# Combined application of parallel membrane permeability assay and Caco-2 permeability assays in drug discovery

ArticleinJournal of Pharmaceutical Sciences 93(6):1440-53 · June 2004with14 Reads
Impact Factor: 2.59 · DOI: 10.1002/jps.20075 · Source: PubMed
Abstract

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.

# Figures

• "...l., 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..."
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).
##### Accelerated Caco-2 cell permeability model for drug discovery
[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.
Full-text · Article · Aug 2013 · Journal of pharmacological and toxicological methods
+3 more authors...
• "...Furthermore, to accurately reproduce the BBB and the g.i. tract (Kerns et al., 2004; Di et al., 2003; Carrara et al., 2007), the permeability of prodrugs was determined at pH 7.4 in the PAM- PA–BBB, and at pH 5..."
Furthermore, to accurately reproduce the BBB and the g.i. tract (Kerns et al., 2004; Di et al., 2003; Carrara et al., 2007), the permeability of prodrugs was determined at pH 7.4 in the PAM- PA–BBB, and at pH 5.0, 6.5 and 7.4 in the PAMPA–GI, respectively. Since our prodrugs were not thoroughly soluble in the conventional solution used for the PAMPA, the permeability tests of compounds 1 and 2 were examined in the presence of cosolvents (2% of Tween 80 in the case of prodrug 1 and 2% of Tween 80 and 5% DMSO in the case of prodrug 2).
##### Memantine-sulfur containing antioxidant conjugates as potential prodrugs to improve the treatment of Alzheimer's disease
[Hide abstract] ABSTRACT: The approved treatments for Alzheimer's disease (AD) exploit mainly a symptomatic approach based on the use of cholinesterase inhibitors or N-Methyl-D-aspartate (NMDA) receptor antagonists. Natural antioxidant compounds, able to pass through the blood-brain barrier (BBB), have been extensively studied as useful neuroprotective agents. A novel approach towards excitotoxicity protection and oxidative stress associated with excess β amyloid (Aβ) preservation in AD is represented by selective glutamatergic antagonists that possess as well antioxidant capabilities. In the present work, GSH (1) or (R)-α-lipoic acid (LA) (2) have been covalently linked with the NMDA receptor antagonists memantine (MEM). The new conjugates, proposed as potential antialzheimer drugs, should act both as glutamate receptor antagonists and radical scavenging agents. The physico-chemical properties and "in vitro" membrane permeability, the enzymatic and chemical stability, the demonstrated "in vitro" antioxidant activity associated to the capacity to inhibit Aβ(1-42) aggregation makes at least compound 2 a promising candidate for treatment of AD patients.
Full-text · Article · Feb 2013 · European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences
+8 more authors...
• "...er layer (UWL) and high implementation costs (Balimane et al., 2000; Deferme et al., 2008; Kerns et al., 2004; Li et al., 2008; Masungi et al., 2008; Reis, Sinko, & Serra, 2010). Intestinal perfusion studies ar..."
Furthermore, there are no metabolism processes in PAMPA (Balimane et al., 2000; Balimane et al., 2006; Deferme et al., 2008; Kerns et al., 2004; Li et al., 2008 ). Cell cultures of Caco-2 and MDCK present limitations such as: expense of cell culture, variable expression of transporters and metabolizing proteins, contamination risk, long cell growth cycle, lack of standardized protocols, use of serum proteins and co-solvents, influence of unstirred water layer (UWL) and high implementation costs (Balimane et al., 2000; Deferme et al., 2008; Kerns et al., 2004; Li et al., 2008; Masungi et al., 2008; Reis, Sinko, & Serra, 2010). Intestinal perfusion studies are closer to in vivo conditions.
##### Determination of lamivudine and zidovudine permeability using a different ex vivo method in Franz cells
[Hide abstract] ABSTRACT: Introduction: The major processes that control the absorption of orally administered drugs are dissolution and gastrointestinal permeation. These processes depend on two main properties: solubility and permeability. Based on these characteristics, the Biopharmaceutical Classification System (BCS) was proposed as a tool to assist in biowaiver and bioavailability prediction of drugs. Methods: The purpose of the present study was to evaluate the permeability of lamivudine (3TC) and zidovudine (AZT) using a different ex vivo method in Franz cells. A segment of jejunum was inserted in a Franz cells apparatus, in order to assess drug permeability in the apical-basolateral (A-B) and basolateral-apical (B-A) directions. Each drug was added to the donor chamber, collected from the acceptor chamber and analyzed by HPLC. Fluorescein (FLU) and metoprolol (METO) were used as low and high permeability markers, respectively. Results: The apparent permeability (Papp) results for the A-B direction were: Papp FLU A-B=0.54×10(-4)cm·s(-1), Papp METO A-B=7.99×10(-4)cm·s(-1), Papp 3TC A-B=4.58×10(-4)cm·s(-1) and Papp AZT A-B=5.34×10(-4)cm·s(-1). For the B-A direction, the Papp results were: Papp FLU B-A=0.56×10(-4)cm·s(-1), Papp METO B-A=0.25×10(-4)cm·s(-1), Papp 3TC B-A=0.24×10(-4)cm·s(-1) and Papp AZT B-A=0.19×10(-4)cm·s(-1). Discussion: For the A-B direction, the Papp results of fluorescein and metoprolol show low and high permeability, respectively, indicating that the membranes were appropriate for permeability studies. For the A-B direction, the Papp results of 3TC and AZT suggest that these antiretroviral drugs have permeability values close to metoprolol. Nevertheless, for the B-A direction the Papp results do not suggest efflux mechanism for any of the drugs. Thereby, the different ex vivo methods using Franz cells can be successfully applied in drug permeability studies, in particular for drug biopharmaceutical classification.
Full-text · Article · Jan 2013 · Journal of pharmacological and toxicological methods
• "...ture of the permeability, it was used mainly for the prediction of the gastrointestinal absorption [10]. Attempts to modify the monolayer to improve the prediction of BBB penetration were done using porc..."
A comparison of the three most used PAMPA models viz., Hexadecane Membrane (HDM), Dioleyoylphosphatidylcholine (DOPC) and Double Sink (DS- PAMPA) were recently carried out by Avdeef and Tsinman [9] and explaining permeability differences. Because of the specific nature of the permeability, it was used mainly for the prediction of the gastrointestinal absorption [10]. Attempts to modify the monolayer to improve the prediction of BBB penetration were done using porcine polar brain lipids [11].
##### Optimization and Validation of an in vitro Blood Brain Barrier Permeability Assay using Artificial Lipid Membrane.
Full-text · Article · Jan 2012 · Journal of Bioequivalence & Bioavailability
• "...rated 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 Cac..."
Contents lists available at ScienceDirect Toxicology in Vitro (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 Artursson et al., , 2001 Rubas et al., 1996; Yamashita et al., 2000).
##### Caco-2/TC7 cell line characterization for intestinal absorption: How reliable is this in vitro model for the prediction of the oral dose fraction absorbed in human?
[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.
Full-text · Article · Feb 2011 · Toxicology in Vitro
+2 more authors...
• "...; 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., 20..."
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.
##### Prediction of the in vitro permeability determined in Caco-2 cells by using artificial neural networks
[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.
Full-text · Article · Sep 2010 · European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences