Assessment of Cell Cytotoxicity and Comet Assay on HER2/neu Positive Cell Line Due to 111In Auger Electrons as DNA-Targeting Radioimmunoconjugate.

BACKGROUND: Breast cancer Auger electron therapy is a growing field of study in radioimmunotherapy and oncology research. Trastuzumab, a high affinity-binding monoclonal antibody against HER2/neu is which is over-expressed in breast tumors, is used in radiopharmaceutical development. OBJECTIVES: In this work, the lethal effects of 111In3+, 111In-DTPA-trastuzumab and 111In-trastuzumab coupled-nuclear localizing sequence peptide (111In-DTPA-NLS-trastuzumab) on malignant cells were studied in vitro. METHODS: DTPA-NLS-trastuzumab was prepared using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) conjugation with NLS peptide in the first step, followed by conjugation with diethylenetriaminepentaacetic acid (DTPA). Both DTPA-trastuzumab and DTPA- NLS-trastuzumab were labeled with 111In followed by purification and quality control techniques. Sk-Br-3 (a HER2/neu+ cell line), was used in the cell viability assessment assay for 111In, 111In-DTPA-trastuzumab and 111In-DTPA-NLS-trastuzumab (3.7 MBq) at 37 ºC. The cytotoxicity of the three species was studied using MTT and comet assay was utilized DNA damage detection. RESULTS: A significant radiochemical purity for 111In-DTPA-NLS-trastuzumab (99.36% ± 0.30%, ITLC) at the DTPA:antibody ratio of 6.90 ± 0.34:1, was obtained. Significant cell viability difference was found for 111In-DTPA-NLS-trastuzumab compared to the other treatments at two-time points. In addition, comet assay demonstrated significant DNA damage at 144 h using 111In-DTPA- NLS-trastuzumab. CONCLUSION: The results of cell viability and cell death using MTT assay and comet assay, respectively, demonstrate the NLS-peptide effectively facilitates 111In-trastuzumab transport into the HER2/neu positive cancer cell nuclei to impose the radiotherapeutic effects of Auger electrons on DNA leading to cell death.

Rapid Response, Dose Assessment, and Clinical Management of a Plutonium-contaminated Puncture Wound.

Internalization of radionuclides occurs not only by inhalation, ingestion, parenteral injection (i.e., administration of radioactive material for a medical purpose), and direct transdermal absorption, but also by contaminated wounds. In June 2010, a glove-box operator at the U.S. Department of Energy's Savannah River Site sustained a puncture wound while venting canisters containing legacy materials contaminated with Pu. To indicate the canisters had been vented, a flag was inserted into the vent hole. The shaft of the flag penetrated the protective gloves worn by the operator. Initial monitoring performed with a zinc-sulfide alpha detector indicated 300 dpm at the wound site. After being cleared by radiological controls personnel, the patient was taken to the site medical facility where decontamination was attempted and diethylenetriaminepentaacetic acid (DTPA) was administered intravenously within 1.5 h of the incident. The patient was then taken to the Savannah River Site In Vivo Counting Facility where the wound was counted with a Canberra GL 2820 high-purity germanium detector, capable of quantifying contamination by detecting low-energy x rays and gamma rays. In addition to the classic 13, 17, and 20 keV photons associated with Pu, the low-yield (0.04%) 43.5 keV peak was also detected. This indicated a level of wound contamination orders of magnitude above the initial estimate of 300 dpm detected with handheld instrumentation. Trace quantities of Am were also identified via the 59.5 keV peak. A 24 h urine sample collection was begun on day 1 and continued at varying intervals for over a year. The patient underwent a punch biopsy at 3 h postincident (14,000 dpm removed) and excisional biopsies on days 1 and 9 (removal of an additional 3,200 dpm and 3,800 dpm, respectively). The initial post-DTPA urine sample analysis report indicated excretion in excess of 24,000 dpm Pu. Wound mapping was performed in an effort to determine migration from the wound site and indicated minimum local migration. In vivo counts were performed on the liver, axillary lymph nodes, supratrochlear lymph nodes, and skeleton to assess uptake and did not indicate measurable activity. Seventy-one total doses of DTPA were administered at varying frequencies for 317 d post intake. After allowing 100 d for removal of DTPA from the body, five 24 h urine samples were collected and analyzed for dose assessment by using the wound model described in National Council on Radiation Protection and Measurements Report No. 156. The total effective dose averted via physical removal of the contaminant and DTPA administration exceeded 1 Sv, demonstrating that rapid recognition of incident magnitude and prompt medical intervention are critical for dose aversion.

In vitro assessment of plutonium uptake and release using the human macrophage-like THP-1 cells.

Plutonium (Pu) intake by inhalation is one of the major potential consequences following an accident in the nuclear industry or after improvised nuclear device explosion. Macrophages are essential players in retention and clearance of inhaled compounds. However, the extent to which these phagocytic cells are involved in these processes highly depends on the solubility properties of the Pu deposited in the lungs. Our objectives were to develop an in vitro model representative of the human pulmonary macrophage capacity to internalize and release Pu compounds in presence or not of the chelating drug diethylenetriaminepentaacetate (DTPA). The monocyte cell line THP-1 was used after differentiation into macrophage-like cells. We assessed the cellular uptake of various forms of Pu which differ in their solubility, as well as the release of the internalized Pu. Results obtained with differentiated THP-1 cells are in good agreement with data from rat alveolar macrophages and fit well with in vivo data. In both cell types, Pu uptake and release depend upon Pu solubility and in all cases DTPA increases Pu release. The proposed model may provide a good complement to in vivo animal experiments and could be used in a first assessment to predict the fraction of Pu that could be potentially trapped, as well as the fraction available to chelating drugs.

Orally administered DTPA di-ethyl ester for decorporation of (241)Am in dogs: Assessment of safety and efficacy in an inhalation-contamination model.

PURPOSE: Currently two injectable products of diethylenetriaminepentaacetic acid (DTPA) are U.S. Food and Drug Administration (FDA)-approved for decorporation of (241)Am; however, an oral product is considered more amenable in a mass casualty situation. The di-ethyl ester of DTPA, named C2E2, is being developed as an oral drug for treatment of internal radionuclide contamination. MATERIALS AND METHODS: Single-dose decorporation efficacy of C2E2 administered 24-h post contamination was determined in beagle dogs using a (241)Am nitrate inhalation contamination model. Single and multiple dose toxicity studies in beagle dogs were performed as part of an initial safety assessment program. In addition, the genotoxic potential of C2E2 was evaluated by the in vitro bacterial reverse mutation Ames test, mammalian cell chromosome aberration cytogenetic assay and an in vivo micronucleus test. RESULTS: Oral administration of C2E2 significantly increased (241)Am elimination over untreated controls and significantly reduced the retention of (241)Am in tissues, especially liver, kidney, lung and bone. Daily dosing of 200 mg/kg/day for 10 days was well tolerated in dogs. C2E2 was found to be neither mutagenic or clastogenic. CONCLUSIONS: The di-ethyl ester of DTPA (C2E2) was shown to effectively enhance the elimination of (241)Am after oral administration in a dog inhalation-contamination model and was well tolerated in toxicity studies.

Tc-99m diethylenetriamine pentaacetic acid (DTPA) renal function reserve estimation: is it a reliable predictive tool for assessment of preclinical renal involvement in scleroderma patients?

Prognosis of systemic sclerosis (SSc) depends on internal organ involvement. We assessed the value of renal function reserve (RFR) for the detection of preclinical nephropathy in scleroderma. Thirty SSc patients with normal serum creatinine and 30 healthy controls were included. Medsger disease severity score, glomerular filtration rate (GFR), and microalbuminuria were measured. Tc-99m DTPA was utilized for GFR measurement at baseline and after oral protein overload (stimulated GFR). RFR was calculated as the percentile increase of stimulated GFR. SSc patients had lower means of baseline GFR (P=0.001), stimulated GFR (P=0.004), RFR (P=0.046), and higher microalbuminuria (P=0.009) than controls. According to baseline GFR, SSc patients showed three categories-normal baseline GFR (n=12), hyperfiltration GFR (n=3), and reduced baseline GFR (n=15). In the former category, RFR was normal in 6/12 patients and abnormal in the remainders (50%). Hyperfiltration patients and those with reduced baseline GFR showed abnormal RFR. A statistically significant negative association was found between microalbuminuria versus stimulated GFR and RFR (r= -0.5, P=0.007 and r= -0.45, P=0.013, respectively). The majority of SSc patients with abnormal RFR had disease duration of ≥48 months (60% vs. 20%, P=0.008). All SSc patients with pulmonary hypertension had abnormal RFR, while reduced baseline GFR was noted in only 60%. A significant negative correlation was found between reduced baseline GFR and cumulative dose of corticosteroids in SSc patients (r= -0.4, P=0.022). RFR estimation could be a useful predictive marker for preclinical renal involvement in SSc patients so that early prophylactic measures and therapy modifications could be considered.

[Antibody radiolabeling with technetium-99m]. / Radiomarcaje de anticuerpos con Tecnecio-99m.

Nowadays, labelled polyclonal and monoclonal antibodies are widely used for immunoscintigraphic diagnosis of different diseases. Technetium-99m is often considered to be the label of choice for radioimmunodiagnosis for reasons of cost, availability and imaging properties, in spite of its relatively short physical half-life (6.01 h). The existing labelling methods may be classified into two types: direct approaches, in which disulphide bridges within are reduced to generate endogenous sulfhydryl groups able to efficiently bind technetium due to their strong chelating capacity and indirect methods, in which an exogenous chelator is covalently attached to the protein to serve as the binding site. All these procedures have their advantages and drawbacks. There is no consensus among the authors about which of the methods is the best. The employed approach depends on the particular situation. The aim of the present work is to show an update about the available procedures for 99mTc-labelling of antibodies and its fragments.

Myocardial infarction: assessment with an intravascular MR contrast medium. Work in progress.

The effect of a new intravascular magnetic resonance (MR) contrast medium (gadolinium diethylenetriaminepentaacetic acid [DTPA] polylysine) was evaluated in acute, subacute, and chronic myocardial infarctions in rats. Signal intensity (SI) was measured before and after intravenous administration of Gd-DTPA polylysine. Before administration of contrast material, chronic infarctions had lower SI than normal myocardium. With Gd-DTPA polylysine, three zones were identified in acute and subacute stages of myocardial infarction, but in the chronic stage, images demonstrated two zones. In acute and subacute infarctions, Gd-DTPA polylysine produced greater enhancement (over 60 minutes) in the peri-infarction zone than in the normal or infarcted myocardium. In chronic infarctions, Gd-DTPA polylysine had no discernible effect on the SI of the central infarction zone. Overall, it caused no significant hemodynamic effects. MR imaging with Gd-DTPA polylysine produced differential tissue enhancement in myocardial infarctions, which varied according to the age of the infarction.