PGlycoprotein Pgp ABCB1

Influencing drug transport in mammalian tumor cells, P-glycoprotein (P-gp) was originally identified as a xenobiotic efflux pump by Juliano and Ling (1976) providing a mechanism for multidrug resistance. Subsequent studies confirmed that P-gp was a 170-180 kDa, ATP-dependent transmembrane glycoprotein, which is formed by the posttranslational glycosylation of a 140 kDa pro-P-gp protein (Kramer et al., 1995). Topological analyses of P-gp showed that the protein comprised four major domains; two membrane-bound domains, each with six transmembrane segments and two cytosolic ATP binding motifs, commonly known as Walker A and B NBDs that bind and hydrolyze ATP. P-gp consists of 1,280 amino acid residues and exhibits a large degree of homology between the car-boxy and amino terminal halves (Leveille-Webster and Arias, 1995). Each half consists of both a hydrophobic and hydrophilic NBD, containing approximately 300 amino acids. Xenobiotics bind to separate sites on P-gp, demonstrating that different drugs and/or the different NBDs can each independently regulate P-gp function (Leveille-Webster and Arias, 1995).

The gene responsible for encoding P-gp belongs to the ABCB family of ABC transporters, and is commonly known as the Multidrug Resistance 1 (MDR1) gene. There are 11 members of the ABCB family; however, the discussion here is restricted to the two commonly considered MDR gene families in humans MDR1 (ABCB1) and MDR2/3 (ABCB4) (Lincke et al., 1991; Germann et al., 1993). The human genome nomenclature details and chromosome location are provided in Table 7.1 and at the Web site http://www.gene.ucl.ac.uk/nomenclature/genefamily/ abc.html. It is worth noting that the homologous MDR3 and Bile-Salt Exporting Protein (ABCB11; sister of P-gp) are also important efflux transporters of ABCB subfamily and mainly involved in the active transport of bile salts across the hepa-tocyte canalicular membrane (Meier and Stieger, 2002). Both MDR3 and sister of P-gp demonstrated low expression and variable ethnicity-based expression in the human small and large intestines, which suggests that they play a minimal role in mediating oral absorption (Wang et al., 2004).

In rodents, the gene responsible for the primary MDR isoforms' expression are depicted by lower case letters as mdr1 (a and b) and mdr2 (Torok et al., 1999). The MDR1 gene product in humans and the mdr1a and mdr1b gene products in rodents confer resistance by effluxing xenobiotics from the cytosolic compartments in cells (Higgins, 1992). The MDR2 and mdr2 genes encode a protein primarily expressed in the bile canalicular membrane that is engaged in the transport of phosphatidylcholine into the liver bile canaliculi (Thiebaut et al., 1987; Smit etal., 1993).

7.2.1.1 The Expression of P-gp

Immunohistological studies with human small intestinal samples indicate that P-gp is localized to the apical brush-border membrane of the intestinal epithelium (Thiebaut et al., 1987). Due to the localized expression of P-gp at the microvillous tip of enterocytes (Terao et al., 1996), P-gp will limit the absorption of compounds by directly effluxing them back into the intestinal lumen. Interestingly, the level of P-gp expression increases from proximal to distal regions of the intestine (Mouly and Paine, 2003).

P-gp expression has also been demonstrated in kidney, adrenal gland, liver, colon, and lungs (Fojo et al., 1987; Gatmaitan and Arias, 1993). Additionally, P-gp expression in endothelial cells is lining the blood-tissue barrier that includes the brain capillaries (Thiebaut et al., 1989), implicating a protective functional role for P-gp through the active efflux of xenobiotics from the endothelial cytoplasm into the capillary lumen, as confirmed by Joly et al. (1995). P-gp is also expressed in the apical membrane of the placental syncytial trophoblasts, which faces the maternal blood compartment (Sugawara et al., 1988), and thus forms a functional barrier between the maternal and fetal blood circulations. Some peripheral blood mononuclear cells, such as cytotoxic T lymphocytes and natural killer cells, also express P-gp, suggesting involvement in cell-mediated cytotoxicity. Moreover, P-gp is expressed and functions in human hematopoietic stem cells, indicating it may contribute to the established chemoresistance of these cells (Chaudhary and Roninson, 1991; Drenou et al., 1993). Low-level P-gp expression is also found in prostate, skin, spleen, heart, skeletal muscle, stomach, and ovary (Fojo et al., 1987; Gatmaitan and Arias, 1993).

7.2.1.2 The Regulation of P-gp Expression

Expectedly, P-gp expression can be modulated by various factors, such as xeno-biotics, environmental stress, differentiating agents, and hormones under cell culture conditions. In the rat liver, P-gp expression was increased after acute treatment by chemical carcinogens including 2-acetylaminofluorene and aflatoxin B1 (Burt and Thorgeirsson, 1988), suggesting xenobiotic-mediated transcriptional induction. Furthermore, cholestasis or carbon tetrachloride intoxication has also resulted in increased P-gp expression in the liver of rodents and nonhuman primates (Schrenk et al., 1993). In addition to anticancer drugs, other xenobiotics, including protein kinase C agonists and chemical carcinogens, have been demonstrated to induce in vitro P-gp expression in several human carcinoma cell lines (Chaudhary and Roninson, 1993). Steroid hormones have also been shown to increase P-gp expression levels, as estradiol treatment has resulted in increased efflux of rhodamine 123 in rat pituitary cells expressing P-gp (Jancis et al., 1993). Moreover, concomitant rifampin therapy (600mg/day for 10 days, p.o.) has also resulted in significant reduction in the area under the plasma concentration time curve (AUC) of oral digoxin (single-dose, 1 mg oral and 1 mg intravenous), a known P-gp substrate. Furthermore, rifampin treatment resulted in a threefold increase in intestinal P-gp levels that correlated with a decrease in the AUC of orally administered digoxin (Greiner et al., 1999). Differentiating agents such as retinoic acid and sodium butyrate also upregulate P-gp expression in human neu-roblastoma and colon carcinoma cells, respectively (Bates et al., 1989; Mickley et al., 1989). Environmental stresses, such as heat, shock, arsenite, and cadmium chloride treatment have also been shown to affect in vitro P-gp expression (Chin etal., 1990).

7.2.1.3 P-gp Mediated Drug Transport

To explain the mechanism by which P-gp actively effluxes xenobiotics, two hypotheses have been postulated: the "hydrophobic vacuum cleaner" (HVC) and the "flippase model" (FM). The HVC model suggests P-gp clears substrates before they enter the cytoplasm (Higgins and Gottesman, 1992; Gottesman and Pastan, 1993). As such P-gp forms a hydrophilic pathway, and the drugs are transported from the cytosol to the extracellular media through the middle of a pore. Alternatively, the FM proposes that P-gp interacts with the xenobiotics as they enter through the lipid membrane and "flips" the drug from the inner leaflet to the outer leaflet and back into the extracellular media. Evidence also supports the presence of at least two allosterically coupled drug-binding sites (Ferry et al., 1992; Martin et al., 1997), although it is not clear if these sites facilitate drug efflux by separate mechanisms, it does suggest that they may convey broader P-gp substrate affinity.

Cornwell et al. (1986) demonstrated the role of P-gp in the specific and saturable binding of vinblastine in membrane vesicles from highly multidrug-resistant human KB carcinoma cell lines. The binding of vinblastine to P-gp is competitively inhibited by vincristine and daunorubicin, suggesting these compounds share the same binding site. Competitive binding studies using colchicine and actinomycin D revealed a lack of competition for the vinblastine-binding site, further supporting the findings of Ferry et al. (1992) and Martin et al. (1997) that P-gp has multiple drug binding domains (Cornwell et al., 1986; Akiyama et al., 1988). Shapiro et al. (1999) demonstrated that progesterone was not effluxed by P-gp, although it was shown to bind P-gp and block the efflux of other substrates. This study further supports the possibility of additional potential binding sites, or high affinity of endogenous progesterone to one of the two binding sites suggested above. There is still confusion about whether these studies have identified the same two binding sites or, in fact there are additional binding sites present in P-gp. As such, considerable research is required to elucidate the functional mechanisms of P-gp active efflux, as well as determine the structural moieties responsible for imparting such broad substrate specificity.

7.2.1.4 The Substrate Specificity of P-gp

Numerous studies have demonstrated that P-gp possesses broad substrate specificity, with a preference for hydrophobic, amphipathic molecules containing a planar ring system ranging in size from 200 to 1,900 Da. P-gp is also involved in the transport of neutral compounds such as digoxin and cyclosporine A, negatively charged carboxyl groups such as those found on atorvastine and fexofenadine, and hydrophilic drugs such as methotrexate (Sharom, 1997). The degree of hydrogen bonding and partitioning into the lipid membrane has been determined to be a rate-limiting step for substrate interactions with P-gp (Seelig and Landwojtowicz, 2000). A representative list of substrates/inhibitors is listed in Table 7.2 and includes anticancer agents, antibiotics, antivirals, calcium channel blockers, and immunosuppressive agents.

Table 7.2. Partial list of compounds that have shown to interact with P-glycoprotein

Group

List of drugs

Anticancer drugs

Doxorubicin, daunorubicin, vinblastine, vincristine, actinomycin D,

paclitaxel, teniposide, etoposide

Immunosuppressive

Cyclosporin A, FK506, valinomycin, gramicidine

Lipid lowering agents

Lovastatin, atorvastatin, pravastatin, simvastatin

Anti-Histaminic

Fexofenadine, terfenadine

Antidiarrheal agents

Loperamide, antiemetics, domperidone, ondansetron

Antibiotic

Ivermectin, itraconazole, dactinomycin, ketoconazole, erythromycin,

valinomycin, grepafloxacin, actinomycin D

HIV protease

Ritonavir, amprenavir, saquinavir, indinavir, nelfinavir

inhibitors

Steroids

Aldosterone, hydrocortisone, cortisol, corticosterone, dexamethasone,

dopamine antagonist, domperidone

Cardiac drugs

Digoxin, digitoxin, quinidine

Analgesics

Morphine, asimadoline, fentanyl

Beta-Adrenoceptor

Bunitrolol, carvedilol, celiprolol, talinolol

antagonists

Food/Herbal

Piperine, quercetin, naringin, curcumin, bergamottin, kaempferol, rutin

Constitutents

Pharmaceutical

Cremophor EL, Tween 20, Tween 80, Nonidet P-40, Acacia, polyethylene

excipients

glycols, Triton-X 100, pluronic block copolymers, Brij 30 & 35, solutol

HS 15, poloxamers, 1-[(3-cholamidopropyl)dimethyla

amino]-1-propanesulfonate (CHAPS)

The table has been modified from the information reported by Fisher et al. (1996), Sikic (1997), Zuylen etal. (2000), and Kunta and Sinko (2004)

The table has been modified from the information reported by Fisher et al. (1996), Sikic (1997), Zuylen etal. (2000), and Kunta and Sinko (2004)

It is important to note that the substrate specificity of P-gp may also vary across populations due to genetic polymorphisms. For example, Kurata et al. (2002) demonstrated a significant difference in oral digoxin bioavailability between two allelically diverged MDR1 populations, which resulted in population specific absorption and/or distribution outcomes. A recent comprehensive review of the clinical implications of P-gp polymorphisms is suggested for additional information on this subject (Ieiri et al., 2004).

While the observed absorption of numerous drugs may be affected by P-gp affinity, there are certain drugs that can also be used to inhibit P-gp activity (Krishna and Mayer, 2000). The P-gp inhibitors include several calcium channel blockers, immunosuppressive agents, and other well-characterized compounds such as SDZ, PSC 833, LY335979, and GF120918 (Table 7.2) (Hyafil et al., 1993; Schinkel and Jonker, 2003). Drug-drug interactions resulting from concomitant administration of P-gp substrates and/or inhibitors may result in an increase in the absorption of one or more of these agents, potentially leading to toxic side effects by virtue of increased plasma levels that rise above the minimum toxic concentration. There are numerous clinical examples of these effects being observed and reported in the literature. For example, a significant therapeutic increase in the plasma levels of digoxin was observed upon coadministration with valspo-dar (PSC833) in healthy patients. The increase in AUC of this narrow therapeutic index compound leads to the conclusion that the digoxin dose should be decreased by 50% and clinical toxicity of digoxin should be monitored upon concomitant administration of these two agents (Kovarik et al., 1999).

In contrast, drug-drug interactions can also result in a lowering of the plasma levels of a therapeutic agent, where the coadministered drug induces P-gp expression that subsequently results in decreased plasma levels due to reduced absorption and increased intestinal clearance. One compelling example of this phenomenon was observed in a study with rifampin and fexofenadine (Hamman et al., 2001), where rifampin induced the intestinal expression of P-gp resulting in increased oral clearance and reduced bioavailability of fexofenadine.

Additionally, common pharmaceutical excipients such as hydrophilic cyclo-dextrin (2,6-di-O-methyl-p-cyclodextrin) (Arima et al., 2001), cosolvents (poly(ethylene)glycol (PEG) 400), and surfactants (Tween 80, Cremophor EL) (Nerurkar et al., 1996; 1997; Hugger et al., 2002) have also been shown to inhibit P-gp activity. Given the obvious adverse effects this may have on drug bioavail-ability and overall therapy in general, excipient selection should be an important factor to be considered in rational formulation design. Surfactants or cosolvents have also been shown to indirectly influence P-gp by inducing changes in cellular membrane fluidity. Changes in membrane fluidity can alter the microenvironment of the apically oriented TM domain, and subsequently alter substrate recognition, binding, and efflux by P-gp (Ferte, 2002).

Dietary constituents and phytochemicals also affect drug absorption and disposition through drug-nutrient P-gp interactions, potentially impacting the clinical pharmacokinetics of therapeutic agents (Walter-Sack and Klotz, 1996). Recently, it has been estimated in the US that approximately 30-50% of the population used a dietary supplement that can alter the absorption of drugs through mechanisms like competitive inhibition or transporter induction (Kauffman et al., 2002). The flavonoids, particularly the flavonols, flavanones, flavones as well as coumarins, and other ingredients present in fruits, vegetables, and herbs have been found to modulate the activity of P-gp function and may cause detrimental effects on drug pharmacokinetics (Izzo, 2005). One of the most recognized interactions is the ingestion of grapefruit juice and/or St John's Wort which results in the change of the pharmacokinetic profiles of cyclosporine A and digoxin due to inhibition or induction of P-gp mediated transcellular intestinal epithelial absorption (Bailey etal., 1998; Diirr etal., 2000). In a representative case, the oral coadministration of grapefruit juice with talinolol (10mg/kg) resulted in an increased talinolol Cmax, AUC and a reduced tmax without significantly affecting the terminal talinolol halflife (Spahn-Langguth and Langguth, 2001). This study indicated that grapefruit juice acted to inhibit intestinal P-gp mediated efflux and resulted in enhanced tali-nolol bioavailability. In a separate study, 1 g of black pepper administered in soup along with phenytoin significantly increased the human plasma levels of pheny-toin (Velpandian et al., 2001). Piperine, an active constituent of black pepper, is an inhibitor of P-gp and CYP3A4 (Bhardwaj et al., 2002). Orange juice components, such as methoxyflavones, increase vinblastine uptake by Caco-2 cells, possibly by interacting with P-gp (Honda et al., 2004). Therefore concomitant intake of herbal extracts or fruits/foods and nutraceuticals may modulate the pharmacokinetic profile of the therapeutic index of drugs, in particular for those agents exhibiting a narrow therapeutic index, resulting in an altered clinical response.

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