Modulatory effects of quercetin and rutin on the activity, expression and inducibility of CYP1A1 in intestinal HCT-8 cells.

Flavonoids, plant secondary metabolites present in fruits and vegetables, show antioxidant and anti-tumorigenic effects in vitro, but their poor absorption from gastrointestinal tract limits their systemic efficacy in humans. On the other hand, flavonoids could protect intestinal cells against carcinogens by their potential to inhibit the enzymes metabolizing pre-carcinogenic compounds (e.g. CYP1A) to reactive ones. This work was designed to test the effect of quercetin (the most abundant flavonoid) and rutin (the most abundant glycosidic form) on the activity, expression and inducibility of CYP1A in intestinal HCT­8 cells. CYP1A enzymatic activity was measured by ethoxyresorufin-O-deethylase (EROD) activity, CYP1A protein expression was detected by western blotting. The effect of flavonoids on viability of cells was examined by neutral red uptake test. No cytotoxic effect of flavonoids up to 50 µM concentration was observed. Quercetin significantly inhibited EROD activity in the cells, where CYP1A had been preinduced by b-naphthoflavone and methylcholanthrene, and it also significantly reduced the CYP1A induction mediated by these model inducers. The effect of rutin was substantially weaker and mostly in significant in all conducted experiments. The results suggest that quercetin may have a potential to limit the CYP1A-mediated activation of pre-carcinogens in intestinal cells.

The involvement of selected membrane transport mechanisms in the cellular uptake of (177)Lu-labeled bombesin, somatostatin and gastrin analogues.

INTRODUCTION: Radiolabeled receptor-targeting peptides are a useful tool for the diagnostic imaging and radiotherapy of some malignancies. However, the retention of radioactivity in the kidney may result in renal radiotoxic injury. This study seeks to evaluate the role of endocytic receptor megalin, renal SLC influx transporters and fluid phase endocytosis (FPE) in the cellular accumulation of radiolabeled peptides. METHODS: In vitro transport cellular studies using megalin ligands (RAP, albumin), fluid phase endocytosis (FPE) inhibitor rottlerin and low temperature were employed to evaluate the transport mechanisms of the peptides. Cells transfected with hOAT1 or hOCT2 were used to analyze the role of these SLC transporters. Somatostatin ((177)Lu-DOTA-[Tyr(3)]octreotate, (177)Lu-DOTA-[1-Nal(3)]octreotide), gastrin ((177)Lu-DOTA-sargastrin) and bombesin ((177)Lu-DOTA-[Pro(1),Tyr(4)]bombesin, (177)Lu-DOTA-[Lys(3)]bombesin, (177)Lu-PCTA-[Lys(3)]bombesin) analogues were involved in the study. RESULTS: RAP, albumin and low temperature decreased the accumulation of all the studied peptides significantly. With one exception, rottlerin caused the concentration dependent inhibition of the cellular accumulation of the radiopeptides. No significant differences in the uptake of the peptides between the control cells and those transfected with hOAT1 or hOCT2 were observed. CONCLUSION: The study showed that active transport mechanisms are decisive for the cellular accumulation in all tested (177)Lu-labeled somatostatin, gastrin and bombesin analogues. Besides receptor-mediated endocytosis by megalin, FPE participates significantly in the uptake. The tested types of renal SLC transporters are not involved in this process.

Pharmacokinetics and renal handling of 99mTc-labeled peptides.

UNLABELLED: 99mTc-labeled peptides, particularly those of a lipophilic nature, are often excreted through the hepatobiliary system, and the subsequent accumulation in the intestine may obscure receptor-mediated uptake in tumor sites in the pelvis. We have therefore explored the route and rate of excretion of a small series of Tc-labeled peptides to shed some light on the mechanisms that influence the clearance of these agents. METHODS: Pharmacokinetic parameters, biodistribution, routes of elimination of 99mTc-complexes of 3 model tetrapeptides--namely, acetyl-N-Gly-Gly-Cys-Gly (AGGCG), acetyl-N-Ser-Ser-Cys-Gly (ASSCG), and acetyl-N-Gly-Gly-Cys-Lys (AGGCL)--were determined in rats in vivo. Renal handling of the complexes was studied in the perfused rat kidney. RESULTS: After intravenous injection, a relatively fast disappearance of the complexes from blood was found. Although the parameters of distribution in all 3 chelates were very similar, the elimination rate of 99mTc-AGGCG was higher than those of 99mTc-ASSCG and 99mTc-AGGCL. The Tc complexes under study were distributed mainly to the excretory organs (kidneys and liver), and no specific accumulation in other organs or tissues was found. Most of the radioactivity after intravenous administration of the chelates was rapidly eliminated through the urine, but a significant amount was also excreted through the feces, in the following order among the 3 chelates: 99mTc-AGGCL < 99mTc-ASSCG < 99mTc-AGGCG. Different proportions of glomerular filtration and secretion in renal tubules of the complexes were found in the perfused rat kidney. Elimination by glomerular filtration was dominant only in the case of 99mTc-AGGCL, whereas the rate of filtration of 99mTc-AGGCG was very low because of its high protein binding. Various rates of secretion into renal tubules were shown for all 3 agents. This renal excretion pathway was decisive in 99mTc-AGGCG and lowest in 99mTc-AGGCL. 99mTc-ASSCG was eliminated by both mechanisms at similar rates. CONCLUSION: These studies show that increasing the hydrophilic nature or reducing the negative charge of the peptides will reduce their hepatobiliary excretion, whereas the incorporation of suitable peptide sequences permits them to exploit efficient routes of renal excretion, such as tubular secretion, thereby optimizing the pattern of biodistribution of these radiopharmaceuticals.

Possible use of excretion of tubular epithelial cells for the study of the nephrotoxic effect of xenobiotics.

The method of determination of the minute excretion of tubular epithelial cells renders it possible to investigate the course of the nephrotoxic effect of the toxin by the influence of which excretion of tubular round epithelial cells is increased. The nephrotoxic effect of repeated administration of amphotericin B (1 mg/kg, i.v.), which produced up to 12-fold increases in the number of excreted epithelial cells, was examined. Repeated administration of cyclosporin A (45 and 56 mg/kg, p.o.) produced up to 23-fold increases in the number of excreted epithelial cells. The degree of excretion of epithelial cells after administration of both drugs was compared with the urinary excretion of alanine aminopeptidase and N-acetyl-beta-D-glucosaminidase, which indicated nephrotoxicity in amphotericin B and cyclosporin A with a lower sensitivity than the increase in the excretion of epithelial cells. In the experiment with cyclosporin A, urinary excretion of epithelial cells was further correlated with renal functional tests (clearance of polyfructosan and hippurate.

N-demethylation activity of renal and hepatic subcellular fractions: an interspecies comparison.

The enzymatic activity of the mixed-function oxidase system in the kidney and liver was evaluated by means of an in vitro N-demethylation activity assay with aminopyrine as the substrate. Renal and hepatic demethylation activity of 9000 x g supernatant fraction was determined in the rat, rabbit, and guinea-pig. In terms of interspecies comparison, the renal tissue demethylation activities were on a similar level with a slight increase in the order guinea-pig, rabbit and rat. In relation to hepatic activity, these relative demethylation activities of renal tissue had the same values in the rat and rabbit, whereas that in the guinea pig was significantly lower. The distribution of demethylation activity in the kidney was determined by comparing the cortex and medullary activity in relation to the total kidney tissue activity in the rabbit and guinea-pig. Although the higher demethylation activities were obtained in rabbit renal preparations and low demethylation activity was detected in the guinea-pig renal medulla only, no significant interspecies differences were found by the statistical evaluation. It may be concluded that the mixed-function oxidase system responsible for renal demethylation activity seems to be concentrated in the renal cortex and its distribution coincides in the rabbit and guinea-pig kidney.

Use of rat hepatocytes immobilized in agarose gel threads for biosynthesis of metabolites of potential cytostatics.

The aim of this work was to evaluate possibility of use of the rat isolated hepatocytes immobilized in agarose gel and continuously perfused for production of needed metabolites of two potential cytostatics, benfluron (5-(2-dimethylamino-ethoxy)-7-oxo-7H-benzo[c]fluorene) and oracin (6-[2-(hydroxyethyl) amino-ethyl]-5,11-dioxo-5,6-dihydro-11H-indeno [1,2-c]isoquinoline). The rat isolated hepatocytes obtained by two-step collagenase perfusion method were immobilized in agarose threads and perfused in a small bioreactor under a recirculation regimes. Biosynthesis of 9-hydroxybenfluron and 3-hydroxyoracin in immobilized rat hepatocytes was studied. Yields of the metabolites of interest in hepatocytes in immobilized and perfused rat hepatocytes was compared to production of metabolites in hepatocyte suspension and in rats in vivo. 9-hydroxybenfluron was presented during perfusion of immobilized rat hepatocytes in a relatively high amounts but total recovery all forms of benfluron was very low due to especially high binding to components of the perfusion system. More effective method remains the production of 9-hydroxybenfluron in rats in vivo. A considerable biosynthesis of 3-hydroxyoracin by immobilized rat hepatocytes in the bioreactor was found. Concentration of the metabolite in the perfusate rose continuously during 6 hours of perfusion. 3-hydroxyoracin production was increased several times with use of immobilized hepatocytes from rats treated for three days with methylcholanthrene. The yield of 3-hydroxyoracin in rats in vivo was comparably high but an advantage of in vitro synthesis is a much shorter interval to obtain the same amount of the metabolite of interest. In spite of some limitations in compounds exerting high trapping in the perfusion system, the method of the immobilized and perfused hepatocytes can be very useful and effective for production of some drug metabolites in biochemistry and pharmacology.

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.

Distribution and elimination characteristics of 111In-DTPA-D-phe1-octreotide and 111In-DTPA-L-phe1-octreotide in rats.

The present study compares distribution and elimination characteristics of 111In-DTPA-D-Phe1-octreotide and 111In-DTPA-L-Phe1-octreotide in rats and evaluated the effect of the replacement of the terminal L-phenylalanine by D-phenylalanine on pharmacokinetic profiles of the radiolabelled peptides. Both agents exhibited rapid radioactivity clearance from the blood and most organs and tissues with no systematic and significant differences in activity accumulation. The long-term retention and high radioactivity concentrations for both compounds under study were found in the kidneys and organs with a high density of somatostatin receptors, such as the pancreas and adrenals. The residence times in these organs were longer for 111In-DTPA-D-Phe1-octreotide in comparison with 111In-DTPA-L-Phe1-octreotide. The major elimination pathway for both radiolabelled peptides was relatively rapid excretion into the urine. Analysis of the renal handling by an employment of the perfused rat kidney showed that both peptides were eliminated mainly by the mechanism of glomerular filtration. Rat liver perfusion experiments confirmed a very low value of bile clearance of radioactivity for both agents under study.

Biodistribution of two octreotate analogs radiolabeled with indium and yttrium in rats.

BACKGROUND: In this study, two octreotate derivatives N-[4-carboxy-4-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]butanoyl]-Tyr(3)-octreotate (DOTAGA-tate) and N-[[4,10-bis(carboxymethyl)-7-(1(1,3-dicarboxypropyl))-1,4,7,10-tetraaza-cyclododec-1-yl]acetyl]-Tyr(3)-octreotate (DOTA-t-GA-tate) were radio-labeled with (111)In or (88)Y and their biodistribution profiles together with their elimination characteristics in rats were compared. MATERIALS AND METHODS: Radiolabeling of the peptides with high radiochemical purity was carried out in an acetate buffer with gentisic acid as radioprotective compound. Biodistribution profiles of the radiolabeled peptides were determined in intact male Wistar rats after an intravenous dose of 1 microg/kg. For elimination pathways analysis, studies in intact rats in metabolic cages and perfused rat kidney and liver were carried out. RESULTS: Fast radioactivity clearance from rat tissues (excepting somatostatin receptor-rich organs and the kidney) was determined for all agents under study. Profound radioactivity uptake in organs with a high density of somatostatin receptors (namely the adrenals and pancreas as biomarkers of somatostatin receptor-positive tissue) was slightly higher for radiolabeled DOTAGA-tate when compared with DOTA-t-GA-tate. Significantly higher accumulation in kidney and somewhat lower urinary elimination of (111)In-labeled peptides in comparison with that of (88)Y-agents were determined. Perfused rat kidney experiments confirmed that glomerular filtration was the main elimination mechanism for the compounds under study; their bile clearances in the perfused rat liver were negligible. CONCLUSION: (111)In((88)Y)-DOTAGA-tates exhibited higher distribution into somatostatin receptor-rich organs when compared with the corresponding radiolabeled DOTA-t-GA-tates. Higher uptake of (111)In-labeled peptides in the kidney is attributed to its different coordination properties.

Analysis of renal handling of radiopharmaceuticals.

Renal excretion is the preferable elimination pathway of radiopharmaceuticals and/or their metabolites from the body. The mechanisms of renal excretion involve glomerular filtration, tubular secretion, tubular reabsorption, and kidney metabolism. Radiopharmaceuticals of a molecular weight of up to 60 kDa are ultrafiltrated in the glomeruli at a rate dependent on their protein binding (the rate of glomerular filtration is a product of glomerular filtration rate and free fraction of the agent in plasma). Exclusive excretion by this route is typical for many chelates such us 99mTc-DTPA (diethylenetriaminepentaacetic acid). Some other radiopharmaceuticals are excreted in the renal tubuli into the tubular fluid by carrier-mediated and active processes involving different transport systems. Examples are ortho-iodohippurate and 99mTc-MAG-3 (mercaptoacetyltriglycine). Tubular reabsorption involves either passive diffusion of lipid-soluble radiopharmaceuticals from the glomerular filtrate back into blood or active, carrier-mediated process. Kidney metabolism and consequent renal uptake of the catabolites is of an outstanding importance for possible therapeutic applications of radiolabeled antibody fragments and peptides. These agents are partially reabsorbed from the ultrafiltrate by the cells of the renal proximal tubules by means of pinocytosis and subsequently degraded in the lysosomes. It limits their therapeutic use due to the potential radiation nephrotoxicity. For the analysis of elimination mechanisms of radiopharmaceuticals in the kidney, different approaches at various experimental levels (the kidney perfusion technique, isolated, functionally intact renal tubules, and isolated membranes) could be successfully employed.

Stereospecificity and stereoselectivity of flobufen metabolic profile in male rats in vitro and in vivo: phase I of biotransformation.

Flobufen (F) is the original nonsteroidal antiinflammatory drug (NSAID) containing two enantiomers. The aim of this investigation was to elucidate the biotransformation pathway of F at chiral level in phase I of biotransformation. Stereoselectivity and stereospecificity of the respective enzymes were studied in male rats in vitro (microsomal and cytosolic fractions, hepatocytes suspension) and in vivo. The rac-F, (+)-R-F and (-)-S-F were used as substrates. Amounts of F enantiomers, 4-dihydroflobufen diastereoisomers (DHF) and other metabolites (M-17203, UM) were determined with a chiral HPLC method in two chromatographic runs on R,R-ULMO and allyl-terguride bonded columns. Stereoselective biotransformation of the two enantiomers of F was observed at all tested levels and significant bidirectional chiral inversion of enantiomers of F was observed in hepatocytes. Mean enantiomeric ratios of F concentrations (S-/R-), after rac-F incubations, ranging from 1.09 in cytosolic fraction to 18.23 in hepatocytes. Stereospecificity of the respective F reductases was also observed. (2R;4S)-DHF and (2S;4S)-DHF are the principal metabolites of F in microsomes and hepatocytes. Neither DHF diastereoisomers nor M-17203 were found in cytosolic fraction. Only the nonchiral metabolite, M-17203, was found in all urine and feces samples after oral administration of F.

Stereoselective pharmacokinetics of flobufen in rats.

Stereoselectivity of the pharmacokinetics of the nonsteroidal anti-inflammatory drug flobufen, 4-(2', 4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in male Wistar rats after intravenous administration. Pharmacokinetic parameters and chiral inversion of flobufen enantiomers were studied after a bolus injection of the racemate and individual enantiomers (5 mg/kg). Determinations of the enantiomers in rat plasma were performed using chiral HPLC (terguride column). After i.v. administration of flobufen racemate, plasma levels of R-enantiomer decreased more rapidly. The S-/R-enantiomer ratio of AUCs after rac-flobufen was 13.3. The total plasma clearance value of S-flobufen was more than 10-fold lower than R-flobufen. The other pharmacokinetic parameters of the enantiomers were also significantly different. While only traces of R-enantiomer (less than 1%) were detected in rat plasma after S-flobufen administration, considerable conversion to the S-enantiomer was found after injection of R-flobufen (R-enantiomer AUC/S-enantiomer AUC = 0.52). The results indicate substantial stereoselectivity in the disposition of flobufen enantiomers in the rat, which is, at least in part, attributed to chiral bioconversion.

Sex differences in stereospecificity of oracin reductases in rat in vitro and in vivo.

In vitro and in vivo experiments to investigate possible stereospecific aspects of oracin reduction in relation to rat gender have been conducted. Incubation of oracin with rat microsomes, cytosol, and hepatocytes in the presence of various coenzymes and under aerobic or anaerobic conditions provided evidence for sex differences in the formation of 11-dihydrooracin (DHO) enantiomers. The greatest sex differences were seen in hepatocytes where females showed higher stereospecificity of the reductases than males. While female biotransformation enzymes preferentially generated approximately 82% of (+)-DHO, male enzymes gave only rise to 63% of (+)-DHO. Males displayed higher stereospecificity than females in the microsomal fraction. However, in the cytosolic fraction females exhibited higher stereospecificity than males. Similarly, in in vivo studies, the ratio of (+)- and (-)-DHO in faeces and urine gave no indication of the significant differences between the male and female rat. Enzyme stereospecificity has been defined as preferential formation of the (+)- or (-)-stereoisomer of 11-DHO by the respective enzyme. HPLC quantitative determinations of both enantiomers were performed using a Chiralcel OD-R column as the chiral stationary phase with excellent resolution and stability.

Study of the biotransformation of a potential benzo[c]fluorene antineoplastic using high-performance liquid chromatography with high-speed-scanning ultraviolet detection.

As the sum of benfluron metabolites found was only a part of the total amount applied, a search for undiscovered metabolites was undertaken in the extracts from isolated rat hepatocytes and in the bile and perfusate in the experiments with an isolated perfused rat liver. To identify the metabolites, high-performance liquid chromatography with UV spectral analysis was used, as benfluron derivatives exhibit characteristic absorption spectra. Administration of known metabolites to experimental animals and selective induction of certain metabolic pathways led to the finding of new metabolites and of the respective conjugates. Fast atom bombardment-mass spectrometry analysis was used to identify the newly found metabolites and conjugates.