Preclinical study of an 18F-labeled glutamine derivative for cancer imaging
Introduction
The metabolisms of glucose, glutamine, and lipids are heterogeneous in different tumors (Scheme 1) [[1], [2], [3], [4]]. This heterogeneity can origin from the intrinsic difference of genetic backgrounds of cancer cells [5,6]. For the cell lines with the same genetic background but locate in different microenvironment [6], the metabolic heterogeneity derived largely from the metabolic reprogramming as a result of adapting to the surrounding microenvironments, such as limited oxygen supply, insufficient nutrition, immune response, and etc. [7]. Combined with the intrinsic needs for biomaterials and bioenergy required during rapid growth, the distinguishing microenvironments force tumor cells to adapt significantly varying uptake of glucose, glutamine, and lipids. Hence, PET imaging with [18F]FDG often behaves false negativity by missing cancerous lesions that are not glucose dependent.
Although FDG-PET is applied for routine diagnosis of cancer and evaluation of cancer treatment, it is still inherently imperfect and cannot meet all practical needs [8]. In certain cases, FDG-PET failed to pinpoint the location of tumor lesions [9]. The false negativity of FDG-PET could be explained if the tumor cells are in resting state when no excess glucose is needed. In addition, the physiological states of patients also influence the results of FDG-PET and lead to a higher rate of false negativity in practice [10].
Characteristic high uptake of glucose and lipids have been exploited as a fundamental guideline for developing PET tracers and accounts for the broad application of [18F]FDG and [11C]acetic acid in both clinic and pre-clinic research (Scheme 1). Unfortunately, the development of PET tracers based on glutamine has been held back partly due to its inevitable difficulty of radiolabeling when conventional 11C-labeling and 18F-labeling radiochemistry is considered [11,12]. Still, 13N-labeled glutamine and 11C-labeled glutamine were radiosynthesized and evaluated in vivo although their application was limited due to the short half-lives of N-13 (t1/2 = 10 min) and C-11 (t1/2 = 20 min) [13,14]. Hank F. Kung et al. established an elegant and sophisticated strategy of radiolabeling glutamine, and subsequently fully evaluated four stereo-isomeric 18F-labeled glutamine analogs as potential tracers [[15], [16], [17]], leaving substantially little space for further optimization using conventional strategies on this restricted structure of glutamine.
Herein, we demonstrated in this work an alternative strategy to radiolabel glutamine-derived PET tracer [18F]Gln-BF3 for tumor imaging and evaluated it in vivo with PET and by biodistribution. Our strategy benefits from the biological similarity between trifluoroborate group (-BF3−) and carboxylate group (COO−) that was demonstrated in previous work [18]. The biological resemblance stems from the isosteric structures of the two functional groups. Boramino acids are indistinguishably transported across cell membranes mediated by amino acids transporters as natural amino acids. Furthermore, the trifluoroborate group is thermally in equilibrium with fluoride ions, which lays the foundation for the isotope exchange between 19F− and 18F− [[19], [20], [21]]. Consequently, a user-friendly, one-pot, HPLC-free, aqueous isotope exchange reaction afforded the late-stage radiosynthesis of [18F]Gln-BF3 [[22], [23], [24]]. Small-animal PET imaging studies were conducted in healthy mice and tumor-bearing mice inoculated with 4T1 xenografts, demonstrating visible and tumor-specific uptake of [18F]Gln-BF3. The rest of [18F]Gln-BF3 cleared rapidly through renal system, giving promising tumor to non-tumor ratios for most organs.
Section snippets
Materials and methods
All chemical reagents except solvents were purchased from J&K, Energy Chemical and Sinopharm, and used without further purification. Solvents were distilled from Na or CaH2 before use. Deuterated solvents were purchased from Cambridge Isotope Laboratories. Nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance 400 MHz spectrometer. High-resolution mass spectroscopy was performed on Bruker Solarix XR Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. High-performance
Chemistry and radiochemistry
The rationale embedded in the synthesis of precursor Gln-BF3 was as follows (Fig. 1A): 1) the amide group was introduced by condensation and was protected throughout the whole synthetic route by the trityl-protective group until the final cleavage in TFA [25]. 2) The pinacol borate group was installed via an asymmetric copper-catalyzed nucleophilic addition reaction of imine [26,27], and then deprotected and converted in KHF2/HCl to trifluoroborate group. The introduction of trityl-protective
Discussion
The compact structure of glutamine and its poor robustness against acid and base has been of great challenge for F-18 radiolabeling. In principle, there are only two possible sites to install fluorine on the side chain of glutamine. Each of the fluorine substitutions would introduce two enantiomers, which are needed to be isolated prior to PET imaging. Kung et al. chose to radiolabel glutamine at the 4-position and successfully identified [18F](2S,4R)4-fluoroglutamine as the most promising PET
Conclusion
A glutamine analog [18F]Gln-BF3 was synthesized and evaluated in the tumor-bearing animal model, demonstrating notable and tumor-selective uptake in 4T1 xenografts at 45 min post injection. This strategy of developing amino acid PET tracer provided a new solution for the future development of glutamine-derived PET tracers. Further investigation would be focused on tuning the side-chain for minimizing the defluoridation and increasing the tumor accumulation.
Acknowledgment
This work was funded by the National Natural Science Foundation of China (No. NSFC 21778003) and the Ministry of Science and Technology of the People's Republic of China (2017YFA0506300). The authors would like to thank Yonghong Dang and Yu Liu of Peking Union Medical College Hospital for their technical support.
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