Exploring the analytical power of the QTOF MS platform to assess monoclonal antibodies quality attributes.

The biopharmaceutical industry is growing at a fast pace, making nowadays 20% of the pharma market. Within this market, therapeutic monoclonal antibodies (mAbs) are the dominant product class. With the patent expirations, biosimilars and, perhaps more relevant, biobetters, are in fast development.

Human carboxylesterase 2: Studies on the role of glycosylation for enzymatic activity.

Human carboxylesterase 2 (hCES2) is a glycoprotein involved in the metabolism of drugs and several environmental xenobiotics, whose crystallization has been proved to be a challenging task. This limitation could partly be due to glycosylation heterogeneity and has delayed the disclosure of the 3D structure of hCES2 which would be of upmost relevance for the development of new substrates and inhibitors. The present work evaluated the involvement of glycans in hCES2 activity and thermo stability in an attempt to find alternative active forms of the enzyme that might be adequate for structure elucidation.

Peptidomimetic β-Secretase Inhibitors Comprising a Sequence of Amyloid-β Peptide for Alzheimer's Disease.

Alzheimer's disease is a grave social problem in an aging population. A major problem is the passage of drugs through the blood-brain barrier. This work tests the hypothesis that the conjugation of peptidomimetic β-secretase inhibitors with a fragment of amyloid-β peptide facilitates entrance into the central nervous system.

Comparison of in vitro methods for carboxylesterase activity determination in immortalized cells representative of the intestine, liver and kidney.

Herein we compare the fluorimetric determination of total and specific carboxylesterase activity in immortalized human derived living cells and in cell lysates. The cell lines chosen are representative of metabolism occurring in the intestine (Caco-2 and HT-29), kidney (HEK-293T) and liver (Hep G2). Caco-2 and HT-29, as cells prone to differentiation, were tested along the differentiation time.

A methodology for detection and quantification of esterase activity.

Carboxylesterases are important enzymes for xenobiotic metabolism and are receiving increasing attention in the context of cancer therapies. Quantification of individual carboxylesterase activity is important since protein levels do not always correlate to activity and significant interorgan, interindividual, and interspecies variations exist. Here we present a methodology enabling the specific quantification of carboxylesterase 2 activity in a pool of other esterases.

Electroosmotic flow modulation in capillary electrophoresis by organic cations from ionic liquids.

This paper describes the ability of several ionic liquids cations for electroosmotic flow modulation in capillary electrophoresis. Organic salts based on phosphonium, sulfonium, cysteinium, ammonium, and guanidinium cations were selected to study this property. In addition, the synergistic effect of these compounds in cyclodextrin chiral separation was also evaluated.

Carboxylesterase 2 production and characterization in human cells: new insights into enzyme oligomerization and activity.

Carboxylesterase 2 (CES2), the main carboxylesterase expressed in human intestine, is an increasingly important enzyme in anti-cancer combined therapies for the treatment of different pathologies like colon adenocarcinoma and malignant glioma. The production of human recombinant CES2, in human embryonic kidney cells (HEK-293T cells) using serum-free media, is herein described. CES2 secretion to the media was achieved by the simple addition of an in-frame C-terminal 10× histidine tag (CES2-10xHis) without the need of addition of extra N-terminal signalling sequences or the mutation or deletion of the C-terminal HTEL motif responsible for retaining the protein in the lumen of endoplasmic reticulum.

Detection and quantification of carboxylesterase 2 activity by capillary electrophoresis.

The purpose of this study was to develop an analytical method to quantify the relative activities of carboxylesterases (CESs) in biological samples. Taking the advantage of loperamide, a specific carboxylesterase 2 (CES2) inhibitor, and bis-p-nitrophenyl phosphate (BNPP), an irreversible CESs inhibitor, we propose for the first time a capillary electrophoresis (CE) method that enables detecting and distinguishing CES2 activity from other CESs in complex biological samples. The capillary electrophoresis method proved to be fast, simple, repeatable, and applicable to the measurement of the specific activity of CESs.

Fullerenol C60(OH)24 prevents doxorubicin-induced acute cardiotoxicity in rats.

Results obtained in vitro suggested that fullerenol's antiproliferative properties and protective effects against doxorubicin (DOX) cytotoxicity are mediated by antioxidative and hydroxyl radical scavenger activity. The aim of this study was to examine the influence of fullerenol on acute cardiotoxicity after the administration of a single high dose of DOX in vivo. The experiment was performed on male Wistar rats randomly divided into five groups, each containing eight individuals, that were treated as follows: I) 0.

Solubilization of fullerene C60 in micellar solutions of different solubilizers.

Fullerene (C(60)), the third carbon allotrope, is a classical engineered material with the potential application in biomedicine. However, extremely high hydrophobicity of fullerene hampers its direct biomedical evaluation and application. In this work, we investigated the solubilization of fullerene using 9 different solubility enhancers: Tween 20, Tween 60, Tween 80, Triton X-100, PVP, polyoxyethylene (10) lauryl ether, n-dodecyl trimethylammonium chloride, myristyl trimethylammonium bromide and sodium dodecyl sulphate and evaluated its antioxidant activity in biorelevant media.

Electrophoretically mediated microanalysis for the evaluation of interspecies variation in cholinesterase metabolism.

This study describes an electrophoretically mediated microanalysis method, suitable for the preclinical evaluation of the hydrolysis of ester drugs by the serum of different animals and for further characterization of human-animal correlation. Dog, cat, cow, horse, sheep, rat and human serum were diluted (25%) in the appropriate buffer and replaced the enzyme solution usually used in electrophoretically mediated microanalysis methods for the study of enzyme kinetics. They were then compared in terms of the ability to hydrolyze acetylthiocholine and butyrylthiocholine (0.

Simultaneous determination of clopidogrel and its carboxylic acid metabolite by capillary electrophoresis.

A capillary electrophoresis method was developed and validated for the first time for the analysis of clopidogrel and its carboxylic acid metabolite. Prior to method optimization, the pH dependence of effective mobility of both compounds was determined in order to define the initial pH of the running buffer. The optimized method demonstrated to be selective, and linear in the concentration range of 2-100 microM for both compounds.

Design, synthesis, and pharmacological effects of a cyclization-activated steroid prodrug for colon targeting in inflammatory bowel disease.

Glucocorticoids are used in the treatment of inflammatory bowel disease. A limitation to their use is that they undergo absorption from the GIT before reaching the colon causing severe systemic side effects. We report here on a novel prodrug approach to targeting corticosteroids to the colon.

Dopamine- and tyramine-based derivatives of triazenes: activation by tyrosinase and implications for prodrug design.

A range of triazene derivatives were synthesized and investigated as prodrug candidates for melanocyte-directed enzyme prodrug therapy (MDEPT). The prodrugs contained a tyramine or dopamine promoiety required for tyrosinase activation and this was joined via a urea functional group to the cytotoxic triazene. The stability of each of the prodrugs in phosphate buffer, human plasma and in mushroom tyrosinase is discussed.

Prodrugs for amines.

The purpose of this work is to review the published strategies for the production of prodrugs of amines. The review is divided in two main groups of approaches: those that rely on enzymatic activation and those that take advantage of physiological chemical conditions for release of the drugs. A compilation of the most important approaches is presented in the form of a table, where the main advantages and disadvantages of each strategy are also referred.

Impregnation of an intraocular lens for ophthalmic drug delivery.

In this work the possibility of impregnating P(MMA-EHA-EGDMA) with flurbiprofen using a clean and environmentally friendly technology, namely supercritical fluid technology was evaluated. P(MMA-EHA-EGDMA) has been proposed as a promising matrix to be used for intraocular delivery of anti-inflammatory drugs used in eye surgery and flurbiprofen is a non-steroidal anti-inflammatory agent. Fundamental studies like, the solubility of the drug in carbon dioxide, as well as the sorption degree of this polymeric matrix in the presence of carbon dioxide have been previously carried out.

Sodium dodecyl sulfate-capillary gel electrophoresis analysis of rotavirus-like particles.

This work describes the application of a sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) method for the analysis of triple 2/6/7 and double-layered 2/6 rotavirus-like particles (RLPs), candidate vaccines against rotavirus infection. SDS-CGE analysis of RLPs resulted in peaks that could be attributed to the viral proteins (VP2, VP6 and VP7) according to their apparent molecular mass (MWapp). Samples containing the glycoprotein VP7 were analysed after deglycosylation with PNGase F.

Prediction of intestinal absorption and metabolism of pharmacologically active flavones and flavanones.

Three glycosilated flavonoids (diosmin, hesperidin and naringin) and respective aglycones were characterized in terms of their apparent ionisation constants and bidirectional permeability using the cellular model Caco-2 as well as the artificial membrane model PAMPA. Ionisation curves were established by capillary electrophoresis. It was confirmed that significant amounts of the aglycones are ionised at physiological pH whereas the glycosides are in the neutral form.

Beta-aminoketones as prodrugs with pH-controlled activation.

N-Mannich bases have been widely applied as prodrugs of amine drugs. The analogous C-Mannich bases (beta-aminoketones) have received rather less attention probably because they are not sufficiently susceptible to elimination at pHs encountered in vivo. Compounds in which there is a thermodynamic advantage to elimination may be an exception.

Chiral separation and identification of beta-aminoketones of pharmacological interest by high performance liquid chromatography and capillary electrophoresis.

This paper describes the development and comparison of chiral methods of analysis for a series of pharmacologically active indane derivatives that have been studied in the context of the evaluation of a promising prodrug system for amines. The methods are intended for studying the differences in the pharmacokinetics of the optical isomers of these compounds. Capillary electrophoresis, using cyclodextrins as chiral selectors, and HPLC, using a Pirkle type stationary phase, were tested.

Preparation of ethyl cellulose/methyl cellulose blends by supercritical antisolvent precipitation.

The supercritical antisolvent (SAS) technique was used to prepare ethyl cellulose/methyl cellulose blends, two biocompatible polymers commonly used as drug carriers in controlled delivery systems. Ethyl cellulose is widely used as a drug carrier. The drug release of the delivery devices can be controlled to some extent by addition of a water-soluble or water swellable polymer, such as methyl cellulose.

Preparation of controlled release microspheres using supercritical fluid technology for delivery of anti-inflammatory drugs.

Ethylcellulose/methylcellulose blends were produced using different precipitation techniques and impregnated with naproxen, a non-steroidal anti-inflammatory drug (NSAID). Solvent-evaporation technique was used not only for the preparation of ethylcellulose/methylcellulose microspheres but also to encapsulate naproxen. Supercritical fluid (SCF) impregnation was also performed to prepare naproxen loaded microspheres.

A new amino-masking group capable of pH-triggered amino-drug release.

The prodrug approach is potentially useful for mitigating pharmaceutical problems--such as poor membrane permeability or stability--which commonly occur with amino drugs. On the other hand there persists a dearth of useful systems for masking amines that satisfy the prodrug requirements of good in vitro stability coupled with predictable and rapid drug release in response to a local tissue condition. This study describes an evaluation of aminoindanes as bioreversibly masked amines poised to undergo elimination to the parent amine.