Preamble

Preparation and Preliminary Biological Evaluation of an [18F]AlF-Labeled Albumin-Binding Agent Modified PARP-Targeted Imaging Probe

Wei Xu¹, Junjie Yan², Donghui Pan², Meng Li², Wei Wu², Min Yang¹,²

¹(School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China; School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China)

²(NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Molecular Imaging Center, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, China)

Abstract

[Background] Poly(ADP-ribose) polymerase (PARP) represents a crucial therapeutic target in cancer treatment, and dynamic assessment of its expression level is essential for achieving precision therapy. [Purpose] This study aimed to design and synthesize a novel 18F-labeled PARP-targeted PET imaging probe by conjugating an albumin-binding moiety to the core structure of the PARP inhibitor olaparib, and to evaluate its imaging performance through in vitro experiments and in vivo microPET imaging in tumor-bearing mice. [Methods] The precursor NOTA-LA was synthesized using 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one and 4-(4-iodophenyl)butanoic acid as starting materials. The target imaging probe [18F]AlF-NOTA-LA was prepared using the [18F]AlF non-covalent radiochemical fluorination method. The radiolabeling yield, radiochemical purity, molar activity, log P value, and stability were subsequently determined. The imaging performance of the probe was evaluated through a combination of in vitro cell uptake studies and in vivo microPET imaging in tumor-bearing mice. [Results] Radiolabeling of [18F]AlF-NOTA-LA was completed in approximately 15 minutes, yielding a radiolabeling efficiency of 49.2 ± 2.5%, with radiochemical purity exceeding 99%, a molar activity of 3.14 GBq/μmol, a log P value of -1.1 ± 0.013, and excellent in vitro stability. After 60 minutes, the cellular uptake of [18F]AlF-NOTA-LA in MDA-MB-453 cells was 3.83 ± 0.26%AD, significantly higher than that in A549 cells (1.89 ± 0.12%AD). MicroPET imaging revealed that in MDA-MB-453 tumor-bearing mice, probe uptake in tumors peaked at 10 minutes post-injection, reaching a maximum value of 5.31 ± 0.20%ID/g. In contrast, A549 tumor-bearing mice and the blocking group showed significantly lower peak uptakes of 3.04 ± 0.03%ID/g and 2.23 ± 0.11%ID/g, respectively. [Conclusion] Preliminary preclinical findings indicate that while the probe exhibited suboptimal pharmacokinetic properties in vivo, structural optimization could enhance its potential as a PARP-targeted PET imaging probe.

Keywords: PARP, olaparib, albumin binding, [18F]AlF, PET imaging, breast cancer

Introduction

Poly(ADP-ribose) polymerase (PARP) is a family of enzymes with diverse cellular functions, primarily involving DNA repair, maintenance of genomic stability, and regulation of cell death. The PARP family comprises at least 17 isoforms, with PARP1 being the most important member responsible for over 90% of PARP activity in cells and playing a critical role in DNA damage repair. PARP1 binds to DNA damage sites (typically single-strand DNA breaks) and catalyzes the synthesis of poly(ADP-ribose) chains on protein substrates, thereby recruiting DNA repair proteins to the damage sites to facilitate DNA repair. Due to the characteristic hyperactivation of DNA damage repair pathways in tumor cells, which leads to significantly enhanced PARP1 activity, PARP1 has emerged as an important therapeutic target in cancer treatment.

PARP inhibitors exert antitumor effects by suppressing PARP enzyme activity and inducing "synthetic lethality" in BRCA1/2-mutated or homologous recombination deficiency (HRD)-positive tumor cells. Since 2014, the U.S. Food and Drug Administration (FDA) has successively approved several PARP inhibitors—including olaparib, niraparib, talazoparib, and rucaparib—for clinical use in various solid tumors such as pancreatic cancer, breast cancer, prostate cancer, and ovarian cancer. Despite the significant role of targeted drug therapy in cancer treatment, clinical applications still face challenges in the effective detection of tumor biomarkers. Real-time and accurate monitoring and evaluation of tumor PARP1 expression levels can help identify patients who would benefit from PARP inhibitor therapy and provide guidance for chemotherapy regimen selection and prognostic assessment.

In recent years, multiple research teams have reported the clinical potential of PARP imaging; however, several challenges remain. For instance, existing probes exhibit relatively low tumor uptake and short retention times within tumors, leading to rapid decay of radioactive signals at tumor sites. These issues limit the imaging window, affect diagnostic accuracy, and hinder the widespread application of radiolabeled PARP inhibitors. Previous studies have demonstrated that albumin-binding agents (ALB) incorporated as functional components of radioligands can effectively enhance tumor uptake of radiopharmaceuticals. Binding of ALB to albumin can prolong the blood half-life of radioligands, thereby increasing tumor uptake and improving probe retention at tumor sites. Based on the core structure of the PARP1 inhibitor olaparib, this study conjugated an ALB moiety (4-(4-iodophenyl)butanoic acid) and a chelator (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA), followed by 18F-Al labeling to obtain [18F]AlF-NOTA-LA (Figure 1 [FIGURE:1]). The feasibility of using this probe for PARP1-specific PET imaging was evaluated through both in vitro and in vivo experiments.

1.1 Experimental Instruments and Materials

A cyclotron (Sichuan Jiuyiyuan Particle Technology Co., Ltd.), high-performance liquid chromatography (HPLC) system (Model 1525, Waters Corporation, USA) equipped with a UV detector (Model 2487, Waters Corporation, USA), a radioactivity detector (PerkinElmer, USA), Sep-Pak C18 columns (4.6 mm × 250 mm, PerkinElmer, USA), a radioisotope calibrator (CRC-55tR, Beijing Pate Biotechnology Co., Ltd.), a microPET scanner (Siemens, Germany), and a small animal anesthesia machine (SAR-830/P model, CWE, USA) were used in this study. 5-[(3,4-dihydro-4-oxo-1-phthalazinyl)methyl]-2-fluorobenzoic acid, 1-Boc-piperazine, 4-(4-iodophenyl)butyric acid, thionyl chloride, N'-tert-butoxycarbonyl-L-2,4-diaminobutyric acid methyl ester, and 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-aza-nonadecan-19-oic acid were purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. NHS-NOTA was obtained from Ganzhou Tanzhen Biomedical Co., Ltd., and all other chemical reagents were purchased from Sinopharm Group Co., Ltd.

1.2 Cell Lines and Experimental Animals

The human breast cancer cell line MDA-MB-453 (high PARP1 expression) was purchased from Wuhan Boster Biological Engineering Co., Ltd. and cultured in L-15 medium supplemented with 15% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO₂ environment. The human non-small cell lung cancer cell line A549 (low PARP1 expression) was obtained from the Cell Bank of the Chinese Academy of Sciences and cultured under similar conditions (37°C, 5% CO₂) in DMEM medium containing 10% fetal bovine serum. Female BALB/c nude mice (18–20 g) were purchased from Changzhou Cavens Experimental Animal Company. MDA-MB-453 or A549 cells (1×10⁷ cells in 0.1 mL saline) were subcutaneously inoculated into the right axilla of mice. Once tumor diameters reached 6–10 mm, microPET imaging and biodistribution studies were performed. All animal experiments were approved by the Ethics Committee of Jiangsu Institute of Nuclear Medicine (JSINM-2024-114) and conducted in accordance with institutional and national regulations for laboratory animal management and use.

1.3 Chemical Synthesis

The synthetic route for the labeling precursor is shown in Figure 1. The synthesis involved multiple steps including HBTU/TEA-mediated coupling in DCM at room temperature, TFA deprotection in DCM, LiOH hydrolysis in water, and final [18F]AlF labeling using H18F, AlCl₃, and CH₃COOH to produce [18F]AlF-NOTA-LA.

1.3.1 Synthesis of tert-butyl (14-(4-{2-fluoro-5-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzoyl}piperazin-1-yl)-14-oxo-3,6,9,12-tetraoxatetradecyl)carbamate

4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one (10 mmol), HBTU (15 mmol), and triethylamine (30 mmol) were dissolved in 100 mL dichloromethane. 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-aza-nonadecan-19-oic acid (12 mmol) was then added, and the mixture was stirred overnight at room temperature. The reaction mixture was washed with 10% hydrochloric acid, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by column chromatography using a dichloromethane:methanol gradient (200:1 to 50:1, V/V) as eluent to afford a white solid (3.2 g, 45.7%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.60 (s, 1H), 8.27 (dd, J=7.7, 1.5 Hz, 1H), 8.00–7.77 (m, 3H), 7.48–7.32 (m, 2H), 7.24 (t, J=9.0 Hz, 1H), 6.74 (t, J=5.8 Hz, 1H), 4.34 (s, 2H), 4.16 (d, J=23.1 Hz, 2H), 3.58 (dd, J=35.5, 13.6 Hz, 8H), 3.44 (s, 2H), 3.33 (s, 8H), 3.19 (d, J=11.3 Hz, 2H), 3.05 (q, J=6.1 Hz, 2H), 1.36 (s, 9H). ESI-MS (m/z): 700.67 [M + H]⁺; 722.68 [M + Na]⁺.

1.3.2 Synthesis of 4-[3-(4-{14-amino-3,6,9,12-tetraoxatetradecanoyl}piperazine-1-carbonyl)-4-fluorobenzyl]-1(2H)-phthalazinone (2)

tert-butyl (14-(4-{2-fluoro-5-[(4-oxo-3,4-dihydrophthalazin-1-yl)methyl]benzoyl}piperazin-1-yl)-14-oxo-3,6,9,12-tetraoxatetradecyl)carbamate (5 mmol) was dissolved in 35 mL dichloromethane, and 10 mL of 4 M HCl in ethyl acetate was added. The reaction was stirred at room temperature for 30 minutes. The reaction mixture was filtered, and the filter cake was adjusted to pH=12 with dilute ammonia solution. After extraction with dichloromethane, concentration, and drying, a white solid was obtained (2.67 g, 89.1%). ESI-MS (m/z): 601.03 [M + H]⁺; 622.59 [M + Na]⁺.

1.3.3 Synthesis of methyl N⁶-(tert-butoxycarbonyl)-N²-(4-(4-iodophenyl)butanoyl)-L-lysinate (4)

4-(4-iodophenyl)butyric acid (10 mmol) was dissolved in 50 mL dichloroethane, followed by addition of thionyl chloride (35 mmol). The mixture was heated at 80°C for 4 hours. The solvent and excess thionyl chloride were removed under reduced pressure, and the residue was diluted with 30 mL dichloromethane for subsequent use. In a separate flask, N'-tert-butoxycarbonyl-L-2,4-diaminobutyric acid methyl ester (10 mmol) and triethylamine (15 mmol) were dissolved in 30 mL dichloromethane. The previously prepared 4-(4-iodophenyl)butyryl chloride solution in dichloromethane was slowly added dropwise at 0°C. After complete addition, the reaction was warmed to room temperature and stirred for 1 hour. The mixture was washed with water, concentrated, and the residue was recrystallized from 70% ethanol to afford a white solid (3.92 g, 73.7%). ¹H NMR (600 MHz, CDCl₃) δ 7.60 (d, J=7.9 Hz, 2H), 6.94 (d, J=7.9 Hz, 2H), 6.06 (d, J=7.7 Hz, 1H), 4.58 (q, J=7.2, 6.7 Hz, 2H), 3.74 (s, 3H), 3.10 (q, J=6.7 Hz, 2H), 2.60 (t, J=7.6 Hz, 2H), 2.22 (t, J=7.5 Hz, 2H), 1.95 (hept, J=6.9 Hz, 2H), 1.69 (h, J=6.7 Hz, 2H), 1.49 (dt, J=14.3, 7.4 Hz, 2H).

1.3.4 Synthesis of N⁶-(tert-butoxycarbonyl)-N²-(4-(4-iodophenyl)butanoyl)-L-lysine (5)

Compound 4 (5 mmol) was dissolved in 20 mL methanol, and 5 mL of 4 M lithium hydroxide solution was added. The reaction was stirred overnight at room temperature. The mixture was then slowly acidified with 2 M dilute hydrochloric acid to pH=4. The precipitate was filtered and dried to give a white solid (2.46 g, 95.3%). ¹H NMR (600 MHz, DMSO-d₆) δ 12.42 (s, 1H), 8.02 (d, J=7.7 Hz, 1H), 7.62 (d, J=7.8 Hz, 2H), 7.02 (d, J=7.9 Hz, 2H), 6.75 (t, J=5.7 Hz, 1H), 4.13 (td, J=8.6, 4.8 Hz, 1H), 2.88 (qd, J=6.8, 3.4 Hz, 2H), 2.11 (t, J=7.4 Hz, 2H), 1.76 (p, J=7.5 Hz, 2H), 1.66 (ddt, J=14.0, 9.9, 5.7 Hz, 1H), 1.54 (dtd, J=14.1, 9.4, 5.1 Hz, 1H), 1.36 (s, 11H), 1.31–1.19 (m, 2H). ESI-MS (m/z): 541.47 [M + Na]⁺.

1.3.5 Synthesis of LA-Boc

Compound 5 (3 mmol), HBTU (4.5 mmol), and triethylamine (9 mmol) were dissolved in 20 mL dichloromethane. Compound 2 (3.6 mmol) was then added, and the reaction was stirred overnight at room temperature. The mixture was washed with 10% hydrochloric acid, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by column chromatography using dichloromethane:methanol (95:5, V/V) as eluent to afford a white solid (1.59 g, 48.2%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.60 (s, 1H), 8.27 (d, J=7.7 Hz, 1H), 7.99–7.81 (m, 5H), 7.61 (d, J=7.8 Hz, 2H), 7.49–7.33 (m, 2H), 7.23 (t, J=9.0 Hz, 1H), 7.00 (d, J=7.8 Hz, 2H), 6.84–6.62 (m, 1H), 4.33 (s, 2H), 4.16 (d, J=23.0 Hz, 3H), 3.62 (d, J=16.0 Hz, 4H), 3.19 (d, J=9.0 Hz, 6H), 2.86 (q, J=6.9 Hz, 2H), 2.11 (t, J=7.3 Hz, 2H), 1.88 (s, 2H), 1.75 (p, J=7.2 Hz, 2H), 1.63–1.42 (m, 2H), 1.35 (s, 9H), 1.21 (s, 2H). ESI-MS (m/z): 1123.21 [M + Na]⁺.

1.3.6 Synthesis of LA

LA-Boc (1 mmol) was dissolved in 10 mL dichloromethane, and 5 mL of 4 M HCl in ethyl acetate was added. The reaction was stirred at room temperature for 15 minutes. The mixture was filtered, and the filter cake was adjusted to pH=12 with dilute ammonia solution. After extraction with dichloromethane, concentration, and drying, a white solid was obtained (2.67 g, 89.1%). ESI-MS (m/z): 1001.17 [M + H]⁺.

1.3.7 Synthesis of NOTA-LA

LA (26.05 nmol) and NHS-NOTA (31.25 nmol) were dissolved in 1 mL DMF, followed by addition of 25 μL N,N-diisopropylethylamine. The reaction was stirred overnight at room temperature. The mixture was purified by semi-preparative HPLC to afford a white solid (11.45 mg, 34.3%). ¹H NMR (400 MHz, DMSO) δ 12.62 (s, 1H), 8.39 (dd, J = 13.5, 6.7 Hz, 3H), 8.26 (dd, J = 7.8, 1.5 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.93–7.79 (m, 2H), 7.61 (dd, J = 9.2, 3.1 Hz, 2H), 7.45 (d, J = 7.8 Hz, 1H), 7.39–7.33 (m, 1H), 7.23 (t, J = 9.0 Hz, 1H), 7.00 (d, J = 8.1 Hz, 2H), 4.33 (s, 2H), 4.21–4.10 (m, 3H), 3.49 (dd, J = 24.6, 14.0 Hz, 28H), 3.17–2.87 (m, 14H), 2.59 (d, J = 22.3 Hz, 6H), 2.48 (d, J = 6.9 Hz, 2H), 2.12 (td, J = 7.3, 2.6 Hz, 2H), 1.91 (s, 2H), 1.74 (p, J = 7.5 Hz, 2H), 1.60–1.23 (m, 6H). ESI-MS (m/z): 1123.21 [M + Na]⁺, HPLC purity >98%.

1.4 [18F]AlF Labeling

NOTA-LA (1 mg/mL, 30 µL) was mixed with 18F target water (1.85 GBq, 10 µL), acetonitrile (200 µL), aluminum chloride (2 mmol/L, 6 µL), and acetic acid (5 µL) to adjust the pH to 4. The reaction was carried out at 90°C for 8 minutes. After completion, the reaction mixture was diluted with 20 mL pure water and passed through a C18 reversed-phase Sep-Pak cartridge. The cartridge was washed with 20 mL pure water, and the product was eluted with 300 µL anhydrous ethanol to obtain [18F]AlF-NOTA-LA. The product was diluted with acetonitrile to 3.7 MBq/mL, and 10 µL was injected into the analytical radio-HPLC to determine radiochemical purity.

1.5 Determination of Lipophilicity (log P)

The probe was added to an equal volume mixture of n-octanol and water, vortexed, and centrifuged (15,000 rpm, 5 minutes). Aliquots of 100 µL were taken from the upper (organic) and lower (aqueous) phases, and radioactivity was measured using a gamma counter to calculate the lipophilicity coefficient (log P).

1.6 In Vitro Stability Studies

[18F]AlF-NOTA-LA (1.48 MBq, 50 µL) was mixed with either phosphate-buffered saline (PBS, 0.01 M, pH 7.4, 450 µL) or mouse plasma (450 µL) and incubated at 37°C for 0, 2, and 4 hours. At each time point, 25 µL samples were taken and analyzed by radio-HPLC to assess stability.

1.7 Western Blot Analysis

Proteins were extracted from MDA-MB-453 and A549 cells and quantified. Samples were loaded onto 10% SDS-polyacrylamide gels for electrophoretic separation and subsequently transferred to hydrophilic polyvinylidene fluoride membranes for antibody incubation. After blocking, membranes were incubated overnight at 4°C with rabbit monoclonal anti-PARP1 antibody (1:5000, Cell Signaling Technology) and mouse monoclonal anti-β-Tubulin antibody (1:2000, Cell Signaling Technology). Finally, species-matched horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) were applied for 1 hour at room temperature. Protein bands were visualized using the BeyoECL Plus Western blot detection system (Beyotime Biotechnology, Shanghai) and quantified using ImageJ software.

1.8 Cell Uptake Assay

MDA-MB-453 and A549 cells were seeded in 24-well plates at a density of 5×10⁵ cells/mL. Radioactive probe diluted in DMEM (74 KBq/mL) was added to each well, and cells were incubated at 37°C for four time points (30, 60, 120, and 240 minutes). In the blocking group, MDA-MB-453 cells were pre-incubated with 0.5 µM olaparib for 30 minutes to inhibit probe uptake before adding the radioactive probe (74 KBq/mL) under the same conditions. At each time point, cells were washed twice with PBS and lysed with 0.1 M sodium hydroxide. Radioactivity in the lysate was measured, and cellular uptake was calculated and expressed as percentage of absorbed dose (%AD).

1.9 MicroPET Imaging

MDA-MB-453 tumor-bearing mice were anesthetized with a mixture of 1.5% isoflurane in oxygen and injected intravenously via the tail vein with [18F]AlF-NOTA-LA (3.7–7.4 MBq) diluted in 200 µL saline. Static microPET scans were performed at 10, 30, 60, 90, and 120 minutes post-injection, with a scan duration of 10 minutes per time point. In the blocking group, mice were pre-injected with olaparib (5 mg/kg body weight) dissolved in 100 µL vehicle (95% saline + 5% DMSO) via the tail vein 30 minutes before probe administration to evaluate competitive inhibition. Regions of interest were drawn to quantify radioactive uptake in various tissues, with results expressed as %ID/g.

1.10 Biodistribution Studies

At 10, 30, and 60 minutes post-injection of [18F]AlF-NOTA-LA, MDA-MB-453 tumor-bearing mice were euthanized and major organs (heart, liver, spleen, lung, stomach, small intestine, muscle, bone, pancreas, brain, blood, tumor, and ovary) were harvested, weighed, and measured for radioactivity, with data expressed as %ID/g. In the blocking group, MDA-MB-453 tumor-bearing mice were pre-injected with olaparib (5 mg/kg body weight) 30 minutes before probe administration, followed by dissection at 10 minutes post-injection for organ collection. To validate PARP-1 expression levels in MDA-MB-453 and A549 cells, immunohistochemical staining was performed on tumor tissues fixed in formalin, embedded in paraffin, and sectioned at 5 µm thickness.

2.4 Western Blot Analysis and Cell Uptake

As shown in Figure 3 [FIGURE:3]A, MDA-MB-453 cells exhibited higher PARP1 protein expression levels compared to A549 cells, with relative expression normalized to β-Tubulin of 1.07 ± 0.21 versus 0.58 ± 0.05 in A549 cells (t = 4.606, p < 0.01). Figure 3C demonstrates that MDA-MB-453 cells showed significantly higher probe uptake than A549 cells at all time points. At 60 minutes, [18F]AlF-NOTA-LA uptake in MDA-MB-453 cells reached a plateau of 3.83 ± 0.26%AD, approximately 2-fold higher than that in A549 cells (1.89 ± 0.12%AD) (t = 11.45, p < 0.001), which was consistent with the Western blot results (1.84-fold difference). In the blocking group, probe uptake in MDA-MB-453 cells was markedly reduced, with uptake of only 0.43 ± 0.06%AD at 60 minutes (t = 21.54, p < 0.0001).

2.5 MicroPET Imaging

As shown in Figures 4 [FIGURE:4]A and D, MDA-MB-453 tumor-bearing mice exhibited significant tumor uptake of [18F]AlF-NOTA-LA at 10 minutes post-injection, reaching a maximum of 5.31 ± 0.20%ID/g, which then gradually decreased to 2.88 ± 0.09, 2.36 ± 0.18, 1.86 ± 0.12, and 1.62 ± 0.05%ID/g at 30, 60, 90, and 120 minutes, respectively. In contrast, the negative control A549 tumor-bearing mice showed a maximum tumor uptake of only 3.04 ± 0.03%ID/g (Figure 4B). In the olaparib blocking group, tumor uptake in MDA-MB-453 tumor-bearing mice was significantly inhibited, with uptake of only 2.23 ± 0.11%ID/g at 10 minutes (Figure 4C), demonstrating strong PARP1 targeting specificity (t = 23.04, p < 0.0001). Probe uptake in muscle of MDA-MB-453 tumor-bearing mice was low, decreasing from 1.77 ± 0.15%ID/g at 10 minutes to 0.05 ± 0.01%ID/g at 120 minutes, resulting in tumor-to-muscle ratios increasing from 2.99 ± 0.13 to 27.73 ± 4.12 over the same period (Figure 4E). In comparison, the tumor-to-muscle ratios at 120 minutes were only 1.66 ± 0.40 (t = 15.39, p < 0.0001) and 8.38 ± 1.84 (t = 10.15, p < 0.001) in A549 tumor-bearing mice and the blocking group, respectively. Compared to previously reported probes such as [64Cu]Cu-DOTA-olaparib (maximum uptake 3.45 ± 0.47%ID/g in MSTO-211H tumors) and [68Ga]Ga-DOTA-Olaparib (maximum uptake 2.83 ± 0.32%ID/g in SKOV3 tumors), [18F]AlF-NOTA-LA demonstrated higher tumor uptake (5.31 ± 0.20%ID/g), indicating that the albumin-binding strategy is effective. Furthermore, compared to other 18F-labeled PARP inhibitors including [18F]olaparib (3.16 ± 0.36%ID/g in PSN1 tumors), [18F]Rucaparib (5.49 ± 0.49%ID/g in PSN1 tumors), and [18F]Talazoparib (3.7 ± 0.7%ID/g in HCC1937 tumors), [18F]AlF-NOTA-LA showed superior performance. Similar to other 18F-labeled PARP inhibitors, this probe exhibited high non-specific uptake in the abdomen and suboptimal pharmacokinetic properties, necessitating structural modifications to reduce lipophilicity and accelerate clearance from non-target tissues. Compared to two clinically advanced probes, [18F]F-PARPi (tumor-to-muscle ratio 5.1 ± 0.9 at 120 min) and [18F]FTT (ratio 1.9 at 120 min), [18F]AlF-NOTA-LA achieved significantly higher tumor-to-muscle ratios of 6.49 ± 0.33 at 60 minutes and 27.73 ± 4.12 at 120 minutes.

2.6 Biodistribution Studies

As shown in Figure 5 [FIGURE:5]A, tumor uptake of [18F]AlF-NOTA-LA in MDA-MB-453 tumor-bearing mice was 5.11 ± 0.32, 3.10 ± 0.76, and 1.76 ± 0.26%ID/g at 10, 30, and 60 minutes post-injection, respectively. In the blocking group, tumor uptake was significantly reduced to 1.83 ± 0.85 and 0.46 ± 0.08%ID/g at 10 and 30 minutes (Figure 5C), further confirming the probe's PARP targeting specificity in vivo. Due to the albumin-binding moiety in [18F]AlF-NOTA-LA, blood uptake was relatively high, with 11.11 ± 0.76 and 13.27 ± 2.03%ID/g in the experimental and blocking groups at 10 minutes, respectively (Figures 5A and C). As the primary metabolic organ, liver uptake was 20.63 ± 0.32 and 6.12 ± 1.24%ID/g at 10 and 30 minutes. Kidney uptake decreased rapidly from 9.12 ± 2.79%ID/g at 10 minutes to 1.97 ± 0.19%ID/g at 30 minutes. Bone uptake remained below 1%ID/g, indicating good in vivo stability without defluorination. Immunohistochemical staining (Figure 5D) confirmed significantly higher PARP-1 expression in MDA-MB-453 xenograft tissues compared to A549 tumors.

Conclusion

In this study, we conjugated an albumin-binding agent to the olaparib core and prepared the PARP-targeted imaging agent [18F]AlF-NOTA-LA using a one-step labeling method. The probe demonstrated convenient labeling, high yield, and good in vitro stability. However, it exhibited high non-specific abdominal uptake and suboptimal pharmacokinetic properties in vivo. Following structural optimization to improve these characteristics, this probe shows promise as a potential PARP-targeted PET imaging agent.

Author Contributions

Wei Xu: Experimental design, chemical synthesis, animal experiments, data analysis, and manuscript writing. Junjie Yan: Design and guidance of chemical synthesis procedures. Donghui Pan: Experimental data analysis. Meng Li: Animal experiments. Wei Wu: Data compilation. Min Yang: Overall experimental design guidance and manuscript revision.

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