Role of the ATP-binding cassette drug transporters in the intestinal absorption of tanshinone IIB, one of the major active diterpenoids from the root of Salvia miltiorrhiza

There is an increasing use of herbal medicines worldwide, and the extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we investigated the mechanism for the intestinal absorption of tanshinone IIB (TSB), a major constituent of S. miltiorrhiza. The oral bioavailability of TSB was about 3% in rats with less proportional increase in its maximum plasma concentration (C_max) and area under the plasma concentration-time curve (AUC) with increasing dosage. The time to C_max (T_max) was prolonged at higher oral dosage. In a single pass rat intestinal perfusion model, the permeability coefficients (P_app) based on TSB disappearance from the lumen (P_lumen) were 6.2- to 7.2-fold higher (p < 0.01) than those based on drug appearance in mesenteric venous blood (P_blood). The uptake and efflux of TSB in Caco-2 cells were also significantly altered in the presence of an inhibitor for P-glycoprotein (PgP) or for multi-drug resistance associated protein (MRP1/2). TSB transport from the apical (AP) to basolateral (BL) side in Caco-2 monolayers was 3.3- to 5.7-fold lower than that from BL to AP side, but this polarized transport was attenuated by co-incubation of PgP or MRP1/2 inhibitors. The P_app values of TSB in the BL-AP direction were significantly higher in MDCKII cells over-expressing MDR1 or MRP1, but not in cells over-expressing MRP2-5, as compared with the wild-type cells. The plasma AUC_0-24hr in mdr1a and mrp1 gene-deficient mice was 10.2- to 1.7-fold higher than that in the wild-type mice. Furthermore, TSB significantly inhibited the uptake of digoxin and vinblastine in membrane vesicles containing PgP or MRP1. TSB also moderately stimulated PgP ATPase activity. Taken collectively, our findings indicate that TSB is a substrate for PgP and MRP1 and that drug resistance to TSB therapy and drug interactions may occur through PgP and MRP1 modulation.

Transport of cryptotanshinone, a major active triterpenoid in Salvia miltiorrhiza Bunge widely used in the treatment of stroke and Alzheimers disease, across the blood-brain

Cryptotanshinone (CTS), a major constituent from the roots of Salvia miltiorrhiza (Danshen), is widely used in the treatment of coronary heart disease, stroke and less commonly Alzheimer's disease. Our recent study indicates that CTS is a substrate for Pglycoprotein (PgP/MDR1/ABCB1). This study has investigated the nature of the brain distribution of CTS across the brain-blood barrier (BBB) using several in vitro and in vivo rodent models. A polarized transport of CTS was found in rat primary microvascular endothelial cell (RBMVEC) monolayers, with facilitated efflux from the abluminal side to luminal side. Addition of a PgP (e.g. verapamil and quinidine) or multi-drug resistance protein 1/2 (MRP1/2) inhibitor (e.g. probenecid and MK-571) in both luminal and abluminal sides attenuated the polarized transport. In a bilateral in situ brain perfusion model, the uptake of CTS into the cerebrum increased from 0.52 ± 0.1% at 1 min to 11.13 ± 2.36 ml/100 g tissue at 30 min and was significantly greater than that of sucrose. Co-perfusion of a PgP/MDR1 (e.g. verapamil) or MRP1/2 inhibitor (e.g. probenecid) significantly increased the brain distribution of CTS by 35.1-163.6%. The brain levels of CTS were only about 21% of those in plasma, and were significantly increased when coadministered with verapamil or probenecid in rats. The brain levels of CTS in rats subjected to middle cerebral artery occlusion and rats treated with quinolinic acid (a neurotoxin) were about 2- to 2.5-fold higher than the control rats. Moreover, the brain levels in mdr1a(-/-) and mrp1(-/-) mice were 10.9- and 1.5-fold higher than those in the wild-type mice, respectively. Taken collectively, these findings indicate that PgP and Mrp1 limit the brain penetration of CTS in rodents, suggesting a possible role of PgP and MRP1 in limiting the brain penetration of CTS in patients and causing drug resistance to Danshen therapy and interactions with conventional drugs that are substrates of PgP and MRP1. Further studies are needed to explore the role of other drug transporters in restricting the brain penetration of CTS and the clinical relevance.

A mechanistic study of the intestinal absorption of crypotanshinone, the major active constituent of Saliva miltiorrhiza

The nature of intestinal absorption of most herbal medicine is unknown. Cryptotanshinone (CTS) is the principal active constituent of the widely used cardiovascular herb Salvia miltiorrhiza (Danshen). We investigated the oral bioavailability of CTS in rats and the mechanism for its intestinal absorption using several in vitro and in vivo models:1) Caco-2 cell monolayers; 2) monolayers of MDCKII cells overexpressing P-glycoprotein
(PgP); and 3) single-pass rat intestinal perfusion with mesenteric vein cannulation. The systemic bioavailabilities of CTS after oral and intraperitoneal administration at 100 mg/kg were 2.05 and 10.60%, respectively. In the perfused rat intestinal model, permeability coefficients based on CTS disappearance from the luminal perfusate (P_lumen) were 6.7- to 10.3-fold higher than permeability coefficients based on drug appearance in venous blood (P_blood). P_blood significantly increased in the presence of the P-gP inhibitor, verapamil. CTS transport across Caco-2 monolayers was pH-, temperature- and ATP-dependent. The transport from the apical (AP) to the basolateral (BL) side was 3- to 9-fold lower than that from the BL to the AP side. Inclusion of verapamil (50 µM) in both AP and BL sides abolished the polarized CTS transport across Caco-2 cells. Moreover, CTS was significantly more permeable in the BL to AP than in the AP to BL direction in MDCKII and MDR1-MDCKII cells. The permeability coefficients in the BL to AP direction were significantly higher in MDCKII cells overexpressing PgP. These findings indicate that CTS is a substrate for PgP that can pump CTS into the luminal side.

Alteration of the pharmacokinetics of COL-3, a matrix metalloproteinase inhibitor, due to actue gastrointestinal tosicity of doxorubicin

Purpose Combination of COL-3, a matrix metalloproteinase inhibitor, and doxorubicin (DOX) might be a promising anticancer regimen. The present study was to examine the potential pharmacokinetic interactions and toxicity profile following their coadministration in rats.
Methods Normal rats were treated with single agent or different combinations with oral or intravenous COL-3 and DOX, and the bile-duct cannulated (BDC) rats received oral COL-3 plus DOX. In a separate disposition study, the effects of DOX on the biliary, urinary, and fecal excretion of COL-3 were examined. In addition, the effects of DOX on in vitro protein binding, metabolism, and transport of COL-3 across Caco-2 monolayers were investigated.
Results COL-3 did not affect the pharmacokinetics of DOX in rats. However, treatment with DOX significantly decreased the oral absorption, and prolonged the elimination, of COL-3 in the normal rats, but not in the BDC rats. DOX did not alter the biliary and urinary excretion of COL-3, but significantly decreased the fecal excretion of COL-3. DOX significantly enhanced the basolateral to apical flux of COL-3 across Caco-2 monolayers, but had no apparent effects on the protein binding and metabolism of COL-3. The combination of DOX with oral COL-3 did not significantly (p > 0.05) increase the acute diarrhea score and intestinal damage compared to rats receiving DOX alone.
Conclusions These results indicated that DOX altered the oral absorption and elimination of COL-3, largely resulting from gastrointestinal toxicity caused by biliary excretion of DOX. Further studies are required to explore the efficacy and optimized dosage regimen of this promising combination.

Mucosal vascular addressin cell adhesion molecule-1 is expressed outside the endothelial lineage on fibroblasts and melanoma cells

Mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) is predominantly expressed on high endothelial venules in inflamed tissues where it assists with leucocyte extravasation. Here we report that MAdCAM-1 has the potential to be more widely expressed outside the endothelial cell lineage than previously appreciated. Thus, MAdCAM-1 RNA transcripts and cell-surface protein were expressed by NIH 3T3 fibroblasts following activation with tumour necrosis factor-alpha (TNF-alpha), and by freshly isolated and cultured primary mouse splenic and tail fibroblasts in the absence of TNF-alpha stimulation. They were constitutively expressed by B16F10 melanoma cells, and expression was enhanced by cell activation with TNF-alpha. Mucosal vascular addressin cell adhesion molecule-1 was expressed on the apical surface of isolated cells, but became predominantly localized to cell junctions in confluent cell monolayers, suggesting it may play a role in the homotypic aggregation of cells. Tumour necrosis factor-alpha enhanced the expression of a firefly luciferase reporter directed by the MAdCAM-1 promoter in NIH 3T3 and B16F10 cells. A DNA fragment extending from nt -1727 to -673 was sufficient to confer cell-type selective expression. Mucosal vascular addressin cell adhesion molecule-1 expressed by NIH 3T3 cells was biologically active, as it supported the adhesion of TK-1 T cells in an alpha4beta7-dependent fashion. The expression of MAdCAM-1 by fibroblasts, and melanomas suggests MAdCAM-1 may play a role in regulating host responses in the periphery, leucocyte transmigration across nonendothelial boundaries, or the homotypic interactions of some malignant melanomas.

Multicellular spheroids in ovarian cancer metastases: Biology and pathology

Epithelial ovarian cancer (EOC) has a relatively high mortality rate (~55%). One of the presiding causes is that the current chemotherapeutic regimes are unable to achieve sustained remission, despite frequently producing a positive response at first treatment. One of the reasons that EOC is difficult to treat is that the mechanism of dissemination is unusual. EOC dissemination characteristically involves local invasion of pelvic and abdominal organs. Unlike many epithelial cancers, initial dissemination rarely requires the vasculature, although the vasculature is often implicated in the advanced stages of disease. Recently, it has become apparent that aggregates of malignant cells (spheroids) contained within malignant ascites represent a significant impediment to efficacious treatment of late stage EOC. In vivo, spheroids are present in the malignant ascites of EOC patients, while in vitro cultured spheroids are capable of tumorgenesis in vivo and display a reduced response to chemotherapeutic drugs when compared to monolayers. A major problem associated with the current generation of chemotherapy agents is that they do not address the anchorage and vascular-independent growth conditions associated with a 3-dimensional structure that has formed and/or grown in suspension. Thus, spheroid formation may represent a key component of platinum/taxanesensitive recurrence. If this is correct, a better understanding of spheroid biology may contribute to the identification of new treatment opportunities for the sustained treatment of metastatic EOC. This review article outlines the key biological features of spheroids, specifically discussing their role in EOC dissemination and chemo-response as well as providing insights into spheroid functionality.

Single molecule conductance through rigid norbornylogous bridges with zero average curvature

A new homologous series of norbornylogous (NB) bridges has been synthesized in which the average curvature of the bridges is very small. The molecules are rigid and have two thiol moieties at each end to allow them to form self-assembled monolayers (SAMs) and to connect to two gold electrodes to form a molecular junction. The SAMs formed were characterized using electrochemistry to determine the surface coverage of molecules on gold surface and to provide an indication of the packing of the NB bridges while ellipsometry and X-ray photoelectron spectroscopy (XPS) were used to provide an indication of the SAM thickness and orientation. Single molecule conductance of NB bridges was measured as a function of the molecular length. The conductance was found to decrease exponentially with the length with a decay constant that is exactly correlated with photoelectron transfer and other studies at the multiple molecule level. The molecule−electrode contact resistance was determined and compared with that of related alkanedithiol molecular junctions.

The self-assembled monolayer modification of electrodes - some recent advances in biological application

The modification of an electrode surface at the molecular level using the technique of depositing self-assembled monolayers (SAM) is a typical example of the techniques used in nanotechnology, from the process "bottom up", which is to create a nanostructure by successive additions of molecular or atomic entities on a surface. This article presents some recent advances in the field, with examples: the development of systems Sat hybridized with biomolecules, nanoparticles or nanotubes in bioelectronics, the use of switchable electrodes to study the adhesion and migration of biological cells , and the integration of molecular son in the SAM to recognize and allow the transduction of a biological response allowing the practice of electrochemistry in a complex biological environment.

Chemisorbed and physisorbed Structures for 1,10-Phenanthroline and Dipyrido[3,2-a:2',3'-c]phenazine on Au(111)

Scanning tunneling microscopy (STM) images of 1,10-phenanthroline (PHEN) and dipyrido[3,2-a:2‘,3‘-c]phenazine (DPPZ) on Au(111) are recorded using both in situ and ex situ techniques. The images of PHEN depict regimes of physisorption and chemisorption, whereas DPPZ is only physisorbed. All physisorbed structures are not pitted and fluctuate dynamically, involving aligned (4 × 4) surface domains with short-range (ca. 20 molecules) order for PHEN but unaligned chains with medium-range (ca. 100 molecules) order for DPPZ. In contrast, the chemisorbed PHEN monolayers remain stable for days, are associated with surface pitting, and form a (4 × √13)R46° lattice with long-range order. The density of pitted atoms on large gold terraces is shown to match the density of chemisorbed molecules, suggesting that gold adatoms link PHEN to the surface. For PHEN, chemisorbed and physisorbed adsorbate structures are optimized using plane-wave density-functional theory (DFT) calculations for the surface structure. Realistic binding energies are then obtained adding dispersive corrections determined using complete-active-space self-consistent field calculations using second-order perturbation theory (CASPT2) applied to cluster-interaction models. A fine balance between the large adsorbate−adsorbate dispersive forces, adsorbate−surface dispersive forces, gold ligation energy, and surface mining energy is shown to dictate the observed phenomena, leading to high surface mobility and substrate/surface lattice incommensurability. Increasing the magnitude of the dispersive forces through use of DPPZ, rather than PHEN, to disturb this balance produced physisorbed monolayers without pits and/or surface registration but with much longer-range order. Analogies are drawn with similar but poorly understood processes involved in the binding of thiols to Au(111).

Recruitment of class I hydrophobins to the air : water interface initiates a multi-step process of functional amyloid formation

Class I fungal hydrophobins form amphipathic monolayers composed of amyloid rodlets. This is a remarkable case of functional amyloid formation in that a hydrophobic:hydrophilic interface is required to trigger the self-assembly of the proteins. The mechanism of rodlet formation and the role of the interface in this process have not been well understood. Here, we have studied the effect of a range of additives, including ionic liquids, alcohols, and detergents, on rodlet formation by two class I hydrophobins, EAS and DewA. Although the conformation of the hydrophobins in these different solutions is not altered, we observe that the rate of rodlet formation is slowed as the surface tension of the solution is decreased, regardless of the nature of the additive. These results suggest that interface properties are of critical importance for the recruitment, alignment, and structural rearrangement of the amphipathic hydrophobin monomers. This work gives insight into the forces that drive macromolecular assembly of this unique family of proteins and allows us to propose a three-stage model for the interface-driven formation of rodlets.

Adsorption, lubrication, and wear of lubricin on model surfaces : polymer brush-like behavior of a glycoprotein

Using a surface force apparatus, we have measured the normal and friction forces between layers of the human glycoprotein lubricin, the major boundary lubricant in articular joints, adsorbed from buffered saline solution on various hydrophilic and hydrophobic surfaces: i), negatively charged mica, ii), positively charged poly-lysine and aminothiol, and iii), hydrophobic alkanethiol monolayers. On all these surfaces lubricin forms dense adsorbed layers of thickness 60–100 nm. The normal force between two surfaces is always repulsive and resembles the steric entropic force measured between layers of end-grafted polymer brushes. This is the microscopic mechanism behind the antiadhesive properties showed by lubricin in clinical tests. For pressures up to ∼6 atm, lubricin lubricates hydrophilic surfaces, in particular negatively charged mica (friction coefficient μ = 0.02–0.04), much better than hydrophobic surfaces (μ > 0.3). At higher pressures, the friction coefficient is higher (μ > 0.2) for all surfaces considered and the lubricin layers rearrange under shear. However, the glycoprotein still protects the underlying substrate from damage up to much higher pressures. These results support recent suggestions that boundary lubrication and wear protection in articular joints are due to the presence of a biological polyelectrolyte on the cartilage surfaces.

Deposition and wetting characteristics of polyelectrolyte multilayers on plasma-modified porous polyethylene

Hydrophilic and chemically reactive porous media were prepared by adsorbing functional polymers at the surface of sintered polyethylene membranes. Modification of the membrane was accomplished by first exposing the membrane to an oxygen glow discharge gas plasma to introduce an electrostatic charge at the membrane surfaces. Cationic polyelectrolyte polyethylenimine (PEI) was adsorbed from solution to the anionic-charged surface to form an adsorbed monolayer. The adsorption of a second anionic polyelectrolyte onto the PEI layer allows further modification of the membrane surface to form a polyelectrolyte-bilayer complex. The conformation and stability of the adsorbed monolayers and bilayers comprising the modified surface are probed as a function of the polymer structure, charge density, and solubility. Using X-ray photoelectron spectroscopy analysis, we demonstrate that the presence of the polyelectrolyte multilayers drastically increases the density and specificity of the functional groups at the surface, more than what can be achieved through the plasma modification alone. Also, using the wicking rate of deionized, distilled water through the porous membrane to gauge the interfacial energy of the modified surface, we show that the membrane wicking rate can be controlled by varying the chemistry of the adsorbing polyelectrolytes and, to a lesser extent, by adjusting the polarity or ionic strength of the polyelectrolyte solution.

Theoretical modeling and experimental validation of surface stress in thrombin aptasensor

Adsorption of target molecules on the immobilized microcantilever surface produced beam displacement due to the differential surface stress generated between the immobilized and non-immobilized surface. Surface stress is caused by the intermolecular forces between the molecules. Van der Waals, electrostatic forces, hydrogen bonding, hydrophobic effect and steric hindrance are some of the intermolecular forces involved. A theoretical framework describing the adsorption-induced microcantilever displacement is derived in this paper. Experimental displacement of thrombin aptamer-thrombin interactions was carried out. The relation between the electrostatic interactions involved between adsorbates (thrombin) as well as adsorbates and substrates (thrombin aptamer) and the microcantilever beam displacement utilizing the proposed mathematical model was quantified and compared to the experimental value. This exercise is important to aid the designers in microcantilever sensing performance optimization.

The influence of graphene on the electrical communication through organic layers on graphite and gold electrodes

The influence of graphene on the electrical communication through organic layers fabricated on graphite and gold electrodes is investigated. These layers were prepared by in situ reductive adsorption of 4-aminobenzoic acid in the presence of NaNO2 and HCl to have surface bound carboxylic acid functionalities, followed by covalent attachment of 1-aminopyrene via an amide coupling reaction to have surface bound pyrene groups for graphene immobilization via noncovalent π-π stacking interaction. The coverage of the layers created via reductive adsorption on graphite electrodes was found to be much higher than that on gold electrodes. It was revealed that graphene significantly enhances the electrical communication through the layers on graphite electrodes but on gold electrodes the enhancement effect through the layers was only minor. However, when gold electrodes were modified with a self-assembled monolayer (SAM) of propanethiol the subsequent immobilization of graphene resulted in a significant enhancement of the electrical communication. It is also found that immobilization of graphene could affect the electron transfer between the redox probe, pyrene and the underlying electrodes. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to characterize the graphene sheets. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS), and X-ray photoelectron spectroscopy (XPS) were also used to characterize the stepwise modified electrodes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.