Caco-2 cell and PAMPA

High-Throughput Screening Using Caco-2 Cell and PAMPA Systems... 

LC/MS/MS techniques with selective and sensitive detection methods make it possible to quantitatively analyze samples from Caco-2 cell and PAMPA buffer matrices. A high-throughput permeability screen with robust LC/MS technology can quickly generate information about structure-permeability relationships that are extremely valuable in the lead optimization phase for the selection of pre-clinical candidates with favorable oral bioavailability properties. 

PAMPA-QSAR-VolSurf To evaiuate absorption of compounds across the membrane via a transceiiuiar route, the permeability of peptide derivatives and related compounds was measured by the PAMPA. The permeability coefficients by PAMPA were analyzed quantitatively using classical QSAR and VolSurf approaches with the physico-chemical parameters. The results from both approaches showed that hydrogen bonding ability of molecules in addition to hydrophobicity at a particular pH were significant in determining variations in PAMPA permeability coefficients. The relationship between Caco-2 cell permeability and artificial lipid membrane permeability was then determined. The compounds were sorted according to their absorption pathway in the plot of the Caco-2 cell and PAMPA permeability coefficients. 

Verma, R.P., Hansch, C. and Selassie, C.D. (2007) Comparative QSAR studies on PAMPA/modified PAMPA for high throughput profiling of drug absorption potential with respect to Caco-2 cells and human intestinal absorption. Journal of Computer-Aided Molecular Design, 21, 3-22. 

Comparative QSAR studies on PAMPA/modified PAM-PA for high-throughput profiling of drug absorption potential with respect to Caco-2 cells and human intestinal absorption... 

Passive diffusion through the lipid bilayer of the epithelium can be described using the partition coefficient between octanol/water (log P) and A log P (the difference between the partition into octanol/water and heptane/ethylene glycol or heptane/ octanol) [157, 158], The lipophilicity of the drug (log P) (or rather log D at a certain pH) can easily be either measured or calculated, and is therefore generally used as a predictor of drug permeability. Recently, a method using artificial membrane permeation (PAMPA) has also been found to describe the passive diffusion in a similar manner to the Caco-2 cell monolayers [159]. 

The suitability and general applicability of an artificial membrane and PAMPA in vitro permeation methods were evaluated for their ability to predict drug absorption potential in comparison to Caco-2 cell literature data [57], A linear correlation (R2 = 0.957) was obtained between artificial membrane Papp and human absorption data, indicating the good predictive ability of the proposed method for HP compounds with greater differentiation of drugs with /a below 50% [57],... 

Complementary use of PAMPA and Caco-2 cells for evaluation of absorption potential. PAMPA measurements are used to discard compounds with clear absorption problems whereas Caco-2 cells would be used to evaluate mechanisms of permeation or reasons for low permeation. It is highly unlikely that PAMPA measurements would be used to select compounds to be tested in vivo. 

Another in vitro method for permeability screening was parallel artificial membrane permeation assay (PAMPA) initially reported by Kansy. In a PAMPA permeability screen, the Caco-2 cell mono-layer membrane is replaced by an artificially generated membrane. Versions of different artificial membranes that lack active transporter systems and pores have been developed to mimic the in vivo transcellular intestinal epithelial cell barrier. Therefore, the PAMPA screen only measures the intrinsic... 

Discrimination of efflux, active or passive transport is already feasible by suitable in vitro experiments. For instance, the PAMPA assay detects passive transport only, while Caco-2 cells include transporters. A comparison between transport in PAMPA and Caco-2 cells by a calibration plot reveals compounds with greater or less transport in Caco-2 cells than in PAMPA. These compounds should be tested in uptake and efflux transport assays in order to gain deeper insight into absorption fate. 

CLperm can be derived from in vitro permeability experiments (Caco-2, MDCK cells, and PAMPA) or in silico from polar surface area and the number of hydrogen-bond donors [4]. Yang et al. found a clear superiority of the Qj, i model over the well-stirred model in terms of accuracy and precision, particularly when plasma protein binding was ignored [4]. This simplified the model to (12.9) ... 

Van Dijck A, Masungi C, MenschJ, Borremans C, Willems B, Mackie C, Brewster M, and Noppe, M. Parallel Artificial Membrane Permeability Assay (PAMPA) Combined with a 10-Day Multiscreen CACO-2 Cell Culture for Prediction of Passive and Active Absorption of Drugs. 2003 American Association of Pharmaceutical Scientists Annual Meeting and Exposition, Salt Lake City, Utah, USA, October 26-30, 2003. 

Drug absorption generally occurs either through passive transcellular or paracellu-lar diffusion, active carrier transport, or active efflux mechanisms. Several methods have been developed to aid in the understanding of the absorption of new lead compotmds. The most common ones use an immortalized cell line (e.g., Caco-2, Madin-Darby canine kidney, and the like) to mimic the intestinal epithelium. These in vitro models provide more predictive permeability information than the artificial membrane systems (i.e., PAMPA and permeability assays, described previously) based on the cells ability to promote (active transport) or resist (efflux) transport. Various in vitro methods are listed in the U.S. FDA guidelines. These are acceptable to evaluate the permeability of a drug substance, and includes a monolayer of suitable epithelial cells, and one such epithelial cell line that has been widely used as a model system of intestinal permeability is the Caco-2 cell line. 

PAMPA-biomimetic-Caco-2-comparison Several in vitro assays have been developed to evaluate the Gl absorption of compounds. Our aim was to compare three of these methods (/) the BAMPA method, which offers a HT, noncellular approach to the measurement of passive transport ( ) the traditional Caco-2 cell assay, the use of which as a HT tool is limited by the long cell differentiation time (21 days) and (// ) The BioCoat HTS Caco-2 assay system, which reduces Caco-2 cell differentiation to three days. The transport of known compounds (such as cephalexin, propranolol, or chlorothiazide) was studied at pH 7.4 and 6.5 in BAMPA and both Caco-2 cell models. Permeability data obtained was correlated to known values of human absorption. Best correlations (f= 0.9) were obtained at pH 6.5 for BAMPA and at pH 7.4 for the Caco-2 cells grown for 21 days. The Caco-2 BioCoat HTS Caco-2 assay system does not seem to be adequate for the prediction of absorption. The overall results indicate that BAMPA and the 21 -day Caco-2 system can be complementary for an accurate prediction of human intestinal absorption. 

The use of the Caco-2 cell line assays provide more physiologically relevant data than the PAMPA assays since they express transporters so that both active and passive transport can be determined. Caco-2 cell monolayers are contained in a 96-well plate format and test compounds can be incubated to model and test absorption [85],... 

The parallel artificial membrane permeability assay (PAMPA) is a recent development in the area of artificial membranes that appears to offer considerable potential. Measuring the flux values (membrane permeation levels) of a range of test compounds by PAMPA and relating these values to the flux curves obtained in Caco-2 studies have shown good correlations, indicating that the PAMPA assay could be a good alternative to Caco-2 cells for the measurement of passively diffusing compounds. 

Besides CYPs, other metabolic enzymes like hydrolases, phosphatases, peptidases, and even some Phase II conjugating enzymes are also found in Caco-2 cells, so careful LCMS analysis of Caco-2 wells in a detailed experiment can provide not only information about the permeability but also some information on the gut wall metabolism, raising early flags for metabolically unstable compounds. Also, unlike PAMPA, Caco-2 results can reflect a para-cellular transport component, at least to a limited extent. Caco-2 monolayers have an average pore size of about 4 A, compared to an estimated 8-13 A found in the intestinal epithelium, but withholding Ca from the medium or chelating it with EDTA can open these tight junctions somewhat to better reflect in vivo pore size." " ... 

Figure 9.10 Relationship between permeability coefficients obtained from Caco-2 and PAMPA screenings. Circles represent passively transported compounds, diamonds are actively transported, and triangles are compounds subject to efflux. (Reprinted from with permission from Fujikawa, M., et al. Relationship between structure and high-throughput screening permeability of diverse drugs with artificial membranes Application to prediction of Caco-2 cell permeability. Bioorg. Med. Chem., 2005,13, 4721 732, copyright 2005, Elsevier).

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