Radiopharmaceutical chemistry of targeted radiotherapeutics, Part 2: radiolytic effects of 211At alpha-particles influence N-succinimidyl 3-211AT-astatobenzoate synthesis.
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A variety of promising targeted radiotherapeutics labeled with alpha-emitters have been developed. Clinical investigation of these radiopharmaceuticals requires the production of high activity levels, which can be hindered by alpha-particle-mediated radiolytic effects on labeling chemistry. The purpose of this study was to investigate the effects of radiation dose on the synthesis of N-succinimidyl 3-(211)At-astatobenzoate (SAB), a compound used in our clinical trials for labeling antibodies with alpha-particle-emitting (211)At. METHODS: Yields for the synthesis of SAB as a function of the radiation dose received by the reaction medium were determined. The variables studied included the radiohalogenation precursors N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB) and N-succinimidyl 3-(trimethylstannyl)benzoate (MeSTB); the solvents chloroform, benzene, and methanol; and the addition of acetic acid and the oxidant N-chlorosuccinimide. The (211)At product spectra were determined from high-performance liquid chromatograms and then plotted against radiation dose. RESULTS: SAB production declined rapidly with increasing dose, consistent with the documented radiolytic decomposition of BuSTB and MeSTB in chloroform. Even though these tin precursors were not appreciably degraded in benzene, SAB could not be produced in this solvent; instead, highly lipophilic (211)At-labeled species were generated in nearly quantitative yields. Although a dose-dependent decline in SAB yield also was observed in methanol, both in the presence and in the absence of an oxidant, the results were better than those obtained with the other solvents. An unexpected observation was that SAB could be obtained at a yield of greater than 30% when the reaction was run in methanol without the addition of acetic acid or an oxidant; these 2 components previously were considered essential for astatodestannylation. CONCLUSION: Radiolytic factors can play an important role in the synthesis of clinical-level activities of (211)At-labeled radiopharmaceuticals, necessitating the development of reaction conditions different from those that are used successfully at lower activity levels. (+info)
Biological effects in unirradiated human tissue induced by radiation damage up to 1 mm away.
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A central tenet in understanding the biological effects of ionizing radiation has been that the initially affected cells were directly damaged by the radiation. By contrast, evidence has emerged concerning "bystander" responses involving damage to nearby cells that were not themselves directly traversed by the radiation. These long-range effects are of interest both mechanistically and for assessing risks from low-dose exposures, where only a small proportion of cells are directly hit. Bystander effects have been observed largely by using single-cell in vitro systems that do not have realistic multicellular morphology; no studies have as yet been reported in three-dimensional, normal human tissue. Given that the bystander phenomenon must involve cell-to-cell interactions, the relevance of such single-cell in vitro studies is questionable, and thus the significance of bystander responses for human health has remained unclear. Here, we describe bystander responses in a three-dimensional, normal human-tissue system. Endpoints were induction of micronucleated and apoptotic cells. A charged-particle microbeam was used, allowing irradiation of cells in defined locations in the tissue yet guaranteeing that no cells located more than a few micrometers away receive any radiation exposure. Unirradiated cells up to 1 mm distant from irradiated cells showed a significant enhancement in effect over background, with an average increase in effect of 1.7-fold for micronuclei and 2.8-fold for apoptosis. The surprisingly long range of bystander signals in human tissue suggests that bystander responses may be important in extrapolating radiation risk estimates from epidemiologically accessible doses down to very low doses where nonhit bystander cells will predominate. (+info)
Mechanism of radiation-induced bystander effect: role of the cyclooxygenase-2 signaling pathway.
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The radiation-induced bystander effect is defined as "the induction of biological effects in cells that are not directly traversed by a charged particle but are in close proximity to cells that are." Although these bystander effects have been demonstrated with a variety of biological endpoints in both human and rodent cell lines (as well as in 3D tissue samples), the mechanism of the phenomenon is not known. Although gap junction communication and the presence of soluble mediator(s) are both known to play important roles in the bystander response, the precise signaling molecules have yet to be identified. By using the Columbia University charged particle beam in conjunction with a strip dish design, we show here that the cyclooxygenase-2 (COX-2, also known as prostaglandin endoperoxide synthase-2) signaling cascade plays an essential role in the bystander process. Treatment of bystander cells with NS-398, which suppresses COX-2 activity, significantly reduced the bystander effect. Because the critical event of the COX-2 signaling is the activation of the mitogen-activated protein kinase pathways, our finding that inhibition of the extracellular signal-related kinase phosphorylation suppressed bystander response further confirmed the important role of mitogen-activated protein kinase signaling cascade in the bystander process. These results provide evidence that the COX-2-related pathway, which is essential in mediating cellular inflammatory response, is the critical signaling link for the bystander phenomenon. (+info)
Differential impact of mouse Rad9 deletion on ionizing radiation-induced bystander effects.
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The cellular response to ionizing radiation is not limited to cells irradiated directly but can be demonstrated in neighboring "bystander" populations. The ability of mouse embryonic stem (ES) cells to express a bystander effect and the role of the radioresistance gene Rad9 were tested. Mouse ES cells differing in Rad9 status were exposed to broad-beam 125 keV/ microm 3He alpha particles. All populations, when confluent, demonstrated a dose-independent bystander effect with respect to cell killing, and the Rad9-/- genotype did not selectively alter that response or cell killing after direct exposure to this high-LET radiation. In contrast, relative to Rad9+/+ cells, the homozygous mutant was sensitive to direct exposure to alpha particles when in log phase, providing evidence of a role for Rad9 in repair of potentially lethal damage. Direct exposure to alpha particles induced an increase in the frequency of apoptosis and micronucleus formation, regardless of Rad9 status, although the null mutant showed high spontaneous levels of both end points. All populations demonstrated alpha-particle-induced bystander apoptosis, but that effect was most prominent in Rad9-/- cells. Minimal alpha-particle induction of micronuclei in bystander cells was observed, except for the Rad9-/- mutant, where a significant increase above background was detected. Therefore, the Rad9 null mutation selectively sensitizes mouse ES cells to spontaneous and high-LET radiation-induced bystander apoptosis and micronucleus formation, but it has much less impact on cell killing by direct or bystander alpha-particle exposure. Results are presented in the context of defining the function of Rad9 in the cellular response to radiation and its differential effects on individual bystander end points. (+info)
Intralesional targeted alpha therapy for metastatic melanoma.
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This paper reports the development and application of intralesional targeted alpha therapy (TAT) for melanoma, being the first part of a program to establish a new systemic therapy. RATIONALE: Labelling the benign targeting vector 9.2.27 with 213Bi forms the alpha-immunoconjugate (AIC), which is highly cytotoxic to targeted melanoma cells. OBJECTIVE: To investigate the safety and efficacy of intralesional AIC in patients with metastatic skin melanoma. FINDINGS: 16 melanoma patients were recruited. All the patients were positive to the monoclonal antibody 9.2.27. AIC doses from 50 to 450 mCi injected into lesions of different sizes resulted in massive cell death, as observed by the presence of tumour debris. The AIC was very effective in delivering a high dose to the tumour while sparing other tissues. There were no significant changes in blood proteins and electrolytes. There was no evidence of a human-antimouse-antibody reaction. Evidence of significant decline in serum marker melanoma-inhibitory-activity protein (MIA) at 2 weeks post-TAT was observed. CONCLUSIONS: Intralesional TAT for melanoma was found to be quite safe up to 450 mCi, and efficacious at a dose of 200 mCi. MIA, apoptosis and ki67 proliferation marker tests all indicated that TAT is a promising therapy for the control of inoperable secondary melanoma or primary ocular melanoma. (+info)
211At radioimmunotherapy of subcutaneous human ovarian cancer xenografts: evaluation of relative biologic effectiveness of an alpha-emitter in vivo.
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The use of alpha-particle emitters in radioimmunotherapy (RIT) appears to be promising. We previously obtained convincing results in the treatment of microscopic intraperitoneal ovarian cancer in nude mice by using the alpha-emitter 211At. This study was performed to evaluate the relative biological effectiveness (RBE) of 211At compared with that of 60Co gamma-irradiation in an RIT model. Our endpoint was growth inhibition (GI) of subcutaneous xenografts. METHODS: GI after irradiation was studied with subcutaneous xenografts of the human ovarian cancer cell line NIH:OVCAR-3 implanted in nude mice. The animals received an intravenous injection of 211At-labeled monoclonal antibody MX35 F(ab')2 at different levels of radioactivity (0.33, 0.65, and 0.90 MBq). Control mice received unlabeled MX35 F(ab')2 only. To calculate the mean absorbed dose to tumor, a separate biodistribution study established the uptake of 211At in tumors and organs at different times after injection. External irradiation of the tumors was performed with 60Co. Tumor growth was monitored, and the normalized tumor volume (NTV) was calculated for each tumor. GI was defined by dividing the NTV values by the fitted NTV curve obtained from the corresponding control mice. To compare the biologic effects of the 2 radiation qualities, the mean value for GI (from day 8 to day 23) was plotted for each tumor as a function of its corresponding absorbed dose. From exponential fits of these curves, the doses required for a GI of 0.37 (D37) were derived, and the RBE of 211At was calculated. RESULTS: The biodistribution study showed the uptake of the immunoconjugate by the tumor (amount of injected radioactivity per gram) to be 14% after 7 h. At 40 h, the ratio of uptake in tumors to uptake in blood reached a maximum value of 6.2. The administered activities of 211At corresponded to doses absorbed by tumors of 1.35, 2.65, and 3.70 Gy. The value (mean+/-SEM) for D37 was 1.59+/-0.08 Gy. Tumor growth after 60Co external irradiation showed a value for D37 of 7.65+/-1.0 Gy. The corresponding RBE of 211At irradiation was 4.8+/-0.7. CONCLUSION: Using a tumor GI model in nude mice, we were able to derive an RBE of alpha-particle RIT with 211At. The RBE was found to be 4.8+/-0.7. (+info)
The use of indwelling medical devices--pacemakers, prosthetic joints, catheters--is rapidly growing and is often complicated by infections with biofilm-forming microbes that are resistant to antimicrobial agents and host defense mechanisms. We investigated for the first time the use of microbe-specific monoclonal antibodies (MAbs) as delivery vehicles for targeting biofilms with cytocidal radiation. MAb 18B7 (immunoglobulin G1 [IgG1]), which binds to capsular polysaccharides of the human pathogenic fungus Cryptococcus neoformans, penetrated cryptococcal biofilms, as shown by confocal microscopy. When the alpha radiation-emitter 213-Bismuth ((213)Bi) was attached to MAb 18B7 and the radiolabeled MAb was added to C. neoformans biofilms, there was a 50% reduction in biofilm metabolic activity. In contrast, when the IgM MAb 13F1 labeled with (213)Bi was used there was no penetration of the fungal biofilm and no damage. Unlabeled 18B7, (213)Bi-labeled nonspecific MAbs, and gamma and beta types of radiation did not have an effect on biofilms. The lack of efficacy of gamma and beta radiation probably reflects the radioprotective properties of polysaccharide biofilm matrix. Our results indicate that C. neoformans biofilms are susceptible to treatment with antibody-targeted alpha radiation, suggesting a novel option for the prevention or treatment of microbial biofilms on indwelling medical devices. (+info)
Radiotoxicity of the alpha-emitting bone-seeker 223Ra injected intravenously into mice: histology, clinical chemistry and hematology.
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BACKGROUND: The alpha-emitter 223Ra, which localizes in osteoblastic active zones, including on skeletal surfaces and in osteoblastic metastases, has recently been introduced as a potential therapeutic agent against skeletal metastases. Here, the adverse effects of high dosages in animals were investigated. MATERIALS AND METHODS: Balb/c mice received intravenously (i.v.) either 1250, 2500, or 3750 kBq/kg of dissolved 223RaCl2 and were followed in the initial toxicity phase. At the 4-week end-point, the animals were sacrificed and blood samples were collected to study the effects on clinical chemistry and hematological parameters. Selected organs were weighed and tissue samples examined by microscopy. RESULTS: Treatment with 223Ra caused a dose-related minimal to moderate depletion of osteocytes and osteoblasts in the bones. Furthermore, a dose-related minimal to marked depletion of the hematopoietic cells in the bone marrow, and a minimal to slight extramedullary hematopoiesis in the spleen and in the mandibular and mesenteric lymph nodes were observed. The LD50 for acute toxicity, defined as death within 4 weeks of receiving the substance, was not reached. CONCLUSION: This study demonstrated that high doses of the bone-seeker 223Ra did not completely inactivate the blood-producing cells. The relatively high tolerance to skeletal alpha doses was probably caused by the surviving pockets of red bone marrow cells beyond the range of alpha particles from the bone surfaces, and the recruitment of peripheral stems cells. (+info)