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[Display omitted] • Polysorbate 20, Brij-L23 and Kolliphor EL inhibit P-gp in their undigested form. • Elements of the standardized in vitro lipolysis are incompatible with cells. • Pre-digestion of polysorbate 20 and Brij-L23 does not affect P-gp inhibition. While various non-ionic surfactants at low concentrations have been shown to increase the transport of P-gp substrates in vitro , in vivo studies in rats have shown that a higher surfactant concentration is needed to increase the oral absorption of e.g. the P-gp substrates digoxin and etoposide. The aim of the present study was to investigate if intestinal digestion of surfactants could be the reason for this deviation between in vitro and in vivo data. Therefore, Kolliphor EL, Brij-L23, Labrasol and polysorbate 20 were investigated for their ability to inhibit P-gp and increase digoxin absorption in vitro. Transport studies were performed in Caco-2 cells, while P-gp inhibition and cell viability assays were performed in MDCKII-MDR1 cells. Polysorbate 20, Kolliphor EL and Brij-L23 increased absorptive transport and decreased secretory digoxin transport in Caco-2 cells, whereas only polysorbate 20 and Brij-L23 showed P-gp inhibiting properties in the MDCKII-MDR1 cells. Polysorbate 20 and Brij-L23 were chosen for in vitro digestion prior to transport- or P-gp inhibiting assays. Brij-L23 was not digestible, whereas polysorbate 20 reached a degree of digestion around 40%. Neither of the two surfactants showed any significant difference in their ability to affect absorptive or secretory transport of digoxin after pre-digestion. Furthermore, the P-gp inhibiting effects of polysorbate 20 were not decreased significantly. In conclusion, the mechanism behind the non-ionic surfactant mediated in vitro P-gp inhibition seemed independent of the intestinal digestion and the results presented here did not suggest it to be the cause of the observed discrepancy between in vitro and in vivo. [ABSTRACT FROM AUTHOR]

In this study we examined the potential of novel biodegradable polymers of polyesterurethane (PU), and its PEGylated (PU-PEG) form as nanocarriers of Infliximab (INF), to treat inflammation in an in-vitro epithelial model. Nanoparticles (NPs) formulated were of average size of 200–287 nm. INF loading of NPs (INF-NPs) resulted in an increase in size and zeta potential. No cytotoxicity was observed for any of the NPs. Cellular interaction and uptake of PU NPs were similar compared with polycaprolactone (PCL) NPs and significantly higher to Poly(lactic- co -glycolic) acid (PLGA) NPs. Cellular interaction was higher for corresponding PEG-NPs. INF-PU and INF-PU-PEG NPs showed a rapid rate and extent of recovery of the epithelial barrier function in inflamed Caco-2 cell monolayers and decreased cytokine levels in inflamed monocytes. Results obtained in this study are promising and the potential of PU and PU-PEG NPs for drug delivery and targeting to treat gastrointestinal inflammation warrants further investigation. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Nonionic surfactants commonly used in pharmaceutical formulations may have P-glycoprotein (P-gp) inhibiting and/or permeation enhancing effects. The present work aims to distinguish these effects and assess the degree of cellular recovery after multiple exposures to nonionic surfactants. The investigated surfactants were polysorbates (PS): PS20, PS40, PS60, PS65, PS80 and PS85; monosaccharide-based: lauroyl methyl glucamide and n-nonyl-β-D-glucopyranoside; or disaccharide-based: lauryl-β-D-maltoside and trehalose 6-laurate. Bi-directional permeability studies of digoxin and mannitol, and calcein-AM efflux assay were performed in cell cultures. Cellular recovery was evaluated by continuous measurements of transepithelial electrical resistance (TEER) in Caco-2 cell monolayers. Polysorbates with one fatty acid chain decreased the efflux of digoxin through P-gp inhibition in MDCKII MDR1 cells. Mono- and di-saccharide-based surfactants, in a dose dependent manner, enhanced digoxin absorptive permeability without decreasing the secretory permeability in Caco-2 cells, suggesting that the surfactants had a transcellular permeation enhancing effect. Caco-2 cell monolayers recovered to different degrees of 60–100% of the initial TEER values. Calcein-AM assay was found to be non-predictive to surfactants influence on digoxin permeability across cell monolayers. In conclusion, these results may assist, in a mechanism-based, selection of suitable surfactants for formulating oral dosage forms to enhance the absorption of low bioavailable P-gp substrates. [ABSTRACT FROM AUTHOR]

The aim of the present work was to investigate the ability of nonionic surfactants to increase the oral absorption of the P-glycoprotein substrate etoposide in vitro and in vivo . Intestinal absorption was investigated by studying bidirectional permeability of etoposide across filter-grown Caco-2 and MDCKII MDR1 cell monolayers. The oral absorption of etoposide was investigated in wild type (WT) and mdr1a deficient (KO) Sprague-Dawley rats. In cell cultures, polysorbate 20 (PS20) decreased P-glycoprotein mediated efflux of etoposide. When PS20 and etoposide were co-administered to WT rats, the oral absorption of etoposide increased significantly in the presence of 5 and 25% (v/v) PS20. However, in KO rats, the exposure of etoposide after oral co-administration with 5% PS20 was similar to control. Unexpectedly, co-administration of etoposide with 25% PS20 significantly reduced the absorption fraction of etoposide in mdr1a KO rats. In vitro dialysis studies performed on PS20-containing etoposide solutions suggested that the reduced bioavailability may be due to etoposide retention in PS20 micelles and/or through increased viscosity. In conclusion, PS20 increases oral bioavailability of etoposide through inhibition of P-glycoprotein. However, the use of the excipient may be challenged by etoposide retention at higher concentrations. [ABSTRACT FROM AUTHOR]

Hydroxypropyl-β-cyclodextrin (HP-β-CD) is known to enable absorption of the lipophilic drug itraconazole. Since the interaction between HP-β-CD and itraconazole is characterized by a non-lineair, A P -type phase-solubility diagram, the present study aimed to investigate the influence of intraluminal dilution (water intake) on the behavior and performance of an orally administered cyclodextrin-based solution of itraconazole. Subsequently, the in vivo behavior was simulated by combining in vitro dilution with permeation assessment. After the administration of a Sporanox ® solution to healthy volunteers with or without a glass of water, gastrointestinal and systemic concentrations of itraconazole were simultaneously monitored. Independently of the intake of water, no gastric precipitation of itraconazole was observed. After transfer to the duodenum, precipitation occurred and was more pronounced in the condition with water, resulting in a 7.6-fold reduction in duodenal AUC 0-3h compared to the condition without water. Nevertheless, plasma concentration-time profiles did not demonstrate any significant differences in AUC 0-8h , C max and t max . Application of freshly aspirated intestinal fluids on Caco-2 cells clearly confirmed that higher intestinal itraconazole concentrations after intake of Sporanox ® without water do not generate a substantially increased itraconazole uptake. A two-stage in vitro dilution test was combined with a permeation compartment to capture this solubility-permeability interplay. In conclusion, this work demonstrates that variations in intraluminal dilution may have a drastic impact on the gastrointestinal behavior of lipophilic drugs in the presence of cyclodextrins. In the case of an AP-type interaction with cyclodextrins, the trade-off between solubility and permeability may be affected. [ABSTRACT FROM AUTHOR]

Oral delivery of apomorphine via prodrug principle may be a potential treatment for Parkinson’s disease. The purpose of this study was to investigate the transport and stability of apomorphine and its esters across Caco-2 cell monolayer and their affinity towards chylomicrons. Apomorphine, monolauroyl apomorphine (MLA) and dilauroyl apomorphine (DLA) were subjected to apical to basolateral (A-B) and basolateral to apical (B-A) transport across Caco-2 cell monolayer. The stability of these compounds was also assessed by incubation at intestinal pH and physiological pH with and without Caco-2 cells. Molecular dynamics (MD) simulations were performed to understand the stability of the esters on a molecular level. The affinity of the compounds towards plasma derived chylomicrons was assessed. The A-B transport of intact DLA was about 150 times lower than the transport of apomorphine. In contrast, MLA was highly unstable in the aqueous media leading to apomorphine appearance basolaterally. MD simulations possibly explained the differences in hydrolysis susceptibilities of DLA and MLA. The affinity of apomorphine diesters towards plasma derived chylomicrons provided an understanding of their potential lymphatic transport. The intact DLA transport is not favorable; therefore, the conversion of DLA to MLA is an important step for intestinal apomorphine absorption. [ABSTRACT FROM AUTHOR]

The relationships between data of passage through Caco-2 cultured cell lines (log P app ), taken from the literature, for 38 structurally unrelated compounds and both n -octanol lipophilicity parameters (log P N and log D 7.4 ) and phospholipid affinity indexes were investigated. Phospholipid affinity ( log k W I A M ) was experimentally determined by HPLC on two different phospholipid stationary phases and the polar/electrostatic interaction component drug/phospholipids ( Δ log   k W IAM ) was calculated according to a method we previously proposed. Log P app moderately related to lipophilicity values measured at pH 7.4 (log D 7.4 ), according to a parabolic pattern, but poorly related with log   k W IAM . Furthermore, a significant inverse linear relationship with Δ log   k W IAM values was only observed for the analytes with m.w. >300 Da, for which paracellular diffusion can be considered a minor transport route in vivo . Indeed, it has been reported that Caco-2 passage data also encode secondary passage mechanisms, which participate in a different extent to the jejunal absorption in vivo and cannot be directly equated to the corresponding human in situ log P eff values, unless a normalization is performed. In an attempt to elucidate this issue, 47 structurally unrelated compounds whose cultured cell line passage data were corrected for the effects of the aqueous boundary layer and paracellular permeability, so as to express transcellular intrinsic permeability, log P 0 Caco-2/MDCK , were also considered. Highly significant inverse linear relationships were observed between log P 0 Caco-2/MDCK and Δ log k W IAM values from both IAM.PC.MG ( r 2 = 0.765) and IAM.PC.DD2 ( r 2 = 0.806) stationary phases whereas the relationships with either lipophilicity in n -octanol or log k W IAM values were very poor. The results of the present study, in complete agreement with those of our recent study on the relationships between jejunal absorption data measured in situ and Δ log k W IAM values, confirm the soundness of Δ log k W IAM parameters in the prediction of the intestinal absorption of drugs. From a mechanistic point of view, they suggest that the polar/electrostatic forces between drugs and phospholipids play a major role in the passage through biomembranes. [ABSTRACT FROM AUTHOR]

Cyclodextrins are widely used excipients in pharmaceutical formulations. They are mainly utilized as solubilizers and absorption enhancers, but recent results revealed their effects on cell membranes and pharmacological barriers. In addition to the growing knowledge on their interaction with plasma membranes, it was confirmed that cyclodextrins are able to enter cells by endocytosis. The number of the tested cyclodextrins was limited, and the role of this mechanism in drug absorption and delivery is not known. Our aim was to examine the endocytosis of fluorescently labeled hydroxypropyl-β-cyclodextrin, random methyl-β-cyclodextrin and soluble β-cyclodextrin polymer, and the cellular uptake of the fluorescent paclitaxel derivative-random methyl-β-cyclodextrin complex. The studied cyclodextrin derivatives were able to enter Caco-2 intestinal cells and localized in vesicles in the cytoplasm, while their permeability was very limited through Caco-2 monolayers. We demonstrated for the first time that the fluorescent paclitaxel derivative and rhodamine-labeled random methyl-β-cyclodextrin were detected in the same intracellular vesicles after treating cells with their inclusion complex. These results indicate that the endocytosis of cyclodextrin complexes can contribute to drug absorption processes. [ABSTRACT FROM AUTHOR]

Abstract: The main objectives of the present study were (i) to evaluate the effect of the mucus layer on saquinavir-loaded nanostructured lipid carriers (SQV-NLCs) uptake and (ii) to evaluate the mucopenetrating properties of dextran–protamine (Dex–Prot) coating on NLCs as per SQV permeability enhancement. Three different NLC formulations differing on particle size and surfactant content were obtained and coated with Dex–Prot complexes. SQV permeability was then evaluated across Caco-2 cell monolayers (enterocyte-like model) and Caco-2/HT29-MTX cell monolayers (mucus model). In the Caco-2 monolayers, Dex–Prot–NLCs increased up to 9-fold SQV permeability in comparison to uncoated nanoparticles. In the Caco-2/HT29-MTX monolayers, Dex–Prot–NLCs presenting a surface charge close to neutrality significantly increased SQV permeability. Hence, Dex–Prot complex coating is a promising strategy to ensure successful nanoparticle mucus-penetration, and thus, an efficient nanoparticle oral delivery. To our knowledge, this is the first time that Dex–Prot coating has been described as a nanoparticle muco-penetration enhancer across the intestinal mucus barrier. [Copyright &y& Elsevier]

[Display omitted] • Over the past two decades, the Caco-2 monolayer model has been used as a powerful permeability screening tool in in vitro pre-clinical drug development. • Due to multiple shortcomings inherently present with this model, the extent of its utility is hampered. • This detailed review presented here reviews these shortcomings and focuses on various innovative modifications applied to this model, they help to overcome many of its drawbacks and in turn increase its efficiency, utility, and overall performance. The Caco-2 cell model has been widely used to assess the permeability of drug candidates. It has provided a high throughput in vitro platform, functionally resembling the enterocytes. Since the oral route is the most preferred for drug administration, the Caco-2 cell model acts as a very important tool to elucidate the oral "druggability" of a molecule by providing a fairly reliable estimate of its permeability through the intestinal membrane. Despite its shortcomings (the lack of a mucus layer, long cultivation period, inter-lab variability, and differences in expression of enzymes, transporters, and tight junction complexes) it remains heavily used due to its reliability, predictive performance, and wide acceptance. Various modifications have been made: co-culturing with other intestinal cells, applying biosimilar mucus, reducing culturing time, combining Caco-2 monolayer with the dissolution apparatus, enhancing protein expression, and redesigning the sampling apparatus. These modifications are intended to overcome some of the shortcomings of the Caco-2 model in order to make its use easier, quicker, economical, and more representative of the intestine. The aim of this review is to discuss such modifications to enhance this model's utility, predictive performance, and reproducibility. [ABSTRACT FROM AUTHOR]

Iron (Fe) loaded solid lipid nanoparticles (SLN's) were formulated using stearic acid and iron absorption was evaluated in vitro using the cell line Caco-2 with intracellular ferritin formation as a marker of iron absorption. Iron loading was optimised at 1% Fe (w/w) lipid since an inverse relation was observed between initial iron concentration and SLN iron incorporation efficiency. Chitosan (Chi) was included to prepare chitosan coated SLN's. Particle size analysis revealed a sub-micron size range (300.3±31.75nm to 495.1±80.42nm), with chitosan containing particles having the largest dimensions. As expected, chitosan (0.1%, 0.2% and 0.4% w/v) conferred a net positive charge on the particle surface in a concentration dependent manner. For iron absorption experiments equal doses of Fe (20μM) from selected formulations (SLN-FeA and SLN-Fe-ChiB) were added to Caco-2 cells and intracellular ferritin protein concentrations determined. Caco-2 iron absorption from SLN-FeA (583.98±40.83ng/mg cell protein) and chitosan containing SLN-Fe-ChiB (642.77±29.37ng/mg cell protein) were 13.42% and 24.9% greater than that from ferrous sulphate (FeSO4) reference (514.66±20.43ng/mg cell protein) (p ≤0.05). We demonstrate for the first time preparation, characterisation and superior iron absorption in vitro from SLN's, suggesting the potential of these formulations as a novel system for oral iron delivery. [ABSTRACT FROM AUTHOR]

Abstract: The pyrido-pyrimidine structure is associated with different biological activities, including kinase inhibition and antibacterial activity. However, drug-like properties of this scaffold have not been explored thoroughly. Therefore, the biopharmaceutical profile of ten pyrido[4,3-d]pyrimidines with different substitution pattern was determined at the intestinal and hepatic level. The compounds’ biopharmaceutical properties were significantly affected by the substitution pattern, resulting in a broad range in fasted state simulated intestinal fluid solubility values (1.9μM–4.2mM) and Caco-2 permeability coefficients (0.17×10−6 cm/s to 52×10−6 cm/s). A phenylhydrazido group was responsible for the low FaSSIF solubility. Caco-2 permeability was impaired by a dimethoxyphenyl substituent. All analogs were metabolically stable in human intestinal microsomes. The hepatic metabolism, reflected by the extraction ratio, was intermediate to high (ER>0.3). Aliphatic chains, methoxy groups on a phenyl substituent, ketone and amine substituents were predicted as most susceptible sites for hepatic metabolism. Correlations were found between polar surface area of the compound and Caco-2 permeability (R =0.86) and metabolic stability (R =0.76). No toxicity was seen for the pyrido[4,3-d]pyrimidines with Caco-2 cells and sandwich-cultured rat hepatocytes. In conclusion, the large diversity of substituents on the pyrido[4,3-d]pyrimidine core highly influenced the compounds’ drug-like properties. [Copyright &y& Elsevier]

The aim of this study was to investigate the multidrug resistance related protein-2 (MRP2) inhibition effect of excipients commonly employed in the formulation of self-emulsifying drug delivery systems (SEDDS). Cytotoxicity and safe dosages of fifteen excipients, including five surfactants, three oils, three co-surfactants and four solid carriers, were compared using MTT assay. Caco-2 cell permeability and MRP2 vesicles transport assays were used in the inhibition analysis. Scutellarin was shown to be a substrate of MRP2 and used as a probe in the inhibition assay. Twelve excipients decreased efflux ratio of scutellarin in Caco-2 model, among them six excipients showed inhibition effect on MRP2 in MRP2 transport model. In Caco-2 model, Cremophor® EL, Maisine® 35-1, PEG 2000 and β-cyclodextrin reduced efflux ratio of scutellarin the most in the four different excipients groups. The efflux reduction mechanism was further validated by MRP2 transport assay in inhibiting MRP2 activity in Cremophor® EL and PEG 2000. It is suggested that scutellarin quantified by LC–MS can be used as a model drug for MRP2 inhibition analysis using Caco-2 permeability and vesicles transport analyses. Together they constitute a sensitive and rapid screening method for assessing interactions between excipients and MRP2 in SEDDS formulation. [ABSTRACT FROM AUTHOR]

Abstract: PLGA-microparticles with 4.7μm in diameter were prepared by the double emulsion technique and loaded with 1.7μg fluorescein/mg PLGA mimicking a hydrophilic API. In an effort to further elucidate the release and bioadhesive characteristics of lectin-grafted formulations in vitro, the particles were coated with wheat germ agglutinin. The microparticles exhibited thermo-responsive release since no free fluorescein was detected at 4°C or room temperature. At body temperature, however, more than 80% of the payload was released within 48h. The adhesion of lectin-grafted particles to Caco-2 monolayers, which were applied as a model for the human intestinal epithelium, exceeded that of plain ones 1.5-fold as also observed by fluorescence microscopy. Furthermore, the amount of model drug bound and taken up into the cells was 5.8-fold higher after incubation for 4h at 37°C as compared to fluorescein in solution. According to fluorescence imaging a considerable amount of the total fluorescein payload was accumulated intracellularily after incubation for 5h at 37°C. These findings not only confirm the utility of bioadhesives of the second generation for improved absorption of low molecular weight hydrophilic compounds but also indicate storage stability of such suspensions at 4°C and room temperature without any premature loss of API. [Copyright &y& Elsevier]

Abstract: This work explored the interaction of chitosan with Calu-3 and Caco-2 cell lines, as models of the airway and intestinal epithelium, respectively. The toxicity, tight junction opening and mucoadhesive effects of chitosan were compared in the two cell lines. Additionally, the role of mucus in the absorption-promoting activity of chitosan was studied systematically. Notably, chitosan exhibited a different degree of toxicity on the Calu-3 and Caco-2 cells. Chitosan''s tight junction-opening effect, observed in terms of reduction of transepithelial electrical resistance and permeability enhancement, was apparent in both cell lines, though somewhat lower in Caco-2 compared to Calu-3 cell layers (though overall permeability was higher in the former). Tight junction opening and association of chitosan with the epithelial cell layers were more prominent in mucus-containing than in mucus-depleted Calu-3 cells and non mucus-excreting Caco-2 monolayers. Overall, the work suggests that chitosan exhibits a different level of toxicity in airway, as compared to intestinal cells and although absorption enhancement is apparent in both cell lines, enabling its potential use as an absorption-promoting excipient in both pulmonary and oral macromolecular delivery, the magnitude and the duration of the effect are dependent on the level of mucus present on the epithelial surfaces. [Copyright &y& Elsevier]

Abstract: In the context of targeted therapy, we addressed the possibility of developing a drug delivery nanocarrier capable to specifically reach cancer cells that express the most prominent marker associated with cancer stem cell (CSC) phenotype, AC133. For this purpose, 100nm lipid nanocapsules (LNCs) were functionalized with a monoclonal antibody (mAb) directed against AC133 according to two distinct methods: firstly, post-insertion within 100nm LNCs of a lipid poly(ethylene glycol) functionalized with reactive-sulfhydryl maleimide groups (DSPE-PEG2000-maleimide) followed by thiolated mAb coupling, and, secondly, creation of a thiolated lipo-immunoglobulin between DSPE-PEG2000-maleimide and AC133, then post-inserted within LNCs. Due to the reduced number of purification steps, lower amounts of DSPE-PEG2000-maleimide that were necessary as well as lower number of free maleimide functions present onto the surface of immuno-LNC, the second method was found to be more appropriate. Thus, 126nm AC133-LNC with a zeta potential of −22mV while keeping a narrow distribution were developed. Use of the IgG1κ isotype control-immunoglobulins produced similar control IgG1-LNCs. Micro-Bradford colorimetric assay indicated a fixation of about 40 immunoglobulins per LNC. Use of human Caco-2 cells that constitutively express AC133 (Caco-2-AC133high) allowed addressing the behavior of the newly functionalized immuno-LNCs. siRNA knockown strategy permitted to obtain Caco-2-AC133low for comparison. Immunofluorescence-combined flow cytometry analysis demonstrated that the epitope-recognition function of AC133 antibody was preserved when present on immuno-LNCs. Although grafting of immunoglobulins onto the surface of LNCs repressed their internalization within Caco-2 cells as evaluated by flow cytometry, AC133-specific cellular binding was obtained with AC133-LNC as assessed by computer-assisted fluorescence microscopy. In conclusion, interest of AC133-LNCs as niche carriers is discussed toward the development of CSC targeted chemo- or radio-nanomedicines. [Copyright &y& Elsevier]

Abstract: Previous in vivo studies using PEG 400 showed an enhancement in the bioavailability of ranitidine. This study investigated the effect of PEG 200, 300 and 400 on ranitidine transport across Caco-2 cells. The effect of PEG polymers (20%, v/v) on the bi-directional flux of 3H-ranitidine across Caco-2 cell monolayers was measured. The concentration dependence of PEG 400 effects on ranitidine transport was also studied. A specific screen for P-glycoprotein (P-gp) activity was used to test for an interaction between PEG and P-gp. In the absence of PEG, ranitidine transport showed over 5-fold greater flux across Caco-2 monolayers in the secretory than the absorptive direction; efflux ratio 5.38. PEG 300 and 400 significantly reduced this efflux ratio (p <0.05), whereas PEG 200 had no effect (p >0.05). In concordance, PEG 300 and 400 showed an interaction with the P-gp transporter, whereas PEG 200 did not. Interestingly, with PEG 400 a non-linear concentration dependence was seen for the inhibition of the efflux ratio of ranitidine, with a maxima at 1%, v/v (p <0.05). The inhibition of ranitidine efflux by PEG 300 and 400 which interact with P-gp provides a mechanism that may account for the observations of ranitidine absorption enhancement by PEG 400 in vivo. [Copyright &y& Elsevier]

Abstract: Microparticle uptake in the small intestine is relevant to both the delivery of pharmaceutics and exposure to environmental pollutants. The Caco-2 enterocyte model is a useful tool to study the parameters that affect epithelial microparticle permeability and the mechanisms controlling them. The current study used this model to explore further the different effects of 10% ethanol v/v or ice on transepithelial resistance (TER), microparticle uptake and immunofluorescent labelling of intercellular junctions. The same exposure times for both treatments were used, rather than those shown in the literature to produce demonstrable changes induced by each. The effects of both pre-treatments were greater after 60min than after 15min. Ethanol pre-treatment for 60min decreased TER, increased particle uptake and was associated with a disorganisation of tight and adhering junctional proteins. Pre-treatment with ice for 60min however, increased TER, decreased particle uptake and was associated with concentration of intercellular junctional proteins in a more constrained manner. These findings on the effects of pre-treatment with ethanol or ice for 60min suggest that the extent of uptake is influenced by changes in the distribution of intercellular junctional proteins. [Copyright &y& Elsevier]

Abstract: Although several in vitro models have been reported to predict the ability of drug candidates to cross the blood–brain barrier, their real in vivo relevance has rarely been evaluated. The present study demonstrates the in vivo relevance of simple unidirectional permeability coefficient (P app) determined in three in vitro cell models (BBMEC, Caco-2 and MDCKII-MDR1) for nine model drugs (alprenolol, atenolol, metoprolol, pindolol, entacapone, tolcapone, baclofen, midazolam and ondansetron) by using dual probe microdialysis in the rat brain and blood as an in vivo measure. There was a clear correlation between the P app and the unbound brain/blood ratios determined by in vivo microdialysis (BBMEC r =0.99, Caco-2 r =0.91 and MDCKII-MDR1 r =0.85). Despite of the substantial differences in the absolute in vitro P app values and regardless of the method used (side-by-side vs. filter insert system), the capability of the in vitro models to rank order drugs was similar. By this approach, thus, the additional value offered by the true endothelial cell model (BBMEC) remains obscure. The present results also highlight the need of both in vitro as well as in vivo methods in characterization of blood–brain barrier passage of new drug candidates. [ABSTRACT FROM AUTHOR]

Abstract: This study examines whether algorithms to predict brain penetration of 88 drug candidates could benefit from inclusion of PAMPA data such as P eff , flux and membrane retention. Specifically the ability to fit experimentally derived LogBB data with PAMPA information and compound related physicochemical and structural parameters was assessed. Collected data were analyzed by partial least square analysis and various regression models for LogBB. Four PAMPA methodologies were evaluated in this study including: (1) a PAMPA-BLM (black lipid membrane) model, (2) a PAMPA-DS (double sink) model, (3) a PAMPA-BBB (blood–brain barrier) model and (4) a PAMPA-BBB-UWL (unstirred water layer). Additionally, plasma protein binding (PPB) experiments and a Caco-2 assay were performed to determine the unbound fraction in plasma and the efflux ratio, respectively, for subsets of the selected compounds. This information was combined with the obtained PAMPA data in an effort to improve the predictions of LogBB. Taken in aggregate, the results presented, suggest that the PAMPA-BLM parameters are the most important contributors to predict the LogBB. The optimized multiple linear regression (MLR) relationship including the PAMPA-BLM properties demonstrated a slightly improved prediction compared to the model without the PAMPA-BLM parameters. Including the plasma protein binding of 15 compounds resulted in a significantly improved PAMPA-BLM prediction of LogBB, while integrating the efflux ratio with PAMPA-BLM or PAMPA-BBB P eff values, resulted in improved classification of brain permeable [BBB+(LogBB≥0)] and impermeable [BBB−(LogBB<0)] compounds. [Copyright &y& Elsevier]

Abstract: Small intestinal microparticle uptake via a paracellular route is relevant to oral drug delivery and environmental pollution. In vitro investigation uses latex microparticle passage across a confluent Caco-2 cell epithelium. This paper examines the influence of culture conditions on transepithelial resistance (TER); cell dimensions from confocal microscopy; and number of particles below the epithelium. Variables investigated include level of initial TER; multiple TER measurements; involvement of medium; cell source; and pretreatment with ethanol or a range of temperatures. Data were collected after exposure to 2μm latex particles for 5–120min: sham groups were exposed to pretreatment but not particles. The results highlight the importance of very precise control of the experimental environment; confirm the pattern of sequential-TER increase/decrease in groups exposed only to particles and show accompanying increases in cell dimensions. Greater particle uptake was associated with ethanol-induced decreased TER, decreased cell height and increased intercellular spaces, similar to previous findings for external irradiation. Low temperatures raised TER but, despite this, cooling did not alter particle uptake. In conclusion, culture microenvironment and sham treatment are crucial considerations in studies of epithelial microparticle uptake in vitro. [Copyright &y& Elsevier]

In vivo systemic absorption of the amino acid prodrugs of acyclovir (ACV) after oral administration was evaluated in rats. Stability of the prodrugs, L-alanine-ACV (AACV), L-serine-ACV (SACV), L-isoleucine-ACV (IACV), γ-glutamate-ACV (EACV) and L-valine-ACV (VACV) was evaluated in various tissues. Interaction of these prodrugs with the transporters on Caco-2 cells was studied. In vivo systemic bioavailability of these prodrugs upon oral administration was evaluated in jugular vein cannulated rats. The amino acid ester prodrugs showed affinity towards various amino acid transporters as well as the peptide transporter on the Caco-2 cells. In terms of stability, EACV was most enzymatically stable compared to other prodrugs especially in liver homogenate. In oral absorption studies, ACV and AACV showed high terminal elimination rate constants (λz). SACV and VACV exhibited approximately five-fold increase in area under the curve (AUC) values relative to ACV (p < 0.05). Cmax(T) (maximum concentration) of SACV was observed to be 39 ± 22 μM in plasma which is 2 times better than VACV and 15 times better than ACV. Clast(T) (concentration at the last time point) of SACV was observed to be 0.18 ± 0.06 μM in plasma which is two times better than VACV and three times better than ACV. Amino acid ester prodrugs of ACV were absorbed at varying amounts (Cmax) and eliminated at varying rates (λz) thereby leading to varying extents (AUC). The amino acid ester prodrug SACV owing to its enhanced stability, higher AUC and better concentration at last time point seems to be a promising candidate for the oral treatment of herpes infections. [Copyright &y& Elsevier]

Abstract: The objective of this study was to synthesize and evaluate novel enzymatically stable dipeptide prodrugs for improved absorption of acyclovir. l-Valine-l-valine-acyclovir (LLACV), l-valine-d-valine-acyclovir (LDACV), d-valine-l-valine-acyclovir (DLACV) and d-valine-d-valine-acyclovir (DDACV) were successfully synthesized. The uptake and transport studies were conducted on a Caco-2 cell line. Buffer stability and metabolism of the prodrugs in Caco-2, rat intestine and liver homogenates were studied. Structure and purity of the all compounds were confirmed with LC–MS/MS and NMR spectroscopy. Uptake and transport of [3H] glycylsarcosine was inhibited by all prodrugs except DDACV. DLACV and DDACV exhibited no measurable degradation in Caco-2 homogenate. Except DDACV other three prodrugs were hydrolyzed in rat intestine and liver homogenates. The order of permeability across Caco-2 was LDACV>LLACV>DDACV>DLACV. A linear correlation between the amount of prodrug transported and over all permeability of acyclovir was established. This study shows that the incorporation of one d-valine in a dipeptide did not abolish its affinity towards peptide transporters (PEPT). Moreover, it enhanced enzymatic stability of prodrug to a certain extent depending on the position in a dipeptide conjugate. This strategy improved both the cellular permeability and the amount of intact prodrug transported which would enable targeting the nutrient transporters at blood ocular barrier (BOB). [Copyright &y& Elsevier]

Abstract: Paclitaxel is widely used to treat several types of solid tumors. The commercially available paclitaxel formulation contains Cremophor/ethanol as solubilizers. This study evaluated the effects of d-α-tocopheryl polyethylene glycol 400 succinate (TPGS 400) on the oral absorption of paclitaxel in mice. Mice were given an intravenous (18mg/kg) or oral (100mg/kg) dose of paclitaxel solubilized in Cremophor/ethanol or in TPGS 400/ethanol formulations. Paclitaxel plasma concentrations and pharmacokinetic parameters were determined. The maximal plasma concentrations of paclitaxel after an oral dose were 1.77±0.17 and 3.39±0.49μg/ml for Cremophor/ethanol and TPGS 400/ethanol formulations, respectively, with a similar time at 40–47min to reach the maximal plasma concentrations. The oral bioavailability of paclitaxel in TPGS 400/ethanol (7.8%) was 3-fold higher than that in Cremophor/ethanol (2.5%). On the other hand, the plasma pharmacokinetic profiles of intravenous paclitaxel demonstrated a superimposition for the two formulations. Furthermore, TPGS 400 concentration-dependently increased the intracellular retention of Rhodamine 123 in Caco-2 cells and enhanced paclitaxel permeability in monolayer Caco-2 cultures. TPGS 400 at concentrations up to 1mM did not inhibit testosterone 6β-hydroxylase, a cytochrome P450 isozyme 3A in liver microsomes metabolizing paclitaxel. Our results indicated that TPGS 400 enhances the oral bioavailability of paclitaxel in mice and the enhancement may result from an increase in intestinal absorption of paclitaxel. [Copyright &y& Elsevier]

Abstract: Proliposomal bead formulations for improved oral delivery of cromolyn (BCS Class III compound) were formulated. Phospholipid (distearylphosphatidylcholine)–cholesterol-surfactant (Tween 80/sodium cholate) systems were spray-coated on beads containing cromolyn sodium and the dosage forms were characterized for vesicle formation and encapsulation efficiency. Delivery of cromolyn sodium from this novel dosage form was evaluated across the Caco-2 and everted rat intestinal sac model. Spontaneous formation of vesicles upon dilution of beads was observed. Enhancement in cromolyn transport was higher with phospholipids-surfactant proliposomal formulations compared to surfactant-free lipid formulations or pure surfactant solutions, most significant enhancement being with formulations with low surfactant concentration. No evidence of cellular damage to Caco-2 monolayers (e.g. significant decrease in the TEER) or change in transport and tissue accumulation of a marker molecule in the intestinal tissue model was observed. This indicated enhancement of transport via transcellular routes and not due to the modulation of the tight junctions or cell disruption. Results suggest that phospholipids-surfactant proliposomal beads offer a good potential for improved oral delivery of cromolyn. [Copyright &y& Elsevier]

Abstract: The objective of the present study was to investigate the reliability of transporter inhibitors in the elucidation of drug–transporter interactions when multiple transporters are present in a test system. The bidirectional permeabilities of digoxin, estrone-3-sulfate (E3S), and sulfasalazine, substrates of P-gp, BCRP/MRP2 and unspecified efflux transporters, respectively, were examined in Caco-2 and MDR-MDCK cells in the absence and presence of transporter inhibitors: CsA (P-gp), FTC (BCRP) and MK571 (MRP). Digoxin showed significant efflux ratios (ER) in both Caco-2 (ER=17) and MDR-MDCK (ER=120), whereas E3S and sulfasalazine only showed significant efflux in Caco-2 (ER=15 and 88, respectively) but not in MDR-MDCK cells (ER=1.1 and 1.3, respectively). CsA at 10μM showed complete inhibition of digoxin efflux, partial inhibition of E3S efflux and no effect on sulfasalazine efflux. FTC and MK571 had different inhibitory effects on the efflux of these compounds. The present study shows evidence of the functional expression of multiple efflux transporter systems in Caco-2 cells. Although the use of Caco-2 cells and selected inhibitors of efflux transporters can provide useful mechanistic information on drug–drug interactions involving efflux transporters, the potential cross-reaction of inhibitors with multiple transporters makes it difficult to discern the role of individual transporters in drug transport or drug–drug interactions. [Copyright &y& Elsevier]

Abstract: Piperacillin, a potent β-lactam antibiotic, is effective in a large variety of Gram+ and Gram− bacterial infections but its administration is limited to the parenteral route as it is not absorbed when given orally. In an attempt to overcome this problem, we have synthesized a novel series of charged liposaccharide complexes of piperacillin comprising a sugar moiety derived from d-glucose conjugated to a lipoamino acid residue with varying side-chain length (cationic entity) and the piperacillin anion. A complete multiple reaction monitoring LC–MS/MS method was developed to detect and characterize the synthesized complexes. The same method was then successfully applied to assess the in vitro apparent permeability values of the charged liposaccharide complexes in Caco-2 monolayers. [Copyright &y& Elsevier]

Abstract: Benzimidazole 2-carbamates, such as albendazole (ABZ) and mebendazole (MBZ), used for the treatment of helmintic infections, have low aqueous solubility and poor bioavailability, both of which lead to high interindividual variability when used for human systemic helmintiosis; therefore, it is necessary to search for new anthelmintics with better biopharmaceutical properties. In the present study the solubility, pK a, log P and apparent permeability in the Caco-2 cells system of four novel anthelmintic (1H-benzimidazol-5(6)-yl)carboxamide derivatives (compounds 1–4) with a 2-methylthyo group were evaluated. Also the pharmacokinetic parameters of compound 1 which in previous studies showed activity similar to ABZ against T. spirallis, was evaluated in BALB/c mice, as a representative molecule of the series. The novel anthelmintics, showed better solubility than ABZ in aqueous acid pH and in organic solvents. The log P, P app and Caco-2 data indicate that the 4 derivatives are highly permeable drugs, but it is possible that an efflux system could be involved in the transport of these compounds. Plasma levels of compound 1 and its sulfoxide (compound 5) were high after the first 5min. This fact strongly suggests that compound 1 is rapidly metabolized in the small intestine. On the other hand, the sulfone metabolite (compound 6) levels were lower than those of compound 5. The half life and mean residence time (MRT) of compound 1 and its main metabolites indicate that their elimination is very rapid. More studies in mammalian species are necessary in order to understand the pharmacokinetic behavior of these novel compounds. [Copyright &y& Elsevier]

Abstract: Curcuma longa and C. zedoaria, belonging to genus Curcuma, have become prevalent as supplements in East Asia. Curcumin is the most well-studied bioactive component isolated from rhizomes of C. longa and other Curcuma species except C. zedoaria. In this study, we investigated the affects of C. longa, C. zedoaria from Japan and curcumin on CYP3A4. Caco-2 cells, in which CYP3A4 expression was induced by 1α,25-(OH)2-D3, were used to mimic the metabolism of small intestine. Caco-2 cells were treated with methanol extracts from two Curcuma rhizomes (0.1mg/ml) or curcumin (30μM) for 72h. Both extracts significantly decreased the activity of CYP3A4 by about 85–98%. The 50% inhibitory concentrations of C. longa and C. zedoaria extracts were 0.019 and 0.014mg/ml, respectively. They caused a 60–70% decrease in CYP3A4 protein. Otherwise, curcumin treatment caused a 30–40% decrease in CYP3A4 catalytic activity and a 38% decrease in CYP3A4 protein expression. Moreover, it was found that both Curcuma extracts and curcumin treatment had no influence on CYP3A4 mRNA expression. Our results suggested that administration of Curcuma drugs might inhibit the catalytic activity of intestinal CYP3A4. However, curcumin was not the major compound responsible for this inhibitory effect. [Copyright &y& Elsevier]

Abstract: The hypothesis that, in vivo in situ, villous uptake of 2μm latex microparticles involves changes at enterocyte tight junctions (TJs) was tested using Caco-2 cells on porous membranes. Epithelial permeability was measured by transepithelial resistance (TER) and particle numbers in surface, intraepithelial and sub-epithelial compartments by microscopy. Apical particle or medium addition initially closed TJs, but this was subsequently reversed in particle-treated groups. Peristaltic onward movement of a bolus was simulated by removing apical particles after an exposure period and leaving the remaining particles to interact with the epithelium: this produced marked TJ loosening during the interaction period. For particle exposure groups, the early similarity with particle numbers in vivo taken up in young adult rats became less marked with time, although bolus removal counteracted this tendency. The TJ response to vasoactive intestinal polypeptide (VIP) was time-dependent. Adsorbed and intraepithelial particle numbers increased with particle exposure time; epithelial-associated microparticle aggregation varied with treatment and submembranous particles were seen in all groups. Correlation between TER changes and particle numbers suggests TJ loosening may be important in microparticle uptake. This Caco-2 model gives epithelial particle numbers that approximate well to published figures for microparticle uptake in vivo and allows effective microenvironmental manipulation. [Copyright &y& Elsevier]

Abstract: As a result of their improved aqueous solubility, the development of phosphate ester prodrugs is an interesting approach to increase intestinal absorption of poorly water-soluble drugs. Absorption of a drug from its phosphate ester prodrug is based on intestinal dephosphorylation of the prodrug which may result in intraluminal supersaturation of the parent drug, followed by an increased absorptive flux across the intestinal mucosa. In this study, we evaluated the behavior of fosamprenavir, a phosphate ester prodrug of amprenavir, in the Caco-2 system and in aspirated human intestinal fluids (HIF), both showing phosphatase activity. Starting from a solution of fosamprenavir in HIF, a supersaturated solution of amprenavir was generated and maintained during a time period sufficient for absorption. Moreover, supersaturation of amprenavir resulted in an enhanced flux across Caco-2 monolayers. To our knowledge, this is the first illustration of supersaturation in real intestinal media. Next, we showed an inhibitory effect of inorganic phosphate on the dephosphorylation of fosamprenavir, both in the Caco-2 model and in HIF. As a consequence, phosphate-buffered media, including fasted state simulated intestinal fluid (FaSSIF), are incompatible with the study of phosphate ester prodrugs and should be replaced with media containing a biorelevant phosphate concentration (0.4–1mM) and another buffering compound such as 2-morpholinoethanesulfonic acid (MES). [Copyright &y& Elsevier]

Abstract: The aim of this study was to correlate the taxonomy of grapefruit, pummelo, orange, lime and lemon with fruit juice-mediated cytotoxicity, modulation of epithelial permeability and P-glycoprotein (P-gp)-mediated efflux using 0–50% juice concentrations. Lime and lemon juices at 30% enhanced the absorption of [14C]-mannitol across Caco-2 cell monolayers by six- and eight-fold, respectively, but grapefruit and pummelo juices did not modulate the paracellular [14C]-mannitol transport even at 50%. Orange juice at 30% increased mannitol absorption to a comparable level as lime juice, but had minimal effects on TEER. All five juices did not modulate the passive diffusional pathway as exemplified by their negligible effects on [3H]-propranolol absorption. Grapefruit, pummelo and orange juices showed P-gp inhibitory activity by reducing rhodamine-123 (R-123) efflux and elevating R-123 cellular accumulation, but lime and lemon juices did not. Lime and lemon juices at ≥30% were cytotoxic towards Caco-2 cells. Grapefruit and pummelo juices at 10% did not affect Caco-2 cell viability, but they enhanced cell growth at concentrations of ≥30%. Orange juice increased cell viability only at lower concentrations. On the basis of these data, lime and lemon juices could be regarded as a group distinct from grapefruit and pummelo juices, while orange juice appeared to belong to a bridging group. This grouping was consistent with the categorization of the citrus fruits according to their dominant flavonoid pattern and taxonomy. [Copyright &y& Elsevier]

Abstract: The effect of nonylphenol ethoxylates (NPEs) on selected barrier functions of biological membranes, such as tight junction and P-gp efflux pump of epithelial membranes, against the transport of xenobiotics was examined. The Caco-2 cell line was used to evaluate the transport of mannitol and daunomycin across the cell monolayer as well as the cellular uptake of daunomycin. In the presence of NPEs, the transport of mannitol was increased, with NP-9 showing a maximal effect, and the transepithelial electrical resistance (TEER) was reduced. The onset of this effect of NP-9 was fairly rapid and reversible for a short term (e.g., 2h) treatment, while irreversible for a long term (e.g., 72h) treatment. In the presence of NP-9, the apical uptake of daunomycin was increased, suggesting competitive inhibition between NP-9 and daunomycin in the efflux via the P-gp system. However, a 72h pretreatment of the cells with NP-9 (up to 1000nM) did not affect the apparent cellular uptake of daunomycin, suggesting no significant effect of NPEs on the expression of P-gp. In conclusion, NPEs appear to rapidly open the tight junction of epithelial cell membranes and to competitively inhibit the efflux of P-gp substrates, thereby reducing the self-protection ability of the organism against xenobiotics or hazardous environmental compounds that are transported via the paracellular pathway (i.e., uptake) or the P-gp system (i.e., efflux). [Copyright &y& Elsevier]

Feasibility to use probiotic bacteria as a living protein delivery system through oral route was assessed in vitro. Lactococcus lactis transformed with a plasmid to express and secret β-lactamase was used to deliver β-lactamase through Caco-2 monolayer, an intestine epithelium. Transport of β-lactamase through Caco-2 monolayer was carried out in the transwells. The viability and integrity of the cell monolayers co-cultured with L. lactis was examined by trypan blue exclusion method and by measuring the transport of mannitol and propranolol as well as the transepithelial electrical resistance (TEER). Results show that it is feasible to use cell culture technique to evaluate the drug delivery by normal flora. The transport rate of β-lactamase when delivered by L. lactis was 2.0 ± 0.1 × 10-2h-1 (n = 9) and through free solution form was 1.0 ± 0.1 × 10-2h-1. When co-cultured with L. lactis, Caco-2 cell viability decreased to 98, 96, and 94% at 6, 8, and 10h, respectively. Transport of mannitol through Caco-2 cell monolayer was significantly increased and the transport of propranolol through Caco-2 cell monolayer was significantly decreased in the presence of L. lactis. Increase in the amount of protein delivered is probably due to the concentrate of the protein by L. lactis on the monolayer (absorption surface) and the opening of the tight junction of Caco-2 monolayer by L. lactis. [Copyright &y& Elsevier]

Presently, the Caco-2 cell culture model is widely used during drug discovery and development as a predictive tool for the oral absorption of drug candidates. For transport experiments in the Caco-2 system, HBSS-like buffered salt solutions are commonly used, although different shortcomings have been associated with the use of these buffers. In this paper, we investigated the effect of using fasted state simulated intestinal fluid (FaSSIF) as potential biorelevant medium for the drug permeability estimation across Caco-2 monolayers. The transport characteristics of 19 model compounds were determined in the Caco-2 cell culture model in the presence of FaSSIF as compared to classic transport medium. A sigmoidal relation was obtained when the estimated Papp, s of the apical to basolateral transport were plotted versus the reported values of the fraction absorbed in man. Although no effect of FaSSIF as compared to classic transport medium (TM) was observed on the total predictability of the model, an impact was demonstrated (1) on the bi-directional transport of actively transported drugs (including talinolol, digoxin and doxorubicin), (2) on recovery and (3) on the solubility and permeability estimation of poorly water-soluble drugs. The observed differences may be attributed to a P-gp inhibitory effect of sodium taurocholate (NaTC), micellar encapsulation by the NaTC/lecithin mixed micelles and/or an increase of the solubility of lipophilic drugs. As the experimental conditions should mimic the physiological in vivo conditions, the use of FaSSIF as medium during Caco-2 experiments may improve the biorelevance of the model. [Copyright &y& Elsevier]

The objective of the present study was to develop a suitable method for evaluation of cytotoxicity of the oils used in Self-Emulsified Drug Delivery Systems (SEDDS) using Coenzyme Q10 (Co Q10) as a model compound. For this purpose, three methods of sample preparation were tested, namely (i) suspensions, (ii) homogenization, and (iii) nanoemulsions of oils in Dulbecco’s Modified Eagle’s Media (DMEM). Studies were carried out by incubating the sample or control with Caco-2 cells grown on transwell insert systems as well as in flat bottom 96-well plates. The cell viability was assessed by using WST-1 and propidium iodide reagents while the monolayer integrity was assessed by mannitol permeability and Trans Epithelial Electrical Resistance (TEER). The cytotoxicity of oils was found to be dependent on the method of sample preparation; nanoemulsions being the least cytotoxic. In conclusion, nanoemulsification is a useful tool for cytotoxicity evaluation of substances, which exhibit poor aqueous/dimethyl sulfoxide (DMSO) solubility. [Copyright &y& Elsevier]

This study was conducted to determine if electroporation can deliver membrane-impermeant molecules intracellularly to intact, physiologically competent monolayers that mimic the intestinal epithelium. In addition, the long-term effects of electroporation on these monolayers were studied to determine the kinetics with which monolayers recover barrier function. Caco-2 and T84 cells were electroporated as monolayers using calcein and fluorescein-labeled bovine serum albumin as marker molecules for measuring delivery into cells. Confocal microscopy and flow cytometry were used, respectively, to visualize and quantify uptake of these molecules. Transepithelial resistance was used as a measure of physiologic barrier function. We found that intracellular uptake of calcein and bovine serum albumin occurred uniformly throughout both types of model epithelia and increased as a function of voltage, pulse length, and pulse number. There was no significant difference in uptake resulting from single and multiple pulses of the same total exposure time. We also observed that monolayers exposed to electroporation that induced uptake of up to 106 molecules/cell were able to recover normal barrier function within one day. These findings suggest that electroporation may be useful for intracellular delivery into monolayers to study epithelial biology and, possibly, for drug delivery to intestinal epithelium. [Copyright &y& Elsevier]

In this study the gastrointestinal absorption and P-glycoprotein (Pgp) efflux transport of heterocyclic drugs was investigated with the Caco-2 cell model.Based on the calculation of the physico-chemical properties a good oral absorption was predicted for all the drugs tested in this study which corresponded well with the measured Caco-2 permeabilities (Papp). Generally a high permeability of the tested heterocyclic drugs was measured being in agreement with earlier published human in vivo absorption data.Based on the transport data of domperidone and verapamil it was found that the Pgp efflux transporter was expressed in the Caco-2 cells. Many of the drugs tested were indicated to be potential Pgp efflux substrates. Since Pgp is expressed at the Blood Brain Barrier (BBB) as well, it was expected that CNS penetration will be impaired if a drug is a Pgp substrate. However, no correlation could be found between brain penetration in rats and the Pgp efflux ratio as measured with the Caco-2 cells.From the data it is concluded that Pgp efflux ratio’s as determined in in vitro High Throughput Screening (HTS) tests, where the transport conditions are fixed (pH gradient, concentration, etc.), cannot routinely be used to predict a possible limited brain penetration. [Copyright &y& Elsevier]

The present study was to investigate the feasibility of oral delivery of recombinant human epidermal growth factor (rhEGF). Polyethylene glycol (PEG)-coated liposomes containing rhEGF was prepared and evaluated for their stability and permeability in Caco-2 cells. In the animal study, we also determined plasma concentration and gastric ulcer healing effect after oral administration of rhEGF liposomes or the solution. Encapsulation of rhEGF into liposomes, suppressed the degradation in Caco-2 cell homogenate compared with the solution. The flux of rhEGF from dipalmitoylphosphatidylcholine (DPPC) liposome across Caco-2 cell monolayer from the apical to basolateral side was three times greater than that from phosphatidylcholine (PC) liposome or the solution. After oral administration of rhEGF liposomes or the solution in rats, the area under the concentration–time curve (AUC) of rhEGF increased 1.7- and 2.5-fold for PC and DPPC liposomes, respectively. The gastric ulcer healing effect was significantly increased in DPPC liposome compared with PC liposome and the solution. The enhanced curative ratio of rhEGF encapsulated into DPPC liposome may be due to the resistance to enzyme degradation, higher permeability and increased plasma AUC. Therefore, PEG-coated liposomes containing rhEGF could be used as an oral delivery formulation with enhanced encapsulation efficiency. [Copyright &y& Elsevier]

Ideally, the amount of enhancer remaining at the donor side during an in vitro transport study should be known, in order to know the true enhancer concentration during a permeability study. The purpose of the present study is to estimate the flux of the enhancer, sodium glycocholate (GC), through Caco-2 cell monolayers, and to study the effect of various enhancer concentrations on the permeability of GC itself, the permeability of mannitol and the transepithelial electrical resistance (TEER).Apical to basolateral permeability was measured with various concentrations 0.50% (10.2 mM), 0.75% (15.5 mM) and 1.00% (20.5 mM) of GC. The GC permeabilities (Papp) were 4.7±1.1, 12.8±2.8 and 25.8±4.3 (×10−7 cm s−1), respectively. Mannitol transport changed accordingly with the Papp; 8.5±0.8, 9.9±2.7, 20.4±2.8 and 31.0±4.9 (×10−7 cm s−1) for GR, 0.50, 0.75 and 1.00% GC, respectively, with a TEER after 120 min, relative to initial, of 86±6, 77±10, 61±11 and 49±7%.In conclusion a low and concentration-dependent permeability was found for GC across the Caco-2 cells. Mannitol transport increased and TEER decreased accordingly with increasing GC concentrations. TEER decreased in less than 10 min to a certain level, without further reduction in a 120 min period, indicating that the enhancer effect is momentarily, rather than time-dependent. The apical GC concentration and enhancer effect may be considered well defined during the experiment, due to the observed low permeability of GC. [Copyright &y& Elsevier]

Solubilizers are often used to enhance the bioavailability of drugs with poor aqueous solubility. This study focuses on the use of the Caco-2 system containing solubilizers to predict the absorption of poorly water-soluble drugs in humans. First, the effects of propylene glycol (PG), hydroxypropyl-β-cyclodextrin (HP-β-CD), polyethylene glycol 400 (PEG 400), and Tween 80 on the viability (transepithelial electrical resistance, TEER) of 3-day cultured Caco-2 monolayers were evaluated. These solubilizers, even at the low concentration, reduce the viability of Caco-2 monolayers; these results indicate the impossibility for 3-day cultured Caco-2 monolayers to be used for this test. Next, the effects of PG, Tween 80, PEG 400, HP-β-CD, Pluronic F-68 (Pluronic), HCO-40, sodium lauryl sulfate (SLS), Gelucire 44/14, Transcutol P, and extract gall powder on the viability of 21-day cultured Caco-2 monolayers and the apparent permeability (Papp) of propranolol (PPL), Nadolol (NDL), and FITC-dextran 4000 (FD-4) were investigated. Five different solubilizing methods (20% PG, 5% Tween 80, 5% PEG 400, 5% HP-β-CD, and 5% Tween 80+5% PEG 400) did not affect the viability of 21-day cultured Caco-2 monolayers. Furthermore, the Papp values of the three compounds containing these solubilizers did not differ from the values for control formulations (without solubilizers). These results clearly suggest that the use of PG, Tween 80, PEG 400, or HP-β-CD as solubilizing excipients and the testing of these formulations on 21-day cultured Caco-2 monolayers can predict intestinal absorption of poorly water-soluble drugs in humans. [Copyright &y& Elsevier]

To examine whether the dietary lectin wheat germ agglutinin (WGA) can facilitate binding and uptake of protein drugs due to its cytoadhesive and cytoinvasive properties, conjugates were prepared by covalent coupling of fluorescein-labeled bovine serum albumin (F-BSA) to WGA using divinylsulfone for crosslinking. Increasing the molar ratio of F-BSA/WGA resulted in 2.6–8.7 times higher Caco-2 binding as compared with glycyl-F-BSA. About 75% of F-BSA-WGA were bound specifically to Caco-2 cells according to inhibition studies in presence of the complementary carbohydrate. The Caco-2 association of F-BSA-WGA was temperature-dependent indicating active uptake of membrane bound conjugate, which was confirmed by confocal microscopy. The conjugate accumulated within lysosomal compartments followed by proteolytic degradation of F-BSA-WGA 1–4 h after conjugate loading as observed by equilibrating the intracellular pH with monensin. Finally low molecular weight degradation products of the proteinaceous prodrug appear in the extracellular medium. Contrary to Caco-2 single cells, a minor part of the conjugate is degraded by brush border proteases already 30 min after exposure to Caco-2 monolayers. But most of the conjugate is taken up into differentiated cells and processed as in single cells. Though the enzymic barrier remains to be surmounted, WGA-mediated drug delivery is a promising strategy for peroral delivery of even high molecular weight drugs to overcome the mucosal barrier. [Copyright &y& Elsevier]

The aim of this study was to determine the intestinal absorption characteristics of the antiviral agent UC-781 and to optimize the experimental conditions of the in vitro system for low solubility compounds. The absorption potential of UC-781 was studied with the Caco-2 system and with the rat intestinal perfusion technique. The low solubility of UC-781 required the use of solubility/dissolution rate enhancing agents (e.g. VitE-TPGS, Gelucire 44/14). The creation of sink conditions in the receiver compartment of the Caco-2 system was a prerequisite to reliably study the transport of this poorly soluble compound. After inclusion of VitE-TPGS in the acceptor solution, UC-781 could be characterized as a class II drug of the Biopharmaceutical Classification System (low solubility, high permeation across membranes). A significant concentration-dependent decrease in transport of UC-781 was observed upon increasing the concentration of VitE-TPGS in the apical compartment. This observation contrasts to the absorption enhancing properties of VitE-TPGS, and can probably be attributed to a decrease in the concentration of free UC-781 when using higher concentrations of the solubility/dissolution rate enhancing agents. The use of Gelucire 44/14 as a solubilizing agent resulted in a batch-dependent degradation of UC-781. The inclusion of the solubility/dissolution rate-enhancing agent VitE-TPGS did not result in absorption enhancement in the intestinal perfusion technique. [Copyright &y& Elsevier]

Abstract: The aim of this work was to investigate the effects of pluronic F68 block copolymer on the P-gp-mediated transport of celiprolol (CEL) and CYP3A4-mediated formation of midazolam (MDZ) metabolite 1′-hydroxymidazolam. Over a range from 0.03 to 0.3%, pluronic F68 increased apical-to-basolateral permeability (AP-BL) and decreased basolateral-to-apical permeability (BL-AP) of the P-gp substrate CEL in Caco-2 cell monolayer with the efflux ratio values of 2.8±0.3 (0.03%), 2.6±0.3 (0.1%), 2.3±0.2 (0.3%), respectively. CEL transport across the intestinal mucosa in the everted gut sac model was also enhanced by the P-gp inhibitor verapamil and the pharmaceutical excipient pluronic F68. Furthermore, CYP3A4-catalyzed formation of 1′-hydroxymidazolam was inhibited by pluronic F68 with IC50 and K i values of 0.11 and 0.16mg/ml, respectively. The results indicate that pluronic F68 is a potent in vitro inhibitor of both P-gp and CYP3A4, suggesting a potential for pluronic F68 to modify the pharmacokinetics of orally administered drugs that are P-gp and/or CYP3A4 substrates in vivo. [Copyright &y& Elsevier]