Using a triphasic model to describe the permeation of dimethyl sulfoxide in porcine corneoscleral discs.

Corneal blindness can be treated by keratoplasty but a lack of readily available corneal donor tissue for this procedure remains a challenge. Cryopreservation can facilitate the long-term storage of tissue but effective protocols for cryopreserving cornea have yet to be developed. Mathematical modelling can guide protocol design, but previously used models are not comprehensive. A comprehensive model should describe the tissue's shrink-swell response and the cryoprotectant concentration throughout the tissue during cryoprotectant loading. Such a model exists for articular cartilage based on a biomechanical triphasic approach. We explored the applicability of this model for describing cryoprotectant permeation in porcine corneas by fitting it to experimental data for the permeation of dimethyl sulfoxide into porcine corneoscleral discs. The model provided as good of a fit for corneoscleral discs data as it did for articular cartilage data, presenting promise for its use in the design of cryopreservation protocols for corneas.

Consideration of Fractional Distribution Parameter fd in the Chen and Gross Method for Tissue-to-Plasma Partition Coefficients: Comparison of Several Methods.

PURPOSE: The tissue-to-plasma partition coefficient (Kp) describes the extent of tissue distribution in physiologically-based pharmacokinetic (PBPK) models. Constant-rate infusion studies are common for experimental determination of the steady-state Kp,ss, while the tissue-plasma concentration ratio (CT/Cp) in the terminal phase after intravenous doses is often utilized. The Chen and Gross (C&G) method converts a terminal slope CT/Cp to Kp,ss based on assumptions of perfusion-limited distribution in tissue-plasma equilibration. However, considering blood flow (QT) and apparent tissue permeability (fupPSin) in the rate of tissue distribution, this report extends the C&G method by utilizing a fractional distribution parameter (fd). METHODS: Relevant PBPK equations for non-eliminating and eliminating organs along with lung and liver were derived for the conversion of CT/Cp values to Kp,ss. The relationships were demonstrated in rats with measured CT/Cp and Kp,ss values and the model-dependent fd for 8 compounds with a range of permeability coefficients. Several methods of assessing Kp were compared. RESULTS: Utilizing fd in an extended C&G method, our estimations of Kp,ss from CT/Cp were improved, particularly for lower permeability compounds. However, four in silico methods for estimating Kp performed poorly across tissues in comparison with measured Kp values. Mathematical relationships between Kp and Kp,ss that are generally applicable for eliminating organs with tissue permeability limitations necessitates inclusion of an extraction ratio (ER) and fd. CONCLUSION: Since many different types/sources of Kp are present in the literature and used in PBPK models, these perspectives and equations should provide better insights in measuring and interpreting Kp values in PBPK.

Effects of Physicochemical Properties of Constituent Ions of Ionic Liquid on Its Permeation through a Silicone Membrane.

Ionic liquids (ILs), defined as liquid salts composed of anions and cations, have the advantage of allowing constituent ions to be stably absorbed through biological membranes, such as skin. However, limited information is currently available on the effects of the physicochemical properties of constituent ions on the membrane permeation of ILs. Therefore, we herein investigated the effects of the polarity of constituent cations on the membrane permeation of each constituent ion from IL. Various ILs were prepared by selecting lidocaine (LID) as a cation and a series of p-alkylbenzoic acids with different n-octanol/water partition coefficients (Ko/w) as anions. These ILs were applied to a skin model, a silicone membrane, and membrane permeability was investigated. The membrane permeabilities of p-alkylbenzoic acids from their single aqueous suspensions were also measured for comparison. The membrane permeability of p-alkylbenzoic acid from the aqueous suspension increased at higher Ko/w. However, the membrane permeability of ILs was similar regardless of the Ko/w of the constituent p-alkylbenzoic acid. Furthermore, the membrane permeability of the counterion LID remained unchanged regardless of the constituent p-alkylbenzoic acid. These results suggest that even when the Ko/w of IL constituents markedly differs, the resulting IL does not affect membrane permeability.

Evaluation of the Influence of Astrocytes on In Vitro Blood-Brain Barrier Models.

In vitro blood-brain barrier (BBB) models are a useful tool to screen the permeability and toxicity of new drugs. Currently, many different in vitro BBB models coexist, but none stands out as being notably better than the rest. Therefore, there is still a need to evaluate the quality of BBB models under various conditions and assess their ability to mimic the in vivo situation. In this study, two brain endothelial cell lines (bEnd.3 and hCMEC/D3) and two epithelial-like cell lines (MDCKII and Caco-2) were selected for BBB modelling purposes. They were grown as monolayers of a single cell type, under the following conditions: in coculture with either primary or immortalised astrocytes; or in the presence of primary or immortalised astrocyte-derived conditioned media. A total of 20 different BBB models were established in this manner, in order to assess the effects of the astroglial components on the BBB phenotype in each case. To this end, six parameters were studied: the expression of selected tight junction proteins; the enzyme activities of alkaline phosphatase and of gamma glutamyl transpeptidase; the transendothelial/transepithelial electrical resistance (TEER); restriction in paracellular transport; and efflux transporter inhibition were each evaluated and correlated. The results showed that coculturing with either primary or immortalised astrocytes led to a general improvement in all parameters studied, evidencing the contribution of this cell type to effective BBB formation. Furthermore, the permeability coefficient (P e) of the tracer molecule, Lucifer Yellow, correlated with three of the six parameters studied. In addition, this study highlights the potential for the use of the Lucifer Yellow P e value as an indicator of barrier integrity in in vitro BBB models, which could be useful for screening the permeability of new drugs.

In Silico Prediction of PAMPA Effective Permeability Using a Two-QSAR Approach.

Oral administration is the preferred and predominant route of choice for medication. As such, drug absorption is one of critical drug metabolism and pharmacokinetics (DM/PK) parameters that should be taken into consideration in the process of drug discovery and development. The cell-free in vitro parallel artificial membrane permeability assay (PAMPA) has been adopted as the primary screening to assess the passive diffusion of compounds in the practical applications. A classical quantitative structure-activity relationship (QSAR) model and a machine learning (ML)-based QSAR model were derived using the partial least square (PLS) scheme and hierarchical support vector regression (HSVR) scheme to elucidate the underlying passive diffusion mechanism and to predict the PAMPA effective permeability, respectively, in this study. It was observed that HSVR executed better than PLS as manifested by the predictions of the samples in the training set, test set, and outlier set as well as various statistical assessments. When applied to the mock test, which was designated to mimic real challenges, HSVR also showed better predictive performance. PLS, conversely, cannot cover some mechanistically interpretable relationships between descriptors and permeability. Accordingly, the synergy of predictive HSVR and interpretable PLS models can be greatly useful in facilitating drug discovery and development by predicting passive diffusion.

Aceclofenac-Galactose Conjugate: Design, Synthesis, Characterization, and Pharmacological and Toxicological Evaluations.

Aceclofenac is a popular analgesic, antipyretic, and nonsteroidal anti-inflammatory drug (NSAID) used for prolonged treatment (at least three months) in musculoskeletal disorders. It is characterized by several limitations such as poor water solubility and low oral bioavailability. The main side-effect of aceclofenac, as well as all NSAIDs, is the gastrotoxicity; among other adverse effects, there is the risk of bleeding since aceclofenac reversibly inhibits platelet aggregation. With the aim to reduce these drawbacks, we have designed, synthesized, and characterized, both in vitro and in vivo, an orally administrable pro-drug of aceclofenac (ACEgal). ACEgal was obtained by conjugating carboxyl group with the 6-OH group of d-galactose; its structure was confirmed by X-ray powder diffractometry. The pro-drug was shown to be stable at 37 °C in simulated gastric fluid (SGF-without pepsin, pH = 1.2) and moderately stable in phosphate buffered saline (PBS, pH = 7.4). However, it hydrolyzed in human serum with a half-life ( t1/2) of 36 min, producing aceclofenac. Furthermore, if compared to its parent drug, ACEgal was four-times more soluble in SGF. To predict human intestinal absorption, cell permeability in a Caco-2 model of aceclofenac and ACEgal was determined. Anti-inflammatory, analgesic, and ulcerogenic activities have been investigated in vivo. In addition, oxidative stress parameters (thiobarbituric acid reactive substances, TBARS, and glutathione, GSH) and platelet antiaggregatory activity both of parent drug and pro-drug were evaluated. Results clearly showed that the conjugation of aceclofenac to a galactose molecule improves physicochemical, toxicological (at gastric and blood level), and pharmacological profile of aceclofenac itself without changing intestinal permeability and antiplatelet activity (in spite the new sugar moiety).

Another interpretation of the Goldman-Hodgkin-Katz equation based on Ling's adsorption theory.

According to standard membrane theory, the generation of membrane potential is attributed to transmembrane ion transport. However, there have been a number of reports of membrane behavior in conflict with the membrane theory of cellular potential. Putting aside the membrane theory, we scrutinized the generation mechanism of membrane potential from the view of the long-dismissed adsorption theory of Ling. Ling's adsorption theory attributes the membrane potential generation to mobile ion adsorption. Although Ling's adsorption theory conflicts with the broadly accepted membrane theory, we found that it well reproduces experimentally observed membrane potential behavior. Our theoretical analysis finds that the potential formula based on the GHK eq., which is a fundamental concept of membrane theory, coincides with the potential formula based on Ling's adsorption theory. Reinterpreting the permeability coefficient in the GHK eq. as the association constant between the mobile ion and adsorption site, the GHK eq. turns into the potential formula from Ling's adsorption theory. We conclude that the membrane potential is generated by ion adsorption as Ling's adsorption theory states and that the membrane theory of cellular potential should be amended even if not discarded.

Evaluation on the reliability of the permeability coefficient (Kp) to assess the percutaneous penetration property of chemicals on the basis of Flynn's dataset.

PURPOSE: The permeability coefficient (Kp) is often used for prediction of the dermal penetration of chemicals. Mathematical models have mostly been derived on Kp data basis. However, confusing Kp values are reported, questioning the general reliability of this parameter. In this study, we tested the plausibility of Kp values expressing the dermal penetration velocity (cm h-1) of chemicals on a larger dataset from literature. METHODS: Kp was applied for the calculation of the time for penetration through skin membranes of defined thickness (tCrossSkin). Kp values were obtained from Flynn's dataset (1990), containing data determined mostly under similar experimental conditions using diffusion cells. Further skin penetration parameters, e.g., times at which the chemicals were firstly measured in the receptor phase, lag times, steady-state times, and exposure duration, where available, were related to Kp values. The data congruence was tested comparing Kp values from Flynn's dataset with those reported in the EDETOX database. Variables, which could bias the results, such as different experimental protocols and research groups were also considered. RESULTS: Kp data for 94 chemicals matched the inclusion criteria were evaluated. According to the Kp values, 21 (22%) compounds would require longer than 100 h, and 20 (21%) further compounds longer than 10 h of exposure to penetrate skin membranes of ~ 0.01-2.5 mm thickness. Obviously, erroneous Kp were found in studies of almost all research groups in Flynn's database, indicating that neither the observer nor the experimental conditions alone biased the values. CONCLUSIONS: Our evaluation demonstrates high implausibility of Kp values to represent the dermal penetration velocity and supports general invalidity of the parameter for implementation in studies using skin membranes. The Kp should not be used to characterize the percutaneous penetration of chemicals or in risk assessment without verification.

MDCKpred: a web-tool to calculate MDCK permeability coefficient of small molecule using membrane-interaction chemical features.

Structure-based models to understand the transport of small molecules through biological membrane can be developed by enumerating intermolecular interactions of the small molecule with a biological membrane, usually a dimyristoylphosphatidylcholine (DMPC) monolayer. This ADME (absorption, distribution, metabolism, and excretion) property based on Madin-Darby Canine Kidney (MDCK) cell line demonstrates intestinal drug absorption of small molecules and correlated to human intestinal absorption which acts as a determining factor to forecast small-molecule prioritization in drug-discovery projects. We present here the development of MDCKpred web-tool which calculates MDCK permeability coefficient of small molecule based on the regression model, developed using membrane-interaction chemical features. The web-tool allows users to calculate the MDCK permeability coefficient (nm/s) instantly by providing simple descriptor inputs. The chemical-interaction features are derived from different parts of the DMPC molecule viz. head, middle, and tail regions and accounts overall intermolecular contacts of the small molecule when passively diffused through the phospholipid-rich biological membrane. The MDCKpred model is both internally (R2 = .76; [Formula: see text]= .68; Rtrain = .87; Rtest = .69) and externally (Rext = .55) validated. Furthermore, we used natural molecules as application examples to demonstrate its utility in lead exploration and optimization projects. The MDCKpred web-tool can be accessed freely at http://www.mdckpred.in . This web-tool is designed to offer an intuitive way of prioritizing small molecules based on calculated MDCK permeabilities.

Effects of Dendrimer-Like Biopolymers on Physical Stability of Amorphous Solid Dispersions and Drug Permeability Across Caco-2 Cell Monolayers.

The potential applications of dendrimer-like biopolymers (DLB) as stabilizing excipients for amorphous solid dispersion (ASD) of niclosamide, celecoxib, and resveratrol were evaluated based on (1) the formation and physical stability of the ASD and (2) the permeability and flux of the agents across Caco-2 cell monolayers. The evaluation was made by comparing the performance of prototype phytoglycogen derivatives (DLB1, DLB2, and DLB3) with commonly used polymers such as HPMCAS, PVPVA, and Soluplus®. PXRD was used to confirm the formation of the dispersions and detect crystallinity peaks formed during 2- and 4-week storage at 40°C/75% RH. At concentrations below 2 g/mL, the viability of Caco-2 cells remained above 80% for all DLB samples compared to untreated cells in the MTT assay. Permeability studies revealed a repeating pattern in which an increase in the initial concentration (C0) was associated with a concomitant decrease in the apparent permeability (Papp) which we theorize is due to differences in drug-polymer interactions. Niclosamide-DLB1 dispersion had the lowest flux due to a significant reduction in Papp. The high increase in the C0 of celecoxib-DLB2, however, made up for the reduction in the Papp and produced the highest flux values compared to other polymers. Resveratrol-DLB3 had a 5× reduction in Papp, but C0 increased from 25.8 to 176 µg/mL led to a higher flux compared to the crystalline drug without polymer. Collectively, these results provide a "proof-of-concept" basis to demonstrate that DLB excipients have the ability to increase apparent solubility (Solapp), most likely due to drug-binding capacity.

Influence of effective stress and dry density on the permeability of municipal solid waste.

A landfill is one of the main sites for disposal of municipal solid waste and the current landfill disposal system faces several problems. For instance, excessive leachate water is an important factor leading to landfill instability. Understanding the permeability characteristics of municipal solid waste is a relevant topic in the field of environmental geotechnical engineering. In this paper, the current research progress on permeability characteristics of municipal solid waste is discussed. A review of recent studies indicates that the research in this field is divided into two categories based on the experimental method employed: field tests and laboratory tests. This paper summarizes test methods, landfill locations, waste ages, dry densities and permeability coefficients across different studies that focus on permeability characteristics. Additionally, an experimental study on compressibility and permeability characteristics of fresh municipal solid waste under different effective stresses and compression times was carried out. Moreover, the relationships between the permeability coefficient and effective stress as well as dry density were obtained and a permeability prediction model was established. Finally, the experimental results from the existing literature and this paper were compared and the effects of effective stress and dry density on the permeability characteristics of municipal solid waste were summarized. This study provides the basis for analysis of leachate production in a landfill.

Elucidation of Compatibility Interactions of Traditional Chinese Medicines: In Vitro Absorptions Across Caco-2 Monolayer of Coptidis Rhizoma and Euodiae Fructus in Zuojin and Fanzuojin Formulas as A Case.

Traditional Chinese medicines are often combined as formulae and interact with each other. As for Coptidis Rhizoma (CR) and Euodiae Fructus (EF), the most classical compatibilities were Zuojin (ZJF) and Fanzuojin formulas (FZJF) with reverse mixture ratios and opposite effects. To compare in vitro absorption interactions between CR and EF, bidirectional transports across Caco-2 cell monolayer of extracts of two formulas and equivalent single herbs were studied. Eighteen alkaloids from CR and EF were determined by liquid chromatography coupled to tandem mass spectrometry. Parameter apparent permeability coefficient (Papp ) and efflux rate (ER) values showed that most alkaloids were well or moderately absorbed and six quaternary protoberberine alkaloids from CR had obvious efflux. ZJF compatibilities reduced both Papp BL→AP and ER values of three indole alkaloids, and increased ER values of two quinolone alkaloids from EF. FZJF compatibilities obviously affected the bidirectional Papp values of CR alkaloids, weakened ERs of five protoberberines from CR and enlarged ERs of two quinolones from EF. Conclusions were drawn that different compatibility ratios of CR and EF led to different interactions on the in vitro absorption of alkaloids. The results may provide a good reference for interaction studies on the compatibilities of traditional Chinese medicines. Copyright © 2017 John Wiley & Sons, Ltd.

New doxorubicin nanoparticles engineered from calcium-crosslinked carbomer and a microemulsion precursor.

The purpose of this study was to investigate a new polymeric system and production process in which self-assembled doxorubicin-loaded nanoparticles were synthetized by using a water-in-oil microemulsion as a template and calcium ions as cross-linkers. The manufacturing process combined cross-linking of carbomer within a W/O microemulsion followed by a phase-separation technique to avoid using organic solvents for extraction. To assess the sustained release behavior of doxorubicin from the nanoparticles, we have developed a new simple method based on the permeability coefficient of a synthetic membrane mounted on Franz diffusion cell system. Franz cells were preferred over the commonly used dialysis tubing because they provide adequate measures of the diffusion area as well as the volumes of the media in both sides of the membrane. The lower permeability values obtained for nanoparticles have shown that the release is a limiting step of the diffusion process, while the calculated straight lines may imply that the apparent release rate of the nanoparticle ensembles is close to a zero-order kinetics. The new drug release method for the evaluation of nano-carriers, utilizing a simple linear model to determine the permeability coefficient, has been proposed for perfect sink and non-sink conditions.

Enhanced Intestinal Permeability of Bufalin by a Novel Bufalin-Peptide-Dendrimer Inclusion through Caco-2 Cell Monolayer.

Bufalin (BFL) has excellent physiological activities such as defending tumors, improving cardiac function, and so on. However, due to its poor water-solubility and bioavailability, the clinical application of BFL remains limited. In order to improve bioavailability of BFL, in our previous research, a novel peptide-dendrimer (PD) was synthesized and applied to encapsulate BFL. In the present study, we investigate the absorption property and mechanism of BFL in free form and BFL-peptide-dendrimer inclusion (BPDI) delivery system by using the Caco-2 cell monolayer model in vitro. The apparent permeability coefficient (Papp) values of BFL in free or BPDI form were over 1.0 × 10-6 cm/s. Meanwhile, their almost equal bi-directional transport and linear transport percentage with time and concentration course indicated that BFL in both forms was absorbed mainly through passive diffusion. The most important result is that the Papp values of BFL increased about three-fold more BPDI than those of its free form, which indicated the intestinal permeability of BFL could be improved while BFL was encapsulated in BPDI form. Therefore, PD encapsulation may be a potential delivery system to increase the bioavailability of BFL.

Carbon nanotube membranes to predict skin permeability of compounds.

In the present study, carbon nanotube (CNT) membranes were prepared to predict skin penetration properties of compounds. A series of penetration experiments using Franz diffusion cells were performed with 16 different membrane compositions for model chemicals. Similar experiments were also carried out with same model molecules using five different commercially available synthetic membranes and human skins for the comparison. Model chemicals were selected as diclofenac, dexketoprofen and salicylic acid. Their permeability coefficients and flux values were calculated. Correlations between permeability values of model compounds for human skins and developed model membranes were investigated. Good correlations were obtained for CNT membrane, isopropyl myristate-treated CNT membrane (IM-CNT membrane) and bovine serum albumin-cholesterol, dipalmitoyl phosphatidyl choline-treated membrane (BSA-Cholesterol-DPPC-IM-CNT membrane). An artificial neural network (ANN) model was developed using some molecular properties and penetration coefficients from pristine CNT membranes to predict skin permeability values and quite good predictions were made.

Hybrid polymeric matrices for oral modified release of Desvenlafaxine succinate tablets.

Purpose: Desvenlafaxine succinate (DSV) is a water soluble anti-depressant drug, which is rapidly absorbed after oral administration exaggerating its side effects. The current work aimed to prepare controllable release DSV matrix to reduce DSV side effects related to its initial burst. Methods: Fifteen DSV matrix formulations were prepared using different polymers, polymer/drug ratios and matrix excipients and characterized using Differential Scanning Calorimetry (DSC), infrared (IR) spectroscopy, water uptake and in vitro DSV release. The release kinetics were calculated to determine the drug release mechanism. Ex-vivo DSV absorption via rat intestinal mucosal cells and the calculation of the apparent permeability coefficient (Papp) were performed using everted sac technique. Results: Maltodextrin was the best matrix excipient (F7 and F10) showing acceptable decrease in the initial burst compared to the innovator. The addition of negatively charged polymers sodium carboxy methyl cellulose (SCMC) or sodium alginate resulted in an interaction that was proved by DSC and IR findings. This interaction slowed DSV release. F10 showed an excellent absorption of more than 80% of DSV after 4 h and the highest similarity factor with the innovator (84.7). Conclusion: A controllable release pattern of DSV was achieved using Methocel, Maltodextrin and SCMC. The obtained results could be used as a platform to control the release of cationic water soluble drugs that suffer from side effects associated with their initial burst after oral administration.

Association of Permeability and Lipid Content of Membrane.

BACKGROUND: Rat skin and goat cul de sac are mostly used in optimization of formulations as the model of human skin and cul de sac. AIM: To explore the correlation between lipid content of rat skin and goat cul de sac and permeability. MATERIALS AND METHODS: Find out wavelength maximum for Sapat plus malam®, Ciplox eye ointment® and chloramphenicol eye caps and the standard curve was also derived. In vitro studies using Cellophane® membrane and ex vivo studies using rat skin or goat cul de sac of the formulations. Permeability coefficient, % dislodgeable dose, lag time, diffusion parameter, and partition coefficient were found for both studies after six and a half hours of penetration studies. Student's unpaired t-test with equal variance was used to find any statistically significant difference in the ex vivo and in vitro diffusion transport studies at 95% level of confidence. RESULTS: Permeability coefficient of Sapat plus malam®, Ciplox eye ointment® and chloramphenicol eye caps were 0.000316 ± 0.0000625, 0.00416 ± 0.0001, 0.0034 ± 0.00004 for Cellophane® membrane and 0.0001 ± 0.000001, 0.002254 ± 0.0002, 0.00303 ± 0.0001 for ex vivo membrane in cm2/min, respectively. For all three formulations, there were calculated t values which were higher than tabulated t values at 95% of confidence level (P<0.05). CONCLUSION: Cellophane® membrane shows a better diffusion than rat skin or goat cul de sac. In the optimization of formulation, only Cellophane® membrane is advisable to use.

[Absorption and transport of isoflavonoid compounds from Tongmai formula across human intestinal epithelial (Caco-2) cells in vitro].

Tongmai formula (TMF) is a drug combination of three components including Puerariae Lobatae Radix [roots of Pueraria lobata], Salviae Miltiorrhizae Radix (roots of Salvia miltiorrhiza) and Chuanxiong Rhizoma (rhizomes of Ligusticum chuanxiong) in a weight ratio of 1∶1∶1. The absorption and transport of isoflavonoid compounds from Tongmai formula across human intestinal epithelial (Caco-2) cells in vitro were studied in this paper. The assay isoflavonoid compounds include daidzein, formononetin, 5-hydroxylononin, ononin, daidzin, 3'-methoxypuerarin, genistin, puerarin, formononetin-8-C-ß-D-apiofuranosyl-(1→6)-O-ß-D-glucopyranoside, formononetin-7-O-ß-D-apiofuranosyl-(1→6)-O-ß-D-glucopyranoside, lanceolarin, kakkanin, daidzein-7,4'-di-O-ß-D-glucopyranoside, mirificin, 3'-hydroxypuerarin, 3'-methoxydaidzin, formononetin-8-C-ß-D-xylopyranosyl-(1→6)-O-ß-D-glucopyranoside, genistein-8-C-ß-D-apiofuranosyl-(1→6)-O-ß-D-glucopyranoside, genistein-7-O-ß-D-apiofuranosyl-(1→6)-O-ß-D-glucopyranoside (ambocin), 3'-hydroxymirificin, 6″-O-ß-D-xylosylpuerarin, biochanin A-8-C-ß-D-apiofuranosyl-(1→6)-O-ß-D-glucopyranoside, 3'-methoxydaidzein-7,4'-di-O-ß-D-glucopyranoside, daidzein-7-O-ß-D-glucopyranosyl-(1→4)-O-ß-D-glucopyranoside, and daidzein-7-O-α-D-glucopyranosyl-(1→4)-O-ß-D-glucopyranoside. By using human Caco-2 monolayer as an intestinal epithelial cell model in vitro, the permeability of above-mentioned 25 isoflavonoids in TMF were studied from the apical (AP) side to basolateral (BL) side or from the BL side to AP side. The assay compounds were determined by reversed phased high-performance liquid chromatography (HPLC) coupled with UV detector. Transport parameters and apparent permeability coefficients (Papp) were then calculated and and compared with those of propranolol and atenolol, which are the transcellular transport marker and as a control substance for high and poor permeability, respectively. The Papp values of daidzein and formononetin were (2.55±0.03) ×10⁻5,(3.06±0.01) ×10⁻5 cm•s⁻¹ from AP side to BL side, respectively, and (2.62±0.00) ×10⁻5, (2.65±0.11) ×10⁻5 cm•s⁻¹ from BL side to AP side, respectively. Under the condition of this experiment, the Papp value was (2.66±0.32) ×10⁻5 cm•s⁻¹ for propranolol and (2.34±0.10) ×10⁻7 cm•s⁻¹ for atenolol. The Papp values of daidzein and formononetin were at a same magnitude with those of propranolol. And the Papp values of other 23 isoflavonoid compounds were at a same magnitude with those of atenolol. On the other hand, the rats of Papp AP→BL/Papp BL→AP of daidzein and formononetin on the influx transport were 0.97 and 1.15, respectively. It can be predicted that daidzein and formononetin can be absorbed across intestinal epithelial cells to go to the body circulation by the passive diffusion mechanism and they were assigned to the well-absorbed compounds. Other 23 isoflavonoid compounds were assigned to the poorly absorbed compounds. Because of the rats of Papp AP→BL/Papp BL→AP of 5-hydroxylononin, genistin, lanceolarin, kakkanin, and genistein-7-O-ß-D-apiofuranosyl-(1→6)-O-ß-D-glucopyranoside were 0.18, 0.28, 0.45, 0.38, 0.49, they may have been involved in the efflux mechanism in Caco-2 cells monolayer model from the BL side to AP side direction.

Transport and metabolism of fumaric acid in Saccharomyces cerevisiae in aerobic glucose-limited chemostat culture.

Currently, research is being focused on the industrial-scale production of fumaric acid and other relevant organic acids from renewable feedstocks via fermentation, preferably at low pH for better product recovery. However, at low pH a large fraction of the extracellular acid is present in the undissociated form, which is lipophilic and can diffuse into the cell. There have been no studies done on the impact of high extracellular concentrations of fumaric acid under aerobic conditions in S. cerevisiae, which is a relevant issue to study for industrial-scale production. In this work we studied the uptake and metabolism of fumaric acid in S. cerevisiae in glucose-limited chemostat cultures at a cultivation pH of 3.0 (pH < pK). Steady states were achieved with different extracellular levels of fumaric acid, obtained by adding different amounts of fumaric acid to the feed medium. The experiments were carried out with the wild-type S. cerevisiae CEN.PK 113-7D and an engineered S. cerevisiae ADIS 244 expressing a heterologous dicarboxylic acid transporter (DCT-02) from Aspergillus niger, to examine whether it would be capable of exporting fumaric acid. We observed that fumaric acid entered the cells most likely via passive diffusion of the undissociated form. Approximately two-thirds of the fumaric acid in the feed was metabolized together with glucose. From metabolic flux analysis, an increased ATP dissipation was observed only at high intracellular concentrations of fumarate, possibly due to the export of fumarate via an ABC transporter. The implications of our results for the industrial-scale production of fumaric acid are discussed.

Validation of an immortalized human (hBMEC) in vitro blood-brain barrier model.

We recently established and optimized an immortalized human in vitro blood-brain barrier (BBB) model based on the hBMEC cell line. In the present work, we validated this mono-culture 24-well model with a representative series of drug substances which are known to cross or not to cross the BBB. For each individual compound, a quantitative UHPLC-MS/MS method in Ringer HEPES buffer was developed and validated according to current regulatory guidelines, with respect to selectivity, precision, and reliability. Various biological and analytical challenges were met during method validation, highlighting the importance of careful method development. The positive controls antipyrine, caffeine, diazepam, and propranolol showed mean endothelial permeability coefficients (P e) in the range of 17-70 × 10(-6) cm/s, indicating moderate to high BBB permeability when compared to the barrier integrity marker sodium fluorescein (mean P e 3-5 × 10(-6) cm/s). The negative controls atenolol, cimetidine, and vinblastine showed mean P e values < 10 × 10(-6) cm/s, suggesting low permeability. In silico calculations were in agreement with in vitro data. With the exception of quinidine (P-glycoprotein inhibitor and substrate), BBB permeability of all control compounds was correctly predicted by this new, easy, and fast to set up human in vitro BBB model. Addition of retinoic acid and puromycin did not increase transendothelial electrical resistance (TEER) values of the BBB model.