Active efflux transporters

In the transport assays, the permeability of a compound in both absorption and secretion directions is measured using polarized epithelial cells that constitutively express high levels of P-gp (e.g. Caco-2) or have been transfected with the gene for a specific P-gp (e.g. MDR1-transfected MDCK or LLC-PK1 cells). Since P-gp is expressed on the apical membrane, ratios ofbasolateral-to-apical (B —> A) permeability versus apical-to-basolateral (A —> B) permeability greater than 1 may indicate an active efflux transport process. Bidirectional permeability measurements can also be performed in the presence of a specific P-gp inhibitor. Thus, apical-to-basolateral permeability increases and basolateral-to-apical permeability decreases such that... 

Active efflux transporters also exist in the placenta, analogous to the gut and blood-brain barrier. These are Pgp, multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP). These transport proteins are located in many tissues but also appear to be expressed in the placenta. Though the substrate specificities of these proteins have not been completely described, they appear to function as efflux transporters, moving endogenous and exogenous chemicals from the placental cells back to the systemic circulation. In this way, they serve as a mechanism to protect the fetus from exposure to unintended chemicals. 

D) Changes in DNA gyrases and active efflux transport system resulting in decreased permeability of drug. 

B. Humans cannot synthesize folic acid (A) diet is their main source. Sulfonamides selectively inhibit microbially synthesized folic acid. Incorporation (B) of PABA into microbial folic acid is competitively inhibited by sulfonamides. The TMP-SMX combination is synergistic because it acts at different steps in microbial folic acid synthesis. All sulfonamides are bacteriostatic. Inhibition of the transpeptidation reaction (C) involved in the synthesis of the bacterial cell wall is the basic mechanism of action of (3-lac-tam antibiotics Changes in DNA gyrases (D) and active efflux transport system are mechanisms for resistance to quinolones. Structural changes (E) in dihydropteroate synthetase and overproduction of PABA are mechanisms of resistance to the sulfonamides. 

The ABC transporter P-gp protects the brain from accumulation of lipophilic compounds by active efflux transport across the BBB. Miillauer et al. [715] investigated the suitability of the radio-labeled Pgp inhibitors [nC]elacridar and [nC]tariquidar to visualize P-gp density in rat brain with PET. The small P-gp binding signals obtained with [nC]elacridar and [nC]tariquidar limit the applicability of these tracers to measure cerebral P-gp density. 

Also can estimate active efflux /transport properties (i.e., P-gp)... 

P-glycoprotein is considered as the most important ABC efflux transporter (ABCBl, MDRl) at the blood-brain barrier [54]. It is a member of the ABC family and consumes ATP during its active efflux transport mechanisms. The major role of P-gp in the phenomenon of multidrug resistance is attributed to its broad substrate specificity. 

Loscher, W. and Potschka, H. (2005) Blood-brain barrier active efflux transporters ATP-binding cassette gene family. NeuroRx, 2, 86-98. 

Although this discussion was related to the active uptake into the cell (or flux across a membrane), many important transporters exist (e.g., P-GP, MRPs, BCRP) that exhibit active efflux transport. The same general mechanisms described above would hold however, the net flux would be in the opposite direction. A combination of passive diffusion into and active transport out of a cell would produce a drug flux that would appear at a slower rate for low drug concentrations. 

Three major mechanisms of elimination of flavanols have been reported elimination by the kidneys in urine, elimination by the liver in bile, and elimination by the small intestine by an active efflux transport mechanism. The final fate of most flavanols after elimination by the intestine or liver is likely to be through metabolism by microflora in the colon. 

Both influx and efflux transporters are located in intestinal epithelial cells and can either increase or decrease oral absorption. Influx transporters such as human peptide transporter 1 (hPEPTl), apical sodium bile acid transporter (ASBT), and nucleoside transporters actively transport drugs that mimic their native substrates across the epithelial cell, whereas efflux transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP) actively pump absorbed drugs back into the intestinal lumen. 

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... 

In conclusion, there are several drawbacks to the use of Caco-2 cells in studies of active drug transport. Despite these drawbacks, we note that a recent comprehensive study comparing various P-glycoprotein drug efflux assays in drug discovery came to the conclusion that the Caco-2 transport assay is the method of choice, since it displays a biased responsiveness towards compounds with low or moderate permeability - in other words, towards compounds whose intestinal permeability is most likely to be significantly affected by drug efflux mechanisms [101]. 

R. T. Borchardt. Effects of poly( ethylene glycol) on efflux transporter activity in Caco-2 cell monolayers,/. Pharm. Sci. 2002, 91, 1980-1990... 

Compounds absorbed by active uptake mechanisms (e.g., glucose and Gly-Pro) and compounds known to be substrates for efflux transport (e.g., digoxin, verapamil) were also included in the list. The applied concentration (10-500 pM) only had minor effects on the permeability values. Thus, the choice of concentration was not critical for this set of compounds with respect to the relationship between permeability and fraction absorbed in humans. Changing the pH on the apical donor side had significant effects on the Papp values of several compounds, the effects being in agreement with the acid-base properties of the compounds. The... 

Several attempts have been made to estimate the dose required in humans in relation to a drug s potency, and to put this into the context of solubility and permeability for an optimal oral drug [2, 3]. A relatively simple example of this is where a 1.0 mg kg-1 dose is required in humans, then 52 pg mL"1 solubility is needed if the permeability is intermediate (20-80%) [3]. This solubility corresponds approximately to 100 pM of a compound with a MW of 400 g mol-1. Most screening activities for permeability determinations in, e.g., Caco-2, are made at a concentration of 10 pM or lower due to solubility restrictions. The first implication of this is that the required potency for these compounds needs to correspond to a dose of <0.1 mg kg-1 in humans if the drug should be considered orally active. Another implication would be the influence of carrier-mediated transport (uptake or efflux), which is more evident at low concentrations. This could result in low permeability coefficients for compounds interacting with efflux transporters at the intestinal membrane and which could either be saturated or of no clinical relevance at higher concentrations or doses. 

Systems to study the role of intestinal oxidative metabolism (CYP3A4) have been developed and appear to have adequate enzyme activity levels. Although there appears to be a relatively limited need for additional system development in this area, there is still a fundamental question as to whether any synergistic interplay exists between metabolic enzymes and transporters (i.e., does the presence of an efflux transporter influence the extent of metabolism ) and co-expression of CYP3A4 and transporters provides a pivotal experimental model. 

Carrier-mediated transport, Active Efflux, Passive (trans and para cellular) diffusion... 

Table 18.2 lists 30 of the molecules used in this study that are known to be substrates for active transport or active efflux. The mechanistic ACAT model was modified to accommodate saturable uptake and saturable efflux using standard Michaelis-Menten equations. It was assumed that enzymes responsible for active uptake of drug molecules from the lumen and active efflux from the enterocytes to the lumen were homogeneously dispersed within each luminal compartment and each corresponding enterocyte compartment, respectively. Equation (5) is the overall mass balance for drug in the enterocyte compartment lining the intestinal wall. 

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