Evaluation of 209At as a theranostic isotope for 209At-radiopharmaceutical development using high-energy SPECT.

The development of alpha-emitting radiopharmaceuticals using 211At requires quantitative determination of the time-dependent nature of the 211At biodistribution. However, imaging-based methods for acquiring this information with 211At have not found wide-spread use because of its low abundance of decay emissions suitable for external detection. In this publication we demonstrate the theranostic abilities of the 211At/209At isotope pair and present the first-ever 209At SPECT images. The VECTor microSPECT/PET/CT scanner was used to image 209At with a collimator suitable for the 511 keV annihilation photons of PET isotopes. Data from distinct photopeaks of the 209At energy spectrum (195 keV (22.6%), 239 keV (12.4 %), 545 keV (91.0 %), a combined 782/790 keV peak (147 %), and 209Po x-rays (139.0 %)) were independently evaluated for use in image reconstructions using Monte Carlo (GATE) simulations and phantom studies. 209At-imaging in vivo was demonstrated in a healthy mouse injected with 10 MBq of free [209At]astatide. Image-based measurements of 209At uptake in organs of interest-acquired in 5 min intervals-were compared to ex vivo gamma counter measurements of the same organs. Simulated and measured data indicated that-due to the large amount of scatter from high energy (>750 keV) gammas-reconstructed images using the x-ray peak outperformed those obtained from other peaks in terms of image uniformity and spatial resolution, determined to be <0.85 mm. 209At imaging using the x-ray peak revealed a biodistribution that matched the known distribution of free astatide, and in vivo image-based measurements of 209At uptake in organs of interest matched ex vivo measurements within 10%. We have acquired the first 209At SPECT images and demonstrated the ability of quantitative SPECT imaging with 209At to accurately determine astatine biodistributions with high spatial and temporal resolution.

In vitro characterization of 211 At-labeled antibody A33--a potential therapeutic agent against metastatic colorectal carcinoma.

The humanized antibody A33 binds to the A33 antigen, expressed in 95% of primary and metastatic colorectal carcinomas. The restricted pattern of expression in normal tissue makes this antigen a possible target for radioimmunotherapy of colorectal micrometastases. In this study, the A33 antibody was labeled with the therapeutic nuclide (211)At using N-succinimidyl para-(tri-methylstannyl)benzoate (SPMB). The in vitro characteristics of the (211)At-benzoate-A33 conjugate ((211)At-A33) were investigated and found to be similar to those of (125)I-benzoate-A33 ((125)I-A33) in different assays. Both conjugates bound with high affinity to SW1222 cells (K(d) = 1.7 +/- 0.2 nM, and 1.8 +/- 0.1 nM for (211)At-A33 and (125)I-A33, respectively), and both showed good intracellular retention (70% of the radioactivity was still cell associated after 20 hours). The cytotoxic effect of (211)At-A33 was also confirmed. After incubation with (211)At-A33, SW1222 cells had a survival of approximately 0.3% when exposed to some 150 decays per cell (DPC). The cytotoxic effect was found to be dose-dependent, as cells exposed to only 56 DPC had a survival of approximately 5%. The (211)At-A33 conjugate shows promise as a potential radioimmunotherapy agent for treatment of micrometastases originating from colorectal carcinoma.

The development of A [211At]-astatinated endoradiotherapeutic drug: Part I. Localization by alpha-particle autoradiography in a murine tumor model.

Alpha-particle track autoradiography has been used to define the in vivo cellular and intracellular distribution of radioactivity from the potential high linear energy transfer endoradiotherapeutic drug, 6-[211At]-astato-2-methyl-1,4-naphthoquinol bis(diphosphate) in tumor and relevant critical normal tissues of mice bearing a transplanted murine rectal carcinoma. A strikingly selective uptake of this compound into tumor cells, particularly into specific tumor cell nuclei, has been demonstrated. Its localization in certain tumor cells appears to depend on the presence of an onco-product, in this case an alkaline phosphatase isoenzyme, which is synthesized in some tumor cells and to which the compound targets. In curable tumors, it selectively concentrates in cells which may be regarded as tumor stem cells. There is low uptake into normal cells, particularly those in bone marrow, colon, and lung, where its sequestration is mainly extranuclear.

Vascular-targeted radioimmunotherapy with the alpha-particle emitter 211At.

Astatine-211, an alpha-particle emitter, was employed in a model system for vascular-targeted radioimmunotherapy of small tumors in mouse lung to compare its performance relative to other radioisotopes in the same system. Astatine-211 was coupled to the lung blood vessel-targeting monoclonal antibody 201B with N-succinimidyl N-(4-[211At]astatophenethyl) succinamate linker. Biodistribution data showed that the conjugate delivered 211At to the lung (260-418% ID/g), where it remained with a biological half-time of about 30 h. BALB/c mice bearing about 100 lung tumor colonies of EMT-6 cells, each about 2000 cells in size, were treated with 211At-labeled monoclonal antibody 201B. The administered activity of 185 kBq per animal extended the life span of treated mice over untreated controls. Injections of 370 kBq, corresponding to an absorbed dose of 25-40 Gy, were necessary to eradicate all of the lung tumors. Mice receiving 740 kBq of 211At-labeled monoclonal antibody 201B developed pulmonary fibrosis 3-4 months after treatment, as did mice treated with 3700 kBq of the alpha-particle emitter 213Bi-labeled monoclonal antibody 201B in previous work. Animals that were injected with 211At bound to untargeted IgG or to glycine, as control agents, also demonstrated therapeutic effects relative to untreated controls. Control groups that received untargeted 211At required about twice as much administered activity for effective therapy as did groups with lung-targeted radioisotope. These results were not consistent with radioisotope biodistribution and dosimetry calculations that indicated that lung-targeted 211At should be at least 10-fold more efficient for lung colony therapy than 211At bound to nontargeting controls. The data showed that 211At is useful for vascular-targeted radioimmunotherapy because lung tumor colonies were eradicated in the mice. Work in this model system demonstrates that vascular targeting of alpha-particle emitters is an efficient therapy for small perivascular tumors and may be applicable to human disease when specific targeting agents are identified.

High-efficiency astatination of antibodies using N-iodosuccinimide as the oxidising agent in labelling of N-succinimidyl 3-(trimethylstannyl)benzoate.

Monoclonal antibodies C215, reactive with colorectal carcinomas, and MOv18, reactive with most of the ovarian carcinomas, were radiohalogenated with [211At]astatine. The radiohalogen was conjugate coupled to antibodies via the intermediate labelling reagent N-succinimidyl-3-(trimethylstannyl)benzoate (m-MeATE) in a two-step, single-pot reaction. Optimisation of the labelling of the reagent was achieved using N-iodosuccinimide, NIS, as the oxidising agent. The yields ranged from 69-95% in the labelling of 0.1-1.0 nmole of the m-MeATE precursor. Subsequent conjugation to antibodies resulted in yields of 58+/-7%. In vitro binding to tumour cells showed that the immunoreactivity of both antibodies was retained after astatine labelling.

Preparation and preliminary evaluation of 4-[211At]astato-N-piperidinoethyl benzamide.

The potential therapeutic agent, 4-[211At]astato-N-piperidinoethyl benzamide (4-APAB) was synthesized via a halodestannylation reaction. Radiochemical yields were 69% for a 5 min reaction and reached 74% by 25 min, whereas 82% radiochemical yields were obtained under similar reaction conditions for radioiodination. A simplified procedure was adopted for the purification of the target compound. In vitro binding of 4-APAB to SK-MEL 28 melanoma and D247 glioma cell lines was 20.7 +/- 1.3% and 12.2 +/- 1.3%, respectively. In comparison, binding of 4-[131I]iodo-N-piperidinoethyl benzamide (4-IPAB) to SK-Mel 28 cells was 13.9 +/- 1.9%. Paired label biodistribution studies were performed in normal Balb/c mice using 4-IPAB and 4-APAB. Thyroid uptake at 1, 2, and 6 h was significantly higher for 4-APAB. Differences in liver accumulation between the two compounds were small but statistically significant at most time points. A higher accumulation of 211At compared with 131I was observed in lungs and spleen at all time points studied. These results indicate that 4-APAB is not stable in vivo, suggesting the need for a better sigma receptor ligand for use in 211At.

Single-cell irradiation from [211At] astatine-labeled C215 monoclonal antibody: improved estimates of radiosensitivity from measurements on cellular uptake and retention.

New data on the biological effect of 211At-C215 monoclonal antibody in a slowly rotating, widely dispersed single-cell suspension of the human cancer cell line Colo-205 is presented. Cell growth curves of each experiment were used to calculate an apparent cell survival after irradiation. Uptake measurements provided the data needed to calculate the average number of 211At decays per cell in the cell suspension. The results from each experiment were then fit to a mono-exponential function. From the exponential fit, an average of 35 +/- 2 (SD) astatine-211 decays per cell are required for 37% apparent cell survival (D0).

Targeting against epidermal growth factor receptors. Cellular processing of astatinated EGF after binding to cultured carcinoma cells.

BACKGROUND: The alpha-emitting nuclide 211At is of great interest for radionuclide therapy when coupled to a tumor-targeting biomolecule, e.g. epidermal growth factor (EGF) the receptors of which are overexpressed in many malignancies. However, almost no information concerning the cellular processing of astatinated targeting agents is available. MATERIALS AND METHODS: We indirectly astatinated EGF ([211At]-benzoate-EGF) and studied its cellular processing in A-431 carcinoma cells in comparison with data concerning [125I]-benzoate-EGF. RESULTS: The biological half-life of astatine (3.5 h) was longer than the half-life of the iodine label (1.5 h). The increase of the half-life was due to longer retention of the internalised astatine radioactivity. The maximum accumulation for the astatine label occurred later (4-6h) than that for the iodine label (2-4h), indicating a slower excretion of astatine that was confirmed in experiment with 211At/1251-benzoate-EGF. CONCLUSION: The long retention of astatine might be advantageous for radionuclide therapy.

Animal studies on the in vivo stability of 211At-labelled albumin particles.

The alpha-emitter astatine-211 appears as one of the most suitable representatives for radiotherapy owing to its excellent decay properties. We report the studies in mice on the stability of the astatine bond to HSA-particles. Microspheres stick fast in the lung capillaries. There is a comparatively very slow decrease of radioactivity in the lungs. The 211At-labelled microspheres appear quite stable in vivo compared with a series of other biomolecules and so give a potential possibility for using the excellent radiophysical features of 211At in endogenous therapy of malignant tumours.

[Effects of At-211 alpha irradiation on Ehrlich ascites tumor cells]. / Deistvie alfa-izlucheniia 211-At na kletki astsitnogo raka Erlikha.

The biological effect of 211At alpha-particles has been investigated using the Chinese hamster fibroblasts and Ehrlich carcinoma cells growth in vitro. The mean energy of 211At alpha-particles is 6.8 MeV, LET in tissue is 70-160 keV/microns; the half-life period of decomposition of 211At is 7.2 h. The end-points used were a decrease in the mitotic activity, an elevation of the number of degenerating cells, cell with chromosome aberrations and the cell survival. The RBE of alpha-particles in comparison with 60Co gamma-rays is close to 3.

Determination of absorbed dose to blood, kidneys, testes and thyroid in mice injected with 211At and comparison of testes mass and sperm number in x-irradiated and 211At treated mice.

Male mice were injected with 211At and the total absorbed dose to the whole blood, kidneys, testes and thyroid was determined. Extrapolation to man shows a close agreement with the predictions of the International Commission on Radiological Protection (ICRP) for the absorbed dose to human blood, kidneys and testes, but the absorbed dose to the thyroid is about 200 times greater than the ICRP prediction. Comparison of the reduction in testes mass and in sperm numbers 28 days after mice were injected with 211At, or exposed to x rays, indicates a factor of about four for the greater effectiveness of 211At alpha particles over 250 kVcp x rays for the induction of effects.

Effect of cetuximab in combination with alpha-radioimmunotherapy in cultured squamous cell carcinomas.

AIM: The monoclonal antibody cetuximab, targeting the epidermal growth factor receptor (EGFR), is a promising molecular targeting agent to be used in combination with radiation for anticancer therapy. In this study, effects of cetuximab in combination with alpha-emitting radioimmunotherapy (RIT) in a panel of cultured human squamous cell carcinomas (SCCs) were assessed. METHODS: SCC cell lines were characterized and treated with cetuximab in combination with anti-CD44v6 RIT using the astatinated chimeric monoclonal antibody U36 ((211)At-cMAb U36). Effects on (211)At-cMAb U36 uptake, internalization and cell proliferation were then assessed in SCC cells. RESULTS: Cetuximab in combination with (211)At-cMAb U36 mediated increased growth inhibition compared to RIT or cetuximab alone in two cell lines. However, cetuximab also mediated radioprotective effects compared to RIT alone in two cell lines. The radioprotective effects occurred in the cell lines in which cetuximab clearly inhibited cell growth during radiation exposure. Cetuximab treatment also influenced (211)At-cMAb-U36 uptake and internalization, suggesting interactions between CD44v6 and EGFR. CONCLUSIONS: Results from this study demonstrate the vast importance of further clarifying the mechanisms of cetuximab and radiation response, and the relationship between EGFR and suitable RIT targets. This is important not only in order to avoid potential radioprotective effects, but also in order to find and utilize potential synergistic effects from these combinations.

Biodistribution of 6-[211At]astato-2-methyl-1,4-naphthoquinol bis(diphosphate salt) and 211At- in mice with a transplanted rectal adenocarcinoma.

6-[211At]astato-MNDP is currently being investigated as a potential high LET endoradiotherapeutic drug. Biodistribution and whole-body radiation retention studies have been carried out with 6-[211At]astato-MNDP and 211At- in a murine rectal tumour model; results indicate that the 211At-C bond in the compound is metabolically stable for at least 6 h. The Mean Biological Concentration of 6-[211At]astato-MNDP in tumour tissue ranged from 170-253% over an initial 12 h period; this was higher than that observed for the [211At]astatide anion. Conversely, the uptake of compound into radiobiologically critical organs was significantly lower.

Alpha-particle track autoradiographic study of the distribution of a [211At]-astatinated drug in normal tissues of the mouse.

The microscopic distribution of the potential endoradiotherapeutic drug, 6-[211At]-astato-2-methyl-1,4- naphthoquinol bis (diphosphate salt) in normal tissues of the mouse has been studied by alpha-particle track autoradiography. The uptake into critical radiosensitive tissues, especially bone marrow, colon and lung, was low.

alpha-Particle track autoradiography for localization of a 211At-astatinated drug.

A potential endoradiotherapeutic drug, 6-211At-astato-2-methyl-1,4-naphthoquinol bis (diphosphate salt), incorporating the alpha-emitting radio-halogen astatine-211 of half-life 7.2 h, is shown to be valuable for localization studies by means of alpha-particle track autoradiography in malignant and normal cells and tissues in the mouse with transplanted adenocarcinoma of the rectum.