Model Of Intestinal Absorption Research Articles

Absorption mechanism of tanshinoneⅡA, cryptotanshinone, tanshinone I and tanshinones extract in rat small intestine in vivo

To study the absorption mechanism of tanshinones in the roots of Salvia miltiorrhiza as well as the interaction between the components in the effective fraction. The model of intestinal absorption in vivo was used and the concentrations of the tanshinones were determined by HPLC, the apparent absorption constant (K(a)) and half life of absorption (t1/2) were obtained, the other components in the extract on the absorption of the there components were also studied. With the increase of dosage of tanshinone II A or cryptotanshinone, K(a) decreased and absorption half life t1/2 increased, the drug absorption rate constant (K(a)) had little significant effect on duodenum, Jejunum and ileum. Tanshinone I has a good absorbing in the rat small intestine. The absorption of tanshinone II A and cryptotanshinone was promoted by the other components in tanshinones extract while tanshinone I' was decreased. When the concentration increases to a certain level, the uptake of tanshinone II A and cryptotanshinone do not increase. The transport mechanism of tanshinone II A and cryptotanshinone in vivo is similar to active transport or facilitated diffusion. They could be well absorbed in general intestinal tract without specific absorption site, and the complex constituents in extract could significant effect the absorption of the pharmaceutical effective constituents.

Water and Solute Absorption From Carbohydrate-Electrolyte Solutions in the Human Proximal Small Intestine: A Review and Statistical Analysis

The purpose of this study is to summarize water, carbohydrate (CHO), and electrolyte absorption from carbohydrate-electrolyte (CHO-E) solutions based on all of the triple-lumen-perfusion studies in humans since the early 1960s. The current statistical analysis included 30 reports from which were obtained information on water absorption, CHO absorption, total solute absorption, CHO concentration, CHO type, osmolality, sodium concentration, and sodium absorption in the different gut segments during exercise and at rest. Mean differences were assessed using independent-samples t tests. Exploratory multiple-regression analyses were conducted to create prediction models for intestinal water absorption. The factors influencing water and solute absorption are carefully evaluated and extensively discussed. The authors suggest that in the human proximal small intestine, water absorption is related to both total solute and CHO absorption; osmolality exerts various impacts on water absorption in the different segments; the multiple types of CHO in the ingested CHO-E solutions play a critical role in stimulating CHO, sodium, total solute, and water absorption; CHO concentration is negatively related to water absorption; and exercise may result in greater water absorption than rest. A potential regression model for predicting water absorption is also proposed for future research and practical application. In conclusion, water absorption in the human small intestine is influenced by osmolality, solute absorption, and the anatomical structures of gut segments. Multiple types of CHO in a CHO-E solution facilitate water absorption by stimulating CHO and solute absorption and lowering osmolality in the intestinal lumen.

Absence of Evidence of Translocation of GLUT2 to the Apical Membrane of Enterocytes in Everted Intestinal Sleeves

Traditional models of intestinal glucose absorption confine GLUT2 to the basolateral membrane. Evidence suggests that GLUT2 is translocated to the apical membrane when the enterocyte is exposed to high luminal glucose concentrations. GLUT2 translocates to the apical membrane by a PKC signaling mechanism dependent on activity of SGLT1 and the cellular cytostructure. Transporter-mediated glucose uptake was studied in rat jejunum using everted sleeves under seven conditions: Control, SGLT1 inhibition (phlorizin), GLUT2 inhibition (phloretin), both SGLT1 and GLUT2 inhibition, PKC inhibition (calphostin C or chelerythrine), and disruption of cellular cytostructure (nocodazole). Each condition was tested in iso-osmotic solutions of 1, 20, or 50 mM glucose for 1 or 5 min incubations (n = 6 rats each). Control rats exhibited a saturable pattern of uptake at both durations of incubation. Phlorizin (P ≤ 0.006 each) inhibited markedly and phloretin (P ≤ 0.01 each) inhibited partially glucose uptake in all concentrations and time. Phloretin and phlorizin together completely inhibited uptake (P = 0.004 each). Calphostin C, chelerythrine, and nocodazole had little effect on glucose uptake at either 1 or 5 min. Inhibition of SGLT1 led to near complete cessation of transporter-mediated glucose uptake, while GLUT2 inhibition led to partial inhibition, suggesting some constitutive expression of GLUT2 in the apical membrane. Disruption of PKC signaling or cytoskeletal integrity partially inhibited transporter-mediated glucose uptake only in 1 mM glucose, suggesting a non-specific effect. Under these conditions, it does not appear that GLUT2 is translocated to the apical membrane on the cellular cytostructure in response to PKC signaling.

The influence of plasma binding on absorption/exsorption in the Caco-2 model of human intestinal absorption.

The Caco-2 cell monolayer has become an increasingly useful in-vitro model of human intestinal absorption. In this study we have determined the effect of plasma on the basolateral side on the absorption as well as exsorption of several drugs that are highly bound to plasma proteins. The drugs used included propranolol and quercetin, which both use the transcellular route of absorption, and taxol and oestradiol 17 beta-D-glucuronide, which are thought to undergo efflux by P-glycoprotein and the multidrug resistance protein MRP, respectively. All experiments were carried out under sink conditions to mimic normal absorption. It was necessary to use heparin anticoagulation for generation of the plasma, as EDTA was found to make the monolayers very leaky. The apparent permeability (P(app)) values for absorption were 1.54 x 10(-6) cm s(-1) for oestradiol 17 beta-D-glucuronide, 3.33 x 10(-6) cm s(-1) for taxol, 20.8 x 10(-6) cm s (-1) for quercetin, and 35.3 x 10(-6) cm s(-1) for propranolol. For these four compounds, plasma on the basolateral side had no influence on absorption. However, plasma on the basolateral side significantly reduced the efflux of oestradiol 17 beta-D-glucuronide by 66%, taxol by 75%, propranolol by 82%, and quercetin by 94%. Failure to consider the effect of plasma binding can result in an overestimate of basolateral to apical efflux and result in misleading net flux calculations.

Gene expression of CYP3A4, ABC-transporters (MDR1 and MRP1-MRP5) and hPXR in three different human colon carcinoma cell lines.

Colon carcinoma cell lines are used widely as screening models for intestinal absorption of drugs. However, the expression of important transport systems and of metabolic enzymes is not completely characterized yet. The expression and inducibility of multidrug resistance gene 1 (MDR1) and cytochrome P450 isoform 3A4 (CYP3A4) was investigated in Caco-2 parental, Caco-2 TC-7 (TC-7) and LS180 cell lines. In the same three cell lines, we investigated the expression of isoforms of the multidrug resistance associated protein family (MRP1-MRP5) and the human pregnane X receptor (hPXR), which may be important for MDR1 and CYP3A4 induction. Cells were treated with rifampicin or 1alpha,25-dihydroxycholecalciferol (1,25(OH)(2)D(3)) for 72 h and the total RNA was extracted. Afterwards reverse transcription real-time polymerase chain reaction (TaqMan) assay was performed to determine the mRNA expression level. We have shown that in LS180 cells, MDR1 and CYP3A4 were inducible with both inducers. In Caco-2 parental and TC-7 cells, CYP3A4 was only inducible with 1,25(OH)(2)D(3). Furthermore, differences were shown in gene expression of several transport proteins (MDR1 and MRP1-MRP5) and CYP3A4 in different human colon carcinoma derived cell lines. hPXR mRNA was expressed in all three cell lines but the amount of mRNA detected was significantly higher in LS180 cells than in Caco-2 and TC-7 cells. We concluded that LS180 cells were a suitable model to study MDR1 and CYP3A4 induction, but for drug transport studies Caco-2 parental and TC-7 cells would be preferred as the more physiological model.

Mechanism of SGLT1-Dependent Translocation of GLUT2 to the Apical Membrane of Enterocytes

Introduction: Traditional models of intestinal glucose absorption confine glucose transporter 2 (GLUT2) to the basolateral membrane. However, considerable evidence suggests that GLUT2 is translocated rapidly to the apical membrane when the enterocyte is exposed to high luminal glucose concentrations. We hypothesized that GLUT2 translocates to the apical membrane of enterocytes by a protein kinase C (PKC) signaling mechanism dependent on activity of the hexose transporter, sodium-glucose co-transporter 1 (SGLT1) and the cellular cytostructure. Methods: Transporter-mediated glucose uptake was studied in Lewis rat jejunum using everted sleeves under 7 conditions: Control, SGLT1 inhibition (phlorizin 0.2 mM), GLUT2 inhibition (phloretin 1 mM), both SGLT1 and GLUT2 inhibition, PKC inhibition (calphostin 500 nM, chelerythrine 50 uM), and disruption of cellular cytostructure (nocodazole 10 μM). Each condition was tested in iso-osmotic solutions of 1, 20, or 50 mM glucose for 1 or 5 min incubations (n=6 rats each). Results: Phlorizin inhibited glucose uptake nearly completely in 1, 20, and 50 mM glucose at both 1 and 5 minutes (p≤0.006 each). Phloretin partially inhibited glucose uptake (p≤0.01 each) in all concentrations and time. Phloretin and phlorizin together almost totally inhibited uptake (p=0.004 each). Calphostin and chelerythrine did not inhibit transporter-mediated glucose uptake except in 1 mM glucose at 5 min (p≤0.05 each). Nocodazole inhibited uptake only in 1 mM glucose at 1 and 5 min (p≤0.01 each) (see figure). Conclusion: Inhibition of SGLT1 led to near complete cessation of transporter-mediated glucose uptake. GLUT2 inhibition led to partial inhibition of glucose uptake suggesting constitutive expression of GLUT2 in the apical membrane. Disruption of PKC signaling or cell cytoskeletal integrity partially inhibited carrier-mediated glucose uptake only in 1 mM glucose, suggesting a non-specific effect rather than inhibition of GLUT2 translocation. In this model, it does not appear that GLUT2 is translocated to the apical membrane on the cellular cytostructure in response to PKC signaling from high luminal concentrations of glucose.

Screening and Identification of Permeable Components of Radix et Rhizoma Rhei Extract by Use of Immobilized Artificial Membrane Chromatography

The objective of this work was to screen and identify permeable components of Radix et Rhizoma Rhei extract by use of immobilized artificial membrane chromatography (IAMC) then to investigate the intestinal absorption behavior of these components and the mechanism of absorption at three concentrations (0.3, 0.6, and 1.5 mg mL−1) by use of the in situ rat intestinal absorption model. The percentage absorption of permeable components was then correlated with their retention behavior by using mobile phases of three different pH (5.0, 6.0, and 7.0) containing buffers of two different ionic strength (10 and 50 mmol L−1). There was better correlation between percentage intestinal absorption and IAMC retention at pH 6.0 (r 2 = 0.82) than at pH 5.0 (r 2 = 0.64) or 7.0 (r 2 = 0.74). The correlation coefficients suggest that most of the permeable components of Radix et Rhizoma Rhei were mainly absorbed by a passive diffusion mechanism. The four main permeable components were identified as rhein, aloe-emodin, chrysophanol, and emodin by comparison of chromatographic and spectrometric behavior with those of reference standards. IAMC seems useful as a promising rapid screening technique for identification of bioactive components in complex systems, e.g. traditional Chinese medicines.

Impact of Transporters in Oral Absorption: A Case Study of in Vitro and in Vivo Organic Anion Absorption

A key determinant for oral bioavailability of a drug candidate is the intestinal epithelial permeation of the drug candidate. This intestinal permeation may be affected by interactions on membrane transporters expressed in the intestinal epithelial cells. The purpose of the present study was to investigate whether transporters were involved in the intestinal absorption of an organic anion A275 and to compare the impact of interactions related to transporters in the Caco-2 cell model versus the in vivo rat model of intestinal absorption. In both models, it was investigated whether intestinal permeation of A275 was concentration dependent and affected by inhibitors or competitive organic anions. Interactions related to transporters in intestinal permeation was clearly demonstrated in the Caco-2 cell model but was not directly evident for in vivo rat absorption. However, an observed biphasic in vivo absorption and a large intervariability between rats might mask a dose-dependent absorption of A275. To avoid these suggested interactions, a dose of at least 10 mg/kg, which saturates the intestinal transporters involved in A275 absorption, should be administered, but at doses below that the risk of such drug interactions should be taken into account.

Nutriomic Analysis of Fresh and Processed Fruit Products. 2. During in Vitro Simultaneous Molecular Passages Using Caco-2 Cell Monolayers

Many studies have used Caco-2 cell monolayers as human intestinal absorption models. However, only a few studied digested foods, instead of pure standard compounds. Moreover, beneficial and nutritional molecules (nutriome) have not been investigated simultaneously. The present study explored nutriome passages from digest solution of fresh, dried, and juiced tomato, mango, and papaya using Caco-2 cell monolayers in apical-->basolateral directions. A validation method using complementary TEER and P(app) values or internal standard caffeine is recommended because physiologically passive diffusion is unlikely to happen. Sugars were transported into basolateral sides, resulting in potential glucose equivalent bioavailability of 2.26-75 mg h(-1)/100 g (WB). Using sugar passage rates (DB) of juices as 100% references, the rate order was tomato (49.8% dried; 89.5% fresh) > mango (56.8% dried; 22.8% fresh) > papaya (18.7% dried; 36.7% fresh). Major indications that phytochemical absorption does not occur in the small intestine were obtained from the bioassay condition selected. Apical organic acid levels decreased, which occasionally were transported into basolateral sides, whereas the disappearances of apical carotenoids and phenolics were not. Pectin substances were predicted to be responsible for the disappearances of bioactive compounds in those pectin-rich fruits. Further investigations on the role of pectin substances in intestinal passages are recommended.

Intestinal absorption of aloin, aloe-emodin, and aloesin; A comparative study using two in vitro absorption models

Aloe products are one of the top selling health-functional foods in Korea, however the adequate level of intake to achieve desirable effects are not well understood. The objective of this study was to determine the intestinal uptake and metabolism of physiologically active aloe components using in vitro intestinal absorption model. The Caco-2 cell monolayer and the everted gut sac were incubated with 5-50 µM of aloin, aloe-emodin, and aloesin. The basolateral appearance of test compounds and their glucuronosyl or sulfated forms were quantified using HPLC. The % absorption of aloin, aloe-emodin, and aloesin was ranged from 5.51% to 6.60%, 6.60% to 11.32%, and 7.61% to 13.64%, respectively. Up to 18.15%, 18.18%, and 38.86% of aloin, aloe-emodin, and aloesin, respectively, was absorbed as glucuronidated or sulfated form. These results suggest that a significant amount is transformed during absorption. The absorption rate of test compounds except aloesin was similar in two models; more aloesin was absorbed in the everted gut sac than in the Caco-2 monolayer. These results provide information to establish adequate intake level of aloe supplements to maintain effective plasma level.

The Expression of Most UDP-Glucuronosyltransferases (UGTs) Is Increased Significantly during Caco-2 Cell Differentiation, whereas UGT1A6 Is Highly Expressed Also in Undifferentiated Cells

The human colon carcinoma cell line Caco-2 is often used as a model for intestinal drug absorption. To better understand xenobiotic glucuronidation in Caco-2 cells, we have examined the expression levels of different UDP-glucuronosyltransferases (UGTs) in them. The effects of two main factors were investigated, namely, passage number and cell differentiation. Hence, the mRNA levels of 15 human UGTs of subfamilies 1A and 2B were assessed in both undifferentiated and fully differentiated cells at four passage levels: P31, P37, P43, and P49. Quantitative reverse transcriptase-polymerase chain reaction was used to determine the mRNA levels of individual UGTs, and the values were normalized using beta-actin as a reference gene. The results indicate that although passage number in the tested range exerts a mild effect on the expression level of several UGTs, the contribution of cell differentiation is much larger. The expression of nearly all the UGTs that were examined in this study was significantly, sometimes greatly, increased during cell differentiation. UGT1A6 was a distinct exception to this rule, however, because it was already highly expressed in the undifferentiated cells. The mRNA findings were confirmed at the enzyme activity level by measuring the glucuronidation of 1-naphthol, a very good substrate for UGT1A6, as well as estradiol that is not glucuronidated by this enzyme. The results revealed that 1-naphthol glucuronidation activity was high in both the differentiated and undifferentiated cells, whereas estradiol glucuronidation was only detected in the differentiated cells. Thus, Caco-2 cell differentiation plays a major role in UGT expression and ensuing metabolic reactions.

In VitroMetabolic Stability and Intestinal Transport of P57AS3 (P57) fromHoodia gordoniiand its Interaction with Drug Metabolizing Enzymes

Hoodia gordonii, a succulent cactus-like plant growing in South Africa, has been used in traditional medicine for its appetite suppressant properties. Its use as a dietary supplement to promote weight loss has recently gained popularity. An oxypregnane steroidal glycoside P57AS3 (P57) is reported to be the active constituent of the sap extract responsible for anorexigenic activity. No information is available about its metabolic stability, intestinal transport and interaction with drug metabolizing enzymes. In the present investigation, the metabolic stability of P57 in human liver microsomes and its interaction with drug metabolizing enzymes (CYP1A2, 2C9, 3A4 and 2D6) were determined. Intestinal transport of P57 was studied in the Caco-2 cell model of intestinal transport and absorption. P57 was metabolically stable in the presence of human liver microsomes. The compound inhibited CYP3A4 activity with an IC50 value of 45 microM, whereas the activity of CYP 1A2, 2C9 and 2D6 was not inhibited. In the Caco-2 model, P57 exhibited a higher transport in the secretory direction than in the absorptive direction with efflux ratios of 3.1 and 3.8 at 100 and 200 microM, respectively. The efflux was inhibited by selective inhibitors of multidrug resistance associated proteins MRP1/MRP2 (MK-571) and P-gp (verapamil). In conclusion, intestinal transport of P57 was mediated by P-gp and MRP transporters. The compound was metabolically stable and showed weak inhibition of CYP 3A4.

Simultaneous LC-MS/MS Determination of Danshensu and Paeoniflorin for Permeability Studies in Caco-2 Intestinal Absorption Model

Abstract A high sensitive method based on liquid chromatography tandem mass spectrometry(LC-MS/MS) was developed and validated for the study of permeability of danshcnsu(DS) and paeoniflorin(PF) in Caco-2 intestinal absorption model. The DS and PF were extracted from cell culture by vacuum-lyophilizing and then separated on a Zorbax Stable Bond C 18 column with 0.1% acetic acid aqueous solution and methanol as mobile phase. Detection was carried out by negative elcctrospray ionization(ESI − ) with selected reaction monitoring(SRM) mode. The apparent permeability cocfficicnts( P app ) of DS and PF in Caco-2 cell medium were calculated and the effects of verapamil on the coefficients P app of the two test compounds were also illustrated. The permeability of PF was much better than that of DS when the two compounds were administrated individually. Co-administration of DS and PF led to the decrease of the transport from apical side to basolateral side for both the compounds. However, the transport in the contrary direction were accelerated. It was also observed that verapamil could accelerate the transport of the test compounds from apical side to basolateral side. However, the absorption-enhanced effect of verapamil was attenuated when DS and PF were co-administrated. These observations suggest that both passive diffusion and active efflux involved in P-gp would effect the passage of DS and PF across Caco-2 cell monolayer. At the same time, the co-administration of DS and PF to an alteration of transport behavior, which suggests that the interaction must be taken into account when ‘ n -in-one’ samples were used in Caco-2 intestinal model.

Applications of Physiologically Based Absorption Models in Drug Discovery and Development

This article describes the use of physiologically based models of intestinal drug absorption to guide the research and development of new drugs. Applications range from lead optimization in the drug discovery phase through clinical candidate selection and extrapolation to human to phase 2 formulation development. Early simulations in preclinical species integrate multiple screening data and add value by transforming these individual properties into a prediction of in vivo absorption. Comparison of simulations to plasma levels measured after oral dosing in animals highlights unexpected behavior, and parameter sensitivity analysis can explore the impact of uncertainties in key properties, point toward factors which are limiting absorption and contribute to assessment of compound developability. Physiological models provide reliable prediction of human absorption and with refinement based on phase 1 data are useful guides to further market formulation development. Improvements in the accuracy of simulations are expected as better in vitro methods generate more in vivo relevant solubility and permeability data, and modeling will play a central role in the development of more predictive methods for transporter-related effects on drug absorption.

Identification of the Active Ingredient Accounting for the Biological Effects of Ligusticum chuanxiong

Ligusticum chuanxiong Hort. (Umbelliferae) is a common Chinese medicinal herb for cardiovascular diseases. Although a number of components have been identified from the herb, the correlation between herbal pharmacological effects and its constituents at comparable quantities is yet to be established; and hence, the active constituents accounting for therapeutic effects of the herb remain unclear. In the current study, using butylidenephthalide, a minor ingredient with a low content in the herb as an example, an integrative approach of chemical analysis, intestinal permeability screening, pharmacokinetics and functional response evaluation to identify the active constituents accounting for the pharmacological effects of Ligusticum chuanxiong is presented. About seventeen constituents were determined in the herbal extract, however, using a Caco-2 cell intestinal absorption model as a first-round screening strategy, including butylidenephthalide, five of the seventeen constituents were observed to have medium to high intestinal permeability. Although it has a low content (~ 0.01–0.05%) in the herb, further pharmacokinetic study demonstrated adequate plasma concentrations of butylidenephthalide in rats. Pharmacological activity studies also revealed its vasorelaxation, anti-platelet aggregation and anti-thrombotic effects. The results demonstrated that butylidenephthalide is one of the main active ingredients accounting for bioactivities of Ligusticum chuanxiong mainly due to, after administration of the herbal extract, 1) significant biotransformation of butylidenephthalide from a co-existing ingredient in the herb and 2) synergistic effects produced by butylidenephthalide with co-existing constituents. Furthermore, our findings suggested that a minor component in the herb may be account for herbal activities and use of the integrated pharmacokinetic and pharmacodynamic approach should be more appropriate for the identification of true bioactive ingredients of herbs.

Improvement of intestinal absorption of water-soluble macromolecules by various polyamines: Intestinal mucosal toxicity and absorption-enhancing mechanism of spermine

The absorption-enhancing effects of three different polyamines, spermine (SPM), spermidine (SPD) and putrescine (PUT) on the intestinal absorption of water-soluble macromolecules were examined in rats. Fluorescein isothiocyanate-labeled dextrans (FDs) with different average molecular weights were chosen as models of water-soluble macromolecules and intestinal absorption of FDs with or without these polyamines was examined by an in situ closed loop method. The intestinal absorption of fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4400 (FD4) was relatively low in the absence of these polyamines. However, its absorption was improved in the presence of 5–10 mM SPM and 10 mM SPD in the jejunum and 10 mM SPM in the colon, while 10 mM PUT had almost no absorption-enhancing effect on the intestinal absorption of FD4. Overall, the enhancing effects of these polyamines were greater in the jejunal membranes than in the colonic membranes. The absorption-enhancing effect of SPM decreased as the molecular weights of FDs increased. The intestinal membrane toxicity of 10 mM SPM was evaluated by measuring the amount of protein and activity of lactate dehydrogenase (LDH) released from the intestinal epithelial cells. We also observed the morphological changes of intestinal mucosa in the presence or absence of SPM. The results indicated that the amount of protein and LDH was not changed in the presence of 10 mM SPM, although we observed a significant increase in these biological markers in the presence of 3% Triton X-100, as a positive control. Furthermore, we found no significant change in the intestinal membrane with 10 mM SPM by the morphological observation. These findings suggested that 10 mM SPM did not cause any significant membrane damage to the intestinal epithelium. To investigate the absorption-enhancing mechanism of SPM, the transepithelial electrical resistance (TEER) of the rat jejunal membranes was studied by using a diffusion chamber method. SPM decreased the TEER values in a concentration dependent manner and 10 mM SPM had almost the same effect to decrease the TEER value compared with 10 mM EDTA as a positive control. These findings suggest that SPM may loosen the tight junction of the epithelium, thereby increasing the intestinal absorption of drugs via a paracellular route. In summary, polyamines, especially SPM would be one of the suitable absorption enhancers with high effectiveness and low intestinal membrane toxicity.

Transport of sennosides and sennidines from Cassia angustifolia and Cassia senna across Caco-2 monolayers – an in vitro model for intestinal absorption

Laxative effects of Senna preparations are mainly mediated by rheinanthrone, a metabolite formed in the intestinal flora from dianthrones. Nevertheless, it was not clear whether dianthrones are bioavailable at all and contribute to the overall effects of this important medicinal plant. Using the Caco-2 human colonic cell line as an in vitro model of the human intestinal mucosal barrier, the bioavailability of dianthrones was studied in apical to basolateral (absorptive) and basolateral to apical (secretive) direction. Permeability coefficients (P(c)) and percent transport were calculated based on quantitations by HPLC. From the data obtained it was concluded that sennosides A and B, as well as their aglycones sennidine A and B are transported through the Caco-2 monolayers in a concentration-dependent manner and their transport was linear with time. The absorption in apical to basolateral direction was poor and P(c) values were comparable to mannitol. The transport was higher in the secretory direction, indicating a significant efflux (e.g. by efflux pumps) of the (poorly) absorbed compounds in the intestinal lumen again. Our findings support the general understanding that the laxative effects of Senna are explainable mainly by metabolites and not by the natively present dianthrones.

Apical GLUT2 and Cav1.3: regulation of rat intestinal glucose and calcium absorption.

We have proposed a model of intestinal glucose absorption in which transport by SGLT1 induces rapid insertion and activation of GLUT2 in the apical membrane by a PKC betaII-dependent mechanism. Since PKC betaII requires Ca(2+) and glucose is depolarizing, we have investigated whether glucose absorption is regulated by the entry of dietary Ca(2+) through Ca(v)1.3 in the apical membrane. When rat jejunum was perfused with 75 mM glucose, Ca(2+)-deplete conditions, or perfusion with the L-type antagonists nifedipine and verapamil strongly diminished the phloretin-sensitive apical GLUT2, but not the phloretin-insensitive SGLT1 component of glucose absorption. Western blotting showed that in each case there was a significant decrease in apical GLUT2 level, but no change in SGLT1 level. Inhibition of apical GLUT2 absorption coincided with inhibition of unidirectional (45)Ca(2+) entry by nifedipine and verapamil. At 10 mM luminal Ca(2+), (45)Ca(2+) absorption in the presence of 75 mM glucose was 2- to 3-fold that in the presence of 75 mM mannitol. The glucose-induced component was SGLT1-dependent and nifedipine-sensitive. RT-PCR revealed the presence of Ca(v)beta(3) in jejunal mucosa; Western blotting and immunocytochemistry localized Ca(v)beta(3) to the apical membrane, together with Ca(v)1.3. We conclude that in times of dietary sufficiency Ca(v)1.3 may mediate a significant pathway of glucose-stimulated Ca(2+) entry into the body and that luminal supply of Ca(2+) is necessary for GLUT2-mediated glucose absorption. The integration of glucose and Ca(2+) absorption represents a complex nutrient-sensing system, which allows both absorptive pathways to be regulated rapidly and precisely to match dietary intake.

Caco-2 cell permeability modelling: a neural network coupled genetic algorithm approach

The ability to cross the intestinal cell membrane is a fundamental prerequisite of a drug compound. However, the experimental measurement of such an important property is a costly and highly time consuming step of the drug development process because it is necessary to synthesize the compound first. Therefore, in silico modelling of intestinal absorption, which can be carried out at very early stages of drug design, is an appealing alternative procedure which is based mainly on multivariate statistical analysis such as partial least squares (PLS) and neural networks (NN). Our implementation of neural network models for the prediction of intestinal absorption is based on the correlation of Caco-2 cell apparent permeability (P (app)) values, as a measure of intestinal absorption, to the structures of two different data sets of drug candidates. Several molecular descriptors of the compounds were calculated and the optimal subsets were selected using a genetic algorithm; therefore, the method was indicated as Genetic Algorithm-Neural Network (GA-NN). A methodology combining a genetic algorithm search with neural network analysis applied to the modelling of Caco-2 P (app) has never been presented before, although the two procedures have been already employed separately. Moreover, we provide new Caco-2 cell permeability measurements for more than two hundred compounds. Interestingly, the selected descriptors show to possess physico-chemical connotations which are in excellent accordance with the well known relevant molecular properties involved in the cellular membrane permeation phenomenon: hydrophilicity, hydrogen bonding propensity, hydrophobicity and molecular size. The predictive ability of the models, although rather good for a preliminary study, is somewhat affected by the poor precision of the experimental Caco-2 measurements. Finally, the generalization ability of one model was checked on an external test set not derived from the data sets used to build the models. The result obtained is of interesting practical application and underlines that the successful model construction is strictly dependent on the structural space representation of the data set used for model development.

Absorption of Dietary Licorice Isoflavan Glabridin to Blood Circulation in Rats

Bioavailability of glabridin was elucidated to show that this compound is one of the active components in the traditional medicine licorice. Using a model of intestinal absorption, Caco-2 cell monolayer, incorporation of glabridin was examined. Glabridin was easily incorporated into the cells and released to the basolateral side at a permeability coefficient of 1.70+/-0.16 cm/s x 10(5). The released glabridin was the aglycone form and not a conjugated form. Then, 10 mg (30 micromol)/kg body weight of standard chemical glabridin and licorice flavonoid oil (LFO) containing 10 mg/kg body weight of glabridin were administered orally to rats, and the blood concentrations of glabridin was determined. Glabridin showed a maximum concentration 1 h after the dose, of 87 nmol/L for standard glabridin and 145 nmol/L for LFO glabridin, and decreased gradually over 24 h after the dose. The level of incorporation into the liver was about 0.43% of the dosed amount 2 h after the dose. These detected glabridins were in the aglycone form and not conjugated forms. The bioavailability was calculated to be AUC(inf) of 0.825 and 1.30 microM.h and elimination T(1/2 )of 8.2 and 8.5 h for standard glabridin and LFO, respectively. Adipocytokine levels were determined in the rats. The secreted amount of monocyte chemoattractant protein-1 was significantly lower in the glabridin group compared to control vehicle group. Thus, dietary glabridin was at least partly incorporated into the body in an unchanged form, though most dietary flavonoids are converted to non-active conjugate forms during intestinal absorption.