Effect of Size and Charge on the Passive Diffusion of Peptides Across Caco-2 Cell Monolayers via the Paracellular Pathway

Purpose. To evaluate the effect of size and charge on the permeation characteristics of peptides across the intestinal mucosa. Methods. The lipophilicities of neutral, positively and negatively charged capped amino acids (Asn, Lys, Asp), tripeptides (Ac-Gly-X-Ala-NH₂; X = Asn, Lys, Asp) and hexapeptides (Ac-Tip-Ala-Gly-Gly-X-Ala-NH₂; X = Asn, Lys, Asp) were estimated using an immobilized artificial membrane. The diffusion coefficients used to calculate the molecular radii were measured by NMR. The transport characteristics of the model peptides were determined across Caco-2 cell monolayers. Results. When model compounds having the same charge were compared, permeation was highly size-dependent (capped amino acids > tripeptides > hexapeptides), suggesting transport predominantly via the paracellular route. For example, the flux of the negatively charged Asp amino acid (P_app = 10.04 ± 0.43 × 10^–8 cm/s) was 3 times greater than that observed for the Asp-containing hexapeptide (P_app = 3.19 ± 0.27 × 10^–8 cm/s). When model compounds of the same size were compared, permeation across the cell monolayer was charge-dependent (negative < positive neutral). For example, the neutral, Asn-containing tripeptide (P_app = 25.79 ± 4.86 × 10^–8 cm/s) was substantially more able to permeate the Caco-2 cell monolayer than the negatively charged Asp-containing tripeptide (P_app = 7.95 ± 1.03 × 10^–8 cm/s) and the positively charged Lys-containing tripeptide (P_app = 9.86 ± 0.18 × 10^–8 cm/s). The permeability of the cell monolayer to peptides became less sensitive to net charge as the size of the peptides increased. Conclusions. A positive net charge of hydrophilic peptides enhances their permeation across the intestinal mucosa via the paracellular pathway. With increasing molecular size, molecular sieving of the epithelial barrier dominates the transport of peptides, and the effect of the net charge becomes less significant.     

不同配比白芍、桂枝多组分大鼠肠渗透特性及其相互影响

目的 考察白芍、桂枝及其不同配伍比例水煎液中的成分在大鼠体外的肠吸收特性及其可能存在的相互作用。方法 采用大鼠外翻肠囊法结合HPLC指纹图谱定性、定量考察肠渗透前后各化学成分的变化以及白芍桂枝不同配比的相互影响。结果 桂枝各成分大多吸收良好（P_app>1×10^-5 cm·s^-1）,组分间渗透前后的相对比例基本未变;白芍中以芍药苷为代表的亲水性成分吸收较好（P_app>1×10^-6 cm·s^-1）,但成分随保留时间延长渗透变差;配伍前后二者各成分的渗透未发生明显的变化。结论 除白芍部分亲脂性成分外,桂枝与白芍中大多成分肠道吸收较好,二者配伍对各成分的吸收影响不大。     

Intestinal Absorption Screening of Mixtures from Combinatorial Libraries in the Caco-2 Model

Purpose. Understanding how chemical structures influence transport across the intestinal mucosa will greatly enhance the discovery of orally available drugs. In an attempt to accelerate defining such relationships between structure and transport, six arbitrary mixtures of N-substituted glycine (NSG) peptoids containing 24 physicochemically diverse compounds were evaluated in the Caco-2 model of intestinal absorption. Methods. Samples were analyzed by HPLC and the areas of the peaks representing the components of each mixture were summed to measure 'aggregate' apparent permeability coefficients (P _app) a score of the influence of the common structural element within each mixture towards absorption. Mass spectrometry was used to identify the chemical structure of Caco-2 permeable compounds. Results. Three linear trimeric mixtures were examined and, for each mixture, none of the components was detected in receiver chambers. It was concluded that the components of these mixtures each had a P _app value less than 0.8 × 10^–6 cm/sec, a permeability less than mannitol. Three dimeric mixtures were examined and they exhibited aggregate P _app values of 9.2 × 10^–6 , 14 × 10^–6 and 6.9 × 10^–6 cm/sec. These transport rates reflected the transport of most of the components of each mixture. Furthermore, the components of the dimeric mixtures which were transported at a rate greater than mannitol were apparently transported by passive mechanisms. Conclusions. This study demonstrated that mixtures can be used to study structure-transport relationships in the Caco-2 model. The information obtained from this type of study will be integrated into the design of future chemical libraries. Other potential uses of chemical mixtures with the Caco-2 model are also discussed.     

Evaluation of Viability of Excised Rat Intestinal Segments in the Ussing Chamber: Investigation of Morphology,Electrical Parameters,and Permeability Characteristics

Purpose. To clarify relations between alterations in electrical and permeability data with time and to elaborate accompanying structural changes of intestinal segments in Ussing chamber experiments. Methods. Excised intestinal segments from the rat were studied in a modified Ussing chamber. Experiments were run up to 180 minutes during which the electrical parameters, PD, SCC, and R, were measured and the permeability coefficients (P_app) of mannitol and propranolol were determined. Each segment was observed under the light microscope for morphological evaluation. Results. PD and SCC values showed a decrease for most segments while the R values remained steady throughout the experiment. The P_app for propranolol increased aborally to the small intestine. For mannitol, the reversed was observed. In some cases, there was a time-dependent change in permeability for these marker molecules. The main morphological changes observed were a decreased nucleo-apical distance, decreased villi amplification factor, initial edema, cell sloughing, and epithelial restitution. Conclusions. The time-dependent changes in permeability coefficients of mannitol and propranolol are suggested to be related to changes in electrical parameters and morphological alterations. Presented data illustrates the importance of information regarding time-dependent structural changes for correct interpretation of permeability data.     

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.     

Permeability Profiles of M-Alkoxysubstituted Pyrrolidinoethylesters of Phenylcarbamic Acid Across Caco-2 Monolayers and Human Skin

Purpose. The purpose of the present research was to study 10 m-alkoxysubstituted pyrrolidinoethylesters of phenylcarbamic acid—potential local anesthetics. The relationships between the structure of the molecule, its physicochemical parameters (log D_oct, log k, R_M, solubility) were correlated to the permeability data obtained from permeation experiments in Caco-2 monolayers and excised human skin in vitro. Methods. The extent and mechanism(s) of permeability of the series were studied through a Caco-2 monolayer in the apical-to-basolateral (a-b) and basolateral-to-apical (b-a) directions. The MTT test was performed to determine cellular damage. In vitro transdermal permeability data were obtained from permeation experiments on excised human skin by using side-by-side chambers. Passive diffusion and iontophoretically enhanced permeability were measured. Results. In Caco-2 monolayers, similar results in the shape of the permeability curves were obtained for the two directions. In the b-a direction, the values of P_app were 2-6 times greater than in the a-b direction. A plot of drug permeability vs. the number of carbons in the alkoxychain plateaued first, after which the permeability decreased by the increasing lipophilicity of the drug. If the log D_oct of the ester was 3.4 and the MW > 385 Da, no measurable Caco-2 permeability was found. Cell damage was also higher by the more lipophilic compounds. In excised human skin, the relationship between the passive diffusion of the drugs and the number of carbons in the alkoxychain was parabolic (r ² = 0.95). Introducing low-level electrical current (iontophoresis), transdermal permeability of the more hydrophilic phenylcarbamic acid esters increased clearly. Conclusions. Lipophilicity and solubility of a compound have crucial roles in the permeation process. A very high lipophilicity has, however, a negative influence on the permeability, both intestinally and transdermally. Iontophoresis significantly increases the diffusion of small and less lipophilic compounds.     

Correlation Between Oral Drug Absorption in Humans,and Apparent Drug Permeability in TC-7 Cells,A Human Epithelial Intestinal Cell Line: Comparison with the Parental Caco-2 Cell Line

Purpose. To determine and compare the relationship between in vivo oral absorption in humans and the apparent permeability coefficients (P _app ) obtained in vitro on two human intestinal epithelial cell lines, the parental Caco-2 and the TC-7 clone. Methods. Both cell lines were grown for 5–35 days on tissue culture-treated inserts. Cell monolayers were analysed for their morphology by transmission electron micrography, and for their integrity with respect to transepithelial electrical resistance, mannitol and PEG-4000 transport, and cyclosporin efflux. P _app were determined for 20 compounds exhibiting large differences in chemical structure, molecular weight, transport mechanisms, and percentage of absorption in humans. Results. The TC-7 clone exhibits morphological characteristics similar to those of the parental Caco-2 cell line, concerning apical brush border, microvilli, tight junctions and polarisation of the cell line. The TC-7 clone however appeared more homogenous in terms of cell size. Both cell lines achieved a similar monolayer integrity towards mannitol and PEG-4000. Monolayer integrity was achieved earlier for the TC-7 clone, mainly due to its shorter doubling time, i.e. 26 versus 30 hours for parental Caco-2 cells. When using cyclosporin A as a P-glycoprotein substrate, active efflux was lower in the TC-7 clone than in the parental Caco-2 cells. The P_app and mechanisms of transport (paracellular or transcellular routes, passive diffusion and active transport) were determined for 20 drugs. A relationship was established between the in vivo oral absorption in humans and P_app values, allowing to determine a threshold value for P_appof 2 10^–6 cm/sec, above for which a 100% oral absorption could be expected in humans. Both correlation curves obtained with the two cell types, were almost completely superimposable. These studies also confirmed that the dipeptide transporter is underexpressed in both cell lines. Conclusions. On the basis of morphological parameters, biochemical activity and drug transport characteristics, the TC-7 clone appeared to be a valuable alternative to the use of parental Caco-2 cells for drug absorption studies.     

Predicting Drug Absorption from Molecular Surface Properties Based on Molecular Dynamics Simulations

Purpose. To develop an efficient method for generating representative conformations for calculation of the conformationally dependent molecular surface area, and to investigate the relation between this parameter and the permeability in Caco-2 cells. Methods. High temperature molecular dynamics (MD) simulations were used to obtain 1000 conformations of six beta-blocking agents and their prodrugs. The Boltzmann averaged (B.a.) polar surface area of the 1000 conformations was correlated to the apparent permeability coefficients (P_app) for transport across filter-grown Caco-2 cells. Results. Sampling of 1000 conformations during the MD simulations was sufficient for obtaining a representative set of conformations. The B.a. polar water accessible surface area (PWASA) yielded an excellent linear correlation with P_app for both series of compounds under study (R² = 0.98). Thus, the improved permeability of the prodrugs could be explained by a reduced PWASA. The improvement of permeability after derivatization correlated positively with the size of the non-polar water accessible surface area—suggesting a synergistic effect of the cyclopropyl and the non-polar parts of the molecule to shield the polar parts from contact with water. Conclusions. An efficient method for generating the representative conformations for calculation of the B.a. polar surface area has been established. An excellent linear correlation explaining the improved permeability of the prodrugs was obtained.     

矢车菊黄素在Caco-2细胞模型中的吸收机制研究

目的 研究矢车菊黄素（centaureidin）在Caco-2细胞单层模型中的吸收机制。方法 以HPLC分析矢车菊黄素浓度,用Caco-2 细胞单层模型评价吸收时间、药物浓度、介质pH值、抑制剂等对矢车菊黄素吸收的影响,研究矢车菊黄素的吸收机制,计算表观渗透系数（apparent permeability coefficient, Papp）。结果 药物的吸收与药物浓度和吸收时间正相关;弱酸性介质条件下有利于药物的吸收;2,4-二硝基酚（DNP）对药物吸收无影响,但异博定（verapamil）可增加药物的吸收;从肠腔侧到基底侧的转运小于基底侧到肠腔侧的转运。结论 矢车菊黄素在Caco-2细胞模型中的吸收主要是被动转运,受P-糖蛋白的外排作用。     

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.     

A Correlation Between the Permeability Characteristics of a Series of Peptides Using an in Vitro Cell Culture Model (Caco-2) and Those Using an in Situ Perfused Rat Ileum Model of the Intestinal Mucosa

In an attempt to establish an in vitro/in situ correlation of intestinal permeability data, the permeability coefficients (P _app) for a series of model peptides, which were determined using an in situ perfused rat ileum model, were compared to the permeability coefficients (P _mono) determined using an in vitro cell culture model (Caco-2). The model peptides, which were all blocked on the N-terminal (acetyl, Ac) and the C-terminal (amide, NH2) ends, consisted of D-phenylalanine (F) residues (e.g., AcFNH₂, AcFFNH₂, AcFFFNH₂). To alter the degree of hydrogen bonding potential, the nitrogens of the amide bonds were sequentially methylated [e.g., AcFF(Me)FNH₂, AcF(Me)F(Me)FNH₂, Ac(Me)F(Me)F(Me)FNH₂, Ac-(Me)F(Me)F(Me)FNH(Me)]. These peptides were shown not to be metabolized in the in situ perfused rat ileum system. The results of the transport experiments showed that there were poor correlations between the apparent permeability coefficients (P _app) determined in an in situ perfused rat ileum model and the octanol–water partition coefficients (r = 0.60) or the hydrogen bonding numbers (r = 0.63) of these peptides. However, good correlations were observed between the in situ P _app values for these peptides and their partition coefficients in heptane–ethylene glycol (r = 0.96) and the differences in their partition coefficients between octanol–water and isooctane–water (r = 0.86). These results suggest that lipophilicity may not be the major factor in determining the intestinal permeability of these peptides and that hydrogen bonding potential may be a major contributing factor. A good correlation (r = 0.94) was also observed between the P _app values determined for these peptides in the in situ perfused ileum model and those P _mono values determined in the in vitro cell culture model (Caco-2) (Conradi et al., Pharm. Res. 8:1453–1460, 1991). These results suggest that the permeability values determined in the Caco-2 cell culture model may be a good predictor of the intestinal permeability of peptides.     

The role of glutathione in the permeation enhancing effect of thiolated polymers

Purpose. To verify or refute the mechanism of permeation enhancement with thiolated polymers via GSH by the use of NaFlu as marker for the paracellular permeation. Methods. The capability of 0.5% polycarbophil cysteine conjugate (PCP-Cys) to reduce 0.02% oxidized glutathione (GSSG) was evaluated via iodometric titration in aqueous solution. Glutathione in its reduced form (GSH; 0.1%-0.4%) and in combination with 0.5% PCP-Cys were tested for their permeation enhancement of sodium fluorescein (NaFlu) and fluorescence labeled bacitracin (bac-FITC) used as paracellular markers. Permeation studies across guinea pig duodenum were carried out in Ussing-type chambers. Opening of the tight junctions was additionally monitored by transepithelial electrical resistance (TEER) measurements. Results. PCP-Cys (0.5%) was shown to reduce 22.0% ± 8.2% of GSSG (0.02%) to GSH in aqueous solution at pH 7.0 and 37°C within 3 h. Permeation of NaFlu was shown to depend on the concentration of GSH. The apparent permeability coefficient (P_app) of NaFlu in buffer only was 4.98 ± 0.5*10^-6, while in the presence of 0.4% GSH a P_app of 9.31 ± 0.92*10^-6 was achieved, representing an enhancement ratio (R = P_app enhancer system/P_app control) of 1.86. The combination of GSH (0.4%) with PCP-Cys (0.5%) led to a significant (p < 0.001) improvement of R for NaFlu up to 2.93 accompanied by a decrease in TEER of 20.3% ± 1.4%. Incubation of bac-FITC with the same GSH / PCP-Cys combination led to an enhancement ratio of 2.06 within 3 h. Conclusion. GSH plays an important role in the opening of tight junctions of intestinal epithelia. It would appear that PCP-Cys is able to reduce GSSG, prolonging the concentration of GSH at the apical membrane, resulting in significantly enhanced paracellular transport.     

在体单向灌流法研究阿哌沙班肾衰大鼠肠吸收特性

目的 研究阿哌沙班在肾衰大鼠体内的肠吸收特性,并考察P糖蛋白(P-glycoprotein,P-gp)抑制剂对阿哌沙班吸收行为的影响。方法 选择肾衰大鼠在体单向灌流法进行肠吸收实验,建立大鼠阿哌沙班肠灌流液HPLC分析方法,以考察大鼠在体肠吸收影响因素。结果 阿哌沙班在各肠段的吸收速率常数（K_a）存在显著性差异（P<0.05）,但表观吸收系数（P_app）未见明显差异（P>0.05）;大鼠回肠段的K_a和P_app值随药物浓度的增加而降低;加入 P-gp抑制剂盐酸维拉帕米（0.1 mmol/L）后,阿哌沙班在空肠和回肠段的K_a和P_app值均明显增加。结论 阿哌沙班在各肠段均有吸收;P-gp抑制剂对阿哌沙班在空肠和回肠段的吸收均有明显的促进作用,表明阿哌沙班为P-gp底物,推测其吸收机制为主动转运。     

Evaluation of Pharmasolve® corneal permeability enhancement and its irritation on rabbit eyes

The aim of this study was to explore the use of Pharmasolve^® as a new kind of permeability enhancer in ophthalmic drug delivery systems. The ocular irritation of different concentrations of Pharmasolve^® on rabbit eyes was evaluated in detail. Four drugs ranging from hydrophilic to lipophilic, namely ribavirin, puerarin, enoxacin, and ibuprofen, were used as model compounds to investigate the effects of different concentrations of Pharmasolve^® on the corneal permeability. The mechanism of ocular permeation enhancement of drugs by Pharmasolve^® was also discussed. The results showed that Pharmasolve^® presented no irritation when the concentration used was lower than 10%. Pharmasolve^® could enhance the ocular permeability of the four test drugs; the maximum enhancement in P_app was 4.04, 2.76, and 2.67-fold for ribavirin, enoxacin, and puerarin, respectively; 2.5% (v/v) Pharmasovle^® increased the P_app by about 1.47-fold for ibuprofen; which suggested that it would have a great potential to be used as a safe and effective penetration enhancer in ocular drug delivery systems in the future.     

Evaluation of an Immortalized Retinal Endothelial Cell Line as an In Vitro Model for Drug Transport Studies Across the Blood-Retinal Barrier

Purpose. To evaluate the growth and barrier properties of an immortalized rat retinal endothelial cell line (TR-iBRB) maintained on permeable membrane for drug transport studies. Methods. TR-iBRB cells were grown on permeable membrane filters. The effect of coating material on cell growth was investigated. Transport of [¹⁴C]-3-O-methyl-D-glucose (3-OMG), AGN 194716, AGN 195127, AGN 197075, acebutolol, alprenolol, atenolol, brimonidine, carbamazepine epoxide (CBZ-E), metoprolol, nadolol, rhodamine 123, and sotalol was measured across the cultured cell layer to determine the apparent permeability coefficients (P_app). Rhodamine 123 uptake into these cells in the presence of these test compounds was evaluated. Western blot was performed to detect the efflux transporter P-glycoprotein (P-gp). Bidirectional transport in MDR1-MDCK cell monolayers overexpressing the human P-gp was measured for AGN 197075. Results. TR-iBRB cells form confluent cell layers when grown on fibronectin-coated membrane and exhibit characteristic spindle-shaped morphology. A good correlation between P_app and cLogD (pH 7.4) of the compounds tested was observed, except for 3-OMG, AGN 197075, and rhodamine 123, which are substrates of carrier-mediated transport systems such as P-gp and a glucose transporter (GLUT1). When grown on permeable membrane, TR-iBRB cells expressed functional P-gp and GLUT1. Conclusions. TR-iBRB cells, when grown on permeable membrane, provide a useful tool for predicting permeability across the BRB. The usefulness of this model for high-throughput screening and rank ordering of drug candidates intended for the back of the eye in treatment of ocular diseases needs further characterization upon correlation with in vivo data.     

金莲花汤中葛根素、牡荆素、迷迭香酸和咖啡酸的肠吸收研究

目的 考察金莲花汤中葛根素、牡荆素、迷迭香酸和咖啡酸的肠吸收特性,确定金莲花汤中活性成分。方法 采用HPLC法测定肠吸收样品中咖啡酸、葛根素、牡荆素、迷迭香酸,以大鼠肠外翻吸收实验和Caco-2细胞模型转运实验计算各成分的累积吸收量、吸收速率常数和表观渗透系数。结果 随着金莲花汤的质量浓度增加,咖啡酸、葛根素、牡荆素、迷迭香酸的吸收速率常数呈线性关系增加,符合零级吸收速率,且吸收率均小于1.4%,在肠道中吸收效果普遍较差,其中咖啡酸的吸收较为明显。各成分的表观渗透系数值大小为迷迭香酸>咖啡酸>葛根素>牡荆素。结论 金莲花汤中葛根素、牡荆素、迷迭香酸和咖啡酸均可在肠道中被吸收,吸收方式为被动扩散,咖啡酸和迷迭香酸可能是金莲花汤中发挥药效的活性物质,葛根素和牡荆素可能是前药。     

Effects of Structural Modifications on the Intestinal Permeability of Angiotensin II Receptor Antagonists and the Correlation of In Vitro,In Situ,and In Vivo Absorption

Purpose. The effects of structural modifications on the membrane permeability of angiotensin II (Ang II) receptor antagonists and the usefulness of in vitro and in situ intestinal absorption models in predicting in vivo absorption or bioavailability were investigated. Methods. Intestinal permeability was determined in vitro using Caco-2 cell monolayers and in situ using a perfused rat intestine method. Several physicochemical parameters were either measured or computed, and correlated with intestinal permeation. Results. Permeation coefficients (Pa) across Caco-2 cell monolayers correlated well with both in situ absorption rate constants (ka) and in vivo bioavailability or % absorption. For these Ang II antagonists, Pa values larger than 3 × 10^–6 cm sec^–1 and in situ ka values of 2 × 10^–4 min^–1 cm^–1 or above were associated with good in vivo absorption. Structural modifications at the R₅ position, where a COOH group was substituted with either a CHO or CH₂OH group, enhanced the permeability of the Ang II receptor antagonists up to 100-fold. There were good correlations between permeability and log P(octanol/buffer), log P_HPLCcharge, solvation/desolvation energy and assigned hydrogen bonding potential. Conclusions. The correlations obtained in this study indicate that both the Caco-2 cell model and the in situ perfused rat intestine could be used to predict intestinal absorption in vivo. Structural modifications of the Ang II antagonists had a significant impact on the intestinal permeability. Charge, solvation energy, and hydrogen bonding are predominant determinants of intestinal permeability and oral bioavailability of these compounds.     

Engineering of Dendrimer Surfaces to Enhance Transepithelial Transport and Reduce Cytotoxicity

Purpose. To evaluate the cytotoxicity, permeation, and transport mechanisms of PAMAM dendrimers and surface-modified cationic PAMAM dendrimers using monolayers of the human colon adenocarcinoma cell line, Caco-2. Methods. Cytotoxicity was determined using the MTT assay. The effect of dendrimers on monolayer integrity was determined from measurements of transepithelial electrical resistance (TEER) and [¹⁴C]mannitol apparent permeability coefficient (P_app). The P_app of dendrimers through monolayers was measured in both the apical (A)-to-basolateral (B) and BA directions at 4°C and 37°C and also in the presence and absence of ethylenediamine tetraacetic acid (EDTA) and colchicine. Results. The cytotoxicity and permeation of dendrimers increased with both concentration and generation. The cytotoxicity of cationic dendrimers (G2, G3, G4) was greater than that of anionic dendrimers (G2.5, G3.5) but was reduced by conjugation with lauroyl chloride; the least cytotoxic conjugates were those with six attached lauroyl chains. At 37°C the P_app of cationic dendrimers was higher than that of anionic dendrimers and, in general, increased with the number of attached lipid chains. Cationic dendrimers decreased TEER and significantly increased the P_app of mannitol. Modified dendrimers also reduced TEER and caused a more marked increase in the P_app of mannitol. The P_app values of dendrimers and modified dendrimers were higher in the presence of EDTA, lower in the presence of colchicine, and lower at 4°C than at 37°C. Conclusions. The properties of dendrimers may be significantly modified by surface engineering. Conjugation of cationic PAMAM dendrimers with lauroyl chloride decreased their cytotoxicity and increased their permeation through Caco-2 cell monolayers. Both PAMAM dendrimers and lauroyl-PAMAM dendrimer conjugates can cross epithelial monolayers by paracellular and transcellular pathways.     

The Effect of β-Turn Structure on the Permeation of Peptides Across Monolayers of Bovine Brain Microvessel Endothelial Cells

Purpose. To investigate the effects of the -turn structure of a peptide on its permeation via the paracellular and transcellular routes across cultured bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the blood-brain barrier (BBB). Methods. The effective permeability coefficients (P_eff) of the model peptides were determined across BBMEC monolayers. The dimensions of the aqueous pores in the tight junctions (TJs) of the BBMEC monolayers were determined using a series of hydrophilic permeants. This value and the molecular radius of each peptide were used to calculate the theoretical paracellular (P_P ^*) and transcellular (P_T ^*) permeability coefficients for each peptide. Results. A comparison of the theoretical P_P ^* values with the observed P_eff values was made for a series of model peptides. For the most hydrophobic peptides (Ac-PheProXaaIle-NH₂ and Ac-PheProXaaIleVal-NH₂; Xaa = Gly, Ile), it was concluded that the Gly-containing peptide of each pair more readily permeates BBMEC monolayers via the transcellular pathway than the Ile-containing analog. In addition, the Gly-containing peptides, which exhibit more -turn structure, were shown to be more lipophilic than the Ile-containing peptides as estimated by the log of their l-octanol:HBSS partition coefficients (log P_o/w). However, the three hydrophilic peptide pairs (Ac-TyrProXaaAspVal-NH₂, Ac-TyrProXaaAsnVal-NH₂, and Ac-TyrProXaaIleVal-NH₂; Xaa = Gly, Ile) were found to permeate BBMEC monolayers predominantly via the paracellular pathway. No differences were observed in the P_eff values of the hydrophilic peptides having higher -turn structures as compared to the peptides lacking these structural features. In addition, the Ile-containing peptides exhibited significantly higher log P_o/w values than the Gly-containing hydrophilic peptides. Conclusions. Hydrophobic peptides that exhibit significant -turn structure in solution are more lipophilic as measured by log P_o/w, and more readily permeate BBMEC monolayers via the transcellular route than hydrophobic peptides that lack this type of solution structure. Similar secondary structural features in hydrophilic peptides do not appear to sufficiently alter the physicochemical properties of the peptides so as to alter their paracellular flux through BBMEC monolayers.     

MDCK (Madin-Darby Canine Kidney) Cells: A Tool for Membrane Permeability Screening

The goal of this work was to investigate the use of MDCK (Madin–Darby canine kidney) cells as a possible tool for assessing the membrane permeability properties of early drug discovery compounds. Apparent permeability (P_app) values of 55 compounds with known human absorption values were determined using MDCK cell monolayers. For comparison, P_app values of the same compounds were also determined using Caco‐2 cells, a well‐characterized in vitro model of intestinal drug absorption. Monolayers were grown on 0.4‐µm Transwell‐COL membrane culture inserts. MDCK cells were seeded at high density and cultured for 3 days, and Caco‐2 cells were cultured under standard conditions for 21 to 25 days. Compounds were tested using 100 µM donor solutions in transport medium (pH 7.4) containing 1% DMSO. The P_app values in MDCK cells correlated well with those in Caco‐2 cells (r² = 0.79). Spearman's rank correlation coefficient for MDCK P_app and human absorption was 0.58 compared with 0.54 for Caco‐2 P_app and human absorption. These results indicate that MDCK cells may be a useful tool for rapid membrane permeability screening.