Permeability assays

Instead of using surrogate measures for oral absorption with a lipophilicity or permeability assay in vitro, oral absorption can also be estimated in silica by using... 

An overview of permeability assays is presented in Table 2.2. As discussed earlier in this chapter, these permeability scales are correlated to each other as well as the various hpophilicity scales via extended Collander equations. 

Kansy, M., Senner, F., Gubemator, K. Physicochemical high throughput screening parallel artificial membrane permeability assay in the description of passive absorption processes. J. Med. Chem. 1998, 41, 1007-1010. 

BBB PAMPA Blood brain barrier parallel artificial membrane permeability assay... 

PARALLEL ARTIFICIAL-MEMBRANE PERMEABILITY ASSAY (PAMPA)... 

The partition coefficient is needed to determine the moles lost to the membrane, VM CM(t). If ionizable compounds are considered, then one must decide on the types of partition coefficient to use -Kp (true pH-independent partition coefficient) or Kd (pH-dependent apparent partition coefficient). If the permeability assay is based on the measurement of the total concentrations, Cn(t) and CA(t), summed over all charge-state forms of the molecule, and only the uncharged molecules transport across the membrane to an appreciable extent, it is necessary to consider the apparent partition (distribution) coefficient, Kd, in order to explain the pH dependence of permeability. 

Zhu, C. Chen, T.-M. Hwang, K., A comparative study of parallel artificial membrane permeability assay for passive absorption screening, in CPS A2000 The Symposium on Chemical and Pharmaceutical Structure Analysis. Milestone Development Services. Princeton, NJ, Sept. 26-28, 2000. 

Sugano, K. Hamada, H. Machida, M. Ushio, H., High throughput prediction of oral absorption Improvement of the composition of the lipid solution used in parallel artificial membrane permeability assay, J. Biomolec. Screen. 6, 189-196 (2001). 

This book is written for the practicing pharmaceutical scientist involved in absorption-distribution-metabolism-excretion (ADME) measurements who needs to communicate with medicinal chemists persuasively, so that newly synthesized molecules will be more drug-like. ADME is all about a day in the life of a drug molecule (absorption, distribution, metabolism, and excretion). Specifically, this book attempts to describe the state of the art in measurement of ionization constants (p Ka), oil-water partition coefficients (log PI log D), solubility, and permeability (artificial phospholipid membrane barriers). Permeability is covered in considerable detail, based on a newly developed methodology known as parallel artificial membrane permeability assay (PAMPA). 

The literature survey in this section suggests that the ideal in vitro permeability assay would have pH 6.0 and 7.4 in the donor wells, with pH 7.4 in the acceptor wells. (Such a two-pH combination could differentiate acids from bases and non-ionizables by the differences between the two Pe values.) Furthermore, the acceptor side would have 3% wt/vol BSA to maintain a sink condition (or some sinkforming equivalent). The donor side may benefit from having a bile acid (i.e., taurocholic or glycocholic, 5-15 mM), to solubilize the most lipophilic sample molecules. The ideal lipid barrier would have a composition similar to those in Table 3.1, with the membrane possessing a substantial negative charge (mainly from PI). Excessive DMSO/other co-solvents would be best avoided, due to their unpredictable effects. 

The trend in the industry has been to automate the Caco-2 permeability assay using semi- or fully automated procedures. With such a system it is possible to obtain a throughput in order of approximately 400-500 compounds per week. Automated Caco-2 assay systems are commercially available through Tecan/BD Bioscience and Bohdan Mettler Toledo. In addition, automated systems for maintenance of cell cultures are commercially available, while totally automated systems for both maintenance and culturing of cells grown on permeable filter supports are under development, e.g., by companies such as The Automation Partnership. 

Alsenz J, Haenel E (2003) Development of a 7-day, 96-well Caco-2 permeability assay with high-throughput of P-gp screening in drug discovery. Eur J Pharm Biopharm 58 99-105. 

Kerns EH, Di L, Petusky S, Farris M, Ley R, Jupp P (2004) Combined application of parallel artificial membrane permeability assay and Caco-2 permeability assays in drug discovery. J Pharm Sci 93 1440-1453. 

Sugano K, Hamada H, Machida M, Ushio H, Saitoh K, Terada K (2001b) Optimized conditions of bio-mimetic artificial membrane permeability assay. Int J Pharm 228 181-188. 

Zhu C, Jiang L, Chen TM, Hwng KK (2002) A comparative study of artificial membrane permeability assay for high-throughput profiling of drug absorption potential. Eur J Med Chem 37 399-407. 

The Caco-2 permeability assay is usually performed in a Transwell device (Figure 18.1). The Transwell contains two compartments a donor and a receiver compartment. The apical donor compartment contains a porous membrane that supports the growth of the Caco-2 monolayer. Caco-2 cells are seeded on the porous membrane. Upon confluency of the cell culture, the compound is added into the donor compartment at a concentration range from one to several hundred micromolar. Samples are collected from the receiver compartment for up to 2 h, then LC-UV or LC-MS methods are used to quantify compound in each sample. The permeability coefficient of the compound is calculated based on the following equation ... 

Table 18.3 Parallel artificial membrane permeability assay (PAMPA) permeability and human absorption for 19 drug compounds.

Table 28.2 IVIVC with Caco-2 Cell Permeability Assays.

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