Search

Vagus nerve stimulation (VNS) is an adjunctive therapy for drug-resistant epilepsy. Noninvasive evoked potential recordings in laryngeal muscles (LMEPs) innervated by vagal branches may provide a marker to assess effective vagal nerve fiber activation. We investigated VNS-induced LMEPs in patients with epilepsy in acute and chronic settings.A total of 17 of 25 patients underwent LMEP recordings at initiation of therapy (acute group); 15 of 25 patients after one year of VNS (chronic group); and 7 of 25 patients were tested at both time points (acute + chronic group). VNS-induced LMEPs were recorded following different pulse widths and output currents using six surface laryngeal EMG electrodes to calculate input/output curves and estimate LMEP latency, threshold current for minimal (IVNS-induced LMEPs were present in all patients. INoninvasive VNS-induced LMEP recording is feasible both at initiation of VNS therapy and after one year. Low output currents (0.25-1.00 mA) may be sufficient to activate vagal Aα-motor fibers. Maximal LMEP amplitudes seemed to decrease after chronic VNS therapy in patients.

The optimal thromboprophylactic strategy for patients affected by Coronavirus disease 2019 (COVID-19) has been debated among experts. This study evaluated the safety and efficacy of a thromboprophylaxis algorithm. This was a retrospective, single-center study in critically ill patients admitted to the intensive care unit (University affiliated Hospital) for acute respiratory failure due to Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2). From March 16 to April 9, 2020, thromboprophylaxis was adjusted according to weight (control group, n = 19) and after this date, thromboprophylaxis depended on an algorithm based on thrombotic and hemorrhagic risk factors (protocol group, n = 13). With regard to safety (number of major bleeding events and blood transfusions), the groups were not significantly different. With regard to efficacy, the number of thrombotic events decreased from 37 to 0%, p = 0.025 after implementation of the algorithm. Also, peak fibrinogen dropped from 8.6 (7.2–9.3) to 6.5 (4.6–8.4) g/L, p = 0.041 and D-dimers from 2194 (1464–3763) to 1486 (900–2582) ng/mL, p = 0.0001. In addition, length of stay declined from 19 (10–31) to 5 (3–19) days, p = 0.009. In conclusion, a tailored thromboprophylaxis algorithm (risk stratification based on clinical parameters and biological markers) reduce thrombotic phenomena in critically ill COVID-19 patients without increasing major bleeding. Supplementary Information The online version of this article (10.1007/s11239-021-02514-3) contains supplementary material, which is available to authorized users.

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical treatment, computed tomography (CT) and magnetic resonance imaging (MRI) were combined during the treatment-planning process. Positron emission tomography (PET) imaging was subsequently added to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI, and PET) using a micro-irradiation system proved to be labor-intensive because multimodal imaging, treatment planning, and dose delivery have to be completed sequentially in the preclinical setting. This also results in a workflow that is more prone to human error. Therefore, a user-friendly algorithm to further optimize preclinical multimodal imaging-based radiation treatment planning was implemented. This software tool was used to evaluate the accuracy and efficiency of dose painting radiation therapy with micro-irradiation by using an in silico study design. The new methodology for dose painting radiation therapy is superior to the previously described method in terms of accuracy, time efficiency, and intra- and inter-user variability. It is also an important step towards the implementation of inverse treatment planning on micro-irradiators, where forward planning is still commonly used, in contrast to clinical systems.

Background: Recent experiments in rats have demonstrated significant effects of VNS on hippocampal excitability but were partially attributed to hypothermia, induced by the applied VNS parameters. Objective: To allow meaningful preclinical research on the mechanisms of VNS and translation of rodent results to clinical VNS trials, we aimed to identify non-hypothermia inducing VNS parameters that significantly affect hippocampal excitability. Methods: VNS was administered in cycles of 30 s including either 0.1, 0.16, 0.25, 0.5, 1.5, 3 or 7 s of VNS ON time (biphasic pulses, 250ms/phase, 1 mA, 30 Hz) and the effect of different VNS ON times on brain temperature was evaluated. VNS paradigms with and without hypothermia were compared for their effects on hippocampal neurophysiology in freely moving rats. Results: Using VNS parameters with an ON time/OFF time of up to 0.5 s/30 s did not cause hypothermia, while clear hypothermia was detected with ON times of 1.5, 3 and 7 s/30 s. Relative to SHAM VNS, the normothermic 0.5 s VNS condition significantly decreased hippocampal EEG power and changed dentate gyrus evoked potentials with an increased field excitatory postsynaptic potential slope and a decreased population spike amplitude. Conclusion: VNS can be administered in freely moving rats without causing hypothermia, while profoundly affecting hippocampal neurophysiology suggestive of reduced excitability of hippocampal neurons despite increased synaptic transmission efficiency. (C) 2020 The Authors. Published by Elsevier Inc.

Introduction Considering the need for rapid change of treatment in recurrent glioblastoma (GB), it is of utmost importance to characterize PET radiopharmaceuticals that allow early discrimination of tumor from therapy-related effects. In this study, we examined the value of 2-[18F]FELP as a LAT1 tumor-specific PET tracer in comparison with [18F]FDG and [18F]FET in a combined orthotopic rat radiation necrosis and glioblastoma model. A second experiment compared 2-[18F]FELP to [18F]FDG in a mouse glioblastoma - inflammation model. Methods Using the small animal radiation research platform (SARRP), radiation necrosis (RN) was induced in the left frontal lobe of the rat brain. When radiation-induced changes were visible on MRI, F98 rat glioblastoma cells were stereotactically inoculated in the contralateral right frontal lobe. When tumor growth was confirmed on MRI, 2-[18F]FELP, [18F]FET and [18F]FDG PET scans were acquired on three consecutive days. In an inflammation experiment, mice were inoculated in the left thigh with U87 human glioblastoma cells. After heterotopic tumor growth was confirmed macroscopically, inflammation was induced by injection of turpentine subcutaneously in the right thigh. Subsequently, 2-[18F]FELP and [18F]FDG scans were acquired on two consecutive days. Results The in vivo PET images demonstrated that 2-[18F]FELP could differentiate glioblastoma and radiation necrosis using SUVmean (p = 0.0016) and LNRmean (p = 0.009), while [18F]FET was only able to differentiate both lesions by means of the SUVmean. (p = 0.047) Delayed [18F]FDGlate PET (4 h postinjection) was also able to distinguish glioblastoma from radiation necrosis, but smaller lesion-to-normal brain ratios were observed (SUVmean: p = 0.009; LNRmean: p = 0.028). In the inflammation study, 2-[18F]FELP showed no significant uptake in the inflammation lesion when compared to the control group (SUVmean: p = 0.149; LNRmean: p = 0.083). In contrast, both conventional and delayed [18F]FDG displayed significant uptake in the turpentine-invoked lesion (SUVmean: p = 0.021; LNRmean: p = 0.021). Conclusion This study suggests that the 2-[18F]FELP PET is able to differentiate glioblastoma from radiation necrosis and that the 2-[18F]FELP uptake is less likely to be contaminated by the presence of inflammation than the [18F]FDG signal. Advances in knowledge These results are clinically relevant for the differential diagnosis between tumor and radiation necrosis because radiation necrosis always contains a certain amount of inflammatory cells. Hence, 2-[18F]FELP is preferred to discriminate tumor from radiation necrosis.

Background and purpose: Intracerebral hemorrhage (ICH) is a known risk factor for the development of seizures, but little is known about the pathophysiology of seizures in the acute phase post-ICH and their influence on functional outcome. With the use of an animal model, the underlying pathophysiology could be further unraveled. The aim of our study was to optimize the rat collagenase stroke model for the detection of acute symptomatic seizures using video-EEG monitoring. Methods: Male Sprague-Dawley rats were implanted with scalp electrodes and a craniotomy was made for later injection of collagenase. After one week of baseline video-EEG recording, rats were injected with 0.6 U collagenase in 0.7 mu L saline in left striatum, in close proximity of the piriform cortex, and immediately reconnected to the video-EEG setup for 7 days. Occurrence of clinical and electrographic seizures was assessed and functional deficits were evaluated on several time points using the cylinder test, Neurological Deficit Scale (NDS) and forelimb placing test. At day 7 post-ICH, animals were euthanized. The volume and cortical involvement of the hemorrhage were assessed by histological examination of the brain tissue, using Cresyl violet stain. Results: Collagenase injection induced ICH in all animals with a mean volume of 27 mm(3) (SEM 7 mm(3), range 4-92 mm(3)). Functional deficits were present in all animals injected with collagenase (pre-ICH vs post-ICH, p< 0.001). Epileptic seizures occurred in 5/11 animals and started between 1 and 61 h after ICH induction. Behavioral changes were observed in 13/15 seizures. Conclusions: Injecting rats with 0.6 U of collagenase is a useful model to study the occurrence of acute symptomatic seizures post-ICH as it results in ICH in all animals without mortality, 45% incidence of ICH-induced acute symptomatic seizures and measurable functional deficits.

Coagulation factor XII (FXII), a S1A serine protease, was discovered more than fifty years ago. However, its in vivo functions and its three-dimensional structure started to be disclosed in the last decade. FXII was found at the crosstalk of several physiological pathways including the intrinsic coagulation pathway, the kallikrein-kinin system, and the immune response. The FXII inhibition emerges as a therapeutic strategy for the safe prevention of artificial surface-induced thrombosis and in patients suffering from hereditary angioedema. The anti-FXII antibody garadacimab discovered by phage-display library technology is actually under phase II clinical evaluation for the prophylactic treatment of hereditary angioedema. The implication of FXII in neuro-inflammatory and neurodegenerative disorders is also an emerging research field. The FXII or FXIIa inhibitors currently under development include peptides, proteins, antibodies, RNA-based technologies, and, to a lesser extent, small-molecular weight inhibitors. Most of them are proteins, mainly isolated from hematophagous arthropods and plants. The discovery and development of these FXII inhibitors and their potential indications are discussed in the review.

Background and purpose In patients treated with vagus nerve stimulation (VNS) for drug resistant epilepsy (DRE), up to a third of patients will eventually not respond to the therapy. As VNS therapy requires surgery for device implantation, prediction of response prior to surgery is desirable. It is hypothesized that neurophysiological investigations related to the mechanisms of action of VNS may help to differentiate VNS responders from non-responders prior to the initiation of therapy. Methods In a prospective series of DRE patients, polysomnography, heart rate variability (HRV) and cognitive event related potentials were recorded. Polysomnography and HRV were repeated after 1 year of treatment with VNS. Polysomnography, HRV and cognitive event related potentials were compared between VNS responders (>= 50% reduction in seizure frequency) and non-responders. Results Fifteen out of 30 patients became VNS responders after 1 year of VNS treatment. Prior to treatment with VNS, the amount of deep sleep (NREM 3), the HRV high frequency (HF) power and the P3b amplitude were significantly different in responders compared to non-responders (P = 0.007; P = 0.001; P = 0.03). Conclusion Three neurophysiological parameters, NREM 3, HRV HF and P3b amplitude, were found to be significantly different in DRE patients who became responders to VNS treatment prior to initiation of their treatment with VNS. These non-invasive recordings may be used as characteristics for response in future studies and help avoid unsuccessful implantations. Mechanistically these findings may be related to changes in brain regions involved in the so-called vagal afferent network.

Seizures are common in patients with high-grade gliomas (30&ndash, 60%) and approximately 15&ndash, 30% of glioblastoma (GB) patients develop drug-resistant epilepsy. Reliable animal models are needed to develop adequate treatments for glioma-related epilepsy. Therefore, fifteen rats were inoculated with F98 GB cells (GB group) and four rats with vehicle only (control group) in the right entorhinal cortex. MRI was performed to visualize tumor presence. A subset of seven GB and two control rats were implanted with recording electrodes to determine the occurrence of epileptic seizures with video-EEG recording over multiple days. In a subset of rats, tumor size and expression of tumor markers were investigated with histology or mRNA in situ hybridization. Tumors were visible on MRI six days post-inoculation. Time-dependent changes in tumor morphology and size were visible on MRI. Epileptic seizures were detected in all GB rats monitored with video-EEG. Twenty-one days after inoculation, rats were euthanized based on signs of discomfort and pain. This study describes, for the first time, reproducible tumor growth and spontaneous seizures upon inoculation of F98 cells in the rat entorhinal cortex. The development of this new model of GB-related epilepsy may be valuable to design new therapies against tumor growth and associated epileptic seizures.

The present study describes the identification of highly potent dimeric 1,2,4-benzothiadiazine 1,1-dioxide (BTD)-type positive allosteric modulators of the AMPA receptors (AMPApams) obtained by linking two monomeric BTD scaffolds through their respective 6-positions. Using previous X-ray data from monomeric BTDs cocrystallized with the GluA2 ligand-binding domain (LBD), a molecular modeling approach was performed to predict the preferred dimeric combinations. Two 6,6-ethylene-linked dimeric BTD compounds (16 and 22) were prepared and evaluated as AMPApams on HEK293 cells expressing GluA2 o(Q) (calcium flux experiment). These compounds were found to be about 10,000 times more potent than their respective monomers, the most active dimeric compound being the bis-4-cyclopropyl-substituted compound 22 [6,6′-(ethane-1,2-diyl)bis(4-cyclopropyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide], with an EC 50 value of 1.4 nM. As a proof of concept, the bis-4-methyl-substituted dimeric compound 16 (EC 50 = 13 nM) was successfully cocrystallized with the GluA2 o-LBD and was found to occupy the two BTD binding sites at the LBD dimer interface.

BACKGROUND Previously, a rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was established. Similarly to the clinic, CT and MRI were combined during the treatment planning process. PET imaging was subsequently added which allowed us to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI and PET) using a micro-irradiation system, proved to be labour intensive because multimodal imaging, treatment planning and dose delivery have to be completed sequentially in the preclinical setting. MATERIAL AND METHODS Two different methodologies were compared in silico for performing preclinical [18F]FET PET based radiation therapy (20 Gy based on MRI, 8 Gy boost based on PET) based on three different cases. Method 1 is based on the previously published methods1,2. However, the process is automated using an in-house developed MATLAB code. Method 2 consists of a more sophisticated method where a series of isocenters and jaw dimensions for the motorised variable collimator were determined based on the [18F]FET PET uptake. Both methods were evaluated by means of the dose volume histograms (DVH) and Q-volume histograms. RESULTS The setup parameters for both methods were calculated. The DVHs for method 2 are systematically closer to the ideal dose distribution compared to method 1. These findings are confirmed by the D90 and D50 values which are considerably lower for method 1. When observing the Q-factor, method 2 always results in dose distributions that are closer to the dose objectives (method 1: 0.141±0.046; method 2: 0.064±0.011). CONCLUSION The described novel method to optimize the preclinical treatment planning process has many advantages in terms of dose delivery, time efficiency and variability, when compared to the previously used methods1,2. These improvements are important to narrow the gap between clinical and preclinical radiation research and for the development of new therapeutics and/or radiation therapy procedures for glioblastoma. 1. Bolcaen, J., Descamps, B., Boterberg, T., Vanhove, C. & Goethals, I. PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator. J. Vis. Exp. 1–10 (2017) doi:10.3791/56601. 2. Verhoeven, J. et al. Technical feasibility of [18F]FET and [18F]FAZA PET guided radiotherapy in a F98 glioblastoma rat model. Radiat. Oncol. 14, (2019).

Magnetic resonance imaging (MRI) is frequently used for preclinical treatment monitoring in glioblastoma (GB). Discriminating between tumors and tumor-associated changes is challenging on in vivo MRI. In this study, we compared in vivo MRI scans with ex vivo MRI and histology to estimate more precisely the abnormal mass on in vivo MRI. Epileptic seizures are a common symptom in GB. Therefore, we used a recently developed GB-associated epilepsy model from our group with the aim of further characterizing the model and making it useful for dedicated epilepsy research. Ten days after GB inoculation in rat entorhinal cortices, in vivo MRI (T2w and mean diffusivity (MD)), ex vivo MRI (T2w) and histology were performed, and tumor volumes were determined on the different modalities. The estimated abnormal mass on ex vivo T2w images was significantly smaller compared to in vivo T2w images, but was more comparable to histological tumor volumes, and might be used to estimate end-stage tumor volumes. In vivo MD images displayed tumors as an outer rim of hyperintense signal with a core of hypointense signal, probably reflecting peritumoral edema and tumor mass, respectively, and might be used in the future to distinguish the tumor mass from peritumoral edema—associated with reactive astrocytes and activated microglia, as indicated by an increased expression of immunohistochemical markers—in preclinical models. In conclusion, this study shows that combining imaging techniques using different structural scales can improve our understanding of the pathophysiology in GB.

Over the last decade, the coagulation factor XIIa (FXIIa) has seen renewed interest as a therapeutic target. Indeed, its inhibition could offer a protection against thrombosis without increasing the risk of bleeding. Moreover, it could answer the need for a safe prevention of blood-contacting medical devices-related thrombosis. Among the FXII and FXIIa inhibitors already described in literature, organic small-molecular-weight inhibitors are rather left behind. In this study, we were focused on the discovery and assessment of water soluble small molecules. First, a search within our library of compounds flagged two promising hits. Indeed, enzymes and plasma assays suggested they have a greater activity on the contact factors (FXIa, plasma kallikrein and FXIIa) than on the TF pathway. Then, simple pharmacomodulations were undertaken with the aim to design more selective FXIIa inhibitors. This afforded compounds having different degrees of selectivity. All compounds were finally screened in whole blood using an 8-channel microfluidic model and thromboelastometry measurements. Interestingly, all molecules interfered with the thrombus formation and one of them could be considered as a small organic contact inhibitor. (C) 2017 Elsevier Ltd. All rights reserved.

AIM. Vagus nerve stimulation (VNS) modulates hippocampal dentate gyrus (DG) electrophysiology and induces hypothermia in freely moving rats. This study evaluated whether hippocampal (CA1) electrophysiology is similarly modulated and to what extent this is associated with VNS-induced hypothermia. METHODS. Six freely moving rats received a first 4[Formula: see text]h session of rapid cycling VNS (7[Formula: see text]s on/18[Formula: see text]s off), while CA1 evoked potentials, EEG and core temperature were recorded. In a second 4[Formula: see text]h session, external heating was applied during the 3rd and 4th[Formula: see text]h of VNS counteracting VNS-induced hypothermia. RESULTS. VNS decreased the slope of the field excitatory postsynaptic potential (fEPSP), increased the population spike (PS) amplitude and latency, decreased theta (4–12[Formula: see text]Hz) and gamma (30–100[Formula: see text]Hz) band power and theta peak frequency. Normalizing body temperature during VNS through external heating abolished the effects completely for fEPSP slope, PS latency and gamma band power, partially for theta band power and theta peak frequency and inverted the effect on PS amplitude. CONCLUSIONS. Rapid cycle VNS modulates CA1 electrophysiology similarly to DG, suggesting a wide-spread VNS-induced effect on hippocampal electrophysiology. Normalizing core temperature elucidated that VNS-induced hypothermia directly influences several electrophysiological parameters but also masks a VNS-induced reduction in neuronal excitability.

Inhibitors of the coagulation factor XIIa (FXIIa) are attractive to detail the roles of this protease in hemostasis and thrombosis, to suppress artifact due to contact pathway activation in blood coagulation assays, and they are promising as antithrombotic therapy. The 3-carboxamide coumarins have been previously described as small-molecular-weight FXIIa inhibitors. In this study, we report a structure-activity relationship (SAR) study around this scaffold with the aim to discover new selective FXIIa inhibitors with an improved physico-chemical profile. To better understand these SAR, docking experiments were undertaken. For this purpose, we built an original hybrid model of FXIIa. This model has the advantage to gather the best features from the recently published crystal structure of FXIIa in its zymogen form and a more classical homology model. Results with the hybrid model are encouraging as they help understanding the activity and selectivity of our best compounds.

With the emergence of more challenging targets, a relatively new approach, fragment-based drug discovery (FBDD), proved its efficacy and gained increasing importance in the pharmaceutical industry. FBDD identifies low molecular-weight (MW) ligands (fragments) that bind to biologically important macromolecules, then a structure-guided fragment growing or merging approach is performed, contributing to the quality of the lead. However, to select the appropriate fragment to be evolved, sensitive analytical screening methods must be used to measure the affinity in the μM or even mM range. In this particular context, we developed a robust and selective partial filling affinity CE (ACE) method for the direct binding screening of a small fragment library in order to identify new thrombin inhibitors. To demonstrate the accuracy of our assay, the complex dissociation constants of three known thrombin inhibitors, namely benzamidine, p-aminobenzamidine and nafamostat were determined and found to be in good concordance with the previously reported values. Finally, the screening of a small library was performed and demonstrated the high discriminatory power of our method towards weak binders compared to classical spectrophotometric activity assay, proving the interest of our method in the context of FBDD.

Cystic fibrosis (CF) is a fatal genetic disease associated with widespread exocrine gland dysfunction. Studies have suggested activating effects of resveratrol, a naturally-occurring polyphenol compound with antioxidant and anti-inflammatory properties, on CF transmembrane conductance regulator (CFTR) protein function. We assayed, in F508del-CFTR homozygous (CF) and in wild-type mice, the effect of resveratrol on salivary secretion in basal conditions, in response to inhibition by atropine (basal β-adrenergic-dependent component) and to stimulation by isoprenaline (CFTR-dependent component). Both components of the salivary secretion were smaller in CF mice than in controls. Two hours after intraperitoneal administration of resveratrol (50 mg/kg) dissolved in DMSO, the compound was detected in salivary glands. As in both CF and in wild-type mice, DMSO alone increased the response to isoprenaline in males but not in females, the effect of resveratrol was only measured in females. In wild-type mice, isoprenaline increased secretion by more than half. In CF mice, resveratrol rescued the response to isoprenaline, eliciting a 2.5-fold increase of β-adrenergic-stimulated secretion. We conclude that the salivary secretion assay is suitable to test DMSO-soluble CFTR modulators in female mice. We show that resveratrol applied in vivo to mice reaches salivary glands and increases β-adrenergic secretion. Immunolabelling of CFTR in human bronchial epithelial cells suggests that the effect is associated with increased CFTR protein expression. Our data support the view that resveratrol is beneficial for treating CF. The salivary secretion assay has a potential application to test efficacy of novel CF therapies.

It has been shown that vagus nerve stimulation (VNS) has an antidepressant-like effect in the forced swim test. The mechanism of action underlying this effect is incompletely understood, but there is evidence suggesting that the locus coeruleus (LC) may play an important role. In this study, noradrenergic LC neurons were selectively lesioned to test their involvement in the antidepressant-like effect of VNS in the forced swim test. Forced swim test behavior was assessed in rats that were subjected to VNS or sham treatment. In half of the VNS-treated animals, the noradrenergic neurons from the LC were lesioned using the selective neurotoxin DSP-4 [ N -(2-chloroethyl)- N -ethyl-2-bromobenzylamine hydrochloride], yielding three experimental arms: sham, VNS and DSP-4-VNS (n = 8 per group). Furthermore, the open field test was performed to evaluate locomotor activity. A dopamine-β-hydroxylase immunostaining was performed to confirm lesioning of noradrenergic LC neurons. VNS significantly reduced the percentage of immobility time in the forced swim test compared to sham treatment (median: 56%, interquartile range: 41% vs. median: 75%, interquartile range: 12%). This antidepressant-like effect of VNS could not be demonstrated in the DSP-4-VNS group (median: 79%, interquartile range: 33%). Locomotor activity in the open field test was not different between the three treatment arms. The absence of hippocampal dopamine-β-hydroxylase immunostaining in the DSP-4-treated rats confirmed the lesioning of noradrenergic neurons originating from the brainstem LC. The results of this study demonstrate that the noradrenergic neurons from the LC play an important role in the antidepressant-like effect of VNS.

Developing an EMMA method for enzymatic assay remains a challenge, particularly using UV detection. Indeed, it is necessary to optimize the separation conditions while allowing the enzymatic reaction to occur within the capillary respecting kinetic constraints and achieving enough sensitivity. In this work, such EMMA methodology was set up to evaluate the inhibitory potency of drugs toward thrombin, a serine protease implicated in the coagulation cascade. To achieve our goal, the separation buffer, the injection sequence, the internal standard and the chromogenic substrate were investigated. The newly developed system was then assessed determining the kinetic Km constant for the selected substrate and compared with the results obtained with a continuous spectrophotometer cell assay. Secondly, the Ki inhibitory constant of the thrombin inhibitor argatroban was determined and found in agreement with the published value.

The “Journées Franco-Belges de Pharmacochimie” is a recognized annual meeting in organic and medicinal chemistry known for the quality of scientific exchange and conviviality. Young researchers were encouraged to present their work and share ideas with senior scientists. Abstracts of plenary lectures, oral communications, and posters presented during the meeting are collected in this report.

63 Resveratrol improves chloride secretion in cystic fibrosis mice homozygous for the F508del mutation A. Palem1, C. Bouckaert1, B. Dhooghe1, A. Leonard2, B. Lubamba1, P. Wallemacq1, P. Lebecque2, T. Leal1. 1Universite Catholique de Louvain, Centre for Toxicology and Applied Pharmacology, Brussels, Belgium; 2Universite Catholique de Louvain, Pediatric Pulmonology & Cystic Fibrosis Unit, Cliniques Saint Luc, Brussels, Belgium

Thrombotic diseases, in which a deregulated clot formation occurs, remain a major cause of death in industrialised countries. Anticoagulants are part of the strategies to address these disorders. However current available drugs are still associated with risk of severe bleeding complications. Novel and safe antithrombotics are thus required. In this context, coagulation factor XIIa (FXIIa), a serine protease implicated in the contact phase of coagulation cascade, recently emerged as a promising target in the development of such agents1. Indeed, it was shown that FXII deficiency as well as FXIIa inhibition protects against thrombosis without causing spontaneous bleeding in mice2.Based on these considerations, the purpose of our project is to develop novel selective FXIIa inhibitors to detail the exact role of this enzyme in thrombotic diseases. These compounds could also be a good starting point for the development of new antithrombotic drugs. The 3-carboxamide coumarins were previously described as nonpetidic and selective inhibitors of FXIIa3. However, their low solubility and poor pharmacokinetics resulted in a lack of activity in in vivo models of thrombosis4. Therefore, we aim to improve these characteristics while keeping the selectivity and potency towards FXIIa.A new series of coumarins with an amino group at position 3 or 6 of the carboxamide coumarin scaffold was first synthesized. The introduction of a negative charge at these strategic positions was also investigated. The solubility and the inhibition potency of these newly synthesized compounds will be presented and the SAR will be fully discussed.

Thrombotic diseases, in which a deregulated haemostatic activity occurs, remain a major concern in medicine. Anticoagulants are part of the strategies to address these disorders. However current available drugs are still associated with risk of severe bleeding complications and thus, novel antithrombotics are required1.In this perspective, coagulation factor XIIa (FXIIa), a serine protease implicated in the coagulation cascade, recently emerged as a promising target in the development of such agents2. Indeed, it was demonstrated that FXII deficiency or inhibition protects against thrombosis without causing spontaneous bleeding in mice3.Based on these considerations, the aim of our project is to develop novel selective FXIIa inhibitors to detail the exact role of this enzyme in thrombotic diseases. These compounds could also be a good starting point for the development of new antithrombotic drugs. The 3-carboxamide coumarins (figure 1) are to date the only nonpetidic and selective inhibitors of FXIIa described in literature4. However, their low solubility and poor pharmacokinetics resulted in a lack of activity in in vivo models of thrombosis. As consequence, we need to improve these characteristics while keeping the selectivity and potency towards FXIIa.In this work, we first synthesized new coumarins with improved solubility. Their inhibition potency was then measured on FXIIa and finally, their stability was evaluated.

Objectives: Expression of the cystic fibrosis transmembrane conductance regulator is altered in individuals with the DF508 CFTR mutation. MicroRNA (miRNA) are differentially expressed in the CF airway epithelium, however their role in regulation of CFTR expression here remains unexplored. Herein we investigated the role of up regulated miRNAs in CFTR regulation in vivo in bronchial brushings from individuals homozygous or heterozygous for DF508 CFTR, validated our observations in vitro and assessed the impact of defective chloride ion conductance, genotype and Pseudomonas status on miRNA expression. Methods: MiRNA target prediction was performed in silico, expression of miRNAs and target genes was measured by qRT-PCR and/or western blotting. Modulation of miRNA was carried out using pre-miRs or antagomirs and a luciferase reporter gene employed to elucidate miRNA/CFTR interaction. MiR-145, -223 and 494 were up regulated in CF versus non-CF bronchial brushings and cell lines, in DF508 homovs. heterozygotes, in subjects positive for P. aeruginosa and in 16HBE14o− cells treated with a CFTR inhibitor. Reciprocal down or up regulation of CFTR gene and/or protein expression was observed following miRNA manipulation and direct miRNA/target relationship demonstrated via a luciferase reporter system containing the full length 3’-untranslated region of CFTR. Conclusions: Increased expression of miR-145, -223 and -494 in vivo in bronchial epithelium of individuals carrying the DF508 CFTR mutation correlates with decreased CFTR expression. Defective CFTR function and Pseudomonas colonisation may affect miRNA expression and contribute to the regulation of DF508 CFTR mutation.