Partition membrane/buffer

In PAMPA measurements each well is usually a one-point-in-time (single-timepoint) sample. By contrast, in the conventional multitimepoint Caco-2 assay, the acceptor solution is frequently replaced with fresh buffer solution so that the solution in contact with the membrane contains no more than a few percent of the total sample concentration at any time. This condition can be called a physically maintained sink. Under pseudo-steady state (when a practically linear solute concentration gradient is established in the membrane phase see Chapter 2), lipophilic molecules will distribute into the cell monolayer in accordance with the effective membrane-buffer partition coefficient, even when the acceptor solution contains nearly zero sample concentration (due to the physical sink). If the physical sink is maintained indefinitely, then eventually, all of the sample will be depleted from both the donor and membrane compartments, as the flux approaches zero (Chapter 2). In conventional Caco-2 data analysis, a very simple equation [Eq. (7.10) or (7.11)] is used to calculate the permeability coefficient. But when combinatorial (i.e., lipophilic) compounds are screened, this equation is often invalid, since a considerable portion of the molecules partitions into the membrane phase during the multitimepoint measurements. 

The apparent membrane-buffer partition (distribution) coefficient Kd, defined at... 

Ketoprofen, a weak-acid drug, with a pKa 4.12 (25°C, 0.01 M ionic strength), was selected to illustrate Eqs. (7.20) and (7.21) in a series of simulation calculations, as shown in Fig. 7.16. The membrane-buffer apparent partition coefficients /(piii were calculated at various pH values, using the measured constants from... 

Each side of the barrier has a different membrane-buffer apparent partition coefficient Kd, defined at f = oo as... 

The role of the ammonium salt anion is not the loading of the amphipathic weak base per se, but rather to control the stability of loading and the profile and rate of release of the amphipathic weak base from the liposome to the external aqueous phase. Two major factors that differentiate the different anions are, firstly, their ability to induce precipitation/crystallization/ gelation in the intraliposome aqueous phase (1,12), and secondly, their effect on the membrane/buffer and octanol/buffer partition coefficient of the amphipathic weak base (1). Regarding the precipitation, the higher the amount of precipitated amphipathic weak base, the more stable is the loading and the slower is its release rate (10-12,18,33,35) and (Wasserman et al.). There are also some risks involved in the precipitation which in some cases reduce the mechanical stability of the liposomes and change liposome shape (36). 

We analyzed the correlation of the membrane/buffer partition coefficient (Pm/ ) obtained by Roth and Seeman (135) for 14 selected anesthetics with octanol/water partition coefficients (112)... 

Interestingly, Roth and Seeman (135) found that the membrane/ buffer partition coefficient is invariably one fifth of the octanol/water partition coefficient, in disagreement with equation 94. 

The toxicity and the affinity of solvents to cell structures increase with hydrophobic properties of solvents, e.g., high toxicities with P, values of l-S. The partition coefficient correlates with the membrane-buffer partition coefficient between membrane and aqueous system. They also depend on membrane characteristics. 

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. 

Barbato, F. La Rotonda, M. I. Quaglia, E, Interactions of nonsteroidal antiinflammatory drugs with phospholipids comparison between octanol/buffer partition coefficients and chromatographic indexes on immobilized artificial membranes, J. Pharm. Sci. 86, 225-229 (1997). 

The method of introduction of the fluorophore into the membrane is also important. Many probes are introduced into preexisting vesicles, natural membranes, or whole cells by the injection of a small volume of organic solvent containing the fluorophore. For DPH, tetrahydrofuran is commonly used, while methanol is often employed for other probes. The amount of solvent used should be the absolute minimum possible to avoid perturbation of the lipids, since the solvent will also partition into the membrane. With lipid vesicles this potential problem can be avoided by mixing the lipids and fluorophore followed by evaporation of the solvent and codispersing in buffer. For fluorophores attached to phospholipids, this is the only way to get the fluorophore into the bilayer with natural membranes, phospholipid exchange proteins or other techniques may have to be employed. 

The octanol/buffer represents a partition coefficient between two bulk phases it is less affected by the structure of the analyte and therefore it cannot be used to predict the exact value of liposome membrane-to-buffer Xp, which is also affected by the geometry of the analyte (41 4). However, it is accepted and established that the octanol-to-buffer can help to predict transmembrane passive diffusion (40). In the case of liposomes such as Doxil, in which the internal aqueous phase (intraliposome aqueous phase) is different from the external liposome aqueous medium due to large differences in the composition and pH of these two aqueous phases, there are two different liposome membrane-to-aqueous phase partition coefficients this is referred to as asymmetry in the membrane-to-aqueous media partition coefficient. 

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