Use Of Caco-2 Cells Research Articles

Use of human Caco-2 cells and HPAE-PAD for α-glucosidase assay

To measure α-glucosidase activity, rat intestinal acetone powder is commonly used as a source of α-glucosidase, and the mutarotase-glucose oxidase (GOD) methods commonly used to quantitate glucose produced by enzymatic hydrolysis of the substrates. In this study, we compared human Caco-2 cell extracts with rat intestinal acetone powder extracts. We also compared high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with the mutarotase-GOD method. The sensitivity of HPAE-PAD was higher than that of mutarotase-GOD. The glucose concentration quantified by HPAE-PAD was similar to that quantified using the mutarotase-GOD method. In the maltase reaction, 1-deoxynojirimycin (1-DNJ) exerted a more potent inhibitory effect on human enzymes than on rat enzymes. This order was reversed during the sucrase reaction. These results suggested that the combined use of Caco-2 cell extracts and HPAE-PAD is advantageous for use in α-glucosidase-related basic research.

Adaptation of the Kirkstall QV600 LLI Microfluidics System for the Study of Gastrointestinal Absorption by Mass Spectrometry Imaging and LC-MS/MS.

Absorption studies on oral drugs can be difficult due to the challenge of replicating the complex structure and environment of the GI tract. Drug absorption studies can be conducted using in vivo and ex vivo animal tissue or animal-free techniques. These studies typically involve the use of Caco-2 cells. However, Caco-2 cells do not incorporate all the cell types found in intestinal tissue and lack P450 metabolizing enzymes. The QV600 LLI system is a microfluidics system designed for use with cell culture. Here, it has been adapted to house appropriate sections of ex vivo porcine tissue to act as a system that models the duodenum section of the small intestine. A pH regulated solution of Atorvastatin was flowed over the apical layer of the GI tissue and a nutrient solution flowed over the basal layer of the tissue to maintain tissue viability. The tissue samples were snap-frozen, cryosectioned, and imaged using MALDI Mass Spectrometry Imaging (MSI). A proof-of-concept study on the effect of excipients on absorption was conducted. Different concentrations of the solubilizing agent were added to the donor circuit of the QV600 LLI. The amount of Atorvastatin in the acceptor circuit was determined to study the effect of the excipient on the amount of drug that had permeated through the tissue. Using these data, Papp, pig values were calculated and compared with the literature.

Effect of Passage Number and Culture Time on the Expression and Activity of Insulin-Degrading Enzyme in Caco-2 Cells

Background:Insulin-degrading enzyme (IDE) is a conserved zinc metallopeptidase. Here, we have evaluated the effect of passage number and culture time on IDE expression and activity in colorectal adenocarcinoma cell line (Caco-2).Methods:Caco-2 cells were cultured with different passage ranges of 5-15, 25-35, 52-63 for 48, 72, and 120 hours. Subsequently, IDE expression and enzyme activity were assessed by Western blot analysis and fluorescent assay, respectively.Results:Our results confirmed that the amount of IDE was higher in cell extract compared to supernatant, and different passage numbers and culture times had small effect on IDE expression. However, when cells were cultured in the passage number range of 5-15 for 72 hours, the IDE activity was 35% higher compared to other passage numbers (p < 0.05).Conclusion:The use of Caco-2 cells at passage number range of 5-15 and culture time of 72 hours provides proper conditions for IDE-related studies.

Toxicokinetics and toxicodynamics of chlorpyrifos is altered in embryos of Japanese medaka exposed to oil sands process-affected water: evidence for inhibition of P-glycoprotein.

Oil sands process-affected water (OSPW) is generated during extraction of bitumen in the surface mining oil sands industry in Alberta, Canada. Studies were performed in vitro by use of Caco-2 cells, and in vivo with larvae of Japanese medaka (Oryzias latipes) to determine if organic compounds from the aqueous phase of OSPW inhibit ATP binding cassette protein ABCB1 (permeability-glycoprotein, P-gp). Neutral and basic fractions of OSPW inhibited activity of P-gp in Caco-2 cells by 1.9- and 2.0-fold, respectively, while the acidic fraction had the least effect. The organophosphate pesticides chlorpyrifos (a substrate of P-gp) and malathion (not a substrate of P-gp), were used as model chemicals to investigate inhibition of P-gp in larvae. Co-exposure to chlorpyrifos and an extract of OSPW containing basic and neutral compounds reduced survival of larvae to 26.5% compared to survival of larvae exposed only to chlorpyrifos, which was 93.7%. However, co-exposure to malathion and the extract of OSPW did not cause acute lethality compared to exposure only to malathion. Accumulation and bioconcentration of chlorpyrifos, but not malathion, was greater in larvae co-exposed with the extract of OSPW. The terminal elimination half-life of chlorpyrifos in larvae exposed to chlorpyrifos in freshwater was 5 days compared with 11.3 days in larvae exposed to chlorpyrifos in OSPW. Results suggest that in non-acute exposures, basic and neutral organic compounds in the water-soluble fraction of OSPW inhibit activity of P-gp, which suggests that OSPW has the potential to cause adverse effects by chemosensitization. Copyright © 2016 John Wiley & Sons, Ltd.

The Use of Caco-2 Cells to Estimate Fe Absorption in Humans - a Critical Appraisal

The Caco-2 cell model is widely used to assess the bioaccessibility/availability of iron from foods and diets. Analysis of iron uptake in this human epithelial cell line is usually preceded by a two-step digestion to simulate the conditions in the stomach and small intestine. Moreover, culturing the cells on inserts permits the measurement of iron transport. The cellular iron uptake is determined by direct measurements using radioisotopes, or indirectly by measurement of ferritin, the intracellular storage form of iron. There is a good correlation between Caco-2 cell uptake and human iron bioavailability for a number of dietary factors known to affect iron absorption. However, recent data suggest that in some cases there is no correlation. Possible reasons for such discrepancies, the benefits, and limitations of the Caco-2 cell model are discussed. In conclusion, in vitro experiments with Caco-2 cells are important tools for ranking foods with respect to bioavailability, for mechanistic studies of iron absorption, and for studies of dietary factors influencing absorption. The results need to be confirmed in humans.

Use of Caco-2 cells based culture systems for improved benefit/risk assessment in human food

Use of Caco-2 cells based culture systems for improved benefit/risk assessment in human food

Time Lapse Monitoring of CaCo-2 Cell Shapes and Shape Dependence of the Distribution of Integrin β1 and F-Actin on their Basal Membrane

CaCo-2 cell line is a model system for cell differentiation. For the effective use of CaCo-2 cells, it is important to understand how their growth depends on environmental conditions. The authors grew them on laminin-1, fibronectin, and collagen-1 adsorbed to glass and polystyrene. The time lapse technique was applied to follow their growth and shape changes for 21.5 h post seeding. The results upgraded the auhtors' previous findings about the series of consecutive shape changes that occur post seeding. Most cells were initially rounded and then they changed shape in two directions. A smaller fraction of cells, which attained cumulus shapes, eventually detached and drifted away. Other cells attained a semispread, transient shape, which was followed by a fully spread shape that was dominant on all protein-coated surfaces. The average time over which cells changed their shape type was different on different surfaces. It was longer on protein-coated glass surfaces than on protein-coated polystyrene surfaces. On collagen-1-coated surfaces, cells spread in the shortest time. Different cell shape types exhibited different spatial distributions of integrin β1, F-actin, and focal adhesions.

Permeability for Intestinal Absorption: Caco-2 Assay and Related Issues

In vitro permeability assays remain a valuable tool of screening scientists for lead compound optimization. As a majority of discovery projects are focused on the development of orally bioavailable drugs, the need for predictability and correlation of in vitro permeability data to in vivo absorption results has never been greater. For more than a decade, the Caco-2 screening assay has remained a popular, in vitro system to test compounds for intestinal permeability and efflux liability. Despite advances in artificial membrane technology and in silico modeling systems, drug compounds still benefit from testing in cell-based epithelial monolayer assays for lead optimization and SAR. This review discusses the strengths and limitations of the Caco-2 permeability assay, and puts into context the power of combining multiple assays and approaches to improve predictability and rank-ordering for lead compound optimization. Technical information for dealing with some of the most pressing issues with in vitro permeability assays (i.e. low aqueous solubility and low post-assay recovery) is also discussed. Insights are offered to help researchers avoid common pitfalls in the interpretation of in vitro permeability data, which can often lead to the perception of misleading results for correlation to in vivo data. In addition, the advantages of addressing the issue of efflux liability early in the drug development process is discussed, detailing the usefulness of Caco-2 cells for this type of screening paradigm.

Application and limitation of inhibitors in drug–transporter interactions studies

The objective of the present study was to investigate the reliability of transporter inhibitors in the elucidation of drug–transporter interactions when multiple transporters are present in a test system. The bidirectional permeabilities of digoxin, estrone-3-sulfate (E3S), and sulfasalazine, substrates of P-gp, BCRP/MRP2 and unspecified efflux transporters, respectively, were examined in Caco-2 and MDR-MDCK cells in the absence and presence of transporter inhibitors: CsA (P-gp), FTC (BCRP) and MK571 (MRP). Digoxin showed significant efflux ratios (ER) in both Caco-2 (ER = 17) and MDR-MDCK (ER = 120), whereas E3S and sulfasalazine only showed significant efflux in Caco-2 (ER = 15 and 88, respectively) but not in MDR-MDCK cells (ER = 1.1 and 1.3, respectively). CsA at 10 μM showed complete inhibition of digoxin efflux, partial inhibition of E3S efflux and no effect on sulfasalazine efflux. FTC and MK571 had different inhibitory effects on the efflux of these compounds. The present study shows evidence of the functional expression of multiple efflux transporter systems in Caco-2 cells. Although the use of Caco-2 cells and selected inhibitors of efflux transporters can provide useful mechanistic information on drug–drug interactions involving efflux transporters, the potential cross-reaction of inhibitors with multiple transporters makes it difficult to discern the role of individual transporters in drug transport or drug–drug interactions.

Analysis of drug transporter expression in human intestinal Caco‐2 cells by real‐time PCR

Expression of drug transporters corresponds to a crucial parameter in intestinal Caco-2 cells widely used for investigating drug absorption. In order to characterize it in an accurate, reproducible and comparative manner, we analyzed mRNA levels of 19 influx and efflux drug transporters through real-time quantitative polymerase chain reaction assays combined with the use of a total RNA reference standard. Profiles of transporter expression were found to be significantly correlated in two independent Caco-2 cell clones and in human small intestine, which may support the use of Caco-2 cells for investigating intestinal drug transport. Several transporters were nevertheless quantitatively expressed at higher (MRP2, MRP3, MRP4, MRP5, MRP6, OATP-A, OATP-B, OCT1 and MCT1) or lower (BCRP) levels in Caco-2 cells comparatively to small intestine. Moreover, MDR1, MRP2, OATP-A and PEPT1 mRNA relative expression were found to differ in the two analyzed Caco-2 cell clones by at least a twofold factor, highlighting that some variations in transporter expression may occur in Caco-2 cells depending on cell origin, and therefore underlining the interest of carefully characterizing transporter levels in any Caco-2 cell clone before its use for drug transport assays.

Impact of P-glycoprotein on clopidogrel absorption

The antiplatelet activity of clopidogrel is characterized by considerable interindividual differences. Variable intestinal absorption is suggested to contribute to the inconsistencies in response. We tested the hypothesis that the intestinal efflux transporter P-glycoprotein (P-gp) limits the oral bioavailability of clopidogrel and that variance in the MDR1 gene encoding P-gp predicts absorption variability. P-gp-mediated transport of clopidogrel was assessed by transflux, influx, and efflux experiments by use of Caco-2 cells. Inhibition of P-gp activity by different modulators increased the absorptive clopidogrel flux across Caco-2 monolayers from 0.51+/-0.19 pmol/cm2 (mean+/-SD) at baseline by a maximum of 5- to 9-fold (P<.001) and the intracellular accumulation from 0.99+/-0.11 pmol/mg protein by a maximum of 2.5-fold (P<.001) in response to 1-micromol/L clopidogrel and decreased clopidogrel efflux to the level of passive diffusion. In 60 patients with coronary artery disease who underwent percutaneous coronary intervention, the peak plasma concentration (Cmax) and the total area under the plasma concentration-time curve (AUC) of clopidogrel and its active metabolite after a single oral loading dose of 300, 600, or 900 mg were tested for correlation with the MDR1 genotype. In the 300-mg and 600-mg groups (but not in the 900-mg group) Cmax and AUC values were lower in subjects homozygous for the MDR1 3435T variant compared with subjects with the 3435C/T and 3435C/C genotypes. After the 600-mg loading dose, Cmax values (mean+/-SD) of clopidogrel and its active metabolite in 3435T/T carriers were 13.3+/-5.2 ng/mL and 2.5+/-1.2 ng/mL, respectively, compared with 49.7+/-41.6 ng/mL (P=.001) and 6.6+/-3.6 ng/mL (P=.011), respectively, in 3435C/T and 3435C/C carriers; AUC values were 1502+/-463 ng/mLxmin for clopidogrel and 209+/-99 ng/mL x min for its active metabolite in 3435T/T carriers compared with 7057+/-5443 ng/mLxmin (P=.0006) and 744+/-541 ng/mLxmin (P=.011), respectively, in 3435C/T and 3435C/C carriers. Clopidogrel absorption and thereby active metabolite formation are diminished by P-gp-mediated efflux and are influenced by the MDR1 C3435T genotype.

Validation of the 96 well Caco-2 cell culture model for high throughput permeability assessment of discovery compounds

The use of Caco-2 cells for permeability screening of discovery compounds is quite well established and serves as the “method-of-choice” across the pharmaceutical industries worldwide. The typical permeability-screening model involves growing cells on a 12 well or 24 well transwell format. In this manuscript, we report the use of Caco-2 cells grown on 96 well transwell plates for screening of discovery compounds to assess their permeability characteristics. A set of standard compounds (marketed compounds) belonging to the various class of Biopharmaceutics Classification System (BCS) were used to assess the utility of the 96 well Caco-2 cells. Extensive validations were also performed with ∼160 Bristol-Myers Squibb (BMS) discovery compounds by comparing the permeability values in the 96 well Caco-2 cells with the in-house 24 well Caco-2 cells. Functional Caco-2 cells with intact monolayers could be established in the 96 well format using optimized seeding and culturing conditions. The permeability of BCS compounds in the 96 well format was found to be comparable to the permeability in 24 well format. Similarly, there was very good correlation ( R 2 = 0.93) between the two formats for the extensive validation performed with in-house discovery compounds. The validated 96 well Caco-2 cell system presents a very attractive permeability screening tool that can perform much more efficiently than the conventional 12 well or 24 well systems while providing the same high quality permeability screening data.

Oral Chemotherapeutic Delivery: Design and Cellular Response

The development of carriers to deliver a variety of cancer therapeutics orally would represent a significant advance in the treatment of this disease. This system is based on hydrophilic polymer carriers to deliver therapeutic agents to the upper region of the small intestine in response to the pH increase when passing from the stomach. Methacrylic acid (MAA) and ethylene glycol (EG) combined in a 1:1 molar ratio were reacted to form P(MAA-g-EG) nanospheres by UV-initiated free radical polymerization. Bleomycin was added prior to polymerization to allow in situ polymerization loading. Release studies were carried out in conditions to model the environment of the stomach and small intestine. Results showed that bleomycin is preferentially released at a higher pH due to the increased mesh size of the swollen hydrogel carrier. The potential cytotoxicity of bleomycin on the small intestine was investigated with the use of Caco-2 cells (human colon adenocarcinoma). Cytotoxicity studies showed maintenance of both viability and proliferation. The presence of the nanospheres decreases the transepithelial electrical resistance across Caco-2 cell monolayers. Complexation hydrogels are promising carriers to expand the number of chemotherapeutics capable of being administered orally.

The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics

The human intestinal Caco-2 cell line has been extensively used over the last twenty years as a model of the intestinal barrier. The parental cell line, originally obtained from a human colon adenocarcinoma, undergoes in culture a process of spontaneous differentiation that leads to the formation of a monolayer of cells, expressing several morphological and functional characteristics of the mature enterocyte. Culture-related conditions were shown to influence the expression of these characteristics, in part due to the intrinsic heterogeneity of the parental cell line, leading to selection of sub-populations of cells becoming prominent in the culture. In addition, several clonal cell lines have been isolated from the parental line, exhibiting in general a more homogeneous expression of differentiation traits, while not always expressing all characteristics of the parental line. Culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often make it extremely difficult to compare results in the literature. This review is aimed at summarizing recent, or previously unreviewed, data from the literature on the effects of culture-related factors and the influence of line sub-types (parental vs. different clonal lines) on the expression of differentiation traits important for the use of Caco-2 cells as a model of the absorptive and defensive properties of the intestinal mucosa. Since the use of Caco-2 cells has grown exponentially in recent years, it is particularly important to highlight these methodological aspects in order to promote the standardization and optimisation of this intestinal model.

Stable Transfection of MDCK Cells with Epitope-Tagged Human PepT1

Establish and characterize a MDCK cell line that stably expressed PepT1. MDCK cells stably transfected with N-terminal HA-tagged hPepT1 were examined by immunostaining using antibodies to HA and hPepT1. HA-hPepT1 expression was verified by immunoprecipitation and Western blotting from cell lysates. The function of the expressed HA-hPepT1 was evaluated by glycyl-sarcosine uptake. The half-life of PepT1 on the cell surface was measured by biotinylation and chase-labeling of the cells, followed by immunoprecipitation with the anti-PepT1 antibody and blotting with HRP-streptavidin. Immunolabeling with antibodies to HA and PepT1 showed fluorescence colocalization. Immunoprecipitation with the anti-HA antibody and Western blotting with the anti-PepT1 antibody showed a broad 90-105 kDa band, and vice versa when the antibodies were utilized in the reverse order. Glycyl-sarcosine uptake was increased in the stably transfected cells by 2-5 fold over untransfected control cells. The Km value of 375 microM accurately reflected the characteristic low affinity of PepT1. Finally, the half-life of the surface biotinylated HA-hPepT1 was measured to be 22 hrs. A MDCK cell line stably expressed intact and functional HA-tagged hPepT1 on its cell surface. This cell line represents an alternative to the use of Caco-2 cells to evaluate PepT1 function.

Utility of 96 well Caco-2 cell system for increased throughput of P-gp screening in drug discovery

The use of Caco-2 cells for screening of discovery compounds for their permeability characteristics and P-glycoprotein interactions is well established and used routinely in pharmaceutical industries world-wide. The screening model involves growing cells on 12 or 24 well transwell format. In this manuscript, we report the use of Caco-2 cells grown on 96 well transwell plates for screening compounds for their potential to interact with P-gp. Bi-directionality studies were performed with known P-gp substrates such as saquinavir, indinavir, vinblastine, vincristine, verapamil, digoxin and taxol. P-gp inhibition studies were also conducted using radiolabeled digoxin as the probe. The results demonstrated that P-gp substrates had efflux ratios ( P c (B to A)/ P c (A to B)) in the 96 well format that were comparable to the ratios seen in 12 and 24 well format. Inhibition of digoxin efflux transport in presence of the test compounds (P-gp substrates) demonstrated that 96 well cells express adequate amounts of efflux transporters and perform as well as the 12 and 24 well Caco-2 cells. Thus, the 96 well Caco-2 cell set-up presents a higher throughput permeability model capable of identifying compounds that interact with P-gp and has the potential to significantly increase the efficiency of P-gp screening in early drug discovery.

Influence of heat treatment of casein in presence of reducing sugars on Zn solubility and Zn uptake by Caco-2 cells after in vitro digestion.

The effect of the heat treatment of casein in presence of reducing sugars on some aspects of Zn availability was investigated. Samples were prepared by mixing casein with glucose-fructose, and were used unprocessed (C) or heated (HC). Changes in Zn speciation after the in vitro digestion of the samples, both as part of a diet and in isolation, were studied. The uptake of soluble Zn from the digested samples was investigated in Caco-2 cells. After in vitro digestion, the percentage of precipitated Zn was significantly higher with the HC sample, both when digested alone and as a part of the diet. In assays with Caco-2 cells, a significant decrease in Zn uptake was observed when the uptake buffer contained the sample C digest, by comparison with the control buffer, without casein digest. When the digested heated mixture was added, Zn uptake by the cells was significantly lower than in either of the two other cases. It may be concluded that the heat treatment of casein in the presence of glucose-fructose has a negative effect on Zn availability because, after in vitro digestion, Zn insolubilization was enhanced and Zn uptake by the enterocyte was impaired, compared with the unheated mixture. In addition, the usefulness of Caco-2 cells in this kind of research has been shown.

Caco-2 Cells Can Be Used to Assess Human Iron Bioavailability from a Semipurified Meal

A Caco-2 cell model with extrinsic radioiron was used to evaluate the effect of dietary factors on nonheme iron bioavailability from a semipurified meal. Study 1 was conducted to evaluate the effect of enhancers (ascorbic acid) and inhibitors (bran, phytate and tea) on iron bioavailability when added to semipurified meal containing egg albumen as a protein source. The effect of various proteins [bovine serum albumin (BSA), casein, beef and soy] on iron bioavailability was evaluated in Study 2 by substituting the above protein sources for egg albumen. Protein solubilization following in vitro digestion for individual test meals was not significantly different from the control. On the other hand, nonheme iron solubilization (0.8+/-0.0 to 5.9+/-0.3 vs. 4.9+/-0.8 mg/L) varied significantly. The total iron uptake for each meal was calculated based on the percentage of radioiron taken up and transported by Caco-2 cells and the amount of nonheme iron present in uptake solutions. Iron uptake ratios represent test/control values. With the exception of BSA and ascorbic acid, the effect of dietary factors was similar to that found in humans reported in the literature. A significant correlation (r = 0.97; P<0.0001) was found between the published human absorption data and the iron uptake by the Caco-2 cells. The results of our study indicate the usefulness of Caco-2 cells in assessing human iron absorption and the feasibility of this cell model in studying iron bioavailability from various food combinations, otherwise not easily performed in humans.

Inhibitory effect of wheat fibre extract on calcium absorption in Caco-2 cells: evidence for a role of associated phytate rather than fibre per se.

In spite of the strong evidence for the beneficial health effects of dietary fibres, one of the potential nutritional disadvantages of high fibre diets is the adverse effect on the bioavailability of micronutrients, especially minerals and trace elements. With regard to Ca, there is considerable evidence that phytate, which is associated with fibre in many foods, such as cereals and soya products, inhibits Ca absorption. However, there is some doubt as to whether fibres per se have an influence on Ca absorption. Therefore, the purpose of this study was to investigate the effect on Ca absorption of two cereal-based fibre extracts (wheat bran and barley hull). In addition, in order to distinguish between the effect of the fibre components per se and the associated phytate content in these fibre extracts, we investigated the effect of dephytinised wheat and barley fibre extracts and the effect of phytate, as sodium phytate at levels present in the fibre extracts, on Ca absorption. Ca absorption was assessed in Caco-2 cells, as a model for studying Ca absorption in humans. The effect of wheat and barley fibre extracts, dephytinised wheat and barley fibre extracts, cellulose, and of sodium phytate on transepithelial 45Ca transport and 45Ca uptake was studied in differentiated Caco-2 cells grown on permeable filter supports. Wheat and barley fibre extracts were dephytinised with wheat phytase. Wheat fibre extract had a 3.2-fold higher phytate content (48.0 mmol/kg) than barley fibre extract (15.1 mmol/kg). Enzymatic dephytinisation of both fibre extracts reduced the phytate content to undetectable levels. The rate of transepithelial 45Ca transport across Caco-2 cell monolayers and the uptake of 45Ca into Caco-2 cells were unaffected by cellulose or barley fibre extract. On the other hand, inclusion of wheat fibre extract in the Ca transport buffer (50 g fibre/l) significantly reduced the rate of 45Ca transport (by 17 and 19% respectively) and the uptake of 45Ca (by 24 and 25% respectively) relative to a fibre-free buffer and a control fibre (cellulose) transport buffer. Increasing the phytate concentration of the transport buffer from 0 to 2 mM (a level close to that in the wheat fibre containing buffer) significantly reduced the rate of 45Ca transport (by 16%) and 45Ca uptake (by 26%). Dephytinisation of the wheat fibre extract removed its inhibitory effects on 45Ca transport and e.g. 45Ca uptake. The results from the present study in Caco-2 cells suggest that it is the phytate in wheat fibre extract which is the major inhibitory factor of Ca absorption and that wheat fibre per se has little if any inhibitory effect on Ca absorption. In addition, the results of this study support the usefulness of Caco-2 cells for investigating the effects of dietary factors on the cellular uptake and transepithelial intestinal transport of Ca.

Use of Caco-2 cells and LC/MS/MS to screen a peptide combinatorial library for permeable structures

The transepithelial transport of a synthetic peptide combinatorial library containing 375 000 individual peptides was assessed using Caco-2 cell monolayers in order to screen for permeability and deliverability. A series of 150 pools, each containing 2500 tripeptide sequences, were applied to the apical side of Caco-2 monolayers. Basolateral side samples were collected after 4 h and screened by capillary high-pressure liquid chromatography. The majority of pools showed no permeable species, due to low solubility, limited permeability and extensive metabolism. Several pools contained permeable structures, and transport proved reproducible with passage number and time. Permeable structures were identified by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). To discriminate between isobaric structures, several tripeptides were resynthesized and tested as discrete compounds. For example, 1–2% d-Phe– d-Ala– d-Ser–OH was transported across the Caco-2 cell monolayer with a P app value of 0.35–0.69×10 –6 cm/s, which is comparable with the permeability of amino acids (Leu, P app=0.30×10 –6 cm/s) and dipeptides ( l-Val– l-Val, P app=0.18×10 –6 cm/s) (Lennernas, H., Palm, K., Fagerholm, U., Artursson, P., 1996. Comparison between active and passive drug transport in human intestinal epithelial (Caco-2) cells in vitro and human jejunum in vivo. Int. J. Pharm. 127, 103–107; Tamura, K., Bhatnagar, P.K., Takata, J.S., Lee, C.P., Smith, P.L., Borchardt, R.T., 1996. Metabolism, uptake, and transepithelial transport of the diastereomers of Val-Val in the human intestinal cell line Caco-2. Pharm. Res. 13, 1213–1218). These studies demonstrate the techniques used to screen combinatorial libraries for permeability across Caco-2 cells and structurally identify the resulting compounds. Such methodology can be of importance in the achievement of structure–permeability relationships, useful in the design of pharmaceuticallly bioavailable drugs.