functional interactions between p-glycoprotein … interactions between p-glycoprotein and cyp3a in...

Author Pro

of

Review

10.1517/17425255.1.4.xxx © 2005 Ashley Publications ISSN 1742-5255 1

Ashley Publicationswww.ashley-pub.com

Functional interactions between P-glycoprotein and CYP3A in drug metabolismUwe Christians†, Volker Schmitz & Manuel Haschke†University of Colorado Health Sciences Center, Clinical Research & Development, Department of Anesthesiology, 4200 East Ninth Ave., Room UH-2122, Campus Box B-113, Denver, Colorado 80262, USA

The interaction between drug-metabolising enzymes and active transportersis an emerging concept in pharmacokinetics. In the gut mucosa, P-glyco-protein and cytochrome P450 (CYP)3A functionally interact in three ways:i) drugs are repeatedly taken up and pumped out of the enterocytes by P-glyco-protein, thus increasing the probability of drugs being metabolised; ii) P-glyco-protein keeps intracellular drug concentrations within the linear range of themetabolising capacity of CYP3A; and iii) P-glycoprotein transports drugmetabolites formed in the mucosa back into the gut lumen. In comparisonwith the gut mucosa, in hepatocytes the spatial sequence of CYP3A andP-glycoprotein is reversed, resulting in different effects when the activity ofone or both are changed. CYP3A and P-glycoprotein are both regulated bynuclear receptors such as the pregnane X receptor (PXR). There is significantgenetic variability of CYP3A, P-glycoprotein and PXR and their expressionand activity is dependent on coadministered drugs, herbs, food, age, hormo-nal status and disease. Future pharmacogenomic and pharmacokineticstudies will have to take all three components into account to allow for validconclusions.

Keywords: cytochrome P450, drug–drug interactions, drug metabolism, drug transport, oral bioavailability, P-glycoprotein, pharmacokinetic variability

Expert Opin. Drug Metab. Toxicol. (2005) 1(4):xxx-xxx

1. Introduction

In a key review article, Wacher et al. [1] suggested the functional interaction betweencytochrome P450 (CYP)3A and P-glycoprotein and coregulation based on theirlocation in intestinal mucosa cells and hepatocytes, an almost complete overlap insubstrates, inhibitors and inducers and adjacent location of their genes.

The CYP3A subfamily is the predominant CYP family in the human liver. Inhumans, the CYP3A subfamily includes CYP3A4, -3A5, -3A7 [2] and -3A43 [3].CYP3A4 represents the predominant CYP in microsomal tissue of adults and isinvolved in the metabolism of > 50% of all currently prescribed drugs [4]. In theliver, CYP3A5 constitutes < 8% of the total CYP3A content and is found in only 10– 20% of adult livers in the Caucasian population [5-7]. In vitro studies have shownan almost complete substrate overlap with CYP3A4, with CYP3A5 having equal orless activity [8]. CYP3A7 is the primary fetal CYP3A enzyme and diminishes duringinfancy [9]. In a study investigating the expression of CYP3A transcription in the liv-ers of 63 Caucasians, CYP3A5 and -3A7 contributes an average of 2% andCYP3A43 an average of 0.3% transcripts to the CYP3A mRNA pool [10]. This studyalso found that CYP3A5 and -3A7, but not CYP3A4, were expressed in the adrenalgland and only CYP3A5 was expressed in the kidney.

P-glycoprotein is a member of the ATP-binding cassette (ABC) transporterfamily. The ABC transporter family is currently the largest known family of

1. Introduction

2. Functional interactions between

CYP3A and P-glycoprotein in

the small intestine

3. Different functional

interactions between CYP3A

and P-glycoprotein in the liver

and small intestine and impact

on drug–drug interactions

4. Coregulation of CYP3A and

P-glycoprotein

5. Sources of variability

6. Impact on drug development

7. Expert opinion

Author Pro

of

Functional interactions between P-glycoprotein and CYP3A in drug metabolism

2 Expert Opin. Drug Metab. Toxicol. (2005) 1(4)

Table 1. The effect of P-glycoprotein inhibition on the extraction ratios of drugs across CYP3A4-overexpressing Caco-2 cell monolayers.

Drug Substrate for Efflux ratio Extraction ratio% ± SD

CYP3A P-gp B to A / A to B Drug alone Drug and cyclosporin

Drug and GG-918

K77 (10 µM) Yes Yes 9 33 ± 3(356 ± 26)

5.7 ± 0.3(1830 ± 40)

14 ± 1(1600 ± 60)

Sirolimus (1 µM) Yes Yes 2.5 60 ± 5(56 ± 10)

15 ± 1(212 ± 19)

45 ± 1(73 ± 3)

Midazolam (3 µM)

Yes No 1 25 ± 2(282 ± 50)

10 ± 1(354 ± 15)

23 ± 2(349 ± 30)

Felodipine (10 µM)

Yes No 1 26 ± 1(3750 ± 130)

14 ± 1(4030 ± 90)

24 ± 2(3710 ± 220)

The table was modified from Benet et al. [19] with permission from Bentham Science Publishers Ltd, and is based on data from references [21] and [22]. Intracellular amounts (pmol) are shown in brackets. All values are means ± SD (n = 3). K77 is an investigational cysteine protease inhibitor currently under development for the treatment of Chagas’ disease. Sirolimus is an immunosuppressive agent, midazolam is a benzodiazepine and felodipine is a Ca2+-channel blocker. Cyclosporin is an immunosuppressant and is a known CYP3A and P-gp substrate and inhibitor. In contrast, GG-918 is a specific P-gp inhibitor that was demonstrated not to affect CYP3A activity [21]. All four drugs have an apparent Km in human microsomes for metabolite formation close to the concentrations tested.The extraction ratio (ER) was calculated using the following equation:

Because intracellular drug concentrations may change when drug transporters are modulated, the parent drug levels have been included in the denominator of the equation [19]. B to A: ratio apical to basolateral; B to A: ratio basolateral to apical, GG-918: N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine; K77: K11777, N-methyl-piperazine-Phe-homoPhe-vinylsulfone-phenyl; P-gp: P-glycoprotein.

ERΣmetabolites apical basolateral intracellular, ,( )

Σparent basolateral intracellular,( ) Σmetabolites apical basolateral intracellular, ,( )+----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------=

transmembrane proteins. Forty-eight genes have beenassigned to this family, which, based on sequence homologiesand domain organisation, has been categorised into seven dis-tinct subfamilies [101]. P-glycoprotein is a 170-kDa transmem-brane glycoprotein and is encoded by the human multipledrug resistance protein (MDR)-1 (ABCB1) gene. It consistsof two homologous halves. Each comprises six transmem-brane domains and a cytoplasmic nucleotide binding domain[11,12]. P-glycoprotein is expressed at high levels on the apicalsurfaces of epithelial cells in the biliary membrane of the liver,small intestine and colon mucosa cells, the proximal tubule ofthe kidney, the adrenal gland and the pancreatic duct [13]. Italso is a key component of the blood–brain barrier [14].

During the last 10 years, a large body of literature has con-firmed Wacher and colleagues’ hypotheses [1]. As discussed inthe following review, the results of those studies are likely torevolutionise our mechanistic understanding of pharmacoki-netics, drug–drug interactions, and intersubject variability forthe pharmacokinetics of drugs where drug transporters anddrug-metabolising enzymes play a significant role. This is alsoreflected by the fact that drug transport is now being viewedas Phase III of drug elimination, in addition to Phase I and IImetabolism [15].

Please note that in this review the term ‘drug’ refersspecifically to drugs that are CYP3A and P-glycoproteinsubstrates. A most recent list of such drugs can be found inreference [16].

2. Functional interactions between CYP3A and P-glycoprotein in the small intestine

P-glycoprotein and CYP3A functionally interact in threeways [17]. First, drugs are repeatedly taken up and pumpedout of the enterocytes by P-glycoprotein. Repeated exposureto CYP3A enzymes increases probability of drugs beingmetabolised. Second, P-glycoprotein keeps intracellular drugconcentrations within the linear range of the metabolisingcapacity of CYP3A enzymes. Third, P-glycoproteintransports drug metabolites formed in the mucosa back intothe gut lumen.

2.1 Effect of P-glycoprotein on intestinal metabolismIn vitro and in vivo studies using specific CYP3A and/orP-glycoprotein substrates and inhibitors demonstrated thatspecific inhibition of P-glycoprotein decreases the metabolicextraction ratio in the small intestine. Those results have beenreviewed in detail by Benet et al. [18-20].

The effect of P-glycoprotein on intestinal drug metabolismwas studied in vitro using CYP3A4-transfected Caco-2 cellsgrown as monolayers [21,22]. The transport and metabolism oftwo P-glycoprotein and CYP3A substrates, K77 and sirolimus,and two CYP3A4-only substrates, midazolam and felodipine,were studied (Table 1). Drugs were given to the apical side ofthe cell culture system to simulate human intestinal absorp-tion. K77 was found to be the best P-glycoprotein substrate

Author Pro

of

Christians, Schmitz & Haschke

Expert Opin. Drug Metab. Toxicol. (2005) 1(4) 3

tested and exhibited a ninefold greater basolateral-to-apicalthan apical-to-basolateral flux [19]. The sirolimus effluxratio was only 2.5, indicating that sirolimus is a weakerP-glycoprotein substrate than K77. Midazolam andfelodipine had efflux ratios of 1 and, thus, were confirmednot to be P-glycoprotein substrates. Both compoundsserved as negative controls. When added to the Caco-2 cellsalone, all compounds tested were significantly metabolisedin the cells (Table 1). As expected, addition of the CYP3Ainhibitor cyclosporin significantly decreased the extractionratios of all four drugs. However, whereas the extractionratios of the CYP3A-only substrates felodipine and mida-zolam were reduced by 46 – 60%, the reduction of theextraction ratios of the CYP3A and P-glycoprotein substratesK77 and sirolimus were greater and found to be 74 – 83%.Those results already suggested a contribution of P-glyco-protein because cyclosporin is also a P-glycoprotein inhibi-tor. The involvement of P-glycoprotein was confirmedwhen K77 and sirolimus were added to the Caco-2 cell cul-tures in combination with the specific P-glycoproteininhibitor GG-918. Specific P-glycoprotein inhibitionresulted in a significant reduction of extraction ratios with-out a change in CYP3A activity. K77, the better P-glyco-protein substrate, was more affected (58% decrease) thansirolimus (25% decrease). Interestingly, a change in intrac-ellular concentration (Table 1) does not explain the results[19]. When P-glycoprotein is inhibited, concentrations ofK77 and sirolimus increase and more substrate is availableto the CYP3A enzymes. Thus, an increase in the extractionratio would be expected. However, when P-glycoproteinwas inhibited, the opposite effect was found. It can be spec-ulated that the observed reduction in metabolism is at leastin part due to saturation of CYP3A enzymes, resulting inmore nonmetabolised drug passing through the cell andinhibition of P-glycoprotein-mediated recycling of thosedrugs that usually results in repeated presentation of drugmolecules to the CYP3A enzymes. These results indicatethat inhibition of intestinal P-glycoprotein may not onlyincrease absorption by blocking efflux transport, but alsodecrease total metabolism, resulting in significantlyenhanced intestinal bioavailability [19].

Analysis of the impact of P-glycoprotein on the intestinalextraction ratio of verapamil, a P-glycoprotein and CYP3Asubstrate, using a four compartment model supported anonlinear relationship between the extent of drug metabo-lism and drug transport [23]. As one of the reasons for thisnonlinearity, saturable drug metabolism in the intestine wasdiscussed.

In vivo, those mechanisms were confirmed using the ratsingle-pass intestinal perfusion model with mesenteric veincannulation [24]. Again, K77, a CYP3A and P-glycoproteinsubstrate, and midazolam, a CYP3A but not a P-glycoproteinsubstrate, were compared in the absence and presence of thespecific P-glycoprotein inhibitor GG-918. In the presence ofGG-918, for K77 both the fraction metabolised (95 ± 3%

versus 85 ± 4% with GG-918) and the extraction ratio (49 ±12% and 37 ± 3%) were decreased. In contrast, the permea-bility and metabolism of midazolam remained unaffected.This study confirmed that also in vivo P-glycoproteinenhances the extent of intestinal metabolism of drugs that aresubstrates of CYP3A and P-glycoprotein [24].

2.2 Interaction between intestinal efflux transporters and drug metabolitesWhen the intestinal metabolism of the immunosuppressantstacrolimus and sirolimus (both substrates of CYP3A andP-glycoprotein) was studied in pig duodenal mucosamounted in an Ussing chamber, it was found that both drugswere metabolised in the small intestine and that, surprisingly,the metabolites were almost exclusively found on the luminalside [25,26]. Because the parent drugs were added to the lumi-nal chamber at a concentration of 10 µM and were present inthe luminal chamber at 100-fold higher concentrations thanthe metabolites, those results could only be explained byactive efflux transport of the metabolites against a concentra-tion gradient, a higher affinity of the metabolites to P-glyco-protein than the parent drugs and/or that the CYP3A-mediated changes resulted in change of affinity to an effluxtransporter other than P-glycoprotein. A latter study inCaco-2 cells confirmed that sirolimus metabolites are trans-ported from the mucosa cell back through the apical (lumi-nal) membrane [22]. The metabolites were confirmed to beP-glycoprotein substrates, but the study also found evidencethat other transporters are involved [22]. The hypothesis thatmetabolism changes the affinity to other transporters thanthose playing a role for the parent was further confirmed inan in vitro study assessing the functional interaction betweentransport and metabolism by comparing the transport oflosartan and its active metabolite EXP-3174 (EXP) across cellmonolayers [27]. Epithelial layers of Caco-2 cells as well asABCB1-overexpressing Madin–Darby canine kidney(MDCK) cells, in comparison with the wild type, were usedto characterise the transcellular transport of losartan andEXP. Losartan was found to be a P-glycoprotein substratewith significantly higher basolateral-to-apical than apical-to-basolateral flux with a ratio of 31 ± 1 in ABCB1-overexpress-ing MDCK cells and a ratio of 4 ± 1 in Caco-2 cells. Themetabolite was only transported in Caco-2 cells with a baso-lateral-to-apical/apical-to-basolateral ratio of 5 ± 1, whilelacking active transport in the ABCB1-overexpressingMCDK cells. Those results suggest a functional interactionbetween CYP3A-dependent metabolism and intestinal effluxin a way, such that drugs are converted to metabolites that aresubstrates for transporters different from those that areinvolved in the intestinal transport of the parent (Figure 1)[27]. It can be hypothesised that inhibitors or inducers of pro-teins, which are involved in the active transport of the meta-bolite, may have an impact on the pharmacokinetics of theparent drug, although the parent itself might not be asubstrate of such transporters.

Author Pro

of



2.3 Local differences in intestinal CYP3A and P-glycoprotein expression and effect on oral bioavailabilityThe small intestine plays a significant role in drug meta-bolism, drug interactions and oral bioavailability of drugsthat are CYP3A and/or P-glycoprotein substrates. Oral bio-availability is also a significant contributor to overall phar-macokinetic variability of such drugs. As discussed above, inthe small intestine drug efflux pumps and CYP enzymes,most importantly P-glycoprotein and CYP3A, form a co-operative barrier against the absorption of xenobiotics(Figures 1 and 2). The following ranking of transportermRNAs in the duodenum was found [28]: MRP3 > > MDR1> MRP2 > MRP5 > MRP4 > MRP1 (ABCC3 > > ABCB1 >ABCC2 > ABCC5 > ABCC4 > ABCC1). In the ileum trans-porter mRNA concentrations were as follows: ABCB1 >ABCC3 > > ABCC1 ≈ ABCC5 ≈ ABCC4 > ABCC2. Moulyand Paine [29] determined P-glycoprotein expression inmucosa scrapings of every other 30-cm segment of the

intestines of four unrelated donors. P-glycoprotein expres-sion increased from proximal to distal. Within-donor varia-tion of P-glycoprotein expression was, depending on theregion compared, 1.5- to 3-fold [29]. In healthy volunteers,Fricker et al. [30] showed that the absorption of the CYP3Aand P-glycoprotein substrate cyclosporin was dependent onthe location of absorption and followed the rank orderstomach > jejunum/ileum > colon. The decrease in absorp-tion was inversely correlated to the expression of ABCB1mRNA (stomach < jejunum < colon) [30]. CYP activity is afunction of the distance along the intestine from the duode-num to the ileum [31,32]. CYP3A concentration and activityincreases slightly from the duodenum to the jejunum andthen decreases towards the ileum [32,33].

Because drug transporters and CYPs are differentlyexpressed in different parts of the human intestine theimportance of their interactions and their effect on the phar-macokinetics of a specific drug will depend on the site ofabsorption from the gut. Again, it is important to consider

Figure 1. Interactions between P-gp-mediated drug transport and CYP3A drug metabolism in the gut mucosa, the effect ofinducers and inhibition of P-gp [17]. Used with permission from CHRISTIANS U: Transport proteins and intestinal metabolism. P-glycoprotein and cytochrome P4503A. Ther. Drug Monit. (2004) 26:104-106. CYP: Cytochrome P450; P-gp: P-glycoprotein; PXR: Pregnane X receptor; SXR: Steroid and xenobiotic receptor.

inducers

PXR/SXR

+ -CYP3ACYP3A CYP3A CYP3A

Mu

cosa

cell

Gu

tlu

men

Po

rtal

vein

CoregulationFunctional

collaborationof P-gp with

CYP3A

Functionalcollaboration

of CYP3A withP-gp

Effect ofP-gp inhibition onCYP3A-mediated

metabolism

P-gp P-gp P-gp P-gp

cytokinesoxygen radicals

P-gp keepsdrug concentrations

in CYP3A linear rangeGene promoter

region (?)

repeated exposureincreases contact time

with CYP3A

Metabolitesbecome

substratesfor other

transporter(s)

Metabolitesbecome better

substratesfor P-gp

P-gp inhibition:-decreased exposure

to CYP3A-drug overwhelms CYP3A

→ Metabolism ↓

→ Oral bioavailability ↑

Author Pro

of



that drugs and/or their metabolites may be substrates fordifferent drug-metabolising enzymes and transporters.

3. Different functional interactions between CYP3A and P-glycoprotein in the liver and small intestine and impact on drug–drug interactions

In a perfused rat liver model, Wu and Benet showed thatspecific inhibition of P-glycoprotein significantly decreasedthe concentration of tacrolimus, a CYP3A and P-glycoproteinsubstrate, in the perfusate, a surrogate for systemic exposure[34]. However, when felodipine, a CYP3A but not a P-glyco-protein substrate, was studied in the same model, there was nodifference in felodipine perfusate concentrations in the absenceand presence of the specific P-glycoprotein inhibitor. This andother studies indicated that, whereas inhibition of P-glyco-protein in the small intestine reduces CYP3A-dependent meta-bolism [21,22,24], it increases hepatic metabolism [34-36]. There

are two major differences between intestinal mucosa cells andhepatocytes that can explain those findings. The abundanceof CYP3A enzymes in hepatocytes is significantly higher thanin mucosa cells. An increase of substrate concentrations in thehepatocytes leads to more CYP3A-mediated metabolism.However, due to the lower metabolic capacity, an increase ofsubstrate concentrations in the mucosa cells may saturateCYP3A relatively fast and then drug can pass the mucosa cellsnonmetabolised (Figure 1). The other difference is that drugenters the mucosa cell through the apical membrane whereP-glycoprotein is located. The drug may come into contactwith P-glycoprotein first and, thus, P-glycoprotein regulatesaccess to CYP3A enzymes (Figure 2). In comparison with theintestinal mucosa, drugs entering the hepatocyte encounterCYP3A and P-glycoprotein in the reverse order [16,19]. Drugspass through the basolateral membrane and may come intocontact with CYP3A before they reach P-glycoprotein locatedin the apical (biliary) membrane (Figure 2).

This leads to significant differences in drug–drug interac-tions with CYP3A and P-glycoprotein substrates in the smallintestine and liver (Table 2) [16,19]. However, as indicated inTable 2, the situation may be more complex for specific drugsas additional transporters such as absorptive efflux transport-ers, and hepatic uptake transporters may play a significant role[16,35]. The clinical relevance of those differences in liver andintestine has not yet been confirmed in clinical trials and, dueto the complexity of mechanisms involved and the differencesof drugs in terms of their affinities to drug-metabolisingenzymes and transporters, will be difficult, if not impossibleto prove, by retrospectively analysing published drug–druginteraction studies.

4. Coregulation of CYP3A and P-glycoprotein

The nuclear receptor family constitutes a large family ofrelated receptors that serve as targets for > 10% of all com-monly prescribed drugs [37]. Important members of the familyare the pregnane X receptor (PXR) and the constitutiveandrostane receptor (CAR). The human orthologue of PXR isalso called steroid and xenobiotics receptor (SXR) and isencoded by NR1I2. PXR is a promiscuous receptor that isactivated by a wide variety of xenobiotics and endogenouscompounds such as progesterone, phytoestrogens, dexametha-sone, plant products such as hyperforin (the active compoundof St John’s Wort), bile acids, insecticides, and drugs such asrifampin, peptide mimetic protease inhibitors and paclitaxel[38-40]. On binding of the activating compound, PXR forms aheterodimer with the retinoid X receptor. The heterodimerfunctions as a transcription factor and interacts with the cog-nate response element in the 5′ regulatory region of theCYP3A4, but not the CYP3A5, gene [41]. Meanwhile, it hasbeen shown that in the human liver and intestine, the PXR aswell as CAR can induce CYP3A4 and -3A5 expression via aneverted repeat separated by a 6-base pair (ER6) motif,100 base pairs upstream from the transcription start site [42].

Figure 2. Sequential differences of efflux transport anddrug metabolism in gut mucosa cells (top) and hepatocytes(bottom) [16]. In the gut, a drug has to pass the efflux transporterfirst in order to enter the mucosa cell and to be absorbed. Drugsrepeatedly come into contact with the efflux transporters and getpumped out of the mucosa cells as they move distal in the gut.This way they have multiple chances of coming into contact withthe second ‘line of defence’, the CYP3A enzymes. Themetabolites again become substrates for active efflux transportersand are transported back into the gut lumen. Onlynonmetabolised drugs and metabolites that did not get in contactwith efflux transporters reach the portal vein. In the liver, drugsget into the hepatocytes from blood through the basolateralmembrane. In the path of a drug through the hepatocytes, incontrast to the gut mucosa cells, drug-metabolising enzymes arelocated in front of the efflux transporters. Please note that this is asimplified model and uptake transporters that may affect theintracellular concentrations of specific drugs are not shown.A: Apical membrane; BL: Basolateral membrane; Efflux: Active efflux transporter.

CYP3A

efflux

CYP3A

efflux

CYP3A

efflux

A

ABL

BL

efflux

gutlumen

blood

portalvein

bile

metabolites

metabolites

drugdrug

drug

Author Pro

of



Among others, the PXR regulates gene expression of the drug-metabolising enzymes CYP2A, -2B, -2C and -3A, various glu-tathione S-transferases, and uridine diphosphate glucuronosyl-transferase (UGT) 1a as well as the drug transporters MDR1Aand -1B, MRP3 and OATP2 in mice [41]. The CAR regulatesthe expression of the drug-metabolising enzymes CYP1A, -2A, -2B and -3A and various glutathione S-transferases as well as thedrug transporters MRP1, -2 and -3 [41]. In mice, there were sev-eral overlaps, but also several genes that were differentially regu-lated by those two receptors [43]. Based on their data, Maglichet al. [43] hypothesised that PXR plays a more important role forthe regulation of drug-metabolising enzymes and drug trans-porters in the small intestine and CAR a more important role forgene regulation of those proteins in the liver. This also meansthat the coordinated regulation of drug-metabolising enzymesand drug transporters may be organ specific [44].

Synold et al. [39] were the first to describe coregulation ofdrug metabolism and efflux via CYP3A and MDR1 in liver andintestine by the human receptor PXR/SXR. This data indicatedthat paclitaxel reduces its own oral bioavailability by activatingthe PXR and induces its own metabolism and biliary elimina-tion. Interestingly, this study also showed that hydroxylatedpaclitaxel metabolites as well as the structurally similardocetaxel did not interact with the PXR [39]. The observationthat PXR interactions are not necessarily associated with certainclasses of drugs was confirmed by another study that showedthat the HIV protease inhibitor ritonavir is a relatively strongactivator of PXR, whereas saquinavir is a relatively weak activa-tor, and nelfinavir and indinavir are unable to activate PXR [40].Ritonavir binds and activates PXR with a half-maximal effectconcentration (EC50) of 2 µM. This is well within the therapeu-tic ritonavir concentrations achieved in patients, indicating thatPXR activation is clinically relevant [40].

5. Sources of variability

5.1 Pharmacogenetics and -genomicsMore than 30 CYP3A4 allelic variants have been identified[101]. Today, there is no consensus as to whether there is a

direct correlation between function and CYP3A4 polymor-phisms. CYP3A4 alleles may have little clinical importance.The reasons are the relatively low allele frequencies and thelimited alterations in enzyme expression and catalytic func-tion [45,46].

The CYP3A5*1 allele is associated with high CYP3A5expression and activity, whereas CYP3A5*3 is associated withirrelevant expression and activity of the enzyme [46,47]. TheCYP3A5*3 allele results in premature termination of transla-tion and a nonfunctional truncated protein. This allele ismainly responsible for differences in CYP3A5 distributionamong ethnic groups.

ABCB1 is highly polymorphic and 49 single nucleotidepolymorphisms (SNPs) and insertion/deletion poly-morphisms in the ABCB1 gene have been described [48,49].Seven SNPs are located in introns and eleven change theamino acid sequence [50]. The distribution of allelic variantsdiffers greatly among ethnic groups [46]; however, most allelicABCB1 variants are silent. The exonic single nucleotide SNPsC3435T and G2677T/A have been shown to correlate withexpression and/or function of P-glycoprotein [50,51]. Subjectswho were homogenous for the 3435T allele in exon 26showed an average of twofold lower P-glycoprotein expressionin the small intestine than the ‘wild type’ [50]. In terms ofCYP3A and ABCB1 polymorphisms, so far the best-studiedgroup of drugs is the immunosuppressants, specifically thecalcineurin inhibitors cyclosporin and tacrolimus [51]. Bothare critical dose drugs. Prediction of adequate dosing can beexpected to facilitate management of immunosuppressivedrug regimens and may improve clinical long-term outcome.However, results have been inconsistent. [52] A link betweenthe polymorphisms of CYP3A4 and -3A5 and ABCB1 genesand the daily dose to achieve adequate tacrolimus blood con-centrations has been shown [53,54]. Another study confirmedthe role of CYP3A4 and -3A5 polymorphisms in tacrolimuspharmacokinetics, but did not support the role of ABCB1polymorphisms [55]. In the case of cyclosporin, a study in livertransplant patients showed that the ABCB1 exon 26 C3435Tis a major determinant of the cyclosporin concentration/dose

Table 2. Differential predicted extraction of drugs that are substrates of both CYP3A and P-glycoprotein after inhibition of CYP3A and/or active transporters in intestine or liver (according to [16,19]).

Intestine Liver

Inhibit P-gp ↓ ↑

Inhibit CYP3A ↓ ↓

Inhibit P-gp + CYP3A ↓↓ ↓↑↔

See also Figures 1 and 2. In both liver and intestine, inhibition of CYP will decrease extraction in those organs. However, possibly due to the reverse sequential locations of P-gp and CYP (Figure 2) in the intestinal mucosa and intestine, inhibition of P-gp in the intestinal mucosa will reduce gut drug metabolism, whereas in the liver inhibition of P-gp was found to increase metabolism. Inhibition of intestinal efflux and metabolism resulted in a more potent inhibition of gut extraction, whereas in the liver the effect was found to go either way, depending on the affinity of substrate and inhibitor to CYP and P-gp. It must be noted that the concepts summarised in this table were based on data from cell and perfused organ models. The relevance for pharmacokinetics and drug–drug interactions in humans still needs to be confirmed. In addition, this table is limited to the CYP3A/P-gp interaction. As soon as a drug is also the substrate of another efflux and/or uptake transporter, and other drug-metabolising enzymes, the transporter interaction will be more complex. CYP: Cytochrome P450; P-gp: P-glycoprotein.

Author Pro

of



ratio [56], whereas a study in kidney graft patients only found aweak correlation between cyclosporin dose requirements andABCB1 SNPs [57]. In renal transplant patients, ABCB1 SNPswere not associated with sirolimus concentration/dose ratios[58]. Population-dependent distribution of CYP3A4, CYP3A5and ABCB1 SNPs is most likely to be responsible for the eth-nic differences in cyclosporin, tacrolimus and possibly alsosirolimus pharmacokinetics [49,59-61].

The CYP3A5*1 allele is associated with high intestinal andhepatic expression and activities of CYP3A5 and with lowconcentration/dose ratios. More than 60% of African-Ameri-cans, compared with < 10% of the Caucasian population,possess this allele [49]. The situation for P-glycoprotein is simi-lar. Only 24% of Caucasians, but 68% of African-Americansare carriers of the CC genotype of the ABCB1 C3435T geneticvariation that is associated with low immunosuppressantexposure [61].

The expression of CYP3A4, CYP3A5, CYP3A43 and PXRmRNA were found closely correlated in 46 Caucasian humanlivers [7]. Thus, variability in expression of CYP3A and possi-bly also ABCB1 may be explained by the allelic variations inthe PXR, one of their transcriptional regulators [46,62]. A totalof 38 SNPs were identified, including 6 SNPs in the codingregion [63]. The frequency of PXR*2 in African-Americanswas 20% and PXR*2 was not found in Caucasians. However,this variant did not affect CYP3A expression. It is yetunknown as to whether the PXR SNPs are of clinicalrelevance.

In summary, studies evaluating the effect of CYP3A andABCB1 polymorphisms on cyclosporin pharmacokineticsshow that ABCB1 seems to be a better predictor than CYP3A[50,64]. This is not surprising as it was shown in a clinical trialthat P-glycoprotein in the small intestine, but not CYP3A,significantly contributes to interpatient pharmacokinetics ofcyclosporin (Table 3) [65]. This can probably be explained bythe fact that cyclosporin after oral administration undergoessignificant intestinal metabolism and that P-glycoprotein-mediated efflux controls cyclosporin concentrations in themucosa cells and thus access of cyclosporin to intestinalCYP3A. In contrast to cyclosporin, CYP3A5 polymorphismwas a better predictor of tacrolimus pharmacokinetics than

ABCB1 [50]. Interestingly, it was found that the results ofCYP3A4 and CYP3A5 genotyping did not sufficiently reflectthe interindividual variability of midazolam, a CYP3A, butnot a P-glycoprotein, substrate [66].

Those studies clearly demonstrate that predictive clinicalvalues of CYP3A and ABCB1 polymorphism will depend onwhether a drug is a good P-glycoprotein and/or CYP3A sub-strate (Table 1). It seems reasonable to expect that the geneticpolymorphism of the transporter or drug-metabolisingenzyme that is first in the elimination pathway will controlaccess of a drug to the subsequent metabolism/transport sitesand will have the most significant effect on overall pharma-cokinetics. Thus, the route of administration will have a sig-nificant impact (Table 2). P-glycoprotein/CYP3A interactionswill also be affected by the presence of drugs potentially inter-acting with PXR, which itself exhibits significant poly-morphism [62]. This is a significant problem in specific patientpopulations such as transplant patients who receive a multi-tude of drugs that are CYP3A and P-glycoprotein substratesand that interact with the PXR.

5.2 AgeFetal activity of CYP3A is 30 – 75% that of adults [67]. CYP3Aactivity increases throughout infancy and commonly exceedsadult activity by the age of 1 year. It continues at this level dur-ing childhood and CYP3A activities decrease gradually to adultlevels by the end of puberty. A study in 90 healthy volunteersaged 0 – 86 years showed that P-glycoprotein activity in periph-eral blood lymphocytes was highest in cord blood and progres-sively declined with age [68]. In human intestinal and livertissues from individuals of different age ranging from neonatalto 85 years, a close correlation between PXR mRNA and themRNAs of CYP3A4 and ABCB1 was shown [69]. PXR mRNAin the liver and intestine reached maximum levels in youngadults (15 – 38 years of age) and decreased to half the maximallevels in older individuals (67 – 85 years) [69].

5.3 GenderThere is evidence in the literature that women have a higherclearance for drugs that are CYP3A and P-glycoproteinsubstrates than men after intravenous administration [70]. Those

Table 3. Contribution of CYP3A and P-gp in the interpatient variation of the oral bioavailability of drugs that are CYP3A and P-gp substrates.

Liver CYP3A Intestinal P-gp Intestinal CYP3A

Apparent oralclearance

56% 17% -

Cmax 32% 30% -

Data were taken from reference [65]. The pharmacokinetics of the CYP3A and P-gp substrate cyclosporin was assessed in 25 kidney transplant patients and was correlated to CYP3A activity in the liver and CYP3A and ABCB1 expression in the small intestine. Forward multiple regression analysis revealed that liver CYP3A and intestinal P-gp contributed to inter-patient variability of apparent oral clearance and the maximal cyclosporin concentration in blood (Cmax), a surrogate marker for the rate of absorption. Although intestinal expression of CYP3A varied 10-fold among the subjects, it did not contribute to the inter-individual pharmacokinetic parameters evaluated. This data suggested that P-gp-mediated efflux, but not CYP3A metabolism is a rate-limiting step in cyclosporin absorption. CYP: Cytochrome P450; P-gp: P-glycoprotein.

Author Pro

of



differences seem to be influenced by the menstrual cycle.Drugs that are only CYP3A, but not P-glycoprotein, sub-strates, did not exhibit gender-dependent differences in phar-macokinetics. Overall, those findings suggested a significantinvolvement of P-glycoprotein [70]. However, analysis of94 well-characterised surgical liver samples revealed twofoldhigher CYP3A concentrations in women and no difference inP-glycoprotein expression [71]. In terms of CYP3A, similar dif-ferences were found for the small intestine when tacrolimusand cyclosporin metabolism rates were assessed in microsomesisolated from human duodenal mucosa [72,73]. However, arecent study in 46 healthy men and 45 healthy women did notfind a gender difference for CYP3A4, CYP3A5 andP-glycoprotein expression in the proximal small intestine [74].When in this study pre- and postmenopausal women werecompared, CYP3A4 was 20% lower in postmenopausalwomen. Expression of the PXR was correlated with CYP3Aexpression, but did not show gender differences [71]. There wasalso no gender difference in ABCB1 expression in this study.

5.4 Herbs and foodThe importance of food–drug interactions and interactionswith herbal medicines has increasingly been recognised dur-ing the last years [75,76]. CYP3A-mediated metabolism andP-glycoprotein transport seem especially vulnerable [76,77].Grapefruit juice and St John’s Wort are the best-studiedfood and herb drug interactions. The interaction betweendrugs and grapefruit juice is mainly driven by depletion ofCYP3A enzymes in the small intestine [78], but effects ofgrapefruit juice components on active transporters in theintestine have also been described [79]. The active com-pound of St John’s Wort, hyperforin, induces CYP3A andP-glycoprotein via the PXR, resulting in reduced drugexposure and efficacy [50]. However, the relative contributionof CYP3A and P-glycoprotein induction to the overall effectof St John’s Wort remains unknown [79]. Recently, it has alsobeen hypothesised that high-fat meals affect pharmacokinet-ics by inhibiting influx and efflux transporters includingP-glycoprotein [16].

Figure 3. Effects of inhibition of the CYP3A/transporter interaction in the liver on the blood metabolite patterns ofcyclosporin in bone marrow transplant patients during GVHD of the liver. Data are taken from reference [84]. Cyclosporin is aknown CYP3A and P-glycoprotein substrate. As shown by the data, during GVHD, transport of drug metabolites from the hepatocytes ismore impaired than CYP3A-mediated metabolism and leads to a shift of the metabolite pattern in blood towards metabolites that havebeen in contact with CYP3A several times, suggesting that exposure of cyclosporin and its metabolites to hepatic CYP3A is increasedwhen transport is reduced (see also Table 2). The nomenclature of the cyclosporin metabolites follows the Hawk’s Cay nomenclature (fordetails see reference [85]). According to this nomenclature ‘A’ stands for cyclosporin A, ‘M’ stands for metabolite and the number showswhich of the 11 amino acids of cyclosporin is modified. A number means hydroxylation, ‘N’ stands for N-demethylation, ‘C’ forcyclisation amino acid 1, and ‘A’ for carboxylic acid. Thus, AM1, the major cyclosporin metabolite in blood in humans, is hydroxylated atamino acid 1, AM19 is hydroxylated in two positions, at amino acids 1 and at amino acid 9. AM1A has a carboxy group at amino acid 1.It is the major hepatic metabolite that is usually not detectable in blood of patients with normal liver function. Formation of AM1A fromcyclosporin requires several oxidation steps via AM1 including oxidation of AM1 to its aldehyde [85].CYP: Cytochrome P450; GVHD: Graft-versus-host disease.

0

50

100

150

200

250

no GVHD (n=23)skin GVHD, unimpaired liver function (n=18)liver GVHD (n=10)

CsAAM1cAM9AM1AM1AAM14N9AM14NAM1c9AM19

ng

/ml

Author Pro

of



5.5 DiseaseThe activity of CYP3A is reduced by high intracellular freeradical concentrations, infectious diseases, inflammation andimmune reactions [80,81]. The activity of ABC transporters isaffected in similar ways [82]. In mice, it was found that pro-inflammatory cytokines suppress the hepatic expression ofABCB1, ABCC2, ABCC3, oatp2, ntcp and bsep in additionto CYP3A enzymes [83]. The results of this study suggestedinvolvement of the PXR. The overall effect of inflammation isincreased exposure to drugs and/or their metabolites due toreduced first-pass effect and inhibited hepatic elimination. Agood example is a clinical trial including 51 bone marrowtransplant patient treated with cyclosporin [84]. Duringimmunoreactions such as rejection of liver transplants andgraft-versus-host disease, hepatic efflux transporters seem tobe more affected than CYP3A-dependent metabolism. In thisstudy, liver graft-versus-host disease led to accumulation ofcyclosporin metabolites and a shift of the blood metabolismpattern from first-generation metabolites (metabolites that arechanged only at one site) to second- and higher-generationmetabolites (metabolites modified at two or more sites) thatare the results of further metabolism of first-generationmetabolites (Figure 3) [85]. Retrospectively, those results can beexplained by CYP3A/efflux transporter interactions as shownin Table 2. When biliary efflux from the hepatocytes is inhib-ited, it is reasonable to assume that cyclosporin and first-gen-eration metabolites will reach higher intracellularconcentrations and the intracellular residence time increases.This leads to more extensive metabolism. Instead of beingeliminated into bile, metabolites show up in relatively highconcentrations in the blood. The effect of inflammation onCYP3A and P-glycoprotein may also explain the observationthat hepatitis C positive liver transplant patients require alower tacrolimus dose than hepatitis C negative patients [86].

6. Impact on drug development

Recently, Wu and Benet [16] suggested a modification of theBiopharmaceutics Classification System (BCS), the Biophar-maceutics Drug Disposition Classification System, to alsoallow for predicting overall drug disposition. At present, theBCS is a scientific framework for classifying drug substancesbased on their aqueous solubility and intestinal permeability.When combined with dissolution of the drug product, theBCS takes into account three major factors that govern the rateand extent of drug absorption from immediate-release solidoral dosage forms: solubility, permeability and dissolution.

The proposed modification is based on the observation thatclass 1 (high solubility and high permeability) and 2 (low sol-ubility and high permeability) drugs are primarily eliminatedvia drug metabolism, whereas class 3 (high solubility and lowpermeability) and 4 (low solubility and low permeability)drugs are primarily eliminated unchanged [16]. Review of theliterature showed that most class 2 compounds are alsosubstrates for active drug transporters and it is reasonable to

expect that drug-metabolising enzyme/transporter interac-tions will play a role in their pharmacokinetics [16].

Changing the BCS permeability component to a route ofelimination component will allow for prediction of routes ofdrug elimination and of when transporter/drug-metabolisingenzyme interactions will result in clinically significant effectssuch as low and variable oral bioavailability and drug–druginteractions [16]. In addition, this modified classification mayalso be relevant for pharmacogenetic predictions. As discussedabove, the impact of polymorphisms depends on whether adrug is a good substrate of drug transporters and/or drug-metabolising enzymes. According to Wu and Benet’s [16]

observations, elimination of class 1 drugs are driven mainly bydrug metabolism. Thus, it can be speculated that for class 1drugs CYP polymorphisms have a significant impact on theirpharmacokinetics. Class 2 drugs, however, have a significantactive transporter component affecting their pharmaco-kinetics. Again, it can be speculated that pharmacokinetics ofthose drugs may be more impacted by polymorphisms oftransporter genes, as those regulate the substrate concentrationsat the CYP enzymes.

Because most of today’s new molecular entities are large-molecular-mass lipophilic compounds that fall into BCSclass 2, a Biopharmaceutics Drug Disposition ClassificationSystem that takes drug-metabolising enzyme/active drugtransporter interactions into account may have significantimpact on drug development and may affect regulatoryrequirements [16].

7. Expert opinion

It was our goal to review emerging concepts of the interactionbetween CYP3A and P-glycoprotein and to give a critical sta-tus report. It is important to note that further experimentalwork will be required to confirm those concepts, some ofwhich, as discussed above, must still be considered specula-tion. Although intriguing, their clinical relevance in humansstill needs further assessment. Most of the key work has beendone in cell and animal models that allow for testing specifichypotheses and for specific targeting of the CYP3A/P-glyco-protein interactions. In addition, clinical studies have beenusing specific probes to evaluate some of the mechanisms inhumans. The situation with drugs that are substrates of a mul-titude of drug-metabolising enzymes and active transportersand for which transport and metabolism in several organs areinvolved in their pharmacokinetics is a lot more complex andit may not be possible to extrapolate in vitro studies solelyassessing the CYP3A/P-glycoprotein interaction to the situa-tion in patients. As mentioned below, this situation is furthercomplicated by the fact that as of yet the transporter field isdeveloping rapidly and it is reasonable to assume that only apart of the transporters relevant for clinical pharmacokineticsare currently known. This review focuses on CYP3A/P-glyco-protein interaction as it is the best studied and most of theaforementioned concepts were developed based on this

Author Pro

of



enzyme/transporter pair. There are other similar interactionsbetween other drug-metabolising enzymes and transporter. Atpresent, the clinically most important example is the interactionbetween the immunosuppressants mycophenolic acid andcyclosporin, which is based on the disruption of the coopera-tion between UGT and MRP-2 (ABCC2) in the liver via inhi-bition of MRP-2 by cyclosporin [87]. Many other relevant drug-metabolising enzyme/active transporter interactions have notyet been studied or discovered. Three components play a keyrole in the absorption and elimination of BCS class 2 drugs:drug-metabolising enzymes, drug transporters and the nuclearxenobiotic receptors. All three, alone or in combination, can bethe target of drug–drug interactions. Again, it must be takeninto account that also our knowledge about nuclear receptors,their networks and their role in drug pharmacokinetics anddrug–drug interactions is probably still in its infancy.

It is common belief that one of the reasons for drug metab-olism is making lipophilic drugs more water soluble, which,therefore, facilitates their elimination. There is a good possi-bility that drug metabolism also transforms drugs into bettersubstrates for related transporters to facilitate their elimina-tion [17]; this hypothesis requires further experimentalconfirmation.

Significant polymorphisms of PXR (NR1I2) have beenreported. However, their clinical relevance is still unknown.One reason is the complexity of the network of genes regu-lated by nuclear receptors. In addition, studies have focusedon using CYP3A expression as a surrogate marker for meta-bolic activity. As discussed above, this may not be correctdepending on the drug and to which extent its pharmaco-kinetics is affected by active transporters. A complete picturerequires the evaluation of drug-metabolising enzymes, uptakeand efflux transporters and may depend on the nature of adrug as well as individual genetics. A complete understandingof PXR (NR1I2) polymorphisms will again require a com-plete understanding of the interaction between drug-meta-bolising enzymes, uptake and efflux transporters. The sameapplies for CYP3A and ABC-transporter gene polymor-phisms. In light of those complex interactions, the generalclinical applicability of diagnostic devices such as a microarraychip designed to routinely identify CYP polymorphisms canonly be expected to be predictable for drugs for which uptakeand efflux transporters have no significant influence on theirpharmacokinetics.

Recent studies have acknowledged the interaction of CYPenzymes, ABC transporters and the PXR and have includedmeasurement of all three in their clinical studies [50,69,83]. Cur-rent regulatory guidance, however, is still mostly focused ondrug-metabolising enzymes [16,88,103]. Those regulatory guide-lines for in vitro drug interaction testing are based solely onassays using isolated microsomes or CYP450 enzymes. Basedon these experiments, they allow for the conclusion that lackof an in vitro drug–drug interaction is reassuring and can

generally eliminate the need for further clinical evaluation[88,103]. As demonstrated in an in vitro study [36] and as dis-cussed in detail by Wu and Benet [16], this can be misleadingfor drugs that are substrates for both intestinal enzymes andintestinal efflux transporters. In addition, inhibition ofhepatic and renal uptake transporters can significantlyincrease systemic concentrations of drugs [16]. Such regulatoryguidances will require critical reevaluation.

Based on the fully sequenced human genome, it can be esti-mated that the number of human transporters and relatedproteins is in the thousands [104]. However, the number ofactive transporter proteins currently known is < 500. Thus,even if a drug-metabolising enzyme–drug transporter interac-tion is well-established, it is possible that a drug may also bethe substrate for yet unknown transporters. It is reasonable toexpect that in the future, as more transporters are identified,more clinically relevant drug-metabolising enzyme/drugtransporter interactions will be described.

In conclusion, the field of drug-metabolising transporter/active transporter interactions in combination with pharma-cogenetics and genomics is fast developing and the complexityof those interactions are still only partially understood.In vivo, drug-metabolising enzyme/transporter interactionsdepend on and can be influenced by:

• the affinity of a drug (and its metabolites) to CYP3A andP-glycoprotein

• the affinity of a drug (and its metabolites) to other drug-metabolising enzymes and other transporters such asuptake transporters

• the interaction of drug-metabolising enzymes and trans-porters with nuclear receptors

• polymorphisms of drug-metabolising enzymes, drug trans-porters and nuclear receptors

• the tissue distribution of a drug (and its metabolites) andexpression patterns of drug-metabolising enzymes, drugtransporters and nuclear receptors in tissues critical forpharmacokinetics

• age, gender, disease, drug–drug, herb–drug and food–druginteractions

Many of the concepts discussed above represent only a firstattempt to assess and explain interaction mechanisms. Futurestudies will have to show as to whether those represent gener-ally applicable concepts or will remain limited to the drug-metabolising enzymes, transporters and tissues based onwhich they have originally been established.

Acknowledgements

This work was supported by the United States National Insti-tutes of Health, grants R01 HL071805-03, R01 DK065094-03, and P30 DK048520-09 (U.C.) and the Merck CompanyFoundation (M.H.).

Author Pro

of



BibliographyPapers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

1. WACHER J, WU CY, BENET LZ: Overlapping substrate specificities and tissue distribution of cytochrome P4503A and P-glycoprotein: implications for drug delivery and cancer chemotherapy. Mol. Carcinogen (1995) 13:129-134.

•• This review article was the first to hypothesise that there is a functional collaboration between and coregulation of CYP3A and P-glycoprotein that is of significant relevance for the pharmacokinetics of most drugs that are known CYP3A substrates.

2. NELSON DR, KOYMANS L, KAMATAKI T et al.: P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics (1996) 6:1-42.

3. DOMANSKI TL, FINTA C, HALPERT JR, ZAPHIROPOULOS PG: cDNA cloning and initial characterization of CYP3A43, a novel cytochrome P450. Mol. Pharmacol. (2001) 59:386-392.

4. RENDIC S, DI CARLO FJ: Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab. Rev. (1997) 29:413-580.

5. AOYAMA T, YAMANO S, WAXMAN DJ et al.: Cytochrome P450 hPCN3, a novel cytochrome P450 IIIA gene product that is differentially expressed in adult human liver. cDNA and deduced amino acid sequence and distinct specificities of cDNA-expressed hPCN1 and hPCN3 for metabolism of steroid hormones and cyclosporine. J. Biol. Chem. (1989) 264:10388-10395.

6. WRIGHTON SA, BRIGHTON WR, SARI MA et al.: Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA5 (HLp3). Mol. Pharmacol. (1990) 38:207-213.

7. WESTLIND-JOHNSSON A, MALMEBO S, JOHANSSON A et al.: Comparative analysis of CYP3A expression in human liver suggests only a minor role for CYP3A5 in drug metabolism. Drug Metab. Dispos. (2003) 31:755-761.

8. PATKI KC, VON MOLTKE LL, GREENBLATT DJ: In vitro metabolism of midazolam, triazolam, nifedipine, and testosterone by human liver microsomes and recombinant cytochromes P450: role of

cyp3a4 and cyp3a5. Drug Metab. Dispos. (2003) 31:938-944.

9. SCHUETZ JD, BEACH DL, GUZELIAN PS: Selective expression of cytochrome P450 CYP3A mRNAs in embryonic and adult human liver. Pharmacogenetics (1994) 4:11-20.

10. KOCH I, WEIL R, WOLBOLD R et al.: Interindividual variability and tissue-specificity in the expression of cytochrome P450 3A mRNA. Drug Metab. Dispos. (2002) 30:1108-1114.

11. GOTTESMAN MM, PASTAN I: Biochemistry of multidrug resistance mediated by the multidrug transporter. Ann. Rev. Biochem. (1993) 62:385-427

12. CHEN CJ CHIN JE UEDA K et al.: Internal duplication and homology with bacterial transport proteins in mdr1 (P-glycoprotein) gene from multi-drug resistant human cells. Cell (1996) 47:381-389.

13. AMBUDKAR SV, DEY S, HRYCYNA CA, RAMACHANDRA M, PASTAN I, GOTTESMAN MM: Biochemical, cellular, and pharmacological aspects of multidrug transporters. Ann. Rev. Pharmacol. Toxicol. (1999) 39:361-398.

14. SCHINKEL AH, WAGENAAR E, VAN DEEMTER L et al.: Absence of the mdr1a P-glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. J. Clin. Invest. (1995) 96:1698-1705.

15. XU C, LI CY, KONG AN: Induction of Phase I, II and III drug metabolism/transport by xenobiotics. Arch. Pharm. Res (2005) 28:249-268.

16. WU CY, BENET LZ: Predicting drug disposition via application of BCS: transport/absorption/elimination interplay and development of a biopharmaceutics drug disposition classification system. Pharm. Res. (2005) 22:11-23.

•• This review article discusses in detail the impact of transporter/drug-metabolising enzyme interactions on regulatory and drug development aspects. It suggests a revised classification system for drugs that will help to develop drugs more efficiently.

17. CHRISTIANS U: Transport proteins and intestinal metabolism. P-glycoprotein and cytochrome P4503A. Ther. Drug Monit. (2004) 26:104-106.

18. BENET LZ, CUMMINS CL, WU CY: Unmasking the dynamic interplay between

efflux transporters and metabolic enzymes. Int. J. Pharm. (2004) 277:3-9.

19. BENET LZ, CUMMINS CL, WU CY: Transport-enzyme interactions: implications for predicting drug–drug interactions from in vitro data. Curr. Drug Metab. (2003) 4:393-398.

20. BENET LZ, CUMMINS CL: The drug efflux-metabolism alliance: biochemical aspects. Adv Drug Deliv Rev (2001) 50:S3- S11.

21. CUMMINS CL, JACOBSEN W, BENET LZ: Unmasking the dynamic interplay between intestinal P-glycoprotein and CYP3A4. J. Pharmacol. Exp. Ther. (2002) 300:1036-1045.

•• This in vitro study showed for the first time that inhibition of efflux transporters without affecting CYP3A results in reduced drug metabolism.

22. CUMMINS CL, JACOBSEN W, CHRISTIANS U, BENET LZ: CYP3A4 transfected Caco-2 cells as a tool for understanding biochemical absorption barriers: studies with sirolimus and midazolam. J. Pharmacol. Exp. Ther. (2004) 308:143-155.

23. JOHNSON BM, CHARMAN WN, PORTER CJH: Application of compartmental modeling to an examination of in vitro intestinal permeability data: assessing the impact of tissue uptake, P-glycoprotein, and CYP3A. Drug Metab. Dispos. (2003) 31:1151-1160.

24. CUMMINS CL, SALPHATI L, REID M, BENET LZ: In vivo modulation of intestinal CYP3A metabolism by P-glycoprotein: studies using the rat single-pass intestinal perfusion model. J. Pharmacol. Exp. Ther. (2003) 305:306-314.

• This study confirmed in vivo that constant efflux of drugs and repeated presentation to the CYP3A in the small intestine enhances drug metabolism and reduces absorption.

25. LAMPEN A CHRISTIANS U GONSCHIOR AK et al.: Metabolism of the macrolide immunosuppressant tacrolimus by the pig mucosa in the Ussing chamber. Br. J. Pharmacol. (1996) 117:1730-1734.

26. LAMPEN A, ZHANG Y, HACKBARTH I, BENET LZ, SEWING KF, CHRISTIANS U: Metabolism and transport of the macrolide immunosuppressant sirolimus in the small

Author Pro

of



intestine. J. Pharmacol. Exp. Ther. (1998) 285:1104-1112.

27. SOLDNER A, BENET LZ, MUTSCHLER E, CHRISTIANS U: Active transport of the angiotensin-II antagonist losartan and its main metabolite EXP 3174 across MDCK-MDR1 and caco-2 cell monolayers. Br. J. Pharmacol. (2000) 129:1235-1243.

• This study provided experimental evidence for the hypothesis that drug metabolism changes the affinity to efflux transporters.

28. ZIMMERMANN C, GUTMANN H, HRUZ P et al.: Mapping of multidrug resistance-associated protein isoforms 1 to 5 mRNA expression along the human intestinal tract. Drug Metab. Dispos. (2005) 33:219-224.

29. MOULY S, PAINE MF: P-glycoprotein increases from proximal to distal regions of human small intestine. Pharm. Res. (2003) 20:1595-1599.

30. FRICKER G, DREWE J, HUWYLER J, GUTMANN H, BEGLINGER C: Relevance of P-glycoprotein for the enteral absorption of cyclosporin A: in vitro–in vivo correlation. Br. J. Pharmacol. (1996) 118:1841-1847.

31. KAMINSKY LS, ZHANG QY: The small intestine as a xenobiotic-metabolizing organ. Drug Metab. Dispos. (2003) 31:1520-1525.

32. PAINE MF, KHALIGHI M, FISHER JM et al.: Characterization of intestinal and intraintestinal variations in human CYP3A-dependent metabolism. J. Pharmacol. Exp. Ther. (1997) 283:1552-1562.

33. ZHANG QY, DUNBAR D, OSTROWSKA A, ZEISLOFT S, YANG J, KAMINSKY LS: Characterization of human small intestinal cytochromes P450. Drug Metab. Dispos. (1999) 27:804-809.

34. WU CY, BENET LZ: Disposition of tacrolimus in isolated perfused rat liver: influence of troleandomycin cyclosporine, and GG918. Drug Metab. Dispos. (2003) 31:1292-1295.

• The results suggested that specific inhibition of P-glycoprotein has the opposite effect on the metabolism of dual P-glycoprotein/CYP3A substrates than in the gut mucosa due to the reverse order of CYP3A and P-glycoprotein in the path of a drug through the hepatocyte than through an intestinal mucosa cell.

35. LAU YY, WU CY, OKOCHI H, BENET LZ: Ex situ inhibition of hepatic uptake and efflux significantly changes

metabolism: hepatic enzyme transporter interplay. J. Pharmacol. Exp. Ther. (2004) 308:1040-1045.

36. LAM JL, BENET LZ: Hepatic microsome studies are insufficient to characterize in vivo metabolic clearance and metabolic drug–drug interactions: studies of digoxin metabolism in primary rat hepatocytes versus microsomes. Drug Metab. Dispos. (2004) 32:1311-1316.

• The results suggested that hepatic drug metabolism is modulated by uptake and influx transporters and that the exclusive use of microsomal systems as supported by current regulatory guidances may not be sufficient to predict or exclude clinically relevant drug–drug interactions.

37. MAGLICH JM, SLUDER AE, WILLSON TM, MOORE JT: Beyond the human genome: examples of nuclear receptor analysis in model organisms and potential for drug discovery. Am. J. Pharmacogenomics (2003) 3:345-353.

38. FRANCIS GA, FAYARD E, PICARD F, AUWERX J: Nuclear receptors and the control of metabolism. Ann. Rev. Physiol. (2003) 65:261-311.

39. SYNOLD TW, DUSSAULT I, FORMAN BM: The orphan nuclear receptor SXR coordinately regulates drug metabolism and efflux. Nat. Med. (2001) 7:584-590.

• The first publication to describe co-regulation of drug-metabolising enzymes (CYP3A and CYP2C8) and drug transporters (MDR1 and MRP2) via the human nuclear receptor SXR.

40. DUSSAULT I, LIN M, HOLLISTER K, WANG EH, SYNOLD TW, FORMAN BM: Peptide mimetic HIV protease inhibitors are ligands for the orphan receptor SXR. J. Biol. Chem. (2001) 276:33309-33312.

41. ROSENFELD JM, VARGAS R, XIE W, EVANS RM: Genetic profiling defines xenobiotics gene network controlled by the nuclear receptor pregnane X receptor. Mol. Endocrinol. (2003) 17:1268-1282.

42. BURK O, KOCH I, RAUCY J et al.: The induction of cytochrome P450 3A5 (CYP3A5) in the human liver and intestine is metadiated by the xenobiotoc sensors pregnane X receptor (PXR) and the constitutively activated receptor (CAR). J. Biol. Chem. (2004) 279:38379-38385.

43. MAGLICH JM, STOLTZ CM, GOODWIN B, HAWKINS-BROWN D, MOORE JT, KLIEWER SA: Nuclear

pregnane X receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotics detoxification. Mol. Pharmacol. (2002) 62:638-646.

44. CHERRINGTON NJ, HARTLEY DP, LI N, JOHNSON DR, KLAASSEN CD: Organ distribution of multidrug resistance proteins 1,2, and 3 (MRP 1,2, and 3) mRNA and hepatic induction of Mrp3 by constitutive androsterane receptor activators in rats. J. Pharmacol. Exp. Ther. (2002) 300:97-104.

45. LAMBA JK, LIN YS, SCHUETZ EG, THUMMEL KE: Genetic contribution to variable human CYP3A-mediated metabolism. Adv. Drug Deliv. Rev. (2002) 54:1271-1279.

46. CHOWBAY B, ZHOU S, LEE EJD: An interethnic comparison of polymorphisms of the genes encoding drug metabolizing enzymes and drug transporters: experience in Singapore. Drug Metab. Rev. (2005) 37:327-378.

47. LIN YS, DOWLING ALS, QUIGLEY SD et al.: Co-regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism. Mol. Pharmacol. (2002) 62:162-172.

48. SAI K, KANIWA N, ITODA M et al.: Haplotype analysis of ABCB1/MDR1 blocks in a Japanese population reveals genotype-dependent renal clearance of irinotecan. Pharmacogenetics (2003) 13:741-757.

49. KUEHL P, ZHANG J, LIN Y et al.: Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat. Genet. (2001) 27:383-391.

50. EICHELBAUM M, FROMM MF, SCHWAB M: Clinical aspects of the MDR1 (ABCB1) gene polymorphism. Ther. Drug Monit. (2004) 26:180-185.

51. ANGLICHEAU D, LEGENDRE C, THERVET E: Pharmacogenetics in solid organ transplantation: present knowledge and future perspectives. Transplantation (2004) 78:311-315.

52. MARLOZINI C, PAUS E, BUCLIN T et al.: Polymorphisms in human MDR1 (P-glycoprotein): recent advances and clinical relevance. Clin. Pharmacol. Ther. (2004) 75:13-33.

53. MACPHEE IA, FREDERICKS S, TAI T et al.: Tacrolimus pharmacogenetics: polymorphisms associated with expression

Author Pro

of



of cytochrome P4503A5 and P-glycoprotein correlate with dose requirement. Transplantation (2002) 74:1486-1489.

54. GOTO M, MASUDA S, KIUCHI T et al.: CYP3A5*1-carrying graft liver reduces the concentration/oral dose ratio of tacrolimus in recipients of living-donor liver transplantation. Pharmacogenetics (2004) 14:471-478.

55. HESSELINK DA, VAN SCHAIK RHN, VAN DER HEIDEN IP et al.: Genetic polymorphisms of the CYP3A4, CYP3A5, and MDR-1 genes and pharmacokinetics of the calcineurin inhibitors cyclosporine and tacrolimus. Clin. Pharmacol. Ther. (2003) 74:245-254.

56. BONHOMME-FAIVRE L, DEVOCELLE A, SALIBA F et al.: MDR-1 C3435T polymorphism influences cyclosporine A dose requirement in liver transplant recipients. Transplantation (2004) 78:21-25.

57. ANGLICHEAU D, THERVET E, ETIENNE I et al.: CYP3A5 and MDR1 genetic polymorphisms and cyclosporine pharmacokinetics after renal transplantation. Clin. Pharmacol. Ther. (2004) 75:422-433.

58. ANGLICHEAU D, LE CORRE D, LECHATON S et al.: Consequences of genetic polymorphisms for sirolimus requirements after renal transplant in patients on primary sirolimus therapy. Am. J. Transplant. (2005) 5:595-603

59. FREDERICKS S, HOLT DW: Pharmacogenomics of immunosuppressive drug metabolism. Curr. Opin. Nephrol. Hypertens. (2003) 12:607-613.

60. DIRKS NL, HUTH B, YATES CR et al.: Pharmacokinetics of immunosuppressants: a perspective on ethnic differences. Int. J. Clin. Pharmacol. Ther. (2004) 42:701-718.

61. AMEYAW MM, REGATEIRO F, LI T et al.: MDR1 pharmacogenetics: frequency of the C3435T mutation in exon 26 is significantly influenced by ethnicity. Pharmacogenetics (2001) 11:217-221.

62. HUSTERT E, ZIBAT A, PRESECAN-SIEDEL E et al.: Natural protein variants of pregnane X receptor with altered transactivation activity toward CYP3A4. Drug Metab. Dispos. (2001) 29:1454-1459.

63. ZHANG J, KUEHL P, GREEN ED et al.: The human pregnane X receptor: genomic structure and identification and functional characterization of natural allelic variants. Pharmacogenetics (2001) 11:555-572.

64. HAUFROID V, MOURAD M, VAN KERCKHOVE V et al.: The effect of CYP3A5 and MDR1 (ABCB1) polymorphisms on cyclosporine and tacrolimus dose requirements and trough blood levels in stable renal transplant patients. Pharmacogenetics (2004) 14:147-154.

65. LOWN KS, MAYO RR, LEICHTMAN AB et al.: Role of intestinal P-glycoprotein (mdr1) in interpatient variation in the oral bioavailability of cyclosporine. Clin. Pharmacol. Ther. (1997) 62:248-260.

66. EAP CB, BUCLIN T, HUSTERT E et al.: Pharmacokinetics of midazolam in CYP3A4- and CYP3A5-genotyped subjects. Eur. J. Clin. Pharmacol. (2004) 60:231-236.

67. LEEDER JS, KEARNS GL: Pharmacogenetics in pediatrics: implications for practice. Pediatr. Clin. North Am. (1997) 44:55-77.

68. MACHADO CG, CALADO RT, GARCIA AB et al.: Age-related changes of the multidrug resistance P-glycoprotein function in normal human peripheral blood T-lymphocytes. Brz J. Med. Biol. Res. (2003) 36:1653-1657.

69. MIKI Y, SUZUKI T, TAZAWA C, BLUMBERG B, SASANO H: Steroid and xenobiotic receptor (SXR), cytochrome P450 3A4 and multidrug resistance gene 1 in human adult and fetal tissues. Mol. Cell. Endocrinol. (2005) 231:75-85.

70. CUMMINS CL, WU CY, BENET LZ: Sex-related differences in the clearance of cytochrome P450 3A4 substrates may be caused by P-glycoprotein. Clin. Pharmacol. Ther. (2002) 72:474-489.

71. WOBOLD R, KLEIN K, BURK O et al.: Sex is a major determinant of CYP3A4 expression in human liver. Hepatology (2003) 38:978-988.

72. PAINE MF, LUDINGTON SS, CHEN ML, STEWART PW, HUANG SM, WATKINS PB: Do men and women differ in proximal small intestinal CYP3A or P-glycoprotein expression? Drug Metab. Dispos. (2005) 33:426-433.

73. LAMPEN A, CHRISTIANS U, GUENGERICH FP et al.: Metabolism of the immunosuppressant tacrolimus in the small intestine: cytochrome P450, drug interactions and interindividual variability. Drug Metab. Dispos. (1995) 23:1315-1324

74. CHRISTIANS U, BLECK JS, LAMPEN A et al.: Are cytochrome P450 3A enzymes in

the small intestine responsible for different cyclosporine metabolite patterns in stable male and female renal allograft recipients after coadministration of diltiazem? Transplant. Proc. (1996) 28:2159-2161.

75. HUANG SM, LESKO LJ: Drug–drug, drug dietary, drug–citrus fruit and other food interactions: what have we learned? J. Clin. Pharmacol. (2004) 44:559-569.

76. FUJITA K: Food-drug interactions via human cytochrome P450 3A (CYP3A). Drug Metab. Interact. (2004) 20:195-217.

77. HARRIS RZ, JANG GR, TSUNODA S: Dietary effects on drug metabolism and transport. Clin. Pharmacokinet. (2003) 42:1071-1088.

78. LOWN KS, BAILEY DG, FONTANA RJ et al.: Grapefruit juice increases felodipine oral availability in humans by decreasing intestinal CYP3A protein expression. J. Clin. Invest. (1997) 99:2545-2553.

79. ZHOU S, LIM LY, CHOWBAY B: Herbal modulation of P-glycoprotein. Drug Metab. Rev. (2004) 36:57-104.

80. MORGAN ET: Regulation of cytochromes P450 during inflammation and infection. Drug Metab. Rev. (1997) 29:1129-1188.

81. IBER H, SEWER MB, BARCLAY TB: Modulation of drug metabolism in infectious and inflammatory diseases. Drug Metab. Rev. (1999) 31:29-41.

82. SUKHAI M, PIQUETTE-MILLER M: Regulation of the multidrug resistance genes by stress signals. J. Pharm. Pharmaceut. Sci. (2000) 3:268-280.

83. TENG S, PIQUETTE-MILLER M: The involvement of the pregnane X receptor in hepatic gene regulation during inflammation in mice. J. Pharmacol. Exp. Ther. (2005) 312:841-848.

84. CHRISTIANS U SPIEKERMANN K BADER A et al.: Cyclosporine metabolite pattern in blood from patients with acute liver GVHD after bone marrow transplantation. Bone Marrow Transplant (1993) 12:27-33.

85. CHRISTIANS U, SEWING KF: Ciclosporin metabolism in transplant patients. Pharmacol. Ther. (1993) 57:291-345.

86. TROTTER JF, OSBORNE JC, HELLER M, CHRISTIANS U: Effect of hepatitis C on tacrolimus doses and blood levels in liver transplantation recipients. Aliment. Pharmacol. Ther. (2005) 22:37-44.

87. HESSELINK DA VAN HEST RM MATHOT RA et al.: Cyclosporine interacts

Author Pro

of



with mycophenolic acid by inhibiting the multidrug resistance-associated protein 2. Am. J. Transplant. (2005) 5:987-994.

88. BJÖRNSSON TD, CALLAGHAN TJ, EINOLF HJ et al.: The conduct of in vitro and in vivo drug–drug interaction studies: a PhRMA perspective. J. Clin. Pharmacol. (2003) 43:443-469.

Websites

101. http://www.ncbi.nlm.nih.gov/booksDEAN M: The human ATP-binding cassette (ABC) transporter superfamily. NCBI Monography, National Library of Medicine, Bethesda, MD, USA (2002).

102. http://www.imm.ki.se/CYPalleles

103. http://www.fda.gov/cder/guidance/clin3.pdf US Department of Health and Human Services, Food and Drug Administration, Centers for Drug Evaluation and Research and Center for Biologics Evaluation and Research. Guidance for the Industry. Drug Metabolism/Drug Interaction Studies in the Drug Development Process: Studies In vitro (1997).

104. http://www.pharmsci.org YAN Q, SADÉE W: Human membrane transporter database: a web-accessible relational database for drug transport studies and pharmacogenomics. AAPS

Pharmsci. (2000) 2:article 20.

AffiliationUwe Christians†, Volker Schmitz & Manuel Haschke†Author for correspondenceUniversity of Colorado Health Sciences Center, Clinical Research & Development, Department of Anesthesiology, 4200 East Ninth Ave., Room UH-2122, Campus Box B-113, Denver, Colorado 80262, USATel: +1 303 315 1845; Fax: +1 303 315 1858;E-mail: [email protected]

functional interactions between p-glycoprotein … interactions between p-glycoprotein and cyp3a in...

Documents