Europium(III) Meets Etidronic Acid (HEDP): A Coordination Study Combining Spectroscopic, Spectrometric, and Quantum Chemical Methods.

Etidronic acid (1-Hydroxyethylidene-1,1-diphosphonic acid, HEDP, H4L) is a proposed decorporation agent for U(VI). This paper studied its complex formation with Eu(III), an inactive analog of trivalent actinides, over a wide pH range, at varying metal-to-ligand ratios (M:L) and total concentrations. Combining spectroscopic, spectrometric, and quantum chemical methods, five distinct Eu(III)-HEDP complexes were found, four of which were characterized. The readily soluble EuH2L+ and Eu(H2L)2- species with log ß values of 23.7 ± 0.1 and 45.1 ± 0.9 are formed at acidic pH. At near-neutral pH, EuHL0s forms with a log ß of ~23.6 and, additionally, a most probably polynuclear complex. The readily dissolved EuL- species with a log ß of ~11.2 is formed at alkaline pH. A six-membered chelate ring is the key motif in all solution structures. The equilibrium between the Eu(III)-HEDP species is influenced by several parameters, i.e., pH, M:L, total Eu(III) and HEDP concentrations, and time. Overall, the present work sheds light on the very complex speciation in the HEDP-Eu(III) system and indicates that, for risk assessment of potential decorporation scenarios, side reactions of HEDP with trivalent actinides and lanthanides should also be taken into account.

Beyond Antiresorptive Activity: Risedronate-Based Coordination Complexes To Potentially Treat Osteolytic Metastases.

Coordination of clinically employed bisphosphonate, risedronate (RISE), to bioactive metals, Ca2+, Mg2+, and Zn2+, allowed the formation of bisphosphonate-based coordination complexes (BPCCs). Three RISE-based BPCCs, RISE-Ca, RISE-Mg, and RISE-Zn, were produced, and their structures were elucidated by single crystal X-ray diffraction. Interestingly, the addition of an auxiliary ligand, etidronic acid (HEDP), resulted in the recrystallized protonated form of the ligand, H-RISE. The pH-dependent structural stability of the RISE-based BPCCs was measured by means of dissolution profiles under neutral and acidic simulated physiological conditions (PBS and FaSSGF, respectively). In comparison to RISE (Actonel), the complexes showed a lower equilibrium solubility (∼70-85% in 18-24 h) in PBS, while a higher equilibrium solubility (∼100% in 3 h) in acidic media. The results point to the capacity to release this BP in a pH-dependent manner from the RISE-based BPCCs. Subsequently, the particle size of RISE-Ca was reduced, from 300 µm to ∼350 d.nm, employing the phase inversion temperature (PIT)-nanoemulsion method, resulting in nano-Ca@RISE. Aggregation measurements of nano-Ca@RISE in 1% fetal bovine serum (FBS):H2O was monitored after 24, 48, and 72 h to study the particle size longevity in physiological media, showing that the suspended material has the potential to maintain its particle size over time. Furthermore, binding assays were performed to determine the potential binding of nano-Ca@RISE to the bone, where results show higher binding (∼1.7×) for the material to hydroxyapatite (HA, 30%) when compared to RISE (17%) in 1 d. The cytotoxicity effects of nano-Ca@RISE were compared to those of RISE against the human breast cancer MDA-MB-231 and normal osteoblast-like hFOB 1.19 cell lines by dose-response curves and relative cell viability assays in an in vitro setting. The results demonstrate that nano-Ca@RISE significantly decreases the viability of MDA-MB-231 with high specificity, at concentrations ∼2-3× lower than the ones reported employing other third-generation BPs. This is supported by the fact that when normal osteoblast cells (hFOB 1.19), which are part of the tissue microenvironment at metastatic sites, were treated with nano-Ca@RISE no significant decrease in viability was observed. This study expands on the therapeutic potential of RISE beyond its antiresorptive activity through the design of BPCCs, specifically nano-Ca@RISE, that bind to the bone and degrade in a pH-dependent manner under acidic conditions.

Comparison of the physical properties of disodium etidronate amorphous forms prepared by different manufacturing methods.

Amorphous forms of disodium etidronate were prepared by three manufacturing methods, heat drying, freeze drying, and anti-solvent precipitation, and the effects of these methods on the physical properties of disodium etidronate amorphous forms were evaluated for the first time. Variable temperature X-ray powder diffraction and thermal analyses revealed that these amorphous forms had different physical properties such as glass transition point, water desorption, and crystallization temperatures. These differences can be explained by the molecular mobility and water content in amorphous forms. The differences in the structural characteristics related to the differences in these physical properties could not be detected clearly by the spectroscopic methods like Raman spectroscopy and X-ray absorption near-edge spectroscopy. Dynamic vapor sorption analyses demonstrated that all amorphous forms were hydrated to form I, a tetrahydrated form, at above 50% relative humidity, and the transition to form I was irreversible. These amorphous forms require strict humidity control to avoid crystallization. Among the three amorphous forms of disodium etidronate, the amorphous form prepared by heat drying was the most suitable for manufacturing the solid formulation, considering the low water content and low molecular mobility.

Investigation of Physical Properties of Disodium Etidronate Tetrahydrate and Application of Phosphorus K-Edge X-Ray Absorption Near-Edge Structure Spectroscopy.

PURPOSE: Disodium etidronate is a bisphosphonate, compounds that are widely used in the treatment of bone disorders such as osteoporosis and Paget's disease. We investigated the physical properties of disodium etidronate tetrahydrate crystal, form I. METHODS: We used X-ray powder diffraction (XRPD), thermal analysis, dynamic vapor sorption (DVS), X-ray single crystal structure analysis, and phosphorus K-edge X-ray absorption near-edge structure (XANES) spectroscopy for the first time. RESULTS: XRPD and thermal analyses demonstrated that form I was dehydrated and transformed to an amorphous form, to a crystalline form II, and finally to a form III by heating. DVS measurements revealed that the amorphous form, form II, and form III were rehydrated to form I by humidification, and form I was stable even at 0% relative humidity. These results indicate that form I is the most stable solid-state under ambient conditions and is suitable as an API for manufacture in solid formulations. The phosphorus K-edge XANES spectra differed among form I, the amorphous form, and form II, which may be ascribed to the difference in the coordinate bond schemes between the phosphate moieties and sodium ions. The results demonstrated that the phosphorus K-edge XANES spectroscopy could be applied to the identification or the discrimination of crystal forms of the APIs containing phosphate moieties. CONCLUSIONS: Acquired information about physical properties are crucial for manufacturing of solid formulations of disodium etidronate. XANES spectroscopy is a promising alternative method for evaluating the solid-state forms of APIs.

Understanding the Protective Effect of Phytate in Bone Decalcification Related-Diseases.

Myo-inositol hexaphosphate (phytate; IP6) is a natural compound that is abundant in cereals, legumes, and nuts, and it can bind to crystal surfaces and disturb crystal development, acting as crystallization inhibitor. The adsorption of such inhibitors to crystal faces can also inhibit crystal dissolution. The binding of phytate to metal cofactors suggests that it could be used for treatment of osteoporosis. Our in-vitro study showed that phytate inhibits dissolution of hydroxyapatite (HAP). The effect of phytate was similar to that of alendronate and greater than that of etidronate. This led us to perform a cross-sectional study to investigate the impact of consumption of IP6 on bone mineral density (BMD) in post-menopausal women. Our data indicate that BMD and t-score of lumbar spine increased with increasing phytate consumption, and a phytate consumption higher than 307 mg/day was associated with a normal BMD (t-score > -1). These data suggest that phytate may have a protective effect in bone decalcification by adsorbing on the surfaces of HAP, and a daily consumption of phytate-rich foods (at least one serving/day of legumes or nuts) may help to prevent or minimize bone-loss disorders, such as osteoporosis. However, further studies are needed to gain a better understanding about the mechanism of inhibition of phytate in bone-related diseases (see graphical abstract).

Mitigation of analyte loss on metal surfaces in liquid chromatography.

The adsorptive loss of acidic analytes in liquid chromatography was investigated using metal frits. Repetitive injections of acidic small molecules or an oligonucleotide were made on individual 2.1 or 4.6 mm i.d. column frits. Losses were observed for adenosine 5'-(α,ß-methylene) diphosphate, 2-pyridinol 1-oxide and the 25-mer phosphorothioate oligonucleotide Trecovirsen (GEM91) on stainless steel and titanium frits. Analyte adsorption was greatest at acidic pH due to the positive charge on the metal oxide surface. Analyte recovery increased when a series of injections was performed; this effect is known as sample conditioning. Nearly complete recovery was achieved when the metal adsorptive sites were saturated with the analyte. A similar effect was achieved by conditioning the frits with phosphoric, citric or etidronic acids, or their buffered solutions. These procedures can be utilized to mitigate analyte loss. However, the effect is temporary, as the conditioning agent is gradually removed by the running mobile phase. Metal frits modified with hybrid organic/inorganic surface technology were shown to mitigate analyte-to-metal surface interactions and improve recovery of acidic analytes. Quantitative recovery of a 15-35 mer oligodeoxythymidine mixture was achieved using column hardware modified with hybrid surface technology, without a need for column conditioning prior to analysis.

Batch Studies of Phosphonate and Phosphate Adsorption on Granular Ferric Hydroxide (GFH) with Membrane Concentrate and Its Synthetic Replicas.

Phosphonates are widely used as antiscalants for softening processes in drinking water treatment. To prevent eutrophication and accumulation in the sediment, it is desirable to remove them from the membrane concentrate before they are discharged into receiving water bodies. This study describes batch experiments with synthetic solutions and real membrane concentrate, both in the presence of and absence of granular ferric hydroxide (GFH), to better understand the influence of ions on phosphonate and phosphate adsorption. To this end, experiments were conducted with six different phosphonates, using different molar Ca:phosphonate ratios. The calcium already contained in the GFH plays an essential role in the elimination process, as it can be re-dissolved, and, therefore, increase the molar Ca:phosphonate ratio. (Hydrogen-)carbonate ions had a competitive effect on the adsorption of phosphonates and phosphate, whereas the influence of sulfate and nitrate ions was negligible. Up to pH 8, the presence of CaII had a positive effect on adsorption, probably due to the formation of ternary complexes. At pH > 8, increased removal was observed, with either direct precipitation of Ca:phosphonate complexes or the presence of inorganic precipitates of calcium, magnesium, and phosphate serving as adsorbents for the phosphorus compounds. In addition, the presence of (hydrogen-)carbonate ions resulted in precipitation of CaCO3 and/or dolomite, which also acted as adsorbents for the phosphorus compounds.

Leaching Behaviour and Enhanced Phytoextraction of Additives for Cadmium-Contaminated Soil by Pennisetum sp.

The leaching behavior of five additives, including citric acid (CA), wood vinegar (WV), 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP), polyaspartic acid (PASP) and FeCl3, was investigated to evaluate the possibility of enhanced phytoextraction of Pennisetum sp. from cadmium-contaminated soil. FeCl3 and CA have the highest leaching potential due to the ability that could convert large amounts of mobile fractions of Cd. The pot experiment showed that HEDP, WV, and PASP treatments could not only significantly increase the biomass of Pennisetum sp., but also maintain high uptake capacity of Cd by activating the stable fractions. HEDP has the highest Cd extraction efficiency and metal extraction ratio (MER) value. The phytoremediation efficiency could be improved mainly by increasing the biomass of the tolerant shoots, and Pennisetum sp. seems to have the maximum potential of phytoextraction to Cd with HEDP which could achieve a higher phytoextraction effect than Cd-hyperaccumulator.

[Basic Studies on the Mechanism, Prevention, and Treatment of Osteonecrosis of the Jaw Induced by Bisphosphonates].

Since the first report in 2003, bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been increasing, without effective clinical strategies. Osteoporosis is common in elderly women, and bisphosphonates (BPs) are typical and widely used anti-osteoporotic or anti-bone-resorptive drugs. BRONJ is now a serious concern in dentistry. As BPs are pyrophosphate analogues and bind strongly to bone hydroxyapatite, and the P-C-P structure of BPs is non-hydrolysable, they accumulate in bones upon repeated administration. During bone-resorption, BPs are taken into osteoclasts and exhibit cytotoxicity, producing a long-lasting anti-bone-resorptive effect. BPs are divided into nitrogen-containing BPs (N-BPs) and non-nitrogen-containing BPs (non-N-BPs). N-BPs have far stronger anti-bone-resorptive effects than non-N-BPs, and BRONJ is caused by N-BPs. Our murine experiments have revealed the following. N-BPs, but not non-N-BPs, exhibit direct and potent inflammatory/necrotic effects on soft-tissues. These effects are augmented by lipopolysaccharide (the inflammatory component of bacterial cell-walls) and the accumulation of N-BPs in jawbones is augmented by inflammation. N-BPs are taken into soft-tissue cells via phosphate-transporters, while the non-N-BPs etidronate and clodronate inhibit this transportation. Etidronate, but not clodronate, has the effect of expelling N-BPs that have accumulated in bones. Moreover, etidronate and clodronate each have an analgesic effect, while clodronate has an anti-inflammatory effect via inhibition of phosphate-transporters. These findings suggest that BRONJ may be induced by phosphate-transporter-mediated and infection-promoted mechanisms, and that etidronate and clodronate may be useful for preventing and treating BRONJ. Our clinical trials support etidronate being useful for treating BRONJ, although additional clinical trials of etidronate and clodronate are needed.

Organic Tissue Dissolution in Clodronate and Etidronate Mixtures with Sodium Hypochlorite.

INTRODUCTION: Mixtures of clodronate with sodium hypochlorite (NaOCl) better maintain free available chlorine (FAC) than etidronate-hypochlorite mixtures. This research aimed to compare organic tissue dissolution and residual FAC between clodronate and etidronate mixtures. Additionally, clodronate-hypochlorite mixtures lose no FAC over several hours. The second aim was to examine how well such mixtures dissolve organic material 6 hours from mixing. METHODS: Soon after mixing, porcine palatal mucosa samples were added to 32°C solutions containing 0.26 mol/L clodronate and 5% NaOCl (0.26 mol/L-5% NaOCl), 0.26 mol/L etidronate-5% NaOCl, 5% NaOCl, 0.26 mol/L clodronate, 0.26 mol/L etidronate, or phosphate-buffered saline. Weights and FAC, where applicable, were recorded initially and at 15 minutes. FAC was measured by iodometric titration. Secondly, 6 hours after mixing, mucosa was added to 0.26 mol/L clodronate-2.5% NaOCl, 2.5% NaOCl, 0.52 mol/L clodronate-5% NaOCl, 5% NaOCl, or phosphate-buffered saline. Sample weights at 0, 5, 10, and 15 minutes were recorded. Analysis of variance was used for statistical analyses (α < .05). RESULTS: Soon after mixing, 0.26 mol/L clodronate-5% NaOCl dissolved mucosa as well as 5% NaOCl and better than 0.26 mol/L etidronate-5% NaOCl compared with which it retained more FAC. At 6 hours after mixing, 0.26 mol/L clodronate-2.5% NaOCl dissolved organic material as well as 2.5% NaOCl. However, 0.52 mol/L clodronate-5% NaOCl dissolved less mucosa than 5% NaOCl. CONCLUSIONS: Soon after mixing, clodronate mixtures better dissolve organic material than etidronate mixtures and have higher residual FAC. Six hours from mixing, 0.26 mol/L clodronate-2.5% NaOCl mixtures dissolve organic material similarly to controls.

The efficacy of different irrigation protocols in removing tricalcium silicate-based sealers from simulated root canal irregularities.

OBJECTIVE: The aim of this study was to evaluate the efficacy of different irrigation protocols in removing two tricalcium silicate-based sealers from simulated root canal irregularities and root canal walls. MATERIAL AND METHODS: Root canals of 140 single-rooted teeth were instrumented. In one-half of each root, an apical groove was created. The samples were divided into two main groups (n = 70) based on the sealer used. In group 1, the grooves were filled with MTA Fillapex; in group 2, BioRoot RCS. The reassembled root halves were divided into six experimental and one control groups: 2.5% NaOCl-17% EDTA (Passive ultrasonic irrigation [PUI]), 5% NaOCl/9% DualRinse HEDP (PUI), 2.5% NaOCl-7% Maleic acid (PUI), 2.5% NaOCl-17% EDTA (Er:YAG laser activated irrigation [LAI]), 2.5% NaOCl/9% DualRinse HEDP (LAI), 2.5% NaOCl-7% Maleic acid (LAI), Distilled water (Control). Specimens were scored using SEM. The data were analysed using Kruskal-Wallis and Mann Whitney U tests. RESULTS: Maleic acid and DualRinse HEDP removed higher amounts of MTA Fillapex from the grooves compared to EDTA, when used with both activation methods (p < .001). CONCLUSIONS: Ultrasonically activated maleic acid or DualRinse HEDP can be an effective irrigation regimen in removing tricalcium silicate-based sealers.

The Effect of Heating to Intracanal Temperature on the Stability of Sodium Hypochlorite Admixed with Etidronate or EDTA for Continuous Chelation.

INTRODUCTION: Tetrasodium etidronate (Na4 etidronate) and tetrasodium EDTA (Na4 EDTA) are chelators that can combine with sodium hypochlorite (NaOCl) as a 1-mix endodontic irrigant in a process called continuous chelation. The therapeutic window of these mixtures is determined by the chemical reaction between NaOCl and the chelator. At room temperature, this window is 60 minutes for Na4 etidronate and 30 minutes for Na4 EDTA. Because reaction kinetics are influenced by heat, this study assesses the influence of heating to an intracanal temperature of 35°C on the therapeutic window in continuous chelation. METHODS: The loss of free available chlorine (FAC) in NaOCl mixtures with Na4 etidronate or Na4EDTA was determined by iodometric titration at 23°C ± 0.7°C (23°C) and 34.6°C ± 0.3°C (35°C) at 1, 20, 40, and 60 minutes after mixing. The pH and temperature of the mixtures were measured. RESULTS: At 23°C, 18% Na4 etidronate/5% NaOCl solutions at 20, 40, and 60 minutes lost 4%, 9% and 18% FAC, and at 35°C, they lost 20%, 68% and 92% FAC; 5% Na4 EDTA/2.5% NaOCl solutions at 20, 40, and 60 minutes at 23°C lost 88%, 94%, and 97% FAC, and at 35°C, they lost 96%, 99%, and 100%. Decreases in FAC were accompanied by pH declines. CONCLUSIONS: The effect of heating to 35°C from a room temperature of 23°C on 18% Na4 etidronate/5% NaOCl solutions reduces its therapeutic window to 20 minutes. Solutions of 5% Na4 EDTA/2.5% NaOCl are not useful in the continuous chelation technique. Tracking pH changes could be used to estimate NaOCl degradation.

Etidronate-zinc Complex Ameliorated Cognitive and Synaptic Plasticity Impairments in 2-Vessel Occlusion Model Rats by Reducing Neuroinflammation.

As one of the bisphosphonate derivatives, etidronate has proved to be beneficial to spatial learning and memory deficits caused by two-vessel occlusion (2-VO). In this study, the novel drug etidronate-zinc complex (Eti-Zn) was used to detect its role in synaptic plasticity and learning and memory functions in a rat model of 2-VO. Chronic cerebral hypoperfusion was induced by permanent occlusion of the common carotid artery bilaterally in adult Sprague-Dawley rats. Eti-Zn (20 mg/kg/day, tail vein injection) was administered for 7 days after a two-week operation. After treatment, a series of tests were carried out. Here, we found that Eti-Zn could reduce spatial learning and memory impairments in 2-VO model rats via the Morris water maze test. We also found that animals treated with Eti-Zn showed preference for the new-object in the novel object recognition test. In addition, the long-term potentiation and depotentiation from the Schaffer collaterals to the CA1 region in the hippocampus were enhanced by Eti-Zn treatment in 2-VO model rats. Furthermore, Eti-Zn significantly up-regulated NMDA receptor (NR) 2A, NR2B, postsynaptic density protein 95 and synaptophysin levels and prevented the destruction of dendritic spines. Moreover, Eti-Zn treatment reduced both the over-activation of microglia and the expressions of neuroinflammatory cytokines (TNF-α, IL-1ß and IL-6) in the hippocampus. The increased NF-κB signaling pathway in the hippocampus of 2-VO rats was reversed after Eti-Zn treatment. In summary, these findings suggest that Eti-Zn could ameliorate the synaptic plasticity and cognitive impairments by reducing neuroinflammation in 2-VO model rats.

Short-term storage stability of NaOCl solutions when combined with Dual Rinse HEDP.

AIM: To assess the stability of NaOCl solutions when combined with a novel product for clinical use, Dual Rinse HEDP, which contains etidronate (1-hydroxyethane 1,1-diphosphonate). METHODOLOGY: Mixtures of NaOCl solutions with Dual Rinse HEDP were prepared so that they initially contained 5.0%, 2.5% or 1.0% NaOCl and always 9.0% of dissolved Dual Rinse HEDP powder per total weight. NaOCl solutions alone were used as controls. The stability of these solutions over 8 h was assessed in transparent borosilicate glass bottles at ambient temperature (23 °C). Subsequently, the effects of heating (60 °C) or storing the solutions at 5 °C were studied in polypropylene syringes. NaOCl concentrations were measured by iodometric titration, that is free available chlorine contents. Experiments were performed in triplicate. RESULTS: In the glass bottles at 23 °C, the 5.0% NaOCl/9.0% Dual Rinse HEDP solution lost 20% of the available chlorine after 1 h, whilst the corresponding 2.5% NaOCl and 1.0% NaOCl solutions retained this relative amount of available chlorine for 2 and 4 h, respectively. Results obtained in the glass bottles were similar to those achieved in the syringes. Heating of the NaOCl/Dual Rinse HEDP mixtures had a detrimental effect on available chlorine, with a complete loss after 1 h. In contrast, storing the NaOCl/Dual Rinse HEDP mixtures in a refrigerator at 5 °C kept the available chlorine high for 7 h, with the expected loss after a further hour of storage at 23 °C. CONCLUSIONS: Initial NaOCl concentration and temperature both affected short-term storage stability of combined solutions containing Dual Rinse HEDP.

Development of bone seeker radiopharmaceuticals by Scandium-47 and estimation of human absorbed dose.

In this study labeling EDTMP (ethylenediamine tetra(methylene phosphonic acid)) and HEDP (Hydroxyethylidene-1, 1-Diphosphonic Acid) as the carrier ligands with Scandium-47 were investigated. The biokinetics of the bone seeking of labeled ligands with Scandium-47 were assessed by measuring the skeletal absorbed dose and then the mice data extrapolated to human absorbed dose and compared with the 186/188Rhenium-HEDP, 153Samarium-EDTMP dosimetry data estimated by other researchers. Because the availability of 47Sc was limited we performed some preliminary studies using 46Sc.

Phosphate functionalized (4,4)-armchair CNTs as novel drug delivery systems for alendronate and etidronate anti-osteoporosis drugs.

The ability of (4,4)-armchair CNT and its three phosphate functionalized forms (CNT-nH2PO4, n=1-3) were evaluated as novel drug delivery systems (DDSs) for the two commercially well-known anti-osteoporosis drugs namely alendronate (AL) and etidronate (ET). For this purpose, the DFT calculations were accomplished at both B3LYP and B3PW91 levels using 6-31g(d) basis set. The binding energy was increased by increasing number of H2PO4 moieties attached on the CNT with the most negative binding energy was measured for the carrier containing three phosphate groups. The dipole moments of all phosphate containing CNTs were much greater (∼2.2-4.4D) than that of pristine CNT (∼0D). The contour maps proved that when the CNT was functionalized by H2PO4 groups, the symmetric distribution of electric charge was vanished with the charge distribution was the highest asymmetric for the CNT-2H2PO4 while it was the lowest asymmetric for CNT-3H2PO4 leading to the greatest dipole moment for the CNT-2H2PO4 (4.177D) while the smallest dipole moment for the CNT-3H2PO4 (1.614D). Among all compounds, those containing the CNT-3H2PO4 exhibited the smallest band gap energy, chemical potential and hardness but the greatest electronegativity and electrophilicity index which were all suitable and effective for the attachment of drugs onto the bone surface (having partial positive charge due to the presence of Ca2+ as CaCO3) and therefore inhibiting the osteoporosis. Consequently, it was established that the drug-CNT-3H2PO4 was the most appropriate drug-carrier compound for both of the AL and ET drugs and it could be used as the most effective drug vehicle. The attachment of AL, ET drugs as well as the AL-CNT-3H2PO4 and ET-CNT-3H2PO4 drug-carrier systems to the bone tissue was modelled by optimization of the structures of these compounds bonded to the hydroxyapatite (HA)-17water (w). It was found that among these four systems, the AL-CNT-3H2PO4 could be suggested as the most suitable DDS for application in the treatment of osteoporosis.

Interactions between the Tetrasodium Salts of EDTA and 1-Hydroxyethane 1,1-Diphosphonic Acid with Sodium Hypochlorite Irrigants.

INTRODUCTION: A clinically useful all-in-one endodontic irrigant with combined proteolytic and decalcifying properties is still elusive. In this study, the chemical effects of dissolving the tetrasodium salts of 1-hydroxyethane 1,1-diphosphonic acid (Na4HEDP) or Na4EDTA directly in sodium hypochlorite (NaOCl) irrigants in polypropylene syringes were assessed during the course of 1 hour. METHODS: The solubility of the salts in water was determined. Their compatibility with 1% and 5% NaOCl was measured by iodometric titration and in a calcium complexation experiment by using a Ca2+-selective electrode. RESULTS: The salts dissolved within 1 minute. The dissolution maximum of Na4HEDP in water (wt/total wt) was 44.6% ± 1.6%. The corresponding dissolution maximum of Na4EDTA was 38.2% ± 0.8%. Na4HEDP at 18% in 5% NaOCl caused a mere loss of 16% of the initially available chlorine during 1 hour. In contrast, a corresponding mixture between NaOCl and the Na4EDTA salt caused 95% reduction in available chlorine after 1 minute. Mixtures of 3% Na4EDTA with 1% NaOCl were more stable, but only for 30 minutes. Na4HEDP lost 24% of its calcium complexation capacity after 60 minutes. The corresponding loss for Na4EDTA was 34%. CONCLUSIONS: The compatibility and solubility of particulate Na4HEDP with/in NaOCl solutions are such that these components can be mixed and used for up to 1 hour. In contrast, short-term compatibility of the Na4EDTA salt with NaOCl solutions was considerably lower, decreasing at higher concentrations of either compound. Especially for Na4HEDP but also for Na4EDTA, the NaOCl had little effect on calcium complexation.

Delineating residues for haemolytic activities of snake venom cardiotoxin 1 from Naja naja as probed by molecular dynamics simulations and in vitro validations.

Cardiotoxins (CTXs) are single polypeptide chain consisting of 59-62 amino acids with four disulfide bridges and globular proteins of simple ß-sheet folds. The CTXs are one of principal toxic components causing haemolysis and damaging various cells and belong to three-finger toxin (TFT) superfamily of snake venoms. However, there is no natural or synthetic small molecular inhibitor to the protein toxins to date. In the present study, modes of interaction of cardiotoxin 1 (CTX1) from Indian cobra (Naja naja) with heterogeneous erythrocyte membrane (EM) model system have been extensively examined by using all-atom molecular dynamics (MD) simulations in near physiological conditions and comprehensive analyses of the MD data revealed two distinct principal regions ('head groove' and 'loop groove') of the protein toxin for establishing structural interactions with the EM system. Moreover, combined analyses of data from high-throughput virtual screening of NCI small molecular database, in vitro haemolytic assays for top-hits of the chemical compounds against crude venom of Naja naja and as well CTXs purified from the venom and pharmacokinetic examinations on the chemical compounds retarding haemolytic activities of CTXs suggested that Etidronic acid and Zoledronic acid are promising prototypic chemical inhibitors to CTXs of snake venoms.

Effects of different irrigation regimes in physico-chemical properties of dentin and consequences of changes in the adhesion of microorganisms and AH Plus sealer / Efeitos de diferentes regimes de irrigação nas propriedades físicoquímicas da dentina e consequências das alterações na adesão de microrganismos e do cimento endodôntico AH Plus

Besides of the desired effects, the chemical solutions used to assist the endodontic instruments in the cleanliness and disinfection of the root canal system can also cause changes in the physicochemical properties of dentin, and consequently affect the adhesion of endodontic sealers and microorganisms to the root canal walls. However, the effects of new irrigators and irrigation protocols remain unknown. The objectives of this thesis were to verify the alterations in the properties of some irrigants when used combined in mixtures, to define the time necessary for the smear layer removal by a new irrigant, to determine the organic matter dissolution capacity and the effects in the physicochemical properties of dentin of some irrigation solutions and protocols, and to evaluate the adhesion of microorganisms and AH Plus sealer to dentin after its submission to different irrigation sequences. In all experiments with dentin, the samples used were obtained from bovine teeth. In the analysis performed in this thesis, the following solutions were tested isolated and combined in different irrigation protocols: saline solution (control), sodium hypochlorite (NaOCl), trisodium (EDTAHNa3), alkaline ethylenediaminetetraacetic acid tetrasodium (EDTANa4), chlorhexidine (CHX), peracetic acid (PAA), and etidronic acid (HEDP). The EDTAHNa3 and EDTANa4 were tested in relation to their effects on the free chlorine content of NaOCl. The solutions were mixed in a 1:1 ratio and the iodometric titration of the mixtures performed in different time intervals. The time necessary for smear layer removal from dentin samples by solutions of EDTAHNa3 and different concentrations of EDTANa4 isolated and mixed with NaOCl was determined with the aid of the scanning electron microscope (SEM). The capacity of NaOCl to dissolve organic matter was determined by weighting fragments of bovine muscle before and after immersion in solutions of 1%, 2.5%, and 5% of NaOCl in different periods of time. Also, the effects of EDTAHNa3, EDTANa4 and HEDP on the organic matter dissolution by NaOCl were evaluated. The alterations produced by all solutions isolated and some irrigation protocols in the organic and inorganic components of the dentin surface were analysed by the attenuated total reflectance of Fourier transform infrared spectroscopy (ATR-FTIR) technique. Absorbance spectra were collected from the dentin surface before and after immersion of samples in the irrigants and the ratios of the amide III/phosphate and carbonate/phosphate bands were calculated. To quantify the adhesion of CHX to mineralized dentin and to dentin demineralized by different irrigation protocols, the areas of the band associated with CHX with the peak in 1492 cm−1 were determined in spectra obtained by ATR-FTIR. The effects of different irrigation protocols in the roughness and wettability of dentin surface were measured with a benchtop roughness tester and the sessile drop technique, respectively. For the assays of microorganisms' adhesion, samples were prepared and treated the same way and with the same irrigation protocols used in the roughness and wettability tests. Following, Candida albicans and Enterococcus faecalis were maintained in contact with the dentin for 2 hours and the samples were analyzed on the confocal laser scanning microscope (CLSM). Tests of push-out were performed to determine the impact of different irrigation protocols on the dentin bonding strength of AH Plus sealer over time. Canals of bovine incisors teeth were instrumented, irrigated and following obturated using only the sealer AH Plus. Half of the samples were submitted to pushout assessment 7 days after the obturation and the other half 20 months later. The results of the experiments showed that the EDTAHNa3 caused an almost complete and immediate loss of free available chlorine from NaOCl, whilst EDTANa4 promoted a slow and concentration-dependent decline. The smear layer was removed only by decalcifying solutions and in about 1 min by the 17% EDTAHNa3 and 5 min by the EDTANa4, both isolated or mixed with NaOCl. The increase in NaOCl concentration and contact time with the samples intensified the dissolution of organic matter. The mixtures of NaOCl with EDTANa4 and HEDP were able to dissolve the fragments of bovine muscle over-time, however, the EDTAHNa3 strongly affected the NaOCl dissolution capacity when they were mixed. The results of ATR-FTIR experiments showed that the increase in the NaOCl concentration intensified the deproteination of the dentin collagen with a reduction in the amide III/phosphate ratio. For the same decalcifying agent, the higher the concentration and immersion time the greater the removal of phosphate, exposure of the collagen matrix and consequently the increases in amide III/phosphate ratio. The PAA caused greater increases in amide III/phosphate ratio, followed by EDTAHNa3, EDTANa4 and HEDP and this order was maintained in the protocols in which NaOCl was used before the decalcifying agents. NaOCl required approximately 0.5 min to deproteinate the collagen matrix exposed after phosphate removal by EDTAHNa3 and PAA. The carbonate/phosphate ratio decreased after 30 s of samples immersion in solutions of NaOCl at 1%, 2.5% and 5% with no more alterations over time. The carbonate of the dentine was removed faster than phosphate by all decalcifying agents employed alone and in the irrigation protocols in which the use of the NaOCl was followed by the use of the EDTAHNa3, PAA and HEDP. For irrigation protocols that associate NaOCl with chelating solutions, the last irrigant used defined the final dentine amide III/phosphate and carbonate/phosphate ratios. For the ATR-FTIR analysis of CHX adhesion, the results showed that the adsorption of this irrigant to the dentin was potentiated when chelating agents were used prior to the CHX. In relation to the experiments of surface roughness, the saline solution, NaOCl, HEDP and CHX did not alter the roughness of the dentin, but EDTAHNa3 and PAA increased it. The wettability of the surface increased after the use of all irrigants, being the HEDP to cause the greater increases. In the assays of microorganisms' adhesion, the smear layer and collagen exposed by the chelating agents favored the adhesion of E. faecalis. The C. albicans adhesion was major in surfaces with smear layer and more mineral. The use of CHX as the final irrigant reduced the adhesion of both microorganisms. The wettability did not influence the microorganisms' adhesion, while increases in roughness seems to potentiate the adherence of E. faecalis. The experiments of bond strength of AH Plus to the dentin showed that the irrigation with NaOCl and mixture of NaOCl + EDTANa4 produced the lowest push-out bond strength values in 7 days compared to NaOCl + EDTAHNa3, NaOCl + EDTAHNa3 + NaOCl, NaOCl + EDTAHNa3 + CHX and the mixture of NaOCl + HEDP. After 20 months the lowest values were obtained in the groups irrigated with NaOCl and NaOCl + EDTAHNa3. The groups of NaOCl + EDTAHNa3 + NaOCl, mixture NaOCl + HEDP, and mixture NaOCl + EDTANa4 presented values of push-out bond strength in 20 months similar to the values in 7 days. It was possible to conclude that the irrigation solutions tested in this study have different effects in the organic and inorganic matter and some of them can affect the action of each other when mixed. Independent of being used isolated or combined in irrigation protocols, these irrigants cause modifications in the dentin physicochemical properties that influence the adhesion of AH Plus sealer in short and long term and the microorganisms' adherence to the surface in cases of recontaminations.(AU)

Além dos efeitos desejados, as soluções químicas utilizadas para auxiliar os instrumentos endodônticos na limpeza e desinfecção do sistema radiculares podem causar alterações nas propriedades físico-químicas da dentina e consequentemente afetar a adesão de cimentos endodônticos e microrganismos às paredes do canal radicular. Contudo, os efeitos de novos irrigantes e protocolos de irrigação ainda são desconhecidos. Os objetivos desta tese foram verificar as alterações nas propriedades de alguns irrigantes quando utilizados combinados em misturas, definir o tempo necessário para a remoção da camada de smear layer por um novo irrigante, determinar a capacidade de dissolução de matéria orgânica e os efeitos de algumas soluções e protocolos de irrigação nas propriedades físico-químicas de dentina e avaliar a adesão de microrganismos e cimento AH Plus à dentina após a submissão desta a diferentes sequências de irrigação. Em todos os experimentos com dentina as amostras utilizadas foram obtidas a partir de dentes bovinos. Nas análise realizadas nesta tese as seguintes soluções foram testadas isoladas e combinadas em diferentes protocolos de irrigação: solução salina (controle), hipoclorito de sódio (NaOCl), ácido etilenodiaminotetraacético trisódico (EDTAHNa3), ácido etilenodiaminotetracético tetrassódico alcalino (EDTANa4), clorexidina (CHX), ácido peracético (PAA) e ácido etidrônico (HEDP). O EDTAHNa3 e o EDTANa4 foram testados em relação aos seus efeitos sobre o teor de cloro livre do NaOCl. As soluções foram misturadas em uma proporção de 1:1 e a titulação iodométrica das misturas realizada em diferentes intervalos de tempo. O tempo necessário para a remoção da smear layer de amostras de dentina pela solução de EDTAHNa3 a 17% e diferentes concentrações de EDTANa4 isoladas e misturadas com NaOCl foi determinado com o auxílio do microscópio eletrônico de varredura (SEM). A capacidade de dissolução de matéria orgânica pelo NaOCl foi determinada pesando fragmentos de músculo bovino antes e depois da imersão em soluções de 1%, 2,5% e 5% de NaOCl em diferentes períodos de tempo. Além disso, os efeitos do EDTAHNa3, EDTANa4 e HEDP na dissolução de matéria orgânica pelo NaOCl foram avaliados. As alterações produzidas por todas as soluções isoladas e alguns protocolos de irrigação nos componentes orgânicos e inorgânicos da superfície da dentina foram analisadas pela técnica de reflexão total atenuada em espectroscopia no infravermelho por transformação de Fourier (ATRFTIR). Espectros de absorbância foram coletados da superfície da dentina antes e após a imersão das amostras nos irrigantes, e foram calculadas as razões das bandas de amida III/fosfato e carbonato/fosfato. Para quantificar a adesão da CHX à dentina mineralizada e à dentina desmineralizada por diferentes protocolos de irrigação, foram determinadas as áreas da banda associada a CHX com pico em 1492 cm−1 em espectros obtidos por ATR-FTIR. Os efeitos de diferentes protocolos de irrigação na rugosidade e molhabilidade da superfície da dentina foram medidos com um rugosímetro de bancada e a técnica de gota séssil, respectivamente. Para os ensaios de adesão de microrganismos, amostras foram preparadas e tratadas da mesma maneira e com os mesmos protocolos de irrigação utilizados nos testes de rugosidade e molhabilidade. Em seguida, Candida albicans e Enterococcus faecalis foram mantidos em contato com a dentina por 2 horas e as amostras foram analisadas no microscópio confocal de varredura laser (CLSM). Testes de push-out foram realizados para determinar o impacto de diferentes protocolos de irrigação na resistência de união à dentina do cimento AH Plus ao longo do tempo. Canais de dentes incisivos de bovinos foram instrumentados, irrigados e em seguida obturados utilizando apenas o cimento AH Plus. Metade das amostras foi submetida a avaliação de push-out 7 dias após a obturação e a outra metade após 20 meses. Os resultados dos experimentos mostraram que o EDTAHNa3 causou uma perda quase completa e imediata do cloro livre do NaOCl, enquanto o EDTANa4 promoveu um declínio lento e concentração dependente. A smear layer foi removida apenas por soluções descalcificantes e em cerca de 1 min pelo EDTAHNa3 a 17% e em 5 min pelo EDTANa4, tanto isolados ou misturados com o NaOCl. O aumento da concentração de NaOCl e do tempo de contato com os fragmentos de músculo bovino intensificou a dissolução da matéria orgânica. As misturas de NaOCl com EDTANa4 e HEDP foram capazes de dissolver as amostras de músculo ao longo do tempo, no entanto, o EDTAHNa3 afetou fortemente a capacidade de dissolução do NaOCl quando eles foram misturados. Os resultados dos experimentos com ATR-FTIR mostraram que o aumento da concentração do NaOCl intensificou a desproteinização do colágeno da dentina com redução da relação amida III/fosfato. Para o mesmo agente de descalcificação, quanto maior a concentração e o tempo de imersão, maior a remoção de fosfato, exposição da matriz de colágeno e consequentemente o aumento da proporção amida III/fosfato. O PAA causou os maiores aumentos na relação amida III/fosfato, seguido de EDTAHNa3, EDTANa4 e HEDP e esta ordem foi mantida nos protocolos em que o NaOCl foi usado antes dos agentes descalcificantes. O NaOCl necessitou aproximadamente 0,5 min para desproteinizar a matriz de colágeno exposta após a remoção de fosfato pelo EDTAHNa3 e o PAA. A relação carbonato/fosfato diminuiu após 30 s de imersão das amostras em soluções de NaOCl a 1%, 2,5% e 5%, sem mais alterações ao longo do tempo. O carbonato da dentina foi removido mais rápido do que o fosfato por todos os agentes descalcificantes empregados sozinhos e nos protocolos de irrigação em que o uso do NaOCl foi seguido pelo uso do EDTAHNa3, PAA e HEDP. Para os protocolos de irrigação que associam o NaOCl com soluções quelantes, o último irrigante utilizado definiu as proporções finais de amida II/fosfato e carbonato/fosfato da dentina. Para as análises da adesão da CHX em ATR-FTIR, os resultados mostraram que a adsorção deste irrigante à dentina foi potencializada quando agentes quelantes foram utilizados antes da CHX. Em relação aos experimentos de rugosidade da superfície, a solução salina, o NaOCl, o HEDP e a CHX não alteraram a rugosidade da dentina, mas o EDTAHNa3 e o PAA a aumentaram. A molhabilidade da superfície aumentou após o uso de todos os irrigantes, sendo que o HEDP causou os maiores aumentos. Nos ensaios de adesão dos microrganismos, a smear layer e o colágeno exposto pelos agentes quelantes favoreceram a adesão de E. faecalis. A adesão da C. albicans foi maior em superfícies com smear layer ou mais mineral. O uso de CHX como irrigante final reduziu a adesão de ambos os microrganismos. A molhabilidade não influenciou a adesão dos microrganismos, enquanto o aumento da rugosidade parece potencializar a adesão do E. faecalis. Os experimentos de resistência de união do AH Plus à dentina mostraram que a irrigação com NaOCl e a mistura de NaOCl + EDTANa4 produziram valores de resistência de união em 7 dias mais baixos em comparação com NaOCl + EDTAHNa3, NaOCl + EDTAHNa3 + NaOCl, NaOCl + EDTAHNa3 + CHX e a mistura de NaOCl + HEDP. Após 20 meses, os valores mais baixos foram obtidos nos grupos irrigados com NaOCl e NaOCl + EDTAHNa3. Os grupos de NaOCl + EDTAHNa3 + NaOCl, mistura de NaOCl + HEDP e mistura de NaOCl + EDTANa4 apresentaram valores de força de união por push-out em 20 meses semelhante aos valores em 7 dias. Foi possível concluir que as soluções de irrigação testadas neste estudo têm diferentes efeitos na matéria orgânica e inorgânica e elas podem afetar as ações umas das outras quando misturadas. Independentemente de serem utilizadas isoladas ou combinadas em protocolos de irrigação, os irrigantes causam modificações nas propriedades físico-químicas dentinárias que influenciam na adesão do cimento AH Plus a curto e longo prazo e na adesão de microrganismos à superfície em casos de recontaminação.(AU)

An IC-MS/MS Method for the Determination of 1-Hydroxyethylidene-1,1-diphosphonic Acid on Uncooked Foods Treated with Peracetic Acid-Based Sanitizers.

A rapid, sensitive, and specific analytical method for the determination of 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP) on uncooked foods after treatment with a peracetic acid-based sanitizer (PAS) was developed. The method involves simple sample preparation steps and analysis using ion chromatography (IC) coupled with tandem mass spectrometry (MS/MS). The quantification limits of HEDP on uncooked foods are 0.007 mg/kg for vegetables and fruits and 0.2 mg/kg for meats. The recovery and relative standard deviation (RSD) of HEDP analyses of uncooked foods ranged from 73.9 to 103.8% and 1.9 to 12.6%, respectively. The method's accuracy and precision were evaluated by inter-day recovery tests. The recovery for all samples ranged from 93.6 to 101.2%, and the within-laboratory repeatability and reproducibility were evaluated based on RSD values, which were less than 6.9 and 11.5%, respectively. Analyses of PAS-treated fruits and vegetables using the developed method indicated levels of HEDP ranging from 0.008 to 0.351 mg/kg. Therefore, the results of the present study suggest that the proposed method is an accurate, precise, and reliable way to determine residual HEDP levels on PAS-treated uncooked foods.