Inhibitory effects of furanocoumarin derivatives in Kampo extract medicines on P-glycoprotein at the blood-brain barrier

Furanocoumarin derivatives, known as components of grapefruit juice, showing inhibitory effects against P-glycoprotein (P-gp) in the intestine are also contained in the plants of rutaceae and umbelliferae families, which are used as components of Kampo extract medicines. In this study, we investigated the inhibitory effects of byakangelicol and rivulobirin A, known as furanocoumarins showing P-gp inhibitory effect using Caco-2 monolayer, against P-gp at the blood-brain barrier (BBB) under both in vitro and in vivo conditions. First we studied the membrane permeability of furanocoumarins to clarify whether they can be absorbed from the intestine. Both furanocoumarins showed high permeability through the Caco-2 monolayer, suggesting that they can easily reach the systemic circulation after oral administration. Then, we evaluated the effect of these furanocoumarins on the uptake of calcein acetoxymethyl ester (calcein-AM), a P-gp substrate, into bovine brain microvascular endothelial cells (BBMEC). Both furanocoumarins significantly increased the uptake amount of calcein-AM into BBMEC by the inhibition of P-gp at the BBB in vitro. Next we also investigated the P-gp inhibitory effect of these furanocoumarins at the rat BBB in vivo using verapamil as a P-gp substrate. Both furanocoumarins increased the B/P ratio of verapamil compared to the control, even under in vivo conditions; however, the extent of the inhibitory effect was much lower than in vitro condition. In conclusion, byakangelicol and rivulobirin A may inhibit P-gp expressed at the BBB even under in vivo conditions. Further studies using Kampo extract medicines under in vivo condition are necessary for safe drug therapy.     

Pluronic P85 Increases Permeability of a Broad Spectrum of Drugs in Polarized BBMEC and Caco-2 Cell Monolayers

Purpose. Previous studies demonstrated that inhibition of P glycoprotein (P-gp) by Pluronic P85 (P85) block copolymer increases apical (AP) to basolateral (BL) transport of rhodamine 123 (R123) in the polarized monolayers of bovine brain microvessel endothelial cells (BBMEC) and Caco-2 cells. The present work examines the effects of P85 on the transport of fluorescein (Flu), doxorubicin (Dox), etoposide (Et), taxol (Tax), 3-azido-3-deoxythymidine (AZT), valproic acid (VPA) and loperamide (Lo) using BBMEC and Caco-2 monolayers as in vitro models of the blood brain barrier and intestinal epithelium respectively. Methods. Drug permeability studies were performed on the confluent BBMEC and Caco-2 cell monolayers mounted in Side-Bi-Side diffusion cells. Results. Exposure of the cells to P85 significantly enhanced AP to BL permeability coefficients of Flu, Tax, Dox and AZT in both cell models. Further, P85 enhanced AP to BL transport of Et, VPA and Lo in Caco-2 monolayers. No changes in the permeability coefficients of the paracellular marker mannitol were observed in the presence of the copolymer. Conclusions. P85 increases AP to BL permeability in BBMEC and Caco-2 monolayers with respect to a broad panel of structurally diverse compounds, that were previously shown to be affected by P-gp and/ or multidrug resistance associated protein (MRP) efflux systems. Broad specificity of the block copolymer effects with respect to drugs and efflux systems appears to be a valuable property in view of developing pharmaceutical formulations to increase drug accumulation in selected organs and overcome both acquired and intrinsic drug resistance that limits the effectiveness of many chemotherapeutic agents.     

Ketoconazole and the modulation of multidrug resistance-mediated transport in Caco-2 and MDCKII-MDR1 drug transport models

The hypothesis tested was that ketoconazole can modulate P-glycoprotein, thereby altering cellular uptake and apparent permeability (P(app)) of multidrug-resistant substrates, such as cyclosporin A (CSA) and digoxin, across Caco-2, MDCKII-MDR1, and MDCKII wild-type cell transport models. (3)H-CSA/(3)H-digoxin transport experiments were performed with and without co-exposure to ketoconazole, and (3)H-ketoconzole transport experiments were performed with and without co-exposure to dietary flavonoids, epigallocatechin-3-gallate, and xanthohumol. Ketoconazole (3 microM) reduced the P(app) efflux of CSA and digoxin from 5.07 x 10(-6) to 2.91 x 10(-6) cm s(-1) and from 2.60 x 10(-6) to 1.41 x 10(-6) cm s(-1), respectively, in Caco-2 cells. In the MDCKII-MDR1 cells, ketoconazole reduced the P(app) efflux of CSA and increased the P(app) absorption of digoxin. Cellular uptake of ketoconazole in the Caco-2 cells was significantly inhibited by CSA and digoxin, whereas epigallocatechin-3-gallate and xanthohumol exhibited biphasic responses. In conclusion, ketoconazole modulates the P(app) of P-glycoprotein substrates by interacting with MDR1 protein. Epigallocatechin-3-gallate and xanthohumol modulate the transport and uptake of ketoconazole.     

Effects of Pluronic P85 on GLUT1 and MCT1 Transporters in the Blood-Brain Barrier

PURPOSE: The amphiphilic block copolymer Pluronic P85 (P85) increases the permeability of the blood-brain barrier (BBB) with respect to a broad spectrum of drugs by inhibiting the drug efflux transporter, P-glycoprotein (Pgp). In this regard, P85 serves as a promising component for CNS drug delivery systems. To assess the possible effects of P85 on other transport systems located in the brain, we examined P85 interactions with the glucose (GLUT1) and monocarboxylate (MCT1) transporters. METHODS: Polarized monolayers of primary cultured bovine brain microvessel endothelial cells (BBMEC) were used as an in vitro model of the BBB. 3H-2-deoxy-glucose and 14C-lactate were selected as GLUT1 and MCT1 substrates, respectively. The accumulation and flux of these substrates added to the luminal side of the BBMEC monolayers were determined. RESULTS: P85 has little effect on 3H-2-deoxy-glucose transport. However, a significant decrease 14C-lactate transport across BBMEC monolayers is observed. Histology, immunohistochemistry, and enzyme histochemistry studies show no evidence of P85 toxicity in liver, kidney, and brain in mice. CONCLUSIONS: This study suggests that P85 formulations do not interfere with the transport of glucose. This is, probably, due to compensatory mechanisms in the BBB. Regarding the transport of monocarboxylates, P85 formulations might slightly affect their homeostasis in the brain, however, without any significant toxic effects.     

Comparison of in vitro BBMEC permeability and in vivo CNS uptake by microdialysis sampling

The studies presented in this report were designed to assess the correlation of the bovine brain microvessel endothelial cell (BBMEC) apparent permeability coefficient (P_app) and in vivo BBB penetration using microdialysis sampling. A mathematical model was developed to describe the relationship of brain extracellular fluid (ECF) concentration to free drug in plasma. The compounds studied have a broad range of physico-chemical characteristics and have widely varying in vitro and in vivo permeability across the blood–brain barrier (BBB). BBMEC permeability coefficients vary in magnitude from a low of 0.9×10⁻⁵ cm/s to a high value of 7.5×10⁻⁵ cm/s. Corresponding in vivo measurements of BBB permeability are represented by clearance (CL_in) into the brain ECF and range from a low of 0.023 μl/min/g to a high of 12.9 μl/min/g. While it is apparent that in vitro data from the BBMEC model can be predictive of the in vivo permeability of a compound across the BBB, there are numerous factors both prior to and following entry into the brain which impact the ultimate uptake of a compound. Even in the presence of high BBB permeability, factors such as high plasma protein binding, active efflux across the BBB, and metabolism within the CNS can greatly limit the ultimate concentrations achieved. In addition, concentrations in the intracellular space may not be the same as concentrations in the extracellular space. While these data show that the BBMEC permeability is predictive of the in vivo BBB permeability, the complexity of the living system makes prediction of brain concentrations difficult, based solely on the in vitro measurement.     

Effects of Pluronic P85 Unimers and Micelles on Drug Permeability in Polarized BBMEC and Caco-2 Cells

Purpose. Using polarized bovine brain microvessel endothelial cells (BBMEC) monolayers as in vitro model of the blood brain barrier and Caco-2 monolayers as a model of the intestinal epithelium, the present work investigates the effects of Pluronic P85 block copolymer (P85) on the transport of the P-gycoprotein (P-gp)- dependent probe, rhodamine 123 (R123). Methods. The permeability and cell efflux studies are performed with the confluent cell monolayers using Side-Bi-Side diffusion cells. Results. At concentrations below the critical micelle concentration, P85 inhibits P-gp efflux systems of the BBMEC and Caco-2 cell monolayers resulting in an increase in the apical to basolateral permeability of R123. In contrast, at high concentrations of P85 the drug incorporates into the micelles, enters the cells and is then recycled back out to the apical side resulting in decrease in Rl 23 transport across the cell monolayers. Apical to basolateral permeability of micelle-incorporated R123 in BBMEC monolayers was increased by prior conjugation of P85 with insulin, suggesting that modified micelles undergo receptor-mediated transcytosis. Conclusions. Pluronic block copolymers can increase membrane transport and transcellular permeability in brain microvessel endothelial cells and intestinal epithelium cells. This suggests that these block copolymers may be useful in designing formulations to increase brain and oral absorption of select drugs.     

Transport of the delta-opioid receptor agonist [D-penicillamine2,5] enkephalin across the blood-brain barrier involves transcytosis1

The delta opioid receptor antagonist [D-penicillamine2,5]enkephalin (DPDPE) is an enzymatically stable peptide analogue of Met-enkephalin. DPDPE uses a saturable transport mechanism to cross the blood-brain barrier (BBB), though the exact mechanism is not fully understood. The aim of the present study was to identify the mechanism by which DPDPE enters the brain. The effect of phenylarsine oxide (PAO), an endocytosis inhibitor, on the transport of [3H]DPDPE was investigated using both in vitro and in situ transport studies. Two in vitro models of the BBB utilizing primary bovine brain microvascular endothelial cells (BBMEC) were studied. [3H]DPDPE permeability across monolayers of BBMEC grown on polycarbonate filters was studied. PAO significantly reduced the permeability of [3H]DPDPE across the monolayer. PAO also reduced the uptake of [3H]DPDPE into BBMEC cells, without affecting binding to the cells. The in situ perfusion model of the BBB was also studied, PAO reduced DPDPE uptake by the brain in a dose-dependent manner. These studies indicate that DPDPE enters the brain via an energy-dependent transcytotic mechanism.     

应用于药物评价的血脑屏障模型研究进展

介绍了几种常见的血脑屏障体内外模型及一种脑渗透性分类方案。其中体外模型主要有溶剂水/分配模型、平行人工膜渗透模型、Transwell细胞模型、微流控芯片血脑屏障模型、永生化内皮细胞系建立的血脑屏障模型、三维血脑屏障模型等。体内模型主要有脑/血浆比率测定法、小鼠脑摄取量分析法、啮齿类动物原位脑灌注法、脑微透析法等。随着血脑屏障模型的不断成熟完善,将有助于筛选中枢神经系统药物,为神经系统疾病的治疗提供更多的理论依据。通过分析和评价各种不同的血脑屏障模型,以期为血脑屏障模型研究及中枢神经系统药物研发提供新的思路。     

Microdialysis in the study of drug transporters in the CNS

Quantitative microdialysis in the central nervous system (CNS) has recently provided evidence for the existence of transporters as they relate to the brain distribution of a variety of drugs. Support for the existence of drug transporters in the blood-brain barrier (or in the blood-CSF barrier) comes from investigations that have found: unbound drug concentrations in brain fluids that are lower than corresponding levels in plasma; saturability of transport clearances across the blood-brain barrier and; the regulation of transport by putative inhibitors. Additional confirmatory evidence for the existence of active transport or carrier-mediated processes has also been derived from models that relate observed drug levels in the CNS with those in plasma or blood. The conclusion that reduced drug levels in brain fluids generally indicate the existence of active efflux transport is questioned. In the case of relatively polar compounds with modest blood-brain barrier permeability, lower unbound concentrations in brain may be a consequence of dilution by turnover of brain fluids. This review summarizes recent reports (grouped by class of compounds) where investigators have used microdialysis to examine the distribution of therapeutic agents to the CNS, and have reached conclusions regarding the functional presence of drug transporters in the brain.     

Transport of amentoflavone across the blood-brain barrier in vitro

The biflavone amentoflavone is an ingredient of Hypericum perforatum L. (Clusiaceae), a plant which is widely used for the treatment of mild to moderate depression. Amentoflavone inhibits the binding of flumazenil to the benzodiazepine binding site of the GABA A -receptor (IC(50) = 14.9 nM). Since it has to pass the blood-brain barrier (BBB) before reaching this receptor, the penetration of [(3)H]-amentoflavone through BBB was studied in an in vitro model consisting of primary cell cultures of porcine brain capillary endothelial cells (BCEC). Concentration-dependent uptake (37-2000 nM) was neither saturable nor temperature-sensitive indicating passive diffusion as the major uptake mechanism. This finding was confirmed by transport experiments through BCEC monolayers (> 2 % of applied dose was transported after 30 min). Co-administration of Hypericum extract increased amentoflavone transport significantly (amentoflavone alone: permeability coefficient P(app) = 4.59.10(-6) cm/s; co-administrated sucrose: P(app) = 3.22.10(-6)cm/s; amentoflavone together with hypericum: P(app) = 6.74.10(-6)cm/s, co-administrated sucrose P(app) = 5.49.10(-6)cm/s) indicating that Hypericum constituents enhance amentoflavone transport possibly by modulating paracellular permeability. Experiments with the P-glycoprotein (P-gp) overexpressing cell line P388-MDR showed that amentoflavone uptake was significantly enhanced by addition of the P-gp inhibitor verapamil, suggesting a P-gp mediated back-transport out of the cells. In conclusion, our findings show, that amentoflavone is able to pass the blood-brain barrier in vitro by passive diffusion.     

The use of microdialysis in CNS drug delivery studies. Pharmacokinetic perspectives and results with analgesics and antiepileptics

Microdialysis can give simultaneous information on unbound drug concentration-time profiles in brain extracellular fluid (ECF) and blood, separating the information on blood-brain barrier (BBB) processes from confounding factors such as binding to brain tissue or proteins in blood. This makes microdialysis suitable for studies on CNS drug delivery. It is possible to quantify influx and efflux processes at the BBB in vivo, and to relate brain ECF concentrations to central drug action. The half-life in brain ECF vs. the half-life in blood gives information on rate-limiting steps in drug delivery and elimination from the CNS. Examples are given on microdialysis studies of analgesic and antiepileptic drugs.     

A microdialysis model to examine nasal drug delivery and olfactory absorption in rats using lidocaine hydrochloride as a model drug

Targeting of the central nervous system by direct drug transport from the nose to the brain has gained increased attention through the last decade. In the present study, a model for olfactory drug absorption has been investigated using intravenous and unilateral nasal administration of lidocaine hydrochloride in rats. To investigate the possible drug delivery aspects of this route of transport to a central part of the brain a microdialysis model using in vivo recovery by calibrator was applied to the systemic blood and to right and left striatum. The integrity of the blood-brain barrier was evaluated following microdialysis probe implantation. The in vivo experiments were carried out as a cross-over study in rats. The drainage from the nasal cavity was not restricted by occlusion. It was found that true unbound lidocaine concentrations could be calculated from in vivo recovery measurements of retrodialysis of prilocaine hydrochloride. The relative in vivo recoveries in striatum (11.3%) and blood (24.0%) were significantly lower than in vitro (31.3 and 44.9%). The blood-brain barrier was found to retain its physical integrity when evaluated one hour after probe implantation. From pharmacokinetic modelling of the time-concentration curves it was found that the absorption rates and area under the curve (AUC) values of lidocaine in left and right striatum were not statistically different following nasal and intravenous administration, respectively. The average nasal bioavailabilities of lidocaine in blood, left and right striatum were 85, 103 and 129%, respectively. It was concluded that no significant olfactory absorption to striatum was evident in the present study. However, the method should be applicable to studies of drug delivery to blood and brain following nasal administration of other drugs.     

Application of an in vivo brain microdialysis technique to studies of drug transport across the blood-brain barrier

There is a wide range of methods available for studying the transport of drugs across the blood-brain barrier (BBB) which is equipped with several systems to transport drugs as well as endogenous nutrients and waste products. The in vivo brain microdialysis technique, which allows direct sampling of the brain interstitial fluid (ISF), is a powerful means of characterizing influx and efflux transport across the BBB. In this paper, we review our results from the successful application of this technique to BBB drug transport studies. The drugs investigated include novel and CNS-active peptides, some agents that are actively removed from the brain ISF across the BBB, and a brain-directed prodrug.     

Efflux transporters in the blood-brain interfaces--in vitro and in vivo methods and correlations

INTRODUCTION: Sufficient brain exposure is crucial to the success of CNS drugs. The twofold greater attrition rate in clinical development of CNS drugs over the respective attrition rate of non-CNS drugs is due to lack of efficacy. It is generally thought that poor brain exposure is at least partly responsible for this, as the concentration-time profile at the brain target site is critical for efficacy. Efflux transporters in the blood-brain interfaces play a crucial role in modulation of permeability of drugs across these interfaces. Validation of preclinical tools to correctly predict brain exposure in humans is essential. AREAS COVERED: This review summarizes in vitro and in vivo tools to detect and characterize interactions of drugs with efflux transporters relevant to blood-brain interfaces. Furthermore, the article discusses the strengths and weaknesses of these methods and the limitations of their application, in addition to covering in vitro - in vivo correlations. EXPERT OPINION: A more detailed validation of in vitro efflux transporter assays employing primary brain endothelial cultures is needed. This should go along with mapping uptake transporters expressed in the blood-brain interfaces. With the availability of specific inhibitors, utilization of in vivo methods such as brain microdialysis is increasing. Once transporter-humanized mice are available, we may witness a further increase in applications of in vivo methods.     

Toward an improved prediction of human in vivo brain penetration

The penetration of drugs into the central nervous system is a composite of both the rate of drug uptake across the blood-brain barrier and the extent of distribution into brain tissue compartments. Clinically, positron emission tomography (PET) is the primary technique for deriving information on drug biodistribution as well as target receptor occupancy. In contrast, rodent models have formed the basis for much of the current understanding of brain penetration within pharmaceutical Drug Discovery. Linking these two areas more effectively would greatly improve the translation of candidate compounds into therapeutic agents. This paper examines two of the major influences on the extent of brain penetration across species, namely plasma protein binding and brain tissue binding. An excellent correlation was noted between unbound brain fractions across species (R(2) > 0.9 rat, pig, and human, n = 21), which is indicative of the high degree of conservation of the central nervous system environment. In vitro estimates of human brain-blood or brain-plasma ratios of marketed central nervous system drugs and PET tracers agree well with in vivo values derived from clinical PET and post-mortem studies. These results suggest that passive diffusion across the blood-brain barrier is an important process for many drugs in humans and highlights the possibility for improved prediction of brain penetration across species.     

Application of in vivo brain microdialysis to the study of blood-brain barrier transport of drugs

Recent advances in blood-brain barrier (BBB) research have led to a new understanding of drug transport processes at the BBB. The BBB acts as a dynamic regulatory interface at which nutrients necessary for neural activity are actively taken up into the brain from the blood circulation, and actively excludes metabolites that might interfere with the maintenance of brain homeostasis. Such influx and efflux transport functions at the BBB would also control the concentrations of various drugs in the brain interstitial fluid (ISF), which are an important determinant of the central nervous system (CNS) effects. Thus, direct measurement of the brain ISF concentration of drugs can provide significant information for clarifying the influx and efflux transport functions of drugs across the BBB. Although several experimental techniques have been developed to investigate transport functions across the BBB, in vivo brain microdialysis seems to be one of the most suitable techniques for characterizing the influx and efflux transport functions across the BBB under physiological and pathological conditions. This review covers studies during the past decade, in which the influx and efflux transport of drugs across the BBB was kinetically and mechanistically evaluated by means of the brain microdialysis technique. Some applications of brain microdialysis to studies on neuronal function and neurotherapeutics are also included.