Biochemical characterization of LR769, a new recombinant factor VIIa bypassing agent produced in the milk of transgenic rabbits.

BACKGROUND: The bypassing agent factor VII (FVIIa) is a first-line therapy for the treatment of acute bleeding episodes in patients with haemophilia and high-titre inhibitors. FVIIa is a highly post-translationally modified protein that requires eukaryotic expression systems to produce a fully active molecule. A recombinant FVIIa was produced in the milk of transgenic rabbits to increase expression and provide an efficient, safe and affordable product after purification to homogeneity (LR769). AIM: To present the biochemical and functional in vitro characteristics of LR769. RESULTS: Mass spectrometric analyses of the intact protein and of heavy and light chains revealed a fully activated, mature and properly post-translationally modified protein notably regarding N/O-glycosylations and γ-carboxylation. Primary structure analysis, performed by peptide mapping, confirmed 100% of the sequence and the low level or absence of product-derived impurities such as oxidized, deamidated and glycated forms. Low levels of aggregates and fragments were observed by different chromatographic methods. Higher order structure investigated by circular dichroism showed appropriate secondary/tertiary structures and conformational change in the presence of Ca2+ ions. Finally, activated partial thromboplastin time and thrombin generation assays showed the ability of LR769 to decrease coagulation time and to generate thrombin in haemophiliac-A-plasmas, even in the presence of inhibitors. CONCLUSION: The innovative expression system used to produce LR769 yields a new safe and effective rhFVIIa for the treatment of haemophilia A or B patients with inhibitors.

Microdosimetric spread for cell-sized targets exposed to 6°Co, ¹9²Ir and ¹²5I sources.

The magnitude of the spread in specific energy deposition per cell may be a confounding factor in dose-response analysis motivating derivation of explicit data for the most common brachytherapy isotopes (125)I and (192)Ir, and for (60)Co radiation frequently used as reference in RBE studies. The aim of this study is to analyse the microdosimetric spread as given by the frequency distribution of specific energy for a range of doses imparted by (125)I, (192)Ir and (60)Co sources. An upgraded version of the Monte Carlo code PENELOPE was used for scoring energy deposition distributions in liquid water for each of the radiation qualities. Frequency distributions of specific energy were calculated according to the formalism of Kellerer and Chmelevsky. Results indicate that the magnitude of the microdosimetric spread increases with decreasing target size and decreasing energy of the radiation quality. Within the clinical relevant dose range (1 to 100 Gy), the spread does not exceed 4 % for (60)Co, 5 % for (192)Ir and 6 % for (125)I. The frequency distributions can be accurately approximated with symmetrical normal distributions at doses down to 0.2 Gy for (60)Co, 0.1 Gy for (192)Ir and 0.08 Gy for (125)I.

Track structure of protons and other light ions in liquid water: applications of the LIonTrack code at the nanometer scale.

PURPOSE: The LIonTrack (Light Ion Track) Monte Carlo (MC) code for the simulation of H(+), He(2+), and other light ions in liquid water is presented together with the results of a novel investigation of energy-deposition site properties from single ion tracks. METHODS: The continuum distorted-wave formalism with the eikonal initial state approximation (CDW-EIS) is employed to generate the initial energy and angle of the electrons emitted in ionizing collisions of the ions with H2O molecules. The model of Dingfelder et al. ["Electron inelastic-scattering cross sections in liquid water," Radiat. Phys. Chem. 53, 1-18 (1998); "Comparisons of calculations with PARTRAC and NOREC: Transport of electrons in liquid water," Radiat. Res. 169, 584-594 (2008)] is linked to the general-purpose MC code PENELOPE/penEasy to simulate the inelastic interactions of the secondary electrons in liquid water. In this way, the extended PENELOPE/penEasy code may provide an improved description of the 3D distribution of energy deposits (EDs), making it suitable for applications at the micrometer and nanometer scales. RESULTS: Single-ionization cross sections calculated with the ab initio CDW-EIS formalism are compared to available experimental values, some of them reported very recently, and the theoretical electronic stopping powers are benchmarked against those recommended by the ICRU. The authors also analyze distinct aspects of the spatial patterns of EDs, such as the frequency of nearest-neighbor distances for various radiation qualities, and the variation of the mean specific energy imparted in nanoscopic targets located around the track. For 1 MeV/u particles, the C(6+) ions generate about 15 times more clusters of six EDs within an ED distance of 3 nm than H(+). CONCLUSIONS: On average clusters of two to three EDs for 1 MeV/u H(+) and clusters of four to five EDs for 1 MeV/u C(6+) could be expected for a modeling double strand break distance of 3.4 nm.

Endothelin-1 levels in patients with Paget's disease of bone.

A locally accelerated bone turnover is the pathophysiological basis of Paget's disease of bone (PD) and may result in severe bone deformations and pain. Affected bone sites are hypervascularized. Secreted endothelial products such as endothelin-1 (ET-1), influence bone metabolism. We investigated a possible correlation between ET-1 plasma concentrations and bone metabolism in patients with PD and whether ET-1 plasma levels are regulated by i. v. bisphosphonate treatment. Plasma ET-1 levels were determined in 22 patients with PD and found to be significantly (p = 0.006) elevated (0.75 +/- 0.48 fmol/ml) compared to 19 healthy controls (0.20 +/- 0.24 fmol/ml). In a group of five patients with PD, plasma ET-1 levels were determined before and after treatment with i. v. pamidronate. On the average, ET-1 levels decreased by 21 % after pamidronate infusions (p = 0.045). The results suggest that bone metabolism in pagetic bone affects endothelial cell metabolism and may also be modulated by endothelial cell products. ET-1 plasma levels may indicate PD activity.

Dose and time dependent apoptotic response in a human melanoma cell line exposed to accelerated boron ions at four different LET.

The aim was to investigate and compare the influence of linear energy transfer (LET), dose and time on the induction of apoptosis in a human melanoma cell line exposed to accelerated light boron ((10)B) ions and photons. Cells were exposed in vitro to doses up to 6 Gy accelerated boron ions (40, 80, 125 and 160 eV nm(-1)) and up to 12 Gy photons (0.2 eV nm(-1)). The induction of apoptosis was measured up to 9 days after irradiation using morphological characterization of apoptotic cells and bodies. In parallel, measurements of cell-cycle distribution, monitored by DNA flow cytometry, and cell survival based on the clonogenic cell survival assay, were performed. In addition, the induction and repair of DNA double-strand breaks (DSB), using pulsed-field gel electrophoresis (PFGE) were studied. Accelerated boron ions induced a significant increase in apoptosis as compared with photons at all time points studied. At 1-5 h the percentage of radiation-induced apoptotic cells increased with both dose and LET. At the later time points (24-216 h) the apoptotic response was more complex and did not increase in a strictly LET-dependent manner. The early premitotic apoptotic cells disappeared at 24 h following exposure to the highest LET (160 eV nm(-1)). A postmitotic apoptotic response was seen after release of the dose-, time- and LET-dependent G2/M accumulations. The loss of clonogenic ability was dose- and LET-dependent and the fraction of un-rejoined DSB increased with increasing LET. Despite the LET-dependent clonogenic cell killing, it was not possible to measure quantitatively a LET-dependent apoptotic response. This was due to the different time course of appearance and disappearance of apoptotic cells.

The influence of RBE variations in a clinical proton treatment plan for a hypopharynx cancer.

Currently, most clinical range-modulated proton beams are assumed to have a fixed overall relative biological effectiveness (RBE) of 1.1. However, it is well known that the RBE increases with depth in the spread-out Bragg peak (SOBP) and becomes about 10% higher than mid-SOBP RBE at 2 mm from the distal edge (Paganetti 2003 Technol. Cancer Res. Treat. 2 413-26) and can reach values of 1.3-1.4 in vitro at the distal edge (Robertson et al 1975 Cancer 35 1664-77, Courdi et al 1994 Br. J. Radiol. 67 800-4). We present a fast method for applying a variable RBE correction with linear energy transfer (LET) dependent tissue-specific parameters based on the alpharef/betaref ratios suitable for implementation in a treatment planning system. The influence of applying this variable RBE correction on a clinical multiple beam proton dose plan is presented here. The treatment plan is evaluated by RBE weighted dose volume histograms (DVHs) and the calculation of tumour control probability (TCP) and normal tissue complication probability (NTCP) values. The variable RBE correction yields DVHs for the clinical target volumes (CTVs), a primary advanced hypopharynx cancer and subclinical disease in the lymph nodes, that are slightly higher than those achieved by multiplying the absorbed dose with RBE=1.1. Although, more importantly, the RBE weighted DVH for an organ at risk, the spinal cord is considerably increased for the variable RBE. As the spinal cord in this particular case is located 8 mm behind the planning target volume (PTV) and hence receives only low total doses, the NTCP values are zero in spite of the significant increase in the RBE weighted DVHs for the variable RBE. However, high NTCP values for the non-target normal tissue were obtained when applying the variable RBE correction. As RBE variations tend to be smaller for in vivo systems, this study-based on in vitro data since human tissue RBE values are scarce and have large uncertainties-can be interpreted as showing the upper limits of the possible effects of utilizing a variable RBE correction. In conclusion, the results obtained here still indicate a significant difference in introducing a variable RBE compared to applying a generic RBE of 1.1, suggesting it is worth considering such a correction in clinical proton therapy planning, especially when risk organs are located immediately behind the target volume.

Endothelin-1 levels in patients with disorders of the thyroid gland.

The endothelium derived peptide endothelin-1 (ET-1) is the major isoform of the endothelin peptide family, which is produced and secreted in the endothelial cell system. We measured plasma levels in patients with thyroid diseases and investigated associations between laboratory and clinical markers of thyroid metabolism and ET-1 plasma levels. ET-1 plasma levels were determined in patients with Graves' disease (n = 54), endemic goiter (n = 26), patients with Hashimoto's thyroiditis (n = 21) and compared to healthy controls (n = 60). ET-1 plasma levels were significantly elevated in patients with Hashimoto's thyroiditis (p < 0.0001) and in patients with Graves' disease (p = 0.003), when compared to healthy controls. In patients with endemic goiter, no significant differences were found compared to healthy controls (p = 0.298) and when compared to patients with Graves' disease (p = 0.16). We did not observe an association between ET-1 plasma levels and parameters of thyroid disease (e.g. thyroidea-stimulating hormone, thyroxine, volume of the thyroid). Furthermore, patients with and without endocrine thyroid disease showed no significantly different ET-1 plasma levels (p = 0.78). These data suggest that the autoimmunologically induced inflammatory response of the thyroid gland in Hashimoto's thyroiditis and Graves' disease is responsible for increased ET-1 plasma levels. Furthermore, our data do not support a role for ET-1 as a valid quantitative indicator for stage or progression in endemic goiter, Graves' disease or Hashimoto's thyroiditis.

Comparison of Monte Carlo calculated electron slowing-down spectra generated by 60Co gamma-rays, electrons, protons and light ions.

When analysing the factors affecting the relative biological effectiveness (RBE) of different radiation qualities, it is essential to consider particularly the low-energy slowing-down electrons (around 100 eV to 1 keV) since they have the potential of inflicting severe damage to the DNA. We present a modified and extended version of the Monte Carlo code PENELOPE that enables scoring of slowing-down spectra. mean local energy imparted spectra and average intra-track nearest-neighbour energy deposition distances of the secondary electrons generated by different radiation qualities, such as electrons, photons, protons and light ions in general. The resulting spectra show that the low-linear energy transfer (LET) beams, 60Co gamma-rays and electrons with initial energies of 0.1 MeV and higher, have as expected approximately the same electron slowing-down fluence per unit dose in the biologically important low-energy interval. Consistent with the general behaviour of the RBE of low-energy electrons, protons and light ions, the low-energy electron slowing-down fluence per unit dose is larger than for low-LET beams, and it increases with decreasing initial projectile energy.

Development of a compact proton scanning system in Uppsala with a moveable second magnet.

A scanned proton beam yields dose distributions that in most cases are superior to passively scattered proton beams and to other external radiation treatment modalities. The present paper gives a description of the scanning system that has been developed at the Svedberg Laboratory (TSL) in Uppsala. The scanning technique and the technical design are described. The solution with a small pole gap of the magnets and a moveable second magnet results in a very compact scanning head, which can therefore be incorporated in a gantry of relatively limited size. A prototype was constructed that has been used to realize various dose distributions with a scanned beam of 180 MeV protons at TSL.

Comparison of cell survival models for mixed LET radiation.

PURPOSE: Biophysical models for predicting survival for mixed LET radiations have been investigated by comparisons with experimental results from heavy ion irradiations. The aim was to choose a model for further theoretical studies on the effects of a variable RBE for protons. METHODS AND MATERIALS: Predicted survival curves by the Katz track-structure model, the linear quadratic model, LQ model, by Kellerer and Rossi and the lesion additivity model of Lam were compared to experimental survival curves for V79 cells that were irradiated with a mixture of nitrogen ions with an LET of either 78 or 165 keV/microm and 60Co gamma-rays. RESULTS: Results showed that all three models could predict survival within the uncertainty of the measurements for the different mixed radiation schedules used in this study. CONCLUSION: The choice of model could be made on other grounds, such as the type of model parameters and the availability of biological data for these parameters. Also, the possibility of including dose-rate effects and repair functions should be considered. For the purpose of carrying out theoretical studies on the effects of a variable RBE for protons, the LQ model was preferred.

Uptake, toxicity and radiation effects of the boron compounds DAAC-1 and DAC-1 in cultured human glioma cells.

PURPOSE: To study the uptake, toxicity and radiation effects in vitro of a diol-amino acid-carborane (DAAC-1) and make comparisons with the previously studied diol-amine-carborane (DAC-1). MATERIALS AND METHODS: Toxicity and radiation effects were studied with clonogenic survival, uptake by measuring the cellular boron content and the subcellular distribution was investigated after organelle separation with centrifugation. The studied cell line was human glioma U343. RESULTS: DAAC-1 showed an accumulation of 1-1.5 times, compared with the culture medium, and was non-toxic up to 47 microg boron/ml. The accumulation of DAC-1 was about 90 times, but toxic effects were detectable already at the concentration 5 microg boron/ml. None of the compounds was localized in the cell nucleus. Following irradiation with thermal neutrons, DAC-1 was about 2.5 times more effective than DAAC-1 and about 4.9 times more effective than neutrons alone, at the survival level 0.2. The dose modifying factors, when compared with the neutron beam alone, were for both DAAC-1 and DAC-1 about 1.5 and about 5 when compared with 60Co-gamma-radiation. CONCLUSIONS: DAAC-1 was less toxic than DAC-1 but gave less accumulation of boron. Both substances gave significant boron-dependent cell inactivation when the test cells were exposed to thermal neutrons.

In vitro determination of toxicity, binding, retention, subcellular distribution and biological efficacy of the boron neutron capture agent DAC-1.

In boron neutron capture therapy (BNCT), 10B is delivered selectively to the tumour cells and the nuclide then forms high-LET radiation (4He2+ and 7Li3+) upon neutron capture. Today much research is focused on development of a variety of boron compounds aimed for BNCT. The compounds must be thoroughly analysed in preclinical tests regarding basic characteristics such as binding and subcellular distribution to enable accurate estimations of dose-modifying factors. DAC-1,2-[2-(3-amino-propyl)-1,2-dicarba-closo-dodecaboran (12)-1-yl-methoxy]- 1,3-propanediol was synthesized at our laboratories and the human colon carcinoma cells LS-174T were used as an in vitro model. The boron compound showed a remarkable intracellular accumulation, 20-100 times higher than the boron content in the culture medium, in cultured cells and was not removed by extensive washes. Approximately half of the boron taken up also remained within the cells for at least 4 days. The DAC-1 compound alone was not toxic at boron concentrations below 2.5 micrograms B/g. The intracellular distribution of the boron compound was investigated by subcellular fractionation experiments and low pH treatments. It is possible that DAC-1 binds to some intracellular molecules or to membranes connected with organelles in the cytoplasm or even to the inside of the outer cell membrane. Another possibility is that the compound, due to the somewhat lipophilic properties, is embedded in the membranes. Thermal neutron irradiations were carried out at the Brookhaven Medical Research Reactor (BMRR). At a survival level of 0.1, DAC-1 + thermal neutrons were about 10.5 times more effective in cell inactivation than the thermal neutrons alone. Monte Carlo calculations gave a mean value of the 10B-dependent specific energy, the dose, of 0.22 Gy. The total physical dose during irradiation of DAC-1-containing cells with a neutron fluence of 0.18 x 10(12) n/cm2 was 0.39 Gy. The dose-modifying factor, at survival level 0.1, when comparing irradiation with thermal neutrons with and without DAC-1 was 3.4, while the dose-modifying factor when comparing neutron irradiations of cells with DAC-1 and irradiation of the cells with 60Co-gamma was 7.3. The results are encouraging and in vivo tests of tissue distributions and tumour uptake should now be carried out.