New grafted PLA-g-PEG polymeric nanoparticles used to improve bioavailability of oral drugs.

Les nanoparticules (NPs) de polymère ont montré des résultats prometteurs pour leur utilisation comme système de transport de médicaments pour une libération contrôlée du médicament, ainsi que pour du ciblage. La biodisponibilité des médicaments administrés oralement pourrait être limitée par un processus de sécrétion intestinale, qui pourrait par la suite être concilié par la glycoprotéine P (P-gp). La dispersion de la Famotidine (modèle de médicament) à l’intérieur des nanoparticules (NPs) pegylées a été évaluée afin d’augmenter la biodisponibilité avec du polyéthylène glycol (PEG), qui est connu comme un inhibiteur de P-gp. L’hypothèse de cette étude est que l’encapsulation de la Famotidine (un substrat de P-gp) à l’intérieur des NPs préparées à partir de PEG-g-PLA pourrait inhiber la fonction P-gp. La première partie de cette étude avait pour but de synthétiser quatre copolymères de PEG greffés sur un acide polylactide (PLA) et sur un squelette de polymère (PLA-g-PEG), avec des ratios de 1% et 5% (ratio molaire de PEG vs acide lactique monomère) de soit 750, soit 2000 Da de masse moléculaire. Ces polymères ont été employés afin de préparer des NPs chargés de Famotidine qui possède une faible perméabilité et une solubilité aqueuse relativement basse. Les NPs préparées ont été analysées pour leur principaux paramètres physicochimiques tels que la taille et la distribution de la taille, la charge de surface (Potentiel Zeta), la morphologie, l’efficacité d’encapsulation, le pourcentage résiduel en alcool polyvinylique (PVA) adsorbé à la surface des NPs, les propriétés thermiques, la structure cristalline et la libération du médicament. De même, les formules de NPs ont été testées in vitro sur des cellules CaCo-2 afin dʼévaluer la perméabilité bidirectionnelle de la Famotidine. Généralement, les NPs préparées à partir de polymères greffés PLA-g-5%PEG ont montré une augmentation de la perméabilité du médicament, ce par l’inhibition de l’efflux de P-gp de la Famotidine dans le modèle CaCo-2 in vitro. Les résultats ont montré une baisse significative de la sécrétion de la Famotidine de la membrane basolatéral à apical lorsque la Famotidine était encapsulée dans des NPs préparées à partir de greffes de 5% PEG de 750 ou 2000 Da, de même que pour d’autres combinaisons de mélanges physiques contenant du PEG5%. La deuxième partie de cette étude est à propos de ces NPs chargées qui démontrent des résultats prometteurs en termes de perméabilité et d’inhibition d’efflux de P-gp, et qui ont été choises pour développer une forme orale solide. La granulation sèche a été employée pour densifier les NPs, afin de développer des comprimés des deux formules sélectionnées de NPs. Les comprimés à base de NPs ont démontré un temps de désintégration rapide (moins d’une minute) et une libération similaire à la Famotidine trouvée sur le marché. Les résultats de l’étude du transport de comprimés à base de NPs étaient cohérents avec les résultats des formules de NPs en termes d’inhibition de P-gp, ce qui explique pourquoi le processus de fabrication du comprimé n’a pas eu d’effet sur les NPs. Mis ensemble, ces résultats montrent que l’encapsulation dans une NP de polymère pegylé pourrait être une stratégie prometteuse pour l’amélioration de la biodisponibilité des substrats de P-gp.

Étude de tour béta actif dans les modulateurs allostériques du récepteur de la prostaglandine F2alpha

La naissance avant terme constitue un important problème de santé périnatale partout dans le monde. Chaque année près de 15 millions de bébés naissent prématurément. Notre laboratoire s’intéresse depuis une décennie à la conception et le développement de nouvelles classes thérapeutiques (appelés agents tocolytiques) capables d’inhiber les contractions utérines et prolonger le temps de gestation. Due à son implication directe dans le déclanchement des contractions utérines, la prostaglandine F2α (FP) est devenue notre cible de choix. Le PDC 113.824 et aza-glycinyl-proline sont apparus comme des inhibiteurs allostériques puissants du récepteur de la prostaglandine F2α, capables de prolonger la durée de gestation chez les souris. Le travail réalisé au cours de ce mémoire a pour objectif d’étudier le tour β nécessaire pour la reconnaissance sur le récepteur de la prostaglandine F2α. Dans la conception de mime peptidique sélectif efficace et puissant, le repliement β est un élément structural essentiel pour le maintien ou l’amélioration de l’activité du mime peptidique. Les études conformationelles du PDC113.824 et l’aza-glycinyl-proline montrent que les squelettes centraux de ces mimes peptidiques pourraient adopter essentiellement un tour β de type I ou II`, ce qui suggère l’implication des deux types de tours dans l’activité biologique. La synthèse de mimes peptidiques adoptant le tour β de type I et II` est devenue une stratégie logique dans la recherche de nouveaux agents tocolytiques. Au cours de ces études quatre analogues du PDC113.824 ont été synthétisés, un azabicyclo[5.3.0]alkanone et un dipeptide macrocyclique (mimes du tours β de type I) pour étudier l’implication du tour β type I. Par la suite la synthèse de glycinyl-proline et le D-alaninyl-proline (des mimes du tour β du type II`) a été réalisée afin de valider l’implication du tour β type II` dans la reconnaissance sur le récepteur. Nous avons utilisé une stratégie de synthèse efficace pour obtenir les deux analogues (azabicyclo [5.3.0]alkanone et dipeptides macrocycles) à partir d’un intermédiaire commun, en procédant par une cyclisation transannulaire diastéréosélective du tétra-peptide.

Inhibition réversible et photomarquage de la transglutaminase tissulaire

La transglutaminase tissulaire est une enzyme dépendante du calcium qui catalyse la formation de liens isopeptidiques, entre les chaînes latérales de résidus glutamine et lysine, permettant, par le fait même, la réticulation des protéines dans les systèmes biologiques. Elle joue un rôle, entre autres, dans l’endocytose, la régulation du développement des cellules, et même dans l’apoptose. Néanmoins, une dérégulation de l’activité biologique de cette enzyme peut entrainer différentes pathologies, comme la formation de cataractes, de plaques amyloïdes dans la maladie d’Alzheimer, ou encore peut mener au développement de la maladie céliaque. C’est pourquoi une meilleure connaissance du mécanisme d’action de cette enzyme et la possibilité de réguler son action à l’aide de substrats ou d’inhibiteurs sont nécessaires. Dans cette optique, une méthode d’expression et de purification de la transglutaminase humaine a été développée, permettant de travailler directement avec la cible pharmacologique désirée. De plus, une étude du mode d’inhibition et de liaison d’une classe d’inhibiteurs réversibles précédemment découverte dans le groupe, soit la famille des trans-cinnamoyles, a permis d’identifier que la puissance de ces molécules est influencée par la présence du calcium et qu’une inhibition dépendante du temps est observée, en lien avec un potentiel équilibre conformationnel lent de la transglutaminase. D’un autre côté, la susceptibilité à une attaque nucléophile par des thiols de cette classe de molécule rend leur potentiel pharmacologique grandement diminué, et c’est pourquoi une nouvelle famille de molécules a été identifiée, basée sur un squelette ynone, avec une valeur d’IC50 très prometteuse de 2,6 μM, en faisant un des meilleurs inhibiteurs réversibles de la transglutaminase développés à ce jour. Finalement, une stratégie de photomarquage jumelée à une analyse de spectrométrie de masse en tandem a été développée pour la découverte du site de liaison du substrat dérivé de la lysine, dans le but de mieux comprendre le mécanisme complexe de cette enzyme.

Planejamento racional de fármacos baseado em produtos naturais

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

Micellar modification of drug stability: analysis of the effect of hexadecyltrimethylammonium halides on the rate of degradation of cephaclor

The intra- and intermolecular rates of degradation of cephaclor were determined with and without hexadecyltrimethylammonium bromide (CTABr). Micellar-derived spectral shifts were used to measure the association of the ionic forms as well as to determine the effect of CTABr on the apparent acid dissociation constant of the antibiotic. The rate of degradation of cephaclor increased with detergent and was salt sensitive. Micellar effects were analyzed quantitatively within the frame-work of the speudophase ion exchange model. All experimental data were fitted to this model which was used to predict the combined effects of pH and detergent concentration. Micelles increased the rate of OH- attack on cephaclor; most of the effect was due to the concentration of reagents in the micellar pseudophase. The intramolecular degradation was catalyzed 25-fold by micelles, and a working hypothesis to rationalize this effect is proposed. The results demonstrate that quantitative analysis can be utilized to assess and predict effects of detergents on drug stability.

Short Communication: In vitro evaluation of the abrasiveness of acidic dentifrices.

AIM: This in vitro study evaluated the abrasiveness of acidic fluoride (F) dentifrices with different F concentrations on bovine enamel. METHODS: Enamel blocks (4.0 x 4.0 mm2, n=120) were selected according to their surface microhardness and divided into 12 groups. Slurries of dentifrices were used containing 0 (placebo), 275, 412, 550 and 1,100 ppm F (pH 4.5 or 7.0), as well as testing two commercial dentifrices (Crest, positive control, 1,100 ppm F and Colgate Baby, 500 ppm F). Enamel blocks were partially protected with an adhesive tape (control area) and then brushed by an automated toothbrushing machine (16,000 strokes). During this process, 0.4 ml of the slurries were injected every 2 mins on the enamel blocks. After toothbrushing, enamel wear was determined by profilometry. STATISTICS: Results were analyzed by ANOVA and Tukey's test (p<0.05). RESULTS: The mean values for pH in the suspensions during treatment were 6.93, 4.32, 7.56 and 8.19 for neutral experimental dentifrices, acidic experimental dentifrices, Crest and Colgate baby, respectively. The abrasiveness of the acidic dentifrices was similar (p<0.05) to the neutral ones, whereas commercial dentifrices yielded lower abrasion (p<0.05). CONCLUSION: It was concluded that a reduction of the pH of dentifrices does not increase their abrasiveness.

Topical use of fluorides for caries control

Since the early findings on the protective effects of fluoride present in drinking water upon caries incidence and prevalence, intensive research has been conducted in order to determine the benefits, safety, as well as the cost-effectiveness of other modalities of fluoride delivery. The present chapter reviews the various forms of topical fluoride use - professionally and self-applied - with special emphasis on clinical efficacy and possible side effects. The most widely used forms of fluoride delivery have been subject of several systematic reviews, providing strong evidence supporting the use of dentifrices, gels, varnishes and mouth rinses for the control of caries progression. Dentifrices with fluoride concentrations of 1,000 ppm and above have been shown to be clinically effective in caries prevention when compared to a placebo treatment, but the evidence regarding formulations with 450-550 ppm is still subject of debate. Therefore, the recommendation for low-fluoride dentifrice use must take into account both risks and benefits. The evidence for the combined use of two modalities of fluoride application in comparison to a single modality is still inconsistent, implying that more studies with adequate methodology are needed to determine the real benefits of each method. Considering the currently available evidence and risk-benefit aspects, it seems justifiable to recommend the use of fluoridated dentifrices to individuals of all ages, and additional fluoride therapy should also be targeted towards individuals at high caries risk. © 2011 S. Karger AG, Basel.

Preparation and characterization of poly(ε-caprolactone) nanospheres containing the local anesthetic lidocaine

The objective of this work was to develop a modified release system for the local anesthetic lidocaine (LDC), using poly(ε-caprolactone) (PCL) nanospheres (NSs), to improve the pharmacological properties of the drug when administered by the infiltration route. In vitro experiments were used to characterize the system and investigate the release mechanism. The NSs presented a polydispersion index of 0.072, an average diameter of 449.6nm, a zeta potential of -20.1mV, and an association efficiency of 93.3%. The release profiles showed that the release of associated LDC was slower than that of the free drug. Atomic force microscopy analyses showed that the spherical structure of the particles was preserved as a function of time, as well as after the release experiments. Cytotoxicity and pharmacological tests confirmed that association with the NSs reduced the toxicity of LDC, and prolonged its anesthetic action. This new formulation could potentially be used in applications requiring gradual anesthetic release, especially dental procedures. © 2012 Wiley Periodicals, Inc.

Biocompatible microemulsion modifies the pharmacokinetic profile and cardiotoxicity of doxorubicin

Doxorubicin (DOX) is an anthracycline antibiotic with a broad antitumor spectrum. However, the clinical use of DOX is limited because of its cardiotoxicity, a dose-dependent effect. Colloidal drug delivery systems, such as microemulsions (MEs), allow the incorporation of drugs, modifying the pharmacokinetic (PK) profile and toxic effects. In this study, we evaluated the PK profile and cardiotoxicity of a new DOX ME (DOX-ME). The PK profile of DOX-ME was determined and compared with that of the conventional DOX after single-dose administration (6mg/kg, intravenous) in male Wistar rats (n = 12 per group). The cardiotoxicity of DOX formulations was evaluated by serum creatine kinase MB (CKMB) activity in both animal groups before and after drug administration. The plasma DOX measurements were performed by high-performance liquid chromatography with fluorescence detection, and the CKMB levels were assayed using the CKMB Labtest® kit. The ME system showed a significant increase in plasma DOX concentrations and lower distribution volume when compared with conventional DOX. Serum CKMB activity increased after conventional DOX administration but was unchanged in the DOX-ME group. These results demonstrate modifications in drug access to susceptible sites using DOX-ME. DOX-ME displayed features that make it a promising system for future therapeutic application. © 2012 Wiley Periodicals, Inc.

Chemical composition and antioxidant activity of dried powder formulations of Agaricus blazei and Lentinus edodes

Several mushroom species have been pointed out as sources of antioxidant compounds, in addition to their important nutritional value. Agaricus blazei and Lentinus edodes are among the most studied species all over the world, but those studies focused on their fruiting bodies instead of other presentations, such as powdered preparations, used as supplements. In the present work the chemical composition (nutrients and bioactive compounds) and antioxidant activity (free radical scavenging activity, reducing power and lipid peroxidation inhibition) of dried powder formulations of the mentioned mushroom species (APF and LPF, respectively) were evaluated. Powder formulations of both species revealed the presence of essential nutrients, such as proteins, carbohydrates and unsaturated fatty acids. Furthermore, they present a low fat content (<2 g/100 g) and can be used in low-calorie diets, just like the mushrooms fruiting bodies. APF showed higher antioxidant activity and higher content of tocopherols and phenolic compounds (124 and 770 μg/100 g, respectively) than LPF (32 and 690 μg/100 g). Both formulations could be used as antioxidant sources to prevent diseases related to oxidative stress. © 2012 Elsevier Ltd. All rights reserved.

The inhibition of lipid transfer protein by negatively charged liposomes modifies the therapeutic index of amphotericin B

We have shown that liposomal amphotericin B (L-AmpB) decreased renal toxicity and maintains the antifungal activity of amphotericin B (AmpB). We have also observed that L-AmpB is predominantly associated with high density lipoproteins (HDL) as compared to Fungizone (AmpB + deoxycholate). The present experiments were designed to assess the biological relevance of transferring AmpB to HDL. We observed that AmpB was less toxic to kidney cells when associated with HDL, however AmpB toxicity was maintained when associated with LDL. To further understand how HDL-associated AmpB reduces renal cell toxicity the presence of HDL and LDL receptors in this cell line was determined. We observed that these cells expressed high and low affinity LDL receptors, but only low affinity HDL receptors. The reduced renal cell toxicity of HDL-associated AmpB may be due to its lack of interaction with renal cells because of the absence of HDL receptors. Since AmpB interacts with cholesteryl esters whose transfer among lipoproteins is regulated by Lipid transfer Protein (LTP), the role of LTP on the distribution of AmpB to HDL and LDL was next examined. We found that negatively charged liposomes significantly reduced LTP-mediated transfer of CE between HDL and LDL, independent of the presence of AmpB, while Fungizone only significantly inhibited CE transfer at one concentration tested (20$\mu$g/ml). Therefore, we believe that the decreased renal toxicity of L-AmpB is related to its predominant distribution to HDL which is regulated by the inhibition of LTP activity. ^

Mechanisms of apoptosis in chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by the accumulation of terminally differentiated, mature B cells that do not progress beyond the G1 stage of cell cycle, suggesting that these cells possess intrinsic defects in apoptosis. Treatment relies heavily on chemotherapy (primarily nucleoside analogs and glucocorticoids) that may initially be effective in patients, but ultimately give rise to refractory, untreatable disease. The purpose of this study was to determine whether key components of the apoptotic machinery were intact in CLL lymphocytes, especially in patients refractory to therapy. ^ Activation of proteases has been shown to be at the core of the apoptotic pathway and this work demonstrates that protease activation is required for glucocorticoid and nucleoside analog-induced apoptosis in CLL cells. Inhibitors of serine proteases as well as caspase inhibitors blocked induced DNA fragmentation, and a peptide inhibitor of the nuclear scaffold (NS) protease completely suppressed both induced and spontaneous apoptosis. However, the NS protease inhibitor actually promoted several pro-apoptotic events, such as caspase activation, exposure of surface phosphatidylserine, and loss of mitochondrial membrane potential. These results suggested that the NS protease may interact with the apoptotic program in CLL cells at two separate points. ^ In order to further investigate the role of the NS protease in CLL, patient isolates were treated with proteasome inhibitors because of previous results suggesting that the ISIS protease might be a β subunit of the proteasome. Proteasome inhibitors induced massive DNA fragmentation in every patient tested, even in those resistant to the effects of glucocorticoid and nucleoside analogs in vitro. Several other features of apoptosis were also promoted by the proteasome inhibitor, including mitochondrial alterations such as release of cytochrome c and drops in mitochondrial membrane potential. Proteasome inhibitor-induced apoptosis was associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in a number of systems. The NS protease inhibitor also caused a decrease in active NFκB, suggesting that the proapoptotic effects of this agent might be due to depletion of NFκB. ^ Given these findings, the role of NFκB, in conferring survival in CLL was investigated. Glucocorticoid hormone treatment was shown to cause decreases in the activity of the transcription factor, while phorbol dibutyrate, which blocks glucocorticoid-induced DNA fragmentation, was capable of upregulating NFκB. Compellingly, introduction of an undegradable form of the constitutive NFκB inhibitor, IκB, caused DNA fragmentation in several patient isolates, some of which were resistant to glucocorticoid in vitro. Transcription of anti-apoptotic proteins by NFκB was postulated to be responsible for its effects on survival, but Bcl-2 levels did not fluctuate with glucocorticoid or proteasome inhibitor treatment. ^ The in vitro values generated from these studies were organized into a database containing numbers for over 250 patients. Correlation of relevant clinical parameters revealed that levels of spontaneous apoptosis in vitro differ significantly between Rai stages. Importantly, in vitro resistance to nucleoside analogs or glucocorticoids predicted resistance to chemotherapy in vivo, and inability to achieve remission. ^

RNA polymerase II large subunit degradation induced by ecteinascidin 743: Molecular characterization and subsequent rational investigation of antitumor mechanisms in cancers with clinical response

Ecteinascidin 743 (Et-743), which is a novel DNA minor groove alkylator with a unique spectrum of antitumor activity, is currently being evaluated in phase II/III clinical trials. Although the precise molecular mechanisms responsible for the observed antitumor activity are poorly understood, recent data suggests that post-translational modifications of RNA polymerase II Large Subunit (RNAPII LS) may play a central role in the cellular response to this promising anticancer agent. The stalling of an actively transcribing RNAPII LS at Et-743-DNA adducts is the initial cellular signal for transcription-coupled nucleotide excision repair (TC-NER). In this manner, Et-743 poisons TC-NER and produces DNA single strand breaks. Et-743 also inhibits the transcription and RNAPII LS-mediated expression of selected genes. Because the poisoning of TC-NER and transcription inhibition are critical components of the molecular response to Et-743 treatment, we have investigated if changes in RNAPII LS contribute to the disruption of these two cellular pathways. In addition, we have studied changes in RNAPII LS in two tumors for which clinical responses were reported in phase I/II clinical trials: renal cell carcinoma and Ewing's sarcoma. Our results demonstrate that Et-743 induces degradation of the RNAPII LS that is dependent on active transcription, a functional 26S proteasome, and requires functional TC-NER, but not global genome repair. Additionally, we have provided the first experimental data indicating that degradation of RNAPII LS might lead to the inhibition of activated gene transcription. A set of studies performed in isogenic renal carcinoma cells deficient in von Hippel-Lindau protein, which is a ubiquitin-E3-ligase for RNAPII LS, confirmed the central role of RNAPII LS degradation in the sensitivity to Et-743. Finally, we have shown that RNAPII LS is also degraded in Ewing's sarcoma tumors following Et-743 treatment and provide data to suggest that this event plays a role in decreased expression of the Ewing's sarcoma oncoprotein, EWS-Fli1. Altogether, these data implicate degradation of RNAPII LS as a critical event following Et-743 exposure and suggest that the clinical activity observed in renal carcinoma and Ewing's sarcoma may be mediated by disruption of molecular pathways requiring a fully functional RNAPII LS. ^

New cytochromes P450: Tale of two variants

Cytochrome P450s, a superfamily of heme enzymes found in most living organisms. They are responsible for metabolism of many therapeutic drugs, industrial pollutants, carcinogens, and additives to foodstuffs, as well as some endogenous compounds including fatty acids and steroids. First pass drug metabolism studies represent mainly liver and small intestine elimination, and are viewed as the standard to predict therapeutic outcome. However, drug plasma levels determined after administration do not always correlate with therapeutic efficacy of the drug. Therefore, a possible explanation may come by understanding drug metabolism in extrahepatic tissues and/or at the site of drug action. Identification and characterization of novel tissue specific isoforms of P450 generated by alternative splicing of known P450 genes or as yet unidentified genes is essential to predict pharmacological outcome of drugs or the fate of a carcinogen that act at sites remote from liver. ^ Using RT-PCR, brain-specific cytochrome P450s were detected in samples of human autopsy brain. So far, we have identified two human brain variants including P450 2D7 and P450 1A1. We have shown the presence of the P450 1A1 brain specific splice variant in African Americans, Caucasians and Indians albeit different patterns of liver to brain variant ratio were seen distributed throughout each population. Interestingly, the splice variant was detected only in the brain but not in any other tissues from the same individual. Homology modeling was used to compare the variant 3D structure to the liver form structure and differences in the substrate access channels and substrate binding sites were noticed. Automated computational docking was used to predict the metabolic fate of the potent carcinogenic substrate, benzo[a]pyrene. P450 1A1 brain variant showed no binding orientations that could produce the active metabolite, whereas P450 1A1 liver form did reveal orientations capable of generating active carcinogenic product. In vitro P32 labeling studies verified the docking predictions. Therefore, the data support the hypothesis that P450 brain splice variants mediate the metabolism of xenobiotics by mechanisms distinct from the well-studied liver counterparts. ^

Use of short term test systems for the prediction of the hazard represented by potential chemical carcinogens

It has been hypothesized that results from the short term bioassays will ultimately provide information that will be useful for human health hazard assessment. Although toxicologic test systems have become increasingly refined, to date, no investigator has been able to provide qualitative or quantitative methods which would support the use of short term tests in this capacity.^ Historically, the validity of the short term tests have been assessed using the framework of the epidemiologic/medical screens. In this context, the results of the carcinogen (long term) bioassay is generally used as the standard. However, this approach is widely recognized as being biased and, because it employs qualitative data, cannot be used in the setting of priorities. In contrast, the goal of this research was to address the problem of evaluating the utility of the short term tests for hazard assessment using an alternative method of investigation.^ Chemical carcinogens were selected from the list of carcinogens published by the International Agency for Research on Carcinogens (IARC). Tumorigenicity and mutagenicity data on fifty-two chemicals were obtained from the Registry of Toxic Effects of Chemical Substances (RTECS) and were analyzed using a relative potency approach. The relative potency framework allows for the standardization of data "relative" to a reference compound. To avoid any bias associated with the choice of the reference compound, fourteen different compounds were used.^ The data were evaluated in a format which allowed for a comparison of the ranking of the mutagenic relative potencies of the compounds (as estimated using short term data) vs. the ranking of the tumorigenic relative potencies (as estimated from the chronic bioassays). The results were statistically significant (p $<$.05) for data standardized to thirteen of the fourteen reference compounds. Although this was a preliminary investigation, it offers evidence that the short term test systems may be of utility in ranking the hazards represented by chemicals which may be human carcinogens. ^