Marilyn E. Morris

Overview of the Proton-coupled MCT (SLC16A) Family of Transporters: Characterization, Function and Role in the Transport of the Drug of Abuse γ-Hydroxybutyric Acid

The transport of monocarboxylates, such as lactate and pyruvate, is mediated by the SLC16A family of proton-linked membrane transport proteins known as monocarboxylate transporters (MCTs). Fourteen MCT-related genes have been identified in mammals and of these seven MCTs have been functionally characterized. Despite their sequence homology, only MCT1–4 have been demonstrated to be proton-dependent transporters of monocarboxylic acids. MCT6, MCT8 and MCT10 have been demonstrated to transport diuretics, thyroid hormones and aromatic amino acids, respectively. MCT1–4 vary in their regulation, tissue distribution and substrate/inhibitor specificity with MCT1 being the most extensively characterized isoform. Emerging evidence suggests that in addition to endogenous substrates, MCTs are involved in the transport of pharmaceutical agents, including γ-hydroxybuytrate (GHB), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins), salicylic acid, and bumetanide. MCTs are expressed in a wide range of tissues including the liver, intestine, kidney and brain, and as such they have the potential to impact a number of processes contributing to the disposition of xenobiotic substrates. GHB has been extensively studied as a pharmaceutical substrate of MCTs; the renal clearance of GHB is dose-dependent with saturation of MCT-mediated reabsorption at high doses. Concomitant administration of GHB and l-lactate to rats results in an approximately two-fold increase in GHB renal clearance suggesting that inhibition of MCT1-mediated reabsorption of GHB may be an effective strategy for increasing renal and total GHB elimination in overdose situations. Further studies are required to more clearly define the role of MCTs on drug disposition and the potential for MCT-mediated detoxification strategies in GHB overdose.

Quercetin pharmacokinetics in humans

The purpose of this study was to examine the pharmacokinetics of quercetin aglycone as well as its conjugated metabolites and to develop a population pharmacokinetic model for quercetin that incorporates enterohepatic recirculation. The stability of quercetin in different matrices at various temperatures and pH, and the quercetin content of six capsules of the herbal preparation Quercetin‐500 Plus® were determined by HPLC. Subjects received quercetin 500 mg three times daily and blood and urine samples were obtained. The concentration of quercetin aglycone and conjugated metabolites were assayed using a liquid chromatography‐tandem mass spectrometry assay. Pharmacokinetic parameters were determined using noncompartmental analysis with WinNonlin. A population compartment model incorporating input from the gallbladder was developed to account for the enterohepatic recirculation observed with quercetin. The oral clearance (CL/F) was high (3.5 × 104l/h) with an average terminal half‐life of 3.5 h for quercetin. The plasma concentration versus time curves exhibited re‐entry peaks. A one‐compartment model that included enterohepatic recirculation best described the plasma data. This represents the first comprehensive evaluation of the pharmacokinetics and enterohepatic recirculation of quercetin in humans. Population pharmacokinetic models adapted for enterohepatic recirculation allowed an assessment of the magnitude and frequency of the enterohepatic recirculation process. Copyright

FLAVONOIDS AS A NOVEL CLASS OF HUMAN ORGANIC ANION-TRANSPORTING POLYPEPTIDE OATP1B1 (OATP-C) MODULATORS

Flavonoids are a class of polyphenolic compounds widely present in the diet and herbal products. The interactions of flavonoids with some major efflux transporters [e.g., P-glycoprotein, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein] have been reported; however, their interactions with uptake transporters are largely unknown. Organic anion-transporting polypeptide OATP1B1 is a liver-specific uptake transporter important in hepatic drug disposition. Our objective was to evaluate the effects of 20 naturally occurring flavonoids, and some of their corresponding glycosides, on the uptake of [3H]dehydroepiandrosterone sulfate (DHEAS) in OATP1B1-expressing and OATP1B1-negative HeLa cells. Many of the tested flavonoids (including biochanin A, genistein, and epigallocatechin-3-gallate) significantly inhibited [3H]DHEAS uptake in a concentration-dependent manner in OATP1B1-expressing cells, with biochanin A being one of the most potent inhibitors with an IC50 of 11.3 ± 3.22 μM. The flavonoids had negligible or small effects in OATP1B1-negative cells. Four of the eight pairs of tested flavonoids and their glycosides, namely, genistein/genistin, diosmetin/diosmin, epigallocatechin/epigallocatechin-3-gallate, and quercetin/rutin, exhibited distinct effects on [3H]DHEAS uptake. For example, genistin did not inhibit DHEAS uptake, whereas genistein did, and rutin stimulated uptake, whereas quercetin had no effect. [3H]Biochanin A uptake was similar in OATP1B1-expressing and OATP1B1-negative cells, suggesting that it is not a substrate for OATP1B1. A kinetic study revealed that biochanin A inhibited [3H]DHEAS uptake in a noncompetitive manner, with a Ki of 10.2 ± 1.89 μM. Taken together, these results indicate that flavonoids are a novel class of OATP1B1 modulators, suggesting the potential for diet-drug interactions.

Combined Effects of Multiple Flavonoids on Breast Cancer Resistance Protein (ABCG2)-Mediated Transport

AbstractPurpose. The purpose of this study was to determine the dynamic parameter (EC50) of flavonoids apigenin, biochanin A, chrysin, genistein, kaempferol, hesperetin, naringenin, and silymarin for breast cancer resistance protein (BCRP) inhibition when used alone, and to evaluate their potential interactions (additive, synergistic, or antagonistic) with regards to BCRP inhibition when used in multiple-flavonoid combinations. Methods. The effects of flavonoids on BCRP-mediated transport were examined by evaluating their effects on mitoxantrone accumulation and cytotoxicity in MCF-7 MX100 cells overexpressing BCRP. The EC50 values of these flavonoids for increasing mitoxantrone accumulation were estimated using a Hill equation. The potential interactions among multiple flavonoids with regard to BCRP inhibition were assessed by isobologram and Berenbaums interaction index methods. Results. The EC50 values of these flavonoids for increasing mitoxantrone accumulation ranged from 0.39 ± 0.13 μM to 33.7 ± 2.78 μM. Quantitative analysis of the combined effects of multiple flavonoids on mitoxantrone accumulation indicated that these flavonoids act additively in inhibiting BCRP when given as 2-, 3-, 5-, or 8-flavonoid combinations with equimolar concentrations of all constituents. The results of the mitoxantrone cytotoxicity studies were consistent with these findings. Conclusions. The additive effects of multiple flavonoids for BCRP inhibition suggests that prediction of BCRP-mediated food (herbal product)-drug interactions should also take into consideration the presence of multiple flavonoids and provides a rationale for using “flavonoid cocktails” as a potential approach for multidrug resistance reversal in cancer treatment.

Gender Differences in the Membrane Transport of Endogenous and Exogenous Compounds

Gender differences have been well described in pharmacokinetics and contribute to the interindividual variation in drug disposition, therapeutic response, and drug toxicity. Sex-related differences in the membrane transport of endogenous substrates and xenobiotics have been reported in various organs of the body including kidney, liver, intestine, and brain. These gender-related differences in transport systems could also contribute to interindividual variability in pharmacokinetics and pharmacodynamics. This review will focus on current knowledge of gender-associated differences in the transport of endogenous and exogenous compounds in a variety of body organs and will discuss the implications and the clinical significance of these observations.

Mechanistic Determinants of Biotherapeutics Absorption Following SC Administration

The subcutaneous (SC) route is of growing interest for the administration of biotherapeutics. Key products on the biotherapeutic market such as insulins, but also several immunoglobulins or Fc-fusion proteins, are administered SC. Despite the importance of the SC route, the available knowledge about the processes involved in the SC absorption of biotherapeutics is limited. This review summarizes available information on the physiology of the SC tissue and on the pharmacokinetic processes after SC administration including “first pass catabolism” at the administration site as well as transport in the extracellular matrix of the SC tissue, followed by absorption into the blood circulation or the lymphatic system. Both monoclonal antibodies and other biotherapeutics are discussed. Determinants of absorption after SC administration are summarized including compound properties such as charge or molecular weight. Scale-up of animal data to humans is discussed, including the current shortcomings of empirical scaling approaches and the lack of suitable mechanistic approaches.

Effect of the flavonoids biochanin A and silymarin on the P-glycoprotein-mediated transport of digoxin and vinblastine in human intestinal Caco-2 cells.

AbstractPurpose. 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. Methods. The cellular accumulation and bidirectional transport of digoxin and vinblastine in Caco-2 cells were determined in the presence and absence of flavonoids. Results. The 1.5-h accumulation of digoxin and vinblastine in Caco-2 cells was significantly increased by 50 μM 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 μM biochanin A or 75 μM silymarin. At 150 μM concentrations of biochanin A or silymarin, mean transport ratios (Papp,B-A/Papp,A-B) of 1.62 and 4.48, respectively, compared with the control ratio of 43.4, were obtained. Conclusion. 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.

Effects of the Flavonoid Chrysin on Nitrofurantoin Pharmacokinetics in Rats: Potential Involvement of ABCG2

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 IC50 of 0.39 μM 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 μM. Compared with the vehicle-treated group, p.o. coadministration of chrysin (200 mg/kg) significantly increased the area under the curve (AUC) and Cmax 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 μg/ml · 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.

Dietary Organic Isothiocyanates Are Cytotoxic in Human Breast Cancer MCF-7 and Mammary Epithelial MCF-12A Cell Lines

Organic isothiocyanates (ITCs) are dietary components present in cruciferous vegetables. The purpose of this investigation was to examine the cytotoxicity of 1-naphthyl isothiocyanate (NITC), benzyl isothiocyanate (BITC), β-phenethyl isothiocyanate (PEITC), and sulforaphane in human breast cancer MCF-7 and human mammary epithelium MCF-12A cell lines, as well as in a second human epithelial cell line, human kidney HK-2 cells. The cytotoxicity of NITC, BITC, PEITC, and sulforaphane, as well as the cytotoxicity of the chemotherapeutic agents daunomycin (DNM) and vinblastine (VBL), were examined in MCF-7/sensitive (wt), MCF-7/Adr (which overexpresses P-glycoprotein), MCF-12A, and HK-2 cells. Cell growth was determined by a sulforhod-amine B assay. The IC50 values for DNM and VBL in MCF-7/Adr cells were 7.12 ± 0.42 μM and 0.106 ± 0.004 μM (mean ± SE) following a 48-hr exposure; IC50 values for BITC, PEITC, NITC, and sulforaphane were 5.95 ± 0.10, 7.32 ± 0.25, 77.9 ± 8.03, and 13.7 ± 0.82 μM, respectively, with similar values obtained in MCF-7/wt cells. Corresponding values for BITC, PEITC, NITC, and sulforaphane in MCF-12A cells were 8.07 ± 0.29, 7.71 ± 0.07, 33.6 ± 1.69, and 40.5 ± 1.25 μM, respectively. BITC and PEITC can inhibit the growth of human breast cancer cells as well as human mammary epithelium cells at concentrations similar to those of the chemotherapeutic drug DNM. Sulforaphane and NITC exhibited higher IC50 values. The effect of these ITCs on cell growth may contribute to the cancer chemopreventive properties of ITCs by suppressing the growth of preclinical tumors, and may indicate a potential use of these compounds as chemotherapeutic agents in cancer treatment.

Serum concentration and renal excretion by normal adults of inorganic sulfate after acetaminophen, ascorbic acid, or sodium sulfate.

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 < 0.001), increased after sodium sulfate (0.513 ± 0.055 mM, P < 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 < 0.005). This saturable reabsorption facilitates sulfate homeostasis.