Preparation of alpha-labeled aldehydes by base-catalyzed exchange reactions

A simple, efficient protocol for the preparation of α-labeled aldehydes based on H/D exchange catalyzed by 4-(N,N-dimethylamino)pyridine or Et3N is described. High chemical yields and ratios of isotope incorporation were obtained even when small amounts (1 mmol) of aldehyde were used.

Systemic adalimumab induces peripheral corneal infiltrates: a case report.

BACKGROUND: Tumor necrosis factor-alpha inhibitors are widely used agents in the treatment of immune disorders such as rheumatoid arthritis and inflammatory bowel disease. Despite their anti-inflammatory action, paradoxical drug-induced inflammatory events have been occasionally associated with the use of infliximab, etanercept, and in a lesser extent adalimumab. However, eye involvement is uncommon and anterior uveitis is the only reported ocular adverse manifestation. It can be induced by etanercept, but has also been described during adalimumab therapy. We present here the first report of recurrent peripheral corneal infiltrates following subcutaneous injections of adalimumab. CASE PRESENTATION: A 34 year-old Caucasian woman with Crohn's disease presented to the emergency department with bilateral red eyes and discomfort 36 hours after she received her bimonthly dose of subcutaneous adalimumab. Examination revealed bilateral peripheral corneal infiltrates with characteristic features of immune infiltrates. Symptoms and infiltrates regressed after topical corticosteroid therapy, but recurred after each adalimumab injection over the following weeks. CONCLUSION: Paradoxical immune reactions associated with tumor necrosis factor-alpha inhibitors may result either from hypersensitivity mechanisms, or from immune-complex deposition via anti-adalimumab antibodies. Both mechanisms could explain this newly described manifestation. Care should be taken to search for corneal infiltrates in the event of red eye symptoms during adalimumab therapy since they respond to topical corticosteroids and do not necessarily prompt the discontinuation of the immunosuppressive therapy.

Drug-eluting stents compared to bare-metal stents improve short-term survival in patients with acute myocardial infarction undergoing primary percutaneous coronary intervention: a nationwide prospective analysis of the AMIS Plus registry.

BACKGROUND: Recently, it has been suggested that the type of stent used in primary percutaneous coronary interventions (pPCI) might impact upon the outcomes of patients with acute myocardial infarction (AMI). Indeed, drug-eluting stents (DES) reduce neointimal hyperplasia compared to bare-metal stents (BMS). Moreover, the later generation DES, due to its biocompatible polymer coatings and stent design, allows for greater deliverability, improved endothelial healing and therefore less restenosis and thrombus generation. However, data on the safety and performance of DES in large cohorts of AMI is still limited. AIM: To compare the early outcome of DES vs. BMS in AMI patients. METHODS: This was a prospective, multicentre analysis containing patients from 64 hospitals in Switzerland with AMI undergoing pPCI between 2005 and 2013. The primary endpoint was in-hospital all-cause death, whereas the secondary endpoint included a composite measure of major adverse cardiac and cerebrovascular events (MACCE) of death, reinfarction, and cerebrovascular event. RESULTS: Of 20,464 patients with a primary diagnosis of AMI and enrolled to the AMIS Plus registry, 15,026 were referred for pPCI and 13,442 received stent implantation. 10,094 patients were implanted with DES and 2,260 with BMS. The overall in-hospital mortality was significantly lower in patients with DES compared to those with BMS implantation (2.6% vs. 7.1%,p < 0.001). The overall in-hospital MACCE after DES was similarly lower compared to BMS (3.5% vs. 7.6%, p < 0.001). After adjusting for all confounding covariables, DES remained an independent predictor for lower in-hospital mortality (OR 0.51,95% CI 0.40-0.67, p < 0.001). Since groups differed as regards to baseline characteristics and pharmacological treatment, we performed a propensity score matching (PSM) to limit potential biases. Even after the PSM, DES implantation remained independently associated with a reduced risk of in-hospital mortality (adjusted OR 0.54, 95% CI 0.39-0.76, p < 0.001). CONCLUSIONS: In unselected patients from a nationwide, real-world cohort, we found DES, compared to BMS, was associated with lower in-hospital mortality and MACCE. The identification of optimal treatment strategies of patients with AMI needs further randomised evaluation; however, our findings suggest a potential benefit with DES.

Drug abuse, brain calcification and glutamate-induced neurodegeneration

Positive and negative reinforcing systems are part of the mechanism of drug dependence. Drugs with abuse potential may change the manner of response to negative emotional stimuli, activate positive emotional reactions and possess primary reinforcing properties. Catecholaminergic and peptidergic processes are of importance in these mechanisms. Current research needs to understand the types of adaptations that underlie the particularly long-lived aspects of addiction. Presently, glutamate is candidate to play a role in the enduring effects of drugs of abuse. For example, it participates in the chronic pathological changes of corticostriatal terminals produced by methamphetamine. At the synaptic level, a link between over-activation of glutamate receptors, [C(a2+)](i) increase and neuronal damage has been clearly established leading to neurodegeneration. Thus, neurodegeneration can start after an acute over-stimulation whose immediate effects depend on a diversity of calcium-activated mechanisms. If sufficient, the initial insult results in calcification and activation of a chronic on-going process with a progressive loss of neurons. At present, long-term effects of drug dependence underlie an excitotoxicity process linked to a polysynaptic pathway that dynamically regulates synaptic glutamate. Retaliatory mechanisms include energy capability of the neurons, inhibitory systems and cytoplasmic calcium precipitation as part of the neuron-glia interactions. This paper presents an integrated view of these molecular and cellular mechanisms to help understand their relationship and interdependence in a chronic pathological process that suggest new targets for therapeutic intervention.

Pharmacogenetic and clinical study on adverse effects induced by psychotropic drugs

Second-generation antipsychotics (SGAs) have become the first-line antipsychotic treatment for psychotic disorders due to their better overall tolerance compared to classical antipsychotics. However, metabolic side effects such as weight gain are frequently described during treatment with SGAs and/or other psychotropic drugs including some antidepressants and mood stabilizers, which may also result in poor adherence to treatment. The aim of this work was to investigate different methods to predict common side effects, in particular weight gain during treatment with weight gain inducing psychotropic drugs. Firstly, clinical data were used to determine the potential predictive power of a one month weight gain on weight increase after three and 12 months of treatment (n=351 patients). A fast and strong weight gain of >5% after a period of one month (>5%WG) was found to be the best predictor for an important weight gain at three (>15%) and 12 months (>20%). Similar analyses in an independent cohort of psychiatric adolescents (n=42), showed that a comparable >4% weight gain at one month is the best predictor for an important weight gain at three months (>15%). Secondly, we aimed to determine whether an extensive analysis of genes could be used, in addition to clinical factors, to predict patients at risk for >5%WG or for type 2 diabetes (T2D). Adding genetic markers to clinical variables to predict >5%WG increased significantly the area under the curve (AUC) of the analysis (AUCfinai:0.92, AUCdmicai:0.75, pcO.OOOl, n=248). Conversely, genetic risk scores were found to be associated with T2D (OR: 2.5, p=0.03, n=285) but without a significant increase of AUC'when compared to the prediction based to clinical factors alone. Finally, therapeutic drug monitoring was used to predict extrapyramidal symptoms during risperidone treatment (n=150). Active moiety (sum of risperidone and of its active metabolite 9- hydroxyrisperidone plasma concentrations) of >40 ng/ml should be targeted only in case of insufficient response. These results highlight different approaches for personalizing psychotropic treatments in order to reduce related side effects. Further research is needed, in particular on the identification of genetic markers, to improve the implementation of these results into clinical practice. Résumé Les antipsychotiques atypiques (APA) sont devenus le traitement antipsychotique de première intention pour le traitement des psychoses, grâce à un profil d'effets secondaires plus favorables comparé aux antipsychotiques typiques. Néanmoins, d'autres effets indésirables d'ordre métabolique (ex. prise pondérale) sont observés sous APA, stabilisateurs de l'humeur et/ou certains antidépresseurs, pouvant aussi limiter l'adhérence au traitement. L'objectif de ce travail est d'explorer différentes méthodes permettant de prédire des effets secondaires courants, en particulier la prise de poids durant un traitement avec des psychotropes pouvant induire un tel effet. Dans une première partie, des données cliniques ont été évaluées pour leurs potentiels prédictifs d'une prise de poids à un mois sur une prise de poids à trois et 12 mois de traitement (n=351 patients). Une prise de poids rapide et forte >5% à un mois (PP>5%) s'est avérée être le meilleur prédicteur pour une prise pondérale importante à trois (>15%) et 12 (>20%) mois de traitement. Des analyses similaires dans une cohorte pédiatrique (n=42) ont indiqué une prise de poids >4% à un mois comme le meilleur prédicteur pour une prise pondérale importante (>15%) à trois mois de traitement. Dans une deuxième partie, des marqueurs génétiques, en complément aux données cliniques, ont été analysés pour leur contribution potentielle à la prédiction d'une PP>5% et au dépistage du diabète de type 2 (DT2). L'ajout de variants génétiques aux données cliniques afin de prédire une PP>5% a augmenté significativement l'aire sous la courbe (ASC) de l'analyse (ASCflnai:0.92, ASCC|inique:0.75, p<0.0001, n=248). Concernant le DT2, un score génétique est associé au DT2 (OR: 2.5, p=0.03, n=285), néanmoins aucune augmentation significative de l'ASC n'a été observée par rapport à l'analyse avec les données cliniques seules. Finalement, des mesures de concentrations plasmatiques de médicaments ont été utilisées pour prédire la survenue de symptômes extrapyramidaux sous rispéridone (n=150). Cette analyse nous a permis d'établir qu'une concentration plasmatique de rispéridone associée à son métabolite actif >40 ng/ml ne devrait être recherchée qu'en cas de réponse clinique insuffisante. Ces différents résultats soulignent différentes approches pour personnaliser la prescription de psychotropes afin de réduire la survenue d'effets secondaires. Des études supplémentaires sont néanmoins nécessaires, en particulier sur l'identification de marqueurs génétiques, afin d'améliorer l'implémentation de ces résultats en pratique clinique. Résumé large publique Les antipsychotiques atypiques et autres traitements psychotropes sont couramment utilisés pour traiter les symptômes liés à la schizophrénie et aux troubles de l'humeur. Comme pour tout médicament, des effets secondaires sont observés. L'objectif de ce travail est d'explorer différentes méthodes qui permettraient de prédire la survenue de certains effets indésirables, en particulier une prise de poids et la survenue d'un diabète. Dans une première partie, nous avons évalué l'effet d'une prise de poids précoce sur une prise de poids au long terme sous traitement psychotrope. Les analyses ont mis en évidence dans une population psychiatrique qu'une prise de poids à un mois >5% par rapport au poids initial permettait de prédire une prise pondérale importante après trois (>15%) et 12 (>20%) mois de traitement. Un résultat semblable a. été observé dans un autre groupe de patients exclusivement pédiatriques. Dans une deuxième partie, nous avons évalué la contribution potentielle de marqueurs génétiques à la prédiction d'une prise pondérale de >5% après un mois de traitement ainsi que dans la survenue d'un diabète de type 2. Pour la prise de poids, la combinaison des données génétiques aux données cliniques a permis d'augmenter de 17% la précision de la prédiction, en passant de 70% à 87%. Concernant la survenue d'un diabète, les données génétiques n'ont pas amélioré la prédiction. Finalement, nous avons analysé la relation possible entre les concentrations sanguines d'un antipsychotique atypique couramment utilisé, la rispéridone, et la survenue d'effets secondaires (ici les tremblements). Il est ressorti de cette étude qu'une concentration plasmatique du médicament supérieure à 40 ng/ml ne devrait être dépassée qu'en cas de réponse thérapeutique insuffisante, au risque de voir augmenter la survenue d'effets secondaires du type tremblements. Ces résultats démontrent la possibilité de prédire avec une bonne précision la survenue de certains effets secondaires. Cependant, en particulier dans le domaine de la génétique, d'autres études sont nécessaires afin de confirmer les résultats obtenus dans nos analyses. Une fois cette étape franchie, il serait possible d'utiliser ces outils dans la pratique clinique. A terme, cela pourrait permettre au prescripteur de sélectionner les traitements les mieux adaptés aux profils spécifiques de chaque patient.

Multigenerational effects of the anticancer drug tamoxifen and its metabolite 4-hydroxy-tamoxifen on Daphnia pulex.

Tamoxifen and its metabolite 4-hydroxy-tamoxifen (4OHTam) are two potent molecules that have anticancer properties on breast cancers. Their medical use is expected to increase with the increasing global cancer rate. After consumption, patients excrete tamoxifen and the 4OHTam metabolite into wastewaters, and tamoxifen has been already detected in wastewaters and natural waters. The concentrations of 4OHTam in waters have never been reported. A single study reported 4OHTam effects on the microcrustacean Daphnia pulex. The effects of tamoxifen and 4OHTam over more than two generations are unknown in aquatic invertebrates. The main goal of this study was to assess the long-term sensitivity of the microcrustacean D. pulex over four generations, based on size, reproduction, viability and the intrinsic rate of natural increase (r). Additional experiments were carried out to observe whether the effects of tamoxifen and 4OHTam were reversible in the next generation after descendants were withdrawn from chemical stress (i.e., recovery experiment), and whether the lowest test concentration of each chemical induced toxic effects when both concentrations were combined (i.e., mixture experiments). Our results showed that tamoxifen and 4OHTam induced the adverse effects at environmentally relevant concentrations. Tamoxifen and 4OHTam impaired size, viability, reproduction and the r in four generations of treated D. pulex, but these effects were not clearly magnified over generations. Tamoxifen was more potent than 4OHTam on D. pulex. When used in a mixture, the combination of tamoxifen and 4OHTam induced effects in offspring, whereas no effects were observed when these chemicals were tested individually. In the recovery experiment, the reproduction and size were reduced in offspring withdrawn from chemical exposures. Our results suggested that tamoxifen and its metabolite may be a relevant pharmaceutical to consider in risk assessment.

Sensitive spectrophotometric determination of lansoprazole in pharmaceuticals using ceric ammonium sulphate based on redox and complex formation reactions

Two simple sensitive and cost-effective spectrophotometric methods are described for the determination of lansoprazole (LPZ) in bulk drug and in capsules using ceric ammonium sulphate (CAS), iron (II), orthophenanthroline and thiocyanate as reagents. In both methods, an acidic solution of lansoprazole is treated with a measured excess of CAS followed by the determination of unreacted oxidant by two procedures involving different reaction schemes. The first method involves the reduction of residual oxidant by a known amount of iron(II), and the unreacted iron(II) is complexed with orthophenanthroline at a raised pH, and the absorbance of the resulting complex measured at 510 nm (method A). In the second method, the unreacted CAS is reduced by excess of iron (II), and the resulting iron (III) is complexed with thiocyanate in the acid medium and the absorbance of the complex measured at 470 nm (method B). In both methods, the amount CAS reacted corresponds to the amount of LPZ. In method A, the absorbance is found to increase linearly with the concentration of LPZ where as in method B a linear decrease in absorbance occurs. The systems obey Beer's law for 2.5-30 and 2.5-25 µg mL-1 for method A and method B, respectively, and the corresponding molar absorptivity values are 8.1×10³ and 1.5×10(4) L mol-1cm-1 . The methods were successfully applied to the determination of LPZ in capsules and the results tallied well with the label claim. No interference was observed from the concomitant substances normally added to capsules.

Spectrophotometric determination of lansoprazole in pharmaceuticals using bromate-bromide mixture based on redox and complexation reactions

Two sensitive spectrophotometric methods are described for the determination of lansoprazole (LPZ) in bulk drug and in capsule formulation. The methods are based on the oxidation of lansoprazole by insitu generated bromine followed by determination of unreacted bromine by two different reaction schemes. In one procedure (method A), the residual bromine is treated with excess of iron (II), and the resulting iron (III) is complexed with thiocyanate and measured at 470 nm. The second approach (method B) involves treating the unreacted bromine with a measured excess of iron (II) and remaining iron (II) is complexed with orthophenanthroline at a raised pH, and measured at 510 nm. In both methods, the amount of bromine reacted corresponds to the amount of LPZ. The experimental conditions were optimized. In method A, the absorbance is found to decrease linearly with the concentration of LPZ (r = -0.9986) where as in the method B a linear increase in absorbance occurs (r = 0.9986) The systems obey Beer's law for 0.5-4.0 and 0.5-6.0 µg mL-1 for method A and method B, respectively. The calculated molar absorptivity values are 3.97µ10(4) and 3.07µ10(4) L mol-1cm-1 for method A and method B, respectively, and the corresponding Sandell sensitivity values are 0.0039 and 0.0013 µg cm-2. The limit of detection (LOD) and quantification (LOQ) are also reported for both methods. Intra-day and inter-day precision, and accuracy of the methods were established as per the current ICH guidelines. The methods were successfully applied to the determination of LPZ in capsules and the results tallied well with the label claim and the results were statistically compared with those of a reference method by applying the Student's t-test and F-test. No interference was observed from the concomitant substances normally added to capsules. The accuracy and validity of the methods were further ascertained by performing recovery experiments via standard-addition method.

Cerimetric determination of simvastatin in pharmaceuticals based on redox and complex formation reactions

Two sensitive spectrophotometric methods are described for the determination of simvastatin (SMT) in bulk drug and in tablets. The methods are based on the oxidation of SMT by a measured excess of cerium (IV) in acid medium followed by determination of unreacted oxidant by two different reaction schemes. In one procedure (method A), the residual cerium (IV) is reacted with a fixed concentration of ferroin and the increase in absorbance is measured at 510 nm. The second approach (method B) involves the reduction of the unreacted cerium (IV) with a fixed quantity of iron (II), and the resulting iron (III) is complexed with thiocyanate and the absorbance measured at 470 nm. In both methods, the amount of cerium (IV) reacted corresponds to SMT concentration. The experimental conditions for both methods were optimized. In method A, the absorbance is found to increase linearly with SMT concentration (r = 0.9995) whereas in method B, the same decreased (r = -0.9943). The systems obey Beer's law for 0.6-7.5 and 0.5-5.0 µg mL-1 for method A and method B, respectively. The calculated molar absorptivity values are 2.7 X 10(4) and 1.06 X 10(5) Lmol-1 cm-1, respectively; and the corresponding sandel sensitivity values are 0.0153 and 0.0039µg cm-2, respectively. The limit of detection (LOD) and quantification (LOQ) are reported for both methods. Intra-day and inter-day precision, and accuracy of the methods were established as per the current ICH guidelines. The methods were successfully applied to the determination of SMT in tablets and the results were statistically compared with those of the reference method by applying the Student's t-test and F-test. No interference was observed from the common excipients added to tablets. The accuracy and validity of the methods were further ascertained by performing recovery experiments via standard addition procedure.

Potential of the Osteoclast’s Proton Pump as a Drug Target in Osteoporosis

Decreasing bone mass during aging predisposes to fractures and it is estimated that every second woman and one in five men will suffer osteoporotic fractures during their lifetime. Bone is an adaptive tissue undergoing continuous remodeling in response to physical and metabolic stimuli. Bone mass decreases through a net negative balance in the bone remodeling process of bone, in which the new bone incompletely replaces the resorbed bone mass. Bone resorption is carried out by the osteoclasts; the bone mineral is solubilized by acidification and the organic matrix is subsequently degraded by proteases. Several classes of drugs are available for prevention of osteoporotic fractures. They act by different mechanisms to increase bone mass, and some of them act mainly as antiresorptives by inhibition of osteoclast formation or their function. Optimally, a drug should act selectively on a specific process, since other processes affected usually result in adverse effects. The purpose of this study was to evaluate whether the osteoclastic vacuolar adenosine trisphosphatases (V-ATPase), which drives the solubilization of bone mineral, can be selectively inhibited despite its ubiquitous cellular functions. The V-ATPase is a multimeric protein composed of 13 subunits of which six possesses two or more isoforms. Selectivity for the osteoclastic V-ATPase could be provided if it has some structural uniqueness, such as a unique isoform combination. The a3 isoform of the 116kDa subunit is inevitable for bone resorption; however, it is also present in, and mainly limited to, the lysosomes of other cells. No evidence of a structural uniqueness of the osteoclastic V-ATPase compared to the lysosomal V-ATPase was found, although this can not yet be excluded. Thus, an inhibitor selective for the a3 isoform would target the lysosomal V-ATPase as well. However, the results suggest that selectivity for bone resorption over lysosomal function can be obtained by two other mechanisms, suggesting that isoform a3 is a valid target. The first is differential compensation; bone resorption depends on the high level of a3 expression, and is not compensated for by other isoforms, while the lower level of a3 in lysosomes of other cells may be partly compensated for. The second mechanism is because the bone resorption process itself is fundamentally different from lysosomal acidification because of the chemistry of bone dissolution and the anatomy of the resorbing osteoclast. By this mechanism, full inhibition of bone resorption is obtained with more than tenfold lower inhibitor concentration than those needed to fully inhibit lysosomal acidification. The two mechanisms are additive. Based on the results, we suggest that bone resorption can be selectively inhibited if VATPase inhibitors that are sufficiently selective for the a3 isoform over the other isoforms are developed.

Molecular Mechanisms and Interactions in Regulation of Photosynthetic Reactions

In photosynthesis, light energy is converted to chemical energy, which is consumed for carbon assimilation in the Calvin-Benson-Bassham (CBB) cycle. Intensive research has significantly advanced the understanding of how photosynthesis can survive in the ever-changing light conditions. However, precise details concerning the dynamic regulation of photosynthetic processes have remained elusive. The aim of my thesis was to specify some molecular mechanisms and interactions behind the regulation of photosynthetic reactions under environmental fluctuations. A genetic approach was employed, whereby Arabidopsis thaliana mutants deficient in specific photosynthetic protein components were subjected to adverse light conditions and assessed for functional deficiencies in the photosynthetic machinery. I examined three interconnected mechanisms: (i) auxiliary functions of PsbO1 and PsbO2 isoforms in the oxygen evolving complex of photosystem II (PSII), (ii) the regulatory function of PGR5 in photosynthetic electron transfer and (iii) the involvement of the Calcium Sensing Receptor CaS in photosynthetic performance. Analysis of photosynthetic properties in psbo1 and psbo2 mutants demonstrated that PSII is sensitive to light induced damage when PsbO2, rather than PsbO1, is present in the oxygen evolving complex. PsbO1 stabilizes PSII more efficiently compared to PsbO2 under light stress. However, PsbO2 shows a higher GTPase activity compared to PsbO1, and plants may partially compensate the lack of PsbO1 by increasing the rate of the PSII repair cycle. PGR5 proved vital in the protection of photosystem I (PSI) under fluctuating light conditions. Biophysical characterization of photosynthetic electron transfer reactions revealed that PGR5 regulates linear electron transfer by controlling proton motive force, which is crucial for the induction of the photoprotective non-photochemical quenching and the control of electron flow from PSII to PSI. I conclude that PGR5 controls linear electron transfer to protect PSI against light induced oxidative damage. I also found that PGR5 physically interacts with CaS, which is not needed for photoprotection of PSII or PSI in higher plants. Rather, transcript profiling and quantitative proteomic analysis suggested that CaS is functionally connected with the CBB cycle. This conclusion was supported by lowered amounts of specific calciumregulated CBB enzymes in cas mutant chloroplasts and by slow electron flow to PSI electron acceptors when leaves were reilluminated after an extended dark period. I propose that CaS is required for calcium regulation of the CBB cycle during periods of darkness. Moreover, CaS may also have a regulatory role in the activation of chloroplast ATPase. Through their diverse interactions, components of the photosynthetic machinery ensure optimization of light-driven electron transport and efficient basic production, while minimizing the harm caused by light induced photodamage.

Functional changes of dendritic cells in hypersensivity reactions to amoxicillin

A better understanding of dendritic cell (DC) involvement in responses to haptenic drugs is needed, because it represents a possible approach to the development of an in vitro test, which could identify patients prone to drug allergies. There are two main DC subsets: plasmacytoid DC (pDC) and myeloid DC (mDC). β-lactams form hapten-carrier conjugates and may provide a suitable model to study DC behavior in drug allergy reactions. It has been demonstrated that drugs interact differently with DC in drug allergic and non-allergic patients, but there are no studies regarding these subsets. Our aim was to assess the functional changes of mDC and pDC harvested from an amoxicillin-hypersensitive 32-year-old woman who experienced a severe maculopapular exanthema as reflected in interleukin-6 (IL-6) production after stimulation with this drug and penicillin. We also aim to demonstrate, for the first time, the feasibility of this method for dendritic cell isolation followed by in vitro stimulation for studies of drug allergy physiopathology. DC were harvested using a double Percoll density gradient, which generates a basophil-depleted cell (BDC) suspension. Further, pDC were isolated by blood DC antigen 4-positive magnetic selection and gravity filtration through magnetized columns. After stimulation with amoxicillin, penicillin and positive and negative controls, IL-6 production was measured by ELISA. A positive dose-response curve for IL-6 after stimulation with amoxicillin and penicillin was observed for pDC, but not for mDC or BDC suspension. These preliminary results demonstrate the feasibility of this methodology to expand the knowledge of the effect of dendritic cell activation by drug allergens.

VAP-1 as drug target in inflammation : structural features determining ligand interactions

Vascular adhesion protein-1 (VAP-1), which belongs to the copper amine oxidases (CAOs), is a validated drug target in inflammatory diseases. Inhibition of VAP-1 blocks the leukocyte trafficking to sites of inflammation and alleviates inflammatory reactions. In this study, a novel set of potent pyridazinone inhibitors is presented together with their X-ray structure complexes with VAP-1. The crystal structure of serum VAP-1 (sVAP-1) revealed an imidazole binding site in the active site channel and, analogously, the pyridazinone inhibitors were designed to bind into the channel. This is the first time human VAP-1 has been crystallized with a reversible inhibitor and the structures reveal detailed information of the binding mode on the atomic level. Similarly to some earlier studied inhibitors of human VAP-1, the designed pyridazinone inhibitors bind rodent VAP-1 with a lower affinity than human VAP-1. Therefore, we made homology models of rodent VAP-1 and compared human and rodent enzymes to determine differences that might affect the inhibitor binding. The comparison of the crystal structures of the human VAP-1 and the mouse VAP-1 homology model revealed key differences important for the species specific binding properties. In general, the channel in mouse VAP-1 is more narrow and polar than the channel in human VAP-1, which is wider and more hydrophobic. The differences are located in the channel leading to the active site, as well as, in the entrance to the active site channel. The information obtained from these studies is of great importance for the development and design of drugs blocking the activity of human VAP-1, as rodents are often used for in vivo testing of candidate drugs. In order to gain more insight into the selective binding properties of the different CAOs in one species a comprehensive evolutionary study of mammalian CAOs was performed. We found that CAOs can be classified into sub-families according to the residues X1 and X2 of the Thr/Ser-X1-X2-Asn-Tyr-Asp active site motif. In the phylogenetic tree, CAOs group into diamine oxidase, retina specific amine oxidase and VAP-1/serum amine oxidase clades based on the residue in the position X2. We also found that VAP-1 and SAO can be further differentiated based on the residue in the position X1. This is the first large-scale comparison of CAO sequences, which explains some of the reasons for the unique substrate specificities within the CAO family.

High-amylose carboxymethyl starch matrices for oral sustained drug-release : in vitro and in vivo evaluation

Les amidons non modifiées et modifiés représentent un groupe d’excipients biodégradables et abondants particulièrement intéressant. Ils ont été largement utilisés en tant qu’excipients à des fins diverses dans des formulations de comprimés, tels que liants et/ou agents de délitement. Le carboxyméthylamidon sodique à haute teneur en amylose atomisé (SD HASCA) a été récemment proposé comme un excipient hydrophile à libération prolongée innovant dans les formes posologiques orales solides. Le carboxyméthylamidon sodique à haute teneur en amylose amorphe (HASCA) a d'abord été produit par l'éthérification de l'amidon de maïs à haute teneur en amylose avec le chloroacétate. HASCA a été par la suite séché par atomisation pour obtenir le SD HASCA. Ce nouvel excipient a montré des propriétés présentant certains avantages dans la production de formes galéniques à libération prolongée. Les comprimés matriciels produits à partir de SD HASCA sont peu coûteux, simples à formuler et faciles à produire par compression directe. Le principal objectif de cette recherche était de poursuivre le développement et l'optimisation des comprimés matriciels utilisant SD HASCA comme excipient pour des formulations orales à libération prolongée. A cet effet, des tests de dissolution simulant les conditions physiologiques du tractus gastro-intestinal les plus pertinentes, en tenant compte de la nature du polymère à l’étude, ont été utilisés pour évaluer les caractéristiques à libération prolongée et démontrer la performance des formulations SD HASCA. Une étude clinique exploratoire a également été réalisée pour évaluer les propriétés de libération prolongée de cette nouvelle forme galénique dans le tractus gastro-intestinal. Le premier article présenté dans cette thèse a évalué les propriétés de libération prolongée et l'intégrité physique de formulations contenant un mélange comprimé de principe actif, de chlorure de sodium et de SD HASCA, dans des milieux de dissolution biologiquement pertinentes. L'influence de différentes valeurs de pH acide et de temps de séjour dans le milieu acide a été étudiée. Le profil de libération prolongée du principe actif à partir d'une formulation de SD HASCA optimisée n'a pas été significativement affecté ni par la valeur de pH acide ni par le temps de séjour dans le milieu acide. Ces résultats suggèrent une influence limitée de la variabilité intra et interindividuelle du pH gastrique sur la cinétique de libération à partir de matrices de SD HASCA. De plus, la formulation optimisée a gardé son intégrité pendant toute la durée des tests de dissolution. L’étude in vivo exploratoire a démontré une absorption prolongée du principe actif après administration orale des comprimés matriciels de SD HASCA et a montré que les comprimés ne se sont pas désintégrés en passant par l'estomac et qu’ils ont résisté à l’hydrolyse par les α-amylases dans l'intestin. Le deuxième article présente le développement de comprimés SD HASCA pour une administration orale une fois par jour et deux fois par jour contenant du chlorhydrate de tramadol (100 mg et 200 mg). Ces formulations à libération prolongée ont présenté des valeurs de dureté élevées sans nécessiter l'ajout de liants, ce qui facilite la production et la manipulation des comprimés au niveau industriel. La force de compression appliquée pour produire les comprimés n'a pas d'incidence significative sur les profils de libération du principe actif. Le temps de libération totale à partir de comprimés SD HASCA a augmenté de manière significative avec le poids du comprimé et peut, de ce fait, être utilisé pour moduler le temps de libération à partir de ces formulations. Lorsque les comprimés ont été exposés à un gradient de pH et à un milieu à 40% d'éthanol, un gel très rigide s’est formé progressivement sur leur surface amenant à la libération prolongée du principe actif. Ces propriétés ont indiqué que SD HASCA est un excipient robuste pour la production de formes galéniques orales à libération prolongée, pouvant réduire la probabilité d’une libération massive de principe actif et, en conséquence, des effets secondaires, même dans le cas de co-administration avec une forte dose d'alcool. Le troisième article a étudié l'effet de α-amylase sur la libération de principe actif à partir de comprimés SD HASCA contenant de l’acétaminophène et du chlorhydrate de tramadol qui ont été développés dans les premières étapes de cette recherche (Acetaminophen SR et Tramadol SR). La modélisation mathématique a montré qu'une augmentation de la concentration d’α-amylase a entraîné une augmentation de l'érosion de polymère par rapport à la diffusion de principe actif comme étant le principal mécanisme contrôlant la libération de principe actif, pour les deux formulations et les deux temps de résidence en milieu acide. Cependant, même si le mécanisme de libération peut être affecté, des concentrations d’α-amylase allant de 0 UI/L à 20000 UI/L n'ont pas eu d'incidence significative sur les profils de libération prolongée à partir de comprimés SD HASCA, indépendamment de la durée de séjour en milieu acide, le principe actif utilisé, la teneur en polymère et la différente composition de chaque formulation. Le travail présenté dans cette thèse démontre clairement l'utilité de SD HASCA en tant qu'un excipient à libération prolongée efficace.

Polimorfismos del gen Butirilcolinesterasa responsables de reacciones adversas en pacientes consumidores de “cocaína”

La butirilcolinesterasa humana (BChE; EC 3.1.1.8) es una enzima polimórfica sintetizada en el hígado y en el tejido adiposo, ampliamente distribuida en el organismo y encargada de hidrolizar algunos ésteres de colina como la procaína, ésteres alifáticos como el ácido acetilsalicílico, fármacos como la metilprednisolona, el mivacurium y la succinilcolina y drogas de uso y/o abuso como la heroína y la cocaína. Es codificada por el gen BCHE (OMIM 177400), habiéndose identificado más de 100 variantes, algunas no estudiadas plenamente, además de la forma más frecuente, llamada usual o silvestre. Diferentes polimorfismos del gen BCHE se han relacionado con la síntesis de enzimas con niveles variados de actividad catalítica. Las bases moleculares de algunas de esas variantes genéticas han sido reportadas, entre las que se encuentra las variantes Atípica (A), fluoruro-resistente del tipo 1 y 2 (F-1 y F-2), silente (S), Kalow (K), James (J) y Hammersmith (H). En este estudio, en un grupo de pacientes se aplicó el instrumento validado Lifetime Severity Index for Cocaine Use Disorder (LSI-C) para evaluar la gravedad del consumo de “cocaína” a lo largo de la vida. Además, se determinaron Polimorfismos de Nucleótido Simple (SNPs) en el gen BCHE conocidos como responsables de reacciones adversas en pacientes consumidores de “cocaína” mediante secuenciación del gen y se predijo el efecto delos SNPs sobre la función y la estructura de la proteína, mediante el uso de herramientas bio-informáticas. El instrumento LSI-C ofreció resultados en cuatro dimensiones: consumo a lo largo de la vida, consumo reciente, dependencia psicológica e intento de abandono del consumo. Los estudios de análisis molecular permitieron observar dos SNPs codificantes (cSNPs) no sinónimos en el 27.3% de la muestra, c.293A>G (p.Asp98Gly) y c.1699G>A (p.Ala567Thr), localizados en los exones 2 y 4, que corresponden, desde el punto de vista funcional, a la variante Atípica (A) [dbSNP: rs1799807] y a la variante Kalow (K) [dbSNP: rs1803274] de la enzima BChE, respectivamente. Los estudios de predicción In silico establecieron para el SNP p.Asp98Gly un carácter patogénico, mientras que para el SNP p.Ala567Thr, mostraron un comportamiento neutro. El análisis de los resultados permite proponer la existencia de una relación entre polimorfismos o variantes genéticas responsables de una baja actividad catalítica y/o baja concentración plasmática de la enzima BChE y algunas de las reacciones adversas ocurridas en pacientes consumidores de cocaína.