Improvement of End-of-Synthesis Radiochemical Purity of 177Lu-DOTA-PSMA-Ligands with Alternative Synthesis Approaches: Conversion Upswing and Side-Products Minimization

Abstract

Background: Radiochemical purity is a key criterion for the quality of radiopharmaceuticals used in clinical practice. The joint improvement of analytical methods capable of identifying related radiochemical impurities and determining the actual radiochemical purity, as well as the improvement of synthesis methods to minimize the formation of possible radiochemical impurities, is integral to the implementation of high-tech nuclear medicine procedures. PSMA-targeted radionuclide therapy with lutetium-177 has emerged as an effective treatment option for prostate cancer, and [¹⁷⁷Lu]Lu-PSMA-617 and [¹⁷⁷Lu]Lu-PSMA^I&T have achieved global recognition as viable radiopharmaceuticals. Recently, it was shown that specific radiochemical impurities can form during the synthesis of [¹⁷⁷Lu]Lu-PSMA-617 because of a spontaneous, thermally mediated condensation of the Glu-C(O)-Lys fragment, resulting in the formation of three different cyclic forms (with no affinity for PSMA). During this study, we identified another impurity, a product of detachment of the Glu-CO fragment from PSMA-617, caused by heating. The total content of all four thermally mediated degradation products may reach 9-11% during classical incubation for 30 min at 95 °C, reducing the radiochemical purity to an unacceptable level (albeit with high levels of radiochemical conversion). It is reasonable to assume that the formation of similar impurities is characteristic of all PSMA-specific vectors that contain Glu-C(O)-Lys pharmacophores. Because the formation of these impurities directly depends on the temperature and incubation time, to reduce their content in the reaction mixture at the end of the synthesis, it is necessary to select conditions to achieve a high level of radiochemical conversion for the minimum possible time and/or at the minimum sufficient temperature.

Methods: In this study, using [¹⁷⁷Lu]Lu-PSMA-617 as an example, we evaluated the efficiency of alternative methods of synthesis with microwave heating and co-solvent (ethanol) addition to ensure radiochemical yield and radiochemical purity in the shortest possible time and at the minimum necessary and sufficient synthesis temperature.

Results: Both approaches achieved a significant reduction in the impurities content, while achieving satisfactory synthesis yields in a short time. In addition to improving the synthesis parameters and radiochemical purity, the use of microwave heating and the addition of ethanol reduces the negative influence of other auxiliaries on labeling kinetics. Notably, the addition of ethanol under certain conditions allowed [¹⁷⁷Lu]Lu-PSMA-617 to be synthesized at room temperature for only 10 min. This makes it possible to achieve exceptionally high real radiochemical purity of the preparations, determined only by the quality of the original precursor. The approaches considered in this study can be successfully applied to improve the synthesis process and quality parameters of the finished product, both for known radiopharmaceuticals and for those under development.

Keywords: DOTA; PSMA; [177Lu]Lu-PSMA-617; ethanol; lutetium-177; microwave heating; mixed media; radiochemical purity; radiochemical yield; related impurities.

Figure 1

Radio-HPLC chromatograms of the model sample [¹⁷⁷Lu]Lu-PSMA-617 synthesized at 95 °C for 15 min (with the addition of 10% free [¹⁷⁷Lu]Lu^III), obtained with Method 1. RCP with correction to free lutetim-177 is 94%.

Figure 2

Radio-HPLC chromatograms of the [¹⁷⁷Lu]Lu-PSMA-617 (a) and [¹⁷⁷Lu]Lu-PSMA^I&T (b) samples obtained at different synthesis temperatures.

Figure 3

Results of HPLC analysis of the [¹⁷⁷Lu]Lu-PSMA-617 sample obtained by prolonged intensive heating for 2 h at 120 °C in Method 2 with radiometric (1) and UV (2) detection (220 nm with baseline correction by blank analysis). The volume of the reaction mixture was 1 mL, the amount of PSMA-617 was 100 μg, the activity of ¹⁷⁷Lu was 150 MBq, and the solution contained 0.03 M sodium acetate at pH 4.5.

Figure 4

Structural formulae of PSMA-617 and its thermal-mediated degradation products: b, c, d—derivatives of hydantoin ‘A’, hydantoin ‘B’, and pyroglutamic acid, respectively; a—cleavage product of the Glu-CO fragment.

Figure 5

(a) Kinetics of [¹⁷⁷Lu]Lu-PSMA-617 formation at different pH values (error bars are omitted for clarity); (b) The magnitude of the radiochemical yield after 5 min of incubation of the reaction mixture at different pH values. RCY values are presented as the mean ± SD, n = 3. Synthesis conditions for (a,b) volume—1 mL, buffer (AcONa/Tris) concentration—0.03 mol/L, 200 MBq/mL of ¹⁷⁷Lu, 20 μg of PSMA-617, ^natLuCl₃ added, [Lu]:[PSMA] = 1:10, ambient (24.5 °C) temperature. (c) Kinetics of [¹⁷⁷Lu]Lu-PSMA-617 formation at different pH values (error bars are omitted for clarity); (d) The magnitude of the radiochemical yield after 5 min of incubation of the reaction mixture at different pH values. RCY values are presented as the mean ± SD, n = 3. Synthesis conditions for (c,d): volume—1 mL, buffer (AcONa/Tris) concentration—0.25 mol/L, 500 MBq/mL of ¹⁷⁷Lu, 20 μg of PSMA-617, ^natLuCl₃ added, [Lu]:[PSMA] = 1:2, C[Lu] = 9.6 μmol/L (equivalent~7.4 GBq/mL of ¹⁷⁷Lu), ambient (24.5 °C) temperature.

Figure 6

Effect of incubation duration and heating intensity of the reaction mixture (1 mL, 20 μg of PSMA-617, 0.03 mol/L sodium acetate, 200 MBq of ¹⁷⁷Lu; pH 4.5 ± 0.1) on accumulation of [¹⁷⁷Lu]Lu-PSMA-617 thermodegradation impurities. The data were corrected for radiochemical conversion values (mean ± SD, n = 5).

Figure 7

Effect of reaction mixture volume on the rate of lutetium-177 incorporation into the PSMA-617 structure at buffer concentrations of 0.1–1.0 M (sodium acetate, pH 4.5 ± 0.1) and 70 °C (20 μg of PSMA-617, 200 MBq/mL of ¹⁷⁷Lu, ^natLuCl₃ added, [Lu]:[PSMA] = 1:10). RCY values are presented as the mean ± SD, n = 3.

Figure 8

Effect of temperature (a) and buffering agent concentration (b) on the rate of lutetium-177 incorporation into the structure of PSMA-617. The volume of the reaction mixture was 5 mL, the amount of PSMA-617 was 20 μg, and the activity of ¹⁷⁷Lu was 1000 MBq (molar ratio of [Lu]:[PSMA] = 1:10). RCY values are presented as the mean ± SD, n = 3.

Figure 9

Kinetics of [¹⁷⁷Lu]Lu-PSMA-617 formation under microwave and convection heating (95 °C) in 0.1 mol/L (a) and 1.0 mol/L (b) sodium acetate media (pH 4.5). The volume of the reaction mixture was 5 mL, the amount of PSMA-617 was 20 μg, the activity of ¹⁷⁷Lu was 1000 MBq, ^natLuCl₃ was added (molar ratio [Lu]:[PSMA] = 1:10); (c) RCY of [¹⁷⁷Lu]Lu-PSMA-617 under microwave and convection heating (95 °C) with the addition of 2.5 μmol 2,3-DHBA. The volume of the reaction mixture was 1 mL, the amount of PSMA-617 was 50 μg, the activity of ¹⁷⁷Lu was 700 MBq, 0.03 M sodium acetate (pH 4.5). RCY values are presented as the mean ± SD, n = 5.

Figure 10

(a): Radio-HPLC chromatograms (method 2) of the [¹⁷⁷Lu]Lu-PSMA-617 preparations obtained after 1 min of microwave heating at 95 °C (1), 1 min of convective heating at 95 °C (2), and 30 min of convective heating at 95 °C (3). (b): Corresponding Radio-TLC chromatograms (method 1). The volume of the reaction mixture was 1 mL, the amount of PSMA-617 was 100 μg, the activity of ¹⁷⁷Lu was 4.8 GBq, 0.03 M sodium acetate (pH 4.5), and no quenchers were added.

Figure 11

The effect of ethanol fraction in the reaction mixture on the rate of lutetium-177 incorporation into the PSMA-617 structure ((a) 0–40 vol.% of ethanol; (b) 40–80 vol.% of ethanol). The volume of the reaction mixture was 1 mL, the amount of PSMA-617 was 20 μg, the activity of ¹⁷⁷Lu was 250 MBq, 0.03 M sodium acetate (pH 4.5), ^natLuCl₃ added, [Lu]:[PSMA] = 1:10. The RCY values are presented as the mean for n = 3 (error bars are omitted for clarity).

Figure 12

Dependence of the radiochemical yield of [¹⁷⁷Lu]Lu-PSMA-617 synthesis at ambient temperature on the ethanol and gentisic acid (GA) content in the reaction mixture. The volume of the reaction mixture was 1 mL, the amount of PSMA-617 was 20 μg, the activity of ¹⁷⁷Lu was 250 MBq, 0.03 M sodium acetate (pH 4.5), ^natLuCl₃ added, [Lu]:[PSMA] = 1:10, 45 min incubation at 24.5 °C. RCY values are presented as the mean ± SD, n = 3.

Figure 13

The results of HPLC analysis (Method 2, a—radioactivity, b—UV) of the [¹⁷⁷Lu]Lu-PSMA-617 sample obtained with the addition of 40 vol.% ethanol after 15 min of incubation at ambient (24.5 °C) temperature. The volume of the reaction mixture was 1 mL, the amount of PSMA-617 was 20 μg, the activity of ¹⁷⁷Lu was 250 MBq/mL, ^natLuCl₃ added, [Lu]:[PSMA] = 1:5, 0.03 M sodium acetate, pH 4.5; 2% of thermocyclization (TC) was determined from the radiochromatogram of the preparation.

Figure 14

Kinetics of [¹⁷⁷Lu]Lu-PSMA-617 formation under different conditions: (a) ethanol-free media, different concentrations of sodium acetate, pH 4.5, [Lu]:[PSMA] = 1:10; (b) 40 vol.% of ethanol, different concentrations of sodium acetate, pH 4.5, [Lu]:[PSMA] = 1:10; (c) different ethanol fractions, 0.25 mol/L sodium acetate, pH 4.5, [Lu]:[PSMA] = 1:10; (d) different ethanol fractions, 0.25 mol/L sodium acetate, pH 4.5, [Lu]:[PSMA] = 1:2; the volume of the reaction mixture was 1 mL, and the mixture was incubated at ambient temperature (24.5 °C).

Figure 15

The kinetics of [¹⁷⁷Lu]Lu-PSMA-617 formation (20 vol.% of ethanol, 0.25 mol/L sodium acetate, pH 4.5) at different temperatures in comparison with ethanol-free media. The reaction mixture volume was 1 mL, the activity of ¹⁷⁷Lu was 250 MBq, ^natLuCl₃ was added, and [Lu]:[PSMA] = 1:2. The RCY values are presented as the mean for n = 3 (error bars are omitted for clarity).