New Bifunctional Chelator 3p-C-NEPA for Potential Applications in Lu(III) and Y(III) Radionuclide Therapy and Imaging

Abstract

We have developed structurally unique bifunctional chelators in the NETA, NE3TA, and DEPA series for potential radiopharmaceutical applications. As part of our continued research efforts to generate efficient bifunctional chelators for targeted radionuclide therapy and imaging of various diseases, we designed a scorpion-like chelator that is proposed to completely saturate the coordination spheres of Y(III) and Lu(III). We herein report the synthesis and evaluation of a new chelator (3p-C-NEPA) with 10 donor groups for complexation with β-emitting radionuclides ⁹⁰Y(III), ⁸⁶Y(III), and ¹⁷⁷Lu(III). The chelator was synthesized and evaluated for radiolabeling kinetics with the readily available radioisotopes ⁹⁰Y and ¹⁷⁷Lu, and the corresponding ⁹⁰Y or ¹⁷⁷Lu-radiolabeled complexes were evaluated for in vitro stability in human serum and in vivo complex stability in mice. The new chelator rapidly bound ⁹⁰Y or ¹⁷⁷Lu and formed a stable complex with the radionuclides. The new chelator 3p-C-NEPA radiolabeled with either ⁹⁰Y or ¹⁷⁷Lu remains stable in human serum without dissociation for 10 days. ¹⁷⁷Lu-labeled 3p-C-NEPA produced a favorable in vivo biodistribution profile in normal mice.

Introduction

β-Particle-emitting radionuclides have been actively explored for the treatment and detection of various diseases.¹⁻³ Among the β-particle emitters, ⁹⁰Y, ⁸⁶Y, and ¹⁷⁷Lu have been labeled with tumor-targeting antibodies and peptides for radionuclide therapy and imaging of cancer.¹⁻⁶ Y-90 (t_1/2 = 2.7 d, E_max = 2.3 MeV) is a highly energetic and pure β^–-emitting radionuclide with the advantage of a longer range of penetration and homogeneous dose distribution in an optimal therapeutic range.^1,2,5 The first antibody-targeted radiotherapeutic agent Zevalin consists of ⁹⁰Y, chelator 1B4M-DTPA, and rituximab (monoclonal anti-CD20 antibody) and has been shown to significantly enhance the overall response rate in the treatment of non-Hodgkin’s lymphoma (NHL) as compared to anti-CD20 therapy alone.^1,7 A positron emitter ⁸⁶Y (t_1/2 = 14.7 h, 33% β⁺) is considered as a suitable radioisotope for positron emission tomography (PET) imaging of molecular targets using biomolecules with relatively long half-lives.^3,686Y has been studied for quantitative PET imaging in targeted radionuclide therapy with ⁹⁰Y.^6,8,9

¹⁷⁷Lu is a β^–-particle emitter with a relatively long half-life (t_1/2 = 6.7 d, E_max = 0.5 MeV) and less energetic and shorter tissue-penetration range (1.6 mm) relative to ⁹⁰Y (11.3 mm).^4,10 Lutathera is a ¹⁷⁷Lu-labeled DOTA-Tyr3-octreotate (DOTA-TATE) conjugate that was approved for the treatment of gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) with elevated expression of somatostatin receptors (SSTRs).¹¹ Lu-177 also possesses an imageable γ-emission and has been investigated for the development of ¹⁷⁷Lu-theranostics and quantitative single photon emission tomography (SPECT) imaging applications.⁴

Successful radionuclide therapy and imaging require the construction of a well-coordinated therapeutic and diagnostic platform consisting of a potent therapeutic or imaging radionuclide, a target-specific molecule, and a bifunctional chelator for radiolabeling of the bioactive molecule.^1,3,4 An effective bifunctional chelator is expected to complex a cytotoxic or imaging radionuclide with a limited half-life rapidly and tightly and possess a high kinetic inertness to trans-chelation by metal cations and natural chelators present in vivo.¹⁻⁴ The β-particle-emitting radionuclides ¹⁷⁷Lu, ⁹⁰Y, and ⁸⁶Y have the prevalent oxidation state (+3) and the respective ionic radius (pm) of 86 and 90 for coordination number 6.¹²

We have developed various bifunctional chelators in the NETA and DEPA series including 3p-C-DEPA and 3p-C-NETA (Figure 1) for potential radiopharmaceutical applications.¹³⁻¹⁸

Figure 1.

Bifunctional chelators in preclinical and clinical evaluations.

As part of our continued research efforts to generate efficient bifunctional chelators, we designed a scorpion-like chelator that can completely saturate the coordination sphere of Y(III) and Lu(III). We herein report the synthesis and in vitro and in vivo evaluation of the new bifunctional chelator 3p-C-NEPA (Figure 1) structured on the 9-membered 1,4,7-triazacyclononane (TACN) ring. 3p-C-NEPA contains 5 amino and 5 carboxylate groups for potential complexation with Y(III) or Lu(III). The potential decadendate chelator 3p-C-NEPA was synthesized and evaluated for radiolabeling kinetics and complex stability with ⁹⁰Y and ¹⁷⁷Lu in human serum and normal mice.

Results and Discussion

Synthesis

The scorpion-like chelator (3p-C-NEPA) containing two acyclic tridentate pendant arms and 10 potential donors is proposed for rapid, tight, and complete saturation of the coordination sphere of a metal cation with a relatively large ionic radius. 3p-C-NEPA (9) was prepared by a convenient synthetic method centered on the regiospecific ring opening of a functionalized aziridinium ion 7 with prealkylated 1,4,9-triazacyclononane (TACN) analogue 5 containing the secondary amine as a nucleophile (Scheme 1).

Scheme 1. Synthesis of a Novel Bifunctional Chelator 3p-C-NEPA (9).

N-Carboxybenzyl (CBZ)-protected TACN 1(19) was selectively alkylated by the reaction of N,N-bisubstituted β-bromoamine 2(20) to produce compound 3 in 86% isolated yield. Compound 3 was further reacted with tert-butylbromoacetate to produce a trisubstituted TACN 4 in a nearly quantitative yield. The CBZ group in 4 was removed by Pd(II)-catalyzed hydrogenation to afford 5 in 89% yield. N,N-Bisubstituted secondary β-iodoamine 6 was prepared by a multistep conversion of a functionalized α-amino acid in the racemic form as we previously reported.¹⁶ The secondary β-iodoamine 6 was converted to aziridinium ion 7 that was further reacted with 5 at the less hindered methylene carbon at 7. The ring-opening reaction of 7 with 5 provided the substitution product 8 that was further treated with 4 M HCl (g) in 1,4-dioxane for the removal of the tert-butyl groups in 8. The acid-promoted reaction afforded the desired chelator 3p-C-NEPA (9).

Radiolabeling Kinetics and in Vitro Serum Stability

The chelator 3p-C-NEPA (9) was evaluated for radiolabeling efficiency with ⁹⁰Y and ¹⁷⁷Lu (Table 1). The new chelator (20 μg) in 0.25 M NH₄OAc buffer solution (pH 5.5) was radiolabeled with ⁹⁰Y or ¹⁷⁷Lu (60 μCi) at room temperature (RT). During the reaction time (1 h), the radiolabeling kinetics was determined by TLC analysis (Supporting Information).

Table 1. Radiolabeling Efficiency (%) of Chelators with ⁹⁰Y or ¹⁷⁷Lu (pH 5.5, RT)^a.

	radiolabeling efficiency (%)
time (min)	90Y-3p-C-NEPA	177Lu-3p-C-NEPA	90Y-C-DOTAb	177Lu-C-DOTAb
1	97.0 ± 1.0	97.4 ± 0.1	77.1 ± 3.7	94.5 ± 3.9
10	99.0 ± 0.4	98.6 ± 0.1	69.4 ± 10.6	99.5 ± 0.5
30	99.7 ± 0.1	99.4 ± 0.1	76.1 ± 9.5	99.9 ± 0.1
60	99.8 ± 0.2	99.5 ± 0.1	83.5 ± 8.1	100.0 ± 0.0

^aRadiolabeling efficiency (mean ± standard deviation %) was measured in triplicate using TLC and a binary mobile phase (CH₃CN/H₂O = 3:2).

^bData are cited for comparison.¹⁵

The data shown in Table 1 indicate that 3p-C-NEPA (1) instantly bound to ⁹⁰Y or ¹⁷⁷Lu (1 min, >97% radiolabeling efficiency, pH 5.5). 3p-C-NEPA was significantly more efficient in binding ⁹⁰Y than C-DOTA¹⁵ (p < 0.05). 3p-C-NEPA with 10 potential donor groups sequestered ⁹⁰Y more rapidly than the octadentate chelator C-DOTA over the time points. Radiolabeling of 3p-C-NEPA with ⁹⁰Y was nearly complete at the 10 min time point (>99% radiolabeling efficiency), while C-DOTA had a lower labeling efficiency with ⁹⁰Y (69% at 10 min). 3p-C-NEPA and C-DOTA displayed similar radiolabeling efficiency with ¹⁷⁷Lu. ⁹⁰Y- or ¹⁷⁷Lu-radiolabeled chelators were further evaluated for in vitro serum stability (Figure 2 and the Supporting Information). ¹⁷⁷Lu-3p-C-NEPA was stable in human serum for at least 14 days, while a small amount of ⁹⁰Y (∼2%) was lost from ⁹⁰Y-3p-C-NEPA over 2 weeks. The radiolabeling and in vitro complex stability data indicate that 3p-C-NEPA with potential 10 donor groups is more effective in sequestering ¹⁷⁷Lu than ⁹⁰Y.

Figure 2.

In vitro complex stability of ⁹⁰Y-3p-C-NEPA and ¹⁷⁷Lu-3p-C-NEPA in human serum at pH 7 and 37 °C (mean ± standard deviation % measured in triplicate).

We previously reported that the hybrid chelator 3p-C-NETA (Figure 1) containing both macrocyclic and acyclic donor groups can rapidly form a stable complex with ⁹⁰Y or ¹⁷⁷Lu.¹⁵⁻¹⁷ 3p-C-NETA instantly bound to ⁹⁰Y or ¹⁷⁷Lu (>97% radiolabeling efficiency, 1 min, RT) and was favorably compared to C-DOTA for radiolabeling kinetics and in vitro and in vivo complex stability with ⁹⁰Y and ¹⁷⁷Lu.¹⁵⁻¹⁷ Although there is no substantial difference between 3p-C-NEPA and 3p-C-NETA as an effective chelator for ¹⁷⁷Lu, the octadentate 3p-C-NETA displayed higher complex stability in serum than the decadentate 3p-C-NEPA. We designed the decadentate chelator 3p-C-DEPA (Figure 1) built on the CYCLEN (1,4,7,10-tetraazacyclododecane) backbone.¹⁴ 3p-C-DEPA was efficient in labeling ⁹⁰Y or ¹⁷⁷Lu (>90% radiolabeling efficiency) at RT. However, the chelator was less effective in holding ⁹⁰Y or ¹⁷⁷Lu than the octadentate 3p-C-NETA. ⁹⁰Y-3p-C-DEPA and ¹⁷⁷Lu-3p-C-DEPA displayed a significant amount of radioactivity (>25%) to human serum.¹⁷

The serum stability data suggest that 3p-C-NEPA containing the smaller macrocyclic backbone TACN can form a more stable complex with ⁹⁰Y or ¹⁷⁷Lu than the larger macrocyclic CYCLEN-based 3p-C-DEPA. It appears that the additional donor groups in the acyclic pendant arms were well tolerated and coordinated to hold ¹⁷⁷Lu(III) on the small TACN ring with high complex kinetics and stability.

In Vivo Biodistribution

The in vivo stability of ¹⁷⁷Lu-radiolabeled complexes was evaluated by performing biodistribution studies in CF-1 normal mice (intravenous injection, n = 3). The mice were euthanized at 1, 4, and 24 h. The selected organs (liver, kidney, muscle, and bone) and blood were harvested and wet-weighed, and the radioactivity was measured in a γ-counter (Figure 3 and the Supporting Information). ¹⁷⁷Lu-3p-C-NEPA displayed excellent in vivo complex stability as evidenced by the low radioactivity level in blood (<0.16% ID/g) over 24 h. Among the organs, the highest accretion of radioactivity was observed in the liver at the 1 h time point (4.6% ID/g) and was rapidly cleared at 4 h (0.5% ID/g) (p < 0.001). The initial high liver uptake might be related to enhanced lipophilicity of the chelator containing an aromatic ring and multiple alkyl chains. ¹⁷⁷Lu-3p-C-NEPA displayed a low radioactivity in bone and muscle which peaked at 1 h (<0.2% ID/g) and decreased at 24 h (<0.1% ID/g). The radioactivity in the kidney remained low at all time points (≤0.7% ID/g). The in vivo data suggest that ¹⁷⁷Lu-3p-C-NEPA displayed rapid blood clearance and low uptake in normal organs and produced an encouraging biodistribution profile in normal mice.

Figure 3.

In vivo biodistribution of ¹⁷⁷Lu-3p-C-NEPA in normal CF-1 mice (n = 3).

Conclusions

The new chelator 3p-C-NEPA was synthesized and evaluated for potential radionuclide therapy and imaging applications using Y(III)- and Lu(III)-based radionuclides. We have shown that 3p-C-NEPA can rapidly produce the radiolabeled complexes at RT. The in vitro complex stability data indicate that ⁹⁰Y- or ¹⁷⁷Lu-radiolabeled complexes of 3p-C-NEPA displayed excellent stability in serum for at least 10 days. The result of the in vivo biodistribution data shows that ¹⁷⁷Lu-3p-C-NEPA produced an excellent biodistribution profile and displayed rapid blood clearance and low uptake in normal organs. The in vitro and in vivo data suggest that 3p-C-NEPA warrants further evaluation for radionuclide therapy and imaging applications using ⁹⁰Y, ⁸⁶Y, and ¹⁷⁷Lu.

Experimental Section

¹H, ¹³C, and DEPT NMR spectra were obtained using a Bruker 300 instrument, and chemical shifts are reported in parts per million (ppm) on the δ scale relative to TMS. Electrospray (ESI) high-resolution mass spectra (HRMS) were obtained on a JEOL double sector JMS-AX505HA mass spectrometer (University of Notre Dame, South Bend, IN). Analytical HPLC was performed on an Agilent 1200 equipped with a diodearray detector (λ = 254 and 280 nm), a thermostat set at 35 °C, and a Zorbax Eclipse XDB-C18 column (4.6 × 150 mm, 80 Å). The mobile phase of a binary gradient (0–100% B/40 min and 100% A/5 min; solvent A, 0.05 M AcOH/Et₃N, pH 6.0; solvent B, CH₃OH for method 1 and 0–100% B/15 min; solvent A, 0.1% TFA in H₂O; solvent B, 0.1% TFA in CH₃CN for method 2) at a flow rate of 1 mL/min was used. Semi-preparative HPLC was performed on an Agilent 1200 equipped with a diodearray detector (λ = 254 and 280 nm), a thermostat set at 35 °C, and a Zorbax Eclipse XDB-C18 column (9.4 × 250 mm, 80 Å). The mobile phase of a binary gradient (0–100% B/80 min; solvent A, 0.05 M AcOH/Et₃N, pH 6.0; solvent B, CH₃OH for method 3) at a flow rate of 3 mL/min was used. All reagents were purchased from Sigma-Aldrich or Acros Organics and used as received unless otherwise noted.

Benzyl 4-(2-{Bis[2-(tert-butoxy)-2-oxoethyl]amino}ethyl)-1,4,7-triazonane-1-carboxylate (3)

To a solution of 1(19) (125 mg, 0.475 mmol) in CH₃CN (15 mL) was added 2(20) (167 mg, 0.475 mmol) and DIPEA (61.4 mg, 0.475 mmol) in CH₃CN (5 mL). The resulting mixture was stirred for 20 h at RT. The reaction mixture was evaporated to dryness and purified via column chromatography on silica gel eluting with 3–5% methanol in CH₂Cl₂ to afford pure 3 (218.8 mg, 86%). ¹H NMR (CDCl₃, 300 MHz): δ 1.38 (s, 18H), 2.40–2.52 (m, 2H), 2.54–2.86 (m, 7H), 2.87–3.06 (m, 2H), 3.13–3.52 (m, 8H), 5.08 (s, 2H), 7.15–7.35 (m, 5H); ¹³C NMR (CDCl₃, 75 MHz): δ 28.1 (q), 47.3 (t), 48.2 (t), 48.39 (t), 52.2 (t), 52.4 (t), 52.6 (t), 53.4 (t), 53.5 (t), 53.9 (t), 54.7 (t), 54.9 (t), 55.5 (t), 55.6 (t), 55.7 (t), 56.1 (t), 56.6 (t), 57.0 (t), 67.0 (t), 67.1 (t), 81.0 (s), 127.9 (d), 127.9 (d), 128.0 (d), 128.0 (d), 128.4 (d), 128.5 (d), 136.7 (s), 136.8 (s), 156.0 (s), 156.0 (s), 170.5 (s). HRMS (positive ion ESI): calcd for C₂₈H₄₇N₄O₆ [M + H]⁺m/z, 535.3490. Found: [M + H]⁺m/z, 535.3517.

Benzyl 4-(2-{Bis[2-(tert-butoxy)-2-oxoethyl]amino}ethyl)-7-[2-(tert-butoxy)-2-oxoethyl]-1,4,7-triazonane-1-carboxylate (4)

To a solution of 3 (150 mg, 0.281 mmol) in CH₃CN (2 mL) at 0 °C was sequentially added K₂CO₃ (40.7 mg, 0.295 mmol) and tert-butyl bromoacetate (54.8 mg, 0.281 mmol) in CH₃CN (1 mL). The resulting mixture was stirred for 16 h at RT while monitoring the reaction progress using TLC. The resulting mixture was evaporated to dryness. Then, 0.1 M HCl solution (30 mL) was added to the residue and extracted with ethyl acetate (30 mL × 3). The combined organic layers were dried over MgSO₄, filtered, and concentrated in vacuo to provide 4 (175 mg, 96%). ¹H NMR (CDCl₃, 300 MHz): δ 1.40 (s, 27H), 2.32–3.07 (m, 12H), 3.21 (d, J = 19.5 Hz, 2H), 3.27–3.50 (m, 8H), 5.08 (s, 2H), 7.15–7.35 (m, 5H); ¹³C NMR (CDCl₃, 75 MHz): δ 28.1 (q), 28.2 (q), 49.6 (t), 49.8 (t), 50.0 (t), 50.5 (t), 52.1 (t), 54.0 (t), 54.5 (t), 54.8 (t), 55.2 (t), 55.8 (t), 56.0 (t), 56.1 (t), 56.3 (t), 56.6 (t), 58.8 (t), 66.7 (t), 80.6 (s), 80.7 (s), 80.8 (s), 127.8 (d), 127.9 (d), 128.4 (d), 137.1 (s), 156.1 (s), 170.6 (s), 170.7 (s), 171.4 (s). HRMS (positive ion ESI): calcd for C₃₄H₅₇N₄O₈ [M + H]⁺m/z, 649.4171. Found: [M + H]⁺m/z, 649.4187.

tert-Butyl 2-{[2-(tert-Butoxy)-2-oxoethyl](2-{4-[2-(tert-butoxy)-2-oxoethyl]-1,4,7-triazonan-1-yl}ethyl)amino}acetate (5)

To a solution of 4 (170 mg, 0.262 mmol) in ethanol (50 mL) at RT was added 10% Pd/C (45 mg) under argon. The reaction mixture was placed under a hydrogenation apparatus set at 20 psi for 14 h. The resulting mixture was filtered via a Celite bed and washed thoroughly with ethanol. The filtrate was concentrated to dryness. The crude product was treated with 0.1 M HCl solution (20 mL) and extracted with CHCl₃ (20 mL × 3). The aqueous layer was further treated with 2 M NaOH solution and adjusted pH to 13 and then extracted with CHCl₃ (20 mL × 3). The combined organic layers from the extractions of the aqueous solution at pH 13 were dried over MgSO₄, filtered, and concentrated in vacuo to dryness to provide the product 5 (120 mg, 89%) as a yellowish oil. ¹H NMR (CDCl₃, 300 MHz): δ 1.45 (s, 27H), 1.96 (s, 1H), 2.52–2.91 (m, 10H), 2.93–3.18 (m, 6H), 3.44 (s, 4H), 3.34 (s, 2H); ¹³C NMR (CDCl₃, 75 MHz): δ 28.2 (q), 44.6 (t), 48.7 (t), 49.6 (t), 51.3 (t), 52.3 (t), 52.8 (t), 53.5 (t), 55.3 (t), 56.5 (t), 81.2 (s), 81.5 (s), 170.4 (s), 170.9 (s). HRMS (positive ion ESI): calcd for C₂₆H₅₁N₄O₆ [M + H]⁺m/z, 515.3803. Found: [M + H]⁺m/z, 515.3814.

tert-Butyl 2-({2-[4-(2-{Bis[2-(tert-butoxy)-2-oxoethyl]amino}-5-(4-nitrophenyl)pentyl)-7-[2-(tert-butoxy)-2-oxoethyl]-1,4,7-triazonan-1-yl]ethyl}[2-(tert-butoxy)-2-oxoethyl]amino) Acetate (8)

To a solution of 7(16) (60.2 mg, 0.107 mmol) in CH₃CN (1 mL) at 0 °C was added compound 11 (55.0 mg, 0.107 mmol) and DIPEA (41.4 mg, 0.321 mmol). The resulting mixture was stirred for stirred for 6 d at RT while monitoring the reaction progress using analytical HPLC (method 1, t_R = 41.5 min). The reaction mixture was concentrated to dryness. The residue was purified via column chromatography on silica gel (60–220 mesh). The column was first eluted with 50% ethyl acetate in hexanes and then dried and eluted with 3% CH₃OH in CH₂Cl₂ to provide the crude product. The crude product was further purified by semiprep HPLC (method 3) to afford 8 (39 mg, 38%). ¹H NMR (CDCl₃, 300 MHz): δ 1.35–1.60 (m, 45H), 1.62–2.25 (m, 6H), 2.48–3.02 (m, 20H), 3.45 (s, 4H), 3.37 (s, 4H), 3.26 (s, 2H), 7.38 (d, J = 8.4 Hz, 2H), 8.12 (d, J = 8.4 Hz, 2H); ¹³C NMR (CDCl₃, 75 MHz): δ 27.7 (t), 28.1 (q), 30.8 (t), 35.8 (t), 52.3 (t), 53.0 (t), 54.9 (t), 55.4 (t), 55.8 (t), 56.2 (t), 56.4 (t), 56.8 (t), 57.0 (t), 59.9 (t), 60.0 (d), 60.8 (t), 80.6 (s), 80.7 (s), 80.9 (s), 123.4 (d), 129.2 (d), 146.2 (s), 151.1 (s), 170.7 (s), 171.4 (s). HRMS (positive ion ESI): calcd for C₄₉H₈₅N₆O₁₂ [M + H]⁺m/z, 949.6220. Found: [M + H]⁺m/z, 949.6213.

2-{[2-(4-{2-[Bis(carboxymethyl)amino]-5-(4-nitrophenyl)pentyl}-7-(carboxymethyl)-1,4,7-triazonan-1-yl)ethyl](carboxymethyl)amino}acetic acid) (9)

To a flask containing compound 8 (17 mg, 0.0179 mmol) at 0–5 °C was added dropwise 4 M HCl (g) in 1,4-dioxane (2 mL) over 5 min. The resulting mixture was gradually warmed to RT and continuously stirred for 24 h. Ether (30 mL) was added to the reaction mixture which was then stirred for 10 min. The resulting precipitate was filtered and washed with ether. The solid product was quickly dissolved in deionized water. The aqueous solution was concentrated in vacuo to provide 9 (15 mg, 98%) as an off-white solid. ¹H NMR (D₂O, 300 MHz): δ 1.19–1.83 (m, 4H), 2.65−2.80 (m, 2H), 2.81–3.78 (m, 21H), 3.89 (s, 4H), 3.91 (s, 2H), 7.35 (d, J = 8.7 Hz, 2H), 8.07 (d, J = 8.7 Hz, 2H). Analytical HPLC (t_R = 7.52 min, Method 2). HRMS (negative ion ESI): calcd for C₂₉H₄₃N₆O₁₂ [M – H]⁻m/z, 667.2944. Found: [M – H]⁻m/z, 667.2976.

Radiolabeling of 3p-C-NEPA with ⁹⁰Y or ¹⁷⁷Lu

All HCl solutions were prepared from the commercially available ultrapure HCl solution (JT baker, #6900-05). For metal-free radiolabeling, plasticware including pipette tips, tubes, and caps was soaked in 0.1 N HCl overnight and washed thoroughly with Milli-Q (18.2 MΩ) water and air-dried overnight. Ultrapure ammonium acetate (Aldrich, #372331) was purchased from Aldrich and used to prepare 0.25 M NH₄OAc buffer solutions (0.25 M, pH 5.5). After adjusting pH using 0.1 M/1 M HCl or NaOH solution, 0.25 M NH₄OAc buffer solutions were treated with Chelex-100 resin (Biorad, #142-2842, 1 g/100 mL buffer solution), shaken overnight at RT, and filtered through a 0.22 μM filter (Corning, #430320) prior to use. ⁹⁰YCl₃ and ¹⁷⁷LuCl₃ were purchased from Perkin Elmer. TLC plates (6.6 × 2 cm, Silica gel 60 F₂₅₄, EMD Chemicals Inc., #5554-7) with the origin line drawn at 0.6 cm from the bottom were prepared. To a buffer solution (0.25 M NH₄OAc, pH 5.5) in a capped microcentrifuge tube (1.5 mL) was sequentially added a solution of 3p-C-NEPA (20 μg/20 μL H₂O). A solution of ⁹⁰YCl₃ or ¹⁷⁷LuCl₃ (0.05 M HCl, 2.22 MBq, 60 μCi) was added to the aqueous solution of the chelator, and the total volume of the resulting solution was brought up to 40 μL by adding the buffer solution. The reaction mixture was agitated on the thermomixer (Eppendorf, #022670549) set at 1000 rpm at RT for 1 h. The labeling efficiency was determined by ITLC eluted with acetonitrile/water (3:2 v/v) as the mobile phase. A solution of radiolabeled complexes (2 μL) was withdrawn at the designated time points, spotted on a TLC plate, and then eluted with the mobile phase. After completion of elution, the TLC plate was warmed and dried on the surface of a heater maintained at 35 °C and scanned using a TLC scanner (Bioscan, #FC-1000). The radiolabeled complex ⁹⁰Y-3p-C-NEPA or ¹⁷⁷Lu-3p-C-NEPA was detected at ∼50 mm from the bottom of the TLC plate, while the unbound radionuclide ⁹⁰Y or ¹⁷⁷Lu moved slower (∼30 mm).

In Vitro Stability of ⁹⁰Y-3p-C-NEPA and ¹⁷⁷Lu-3p-C-NEPA

Human serum was purchased from Gemini Bioproducts (#100110). ⁹⁰Y-3p-C-NEPA and ¹⁷⁷Lu-3p-C-NEPA was prepared by the reaction of 3p-C-NEPA (50 μg/50 μL H₂O) with ⁹⁰Y or ¹⁷⁷Lu (5.55 MBq, 150 μCi) in 0.25 M NH₄OAc buffer (pH 5.5), respectively. Completion of radiolabeling was determined by ITLC, and the resulting complexes ⁹⁰Y-3p-C-NEPA and ¹⁷⁷Lu-3p-C-NEPA were directly used for serum stability studies without further purification. ⁹⁰Y-3p-C-NEPA (6.96 MBq, 188 μCi, 130 μL) was added to human serum (660 μL) in a microcentrifuge tube. ¹⁷⁷Lu-3p-C-NEPA (5.40 MBq, 146 μCi, 100 μL) was added to human serum (500 μL) in a microcentrifuge tube. The stability of the radiolabeled complexes in human serum was evaluated at 37 °C for 14 days. The serum stability of the radiolabeled complexes was assessed by measuring the transfer of the radionuclide from each complex to serum proteins using ITLC (acetonitrile/water = 3:2 v/v). A solution of the radiolabeled complex in serum (5–16 μL for ITLC) was withdrawn at the designated time point and evaluated by ITLC. At each of the time points, the percentage of ⁹⁰Y released from each of the radiolabeled complexes into serum was assessed by ITLC. The radiolabeled complex ⁹⁰Y-3p-C-NEPA or ¹⁷⁷Lu-3p-C-NEPA was detected at ∼50 mm from the bottom of the TLC plate, while the unbound radionuclide ⁹⁰Y or ¹⁷⁷Lu moved slower (∼30 mm).

In Vivo Biodistribution Studies

All animal experiments were conducted in accordance with the guidelines established by the Animal Care and Use Committee of the University of Missouri and the Harry S. Truman Memorial Veterans’ Hospital Subcommittee for Animal Studies. Six to eight-week-old CF-1 mice were obtained from Charles River Laboratories and housed one week prior to the studies. An aliquot of ¹⁷⁷Lu-3p-C-NEPA (2.22 MBq, 60 μCi) that was prepared as described above was intravenously injected via the tail vein in phosphate-buffered saline (100 μL). At 1 h, 4 h, and 24 h postinjection, mice were sacrificed, and blood, liver, kidney, muscle, and bone were collected, weighed, and counted in a gamma counter. The radioactivity from each tissue/organ was decay-corrected using a known aliquot of the injected dose, and the percent-injected dose per gram of tissue (% ID/g) and percent-injected dose per organ (% ID/O) was calculated. Values were presented as mean ± SD for each group of three mice.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c03551.

• Copies of TLC chromatograms for assessment of radiolabeling reaction kinetics and serum stability and in vivo biodistribution data (PDF)

The authors declare no competing financial interest.