The radiolytic degradation of vector molecules is a major factor affecting the shelf life of therapeutic radiopharmaceuticals. The development of time-stable dosage forms of radiopharmaceuticals is the key to their successful implementation in clinical practice. Using [¹⁷⁷Lu]Lu-PSMA-617 molecule as an example, the time dependence of the change in radiochemical purity (RCP, %) under radiolysis conditions was studied. The dependence of [¹⁷⁷Lu]Lu-PSMA-617 radiolysis on parameters such as time, radionuclide activity, buffer agent concentration, precursor amount, and preparation volume was evaluated. It was shown that the absorbed dose was the dominant factor influencing the RCP. The RCP value is inversely proportional to the absorbed dose in the [¹⁷⁷Lu]Lu-PSMA-617 preparation and has an exponential dependence. The lutetium-177 dose factor ψ (Gy·mL·MBq⁻¹) and PSMA-617 concentration-dependent dose constant κ (Gy⁻¹) were evaluated for absorbed dose estimation via computer modeling, chemical dosimetry, and radiochemical purity monitoring under various conditions. The further refinement and application of the dependencies found can be useful for predicting the RCP value at the stage of optimizing the composition of the finished dosage form of therapeutic radiopharmaceuticals. The influence of the buffer agent (sodium acetate) concentration on [¹⁷⁷Lu]Lu-PSMA-617 radiolytic degradation was shown and should be considered both when developing a dosage form, and when comparing the results of independent studies. The effectiveness of the addition of various stabilizing agents, such as DMSA, cysteine, gentisic acid, vanillin, methionine, adenine, dobesilic acid, thymine, uracil, nicotinamide, meglumine, and mannitol, in suppressing the effects of radiolysis was evaluated.

The folate receptor (FR) is a promising cell membrane-associated target for molecular imaging and radionuclide therapy of cancer (FR-α) and potentially also inflammatory diseases (FR-β) through use of folic acid-based radioconjugate. FR is often overexpressed by cells of epithelial tumors, including tumors of ovary, cervix, endometrium, lungs, kidneys, etc. In healthy tissues, FR can be found in small numbers by the epithelial cells, mainly in the kidneys. Extremely high undesired accumulation of the folate radioconjugates in the renal tissue is a main drawback of FR-targeting concept. In the course of this work, we aimed to reduce the undesirable accumulation of folate radioconjugates in the kidneys by introducing a histidine/glutamic acid tag into their structure. Two folic acid based compounds were synthesized: NODAGA-1,4-butanediamine-folic acid (FA-I, as control) and NODAGA-[Lys-(HE)₂]-folic acid (FA-II) which contains a (His-Glu)₂ fragment. In vitro studies with FR (+) cells (KB and others) showed that both compounds have specificity for FR. Introduction of (HE)₂-tag does not affect FR binding ability of the conjugates. In vivo biodistribution studies with normal laboratory animals, as well as with KB tumor bearing animals, were carried out. The results showed that introduction of the (HE)₂ tag into the structure of folate radioconjugates can significantly reduce the accumulation of these compounds in non-target tissues and important organs (the accumulation in the kidneys is reduced 2–4 times), leaving the accumulation in tumor at least at the same level, and even increasing it.

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.

Особенности и практические аспекты определения радиохимической чистоты рецепторспецифичных препаратов лютеция-177 на примере [ 177 Lu]Lu–PSMA-617

In recent years, folate receptors have been considered as a target for radiopharmaceutical drugs. For this purpose, new, previously unexplored com-pounds based on folic acid were synthesized. Objective. Study of the accumulation of gallium-68-labeled folic acid conjugates in various etiologies inflammation foci. Material and methods. The objects of the research were folic acid derivatives labeled with gallium-68. Systemic juvenile idiopathic arthritis was chosen as a pathology model. Acute inflammatory processes of soft tissues of septic and aseptic etiology were selected as differential models. Results. The results obtained in the study showed a significantly elevated level of accumulation in the juvenile idiopathic arthritis focus compared to healthy rat paws and accumulation in the foci of differential models of the inflammatory process, which confirms the macrophage-mediated pathway of accumulation of the studied compounds. Conclusion. The data obtained allow us to conclude about the diagnostic potential of radiolabeled conjugates based on folic acid in the radionuclide di-agnosis of rheumatoid and other diseases characterized by a pronounced macrophage immune response.

Preclinical models play a critical role in the development of PSMA-targeted radiopharmaceuticals for prostate cancer. However, many existing models have practical limitations, including slow tumor growth, low engraftment rates, and restricted availability, and all human PSMA-positive lines are confined to immunodeficient hosts. We developed and characterized a novel PSMA-expressing transgenic cell line, CT26-PSMA, as a practical tool for preclinical screening of PSMA-targeting agents. The CT26-PSMA cell line was established by stable transfection of the murine colon carcinoma CT26 cell line with human PSMA using the Sleeping Beauty transposon system. PSMA expression was confirmed by RT-qPCR (reverse transcription quantitative polymerase chain reaction), flow cytometry, and radioligand saturation binding on intact cells. Two [⁶⁸Ga]Ga-labeled radioconjugates—the well-established PSMA-617 and a newly synthesized conjugate (Conjugate-1)—were used to validate the functionality of the model through in vitro binding, uptake and internalization studies, and through ex vivo biodistribution in CT26-PSMA tumor-bearing athymic male nu/nu mice. The CT26-PSMA cell line demonstrated high and stable PSMA expression, with approximately 95% of cells expressing the biomarker and no measurable loss over 16 passages in antibiotic-free medium. Saturation binding gave a receptor density of ∼3.5 × 10⁶ sites per cell, approximately four-fold higher than that of LNCaP cells (∼0.8 × 10⁶), with dissociation constants that were indistinguishable between the two radioconjugates and between the two cell lines (K_d 9.0–11.6 nM). Subcutaneous tumors reached ~300 mm³ within 8–10 days of inoculation, with a take rate of 10/10 versus 1/10 for LNCaP (Fisher’s exact test, p = 1.2 × 10⁻⁴). Both radiotracers showed saturable, 2-PMPA-blockable binding and uptake in CT26-PSMA cells, confirming the functional activity of the recombinant receptor. Biodistribution studies revealed accumulation of both conjugates in CT26-PSMA tumors, with generally comparable tumor-to-background profiles. The CT26-PSMA cell line represents a robust, rapid, and reproducible platform for preclinical evaluation of PSMA-targeting radiopharmaceuticals, and its murine BALB/c origin permits engraftment in immunocompetent or minimally immunosuppressed hosts, whereas existing human PSMA-positive lines do not. It is intended as a screening platform rather than as a model of prostate cancer biology. The validation data obtained with [⁶⁸Ga]Ga-labelled conjugates confirm the suitability of this cell line for future studies of PSMA-directed compounds.