Études pharmacocinétiques exploratoires de certains médicaments utilisés en analgésie post-opératoire

La douleur post-opératoire chez les patients constitue un défi thérapeutique important pour les cliniciens. Le traitement de la douleur post-opératoire n’est pas accessoire ni optionnel, puisqu’il permet de donner un congé de l’hôpital plus rapide aux patients et ainsi, il contribue à des économies importantes pour notre système de santé. Parmi les approches thérapeutiques utilisées pour la prise en charge de la douleur post-opératoire, cette thèse s’intéresse particulièrement aux blocs de nerfs périphériques par les anesthésiques locaux et à l’administration de la néostigmine par voie épidurale. Ces médicaments sont utilisés en clinique sans avoir préalablement établi, en se basant sur leur propriétés pharmacocinétiques et pharmacodynamiques spécifiques, leurs doses optimales. Ces doses devraient également tenir en considération les particularités anatomiques du site d’injection par rapport au site d’action. Cette thèse inclut des études exploratoires qui ont contribué à caractériser la pharmacocinétique de la ropivacaïne et de la bupivacaïne ainsi que la pharmacocinétique et la pharmacodynamie de la néostigmine. La première étude portait sur seize patients subissant une chirurgie orthopédique avec un bloc combiné des nerfs fémoral et sciatique par la ropivacaïne (n=8) ou la bupivacaïne (n=8). C’était la première étude qui a inclu des temps d’échantillons pharmacocinétiques allant jusqu’à 32 h après le bloc et ces résultats ont démontré une variabilité interindividuelle considérable. La modélisation par approche de population a aidé à expliquer les sources de la variabilité et démontré que l’absorption systémique des anesthésiques locaux était très lente. De plus, les concentrations plasmatiques demeuraient mesurables, et dans certains cas présentaient un plateau, 32 h après le bloc. Dans les prochaines études, un échantillonnage allant jusqu’à 4 ou 5 jours sera nécessaire afin d’atteindre la fin de l’absorption. La deuxième étude a établi le développement d’un modèle animal en étudiant la pharmacocinétique de la ropivacaïne après administration intraveineuse ainsi que son degré de liaison aux protéines plasmatiques chez le lapin (n=6). Les résultats ont démontré que, chez le lapin la ropivacaïne est beaucoup moins liée aux protéines plasmatiques comparativement à l’humain. Ce résultat important sera utile pour planifier les prochaines études précliniques. La troisième étude a exploré, pour la première fois, la pharmacocinétique et la pharmacodynamie de la néostigmine administrée par voie épidurale et a essayé de caractériser la courbe dose-réponse en utilisant trois doses différentes : 0.5, 1 et 1.5 mg. Bien que les concentrations de la néostigmine dans le liquide céphalo-rachidien fussent très variables une relation inverse entre la consommation de mépéridine et la dose de néostigmine a été démontrée. La dose de 1.5 mg a donné une meilleure réponse pharmacodynamique sur la douleur, mais elle a été considérée comme dangereuse puisqu’elle a résulté en deux cas d’hypertension. Nous avons conclu que des doses plus faibles que 1.5 mg devront être utilisées lors de l’utilisation de la néostigmine par voie épidurale. En conclusion, les études rapportées dans cette thèse ont exploré les propriétés pharmacocinétiques et/ou pharmacodynamiques de certains médicaments utilisés pour le traitement de la douleur post-opératoire. Ceci mènera au but ultime qui est la meilleure prise en charge de la douleur post-opératoire chez les patients.

TDP-43 régule la dynamique et la fonction des Granules de Stress via G3BP1

Les Granule de Stress (GS) sont des inclusions cytoplasmiques contenant des protéines et des ARNm qui s’assemblent en réponse à l’exposition à un stress. Leur formation fait partie intégrante de la réponse cellulaire au stress et est considérée comme une étape déterminante pour la résistance au stress et la survie cellulaire. Actuellement, les GS sont reliés à divers pathologies allant des infections virales aux maladies neurovégétatives. L’une d’entre elle, la Sclérose Latérale Amyotrophique (SLA) est particulièrement agressive, caractérisée par une perte des neurones moteurs aboutissant à la paralysie et à la mort du patient en cinq ans en moyenne. Les mécanismes de déclenchement de la pathologie restent encore à déterminer. TDP-43 (TAR DNA binding protein 43) et FUS (Fused in liposarcoma) sont deux protéines reliées à la pathologie qui présentent des similarités de structure et de fonction, suggérant un mécanisme commun de toxicité. TDP-43 et FUS sont toutes les deux recrutées au niveau des GS en condition de stress. Nous avons démontré pour la première fois que la fonction des GS est de protéger les ARNm de la dégradation induite par l’exposition au stress. Cette fonction n’était que suspectée jusqu’alors. De plus nous avons mis en évidence que G3BP1 (Ras GTPase-activating protein-binding protein 1) est l’effectrice de cette fonction via son implication dans la dynamique de formation des GS. TDP-43 étant un régulateur de G3BP1, nous prouvons ainsi que la perte de fonction de TDP-43/G3BP1 aboutit à un défaut de réponse au stress aboutissant à une vulnérabilisation cellulaire. Le mécanisme de toxicité emprunter par FUS diffère de celui de TDP-43 et ne semble pas passer par une perte de fonction dans le cadre de la réponse au stress.

Copper complexes of new benzodioxotetraaza macrocycles with potential applications in nuclear medicine

Two novel benzodioxotetraaza macrocycles [2,9-dioxo-1,4,7,10-tetraazabicyclo[10.4.0]1,11-hexadeca-1(11),13,15-triene (H(2)L1) and 2,10-dioxo-1,4,8,11-tetraazabicyclo[11.4.0]1,12-heptadeca-1(12),14,16-triene (H(2)L2)] were synthesized by a [1 + 1] crablike cyclization. The protonation constants of both ligands were determined by H-1 NMR titration and by potentiometry at 25.0 degrees C in 0.10 M ionic strength in KNO3. The latter method was also used to ascertain the stability constants of their copper(II) complexes. These studies showed that the CuL1 complex has a much lower thermodynamic stability than the CuL2, and the H(2)L2 displays an excellent affinity for copper(II), due to the good fit of copper(II) into its cavity. The copper complexes of the novel ligands were characterized by electronic spectroscopy in solution and by crystal X-ray diffraction. These studies indicated that the copper center in the CuL1 complex adopts a square-pyramidal geometry with the four nitrogen atoms of the macrocycle forming the equatorial plane and a water molecule at axial position, and the copper in the CuL2 complex is square-planar. Several labeling conditions were tested, and only H(2)L2 could be labeled with Cu-67 efficiently (> 98%) in mild conditions (39 degrees C, 15 min) to provide a slightly hydrophilic radioligand (log D = -0.19 +/- 0.03 at pH 7.4). The in vitro stability was studied in the presence of different buffers or with an excess of diethylenetriamine-pentaethanoic acid. Very high stability was shown under these conditions for over 5 days. The incubation of the radiocopper complex in human serum showed 6% protein binding.

Selective separation of the major whey proteins using ion exchange membranes

Synthetic microporous membranes with functional groups covalently attached were used to selectively separate beta-lactoglobulin, BSA, and alpha-lactalbumin from rennet whey. The selectivity and membrane performance of strong (quaternary ammonium) and weak (diethylamine) ion-exchange membranes were studied using breakthrough curves, measurement of binding capacity, and protein composition of the elution fraction to determine the binding behavior of each membrane. When the weak and strong anion exchange membranes were saturated with whey, they were both selective primarily for beta-lactoglobulin with less than 1% of the eluate consisting of alpha-lactalbumin or BSA. The binding capacity of a pure alpha-lactoglobulin solution was in excess of 1.5 mg/cm(2) of membrane. This binding capacity was reduced to approximately 1.2 mg/cm(2) when using a rennet whey solution (pH 6.4). This reduction in protein binding capacity can be explained by both the competitive effects of other whey proteins and the effect of ions present in whey. Using binary solution breakthrough curves and rennet whey breakthrough curves, it was shown that alpha-lactalbumin and BSA were displaced from the strong and weak anion exchange membranes by beta-lactoglobulin. Finally, the effect of ionic strength on the binding capacity of individual proteins for each membrane was determined by comparing model protein solutions in milk permeate (pH 6.4) and a 10 mM sodium phosphate buffer (pH 6.4). Binding capacities of beta-lactoglobulin, alpha-lactalbumin, and BSA in milk permeate were reduced by as much as 50%. This reduction in capacity coupled with the low binding capacity of current ion exchange membranes are 2 serious considerations for selectively separating complex and concentrated protein solutions.

Molecular mechanisms by which dietary isoflavones potentially prevent atherosclerosis

Dietary isoflavones are currently receiving much attention because of their potential role in preventing coronary artery disease and other chronic diseases. Accumulating evidence from cell culture and laboratory animal experiments indicates that isoflavones have the potential to prevent or delay atherogenesis. Suggested mechanisms of action include: a reduction in low-density lipoprotein (LDL) cholesterol and a potential reduction in the susceptibility of the LDL particle to oxidation; (2) an improvement in vascular reactivity; (3) an inhibition of pro-inflammatory cytokines, cell adhesion proteins and nitric oxide (NO) production; and (4) an inhibition of platelet aggregation. These mechanisms are consistent with the epidemiological evidence that a high consumption of isoflavone-rich soy products is associated with a reduced incidence of coronary artery disease. Biological effects of isoflavones are dependent on many factors, including dose consumed, duration of use, protein-binding affinity, and an individual's metabolism or intrinsic oestrogenic state. Further clinical studies are necessary to determine the potential health effects of isoflavones in specific population groups as we currently know little about age-related differences in exposure to these compounds and there are few guidelines on optimal dose for cardiovascular health benefits.

Genetic diversity at the Dhn3 locus in Turkish Hordeum spontaneum populations with comparative structural analyses

We analysed Hordeum spontaneum accessions from 21 different locations to understand the genetic diversity of HsDhn3 alleles and effects of single base mutations on the intrinsically disordered structure of the resulting polypeptide (HsDHN3). HsDHN3 was found to be YSK2-type with a low-frequency 6-aa deletion in the beginning of Exon 1. There is relatively high diversity in the intron region of HsDhn3 compared to the two exon regions. We have found subtle differences in K segments led to changes in amino acids chemical properties. Predictions for protein interaction profiles suggest the presence of a protein-binding site in HsDHN3 that coincides with the K1 segment. Comparison of DHN3 to closely related cereals showed that all of them contain a nuclear localization signal sequence flanking to the K1 segment and a novel conserved region located between the S and K1 segments [E(D/T)DGMGGR]. We found that H. vulgare, H. spontaneum, and Triticum urartu DHN3s have a greater number of phosphorylation sites for protein kinase C than other cereal species, which may be related to stress adaptation. Our results show that the nature and extent of mutations in the conserved segments of K1 and K2 are likely to be key factors in protection of cells.

Proanthocyanidins inhibit Ascaris suum glutathione-S-transferase activity and increase susceptibility of larvae to levamisole and ivermectin in vitro

Proanthocyanidins (PAC) are a class of plant secondary metabolites commonly found in the diet that have shown potential to control gastrointestinal nematode infections. The anti-parasitic mechanism(s) of PAC remain obscure, however the protein-binding properties of PAC suggest that disturbance of key enzyme functions may be a potential mode of action. Glutathione-S-transferases (GSTs) are essential for parasite detoxification and have been investigated as drug and vaccine targets. Here, we show that purified PAC strongly inhibit the activity of both recombinant and native GSTs from the parasitic nematode Ascaris suum. As GSTs are involved in detoxifying xenobiotic substances within the parasite, we hypothesised that this inhibition may render parasites hyper-susceptible to anthelmintic drugs. Migration inhibition assays with A. suum larvae demonstrated that the potency of levamisole (LEV) and ivermectin (IVM) were significantly increased in the presence of PAC purified from pine bark (4.6-fold and 3.2-fold reduction in IC50 value for LEV and IVM, respectively). Synergy analysis revealed that the relationship between PAC and LEV appeared to be synergistic in nature, suggesting a specific enhancement of LEV activity, whilst the relationship between PAC and IVM was additive rather than synergistic, suggesting independent actions. Our results demonstrate that these common dietary compounds may increase the efficacy of synthetic anthelmintic drugs in vitro, and also suggest one possible mechanism for their well-known anti-parasitic activity.

Chitosan as a removing agent of beta-lactoglobulin from membrane models

Many chitosan biological activities depend on the interaction with biomembranes, but so far it has not been possible to obtain molecular-level evidence of chitosan action. In this article, we employ Langmuir phospholipid monolayers as cell membrane models and show that chitosan is able to remove beta-lactoglobulin (BLG) from negatively charged dimyristoyl phosphatidic acid (DMPA) and dipalmitoyl phosphatidyl glycerol (DPPG). This was shown with surface pressure isotherms and elasticity and PM-IRRAS measurements in the Langmuir monolayers, in addition to quartz crystal microbalance and fluorescence spectroscopy measurements for Langmuir-Blodgett (LB) films transferred onto solid substrates. Some specificity was noted in the removal action because chitosan was unable to remove BLG incorporated into neutral dipalmitoyl phosphatidyl choline (DPPC) and cholesterol monolayers and had no effect on horseradish peroxidase and urease interacting with DMPA. An obvious biological implication of these findings is to offer reasons that chitosan can remove BLG from lipophilic environments, as reported in the recent literature.

PK/DB: database for pharmacokinetic properties and predictive in silico ADME models

The study of pharmacokinetic properties (PK) is of great importance in drug discovery and development. In the present work, PK/DB (a new freely available database for PK) was designed with the aim of creating robust databases for pharmacokinetic studies and in silico absorption, distribution, metabolism and excretion (ADME) prediction. Comprehensive, web-based and easy to access, PK/DB manages 1203 compounds which represent 2973 pharmacokinetic measurements, including five models for in silico ADME prediction (human intestinal absorption, human oral bioavailability, plasma protein binding, bloodbrain barrier and water solubility).

Menezesite, the first natural heteropolyniobate, from Cajati, Sao Paulo, Brazil: Description and crystal structure

Menezesite, ideally Ba2MgZr4(BaNb12O42)center dot 12H(2)O, occurs as a vug mineral in the contact zone between dolomite carbonatite and ""jacupirangite"" (=a pyroxenite) at the Jacupiranga mine, in Cajati county, Sao Paulo state, Brazil, associated with dolomite, calcite, magnetite, clinohumite, phlogopite, ancylite-(Ce), strontianite, pyrite, and tochilinite. This is also the type locality for quintinite-2H. The mineral forms rhombododecahedra up to I mm, isolated or in aggregates. Menezesite is transparent and displays a vitreous luster; it is reddish brown with a white streak. It is non-fluorescent. Mohs hardness is about 4. Calculated density derived from the empirical formula is 4.181 g/cm(3). It is isotropic, 1.93(1) (white light); n(calc) = 2.034. Menezesite exhibits weak anomalous birefringence. The empirical formula is (Ba1.47K0.53Ca0.3,Ce0.17Nd0.10Na0.06La0.02)(Sigma 2.66)(Mg0.94Mn0.23Fe0.23Al0.03)(Sigma 1.43)(Zr2.75Ti0.96Th0.29)(Sigma 4.00)[(Ba0.72Th0.26U0.02)(Sigma 1.00)(Nb9.23Ti2.29Ta0.36Si0.12)Sigma O-12.00(42)]center dot 12H(2)O. The mineral is cubic, space group 10 (204), a = 13.017(1) angstrom, V = 2206(1) angstrom(3), Z = 2. Menezesite is isostructural with the synthetic compound Mg-7[MgW12O42](OH)(4)center dot 8H(2)O. The mineral was named in honor of Luiz Alberto Dias Menezes Filho (born 1950), mining engineer, mineral collector and merchant. Both the description and the name were approved by the CNMMN-IMA (Nomenclature Proposal 2005-023). Menezesite is the first natural heteropolyniobate. Heteropolyanions have been employed in a range of applications that include virus-binding inorganic drugs (including the AIDs virus), homogeneous and heterogeneous catalysts, electro-optic and electrochromic materials, metal and protein binding, and as building blocks for nanostructuring of materials.

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide ROLE IN ARGININE AND NITRIC OXIDE PRODUCTION

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, alpha-methyl-DL-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

Interaction of bovine serum albumin (BSA) with ionic surfactants evaluated by electron paramagnetic resonance (EPR) spectroscopy

EPR spectra of 5- and 16-doxyl stearic acid nitroxide probes (5-DSA and 16-DSA, respectively) bound to bovine serum albumin (BSA) revealed that in the presence of ionic surfactants, at least, two label populations coexist in equilibrium. The rotational correlation times (tau) indicated that component I displays a more restricted mobility state, associated to the spin labels bound to the protein; the less immobilized component 2 is due to label localization in the surfactant aggregates. For both probes, the increase of surfactant concentration leads to higher motional levels of component 1 followed by a simultaneous decrease of this fraction of nitroxides and its conversion into component 2. For 10 mM cethyltrimethylammonium chloride (CTAC), the nitroxides are 100% bound to the protein, whereas at 10mM N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and sodium dodecyl sulfate (SDS) the fractions of bound nitroxides are reduced to 18% and 86%, respectively. No significant polarity changes were observed in the whole surfactant concentration range for component 1. Moreover, at higher surfactant concentration, component 2 exhibited a similar polarity as in the pure surfactant micelles. For 16-DSA the surfactant effect is different: at 10mM of HPS and CTAC the fractions of bound nitroxides are 76% and 49%, respectively, while at 10 mM SDS they are present exclusively in a micellar environment, consistent with 100% of component 2. Overall, both SDS and HPS are able to effectively displace the nitroxide probes from the protein binding sites. while CTAC seems to affect the nitroxide binding to a significantly smaller extent. (C) 2008 Elsevier B.V. All rights reserved.

Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes

Interferon regulatory factor 6 (IRF6) belongs to a family of nine transcription factors that share a highly conserved helix-turn-helix DNA-binding domain and a less conserved protein-binding domain. Most IRFs regulate the expression of interferon-alpha and -beta after viral infection(1), but the function of IRF6 is unknown. The gene encoding IRF6 is located in the critical region for the Van der Woude syndrome (VWS; OMIM 119300) locus at chromosome 1q32-q41 (refs 2,3). The disorder is an autosomal dominant form of cleft lip and palate with lip pits(4), and is the most common syndromic form of cleft lip or palate. Popliteal pterygium syndrome (PPS; OMIM 119500) is a disorder with a similar orofacial phenotype that also includes skin and genital anomalies(5). Phenotypic overlap(6) and linkage data(7) suggest that these two disorders are allelic. We found a nonsense mutation in IRF6 in the affected twin of a pair of monozygotic twins who were discordant for VWS. Subsequently, we identified mutations in IRF6 in 45 additional unrelated families affected with VWS and distinct mutations in 13 families affected with PPS. Expression analyses showed high levels of Irf6 mRNA along the medial edge of the fusing palate, tooth buds, hair follicles, genitalia and skin. Our observations demonstrate that haploinsufficiency of IRF6 disrupts orofacial development and are consistent with dominant-negative mutations disturbing development of the skin and genitalia.

Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Real-time monitoring and kinetic parameter estimation of the affinity interaction of jArtinM and rArtinM with peroxidase glycoprotein by the electrogravimetric technique

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)