Efflux ratio

Efflux ratio (transport basolateral to apical divided by transport apical to basolateral)... 

The applicability range of any model should be limited to molecules having a similar mechanism of transport. Therefore, we have selected from the literature only those compounds with well-characterized Caco-2 cell permeability and excluded compounds with a high efflux ratio. Known P-gp substrates and actively transported compounds were also excluded from the list. 

Fluorine has been used to modulate the basicity of amines which may lead to an improvement in brain exposure. Recently, the discovery of a series of a4(32 nicotinic acetylcholine receptor (nAChR) potentiators as possible treatment for Parkinson s disease and schizophrenia was were disclosed [40]. Optimization of isoxazole 40 included the bioisosteric replacement of the central amide by an imidazole ring. Introduction of a fluorine at the 6-position of the phenyl ring provided compound 41. This compound had excellent potency but was determined to be a substrate for P-gp (efflux ratio >10). In an attempt to reduce amine basicity and decrease the efflux propensity, the 4-fluoropiperidine 42 was identified which retained potency and had significantly reduced P-gp efflux liability (efflux ratio 1). CNS penetration of 42 was observed in rodents following intraperitoneal (IP) treatment at 5mg/kg and showed a brain concentration of 6.5 gM. 

Either Transwell inserts or side-by-side diffusion chambers can be used for transport studies. Bode et al. have provided an excellent review on this subject [60], Briefly, cells are incubated for 30-60 min with a buffer solution. To initiate the transport study, a transport buffer containing the drug under investigation is added to either the apical or the basal chamber depending on the transport direction of interest. At predetermined time points, the respective receiver chamber is sampled and the withdrawn volume is replaced with the same volume of fresh buffer. The permeability coefficient (Papp) is calculated and the ratio of /apP in the basolateral-to-apical direction versus that in the apical-to-basolateral direction gives the efflux ratio. These sort of transport experiments are well suited to determine if drugs/xenobiotics are substrates of the placental efflux proteins. 

Caco2-ER are the Caco-2 permeability values in the apical to basolateral, basoletaral to apical and the Caco-2 efflux ratio respectively. ClogP and ClogD values are the calculated logP and D (at pH7.4) values. % fun pH 7.4 is the fraction unionized at pH7.4. PAMPA pH6.8 is the effective permeability measured in a PAMPA assay [19] and PAMPA%FA is the fraction absorbed extrapolated from the PAMPA permeability assay Blood 2hrs is the blood concentration 2 hours after p.o. administration of the compound and % inhibition is the functional readout. 

Figure 3.3 Impact of passive permeability on the efflux ratio (ER). Passive permeability (x-axis) was measured in a PAMPA assay [19]. Efflux ratios were derived from permeability measurements in a Caco-2 monolayer assay [44] and are expressed as the basolateral to apical/ apical to basolateral permeability ratios. The loading concentration was 5 XM in the PAMPA assay and 10 xM in the Caco assay. LC-MS/MS readout was used for both assays. The y-axis...

An interesting and more mechanistic approach was taken by Stoner et al. [98] who collected the largest self consistent Caco-2 data set known to date and used the efflux ratio values (i.e., the basal to apical divided by the apical to basal apparent permeability)... 

The most recent example of in silico efflux modeling has been based on Caco-2 permeability measured in the basal to apical direction [100]. This model can be very effective at ruling out compounds that most likely will show low in vivo intestinal absorption - however it carmot indicate which efflux pump(s) is/are responsible for that, making it more difficult for designers to circumvent the problem. Johnson [92] also included in his review an excellent summary of QSAR models and rules of thumb developed for P-gp substrates and inhibitors. These models are normally based on efflux ratios from MDCK/MDRl or Caco-2 cell lines - in the latter case it is important to notice that the data is combined with inhibition values from the calcein-AM assay, as the observed efflux might not be exclusively due to P-gp. 

Troutman MD, Thakker DR. Efflux ratio cannot assess P-glycoprotein-mediated attenuation of absorptive transport asymmetric effect of P-glycoprotein on absorptive and secretory transport across Caco-2 cell monolayers. Pharm Res 2003 20(8) 1200-1209. 

The polysorbates used most regarding efflux pump inhibition are polyoxyethylene sorbitan monolaurates (Tween 20), polyoxyethylene sorbitan monopalmi-tates (Tween 40) and polyoxyethylene sorbitan monooleates (Tween 80). Various studies demonstrate the ability of polysorbates to inhibit efflux pumps. In transport experiments across intestinal mucosa, the efflux ratio (basolateral to apical drug transport/apical to basolateral drug transport) of rhodamine 123 was reduced in the presence of Tween 80 (Shono et al. 2004). In another study, Zhang et al. demonstrated enhanced absorption of the P-glycoprotein substrate digoxin in rats in the presence of Tween 80 (Zhang et al. 2003). 

As standards digoxin, etoposide, Cyclosporin A, Colchicine (all for MDR 1), Bromosulfophthalein, Furosemide (for MRP2) and Metothrexate, topotecan (for ABCG2) can be used. Efflux ratio of the compound is compared to ratio of standards. 

Ratio of B-A/A-B is calculated and compared to the ratio of standard compounds. In addition B-A/A-B ratio determined in the inhibition experiment is compared to efflux ratios determined without inhibition. Given that efflux of a compound can not be inhibited by an inhibitor one should conclude that more than one efflux transporter are involved in the efflux. Adding two or more inhibitors at the same time provides maximal inhibition. 

A dataset comprised of 129 molecules, 100 Sanofi-Aventis proprietary compounds and 29 publicly available compounds, was studied in order to obtain a 3D quantitative structure-property relationship (3D-QSPR) model able to identify the structural features that a molecule should possess in order to be recognized as a substrate of Pgp. All the chosen compounds have an efflux ratio in a Caco-2 assay greater than one, which normally implies that the molecules are Pgp substrates. 

A1 ABC ATCC BCRP BSA Caco-2 cDNA CYP DMSO ECACC ER Conditionally immortalized cell line derived from fetal rat intestine ATP-binding cassette American-type culture collection Breast cancer-resistance protein (ABCG2) Bovine serum albumin Adenocarcinoma cell line derived from human colon Complementary DNA Cytochrome P450 Dimethyl sulfoxide European Collection of Cell Cultures Efflux ratio (transport basolateral-to-apical divided by transport a pical-to-basolateral)... 

Kalvass JC, Maurer TS, Pollack GM (2007) Use of plasma and brain unbound fractions to assess the extent of brain distribution of 34 drugs comparison of unbound concentration ratios to in vivo p-glycoprotein efflux ratios. Drug Metab Dispos 35(4) 660-666... 

Drug Substrate for Efflux ratio Extraction ratio (%) SD ... 

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