Carbazochrome carbon nanotube as drug delivery nanocarrier for anti-bleeding drug: quantum chemical study.

The interaction between drugs and single-walled carbon nanotubes is proving to be of fundamental interest for drug system of delivery and nano-bio-sensing. In this study, the interaction of pristine CNT with carbazochrome, an anti-hemorrhagic or hemostatic agent, was investigated with M06-2X functional and 6-31G* basis set. All probable positions of related adsorption for these kind drugs were thought-out to find out which one is energetically suitable. Based on the achieved data, the stronger interactions appeared the oxygen atom of C = O group and nitrogen atom of imine groups. The topology analysis of QTAIM (quantum theory of atoms in a molecule) method was accomplished to understand the properties of interactions between the CNT and carbazochrome. Frontier molecular orbital energies of all systems, global index including stiffness, softness, chemical Gibbs energies, and electrophilicity parameters, as well as some other important physical data such as dipole moment, polarizability, anisotropy polarisibility, and hyperpolaribility were calculated, evaluated, and then compared together. The essence of the formed bonding model progress along the reaction roots was further validated using electron localization function (ELF) calculations. The highest values of adsorption energies were determined in the range of 18.24 up to 22.12 kcal mol-1 for these kind systems. The acceptable recovery time of 849 s was obtained for the desorption of carbazochrome from the CNT surface under UV-light. The final results exhibit that carbazochrome can serve as a promising carrier and also as sensitive sensors in any kind of practical application.

Dynamic agent of an injectable and self-healing drug-loaded hydrogel for embolization therapy.

Embolic agents are crucial for trans-catheter arterial embolization (TAE) in the treatment of various unresectable malignant tumors. Although solid particles, liquid oils, and some polymeric hydrogels have proved their capacities for embolic therapies, the low efficiency, time sensitivity, and cytotoxicity are still considered as challenges. In this study, we developed a three-component dynamic self-healing hydrogel to overcome these limitations. With the help of the Schiff-base bonding, both glycol-chitosan and carbazochrome, containing amine groups, react with dibenzaldehyde-terminated poly(ethylene-glycol) (DF-PEG), forming the dynamic self-healing hydrogels under a mild condition within 200 s. 1H NMR and rheology test were used to characterize the Schiff-base formation and mechanical strength. Controlled-release of carbazochrome from different gelator concentrations of DF-PEG was also studied. Furthermore, in vivo evaluation of the embolization on rats showed the superior embolic effects of the injectable and self-healing hydrogel. Therefore, this new dynamic agent demonstrated the potential for application as a simple, inexpensive, and tunable embolic agent for cancer treatment and drug delivery system.

ANTIOXIDANT ACTIVITY OF ALKOXY DERIVATIVES OF (QUINOLINE-4-YLTHIO)CARBOXYLIC ACIDS.

A selection of potential bioactive molecules including alkoxy derivatives of (quinoline-4-ylthio)carboxylic acids using virtual screening has been carried out and their biological activity was determined. The studied substances proved to be low-toxic, biologically active compounds. It was established that 4-thio derivatives of quinoline exhibit pronounced antiradical, antioxidant-effects and can act as preventive antioxidants, radio and cytoprotectors.

Validated spectrophotometric methods for simultaneous determination of troxerutin and carbazochrome in dosage form.

Four simple, accurate, sensitive and precise spectrophotometric methods were developed and validated for simultaneous determination of Troxerutin (TXN) and Carbazochrome (CZM) in their bulk powders, laboratory prepared mixtures and pharmaceutical dosage forms. Method A is first derivative spectrophotometry (D(1)) where TXN and CZM were determined at 294 and 483.5 nm, respectively. Method B is first derivative of ratio spectra (DD(1)) where the peak amplitude at 248 for TXN and 439 nm for CZM were used for their determination. Method C is ratio subtraction (RS); in which TXN was determined at its λmax (352 nm) in the presence of CZM which was determined by D(1) at 483.5 nm. While, method D is mean centering of the ratio spectra (MCR) in which the mean centered values at 300 nm and 340.0 nm were used for the two drugs in a respective order. The two compounds were simultaneously determined in the concentration ranges of 5.00-50.00 µg mL(-1) and 0.5-10.0 µg mL(-1) for TXN and CZM, respectively. The methods were validated according to the ICH guidelines and the results were statistically compared to the manufacturer's method.

A high-throughput adrenaline test for the exploration of the catalytic potential of halohydrin dehalogenases in epoxide ring-opening reactions.

The principle of the adrenaline test for enzymes is based on the quantification of periodate-sensitive reaction products with adrenaline to produce a chromogenic compound adrenochrome that can be easily detected. Here, a rapid whole-cell -based adrenaline assay for the activity measurement of halohydrin dehalogenases (HHDHs) in nucleophile-mediated epoxide ring-opening reactions is presented. The assay was validated using two types of model reactions (glycidol with nucleophiles and nitrite with epoxides). Moreover, the reliability of the assay was confirmed by gas chromatography analysis. Our results demonstrated that the developed assay is efficient in both library screening and the evaluation of catalytic diversity and specificity of HHDHs. Thus, the assay represents a valuable tool in the evolution of HHDHs for its industrial applications. Moreover, the adrenaline test exhibits a great potential for enzyme assay and could be easily adopted for other suitable enzymes.

Optimization of solid phase extraction clean up and validation of quantitative determination of carbazochrome sodium sulfonate in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry.

A mixed-mode anion exchange solid phase extraction (SPE) method for extraction and clean up of carbazochrome sodium sulfonate (CSS) and (1S)-(+)-10-camphorsulfonic acid (IS) was optimized for quantification by high-performance liquid chromatography/negative electrospray ionization mass spectrometry. The analytes were extracted from 1mL of human plasma via SPE on Oasis(®) WAX cartridge. Chromatographic separation was achieved on a Zorbax SB-Aq (4.6×250mm, 5µm) column under an isocratic condition. Detection was performed using electrospray ionization in negative ion multiple reaction monitoring (MRM) mode. The deprotonated precursor to product ion transitions monitored for CSS and IS was at m/z 299.0→256.0 and m/z 230.9→79.8, respectively. The method was fully validated for its selectivity, sensitivity, precision, accuracy, recovery, matrix effect and stability. Linear range was 0.189-37.8ng/mL with a high square regression coefficient (r=0.9995). The intra-and inter-day precision (RSD, %) ranged from 0.95% to 4.17%, and the intra-and inter-day accuracy was between 95.03% and 105.9%. This method was successfully applied to a bioequivalence study of 90mg CSS formulation in 18 healthy Chinese male subjects under fasting condition.

A novel blending method for dispensing powdered medicine.

We introduced the application of a planetary centrifugal mixer to dispensing powdered medicines to prevent from individual variation in the skills of pharmacists with a manual blending. The blending performance of the mixer was explored in terms of four operational variables, namely, operation speed (400-1000 rpm), operation time (10-60 s), charging rate in vessel (20-50%), and size of vessel (35, 58, 125, 550 mL), using colored lactose and crystalline lactose as the principle model medicine and diluent, respectively. The blending degree was assessed by image analysis, so the extent of uniformity was expressed as the relative standard deviation of the color difference signal Cb value of YCrCb color space. Application of the mixer to blending three commercial medicines with diluents was carried out. Sufficient blending was achieved at 10 s using a 20% charging rate and 35 mL vessel irrespective of operation speed. As the charging rate was increased, a higher operation speed was needed to obtain uniform blending. A larger sized vessel also required a higher operation speed. Uniform blending was achieved in all of the mixtures of colored lactose and crystalline lactose at the weight ratio of 1 : 9-9 : 1. In the application studies using Adona®, Anginal® and Neophylline® powder, the blending performance of the mixer was equivalent to that of the manual blending method, showing relative standard deviations of 2.2-3.3% and 1.8-3.8%, respectively. These results revealed that the planetary centrifugal mixer was suitable for blending powdered medicine.

Multiple targeted drugs carrying biodegradable membrane barrier: anti-adhesion, hemostasis, and anti-infection.

A multiple targeted drug carrying bilayer membrane for preventing an abdominal adhesion is prepared by electrospinning. Two bioactive drugs were successfully incorporated into this bilayer membrane and can be independently released from nanofibrous scaffolds without losing structural integrity and functionality of the anti-adhesion membrane. Besides, the drug release profile could be easily adjusted by optimizing the swelling behavior of the fibrous scaffold. The inner layer of the bilayered fibrous membranes loaded with carbazochrome sodium sulfonate (CA) showed an excellent vascular hemostatic efficacy and formed little clot during in vivo experiment. The outer layer loaded with tinidazole (TI) had outstanding antibacterial effect against the anaerobe. We believe this approach could serve as a model technique to guide the design of implants with drug delivery functions.

On the mechanism of action of antipsychotic drugs: a chemical reaction not receptor blockade.

Over forty years ago, biochemist Lauro Galzigna conducted an in-vitro experiment showing that the antipsychotic chlorpromazine reacted with the putative psychotogen adrenochrome to form a polymer resembling melanin. The field of psychopharmacology has essentially ignored that simple but illustrative experiment in the intervening time. The present study reproduces principle elements of Galzigna's experiment and expands the scope to include the antipsychotic medications olanzapine and minocycline. The rate of reaction was slow, with maximal yield of black polymer being achieved by 4, 10 and 7 days with chlorpromazine, olanzapine and minocycline, respectively. Changing the pH was most informative for chlorpromazine and minocycline reactions, where yield increased sharply between pH 6.1 and 6.9, and decreased slightly between pH 6.9 and 7.8, consistent with reaction profiles expected for aromatic substitution. Preincubation of olanzapine with iodine doubled the polymer yield, facilitated by the addition of iodine to the aromatic ring and presumably followed by its departure as a "leaving group". Increasing the salt concentration 1.5-fold depressed yields for all three drugs, most likely via ionic shielding of charged functional groups, diminishing reactivity. The results are discussed in regards to the mechanism of action of antipsychotic medications, casting doubt on commonly held theories. The time course of the chemical reactions presented here and the concentrations required, are much more consistent with clinical results than are models concerning receptor-mediated mechanisms. Furthermore, minocycline was effective in this model, but does not appear to have affinity for the primary receptor families thought by many to mediate antipsychotic efficacy.

Fluorescence quenching method for the determination of carbazochrome sodium sulfonate with aromatic amino acids.

In Britton-Robinson (BR) buffer medium (pH 3.3), carbazochrome sodium sulfonate (CSS) can react with some aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) to form a 1:1 complex by electrostatic attraction, aromatic stacking interaction and Van der Waals' force, resulting in fluorescence quenching of these amino acids. Maximum quenching wavelengths were located at 352 nm (CSS-Trp system), 303 nm (CSS-Tyr system) and 284 nm (CSS-Phe system), respectively. The fluorescence quenching value (ΔF) was proportional to the concentration of CSS in a certain range. The fluorescence quenching method for the determination of CSS showed high sensitivity, with detection limits of 31.3 ng/mL (CSS-Trp system), 44.6 ng/mL (CSS-Tyr system) and 315.0 ng/mL (CSS-Phe system), respectively. The optimum conditions of the reaction conditions and the effect of coexisting substances were investigated and results showed that the method had good selectivity. The method was successfully applied for the rapid determination of CSS in blood and urine samples. Based on the bimolecular quenching constant Kq , the effect of temperature and Stern-Volmer plots, this study showed that quenching of fluorescence of amino acids by CSS was a static quenching process.

Determination of carbazochrome by fluorescence quenching method.

A sensitive, simple and selective spectrofluorimetric method for the reaction of carbazochrome (CBZC) and Eosin Y (EY) or Phloxine B (PB) in acidic medium is developed for the determination of carbazochrome in biological fluids, which gives a highly fluorescent derivative measured at 545 and 565 nm at excitation wavelengths of 301 and 305 nm. The fluorescence quenching extent (ΔF) is proportional to the concentration of CBZC for CBZC-EY and CBZC-PB system at the range of 0.03-1.50 µg/mL and 0.08-1.25 µg/mL, respectively. The detection limit is 9.1 ng/mL for EY system and 22.7 ng/mL for PB system. The intra-day and inter-day reproducibility (RSD values) are less than 8.3% under three concentrations. Moreover, the affecting factors of fluorescence intensity of the product are carefully investigated and optimized, as well as the effect of coexisting substances. Judging from temperature, the Stern-Volmer plots and fluorescence emission decay curves, the quenching of fluorescence of EY and PB by CBZC is a static quenching process, caused by electrostatic attraction and aromatic stacking interaction.

[A new approach to studying the autoxidation of adrenaline: possibility of the determination of superoxide dismutase activity and the antioxidant properties of various preparations by polarography].

The reaction of adrenaline autoxidation in an alkaline buffer with the formation of superoxide radicals and the product of its oxidation, adrenochrome, which models the quinoid pathway of adrenaline conversion in the body, is accompanied by oxygen consumption. This reaction is applicable for polarographic determination of the activity of superoxide dismutase and the antioxidant properties of biological and chemical compounds, it is based on evaluation of the latent period and the rate of oxygen consumption, which are measured in the presence of the compounds examined. It was assumed that the neuro- and cardiotoxicity of quinone products of adrenaline oxidation is related not only to their "own" properties and reactive oxygen species formed but also the hypoxia of those regions of the cell and tissue where the quinoid oxidation of adrenaline occurs.

Accelerated oxidation of epinephrine by silica nanoparticles.

We have measured the influence of mesoporous silica (MCM-41 and SBA-15) nanoparticles and dense silica nanoparticles on epinephrine oxidation, a pH-dependent reaction, whose rate is small in acidic or neutral solutions but much greater at higher pH. The reaction was measured by monitoring adrenochrome at 480 nm, the product of epinephrine oxidation. In distilled water (dH(2)O) with no particles present, the oxidation of epinephrine occurs slowly but more rapidly at higher pH. The presence of MCM-41 or silica spheres does not accelerate the oxidation, but SBA-15 does, showing that the difference in the structures of nanomaterials leads to differing effects on the epinephrine oxidative process. In phosphate buffered saline (PBS, pH = 7.4), epinephrine undergoes a much quicker oxidation, and, in this case, the presence of SBA-15 and MCM-41 makes it even more rapid. Silica spheres have no noticeable influence on the oxidation in PBS or in dH(2)O. The possibility that the catalytic effect of mesoporous silica nanoparticles (MSN) could result from the residue of templating chemicals, however, can be excluded due to the postsynthesis calcinations. Experiments with dithionite, added either earlier than or at the same time as the epinephrine addition, show that fast oxidation takes place only when dithionite and epinephrine are simultaneously added into PBS solution. This confirms a vital role of oxygen radicals (probably *O(2)(-)) in the oxidation of epinephrine. These oxygen radicals are likely to form and accumulate within the phosphate buffer or in the presence of MSN. Comparing the three kinds of silica nanoparticles applied, we note that mesoporous SBA-15 and MCM-41 materials own much larger surface area than solid silica particles do, whereas MCM-41 possesses a much narrower pore size (0.4-fold) than SBA-15. It seems, therefore, that large surface area, characteristic mesoporosity, and surface structures aid in the deposit of oxygen radicals inside MSN particles, which catalyze the epinephrine oxidation in a favorable phosphate environment.

Enzyme assay and activity fingerprinting of hydrolases with the red-chromogenic adrenaline test.

The adrenaline test for enzymes is a colorimetric enzyme assay based on the quantification of periodate-sensitive reaction products such as 1,2-diols and 1,2-aminoalcohols by back-titration of the oxidant with adrenaline to produce adrenochrome as an easily detectable red product. The test uses commercial reagents and is suitable for screening the activity of various hydrolases. It is demonstrated here for testing epoxide hydrolases, lipases and esterases, and for activity fingerprinting of these enzymes across substrate series. The complete assay requires 2-3 h.

Quinone reductase 2 is a catechol quinone reductase.

The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference between quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.

Short-term delayed-release microcapsules spraycoated with acrylic terpolymers.

A series of poly(ethyl acrylate (EA)/methyl methacrylate (MMA)/2-hydroxyethyl methacrylate (HEMA)) lattices were synthesized to prepare short-term delayed-release microcapsules by employing the Wurster coating process. Latex with a HEMA molar fraction exceeding 60% could not be synthesized as an aqueous suspension due to latex particle precipitation. The effects of monomer composition on the particle size of latex and the water-uptake and glass transition temperature (T(g)) of cast films were investigated. Lattices whose T(g) ranged from 40 to 80 degrees C were used to prepare the microcapsules. Most of the lattices exhibited excellent process performance while coating particles that were smaller than 100 microm: the product yields were 85.1-90.6% and the mean particle sizes were 82-85 microm. However, since the lattices with high molar ratios of EA and HEMA were highly hydrophilic and strongly adhesive, the core particles in the coating were severely agglomerated. The microcapsules coated with lattices whose HEMA molar fractions were higher than 50% were unable to retard the release of carbazochrome sodium sulfonate, a water-soluble model drug, during the initial 0.5 min. Poly(EA/MMA/HEMA) with a molar ratio of 9:9:10 appeared to be suitable for the preparation of short-term delayed-release microcapsules by the Wurster coating process.

Photostability of epinephrine - the influence of bisulfite and degradation products.

Sulfites are previously demonstrated to increase the photodegradation of epinephrine. The aim of this study was to clarify the factors responsible for this effect. Adrenochrome sulfonate seems to be the important substance. Photoproduction of singlet oxygen is indicated to be the mechanism by which adrenochrome sulfonate acts. Protection of epinephrine solutions from irradiation <418 nm prevented the photodegradation. A reaction pathway for thephotochemical decomposition of epinephrine in the presence of bisulfite is suggested.

Flow injection spectrophotometric determination of adrenaline in pharmaceutical formulations using a solid-phase reactor containing lead(IV) dioxide immobilized in a polyester resin.

A flow injection spectrophotometric procedure is proposed for determining adrenaline in pharmaceutical formulations. In this work, the adrenaline in acetate buffer reacts with a solid-phase reactor containing lead(IV) dioxide immobilized in a polyester resin and the adrenochrome yielded was continuously monitored at 486 nm. The analytical curve was linear in the adrenaline concentration range from 0.1 to 0.8 mmol l(-1) with a detection limit of 8 x 10(-3) mmol l(-1). Recoveries of 96.5-105% and relative standard deviation of 0.2% for a solution containing 0.4 mmol l(-1) adrenaline (n = 10) were obtained. The analytical frequency was 130 determinations per hour and the results obtained for adrenaline in pharmaceutical formulations using this procedure and those obtained using a pharmacopoeia procedure are in agreement at the 95% confidence level.

The fallacy of using adrenochrome reaction for measurement of reactive oxygen species formed during cytochrome p450-mediated metabolism of xenobiotics.

The adrenochrome reaction (oxidation of epinephrine to adrenochrome) has been widely employed as a standard assay for reactive oxygen species, produced under a variety of conditions, including those produced during cytochrome P450 (CYP)-mediated oxidation of substrates such as cyclosporine. However, it has been reported that epinephrine and adrenochrome can be metabolized by hepatic microsomes and that adrenochrome can also be metabolized by NADPH-CYP reductase. Thus, in the present report, we provide evidence that measurement of adrenochrome cannot be used as an index of reactive oxygen species generated during CYP-mediated metabolism of xenobiotics because adrenochrome and its precursor, epinephrine, interact with the CYP enzyme system as substrates and inhibitors. Our results indicated that adrenochrome was moderately stable in phosphate buffer but degraded rapidly (over 50% consumed in less than 2 min) by (cloned and expressed) CYP3A4 and CYP reductase in the presence of NADPH. Furthermore, both epinephrine and adrenochrome were found to be inhibitors of CYP3A4-mediated oxidation of testosterone. Together, these results lead to the conclusion that the use of adrenochrome reaction for measurement of reactive oxygen species formed during CYP3A4-mediated metabolism of xenobiotics is inappropriate.

Spectroscopic detection of adrenaline-quinone formation in micelles.

Spectral changes, from 200 nm to 600 nm, of the oxidation of adrenaline to adrenochrome induced by periodate in electrically charged and neutral micelles at pH 3.77 were studied. The observed variations of the peak position, intensity and shape of the fluorescence spectra indicated that depending on the charge of the micelle adrenaline ion is partially embedded into the micellar core. Fluorescence lifetime measurements using Omnilyzer allowed to calculate partition coefficients of 0.36, 0.05 and 0.01 in sodium dodecyl sulphate, tetradodecyltrimethylammonium bromide and Triton X-100, respectively. Kinetics of adrenaline decay during oxidation were followed by its fluorescence what overcame spectral interference in the absorption spectra of adrenaline from the formed intermediates. Scanning absorption spectroscopy, with 100 ms resolution, allowed the recording of spectral changes during the transformation. With this method, the formation of adrenaline-quinone with absorption maxima at 388 nm and 274 nm was detected. The calculated rate constants of the observed kinetics during oxidation were significantly lowered in both charged micelles compared to buffer solution and in Triton X-100 neutral micelles. The observed phenomena are discussed in terms of the electrostatic forces mechanism and in the frame of the Raper-Mason scheme of adrenaline transformation.