BBB PAMPA

Fig. 3.4 Permeability profiles for (a) warfarin (acid), (b) propranolol (base) and (c) morphine (ampholyte) based on a BBB PAMPA model (plON) composed of animal brain extract of lipids. The data (unpublished) were analyzed with the pCEL-X program (plON), with the refined parameters indicated in the three frames. In all three cases, there was evidence for the permeation of charged...

In the BBB-PAMPA lipid formulation illustrated in Fig. 3.4, the diff values, defined as the difference log Pq-log Pi, range from 2.9 (morphine) to 4.2 (warfarin), somewhat similar to the values observed in the octanol-water system. However, it is premature to propose a pdiffi-A approximation, given the limited amount of data reported. With other lipid formulations, larger differences are usually observed. In Double-Sink PAM PA, and especially in hexadecane-PAMPA, transport of ionized drugs has not been reported [84]. 

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

In addition to the data generated from plasma samples in a standard PK study, for targets that are contained within the brain, it is also important to determine brain levels of compound, so that brain/plasma ratios can be determined. Again, these data should be correlated with in vitro assays and calculated properties such as BBB PAMPA [39]... 

Composition of the PAM PA membrane varies from a purely organic solvent membrane to a purely phospholipid membrane. At the first international conference of PAM PA in 2002 (www.pampa2002.com/), it was agreed that these variations would be notated as initials or a short adjective at the head of PAMPA (e.g., BM-PAMPA for biomimetic PAMPA). The original PAMPA (Egg-PAMPA) [47], hexadecane membrane PAMPA (HDM-PAMPA) [48], BM-PAMPA [49], double sink PAMPA (DS-PAMPA) and blood-brain barrier PAMPA (BBB-PAMPA) [51] are reviewed elsewhere [3]. 

Artificial membrane permeability assays such as PAMPA (Chen et al., 2008) and BBB PAMPA offer a cost-effective... 

The examination of over 50 PAMPA lipid models has led to an optimized model for gastrointestinal tract (GIT) absorption. Table 7.22 shows six properties of the GIT, which distinguish it from the blood-brain barrier (BBB) environment. 

The in vitro measurements of permeability by the cultured-cell or PAMPA model underestimate true membrane permeability, because of the UWL, which ranges in thickness from 1500 to 2500 pm. The corresponding in vivo value is 30-100 pm in the GIT and nil in the BBB (Table 7.22). The consequence of this is that highly permeable molecules are (aqueous) diffusion limited in the in vitro assays, whereas the membrane-limited permeation is operative in the in vivo case. Correcting the in vitro data for the UWL effect is important for both GIT and BBB absorption modeling. 

TABLE 7.22 In Vitro Double-Sink PAMPA Models for GIT and BBB Targets... 

Many organizations use colon adenocarcinoma (Caco-2) for detailed study of permeability however, this method can be resource intensive. Parallel artificial-membrane permeability (PAMPA) [19] has proven to be a reliable predictor of passive transcellular permeability for intestinal absorption prediction. It is also useful to interpret results of cell-based discovery assays, in which cell-membrane permeability is limiting. Finally, pTf provides insight into the pH dependence of solubility and permeability. It can be measured [20] or calculated to get an understanding of the regions of the intestine in which the compound will be best absorbed, as well as to anticipate the effect of pH on solubility and pemieability. Permeability at the blood-brain barrier (BBB) also can be rapidly profiled [21]. 

The permeability and PAMPA assays as described are robust and reproducible assays for determining passive, transcellular compound permeability. Permeability and PAMPA are automation compatible, relatively fast (4-16 hours), inexpensive, straightforward, and their results correlate with human drug absorption values from published methods. The PAMPA assay provides the benefits of a more biologically relevant system. It is also possible to tailor the lipophilic constituents so that they mimic specific membranes, such as the blood-brain barrier (BBB). Optimization of incubation time, lipid mixture, and lipid concentration will also enhance the assay s ability to predict compound permeability. 

HT-solubility/permeability First, solubility is determined at four pH values by comparing the concentration of a saturated compound solution with its dilute, known as the concentration. The filtered, saturated solution from the solubility assay is then used as input material for the membrane permeability determination. The permeability assay is a parallel artificial membrane technique whereby a membrane is created on a solid support, PAMPA. The two artificial membranes presented here model the GIT and the BBB. Data are presented for control compounds, which are well documented in the literature and exemplify a range of solubility and membrane permeability. The advantages of the combination method are (/) reduction of sample usage and preparation time, ( /) elimination of interference from compound precipitation in membrane permeability determination, Hi) maximization of input concentration to permeability assay for improved reproducibility, and (/v) optimization of sample tracking by streamlining data entry and calculations. BBB permeability ranking of compounds correlates well with literature CNS activity. 

The development of a new coculture-based model of human BBB that enables the prediction of passive and active transport of molecules into the CNS has recently been reported (Josserand et al., 2006). This new model consists of primary cultures of human brain capillary endothelial cells cocultured with primary human glial cells (Megard et al., 2002 Josserand et al., 2006). The advantage of this systan includes the use of human primary cells, avoiding species, age, and interindi-vidual differences since the two cell types are removed from the same human donor and because of the danonstrated expression of functional efflux transporters such as P-gp, MRP-1, MRP-4, MRP-5, and BCRP. Such models have potential for the assessment of permeability of drug and specific transport mechanisms, which is not possible in artificial membrane assays (e.g., PAMPA) or other cell models due to incomplete expression of active transporters. 

Di L, Kerns EH, Bezar IF, Petusky SL, Huang Y. Comparison of blood-brain barrier permeability assays in situ brain perfusion, MDRl-MDCKII and PAMPA-BBB. J Pharm Sci 2009 98 1980-1991. 

A Key Experiment PAMPA-BBB, a Lipid-Based Model for the Blood-Brain Barrier 357... 

A KEY EXPERIMENT PAMPA-BBB, A LIPID-BASED MODEL FOR THE BLOOD-BRAIN BARRIER... 

From a methodological point of view, tite PAMPA-BBB system is quite simple. The lipids, dissolved in dodecane, are soaked with a filter mounted in a two-compartment chamber. The drug is added to the donor compartment (which can be either the upper or lower chamber), and its passage through the artificial membrane is measured in the acceptor compartment (Fig. 14.17). A standard compound with well-characterized permeability proper-hes (e.g., verapamil) is tested in parallel. Compounds that readily cross the blood-brain barrier have an in vitro permeability (P,) > 2.7 10" cm s in the PAMPA assay. On the opposite, drugs with low blood-brain barrier permeation have a < 0.710 cm s". Beside... 

FIGURE 14.17 Schematic description of the PAMPA-BBB assay. The compounds of interest (red disks) are introduced in one compartment (referred to as donor) of a two-compartment chamber separated by a reconstituted bilayer of porcine brain lipids spread on a porous membrane. At the end of incubation, the compounds that have crossed the barrier are harvested in the "acceptor" compartment and dosed. Standard compounds with well-characterized permeability properties are tested in parallel. 

---