Influence of Morphometric Factors on Quantitation of Paracellular Permeability of Intestinal Epithelia In Vitro

Purpose. The relative contribution of the small and large intestine to paracellular absorption is a subject of some controversy. Direct comparison of paracellular permeability in different epithelia is complicated by variations in junctional density and/or the absorptive surface area. Methods. This study used a combination of morphometric analyses and in vitro absorption studies to define permeability characteristics in relation to the amount of paracellular pathway present in rat ileum, colon and the model epithelium, Caco-2. Results. Mucosal to serosal amplification was higher in ileum (3.9) than colon (1.9) or Caco-2 (1). Tight junctional density (1p) of ileal crypts was 3 fold greater (91 m/cm²) than that measured in ileal villi, colonic surface and crypt cells or Caco-2 monolayers (34–37 m/ cm²). However, when the relative contributions of the crypts and villi was taken into account there was no significant difference in the mean 1p per mucosal area for the three epithelia studied. Using these data to correct for morphometric differences the permeabilities of a range of small hydrophilic molecules (atenolol, D-PheAsp and PEG oligomers MW 282-634) was measured. Permeability of rat ileum and colon were virtually identical for all compounds studied. In contrast, Caco-2 monolayers showed a significantly lower permeability than intestinal tissues with the difference increasing markedly with molecular size. Conclusions. These studies suggest the importance of accounting for morphological variation when comparing the permeability characteristics of different epithelial systems.     

In Vitro Permeability Across Caco-2 Cells (Colonic) Can Predict In Vivo (Small Intestinal) Absorption in Man—Fact or Myth

Purpose. To evaluate and optimize the use of Caco-2 cell monolayers to predict thein vivo absorption of a broad range of compounds in man. Methods. Caco-2 cells are derived from human adenocarcinoma colon cells and spontaneously differentiate when grown on porous polyethylene terephthalate membranes (PETP) in a 12 well format to form monolayers of polarized cells possessing function similar to intestinal enterocytes. Transport experiments were conducted using 21 day cultured cells in a shaking water bath at 37°C. Radiolabeled mannitol was used to determine monolayer integrity. Apparent permeability coefficient (P_app) was calculated from the appearance of drug in the receiver side. Results. A strong correlation was observed between in vivo human absorption and in vitro P_app for a variety of compounds (R = 0.95, N = 35). For compounds that are substrates of p-glycoprotein (Pgp), use of a Pgp inhibitor resulted in a better estimate of absorption in humans. The results of this study suggest that the overall ranking of compounds with P_app < 1 × 10^–6 cm/sec, between 1–10 × 10^–6 cm/ sec, and > 10 × 10^–6 cm/sec can be classified as poorly (0–20%), moderately (20–70%) and well (70–100%) absorbed compounds, respectively. Conclusions. These data suggest that Caco-2 cells developed under the culturing and transport conditions defined herein can be used to predict in vivo human absorption of compounds regardless of transport mechanism, viz., transcellular, paracellular and carrier-mediated.     

Effects of Peptide Structure on Transport Properties of Seven Thyrotropin Releasing Hormone (TRH) Analogues in a Human Intestinal Cell Line (Caco-2)

Pharmaceutical Research -     

Selective Paracellular Permeability in Two Models of Intestinal Absorption: Cultured Monolayers of Human Intestinal Epithelial Cells and Rat Intestinal Segments

New data on the permeabilities of hydrophilic markers in two commonly used in vitro models, i.e., excised intestinal segments from the rat and monolayers of Caco-2 cells, are presented. The results are compared to human in vivo data. Two groups of hydrophilic marker molecules were tested: (1) monodisperse polyethylene glycols of molecular weights ranging from 194 to 502 g/mol and (2) a heterogeneous group of molecules consisting of urea, creatinine, erythritol, and mannitol (60–182 g/mol). The permeabilities of the marker molecules showed a nonlinear dependence on the molecular weight and decreased in the order rat ileum > rat colon > Caco-2 cells. Surprisingly, the polyethylene glycols permeated more easily than the other marker molecules, indicating that characteristics other than molecular weight, e.g., the flexibility of the structure, may also be important for permeation through the membrane. Comparisons with the published permeability profiles of polyethylene glycols in human intestinal segments in vivo (i.e., calculated permeability coefficients as a function of molecular weight) indicate that the human intestine is more permeable than the in vitro models. However, the permeability profiles of the corresponding segments in the human intestine and the in vitro models were comparable. Thus, good correlations were established between permeabilities of the human ileum and rat ileum and between those of human colon, rat colon, and the Caco-2 cells. We conclude that the paracellular absorption in humans can be studied mechanistically in these in vitro models.     

In Vitro Intestinal Permeability of Factor Xa Inhibitors: Influence of Chemical Structure on Passive Transport and Susceptibility to Efflux

Purpose. To study the in vitro intestinal permeability of a number of newly synthesised factor Xa inhibitors to better understand the poor oral absorption of these compounds. Methods. The bidirectional transport of the fXa inhibitors was studied in the Caco-2 cell model and isolated rat ileal tissue. An attempt was made to characterize efflux mechanisms with the help of commonly used substrates and inhibitors of various transport proteins. In addition, the transport of the fXa inhibitors was studied in MDCK cells transfected with the human MDR1 gene and expressing large amounts of P-glycoprotein (Pgp). Results. The in vitro absorptive permeability was low for all but one of the fXa inhibitors. For compounds with non-substituted amidine, a charge (due to ionisation at neutral pH) may have resulted in poor membrane partitioning. Neutral compounds with substituted amidines were effluxed from the epithelial cells. The significance of the secretion process was illustrated by the results obtained for a neutral analogue showing high absorptive Caco-2 cell permeability that was not obviated by efflux. Transport inhibition studies in Caco-2 and permeability studies in the MDR1-transfected MDCK cells consistently showed that Pgp is not involved in the secretion of fXa inhibitors. Besides efflux, metabolic liability limited the permeation of the neutral lipophilic analogues with a carbamate ester. Conclusions. Poor intestinal permeability may be an important factor in the incomplete oral absorption of the bisbenzimidazole-type fXa inhibitors. Poor permeability may be related to poor membrane partitioning for hydrophilic analogues, whereas susceptibility to efflux transports and gastro-intestinal enzymatic degradation may limit the permeability of some of the neutral less hydrophilic derivatives.     

Desolvation Energy: A Major Determinant of Absorption,But Not Clearance,of Peptides in Rats

The oral delivery of peptidic drugs is problematic because of their degradation in the gastrointestinal tract and low absorption through the intestinal mucosa. Earlier in vitro studies with two series of digestion-resistant, radiolabeled peptides that varied in physical properties (molecular weight, lipophilicity, and hydrogen bonding sites) had suggested that intestinal transport of these peptides was most influenced by the number of hydrogen bonding sites, the major determinant of desolvation energy. To determine whether this correlation could be confirmed in vivo, intestinal absorption was determined by comparing the biliary and urinary recovery of these radiolabeled peptides in rats given intravenous or intraduodenal doses. Absorption was inversely correlated to the number of calculated hydrogen bonding sites for the model peptides, similar to what had been found in vitro. Clearance by liver and kidneys appeared to be unaffected by desolvation energy but was well correlated with lipophilicity.     

Heparin Absorption Across the Intestine: Effects of Sodium N-[8-(2-Hydroxybenzoyl)Amino]Caprylate in Rat In Situ Intestinal Instillations and in Caco-2 Monolayers

Purpose. The effects of sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) on heparin intestinal absorption were studied using rat in situ ileal and colonic instillations and Caco-2 monolayers. Methods. The flux of heparin was tested in the following groups: i) heparin alone, ii) heparin in the presence of SNAC, iii) heparin in the presence of propylene glycol (PG), and iv) heparin in the presence of SNAC and PG. Heparin absorption was measured by the APTT assay in the in situ models and by the anti-Factor Xa assay in Caco-2. SNAC and [³H]-SNAC fluxes were assessed by HPLC and by scintillation counting respectively. Results. In the rat ileal and colonic in situ instillations SNAC (17–35mg) promoted heparin absorption in the presence and absence of PG without damaging the tissue. PG alone did not alter heparin absorption in situ, but it amplified the effect of SNAC. In Caco-2, enhanced heparin fluxes were variable in the presence of non-cytotoxic concentrations of SNAC (< l0mg/ml) and these effects could not be discriminated from those of PG. Papp values for SNAC alone were 2.2 × 10^–5 cm/s and 2.0 × 10^–5 cm/s in the mucosal-to-serosal and serosal-to-mucosal directions respectively, suggesting a substantial passive transcellular flux. Transport of SNAC was significantly reduced in the presence of heparin and/or PG, perhaps indicating physical association between the agents. Conclusions. SNAC augmented heparin absorption alone and in combination with PG in the rat in situ models without causing toxicity. Caco-2 had limitations for testing increased heparin absorption due to cytotoxic effects of high concentrations of SNAC and PG. However, SNAC itself was well absorbed across Caco-2 and its mechanism of permeation was determined.     

In Vitro and In Situ Permeability of a 'Second Generation' Hydroxypyridinone Oral Iron Chelator: Correlation with Physico-Chemical Properties and Oral Activity

Purpose. The in vitro and in situ transport of CGP 65015 ((+)-3-hydroxy-1-(2-hydroxyethyl) -2-hydroxyphenyl-methyl-1 H-pyridin-4-one), a novel oral iron chelator, is described. The predictive power of these data in assessing intestinal absorption in man is described. Methods. Caco-2 epithelial monolayer and in situ rat jejunum perfusion intestinal permeability models were utilized. In vivo iron excretion and preliminary animal pharmacokinetic experiments were described, lonization constants and octanol/aqueous partition coefficients were measured potentiometrically. Solubilities and intrinsic dissolution rates were determined using standard procedures. Results. Caco-2 cell (P_app 0.25 X 10^–6 cm.s^–1) and rat jejunum (P_w 0.4) permeabilities of CGP 65015 were determined. The log D(pH 7.4) of CGP 65015 was 0.58 and its aqueous solubility was > 0.5 mg.ml^–1 (pH 3–9). The intrinsic dissolution rate of CGP 65015 in USP simulated intestinal fluid was 0.012 mg.min^–1.cm^–2. CGP 65015 promotes iron excretion effectively and dose dependently in animals. Conclusions. Caco-2 and rat intestinal permeabilities predict incomplete oral absorption of CGP 65015 in man. Preliminary rat pharmacokinetics support this. Physico-chemical data are, also, in line and suggest that CGP 65015 may, in addition, be solubility/dissolution rate limited in vivo. Nevertheless, early animal pharmacological data demonstrate that CGP 65015 is a viable oral iron chelator candidate.     

In Vitro/in Vivo Models for Peptide Oral Absorption: Comparison of Caco-2 Cell Permeability with Rat Intestinal Absorption of Renin Inhibitory Peptides

Pharmaceutical Research -     

Polar Molecular Surface Properties Predict the Intestinal Absorption of Drugs in Humans

Purpose. A theoretical method has been devised for prediction of drug absorption after oral administration to humans. Methods. Twenty structurally diverse model drugs, ranging from 0.3 to 100% absorbed, were investigated. The compounds also displayed diversity in physicochemical properties such as lipophilicity, hydrogen bonding potential and molecular size. The dynamic molecular surface properties of the compounds were calculated, taking into account their three-dimensional shape and flexibility. Results. An excellent sigmoidal relationship was established between the absorbed fraction after oral administration to humans (FA) and the dynamic polar molecular surface area (PSA_d) (r² = 0.94). The relationship was stronger than those obtained for more established predictors of drug absorption. Drugs that are completely absorbed (FA > 90%) had a PSA_d 60 ² while drugs that are < 10% absorbed had a PSA_d > 140 ². Conclusions. The results indicate that PS Ad can be used to differentiate poorly absorbed drugs at an early stage of the drug discovery process.     

Evidence for Intestinal Secretion as an Additional Clearance Pathway of Talinolol Enantiomers: Concentration- and Dose-dependent Absorption in Vitro and in Vivo

Purpose. To evaluate carrier-mediated intestinal secretion of talinolol enantiomers in vivo and in vitro. Methods. In clinical studies with i.v. and p.o. dosage of rac-talinolol (30 mg and 100 mg, resp.) performed in a small number of cholecystectomized patients total and partial clearances were determined on the basis of plasma, bile and urine concentrations. The dose-dependence of AUC was investigated in 12 healthy volunteers (25, 50, 100, and 400 mg rac-talinolol as single p.o. doses). Concentration-dependence of the permeability across Caco-2 cell monolayers included concentrations from 0.1 to 2.0 mM, inhibition by verapamil was tested at 0.5 mM. Results. The total clearance as well as the apparent oral clearance (CL/F) were slightly higher for S-(–)-than for R-(+)-talinolol. Calculation of the partial clearances showed that also the residual clearance was higher for the S- than for the R-enantiomer. In the healthy volunteers, CL/F increased with increasing doses, while the S/R ratio decreased approaching unity for the highest dose. Also the results from Caco-2 cell permeation studies yielded a clear concentration-dependence with decreasing stereoselectivity for the higher concentration range. Permeability of both enantiomers was considerably higher for ba than ab transport, however, this difference disappeared when verapamil was added. Conclusions. Although not very expressed, the detected stereoselectivities indicate a preferential absorption of R-(+)-talinolol in a lower concentration and dose range, which is most probably due to a moderate stereoselectivity at the carrier system involved in intestinal secretion.     

N-Trimethylated Chitosan Chloride (TMC) Improves the Intestinal Permeation of the Peptide Drug Buserelin In Vitro (Caco-2 Cells) and In Vivo (Rats)

Purpose. To evaluate N-trimethyl chitosan chloride (TMC) of highdegrees of substitution as intestinal permeation enhancers for thepeptide drug buserelin in vitro using Caco-2 cell monolayers, and toinvestigate TMCs as enhancers of the intestinal absorption of buserelinin vivo, in rats. Methods. TMCs were tested on Caco-2 cells for their efficiency toincrease the paracellular permeability of the peptide buserelin. For thein vivo studies male Wistar rats were used and buserelin wasadministered with or without the polymers intraduodenally. Both types ofexperiments were performed at pH 7.2. Results. Transport studies with Caco-2 cell monolayers confirmed thatthe increase in buserelin permeation is dependent on the degree oftrimethylation of TMC. In agreement with the in vitro results, in vivodata revealed highly increased bioavailability of buserelin followingintraduodenal co-administration with 1.0% (w/v) TMCs.Intraduodenally applied buserelin resulted in 0.8% absolute bioavailability,whereas co-administrations with TMCs resulted in mean bioavailabilityvalues between 6 and 13 %. Chitosan HCl (1.0% pH = 7.2) did notsignificantly increase the intestinal absorption of buserelin. Conclusions. Both the in vitro and in vivo results indicate that TMCsare potent mucosal permeation enhancers of the peptide drug buserelinat neutral pH values.     

Suitability of Enalapril as a Probe of the Dipeptide Transporter System: In Vitro and In Vivo Studies

Purpose. Previous in situ and in vitro studies indicated that the intestinal absorption of enalapril is a saturable carrier-mediated process via the dipeptide transporter system (DTS); however, the oral absorption of enalapril has not been reported to be a saturable process in vivo. Our objectives were to: 1) evaluate the suitability of enalapril as a probe of the DTS, and 2) compare various experimental models as they pertain to studying the DTS. Methods. The in vitro uptake of enalapril by rat intestinal rings and permeability across Caco-2 cells were studied as a function of concentration and in the presence of compounds that are known substrates of the DTS. The effect of enalapril on the uptake of [³H]-glycyl-L-proline (gly-L-pro) by Caco-2 cells was also examined. In vivo studies were conducted in rats (1 to 50 mg/kg) and dogs (0.06 to 6 mg/kg) to evaluate the oral absorption of enalapril over a wide dose range. Results. In vitro intestinal uptake/permeability of enalapril was not saturable nor inhibited by p-lactam antibiotics, gly-L-pro, or SQ-29852. Moreover, a 20,000-fold molar excess of enalapril did not inhibit the uptake of [³H]-gly-L-pro by Caco-2 cells. The in vivo studies in rats and dogs did not demonstrate saturable absorption. Conclusions. The present in vitro and in vivo results indicated that enalapril is primarily absorbed by a non-saturable, passive diffusion process and it is not a suitable model compound for studying the DTS.     

柚皮素、橙皮素的大鼠在体肠吸收特性研究

目的 考察柚皮素、橙皮素在大鼠肠道的吸收特性,并考察P-糖蛋白(P-gp)对这2种成分吸收的影响。方法 采用大鼠在体肠灌流模型,以超高效液相色谱(UPLC)测定灌流液中柚皮素、橙皮素的含量,计算有效渗透系数(P_eff*)及10 cm肠段吸收百分比。结果 20 μmol·L^-1柚皮素在十二指肠、空肠、回肠、结肠4个肠段的P_eff*值分别为2.77±0.43,2.39±0.30,1.90±0.53,3.15±0.30;20 μmol·L^-1橙皮素分别为2.51±0.18,2.29±0.12,1.99±0.14,3.38±0.20。当两者加了P-gp抑制剂维拉帕米后,柚皮素、橙皮素在4个肠段的吸收均显著增加(P<0.05)。结论 柚皮素、橙皮素在大鼠肠道内吸收较好,但两者均可被肠黏膜上的P-gp外排。     

Improved Prediction of In Vivo Peroral Absorption from In Vitro Intestinal Permeability Using an Internal Standard to Control for Intra- and Inter-Rat Variability

Purpose. To evaluate the use of an in vitrointestinal permeability model to predict rat and human absorption as well as to evaluate the use of an internal standard to control for intra- and inter-rat variability. Methods. In vivoperoral absorption and in vitro steady-state intestinal permeability coefficients were determined in the rat for a variety of structurally different compounds with different physicochemical properties including: progesterone, hydrocortisone, salicylic acid, caffeine, clonidine, p-aminoclonidine, UK-14304, oxymetazoline, mannitol, PEG 900, PEG 4000, and a number of novel hydrophilic chemical entities. Results. The intestinal permeability coefficients determined in vitro could be used to predict the peroral absorption of a compound in both the rat and human. Normalizing the permeability of a test compound to an internal standard, e.g. mannitol, greatly improved the prediction of peroral absorption. Conclusions. The use of an internal standard can aid in the prediction of the peroral absorption of a test compound, in particular, for one that has moderate absorption in the range of 20–80%. Moreover, these methods would appear to be a useful means to improve the prediction of other absorption models as well, such as the Caco-2 cell systems and in-situ perfusion methods.     

Effect of Salt Forms and Molecular Weight of Chitosans on In Vitro Permeability Enhancement in Intestinal Epithelial Cells (Caco-2)

The purpose of this study was to investigate the effect of molecular weight (MW) and salt forms of chitosans (aspartate; CS A, glutamate; CS G, lactate; CS L and hydrochloride, CS HCl) on the transepithelial electrical resistance (TEER) and permeability of Caco-2 cells monolayer, using fluorescein isothiocyanate dextran 4000 (FD-4) as the model compound for paracellular tight junction transport. Chitosan salts were prepared by spray-drying method. FTIR and solid-state ¹³C NMR spectra showed the functional groups of salts in their molecular structures. Salt form, MW of chitosan, and amount of chitosan influenced the permeation-enhancing effects. These studies showed that chitosan salts appeared to increase cell permeability in a dose-dependent manner and caused relatively reversible effects only at the lower doses of 0.001–0.01% w/v. As the MW of chitosan increased from 20 to 460 kDa, the reduction in TEER significantly decreased in the following order: 20 < 45 < 200 < 460 kDa, observed in CS L and CS HCl. In CS A and CS G, the decrease in TEER was not significantly different in all MW because both chitosan salts showed rapid reduction in TEER within 20 min after the start of the experiment. Among chitosan salts, CS A was the most potent absorption enhancer in acidic (pH 6.2) environment. Cytotoxicity of chitosan salts was concentration dependent and varied slightly among the salt forms of chitosan used. CS HCl (MW 45 kDa) was the most toxic having an IC₅₀ of 0.22 ± 0.06 mg/mL. The ranking of chitosan salts cytotoxicity was CS HCl > CS L> CS G > CS A.     

Effect of Methotrexate on Drug Absorption from the Rat Small Intestine in Situ and in Vitro

Abstract The effect of methotrexate (20 mg/kg intramuscularly) on the absorption of phenobarbitone, sulphafurazole, mecamylamine, quinidine and isoniazid from the rat small intestine was studied in situ and in vitro. The disappearance of all drugs studied from the intestinal fluid in situ was retarded on the third day after methotrexate administration. The fluid transfer and the amount of drugs passed through the intestinal wall in vitro were also decreased. The absorption of phenobarbitone was reversible within six days, whereas the absorption of quinidine was still retarded on the sixth day after methotrexate administration. Methotrexate did not modify the amount of quinidine excreted into the intestinal lumen after intravenous administration. The levels of other drugs except isoniazid in the blood at the end of the experiment showed changes corresponding to their disappearance from the intestinal lumen. In situ the drug levels in the intestinal wall were much lower than in vitro. Their levels in the intestinal wall reflected drug absorption in vitro but not in situ. The methotrexate-induced reversible decrease in absorption seems to be attributable at least partly to diminished water flux through the intestinal wall, although other mechanisms may also exist.