Efflux pumps substrates

This attachment includes model drugs suggested for use in establishing suitability of a permeability method as described in section III. The permeability of these compounds was determined based on data available to the FDA. Potential internal standards (IS) and efflux, pump substrates (ES) are also identified. 

Table 7.1 Summary of various efflux pump substrates...

In this review efflux pump inhibitors are classified into two groups low molecular mass inhibitors and polymeric inhibitors, because the high molecular mass of the polymeric excipients prevents absorption into systemic circulation after oral administration. In some cases, just a local inhibition of efflux transporters in the intestine is desired, whereas in other cases also an additional systemic modulation of efflux pumps can be of advantage. For chronical treatments, impact on the complex systemic efflux transporter system can result in severe complications. In this case, an enhanced intestinal absorption of efflux pump substrates can be achieved by using drug delivery systems based on polymeric inhibitors. On the other hand, in cancer therapy it would be of advantage to reduce efflux of anticancer compounds also in the systemic system because tumour tissues often overexpress these transporters. Then a low molecular mass efflux inhibitor could be useful. 

The Calu-3 human submucosal gland cell line forms polarized cell monolayers with tight junctions, produces mucus, and develops apical cilia when grown at an air interface. Transport studies can be performed after 10-14 days in culture, and it has been shown that Calu-3 cells express P-gp and actively transport amino acids, nucleosides, and dipeptide analogs organic anions, organic cations, polyamines, and efflux pump substrates are not actively transported.43,51,53-56 Because Calu-3 cells are not subject to the influence of multiple in vivo cell types, the expression of carrier proteins and enzymes may not reflect in vivo levels. Nevertheless, values obtained in Calu-3 permeability studies correlate well with those obtained from primary cultured rabbit tracheal epithelial cells and in vivo rat lung absorption studies.54 Mannitol permeation in Calu-3 cells is about 10 times less than that in vivo, but this is the same ratio difference between Caco-2 cells and in vivo intestinal epithelium.51... 

The ability of amphiphilic block copolymers to modulate multi drag resistance related processes has been demonstrated the first time more than 10 years ago. Nowadays, the efflux pump inhibitory activity of amphiphilic block copolymers is used in two main areas. First, to improve the transport of efflux pump substrates across the blood brain barrier (BBB) and second, in cancer therapy. It has been shown that in the presence of amphiphilic block copolymers higher concentrations of certain anhcancer drugs, which are known as efflux pump substrates, can be found in the brain. Within the current chapter, recent developments in the field of amphiphilic block copolymer mediated efflux pump inhibition are discussed. Besides presenting data from in vitro and vivo studies, also the mechanisms involved in efflux pump inhibition are addressed. In addition, the influence of hydrophilicity/lipophilicity of various amphiphilic block copolymers as well as factors such as micelle formation on the efflux pump inhibitory activity are explained. 

Acquired resistance has been observed by constitutive upregulation of mdr efflux pump expression due to a mutation inactivating a respective repressor or inducibly, caused by molecules transiently inactivating repressor molecules upon binding. Depending upon the substrate spectra of the respective subset of efflux pumps upregulated, a multiple drug resistance (mdr) phenotype is expressed, which in combination with a specific resistance mechanism can contribute to a clinically relevant level of resistance. 

Although nucleoside analogs are not substrates for P-gp or CYP3A4, most protease inhibitors and NNl are substrates for both the P-gp efflux pump (Aungst 1999 ... 

Breast Cancer Resistance Protein (BCRP, also known as MXR or ABCP), first cloned from mitoxantrone and anthracycline-resistant breast and colon cancer cells [188, 189] is a half-transporter efflux pump believed to function as a homo-or hetero-dimer. Following its identification, BCRP-mediated drug resistance was observed for topoisomerase inhibitors including camptothecins [190, 191] and in-dolocarbazoles [192]. In normal tissues, BCRP was detected in placental syncytio-trophoblasts, hepatocyte canalicular membrane, apical intestinal epithelia and vascular endothelial cells [193]. These findings support the important role BCRP plays in modulating topotecan bioavailability, fetal exposure and hepatic elimination [194]. Considering that the substrates and tissue distributions for BCRP overlap somewhat with MDR1 and MRPs [195], additional studies will be required to define the relative contribution of each of these transporters in the overall and tis-... 

There are several efflux pumps which may affect absorption, blood-brain-barrier penetration, and reabsorption from kidney microtubules. The most commonly tested efflux pump in early drug discovery is the P-glycoprotein. Assays to identify P-glycoprotein substrates or inhibitors can be run using a variety of cell lines. 

Figure 3.2. A representative RND efflux pump system. This system is comprised of three essential components The RND pump (e.g., AcrD and MexX) is located on the cytoplasmic membrane and is responsible for the recognition of substrates in the cytosol, including aminoglycosides, and moving them into the periplasmic space. The membrane fusion protein (MFP) (e.g., AcrA and MexY) is responsible for moving the substrate across the periplasm the final component is the outer membrane factor (OMF) (e.g., TolC and OprM) that provides a conduit for the substrate to the extracellular region of the cell.

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. 

S pneumoniae, S aureus, H influenzae, Moraxella catarrhaiis, mycoplasmas, Legionella, Chlamydia, H pylori, N gonorrhoeae, fragilis, T gondii, and nontuberculosis mycobacteria. Many macrolide-resistant strains are susceptible to ketolides because the structural modification of these compounds renders them poor substrates for efflux pump-mediated resistance and they bind to ribosomes of some bacterial species with higher affinity than macrolides. 

Permeation enhancement by excipients has generated some interest, but there is still much research that needs to be done to elucidate the mechanism of these excipients. PEG-400 (and many other excipients such as polyethylene glycol, poloxamers, polysorbates, and vitamin E) is known to inhibit p-glycoprotein, which may increase the bioavailability of the API, which was a substrate for this efflux pump. On the other hand, it has been demonstrated that PEG-400 can accelerate small intestinal transit, and thereby reduce the bioavailability of some drugs (e.g., ranitidine) (5). 

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