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ther inflammatory cells or accessory cells. As accessory cells they express major histocompatibility complex (MHC) class II molecules on their surface , synthesize IL-1β and present antigens to T cells [51]. As inflammatory cells, KCs enhance chemotaxis, phagocytosis, and oxidative metabolism of inflammatory cells [52] by producing cytokines, eicosanoids and reactive oxygen species (ROS). After LPS stimulation, KCs produce chemokines such as monocyte chemotactic protein (MCP-1), macrophage inflammatory protein-1α/β (MIP-1α/β), RANTES (Regulated upon Activation, Normal T-cell Expressed and Secreted) and IL-8 [53,54], in addition to TNFα, IL-1α/β, interferon alpha/beta (IFNα/β), and IL-6 [55]. Release of these mediators will lead to activation and local infiltration of inflammatory cells and activation of other resident hepatic cells. KCs also produce transforming growth factor beta (TGFβ), which stimulates collagen synthesis by HSCs, while inhibiting proliferation of these cells [56].

Besides LPS, other particulate and soluble agents are known to stimulate the formation of eicosanoids, e.g. PGE2, PGD2, and thromboxane [57]. These agents also elicit nitric oxide and superoxide anion formation, which may help to destroy phagocytosed microorganisms or particles [58].

4.2.4 The Hepatic Stellate Cell (HSC)

Another resident hepatic cell that is important in the pathogenesis of chronic liver diseases is the hepatic stellate cell (also known as fat-storing cell, Ito cell, lipocyte, perisinusoidal cell). They are located in the space of Disse and represent 5–8% of all liver cells. With cytoplasmatic extensions encircling the sinusoid they regulate blood flow through the sinusoidal lumen, in response to endothelin-1, nitric oxide, angiotensin-II, thromboxane A2, and the prostaglandins F2α, I2, and E2 [59]. They also contain many vitamin A-rich lipid droplets which account for 75% of the total amount of retinoids stored in the body. As well as controlling the uptake, storage, and release of retinoids, HSCs are the major regulators of the extracellular matrix composition after activation. They produce and secrete matrix proteins such as collagens I, III, IV, V and VI, fibronectin, laminin, tenascin, undulin, hyaluronic acid and proteoglycans [60], as well as extracellular matrix degrading metalloproteinases and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) [61].

HSCs have a dual phenotype. In healthy livers they have the quiescent phenotype, regulating retinoid storage and blood flow. In response to liver injury, however, they acquire an activated myofibroblast-like phenotype. During the transition to the activated phenotype there is a gradual loss of lipid droplets and an increased expression of α-smooth muscle actin (αSMA). In rat livers this is also accompanied by a loss of desmin expression [62]. A consequence of HSC activation is the change in synthetic activity towards production of excess collagen I and III molecules and other matrix molecules. These matrix proteins are deposited in the space of Disse obstructing efficient exchange of proteins and reducing the diameter of the sinusoids, thereby impeding blood flow. This process is called capillarization. It is also accompanied by a loss of fenestration of the sinusoidal endothelial lining, which further hampers the diffusion of proteins between plasma and hepatic cells. The alterations in the appearance of the sinusoid are the hallmark of fibrosis.

The transdifferentiation of HSCs to myofibroblasts, producing extracellular matrix constituents is characterized by an increased expression of several receptors, including the

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platelet derived growth factor (PDGF) receptor, the collagen type VI receptor, and the in- sulin-like growth factor II/mannose-6-phophate receptor (IGFII/M6P). For reviews on this subject see Beljaars et al. [63], Li et al. [64], and Bissell [65].

4.3 Hepatic Inflammation and Fibrosis

Virtually any insult to the liver can cause hepatocyte destruction and parenchymal inflammation. If the insult is minor and occurs only once, local restoration mechanisms will suffice to repair the damage. If, however, the insult is major or persistent, an inflammatory response will be generated. This inflammation is the result of cytokine-mediated activation of sinusoidal cells, their subsequent release of pro-inflammatory cytokines and their expression of adhesion molecules for the recruitment of circulating leucocytes. Once the damage is under control and the inciting insult has been eliminated, the inflammatory process will end and local mechanisms will proceed until the damage is repaired. Usually little scar tissue will be detectable, because of extracellular matrix remodelling. During conditions of chronic liver injury, however, the repair process does lead to scar tissue formation, which is deposited within the liver until impairment of liver function occurs. This process is called liver fibrogenesis and the end stage, or irreversible stage, is referred to as liver cirrhosis (Figure 4.2).

Figure 4.2. Diagram outlining the pathogenesis of liver fibrosis. Injury to parenchymal cells (PC) results in the activation of Kupffer cells (KC) and sinusoidal endothelial cells (SEC) and the recruitment of inflammatory cells (IC). These cells release cytokines, growth factors and reactive oxygen species that induce activation and proliferation of hepatic stellate cells (HSC). HSCs gradually transform into myofibroblasts (MF), the major producers of extracellular matrix (ECM) proteins.

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After damage or infection, monocytes and KCs in the area detect the damaged cells or infectious agent and respond with release of primary mediators such as TNFα, IL-1 and some IL-6. These cytokines activate the surrounding cells, that respond with a secondary, amplified release of cytokines. This second wave includes large amounts of IL-6, which induce the synthesis of acute phase proteins in hepatocytes and chemoattractants such as IL-8 and MCP-1. These events will then lead to the typical inflammatory reactions. Both IL-1 and TNFα activate the central regulatory protein of many reactions involved in immunity and inflammation, nuclear factor kappa B (NFκB).These cytokines cause dissociation of NFκB from its inhibitor IκB, which makes translocation of NFκB to the nucleus possible. In the nucleus active NFκB induces the transcription of the ‘second wave’ cytokines (see also Chapter 7 for the molecular mechanisms of cytokine-mediated cell activation).

The release of TNFα and IL-1 also upregulates adhesion molecules like ICAM-1 and VCAM-1 on SECs, that are subsequently responsible for the adhesion and recruitment of circulating neutrophils. KCs and PCs release IL-8, which is a potent neutrophil chemoattractant. The attracted neutrophils and KCs are stimulated to release large amounts of reactive oxygen species (ROS: hydrogen peroxide, superoxide anion and nitric oxide (NO) radicals). The production of NO is also mediated through the NFκB pathway. The enzyme responsible for the increased synthesis of NO, inducible NO synthetase (i-NOS), is increasingly expressed through NFκB-mediated stimulation of the i-NOS promotor region.

TGFβ and TNFα produced by KCs and PDGF produced by SECs subsequently play an important role in the activation of HSCs.TGFβ appears to be the most important cytokine in stimulating the production of scar tissue components like collagens by HSCs.The mechanism of activation is probably via the IGF-II/M6P receptor, which is also increasingly expressed on activated HSCs. As yet unknown factors produced by KCs [66] stimulate expression of PDGF receptors on the surface of HSCs. In the presence of PDGF the HSC will now proliferate as well. On chronic stimulation, HSC stimulation and proliferation will result in production of excess extracellular matrix and the onset of fibrosis. KC-produced mediators appear to be important for HSC stimulation, but substances directly released by PCs are also found to be mitogenic [67].

Since not every insult necessarily results in liver fibrosis, counter-regulatory mechanisms must also exist. During inflammation, elimination of ROS by SECs and KCs is enhanced via increased expression of radical scavengers like superoxide dismutases and glutathione peroxidase. The radical nitric oxide itself also has an anti-inflammatory role. It has been described to prevent leucocyte adhesion to the endothelium [68] and to block an activation pathway of thrombocytes by stimulating guanylyl cyclase [69]. Furthermore, both PGE2 and IL-10 can downregulate cytokine production by macrophages [70,71] and can also inhibit the antigen-presenting properties of SECs and KCs [18,72]. HGF produced by KCs, SECs, and quiescent HSCs is a potent mitogen for PCs and stimulates liver regeneration. It is probably aided by PGE2 which also stimulates DNA synthesis in PCs [73]. Finally, scar tissue formation is not only regulated by production of extracellular matrix components, but also by the degradation of matrix components. Activated and quiescent HSCs, KCs, and SECs produce matrix metalloproteinases that are responsible for matrix degradation [74].

Whether liver regeneration will dominate over scar tissue formation depends on many factors, including the nature and the duration of the injury and the genetic background of the individual. It is still unclear at which point liver regeneration is no longer possible and fibroge-

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nesis will progress to cirrhosis. When fibrogenesis takes over, however, collagens type I and III which are normally concentrated in the portal tracts and around central veins, are deposited throughout the liver. Collagen IV and VI and other components of the extracellular matrix are also increasingly expressed. The normal liver only contains 1–2% connective tissue, but in patients with cirrhosis this can increase up to a maximum of 50% [75]. The increased amount of extracellular matrix results in severe disruption of blood flow and impaired diffusion of solutes between PCs and plasma, which may have implications for drug targeting preparations to hepatic cells. Deposition of collagens in the space of Disse is also accompanied by the loss of fenestrations in SECs, which further impairs the movement of proteins between PCs and plasma. The subsequent resistance to portal flow induces portal hypertension and together with the reduced metabolic capacity of the liver this leads to four major clinical consequences: development of ascites, the formation of portosystemic venous shunts leading to dangerous esophagogastric varices, congestive splenomegaly causing haematologic abnormalities, and hepatic encephalopathy because of the exposure of the brain to an altered metabolic milieu. Other complications arising from the progressing fibrosis of the liver are the appearance of renal failure (hepatorenal syndrome), endotoxemia and hepatic failure. When loss of the hepatic functional capacity exceeds 80–90%, liver transplantation is usually the only option for survival. Many new pharmacological approaches to the therapy of fibrosis are being explored, but lack of effectiveness or a small therapeutic window remain major obstacles. These approaches may therefore benefit from drug targeting strategies [3].

4.4 Liver Cirrhosis: Causes and Therapy

Cirrhosis is among the top 10 causes of death in the Western World. This is largely the result of alcohol abuse, viral hepatitis and biliary diseases [75]. The causes for cirrhosis can be roughly divided into six categories:

1.Chronic exposure to toxins such as alcohol, drugs or chemicals,

2.Viral hepatitis resulting from infection with the hepatitis B, C or D viruses,

3.Metabolic disorders such as Wilson’s disease (copper storage disease) and haemochromatosis (iron overload disease),

4.Autoimmune diseases such as primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and autoimmune hepatitis,

5.Venous outflow obstruction,

6.Cirrhosis of unknown causes.

Obviously the best treatment for cirrhosis is removal of the injurious event. In the case of viral hepatitis, viral load can at least be temporarily reduced with anti-viral agents such as lamivudine, ribavirin and/or IFNα [76]. Unfortunately, complete removal of the injurious event is frequently not possible. Moreover, by the time cirrhosis is diagnosed the fibrotic process has usually progressed beyond ‘the point of no return’ and removal of the injurious event will have little effect. Successful pharmacological treatment to reverse the fibrotic

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process is not yet available. Several drugs have been tested in clinical trials, but the most effective treatment remains a liver transplantation.

The bile acid ursodeoxycholic acid has shown some promise in slowing down the fibrotic process in cholestatic patients, especially those suffering from PBC and PSC [77,78]. Its mechanism of action, however, is still a matter of debate.

Penicillamine, an inhibitor of collagen crosslinking, was evaluated in PBC, but failed to demonstrate any efficacy [79]. More promising results were found for colchicine, which inhibits collagen synthesis and secretion and enhances collagenase activity. Long-term use of colchicine prolonged survival in patients with mild to moderate cirrhosis, regardless of the cause [77,80]. Other types of collagen synthesis inhibitors, like the prolyl hydroxylase inhibitors, have been studied in experimental animal models [81], but have not yet found their way into the clinic.

Several types of immunosuppression have also been tried. Azathioprine alone was found to have no effect on PBC [82], but additional benificial effects were found in combination with ursodeoxycholic acid and corticosteroids [78]. Cyclosporin showed some success, especially in corticosteroid-resistant autoimmune hepatitis [83], but its use is generally considerably limited by severe side-effects. Corticosteroids were effective in the management of several types of autoimmune chronic active hepatitis [84,85] and in the management of acute alcoholic hepatitis [86]. Their use, however, has to be brief in order to minimize side-effects. In the treatment of PBC, corticosteroids alone were found to be toxic and had only limited efficacy [77].

A promising new development in drug therapy is the endothelin-antagonists [87,88]. Though not yet clinically tested, these compounds show potential in the management of portal hypertension, a hallmark of cirrhosis. Again, uptake of these antagonists by other parts of the body hampers their applicability [89], which might be circumvented by drug targeting.

4.5 Drug Targeting to the Liver

With no effective drugs available and the unacceptable side-effect profile of those drugs which have been studied so far, liver cirrhosis might benefit from the targeting of drugs to cells within the liver. There are several ways to intervene in the fibrotic process. One way is the targeting of drugs to SECs and KCs to modulate their release of pro-inflammatory mediators. This may arrest the inflammatory process leading to cirrhosis. Another way is the delivery of drugs to HSCs to inhibit collagen production or to enhance their extracellular matrix degrading capabilities. This chapter will focus on targeting to KCs and SECs to influence the inflammatory process that is the basis of most forms of liver cirrhosis. As mentioned before these cells have a number of specific entry mechanisms that could be used for cell-spe- cific delivery of drugs. By either enclosing drugs in particles or by coupling drugs to macromolecular carriers with high affinity for certain uptake mechanisms, drugs can be concentrated in the target cells without causing side-effects elsewhere in the body. The choice for a type of carrier is determined by a number of considerations, depending on the specificity of the carrier, the potency of the drug and the entry mechanism during pathological conditions. The possible carriers directed to KCs and SECs show a considerable overlap, because these

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cells share many receptor-mediated endocytotic uptake mechanisms, such as uptake mediated by scavenger receptors or mannose receptors. Most of the carriers directed to SECs and KCs are designed for these receptors and are reviewed below.

4.5.1 Carriers Directed at SECs and KCs

4.5.1.1 Albumins

Albumin is one of the soluble macromolecular carriers available for drug targeting purposes. With a molecular weight of approximately 67 kDa, it is small in size as compared to other potential carriers. It can be derivatized with molecules that will determine its cell specificity, and with drug molecules. A maximum of about 60 molecules can be coupled to albumin through the ε-NH2 of the lysine residues. Table 4.1 shows the modified albumins that have been used for targeting to SECs and KCs.

Albumin modified with negatively charged groups like succinic acid (Suc-HSA) and aconitic acid (Aco-HSA) are avidly taken up by SECs via the scavenger receptors type A. These receptors are also present on KCs, but have a slightly different substrate specificity. This was elegantly shown with formaldehyde-treated HSA (Form-HSA). The scavenger receptors on SECs take up the monomeric negatively-charged Form-HSA, whereas these receptors on KCs take up the polymeric Form-HSA [90]. Scavenging receptors are also involved in the uptake of maleylated albumin (Mal-BSA), which was designed for the targeting of chemotherapeutics to macrophages. It was found to be taken up by the non-parenchy- mal cells of the liver and by peritoneal macrophages, uptake by SECs was not determined [91].

The subtle differences in substrate specificity were found for the mannose receptor as well. Both SECs and KCs have mannose receptors, but mannosylated albumin (Man10-HSA) is almost exclusively taken up by KCs. The relatively low mannose substitution (10 molecules of mannose per HSA molecule) combined with the extra negative charge added to the albumin molecule by the coupling of the mannose molecules to the lysine-residues of HSA, directs

Table 4.1. Albumin carriers designed for targeting to SEC and KC.

Carrier	SEC	KC
Suc-HSA	+++	–
Aco-HSA	+++	–
Form-HSA	++	++
Man10-HSA	+	+++
Mal-BSA	ND	+++
Nap20-HSA	+++	+
Dexa10-HSA	+++	+

HAS, human serum albumin; BSA, bovine serum albumin; Suc, succinic acid; Aco, aconitic acid; Form, formaldehyde; Man, mannose; Mal, maltose; Nap, naproxen; Dexa, dexamethasone. ND, not done; –, no uptake; +, small uptake; ++, moderate uptake; +++, abundant uptake.

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this carrier to the KCs. When the mannose substitution is increased, the uptake by SECs also increases [92]. For a long time this carrier has been assumed to be inert. However, recent studies from our laboratory indicate that the carrier Man10-HSA may activate KCs and induce an immunological response [93].Whether this limits the use of this carrier remains to be established. The subsequent coupling of dexamethasone to Man10-HSA attenuated this immunological response [93].

Direct modification of albumin with drugs like naproxen (Nap20-HSA) and dexamethasone (Dexa10-HSA) changes the protein into a substrate for the scavenging receptors type A. These drugs are coupled to the free ε-NH2-groups of the lysine residues in albumin. Normally these NH2-groups are positively charged through protonation. Coupling of a drug molecule inhibits this protonation. The albumin molecule is left with a relative negative charge and becomes a substrate for the scavenger receptors. Apart from the net negative charge, it has been postulated that the added hydrophobicity of these drug molecules is an important feature in determining their affinity for the scavenger receptors [94].

After interaction of the aforementioned carriers with specific receptors, the carrier is then taken up by endocytosis and transported intracellularly to acidified endosomes and lysosomes. The carrier is proteolytically degraded in the lysosomes and if a drug is coupled to the carrier, it is then released to diffuse into the cytoplasmic compartment.

4.5.1.2 Liposomes

Liposomes are small vesicles composed of unilamellar or multilamellar phospholipid bilayers enclosing an aqueous space. Soluble drugs can readily be incorporated into this aqueous space and lipophilic drugs can be incorporated into the lipid bilayer. The loading capacity for drugs is therefore much greater than that of the modified albumins. Elimination from the circulation is dependent on the lipid composition, charge, and size of the liposomes. Common liposomes such as neutral and negatively-charged liposomes, are however, primarily cleared by the phagocytotic processes of the cells of the reticuloendothelial system (RES), the KCs having the greatest responsibility for this process.This feature of liposomes can seriously limit the use of liposomes in targeting other sites in the body [95]. It has been shown for instance that the targeting of cytostatic agents such as adriamycine to tumours is associated with loss of KC function [96], thereby contributing to the immuno-suppressed status of patients. The high KC uptake has been suprisingly under-exploited in drug targeting approaches to treat liver diseases. Liposomes have been used for the targeting of anti-Leishmania drugs [97,98] and immunomodulators [99] and have greatly increased the efficacy of these drugs in Leishmania infections and metastatic tumour growth, respectively. However, intervening in the fibrotic process by modulating KC or SEC functions with liposome-encapsuled drugs has not yet been attempted.

The exact mechanism responsible for the uptake of liposomes by KCs and SECs is not clear. Most studies confirm internalization of whole liposomes in an energy-dependent phagocytic process in which the liposomes are delivered to the lysosomes. The liposomal lipids are completely degraded and the encapsulated solutes released. Neutral liposomes consisting of lipids such as cholesterol and phosphatidylcholine are probably cleared by re- ceptor-mediated mechanisms, due to the adsorption of opsonizing proteins onto the lipid bi-

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layer. Some of the opsonizing proteins that have been found to play a role are complement factors [100] and fibronectin [101]. The opsonization of liposomes by plasma proteins, in particular complement factors C3bi and C5a, affects the cell selectivity of this carrier, causing uptake by the RES and by neutrophils [102]. The uptake of liposomes containing the nega- tively-charged phospholipid phophatidylserine (PS) is still a matter of debate. These liposomes may be taken up by the RES via specific PS receptors or via scavenger receptors, but here too uptake appears to be mediated predominantly by plasma proteins that bind in a PSspecific manner to liposomes [103]. The influence of plasma proteins on the uptake route of PS-containing liposomes was shown by Kamps et al. In vitro studies with liposomes containing 30% PS showed scavenger receptor-mediated uptake in KCs and SECs, but subsequent in vivo studies did not reveal a significant contribution of scavenger receptors to the KC uptake of these liposomes [104]. Specific scavenger-mediated uptake of liposomes by SECs was achieved by coating liposomes with negatively-charged albumins [105].

Lipoproteins are endogenous carriers for the transport of cholesterol and other lipids in the blood circulation and can be regarded as ‘natural liposomes’. Because they are endogenous, they are not immunogenic and escape recognition by the RES. They are cleared from the circulation by specific lipoprotein receptors that recognize the apolipoproteins [106]. They can be directed to non-lipoprotein receptors as well, by chemical modification of the apolipoprotein moiety. Specific scavenger receptor-mediated uptake by SECs was achieved by the acetylation of LDL, whereby oxidized LDL was specifically taken up by KCs via the scavenger receptors and lactosylated LDL via the galactose-particle receptors [106–108].

The lipid core can be used to incorporate lipophilic drugs, whereas more hydrophilic drugs have to be provided with a lipophilic anchor to enable incorporation. Oleyl, retinyl and cholesteryl residues have been used for this purpose [109]. Chemical derivatization will however, alter the pharmacological activity of the parent drug in most cases. The anchors should therefore be easily removable inside the cell, yielding the original drugs. These liposomes have not as yet been used much to target drugs to KCs and SECs. Just one study described the enhancement of the tumouricidal activity of KCs with the immunomodulator muramyldipeptide incorporated in lactosylated LDL [110].

4.5.1.3 Carriers with Intrinsic Anti-inflammatory Activity

Another approach to drug targeting is the use of carriers with an intrinsic pharmacological activity. In this ‘dual targeting’ strategy a beneficial effect is achieved both from the carrier itself and the drug it carries. The negatively-charged HSA carriers, for instance, developed for the targeting of drugs to HIV-infected cells, exert strong antiviral activity themselves [111]. Possible carriers with intrinsic anti-inflammatory activity are superoxide dismutase (SOD) and alkaline phosphatase (AP).

SOD is a major oxygen free radical scavenging enzyme, which may therefore have beneficial effects in liver fibrosis. Through mannosylation or coupling to the polyanion DIVEMA, SOD was made more liver specific. Both conjugates showed superior inhibition of intrahepatic ROS production in fibrotic rats as compared to unmodified SOD. DIVEMA-SOD, however, exhibited the most potent inhibitory effects [112].Although their mode of action is most probably extracellular free radical scavenging, Man-SOD and DIVEMA-SOD are likely to

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be taken up rapidly by mannose receptors and scavenger receptors, respectively. However, depending on the dose, a considerable fraction may be present on the cell surface, either bound to the receptor or through re-exposure via retroendocytosis after prior internalization [113]. Therefore, sufficient enzymatic activity might still be obtained in the extracellular space.

AP is a membrane-anchored protein, that can be shed into the general circulation, which was shown to be able to detoxify LPS in vivo through dephosphorylation [114]. This dephosphorylating activity could be enhanced by increasing the negative charge of the enzyme through succinylation [115]. Using AP as a carrier for anti-inflammatory drugs to KCs, the main site of LPS uptake, it could intrinsically contribute to therapeutic efficacy in cirrhosis through detoxicification of LPS. The LPS-detoxifying activity of KCs in cirrhotic livers is impaired and consequently LPS may promote the fibrotic process [116].

4.5.2 Targeting to other Hepatic Cells

Selective delivery of anti-fibrotic drugs to HSCs would be an elegant option in the design of effective anti-fibrotic therapy. Only recently the first carriers targeted to HSCs were developed: albumin modified with mannose 6-phosphate groups for uptake via mannose 6-phos- phate/insulin-like growth factor II receptor and albumin derivatized with cyclic peptides containing amino acid sequences that mimic the binding site of either collagen type VI or PDGF to their receptors [117,118]. In addition to being used as drug carriers, these carriers could also be intrinsically active. The mannose 6-phosphate/insulin-like growth factor II receptor is involved in the activation of the fibrogenic mediator TGFβ [119,120], which, in theory, could be competively inhibited by the mannose 6-phosphate-modified albumin. The same competition between carrier and endogenous ligands can be anticipated for collagen type VI and PDGF receptors. The approach of using cyclic peptides with the receptor-recognizing domains of various cytokines or growth factors, that will mediate binding to their respective receptors, can also be exploited for the design of other dual active carriers to cell types such as SEC and KC.

4.6 Anti-inflammatory Drugs

Several classes of drugs can be potentially used to reduce the release of pro-inflammatory mediators by SECs and KCs in the fibrotic process.

4.6.1 Nonsteroidal Anti-inflammatory Drugs (NSAIDs)

NSAIDs are drugs related to acetylsalicylic acid which inhibit cyclooxygenase (COX), the enzyme in the synthesis of PGs and thromboxanes from arachidonic acid. There are two isoforms of cyclooxygenase, COX-1 and COX-2 [121]. The former is constitutively expressed in blood vessels, stomach and kidney, while COX-2 is normally not present at these sites. It can,

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however, be induced under inflammatory conditions by certain serum factors, cytokines, and growth factors [122]. Most of the currently used NSAIDs non-selectively inhibit both COX- 1 and COX-2. They are widely used in inflammatory disorders of the joints such as arthritis, of the tendons and of the bursae. The side-effects are to some extent the result of the non-se- lective inhibition of the constitutive COX-1 production by PGs in other tissues. In the kidneys this may lead to renal insufficiency and in the gastrointestinal tract to the formation of ulcers and bleeding [123,124].

Acetylsalicylic acid was shown to prevent cirrhosis under certain experimental conditions [125]. Naproxen and indomethacin partially protected against LPS and D-galactosamine-in- duced hepatotoxicity [126] Acetylsalicylic acid and ibuprofen were also protective in endotoxic shock [127]. Endotoxaemia is one of the complications in cirrhotic patients [128] and is probably caused by an impaired ability of the liver to take up and detoxify gut-derived LPS [116]. The presence of portosystemic shunts in cirrhotic patients may also contribute to this spill-over of LPS into the systemic circulation [129]. NSAIDs, however, are also reported to provoke deleterious effects on renal function in cirrhosis [130], and can therefore not be used in cirrhotic patients. Cell-specific delivery of NSAIDs to SECs and/or KCs may make application of these drugs in cirrhosis feasible by circumventing the renal side-effects.

4.6.2 Glucocorticosteroids

Glucocorticosteroids are the synthetic derivatives of the adrenal gland hormone cortisol. At pharmacological doses they prevent or suppress inflammation and other immunologically mediated processes. These drugs are therefore used for a variety of inflammatory diseases such as allergic diseases, rheumatic disorders, renal diseases, bronchial asthma, skin and gastrointestinal diseases [122]. The anti-inflammatory and immunosuppressive activities of glucocorticosteroids are most likely due to the inhibition of the production of a wide range of cytokines, chemokines, eicosanoids, and metalloproteinases in many cell types. In macrophages they block the release of numerous cytokines (IL-1, IL-6, TNFα), inhibit the expression of the MHC class II antigens, depress production and release of pro-inflammato- ry PGs and LTs, and depress tumouricidal and microbicidal activities of activated macrophages [131]. In the case of neutrophils they inhibit neutrophil adhesion to endothelial cells, thereby reducing the infiltration of neutrophils at inflamed sites. At pharmacological doses they only modestly block neutrophil functions such as lysosomal enzyme release and respiratory burst [132]. Glucocorticosteroids also have profound effects on the activation and subsequent function of endothelial cells. Besides inhibiting cytokine and eicosanoid release, they depress vascular permeability, LPS-induced upregulation of adhesion molecules ICAM- 1 and endothelial leucocyte adhesion molecule 1 or E-selectine, and expression of MHC class II antigens [133,134]. Moreover, they inhibit the secretion of the complement pathway proteins C3 and factor B [135].

The molecular mechanisms underlying glucocorticosteroid inhibition of inflammatory responses are slowly being unravelled (see also Chapter 7). After entering the cell, glucocorticosteroids bind to the glucocorticoid receptor (GR) present in the cytoplasm. Following ligand binding, the GR is redirected to the nucleus where it can interact with specific DNA sequences. The expressions of many proteins involved in inflammatory reactions are regulated