Controlled Release Electron Donors: Hydrogen Release Compound (HRC)—An Overview of a Decade of Case Studies

Hydrogen Release Compound (HRC) has been a commercially available product for engineered bioremediation of anaerobically biodegradable contaminants since 1999. HRC is a polylactate ester that, upon hydration or microbial cleavage of its ester bonds, slowly releases lactic acid. Lactic acid serves as an electron donor for microbial reductive biodegradation, while also providing hydrogen and carbon where required. HRC is a viscous amber-colored liquid that is typically injected into a contaminated aquifer using direct push technology or backfill injection into boreholes created by traditional drilling methods. Once in place, HRC creates a plume of lactic acid and its fermentation products (other organic acids and hydrogen) downgradient of the injection area and serves to accelerate anaerobic bioremediation processes. In this review of HRC field application results, the authors summarize application types, contaminants treated, site types, application locations, injection methods, site lithology and hydrology, and concentration ranges of geochemical species. The source of this information is a database of more than 850 HRC field applications, a series of 80 HRC publications that are publicly available, and 44 detailed site case histories that are available electronically.     

Measurement of Slow Spontaneous Release of 11-cis-Retinal from Rhodopsin

The vertebrate visual photoreceptor rhodopsin (Rho) is a unique G protein-coupled receptor as it utilizes a covalently tethered inverse agonist (11-cis-retinal) as the native ligand. Previously, electrophysiological studies showed that ligand binding of 11-cis-retinal in dark-adapted Rho was essentially irreversible with a half-life estimated to be 420 years, until after thermal isomerization to all-trans-retinal, which then slowly dissociates. This long lifetime of 11-cis-retinal binding was considered to be physiologically important for minimizing background signal (dark noise) of the visual system. However, in vitro biochemical studies on the thermal stability of Rho showed that Rho decays with a half-life on the order of days. In this study, we resolve the discrepancy by measuring the chromophore exchange rate of the bound 11-cis-retinal chromophore with free 9-cis-retinal from Rho in an in vitro phospholipid/detergent bicelle system. We conclude that the thermal decay of Rho primarily proceeds through spontaneous breaking of the covalent linkage between opsin and 11-cis-retinal, which was overlooked in the electrophysiological recording. We estimate that this slow spontaneous release of 11-cis-retinal from Rho should result in 10⁴ to 10⁵ free opsin molecules in a dark-adapted rod cell—a number that is three orders of magnitude higher than previously expected. We also discuss the physiological implications of these findings on the basal activity of opsins and the associated dark noise in the visual system.     

Release of galactosyl oligosaccharides by endo-β-N-acetylglucosaminidase D

Endo-β-N-acetylglucosaminidase D from Diplococcus pneumoniae released galactosyl oligosaccharides from IgG glycopeptides treated with β-N-acetylglucosaminidase. The structure of the major oligosaccharide was proposed to be as follows.

Since α-mannosidase digestion of the β-N-acetylglucosaminidase-treated glycopeptides made them again resistant to the endoglycosidase, we concluded that an unsubstituted α-mannosyl residue was required for the enzymic action.     

Release of carboxymethyl-cellulase and β-glucosidase from cell walls of Trichoderma reesei

Summary Carboxymethyl-cellulase and -glucosidase activities were determined in the cytosole, cell walls and extracellular culture fluid of Trichoderma reesei QM 9414 cultivated on cellulose and cellobiose. By means of carboxymethylcellulose as a specific desorbens for cellulose bound CM-cellulase and -glucosidase it was found that these enzymes are cell wall bound during consumption of the carbon source, but are excreted during the subsequent cultivation stage. Treatment of intact cell walls with various chemical agents could not cause a release of the enzyme. Treatment of intact cell walls with -mannanase or trypsin released CM-cellulase, whereas, treatment with laminarinase or chitinase released -glucosidase. Both enzymes were also released during autolysis in phosphate buffer. This autolysis was accompanined by the appearance of extracellular mannanase, laminarinase and proteinase. The results suggest that cleavage of chemical bonds of certain cell wall polymers of T. reesei could be responsible for the appearance of CM-cellulase and -glucosidase in the culture fluid during later stages of growth.     

Investigation into the Effect of Ethylcellulose Viscosity Variation on the Drug Release of Metoprolol Tartrate and Acetaminophen Extended Release Multiparticulates—Part I

Ethylcellulose is one of the most commonly used polymers to develop reservoir type extended release multiparticulate dosage forms. For multiparticulate extended release dosage forms, the drug release is typically governed by the properties of the barrier membrane coating. The ICH Pharmaceutical Development Guideline (ICH Q8) requires an understanding of the influence of critical material attributes and critical process parameters on the drug release of a pharmaceutical product. Using this understanding, it is possible to develop robust formulations with consistent drug release characteristics. Critical material attributes for ethylcellulose were evaluated, and polymer molecular weight variation (viscosity) was considered to be the most critical attribute that can impact drug release. To investigate the effect of viscosity variation within the manufacturer’s specifications of ethylcellulose, extended release multiparticulate formulations of two model drugs, metoprolol tartrate and acetaminophen, were developed using ETHOCEL? as the rate controlling polymer. Quality by Design (QbD) samples of ETHOCEL Std. 10, 20, and 100 Premium grades representing the low, medium, and high molecular weight (viscosity) material were organically coated onto drug layered multiparticulates to a 15% weight gain (WG). The drug release was found to be similar (f ₂?>?50) for both metoprolol tartrate and acetaminophen multiparticulates at different coating weight gains of ethylcellulose, highlighting consistent and robust drug release performance. The use of ETHOCEL QbD samples also serves as a means to develop multiparticulate dosage formulations according to regulatory guidelines.     

Development of Sustained Release Capsules Containing “Coated Matrix Granules of Metoprolol Tartrate”

The objective of this investigation was to prepare sustained release capsule containing coated matrix granules of metoprolol tartrate and to study its in vitro release and in vivo absorption. The design of dosage form was performed by choosing hydrophilic hydroxypropyl methyl cellulose (HPMC K100M) and hydrophobic ethyl cellulose (EC) polymers as matrix builders and Eudragit® RL/RS as coating polymers. Granules were prepared by composing drug with HPMC K100M, EC, dicalcium phosphate by wet granulation method with subsequent coating. Optimized formulation of metoprolol tartrate was formed by using 30% HPMC K100M, 20% EC, and ratio of Eudragit® RS/RL as 97.5:2.5 at 25% coating level. Capsules were filled with free flowing optimized granules of uniform drug content. This extended the release period upto 12 h in vitro study. Similarity factor and mean dissolution time were also reported to compare various dissolution profiles. The network formed by HPMC and EC had been coupled satisfactorily with the controlled resistance offered by Eudragit® RS. The release mechanism of capsules followed Korsemeyer–Peppas model that indicated significant contribution of erosion effect of hydrophilic polymer. Biopharmaceutical study of this optimized dosage form in rabbit model showed 10 h prolonged drug release in vivo. A close correlation (R² = 0.9434) was established between the in vitro release and the in vivo absorption of drug. The results suggested that wet granulation with subsequent coating by fluidized bed technique, is a suitable method to formulate sustained release capsules of metoprolol tartrate and it can perform therapeutically better than conventional immediate release dosage form.Key words: biopharmaceutical evaluation, coated granules, metoprolol tartrate, sustained release     

Mechanisms of VEGF- and Glutamate-Induced Inhibition of Osmotic Swelling of Murine Retinal Glial (Müller) Cells: Indications for the Involvement of Vesicular Glutamate Release and Connexin-Mediated ATP Release

We determined the mechanisms of glutamate and ATP release from murine retinal glial (Müller) cells by pharmacological manipulation of the vascular endothelial growth factor (VEGF)- and glutamate-induced inhibition of cellular swelling under hypoosmotic conditions. It has been shown that exogenous glutamate inhibits hypoosmotic swelling of rat Müller cells via the induction of the release of ATP (Uckermann et al. in J Neurosci Res 83:538–550, 53). VEGF was shown to inhibit hypoosmotic swelling of rat Müller cells by inducing the release of glutamate (Wurm et al. in J Neurochem 104:386–399, 55). The swelling-inhibitory effect of VEGF in murine Müller cells was blocked by an inhibitor of vesicular exocytosis, by a modulator of the allosteric site of vesicular glutamate transporters, and by inhibitors of phospholipase C and protein kinase C. The swelling-inhibitory effect of glutamate in murine Müller cells was prevented by inhibitors of connexin hemichannels. The effects of both VEGF and glutamate were blocked by tetrodotoxin and by an inhibitor of T-type voltage-gated calcium channels. Murine Müller cells display connexin-43 immunoreactivity. The data suggest that Müller cells of the murine retina may release glutamate by vesicular exocytosis, whereas ATP is released through connexin hemichannels.     

Release of Interleukin-1α or Interleukin-1β Depends on Mechanism of Cell Death

The cytokine interleukin-1 (IL-1) has two main pro-inflammatory forms, IL-1α and IL-1β, which are central to host responses to infection and to damaging sterile inflammation. Processing of IL-1 precursor proteins to active cytokines commonly occurs through activation of proteases, notably caspases and calpains. These proteases are instrumental in cell death, and inflammation and cell death are closely associated, hence we sought to determine the impact of cell death pathways on IL-1 processing and release. We discovered that apoptotic regulation of caspase-8 specifically induced the processing and release of IL-1β. Conversely, necroptosis caused the processing and release of IL-1α, and this was independent of IL-1β processing and release. These data suggest that the mechanism through which an IL-1-expressing cell dies dictates the nature of the inflammatory mechanism that follows. These insights may allow modification of inflammation through the selective targeting of cell death mechanisms during disease.     

Release of [3H]noradrenaline by α1-adrenoceptor agonists

Mouse isolated vas deferens preincubated with [³-H]noradrenaline was superfused and the effect of ₁-adrenoceptor agonists was studied on the release of total radioactivity ([³H]noradrenaline +³H-metabolites) and [³H]noradrenaline. Reverse phase high pressure liquid chromatography (HPLC) combined with scintillation spectrometry was used to separate [³H]noradrenaline from its metabolites. Among the ₁-adrenoceptor agonists (1-phenylephrine, ST-587(2-(2-chloro-5-trifluoromethyl phenylimino)-imidazole), (–)-amidephrine, methoxamine, cirazoline and l-noradrenaline) studied l-phenylephrine, ST-587 and l-noradrenaline were capable of releasing³H-noradrenaline. The effect of noradrenaline was stereospecific. As determined by HPLC combined with scintillation spectrometry the release of total radioactivity in response to l-noradrenaline is mainly due to [³H]noradrenaline. It is suggested that l-noradrenaline, l-phenylephrine, and ST-587 in addition to their direct effect on different receptors they also have indirect action through the release of noradrenaline which might be partly involved in the pharmacological responses. The mechanisms whereby l-noradrenaline and l-phenylephrine release noradrenaline would appear to involve a saturable Ca-independent and a cocaine and temperature sensitive process. On the basis of our findings among the ₁-adrenoceptor agonist studied (–)-amidephrine, methoxamine and cirazoline is a better choice than l-phenylephrine or ST-587 for selective stimulation of postjunctional ₁-adrenoceptor, they do not release noradrenaline.     

Modulation of Starch Digestion for Slow Glucose Release through "Toggling" of Activities of Mucosal α-Glucosidases

Starch digestion involves the breakdown by α-amylase to small linear and branched malto-oligosaccharides, which are in turn hydrolyzed to glucose by the mucosal α-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). MGAM and SI are anchored to the small intestinal brush-border epithelial cells, and each contains a catalytic N- and C-terminal subunit. All four subunits have α-1,4-exohydrolytic glucosidase activity, and the SI N-terminal subunit has an additional exo-debranching activity on the α-1,6-linkage. Inhibition of α-amylase and/or α-glucosidases is a strategy for treatment of type 2 diabetes. We illustrate here the concept of "toggling": differential inhibition of subunits to examine more refined control of glucogenesis of the α-amylolyzed starch malto-oligosaccharides with the aim of slow glucose delivery. Recombinant MGAM and SI subunits were individually assayed with α-amylolyzed waxy corn starch, consisting mainly of maltose, maltotriose, and branched α-limit dextrins, as substrate in the presence of four different inhibitors: acarbose and three sulfonium ion compounds. The IC(50) values show that the four α-glucosidase subunits could be differentially inhibited. The results support the prospect of controlling starch digestion rates to induce slow glucose release through the toggling of activities of the mucosal α-glucosidases by selective enzyme inhibition. This approach could also be used to probe associated metabolic diseases.     

Caspase-11 Controls Interleukin-1β Release through Degradation of TRPC1

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Developing In Vitro–In Vivo Correlation of Risperidone Immediate Release Tablet

The present study was aimed to predict the absorption profile of a risperidone immediate release tablet (IR) and to develop the level A in vitro–in vivo correlation (IVIVC) of the drug using the gastrointestinal simulation based on the advanced compartmental absorption and transit model implemented in GastroPlus™. Plasma concentration data, physicochemical, and pharmacokinetic properties of the drug were used in building its absorption profile in the gastrointestinal tract. Since the fraction absorbed of risperidone in simulation was more than 90% with low water solubility, the drug met the criteria of class II of the Biopharmaceutics Classification System. The IVIVC was developed based on the model built using the plasma data and the in vitro dissolution data in several dissolution media based on the Japanese Guideline for Bioequivalence Studies of Generic Products. The gastrointestinal absorption profile of risperidone was successfully predicted. A level A IVIVC was also successfully developed in all dissolution media with percent prediction error for Cmax and the area under the curve less than 10% for both reference and test drug.Key words: GastroPlus™, immediate release tablet, in vitro–in vivo correlation, risperidone     

Release of ATP from avian Müller glia cells in culture

ATP can be released from neurons and act as a neuromodulator in the nervous system. Besides neurons, cortical astrocytes also are capable of releasing ATP from acidic vesicles in a Ca(2+)-dependent way. In the present work, we investigated the release of ATP from Müller glia cells of the chick embryo retina by examining quinacrine staining and by measuring the extracellular levels of ATP in purified Müller glia cultures. Our data revealed that glial cells could be labeled with quinacrine, a reaction that was prevented by incubation of the cells with 1μM bafilomycin A1 or 2μM Evans blue, potent inhibitors of vacuolar ATPases and of the vesicular nucleotide transporter, respectively. Either 50mM KCl or 1mM glutamate was able to decrease quinacrine staining of the cells, as well as to increase the levels of ATP in the extracellular medium by 77% and 89.5%, respectively, after a 5min incubation of the cells. Glutamate-induced rise in extracellular ATP could be mimicked by 100μM kainate (81.5%) but not by 100μM NMDA in medium without MgCl(2) but with 2mM glycine. However, both glutamate- and kainate-induced increase in extracellular ATP levels were blocked by 50μM of the glutamatergic antagonists DNQX and MK-801, suggesting the involvement of both NMDA and non-NMDA receptors. Extracellular ATP accumulation induced by glutamate was also blocked by incubation of the cells with 30μM BAPTA-AM or 1μM bafilomycin A1. These results suggest that glutamate, through activation of both NMDA and non-NMDA receptors, induces the release of ATP from retinal Müller cells through a calcium-dependent exocytotic mechanism.     

Sustained Release of Amoxicillin from Ethyl Cellulose-Coated Amoxicillin/Chitosan–Cyclodextrin-Based Tablets

Sustained release mucoadhesive amoxicillin tablets with tolerance to acid degradation in the stomach were studied. The sustained-release tablets of amoxicillin were prepared from amoxicillin coated with ethyl cellulose (EC) and then formulated into tablets using chitosan (CS) or a mixture of CS and beta-cyclodextrin (CD) as the retard polymer. The effects of various (w/w) ratios of EC/amoxicillin, the particle sized of EC coated amoxicillin and the different (w/w) ratios of CS/CD for the retard polymer, on the amoxicillin release profile were investigated. The physicochemical properties of the EC coated amoxicillin particles and tablets were determined by scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, and differential scanning calorimetry. The result showed that the release profiles of amoxicillin were greatly improved upon coating with EC, while the inclusion of CD to the CS retardant additionally prolonged the release of the drug slightly. Overall, a sustained release of amoxicillin was achieved using amoxicillin coated with EC at a (w/w) ratio of 1:1 and a particle size of 75–100 μm. Therefore, the tablet formulation of amoxicillin may be an advantageous alternative as an orally administered sustained-release formulation for the treatment of peptic ulcers.     

Costs and Consequences of Using Interferon-γ Release Assays for the Diagnosis of Active Tuberculosis in India

Background

There is growing concern that interferon-γ release assays (IGRAs) are being used off-label for the diagnosis of active tuberculosis (TB) disease in many high-burden settings, including India, where the background prevalence of latent TB infection is high. We analyzed the costs and consequences of using IGRAs for the diagnosis of active TB in India from the perspective of the Indian TB control sector.

Methods and Findings

We constructed a decision analytic model to estimate the incremental cost and effectiveness of IGRAs for the diagnosis of active TB in India. We compared a reference scenario of clinical examination and non-microbiological tests against scenarios in which clinical diagnosis was augmented by the addition of either sputum smear microscopy, IGRA, or Xpert MTB/RIF. We examined costs (in 2013 US dollars) and consequences from the perspective of the Indian healthcare sector. Relative to sputum smear microscopy, use of IGRA for active TB resulted in 23,700 (95% uncertainty range, UR: 3,800 – 38,300) additional true-positive diagnoses, but at the expense of 315,700 (95% UR: 118,300 – 388,400) additional false-positive diagnoses and an incremental cost of US$49.3 million (95% UR: $34.9 – $58.0 million) (2.9 billion Indian Rupees). Relative to Xpert MTB/RIF (including the cost of treatment for drug resistant TB), use of IGRA led to 400 additional TB cases treated (95% UR: [-8,000] – 16,200), 370,600 (95% UR: 252,200 – 441,700) more false-positive diagnoses, 70,400 (95% UR: [-7,900] – 247,200) fewer disability-adjusted life years averted, and US$14.6 million (95%UR: [-$7.2] – $28.7 million) (854 million Indian Rupees) in additional costs.

Conclusion

Using IGRAs for diagnosis of active TB in a setting like India results in tremendous overtreatment of people without TB, and substantial incremental cost with little gain in health. These results support the policies by WHO and Standards for TB Care in India, which discourage the use of IGRAs for the diagnosis of active TB in India and similar settings.     

Prejunctional α2-Adrenoceptors and Peroxide-Induced Potentiation of Norepinephrine Release from the Bovine Iris

Peroxides can enhance field-stimulated [³H]norepinephrine ([³H]NE) release in isolated irides from several mammalian species. In the present study, we investigated the role of prejunctional ₂-adrenoceptors in peroxide-induced potentiation of sympathetic neurotransmission in bovine isolated irides. Isolated hemi-irides were incubated in a Krebs buffered-solution containing [³H]NE and prepared for studies of neurotransmitter release using the superfusion method. ₂-Adrenoceptor agonists, oxymetazoline, UK-14304 and clonidine inhibited field-stimulated [³H]NE overflow without affecting basal tritium efflux. Pretreatment of tissues with H₂O₂ (300 M) had no effect on inhibition of evoked [³H]NE release caused by the ₂-adrenergic agonists. However, H₂O₂ (300 M) caused significant (P < 0.01) leftward shifts of excitatory concentration-response curves to yohimbine (10 nM–1 M). In contrast, yohimbine (1 M) did not prevent the enhancement of evoked [³H]NE overflow induced by H₂O₂ (300 M). In conclusion, excitatory effects of peroxides on sympathetic neurotransmission in bovine irides are not mediated by prejunctional ₂-adrenoceptors.