Drug-Carrier Miscibility in Solid Dispersions of Glibenclamide and a Novel Approach to Enhance Its Solubility Using an Effervescent Agent.

The present research aims to investigate the miscibility, physical stability, solubility, and dissolution rate of a poorly water-soluble glibenclamide (GLB) in solid dispersions (SDs) with hydrophilic carriers like PEG-1500 and PEG-50 hydrogenated palm glycerides (Acconon). Mathematical theories such as Hansen solubility parameters, Flory Huggins theory, Gibbs free energy, and the in silico molecular dynamics simulation study approaches were used to predict the drug-carrier miscibility. To increase the solubility further, the effervescence technique was introduced to the conventional solid dispersions to prepare effervescent solid dispersions (ESD). Solid dispersions (SDs) were prepared by microwave, solvent evaporation, lyophilization, and hot melt extrusion (HME) techniques and tested for different characterization parameters. The theoretical and in silico parameters suggested that GLB would show good miscibility with the selected carriers under certain conditions. Intermolecular hydrogen bonding between the drug and carrier(s) was confirmed by Fourier transform infrared spectroscopy and proton nuclear magnetic resonance spectroscopy. Solid-state characterizations like powder X-ray diffraction, differential scanning calorimetry, and microscopy confirm the amorphous nature of SDs. The addition of the effervescent agent improved the amorphous nature, due to which the solubility and drug release rate was increased. In vitro and ex vivo intestinal absorption studies showed improved flux and permeability than the pure drug, suggesting an enhanced drug delivery. The GLB solubility, dissolution, and stability were greatly enhanced by the SD and ESD technology.

Analysis of molecular mechanisms of ATP synthesis from the standpoint of the principle of electrical neutrality.

Theories of biological energy coupling in oxidative phosphorylation (OX PHOS) and photophosphorylation (PHOTO PHOS) are reviewed and applied to ATP synthesis by an experimental system containing purified ATP synthase reconstituted into liposomes. The theories are critically evaluated from the standpoint of the principle of electrical neutrality. It is shown that the obligatory requirement to maintain overall electroneutrality of bulk aqueous phases imposes strong constraints on possible theories of energy coupling and molecular mechanisms of ATP synthesis. Mitchell's chemiosmotic theory is found to violate the electroneutrality of bulk aqueous phases and is shown to be untenable on these grounds. Purely electroneutral mechanisms or mechanisms where the anion/countercation gradient is dissipated or simply flows through the lipid bilayer are also shown to be inadequate. A dynamically electrogenic but overall electroneutral mode of ion transport postulated by Nath's torsional mechanism of energy transduction and ATP synthesis is shown to be consistent both with the experimental findings and the principle of electrical neutrality. It is concluded that the ATP synthase functions as a proton-dicarboxylic acid anion cotransporter in OX PHOS or PHOTO PHOS. A logical chemical explanation for the selection of dicarboxylic acids as intermediates in OX PHOS and PHOTO PHOS is suggested based on the pioneering classical thermodynamic work of Christensen, Izatt, and Hansen. The nonequilibrium thermodynamic consequences for theories in which the protons originate from water vis-a-vis weak organic acids are compared and contrasted, and several new mechanistic and thermodynamic insights into biological energy transduction by ATP synthase are offered. These considerations make the new theory of energy coupling more complete, and lead to a deeper understanding of the molecular mechanism of ATP synthesis.

Double differential cross sections for proton induced electron emission from molecular analogues of DNA constituents for energies in the Bragg peak region.

For track structure simulations in the Bragg peak region, measured electron emission cross sections of DNA constituents are required as input for developing parameterized model functions representing the scattering probabilities. In the present work, double differential cross sections were measured for the electron emission from vapor-phase pyrimidine, tetrahydrofuran, and trimethyl phosphate that are structural analogues to the base, the sugar, and the phosphate residue of the DNA, respectively. The range of proton energies was from 75 keV to 135 keV, the angles ranged from 15° to 135°, and the electron energies were measured from 10 eV to 200 eV. Single differential and total electron emission cross sections are derived by integration over angle and electron energy and compared to the semi-empirical Hansen-Kocbach-Stolterfoht (HKS) model and a quantum mechanical calculation employing the first Born approximation with corrected boundary conditions (CB1). The CB1 provides the best prediction of double and single differential cross section, while total cross sections can be fitted with semi-empirical models. The cross sections of the three samples are proportional to their total number of valence electrons.

The alanine detector in BNCT dosimetry: dose response in thermal and epithermal neutron fields.

PURPOSE: The response of alanine solid state dosimeters to ionizing radiation strongly depends on particle type and energy. Due to nuclear interactions, neutron fields usually also consist of secondary particles such as photons and protons of diverse energies. Various experiments have been carried out in three different neutron beams to explore the alanine dose response behavior and to validate model predictions. Additionally, application in medical neutron fields for boron neutron capture therapy is discussed. METHODS: Alanine detectors have been irradiated in the thermal neutron field of the research reactor TRIGA Mainz, Germany, in five experimental conditions, generating different secondary particle spectra. Further irradiations have been made in the epithermal neutron beams at the research reactors FiR 1 in Helsinki, Finland, and Tsing Hua open pool reactor in HsinChu, Taiwan ROC. Readout has been performed with electron spin resonance spectrometry with reference to an absorbed dose standard in a (60)Co gamma ray beam. Absorbed doses and dose components have been calculated using the Monte Carlo codes fluka and mcnp. The relative effectiveness (RE), linking absorbed dose and detector response, has been calculated using the Hansen & Olsen alanine response model. RESULTS: The measured dose response of the alanine detector in the different experiments has been evaluated and compared to model predictions. Therefore, a relative effectiveness has been calculated for each dose component, accounting for its dependence on particle type and energy. Agreement within 5% between model and measurement has been achieved for most irradiated detectors. Significant differences have been observed in response behavior between thermal and epithermal neutron fields, especially regarding dose composition and depth dose curves. The calculated dose components could be verified with the experimental results in the different primary and secondary particle fields. CONCLUSIONS: The alanine detector can be used without difficulty in neutron fields. The response has been understood with the model used which includes the relative effectiveness. Results and the corresponding discussion lead to the conclusion that application in neutron fields for medical purpose is limited by its sensitivity but that it is a useful tool as supplement to other detectors and verification of neutron source descriptions.

XYLH encodes a xylose/H+ symporter from the highly related yeast species Debaryomyces fabryi and Debaryomyces hansenii.

The closely related yeasts Debaryomyces fabryi and Debaryomyces hansenii are excellent xylose consumers. We previously described the activity of a high-affinity xylose/H(+) symport from an industrial strain of D. hansenii subsequently reclassified as D. fabryi. We now report the identification of the gene encoding this permease, AY347871.2. This was retrieved from D. fabryi gDNA using a degenerate primer PCR strategy, based on conserved regions from the amino acid sequences of three well-characterized bacterial xylose/H(+) symporters. This sequence is 86% identical to another, DEHA2C11374p from D. hansenii type strain. DEHA2C11374p was conceptually ascribed to the major facilitator superfamily. The putative amino acid sequence of AY347871.2 and DEHA2C11374p presented a hydrophobicity pattern compatible with plasma membrane proteins. The last was functionally expressed in Saccharomyces cerevisiae. The sensitivity of transport activity to a protonophore confirmed its dependence on proton motive force, as expected from a symporter. We named D. fabryi AY347871.2 and D. hansenii DEHA2C11374p as XYLH from Xylose/H(+) symport. Based on the very high similarity, we suggested that Scheffersomyces stipitis Xut3 and Aspergillus nidulans AN8400.2 may also encode xylose high-affinity permeases.

An experimental charge density of HEPES.

We report the experimental charge density of HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], which is a common buffering agent. The structure was refined using the Hansen-Coppens formalism. The ability of the HEPES molecule to form stable intermolecular interactions and intermolecular hydrogen bonds in the crystal structure is discussed in terms of its buffering properties. The protonation mode observed in the crystal structure is different from that expected in solution, suggesting that additional factors must be taken into consideration in order to explain the solution properties of the compound. As ordered HEPES molecules are found in the active sites of proteins in several protein crystal structures, our results will allow for quantitative analysis of the electrostatic potential of the interacting surfaces of those proteins.

Monte Carlo simulation and analysis of proton energy-deposition patterns in the Bragg peak.

The spatial pattern of energy depositions is crucial for understanding the mechanisms that modify the relative biological effectiveness of different radiation qualities. In this paper, we present data on energy-deposition properties of mono-energetic protons (1-20 MeV) and their secondary electrons in liquid water. Proton-impact ionization was described by means of the Hansen-Kocbach-Stolterfoht doubly differential cross section (DDCS), thus modelling both the initial energy and angle of the emitted electron. Excitation by proton impact was included to account for the contribution of this interaction channel to the electronic stopping power of the projectile. Proton transport was implemented assuming track-segment conditions, whereas electrons were followed down to 50 eV by the Monte Carlo code PENELOPE. Electron intra-track energy-deposition properties, such as slowing-down and energy-imparted spectra of electrons, were calculated. Furthermore, the use of DDCSs enabled the scoring of electron inter-track properties. We present novel results for 1, 5 and 20 MeV single-proton-track frequencies of distances between the nearest inter- (e(-)-e(-), e(-)-H+) and intra-track (e(-)-e(-), e(-)-H+, H+-H+) energy-deposition events. By setting a threshold energy of 17.5 eV, commonly employed as a surrogate to discriminate for elementary damage in the DNA, the variation in these frequencies was studied as well. The energy deposited directly by the proton represents a large amount of the total energy deposited along the track, but when an energy threshold is adopted the relative contribution of the secondary electrons becomes larger for increasing energy of the projectile. We found that the frequencies of closest energy-deposition events per nanometre decrease with proton energy, i.e. for lower proton energies a denser ionization occurs, following the trend of the characteristic LET curves. In conclusion, considering the energy depositions due to the delta electrons and at the core of the track, 1 MeV protons have an intrinsic capability of generating about five times more dual depositions within the characteristic 2 nm of the DNA-chain structure than 20 MeV protons.

Revisiting the structure of the anti-neoplastic glucans of Mycobacterium bovis Bacille Calmette-Guerin. Structural analysis of the extracellular and boiling water extract-derived glucans of the vaccine substrains.

The attenuated strain of Mycobacterium bovis Bacille Calmette-Guérin (BCG), used worldwide to prevent tuberculosis and leprosy, is also clinically used as an immunotherapeutic agent against superficial bladder cancer. An anti-tumor polysaccharide has been isolated from the boiling water extract of the Tice substrain of BCG and tentatively characterized as consisting primarily of repeating units of 6-linked-glucosyl residues. Mycobacterium tuberculosis and other mycobacterial species produce a glycogen-like alpha-glucan composed of repeating units of 4-linked glucosyl residues substituted at some 6 positions by short oligoglucosyl units that also exhibits an anti-tumor activity. Therefore, the impression prevails that mycobacteria synthesize different types of anti-neoplastic glucans or, alternatively, the BCG substrains are singular in producing a unique type of glucan that may confer to them their immunotherapeutic property. The present study addresses this question through the comparative analysis of alpha-glucans purified from the extracellular materials and boiling water extracts of three vaccine substrains. The polysaccharides were purified, and their structural features were established by mono- and two-dimensional NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the enzymatic and chemical degradation products of the purified compounds. The glucans isolated by the two methods from the three substrains of BCG were shown to exhibit identical structural features shared with the glycogen-like alpha-glucan of M. tuberculosis and other mycobacteria. Incidentally, we observed an occasional release of dextrans from Sephadex columns that may explain the reported occurrence of 6-substituted alpha-glucans in mycobacteria.

LC-1H NMR used for determination of the elution order of S-naproxen glucuronide isomers in two isocratic reversed-phase LC-systems.

The reactive metabolite S-naproxen-beta-1-O-acyl glucuronide was purified from human urine using solid phase extraction (SPE) and preparative HPLC. The structure was confirmed by 600 MHz 1H NMR. Directly coupled 600 MHz HPLC-1H NMR was used to assign the peaks in chromatograms obtained when analysing a sample containing S-naproxen aglycone and the 1-, 2-, 3-, and 4-isomers of S-naproxen-beta-1-O-acyl glucuronide in two simple isocratic reversed phase HPLC-systems. Using mobile phase 1 (50 mM formate buffer pH 5.75/acetonitrile 75:25 v/v) the elution order was: 4-O-acyl isomers, beta-1-O-acyl glucuronide, 3-O-acyl isomers, 2-O-acyl isomers, and S-naproxen aglycone. Using mobile phase II (25 mM potassium phosphate pH 7.40/acetonitrile 80:20 v/v) the elution order was: alpha/beta-4-O-acyl isomers, S-naproxen aglycone, beta-1-O-acyl glucuronide, 3-O-acyl isomers, and alpha/beta-2-O-acyl isomers. In both systems the elution order for the 2-, 3- and 4-O-acyl isomers corresponded with previously published results for 2-, 3-, and 4-fluorobenzoic acid glucuronide isomers determined by reversed phase HPLC-1H NMR (U.G. Sidelmann, S.H. Hansen, C. Gavaghan, A.W. Nicholls, H.A.J. Carless, J.C. Lindon, I.D. Wilson, J.K. Nicholson, J. Chromatogr. B Biomed. Appl. 685 (1996) 113-122]. The alpha-1-O-acyl isomer was found to be present at approximately 3% of the initial S-naproxen-beta-1-O-acyl glucuronide concentration in the glucuronide isomer mixture after 6 h of incubation at pH 7.40 and 37 degrees C. In both HPLC systems it eluted just before the beta-1-O-acyl glucuronide well separated from other isomers. Investigators should consider the possible formation of a alpha-1-O-acyl isomer when studying glucuronide reactivity and degradation.

Active glycerol uptake is a mechanism underlying halotolerance in yeasts: a study of 42 species.

A comparison of 42 yeast species with respect to growth in the presence of high NaCl concentration and characteristics of glycerol uptake is presented. The yeast species were classified into four classes on the basis of their ability to grow in the presence of 1, 2, 3 or 4 M NaCl. Considering that two different types of active-transport systems for glycerol uptake have been described, Na+/glycerol and H+/glycerol symports, glycerol transport was investigated by testing for proton uptake upon glycerol addition in cells incubated in the absence and in the presence of NaCl. Only strains belonging to the two higher classes of salt tolerance showed constitutive active glycerol uptake, and could accumulate glycerol internally against a concentration gradient. Five of these strains exhibited a H+/glycerol symport. All the other strains showed evidence of the activity of a salt-dependent glycerol uptake similar to that described in the literature for Debraryomyces hansenii. The strains within the two lower classes of salt tolerance showed, to varying degrees, glycerol active uptake only when glycerol was used as the carbon and energy source, suggesting that this uptake system is involved in glycerol catabolism. The results within this work suggest that active glycerol uptake provides a basis for high halotolerance, helping to maintain a favourable intracellular concentration of glycerol. The relation between the constitutive expression of such carriers and a higher level of salt-stress resistance suggests that this may be an evolutionary advantage for growth under such conditions.

Thioredoxin reductase-thioredoxin fusion enzyme from Mycobacterium leprae: comparison with the separately expressed thioredoxin reductase.

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. A unique gene organization of TrxR and Trx has been found in Mycobacterium leprae, where TrxR and Trx are encoded by a single gene and, therefore, are expressed as a fusion protein (MlTrxR-Trx). This fusion enzyme is able to catalyze the reduction of thioredoxin or 5,5'-dithiobis(2-nitrobenzoic acid) or 1, 4-naphthoquinone by NADPH, though the activity is much lower than that of Escherichia coli TrxR. It has been proposed that a large conformational change is required in catalysis of E. coli TrxR. Because the reductase portion of the enzyme from M. leprae shows significant primary structure similarity with E. coli TrxR, it is possible that MlTrxR-Trx may require a similar conformational change and that the change in conformation may be affected by the tethered Trx. The reductase has been expressed without Trx attached (MlTrxR). As reported here, comparison of the steady-state and pre-steady-state kinetics of MlTrxR-Trx with those of MlTrxR suggests that the low reductase activity of the fusion enzyme is an inherent property of the reductase, and that any steric limitation caused by the attached thioredoxin in the fusion protein makes only a minor contribution to the low activity. Titration of MlTrxR-Trx and MlTrxR with 3-aminopyridine adenine dinucleotide phosphate (AADP+), an NADP(H) analogue, results in only slight quenching of FAD fluorescence, suggesting an enzyme conformation in which the binding site of AADP+ is not close to the FAD, as in one of the conformations of E. coli TrxR.

The expanded Hansen approach to solubility parameters. Paracetamol and citric acid in individual solvents.

In this study two solubility-parameter models have been compared using as dependent variables the logarithm of the mole fraction solubility, lnX2e, and ln(alpha)/U (originally used in the extended Hansen method), where alpha is the activity coefficient and U is a function of the molar volume of the solute and the volume fraction of the solvent. The results show for the first time the proton-donor and -acceptor hydrogen-bonding capacities of paracetamol, as measured by the acidic and basic partial-solubility parameters. The influence of solvents on the differential scanning calorimetry (DSC) pattern of the solid phases was also studied in relation to the solubility models tested. Citric acid was chosen as a test substance because of its high acidity and its proton donor capacity to form hydrogen bonds with basic solvents. The partial acidic and basic solubility parameters obtained from multiple regression were consistent with this property, validating the model chosen. The results show that the more direct lnX2e variable was more suitable for fitting both models, and the four-parameter model seemed better for describing the interactions between solvent and solute.

Proton transfer in catalysis by fumarase.

Using 3T[14C]malate it was possible to show intermolecular T-transfer to unlabeled fumarate. The rate of dissociation of ET derived from the malate was not rapid, only about as fast as required for KMcat. Because of the slow dissociation of ET derived from T-malate, the awkward complex ET-malate is readily formed. As shown by the effect of added malate on the partition of ET, otherwise captured by fumarate, ET.malate must be functional. Its rate of dissociation to E.M determines the V/Km value of malate. Hydrogen dissociation of the complex ET.F was linearly related to the concentration and basicity of the buffer provided, varying from < 10% to > 60% of the overall rate with alkyl phosphonates. Partition of EH.F to free malate or fumarate occurs in a ratio approximately 2:1 at both low and high buffer. This agrees well with the comparison of the equilibrium exchange rates: malate with [18O]water to malate with [14C]-fumarate [Hansen, J.N., Dinovo, E.C., & Boyer, P.D. (1969) J. Biol. Chem. 244, 6270-6279]. Therefore, the abstracted hydroxyl group is fully exchanged from the enzyme when the bound hydrogen and fumarate return to malate and must be much more accessible to the medium than the abstracted proton. The fact that buffer increases the rate of proton transfer to the medium in the central complex makes it appear that a proton relay connects the active site donor with a remote site that interfaces with the ultimate proton source, water.