Passive Transcellular Diffusion Research Articles

Intestinal permeability and gut microbiota interactions of pharmacologically active compounds in valerian and St. John’s wort

Phytomedicines such as valerian and St. John’s wort are widely used for the treatment of sleeping disorders, anxiety and mild depression. They are perceived as safe alternatives to synthetic drugs, but limited information is available on the intestinal absorption and interaction with human intestinal microbiota of pharmacologically relevant constituents valerenic acid in valerian, and hyperforin and hypericin in St. John’s wort. The intestinal permeability of these compounds and the antidepressant and anxiolytic drugs citalopram and diazepam was investigated in the Caco-2 cell model with bidirectional transport experiments. In addition, interaction of compounds and herbal extracts with intestinal microbiota was evaluated in artificial human gut microbiota. Microbiota-mediated metabolisation of compounds was assessed, and bacterial viability and short-chain fatty acids (SCFA) production were measured in the presence of compounds or herbal extracts. Valerenic acid and hyperforin were highly permeable in Caco-2 cell monolayers. Hypericin showed low-to-moderate permeability. An active transport process was potentially involved in the transfer of valerenic acid. Hyperforin and hypericin were mainly transported through passive transcellular diffusion. All compounds were not metabolized over 24 h in the artificial gut microbiota. Microbial SCFA production and bacterial viability was not substantially impaired nor promoted by exposure to the compounds or herbal extracts.

Analysis of Antioxidant Constituents from Ilex rotunda and Evaluation of Their Blood-Brain Barrier Permeability.

Ilex rotunda Thunb., has been used to treat common cold, tonsillitis, and eczema. It is also a source of antioxidants. However, information regarding its antioxidative phytochemical composition is still incomplete and limited. In this present study, we initially determined DPPH radical scavenging activity of the extracts of I. rotunda fruits, twigs, and leaves. Among them, the twig extract exhibited a potential of antioxidant capacity. Based on antioxidant effect guided experiments, extraction condition using 80% EtOH was then optimized. DPPH and ABTS radical scavenging assays were also performed for fractions. The n-butanol fraction showed the highest antioxidant effect. Using chromatographic methods, eight marker compounds (1–8) were further isolated. Their structures were determined by spectroscopic and mass data. Method validation was employed to quantitate contents of these eight marker compounds. Subsequently, the HPLC-DPPH method was used to evaluate the contribution of certain compounds to total antioxidant activity of the extract. Lastly, parallel artificial membrane permeability assay for blood–brain barrier (PAMPA-BBB) was applied to investigate brain-penetrable antioxidants from I. rotunda extract. As a result, compound 7 (4,5-dicaffeoylquinic acid) showed significant antioxidant activity and penetration across the BBB via transcellular passive diffusion. Our findings suggested that compound 7 can be used as a therapeutic potential candidate in natural product-based central nervous system (CNS) drug discovery.

In silico-in vitro estimation of lipophilicity and permeability association for succinimide derivatives using chromatographic anisotropic systems and parallel artificial membrane permeability assay.

Passive permeability is one of the key features that determine absorbability and one of the most studied properties in the early phases of drug development. Newly synthesized succinimide derivatives from two different series (1-aryl-3-methylsuccinimides and 1-aryl-3-ethyl-3-methylsuccinimides) with high biological potential have been subjected to estimation of their passive permeability and their association with (a) experimentally obtained anisotropic lipophilicity, (b) in silico-calculated lipophilicity and (c) in silico-predicted permeability and absorbability. Non-cellular-based parallel artificial membrane permeability assay was applied for quantifying their passive permeation, expressed as logPapp . Passive permeation was governed by the lipophilicity of the analysed compounds, and anisotropic lipophilicity was related with statistically significant passive transcellular diffusion (r2 = 0.614, P < 0.001). Moreover, experimentally determined passive permeability, logPapp , was statistically significantly associated with both in silico-predicted absorption constant, ka (r2 = 0.7886, P < 0.001), and human intestinal absorption (HIA) in percentage (r2 = 0.484, P < 0.001), respectively. However, there was no statistically significant relationship between experimentally obtained permeability on non-cellular-based model and in silico-predicted Caco-2 permeability based on the predictions conducted on two different software. Based on the obtained results, anisotropic systems are promising surrogates for determining lipophilicity, except for compounds with acidic functional groups that are completely ionized under (pH = 7.4).

UHPLC-DPPH method reveals antioxidant tyramine and octopamine derivatives in Celtis occidentalis

Celtis occidentalis L. (common Hackberry, Cannabaceae) has been applied in the traditional medicine for a long time as a remedy for sore throat, aid during menstruation and for treating jaundice. Nevertheless, the phytochemical exploration of the plant is still incomplete, literature data is limited to flavonoid derivatives isolated from the leaves.The present study reports screening approaches for bioactive compounds in C. occidentalis by fast and simple UHPLC-coupled assays. The UHPLC-DPPH method revealed six constituents in the methanolic extract of the twigs that had not been reported in C. occidentalis before. The antioxidant compounds were isolated by the means of flash chromatography and semi-preparative HPLC and identified by Orbitrap® MS and NMR spectroscopy as N-trans-p-coumaroyloctopamine (1), N-trans-feruloyloctopamine (2), N-trans-caffeoyltyramine (3), 2-trans-3-(4-hydroxyphenyl)-N-[2-(4-hydroxyphenyl)-2-oxoethyl] prop-2-enamide (4), N-trans-p-coumaroyltryramine (5) and N-trans-feruloyltyramine (6).Despite the high antioxidant activity measured in the present study and literature data suggesting potential positive effects of the compounds in the central nervous system, the PAMPA-BBB assay performed with the Celtis extract revealed that none of the aforementioned compounds are able to penetrate across the blood-brain barrier via transcellular passive diffusion.

Ultrathin, Large-Area Membrane Diffusion Cell for pH-Dependent Simultaneous Dissolution and Absorption Studies.

Preclinical evaluation of modern oral dosage forms requires more advanced in vitro devices as the trend of selecting low solubility, high permeability compounds for commercial development continues. Current dissolution methodologies may not always be suitable for such compounds due to excessive fluid volume, high fluid shear rates, heterogeneity of shear rates, suboptimal fluid flow, and, ultimately, the lack of absorption ability (Gray The Science of USP 1 and 2 Dissolution: Present Challenges and Future Relevance; Pharmaceutical Research, 2009; Vol. 26; pp 1289-1302). Herein, a new dissolution apparatus is introduced in combination with an ultrathin, semipermeable polymer membrane that mimics human passive absorption for lipophilic compounds. The ultrathin large-area polydimethylsiloxane (PDMS) membrane (UTLAM) absorption system is designed to mimic the dissolution and passive transcellular diffusion process representing the oral absorption pathway. A simple spin-casting method was developed to fabricate the ultrathin highly uniform membranes. To minimize membrane resistance to diffusion and maximize transport across the polymer membrane, 10-40 μm PDMS membranes were successfully prepared. A new diffusion cell was designed and tested to support the UTLAM and incorporates a hydrofoil impeller for more desirable hydrodynamics and mixing, using ibuprofen as a model weak acidic drug. UTLAM permeability was sufficiently high that the aqueous boundary layer contributed to the overall permeability of the system. This diffusion cell system demonstrated that, when the aqueous diffusion layer contributes to the overall resistance to transport, the pH at which absorption is 50% of maximum (pH50%) shifts from the pKa to higher values, demonstrating why weak acid drugs can exhibit high absorption at pH's significantly greater than their pKa. High rates of transport across the UTLAM are possible for drugs with high partition coefficients (i.e., BCS II compounds even under mostly ionized conditions), and PDMS UTLAMs may be tailored to simulate human intestinal passive absorption rates.

Blood-brain barrier permeability study of ginger constituents

Ginger, the rhizome of Zingiber officinale Roscoe is of great importance in the traditional medicine for the treatment of various diseases. More than 400 constituents have been reported in the plant, the most important ones being the gingerol and shogaol derivatives. Positive effects of ginger extracts and isolated [6]-gingerol have been proved in animal models of anxiety, Alzheimer’s disease, Parkinson’s disease and epilepsy. Taken in consideration these promising positive effects of ginger and its constituents in the central nervous system, the isolation of gingerol and shogaol derivatives ([6]-gingerol (1), [8]-gingerol (2), [10]-gingerol (3), [6]-shogaol (4), [10]-shogaol (5), 1-dehydro-[6]-gingerdione (6), 1-dehydro-[10]-gingerdione (7)) and investigation of their transcellular passive diffusion across the blood-brain barrier (BBB) were carried out. For this purpose, a Parallel Artificial Membrane Permeability Assay for the Blood-Brain Barrier (PAMPA-BBB) was chosen that had previously been validated for natural compounds. Based on our results, [6]-gingerol, [8]-gingerol and [6]-shogaol were found to be able to penetrate the BBB via passive diffusion, suggesting them to contribute to the positive effects of ginger extracts in the central nervous system.

Current understanding of transport and bioavailability of bioactive peptides derived from dairy proteins: a review

SummaryDairy protein‐derived bioactive peptides (DBPs) have potential benefits for human health. However, their transport mechanisms and bioavailability from intestinal lumen to bloodstream are not well understood. This review summarises current understanding of their transport mechanisms across the intestinal membrane (peptide transport 1, paracellular route, transcytosis and passive transcellular diffusion) and the bioavailability of DBPs in animal and human studies. Some DBPs can escape the degradation of peptidase and reach the bloodstream at concentrations of micromolar range, and keep intact for several minutes to hours. The presences of brush‐border peptidases at the site of absorption and other peptidases in the blood, along with the peptide properties, such as molecular size and weight, stability to proteases, hydrophobicity and charge, determine their bioavailability. Developing novel analytical tools for accurate measurement and study of the transport, metabolism, and bioavailability of DBPs in vivo are expected.

Pharmacokinetic Properties of Saffron and its Active Components.

Saffron as a medicinal plant has many therapeutic effects. Phytochemical studies have reported that saffron is composed of at least four active ingredients which include crocin, crocetin, picrocrocin and safranal. The carotenoids of saffron are sensitive to oxygen, light, heat and enzymatic oxidization. However, regulation of these factors is required for saffron quality. Some pharmacologic effects of saffron and its active compounds include cardioprotective, neuroprotective, memory enhancer, antidepressant and anxiolytic. Among more than 150 chemicals of saffron, the most biologically active components are two carotenoids including crocin and crocetin. Most of the pharmacokinetic studies are related to these compounds. The pharmacokinetic studies have shown that crocin is not available after oral administration in blood circulation. Crocin is converted to crocetin in intestine but after intravenous injection, the level of crocetin in plasma is low. Crocetin can distribute in different tissues because of weak interaction between crocetin and albumin. Also it can penetrate blood-brain barrier and reach CNS by passive transcellular diffusion; thus it can be effective in neurodegenerative disorders. The large portion of crocin is eliminated via feces.

Exposure Characteristics of the Analogous β-Carboline Alkaloids Harmaline and Harmine Based on the Efflux Transporter of Multidrug Resistance Protein 2.

Harmaline and harmine occur naturally in plants and are distributed endogenously in human and animal tissues. The two β-carboline alkaloids possess potential for treating Alzheimer's disease, Parkinson's disease, depression and other central nervous system diseases. However, studies have showed that the two compounds have similar structures but with quite different bioavailability. The aim of this study was to elucidate the exposure difference and characterize the in vitro transport, metabolism, and pharmacokinetic properties of harmaline and harmine. The results showed that the harmaline and harmine transport across the Caco-2 and MDCK cell monolayers was varied as the time, concentration, pH and temperature changed. The absorption of harmaline and harmine was significantly decreased when ES (OATPs inhibitor), TEA (OCTs/OCTNs substrate), NaN3 (adenosine triphosphate inhibitor), or sodium vanadate (ATPase Na+/K+-dependent inhibitor) was added. However, when given MK571 and probenecid (the typical MRP2 inhibitor), the PappAB of harmine was increased (1.62- and 1.27-folds), and the efflux ratio was decreased from 1.59 to 0.98 and from 1.59 to 1.19, respectively. In addition, the uptake ratio of harmine at 1 μM was >2.65 in the membrane vesicles expressing human MRP2. Furthermore, harmine could slightly up-regulate the expression of MRP2, which implying harmine might be the substrate of MRP2. Particularly, the CLint-value for harmine was ~1.49-folds greater than that of harmaline in human liver microsomes. It was worth noting that the F-value of harmine was increased 1.96-folds after harmine co-administration with probenecid. To summarize, comprehensive analysis indicated that harmaline and harmine were absorbed by transcellular passive diffusion and a pH- and Na+-dependent mechanism might be mediated by OATPs and OCTs/OCTNs. MRP2 but MDR1 or BCRP might be involved in the transport of harmine. Furthermore, harmine was more unstable and easily metabolized than harmaline. All these findings suggested that harmine not only appears be an MRP2 substrate, but also possesses weak metabolic stability, and eventually leads to a low oral bioavailability. Taken together, the elucidated absorption, transport, metabolism as well as pharmacokinetic characteristics of harmaline and harmine provide useful information for designing delivery systems, pharmacological applications and avoiding drug-drug interactions.

Acute Effects of Vitamin C Exposure On Colonic Crypts: Direct Modulation of pH Regulation

Background/Aim: Colorectal cancer is still considered a leading cause of death in the United States and worldwide. One potential way to improve survival besides detection is to look to new therapeutic agents that can be taken prophylactically to reduce the risk of tumor formation. For cancer cells to grow and invade, a higher (more alkaline) intracellular pH must occur. We chose to examine a specific nutraceutical agent, which is Vitamin C. The acute effect of Vitamin C exposure on normal colonic crypts has been studied, providing some insight into how Vitamin C achieve its effect. Methods: Distal colon was excised from rats. Following enzymatic digestion single colonic crypts were isolated. Colonic crypts were loaded with pH sensitive dye to measure the intracellular pH changes. Crypts were exposed to solutions +/- Vitamin C. Results: 10 mM Vitamin C decreased Na⁺-dependent intracellular pH recovery. Vitamin C modulates SVCT leading to changes in proton extrusion. Vitamin C entry occurs via either SVCT2 on the basolateral membrane or by transcellular passive diffusion through tight junctions to the apical membrane and then active transport via SVCT1. Conclusion: Acute addition of Vitamin C to the basolateral membrane maintains low intracellular pH for a longer period which could halt and/or prevent tumor formation.

Intestinal Absorption of Ergostane and Lanostane Triterpenoids from Antrodia cinnamomea Using Caco-2 Cell Monolayer Model.

Antrodia cinnamomea is a precious medicinal mushroom. It exhibits promising therapeutic effects on cancer, intoxication, hypertension, hepatitis, and inflammation. Its major bioactive constituents are ergostane and lanostane triterpenoids. In this study, we used intestinal Caco-2 cell monolayer model to reveal the intestinal absorption property of 14 representative triterpenoids from A. cinnamomea. The bidirectional transport through the monolayer at different time points was monitored by a fully validated LC/MS/MS method. In the case of pure compounds, ergostanes 5 (25R-antcin H), 6 (25S-antcin H) and 10 (25R-antcin B) could readily pass through the Caco-2 cell layer, whereas lanostanes 13 (dehydroeburicoic acid) and 14 (eburicoic acid) could hardly pass through. When the cells were treated with A. cinnamomea extract, antcins A, B, C, H and K (1–6 and 9–11) were absorbed via passive transcellular diffusion, and showed high PAB and PBA values (> 2.5 × 10−5 cm/s). Meanwhile, the lanostanes dehydrosulphurenic acid (8), 15α-acetyldehydrosulphurenic acid (12), 13 and 14 exhibited poor permeability. Transport features of these compounds were consistent with their pharmacokinetic behaviors in rats. This study could also be helpful in predicting the intestinal absorption of A. cinnamomea in human.Graphical Electronic supplementary materialThe online version of this article (doi:10.1007/s13659-015-0072-4) contains supplementary material, which is available to authorized users.

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 7.5–480μM. 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.

Computational strategy for quantifying human pesticide exposure based upon a saliva measurement.

Quantitative exposure data is important for evaluating toxicity risk and biomonitoring is a critical tool for evaluating human exposure. Direct personal monitoring provides the most accurate estimation of a subject’s true dose, and non-invasive methods are advocated for quantifying exposure to xenobiotics. In this regard, there is a need to identify chemicals that are cleared in saliva at concentrations that can be quantified to support the implementation of this approach. This manuscript reviews the computational modeling approaches that are coupled to in vivo and in vitro experiments to predict salivary uptake and clearance of xenobiotics and provides additional insight on species-dependent differences in partitioning that are of key importance for extrapolation. The primary mechanism by which xenobiotics leave the blood and enter saliva involves paracellular transport, passive transcellular diffusion, or transcellular active transport with the majority of xenobiotics transferred by passive diffusion. The transcellular or paracellular diffusion of unbound chemicals in plasma to saliva has been computationally modeled using compartmental and physiologically based approaches. Of key importance for determining the plasma:saliva partitioning was the utilization of the Schmitt algorithm that calculates partitioning based upon the tissue composition, pH, chemical pKa, and plasma protein-binding. Sensitivity analysis identified that both protein-binding and pKa (for weak acids and bases) have significant impact on determining partitioning and species dependent differences based upon physiological variance. Future strategies are focused on an in vitro salivary acinar cell based system to experimentally determine and computationally predict salivary gland uptake and clearance for xenobiotics. It is envisioned that a combination of salivary biomonitoring and computational modeling will enable the non-invasive measurement of chemical exposures in human populations.

Intestinal formation of trans-crocetin from saffron extract (Crocus sativus L.) and in vitro permeation through intestinal and blood brain barrier

Aims: Extracts of saffron (Crocus sativus L.) have traditionally been used against depressions. Recent preclinical and clinical investigations have rationalized this traditional use. Trans-crocetin, a saffron metabolite originating from the crocin apocarotenoids, has been shown to exert strong NMDA receptor affinity and is thought to be responsible for the CNS activity of saffron. Pharmacokinetic properties of the main constituents from saffron have only been described to a limited extent. Therefore the present in vitro study aimed to determine if crocin-1 and trans-crocetin are able to pass the intestinal barrier and to penetrate the blood brain barrier (BBB). Additionally, the intestinal conversion of glycosylated crocins to the lipophilic crocetin had to be investigated. Experiments with Caco-2 cells and two different porcine BBB systems were conducted. Further on, potential intestinal metabolism of saffron extract was investigated by ex vivo experiments with murine intestine.Methodology: In vitro Caco-2 monolayer cell culture was used for investigation of intestinal permeation of crocin-1 and trans-crocetin. In vitro models of porcine brain capillary endothelial cells (BCEC) and blood cerebrospinal fluid barrier (BCSFB) were used for monitoring permeation characteristics of trans-crocetin through the blood brain barrier (BBB). Intestine tissue and feces homogenates from mice served for metabolism experiments.Results: Crocin-1, even at high concentrations (1000 µM) does not penetrate Caco-2 monolayers in relevant amounts. In contrast, trans-crocetin permeates in a concentration-independent manner (10–114 µM) the intestinal barrier by transcellular passage with about 32% of the substrate being transported within 2 h and a permeation coefficient of Papp 25.7 × 10−6 ± 6.23 × 10−6 cm/s. Trans-crocetin serves as substrate for pGP efflux pump. Trans-crocetin permeates BBB with a slow but constant velocity over a 29 h period (BCEC system: Papp 1.48 × 10−6 ± 0.12 × 10−6 cm/s; BCSFB system Papp 3.85 × 10−6 ± 0.21 × 10−6 cm/s). Conversion of glycosylated crocins from saffron extract to trans-crocetin occurs mainly by intestinal cells, rather than by microbiological fermentation in the colon.Conclusion: The here described in vitro studies have shown that crocins from saffron are probably not bioavailable in the systemic compartment after oral application. On the other side the investigations clearly have pointed out that crocins get hydrolyzed in the intestine to the deglycosylated trans-crocetin, which subsequently is absorbed by passive transcellular diffusion to a high extend and within a short time interval over the intestinal barrier. Crocetin will penetrate in a quite slow process the blood brain barrier to reach the CNS. The intestinal deglycosylation of different crocins in the intestine is mainly due to enzymatic processes in the epithelial cells and only to a very minor extent due to deglycosylation by the fecal microbiome. On the other side the fecal bacteria degrade the apocarotenoid backbone to smaller alkyl units, which do not show any more the typical UV absorbance of crocins. As previous studies have shown strong NMDA receptor affinity and channel opening activity of trans-crocetin the use of saffron for CNS disorders seems to be justified from the pharmacokinetic and pharmacodynamic background.

In VitroCharacterization of the Intestinal Absorption of Methylmercury using a Caco-2 Cell Model

Methylmercury (CH3Hg) is one of the forms of mercury found in food, particularly in seafood. Exposure to CH3Hg is associated with neurotoxic effects during development. In addition, methylmercury has been classified by the International Agency for Research on Cancer as a possible human carcinogen. Although the diet is known to be the main source of exposure, few studies have characterized the mechanisms involved in the absorption of this contaminant. The present study examines the absorption process using the Caco-2 cell line as a model of the intestinal epithelium. The results indicate that transport across the intestinal cell monolayer in an absorptive direction occurs mainly through passive transcellular diffusion. This mechanism coexists with carrier-mediated transcellular transport, which has an active component. The participation of H(+)- and Na(+)-dependent transport was observed. Inhibition tests point to the possible participation of amino acid transporters (B(0,+) system, L system, and/or y(+)L system) and organic anion transporters (OATs). Our study suggests the participation in CH3Hg absorption of transporters that have already been identified as being responsible for the transport of this species in other systems, although further studies are needed to confirm their participation in intestinal absorption. It should be noted that CH3Hg experiences important cellular acumulation (48-78%). Considering the toxic nature of this contaminant, this fact could affect intestinal epithelium function.

Elucidation of the transport mechanism of baicalin and the influence of a Radix Angelicae Dahuricae extract on the absorption of baicalin in a Caco-2 cell monolayer model

Ethnopharmacological relevanceAngelicae Dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav combined with Radix Scutellariae baicalensis Georgi has been widely used in traditional Chinese medicine (TCM) as an antipyretic analgesic and anti-inflammatory drug. Modern pharmacological studies have demonstrated that the compatible application of these two drugs is an effective treatment for hepatitis. A previous study indicated that a Radix Angelicae Dahuricae extract enhanced the intestinal absorption of the baicalin found in Radix Scutellariae; however, the underlying compatibility mechanism of these two herbs remains unknown. In this study, we further examined the effect of a Radix Angelicae Dahuricae extract on the absorption and transport properties of baicalin in a Caco-2 cell model to determine the compatibility mechanism of these two herbs. Aim of the studyThe aim of this work was to study the transport properties of baicalin in Radix Scutellariae across cell membranes and the effects of a Radix Angelicae Dahuricae extract on baicalin absorption using the well-characterized, human-based intestinal Caco-2 cell model. Materials and methodsWe assessed the absorption, bidirectional transport and toxicity of baicalin using a range of parameters, including drug concentration, pH, a P-glycoprotein (P-gp) inhibitor (Verapamil), an MRP inhibitor (MK-571) and EDTA-Na2 (tight junction modulator). Next, we studied the influence of a Radix Angelicae Dahuricae extract on the transport of baicalin under the same conditions. Drug concentration was measured by HPLC, and the apparent permeability coefficient (Papp) and apparent permeability ratio (PDR) were subsequently calculated. ResultsThe results showed that baicalin is non-toxic within a concentration range of 800µg/mL to 4800µg/mL. The transport of baicalin showed time and concentration dependence. The absorption of baicalin was optimal at pH 7.4 in 37°C; however, the absorption decreased at 4°C. The Papp of baicalin transport through the Caco-2 cell monolayer model was altered when specific inhibitors of P-gp or MRP were added to the cells. However, there was no significant difference in the PDR value. The Papp of baicalin improved when it was combined with the Radix Angelicae Dahuricae extract. The influence of EDTA-Na2 on the transport of baicalin showed that the permeability of baicalin significantly increased. The result further indicated that the mechanism of baicalin intestinal absorption in the Caco-2 cell monolayer involves passive transcellular diffusion. ConclusionsPassive diffusion is the main mode of intestinal absorption of bacalin and it involved in the efflux of proteins. The enhanced intestinal absorption of baicalin by Radix Angelicae Dahuricae can be due to opening of the tight junctions between cells and inhibition of MRP efflux protein expression or function.

Transepithelial Transport of a Natural Cholinesterase Inhibitor, Huperzine A, along the Gastrointestinal Tract: the Role of Ionization on Absorption Mechanism

During recent years there has been increasing interest in the Lycopodium alkaloid huperzine A as a potential therapeutic agent for neurodegenerative diseases. This study aimed to characterize huperzine A's permeability across the enterocyte barrier along the gastrointestinal tract with an emphasis on the effect of ionization on the drug absorption. Intestinal permeability of huperzine A was evaluated by in vitro Caco-2 and parallel artificial membrane permeation assay models and by the ex vivo Ussing chamber model. The permeability rate was strongly dependent on the degree of ionization and increased with elevation of the donor medium pH in all studied models. The transport of the unionized fraction was similar to the permeability of the markers for passive transcellular diffusion. Addition of the paracellular permeability modulator palmitoylcarnitine in the Caco-2 model led to significant enhancement in the permeability of the ionized huperzine A fraction. No evidence of active transport of huperzine A was detected in this study. The Ussing chamber model experiments showed similar drug permeability along the entire rat intestine. In conclusion, huperzine A permeates the intestinal border mainly by passive transcellular diffusion whereas some fraction, dependent on the degree of huperzine A ionization, is absorbed by the paracellular route. Huperzine A's permeability characteristics pave the way to the development of its oral extended release dosage form. The specific population of the potential users of huperzine A and the high potency of this molecule support the rationale for such a delivery.

Absorption Mechanism of Ginsenoside Compound K and Its Butyl and Octyl Ester Prodrugs in Caco-2 Cells

Ginsenoside compound K (CK) is a bioactive compound with poor oral bioavailability due to its high polarity, while its novel ester prodrugs, the butyl and octyl ester (CK-B and CK-O), are more lipophilic than the original drug and have an excellent bioavailability. The aim of this study was to examine the transport mechanisms of CK, CK-B, and CK-O using human Caco-2 cells. Results showed that CK had a low permeability coefficient (8.65 × 10(-7) cm/s) for apical-to-basolated (AP-BL) transport at 10-50 μM, while the transport rate for AP to BL flux of CK-B (2.97 × 10(-6) cm/s) and CK-O (2.84 × 10(-6) cm/s) was significantly greater than that of CK. Furthermore, the major transport mechanism of CK was found as passive transcellular diffusion with active efflux mediated by P-glycoprotein (P-gp). In addition, it was found that CK-B and CK-O were not the substrate of efflux transporter since the selective inhibitors (verapamil and MK-571) of efflux transporter had little effects on the transport of CK-B and CK-O in the Caco-2 cells. These results suggest that improving the lipophilicity of CK by acylation can significantly improve the transport across Caco-2 cells.

Optimization and Validation of an In Vitro Blood Brain Barrier Permeability Assay Using Artificial Lipid Membrane

Blood-Brain Barrier (BBB) is one of the key issues in the pharmaceutical industry since the Central Nervous System (CNS) drugs need to penetrate the barrier, while the peripherally acting drugs should be impaired in the passage. Most of the CNS drugs enter the brain by transcellular passive diffusion mechanism due to the presence of zonula occludens and limited transport pathways. In the present study two different in-vitro methods to predict BBB permeability of drugs were compared and evaluated. We focused our attention on the effect of time on the permeability in PAMPA model to maximize the high through put nature by decreasing the incubation time. Moreover, we have compared the permeability of 16 structurally diverse, commercially available drugs assessed in two different PAMPA models: (1) a PAMPA-PBL (Porcine brain lipid) (2) a PAMPA- Phosphatidylcholine lipid. Both the models successfully identify CNS+ (High brain penetration) and CNS - (Low brain penetration) drugs. A comparison of the permeability by plotting P app values from both methods allows forecasting capacity of the assays. The correlation of the P app value of the both assays with the literature reports showed good correlation of r 2 of 0.9487 and 0.930. The robustness of the established models was further evaluated by establishing correlation of in silico generated logBB values and the experimental logBB values (r 2 0.915). Thus, the developed models have the ability to identify the CNS penetration with reduced incubation times, which in turn will shorten the assay time especially when high throughput screening is employed.

Transport mechanisms of flavanone aglycones across Caco-2 cell monolayers and artificial PAMPA membranes

We recently reported that flavanone aglycones (hesperetin, naringenin and eriodictyol) are efficiently absorbed via proton-coupled active transport, in addition to transcellular passive diffusion, in Caco-2 cells. Here, we aimed to evaluate in detail the absorption mechanisms of these flavanones, as well as homoeriodictyol and sakuranetin. We evaluated the absorption mechanisms of the above compounds by means of in vitro studies in Caco-2 cells in parallel with an artificial membrane permeation assay (PAMPA) under pH-gradient and iso-pH conditions. Comparison of the permeability characteristics of flavanones in Caco-2 cells and in PAMPA under these conditions, as well as a consideration of the physicochemical properties, indicated that hesperetin, naringenin, eriodictyol and homoeriodictyol were efficiently transported by passive diffusion according to the pH-partition hypothesis, except in the case of sakuranetin. However, transport of all flavanones were remarkably temperature-dependent, and was significantly reduced when Caco-2 cells were treated with amino acid-modifying reagents. Our data confirm that both passive diffusion and an active transport mechanism contribute to flavanone absorption through human intestinal epithelium.