Standard “cold” compounds L-FMA and D-FMA were synthesized from commercially available Boc-Asp(OBzl)-OH 1 (as shown in ). After esterification of carboxylic group and removal of benzyl group via hydrogenolysis, partially protected aspartic acid derivative Boc-Asp-OtBu 3 was reduced to desired alcohol 5 following a previously reported mixed anhydride reduction method. Tosylation of alcohol 5 provided the labeling precursors in 83 to 90% yield. The fluoride intermediate 10 was prepared by following our recently developed fluorination method using a “neutralized” tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF) (mixture of TASF and Et3N(HF)3). After fluorination and then deprotection with TFA, final compounds L-FMA and D-FMA could be obtained with an overall yield of 37 to 39%. The synthesis of L-FEA started from protected partially protected glutamic acid derivative, Boc-Glu-OtBu 4 (). Tosylate intermediate 8 was prepared through reduction of 4 and then tosylation of alcohol 6. The labeling precursor iodide 9 was then produced in 77% yield through the exchange of tosyl group to iodo group via Finkelstein type reaction. It is noteworthy to point out that the desirable labeled product could not be prepared from tosylate 8, probably due to its fast rate of cyclization in labeling process. The standard compound for radiosynthesis L-FEA was prepared from tosylate 8 after fluorination and deprotection, similar to FMA.
Scheme 1 Synthesis of labeling precursors and standards for FMA and FEA. Reagents and conditions: (a) tert-butyl 2,2,2-trichloroacetamidate, BF3.Et2O, rt; (b) 10% Pd/C, H2, rt; (c) i. ethyl chloroformate, Et3N, −5 ~ −10 °C; ii. NaBH4, H (more ...)
[18F]FMA and [18F]FEA were conveniently prepared via a two-step reaction — fluorination using 18-Crown-6/KHCO3/K[18F]F followed by deprotection in TFA. summarizes the labeling results of the new alanine derivatives under the condition described in , including synthesis time, non-decay corrected radiochemical yield (RCY), radiochemical purity (RCP) and enantiomeric purity.
Summary of radiolabeling of [18F]FMA and [18F]FEA*
Radiolabeling of FMA and FEA. Reagents and conditions: (a) 18-Crown-6/KHCO3/K[18F]F, for L- and D-[18F]FMA: DMSO, 80, 15 min; for L-[18F]FEA: CH3CN, 60, 10 min; (b) TFA, 60, 10 min.
One potential problem in labeling amino acid derivatives is the racemization occurring during radiofluorination. The L- and the D-isomer of amino acids usually display a very different biological activity. There is a great disparity in tumor cell uptake between L- and D-isomers: the naturally occurring L-isomers are more readily transported than their corresponding D-isomers. Thus, obtaining the enantiomerically pure 18F labeled amino acids is important for accurate biological evaluation. To establish the optimal condition for preparing enantiomerically pure [18F]FMA, we have carefully studied the conditions of fluorination for radiosynthesis of L-[18F]FMA. The results demonstrated that in CH3CN at 80 °C using 18-Crown-6 as the phase transfer catalyst and KHCO3 as the base led to enantiomerically pure intermediate (S)-[18F]10 (ee >99%). In contrast, using reagents K222/K2CO3 (ee 93–95%) and TBAHCO3 (ee 79–84%) led to a lower enantiomeric purity (). Effects of solvent and temperature on enantiomeric purity and labeling yield were also evaluated (see ). (S)-[18F]10 fluorinated with 18-Crown-6/KHCO3/K[18F]F was prepared with the highest yield and optical purity in DMSO at 80 °C. Synthesis of (S)-[18F]11, the intermediate of L-[18F]FEA, required lower temperature (60 °C) to reduce cyclization, a side reaction.
Fig. 2 Effects of fluorination reagents and catalysts on the purity and yield of radiosynthesis of L-[18F]FMA were evaluated using HPLC. Top three HPLC profile is the radiotrace of intermediate [18F]10. Bottom HPLC profile (in Black) is the UV tracer of cold (more ...)
Effect of solvent and temperature on radiofluorination of L-[18F]FMA
3.3 Cell uptake studies
To evaluate these new 18F-labeled alanine derivatives, we first conducted the cell uptake studies in 9L gliosarcoma and PC-3 prostate cancer cells. The in vitro cell uptake in phosphate buffered saline (PBS) was measured at selected time points up to 2 h (). FDG was used as the reference since it is the most commonly used PET tracer. The results demonstrated that L[18F]FMA showed the highest uptake among the three new alanine derivatives. Its uptake at 1 h is 2.4 and 1.2 fold higher than those of FDG in 9L and PC-3 cells, respectively. This observation is highly important because it suggests that amino acid transport system(s) (potentially system ASC as indicated by transport characterization studies) is capable of moving the alanine analog, L[18F]FMA, across tumor membrane in a higher speed than that observed for FDG via the glucose transporter. This fast tumor cell uptake fulfills the first requirement for developing biomarker for detecting tumor proliferation.
Fig. 3 Time vs uptake of 18F alanine derivatives into 9L (A) and PC-3 (B) are presented. A standard agent, FDG, was also measured in the same uptake conditions. It is important to note that the uptake of L-[18F]FMA is higher than that of FDG at 60 min. Uptake (more ...)
Uptake of the D-isomer (D-[18F]FMA) was very low (< 1% ID/100 g protein). Maximum uptake of D-[18F]FMA was less than 5% of that of its L- isomer in both cell lines. The data suggest that the L configuration is essential for the tumor cell uptake for this series of alanine derivative. Derivative with a longer alkyl chain, L-[18F]FEA, showed a faster kinetics, reaching the peak within 30 min and then washes out from the tumor cells thereafter. Maximum uptake of L-[18F]FEA could only reach 26 and 63% of that of L-[18F]FMA in 9L and PC-3 cells, respectively. These data suggested that among the new alanine analogs, L-[18F]FMA was the most promising candidate. Its biological properties were further investigated.
In addition to 9L and PC-3 cells, uptake of L-[18F]FMA in PANC-1 human pancreatic epithelial carcinoma and U-87 MG human glioma was measured, as well (). High uptake, 25 to 60 % ID/100 μg protein at 60 min, was observed in these cancer cell lines. Although uptake value varies in different cell lines, similar kinetics was observed – uptake reached a maximum around 60 min. It is reasonable to conclude that L-[18F]FMA is taken up efficiently by both fast and slow growing tumor cells.
Time course of L-[18F]FMA in various cancer cells was evaluated in four different tumor cells. Uptake value are represented as % uptake/100 μg protein. Each point represents mean ± SD (n = 3).
3.4 Transport characterization studies
To examine the underlying mechanism(s) of uptake of L-[18F]FMA, we have conducted a series of competitive uptake inhibition studies in 9L glioma cells. A number of inhibitors specific for system A, ASC and L were used. The uptake of L-[18F]FMA was measured in absence of inhibitors as well as in the presence of concentration from ranging 0.5 to 5 mM of system A inhibitor MeAIB, system L inhibitor BCH, and system ASC inhibitors L-Ala and L-Ser. Sodium and pH dependence studies were carried out as well for better characterization of the transport system(s) responsible for uptake of L-[18F]FMA. These studies were performed with a 30 min incubation time and the results were normalized to uptake of L-[18F]FMA in PBS (pH 7.4) in absence of inhibitors as summarized in . The results showed that MeAIB had no inhibitory effect on the uptake of L-[18F]FMA (); This indicates system A does not contribute to its transportation across the membrane. System L inhibitor BCH could reduce L-[18F]FMA’s uptake up to 20%. In contrast, system ASC inhibitor L-Ala and L-Ser could reduce >95% of the uptake at 5 mM concentration (). Furthermore, in Na+ free media, uptake of L-[18F]FMA was reduced by 85%. Changes in pH did not have significant effect on the uptake of L-[18F]FMA (110 ± 13% and 86 ± 5% of control at pH 6 and 8, respectively), similar to L-[3H]Ala (93 ± 4% and 99 ± 3% of control at pH 6 and 8, respectively). These data indicate that uptake of L-[18F]FMA is predominately through a sodium dependent and pH insensitive system ASC. Sodium independent system L plays a minor role and it may be responsible for the observed sodium independent cell uptake.
Fig. 5 Transportation of L-[18F]FMA in 9L glioma cells across tumor cells were evaluated in the presence of various amino acid transporter inhibitors. Uptake of L[18F]FMA in presence of inhibitors for system ASC (A), A and L (B), and media free of Na+ and at (more ...)
We also examined the selectivity of L-[18F]FMA towards a system ASC subtype - ASCT2. Between two subtypes, ASCT1 and ASCT2, the latter demonstrated overexpression in a variety of cancer cells and plays essential role for tumor growth and survival. Inhibitory effect of GPNA, a potent ASCT2 inhibitor, was compared to its close analog GA that is inactive towards ASCT2 (). There was no apparent difference between inhibition result of GPNA and GA and they could only reduce uptake of L-[18F]FMA up to 28% at 1 mM concentration. This result indicate that L-[18F]FMA is not selective for ASCT2 and ASCT1.
Furthermore, since L-[18F]FMA is a close analog of L-Ala, we compared the transport of L[18F]FMA and L-[3H]Ala to examine the effect of fluroalkyl chain on the transport mechanism. The results () demonstrated that L-[18F]FMA and L-[3H]Ala had similar transport characteristics. While L-[3H]Ala is more sensitive towards inhibitors of system A and system L, both tracers appear mainly transported via system ASC and they are not selective towards ASCT2. To quantitatively evaluate the involvement of ASCT1 and ASCT2 in L-[18F]FMA uptake, more extensive transport assays are required such as transporter mRNA expression profiling for 9L glioma cells and studies in ASCT1/2 knock-down cell lines.
Comparison of tumor cell uptake of L-[18F]FMA and [3H]Ala in the presence of various inhibitors of amino acid transporter as well as in Na+ free and different pH buffers. Data are shown as mean ± SD (n = 3).
3.5. In vitro metabolism studies
We tested the stability of L-[18F]FMA in 9L cells by measuring the metabolites after 0, 30, 60, 120 min (). The HPLC result of L-[18F]FMA in PBS for 2 h without incubating with 9L cells was used as the control. No metabolite other than L-[18F]FMA itself was found in 2 h. This suggests that L-[18F]FMA is metabolically stable within 2 h after being transported into 9L cells. Additionally, protein incorporation of L-[18F]FMA was examined in 9L cells. No significant incorporation of L-[18F]FMA into protein was observed. The protein bound fraction of L-[18F]FMA was < 0.5%, in contrast to L-[3H]Ala, which had significant incorporation (78.9 ± 2.9 %) into protein after 2 h. These result indicate that L-[18F]FMA remained in its native form and was not further metabolized after transported into tumor cells.
Metabolic stability of L-[18F]FMA in 9L glioma cells was measured by HPLC.
3.6. In vivo biodistribution studies
The in vivo biodistribution studies of L-[18F]FMA was carried out in Fisher rats bearing 9L tumors. The distribution of radioactivity in tissues after intravenous injection of L-[18F]FMA at selected time points is summarized in . Uptake of L-[18F]FMA in tumor was very fast and reached maximum uptake within 30 min. Rapid clearance of radioactivity in blood, heart, lung, pancreas, spleen and liver was observed within the first 30 min. Apart from bone, other examined organs had continuously decreasing radioactivity after 30 min. Bone uptake increased as a function of time from 0.48 %ID/g at 2 min to 1.91 %ID/g at 2 h, indicating substantial defluorination occurred. Although tumor tissue uptake was not very high, an adequate tumor-to-background ratio was achieved after 30 min. At 1 h, tumor-to-blood and tumor-to-muscle, and tumor-to-brain ratios reached 1.9, 2.2 and 3.0 respectively. The discrepancy between in vitro and in vivo tumor uptake is possibly the result of competition from natural amino acids present in the blood circulation of animals. Total concentration of system ASC substrates, including alanine, serine and threonine, is over 1 mM in rat plasma. In conjunction with in vitro inhibition data, this suggests it is possible to reduce the uptake of L-[18F]FMA by >80%. In comparison, the tumor uptake of [18F]FACBC appears to be less susceptible to the presence of natural amino acids in the blood circulation. The reason might be that FACBC is transported via multiple amino acid transport systems; besides system ASC, system L also plays an important role in its transport, especially in acidic environment that tumor cells live in. The significance of this observation will require additional studies to fully clarify the mechanisms responsible for the tumor cell uptake of these amino acid based imaging agents.
Biodistribution result of L-[18F]FMA in Fisher rats bearing 9L tumors after intravenous injectiona
Highest uptake of L-[18F]FMA in normal tissues was in liver followed by kidney, suggesting excretion through both biliary and renal routes. L-[18F]FMA had very low pancreatic uptake after 30 min (< 0.3% ID/g). This pattern of biodistribution is quite different from those of many other amino acid tracers, which are excreted mainly via renal route and have high pancreatic uptake. This may relate to distribution of system ASC in tissues of rats. Further studies will be required to find out the underlying mechanism.
3.7. Small animal PET imaging studies
Small animal PET imaging studies of L-[18F]FMA were carried out in two rodent tumor models: rats bearing 9L tumors and transgenic mice bearing spontaneous M/tomND tumors. The latter is a transgenic mouse model with spontaneously generated mammary gland adenocarcinoma. The 2 h summed images from coronal sections ( and ) were selected for visualization. Time-activity curves generated by drawing region of interest for assessing kinetics were shown in and . Tumors were visualized with L-[18F]FMA in both animal models. Uptake in tumors in both animal models quickly reached the peak with 10 to 20 min. High activity in the skeleton (sternum, ribs et al) and gut regions was observed. Bone uptake suggests that there is apparent defluorination, consistent to that observed in biodistribution studies by dissection method (). Imaging analysis showed that tumor-to-muscle ratio in rat bearing 9L tumor increased from 2.6 at 30 min to 3.8 at 120 min, in agreement with the results from biodistribution studies. During the same time period, this ratio in transgenic mouse had small decrease from 2.3 to 2.0.
Small animal PET imaging of L-[18F]FMA in Fisher rat bearing 9L tumor model was performed: (A) Color-coded PET image from summed 2 h data in coronal view. The arrowhead points to the 9L tumor. (B) Time activity curves in 9L tumors and muscle.
Fig. 9 Small animal PET imaging of L-[18F]FMA in transgenic mice bearing spontaneously generated mammary gland tumor (or m/tomND tumor) was performed: (A) Color-coded PET image from summed 2 h data in coronal view. The arrowhead points to the m/tomND tumor. (more ...)