Novel 18F-Labeled α-Methyl-Phenylalanine Derivative with High

Exclusive uptake via L-type amino acid transporter 1 (LAT1), a tumor-specific transporter, ... These findings suggest that L-2-18F-FAMP constitutes a ...
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Novel 18F‑Labeled α‑Methyl-Phenylalanine Derivative with High Tumor Accumulation and Ideal Pharmacokinetics for Tumor-Specific Imaging Hirofumi Hanaoka,*,† Yasuhiro Ohshima,‡ Aiko Yamaguchi,† Hiroyuki Suzuki,§ Noriko S. Ishioka,‡ Tetsuya Higuchi,∥ Yasushi Arano,§ and Yoshito Tsushima∥ †

Department of Bioimaging Information Analysis, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan Project “Medical Radioisotope Application”, Department of Radiation-Applied Biology Research, Takasaki Advanced Radiation Research Institute, Quantum Beam Advanced Research Directorate, National Institutes for Quantum and Radiological Science and Technology (QST), Takasaki 370-1292, Japan § Department of Molecular Imaging and Radiotherapy, Graduate School of Pharmaceutical Science, Chiba University, Chiba 260-8675, Japan ∥ Department of Diagnostic Radiology and Nuclear Medicine, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan

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S Supporting Information *

ABSTRACT: Positron emission tomography (PET) imaging with 18F-labeled α-methyl-substituted amino acids exerts significant influence on differential diagnosis of malignant tumors and tumor-like lesions. Exclusive uptake via L-type amino acid transporter 1 (LAT1), a tumor-specific transporter, accounts for their excellent tumor specificity and low background accumulation. However, further refinement and optimization in their tumor accumulation and pharmacokinetics are sorely needed. To address these issues, we newly designed 18F-labeled α-methyl-phenylalanine (18F-FAMP) regioisomers (2-, 3-, or 4-18F-FAMP) and stereoisomers (L- or D-form), and we comprehensively evaluated their potential as tumor-imaging agents. 18 F-FAMPs were prepared from α-methyl phenylalanine by electrophilic radiofluorination and purified by reversed-phase HPLC. In biodistribution studies on normal mice, L-2-18F-FAMP and the three D-18F-FAMPs showed faster blood clearance and lower renal accumulation than L-3-18F-FAMP or L-4-18F-FAMP. In LS180 human colorectal cancer cell line xenograft mice, L-2-18F-FAMP exhibited significantly higher tumor accumulation than the D-18F-FAMPs or a clinically relevant tracer, L-3-18Fα-methyl-tyrosine (18F-FAMT) (p < 0.05). The renal accumulation levels of L-2-18F-FAMP were significantly lower than that of 18 F-FAMT (p < 0.01). LAT-1 specificity of L-2-18F-FAMP was validated in the cellular uptake studies. The PET imaging with L2-18F-FAMP clearly visualized the tumor as early as 1 h after injection, and the high tumor accumulation level was retained for 3 h. These findings suggest that L-2-18F-FAMP constitutes a potential PET tracer for tumor-specific imaging. KEYWORDS: 18F-labeled α-methyl-phenylalanine, tumor-specific imaging, PET, high tumor uptake, low kidney uptake

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INTRODUCTION

Positron emission tomography (PET) with 2-18F-fluoro-2deoxy-glucose (18F-FDG) has had a significant impact on the management of oncology patients by diagnosis, staging, restaging, and monitoring. The high uptake of 18F-FDG in the tumor cells relies on the increased rate of glucose transport © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

April 26, 2019 June 18, 2019 June 26, 2019 June 26, 2019 DOI: 10.1021/acs.molpharmaceut.9b00446 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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Article

MATERIALS AND METHODS General. The L- and D-form of α-methyl-phenylalanine (AMP) and 2-, 3-, or 4-fluorinated L-α-methyl-phenylalanine were purchased from NAGASE & Co (Osaka, Japan, optical purity: ≥ 98%). 18F−F2 was produced on a biomedical cyclotron, CYPRIS HM-18 (Sumitomo Heavy Industries, Tokyo, Japan). 18F-FAMT was synthesized according to the method developed by Tomiyoshi et al.9 All other chemicals used were of the highest purity available. A human colon adenocarcinoma cell line, LS180, a human glioblastoma cell line, U87MG, and a human lung carcinoma cell line, A549, were purchased from the American Type Culture Collection (Manassas, VA) and were used within short-term cultures. Preparation of 18F-FAMPs. 18F-FAMP was synthesized according to the procedure described by Tomiyoshi et al.9 with subtle modifications (Figure 1). 18F−F2 gas (1 GBq) was

and glycolysis, compared with that in normal cells. However, unfavorable accumulations of 18F-FDG in some physiological (e.g., cerebral gray matter and urinary tract) and pathological (e.g., inflammatory site) conditions leads to suboptimal sensitivity or specificity in certain cases. This limitation invokes the need for other PET tracers that could complement or replace 18F-FDG.1,2 Among them, amino acid tracers with high tumor specificity play crucial roles. Clinical and experimental studies demonstrated that amino acid tracers accumulate less in the brain and can differentiate malignant tumors from benign tumors or inflammation.3−5 While 11C-methionine has been used most frequently,4 a major limitation of this tracer is the shorter halflife of 11C compared with 18F (20 and 110 min, respectively); consequently, this tracer cannot be stored for a long time before use. These facts have led to the development of a variety of 18F-labeled amino acid tracers such as O-18F-fluoromethyl-Ltyrosine, O-18F-fluoroethyl-L-tyrosine, and 3-18F-fluoro-αmethyl-L-tyrosine (18F-FAMT).6−8 Even now, the effort to seek amino acid tracers with dominant properties that combine better tumor accumulation and lower nonspecific accumulation continues. At our university hospital, 18F-FAMT has been routinely used for tumor diagnosis since its development in the late 1990s.9,10 The specific accumulation of 18F-FAMT in malignant tumors is exclusively promoted by L-type amino acid transporter 1 (LAT1), the expression of which is highly upregulated in many types of cancer cells. More than 2000 patients with many kinds of cancer such as brain cancer, lung cancer, maxillofacial cancer, and thoracic cancer11−14 have received the benefits of 18F-FAMT PET imaging. The accumulation of clinical evidence exceeds other 18F-labeled amino acid tracers by far. However, a major drawback of 18FFAMT PET is the relatively high frequency of false-negative results because of its low tumor accumulation level.12,13 These clinical findings have prompted the further development of 18 F-FAMT analogues with higher tumor accumulation and improved pharmacokinetics. While LAT1 accepts a wide variety of chemical modifications on the aromatic side chain of natural substrate amino acids,15,16 even a single chemical transformation or chirality may greatly impact the pharmacokinetics of the tracers.17,18 We previously designed and evaluated LAT1-specific 76Brlabeled amino acid derivatives: 2- or 4-76Br-α-methyl-Lphenylalanine (2-76Br-BAMP and 4-76Br-BAMP, respectively).19 In comparison with 18F-FAMT, 2-76Br-BAMP showed comparable tumor accumulation and markedly decreased renal accumulation. In contrast, 4-76Br-BAMP showed higher tumor accumulation and extended blood halflife compared with those of 18F-FAMT. These results suggest the potential of 18F-labeled α-methyl-phenylalanine (18FFAMP), the 18F-labeled derivatives of 76Br-BAMPs, as tumorspecific amino acid tracers. The influence of structural and optical isomerism on tumor accumulation levels and pharmacokinetics awaits comprehensive evaluation. In this study, we prepared 18F-FAMP regioisomers (2-, 3-, or 18 4- F-FAMP) and stereoisomers (L- or D-form), and compared their biodistribution patterns in mice xenograft models. On the basis of the results, the potentials of the 18FFAMPs as tumor-imaging agents were evaluated.

Figure 1. Radiosynthesis of 18F-FAMPs. 18F−F2 gas was converted CH3COO18F by passing the gas through a column of CH3COOK/ CH3COOH. Then, CH3COO18F gas was bubbled into a mixture of acetic acid (AcOH) and trifluoroacetic acid (TFA) (1:1) containing L- or D-form AMP at a flow rate of 250 mL/min for 5 min and incubated for 5 min at 90 °C.

converted to 18F-labeled acetylhypofluorite (CH3COO18F) by passing the gas through a column of CH3COOK/CH3COOH. Then, CH3COO18F gas was bubbled into a mixture of acetic acid and trifluoroacetic acid (TFA) (1:1, 2.5 mL) containing 25 mg of L- or D-form AMP at a flow rate of 250 mL/min for 5 min and then incubated for 5 min at 90 °C. Purification was performed by reversed-phase HPLC (RP-HPLC) with a C-18 column (Capcell Pak C18 AQ, 10 × 250 mm, Shiseido Co., Tokyo, Japan) at a flow rate of 5 mL/min and eluted with a linear gradient of water containing 0.1% TFA and acetonitrile containing 0.1% TFA from 95:5 to 90:10 in 30 min. With these conditions, each FAMP regioisomer and AMP could be isolated (Figure 2A). Retention times of AMP, 2-FAMP, 3FAMP, and 4-FAMP were 20.9, 22.0, 24.9, and 26.2 min, respectively. Each 18F-FAMP regioisomer was collected according to the retention time of nonradioactive FAMP. The solvent was removed using an evaporator (Smart Evaporator C1, BioChromato, Fujisawa, Japan), and then phosphate-buffered saline (PBS) was added to the residue. Finally, each tracer was obtained as a PBS solution (0.5−1 mL, pH 6.5−7.5). The radiochemical purity of 18F-FAMPs was determined by RP-HPLC. Lipophilicity Measurement. The lipophilicity of FAMPs was estimated by measuring the coefficients of partition between 1-octanol and 0.1 M of phosphate buffer (pH 7.4) as follows: a 10-μL aliquot of each 18F-FAMP (100 kBq) was mixed with 3 mL each of 1-octanol and 0.1 M of phosphate buffer in a test tube. The mixture was vortexed (3 × 1 min), and this mixture was then left to stand for 20 min. After the procedure had been repeated three times, the mixture was centrifuged for 5 min. Two 1 mL aliquots of each phase were B

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Figure 2. Analytical RP-HPLC profiles of FAMPs. (A) Typical chromatogram of nonradioactive standard of AMP, 2-FAMP, 3-FAMP, and 4FAMP. (B) UV absorbance (254 nm) and (C) radioactivity profiles of reaction solution after the electrophilic radiofluorination of AMP.

versus time after injection. The formula is as follows: LnCt = LnC0 − kel × t (Ct: concentration after time t, C0: initial concentration). PET Imaging. Tumor-bearing mice were prepared by the implantation of LS180 cells, A549 cells, or U87MG cells (2 × 106 cells/head) into the shoulder of mice. L-2-18F-FAMP (3.5−6.5 MBq, approximately 1 MBq/μmol) was injected intravenously into tumor-bearing mice (n = 3−4 per group, weight: 18−20 g). PET scanning in list mode was performed at 1 and 3 h after injection using an animal PET scanner (Inveon, Siemens, Knoxville, TN) with a 10 min acquisition time under isoflurane anesthesia. The list mode data were reconstructed using an iterative OSEM3D/MAP procedure with the matrix size 128 × 128 × 159, including attenuation correction. MeanSUV was determined by placing the region of interest (ROI) on the whole tumor using an Inveon Research Workplace workstation (Siemens). The maximum intensity projection (MIP) images were displayed using AMIDE 1.0.4 (Stanford University, Stanford, CA). 18F-FAMT-PET (dose of 18FFAMT: 10−12 MBq, approximately 2 MBq/μmol) was also performed in LS180 tumor-bearing mice (n = 4, weight, 18−20 g) using same protocol with L-2-18F-FAMP. Immunohistochemistry. The expression levels of LAT1 in the xenograft tumors were analyzed by immunohistochemical staining. Tumor xenografts excised from nude mice were embedded in O.C.T. compound (Sakura Finetek Japan, Tokyo) and frozen at −80 °C. Cryosections (10 μm) were immunostained with a rabbit anti-LAT1 primary antibody (ab111106, Abcam, Cambridge, U.K.). Immunostaining was detected with a goat antirabbit IgG HRP (ab6721, Abcam). Statistical Analyses. Statistical analyses were performed using SYSTAT 13 software (Systat Software, San Jose, CA). Results are expressed as mean ± standard deviation (SD). The

removed, and their radioactivity was measured with a well-type gamma counter (ARC-7001; Hitachi Aloka Medical, Tokyo, Japan). The partition coefficient at pH 7.4 was determined by calculating the ratio of radioactivity of 1-octanol to that of buffer and then expressed as a common logarithm (logD7.4). Cellular Uptake Studies. LS180 cells were incubated with L-2-18F-FAMP (10 kBq, 10 nmol) at 37 °C for 1 min in Hank’s balanced salt solution (HBSS) or Na+-free HBSS. After the incubation, the cells were lysed, and the radioactivity was measured by a well-type gamma counter. For the inhibition assay, cells were incubated in Na+-free HBSS with various inhibitors (amino acids and their analogues) at 1 mM for 1 min. After the incubation, cells were lysed and the radioactivity in the cell lysate was measured by a well-type gamma counter. Biodistribution Studies. All animal experiments were approved by the animal experiments committee of Gunma University. Biodistribution studies were performed on 6-wkold ddY male mice (weight, 27−30 g, Japan SLC, Shizuoka, Japan) or tumor-bearing mice (weight, 18−22 g). Tumorbearing mice were prepared by the implantation of LS180 cells (2 × 106 cells/head) into the flanks of BALB/c nude mice (5wk-old, Japan SLC). When palpable tumors had developed, the mice were used for biodistribution experiments. For the biodistribution studies, one of the six 18F-FAMPs or 18FFAMT (15 kBq, approximately 15 nmol in 100 μL of saline) was injected into the tail vein of mice (n = 4−5 per group). At selected time points after the injection, mice were sacrificed, and the tissues of interest were dissected out and weighed. The radioactivity was measured by a well-type gamma counter. The uptake of the tracers is expressed as a percentage of the injected dose per gram of organ. The elimination rate constant (kel) of each 18F-FAMP was calculated as the slope of the least-squares linear regression line of log blood radioactivity C

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Figure 3. Radioactivity profiles in the blood, kidney, and pancreas after injection of L-18F-FAMP or D-18F-FAMP into normal mice. L-2, L-2-18FFAMP; L-3, L-3-18F-FAMP; L-4, L-4-18F-FAMP; D-2, D-2-18F-FAMP; D-3, D-3-18F-FAMP; D-4, D-4-18F-FAMP.

Figure 4. Biodistribution studies of L-2-18F-FAMP, D-18F-FAMPs, and 18F-FAMT in LS180-bearing mice. (A) Accumulation levels in the organs and (B) tumor-to-organ ratio in LS180-bearing mice at 1 h after injection (mean ± SD, 4−5 per group). T/B, tumor-to-blood ratio; T/M, tumorto-muscle ratio; T/K, tumor-to-kidney ratio. #p < 0.05 and *p < 0.01 compared with 18F-FAMT.

2-18F-FAMP: 0.63 h−1, L-3-18F-FAMP: 0.56 h−1, L-4-18FFAMP: 0.26 h−1, D-2-18F-FAMP: 1.39 h−1, D-3-18F-FAMP: 1.19 h−1, and D-4-18F-FAMP: 1.11 h−1. All 18F-FAMPs accumulated in the pancreas, which expresses LAT1.20 Accumulation levels in the pancreas of each L-form 18FFAMP were more than 2 times higher than those of the respective D-form 18F-FAMPs at 30 min and 1 h. In contrast, all 18F-FAMPs showed similarly low renal accumulation levels compared with that of 18F-FAMT.21 Among L-form FAMPs, L-4-18F-FAMP showed the slowest blood clearance and longest pancreas retention, whereas L-2-18F-FAMP showed the fastest blood clearance. In contrast, all D-form 18F-FAMPs showed similar biodistribution profiles. Because rapid blood clearance is a favorable factor for an amino acid tracer, we did a subsequent biodistribution study in LS180 tumor-bearing mice with L-2-18F-FAMP and all three D-18F-FAMPs. The immunohistochemical analysis revealed the high expression level of LAT1 in the LS180 tumors (Figure S2). L-2-18F-FAMP showed tumor accumulation significantly higher than that of 18 F-FAMT or the D-18F-FAMPs (Figure 4A). However, L2-18F-FAMP showed a tumor-to-blood ratio comparable to those of 18F-FAMT or the D-18F-FAMPs because of its relatively high radioactivity levels in blood (Figure 4B). The renal accumulation levels of L-2-18F-FAMP and the three D-18F-FAMPs were significantly lower than that of 18F-FAMT (p < 0.01). Of note, the tumor-to-kidney ratio of L-2-18FFAMP, D-2-18F-FAMP, and D-4-18F-FAMP exceeded 1.0 at 1 h after injection. Cellular Uptake Studies. On the basis of the high tumor uptake level and tumor-to-organ ratios, we did further experiments with L-2-18F-FAMP. L-2-18F-FAMP was taken up by LS180 cells in a Na+-independent manner (Figure 5A).

results were analyzed using the unpaired t-test for comparing differences between two groups, or one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) test for comparing differences among multiple groups. Differences were considered significant when the p-value was less than 0.05.

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RESULTS Radiolabeling. The electrophilic radiofluorination reaction of AMP preferentially produced ortho-substituted 18F-FAMP (2-18F-FAMP) in decay-corrected radiochemical yield of 20− 30% (calculated from the obtained radioactivity of CH3COO18F). The isomeric ratio of 2-18F-FAMP: 3-18FFAMP: 4-18F-FAMP was 20:3.5:1 (Figure 2). More than 50% of AMP remained in an unreacted form. The RP-HPLC purification yielded each 18F-FAMP with radiochemical purity >95% (Figure S1, Supporting Information). Total synthesis time including HPLC purification was 1 h from 18F−F2 gas. The specific activity of each18F-FAMP was 2−3 MBq/μmol. Lipophilicity Measurement. From the octanol/water partition coefficient measurement, the logD7.4 values of L2-18F-FAMP, L-3-18F-FAMP and L-4-18F-FAMP were found to be −1.32 ± 0.01, −1.12 ± 0.01, and −1.08 ± 0.01, respectively (p < 0.05), indicating that the lipophilicity of 2-FAMP was lower than those of 3-FAMP and 4-FAMP. This result was consistent with that of the RP-HPLC analysis of 18F-FAMPs (Figure 2). Biodistribution Studies. In biodistribution studies in normal mice, D-form 18F-FAMPs showed rapid blood clearance compared with the corresponding L-form analogues (Figure 3 and Table S1, Supporting Information). The elimination rate constant (kel) of each 18F-FAMP was LD

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Figure 5. Cellular uptake studies. (A) Cellular uptake of L-2-18F-FAMP into LS180 cells in HBSS or Na+-free HBSS. (B) Inhibition of the cellular uptake of L-2-18F-FAMP into LS180 cells by L-amino acids or these analogues. Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartic acid; BCH, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid; Cys, cysteine; Gln, glutamine; Glu, glutamic acid; Gly, glycine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; MeAIB, α-methylaminoisobutyric acid; Met, methionine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Trp, tryptophan; Tyr, tyrosine; Val, valine.

Consistent with the result of 18F-FAMT,19 coincubation with LAT1 substrate natural amino acids and the LAT1-specific inhibitors α-methyl-tyrosine (AMT) and FAMT markedly reduced the uptake level of L-2-18F-FAMP (Figure 5B). PET Imaging. As shown in Figure 6, L-2-18F-FAMP clearly visualized the LS180-tumor at 1 h after injection, and the high

isomer simultaneously, we used an electrophilic radiofluorination method for the production of the 18F-FAMPs. Because of the addition of carrier nonradioactive fluoride, the obtained 18 F-FAMPs contain a certain degree of nonradioactive FAMPs. However, the low specific activity would not have affected the comparative evaluation of 18F-FAMP derivatives, because (1) LAT1 has high tracer uptake capacity, (2) LAT1 uptakes amino acid tracers in obligately exchange mechanism, and (3) all derivatives have comparable specific activities. Each 18FFAMP isomer was readily isolated by using RP-HPLC with high radiochemical purity. Considering patient scale synthesis, the current production method of the most potent analogue, L2-18F-FAMP, will fulfill the radioactivity requirement for imaging a few patients. To improve the availability of L2-18F-FAMP, the development of a production method via nucleophilic radiofluorination is underway in our laboratory. The use of this method will likely allow the production of L2-18F-FAMP in a scale sufficient to image more than 20 patients. The six 18F-FAMP isomers showed different biodistribution profiles. Faster blood clearance of the D-form compared to the L-form was also observed with 18F-FAMT,18 while such enantiomeric differences were not observed with 11Cmethionine, O-18F-fluoromethyl-tyrosine, or 2-123I-iodo-phenylalanine.22,23 These results indicate that the conformational rigidity and/or lipophilicity provided by the α-methyl group may influence the differences in blood clearance of the enantiomers. Among the L-form 18F-FAMPs, L-2- and L-4-18FFAMP showed the fastest and slowest blood clearance, respectively, which is consistent with the results for bromine77 (77Br) and iodine-125 (125I)-labeled phenylalanine derivatives.17,19 This difference would be partially attributable to the hydrophilicity of tracers. Further studies are necessary to elucidate the role of the halogenation position on the blood clearance of phenylalanine derivatives. The results from the biodistribution study in tumor-bearing mice indicated the surpassing potential of L-2-18F-FAMP to overcome a major limitation (i.e., low tumor accumulation) of 18 F-FAMT. Although L-2-18F-FAMP, 18F-FAMT, and the three D-18F-FAMPs showed comparable tumor-to-blood ratios, L2-18F-FAMP showed a significantly higher tumor accumulation level than those of 18F-FAMT and D-18F-FAMPs. High tumor accumulation level would improve diagnostic accuracy by providing clearer tumor delineation and hence eliminate false negatives. Alternatively, L-2-18F-FAMP can achieve image

Figure 6. A typical PET image of the tumor in LS180 tumor-bearing mice injected with L-2-18F-FAMP or 18F-FAMT. Orange circles indicate the implanted tumors. B, bladder; K, kidney; P, pancreas. Mean-SUV represents means ± SD of 4 mice. #p < 0.05 and *p < 0.01 compared with 18F-FAMT.

tumor accumulation level was retained for 3 h. The meanSUVs of L-2-18F-FAMP were significantly higher than those of 18 F-FAMT. Reflecting the high LAT1 specificity, L-2-18FFAMP also clearly visualized the pancreas. Although high uptake in the kidney was apparent at 1 h after injection, the levels rapidly decreased with time. Consistent with the biodistribution studies, 18F-FAMT showed mean-SUV values in the kidney significantly higher than that of L-2-18F-FAMP. L-2-18F-FAMP also clearly visualized the other types of tumor with high LAT-1 expression levels (U87MG and A549, Figure S2).

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DISCUSSION Aiming for the development of a tumor-specific amino acid tracer, we synthesized and evaluated six 18F-FAMP isomers. To comprehensively compare the properties of each 18F-FAMP E

DOI: 10.1021/acs.molpharmaceut.9b00446 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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quality similar to that of 18F-FAMT in smaller activity dose administration, which would increase the number of patients per day. In addition, L-2-18F-FAMP would substantially decrease radiation exposure to the kidney because of its significantly higher tumor-to-kidney ratio than that with 18FFAMT (1.60 ± 0.27 vs 0.19 ± 0.05 at 1 h postinjection, p < 0.001). High pancreatic accumulation of 18F-FAMPs as observed in mice would not be expected in patients since the human pancreas lacks LAT1 expression.24 Indeed, in clinical studies, 18F-FAMT accumulated less in pancreas,11 despite the high accumulation in the murine pancreas.18 The high specificity toward LAT1 is essential for the development of tumor-specific amino acid tracers.24−27 Uptake inhibition patterns of L-2-18F-FAMP by LAT1 substrates are comparable to that of 18F-FAMT;16,19 thus, L-2-18F-FAMP would be taken up by tumor cells via LAT1 transporters. Taken together, the results of cellular uptake and biodistribution studies suggest that L-2-18F-FAMP would be an alternative for 18F-FAMT in clinical practice. There has accumulated evidence regarding the relationship between 18 F-FAMT accumulation levels and the LAT1 expression levels in various types of tumor from the clinical studies so far performed in our university hospital.28−30 These data will facilitate clinical translation of L-2-18F-FAMP by providing information about the suitable clinical indications, which would constitute a significant advantage of L-2-18F-FAMP over the other new amino acid tracers in the development phase. The PET images of L-2-18F-FAMP in LS180-tumor bearing mice consolidated the result of the biodistribution study. The low renal accumulation level of L-2-18F-FAMP enabled more precise visualization of the tumor than that with 18F-FAMT.19 L-2-18F-FAMP also clearly depicted LAT1-positive brain cancer (U87MG31) and lung cancer (A54932), the tumors of which clinical application of highly tumor-specific amino acid tracers is sorely needed.3,33 Although the lack of side-by-side comparisons of 18F-FAMPs with other clinically used amino acid tracers is a limitation of this study, an advantage of 18FFAMPs is their tumor specificity, which is similar to that of 18 F-FAMT. Further comparative clinical studies are required to establish the advantages of using L-2-18F-FAMP instead of 18FFAMT. An emerging role that LAT1 specific tracers are anticipated to play is companion diagnostics. Considering tumor-specific expression of LAT1, an α-emitter-labeled LAT1 substrate will be a good candidate as an agent for targeted-alpha therapy, a promising anticancer therapy that exploits high-energy transfer of α-particle. Phenylalanine derivatives labeled with astatine211 indeed demonstrated significant therapeutic effect in a rat model of intracranial glioma.34,35 In addition, since a number of studies suggested potential involvement of LAT1 in metastasis of cancer, LAT1 inhibition constitutes an attractive therapeutic strategy to prevent metastasis.36 Thus, as a companion diagnostics, L-2-18F-FAMP will likely benefit in the realization of personalized medicine in the near future. In conclusion, among the six 18F-FAMP isomers, L-2-18FFAMP showed the most suitable pharmacokinetics and especially high tumor uptake via LAT1. L-2-18F-FAMP clearly visualized the tumor as early as 1 h after injection by PET imaging. These findings suggest translational potential of L2-18F-FAMP as a new amino acid tracer. This promising PET tracer will eventually contribute to improvement in diagnostic accuracy for tumor.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.9b00446.

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Analytical RP-HPLC profiles of FAMPs (Figure S1), biodistribution of L-18F-FAMPs and D-18F-FAMPs in normal mice (Table S1), and PET image, mean-SUV of the tumor injected with L-2-18F-FAMP and immunohisitochemical staining of LAT1 in LS180, U87MG and A549 tumor-bearing mice (Figure S2) (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-27-220-8403; Fax: +81-27-220-8409. E-mail:hanao [email protected]. ORCID

Hirofumi Hanaoka: 0000-0003-2421-7397 Yasushi Arano: 0000-0001-6091-5382 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mr. Takashi Ogasawara (Cyclotron Facility, Gunma University Hospital) for producing 18F-FAMT and 18FFAMPs.

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ABBREVIATIONS PET, positron emission tomography; 18F, fluorine-18; 18FFAMP, 18F-labeled α-methyl-phenylalanine; 18F-FAMT, L3-18F-α-methyl-tyrosine; LAT1, L-type amino acid transporter 1; 18F-FDG, 2-18F-fluoro-2-deoxy-glucose; 76Br-BAMP, 76Br-αmethyl-L-phenylalanine; AMP, α-methyl-phenylalanine; TFA, trifluoroacetic acid; RP-HPLC, reversed-phase HPLC; HBSS, Hank’s balanced salt solution; SD, standard deviation; ANOVA, analysis of variance; HSD, honestly significant difference; AMT, α-methyl-tyrosine; Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartic acid; BCH, 2aminobicyclo-(2,2,1)-heptane-2-carboxylic acid; Cys, cysteine; Gln, glutamine; Glu, glutamic acid; Gly, glycine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; MeAIB, α-methylaminoisobutyric acid; Met, methionine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Trp, tryptophan; Tyr, tyrosine; Val, valine.

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REFERENCES

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Molecular Pharmaceutics

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DOI: 10.1021/acs.molpharmaceut.9b00446 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.molpharmaceut.9b00446 Mol. Pharmaceutics XXXX, XXX, XXX−XXX