| Fibroblast activation protein(FAP)is a serine hydrolase and has been identified in fibroblasts cultured with monoclonal antibody F19 as early as 1986.As a specific marker,FAP is widely expressed in tumor-related fibroblasts(cancer-associated fibroblasts,CAFs),and because of its special biological characteristics and gene stability,it plays an important role in the growth,invasion,metastasis and chemotherapy resistance of tumor cells.Compared to normal subjects,FAP is differentially highly expressed in a variety of tumors and fibrosis-related diseases,making it a very promising target.In recent years,the use of FAP as a target for imaging and therapy has become a hot topic of interest for researchers.Since FAP possesses gelatinase and dipeptidase activities,it can degrade substrates such as gelatin,collagen and dipeptidase to promote tumor growth,invasion,metastasis and ECM degradation.Therefore,tumor progression can be controlled by selective inhibition of FAP enzymatic activity,and the in-depth study of the biological functions of FAP in the tumor microenvironment in recent years has led to the application of fibroblast activation protein inhibitor(FAPI)in tumor FAP-targeted therapy.The application of FAPI in tumor FAP targeting therapy has attracted much attention.At present,FAPI based on radiolabeled nuclides such as 18F,68Ga,etc.are usually used,but their expensive cost restricts their widespread use.However,the low cost and suitable halflife(T1/2=6.02h)of single-photon nuclide such as technetium-99m make it promising for imaging.The main focus of this thesis is on the "synthesis-evaluation-validation" of 99mTcGlc-FAPT,a small molecule inhibitor of 99mTc-labeled FAP.In the first part,a small molecule inhibitor targeting FAP,HYNIC-Glc-FAPT,was constructed,labeled with the radionuclide 99mTc and quality controlled to investigate the feasibility and ease of labeling with the radionuclide.In the second part,we screened and evaluated the best performing single photon probes for targeting FAP by cell and animal experiments and applied for national invention patents.In the third part,we investigated the value of 99mTc-Glc-FAPT in preclinical stage for tumor and non-tumor models,using tumor animal models and renal fibrosis animal models as the main research subjects.Part Ⅰ:SynthesisMethodology:FAPI-04,a widely used targeting moiety,was selected as the main body of the drug construct,and a precursor compound consisting of a pharmacodynamic moiety ligand(FAPI-04),a pyranoglucosaminyl ligand,a hydrazineglutamyl group(HYNIC)and a linkage group,in which the linkage group includes polyethylene glycol group,aspartic acid group and glutamic acid group,was constructed by solid-phase chemical synthesis(Fmoc method).The compounds were identified by mass spectrograph(MS)and analyzed for chemical purity by high performance liquid chromatography(HPLC).Result:The precursor compounds were successfully synthesized and identified by mass spectrometry with accurate structures,and the chemical purity was>99%by HPLC.Conclusion:The precursor compound obtained by Fmoc solid-phase synthesis method has a mature process,high chemical purity,and stable chemical properties in the solid state of small molecule drugs,which can achieve the goal of ready-to-use dissolution and facilitate the subsequent experiments.Part Ⅱ:EvaluationMethodology:Ethylenediamine-N,N’-diacetic acid(EDDA)and Tricine were selected as the radiolabeled co-ligands,and stannous chloride(SnCl2)was used as the reducing agent,and the precursor compounds were radiolabeled in combination with freshly washed pertechnetate solution.EDDA was dissolved in NaOH solution and Tricine was dissolved in PBS buffer as preliminary preparation,and the reaction was carried out by adding the pertechnetate solution,EDDA,Tricine and SnCl2 to an EP tube(Eppendorf tube)and heating for 10 min,then the reaction solution was cooled,diluted and purified by a C18 column,followed by a 50%ethanol-water solution.Similarly,the target product 99mTc-Tricine(2)-FAPT was obtained by adjusting the coligand formula.The uptake of the two imaging agents in different organs and tissues was evaluated in normal Kunming mice,especially in the kidneys.SPECT imaging in small animals was performed to select the imaging agent with more stable function.Result:The radiochemical purity of the unpurified end-product was>97%,and the radiochemical purity of the product purified by C18 column was>99.5%with specific activity>36.5 GBq/μmol.The biological distribution of normal Kunming mice and small animal SPECT/CT imaging showed that the two imaging agents were rapidly excreted by the kidney and cleared in the blood,and no obvious organ physiologic uptake was observed.However,compared with the co-ligand Tricine labeling,the end product produced by the combined Tricine/EDDA labeling strategy has higher water solubility and faster renal clearance.However,the combined Tricine/EDDA labeling strategy resulted in higher water solubility and faster renal clearance than the co-ligand Tricine labeling approach.Conclusion:It was concluded that the two labeling strategies used were simple and efficient,and the radiopurity of the unpurified product was high,>97%,which met the requirements of animal experiments and could not be further purified.The purity of the purified product was greater than 99.5%,indicating that this purification strategy is simple and feasible,and is conducive to clinical translation.Normal animal studies showed that 99mTc[Tc]-Tricine/EDDA-FAPT possessed more promising chemical properties and could be used in subsequent experimental studies.It is concluded that the two labeling strategies used are simple and efficient with radiolabelling rate of more than 97%and the specific activity of more than 36.5GBq/μmol,which met the requirements of animal experiments and cannot need to be further purified.The radiochemical purity is greater than 99.5%,indicating that this purification strategy by a C18 column is simple and feasible,and contributes to clinical translation.Normal mice experiment studies show that 99mTc-Tricine/EDDA-FAPT possesses more promising chemical properties and can be used in subsequent experimental studies.Part Ⅲ:ValidationMethodology:A549-FAP cells with positive FAP expression were constructed,and cell uptake inhibition,endoflux and efflux,competitive inhibition and other experiments were conducted to verify the targeting characteristics of the imaging agent at the cellular level.The location and level of FAP protein expression in cellular were verified by immunofluorescence staining.U87MG,A549-FAP and A549 tumorbearing nude mouse models were constructed.The targeting and safety of 99mTcTricine/EDDA-FAPT in tumor animal models were analyzed by SPECT/CT imaging in vivo,inhibition study,biodistribution and immunohistochemical experiments.The model of renal fibrosis in SD rats was established,and the diagnostic value of the imaging agent in non-tumorigenic lesions was verified by similar study methods.Result:Cell experiments showed that the two imaging agents can bind to FAP specifically,and had rapid influx level,very small outflow level and high affinity.Immunofluorescence staining verified the expression of FAP protein in the cell membrane and cytoplasm,which possessed a higher degree of expression in the membrane of A549-FAP cells transfected with the FAP gene and was more pronounced in the cytoplasm than in the cytomembrane of A549 cells.Significant tumor uptake was observed in the A549-FAP and U87MG tumor-bearing nude mouse model,with significantly lower uptake values in the A549 tumor-bearing model than in the other two tumors.Immunohistochemical studies showed significantly high expression of FAP in U87MG tumors and moderate to low expression in the mesenchyme of A549 tumors.In the SD rat model of renal fibrosis,significant renal uptake was observed compared to controls and Masson staining of the kidneys suggested significant interstitial fibrosis.Conclusion:It is concluded that 99mTc-TE-FAPT is highly targeted at both cellular and living animal levels and can be used for rapid and high-contrast imaging of tumors,and has corresponding value in non-tumor models such as non-invasive diagnosis of renal fibrosis. |
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