Data from permeability profiling using the parallel artificial membrane permeability assay (PAMPA) and cell monolayer (Caco-2 and MDR1-MDCKII) methods were compared for two published compound sets and one in-house set. A majority of compounds in each set correlated (R(2) = 0.76-0.92), indicating the predominance of passive diffusion in the permeation of these compounds. Compounds that did not correlate grouped into two subsets. One subset had higher PAMPA permeability than cell monolayer permeability and consisted of compounds that are subject to secretory mechanisms: efflux or reduced passive diffusion of bases under Caco-2 when run under a pH gradient. The other subset had higher cell monolayer permeability than PAMPA permeability and consisted of compounds that are subject to absorptive mechanisms: paracellular, active transport, or increased passive diffusion of acids under Caco-2 when run under a pH gradient. Given the characteristics of the two methods, these studies suggest how PAMPA and Caco-2 can be synergistically applied for efficient and rapid investigation of permeation mechanisms in drug discovery. During early discovery, all compounds can be rapidly screened using PAMPA at low pH and neutral pH to assess passive diffusion permeability to indicate potential for gastrointestinal and cell assay permeation. During intermediate discovery, selected compounds can be additionally assayed by apical-to-basolateral Caco-2, which, in combination with PAMPA data, indicates susceptibility to additional permeation mechanisms (secretory and absorptive). During mid-to-late discovery, selected candidates can be examined in detail via multiple directional Caco-2 experiments and with transporter inhibitors for complete characterization of permeation mechanisms.
"However, a major practical shortcoming of these cells is the long culturing period of at least 21 days with typically a minimum of 9 laborious cell-feeding steps to allow for full differentiation of the Caco-2 cell monolayers (Yamashita et al., 2002a). This long culturing period limits the throughput and usefulness of the model (Kerns et al., 2004). It has been estimated that a typical Caco-2 cell permeability study, (culturing the cells for 21-days prior to a uni-directional (A → B) permeability assessment), costs ~ US$19 per compound (Balimane & Chong, 2005). "
[Show abstract][Hide abstract] ABSTRACT: By culturing Caco-2 cells according to a new and optimized protocol, it has been possible to accelerate the cell culture process in such a way that the cells can be used for experiments after only 6days. The accelerated Caco-2 model has been compared to the traditional model (requiring 21-25days of culture) in terms of tightness of the junctions, ability to rank chemical compounds for apparent permeability, active efflux and to discriminate P-gp substrates.
In the new protocol, Caco-2 cells were cultured with the classical Caco-2 medium supplemented with puromycin. The initial cell seeding density was increased two times compared to the traditional procedure and the presence of a low concentration of puromycin in the culture medium reduced the Caco-2 permeability of mannitol. Bi-directionally studies were performed with known P-gp substrates (rhodamine 123, digoxin and saquinavir) and with a total of 20 marketed drugs covering a wide range of physicochemical characteristics and therapeutic indications. Strong correlations were obtained between the apparent permeability in absorptive (Papp A→B) or secretory (Papp B→A) of the drugs in the accelerated model and in the traditional models and comparable efflux ratios were observed in the two studied models.
The new protocol reduces costs for screening and leads to higher throughput compared to traditional Caco-2 cell models. This accelerated model provides short time-feedback to the drug design during the early stage of drug discovery.
Journal of pharmacological and toxicological methods 08/2013; 68(3). DOI:10.1016/j.vascn.2013.07.004 · 2.39 Impact Factor
"As reviewed in some papers Author's personal copy (Balimane et al., 2000; Nigsch et al., 2007), a number of in vitro models for intestinal absorption are available, which can be classified in physicochemical and biological methods. The advantages of integrated approaches, by using a combination of models have been presented (Miret et al., 2004; Kerns et al., 2004). However, the cell-based methods alone are at present the most widely used, and among them the Caco-2 cell line has been extensively studied (Artursson, 1991; Artursson et al., 1994, 2001; Rubas et al., 1996; Yamashita et al., 2000). "
[Show abstract][Hide abstract] ABSTRACT: Caco-2 cell line is one of the most used in vitro model to study intestinal absorption of compounds at screening level. Several clones have been isolated from Caco-2 cell line and characterized for their activities. Among them, TC7 clone was isolated from a late passage of the parental Caco-2 line and has shown to consist of a more homogeneous population with respect to the most representative functions of the small intestinal enterocytes, with more developed intercellular junctions. On the basis of these characteristics, it was selected within the framework of the EU A-Cute-Tox project to check its suitability to predict intestinal transport. In the present study, drugs, synthetic or natural chemicals have been characterized for their absorption profile in TC7 cells cultivated on semi-permeable filters for 21 days. The absorption experiments have been performed with the highest nontoxic concentration as determined in a preliminary set of cytotoxicity tests. The apparent permeability coefficient (P(app)) has been extrapolated by calculating the passage of the test compound from the donor to the receiver compartment as a time function. The samples have been collected at different time intervals and the concentration of the test compounds analyzed by analytical methods (HPLC, GC, GC/MS). The P(app) obtained with the TC7 clone are comparable to those obtained with the parental cell line. However, some drawbacks related to the experimental system have been highlighted (i.e. low mass balance, adsorption to the plastics), on the basis of which some compounds were excluded from the analysis. In order to check the predictability of the model, a regression analysis has been performed by plotting P(app) values vs. the fraction absorbed in humans (FA, expressed as % of the administered dose). Additional elaborations have highlighted that the specific absorption pathway (passive, active and carrier-mediated) and other factors (i.e. efflux proteins and/or metabolic activity) can strongly affect the robustness of the prediction model. On the basis of the obtained results, TC7 clone has shown to be a model for passive diffusion as reliable as the parental cell line. However, we have remarked the non-suitability of the TC7 cells to predict intestinal absorption: (i) for highly lipophilic compounds; (ii) for poorly absorbed compounds; or (iii) when transporter-mediated routes and/or first pass metabolism are involved. The preliminary study of those factors likely influencing compound biokinetics, as well as the characterization of the cellular model with respect to metabolic and transporter competence, would help in the interpretation of data.
Toxicology in Vitro 02/2011; 25(1):13-20. DOI:10.1016/j.tiv.2010.08.009 · 2.90 Impact Factor
"Predicted values and the residuals between observed and predicted values, using the 12-6- 3-3-1 network are also presented. Data values were obtained from references (Alt et al., 2004; Anand et al., 2003; Artursson et al., 1993; Behrens and Kissel, 2003; Bhardwaj et al., 2005; Caldwell et al., 1998; Cavet et al., 1997; Crowe, 2002; Crowe and Lemaire, 1998; Da Violante et al., 2004; Ekins et al., 2001; Ertl et al., 2000; Fujiwara et al., 2002; Fukada et al., 2002; Furfine et al., 2004; Hartter et al., 2003; He et al., 2004; Hilgendorf et al., 2000; Hu and Borchardt, 1990; Hunter et al., 1993; Irvine et al., 1999; Karlsson et al., 1999; Kerns et al., 2004; Khan et al., 2004; Lalloo et al., 2004; Lee et al., 2005; Marino et al., 2005; Markowska et al., 2001; Martel et al., 2003; Masungi et al., 2004; Matthias et al., 2004; Neuhoff et al., 2003; Nishimura et al., 2004; Nordqvist et al., 2004; Oka et al., 2002; Pachot et al., 2003; Pade and Stavchansky, 1998; Polli and Ginski, 1998; Saitoh et al., 2004; Soldner et al., 2000; Stenberg et al., 2001; Sun et al., 2003; Tammela et al., 2004; Tannergren, 2004; Tronde, 2002; Vaidyanathan and Walle, 2003; Watson et al., 2001; Wong et al., 2002; Yang and Wang, 2003; Zhou et al., 2004; Camenisch et al., 1998; Gan et al., 1993; Hou et al., 2004; Laitinen et al., 2003; Miret et al., 2004; Raoof et al., 1996; Ribadeneira et al., 1996; Yazdanian et al., 1998). error decreases in an early period of training, reaches a minimum and then increases as training goes on, while the training error monotonically decreases. "
[Show abstract][Hide abstract] ABSTRACT: Caco-2 cells are currently the most used in vitro tool for prediction of the potential oral absorption of new drugs. The existence of computational models based on this data may potentiate the early selection process of new drugs, but the current models are based on a limited number of cases or on a reduced molecular space. We present an artificial neural network based only on calculated molecular descriptors for modelling 296 in vitro Caco-2 apparent permeability (P(app)) drug values collected in the literature using also a pruning procedure for reducing the descriptors space. LogP(app) values were divided into a training group of 192 drugs for network optimization and a testing group of another 59 drugs for early stop and internal validation resulting in correlations of 0.843 and 0.702 and RMSE of 0.546 and 0.791 for the training and testing group, respectively. External validation was made with an additional group of 45 drugs with a correlation of 0.774 and RMSE of 0.601. The selected molecular descriptors encode information related to the lipophilicity, electronegativity, size, shape and flexibility characteristics of the molecules, which are related to drug absorption. This model may be a valuable tool for prediction and simulation in the drug development process, as it allows the in silico estimation of the in vitro Caco-2 apparent permeability.
European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences 09/2010; 41(1):107-17. DOI:10.1016/j.ejps.2010.05.014 · 3.35 Impact Factor
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