Diuretics prime plant immunity in Arabidopsis thaliana.

Plant activators are agrochemicals that activate the plant immune system, thereby enhancing disease resistance. Due to their prophylactic and durable effects on a wide spectrum of diseases, plant activators can provide synergistic crop protection when used in combination with traditional pest controls. Although plant activators have achieved great success in wet-rice farming practices in Asia, their use is still limited. To isolate novel plant activators applicable to other crops, we screened a chemical library using a method that can selectively identify immune-priming compounds. Here, we report the isolation and characterization of three diuretics, bumetanide, bendroflumethiazide and clopamide, as immune-priming compounds. These drugs upregulate the immunity-related cell death of Arabidopsis suspension-cultured cells induced with an avirulent strain of Pseudomonas syringae pv. tomato in a concentration-dependent manner. The application of these compounds to Arabidopsis plants confers disease resistance to not only the avirulent but also a virulent strain of the pathogen. Unlike salicylic acid, an endogenous phytohormone that governs disease resistance in response to biotrophic pathogens, the three diuretic compounds analyzed here do not induce PR1 or inhibit plant growth, showing potential as lead compounds in a practical application.

Binding of diuretic antihypertensive bendroflumethiazide to human serum albumin studied by ¹9F nuclear magnetic resonance method.

Simultaneous specific and nonspecific binding of bendroflumethiazide (BFZ) to human serum albumin (HSA) and concentration profile of BFZ in HSA buffer (pH 7.40) solution were investigated by ¹9F nuclear magnetic resonance (NMR) method. The ¹9F NMR spectrum of BFZ (200 µM) in a buffer solution showed a sharp signal of its CF3 group at 17.8 ppm from the reference trifluoroethanol. Addition of 0.60mM HSA to the sample solution caused the CF(3) signal splitting into three broadened peaks at 18.4 (A), 17.9 (B) and 17.4 ppm (C). By its chemical shift and spectral behavior, B was assigned to unbound BFZ. Competition experiments with Site I and II ligands lead to C being assigned to Site II bound BFZ. However, the peak intensity (areas) of A was not reduced by these ligands, suggesting that A arises from nonspecific binding. Using the peak intensities at several total concentrations of BFZ, Scatchard plot was performed. The plot for A provided a straight line parallel to the x-axis confirming nonspecific binding and that for C was consistent with specific binding. The binding constants for nonspecific and specific Site II binding were 1.02 and 1.00 × 104 (M⁻¹) (n=1.1), respectively. The presence of 0.10 M Cl⁻ in the sample solution affected the binding constant of Site II binding, but not that of nonspecific binding. The concentration profile of BFZ calculated using the binding constants revealed that nonspecific binding is more effective than Site II binding for the binding of BFZ to HSA. It was also confirmed that considerable amounts of BFZ liberated from Site II by the Site II ligands or Cl⁻ ions bind again nonspecifically.

Mechanism of [m+h]+ formation in atmospheric pressure photoionization mass spectrometry: identification of propionitrile in acetonitrile with high mass accuracy measurement and tandem mass spectrometry and evidence for its involvement in the protonation phenomenon.

The role of propionitrile in the production of [M+H]+ under atmospheric pressure photoionization (APPI) was investigated. In dopant-assisted APPI using acetone and anisole, protonated acetone and anisole radical cations were the most prominent ions observed. In dopant-free or direct APPI in acetonitrile, however, a major ion in acetonitrile was detected and identified as propionitrile, using high accuracy mass measurement and collision induced dissociation studies. Vaporizing ca. 10(-5) M althiazide and bendroflumethazide under direct APPI in acetonitrile produced their corresponding protonated species [M+H]+. In addition to protonated acetonitrile, its dimers, and acetonitrile/water clusters, protonated propionitrile, propionitrile dimer, and propionitrile/water clusters were also observed. The role of propionitrile, an impurity in acetonitrile and/or a possible product of ion-molecule reaction, in the production of [M+H]+ of althiazide and bendroflumethazide was further investigated in the absence of dopant using propionitrile-d5. The formation of [M+D]+ species was observed, suggesting a possible role of propionitrile in the protonation process. Additionally, an increase in the [M+H]+ signal of althiazide and bendroflumethazide was observed as a function of propionitrile concentration in acetonitrile. Theoretical data from the literature supported the assumption that one possible mechanism, among others, for the formation of [M+H]+ could be attributed to photo-initiated isomerization of propionitrile. The most stable isomers of propionitrile, based on their calculated ionization energy (IE) and relative energy (DeltaE), were assumed to undergo proton transfer to the analytes, and mechanisms were proposed.

Characterisation of excipient-free nanoporous microparticles (NPMPs) of bendroflumethiazide.

The purpose of this study was to prepare excipient-free porous microparticles of bendroflumethiazide by spray drying and to characterise the physicochemical properties of the particles produced. Solutions of bendroflumethiazide in ethanol/water, ethanol/water/ammonium carbonate or methanol/water/ammonium carbonate were spray dried using a laboratory spray dryer. Spray dried products were characterised by scanning electron microscopy, X-ray powder diffraction, differential scanning calorimetry, FTIR, laser diffraction particle sizing and density measurement. Nanoporous microparticles (NPMPs) were prepared from the alcoholic solutions containing ammonium carbonate. NPMPs were amorphous in nature, had median particles sizes less than 3 microm and densities that were significantly reduced compared to non-porous spray dried bendroflumethiazide powder. The novel process may be used to produce excipient-free amorphous microparticles with desirable physical properties such as amorphous solid state, porosity and low bulk density. This new engineering technology has applications in the design of other therapeutic agents such as those used in pulmonary delivery.

Hirshfeld surface analysis of two bendroflumethiazide solvates.

Bendroflumethiazide, or 3-benzyl-6-(trifluoromethyl)-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide, is reported to crystallize as 1:1 solvates with acetone, C(15)H(14)F(3)N(3)O(4)S(2).C(3)H(6)O, and N,N-dimethylformamide, C(15)H(14)F(3)N(3)O(4)S(2).C(3)H(7)NO. A detailed investigation of the crystal packing and intermolecular interactions is presented by means of Hirshfeld surface analysis. This analysis confirms the atomic positions of methyl H atoms of the solvent molecules that were inferred from the X-ray data and provides a useful tool for structure validation.

Interaction of human serum albumin with bendroflumethiazide studied by fluorescence spectroscopy.

The interactions between bendroflumethiazide (BFTZ) and human serum albumin (HSA) have been studied by fluorescence spectroscopy. Binding constants for drug attachment to the various binding sites of HSA have been measured at different temperatures in physiological buffer solution. The effect of metal ions on BFTZ interaction with HSA was also investigated. The thermodynamic parameters, DeltaH and DeltaS, have been calculated to be 49.28kJmol(-1)>0, and 258.83Jmol(-1)K(-1)>0, respectively. The distance between HSA and BFTZ, r, was determined to be 1.47nm based on Förster's non-radiative energy transfer theory. The experimental results reveal that BFTZ has a strong ability to quench the intrinsic fluorescence of HSA through a static quenching mechanism. Furthermore, the binding constants between BFTZ and HSA are remarkably independent of temperature, and decrease in the presence of various ions, usually by about 30-55%. Hydrophobic interaction occurs between BFTZ and the sub-domain II A of HSA.

The effect of spray drying solutions of bendroflumethiazide/polyethylene glycol on the physicochemical properties of the resultant materials.

The physicochemical properties of co-spray dried bendroflumethiazide (BFMT)/polyethylene glycol (PEG) 4000 composites were investigated. The co-spray dried composites produced from all BFMT/polymer solutions were amorphous. BFMT/PEG 4000 10 and 20% systems consisted of smooth spherical particles approximately 0.5-4mm in diameter. Spray drying resulted in no significant production of the main BFMT degradant, 5-trifluoromethyl-2,4-disulphamoylaniline (TFSA), and for composites consisting of 90% PEG 4000 by weight of total solids, spray drying appeared a superior method of production than the melt method which resulted in significant BFMT degradation. All BFMT/PEG compressed discs showed initial increased release of BFMT compared to discs of micronised BFMT alone, with the spray dried BFMT/PEG 4000 10% system showing initial rates two to three times that of BFMT alone. The physical stability of amorphous BFMT was reduced on inclusion of PEG 4000, recrystallisation occurring more quickly with increasing amount of PEG 4000 in the composites. PEG in the co-spray dried systems appeared to degrade on storage and recrystallised samples failed to show the presence of PEG by differential scanning calorimetry (DSC), X-ray powder diffraction (XRD) or GPC. DSC results were consistent with BFMT/PEG forming a eutectic combination rather than a monotectic system.

Interactions of diuretics with a neutral temperature-responsive polymer: study by capillary electroporesis and dynamic light scattering.

Interactions between diuretics and a recently synthesized temperature-responsive neutral copolymer, poly(N-isopropyl acrylamide) (PNIPA) grafted with poly(ethyleneoxide) (PEO) (PNIPA-g-PEO) were investigated by capillary electrophoresis and dynamic light scattering (DLS). At ambient temperatures, the copolymer takes an open, random coil conformation, but above the lower critical solution temperature (LCST), the polymer shrinks and forms large, stable aggregates. Among the diuretics studied, hydrochlorothiazide was detected to bind to PNIPA-g-PEO.

Photodegradation products of sulphonamide-derived oral antidiabetics and diuretics are not phototoxic in vitro.

The oral antidiabetics glibenclamide and glipizide, and the diuretics bendroflumethiazide and furosemide, all sulphonamide derived drugs, were investigated in vitro for phototoxic properties. Irradiation with broad-band UV induced phototoxic inhibition of colony forming ability in cell cultures. During irradiation, the substances lost one absorption maximum in the UVA region, demonstrated by UV spectroscopy. These findings correlate well with the UV applied, the action spectrum being in the UVA region. Photoproducts detected during and after irradiation showed a decomposition of the substances due to ionization and fragmentation. Incubation of these preirradiated drugs with the cell cultures revealed no phototoxic effects.

Stability of bendrofluazide in a low-dose extemporaneously prepared capsule.

The stability of bendrofluazide 1.25 mg in capsules was studied. The formulation is intended to provide a unit dose powder for administration by dispersing the contents in liquid. The capsules were prepared from ground bendrofluazide tablets 5 mg from two different manufacturers, diluted with lactose and packed manually by weight into hard gelatin capsule shells. The capsules were stored at ambient temperature exposed to light or 75% relative humidity, and at 45 or 60 degrees C. The ambient temperature and 60 degrees C conditions were studied for up to 1 year. Bendrofluazide was determined by a stability-indicating HPLC method. The capsule contents remained easy to disperse in liquid by the end of the study. The weight of contents which could be extracted from the capsules also remained satisfactory. A 7-month storage life at ambient temperature would be feasible, based on the lower 95% confidence limits of the time to 5% degradation.