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Current concepts on hepatic transport of drugs
Journal of Hepatology, 1987;4:259-268 Elsevier
259
HEP 00272
Review
Current concepts on hepatic transport of drugs
Dirk K.F. Meijer Department of Pharmacology and Therapeutics, Universityof Groningen, Groningen (The Netherlands)
Introduction: the hepatic transport system Traffic in the liver is heavy: substances come and go, they are taken up, degraded, synthesized, and released via the bile, the lymphatic system or the general circulation. The ultrastructure of this flow-dynamic system (Fig. 1) shows the unique features necessary for these interactive processes: the liver sinusoid with endothelial lining, possessing fenestrae that allow drugs to pass in their unbound but also in their protein-bound form, exposing them to the large surface of the villous plasma membrane. Hepatic uptake often involves simple passive lipid-permeation. In the case of hydrophilic or charged drug-molecules, however, carrier-mediated transport processes are operating that in the liver are probably quite typical for the parenchymal cell [1,9,12,17]. Bidirectional carrier transport at the sinusoidal membranes is important for the distribution of drugs to the liver. In between the hepatocytes (see Fig. 1) is the bile canaliculus: a site for specialized transport processes related to drug elimination. The study of hepatic membrane transport over the past 30 years has largely progressed through the application of powerful in vitro techniques. Many classical studies approached liver function in the complex integrated physiological system of the intact orga-
nism, but soon the advantage of the isolated perfused organ was recognized. With this versatile technique hepato-biliary kinetics could be studied in detail and even a functional differentiation within the hepatic acini could be made. The intact architecture of the liver was then left and cell populations highly enriched in the various cell types were isolated [1,2]. Subcellular fractions such as plasma membrane vesicles, and finally isolation and purification of potential carrier proteins, brought the study of drug transport to the membrane level [9,12,13,15,17,18]. This short review will start with the whole liver in the intact body and finally will arrive at the macromolecules responsible for transport at the plasma membrane.
Drug transport in man: a few examples Discussing molecular mechanisms in drug transport, we should not forget about the patient. It seems appropriate therefore to start this review with some examples of clinical transport studies. In a series of studies we looked at the relation between biliary excretion rate of drugs and their plasma decay patterns in man [3]. One recent example is that of the organic anion ICG, of which much is known with regard to
Correspondence: D.K.F. Meijer, Department of Pharmacology and Therapeutics, University of Groningen, Ant. Deusinglaan 2, 9713 AW Groningen, The Netherlands. 0168-8278/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
260
D.K.F, MEIJER
- Intact l i v e r
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(organelles)
- Isolated membrane
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Fig. 1. Ultrastructure of the liver (right) and techniques to study hepatic transport function, ranged in an order of decreasing structural organization. The scanning electronmicrograph (5000 x) shows the sinusoidal wall with large fenestrae and a plate of parenchymal cells (hepatocytes) with microvilli at the sinusoidal and canalicular poles of the cell representing sites for carrier-mediated transport of drugs.
plasma clearance but surprisingly little of its exact biliary excretion pattern. If plasma decay is followed for only 15 min after injection, removal from plasma seems mono-exponential. The initial half life of 3-5 min indicates a fast process: initial clearance is practically limited by hepatic blood-flow. However, looking at lower concentrations a secondary slow component is seen with a tt/2 of about 70 min. The biliary excretion rate of unchanged ICG in a postcholecystectomy patient with complete bile drainage revealed curves of which the descending part has an identical terminal tl/2. The first phase in the plasma disappearance curve therefore is mainly due to distribution of ICG to the liver, while the second component probably reflects the rate of biliary excretion instead of ICG metabolism as has been suggested in some studies. Both uptake into the liver and biliary excretion can be partly saturated at higher doses, providing one clue for the involvement of the carrier-mediated mechanisms in both of these transport steps. A second clinical example is hepatic transport of cationic drugs. Agoston and co-workers [4] reported on two bivalent steroidal organic cations, vecuronium and its methylated derivative pancuronium, that are broadly used in anesthesia for peripheral muscle relaxation. Since only in the vecuronium molecule a
proton can dissociate from one of the nitrogen groups, a large uncharged ring system can be formed resulting in a proper balance between the remaining hydrophilic positively charged group and a heterocyclic hydrophobic part of the molecule. With regard to hepato-biliary transport, the liver discriminates especially sharply between drugs concerning this molecular balance [5]. Indeed a marked difference in hepatic uptake rate was observed in various species including man [4]. In remainders of liver biopsies taken from patients for diagnostic reasons, the authors were able to determine both compounds. It could be estimated that 15 min after injection the human liver contains about 60% of the injected dose of vecuronium, while this figure is much lower for pancuronium. This is also reflected in the plasma disappearance patterns in man; that for vecuronium is initially much more rapid. Taking into account the effective concentration range of these curare-like agents, these combined data imply that distribution to the liver will terminate the action of the compound. Indeed, the clinical duration of action of equipotent doses of the compounds are a factor of 3-4 different, and also the final elimination of vecuronium compared with pancuronium is much less affected by renal function.
CURRENT CONCEPTS ON HEPATIC TRANSPORT OF DRUGS
Chemical structure and hepato-biliary clearance The results on the cationic muscle relaxants raise the question about the relation between chemical structure and hepato-biliary clearance. The two agents mentioned showed almost absolute difference in hepatic disposition in spite of the fact that their molecular weight is almost identical. This seems to negate the well-known molecular weight-threshold hypothesis postulating that drugs with a molecular weight exceeding a (species-dependent) threshold of 200-300 are mainly excreted in bile while drugs with a lower MW are predominantly excreted in urine [1]. A better parameter is probably the balance between hydrophilic and hydrophobic properties of the agents. In a study with a homologous series of organic cations with increasing molecular weights ranging from 74 to 315, lipid solubility was correlated with the relative clearance of each of these compounds in liver, intestine and kidney in the intact rat [5]. Small organic cations of low lipid solubility were poorly excreted in bile, however, a change of a factor of 5 in lipophilicity increased biliary clearance 1000 times, while carrier-mediated secretion from blood into the intestinal lumen increased 100-fold. In contrast there was no evident relation between lipid solubility of organic cations and their renal tubular secretion. In a more biological context: the carrier-systems in the liver involved in hepatic uptake and biliary excretion may preferentially recognize relative lipophilic or amphipathic cations.
Acinar heterogeneity in transport How are such highly cleared drugs distributed in the liver? For this we look at another level of magnification: the microcirculatory unit, also called the hepatic acinus. It became evident in the last decade that hepatocytes in the acinar zones, apart from being heterogeneous in, for instance, carbohydrate-, drugand lipid-metabolism, are also unequal in transport function [1,6,9,19]. This dynamic situation may be due to their localization in the blood stream along the sinusoidal axis or to intrinsic differences between the
261
cells which in turn could be secondary to concentration gradients of 0 2, CO 2, hormones or other substrates. How can we experimentally differentiate between these types of heterogeneity? A very adequate method is again the isolated perfused liver. The isolated organ can be perfused in the normal direction or in a retrograde direction allowing manipulation of the localization of the drug in this system: with shortterm perfusions with high extraction types of drug at low protein medium concentration, zone 1 or zone 3 can be loaded. Subsequently the normal flow can be restored and the kinetics of efflux to plasma and bile can be monitored. This can be combined with freezeautoradiographic or fluorescent methods in which the localization of the test compound can be visualized as shown previously [6,10,19]. The acinar distribution of taurocholate a few minutes after injection, for instance, showed a sharp gradient with a clear zone 1 localization [6]. If the direction of the perfusate flow is reversed the taurocholate is predominantly found in zone 3; it is only a matter of presentation. Kinetic studies confirmed that the uptake rate in these zones seems to be equal. However, transport to bile is considerably slower from zone 3, and this indicates an intrinsic cellular difference in excretion efficiency for the bile acid. Such intrinsic differences may even lead to intrahepatic redistribution of drugs in time. Recently we performed a similar study with rhodamine B, that has a net positive charge at physiological pH. Ten minutes after injection almost all of this highly extracted drug is in the periportal zone 1, but interestingly, keeping the same direction of flow, 50 min later the picture seems to be reversed: most of it is in zone 3 of the acinus. Computer simulations using distributional perfusion models and zonalwashout studies indicated that these patterns can be explained by unequal rates of efflux from cell to blood in zone 1 and zone 3. This again indicates an intrinsic zonal difference in transport function or intracellular binding. Similar acinar concentration gradients have been reported, for instance, for propranolol and galactose (for references see Ref. 6). The general conclusion should be that the liver as a clearance organ should not be analyzed as a well-stirred compartment since both the rate of distribution and
262
D.K.F. MEIJER
ELIMINATION OF DRUGS FROM THE BODY
the rate of elimination of drugs in the various zones o! the hepatic acini can be quantitatively different [6,111.
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One of the factors determining such concentration gradients is the extent of protein binding. Protein binding may be a very important factor in the hepatic clearance of drugs. Basic drugs are mainly bound to orosomucoid or al-acid-glycoprotein, and acidic drugs are predominantly bound to albumin. Clearance of the organic anion DBSP in isolated perfused rat liver is inversely related to the albumin concentrations, as is that of BSP in man [7]. This phenomenon evidently may interfere with the diagnosis of liver disease. A lowered plasma albumin concentration can lead to a rise in unbound fraction that may partly compensate for the decreased intrinsic transport function. If DBSP is infused in perfused liver until steady state concentrations are reached, addition of albumin to the perfusate leads to an efflux of the organic anion from the liver into plasma. This demonstrates two points: hepatic storage of the organic anion, apart from the membrane transport, is determined by the relative binding capacity inside and outside the liver, and transport between plasma and liver of organic anions is bidirectional. This latter phenomenon is very likely related to the well-known fact that the parenchymal cells do not only secrete organic anions into bile but also into the general circulation (Fig. 2). For instance, how do anionic sulfate- or glucuronide conjugates leave the parenchymal cell? One example is the phenolic drug harmol that was constantly infused in rats. In the steady state mainly sulfate conjugate is formed, 60% of which is eliminated via urine and 30% via bile. At steady state the anionic dye DBSP was injected. It was anticipated that DBSP would inhibit the biliary output of harmol sulfate. Surprisingly, the biliary excretion of the sulfate conjugate immediately increased after addition of DBSP
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Fig. 2. Schematic representation of drug disposition by the liver. Hepatic uptake can occur passively or by carrier transport. Phase 1 and phase 2 biotransformation may produce polar conjugates that can be excreted into the general circulation or into bile, both via carrier-mediated transport. Conjugates or polar drugs may also be excreted via urine or via intestinal secretion. In the gut, conjugates can be deconjugated and reabsorbed entering an enterohepatic cycle.
while, in contrast, urinary excretion decreased. The combined data were explained by competitive inhibition of harmol sulfate excretion from the liver into plasma by DBSP, decreasing the driving force for excretion of harmol sulfate in the urine but increasing it for its biliary output. Such an inhibitory effect on liver-to-plasma transport was subsequently clearly demonstrated by loading the liver with preformed sulfate conjugate and measuring efflux in single pass perfusion, showing that hepatic efflux also is carrier-mediated [8]. Similar types of competitive phenomena at the sinusoidal level have been recently described for glutathione and glutathione conjugates by Sies et al. and Kaplowitz et al. (see Ref. 9).
CURRENT CONCEPTS ON HEPATIC TRANSPORT OF DRUGS Mechanisms of hepatic drug-plasmaprotein dissociation Another interesting point is hidden in the abovementioned data on DBSP transport and albumin binding: if clearance of DBSP is related to the unbound fraction the relation is essentially non-linear. It is as if the liver removes more DBSP than is predicted from its unbound fraction. How can we explain such a non-linear relation between unbound fraction and clearance in general? First of all, clearance may not entirely follow changes in unbound fraction if blood flow becomes rate-limiting or if saturation of membrane transport occurs [7]. Secondly, binding of drugs to albumin may facilitate passage of unstirred layers at the sinusoidal domain of the plasma membrane or even directly affect affinity for the membrane transport. The latter phenomenon was recently found for Na÷-coupled taurocholate transport in membrane vesicles (see Ref. 9). However, possible explanations are also present at the level of protein binding itself: the drug may spontaneously dissociate due to effective removal of unbound drug by the transport system during passage to the liver in nonequilibrium conditions. Alternatively, debinding may occur due to binding of albumin to a membrane receptor or due to aspecific surface interaction, both of which could lead to conformational changes in the protein and facilitation of dissociation of the complex (for an interesting discussion see Refs. 10 and 11). Finally, the liver is a metabolically very active organ, and at the microclimate of the plasma membrane competition may occur with endogenous substrates secreted from the liver. In principle all of these mechanisms may play a role, although the relative contribution of each of these processes may be different for each compound. Such mechanisms anyhow may explain that the liver can deal with highly proteinbound drugs.
Organic cation transport: influence of cofactors on uptake and subcellular distribution It should be mentioned here that the extraction of
263
drugs during passage through the liver is not only dependent on their extent of protein binding but also on the presence of cofactors which may help in the transport process. For instance, there is evidence that some organic cations in a physiological milieu can form electro-neutral ion-pairs with inorganic or organic counter anions. Such an interaction at the lipidwater interphase may help transport across the membrane, either due to passive diffusion or by interaction with carrier-systems for uncharged organic compounds. An example is the organic cation d-tubocurarine that normally is quite slowly removed from the circulation. Uptake into the liver is rate-limiting in hepato-biliary clearance. Addition of iodide that readily forms ion-pairs with organic cations immediately results in an increased disappearance rate and an elevated biliary excretion rate of the compound, providing a procedure to accelerate hepatic clearance of this drug. Similar effects can be found with H C O 3- as well as bile acid counter anions, suggesting that basic drugs are not necessarily transported as organic cations. Uptake into the liver of some cationic drugs can lead to rapid biliary excretion; however, it may also partly lead to persistent hepatic storage of such compounds, in which accumulation in lysosomes plays a major role. We arrive now at the level of intracellular distribution. The presence of such a drug in the lysosomes was demonstrated by classical subfractionation studies and also could be visualized with the electron microscope by making electron-dense precipitates with molybdate. What is the mechanism of this lysosomal accumulation? One speculation could be that such drugs are co-endocytosed with glycoproteins taken up in the liver via receptor-mediated endocytosis and trafficked to the lysosomes together with the protein. A candidate here is orosomucoid which both in sialated and desialated form binds cationic drugs much more strongly than albumin does. However, observations in the author's laboratory showed that addition of asialo-orosomucoid to perfusion media of isolated livers increases binding of such basic drugs about 5-10-times but does not affect clearance or the intracellular distribution, This must imply that within the liver the protein-drug complex
264
D.K.F. MEIJER Hepato-biliary transport of or.q.cations
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Separate transport processes for anionic, cationic and uncharged drugs? So far we have discussed membrane transport of various classes of drugs: organic anions like DBSP and taurocholate as well as hepatobiliary transport of organic cations. How separate are these transport systems? The classical subdivision of the processes is based on the supposed charge of the compounds. For organic anions like DBSP a Na*-independent uptake process is present. A separate Na+-dependent mechanism for the anionic bile acids has been demonstrated, while one assumes additional processes for organic cations and uncharged drugs (cardiac glycosides such as ouabain and other steroids). How useful is the above-mentioned classification of hepatic transport processes? It may be useful from a mechanistic point of view but it is probably of little value if we want to predict drug transport interactions in the liver. One reason for this could be that there is a broad overlapping substrate specificity, especially for the hepatic uptake process: one compound can often be transported by various systems depending on its concentration relative to the K m of the particular process. Studies with isolated hepatocytes have largely contributed to this view [1]. Alternatively one or two very broad unspecific uptake systems for the above-mentioned classes of organic compounds could operate. In fact, recent studies combining photoaffinity label data with kinetic studies from the group of Kurz et al. [13] indicate that probably a general transport system is present for amphipathic compounds that can accommodate drugs of various charge such as the anionic unconjugated bile acids, the uncharged steroidal compounds, as wall as lipophilic bivalent and monovalent organic cations. However, even the Na÷-dependent uptake system for bile acids seems to be quite unspecific: it has been reported to transport conjugated bile acids, fusidic acid, which is structurally related, but also dissimilar structures such as the dicarboxylic acids iodipamide and methotrexate, as well as large cyclopeptides such as phalloidin and cyclosporin [9,13]. The particular processes thus may be quite aspecific and are able to recognize largely varying chemical
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Fig. 3. Transport mechanisms for hepatobiliary transport of organic cations (indicated by ¢r). Uptake can occur by two systems, only one is inhibitable by cardiac glycosides that may share a carrier process transporting large-MW lipophilic organic cations, possibly as ion-pairs with inorganic counter anions (2). Lipophilic organic cations bind in plasma to at-acid glycoprotein (orosomucoid), but are not co-endocytosed with asialo forms of this glycoprotein. Accumulation in lysosomes may occur via aspecific fluid-phase endocytosis. Direct transport from lysosomes to bile is unknown. Biliary excretion may involve carrier-mediated transport, possibly by antiport with protons. Binding to mixed biliary micelles facilitates net transport into the bile canaliculus.
completely dissociates before endocytosis as is pictured in Fig. 3. Other mechanisms of lysosomal uptake may resemble transport systems at the plasma membrane level: ion-pair transport or antiport with protons. The latter type of transport could also explain net canalicular secretion operating against an electrochemical gradient that further could be helped by micelle binding in the bile canalicular lumen. Accumulation of positively charged drugs in lysosomes can alternatively be explained by binding to negatively charged groups in the plasma membrane which triggers aspecific fluid-phase endocytosis and leads to vectorial transport to lysosomes. The particular lysosomal accumulative process may resemble that for aminoglycosides in kidney tubular cells and could have consequences for inhibition of proteolytic processes and hepatotoxicity of cationic drugs in the liver.
CURRENT CONCEPTS ON HEPATIC TRANSPORT OF DRUGS structures. However, we should also realize that model drugs that are commonly used are quite complex structures. Indocyanine green, for instance, is used as an anionic model compound. However, inspection of its molecular structure indicates it to be a zwitter-ion which also has a cationic group. In fact the compound is a very strong inhibitor of organic cation transport in isolated hepatocytes [1]. As mentioned earlier, cationic drugs can be present also as uncharged ion-pairs and may be recognized by systems normally transporting uncharged organic compounds. Ouabain in its turn has a steroid structure which could well fit the bile acid carrier and, in fact, (partly) Na+-dependent transport has been reported in perfused livers. Finally, the bile acids and especially the unconjugated ones can even be present in three forms: dissociated as anions, undissociated molecules resembling the uncharged cardiac glycosides, and complexed with ions such as Ca 2÷, thus having a net positive charge. This might explain the observation that bile acids in relatively high concentrations are very potent but completely reversible inhibitors of anionic, cationic and neutral transport pathways [14].
Concentrative membrane transport-what is the driving force? Gradients across both the sinusoidal and canalicular membranes can be calculated. These gradients can only be partly explained by electrical potential differences across these compartments, meaning that other driving forces should be present. The existence of concentrative transport steps is generally confirmed now in experiments with isolated hepatocytes and membrane vesicles from basolateral and canalicular portions of the hepatocyte plasma membrane [9,13,15]. If concentrations of unbound drugs are determined by ultrafiltration of plasma- and liver-cytosol fractions and if one estimates the extent of binding of drugs to biliary micelles by ultracentrifugation of bile, uphill transport against an electrochemical gradient can be calculated [1,7,9]. In addition, the question as to whether drugs are transported as
265
charged molecules can be approached by study of isolated membrane vesicles. Highly purified preparations enriched in either sinusoidal and canalicular elements can now be prepared. Examples are the studies of Inoue and Arias, the work from Blitzer and Boyer and recent studies from Meier and co-workers (see for references Refs. 9 and 17). The obvious advantage of this preparation is that in contrast to intact cells both the external and internal milieu can be easily manipulated: it is possible to impose ion-gradients and even induce short-lasting membrane diffusion potentials. For instance, by adding NaNO 3, in a non-equilibrium situation the negative NOs--ions will enter more rapidly than Na ÷ so that a transient inside negative membrane potential is produced. Typical uptake curves for taurocholate in sinusoidal membrane vesicles show overshoot phenomena that are due both to the presence of a Na÷-gradient and a short-lasting inside negative diffusion potential produced by addition of NaNO s in non-equilibrium conditions [9,12]. Co-transport of one taurocholate and more than one Na÷-ion is found in several systems and explains the electrogenic character. In contrast, uptake of the organic cation methyldeptropine (an anticholinergic drug) is not stimulated by such a NaNOa-induced negative membrane potential, indicating an electroneutral uptake system for this quaternary ammonium compound [12]. This confirms the idea of transport as uncharged ion-pairs or alternatively counter transport with protons. All of these mechanisms strongly suggest a relation between ion-gradients and drug transport. This brings us finally to the molecular level and especially to the question of driving forces for these transport processes for drugs. Based on the work of Arias et al. (see Refs. 9 and 17), Scharschmidt et al. (see Refs. 9 and 17) and Meier and co-workers [9,15] the following picture of inorganic ion transport in the hepatocyte can be made (Fig. 4). The initial event is electrogenic transport of Na + and K + by the ATPase system. The created Na÷-gradient then induces antiport of Ca 2÷ and of protons. Protons are formed within the cell from CO 2 and water and their removal from the cell therefore results in an intracellular concen-
266
D.K.F. MEIJER
T R A N S P O R T M E C H A N I S M S FOR DRUGS IN THE LIVER
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terns are quite unspecific and show overlapping substrate specificity. Looking at carrier-mediated transport at the canalicular pole of the cells, the general impression is that the systems are more specific. Consequently the above-mentioned classes of compounds have less interaction at this level. This is also suggested by earlier transport studies in mutant sheep [1,9,17], and was confirmed in mutant rats with deficient canalicular transport of organic anions and uncharged compounds but, in contrast, unchanged bile acid and cation transport into bile [16].
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Fig. 4. Coupling between gradients of inorganic ions with cartier-mediated transport of organic anions (OA-), bile acids (BA-), organic cations (OC +) and uncharged compounds (UC) in the hepatocyte. Inorganic ion-pumps include electrogenic Na+/K÷ exchange, Na+/H+, OH-/SO4--antiport (sinusoidal) and Cl-/HCO3--antiport (canalicular). Na+-coupled bile acid transport (1) and OH-/OA--antiport (2) are anionic systems. Organic cations are taken up by system 1 and may also share a transport system for uncharged compounds (2). In the latter system ion-pair formation may play a role; at the canalicular level antiport of organic cations with protons is tentatively assumed. The transport processes at sinusoidal level have more overlapping substrate specificity than the projected four canalicular carrier systems (adapted from Hugentobler and Meier, Ref. 15).
tration of bicarbonate (or hydroxyl-ions) exceeding the normal electrochemical equilibrium. Bicarbonate in its turn may drive CI- antiport across the canalicular membrane and hydroxyl-ions may induce sinusoidal sulfate uptake also by antiport [15]. For drugs at least four transport systems related to ion gradients may operate: firstly, co-transport with Na ÷ (conjugated bile acids); secondly, a Na+-indepen dent anion uptake system for compounds such as BSP, possibly driven by hydroxyl antiport; thirdly, canalicular cation transport for relatively non-lipophilic basic drugs which could operate via antiport with protons and therefore is coupled to Na+/H ÷ exchange, and finally, a broad unspecific uptake system that transports uncharged steroid-like compounds as well as amphipathic cations probably in the form of ion-pairs with inorganic anions. These uptake sys-
Carrier proteins: the responsible transport modalties? How can we define carrier transport function at the membrane level other than by kinetic studies? One answer is isolation or specific labeling of the carrier proteins involved. A number of groups have succeeded now to characterize potential carrier proteins (Fig. 5). The group of Tiribelli and Sottocasa isolated a 110 kDa protein, binding organic anions, which they called bilitranslocase (BTL), having an a2fl structure with three subunits of about 35 000. Berk and Stremmel [17] as well as Wolkoff et al. (see Refs. 9 and 16 for references) isolated a protein with subunits of about 55 000 mol. weight which bound organic anions but not bile acids, and also raised antibodies against it. In spite of the identical molecular weight, they may represent different carrier systems since the particular antibodies do not clearly crossreact and the iso-electric points are reported to be different. At least three groups (see Fig. 5) approached the carrier function by photo-affinity labeling techniques and generally found two macromolecules, one being of mol. weight 48 000 that binds photo-affinity probes of especially conjugated bile acids. There is good evidence that this represents the Na+-dependent system; as shown by Kurz et al. and Petzinger and Frimmer, it also transports cyclo-peptides such as phalloidin and cyclosporin [13]. These investigators also found a 55 000 mol. weight protein that preferentially binds unconjugated bile acids, but also may transport neutral steroidal compounds and amphipathic ca-
267
CURRENT CONCEPTS ON HEPATIC TRANSPORT OF DRUGS
POTENTIAL CARRIER-PROTEINS FOR HEPATIC TRANSPORTOF DRUGS Tlrlbelll Sottocasa et al. Mol Weight Proteln-lsolatlon
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Fig. 5. Experimental work reported on potential carrier proteins responsible for hepatic uptake of organic anions and other drugs. Estimated mol. weight, use of photoaffinity techniques, preparation of antibody with measurement of its effect on uptake (in brackets), reconstitution experiments performed, cofactors established and binding or transport of substrates are indicated. The scheme suggests separate carrier-processes; however, some may turn out to be identical (for instance 2nd, 3rd and 5th from left to right).
tions: a good candidate for the broad unspecific transport system that was mentioned above [13]. One crucial point here is to prove that the isolated protein is not only binding the transported drugs but also is able to translocate or 'channel' a drug across the membrane. This can be approached in at least two ways. One is to study the effect of monospecific antibodies raised against such purified proteins on drug transport. Berk and Stremmel recently reported inhibitory effects of antibody preparations in isolated hepatocytes [17]. In the author's laboratory an inhibitory effect of a BTL-antibody preparation on DBSP transport from liver into perfusate of isolated perfused livers was found but there was essentially no effect on hepatic uptake of this model compound. A second approach is reconstitution of transport with the isolated carrier proteins. In a preliminary study [18] the potential carrier protein bilitranslocase was inserted in monolaminar liposomes. It was demonstrated that this protein clearly supported BSP trans-
port out of such liposomes as driven by a K+-valino mycin-induced negative membrane potential. This facilitatory effect was not seen with other anion-binding proteins such as ligandin and albumin. The study of intramembrane mobility of the carrier proteins (rotating units, flip-flop movements or ion-selective channel conformation) will hopefully provide more insight into the precise membrane transport mechanisms involved. The study of the regulation of the cellular synthesis of such carrier proteins in individuals with genetic transport defects or following adaptive changes in hepato-biliary transport under various conditions may further clarify their crucial role in health and disease. For now, here our travel through the hepatic transport system ends: indeed, traffic in the liver is heavy, but fascinating, confronting us with the amazing number of secrets and surprises that nature has to offer.
268
Acknowledgements
D.K.F. MEIJER Drs. W . E . M . M o l . I a m g r a t e f u l for the c o o p e r a t i o n of Dr. R . J . V o n k , D r . P . L . M . J a n s e n , Dr. M . J .
This r e v i e w is d e d i c a t e d to the m e m b e r s of the ' E u -
H a r d o n k , D r . S. A g o s t o n , D r . A . H . J . S c a f and D r .
r o p e a n Study G r o u p for H e p a t o - b i l i a r y T r a n s p o r t ' ,
G . J . M u l d e r . P a r t of the m a t e r i a l in this r e v i e w was
with m a n y thanks for advice and friendship. I ac-
p r e s e n t e d in a ' s t a t e - o f - t h e - a r t ( p r e s i d e n t i a l ) l e c t u r e '
k n o w l e d g e the active i n v o l v e m e n t of m y c o - w o r k e r s
during the 36th m e e t i n g of t h e A m e r i c a n A s s o c i a t i o n
Dr. G . M . M . G r o o t h u i s , D r . G . A . V e r m e e r , D r . C.
for the Study of L i v e r D i s e a s e s , C h i c a g o , N o v e m b e r
N e e f , Dr. P . G . R u i f r o k , Dr. P. W e s t r a , Dr. A . F .
5, 1985.
B e n c i n i , Drs. P. van d e r Sluijs, D r s . I. B r a a k m a n and
References 1 Klaassen CD, Watkins JB. Mechanisms of bile formation, hepatic uptake and biliary excretion. Pharmacol Rev 1984; 36: 1-67. 2 Biota A, Scaf AHJ, Meijer DKF. Hepatic drug transport in the rat. A comparison between isolated hepatocytes, the isolated perfused liver and the liver in vivo. Biochem Pharmacol 1982; 31: 1553-1565. 3 Meijer, DKF, Weitering JG, Vermeer GA. Pharmacokinetics of hiliary excretion in man. V. Dibromosulfthalein. Eur J Clin Pharmacol 1983; 24: 549-556. 4 Sohn Y J, Bencini A, Scar AH,I, et al. Comparative pharmacokinetics and dynamics of vecuronium and pancuronium in anesthetized patients. Anesth Analg 1985; 65: 233-239. 5 Neef C, Meijer DKF. Structure-pharmacokinetics relationship of quaternary ammonium compounds. Correlation of physicochemical and pharmacokinetic parameters. Naunyn-Schmiedeberg's Arch Pharmacol 1984; 328: l 1 l - 118. 6 Groothuis GMM, Hardonk MJ, Meijer DKF. Hepatobiliary transport of drugs: do periportal and perivenous hepatocytes perform the same job? Trends Pharmacol Sci 1985 (Aug): 322-327. 7 Meijer DKF, BIom A, Weitering JG, et al., Pharmacokinetics of the hepatic transport of organic anions: Influence of extra- and intracellular binding on hepatic storage of dibromosulfophthalein and interactions with indocyanine green. ,l Pharmacokin Biopharm 1984; 12: 43-65. 8 De Vries MH, Groothuis GMM, Mulder G.l, et al. Secretion of the organic anion harmol sulfate from liver into blood. Evidence for a carrier-mediated mechanism. Biochem Pharmacol 1985; 34: 2129-2135. 9 Boyer .IL. Mechanisms of bile secretion and hepatic transport. In: TC Andreoli, JF Hoffman, DD Fanestil, SG Schulz, eds. Physiology of Membrane Disorders. 2nd ed. New York: Plenum Press, 1986; 609-636.
10 Morgan D,l, Jones DB, Smallwood RA. Modeling of substrate elimination by the liver: has the albumin receptor model superseded the well stirred model? Hepatology 1985; 5: 1231-1235. 11 Forker EL, Luxon B. Lumpers vs distributors. Hepatology 1985; 5: 1236-1237. 12 Meijer DKF, Neef C, Groothuis GMM. Carrier-mediated transport in the handling of drugs by the liver. In: DD Breimer, P Speiser, eds. Topics in Pharmaceutical Sciences. Amsterdam: Elsevier, 1983; 167-189. 13 Buscher HP, Fricker G, Gerok W, et al. Membrane transport of amphiphilic compounds by hepatocytes. In: H Greten, E Windier, U Beisiegel, eds. Receptor-Mediated Uptake in the Liver. Heidelberg: Springer-Verlag, 1986; 189-199. 14 Vonk R,l, Van Doorn ABD, Mulder G J, et al. The influence of bile salts on hepatocellular transport, Falk Symposium no. 26, V. Bile acid meeting. Biological effects of bile salts. 1978; 121-126. 15 Hugentobler G, Meier PJ. Multispecific anion exchange in basolateral (sinusoidal) rat liver plasma membrane. Am J Physiol 1986; 251: G656-G664. 16 Jansen PLM, Groothuis GMM, Peters WHM, et al. Selective defect in the hepatobiliary transport of organic compounds in mutant rats with heriditary conjugated hyperbilirubinaemia. Heterogeneity of hepatobiliary transport. Hepatology 1987; (in press). 17 Berk PD, Stremmel W. Hepatocellular uptake of organic anions. In: H Poppen, F Schaffner, eds. Progress in Liver Diseases, Vol. VIII. New York: Grune & Stratton, 1986; 125-144. 18 Sottocasa GL, Baldini G, Sandri G, et al. Reconstitutions in vitro of sulfobromophthalein transport by bilitranslocase. Biochim Biophys Acta 1982; 685: 123-128. 19 Gumucio ,l,l. Functional and anatomic heterogeneity in the liver acinus: impact on transport. Am ,l Physiol 1983; 244: G578-G582.
259
HEP 00272
Review
Current concepts on hepatic transport of drugs
Dirk K.F. Meijer Department of Pharmacology and Therapeutics, Universityof Groningen, Groningen (The Netherlands)
Introduction: the hepatic transport system Traffic in the liver is heavy: substances come and go, they are taken up, degraded, synthesized, and released via the bile, the lymphatic system or the general circulation. The ultrastructure of this flow-dynamic system (Fig. 1) shows the unique features necessary for these interactive processes: the liver sinusoid with endothelial lining, possessing fenestrae that allow drugs to pass in their unbound but also in their protein-bound form, exposing them to the large surface of the villous plasma membrane. Hepatic uptake often involves simple passive lipid-permeation. In the case of hydrophilic or charged drug-molecules, however, carrier-mediated transport processes are operating that in the liver are probably quite typical for the parenchymal cell [1,9,12,17]. Bidirectional carrier transport at the sinusoidal membranes is important for the distribution of drugs to the liver. In between the hepatocytes (see Fig. 1) is the bile canaliculus: a site for specialized transport processes related to drug elimination. The study of hepatic membrane transport over the past 30 years has largely progressed through the application of powerful in vitro techniques. Many classical studies approached liver function in the complex integrated physiological system of the intact orga-
nism, but soon the advantage of the isolated perfused organ was recognized. With this versatile technique hepato-biliary kinetics could be studied in detail and even a functional differentiation within the hepatic acini could be made. The intact architecture of the liver was then left and cell populations highly enriched in the various cell types were isolated [1,2]. Subcellular fractions such as plasma membrane vesicles, and finally isolation and purification of potential carrier proteins, brought the study of drug transport to the membrane level [9,12,13,15,17,18]. This short review will start with the whole liver in the intact body and finally will arrive at the macromolecules responsible for transport at the plasma membrane.
Drug transport in man: a few examples Discussing molecular mechanisms in drug transport, we should not forget about the patient. It seems appropriate therefore to start this review with some examples of clinical transport studies. In a series of studies we looked at the relation between biliary excretion rate of drugs and their plasma decay patterns in man [3]. One recent example is that of the organic anion ICG, of which much is known with regard to
Correspondence: D.K.F. Meijer, Department of Pharmacology and Therapeutics, University of Groningen, Ant. Deusinglaan 2, 9713 AW Groningen, The Netherlands. 0168-8278/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
260
D.K.F, MEIJER
- Intact l i v e r
~n v t v o
- Isolated perfused liver - Liver slices - Isolated hepatocytes - H~ogenate/subfractions
(organelles)
- Isolated membrane
fractions
(vesicles)
- Isolated enzymes;
carrier proteins
lW',," U°
; • .
Fig. 1. Ultrastructure of the liver (right) and techniques to study hepatic transport function, ranged in an order of decreasing structural organization. The scanning electronmicrograph (5000 x) shows the sinusoidal wall with large fenestrae and a plate of parenchymal cells (hepatocytes) with microvilli at the sinusoidal and canalicular poles of the cell representing sites for carrier-mediated transport of drugs.
plasma clearance but surprisingly little of its exact biliary excretion pattern. If plasma decay is followed for only 15 min after injection, removal from plasma seems mono-exponential. The initial half life of 3-5 min indicates a fast process: initial clearance is practically limited by hepatic blood-flow. However, looking at lower concentrations a secondary slow component is seen with a tt/2 of about 70 min. The biliary excretion rate of unchanged ICG in a postcholecystectomy patient with complete bile drainage revealed curves of which the descending part has an identical terminal tl/2. The first phase in the plasma disappearance curve therefore is mainly due to distribution of ICG to the liver, while the second component probably reflects the rate of biliary excretion instead of ICG metabolism as has been suggested in some studies. Both uptake into the liver and biliary excretion can be partly saturated at higher doses, providing one clue for the involvement of the carrier-mediated mechanisms in both of these transport steps. A second clinical example is hepatic transport of cationic drugs. Agoston and co-workers [4] reported on two bivalent steroidal organic cations, vecuronium and its methylated derivative pancuronium, that are broadly used in anesthesia for peripheral muscle relaxation. Since only in the vecuronium molecule a
proton can dissociate from one of the nitrogen groups, a large uncharged ring system can be formed resulting in a proper balance between the remaining hydrophilic positively charged group and a heterocyclic hydrophobic part of the molecule. With regard to hepato-biliary transport, the liver discriminates especially sharply between drugs concerning this molecular balance [5]. Indeed a marked difference in hepatic uptake rate was observed in various species including man [4]. In remainders of liver biopsies taken from patients for diagnostic reasons, the authors were able to determine both compounds. It could be estimated that 15 min after injection the human liver contains about 60% of the injected dose of vecuronium, while this figure is much lower for pancuronium. This is also reflected in the plasma disappearance patterns in man; that for vecuronium is initially much more rapid. Taking into account the effective concentration range of these curare-like agents, these combined data imply that distribution to the liver will terminate the action of the compound. Indeed, the clinical duration of action of equipotent doses of the compounds are a factor of 3-4 different, and also the final elimination of vecuronium compared with pancuronium is much less affected by renal function.
CURRENT CONCEPTS ON HEPATIC TRANSPORT OF DRUGS
Chemical structure and hepato-biliary clearance The results on the cationic muscle relaxants raise the question about the relation between chemical structure and hepato-biliary clearance. The two agents mentioned showed almost absolute difference in hepatic disposition in spite of the fact that their molecular weight is almost identical. This seems to negate the well-known molecular weight-threshold hypothesis postulating that drugs with a molecular weight exceeding a (species-dependent) threshold of 200-300 are mainly excreted in bile while drugs with a lower MW are predominantly excreted in urine [1]. A better parameter is probably the balance between hydrophilic and hydrophobic properties of the agents. In a study with a homologous series of organic cations with increasing molecular weights ranging from 74 to 315, lipid solubility was correlated with the relative clearance of each of these compounds in liver, intestine and kidney in the intact rat [5]. Small organic cations of low lipid solubility were poorly excreted in bile, however, a change of a factor of 5 in lipophilicity increased biliary clearance 1000 times, while carrier-mediated secretion from blood into the intestinal lumen increased 100-fold. In contrast there was no evident relation between lipid solubility of organic cations and their renal tubular secretion. In a more biological context: the carrier-systems in the liver involved in hepatic uptake and biliary excretion may preferentially recognize relative lipophilic or amphipathic cations.
Acinar heterogeneity in transport How are such highly cleared drugs distributed in the liver? For this we look at another level of magnification: the microcirculatory unit, also called the hepatic acinus. It became evident in the last decade that hepatocytes in the acinar zones, apart from being heterogeneous in, for instance, carbohydrate-, drugand lipid-metabolism, are also unequal in transport function [1,6,9,19]. This dynamic situation may be due to their localization in the blood stream along the sinusoidal axis or to intrinsic differences between the
261
cells which in turn could be secondary to concentration gradients of 0 2, CO 2, hormones or other substrates. How can we experimentally differentiate between these types of heterogeneity? A very adequate method is again the isolated perfused liver. The isolated organ can be perfused in the normal direction or in a retrograde direction allowing manipulation of the localization of the drug in this system: with shortterm perfusions with high extraction types of drug at low protein medium concentration, zone 1 or zone 3 can be loaded. Subsequently the normal flow can be restored and the kinetics of efflux to plasma and bile can be monitored. This can be combined with freezeautoradiographic or fluorescent methods in which the localization of the test compound can be visualized as shown previously [6,10,19]. The acinar distribution of taurocholate a few minutes after injection, for instance, showed a sharp gradient with a clear zone 1 localization [6]. If the direction of the perfusate flow is reversed the taurocholate is predominantly found in zone 3; it is only a matter of presentation. Kinetic studies confirmed that the uptake rate in these zones seems to be equal. However, transport to bile is considerably slower from zone 3, and this indicates an intrinsic cellular difference in excretion efficiency for the bile acid. Such intrinsic differences may even lead to intrahepatic redistribution of drugs in time. Recently we performed a similar study with rhodamine B, that has a net positive charge at physiological pH. Ten minutes after injection almost all of this highly extracted drug is in the periportal zone 1, but interestingly, keeping the same direction of flow, 50 min later the picture seems to be reversed: most of it is in zone 3 of the acinus. Computer simulations using distributional perfusion models and zonalwashout studies indicated that these patterns can be explained by unequal rates of efflux from cell to blood in zone 1 and zone 3. This again indicates an intrinsic zonal difference in transport function or intracellular binding. Similar acinar concentration gradients have been reported, for instance, for propranolol and galactose (for references see Ref. 6). The general conclusion should be that the liver as a clearance organ should not be analyzed as a well-stirred compartment since both the rate of distribution and
262
D.K.F. MEIJER
ELIMINATION OF DRUGS FROM THE BODY
the rate of elimination of drugs in the various zones o! the hepatic acini can be quantitatively different [6,111.
gl0m. tub. filtration seer. i}
Bidirectional sinusoidal transport
Liver
:I
One of the factors determining such concentration gradients is the extent of protein binding. Protein binding may be a very important factor in the hepatic clearance of drugs. Basic drugs are mainly bound to orosomucoid or al-acid-glycoprotein, and acidic drugs are predominantly bound to albumin. Clearance of the organic anion DBSP in isolated perfused rat liver is inversely related to the albumin concentrations, as is that of BSP in man [7]. This phenomenon evidently may interfere with the diagnosis of liver disease. A lowered plasma albumin concentration can lead to a rise in unbound fraction that may partly compensate for the decreased intrinsic transport function. If DBSP is infused in perfused liver until steady state concentrations are reached, addition of albumin to the perfusate leads to an efflux of the organic anion from the liver into plasma. This demonstrates two points: hepatic storage of the organic anion, apart from the membrane transport, is determined by the relative binding capacity inside and outside the liver, and transport between plasma and liver of organic anions is bidirectional. This latter phenomenon is very likely related to the well-known fact that the parenchymal cells do not only secrete organic anions into bile but also into the general circulation (Fig. 2). For instance, how do anionic sulfate- or glucuronide conjugates leave the parenchymal cell? One example is the phenolic drug harmol that was constantly infused in rats. In the steady state mainly sulfate conjugate is formed, 60% of which is eliminated via urine and 30% via bile. At steady state the anionic dye DBSP was injected. It was anticipated that DBSP would inhibit the biliary output of harmol sulfate. Surprisingly, the biliary excretion of the sulfate conjugate immediately increased after addition of DBSP
"'",'2-;_-_~ Drug-conj.
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Fig. 2. Schematic representation of drug disposition by the liver. Hepatic uptake can occur passively or by carrier transport. Phase 1 and phase 2 biotransformation may produce polar conjugates that can be excreted into the general circulation or into bile, both via carrier-mediated transport. Conjugates or polar drugs may also be excreted via urine or via intestinal secretion. In the gut, conjugates can be deconjugated and reabsorbed entering an enterohepatic cycle.
while, in contrast, urinary excretion decreased. The combined data were explained by competitive inhibition of harmol sulfate excretion from the liver into plasma by DBSP, decreasing the driving force for excretion of harmol sulfate in the urine but increasing it for its biliary output. Such an inhibitory effect on liver-to-plasma transport was subsequently clearly demonstrated by loading the liver with preformed sulfate conjugate and measuring efflux in single pass perfusion, showing that hepatic efflux also is carrier-mediated [8]. Similar types of competitive phenomena at the sinusoidal level have been recently described for glutathione and glutathione conjugates by Sies et al. and Kaplowitz et al. (see Ref. 9).
CURRENT CONCEPTS ON HEPATIC TRANSPORT OF DRUGS Mechanisms of hepatic drug-plasmaprotein dissociation Another interesting point is hidden in the abovementioned data on DBSP transport and albumin binding: if clearance of DBSP is related to the unbound fraction the relation is essentially non-linear. It is as if the liver removes more DBSP than is predicted from its unbound fraction. How can we explain such a non-linear relation between unbound fraction and clearance in general? First of all, clearance may not entirely follow changes in unbound fraction if blood flow becomes rate-limiting or if saturation of membrane transport occurs [7]. Secondly, binding of drugs to albumin may facilitate passage of unstirred layers at the sinusoidal domain of the plasma membrane or even directly affect affinity for the membrane transport. The latter phenomenon was recently found for Na÷-coupled taurocholate transport in membrane vesicles (see Ref. 9). However, possible explanations are also present at the level of protein binding itself: the drug may spontaneously dissociate due to effective removal of unbound drug by the transport system during passage to the liver in nonequilibrium conditions. Alternatively, debinding may occur due to binding of albumin to a membrane receptor or due to aspecific surface interaction, both of which could lead to conformational changes in the protein and facilitation of dissociation of the complex (for an interesting discussion see Refs. 10 and 11). Finally, the liver is a metabolically very active organ, and at the microclimate of the plasma membrane competition may occur with endogenous substrates secreted from the liver. In principle all of these mechanisms may play a role, although the relative contribution of each of these processes may be different for each compound. Such mechanisms anyhow may explain that the liver can deal with highly proteinbound drugs.
Organic cation transport: influence of cofactors on uptake and subcellular distribution It should be mentioned here that the extraction of
263
drugs during passage through the liver is not only dependent on their extent of protein binding but also on the presence of cofactors which may help in the transport process. For instance, there is evidence that some organic cations in a physiological milieu can form electro-neutral ion-pairs with inorganic or organic counter anions. Such an interaction at the lipidwater interphase may help transport across the membrane, either due to passive diffusion or by interaction with carrier-systems for uncharged organic compounds. An example is the organic cation d-tubocurarine that normally is quite slowly removed from the circulation. Uptake into the liver is rate-limiting in hepato-biliary clearance. Addition of iodide that readily forms ion-pairs with organic cations immediately results in an increased disappearance rate and an elevated biliary excretion rate of the compound, providing a procedure to accelerate hepatic clearance of this drug. Similar effects can be found with H C O 3- as well as bile acid counter anions, suggesting that basic drugs are not necessarily transported as organic cations. Uptake into the liver of some cationic drugs can lead to rapid biliary excretion; however, it may also partly lead to persistent hepatic storage of such compounds, in which accumulation in lysosomes plays a major role. We arrive now at the level of intracellular distribution. The presence of such a drug in the lysosomes was demonstrated by classical subfractionation studies and also could be visualized with the electron microscope by making electron-dense precipitates with molybdate. What is the mechanism of this lysosomal accumulation? One speculation could be that such drugs are co-endocytosed with glycoproteins taken up in the liver via receptor-mediated endocytosis and trafficked to the lysosomes together with the protein. A candidate here is orosomucoid which both in sialated and desialated form binds cationic drugs much more strongly than albumin does. However, observations in the author's laboratory showed that addition of asialo-orosomucoid to perfusion media of isolated livers increases binding of such basic drugs about 5-10-times but does not affect clearance or the intracellular distribution, This must imply that within the liver the protein-drug complex
264
D.K.F. MEIJER Hepato-biliary transport of or.q.cations
~,
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Separate transport processes for anionic, cationic and uncharged drugs? So far we have discussed membrane transport of various classes of drugs: organic anions like DBSP and taurocholate as well as hepatobiliary transport of organic cations. How separate are these transport systems? The classical subdivision of the processes is based on the supposed charge of the compounds. For organic anions like DBSP a Na*-independent uptake process is present. A separate Na+-dependent mechanism for the anionic bile acids has been demonstrated, while one assumes additional processes for organic cations and uncharged drugs (cardiac glycosides such as ouabain and other steroids). How useful is the above-mentioned classification of hepatic transport processes? It may be useful from a mechanistic point of view but it is probably of little value if we want to predict drug transport interactions in the liver. One reason for this could be that there is a broad overlapping substrate specificity, especially for the hepatic uptake process: one compound can often be transported by various systems depending on its concentration relative to the K m of the particular process. Studies with isolated hepatocytes have largely contributed to this view [1]. Alternatively one or two very broad unspecific uptake systems for the above-mentioned classes of organic compounds could operate. In fact, recent studies combining photoaffinity label data with kinetic studies from the group of Kurz et al. [13] indicate that probably a general transport system is present for amphipathic compounds that can accommodate drugs of various charge such as the anionic unconjugated bile acids, the uncharged steroidal compounds, as wall as lipophilic bivalent and monovalent organic cations. However, even the Na÷-dependent uptake system for bile acids seems to be quite unspecific: it has been reported to transport conjugated bile acids, fusidic acid, which is structurally related, but also dissimilar structures such as the dicarboxylic acids iodipamide and methotrexate, as well as large cyclopeptides such as phalloidin and cyclosporin [9,13]. The particular processes thus may be quite aspecific and are able to recognize largely varying chemical
ii;/o"
Fig. 3. Transport mechanisms for hepatobiliary transport of organic cations (indicated by ¢r). Uptake can occur by two systems, only one is inhibitable by cardiac glycosides that may share a carrier process transporting large-MW lipophilic organic cations, possibly as ion-pairs with inorganic counter anions (2). Lipophilic organic cations bind in plasma to at-acid glycoprotein (orosomucoid), but are not co-endocytosed with asialo forms of this glycoprotein. Accumulation in lysosomes may occur via aspecific fluid-phase endocytosis. Direct transport from lysosomes to bile is unknown. Biliary excretion may involve carrier-mediated transport, possibly by antiport with protons. Binding to mixed biliary micelles facilitates net transport into the bile canaliculus.
completely dissociates before endocytosis as is pictured in Fig. 3. Other mechanisms of lysosomal uptake may resemble transport systems at the plasma membrane level: ion-pair transport or antiport with protons. The latter type of transport could also explain net canalicular secretion operating against an electrochemical gradient that further could be helped by micelle binding in the bile canalicular lumen. Accumulation of positively charged drugs in lysosomes can alternatively be explained by binding to negatively charged groups in the plasma membrane which triggers aspecific fluid-phase endocytosis and leads to vectorial transport to lysosomes. The particular lysosomal accumulative process may resemble that for aminoglycosides in kidney tubular cells and could have consequences for inhibition of proteolytic processes and hepatotoxicity of cationic drugs in the liver.
CURRENT CONCEPTS ON HEPATIC TRANSPORT OF DRUGS structures. However, we should also realize that model drugs that are commonly used are quite complex structures. Indocyanine green, for instance, is used as an anionic model compound. However, inspection of its molecular structure indicates it to be a zwitter-ion which also has a cationic group. In fact the compound is a very strong inhibitor of organic cation transport in isolated hepatocytes [1]. As mentioned earlier, cationic drugs can be present also as uncharged ion-pairs and may be recognized by systems normally transporting uncharged organic compounds. Ouabain in its turn has a steroid structure which could well fit the bile acid carrier and, in fact, (partly) Na+-dependent transport has been reported in perfused livers. Finally, the bile acids and especially the unconjugated ones can even be present in three forms: dissociated as anions, undissociated molecules resembling the uncharged cardiac glycosides, and complexed with ions such as Ca 2÷, thus having a net positive charge. This might explain the observation that bile acids in relatively high concentrations are very potent but completely reversible inhibitors of anionic, cationic and neutral transport pathways [14].
Concentrative membrane transport-what is the driving force? Gradients across both the sinusoidal and canalicular membranes can be calculated. These gradients can only be partly explained by electrical potential differences across these compartments, meaning that other driving forces should be present. The existence of concentrative transport steps is generally confirmed now in experiments with isolated hepatocytes and membrane vesicles from basolateral and canalicular portions of the hepatocyte plasma membrane [9,13,15]. If concentrations of unbound drugs are determined by ultrafiltration of plasma- and liver-cytosol fractions and if one estimates the extent of binding of drugs to biliary micelles by ultracentrifugation of bile, uphill transport against an electrochemical gradient can be calculated [1,7,9]. In addition, the question as to whether drugs are transported as
265
charged molecules can be approached by study of isolated membrane vesicles. Highly purified preparations enriched in either sinusoidal and canalicular elements can now be prepared. Examples are the studies of Inoue and Arias, the work from Blitzer and Boyer and recent studies from Meier and co-workers (see for references Refs. 9 and 17). The obvious advantage of this preparation is that in contrast to intact cells both the external and internal milieu can be easily manipulated: it is possible to impose ion-gradients and even induce short-lasting membrane diffusion potentials. For instance, by adding NaNO 3, in a non-equilibrium situation the negative NOs--ions will enter more rapidly than Na ÷ so that a transient inside negative membrane potential is produced. Typical uptake curves for taurocholate in sinusoidal membrane vesicles show overshoot phenomena that are due both to the presence of a Na÷-gradient and a short-lasting inside negative diffusion potential produced by addition of NaNO s in non-equilibrium conditions [9,12]. Co-transport of one taurocholate and more than one Na÷-ion is found in several systems and explains the electrogenic character. In contrast, uptake of the organic cation methyldeptropine (an anticholinergic drug) is not stimulated by such a NaNOa-induced negative membrane potential, indicating an electroneutral uptake system for this quaternary ammonium compound [12]. This confirms the idea of transport as uncharged ion-pairs or alternatively counter transport with protons. All of these mechanisms strongly suggest a relation between ion-gradients and drug transport. This brings us finally to the molecular level and especially to the question of driving forces for these transport processes for drugs. Based on the work of Arias et al. (see Refs. 9 and 17), Scharschmidt et al. (see Refs. 9 and 17) and Meier and co-workers [9,15] the following picture of inorganic ion transport in the hepatocyte can be made (Fig. 4). The initial event is electrogenic transport of Na + and K + by the ATPase system. The created Na÷-gradient then induces antiport of Ca 2÷ and of protons. Protons are formed within the cell from CO 2 and water and their removal from the cell therefore results in an intracellular concen-
266
D.K.F. MEIJER
T R A N S P O R T M E C H A N I S M S FOR DRUGS IN THE LIVER
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terns are quite unspecific and show overlapping substrate specificity. Looking at carrier-mediated transport at the canalicular pole of the cells, the general impression is that the systems are more specific. Consequently the above-mentioned classes of compounds have less interaction at this level. This is also suggested by earlier transport studies in mutant sheep [1,9,17], and was confirmed in mutant rats with deficient canalicular transport of organic anions and uncharged compounds but, in contrast, unchanged bile acid and cation transport into bile [16].
......
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Fig. 4. Coupling between gradients of inorganic ions with cartier-mediated transport of organic anions (OA-), bile acids (BA-), organic cations (OC +) and uncharged compounds (UC) in the hepatocyte. Inorganic ion-pumps include electrogenic Na+/K÷ exchange, Na+/H+, OH-/SO4--antiport (sinusoidal) and Cl-/HCO3--antiport (canalicular). Na+-coupled bile acid transport (1) and OH-/OA--antiport (2) are anionic systems. Organic cations are taken up by system 1 and may also share a transport system for uncharged compounds (2). In the latter system ion-pair formation may play a role; at the canalicular level antiport of organic cations with protons is tentatively assumed. The transport processes at sinusoidal level have more overlapping substrate specificity than the projected four canalicular carrier systems (adapted from Hugentobler and Meier, Ref. 15).
tration of bicarbonate (or hydroxyl-ions) exceeding the normal electrochemical equilibrium. Bicarbonate in its turn may drive CI- antiport across the canalicular membrane and hydroxyl-ions may induce sinusoidal sulfate uptake also by antiport [15]. For drugs at least four transport systems related to ion gradients may operate: firstly, co-transport with Na ÷ (conjugated bile acids); secondly, a Na+-indepen dent anion uptake system for compounds such as BSP, possibly driven by hydroxyl antiport; thirdly, canalicular cation transport for relatively non-lipophilic basic drugs which could operate via antiport with protons and therefore is coupled to Na+/H ÷ exchange, and finally, a broad unspecific uptake system that transports uncharged steroid-like compounds as well as amphipathic cations probably in the form of ion-pairs with inorganic anions. These uptake sys-
Carrier proteins: the responsible transport modalties? How can we define carrier transport function at the membrane level other than by kinetic studies? One answer is isolation or specific labeling of the carrier proteins involved. A number of groups have succeeded now to characterize potential carrier proteins (Fig. 5). The group of Tiribelli and Sottocasa isolated a 110 kDa protein, binding organic anions, which they called bilitranslocase (BTL), having an a2fl structure with three subunits of about 35 000. Berk and Stremmel [17] as well as Wolkoff et al. (see Refs. 9 and 16 for references) isolated a protein with subunits of about 55 000 mol. weight which bound organic anions but not bile acids, and also raised antibodies against it. In spite of the identical molecular weight, they may represent different carrier systems since the particular antibodies do not clearly crossreact and the iso-electric points are reported to be different. At least three groups (see Fig. 5) approached the carrier function by photo-affinity labeling techniques and generally found two macromolecules, one being of mol. weight 48 000 that binds photo-affinity probes of especially conjugated bile acids. There is good evidence that this represents the Na+-dependent system; as shown by Kurz et al. and Petzinger and Frimmer, it also transports cyclo-peptides such as phalloidin and cyclosporin [13]. These investigators also found a 55 000 mol. weight protein that preferentially binds unconjugated bile acids, but also may transport neutral steroidal compounds and amphipathic ca-
267
CURRENT CONCEPTS ON HEPATIC TRANSPORT OF DRUGS
POTENTIAL CARRIER-PROTEINS FOR HEPATIC TRANSPORTOF DRUGS Tlrlbelll Sottocasa et al. Mol Weight Proteln-lsolatlon
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Steroids Amph, cat Ions SO~-
Fig. 5. Experimental work reported on potential carrier proteins responsible for hepatic uptake of organic anions and other drugs. Estimated mol. weight, use of photoaffinity techniques, preparation of antibody with measurement of its effect on uptake (in brackets), reconstitution experiments performed, cofactors established and binding or transport of substrates are indicated. The scheme suggests separate carrier-processes; however, some may turn out to be identical (for instance 2nd, 3rd and 5th from left to right).
tions: a good candidate for the broad unspecific transport system that was mentioned above [13]. One crucial point here is to prove that the isolated protein is not only binding the transported drugs but also is able to translocate or 'channel' a drug across the membrane. This can be approached in at least two ways. One is to study the effect of monospecific antibodies raised against such purified proteins on drug transport. Berk and Stremmel recently reported inhibitory effects of antibody preparations in isolated hepatocytes [17]. In the author's laboratory an inhibitory effect of a BTL-antibody preparation on DBSP transport from liver into perfusate of isolated perfused livers was found but there was essentially no effect on hepatic uptake of this model compound. A second approach is reconstitution of transport with the isolated carrier proteins. In a preliminary study [18] the potential carrier protein bilitranslocase was inserted in monolaminar liposomes. It was demonstrated that this protein clearly supported BSP trans-
port out of such liposomes as driven by a K+-valino mycin-induced negative membrane potential. This facilitatory effect was not seen with other anion-binding proteins such as ligandin and albumin. The study of intramembrane mobility of the carrier proteins (rotating units, flip-flop movements or ion-selective channel conformation) will hopefully provide more insight into the precise membrane transport mechanisms involved. The study of the regulation of the cellular synthesis of such carrier proteins in individuals with genetic transport defects or following adaptive changes in hepato-biliary transport under various conditions may further clarify their crucial role in health and disease. For now, here our travel through the hepatic transport system ends: indeed, traffic in the liver is heavy, but fascinating, confronting us with the amazing number of secrets and surprises that nature has to offer.
268
Acknowledgements
D.K.F. MEIJER Drs. W . E . M . M o l . I a m g r a t e f u l for the c o o p e r a t i o n of Dr. R . J . V o n k , D r . P . L . M . J a n s e n , Dr. M . J .
This r e v i e w is d e d i c a t e d to the m e m b e r s of the ' E u -
H a r d o n k , D r . S. A g o s t o n , D r . A . H . J . S c a f and D r .
r o p e a n Study G r o u p for H e p a t o - b i l i a r y T r a n s p o r t ' ,
G . J . M u l d e r . P a r t of the m a t e r i a l in this r e v i e w was
with m a n y thanks for advice and friendship. I ac-
p r e s e n t e d in a ' s t a t e - o f - t h e - a r t ( p r e s i d e n t i a l ) l e c t u r e '
k n o w l e d g e the active i n v o l v e m e n t of m y c o - w o r k e r s
during the 36th m e e t i n g of t h e A m e r i c a n A s s o c i a t i o n
Dr. G . M . M . G r o o t h u i s , D r . G . A . V e r m e e r , D r . C.
for the Study of L i v e r D i s e a s e s , C h i c a g o , N o v e m b e r
N e e f , Dr. P . G . R u i f r o k , Dr. P. W e s t r a , Dr. A . F .
5, 1985.
B e n c i n i , Drs. P. van d e r Sluijs, D r s . I. B r a a k m a n and
References 1 Klaassen CD, Watkins JB. Mechanisms of bile formation, hepatic uptake and biliary excretion. Pharmacol Rev 1984; 36: 1-67. 2 Biota A, Scaf AHJ, Meijer DKF. Hepatic drug transport in the rat. A comparison between isolated hepatocytes, the isolated perfused liver and the liver in vivo. Biochem Pharmacol 1982; 31: 1553-1565. 3 Meijer, DKF, Weitering JG, Vermeer GA. Pharmacokinetics of hiliary excretion in man. V. Dibromosulfthalein. Eur J Clin Pharmacol 1983; 24: 549-556. 4 Sohn Y J, Bencini A, Scar AH,I, et al. Comparative pharmacokinetics and dynamics of vecuronium and pancuronium in anesthetized patients. Anesth Analg 1985; 65: 233-239. 5 Neef C, Meijer DKF. Structure-pharmacokinetics relationship of quaternary ammonium compounds. Correlation of physicochemical and pharmacokinetic parameters. Naunyn-Schmiedeberg's Arch Pharmacol 1984; 328: l 1 l - 118. 6 Groothuis GMM, Hardonk MJ, Meijer DKF. Hepatobiliary transport of drugs: do periportal and perivenous hepatocytes perform the same job? Trends Pharmacol Sci 1985 (Aug): 322-327. 7 Meijer DKF, BIom A, Weitering JG, et al., Pharmacokinetics of the hepatic transport of organic anions: Influence of extra- and intracellular binding on hepatic storage of dibromosulfophthalein and interactions with indocyanine green. ,l Pharmacokin Biopharm 1984; 12: 43-65. 8 De Vries MH, Groothuis GMM, Mulder G.l, et al. Secretion of the organic anion harmol sulfate from liver into blood. Evidence for a carrier-mediated mechanism. Biochem Pharmacol 1985; 34: 2129-2135. 9 Boyer .IL. Mechanisms of bile secretion and hepatic transport. In: TC Andreoli, JF Hoffman, DD Fanestil, SG Schulz, eds. Physiology of Membrane Disorders. 2nd ed. New York: Plenum Press, 1986; 609-636.
10 Morgan D,l, Jones DB, Smallwood RA. Modeling of substrate elimination by the liver: has the albumin receptor model superseded the well stirred model? Hepatology 1985; 5: 1231-1235. 11 Forker EL, Luxon B. Lumpers vs distributors. Hepatology 1985; 5: 1236-1237. 12 Meijer DKF, Neef C, Groothuis GMM. Carrier-mediated transport in the handling of drugs by the liver. In: DD Breimer, P Speiser, eds. Topics in Pharmaceutical Sciences. Amsterdam: Elsevier, 1983; 167-189. 13 Buscher HP, Fricker G, Gerok W, et al. Membrane transport of amphiphilic compounds by hepatocytes. In: H Greten, E Windier, U Beisiegel, eds. Receptor-Mediated Uptake in the Liver. Heidelberg: Springer-Verlag, 1986; 189-199. 14 Vonk R,l, Van Doorn ABD, Mulder G J, et al. The influence of bile salts on hepatocellular transport, Falk Symposium no. 26, V. Bile acid meeting. Biological effects of bile salts. 1978; 121-126. 15 Hugentobler G, Meier PJ. Multispecific anion exchange in basolateral (sinusoidal) rat liver plasma membrane. Am J Physiol 1986; 251: G656-G664. 16 Jansen PLM, Groothuis GMM, Peters WHM, et al. Selective defect in the hepatobiliary transport of organic compounds in mutant rats with heriditary conjugated hyperbilirubinaemia. Heterogeneity of hepatobiliary transport. Hepatology 1987; (in press). 17 Berk PD, Stremmel W. Hepatocellular uptake of organic anions. In: H Poppen, F Schaffner, eds. Progress in Liver Diseases, Vol. VIII. New York: Grune & Stratton, 1986; 125-144. 18 Sottocasa GL, Baldini G, Sandri G, et al. Reconstitutions in vitro of sulfobromophthalein transport by bilitranslocase. Biochim Biophys Acta 1982; 685: 123-128. 19 Gumucio ,l,l. Functional and anatomic heterogeneity in the liver acinus: impact on transport. Am ,l Physiol 1983; 244: G578-G582.