Intestinal Perfusion Experiments Research Articles

Nanocrystal-Loaded Lipid Carriers for Improved Oral Absorption and Anticancer Efficacy of Etoposide: Formulation Development, Transport Mechanism, In Vitro and In Vivo Evaluation.

To improve the oral absorption and anticancer efficacy of the BCS-IV drug etoposide (ETO), oral nanocrystal-loaded lipid carriers (Lipo@NCs) were developed in this study by modifying the BCS-IV drug nanocrystal with the lipid bilayer. The ETO-Lipo@NCs were prepared by the thin film hydration high-pressure homogenization method, and the core of positively charged ETO nanocrystals was prepared by the sonoprecipitation-high pressure homogenization method. The optimized ETO-Lipo@NCs were spherical particles with an average particle size of 220.3 ± 14.2 nm and a zeta potential of -9.95 ± 0.81 mV, respectively. The successful coating of a lipid bilayer on the surface of nanocrystals in ETO-Lipo@NCs was confirmed by several characterization methods. Compared to nanocrystals, the release rate and degree of Lipo@NCs in SIF were significantly decreased, indicating that the lipid bilayer can effectively prevent the rapid dissolution of core nanocrystals. ETO-Lipo@NCs demonstrated a significant improvement in the intestinal permeability and absorption of ETO in a single intestinal perfusion experiment. In the cells, ETO-Lipo@NCs showed enhanced cellular uptake and transepithelial transport compared with ETO nanocrystals. Pharmacokinetic analysis indicated that ETO-Lipo@NCs had a longer plasma half-life than ETO solution, and the oral bioavailability of ETO-Lipo@NCs was 1.96- and 10.92-fold higher than that of ETO nanocrystals and ETO coarse crystals, respectively. Moreover, the ETO-Lipo@NCs orally dosed at 10 mg/kg exhibited an excellent inhibitory effect against tumors in a subcutaneous Lewis lung carcinoma (LLC) xenograft model compared with other preparations. These results indicate that the Lipo@NCs formulation has an oral absorption-promoting effect of the BCS-IV drug ETO, which could warrant further application in the oral delivery of other poorly bioavailable drugs.

D-allulose reduces postprandial glucose absorption in small intestine by competitive binding to glucose transport sites

d-allulose, a C3-epimer of d-fructose, is a naturally occurring non-metabolic sugar found in small amounts. Since d-allulose can inhibit the intestinal absorption of glucose,it is necessary to investigate the role of the sodium-coupled glucose cotransporter 1 (SGLT1) in intestinal transport of d-allulose. In this study, the transcellular pathway of d-allulose in small intestinal epithelial cells was investigated by constructing a monolayer model of Caco-2 cells and in situ single-pass intestinal perfusion experiment. We observed a notable decrease in the permeation rate of d-allulose with the addition of phlorizin, a SGLT1 inhibitor, and that phloretin, a GLUT2 inhibitor, leads to a moderate decrease in the permeation rate. This suggests that both SGLT1 and GLUT2 can mediate the transport of d-allulose in small intestine. These observations were supported by computer molecular simulation. Additionally, the mRNA abundance of sugar transporters in Caco-2 cells treated with d-allulose were decreased, determined by quantitative real-time PCR. This study suggests that d-allulose can competitively bind to the sugar transport sites and reduce the mRNA abundance of sugar transporter proteins within the small intestine to effect postprandial glucose absorption.

Effects of schisandra lignans on the absorption of protopanaxadiol-type ginsenosides mediated by P-glycoprotein and protopanaxatriol-type ginsenosides mediated by CYP3A4

Ethnopharmacological relevanceGinseng Radix et Rhizoma (GRR) and Schisandrae Chinensis Fructus (SCF) are frequently used as herb pairs in traditional herbal formulas especially for the synergetic beneficial effects on lung and heart. Shengmai-yin (SMY), a noted formula, was first published in the traditional Chinese medicine classic named Yixue Qiyuan written by Zhang Yuansu in the Jin Dynasty, and has been used for deficiency of both qi and yin, palpitation, shortness of breath and spontaneous sweating. In SMY, GRR, a sovereign herb, plays an essential role in tonifying lung and supplementing qi, and SCF as an adjuvant herb contributes to the effects of nourishing yin and promoting fluid production, both of which are traditionally used as invigorants in China, Korea, Japan, and Russia. However, the underlying compatibility mechanism of GRR-SCF has remained unknown. Aim of the studyIn order to explore the impact and underlying mechanism of schisandra chinensis extract (SCE) on the absorption of ginsenosides Rb1, Rc, Rb2 and Rd belonging to protopanaxdiol (PPD)-type and ginsenosides Rg1 and Re belonging to protopanaxtriol (PPT)-type, pharmacokinetic studies, molecular docking technique and single-pass intestinal perfusion (SPIP) experiment were conducted. Material and methodsPreliminarily, pharmacokinetic characteristics of ginseng extract (GE) in the presence and absence of SCE were studied. Thereafter, molecular docking was used to predict whether ginsenosides were P-glycoprotein (P-gp) or cytochrome P450 isoenzyme 3A4 (CYP3A4) substrates. Finally, the effects and underlying mechanism of SCE on the absorption of GE were further investigated by in situ SPIP experiment. ResultsOur findings indicated that SCE could increase exposure in vivo and the intestinal absorption of distinct ginsenosides. Additionally, we found that the PPD-type ginsenosides Rb1, Rc, Rb2, and Rd were substrates for P-gp, and the PPT-type ginsenosides Rg1 and Re were substrates for CYP3A4 rather than P-gp. SCE, which has been found with extensive inhibitory effects on P-gp and CYP3A4, could remarkably promote the intestinal absorption of ginsenosides Rg1, Re, Rb1, Rc, Rb2, and Rd, obtaining similar effects comparable with ketoconazole known as a classic dual inhibitor of P-gp and CYP3A4. ConclusionsThe study demonstrated that SCE could improve the absorption of GE, and revealed the underlying compatibility mechanism of GRR and SCF from the perspective of P-gp and CYP3A4-mediated interactions to some extent, which provided a certain scientific reference for the compatibility and clinical practice of GRR-SCF as common herb pairs in traditional prescriptions such as SMY. Moreover, this study also furnished a strategy for improving the oral bioavailability of different types of ginsenosides by drug combinations.

Strain differences in the drug transport capacity of intestinal glucose transporters in Sprague–Dawley versus Wistar rats, C57BL/6J versus Kunming mice

Designing oral drug delivery systems using intestinal glucose transporters (IGTs) may be one of the strategies for improving oral bioavailability of drugs. However, little is known about the biological factors affecting the drug transport capacity of IGTs. Gastrodin is a sedative drug with a structure very similar to glucose. It is a highly water-soluble phenolic glucoside. It can hardly enter the intestine through simple diffusion but exhibits good oral bioavailability of over 80%. We confirmed that gastrodin is absorbed via the intestinal glucose transport pathway. It has the highest oral bioavailability among the reported glycosides’ active ingredients through this pathway. Thus, gastrodin is the most selective drug substrate of IGTs and can be used to evaluate the drug transport capacity of IGTs. Obviously, strain is one of the main biological factors affecting drug absorption. This study firstly compared the drug transport capacity of IGTs between SD rats and Wistar rats and between C57 mice and KM mice by pharmacokinetic experiments and single-pass intestinal perfusion experiments of gastrodin. Then, the sodium-dependent glucose transporter type 1 (SGLT1) and sodium-independent glucose transporters type 2 (GLUT2) in the duodenum, jejunum, ileum and colon of these animals were quantified using RT-qPCR and Western blot. The results showed that the oral bioavailability of gastrodin in Wistar rats was significantly higher than in SD rats and significantly higher in KM mice than in C57 mice. Gastrodin absorption significantly differed among different intestinal segments in SD rats, C57 mice and KM mice, except Wistar rats. RT-qPCR and Western blot demonstrated that the intestinal expression distribution of SGLT1 and GLUT2 in SD rats and C57 mice was duodenum ≈ jejunum > ileum > colon. SGLT1 expression did not differ among different intestinal segments in KM mice, whereas the intestinal expression distribution of GLUT2 was duodenum ≈ jejunum ≈ ileum > colon. However, the expression of SGLT1 and GLUT2 did not differ among different intestinal segments in Wistar rats. It was reported that the intestinal expression distribution of SGLT1 and GLUT2 in humans is duodenum > jejunum > ileum > colon. Hence, the intestinal expression distribution of SGLT1 and GLUT2 of SD rats and C57 mice was more similar to that in humans. In conclusion, the drug transport capacity of IGTs differs in different strains of rats and mice. SD rats and C57 mice are more suitable for evaluating the pharmacokinetics of glycosides’ active ingredients absorbed via the intestinal glucose transport pathway.

Transcellular Transport Behavior of the Intact Polymeric Mixed Micelles with Different Polymeric Ratios.

In order to better promote the application of the polymeric mixed micelles (PMMs) in oral delivery, in addition to focusing on the improvement of micellar structural stability, it is necessary to obtain the absorption characteristics of the intact micellar particles. In this work, the transport behavior across Caco-2 cells of FS/PMMs composed of Pluronic F127 and Solutol HS15 was tracked by encapsulating an environment-responsive probe into the particles. The specific property of the probe is the water-initiated aggregation-caused quenching (ACQ) ability, by which integral particles can be identified accurately. The influence of polymeric ratios (FS) on the transcellular behavior of FS/PMMs was explored and the single pass intestinal perfusion experiment was used to further illustrate it. Moreover, pharmacokinetics parameters were detected to analyze the relationship among FS ratios, transport behavior, and pharmacokinetic parameters. FS ratios were found to hardly affect the endocytosis pathways and intracellular itinerary of FS/PMMs, but do affect the proportion of each path. FS/PMMs with high HS15 content, namely System-I, were found to primarily undergo receptor-mediated endocytosis pathway and be less susceptible to lysosomal degradation, which would lead to more absorption and higher Cmax and AUC than drug suspension. In contrast, despite System-II with high F127 content cannot contribute to drug plasma concentration, it can prolong the in vivo retention time. These findings provided evidence for the role of polymeric ratios in modulating the transcellular absorption and pharmacokinetic parameters of the drug-loaded PMMs, and would be a step forward in helping PMMs' design to enhance oral drug delivery.

Encapsulation of vitexin-rhamnoside based on zein/pectin nanoparticles improved its stability and bioavailability

To improve the solubility, stability, and bioavailability of vitexin-rhamnoside (VR) isolated from hawthorn, it was encapsulated by the zein-pectin nanoparticles system. When the mass ratio of zein to pectin was 1:4, the particle size of nanoparticles was 222.7 nm, and the encapsulation efficiency of VR was 67%. Analysis with the scanning electron microscope (SEM), fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM) revealed that the zein-VR-pectin nanoparticles were spherical and uniformly distributed. The hydrogen bonding and electrostatic interactions were the main forces to assemble the nanoparticles. The nanoparticle had good stability at pH 3–8.5 with particle sizes ranging from 234 to 251 nm, and the nanoparticles were able to resist the relatively lower ionic strength. In vitro simulated digestion and rat in vivo intestinal perfusion experiments showed that the nanoparticles exhibited significant slow-release properties and the highest absorption rate in the duodenal segment of rats, with Ka and Papp of 0.830 ± 0.11 and 17.004 ± 1.09. These results provided a theoretical and technological approach for the construction of flavonoids delivery system with slow-release properties and improved bioavailability.

Lactoferrin Decreases Enterotoxigenic Escherichia coli-Induced Fluid Secretion and Bacterial Adhesion in the Porcine Small Intestine.

Enterotoxigenic Escherichia coli (ETEC) infections are one of the most prevalent causes of post-weaning diarrhea in piglets, resulting in morbidity, mortality and elevated use of antibiotics. The emergence and further spread of antimicrobial resistance together with the growing demand for high quality animal protein requires the identification of novel alternatives for antimicrobials. A promising alternative is lactoferrin, as we previously showed that it can both inhibit the growth and degrade bacterial virulence factors of porcine ETEC strains in vitro. Aiming to confirm these findings in vivo, we performed a small intestinal segment perfusion experiment in piglets. Here, we showed that lactoferrin could not only decrease ETEC-induced fluid secretion, but also their ability to colonize the small intestinal epithelium. Furthermore, while ETEC infection induced pro-inflammatory cytokine mRNA expression in this experiment, lactoferrin was not able to counteract these responses. In addition, a bacterial motility assay showed that lactoferrin can reduce the motility of ETEC. Our findings further support the use of lactoferrin as an alternative for antimicrobials and also show its potential for the prevention of ETEC infections in pigs.

Comparision of the phenol red, gravimetric, and synthesized mPEG-PR methods for correcting water flux using the single-pass intestinal perfusion method

Phenol red and PEG-4000, the usual non-absorbable indicators, have non-negligible absorption problems in measuring water flux. mPEG-PR, combined phenol red with mPEG-4000, was first synthesized and could decrease absorption. However, its application has not been confirmed. The purpose of this study was to explore the applicability of mPEG-PR as a novel non-absorption indicator in the in situ single-pass intestinal perfusion (SPIP) experiment. Six model drugs (atenolol ranitidine, ibuprofen, ketoprofen, antipyrine, hydrochlorothiazide) were used to compare the accuracy of four measuring methods including phenol red, mPEG-PR, gravimetric, and non-corrected methods of correcting intestinal fluid transport. Moreover, we evaluated the correlations between the effective permeability coefficients (Peff) in rat and fraction dose absorbed (Fabs) in human, Peff in human, and apparent permeability coefficients (Papp) by the Ussing Chamber system using human tissue. Among these methods, mPEG-PR was the most reliable approach, which avoided the absorption of phenol red method and mucous shedding or water evaporation of gravimetric method. An excellent correlation was obtained between the Peff of rat and Fabs of human. Our results of this study indicated that mPEG-PR was a stable and accurate non-absorbable indicator to correct water flux in the in situ SPIP model, which could be developed to predict the human Fabs.

Investigating the Mechanisms behind the Positive Food Effect of Abiraterone Acetate: In Vitro and Rat In Situ Studies.

The anticancer agent abiraterone suffers from an extensive positive food effect after oral intake of the prodrug abiraterone acetate (Zytiga). The underlying processes determining postprandial abiraterone absorption were investigated in this study. The impact of lipids and lipid digestion products on (i) the solubility of abiraterone acetate and abiraterone, (ii) the conversion of abiraterone acetate to abiraterone, and (iii) the passive permeation of abiraterone was determined in vitro. The interaction of abiraterone acetate and abiraterone with vesicles and colloidal structures in the simulated fed state media containing undigested lipids and lipid digestion products enhanced the solubility of both compounds but limited the esterase-mediated hydrolysis of abiraterone acetate and the potential of abiraterone to permeate. Rat in situ intestinal perfusion experiments with a suspension of abiraterone acetate in static fed state simulated media identified abiraterone concentrations in the perfusate as the main driving force for absorption. However, experiments with ongoing lipolysis in the perfusate highlighted the importance of including lipid digestion as a dynamic process when studying postprandial abiraterone absorption. Future research may employ the in situ perfusion model to study postprandial drug absorption from a dynamic lipolysis-mediated intestinal environment to provide reference data for the optimisation of relevant in vitro models to evaluate food effects.

Enhanced therapeutic effect on colitis with powder formulations of Painong San associated with the promotion of intestinal adhesion and absorption

Ethnopharmacological relevancePainong San is a prescription composed of traditional Chinese medicine, which has been used to treat colitis. The Painong San's usage recorded in “Jingui Yaolve” by Zhongjing Zhang in the Later Han Dynasty is powder. However, the decoction is often used in reality. It's unclear which dosage form of Painong San is more suitable for colitis treatment and why? Aim of the studyThis study aims to evaluate the different therapeutic effects of Painong San (a powder of Painong San) and Painong Decoction (a decoction of Painong San) on a dextran sulfate sodium salt-induced colitis model and the possible reasons of these different effects. Materials and methodsThe contents of paeoniflorin, naringin, heperidin and neohesperidin in Painong San and Painong decoction were determined by an ultra-performance liquid chromatography system. The therapeutic effect on colitis was evaluated by intragastric administration of Painong San or Painong Decoction in dextran sulfate sodium salt-induced mouse model. The accumulated release rate of Painong San in vitro was analyzed with artificial gastric juice, artificial intestinal juice, and artificial colon juice. The concentrations of four compounds in the blood, intestinal contents, and intestinal fluids were detected by an-ultra performance liquid chromatography-tandem quadrupole mass spectrometry system. In situ intestinal perfusion experiments were used to observe the adhesion of Painong San to the intestine's surface. The expression of Mucin-2 and Trefoil Factor 3 in the colon was studied by immunohistochemistry. ResultsThere was no significant difference in the contents of the four compounds in Painong San and Painong Decoction. In vivo, Painong San has a better therapeutic effect than Painong Decoction in the treatment of colitis. Painong San could be released slowly in the simulated human digestion environment in vitro, and more Painong San particles were released on the intestinal surface in the colitis state in the healthy state. Painong San could increase the bioavailability of hesperidin and neohesperidin, and their concentrations in local intestinal tissue, intestinal fluid and intestinal contents. The expression of trefoil Factor 3 protein on the surface of the colon tended to be in the intestinal cavity and overlapped with the expression of mucin-2. Trefoil Factor 3 accumulates in the intestinal cavity of the colon in the state of colitis, which may increase the adhesion of Painong San particles. ConclusionIn conclusion, this experiment proved that Painong San is a more suitable dosage form for the treatment of dextran sulfate sodium salt-induced colitis than Painong Decoction, which may be related to the enhancement of Painong San particle adhesion to the intestine in colitis. This study provides a reference for the selection of clinical dosage forms for traditional Chinese medicine.

Preparation and evaluation of oral self-microemulsifying drug delivery system of Chlorophyll

Objective This study was aimed at improving the water solubility and oral bioavailability of Chl by self-microemulsifying drug delivery system (Chl-SMEDDS). Methods Compatibility experiments, pseudo-ternary phase diagram and central composite design were used to optimize the formulation. The selected systems were further evaluated for physical characteristics, including particle size, zeta potential, and appearance. The stability, in vitro dispersion test, and in vivo intestinal perfusion experiments were used to evaluate the SMEDDS. Results The optimal composition of Chl-SMEDDS included: Labrafil M 1944 CS (35%), kolliphor RH 40 (46%), Transcutol HP (19%) and 60 mg/g Chl. The appearance of water emulsified Chl-SMEDDS was green and transparent. The particle size, ζ-potential, and transmission electron microscopy studies showed that spherical globules of Chl-SMEDDS with a size of about 22.82 ± 1.29 nm and a negative surface charge of −24.21 ± 3.45 mV were obtained. Chl-SMEDDS could remain stable at 25 °C and 4 °C for at least 6 months. The dispersion test showed that Chl-SMEDDS dispersed spontaneously to form microemulsion after disintegration of capsule shell and 90% drug dispersed in just 30 min in pH 1.2 HCl without any drug precipitation during the test period. In vivo intestinal perfusion experiment revealed that the main absorption site for Chl-SMEDDS was duodenum. Conclusions This study indicates that SMEDDS formulation could be an effective strategy for the oral administration of Chl.

Comparative study on the intestinal absorption of three gastrodin analogues via the glucose transport pathway

Gastrodin is the main active constituent of Tianma, a famous traditional Chinese herbal medicine. Our previous research has found that gastrodin is absorbed rapidly in the intestine by the sodium-dependent glucose transporter 1 (SGLT1). In the current report, gastrodin is the best glycoside compound absorbed via the glucose transport pathway. This study aimed to investigate the effect of the slight difference in chemical structure on the drug intestinal absorption via the glucose transport pathway. Traditional biopharmaceutical and computer-aided molecular docking methods were used to evaluate the intestinal absorption characteristics of three gastrodin analogues, namely, salicin, arbutin and 4-methoxyphenyl-β-D-glucoside (4-MG). The oil–water partition coefficient (logP) experiments showed that the logP values of the gastrodin analogues followed the order: 4-MG > salicin > arbutin. In vitro Caco-2 cell transport experiments demonstrated that the apparent permeability coefficient (Papp) value of arbutin was higher than those of salicin and 4-MG. In situ single-pass intestinal perfusion experiments showed that the absorption of arbutin and 4-MG was better than that of salicin and that the absorption of the three compounds in the colon was lower than that in the small intestine. Quantitative real-time polymerase chain reaction results confirmed that the SGLT1 mRNA expression in the small intestine of rats was obviously higher than that in the colon of rats. In vivo pharmacokinetic experiments demonstrated that the oral bioavailability of salicin was lower than those of arbutin and 4-MG. In vitro and in vivo experiments showed that glucose or phlorizin (SGLT1 inhibitor) could decrease the intestinal absorption of the three compounds. Contrary to the above biopharmaceutical experiments, the computer-aided molecular docking test showed that the affinity of salicin to the vSGLT receptor was stronger than those of arbutin and 4-MG. In conclusion, the SGLT1 can facilitate the intestinal absorption of salicin, arbutin and 4-MG, and the slight difference in chemical structure can affect absorption.

Preparation and invitro evaluation of amphiphilic paclitaxel small molecule prodrugs and enhancement of oral absorption.

A series of novel amphiphilic paclitaxel (PTX) small molecule prodrugs, PTX-succinic anhydride-cystamine (PTX-Cys), PTX-dithiodipropionic anhydride (PTX-SS-COOH) and PTX-succinic anhydride-cystamine-valine (PTX-SS-Val) were designed, synthesized and evaluated against cancer cell lines. Compared with paclitaxel, these prodrugs contained water-soluble groups such as amino, carboxyl and amino acid, which improved the aqueous solubility of the prodrugs. More importantly, the valine was introduced in PTX-SS-Val molecule and made the molecule conform to the structural characteristics of intestinal oligopeptide transporter PEPT1 substrate. Thus the oral bioavailability of prodrug could be improved because of the mediation of PEPT1 transporter. These small molecule paclitaxel prodrugs could self-assemble into nanoparticles in aqueous solution, which effectively improved the solubility of paclitaxel, and had certain stability in pH 6.5, pH 7.4 buffer solutions and simulated gastrointestinal fluids. Some of these prodrugs, especially for PTX-Cys and PTX-SS-Val, exhibited nearly equal or slightly better anticancer activity when compared to paclitaxel. Further studies on PTX-Cys and PTX-SS-Val showed that both had good intestinal absorption in the rat single-pass intestinal perfusion (SPIP) experiments. Oral pharmacokinetic experiments showed that PTX-SS-Val could effectively improve the oral bioavailability of PTX.

The Effect of Anesthetic Regimens on Intestinal Absorption of Passively Absorbed Drugs in Rats.

Different anesthetic regimens are used during single pass intestinal perfusion (SPIP) experiments for the study of intestinal drug absorption in rats. We examined the ketamine/xylazine anesthetic combination to evaluate its influence on drug absorption compared to older regimens. Additionally, we examined whether supplementary analgesia has any effect on drug absorption and the effect of the different anesthetic regimens on induction time and stress response. Rats were anesthetized using four different anesthetic regimens; ketamine/midazolam, pentobarbital, ketamine/xylazine and ketamine/xylazine/butorphanol. Three model drugs were administered to rat intestines and Peff was calculated. Stress response was evaluated by quantifying blood corticosterone levels and induction time was recorded. We found absorption under pentobarbital to be higher or similar to absorption under ketamine/midazolam. These results partly correlate with past literature data. Ketamine/xylazine was found to give similar or higher Peff compared to pentobarbital and ketamine/midazolam. Addition of butorphanol did not affect absorption and reduced induction time and stress. In studies of intestinal drug absorption, the ketamine/xylazine combination is superior to other anesthetic regimens as it is more convenient and seems to affect absorption to a lesser extent. Addition of butorphanol is highly recommended as it did not affect absorption but led to a more effective and less stress inducing experiment.

In vitro and in situ experiments to evaluate the biodistribution and cellular toxicity of ultrasmall iron oxide nanoparticles potentially used as oral iron supplements

Well-absorbed iron-based nanoparticulated materials are a promise for the oral management of iron deficient anemia. In this work, a battery of in vitro and in situ experiments are combined for the evaluation of the uptake, distribution and toxicity of new synthesized ultrasmall (4 nm core) Fe2O3 nanoparticles coated with tartaric/adipic acid with potential to be used as oral Fe supplements. First, the in vitro simulated gastric acid solubility studies by TEM and HPLC-ICP-MS reveal a partial reduction of the core size of about 40% after 90 min at pH 3. Such scenario confirms the arrival of the nanoparticulate material in the small intestine. In the next step, the in vivo absorption through the small intestine by intestinal perfusion experiments is conducted using the sought nanoparticles in Wistar rats. The quantification of Fe in the NPs suspension before and after perfusion shows Fe absorption levels above 79%, never reported for other Fe treatments. Such high absorption levels do not seem to compromise cell viability, evaluated in enterocytes-like models (Caco-2 and HT-29) using cytotoxicity, ROS production, genotoxicity and lipid peroxidation tests. Moreover, regional differences in terms of Fe concentration are obtained among different parts of the small intestine as duodenum > jejunum > ileum. Complementary transmission electron microscopy (TEM) images show the presence of the intact particles around the intestinal microvilli without significant tissue damage. These studies show the high potential of these NP preparations for their use as oral management of anemia.

A Newly Integrated Model for Intestinal Cholesterol Absorption and Efflux Reappraises How Plant Sterol Intake Reduces Circulating Cholesterol Levels.

Cholesterol homeostasis is maintained through a balance of de novo synthesis, intestinal absorption, and excretion from the gut. The small intestine contributes to cholesterol homeostasis by absorbing and excreting it, the latter of which is referred to as trans-intestinal cholesterol efflux (TICE). Because the excretion efficiency of endogenous cholesterol is inversely associated with the development of atherosclerosis, TICE provides an attractive therapeutic target. Thus, elucidation of the mechanism is warranted. We have shown that intestinal cholesterol absorption and TICE are inversely correlated in intestinal perfusion experiments in mice. In this review, we summarized 28 paired data sets for absorption efficiency and fecal neutral sterol excretion, a surrogate marker of TICE, obtained from 13 available publications in a figure, demonstrating the inverse correlation were nearly consistent with the assumption. We then offer a bidirectional flux model that accommodates absorption and TICE occurring in the same segment. In this model, the brush border membrane (BBM) of intestinal epithelial cells stands as the dividing ridge for cholesterol fluxes, making the opposite fluxes competitive and being coordinated by shared BBM-localized transporters, ATP-binding cassette G5/G8 and Niemann-Pick C1-like 1. Furthermore, the idea is applied to address how excess plant sterol/stanol (PS) intake reduces circulating cholesterol level, because the mechanism is still unclear. We propose that unabsorbable PS repeatedly shuttles between the BBM and lumen and promotes concomitant cholesterol efflux. Additionally, PSs, which are chemically analogous to cholesterol, may disturb the trafficking machineries that transport cholesterol to the cell interior.

The Circadian Clock Gene Bmal1 Controls Intestinal Exporter MRP2 and Drug Disposition

The intestinal exporter MRP2 plays an important role in disposition and elimination of a wide range of drugs. Here, we aimed to clarify the impact of circadian clock on intestinal MRP2, and to determine the molecular mechanisms for generation of diurnal MRP2 expression.Methods: The regulatory effects of Bmal1 on intestinal MRP2 expression were assessed using intestine-specific Bmal1 knockout (Bmal1iKO) mice and colon cancer cells. The relative mRNA and protein levels were determined by qPCR and Western blotting, respectively. Everted gut sac, cell viability and in situ intestinal perfusion experiments were performed to evaluate intestinal efflux of the MRP2 substrate methotrexate (MTX). Toxicity and pharmacokinetic experiments were performed with Bmal1iKO mice and control littermates (Bmal1fl/fl mice) after oral gavage of MTX. Transcriptional gene regulation was investigated using luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays.Results: Bmal1iKO mice were generated by inter-crossing the mice carrying a Bmal1 exon 8 floxed allele (Bmal1fl/fl) with Villin-Cre mice. Intestinal MRP2 expression exhibited a diurnal oscillation in Bmal1fl/fl mice with a zenith value at ZT6. Bmal1 ablation caused reductions in Mrp2 mRNA and protein levels [as well as in transport activity (measured by MTX)], and blunted their diurnal rhythms. Intestinal ablation of Bmal1 abrogated circadian time-dependency of MTX pharmacokinetics and toxicity. Bmal1/BMAL1 regulation of rhythmic Mrp2/MRP2 expression was also confirmed in the colon cancer CT26 and Caco-2 cells. Based on a combination of luciferase reporter, mobility shift and ChIP assays, we found that Dbp activated and E4bp4 repressed Mrp2 transcription via specific binding to a same D-box (-100/-89 bp) element in promoter region. Further, Bmal1 directly activated the transcription of Dbp and Rev-erbα through the E-boxes, whereas it negatively regulated E4bp4 via the transcriptional repressor Rev-erbα. Positive regulation of Mrp2 by Rev-erbα was also observed, and attained through modulation of E4bp4.Conclusion: Bmal1 coordinates temporal expressions of DBP (a MRP2 activator), REV-ERBα (an E4BP4 repressor) and E4BP4 (a MRP2 repressor), generating diurnal MRP2 expression.

Intestinal absorption of the wheat allergen gliadin in rats

BackgroundAspirin enhances food allergy symptoms by increasing absorption of ingested allergens. The objective of this study is to elucidate the role of aspirin in facilitating intestinal absorption of the wheat allergen, gliadin, in rats. MethodsPlasma concentrations of gliadin were determined after oral administration by gavage or administration into a closed intestinal loop in rats. We used an in situ intestinal re-circulating perfusion experiment to examine the effect of pepsin on aspirin-facilitated gliadin absorption. Fluorescein isothiocyanate (FITC)-labeled dextran-40 (FD-40) was used as a marker of non-specific absorption. The molecular size of gliadin and its allergenicity in plasma were examined using immunoblot analysis and intradermal reaction tests with Evans blue dye (EBD) extravasation, respectively. ResultsAspirin increased plasma concentrations of gliadin after oral administration but had no effect in the closed intestinal loop study. An in situ intestinal re-circulating perfusion study showed that FITC-labeled gliadin was absorbed similarly to FD-40. Aspirin increased absorption of both intact and pepsin-digested gliadin, with a more significant effect on absorption of pepsin-treated gliadin. Immunoblotting showed that most gliadin was absorbed in intact form. When the gliadin fraction was extracted from rat plasma after gavage and injected intradermally into gliadin-sensitized rats, EBD extravasation was observed at injection sites in a gliadin dose-dependent manner. ConclusionsAspirin increased the absorption of intact and pepsin-digested gliadin via the paracellular pathway, maintaining their allergenicity. Moreover, the effect of aspirin on gliadin absorption was enhanced by modification and digestion of gliadin in the stomach.

Preparation and characterization of nimodipine-loaded nanostructured lipid systems for enhanced solubility and bioavailability.

PurposeNimodipine (NMP) is a clinical dihydropyridine calcium antagonist. However, the clinical application of NMP is limited by poor water solubility and low oral bioavailability. To overcome these drawbacks, this study designed optimal NMP-incorporated nanostructured lipid carriers (NLCs).MethodsHigh-pressure homogenization was successfully applied to prepare NMP-NLC, and the nanoparticle morphology was observed by a transmission electron microscope. The existence form of NMP in NMP-NLC was investigated by powder X-ray diffraction, differential scanning calorimetry, and Fourier transform infrared spectroscopy, respectively. The in vitro release study was performed by the dialysis method, and in vivo studies including in situ intestinal perfusion and pharmacokinetics were investigated in rats with NMP detected by high-performance liquid chromatography.ResultsThe obtained NMP-NLC shared a spherical shape of ~70 nm with a smooth surface and high encapsulation efficiency of 86.8%±2.1%. Spectroscopy indicated that the drug was in an amorphous state. The NMP-NLC exhibited a sustained release and diverse release profiles under different release medium, which mimicked the physiological environment. Moreover, an in situ intestinal perfusion experiment revealed that NMP-NLC could be mainly absorbed by the small intestine. Remarkable improvements in Cmax and AUC0–∞ from NMP-NLC were obtained from pharmacokinetic experiments, and the relative bioavailability of NMP-loaded nanostructured lipid systems was 160.96% relative to NMP suspensions.ConclusionCollectively, the NLCs significantly enhanced the oral bioavailability of NMP and might provide a promising nanoplatform for hydrophobic drug delivery.

Functional Calcium Binding Peptides from Pacific Cod (Gadus macrocephalus) Bone: Calcium Bioavailability Enhancing Activity and Anti-Osteoporosis Effects in the Ovariectomy-Induced Osteoporosis Rat Model.

Calcium binding peptides from Pacific cod (Gadus macrocephalus) bone have attracted attention due to their potential effects on bone health. In this study, calcium binding peptides (CBP) were prepared from Pacific cod bone by trypsin and neutral protease. Ultraviolet spectra, circular dichroism (CD), and Fourier transform infrared spectroscopy (FTIR) revealed that carboxyl and amino groups in CBP could bind to Ca2+, and form the peptide-calcium complex (CBP-Ca). Single-pass intestinal perfusion (SPIP) experiments indicated that the intestinal calcium absorption was significantly enhanced (p < 0.01) in CBP-Ca treated Wistar rats. The anti-osteoporosis activity of CBP-Ca was investigated in the ovariectomized (OVX) Wistar rat model. The administration of CBP-Ca significantly (p < 0.01) improved the calcium bioavailability, trabecular bone structure, bone biomechanical properties, bone mineral density, and bone mineralization degree. CBP-Ca notably (p < 0.01) increased serum calcium, however, it remarkably (p < 0.01) reduced the levels of osteocalcin (OCN), bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase isoform 5b (TRAP5b), and C-telopeptide of type I collagen (CTX-1) in serum. Results suggested that the cod bone derived CBP could bind with calcium, improve the intestinal calcium absorption, calcium bioavailability, and serum calcium, then reduce the bone turnover rate, and thus ameliorate osteoporosis.