Assessment of the eye irritation potential of chemicals: A comparison study between two test methods based on human 3D hemi-cornea models.

We have recently developed two hemi-cornea models (Bartok et al., Toxicol in Vitro 29, 72, 2015; Zorn-Kruppa et al. PLoS One 9, e114181, 2014), which allow the correct prediction of eye irritation potential of chemicals according to the United Nations globally harmonized system of classification and labeling of chemicals (UN GHS). Both models comprise a multilayered epithelium and a stroma with embedded keratocytes in a collagenous matrix. These two models were compared, using a set of fourteen test chemicals. Their effects after 10 and 60 minutes (min) exposure were assessed from the quantification of cell viability using the MTT reduction assay. The first approach separately quantifies the damage inflicted to the epithelium and the stroma. The second approach quantifies the depth of injury by recording cell death as a function of depth. The classification obtained by the two models was compared to the Draize rabbit eye test and an ex vivo model using rabbit cornea (Jester et al. Toxicol in Vitro. 24, 597-604, 2010). With a 60 min exposure, both of our models are able to clearly differentiate UN GHS Category 1 and UN GHS Category 2 test chemicals.

Development of an in vitro ocular test system for the prediction of all three GHS categories.

In the present study we considered a new approach that allows the individual quantification of damages induced in the epithelium and stroma of an in vitro hemi-cornea model after chemical treatment. We aimed at a stand-alone test system for classification according to the classification of the globally harmonized system of classification and labelling of chemicals (GHS). We have modified a previously developed 3D hemi-cornea model by the insertion of a collagen membrane between epithelium and stroma. This membrane allows the separation and independent assessment of these compartments after topical exposure to potential eye irritants. The cell viability quantified by MTT assay was used as the toxicological endpoint. The prediction model based on the results obtained from 30 test chemicals uses a single exposure period and the combination of cut-off values in tissue viability from both epithelium and stroma. The in vitro-in vivo concordance of the test system is 77%. All of the GHS category 1, 80% of the GHS category 2 and 50% of the GHS not categorized chemicals are predicted correctly. In conclusion, the test system predicts and discriminates GHS category 1 and GHS category 2 chemicals, but is over-predictive for GHS not categorized materials.

A human hemi-cornea model for eye irritation testing: quality control of production, reliability and predictive capacity.

We have developed a 3-dimensional human hemi-cornea which comprises an immortalized epithelial cell line and keratocytes embedded in a collagen stroma. In the present study, we have used MTT reduction of the whole tissue to clarify whether the production of this complex 3-D-model is transferable into other laboratories and whether these tissues can be constructed reproducibly. Our results demonstrate the reproducible production of the hemi-cornea model according to standard operation procedures using 15 independent batches of reconstructed hemi-cornea models in two independent laboratories each. Furthermore, the hemi-cornea tissues have been treated with 20 chemicals of different eye-irritating potential under blind conditions to assess the performance and limitations of our test system comparing three different prediction models. The most suitable prediction model revealed an overall in vitro-in vivo concordance of 80% and 70% in the participating laboratories, respectively, and an inter-laboratory concordance of 80%. Sensitivity of the test was 77% and specificity was between 57% and 86% to discriminate classified from non-classified chemicals. We conclude that additional physiologically relevant endpoints in both epithelium and stroma have to be developed for the reliable prediction of all GHS classes of eye irritation in one stand alone test system.

Boron determination in liver tissue by combining quantitative neutron capture radiography (QNCR) and histological analysis for BNCT treatment planning at the TRIGA Mainz.

The typical primary malignancies of the liver are hepatocellular carcinoma and cholangiocarcinoma, whereas colorectal liver metastases are the most frequently occurring secondary tumors. In many cases, only palliative treatment is possible. Boron neutron capture therapy (BNCT) represents a technique that potentially destroys tumor tissue selectively by use of externally induced, locally confined secondary particle irradiation. In 2001 and 2003, BNCT was applied to two patients with colorectal liver metastases in Pavia, Italy. To scrutinize the rationale of BNCT, a clinical pilot study on patients with colorectal liver metastases was carried out at the University of Mainz. The distribution of the (10)B carrier (p-borono-phenylalanine) in the liver and its uptake in cancerous and tumor-free tissue were determined, focusing on a potential correlation between the uptake of p-borono-phenylalanine and the biological characteristics of cancerous tissue. Samples were analyzed using quantitative neutron capture radiography of cryosections combined with histological analysis. Methodological aspects of the combination of these techniques and results from four patients enrolled in the study are presented that indicate that the uptake of p-borono-phenylalanine strongly depends on the metabolic activity of cells.

Cell type selective accumulation of mercaptoundecahydro- closo-dodecaborate (BSH) in glioblastoma multiforme.

BACKGROUND: Boron uptake in glioblastoma tissue for boron neutron capture therapy is of great importance for the clinical outcome of the treatment. METHODS: The cell type specific distribution of mercaptoundecahydro-closo-dodecaborate (BSH) in glioblastoma multiforme tissue sections of seven patients having received BSH prior to surgery was investigated by light and fluorescence microscopy. FINDINGS: With use of specific antibodies against different tumour specific epitopes and BSH, BSH was found predominantly (approx. 90%) in the cytoplasm of GFAP-positive cells of all but two patients. The latter were younger (33 and 38 years versus 46-71 (mean 60) years). There was no correlation between BSH uptake and expression of EGFR, p53, CD44 and Ki-67. INTERPRETATION: GFAP-positive cells appear to be the primary cell type for BSH uptake in primary glioblastoma, and an important cell type for localisation of BSH in secondary glioblastoma. The molecular basis and the selective uptake mechanism require further work. If a correlation between histologically distinct patterns and clinical outcome for patients undergoing boron neutron capture therapy could be derived, prognostic factors for the treatment could be developed.

Optimal timing of neutron irradiation for boron neutron capture therapy after intravenous infusion of sodium borocaptate in patients with glioblastoma.

PURPOSE: A cooperative study in Europe and Japan was conducted to determine the pharmacokinetics and boron uptake of sodium borocaptate (BSH: Na(2)B(12)H(11)SH), which has been introduced clinically as a boron carrier for boron neutron capture therapy in patients with glioblastoma. METHODS AND MATERIALS: Data from 56 patients with glioblastoma who received BSH intravenous infusion were retrospectively reviewed. The pharmacokinetics were evaluated in 50 patients, and boron uptake was investigated in 47 patients. Patients received BSH doses between 12 and 100 mg/kg of body weight. For the evaluation, the infused boron dose was scaled linearly to 100 mg/kg BSH. RESULTS: In BSH pharmacokinetics, the average value for total body clearance, distribution volume of steady state, and mean residence time was 3.6 +/- 1.5 L/h, 223.3 +/- 160.7 L, and 68.0 +/- 52.5 h, respectively. The average values of the boron concentration in tumor adjusted to 100 mg/kg BSH, the boron concentration in blood adjusted to 100 mg/kg BSH, and the tumor/blood boron concentration ratio were 37.1 +/- 35.8 ppm, 35.2 +/- 41.8 ppm, and 1.53 +/- 1.43, respectively. A good correlation was found between the logarithmic value of T(adj) and the interval from BSH infusion to tumor tissue sampling. About 12-19 h after infusion, the actual values for T(adj) and tumor/blood boron concentration ratio were 46.2 +/- 36.0 ppm and 1.70 +/- 1.06, respectively. The dose ratio between tumor and healthy tissue peaked in the same interval. CONCLUSION: For boron neutron capture therapy using BSH administered by intravenous infusion, this work confirms that neutron irradiation is optimal around 12-19 h after the infusion is started.

Spectromicroscopy of boron in human glioblastomas following administration of Na2B12H11SH.

Boron neutron capture therapy (BNCT) is an experimental, binary treatment for brain cancer which requires as the first step that tumor tissue is targeted with a boron-10 containing compound. Subsequent exposure to a thermal neutron flux results in destructive, short range nuclear reaction within 10 microm of the boron compound. The success of the therapy requires than the BNCT agents be well localized in tumor, rather than healthy tissue. The MEPHISTO spectromicroscope, which performs microchemical analysis by x-ray absorption near edge structure (XANES) spectroscopy from microscopic areas, has been used to study the distribution of trace quantities of boron in human brain cancer tissues surgically removed from patients first administered with the compound Na2B12H11SH (BSH). The interpretation of XANES spectra is complicated by interference from physiologically present sulfur and phosphorus, which contribute structure in the same energy range as boron. We addressed this problem with the present extensive set of spectra from S, B, and P in relevant compounds. We demonstrate that a linear combination of sulfate, phosphate and BSH XANES can be used to reproduce the spectra acquired on boron-treated human brain tumor tissues. We analyzed human glioblastoma tissue from two patients administered and one not administered with BSH. As well as weak signals attributed to BSH, x-ray absorption spectra acquired from tissue samples detected boron in a reduced chemical state with respect to boron in BSH. This chemical state was characterized by a sharp absorption peak at 188.3 eV. Complementary studies on BSH reference samples were not able to reproduce this chemical state of boron, indicating that it is not an artifact produced during sample preparation or x-ray exposure. These data demonstrate that the chemical state of BSH may be altered by in vivo metabolism.

Transcervical or intraperitoneal analgesia for laparoscopic tubal sterilization: a randomized controlled trial.

OBJECTIVE: To test the effectiveness of analgesia administered transcervically through a uterine manipulator compared with direct topical application to the fallopian tubes for relief of postoperative pain after interval laparoscopic tubal sterilization. METHODS: Sixty-one women who had laparoscopic sterilization were enrolled in a randomized, double-masked clinical trial comparing analgesia with 75 mg of bupivacaine administered through a uterine manipulator with 75 mg of bupivacaine applied directly to the fallopian tubes through a secondary trocar. Results were evaluated using visual analog scale pain levels, time of administration of analgesics, total analgesics required, and recovery room times. We calculated that a sample size of 60 women would detect a 30% difference in pain levels with a power of 80% at a significance level of .05. RESULTS: In the 59 women who completed the study, there were no differences in the two groups in pain levels, amounts of medications used, or times to administration of postoperative analgesia. Mean recovery room time was shorter in the group given analgesia transcervically, but that difference was not statistically significant. CONCLUSION: There were no significant differences in postoperative pain relief between transcervical administration and topical application of analgesia for laparoscopic tubal sterilization.

Postoperative treatment of glioblastoma with BNCT at the petten irradiation facility (EORTC protocol 11,961).

The boron neutron capture therapy is based on the reaction occurring between the isotope 10B and thermal neutrons. A low energy neutron is captured by the nucleus and it disintegrates into two densely ionising particles, Li nucleus and He nucleus (alpha particle), with high biological effectiveness. On the basis of comprehensive preclinical investigations in the frame of the European Collaboration with Na2B12H11SH (BSH), as boron delivery agent, the first European phase I, clinical trial was designed at the only available epithermal beam in Europe, at the High Flux Reactor, Petten, in the Netherlands. The goal of this study is to establish the safe BNCT dose for cranial tumors under defined conditions. BNCT is applied as postoperative radiotherapy in 4 fractions, after removal of the tumor for a group of patients suffering from glioblastoma, who would have no benefit from conventional treatment, but have sufficient life expectancy to detect late radiation morbidity due to BNCT. The starting dose is set at 80% of the dose where neurological effects occurred in preclinical large animal experiments following a single fraction. The radiation dose will be escalated, by constant boron concentration in blood, in 4 steps for cohorts of ten patients, after an observation period of at least 6 months after the end of BNCT of the last patient of a cohort. The adverse events on healthy tissues due to BSH and due to the radiotherapy will be analysed in order to establish the maximal tolerated dose and dose limiting toxicity. Besides of the primary aim of this study the survival will be recorded. The first patient was treated in October 1997, and further four patients have been irradiated to-date. The protocol design proved to be well applicable, establishing the basis for scientific evaluation, for performance of safe patient treatment in a very complex situation and for opening the possibility to perform further clinical research work on BNCT.

Interaction of mercaptoundecahydrododecaborate (BSH) with phosphatidylcholine: relevance to boron neutron capture therapy.

The interaction of mercaptoundecahydrododecaborate (B12H11SH2-, BSH) with phosphatidylcholine was investigated in this study in order to illuminate possible uptake mechanisms of BSH in tumor cells. BSH has been used clinically in Japan as a boron containing agent in patients with malignant brain tumors for boron neutron capture therapy (BNCT). After infusion, BSH accumulates selectively in tumor tissue. Little is known for the mechanism of boron uptake to tumor cells. Fourier transform infrared (FTIR) spectrometry was used to quantify BSH (at wavenumber 2490 cm-1) and phosphatidylcholine (at wavenumber 2850-2970 cm-1). After extraction into carbon tetrachloride (CCl4), we could find an absorbance maximum at 2490 cm-1 as a B-H band in the mixture of BSH with phosphatidylcholine, which is attributed to a BSH-phosphatidylcholine complex, which could dissolve well in CCl4. The molar ratio of BSH to phosphatidylcholine in the CCl4 solution was at most one mole of BSH to two moles of phosphatidylcholine independent of the excess BSH. The doubly negatively charged BSH can interact with two phosphatidylcholine molecules through their singly positively charged choline residues. These ion pairs could be responsible for membrane binding and penetration, and for cell internalization.

Selective delivery of 10B to soft tissue sarcoma using 10B-L-borophenylalanine for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) may improve the locoregional control of radio/chemoresistant tumours like soft tissues sarcomas (STS). This technique uses the 10B(n,alpha)7Li nuclear reaction to destroy tumour cells, provided that a sufficient amount of 10B may be carried selectively into them. In order to evaluate the targeting potential of 10B-L-borophenylalanine (BPA) a 10B biodistribution study was carried out in 24 Wistar rats bearing Yoshida sarcoma. Six animals received increasing intraperitoneal doses of BPA (300, 600 and 1200 mg kg-1), while the remainder received a BPA dose of 600 mg kg-1 but with a sacrifice at six different time points: 1, 2, 4, 6, 9 and 12 h. The 10B concentrations in the tumours, normal tissues and blood were analysed with neutron capture radiography (NCR). The analysis shows that 36 micrograms g-1 (+/- 4 SD) of 10B may be incorporated into the tumour, with a ratio of 13 (+/- 4 SD) versus the muscle and a ratio of 15 (+/- 3 SD) versus the blood, 6 h after an intraperitoneal injection of 600 mg kg-1 of BPA. The BPA appears to be abundantly incorporated in the tumour, and the kidney proximal tubule area. These data suggest that BNCT using BPA may provide an improved therapeutic ratio for the treatment of STS.

The compound factor of the 10B(n,alpha)7Li reaction from borocaptate sodium and the relative biological effectiveness of recoil protons for induction of brain damage in boron neutron capture therapy.

To make clinical trials of boron neutron capture therapy safe for patients, it is necessary to know the relative biological effectiveness (RBE) of the radiation components and the compound factor of the boron carrier to be used. Here a method is derived to determine the RBE of recoil protons and the compound factor of compounds from in vivo experiments with different concentrations of boron. The method uses a simultaneous fit of both these parameters to all experimental data. This method is applied to the studies of tolerance of healthy tissue in dogs at the High Flux Reactor in Petten, The Netherlands. The RBE for the recoil protons generated by the neutrons present in the epithermal neutron beam [together with the RBE of the protons from the 14N(n,p)14C reaction] for induction of severe neurological symptoms was found to be 3.93+/-0.43 (95% confidence limits 3.06-4.79), and 2.33+/-0.14 (2.04-2.61) for induction of changes detectable by magnetic resonance imaging. The compound factor for Na2B12H11SH in brain tissue, using severe neurological symptoms as end point, was determined to be 0.37+/-0.06 (95% confidence limits 0.24-0.50). For changes detectable by magnetic resonance imaging, the value was found to be 0.65+/-0.04 (0.58-0.73).

Binding and distribution of Na2B12H11SH on cellular and subcellular level in tumor tissue of glioma patients in boron neutron capture therapy.

To determine binding and distribution of Na2B12H11SH (BSH) in glioma tissue in case of boron neutron capture therapy, an antibody to this compound was produced and used in immunohistochemical investigations. It is possible to trace BSH in immunohistochemistry, because BSH is firmly bound to the glioma tissue. The antibody against BSH is specific for that antigen, as tumor tissue from patients without BSH administration did not stain. In areas of healthy brain from BSH infused patients, no staining of tissue was detectable. In tumor tissues, BSH is presenting as a strong staining in cytoplasm and nucleus areas.

Pharmacokinetics of Na2B12H11SH (BSH) in patients with malignant brain tumours as prerequisite for a phase I clinical trial of boron neutron capture.

The disposition of Na2B12H11SH (BSH) in patients with malignant glioma has been investigated, in preparation for a Phase I clinical trial of boron neutron capture therapy. BSH was found to possess a linear disposition over the dosage interval investigated (up to 75 mg/kg). A bi-phasic blood pharmacokinetics was observed. Tumour-to-blood ratios showed variations between patients between 0.08 and 5.1. The data allow the definition of amount of BSH and timing of infusion for a Phase I clinical trial protocol.

Progress in the field of compounds for boron neutron capture therapy.

For successful application of boron neutron capture therapy to the treatment of cancer, selective accumulation of boron is required. This can be achieved by the preparation of suitable substances, which must contain boron and at the same time accumulate or be retained in tumor tissue. The radiobiological rationale for selection of suitable compounds is discussed. Examples of useful new compounds are given for which boronated analogues exist.

Present status and perspectives of boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is a mode of radiotherapy with great attractiveness, but also with a burden of past failure. In this review, the principles of BNCT, the reasons for its past failure, its present clinical application, and the on-going developmental work towards clinical trials are described.

Clinical phase-I study of Na2B12H11SH (BSH) in patients with malignant glioma as precondition for boron neutron capture therapy (BNCT).

PURPOSE: Within the European collaboration on boron neutron capture therapy (BNCT), a clinical Phase I study is being carried out to establish BNCT as an alternative treatment modality for malignant glioma (WHO III/IV). Data about the pharmacokinetics, biodistribution and toxicity of the boron compound Na2B12H11SH (BSH) are of great importance to avoid radiation damage of healthy tissue and to deliver a sufficient radiation dose. METHODS AND MATERIALS: Twenty four patients suffering from a glioblastoma multiforme entered the study to date, infused with a maximum concentration of up to 50 mg BSH/kg. Boron concentration measurements in tissues, urine, and blood were carried out, using inductively coupled plasma-atomic spectroscopy (ICP-AES) and quantitative neutron capture radiography (QNCR). A cross-calibration of these boron determination techniques was carried out. RESULTS: In tumor tissue, confirmed by histopathology of small biopsies, we found a consistently high but heterogeneous boron uptake. Necrotic parts contain much lower amounts of boron; normal brain tissue has shown no significant uptake. In skin, bone, muscle, and dura mater only small amounts of boron were found. In blood samples, we found biphasic kinetics, but with variations of the half-lives from patient to patient. The compound is mainly excreted through the urine, but an additional entero-hepatic pathway can be demonstrated. Systematic investigations revealed no toxic side effect of the intravenously administered BSH. Comparable data were obtained by using ICP-AES and QNCR for boron concentration measurements. CONCLUSION: Taking into account the radiobiological considerations of the neutron beam source, we found promising facts that BNCT could be a useful irradiation method for highly malignant brain tumors. Favorable amounts of the boron compound BSH were found in tumor tissue, whereas healthy brain tissue has shown no significant uptake.

Radiobiological considerations concerning the development of compounds for boron neutron capture therapy.

An analysis is carried out to evaluate the suitability of compounds for boron neutron capture therapy (BNCT). Suitable boron compounds are not necessarily those that show a high uptake ratio between tumor and healthy tissue. Compounds with lower uptake ratios, but higher concentrations in both healthy tissue and tumor, can be as effective in increasing the dose to the tumor over that of healthy tissue. In compound synthesis and evaluation, the parameters for optimization should therefore not be limited to the uptake ratio. A final assessment of the question whether and how well a given compound is suitable for BNCT can only be made after a study of its radiobiological effects on tissue. The analysis also illustrates the importance of beam quality in thermal and epithermal BNCT. An increase of adventitious radiation must be compensated by higher absolute tissue levels of a given boron compound and/or a higher uptake ratio, in order to arrive at the same dose differential between tumor and healthy tissue.

Biodistribution and toxicity of 2,4-divinyl-nido-o-carboranyldeuteroporphyrin IX in mice.

BALB/c mice with transplanted subcutaneous KHJJ mammary carcinomas were given 2,4-divinyl-nido-o-carboranyldeuteroporphyrin IX (VCDP), a prospective boron carrier for boron neutron-capture therapy, to determine the dose schedule that results in maximal boron uptake in tumor. A total dose of 270 +/- 10 micrograms/g body weight given in a 4-day multiple intraperitoneal injection schedule (3/day) resulted in 30-50 micrograms boron/g tumor. After such a dose, thrombocytopenia, granulocytosis and altered liver enzyme levels were measured in the blood. Blood boron clearance was followed for an 18 hr to 6 day post-injection period. Toxic effects of VCDP subsided within 4-6 days after the last injection. In view of the greater than 30 micrograms/g peak accumulation of boron in tumor from VCDP and the subsequent rapid reversal of VCDP toxicity, further studies of VCDP in small mammals relevant to its distribution, toxicity and potential clinical use for neutron-capture therapy of tumors appear warranted.

The influence of periodate oxidation on monoclonal antibody avidity and immunoreactivity.

Eight monoclonal antibodies of different classes and isotypes and rabbit IgG were oxidized under a variety of conditions with 5-50 mM periodate. The number of aldehyde groups generated per immunoglobulin were measured by reduction with tritiated sodium borohydride or coupling of fluoresceinthiosemicarbazide. There were up to 25.5 aldehyde groups detected on the periodate-oxidized antibody 96.5, measured by borohydride reduction whereas the same conditions led to only 9.6 aldehydes per IgG on the antibody L6 of the same IgG2A isotype. Fluoresceinthiosemicarbazide bound to oxidized antibodies but not to the same extent as tritium. On mildly oxidized IgMs it was possible to generate more than 200 aldehyde groups per antibody molecule. Depending on the conditions and the antibody used periodate oxidation could lead to antibody crosslinking. The avidities of the modified antibodies were determined by Scatchard analyses and inhibition assays. A new mathematical method to evaluate the immunoreactivities of modified antibodies relative to the unlabeled native antibody from inhibition binding data was established. Periodate concentrations higher than 50 mM decreased the avidities and immunoreactivities of all IgGs tested. This effect is more pronounced if the oxidation is performed at pH 5.6 and 25 degrees C instead of pH 4.6 and 0 degree C. The BR96 antibody is inactivated even under mild oxidation conditions.