A 18F-labeled Alanine Derivative Serve as An ASCT2 Marker for

Subscriber access provided by READING UNIV

Article

A 18F-labeled Alanine Derivative Serve as An ASCT2 Marker for Cancer Imaging Hui Liu, Yuxiang Han, Jiyuan Li, Ming Qin, Qunfeng Fu, Chunhong Wang, and Zhibo Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00884 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

An 18F-Alanine Derivative Serves as An ASCT2 Marker for Cancer Imaging Hui Liu,† Yuxiang Han,† Jiyuan Li,† Ming Qin,† Qunfeng Fu,† Chunhong Wang,† Zhibo Liu†‡* † Radiochemistry and Radiation Chemistry Key Laboratory of Fundamental Science, Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China ‡Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China

ABSTRACT Amino acids derivative are well established molecular probes for diagnosis of a variety of cancer using positron emission tomography (PET). Recently, boramino acid (BAAs) was found as a prospective molecular platform for developing PET tracer. The objective of this study was to develop a

18

F-labeled alanine derivative through

displacing its carboxylate by trifluoroborate as a selective ASCT2 marker for cancer imaging. 18F-Ala-BF3 was firstly evaluated in healthy FVB/N mice in vivo, exhibiting rapid renal clearance with almost negligible uptake in stomach (1.53 ± 0.31 %ID/g). Notable uptake was observed in thyroid (3.71 ± 0.49 %ID/g, 40 min post injection), of which the uptake was significantly inhibited by co-injecting with natural L-alanine. In addition, we further established 18F-Ala-BF3 on a human gastric cancer cell (BGC-823) xenograft bearing mouse model. Dynamic PET-CT scan revealed the optimal time window for tumor imaging, it was between 40 min and 60 min post injection, when the BGC-823 xenograft uptake was 5.49 ±1.47 %ID/g (n=4), and the tumor-to-stomach, tumor-to-blood, tumor-to-muscle, and tumor-to-brain ratios were 3.27 ± 1.53, 3.80 ± 1.48, 3.47 ± 1.48, and 6.20 ± 1.47, respectively.

KEY WORDS: Boramino acid, ASCT2, positron emission tomography, cancer imaging

INTRODUCTION Gastric cancer is a great challenge for cancer therapy because of its high lethality and morbidity.1-4 Though incidence has declined worldwide, gastric cancer remains

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 23

epidemic in East Asia, and its corresponding incidence is twice than the world average level in China.3 In most cases, gastric cancer is diagnosed in advanced stages, due to the lack of an effective technique for early cancer diagnosis. Gastroscopy is most widely used for gastric cancer diagnosis yet is not sufficiently effective in many cases.5-7 Many patients cannot endure the discomfort caused by gastroscopy, and malignant lesions on hypogastric and iliac regions could be missed owing to lack of experiences.8 In last two decades,

18

F-FDG PET scan has been successfully used in

the diagnosis, staging, and monitoring the treatment of many types of cancers.9-12 However, gastric cancer is usually accompanied by chronic gastritis,13 and inflammation commonly resolved by FDG results in false positive cancer diagnosis. Therefore, to improve clinical diagnostic and prognostic approaches, novel biomarkers with better specificity are in urgent need for gastric cancer imaging. Alanine-Serine-Cysteine-Threonine transporter type 2 (ASCT2), also known as SLC1A5, is a neutral amino acids transporter, which uphill transports neutral amino acids into cells to provide nutrients for metabolism and also maintains the concentration of neutral amino acid in cell plasma.14, 15 The substrates of ASCT2 are various, among which L-alanine is one of dominants.16 Interestingly, tumor cell is well known of overexpressing ASCT2 for uptaking amino acids to fulfill their exceptional demanding of nutrients (Fig. 1). Herein, ASCT2 is regarded as a promising biomarker for cancer, with potentially great pharmacological importance for cancer treatment.17 Aberrant amino acids uptake is exhibited in many kinds of cancer and represent a promising source of potential biomarkers.18-22 A variety of amino acid analogues have been synthesized for PET imaging.23-27 Recently, boramino acids (BAAs) have been discovered as promising candidates for imaging transporters or receptors. Substitution of the carboxylate group (-COO-) of the natural amino acids with its isosteric trifluroborate (-BF3-) results in highly resemble amino acids derivative, exhibiting well stability in animals.28 BAAs share many similar properties with natural amino acids, and its uphill transportation into cancer cells is also amino acid transporter dependent.29 Meanwhile, trifluoroborate could be readily labeled with ACS Paragon Plus Environment

18

F-fluoride

Page 3 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


according to the one-step

18

F-19F exchange approach,30,

31

resulting a valuable

advantage for the future potential clinical application comparing with other radioactive PET tracers. In this study, we synthesized Ala-BF3 as an alanine derivative for cancer imaging. The electron distributions of Ala-BF3 and Alanine highly resemble to each other. Both subcutaneous BGC-823 human stomach cancer model in mice and in healthy mice were employed for preclinical assessment of 18

F-Ala-BF3 in gastric cancer diagnosis.

EXPERIMENTAL SECTION Reagents and solvents were purchased from Aladdin, Energy Chemical, Sinopharm, Xilong Scientific. High-resolution mass spectroscopy was performed on Waters e2695. Nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance Spectrometers: 400 MHz and 500 MHz. Signals are showed in parts per million (ppm), and multiplicity is presented as single (s), doublet (d), quartet (q) and multiplet (m); coupling constants are showed in hertz. Ala-BF3 was lyophilized under high vacuum (0.01 to 0.05 torr) after purification. Chemistry and radiochemical yields refer to isolated pure chemicals and non-decay-corrected radio-chemically pure products respectively.

Synthesis of Ala-BF3 To a solution of (S)-2-methylpropane-2-sulfinamide (245 mg, 2 mmol) in THF (0.6mL), Ti(OPri)4 (1.2 mL, 4 mmol) and 6 mmol acetaldehyde were added, the reaction was performed at room temperature. The reaction was monitored by TLC, then quenched with 1 mL of water using stirring. 10 mL of dichloromethane was added to the solution followed by sodium sulphate filtration. The solid phase was washed by 5 mL of petroleum ether. The mixed phases were purified through silica gel column chromatography with petroleum ether/ethyl acetate (1:5 to 1:10) following desiccation by anhydrous Mg2SO4 to give compound 1.14 Data for the compound 1 was consist with published data (Fig. S2). NMR spectroscopy: 1H NMR (400 MHz, CDCl3, 20℃, δ, ppm): δ 8.07 (q, J = 5.1 Hz, 1H), 2.22 (d, J = 5.1 Hz, 3H), 1.18 (s, 9H). To a solution of PCy3·HBF4 (1.1 mg, 3.0 µmol, 1.2 mol %) were added toluene



(0.05 mL), aqueous CuSO4 (0.10 mL, 30 mM, 3.0 µmol, 1.2 mol %), and benzylamine (1.4 µL, 12.5 µmol, 5.0 mol %), sequentially, in a dram vial. The mixture was stirred for 10 min with cap. Then uncapped the vial, and toluene (0.45 mL), the appropriate acetaldimine (0.25 mmol, 1.0 equiv), and bis(pinacolato)diboron (127 mg, 0.5 mmol, 2.0 equiv) were added. Recapped the vial, and the reaction mixture was stirred rapidly for 24 h.32 The resulting solution was concentrated under vacuum. To alanine boronic ester (20.3 mg, 0.08 mmol), potassium fluoride (3 M; water solution, 0.05 mL), hydrogen chloride (4M; water solution, 0.01 mL), and acetonitrile (MeCN, 0.04 mL) were added. The reaction was performed at 45 °C for 1 h in a 1.5 ml Eppendorf tube. Then purified through silica gel column chromatography with dichloromethane/ acetonitrile (1:1) to give compound 2.29 Data for the compound 2 was consist with the published data (Figs. S3 to S4). NMR spectroscopy: 19F NMR (500 MHz, CD3CN, 20 °C, δ, ppm): −153.58. LC-MS (m/z): calculated for C2H6BF3N, [M-H]−112.07. Found: 112.

Radiochemistry Ala-BF3 (25 nmol) was resuspended in 5 µL of pyridazine-HCl buffer (pH 2.0 to 2.5, pyridazine 1 M, N, N′-dimethylformamide 6 M). The radiolabeling reaction was started by adding 30 µL of

18

F-fluoride water solution (~20 mCi). The reaction was

incubated at 85 °C for 15 min, then quenched by 0.5 ml of Saline. The remaining free 18 -

F was removed by neutral alumina column.30 And the preparing Ala-BF3 was

confirmed by radioactive HPLC chromatography.

Animal study Small-animal model of BGC-823 xenografts was established. All animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals, after approval from the Peking University animal ethics committee. The BGC-823 human gastric carcinoma cell line grow in RPMI 1640, which was supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 mg/mL) (Gibco), 5% CO2, 37°C. The tumor model was established in 6-8 weeks female athymic nude mice (Charles River) by injection of 5 × 106 cells


Page 4 of 23

Page 5 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


into their right shoulder subcutaneously. The micro-PET study was conducted when the tumor volume reached 100 to 300 mm3 (10 to 15 days after inoculation). Small-animal model of inflammation. The inflammation model was established by intramuscular injection of turpentine into the mice implanted with subcutaneous BGC-823 xenografts. Up to 30 µL of turpentine was injected in the caudal thigh muscles of the left hindlimb. PET imaging was implemented 48 hours (18F-FDG) and 72 hours (18F-Ala-BF3) following turpentine injection.

PET study PET scans were performed on Nanoscan PET-CT 122s (Mediso Medical Imaging Systems). About 7.4 MBq of

18

F-Ala-BF3 was injected via intravenous route under

isoflurane anesthesia. For acquisition, 15-min PET static acquisition was performed at 45 min after veil injection for static reconstruction. 120-min PET acquisition was acquired for dynamic reconstruction. The images were reconstructed using Tera-Tomo 3D method, Variance Reduced D.W. random correction and scatter were used for reconstruction. For each acquisition, ROIs were acquired by Nucline NanoScan software (InterViewTM FUSION, Mediso Medical Imaging Systems) on decay-corrected recontructed images. The concentrations of radioactivity in relevant tissues were obtained from mean and maximum SUV values of the ROIs and then converted to percentage injected dose per gram (% ID/g, assuming a tissue density of 1 g/mL).

Statistic analysis All data were expressed as mean ± SD. Statistical significance was determined by one-way analysis of variance (ANOVA) and Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).

RESULTS Synthesis of Alanine derivative 18F-Ala-BF3 18

F-Ala-BF3 was prepared by incubating it with 18F- water solution under pH = 2.5 in

a pyridazine buffer, resulting the product with the radiochemical yield of 45 ± 5.0%,



Page 6 of 23

radiochemical purity of >98%, and a specific activity of 7.0 ± 2.6 GBq/µmol (n=12). The validation of the radiochemistry precursor and cold standard was confirmed through mass spectroscopy and nuclear magnetic resonance (NMR) spectroscopy.

Pharmacokinetics of 18F-Ala-BF3 in healthy mice 18

F-Ala-BF3 was firstly evaluated by PET-CT scan in healthy FVB/N mice (Fig.

2A). The ex vivo bio-distribution data of 18F-Ala-BF3 at 40 min (Fig. 2B) was found to show good correlation to the scanning data. Uptake of thyroid in this animal model was 3.71 ± 0.49 %ID/g, while the uptake of stomach was 1.53 ± 0.31 %ID/g. Besides, the uptake in the blood, brain, muscle was 2.44 ± 0.73 %ID/g, 0.29 ± 0.04 %ID/g, 1.19 ± 0.21 %ID/g，respectively (Table 1). Moreover, the excretion of

18

F-Ala-BF3

radiotracer was mainly through renal route, accompanied obvious clearance to kidney and bladder. 18

F-Ala-BF3 demonstrated specific uptake in thyroid which can be

effectively blocked by natural Ala Co-injection of L-Alanine (5 mg) together with 18F-Ala-BF3, resulted in obviously blocking thyroid uptake in healthy mice (Fig. 3A). The uptake of thyroid of 18

F-Ala-BF3 tracer and co-injection natural L-alanine in healthy mice at 20 min post

injection shows visible contrast, which was consistent in the bio-distribution data of thyroid. Uptake of thyroid in the healthy unblocked mice using 18F-Ala-BF3 was 3.71 ± 0.49 %ID/g, as expected, co-injection excess natural L-alanine caused an appropriate reduction in thyroid uptake: 2.35±0.16 %ID/g (Fig. 3B), resulting 37% blocking because ASCT2 transporter on thyroid was inhibited by the high blood concentration of natural Ala. In addition, the uptake values in brain and muscle of 18

F-Ala-BF3 were insignificant, exhibiting the potential of imaging brain metastasis

with 18F-Ala-BF3. 18

F-Ala-BF3 is in favor of tumor imaging in vivo Given the favorable tumor cell retention and specificity of 18F-Boron amino acid in

cell culture study, we established small-animal

18

F-Ala-BF3 PET in a Nu/Nu mouse


Page 7 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


model implanted with subcutaneous BGC-823 human stomach xenograft. PET-CT dynamic scan was carried out in this model to study its pharmacokinetics in vivo (Fig. 4). During the first 30 min, 18F-Ala-BF3 was circulating in blood, with notable uptake in heart, liver and kidney, followed by clearance through the bladder. Note worthily, 18

F-Ala-BF3 radiotracer cleared out from most organs at 40 min post injection.

Moreover, mean and maximum accumulation of liver, heart, muscle and tumor determined by dynamic PET scan further validated the optimal time of imaging in mice (Fig. 5). Tumor accumulation was clearly visible at 40 min post injection in Nu/Nu mice bearing BGC-823 xenografts. Ex vivo bio-distribution study corroborated the PET imaging data. At 40 min post injection, BGC-823 tumor uptake was found to be

5.49±1.47

%ID/g

(n=

4),

and

the tumor-to-stomach,

tumor-to-blood,

tumor-to-muscle, and tumor-to-brain ratios were 3.27 ± 1.53, 3.80 ± 1.48, 3.47 ± 1.48, and 6.20 ± 1.47, respectively (Fig. 6B). 18

F-Ala-BF3 distinguish tumor from inflammation in vivo Though in vitro assays validated ASCT2 for Ala-BF3 specific transportation, it is

necessary to illustrate its pathway in vivo as well. Therefore, 18

18

F-FDG and

F-Ala-BF3 PET-CT was tested in a Nu/Nu mouse model implanted with

subcutaneous BGC-823 human stomach xenografts and inflammation model. As shown in Figure 7,

18

F-Ala-BF3 accumulated specifically in BGC-823 xenografts to

give high tumor-to-background contrast at 45 min post injection. The average uptake of tumor and inflammation tissue was 5.23 ± 0.93 %ID/g and 2.70 ± 1.03 %ID/g (n=4), while

18

F-FDG exhibited high uptake both in tumor and inflammation tissue,

and the corresponding uptake was 4.93 ± 0.85 %ID/g and 5.83 ± 1.94 %ID/g (n=4). Compared to 18F-FDG, the gold standard PET tracer in cancer diagnosis, 18F-Ala-BF3 exhibited high tumor uptake but obviously lower uptake in inflammation region (Fig. 8). Herein,

18

F-Ala-BF3 is a useful tracer as an ASCT2 marker for gastric cancer

imaging, making it possible to diagnose early gastric cancer in clinical.

DISCUSSION The increasing incidence of gastric cancer has become a major health concern in



Page 8 of 23

east Asia,3 and the lack of an effective treatment for late-stage cancer has promoted a great deal of research to develop new methods for early cancer diagnosis.1 Metabolism in tumor cells is significantly different from normal cells. In tumor tissues, both glucose and amino acids uptake increase, resulting from additional need for proliferation.33,

34

Until now,

18

F-labeling of amino acids with aromatic ring

became achievable through many approaches.29, 35 However, radiolabeling of amino acids without aromatic ring, specially alanine and serine, is still of great difficulty. 11

C-labeled alanine has been synthesized previously, but the relative short half-life of

11

C (t1/2 = 20.4 min) limited its clinical application.36 Due to the similarity in chemical

and biological properties between fluorine (-F) and hydroxyl group (-OH), 18F-labeled Alanine behaved more similar to serine rather than alanine.37 Recently, substitution of a carboxylate group (-COO-) with its isosteric trifluroborate (-BF3-) is used for 18

F-labeling amino acids. Therefore, we synthesized the tracer Ala-BF3, which mimics

the natural L-alanine by substituting of carboxylate group (-COO-) with its isosteric trifluoroborate (-BF3-).29 Due to the interaction between trifluoroborate and its adjacent ammonium group, Ala-BF3 exhibited high stability.28 Moreover, we got relatively high radiochemical yield of

18

F-Ala-BF3 through one-step

18

F-19F isotope

exchange approach.30 The in vivo experiments did show defluorination of the tracer, for there was bone signal in PET imaging and bio-distribution data. Nevertheless, the stability of tracer in vitro (Fig. S5 and Fig. S6) and cold standard was relatively high, arguing the self-decomposing of the tracer. Since

18

F-Ala-BF3 defluorinated faster in vivo,

metabolic effects may take part in this process. Blood alanine aminotransferase is broadly spread in hematological system, catalyzing deamination of L-alanine.38 Substitution of carboxylate group (-COO-) didn’t affect the complex formation between 18F-Ala-BF3 and pyridoxal 5'-phosphate (PLP), which served as substrate of blood alanine aminotransferase.39, 40 Deamination of 18F-Ala-BF3 resulted in unstable trifluroborate product, which may account for bone signal.28 Alanine involves in many metabolic pathways, such as transamination in liver and muscle,41-43 which was also consisted with our experimental results.

18

F-Ala-BF3 showed uptake on many


Page 9 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


different tissues, such as muscle, heart, spleen and liver, at 1.57 ± 0.33 %ID/g higher than background (0.05 %ID/g). Besides, it also exhibited specific accumulation, such as tumor and thyroid, whose uptake were significantly higher than other organs. In addition, the relatively lower uptake in whole body was reminiscent of the broad metabolic participation of alanine. Meanwhile,

18

F-Ala-BF3 exhibited significant

higher uptake in tumor than inflammation region, which makes it possible to be a potential tumor- specific tracer. ASCT2 has been developed as a tracer target for 18F-FACBC (fluciclovine), which was approved by FDA for prostate cancer PET imaging.44 More importantly, Okudaira et al. reported that

14

C-FACBC can be transported by ASCT2 ,with high

Michaelis–Menten kinetics Km value of 92.0 ± 32.3 µM.45 In addition, Ono et al. reported that in NHA and low-grade glioma cell lines, uptake than

14

14

C-FACBC showed higher

C-Met, showing the transporter of ASCT2 was a potential cancer

marker.46 According to previous study, amino acid transporter cannot distinguish natural amino acid from BAAs, in which carboxylate group is substituted by trifluoroborate. Thus, ASCT2 positive tumors can uptake since

18

18

F-Ala-BF3 specifically,

F-Ala-BF3 can be transported by ASCT2 along with natural L-alanine. To

attest this hypothesis, both subcutaneous BGC-823 human stomach cancer model mice and healthy mice were applied in evaluating the behavior of 18F-Ala-BF3 in vivo, which were favor in high tumor accumulation of

18

F-Ala-BF3. Nowadays, PET

imaging has been widely used in cancer diagnosis in clinical currently, but biomarkers for cancer other than glycolysis is urgently needed. Our study suggests that 18

F-Ala-BF3 is a useful tracer as an ASCT2 marker for gastric cancer imaging, with

negligible uptake in inflammation regions, paving the way to diagnose early gastric cancer in clinical.

CONCLUSION The

18

F-Ala-BF3 was successfully synthesized through one-step

18

F-19F isotope

exchange with fine specific activity and good radiochemical yield. Following in vitro bio-distribution and in vivo PET imaging in gastric tumor xenograft bearing mice and



healthy mice revealed high tumor to normal tissue rate consist with fine tumor imaging of

18

F-Ala-BF3. This study shed light on ASCT2 as a cancer biomarker for

clinical diagnosis, and emphasized the usefulness of BAAs in tumor PET imaging. 18

F-Ala-BF3 represents a useful molecular platform for tumor diagnosis.

ASSOCIATED CONTENT Supporting Information Fig. S1. Synthetic route of Ala-BF3 and radiolabeling reaction. Fig. S2. 1H NMR spectrum of Ala-BF3 precursor (compound 1). Fig. S3. 19F NMR spectrum of HPLC-purified Ala-BF3 (δ = −153.58 ppm). Fig. S4. The LC-MS spectrum of HPLC-purified Ala-BF3. Fig. S5. In vitro stability assay of 18F-Ala-BF3 in PBS. Fig. S6. In vitro stability assay of 18F-Ala-BF3 in FBS. Fig. S7. Time−activity curves of bone and joint from female Nu/Nu mice bearing BGC-823 xenografts. Fig. S8. High-resolution mass spectrum (HRMS) of Ala-BF3. Fig. S9. HPLC analysis of enantiomeric purity of L-isomer of Ala-BF3. Fig. S10. Competitive inhibition of BGC-823 cell uptake of 18F- Ala-BF3.

AUTHOR INFORMATION Corresponding Author *Radiochemistry and Radiation Chemistry Key Laboratory of Fundamental Science, Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China. E-mail: [email protected]

Notes The authors declare no competing financial interest.

ABBREVIATIONS PET, positron emission tomography; LC-MS, liquid chromatography-Mass Spectrum; p.i., post injection


Page 10 of 23

Page 11 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES 1.

J. Ferlay, I. S., M. Ervik, D. Forman, F. Bray, R. Dikshit, S. Elser, C. Mathers, M. Rebelo, DM. Parkin.

International Agency for Research on Cancer. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11. globocan.iarc.fr (Accessed July 14 2016) 2013. 2.

Petersen, P. E.; Ogawa, H.

The global burden of periodontal disease: towards integration with

chronic disease prevention and control. Periodontology 2000 2012, 60, (1), 15-39. 3.

Balakrishnan, M.; George, R.; Sharma, A.; Graham, D. Y.

Changing Trends in Stomach Cancer

Throughout the World. Current gastroenterology reports 2017, 19, (8), 36. 4.

Torre, L. A.; Bray, F.; Siegel, R. L.; Ferlay, J.; Lortet-Tieulent, J.; Jemal, A.

Global cancer statistics,

2012. CA: a cancer journal for clinicians 2015, 65, (2), 87-108. 5.

Yao, K.

The endoscopic diagnosis of early gastric cancer. Annals of gastroenterology 2013, 26,

(1), 11-22. 6.

Liedlgruber, M.; Uhl, A.

Computer-aided decision support systems for endoscopy in the

gastrointestinal tract: a review. IEEE reviews in biomedical engineering 2011, 4, 73-88. 7.

Liu, D. Y.; Gan, T.; Rao, N. N.; Xing, Y. W.; Zheng, J.; Li, S.; Luo, C. S.; Zhou, Z. J.; Wan, Y. L.

Identification of lesion images from gastrointestinal endoscope based on feature extraction of combinational methods with and without learning process. Medical image analysis 2016, 32, 281-94. 8.

Allen, P.; Shaw, E.; Jong, A.; Behrens, H.; Skinner, I.

Severity and duration of pain after

colonoscopy and gastroscopy: a cohort study. Journal of clinical nursing 2015, 24, (13-14), 1895-903. 9.

Som, P.; Atkins, H. L.; Bandoypadhyay, D.; Fowler, J. S.; MacGregor, R. R.; Matsui, K.; Oster, Z. H.;

Sacker, D. F.; Shiue, C. Y.; Turner, H.; Wan, C. N.; Wolf, A. P.; Zabinski, S. V.

A fluorinated glucose

analog, 2-fluoro-2-deoxy-D-glucose (F-18): nontoxic tracer for rapid tumor detection. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 1980, 21, (7), 670-5. 10. de Geus-Oei, L. F.; Ruers, T. J.; Punt, C. J.; Leer, J. W.; Corstens, F. H.; Oyen, W. J.

FDG-PET in

colorectal cancer. Cancer imaging : the official publication of the International Cancer Imaging Society 2006, 6, S71-81. 11. Jadvar, H.

FDG PET in Prostate Cancer. PET clinics 2009, 4, (2), 155-61.

12. Rosen, E. L.; Eubank, W. B.; Mankoff, D. A.

FDG PET, PET/CT, and breast cancer imaging.

Radiographics : a review publication of the Radiological Society of North America, Inc 2007, 27 Suppl 1, S215-29. 13. Kong, D.; Piao, Y. S.; Yamashita, S.; Oshima, H.; Oguma, K.; Fushida, S.; Fujimura, T.; Minamoto, T.; Seno, H.; Yamada, Y.; Satou, K.; Ushijima, T.; Ishikawa, T. O.; Oshima, M.

Inflammation-induced

repression of tumor suppressor miR-7 in gastric tumor cells. Oncogene 2012, 31, (35), 3949-60. 14. Yao, Q.; Yuan, C.

Enantioselective synthesis of H-phosphinic acids bearing natural amino acid

residues. The Journal of organic chemistry 2013, 78, (14), 6962-74. 15. Utsunomiya-Tate, N.; Endou, H.; Kanai, Y.

Cloning and functional characterization of a system

ASC-like Na+-dependent neutral amino acid transporter. The Journal of biological chemistry 1996, 271, (25), 14883-90. 16. Kekuda, R.; Prasad, P. D.; Fei, Y. J.; Torres-Zamorano, V.; Sinha, S.; Yang-Feng, T. L.; Leibach, F. H.; Ganapathy, V.

Cloning of the sodium-dependent, broad-scope, neutral amino acid transporter Bo

from a human placental choriocarcinoma cell line. The Journal of biological chemistry 1996, 271, (31), 18657-61. 17. Colas, C.; Grewer, C.; Otte, N. J.; Gameiro, A.; Albers, T.; Singh, K.; Shere, H.; Bonomi, M.; Holst, J.;



Schlessinger, A.

Page 12 of 23

Ligand Discovery for the Alanine-Serine-Cysteine Transporter (ASCT2, SLC1A5) from

Homology Modeling and Virtual Screening. PLoS computational biology 2015, 11, (10), e1004477. 18. Laverman, P.; Boerman, O. C.; Corstens, F. H.; Oyen, W. J.

Fluorinated amino acids for tumour

imaging with positron emission tomography. European journal of nuclear medicine and molecular imaging 2002, 29, (5), 681-90. 19. Kirwan, A.; Utratna, M.; O'Dwyer, M. E.; Joshi, L.; Kilcoyne, M.

Glycosylation-Based Serum

Biomarkers for Cancer Diagnostics and Prognostics. BioMed research international 2015, 2015, 490531. 20. Tai, W.; Chen, Z.; Cheng, K.

Expression profile and functional activity of peptide transporters in

prostate cancer cells. Molecular pharmaceutics 2013, 10, (2), 477-87. 21. Thakkar, N.; Kim, K.; Jang, E. R.; Han, S.; Kim, K.; Kim, D.; Merchant, N.; Lockhart, A. C.; Lee, W. A cancer-specific variant of the SLCO1B3 gene encodes a novel human organic anion transporting polypeptide 1B3 (OATP1B3) localized mainly in the cytoplasm of colon and pancreatic cancer cells. Molecular pharmaceutics 2013, 10, (1), 406-16. 22. Fung, K. L.; Tepede, A. K.; Pluchino, K. M.; Pouliot, L. M.; Pixley, J. N.; Hall, M. D.; Gottesman, M. M.

Uptake of compounds that selectively kill multidrug-resistant cells: the copper transporter

SLC31A1 (CTR1) increases cellular accumulation of the thiosemicarbazone NSC73306. Molecular pharmaceutics 2014, 11, (8), 2692-702. 23. Wang, L.; Zha, Z.; Qu, W.; Qiao, H.; Lieberman, B. P.; Plossl, K.; Kung, H. F.

Synthesis and

evaluation of 18F labeled alanine derivatives as potential tumor imaging agents. Nuclear medicine and biology 2012, 39, (7), 933-43. 24. Wang, L.; Lieberman, B. P.; Ploessl, K.; Kung, H. F.

Synthesis and evaluation of (1)(8)F labeled

FET prodrugs for tumor imaging. Nuclear medicine and biology 2014, 41, (1), 58-67. 25. Wu, Z.; Zha, Z.; Li, G.; Lieberman, B. P.; Choi, S. R.; Ploessl, K.; Kung, H. F. [(18)F](2S,4S)-4-(3-Fluoropropyl)glutamine as a tumor imaging agent. Molecular pharmaceutics 2014, 11, (11), 3852-66. 26. Bhutia, Y. D.; Babu, E.; Ramachandran, S.; Ganapathy, V.

Amino Acid transporters in cancer and

their relevance to "glutamine addiction": novel targets for the design of a new class of anticancer drugs. Cancer research 2015, 75, (9), 1782-8. 27. Nodwell, M. B.; Yang, H.; Colovic, M.; Yuan, Z.; Merkens, H.; Martin, R. E.; Benard, F.; Schaffer, P.; Britton, R.

18F-Fluorination of Unactivated C-H Bonds in Branched Aliphatic Amino Acids: Direct

Synthesis of Oncological Positron Emission Tomography Imaging Agents. Journal of the American Chemical Society 2017, 139, (10), 3595-3598. 28. Liu, Z.; Chao, D.; Li, Y.; Ting, R.; Oh, J.; Perrin, D. M.

From minutes to years: predicting

organotrifluoroborate solvolysis rates. Chemistry 2015, 21, (10), 3924-8. 29. Liu, Z.; Chen, H.; Chen, K.; Shao, Y.; Kiesewetter, D. O.; Niu, G.; Chen, X.

Boramino acid as a

marker for amino acid transporters. Science advances 2015, 1, (8), e1500694. 30. Liu, Z.; Pourghiasian, M.; Radtke, M. A.; Lau, J.; Pan, J.; Dias, G. M.; Yapp, D.; Lin, K. S.; Benard, F.; Perrin, D. M.

An organotrifluoroborate for broadly applicable one-step 18F-labeling. Angewandte

Chemie 2014, 53, (44), 11876-80. 31. Liu, Z.; Lin, K. S.; Benard, F.; Pourghiasian, M.; Kiesewetter, D. O.; Perrin, D. M.; Chen, X. One-step (18)F labeling of biomolecules using organotrifluoroborates. Nature protocols 2015, 10, (9), 1423-32. 32. Buesking, A. W.; Bacauanu, V.; Cai, I.; Ellman, J. A.

Asymmetric synthesis of protected


Page 13 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


alpha-amino boronic acid derivatives with an air- and moisture-stable Cu(II) catalyst. The Journal of organic chemistry 2014, 79, (8), 3671-7. 33. Hanahan, D.; Weinberg, R. A.

Hallmarks of cancer: the next generation. Cell 2011, 144, (5),

646-74. 34. Koppenol, W. H.; Bounds, P. L.; Dang, C. V.

Otto Warburg's contributions to current concepts of

cancer metabolism. Nature reviews. Cancer 2011, 11, (5), 325-37. 35. Preshlock, S.; Tredwell, M.; Gouverneur, V.

(18)F-Labeling of Arenes and Heteroarenes for

Applications in Positron Emission Tomography. Chemical reviews 2016, 116, (2), 719-66. 36. Ropchan, J. R.; Barrio, J. R.

Enzymatic synthesis of [1-11C]pyruvic acid, L-[1-11C]lactic acid and

L-[1-11C]alanine via DL-[1-11C]alanine. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 1984, 25, (8), 887-92. 37. Yang, D.; Kuang, L. R.; Cherif, A.; Tansey, W.; Li, C.; Lin, W. J.; Liu, C. W.; Kim, E. E.; Wallace, S. Synthesis of [18F]fluoroalanine and [18F]fluorotamoxifen for imaging breast tumors. Journal of drug targeting 1993, 1, (3), 259-67. 38. Galvin, Z.; McDonough, A.; Ryan, J.; Stewart, S.

Blood alanine aminotransferase levels >1,000

IU/l - causes and outcomes. Clinical medicine 2015, 15, (3), 244-7. 39. Cooper, A. J.

Proton magnetic resonance studies of glutamate-alanine transaminase-catalyzed

deuterium exchange. Evidence for proton conservation during prototropic transfer from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate. The Journal of biological chemistry 1976, 251, (4), 1088-96. 40. Duff, S. M.; Rydel, T. J.; McClerren, A. L.; Zhang, W.; Li, J. Y.; Sturman, E. J.; Halls, C.; Chen, S.; Zeng, J.; Peng, J.; Kretzler, C. N.; Evdokimov, A.

The enzymology of alanine aminotransferase (AlaAT)

isoforms from Hordeum vulgare and other organisms, and the HvAlaAT crystal structure. Archives of biochemistry and biophysics 2012, 528, (1), 90-101. 41. Cornelius, C. E.

Relation of Body-Weight to Hepatic Glutamic Pyruvic Transaminase Activity.

Nature 1963, 200, 580-1. 42. Sousa, C. M.; Biancur, D. E.; Wang, X.; Halbrook, C. J.; Sherman, M. H.; Zhang, L.; Kremer, D.; Hwang, R. F.; Witkiewicz, A. K.; Ying, H.; Asara, J. M.; Evans, R. M.; Cantley, L. C.; Lyssiotis, C. A.; Kimmelman, A. C.

Pancreatic stellate cells support tumour metabolism through autophagic alanine

secretion. Nature 2016, 536, (7617), 479-83. 43. Bornstein, J.

Inhibition of alanine transaminase by the hypoglycaemic sulphonylurea

derivatives. Nature 1957, 179, (4558), 534-5. 44. Okudaira, H.; Shikano, N.; Nishii, R.; Miyagi, T.; Yoshimoto, M.; Kobayashi, M.; Ohe, K.; Nakanishi, T.; Tamai, I.; Namiki, M.; Kawai, K.

Putative transport mechanism and intracellular fate of

trans-1-amino-3-18F-fluorocyclobutanecarboxylic acid in human prostate cancer. Journal of nuclear medicine : official publication, Society of Nuclear Medicine 2011, 52, (5), 822-9. 45. Okudaira, H.; Nakanishi, T.; Oka, S.; Kobayashi, M.; Tamagami, H.; Schuster, D. M.; Goodman, M. M.;

Shirakami,

Y.;

Tamai,

I.;

Kawai,

K.

Kinetic

analyses

of

trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid transport in Xenopus laevis oocytes expressing human ASCT2 and SNAT2. Nuclear medicine and biology 2013, 40, (5), 670-5. 46. Ono, M.; Oka, S.; Okudaira, H.; Schuster, D. M.; Goodman, M. M.; Kawai, K.; Shirakami, Y. Comparative

evaluation

of

transport

mechanisms

of

trans-1-amino-3-[(1)(8)F]fluorocyclobutanecarboxylic acid and L-[methyl-(1)(1)C]methionine in human glioma cell lines. Brain research 2013, 1535, 24-37.



Figure 1. (A) Schematic depiction of biological mechanism of Ala-BF3 tumor specificity. ASCT2 is overexpressed in many kinds of tumors. Cancer cells (red) can uptake much more Alanine than normal cells (orange) around. A new tracer was labelled the tracer with radioactive nuclide, which can mimic Alanine activity and make a new PET tracer for cancer in vivo. (B) Structure of L-Alanine and Ala-BF3.


Page 14 of 23

Page 15 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. (A) Representative PET/CT of 18F-Ala-BF3 in healthy FVB mice at 40 min post injection. FVB/N mice received 200 µCi of

18

F-Ala-BF3 via tail vein injection,

with PET/CT images acquired 40 min post injection. Color bar is calibrated in % ID/g, with no background subtracted. (B) Corresponding bio-distribution of 18F-Ala-BF3 in healthy FVB mice at 40 min post injection. Data are means ± SD (n=3 animals).



Figure 3. (A) Representative PET imaging of

18

F-Ala-BF3 tracer and co-injection

natural L-alanine in healthy mice at 40 min post injection. Color bar is calibrated in %ID/g, with no background subtracted. (B) The uptake of thyroid of 18F-Ala-BF3 tracer and co-injection natural L-alanine in healthy mice at 40 min post injection shows obvious contrast. Data are means ± SD (n=3 animals). *p ＜ 0.05.


Page 16 of 23

Page 17 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Representative dynamic scan PET-CT imaging of

18

F-Ala-BF3 in Nu/Nu

mice bearing BGC-823 xenograft. Nude mice received 300 µCi of

18

F-Ala-BF3 via

tail vein injection, with dynamic PET/CT images acquired after injection. t,tumor. Color bar is calibrated in % ID/g, with no background subtracted.



Figure 5. Time−activity curves of tumors and major organs from female Nu/Nu mice bearing BGC-823 xenografts. The data are from 10 min dynamic scans following intravenous injection of 18F-Ala-BF3 (200 µCi/mouse).


Page 18 of 23

Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. (A) Representative PET imaging of

18

F-Ala-BF3 in Nu/Nu mice bearing

BGC-823 tumor at 45 min post injection. Nude mice received 200 µCi of 18F-Ala-BF3 via tail vein injection, with PET/CT images acquired 45 min post injection. Color bar is calibrated in %ID/g, with no background subtracted. (B) Corresponding bio-distribution of

18

F-Ala-BF3 in Nu/Nu mice bearing BGC-823 tumor at 45 min

post injection. Data are means ± SD (n=4 animals). t, tumor; k, kidney; b, bladder.



Figure 7. Representative PET imaging of

18

F-FDG and

Page 20 of 23

18

F-Ala-BF3 in Nu/Nu mice

bearing BGC-823 xenografts（right shoulder）and inflammation model (left hindlimb) at 45 min post injection. Nude mice received 200 µCi of

18

F-FDG and

18

F-Ala-BF3

via tail vein injection, with PET/CT images acquired 45 min post injection. Color bar is calibrated in %ID/g, with no background subtracted. t, tumor; i, inflammation.


Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. The uptake of tumors and inflammatory tissues from female Nu/Nu mice bearing BGC-823 xenografts and inflammation model. The data are from 15-min static scans following intravenous injection of

18

F-Ala-BF3 (200 µCi/mouse). Data

are means ± SD (n=4 animals). *p ＜ 0.05.



Page 22 of 23

Table 1

healthy mice (n=3) organ unblocked blocked Blood 2.44 ± 0.73 1.17 ± 0.33 Adrenal 5.07 ± 0.25 3.82 ± 0.22 Pancreas 2.84 ± 0.34 1.77 ± 0.16 Spleen 2.74 ± 0.41 1.73 ± 0.15 Intestine/s 2.54 ± 0.43 1.56 ± 0.09 Intestine/l 2.74 ± 0.32 1.74 ± 0.10 Fat 0.76 ± 0.16 0.38 ± 0.05 Liver 2.02 ± 0.57 0.90 ± 0.09 Kidney 23.22 ± 0.58 22.47 ± 0.76 Stomach 1.53 ± 0.31 0.93 ± 0.33 Heart 1.49 ± 0.22 1.05 ± 0.10 Lung 3.58 ± 0.57 3.23 ± 1.70 Muscle 1.19 ± 0.21 0.75 ± 0.02 Bone 2.88 ± 0.55 1.96 ± 0.25 Brain 0.29 ± 0.04 0.30 ± 0.02 Thyroid 3.71 ± 0.49 2.35 ± 0.16 Tumor The data are represented as %ID/g ± SD.

BGC-823 tumor-bearing mice (n=4)


1.45 ± 0.16 7.22 ± 1.76 2.59 ± 0.30 2.05 ± 0.27 1.77 ± 0.30 7.78 ± 0.81 1.61 ± 0.64 1.39 ± 0.30 16.52 ± 0.88 1.68 ± 0.42 1.51 ± 0.08 2.69 ± 0.20 1.58 ± 0.17 1.87 ± 0.36 0.45 ± 0.03 5.49 ± 1.47

Page 23 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For table of contents use only:

An 18F-Alanine Derivative Serves as An ASCT2 Marker for Cancer Imaging Hui Liu, Yuxiang Han, Jiyuan Li, Ming Qin, Qunfeng Fu, Chunhong Wang, Zhibo Liu *To whom correspondence should be addressed. E-mail: Z.Liu ([email protected]).

The PDF file include: Table of contents graphic


A 18F-labeled Alanine Derivative Serve as An ASCT2 Marker for

Recommend Documents