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Our objective was to determine the effect of the flavonoid biochanin A (BCA), administered alone or in combination with the flavonoids quercetin and epigallocatechin-3-gallate (EGCG), on the growth of human breast cancer MCF-7 cells in a murine xenograft animal model.MCF-7 tumors were implanted into mice and groups of mice were treated with vehicle, BCA at 2 doses (5 or 15 mg/kg), quercetin and EGCG (5 mg/kg each), or BCA combined with quercetin and EGCG (5 mg/kg each). The flavonoids were injected once daily intraperitoneally, with treatment starting 4 weeks prior to cell inoculation.Treatment with 15 mg/kg of BCA or the mixture of the 3 flavonoids resulted in a reduction in tumor incidence. Tumor size in xenograft mice treated with 15 mg/kg BCA was significantly smaller than in the control group. Although quercetin/EGCG administration did not affect tumor size, treatment with the mixture of the 3 flavonoids at doses of 5 mg/kg produced similar effects as seen with 15 mg/kg BCA.Our findings indicate that BCA inhibits tumor growth in a xenograft animal model; BCA may represent a breast cancer preventive agent, either administered alone or in combination with other flavonoids.

Little is known regarding pharmacokinetic (PK) or pharmacodynamic interactions of flavonoids with each other: this is of significance since multiple flavonoids are present in the diet and in dietary supplements. Our objective was to determine the effect of quercetin and (-)-epigallocatechin-3-gallate (EGCG), major flavonoids present in the diet, on the PK and bioavailability of biochanin A, a flavonoid with chemopreventive properties. BCA was administered to rats intravenously (5 mg/kg) or orally (16.67 or 50 mg/kg) with or without concomitant EGCG and quercetin. In vitro studies with the human intestinal Caco-2 and human hepatic HepG2 cell lines were performed to evaluate the effects of quercetin and EGCG on the cellular metabolism of BCA, and studies with human breast cancer MCF-7 cells that overexpress P-glycoprotein or BCRP (MCF-7/ADR and MCF-7/MX100 cells, respectively) or MDCK cells that express MRP2 (MDCK-MRP2) were performed to evaluate the effects of cellular efflux. An HPLC assay was used to determine plasma, urine, and cellular concentrations of BCA and the conjugated metabolites of BCA (following enzymatic hydrolysis). The coadministration of quercetin and EGCG significantly increased the BCA area under the plasma concentration vs time curve (AUC) in rats, after both iv and oral administration of BCA. The AUC of total BCA (unchanged + conjugated) was also increased. The increases in BCA AUC reflected predominantly increased bioavailability; this was true even after iv administration due to an apparent increase in the enterohepatic cycling of BCA. Our findings demonstrate for the first time that the administration of multiple flavonoids results in increased flavonoid bioavailability, as well as a decrease in clearance, potentially due to increased enterohepatic cycling.

Contributors. Preface. 1 Overview of Drug Transporter Families (Guofeng You and Marilyn E. Morris). 2 Organic Cation Transporters (Thomas J. Urban and Kathleen M. Giacomini). 3 Organic Cation/Carnitine Transporters (Ijaz A. Qureshi, Ihsan Elimrani, Ernest Seidman, and Grant Mitchell). 4 Organic Anion Transporters (Megha Nagle, David M. Truong, Vibha Bhatnagar, Gregory Kaler, Kevin T. Bush, Wei Wu, Satish A. Eraly, and Sanjay K. Nigam). 5 Organic Anion-Transporting Polypeptides (Rommel G. Tirona and Richard B. Kim). 6 Mammalian Oligopeptide Transporters (Stephen M. Carl, Dea Herrera-Ruiz, Rajinder K. Bhardwaj, Olafur Gudmundsson, and Gregory T. Knipp). 7 Monocarboxylate Transporters (Justin A. Spanier and Lester R. Drewes). 8 Nucleoside Transporters: CNTs and ENTs (Li Xia, Mingyan Zhou, and Joanne Wang). 9 Bile Acid Transporters (Jyrki J. Eloranta and Gerd A. Kullak-Ublick). 10 Multidrug Resistance Protein: P-Glycoprotein (Frances J. Sharom). 11 Multidrug Resistance Proteins of the ABCC Subfamily (Anne T. Nies, Maria Rius, and Dietrich Keppler). 12 Breast Cancer Resistance Protein (Robert W. Robey, Orsolya Polgar, John Deeken, Kenneth K. W. To, and Susan E. Bates). 13 Drug Transport in the Liver (Pieter Annaert, Brandon Swift, Jin Kyung Lee, and Kim L. R. Brouwer). 14 Drug Transport in the Brain (Patrick T. Ronaldson, Karlo Babakhanian, and Reina Bendayan). 15 Drug Transport in the Kidney (Naohiko Anzai and Hitoshi Endou). 16 Drug Transporters in the Intestine (Yaming Su and Patrick J. Sinko). 17 Regulation of Drug Transporter Activity (Mei Hong and Guofeng You). 18 Experimental Approaches to the Study of Drug Transporters (Yoshiyuki Kubo, Yukio Kato, and Akira Tsuji). 19 In Vitro-In Vivo Scale-up of Drug Transport Activities (Kazuya Maeda and Yuichi Sugiyama). 20 Age- and Gender-Related Differences in Xenobiotic Transporter Expression (Curtis D. Klaassen and Xingguo Cheng). 21 Polymorphisms of Drug Transporters and Clinical Relevance (Leslie W. Chinn, Jason M. Gow, and Deanna L. Kroetz). 22 Diet/Nutrient Interactions with Drug Transporters (Xiaodong Wang and Marilyn E. Morris). 23 Interplay of Drug Transporters and Enzymes on Hepatic Drug Processing (K. Sandy Pang, Huadong Sun, and Shanjun Liu). 24 Clinical Relevance: Drug-Drug Interactions, Pharmacokinetics, Pharmacodynamics, and Toxicity (Serena Marchetti, Roberto Mazzanti, Jos H. Beijnen, and Jan H. M. Schellens). Index.

The objective of this study was to determine the effects of flavonoids on the in vitro monocarboxylate transporter 1 (MCT1)-mediated transport and in vivo disposition of the drug of abuse, gamma-hydroxybutyrate (GHB). The uptake of GHB in rat MCT1 gene-transfected MDA-MB231 cells was significantly decreased in the presence of the flavonoids apigenin, biochanin A, chrysin, diosemin, fisetin, genistein, hesperitin, kaempferol, luteolin, morin, narigenin, phloretin, and quercetin, but was not affected by the flavonoid glycosides phloridzin and rutin. The IC(50) values for luteolin, morin, and phloretin were 0.41 +/- 0.14, 6.41 +/- 2.01, and 2.57 +/- 0.48 microM, with the inhibition mechanism for luteolin being competitive. [(3)H]Kaempferol and [(3)H]biochanin A did not exhibit MCT1-mediated uptake, suggesting that these flavonoids are not substrates for MCT1. The combination of luteolin and phloretin inhibited the uptake of GHB in a synergistic manner; however, the combination of luteolin and morin was antagonistic. GHB 1000 mg/kg was administered to rats by i.v. bolus, with or without the concomitant administration of luteolin 10 mg/kg i.v. After luteolin treatment, the renal and total clearances of GHB were significantly increased, probably because of inhibition of the MCT1-mediated renal reabsorption of GHB, and the sleep time significantly decreased (121 +/- 5 min versus 165 +/- 10 min) compared with control rats. Overall, the results of this study indicate that flavonoids from food or herbal products may significantly alter the pharmacokinetics and pharmacodynamics of MCT substrates.

Breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette efflux transporter, important in drug disposition and in the development of multidrug resistance in cancer. Flavonoids, a large class of natural compounds widely present in the diet and herbal products, have been shown in vitro to be BCRP inhibitors. The flavonoid chrysin is a potent inhibitor of BCRP, inhibiting the efflux of mitoxantrone with an IC(50) of 0.39 microM in BCRP-overexpressing human MCF-7 breast cancer cells. The purpose of this study was to investigate the potential pharmacokinetic interactions between chrysin and nitrofurantoin (a specific BCRP substrate) in rats. In Madin-Darby canine kidney cells expressing human BCRP or murine Bcrp1, the polarized transport of nitrofurantoin was effectively inhibited by chrysin at concentrations of 20 and 100 microM. Compared with the vehicle-treated group, p.o. coadministration of chrysin (200 mg/kg) significantly increased the area under the curve (AUC) and C(max) of nitrofurantoin (10 mg/kg) by 1.76- (p < 0.01) and 1.72-fold (p < 0.05), respectively. When nitrofurantoin (2 mg/kg) was given i.v., administration of chrysin (50 mg/kg i.p.) significantly increased the AUC of nitrofurantoin (123 +/- 34.0 versus 91.5 +/- 18.0 microg/ml x min in controls, p < 0.05). Moreover, the cumulative hepatobiliary excretion of nitrofurantoin (1.5 mg/kg i.v.) was significantly decreased by approximately 75% at the end of 120 min after the coadministration of chrysin (50 mg/kg i.p.). Taken together, these results indicate that the flavonoid chrysin significantly inhibits nitrofurantoin transport mediated by human BCRP and murine Bcrp1. Bcrp1 inhibition by chrysin is likely one potential mechanism for the observed chrysin-nitrofurantoin pharmacokinetic interactions in rats.

Study Objectives. To determine if quercetin, a bioflavonoid that inhibits p-glycoprotein, alters plasma saquinavir concentrations, and to explore the potential influence on intracellular concentrations. Design. Prospective pharmacokinetic analysis. Setting. University-affiliated general clinical research center. Subjects. Ten healthy adults (four women, six men) with a mean ± SD age of 30.7 ± 9.4 years. Intervention. All subjects received saquinavir 1200 mg 3 times/day with food on days 1–11 and quercetin 500 mg 3 times/day with food on days 4–11. Measurements and Main Results. On days 4 and 11, nine blood samples and four peripheral blood mononuclear cell samples were drawn during a steady-state dosing interval. Pharmacokinetic parameters were calculated by using standard noncompartmental techniques. Plasma saquinavir concentrations were similar regardless of quercetin administration. Geometric mean ratios for the area under the concentration-time curve during an 8-hour dosing interval (AUC0–8), maximum concentration in the dosing interval, and minimum concentration in the dosing interval were 0.99 (95% confidence interval [CI] 0.65–1.50), 0.99 (95% CI 0.64–1.54), and 1.06 (95% CI 0.68–1.67), respectively. Intracellular saquinavir concentrations displayed substantial intra- and intersubject variability, which limited the ability to determine the influence of quercetin coadministration (geometric mean ratio for AUC0–8 = 0.51 [95% CI 0.14–1.95], six patients). Conclusion. Quercetin coadministration did not influence plasma saquinavir concentrations. Because of substantial inter- and intrasubject variability, more study is necessary to determine if saquinavir intracellular concentrations are altered by coadministration of quercetin.

Previous studies in our laboratory have suggested that GHB may undergo renal reabsorption mediated by monocarboxylic acid transporters (MCT). The objectives of this study were to characterize the renal transport of GHB using HK-2 cells and the role of MCT in the renal transport of GHB.Western blot was used to detect the protein expression of MCT1, 2, and 4. Cellular uptake and directional flux studies were conducted to investigate the transport of GHB and L-lactate. RNA interference assay was used to investigate the involvement of MCT isoforms in the transport of GHB.MCT1, 2 and 4 were present in HK-2 cells. The cellular uptake of L-lactate and GHB exhibited pH- and concentration-dependence (L-lactate: K (m) of 6.5 +/- 1.1 mM and V (max) of 340 +/- 60 nmol mg(-1)min(-1); GHB: K (m) of 2.07 +/- 0.79 mM, V (max) of 27.6 +/- 9.3 nmol mg(-1)min(-1), and a diffusional clearance of 0.54 +/- 0.15 microl mg(-1)min(-1)), but not sodium-dependence. alpha-Cyano-4-hydroxycinnamate (CHC) competitively inhibited the uptake of GHB and L-lactate with inhibition constants (K (i)) of 0.28 +/- 0.1 mM, and 0.19 +/- 0.03 mM, respectively. Using small-interference RNA (siRNA) for MCT1, the protein expression of MCT1 and the uptake of L-lactate and GHB were significantly decreased. The siRNA treatment of MCT2 in HK-2 cells inhibited the uptake of GHB by 17%, and the siRNA treatment of MCT4 demonstrated no inhibition of GHB uptake. GHB exhibited a directional flux across HK-2 monolayer from apical to basal chambers in the presence of a pH gradient of pH 6.0 to pH 7.4.These data suggest that MCT1 represents an important transporter for GHB transport in renal tubule cells, responsible for the reabsorption of GHB in the kidney.

Many naturally occurring and synthetic isothiocyanates can inhibit chemical carcinogenesis in animal models. Recently, we found that alpha-naphthyl isothiocyanate (1-NITC) inhibited P-glycoprotein- and multidrug resistance associated protein 1-mediated efflux, indicating the potential application of 1-NITC as a chemosensitizing agent for cancer chemotherapy. The objective of this study was to explore the pharmacokinetic characteristics of 1-NITC in rats. A single dose of 10, 25, 50, or 75 mg/kg of 1-NITC was administered intravenously or orally to female Sprague-Dawley rats (n = 4 for each group). Dose-normalized concentration-time profiles were not superimposable following intravenous or oral dosing, indicating that the disposition of 1-NITC in rats was nonlinear. As doses increased from 10 to 75 mg/kg following iv administration, the total clearance decreased from 2.2 +/- 0.9 to 0.8 +/- 0.3 L/h/kg; oral availability averaged 0.46 for oral doses of 10-75 mg/kg. A nonlinear two-compartment open model with capacity-limited absorption and capacity-limited elimination from the central compartment best fit the data, based on goodness-of-fit criteria. The mechanism underlying the nonlinear elimination of 1-NITC in rats is most likely due to the capacity-limited metabolism of 1-NITC. This study represents the first report of the pharmacokinetics of 1-NITC.

(+/−)3-(3-(4,4-diphenylpiperidin-1-yl)propyl) 5-methyl 4-(3,4-dimethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate ((±)-DHP-014), is a new 4-aryl-1,4-dihydropyridine that can reverse multidrug resistance mediated by the ATP-binding cassette (ABC) transport proteins, P-glycoprotein, multidrug resistance-associated protein 1 and breast cancer resistance protein; it exhibits negligible calcium channel blocking activity. The objective of this work was to investigate the pharmacokinetics of this new compound in rats. Three intravenous (1, 2 and 5 mg/kg) and two oral (25 and 50 mg/kg) doses were administered to female Sprague-Dawley rats. A two-compartment model with nonlinear elimination best characterized the pharmacokinetic profiles after intravenous and oral administration in rats. The terminal half-life of (±)-DHP-014 increased and the systemic clearance significantly decreased at higher doses, indicating nonlinear elimination. The dose-dependent clearance is likely due to saturation of metabolism. The apparent volume of distribution of (±)-DHP-014 was 2.0 L/kg in rats and was unchanged with increasing intravenous doses of (±)-DHP-014. The estimated oral bioavailability of (±)-DHP-014 was 8.2%. The poor bioavailability is likely due to the poor solubility of the compound, as well as to substantial first-pass elimination. Copyright © 2005 John Wiley & Sons, Ltd.

A simple and reliable reversed-phase high-performance liquid chromatography method was developed and validated for the determination of DHP-014, a niguldipine analogue with potent P-glycoprotein inhibitory and negligible calcium channel blocking properties, in rat plasma. DHP-014 and niguldipine hydrochloride (the internal standard) were extracted from rat plasma by liquid extraction using hexane. DHP-014 was then separated by HPLC on a C18 column and quantified by ultraviolet detection at 238 nm. The mobile phase consisted of acetonitrile–aqueous 5 mm phosphate buffer (65:35, v[sol ]v) containing 0.4% (v[sol ]v) triethylamine adjusted to pH 7.0. The mean extraction efficiency of DHP-014 was 109.0 ± 12.9, 97.7 ± 8.0 and 102.9 ± 7.5% for DHP-014 concentrations of 10, 50 and 100 nm, respectively (n = 5). The method was linear over the concentration range 2.5–200 nm with a regression coefficient of 0.998. The limit of detection of DHP-014 in rat plasma was 1.0 nm. The intra- and inter-day coefficients of variation for DHP-014 in rat plasma were 4.7–7.9 and 6.9–9.9%, respectively. The intra- and inter-day accuracy was 98.2–99.5 and 97.9–103%, respectively. The bioanalytical technique was used to determine DHP-014 in plasma samples in a pharmacokinetic study of DHP-014 administered to female Sprague–Dawley rats. Copyright © 2005 John Wiley & Sons, Ltd.

Breast cancer resistance protein (BCRP, ABCG2) is a recently identified member of the ATP-binding cassette family of cell surface transport proteins. This study was conducted to investigate the effect of a series of newly synthesized 1,4-dihydropyridines and pyridines, designed as potent P-glycoprotein inhibitors, on BCRP-mediated drug efflux both in vitro and in vivo. The effects of 25 synthesized dihydropyridines and corresponding pyridines along with 4 commercially available dihydropyridines (niguldipine, nicardipine, nifedipine, and nitrendipine) on the intracellular accumulation of the BCRP substrate mitoxantrone were evaluated in BCRP-expressing human breast cancer MCF-7/MX100 and human non-small cell lung cancer H460/MX20 cells. At a 2.5 microM concentration, 24 of 25 newly synthesized dihydropyridines and pyridines produced a significant increase of mitoxantrone accumulation in both cell lines. The most potent compound was able to enhance mitoxantrone accumulation approximately 4.5-fold, greater than that obtained with 10 microM fumitremorgin C, which is a specific BCRP inhibitor. The results from the two cell lines showed good correlation (r(2) = 0.71, p < 0.01). Niguldipine, nicardipine, and nitrendipine also demonstrated potent BCRP inhibition, whereas nifedipine had no effect. The effects of the dihydropyridine and pyridine compounds on mitoxantrone cytotoxicity paralleled their effects on mitoxantrone accumulation. Coadministration of a selected dihydropyridine compound, I(m) [DHP-014; 3-(3-(4,4-diphenylpiperidin-1-yl)propyl) 5-methyl 4-(3,4-dimethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate)] with topotecan, a good BCRP substrate and a moderate to poor P-glycoprotein substrate, resulted in significant increases in the systemic exposure and peak concentration of topotecan in Sprague-Dawley rats when oral topotecan (2 mg/kg) was combined with 20 mg/kg DHP-014. The observed increase of topotecan exposure provides proof-of-concept for in vivo inhibition of BCRP by these agents.

We demonstrated recently that phenethyl isothiocyanate (PEITC), a potent anticarcinogen present in cruciferous vegetables, inhibited P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1) and that MRP1 can transport PEITC and/or its metabolites. In this study, we have examined whether PEITC is transported by P-gp and MRP2, two transporters with high expression in human intestine, liver and kidney. Using (14)C-PEITC, no significant difference was observed for the intracellular accumulation of PEITC in human breast cancer MCF-7/sensitive (control) and MCF-7/ADR (P-gp overexpressing) cells at PEITC concentrations of 1, 10 and 50 microM. Moreover, the presence of verapamil or PSC833, two P-gp inhibitors, had no significant effect on the intracellular accumulation of PEITC in P-gp overexpressing MCF-7/ADR and MDA435/LCC6MDR1 cells, indicating that PEITC may not be a substrate for P-gp. In contrast, (14)C-PEITC intracellular accumulation in the kidney epithelial MDCK II/MRP2 cells (transfected with human MRP2) was significantly lower than in the wild-type MDCK II/wt cells at PEITC concentrations of 1, 5, 10 and 50 microM. The presence of MK571, an MRP inhibitor, significantly enhanced (14)C-PEITC accumulation in MDCK II/MRP2 but not MDCK II/wt cells. Furthermore, depletion of intracellular glutathione (GSH) following treatment with buthionine sulphoximine, an inhibitor of GSH biosynthesis, significantly increased (14)C-PEITC intracellular accumulation in a concentration-dependent manner. Transcellular transport studies also demonstrated that depletion of intracellular GSH reduced the mean ratio of basal-to-apical transport to apical-to-basal transport of PEITC in MDCK II/MRP2, but not MDCK II/wt cell monolayers. These results indicate that GSH plays an important role in the MRP2-mediated transport of PEITC. The findings provide new information concerning the interactions between PEITC and membrane transporters and suggest the possibility of PEITC interactions with xenobiotics that are MRP2 substrates.

Flavonoids are present in fruits, vegetables and beverages derived from plants (tea, red wine), and in many dietary supplements or herbal remedies including Ginkgo Biloba, Soy Isoflavones, and Milk Thistle. Flavonoids have been described as health-promoting, disease-preventing dietary supplements, and a high intake of flavonoids has been associated with a reduced risk of cancer, cardiovascular diseases, osteoporosis and other age-related degenerative diseases. Due to an increased public interest in alternative medicine and disease prevention, the use of herbal preparations containing high doses of flavonoids for health maintenance has become very popular, raising the potential for interactions with conventional drug therapies. This review will summarize the current literature regarding the interactions of flavonoids with ATP-binding cassette (ABC) efflux transporters, mainly P-glycoprotein, MRP1, MRP2 and BCRP and discuss the potential consequences for flavonoid-drug transport interactions.

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography–tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within ±15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with β-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.

Breast cancer resistance protein (BCRP) is a recently identified ATP-binding cassette transporter, important in drug disposition and in the development of multidrug resistance in cancer. Flavonoids, a major class of natural compounds widely present in foods and herbal products, have been shown to be human BCRP inhibitors. The objective of the present study was to evaluate the potential for in vivo pharmacokinetic interactions by comparing the pharmacokinetics of topotecan (a model BCRP substrate) after oral administration of 2 mg/kg topotecan with and without different doses of the flavonoids chrysin or 7,8-benzoflavone (BF) in rats and mdr1a/1b (-/-) mice. Coadministration of 50 mg/kg GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9, 10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] with 2 mg/kg topotecan significantly increased the area under the plasma concentration-time curve and bioavailability of topotecan by more than 4-fold in these animals, indicating the importance of BCRP in the bioavailability and disposition of topotecan in rats. Chrysin (50 microM) and BF (5 microM) significantly inhibited the BCRP-mediated transport of topotecan in BCRP-overexpressing MCF-7 MX100 cells (MCF-7 cells selected with mitoxantrone) to a level comparable to that observed with 10 microM fumitremorgin C (a potent BCRP inhibitor). However, neither chrysin nor BF significantly altered topotecan pharmacokinetics in rats or in mdr1a/1b (-/-) mice after oral coadministration of doses up to 50 mg/kg. The reason(s) for this lack of in vitro-in vivo association may be the lack of potent inhibition activity of the flavonoids against mouse or rat BCRP, as evidenced by our observation that these flavonoids have only weak, if any, inhibition activity against mouse Bcrp1-mediated transport of topotecan in MDCK-Bcrp1 cells.

Efflux of cytotoxic agents mediated by P-glycoprotein is believed to be an important mechanism of multidrug resistance, which remains a serious limitation to successful chemotherapy in cancers such as metastatic breast cancer. A series of 4-aryl-1,4-dihydropyridines and corresponding aromatized 4-arylpyridines have been synthesized based on structure modifications of niguldipine to enhance multidrug resistance reversal activity, while minimizing calcium channel binding. Thirty new compounds were characterized. [(3)H]Vinblastine accumulation studies indicated that at a concentration level of 3 muM, 15 of 18 4-aryl-1,4-dihydropyridines and all 4-arylpyridines can successfully restore intracellular accumulation of vinblastine in a resistant human breast adenocarcinoma cell line, MCF-7/adr, which overexpresses P-glycoprotein. The most potent compounds led to an approximately 15-fold increase of vinblastine accumulation. All of the test compounds that significantly increased vinblastine accumulation in MCF/adr cells were able to substantially reduce IC(50) values of daunomycin and increase its cytotoxicity in MCF-7/adr-resistant cells, confirming the results of the vinblastine accumulation studies. Calcium channel binding assays for these newly synthesized compounds were conducted using rat cerebral cortex membrane. All but eight compounds demonstrated negligible calcium channel binding over the concentration range from 15 to 2500 nM. The results demonstrate that the newly synthesized series of 1,4-dihydropyridines and pyridines represent P-glycoprotein modulators with negligible calcium channel blocking activity.

Our hypothesis is that PEITC is an effective preventive agent in breast cancer. Although a number of mechanisms may be involved, one mechanism of PEITC effects that has not been explored is the potential for PEITC to alter the synthesis and elimination of estrogens. Our specific aims are: (1) to evaluate the efficacy of dietary PEITC, at relevant doses, in preventing or delaying the onset of breast cancer in estrogen-dependent breast cancer animal models, (2) to evaluate the effects of PEITC on 1 Theta-estradiol (E2) and metabolite (E2 sulfate, E2 glucuronide, estrone, 2-hydroxyE2, 4-hydroxyE2, 2- methoxyE2, and 4-methoxyE2) concentrations in plasma and tumor samples, and (3) to determine changes in enzyme activity and/or mRNA/protein expression in tumor and liver samples, for metabolic pathways which may be altered based on the E2 and E2 metabolite profiles. Our studies to date have characterized the pharmacokinetics of PEITC in rats (our animal model) and demonstrated in preliminary studies that PEITC can alter estrogen metabolism in the liver. Additionally, we found that low concentrations of PEITC can alter the expression of some genes important in breast cancer. These studies will provide new findings regarding the chemopreventive effects of the dietary compound PEITC in estrogen-dependent breast cancer.

The objective of this investigation was to evaluate the effects of two dietary isothiocyanates (ITCs), benzyl‐ (BITC) and phenethyl isothiocyanate (PEITC), and one synthetic ITC, α‐naphthyl isothiocyanate (1‐NITC), on the P‐glycoprotein (P‐gp)‐ and multidrug‐resistance protein 1 (MRP1)‐mediated efflux of daunomycin (DNM), determine whether PEITC is a substrate of P‐gp and/or MRP1, and elucidate the mechanism(s) involved in the inhibition of transport. BITC, PEITC, and 1‐NITC significantly increased the 2‐h accumulation of DNM in MCF‐7/ADR (P‐gp overexpression), PANC‐1 (MRP1 overexpression), and human colon adenocarcinoma Caco‐2 cells (except for 1‐NITC). The accumulation of 14 C‐PEITC was not changed in Caco‐2, human breast cancer MDA435/LCC6 and MDA435/LCC6MDR1 (P‐gp overexpression) cells in the absence and presence of the P‐gp inhibitor verapamil, but significantly increased with the MRP inhibitor MK571 in PANC‐1 cells. The isocyanate and amine metabolites had no effect on DNM accumulation in any cell line. After 2‐ and 24‐h ITC treatments, cellular concentrations of glutathione (GSH) in PANC‐1 and Caco‐2 cells were depleted by BITC and PEITC, but not by 1‐NITC; glutathione‐S‐transferase activity exhibited small changes. Our results suggest that (1) BITC, PEITC, and 1‐NITC inhibit the P‐gp‐ and MRP1‐mediated efflux of DNM; (2) PEITC and/or its conjugates do not represent P‐gp substrates; (3) BITC and PEITC, but not 1‐NITC, inhibit MRP1 through the depletion of intracellular GSH, which acts as a cosubstrate for DNM efflux via MRP1; and (4) PEITC and/or its conjugates are MRP1 substrates so binding interactions with DNM represent a second potential mechanism involved in MRP1 inhibition. © 2004 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1901–1911, 2004

Drug resistance is the main cause for therapeutic failure and death in breast cancer. Our goal is to evaluate dietary organic isothiocyanates (ITCs) as inhibitors of MDR. The first two specific aims of our proposal are: 1) To determine the concentration-dependent effects of benzyl ITC (BITC), phenethyl ITO (PEITC) and naphthyl ITC (NITC) on the accumulation of 3H-daunomycin (DNM) in sensitive and resistant human breast cancer cell lines; and 2) To evaluate the pharmacokinetics and toxicity of NITC, PEITC and BITC in the rat following oral and intravenous administration. NITC, PEITC and BITC significantly increased the cellular accumulation of DNM and vinblastine (VBL) in resistant human breast cancer MCF-7 cells at 20-100 micrometers concentrations, without affecting accumulation in MCF-7 sensitive cells. Cytotoxicity studies revealed that PEITC and BITC inhibited the growth of both the MCF-7 and normal mammary MCF-12A cell lines. Assays to determine PEITC, BITC and NITC in biological fluids were developed, and stability studies demonstrated limited stability of NITC in biological fluids at RT, while PEITC and BITC degraded with half-lives of 36 and 40 h, respectively, at pH 7.4. Characterization of the pharmacokinetics of NITC in rats revealed a high clearance (2.29+/-0.81 L/kg/h) and large volume of distribution (16.8+/-3.6 L/kg). The ITCs may represent a new class of inhibitors of MDR in breast cancer.

Flavonoids are present in fruits, vegetables and beverages derived from plants (tea, red wine), and in many dietary supplements or herbal remedies including Ginkgo Biloba, Soy Isoflavones, and Milk Thistle. Flavonoids have been described as health-promoting, disease-preventing dietary supplements, and have activity as cancer preventive agents. Additionally, they are extremely safe and associated with low toxicity, making them excellent candidates for chemopreventive agents. The cancer protective effects of flavonoids have been attributed to a wide variety of mechanisms, including modulating enzyme activities resulting in the decreased carcinogenicity of xenobiotics. This review focuses on the flavonoid effects on cytochrome P450 (CYP) enzymes involved in the activation of procarcinogens and phase II enzymes, largely responsible for the detoxification of carcinogens. A number of naturally occurring flavonoids have been shown to modulate the CYP450 system, including the induction of specific CYP isozymes, and the activation or inhibition of these enzymes. Some flavonoids alter CYPs through binding to the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, acting as either AhR agonists or antagonists. Inhibition of CYP enzymes, including CYP 1A1, 1A2, 2E1 and 3A4 by competitive or mechanism-based mechanisms also occurs. Flavones (chrysin, baicalein, and galangin), flavanones (naringenin) and isoflavones (genistein, biochanin A) inhibit the activity of aromatase (CYP19), thus decreasing estrogen biosynthesis and producing antiestrogenic effects, important in breast and prostate cancers. Activation of phase II detoxifying enzymes, such as UDP-glucuronyl transferase, glutathione S-transferase, and quinone reductase by flavonoids results in the detoxification of carcinogens and represents one mechanism of their anticarcinogenic effects. A number of flavonoids including fisetin, galangin, quercetin, kaempferol, and genistein represent potent non-competitive inhibitors of sulfotransferase 1A1 (or P-PST); this may represent an important mechanism for the chemoprevention of sulfation-induced carcinogenesis. Importantly, the effects of flavonoids on enzymes are generally dependent on the concentrations of flavonoids present, and the different flavonoids ingested. Due to the low oral bioavailability of many flavonoids, the concentrations achieved in vivo following dietary administration tend to be low, and may not reflect the concentrations tested under in vitro conditions; however, this may not be true following the ingestion of herbal preparations when much higher plasma concentrations may be obtained. Effects will also vary with the tissue distribution of enzymes, and with the species used in testing since differences between species in enzyme activities also can be substantial. Additionally, in humans, marked interindividual variability in drug-metabolizing enzymes occurs as a result of genetic and environmental factors. This variability in xenobiotic metabolizing enzymes and the effect of flavonoid ingestion on enzyme expression and activity can contribute to the varying susceptibility different individuals have to diseases such as cancer. As well, flavonoids may also interact with chemotherapeutic drugs used in cancer treatment through the induction or inhibition of their metabolism.

Phenethyl isothiocyanate (PEITC) is a dietary compound present in cruciferous vegetables that has cancer-preventive properties. Our objective was to develop and validate a novel liquid chromatography-tandem mass spectrometry procedure to analyze PEITC concentrations in human plasma and urine. Following hexane extraction, ammonia was added to samples to derivatize PEITC to phenethylthiourea. Chromatographic separation was achieved on a C(18) column with acetonitrile/5 mM formic acid (60:40, v/v) as the mobile phase followed by tandem mass spectrometry detection in multiple reaction monitoring mode. Deuterium-labeled PEITC was used as the internal standard. The detection limit was 2 nM and calibration curves were linear from 7.8 to 2000 nM. The intra- and inter-day coefficients of variation were less than 5 and 10%, respectively. The intra- and inter-day accuracies ranged from 101.0 to 104.2% and from 102.8 to 118.6%, respectively. The recovery from spiked human plasma and urine ranged from 100.3 to 113.5% and from 98.3 to 103.9%, respectively. The assay was used to measure PEITC in plasma and urine samples obtained from subjects after consumption of 100g of watercress. This novel assay represents the first analytical method with the sensitivity and specificity to determine plasma and urine concentrations of PEITC.

The purpose of this study was to investigate the effects of flavonoids biochanin A and silymarin on intestinal absorption of P-gp substrates by determining their effects on P-gp-mediated efflux in Caco-2 cells.The cellular accumulation and bidirectional transport of digoxin and vinblastine in Caco-2 cells were determined in the presence and absence of flavonoids.The 1.5-h accumulation of digoxin and vinblastine in Caco-2 cells was significantly increased by 50 microM biochanin A or silymarin, and this effect was flavonoid-concentration dependent. The AP-to-BL transport of digoxin was significantly increased, whereas the BL-to-AP transport was significantly decreased by 50 microM biochanin A or 75 microM silymarin. At 150 microM concentrations of biochanin A or silymarin, mean transport ratios (P(app,B-A)/P(app,A-B)) of 1.62 and 4.48, respectively, compared with the control ratio of 43.4, were obtained.These results indicate that biochanin A and silymarin can inhibit P-gp-mediated efflux in Caco-2 cells, suggesting they could potentially increase the absorption/bioavailability of coadministered drugs that are P-gp substrates.

The objective of the current investigation was to examine the transport characteristics of choline, an endogenous quaternary ammonium compound, into human intestinal Caco-2 cells; the transport of choline has not been characterized in human intestine. The cellular accumulation of choline was independent of an inwardly directed Na(+) gradient and demonstrated temperature dependence and saturability. Using the initial uptake rates, choline accumulation was best characterized by a Michaelis-Menten equation and a diffusion component with a K(m) and V(max) of 110 +/- 3 micro mol/L and 2800 +/- 250 pmol/(mg protein. 10 min), respectively. Choline uptake was significantly inhibited by an excess of choline itself and by hemicholinium-3, a structural analog of choline. However other hydrophilic organic cations, such as tetraethylammonium (TEA) and N-methylnicotinamide (NMN), did not affect choline uptake in Caco-2 cells. Additionally, two typical p-glycoprotein substrates, daunomycin and verapamil, both inhibited choline accumulation. However the opposite was not true: choline did not inhibit DNM accumulation in Caco-2 cells. These results indicate the presence of a carrier-mediated transport system for choline in Caco-2 cells. The substrate specificity of this carrier is unlike that seen in the rat intestinal epithelium, and the human transport protein is distinct from those for TEA and NMN. P-glycoprotein substrates may inhibit choline uptake through specific or nonspecific interactions with the choline transporter.

Flavonoids are constituents of fruits, vegetables, and plant-derived beverages, as well as components in herbal-containing dietary supplements. The objective of this investigation was to characterize the effect of flavonoids on P-glycoprotein (P-gp)-mediated cellular efflux and to determine the molecular mechanism(s) of the flavonoid-drug interaction. Studies were conducted in the sensitive and multidrug resistant human breast cancer cell lines MCF-7 and MDA435/LCC6 and examined the effects of the flavonoids biochanin A, morin, phloretin, and silymarin on daunomycin (DNM) accumulation and doxorubicin cytotoxicity. The potential mechanism(s) involved in the interaction was evaluated by determining flavonoid effects on 1) P-gp ATPase activity, 2) [ 3 H]azidopine photoaffinity labeling of P-gp, and 3) cellular P-gp levels. The flavonoids increased [ 3 H]DNM accumulation in P-gp positive cells, but not P-gp negative cells, and these effects were both flavonoid concentration- and P-gp expression level-dependent. Biochanin A and silymarin potentiated doxorubicin cytotoxicity in P-gp positive cells. Biochanin A and phloretin stimulated, whereas morin and silymarin inhibited P-gp ATPase activity, confirming that these flavonoids interact with P-gp. Morin and silymarin significantly inhibited [ 3 H]azidopine photoaffinity labeling of P-gp, suggesting a direct interaction with P-gp substrate binding. A 24-h preincubation with all flavonoids, followed by flavonoid removal, did not alter cellular P-gp level in P-gp positive cells. In conclusion, biochanin A, morin, phloretin, and silymarin all inhibited P-gp-mediated cellular efflux and the mechanism of the interaction involved, at least in part, a direct interaction. The findings of this study indicate a potential for significant flavonoid-drug interactions with P-gp substrates.

Serum sulfate concentrations are elevated in infants, young children, and pregnant women due, at least in part, to increased renal sulfate reabsorption. Little is known about the effects of hormones, particularly those involved in growth, development, and pregnancy, on renal sulfate reabsorption. The objective of this investigation was to examine the effects of growth hormone (GH), insulin-like growth factor 1 (IGF-1), progesterone (PG), and 17beta-estradiol (EST) on renal sodium/sulfate co-transport. 35S-sulfate uptake was determined in Madin-Darby canine kidney (MDCK)/NaSi-1 cells (MDCK cells that have been stably transfected with rat sodium/sulfate co-transporter (NaSi-1) cDNA) and in opossum kidney (OK) cells. NaSi-1 mRNA was determined by RT-PCR and protein levels by ELISA. GH (0.1 nM) significantly increased the sodium/sulfate co-transport in MDCK/NaSi-1 cells up to 35%. IGF-1 induced a concentration-related stimulation of the sodium/sulfate co-transport with a maximal response observed at 1000 nM (59% increase). Sodium-dependent sulfate uptake was significantly increased when cells were preincubated with 10 nM PG, 10 nM EST, or 10 nM PG/10 nM EST up to 41%, 46%, or 39%, respectively. OK cells exhibited endogenous sodium-dependent sulfate transport; significantly increased sodium/sulfate co-transport was also observed in OK cells that were preincubated with GH, IGF-1, and PG/EST, although not with EST alone. The NaSi-1 mRNA and NaSi-1 protein levels were significantly increased in MDCK/NaSi-1 cells treated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST compared with control. These results suggest that the increased renal sulfate reabsorption that occurs in neonates, young and pregnant humans, and animals could be mediated by the increased steady-state levels of NaSi-1 mRNA produced by the higher plasma concentrations of GH, IGF-1, or PG/EST.

Serum sulfate concentrations are increased in infants, young children, and pregnant women, compared with adult values. The objective of this investigation was to examine the influences of age, gender, and pregnancy on renal sulfate transport using guinea pigs as an animal model. Membrane vesicles were isolated from the kidney cortex of male animals at four different ages, from male and female adult animals, and from pregnant and nonpregnant female animals. There were no significant differences in marker enzymes for the brush-border membrane (BBM) or basolateral membrane (BLM) among all groups examined. Uptake was determined by a rapid filtration method and membrane fluidity by measuring the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The Vmax values for Na+ /sulfate co-transport in BBM were significantly increased with decreasing age, whereas the Km for this process was unchanged. The Vmax and Km for Na + /sulfate co-transport in BBM of pregnant animals were significantly higher than the values in the nonpregnant group. Bicarbonate-driven anion exchange of sulfate in BLM was not different among the different age groups. The Vmax for the bicarbonate/sulfate exchange process in BLM was not different between pregnant and nonpregnant groups; however, the Km for this process in BLM of pregnant animals was significantly greater than the value in nonpregnant animals. There were no gender-related differences in sulfate transport in BBM or BLM isolated from adult male and female animals. Renal BBM fluidity was increased with decreasing age and in pregnant animals, suggesting that altered membrane fluidity may represent one possible mechanism to explain the increased sodium/sulfate uptake in young and pregnant animals. The higher Vmax for Na+/sulfate co-transport in young and pregnant animals suggests that there is an increased density of co-transporter protein or an increase in the rate of movement of the carrier protein (i.e., turnover) once loaded with sodium and sulfate. This increased conservation of inorganic sulfate in young and pregnant guinea pigs may be related to the increased demand for sulfated substrates, such as sulfated glycosaminoglycans, during growth and development.

P-glycoprotein (P-gp), the multidrug resistance gene product, is expressed in a normal liver exclusively on the canalicular membrane of the hepatocyte. The objective of this study was to examine the effect of age on the P-gp transport system using canalicular membrane (cLPM) vesicles isolated from the liver of developing (22 days old) and adult rats. No differences in protein yield, intravesicular volumes, and enrichments of cLPM enzymes or enzymes representing contamination of subcellular organelles were found for vesicles isolated from both groups, demonstrating the isolation of similar cLPM vesicle preparations. The transport of daunomycin (DNM), a P-gp substrate, was used to study age-related functional differences in P-gp. DNM uptake in the presence of ATP was greater than uptake in the absence of ATP in both young and adult cLPM vesicles, showing that P-gp is functional in both groups. In young and adult groups only ATP was a potent stimulator of transport when compared with ATP degradation products and a nonhydrolyzable ATP analogue. Although ATP-dependent uptake tended to be greater in the adult compared to the young, there was no statistically significant difference in DNM kinetics (Vmax, km, gamma) between groups. Canalicular membrane from the young rats showed decreased fluidity, as assessed by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene; however there was no significant difference between groups. Examination of P-gp expression using the monoclonal antibody C219 revealed similar levels of expression in the young as in the adult. Our results suggest that P-gp in the bile canaliculus of developing rats is functional with similar levels of function and expression as observed in the adult.

Salicylic acid (SA) can inhibit the facilitated transport of inorganic sulfate in the kidney, placenta, and erythrocytes. One mechanism of this inhibition could involve the interaction of SA with membranes, resulting in altered function of transporter protein(s) due to changes in membrane fluidity. Such membrane effects could result in altered membrane transport and consequently in changes in the pharmacokinetics and the therapeutic activity of both xenobiotics and endogenous substrates. We investigated the effect of SA on the fluidity of brush border membrane (BBM) and basolateral membrane (BLM) isolated from rat kidney and also on the physical properties (such as phase transition temperature and fluidity) of model membranes by fluorescence polarization and differential scanning calorimetry (DSC) techniques. SA decreased the lipid order parameter (S) of BBM and BLM membranes in a concentration-dependent manner, indicating that the addition of SA makes the membrane more fluid. The fluidizing effect of SA was more pronounced than that of benzyl alcohol. Studies were carried out with protein-free model membranes composed of dipalmi-toylphosphatidylcholine (DPPC) to investigate the effects of SA on the bilayer membrane lipids. SA decreased the fluorescence polarization of DPH (1,6-diphenyl 1,3,5- hexatriene) incorporated in DPPC vesicles. DSC studies demonstrated that SA broadened the phase transition temperature of DPPC vesicles and suggested that SA is located in the C1-C8 region of the acyl chain. In protein-free model membranes, SA exerted fluidizing effects through its incorporation into the cooperative hydrophobic region of the bilayer. The perturbation of membrane physical properties induced by SA and its hydrophobic localization in the membrane bilayer may be important in the SA-induced alteration of sulfate membrane transport.

P-glycoprotein (P-gp), the multidrug resistance (MDR) gene product, is exclusively located on the canalicular membrane of hepatocytes. Recent studies using isolated rat canalicular liver plasma membrane (cLPM) vesicles indicate that daunomycin (DNM) is a substrate for the ATP-dependent P-gp efflux system in the rat liver. The isoforms of P-gp present in cLPM and in cancer cell lines differ in that the major form present in the liver represents the gene product of mdr2 in mice (MDR3 in humans; class III) while the isoform of P-gp in cancer cells is the gene product of mdr1 in mice (MDR1 in humans, class I). The objective of this study was to examine the inhibitory effects of various organic compounds, most of which have been studied previously in MDR cancer cells, on P-gp-mediated [3H]DNM uptake into cLPM. Also, the stereospecificity of P-gp for its substrates was investigated by comparing the inhibitory effects of the enantiomers and the racemic mixtures of verapamil and propranolol. DNM exhibited ATP-dependent active transport into rat liver cLPM with a Km of 26.8 +/- 13.4 microM and a Vmax of 4.9 +/- 0.8 nmol/45 s/mg of protein (n = 4). ADP, AMP, and a nonhydrolyzable ATP analogue did not increase DNM transport over the control value. Thirty-one potential inhibitors were examined; only acridine orange, doxorubicin, verapamil, propranolol, phosphatidylcholine, beta-estradiol glucuronide, and DNM itself showed statistically significant inhibition of [3H]DNM uptake into cLPM. These results suggest that only a limited number of substrates bind to or are transported across the hepatic canalicular membrane via P-gp. Phosphatidylcholine, a substrate for the gene product of the class III P-gp gene, produced significant inhibition of [3H]DNM transport (30.6% at a 10-fold-higher substrate concentration), suggesting that transport may be mediated, at least in part, by this P-gp gene product. There were no statistically significant differences in the inhibitory effects of the enantiomers and racemate of verapamil on [3H]DNM transport into cLPM, but the enantiomers of propranolol exhibited stereospecific inhibition of DNM transport. (R)-(+)-Propranolol produced a statistically significant inhibition of [3H]DNM transport similar to that observed with the racemic mixture, while (S)(-)-propranolol showed no inhibition. These findings suggest that bile canalicular P-gp may exhibit stereospecificity of binding or transport for its substrates.

Inorganic sulfate is a physiological anion which is utilized in the metabolism of both endogenous compounds and xenobiotics. Its homeostasis is maintained predominantly by facilitated reabsorptive processes in the kidneys. The objectives of the present investigation were to evaluate the effects of menopausal status and caffeine ingestion on the serum concentrations and clearance of inorganic sulfate. Thirty-nine women who were classified as premenopausal, postmenopausal with or without estrogen treatment, and postmenopausal with osteoporosis participated in the study. The women were studied on two separate occasions following the ingestion of a decaffeinated beverage to which 6 mg caffeine/kg lean body mass or no caffeine was added. All women were habitual caffeine users (mean ingestion of 588 mg caffeine per day) but abstained from all caffeine sources for 2 weeks prior to the control study day. Postmenopausal women with estrogen supplementation exhibited significantly lower sulfate serum concentrations (0.24 +/- 0.02 mM vs. 0.32 +/- 0.04 mM in premenopausal women, mean +/- SD, p < 0.05) and a decreased renal reabsorption of sulfate for the control (no caffeine) period. There was no difference in serum sulfate or sulfate reabsorption in estrogen supplemented postmenopausal women, compared with women not taking estrogen. Postmenopausal women with osteoporosis had significantly lower creatinine and sulfate clearances than postmenopausal women with estrogen supplementation which may be related to their older age, or factors related to the disease process. The 6 mg/kg dose of caffeine caused a diuresis, but no change in GFR, as indicated by urine volume and creatinine clearance values, respectively. Caffeine administration resulted in an increase in the sulfate excretion rate; there was no change in sulfate serum concentrations. The results of this investigation indicate that menopause results in decreased sulfate serum concentrations that may be the consequence of a decreased renal reabsorption of sulfate. Secondly, this investigation demonstrated that caffeine ingestion increases the urinary excretion of sulfate, an effect that may be related to the diuretic effect of caffeine or due to a caffeine-induced alteration in the renal reabsorption of sulfate.

Renal proximal tubule dysfunction has been reported in patients treated with the chemotherapeutic agent ifosfamide. The present study investigated whether ifosfamide or its metabolites acrolein and chloroacetaldehyde would impair function in the isolated perfused rat kidney. Renal function was monitored before and after these chemicals were added to a modified Krebs-Ringer-bicarbonate perfusion medium containing 6.6 g/dl albumin and a mixture of substrates. No functional changes were observed when ifosfamide (470 microM) or acrolein (470 microM) was added to the perfusate. Addition of chloroacetaldehyde (210 microM) resulted in significant decreases in the fractional reabsorption of sodium (from 92 to 32%), glucose (from 97 to 46%), inorganic phosphate (from 88 to 22%), and inorganic sulfate (from 94 to 86%). There were no changes in glomerular filtration rate. PAH clearance also significantly decreased from 4.1 to 0.7 ml/min per gram of kidney weight, indicating impairment of proximal tubule organic acid secretion. This impairment was associated with a significant decline in the extraction ratio for PAH, suggesting abnormal PAH uptake at the basolateral membrane. These results show that chloroacetaldehyde causes generalized renal proximal tubule dysfunction and that it may be the ifosfamide metabolite responsible for nephrotoxic side effects.

Gentisamide (2,5-dihydroxybenzamide, GAM), a substrate that forms two monosulfates at the 2 and 5 positions (GAM-2S and GAM-5S) and a monoglucuronide at the 5 position (GAM-5G), was delivered at 8 or 80 microM by normal (N) and retrograde (R) flows to the once-through rat liver preparation. At the lower (8 microM) input concentration, ratios of conjugate formation rate, GAM-5S/GAM-5G and GAM-2S/GAM-5G, were decreased significantly (4.01 +/- 1.42 to 2.93 +/- 0.99, and 1.13 +/- 0.65 to 0.66 +/- 0.41, respectively) whereas a small but significant increase in the steady-state extraction ratio, E (0.89 +/- 0.029 to 0.94 +/- 0.016), was observed upon changing the flow direction from N to R. At the higher input GAM concentration (80 microM), conjugate formation rate ratios were relatively constant for GAM-5S/GAM-5G (1.18 +/- 0.08 to 1.11 +/- 0.12) and GAM-2S/GAM-5G (0.33 +/- 0.05 to 0.31 +/- 0.05) upon changing flow direction from N to R, despite a slight increase in E from 0.87 +/- 0.023 to 0.91 +/- 0.016 was observed. These results suggest that the sulfotransferase activities responsible for 2- and 5-sulfoconjugations are identically distributed and localized anterior to 5-glucuronidation activities during a normal flow of substrate into the rat liver (entering the portal vein and exiting the hepatic vein), and that the presence of uneven distribution of conjugation activities is discriminated only at the lower input drug concentration. At high concentration (greater than Km for all systems), saturation of all pathways occurs, and other anteriorly/identically distributed competing pathways would fail to perturb downstream intrahepatic drug concentrations and the resultant conjugation rates. The lack of change in metabolic profiles renders the condition unsuitable for examination of uneven distribution of enzymes in the liver. These observations were generally predicted by theoretical enzymic models of consistent distribution patterns. Because 2- and 5-sulfation were mediated by systems of similar Km but different Vmax values, two possibilities, the same isozyme of sulfotransferase being involved in the formation of two enzyme-substrate complexes to form two distinctly different products or two isozymes of sulfotransferases of identical distribution, were discussed.

Depletion of endogenous inorganic sulfate can have pronounced effects on the elimination kinetics and metabolic fate of phenolic drugs. Our purpose was to determine the effects of acetaminophen (which is partly metabolized to acetaminophen sulfate), ascorbic acid (subject to more limited sulfation than acetaminophen), and sodium sulfate (useful for sulfate repletion by the oral route) on the serum concentration and renal excretion of inorganic sulfate in healthy adults. Six men and two women, 26 to 35 yr old, were studied on four occasions that were at least 4 days apart. They received no medication, 1.5 gm acetaminophen, 6 gm ascorbic acid, or 9 gm sodium sulfate decahydrate orally, in aqueous solution. A blood sample was obtained 2 hr later and urine was collected from 1 to 3 hr. Serum inorganic sulfate concentrations (mean +/- SD), 0.410 +/- 0.043 mM in the control period, were decreased after acetaminophen (0.311 +/- 0.043 mM, P less than 0.001), increased after sodium sulfate (0.513 +/- 0.055 mM, P less than 0.001), and apparently unchanged after ascorbic acid (0.417 +/- 0.059 mM). The urinary excretion of inorganic sulfate was decreased after acetaminophen and increased after sodium sulfate. The renal clearance of endogenous creatinine was not affected by any of the treatments. The renal tubular reabsorption of inorganic sulfate is capacity limited, as evidenced by the decrease of the reabsorbed fraction with increasing glomerular filtration rate of the anion (r = -0.54, P less than 0.005). This saturable reabsorption facilitates sulfate homeostasis.

The use of magnesium sulfate (Epsom salt) as a cathartic in patients with impaired renal function can lead to severe toxicity due to hypermagnesemia. Although toxicity is uncommon in healthy subjects, little is known concerning the extent of absorption of magnesium after a cathartic dose of magnesium sulfate. The bioavailability of magnesium following a large oral dose of magnesium sulfate in normal volunteers was examined in the present investigation. Baseline 24-hour urinary excretion rates of magnesium and creatinine were determined over 3 consecutive days in 6 healthy men. The oral administration of 13.9 g (56.5 mmoles) magnesium sulfate U.S.P., in 4 equal hourly increments, resulted in the urinary excretion (corrected for baseline excretion rate) of 4.0 +/- 2.9% (mean +/- SD) of the dose of magnesium during the first 24 hours and 6.9 +/- 7.0% of the dose during a 72-hour interval. Magnesium sulfate administration had no effect on the 24-hour urinary excretion rate of creatinine. The baseline excretion rate of magnesium was significantly correlated with that of creatinine (r = 0.875) and inorganic sulfate (r = 0.921). All of the subjects experienced mild or moderate diarrhea. Therefore, magnesium is absorbed to a limited and variable extent in healthy adults following a cathartic dose of magnesium sulfate.

In a double-blind, crossover study involving 40 post-cholecystectomy patients, the analgesic activity of a new oral nonnarcotic agent, floctafenine, was compared with that of oral meperidine and oral propoxyphene. A behavioral approach to the measurement of pain relief, i.e., the measurement of pain on movement or coughing, was used in addition to the accepted subjective method. Floctafenine 200 mg was found to have analgesic activity intermediate between that of meperidine 75 mg and propoxyphene 65 mg. Patients reported the fewest side effects after floctafenine. Initial pain level was found to affect the final pain score. Effects on pain relief due to age, sex, time of the day, and carryover effects were also examined and not found to be significant.

A case report of subacute reversible ischemic colitis associated with use of oral contraceptives (OCs) is reported. A 19-year-old woman was admitted to the hospital with chief complaints of abdominal cramps nausea vomiting diarrhea and rectal bleeding of 2 days duration. Past medical history and family history were noncontributory. The patient was receiving no medication other than Norinyl 2 (2 mg of norethindrone and .1 mg of mestranol) which she had been taking for 6 months. 2 days before admission the patient had taken 100 mg of dimenhydrinate and 2 ExLax tablets (90 mg of phenolphthalein) for constipation. Colonic roentgenograms revealed impaired mesenteric circulation and bowel ischemia; OC-induced ischemic bowel disease was diagnosed. Patient symptoms subsided within 96 hours of discontinuing the OC and initiating supportive therapy (including intravenous fluid infusion nasogastric suction analgesics and antiemetics). When a repeat barium enema was performed it showed resolution of the ischemia. In a short review following the case report these drugs were indicted in causation of colitis-like syndrome: amoxicillin ampicillin cephazolin chloramphenicol chlorpropamide clindamycin cloxacillin cotrimoxasole cyclophosphamide digitalis ergotamine tartrate flucytosine fluorouracil gold salts laxative and cathartic abuse mercurous chloride methyldopa penicillin V and tetracycline. Ischemic bowel disease secondary to OC use is a rare but important complication because of its significant morbidity and potential mortality and because of the widespread use of the drugs. The case report emphasizes the need to consider the differential diagnosis of acute vascular insult with bowel ischemia when acute abdominal pain progressing to bloody diarrhea occurs in young women taking OCs.

An assay using nonsuppressed (single-column) anion chromatography was developed to determine the concentration of inorganic sulfate in biologic fluids. A conventional HPLC system with an anion-exchange column and conductimetric detector interfaced with an automatic injector and integrator was used. The mobile phase for the chromatography of urine and serum samples is 4 m m potassium hydrogen phthalate, pH 4.5, and potassium iodide is used as the internal standard. For cerebrospinal fluid samples, the mobile phase is modified by addition of 10% of a 4 m m phthalic acid solution. Results of the HPLC assay were found to correlate well (r = 0.991 and 0.999) with those of two commonly used spectrophotometric methods for urine and serum inorganic sulfate determinations. However, the concentrations determined by ion chromatography were 2.5 to 10% lower, possibly due to less assay interference by other substances following chromatographic separation of sulfate. Anion chromatography using a single-column system is a convenient and relatively inexpensive method with sufficient sensitivity for the determination of inorganic sulfate concentrations in urine, serum, and cerebrospinal fluid.

The compatibility and stability of diazepam injection were studied following dilution to 10 different concentrations in dextrose 5% in water, normal saline, Ringer's injection and lactated Ringer's injection. Prepared solutions were examined for clarity and pH throughout a 24-hour period. Clear solutions were filtered and then assayed 24 hours after preparation using UV spectrophotometry. Solutions which formed visible precipitates before 24 hours or which did not demonstrate acceptable potency (greater than or equal to 90%) were assayed after shorter time periods. Dilutions lower than 1:20 (5 mg in 20 ml) resulted in immediate visible precipitates in all four diluents. The 1:20 dilution was compatible with all four diluents and maintained acceptable potency for four hours, whereas at 1:40 dilution (5 mg in 40 ml) diazepam was stable for at least 6-8 hours in the same diluents. At higher dilutions of 1:50, 1:75 and 1:100 (5 mg in 50, 75 and 100 ml, respectively) the injection was compatible with and stable in all four diluents for 24 hours. Further studies are required before the use of diazepam injection as an infusion can be recommended. However, if in some circumstances it is necessary to administer diazepam as an infusion, it is recommended that it be diluted in dextrose 5% in water, normal saline, Ringer's injection or lactated Ringer's injection to a dilution of at least 1:40 and used within 6 hours or to a dilution of 1:50 and used within 24 hours.