Towards Green Titration: Batchwise Titration with Reusable Solid Sorbed Indicators.

We propose the creation of reusable indicator-sorbed-solids, using anion-exchange resins or kaolin as supports, with the aim to reduce chemical use towards green analytical chemistry. Indicators (phenolphthalein, thymol blue and butterfly pea flower extract as a natural indicator) sorbed on a solid support, were employed in acid-base titration, in both homogenous aqueous and heterogenous aqueous organic phases. Applications of the developed techniques to some real samples, such as vinegar, colored fruit juice and vegetable oil, have been demonstrated.

Complete Kinetic Characterization of Enzyme Inhibition in a Single Isothermal Titration Calorimetric Experiment.

Techniques for rapidly measuring both the strength and mode of enzyme inhibitors are crucial to lead generation and optimization in drug development. Isothermal titration calorimetry (ITC) is emerging as a powerful tool for measuring enzyme kinetics with distinct advantages over traditional techniques. ITC measures heat flow, a feature of nearly all chemical reactions, and gives an instantaneous readout of enzyme velocity, eliminating the need for artificial substrates or postreaction processing. In principle, ITC is an ideal method for characterizing enzyme inhibition. However, existing ITC experiments are not well-suited to rapid throughput and few studies to date have employed this approach. We have developed a new ITC experiment, in which substrate and inhibitor are premixed in the injection syringe, that yields complete kinetic characterization of an enzyme inhibitor in an hour or less. This corresponds to savings in time and material of 5-fold or greater compared to previous ITC methods. We validated the approach using the trypsin inhibitor benzamidine as a model system, recapitulating both its competitive inhibition mode and binding constant. Our approach combines the rapid throughput of optimized spectroscopic assays with the universality and precision of ITC-based methods, providing substantially improved inhibitor characterization for biochemistry and drug development applications.

Development of a Platform to Enable Fully Automated Cross-Titration Experiments.

In the triage of hits from a high-throughput screening campaign or during the optimization of a lead compound, it is relatively routine to test compounds at multiple concentrations to determine potency and maximal effect. Additional follow-up experiments, such as agonist shift, can be quite valuable in ascertaining compound mechanism of action (MOA). However, these experiments require cross-titration of a test compound with the activating ligand of the receptor requiring 100-200 data points, severely limiting the number tested in MOA assays in a screening triage. We describe a process to enhance the throughput of such cross-titration experiments through the integration of Hewlett Packard's D300 digital dispenser onto one of our robotics platforms to enable on-the-fly cross-titration of compounds in a 1536-well plate format. The process handles all the compound management and data tracking, as well as the biological assay. The process relies heavily on in-house-built software and hardware, and uses our proprietary control software for the platform. Using this system, we were able to automate the cross-titration of compounds for both positive and negative allosteric modulators of two different G protein-coupled receptors (GPCRs) using two distinct assay detection formats, IP1 and Ca2+ detection, on nearly 100 compounds for each target.

Manejo de opioides para el dolor basal e irruptivo oncológico / Opioid management for background and breakthrough cancer pain

El dolor relacionado con cáncer es experimentado por el 90% de los pacientes oncológicos. La base del tratamiento son los opioides y la escalera analgésica, de la Organización Mundial de la Salud. El dolor irruptivo oncológico es una forma de dolor relacionado con el cáncer particularmente difícil de gestionar con los opioides clásicos. En esta revisión se cubre la titulación y la rotación de opioides, tanto para dolor basal como irruptivo. También se revisa la contribución de los preparados de fentanilo de acción rápida para el tratamiento del dolor irruptivo oncológico, su indicación y titulación. El citrato de fentanilo oral transmucoso y los comprimidos bucales de fentanilo fueron los primeros fármacos desarrollados específicamente para el tratamiento del dolor irruptivo oncológico. Dado que la administración oral de fentanilo no es una opción en muchos pacientes oncológicos, el desarrollo de un espray intranasal de fentanilo con pectina surgió como un método más eficaz de administración

Cancer-related pain is experienced by 90% of patients with cancer. The mainstays of treatment are opioids and the World Health Organization's analgesic ladder. Breakthrough cancer pain (BCP) is a form of cancer-related pain that is particularly difficult to manage with classical opioids. In this review, we discuss titration and opioid rotation in both background and breakthrough pain. We also review the contribution of fast-acting fentanyl preparations for the treatment of BCP and its indications and dose titration. The first drugs developed specifically for the treatment of BCP were oral transmucosal fentanyl citrate (CFOT) and fentanyl buccal tablets. Because oral fentanyl administration is not an option in many cancer patients, the development of a fentanyl nasal spray (FNS) has emerged as a more effective method of administration

Assessment of phytoestrogen and mycoestrogen recognition by recombinant human estrogen receptor-α using ligand titration arrays.

INTRODUCTION: Exposure to phytoestrogens and mycoestrogens has emerged as a public health issue due to their potentially endocrine disruption activities resulting from direct interaction with sex-steroid hormone receptors. There is a significant requirement for comprehensive, reproducible methods to determine the extent of estrogen mimicry by compounds encountered in the environment to estimate risk:benefit ratios, particularly in humans. OBJECTIVE: To develop a systematic approach for assessing recognition of chemically diverse compounds by human estrogen receptor proteins to aid in their assessment as endocrine disruptor compounds (EDCs). METHODS: Recombinant human estrogen receptor-α protein (rhERα) was expressed in Saccharomyces cervisiae as an ubiquitin fusion under control of a CUP1 promoter and partially purified with heparin affinity chromatography in the unliganded state. A novel radio-ligand binding array was developed to evaluate structurally diverse compounds, both naturally occurring and synthetic, for estrogen binding activity and affinity. RESULTS: Binding affinities of suspected estrogen mimics for rhERα were calculated over a range of [(3) H]estradiol-17ß concentrations using Lundon OneSite® and Compete® software. CONCLUSION: ß-zearalanol, a mycoestrogen similar to zearalenone used as an ICCVAM validation substance for the in vitro estrogen receptor binding assays (ICCAM report), was employed as a model estrogen mimic to illustrate the approach, methods and calculations using these techniques.

[Research on TLC identification and anti-coagulant activity quantitatively methods of Scolopendra subspinipes mutilans].

OBJECTIVE: To improve the quality standard of Scolopendra subspinipes mutilans by researching the methods of the TLC identification and anti-coagulant activity quantitatively. METHODS: Identified the free arginine (Arg) and serine (Ser) in scolopendra by TLC, screened the samples preparation process and developed solvent systems; Determined the anti-coagulant activity by method of titration with thrombin and screened the pretreatment methods. RESULTS: When medicinal materials was extracted by formic acid and 95% ethanol (1:1) with ultrasonic method and developed by n-butanol-acetic acid-water (12:5:4), the spots of Arg and Ser were well separated. Ultrasonic method was suitable for preparation of the anti-coagulant components in Scolopendra subspinipes mutilans and their anti-coagulant activity was determined by method of titration with thrombin could get a well reproducibility, the anti-thrombin activity of testing sample was (14.00 +/- 1.53) U/g and those of three different batch were (13.00 +/- 0.58) U/g, (17.00 +/- 1.15) U/g, (15.67 +/- 1.53) U/g respectively. CONCLUSION: The methods of TLC identification and anti-coagulant activity quantitatively could be used as a basis for improving the quality standard of Scolopendra subspinipes mutilans.

Genome-wide in vitro reconstitution of yeast chromatin with in vivo-like nucleosome positioning.

Recent genome-wide mapping of nucleosome positions revealed that well-positioned nucleosomes are pervasive across eukaryotic genomes, especially in important regulatory regions such as promoters or origins of replication. As nucleosomes impede access to DNA, their positioning is a primary mode of genome regulation. In vivo studies, especially in yeast, shed some light on factors involved in nucleosome positioning, but there is an urgent need for a complementary biochemical approach in order to confirm their direct roles, identify missing factors, and study their mechanisms. Here we describe a method that allows the genome-wide in vitro reconstitution of nucleosomes with very in vivo-like positions by a combination of salt gradient dialysis reconstitution, yeast whole cell extracts, and ATP. This system provides a starting point and positive control for the biochemical dissection of nucleosome positioning mechanisms.

Using spectrophotometric titrations to characterize humic acid reactivity at environmental concentrations.

Potentiometric titration is a common method to characterize dissolved organic matter (DOM) reactivity. Because of the sensitivity of pH electrodes, it is necessary to work with very high DOM (>1 g/L) concentrations that are unrealistic compared to those found in natural waters (0.1 to 100 mg/L). To obtain proton binding data for concentrations closer to environmental values, spectroscopic titration methodology is a viable alternative to traditional potentiometric titrations. Spectrophotometric titrations and UV-visible spectra of a diluted solution of purified Aldrich humic acid (5 mgDOC/L) are used to estimate changes in proton binding moieties as function of pH and ionic strength after calculation of differential absorbance spectra variations. After electrostatic correction of spectrophotometric data, there is a linear operational correlation between spectrophotometric and potentiometric data which can be used as a transfer function between the two properties. Spectrophotometric titrations are then used to determine the changes of humic acid protonation after adsorption onto alpha-alumina.

pH-mediated fluorescence and G-quadruplex binding of amido phthalocyanines.

A new family of G-quadruplex ligands termed "amido phthalocyanines" (APcs) was synthesized by reacting variable amino acids with tetraamino zinc phthalocyanine. Variation in the number of methylene units separating the APc scaffold from terminal ammonium groups systematically modulated ammonium pK(a) values that, in turn, mediated APc aggregation and DNA binding. Certain APcs exhibited nearly 1000-fold enhancements in fluorescence quantum yield upon binding G-quadruplex DNA under physiological conditions of pH and ionic strength. G-quadruplexes derived from the c-myc and c-kit promoters and the human telomeric repeat were evaluated for APc affinity and specificity using two complementary and direct fluorescence binding assays that revealed apparent dissociation constants ranging from 20 to 200 nM. Approximately 500-fold lower affinities for duplex and single-stranded DNAs were observed. Interestingly, APc-quadruplex binding was relatively insensitive to ionic strength (0.03-1 M KCl) but highly dependent on the pH of the solution. Our results provide a mechanism for the "turn-on" fluorescence properties exhibited by these compounds that will assist in future rational design of new G-quadruplex-specific fluorescent probes and drug candidates.

Phage display screening in low dielectric media.

Here we report the first application of phage display screening in low dielectric media. Two series of phage clones with affinity for alpha-chymotrypsin (CT) were selected from a Ph.D.(TM)-C7C library, using either a buffer or acetonitrile in buffer (50%, v/v). The affinity of lysates, individual clones or selected cyclic peptides for the enzyme was studied by examining their influence on CT activity. Peptides displayed on phage selected in buffer provided significant protection from enzyme autolysis resulting in marked increase in CT activity (>100%). Phage selected in ACN provided some, albeit weak, protection from the detrimental influence on CT from ACN. In conclusion, the results demonstrate the potential for the application of phage display screening protocols to targets in media of low dielectricity.

Differential potentiometric titration: development of a methodology for determining the point of zero charge of metal (hydr)oxides by one titration curve.

A new methodology is presented, called differential potentiometric titration (DPT), which allows the determination of the point of zero charge (pzc) of metal (hydr)oxides using only one potentiometric curve. By performing extensive simulations of potentiometric titrations for various model (hydr)oxides, we found that an inflection point in a H+(cons,surf) versus pH potentiometric curve (H+(cons,surf): hydrogen ions consumed on the surface of the (hydr)oxide) and a peak in the corresponding differential curve, dH+(cons,surf)/dpH versus pH, appear at a pH equal to the pzc assumed for a model (hydr)oxide. This distinguishable peak appears at the same position irrespective of the surface ionization and the interfacial model adopted as well as the assumed ionic strength. It was found that the aforementioned peak also appears in the high-resolution differential potentiometric curves experimentally determined for four oxides (SiO2, TiO2, gamma-Al2O3, and MgO) that are widely used in various environmental and other technological applications. The application of DPT to the above-mentioned oxides provided practically the same pzc values as the corresponding ones achieved by using four different techniques as well as the corresponding isoelectric point (iep) values determined by microelectrophoresis. Differences between the pzc and iep values determined using various techniques in the case of MgO were attributed to the increasing dissolution of this oxide as pH decreases and the adsorption of cations (Mg2+, Na+) on the MgO/electrolytic solution interface.

[Analysis of vitamin C in the biologically active supplements to food and food products enriched with micronutrients].

Peculiarities in Vitamin C analysis of biologically active supplements to food and food products enriched with micronutrients are discussed. Visual titration method and spectrophotometric determination based on the utilization of sodium 2,6-dichlorophenolindophenolate are shown to be the most favourable oner. A special attention is paid to the nature of extragents used and protection against undesirable effect of some food components.

[Comparative titration of three antivenin serums used against sub-Saharan African snakes]. / Titrage comparatif de trois sérums antivenimeux utilisés contre les serpents d'Afrique sub-saharienne.

The standardisation of serotherapy is necessary in Africa mainly because of the frequency of envenomations and the lack of alternative treatments. Comparative titrations of FAV-Afrique (Aventis Pasteur), Polyvalent serum (Serum Institute of India = SII) and Polyvalent antivenin (South African Vaccine Fabricants Ltd = SAIMR) were carried out on venoms of Echis ocellatus from Cameroun, E. ocellatus from Mali, E. leucogaster and Naja melanoleuca. The 50% protective doses (ED50) of the antivenoms were given according either to i) the in vitro method which consists of inoculating 5 batches of 5 mice with a mixture containing 3 DL50 of venom and increasing volumes of antivenom incubated for 30 mn at 37 degrees C and ii) the in vivo method which consists of successive administration of venom and then antivenom after a 30 to 60 mn interval. The three antivenoms showed a similar efficacy against all the Echis venoms. Interestingly, the SAIMR proved to be effective against the venom of E. leucogaster and E. ocellatus although no venom of Echis was used to immunise horses during the preparation of antivenom. Conversely, this paraspecificity did not exist with the Naja melanoleuca venom against which FAV Afrique showed a higher efficacy. The electrophoresis pattern of FAV-Afrique performed on acetate gel strips showed only one protein fraction (76 g.l-1), whereas both the SII and SAIMR antivenoms showed four fractions whose protein concentrations was respectively 64 g.l-1 and 145 g.l-1.

A label-free optical technique for detecting small molecule interactions.

A novel approach for the label-free detection of molecular interactions is presented in which a colorimetric resonant grating is used as a surface binding platform. The grating, when illuminated with white light, is designed to reflect only a single wavelength. When molecules are attached to the surface, the reflected wavelength (color) is shifted due to the change of the optical path of light that is coupled into the grating. By linking receptor molecules to the grating surface, complementary binding molecules can be detected without the use of any kind of fluorescent probe or radioactive label. The detection technique is capable of detecting the addition and removal of small molecules as they interact with receptor molecules on the sensor surface or enzymes in the solution surrounding the sensor. Two assays are presented to exemplify the detection of small molecule interactions with the biosensor. First, an avidin receptor layer is used to detect 244 Da biotin binding. Second, a protease assay is performed in which a 136 Da p-nitroanilide (pNA) moeity is cleaved from an immobilized substrate. Because the sensor structure can be embedded in the plastic surfaces of microtiter plates or the glass surfaces of microarray slides, it is expected that this technology will be most useful in applications where large numbers of biomolecular interactions are measured in parallel, particularly when molecular labels will alter or inhibit the functionality of the molecules under study. Screening of pharmaceutical compound libraries with protein targets, and microarray screening of protein-protein interactions for proteomics are examples of applications that require the sensitivity and throughput afforded by this approach.

A dynamic in vitro lipolysis model. I. Controlling the rate of lipolysis by continuous addition of calcium.

Lipolysis by pancreatic lipase was investigated with the aim to establish an in vitro lipolysis model, which can be used to investigate the dissolution of poorly soluble lipophilic drug substances at controlled hydrolysis rates. The effects of three experimental parameters -- the concentrations of bile salts and Ca(2+) and the lipase activity -- were investigated. The effect on the rate of hydrolysis of emulsified soybean oil was investigated in experiments in a pH-stat at pH 6.5 and 37 degrees C. The free fatty acids produced by the hydrolysis were titrated at pH 6.5. It was shown that all three investigated parameters influence the initial rate of hydrolysis, whereas only the lipase activity and the concentration of Ca(2+) affect the subsequent stages. It was also shown that the rate of lipolysis can be controlled by the rate of adding Ca(2+). Thus, it is possible to design an in vitro model using readily available and inexpensive materials in which the hydrolysis rate can be controlled by the continuous addition of Ca(2+).

New in vitro fluorimetric microtitration assays for toxicological screening of drugs.

Flow cytometry has been widely used to quantify fluorescent probes in cell culture. However, FCM is not adapted to toxicological screenings due to the cost, the length and the poor reproducibility of this technique. Moreover, several multicenter studies have preferred microtitration methodologies for drug screening. A new fluorimetric technology has been designed that is sensitive and adapted to direct screening in 96-well microplates. This fluorimeter uses cold light technology (CLF) with chemical and physical modifications of the lighting system (Rat et al., 1995). CLF allows reading of UV, visible and near infrared fluorescence by increasing light energy (from 1000 to 2300 lumens) and reducing the calorific part of light (IR > 900 nm, Joule effect). It induces a decrease in background and a 500- to 1000-fold improvement of detection limit of probes in comparison with classical fluorimeters and permits detection of pg/ml to fg/ml. CLF allows easy evaluation of cell injury induced by physical agents (UVA) or chemical toxins (CCl4). Four biological endpoints for cytotoxicity evaluation have been tested with several probes: proliferation (H33258); viability (fluorescent Neutral Red); cell-cell adhesion (calcein-AM); and mitochondrial metabolic effects (Rhodamine 123). Rh123 assay appeared more sensitive than fluorimetric or photometric detection of Neutral Red assay. Cold light fluorimetry (CLF) permits direct detection of low concentrations of probes (pg/ml to fg/ml). CLF is shown to improve classical cytotoxicity assays and, owing to its adaptability to microtitration (in 6-, 12- or 96-well plates and in Petri dishes), it is thus a promising alternative to flow cytometry for drug cytotoxicity screening.