Drug Targeting Organ-Specific Strategies

The mechanisms of uptake and excretion of drugs by the liver has been widely studied using isolated hepatocytes and isolated perfused livers of rodents. The subject was extensively discussed by Oude Elferink et al. [82], summarized in a comprehensive special issue of the Journal of Hepatology in 1996 [83] and reviewed in several papers and chapters by our group [84,85]. In general, the rate and mechanism of drug uptake in isolated rat hepatocytes are very similar to those found in vivo. Only a few studies have investigated transport of drugs in human hepatocytes, and even fewer have used liver slices. However, studies in human and rat hepatocytes are hampered by the fact that the isolation procedures involve collagenase digestion for the disruption of cell-to-cell contacts. Clearly, this proteolytic enzyme may also damage plasma membranes and transport systems therein.

Jansen et al. [86] published an example of transport of a drug targeting preparation in human and rat hepatocytes. They found that the anti-viral drug ara-AMP coupled to lactosaminated human serum albumin, was taken up to the same extent by human and rat hepatocytes. This is one of the few examples where an equal rate of transport was found in both species. In general the uptake of drugs in human hepatocytes is slower than that in rat hepatocytes. In vit- ro–in vivo scaling calculations [87,88] showed that these differences in the uptake rate of drugs between rat and human cells, actually reflect inter-species differences rather than being due to differences in viability. Moreover, the differences described in rate and mechanism of drug transport in rat and man emphasize again that extrapolation to man of pharmacokinetic data obtained in rat, is hazardous. Most of the current knowledge on drug transport carriers is derived from experiments with rats. Therefore, more studies need to be carried out in human liver preparations to further elucidate the mechanisms of drug transport in the human liver and hence the relevance of animal data.

These results also indicate that human hepatocytes are an appropriate model in which to study inter-species differences and the mechanisms of hepatic transport in man.

In contrast to isolated hepatocytes, liver slices retain the cellular architecture of the liver without prior digestion with collagenase. This makes a systematic comparison of the data relating to transport of free drugs as well as drug targeting moieties from isolated hepatocytes and liver slices, an attractive model for studying the potential and limitations of the liver slice model in this area of research.

12.5.2 Transport in Liver Slices

Mechanisms of drug uptake in liver slices were studied in vitro as early as in 1963 by Schanker and Solomon [89]. The results obtained in these experiments are still valuable and show that the influence of temperature, anoxia, metabolic inhibitors and substrate inhibition can be successfully studied in this preparation. However, as mentioned before, at that time the preparation of reproducible precision-cut slices was not feasible. Therefore, the slice incubation technique was virtually abandoned in transport studies after the introduction of the successful isolation of rat hepatocytes.

In order to investigate the possibilities and limitations of the use of precision-cut liver slices prepared with a mechanical slicer in drug transport studies, different aspects of the mechanism of uptake of several classes of drugs in human and rat liver slices were investigated in our laboratory. Four model compounds which enter hepatocytes via entirely differ-

320 12 Use of Human Tissue Slices in Drug Targeting Research

Figure 12.4. Rhodamine B (25 M) distribution in the cross-section of a rat liver slice (± 250 M) after 5 min incubation. Fluorescence microscopy, bar = 100 M.

ent membrane transport mechanisms were investigated: the fluorescent dyes rhodamine B and lucigenin, the cardiac glycoside digoxin and the neo-glycoprotein lactosylated albumin. Receptor-mediated endocytosis into endothelial cells was studied with succinylated and aconylated albumin. The rate of penetration into the rat liver slice was studied with the lipophilic cationic compound rhodamine B (RB) and the hydrophilic organic cation lucigenin (LU). RB, which enters hepatocytes by passive diffusion was homogeneously distributed throughout the rat liver slice (250 m thickness) within 5 min [81] (Figure 12.4). These results indicate that for very lipophilic components both the penetration rate into the slice and the diffusion rate into all the cells are rapid processes. In contrast, after incubation with LU, which is taken up by hepatocytes through adsorptive endocytosis, fluorescence in the inner cell layers could only be detected after 15 min. If the rates of uptake of drugs in liver slices are to be compared with parameters obtained in vivo, some difficulties may arise. The uptake rate of compounds into the slice may not only reflect the uptake rate of the cells involved, but may also be influenced by the rate of penetration of the substrates (i.e. diffusion through sinusoidal spaces) into the slice. From our results with the lipophilic agent rhodamine B it is clear that the penetration process into the slice takes at least 5 min. Therefore, for substrates that are taken up into hepatocytes relatively fast, penetration into the slice may limit the uptake of the drug by the inner cell layers in the slice.

Digoxin uptake into rat liver slices showed a temperature-dependent component, compatible with the involvement of carrier-mediated uptake mechanisms. Quinine markedly inhibited the uptake of digoxin, in contrast to its diastereomer quinidine, which only slightly inhibited the digoxin uptake in rat liver slices. This stereoselective inhibition is in line with results obtained in isolated rat hepatocytes and isolated perfused rat livers [90,91]. These results were also found after cryopreservation of the slices, indicating that carrier-specific phenomena can be studied after cryopreservation [92].

From these results it can be concluded that liver slices are a powerful tool for studying the mechanisms and specificity of carrier-mediated uptake of drugs and drug interactions which occur at the transport level.

Many drugs that are taken up and metabolized by hepatocytes are excreted via the bile canaliculi into the bile. One of the remaining topics in liver slice research is the question of whether liver slices are capable of bile excretion via the bile canaliculus. Thompson et al.[93] showed that slices are capable of excreting bile acids, however, there is a need for more experiments to determine whether this excretion takes place across the bile canalicular membrane.

12.6 The Use of Liver Slices in Drug Targeting Research

12.6.1 Distribution and Transport of Drug Targeting Devices

In our Institute, drug targeting devices are predominantly developed for the treatment off inflammatory diseases of the liver, such as fibrosis and cirrhosis. In order to increase the therapeutic efficacy of drugs, human serum albumin is used as a protein backbone; modifying this protein by the attachment of different sugar groups or peptide molecules targets these modified proteins to specific cell types in the liver as described in more detail in Chapter 4. Antiinflammatory agents are subsequently coupled to the protein backbone to serve as effector moieties.

Further studies are needed to determine whether these liver-directed drug targeting preparations are actually delivered to the specific liver cells. In addition, experiments should be carried out to ascertain whether the drug coupled to a drug targeting device is released in the target cell in the liver, and to ensure that the drug is still active in these cells. Up until now the distribution of these modified proteins has been tested in vivo mainly in rats, and in vitro in the perfused rat liver or isolated liver cells (both parenchymal and non-parenchymal cells). Almost no studies have been performed in the target species, man. As the main aim of drug targeting research is the development of preparations for clinical use, we investigated whether precision-cut liver slices could be used to study the uptake of these modified proteins into target cells of the human liver.

The temperature-dependent uptake and immunohistochemical localisation of modified proteins, Lactose27-Human Serum Albumin (Lact27-HSA), Succinylated-Human Serum Albumin (Suc-HSA) and Aconylated Human Serum Albumin (Aco-HSA), in rat and human liver slices showed that large molecules can enter the slice and are probably taken up by re- ceptor-mediated endocytosis (Figure 12.5). These large modified proteins were found distributed all over the liver slice, as was determined by immuno-histochemistry [81] (Figure 12.6). Recently, Beljaars et al. [94] showed that mannose-6-phosphate21-Bovine Serum Albumin (M6P21-BSA) is taken up by the non-parenchymal cells of human liver using slices from cirrhotic human livers. The non-parenchymal cells were identified as hepatic stellate cells (HSC) or endothelial cells. The involvement of hepatocytes, Kupffer cells, and bile duct epithelial cells was excluded. Until now, there has been little information concerning the presence of the mannose-6-phosphate receptor in human adult livers. The accumulation of M6P-modified albumin in human cirrhotic livers demonstrates, however, that M6P receptors are present in the human liver and that M6P-modified albumin may be useful as a drug carrier for the targeting of anti-fibrotic drugs in patients. In another study by Beljaars et al. [95],

Figure 12.6. Fluoresceinlabelled aconylated human serum albumin distribution in the cross-section of a rat liver slice (± 250 M) after 120 min incubation. Bar = 100 M.

parenchymal cells from diseased livers is experimentally very difficult, therefore the liver slice technique offers a unique opportunity to study drug targeting preparations in diseased liver.

12.7 Efficacy Testing of the Drug Targeting Device in the Liver

It is of paramount importance to ensure that the drug targeting device with the drug attached or incorporated, is not only taken up by the target cells in the liver, but is also released from the device and remains active within the target cell. In the case of the development of drug targeting strategies for inflammatory liver diseases, an in vitro system was needed that could be used to test the anti-inflammatory effect of the drug-targeting preparations in the human liver.

To set up and validate the in vitro systems we initiated a study with rat liver slices. Stimulation by lipopolysaccharide (LPS) in liver slices was used to evoke a pro-inflammatory response in the liver. Lipopolysaccharide (LPS), a component of Gram-negative bacterial cell walls (also called endotoxin), has been associated with tissue injury and sepsis. In the liver LPS activates the resident macrophages, the Kupffer cells, which results in cytokine release [96]. Furthermore, LPS is cleared by the liver, mainly by Kupffer cells [97]. One of the major features of endotoxic shock is the induction of nitric oxide synthase in the liver [98]. Inducible nitric oxide synthase (iNOS), the expression of which is induced by LPS and cytokines, produces nitric oxide (NO) in large quantities [99].

The induction of iNOS by LPS, as observed in the hepatocytes in vivo [99], cannot be achieved in pure cultures of isolated hepatocytes. In fact, the induction of hepatocyte-associ- ated iNOS is found in pure cultures only after incubation with a mix of LPS and cytokines [100]. Induction of iNOS by LPS alone can be accomplished in co-cultures of hepatocytes and Kupffer cells with LPS [98]. These data indicate that induction of iNOS by LPS is mediated by cytokines released by the Kupffer cells in the liver and is thus a result of intercellular

Time (hours)

Figure 12.8. NO production in rat liver slices after incubation in the absence or presence of 100 g ml–1 LPS for different time periods. The NO production is measured as nitrate/nitrite (NOx) concentrations in the medium ( M). Control (■) and + 100 g ml–1 LPS ( ). Data are expressed as mean ± SEM of four experiments. *p < 0.005 represents a significant increase in NO production by liver slices due to stimulation by LPS.

duction (Figure 12.9) in the liver slice model. These results show that the conjugate dexa- methasone10–HSA is taken up intracellularly and that active dexamethasone is released.

Studies similar to those described above are now being carried out in human liver slices. LPS induction in human liver slices also increased TNFα production to the same extent as was found in rat liver slices [104] (Figure 12.10). Human liver slices also produced IL-6, IL-8 and IL-1β, although the latter to a lesser extent than that observed in the liver slices of rat origin. However, human liver slices produced less NO after LPS stimulation than those of the rat. More experiments will be undertaken to elucidate this species difference.

Taken together these results indicate that non-parenchymal cells are still active in the slice preparations and that intercellular communication is still intact. Furthermore, pharmacological intervention by anti-inflammatory drugs can be successfully studied in liver slices. Together with the results obtained in regard to drug transport, liver slices seem to be a very promising in vitro system for studying intercellular distribution, cellular processing and effectiveness of anti-inflammatory drugs coupled to a targeting device.

328 12 Use of Human Tissue Slices in Drug Targeting Research

specificity of carrier-mediated uptake of drugs. In addition, the distribution of the drug into different cell types in the tissue can easily be studied in preparations of organ slices, as can the efficacy of the drug which is coupled to the targeting device. Furthermore, metabolism and toxicity of the drug targeting device or the released drug can be determined in the human liver. And finally, an important aspect of this type of in vitro research in man is, that it will ultimately lead to a reduction in the use of animal experiments.

In future, drug targeting devices aimed at other human organs may also be studied using precision-cut tissue slices.The latest data/literature on precision-cut tissue slices can be found at http://www.farm.rug.nl/slice/open.html.

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