Transporter expression levels

The maximal efflux velocity (Vm) depended on the P-gp secretion transporter expression level. A baseline value was taken to be the expression level in ileum (Vmi). The value of maximal velocity in duodenum (Vmn) and jejunum (Vmj) was computed from the baseline value and a correction factor (Ej), based on... 

The same quinolone CNV97100 was used to perform bidirectional experiments in Caco-2 cells to obtain the apical to basal Pai, and basal to apical Pya permeabilities at different initial concentrations. The relationship between previously obtained in situ rat permeability values and in vitro Caco-2 of CNV97100 was examined. In each system the in vitro and in situ paracellular permeability of CNV97100 was considered to be negligible. The systems differed in effective surface area (Sj) and in efflux transporter expression level. Although the Caco-2 expression level of P-gp is similar to the expression levels in rat ileum, the model allows for system differences (EJj. 

Neither chemical nor pharmacological chaperones lead to wild-type expression levels of the mutant proteins at the cell surface. Alternative or additional strategies are needed to improve the intracellular transport of the mutant proteins. In the future, dtugs may also be developed that influence those components of the quality control system that are involed in the retention of misfolded proteins. 

In addition to the described lipid pathways mainly operative in macrophages, two further ABC-transporteis, ABCG5 and ABCG8 have been implicated in the efflux of dietary sterols from intestinal cells back into the gut lumen and from liver to the bile duct (Fig 1). Both ABC-transporters form a functional heterodimer with highest expression levels in liver and intestine and are regulated... 

Another limitation is that there is no quantitative relationship between active drug transport in the cell culture models and in vivo e.g. [92, 93]. The reason may be that the expression level of the transporter in Caco-2 cells is not comparable to that in vivo or that there is a difference in effective surface area (see Section 4.3.2.2 below). One solution to this problem is to determine the apparent transport constants, Km and Vmax, for each transporter and subsequently, to determine a scaling factor. However, this is not readily done. In addition these studies are further complicated by the lack of specific substrates. For example, there are almost no specific substrates for the drug efflux transporters [18]. Therefore, other epithelial... 

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. 

From the above, it is clear that the gut wall represents more than just a physical barrier to oral drug absorption. In addition to the requirement to permeate the membrane of the enterocyte, the drug must avoid metabolism by the enzymes present in the gut wall cell as well as counter-absorptive efflux by transport proteins in the gut wall cell membrane. Metabolic enzymes expressed by the enterocyte include the cytochrome P450, glucuronyltransferases, sulfotransferases and esterases. The levels of expression of these enzymes in the small intestine can approach that of the liver. The most well-studied efflux transporter expressed by the enterocyte is P-gp. 

The expression of metabolic enzymes in the enterocyte can lead to a profound gut wall first-pass extraction ratio for substrate drugs. In addition, efflux transporters can slow the passage of drugs across the enterocyte in a cycling fashion. This allows the metabolic enzymes several opportunities to metabolize their substrates, and in this way a low expression level of an enzyme can exhibit a significant extraction. 

Some drugs with low intrinsic permeability achieve acceptable oral bioavailability because they are substrates for uptake transporters, which normally function in nutrient uptake. The most prominent example is the peptide transporter, PepTl, which is active toward peptidomimetic antibiotics such as cephalexin, the antiviral agent valacyclovir [24] and other drugs. PepTl is natively expressed in Caco-2 cells, and adenovirus transduction has been used to increase PepTl expression levels [25]. However, the expression of PepTl was not polarized in this system and this expressed system appears to be of limited value as an improved screening model. PepTl has also been expressed in Chinese hamster ovary cells and a variety of other mammalian systems [26, 27]. 

When applying any of these models it is crucial to understand the main transport mechanisms as well as the metabolic route and characterization of the activity of the transporter/enzyme involved. It is well recognized that the activities of carrier-mediated processes in Caco-2 cells are considerably lower than in vivo [20, 42, 48] therefore, it is crucial to extrapolate in vitro cell culture data to the in vivo situation with great care [18, 20, 42, 48], This is especially important when carrier-mediated processes are involved, as evidenced by a recent report which showed significant differences in gene expression levels for transporters, channels and metabolizing enzymes in Caco-2 cells than in human duodenum [48], If an animal model is used, then potential species differences must also be considered [18, 20, 45],... 

Within the OAT family, OAT4 is the only transporter expressed at appreciable levels in both the placenta and in the kidney [54]. The membrane localization of OAT4 within these tissues has not been examined. Steroid sulfates, and ochratoxinA are efficient transport substrates of OAT4, whereas PAH is weakly transported [54]. The functional importance of OAT4 in regulating placental permeability and renal drug elimination is currently unknown. 

Figure 2.12 Correlation plots of (A) transporter expression and (B) metabolic enzyme expression in duodenum between rat and human. The expression levels of transporters and metabolic enzymes are normalised by GADPH expression, transformed by natural logarithm and absolute values were used in the correlation analysis [121].

MDCK Madin-Darby canine kidney (MDCK) cells have received attention as an alternative to Caco-2 cells for permeability measurements. When grown under standard culture conditions, MDCK cells develop tight junctions and form monolayers of polarized cells. The main advantage over Caco-2 cells is the shorter culture time to confluence (3-5 days). The transep-ithelial electrical resistance of MDCK cells is lower than that of Caco-2 cells and thus, closer to the TEER of the small intestine in vivo. The permeability coefficients of hydrophilic compounds are usually lower in Caco-2 cells than in MDCK cells, which is consistent with the lower TEER values for MDCK cell monolayers. The nonhuman (canine) and nonintestinal (renal) origin of MDCK cells is considered as a disadvantage. They have low expression levels of transporter proteins and low metabolic activity [34], MDCK cells that are stably transfected with P-gp/MDRl are often proposed as an alternative for Caco-2 cells to study bidirectional transport of compounds and, more... 

Ex Vivo Transporter Gene Expression Levels at the Inner Blood-Retinal Barrier (Magnetic Isolation of Retinal Vascular Endothelial Cells)... 

Figure 14.5 Schematic diagram of the magnetic isolation method for rat retinal vascular endothelial cells (RVEC) (A) and the transcript level of organic anion-transporting polypeptides (Oatps) in RVEC (B). A Endothelial cells (RVEC) EC, Nonendothe-lial cells (Non-RVEC) Non-EC. B Not detected N.D. Data taken from Journal of Neurochemistry, 91, Tomi and Hosoya, Application of magnetically isolated rat retinal vascular endothelial cells for the determination of transporter gene expression levels at the inner blood-retinal barrier. 1244-1248, 2004, with permission from Blackwell Publishing.

M. Tomi and K. Hosoya. Application of magnetically isolated rat retinal vascular endothelial cells for the determination of transporter gene expression levels at the inner blood-retinal barrier. J. Neurochem. 91 1244—1248 (2004). 

Y. Sakurai, H. Motohashi, H. Ueo, S. Masuda, H. Saito, M. Okuda, N. Mori, M. Matsuura, T. Doi, A. Fukatsu, O. Ogawa, and K. Inui. Expression levels of renal organic anion transporters (OATs) and their correlation with anionic drug excretion in patients with renal diseases. Pharm Res 21 61-67 (2004). 

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