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Clin Pharmacokinet 2003; 42 (1): 59-98 0312-5963/03/0001-0059/$30.00/0 © Adis International Limited. All rights reserved.
Role of P-Glycoprotein in Pharmacokinetics
Jiunn H. Lin and Masayo Yamazaki
Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania, USA
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Structure and Mechanism of Drug-Transporting P-Glycoprotein . . . . . . . . . . 1.1 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 ATP- and Substrate-Binding Sites . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Substrate Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Polymorphisms of P-Glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. In Vitro/In Vivo Extrapolation and Species Differences . . . . . . . . . . . . . . . 4. Role of P-Glycoprotein in Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . 4.1 Drug Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1 Distribution of Intestinal P-Glycoprotein . . . . . . . . . . . . . . . . . . 4.1.2 Interindividual Variability of Intestinal P-Glycoprotein . . . . . . . . . . 4.1.3 Evidence of Intestinal P-Glycoprotein Involvement in Drug Absorption 4.1.4 Saturable Efflux Transport by Intestinal P-Glycoprotein . . . . . . . . . 4.2 Drug Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Blood-Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2 Placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Drug Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Drug Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1 Biliary Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2 Renal Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. P-Glycoprotein-Mediated Drug-Drug Interactions . . . . . . . . . . . . . . . . . . 5.1 P-Glycoprotein Inhibition Does Not Follow Simple Kinetics . . . . . . . . . . 5.2 Drug Interactions Caused by P-Glycoprotein Inhibition . . . . . . . . . . . . 5.3 P-Glycoprotein Induction is a Complex Process . . . . . . . . . . . . . . . . 5.4 Drug Interactions Caused by P-Glycoprotein Induction . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 61 61 62 63 64 66 68 69 70 70 71 72 75 75 79 80 81 82 83 84 84 86 88 90 91
P-glycoprotein, the most extensively studied ATP-binding cassette (ABC) transporter, functions as a biological barrier by extruding toxins and xenobiotics out of cells. In vitro and in vivo studies have demonstrated that P-glycoprotein plays a significant role in drug absorption and disposition. Because of its localisation, P-glycoprotein appears to have a greater impact on limiting cellular uptake of drugs from blood circulation into brain and from intestinal lumen into epithelial cells than on enhancing the excretion of drugs out of hepatocytes and renal tubules into the adjacent luminal space. However, the relative contribution of intestinal
Lin & Yamazaki
P-glycoprotein to overall drug absorption is unlikely to be quantitatively important unless a very small oral dose is given, or the dissolution and diffusion rates of the drug are very slow. This is because P-glycoprotein transport activity becomes saturated by high concentrations of drug in the intestinal lumen. Because of its importance in pharmacokinetics, P-glycoprotein transport screening has been incorporated into the drug discovery process, aided by the availability of transgenic mdr knockout mice and in vitro cell systems. When applying in vitro and in vivo screening models to study P-glycoprotein function, there are two fundamental questions: (i) can in vitro data be accurately extrapolated to the in vivo situation; and (ii) can animal data be directly scaled up to humans? Current information from our laboratory suggests that in vivo P-glycoprotein activity for a given drug can be extrapolated reasonably well from in vitro data. On the other hand, there are significant species differences in P-glycoprotein transport activity between humans and animals, and the species differences appear to be substrate-dependent. Inhibition and induction of P-glycoprotein have been reported as the causes of drug-drug interactions. The potential risk of P-glycoprotein-mediated drug interactions may be greatly underestimated if only plasma concentration is monitored. From animal studies, it is clear that P-glycoprotein inhibition always has a much greater impact on tissue distribution, particularly with regard to the brain, than on plasma concentrations. Therefore, the potential risk of P-glycoproteinmediated drug interactions should be assessed carefully. Because of overlapping substrate specificity between cytochrome P450 (CYP) 3A4 and P-glycoprotein, and because of similarities in P-glycoprotein and CYP3A4 inhibitors and inducers, many drug interactions involve both P-glycoprotein and CYP3A4. Unless the relative contribution of P-glycoprotein and CYP3A4 to drug interactions can be quantitatively estimated, care should be taken when exploring the underlying mechanism of such interactions.
P-glycoprotein was first identified by Juliano and Ling as a surface phosphoglycoprotein expressed in drug-resistant Chinese hamster ovary cells. This discovery led to the finding that Pglycoprotein is an energy-dependent efflux transporter driven by ATP hydrolysis. In humans, two members of the P-glycoprotein gene family (MDR1 and MDR3) exist, while three members of this family (mdr1a, mdr1b and mdr2) are found in mice.[2,3] The P-glycoprotein encoded by the human MDR1 and mouse mdr1a/1b genes functions as a drug efflux transporter, whereas human MDR3 P-glycoprotein and mouse mdr2 P-glycoprotein are believed to be functional in phospholipid transport.[4,5] However, the involvement of human MDR3 P-glycoprotein in drug transport has been
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recently reported. An increased directional transport of digoxin, paclitaxel and vinblastine across polarised monolayers of MDR3-transfected cells has recently been reported by Smith et al. These results suggest that MDR3 P-glycoprotein is also able to transport a range of drugs in addition to phospholipids. In addition to the expression in tumour cells, human P-glycoprotein is also highly expressed in normal tissues. This transporter is localised on the canalicular surface of hepatocytes in liver, the apical surface of epithelial cells of proximal tubules in kidneys, columnar epithelial cells of intestine, epithelial cells of placenta, and the luminal surface of capillary endothelial cells in brain in humans.[7,8] The anatomical localisation of P-glycoClin Pharmacokinet 2003; 42 (1)
protein expression suggests that the efflux transporter can functionally protect the body against toxic xenobiotics by excreting these compounds into bile, urine and the intestinal lumen, and by preventing their accumulation in brain. Because of its localisation, it is believed that P-glycoprotein may play a significant role in the processes of absorption, distribution, metabolism and excretion of drugs in humans and animals. Indeed, the role of P-glycoprotein in drug absorption and disposition has been demonstrated in vivo using mdr1a and mdr1a/1b knockout mice.[9,10] A lack of either one of the two murine P-glycoproteins (mdr1a or mdr1b) results in significant changes in drug absorption and disposition. The purpose of this review is to briefly summarise the current knowledge regarding the structure and mechanism of drug-transporting P-glycoprotein and its role in drug absorption, distribution, metabolism and excretion. In addition, the potential for P-glycoprotein-mediated drug-drug interactions and its clinical implications will be discussed. 1. Structure and Mechanism of Drug-Transporting P-Glycoprotein
Since the identification of P-glycoprotein, major efforts have been made to elucidate the structure of the protein to gain better insight into the mechanism of its action. The cloning of genes and structure-function analysis of the protein by genetic and biochemical studies have contributed to a better understanding of the transporter. P-glycoprotein is composed of two homologous and symmetrical halves (cassettes), each of which contains six transmembrane domains that are separated by an intracellular flexible linker polypeptide loop with an ATP-binding motif. Interestingly, the two halves of human P-glycoprotein are not identical, and of the amino acids aligned, only 43% are identical, suggesting that the molecules of the two halves might have either evolved indepen Adis International Limited. All rights reserved.
dently or have undergone major intron movement after a duplication event. Site-directed mutagenesis and antibody mapping studies suggest that the two cassettes of human P-glycoprotein interact cooperatively to form a single functional unit.[12,13] The direct evidence that supports the hypothesis of a single functional unit is from the study that co-expressed each cassette of human P-glycoprotein. The cDNA coding for human P-glycoprotein was divided in half and subcloned into separate plasmids in order to express each half as a separate polypeptide and to characterise its contribution to function. No drugstimulative ATPase activity was observed when Sf9 cells were transfected separately with cDNA coding for each cassette. The hypothesis of the single functional unit is further supported by the mutation studies carried out by Takada et al. In this study, the key lysine and cysteine residues in the Walker A motifs of ATP-binding domains were substituted by methionine and alanine, respectively. The results of this study clearly demonstrate that if one ATP-binding domain is not functional, there is no ATP hydrolysis even when ATP binds to the other ATP-binding domain. In addition to the ATP-binding domains, the intracellular flexible linker loops also play a key role in ATPase and transport activity. Deletion of the central core of the intracellular flexible linker region of human P-glycoprotein resulted in a protein without functional ATPase and transport activity. Collectively, these data strongly suggest that the two cassettes of P-glycoprotein interact as a single transporter and that the flexible linker region is important for the proper interaction of the two cassettes. Several mechanistic models have been proposed to describe the mechanism for drug transport activity. The initial mechanistic model hypothesises that, similar to ion channel proteins, hydrophobic membrane-spanning regions and hydrophilic elements of P-glycoprotein form an aqueous transmembrane pore through which drugs are transported from the cytosol to the extracellular media. However, another model suggests that
Clin Pharmacokinet 2003; 42 (1)
Lin & Yamazaki
P-glycoprotein might extrude drugs directly from the cell membrane even before they enter the cytoplasm. This second model is supported by a study of fura-2 acetoxymethyl ester in NIH-3T3 mouse fibroblasts. Fura-2 acetoxymethyl ester, a fluorescent indicator, is hydrophobic and actively extruded by P-glycoprotein, whereas the hydrophilic free acid form of the indicator, to which the ester is rapidly hydrolysed once in the cellular cytoplasm, is not exported by P-glycoprotein. The intracellular trapping of Fura-2 free acid was remarkably reduced in P-glycoprotein-expressing NIH-3T3 mouse fibroblasts as compared with that in the control fibroblasts (no P-glycoprotein expression) by a factor of 10. Addition of verapamil did not alter the intracellular concentration of Fura-2 free acid in control cells, whereas it increased the amount of trapped Fura-2 free acid in the P-glycoprotein-expressing cells up to the level found in the control cells. From these results, the investigators concluded that hydrophobic molecules of Fura-2 acetoxymethyl ester interacted with the P-glycoprotein in the cell membrane before entering the cytosol, and hence cytoplasmic esterases had no chance to ‘see’ Fura-2 acetoxymethyl ester. However, their conclusion is valid only if the rate of P-glycoprotein transport is the ratelimiting step in trapping of Fura-2 free acid, i.e. the rate of ester hydrolysis is much faster than the rate of transport. More convincing evidence that supports the concept of the interaction of substrates with Pglycoprotein in the lipid membrane of cells came from the study by Shapiro and Ling. These investigators measured the kinetics of Hoechst 33 342 in P-glycoprotein-enriched plasma membrane vesicles from Chinese hamster ovary cells. Hoechst 33 342 is fluorescent only when bound to the membrane, but not when in the aqueous medium. Therefore, the movement of Hoechst 33 342 in and out of the membrane can be directly monitored by the fluorescence intensity. Using the fluorometric assay, the results revealed that the initial rate of transport was directly proportional to the amount of dye in the lipid phase, but not to the
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concentration in the aqueous phase, suggesting that P-glycoprotein extruded Hoechst 33342 from the lipid membrane before it entered the cytosol. With some modifications of the second model, a recent and more favoured model proposes that P-glycoprotein intercepts lipophilic drugs as they move through the lipid membrane and flips the drugs from the inner leaflet to the outer leaflet and into the extracellular medium. This model is consistent with the notion that the lipophilicity of a drug is an important determinant in its interaction with P-glycoprotein. Recently, Rosenberg et al. used high-resolution electron microscopy and image analysis to obtain the first three-dimensional architecture of a P-glycoprotein purified from Chinese hamster ovary CHrB30 cells that retained the ability to bind substrates and hydrolyse ATP. When viewed from above the membrane plane, the P-glycoprotein is toroidal, with six-fold symmetry and a diameter of ~10nm. There is a large central pore of about 5nm in diameter, which is closed on the cytoplasmic surface of the plasma membrane forming an aqueous chamber within the membrane. Overall, the observed microscopic image appears to be consistent with the proposed models. Although in recent years there has been a great advancement in our understanding of the structure of P-glycoprotein through mutational and biochemical studies, the precise molecular mechanism of drug transport by P-glycoprotein is still not fully understood.
1.2 ATP- and Substrate-Binding Sites
There are two ATP-binding domains of P-glycoprotein, located in the cytosol side. Each ATPbinding domain contains three regions: Walker A and B and Signature C motifs. The sequences of amino acid residues for the first ATP-binding domain are: from 427–435 for Walker A motif, from 531–542 for Walker B motif, and from 551–556 for Signature C motif. The corresponding amino acid residues for the second ATP-binding domain are from 1070–1078, from 1176–1182 and from 1196–1201, respectively. Recently, Hung et al.
Clin Pharmacokinet 2003; 42 (1)
TM11 and TM12. It is clear now that ATP binding and subsequent hydrolysis are essential for drug transport. ranging from a Clin Pharmacokinet 2003. Gly288 between TM4 and TM5. Gly64 and Leu65) in TM1 are involved in the formation of a binding pocket that plays a key role in determining the suitable substrate sizes for P-glycoprotein. respectively.3 Substrate Recognition One of the most intriguing aspects of P-glycoprotein is that a single integral membrane protein can recognise and transport so many drugs with a wide array of chemical structures. 42 (1) . Based on the data from vanadate trapping studies.32] In addition to the transmembrane domains. TM6 and TM12 of P-glycoprotein. Mutations in either one of these residues result in nonfunctional activity of histidine permease. an ABC transporter. molecular weight 811).[33. whereas substitution of an amino acid with a long side chain increased resistance to colchicine (small molecular size. All rights reserved. including the transmembrane domains. only one site participates in the catalysis at a given time.28] After complete digestion of the Pglycoprotein with trypsin. Consistent with the studies of photoaffinity probes. Two substrate-binding sites were found in TM6 and TM12 by using photoaffinity probes.[31. TM6. a region that includes TM7 and TM8 was also reported to be photolabelled specifically by an analogue of paclitaxel. intracellular loops and even the ATPbinding domains. the last transmembrane domain of each cassette. These results of histidine permease suggest that the ATP-binding sites may also be restricted to the Walker A motifs of P-glycoprotein. the 4kD fragment includes residues from 979– Adis International Limited.6–3 molecules of ATP are hydrolysed for every molecule of drug transported out the cell. TM4. Unlike the ATP-binding sites that are restricted to the Walker A motifs of ATP-binding domains. extending a few residues beyond the Walker A motif of the first ATP-binding site. The stoichiometry of ATP hydrolysis to drug transport has been studied.[27.26] The reason for the substrate-dependent ATP stoichiometry is still unknown. and the data indicate that. many substrate-binding sites have been identified throughout the transmembrane domains (TM) of P-glycoprotein. studies of many mutant P-glycoprotein molecules suggest that the major drug-binding sites reside in or near TM6 and TM12. 0. mutational analyses have suggested that the intracellular linker loops of P-glycoprotein are also important for substrate recognition.34] The systematic mutagenesis of 20 Gly residues in the cytoplasmic loops revealed that Gly141 and Gly187 between TM2 and TM3. and a highly conserved Asp residue within the Walker B motif serves to bind the Mg2+ ion. 1. molecular weight 399).32] suggested that three amino acids (His61. and conformation of this catalytic site precludes the other site from hydrolysing ATP. is directly involved with the binding of ATP. which explains that although both ATPbinding sites are capable of binding ATP. two major photolabelled fragments (5 and 4kD) were mapped by immunological analysis.P-Glycoprotein 63 reported that a highly conserved Lys residue within the Walker A motif of histidine permease. For example. depending on substrate. 1048. these data suggest that drug-binding sites are scattered throughout the P-glycoprotein molecule. Senior and Gadsby have proposed a so-called alternate ATP-binding site model. but not including the Walker A motif of the second ATP-binding site. Recent studies by Taguchi et al. and Gly812 and Gly830 between TM8 and TM9 are important in determining substrate specificity. substitution of His61 by an amino acid with a short side-chain increased resistance to vinblastine (large molecular size.[31. In summary. The 5kD fragment includes amino acid residues from 311– 456. Studies with photoactive analogues of ATP have shown that these analogues bind to the ATP-binding domains. These two fragments are located within. TM10. However.[25. In addition to the regions of TM6 and TM12. including TM1. mutational studies also suggest that amino acid substitutions that affect substrate specificity are scattered throughout P-glycoprotein. or immediately next to. In contrast.
The 50% lethal dose (LD50) values were 0. was very sensitive to neurotoxicity following exposure to avermectin. suggesting that partitioning of the lipid membrane of cells is the first step for the interaction of a substrate with the active sites of P-glycoprotein. Additionally. All rights reserved. there is a remarkable degree of genetic variability built into the population. some essential structural elements of substrates are required for an interaction with P-glycoprotein.40] have shown that both the lipophilicity and number of hydrogen bonds of compounds are probably the most important parameters in determining the affinity of compounds to P-glycoprotein. The only common feature is that most of the P-glycoprotein substrates are hydrophobic in nature. a clear structure-activity relationship for predicting P-glycoprotein substrates still cannot be established. 2. The lack of clear structure-activity relationship for substrate recognition is attributed mainly to the structural complexity of P-glycoprotein.3Å or 4. For example. there are many exceptions. aldosterone and dexamethasone. resulting in a more than 80-fold higher accumulation of avermectin in the brain. The higher the lipophilicity or the larger the number of hydrogen bonds. it was believed that a basic nitrogen atom was a prerequisite for the interaction of substrate and P-glycoprotein.5 ± 0. Seelig and Landwojtowicz have also suggested that both lipophilicity and number of hydrogen bonds are important determinants for substrates and P-glycoprotein interaction. The recognition elements of substrates are formed by two or three electron donor (hydrogen–bonding acceptor) groups Adis International Limited. Seelig further proposed that in addition to lipophilicity and number of hydrogen bonds. approximately 25%. compounds lacking a nitrogen atom. with a fixed spatial separation: 2.64 Lin & Yamazaki molecular weight of 250 (cimetidine) to 1202 (cyclosporin). Originally. they suggested that dissociation rate of the P-glycoproteinsubstrate complex is controlled by the number of hydrogen bonds. The P-glycoprotein-deficient CF-1 mice Clin Pharmacokinet 2003.6Å. Furthermore. an antiparasitic agent. In normal (wild-type) CF-1 mice the abundant P-glycoprotein in the BBB pumps avermectin efficiently out of the brain.3 and 120 mg/kg for the sensitive and insensitive groups. However. such as cortisol.47] A subpopulation of CF-1 mice. the genetic polymorphisms of P-glycoprotein may also represent a major source of individual variability in the potential toxicity and pharmacokinetics of drugs. this protective function is absent.[39. are recognised to be good substrates for P-glycoprotein. They concluded that partitioning of the lipid membrane is the rate-limiting step for the interaction of a substrate with P-glycoprotein. Studies by Ecker et al. Although a wealth of information on the relationship between physicochemical properties of substrates and P-glycoprotein activity has been generated in recent years. Subsequently. The genetic polymorphism of P-glycoprotein was first reported in CF-1 mice by Lankas and Umbenhauer at Merck in 1997. Polymorphisms of P-Glycoprotein Humans are not necessarily created equal in terms of biological make-up. Thus. Similarly.[46. Various efforts have been made to establish the structure-activity relationship for P-glycoprotein substrates. 42 (1) .36] Although most of the drugs transported by P-glycoprotein are basic or uncharged. studies of colchicine and its analogues suggested that the nitrogen atom of the acetamido group at the 7 position was essential for P-glycoprotein recognition. respectively. Because of evolutionary and environmental factors. Based on structural analysis of 100 P-glycoprotein substrates. the better the substrates are for the Pglycoprotein transporter.6 ± 0. Like many cytochrome P450 (CYP) isoenzymes. the surface area and amphiphilic characteristic of the substrate also appear to play a significant role in determining its P-glycoprotein activity. but in mdr1a-deficient mice. genetic polymorphisms of P-glycoprotein in animals and humans have been reported.[35. it is now known that this avermectininduced neurotoxicity is the result of a deficiency in mdr1a P-glycoprotein that normally contributes to a functional blood-brain barrier (BBB).
and 1.51] Recently. The birth defect is attributed to fetal exposure to avermectin. the impact of C3534T polymorphism on drug absorption is still not clear. Additionally. suggests that there are no statistically significant differences in the Cmax and 24-hour area under the curve (AUC24) of digoxin between individuals carrying wild-type C-allele or homozygous mutant T-allele.60] Therefore. A strong association between C3435T allele and G2677A/G2677T alleles was observed when MDR1 polymorphisms were investigated in 100 placentas from Japanese women. while their heterozygote litters (+/-) were less sensitive. who identified a single nucleotide polymorphism (SNP) in exon 26 (C3435T) of MDR1. heterozygotes and homozygotes of G2677A/G2677T mutant allele were 2. The frequency in exon 21 occurred in 58% of the sample as heterozygosity Clin Pharmacokinet 2003.97. The homozygous T-allele (mutant) is associated with more than 2-fold lower intestinal P-glycoprotein expression levels compared with homozygous Adis International Limited.[50. a recent study by Sakaeda et al.45 (arbitrary units). The molecular basis of mdr1a deficiency in CF-1 mice was further studied at the RNA and DNA level. 1..51 (arbitrary units). When female CF-1 mice were treated with avermectin during pregnancy. C-allele (wild type). This insertion results in the aberrant splicing of the mRNA and loss of exon 23 during RNA processing. and 1. C/T and T/T genotypes at position 3435 were 2. The above cases of neurotoxicity and teratogenesis demonstrate the importance of P-glycoprotein in protecting the brain and fetus against toxic xenobiotics. probably through an increase in digoxin absorption as a result of decreased intestinal P-glycoprotein. Kim et al. All rights reserved. Genetic polymorphisms of human P-glycoprotein were first reported from in vitro studies with cancer cells.[53. 61 (93. conflicting results of the effect of C3534T polymorphism on the absorption of fexofenadine have also been reported.54] However. respectively. In contrast. using reverse transcription-polymerase chain reaction (RT-PCR) and long PCR with oligonucleotides specific for mdr1a. However. there appeared to be a correlation between the level of Pglycoprotein expression and G2677T/G2677A in exon 21.[59. fetuses deficient in P-glycoprotein (–/–) were 100% susceptible to cleft palate. The homozygous fetuses (+/+) with abundant P-glycoprotein were totally insensitive at the dose tested. a substrate of Pglycoprotein. Drescher et al. Interestingly.35kb of DNA at the exon 23 intron-exon junction. the kinetic impact of polymorphism on P-glycoprotein function in vivo remained unclear until the recent report by Hoffmeyer et al.84.8%) also had a mutant G2677T/G2677A allele. Sequencing of the intron between exon 22 and 23 in P-glycoprotein-deficient CF-1 mice revealed an insertion of approximately 8. This was the first example that indicated that P-glycoprotein polymorphism can directly affect drug absorption in humans.11. 42 (1) . claimed that there were no significant differences between T/T and C/T genotypes. There was a significant correlation of the SNP and the functional activity of P-glycoprotein. while the corresponding mean P-glycoprotein expression levels for the C/C. The placental P-glycoprotein expression levels for wild-type. showed that individuals harbouring homozygous T-allele mutation tended to have lower plasma AUC24 of fexofenadine.P-Glycoprotein 65 are also at higher risk of birth defects caused by avermectin. RT-PCR studies revealed a deletion mutation of the MDR1 gene in avermectin-sensitive collie dogs. The 4-bp deletion results in a frame shift. and between the P-glycoprotein level and C3435T in exon 26. A subpopulation of collie dogs is also known to be very sensitive to avermectin and it has been speculated that the avermectininduced neurotoxicity in the dogs is due to P-glycoprotein genetic polymorphisms. 1. kinetic studies in healthy subjects and renal transplant patients suggested that C3534T polymorphism had little effect on the absorption of cyclosporin. Similarly. Of 65 samples with a C3435T allele. generating several stop codons that prematurely terminate P-glycoprotein synthesis.44. Individuals carrying homozygous T-allele showed a lower duodenal P-glycoprotein level and consequently higher peak plasma concentrations (Cmax) of digoxin.
42 (1) . The availability of transgenic mdr knockout mice and in vitro cell systems has paved the way for studies of the role of P-glycoprotein in drug absorption and disposition. 1280 subjects from 10 different ethnic groups were evaluated for the C3435T polymorphism in exon 26. including C3435T. It is interesting to note that ivermectin. African-American and Sudanese populations have frequencies of 83. a potent anthelmintic agent used for the prevention and treatment of river blindness (onchocerciasis) in Africa. In contrast. interethnic differences in MDR1 polymorphisms are observed. To date.. respectively. In summary. Similar to the ethnic variation in the polymorphism of CYP. variation in P-glycoprotein expression resulting from MDR1 polymorphism is one of the major sources contributing to interindividual variability in drug absorption and disposition. Hoffmeyer’s group detected both C3435T and G2677A/G2677T polymorphisms with high frequencies in a relatively larger sample population of 461. we have conducted a study to measure in vitro and in vivo P-glycoprotein activity of ten model compounds. The lack of neurotoxicity might be Adis International Limited. Chinese. even though this drug causes neurotoxicity in animals with low P-glycoprotein expression. metabolism and excretion of many drugs. distribution. Given the high frequency for the mutant allele G2677A/G2677T in this study from 100 placentas. 83. not a single case of neurotoxicity has been reported. In Vitro/In Vivo Extrapolation and Species Differences As will be discussed in section 4. The in vitro P-glycoprotein activity of these compounds was determined using mdr1aClin Pharmacokinet 2003. Undoubtedly. To date. for the C/C (wild type) allele. On the other hand. at least 16 SNPs have been identified in the MDR1 gene. and it is anticipated that more SNPs will be found in the future (table I). attributed to the high P-glycoprotein expression in the African population. Because of the importance of P-glycoprotein in pharmacokinetics. When applying in vitro and in vivo screening models to study P-glycoprotein function. Kenyan. and (ii) can animal data be directly scaled to humans? To test whether in vitro P-glycoprotein activity of drugs can be extrapolated to the in vivo situation. it is quite puzzling why no G2677A/G2677T mutant allele was observed from a sample population of 188 in the aforementioned study by Hoffmeyer et al. 3. only three SNPs (T–129C in exon 1b. Although the level of P-glycoprotein expression was not determined in this study. Portuguese. there are two fundamental questions that industrial drug metabolism scientists must confront daily: (i) can in vitro data be accurately extrapolated to the in vivo situation. So far. even though a total of 15 different SNPs. ranging from 34–55%. 84 and 73%.66 Lin & Yamazaki and 28% as homozygosity for the mutant allele. Marked differences in genotype and allele frequency were observed between the African and the Caucasian/Asian populations. Although some variants lead to amino acid changes. is a very safe drug. the high expression of P-glycoprotein might contribute to the high incidence of drug resistance to cancer treatment in individuals of African origin. these results suggest that P-glycoprotein expression in African populations may be higher than that in the Caucasian/Asian populations. Southwest Asian. were identified in their study. most of the detected polymorphisms are intronic or silent. of more than 20 million patients in Africa who had been treated with ivermectin. Recently. the MDR1 gene is highly polymorphic. G2677A/G2677T in exon 21 and C3435T in exon 26) have been demonstrated to be associated with variation in P-glycoprotein expression. while the frequency in exon 26 occurred in 46% of the sample as heterozygosity and 19% as homozygosity for the mutant allele. All rights reserved. The Ghanaian. many pharmaceutical companies have begun to incorporate P-glycoprotein drug transport screening into the drug discovery process. Filipino and Saudi populations have lower frequencies of the C/C allele compared with the African groups. Using a polymerase chain reaction-restriction fragment length polymorphism assay. P-glycoprotein plays an important role in absorption. the British Caucasian.
On the other hand.9 49. All rights reserved.93.0 11. Although the kinetics and substrate specificities are generally similar between mouse mdr1a and mdr1b P-glycoprotein. and a strong correlation between in vitro B-to-A/A-to-B ratio and the in vivo brain AUC ratio was observed again (unpublished data).5 41.5 41.3 48.6 1. 7.3 Mickley et al. and expressed as the ratio of basolateral-to-apical transport to apical-tobasolateral transport (B-to-A/A-to-B).001) when the in vitro B-to-A/A-to-B ratio was plotted against the in vivo brain AUC ratio for these ten compounds. Following intravenous administration. vivo P-glycoprotein activity of a given drug can be reasonably well extrapolated from in vitro data.6 9. transfected LLC-PK1 cells.2 0 0 5.[68-70] species differences in functional activity between human MDR1 P-glycoprotein and these two mouse P-glycoproteins have Clin Pharmacokinet 2003.8 5. and just one in humans (MDR1). In our laboratory. p < 0.7 56. There was a strong positive correlation (r2 = 0. Tanabe et al. The most obvious species differences in the drug-transporting P-glycoprotein between mice and humans is that there are two members of drugtransporting P-glycoprotein (mdr1a and mdr1b) for mice.9 45.3 0.5 (T>C) 5.0 8. the drug concentration in brain and plasma was measured every 15 minutes up to 60 minutes. the in vivo P-glycoprotein activity was determined using CF-1 mdr1a (+/+) and mdr1a (–/–) mice.6 0 38.0 Cascorbi et al.4 (T>C) 0 37.2 9.6 56.5 37. A strong correlation was also observed when the brain concentration was normalised by plasma concentration. 42 (1) .3 16.2 Mutant allele frequency (%) Hoffmeyer et al.6 1.6 0.9 36.5 0.0 0 25. the in vitro and in vivo correlation was further evaluated with an additional 20 compounds.3 1. These results suggest that in Adis International Limited. Mutation A/G C/G T/C G/A A/G G/T G/C T/C C/T C/T G/A C/T C/T T/A A/G G G/T G/A A/G G/A A/C C/T C/T G/C A/G 0.2 6.2 35.8 0 6. Ito et al.0 4.P-Glycoprotein 67 Table I. 9.4 43.4 1. The ratio of brain AUC in mdr1a (–/–) mice to that in mdr1a (+/+) mice was used as an index of in vivo P-glycoprotein activity.6 40.0 0.5 41.9 46.9 5.7 21.1 53. Single nucleotide polymorphisms (SNPs) in the MDR1 gene SNPa 5′-flanking/-41 1a/–145 1b/-129 2/–1 2/61 5/–25 5/–35 5/307 6/+139 6/+145 11/1199 12/1236 12/+44 17/–76 17/+137 21/2677 21/2677 21/2677 24/2956 24/2995 26/3220 26/3396 26/3435 28/4030 28/4036 a Exon/position.
to 20-fold) in MDR1 transfected cells than in mdr1a cells. with the exception of dactinomycin. rat and canine P-glycoprotein. The three transfected cell lines were tested for their cellular resistance (cell survival) to dactinomycin. the apical surface of epithelial cells of placenta and the apical surface of endothelial cells in blood capillaries of the brain. We have recently compared the P-glycoprotein activity of marker P-glycoprotein substrates using cell lines expressing human. In humans. Approximately 35% of the compounds exhibited substantial differences (>3-fold) between mdr1a and MDR1 gene transfected cells. the P-glycoprotein transporter functionally can limit Clin Pharmacokinet 2003. The observed species difference in the P-glycoprotein transport was not due to the differences in the level of the P-glycoprotein expression. A-to-B = apical to basal. mouse provides a very useful model for the study of the role of P-glycoprotein in pharmacokinetics of drugs. B-to-A = basal to apical. the biliary canalicular membrane of hepatocytes. Role of P-Glycoprotein in Pharmacokinetics Although the physiological function for P-glycoprotein is still not fully understood. the mdr1a/1b knockout Adis International Limited. As shown in figure 1. Likewise. significant species differences in P-glycoprotein activity were found among these animal species (unpublished data). Although these in vitro studies strongly suggest the possibility of species differences in P-glycoprotein activity. Species differences in transport activity were also observed between human P-glycoprotein and P-glycoprotein of other animal species.[7. 1. Western blotting data revealed that the P-glycoprotein level was similar in both mdr1a and MDR1transfected cells. extrapolation from knockout mice to humans should be carried out with discretion. Correlation of transcellular transport ratios for 642 structurally diverse compounds in monolayers of LLC-PK1 cells transfected with mouse mdr1a or human MDR1 (L-mdr1a and L-MDR1. the apical surface of epithelial cells of the proximal tubules of kidney. colchicine and vinblastine. 4. However. All rights reserved. the IC50 values were lower in human MDR1 cells than in mdr1b cells. more than 640 compounds have been evaluated for their P-glycoprotein activity in mouse mdr1a and human MDR1 transfected LLC-PK1 cells (L-mdr1a and L-MDR1. These results suggest that there are species differences in functional capacity between human MDR1 P-glycoprotein and murine P-glycoproteins.68 Lin & Yamazaki been reported by Tang-Wai et al. there are still no in vivo data to support the potential of species differences. respectively). 42 (1) . respectively). because of the potential species differences in P-glycoprotein activity. In our laboratory.44). there is a poor correlation between mdr1a and MDR1 transcellular transport activity as measured by B-to-A/A-to-B transport ratio (r2 = 0.8] Because of its strategic localisation. The 50% inhibitory concentrations (IC50 values) for all of the drugs were much lower (3. mouse. the role of this efflux transporter in pharmacokinetics is becoming increasingly appreciated. Again. Pglycoprotein is found on the apical surface of columnar epithelial cells of small and large intestines. mdr1b and MDR1 genes. Stably transfected cells were developed that expressed similar amounts of P-glycoprotein encoded by mdr1a. doxorubicin. As will be discussed in section 4. 120 B-to-A/A-to-B in L-MDR1 (human) 100 80 60 40 20 0 0 20 40 60 80 100 120 B-to-A/A-to-B in L-mdr1a (mouse) Fig.
the relative contribution of the transcellular pathway was determined to be 25. Therefore. it is very important to distinguish the localisation of P-glycoprotein in cells in relation to drug movement – either uptake of drugs into cells or excretion of drugs out of cells.4 and 3. which can be broadly categorised as physicochemical and biological factors. the logP values of which were –0. drug absorption occurs predominantly within the small intestine. Most orally administered drugs enter the systemic circulation by passive transcellular diffusion. have reported that both the protein expression and catalytic activity of CYP is significantly increased in the mdr1a. and from the gastrointestinal lumen into the enterocyte. The values correlated fairly well with the lipophilicity of the drugs. which are expressed in a tissue-specific manner. Therefore. or a combination of both. During oral absorption. 4.2.6. for the ge Adis International Limited. netically identical mdr1a and mdr1a/1b knockout mice housed in the US. because of their lipophilicity. Recently. which implies that lipophilic drugs are likely to be P-glycoprotein substrates. molecular size. Since mdr knockout mice became available. The relative contribution of the transcellular pathway to overall absorption is highly dependent on the lipophilicity of drugs. Schuetz et al. only mdr1a P-glycoprotein is expressed in the brain and intestine of mice. 42 (1) . For example. As will be discussed later. this transporter can also enhance the elimination of drugs from hepatocytes. drugs can be transported by either the transcellular or paracellular pathway across the epithelial cells. 85 and 99% for chlorothiazide. renal tubules and intestinal epithelial cells into the adjacent luminal space. All rights reserved. Because of tissuespecific expression. As noted earlier. mdr1b and mdr1a/1b knockout mice housed in The Netherlands. respectively. there is a tendency for P-glycoprotein to have a greater impact on drug uptake than on drug excretion. it is known that the mdr1b gene is upregulated in mdr1a knockout mice. In an in vitro study with Caco-2 cells. For example. In addition. such as pKa. it should be noted that genetic disruption of one or both of the mdr genes might affect the expression and function of other transporter systems or even drug-metabolising enzyme systems in mice. it is expected that genetic disruption of the mdr1a gene would have a greater impact on drug uptake into the brain and intestine than drug excretion from the liver and kidney. Therefore. Interestingly.P-Glycoprotein 69 cellular uptake of drugs from the blood circulation into the brain and placenta. –0.1 Drug Absorption There are many factors that influence the bioavailability of drugs. there were no significant changes in their protein expression and catalytic activity of CYP. 45. mucosa blood flow rate and first-pass metabolism. absorption Clin Pharmacokinet 2003. mice have two types of drug-transporting P-glycoprotein (mdr1a and mdr1b). respectively. 0. because of its large surface area provided by epithelial folding and the villous structures of epithelial cells. However. suggesting that mdr1a and mdr1b together fulfil the same function as the single P-glycoprotein in humans. On the other hand. and the latter include gastric and intestinal transit time.08. data derived from mdr1a single knockout mice or from mdr1a/1b double knockout mice have to be interpreted with caution.[74. while both mdr1a and mdr1b P-glycoprotein are expressed in the liver and kidney. Perhaps the most important milestone in P-glycoprotein research was the development of mdrknockout mice. lumen pH.75] The former comprise the intrinsic properties of the drug. As discussed above. the intestinal absorption of a drug is often predicted on the basis of its lipophilicity. mdr1a and mdr1b P-glycoprotein together appear to cover the same tissues as the single human MDR1 P-glycoprotein. our understanding of the role of P-glycoprotein in pharmacokinetics has increased exponentially. cimetidine and propranolol. After oral administration. membrane permeability. lipophilicity and solubility. Because of the possible existence of unrecognised factors that are associated with the genetic disruption. furosemide. the most common physicochemical property for P-glycoprotein substrates identified so far is that they are mostly lipophilic.
However. Because P-glycoprotein is expressed exclusively in mature epithelial cells in the villous tip of intestinal mucosa. The oral AUC of cyclosporin was in the rank order stomach > jejunum > colon. All rights reserved. There was a negative correlation between MDR1 mRNA expression and oral AUC of cyclosporin. The results from this study indicate that there is a significant interindividual variability in the intestinal P-glycoprotein expression. The distribution of P-glycoprotein is also not uniform along the length of intestine. The influence of uneven distribution of P-glycoprotein in intestine was demonstrated in a clinical study with cyclosporin. ranging from 30–60% of the dose in 30 minutes. a portion of drug molecules will be removed by the efflux Pglycoprotein transporter out of cells back into the lumen and another part of the drug molecules is subject to intestinal metabolism. Whether the programmed life span and rapid migration will have impact on the regulation of intestinal P-glycoprotein expression is an open question.2 Interindividual Variability of Intestinal P-Glycoprotein Although interindividual variability in drugmetabolising enzymes is well documented. which is highly expressed on the apical surface of epithelial cells. The levels of mRNA appear to increase progressively from the stomach to the colon Adis International Limited. Consequently. Unlike hepatocytes. a fraction of the drug molecules continues to diffuse along the concentration gradient into capillary blood. jejunum and colon). expression level of intestinal P-glycoprotein was measured using immunoblotting. as an internal standard. In the intestinal lumen. More than 8-fold differences in the P-glycoprotein expression were observed in a small popuClin Pharmacokinet 2003. 42 (1) .1 Distribution of Intestinal P-Glycoprotein The distribution of intestinal P-glycoprotein is not uniform among cells along the epithelial villi. The villous epithelial cells are mature and nondividing. The uneven distribution of intestinal P-glycoprotein is expected to have a significant impact on the absorption of P-glycoprotein substrates. Once inside the cells. limited life span.1. the investigators used villin. with a low level in the stomach (5 arbitrary units). have measured the content of MDR1 mRNA expression over the total length of human gastrointestinal tract. a constitutively expressed protein in mature epithelial cells. vinblastine. which regenerate only when untimely death occurs. Biopsy specimens were obtained from the second portion of the duodenum of each patient. Cyclosporin was given to ten healthy volunteers at different parts of the gastrointestinal tract (stomach. Uneven distribution of P-glycoprotein has also been observed in rats. suggesting a high level of P-glycoprotein expression in rat jejunum. an intermediate level in the jejunum (20 arbitrary units) and a high level in the colon (30 arbitrary units). differences in the number of total mature cells in individual biopsies might contribute to the variability. whereas the crypt cells continue to mature as they ascend toward the villus and are extruded at its tip. To correct this practical problem. In a clinical study of 25 kidney transplant recipients. and requires further investigation. Immunohistological studies with human jejunum and colon using MRK16 antibody revealed that high levels of P-glycoprotein were only observed in the apical surface of columnar epithelial cells. The time required for migration from the crypt base to the villous tip has been estimated to be 2– 6 days. 4. Using the rat intestinal loop technique. was absorbed fairly well from ileal loops.1. only a few papers deal with the issue of interindividual variability of P-glycoprotein. Fojo et al.70 Lin & Yamazaki of drugs is further complicated by the existence of P-glycoprotein efflux transporter. the net amount of drug absorbed into the mesenteric blood circulation is the difference between the amount absorbed by the influx process and the summation of the amount extruded by efflux transport together with the amount metabolised by enzymes. 4. but not in crypt cells. a well-known P-glycoprotein substrate. whereas absorption of vinblastine from the jejunal loop was almost negligible. drugs that are P-glycoprotein substrates will be absorbed and cross the epithelial cell membrane by simple diffusion. intestinal epithelial cells have a programmed.
studies with Caco-2 cells revealed that active B-to-A transport of peptides was inhibited by verapamil. and T/T homozygotes (n = 5) at position 3435 were 1275. suggesting the involvement of P-glycoprotein in the absorption of peptides. Collectively.3 Evidence of Intestinal P-Glycoprotein Involvement in Drug Absorption Evidence of the involvement of intestinal Pglycoprotein in drug absorption was first demonstrated in vitro with Caco-2 cells in which P-glycoprotein was highly expressed. n = 6). P-glycoprotein efflux function. it is clear that intestinal P-glycoprotein does limit drug absorption by extruding drugs from epithelial cells back into the intestinal lumen. Similarly. Although complete P-glycoprotein deficiency has not been reported for these polymorphisms.and 20-fold.[83.1. At least 16 SNPs have been identified (table I). The variation in P-glycoprotein expression inversely related fairly well to the variation of tacrolimus concentrations after oral administration. the B-to-A transport of cyclosporin was much greater than the A-to-B transport. All rights reserved. there was a 4-fold variation in MDR1 mRNA expression level and a 2-fold variation in trough plasma concentration of tacrolimus. the SNP at 3435 in exon 26 does influence the expression level of intestinal P-glycoprotein.84] Caco-2 cells have also been used to study the intestinal transport of cyclosporin.P-Glycoprotein 71 lation of 25 patients. These results from in vitro studies clearly suggest that P-glycoprotein plays a significant role in drug absorption by limiting drug transport from intestinal lumen. As discussed earlier. 4. greater than the A-to-B transport. these results suggest that interindividual and intraindividual variability in intestinal P-glycoprotein expression may contribute to variability of oral absorption of drugs that are Pglycoprotein substrates. Both the mRNA expression and plasma concentration of tacrolimus were measured periodically during the immunosuppressant therapy. Based on the AUC values after intravenous and oral administration. 956 and 627 (arbitrary units). 42 (1) . C/T heterozygotes (n = 10).and 6-fold. Furthermore. The increased AUC of paclitaxel after intravenous administration in mdr1a (–/–) mice reflected a decrease in elimination clearance. Direct evidence for the role of intestinal P-glycoprotein in drug absorption was derived from in vivo studies with mdr1a (–/–) knockout mice. higher in mdr1a (–/–) mice than mdr1a (+/+) mice after intravenous and oral drug administration. respectively. Interestingly. In a period of 120 days. the B-to-A transport of vinblastine and docetaxel was 10. The efflux function of intestinal P-glycoprotein is further supported by the observations that a sigClin Pharmacokinet 2003. respectively. The mean values of intestinal P-glycoprotein expression for the C/C homozygotes (wild type. A profound intraindividual variability in intestinal P-glycoprotein expression (mRNA) was observed in a young patient during tacrolimus therapy after small bowel transplantation. respectively. The plasma AUC of paclitaxel was 2. the bioavailability of paclitaxel was calculated to be 11 and 35% for mdr1a (+/+) and mdr1a (–/–) mice. Further evidence for the involvement of intestinal P-glycoprotein in drug absorption in humans is provided by the clinical study by Hoffmeyer et al. Using Caco-2 cells. the MDR1 gene is highly polymorphic. and the A-to-B transport was enhanced significantly in the presence of verapamil and MRK16 by blocking the Adis International Limited. the intestinal P-glycoprotein level of duodenal biopsies ranged from 31–263 (arbitrary units).. The A-to-B cyclosporin transport increased and the B-to-A transport decreased after treatment with the P-glycoprotein inhibitors progesterone and chlorpromazine. The oral absorption of paclitaxel was studied in mdr1a (–/–) and mdr1a(+/+) mice. From this study. intraindividual variability in intestinal P-glycoprotein expression has also been reported. respectively. who showed a negative correlation between duodenal P-glycoprotein expression and plasma level of digoxin. whereas the higher AUC after oral administration in mdr1a (–/–) mice resulted from a combination of a decrease in the elimination clearance and an increase in the extent of drug absorption from intestinal lumen.
the inhibition study can only provide a qualitative assessment as to whether P-glycoprotein is involved in drug absorption. In a clinical study. respectively.4 and 1. and increased to 50% when cyclosporin was coadministered. In a clinical study. P-glycoprotein as an intestinal barrier in limiting the absorption of paclitaxel and docetaxel. Pglycoprotein-mediated intestinal excretion in mice was also reported for digoxin. concluded that intestinal P-glycoprotein plays a significant role in the absorption of cyclosporin. jejunum/ileum and colon) in a crossover manner. The bioavailability of docetaxel in cancer patients increased from 8% without cyclosporin to 88% in combination with cyclosporin. Another way by which evidence can be shown for intestinal P-glycoprotein involvement in drug absorption is to establish the correlation between the absorption profile (oral AUC) of drugs and the expression of intestinal P-glycoprotein (or mRNA). Moreover. Because cyclosporin is known to be a potent P-glycoprotein inhibitor. Similarly.2. it is difficult to estimate the quantitative contribution of P-glycoprotein to drug absorption by the inhibition approach. The effect of P-glycoprotein on intestinal excretion can be experimentally determined in mice after interruption of bile flow by gallbladder cannulation. Lown et al. was excreted into the intestinal lumen of mdr1a (+/+) and mdr1a (–/–) mice with a cannulated gallbladder within 90 min after intravenous administration. Similarly. involvement of P-glycoprotein in intestinal excretion has only been recognised recently. based on the observation that a highly significant correlation exists between enterocyte P-glycoprotein content and cyclosporin absorption kinetics. cyclosporin is a potent inhibitor of other transporters.72 Lin & Yamazaki nificant amount of paclitaxel was excreted directly from blood circulation into intestinal lumen after intravenous administration. the role of intestinal Pglycoprotein in drug absorption in humans is often derived indirectly from inhibition studies. Although the phenomenon of intestinal excretion of drugs has been known for more than two decades. There was a strong negative correlation between the plasma AUC of cyclosporin after administration at different locations of the gastrointestinal tract and the local mRNA expression of intestinal P-glycoprotein. Unlike the direct evidence obtained from mdr1a knockout mice. 3. A fraction of 11% of the dose was excreted into the intestinal lumen within 90 minutes after intravenous administration of paclitaxel to mdr1a (+/+) mice with a cannulated gallbladder.5 µmol/L. respectively. five patients received a safe oral dose of paclitaxel 60 mg/m2.5% of the dose in the intestinal lumen of mdr1a (–/–) mice. Clin Pharmacokinet 2003. but only 2. Due to the lack of a specific P-glycoprotein inhibitor. leukotriene C4 (cMOAT) and daunorubicin (P-glycoprotein) were 0. Since cyclosporin is also an inhibitor of CYP3A4 and other CYP enzymes. All rights reserved. From a mechanistic point of view. Approximately 16 and 2% of the digoxin dose. Thus. it is likely that the observed increase in oral bioavailability of these drugs could also be partly attributed to reduced metabolism by the inhibition of CYP enzymes by cyclosporin. Clearly. these results suggest the involvement of Adis International Limited. intestinal excretion should also be considered as an additional pathway for the elimination clearance of P-glycoprotein substrates. Therefore. the increased bioavailability of paclitaxel and docetaxel caused by cyclosporin cannot be explained by P-glycoprotein inhibition alone. Similar results were found in another clinical study with docetaxel. with a washout period between the administrations of at least 7 days. the involvement of MDR1 P-glycoprotein in drug absorption is difficult to prove directly in humans. such as canalicular bile salt transporter and canalicular multispecific organic anion transporter (cMOAT). cyclosporin was given by gavage to ten male volunteers at different parts of the gastrointestinal tract (stomach. 42 (1) . and nine other patients received the same oral dose of paclitaxel combined with one single oral dose of cyclosporin 15 mg/kg. The inhibitory Ki values of cyclosporin for taurocholate (bile salt transporter). The oral bioavailability of paclitaxel when given without cyclosporin was less than 5%.
There is a widespread misconception that the extent of oral absorption of a drug is always markedly limited by intestinal P-glycoprotein when the drug is a P-glycoprotein substrate. It is evident that the P-glycoprotein-mediated efflux and CYP-mediated metabolism are saturable processes.4 Saturable Efflux Transport by Intestinal P-Glycoprotein Like that of drug-metabolising enzymes. All rights reserved. The extent of cyclosporin absorption increased with increasing doses in rats. saturable P-glycoproteinmediated efflux has also been reported for cyclosporin in rats. rat colon and human colon. However. rat ileum. concentration-dependent permeability across Caco2 cell monolayers was observed when the concentration of talinolol was increased from 0. These results indicate that P-glycoprotein-mediated efflux transport can be saturated when higher oral doses are given. Therefore. it should be noted that a good correlation itself does not prove a causal relationship. at least in part. Collectively. The Km values for digoxin were 81. explain the observed dose-dependent absorption of talinolol (a P-glycoprotein substrate) in healthy volunteers.4 µmol/L). At these doses. which is well below the Km value (58 µmol/L) derived from Caco-2 cells or human colon. the conclusion from these two correlation studies that P-glycoprotein transport is involved in the absorption of cyclosporin appears valid and appropriate. 42 (1) . these results strongly suggest that the efflux function of intestinal P-glycoprotein may be saturated when drug concentrations in the intestinal lumen exceed the Km values after high oral doses.8 µmol/L.1–2 mmol/L. respectively. By using the Ussing chamber technique. a well-known P-glycoprotein substrate. the efflux of vinblastine and digoxin by P-glycoprotein have been demonstrated to be saturable. Interestingly. A similar Km value for vinblastine (18 µmol/L) was reported by other investigators using Caco-2 cells. which undergoes minimal metabolism. This is only true for a few P-glycoprotein substrate drugs that are given at low doses. For both enantiomers. In a recent kinetic study involving Caco-2 cells. Similar results were observed for (R)-(+)talinolol. is given orally at a very low oral dose of 0. the bioavailability increased from 13% at an oral dose of 6 mg/kg to 25% at 18 mg/kg. the functional activity of P-glycoprotein is saturable. This Km value for cyclosporin was comparable to that found in other cell lines expressing human P-glycoprotein (8.5 to 1mg. The dose-normalised AUC of (S)-(–)-talinolol increased from 18 µg • h/L at a 12. the Michaelis-Menten constants (Km values) for vinblastine and digoxin were 26 and 58 µmol/L. The saturable P-glycoprotein efflux may. transport of cyclosporin was shown to be saturable with a Km of 3. 4.5mg dose to 36 µg • h/L at a 200mg dose. the dosenormalised AUC increased with increasing doses after oral administration. the transport of digoxin was studied in human and rat intestinal tissues.P-Glycoprotein 73 With several lines of supportive evidence. The reported low and variable absorption of digoxin can most probably be attributed to the efflux transport of intestinal P-glycoprotein. it is highly desirable to have other supportive in vitro and/or animal data when applying the correlation approach.1. Consistent with the in vivo observations. the Km values for digoxin derived from human colon tissue and Caco-2 cells are almost identical (59 vs 58 µmol/L). Similarly. As discussed previously. Clin Pharmacokinet 2003. efflux transport together with the amount metabolised by enzymes. Digoxin. for drugs that are Pglycoprotein substrates the net amount of drug passing through the intestinal epithelial cells is the difference between the amount absorbed by influx processes (passive diffusion and/or active uptake) and the summation of the amount extruded by Adis International Limited. 74. Saturable transport for digoxin was also observed in both human and rat intestinal tissues. Thus. 51 and 59 µmol/L. Using Caco-2 cells. for rat jejunum. the concentration of digoxin in intestinal lumen is estimated to be less than 10 µmol/L. respectively. Absorption of digoxin is a good example. P-glycoprotein plays a quantitatively significant role in the absorption of digoxin.
82. in spite of being good P-glycoprotein substrates. Indinavir. an HIV protease inhibitor. Apparent values of the Michaelis-Menten constant (Km) for P-glycoprotein substrates Compound Cyclosporin Digoxin Material (flux evaluated) Caco-2 (net B-to-A) Caco-2 (net B-to-A) Stripped rat jejunum (net B-to-A) Stripped rat ileum (net B-to-A) Stripped rat colon (net B-to-A) Stripped human colon (net B-to-A) Etoposide Caco-2 (B-to-A) Stripped rat jejunum (B-to-A) Stripped rat colon (B-to-A) Indinavir Verapamil Caco-2a (net B-to-A) Stripped rat jejunum (B-to-A) Stripped rat ileum (B-to-A) Stripped rat colon (B-to-A) Vinblastine Caco-2 (net B-to-A) Caco-2 (net B-to-A) Stripped rat ileum (net B-to-A) Stripped rat colon (net B-to-A) a Treated with calcitriol. However. but clinical studies clearly indicate that P-glycoprotein does play a significant role in limiting their oral absorption. even though it is a good P-glycoprotein substrate.101] This can be explained by the fact that both cyclosporin and paclitaxel have very poor water solubility. Given the low Km values for P-glycoprotein drugs. This can explain why indinavir has a reasonably good bioavailability (>60%) in patients. is a Pglycoprotein substrate and is given orally at a dose of 800mg.74 Lin & Yamazaki However.87. Apparent Km (µmol/L) 3. The notion of slow dissolution rate and/or slow membrane diffusion rate of cyclosporin in intestine is supported by the fact that peak concentrations of the drug occur slowly at 3–4 hours after administration to patients. even when they are given at high doses.84 93 93 93 Adis International Limited. Interestingly. slow dissolution rate and large molecular weight (1202 for cyclosporin and 854 for paclitaxel). and hence the role of intestinal P-glycoprotein in drug absorption becomes quantitatively less significant. the oral dose for most drugs is high (>50mg) and drugs in the intestinal lumen can easily reach the mmol/L concentration range. A detailed literature survey revealed that all of the reported Km values for P-glycoprotein drugs are relatively low. ranging from 4–213 µmol/L (table II). The poor water solubility and slow dissolution rate can result in low drug concentration in the intestinal lumen in relation to their Km value for P-glycoprotein transport. B-to-A = basal to apical. P-glycoprotein activity can readily be saturated when drugs are administered at high doses. Clin Pharmacokinet 2003. and the large molecular size can impede the rate of passive diffusion across the cell membranes. Therefore.8 58 81 74 51 59 213 94 119 140 31 29 4. at high doses. At this high dose. Table II. there are exceptions that intestinal Pglycoprotein still plays a significant role in absorption for some drugs. in a recent literature survey.4 19 27 48 ~100 References 79 93 93 93 93 93 100 100 100 97 100 100 100 83. 42 (1) . Chiou et al. the indinavir concentration in the intestinal lumen is expected to be greater than 1 mmol/L. All rights reserved. For example. the clinical oral dose is 200–700mg for cyclosporin and 100–200mg for paclitaxel. which is much higher than the Km value for P-glycoprotein transport (140 µmol/L) derived from Caco-2 cells. the effect of intestinal P-glycoprotein on indinavir absorption becomes quantitatively less important.[79. have concluded that the in vivo oral absorption of 13 drugs is not significantly impeded by efflux transport.
such as hydrophobicity.[8. drug distribution has historically received much less attention than the other processes. molecular size and number of hydrogen bonds. several in vitro systems have now been developed for studying drug distribution. The physical and biochemical properties of membranes. the lower the permeability. A strong positive correlation between lipophilicity and brain penetration of drugs has been reported by many investigators. In addition. a negative correlation was found between the BBB permeability of lipophilic compounds (steroid hormones and peptides) and the total number of hydrogen bonds. However. with recent advances in the molecular biology and biochemistry of transporter systems. the importance of drug distribution is becoming increasingly recognised. All rights reserved. such as lipid bilayer structure and dynamics. or the dissolution and/or membrane diffusion rates of the drug are very slow. the greater the total number of hydrogen bonds.6.2. in spite of its importance as a key factor in determining drug response. leaving no space between cells. The lack of attention stems partly from a lack of useful experimental tools in studying drug distribution. For example.106] In addition. Levin reported a good correlation between the in vivo BBB permeability coefficient of 22 compounds and their lipophilicity. ionisation profile. However.107] In addition. it is clear that the effect of intestinal P-glycoprotein on drug absorption is unlikely to be quantitatively important unless a very small oral dose is given. despite relatively high lipophilicity (logP value of 2. These newly developed tools and experimental methodologies will Adis International Limited. the drug must be absorbed and transported from the site of administration across several biomembranes to reach the target tissue and the site of action. For example. 4. To be effective. many other lipophilic compounds also exhibit poor BBB penetration. they found that vincristine and epipodophylotoxin displayed poor BBB permeability. 42 (1) . Consequently. All other organs are perfused by capillaries lined with endothelial cells that have small pores to allow for movement of drugs into the organ interstitial fluid from the circulation. Additionally. the physicochemical properties of drugs. One of the most important features is that the brain is anatomically separated from the blood circulation by the BBB. Because of this lack of attention. Although extensive efforts have been made to study the molecular mechanisms of the processes of drug absorption. the molecular size of drugs is also an important determinant for brain penetration. In a rat study. However.[105. metabolism and excretion. drug distribution has been regarded as a forgotten relative in clinical pharmacokinetics. The poor BBB penetration of these drugs cannot be explained by the number of hydrogen bonds and Clin Pharmacokinet 2003. Penetrating cell membranes is a complex process that is highly dependent on the nature of the membrane and the physicochemical properties of the drug. the endothelial cells in brain capillary blood vessels are closely joined to each other.P-Glycoprotein 75 In conclusion. Schinkel et al. and it has poor brain penetration.2 Drug Distribution certainly provide important insights into the mechanisms of drug distribution in the very near future. factors other than lipophilicity may also play an important role in the transport of drugs across the BBB.9] demonstrated large differences in drug distribution into the brain and other tissues between mdr1a (–/–) and mdr1a (+/+) mice.[105. also play a significant role in membrane penetration. 4. Although lipophilicity is an important factor in determining the BBB penetration of drugs. In addition. play an important role in drug penetration. The brain is different from other organs of the body in many aspects.8). many lipophilic drugs have exhibited poor BBB penetration. For example. only lipophilic drugs can cross endothelial cells and enter the BBB by way of passive diffusion. a potent CCKB receptor antagonist candidate is a lipophilic compound with a logP value of 3.1 Blood-Brain Barrier Lipophilicity and Brain Penetration Drugs are often administered at a location distant from their intended site of action.
Similarly. and Cordon-Cardo et al. In this study. these compounds were regarded as ‘outlier compounds’ for brain penetration without knowing the exact cause. Adis International Limited. a recent study by Decleves et al. The apparently discontinuous and abluminal localisation of MRK16 staining in isolated brain capillaries and the similarity in immunostaining patterns by MRK16 and anti-GFAP antibodies led the investigators to conclude that P-glycoprotein is expressed in astrocyte foot processes. (b) an antiserum to GFAP and (c) an antiserum to GLUT1. Pardridge et al. using P-glycoprotein and GFAP double-immunolabelling technique. using a dual immunostaining approach in combination with confocal microscopy. suggesting a very minor contamination of astrocytes in the luminal membrane preparations. have demonstrated that P-glycoprotein is localised in the brain capillaries of rats. Localisation of P-Glycoprotein in Brain Localisation of P-glycoprotein in the brain is a widely disputed issue that has become the centre of much controversy in recent years. Using monoclonal antibodies. The possible efflux function of P-glycoprotein in the BBB was not connected with the observed poor BBB permeability of lipophilic drugs until the findings of P-glycoprotein in brain capillaries by Thiebaut et al. Western blotting analysis revealed much higher expression level of P-glycoprotein in endothelial cells compared with astrocytes. Furthermore. In this study. Although most immunohistochemical studies indicate that P-glycoprotein is predominantly localised on the surface of endothelial cells facing the luminal side. The conclusion was further supported by their subsequent findings that staining of the endothelial membrane marker protein GLUT1 was continuous and showed only minimal overlap with Clin Pharmacokinet 2003. it is now clear that the observed poor BBB permeability of those lipophilic drugs is due mainly to the efflux function of P-glycoprotein. Twenty years ago. Beaulieu et al. Enrichment of GLUT1 and P-glycoprotein relative to whole brain membrane preparations was 280. was enriched only 1. a specific marker for astrocytes. However.9-fold enrichment of the endothelial membrane marker protein GLUT1 (glucose transporter 1) and a 17-fold enrichment of P-glycoprotein relative to isolated brain capillaries. claim that Pglycoprotein is localised mainly to astrocyte foot processes. the investigators were able to selectively isolate the luminal membrane of endothelial cells of rat brain capillaries. they demonstrated that P-glycoprotein is highly expressed on the apical surface of the endothelial cells of the brain capillaries. They observed that the P-glycoproteinspecific antibody MRK16 bound to microvessels with a similar. discontinuous staining pattern as an antiserum directed against GFAP. whereas both mdr1a and mdr1b mRNA were detected in endothelial cells. Barrand et al. Matsuoka et al. provided very convincing evidence for Pglycoprotein being predominantly localised in the luminal membrane of endothelial cells of rat brain capillaries facing blood circulation. respectively. All rights reserved.76 Lin & Yamazaki molecular weight. concluded that endothelial marker C219 staining did not colocalise with astrocyte marker GFAP staining in rat brain microvessels. glial fibrillary acidic protein (GFAP).4-fold relative to brain capillary. Using a novel technique with cationic colloidal silica. With these findings. these results consistently suggest that brain P-glycoprotein is predominantly expressed on the apical surface of endothelial cells of capillaries. Consistent with Beaulieu’s results. The co-enrichment of P-glycoprotein (500-fold) and GLUT1 (280-fold) in brain capillary luminal membranes compared with whole brain membrane preparations strongly suggests that P-glycoprotein is expressed predominantly in the luminal membrane of brain endothelial cells. 42 (1) . showed that P-glycoprotein is expressed in both cultured rat endothelial cells and astrocytes. RT-PCR analysis showed that mdr1b mRNA was preferentially expressed in astrocytes. rather than in endothelial cells.. They conducted an immunochemical study with human brain microvessels using (a) the MRK16 antibody to human P-glycoprotein. The isolation procedures resulted in a membrane preparation with a 9. Collectively.and 500-fold.
 showed that a deficiency of P-glycoprotein in mice resulted in the same degree of increase in drug concentrations of rhodamine-123 (a P-glycoprotein substrate) in the brain as well as ISF. 42 (1) . but would cause a decrease in the drug concentration in the interstitial fluid (ISF) of brain. Consistent with these findings. Golden and Pardridge have proposed a revised kinetic model of P-glycoprotein in the brain in contrast to the classic kinetic model. vestigators. a potent P-glycoprotein inhibitor. These results appear to be consistent with the classic concept that in mice the P-glycoprotein transporter is expressed in the brain capillary endothelial cells. the EC50 (concentration producing half-maximal antinociception effect) of DPDPE was 13 times lower in mdr1a (–/–) mice compared with wild-type mice (12 versus 160 ng/g). Furthermore. Recently. Based on brain concentrations. the dose required to elicit comparable antinociception was more than 30-fold lower in mdr1a (–/–) mice compared with mdr1a (+/+) mice. Kinetic studies showed that efflux transport of vincristine from the bovine brain endothelial cells was inhibited by verapamil. a deficiency (or inhibition) of P-glycoprotein would result in an increase in BBB permeability as well as an increase in drug concentration in the ISF of brain. have demonstrated that P-glycoprotein is expressed and functional in brain microglia. They showed by immunochemical studies that P-glycoprotein was Clin Pharmacokinet 2003. P-glycoprotein has also been detected in mixed glial cells. according to the classic model. de Lange et al. These results suggest either differences in DPDPE distribution within the brain or differences in the intrinsic activity of δ-opioid receptors between mdr1a (–/–) mice and mdr1a (+/+) mice. Similar results were also reported by Tsuruo and colleagues with mouse brain capillary endothelial cells. but not due to differences in intrinsic response. resulting in a significant increase in intracellular drug concentration.to 4-fold higher in mdr1a (–/–) mice than in mdr1a (+/+) mice after intravenous administration. Immunostaining with a P-glycoprotein antibody (MRK16) demonstrated an exclusively apical localisation of P-glycoprotein in the cultured bovine brain endothelial cells.5]enkephalin (DPDPE) in mdr1a (–/–) and mdr1a (+/+) mice demonstrated by Chen and Pollack. According to the revised kinetic model of Golden and Pardridge. Using an intracerebral microdialysis technique. All rights reserved. the rhodamine-123 concentrations in ISF were also about four times higher in mdr1a (–/–) mice than in mdr1a (+/+) mice. the accumulation of digoxin by microglia was significantly enhanced by valspodar (PSC-833). Lee et al. Although the brain concentrations of DPDPE were 2. but not in primary cultured neurons. On the other hand. Similarly. by other in Adis International Limited. immunocytochemistry studies revealed the location of P-glycoprotein along the nuclear envelope and plasma membrane of microglia. Evidence of P-Glycoprotein Involvement in Brain Uptake The first experimental evidence that P-glycoprotein is involved in drug transport in the BBB came from Tsuji and coworkers.P-Glycoprotein 77 MRK16 staining. After an intravenous infusion of rhodamine-123. The expression of P-glycoprotein in glial cells may partly explain the intriguing pharmacokinetics and pharmacodynamics of [Dpenicillamine2. the total brain concentrations were about four times higher in the mdr1a (–/–) mice compared with wild-type mice. Using a continuous rat brain microglia cell line (MLS-9). With their observations. a deficiency (or inhibition) of P-glycoprotein would have no effect on BBB permeability. These results are consistent with the notion that P-glycoprotein is expressed in other type of brain cells in addition to the endothelial cells of capillaries. Therefore. comparison of brain uptake of P-glycoprotein substrates in normal and P-glycoprotein-deficient mice can be used to address the question of whether the P-glycoprotein transporter is localised in the capillary endothelial cells or in the astrocytes. Subsequent pharmacokinetic and pharmacodynamic modelling suggested that the difference in antinociception between mdr1a (–/–) and mdr1a (+/+) mice was due to the distribution of DPDPE within the brain as well as between the blood and brain.
 These in vitro studies provide evidence of functional involvement of P-glycoprotein in the BBB penetration of drugs. Experiments performed using fluorescein and fluorescein-dextran-4000 as integrity markers showed that there were no differences in brain/plasma concentration ratio of these compounds between mdr1a (–/–) and wild-type mice. increases in drug concentrations in the liver and kidney are also much lower than that in the brain. markedly higher brain levels of radioactivity (17and 55-fold. when [3H]digoxin Adis International Limited. the mdr gene in mice is expressed in a tissue-specific manner.43 µmol/g brain). kidney. and [3H]cyclosporin were given intravenously. the ATP content in the rats with transient brain ischaemia was only 3% of that in normal rats (0. while only mdr1a gene is expressed in the brain of mice. small intestine and plasma of mdr1a (–/–) mice. indicating maintenance of BBB integrity in the absence of P-glycoprotein. The unidirectional transport of vincristine from basolateral side to apical side was demonstrated in the polarised monolayer of mouse endothelial cells. because the BBB permeability decreases when P-glycoprotein function is impaired. small intestine and plasma were increased by less than 4-fold. It is puzzling why the most marked increase in drug concentration of P-glycoprotein substrates in mdr1a (–/–) knockout mice is always observed in the brain.3 and 1. only a moderate increase in radioactivity levels (2to 3-fold) of these two drugs was observed for liver. efflux transport of cyclosporin was also observed in cultured endothelial cells of bovine and mouse brain capillaries. are relatively modest. being 2.5fold increase in digoxin concentration in the liver.04 versus 1. At first glance.[126. kidney and plasma of double knockout mice. These results from the ATPdepleted rat studies are consistent with the classic concept that the P-glycoprotein is expressed in the brain capillary endothelial cells. As mentioned earlier.[123. whereas the levels in liver. A 27-fold increase in the brain concentration of digoxin was observed in mdr1a/1b (–/–) double knockout mice compared with the wild-type mice. Oral administration of [3H]ivermectin in mdr1a (–/–) and mdr1a (+/+) mice resulted in 87-fold higher levels of radioactivity in the brain of mdr1a (–/–) mice as compared with wild-type mice. Similarly. even in mdr1a/1b (–/–) double knockout mice. an increase in the BBB permeability coefficient of cyclosporin in ATP-depleted rats was also reported by Tsuji’s group.124] Immunostaining with P-glycoprotein antibody also demonstrated an exclusively apical localisation of P-glycoprotein in human brain endothelial cells. while the increases in drug concentration in other tissues in which P-glycoprotein is also highly expressed. The knockout animal model of mdr1a(–/–) mice also provides a powerful tool for studying brain uptake of drugs. The marked increases in drug concentration in the brain of P-glycoprotein-deficient mice also cannot be explained by the loss of BBB integrity.4%. 42 (1) . respectively) were observed in mdr1a (–/–) mice than in mdr1a (+/+) mice. They demonstrated that the BBB permeability coefficient of doxorubicin increased from 14 µl/min/g brain in control rats to 243 µl/min/g brain in rats ATPdepleted by occlusion of vertebral and common carotid arteries.127] the involvement of ATP in P-glycoprotein-mediated transport was first demonstrated in vivo by Tsuji and colleagues. However. respectively. Similarly.78 Lin & Yamazaki localised on the apical surface of endothelial cells of mouse brain. with only a 2. All rights reserved. The underlying mechanism for the marked increases in brain concentration remains Clin Pharmacokinet 2003. The BBB permeability coefficient of doxorubicin was determined by using in situ brain perfusion technique. Under the experimental conditions. kidney. one might assume that the less profound increases in drug concentration in the liver and kidney in mdr1a (–/–) mice are due to the extra protective functions of mdr1b P-glycoprotein in these tissues. such as liver and kidney. In another study. The large differences in brain concentration were also observed between mdr1a/1b (–/–) double knockout and normal mice. in vitro studies have shown that ATP is essential for P-glycoprotein transport function. Although. Again. Both mdr1a and mdr1b genes are expressed in the liver and kidney.
P-Glycoprotein 79 unknown. one should take the rate of influx diffusion. P-glycoprotein inhibitors should be used with caution to avoid potential neurotoxicity. for digoxin.2. the investigators further demonstrated that the P-glycoprotein inhibitors valspodar and FG-120918 were able to completely block the placental Pglycoprotein function. were observed in mdr1a (+/+) mice. Lankas et al. rate of P-glycoprotein efflux transport and nonspecific binding of compounds into consideration when predicting brain penetration. Heterozygous mdr1a/1b (+/–) female mice were mated with heterozygous male mice to produce fetuses of three genotypes: mdr1a/1b (–/–). respectively. ranging from 30–70% of the corresponding plasma concentration. these results clearly demonstrate that placenta is very sensitive to changes in P-glycoprotein function.5. The ratio of drug concentration in the mdr1a/1b (–/–) fetus to that in the wildtype fetus was 2. fetal drug exposure was much higher in the mdr1a/1b (–/–) fetus than the wild-type mdr1a/1b (+/+) fetus. the data from the mdr1a and mdr1a/1bknockout mice studies suggest that the brain is more sensitive to changes in P-glycoprotein function than other tissues. the drug concentrations in the heterozygous mdr1a/1b (+/–) fetus were similar to those in the wild-type fetus. Adis International Limited. saquinavir and paclitaxel.2 Placenta As noted earlier. For example. Since P-glycoprotein is exClin Pharmacokinet 2003. In the BeWo monolayer. immunostaining showed a high expression of Pglycoprotein in trophoblasts of human placenta. Similarly. These results suggest that the placental P-glycoprotein acts as an efflux transporter by removing xenobiotics from cells. Western blotting studies with monoclonal antibody C219 or JSB-1 indicated that P-glycoprotein is highly expressed in BeWo cells. All rights reserved. Regardless of the underlying mechanisms. A fraction of drug molecules can reach the brain tissue if the influx diffusion rate is greater than the P-glycoprotein efflux rate. Following intravenous administration of digoxin. vincristine and digoxin was significantly greater than the A-to-B transport. 5 and 16. Another important observation from these mdr1a and mdr1a/1b knockout mice studies is that for certain P-glycoprotein substrates. Functional P-glycoprotein activity has been demonstrated in cultured human placenta choriocarcinoma epithelial cells (BeWo cells). appreciable brain concentrations of cyclosporin. mdr1a/1b (+/–) and mdr1a/1b (+/+). the drug concentration in the brain is determined by the difference between the amount of drug transported by influx processes (passive diffusion and active uptake) and the amount of drug extruded by the P-glycoproteinmediated efflux process. the B-to-A transport of vinblastine. it is recommended that P-glycoprotein inhibitors should not be used in women during pregnancy to avoid excessive fetal exposure to xenobiotics. 4. 42 (1) . Using monoclonal antibody C219. On the other hand. As in the brain. P-glycoprotein is also highly expressed in human placenta. In this study. have clearly demonstrated the protective role of placental Pglycoprotein in reducing fetal exposure to xenobiotics in CF-1 mice. an appreciable amount of drug is still observed in the brain of wild-type mdr1a (+/+) mice. Kinetically. The role of placental P-glycoprotein in protection of the fetus has been further evaluated using mdr1a/1b (–/–) double knockout mice.7fold higher than the corresponding plasma concentrations at the same time point in mdr1a(+/+) mice after intravenous administration. Therefore. Because of potential function blockade. The fetal drug concentrations in the wild-type fetus were increased and were comparable to those in the mdr1a/1b (–/–) fetus after oral administration of the P-glycoprotein inhibitors to heterozygous mothers. suggesting that the P-glycoprotein level in the placenta of the heterozygous fetus is still sufficient to protect the fetus. Nonspecific binding to brain tissue may also be a contributing factor in determining drug concentration in the brain. Therefore. saquinavir and paclitaxel to pregnant heterozygous dams. the brain concentrations of tacrolimus were about 2. Addition of cyclosporin resulted in an increase in the A-to-B transport of the drugs and a decrease in the B-to-A transport.
 who investigated the CYP3A content at ten different locations in a human liver. distal jejunum. Because of overlapping substrate specificity. a portion of the extruded drugs then can be reabsorbed into the epithelial cells. The data of Watkins et al. All rights reserved. the columnar absorptive epithelial cells of the villi exhibited the strongest immunoreactivity. Of these SNPs.45 (arbitrary units). however. it is conceivable that P-glycoprotein may play an important role in drug metabolism. Unlike the liver. 23 and 17 pmol/mg microsomal protein were found in human duodenum. Drug molecules are exposed to P-glycoprotein prior to intracellular distribution and metabolism. the expression of P-glycoprotein appears to increase progressively along the length of intestine. In the liver and kidney. arbitrary units). Although the clinical implications of interindividual variability in placental P-glycoprotein remains to be investigated. the magnitude of the effect of P-glycoprotein on metabolism appears to be dependent on the spatial relationship between P-glycoprotein and CYP3A enzymes. intracellular distribution and metabolism in both the liver and kidney. CYP3A4 is the principal enzyme involved in the hepatic and intestinal metabolism of drugs. Genetic variation in the expression level of placental P-glycoprotein was studied by Western blotting in 100 placentas obtained from Japanese women.39) in exon 21 were associated with an amino acid conversion from Ala to Thr and to Ser. 1. nine SNPs were identified with an allelic frequency of 0. Using a monoclonal antibody to CYP3A. respectively. A large fraction of drug molecules is extruded by intestinal P-glycoprotein from the inside of the epithelial cells back into the intestinal lumen after the drug molecules gain access across the luminal surface of the epithelial cells. G2677A (allelic frequency 0. also indicate that hepatic CYP3A is homogeneously distributed. Individuals with heterozygous T–129C (T/C) had significantly lower levels of P-glycoprotein than the wild-type (T/T) individuals (1. Through the repetitive processes of extrusion Clin Pharmacokinet 2003.44. high placental levels of P-glycoprotein may provide a better protection for the fetus against xenobiotics. and distal ileum. kidney and liver. In contrast to CYP3A4.99. Therefore. However.005–0. 42 (1) . In this study. and because of coexpression of CYP3A enzymes and P-glycoprotein in the intestine. the distribution of CYP3A4 is not uniform along the length of the small intestine. or Adis International Limited. P-glycoprotein is localised at the entrance site of epithelial cells of intestines. In contrast to the situation in the liver and kidney. In humans.3 Drug Metabolism It has been widely accepted that the liver is the major site of drug metabolism because of its size and high content of drug-metabolising enzymes.80 Lin & Yamazaki pressed in human placental trophoblasts.07 vs 1. This means that P-glycoprotein is localised at the exit site of hepatocytes and renal epithelial cells. respectively. suggest that the CYP3A enzymes in the liver are evenly distributed. along the villi within a cross-section of mucosa.. P-glycoprotein only ‘sees’ drug molecules after cellular uptake. whereas no immunostaining was detectable in the goblet and crypt cells. Studies in rats by Debri et al.18) and G2677T (0. In addition to the liver. There is a striking overlap between CYP3A4 substrates and P-glycoprotein substrates.97 and 1. it is likely that placental P-glycoprotein also protects fetuses from xenobiotics in humans as well. It has also been shown that CYP3A4 expression varied along the length of small intestine: median values of 31. Comparison of the MDR1genotyping and corresponding placental P-glycoprotein level revealed a correlation between the Pglycoprotein expression level and SNPs in exon 1b (T–129C) and exon 21 (G2677A/G2677T). The expression levels of placental P-glycoprotein in homozygotes for wild-type allele. the small intestine and kidney may contribute significantly to overall metabolism in the body.42 (table I). 4. heterozygotes and homozygotes of mutant allele in exon 21 were 2. P-glycoprotein is localised on the luminal membrane of hepatic canaliculi facing the bile duct lumen or on the luminal brush-border membrane of renal proximal tubular cells facing the renal tubule lumen.
 4. it should be reemphasised that the effect of P-glycoprotein on drug metabolism becomes quantitatively less significant when high doses are given. biliary excretion and renal tubular excretion share certain characteristics.5-fold increase in both CYP3A and P-glycoprotein levels in the intestine. as well as other efflux transporter systems. have to be taken into consideration when biliary excretion of drugs is evaluated. Often. biotransformation occurs when the drug molecules are passing through the hepatocytes.5-fold increase in intestinal CYP3A level alone. The 6-fold increase in intestinal first-pass metabolism of indinavir cannot be explained by the 2. hepatic uptake. the drug molecules continue to diffuse and reach the canalicular membrane. The effect of P-glycoprotein on CYP3A4mediated intestinal metabolism of indinavir. the metabolism of cyclosporin in Caco-2 cells was higher from the apical side than from the basolateral side. P-glycoprotein may enhance intestinal metabolism of drugs. The increased intestinal first-pass metabolism is most probably due to a combination of increased intestinal CYP3A and P-glycoprotein levels.[97. In this study. Therefore. These results strongly suggest a role of P-glycoprotein in enhancement of CYP3A4mediated intestinal metabolism of drugs. The luminal brush-border membrane also contains numerous active transporters. P-glycoprotein prolongs the intracellular residence time of drug molecules and increases the probability of exposure to drug-metabolising enzymes. All rights reserved. Once in the hepatocytes. where P-glycoprotein and other efflux transporter systems will pump the drug molecules into bile. Pretreatment of rats with dexamethasone (40 mg/kg orally for 3 days) resulted in a 2. a substrate for both CYP3A4 and P-glycoprotein. a drug must first traverse the sinusoidal (basolateral) membrane of the hepatocytes by passive diffusion and/or hepatic uptake transporters. In principle. has been carefully evaluated in our laboratory using calcitriol-treated Caco-2 cells expressing both CYP3A4 and P-glycoprotein.4 Drug Excretion Drugs are generally eliminated from the body by metabolism and/or excretion. The intestinal firstpass metabolism of indinavir increased from 6% in control rats compared with 34% in dexamethasone-treated rats. uptake of drugs across the basolateral membrane of renal epithelial cells is the first step in renal excretion. The sinusoidal membrane of the hepatocyte contains a number of active transporters responsible for the uptake of cations. Similarly. and the increased hepatic CYP3A enzyme activity alone Adis International Limited. The effect of P-glycoprotein on intestinal metabolism was also shown in vivo. Pretreatment of rats with dexamethasone also induced hepatic first-pass metabolism of indinavir. the effect of P-glycoprotein on hepatic metabolism was also investigated. However.P-Glycoprotein 81 and reabsorption. was more than 6-fold greater when the drug was applied at the apical side than when the drug was applied at the basolateral side. Similarly.141] The formation of the major metabolite (M6). suggesting that P-glycoprotein plays a less significant role in hepatic metabolism as compared with intestinal metabolism. appeared to be able to explain the increased hepatic first-pass metabolism. The basolateral membrane contains a number of active transporters responsible for drug uptake. and biotransformation may occur. Consequently. which is responsible for the last step of Clin Pharmacokinet 2003. expressed as the ratio of the amount of the metabolite formed to the amount of the parent drug transported across the monolayer. anions and endogenous substances into hepatocytes from the circulation. These results clearly suggest that P-glycoprotein may play an important role in intestinal metabolism of drugs. 42 (1) . intracellular diffusion and metabolism. Both the liver and kidney play an important role in the excretion of unchanged drugs and their metabolites. providing in vivo evidence that P-glycoprotein enhances the intestinal firstpass metabolism of indinavir. including P-glycoprotein. whereas it has less of an effect on drug metabolism in liver and kidney. For biliary excretion.
suggesting that additional carrier systems are involved in the biliary excretion of cationic drugs. they showed that the transport of daunomycin was inhibited by verapamil. is eliminated predominantly by biliary excretion in mice. It was found that the complete absence of both mdr1a and mdr1b P-glycoprotein at the canalicular membrane results in a greater decrease in biliary excretion of a number of basic drugs compared with mdr1a (–/–) single knockout and wild-type mice. Kamimoto et al. Vincristine is eliminated in rats mainly by biliary excretion.to 5-fold greater in mdr1a (+/+) mice than in mdr1a (–/–) mice. All rights reserved.84 ml/min/kg). it is important to note that not all P-glycoprotein substrates are subject to significant biliary excretion. additional factors. However. respectively.152] Vecuronium. the role of P-glycoprotein in biliary excretion has also been reported for doxorubicin and vinblastine. biliary excretion of drugs was further investigated in a double knockout mouse model in which both mdr1a and mdr1b genes were disrupted. The biliary clearance of vecuronium in mdr1a/1b (–/–) double knockout mice was found to be about six times lower than in wild-type mice. appreciable residual biliary excretion of vecuronium was still observed in mdr1a/1b (–/–) double knockout mice. only a minor fraction of digoxin (<3%) is metabolised. and the biliary excretion of vecuronium is profoundly reduced in mdr1a/1b(–/–) double knockout mice compared with mdr1a (–/–) single knockout and wild-type mice. Similarly. tine into bile is 3. intracellular distribution and metabolism have to be taken into consideration when quantitatively assessing the role of P-glycoprotein in biliary excretion. Digoxin is mainly excreted as unchanged drug in the bile and urine of mice. studies in isolated perfused rat liver also suggest that P-glycoprotein plays a significant role in the biliary excretion of doxorubicin. The biliary clearance of digoxin is substantially greater in mdr1a (+/+) mice (2.5 times smaller than in the wildtype mice. Clin Pharmacokinet 2003. These results clearly indicate that Pglycoprotein plays a significant role in the biliary excretion of digoxin in mice. Furthermore. The biliary excretion of vincristine has been shown to be saturable and inhibited by verapamil. 4. These results suggest the involvement of P-glycoprotein in the biliary excretion of daunomycin. such as hepatic uptake. demonstrated ATP-dependent transport of daunomycin in canalicular membrane vesicles. a neuromuscular blocking agent.1 Biliary Excretion The involvement of P-glycoprotein in the biliary excretion of drugs was first suggested by immunohistochemical studies showing that P-glycoprotein is highly expressed on the canalicular membrane of hepatocytes. From these two studies. 42 (1) . More direct evidence for the involvement of Pglycoprotein in biliary excretion of drugs has come from studies with mdr1a knockout mice.[149. The excretion of unchanged doxorubicin and vinblas Adis International Limited. The involvement of P-glycoprotein in the biliary excretion of vincristine has also been demonstrated by using isolated perfused rat liver.3 ml/min/kg) than in mdr1a (–/–) mice (0. the absolute biliary recovery of unchanged drugs in mdr1a (+/+) mice is only 5 and 13% of the dose for vinblastine and doxorubicin. Earlier experimental evidence of the potential involvement of P-glycoprotein in biliary excretion had come from in vitro studies with highly purified canalicular membrane vesicles and isolated perfused rat liver. the results from both in vitro and in vivo studies clearly demonstrate that P-glycoprotein plays a significant role in biliary excretion of P-glycoprotein substrates. However.82 Lin & Yamazaki excretion of P-glycoprotein substrates into the urine. Because both mdr1a and mdr1b genes are expressed on the canalicular membrane. Similarly.150] Interestingly. Approximately 45% of the dose is excreted as unchanged digoxin in the bile of mdr1a (+/+) mice.[151. a potent P-glycoprotein inhibitor.4. approximately 50% of the dose is excreted as unchanged drug into the bile. The low biliary excretion of doxorubicin and vinblastine in mice is partly attributed to their high hepatic metabolism. Collectively. whereas the biliary clearance in mdr1a (–/–) mice was only 2.
Although quantification of each process of renal excretion is difficult due to practical limitations. The B-to-A transepithelial transport of digoxin across LLC-PK1 monolayers expressing human Pglycoprotein is much greater than the A-to-B transport by a factor of 7. showed that digoxin was actively secreted in the isolated perfused rat kidney with a CLR/(fu • GFR) ratio of 2. The relationship between renal clearance (CLR) and these processes can be expressed as equation 1: CLR = fu • GFR + CLS – CLRA Rearrangement of equation 1 yields equation 2: CLR/(fu • GFR) = 1 + (CLS – CLRA)/(fu • GFR) where GFR. 42 (1) . the renal clearance of digoxin in mdr1a (+/+) mice was three times greater than that in mdr1a (–/–) mice. respectively. As shown in equation 2.15 and 0. These results suggest that the Pglycoprotein functions as an efflux transporter at the apical membrane of epithelial cells of the renal tubule. Non-filtered drugs must first cross the basolateral membrane and then the apical membrane of epithelial cells of the renal tubule. the CLR/(fu • GFR) ratio became unity. In vitro systems have proven to be very useful tools for the study of the P-glycoprotein role in tubular secretion. CLS. respectively. at 3. when the ratio is less than unity. the CLR/(fu • GFR) ratio can be used as a simple way to assess the relative contribution of each process to overall renal excretion. As expected. the results from these mouse studies have remained controversial. GFR is a passive process by which only unbound drugs can be filtered. and reabsorption from the renal tubular lumen.4. Addition of cyclosporin results in a marked decrease in the B-to-A transport and an increase in the A-to-B transport. suggesting minimal reabsorption process of digoxin.99 ml/min/kg. In the presence of quinidine and verapamil at a concentration of 8 µmol/L. either by passive diffusion or carrier-mediated processes. Similarly. All rights reserved. this means that tubular secretion of the drug occurs. secretion clearance and reabsorption clearance. Although transgenic mice have also been used to study the role of P-glycoprotein in renal excretion. which is the major site of renal secretion. reabsorption of the drug from the tubular lumen occurs. Renal clearance of digoxin was compared in mdr1a (+/+) and mdr1a (–/–) mice following intravenous administration. Hori et al. Because doxorubicin is a P-glycoClin Pharmacokinet 2003. there are three processes involved in renal excretion. The P-glycoprotein inhibitors quinidine and verapamil inhibited tubular secretion and decreased the ratio. When the CLR/(fu • GFR) ratio of a drug is greater than unity. while tubular secretion and reabsorption often involve active transporters. Immunohistochemical studies reveal that Pglycoprotein is localised at the apical brush-border membrane of the proximal renal tubule. and CLRA are glomerular filtration rate. The finding of the localisation of renal P-glycoprotein has led to recognition of the importance of this transporter in tubular secretion of drugs.P-Glycoprotein 83 4. Transepithelial transport of vinblastine has also been observed in Adis International Limited. The B-to-A transport of vinblastine has been observed to be about six times higher than A-to-B transport. The isolated perfused kidney technique has also been used to investigate the role of P-glycoprotein in tubular secretion. These results provide further evidence that digoxin is actively secreted into the renal tubular lumen by P-glycoprotein.5. renal tubular secretion. MDCK monolayers expressing human P-glycoprotein. LLC-PK1 cells expressing human P-glycoprotein exhibit greater B-to-A transport of vinblastine than A-to-B transport. Similar results were also observed for digoxin when the drug was studied in dog isolated perfused kidney using the single-pass multiple indicator dilution method. Human MDR1 gene-transfected Madin-Darby canine kidney (MDCK) and porcine kidney (LLC-PK1) epithelial cell lines are the two most widely used models for the study of renal P-glycoprotein function. Conversely. and fu is the unbound fraction of drug in plasma.2 Renal Excretion Renal excretion of drugs usually involves three processes: glomerular filtration. Using the CLR/(fu • GFR) ratio approach.
in vitro studies with multidrug-resistant P388 leukaemia cells reveal that verapamil competitively inhibits P-glycoproteinmediated daunomycin uptake. it is expected that the absence of P-glycoprotein in renal tubule will result in a decrease in renal excretion of doxorubicin. In addition. P-Glycoprotein-Mediated Drug-Drug Interactions Inhibition and induction of CYP enzymes. One possible explanation is that other transporter systems may be involved in the renal excretion of doxorubicin and these basic compounds. Likewise.162] Therefore. CYP-mediated drug interactions have always been a major concern for clinicians and patients. Pglycoprotein-mediated drug interactions may be anticipated when P-glycoprotein substrates and P-glycoprotein inhibitors (or inducers) are coadministered. lower renal excretion in mdr1a/1b (+/+) mice was not expected. However. On the other hand. the pattern of P-glycoprotein inhibition appears to be substrate-dependent. the P-glycoproClin Pharmacokinet 2003. and by dideoxyforskolin in a noncompetitive manner. but digoxin surprisingly did not inhibit the P-glycoprotein-mediated transepithelial transport of cyclosporin. the renal excretion of unchanged doxorubicin was found to be higher in mdr1a (–/–) mice (15% of the dose) than in mdr1a(+/+) mice (10%) after intravenous administration. 5. although less frequently than for CYP enzymes. even though the exact number of binding sites is not yet known.[163-166] These results clearly indicate that multiple mechanisms are responsible for P-glycoprotein inhibition. Similarly. Adis International Limited. two ATP-binding domains are also involved in the P-glycoprotein function of drug transport. For example. Inhibition and induction of P-glycoprotein in animals and humans have been reported. are probably the most common causes for documented drug interactions. 42 (1) . in contrast with the expectation. azidoprocainamide and vecuronium) in mdr1a/1b (–/–) double knockout mice was significantly higher than that in mdr1a/1b (+/+) mice.[159. vanadate interacts with the ATPbinding domains of P-glycoprotein without interacting with the substrate-binding sites. Since these three basic compounds are P-glycoprotein substrates.84 Lin & Yamazaki protein substrate. verapamil inhibits the transport function in a competitive manner without interrupting the cyclic activity (ATP hydrolysis) of P-glycoprotein. 5.160] Several prominent drugs have been withdrawn from the market because of serious adverse effects as a result of CYP-mediated interactions. All of the drug-binding sites and ATPbinding domains interact cooperatively as a functional unit. inhibition of P-glycoprotein transport of a drug by other drugs could potentially result from either competition for drug-binding sites or from blockage of the ATP hydrolysis process. it is difficult to assess the mechanism and type of P-glycoprotein inhibition when P-glycoprotein substrate drugs and P-glycoprotein inhibitors are given simultaneously. As shown in the following examples. and cyclosporin inhibits transport function by interfering with both substrate recognition and ATP hydrolysis. The reason for the conflicting results is presently not known. Furthermore. whereas vinblastine shows noncompetitive inhibition with daunomycin. Thus. Like CYP-mediated drug interactions. and disruption of mdr1a and mdr1b genes may increase the expression of other transporter systems. particularly CYP3A4. P-glycoprotein has more than one drug-binding site. All rights reserved. the renal excretion of three basic compounds (tributylmethylammonium. the P-glycoproteinmediated transepithelial transport of digoxin in LLC-PK1 cells was inhibited by cyclosporin. P-glycoprotein-mediated transepithelial transport of vinblastine in Caco-2 cells was inhibited by verapamil in a competitive manner.[161. Because of the complexity. For instance. and their pharmacokinetic consequences are similar to those observed for inhibition and induction of CYP enzymes.1 P-Glycoprotein Inhibition Does Not Follow Simple Kinetics As discussed in section 1.
 Stimulation was also observed in ATPase activity studies. the transport of Hoechst 33 342 was stimulated by daunorubicin and doxorubicin. 42 (1) . Similarly. whereas vinblastine uptake into plasma membrane vesicles was competitively inhibited by cyclosporin. partial inhibition and activation have been observed in the interactions of testosterone with terfenadine. in another ATPase activity study.168] P-glycoprotein-mediated interactions have also been investigated by measuring the displacement of reversible binding to P-glycoprotein using membrane vesicles in the absence of ATP. Competitive inhibition suggests that two substrates act on the same sites of P-glycoprotein and that only one or the other can bind at any one time. The pattern of P-glycoprotein interaction can be classified into at least three major categories: competitive inhibition. In summary. Shou et al. verapamil-induced ATPase activity was Adis International Limited. nicardipine displayed competitive interaction with vinblastine but noncompetitive interaction with verapamil.P-Glycoprotein 85 tein-mediated uptake of azidopine into plasma membrane vesicles obtained from P-glycoproteinexpressing multidrug-resistant cells was noncompetitively inhibited by vinblastine or by cyclosporin. testosterone with midazolam and terfenadine with midazolam. All rights reserved. Collectively.[164. Wang et al. Furthermore. The complexity of the molecular mechanism for P-glycoprotein inhibition prevents our ability to predict the potential of Pglycoprotein-mediated drug-drug interactions. To test the hypothesis of multiple binding sites. Noncompetitive inhibition means that two substrates are able to bind simultaneously to P-glycoprotein molecule at distinct sites that are functionally independent. The P-glycoprotein-mediated doxorubicin efflux out of multidrug-resistant HCT-15 colon cells was significantly increased by some flavonoids. respectively. stimulated by progesterone. The displacement of morphine binding by verapamil was only partial. but the transport of rhodamine 123 was inhibited by daunorubicin and doxorubicin. activation of CYP3A4 has also been reported. the concept of multiple binding sites has also been proposed to explain many unusual enzyme kinetics observed for CYP3A4. For example. either quantitatively or qualitatively. The situation can be even more complicated if allosteric effects are involved in interaction between substrate and inhibitor. rhodamine 123 and Hoechst 33 342 stimulated the rate of P-glycoprotein-mediated transport of each other in P-glycoprotein-enriched plasma membrane vesicles isolated from Chinese hamster ovary CHrB30 cells. It is of interest to note that activation is not a phenomenon limited to P-glycoprotein interaction. A systematic analysis of inhibition patterns of verapamilinduced P-glycoprotein ATPase activity revealed that noncompetitive inhibition of verapamilstimulated ATPase activity was found with vanadate. The interaction between substrates and inhibitors of CYP3A4 does not always follow simple enzyme kinetics. these results suggest that two substrates are able to bind simultaneously to P-glycoprotein at different sites that may interact allosterically. whereas competitive inhibition was found with cyclosporin. have demonstrated that in vitro interaction patterns of CYP 3A4 substrates are substrate-dependent. noncompetitive inhibition and co-operative stimulation. Mutual inhibition. amitriptyline and propranolol. although vinblastine binding was completely displaced by verapamil. diltiazem. The ATPase activity assay has also been used as a convenient tool for determining the type of interaction between P-glycoprotein substrates. the interaction between P-glycoprotein substrates does not always follow simple kinetics. Similarly. Clin Pharmacokinet 2003. Similarly. Although the competition of two substrates for the same P-glycoprotein usually results in an inhibitory effect on the P-glycoprotein-mediated transport of the substrates. have successfully described the enzyme kinetics of CYP3A4-mediated metabolism of diazepam and its derivatives with a kinetic model which consists of two substrate-binding sites (apoprotein) and one catalytic site (prosthetic haem). activation of P-glycoprotein-mediated efflux transport has been reported in some cases.
the deliver of loperamide to the brain increases. resulting in increased toxicity. of BBB P-glycoprotein function was also observed when valspodar or GF-120918 (potent P-glycoprotein inhibitors) were coadministered with other drugs in mice. a 10-fold increase in brain concentrations of digoxin was also observed when valspodar 50 mg/kg was given orally to mdr1a (+/+) mice. 42 (1) . respectively. From the ratio of IC50 values (208 for LY-335979 and 0. However. If the ratio is much greater than unity. The IC50 values for ketoconazole to inhibit digoxin transport and nifedipine metabolism were 1. resulting in serious neurotoxicity. Loperamide 16mg was administered to healthy male volunteers with or without coadministration of quinidine 600mg. coadministration of ketoconazole (50 mg/kg intravenously) caused an 8.86 Lin & Yamazaki 5. have proposed to use the ratio of IC50 for CYP3A4 to IC50 for P-glycoprotein as an index of the relative selectivity of a drug for P-glycoprotein-mediated inhibition versus CYP3A4-mediated inhibition. Relative selectivity is best exemplified by the following study by Choo et al. valspodar and FG-120918. such as LY335979. but only a modest increase (2-fold) in plasma concentrations. but respiratory depression occurred when loperamide was given with quinidine. the inhibitory effect of ketoconazole on the brain and plasma concentrations of nelfinavir is attributed mainly to CYP3A-mediated inhibition. and the corresponding values for LY-335979 were 0.5-fold increase in plasma concentrations. Inhibition of hepatic and intestinal P-glycoprotein has also been reported. Loperamide produced no respiratory depression when administered alone.2 Drug Interactions Caused by P-Glycoprotein Inhibition Because of overlapping substrate specificities and inhibitors between CYP3A4 and P-glycoprotein. For this reason. under normal conditions the brain penetration of this drug is limited as a result of P-glycoprotein extrusion. Although the use of effective P-glycoprotein modulators (P-glycoprotein inhibitors). it is important to distinguish CYP3A4-mediated inhibition from P-glycoprotein-mediated inhibition in order to make appropriate interpretation of drug interaction data. This is particularly true for the brain. it is clear that the increased brain concentrations of nelfinavir by LY-335979 are caused mainly by P-glycoprotein inhibition. may improve the treatment of cancers. On the other hand. These experimental findings present a major problem that may confound attempts to use P-glycoprotein modulators in the clinical setting. Pretreatment with GF-120918 (250 mg/kg/day for 4 days. it means that the relative contribution by P-glycoproteinmediated inhibition is quantitatively more significant. and to a lesser extent to P-glycoprotein inhibition. In contrast. The increased neurotoxicity caused by P-glycoprotein modulators is best exemplified by a clinical study with loperamide. Similarly. Since loperamide is a P-glycoprotein substrate. an antidiarrhoeal agent. In another study. Extensive inhibition Adis International Limited. pretreatment with LY335979 (25 mg/kg intravenously) resulted in a 15fold increase in brain concentrations of nelfinavir. of amprenavir in mdr1a (+/+) mice. but had little effect on plasma concentrations.2 and 0. orally) led to a 13-fold increase in brain concentrations. Wandel et al.15 µmol/L. the profound increase of drug concentration in the brain by these P-glycoprotein modulators increases the risk of neurotoxicity. As shown in the above study. Therefore. The inhibitory effect Clin Pharmacokinet 2003.13 for ketoconazole). many drug interactions may involve both CYP 3A4 and P-glycoprotein. All rights reserved. in the presence of quinidine (a potent P-glycoprotein inhibitor). In mdr1a (+/+) mice.5-fold increase in brain concentrations of nelfinavir and a 3.024 and 5 µmol/L. these P-glycoprotein modulators also inhibit P-glycoprotein function in normal cells. it is clear that coadministration of a P-glycoprotein inhibitor causes a much greater increase in drug concentration in brain than in plasma. pretreatment with intravenous valspodar 25 mg/kg resulted in an 80-fold increase in brain concentrations of nelfinavir in mdr1a (+/+) mice.
Perhaps the most compelling clinical evidence of P-glycoprotein-mediated drug interactions in humans is the interaction of digoxin with other cardiac drugs. the lack of improvement of toxicity profiles might result from the increased tissue distribution of drugs caused by P-glycoprotein modulators. All rights reserved. resulting in increased absorption and decreased elimination of digoxin. is able to restore the in vitro sensitivity to vincristine in multidrug-resistant cell lines by inhibiting P-glycoprotein-mediated drug transport. An important issue related to P-glycoprotein inhibition is the concept of P-glycoprotein modulation in cancer research. oral pretreatment with valspodar 50 mg/kg led to a marked decrease in both intestinal and biliary excretion of digoxin in wild-type mice. For example. and that these unidentified transporters can be inhibited by valspodar. appreciable residual biliary excretion of digoxin (14% of an intravenous dose) was still observed in mdr1a/1b (–/–) double knockout mice. This finding implies that clinical drug resistance can be circumvented through the concomitant administration of P-glycoprotein inhibitors and anticancer drugs. Because digoxin is exclusively eliminated in humans by renal excretion as unchanged drug. a potent P-glycoprotein inhibitor. both valspodar and FG-120918 caused an increase in systemic exposure to doxorubicin associated with a decrease in doxorubicin clearance in patients. and care should be exercised to make appropriate interpretations. In 1981. The digoxin intoxication by quinidine and verapamil Clin Pharmacokinet 2003. whereas a daily dosage of 240mg caused a 60–80% increase.P-Glycoprotein 87 of valspodar on hepatic and intestinal P-glycoprotein was compared in wild-type mice and mdr1a/1b (–/–) double knockout mice. Although the P-glycoprotein modulators might completely inhibit P-glycoprotein function in tumour cells and restore drug sensitivity. An important lesson to be learned from these studies is that the underlying mechanisms for drug interactions can be very complicated. and pretreatment with valspodar caused an unexpected decrease in biliary excretion of digoxin (<5%) in these animals. suggesting dose-dependent P-glycoprotein inhibition. such as valspodar. These results suggested that other unidentified transport systems. rather than P-glycoprotein. it is highly likely that the observed drug interaction between digoxin and verapamil (or quinidine) is due to inhibition of P-glycoprotein activity.[187. The idea of reversing P-glycoprotein-mediated drug resistance has led to intensive efforts to develop potent and specific P-glycoprotein modulators.[156.157] Adis International Limited. FG-120918 and LY-335979. Tumour cells that exhibit multidrug resistance are often associated with overexpression of P-glycoprotein. Consistent with the clinical data. using digoxin as a model compound. As expected. 42 (1) . the pharmacodynamic results from clinical modulation studies are less than encouraging. the modulators could also inhibit the P-glycoprotein protective function of normal cells. However. such as verapamil and quinidine. reported that verapamil. Therefore. Tsuruo et al. As expected. One of the main reasons for the clinical disappointment is that the coadministration of P-glycoprotein modulators with anticancer drugs fails to improve the toxicity profiles of the chemotherapeutic agents. increased absorption of digoxin by verapamil and quinidine has been shown in rats. Resistance of tumour cells to chemotherapeutic agents is a major problem in the treatment of human cancers. may also be involved in the biliary excretion of digoxin in mice. Pglycoprotein-mediated interactions were observed when anticancer drugs were given with these potent P-glycoprotein modulators in cancer patients. treatment with P-glycoprotein modulators resulted in a much greater increase in drug concentration in tissues than in plasma.188] and decreased renal excretion of digoxin by verapamil and quinidine has been demonstrated in in vitro studies using isolated perfused rat and dog kidney. leading to cytotoxicity.[182-185] A daily dosage of verapamil 160mg caused a 40% increase in digoxin plasma concentration.[190. As shown in the mouse studies discussed above.191] However. P-glycoprotein inhibition as a cause of drug interaction has also been reported in humans.
 Western blot analysis showed that cyclosporin induced renal P-glycoprotein in a dosedependent manner. These results suggest species differences in inductive response to P-glycoprotein inducers. a 5-fold increase of rat hepatic P-glycoprotein level was reported after intraperitoneal administration of dexamethasone at 100 mg/kg/day for 4 days. the inductive response to P-glycoprotein inducers also appears to be tissuedependent. that both defensive systems possess adaptive manoeuvres to reinforce their protective role. Consistent with in vitro observations.[184. Similarly. As will be discussed below. In kidneys.88 Lin & Yamazaki can also be explained by the increased drug concentration in tissues. the increase in P-glycoprotein expresClin Pharmacokinet 2003. Species differences are well known for the induction of CYP enzymes. such as doxorubicin. In heart. 42 (1) . In another study. daunomycin and mitoxantrone. After administration of cyclosporin 10 mg/kg/day for 15 days. 10 or 30 mg/kg/day. an increase in the expression of P-glycoprotein was detected in many.194] Unless a specific P-glycoprotein modulator that selectively inhibits tumour cell P-glycoprotein can be developed. 5. although these cytotoxic drugs induced mdr RNA in all rat and mouse cell lines. respectively. 239. During combination therapy with digoxin and verapamil (or quinidine). for 28 days. kidney and lungs was maximal after 10 days of treatment in most tissues of rats. intestine. intestine. Similarly. Because CYP enzymes and P-glycoprotein function together to protect the body from accumulation of toxins and xenobiotics. the increase of the P-glycoprotein levels in liver. both verapamil and quinidine enhanced digoxin toxicity even when digoxin plasma concentrations were in the therapeutic range observed in the absence of the P-glycoprotein inhibitors. When cyclosporin was given subcutaneously at 10 mg/kg/day for 5. Dexamethasone has been shown to induce the mdr1a and mdr1b P-glycoproteins in a mouse cell line. and spleen. Regulation of P-glycoprotein expression in response to inducers has been extensively studied in vitro using cell lines derived from animals and humans.193. 10 and 15 days. In another study. but not all. but also time-dependent. reported that 3-methylcholanthrene strongly induced functional P-glycoprotein levels in a dose-dependent manner through an increased expression of the mdr gene in rat liver epithelial cells.3 P-Glycoprotein Induction is a Complex Process Like some of the CYP isoenzymes. mdr RNA levels were found to increase substantially in rat and mouse cell lines following acute exposure to cytotoxic drugs. Adis International Limited. the amount of P-glycoprotein detected was increased by 256. 161.to 3-fold. testis. the expression of P-glycoprotein is inducible. In another study. manipulation of the balance between tumour cell kill and toxicity to normal tissue will be very difficult to achieve. 5 or 15 mg/kg/day. Fardel et al. and recently it has been demonstrated that the species differences in CYP3A induction are due mainly to sequence differences in the ligand-binding domain of pregnane X receptor (PXR). from an evolutionary point of view. All rights reserved. it is not surprising. P-glycoprotein is also likely to be regulated by PXR. 144 and 69% compared with control groups. the increase was maximal after 10 days of treatment. Interestingly. In all of these tissues. These results suggest that the induction of P-glycoprotein expression is a dose-dependent process. stomach. and therefore it is possible that the observed species differences in CYP3A induction and P-glycoprotein induction have a similar molecular basis. or subcutaneously at 1. liver and lungs. the induction of P-glycoprotein expression by cyclosporin was demonstrated to be not only dose-dependent. Interestingly. they had no effects in human cell lines. tissues of rats. pretreatment of rats with dexamethasone (40 mg/kg/day orally for 3 days) resulted in significant increases in both intestinal and hepatic P-glycoprotein expression by approximately 2. dose-dependent induction of P-glycoprotein expression was observed in rats receiving cyclosporin orally at 2. the inductive effect of dexamethasone on P-glycoprotein expression in cultured rat hepatocytes has been reported.
However. has been shown to induce Pglycoprotein in human hepatoma. Consistent with in vivo observations. including phenobarbital. respectively.5-fold increase of hepatic CYP3A2 expression level.[211-213] It has been speculated that induction of P-glycoprotein is also attributed to the same PXR activation. ferences exist in the susceptibility to P-glycoprotein induction. because the rats received a dose of St John’s Wort that was approximately 50 times higher per kilogram bodyweight than that received by the volunteers. An alternative explanation could be that species dif Adis International Limited. and the induction was maximal after 5 days of treatment in heart and after 15 days of treatment in testis and spleen. 42 (1) . phenobarbital.and 1. Paclitaxel and rifampicin also induce CYP3A4 and Clin Pharmacokinet 2003. Oral administration of St John’s Wort extract to eight healthy male volunteers for 14 days at a dose of 300mg three times daily resulted in a 1. However.P-Glycoprotein 89 sion was 82. These results suggest the coordinated regulation of P-glycoprotein and CYP3A enzymes in both rats and humans. The high level of P-glycoprotein in this tissue could cause a rapid extrusion of cyclosporin from the capillary endothelial cells. The possible coordinate regulation of P-glycoprotein and CYP3A4 gene expression was further hypothesised by Wacher et al. including rifampicin. whereas the CYP3A4 gene is located at 7q22. co-induction of P-glycoprotein and CYP3A enzymes by dexamethasone was also observed in rats.5fold increase of duodenal P-glycoprotein and CYP3A4. rifampicin (rifampin). direct evidence was only made available recently.4. and this activation results in an increase in MDR1 mRNA and P-glycoprotein expression in primary human hepatocytes and LS 180 colon cancer cells. 74 and 50% compared with control groups. both P-glycoprotein and CYP3A4 were induced after treatment with many known inducers. In a cell line derived from human colon adenocarcinoma LS 180/WT and its doxorubicinresistant subline (LS 180/AD 50). All rights reserved. tissue differences in P-glycoprotein induction were also observed after administration of cycloheximide to rats. The human MDR gene has been mapped to chromosome locus 7q21. Durr et al. clotrimazole and hyperforin (one of the constituents of St John’s Wort) are known to activate the nuclear receptor PXR and induce CYP3A4 expression. but only a 1.[199. coordinated regulation between CYP3A and P-glycoprotein has been reported in vitro in animal and human cell lines. Recently.1. A diverse array of drugs. Similarly. Recent in vitro studies have demonstrated that the PXR (also known as SXR) plays a central role in regulating CYP3A4 transcription. when it was shown that the PXR activates the expression of the MDR1 gene. Similarly. The lack of P-glycoprotein induction in the brain could be due to the high basal level of P-glycoprotein in the brain capillaries. These differences most probably reflect a dose-dependent effect. based on the proximity of the chromosomal loci of these two genes. Collectively. The inducing effect of St John’s Wort on intestinal P-glycoprotein was considerably more pronounced in rats than in humans. dexamethasone. the mechanisms underlying tissue differences in the inductive response require further investigation. Administration of St John’s Wort extract to rats at an oral dose of 1000 mg/kg/day for 14 days resulted in a 3. Synold et al.[214.5fold increase was observed for small intestine. leaving no opportunity for this compound to induce P-glycoprotein. have reported that St John’s Wort induces P-glycoprotein and CYP3A enzymes in rats and humans. showed that the PXR is activated by paclitaxel and rifampicin. no change in the level of P-glycoprotein expression was found in the brain during treatment. these results clearly point to a tissuedependent inductive response to P-glycoprotein inducers.8-fold increase of intestinal P-glycoprotein and a 2. Similarly. these in vitro and in vivo observations have led to the speculation that regulation of CYP3A4 and MDR1 gene expression is coordinated through a similar mechanism. However. clotrimazole and reserpine.215] Using pharmacological and genetic approaches. respectively. Cycloheximide caused an 8-fold increase in mRNA of mdr1a P-glycoprotein in lung. a potent CYP3A4 inducer.206] Collectively.1.
pretreatment with rifampicin had little effect on the AUC and renal clearance of digoxin after intravenous administration. respectively. whereas the induction of mdr1b P-glycoprotein is post-transcriptionally regulated. and since digoxin is given orally at a very low dose. The induction of mdr1a P-glycoprotein is transcriptionally regulated. Since digoxin is eliminated exclusively by renal excretion. The decreased plasma AUC correlated reasonably well with the increased intestinal P-glycoprotein expression. a detailed knowledge of the inductive processes for each inducer is required in order to understand its implications and consequences. although it is currently not known how the mRNA is stabilised. Rifampicin treatment increased intestinal P-glycoprotein content 3.4 µg/L and 55 µg • h/L before rifampicin pretreatment to 2. 42 (1) . All rights reserved.6 µg/L and 38 µg • h/L. Interestingly. The molecular mechanism of PXR-mediated Pglycoprotein induction by rifampicin has been studied in detail by Geick et al. These findings strongly suggest that PXR plays an important role in regulating drug-metabolising enzymes and the P-glycoprotein transporter.90 Lin & Yamazaki CYP2C8 mRNA and enzyme protein expressions in primary human hepatocytes through PXRmediated transcriptional effects. the regulatory processes of P-glycoprotein induction are very complex and every inducer has its own pattern of induction. it was shown that the induction of MDR1 is mediated by a DR4 nuclear response element at about –8 kilobase pairs of the MDR1 upstream region to which PXR binds. Similarly.[211.216] The question as to whether the tissue-dependent differences in P-glycoprotein induction are attributed to the tissue differences in the expression level of PXR. remains to be explored. administration of St John’s Wort extract (three doses of 300mg per day for 14 days) resulted in an 18% decrease of plasma AUC after a single digoxin dose of 0. which correlated inversely with the oral AUC of digoxin. including the kidney and placenta.5-fold. Clearly. However. and not by metabolism. By using DNA binding assay and transfections. Lee reported that the induction of rat mdr1a and mdr1b mRNA transcripts by cycloheximide is mediated by different mechanisms. Although it is evident that the nuclear receptor PXR plays a central role in P-glycoprotein induction. Recently. Twenty volunteers received a 60mg oral dose of fexofenadine before and after treatment with oral rifampicin 600mg for 6 days. 5. Pretreatment with rifampicin also significantly decreased systemic exposure to fexofenadine in healthy volunteers. during rifampicin pretreatment when the volunteers received a single oral dose of digoxin 1mg. the inductive effects of a single drug may be mediated by multiple mechanisms. in addition to the liver and intestine.4 Drug Interactions Caused by P-Glycoprotein Induction The most compelling evidence to date for P-glycoprotein induction as a cause of drug interactions was provided by Greiner et al. The Cmax and AUC of fexofenadine decreased by Clin Pharmacokinet 2003. The post-transcription is probably due to mRNA stabilisation. Treatment with the extract resulted in a 40% increase in the expression of duodenal P-glycoprotein. using the human colon carcinoma cell line LS174T. The plasma Cmax and AUC of digoxin decreased from 5. Therefore. Duodenal biopsies were obtained from each volunteer before and after administration of rifampicin. in a clinical study comparing the pharmacokinetics of digoxin before and during coadministration of rifampicin 600 mg/day for 10 days in eight healthy volunteers. Adis International Limited.5mg in healthy volunteers. This means that the decreased plasma concentration of digoxin during rifampicin treatment is caused by reduced bioavailability of digoxin as a result of induction of intestinal Pglycoprotein. The above two studies provide direct evidence that a similar mechanism is responsible for induction of CYP3A4 and MDR1 by xenobiotics. these results strongly suggest that the digoxin-rifampicin interaction mainly occurs at the level of the intestine through P-glycoproteinmediated induction. the nuclear receptor PXR and P-glycoprotein are co-expressed in a number of other tissues. or to different mechanisms in different tissues.
From a human mass balance study. If based on the urinary recovery. Thus. it appears that P-glycoprotein has a greater impact on limiting cellular uptake of drugs from the blood circulation into the brain and placenta. respectively. and because of the similarities in the inhibitors and inducers between these two proteins.3 L/h/kg (5 ml/min/kg) before rifampicin treatment to 0. It is possible that rifampicin is able to induce OATP. and from intestinal lumen into epithelial Clin Pharmacokinet 2003. and bioavailability decreased from 27% without rifampicin to 10% with rifam Adis International Limited. but not by metabolism. picin. Therefore.42 L/h/kg (7 ml/min/kg) during rifampicin treatment. accounted for about 20% of the radioactivity in urine. it is possible that the observed interaction between fexofenadine and rifampicin is due to a combination of both CYPmediated and P-glycoprotein-mediated induction. Conclusions Although the physiological function of P-glycoprotein is still not fully understood. particularly in terms of the underlying mechanisms. metabolism and excretion) is a very important factor in determining P-glycoprotein function.829. but also decreased its bioavailability to a greater extent than would have been predicted by the increased clearance. Since cyclosporin is a substrate for both CYP3A4 and P-glycoprotein. a metabolite of fexofenadine. 42 (1) . Because of overlapping substrate specificity between CYP3A4 and P-glycoprotein. All rights reserved. Although attempts have been made to quantify the relative contribution of CYP3A4 and P-glycoprotein to overall interaction. From transgenic mouse studies. there is still no simple way by which the relative contribution of these two systems can be quantified because of the complexity of the interplay involved between intestinal and hepatic CYP3A4 and P-glycoprotein. care should be taken in interpreting data for drug interactions. Rifampicin not only increased elimination clearance of cyclosporin. 6.and 3-fold. However. and since rifampicin can induce both CYP3A4 and P-glycoprotein. the role of this efflux transporter in drug absorption and disposition is becoming increasingly defined. the involvement of OATP in the hepatobiliary excretion of fexofenadine may further complicate the interpretation of the observed interaction between fexofenadine and rifampicin. many drug interactions may involve both P-glycoprotein and CYP3A4. and information on the metabolites in faeces is not available. respectively. the investigators concluded that the decreased plasma profiles are the result of a reduced bioavailability caused by induction of intestinal P-glycoprotein.P-Glycoprotein 91 2. The observed interaction between cyclosporin and rifampicin in healthy volunteers is also probably due to a combination of both CYP3A4 and P-glycoprotein induction. metabolism of fexofenadine is not insignificant. P-glycoprotein is highly expressed in various tissues. With the assumption that fexofenadine is predominantly eliminated by biliary excretion. However. it is unknown whether the faecal component represents unabsorbed drug or the result of biliary excretion. Furthermore. intracellular distribution. Blood clearance of cyclosporin increased from 0. MDL-4. Unless the relative contribution of Pglycoprotein and CYP3A4 to overall drug interactions can be quantitatively differentiated. and the anatomical localisation of P-glycoprotein in relation to the sequences of drug movement (cellular uptake. the increased clearance and decreased bioavailability of cyclosporin during rifampicin treatment is most probably due to a combination of CYP3A4 and P-glycoprotein induction. the assumption of lack of metabolism may not be valid. The pharmacokinetics of cyclosporin were studied in six healthy volunteers after administration of cyclosporin orally (10 mg/kg) and intravenously (3 mg/kg) with and without rifampicin pretreatment (600 mg/day for 11 days). it was shown that approximately 80 and 11% of an oral dose of [14C]fexofenadine were recovered in the faeces and urine. in volunteers after rifampicin treatment. it has recently been demonstrated that organic anion transporting polypeptide (OATP) is involved in the hepatic uptake of fexofenadine.
Although the transgenic mdr1a (–/–) and mdr1a/1b (–/–) mice provide a powerful tool for studying drug absorption and disposition. the potential risk of P-glycoproteinmediated drug interactions might be underestimated if only the plasma concentration is monitored. Cellular localization of the multidrug resistance gene product P-glycoprotein in normal human tissues. J Biol Chem 1996. Cell 1994. 271: 1877-83 14. Schinkel AH. care should be taken when exploring the underlying mechanism of drug interactions. Germann UA. 47: 381-9 12. J Biol Chem 1997. Eur J Biochem 1997. 62: 385-427 3. et al. Structure of multidrug resistance P-glycoprotein to 2. than on plasma concentration of drugs. Clin Pharmacokinet 2003. et al. Juliano RL. Chen C-J. Clarke DM. Smith AJ. 8: 161-70 4. Cell 1994. Schinkel AH. et al. et al. MDR3 P-glycoprotein. while MDR3 P-glycoprotein specifically translocates phosphatidylcholine. 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