| The incidences of chronic hepatitis, hepatic cirrhosis and related liver cancer are all very high in China. For these malignant diseases, the accurate assessment of hepatic function is not only of great significance in the perioprative evaluation of the liver, but it also provides guide to treatment and prognosis. At present, commonly used means of liver function evaluation, such as Child-Pugh classification, blood detection of liver removing and loading test and so on, can reflect the preoperative total hepatic function, but no test exists to predict hepatic functions of the excised and residual parts after liver resection. Fortunately, recent 3D imaging technologies, such as single photon emission computed tomography (SPECT) and PET—CT, make it possible to evaluate the preoperative hepatic functions of liver sections. In this way, the liver imaging agents that can demonstrate liver function becomes a key factor.In our preliminary studies, by combining the liver imaging agent of 99mTc—EHIDA that can demonstrate the bile metabolism and the SPECT imaging technology, both animal experiments and clinical experiments were carried out. If the liver is divided into several voxels, then the relation between the hepatic function of any part of the liver and that of the whole liver can be denoted by dividing the total radioactive counts of any part of the liver by those of the whole liver. In this way, the postoperative residual hepatic function of any part of the liver can be computed. However, there haven't been similar reports of this study in China. As a liver imaging agent, EHIDA has some disadvantages: first, as it is metabolized through the biliary tract, the imaging results may be affected by the biliary tract diseases; second, after the hepatic uptake, metabolism and excretion is processed at the same time, and it cannot fully demonstrate the function of liver cells; due to the quick metabolism of EHIDA, its concentration may decrease rapidly from 30 minutes after the intravenous injection, and this may lead to poor imaging results in the late stage. Therefore, in this study, the imaging agent that is more relevant to the hepatic function is used to replace EHIDA.It is found by Kokudo, et al that, when the mammals are suffering the hepatitis, cirrhosis of the liver or liver cancer, the amount and activity of Asialoglycoprotein receptors (ASGPR) on the cell membrane are both damaged. On this basis, the Japanese scholars have synthesized diethylenetriamine pentaacetic acid-galactosyl-human serum albumin (GSA), which is an analog of asialoglycoprotein (ASGP), and developed the clinical imaging technologies. As these technologies have not been reported in China, America and Europe, we synthesized the GSA with patent right, produced one-step freeze-dried kits (patent number: 200810057222.1), and carried out the researches on liver imaging by using 99mTc as the marked element. The uptake and distributing of it in the normal mice and the mice with liver fibrosis, gallbladder stasis type hepatitis or liver cancer were determined, and its bioavailability was also verified. Then, it was used in the normal New Zealand Rabbits, and the imaging effectiveness and biological metabolism were both determined by SPECT imaging. After that, we first applied it to the clinical treatment, observed its effectiveness, further evaluated the hepatic function of any section of the liver with the help of computer programs, and finally expected to complete the digital demonstration of the risk of liver resection.Part 1 The preparation of 99mTc-GSA and the formula of its kits for clinical useObjectiveIn order to accurately evaluate the preoperative hepatic function, help predict the risk of the surgeries, as well as reduce the mortality in the perioprative period of liver, we prepared the 99mTc-GSA, which is an ASGPR imaging agent. In order to facilitate its clinical research and application, we prepared the sterile one-step freeze-dried kits. And besides, the biological evaluation in normal mice of 99mTc-GSA was also carried out.MethodIntroduce the dual-function coupling agent to the human serum albumin (HSA) molecule by DTPA anhydride ring, synthesize the 2-sub-amido-2- methoxy-ethyl-1-β-D- thiogalactopyranoside, and then the GSA is acquired by introducing the thiosulfate galactose though the reaction with DTPA-HAS.Use stannous chloride as the reducing agent, and determine the best conditions for marking. On this basis, further prepare the sterile one-step freeze-dried kits for GSA, and compare the marked compound prepared with the wet method with that prepared with the freeze-dried method. Finally, carry out the experiments to determine its biodistribution in normal mice. ResultsSuccessfully synthesized the 2-sub-amido-2-methoxy-ethyls-1-β-D-thiogalactopyranoside with a high chemical yield, and all the intermediate products were identified through IR, NMR and elemental analysis. The saccharide density, determined by the phenol-sulfuric acid method was 31, which is in the saccharide density range (30-40) of GSA in kits as reported in the literature. By assaying, the best protocal for imaging were determined as follows: GSA 3mg, SnCl2·2H2O 20μg and 30 min (reaction time). The final formula of the one-step freeze-dried kits is: GSA 3.0mg, SnCl2·2H2O 20μg, pH=3.5 and suitable excipients. It is demonstrated by the biodistribution experiments that, 99mTc-GSA is highly incepted by the liver of a mouse, the intake is still more than 70%ID·g-1 at 30 min after the experiment begins, and it can be saturated.ConclusionThe production of liver receptor imaging agent lays a good foundation for the nuclear medicine imaging technologies, such as SPECT, to evaluate the hepatic function three-dimensionally. The synthetic GSA, which is an analogue of ASGP and can be combined with the specific receptors on the surface of a liver cell, can not only evaluate the morphology and function of the liver to provide parameters for the liver surgeries, but also is very helpful to the researches in vivo metabolism, liver physiology and pathophysiology, etc. Some Japanese scholars have developed the imaging technologies for clinical use by GSA, while the similar studies haven't been reported in Europe and America yet. Based on the considerations above, we successfully prepared the ASGPR imaging agent-99mTc-GSA, and in accordance with the best protocal for iamging, we prepared the 99mTc-GSA, which is a marker compound with a labeling rate more than 96%; and especially, we invented the sterile one-step freeze-dried kits (patent number: 200810057222.1). It was demonstrated by the biodistribution experiments that 99mTc-GSA can be specifically bound to the liver of a mouse, and the imaging time is longer than half an hour. This demonstrates that the marking of the GSA one-step freeze-dried kits is simple and reliable, and at the same time, thanks to its good biological properties, its clinical use is worth being further researched and applied. SummaryThis study first synthesized the 99mTc-GSA with patent right and prepared a freeze-fried kits for it. After its biological effect being further verified, it can be used in the researches on cirrhosis of the liver and liver cancer as well as the experimental studies on the three-dimensional evaluation of the residual liver function.Part 2 Application of 99mTc-GSA in different liver injury models inmiceObjectiveOur newly synthesized liver imaging agent 99mTc-GSA can indicate liver function according to its distribution in the liver. This study was to identify the uptake and biological distribution of 99mTc-GSA in three mouse models with different degrees of hepatic injuries.MethodsMouse models included hepatic fibrosis, hepatic cholestasis, and liver cancer. Hepatic fibrosis model was established by intraperitoneal injection of carbon tetrachloride, 0.4ml 10% ,every 48 hours for 48 days. Cholestasis model was setup by ligature of the common bile duct for 72 hours, and liver cancer model by implantation of H22 tumor cells underneath liver capsule for 10 days. On measurement, each mouse in different models and normal controls was injected 0.1 mL (0.37MBq) 99mTc-GSA (2μg) into vena caudalis, and 5 minutes later sacrificed by decapitation. Important organs including liver, heart, lungs, kidney, spleen, stomach, blood, bones, muscles, and intestines were taken and their different radiocountings were measured. The hepatic injuries were evaluated with serum and pathological examinations.Result99mTc-GSA was concentrated in the liver in all three mice models and the control mice (%ID·g-1>40) . Compared with control (%ID·g-1=90.05±10.55) , the density of 99mTc-GSA was lower in the models with hepatic injuries (P<0.001) . Among the three models, the concentration of 99mTc-GSA in hepatic fibrosis model (%ID·g-1 = 72.20±2.13) was significantly higher than that in the models with cholestasis (%ID·g-1 = 56.72±5.92) and liver cancer (%ID·g-1=42.80±6.05)(P<0.001, P<0.001).ConclusionNewly synthesized 99mTc-GSA appears to concentrate in the liver and its concentration is invensly related with hepatic injuries. Thus it could be used as liver imaging in the clinic indicating of liver function. When combined with three dimensional scanning technique, it might be able to help constructing a new three dimensional imaging method that demonstrates function of designed liver segments.SummaryIn this study, the bioavailability of 99mTc-GSA is verified by using it in three different injury models in mice, and this kit technology may be used in clinical treatment if its safety can be determined.Part 3 Preclinical study of animal imaging of 99mTc-GSAObjectiveOn the basis of that the bioavailability of 99mTc-GSA is verified, further prove that it can clearly display the liver while being used with the SPECT technology, and observe its metabolism in vivo.MethodSeven anesthized New Zealand rabbits were fixed onto plates, then injected with 0.1ml (about 0.37MBq) 99mTc-GSA (0.1mg/Kg) marked by the kit method into the ear vein of the rabbits. After that, immediately acquire the dynamic data for 1 minute by using a SPECT Instrument with a speed of 2S/frame; then acquire the systemic data for 5 min, 10min, 20min, 30min, 60min and 120min respectively; immediately carry out the cross-sectional acquisition when the systemic data is acquired for 30 min, rotate the probe around the liver by 180°, and acquire a frame every 6°with a speed of 30s/frame.ResultThe Time- Radioactivity Curve of the heats of New Zealand rabbits shows that the 99mTc-GSA inside the heart is quickly discharged, and the ID% of the heart at 1 min is only 7.96%. The acquired systemic data shows that, in 30 min, the ID% of the 99mTc-GSA insides the liver can be over 50%, and the 99mTc-GSA can well image the liver three-dimensionally. Besides liver, 99mTc-GSA mainly concentrates in bladder, stomach and intestine, in which the concentration is proportional to the time, while there is almost no concentration in brain.ConclusionAfter entering the organism, the 99mTc-GSA can be specifically incepted by the liver immediately, and it generally doesn't accumulate in the circulatory system. It will stay in the liver for more than 30 min, and can image the liver three-dimensionally very well. It is metabolized mainly by the urinary system and digestive system while blocked by the blood-brain barrier.SummaryThis study demonstrates that the 99mTc-GSA has a good efficacy of liver imaging and can be metabolized safely, and it can be used in the three-dimensional evaluation of human liver clinically.Part 4 The clinical application of 99mTc-GSAObjectiveAt the conclusion of the preliminary synthesis and animal imaging studies of 99mTc-GSA, we apply it to normal human bodies and a patient with liver occupying lesions to observe the imaging effect of 99mTc-GSA, thereby laying a foundation for the clinically three-dimensional evaluation of liver function by use of 99mTc-GSA.MethodIntravenously inject 99mTc-GSA (5mCi/1ml) into four normal human bodies and one patient with liver occupying lesions, and then carry out the dynamic SPECT data acquisition, liver section acquisition and whole body scan. Outline the region of interest (ROI) according to the dynamic data acquisition images, evaluate the concentration of imaging agent in liver and conduct 3D imaging by use of SPECT technology.ResultThe concentration of 99mTc-GSA in liver was obvious, and it can clearly show the outline of liver and stay in liver for more than 20 minutes. Obvious cold regions were observed in the section and three-dimensional imaging of patient with liver occupying lesions.ConclusionIt is shown by the clinical trials that, 99mTc-GSA can be specifically combined with the receptors on the surface of liver cells to achieve a significant concentration in liver, thereby making the imaging of liver clearer; moreover, it can stay in liver for a relatively longer time, so that a better evaluation on liver function can be achieved. The occupying tissue can't be combined with imaging agent, so it will become a cold region where the concentration is rather low.SummaryThe result of small-scale clinical application of 99mTc-GSA is in line with the expected imaging effect, and this can lay a foundation for the clinical three-dimensional evaluation of liver and establishment of the risk evaluation system that is used to determine the excision scope of liver. |
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