Loss of Akt activity increases circulating soluble endoglin release in preeclampsia:identification of inter-dependency between Akt-1 and heme oxygenase-1

Aims - Endothelial dysfunction is a hallmark of preeclampsia. Desensitization of the phosphoinositide 3-kinase (PI3K)/Akt pathway underlies endothelial dysfunction and haeme oxygenase-1 (HO-1) is decreased in preeclampsia. To identify therapeutic targets, we sought to assess whether these two regulators act to suppress soluble endoglin (sEng), an antagonist of transforming growth factor-ß (TGF-ß) signalling, which is known to be elevated in preeclampsia. Methods and results - Vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), angiopoietin-1 (Ang-1), and insulin, which all activate the PI3K/Akt pathway, inhibited the release of sEng from endothelial cells. Inhibition of the PI3K/Akt pathway, by overexpression of phosphatase and tensin homolog (PTEN) or a dominant-negative isoform of Akt (Aktdn) induced sEng release from endothelial cells and prevented the inhibitory effect of VEGF-A. Conversely, overexpression of a constitutively active Akt (Aktmyr) inhibited PTEN and cytokine-induced sEng release. Systemic delivery of Aktmyr to mice significantly reduced circulating sEng, whereas Aktdn promoted sEng release. Phosphorylation of Akt was reduced in preeclamptic placenta and this correlated with the elevated level of circulating sEng. Knock-down of Akt using siRNA prevented HO-1-mediated inhibition of sEng release and reduced HO-1 expression. Furthermore, HO-1 null mice have reduced phosphorylated Akt in their organs and overexpression of Aktmyr failed to suppress the elevated levels of sEng detected in HO-1 null mice, indicating that HO-1 is required for the Akt-mediated inhibition of sEng. Conclusion - The loss of PI3K/Akt and/or HO-1 activity promotes sEng release and positive manipulation of these pathways offers a strategy to circumvent endothelial dysfunction.

Amino acids in oral drug delivery:salts, ion-pairs and transcriptomics

Oral drug delivery is considered the most popular route of delivery because of the ease of administration, availability of a wide range of dosage forms and the large surface area for drug absorption via the intestinal membrane. However, besides the unfavourable biopharmaceutical properties of the therapeutic agents, efflux transporters such as Pglycoprotein (P-gp) and multiple resistance proteins (MRP) decrease the overall drug uptake by extruding the drug from the cells. Although, prodrugs have been investigated to improve drug partitioning by masking the polar groups covalently with pre-moieties promoting increased uptake, they present significant challenges including reduced solubility and increased toxicity. The current work investigates the use of amino acids as ion-pairs for three model drugs: indomethacin (weak acid), trimethoprim (weak base) and ciprofloxacin (zwitter ion) in an attempt to improve both solubility and uptake. Solubility was studied by salt formation while creating new routes for uptake across the membranes via amino acids transporter proteins or dipeptidyl transporters was the rationale to enhance absorption. New salts were prepared for the model drugs and the oppositely charged amino acids by freeze drying and they were characterised using FTIR, 1HNMR, DSC, SEM, pH solubility profile, solubility and dissolution. Permeability profiles were assessed using an in vitro cell based method; Caco-2 cells and the genetic changes occurring across the transporter genes and various pathways involved in the cellular activities were studied using DNA microarrays. Solubility data showed a significant increase in drug solubility upon preparing the new salts with the oppositely charged counter ions (ciprofloxacin glutamate salt exhibiting 2.9x103 fold enhancement when compared to the free drug). Moreover, permeability studies showed a 3 fold increase in trimethoprim and indomethacin permeabilities upon ion-pairing with amino acids and more than 10 fold when the zwitter ionic drug was paired with glutamic acid. Microarray data revealed that trimethoprim was absorbed actively via OCTN1 transporters while MRP7 is the main transporter gene that mediates its efflux. The absorption of trimethoprim from trimethoprim glutamic acid ion-paired formulations was affected by the ratio of glutamic acid in the formulation which was inversely proportional to the degree of expression of OCTN1. Interestingly, ciprofloxacin glutamic acid ion-pairs were found to decrease the up-regulation of ciprofloxacin efflux proteins (P-gp and MRP4) and over-express two solute carrier transporters; (PEPT2 and SLCO1A2) suggesting that a high aqueous binding constant (K11aq) enables the ion-paired formulations to be absorbed as one entity. In conclusion, formation of ion-pairs with amino acids can influence in a positive way solubility, transfer and gene expression effects of drugs.

Role of endothelial Nox2 NADPH oxidase in angiotensin II-induced hypertension and vasomotor dysfunction

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are known to be involved in angiotensin II-induced hypertension and endothelial dysfunction. Several Nox isoforms are expressed in the vessel wall, among which Nox2 is especially abundant in the endothelium. Endothelial Nox2 levels rise during hypertension but little is known about the cell-specific role of endothelial Nox2 in vivo. To address this question, we generated transgenic mice with endothelial-specific overexpression of Nox2 (Tg) and studied the effects on endothelial function and blood pressure. Tg had an about twofold increase in endothelial Nox2 levels which was accompanied by an increase in p22phox levels but no change in levels of other Nox isoforms or endothelial nitric oxide synthase (eNOS). Basal NADPH oxidase activity, endothelial function and blood pressure were unaltered in Tg compared to wild-type littermates. Angiotensin II caused a greater increase in ROS production in Tg compared to wild-type aorta and attenuated acetylcholine-induced vasorelaxation. Both low and high dose chronic angiotensin II infusion increased telemetric ambulatory blood pressure more in Tg compared to wild-type, but with different patterns of BP change and aortic remodeling depending upon the dose of angiotensin II dose. These results indicate that an increase in endothelial Nox2 levels contributes to angiotensin II-induced endothelial dysfunction, vascular remodeling and hypertension. © 2011 The Author(s).

Modelling and simulation of biomass gasification in a circulating fluidized bed reactor

Computational Fluid Dynamics (CFD) has found great acceptance among the engineering community as a tool for research and design of processes that are practically difficult or expensive to study experimentally. One of these processes is the biomass gasification in a Circulating Fluidized Bed (CFB). Biomass gasification is the thermo-chemical conversion of biomass at a high temperature and a controlled oxygen amount into fuel gas, also sometime referred to as syngas. Circulating fluidized bed is a type of reactor in which it is possible to maintain a stable and continuous circulation of solids in a gas-solid system. The main objectives of this thesis are four folds: (i) Develop a three-dimensional predictive model of biomass gasification in a CFB riser using advanced Computational Fluid Dynamic (CFD) (ii) Experimentally validate the developed hydrodynamic model using conventional and advanced measuring techniques (iii) Study the complex hydrodynamics, heat transfer and reaction kinetics through modelling and simulation (iv) Study the CFB gasifier performance through parametric analysis and identify the optimum operating condition to maximize the product gas quality. Two different and complimentary experimental techniques were used to validate the hydrodynamic model, namely pressure measurement and particle tracking. The pressure measurement is a very common and widely used technique in fluidized bed studies, while, particle tracking using PEPT, which was originally developed for medical imaging, is a relatively new technique in the engineering field. It is relatively expensive and only available at few research centres around the world. This study started with a simple poly-dispersed single solid phase then moved to binary solid phases. The single solid phase was used for primary validations and eliminating unnecessary options and steps in building the hydrodynamic model. Then the outcomes from the primary validations were applied to the secondary validations of the binary mixture to avoid time consuming computations. Studies on binary solid mixture hydrodynamics is rarely reported in the literature. In this study the binary solid mixture was modelled and validated using experimental data from the both techniques mentioned above. Good agreement was achieved with the both techniques. According to the general gasification steps the developed model has been separated into three main gasification stages; drying, devolatilization and tar cracking, and partial combustion and gasification. The drying was modelled as a mass transfer from the solid phase to the gas phase. The devolatilization and tar cracking model consist of two steps; the devolatilization of the biomass which is used as a single reaction to generate the biomass gases from the volatile materials and tar cracking. The latter is also modelled as one reaction to generate gases with fixed mass fractions. The first reaction was classified as a heterogeneous reaction while the second reaction was classified as homogenous reaction. The partial combustion and gasification model consisted of carbon combustion reactions and carbon and gas phase reactions. The partial combustion considered was for C, CO, H2 and CH4. The carbon gasification reactions used in this study is the Boudouard reaction with CO2, the reaction with H2O and Methanation (Methane forming reaction) reaction to generate methane. The other gas phase reactions considered in this study are the water gas shift reaction, which is modelled as a reversible reaction and the methane steam reforming reaction. The developed gasification model was validated using different experimental data from the literature and for a wide range of operating conditions. Good agreement was observed, thus confirming the capability of the model in predicting biomass gasification in a CFB to a great accuracy. The developed model has been successfully used to carry out sensitivity and parametric analysis. The sensitivity analysis included: study of the effect of inclusion of various combustion reaction; and the effect of radiation in the gasification reaction. The developed model was also used to carry out parametric analysis by changing the following gasifier operating conditions: fuel/air ratio; biomass flow rates; sand (heat carrier) temperatures; sand flow rates; sand and biomass particle sizes; gasifying agent (pure air or pure steam); pyrolysis models used; steam/biomass ratio. Finally, based on these parametric and sensitivity analysis a final model was recommended for the simulation of biomass gasification in a CFB riser.

Challenges and emerging solutions in the development of compressed orally disintegrating tablets

Areas covered: The review discusses the main challenges of ODT manufacturing process and the emerging solutions featured at early drug development stages. The research specifically describes the methods reported for taste masking/assessment and solubilisation of unpalatable and poorly soluble drugs, respectively. Furthermore, this review highlights the techniques used for developing modified-release ODTs, an emerging area in the field. In addition, it also discusses the poor flowability and segregation problems of directly compressed powders. Moreover, the review describes the tests reported in the literature for ODT disintegration time assessment since a universal technique is still non-existent. Expert opinion: The approaches used to overcome the manufacturing challenges often have a bearing on the price of the end product. However, despite the technical and regulatory challenges, ODTs can offer many advantages over the conventional dosage forms if accompanied by suitable adjuvant technologies and in vitro analytical tools. © 2014 Informa UK, Ltd. Introduction: Orally disintegrating tablets (ODTs) provide several advantages over conventional tablets such as suitability for patients with swallowing difficulties and faster onset of action. The manufacture of ODTs by compression/tableting offers a practical and cost-effective strategy over the freeze drying (lyophilisation) method. Nonetheless, the FDA recommends a disintegration time of 30 s and a maximum weight of 500 mg for a tablet to be labelled as an ODT. These requirements, alongside other desirable product properties, have created a number of challenges for the formulator to overcome while developing compressed ODTs.