Active Transports and Efflux

Palm and co-workers, as part of their research regarding the applicability of PSA, selected 20 compounds (Table 16.1) with quite reliable values for the percent fraction absorbed (%FA) in humans that were devoid of, or compensated for, problems such as active transport and efflux mechanisms [2]. The dataset consists of a variety of compounds drugs, drug-like compounds, as well as sugar-like structures. A number of studies based on this dataset using various structural descriptions have been performed [2, 17-22],... 

Secondary Active transports and efflux hERG patch clamp... 

Choroid-Bruch s membrane-RPE 7) Passive diffusion across these tissues 8) Active transport and efflux in RPE 9) Clearance via choroidal circulation 10) Binding to melanin 7) In vitro permeabiUty of human and bovine tissues in vivo permeabiUty of rabbit RPE 8) In vitro permeabiUty in rabbit and porcine RPE in vivo permeabiUty of rabbit RPE 9) Choroidal blood flow in humans and several animal species in vivo permeabiUty of rabbit and cat choriocapiUaris 10) Melanin amount in human choroid-RPE and binding parameters of drugs to melanin... 

The advantage of cell culture models is that they are able to measure active transport processes across the cell membranes and not just the interaction of a drug with a lipid bilayer. They can also be used to study passive and active transport routes indeed, much of the knowledge as to the active transport mechanisms in the intestine has been derived from cell culture studies. Despite the predominant route being passive diffusion, the research into transport mechanisms indicates that there are a large number of drugs that are used as substrates for active transporter and efflux systems, and it must therefore be appreciated that multiple transport routes may be involved in the intestinal drug transport. 

FIG. 2 Mechanisms of drug transfer in the cellular layers that line different compartments in the body. These mechanisms regulate drug absorption, distribution, and elimination. The figure illustrates these mechanisms in the intestinal wall. (1) Passive transcellular diffusion across the lipid bilayers, (2) paracellular passive diffusion, (3) efflux by P-glycoprotein, (4) metabolism during drug absorption, (5) active transport, and (6) transcytosis [251]. 

Culture protocols have been published which describes an accelerated differentiation process where monolayers are ready to be used after 3-7 days of culture [90-92]. One of these systems, the so-called BD BioCoat Intestinal Epithelium Differentiation Environment, is commercially available through BD Bioscience. This system is described to produce monolayers of a quality that are comparable with the typical Caco-2 cells with respect to permeability for drugs transported transcellularly. The paracellular barrier function is however low, as indicated by high mannitol permeability and low TER. The functional capacity for active uptake and efflux is not as thoroughly characterized as for the standard Caco-2 mono-layers. 

The availability of human Pgfr data is more limited than Fa% data and is biased towards highly absorbed compounds. Therefore, the use of in vivo P ff data is more limited for understanding incompletely absorbed compounds which may be subject to paracellular transport or active uptake and efflux mechanisms. 

CRITICAL ASSESSMENT OF THE METHOD VolSurf descriptors are able to predict absorption for a diverse set of drugs. The presented model is derived using a consistent frame of relevant chemically interpretable descriptors, which find applications in different local and general models. However, absorption is not only controlled by passive membrane permeability. There are other factors influencing in vivo human absorption namely the in vivo dissolution rate in small intestinal fluid and the dose used for the human study. Furthermore, active transport or efflux mechanisms are difficult to rule out but can only be partially monitored by in vitro experiments. These important pieces of information should be known before any QSAR analysis is attempted on human absorption. This lack of consistent information throughout the literature is difficult to overcome, in particular for human studies. Hence, this study for the dataset from Zhao et al. (2001) provides a reasonable attempt to address these problems to carefully selecting members of the final dataset. 

Also the fact that only transcellular permeation is regarded in PAMPA experiments is valuable as it allows comparing PAMPA results to cellular permeation experiments that feature all possible permeation mechanism including paracellular or active transport and active efflux. Kerns (2004) recommended this comparison to get an insight into the permeation mechanism applied by a compound under investigation. The possibility to obtain permeability pH profiles is also really helpful to identify the relevant permeability value of a compound and cannot be determined by cellular assays due to the limited pH range usable with living cells. 

Three processes are involved in transcellular transport across the intestinal epithelial cells simple passive trans-port, passive diffusion together with an efflux pump, and active transport and endocytosis. Simple passive transport is the diffusion of molecules across the membrane by thermodynamic driving forces and does not require direct expenditure of metabolic energy. In contrast, active transport is the movement of molecules across the mem-brane resulting directly from the expenditure of metabolic energy and transport against a concentration gradient. Endocytosis processes include three mechanisms fluid-phase endocytosis (pinocytosis), receptor-mediated endocytosis, and transcytosis (Fig. 6). Endocytosis processes are covered in detail in section Absorption of Polypeptides and Proteins, later. 

In the assessment of placental toxicology of any foreign chemical substances, there are two major areas of concern what the placenta does to xenobiotics and what xenobiotics do to the placenta (Myllynen et ai, 2005). In the former area the major topics of concern are the entry and possible storage of substances in placental cells and through the placenta, aided perhaps by various transporters and efflux pumps the distribution and binding of compounds in placental cells and biotransformafion of substances by intracellular enzymes. Metabolic activation and production of reactive intermediates by placental enzymes link these areas with toxicodynamics of placental toxicants. In the latter area, effects of compounds on placental blood flow and vasculature and the presence of membrane and intracellular receptors, enzymes, and other potential targets for foreign substances are important areas of inquiry for placental toxicity. 

Figure 9.10 Relationship between permeability coefficients obtained from Caco-2 and PAMPA screenings. Circles represent passively transported compounds, diamonds are actively transported, and triangles are compounds subject to efflux. (Reprinted from with permission from Fujikawa, M., et al. Relationship between structure and high-throughput screening permeability of diverse drugs with artificial membranes Application to prediction of Caco-2 cell permeability. Bioorg. Med. Chem., 2005,13, 4721 732, copyright 2005, Elsevier).

On the other hand, artificial membranes represent a further simplification of the human GIT than Caco-2 monolayers as a number of features are not present, like the paracellular pathway, active transporters, or efflux systems. Caco-2 is a useful model to study actively transported compounds as well as substrates and inhibitors of efflux systems (PgP, MRP). Caco-2 monolayers also have some potential to model paracellular transport, although the junctions appear tighter than in the upper GIT. The consequence is that some paracellular transported compounds are properly transported while others are underestimated (Figure 15.6R). 

It appears then that the active transport and accumulation of HCO3 followed by intracellular conversion to CO2 and leakage back to the medium is the mechanism responsible for the rise in extracellular [C02] Once in the medium, CO2 will be converted to HCO3 but presumably at a rate which is initially lower than the CO2 efflux rate. Normally, leaked CO2 is efficiently transported back into the cells keeping [CO2] near zero. 

Radionuclide uptake in and efflux from isolated cells can detect enzyme activity, transport, and permeability of the apical and basolateral membranes. The weakness of this method is that it cannot differentiate a carrier from a channel (Sim and Gondos 1986), nor identify the exact permeability route (Todd, Inman et al. 2000). To circumvent this problem hydrophilic permeability probes such as [ Cr]-EDTA or [ " C] -maimitol can be used, which are markers that permeate mainly through the paracellular pathway (Bjamason, MacPherson et al. 1995 Nylander, Pihl et al. 2003). The transepithelial transport of [ " CJ-maimitol across the epithelial cell monolayer was assessed by the apparent permeability coefficient, Papp, according to the following equation ... 

Picard, M. Verchere, A. Broutin, I. Monitoring the active transport of efflux pumps after their reconstitution into proteoliposomes Caveats and keys. Anal. Biochem. 2012,420,194-196. 

Permeation across the intestinal wall involves both passive and facilitated processes. Passive transport includes passive transcellular permeation and paracellular diffusion across cell junctions, while facilitated transport includes active influx and efflux processes that pump molecules in and out of the cells. In order to improve absorption of a molecule, engineering passive permeability is a preferred strategy over engineering affinity to an influx transporter. The expression levels of active transporters vaiy significantly between different tissues and individuals, and the specificities and expression levels vaiy among mammals, which decreases the ability to predict human PK based on animal studies. In contrast, passive membrane permeability basically works the same with any eukatyotic membrane, although there may be minor quantitative differences due to different membrane compositions. As paracellular permeation is mainly pertinent to small and polar molecules, passive membrane permeability, which is crucial to transcellular diffusion, is therefore one of the kty properties that needs to be optimized for developing bioavailable macrocycles. 

---