Transcellular Uptake

It has been suggested that, since A1 binds to both transferrin (Trapp 1983) and ferritin (Fleming and Joshi 1987), the Fe uptake system might be involved in A1 absorption, and some evidence exists that Fe competes with A1 for uptake (Fernandez Mendez et al. 1991 Van der Voet and De Wolff 1987 Van der Voet 1992). Whether or not Al interacts with the Fe uptake system at the intestinal mucosa, there is strong evidence that Al interacts with Fe transport and metabolism (see Sects. C.V, C.VII, D). These possibilities do not preclude uptake via other, as yet unidentified, systems also. For example, there are no studies on the interaction between Al and Mg uptake. This may be because out knowledge of Mg uptake at the intestinal mucosal membrane is far from complete (Shils 1988). 

---

Conner SD, Schmid SL (2003) Regulated portals of entry into the cell. Nature 422 37-44 Daugherty AL, Mrsny RJ (1999) Transcellular uptake mechanisms of the intestinal epithelial barrier. Part one. PSTT 2 144-151... 

Daugherty, A. L., Mrsny, R. J. Transcellular uptake mechanisms of the intestinal epitheUal barrier - Part one. PSTT 1999, 2(4), 144-151. 

In many epithelia Cl is transported transcellularly. Cl is taken up by secondary or tertiary active processes such as Na 2Cl K -cotransport, Na Cl -cotransport, HCOJ-Cl -exchange and other systems across one cell membrane and leaves the epithelial cell across the other membrane via Cl -channels. The driving force for Cl -exit is provided by the Cl -uptake mechanism. The Cl -activity, unlike that in excitable cells, is clearly above the Nernst potential [15,16], and the driving force for Cl -exit amounts to some 2(f-40mV. 

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. 

Table 20 Comparison of Transcellular (AP to BL) Permeability Coefficients with Permeability Coefficients from Independent Uptake and Efflux Kinetic Studies... 

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. 

It is also important to predict the in vivo biliary excretion clearance in humans, and for this purpose MDCK II cell lines expressing both uptake and efflux transporters may be used (Fig. 12.3) [92, 93]. It has been shown that MRP2 is expressed on the apical membrane, whereas OATP2 and 8 are expressed on the basolateral membrane after cDNA transfection (Fig. 12.3) [92, 93]. The transcellular transport across such double-transfected cells may correspond to the excretion of ligands from blood into bile across hepatocytes. Indeed, the vectorial transport from the basal to apical side was observed for pravastatin only in OATP2- and MRP2-expressing... 

MDCK II cells (Fig. 12.3) [93], Kinetic analysis revealed that the Km value for transcellular transport (24 pM) was similar to the Km for OATP2 (34 pM) [93], Moreover, the efflux across the bile canalicular membrane was not saturated under these experimental conditions. These in vitro observations are consistent with in vivo experimental results in rats which showed that the rate-determining process for the biliary excretion of pravastatin is uptake across the sinusoidal membrane. By normalizing the expression level between the double transfectant and human hepatocytes, it might be possible to predict in vivo hepatobiliary excretion. 

Bourdet DL, Pollack GM, Thakker DR (2006) Intestinal absorptive transport of the hydrophilic cation ranitidine A kinetic modeling approach to elucidate the role of uptake and efflux transporters and paracellular vs. transcellular transport in Caco-2 cells. Pharm Res 23 1178-87. 

Passive transcellular transport across the intestinal epithelium involves three discrete steps (1) uptake across the apical membrane, (2) diffusion through the cytoplasm, and (3) efflux across the basolateral membrane. Occasionally, drug molecules without favorable physicochemical properties traverse the intestinal epithelium using endogenous membrane transporters.6-8 In addition, the intestinal mucosa, with its numerous drug-metabolizing enzymes and efflux transporters, such as P-glycoprotein (Pgp), functions as a biochemical barrier.9... 

Major transport pathways in Caco-2 monolayers. A Passive transcellular B Passive paracellular C Transporter-mediated apical uptake D Transporter-mediated apical efflux E Transporter-mediated basolateral efflux F Transporter-mediated basolateral uptake. 

Cell culture models are routinely used to assess permeability of new potential drug candidates. The simplicity and higher throughput of these models makes them a useful alternative to in vivo studies. These models are used to predict absorption in vivo, rank order compounds and examine absorption mechanism. Transcellular, paracellular, active uptake and efflux mechanisms can be studied with these models. 

The use of in vitro models for prediction of compounds that are predominantly absorbed passively by the transcellular route is generally good with these models. Predicting compounds which are absorbed paracellularly or via active uptake or efflux mechanisms is more difficult. There is a lack of understanding of expression levels of transporters in the gut, which makes in vivo predictions difficult. 

The most efficient rectal absorption enhancers, which have been studied, include surfactants, bile acids, sodium salicylate (NaSA), medium-chain glycerides (MCG), NaCIO, enamine derivatives, EDTA, and others [45 17]. Transport from the rectal epithelium primarily involves two routes, i.e., the paracellular route and the transcellular route. The paracellular transport mechanism implies that drugs diffuse through a space between epithelial cells. On the other hand, an uptake mechanism which depends on lipophilicity involves a typical transcellular transport route, and active transport for amino acids, carrier-mediated transport for (3-lactam antibiotics and dipeptides, and endocytosis are also involved in the transcellular transport system, but these transporters are unlikely to express in rectum (Figure 8.7). Table 8.3 summarizes the typical absorption enhancers in rectal routes. 

Permeation of mAbs across the cells or tissues is accomplished by transcellular or paracellular transport, involving the processes of diffusion, convection, and cellular uptake. Due to their physico-chemical properties, the extent of passive diffusion of classical mAbs across cell membranes in transcellular transport is minimal. Convection as the transport of molecules within a fluid movement is the major means of paracellular passage. The driving forces of the moving fluid containing mAbs from (1) the blood to the interstitial space of tissue or (2) the interstitial space to the blood via the lymphatic system, are gradients in hydrostatic pressure and/or osmotic pressure. In addition, the size and nature of the paracellular pores determine the rate and extent of paracellular transport. The pores of the lymphatic system are larger than those in the vascular endothelium. Convection is also affected by tortuosity, which is a measure of hindrance posed to the diffusion process, and defined as the additional distance a molecule must travel in a particular human fluid (i. e., in vivo) compared to an aqueous solution (i. e., in vitro). 

Generally, low molecular mass permeation enhancers can be divided into transcellular and paracellular permeation enhancers. On the one hand the potential of permeation enhancers to open the paracellular route of uptake can be determined by the reduction in the transepithelial electrical resistance (TEER) (enhancement potential = EP). On the other hand the potential of permeation enhancers to open the transcellular route of uptake can be determined by the lactate dehydrogenase (LDH) assay (LDH potential = LP). The parameter K = (EP—LP)/EP represents the relative contribution of the paracellular pathway. Consequently, a K value of 0 means predominantly transcellular and a K value of 1 means predominantly paracellular. Based on this classification system Whitehead and Mitragotri classified over 50 low molecular mass permeation enhancers showing that most of them are paracellular and only a few of them are transcellular permeation enhancers (2008). 

The oral bioavailability of a peptide was improved from 0.5 to 27% due to the co-administration of MCG (Constantinides et al. 1994). Lundin et al. (1997) demonstrated that monohexanoin is even more effective than MCG in increasing oral absorption of the therapeutic peptide dDAVP in rats. Furthermore, due to the co-administration of a monoolein/sodium taurocholate combination the colonic uptake of polyethylene glycol 4000, calcitonin and horseradish peroxidase in rats was significantly increased without causing morphologic damage to the mucosa. The appearance of horseradish peroxidase in the cytoplasm suggested enhancement via the transcellular route of uptake (Hastewell et al. 1994). 

The second major obstacle of the oral delivery of proteins is the low permeability of proteins in the intestinal epithelium. The uptake of proteins is mediated by passive diffusion across the enterocytes (transcellular diffusion), paracellular diffusion (through intercellular spaces) and mostly by transcytosis (facilitated by receptor-mediated endocytosis). Erodible microcapsules and nanoparticles were shown to be absorbed intact through the GI tract and have opened the pos-... 

---