Permeability

Comparison

PAMPA

Caco-2

Membrane

Phospholipid

Cell Monolayer

Mechanisms

Passive

Passive, Influx, Efflux, Metabolism

Throughput

500 / week

30 / week

Cost

< $1 / sample

~ $30 / sample

Manpower

035 FTE

2 FTE

Table 3. Comparison of PAMPA and Caco-2 Assay

Table 3. Comparison of PAMPA and Caco-2 Assay works. Compounds are diluted with pH buffer (e.g., pH 7.4) from DMSO stock solution and added to the donor wells. The acceptor is a filter plate, coated with lipid (PC in dodecane) and filled with buffer (pH 7.4). The acceptor plate is placed carefully on the donor plate to form the PAMPA sandwich. Compounds will diffuse through the lipid membrane and enter the acceptor. After a certain period of time, the acceptor plate is removed from the donor plate. The concentration in both the acceptor and the donor is determined using a UV plate reader. From these concentrations the effective permeability (Pe) is derived. There are several variations of the PAMPA assay being used in the industry, including different lipid compositions, different filter membranes and thickness, different buffer pHs, acceptor sink conditions, sink composition, and different quantitation methods (e.g., UV plate reader, LC-UV, LC-MS-MS). The variations are intended to mimic in vivo conditions to produce higher correlations. For example, sink conditions trap molecules on the acceptor side, mimicking in vivo trapping by protein binding and blood flow away from the intestine.

Before PAMPA came along, the Caco-2 assay was commonly used in the industry for permeability measurement. The comparison of PAMPA and Caco-2 is shown in Table 3. The membrane in PAMPA is composed of phospholipid in alkane, and Caco-2 is composed of a Caco-2 cell monolayer. The transport mechanism of PAMPA is passive diffusion. Caco-2 is a multiple mechanism assay: passive diffusion, active influx and efflux. Compounds can also be metabolized in Caco-2. The throughput of PAMPA is approximately 500 compounds / week and Caco-2 is approximately 30 compounds / week. The cost of PAMPA is less than $1 / sample and Caco-2 is about $30 / sample. It takes 0.35 FTE to run PAMPA assay, but 2 FTE to run Caco-2 assay. Thus, PAMPA is more applicable for early drug discovery and Caco-2 is more applicable for late-stages of drug discovery and development. The combined use of the two assays can help diagnose transport mechanisms. It has been reported that PAMPA has good correlation with cell-based assays, such as Caco-2 and MDCK, for compounds that are passively absorbed (Kerns et al., 2004). For compounds that are actively influxed, Caco-2 gives higher permeability relative to PAMPA. For compounds that are effluxed, Caco-2 or MDR1-MDCKII gives comparatively lower permeability than PAMPA.

Structure-Permeability Relationship

Increasing lipophilicity and decreasing H-bonds are two major approaches to improve permeability. Scheme 5 shows an example from a discovery project. The substituents are for a common core structure. The compounds with methyl or chlorine substituents have high PAMPA permeability, the compounds with methoxyl or fluorine atoms showed low permeability. This is consistent with an increase in permeability that results from a decrease in H-bonding and polarity.

/CH3
0.4

Scheme 5. Examples of Increasing Permeability by Decreasing H-Bonds and Polarity. PAMPA Permeability is in Pe x10-6 cm/s.

Scheme 6 shows the effect of lipophilicity on Caco-2 permeability for a series of phenylalanine dipeptides. As the substituents change through the progression H, methyl, i-propanyl, i-butyl, benzyl and cyclohexanylmethyl groups, the lipophilicity increases and results in increasing permeability (Goodwin et al., 2001).

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