Sink ionization-maintained

For ionizable sample molecules, it is possible to create an effective sink condition in PAMPA by selecting buffers of different pH in the donor and acceptor compartments. For example, consider salicylic acid (v>Ka 2.88 see Table 3.1). According to the pH partition hypothesis, only the free acid is expected to permeate lipophilic membranes. If the donor pH < 2 and the acceptor pH is 7.4, then as soon as the free acid reaches the acceptor compartment, the molecule ionizes, and the concentration of the free acid becomes effectively zero, even though the total concentration of the species in the acceptor compartment may be relatively high. This situation may be called an ionization-maintained sink. 

Donor wells pH 6 and pH 7 (ionization-maintained sink), 5-10 mm bile acid, such as taurocholic acid or glycocholic acid to solubilize lipophilic molecules (binding-... 

Ionization-maintained sink conditions Owing to the dependence of ionization on pH, weak acids will be more permeable in a gradient system where the pH of the donor is below that of the acceptor, while weak bases will be more permeable in an iso-pH system. Uncharged species will show the same results using either system. If the pH gradient is wide enough, a neutral compound will become ionized once it reaches the receiver wells. 

In this chapter we use the term sink to mean any process that can significantly lower the concentration of the neutral form of the sample molecule in the acceptor compartment. Under the right conditions, the ionization and the binding sinks serve the same purpose as the physically maintained sink often used in Caco-2 measurements. We will develop several transport models to cover these chemical sink conditions. When both of the chemical sink conditions (ionization and binding) are imposed, we will use the term double sink in this chapter. 

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. 

ThepION Inc. method uses double-sink conditions, both ionization and binding maintained. 

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