FAPI-34   中文名称: FAPI-34

FAP/Fibroblast activation protein

附加亲水性氨基酸对肝脏蓄积和肝胆排泄的抑制作用[2]
为了改善体内特性，用Asn、Glu和Gla （γ-羧谷氨酸）修饰了螯合部分。此外，在哌嗪基和喹啉基之间合成了一种以三甘醇为间隔剂的前体，以增加FAPI-29的亲水性，而无需在螯合部分进行进一步修饰。与最初合成的99mTc-FAPI-19相比，衍生物99mTc-FAPI-33和-34在HT-1080-FAP细胞上的摄取率分别高达45.8%±1.3%和41.86%±1.07%（图2A）。此外，内部化率在95%以上(图2A；补充表2)和对FAP的高亲和力，观察到FAPI-33的IC50值为10.9 nM， FAPI-34的IC50值为6.9 nM（图1），通过竞争实验评估（图2B）。相比之下，99mTc-FAPI-27（≤12.94%±0.77%）、99mTc-FAPI-28（≤37.52%±1.62%）、99mTc-FAPI-29（≤39.34%±1.01%）和99mTc-FAPI-43（≤28.83%±0.88%）暴露于HT-1080-FAP细胞4小时后的结合率较低（图2A）。此外，竞争实验显示这些衍生物对FAP的亲和力略有降低，FAPI-29的IC50值为12.0 nM， FAPI-28的IC50值为12.7 nM（图1）。

99mtc标记的FAPI衍生物的体内靶向特性和药代动力学[2]
为了比较FAPI-28、-29、-33、-34和-43与FAPI-19的体内靶向特性和药代动力学，我们在ht -1080- fap异种移植小鼠体内进行了生物分布实验。扫描图像显示FAPI衍生物的药代动力学有所改善。与FAPI-19比较(图3A；补充图3A)，在注射化合物后60分钟观察到肿瘤病变中的放射性积累和肝胆排泄比例的减少，并持续到至少120分钟（图3A）。99mTc-FAPI-34在小鼠的肝脏、胆管和肠道中摄取最低，在肿瘤病变中摄取显著(图3A；补充图3B)，通过同时注射未标记的类似物来防止这种情况，并证实了该化合物的靶特异性（图3B）。 [2]
根据这些结果，99mTc-FAPI-34的生物分布实验显示，在注射示踪剂后1和4小时，肿瘤摄取分别为5.4±2.05和4.3±1.95% ID/g，肝脏摄取分别为0.91±0.25和0.73±0.18% ID/g（图4A）。除肾脏外，在异种移植物的血液和器官中检测到的FAPI-34活性低于1% ID/g，占肿瘤与组织的比例大于1（图4B）。相比之下，我们在注射示踪剂后1和4小时测得99mTc-FAPI-29（2.79±1.19和1.43±1.13 %ID/g）和99mTc-FAPI-43（2.41±0.34和2.57±0.32% ID/g）的肿瘤摄取较低（补充图4）。然而，这些衍生物的肝脏摄取分别在1和4小时后从0.63±0.06增加到1.73±1.33 %ID/g （FAPI-29）或从1.74±0.28略微下降到1.56±0.03 %ID/g （FAPI-43）。综上所述，99mTc-FAPI-34在异种移植物中提供了最佳的药代动力学，因此可用于临床显像和SPECT。

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FAPI-34在人肿瘤中的积累[2]
2例转移性卵巢癌和胰腺癌患者接受了68Ga-FAPI-46的PET和90Y-FAPI-46作为最后一线治疗，99mTc-FAPI-34的显像或SPECT。转移性卵巢癌患者于2018年7月7日行68Ga-FAPI-46 PET/CT检查，并于2018年7月25日行6gbq 90Y-FAPI-46治疗。2018年9月19日使用99mTc-FAPI-34进行治疗随访，病情稳定。该胰腺癌患者于2018年6月接受了FAPI治疗。99mTc-FAPI-34荧光显像进行随访。扫描后1天，用6 GBq 90Y-FAPI-46进行另一次治疗。治疗是用90Y进行的，因为当时没有188Re。6周后，随访FAPI-46 PET/CT成像。在这两种情况下，都可以看到肿瘤病变(图5和6；补充图5和图6)。虽然在动物实验中发现了胆道分泌物进入肠道的证据，但在这些患者中没有发现这种情况。

细胞培养[2]
用稳定转染人FAP基因（HT-1080-FAP）的HT-1080细胞，以及转染小鼠FAP基因（HEK-muFAP）和人CD26 （HEKCD26）的人胚胎肾细胞来评价99mtc标记的FAPI衍生物的结合特性。细胞在含有10%胎牛血清的Dulbecco修饰Eagle培养基中培养，温度37℃，二氧化碳5%。[2]
放射性配体结合研究如前所述进行。简而言之，将重组细胞接种于6孔板中，培养48 h，最终汇合率约为80%-90%（每孔1.2-2 × 106个细胞）。用1 mL不含胎牛血清的新鲜培养基代替培养基。将放射性标记的化合物加入细胞培养中，孵育10至240分钟不等。竞争实验通过同时暴露于未标记（10−5-10 M）和放射性标记的化合物60分钟进行。在所有实验中，细胞用1ml pH 7.4的磷酸盐缓冲盐水洗涤两次，随后用1.4 mL裂解缓冲液（0.3 M NaOH， 0.2%十二烷基硫酸钠）裂解。[2]
在内化实验中，细胞与放射性标记的化合物在37℃下孵育60和240 min。从细胞中去除培养基，用1ml磷酸盐缓冲盐水洗涤两次，终止细胞摄取。随后，将细胞与1ml甘氨酸-盐酸（1m, pH 2.2）在室温下孵育10分钟，以收获表面结合的肽（甘氨酸部分）。之后，用2ml的冷冻磷酸盐缓冲盐水洗涤细胞，并按（4,5,11）所述进行裂解，以确定内化（裂解）部分。在Wizard γ-计数器 中测定放射性，归一化为1 × 106个细胞，并以施加剂量的百分比计算。每个实验进行3次，每个独立实验进行3次重复。[2]

Animal Studies[2]
For in vivo experiments, 5 × 106 HT-1080-FAP cells were subcutaneously inoculated into the right trunk of 8-wk-old BALB/c nu/nu mice. When the size of the tumor reached approximately 1 cm3, the radiolabeled compound was injected via the tail vein (2–5 MBq in 100 μL of 0.9% saline for small-animal imaging and 1 MBq in 100 μL of 0.9% saline for organ distribution). For organ distribution, the animals (n = 6 or 3 for each time point) were sacrificed at 1 and 4 h or at different time points (30 min–24 h) after tracer administration. The distributed radioactivity was measured in all dissected organs and in blood using a γ-counter (Cobra Autogamma; Packard). The values are expressed as percentage injected dose per gram of tissue (%ID/g). Scintigraphic images were obtained using a γ-camera (γ-Imager) with a recording time of 10 min per image. For the in vivo blockade experiments, 30 nmol of unlabeled FAPI were added to the radiolabeled compound directly before injection. [2]
All animal experiments were conducted in compliance with the German animal protection laws (permission 35-91185.81/G-158/15).

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Scintigraphy and SPECT/CT Imaging[2]
The patients gave written informed consent to undergo FAPI PET/CT, FAPI therapy, and FAPI scintigraphy following the regulations of the German Pharmaceuticals Act §13(2b). All patients were referred for the experimental diagnostics by their oncologists, who were facing an unmet diagnostic challenge that could not be solved sufficiently with standard diagnostic means. The data were analyzed retrospectively with approval of the local ethics committee (approval S016/2018). [2]
The 99mTc-FAP-34 was applied via intravenous catheter as a bolus injection of 660 MBq via a sterile filter system. Whole-body planar scintigraphy was performed at 10 min, 1 h, 4 h, and 20 h, and 2-bed-position SPECT/CT was performed at 4 h after tracer administration. [2]
Scintigraphic images were obtained using a low-energy high-resolution collimating system with an acquisition time of 1 min/15 cm of body height in a 1,025 × 256 matrix. The SPECT acquisition was performed on an Infinia scanner system using a 128 × 128 matrix, a zoom of 1, step-by-step scanning at 30 s per step, and 120 images with a 3° angle cut in a 128 × 128 matrix. For FAPI-34 imaging, a 4-slice low-dose CT scan (as a part of SPECT/CT) was performed for attenuation correction and general localization of FAPI-positive lesions. [2]
PET/CT imaging was performed on a Biograph mCT Flow scanner. After non–contrast-enhanced low-dose CT (130 keV, 30 mAs, CareDose; reconstructed with a soft-tissue kernel to a slice thickness of 5 mm), PET was acquired in 3-dimensional mode (matrix, 200 × 200) using FlowMotion. The emission data were corrected for randoms, scatter, and decay. Reconstruction was performed with ordered-subset expectation maximization using 2 iterations and 21 subsets, along with Gauss filtering to a transaxial resolution of 5 mm in full width at half maximum. Attenuation correction was performed using the nonenhanced low-dose CT data. The FAPI-46 was synthesized and labeled as described previously. The injected activity for the 68Ga-FAPI-46 (11) examinations was 260 MBq, and the PET scans began 1 h after injection. A 500-mL volume of saline with 20 mg of furosemide was infused from 15 min before to 30 min after tracer application. The patients were asked to self-report any side effects 30 min after finishing the examination.

[1]. Fap inhibitor. WO2019154886A1.

Tumor growth and spread are not only determined by the cancer cells, but also by the non-malignant constituents of the malignant lesion, which are subsumed under the term stroma. The stroma may represent over 90% of the tumor mass in tumors with desmoplastic reaction such as breast, colon and pancreatic carcinoma. Especially a subpopulation of fibroblasts called cancer-associated fibroblasts (CAFs) is known to be involved in tumor growth, migration and progression. Therefore, these cells represent an attractive target for diagnosis and anti-tumor therapy.
A distinguishing feature of CAFs is the expression of seprase or fibroblast activation protein a (FAP-a), a type II membrane bound glycoprotein belonging to the dipeptidyl peptidase 4 (DPP4) family. FAP-a has both dipeptidyl peptidase and endopeptidase activity. The endopeptidase activity distinguishes FAP-a from the other members of the DPP4 family. Identified substrates for the endopeptidase activity so far are denatured Type I collagen, al- antitrypsin and several neuropeptides. FAP-a has a role in normal developmental processes during embryogenesis and in tissue modelling. It is not or only at insignificant levels expressed on adult normal tissues. However, high expression occurs in wound healing, arthritis, artherosclerotic plaques, fibrosis and in more than 90% of epithelial carcinomas.
The appearance of FAP-a in CAFs in many epithelial tumors and the fact that overexpression is associated with a worse prognosis in cancer patients led to the hypothesis that FAP-a activity is involved in cancer development as well as in cancer cell migration and spread. Therefore, the targeting of this enzyme for imaging and endoradiotherapy can be considered as a promising strategy for the detection and treatment of malignant tumors. The present inventors developed a small molecule based on a FAP-a specific inhibitor and were able to show specific uptake, rapid internalization and successful imaging of tumors in animal models as well as in tumor patients. A comparison with the commonly used radiotracer 18F-fluorodeoxyglucose (18F-FDG) revealed a clear superiority of the new FAP-a ligand in patients with locally advanced lung adenocarcinoma. Thus, the present invention provides inter alia: (i) detection of smaller primary tumors and, thus the possibility of earlier diagnosis, (ii) the detection of smaller metastasis and, thus a better assessment of tumor stage, (iii) precise intra-operative guidance facilitating complete surgical removal of tumor tissue, (iv) better differentiation between inflammation and tumor tissue, (v) more precise staging of patients with tumors, (vi) better follow up of tumor lesions after antitumor therapy, (vii) the opportunity to use the molecules as theranostic agents for diagnosis and therapy. Furthermore, the molecules can be used for the diagnosis and treatment of non-malignant diseases such as chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid disorders.[1]

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Most epithelial tumors recruit fibroblasts and other nonmalignant cells and activate them into cancer-associated fibroblasts. This often leads to overexpression of the membrane serine protease fibroblast-activating protein (FAP). It has already been shown that DOTA-bearing FAP inhibitors (FAPIs) generate high-contrast images with PET/CT scans. Since SPECT is a lower-cost and more widely available alternative to PET, 99mTc-labeled FAPIs represent attractive tracers for imaging applications in a larger number of patients. Furthermore, the chemically homologous nuclide 188Re is available from generators, which allows FAP-targeted endoradiotherapy. Methods: For the preparation of 99mTc-tricarbonyl complexes, a chelator was selected whose carboxylic acids can easily be converted into various derivatives in the finished product, enabling a platform strategy based on the original tracer. The obtained 99mTc complexes were investigated in vitro by binding and competition experiments on FAP-transfected HT-1080 (HT-1080-FAP) or on mouse FAP-expressing (HEK-muFAP) and CD26-expressing (HEKCD26) HEK cells and characterized by planar scintigraphy and organ distribution studies in tumor-bearing mice. Furthermore, a first-in-humans application was done on 2 patients with ovarian and pancreatic cancer, respectively. Results: 99mTc-FAPI-19 showed specific binding to recombinant FAP-expressing cells with high affinity. Unfortunately, liver accumulation, biliary excretion, and no tumor uptake were observed on planar scintigraphy for a HT-1080-FAP–xenotransplanted mouse. To improve the pharmacokinetic properties, hydrophilic amino acids were attached to the chelator moiety of the compound. The resulting 99mTc-labeled FAPI tracers revealed excellent binding properties (≤45% binding; >95% internalization), high affinity (half-maximal inhibitory concentration, 6.4–12.7 nM), and significant tumor uptake (≤5.4% injected dose per gram of tissue) in biodistribution studies. The lead candidate 99mTc-FAPI-34 was applied for diagnostic scintigraphy and SPECT of patients with metastasized ovarian and pancreatic cancer for follow-up to therapy with 90Y-FAPI-46. 99mTc-FAPI-34 accumulated in the tumor lesions, as also shown on PET/CT imaging using 68Ga-FAPI-46. Conclusion: 99mTc-FAPI-34 represents a powerful tracer for diagnostic scintigraphy, especially when PET imaging is not available. Additionally, the chelator used in this compound allows labeling with the therapeutic nuclide 188Re, which is planned for the near future.[2]

C50H57F2N13O18

1166.06

1165.391

2374782-07-5

156060696

White to light yellow solid powder

-6.5

440

9

26

30

83

2330

3

N1(CC(=O)N[C@H](C(O)=O)CC(C(=O)O)C(=O)O)C(CN(CC(=O)N2CCN(CCCOC3=CC=C4C(=C3)C(C(=O)NCC(=O)N3CC(F)(C[C@H]3C#N)F)=CC=N4)CC2)CC2N(CC(=O)N[C@H](C(O)=O)CC(C(=O)O)C(=O)O)C=CN=2)=NC=C1

FPOZMWSPTRNDPQ-UVXHQIPUSA-N

InChI=1S/C50H57F2N13O18/c51-50(52)19-28(20-53)65(27-50)41(68)21-57-43(70)30-4-5-54-34-3-2-29(16-31(30)34)83-15-1-8-60-11-13-62(14-12-60)42(69)26-61(22-37-55-6-9-63(37)24-39(66)58-35(48(79)80)17-32(44(71)72)45(73)74)23-38-56-7-10-64(38)25-40(67)59-36(49(81)82)18-33(46(75)76)47(77)78/h2-7,9-10,16,28,32-33,35-36H,1,8,11-15,17-19,21-27H2,(H,57,70)(H,58,66)(H,59,67)(H,71,72)(H,73,74)(H,75,76)(H,77,78)(H,79,80)(H,81,82)/t28-,35-,36-/m0/s1

(3S)-3-[[2-[2-[[[2-[4-[3-[4-[[2-[(2S)-2-cyano-4,4-difluoropyrrolidin-1-yl]-2-oxoethyl]carbamoyl]quinolin-6-yl]oxypropyl]piperazin-1-yl]-2-oxoethyl]-[[1-[2-oxo-2-[[(1S)-1,3,3-tricarboxypropyl]amino]ethyl]imidazol-2-yl]methyl]amino]methyl]imidazol-1-yl]acetyl]amino]propane-1,1,3-tricarboxylic acid

FAPI-34; 2374782-07-5; SCHEMBL22966423;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 50 mg/mL (42.88 mM)

配方 1 中的溶解度: ≥ 5 mg/mL (4.29 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 50.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 5 mg/mL (4.29 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 50.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。