Passive transcellular permeability

Let us conclude this section by proposing that provided that the drug is sufficiently soluble in the gastrointestinal fluids, the complex process of intestinal drug absorption can often be satisfactorily described by focusing on passive transport across the cell membrane, and that the development of models that predict passive transcellular permeability is particularly important. Such models are the focus of the remaining part of this chapter. 

The advantages of PAMPA over Caco-2 permeability measurements are higher throughput, lower cost, and shorter planning as there is no cell culture involved. PAMPA measurements help identify compounds that have high passive transcellular permeability and... 

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]. 

PAMPA is typically used to make a prediction of the passive, transcellular absorption of a compound. Compounds which may be absorbed by a paracellular mechanism or may be substrates for active transport (uptake or efflux) are usually better assessed in a cell based system. A combination of assays can be applied to gain a greater understanding of the permeability and transport properties of a compound. 

The successful application of in vitro models of intestinal drug absorption depends on the ability of the in vitro model to mimic the relevant characteristics of the in vivo biological barrier. Most compounds are absorbed by passive transcellular diffusion. To undergo tran-scellular transport a molecule must cross the lipid bilayer of the apical and basolateral cell membranes. In recent years, there has been a widespread acceptance of a technique, artificial membrane permeation assay (PAMPA), to estimate intestinal permeability.117118 The principle of the PAMPA is that, diffusion across a lipid layer, mimics transepithelial permeation. Experiments are conducted by applying a drug solution on top of a lipid layer covering a filter that separates top (donor) and bottom (receiver) chambers. The rate of drug appearance in the bottom wells should reflect the diffusion across the lipid layer, and by extrapolation, across the epithelial cell layer. 

Sugano et al. studied the membrane permeation of 51 benzamidine-based thrombin inhibitors in a rat everted sac permeability model [197]. They reported significant membrane permeabilities in this in vitro model, which they attributed to passive paracellular transport, a different absorption mechanism to transcellular permeability. 

Passive Transcellular Pathway pH Partition Theory as the Basis of Understanding Membrane Permeability... 

Recently, there is some negativity towards PAMPA [52], seemingly due to an overexpectation and misunderstanding of PAM PA and the science of passive membrane permeation [53]. PAMPA is a refined descendant of log Poet and is an improved surrogate measurement for passive transcellular permeation. PAMPA permeability usually correlates well with passive transcellular permeation. It is important to correctly understand the pros and cons of this tool and to use it appropriately in drug discovery. 

Action on the membrane components Numerous studies have shown that the passive transcellular transport of hydrophilic compounds, including macromolecules such as peptides, can be enhanced by interaction of the penetration enhancers with both the phospholipid bilayer and the integrated proteins, thereby making the membrane more fluid and thus more permeable to both lipophilic and hydrophilic compounds. 

There is one major caveat of using the tissue culture transport experiment to study P-gp efflux that cannot be overlooked—P-gp efflux is not directly determined in this experiment. Rather, the effects of P-gp-mediated efflux activity and changes to this activity are inferred from the resulting overall transport data. Particularly with regards to substrate identification, there is the potential for false negatives. For a compound to be affected by P-gp-mediated efflux, it must reach P-gp s binding site that is within the cell. Compounds with poor membrane (transcellular) permeability are not likely to be identified as substrates (395,397). Conversely, compounds with very high passive membrane permeability can saturate P-gp efflux at low micromolar concentrations and are often not identified as substrates... 

The surface area of brush border membranes is 1000 fold larger than paracellular surface area (Pap-penheimer and Reiss 1987). Therefore, the probability for transcellular permeability is much higher than for paracellular permeability. Indeed, lipophilic drugs with rapid and complete absorption have a high probability for passive transcellular route. Hydrophilic drags tend to pass cellular membranes via water filled pores in the paracellular pathway (for review, see Lee et al. 1991). However, there is also a part of hydrophilic molecules passing membranes by transcellular route (Nellans 1991). The paracellular pathway is used by some positively charged compounds whereas transcellular pathway is preferred with unionised compounds. 

Prognosis of a compounds permeability should be made stressing limitations of the model. There is no bioavailability prognosis from in vitro data - a cellular assay can provide only permeability potential through a biological membrane. The membrane, in most cases CACO-2 cells, is very similar to what we observe in vivo in the small intestine and resembles many characteristics to in vivo enterocytes. CACO-2 cells can be used for prediction of different pathways across intestinal cells. Best correlation occurs for passive transcellular route of diffusion. Passive paracellular pathway is less permeable in CACO-2 and correlations are rather qualitative than quantitative for that pathway. CACO-2 cells are an accepted model for identification of compounds with permeability problems, for ranking of compounds and selection of best compounds within a series. Carrier-mediated transport can be studied as well using careful characterization of transporters in the cell batch or clone as a prerequisite for transporter studies. 

Several authors have reviewed the strengths and weaknesses of various modeling approaches for predictive ADMET in early discovery. These approaches involve computational models for permeability in vivo, in vitro, and in situ. In general such models have broad applicability for estimation of both passive transcellular and paracellular permeability. However, we still have not developed a general model that accurately predicts the role of transport proteins in active and facilitated uptake of drugs following oral administration. 

In general, the physicochemical properties of a compound can greatly affect its capacity for passive transcellular BBB permeation. General strategies for modifying drug structures to improve BBB permeability include the following [37, 38] ... 

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. 

Drug absorption generally occurs either through passive transcellular or paracellu-lar diffusion, active carrier transport, or active efflux mechanisms. Several methods have been developed to aid in the understanding of the absorption of new lead compotmds. The most common ones use an immortalized cell line (e.g., Caco-2, Madin-Darby canine kidney, and the like) to mimic the intestinal epithelium. These in vitro models provide more predictive permeability information than the artificial membrane systems (i.e., PAMPA and permeability assays, described previously) based on the cells ability to promote (active transport) or resist (efflux) transport. Various in vitro methods are listed in the U.S. FDA guidelines. These are acceptable to evaluate the permeability of a drug substance, and includes a monolayer of suitable epithelial cells, and one such epithelial cell line that has been widely used as a model system of intestinal permeability is the Caco-2 cell line. 

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