Chemosynthesis,Radiolabeling Of Positron Imaging Agent ¹¹C-Fethypride And It's Applications In Parkinson's Disease

As basic chemistry and core chemistry,organic synthetic chemistry is widely used in medicine,agriculture,industry and other fields.With the continuous development of life science,organic synthesis technology has penetrated into various fields of medicine,such as physiology,pathology,pharmacology,imaging medicine,which has greatly promoted the development of medicine.As a new imaging technology,molecular imaging can diagnose human body with non-invasive technology,which greatly improves the accuracy of diagnosis and brings good news to patients.Among them,radioimaging agents are widely used in clinical medicine because of their specific binding with target organs or tissues.Imaging medicine will be the world of Molecular Imaging(MI)technology,in the future,the research and development of specific imaging agents will be the most promising field.Chemical synthesis is based on the simple reagent as the basic raw material,connecting a functional group or carbon chain through organic reaction,and then using the functional group on the intermediate to assist the reaction,finally synthesizing the target compound.The reaction conditions of each step should be mild,the reaction yield should be high,the raw materials used should be low toxic,low pollution,cheap and easy to obtain,the synthesis reaction should be fast and convenient,and the waste pollution should be less.Positron imaging agent is a kind of radioactive imaging agent,which can label positron nuclide to target organic compounds,detect biological system in vitro,and study the physiological and pathological changes of target tissue in vivo.Because of its high specificity and high positive detection rate for specific target tissue,it greatly promotes the development of imaging information technology of related molecules,and has become a hot topic in nuclear medicine.1.ObjectiveThe main objective of this work is to develop a new type of Positron imaging agent¹¹C-Fethypride for the early diagnosis of Parkinson's Disease(PD).Each product in the process of the precursor synthesis will undergo nuclear magnetic resonance(NMR)and high resolution mass spectrometry analysis in order to determine whether the products are target products.Grope for the radioactive method by¹¹C labeling,the optimum reaction conditions and a series of quality control tests of the final products.Investigate the clinical application feasibility of radioactive imaging agent¹¹C-Fethypride through pre-clinical fat solubility test,acute toxicity test,stability test,biodistribution test and pharmacokinetic test.Established an animal model of PD in rats.PET/CT scan was performed after intravenous injection of¹¹C-Fethypride,to explore the feasibility of¹¹C-Fethypride as a PET/CT dopamine receptor imaging agent in the diagnosis of PD,and to further explore the feasibility of¹¹C-Fethypride as a platform for etiological evaluation and drug screening of PD.2.Methods2.1 A new type of Positron imaging agent with vanillic acid methyl ester as raw material was prepared,involving 4-step conventional reactions and 1 step radioactive¹¹C labeling.This new agent will have great potential application prospect was for the simple synthesis method and the cheap and widely available raw material.What's more,the substituent on nitrogen can be further optimized.2.2 Positron Radiopharmaceutical ¹¹C-Fethypride Quality ControlPhysical identification:Clarity Detection:Detect the clarity by turbidimetric method.It was clear and qualified when the turbidity is below 0.5 turbidity standard solution.Purity Detection:Detect the radiochemical purity of ¹¹C-Fethypride solution by HPLC method and TLC method.Chemical identification:p H Detection:Determine p H value of the solution by using p H test strips.Radiochemical.The Stability Test:To detect,analyze and evaluate its stability,¹¹C-Fethypride solution was placed at room temperature and in static state for 5 min,15 min,30 min,60 min,90 min,120 min respectively after radioactive labeling.It was proved to be stable if the radioactive chemical purity(RCP)was not less than 90%.Biological identification:Bacterial Endotoxin Detection:Measure the bacterial endotoxin of ¹¹C-Fethypride solution by Tachypleus Amebocyte Lysate(TAL)reagent method.It was qualified when the content of bacterial endotoxin was below10 EU/m L.Sterility Test:¹¹C-Fethypride solution was vaccinated in two different medium respectively and placed at different temperatures to cultivate for 14 days.It was qualified if no bacterial grew in the medium tube.Abnormal Toxicity Test:Lab animals,including mice and guinea pigs,were intraperitoneally injected with different doses of radiopharmaceutical ¹¹C-Fethypride and were observed for seven days.During the period of observation,the test was qualified if the animals were in good health,without abnormal reactions observed and gaining in weight.2.3 ¹¹C-Fethypride Biodistribution experimentsLipo-hydro Partition Coefficient:Determine the radiocounting of Radiopharmaceutical ¹¹C-Fethypride with?-radioactive counter when it was in n-butyl alcohol phase and water phase.The lipo-hydro partition coefficient(Log P)was the logarithm of both radiocountings ratios.Acute Toxic Test:Kunming mice were injected with different doses of radiopharmaceutical ¹¹C-Fethypride into the tail vein.After 7-day's observation,they were killed and pathological section was done to check whether the main organs were normal.¹¹C-Fethypride Biodistribution Test in the Main Organs in Rat:Rats were injected with radiopharmaceutical ¹¹C-Fethypride solution into the tail vein and were killed 10 min,30 min,60 min,90 min and 120 min respectively after injection.Adequate amount of samples from Brain,heart,lung,liver,spleen,stomach,intestine,kidney and muscle were rinsed in saline,blotted up moisture with filter paper.Weigh the samples with the balance and determine the radiocounting with?-radioactive counter.(%ID/g)was calculated.¹¹C-Fethypride Biodistribution Test of Brain Tissue in Rat:Rats were injected with radiopharmaceutical ¹¹C-Fethypride solution into the tail vein,were killed 10 min,30min,60 min and 90 min respectively after injection.Adequate amount samples from frontal lobe,parietal lobe,temporal lobe,occipital lobe,striatum,hippocampus,thalamus and cerebellum were taken and rinsed in saline,blotted up moisture with filter paper.Weigh the samples with balance and determine the radiocounting with?-radioactive counter.(%ID/g)was calculated.2.4 ¹¹C-Fethypride Pharmacokinetic TestRats were injected with radiopharmaceutical ¹¹C-Fethypride solution into the tail vein.50 m L blood sample was taken at different time and was analyzed by using liquid chromatography-mass spectrometry(LC-MS).2.5 PET/CT ScanEstablishment of rat model of Parkinson's disease:MPTP rat model and negative control group were established,and the success or failure of the model was evaluated by rotating bar test.Normal rats and PD rats were injected with radiopharmaceutical ¹¹C-Fethypride solution into the tail vein and were fixed in center field of examination bed after anesthesia with 10%chloral hydrate,supine position for PET/CT scanning.2.6 Biological study and PET/CT correlation study of Parkinson's disease rats:Correlation between LncRNA HOTAIR expression and PET/CT imaging in rats with Parkinson's disease:QRT PCR,CCK-8 test and flow cytometry were used to observe the expression of LncRNA HOTAIR,cell proliferation and apoptosis in midbrain cells of normal rats and Parkinson's disease rats,and to explore the correlation with ¹¹C-Fethypride PET/CT imaging results.Evaluation of the therapeutic effect of Monascus on rats with Parkinson's disease by PET/CT imaging:The rats in the treatment group were intraperitoneally injected with Monascus solution(400 mg/kg)for 7 days from the first day of establishment of Parkinson's disease model,followed by injection of ¹¹C-Fethypride via tail vein for PET/CT imaging.After PET/CT scanning,the normal rats,PD treatment group and PD control group were killed after anesthesia.Tyrosine hydroxylase immunopositive(TH+)cells were observed.The PET/CT images and th+cells of treatment group and control group were compared,and their correlation was discussed.3.Results3.1 A new type of PET/CT imaging agent with vanillic acid methyl ester as rawThe total activity of¹¹C-Fethypride was 1536 mbq,the total yield was 33.8%,the total volume was 2 m L,and the specific activity was 714 mbq/m L.The method is simple,rapid,time-saving and labor-saving,which is suitable for promotion.3.2 Positron Radiopharmaceutical ¹¹C-Fethypride Quality ControlPhysical identification:¹¹C-Fethypride solution was colorless,clear,transparent,without impurities comforming to the rules.RCP was 100%and 99%by TLC and HPLC method,respectively.Chemical identification:p H value of 7.The Stability Test:Radioactive chemical purity(RCP)still exceeded 95%by TLC method 2 hours after keeping at room temperature.So the stability of radiopharmaceutical¹¹C-Fethypride was good,and suitable for clinical application.Biological identification:The content of bacterial endotoxin was below 5 EU/m L by Tachypleus Amebocyte Lysate reagent method through the conversion,conforming to the rules.Traceability sterility test result showed that¹¹C-Fethypride solution was qualified and no bacterial grew in two different medium at different culture temperature.Animal abnormal toxicity test showed that mice and guinea pigs were alive after 7-day's experiment.The animals had increase in weight and no abnormal reactions were observed.Quality control tests indicated that positron radio-pharmaceutical¹¹C-Fethypride was qualified and safe.3.3 ¹¹C-Fethypride Biodistribution experimentsLipo-hydro Partition Coefficient:Log P of radiopharmaceutical ¹¹C-Fethypride were 2.3 and 2.1,which indicated that¹¹C-Fethypride had good fat-solubility which helped radiopharmaceutical¹¹C-Fethypride to pass through the blood-brain barrier (BBB)into the brain tissue.Acute Toxic Test:Compared with negative control group,no abnormal phenomenon were observed in livers and kidneys through pathological section.The maximum injection doses for mice was as many as 500 times of that for the human being,which indicated that radiopharmaceutical ¹¹C-Fethypride had less toxic effects and was safe in use.¹¹C-Fethypride Biodistribution Test of Main Organs in Rats:Radiopharmaceutical ¹¹C-Fethypride was mainly distributed in the liver and kidney and radioactivity gradually decreased with the passage of time.It indicated that¹¹C-Fethypride was metabolized through the liver,and excreted through the kidney,which was coincided with benzamide compounds.¹¹C-Fethypride Biodistribution Test of Brain Tissue in Rats:Radiopharmaceutical¹¹C-Fethypride was mainly distributed in the thalamus.Clearance rate in the cerebellum was the fastest,with the slowest clearance rate in the striatum Striatum gradually increased with the extension of time,which indicated that ¹¹C-Fethypride had specific and high affiliative bind with the dopamine D₂receptor in the striatum.3.4 ¹¹C-Fethypride Pharmacokinetic TestWhen the weight is 1/cc,the AIC value is-15.36,the goodness of fit is 0.1087,and the square of regression coefficient is 1.According to the principle of minimum AIC and maximum R2,the fitting result is a open two artment model.The distribution half-life T_1/2alpha and elimination half-life T_1/2beta of ¹¹C-Fethypride were1.453±0.312 min,25.245±3.892 min,respectively;The apparent distribution volume V(c)was 0.403±0.064 m L/mg,and the drug clearance rate Cl(s)was 0.072±0.030m L/min/mg,indicating that ¹¹C-Fethypride was rapidly distributed and eliminated in vivo.3.5 PET/CT ScanThe results of rotating bar test showed that there was significant difference in motor balance ability between MPTP treatment group and negative control group(P<0.05),suggesting that MPTP treatment group was successful in modeling Parkinson's disease rats.PET/CT scan showed that radiopharmaceutical ¹¹C-Fethypride rapidly entered into all the major organs of rats and quickly cleared.The liver was the major metabolic organ,and the kidney was the major excretory organ.Radiopharmaceutical¹¹C-Fethypride could quickly pass through the blood brain barrier into the brain tissue and specific uptake was observed in the striatum.It was suggested that ¹¹C-Fethypride could be specifically bound to dopamine receptor in striatum.3.6 Biological study and PET/CT correlation study of Parkinson's disease rats:Correlation between LncRNA HOTAIR expression and PET/CT imaging in rats with Parkinson's disease:The results of q RT-PCR showed that the expression of LncRNA HOTAIR in midbrain cells of Parkinson's disease rats was significantly higher than that of normal group;CCK-8 test showed that hotair significantly reduced cell proliferation;flow cytometry showed that overexpression of HOTAIR increased cell apoptosis.These results suggest that LncRNA HOTAIR plays a role in the pathogenesis and progression of Parkinson's disease through gene regulation,which is consistent with the results of ¹¹C-Fethypride PET/CT imaging,reflecting that¹¹C-Fethypride can reveal the underlying pathological mechanism of Parkinson's disease and the interaction between it and molecular targets to a certain extent.Evaluation of the therapeutic effect of Monascus on rats with Parkinson's disease by PET/CT imaging:The number of TH+cells in the PD control group was significantly less than that in the normal rats,while the number of TH+cells in the PD treatment group was significantly more than that in the PD control group,which was close to that in the normal rats,suggesting that Monascus has a protective effect on tyrosine hydroxylase,and then has a certain curative effect on Parkinson's disease.The uptake of ¹¹C-Fethypride in striatum of PD control group was slightly lower than that of PD treatment group.The uptake of ¹¹C-Fethypride in striatum of PD treatment group was higher than that of control group,which indicated that the lesion in striatum of treatment group was significantly improved than that of control group.It also proved that Monascus has certain therapeutic effect on Parkinson's disease,and confirmed that our self-developed ¹¹C-Fethypride can well bind to dopamine receptor,Objective to evaluate the therapeutic effect of drugs for Parkinson's disease by noninvasive method.4.Conclusions4.1 A new precursor compound of ¹¹C-Fethypride with vanillic acid methyl ester as a raw material was successfully synthetized after 4-step conventional reaction. Products at different stages all undergo NMR and high resolution mass spectrometry analysis.The precursor compound was 3-allyl-N-((1-ethylpyrrolidin-2-yl)methyl)-4-hydroxy-5-methoxybenzamide.4.2 Positron radiopharmaceutical ¹¹C-Fethypride was successfully labelled through C11-Multifunctional synthesis module.Quality control tests indicated it was qualified and safe.4.3 Lipo-hydro partition coefficient of radiopharmaceutical ¹¹C-Fethypride was appropriate.It could quickly pass through the blood-brain barrier and specifically bind with the corresponding receptor.It was stable,and had low toxic effect.So it is suitable for clinical intravenous injection.4.4 Radiopharmaceutical ¹¹C-Fethypride had specific and high affiliative bind with the dopamine D₂receptor in the striatum,with low clearance,is an ideal imaging agent for PD.4.5 The kinetic courses of ¹¹C-Fethypride in rats were described by an open two artment model,metabolic mainly through liver and kidney excretion;the distribution and elimination are fast in the body,showing a first-order linear dynamic process.4.6 ¹¹C-Fethypride can reveal the underlying pathophysiological mechanism of Parkinson's disease and the interaction between¹¹C-Fethypride and molecular targets to a certain extent.It is an ideal drug screening platform to evaluate the efficacy of drugs for Parkinson's disease by noninvasive methods.This research involved a series of pre-clinical experimental tests for radiopharmaceutical¹¹C-Fethypride,including precursor design,chemical synthesis,radioactive labeling,quality control,biodistribution test,pharmacokinetic test and PET/CT scan,Biological study,etc.It laid an experimental foundation for the application of radiopharmaceutical ¹¹C-Fethypride in clinical diagnosis of Parkinson's Disease.