Encapsulation of Lipophilic Bioactive Molecules by Microchannel Emulsification

Currently, much effort is being invested in novel formulations of bioactive molecules, such as emulsions, for pharmaceutical, food, and cosmetic applications. Therefore, methods to produce emulsions with controlled-size droplets of uniform size distribution have been developed. On this concern, a microfluidic device called the microchannel (MC) was used in this work for emulsification. This is a novel method for producing monodispersed emulsion droplets with very narrow droplet size distribution and low energy input, due to the spontaneous droplet generation basically driven by the interfacial tension, unlike other conventional emulsification processes. This technology provides the formulation of oil-in-water (O/W) emulsions containing lipophilic active molecules with increased bioavailability, which may be readily absorbed by the human body. MC emulsification enables the preparation of highly monodispersed O/W emulsions, which may be applied as enhancer on active molecules delivery systems, as well as in foodstuff. In this study, formulations of O/W emulsions loaded with bioactive molecules, such as β-carotene and γ-oryzanol, were prepared by the MC emulsification process. Refined soybean oil containing the dissolved lipophilic molecule and either sugar ester or gelatin solution (1 wt.%) were used as the dispersed and continuous phases, respectively. The emulsification process conducted using the asymmetric straight-through MC plate enabled the production of monodispersed O/W emulsions, resulting in β-carotene-loaded O/W emulsions with average droplet size (d _av) of 27.6 μm and coefficient of variation (CV) of 2.3% and γ-oryzanol-loaded droplets with d _av of 28.8 μm and CV of 3.8%. The highly monodisperse β-carotene-loaded droplets were physically stable throughout the storage period observed, resulting in droplets with d _av 28.2 μm and CV of 2.9% after 4 months storage in darkness at 5 °C. Single micrometer-sized monodisperse emulsions loaded with β-carotene were successfully formulated using the grooved MC emulsification, resulting in droplets with d _av of 9.1 μm and CV of 6.2%. This work was funded by The Ministry of Agriculture, Forestry and Fisheries of Japan, through the Food Nanotechnology Project, and the Japan Society for the Promotion of Science.     

Ebselen is a potent non-competitive inhibitor of extracellular nucleoside diphosphokinase

Nucleoside di- and triphosphates and adenosine regulate several components of the mucocilairy clearance process (MCC) that protects the lung against infections, via activation of epithelial purinergic receptors. However, assessing the contribution of individual nucleotides to MCC functions remains difficult due to the complexity of the mechanisms of nucleotide release and metabolism. Enzymatic activities involved in the metabolism of extracellular nucleotides include ecto-ATPases and secreted nucleoside diphosphokinase (NDPK) and adenyl kinase, but potent and selective inhibitors of these activities are sparse. In the present study, we discovered that ebselen markedly reduced NDPK activity while having negligible effect on ecto-ATPase and adenyl kinase activities. Addition of radiotracer [γ ³²P]ATP to human bronchial epithelial (HBE) cells resulted in rapid and robust accumulation of [³²P]-inorganic phosphate (³²Pi). Inclusion of UDP in the incubation medium resulted in conversion of [γ ³²P]ATP to [³²P]UTP, while inclusion of AMP resulted in conversion of [γ ³²P]ATP to [³²P]ADP. Ebselen markedly reduced [³²P]UTP formation but displayed negligible effect on ³²Pi or [³²P]ADP accumulations. Incubation of HBE cells with unlabeled UTP and ADP resulted in robust ebselen-sensitive formation of ATP (IC₅₀ = 6.9 ± 2 μM). This NDPK activity was largely recovered in HBE cell secretions and supernatants from lung epithelial A549 cells. Kinetic analysis of NDPK activity indicated that ebselen reduced the V _max of the reaction (K _i = 7.6 ± 3 μM), having negligible effect on K _M values. Our study demonstrates that ebselen is a potent non-competitive inhibitor of extracellular NDPK.     

Comparison of metabolite levels in callus of <Emphasis Type="Italic">Tecoma stans</Emphasis> (L.) Juss. ex Kunth. cultured in photoperiod and darkness

Tecoma stans is a tropical plant from the Americas. Antioxidant activity and both phenolic compound and flavonoid total content were determined for callus tissue of T. stans cultured in either a set photoperiod or in darkness. Callus lines from three explant types (hypocotyls, stem, and leaf) were established on B5 culture medium supplemented with 0.5 μM 2,4-D and 5.0 μM kinetin. While leaf-derived callus grew slower under a 16-h photoperiod (specific growth rate, μ = 0.179 d⁻¹, t _D = 3.9 d) than in darkness (μ = 0.236 d⁻¹, t _D = 2.9 d), it accumulated the highest amount (p < 0.05) of both phenolics (86.6 ± 0.01 mg gallic acid equivalents/g) and flavonoids (339.6 ± 0.06 mg catechin equivalents/g). Similarly, antioxidant activity was significantly higher (p < 0.05) when callus was cultured in period light than when grown in extended darkness. Antioxidant activity measured with a 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS)-based assay was 350.5 ± 15.8 mmol Trolox/g extract for callus cultured under a defined photoperiod compared to 129.1 ± 7.5 mmol Trolox/g extract from callus cultured in darkness. Content of phenolic compounds and flavonoids was in agreement with a better antioxidant power (EC₅₀ = 450 μg extract/mg 1,1-diphenyl-2-picrylhydrazyl) and antiradical efficiency. Results of the present study show that calli of T. stans are a source of compounds with antioxidant activity that is favored by culture under a set photoperiod.     

Improved Bioavailability of Poorly Water-Soluble Drug Curcumin in Cellulose Acetate Solid Dispersion

Curcumin (Cur), one of the most widely used natural active constituents with a great variety of beneficial biological and pharmacological activities, is a practically water-insoluble substance with a short biologic half-life. The aim of this study was to develop a sustained-release solid dispersion by employing water-insoluble carrier cellulose acetate for solubility enhancement, release control, and oral bioavailability improvement of Cur. Solid dispersions were characterized by solubility, in vitro drug release, Fourier transform infrared spectroscopy, X-ray diffractometry, and differential scanning calorimetry studies. The in vivo performance was assessed by a pharmacokinetic study. Solid-state characterization techniques revealed the amorphous nature of Cur in solid dispersions. Solubility/dissolution of Cur was enhanced in the formulations in comparison with pure drug. Sustained-release profiles of Cur from the solid dispersions were ideally controlled in vitro up to 12 h. The optimized formulation provided an improved pharmacokinetic parameter (C _max = 187.03 ng/ml, t _max = 1.95 h) in rats as compared with pure drug (C _max = 87.06 ng/ml, t _max = 0.66 h). The information from this study suggests that the developed solid dispersions successfully enhanced the solubility and sustained release of poorly water-soluble drug Cur, thus improving its oral bioavailability effectively.     

Isochorismate hydroxymutase from a cell-suspension culture of Galium mollugo L.

Isochorismate hydroxymutase (i.e. isochorismate synthase, EC 5.4.99.6) was purified from an anthraquinone-producing cell-suspension culture of Galium mollugo L. Although attempts to stabilize the labile enzyme met with little success, a substantial increase in enzyme activity was observed in the presence of glycine betaine (500 mM). Column chromatography on solid supports other than diethylaminoethyl (DEAE)-Sephacel, Phenylsepharose Cl-4B or Cibacron Blue 3G-A did not give active enzyme preparations. In spite of these drawbacks the enzyme was purified 573-fold. Enzyme activity depended strictly on the presence of Mg²⁺. Kinetic data for chorismate in the forward reaction (K _m = 807 μM, V _max = 6.2 pkat · mg⁻¹) and for isochorismate in the reverse reaction (K _m = 675 μM, V _max = 5.9 pkat · mg⁻¹) were determined. Received: 18 November 1996 / Accepted: 28 December 1996     

Influence of Interfacial Composition on in Vitro Digestibility of Emulsified Lipids: Potential Mechanism for Chitosan's Ability to Inhibit Fat Digestion

The objective of this study was to investigate the influence of interfacial composition and electrical charge on the in vitro digestion of emulsified fats by pancreatic lipase. An electrostatic layer-by-layer deposition technique was used to prepare corn oil-in-water emulsions (3 wt% oil) that contained droplets coated by (1) lecithin, (2) lecithin–chitosan, or (3) lecithin–chitosan–pectin. Pancreatic lipase (1.6 mg mL⁻¹) and/or bile extract (5.0 mg mL⁻¹) were added to each emulsion, and the particle charge, droplet aggregation, and free fatty acids released were measured. In the presence of bile extract, the amount of fatty acids released per unit amount of emulsion was much lower in the emulsions containing droplets coated by lecithin–chitosan (38 ± 16 μmol mL⁻¹) than those containing droplets coated by lecithin (250 ± 70 μmol mL⁻¹) or lecithin–chitosan–pectin (274 ± 80 μmol mL⁻¹). In addition, there was much more extensive droplet aggregation in the lecithin–chitosan emulsion than in the other two emulsions. We postulated that lipase activity was reduced in the lecithin–chitosan emulsion as a result of the formation of a relatively thick cationic layer around each droplet, as well as the formation of large flocs, which restricted the access of the pancreatic lipase to the lipids within the droplets. Our results also suggest that droplets initially coated by a lecithin–chitosan–pectin layer did not inhibit lipase activity, which may have been because the chitosan–pectin desorbed from the droplet surfaces thereby allowing the enzyme to reach the lipids; however, further work is needed to establish this. This information could be used to create food emulsions with low caloric level, or to optimize diets for individuals with lipid digestion problems.     

Rheological Behavior of Food Emulsions Mixed with Saliva: Effect of Oil Content, Salivary Protein Content, and Saliva Type

In this paper, we studied the effect of saliva on the rheological properties of β-lactoglobulin- and lysozyme-stabilized emulsions, prepared at pH = 6.7 in relation to variation of emulsions- and saliva-related parameters. The effect of oil–volume fraction (2.5% w/w to 10% w/w), salivary protein concentration (0.1 to 0.8 mg ml⁻¹), and the use of both stimulated and unstimulated saliva was investigated. Viscosity and storage modulus were measured before (η _emul and G′_emul, respectively) and after addition of saliva (η _mix and G′_mix). To better estimate the changes due to saliva-induced flocculation of the emulsions, the ratios η _mix/η _emul, G′_mix/G′_emul were calculated. In addition, tan δ (=the ratio of the loss and storage moduli) was investigated to evaluate the viscoelastic behavior of the emulsion/saliva mixtures. Increasing the oil–volume fraction and salivary protein concentration resulted in an increase in η _mix/η _emul and G′_mix/G′_emul, while a decrease in tan δ of the emulsion/saliva mixtures is occurring. When compared with unstimulated saliva, mixing β-lactoglobulin-stabilized emulsions with stimulated saliva led to a reduction in η _mix/η _emul and G′_mix/G′_emul, and an augment of tan δ at all measured deformations. In case of lysozyme-stabilized emulsions, the use of stimulated saliva increased G′_mix/G′_emul for γ < 3 when compared to unstimulated saliva. The effect of stimulated saliva on the η _mix/η _emul and tan δ in this mixture is similar to that of unstimulated saliva. These results indicate that the influence of stimulated saliva on the rheological parameters of emulsion/saliva mixtures largely depends on the type of emulsions. To conclude, our findings demonstrate that the rheological behavior of emulsions upon mixing with saliva is greatly affected by both saliva and emulsion properties.     

Effects of saturated fatty acids on amylase release from exocrine pancreatic segments of sheep,rats, hamsters,field voles and mice

 Stimulatory effects of saturated fatty acids consisting of 4 (butyrate), 8 (octanoate), 12 (laurate) and 16 (palmitate) carbon atoms, as well as acetylcholine on pancreatic amylase release were assessed in tissue segments isolated from sheep, rats, hamsters, field voles and mice. The amount of amylase release induced by the fatty acids (1 μmol ⋅ l^-1 to 10 mml ⋅ l^-1) and by acetylcholine (10 nmol ⋅ l^-1 to 100 μmol ⋅ l^-1) increased in a concentration-dependent manner, and the maximum response in response to the fatty acids was obtained at the maximal dose used. The maximum increase in amylase release in response to butyrate or octanoate was highly and significantly (r=0.974, P<0.001) dependent on the log value of the mean body mass in the following order: sheep>rats>hamsters>field voles>mice. On the other hand, the response to laurate and palmitate was variable among animal species. Addition of atropine (1.4 μmol ⋅ l^-1) to the medium did not reduce the responses to octanoate stimulation, but significantly reduced acetylcholineinduced responses, implying that the effects of the fatty acids were not mediated through activation of muscarinic acetylcholine receptors. Reduction of calcium ion concentration in the medium significantly inhibited the responses induced by the fatty acids and acetylcholine, suggesting that amylase release depends on extracellular calcium ions. Accepted: 14 May 1996     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Identification of novel 5-hydroxy-1<Emphasis Type="Italic">H</Emphasis>-indole-3-carboxylates with anti-HBV activities based on 3D QSAR studies

Infection with hepatitis B virus (HBV) is a major cause of liver diseases such as cirrhosis and hepatocellular carcinoma. In our previous studies, we identified indole derivatives that have anti-HBV activities. In this study, we optimize a series of 5-hydroxy-1H-indole-3-carboxylates, which exhibited potent anti-HBV activities, using three-dimensional quantitative structure-activity relationship (3D QSAR) studies with comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The lowest energy conformation of compound 3, which exhibited the most potent anti-HBV activity, obtained from systematic search was used as the template for alignment. The best predictions were obtained with the CoMFA standard model (q ² = 0.689, r ² = 0.965, SEE = 0.082, F = 148.751) and with CoMSIA combined steric, electrostatic, hydrophobic and H-bond acceptor fields (q ² = 0.578, r ² = 0.973, SEE = 0.078, F = 100.342). Both models were validated by an external test set of six compounds giving satisfactory prediction. Based on the clues derived from CoMFA and CoMSIA models and their contour maps, another three compounds were designed and synthesized. Pharmacological assay demonstrated that the newly synthesized compounds possessed more potent anti-HBV activities than before (IC₅₀: compound 35a is 3.1 μmol/l, compound 3 is 4.1 μmol/l). Combining the clues derived from the 3D QSAR studies and from further validation of the 3D QSAR models, the activities of the newly synthesized indole derivatives were well accounted for. Furthermore, this showed that the CoMFA and CoMSIA models proved to have good predictive ability.     

Biodegradation kinetics of monoterpenes in liquid and soil-slurry systems

Batch experiments were conducted to evaluate the biodegradation rates of limonene, α-pinene, γ-terpinene, terpinolene and α-terpineol at 23 °C under aerobic conditions. Biodegradation was demonstrated by the depletion of monoterpene mass, CO₂ production and a corresponding increase in biomass. Monoterpene degradation in liquid cultures devoid of soil followed Monod kinetics. The maximum specific growth rate (μ_max) was 0.02 h⁻¹ and 0.06 h⁻¹ and the half-velocity constant (K _s ) varied from 32 mg/l to 3 mg/l for the limonene and α-terpineol respectively. The recovery of monoterpenes by solvent extraction from autoclaved and azide-amended soil-slurry samples decreased over time and ranged from 69% to 73% for 120 h of incubation period. Although a significant fraction of monoterpene hydrocarbon could not be extracted, mineralization of these compounds in the soil-slurry systems took place, as shown by CO₂ production. The soil-normalized degradation rates for the hydrocarbon monoterpenes ranged from 0.6 μg g⁻¹ h⁻¹ to 2.1 μg g⁻¹ h⁻¹. A kinetic model – which combined monoterpene biodegradation in the liquid phase and net desorption – was developed and applied to data obtained from soil-slurry assays. Received: 10 September 1996 / Received revision: 16 December 1996 / Accepted: 10 January 1997     

Cometabolic biodegradation of methyl t-butyl ether by Pseudomonas aeruginosa grown on pentane

A bacterial strain identified as Pseudomonas aeruginosa was isolated from a soil consortium able to mineralize pentane. P. aeruginosa could metabolize methyl t-butyl ether (MTBE) in the presence of pentane as the sole carbon and energy source. The carbon balance for this strain, grown on pentane, was established in order to determine the fate of pentane and the growth yield (0.9 g biomass/g pentane). An inhibition model for P. aeruginosa grown on pentane was proposed. Pentane had an inhibitory effect on growth of P. aeruginosa, even at a concentration as low as 85 μg/l. This resulted in the calculation of the following kinetic parameters (μ_max = 0.19 h⁻¹, K _s = 2.9 μg/l, K _i = 3.5 mg/l). Finally a simple model of MTBE degradation was derived in order to predict the quantity of MTBE able to be degraded in batch culture in the presence of pentane. This model depends only on two parameters: the concentrations of pentane and MTBE. Received: 16 July 1998 / Received revision: 11 November 1998 / Accepted 31 November 1998     

Effect of Polyglycerol Esters of Fatty Acids on the Physicochemical Properties and Stability of β-Carotene Emulsions during Digestion in Simulated Gastric Fluid

The effects of six different polyglycerol esters of fatty acids (PGEs) and two different particle sizes produced using various processing parameters on the physicochemical properties and stability of the β-carotene emulsions during digestion in simulated gastric fluid (SGF) were investigated. β-Carotene emulsions were prepared by high-pressure homogenization using β-carotene (0.1% w/w) in soybean oil as the oil phase and 1% (w/w) PGE in Milli-Q water as the water phase. The particle size of β-carotene emulsions was measured by a laser diffraction technique, and the stability of emulsions was interpreted in terms of the increase in particle size and span value of emulsion droplets and the retention of β-carotene during digestion in SGF. The average particle size ranges of emulsions were 0.17 to 0.27 μm for fine emulsions and 1.16 to 1.59 μm for coarse emulsions. In the prepared β-carotene emulsions, the particle size decreased with increasing polymerization of the glycerol in PGEs, and the higher polymerization of the glycerol also increased the stability of emulsions during digestion in SGF. Although the β-carotene content in the emulsions significantly decreased with increasing digestion period, loss of β-carotene was more severe in unstable emulsions than in stable emulsions, suggesting that the particles incorporated into droplets could provide some protective barrier for decreasing the β-carotene degradation. Therefore, β-carotene emulsions stabilized by PGEs with high polymerization of the glycerol may be useful for further applications in food and drug formulations. Decaglycerol monooleate (MO750) was demonstrated to be the most effective emulsifier in stabilizing β-carotene emulsions in this study.     

Pyranose 2-oxidase from Phanerochaete chrysosporium– further biochemical characterisation

Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (k _cat) of 111.0 s⁻¹ and an affinity constant (K _m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k _cat/K _m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k _cat decreased to 60.9 s⁻¹ and the K _m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl₂, AgNO₃ and ZnCl₂, and 50% by lead acetate, CuCl₂ and MnCl₂. Received: 28 August 1996 / Received revision: 25 November 1996 / Accepted: 29 November 1996     

Sensitive and rapid HPLC quantification of tenofovir from hyaluronic acid-based nanomedicine

The purpose of this study was to develop and validate a rapid, sensitive, and specific reversed-phase high-performance liquid chromatography method for the quantitative determination of native tenofovir (TNF) for various applications. Different analytical performance parameters such as linearity, precision, accuracy, limit of quantification (LOQ), limit of detection (LOD), and robustness were determined according to International Conference on Harmonization (ICH) guidelines. A Bridge™ C18 column (150 × 4.6 mm, 5 μm) was used as stationary phase. The retention time of TNF was 1.54 ± 0.03 min (n = 6). The assay was linear over the concentration range of 0.1–10 μg/mL. The proposed method was sensitive with LOD and LOQ values equal to 50 and 100 ng/mL, respectively. The method was accurate with percent mean recovery from 95.41% to 102.90% and precise as percent RSD (relative standard deviation) values for intra-day, and inter-day precision were less than 2%. This method was utilized for the estimation of molar absorptivity of TNF at 259 nm (ε ₂₅₉ = 12,518 L/mol/cm), calculated from linear regression analysis. The method was applied for determination of percentage of encapsulation efficiency ( 22.93 ± 0.04%), drug loading (12.25 ± 1.03%), in vitro drug release profile in the presence of enzyme (43% release in the first 3 h) and purification analysis of hyaluronic acid-based nanomedicine.     

Recent improvements in the development of A<Subscript>2B</Subscript> adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis.     

Recent improvements in the development of A2B adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis. This article has previously been published in issue 4/4, under doi:.     

Production of flavour compounds by yogurt starter cultures

The present work studied the production of carbonyl compounds and saturated volatile free fatty acids by pure cultures of Streptococcus thermophilus and Lactobacillus bulgaricus, and by starter cultures for Bulgarian yogurt during cultivation and cooling. The mixed cultures formed volatile aromatic compounds more actively than the pure cultures. A guiding factor in the preparation of the starter cultures was the biochemical activity of Lactobacillus bulgaricus in synthesizing the major carbonyl compounds, acetaldehyde, diacetyl and the volatile fatty acids C₂–C₁₀. The activity of the yogurt cultures in synthesizing carbonyl compounds was at its highest during milk coagulation and cooling, up to 7 h. However, maximum concentration was reached by 22–31 h. In the cooled 22–h starter cultures, acetaldehyde predominated (1415.0–1734.2 μg per 100 g) followed by diacetyl (165.0–202.0 μg per 100 g), acetoin (170.0–221.0 μg per 100 g), acetone (66.0–75.5 μg per 100 g), ethanol (58.0 μg per 100 g), and butanone-2 (3.6–3.8 μg per 100 g). The thermophilic streptococcus and lactobacillus cultures, and the starter cultures contained predominantly acetic, butyric and caproic acids. Received 19 June 1997/ Accepted in revised form 10 January 1998     

Valsartan Orodispersible Tablets: Formulation, <Emphasis Type="Italic">In vitro</Emphasis>/<Emphasis Type="Italic">In vivo</Emphasis> Characterization

Valsartan orodispersible tablets have been developed at 40-mg dose, with the intention of facilitating administration to patients experiencing problems with swallowing and hopefully, improving its poor oral bioavailability. Work started with selecting drug compatible excipients depending on differential scanning calorimetric analysis. A 3³ full factorial design was adopted for the optimization of the tablets prepared by freeze-drying technique. The effects of the filler type, the binder type, and the binder concentration were studied. The different tablet formulas were characterized for their physical properties, weight variation, disintegration time, surface properties, wetting properties, and in vitro dissolution. Amongst the prepared 27 tablet formulas, formula number 6 (consisting of 4:6 valsartan:mannitol and 2% pectin) was selected to be tested in vivo. Oral bioavailability of two 40 mg valsartan orodispersible tablets was compared to the conventional commercial tablets after administration of a single dose to four healthy volunteers. Valsartan was monitored in plasma by high-performance liquid chromatography. The apparent rate of absorption of valsartan from the prepared tablets (C _max = 2.879 μg/ml, t _max = 1.08 h) was significantly higher than that of the conventional tablets (C _max = 1.471 μg/ml, t _max = 2.17 h), P ≤ 0.05. The relative bioavailability calculated as the ratio of mean total area under the plasma concentration–time curve for the orodispersible tablets relative to the conventional ones was 135%. The results of the in vivo study revealed that valsartan orodispersible tablets would be advantageous with regards to improved patient compliance, rapid onset of action, and increase in bioavailability.     

Discovery of nigerose phosphorylase from Clostridium phytofermentans

A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of d-glucose and β-d-glucose 1-phosphate (β-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k _cat = 67 s⁻¹ and K _m = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using d-glucose as the acceptor and β-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on d-xylose, 1,5-anhydro-d-glucitol, d-galactose, and methyl-α-d-glucoside. All major products were α-1,3-glucosyl disaccharides, although the reaction with d-xylose and methyl-α-d-glucoside produced significant amounts of α-1,2-glucosides as by-products. We propose 3-α-d-glucosyl-d-glucose:phosphate β-d-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.