The feasibility of enzyme targeted activation for amino acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential targeted enzyme

The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5'-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0-105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5'-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5'-O-L-phenylalanyl-L-tyrosylfloxuridine and 5'-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enzymes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5'-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.     

Nonlinear studies of Pararosanilin dye in liquid and solid media

Solid-state dye-doped polymers are attractive alternative to the conventional liquid dye solutions. In this paper, nonlinear properties of the dye Pararosanilin has been studied. The third-order nonlinear optical properties of Pararosanilin dye in 1-butanol and dye-doped polymer film were measured by the Z-scan technique using 532 nm diode pumped Nd:Yag laser. This material exhibits negative optical nonlinearity. The dye at 0.4 mM concentration exhibited nonlinear refractive coefficient (n(2) = -6.8 x 10(-8) and -7.11 x 10(-8) (cm(2)/W) in liquid and solid media, respectively), nonlinear absorption coefficient (beta = -7.7 x 10(-4) and -7.93 x 10(-4)cm/W in liquid and solid media, respectively) and susceptibility (chi((3))=3.38 x 10(-6) and 3.53 x 10(-5)esu in liquid and solid media, respectively). These results show that Pararosanilin dye has potential applications in nonlinear optics.     

Nonlinear studies of acid fuchsin dye in liquid and solid media

Solid-state dye-doped polymers are attractive alternative to the conventional liquid dye solutions. In this paper, nonlinear properties of the dye Acid Fuchsin has been studied. The third-order nonlinear optical properties of Acid Fuchsin dye in 1-butanol and dye-doped polymer film were measured by the Z-scan technique using 532 nm diode pumped Nd:Yag laser. This material exhibits negative optical nonlinearity. The dye at 0.4 mM concentration exhibited nonlinear refractive coefficient (n(2)=-8.72 x 10(-8) and -10.308 x 10(-8) (cm(2)/W) in liquid and solid media, respectively), nonlinear absorption coefficient (beta=-7.69 x 10(-4) and -8.294 x 10(-4)cm/W in liquid and solid media, respectively) and susceptibility (chi(3)=4.33 x 10(-6) and 5.13 x 10(-5)esu in liquid and solid media, respectively). These results show that Acid Fuchsin dye has potential applications in nonlinear optics.     

Non-destructive neutron-activation analysis for determining the chlorine content of paper-pulp

Non-destructive neutron-activation analysis is used for determining chlorine in paper-pulp. Numerical data have been obtained for bleached and unbleached paper-pulps of different types and origins. The sensitivity of this method is 100 ppm for an irradiation time of 30 min and a neutron flux of 6 x 10(10) neutrons.cm(-2).sec(-1) and 10 ppm for an irradiation time of 1 min and a neutron flux of 2 x 10(12) neutrons.cm(-2).sec(-1). In both cases the amount of chlorine that can be determined depends on the presence of the interfering elements manganese and sodium in the paper-pulp. The time required for a complete analysis, after irradiation, is 5 min.     

NMR diffusion spectroscopy as a measure of host-guest complex association constants and as a probe of complex size

The complexes of cyclohexylacetic acid and cholic acid with beta-cyclodextrin were studied by NMR diffusion coefficient measurements. The diffusion coefficient of the 1:1 cyclohexylacetic acid/beta-cyclodextrin complex, K(a) = 1800 +/- 100 M(-1), is slightly slower (3.23 +/- 0.07 x 10(-6) cm(2) s(-1)) than that of beta-cyclodextrin (3.29 +/- 0.07 x 10(-6) cm(2) s(-1)). The diffusion coefficient of the 1:1 cholic acid/beta-cyclodextrin complex, K(a) = 5900 +/- 800 M(-1), is significantly slower (2.93 +/- 0.07 x 10(-6) cm(2) s(-1)) than that of beta-cyclodextrin. The results indicate that caution should be exercised when studying host-guest complexation by the so-called 'single point' technique. A novel data treatment is introduced which takes into account the diffusion behavior of all of the species when determining K(a). Experimentally determined diffusion coefficients of complexes are also a useful probe of the size of host-guest complexes.     

Increasing the throughput and productivity of Caco-2 cell permeability assays using liquid chromatography-mass spectrometry: application to resveratrol absorption and metabolism

The Caco-2 cell monolayer permeability assay has become a standard model of human intestinal absorption and transport. This paper reviews recent progress in increasing the throughput of Caco-2 cell monolayer assays and in expanding the scope of this assay to include modeling intestinal drug metabolism. The state-of-the-art in Caco-2 cell monolayer permeability assays combines multi-well plates fitted with semi-permeable inserts on which Caco-2 cells have been cultured with liquid chromatography-mass spectrometry (LC-MS) or LC-tandem mass spectrometry (LC-MS-MS) for the quantitative analysis of test compounds and the identification of their intestinal metabolites. After reviewing the progress in increasing the throughput of Caco-2 cell monolayer assays for both modeling human intestinal permeability or transport and the metabolism of xenobiotic compounds, we demonstrate the application of LC-MS and LC-MS-MS to the measurement of resveratrol permeability and metabolism in the Caco-2 model. trans-Resveratrol (trans-3,5,4'-trihydroxystilbene) is a polyphenolic compound occurring in grapes, peanuts and other food sources, that is under investigation as a cancer chemoprevention agent. The apparent permeability coefficient for apical (AP) to basolateral (BL) movement of resveratrol was 2.0 x 10(-5)cm/sec. Resveratrol was not a substrate for P-glycoprotein or the multi-drug resistance associated proteins (MRP). No phase I metabolites were observed, but the phase II conjugates resveratrol-3-glucuronide and resveratrol-3-sulfate was identified based on LC-MS and LC-MS-MS analysis and comparison with synthetic standards. Although these data indicate that resveratrol diffuses rapidly across the intestinal epithelium, extensive phase II metabolism during absorption might reduce resveratrol bioavailability.     

High efficiency micellar electrokinetic chromatography of hydrophobic analytes on poly(dimethylsiloxane) microchips

This paper describes a simple method for the effective and rapid separation of hydrophobic molecules on polydimethylsiloxane (PDMS) microfluidic devices using Micellar Electrokinetic Chromatography (MEKC). For these separations the addition of sodium dodecyl sulfate (SDS) served two critical roles - it provided a dynamic coating on the channel wall surfaces and formed a pseudo-stationary chromatographic phase. The SDS coating generated an EOF of 7.1 x 10(-4) cm(2) V(-1) s(-1) (1.6% relative standard deviation (RSD), n = 5), and eliminated the absorption of Rhodamine B into the bulk PDMS. High efficiency separations of Rhodamine B, TAMRA (6-carboxytetramethylrhodamine, succinimidyl ester) labeled amino acids (AA), BODIPY FL CASE (N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester) labeled AA's, and AlexaFluor 488 labeled Escherichia coli bacterial homogenates on PDMS chips were performed using this method. Separations of Rhodamine B and TAMRA labeled AA's using 25 mM SDS, 20% acetonitrile, and 10 mM sodium tetraborate generated efficiencies > 100,000 plates (N) or 3.3 x 10(6) N m(-1) in <25 s with run-to-run migration time reproducibilities <1% RSD over 3 h. Microchips with 30 cm long serpentine separation channels were used to separate 17 BODIPY FL CASE labeled AA's yielding efficiencies of up to 837,000 plates or 3.0 x 10(6) N m(-1). Homogenates of E. coli yielded approximately 30 resolved peaks with separation efficiencies of up to 600,000 plates or 2.4 x 10(6) N m(-1) and run-to-run migration time reproducibilities of <1% RSD over 3 h.     

Novel Dendritic Naproxen Prodrugs with Poly(aspartic Acid) Oligopeptide: Synthesis and Hydroxyapatite Binding in Vitro

Abstract

Novel bone-targeting prodrugs containing dendritic naproxen and poly(aspartic acid) oligopeptide were synthesized in a convergent approach and were characterized by NMR, mass spectral, and elemental analysis techniques. The modified naproxen prodrugs showed a high affinity to hydroxyapatite in vitro and provided an effective entry for the synthesis of a dendritic naproxen–poly(aspartic acid) oligopeptide conjugates used for bone targeting.     

Separation and determination of norepinephrine, epinephrine and isoprinaline enantiomers by capillary electrophoresis in pharmaceutical formulation and human serum

A capillary electrophoresis method with ultraviolet (UV) detection was developed and optimized for the enantiomer separation of norepinephrine (NE), epinephrine (EP) and isoprenaline (IP) using dual cyclodextrins (CDs) of 2-hydroxypropyl-beta-CD (HP-beta-CD) and heptakis (2,6-di-o-methyl)-beta-CD (DM-beta-CD) as chiral selectors. Optimal separation was obtained using a running buffer of 50mM phosphate containing 30mM HP-beta-CD and 5mM DM-beta-CD at pH 2.90 and a field strength of 20kV in 45cmx75mum (40cm effective length) uncoated capillary. The UV absorbance detection was set at 205nm. A 0.1% (w/w) polyethylene glycol or 0.1% (v/v) acetonitrile was used to enhance the detection sensitivity. There was a wide and excellent linear calibration graph for each enantiomer in the range 1.0x10(-3) to 1.0x10(-6)M and the detection limit (S/N=3) was found from 8.5x10(-7) to 9.5x10(-7)M. The method has been applied for the determination of isoprenaline in isoprenaline hydrochloride aerosol and to the analysis of serum samples. The recoveries of NE and EP in serum and IP in drug were ranged from 90 to 110%. The relative standard deviations of all the analyte peaks were less than 2.8% for migration time and less than 4.8% for peak area.     

High-performance liquid chromatographic determination of 1,2-diethyl-3-hydroxypyridin-4-one and its 2-(1-hydroxyethyl) metabolite in rat blood.

A sensitive and selective high-performance liquid chromatographic (HPLC) method for the analysis of 1,2-diethyl-3-hydroxypyridin-4-one (CP94, I) and its 2-(1-hydroxyethyl) metabolite (II) in rat blood is described. I, II and the internal standard, 1-propyl-2-ethyl-3-hydroxypyridin-4-one (CP95, III) were extracted into dichloromethane (3 x 5 ml, with the addition of 1 g of sodium chloride) from blood (0.25 ml plus 0.75 ml of pH 7.0 morpholinopropanesulphonic acid buffer). Extractability approached 100% for I and III, and approximately 65% for II under these conditions. Chromatographic analysis was carried out using a Hypercarb porous graphitised carbon HPLC column (10 cm x 0.46 cm). The mobile phase was 14:86 (v/v) acetonitrile-NaH2PO4 buffer (10 mM, containing 2 mM EDTA, pH adjusted to 3 with phosphoric acid) and detection was by ultraviolet at 280 nm. Calibration curves were linear (correlation coefficient greater than 0.99) and reproducible over the concentration range 0-80 micrograms/ml and the coefficient of variation was less than 16% even at low (1 microgram/ml) concentrations. The minimum quantifiable level was 0.5 microgram/ml for both I and II.     

Fibre-optic pesticide biosensor based on covalently immobilized acetylcholinesterase and thymol blue

A fibre-optic based on immobilized acetylcholinesterase is described and its application in the detection of carbamate and organophosphate pesticides through enzyme inhibition measurements is discussed. The bioactive component of the sensor consists of acetylcholinesterase covalently immobilized on preactivated isothiocyanate glass mixed with thymol blue indicator bound on aminopropyl glass and the sensor was constructed by packing a thin layer of the glass bead mixture at the tip of a bifurcated fibre-optic sensor head, which was then integrated with a flow-through cell. The response of the sensor to acetylcholine was highly reproducible (RSD<2%) and readily reversible. The sensor exhibited a linear response to acetylcholine in the concentration range 2.5-25 mM (r(2)=0.992). Inhibition plots obtained for test organophosphate (paraoxon) and carbamate (carbofuran) pesticides exhibited concentration-dependent behaviour and showed linear profiles in the concentration ranges 5x10(-8)-5x10(-7) M for carbofuran and 5x10(-7)-5x10(-6) M for paraoxon. The detection limits, calculated at I(10%), are 1.5x10(-8) M (3.1 ppb) and 1.1x10(-7) M (24.7 ppb) for carbofuran and paraoxon, respectively. The regeneration of paraoxon-inhibited sensor was possible using 2-pyrimidine aldoxime, while repetitive substrate injection was necessary to reactivate the carbofuran-inhibited optrode. The factors affecting the inhibition and reactivation processes were investigated.     

Characterization of procaine metabolism as probe for the butyrylcholinesterase enzyme investigation by simultaneous determination of procaine and its metabolite using capillary electrophoresis with electrochemiluminescence detection

Capillary electrophoresis with electrochemiluminescene detection was used to characterize procaine hydrolysis as a probe for butyrylcholinesterase by in vitro procaine metabolism in plasma with butyrylcholinesterase acting as bioscavenger. Procaine and its metabolite N,N-diethylethanolamine were separated at 16 kV and then detected at 1.25 V in the presence of 5.0 mM Ru(bpy)(3)2+, with the detection limits of 2.4x10(-7) and 2.0x10(-8) mol/L (S/N=3), respectively. The Michaelis constant Km value was 1.73x10(-4) mol/L and the maximum velocity Vmax was 1.62x10(-6) mol/L/min. Acetylcholine bromide and choline chloride presented inhibition effects on the enzymatic cleavage of procaine, with the 50% inhibition concentration (IC50) of 6.24x10(-3) and 2.94x10(-4) mol/L.     

Preparation of polypyrrole-coated platinum modified electrode in chloroform in the presence of various supporting electrolytes and its use for the catalytic oxidation of hydroquinone in aqueous and chloroform solutions

Polypyrrole (PPy)-coated platinum modified electrode was prepared electrochemically in chloroform in the presence of tetrabutylammonium perchlorate, tosylate, tetrafluoroborate, periodate, and hydrogen sulphate. Evidence in favour of the occurrence of a redox reaction at the film/solution interface was given by ferrocene oxidation. The electrocatalytic effect of the PPy-coated electrode was revealed by oxidation of hydroquinone on it in water and chloroform. The influence of dopant anion, method of electrosynthesis and operation temperature were demonstrated. The calculated heterogeneous electron transfer rate constant (k(o)) at the PPy-modified Pt electrode at 25 degrees C was 1.4 x 10(-3) cm/sec which is 1000-fold greater than the value (< 10(-6) cm/sec) reported for bare electrode.     

New reagent for spectrophotometric determination of salicylic acid

In 6M hydrochloric acid solution, salicylic acid reacts with pentachloronitrosyliridate [Ir(NO)Cl(5)](-) to form a new 1:1 complex giving two absorption maxima at 369 and 446 nm with molar absorptivities both of 1.1 x 10(4) l.mole(-1) cm(-). The reaction is second order with apparent energy, E(a), of 16.8 kJ/mole. At constant temperature, the apparent rate constant k increases to a maximum when the concentration of hydrogen ion is decreased to 4-5M and the concentration of chloride ion is kept at approximately 6M. The mechanism of the reaction is also discussed. In alkaline solution, the absorption maxima shift to 381 and 506 nm with molar absorptivities of 6.0 x 10(3) and 2.4 x 10(4) l.mole(-1) cm(-1), respectively. Salicylic acid can be determined spectrophotometrically with high sensitivity and precision. Benzoic and boric acids do not interfere.     

Direct oxidation of L-cysteine by [FeIII(bpy)2(CN)2]+ and [FeIII(bpy)(CN)4]-

The oxidation of L-cysteine by the outer-sphere oxidants [Fe(bpy)2(CN)2]+ and [Fe(bpy)(CN)4]- in anaerobic aqueous solution is highly susceptible to catalysis by trace amounts of copper ions. This copper catalysis is effectively inhibited with the addition of 1.0 mM dipicolinic acid for the reduction of [Fe(bpy)2(CN)2]+ and is completely suppressed with the addition of 5.0 mM EDTA (pH<9.00), 10.0 mM EDTA (9.010.0) for the reduction of [Fe(bpy)(CN)4]-. 1H NMR and UV-vis spectra show that the products of the direct (uncatalyzed) reactions are the corresponding Fe(II) complexes and, when no radical scavengers are present, L-cystine, both being formed quantitatively. The two reactions display mild kinetic inhibition by Fe(II), and the inhibition can be suppressed by the free radical scavenger PBN (N-tert-butyl-alpha-phenylnitrone). At 25 degrees C and micro=0.1 M and under conditions where inhibition by Fe(II) is insignificant, the general rate law is -d[Fe(III)]/dt=k[cysteine]tot[Fe(III)], with k={k2Ka1[H+]2+k3Ka1Ka2[H+]+k4Ka1Ka2Ka3{/}[H+]3+Ka1[H+]2+Ka1Ka2[H+]+Ka1Ka2Ka3}, where Ka1, Ka2, and Ka3 are the successive acid dissociation constants of HSCH2CH(NH3+)CO2H. For [Fe(bpy)2(CN)2]+, the kinetics over the pH range of 3-7.9 yields k2=3.4+/-0.6 M(-1) s(-1) and k3=(1.18+/-0.02)x10(6) M(-1) s(-1) (k4 is insignificant in the fitting). For [Fe(bpy)(CN)4]- over the pH range of 6.1-11.9, the rate constants are k3=(2.13+/-0.08)x10(3) M(-1) s(-1) and k4=(1.01+/-0.06)x10(4) M(-1) s(-1) (k2 is insignificant in the fitting). All three terms in the rate law are assigned to rate-limiting electron-transfer reactions in which various thiolate forms of cysteine are reactive. Applying Marcus theory, the self-exchange rate constant of the *SCH2CH(NH2)CO2-/-SCH2CH(NH2)CO2- redox couple was obtained from the oxidation of L-cysteine by [Fe(bpy)(CN)4]-, with k11=4x10(5) M(-1) s(-1). The self-exchange rate constant of the *SCH2CH(NH3+)CO2-/-SCH2CH(NH3+)CO2- redox couple was similarly obtained from the rates with both Fe(III) oxidants, a value of 6x10(6) M(-1) s(-1) for k11 being derived. Both self-exchange rate constants are quite large as is to be expected from the minimal rearrangement that follows conversion of a thiolate to a thiyl radical, and the somewhat lower self-exchange rate constant for the dianionic form of cysteine is ascribed to electrostatic repulsion.     

Ion-exchange voltammetry of dopamine at Nafion-coated glassy carbon electrodes: quantitative features of ion-exchange partition and reassessment on the oxidation mechanism of dopamine in the presence of excess ascorbic acid

The incorporation/exclusion features of dopamine (DA), ascorbic acid (AA) and uric acid (UA) are evaluated for Nafion (NA)-coated glassy carbon electrodes (GCE) of different thicknesses. The ion-exchange partition of DA(+) between the NA film and the sodium phosphate electrolyte is evaluated by determining the partition coefficient (k(D)) and the apparent diffusion coefficient (D(app)) in thick NA films which were 401 and 1.5 x 10(-9) cm(2) s(-1), respectively. The solution diffusion coefficient was found to be 6.0 x 10(-6) cm(2) s(-1). Also, the effect of NA loading and of the voltammetric timescale on DA voltammetry in the presence of excess AA is assessed, at physiologic like conditions. It is demonstrated that, although AA is excluded at the NA coating, a catalytic regeneration of DA, induced by AA, occurs at the interface NA film/electrolyte resulting from the diffusion of the o-quinone product of DA oxidation from the electrode surface to that interface. The interference of AA in the voltammetric signal of DA is eliminated using 18 microg mm(-2) NA films and v> or =0.5 V s(-1). Therefore, fast, selective and sensitive voltammetric analysis of DA at concentrations<100 microM in the presence of excess AA, e.g., 1 mM is achieved.     

An unpaired electron-based hole-transporting molecule: triarylamine-combined nitroxide radicals

A durable nitroxide radical combined with a triarylamine moiety exhibited a hole-drift mobility of 6 x 10(-3) cm(2) V(-1) s(-1), to which the aminophenyl nitroxide structure contributed.     

Comparative kinetic study for rate constant determination of the reaction of ascorbic acid with 2,6-dichlorophenolindophenol

The rate constant k of the reaction of ascorbic acid with 2,6-dichlorophenolindophenol (DCPI) in oxalic acid solutions is determined, by stopped-flow techniques. Four different methods are used to evaluate the results. The values and errors are compared statistically. The average of the rate constant is 56.5 x 10(3) l. mole(-1), sec(-1) and the overall standard deviation is 0.6 x 10(3) l. mole(-1), sec(-1) or 1.0% relative. The pH-dependence of the rate constant suggests that DCPI reacts with undissociated ascorbic acid.     

Stability study of simvastatin under hydrolytic conditions assessed by liquid chromatography

In this work, a liquid chromatography stability-indicating method was developed and applied to study the hydrolytic behavior of simvastatin in different pH values and temperatures. The selected chromatographic conditions were a C18 column; acetonitrile-28 mM phosphate buffer solution, pH 4 (65 + 35) as the mobile phase; 251 degrees C column temperature; and flow rate 1 mL/min. The developed method exhibited an adequate repeatability and reproducibility (coefficient of variation 0.54 and 0.74%, respectively) and a recovery higher than 98%. Furthermore, the detection and quantification limits were 9.1 x 10(-7) and 2.8 x 10(-6) M, respectively. The degradation of simvastatin fitted to pseudo-first order kinetics. The degradation was pH dependent, being much higher at alkaline pH than at acid pH. Activation energy, kinetic rate constants (k) at different temperatures, the half life (t1/2) and the time for 10% degradation to occur (t90) values are also reported.     

High-performance liquid chromatographic method for the simultaneous measurement of floxuridine and fluorouracil in human body fluids.

A method for the simultaneous measurement of floxuridine (5-fluorodeoxyuridine) and fluorouracil in human plasma and peritoneal fluid has been developed. This method utilizes high-performance liquid chromatographic analysis with a Zorbax RX column (25 cm x 4.6 mm I.D.) plus a guard cartridge of the same material. The sensitivity limits for this assay are 0.25 mumol/l in a 20-microliters sample. The detection limit at a signal-to-noise ratio of 3 is 2.5 nmol/l. This procedure has been used to quantitatively measure concentration versus time profiles of floxuridine and fluorouracil in plasma and peritoneal fluid of human patients after receiving intraperitoneal administration of floxuridine.