Intestinal secretion of drugs. The role of P-glycoprotein and related drug efflux systems in...

advanced drug delivery reviews

ELSEVIER Advanced Drug Delivery Reviews 25 ( 1997) I29- I57

Intestinal secretion of drugs. The role of P-glycoprotein and related drug efflux systems in limiting oral

drug absorption

Janice Hunter, Barry H. Hirst”

Abstract

Oral bioavailability requires absorption of drugs across the intestinal epithelium. This may be mediated by either the paracellular and/or transcellular routes. Passive transcellular absorption requires the appropriate physicochemical properties to allow permeation across the apical and basolateral membrane domains. Compounds demonstrating these properties are more likely to be recognised as substrates for intracellular metabolism, such as by cytochrome P450 isozymes, and/or secretory drug efflux systems, including P-glycoprotein, such that oral bioavailability will be limited. P-glycoprotein, which leads to multidrug resistance in tumour cells, is an ATP-dependent secretory drug efflux pump, encoded by the MDRI gene in humans. It acts to clear the membrane lipid bilayer of lipophilic drugs, in the manner of a flippase. In the intestine, as well as at specific other epithelial and endothelial sites, P-glycoprotein expression is jocalised to the apical membrane, consistent with secretory detoxifying and absorption limitation functions. Other secretory efflux systems, such as multidrug-resistance associated protein (a glutathione S-conjugate transporter), fluorochrome efflux systems and the methotrexate efflux system, together with drug ionic charge and the intestinal pH microclimate, may mediate intestinal secretion of a wide variety of

drugs. Direct evidence for P-glycoprotein limiting drug absorption comes from studies in vitro with human Caco-2 cells and includes non-linear dependence of absorption on substrate (vinblastine) concentration, increased absorption upon saturation

of secretion and increased absorption upon inhibition of P-glycoprotein function, with modulators such as verapamil. I’-glycoprotcin-like mechanisms are implicated in the intestinal secretion of a variety of drugs, in addition to classical

P-glycoprotein substrates, including cyclosporin, certain peptides, digoxin, fluoroquinolones, ranitidine and /3-adrenoceptor antagonists. These drug interactions with P-glycoprotein may explain the pharmacokinetics of absorption in vivo. P-glycoprotein function may be integrated with drug metabolism, with several drugs being common substrates for P-glycoprotein and cytochrome P-450 3A. Recognising the interactions of drugs with intestinal secretory and metabolic systems that limit absorption will lead to novel strategies of overcoming problems of poor oral bioavailability.

Kqwords: P-glycoprotein; multidrug resistance; Cytochrome P-450; Intestine; Oral bioavailability; Drug absorption

Contents

I. Introduction ._..__........._____.............................................................,,..........,,..........,,,..........,,,,.......,....,...,,,.,,.....,.,.,......,,,,,,....,. 130

2. Factors affecting the oral bioavailability of drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I31

3. Multidrug resistance and P-glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........... 132 3. I. Structure and function of P-glycoprotein .._............_................ .._....... .._........_..___........... _........._._ 132

3.2. Pharmacological reversal of multidrug resistance . . ..___.........................................................................,..........,.........,,,.,,. 134

3.3. P-glycoprotein in normal tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................ 134 4. Non-P-glycoprotein efflux mechanisms .._...................................................,...........,,..........,,,,......,..,,,.......,,..........,,,.......,,,...... 135

*Corresponding author. Fax: + 44-191-222-6706; e-mail: [email protected]

0169-409X/97/$32.00 0 1997 Elsevicr Science B.V. All rights reserved

PII so 169-409X(97)00497-3

130 J. Hunter, B.H. Hirst I Advanced Drug Delivery Reviews 25 (1997) 129-157

4.1. Multidrug resistance-associated protein (MRP) ..................................................................................................................

4.2. BCECF and other fluorochrome efflux ............................................................................................................................... 4.3. Methotrexate efflux .......................................................................................................................................................... 4.4. Role of ionic charge and pH microclimate in intestinal secretion .........................................................................................

5. Direct evidence for P-glycoprotein-mediated intestinal secretion.. ...............................................................................................

5.1. Studies with cultured intestinal epithelial cells .................................................................................................................... 5.2. Direct evidence for P-glycoproteins as secretory detoxifying mechanisms limiting drug absorption ........................................

5.3. Peptide secretion by Caco-2 intestinal epithelia ..................................................................................................................

5.4. Implication of P-glycoprotein-like mechanisms in intestinal secretion of other drugs.. ...........................................................

5.4.1. Digoxin ................................................................................................................................................................. 5.4.2. Ranitidine.. ............................................................................................................................................................

5.4.3. Fluoroquinolones ................................................................................................................................................... 5.4.4. B-adrenoceptor antagonists .....................................................................................................................................

5.4.5. Amoxicillin ........................................................................................................................................................... 5.4.6. Hydrophihc compounds ..........................................................................................................................................

6. Pharmacokinetic interactions between P-glycoprotein modulators and drugs known to interact with P-glycoprotein in vivo ............

7. Interactions between P-glycoprotein and drug metabolism.. ........................................................................................................

8. Conclusions and future perspectives ................................................

References .........................................................................................

1. Introduction

The intestinal epithelium was long thought to

be rather a passive barrier to drug absorption. However, from a recognition that the barrier function is selective, greater insight into the pos-

sible mechanisms whereby drug absorption may be facilitated or hindered has developed. The

selective nature of the intestinal barrier to drugs

is borne of the necessity of the intestinal epithelium to allow and indeed facilitate absorption

of nutrients and other essential constituents of the diet, including sugars, amino acids, small

peptides, nucleosides, vitamins and trace ele- ments. Absorption of other luminal contents, including bile salts are also facilitated. The facili-

tation of absorption of these essential components of the diet is mediated by numerous spe-

cific membrane transport systems, localised to

both the luminal (apical) and blood-facing (basolateral) membrane domains of the absorp-

tive enterocytes [l]. The genes encoding these transporter systems are rapidly being identified, leading to further knowledge of their functions

and modes of operation. Although these systems have evolved to supply the body with necessary nutrients, the substrate specificity of these systems is sometimes broad enough to allow recognition of non-nutrient substrates. Similarly, the intercellular tight junctions that connect together adjacent epithelial cells were, until recently,

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thought to be rather static structures [2]. Recog- nition of the dynamic nature of these junctional complexes has led to concepts of enhancing ab-

sorption of certain, hydrophilic drugs, by junctional permeability modulation [3-51.

Although, the role of the intestine in the metab-

olism and secretion of drugs has been recognised

for many years, it is often overlooked, or its importance is understated. First-pass metabolism of

drugs administered by the oral route is greater in the liver, but still may be highly significant in the

intestinal enterocytes. Similarly, phase 2 reactions occur in enterocytes and may be an overlooked contribution to drug elimination. Non-biliary intestinal excretion is a significant route for drug elimination, while in anephric conditions, it may be the major route. This elimination is now rec-

ognised to be, in part, mediated by secretory ef-

flux systems, similar to those expressed in the kidney and liver where they also contribute to

drug elimination [6,7]. This article reviews intestinal secretory func-

tions, the underlying mechanisms and how they contribute towards limiting drug absorption and the characteristics of drug pharmacokinetics and elimination. The focus is a discussion of the function and role of P-glycoprotein and P-glycoprotein-like mechanisms in the intestine. Possible

interactions between such secretory systems and drug metabolic pathways are highlighted towards the end of the article.

J. Hunter, B.H. Hirst I Advanced Drug Delivery Reviews 25 (1997) 129-157 131

2. Factors affecting the oral bioavailability of drugs

The enteral (oral) route of drug administration is the most convenient and economical. For orally administered drugs, the small intestine represents the major site of absorption because of the functional specialisation of the cells lining the mucosa, combined with the significant transit time [S]. Prediction

of the likely oral bioavailability of drug candidates is

often approximated by diffusion-solubility models

utilising correlations with octanol! water partition coefficients [9]. These models are sometimes modi-

fied by use of other solvent mixtures, which might better model intestinal membrane permeability, in-

cluding measurement of the partition coefficient between intestinal brush-border membrane vesicles and water [lo]. However, these simple parameters do

not take into account many of the problems involved in drug delivery, including intrinsic intestinal transport systems for which the drug may have affinity.

lism by luminal digestive enzymes, or by luminal

micro-organisms. Examples of compounds that are

unstable at the acidic pH of the stomach include some penicillins, omeprazole and peptide drugs [ 111. Degradation in acid is a relatively easy problem to solve, by the use of enteric coated drug particles, or by the formulation of the drug with antacids [12]. A

more difficult problem to solve, a particular problem for peptide drugs, is metabolism by the digestive enzymes within the gastrointestinal tract. Intralumi-

nal metabolism of certain types of non-peptide

compounds may also be mediated by the luminal microflora in the distal small intestine and the colon

[ 13,141. Another potential site of pre-systemic me-

tabolism is the intestinal luminal brush-border mem-

brane, with its associated hydrolases. Quantitatively, the liver is usually the most important site of

xenobiotic metabolism, but the intestinal enterocytes are qualitatively similar, capable of performing many of the same enzymic reactions, including oxidation,

dealkylation, hydrolysis and conjugation [ 15,161. Several factors can influence the bioavailability of The final type of bioavailability problem is poor

a drug when given orally. Aqueous solubility and membrane permeation. Paracellular and transcellular aqueous dissolution rates are important; if a drug routes are available for compounds to cross the does not dissolve within the gastrointestinal tract intestinal epithelium (Fig. 1 [17]). Transport across transit time, it will simply be eliminated in the faeces healthy intestinal epithelium by the paracellular route [ 111. Degradation within the gastrointestinal lumen (pathway A) is minimal due to the presence of the could be due to instability in acidic pH or metabo- tight junctions. Only small hydrophilic molecules are

APICAL

B c

BASAL Fig. 1. Pathways of intestinal absorption. (A) paracellular diffusion; (B) paracellular diffusion enhanced by a modulator of tight junctions;

(C) transcellular passive diffusion, (C *, intracellular metabolism); (D) carrier-mediated transcellular transport; (E) transcellular diffusion modified by an apically polarised efflux mechanism; (F) transcellular vesicular transcytosis.

132 J. Hunter, B.H. Him I Advanced Drug Delivery Reviews 25 (1997) 129-157

allowed to pass between cells, unless a modulator of tight junctions (pathway B) is present 121. Trans- cellular transport of a molecule can take place by a passive mechanism (pathway C), or be mediated by a specific carrier (pathway D). For the passive

transcellular flux of a molecule to occur, it must have

the appropriate physicochemical properties (e.g. size, charge, ’ lipophilicity, hydrogen bonding potential,

solution conformation) to cross both the apical and basolateral membrane, lipophilic barriers [8,17].

Separating the two membrane domains is the aque-

ous cytoplasmic environment and transport across this compartment may be facilitated by binding to

cytoplasmic components. However, compounds that are adapted for absorption by this passive transcellular mechanism may be substrates for enterocytic intracellular metabolism (pathway C*), while the same general characteristics are more likely to make

them substrates for apically polarised efflux mecha-

nisms (pathway E), such as P-glycoprotein [18-211. Most orally administered drugs are absorbed by this

passive transcellular mechanism. Transcellular ab-

sorption using naturally occurring carriers (pathway D), such as those for nutrients, vitamins and bile

salts [22], is also important for specific classes of drugs. These carrier-mediated pathways are important for the absorption of some hydrophilic drugs. For example, L-dopa [23] and o-cycloserine [24] are absorbed by intestinal amino acid transport systems,

while orally available cephalosporins [25], angiotensin-converting enzyme inhibitors and renin antago-

nists are substrates for the intestinal oligopeptide

(di/tri-peptide) transporter [26-281. Endocytosis of compounds (pathway F) is minimal in adult small

intestine and is not a quantitatively significant mechanism for drug absorption in the intestine. However,

a transcytotic pathway is important for delivery of antigens through the specialised intestinal M-cells that are localised to the follicle-associated epithelium overlying the gut-associated lymphoid tissues. This pathway may be exploited for vaccine delivery and, perhaps, for delivery of macromolecules, particularly

those where targeting to the mucosal immune system

is relevant [29]. In general terms, for oral drug delivery, the drug

must either be small (M, approximately < 350) and hydrophilic to access the diffusive paracellular pathway, or lipophilic (while still exhibiting significant water-solubility to allow dissolution) for passive

transcellular absorption, or be transported across the intestinal enterocyte by specific carrier-mediated mechanisms.

3. Multidrug resistance and P-glycoprotein

Clinical resistance to chemotherapeutic drugs is a

major problem in the treatment of cancer. One form

of drug resistance, termed multidrug resistance (MDR), is defined as the ability of cells exposed to a

single drug to develop resistance to a broad range of structurally and functionally unrelated drugs, due to

enhanced outward transport (efflux) of drugs, mediated by a membrane glycoprotein “drug transport pump” [30-331. Most experimental models of MDR

have been obtained by growing cell lines in pro- gressively increasing concentrations of cytotoxic drugs in culture. Cells selected for resistance with

one cytotoxic drug display significant cross-resist-

ance to the other drugs, including natural products such as the anthracyclines, Vinca alkaloids, epi-

podophyllotoxins, colchicine and actinomycin D, but not to drugs such as bleomycin, methotrexate, nor

alkylating agents [34]. This fairly consistent pattern of cross-resistance is termed the MDR phenotype

130-331. The degree of cross-resistance displayed by MDR

cells to the individual drugs varies among cell lines. However, the similarity in the pattern of resistance to this set of chemotherapeutic agents suggests a single

underlying mechanism that is responsible for MDR.

The most consistent alteration found in MDR cell lines is an increased expression of a high molecular

weight cell surface glycoprotein (P-glycoprotein), with a concomitant decrease in the accumulation and

retention of cytotoxic drugs [35].

3. I. Structure and function of P-glycoprotein

The human MDRl gene, which encodes P- glycoprotein, was cloned and sequenced in 1986 [36]. It was shown to encode a protein of 1280

amino acids, and transfection of this gene into drug- sensitive cells resulted in acquisition of resistance to drugs of the MDR family [37,38]. A transporter function for P-glycoprotein is consistent with the long observed reduced intracellular drug accumulation that is characteristic of multidrug resistant cells

J. Hunter. B.H. Hirst I Advattced Drug Delivery Reviews 25 (1997) 129-157 133

[39]. P-glycoprotein is recognised as a member of

the ATP-binding cassette (ABC) super-family of

membrane transport proteins, with overall homology with a number of bacterial, yeast, insect and other mammalian transport systems [40,4 1 I.

Direct evidence that P-glycoprotein binds drugs is

provided by the observation that photoaffinity label- ling analogues of vinblastine and colchicine bind to

P-glycoprotein in a manner that can be competitively

inhibited by vinblastine and other drugs of the MDR

family [42] (see Table 1). Isolated membrane vesicles from MDR cells have been shown to transport vinblastine [43-4.51, while transfection of the MDRl

gene into cultured canine kidney cells results in enhanced transepithelial transport of drug [46]. Purified preparations of P-glycoprotein possess adenosine triphosphatase (ATPase) activity, as pre-

dicted by the sequence of nucleotide binding consen- sus sequences [47-491. These data support a model

of MDR in which P-glycoprotein functions as an energy-dependent drug efflux pump of broad spe-

cificity, resulting in reduced intracellular drug accumulation.

Speculation on the mechanism by which the

multidrug transporter reduces accumulation of cytotoxic drugs in multidrug resistant cells has evolved as the physiology, biochemistry and pharmacology

Table I

Agents that interact with P-glycoprotein

of the transporter have been elucidated. The current

model of the mechanism of action of P-glycoprotein

is that drugs can be detected and expelled as they enter the plasma membrane in the manner of a “hydrophobic vacuum cleaner” (501. This effect accounts for the decreased accumulation in the

cytosol. Several lines of evidence support the conclu-

sion that drugs are removed directly from the plasma membrane: (a) Kinetic data [51,52], which display

both increased drug efflux and decreased drug influx.

(b) Virtually every study of a series of structurally

related drugs differing in their affinity for P- glycoprotein demonstrates that the most important determinant is their relative hydrophobicity and that substrates for the transporter have a partition coefficient (octanol/water) of approximately + 1 or great-

er [53,54]. However, very hydrophobic agents, such as camptothecin, which are sparingly soluble in

water, are not substrates, indicating that some water solubility is required for the recognition by P-

glycoprotein. (c) Rhodamine 123, a highly fluorescent dye and MDR substrate, was shown by Kessel

1551 to have different fluorescence spectra in drug- resistant compared to drug-sensitive cells. In drug-

sensitive cells, the fluorescence was similar to that of rhodamine 123 in the presence of octanol, suggesting a hydrophobic environment, whereas with the drug-

Antiarrhythmics e.g. amioderone, lidocaine, propranolol, quinidine

Antibiotics and antifungals e.g. cefoperazone, ceftriaxone, erythromycin, itraconizole

Anticoagulants e.g. dipridamole

Antimalarials and antiparasites e.g. chloroquine, emetine, hydroxychloroquine, quinacrine, quinine

Calcium channel blockers e.g. bepridil, diltiarem, felodipine, nifedipine, nisoldipine, nitrendipine, tiapamil, verapamil

Calmodulin antagonists e.g. chlorpromazine, trifluoperaaine

Cancer chemotherapeutics, combination regimens u.~. actinomycin D, colchicine, daunorubicin, doxorubicin, etoposide, mitomycin C.

mithramycin, podophyllotoxin, puromycin, taxol, topotecan, trimetrexate, vinblastine, vincristine

Cyclosporins e.g. CsA, CsH, SDZ PSC 833

DNA intercalators e.g. ethidium bromide

Fluorescent dyes e.g. BCECF-AM, Flue-3, Fura-2, rhodamine 123

Hormones e.g. aldosterone, clomiphene, cortisol, deoxycorticosterone, dexamethasone, prednisone. progesterone, tamoxifen

Indole alkaloids e.g. reserpine, yohimbine

Local anaesthetics e.g. bupivacaine

Phenothiazines

SurfactanWsolvents e.g. cremophor-EL, triton X-100, Tween 80

Toxic peptides e.g. N-acetyl-leucyl-leucinal (ALLN), gramicidin D, valinomycin

Tricyclic antidepressants e.g. desipramine, trazodone

Miscellaneous e.g. components of fruit juice, liposomes, quercetin, terfenadine, tumour necrosis factor, vitamin A

(From Gottesman and Pastan [3l] and Ford and Hait 1531)

134 .I. Hunter, B.H. Hint I Advanced Drug Delivery Reviews 25 (1997) 129-157

resistant cells, the spectrum indicated a hydrophilic environment, suggesting that in these cells the dye had been removed from the plasma membrane. (d) The final evidence for the hydrophobic vacuum

cleaner model rests on the demonstration that drugs

such as doxorubicin are removed directly from the

plasma membrane by the action of a transporter.

Utilising the transfer of energy from doxorubicin to iodinated naphthaline azide (INA), a highly hydrophobic label of membrane constituents including transmembrane proteins, the presence of doxorubicin within the membranes of drug-sensitive cells can be

easily detected. However, in drug-resistant cells, doxorubicin is only found in the plasma membrane associated with P-glycoprotein. This result indicates

that the transporter has removed doxorubicin from

the plasma membrane [50]. How is the energy of ATP transduced to result in

the removal of drug from the plasma membrane of multidrug-resistant cells? Although we know that both ATP sites are needed for this activity to occur

efficiently, and that the drugs themselves stimulate the ATPase activity [56], very little is known about how the energy of ATP is harnessed in this transporter [57]. One idea is that the P-glycoprotein is

essentially a “flippase” that detects drug within the inner leaflet of the membrane and “flips” it into the outer leaflet (from which it can diffuse away from

the cell) or directly into the extracellular space.

Recent data from van Helvoort et al. [58] using LLC-PKl cells transfected with either MDRl or

MDR3 show that, at low temperatures ( 15°C) newly synthesised short-chain analogues of various membrane lipids were recovered in the apical albumin- containing medium of MDR 1 -transfected cells but not of control cells. MDR inhibitors and energy

depletion reduced apical release. MDR3-transfected cells exclusively released a short-chain phosphatidyl- choline. As no vesicular secretion occurs at 15°C the

short-chain lipids must have been translocated by the

P-glycoproteins, across the plasma membrane, before extraction into the medium, by the lipid-acceptor,

albumin. In the absence of a suitable acceptor, such as albumin, the short-chain lipids ended up in the outer leaflet of the plasma membrane of MDRl- transfected cells. These data provide direct support for the flippase hypothesis as the mechanism of multidrug-transport by the MDRl P-glycoprotein.

3.2. Pharmacological reversal of multidrug

resistance

A major goal in clinical, as well as experimental,

investigations of drug resistance is to discover

unique methods by which it may be reversed or

circumvented. The first report of the pharmacological

reversal of MDR came from Tsuruo et al. [59], who showed that the calcium channel blocker, verapamil, and the calmodulin (CaM) antagonist, tri- fluoperazine, greatly potentiated the anti-proliferative activity of vincristine and produced an increased cellular accumulation of vincristine in an MDR

murine leukaemia cell line in vitro and in vivo. Since this original observation, many compounds have

been shown to antagonise MDR in a variety of cell lines and in vivo tumour models.

The chemosensitisers described to date may be grouped into six broad categories: (a) Calcium

channel blockers, (b) CaM antagonists, (c) non-cytotoxic anthracycline and Vinca alkaloid analogues, (d) steroids and hormonal analogues, (e) miscellaneous

hydrophobic [60], cationic compounds and (f) cyclosporins (Cs) (Table 1). These pharmacological tools have been used to explore the role of MDR-

related secretory systems in drug absorption. For an in depth review of these compounds, readers are

referred to Ford and Hait [53].

3.3. P-glycoprotein in normal tissues

After the description of P-glycoprotein in MDR cell lines, expression in normal human tissues was documented. Initial examination of normal human tissues for P-glycoprotein expression employed slot blot analysis for measurement of MDRl RNA [61] and reported high levels of expression in the adrenal

gland and kidney, intermediate levels in lung, liver, jejunum and colon, and low levels in prostate, skin,

spleen, heart and skeletal muscle. Although this report provided important initial insight into differen- tial tissue expression of P-glycoprotein, this method is not well suited to the examination of highly

heterogenous tissues and to the correlation of expression with microanatomic detail. Thiebaut et al. [62] employed immunohistochemistry with the anti-P- glycoprotein monoclonal antibody, MRK16, to examine frozen normal tissues. P-glycoprotein expres-

.I. Hunter, B.H. Hint I Advanced Drug Deliveq Reviewzs 25 (1997) 129-157 135

sion was found on the biliary canalicular surface of

hepatocytes and the apical surface of small biliary ductules of the liver, the apical surface of proximal tubular epithelial cells of the kidney, the epithelial cells of small pancreatic ductules and the luminal

surface of columnar epithelial cells of the jejunum and colon. The adrenal gland expressed P-glycopro-

tein in both the cortex and medulla, while no expression was identified in stomach, lung, ovary,

uterus, spleen, skin or central nervous system tissues. Using this same antibody, Sugawara et al. [63] reported P-glycoprotein expression in the placenta and adult adrenal gland, but not in foetal or neonatal adrenal tissues. The mouse homologue of the human

MDRI gene was found to be expressed in the mouse gravid uterus [64]. Induction of high levels of

expression occurred at a specific point during gesta-

tion, suggesting that P-glycoprotein expression is tightly regulated and may serve some important, as

yet undetermined, physiological function at the maternal-foetal interface. Expression of P-glycoprotein in these selective tissues, together with some other specific sites, has since been confirmed with a panel of monoclonal antibodies (Table 2). Currently, P- glycoprotein expression is recognised in three dis-

tinct types of normal human tissues; a subset of columnar epithelial cells, endothelial cells of capil- lary beds in specific anatomic locations and placental

trophoblasts [65]. The pattern of epithelial cell expression has been

extensively studied, using in situ hybridisation tech-

niques, and found to be primarily associated at the villus epithelium of the small intestine [66] and displays complementary expression with CFTR (cys-

tic fibrosis transmembrane regulator protein, another member of the ABC gene family). Thus, CFTR expression is observed in the crypts, but switches to P-glycoprotein expression as enterocytes differentiate

and move up the crypt-villus axis, to be maximal at the villus tips. This pattern of epithelial cell expression has led to the suggestion that P-glycoprotein

functions in tissues such as kidney, bowel and the

biliary tree to facilitate the excretion and/or mini- mise the absorption of toxic natural products that are ubiquitous in our diet and environment. The expression of P-glycoprotein in capillaries of the brain, endoneurium, testis and papillary dermis of the skin, but not in mid-sized and large blood vessels, is

associated with the continuous non-fenestrated ar-

rangement of endothelial cells at these sites and the recognition of these anatomic locations as blood- tissue barriers [65]. This may limit the penetration of

cytotoxic agents into these tissues, resulting in the creation of pharmacological sanctuaries. P-glycopro-

tein in the placenta suggests that it may be functioning as a component of the maternal-foetal barrier

1641. Alternatively, the closely regulated expression of the mdr-1 (mouse P-glycoprotein) gene in glandu-

lar epithelial cells of the gravid uterus suggests that P-glycoprotein expression in the uterus and placenta may be involved in some normal physiological

process that is important during gestation [64]. The specific location of P-glycoprotein expression,

therefore, indicates that it could be a factor that

limits intestinal absorption and diffusion, for in-

stance, across the blood-brain barrier, of xenobiot- its, as well a feature that participates in the biliary,

renal and intestinal clearance of drugs. Many of these non-intestinal aspects of P-glycoprotein function are covered elsewhere in this volume.

4. Non-P-glycoprotein efflux mechanisms

It is recognised that the intestinal secretion of

some drugs cannot be explained by P-glycoprotein.

Instead, a variety of other secretory systems may function as additional secretory, detoxifying systems and influence oral bioavailability.

4. I. Multidrug resistance-associated protein (MRP)

During the years following the experimental description of the phenomenon of multidrug resistance, its association with decreased cellular accumulation of the involved drug and the identification of P-

glycoprotein as the underlying mechanism, many laboratories around the world began to isolate their

own drug-resistant cell lines. Several laboratories succeeded in isolating MDR cell lines, which shared

many features with lines having a classical MDR mechanism (that is, involving P-glycoprotein), but in which overexpression of neither P-glycoprotein nor of mRNA from the encoding MDRl gene could be detected [67]. A line isolated by Danks et al. [68], referred to as showing atypical MDR, was found to

136 .I. Hunter, B.H. Him I Advanced Drug Delivery Reviews 25 (1997) 129-157

Table 2

Expression of P-glycoprotein in normal human tissues

Tissue

Nervous system

Neurons

Glial cells

Peripheral nerves

Reactivity

_

_

Tissue

Urinary system

Kidney glomeruli

Proximal tubules

Collecting ducts

Urothelium

Prostate

Reactivity

+ _

_

+ _

Haemopoietic system

Thymocytes

T-Lymphocytes

B-Lymphocytes

Leucocytes

Macrophages

Respiratory system

Bronchial cells

Pneumocytes

Gastrointestinal system

Oesophageal mucosa

Stomach

Small intestine

Large intestine

Pancreatic ducts

Pancreatic acini

Liver hepatocytes

Biliary canalicular

Vascular tissues

Heart-myocardium

Endothelium#

_

+

*

Skin

Keratinocytes

Sweat glands

Melanocytes

_

+ _

Adrenal gland

Cortical cells

Chromaffin cells

+

Endocrine system

Thyroid

Pancreatic islet cells

Reproductive system

Breast epithelia

Cervical mucosa

Endometrium

Ovary germ cells

Testes germ cells

Placenta trophoblasts

Connective tissues

Skeletal muscle

Smooth muscle

Fibroblasts

Chondrocytes

Adipocytes

+

-*

Immunoreactivities: + = homogenous staining; ? = weak and heterogeneous staining; - = undetectable levels. * = Distinctive patterns of

immunoreactivities are observed when HYB-241 and C,,, antibodies are used to examine the liver and skeletal muscle. HYB-241 stains

biliary ductal epithelium, but is unreactive with hepatocytes and skeletal muscle. However, Cz,s stains the biliary pole of hepatocytes,

myocardium and a subset of skeletal muscle fibres. #, Endothelial staining is extensive and covered in other reviews in this issue.

demonstrate abnormalities in the functioning of the nuclear enzyme topoisomerase II [69]. However, many other lines showed a clear deficit in drug accumulation and a full spectrum of cross-resistance, this phenotype became referred to as non-P-glycoprotein-mediated MDR [70].

A protein band of 190 kDa was detected in these cell lines and designated MRP [71] (for multidrug resistance-associated protein) [72]. Subsequent clon-

ing of the gene, and transfection studies, confirmed that the MRP gene was itself able to confer an MDR phenotype to the resistant cells [73]. More recently, data supporting identity between MRP and the glutathione S-conjugate transporter present in a variety of normal cell types has been produced [72,74,75]. Natural substrates for the transporter include leukotriene LTC, and reduced glutathione, GSH [76].

.I. Hunter. B.H. Hirst I Advanced Drug Delivery Reviews 25 (1997) 129-157 137

The tissue distribution of MRP is broadly similar

to that described for P-glycoprotein. Expression has been described in liver (canaliculus), erythrocyte membranes, heart [77], kidney and intestinal brush borders (781, and lung (including many lung tumour cell lines [67]), in addition to many selected tumour

cell lines 1721. MRP is a member of the ABC family of transpor-

ters [57], along with P-glycoprotein and CFTR, its substrate specificity, like that of P-glycoprotein, is

diverse, with many overlaps in substrates with P-

glycoprotein. Calcein and LTC, transport [76], how-

ever, appear to be good markers of MRP activity.

Modulators of MRP-mediated drug transport, also, include many of the modulators of P-glycoprotein, including verapamil and cyclosporin A. However,

these compounds appear to be less effective at inhibiting MRP, compared to MDR. Other com-

pounds found to inhibit MRP activity include BSO (buthionine sulphoximine, which reduces cellular glutathione stores), genistein, probenicid (an inhibitor of the organic anion transporter), vanadate and

ouabain [ 761.

4.2. BCECF md other ,fluorochrmne qf’hx

The fluorochrome 2’,7’-bis(2-carboxyethyl)-5(6)-

carboxyfluorescein (BCECF) is widely used as an indicator of intracellular pH, but its use may be

limited by dye loss from cells. This dye loss is mediated by an ATP-dependent transport mechanism and is inhibitable with a novel pharmacological

profile 1791. BCECF transport is present in several epithelial cell lines, including Caco-2, HCT-8, T84,

MDCK, which display P-glycoprotein-mediated drug transport, however, BCECF transport does not follow

the same pharmacological profile. Indomethacin is an effective inhibitor, along with other non-steroidal

anti-inflammatory drugs; however, the concentrations of these drugs that are needed to inhibit BCECF

transport are much greater than those used to inhibit cyclooxygenase activity, indicating that cyclooxygenase is unlikely to be involved in this transport

mechanism. The anion channel blocker, NPPB, inhibits BCECF transport, as does probenecid [79], the latter also inhibits fura- efflux and is an inhibitor of MRP. All of these compounds are agents that interfere with anion transport. However, Cl replacement experiments, and lack of effect of

stimulation of Cl- secretion, makes it unlikely that

secretory Cl channels are involved in BCECF transport. Further experiments showed the inhibition of BCECF efflux by P-glycoprotein substrates such

as vinblastine and actinomycin D, however, efflux was unaffected by the potent modulators of P- glycoprotein, nifedipine and reserpine. Thus, BCECF

efflux cannot be equated with P-glycoprotein or MRP function, thus, it could be speculated that it is mediated by an indomethacin-sensitive ABC trans-

port protein [21]. This efflux system is polarised in

intestinal epithelia, located to the apical membrane, analogous to P-glycoprotein. In contrast to the

anionic fluorochromes, the acetoxymethyl ester de-

rivatives (including BCECF, fura- and calcein) are

recognised to be substrates for P-glycoprotein [SO]. Other fluorochromes, such as Fluo-3, have been proposed as a simple assay for P-glycoprotein activi-

ty in single cell suspensions, especially in leukaemia

samples [81,X2], while calcein is recognised as a substrate for MRP (801.

4.3. Methhotrexnte qjj7ux

Methotrexate (MTX), a synthetic analogue of folic

acid, is a potent inhibitor of dihydrofolate reductase,

the enzyme that reduces dihydrofolic acid to the metabolically active form, tetrahydrofolate [83]. This

drug, initially employed in the treatment of

choriocarcinoma and leukaemia 1841, has been intro-

duced as a therapeutic agent in a wide range of non-malignant diseases, such as psoriasis [83], rheumatoid arthritis [SS], bronchial asthma [86],

primary biliary cirrhosis [87] and inflammatory bowel diseases [SS]. Because of the widespread use

of this drug, its transport has been subjected to extensive studies. Distinct membrane carriers that

mediate influx and efflux of this drug have been

described in various cell systems 1891. Altered transport of MTX across the membrane of leukaemic

cells may result in resistance to this drug, a common problem in cancer chemotherapy 1901. MTX has been widely used as a substrate in studies of intestinal transport of folates 1911. Competitive inhibition of the uptake of folic acid and 5-methyl tetrahydrofolate by MTX has been demonstrated in various preparations of intestinal tissue. These observations have suggested that all folates share a


common carrier in the intestinal luminal membrane

[92-941. Variations in the energetic competence of the cells

profoundly affects the expression of efflux multiplicity for MTX in L1210 (leukaemic) cells [95]. In

ATP-replete cells, a probenicid-, bromosul- phophthalein- [96] and &apamil-sensitive route

accounted for nearly 90% of [3H]MTX efflux,

indicating a MRP-like efflux mechanism. Whereas

efflux by a separate one-carbon reduced folate system (inhibitable by the folate analogue, NHS-

MTX) accounted for the remaining efflux seen. In

cells depleted of ATP, the NHS-MTX inhibitable system was responsible for all MTX efflux mea-

sured. Thus, it appears that two separate efflux systems for this drug exist, along with several

different carriers for folate [93] uptake into cells. Therefore, MTX absorption appears to be a complex

balance of several systems.

compound (ionised and unionised form) through the

unstirred water layer, a clearance based on the serum concentrations will be high. The intestinal clearance of acidic compounds, such as barbituric acid or

salicylic acid, calculated on the serum drug concentration will be correspondingly small.

The theory of ion trapping in the unstirred water

layer may explain the discrepancies in intestinal

clearance between these acidic and basic compounds, but it does not explain why theophylline also shows

a high intestinal clearance. In addition, the intestinal

clearance of quinidine appears to be too high to be explained by ion-trapping alone. Secretion of organic

bases and quaternary ammonium compounds has been reported [ 102,103] and there may be some non-diffusional pathways in exorption of theophyl-

line and the basic compounds to account for these high clearances [ 1041.

4.4. Role of ionic charge and pH microclimate in 5. Direct evidence for P-glycoprotein-mediated intestinal secretion intestinal secretion

Ionic charge at physiological pH appears to be a

major factor determining the intestinal clearance of charged drugs [8,97]. The ratio, in vitro, of trans-

epithelial flux from mucosal to serosal side against the flux from serosal to the mucosal side deviates

from unity for a number of organic acids and bases. Considering the luminal flow as the sum of laminar flows, the flow rate near the intestinal brush border is

slow and the fluid layer at the membrane boundary is essentially unstirred 1981. This layer can often be the

major resistance to the intestinal absorption of a

solute [99]. An acidic microclimate adjacent to the intestinal brush border [ 1001 may, in part, explain the

high clearance of basic compounds and the low clearance of acidic compounds in terms of ion-

trapping. The estimate of the pH microclimate in the unstirred water layer is 5.7 in duodenum [loll, with slightly higher values in the jejunum and the ileum. The unbound, unionised drug concentration in the unstirred water layer is assumed to be the same as that in serum at steady state. If the average microclimate-pH is one unit lower than the serum pH, the ionised concentration of the basic compounds such

as S-disopyramide and quinidine will be ten-fold greater in the unstirred water layer. If the rate of drug exorption is limited by the diffusion of the

5.1. Studies with cultured intestinal epithelial cells

Epithelial cells from different organs can be grown in culture to maintain polarised morphological and

functional characteristics that are typical of their role in vivo, where they form a barrier between the external and the internal environments [l]. The

MDCK cell line is frequently used to study the

development and maintenance of epithelial polarised morphology and functions [105]. In addition, the

more recent observation that intestinal cell lines, derived from colon carcinomas, can be induced, under controlled culture conditions, to differentiate

into mature absorptive enterocytes, has opened up

the possibility of developing new in vitro models for intestinal absorption and metabolism [106]. Some of these cell lines, such as Caco-2 [105,107-1091, HT29 [l lo], T84 [I 111, etc., which were obtained from human colonic adenocarcinomas, grow as monolayers of cells that, at confluency, initiate a process of differentiation. In Caco-2 cells, this leads to the formation of a brush-border with well developed microvilli, apical tight junctional complexes

[105] and a polarised distribution of membrane components, including enzymes [ 1121, receptors, transport systems [113,114], ion channels [115] and

J. Hunter, B.H. Hirst f Advanced Drug Delivery Reviews 25 (1997) 129-157 139

lipid molecules [l], similar to those found in absorp

tive small intestinal epithelial enterocytes in vivo

[ 1081. The transport and metabolism of several substances, both components of the diet and xenobiotic compounds, have been studied in Caco-2 cells [107-109,116,117]. More recently, Caco-2 and HT29 cells have been proposed as models for the intestinal transport of different classes of drugs

[17,25,107,108,118].

5.2. Direct evidence for P-glycoproteins us

secretory detoxibing mechanisms limiting drug

absorption

Immunohistochemical localisation of P-glycoprotein to the apical membrane of Caco-2 cells dem-

onstrates an additional membrane transport protein

with polarised expression in this cell line (Fig. 2). This polarised expression is consistent with the proposed role of P-glycoprotein as a secretory detoxifying system. The heterogenous staining pattern for P-glycoprotein, exhibited by Caco-2 cells, is typical of the pattern observed with a variety of brush border hydrolases, although, in our laboratory, we have not investigated if high brush-border hydrolase activity is

correlated with P-glycoprotein expression [ 181. The polarised expression of P-glyioprotein in

Caco-2 cell monolayers is accompanied by net

secretory (basal-to-apical) transport of [ ‘H]vinblastine sulphate (a typical MDR substrate) (Fig. 3). Thus, there is close agreement between the sub- cellular localisation and functional measurements.

Fig. 2. Immunohistochemical detection of P-glycoprotein in Caco-2 cell monolayers. Panel A shows indirect fluorescence staining with

MRK16 anti-P-glycoprotein antibodies of a Caco-2 cell monolayer viewed in the plane of the apical brush-border using confocal laser

scanning microscopy. Panels B-D are optical (x-z) sections perpendicular to the plane of the cell layers obtained by confocal laser scanning

microscopy imaging. Panel B illustrates an x-z section of a layer stained with MRK16, confirming the apical localisation of P-glycoprotein

approximately 20 (*m above the plane of the tissue culture insert filter (arrow). Panel C illustrates an x-z section of an identical layer stained

with a control, non-specific antibody and imaged using the same parameters as in panel B (arrow indicates the plane of the filter). Panel D is

an x-z section from the same cell layer as in panel C, imaged by increasing the detection sensitivity to reveal autofluorescence. The outline

of the cells, above the plane of the filter, and lack of fluorescence from the basally situated nuclei can clearly be seen. (From [IS]).

140 J. Hunter, B.H. Hirst I Advanced Drug Delivery Reviews 2.5 (1997) 129-l-57

z ~ 0.50

a n 0.25 .$

0.00 I

MRK16

0 60 120 180 240

Time lmin)

Fig. 3. Transepithelial flux of [‘Hlvinblastine across human

intestinal Caco-2 cell layers. Vinblastine flux, as a function of

time, in the secretory (Jh_.,, n ) direction exceeds that in the

absorptive (J,,+h, 0) direction. Secretory fluxes are reduced when

P-glycoprotein is inhibited by the apical addition of 50 pg ml-’

MRK16, anti-P-glycoprotein monoclonal antibody (0). Concen-

tration of vinblastine was IO nM. Data are illustrated as

meankS.E. (n = 3). (From [18]).

That this vectorial transport of vinblastine is the direct consequence of P-glycoprotein expression has been demonstrated by its reduction after treatment with monoclonal antibodies against P-glycoprotein, MRK16 [18].

These results from our laboratory provide direct evidence in support of the hypothesis that the drug

efflux mechanism at the apical surface of an intesti-

nal epithelium sustains a secretory detoxifying function and renders that epithelium relatively imperme-

able to a substrate [ 1191. The absorption kinetics of

substrate (vinblastine) from the apical (lumen) compartment to the basal (blood) compartment show: (a) a non-linear dependence of vinblastine absorption upon vinblastine concentration; (b) an increase in absorption correlated with the saturation of the net secretory vinblastine flux; (c) a similar increase in

absorption observed after inhibition of P-glycopro-

tein flux by a variety of inhibitors, including verapamil (Fig. 4), 1,9-dideoxyforskolin, nifedipine and

taxotere [19] and (d) a linear dependence of vinblastine absorption upon vinblastine concentration after P-glycoprotein inhibition. That P-glycoprotein

explains this drug efflux mechanism is shown by; (a) kinetics for substrate (vinblastine) transport (K, = 19 PM [ 18]), in Caco-2 cells, similar to those reported for P-glycoprotein in other systems. For example, a

1000 -

i z 800 - 7 E 3 g 600 -

,P z 400 - E

z 200 -

o-

0 10 20

[vinblastinel pM

30

Fig. 4. Vinblastine absorption is increased by verapamil. Kinetics

of vinblastine absorptive (A-B) flux across Caco-2 cell mono-

layers in the presence (@) or absence (0) of 100 pM verapamil.

Data are illustrated as mean2S.E. (n = 3). (From [I 191).

K,,, of 0.5 FM was reported for THP-adriamycin

efflux from K562 leukaemia cells [120]. Horio et al. [ 1211 estimated the K,,, to be 1 .l pM for trans-

epithelial transport of vinblastine by MDCK cells. Daunorubicin transport by various cell lines, using a flow through system [ 1221, gave a K, of 1.5 p,M. Vincristine transport across membrane vesicles pre-

pared from DC-3F/VCRd-5L Chinese hamster ovary cells selected for vincristine resistance gave a K,,, of 0.14 pM. Also, using rat liver canalicular membrane

vesicles K, values of 44 and 26 pM were observed

for [3H]daunomycin and [3H]vinblastine, respectively [60]; (b) inhibition of vinblastine secretion by

recognised pharmacological modulators of P- glycoprotein, including verapamil [ 123,124] and 1,9- dideoxyforskolin [ 191; (c) inhibition of vinblastine secretion by anti-P-glycoprotein antibodies [ 181 and

(d) localisation of P-glycoprotein to the apical membrane domain of the epithelial cells [ 181.

In the simplest model, P-glycoprotein is an additional component influencing the apical drug per-

meability (Fig. 5). Drug transport across an epithelial layer may be described as the product of the individual drug permeabilities across each membrane face, apical and basolateral, resulting in the observed unidirectional transepithelial fluxes. P-glycoprotein is

an additional “permeability”, contributing to the outward flux across the apical membrane, P,-,, resulting in the transepithelial flux in the secretory direction, Jb_a, exceeding that in the absorptive

.I. Hunter, B.H. Him I Advanced Drug Delivery Reviews 2.5 (1997) 129-157 141

b

J Jc-a a-c

J Jc-b b-c J a-b

Ja-b < Jb-a

C

inhib J b-a

J a-b

J Jb-a a-b =

Fig. S. Model of P-glycoprotein substrate absorption. (a) Transepithehal permeabilities for a drug. J,$.., and .I.,% are the sums of

transmembrane permeabilities, J,. L plus J,_, and J,_, plus J,_,, respectively. (b) In the presence of P-glycoprotein, JC .,l is increased as the

drug is actively pumped out across the apical membrane, illustrated by increased secretory flux, Jbm4. This active removal of drug results in

reduced J,)_,, i.e., reduced absorption. (c) Inhibition of P-glycoprotein activity results in a decrease in J,_, as efflux (JL_,,) is reduced, while

absorption is no longer limited by secretion, such that J,_, increases.

direction, J,_,. Thus, the absorption of P-glycoprotein substrates is limited to an extent that is depen-

dent upon their passive permeability balanced against their affinity for P-glycoprotein (see below). Inhibi-

tion of P-glycoprotein function reduces P,_,, reduc-

ing the flux in the secretory direction, J,_,, such that, assuming that there are no other transport systems in this simple model, it is equal and opposite to the flux absorptive direction, J,_,. In “normal” absorptive

situations, where luminal drug concentrations are greater than plasma concentrations, this will result in an observed increase in drug absorption.

The absorptive permeability of vinblastine (mea-

sured at 10 nM) across Caco-2 cell monolayers, with functional P-glycoprotein activity, is around 0.4.

IO-’ cm hh’. This value is comparable to those permeability values obtained for acetylsalicylic acid

(0.9. lo-* cm h-l) and practolol (0.3 * lop2 cm

hh’) in the identical Caco-2 cell model [125]. The octanol /water partition coefficients [log D (log octanol/water partition coefficient [ 1251); - 2.14 and - 2.57, respectively] emphasise the relative

hydrophilicity of both acetylsalicylic acid and prac-

tolol. In the presence of P-glycoprotein inhibitors, Caco-2 cell monolayers have an increased per-

meability to vinblastine (Fig. 4). Comparison of the

passive permeability of vinblastine, in the absence of

P-glycoprotein function, (2.83 * lo-* cm h-‘) to lipophilic drugs such as felodipine (8.17 * lo-” cm

hh’) and dexamethasone (7.7. lo-’ cm h-‘) (log D

values of + 1.89 and + 3.31, respectively) empha- sises the native permeability of the apical cell border

of the intestinal Caco-2 to vinblastine, as would be expected from a priori considerations of its molecu-

lar structure, indicated by its log D value of + 2.9

[126]. The transepithelial permeability of a substrate for

P-glycoprotein will be dependent not only on the

passive permeability of the apical membrane to the substrate, but also on its affinity for the active transport site and the maximal capacity of P- glycoprotein contained in the apical membrane. A

simple pump-leak balance will exist at the apical membrane (Fig. 5). The function of P-glycoprotein

142 .I. Hunter, B.H. Him I Advanced Drug Delivery Reviews 25 (1997) 129-157

10-4 -I

c 0

10-s - v

‘;;

‘i s

t 10-e - A

t

Ii 10-l - 0

0

10-I 2

I - I - I1 I .I I (

-6 -4 -2 0 2 4

log D

Fig. 6. Apparent permeability coefficients in the Caco-2 model as

a function of lipophilicity. The apparent permeability across Caco-

2 cell monolayers for a range of drugs (0) with differing

lipophilicity were determined and plotted as a function of octanoll

water partition coefficient (log D). Values are taken from Artur-

sson and Karlsson [125]. Data for vinblastine (A), from Hunter et

al. [I 191, are plotted on the same scale. Verapamil (100 PM)

increased the apparent permeability of vinblastine (V) to values

closer to those predicted on the basis of its log D value.

may explain divergence from the predicted permeability based upon drug octanol/ water permeabili-

ty (log D) values (Fig. 6). Inhibition of P-glycopro-

tein function normalises this relationship based on the drug’s physicochemical properties. Limitation of

absorptive uptake will be most pronounced with P-glycoprotein substrates when diffusional influx is

low (at low luminal concentrations and/or with substrates with an intrinsically low passive permeability to the apical membrane lipids) and when ATP-dependent drug efflux is not limiting with respect to diffusional input. This condition is met if

the drug has a high affinity with respect to ATP-

dependent export and the cytosolic drug concentrations are non-saturating. A minimal effect of P- glycoprotein in limiting absorption will be apparent

with high diffusional fluxes (resulting from high intrinsic passive permeability or elevated external concentration) with respect to drug efflux (when the

pump is saturated with respect to substrate or when substrates are of a low affinity with respect to export).

Studies reported from our laboratory have concen- trated on vinblastine absorption [119], a typical MDR drug and P-glycoprotein substrate. Multidrug

resistance is associated with cross-resistance to a number of cytotoxic drugs, which include natural

products such as anthracylines, other Vinca alkaloids, epipodophylotoxins, colchicine, and actinomycin D,

but not to drugs such as bleomycin, methotrexate, or

alkylating agents. Thus, those natural products that demonstrate cross-resistance may also interact with

intestinal P-glycoprotein, with limited absorption at subsaturating concentrations. Characterisation of

cyclosporin A transport across Caco-2 cell layers has suggested that it is transported primarily by passive diffusion. However, a polarised efflux (presumably a

P-glycoprotein) located at the apical membrane can attenuate the net apical-to-basal transport. It was,

therefore, concluded that the presence of P-glycoprotein in the intestinal mucosa could contribute to the

reduction in oral bioavailability that is frequently observed with CsA therapy [127]. This hypothesis was further investigated in a recent study by Fricker

et al. [128], who, along with Caco-2 monolayer

studies, found a strong correlation between the decreased absorption of cyclosporin A in healthy volunteers and mRNA expression levels for P- glycoprotein along the gastrointestinal tract. Ver-

apamil is orally active and it has a low affinity with respect to inhibition of ATP-dependent drug export, epithelial vinblastine secretion, and potentiation of

vinblastine-mediated cytotoxicity [45,53,129]. Ver-

apamil is itself transported by P-glycoprotein [ 1301. Studies using rat intestine [26] have shown that

verapamil is efficiently secreted as a substrate of intestinal P-glycoprotein in the upper intestine and colon. Rat ileal P-glycoprotein does not transport

verapamil efficiently, but transports propantheline. The rat intestine thus appears to have multiple P- glycoprotein transport systems with distinct substrate

specificities, depending on intestinal site [26]. Thus, bioavailability problems may be due to P-glycoprotein systems at different intestinal sites with different

drug specificities. The inhibition of P-glycoprotein by forskolin and its analogue, 1,9-dideoxyforskolin, has been documented [19]. These agents are highly lipophilic and their relatively low affinity for P-

glycoprotein suggests that cellular forskolin accumulation will not be limited by such a mechanism. Similar considerations apply to the whole range of P-glycoprotein substrates, e.g., nifedipine and taxotere, a more potent analogue of the anticancer agent, taxol [117]. Non-competitive mechanisms of P-

.I. Hunter, B.H. Hirsr I Advanced Drug Delivery Reviews 25 (1997) 129-157 143

glycoprotein inhibition would imply that such agents

would not be subject to absorption restriction, de- spite interacting with P-glycoprotein.

The general principle illustrated by absorption

limitation of a single substrate of one ATP-dependent export pump may be applicable to other sys-

tems. For drugs with unexpectedly poor oral absorption profiles, investigation as to whether ATP-depen-

dent export functions render the intestinal membrane effectively impermeable is indicated.

5.2. Peptide secretion by Caco-2 intestinal

epithelia

An anomalous increase in the permeability with

increasing concentration of some peptides, prepared as an homologous series of peptides for investigation

of oral bioavailability, led to the investigation of

peptide transport across Caco-2 cell monolayers by Burton et al. [ 13 11. Using similar techniques to those described above, they described results which

showed that this increase in permeability was apparently due to saturation of an apically located transporter, which, at low peptide concentrations, opposes

transport by returning substrate to the luminal medium. Two peptides were investigated, [‘“C]AcPheNH, (a non-substrate for the efflux system) and [ ‘“C]AcPhe(N-MePhe),NH, (as a substrate

for the efflux system). The permeability of [‘4C]AcPhe(N-MePhe),NH, was enhanced by addi-

tion of the MDR-modulator, verapamil (Fig. 7). The evidence presented supports the involvement of an

apically polarised transporter in Caco-2 cells for the peptide [ “C]AcPhe(N-MePhe),NH,. Thus, the increase in permeability of the peptide with increasing

concentration is consistent with saturation of this efflux component. Similarly, the increase in permeability, in the presence of verapamil, is suggestive of a specific resistance to the transporter which can

be competitively inhibited. A more recent study of transepithelial transport of these peptides [132] has shown the inhibition of basal-to-apical flux of

[“C]AcPhe(N-MePhe),NH, by the surfactants Polysorbate-80 and Cremophor-EL, again, as with the verapamil experiments, only [‘4C]AcPhe(Ne- MePhe)? transport was affected at low surfactant concentrations, thus, a further indicator of P- glycoprotein involvement. Although the identity. of the putative transporter could not be proved through

I AZ

Ei 2 z 0.0010 ‘- z

3 E t = 0.0005

S :

B a a

IA) (I31 ICI

Fig. 7. Apparent permeability coefficients of peptides across

Caco-2 cell monolayers. AcPhe(NMePhe),NH? is secreted by

Caco-2 cell layers by a verapamil-sensitive mechanism, which

limits absorption. (A) apparent permeability of 50 FM AcPheNH,;

(B) apparent permeability of 20 pM AcPhe(NMePhe)zNH,; (C)

apparent permeability of 20 (LM AcPhe(NMePhe),NHz +

verapamil (265 )*M in the J,,, direction, and 170 FM in the .I,,,,,

direction). Absorptive flux, J,,, (open bars), secretory Rux, _I,,_

(hatched bars). Values are mean (kS.D.) for at least three

experiments. (From [ 1311).

these studies, several lines of evidence suggest that it

may be related to P-glycoprotein; i.e., its presence in

the Caco-2 cells and inhibition by verapamil, at the same concentration as vinblastine transport. How- ever, involvement of MRP and/or other efflux

systems cannot be excluded at this stage. With respect to structural specificity of substrates, several

reports have shown peptides to be substrates for

P-glycoprotein. Among these, cyclosporin is known to be an effective inhibitor (as mentioned earlier

[53]). Similarly, the cytotoxic peptide acetyl-leucyl- leucyl-norlucinal (ALLN) was recently shown to be transported by P-glycoprotein in Chinese hamster

ovary cells [ 1331. These experiments point to the potential importance of an apically polarised active resistance to peptide absorption in Caco-2 cells.

5.4. Implication of P-glycoprotein-like mechanisms

in intestinal secretion of other drugs

5.4.1. Digoxin

The cardiac glycoside, digoxin, shows altered pharmacokinetic and dynamic changes in the presence of P-glycoprotein modulators. Oral bioavailability of this drug is high, however, several studies have shown drug interactions between digoxin and

verapamil [ 1341, nifedipine and quinidine [ 1351. Major alterations in plasma digoxin levels, increased

by 60-SO%, and increased plasma half-life, from 34

to 41 h, were evident [134,136]. The intestinal permeation of cardiac glycosides,

such as digoxin and digitoxin, has been thought to be solely determined by the relative polarity of the glycoside molecule; thus, absorption inversely paral-

lels polarity [ 1371. Lauterbach [ 1381 has discussed evidence that shows that oral absorption of cardiac

glycosides is not simply described by simple diffusive processes. A strict inverse relationship between

polarity and absorption rate does not hold for all

glycosides. Indeed, the absorption coefficient of

digoxin decreased as a function of time after con- tinued oral dosage in the rat [ 1381. Such discrepancies may be explained by the observations of intestinal secretion of digoxin in isolated preparations of

intestinal mucosae maintained in vitro. [ 1381 An important problem encountered in digitalis

therapy is that associated with a substrate-specific

elimination pathway that is subject to physiological variability and changes in disease [ 1361. Thus,

maintenance of stable plasma concentrations at con- stant dosage has been problematic. For digoxin and

other renal-dependent glycosides, it has been assumed that the only pharmacokinetically significant

route of elimination is renal. Intestinal secretory capacity will not only affect absorption kinetics, but

will also contribute to whole body clearance of cardiac glycosides, particularly with renal insuf-

ficiency and in the elderly. A recent study, using Caco-2 cell monolayers,

demonstrated that transepithelial basal-to-apical

[3H]digoxin flux exceeds apical-to-basal flux, resulting in net basal-to-apical digoxin secretory trans-

port [139]. Cellular uptake of digoxin was greater across the basal surface of the cells. Net secretion of [3H]digoxin was subject to inhibition by digitoxin

and bufalin, but was not inhibited by ouabain, convallatoxin and strophanthidine (all at 100 mM). The P-glycoprotein inhibitors, verapamil, nifedipine and vinblastine, all abolished net secretion of digoxin. The increase in absorptive permeability, and cellular digoxin uptake upon P-glycoprotein inhibition, showed that the intestinal epithelium was rendered effectively impermeable by ATP-dependent extrusion at the apical surface. A model of [“Hldigoxin secretion by the intestinal epithelium is

likely to involve both diffusional uptake and Na+- K+ pump-mediated endocytosis, followed by active

extrusion at the apical membrane. Data using LLC-PKl cells, transfected with the

human MDRl gene, showed that digoxin is a

substrate for P-glycoprotein [140]. Digoxin is a drug of high bioavailability, due to its lipophilicity, thus the effects of P-glycoprotein on absorption may be difficult to detect if its concentration in the gastroin-

testinal tract is high, saturating for P-glycoprotein, since any efflux would be masked by high passive

permeability. Thus, the major effects on digoxin

pharmacokinetics by the use of modulators, is its decreased renal elimination. Intestinal exorption,

however, cannot be ruled out as an elimination pathway, along with renal and biliary excretion, resulting in its rapid elimination from the body and,

hence, its short half life.

5.4.2. Ranitidine

The histamine Hz-receptor antagonist, ranitidine,

exhibits secondary peaks in the oral concentration- time profile after a single dose in humans and in rats.

Proposed mechanisms responsible for this phenomenon include enterohepatic recirculation and delayed gastric emptying of a portion of the oral dose.

However, less than 2% of an oral dose of ranitidine is recovered in the bile. Double peaks in the con-

centration-time profiles after direct administration of ranitidine into the duodenum and jejunum of human

subjects indicated that factors other than gastric emptying are involved. Other mechanisms have been suggested, including post-absorptive storage and release, and discontinuous or site-specific absorption

of drug [141]. Significant pharmacokinetic interactions have also

been demonstrated between ranitidine and several

drugs, including the P-glycoprotein modulators, nifedipine and theophylline (for a review see Kirch et al. [142]). Thus, transepithelial transport of

ranitidine across Caco-2 cell monolayers has been investigated. Transepithelial bi-directional fluxes of

ranitidine showed marked asymmetry, flux in the basal-to-apical direction being five times greater than that in the absorptive apical-to-basal direction. Cel- lular accumulation across the basal surface was also greater (three-fold) than that across the apical surface [ 1431. Inhibitors of P-glycoprotein-mediated transport, 1,9 dideoxyforskolin, verapamil and

J. Hunter, B.H. Hirst I Advanced Drug Delivery Reviews 25 (1997) 129-157 145

MRK16 monoclonal antibodies (Hunter and Hirst, unpublished results), reduced net ranitidine secretion. Also ranitidine inhibited transepithelial secretion of the P-glycoprotein substrate, vinblastine. These results are consistent with ranitidine being a substrate for P-glycoprotein and, thus, being subject to reduced oral bioavailability by this mechanism.

5.4.3. Fluoroquinolones

The 4-quinolone and related fluoroquinolones are

a group of synthetic antibacterial agents in which

clearance in humans is due primarily to renal excretion. However, gastrointestinal secretion into the

intestinal lumen is a second quantitatively important route for elimination [144]. In human volunteers,

18% of an i.v. dose of ciprofloxacin is eliminated by intestinal secretion. In as much as biliary elimination of ciprofloxacin and its metabolites is small and

because only a minor component of ciprofloxacin in faeces are metabolites, it is apparent that an intestinal

secretory mechanism for this drug must exist. Intesti- nal secretion is not restricted to ciprofloxacin. Both

fleroxacin and temofloxacin show significant gastrointestinal secretion into faeces in humans [14.5],

whereas, with ofloxacin, only approximately 4% of a p.o. dose is eliminated via faeces and 80-95% is

recovered via urine. Thus, the relative capacity and

mechanism of intestinal secretion of the various fluoroquinolones remains to be defined.

In 1993, Griffiths et al. [20] demonstrated the active transepithelial secretion of the fluoro-

quinolone, ciprofloxacin, by human intestinal Caco-2 cell layers in vitro, demonstrating some similarities with a P-glycoprotein-like mechanism. A later study

demonstrated similar secretion of norfloxicin and perfloxacin in Caco-2 intestinal epithelia [ 1461.

Active net secretion of norflaxacin displayed satura-

tion kinetics with V,,, and K,, values of 36 nmol cm -’ h-’ and 1.4 mM, respectively. In contrast, transepithelial pefloxacin fluxes were large, showed marked saturation, while the direction of the net flux

was variable and small relative to the transepithelial fluxes. Norfloxacin, perfloxacin and ciprofloxacin were all subject to accumulative transport across the basal surface of Caco-2 cell layers. A number of 4-quinolones and fluoroquinolones are capable of inhibition of both net secretion of ciprofloxacin and cellular accumulation across the basal-lateral cell surface. Cinoxacin, a 4-quinolone, may selectively

inhibit exit from the cell across the apical membrane. Cross-competition studies suggest that fluoroquinolones may compete for a common carrier at the

basolateral membrane. It is likely that the mechanism of transepithelial secretion involves a common accumulative transport at the basal membrane, followed by facilitated exit across the apical membrane. Pefloxacin may interact with a brush border carrier,

for which norfloxacin and ciprofloxacin are poor

substrates, enhancing the absorptive flux of this

fluoroquinolone.

5.4.4. P-adrenoceptor antagonists

Celiprolol is a “cardioselective” P-adrenoceptor blocking drug with intrinsic sympathomimetic activi-

ty and a weak vasodilator effect [ 1471. The drug exhibits dose-dependent bioavailability in rats and

man after oral administration [147,148]. In man, the bioavailability was 30% after an oral dose of 100 mg

and 74% after a 400-mg dose [ 149-1.5 I]. The differences in bioavailability cannot be explained by

altered dissolution, first pass metabolism or changes in excretion. Therefore, the possibility that celiprolol

is actively transported across the intestinal epi-

thelium has been considered.

The transepithelial transport of celiprolol has been

investigated in Caco-2 cell monolayers [ 1521. The basal-to-apical transport of [ “C]celiprolol (50 mM)

was five-times greater than apical-to-basal transport. In the presence of an excess of unlabelled celiprolol, the basal-to-apical transport was reduced by more than 80%, whereas the apical-to-basal transport remained unchanged. Net celiprolol secretion, ob-

tained in the concentration range 0.01 to 5 mM,

displayed saturable kinetics with an apparent K,, of 1.0 mM. This result is consistent with saturable

active secretion and provides an explanation for the

dose-dependent bioavailability of celiprolol. The secretion of celiprolol is sensitive to pH and decreased in the absence of sodium and in the presence

of ouabain, suggesting that the transport was coupled to proton and sodium gradients. The secretion is inhibited by substrates for P-glycoprotein (vinblastine, verapamil and nifedipine) and either inhibited or stimulated by typical substrates for the renal organic cation-H+ exchanger (cimetidine, N-methyl- nicotinamide, tetraethylammonium and choline), suggesting that there are at least two distinct transport systems. The secretion of celiprolol is also

146 J. Hunter. B.H. Hirst I Advanced Drug Delivey Reviews 25 (1997) 129-157

inhibited by other P-adrenoceptor blocking drugs (acebutolol, atenolol, metoprolol, pafenolol and propranolol) and by diuretics, acetazolamide, chlorthalidone and hydrochlorothiazide, suggesting that

the clinically observed effect of chlorthalidone on the bioavailability of celiprolol occurs at the level of the

intestinal epithelium.

Celiprolol has intermediate lipophilic properties in

comparison with other P-adrenoceptor blocking

drugs, with an apparent partition coefficient of + 0.2 at pH 7.4. It is a weak base with a pK, value of 9.7 and, consequently, it is present mainly as an organic

cation at physiological pH. Intestinal secretion of organic cations, including other P-blockers, have

been observed in vivo, but so far the mechanisms behind the secretion have not been elucidated

[103,153-1551. The P-blockers, acebutolol, propran- 0101 and pafenolol, were found to be strong inhibitors

of celiprolol transport when added to the apical side of the Caco-2 monolayers. These results support the

hypothesis that various P-blocking drugs are actively

secreted across the intestine. With the exception of

celiprolol itself, none of the P-blockers affected the

basal-to-apical transport of celiprolol after basal addition. This suggests that their inhibitory effects were different from those of the typical substrates for the organic cation-H+ exchanger and P-glycoprotein.

The study by Karlsson et al. [152] shows that celiprolol is actively transported across the human

intestinal epithelium and that the transport in the

basal-to-apical direction (secretion) is larger than that in the apical-to-basal direction (absorption). These

results offer an explanation for the enhanced absorption of celiprolol at high doses and its non-linear absorption. The secretion of celiprolol is altered by

typical substrates for P-glycoprotein and the organic cation-H+ exchanger, suggesting that the secretion is dependent on multiple transporters.

Diuretics such as chlorthalidone are often co-

administered with P-blocking drugs. Since many diuretics are cations, it is likely that these drugs would also interact with the transport of celiprolol. Indeed the three diuretics, chlorthalidone, acetazolamide and hydrochlorothiazide, all inhibited celiprolol

secretion after apical addition. In addition, hydrochlorothiazide stimulated secretion after basal addition, suggesting that this drug may reduce the absorption of celiprolol. These results may be of clinical significance since the absorption of celiprolol

is altered by co-administration of chlorothadone

[ 1561. Further studies are needed to confirm this hypothesis.

In preliminary bioanalytical/pharmacokinetic studies, expressed double peaks have been detected

in several volunteers following oral administration of

talinolol. Another aspect which also turned out to be

of relevance was the phenomenon of a dose dependence of absorption, with increased absorption seen

at higher doses. This has also been observed with celiprolol (see above). For the P-adrenoceptor agon-

ist, pafenolol, the dose-dependent bioavailability was found to be due to non-linear intestinal uptake. For

pafenolol, it was found that intestinal secretion (exorption) accounts for approximately 25% of an iv. dose [155].

The occurrence of two “absorption windows” appears to be the most plausible explanation for the

observed phenomenon of discontinuous drug input, with a minor fraction of the dose being absorbed in

the more proximal part of the gastrointestinal tract

(stomach or upper region of the small intestine), followed by cessation or substantial reduction of the

amount absorbed per time unit until the drug reaches the ileo-caecal region, where probably the major

amount is absorbed. This may be explained by a varying extent of intestinal secretion due to varying

amounts or activities of the respective carrier system in different regions. However, although discontinuous input models are applicable to most of the profiles, the extent of discontinuity is highly variable,

indicating that the involved carrier system shows considerably higher variability than that observed for

other “secretion” processes. At low doses, secretion of talinolol in the intestinal tract serves to limit the absorption of talinolol. As the secretion system

begins to saturate, overall absorption increases, resulting in non-linear increases in bioavailability. Once secretion is totally saturated, passive diffusion

dominates and a new equilibrium is reached, with linear absorption now being evident. It appears highly probable that talinolol is transported by P- glycoprotein, as, in the presence of verapamil, basal- to-apical transport of talinolol was clearly inhibited, using Caco-2 cells [ 1571.

5.4.5. Amoxicillin

Pharmacokinetic results of the study by Westphal et al. [135] showed that p.o. preadministration of nifedipine (20 mg) induced a significant increase in

J. Hunter, B.H. Him I Advanced Drug Delivery Reviews 25 (1997) 129-1.57 147

both the amount of amoxicillin absorbed and its rate

of absorption. The mean values for C, (clearance) and V,, (volume of distribution) were unchanged by nifedipine. Thus, it was proposed that neither metabolism nor excretion (renal or biliary) of amoxicillin were modified by nifedipine. Hence, it was postulated that enhancement of amoxicillin bioavailability was related to a promoting effect of the calcium channel blocker on the intestinal absorption

efficiency of this antibiotic.

5.4.6. Hydrophilic compounds

Unlike the classical MDR substrates, which are

lipophilic, the other examples of drugs that undergo intestinal secretion, described above, display con-

siderably greater water solubility. This highlights that, in addition to a P-glycoprotein-like apical exit pathway, when undergoing transepithelial secretion, it is likely that these drugs are recognised by

basolateral transport systems, allowing access to the enterocytes (Fig. 7) as in the case of the fluoroquinolones described above [146]. These may show

similarities to the various ion-coupled symport and antiport systems that are most completely described

in renal tissues. Thus, secretion of these hydrophilic drugs, in particular, is best described by a multi-step

process.

6. Pharmacokinetic interactions between P- glycoprotein modulators and drugs known to interact with P-glycoprotein in vivo

The specific distribution of P-glycoprotein in human tissues raises the potential for kinetic interac-

tions between P-glycoprotein modulators and its

substrates, anticancer drugs (MDR-related drugs). Unfortunately, few clinical trials combining P- glycoprotein modulators and MDR-related drugs have involved pharmacokinetic monitoring of both agents [ 1581. Most studies have been concerned with obtaining clinically relevant levels of modulators in the patients involved. Hence, it seems important to

know first whether target concentrations of the modulator obtained in vitro are achievable in vivo, and second whether the co-administration of the reversing agent leads to kinetic alterations of the anticancer drug and, thereby, whether it can be associated with clinical events (therapeutic or toxic). In general, results from these studies, even when

consistent with modulation of P-glycoprotein func-

tion, do not allow definition of the tissue involved

[159,160]. Among the modulators, verapamil was the first

agent that was shown to reverse the MDRl phenotype in laboratory models [59] and that was intro- duced into the clinical setting. Unfortunately, as for many of the known modulators, its use is severely limited by toxic events, in particular its cardiovascu-

lar effects, which stimulated the development of the dex- (D-) isomer, which is less cardiotoxic [161] but

equally potent as a P-glycoprotein modulator [ 162-

1641. Nevertheless, some pharmacokinetic interactions between verapamil and anticancer drugs have been reported in animals and man, as highlighted by

the studies of patients that are summarised below. Co-administration of verapamil in five patients resulted in a 30% decrease in doxorubicin clearance, associated with a prolonged half-life [165]. On the other hand, verapamil had no effect on epirubicin

kinetics, although the anthracycline metabolism was altered; resulting in an increase in the area under the curve (AUC) for these compounds [166]. n-Ver-

apamil, in combination with etoposide and doxorubi- tin, resulted in a 10% increase in etoposide steady-

state concentration and a 2-fold increase in the concentrations of doxorubicin and doxorubicinol

[ 1671. Similarly, n-verapamil, in combination with paclitaxel (i.v.), resulted in a 2-fold increase in AUC and a 50% decrease in paclitaxel clearance [168].

Nifedipine, another commonly used MDR

modulator, produced a large (30%) increase in the terminal half-life of vincristine, combined with a

reduction in total clearance [169]. Bepridil, another calcium channel blocker, increased plasma concen-

trations of vinblastine compared to administration of the alkaloid alone [ 1701.

As regards the results with these calcium channel blockers, it is difficult to speculate whether the interactions involve modulation of P-glycoprotein in vivo and, if so, modulation at which sites of expression are important for the kinetic alterations observed. Anthracyclines (doxorubicin, epirubicin) and

Vinca alkaloids (vincristine, vinblastine) are mainly eliminated by the liver [ 171,172]. The modulation may thus be, in part, a reflection of inhibition of hepatic and biliary P-glycoprotein function. This

does not exclude intestinal exorption as a clearance mechanism, which may be modulated by the calcium antagonists. Nevertheless, it should be recognised


that the interactions noted in these studies of patients may be explained by other features, such as haemo- dynamic alterations, metabolic inhibition or effects on ion channels, either directly or indirectly.

Cyclosporin is one of the most potent MDR reversing agents and clinically it is the modulator that raises most interest [53]. Alterations of etoposide kinetics by cyclosporin were reported in

patients administered paired courses of etoposide and both drugs [173]. In 10 patients whose cyclosporin

plasma concentrations exceeded 2 kg/ml, systemic exposure of etoposide (AUC) increased by 80%, with a 46% decrease in total clearance and a 108%

increase in plasma half-life. In addition, both renal and non-renal clearances of epipodophyllotoxin were

altered (38 and 52% decreases, respectively). These kinetic modifications were associated with a more

severe leukopaenia. The authors of this study concluded that this pharmacokinetic interaction was consistent with alterations of P-glycoprotein activity in normal tissues. A similar observation has been reported with doxorubicin in 12 patients for whom

paired pharmacokinetics of the anthracycline were carried out [ 1741. The addition of cyclosporin re-

sulted in a 55% increase of doxorubicin AUC and a

350% increase of the doxorubicinol metabolite [ 1751. Adverse neurological symptoms have been observed,

which, upon investigation in the rat .[176], were postulated to be indicative of P-glycoprotein modulation at the blood-brain barrier.

When cyclosporin A was recognised as the most potent of the first generation of MDR modulators, numerous cyclosporin analogues were screened for their ability to inhibit the P-glycoprotein efflux

pump. PSC 833 emerged as the leading pre-clinical candidate, with greater potency as a modulator than

cyclosporin A and no immunosuppressive activity or nephrotoxicity. The latter point is of interest in

considering a possible nephrotoxic mechanism related to renal P-glycoprotein inhibition (Simmons et

al., this volume). In a phase I clinical study [177], PSC 833 was

administered with etoposide [178], resulting in in-

creased toxicity, including severe ataxia. Haematological toxicity required dose reduction of the etoposide by 25%, as the dose of PSC 833 was

increased. Among patients in whom serum concentrations of PSC 833 greater than 1.7 PM (2000 ng/ml) were achieved, the average increase in

etoposide AUC was 89%, with clearance decreased

by 46%. Additional phase I studies have been conducted at Stanford [179,180]. These used a combination of PSC 833, in two separate trials, with either etoposide or paclitaxel. The effects on

etoposide kinetics were dramatic; AUC increased 2.2-fold, clearance fell by 57% and serum half-life increased by 57%. In both of these trials, pharmacokinetic parameters were similar, with a reduc-

tion in drug dose of 50-60% yielding equivalent levels of myelosuppression when compared to full dose chemotherapy alone. Ataxia was again ob-

served, but was found to be due to PSC 833 alone rather than modulator effects on the blood-brain barrier P-glycoprotein [ 176,18 11.

The absorption and exorption of etoposide has also been studied in animal models. The oral bioavailability of etoposide is about 10% in rats [182],

indicating that the rat may be a useful model in which to evaluate modulators. In vitro data, with rat everted gut sacs, support the hypothesis that etoposide is secreted by P-glycoprotein after its

absorption across the intestinal epithelium [ 1831. Inhibition of P-glycoprotein by quinidine, the unhy-

drolyzable ATP-analogue (AMPPNP), or the mono-

clonal antibody, C,,,, reduced etoposide exorption

into everted gut sacs, with a substantial increase in its absorption. In a rat perfused intestine in vivo

model, quinidine infusion (i.v. 1 mg/h) decreased the total clearance of etoposide by 38% [183]. In the absorption study, the etoposide concentration in

serum increased by more than 2-fold during quinidine infusion, This suggests that quinidine not only decreased the rate of etoposide elimination but also

increased the rate of etoposide absorption. Both the total and the intestinal clearance of etoposide were decreased by the infusion of quinidine. The change

noted in intestinal clearance (24 mll ’ h- ’ kg- ’ ) was too small to explain the magnitude of change observed in total clearance (- 100 ml-’ h-’ kg-‘). Apparently, quinidine had inhibited the other path-

ways of etoposide elimination. Concentration-dependent exorption of other com-

pounds from the rat intestine have been investigated by Bair et al. [ 184,185]. Quinidine itself, theophylline and S-disopyramide have all been implicated as

having saturable efflux mechanisms. This may be due to the presence of an active transport process for these compounds, however, the nature of the carrier

.I. Hunter, B.H. Him I Advtmced Drug Deliver?: Reviews 25 (1997) 129%IS7 149

in this study was not elucidated. Similarly, digoxin is

secreted by the intestine in vitro [ 138,186], influencing its oral absorption. Recent studies with Caco-2 cell layers (see above) suggest that intestinal P- glycoprotein may be a significant component of this intestinal clearance of digoxin [ 1391.

It is worth noting that among MDR-related drugs, only etoposide is available as an oral formulation, with two others under investigation (vinorelbine and,

to a lesser degree, idarubicin). It may be speculated that P-glycoprotein is a factor that limits the oral

bioavailability of the majority of MDR compounds, in addition to their physico-chemical properties, or first pass metabolism effects. Modulation of intestinal P-glycoprotein via pharmacological agents, or pharmaceutical formulations, including encapsulation or use of excipients with recognised MDR-modulator activity, such as Cremophor-EL, is an attractive mechanism to enhance the absorption of MDR-related drugs. Hence, increased oral absorption of etoposide in rats was noted upon co-administration

of SDZ PSC 833 [ 1781.

7. Interactions between P-glycoprotein and drug metabolism

There is a growing awareness of the potential for

intestinal metabolism of drugs. The implications of the many and varied metabolic possibilities have yet to be elucidated. The potential contribution of glutathione conjugation, via glutathione S-transferase, with subsequent transport by an S-glutathione-conjugate ATPase is recognised [ 1871. Cytochrome P450 (CUP) phase I enzymes are also expressed in the intestine, particularly members of the CYP3A gene family, which constitute 70% of total P450 activity

in the human intestine [ 1881. There is some con- cordance of P-glycoprotein and CYP3A substrates

and modulators, including verapamil, nifedipine, cyclosporin A and taxol, although the complemen- tarity of substrates/modulators is incomplete [189]. Not only may these two genes, involved in drug metabolism and elimination, be expressed in the same cells and share substrates, there is evidence for coordinated regulation. Thus CYP3A4 and P- glycoprotein are both up-regulated by treatment of colonic carcinoma cells with numerous drugs, including reserpine, isosafrole, rifampicin and pheno-

barbital [ 1881. This coordinated regulation is incomplete, as nifedipine only up-regulated P-glycoprotein and not CYP3A4.

Recognition that interactions between CYP3A and P-glycoprotein has led to additional concepts on the mode of drug secretion/elimination (Fig. 8). For example, rifampicin-induced decreases in the bio-

availability of cyclosporin A have been explained by means of CYP3A induction and, hence, increased metabolism [ 1891. However, recognitiop that rifampicin increases P-glycoprotein expression provides

the alternate hypothesis that the reduced oral bioavailability may be mediated by enhanced P-glycoprotein-mediated intestinal secretion, limiting oral absorption [188]. These two hypotheses are not

Fig. 8. Model of drug absorbtion in the presence of both P-

glycoprotein and cytochrome P450 3A. A drug (D) entering the

epithelium may be effluxed via P-glycoprotein (Pgp) and/or

metabolised by CYP3A to D*, with the residual native drug

absorbed across the epithelium (D). The recirculation of the drug

(D) out of the cell via Pgp and reabsorption allows greater access

to a limited amount of CYP3A, hence, a greater proportion will be

metabolised and reduced absorption of drug (D) will ensue.

150 .I. Hutzter, B.H. Him I Advanced Drug Delivery Reviews 2-5 (1997) 129-157

mutually exclusive and recent studies provide an intriguing link [ 1901.

Thus, substances which either modulate metabolism and/or secretion are an attractive avenue to

explore in the optimisation of oral drug bioavailability. The bioavailability could be maximised by

increasing net drug absorption and reducing drug

biotransformation in the gut, by using either cytochrome P-450 drug metabolism inhibitors, or P-

glycoprotein drug transport inhibitors. Several of these inhibitors show overlap in their inhibition

profiles. For example, the calcium channel blockers,

verapamil, diltiazem, felodipine and nifedipine,

along with the steroid hormones, cortisol, predni- sane, and progesterone and other compounds such as

quercitin, all inhibit both systems [191]. Thus, this leads to the possibility of co-administration of oral drugs with these compounds to increase bioavailability. However, toxic effects of these modulators,

and increased toxicity of the co-administered drug in locations other than the gut (e.g. in high P-glycopro-

tein- and cytochrome P450-expressing tissues, such

as the liver), would have to be considered. The potential use of modulators of these systems has yet

to be investigated, as has their usefulness in the clinical setting to be determined. In some cases, however, this mode of increasing bioavailability may

already be, in a less planned manner, taking place. For example, the increased digoxin bioavailability when co-administered with calcium channel blockers such as verapamil [ 1341. In addition, this phenom-

enon may explain some of the drug interaction problems that occur with females on the contracep-

tive pill [192].

8. Conclusions and future perspectives

Intestinal secretion of drugs is an important mech-

anism of clearance which also affects oral bioavailability of a wide variety of drugs. In extreme situations, these secretory systems may compromise oral bioavailability. No single efflux system explains the diversity of drug secretion displayed by the intestine, while interactions with intestinal drug metabolism systems add an extra dimension to the

equation describing oral bioavailability. Isolated first as one feature associated with a

phenotype of cross-resistance to antitumoral agents,

P-glycoprotein is receiving great attention as a possible physiological determinant of the disposition of drugs. Anticancer drugs were originally identified

as substrates, but other pharmacological classes of drugs are now being investigated. In the near future, more information will be available on the physiologi-

cal functions of P-glycoprotein, which will lead to a

better understanding of the disposition of drugs and pharmacokinetic interactions. Intestinal expression of

P-glycoprotein is only one contributing factor to such interactions, but may play a central role in limiting

oral bioavailability of specific and varied classes of

drugs. Identification of the role of other secretory efflux systems and their molecular characterisation

will be required before the importance of intestinal secretory systems in drug absorption and elimination can be fully described. A further fruitful avenue will be the understanding of the interactions between

those secretory efflux systems and cellular drug metabolism pathways. Understanding the fundamen-

tal mechanisms underlying drug secretion and metabolism limiting drug absorption will allow develop-

ment of optimal oral dosing protocols, including the

use of agents to specifically enhance absorption, by

reducing specific secretory efflux systems.

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