Stoichiometric Molecularly Imprinted Polymers for the Recognition of Anti-Cancer Pro-drug Tegafur

Molecularly Imprinted Polymers (MIPs) targeting tegafur, an anti-cancer 5-fluorouracil pro-drug, have been prepared by stoichiometric imprinting using 2,6-bis(acrylamido)pyridine (BAAPy) as the functional monomer. Solution association between tegafur and BAAPy was studied by 1H NMR titration, which confirmed the formation of 1:1 complexes with an affinity constant of 574±15 M-1 ¬in CDCl3. Evaluation of the synthesised materials by HPLC and equilibrium rebinding experiments revealed high selectivity of the imprinted polymer for the pro-drug versus 5-fluorouracil and other competing analytes, with maximum imprinting factors of 25.3 and a binding capacity of 45.1 μmol g-1. The synthesised imprinted polymer was employed in solid-phase extraction of the pro-drug using an optimised protocol that included a simple wash with the porogen used in the preparation of the material. Tegafur recoveries of up to 96% were achieved from aqueous samples and 92% from urine samples spiked with the template and three competing analytes. The results demonstrate the potential of the prepared polymers in the pre-concentration of tegafur from biological samples, which could be an invaluable tool in the monitoring of patient compliance and drug uptake and excretion.

Proteomic analysis of Artemisia annua – towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin

Background: MS-based proteomics was applied to the analysis of the medicinal plant Artemisia annua, exploiting a recently published contig sequence database (Graham et al. (2010) Science 327, 328–331) and other genomic and proteomic sequence databases for comparison. A. annua is the predominant natural source of artemisinin, the precursor for artemisinin-based combination therapies (ACTs), which are the WHO-recommended treatment for P. falciparum malaria. Results: The comparison of various databases containing A. annua sequences (NCBInr/viridiplantae, UniProt/ viridiplantae, UniProt/A. annua, an A. annua trichome Trinity contig database, the above contig database and another A. annua EST database) revealed significant differences in respect of their suitability for proteomic analysis, showing that an organism-specific database that has undergone extensive curation, leading to longer contig sequences, can greatly increase the number of true positive protein identifications, while reducing the number of false positives. Compared to previously published data an order-of-magnitude more proteins have been identified from trichome-enriched A. annua samples, including proteins which are known to be involved in the biosynthesis of artemisinin, as well as other highly abundant proteins, which suggest additional enzymatic processes occurring within the trichomes that are important for the biosynthesis of artemisinin. Conclusions: The newly gained information allows for the possibility of an enzymatic pathway, utilizing peroxidases, for the less well understood final stages of artemisinin’s biosynthesis, as an alternative to the known non-enzymatic in vitro conversion of dihydroartemisinic acid to artemisinin. Data are available via ProteomeXchange with identifier PXD000703.

Differential Cocrystallization Behavior of Isomeric Pyridine Carboxamides toward Antitubercular Drug Pyrazinoic Acid

Pyrazinoic acid, the active form of the antitubercular pro-drug Pyrazinamide, is an amphiprotic molecule containing carboxylic acid and pyridine groups and therefore can form both salts and cocrystals with relevant partner molecules. Cocrystallization of pyrazinoic acid with isomeric pyridine carboxamide series resulted in a dimorphic mixed-ionic complex with isonicotinamide and in eutectics with nicotinamide and picolinamide, respectively. It is observed that with alteration of the carboxamide position, steric and electrostatic compatibility issues between molecules of the combination emerge and affect intermolecular interactions and supramolecular growth, thus leading to either cocrystal or eutectic for different pyrazinoic acid-pyridine carboxamide combinations. Intermolecular interaction energy calculations have been performed to understand the role of underlying energetics on the formation of cocrystal/eutectic in different combinations. On the other hand, two molecular salts with piperazine and cytosine and a gallic acid cocrystal of the drug were obtained, and their X-ray crystal structures were also determined in this work.

Scope and Limitations of The Co-Drug Approach to Topical Drug Delivery

Many currently available drugs show unfavourable physicochemical properties for delivery into or across the skin and temporary chemical modulation of the penetrant is one option to achieve improved delivery properties. Pro-drugs are chemical derivatives of an active drug which is covalently bonded to an inactive pro-moiety in order to overcome pharmaceutical and pharmacokinetic barriers. A pro-drug relies upon conversion within the body to release the parent active drug (and pro-moiety) to elicit its pharmacological effect. The main drawback of this approach is that the pro-moiety is essentially an unwanted ballast which, when released, can lead to adverse effects. The term ‘co-drug’ refers to two or more therapeutic compounds active against the same disease bonded via a covalent chemical linkage and it is this approach which is reviewed for the first time in the current article. For topically applied co-drugs, each moiety is liberated in situ, either chemically or enzymatically, once the stratum corneum barrier has been overcome by the co-drug. Advantages include synergistic modulation of the disease process, enhancement of drug delivery and pharmacokinetic properties and the potential to enhance stability by masking of labile functional groups. The amount of published work on co-drugs is limited but the available data suggest the co-drug concept could provide a significant therapeutic improvement in dermatological diseases. However, the applicability of the co-drug approach is subject to strict limitations pertaining mainly to the availability of compatible moieties and physicochemical properties of the overall molecule.

Design, synthesis and in vitro degradation of a novel co-drug for the treatment of psoriasis

Psoriasis is a common, chronic and relapsing inflammatory skin disease. It affects approximately 2% of the western population and has no cure. Combination therapy for psoriasis often proves more efficacious and better tolerated than monotherapy with a single drug. Combination therapy could be administered in the form of a co-drug, where two or more therapeutic compounds active against the same condition are linked by a cleavable covalent bond. Similar to the pro-drug approach, the liberation of parent moieties post-administration, by enzymatic and/or chemical mechanisms, is a pre-requisite for effective treatment. In this study, a series of co-drugs incorporating dithranol in combination with one of several non-steroidal anti-inflammatory drugs, both useful for the treatment of psoriasis, were designed, synthesized and evaluated. An ester co-drug comprising dithranol and naproxen in a 1:1 stoichiometric ratio was determined to possess the optimal physicochemical properties for topical delivery. The co-drug was fully hydrolyzed in vitro by porcine liver esterase within four hours. When incubated with homogenized porcine skin, 9.5% of the parent compounds were liberated after 24 h, suggesting in situ esterase-mediated cleavage of the co-drug would occur within the skin. The kinetics of the reaction revealed first order kinetics, Vmax = 10.3 μM/min and Km = 65.1 μM. The co-drug contains a modified dithranol chromophore that was just 37% of the absorbance of dithranol at 375 nm and suggests reduced skin/clothes staining. Overall, these findings suggest that the dithranol-naproxen co-drug offers an attractive, novel approach for the treatment of psoriasis.

Characterization of inhibition of platelet function by paracetamol and its interaction with diclofenac and parecoxib

Aim: To characterize the inhibition of platelet function by paracetamol in vivo and in vitro, and to evaluate the possible interaction of paracetamol and diclofenac or valdecoxib in vivo. To assess the analgesic effect of the drugs in an experimental pain model. Methods: Healthy volunteers received increasing doses of intravenous paracetamol (15, 22.5 and 30 mg/kg), or the combination of paracetamol 1 g and diclofenac 1.1 mg/kg or valdecoxib 40 mg (as the pro-drug parecoxib). Inhibition of platelet function was assessed with photometric aggregometry, the platelet function analyzer (PFA-100), and release of thromboxane B2. Analgesia was assessed with the cold pressor test. The inhibition coefficient of platelet aggregation by paracetamol was determined as well as the nature of interaction between paracetamol and diclofenac by an isobolographic analysis in vitro. Results: Paracetamol inhibited platelet aggregation and TxB2-release dose-dependently in volunteers and concentration-dependently in vitro. The inhibition coefficient was 15.2 mg/L (95% CI 11.8 - 18.6). Paracetamol augmented the platelet inhibition by diclofenac in vivo, and the isobole showed that this interaction is synergistic. Paracetamol showed no interaction with valdecoxib. PFA-100 appeared insensitive in detecting platelet dysfunction by paracetamol, and the cold-pressor test showed no analgesia. Conclusions: Paracetamol inhibits platelet function in vivo and shows synergism when combined with diclofenac. This effect may increase the risk of bleeding in surgical patients with an impaired haemostatic system. The combination of paracetamol and valdecoxib may be useful in patients with low risk for thromboembolism. The PFA-100 seems unsuitable for detection of platelet dysfunction and the cold-pressor test seems unsuitable for detection of analgesia by paracetamol.

Conformational basis for substrate recognition and regulation of catalytic activity in Staphylococcus aureus nucleoside di-phosphate kinase

Nucleoside diphosphate kinases (NDK) are characterized by high catalytic turnover rates and diverse substrate specificity. These features make this enzyme an effective activator of a pro-drug an application that has been actively pursued for a variety of therapeutic strategies. The catalytic mechanism of this enzyme is governed by a conserved histidine that coordinates a magnesium ion at the active site. Despite substantial structural and biochemical information on NDK, the mechanistic feature of the phospho-transfer that leads to auto-phosphorylation remains unclear. While the role of the histidine residue is well documented, the other active site residues, in particular the conserved serine remains poorly characterized. Studies on some homologues suggest no role for the serine residue at the active site, while others suggest a crucial role for this serine in the regulation and quaternary association of this enzyme in some species. Here we report the biochemical features of the Staphylococcus aureus NDK and the mutant enzymes. We also describe the crystal structures of the apo-NDK, as a transition state mimic with vanadate and in complex with different nucleotide substrates. These structures formed the basis for molecular dynamics simulations to understand the broad substrate specificity of this enzyme and the role of active site residues in the phospho-transfer mechanism and oligomerization. Put together, these data suggest that concerted changes in the conformation of specific residues facilitate the stabilization of nucleotide complexes thereby enabling the steps involved in the ping-pong reaction mechanism without large changes to the overall structure of this enzyme. (C) 2011 Elsevier B.V. All rights reserved.

Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils

Short chain fatty acids (SCFAs) are fermentation products of anaerobic bacteria. More than just being an important energy source for intestinal epithelial cells, these compounds are modulators of leukocyte function and potential targets for the development of new drugs. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate and butyrate) on production of nitric oxide (NO) and proinflammatory cytokines [tumor necrosis factor alpha (TNF-alpha) and cytokine-induced neutrophil chemoattractant-2 (CINC-2 alpha beta)] by rat neutrophils. The involvement of nuclear factor kappa B (NF-kappa B) and histone deacetylase (HDAC) was examined. The effect of butyrate was also investigated in vivo after oral administration of tributyrin (a pro-drug of butyrate). Propionate and butyrate diminished TNF-alpha, CINC-2 alpha beta and NO production by LPS-stimulated neutrophils. We also observed that these fatty acids inhibit HDAC activity and NF-kappa B activation, which might be involved in the attenuation of the LPS response. Products of cyclooxygenase and 5-lipoxygenase are not involved in the effects of SCFAs as indicated by the results obtained with the inhibitors of these enzymes. The recruitment of neutrophils to the peritonium after intraperitoneal administration of a glycogen solution (1%) and the ex vivo production of cytokines and NO by neutrophils were attenuated in rats that previously received tributyrin. These results argue that this triglyceride may be effective in the treatment of inflammatory conditions. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Updates on naringinase : structural and biotechnological aspects

Naringinases has attracted a great deal of attention in recent years due to its hydrolytic activities which include the production of rhamnose, and prunin and debittering of citrus fruit juices. While this enzyme is widely distributed in fungi, its production from bacterial sources is less commonly known. Fungal naringinase are very important as they are used industrially in large amounts and have been extensively studied during the past decade. In this article, production of bacterial naringinase and potential biotechnological applications are discussed. Bacterial rhamnosidases are exotype enzymes that hydrolyse terminal non-reducing α-l-rhamnosyl groups from α-l-rhamnose containing polysaccharides and glycosides. Structurally, they are classified into family 78 of glycoside hydrolases and characterized by the presence of Asp567 and Glu841 in their active site. Optimization of fermentation conditions and enzyme engineering will allow the development of improved rhamnosidases for advancing suggested industrial applications.

Synthesis, Characterization and Pharmacological Evaluation of 1-(2-Chloro-6-Fluorophenyl)-5-Methylindolin-2-One: A New Anti-Inflammatory Compound with Reduced Gastric Ulceration Properties

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Synthesis, ex Vivo and in Vitro Hydrolysis Study of an Indoline Derivative Designed as an Anti-Inflammatory with Reduced Gastric Ulceration Properties

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Theoretical study of omeprazole behavior: Racemization barrier and decomposition reaction

omeprazole is a substituted benzimidazole which suppresses gastric-acid secretion by means of H+, K+-ATPase inhibition. It is an optically active drug with the sulfur of the sulfoxide being the chiral center. This pro-drug can be easily converted into its respective sulfenamide at low pH. In this work, omeprazole has been studied in relation to racemization barrier and decomposition reaction. Quantum chemistry coupled to PCA chemometric method were used to find all minimum energy structures. Conformational analysis and calculation of racemization barriers were carried out by PM3 semiempirical method (Gaussian 98). The average racemization energy barrier for all minimum energy structures (43.56 kcal mol(-1)) can be related to the velocity constant in Eyring's equation. The enormous half-life time at 100 degrees C (9.04 x 10(4) years) indicates that the process cannot be observed in human time scale. on the other hand, the difference of free energy change (Delta(Delta G) = -266.78 kcal mol(-1)) for the decomposition reaction shows that the process is favorable to the sulfenamide formation. The highly negative Delta(Delta G) obtained for the decomposition reaction shows that this process is extremely exothermic. This result explains why omeprazole decomposes and does not racemize. (C) 2008 Wiley Periodicals, Inc.

Crystallographic studies on the binding of isonicotinyl-NAD adduct to wild-type and isoniazid resistant 2-trans-enoyl-ACP (CoA) reductase from Mycobacterium tuberculosis

The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (121V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents. (c) 2007 Elsevier B.V. All rights reserved.