Cellular lethal damage of 64Cu incorporated in mammalian genome evaluated with Monte Carlo methods.

Purpose: Targeted Radionuclide Therapy (TRT) with Auger Emitters (AE) is a technique that allows targeting specific sites on tumor cells using radionuclides. The toxicity of AE is critically dependent on its proximity to the DNA. The aim of this study is to quantify the DNA damage and radiotherapeutic potential of the promising AE radionuclide copper-64 (64Cu) incorporated into the DNA of mammalian cells using Monte Carlo track-structure simulations. Methods: A mammalian cell nucleus model with a diameter of 9.3 µm available in TOPAS-nBio was used. The cellular nucleus consisted of double-helix DNA geometrical model of 2.3 nm diameter surrounded by a hydration shell with a thickness of 0.16 nm, organized in 46 chromosomes giving a total of 6.08 giga base-pairs (DNA density of 14.4 Mbp/µm3). The cellular nucleus was irradiated with monoenergetic electrons and radiation emissions from several radionuclides including 111In, 125I, 123I, and 99mTc in addition to 64Cu. For monoenergetic electrons, isotropic point sources randomly distributed within the nucleus were modeled. The radionuclides were incorporated in randomly chosen DNA base pairs at two positions near to the central axis of the double-helix DNA model at (1) 0.25 nm off the central axis and (2) at the periphery of the DNA (1.15 nm off the central axis). For all the radionuclides except for 99mTc, the complete physical decay process was explicitly simulated. For 99mTc only total electron spectrum from published data was used. The DNA Double Strand Breaks (DSB) yield per decay from direct and indirect actions were quantified. Results obtained for monoenergetic electrons and radionuclides 111In, 125I, 123I, and 99mTc were compared with measured and calculated data from the literature for verification purposes. The DSB yields per decay incorporated in DNA for 64Cu are first reported in this work. The therapeutic effect of 64Cu (activity that led 37% cell survival after two cell divisions) was determined in terms of the number of atoms incorporated into the nucleus that would lead to the same DSBs that 100 decays of 125I. Simulations were run until a 2% statistical uncertainty (1 standard deviation) was achieved. Results: The behavior of DSBs as a function of the energy for monoenergetic electrons was consistent with published data, the DSBs increased with the energy until it reached a maximum value near 500 eV followed by a continuous decrement. For 64Cu, when incorporated in the genome at evaluated positions (1) and (2), the DSB were 0.171 ± 0.003 and 0.190 ± 0.003 DSB/decay, respectively. The number of initial atoms incorporated into the genome (per cell) for 64Cu that would cause a therapeutic effect was estimated as 3,107 ± 28, that corresponds to an initial activity of 47.1 ± 0.4 × 10-3 Bq. Conclusion: Our results showed that TRT with 64Cu has comparable therapeutic effects in cells as that of TRT with radionuclides currently used in clinical practice.

Preclinical Efficacy of a PARP-1 Targeted Auger-Emitting Radionuclide in Prostate Cancer.

There is an unmet need for better therapeutic strategies for advanced prostate cancer. Poly (ADP-ribose) polymerase-1 (PARP-1) is a chromatin-binding DNA repair enzyme overexpressed in prostate cancer. This study evaluates whether PARP-1, on account of its proximity to the cell's DNA, would be a good target for delivering high-linear energy transfer Auger radiation to induce lethal DNA damage in prostate cancer cells. We analyzed the correlation between PARP-1 expression and Gleason score in a prostate cancer tissue microarray. A radio-brominated Auger emitting inhibitor ([77Br]Br-WC-DZ) targeting PARP-1 was synthesized. The ability of [77Br]Br-WC-DZ to induce cytotoxicity and DNA damage was assessed in vitro. The antitumor efficacy of [77Br]Br-WC-DZ was investigated in prostate cancer xenograft models. PARP-1 expression was found to be positively correlated with the Gleason score, thus making it an attractive target for Auger therapy in advanced diseases. The Auger emitter, [77Br]Br-WC-DZ, induced DNA damage, G2-M cell cycle phase arrest, and cytotoxicity in PC-3 and IGR-CaP1 prostate cancer cells. A single dose of [77Br]Br-WC-DZ inhibited the growth of prostate cancer xenografts and improved the survival of tumor-bearing mice. Our studies establish the fact that PARP-1 targeting Auger emitters could have therapeutic implications in advanced prostate cancer and provides a strong rationale for future clinical investigation.

How to explain the sensitivity of DNA double-strand breaks yield to 125I position?

PURPOSE: Auger emitters exhibit interesting features due to their emission of a cascade of short-range Auger electrons. Maximum DNA breakage efficacy is achieved when decays occur near DNA. Studies of double-strand breaks (DSBs) yields in plasmids revealed cutoff distances from DNA axis of 10.5 Å-12 Å, beyond which the mechanism of DSBs moves from direct to indirect effects, and the yield decreases rapidly. Some authors suggested that the average energy deposited in a DNA cylinder could explain such cutoffs. We aimed to study this hypothesis in further detail. MATERIALS AND METHODS: Using the Monte Carlo code CELLDOSE, we investigated the influence of the 125I atom position on energy deposits and absorbed doses per decay not only in a DNA cylinder, but also in individual strands, each modeled as 10 spheres encompassing the fragility sites for phosphodiester bond cleavage. RESULTS: The dose per decay decreased much more rapidly for a sphere in the proximal strand than for the DNA cylinder. For example, when moving the 125I source from 10.5 Å to 11.5 Å, the average dose to the sphere dropped by 43%, compared to only 13% in the case of the cylinder. CONCLUSIONS: Explaining variations in DSBs yields with 125I position should consider the probability of inducing damage in the proximal strand (nearest to the 125I atom). The energy received by fragility sites in this strand is highly influenced by the isotropic (4π) emission of 125I low-energy Auger electrons. The positioning of Auger emitters for targeted radionuclide therapy can be envisioned accordingly.

An approach to assessing the contribution of the high LET effect in strategies for Auger endoradiotherapy.

Purpose: The interest in exploiting Auger emitters in cancer therapy stems from their high linear energy transfer (LET)-type radiation damage to DNA. However, the design of Auger-emitter labeled vehicles that target the Auger cascade specifically to the DNA of tumour cells is challenging. Here we suggest a possible approach to evaluate tumour-targeting Auger-labeled conjugates by assessing the impact of a radioprotector known to be effective in protecting from low LET radiation, but not high LET radiation. Given some similarity between the energy spectrum of Auger electrons and that of secondary electrons from soft X-rays, we report the results of radioprotection experiments with 25 kVp X-rays. Materials and methods: Clonogenic survival curves for cultured human keratinocytes were established for three different irradiation conditions: 137Cs γ-rays, 25 kVp X-rays and 320 kVp X-rays, and the effect of including a new radioprotector, denoted "2PH", was investigated.Results: The extent of radioprotection by 2PH was comparable for all radiation conditions, although RBE was higher (about 1.7) for soft X-rays. Conclusions: Radioprotectors like 2PH will help to evaluate Auger endoradiotherapy strategies, by determining the relative contributions of the high-LET effects (not protected), compared to other components, such as Auger electrons not effectively targeted to DNA.

Improved radiosynthesis of 123I-MAPi, an auger theranostic agent.

PURPOSE: 123I-MAPi, a novel PARP1-targeted Auger radiotherapeutic has shown promising results in pre-clinical glioma model. Currently, 123I-MAPi is synthesized using multistep synthesis that results in modest yields and low molar activities (MA) that limits the ability to translate this technology for human studies where high doses are administered. Therefore, new methods are needed to synthesize 123I-MAPi in high activity yields (AY) and improved MA to facilitate clinical translation and multicenter trials. MATERIALS AND METHODS: 123I-MAPi was prepared in a single step via 123I-iododetannylation of the corresponding tributylstannane precursor. In vitro internalization assay, subcellular fractionation and confocal microscopy where used to evaluate the performance of 123I-MAPi in a small cell lung cancer model. RESULTS: 123I-MAPi was synthesized in a single step from the corresponding stannane precursor in AY of 45 ± 2% and MA of 11.8 ± 4.8 GBq µmol-1. In vitro in LX22 cells showed rapid internalization (5 min) with accumulation found predominantly in the membrane, nucleus and chromatin of the cell as determined by subcellular fractionation. CONCLUSIONS: Here, we have developed an improved radiosynthesis of 123I-MAPi, an Auger theranostic agent. This process was achieved using a single step, 123I-iododestannylation reaction from the corresponding stannane precursor in good AY and MA. 123I-MAPi was evaluated in vitro in a small cell lung cancer model with high PARP expression, rapid internalization and high nuclear uptake shown.

Cs-131 as an experimental tool for the investigation and quantification of the radiotoxicity of intracellular Auger decays in vitro.

PURPOSE: In this work, we set out to provide an experimental setup, using Cs-131, with associated dosimetry for studying relative biological effectiveness (RBE) of Auger emitters. MATERIAL AND METHODS: Cs-131 decays by 100% electron capture producing K- (9%) and L- (80%) Auger electrons with mean energies of 26 keV and 3.5 keV, respectively, plus ≈ 9.4 very low energy electrons (<0.5 keV) per decay. Cs-131 accumulates in the cells through the Na+/K+-ATPase. By this uptake mechanism and the alkali chemistry of Cs+, we argue for its intracellular homogeneous distribution. Cs-131 was added to the cell culture medium of HeLa and V79 Cells. The bio-kinetics of Cs-131 (uptake, release, intracellular distribution) was examined by measuring its intracellular activity concentration over time. Taking advantage of the 100% confluent cellular monolayer, we developed a new and robust dosimetry that is entrusted to a quantity called SC-value. RESULTS: The SC-values evaluated in the cell nucleus are almost independent of the nuclear size and geometry. We obtained dose-rate controlled RBE-values for intracellular Cs-131 decay. Using the γH2AX assay, the RBE was 1 for HeLa cells. Using the clonogenic cell survival, it was 3.9 for HeLa cells and 3.2 for V79 cells. CONCLUSION: This experimental setup and dosimetry provides reliable RBE-values for Auger emitters in various cell lines.

Experimental study of α-particle induced reactions on natural erbium for the production of Auger-emitters 167Tm, 165Er and 169Yb.

The cross sections for nuclear reactions natEr(α,x) were measured in the energy range 60 â†’ 10 MeV using the stacked-foil technique. The experiments were carried out in a wider energy range in comparison with previous works. The results are consistent with other studies and modeling using TENDL-2019. The 167Tm yield was 5.4 MBq/µAh in the range 60 â†’ 30 MeV, and the main long-lived impurity is 168Tm (0.78% in terms of activity). The 165Tm yield is 4.6 MBq/µAh (60 â†’ 40 MeV). 169Yb is formed with a yield of 1.0 MBq/µAh in the energy range 60 â†’ 20 MeV.

Microdosimetry modeling with auger emitters in generalized cell geometry.

A microdosimetry model was developed for the prediction of cell viability for irregular non-spherical cells that were irradiated by low energy, short range auger electrons. Measured cell survival rates for LNCaP prostate cancer were compared to the computational results for the radioisotopes177Lu and161Tb (conjugated to PSMA). The cell geometries used for the computations were derived directly from the cell culture images. A general computational approach was developed to handle arbitrary cell geometries, based on distance probability distribution functions (PDFs) derived from basic image processing. The radiation calculations were done per coarse grained PDF bin to reduce computation time, rather than on a pixel/voxel basis. The radiation dose point kernels over the full electron spectrum were derived using Monte Carlo simulations for energies below 50 eV to account for the propagation of auger electrons over length scales at and below a cellular radius. The relative importance of short range auger electrons were evaluated between the two nuclide types. The microdosimetry results were consistent with the cell viability measurements, and it was found that161Tb was more efficient than177Lu primarily due to the short range auger electrons. We foresee that imaging based microdosimetry can be used to evaluate the relative therapeutic effect between various nuclide candidates.

Synthesis and Biological Evaluation of 99mTc(I) Tricarbonyl Complexes Dual-Targeted at Tumoral Mitochondria.

For effective Auger therapy of cancer, the Auger-electron emitters must be delivered to the tumor cells in close proximity to a radiosensitive cellular target. Nuclear DNA is considered the most relevant target of Auger electrons to have augmented radiotoxic effects and significant cell death. However, there is a growing body of evidence that other targets, such as the mitochondria, could be relevant subcellular targets in Auger therapy. Thus, we developed dual-targeted 99mTc(I) tricarbonyl complexes containing a triphenylphosphonium (TPP) moiety to promote accumulation of 99mTc in the mitochondria, and a bombesin peptide to provide specificity towards the gastrin releasing peptide receptor (GRPr) overexpressed in prostate cancer cells. The designed dual-targeted complex, 99mTc-TPP-BBN, is efficiently internalized by human prostate cancer PC3 cells through a specific GRPr-mediated mechanism of uptake. Moreover, the radioconjugate provided an augmented accumulation of 99mTc in the mitochondria of the target tumor cells, most probably following its intracellular cleavage by cathepsin B. In addition, 99mTc-TPP-BBN showed an enhanced ability to reduce the survival of PC3 cells, in a dose-dependent manner.

Radiobiological and dosimetric assessment of DNA-intercalated 99mTc-complexes bearing acridine orange derivatives.

BACKGROUND: Recently, a new family of 99mTc(I)-tricarbonyl complexes bearing an acridine orange (AO) DNA targeting unit and different linkers between the Auger emitter (99mTc) and the AO moiety was evaluated for Auger therapy. Among them, 99mTc-C3 places the corresponding radionuclide at a shortest distance to DNA and produces important double strand breaks (DSB) yields in plasmid DNA providing the first evidence that 99mTc can efficiently induce DNA damage when well positioned to the double helix. Here in, we have extended the studies to human prostate cancer PC3 cells using the 99mTc-C3 and 99mTc-C5 complexes, aiming to assess how the distance to DNA influences the radiation-induced biological effects in this tumoral cell line, namely, in which concerns early and late damage effects. RESULTS: Our results highlight the limited biological effectiveness of Auger electrons, as short path length radiation, with increasing distances to DNA. The evaluation of the radiation-induced biological effects was complemented with a comparative microdosimetric study based on intracellular dose values. The comparative study, between MIRD and Monte Carlo (MC) methods used to assess the cellular doses, revealed that efforts should be made in order to standardize the bioeffects modeling for DNA-incorporated Auger electron emitters. CONCLUSIONS: 99mTc might not be the ideal radionuclide for Auger therapy but can be useful to validate the design of new classes of Auger-electron emitting radioconjugates. In this context, our results highlight the crucial importance of the distance of Auger electron emitters to the target DNA and encourage the development of strategies for the fine tuning of the distance to DNA for other medical radionuclides (e.g., 111In or 161Tb) in order to enhance their radiotherapeutic effects within the Auger therapy of cancer.

The quest to exploit the Auger effect in cancer radiotherapy - a reflective review.

To identify the emergence of the recognition of the potential of the Auger effect for clinical application, and after tracing the salient milestones towards that goal, to evaluate the status quo and future prospects. It was not until 40 years after the discovery of Auger electrons, that the availability of radioactive DNA precursors enabled the biological power, and the clinical potential, of the Auger effect to be appreciated. Important milestones on the path to clinical translation have been identified and reached, but hurdles remain. Nevertheless the potential is still evident, and there is reasonable optimism that the goal of clinical translation is achievable.

A stochastic cascade model for Auger-electron emitting radionuclides.

To benchmark a Monte Carlo model of the Auger cascade that has been developed at the Australian National University (ANU) against the literature data. The model is applicable to any Auger-electron emitting radionuclide with nuclear structure data in the format of the Evaluated Nuclear Structure Data File (ENSDF). Schönfeld's algorithms and the BrIcc code were incorporated to obtain initial vacancy distributions due to electron capture (EC) and internal conversion (IC), respectively. Atomic transition probabilities were adopted from the Evaluated Atomic Data Library (EADL) for elements with atomic number, Z = 1-100. Atomic transition energies were evaluated using a relativistic Dirac-Fock method. An energy-restriction protocol was implemented to eliminate energetically forbidden transitions from the simulations. Calculated initial vacancy distributions and average energy spectra of 123I, 124I, and 125I were compared with the literature data. In addition, simulated kinetic energy spectra and frequency distributions of the number of emitted electrons and photons of the three iodine radionuclides are presented. Some examples of radiation spectra of individual decays are also given. Good agreement with the published data was achieved except for the outer-shell Auger and Coster-Kronig transitions. Nevertheless, the model needs to be compared with experimental data in a future study.

Strand breakage by decay of DNA-bound 124I provides a basis for combined PET imaging and Auger endoradiotherapy.

Purpose DNA ligands labelled with 125I induce cytotoxic DNA double-strand breaks (DSB), suggesting a potential for Auger endoradiotherapy. Since the 60-day half-life of 125I is suboptimal for therapy, we have investigated another Auger-emitter 124I, with shorter half-life (4.18 days), and the additional feature of positron-emission, enabling positron emission tomography (PET) imaging. The purpose of this study was to compare the two radionuclides on the basis of DNA DSB per decay. Materials and methods Using a 124I- (or 125I)-labelled minor groove binding DNA ligand, we investigated DNA breakage using the plasmid DNA assay. Biodistribution of the conjugate of the labelled ligand with transferrin was investigated in nude mice bearing a K562 human lymphoma xenograft. Results The probability of DSB per decay was 0.58 and 0.85 for 124I and 125I, respectively, confirming the therapeutic potential of the former. The crystal structure of the ligand DNA complex shows the iodine atom deep within the minor groove, consistent with the high efficiency of induced damage. Biodistribution studies, including PET imaging, showed distinctive results for the conjugate, compared to the free ligand and transferrin, consistent with receptor-mediated delivery of the ligand. Conclusions Conjugation of 124I-labelled DNA ligands to tumor targeting peptides provides a feasible strategy for Auger endoradiotherapy, with the advantage of monitoring tumor targeting by PET imaging.