Functional role of P-glycoprotein in limiting peroral drug absorption: optimizing drug delivery Manthena VS Varma1, Omathanu P Perumal2 and Ramesh Panchagnula3 P-glycoprotein (P-gp) associated multi-drug resistance is one of the major challenges in the chemotherapy of various cancers. On the other hand, it is now widely recognized that P-gp influences drug transport across various biological membranes. To this end, there is an increasing trend to optimize pharmacokinetics and drug delivery right from the initial stages of drug discovery by exploring all the possible mechanisms involved in ‘deliverability’. Recent advances in molecular biology techniques and biochemical characterization methodologies have helped in identification of various transporters involved in absorption or secretion of drugs. P-gp, an efflux pump expressed along the gastrointestinal tract, limits the permeability of many drugs and thus affects their peroral absorption and bioavailability. A fundamental insight and thorough understanding of P-gp and its functional role in limiting drug absorption is critical to improve predictability of dynamic absorption models and aid in selection of new candidates for development, and also widen the scope of peroral delivery for ‘challenging’ molecules. Addresses 1 Department of Pharmaceutics, College of Pharmacy, University of Minnesota, 308 Harvard Street SE, Minneapolis, MN55455, USA 2 South Dakota State University, College of Pharmacy, PHA 125, Box 2202C, Brookings, SD-57007, USA 3 School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, BT52 1SA, UK Corresponding author: Panchagnula, Ramesh ([email protected])

Current Opinion in Chemical Biology 2006, 10:367–373 This review comes from a themed issue on Next-generation therapeutics Edited by Clifton E Barry III and Alex Matter Available online 30th June 2006 1367-5931/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2006.06.015

understanding of P-gp molecular mechanisms. Interested readers are referred to the extensive reviews on the biology and pharmacology of P-gp [4,5,6

]. P-gp is expressed on one membrane domain of a differentiated functional cell such that the bidirectional transport of substrates is asymmetric. It is now recognized that P-gp is localized in a wide range of tissues, particularly in columnar epithelial cells (enterocytes) of lower gastrointestinal tract (GIT), capillary endothelial cells of brain and testis, canalicular surface of hepatocytes and apical surface of proximal tubules in kidney [7]. Due to this selective distribution at the site of drug absorption and elimination, P-gp poses a major physiological barrier in dictating the pharmacokinetics of drugs [8]. The efflux pump limits drug entry into systemic circulation from the intestine, pumps out hepatocytic drug into the canalicular system, prevents distribution into vital organs such as the brain, and limits reabsorption of drug into systemic circulation from renal tubules [2,9]. These effects of P-gp on drugs have essentially forced discovery scientists to screen molecules for P-gp involvement during pharmacokinetic optimization. Additionally, many drug–drug or drug–food interactions in preclinical and clinical studies have been associated with transportermediated efflux [10,11]. P-gp mediated efflux of a compound is more likely to have an impact on drug exposure through a decrease in intestinal absorption rather than an increase in systemic elimination through bile or urine. However, the contribution of P-gp efflux to oral drug absorption is not likely to be quantitatively important for all P-gp substrates (P-gpS) [12
]. A thorough understanding of drug efflux and its influence by physiological variables can aid in developing predictive models and suggest approaches to improve peroral drug delivery. This article gives an overview and update on the knowledge of recognizing the relevance of P-gp efflux to in vivo intestinal absorption vis-a`-vis the biopharmaceutical factors that influence P-gp mediated efflux.

Introduction

Drug efflux and in vivo absorption

The efflux pump P-glycoprotein (P-gp) plays a major role in altering the pharmacokinetics of a wide variety of drugs. P-gp affinity screening using various in vitro culture models has become an integral part of discovery programs [1,2,3
]. Molecular biology and biochemical methodologies, especially immunohistochemical analysis, imaging and cloning studies have considerably increased our

An increasing number of drugs, including HIV protease inhibitors such as indinavir, ritonavir and saquinavir, and anticancer drugs such as paclitaxel and docetaxel, have been reported to be substrates for P-gp [8]. At the same time, it should also be realized that P-gp does not have an effect on the bioavailability of a number of drugs from the above therapeutic class. P-gpS such as etoposide,

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indinavir, ritonavir and verapamil, which exhibit varying degrees of efflux, show dose-independent in vivo absorption kinetics [13]. On the contrary, P-gpS such as digoxin, paclitaxel, talinolol and saquinavir, which exhibit high efflux, show improved bioavailability in the presence of P-gp inhibitors (P-gpI) [8,14]. This leads to the fact that both passive permeability and the P-gp efflux process operate in mutually opposite directions, contributing to overall absorptive permeability and bioavailability. The permeability of a P-gpS is the net result of passive influx rate minus the P-gp mediated active efflux rate. For a number of P-gpS the drug absorption is not affected, despite a high drug efflux. This can be ascribed to a lower control by P-gp, caused by higher passive transmembrane movement rate and/or lower intrinsic activity of P-gp. Efflux ratio (ratio of secretory permeability to absorptive permeability) is the most common parameter used in in vitro studies to indicate the possible involvement of P-gp mediated efflux. Although the affinity of a molecule to Pgp is the main determinant for its efflux, the correlation between efflux ratio and MDRI-MDCKII (multidrug resistance transfected MDCK type II) monolayer permeability for a set of P-gpS indicates that the rate of passive transport plays a major role in P-gp mediated efllux [12
,15,16]. If the drug has a moderate passive transport, it tends to show significant P-gp mediated efflux. On the other hand, for a P-gpS with high passive transport, the secreted molecules can rapidly reabsorb into the enterocytes and hence the influence of P-gp mediated efflux will be negligible. Troutman and Thakker proposed a new parameter, absorption quotient (AQ), which can predict how P-gp mediated efflux activity attenuates intestinal permeability in vivo [17,18]. AQ ¼

PPg p;AB PPD;AB

This ratio indicates the contribution of P-gp efflux to the overall passive transport from the apical to basolateral side. PP–gp,AB expresses the effect P-gp would have in attenuating absorption transport. PPD,AB is the overall passive transport that takes into account passive permeability (Papp,AB) in the presence of P-gp activity (PP–gp,AB). AQ quantifies the functional activity of P-gp observed during absorptive permeability, and does not consider the asymmetric effect of P-gp on absorptive (PPD,AB) versus secretory transports (PPD,BA). A linear relationship between AQ and permeability was reported with a subset of P-gpS, where P-gpS with high AQ were found to have less absorptive permeability [12
]. A model was developed that takes into account AQ, in vitro permeability coefficient and fraction absorbed in humans to quantitatively predict the functional role of P-gp in limiting oral drug absorption for in vivo absorption enhancement on P-gp inhibition [19]. The model demonstrated that complete inhibition of P-gp has a profound effect on the fraction absorbed for low and moderately permeable drugs, which is quantitatively Current Opinion in Chemical Biology 2006, 10:367–373

dictated by their affinity for P-gp (Figure 1). On the basis of these principles, a classification system was proposed to differentiate substrates for which P-gp mediated efflux has a significant effect from those that have an insignificant effect on in vivo absorption in humans [19].

Physicochemical properties versus drug efflux Simple empirical rules and predictive models based on molecular descriptors and physicochemical properties aid in rank-ordering oral activity of the molecules from chemical libraries [20,21]. Lipinski’s ‘rule-of-five’ is the first qualitative attempt to develop tools to help chemists design orally active compounds, and is now extensively used in selecting drug molecules for further development [22]. As implemented in the Pfizer registration systems, the rule-of-five generates an alert for compounds when any two or more of the following conditions are not satisfied: 1. 2. 3. 4.

Molecular weight (MW) <500 Da Number of hydrogen bond acceptors <10 Number of hydrogen bond donors <5 Calculated n-octanol-water partition coefficient (Clog P) <5

The dependence of permeability on molecular size, hydrogen bonding and polar surface area has been demonstrated on a number of occasions [23,24]. Didziapetris et al. analyzed the classification of P-gp substrate specificity using MW, hydrogen bonding and pKa to differentiate P-gpS from non-substrates. They reported that compounds with MW and hydrogen bonding above certain limits are likely to be P-gpS [25]. The rule-of-five seems to hold equally well in identifying P-gpS, which show limited absorption in vivo. This may be because the drug efflux is directly related to the passive permeability, which in turn is determined by these rules. As shown in Figure 2a, a set of PgpS with MW 500 and Clog P in the range of 1–5 shows high permeability in MDCK cell monolayer, whereas compounds with MW >500 and Clog P in the above range show less permeability [12
]. With very few exceptions, P-gpS with MW >500 and total polar surface area (TPSA) >75 A˚2 fail to show good permeability (Figure 2b). Although P-gpS have lipophilicity in the desirable range for exhibiting high permeability, MW and TPSA of these compounds significantly limit passive transport. Because P-gpS with low or moderate passive permeability are more attenuated by P-gp, this dataset implies that unfavorable physicochemical properties limit their passive transport leading to more susceptibility to efflux pump. Hence, it is important to keep the molecular properties of the candidates in the chemical libraries within the limits of the rule-of-five.

GI physiological variables and drug efflux Several reports have shown regional variability in intestinal P-gp expression and/or function in human and www.sciencedirect.com

Functional role of P-glycoprotein Varma, Perumal and Panchagnula 369

Figure 1

Quantitative estimation of the functional role of P-gp in intestinal drug absorption, based on the combined effects of P-gp activity and intestinal permeability. Effect of complete P-gp inhibition on the fraction absorbed in human as a function of Peff,control (rat in situ permeability in the presence of P-gp activity) for P-gp substrates with different AQ. Inset shows the maximum achievable in vivo human intestinal absorption enhancement for drugs with different Peff,control and AQ, on P-gp inhibition. Human intestinal absorption enhancement ratio, the ratio of Fa,human in the absence and presence of P-gp efflux, was found to be more than 1.5-fold for P-gpS with AQ = 0.75 and moderate Peff,control (between 0.002  104 and 0.1  104cm/s). However, P-gpS with high Peff,control (>0.28  104cm/s) are not affected by P-gp inhibition or induction. Reproduced from [19] with the permission. Copyright 2005, Wiley Interscience.

rodents [26–28]. Mouly and Paine reported that relative Pgp levels increased gradually from jejunum to distal ileum (roughly twofold) in human donor intestine [29]. Gradient P-gp expression may significantly affect drug absorption for P-gpS with low solubility and/or permeability (Class II– IV of biopharmaceutic classification system (BCS)) [19,23]. Jejunum has a relatively shorter transit time (40 min) favoring absorption of compounds that exist in solution, and has a high membrane permeability (BCS class I). Such compounds may rapidly partition into the membrane and, therefore, P-gp mediated efflux is counterbalanced by the higher passive influx. On the contrary, the poor solubility and/or permeability of class II–IV drugs of BCS, shifts their absorption site more towards the ileum, which has a transit time of 140 min. The high P-gp expression levels in the lower intestine make them more susceptible to P-gp mediated efflux [19]. Inherent differences in passive transport along the gut, mainly due to differences in lipid bilayer composition, may regulate the net absorption of P-gpS from various segments [28]. Further, P-gp displays entangled connections with its membrane lipids and proteins since it recognizes its substrates within the cytosolic leaflet, and also translocates some endogenous lipids to the exoplasmic leaflet (see Update). For protic and ampholytic drugs, the charge state is an important factor associated with their passive permeability [30]. When an www.sciencedirect.com

ionizable P-gpS moves down the gut, it experiences various pH conditions that change its degree of ionization. This can influence its passive permeability vis-a`-vis the efflux activity [31–34]. Recently, for basic drugs, Varma and Panchagnula [33] demonstrated that high passive permeability at higher pH leads to decreased P-gp efflux in situ (Figure 3). The study also showed the dependence of efflux activity on inherent regional differences in passive permeability. Although the apparent P-gp expression level is high in ileum, the inherent high regional passive influx of quinidine counter-balanced the high Pgp mediated efflux. By integrating all these factors, it is possible to estimate the change in absorption rate along the gut and also define drug ‘absorption window(s)’.

Drug design to overcome P-gp efflux Structural modifications during lead optimization should carefully consider the substrate specificity to P-gp. For example, the optimization of pro-drugs of opioid peptide DADLE led to the development of an oxymethyl-modified coumarinic acid-based cyclic pro-drug, which appeared to be sensitive to esterases [35]. However, this prodrug exhibited substrate specificity for efflux transporters, which in vivo would limit its permeation across the intestinal mucosa. Similarly, restricted permeability was also shown for acyloxyalkoxy-based cyclic pro-drugs of DADLE [36]. Other complications involved with P-gp during structural modifications have been illustrated with Current Opinion in Chemical Biology 2006, 10:367–373

370 Next-generation therapeutics

Figure 2

Analysis of relationship between physicochemical properties and MDRI-MDCKII monolayer permeability of P-gpS. (a) ClogP versus permeability, and (b) TPSA versus permeability of P-gpS with different MW range. Data taken from [12
,15,16]. Permeability criteria were set as low (Papp,AB  20 nm/s) and high (Papp,AB  100 nm/s) permeability (shaded area) on the basis of the discussion in [12
]. In the dataset, about 30% of molecules had MW above 500. The mean MW and TPSA of P-gpS are 488.4 A˚ and 92.3 A˚. It is also interesting to note that P-gpS with MW >500 and TPSA >100 A˚ have an average efflux ratio of 49.5, indicating that bidirectional transport of P-gpS with the above physicochemical properties is highly asymmetric. Two possible reasons for this trend are (i) limited passive influx due to unfavorable physicochemical features and/or (ii) high substrate–transporter interaction determined by these features. The data points in the graph indicate P-gpS with differing molecular weight (Da).

ME3229, an ester-type prodrug of a hydrophilic glycoprotein IIb/IIIa antagonist. ME3229, developed for increasing the bioavailability of the parent compound, showed sufficient lipophilicity to cross the apical membrane, but only a small fraction of the active metabolites formed in the enterocytes reached the mesenteric veins, Current Opinion in Chemical Biology 2006, 10:367–373

primarily attributed to the efflux of metabolite [37]. A simple approach to evade P-gp efflux in vivo is to improve the passive permeability of the molecule by carefully manipulating the lipophilicity, molecular size and hydrogen binding capacity to effectively saturate P-gp and/or reduce access to the protein. www.sciencedirect.com

Functional role of P-glycoprotein Varma, Perumal and Panchagnula 371

Figure 3

Effect of luminal pH on the in situ permeability and P-gp functional activity (AQ) of quinidine in the absence (control) and presence (+ Ver) of P-gp inhibitor, in jejunum and ileum. P-gp functional activity was found to decline with increase in pH in both jejunum and ileum, and this trend was attributed to the increased passive flux of quinidine as the pH increases. The difference in jejunum and ileum P-gp activity at a fixed pH can infer the intrinsic regional variability in quinidine passive transport. AQ is absorption quotient; ver, verapamil. A star indicates significant difference ( p < 0.05) from the control at corresponding pH and a hash indicates significant difference ( p < 0.05) from permeability at pH 4.5. Reproduced from [33], with permission. Copyright 2005, Wiley Interscience.

Unlike the use of well-defined structure–activity relationships (SARs) for identifying the structural features necessary for activity, designing molecules that circumvent Pgp efflux is still hampered by lack of information on the 3-D structure of P-gp [38]. Nevertheless, certain qualitative and semi-quantitative rules can aid in drug design to overcome susceptibility to P-gp mediated efflux [39]. Substrates and non-substrates are characterized by specific spatial separation of pairs of recognition elements that have different strengths to act as electron donors or acceptors [40]. This was exemplified by chemical modification of paclitaxel, where succinate added at the C10 position of paclitaxel resulted in a lesser interaction with P-gp [41]. Further, it was found that simple hydrolysis or epimerization of the C10 acetate of paclitaxel can also reduce P-gp interaction [42]. The prodrug approach was also successful in circumventing P-gp mediated efflux [43,44].

Design of inhibitors The role of P-gp in limiting oral absorption was first demonstrated for paclitaxel in a preclinical study showing an increase in the apparent oral bioavailability from 11% in control mice to 35% in mdr1a/ mice [45]. Several studies have since demonstrated the possible use of P-gpI that reverse the MDR phenotype-associated P-gp mediated efflux in an attempt to improve the efficacy of chemotherapy and pharmacokinetic profiles for a number of challenging molecules. To this end, clinical trials have began to test the clinical potential of existing drugs that were found to be effective P-gpI (first-generation inhibitors). Improved clinical efficacy achieved through www.sciencedirect.com

P-gp inhibition for various P-gp substrate drugs encouraged the development of inhibitors that specifically block P-gp efflux. Second-generation inhibitors are mostly analogs of the previous generation that lack the pharmacological activity and usually have a higher P-gp affinity. But, the affinity towards two or more ABC transporters and CYP enzymes resulted in complicated drug interactions involving metabolism and clearance. Several P-gpI, such as elacridar (GF120918) and biricodar, also act on other ABC transporters such as BCRP and MRP1, increasing the scope of possible side effects and drug interactions. On the positive side, these may provide improved absorption for molecules effluxed by multiple transporters. However, the clinical applications depend on the affinity of P-gpS for P-gp and/or other ABC transporters and the toxicity profile of the drug–inhibitor combination [46]. Third-generation P-gpI are designed primarily to improve the chemotherapy of multidrug-resistant tumors and to inhibit P-gp with high specificity. The inhibitors zosuquidar (LY335979), oc144093 (ONT-093), tariquidar (XR9576) and laniquidar (R101933) were selective and more potent than the earlier inhibitors. LY335979 has been extensively characterized for specificity and found not to inhibit MRP2, MRP3 or BCRP [47,48]. It was also found that LY335979 is 60 fold more specific for P-gp over CYP enzymes. Overall, the third-generation P-gpI exhibit the characteristics of an ideal P-gp inhibitor; they are very effective inhibitors and also do not show pharmacokinetic interactions involving other transporters of the same family or with the metabolic enzymes [49]. It is Current Opinion in Chemical Biology 2006, 10:367–373

372 Next-generation therapeutics

also important to consider the biopharmaceutical properties including solubility and stability of P-gpI that ultimately determine their local concentrations in the gut to act as an effective inhibitor. In addition, pharmaceutical surfactants (tween 80, pluronic P85, TPGS, tritonX-100, cremophor EL) and food/ herbal extracts (grapefruit juice, orange juice, St John’s wort) are emerging as a separate class of P-gpI. Recent studies [50] suggested that pluronic block copolymers are potent non-ionic surfactant inhibitors of both P-gp and MRP efflux. It was found that the pluronic P85 caused membrane fluidization leading to a decrease in P-gp ATPase activity. Preclinical studies in rats showed about six-fold increase in the bioavailability of paclitaxel when administered with water-soluble vitamin E derivative TPGS [51]. The authors observed that the increase in bioavailability is due to micellar solubilization and inhibition of P-gp efflux by TPGS. These properties of TPGS have been effectively translated into a clinically effective oral formulation for amprenavir (AgeneraseTM). Clearly, the contribution of P-gpI in oral drug delivery is evident; and at least a few P-gpI under development are likely to emerge as delivery tools in near future.

Conclusions The contribution of P-gp in limiting intestinal absorption is determined by (i) affinity of drugs towards P-gp, (ii) the passive permeability of the drug molecules across the enterocytes, (iii) expression levels of P-gp and variability in expression levels along the gut, and (iv) physiological variables that influence the solubility and passive transport along the gut. Estimating the quantitative involvement of these factors can help in drug design and assist formulation scientists to explore the possible means to evade P-gp mediated efflux in vivo (Viz. design molecules with improved passive permeability and/or molecules with desired affinity to P-gp, and appropriate use of P-gpI). With current knowledge, it is possible to reasonably estimate the functional activity of P-gp in limiting intestinal absorption. However, attention should be focused to develop better models that incorporate all relevant physicochemical and physiological determinants. This would provide improved biopharmaceutic screens in drug discovery and aid in devising strategies to address the ‘permeability dogma’ in peroral drug delivery.

Update It is now recognized that membrane bilayer composition determines the P-gp functional activity independent of passive influx rate. The recent literature analysis by Orlowski et al. on the P-gp relationships with membrane micro-domains concluded that P-gp expression, substrate recognition and functional activity are determined by the cholesterol composition and the other protein partners within the membrane bilayer of cells [52]. Current Opinion in Chemical Biology 2006, 10:367–373

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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Current Opinion in Chemical Biology 2006, 10:367–373