Metabolic pathways of flobufen-a new antirheumatic and antiarthritic drug. Interspecies comparison.

Metabolic transformations of flobufen, [4-(2',4'-difluoro-biphenyl-4-y1)-4-oxo-2-methylbutanoic acid], a non-steroid antiinflammatory agent, were studied in vitro using the following biological models and species: rat and mouse liver homogenates and liver subcellular fractions (5 000 g and 100 000 g supernatant, mitochondria); rat, mouse, rabbit, guinea-pig and mini-pig liver microsomes; isolated rat hepatocytes; perfused rat liver and 5000 g rat muscle tissue supernatant. Reduced flobufen [4-(2',4'-difluorobiphenyl-4-yl)-4-hydroxy-2-methylbutanoic acid] is the major metabolite generated by the subcellular fractions (in the mild acidic extraction conditions during subsequent laboratory processing is converted to its lactone form). It was detected upon the incubation of flobufen with liver microsomes isolated from all the animals tested. Maximum yield of reduced flobufen in experiments with rat and mouse liver microsomes was found after anaerobic incubation with NADPH. This finding combined with the knowledge of subcellular distribution of enzymes suggest that metabolite formation depends on the activity of microsomal reductases and, probably, also on the activity of the important microsomal reductase, cytochrome P-450. Another flobufen metabolite, arylacetic acid [(2',4'-difluorobiphenyl-4-yl)ethanoic acid], is generated from the reduced metabolite by the cleavage of its side chain, and was detected in isolated hepatocytes - it was the only metabolite found in urine and faeces upon oral administration of the drug. All these metabolites were identified and quantified.

Separation of the stereoisomers of the main metabolite of a non-steroidal anti-inflammatory drug, flobufen, by chiral high-performance liquid chromatography.

The major metabolite of a novel non-steroidal anti-inflammatory drug, DL-4-(2',4'-difluorobiphenyl-4-yl)-2-oxo-2-methylbutanoic acid (flobufen, I), namely 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-gamma-butyrolactone (4-dihydroflobufen lactone, III), has four stereoisomers consisting of two racemic pairs of enantiomers. Of three chiral stationary phases tested, Cyclobond I beta-RSP (Astec) (beta-cylodextrin derivatized with R,S-hydroxypropyl) was best able to separate the (+2)(--) racemate, with a liquid phase containing acetonitrile as modifier and triethylamine acetate as buffer. Using the Box-Wilson Central Composite Design for three factors, an optimum combination of pH and concentrations of the modifier and buffer was eventually obtained. A chromatographic response function based on a combination of the Kaiser peak separation function, Pi, and retention time of the second eluting enantiomer, tRL, served as a response criterion for the process of optimization. The optimum conditions developed for the (+2)(--) racemate were also found to be suitable for separating the (+-)(-+) racemate, for which earlier studies had shown the separation to be more facile. Separation of the four stereoisomers of III, for which the chiral chromatographic system optimized in this study is proposed as the second stage, is targeted at a biochemical study of the stereoisomeric metabolism of I.

High-performance liquid chromatographic assay for the separation and characterization of metabolites of the potential cytostatic drug oracine.

Oracine (I), a potential cytostatic drug, is enzymically converted to a number of metabolites whose formation has been studied in vitro and in vivo. The metabolites were separated by reversed-phase HPLC and characterized by UV spectra. Preparative TLC served for the isolation of the individual metabolites to allow their identification. Two metabolites were identified by Fourier transform NMR as 11-dihydrooracine (II) and a phenolic product (III). Two further metabolites (IV,V) were characterized. Some minor, presumably 11-dihydro metabolites and an 11-oxo metabolite produced in vitro and in vivo were revealed.

Urinary excretion of indolylacryloylglycine, indolylacetic acid and 5-hydroxyindolylacetic acid in burn patients.

Indolylacryloylglycine (IAcrGly) is a regular constituent of human urine. Changes in its excretion have been observed, among other conditions, in some skin diseases. Skin lesions in burns are dealt with in the present paper. IAcrGly excretion has been followed during several weeks of hospital treatment and compared with that of two other tryptophan metabolites, namely indolylacetic acid (IAA) and 5-hydroxyindolylacetic acid (5-HIAA). The average excretion of IAcrGly was significantly lower especially between day 3 and 15 post injury. Some alternative explanations of these results are put forward.

Biotransformation of the sympathomimetic tetraminol in vitro.

On incubation with the postmitochondrial fraction of the liver homogenate of rabbits, guinea-pigs, rats, and mice in the presence of NADPH and oxygen, the alpha-sympathomimetic trans-3-(2-hydroxyethylamino)-5,8-dimethoxy-1,2,3,4-tetrahydro-2-n aphthol (Tetraminol, 1) is preferentially O-demethylated in position 8, yielding metabolite 3. In male rats O-demethylation is stronger than in females.

Elimination of benfluron and its metabolites in the faeces and urine of rats.

The study of the biotransformation of the potential cytostatic benfluron has been continued. The elimination of benfluron and of nine of its metabolites whose structure had been established, mainly on the basis of the comparison of their IR, MS and NMR spectra with those of standards, was studied. After oral administration of 500 mg.kg-1 to rats, the amounts of these substances in the faeces and urine were followed up by high-performance liquid chromatography for five days. Striking qualitative and quantitative differences were observed in the elimination of benfluron and its metabolites by both routes.

Interaction of Z-4,4-bis(4-ethylphenyl)-2,3-dibromo-2-butenoic acid with aminotransferases: changes in absorption and circular dichroism spectra.

The influence of Z-4,4-bis(4-ethylphenyl)-2,3-dibromo-2-butenoic acid, the compound originally synthetized as a cytostatic edikron and showing inhibitory effect on several pyridoxal enzymes, on absorption and circular dichroism spectra of alanine and aspartate aminotransferases (ALT, AST) in the region of coenzyme absorption characteristics was studied. In the case of AST, the compound decreased absorption and CD maxima at 360 nm, which represents the active form of the enzyme, but it did not seem to prevent formation of the pyridoxamine form of the enzyme, produced in the presence of L-aspartate. Edikron caused insignificant spectral changes of ALT, but it partially denatured the enzyme. Circular dichroism measurement of both enzymes uncovered some effects of edikron at 250-300 nm, which suggests conformational changes in the aromatic amino acids of the apoenzymes due to the compound studied.

Evaluation of photometric high-performance liquid chromatographic data for the determination of benflurone metabolites in biological materials with and without concomitant use of a standard.

The results produced by a new method of calculation based on the knowledge of the absorption coefficient and instrumental parameters without the concomitant use of a standard were compared with those calculated by the routine external standard method for a model system utilizing the quantification of benzofluorene derivatives. These were present in the incubation mixture of 5-[2-(dimethylamino)ethoxy]-7-oxo-7H-benzo[c]fluorene (benflurone) with the microsomal fraction of rat liver homogenate. Reasonable agreement between the two methods was observed. The potential utility of the new method of calculation is discussed.

[Inhibition of aromatic amino acid decarboxylases by the anti-inflammatory agent flobufen and 2',4'-difluorbiphenyl-4-acetic acid]. / Inhibice dekarboxylázy aromatických aminokyselin protizánetlivými látkami flobufenem a kyselinou 2',4'-difluorbifenyl-4-yloctovou.

The effect of the novel Czechoslovak antiphlogistic agent flobufen (VUFB 16066, 4-[2',4'-difluorbiphenyl]-4-oxo-methyl-butanic acid) and its metabolite 2',4'-difluorbiphenyl-4-yl acetic acid (VUFB 17203) on decarboxylase of aromatic amino acids (DAAK, E. C. 4.1.1.28) of the rat liver (supernatant 20,000 x g) was studied in vitro. The concentrations of 3.8 x 10(-4) mol x 1(-1) of flobufen and 4.4 x 10(-4) mol x 1(-1) of difluorbiphenylyl acetic acid produced 50% inhibition n the enzyme. Ki for flobufen is approximately 1.5 x 10(-4) mol x 1(-1), the reaction with the inhibitor takes place in the ratio 1:1 and the enzyme-inhibitor complex does not exert any catalytic activity. In the tissues with a low activity of DAAK, inhibition could be manifested by decreased synthesis of serotonin and dopamine.

Chromatography beyond analytical chemistry.

Although chromatography is one of the most important branches of analytical chemistry, it also serves purposes that cannot strictly be considered as part of analytical chemistry: it may be a model of natural processes, a method for the study of surface properties of molecules, for the collection of data in quantitative structure-activity relationship studies and for preparative separations, a teaching aid and sometimes even a kind of visual art. Some speculations and proposals are summarized.

Direct absorptiometric quantitation in high-performance liquid chromatography in the absence of concomitant reference standards.

A method for absorptiometric quantification in high-performance liquid chromatography that utilizes the known molar absorption coefficients of the individual components of a mixture of analytes to enable a series of determinations to be carried out without the concomitant use of reference standards is described. An equation is derived that describes the dependence of the molar amount of a defined analyte on the parameters influencing the detector response and on the peak area of the analyte in the chromatogram. The equation can be used for the direct calculation of the molar amount of individual analytes in a well resolved mixture. A method for the analysis of two analytes, the peaks of which totally overlap but which differ by their molar absorption coefficients, is also described. The validity of the equations and the applicability of the proposed method was examined in the analysis of 5-(2-dimethylaminoethoxy)-7-oxo-7H-benzo[c]fluorene hydrochloride (benflurone) and its metabolites. Some examples of the application of this approach are considered.

Biospecific or non-specific adsorption of amylase on starch in Starkenstein's experiments (1910)?

A paper by Starkenstein on the influence of chloride on the enzymatic activity of liver amylase, including a description of the adsorption of the enzyme on starch, is discussed with reference to the early concepts of enzyme-substrate binding and to later work on specific adsorption of amylase on starch.

Biospecific sorption, Prague, 1910: Emil Starkenstein (1884-1942).

Emil Starkenstein's paper (1910) on the influence of chloride on the enzymatic activity of liver amylase has been considered generally as the first experimental demonstration of the biospecific adsorption of an enzyme on a solid substrate. Emil Starkenstein's life is also briefly mentioned.

Chromatographic characterization of in vitro metabolites of 5-[2-(N,N-dimethylamino)ethoxy]-7-oxo-7H-benzo[c]fluorene.

Detection reactions and RF values in thin-layer chromatography on silica gel were studied for the antineoplastic drug Ih (benfluron) and related substances. On incubation of Ih with homogenate fractions of mammalian livers the N-oxide Ii and 5-[2-(N,N-dimethylamino)ethoxy]-7-hydroxy-7 H-benzo[c]fluorene (IIh) were established as products, and 5-[2-(N-methylamino)ethoxy]-7-oxo-7 H-benzo[c]fluorene (Ig), 5-[2-(N-methylamino)ethoxy]-7-hydroxy-7 H-benzo[c]fluorene (IIg) and a phenolic product of benfluron (IV) were tentatively identified.