Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids.

Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide an unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using colon organoids generated from healthy tissue of colorectal cancer patients. Culture of these organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1, FABP2) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the γ-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 showing only weak signals of cell differentiation, and were unresponsive to DBZ. We also investigated the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of BMP4 and DBZ on colon normal organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

Microliquid/Liquid Interfacial Sensors: Biomimetic Investigation of Transmembrane Mechanisms and Real-Time Determinations of Clemastine, Cyproheptadine, Epinastine, Cetirizine, and Desloratadine.

Antihistamines relieve allergic symptoms by inhibiting the action of histamine. Further understanding of antihistamine transmembrane mechanisms and optimizing the selectivity and real-time monitoring capabilities of drug sensors is necessary. In this study, a micrometer liquid/liquid (L/L) interfacial sensor has served as a biomimetic membrane to investigate the mechanism of interfacial transfer of five antihistamines, i.e., clemastine (CLE), cyproheptadine (CYP), epinastine (EPI), desloratadine (DSL), and cetirizine (CET), and realize the real-time determinations. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques have been used to uncover the electrochemical transfer behavior of the five antihistamines at the L/L interface. Additionally, finite element simulations (FEMs) have been employed to reveal the thermodynamics and kinetics of the process. Visualization of antihistamine partitioning in two phases at different pH values can be realized by ion partition diagrams (IPDs). The IPDs also reveal the transfer mechanism at the L/L interface and provide effective lipophilicity at different pH values. Real-time determinations of these antihistamines have been achieved through potentiostatic chronoamperometry (I-t), exhibiting good selectivity with the addition of nine common organic or inorganic compounds in living organisms and revealing the potential for in vivo pharmacokinetics. Besides providing a satisfactory surrogate for studying the transmembrane mechanism of antihistamines, this work also sheds light on micro- and nano L/L interfacial sensors for in vivo analysis of pharmacokinetics at a single-cell or single-organelle level.

2-deoxy-D-glucose mitigates Citrobacter rodentium and dibenzazepine-induced gastrointestinal damage and colitis: novel implications of 2-DG polypharmacopea.

PURPOSE: Citrobacter rodentium (CR) infection coupled with blocking Notch/Wnt signaling via γ-secretase inhibitor dibenzazepine (DBZ) disrupts the gastro-intestinal (GI) barrier and induces colitis, akin to ionizing radiation (IR)-induced GI-injury. We investigated the effects of 2-deoxy-D-glucose (2-DG) to ameliorate the CR-DBZ-induced GI damage. MATERIALS AND METHODS: NIH:Swiss outbred mice were inoculated with 109CFUs of CR orally. DBZ was administered intraperitoneally (10 µM/kg b.wt; for 10 days 2 days post-CR infection). Mice were fed with 0.4% 2-DG (w/v) daily in drinking water. For microbiota depletion, antibiotics (Abx), 1 g/l metronidazole, and 0.2 g/l ciprofloxacin were administered for 10 days in drinking water. Oxidative stress, survival assay, colonic crypt hyperplasia, Notch/Wnt downstream signaling, immunomodulation, and bacterial dysbiosis were measured. RESULTS: We show that real-time visualization of reactive oxygen species (ROS) is similar during CR-induced colonic infection and IR-induced GI-damage. The histology revealed that dietary 2-DG mitigates CR + DBZ-induced colitis and improves survival compared with CR + DBZ alone. These changes were phenocopied in Abx-treated mice. Both 2-DG and Abx reduced dysbiosis, increased proliferation, inhibited pro-inflammatory response, and restored Hes-1 and ß-catenin protein levels, in the crypts. CONCLUSION: The energy disruptor 2-DG mitigates bacterial infection and its responsive hyperplasia/colitis, indicating its utility as a mitigator of infection/IR-induced GI-damage.

Effects of Notch signaling pathway inhibition by dibenzazepine in acute experimental toxoplasmosis.

Notch signaling pathway plays a crucial role in cellular fate across species, being important for the differentiation and development of several cell types. The aim of this study was to evaluate the effect of Notch inhibition pathway by dibenzazepine (DBZ) in histological and inflammatory alterations and, tissue parasitism in acute Toxoplasma gondii infection. For this, C57BL/6 mice were treated with DBZ before infection with T. gondii, and the small intestine, lungs and liver were analyzed. The genes related to Notch signaling pathway were assayed through qPCR in the organs, and cytokine measurement was performed in serum samples. In the small intestine, T. gondii infection impaired the Hes1 and Math1 mRNA expressions, increased the inflammation and decreased goblet and Paneth cell numbers. The DBZ-treatment was able to partially preserve these cells, however, the parasitism and inflammation were not altered. In parallel, the high IL-2, IL-6, TNF and, IFN-γ levels induced by infection were not changed with the DBZ treatment, with the IFN-γ levels even higher. In contrast, in the liver and lungs, the DBZ-treatment diminished parasitism and inflammation. Our results highlight that Notch pathway inhibition in T.gondii infection results in different parasitological and inflammatory outcomes depending on the organ analyzed.

Synthesis and biological evaluation of BU-4664L derivatives as potential anticancer agents.

BU-4664L is a naturally occurring N-farnesylated dibenzodiazepinone with important biological activities. Herein, we report the synthesis and antitumor evaluation of two series of BU-4664L derivatives bearing different substituent patterns on the dibenzodiazepinone core and with diverse side chains. All of the derivatives displayed micromolar activity against the human prostate cancer PC-3 cells, while lower or no activity against the human lung H460 cells. The most active derivatives were 10a and 16c which exerted antiproliferative activity against PC-3 cells with GI50 values of 5.66 and 5.94 µM, respectively, and thus represent promising lead compounds for further development.

Cell differentiation is disrupted by MYO5B loss through Wnt/Notch imbalance.

Functional loss of myosin Vb (MYO5B) induces a variety of deficits in intestinal epithelial cell function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). The impact of MYO5B loss on differentiated cell lineage choice has not been investigated. We quantified the populations of differentiated epithelial cells in tamoxifen-induced, epithelial cell-specific MYO5B-knockout (VilCreERT2 Myo5bfl/fl) mice utilizing digital image analysis. Consistent with our RNA-sequencing data, MYO5B loss induced a reduction in tuft cells in vivo and in organoid cultures. Paneth cells were significantly increased by MYO5B deficiency along with expansion of the progenitor cell zone. We further investigated the effect of lysophosphatidic acid (LPA) signaling on epithelial cell differentiation. Intraperitoneal LPA significantly increased tuft cell populations in both control and MYO5B-knockout mice. Transcripts for Wnt ligands were significantly downregulated by MYO5B loss in intestinal epithelial cells, whereas Notch signaling molecules were unchanged. Additionally, treatment with the Notch inhibitor dibenzazepine (DBZ) restored the populations of secretory cells, suggesting that the Notch pathway is maintained in MYO5B-deficient intestine. MYO5B loss likely impairs progenitor cell differentiation in the small intestine in vivo and in vitro, partially mediated by Wnt/Notch imbalance. Notch inhibition and/or LPA treatment may represent an effective therapeutic approach for treatment of MVID.

T-Cell Activation-Inhibitory Assay to Screen Caloric Restriction Mimetics Drugs for Drug Repositioning.

We previously reported a screening method for caloric restriction mimetics (CRM), a group of plant-derived compounds capable of inducing good health and longevity. In the present study, we explored the possibility of using this method to screen CRM drugs for drug repositioning. The method, T-cell activation-inhibitory assay, is based on inductive logic. Most of CRM such as resveratrol have been reported to suppress T-cell activation and have anti-inflammatory functions. Here, we assessed the activity of 12 antiallergic drugs through T-cell activation-inhibitory assay and selected four that showed the lowest IC50 values-ibudilast (IC50 0.97 µM), azelastine (IC50 7.2 µM), epinastine (IC50 16 µM), and amlexanox (IC50 33 µM)-for further investigation. Because azelastine showed high cytotoxicity, we selected only the remaining three drugs to study their biological functions. We found that all the three drugs suppressed the expression of interleukin (IL)-6, an inflammatory cytokine, in lipopolysaccharide-treated macrophage cells, with ibudilast being the strongest suppressor. Ibudilast also suppressed the secretion of another inflammatory cytokine, tumor necrosis factor (TNF)-α, and the expression of an inflammatory enzyme, cyclooxygenase-2, in the cells. These results suggest that T-cell activation-inhibitory assay can be used to screen potential CRM drugs having anti-inflammatory functions for the purpose of drug repositioning.

Iminostilbene, a novel small-molecule modulator of PKM2, suppresses macrophage inflammation in myocardial ischemia-reperfusion injury.

Introduction: Inflammation is a key factor in myocardial ischemia/reperfusion (MI/R) injury. Targeting leucocyte-mediated inflammation is an important strategy for MI/R therapy. Iminostilbene (ISB), a simple dibenzoazepine small molecule compound, has a strong anti-neurodegenerative effect. However, no study has shown the cardioprotective effect of ISB. Objectives: This study aimed to investigate the role of ISB against MI/R injury and identify its molecular target. Methods: To verify the cardiac protection of ISB in vivo and in vitro, we performed rat MI/R surgery and subjected inflammatory modeling of macrophages. In terms of molecular mechanisms, we designed and synthesized a small molecular probe of ISB and employed it on the click chemistry-activity-based protein profiling technique to fish for ISB targets in macrophages. To identify the target, we applied the competitive inhibition assay, surface-plasmon resonance (SPR), cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay. Results: In vivo, ISB showed robust anti-myocardial injury activity by improving cardiac function, reducing myocardial infarction, and inhibiting macrophage-mediated inflammation. In vitro, ISB strongly inhibited the transcription and the expression levels of inflammatory cytokines in macrophages. The pyruvate kinase isozyme type M2 (PKM2) was identified as the potential target of ISB through proteomic analysis and the competitive assay was performed for specific binding verification. Further thermodynamic and kinetic experiments showed that ISB was bound to PKM2 in a dose-dependent manner. Moreover, in terms of the biological function of ISB on PKM2, ISB reduced the expression of PKM2, thereby reducing the expression of HIF1α and the phosphorylation of STAT3. Conclusion: This study for the first time demonstrated that ISB targeted PKM2 to reduce macrophage inflammation thereby significantly alleviating MI/R injury.

Role of Human Arylacetamide Deacetylase (AADAC) on Hydrolysis of Eslicarbazepine Acetate and Effects of AADAC Genetic Polymorphisms on Hydrolase Activity.

Human arylacetamide deacetylase (AADAC) plays a role in the detoxification or activation of drugs and is sometimes involved in the incidence of toxicity by catalyzing hydrolysis reactions. AADAC prefers compounds with relatively small acyl groups, such as acetyl groups. Eslicarbazepine acetate, an antiepileptic drug, is a prodrug rapidly hydrolyzed to eslicarbazepine. We sought to clarify whether AADAC might be responsible for the hydrolysis of eslicarbazepine acetate. Eslicarbazepine acetate was efficiently hydrolyzed by human intestinal and liver microsomes and recombinant human AADAC. The hydrolase activities in human intestinal and liver microsomes were inhibited by epigallocatechin gallate, a specific inhibitor of AADAC, by 82% and 88% of the control, respectively. The hydrolase activities in liver microsomes from 25 human livers were significantly correlated (r = 0.87, P < 0.001) with AADAC protein levels, suggesting that the enzyme AADAC is responsible for the hydrolysis of eslicarbazepine acetate. The effects of genetic polymorphisms of AADAC on eslicarbazepine acetate hydrolysis were examined by using the constructed recombinant AADAC variants with T74A, V172I, R248S, V281I, N366K, or X400Q. AADAC variants with R248S or X400Q showed lower activity than wild type (5% or 21%, respectively), whereas those with V172I showed higher activity than wild type (174%). Similar tendencies were observed in the other four substrates of AADAC; that is, p-nitrophenyl acetate, ketoconazole, phenacetin, and rifampicin. Collectively, we found that eslicarbazepine acetate is specifically and efficiently hydrolyzed by human AADAC, and several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response. SIGNIFICANCE STATEMENT: This is the first study to clarify that arylacetamide deacetylase (AADAC) is responsible for the activation of eslicarbazepine acetate, an antiepileptic prodrug, to eslicarbazepine, an active form, in the human liver and intestines. In addition, we found that several AADAC polymorphic alleles would be a factor affecting the enzyme activity and drug response.

Complex effects of eslicarbazepine on inhibitory micro networks in chronic experimental epilepsy.

OBJECTIVE: Many antiseizure drugs (ASDs) act on voltage-dependent sodium channels, and the molecular basis of these effects is well established. In contrast, how ASDs act on the level of neuronal networks is much less understood. METHODS: In the present study, we determined the effects of eslicarbazepine (S-Lic) on different types of inhibitory neurons, as well as inhibitory motifs. Experiments were performed in hippocampal slices from both sham-control and chronically epileptic pilocarpine-treated rats. RESULTS: We found that S-Lic causes an unexpected reduction of feed-forward inhibition in the CA1 region at high concentrations (300 µM), but not at lower concentrations (100 µM). Concurrently, 300 but not 100 µM S-Lic significantly reduced maximal firing rates in putative feed-forward interneurons located in the CA1 stratum radiatum of sham-control and epileptic animals. In contrast, feedback inhibition was not inhibited by S-Lic. Instead, application of S-Lic, in contrast to previous data for other drugs like carbamazepine (CBZ), resulted in a lasting potentiation of feedback inhibitory post-synaptic currents (IPSCs) only in epileptic and not in sham-control animals, which persisted after washout of S-Lic. We hypothesized that this plasticity of inhibition might rely on anti-Hebbian potentiation of excitatory feedback inputs onto oriens-lacunosum moleculare (OLM) interneurons, which is dependent on Ca2+ -permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Indeed, we show that blocking Ca2+ -permeable AMPA receptors completely prevents upmodulation of feedback inhibition. SIGNIFICANCE: These results suggest that S-Lic affects inhibitory circuits in the CA1 hippocampal region in unexpected ways. In addition, ASD actions may not be sufficiently explained by acute effects on their target channels, rather, it may be necessary to take plasticity of inhibitory circuits into account.

Dibenzazepine combats acute liver injury in rats via amendments of Notch signaling and activation of autophagy.

Paracetamol is a commonly used over-the-counter analgesic and antipyretic drug. Nevertheless, an overdose of paracetamol leads to hepatic necrosis that can be lethal. This study aimed to assess the potential hepatoprotective effects of dibenzazepine, a Notch inhibitor, against acute liver injury in rats via interfering with oxidative stress, inflammation, apoptosis, autophagy, and Notch signaling. Silymarin (200 mg/kg, p.o.) or dibenzazepine (2 mg/kg, i.p.) were administered to rats for 5 days before a single hepatotoxic dose of paracetamol (800 mg/kg, i.p.). Pretreatment with silymarin and dibenzazepine significantly mitigated oxidative stress, inflammatory and apoptotic markers induced by paracetamol hepatotoxicity where dibenzazepine showed greater repression of inflammation. Furthermore, dibenzazepine was found to be significantly more efficacious than silymarin in inhibiting Notch signaling as represented by expression of Notch-1 and Hes-1. A significantly greater response was also demonstrated with dibenzazepine pretreatment with regard to the expression of autophagic proteins, Beclin-1 and LC-3. The aforementioned biochemical results were confirmed by histopathological examination. Autophagy and Notch signaling seem to play a significant role in protection provided by dibenzazepine for paracetamol-induced hepatotoxicity in rats, which could explain its superior results relative to silymarin. Graphical abstract.

Generation of intestinal chemosensory cells from nonhuman primate organoids.

Several gastrointestinal epithelial cells are involved in taste signal transduction. Although rodent tissues are extensively used as a human gut model, recent studies show that the chemical sensing system in rodents differs from that in humans. Nonhuman primates in biomedical research are valuable animal models to advance our understanding of biological responses in humans. The 3D organoid culture produces functional gastrointestinal epithelial cells in vitro and can be generated from animal and human tissues. Here, we report the generation of intestinal chemosensory cells from nonhuman primates, macaques, using an organoid culture system. We were able to maintain macaque intestinal organoids in the proliferation medium for more than six months. Upon switching to differentiation medium, we observed a drastic change in organoid morphology and chemosensory cell marker protein expression. This switch from proliferation to differentiation was confirmed by transcriptome analysis of the duodenum, jejunum, and ileum organoids. We further observed that the supplementation of culture media with interleukin (IL)-4 or the Notch inhibitor dibenzazepine (DBZ) accelerated terminal cell differentiation into chemosensory cells. Overall, we generated monkey intestinal organoids for the first time. These organoids are suitable for studying the function of primate chemosensory cells.

Distinct effects on cAMP signaling of carbamazepine and its structural derivatives do not correlate with their clinical efficacy in epilepsy.

The antiepileptic sodium channel blocker, carbamazepine, has long been known to be able to attenuate cAMP signals. This could be of clinical importance since cAMP signaling has been shown to be involved in epileptogenesis and seizures. However, no information on the ability to affect cAMP signaling is available for the marketed structural derivatives, oxcarbazepine and eslicarbazepine acetate or their dominating metabolite, licarbazepine. Thus, we employed a HEK293 cell line stably expressing a cAMP biosensor to assess the effect of these two drugs on cAMP accumulation. We find that oxcarbazepine does not affect cAMP accumulation whereas eslicarbazepine acetate, surprisingly, is able to enhance cAMP accumulation. Since the transcription of ADCY8 (adenylyl cyclase isoform 8; AC8) has been found to be elevated in epileptic tissue from patients, we subsequently expressed AC8 in the HEK293 cells. In the AC8-expressing cells, oxcarbazepine was now able to attenuate whereas eslicarbazepine maintained its ability to increase cAMP accumulation. However, at all concentrations tested, licarbazepine demonstrated no effect on cAMP accumulation. Thus, we conclude that the effects exerted by carbamazepine and its derivatives on cAMP accumulation do not correlate with their clinical efficacy in epilepsy. However, this does not disqualify cAMP signaling per se as a potential disease-modifying drug target for epilepsy since more potent and selective inhibitors may be of therapeutic value.

Dibenzazepine promotes cochlear supporting cell proliferation and hair cell regeneration in neonatal mice.

OBJECTIVES: To investigate the role of dibenzazepine (DBZ) in promoting supporting cell (SC) proliferation and hair cell (HC) regeneration in the inner ear. MATERIALS AND METHODS: Postnatal day 1 wild-type or neomycin-damaged mouse cochleae were cultured with DBZ. Immunohistochemistry and scanning electron microscopy were used to examine the morphology of cochlear cells, and high-throughput RNA-sequencing was used to measure gene expression levels. RESULTS: We found that DBZ promoted SC proliferation and HC regeneration in a dose-dependent manner in both normal and damaged cochleae. In addition, most of the newly regenerated HCs induced by DBZ had visible and relatively mature stereocilia bundle structures. Finally, RNA sequencing detected the differentially expressed genes between DBZ treatment and controls, and interaction networks were constructed for the most highly differentially expressed genes. CONCLUSIONS: Our study demonstrates that DBZ can significantly promote SC proliferation and increase the number of mitotically regenerated HCs with relatively mature stereocilia bundles in the neonatal mouse cochlea by inhibiting Notch signalling and activating Wnt signalling, suggesting the DBZ might be a new therapeutic target for stimulating HC regeneration.

[Long-term effects of dibenzazepines on metabolic parameters: retrospective comparison of carbamazepine, oxcarbazepine and eslicarbazepine acetate in the real world]. / Efectos a largo plazo de las dibenzacepinas sobre los parámetros metabólicos: comparación retrospectiva de carbamacepina, oxcarbacepina y acetato de eslicarbacepina en el mundo real.

INTRODUCTION: Although carbamazepine (CBZ) has strong enzyme-inducing properties, oxcarbazepine (OXC) and eslicarbazepine acetate (ESL) are thought to have a milder effect. These drugs are known to have effects on lipid metabolism and may cause hyponatremia and changes in blood cell counts and liver function tests. AIM: To compare the long-term effects of three antiepileptic drugs (CBZ, OXC and ESL) on these variables. PATIENTS AND METHODS: Retrospective cohort study of consecutive patients treated with CBZ, OXC or ESL. Natremia, lipid concentrations, blood cell counts and liver function tests were compared before, during and at the end of the study period. RESULTS: A total of 292 patients were included. Of these, 143 were treated with CBZ, 55 with OXC and 94 with ESL. CBZ showed a greater impact on lipid metabolism, while OXC was correlated with lower mean sodium levels and a higher frequency of hyponatremia. Lifestyle recommendations related to diet, physical activity and water intake were helpful in overcoming these side effects. No other statistically significant differences were detected. CONCLUSIONS: While CBZ showed a greater impact on lipid metabolism, OXC displayed a higher frequency of hyponatremia. Lifestyle recommendations may be helpful in overcoming these side effects. No other statistically significant differences were found.

TITLE: Efectos a largo plazo de las dibenzacepinas sobre los parámetros metabólicos: comparación retrospectiva de carbamacepina, oxcarbacepina y acetato de eslicarbacepina en el mundo real.Introducción. Aunque la carbamacepina (CBZ) tiene fuertes propiedades de inducción enzimática, se cree que la oxcarbacepina (OXC) y el acetato de eslicarbacepina (ESL) ejercen un efecto más leve. Se sabe que estos fármacos tienen efectos sobre el metabolismo lipídico, pueden causar hiponatremia y cambios en el recuento de células sanguíneas y en las pruebas de función hepática. Objetivo. Comparar los efectos a largo plazo de tres medicamentos antiepilépticos (CBZ, OXC y ESL) en estas variables. Pacientes y métodos. Estudio de cohorte retrospectivo de pacientes consecutivos tratados con CBZ, OXC o ESL. La natremia, las concentraciones de lípidos, el recuento de células sanguíneas y las pruebas de función hepática se compararon antes, durante y al final del período de estudio. Resultados. Se incluyó a 292 pacientes. De ellos, 143 fueron tratados con CBZ, 55 con OXC y 94 con ESL. La CBZ mostró un mayor impacto en el metabolismo de los lípidos, mientras que la OXC se correlacionó con niveles medios de sodio más bajos y una frecuencia mayor de hiponatremia. Las recomendaciones de estilo de vida relacionadas con la dieta, la actividad física y la ingesta de agua fueron útiles para superar estos efectos secundarios. No se detectaron otras diferencias estadísticamente significativas. Conclusiones. Mientras que la CBZ mostró un mayor impacto en el metabolismo de los lípidos, la OXC mostró una mayor frecuencia de hiponatremia. Las recomendaciones de estilo de vida pueden ser útiles para superar estos efectos secundarios. No se encontraron otras diferencias estadísticamente significativas.

Canonical Notch signaling is required for bone morphogenetic protein-mediated human osteoblast differentiation.

Osteoblast differentiation of bone marrow-derived human mesenchymal stem cells (hMSC) can be induced by stimulation with canonical Notch ligand, Jagged1, or bone morphogenetic proteins (BMPs). However, it remains elusive how these two pathways lead to the same phenotypic outcome. Since Runx2 is regarded as a master regulator of osteoblastic differentiation, we targeted Runx2 with siRNA in hMSC. This abrogated both Jagged1 and BMP2 mediated osteoblastic differentiation, confirming the fundamental role for Runx2. However, while BMP stimulation increased Runx2 and downstream Osterix protein expression, Jagged1 treatment failed to upregulate either, suggesting that canonical Notch signals require basal Runx2 expression. To fully understand the transcriptomic profile of differentiating osteoblasts, RNA sequencing was performed in cells stimulated with BMP2 or Jagged1. There was common upregulation of ALPL and extracellular matrix genes, such as ACAN, HAS3, MCAM, and OLFML2B. Intriguingly, genes encoding components of Notch signaling (JAG1, HEY2, and HES4) were among the top 10 genes upregulated by both stimuli. Indeed, ALPL expression occurred concurrently with Notch activation and inhibiting Notch activity for up to 24 hours after BMP administration with DAPT (a gamma secretase inhibitor) completely abrogated hMSC osteoblastogenesis. Concordantly, RBPJ (recombination signal binding protein for immunoglobulin kappa J region, a critical downstream modulator of Notch signals) binding could be demonstrated within the ALPL and SP7 promoters. As such, siRNA-mediated ablation of RBPJ decreased BMP-mediated osteoblastogenesis. Finally, systemic Notch inhibition using diabenzazepine (DBZ) reduced BMP2-induced calvarial bone healing in mice supporting the critical regulatory role of Notch signaling in BMP-induced osteoblastogenesis.

Eslicarbazepine for focal epilepsy and acute intermittent porphyria.

Porphyrias are rare genetic disorders which cause a deficiency in the enzymes involved in the biosynthesis of heme. The treatment of epilepsy in patients with acute intermittent porphyria can be difficult since many anticonvulsants can increase heme synthesis and trigger porphyric attacks. We report a patient with focal epilepsy successfully treated with eslicarbazepine without exacerbation of porphyria.

Retinoic acid receptor-beta prevents cisplatin-induced proximal tubular cell death.

Cisplatin's toxicity in renal tubular epithelial cells limits the therapeutic efficacy of this antineoplastic drug. In cultured human proximal tubular HK-2 cells (PTC) a prostaglandin uptake transporter (PGT)-dependent increase in intracellular prostaglandin E2 (iPGE2) mediates cisplatin's toxicity (i.e. increased cell death and loss of cell proliferation) so that it is prevented by PGT inhibitors. Here we found in cisplatin-treated PTC that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a PGT inhibitor, prevented cisplatin's toxicity but not the increase in iPGE2. Because expression of retinoic acid receptor-ß (RAR-ß) is dependent on iPGE2 and because RAR-ß is a regulator of cell survival and proliferation, we hypothesized that RAR-ß might mediate the protective effect of DIDS against cisplatin's toxicity in PTC. Our results confirmed this hypothesis because: i) protection of PTC by DIDS was abolished by RAR-ß antagonist LE-135; ii) DIDS increased the expression of RAR-ß in PTC and prevented its decrease in cisplatin-treated PTC but not in cisplatin-treated human cervical adenocarcinoma HeLa cells in which DIDS failed to prevent cisplatin's toxicity; iii) while RAR-ß expression decreased in cisplatin-treated PTC, RAR-ß over-expression prevented cisplatin's toxicity. RAR-ß agonist CH55 or RAR pan-agonist all-trans retinoic acid did not prevent cisplatin's toxicity, which suggests that RAR-ß does not protect PTC through activation of gene transcription. In conclusion, RAR-ß might be a new player in cisplatin-induced proximal tubular injury and the preservation of its expression in proximal tubules through treatment with DIDS might represent a novel strategy in the prevention of cisplatin's nephrotoxicity without compromising cisplatin's chemotherapeutic effect on cancer cells.

Effects of carbamazepine, eslicarbazepine, valproic acid and levetiracetam on bone microarchitecture in rats.

BACKGROUND: Metabolic bone disease and fractures are a great problem for patients with epilepsy. The use of antiepileptic drugs (AEDs) is known to play an essential role in the progression of bone loss by various pathophysiological mechanisms. The aim of this study was to evaluate the effects of AEDs on bone microstructure as an additional cause of an increased fracture risk in patients with epilepsy. METHODS: Five groups of each of 12 female rats were orally dosed daily for 8 weeks with either carbamazepine (CBZ) (60 mg/kg), eslicarbazepine (ESL) (80 mg/kg), valproic acid (VPA) (300 mg/kg), levetiracetam (LEV) (50 mg/kg) or saline (control (CTL)). Following killing, dissected femurs were analyzed using X-ray micro-computed tomography (µCT), dual-energy X-ray absorptiometry (DXA) and biomechanical testing. In addition, serum bone turnover markers (BTM) were monitored throughout the experiment. RESULTS: Compared to CTL treatment, VPA decreased bone volume fraction by 19%, decreased apparent density by 14% and increased structural model index by 41%. No changes were observed in bone biomechanics nor mineral density evaluated by DXA or in levels of BTM. CONCLUSIONS: Our findings suggest that VPA affects the microarchitectural properties of the bone. The AEDs CBZ, ESL and LEV appear to have less adverse effects on bone biology and may be a better choice when treating patients with respect to bone health.

Safety, tolerability and effectiveness of transition to eslicarbazepine acetate from carbamazepine or oxcarbazepine in clinical practice.

PURPOSE: To assess the efficacy, safety and tolerability of eslicarbazepine acetate (ESL) in patients transitioning from carbamazepine or oxcarbazepine to ESL in clinical practice, by analysing data from the Euro-Esli study. METHODS: Euro-Esli was a pooled analysis of 14 European clinical practice studies. Effectiveness assessments included responder rate (≥50 % seizure frequency reduction) and seizure freedom rate (seizure freedom at least since prior visit), assessed after 3, 6 and 12 months of ESL treatment, and at the last visit. Safety and tolerability were assessed throughout follow-up by evaluating adverse events (AEs) and ESL discontinuation due to AEs, respectively. Data were analysed for cohorts of patients who transitioned from carbamazepine and oxcarbazepine to ESL either due to lack of efficacy or poor tolerability. RESULTS: Euro-Esli included 2058 patients, of whom 233 (11.3 %) transitioned from carbamazepine to ESL and 134 (6.5 %) transitioned from oxcarbazepine to ESL. After 12 months of ESL treatment, responder and seizure freedom rates for patients transitioning from carbamazepine due to lack of efficacy (n = 163) were 70.0 % and 30.9 %, respectively. Corresponding values for patients transitioning from oxcarbazepine due to lack of efficacy (n = 90) were 57.1 % and 25.0 %, respectively. Among patients who transitioned from carbamazepine and oxcarbazepine to ESL due to poor tolerability (n = 64 and n = 61, respectively), 26.6 % and 39.5 % experienced AEs, and 8.3 % and 6.8 % discontinued ESL due to AEs, respectively. CONCLUSION: ESL was efficacious and generally well tolerated in patients transitioning from carbamazepine or oxcarbazepine in clinical practice due to inadequate seizure control or intolerable AEs with these agents.