| Nervous and mental diseases pose a serious impact on human health and social development. Schizophrenia brings heavy dysfunction to the human body and has a chronic course of the disease and a high disease rate in lifecycle. As a result, early diagnosis and treatment of schizophrenia is one of the thorny problems in the 21st century. Central N-methyl-D-aspartate receptor (NMDAR) is a major receptor of ionotropic glutamate receptors (iGluRs). The sustained depolarization of excitatory neurons resulted from NMDAR's over-activation may lead to Ca2+ influx which cause cell death. This may play an important role during the development of the neurological diseases such as Parkinson's disease, Alzheimer's disease, Huntington's, pain, stroke and the mental illnesses such as schizophrenia, bipolar disorder. Studies have shown that the reduction of NMDAR activity was the main reason of causing schizophrenia. As to detect the variance in distribution, the number (density) and function (affinity) of NMDAR in specific regions of nervous system, to show the physiological and pathological states of brain in terms of the molecular level, to make early diagnosis of schizophrenia and to observe the disease progress, guide the rational use of drugs and the efficacy evaluation and prognosis, NMDAR imaging has an important theoretical and practical value. With irreplaceable advantages to other research methods, brain receptor imaging of nuclear medicine is today's hot researches and topics.As a medical tracer,99mTc has incomparable advantages to other nuclides in that simple radiopharmaceutical equipment, low-price and full availability of the stuff, energy (140KeV), half-life (6h) is moderate. By far, technetium labeled radiopharmaceuticals about NMDAR are still blank in the market at homeland and abroad, hence studying targeted NMDAR must significantly promote the study of schizophrenia pathogenesis and its early diagnosis. Due to the special structure of pyrroloquinoline quinine (PQQ), PQQ can be used as electron donors and receptors in various reactions at the same time. So we chose PQQ as ligand to bind with 99mTc for the preparation of 99mTc-PQQ molecular probe.99mTc labeled PQQ specific receptor's imaging drugs are expected to apply to observe the density changes of NMDAR caused by these diseases, and help to clarify the pathological mechanism of these changes. In order to study whether 99mTc-PQQ can be as a potential NMDAR imaging agent and whether PQQ is helpful to the diagnosis and treatment of schizophrenia, this experiment, which has not been reported at home and abroad, was designed.The contents of experiment include PQQ labeled with technetium, analyzed the radioactive receptor binding, in vitro stability, partition coefficient, plasma-protein binding and other biological characteristics, in vivo distribution, preliminary pharmacokinetics, acute toxicity and in vivo and vitro brain imaging research.With the reduction of stannous fluoride,99mTc-PQQ was acquired by chelating PQQ with technetium in ligand exchange method. Radiochemical purity was determined with paper chromatography and HPLC. Through the optimizing of labeling conditions, high radiochemical purity of 99mTc-PQQ, which was greater than 90%, was obtained. It was showed that radiochemical purity of 99mTc-PQQ remained more than 90% after 6h at room temperature by stability experiments in vitro.Receptor binding analysis showed that labeled compound 99mTc-PQQ can specifically combine with the NMDAR and take on a single-saturation trend. The combination between 99mTc-PQQ and NMDAR has a equilibrium dissociation constant Kd of 6.648μmol/L and a maximum binding capacity Bmax of 2.381 nmol/mg. EB and MK-801 are two types of antagonist with different affinities of NMDAR, and Haloperidol is a antagonist of dopamine D-2 receptor and the a receptor. With three compounds above as inhibitors, the experiment was taken on to compete with 99mTc-PQQ, and the results showed that EB and MK-801 had an effect of inhibition on 99mTc-PQQ and NMDAR to varying degrees, while haloperidol had no obvious effect. In addition, it was showed that there was targeting characteristics in the combination between 99mTc-PQQ and NMDAR which provided good evidence for the fact that 99mTc-PQQ can become a NMDAR's perfect imaging agent.99mTc-PQQ has certain characteristics of binding specificity and targeting to NMDAR in vitro. Moreover, brain receptor's imaging agent should have good fat-soluble attribution and suitable electrical attribution so that it can enter into the brain and play a important role. The experimental results showed that the lipid -water partition coefficient (1gP), when 99mTc-PQQ was in octanol/water system, was-1.49±0.16, and it was of negative charge in pH=7.4 phosphate/ethanol system. The binding rates of plasma protein were 6.02%,5.43%,6.15% and 7.23% respectively in four doses and without the concentration-dependence. The experiments of biodistribution in vivo showed that 99mTc-PQQ's distribution in vivo had high level in the stomach, liver, kidneys and lung, and the metabolism was mainly through the liver and kidney. The brain had certain uptake, and the hippocampus, cortex and brain stem which occupied relevantly abundant NMDAR distribution had the most of uptake. It demonstrated that the distribution and elimination of 99mTc-PQQ was in line with the two-compartment model, and the term of absorption half-life was 18.16min and the term of organized half-life 100.45min in the blood kinetic experiments. Brain imaging showed that the radioactive ligand had a higher concentration in the hippocampus and cortex than that of the other brain regions. No significant response showed in acute toxicity test.Dizocilpine (MK-801)-indued schizophrenia-like mouse model was established for further study of 99mTc-PQQ. Through treatment of inbred strain ICR mouse by MK -801 maleate (0.6 mg/kg, i.p., single injection) products acute schizophrenia animal model. Subacute model was created 7 days after using MK-801 one time per day. Representative hyperlocomotion, stereotypy behavior and ataxia actions would be observed to determine the validity of animal model. Vacant group and Risperidone (0.9 mg/kg, i.p.,and one time per day) treatment group were used as control groups. The effect of intervention on SZ with different doses of PQQ and PQQ ester was evaluated with 18F-FDG/PET and autoradiography.Preliminary studies showed that 99mTc-PQQ is a potential SPECT imaging agent for NMDAR. It provides a possible new basis for pathogenesis of schizophrenic, which also lays down a good foundation for subsequent research. Futher study is to focus on reforming of PQQ structure and developing a series of analogues so as to increase fat-soluble. As a result, we will improve the brain volume for technological breakthrough and develop more specific NMDAR imaging agents. |
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