Intestinal transport of HDND-7, a novel hesperetin derivative, in in vitro MDCK cell and in situ single-pass intestinal perfusion models.

1. Hesperetin (HDND) possesses extensive bioactivities, however, its poor solubility and low bioavailability limit its application. HDND-7, a derivative of HDND, has better solubility and high bioavailability. In this study, we investigated the intestinal absorption mechanisms of HDND-7. 2. MDCK cells were used to examine the transport mechanisms of HDND-7 in vitro, and a rat in situ intestinal perfusion model was used to characterize the absorption of HDND-7. The concentration of HDND-7 was determined by HPLC. 3. In MDCK cells, HDND-7 was effectively absorbed in a concentration-dependent manner in both directions. Moreover, HDND-7 showed pH-dependent and TEER-independent transport in both directions. The transport of HDND-7 was significantly reduced at 4 °C or in the presence of NaN3. Furthermore, the efflux of HDND-7 was apparently reduced in the presence of MRP2 inhibitors MK-571 or probenecid. However, P-gp inhibitor verapamil had no effect on the transport of HDND-7. The in situ intestinal perfusion study indicated HDND-7 was well-absorbed in four intestinal segments. Furthermore, MRP2 inhibitors may slightly increase the absorption of HDND-7 in jejunum. 4. In summary, all results indicated that HDND-7 might be absorbed mainly by passive diffusion via transcellular pathway, MRP2 but P-gp may participate in the efflux of HDND-7.

The permeability characteristics and interaction of the main components from Zhizi Bopi decoction in the MDCK cell model.

1. Although emerging evidence indicates the therapeutic effects of Zhizi Bopi Decoction, the extent to which essential ingredients are absorbed and the possible synergistic actions are poorly understood. 2. In this study, MDCK cell model was used to determine the bi-directional permeability and interaction between the main components (geniposide, berberine and glycyrrhizic acid) of Zhizi Bopi Decoction. 3. The transport of the active ingredients was concentration-dependent in both directions. Moreover, the Papp (AP-BL) values of berberine and glycyrrhizic acid were significantly reduced when co-incubation with an ATP inhibitor. Additionally, uptake of berberine, glycyrrhizic acid were clearly inhibited by the inhibitors of P-glycoprotein and MRP2, indicating that P-gp and MRP2 may be involved in the transport of berberine and glycyrrhizic acid, respectively. However, it was found that geniposide may be purely passive diffusion. Furthermore, the combined incubation of geniposide with berberine and glycyrrhizic acid had a powerful sorbefacient effect than use of a single drug alone which may be regulated by tight junctions. 4. In summary, our study provides useful information for pharmacological applications of Zhizi Bopi Decoction and offers new insights into this ancient decoction for further researches, especially in drug synergism.

Single Dose Oral and Intravenous Pharmacokinetics and Tissue Distribution of a Novel Hesperetin Derivative MTBH in Rats.

BACKGROUND: MTBH, a novel hesperetin derivative, possesses in vivo hepatoprotective effects against carbon tetrachloride (CCl4)-induced acute liver injury in Institute of Cancer Research (ICR) mice. OBJECTIVES: This study investigated the pharmacokinetics and tissue distribution of MTBH and its conjugated metabolites in rats after a single dose of MTBH. METHODS: Male Sprague-Dawley (SD) rats were orally administered (25, 50, 100 mg/kg) or intravenously administered (25 mg/kg) MTBH and blood samples were withdrawn at specific times. Moreover, after a single oral dose of MTBH (200 mg/kg), tissues (heart, liver, spleen, lung, kidney, stomach, intestine, brain and muscle) were collected at scheduled time points. RESULTS: The concentration of MTBH in plasma and tissues was assayed by HPLC before and after hydrolysis with ß-glucuronidase or sulfatase. The glucuronides/sulfates were extensively present in the plasma, moreover, the free form was detectable in the plasma, but in a small amount equivalent to nearly 0.85-1.46 % of the amount of glucuronides/sulfates, the absolute bioavailability of MTBH was approximately 31.27 %. In tissues, the free form appeared in all tissues examined, with trace amount in brain and muscle, and considerable concentration in stomach and lung. Glucuronides/sulfates were the major forms in intestine, kidney and liver, whereas not detectable in heart, brain and muscle. The liver and intestine were found likely to accumulate MTBH at a high concentration among all tissues. CONCLUSIONS: The free form of MTBH was present in the circulation and all assayed organs, whereas its glucuronides/sulfates were the major forms in plasma and intestine, kidney and liver after a single dose.

A HPLC-MS/MS method for the quantitation of free, conjugated, and total HDND-7, a novel hesperetin derivative, in rat plasma and tissues: Application to the pharmacokinetic and tissue distribution study.

A sensitive and reliable HPLC-MS/MS method was developed and validated for the determination of free (unconjugated), glucuronidated, sulfated, and total (free and conjugated) HDND-7 in rat plasma and tissues. Plasma and tissues samples were treated prior to and after the enzyme hydrolysis. Chromatographic separation was achieved on a Phenomenex Luna C18 column (150 × 4.6mm, 3 µm), using isocratic mobile phase consisting of 0.1% formic acid-acetonitrile (50:50, v/v) at a flow rate of 300 µl/min. The detection was performed on a triple quadruple tandem mass spectrometer using positive electrospray ionization (ESI) source with a chromatographic run time of 5.0 min. The detection was operated by multiple reaction monitoring (MRM) of the transitions of m/z 429.3 → 223.9 for HDND-7 and 272.9 → 152.9 for naringenin (IS), respectively. This method was validated in terms of specificity, linearity, precision, accuracy, and stability. The calibration curves for plasma and tissues were linear over a wide concentration range of 0.02-40 µg/ml with a lower limit of quantification (LLOQ) of 0.02 µg/ml. Mean extraction recoveries in plasma and tissues ranged from 87.4 to 97.1% and from 54.2 to 70.5%, respectively. The intra- and inter-day precision values were below 15% and the accuracy was within ± 15%. The samples were stable under all the tested conditions. This method has been successfully applied to the pharmacokinetic study following oral doses of 25, 50 and 100mg/kg and intravenous dose of 25mg/kg, and tissue distribution study following oral dose of 50mg/kg.

Intestinal absorption mechanisms of MTBH, a novel hesperetin derivative, in Caco-2 cells, and potential involvement of monocarboxylate transporter 1 and multidrug resistance protein 2.

Hesperetin, the aglycone of hesperidin, occurs naturally in citrus fruits. It exerts extensive pharmacological activities. However, hesperetin's poor solubility and low bioavailability limit its wide application. In order to overcome these limitations, recently a series of novel hesperitin derivatives containing Mannich base moieties were synthesized and the anti-inflammatory activity was evaluated, among which MTBH (8-methylene-tert-butylamine-3',5,7-trihydroxy-4'-methoxyflavanone) showed a significantly improved water solubility, and promising anti-inflammatory activity in vitro and in vivo compared with hesperitin. Thus, the aim of this study was to investigate the permeability and transport mechanisms of MTBH, using Caco-2 cell monolayer. MTBH was effectively absorbed by Caco-2 cells in a concentration-dependent manner in both directions at 7.5-480 µM. Moreover, MTBH showed pH dependent and TEER values independent transport in both directions. Transport of MTBH was obviously decreased in the presence of sodium azide (an ATP inhibitor) or CCCP (a proton-ionophore). MTBH transport was markedly reduced by MCT inhibitors quercetin or phloretin, and the substrate analogs l-lactate or benzoic acid. We verified MCT1, MCT3, MCT4, MCT5, and MCT6 were expressed in Caco-2 cells by western blot. Silence MCT1 with siRNA resulted in significant inhibition of MTBH uptake. The verapamil, a P-gp inhibitor, and Ko143, a BCRP inhibitor, had no effect on the transport of MTBH. However, MK-571 or probenecid, MRP2 inhibitors, led to an apparently decrease in the efflux of MTBH. In summary, MTBH was absorbed by transcellular passive diffusion and a pH dependent mechanism mediated by MCT1. MRP2 but P-gp or BCRP may be involved in the transport of MTBH.