more accurately reflected, so that the degree of the neuroinflammation in various nervous system diseases can be more accurately evaluated, and the significance of the neuroinflammation on the diagnosis and treatment of the diseases can be evaluated on the basis.
Disclosure of Invention
The invention aims to solve the technical problem of how to more accurately reflect the expression condition of TSPO in nervous system diseases so as to more accurately evaluate the progression degree of neuroinflammation in various nervous system diseases.
In order to solve the problems, the technical scheme adopted by the invention is to provide the application of the TSPO translocator probe in nervous system diseases, and the PET image co-registration and dynamic PET data analysis are carried out; performing imaging analysis by using an arterial plasma input function AIF (advanced immune function), and generating a Logan graph and a parameter image of a total distribution volume VT of each radioactive tracer; a parametric image for the binding capacity constant BPND is generated by using a reference Logan model of a healthy brain as a reference region.
Preferably, the TSPO translocator probes are set to 18F-VUIIS1009A and 18F-VUIIS 1009B.
The invention provides a method for developing a TSPO translocator PET probe in neuroinflammation, which comprises the following steps:
step 1: injecting the TSPO translocator probes 18F-VUIIS1009A, 18F-VUIIS1009B and 18F-DPA-714 into a cerebral ischemic rat model, and carrying out PET imaging treatment on the cerebral ischemic rat by using a small animal PET scanner;
step 2: the binding specificity of 18F-VUIIS1009A and 18F-VUIIS1009B, respectively, was assessed by displacement of the non-radioactive analogs;
and step 3: carrying out PET image co-registration and dynamic PET data analysis; performing imaging analysis by using an arterial plasma input function AIF (advanced immune function), and generating a Logan graph and a parameter image of a total distribution volume VT of each radioactive tracer;
and 4, step 4: generating a parametric image for a binding capacity constant BPND by using a reference Logan model with a healthy brain as a reference region;
and 5: carrying out statistical analysis; all quantitative data are expressed as mean ± standard deviation and the relationship between regional outcome parameters under test conditions and re-test conditions was estimated using GraphPad Prism 5 software.
Preferably, the PET images of the image analysis in step 3 are co-registered by: firstly, registering corresponding CT images by using Inveon Research Workplace 4.0 software, carrying out spatial alignment on brain areas, and generating a conversion file for co-registration; the generated conversion file is used as an input to register a corresponding PET image, the registered PET image is used to draw an ROI, the voxel values are analyzed, and a parameter map is calculated.
Preferably, the method for performing dynamic PET data analysis in step 3 is as follows: analyzing the PET image by using PMOD 3.4 layout image analysis software and Inveon Research Workplace 4.0; ROIs were manually drawn ipsilateral and contralateral to striatum and cortex; generation of parameter V Using Logan analysis based on plasma inputsTImage, plasma contribution factor vp for all analyses set to 0.05; for the same animal, a Logan reference tissue model was also used to generate a parameterized BP with contralateral brain TAC as inputNDAn image; for DVR or BPNDDetermining and parameterizing image generation, using the SRTM method, with the contralateral brain as reference tissue to calculate k2_REF(ii) a For each activityTracer, analysis% ID/cc, BP using parameters determined from the same voxels in normal and parametric PET imagesNDAnd VTThe correlation of (c); correlation was analyzed using Pearson correlation coefficient r and p-values were generated using GraphPad Prism 5 software.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a TSPO translocator probe for analyzing neuroinflammation, and adopts a method of measuring three TSPO translocator probe radiotracers, so as to evaluate the performances of the high-specificity TSPO radiotracers 18F-VUIS 1009A and 18F-VUIS 1009B in a mild neuroinflammation model, and directly compare the performances with the performances of the existing TSPO radiotracer 18F-DPA-714 in the same neuroinflammation animal model. The in vitro TSPO binding affinity of VUIIS1009A (IC 50: 14.4pM) was 750-fold higher than that of DPA-714(IC 50: 10.9 nM). VUIIS1009B (IC 50: 19.4pM) had 560-fold higher in vitro TSPO binding affinity than DPA-714. The present invention uses graphs to assess their semi-quantitative parameters (% ID/cc or SUV) and include the Binding Potential (BP) by in vivo PET dynamic scanningND) Total distribution volume (V)T) And analysis of macroscopic parameters including volume-to-volume-ratio-of-Distribution (DVR). A specific parametric image (BP) is also generatedND，VT) And compared to identify appropriate quantitative methods and parameters. The invention proves that the 18F-VUIIS1009B is better than the 18F-VUIIS1009A and the 18F-DPA-714 in the aspect of neuroinflammation imaging; 18F-VUIIS1009B PET imaging and parameter mapping (V)TAnd BPND) And the pattern analysis is combined, so that the method has wide prospect for neuroinflammation characterization and TSPO density measurement.
Experiments show that compared with 18F-DPA-714, 18F-VUIIS1009B has higher in vitro binding specificity and Binding Potential (BP)ND) And a Distribution Volume Ratio (DVR). BP (Back propagation) ofNDAnd VTAlso, the parametric images of (a) also show that 18F-VUIIS1009B has better imaging properties than the other two radiotracers in TSPO imaging. BP at 18F-VUIIS1009B and 18F-DPA-714NDCorrelation analysis between the two also showed that 18F-VUIIS1009B was more sensitive than 18F-DPA-714 in TSPO density measurements.
Drawings
Fig. 1 is a schematic structural diagram of the DPA-714, VUIIS1009A, VUIIS1009B and their corresponding ICs 50 according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of a 45 minute PET dynamic scan of 18F-DPA-714, 18F-VUIIS1009A and 18F-VUIIS1009B as provided by an embodiment of the invention;
wherein: (a) FIG. is a coronary artery of a cerebral ischemic rat dynamically scanned with 18F-DPA-714; (b) FIG. is a transverse PET image of the same coronary artery of a dynamically scanned cerebral ischemic rat using 18F-DPA-714; (c) FIG. is a dynamic scan of the coronary arteries of the same cerebral ischemic rat with 18F-VUIIS 1009A; (d) FIG. is a coronal transverse PET image of the same cerebral ischemic rat dynamically scanned with 18F-VUIIS 1009A; (e) FIG. is a coronary artery from the same cerebral ischemic rat dynamically scanned by 18F-VUIIS 1009B; (f) FIG. is a coronary artery transverse PET image of the same cerebral ischemic rat by dynamic scanning with 18F-VUIIS 1009B; (g) graphs are Time Activity Curves (TAC) plots for inflammation and contralateral brain for 18F-DPA-714 in a 45 minute dynamic scan (n ═ 6); (h) graphs are Time Activity Curves (TAC) plots for inflammation and contralateral brain for 18F-VUIIS1009A in a 45 minute dynamic scan (n ═ 6); (i) graphs are Time Activity Curves (TAC) plots for inflammation and contralateral brain for 18F-VUIIS1009B in a 45 minute dynamic scan (n ═ 6); % ID/cm in the figure3Percent of injected dose per cubic centimeter. In TAC, data are mean ± SD. The inflamed area is marked with a white arrow on the image.
FIG. 3 is a graphical representation of the displacement analysis of rats imaged with 18F-VUIIS1009A and 18F-VUIIS1009B injected at 20 minutes with their corresponding non-radioactive analogs (10mg/kg) as provided by the present examples;
wherein: (a) the figure summarizes the first 20 minutes of PET imaging (coronal) with 18F-VUIIS 1009A; (b) the figure summarizes the last 25 minutes of PET imaging (coronal) with 18F-VUIIS 1009A; (c) FIG. TAC for brain and neuroinflammation in the 18F-VUIIS1009A displacement assay; (d) the figure summarizes the first 20 minutes of PET imaging (coronal) with 18F-VUIIS1009B (n ═ 3); (e) the figure summarizes the last 25 minutes of PET imaging (coronal) with 18F-VUIIS 1009B; (f) fig. TAC (n-3) for brain and neuroinflammation in 18F-VUIIS1009B displacement analysis. The inflammatory area is marked with a white arrow. Data are mean ± SD.
FIG. 4 is a pharmacokinetic profile of an embodiment of the invention;
wherein (a) is a 2 tissue 4 kinetic parameter model provided by an embodiment of the present invention to describe the kinetics of 18F-DPA-714, 18F-VUIIS1009A and 18F-VUIIS1009B in reference tissue (including inflamed areas and healthy contralateral brain). The four parameters of K1, K2, K3 and K4 reflect the rate of transport or binding of the radiotracer. In this particular experiment, K1 represents the rate of perfusion of the probe from plasma to tissue, while K2 represents the rate of clearance of the tracer from tissue to plasma; k3 and k4 represent the specific binding rate and dissociation rate of the tracer, respectively; (b) FIG. is a graphical diagram of the Logan reference organization of 18F-DPA-714; (c) FIG. is a graphical illustration of the Logan reference organization of the 18F-VUIIS 1009A; (d) FIG. is a graphical illustration of the Logan reference organization of the 18F-VUIIS 1009B.
FIG. 5(a) is a diagram of a parametric image of 18F-DPA-714, 18F-VUIIS1009A and 18F-VUIIS1009B using the Logan method with AIF input as provided by an embodiment of the present invention; the graph in fig. 5(b) is a parametric image of 18F-DPA-714, 18F-VUIIS1009A and 18F-VUIIS1009B using the Logan reference tissue method with TAC input to the contralateral brain as the reference region.
FIG. 6 is a pair of voxel levels provided by an embodiment of the present invention;
wherein (a) is the% ID/cc, BP determined by 18F-DPA-714NDAnd VTThe correlation of (c).
(b) FIG. 18F-VUIIS1009A for% ID/cc, BPNDAnd VTThe correlation of (c).
(c) FIG. 18F-VUIIS1009B for% ID/cc, BPNDAnd VTThe correlation of (c).
FIG. 7(a) is a graph of BP determined at the voxel level of the same rat from the 18F-VUIIS1009B and 18F-DPA-714 dynamic PET scans provided by an example of the present inventionNDThe correlation of (c); (b) the graph is the region BP determined by 18F-VUIIS1009B and 18F-DPA-714 dynamic PET scans in a group of ratsNDCorrelation (n ═ 6).
FIG. 8 is a flow chart of a method for in vivo imaging of TSPO translocator probes as provided by embodiments of the present invention.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings:
the present invention provides a method for analyzing neuroinflammation and measuring with three TSPO radiotracers, as shown in figures 1-8, which is described in detail below with reference to the accompanying drawings.
As shown in fig. 8, the method for in vivo imaging of TSPO translocator probe provided in the embodiments of the present invention includes the following steps:
s101, rat models of cerebral ischemia were created and imaged using 18F-VUIIS1009A, 18F-VUIIS1009B and 18F-DPA-714.
S102, the binding specificity of 18F-VUIIS1009A and 18F-VUIIS1009B, respectively, was assessed by displacement of the non-radioactive analogs.
And S103, carrying out PET image co-registration and analysis, and carrying out imaging analysis by using an arterial plasma input function AIF (automated optical tomography), so as to generate a Logan graph of each radioactive tracer and a parameter image of the total distribution volume VT.
S104, generating a parametric image for the binding ability constant BPND by using the reference Logan model with the healthy brain as a reference region.
S105, statistical analysis: all quantitative data are expressed as mean ± standard deviation and the relationship between regional outcome parameters under test conditions and re-test conditions was estimated using GraphPad Prism 5 software.
The technical solution of the present invention will be further described with reference to the following examples.
Transporter protein (TSPO) is a mitochondrial membrane protein that is considered to be a key biomarker of neuroinflammation in a variety of neurodegenerative diseases. In the examples, the present invention was directed to the evaluation of two highly specific TSPO radiotracers 18F-VUIIS1009A and 18F-VUIIS1009B in a rat model of mild cerebral ischemia and their in vivo performance compared to neuroinflammatory imaging of the mature TSPO probe 18F-DPA-714. By testing multiple pattern analysis methods and determining macroscopic parameters, the present invention provides a suitable optimal quantitative method to analyze neuroinflammation and measure TSPO density using three TSPO radiotracers.
1.1 methods
Rat models of cerebral ischemia were created and imaged using 18F-VUIIS1009A, 18F-VUIIS1009B and 18F-DPA-714. Displacement experiments with non-radioactive analogs were performed to assess the binding specificity of 18F-VUIIS1009A and 18F-VUIIS1009B, respectively. Imaging analysis using arterial plasma import function (AIF) can generate a Logan plot and a parametric image of total distribution Volume (VT) for each radiotracer. A reference Logan model using a healthy brain as a reference region to generate a parametric image for the binding capacity constant (BPND).
1.2 results
Compared with 18F-DPA-714, 18F-VUIIS1009B has higher in vitro binding specificity, Binding Potential (BPND) and Distribution Volume Ratio (DVR). Parametric images of BPND and VT also show that 18F-VUIIS1009B has better imaging properties than the other two radiotracers in TSPO imaging. Correlation analysis between BPND for 18F-VUIIS1009B and 18F-DPA-714 also showed that 18F-VUIIS1009B was more sensitive than 18F-DPA-714 in TSPO density measurements.
This experiment demonstrates that 18F-VUIIS1009B is superior to 18F-VUIIS1009A and 18F-DPA-714 in neuroinflammatory imaging. It also shows 18F-VUIIS1009B PET imaging and parameter mapping (V)TAnd BPND) And the pattern analysis is combined, so that the method has wide prospect for neuroinflammation characterization and TSPO density measurement.
Indeed, radiotracers with higher binding specificities generally exhibit higher values of macroscopic parameter (such as BPND), higher SNR and therefore better visual effect in PET imaging, and higher sensitivity to TSPO density variations.
In this regard, the present invention first evaluated the performance of the highly specific TSPO radiotracers 18F-VUIIS1009A and 18F-VUIIS1009B in a mild neuroinflammatory model and directly compared to the performance of the existing TSPO radiotracer 18F-DPA-714 in the same neuroinflammatory animal model. The in vitro TSPO binding affinity of VUIIS1009A (IC 50: 14.4pM) was 750-fold higher than that of DPA-714(IC 50: 10.9 nM). VUIIS1009B (IC 50: 19.4pM) had 560-fold higher in vitro TSPO binding affinity than DPA-714. By in vivo PET dynamic scanning, the present invention uses graphs to assess their semi-quantitative parameters (% ID/cc or SUV) as well as macroscopic parameter analysis including Binding Potential (BPND), total volume of distribution (VT) and volume of distribution ratio (DVR). Specific parametric images (BPND, VT) were also generated and compared to identify appropriate quantitative methods and parameters to describe neuroinflammation in future experiments.
2.1 materials and methods
All chemicals were purchased from commercial sources. Unlabeled DPA-714, VUIIS1009A/B and its precursors for radiosynthesis were synthesized internally and 18F was produced using an IBA cyclotron (Belgium). The radioactivity of the wastewater was monitored using a NaI (Tl) scintillation detector system. Unless otherwise indicated, all other synthetic reagents and solvents were purchased from Sigma-Aldrich (st. louis, missouri) and used without further purification.
2.2 radiosynthesis
The radiosynthesis of probes 18F-DPA-714, 18F-VUIIS1009A and 18F-VUIIS1009B was carried out according to known methods. In detail, the precursor is nucleophilic fluorinated with fluorine-18. The 18F-labelled radiotracer was purified by preparative HPLC. The retention time of all radiotracers determined according to the gamma detection method was checked again to ensure that it was consistent with the UV retention time determined using the corresponding non-radioactive analogue when HPLC analysis was performed under the same conditions. The radiochemical purity, determined by HPLC, was consistently above 99% and the specific activity was consistently above 4203Ci/mmol (156 TBq/mmol).
2.3 animals
Animals were raised and treated according to the recommendations of the national institutes of health in china. Animal experiments were approved by local university and hospital ethics committees. All experiments conducted at the Shanghai university of transportation medical school were approved by the animal ethics Committee. Male Wistar rats (7 weeks old, 230-.
2.4 ischemia rat model
According to the intraluminal model, intraluminal occlusion of the middle cerebral artery for 30 minutes induced mild focal ischemia. After ischemic surgery, rats were used for PET imaging and metabolite analysis was performed on days 5-7. Rats (n-12) were fixed on arterial catheters before MRI and PET/CT experiments were performed.
2.5 in vivo dynamic PET imaging experiment
PET imaging was performed using a small animal PET scanner (Siemens Medical Solutions USA, noksville, tennessee, USA) according to the protocol previously reported. Rats (n ═ 6) were first anesthetized with isoflurane (induction rate 3.5%, maintenance rate 1.5-2%). Immediately after intravenous injection of a specific radiotracer into rats via the tail vein, a 45 minute list mode emission scan was performed. Experiments were performed on consecutive days using 18F-VUIIS1009A (46.0. + -. 8.0MBq), 18F-VUIIS1009B (48.8. + -. 10.7MBq) and 18F-DPA-714 (47.2. + -. 9.9 MBq). During the scan, blood samples were drawn according to the following schedule: 15 μ L were withdrawn every 10s for the first 90s, 5, 8, 20 and 45 min, while 200 μ L were withdrawn at 2, 12, 30 and 60min for metabolite correction. The time frame reconstruction of PET is as follows: 10 sx 12 frames, 1min × 3 frames, 5min × 8 frames. In addition, metabolite corrected maternal plasma import function (AIF) was measured according to the previously published protocol of the present invention.
For displacement experiments, unlabeled TSPO compounds VUIS 1009A (10.0mg/kg) or VUIS 1009B (10.0mg/kg) were dissolved in 1mL saline containing 10% ethanol and 5% Tween-80 and infusion was initiated 20 minutes after the PET scan. Three independent experiments (in triplicate) were performed per group. PET scan data was modeled using a 3-dimensional sinogram, which was then converted to a two-dimensional sinogram by fourier reconstruction. Dynamic image reconstruction is achieved by filtered back-projection using a hanning filter (with a nyquist cut-off frequency of 0.5 cycles/pixel).
2.6PET image co-registration:
in order to analyze the voxel values of the region of interest (ROI) and to calculate the parameter map, the X-ray images of the same rat were registered with different radiotracers. In detail, using Inveon Research Workplace 4.0 (Siemens medical solutions, Inc., N.S. of Nox, Tenn.), the corresponding CT images are first registered for spatial alignment of the brain regions. A conversion file for this co-registration is generated and then used as input to register the corresponding PET image. The ROI was rendered using the registered PET image and the values of the voxels were analyzed.
2.7 dynamic PET data analysis
PET images were analyzed using PMOD 3.4 domain image analysis software (PMOD Technologies, zurich, switzerland) and inventon Research Workplace 4.0 (U.S. siemens medical solutions, noksville, tennessee, usa). ROIs were manually drawn ipsilateral and contralateral to the striatum and cortex.
In this experiment, parameter V was generated using Logan analysis based on plasma inputTImage, plasma contribution factor vp for all analyses was set to 0.05. For the same animal, a Logan reference tissue model was also used to generate a parametric BPND image with contralateral brain TAC as input. For DVR or BPNDDetermination and parametric image generation k2_ REF was calculated using the SRTM method with the contralateral brain as the reference tissue. The DVRLogan in table 1 was generated directly from the ratio of the volume of inflammation distribution to the brain distribution by Logan analysis based on plasma input.
For each radiotracer,% ID/cc, BP was analyzed using parameters determined from the same voxels in normal and parametric PET imagesNDAnd VTThe correlation of (c). Then, the correlation was analyzed using Pearson correlation coefficient (r) and p-values were generated using GraphPad Prism 5 Software (GraphPad Software, la, ca, usa).
2.8 statistical analysis
All quantitative data are expressed as mean ± Standard Deviation (SD). The relationship between the regional outcome parameters under the test conditions and the retest conditions was estimated using GraphPad Prism 5 Software (GraphPad Software, La Jolla, CA, usa).
3. Results
3.1 in vivo 45 min dynamic Scan
The performance of 18F-DPA-714, 18F-VUIIS1009A and 18F-VUIIS1009B was evaluated during a 45 minute dynamic PET scan using a rat model of focal cerebral ischemia, in which activated microglia upregulated TSPO expression, while the Blood Brain Barrier (BBB) was intact or not severely disrupted. As expected, in PET imaging of all three radiotracers, the ipsilateral (ischemic) radiotracer uptake was higher than the contralateral brain, as shown in fig. 2(a), fig. 2(b) of fig. 18F-DPA-714; FIG. 2(c) and FIG. 2(d) of the 18F-VUIIS 1009A; FIG. 2(e) and FIG. 2(F) of the 18F-VUIIS 1009B. From analysis of the ipsilateral time-activity curve (TAC), rapid uptake of the radiotracer was demonstrated (FIG. 2(g) plot of 18F-DPA-714, FIG. 2(h) plot of 18F-VUIIS1009A and FIG. 2(i) plot of 18F-VUIIS 1009B). In addition, as shown by TAC, the ipsilateral radiotracer uptake levels were also consistent 2 minutes after radiotracer injection (FIG. 2(g) for 18F-DPA-714; FIG. 2(h) for 18F-VUIIS 1009A; FIG. 2(i) for 18F-VUIIS 1009B). Compared to 18F-DPA-714 (0.73% ID/cc at 45 min), 18F-VUIIS1009B (0.70% ID/cc at 45 min) had comparable% ID/cc in the neuro-inflammatory region, whereas 18F-VUIIS1009A (0.50% ID/cc in the same region, as shown in fig. 2c, d and h) had significantly lower absorption in the same region.
Contralateral has relatively low uptake and consistent% ID/cc levels were achieved 10 minutes after radiotracer injection for all three radiotracers (fig. 2(g), fig. 2(h) and fig. 2 (i)). Compared to 18F-VUIIS1009A, 18F-VUIIS1009B and 18F-DPA-714 showed higher contralateral healthy brain uptake, as shown by 45 min uptake (0.20vs 0.15, 45% ID%/cc). For 18F-DPA-714, in this experiment, the% ID/cc ratio at 45 minutes was 3.50 for the ipsilateral and contralateral (as indicated by TAC) (0.73 vs.0.20% ID/cc at 60 minutes; FIG. 2(g) panel), 3.33 for 18F-VUIIS1009A (0.50 versus 0.15% ID/cc at 45 minutes) and 3.50 for 18F-VUIIS1009B (0.70 versus 0.2% ID/cc at 45 minutes), indicating a high signal to noise ratio for all three TSPO radiotracers in this experiment.
3.2 in vivo replacement experiments
In vivo displacement experiments were also performed to assess TSPO binding specificity of 18F-VUIIS1009A and 18F-VUIIS1009B in the same rat. Displacement measurements were performed using 19F-VUIIS1009A and 19F-VUIIS1009B and injected at 20 minutes during a 45 minute dynamic scan. According to imaging results, both TSPO compounds can replace the ipsilateral radio uptake of the brain. As shown in FIG. 3(a) (18F-VUIIS1009A) and FIG. 3(c) (18F-VUIIS1009B), both radiotracers had significantly higher ipsilateral uptake than contralateral uptake prior to injection of the nonradioactive analog. However, after displacement, the absorption of the ipsilateral radiotracer dropped dramatically (fig. 3(b) and 3 (d)), indicating that the absorption of the tracer in both radiotracers TAC dropped by more than 66% (as shown in fig. 3 (e) and (f)). The ipsilateral uptake levels were nearly comparable to the contralateral after replacement with their nonradioactive analogs, as demonstrated in both the imaging plot and TAC in figure 3. All of these indicate that the binding specificity of both radiotracers is high in areas of neuroinflammation.
3.3 determining macro parameters using graphical analysis:
although the in vitro binding affinities of 18F-VUIIS1009A and 18F-VUIIS1009B were much higher compared to 18F-DPA-714, they did not show more promising imaging characterized by semi-quantitative uptake (e.g.,% ID/cc). To further evaluate their in vivo performance, graphical analysis using AIF or reference tissue TAC inputs determined such as VTDVR and BPNDAnd the like. In accordance with previous experiments of the present invention, TSPO PET imaging can be analyzed using a two tissue, four parameter model as shown in a-plot in fig. 4. Based on this model, graphical analysis using AIF can also be used to determine V for neuroinflammatory and normal brain regionsT. As shown in Table 1, all three radiotracer-determined inflammations VTThe values were all higher than those for normal brain, indicating that all these radiotracers tend to accumulate in areas of inflammation rather than healthy brain. Direct comparison of inflammation of three radiotracers VTV of 18F-VUIIS1009B, as shown in Table 1THigher than the other two radiotracers. Furthermore, V of 18F-VUIIS1009B for contralateral healthy brainTAnd also higher than the other two radiotracers. V between the inflamed region and the contralateral healthy brain compared to 18F-DPA-714(6.00 + -0.41) and 18F-VUIIS1009A (18F-VUIIS1009B (6.00 + -0.41)TThe ratio of values (denoted DVR) indicated a higher DVR (8.53. + -. 1.06vs. 4.37. + -. 0.82) for 18F-VUIIS 1009B. Further experiments were also performed using the contralateral healthy brain as a reference region to determine DVR and BP for all three radiotracersND(as shown in fig. 4 and table 1). Logan plots using the contralateral brain as a reference region and determination of BPNDAnd DVR (FIG. 4(b), FIG. 4(c), and FIG. 4 (d)), showing r>Good fitness and linearity for all three radiotracers of 0.96. As shown in Table 1, the DVR of 18F-VUIIS1009B (7.55 + -0.65) was much higher than that of 18F-DPA-714(5.37 + -0.36) and 18F-VUIIS1009A (3.91 + -0.50). Similarly, with respect to DVR and BPNDRelationship between (recorded as DVR ═ BP)ND+1), as shown in table 1, the 18F-VUIIS1009B also showed a much higher BP than the other two radiotracersNDThe value is obtained. In summary, macro-parametric analysis showed that 18F-VUIIS1009B had a ratio of VTDVR and BPNDThe other two radiotracers above have higher imaging potential. FIG. 4(a) is a drawing
Table 1 macro parameters determined for 18F-DPA-714, 18F-VUIIS1009A and 18F-VUIIS1009B in rats with the same cerebral ischemia (n ═ 6) (data ═ mean ± SD)
3.4 parametric image analysis:
macro-parametric analysis using Logan plots showed that dynamic PET imaging showed 18F-VUIIS1009B to be superior to 18F-DPA-714 and 18F-VUIIS 1009A. In this experiment, the present invention further compared the performance of the three radiotracers with the generated parametric images (VT and BPND) (see fig. 5). In detail, the voxel direction VT image is generated by a Logan graph method with AIF input. A Logan reference model is used to generate a BPND image classified by pixels with contralateral healthy brain input as a reference region. As expected, the 18F-VUIIS1009B has similar biodistribution characteristics in VT and BPND parameter images for neuroinflammatory regions (as in fig. 5) compared to 18F-DPA-714, but higher VT and BPND in neuroinflammatory regions, demonstrating its good foreground imaging for TSPO. Although 18F-VUIIS1009A and 18F-VUIIS1009B have relatively high in vitro binding affinities, 18F-VUIIS1009A does not exhibit similar imaging characteristics in both VT and BPND parametric images as 18F-VUIIS1009B (see FIG. 5). The imaging analysis of the 18F-VUIIS1009A produced noisy parametric images compared to the 18F-VUIIS1009B parametric images, as shown in FIG. 5(a) and FIG. 5 (b).
3.5％ID/cc，BPNDAnd VTCorrelation between voxel levels:
this experiment also determined and evaluated the% ID/cc, BP at the voxel levelNDAnd VTThe aim is to better elucidate the relationship between these parameters derived from dynamic PET images. As shown in FIG. 6, the% ID/cc, BP of voxels in the region of defined neuroinflammationNDAnd VTThe invention then plots the three probes differently and measures the correlation coefficient r and p values for each data set. As shown in FIG. 6(a) and FIG. 6(c), BP is measured at all three parameters (% ID/cc)NDAnd VT) The correlation between the 18F-DPA-714 and the 18F-VUIIS1009B is clarified. The 18F-VUIIS1009B has a higher r value (0.99 vs. 0.78) than 18F-DPA-714, so VTAnd BPNDThe linear relationship therebetween is corrected as shown in fig. 6(a) and fig. 6 (c). In addition, as shown in FIG. 6(a) and FIG. 6(c), 18F-VUIIS1009B also shows the semi-quantitative parameters% ID/cc and BPNDOr VTA stronger correlation between this, which may be due to the 18F-VUIIS1009B instead of the 18F-DPA-714. For the 18F-VUIIS1009A, as shown in FIG. 6(b), at% ID/cc and two other parameters (BP)NDAnd VT) A weaker correlation was found between.
BP of 3.618F-VUIIS 1009BNDCorrelation analysis with 18F-DPA-714:
in this experiment, 18F-VUIIS1009B and 18F-DPA-714PET imaging were performed using the same rat, with the aim of more accurately reflecting its BPNDAnd in vivo performance. To compare the performance of the 18F-VUIIS1009B and 18F-DPA-714, the present invention co-registered PET images of the same rat and calculated BPND at the region and voxel levels for 18F-VUIIS1009B and 18F-DPA-714, with the same return on investment. As shown in fig. 7(a), at the voxel level of the same rat, 18F-VUIIS1009B showed a strong positive correlation with 18F-DPA-714 (r ═ 0.88). The slope of the fitted curve was 1.35, indicating that BPND at 18F-VUIIS1009B is more sensitive to TSPO expression than 18F-DPA-714. For the same rat (n ═ 6), region BP of 18BP-VUIIS1009B and 18F-DPA-714 was also obtainedNDAnd analyzed. As shown in fig. 7(b), for a group of rats (n ═ 6), 18F-VUIIS1009B also shows 18F-DPA-714 in region BPNDThe stronger correlation (r ═ 0.89). The slope of the fitted curve was 1.45, which also indicates a higher sensitivity of 18F-VUIIS1009B to TSPO expression compared to 18F-DPA-714.
4. As a result:
TSPO is poorly expressed in the normal brain and is often overexpressed in neuropathological conditions such as stroke, brain trauma, AD and PD. TSPO-PET imaging has now become a useful tool in neuroinflammatory assessment as well as in the diagnostic and therapeutic assessment of many neurological diseases. In longitudinal experiments with neurological disease, TSPO density assessments have been performed using different radiotracers and assays to increase the sensitivity of TSPO expression profiles to elucidate the relationship between disease progression and TSPO density. In these experiments, semi-quantitative parameters like% ID/cc and SUV were extensively evaluated in both clinical and preclinical experiments. Macro-parameters (e.g. BP) in contrast to semi-quantitative parametersND，DVR，VT) More detailed information about radiotracer pharmacokinetics and TSPO expression can be provided, which is more meaningful in longitudinal monitoring of neuroinflammatory diseases. In addition, TSPO radiotracers with higher binding affinity tend to have higher Binding Potential (BP)ND) This may be more sensitive to measuring changes in TSPO density in a longitudinal analysis of disease progression. Over the past 20 years, many experiments were performed to develop novel TSPO probes with higher binding affinity, specificity, binding potential and good pharmacokinetics. To date, 11C-PK11195 is the most widely used probe for TSPO imaging. However, it still has many limitations in brain imaging due to its low brain permeability, high non-specific binding rate, low sensitivity, low signal-to-noise ratio and poor quantification applicability. To overcome these limitations, a variety of novel radiotracers have been developed. The second generation radiotracer 18F-DPA-714 developed by James et al demonstrates its superiority in neuroinflammation imaging with higher binding potential, binding specificity and SNR. Golla et al generated quantitative images of 18F-DPA-714 in a clinical trial involving healthy subjects and AD patients using a variety of methods. In this experiment, they concluded that both the Roots analysis and the spectral analysis are plasma input based methods that can generate quantitative and accurate parameters VTAnd (4) an image. Also, in the reference organization approach, a reference Logan analysis or SRTM2 can be used to generate the parameterized BPNDAnd (4) an image.
The present invention further modifies 18F-DPA-714 and results in several novel TSPO imaging radiotracers, including 18F-VUIIS1008, 18F-VUIIS1018A and 18F-VUIIS 1009A/B. These novel radiotracers have higher in vitro TSPO binding affinity and lipophilicity suitable for brain imaging compared to the parent radiotracer 18F-DPA-714. In addition, the great potential of TSPO PET imaging is demonstrated, including high signal-to-noise ratio, higher binding potential and enhanced visual effect. In experiments, the present invention further evaluated the performance of 18F-VUIIS1009A and 18F-VUIIS1009B in preclinical models of neuroinflammation. PET imaging shows that both radiotracers can be used to detect areas of neuroinflammation with high SNR, as well as rapid distribution and equilibrium of contralateral and neuroinflammatory areas. To further confirm the binding specificity of 18F-VUIIS109A and 18F-VUIIS1009B to TSPO, the present invention performed in vivo displacement analysis by treating animals with unlabeled VUIIS1009A and VUIIS1009B during a 45 minute dynamic scan. The results show that the rate of displacement was high for both radiotracers (up to 66%), reflecting the high TSPO binding specificity of both radiotracers.
Although the binding affinities of 18F-VUIIS1009A and 18F-VUIIS1009B were enhanced by more than 500-fold compared to 18F-DPA-714, they did not exhibit superior semi-quantitative imaging properties (% ID/cc) compared to 18F-DPA-714. In experiments, graphical analysis was also performed to determine the macro-parameters (BPND, VT and DVR) of these radiotracers. The results indicate that 18F-VUIIS1009B has a higher VT, and higher BP in both the inflammatory and normal brain regions than 18F-DPA-714 doesND，VTAnd DVR values to highlight performance over 18F-DPA 714. Parametric image (V) generated using Logan method compared to 18F-DPA-714TAnd BPND) The enhanced visual effect of the 18F-VUIIS1009B is also shown. In pharmacokinetics, BPNDIs a comprehensive measure of the "available" neuroreceptor density and the affinity of the drug or radiotracer for that neuroreceptor. In the experiment, PET imaging was performed on rats with the same TSPO density for each radiotracer. Thus, the BP determined by the present inventionNDCan reflect the binding affinity of the radiotracer and the sensitivity of the TSPO density measurement. As expected, BP for 18F-VUIIS1009B, whether at region ROI or voxel levelNDAre much higher than 18F-DPA-714 and are in contact with BP of 18F-DPA-714NDAre closely related. Furthermore, the linear slope is greater than in fig. 7, reflecting the superior imaging potential and greater sensitivity of the 18F-VUIIS1009B to determine TSPO expression in PET imaging compared to 18F-DPA-714. Also for each radiotracer% ID/cc, BP at the voxel levelNDAnd VTCorrelation analysis was performed to show that all three parameters were present for 18F-DPA-714 and 18F-VUIIS1009BThere is a strong correlation. Furthermore, the 18F-VUIIS1009B is in BP in comparison to 18F-DPA-714NDAnd VTExhibit a stronger positive linear relationship therebetween. This shows that in this experiment, 18F-VUIIS1009B can be dissected more accurately than 18F-DPA-714 using the AIF-based Logan and Logan reference tissue methods. As shown in this experiment, for 18F-VUIIS1009A,% ID/cc and BPNDAnd VTThe correlation of (a) is weak. Furthermore, 18F-VUIIS1009A did not exhibit higher BP than 18F-DPA-714 and 18F-VUIIS1009BNDOr a DVR. This behavior was due to the higher plasma binding affinity of 18F-VUIIS1009A compared to 18F-DPA-714 and 18F-VUIIS1009B, which was also noted in previous experiments of the present invention. For the 18F-VUIIS1009A, the higher plasma binding limits the distribution of the radiotracer from blood to tissue, thus reducing its tissue uptake and image SNR, which can affect subsequent macroparametric determinations.
By evaluating two highly specific TSPO radiotracers 18F-VUIIS1009A and 18F-VUIIS1009B in a neuroinflammatory model, a more sensitive radiotracer with superior macroscopic parameters was obtained to reflect TSPO expression. The 18F-VUIIS1009B provided by the present invention would be a suitable PET tracer to achieve this goal. The invention provides 18F-VUIIS1009B PET imaging and parameter images (V)TAnd BPND) And the combination of graph/kinetic analysis has wide application prospect on the characterization of the longitudinal neuroinflammation.
The present invention focuses on the radiosynthesis and evaluation of a novel TSPO probe 18F-VUIIS1009A/B for neuroinflammatory imaging in ischemic rats by performing a series of in vivo assays and graphical analyses. The results confirmed 18F-VUIIS1009B PET imaging and parametric images (V)TAnd BPND) And the pattern analysis is combined, so that the method has wide application prospect for characterization of neuroinflammation. The present invention contemplates that 18F-VUIIS1009B will be a suitable PET tracer for imaging neuroinflammation in a number of diseases.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Claims (5)
- Use of a TSPO translocator probe in a neurological disease, characterized in that: co-registering through PET images and analyzing dynamic PET data; performing imaging analysis by using an arterial plasma input function AIF (advanced immune function), and generating a Logan graph and a parameter image of a total distribution volume VT of each radioactive tracer; a parametric image for the binding capacity constant BPND is generated by using a reference Logan model of a healthy brain as a reference region.
- 2. The use of a TSPO translocator probe according to claim 1 in a neurological disease, wherein: TSPO translocator probes were set to 18F-VUIIS1009A and 18F-VUIIS 1009B.
- 3. A method for imaging TSPO translocator PET probe in neuroinflammation, which is characterized in that: the method comprises the following steps:step 1: injecting the TSPO translocator probes 18F-VUIIS1009A, 18F-VUIIS1009B and 18F-DPA-714 into a cerebral ischemic rat model, and carrying out PET imaging treatment on the cerebral ischemic rat by using a small animal PET scanner;step 2: the binding specificity of 18F-VUIIS1009A and 18F-VUIIS1009B, respectively, was assessed by displacement of the non-radioactive analogs;and step 3: carrying out PET image co-registration and dynamic PET data analysis; performing imaging analysis by using an arterial plasma input function AIF (advanced immune function), and generating a Logan graph and a parameter image of a total distribution volume VT of each radioactive tracer;and 4, step 4: generating a parametric image for a binding capacity constant BPND by using a reference Logan model with a healthy brain as a reference region;and 5: carrying out statistical analysis; all quantitative data are expressed as mean ± standard deviation and the relationship between regional outcome parameters under test conditions and re-test conditions was estimated using GraphPad Prism 5 software.
- 4. A method of imaging a TSPO translocator PET probe in neuroinflammation as claimed in claim 3, wherein: the PET images of the image analysis in the step 3 are co-registered, and the method comprises the following steps: firstly, registering corresponding CT images by using Inveon Research Workplace 4.0 software, carrying out spatial alignment on brain areas, and generating a conversion file for co-registration; the generated conversion file is used as an input to register a corresponding PET image, the registered PET image is used to draw an ROI, the voxel values are analyzed, and a parameter map is calculated.
- 5. A method of imaging a TSPO translocator PET probe in neuroinflammation according to claim 4, wherein: the method for analyzing the dynamic PET data in the step 3 comprises the following steps: analyzing the PET image by using PMOD 3.4 layout image analysis software and Inveon Research Workplace 4.0; ROIs were manually drawn ipsilateral and contralateral to striatum and cortex; generation of parameter V Using Logan analysis based on plasma inputsTImage, plasma contribution factor vp for all analyses set to 0.05; for the same animal, a Logan reference tissue model was also used to generate a parameterized BP with contralateral brain TAC as inputNDAn image; for DVR or BPNDDetermining and parameterizing image generation, using the SRTM method, with the contralateral brain as reference tissue to calculate k2_REF(ii) a For each radiotracer,% ID/cc, BP was analyzed using parameters determined from the same voxels in normal and parametric PET imagesNDAnd VTThe correlation of (c); correlation was analyzed using Pearson correlation coefficient r and p-values were generated using GraphPad Prism 5 software.
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