Mechanism of action of SNF472, a novel calcification inhibitor to treat vascular calcification and calciphylaxis.

BACKGROUND AND PURPOSE: No therapy is approved for vascular calcification or calcific uraemic arteriolopathy (calciphylaxis), which increases mortality and morbidity in patients undergoing dialysis. Deposition of hydroxyapatite (HAP) crystals in arterial walls is the common pathophysiologic mechanism. The mechanism of action of SNF472 to reduce HAP deposition in arterial walls was investigated. EXPERIMENTAL APPROACH: We examined SNF472 binding features (affinity, release kinetics and antagonism type) for HAP crystals in vitro, inhibition of calcification in excised vascular smooth muscle cells from rats and bone parameters in osteoblasts from dogs and rats. KEY RESULTS: SNF472 bound to HAP with affinity (KD ) of 1-10 µM and saturated HAP at 7.6 µM. SNF472 binding was fast (80% within 5 min) and insurmountable. SNF472 inhibited HAP crystal formation from 3.8 µM, with complete inhibition at 30.4 µM. SNF472 chelated free calcium with an EC50 of 539 µM. Chelation of free calcium was imperceptible for SNF472 1-10 µM in physiological calcium concentrations. The lowest concentration tested in vascular smooth muscle cells, 1 µM inhibited calcification by 67%. SNF472 showed no deleterious effects on bone mineralization in dogs or in rat osteoblasts. CONCLUSION AND IMPLICATIONS: These experiments show that SNF472 binds to HAP and inhibits further HAP crystallization. The EC50 for chelation of free calcium is 50-fold greater than a maximally effective SNF472 dose, supporting the selectivity of SNF472 for HAP. These findings indicate that SNF472 may have a future role in the treatment of vascular calcification and calcific uraemic arteriolopathy in patients undergoing dialysis.

GlutenTox® Pro Test for the Detection of Gluten in Select Foods and Surfaces.

The GlutenTox® Pro Test is an immunochromatographic test for the detection of gluten in foods and on surfaces with varying compositions and levels of processing, from raw foods/ingredients to final product testing. The Method Developer evaluation for the validation of the GlutenTox Pro Test Kit (Biomedal Diagnostics, Sevilla, Spain) for the detection of gluten in foods and on surfaces was conducted at Biomedal, S. L., Camas, Sevilla, Spain. The GlutenTox Pro test method was evaluated by testing the following: cross-reactivity, interference, specificity and sensitivity, robustness, stability, lot-to-lot variation, food matrix, and environmental surface. To evaluate the performance of the GlutenToxPro test for the detection of gluten, 10 matrixes were selected: rice flour, bread/biscuit, rolled oat, pâté, and yogurt (and a second bread matrix for incurred sampled testing) for the food matrix study and food-grade painted wood, plastic, rubber, sealed ceramic, and stainless steel for the environmental surface matrix study. For the food matrix study, 30 replicates were evaluated at six spiked levels of gluten (0, 3, 8, 15, 25, and 45 ppm) against four detection thresholds (5, 10, 20, and 40 ppm) for each food matrix. Additionally, 10 replicates were evaluated at a concentration of 10,000 ppm using all four detection thresholds only for rice flour matrix. Three replicates of each concentration level of gluten were analyzed using paired samples by the AOAC OMA 2012.01 reference method for each food matrix. For the environmental surface study, 30 replicates were evaluated at a low spike level of gluten (16 ng/16 cm2), five replicates at a high spike level of gluten (400 ng/16 cm2), and five replicates at an unspiked control level (0 ng/16 cm2) for each surface matrix. Upon completion of testing, the probability of detection values and confidence intervals were calculated and plotted versus the concentration level as determined by the reference method when applicable. An independent laboratory evaluation of the GlutenTox Pro Test Kit with rice flour and stainless steel environmental surface was conducted at Q Laboratories, Inc. (Cincinnati, OH). The GlutenTox Pro Test Kit demonstrated reliability as an effective rapid method for the detection of gluten in food matrixes (LOD 5 ppm gluten; threshold limits 5, 10, 20, and 40 ppm gluten) and on environmental surfaces (amount of detection 16 ng/16 cm2).

Investigating a novel nanostructured fibrin-agarose biomaterial for human cornea tissue engineering: rheological properties.

In this work, the rheological properties of the biomaterial fibrin with different agarose concentrations, used for the generation of a bioengineered human corneal stroma by tissue engineering, before and after using a nanostructuring technique, were analyzed. The transparency of these artificial human stromas was also investigated. The temporal evaluation of the properties of these biomaterials is essential for the design of functional biological human corneal replacements. The nanostructuring technique used for the generation of nanostructured corneal constructs (NCCs) had a major influence on the rheological properties of the fibrin-agarose corneal equivalents. For an oscillatory shear stress of 1 Hz, well in the order of the natural oscillations of the human cornea, the NCCs had viscoelasticity values higher than those of non-nanostructured corneal constructs (N-NCCs), but similar to those of an ex vivo native cornea. The model that most resembled the rheological behavior of the native cornea was a fibrin-0.1% agarose concentration nanostructured construct. In addition, this artificial cornea model displayed optimal levels of transparency, similar to the native tissue. All these properties indicate that the fibrin-0.1% agarose concentration nanostructured construct might serve as an adequate candidate for the generation of an artificial complete cornea, not only for transplanting use but also for conducting pharmaceutical testing and biomedical research.

Generation of bioengineered corneas with decellularized xenografts and human keratocytes.

PURPOSE: Decellularization of animal corneas is a promising method for the development of artificial human corneas by tissue engineering. In this study, two different decellularization protocols were evaluated to determine which one is able to best preserve the histologic structure, composition, and optical behavior of decellularized porcine corneas. Then, these corneas were recellularized with human keratocytes to obtain a partial human corneal substitute. METHODS: Two different decellularization protocols were applied, using NaCl and SDS, to determine which one is able to best preserve the histologic structure, composition, and optical behavior of the decellularized corneas. Then, those decellularized corneas that showed the most appropriate results were recellularized with human keratocytes and evaluated at the histologic, biochemical, and optical levels for use in regenerative medicine. RESULTS: The results showed that 1.5 M NaCl treatment of porcine corneas is able to generate an acellular corneal stroma with adequate histologic and optical properties and that human keratocytes are able to penetrate and spread within this scaffold with proper levels of cell differentiation. In contrast, 0.1% SDS treatment of porcine corneas resulted in high levels of fibril disorganization and poor optical behavior of these corneas. CONCLUSIONS: In conclusion, the authors suggest that the decellularization of animal corneas with 1.5 M NaCl represents a useful method for the development of human bioengineered corneas with therapeutic potential.

UV absorbance of a bioengineered corneal stroma substitute in the 240-400 nm range.

PURPOSE: To determine the UV absorbance of a bioengineered human corneal stroma construct based on fibrin and fibrin-agarose scaffolds in the 240-400 nm range. METHODS: Three types of artificial substitutes of the human corneal stroma were developed by tissue engineering using fibrin and fibrin with 0.1% and 0.2% agarose scaffolds with human keratocytes immersed within. After 28 days of culture, the UV absorbance of each sample was determined using a spectrophotometer. The thickness of corneal stroma samples was determined by light microscope. RESULTS: For all the 3 types of corneal stroma substitutes studied, the range of the UV absorbance values was similar to that of the native human corneal stroma, although the fibrin with 0.1% agarose stroma substitute had the best UV filtering properties. The higher UV absorbance of the artificial substitute of the human corneal stroma was in the UV-B and -A ranges, suggesting that these artificial tissues could have potential clinical usefulness and proper UV light-absorption capabilities. CONCLUSION: Our data suggest that the bioengineered human corneal substitute of fibrin with 0.1% agarose is an effective absorber of harmful UV radiation and could therefore be potentially useful.

Light polymerization-dependent changes in color and translucency of resin composites.

PURPOSE: To determine the influence of type of polymerization light on changes in color and translucency of resin composites. METHODS: 16 shades of commercial composites were analyzed. Specimens were polymerized with quartz-tungsten-halogen (QTH) and light-emitting diode (LED) polymerization light. Color of non-polymerized and polymerized composites was measured against white and black backgrounds using a spectroradiometer. Changes in color (deltaE'), translucency (deltaTP') and color parameters (deltaL', deltaC' and deltah') were calculated for each polymerization light. The differences among deltaE' values and among deltaTP' values obtained for each device were analyzed by two-way ANOVA. Multiple regression analysis was used to determine the influence of color parameters on deltaE' and deltaTP'. RESULTS: The results indicated that there was significant difference among the deltaE' and deltaTP' values obtained using QTH and LED light polymerization (P< 0.05). The prediction equations for deltaE' and deltaTP' based on the multiple regression analyses indicated that deltaE' was mainly caused by deltaC' for both lights. However deltaTP' was mainly caused by deltah' for LED light and by the deltaC' for QTH light. Changes in translucency significantly influenced the overall color changes after polymerization.

Color difference thresholds of maxillofacial skin replications.

PURPOSE: The purpose of this study was to determine perceptibility and acceptability thresholds for color differences in light and dark skin-colored maxillofacial elastomers. MATERIALS AND METHODS: A total of 15 pairs of light specimens (mimicking white, Asian, and Hispanic skin) and 15 pairs of dark specimens (mimicking African-American skin) were made using skin-colored maxillofacial silicone elastomers, combined with opacifiers and pigments. Color match/mismatch and acceptable/unacceptable mismatch of each pair of specimens were visually evaluated by 45 evaluators under controlled conditions of a viewing booth. Color differences were calculated using CIELAB and CIEDE2000 formulae. After calculating the model parameters, receiver operating characteristics (ROC) curves and area under the ROC curve (AUC) were analyzed. Repeated measures ANOVA and Tukey's HSD test were used in a statistical analysis (alpha= 0.05). RESULTS: CIELAB/CIEDE2000 perceptibility and acceptability thresholds for light specimens were 1.1/0.7 and 3.0/2.1, respectively. Corresponding values for dark specimens were 1.6/1.2 and 4.4/3.1, respectively. Differences in primary specimen color and type of threshold were found to be significant (p < 0.001). Only the primary specimen color effect was found to be significant in AUC comparisons. CONCLUSIONS: Within the limitations of this study, both main effects of threshold type (perceptibility and acceptability) and primary color (light and dark) on 50:50% color-difference thresholds of colored maxillofacial elastomers were found significant for both color-difference formulae used (CIELAB, CIEDE2000). In addition, significant interaction between the two main effects was found, indicating a stronger effect of skin type on acceptability than perceptibility thresholds. Primary specimen color (light vs. dark) was found to be the only significant main effect on the AUC of ROC curves constructed from logistic regression.

Phase II trial of vinorelbine and estramustine in the treatment of metastatic hormone-resistant prostate cancer.

The purpose of this study was to investigate the efficacy and safety of combination chemotherapy using estramustine and vinorelbine in chemotherapy-naïve patients with hormone-resistant prostate cancer (HRPC). The patients (n = 54) received oral estramustine 840 mg/day on Days 1 to 14 and IV vinorelbine 25 mg/m(2) on Days 1 and 8 of every 3 wk cycle. The median number of cycles per patient was 9 (range, 1 to 27). Fifty-three patients were evaluable for toxicity and survival and 52 for prostate specific antigen (PSA) response. Median age was 68 (range, 46-80). PSA sustained decrease >50% was seen in 52% of patients (95% CI: 38-66%). A complete response was seen in 3 and a partial response in 12 of 25 patients with measurable disease, for an overall objective response of 60% (95% CI: 41-79%). Improvement in performance status was observed in 30 out of 43 evaluable for clinical benefit response. The median duration of response was 7 mo and median time to progression was 6 mo. The median survival time was 15 mo. The most common adverse event was mild gastrointestinal toxicity. In general, toxicity G3-4 was low: granulocytopenia Grade 3-4 (8%), thrombocytopenia Grade 3 (6%), and anemia Grade 3 (13%). Other Grade 3 toxicities included deep vein thrombosis (4%), hepatic (2%), cardiac ischemia (2%), fatigue (6%), and sensory neuropathy (2%). There were 2 treatment-related deaths (4%). We conclude that vinorelbine and estramustine as used in this trial is an efficacious and well-tolerated therapeutic regimen in the management of HRPC.