Chromatid breaks in cells exposed to low dose irradiation are thought to be initiated by DNA double-strand breaks (DSB), and the frequency of chromatid breaks has been shown to increase in DSB rejoining deficient cells. However, the underlying causes of the wide variation in frequencies of G2 chromatid breaks (or chromatid ‘radiosensitivity') in irradiated T-lymphocytes from different normal individuals and cancer cases are as yet unclear. Here we report evidence that topoisomerase IIα expression level is a factor determining chromatid radiosensitivity. We have exposed the promyelocytic leukaemic cell line (HL60) and two derived variant cell lines (MX1 and MX2) that have acquired resistance to mitoxantrone and low expression of topoisomerase II α, to low doses of γ-radiation and scored the induced chromatid breaks. Chromatid break frequencies were found to be significantly lower in the variant cell lines, compared with their parental HL60 cell line. Rejoining of DSB in the variant cell lines was similar to that in the parental HL60 strain. Our results indicate the indirect involvement of topoisomerase IIα in the formation of radiation-induced chromatid breaks from DSB, and suggest topoisomerase IIα as a possible factor in the inter-individual variation in chromatid radiosensitivity.

One of the most prominent cell populations playing a role in rheumatoid arthritis (RA) is activated fibroblast-like synoviocytes. Among many other proteins, fibroblast-like synoviocytes dominantly express fibroblast activation protein (FAP). Because of the high expression of FAP in arthritic joints, radioimmunoimaging of activated fibroblasts with anti-FAP antibodies might be an attractive noninvasive imaging tool in RA. Methods: SPECT and PET with ¹¹¹In- and ⁸⁹Zr-labeled anti-FAP antibody 28H1 was performed in mice with CIA. The radioactivity uptake in joints was quantified and correlated with arthritis score. Results: Both ¹¹¹In-28H1 and ⁸⁹Zr-28H1 showed high uptake in inflamed joints, being 3-fold higher than that of the irrelevant isotype-matched control antibody DP47GS, clearly indicating specific accumulation of 28H1. Uptake of ¹¹¹In-28H1 ranged from 2.2 percentage injected dose per gram (%ID/g) in noninflamed joints to 32.1 %ID/g in severely inflamed joints. DP47GS accumulation ranged from 1.6 %ID/g in noninflamed tissue to 12.0 %ID/g in severely inflamed joints. Uptake of 28H1 in inflamed joints correlated with arthritis score (Spearman ρ, 0.69; P < 0.0001) and increased with severity of arthritis. Conclusion: SPECT/CT imaging with the anti-FAP antibody ¹¹¹In-28H1 specifically visualized arthritic joints with high resolution, and tracer accumulation correlated with the severity of the inflammation in murine experimental arthritis. Background uptake of the radiolabeled antibody was low, resulting in excellent image quality. ⁸⁹Zr-28H1 was less favorable for RA imaging because of an elevated bone uptake of ⁸⁹Zr. Future studies will focus on the potential role of 28H1 as a tool to monitor therapy response early on.

The clinical impact and accessibility of ⁶⁸Ga tracers for the prostate-specific membrane antigen (PSMA) and other targets would be greatly enhanced by the availability of a simple, 1-step kit-based labeling process. Radiopharmacy staff are accustomed to such procedures in the daily preparation of ^99mTc radiopharmaceuticals. Currently, chelating agents used in ⁶⁸Ga radiopharmaceuticals do not meet this ideal. The aim of this study was to develop and evaluate preclinically a ⁶⁸Ga radiotracer for imaging PSMA expression that could be radiolabeled simply by addition of ⁶⁸Ga generator eluate to a cold kit. Methods: A conjugate of a tris(hydroxypyridinone) (THP) chelator with the established urea-based PSMA inhibitor was synthesized and radiolabeled with ⁶⁸Ga by adding generator eluate directly to a vial containing the cold precursors THP-PSMA and sodium bicarbonate, with no further manipulation. It was analyzed after 5 min by instant thin-layer chromatography and high-performance liquid chromatography. The product was subjected to in vitro studies to determine PSMA affinity using PSMA-expressing DU145-PSMA cells, with their nonexpressing analog DU145 as a control. In vivo PET imaging and ex vivo biodistribution studies were performed in mice bearing xenografts of the same cell lines, comparing ⁶⁸Ga-THP-PSMA with ⁶⁸Ga-HBED-CC-PSMA. Results: Radiolabeling was complete (>95%) within 5 min at room temperature, showing a single radioactive species by high-performance liquid chromatography that was stable in human serum for more than 6 h and showed specific binding to PSMA-expressing cells (concentration giving 50% inhibition of 361 ± 60 nM). In vivo PET imaging showed specific uptake in PSMA-expressing tumors, reaching 5.6 ± 1.2 percentage injected dose per cubic centimeter at 40–60 min and rapid clearance from blood to kidney and bladder. The tumor uptake, biodistribution, and pharmacokinetics were not significantly different from those of ⁶⁸Ga-HBED-CC-PSMA except for reduced uptake in the spleen. Conclusion: ⁶⁸Ga-THP-PSMA has equivalent imaging properties but greatly simplified radiolabeling compared with other ⁶⁸Ga-PSMA conjugates. THP offers the prospect of rapid, simple, 1-step, room-temperature syringe-and-vial radiolabeling of ⁶⁸Ga radiopharmaceuticals.

Despite its desirable half-life and low energy Auger electrons that travel further than for other radionuclides, 67Ga has been neglected as a therapeutic radionuclide. Here, 67Ga is compared with Auger electron emitter 111In as a potential therapeutic radionuclide. Plasmid pBR322 studies allowed direct comparison between 67Ga and 111In (1MBq) in causing DNA damage, including the effect of chelators (EDTA and DTPA) and the effects of a free radical scavenger (DMSO). The cytotoxicity of internalized (by means of delivery in the form of oxine complexes) and non-internalized 67Ga and 111In was measured in DU145 prostate cancer cells after a one-hour incubation using cell viability (trypan blue) and clonogenic studies. MDA-MB-231 and HCC1954 cells were also used. Plasmid DNA damage was caused by 67Ga and was comparable to that caused by 111In; it was reduced in the presence of EDTA, DTPA and DMSO. The A50 values (internalized activity of oxine complexes per cell required to kill 50% of cells) as determined by trypan blue staining was 1.0Bq/cell for both 67Ga and 111In; the A50 values determined by clonogenic assay were 0.7Bq/cell and 0.3Bq/cell for 111In and 67Ga respectively. At the concentrations required to achieve these uptake levels, non-internalized 67Ga and 111In caused no cellular toxicity. Qualitatively similar results were found for MDA-MB-231 and HCC1954 cells. 67Ga causes as much damage as 111In to plasmid DNA in solution and shows similar toxicity as 111In at equivalent internalized activity per cell. 67Ga therefore deserves further evaluation for radionuclide therapy. The data presented here is at the basic level of science. If future in vivo and clinical studies are successful, 67Ga could become a useful radionuclide with little healthy tissue toxicity in the arsenal of weapons for treating cancer.

Two new bifunctional tris(hydroxypyridinone) (THP) chelators designed specifically for rapid labeling with ⁶⁸Ga have been synthesized, each with pendant isothiocyanate groups and three 1,6-dimethyl-3-hydroxypyridin-4-one groups. Both compounds have been conjugated with the primary amine group of a cyclic integrin targeting peptide, RGD. Each conjugate can be radiolabeled and formulated by treatment with generator-produced ⁶⁸Ga³⁺ in over 95% radiochemical yield under ambient conditions in less than 5 min, with specific activities of 60–80 MBq nmol^–1. Competitive binding assays and in vivo biodistribution in mice bearing U87MG tumors demonstrate that the new ⁶⁸Ga³⁺-labeled THP peptide conjugates retain affinity for the α_vβ₃ integrin receptor, clear within 1–2 h from circulation, and undergo receptor-mediated tumor uptake in vivo. We conclude that bifunctional THP chelators can be used for simple, efficient labeling of ⁶⁸Ga biomolecules under mild conditions suitable for peptides and proteins.

Molecular radiotherapy using ¹⁷⁷Lu-DOTATATE is a most effective treatment for somatostatin receptor–expressing neuroendocrine tumors. Despite its frequent and successful use in the clinic, little or no radiobiologic considerations are made at the time of treatment planning or delivery. On positive uptake on octreotide-based PET/SPECT imaging, treatment is usually administered as a standard dose and number of cycles without adjustment for peptide uptake, dosimetry, or radiobiologic and DNA damage effects in the tumor. Here, we visualized and quantified the extent of DNA damage response after ¹⁷⁷Lu-DOTATATE therapy using SPECT imaging with ¹¹¹In-anti-γH2AX-TAT. This work was a proof-of-principle study of this in vivo noninvasive biodosimeter with β-emitting therapeutic radiopharmaceuticals. Methods: Six cell lines were exposed to external-beam radiotherapy (EBRT) or ¹⁷⁷Lu-DOTATATE, after which the number of γH2AX foci and the clonogenic survival were measured. Mice bearing CA20948 somatostatin receptor–positive tumor xenografts were treated with ¹⁷⁷Lu-DOTATATE or sham-treated and coinjected with ¹¹¹In-anti-γH2AX-TAT, ¹¹¹In-IgG-TAT control, or vehicle. Results: Clonogenic survival after external-beam radiotherapy was cell-line–specific, indicating varying levels of intrinsic radiosensitivity. Regarding in vitro cell lines treated with ¹⁷⁷Lu-DOTATATE, clonogenic survival decreased and γH2AX foci increased for cells expressing high levels of somatostatin receptor subtype 2. Ex vivo measurements revealed a partial correlation between ¹⁷⁷Lu-DOTATATE uptake and γH2AX focus induction between different regions of CA20948 xenograft tumors, suggesting that different parts of the tumor may react differentially to ¹⁷⁷Lu-DOTATATE irradiation. Conclusion: ¹¹¹In-anti-γH2AX-TAT allows monitoring of DNA damage after ¹⁷⁷Lu-DOTATATE therapy and reveals heterogeneous damage responses.