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Preformulation studies with melanotan-II: a potential skin cancer chemopreventive peptide.

Melanotan-II (1) is a cyclic heptapeptide analogue of alpha-melanocyte-stimulating hormone (alpha-MSH) which tans the skin and is currently being evaluated for the prevention of sunlight-induced skin cancers. The dissociation constants of 1 were determined using potentiometric titration and ultraviolet spectrophotometry. The pKa1 (histidine) and pKa2 (arginine) were estimated to be 6.54 and 11.72, respectively. The apparent partition coefficient (PC) was measured at three pH values using both n-octanol and isooctane as the nonpolar phase. The PC(octanol) and delta log PC at pH 7.35 were 2.82 and 1.05, respectively. These data, together with the observance of a bioavailability of 4.6% in the rat, indicate that 1 may be a suitable candidate for oral delivery. The data presented here are useful in developing an appropriate dosage form for 1.

Amino Acid Sequence↗

Structure and biosynthesis of novel conjugated polyene fatty acids from the marine green alga Anadyomene stellata.

Novel polyunsaturated fatty acids with four conjugated double bonds were found in extracts of the green macroalga, Anadyomene stellata. The isolation of five of these with different chain lengths and varying degrees of unsaturation--16:5, 18:4, 20:5, 20:6, and 22:7--was accomplished by organic extraction followed by a combination of vacuum and high-performance liquid chromatography. One of these that was a novel substance (22:7) was characterized as 4Z,7Z,9E,11E,13Z,16Z,19Z-do cosaheptaenoic acid and assigned the trivial name stellaheptaenoic acid. The structure of this new compound, isolated as its methyl ester derivative, was deduced from detailed nuclear magnetic resonance, gas chromatography/mass spectrometry (GC/MS), and other spectroscopic methods. Incubation of a chloroplast preparation, isolated from a crude algal homogenate by differential centrifugation, with six unsaturated fatty acids (palmitoleic, 6Z,9Z,12Z,15Z-octadecatetraenoic acid, arachidonic acid, eicosapentaenoic acid, 7Z,10Z,13Z,16Z-docosatetraenoic acid, and 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoi c acid) resulted in substantially increased synthesis of unique tetraene compounds as detected by ultraviolet spectrophotometry and tentatively identified by GC/MS.

Chlorophyta↗

The effect of pancuronium on the solubility of aqueous thiopentone.

In this study we identified the precipitate formed when pancuronium bromide (pH 3.9) and sodium thiopentone (pH 10.6) are combined in vitro. A precipitate formed when sufficient pancuronium was added to thiopentone to decrease the pH below 9.25. The precipitate was isolated, redissolved in alkaline solution and identified as thiopentone using ultraviolet spectrophotometry. Pancuronium was soluble in water in the pH range of 2.0 to 12.0, whereas thiopentone was soluble in water only at a pH greater than 9.90. We conclude that thiopentone precipitates when combined with pancuronium because of a pH-dependent decrease in the solubility of thiopentone, rather than a chemical interaction.

Anesthesia↗

Colorimetric determination of agarose-immobilized proteins by formation of copper-protein complexes.

A simple, reliable, and sensitive colorimetric procedure for the determination of proteins bound to agarose is described. The procedure utilizes the capacity of diethyldithiocarbamate to react with cupric ions resulting in a complex of dark yellow color. The extent of reduction in the color, due to chelation of Cu2+ by the immobilized proteins, indicates the amount of protein. The optimum conditions for the determination of immobilized proteins are investigated. The formation of Cu2+-protein complex proceeds stepwise until enough excess of Cu2+ is present to form the final complex. The reliability of the procedure requires that all the protein species, samples and standards, are in the final Cu2+-protein complex form. A comparative study on the determination of different proteins bound to agarose using this method as well as other methods, such as protein balance, modified Lowry reaction, and direct ultraviolet spectrophotometry, is described. The method is substantially more reliable, accurate, and simple than previously described methods.

Biuret Reaction↗

Formation of alpha-tocopherol complexes with fatty acids. Nature of complexes.

Using ultraviolet spectrophotometry and 1H-NMR high-resolution spectroscopy, it has been demonstrated that the formation of alpha-tocopherol complexes with free fatty acids occurs via two types of interaction, namely formation of a hydrogen bond between the alpha-tocopherol chromanol nucleus hydroxyl and the carboxyl group of a fatty acid, and interaction of the fatty acid acyl chains with the chromanol nucleus methyl groups. The second interaction is significantly enhanced by an increase in the number of double bonds in the fatty acid molecule, which results in restriction of the molecular mobility of alpha-tocopherol. The proposed structural model of alpha-tocopherol-fatty acid complexes has been confirmed by the use of molecular models. It has been assumed that the efficiency of complex formation of natural tocopherols with fatty acids is correlated with their biological activity.

Fatty Acids↗

Partition of DDE in synthetic and native membranes determined by ultraviolet derivative spectroscopy.

Partition coefficients of DDE (2,2-bis(p-chlorophenyl)-1,1-dichloroethylene) were determined, in model and native membranes, as a function of temperature, lipid chain length, cholesterol content and DDE concentration, by means of second derivative ultraviolet spectrophotometry. DDE incorporation increases with the temperature, since the partition values in dimyristoylphosphatidylcholine (DMPC), at 24, 30 and 37 degrees C, are 5722 +/- 138, 10356 +/- 763 and 14006 +/- 740, respectively. The insecticide incorporates better into bilayers of DMPC as compared with DPPC (dipalmitoylphosphatidylcholine). The partition decreases from 10355 +/- 763 in DMPC to 6432 +/- 613 in DPPC, at temperatures 5-7 degrees C above the midpoint of their transitions. The addition of cholesterol to fluid membranes of DMPC depresses the partition of DDE. In agreement with the results in models of synthetic lipids, the partition of DDE into native membranes increases with the temperature and decreases with the intrinsic cholesterol. It is concluded that a fluid membrane favors the accumulation of DDE.

Cholesterol↗

Cholesterol oxidase: thermochemical studies and the influence of hydroorganic solvents on enzyme activity.

Thermal and binary cosolvent studies of the cholesterol oxidase (cholesterol: oxygen oxidoreductase, EC 1.1.3.6) reaction have been carried out using batch microcalorimetry and ultraviolet spectrophotometry respectively. Heat conduction measurements are shown to provide the basis for a serum cholesterol assay yielding results comparable to conventional automated clinical assay. The enthalpy of the reaction for cholesterol oxidation, measured with different sources of the enzyme in the presence and absence of catalase is -113 +/- 7.2 mJ/mumol. The value is agreement with calculated estimates based on bond energies, enthalpies of formation and trigonal additivity contribution calculations. From this heat of reaction the deltaHf0 of cholestenone (c) is calculated to be -490 kJ . mol-1. No evidence for the reverse reaction could be adduced. Enzyme activation with detergent (Surfal) is attributed to the formation of mixed micelles of cholesterol with detergent molecules. The detergent concentration at which the enzyme is half activated corresponds to the critical micelle concentration of Surfal. The enhanced enzyme activity found when ethanol, acetonitrile and dioxane were examined as binary cosolvents with water is ascribed to a conformational change in the enzyme mediated through the altered structuredness of water. This cosolvent effect is abolished in the presence of 0.18% Surfal due to the formation of inverted mixed micelles of detergent with cholesterol.

3-Hydroxysteroid Dehydrogenases↗

Novel transformations of arachidonic acid by the rat thyroid in vitro.

The transformation of arachidonic acid by the rat thyroid in vitro has been investigated. At least two metabolites have been partially characterized: they differed from known metabolites of arachidonic acid in terms of retention volume in liquid chromatography, ultraviolet spectrophotometry and pharmacology (formation not inhibited by indomethacin and enhanced by eicosatetraynoic acid). The analysis by chemical ionization mass spectrometry suggested that these metabolites might be diketo-monohydroxy- and monoketo-dihydroxy-compounds. The conversion of arachidonic acid into these compounds was stimulated by ionophore A23187, decreased by the peroxidase inhibitor methimazole and potentiated by iodide, suggesting that this pathway is under the control of Ca2+ and of a peroxidase product.

Animals↗

A new metabolic pathway for N,N-dimethyltryptamine.

N,N-Dimethyltryptamine (DMT) undergoes a major structural alteration when added to whole human blood or its red blood cells in vitro. A new high-pressure liquid chromatography (HPLC) peak is present in extracts of these treated tissues. The compound responsible for this peak has been identified by ultraviolet spectrophotometry and by mass spectrometry as dimethylkynuramine (DMK). The enzyme responsible for this appears to be different from tryptophan 2,3-dioxygenase and also from indoleamine 2,3-dioxygenase.

Carbon Radioisotopes↗

A modified ultraviolet spectrophotometric method for the determination of theophylline in serum in the presence of barbiturates.

Ultraviolet spectrophotometry is the most commonly used technique for the determination of theophylline levels for therapeutic monitoring. Common interferences in most methods are barbiturates and xanthines. A modified method which eliminates interferences from barbiturates and most xanthines is presented. Theophylline is extracted from serum with chloroform/isopropanol at pH 7.4 back extracted into dilute hydrochloric acid and then the solution is made alkaline with sodium hydroxide. Barbiturate interferences are eliminated. Interference from caffeine, uric acid, 7-(2,3-dihydroxypropyl)theophylline, xanthine, and hypoxanthine are not observed. Theobromine, and the metabolite 3-methylxanthine interfere. Interference is not observed from quinidine, diazepam, salicylate, glutethimide, methylprylon, propranolol, methaqualone, dilatin and ethchlorvynol. Sulfanilamide, procainamide and chlordiazepoxide interfere.

Barbiturates↗

The binding of Ni2+ to adenylyl-3',5'-adenosine and to poly(adenylic acid).

Studies of the binding of Ni2+ to adenylyl-3',5'-adenosine (ApA) at pH 6.0 by ultraviolet spectrophotometry indicate the formation of a 1:1 complex in the presence of a large excess of metal ion. At 25 degrees C, and ionic strength mu = 0.5 M, the stability constant of Ni(ApA) is evaluated to be K = 2.6 (+/- 0.6) M-1. The low stability is taken as evidence that the predominant complex species is one in which the ApA acts as a monodentate ligand, mainly through the adenine group. The rate constants for complex formation and dissociation, kf = 1430 M-1 s-1 and kb =665 s-1 (25 degrees C, mu=0.5 M), determined by the temperature-jump relaxation technique, are consistent with this interpretation. The binding strength of Ni2+ to poly(adenylic acid) [poly(A)] has been studied at pH 7.0 using murexide as an indicator of the concentration of free Ni2+. Within the concentration range [Ni2+]free = 1 x 10(-5) -1 x 10(-3) M the data can be represented in the form of a linear Scatchard plot, i.e., the process can be described as the binding of Ni2+ to one class of independent binding sites. The number of binding sites per monomer is 0.26, and the stability constant K=8.2 x 10(3) M-1 (25 degrees C, mu=0.1 M). In kinetic studies of the reaction of Ni2+ with poly(A), two relaxation effects due to complex formation were detected, one with a concentration-independent time constant of about 0.4 ms, the other with a concentration-dependent time constant in the millisecond range. The concentration dependence of the longer relaxation time can be accounted for by a three-step mechanism which consists of a fast second-order association reaction followed by two first-order steps. There is evidence, however, that the overall process is more complicated than expressed by the three-step mechanism.

Adenosine Monophosphate↗

A hydrogen exchange study of the open segment in a DNA double helix.

The kinetics of the hydrogen-deuterium exchange reactions of double-helical poly[d(A-T)] X poly[d(A-T)], poly(dA) X poly(dT), and constituent nucleosides (deoxyadenosine and thymidine) have been examined at various temperatures by stopped-flow ultraviolet spectrophotometry, in the spectral region 240-300 nm. The results were interpreted on the basis of a mechanism of the hydrogen exchange reaction of a helical polynucleotide, proposed by Englander and colleagues as well as by the Tsuboi and Nakanishi group. It was concluded that the rates of the base-pair opening reactions are nearly equal to one another in double-helical DNAs, irrespective of the base sequence. On the other hand, the free energy required for bringing the open segment at a particular base-pair was found to be much greater for poly(dA) X poly(dT) than for poly[d(A-T)] X poly[d(A-T)].

DNA↗

Determination of penicillin-V in human plasma by high-performance liquid chromatography and solid-phase extraction.

A high-performance liquid chromatographic method has been developed for the determination of penicillin-V concentrations between 0.1 and 19 micrograms/ml in human plasma. Penicillin-V was isolated from plasma by solid-phase extraction on a C18/OH cartridge. The extracts were injected onto a reversed-phase HPLC system. A 125 x 4 mm C18 column was used to separate penicillin-V from its main metabolites, 5R- and 5S-penicilloic acid and endogenous compounds. The eluent consisted of 66% 0.02 M phosphoric acid buffer, to which tetrabutylammonium dihydrogenphosphate and 34% acetonitrile were added. The column effluent was monitored by ultraviolet spectrophotometry at 269 nm. Using this method, penicillin-V concentrations in plasma could be determined with an accuracy between -5.4 and 5.2% and a precision between 0.8 and 1.6%. The method has proved to be reliable and was used in bioavailability studies for the development of a new oral penicillin-V formulation.

Calibration↗

Determination of citalopram and escitalopram together with their active main metabolites desmethyl(es-)citalopram in human serum by column-switching high performance liquid chromatography (HPLC) and spectrophotometric detection.

We established a method for automated quantitative analysis of (es-)citalopram and desmethyl(es-)citalopram in serum using column-switching high performance liquid chromatography (HPLC). For sample clean-up serum was injected onto a LiChrospher CN 20 microm precolumn using 8% acetonitrile in deionized water. Drugs were eluted by back-flush flow onto the analytical column (LiChrospher CN 5 microm) at a flow rate of 1.5 ml/min with phosphate buffer 8 mmol/l pH 6.4/acetonitrile (50/50, v/v). Haloperidol was used as internal standard. Analytes were detected by ultraviolet spectrophotometry at 210 nm. Detection limit of (es-)citalopram was 6 ng/ml. The method was found to be suitable for therapeutic drug monitoring of patients treated with citalopram or escitalopram.

Chromatography, High Pressure Liquid↗

Characterization of hydroxypropylmethylcellulose films using microwave non-destructive testing technique.

The applicability of microwave non-destructive testing (NDT) technique in characterization of matrix property of pharmaceutical films was investigated. Hydroxypropylmethylcellulose and loratadine were selected as model matrix polymer and drug, respectively. Both blank and drug loaded hydroxypropylmethylcellulose films were prepared using the solvent-evaporation method and were conditioned at the relative humidity of 25, 50 and 75% prior to physicochemical characterization using microwave NDT technique as well as ultraviolet spectrophotometry, differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR) techniques. The results indicated that blank hydroxypropylmethylcellulose film exhibited a greater propensity of polymer-polymer interaction at the O-H and C-H domains of the polymer chains upon conditioned at a lower level of relative humidity. In the case of loratadine loaded films, a greater propensity of polymer-polymer and/or drug-polymer interaction via the O-H moiety was mediated in samples conditioned at the lower level of relative humidity, and via the C-H moiety when 50% relative humidity was selected as the condition for sample storage. Apparently, the absorption and transmission characteristics of both blank and drug loaded films for microwave varied with the state of polymer-polymer and/or drug-polymer interaction involving the O-H and C-H moieties. The measurement of microwave NDT test at 8GHz was sensitive to the chemical environment involving O-H moiety while it was greatly governed by the C-H moiety in test conducted at a higher frequency band of microwave. Similar observation was obtained with respect to the profiles of microwave NDT measurements against the state of polymer-polymer and/or drug-polymer interaction of hydroxypropylmethylcellulose films containing chlorpheniramine maleate. The microwave NDT measurement is potentially suitable for use as an apparent indicator of the state of polymer-polymer and drug-polymer interaction of the matrix.

Administration, Cutaneous↗

High temperature stabilization of DNA in complexes with cationic lipids.

The influence on the melting of calf thymus and plasmid DNA of cationic lipids of the type used in gene therapy was studied by ultraviolet spectrophotometry and differential scanning calorimetry. It was found that various membrane-forming cationic lipids are able to protect calf thymus DNA against denaturation at 100 degrees C. After interaction with cationic lipids, the differential scanning calorimetry melting profile of both calf thymus and plasmid DNA revealed two major components, one corresponding to a thermolabile complex with transition temperature, T(m(labile)), close to that of free DNA and a second corresponding to a thermostable complex with a transition temperature, T(m(stable)), at 105 to 115 degrees C. The parameter T(m(stable)) did not depend on the charge ratio, R(+/-). Instead, the amount of thermostable DNA and the enthalpy ratio Delta H((stable))/Delta H((labile)) depended upon R(+/-) and conditions of complex formation. In the case of O-ethyldioleoylphosphatidylcholine, the cationic lipid that was the main subject of the investigation, the maximal stabilization of DNA exceeded 90% between R(+/-) = 1.5 and 3.0. Several other lipids gave at least 75% protection in the range R(+/-) = 1.5 to 2.0. Centrifugal separation of the thermostable and thermolabile fractions revealed that almost all the transfection activity was present at the thermostable fraction. Electron microscopy of the thermostable complex demonstrated the presence of multilamellar membranes with a periodicity 6.0 to 6.5 nm. This periodic multilamellar structure was retained at temperatures as high as 130 degrees C. It is concluded that constraint of the DNA molecules between oppositely charged membrane surfaces in the multilamellar complex is responsible for DNA stabilization.

Animals↗

An ultraviolet spectrophotometric procedure for the routine determination of naproxen.

1. A procedure for the routine assay of Naproxen in serum by ultraviolet spectrophotometry is presented. The absorption coefficient (A1 1% cm) of Naproxen in methanol at 261 nm was found to be 216. 2. The Naproxen procedure appears to be relatively free from interferences of commonly coadministered medications. Salicylate interference is eliminated by a modified procedure. 3. Concentrations of Naproxen in a limited number (40) of clinical samples ranged from non-detected to 12.5 mg/dl. The therapeutic concentration appears to be 4 to 6 mg/dl (174-260 mumol/L).

Humans↗

Studies on the pharmacological properties of novel arylene bis(methylketone) compounds using solid-phase extraction and high-performance liquid chromatography.

A method utilising solid-phase extraction followed by high-performance liquid chromatography has been developed to quantify novel arylene bis(methylketone) chemotherapeutics present in biological samples. The samples are extracted over cyanopropylsilane solid-phase extraction cartridges using 10 mM heptanesulfonate-10 mM tetramethylammonium chloride-4.2 mM H3PO4-95% CH3CN as the eluent. Analytical chromatography utilises a diisopropyl-C8 reversed-phase column and a 7.5-45% CH3CN gradient in 10 mM heptanesulfonate-10 mM tetramethylammonium chloride-4.2 mM H3PO4-H2O. Detection was by ultraviolet spectrophotometry at 300 or 240 nm. The linear response of the assay was found to extend from at least 100 microg/ml down to 97.66 ng/ml for a 100 microl injection. The assay system was utilised to determine the plasma kinetics of the compounds in mice, where all the drugs were found to display rapid absorption and elimination following intraperitoneal dosing. In vitro and in vivo studies of metabolism demonstrated that each of the compounds produced several metabolites, and that this conversion could be extensive in vivo.

Aniline Compounds↗