PDF vOl.9 no. 1 January-June 2022

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JOURNAL OF SIRIRAJ RADIOLOGY [VOL.9 NO. 1 JANUARY-JUNE 2022]

34 | Development of 18F-PSMA1007 Radiosynthesis for Prostate Cancer Diagnosis

การพัฒนาการสังเคราะห์ติดฉลาก

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F-PSMA1007 ส าหรับ ตรวจวินิจฉัยโรคมะเร็งต่อมลูกหมาก Development of

¹⁸

F-PSMA1007 Radiosynthesis for Prostate Cancer Diagnosis

ญาณี กาฬกาญจน์ วท.บ.เคมี

บันฑิตา อรุณวิภาดา วท.บ.เคมี

Received March 15, 2022; Revised April 30, 2022; Accepted May 20, 2022

สาขาวิชาเวชศาสตร์นิวเคลียร์ ภาควิชารังสีวิทยา คณะแพทยศาสตร์ศิริราชพยาบาล มหาวิทยาลัยมหิดล

วัตถุประสงค์ เพื่อพัฒนาวิธีการสังเคราะห์สารเภสัชรังสีชนิด ¹⁸F-PSMA1007 ส าหรับตรวจวินิจฉัยโรคมะเร็งต่อมลูกหมากโดย การติดฉลากสารรังสีฟลูออรีน-18 เข้ากับสารประกอบที่มีความจ าเพาะต่อการตรวจระดับการท างานของเซลล์มะเร็งต่อม ลูกหมากที่เรียกว่า PSMA Ligand ชนิด PSMA1007 ด้วยเครื่องสังเคราะห์สารเภสัชรังสีอัตโนมัติ CFN-MPS200 ที่ศูนย์ไซโค ตรอนศิริราช

วิธีด าเนินการ การสังเคราะห์ ¹⁸F-PSMA1007 เริ่มต้นจากการรับสารรังสีฟลูออรีน-18 ที่ผลิตด้วยเครื่องไซโคลตรอนเข้าสู่เครื่อง CFN-MPS200 ท าการแยกฟลูออรีน-18 ออกจากออกซิเจน-18 โดยใช้ QMA cartridge และชะออกด้วย TBA.HCO3 ความเข้มข้น 0.075M แล้วท าให้แห้ง เติมสารตั้งต้น PSMA1007 ในสารละลายไดเมททิลซัลฟอกไซด์ จะเกิดปฏิกิริยาฟลูออริเนชันระหว่าง ฟลูออรีน-18 กับสารตั้งต้น จากนั้นเจือจางด้วยเอทานอลความเข้มข้นร้อยละ 5 โดยปริมาตร แล้วท าให้สารเภสัชรังสีบริสุทธิ์ด้วย การผ่านตัวดูดซับของแข็งและชะออกด้วยเอทานอลความเข้มข้นร้อยละ 30 โดยปริมาตร ท าให้ปราศจากเชื้อด้วย Cathivex-GV filter

ผลการด าเนินงาน ¹⁸F-PSMA1007 ที่สังเคราะห์ได้มีปริมาณสารผลิตภัณฑ์ร้อยละ 41.4-51.6% ใช้เวลาในสังเคราะห์ทั้งหมด 40 นาที ด้วยปริมาณรังสีในช่วง 20-100 GBq มีค่าความบริสุทธิ์ทางเคมีรังสีมากกว่าร้อยละ 99.83 มีสารเคมีปนเปื้อนน้อยกว่า 0.1 มิลลิกรัมต่อปริมาตรทั้งหมดและมีค่าตัวท าละลายที่ตกค้างไม่เกินจากค่าที่มาตรฐานก าหนด มีค่าความเป็นกรดเบสที่ 7.0±0.4 มีปริมาณสารที่ก่อให้เกิดไข้และความปราศจากเชื้อเป็นไปตามเกณฑ์มาตรฐาน

สรุปผล สามารถสังเคราะห์สารเภสัชรังสีชนิด ¹⁸F-PSMA1007 ด้วยเครื่อง CFN-MPS200 ที่ศูนย์ไซโคลตรอนศิริราชที่มีคุณภาพ เป็นไปตามมาตฐานตามมาตรฐานก าหนดและสามารถใช้ในการตรวจวินิจฉัยโรคมะเร็งต่อมลูกหมากโดยการถ่ายภาพด้วย เครื่อง PET/CT ได้

ค าส าคัญ มะเร็งต่อมลูกหมาก, สารเภสัชรังสี, CFN-MPS200 , ¹⁸F-PSMA1007, PSMA-1007

บทความวิจัย

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ญาณี กาฬกาญจน์

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Introduction

Prostate cancer (PCa) is the second most commonly diagnosed cancer in men worldwide [1]. Prostate- specific membrane antigen (PSMA) is overexpressed on prostate cancer cells, as known as N-acetylated-alpha- linked acidic dipeptidase (NAALADase), folate hydrolase (FOLH1) or glutamate carboxypeptidase II (GCPII) is type II integral membrane glycoprotein expressed in various tissues, in central nervous system, which cleavage of peptide neurotransmitter N-acetyl-L- aspartyl-L-glutamate (NAAG) into N-acetyl-Laspartate

and L-glutamate [2,3,4]. Therefore, development of PSMA-specific based ligands has been labeled with radionuclide for diagnostic and therapy of prostate cancer. The interested things is urea-PSMA based inhibitors which possess the Glu-NH-CO-NH-Lys/Glu (Glu-urea-motif) pharmacophore [2].

In recent years, the Ga-68 radiometal labeled in urea-PSMA based inhibitors, using Glu-urea-lys(Ahx)- HBED-CC (also called PSMA-11) has successfully for positron emission tomography ( PET) imaging of diagnosing prostate cancer that can detection of Abstract

Propose Prostate-specific membrane antigen (PSMA) is target for the positron emission tomography of prostate cancer (PCa).

18F-labelled PSMA-1007 was introduced from 68Ga-PSMA-11, urea-PSMA based inhibitors. The aim of this study is describe and report novel one-step procedure and the quality control of ¹⁸F-PSMA1007 using CFN-MPS200 automation radiosynthesizer.

Method The novel one-step procedure of ¹⁸F-PSMA1007 using CFN-MPS200 synthesizer module from Sumitomo (Japan).

18F-Fluoride was trapped on QMA Cartridge and eluted with TBA.HCO3 solution. Drying was accomplished by azeotropic distillation using acetonitrile. Then PSMA-1007 precursor/DMSO was added and heated for fluorination reaction. The reaction mixture was diluted with 5% EtOH solution and passed through SPE cartridges. The final product was eluted with 30% EtOH into product vial by passed through Cathivex GV filter and diluted with saline containing phosphate buffer.

Result 18F-PSMA1007 was produced in radiochemical yields 42.4 - 51.6% after a total synthesis time of approximately 40 min, initial activities from 20-100 GBq. Radiochemical purity determined by HPLC was ≥ 99.8%. Chemical impurities were

≤ 0.1 mg/Vmax. The amounts of the residual solvent were below the pre-defined limits (acetonitrile 0.1 µg/mL; DMSO 0.1 µg/mL; and ethanol content was in the range 2.6-7.7% v/v.). The pH of the final product was 7.0±0.4. Bacterial endotoxin testing was ≤ 175 IU/Vmax and sterility testing was performed post release.

Conclusion A fully automated production of ¹⁸F-PSMA1007 on CFN-MPS200 synthesizer module (Sumitomo) has successful with high reproducible radiochemical yields and use as diagnostic PET tracer for prestaging and monitoring of prostate cancer.

Keywords: CFN-MPS200, ¹⁸F-PSMA1007, Prostate cancer, PSMA-1007, Radiopharmaceutical

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36 | Development of 18F-PSMA1007 Radiosynthesis for Prostate Cancer Diagnosis biochemical recurrence and improving primary staging

of prostate cancer patients [ 5 , 6 ] . Thereafter, development and evaluation of PSMA radiotracer for therapy according to theragnostic concept. However, PSMA-11 cannot labeled with therapeutic radiometals such as Lu-177 or Ac-225. Thus, the linker modification to pharmacokinetic properties for the treatment of prostate cancer, the DOTA-conjugated PSMA Inhibitors [7] that radioligands is PSMA I&T and PSMA-617.

¹⁸F-PSMA1007 is a novel urea-PSMA based radiopharmaceutical was modified from PSMA-617 [8]

that has several advantages over 68Ga-PSMA-11, Ga- 68 (88.9% ^β+; half-life, 67.7 min) obtain from a Ge- 68/Ga-68 generator systems. The short half-life is the limitation of the batch production approximately 2-4

patients doses per generator elution, the stability to produce and use in-house, and higher positron energy (Ga-68, 1.90 MeV vs. F-18, 0.65 MeV), reducing the theoretical maximum spatial resolution [ 1 0 , 1 1 ] . Consequently, ¹⁸F-PSMA1007 is alternative radiotracer for PET/CT imaging with 177Lu-PSMA-617radiotracer for therapy under theragnostic concept [ 1 2 , 1 3 ] . Recently, ¹⁸F-PSMA1007 can be synthesized by two- step and the novel one-step procedure, which have been performed on different radio-synthesizers (Table 1.) [10,14-17]. The aim of the present study is described and report the process on the automation radiosynthesizer, the novel one- step procedure and the quality control of ¹⁸F-PSMA1007 according to GMP- compliant follow Cardinale J. [9].

Figure 1. Comparison of different PSMA-ligands (PSMA-11, PSMA-617 and PSMA-1007). [9]

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ญาณี กาฬกาญจน์ 37 Figure 2. CFN-MPS200 multipurpose synthesizer.

Materials and Methods

HM-20S medical cyclotron and CFN-MPS200 multipurpose synthesizer from Sumitomo Heavy Industries, Ltd. (see Figure 2).

Chemicals, solvents and reagents for synthesis were purchased from ABX advanced biochemical compounds (Germany). Acetonitrile for DNA synthesis were purchased from Sigma-Aldrich (Singapore). The synthesis cassette for CFN- MPS2 0 0 module type CA005F purchased from Sumitomo Heavy Industries

(Japan). ¹⁸F-Fluoride ion was produced by irradiation of 18O enriched water (≥ 98% purity; Taiyo Nippon Sanso, Tokyo, Japan) with a 20.0 MeV via 18O(p,n)18F nuclear reaction.

Setup of the Radiosynthesizer

The radiosynthesizer CFN- MPS200 (Sumitomo, Japan) was used for one-step procedure of 18F- PSMA1007. First, installed the cassette type

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38 | Development of 18F-PSMA1007 Radiosynthesis for Prostate Cancer Diagnosis CA005F and connected line to synthesis equipment

main unit. Check leak test and gas flow before actual synthesis for check that the equipment and devices have been appropriately installed. After that, installed reagents vial on the reagent holder and connected lines to the synthesis equipment main unit (0.6 mL of 0.075M Tetrabutylammonium Hydrogen Carbonate (TBA.HCO3); 1.0 mL of anhydrous acetonitrile; 1.6 mg of PSMA- 1007 precursor dissolved in 2. 0 mL of dimethyl sulfoxide (DMSO); 3.0 mL, 7.0mL and 23.0

mL of 5% Ethanol solution; 3.0 mL and 4.0 mL of 30%

Ethanol solution and 11.0 mL of Phosphate buffered Saline). Finally, a pre-conditioned Sep-PAK® Light QMA Cartridge (pre-activated with 20.0 mL of water), PS-H+ cartridge (top) connected with C18ec cartridge (bottom) (pre-condition both cartridges with 3.0 mL of ethanol solution and then 25. 0 mL of 5% ethanol solution) and Cathivex GV filter were installed on the radiosynthesizer. The general set up depicted in Figure 3.

Figure 3. Schematic of the Sumitomo CFN MPS-200 radio-synthesizer module for novel one-step production of ¹⁸F- PSMA1007.

Radiosynthesis of ¹⁸F-PSMA1007

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ญาณี กาฬกาญจน์ 39 The target water containing 18F-Fluoride from

cyclotron transferred to synthesizer module and was trapped on QMA Cartridge.18F-Fluoride was eluted with 0.075M Tetrabutylammonium Hydrogen Carbonate solution into the reactor vial using vacuum.

Solvent in reactor was removed at a temperature 120°C under a stream of nitrogen and using vacuum pump for 10 minutes. Then, 1.0 mL of acetonitrile was added for azeotropic distillation and heated at a temperature 120°C under a stream of nitrogen and using vacuum pump for 10 minutes. After drying process, PSMA-1007 precursor solution was added into reactor vial and heated for fluorination reaction at a temperature 95°C for 10 minutes in closed system. The mixture solution was transferred to the reaction mixture vial containing 7.0 mL of 5% ethanol solution and washed line with 3.0 mL of 5% ethanol solution. After that, diluted solution was passed through the PS-H+ cartridge and C18ec cartridge. Both cartridges were washed with 23.0 mL of 5% ethanol solution and then 3.0 mL of 30% ethanol solution into waste vial to remove chemical and radiochemical impurity. Finally, the final product was eluted with 4.0 mL of 30% ethanol solution into product vial by passed through Cathivex GV filter and diluted with saline containing 11.0 mL of phosphate buffer.

Quality Control

A Quality control analysis method of ¹⁸F- PSMA1007 according to GMP-compliant follow Cardinale J. [9]. Acceptance criteria of radiochemical

purity more than 95.0% determined by HPLC (Agilent1260) and TLC. Chemical impurities not more than 0.5 mg/Vmax. The amounts of the residual solvent below the pre-defined limits (TBA 2.6 mg/V_max; acetonitrile 4.1 mg/V_max; DMSO 50 mg/V_max; and ethanol content not more than 10% v/v.) determined by GC (Agilent7890). The pH of the final product between 4.5-7.5. Bacterial endotoxin testing not more than 175 IU/V_max and sterility testing was performed post release (Table 1.).

HPLC system performed on Agilent 1260 system both equipped with US-300 RI detector (Japan) and Chromolith® High Resolution RP-18 endcapped 100-4.6 mm. (Germany) HPLC column. A multi-step gradient was applied using acetonitrile (Solvent A) and 0.1% TFA (Solvent B): 5% A to 15% B 0 – 1.5 min;

then 15% A to 35% B 1.5 – 10.5 min; then 35% A to 95% B 10.5 – 13 min; then 95% A to 5% B 13 – 19 min, wavelength 254 nm., flowrate 3 mL/min, total run time 19 min.

TLC method using TLC Silica gel 60G F254 glass plates 1.5 x 13.5 cm (Merck.). Apply about 1.0 µL of ¹⁸F-PSMA1007. Mobile phase condition was acetonitrile/water (60:40 v/v) and analyzed using Gamma radioactivity TLC-Scanner (MiniGita).

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40 | Development of 18F-PSMA1007 Radiosynthesis for Prostate Cancer Diagnosis Table 1. Quality control specification of 18F-PSMA1007 follow Cardinale J. [9]

Parameter Test method Acceptance Criteria

Appearance Visual inspection Clear and colorless

Radionuclidic Identity Haft-life measurement 105-115 min

Bacteria Endotoxin Endotoxin test < 175 EU/V_max

pH pH meter 4.5-7.5

Identity HPLC Rt ± 0.5 min of reference standard

Radiochemical purity TLC > 95.0%

HPLC > 95.0%

Radionuclidic purity

Multichannel analyzer 511 Kev

Yield ≥ 99.5%

(post-release)

Residual solvents

GC Acetonitrile < 4.1 mg/V_max

DMSO < 50 mg/ V_max Ethanol < 10%v/v

TLC TBA < 2.6 mg/ V_max

Sterility Contracted individual

laboratory Sterile (post-release)

Result and Discussion

18F-PSMA1007 using CFN-MPS200 synthesizer module was produced in good radiochemical yields 42.4 - 51.6% after a total synthesis time of approximately 40 min, upscaling to started activities of approximately from 20 to 100 GBq.

In the process of quality control of ¹⁸F- PSMA1007 with HPLC, it was found that radiochemical purity was ≥99.8% and radiochemical identity from chromatogram showed R_t (retention time) at 8.183 min

compared with R_tof reference standard ¹⁸F-PSMA1007 at 8.196 min, the difference value is 0.013 min within the range Rt ± 0.5 min of reference standard, therefore confirmed that the product is ¹⁸F-PSMA1007. (see Figure 4.)

Chemical impurities were ≤ 0.5 mg/Vmax. The amounts of the residual solvent were below the pre- defined limits ( acetonitrile 0. 1 µg/ mL; DMSO 0. 1 µg/mL; and ethanol content was in the range 2.6-7.7%

v/v.). The pH of the final product was 7.0±0.4. Bacterial

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ญาณี กาฬกาญจน์ 41 endotoxin testing was ≤ 175 IU/ V_max and sterility

testing was performed post release.

Conclusions

A fully automated one-step procedure of ¹⁸F- PSMA1007 was successfully on CFN- MPS200 synthesizer module ( Sumitomo) with good

radiochemical yields and all batches passed quality control according to European Pharmacopoeia. ¹⁸F- PSMA1007 can be produced in large amount per batch in PET/CT with on-site cyclotron and can detection of biochemical recurrence and improving primary staging of prostate cancer patients.

Figure 4. HPLC radio-chromatogram of the ¹⁸F-PSMA1007 was produced (1) and reference standard (2)

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42 | Development of 18F-PSMA1007 Radiosynthesis for Prostate Cancer Diagnosis Acknowledgement

We also would like to thank Assoc. Prof.

Benjapa Khiewvan, Assoc.Prof. Shuichi Shiratori, Miss Pornpun Jiemwutthisak and all staffs of Siriraj Cyclotron Center, Division of Nuclear Medicine, Department of Radiology, Faculty of Medicine Siriraj Hospital for supported and advised in conducting this research.

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