Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells.

Monolayers of a well differentiated human intestinal epithelial cell line, Caco-2, were used as a model to study passive drug absorption across the intestinal epithelium. Absorption rate constants (expressed as apparent permeability coefficients) were determined for 20 drugs and peptides with different structural properties. The permeability coefficients ranged from approximately 5 x 10(-8) to 5 x 10(-5) cm/s. A good correlation was obtained between data on oral absorption in humans and the results in the Caco-2 model. Drugs that are completely absorbed in humans had permeability coefficients greater than 1 x 10(-6) cm/s. Drugs that are absorbed to greater than 1% but less than 100% had permeability coefficients of 0.1-1.0 x 10(-6) cm/s while drugs and peptides that are absorbed to less than 1% had permeability coefficients of less than or equal to 1 x 10(-7) cm/s. The results indicate that Caco-2 monolayers can be used as a model for studies on intestinal drug absorption.     

Membrane permeability parameters for some amino acids and beta-lactam antibiotics: application of the boundary layer approach

The boundary layer approach to analyzing the results of the perfused intestinal segment method of measuring membrane permeabilities is applied to the amino acids; leucine, valine, phenylalanine, lysine and aspartic acid and the beta-lactam antibiotics, cephalexin and penicillin V. The analysis indicates that in determining the membrane parameters, Pw vs. Cw data are preferable to using Jss = PwCw vs. Cw data. It is further shown that the carrier permeability, Pc* = Jmax*/Km, may be the most significant parameter to consider since luminal amino acid or drug concentration may generally be below the Km value. A comparison of P*c values for the beta-lactams with results for passively absorbed compounds indicates that the cephalosporins would be expected to be well absorbed orally based on the perfusion results. This suggests that this approach may be useful in estimating oral drug absorption for compounds that are absorbed passively as well as by a carrier-mediated mechanism.     

Differentiated intestinal epithelial cell lines asin vitro models for predicting the intestinal absorption of drugs

The oral absorption of a compound is a critical factor for the future of the compound as a drug. This absorption is mainly controlled by the passage across, the intestinal epithelium. Thus, the prediction of the intestinal absorption by means of anin vitro model may represent a powerful tool for the early selection of molecules during the process of drug development. In the present study, the differentiated human intestinal epithelial cell line HT29-18-C₁, was grown on permeable filters in dual chambers. These cells formed tight monolayers that were used to measurein vitro the transepithelial permeability coefficient (P _c) of various molecules. The results were compared within vivo data of oral absorption. A threshold value ofin vitro permeability of 2×10^–6 cm/s was found. Molecules having a permeability coefficient higher than this value were absorbed orally more than 80%, while drugs withP _c values lower than 2×10^–6 cm/s were poorly absorbed. By mathematical simulation, it was found that thisP _c value, when extrapolated to the surface area and volume of the small intestine, corresponds to an absorption of 80% for a compound with a transit time through the small intestine of 5 h. This demonstrates the predictive utility of the threshold value of the permeability coefficient derived from thein vitro model of intestinal epithelium.Abbreviations P _c transepithelial permeability coefficient - MTX methotrexate     

Pharmacokinetic properties of crocin (crocetin digentiobiose ester) following oral administration in rats

This study investigated the pharmacokinetic properties of crocin following oral administration in rats. After a single oral dose, crocin was undetected while crocetin, a metabolite of crocin, was found in plasma at low concentrations. Simultaneously, crocin was largely present in feces and intestinal contents within 24h. After repeated oral doses for 6 days, crocin remained undetected in plasma and plasma crocetin concentrations were comparable to the corresponding data obtained after the single oral dose. Furthermore, the absorption characteristics of crocin were evaluated in situ using an intestinal recirculation perfusion method. During recirculation, crocin was undetected and low concentrations of crocetin were detected in plasma. The concentrations of crocin in the perfusate were reduced through different intestinal segments, and the quantities of drug lost were greater throughout the colon. These results indicate that (1) orally administered crocin is not absorbed either after a single dose or repeated doses, (2) crocin is excreted largely through the intestinal tract following oral administration, (3) plasma crocetin concentrations do not tend to accumulate with repeated oral doses of crocin, and (4) the intestinal tract serves as an important site for crocin hydrolysis.     

Non-everted oxygenated rat intestinal segments as a measure of neutral detergent fiber effects on iron absorption

Iron absorption in the presence of varying amounts and sizes of dietary fiber was measured. A method using non-everted rat intestinal segments perfused in oxygen was refined. Neutral detergent fiber (NDF), a component of dietary fiber, was extracted from cooked pinto bean (Phaseolus vulgaris). The NDF did not affect iron absorption in intestinal segments from iron replete rats. However, 4 and 6 mg of NDF/ml significantly decreased iron absorption in the intestinal segments from anemic rats. NDF with a smaller particle size of 0.125 mm increased iron absorption relative to that absorbed with 0.180 mm particles. Histological examination validated using non-everted intestinal segments perfused with oxygen as a method for studying dietary effects on iron absorption. Segments which are not everted are less prone to damage. Perfusion with oxygen maintained metabolic activity in the tissue during the experiment.     

Correlation of in vitro and in vivo models for the oral absorption of peptide drugs

The aim of this study was to evaluate two in vitro models, Caco-2 monolayer and rat intestinal mucosa, regarding their linear correlation with in vivo bioavailability data of therapeutic peptide drugs after oral administration in rat and human. Furthermore the impact of molecular mass (Mm) of the according peptides on their permeability was evaluated. Transport experiments with commercially available water soluble peptide drugs were conducted using Caco-2 cell monolayer grown on transwell filter membranes and with freshly excised rat intestinal mucosa mounted in Using type chambers. Apparent permeability coefficients (P (app)) were calculated and compared with in vivo data derived from the literature. It was shown that, besides a few exceptions, the Mm of peptides linearly correlates with permeability across rat intestinal mucosa (R (2) = 0.86; y = -196.22x + 1354.24), with rat oral bioavailability (R (2) = 0.64; y = -401.90x + 1268.86) as well as with human oral bioavailability (R (2) = 0.91; y = -359.43x + 1103.83). Furthermore it was shown that P (app) values of investigated hydrophilic peptides across Caco-2 monolayer displayed lower permeability than across rat intestinal mucosa. A correlation between P (app) values across rat intestinal mucosa and in vivo oral bioavailability in human (R (2) = 0.98; y = 2.11x + 0.34) attests the rat in vitro model to be a very useful prediction model for human oral bioavailability of hydrophilic peptide drugs. Presented correlations encourage the use of the rat in vitro model for the prediction of human oral bioavailabilities of hydrophilic peptide drugs.     

Quantitative aspects of the intestinal absorption and metabolism of cholesterol and beta-sitosterol in the rat

The quantitative aspects of intestinal absorption and metabolism of cholesterol and -sitosterol have been studied in the rat after a single feeding of radioactive sterols. When increasing amounts of cholesterol were fed in a constant amount of triolein, the percentage absorbed decreased only gradually and the total amounts absorbed increased to a maximum. Solubility in the fat component fed is one limiting factor in the absorption of cholesterol. At the lowest dose fed, only about 50% of dietary cholesterol was absorbed even though increasing the amount fed led to a 10- to 15-fold increase in total absorption. Sitosterol, when fed in triolein, was absorbed in amounts only one-tenth of the corresponding dose of cholesterol. Intestinal transit studies indicate that the distinction between sitosterol and cholesterol, when fed together, took place during the process of uptake into the intestinal mucosa. Once taken up by the intestinal mucosal cells, cholesterol and sitosterol did not differ in their subsequent rate of transit out of the mucosal cell. Feeding sitosterol with cholesterol seems to have the same effect on cholesterol absorption as feeding the same additional dose of cholesterol, the difference being that sitosterol is taken up by the intestinal wall in amounts only one-tenth to one-fifth of that of cholesterol. The rapid and complete absorption of the triglyceride fat and the subsequent transit of the intestinal content to the large intestine are most probably important factors in the determination of the extent of absorption of nonglyceride fat. The mechanism behind the difference in extent of absorption of the closely related sterols is not explained.     

An exploratory study of two Caco-2 cell models for oral absorption: a report on their within-laboratory and between-laboratory variability, and their predictive capacity

In 2005, the European Centre for the Validation of Alternative Methods (ECVAM) sponsored a study aimed at evaluating the reproducibility (between-laboratory and within-laboratory variability) and the predictive capacity of two in vitro cellular systems--the Caco-2/ATCC parental cell line and the Caco- 2/TC7 clone--for estimating the oral fraction absorbed (Fa) in humans. Two laboratories, both of which had experience with Caco-2 cultures, participated in the study. Ten test chemicals with documented in vivo oral absorption data were selected. Atenolol, cimetidine and propranolol were included as reference compounds for low, medium and high intestinal absorption, respectively. Transport experiments were independently carried out in the two laboratories, according to an agreed protocol. The apparent permeability coefficient (Papp) was calculated in either the apical to basolateral (absorption) or the basolateral to apical (efflux) direction. To investigate the involvement of possible active transport processes, experiments were also performed in the presence of sodium azide plus 2-deoxy-D-glucose in the donor compartment. Before performing the permeability experiments, the highest concentration that did not impair barrier integrity was identified for each test chemical in both cell models, by applying the chemicals together with a marker of the paracellular pathway. In addition, barrier integrity was assessed by measuring the trans-epithelial electrical resistance. All the permeability data obtained were independently analysed. Reproducibility was assessed for the seven substances for which sufficient data were available. Within-laboratory variability was based on coefficient of variation (CV) values. Median CV values of 10.4% and 14.7% were found for the two laboratories. Concerning between-laboratory reproducibility, comparable response levels were obtained for the three reference compounds and for paracetamol, while, for the other chemicals, the results were less reproducible--in particular, for compounds known to be actively transported. The Papp values obtained for both cell lines were comparable for identical experimental conditions. Despite the limited number of substances tested, the predictive capacity was investigated by using two mathematical models available in the literature. Good estimations of the human Fa were obtained for five well-absorbed compounds, while moderately and poorly absorbed compounds were overestimated. It is proposed that a confirmatory study addressing the main results, including power considerations, would now be useful.     

Further mechanistic study on intestinal absorption enhanced by unsaturated fatty acids: reversible effect by sulfhydryl modification.

In order to study the relationship between the sulfhydryl (SH) modification of membrane-associated proteins and the oleic acid-induced permeability enhancement of the colonic mucosa, in vitro and in situ absorption studies were performed using rat colon and carboxyfluorescein as an impermeable dye. The pretreatment of the mucosa with diamide, a bifunctional sulfhydryl modifier, in in vitro experiments with the everted colonic loops reduced the absorption enhancing effect of oleic acid in a concentration-dependent manner, less inhibitory effect, though just a little, was observed as compared to N-ethylmaleimide. The inhibition caused by the addition of diamide was absolutely restored by exposure of the mucosa to dithiothreitol. On the other hand, these SH modifiers showed no pronounced effect on the in vivo permeability of quinine which is well-known to be absorbed by a passive transport system mainly via the membrane lipid bilayer. These results obtained in the present study have identified an important role of the functional SH groups of membrane proteins on modulating the permeability alteration of the mucosal epithelium provoked by oleic acid. Furthermore, the SH proteins have been revealed as being unimportant in the intestinal absorption of lipoid-soluble compounds.     

Absorption of bovine angiogenin into peripheral blood of rats orally administered milk basic protein

Bovine angiogenin is a major component of the bone resorption inhibitory activity of milk basic protein (MBP). The intestinal absorption of bovine angiogenin was investigated in a rat model, where it was detected in an intact form in the peripheral blood after the oral administration of MBP. This finding demonstrates that orally administered bovine angiogenin is absorbed without being degraded.     

Biopartitioning micellar chromatography: an in vitro technique for predicting human drug absorption

The main oral drug absorption barriers are fluid cell membranes and generally drugs are absorbed by a passive diffusion mechanism. Biopartitioning micellar chromatography (BMC) is a mode of micellar liquid chromatography that uses micellar mobile phases of Brij35 under adequate experimental conditions and can be useful to mimic the drug partitioning process in biological systems. In this paper the usefulness of BMC for predicting oral drug absorption in humans is demonstrated. A hyperbolic model has been obtained using the retention data of a heterogeneous set of 74 compounds, which shows predictive ability for drugs absorbed by passive diffusion. The model obtained in BMC is compared with those obtained using the well-known systems (Caco-2 and TC-7) that use intestinal epithelium cell lines. The use of BMC is simple, reproducible and can provide key information about the transport properties of new compounds during the drug discovery process.     

Evaluation of intestinal absorption of calcium by means of double-isotope methods and oral administration of the tracer

The percent intestinal absorption of calcium was measured in normal volunteers and in patients with idiopathic hypercalciuria employing the deconvolution method, the ratio of the two administered isotopes at equilibrium and the percent of dose present in plasma 2 hr after oral administration of the tracer. Comparison of results obtained showed that the technique based on the ratio between the two radioisotopes overestimates intestinal absorption by about 9% with respect to values calculated with the deconvolution method, but gives results comparable to those determined by oral administration of the isotope. The percent dose of the tracer 2 h after i.v. administration is closely correlated with the size of the miscible calcium pool. A less significant correlation exists between the size of the pool and percent of the dose 2 h after oral administration.     

Elucidation of intestinal absorption of D,L-amino acid enantiomers and aging in rats

At the present time, the origin of protein bound D-amino acid (AA) has been fairly well elucidated, but that of free D-AA is still not well understood. To gain greater understanding of this, intestinal absorption in rats of free D,L-AA enantiomers (arginine, alanine and aspartic acid as models for basic, neutral and acidic AAs, respectively, in this study) and the relationship between age and absorption were investigated. The degree of rat intestinal absorption of free D,L-AAs was evaluated using apparent membrane permeability coefficients (Papp) which were obtained from an in situ intestinal single-pass perfusion method with Krebs-Ringer bicarbonate buffer (pH 7.4) solution containing D,L-AA enantiomers. Determinations of D,L-AA enantiomers in perfusion (in- and outflow) solutions were carried out by the in-capillary derivatization high-performance capillary electrophoretic methods (ICD-HPCE methods) that were previously developed by our group. Collectively, our observations suggest: (1) that the Papp of L-AA is higher than that of the D-isomer; (2) that D-AA can be absorbed as well as L-AA using a sodium ion-dependent transporter that is located on the brush border membrane of rat intestinal epithelial cells; (3) that Papp reached a maximum at 8 weeks of age, but were measured at decreased amounts at 52 and 104 weeks of age. These results suggest that free D-AA in a mammalian body originates from 'exogenous sources'.     

Biowaiver or Bioequivalence: Ambiguity in Sildenafil Citrate BCS Classification

The aim of the present study is to contribute to the scientific characterization of sildenafil citrate according to the Biopharmaceutics Classification System, following the World Health Organization (WHO) guidelines for biowaivers. The solubility and intestinal permeability data of sildenafil citrate were collected from literature; however, the experimental solubility studies are inconclusive and its “high permeability” suggests an API in the borderline of BCS Class I and Class II. The pH-solubility profile was determined using the saturation shake-flask method over the pH range of 1.2–6.8 at a temperature of 37 °C in aqueous media. The intestinal permeability was determined in rat by a closed-loop in situ perfusion method (the Doluisio technique). The solubility of sildenafil citrate is pH-dependent and at pH 6.8 the dose/solubility ratio obtained does not meet the WHO criteria for “high solubility.” The high permeability values obtained by in situ intestinal perfusion in rat reinforce the published permeability data for sildenafil citrate. The experimental results obtained and the data available in the literature suggest that sildenafil citrate is clearly a Class II of BCS, according to the current biopharmaceutics classification system and WHO guidance.     

Intestinal handling of an oral oleoyl-estrone gavage by the rat

Adult Zucker lean (Fa/?) female rats received a single 250 nmol oral gavage of 3H-labelled oleoylestrone in 0.2 ml of sunflower oil. After one hour, samples of arterial, portal and suprahepatic blood, and lymph were obtained and fractioned to determine the amount of radioactivity present in the form of free estrone, acyl-estrone and hydrophilic estrone esters in the blood of each vessel. Lipoprotein fractions (chylomicra + VLDL, LDL, HDL and lipoprotein-depleted plasma) were also analysed as well as the distribution of absorbed 3H-estrone in the intestine, specific organs and carcass. About one third of the oleoyl-estrone dose recovered was found in the tissues, mainly in the blood, the rest remaining relatively untouched in the intestinal content. High hypothalamic estrone uptake (compared with the rest of the brain) was observed. Data from non-radioactive estrone measurements showed a similar pattern of absorption and tissue distribution to that obtained by 3H-estrone tracking alone. In both cases, most of the estrone present in the intestinal lumen was absorbed as intact oleoyl-estrone, but a significant part was absorbed as free estrone. There is a net transfer of 3H-estrone into portal blood HDL, and part of the 3H-estrone is also loaded into lymph-carried chylomicra. A large share of free estrone is filtered by the liver, but most of the acyl-estrone absorbed passes unaltered. The oral administration of oleoyl-estrone results in significant absorption of the unaltered molecule, which is transferred to lymph-carried chylomicra and also directly to plasma HDLs. It may be inferred that the HDL fraction contains the physiological carrier of oleoyl-estrone in its role of ponderostat signal.     

Effect of oral aluminum and aluminum citrate on blood level and short-term tissue distribution of aluminum in the rat

Aluminum (Al) absorption seems to be very low, but many factors can enhance it in animals and humans. In the present study, we investigated the acute effect of Na citrate on Al absorption by monitoring Al levels in blood and several tissues. For this purpose, 18 Wistar male rats were divided into 3 groups: control, Al, and Al + Na citrate. After a 14-h fasting period, animals were dosed orally with deionized water, or 2 mmol Al chloride, or 2 mmol Al chloride plus 2 mmol Na citrate. Blood samples were taken before and 1, 2, 4, and 6 h after the gavage. Al concentrations in blood, liver, tibia, kidney, and intestinal wall were determined by ICP-OES. In the Al and Al + citrate groups, Al blood concentrations peaked at 1 h and 2 h with higher levels in the Al + citrate group. Al gavage resulted in an increase in Al level in intestinal wall, but not in the other investigated tissues. Simultaneous gavage of citrate with Al significantly increased its tissue levels in tibia, kidney, and in intestinal wall. Our data show clearly that Al as chloride can be absorbed, but not well retained by the organism tissues. Furthermore, the model used in the present study is appropriate for acute studies to investigate the effect of various compounds on Al absorption in the rat.     

Enhanced Intestinal Absorption of Daidzein by Borneol/Menthol Eutectic Mixture and Microemulsion

In the present study, the effect of a borneol/menthol eutectic mixture (25:75) and microemulsion on the absorption of daidzein in rat intestinal membrane was evaluated. The microemulsion formulation was composed of ethyl oleate (oil), Cremophor RH40 (surfactant), PEG400 (co-surfactant), and water. The borneol/menthol eutectic mixture and its microemulsion were found to enhance the intestinal absorption of daidzein in vitro. A diffusion chamber system with isolated rat intestinal membranes was used. In contrast, verapamil (0.3 mM), a typical P-glycoprotein inhibitor, showed no effect on the absorption of daidzein by this system. A pharmacokinetic study was conducted in rats. After oral administration of daidzein at a dose of 10 mg/kg in the form of either borneol/menthol eutectic mixtures or suspension, the relative bioavailability of borneol/menthol eutectic mixtures and microemulsion was enhanced by about 1.5- and 3.65-fold, respectively, compared with a daidzein suspension. In conclusion, a borneol/menthol eutectic mixture can enhance the absorption of daidzein, although the mechanism of absorption enhancement is still unclear.     

Bioavailability and intestinal absorption of aluminum in rats

In the present investigation, the deposition of aluminum in intestinal fragment and the appearance in blood were studied in a perfused rat intestine in situ for 1 h with several aluminum forms (16 mM). We observed that aluminum absorption was positively correlated with the theoretic affinity of aluminum and the functional groups of the chelating agent. The absorption of aluminum after ingestion of organic compounds is more important than after ingestion of mineral compounds, with the following order: Al citrate > Al tartrate, Al gluconate, Al lactate > Al glutamate, Al chloride, Al sulfate, Al nitrate. Absorption depends on the nature of the ligands associated with the Al3+ ion in the gastrointestinal fluid. The higher the aluminum retention in intestinal fragment, the lower the absorption and appearance in blood. However, the higher aluminum concentration is always in the jejunal fragment because of the influence of pH variation on this fragment. Another objective of the present study was to determine the influence of several parameters on aluminum citrate absorption: with or without 0.1 mmol dinitrophenol/L, with aluminum concentration from 3.2, 16, 32, and 48, to 64 mmol/L, media containing 0, 3, or 6 mmol Ca/L, with or without phosphorus or glucose. It is concluded that aluminum is absorbed from the gastrointestinal tract by (1) a paracellular energy independent and nonsaturable route, mainly used for high aluminum concentration, which is modified by extracellular calcium, and (2) a transcellular and saturable route, the aluminum level was not modified with enhancement of aluminum quantity in intestinal lumen. This pathway can be similar with calcium transfer through the intestine and is energy dependent because of a decrease of aluminum absorption that follows the removal of glucose and phosphorus.     

Green tea extract markedly lowers the lymphatic absorption and increases the biliary secretion of 14C-benzo[a]pyrene in rats

Previously, we have shown that green tea extract (GTE) lowers the intestinal absorption of lipids and lipophilic compounds in rats. This study was conducted to investigate whether GTE inhibits the intestinal absorption and biliary secretion of benzo[a]pyrene (BaP), an extremely lipophilic potent carcinogen, present in foods as a contaminant. Male rats with lymph or bile duct cannula were infused at 3.0 ml/h for 8 h via a duodenal catheter with lipid emulsion containing (14)C-BaP with or without GTE in PBS buffer. Lymph and bile were collected hourly for 8 h. The (14)C-radioactivities in lymph, bile and intestine were determined and expressed as % dose infused. Results showed that GTE drastically lowered the lymphatic absorption of (14)C-BaP (7.6±3.2% in GTE-infused vs. 14.4±2.7% dose/8 h in control rats), with a significantly higher amount of (14)C-radioactivity present in the small intestinal lumen and cecum in rats infused with GTE. GTE also markedly increased the hourly rate (3.9±0.1% dose/h in GTE-infused vs. 3.0±0.1% dose/h in control rats) and the total biliary secretion of (14)C-BaP (31.5±0.8% dose/8 h in GTE-infused vs. 24.3±0.4% dose/8 h in control rats). The findings provide first direct evidence that GTE has a profound inhibitory effect on the intestinal absorption of BaP and promotes the excretion of absorbed BaP via the biliary route. Further studies are warranted to investigate whether green tea could be recommended as a dietary means of ameliorating the toxicity and carcinogenic effect of BaP.     

Study on the Absorbed Fingerprint-Efficacy of Yuanhu Zhitong Tablet Based on Chemical Analysis,Vasorelaxation Evaluation and Data Mining

Yuanhu Zhitong Tablet (YZT) is an example of a typical and relatively simple clinical herb formula that is widely used in clinics. It is generally believed that YZT play a therapeutical effect in vivo by the synergism of multiple constituents. Thus, it is necessary to build the relationship between the absorbed fingerprints and bioactivity so as to ensure the quality, safety and efficacy. In this study, a new combinative method, an intestinal absorption test coupled with a vasorelaxation bioactivity experiment in vitro, was a simple, sensitive, and feasible technique to study on the absorbed fingerprint-efficacy of YZT based on chemical analysis, vasorelaxation evaluation and data mining. As part of this method, an everted intestinal sac method was performed to determine the intestinal absorption of YZT solutions. YZT were dissolved in solution (n = 12), and the portion of the solution that was absorbed into intestinal sacs was analyzed using rapid-resolution liquid chromatography coupled with quadruple time-of-flight mass spectrometry (RRLC-Q-TOF/MS). Semi-quantitative analysis indicated the presence of 34 compounds. The effect of the intestinally absorbed solution on vasorelaxation of rat aortic rings with endothelium attached was then evaluated in vitro. The results showed that samples grouped by HCA from chemical profiles have similar bioactivity while samples in different groups displayed very different. Moreover, it established a relationship between the absorbed fingerprints and their bioactivity to identify important components by grey relational analysis, which could predict bioactive values based on chemical profiles and provide an evidence for the quantification of multi-constituents.