The influence of different drug transport routes in intestinal drug permeability screening assays was studied. Three experimental models were compared: the small-intestine-like 2/4/A1 cell model, which has a leaky paracellular pathway, the Caco-2 cell model, which has a tighter paracellular pathway, and artificial hexadecane membranes (HDMs), which exclusively model the passive transcellular pathway. The models were investigated regarding their ability to divide passively and actively transported compounds into two permeability classes and to rank compounds according to human intestinal absorption. In silico permeability models based on two-dimensional (2D) and three-dimensional (3D) molecular descriptors were also developed and validated using external test sets. The cell-based models classified 80% of the acceptably absorbed compounds (FA >/= 30%) correctly, compared to 60% correct classifications using the HDM model. The best compound ranking was obtained with 2/4/A1 (r(s) = 0.74; r(s) = 0.95 after removing actively transported outliers). The in silico model based on 2/4/A1 permeability gave results of similar quality to those obtained when using experimental permeability, and it was also better than the experimental HDM model at compound ranking (r(s) = 0.85 and 0.47, respectively). We conclude that the paracellular transport pathway present in the cell models plays a significant role in models used for intestinal permeability screening and that 2/4/A1 in vitro and in silico models are promising alternatives for drug discovery permeability screening.
"Since Caco-2 monolayers can withstand quite vigorous stirring conditions, good estimates of passive permeability coefficients in the vicinity of those observed in human in vivo can be obtained (Artursson and Karlsson, 1991; Lennernäs et al., 1996). For compounds displaying an intermediate passive permeability , it is more difficult to predict the intestinal permeability in vivo since in all systems, including the perfused human intestine, there is a steep relationship between permeability and absorption (Lennernäs, 1998; Matsson et al., 2005). In this permeability range, compounds tend to be more polar and have a slower distribution into the cell membranes and/or they may be subjected to active transport processes. "
[Show abstract][Hide abstract] ABSTRACT: Preformulation measurements are used to estimate the fraction absorbed in vivo for orally administered compounds and thereby allow an early evaluation of the need for enabling formulations. As part of the Oral Biopharmaceutical Tools (OrBiTo) project, this review provides a summary of the pharmaceutical profiling methods available, with focus on in silico and in vitro models typically used to forecast active pharmaceutical ingredient's (APIs) in vivo performance after oral administration. An overview of the composition of human, animal and simulated gastrointestinal (GI) fluids is provided and state-of-the art methodologies to study API properties impacting on oral absorption are reviewed. Assays performed during early development, i.e. physicochemical characterization, dissolution profiles under physiological conditions, permeability assays and the impact of excipients on these properties are discussed in detail and future demands on pharmaceutical profiling are identified. It is expected that innovative computational and experimental methods that better describe molecular processes involved in vivo during dissolution and absorption of APIs will be developed in the OrBiTo. These methods will when successful provide early insights into successful pathways (medicinal chemistry or formulation strategy) and are anticipated to increase the number of new APIs with good oral absorption being discovered.
European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences 11/2013; 57(1). DOI:10.1016/j.ejps.2013.10.015 · 3.35 Impact Factor
"compounds used here , the variation of FA data from different sources was quite limited ( less than 15% ) . The only exception was verapamil , for which FA was generally identified in the range 95 – 100% ( Matsson et al . , 2005 ; Zhao et al . , 2001 ; Kansy et al . , 1998 ; Shin et al . , 2009 ) , except for one value of 28% ( Varma et al . , 2004 ) . Being verapamil a substrate for P - gp , its FA may be affected by individual differences in the P - gp activity . Indeed , in vivo data related to actively transported chemicals could be strongly dependent on th"
[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
"The permeability of confluent cell monolayers to soluble factors is also studied using transwell devices . For example, the ability of small molecules to pass culture models of the intestinal lining is one important indicator of the efficacy of drug candidates . The membranes used in transwells are typically 'track-etched' membranes created by bombarding sheets of dense polycarbonate (PC) or polyester-sulfone (PES) with subatomic particles to fracture the polymer backbone. "
[Show abstract][Hide abstract] ABSTRACT: Porous nanocrystalline silicon (pnc-Si) is new type of silicon nanomaterial with potential uses in lab-on-a-chip devices, cell culture, and tissue engineering. The pnc-Si material is a 15 nm thick, freestanding, nanoporous membrane made with scalable silicon manufacturing. Because pnc-Si membranes are approximately 1000 times thinner than any polymeric membrane, their permeability to small solutes is orders-of-magnitude greater than conventional membranes. As cell culture substrates, pnc-Si membranes can overcome the shortcomings of membranes used in commercial transwell devices and enable new devices for the control of cellular microenvironments. The current study investigates the feasibility of pnc-Si as a cell culture substrate by measuring cell adhesion, morphology, growth and viability on pnc-Si compared to conventional culture substrates. Results for immortalized fibroblasts and primary vascular endothelial cells are highly similar on pnc-Si, polystyrene and glass. Significantly, pnc-Si dissolves in cell culture media over several days without cytotoxic effects and stability is tunable by modifying the density of a superficial oxide. The results establish pnc-Si as a viable substrate for cell culture and a degradable biomaterial. Pnc-Si membranes should find use in the study of molecular transport through cell monolayers, in studies of cell-cell communication, and as biodegradable scaffolds for three-dimensional tissue constructs.
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