Transport of quercetin di-sodium salt in the human intestinal epithelial Caco-2 cell monolayer 139.

Quercetin di-sodium salt (QDS), a water-soluble derivative of quercetin (Q), is a potent free radical scavenger. The aim of this study was to examine the in vitro intestinal transport of QDS compared to that of Q using the Caco-2 human intestinal epithelial cell line. The apical (A) to basolateral (B) transport of QDS was found to be higher than the B to A transport of this compound. This polarized transport involved the presence of a carrier protein system. The involvement of the sodium/glucose transporter-1 (SGLT-1) was shown by using phloridzin, a selective inhibitor of this conveyor system. However, the transport of Q was not affected by this inhibitor. Moreover, the influx of QDS was pH-sensitive and decreased at pH 5.5 compared with that observed at pH 7.4 and 6.5. The permeability of QDS was 10-fold higher than that of Q. This could be explained by the involvement of SLGT-1 and the absence of an active efflux pump in the absorption of QDS in comparison with Q. This finding was supported by comparing the solubility of Q with that of QDS. This study indicates that both the higher solubility of QDS and its dependence on the SGLT-1 transport system resulted in more efficient permeability compared to Q.

Inhibition of in-vitro simvastatin metabolism in rat liver microsomes by bergamottin, a component of grapefruit juice.

Grapefruit juice can modify the pharmacokinetic parameters of many drugs, in particular simvastatin, an orally active cholesterol-lowering agent. The exact components in grapefruit juice responsible for drug interactions are not perfectly known. However, it seems that bergamottin, a furocoumarin derivative, is one of the main active components within grapefruit juice. The objective of this paper was to quantify and to characterize in-vitro the inhibitory effect of bergamottin on simvastatin metabolism by using rat and human liver microsomes. In rat liver microsomes, the incubation conditions (+/-NADPH) of bergamottin were found to influence its inhibiting capacity. In co-incubation with simvastatin, the Ki value (the equilibrium dissociation constant for the enzyme-inhibitor complex) was higher (Ki = 174 +/- 36 microM) than in pre-incubation (Ki = 45 +/- 6 microM and 4 +/- 2 microM, without and with NADPH, respectively). It thus seems that the pre-incubation of bergamottin (in particular with NADPH) increases its inhibiting capacity on simvastatin metabolism. Bergamottin metabolism study in rat liver microsomes showed the formation of two metabolites that were CYP-450 dependent. In contrast, in human liver microsomes, the incubation conditions of bergamottin did not influence its inhibiting capacity of simvastatin metabolism (Ki = 34 +/- 5 microM, Ki = 22 +/- 5 microM, Ki = 27 +/- 11 microM in coincubation and pre-incubation without and with NADPH, respectively). In rat and man, bergamottin was found to be a mixed-type inhibitor of simvastatin hepatic metabolism. However, in rat, bergamottin was partially a mechanism-based inhibitor by involvement of either bergamottin alone or one of its metabolites. The results highlight the importance of validating in-vitro models to help verify the suitability of the in-vitro model for predicting the nature and degree of metabolic drug interactions.

Free radical scavenging and skin penetration of troxerutin and vitamin derivatives.

BACKGROUND: By its 'protective function', human skin is a potential target for the production of free radicals. The role played by topically applied antioxidants as inhibitors of oxidative stress damage was felt to be worth investigation. OBJECTIVE: To investigate the free radical scavenging (superoxide, hydroxyl and peroxyl radicals) and skin penetration of troxerutin in association with ascorbyl palmitate and alpha-tocopheryl succinate, esters of two vitamins commonly used in skin care products. METHODS: The compounds' scavenging activities, in a concentration-dependent manner, were as follows: hydroxyl radicals in a Fenton-based assay; superoxide radicals in a hypoxanthine/xanthine oxidase system; and lipid peroxidation inhibition of liver microsomes was induced by 2,2'-azobis-(2-amidinopropane) dihydrochloride (ABAP). RESULTS: A synergic action was observed between alpha-tocopheryl succinate and troxerutin for hydroxyl radical scavenging, between the three compounds for superoxide scavenging and between troxerutin and ascorbyl palmitate in lipid peroxidation inhibition. CONCLUSION: Using a stripping method, it was shown that the three substances, incorporated in a pharmaceutical preparation, permeated through human epidermis. Thus, this association can improve skin care products for preventing free radical-mediated damage.

Comparison of in vitro hepatic models for the prediction of metabolic interaction between simvastatin and naringenin.

The study of potential interaction at a very early stage of drug development requires suitable in vitro models that describe drug interactions both qualitatively and quantitatively. The purpose of this work was to help assessing the predictive value of in vitro drug interaction test with liver microsomal fractions and hepatocytes by determination of enzymatic parameters such as the inhibition constant (Ki) and the intrinsic clearance (Clint). This study was conducted to compare different methods of Ki calculation and to determine the most suitable parameter for describing drug interactions. The metabolic interaction between SV and NRG was used as a model to help verifying the suitability of the in vitro model for predicting the kind and degree of metabolic drug interactions. The method of Ki calculation using linearized versions of Michaelis Menten equations based on the simultaneous non linear regressions and the "km app" approach accurately estimated the Ki values. The linear representation of an inherently non linear relationship was only used to establish the mechanism of inhibition and the Clint could be used to predict drug interactions. To further prediction in humans, it seems likely that the simultaneous application of both systems, microsomal fractions and hepatocytes will yield conclusions.

Comparison of the stability of some major cytochrome P450 and conjugation reactions in rat, dog and human hepatocyte monolayers.

The stability of four major cytochrome P450 isoenzymes (CYPIA, CYP2B, CYP2E1 and CYP3A) and of two phase II conjugation enzymes (glucuronyl- and sulfotransferases) was investigated in primary cultures of rat, dog and human hepatocytes in the same conditions. 7-ethoxyresorufin deethylation (EROD), 7-methoxycoumarin demethylation (MCOD), chlorzoxazone (CLOX) 6-hydroxylation, 1'- and 4-hydroxylation of midazolam (MDZ), and p-nitrophenol glucuronidation and sulfation, were used respectively. The EROD activity was stable over 72 hours in rat and dog and only 48 hours in human hepatocytes. The MCOD activity was also stable in rat but decreased in dog by 30% within 72 hours The CLOX hydroxylase activity was most stable in human whereas in rat and dog it fell down to 30% within 72 and 24 hours, respectively. The MDZ hydroxylase activity showed the same unstability profile in the three species investigated. Both conjugation reactions were either stable or showed an increase by up to 60-70% in all three species over 72 hours. The enzymes tested showed different stabilities in rat, dog and human hepatocytes over 72 hours, thus demonstrating the limitations of hepatocyte monolayers as models for metabolic investigations and emphasising the need for validation/characterization studies before routine use.

In vitro inhibition of simvastatin metabolism in rat and human liver by naringenin.

We demonstrated that naringenin (NRG), the aglycon form of naringin present in grapefruit juice inhibits in vitro the metabolism of simvastatin (SV), a HMG-CoA reductase inhibitor. SV undergoes an important first pass metabolism and this is thought to be partly responsible for its low bioavailability after oral administration. SV is a prodrug that requires metabolic activation through hydrolysis by esterases. In addition, SV is a substrate for cytochrome P450 enzymes. NRG, a potent inhibitor of cytochrome P450 enzymes, interferes with the isoenzymes of cytochrome P450 involved in the hepatic metabolism of SV. NRG inhibits the metabolism of SV in rat hepatocytes (the intrinsic clearance of SV decreases from 26.2 microl/min/10(6) cells in absence of NRG to 4.15 microl/min/10(6) cells in presence of 50 microM NRG). This inhibition is more pronounced in hepatocytes (Ki value approximately 5 microM) than in liver microsomes (Ki approximately 23 microM and approximately 30 microM in human and rat liver microsomes respectively). Therefore, the hepatocytes seem to be the best approach for in vitro interaction study between SV and NRG ; and this should be taken into account in the in vitro/in vivo extrapolation. If this interaction were confirmed in man, the doses of SV should be reduced when co-administered with grapefruit juice because of increased bioavailability of SV.

Estimation of flavin-containing monooxygenase activity in intact hepatocyte monolayers of rat, hamster, rabbit, dog and human by using N-oxidation of benzydamine.

The flavin-containing monooxygenase (FMO)-dependent N-oxidation of benzydamine has been assessed as a method for monitoring the activity of FMOs in monolayer cultures of hepatocytes from rat, dog, rabbit, hamster and human. The advantage of this substrate is that benzydamine N-oxide formation can be measured directly in extracts of cellular incubations without an intensive work-up procedure. Benzydamine and its N-oxide are readily separated by HPLC with fluorometric detection. This assay proved sensitive enough to monitor FMOs activity in intact monolayer of cultured hepatocytes. The formation of benzydamine N-oxide was inhibited when hepatocytes were coincubated with methimazole (another FMO substrate) in a dose-dependent manner, whereas N-octylamine (an inhibitor of cytochrome P450) had no inhibitory effect. In contrast to cytochrome P450, FMO activity assessed by benzydamine N-oxidation was relatively stable for all species studied during 72-h cultures.

In vitro biotransformation of SDZ RAD: a new immunosuppressive macrolide in human liver microsomal preparations.

In the present biotransformation study using human in vitro liver samples, metabolites of SDZ RAD were characterized by LC-MS. The major metabolites resulted from single hydroxylation and demethylation pathways and corresponded to the class of first-generation metabolites; 39-O-demethyl-RAD was identified as metabolite. The potential ring-opened degradation product resulting from ester hydrolysis and its dehydrated analogue (seco acid) was detected to comparable amounts in both the incubations and in controls without the presence of NADPH. The direct formation of rapamycin from SDZ RAD could not be detected in this study.

Concentrations of pefloxacin in plasma and tissue after administration as surgical prophylaxis.

Plasma and epiploic-fat drug concentrations determined by high-performance liquid chromatography and fat penetration of pefloxacin and its metabolite (norfloxacin) given for antimicrobial prophylaxis were studied in patients scheduled for colorectal surgery. Concentrations of pefloxacin in plasma decreased about 40% from the beginning of the operation to closure of the peritoneum, and corresponding levels in epiploic fat stayed stable. The plasma and tissue norfloxacin concentrations were very low. Concentrations of pefloxacin in tissue were greater than MIC at which 90% of isolates are inhibited for sensitive bacteria (members of the family Enterobacteriaceae). The penetration of pefloxacin into epiploic fat was about 32%.

The use of human hepatocytes to select compounds based on their expected hepatic extraction ratios in humans.

PURPOSE: The present investigation retrospectively evaluates the use of human hepatocytes to classify compounds into low, intermediate or high hepatic extraction ratio in man. METHODS: A simple approach was used to correlate the in vivo hepatic extraction ratio of a number of compounds in man (literature and in-house data) with the corresponding in vitro clearance which was determined in human hepatocytes. The present approach assumes that, for compounds eliminated mainly through liver metabolism, intrinsic clearance is the major determinant for their in vivo hepatic extraction ratio and subsequently their bioavailability in man. The test compounds were selected to represent a broad range of extraction ratios and a variety of metabolic pathways. RESULTS: The present data show that in vitro clearances in human hepatocytes are predictive for the hepatic extraction ratios in vivo in man. Most of the test compounds (n = 19) were successfully classified based upon human hepatocyte data into low, intermediate or high hepatic extraction compounds, i.e. compounds with potential for high, intermediate or low bioavailabilities in humans. CONCLUSIONS: The present approach, validated so far with 19 test compounds, appears to be a valuable tool to screen for compounds with respect to liver first-pass metabolism at an early phase of drug discovery.

Interspecies scaling of bosentan, a new endothelin receptor antagonist and integration of in vitro data into allometric scaling.

PURPOSE: The goal of this study was to find a rational and reliable method of using animal data to predict the clearance of metabolised drugs in humans. METHODS: One such approach is to use in vitro liver models (e.g. hepatocytes and microsomes) to determine the relative capacities of the various animal species and humans to metabolise the test compound. These data can then be combined with the in vivo clearances in animals, to calculate the in vivo clearance in humans using allometric scaling techniques. In this study, this approach was evaluated with a new endothelin receptor antagonist, bosentan, which is eliminated mainly through metabolism and is characterized by very large interspecies differences in clearance. Therefore, this compound provided a stringent test of our new extrapolation method for allometric scaling. RESULTS: The results obtained with bosentan showed that adjusting the in vivo clearance in the different animal species for the relative rates of metabolism in vitro gave a far better prediction of human clearance than an empirical correcting factor (brain weight). CONCLUSIONS: This approach provided a more rational basis for predicting the clearance of metabolised compounds in humans.

Bosentan, a new endothelin receptor antagonist: prediction of the systemic plasma clearance in man from combined in vivo and in vitro data.

1. Accurately predicting the kinetics in man greatly improves the design of the phase I clinical studies. This was particularly crucial in the case of bosentan, a new endothelin receptor antagonist, as very large interspecies differences in systemic clearance were observed in the animal species investigated, namely from 1.5ml/min/kg in the dog up to 70 ml/min/kg for the rabbit. 2. Bosentan was shown to be metabolized by the hepatic cytochrome P450, therefore the rate of metabolism was investigated in vitro in liver microsomes and hepatocytes, across the species which had been tested in vivo. The same rank-order of metabolism was found for the laboratory animals both in vitro and in vivo, and hepatocytes appeared to be more representative of the in vivo situation than liver microsomes. The in vitro clearance in human hepatocytes was very close to that observed in dog hepatocytes. 3. A plasma clearance for bosentan in man of 1-2 ml/min/kg was predicted by combining the in vivo and in vitro data from a few animal species with the in vitro data in man. This expectation was subsequently found to agree reasonably well with the plasma clearance observed in healthy volunteers: ca 2 ml/min/kg. Integrating this prediction into the design of the first clinical protocols substantially improved the quality of the human pharmacokinetic data obtained.

A new extrapolation method from animals to man: application to a metabolized compound, mofarotene.

Allometric scaling (a technique which uses data obtained in laboratory animals to predict human pharmacokinetics) works well for drugs that are cleared intact, but is less successful with extensively metabolised compounds. This paper describes a new method to improve the accuracy of such projections, by integrating metabolic data obtained in vitro (e.g. with liver microsomes or hepatocytes) into these calculations. The approach was used prospectively, to predict the clearance of mofarotene (Ro 40-8757) in humans from in vivo kinetic data obtained in mouse, rat and dog. This compound was selected to illustrate this approach because it is exclusively eliminated through metabolism. Without the metabolic correction or using empirical correcting factors, the values predicted for man were 2.7 and 0.6 ml/min/kg. This fell outside the range subsequently obtained in healthy volunteers dosed orally with 300 mg of mofarotene (7.5 +/- 4.0 ml/min/kg, n = 12). However, inclusion of the microsomal or hepatocyte data gave values of 5.1 and 4.2 ml/min/kg, respectively, illustrating that the integration of in vitro metabolic data improves the accuracy of kinetic extrapolations. In contrast to the existing empirical techniques, this approach offers a rational basis to predict clearance of metabolized compounds in human.

Calcium anti-ionophoretic effect of some calcium channel blockers in rat liver mitochondria.

Beyond their classical action on calcium channels, some calcium channel blockers also exhibit a calcium anti-ionophoretic effect. We studied this effect on respiratory control and Ca2+ fluxes in a mitochondrial model to compare calcium antagonists chosen among three clinical classes: vascular, cardiac, and mixed effects. Synthetic calcium ionophore A23187 decreases respiratory control and modifies Ca2+ fluxes. We show that calcium antagonists partially restore the parameters altered by A23187. By calculating the percentage of restoration, we found that vascular drugs exhibit a strong anti-ionophoretic effect, cardiac drugs exert no significant effect, and mixed calcium antagonists exert an intermediate effect. Thus, it appears possible to link the intensity of calcium anti-ionophoretic effect with the clinical interest of a calcium antagonist.

Counteraction by nicardipine of the ionophoretic effect of gliclazide: a mitochondrial study.

Using mitochondria, we demonstrate that gliclazide exhibits a calcium ionophoretic activity. Indeed, gliclazide induces a decrease in the respiratory control of mitochondria; this effect is increased by addition of Ca2+ and corrected by ruthenium red, all characteristics of a calcium ionophoretic compound. Our results, on a biological membrane, confirm previous findings in vitro. Moreover, we show that nicardipine counteracts the action of gliclazide. Besides the effect on ATP-stimulated K+ channels, the ionophoretic effect of gliclazide may play a role in its hypoglycaemic effect, thus this counteracting action of nicardipine might induce drug interaction during the concomitant clinical administration of gliclazide and nicardipine.