Transdermal iontophoretic delivery of ketoprofen in cadaver skin and healthy volunteers was examined. In vitro anodic and cathodic iontophoresis (0.5 mA/cm2, 3 h) of ketoprofen (75 mg/ml) resulted in equivalent intracutaneous ketoprofen permeation (232.1 ± 27.1 vs. 275.0 ± 141.0 μg/cm2, respectively), which in turn was higher than passive intracutaneous uptake of ketoprofen (40.7 ± 42.1 μg/cm2). In contrast, only cathodic iontophoresis resulted in transcutaneous ketoprofen permeation across cadaver skin, under these conditions. The in vitro study was then repeated to achieve transcutaneous permeation of ketoprofen at clinical iontophoretic parameters (0.28 mA/cm2, 40 min) by increasing drug concentration to 300 mg/ml. No stereo-selective permeation of R- and S-ketoprofen enantiomers was observed in vitro. In humans, cathodic iontophoresis of 300 mg/ml ketoprofen (0.28 mA/cm2, 40 min) was performed at the wrist. Ketoprofen was detected at 40 min (0.88 ± 0.42 μg/ml) from the forearm veins of the ipsilateral arm. Urinary excretion of ketoprofen totaled 790 ± 170 μg at 16 h post iontophoresis. This investigation is the first to clearly demonstrate transcutaneous iontophoresis of an antiinflammatory agent in humans utilizing a commercially cleared iontophoretic device. The investigation also adds to the very limited number of publications in the area of iontophoretic delivery of drugs to humans.

Yes / The use of methylmalonyl-CoA epimerase (MCEE) to improve stereoselectivity in crotonase-mediated biocatalysis is exemplified by the coupling of MCEE, crotonyl-CoA carboxylase reductase and carboxymethylproline synthase in a three-enzyme one-pot sequential synthesis of functionalised C-5 carboxyalkylprolines starting from crotonyl-CoA and carbon dioxide. / Biotechnology and Biological Sciences Research Council, The Wellcome Trust, and CONACyT and FIDERH (Mexico, RGC) The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from AbbVie, Boehringer Ingelheim, the Canada Foundation for Innovation, the Canadian Institutes for Health Research, Genome Canada, GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda, and the Wellcome Trust (092809/Z/10/Z).

Les héparanes sulfates (HS) sont des polysaccharides linéaires et sulfatés exprimés sur la surface cellulaire où ils interagissent et régulent l’activité de nombreuses proteines, en particulier les cytokines et chimiokines. Ils sont à ce titre de bons candidats médicaments dans des pathologies inflammatoires, autoimmunes ou en oncologie. L’unité répétitive de ce biopolymère est constituée d’un résidu de D-glucosamine lié à un acide uronique par une liaison 1,2-cis. Malheureusement, la formation d’un glycoside 1,2-cis dans la série 2-azido-2-déoxy-D-glucose avec une haute stéréosélectivité reste un des plus grands défis de la glycochimie. Parmi les nombreuses méthodologies permettant d’accéder à la synthèse des fragments d’HS avec de bons rendements et une bonne stéréosélectivité, nous avons été particulièrement intéressés par l’assistance anchimérique d’un groupement protecteur en position 6 du donneur. L’objectif de ce travail était de trouver des groupements protecteurs qui favoriseront la stéréosélectivité 1,2-cis. Nous avons préparés plusieurs donneurs thioglycosides modifiés en position 6 par des différents groupements protecteurs. L’activation des thioglycosides passe par une étape de formation des triflates anomériques. Nous avons élaboré un protocole de suivi de l’activation sur un donneur modèle afin de suivre la formation du triflate anomérique, sa plage de stabilité, ses produits de dégradation ainsi que les produits secondaires d’activation par RMN à basse température. Ensuite, ce protocole d’activation a été utilisé avec tous les donneurs synthétisés afin d’ajuster les conditions de glycosylation. Les tests de glycosylation nous ont permis de décéler plusieurs groupes capables de favoriser la stéréosélectivité 1,2-cis. Certains groupements protecteurs ont manifesté une incompatibilité avec les conditions d’activation des thioglycosides. Pour contourner ce problème, nous avons remplacé les thioglycosides par les donneurs N-phényltrifluoroacétimidates. Après avoir effectué des études d’activation sur ces donneurs toujours par RMN à basse température, les glycosylations ont été effectuées. Finalement, les groupements protecteurs favorisant la stéréosélectivité 1,2-cis ont été testés dans différentes conditions de déprotection afin d’établir la compatibilité de ces groupements protecteurs avec les conditions de synthèse des oligosaccharides d’HS. / Heparin sulfate (HS) are linear and sulfated polysaccharides present at the cell surface. HS interact and regulate activity of numerous proteins, especially cytokines and chemokines. Therefore, HS oligosaccharides are targeted as potential drugs in inflammation, autoimmune disease or tumor treatment. The basic disaccharide unit of HS consists in D-glucosamine residue linked to an uronic acid by 1,2-cis glycosidic linkage. Unfortunately, the formation of highly stereoselective 1,2-cis glycosidic bond in 2-azido-2-deoxy-D-glucose series is still a major concern in glycochemistry. Amongst the numerous methodologies favoring the stereoselective 1,2-cis linkage formation, we were particularly interested in 6-O-anchimeric assistance. Several thioglycoside donors with different protecting groups in position 6 were prepared to find some 1,2-cis stereodirecting protecting groups. Some thioglycoside activation related in literature yields a reactive anomeric triflate intermediate. In order to observe its formation and to determine the limits of its stability and by-product formation, a new low temperature NMR experiment protocol was elaborated. All synthesized donors were tested using this protocol in order to adjust their glycosylation conditions. The glycosylation tests revealed several 1,2-cis stereodirecting protecting groups. Since certain protecting groups were incompatible with thioglycoside activation conditions, corresponding NPTFA donors were used as an alternative. Their activations were monitored by low temperature NMR techniques and followed by their glycosylations. Finally, all 1,2-cis stereodirecting protecting groups were tested in different deprotection conditions to determine the compatibility of chosen protecting groups with our HS oligosaccharide design synthesis.

Enantiopure alcohols are valuable intermediates in fine organic synthesis, in particular for preparation of biologically active compounds. The necessity of preparing single enantiomer drugs in an optically pure form has triggered much research, especially in the pharmaceutical industry. The biocatalytical production of chiral alcohols by alcohol dehydrogenase enzymes is characterized by the asymmetric reduction of the corresponding ketones, usually with high degree of stereoselectivity. The commercial value of the enzymes as stereoselective biocatalysts has been a significant driving force in understanding features that control their mechanism of catalysis and stereoselectivity. This work focuses on two enantiocomplementary dehydrogenase enzymes ((R)- and 2-(S)-hydroxypropyl-CoM (HPC) dehydrogenases (DH)) of the epoxide carboxylation pathway in Xanthobacter autotrophicus strain Py2. The main goal of this dissertation is to kinetically, mechanistically and structurally characterize S-HPCDH and through the comparison studies with R-HPCDH reveal the basis for high degree of stereoselectivity exhibited by both enzymes. Analysis of the molecular structure of R-HPCDH and the homology model of S-HPCDH suggests a mechanism of substrate specificity in which the binding of the substrate sulfonate moiety at distinct sites on each stereoselective enzyme directs the orientation of the appropriate substrate enantiomer for the hydride abstraction. The positively charged residues responsible for binding the CoM moiety of the substrate were identified in R-HPCDH (Arg152 and Arg196), and in S-HPCDH (Arg211 and Lys214). Site-directed mutagenesis confirmed their importance in binding and orienting physiological substrates, but not the substrates lacking the CoM moiety. Extensive kinetic and mechanistic characterization of S-HPCDH reveals its key catalytic features similar to those of R-HPCDH, but also points out a few important differences. Furthermore, the role of the methionine residues flanking the substrate in the active site of both dehydrogenases was investigated. Substitution of these residues to alanine resulted in enzymes with significantly altered catalytic parameters and suggested their importance in binding and catalysis. Additionally, the X-ray crystal structures of the Met187Ala and Met192Ala mutants of R-HPCDH have revealed their role as "gate keepers," protecting the active site from the surrounding solvent. Kinetic analysis of Met187Leu and Met192Leu mutants implied a structural, rather than catalytic function of the methionines. It is proposed that steric clashes of the terminal methyl group of the HPC substrates with the nicotinamide ring of NAD+ are a major determinant of the enantioselectivity in S-HPCDH. This research provides the first side-by-side characterization of a pair of short-chain dehydrogenase/reductase (SDR) enzymes expressed simultaneously to act on two enantiomers of the same alcohol produced in a metabolic pathway. The R-HPCDH and S-HPCDH enzymes are distinguished from all other known members of the SDR family in using the novel sulfonate functional group of coenzyme M as a handle for chiral discrimination. These results provide a standard for examining the molecular basis of stereoselectivity in other such enzyme pairs.

<p>Glutathione transferases (GSTs) constitute a family of multifarious enzymes that conjugate glutathione (GSH) with a wide range of electrophiles. GSTs are grouped into different classes based on protein sequence similarities. Despite high sequence identities between GSTs of the same class they often display different substrate specificites. Human GST M1-1 is efficiently catalyzing the conjugation of GSH and various epoxide substrates, whereas the 84% sequence-identical GST M2-2 has low activities with the same substrates.</p><p>Evolutionary rate analysis was used to identify hypervariable amino acid positions among GST Mu class sequences. A Thr to Ser conversion of the variable residue 210 in GST M2-2 elicited a drastic increase in catalytic activity with epoxides, which is the characteristic activity of GST M1-1. This provides support for the usefulness of evolutionary analysis in identifying functionally important residues, although the additional mutations of two other variable residues did not confer any noteworthy changes in activity.</p><p>To further investigate the functional importance of residue T210 in GST M2-2 it was replaced by all other commonly occurring amino acids. The replacements caused marked changes in substrate specificity, stability, and expressivity, indicating how functionalities of a duplicated Mu class GST may easily be altered by point mutations. </p><p>The stereo- and regioselectivity in epoxide-conjugation catalyzed by GSTs M1-1 and M2-2 was investigated. The results show that a serine in position 210 is beneficial for high enantioselectivity with trans-stilbene oxide. However, an alanine in position 210 is more favorable for stereo- and regioselectivity with the smaller epoxide substrate styrene-7,8-oxide. </p><p>The low enantioselectivity of GST M1-1 was improved 10- and 9- fold with styrene-7,8-oxide and 1-phenylpropylene oxide, respectively, through different combination of site-specific mutations and posttranslational chemical modifications. The approach can be employed in more extensive screening experiments where a large variety of modifications easily can be tested.</p>

The influence of hyperlipidemia on the pharmacodynamic and pharmacokinetic aspects of lipophilic drugs was explored. The antiarrhythmic, amiodarone, and the antifungal, ()-ketoconazole, were used as model drugs. Experimental hyperlipidemia was induced in rat using poloxamer 407 and two sensitive novel HPLC assays were developed. In a multiple dosing study, hyperlipidemia increased amiodarone plasma concentrations, heart concentrations and electrocardiographic changes. The amiodarone heart uptake could not be totally attributed to its unbound fraction, where the cardiac very low density lipoprotein receptors seemed to play a role in the uptake of bound drug. Amiodarone liver metabolism in presence and absence of hyperlipidemia was studied using isolated primary rat liver hepatocytes. The metabolism of amiodarone was lower in hepatocytes isolated from hyperlipidemic than those from normolipidemic rats. Hyperlipidemic serum resulted in a decrease in amiodarone metabolism and when coincubated, the expected decrease in unbound fraction seemed to resulted in greater inhibition of metabolism. ()-Ketoconazole showed stereoselectivity in its pharmacokinetics in rat with (+)-ketoconazole showing higher plasma concentrations than its antipode. This was attributed to its higher protein binding. There was no difference in the total bioavailability of the two enantiomers. Ketoconazole enantiomers exhibited nonlinear pharmacokinetics. In normolipidemic rat plasma ketoconazole enantiomers were more than 95% bound to lipoprotein deficient fraction. Hyperlipidemia resulted in shifting both enantiomers 20% to very low density and low density lipoprotein fractions. In a pharmacokinetic assessment, hyperlipidemia was found to increase ketoconazole enantiomer volume of distribution. Moreover, the stereoselectivity ratios of most pharmacokinetic parameters were changed. After oral dosing, the uptake of (-)-ketoconazole was significantly decreased. Since ketoconazole is used as a potent CYP3A inhibitor, alteration in liver concentrations of (-)-ketoconazole, the more potent inhibitory enantiomer, could decrease its CYP inhibitory potential. Hyperlipidemia potentiated the CYP-mediated interaction between ketoconazole and midazolam with significantly higher midazolam AUC and lower clearance. This was attributed to the inhibitory action of ketoconazole and the effect of hyperlipidemia on the binding of midazolam. Hyperlipidemia was found to unexpectedly decrease midazolam unbound fraction in plasma. In conclusion, the findings could explain some unexpected dose versus effect outcomes in hyperlipidemic patients receiving amiodarone or ketoconazole. / Pharmaceutical Sciences

Glutathione transferases (GSTs) constitute a family of multifarious enzymes that conjugate glutathione (GSH) with a wide range of electrophiles. GSTs are grouped into different classes based on protein sequence similarities. Despite high sequence identities between GSTs of the same class they often display different substrate specificites. Human GST M1-1 is efficiently catalyzing the conjugation of GSH and various epoxide substrates, whereas the 84% sequence-identical GST M2-2 has low activities with the same substrates. Evolutionary rate analysis was used to identify hypervariable amino acid positions among GST Mu class sequences. A Thr to Ser conversion of the variable residue 210 in GST M2-2 elicited a drastic increase in catalytic activity with epoxides, which is the characteristic activity of GST M1-1. This provides support for the usefulness of evolutionary analysis in identifying functionally important residues, although the additional mutations of two other variable residues did not confer any noteworthy changes in activity. To further investigate the functional importance of residue T210 in GST M2-2 it was replaced by all other commonly occurring amino acids. The replacements caused marked changes in substrate specificity, stability, and expressivity, indicating how functionalities of a duplicated Mu class GST may easily be altered by point mutations. The stereo- and regioselectivity in epoxide-conjugation catalyzed by GSTs M1-1 and M2-2 was investigated. The results show that a serine in position 210 is beneficial for high enantioselectivity with trans-stilbene oxide. However, an alanine in position 210 is more favorable for stereo- and regioselectivity with the smaller epoxide substrate styrene-7,8-oxide. The low enantioselectivity of GST M1-1 was improved 10- and 9- fold with styrene-7,8-oxide and 1-phenylpropylene oxide, respectively, through different combination of site-specific mutations and posttranslational chemical modifications. The approach can be employed in more extensive screening experiments where a large variety of modifications easily can be tested.

The following study investigates the intramolecular and intermolecular interactions responsible for invoking stereoselectivity and stereospecificity in the synthesis of a chiral original species and amino acid receptor. The former commences with a brief overview of the nature, scope and applications of helical chirality, and culminates in the formation of a permanent helix via the synthesis of a novel chiral lactone. The latter is discussed as an extension of a naturally occurring cofactor whose identity is modulated to furnish a tailored receptor selective to the binding of amino-acid enantiomers. The study and analysis is executed via both synthetic and computational methods.

The following study investigates the intramolecular and intermolecular interactions responsible for invoking stereoselectivity and stereospecificity in the synthesis of a chiral original species and amino acid receptor. The former commences with a brief overview of the nature, scope and applications of helical chirality, and culminates in the formation of a permanent helix via the synthesis of a novel chiral lactone. The latter is discussed as an extension of a naturally occurring cofactor whose identity is modulated to furnish a tailored receptor selective to the binding of amino-acid enantiomers. The study and analysis is executed via both synthetic and computational methods.

No description available.