Yoshihiro Uesawa

Effects of furanocoumarin derivatives in grapefruit juice on nifedipine pharmacokinetics in rats

AbstractPurpose. It has been reported that grapefruit juice (GJ) causes a pharmacokinetic interaction with many drugs after co-ingestion. It is postulated that the substances in GJ may inhibit the first-pass metabolism during the intestinal absorption process. In recent years, several furanocoumarin derivatives that inhibit P450 activity in intestinal microsomes were isolated from GJ. In this study, we report the effects of the furanocoumarin derivatives in GJ on the nifedipine (NFP) pharmacokinetics in rats. Methods. Three furanocoumarin derivatives (bergaptol [BT], bergamottin [BG], and 6′,7′-dihydroxybergamottin [DHB]) found in GJ were used in this study. Each furanocoumarin was reconstituted in orange juice at the same concentration as in the GJ. Two milliliters of each sample was administered into the rat duodenum. After 30 min, NFP was intraduodenally administered at a dose of 3 mg/kg body weight. The NFP concentrations in the plasma samples were determined by HPLC. Results. A significant increase in the AUC of NFP was observed only in the rats administered BG; 1.5 times that of the control group. The result was quite identical with that of the group that was administered GJ. BT and DHB had no significant effects on the NFP pharmacokinetics. Conclusions. The results strongly suggested that BG in GJ might be the substance that elevates the NFP plasma concentrations.

Effects of cranberry juice on nifedipine pharmacokinetics in rats.

Little information is available about drug interactions with cranberry juice (CJ). Using microsomes from the human liver and rat small intestine, this study was designed to determine whether CJ could inhibit CYP3A‐mediated nifedipine (NFP) oxidase activity; it showed that CJ was a potent inhibitor of human and rat CYP3A. Preincubation with 10% vol/vol of CJ and 1 mM NADPH for 10 min resulted in significant inhibition of the NFP oxidation activity of human and rat CYP3A (18.2 and 12.6% decreases, respectively, compared with preincubation experiments without NADPH). In addition, the pharmacokinetic interaction between CJ and NFP in vivo was confirmed in rats. In comparison with a control group, the area under the concentration‐time curve (AUC) of NFP was approximately 1.6‐fold higher when CJ (2 mL) was injected intraduodenally 30 min before the intraduodenal administration of NFP (30 mg kg−1). However, the mean residence time, the volume of distribution and the elimination rate constant were not changed significantly. These data suggest that CJ component(s) inhibit the function of enteric CYP3A. In conclusion, it was found that CJ inhibits the CYP3A‐mediated metabolism of NFP in both rats and humans. Furthermore, CJ alters NFP pharmacokinetics in rats.

Substrate Specificity of Human Hepatic Udp‐Glucuronosyltransferases

Five human hepatic UDP-glucuronosyltransferases (UGTs) catalyze the facilitated excretion of more than 90% of drugs eliminated by glucuronidation. The substrate specificity of these UGTs has been examined using cloned expressed enzymes and liquid chromatography-mass spectrometry assays to determine the intrinsic clearance of drug glucuronidation in vitro. Specific substrates for the five individual UGTs have been identified. These five probe substrates could be used to predict the drug clearance catalyzed by individual UGTs in vivo.

Enzymatic Activities in the Microsomes Prepared from Rat Small Intestinal Epithelial Cells by Differential Procedures

In 1982, it was reported that the small intestinal extraction ratio of phenacetin in rats coincided with the result of a metabolic investigation using epithelial cells (enterocytes) of the rat small intestine (1). That is, when Klippert et al. administered phenacetin into the jugular vein, the portal vein, and the duodenum of rats pretreated with 3-methylcholanthrene, the extraction ratio by the gut was calculated to be 0.53. However, the extraction ratio obtained from the in vitro data on phenacetin metabolism using isolated mucosal cells of 3-methylcholanthrene-pretreated rats was between 0.31 and 0.53. Gupta et al. (2) first reported in humans that the contribution of the small intestine in the first pass metabolism of cyclosporine was significantly large. In recent years, the metabolism of a drug when it passes through enterocytes has been the focus of attention (3–8). Although drug metabolism studies using rat intestinal microsomes have been increasing, their results vary quite widely among the researchers. It is conceivable that different procedures in the preparation of intestinal microsomes contribute severely to enzymatic stability; however, a comparative study of the preparation procedures of the microsomes from rat enterocytes has never been reported. In this report, we describe the activities of cytochrome P450 and UDP-glucuronosyltransferase in the rat small intestinal microsomes prepared by four different procedures and the stability of the enzyme activities in the microsomes during storage at 4°C, −20°C, and −80°C for 30 days.

Hesperidin in orange juice reduces the absorption of celiprolol in rats

It has been reported that the intestinal absorption of celiprolol, an antihypertensive drug, is inhibited when it is taken with orange juice; it has been suggested that element(s) in citrus juice are responsible for this. In the present study, the pharmacokinetic interaction between celiprolol and orange juice was characterized through in vivo experiments with rats. Celiprolol 5 mg/kg was injected into the rat duodenum together with 5 ml/kg of neutralized orange juice or the same concentration of hesperidin as in the orange juice. Plasma celiprolol concentrations were measured by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Concomitant administration of orange juice or hesperidin with celiprolol significantly decreased the area under the plasma concentration-time curve (AUC) by 74% and 75%, respectively, compared with control. These findings suggest that hesperidin is responsible for the decreased absorption of celiprolol and that orange juice taken with celiprolol has an inhibiting effect on intestinal absorption of the drug.

IDENTIFICATION OF THE RABBIT LIVER UDP-GLUCURONOSYLTRANSFERASE CATALYZING THE GLUCURONIDATION OF 4-ETHOXYPHENYLUREA (DULCIN)

Dulcin (DL), 4-ethoxyphenylurea, a synthetic chemical about 200 times as sweet as sucrose, has been proposed for use as an artificial sweetener. DL is excreted as a urinary ureido-N-glucuronide after oral administration to rabbits. The phenylurea N-glucuronide is the only ureido conjugate with glucuronic acid known at present; therefore, DL is interesting as a probe to search for new functions of UDP-glucuronosyltransferases (UGTs). Seven UGT isoforms (UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT2B13, UGT2B14, and UGT2B16) have been identified from rabbit liver, but these UGTs have not been investigated using DL as a substrate. In this work, the identities of UGT isoforms catalyzing the formation of DL glucuronide were investigated using rabbit liver microsomes (RabLM) and cloned/expressed as rabbit UGT isoforms. DL-N-glucuronide (DNG) production was determined quantitatively in RabLM and homogenates of COS-7 cells expressing each UGT isoform by using electrospray liquid chromatography-tandem mass spectrometry. Analysis of DNG formation using RabLM, by Eadie-Hofstee plot, gave a Vmax of 0.911 nmol/min/mg protein and the Km of 1.66 mM. DNG formation was catalyzed only by cloned expressed rabbit UGT1A7 and UGT2B16 (Vmax of 3.98 and 1.16 pmol/min/mg protein and a Km of 1.23 and 1.69 mM, respectively). Substrate inhibition of UGT1A7 by octylgallate confirmed the significant contribution of UGT1A7 to the formation of DNG. Octylgallate was further shown to competitively inhibit DNG production by RabLM (Ki = 0.149 mM). These results demonstrate that UGT1A7 is the major isoform catalyzing the N-glucuronidation of DL in RabLM.

Asp73-dependent and -independent regulation of the affinity of ligands for human histamine H1 receptors by Na().

ABSTRACT The affinity of ligands for G‐protein‐coupled receptors (GPCRs) is allosterically regulated by Na+ via a highly conserved aspartate residue (Asp2.50) in the second transmembrane domain of GPCRs. In the present study, we examined the Na+‐mediated regulation of the affinity of ligands for Gq/11‐protein‐coupled human histamine H1 receptors in Chinese hamster ovary cells. The affinities of 3 agonists and 20 antihistamines were evaluated by their displacement curves against the binding of [3H]‐mepyramine to membrane preparations in the presence or absence of 100 mM NaCl. The affinities of most drugs including histamine, an agonist, and d‐chlorpheniramine, a first‐generation antihistamine, were reduced by NaCl, with the extent of NaCl‐mediated changes varying widely between drugs. In contrast, the affinities of some second‐generation antihistamines such as fexofenadine were increased by NaCl. These changes were retained in intact cells. The mutation of Asp2.50 (Asp73) to asparagine abrogated NaCl‐induced reductions in affinities for histamine and d‐chlorpheniramine, but not NaCl‐induced increases in the affinity for fexofenadine. Quantitative structure‐activity relationship (QSAR) analyses showed that these Na+‐mediated changes were explained and predicted by a combination of the molecular energies and implicit solvation energies of the compounds. These results suggest that Na+ diversely regulates the affinity of ligands for H1 receptors from the extracellular sites of receptors via Asp73‐dependent and ‐independent mechanisms in a manner that depends on the physicochemical properties of ligands. These results may contribute to a deeper understanding of the fundamental mechanisms by which the affinity of ligands for their receptors is allosterically regulated by Na+.

Establishment of a Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics Method and Its Application to Lichen Profiling

Direct-injection electron ionization-mass spectrometry (DI-EI-MS) is a multivariate analysis method useful for characterizing biological materials. We demonstrated the use of DI-EI-MS for metabolic profiling using several closely related lichen species: Cladonia krempelhuberi, C. gracilis, C. pseudogymnopoda, and C. ramulosa. The methodology involves conversion of total ion chromatograms to integrated chromatograms and assessment of reproducibility. The qualitative DI-EI-MS method was used to profile the major and/or minor constituents in extracts of lichen samples. It was possible to distinguish each lichen sample by altering the electron energy in DI-EI-MS and examining the resulting data using one-way analysis of variance. Previously undetectable peaks, which are easy to fragment could be revealed by varying the electron energy. Our results suggest that metabolic profiling using DI-EI-MS would be useful for discriminating between subgroups within the same species. This is the first study to report the use of DI-EI-MS in a metabolomics application.

Predictive performance of three practical approaches for grapefruit juice-induced 2-fold or greater increases in AUC of concomitantly administered drugs

Clinical pharmacists have a challenging task when answering patients’ question about whether they can take specific drugs with grapefruit juice (GFJ) without risk of drug interaction. To identify the most practicable method for predicting clinically relevant changes in plasma concentrations of orally administered drugs caused by the ingestion of GFJ, we compared the predictive performance of three methods using data obtained from the literature.

Metabolism of bucolome in rats Stability and biliary excretion of bucolome N-glucuronide

Bucolome (BCP) is a non-steroidal anti-inflammatory drug, which is used in the treatment of chronic articular rheumatism. Bucolome N-glucuronide (BCP-NG), a metabolite of BCP, is the first unique N-glucuronide of barbituric acid derivatives. First, the stability of BCP-NG in various pH aqueous solutions was studied. BCP-NG was quite unstable under neutral and acidic conditions, and is easily hydrolyzed to BCP. Based on these characteristics of BCP-NG, a simple, rapid and highly sensitive method for the simultaneous determination of BCP and BCP-NG with phenylbutazone (I.S.) in biological fluids was developed using high-performance liquid chromatography (HPLC). A reversed-phase ODS column was used for the separation of BCP, BCP-NG and I.S. A pharmacokinetic study for BCP and BCP-NG was carried out in male Wistar/ST rats following i.v. administration of BCP at a dose of 10 mg/kg body weight. The slow plasma elimination of BCP with time was shown. A major metabolite of BCP in bile was N-glucuronide. The cumulative amounts of BCP and BCP-NG in the bile over 8 h were approximately 2.4 +/- 1.4% and 12.6 +/- 2.3% of the dose, respectively. BCP and BCP-NG in the urine were 2.7 +/- 0.7% and 3.2 +/- 0.3% of the dose. Although BCP had a long half-life (over 8.5 h), the preliminary pharmacokinetic parameters (0-8 h) were determined: t 1/2, 8.52 +/- 1.96 h; AUC, 419.9 +/- 45.2 microg x h/ml; MRT, 3.29 +/- 0.11 h; CLtot, 5.93 +/- 0.54 ml/h; and Vdss, 19.5 +/- 1.3 l. These observations are the first pharmacokinetic findings for the N-glucuronide of the barbituric acid derivatives.