20780_ftp

REVIEW

Lipid Formulation Strategies for Enhancing IntestinalTransport and Absorption of P-Glycoprotein (P-gp)Substrate Drugs: In vitro/In vivo Case Studies

PANAYIOTIS P. CONSTANTINIDES,1 KISHOR M. WASAN2

1Biopharmaceutical & Drug Delivery Consulting, LLC, Gurnee, IL 60031

2Division of Pharmaceutics and Biopharmaceutics, University of British Columbia, Vancouver, Canada V6T123

Received 23 May 2006; revised 23 August 2006; accepted 24 August 2006

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20780

ABSTRACT: The intestinal efflux pump, P-glycoprotein (P-gp), located in the apicalmembranes of intestinal absorptive cells, can reduce the bioavailability of awide range ofdrugs which are substrates for thismembrane transporter. In addition to anticancer andanti-HIV drugs, NCEs for other disease indications are P-gp substrates and thereis considerable interest in inhibiting P-gp and thus increasing the bioavailability of thesemolecules. In this review article, an overview of P-gp and its role in drug transportand absorption will be presented first and then formulation strategies to effectivelyinhibit P-gp will be discussed and compared. These strategies independently and incombination, are: (a) coadministration of another P-gp substrate/specific inhibitor, and(b) incorporation of a nonspecific lipid and/or polymer excipient in the formulation. Thefirst approach, althoughveryeffective in inhibitingP-gp,utilizes a secondactive compoundin the formulation and thus imposes regulatory constraints and long developmenttimelines on such combination products. Excipient inhibitors appear to have minimalnonspecific pharmacological activity and thus potential side effects of specific active com-pound inhibitors can be avoided. Case studies will be presented where specific activecompounds, surfactants, polymers, and formulations incorporating these molecules areshown to significantly improve the intestinal absorption of poorly soluble and absorbeddrugs asa result of P-gp inhibitionand enhanced drug transport in vitro. �2006Wiley-Liss,

Inc. and the American Pharmacists Association J Pharm Sci 96:235–248, 2007

Keywords: biopharmaceutics; drug transport; P-glycoprotein inhibition; activecompounds; surfactants; lipid formulations; self-emulsifying drug delivery systems;poorly soluble drugs; case studies

OVERVIEW OF P-GLYCOPROTEIN AND ITSROLE IN DRUG TRANSPORTAND ABSORPTION

P-glycoprotein (P-gp) is a membrane-bound trans-porter that mediates active transport, ‘‘efflux,’’ of

a wide range of structurally unrelated drugs andother xenobiotics out of the cells.1 It is expressedalong the entire length of the gut and also in theliver (canalicular membrane), kidney, blood-brainbarrier, and placenta.1,2 Intestinal P-gp is locatedon the apical membranes of the epithelial cells.Utilizing the energy that is generated by hydro-lysis of ATP,3 P-gp drives the efflux of varioussubstrates against a concentration gradient andthus reduces their intracellular concentration andin the case of drugs their oral bioavailability

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 2, FEBRUARY 2007 235

Correspondence to: Panayiotis P. Constantinides (Tel: 847-599-9496; Fax: 847-599-9496; E-mail: [email protected])

Journal of Pharmaceutical Sciences, Vol. 96, 235–248 (2007)� 2006 Wiley-Liss, Inc. and the American Pharmacists Association

(Fig. 1A and B). The proposed models of P-gpaction are shown in Figure 1A. In the Pore model(a), drugs associate with P-gp in the cytosoliccompartment and are transported out through aprotein channel. A main feature of the Flippasemodel (b) is that P-gp acts as a drug exporter byflipping drugs from the inner leaflet of the plasmamembrane to the outer leaflet against a concen-tration gradient. Finally, in regard to VacuumCleaner Model (c), intramembranous moleculeswhich do not belong to the membrane, arerecognized by P-gp, enter P-gp from the membra-nous site and leave the cell. Amongst the proposedthree mechanistic models, it appears that theFlippase model is the most widely accepted. Aschematic representation of the structure of P-gp

with the membrane spanning, endo- and exo-domains, is shown in Figure 1B.3

This complex glycoprotein (MW of 170 kDa;Fig. 1A) is the product of the MDR1 gene andeffluxes drugs without modification thereby con-ferring multidrug resistance. Approximately 50%of the anticancer drugs used clinically today aresubstrates for P-gp and include anthracyclines,vinca alkaloids, taxanes, epidophyllotoxins, mito-mycins, camptothecins, and other.4–6 Given itsbroad specificity for anticancer drugs, overexpres-sion of P-gp in many types of tumors appears to beprevalent and may be induced rapidly in responseto chemotherapy.

The link of P-gp inhibition to drug transportand absorption is shown in Figure 2 where the

Figure 1. (A)Proposedmodels ofP-gp action: (a)Poremodel: drugs associatewithP-gpin the cytosolic compartment and are transported out through a protein channel.(b) Flippasemodel: P-gp acts as a drug exporter by flipping drugs from the inner leaflet ofthe plasma membrane to the outer leaflet against a concentration gradient. (c) VacuumCleaner Model: intramembranous molecules that do not belong to the membrane,are recognized by P-gp, enter P-gp from the membranous site and leave the cell.(B) Schematic representation of the structure of P-gp (adapted with permission from thepublisher, Ref. 3).

236 CONSTANTINIDES AND WASAN

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 2, FEBRUARY 2007 DOI 10.1002/jps

intersected area in the Venn diagram representsin vitro/in vitro correlations. Caco-2 and MDCKcells expressP-gpandareamongst themostwidelyused cellular models to study drug absorption.7

Since Caco-2 cells are derived from human coloncarcinoma cells, P-gp expression and inhibitionmay closely resemble in vivo P-gp expression andinhibition; although such correlations have not yetstrictly established. MDCK cells originate fromdog kidney and thus there may be significantdifferences in their substrate and inhibitor specifi-cities from those of the human transporter.7 There

are other advantages and disadvantages in the useof the Caco-2 versus theMDCKmodel in referenceto their culturing times and transepithelial elec-trical resistance (TEER) with the former cellsexhibiting longer culturing times (2–3 weeks) andforming very tight junctions with higher TEERvalues. However, it should be kept in mind thatany cell culturemodelmaybehavedifferently fromthe in vivo situation particularly due to the lack ofgut content flow and basolateral blood flow.7 Thus,it not surprising that when the permeability ofvarious compounds was plotted against lipophili-city, the resulting curve was found to be sigmoidalwith a very sharp slope and this was correlated tovariability in the actual drug absorption in vivo.7

Regardless of their limitations, cellular modelscontinue to be a valuable tool to study drugtransport and absorption of multiple compoundsand formulations in a timely fashion.

P-GLYCOPROTEIN SUBSTRATES ANDINHIBITORS: CLASSIFICATIONSAND COMPARISONS

In recent years, identifying and developing selec-tive and potent P-gp inhibitors has been a majorfocus area in drug development particularly intreating drug-resistant tumors. P-gp inhibitorsare noncytotoxic compounds and when combinedwith drugs that are effluxed by P-gp, theirintracellular concentration is maintained andsensitivity to these therapeutics is restored.6–9

The P-gp inhibitors developed and used clinicallyto date can be divided into two major classes,specific and nonspecific (Tabs. 1 and 2).

There are three generations of specific P-gpinhibitors (Tab. 1). The first generation incorpo-rates compounds which tend to be less potent and

Figure 2. P-gp inhibition, drug transport, andabsorption interrelationships. This diagram representsa simple way to emphasize the link between P-gpinhibition and drug transport and absorption. Thecommon intersection area represents in vitro/in vivocorrelations. Drug absorption is defined as prehepaticconcentration of the drug.

Table 1. P-gp Inhibitors: Approved Actives and New Chemical Entities

1st Generation: Developed for other indications (less potent and not selective)

Examples: Verapamil, Cyclosporine

2nd Generation: Developed initially to reduce toxicity, more selective than 1stgeneration due to reduced toxicity

Examples: PSC 833 (CsA analog) without immunosuppressive effect

Dexverapamil without the cardiac effect of verapamil

3rd Generation: Designed to be more potent and selective inhibitors

Examples: VX-710 (biricodar)GF120918 (elacridar)LY335979 (zosuquidar.3HCl)OC144-093

LIPID FORMULATION STRATEGIES FOR P-gp INHIBITION 237

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 2, FEBRUARY 2007

not selective and have primarily been developedfor other indications with unwanted side effects atlevels necessary to inhibit P-gp clinically.8 Thesecompounds include, the calcium channel blocker,verapamil, and the immunosuppressive drug,CsA. PSC 833 an analog of cyclosporine withoutthe immunosuppressive effect and dexverapamil,a verapamil analog without its cardiac effect,belong to the second generation P-gp inhibitorswhich were developed primarily to reduce thetoxicity of the parent compound/drug.8Finally, thethird generation inhibitors are characterized asbeing more potent and selective. The followingthird generation inhibitors are at least 10-foldmore potent than the earlier compounds andinhibit P-gp in the 30–100 nM range:8 VX-710(biricodar), GF 120918 (elacridar), LY335979(zosuquidar.HCl), XR9576 (tariquidar), and OC144-093.

Coadministration of specific pharmacologicallyactive compounds can effectively enhance the oralbioavailability of P-gp substrate drugs. Thesecompounds, however, have their own pharmaco-logical activity and thus potential drug–druginteractions and enhanced side effects in suchcombination drug products need to be consideredand properly addressed. In addition, active com-pound inhibitors may also interact with P-gpin other organs/tissues as it has been shown withthe new generation P-gp inhibitors.8 Thus, dueto anticipated product development challengesand regulatory constraints with dosage formsincorporating two active compounds, there is aneed to identify other P-gp inhibitors which haveminimal nonspecific pharmacological activity.

Nonspecific inhibitors of P-gp include surface-active compounds, suchas lipids/surfactantsand/orpolymers (Tab. 2). These agents exhibit low non-specific pharmacological activity unlike the poten-tially more serious side effects of the active

compound inhibitors. Several excipients whichare present in pharmaceutical formulationscan indirectly inhibit P-gp through effects on thelipid membrane and thus enhance the intestinalmembrane permeability and oral absorption ofthe substrate drug.10 Surfactant inhibitors, pri-marily nonionic ones, include polyoxyethylated/pegylated surfactants, such as, polyoxyl 35 castoroil (Cremophor), PEG-15-hydoxystearate (SolutolHS-15), medium-chain (C8/C10) glycerol and PEGesters (Labrasol, Softigen 767 and Acconon E),polysorbates such as Polysorbate 20 and 80,sucrose esters such as sucrose monolaurate, ana-tocopheryl polyethylene glycol-1000-succinate(TPGS). Amongst polymers, effective P-gp modula-tors are pluronic block copolymers which includepoly(ethylene oxide)/poly-(propylene oxide) blockcopolymers and amphiphilic diblock copolymers,such as methoxypolyethylene glycol-block-polyca-prolactone (MePEG-b-PCL).11

Structurally as discussed earlier, P-gp is amembrane-bound protein composed of 12 trans-membrane domains and 2 cytoplasmic nucleotidebinding domains. Based on the widely acceptedFlippase model (Fig. 1A), P-gp translocates hydro-phobicmolecules/drugs from the inner to the outerleaflet of the lipid bilayer upon a conformationalchange induced by ATP binding to a cytoplasmicbinding domain (Fig. 1B). Thus, its activity ismodulated by the physical state of the lipid bilayerwhere the protein resides as it has been demon-strated by several studies.4,6,12,13 Hydrophobicsubstrates are first partitioned into the lipidbilayer and then by lateral diffusion interact withthe P-gp binding domain within the lipid mem-brane.12,13 Molecules which can interact withthe lipid bilayer, such as, surfactants and blockcopolymers can modulate the activity of P-gpindirectly or nonspecifically. An alteration in thefluidity of the lipidmembrane environment of P-gp

Table 2. P-gp Inhibitors: Lipidic and Polymeric Excipients

(A) SurfactantsC8/C10 glycerol and PEG esters: Cremophor, Solutol HS-15, Labrasol, Softigen

767, Aconnon ESucrose esters: Sucrose monolauratePolysorbates: Tween 80, Tween 20Tocopherol esters: a-tocopheryl-PEG-1000-succinate (TPGS)

(B) PolymersPluronic block copolymers (poloxamers): poly-(ethyleneoxide)/poly-(propyleneoxide)

block copolymersAmphiphilic diblock copolymers: Methoxypolyethylene glycol-block-polycaprolactone

(MePEG-b-PCL)



by surfactants was shown to modulate drug effluxfrom MDR cells by reduction in the P-gp ATPaseactivity.13 The mechanisms by which amphiphilicpluronic block copolymers modulate P-gp activityinclude both membrane permeability changesthrough a reduction in membrane microviscosity,and cellular depletion of ATP levels.10 WhileP-gp inhibition by certain surfactants10,14–22 wasobserved at concentrations below or at theircritical micelle concentration (CMC), for theMePEG-b-PCL copolymers modulation of the P-gp activity was observed at concentrations abovetheir CMC with little or no activity below theCMC.11 It has been suggested that vesiculartransport of micellized substrate may be one ofthe possiblemechanisms bywhich thesemoleculesmodulate P-gp activity.11

Table 3 summarizes several biopharmaceuticaleffects of lipid-based systems on the absorption ofthe various classes of drug molecules,23 accordingto the Biopharmaceutics Classification Scheme.24

In addition to improving the solubilization ofpoorly soluble drugs, other biopharmaceuticaleffects of lipids include inhibition of enzymatichydrolysis and drug efflux. Due to these effects,intestinal absorption and oral bioavailabilityenhancement have been well established andrepresentative case studies will be presented insubsequent sections of this Review article.

P-gp INHIBITION BY LIPID EXCIPIENTSAND FORMULATIONS: IN VITROCASE STUDIES

As discussed in the preceding section, there aremany reports in the literature on the modulation

of the activity of P-gp using lipidic and polymericexcipients. The following case studies are repre-sentative of this rapidly growing field of biophar-maceutics.

Paclitaxel

Proprietary lipid polymer emulsions (LPETM)incorporating P-gp inhibitors were developedand assessed for their ability to enhance pacli-taxel transport in Caco-2 cells and to correlatethis effect to intestinal drug absorption enhance-ment in rats.25,26 The ability of LPETM formula-tions to inhibit P-gp in Caco-2 cell monolayers wasassessed using a calcein extrusion assay.27 Mono-layers of Caco-2 cells were incubated with testformulations and controls and then with calcein-AM, a fluorescent P-gp substrate. After washing,the amount of intracellular calcein was measuredwith a fluorescent plate reader. The data inFigure 3A demonstrated that both LPETM-S,incorporating C8/C10 glycerol and PEG esters(Softigen 767) and LPETM-CsA formulations wereeffective inhibitors of P-gp. Incorporation of CsAinto LPETM did not affect its P-gp inhibitoryactivity. LPETM formulations incorporating Softi-gen 767 inhibited P-gp at 50%–60% of the levelof inhibition observed by CsA alone (positivecontrol).

Formulations containing a constant amountof paclitaxel were incubated with Caco-2 cellsgrown on Transwell filters. The transwells wereremoved after the transport period and Jurkatcells in the receiver wells were incubated for anadditional 48 h. The viability of the Jurkat cellswas then assessed by their ability to reduce thetetrazolium dye WST-I.28,29 Figure 3B shows the

Table 3. Biopharmaceutical Effects of Oral Lipid-Based Systems (Modified fromRef. 23)

BCS Aqueous SolubilityMembranePermeability

Potential Effect ofLipid-Based Systems

I High High Enzymatic degradation #Gut wall efflux #

II Low High Solubilization "Bioavailability "

III High Low Enzymatic degradation #Gut wall efflux #Bioavailability "

IV Low Low Solubilization "Enzymatic degradation #Gut wall efflux #Bioavailability "



concentrations of paclitaxel transported fromLPETM-S and LPETM-CsA formulations and com-pared these values to those produced by mediaalone. Under the assay conditions there was noloss of monolayer integrity as determined bytransepithelial resistance measurements datanot shown). This data indicates that LPETM-CsAand LPETM-S formulations were effective in enhan-cing transport of paclitaxel across the Caco-2monolayer although some transport was observedin the absence of these lipidic formulations.

Enhancement of paclitaxel solubility and per-meability by TPGS in vitro/in situ and effects onin vivo drug absorption has recently been reportedby Varma and Panchagnula.30 In situ studiesusing rat ileum tissue in Ussing chambers, thebi-directional transport of 14C-paclitaxel was alsomonitored in the presence of increasing concentra-

tions of TPGS. The apical-to-basolateral (A!B)permeability of paclitaxel was found to increase inthe presence of TPGS while the basolateral-to-apical (B!A)wasdecreasedwithmaximumeffectat 0.1 mg/mL of TPGS.30 Increasing TPGSconcentrations above 0.1 mg/mL, resulted in adecrease in A!B permeability with no change inthe B!A permeability, which suggests the invol-vement of monomeric and not micellar TPGS inP-gp inhibition.30

HIV Protease Inhibitors

TPGS with a CMC of 0.2 mg/mL was alsoshown to improve the solubility and intestinalpermeability of amprenavir, a protease inhibitormarketed as Agenerase1 by GSK.31 When theconcentration of TPGS was increased above0.1 mg/mL, an improvement in paclitaxel solubi-lity was observed presumably via micellar solubi-lization similarly to the reported effect of TPGS onpaclitaxel solubility.30 The effects of TPGS werereported to be: (a) improvement in the solubility(S) and thus dissolution of amprenavir throughmicellar solubilization, (b) enhancement of thepermeability (Peff) of amprenavir across the gutwall, presumably through drug efflux inhibition.In one particular study, TPGS enhanced theoverall absorption flux (J¼Peff�S) of the drug(Fig. 4) by increasing its solubility and intestinalpermeability.31

To correlate the inhibitory effects of TPGS ondrug efflux to the structure of TPGS and specifi-cally to the chain-length of the PEG group, several

Figure 3. (A) Inhibition of P-glycoprotein by lipidexcipient alone and in a lipid polymer emulsion(LPETM). S stands for Softigen 767, a C8/C10 mixtureof glycerol and pegylated esters (B). Paclitaxel transportin Caco-2 cells in the presence of LPETM formulationsincorporating P-gp inhibitors. S stands for Softigen 767,a C8/C10 mixture of glycerol and PEG esters.

Figure 4. Amprenavir flux in the presence of TPGS.Absorption flux levels off above 0.5 mg/mL of TPGSsuggesting P-gp inhibition by monomeric TPGS(adapted from Ref. 31).



analogs incorporating PEGs with an average MWin the range of 200–6000 were synthesized andevaluated in vitro.32 The commercially availablegrade of TPGS contains PEG 1000 and its effect onthe efflux transport of the P-gp substrate Rhoda-mine 123 was compared against the other syn-thetic analogs in Caco-2 monolayers.32 The abilityof these molecules to inhibit P-gp and enhance theapical to basolateral transport of Rhodamine 123was also correlated to their cytotoxicity based onTEER measurements and lactate dehydrogenase(LDH) release. Interestingly, the results indicatedan optimum PEG chain-length near 1000 whichcorresponds to the commercial TPGS product.Further mechanistic studies on these TPGSanalogs are being pursued by the authors.32

Although TPGS is widely used as a drug solventandoral bioavailability enhancer for poorly solubledrugs primarily by inhibiting P-gp, there havebeen cases where TPGS had aminimal or no effecton P-gp inhibition and drug absorption.19,32–34

Differences in the physicochemical properties ofthe investigated compounds/drugs may account atleast in part for the lack of a significant P-gpinhibition in these studies.

Celiprolol and Digoxin

Studies reported by Cornaire et al.10 used theeverted gut sac technique to study transport oftwo P-gp substrate drugs, Digoxin and Celiprolol.Digoxin (logP¼ 1.51) undergoes intestinal andliver metabolism whereas celiprolol (logP¼ 0.31)transport is stable in the intestine.10 The excipi-ents were tested at 0.05, 0.1, and 0.5%, w/w andtheir effect on celiprolol tansport in the evertedgut sac model is shown in Table 4.10 CremophorEL and Acconon E had no effect whereas drugtransport was enhanced by other surfactants inthe order of Softigen 767>TPGS> Imwitor 742.10

In the case of digoxin, the rank order wasLabrasol> Imwitor 742>Acconon E¼Softigen767>Cremophor EL>Miglyol>Solutol HS-15>Sucrose Monolaurate>Polysorbate 20>TPGS>Polysorbate 80.10 The relative effects of theinvestigated excipients appear to be dependenton the physicochemical properties of the P-gpsubstrate with differences in the lipophilicity ofthe P-gp substrate being a potential determiningfactor.10 Other properties such as the hydrophilic-lipophilic balance (HLB) of the lipidic excipientmay also be a contributing factor.10 It should alsobe mentioned that potential toxicity of the lipidicexcipients should be considered in addressing

their clinical use in various formulations. In theaforementioned studies, it was found that sucrosemonolaurate and to a lesser extent Labrasol weretoxic to the intestinal cells.10 Under the employedexperimental conditions,10 these excipients pro-duce 378% and 239% release of the cytosolicenzyme LDH under the experimental conditionsused, respectively, relative to the 100% controlvalue in the absence of any excipient.

Amphotericin B (AmpB)

Amphotericin B (cLogP¼ 1.29) is a heptaenemacrolide antifungal derived from a strain ofStreptomyces nodosus and used in the treatmentof systemic fungal infections.35,36 AmpB is anamphoteric molecule with a carboxyl pKa of 5.7and an amino pKa of 10. Due to its poor watersolubility, AmpB is usually formulated as either acolloidal dispersion or in a lipid-based vehicle.

Further studies were conducted by Wasan andcolleagues to ascertain that the incorporation ofAmpB into a glyceride-rich excipient Peceol (gly-ceryl monooleate) significantly increased gastro-intestinal absorption of AmpB in white maleSprague-Dawley rats.35,36 Based on preliminarystudies,35 it was proposed that incorporation ofAmpB into mixed micelles composed of Peceol

Table 4. Effect of Surfactant Excipients on CeliprololAbsorption in the Everted Gut Sac (Adapted fromRef. 10)

Excipient

Excipient Concentration (%, w/w)

0.05 0.1 0.5

Control 100% 100% 100%Cremophor EL 91� 58 87� 68 66� 18TPGS 52� 11* 53� 15 215� 20**Acconon E 92� 17 50� 12 96� 34Imwitor 742 141� 22 123� 20 189� 24**Softigen 767 120� 22 96� 13 282� 32**

Uptake is expressed as percentage of celiprolol uptake intothe serosal contents of everted gut sacs in the presence ofexcipients compared with the control (no excipient). Reportedvalues are means�SE (n¼ 3).

*p<0.05., the rest of the values are not significant bycomparisonwith the control. A two-way in reference to the timeand concentrations of excipients ANOVA method with mul-tiple comparisons (Fisher’s pairwise comparisons) was used forthe statistical analysis of the data.10

**p< 0.01, the rest of the values are not significant bycomparisonwith the control. A two-way in reference to the timeand concentrations of excipients ANOVA method with mul-tiple comparisons (Fisher’s pairwise comparisons) was used forthe statistical analysis of the data.10



would significantly enhance gastro-intestinal (GI)tract absorption by decreasing P-glycoprotein (P-gp)-mediated drug efflux. Caco-2 cells were seededat 10000 cells/cm2 in T-75 flasks. When the cellsreached 80% confluency, they were treated for1 day and 7 days with 0.1% to 1.0% (v/v) Peceol ormedia alone (control). Following treatment, totalRNAwas isolated using TRIzol1 reagent, followedby reverse transcription into single-strandedcDNA. Polymerase chain reactions (PCR) wereperformed with specific primers for mdr-1. The P-gp protein content was determined by WesternBlot Analysis. A significant lower mdr-1 mRNAand P-gp protein expression within Caco-2 cellswas observed following 1- and 7-day treatmentwith Peceol 0.1% to 1.0% (v/v) compared tonontreated controls.36 Taken together, these find-ings suggest that Peceol may increase the gastro-intestinal absorption of AmpB by decreasing mdr-1 mRNA and P-gp protein expression, resulting inlower PGP-mediated AmpB efflux.36 Additionalin vitro AmpB transport studies are warranted tofurther support this hypothesis.

EFFECTS OF LIPID FORMULATIONSINCORPORATING SPECIFIC ANDNONSPECIFIC P-gp INHIBITORSON DRUG TRANSPORT ANDABSORPTION: IN VIVO CASE STUDIES

There is increasing interest in the use of self-emulsifying drug delivery systems (SEDDS) todeliver orally poorly soluble drugs, including thosewhich are substrates for P-gp.37–39 Their compat-ibility with a variety of molecular structures ofactive compounds, oils, surfactants, and cosolventsand ease of processing and manufacture makeSEDDS an attractive vehicle for pharmaceuticaldevelopment. One advantage in using SEDDSwithoral drugs that are P-gp substrates is their capacityto incorporate/solubilize high levels of specific andnonspecific P-gp inhibitors. This property will beillustrated in the subsequent case studies. In thecase of TPGS and other tocols, advances in theiruse as drug delivery vehicles and specific in vitro/in vivo case studies, including P-gp inhibition andoral absorption can also be found in a recentlypublished review article.40

Cyclosporin A

Absorption of CsA is affected by several factorssuch as poor solubility in aqueous solutions and

GI fluids, impaired bile flow and fat content in thediet.41 Sokol et al. presented clinical data obtainedfrom liver transplantation patients, showing thatTPGS enhances CsA absorption.42 Enhancementof the absorption of CsA was achieved when TPGSat 65 mg/kg/day was coadministered with the drugat doses in the range of 12.5–40 mg/kg/daycompared to 29–136 mg/kg/day in the absence ofTPGS. Thus, coadministration of TPGS resulted inapproximately 40%–70% reduction in the dailydose of CsA.

The role of TPGS in enhancing CsA absorptionwas further confirmed in clinical studies with livertransplantation patients.43–45 A reduction in CsAdaily dose and associated costs by TPGS withoutany clinical or biochemical evidence of TPGSinduced toxicity, was reported by Pan et al.43

Boudreaux et al. reported on increased plasmaconcentration of CsA upon TPGS administrationand this effect was attributed to enhanced drugsolubilization through micelle formation.44 CsApharmacokinetics in healthy subjects was affectedby TPGS and a significant increase in plasmaAUCwas reported.44 Independent studies have shownthat the improvements in CsA pharmacokineticswere the result of the effects of TPGS in enhancingdrug solubility and inhibiting P-gp and/or intest-inal metabolism.46

Paclitaxel

Paclitaxel, a highly hydrophobic and poorlywater-soluble molecule and key chemotherapeu-tic agent with other disease indications in clinicaldevelopment, is another oral drug with P-gplimited absorption. Reduction in the oral bioavail-ability of paclitaxel due to efflux by P-gp has beendemonstrated earlier by Sparreboom et al. in micehomozygous for a disruption of the mdr1a gene incomparison with normal mice.47 It was reportedrecently by Woo et al.48 that in the rat, approxi-mately 54% of the dose of the orally administeredpaclitaxel is lost due to efflux by P-gp, 38% due togut lumen metabolism, and 3.5% by gut wall andliver metabolism.

Improvement of the oral bioavailability ofpaclitaxel has been widely reported in theliterature.25,26,47–54 The most commonly usedapproaches were the coadministration of a specificP-gp substrate and/or incorporation of a nonspe-cific inhibitor in the formulation.Woo et al.48 usingKR-30031, a verapamil analog with fewer cardio-vascular effects than verapamil, showed that inCaco-2 cells the apical-to-basolateral transport of



paclitaxel (5 mM) was increased in the presence ofKR-30031 (25 mM) and this effect on drug effluxwas similar to that of verapamil (25 mM). Amicroemulsion formulation containing dimethyli-sosorbide, Tween 80 and DL-a-tocopherol andincorporating 6 mg/mL paclitaxel was adminis-tered by oral gavage to anesthetized rats.48 A doseof 25 mg/kg of paclitaxel was used with andwithout 5,10, 20, and 30 mg/kg of KR-30031 or 20mg/kg of Verapamil. KR-30031 increased the oralbioavailability of paclitaxel from 4.6% to 41% (34%P-gp and 7% livermetabolism inhibition) in a dose-dependent manner and this effect was saturableabove 20 mg/kg of KR-30031.48

Earlier studies in improving the oral absorptionof paclitaxel were focused on the coadministrationof CsA or one of its analogs. By combining themarketed lipid formulations of paclitaxel (Taxol1)and CsA (Sandimmune1), the peroral bioavail-ability of paclitaxel in mice increased from 9.5% to67%. Increase was 10-fold when CsA was sub-stituted by its nonimmunosuppressive analogPSC833.54

In Phase II studieswith cancer patients, weeklyoral paclitaxel using the i.v.Taxol1 formulation,was administered in two doses of 90 mg/m2 on thesame day proceeding with 10 mg/kg of CsA(Neoral1) given orally 30 min before.49–51 It wasfound that oral paclitaxel was safe and active withplasma concentrations maintained for at least150 ng/mL over 4 h. The Cmax and AUC did notincrease as the absolute oral dose of paclitaxel wasincreased from 180 to 540 mg indicating a satur-able process.

The use of self-microemulsifying drug deliverysystem (SMEDDS) based on a-tocopherol andTPGS for enhancing the oral bioavailability ofpaclitaxel in rats has been reportedbyYang et al.52

These SMEDDS are composed of a-tocopherol,TPGS, propylene glycol, sodium deoxycholate(DOC-Na), and Cremophor RH40. TPGS andDOC-Na in SMEDDS showed modest differencesin thePKparameters of paclitaxel in rats (Table 5).However, when other specific P-gp inhibitors wereincluded in the formulation, particularly CsA,significant effects in these PK parameters wereobserved (Tab. 5). Thus, in these particularstudies, significant improvement in paclitaxelabsorption was observed only upon a combinationof lipidic excipient(s) with specific P-gp inhibitingdrug. This in fact reflects the actual clinicalformulations of both Paclitaxel and CsA oranotherP-gp drug inhibitor,whereP-gp inhibitinglipidic excipients are present in the formulationsin order to solubilize and effectively deliver thedrugs.

Gao et al.53 developed a supersaturable self-emulsifying drug delivery system (S-SEDDS) forpaclitaxel and other lipophilic drugs by incorpor-ating hydroxypropyl methylcellulose (HPMC) inthe formulation. By prolonging the supersatu-rated state, the presence of HPMC prevents drugprecipitation that was observed from regularSEDDS formulations upon dilution with aqueousmedia to form a fine emulsion/microemulsion. Thepharmacokinetics of paclitaxel from SEDDS andS-SEDDS was compared in rats upon oral admin-istration of a paclitaxel dose of 10 mg/kg.53 A 10-fold higher Cmax and a fivefold higher AUC wasobtained from S-SEDDS compared to SEDDS orthe marketed Taxol1 formulation (9.5% absolutebioavailability from S-SEDDS vs. 2% from Taxol1

and 1% from SEDDS). In order to see furtherenhancement in the oral bioavailability of pacli-taxel it was necessary to include cyclosporine inthe formulation. In the presence of CsA (5 mg/kg)

Table 5. Pharmacokinetic Parameters of Paclitaxel after Oral Administration in Rats with P-gp Inhibitors(Modified from Ref. 52)

Parameters

2.0 mg Paclitaxel/kg 5.0 mg Paclitaxel/kg10.0 mg

Paclitaxel/kg

SMEDDS(0.5% w/w)

SMEDDS(0.5% w/w)

SMEDDS(0.5% w/w)

Taxol1

(0.6% w/v)SMEDDS

(1.25% w/w)SMEDDS

(1.25% w/w)SMEDDS(2.5% w/w)

CsA(40.0 mg/kg)

Etoposide(20.0 mg/kg)

Tacrolimus(8.0 mg/kg)

CsA(40.0 mg/kg)

CsA(40.0 mg/kg)

Tacrolimus(8.0 mg/kg)

CsA(40.0 mg/kg)

Tmax (h) 1.0 1.0 1.0 2.0 4.0 1.0 4.0Cmax (ng/mL) 164� 23 84� 10.2 58� 9 175� 25 225� 27 67� 12 239� 24AUC0–24 (ng �h/mL) 1135.5 837.5 838.0 1134.0 1698.5 921.0 1746.0Fr0! 24 (%) 188.8 139.2 139.3 168.2 252.0 136.6 250.5



in the S-SEDDS, the oral bioavailability of pacli-taxel increased to about 23%.53

Table 6 summarizes bioavailability data ofpaclitaxel upon peroral (p.o.) or intraduodenal(i.d.) administration to rats of LPETM formulationsincorporating P-gp inhibitors.25,26 The absolutebioavailabilities (%F) of paclitaxel in rats upon i.d.administration of LPETM-CsA and LPETM-S for-mulationswere in the range of 15%–20%using i.v.Taxol1 as a reference formulation, although withlarge interanimal variability (Tab. 6). The datasuggest that LPETM formulations with and with-out CsA were at least as effective in improving theintestinal absorption of paclitaxel in rats as thecombination of themarketed oral and i.v. formula-tions of these drugs, respectively. The peroralbioavailability of paclitaxel from the i.v. Taxol1

formulation was not investigated in these studiesbut it is expected to have a mean value of about4%.30,47–51 The effects of the LPETM to inhibit P-gpactivity and to enhance transport of paclitaxelacross Caco-2 cells (Figs. 3A and B) correlatedfairly well with the in vivo absorption of paclitaxel.These effectsweremore discriminatory in theP-gpinhibition data (Fig. 3A) than in drug transportdata (Fig. 3B) possibly due to certain limitations ofthe employed drug transport system and assay. Inthe absence of additional studies, it is difficult tomake absolute in vitro/in vivo correlations. Oneadvantage of theLPETMdrug delivery system is itsability to inhibit P-gp and improve drug transportand absorption in the absence of a second drug. Assuch, drug side effects and drug–drug interactionscan be reduced. LPETM can be applied to otherpoorly absorbed water-insoluble drugs with P-gplimited intestinal absorption.

In a recent study,30 14C-paclitaxel in a solutioncontaining Cremophor: ethanol (1:1) that wasfurther diluted with saline was administeredperorally to anesthetized rats with and withoutthe coadministration of TPGS (50 mg) or Verapa-mil (25 mg). Both TPGS and Verapamil increased

theCmax and AUC of paclitaxel although the effectof TPGS on both parameters was greater. Thebioavailability of paclitaxel increased 6.3-fold and4.2-fold with TPGS or Verapamil, respectively.30

Mechanistically, however, further work is neededto support whether TPGS enhances the oralbiovailability of paclitaxel by improving drugdissolution through micellar solubilization and/orby othermechanisms. As discussed in the previoussection, the presence of TPGS micelles diminishesP-gp inhibition in vitro.30,31

HIV Protease Inhibitors

A soft gelatin formulation using a SEDDS andincorporating TPGS, PEG 400, and propyleneglycol was used to increase the bioavailability ofamprenavir.31 The softgel formulation containing20% TPGS produced 69� 8% absolute bioavail-ability in beagle dogs at a drug dose of 25 mg/kg.When the concentration of TPGS in the formula-tion was increased from 20% to 50% the absolutebioavailability was increased to 80� 16%.31

Although the difference in the bioavailability ofamprenavir between softgel formulations incor-porating 20% TPGS versus higher levels is small,the overall bioavailibility data in dogs were foundto be correlated well with the in vitro Caco-2permeability data as discussed earlier. It shouldalso be pointed out that the 20%–50% w/v TPGSor 200–500 mg/mL is outside of the concentrationrange used in the study shown in Figure 4.31

However, this is in general the case with otherstudies, where the concentrations of surfactantsused in in vivo absorption studies are muchhigher than those used in in vitro drug transportand permeability studies.

Saquinavir, is a poorly soluble HIV proteaseinhibitor and it ismarketed asFortovase1 byRochein a softgel formulation, 200mgstrength,where thedrug is solubilized in a mixture of vitamin E andmedium-chain mono- and diglycerides.55 A three-fold increase in the oral bioavailability of saquinavirfromthe softgel formulationwas reported comparedto the solid dosage form Invarase1. With thereported oral bioavailability of saquinavir in Invar-ase1beingvariable (1%–9%)andaveragedat about4%, the drug bioavailability from the Fortovase1

formulation which is not reported can be estimatedto be about 15%.56

Celiprolol and Digoxin

Absorption studies of digoxin and celiprolol werereported by Cornaire et al.10 using the rat model

Table 6. Bioavailability of Paclitaxel in Rats

Formulation% Fa (Mean�SD,

n¼ 3)

Taxol1 i.v. 100CsA (Neoral1)þTaxol1 p.o.b 11.8þ 3.3LPETM-S, i.d.c 14.3� 7.3LPETM-CsA, i.d. 19.8� 12.2

a% F¼ (AUCi.d./p.o./AUCi.v.)� (Doseiv/Dosei.d./p.o.)� 100.bPeroral.cIntraduodenal.



and administering the formulations by oralgavage. Digoxin, dosed at 0.25 mg/kg, wasdissolved at 0.025% (w/v) in water, propyleneglycol, and ethanol (50:40:10, v/v/v) whereasceliprolol dosed at 30 mg/kg, was dissolved inwater to produce a 1% w/v solution.10 Thecoadministered excipients were added to the drugsolutions at the concentrations of 1 and 120 mg/kgfor digoxin and celiprolol, respectively, to give thesame drug:excipient ratio for each.10 The evalu-ated excipients changed the PK profile of theorally administered digoxin or celiprolol butwithout changing the overall AUC.10 Interest-ingly, only Softigen 767 produced amajor increasein the AUC for the total absorption time course(0–480 min). They observed an early peak ofabsorption (1–40 min), in the presence of certainexcipients (TPGS, Imwitor 742, and Softigen 767)and interpreted it as being the result of higherexcipient concentration in the proximal intestinewhere P-gp expression is lower.10 In the presenceof these excipients, the AUC for the time course of0–40 min for digoxin absorption was increasedfrom 252.2 mg �min/L of the control value to about440 mg �min/L.10 It is apparent from these andother results that the effects of lipid excipients/surfactants on efflux transport and the pharma-cokinetic parameters of the administered drugare complex with multiple mechanisms beinginvolved. The excipients are also mixtures ofvarious chemical species with varying degree ofpurity and lot-to-lot variability and these char-acteristics should also be considered in interpret-

ing the results from in vitro and in vivo studiesand ranking the excipients in terms of theirrelative effects.

Amphotericin B

Wasan and colleagues recently reported on the oralabsorption of the polyene macrolide antifungalagent, AmpB, when incorporated into Peceol.35

The purpose of this study was to determine theeffects of various lipid and mixed-micelle formu-lations on the oral absorption and renal toxicityof AmpB in rats. The maximum concentration ofAmpB in plasma and the AUC0�24h for AmpBwere elevated in rats administered glyceride-richAmpB formulations in comparison to those inrats given (i) AmpB preformulated as a micellecontaining sodium deoxycholate with sodiumphosphate as a buffer (DOC-AmpB), (ii) anAmpB-lipid complex suspension, or (iii) AmpBsolubilized in methanol (Table 7). Furthermore,these findings suggest that AmpB incorporatedinto glyceride-based oral formulations has lessrenal toxicity than DOC-AmpB.

CONCLUSIONS AND FUTURE PERSPECTIVES

Lipid excipients and formulations incorporatingthese excipients and/or specific active compoundscontinue to serve as a useful approach to inhibitP-gp and improve the oral bioavailability of drugs

Table 7. Plasma Creatinine and Amphotericin B (AmpB) Concentrations after Administration of a SingleIntravenous Dose of Deoxycholate (DOC)-AmpB and a Single Oral Gavage of DOC-AmpB and Various AmpB LipidFormulations to Male Sprague-Dawley Rats (Modified from Ref. 35)

Treatment GroupsDose(mg/kg)

Plasma Creatinine Level

Tmax (h) Cmax (ng/mL)AUC0–24 h

(ng �h/mL)Prior to dose

(mg/dL) 24 h

DOC-AmpBIV 1 0.38� 0.13 0.71� 0.10* — —- 4.3� 1.1Oral 5 0.34� 0.07 0.29� 0.05 ND ND NDOral 50 0.29� 0.07 0.42� 0.02* 8 39.8� 22 519� 209**

AmpB lipid formulationsABLC 50 0.45� 0.01 0.24� 0.07* 10 48.5� 26 542� 271Intralipid-AmpB 50 0.27� 0.02 0.38� 0.02* 2 769� 213*** 5984� 3461***Peceol/AmpB 50 0.48� 0.10 0.65� 0.07 4 1469� 891*** 11407� 4971***Peceol/AmpB 5 0.44� 0.06 0.39� 0.06 2 1187� 409*** 4415� 2411***

Data presented as mean� standard deviation n¼ 6 weighted 350–400 g.*p< 0.05 versus prior to dose.**p< 0.05 versus IV DOC-AmpB.***p< 0.05 versus AUC0–24 h for ABLC.



with P-gp limited absorption. Based on the datareported in this review article, there appears to bea reasonably good correlation between the in vitroeffects of lipidic excipients on P-gp inhibitionand drug transport and in vivo drug absorption.In vitro/in vivo correlations, however, are notalways clear and applicable to all classes of drugswith P-gp limited absorption. In cases where invtro/in vivo correlations are established, there arecertain product development and clinical require-ments that also need to be considered such as:(a) the molecule must be specific and pharmacoki-netic interactions must be minimal to reducepotential toxicity and (b) clinically relevant dosesof the inhibitor and optimum duration of P-gpinhibition need to be considered. There areadditional needs in the case of excipient inhibitorswhich include better understanding of the inhibi-tion mechanism(s), and establishment of excipi-ent inhibitor-P-gp substrate structure–activityrelationships. To this end, the development ofnew and safe excipients with a wide spectrum ofphysicochemical and biopharmaceutical proper-ties should be very beneficial. It is evident that thefunction of excipients present in pharmaceuticalformulations goes well beyond their use asinactive components to solubilize and stabilizedrug molecules. Their expanding role as P-gpmodulators requires that their function in theformulation is properly reassessed as morein vitro/in vivo data emerge using a wide rangeof excipients and P-gp substrates. Product devel-opment challenges, regulatory quality and accep-tance of formulations incorporating theseexcipients can be assured once the above require-ments are met.

ABBREVIATIONS

P-gp P-glycoproteinATP adenosine triphosphateMW molecular weightMDR multi-drug resistanceCaco-2 human colon adenocarcinoma cellsMDCK Madin-Darby canine kidney cellsTEER transepithelial electrical resistanceNCE new chemical entityPEG polyethylene glycolCMC critical micelle concentrationLPETM lipid polymer emulsionsCalcein-AM 6-carboxyfluorescein-

acetoxymethylesterA!B apical to basolateral transport

B!A basolateral to apical transportCsA cyclosporin ATPGS a-tocopheryl-PEG-1000-succinateGSK GlaxoSmithKlineAmpB amphotericin BGI gastro-intestinalSEDDS self-emulsifying drug delivery

systemAUC area-under-the-curveAUC0! 24 area-under-the-curve from 0 to 24 hCmax maximum plasma concentrationTmax time to reach peak plasma

concentration (Cmax)Fr relative bioavailabilitySMEDDS self-microemulsifying drug delivery

systemDOC-Na sodium deoxycholateS-SEDDS supersaturable self-emulsifying

drug delivery systemHPMC hydroxypropyl methylcelluloseHIV human immunodeficiency virusPK pharmacokinetic

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