Targeting fibrosis with selective drug carriers

Hepatic stellate cells (HSCs) as a drug target during fibrogenesis 

HSCs play a central role in the progression of liver fibrosis, independent of the aetiology of the underlying disease [1]. Thus, HSCs form an attractive cellular target for the treatment of hepatic fibrogenesis. In response to liver injury, HSCs transdifferentiate from a quiescent, vitamin A-storing cell type into a cell with fibrogenic properties. This takes place under the influence of a large number of mediators such as transforming growth factor-β1 (TGF-β1), tumour necrosis factor-α (TNF-α), epidermal growth factor (EGF) and insulin-like growth factor (IGF) [2]. These cytokines induce the activation of HSCs and also stimulate the secretion of additional inflammatory and fibrogenic cytokines by Kupffer cells, thus creating autocrine and paracrine loops by which the fibrotic process can perpetuate itself.

In addition, HSC proliferation is a hallmark of the fibrotic process. This is accompanied by a high expression of the platelet-derived growth factor (PDGF) receptors [3] of other growth factor receptors, such as the IGFII-receptor [4] on HSC membranes.

The fate of HSCs during the resolution of liver fibrosis 

In recent years, an increasing number of research papers on liver fibrosis show that, at least in an experimental setting, it is reversible [5]. Bile duct ligation-induced fibrosis [5] and CCl4-induced liver fibrosis can be reversed after the withdrawal of the inciting stimulus [6].

During the resolution of fibrosis, the activated HSC population decreases to the level found in normal livers. In animal models of liver fibrogenesis, the HSC apoptosis has been found to be a central event during the resolution process [7], [8].

Inhibiting HSC proliferation and inducing HSC apoptosis as a therapeutic intervention 

Clearly, HSC activation, proliferation, transformation and apoptosis are very important processes in the development of fibrosis. Inhibition of these processes therefore is a logical and attractive strategy to interfere with fibrogenesis on the level of HSCs. Indeed, recent studies have shown that the treatment of rats with experimental liver fibrosis with the antiproliferative drug doxorubicin inhibits the fibrotic process [9], [10]. Moreover, many studies show that it is possible to induce HSC apoptosis in vitro and in vivo and this is associated with a reduction of fibrosis. Gliotoxin [11], [12] and 15-deoxy-Δ [12,14-prostaglandin J2 [13] are able to induce HSC apoptosis. However, for apoptosis-inducing drugs, cell specificity is crucial because, in the damaged liver, hepatocyte restoration and proliferation of immune-competent cells should not be inhibited. Gliotoxin, for example, seriously affected the viability of experimental animals. For mice, the 50% lethal dose (LD50) of GTX was found to be 10mgkg−1 [14]. In this same study, it was demonstrated that intra-peritoneal injections of GTX also caused apoptosis in the thymus, spleen and mesenteric lymph nodes [14]. This apoptotic effect was possibly a direct action of the toxin, since relatively low concentrations of GTX were measured in these organs. Also, within the liver, GTX affected the Kupffer cells, endothelial cells and hepatocytes, besides its beneficial effects on HSCs [11], [12], [15].

The key to HSC selectivity 

The HSC-selective targeting strategies may offer a solution to this problem. In activated HSCs, the receptor expression for some growth factors on the cell surface is drastically upregulated. Examples of receptors that are upregulated on activated cells are the PDGF receptors [3], the mannose-6-phosphate/insulin-like growth factor II (M6P/IGF-II) receptor [4] and many receptors that allow HSCs to interact with the surrounding extracellular matrix (ECM), for example, the collagen type-VI receptor [16]. Also, the membrane protein synaptophysin appears to be expressed in quiescent as well as in activated HSCs but not in many other cell types [17]. The strong upregulation of these receptors on the cell surface of activated HSCs make them suitable targets for the selective delivery of drugs.

Drug carriers for selective targeting to the HSCs 

In 1999, the first HSC-selective drug carriers were developed [18]. These first-generation HSC-selective drug carriers consist of an albumin core protein to which the targeting devices are coupled. Drug carriers were designed that bind to the M6P/IGF-II receptor [18], [19], the PDGF-β receptor [20] and the collagen-VI receptor [16]. M6P/IGF-II receptor specificity was obtained by coupling M6P groups to the protein backbone, whereas the PDGF receptor and collagen-VI receptor specificities were obtained by coupling receptor-recognising cyclic peptides to the core protein [21], [22]. On administering these proteins to fibrotic rats intravenoulsy, 60–70% of the dose accumulated in the liver. Within the liver, approximately 70% of the hepatic cells that were positive for the constructs after immunohistochemical staining were also positive for HSC markers. The hepatic uptake and intrahepatic distribution were roughly similar for all carriers, although in vitro differences were observed with respect to cellular internalisation after binding to the receptor [16], [18], [19], [20], [21], [22]. In several recent studies, these modified albumins have been used to target drugs to HSCs. To that end, drugs were chemically coupled to the protein backbone of the carrier. Losartan, mycophenolic acid [23], [24], doxorubicin [9], [10], 15-d-prostaglandin J2 [25], gliotoxin [26], the viral vector HVJ [27], pentoxifylline [28], interleukin-10 [29] and a kinase inhibitor [30] have been targeted in recent years to HSCs, using these drug carriers. Most of these constructs displayed antifibrotic effects in vivo, although complete inhibition of fibrosis was not obtained. It remains to be established which activation pathways are the most relevant in vivo, in different experimental animal models. The application of the drug-targeting approach, which allows cell-selective elimination of specific pathways, may be used to identify the most crucial activation pathways in HSCs.

In addition to the protein-based drug carriers, Adrian et al. incorporated M6P–albumin into the lipid bilayer of liposomes [31], [32], [33], [34], which yielded a liposomal drug-targeting preparation that binds to HSCs and endothelial cells in vitro and, upon administration to fibrotic animals, distributed to the liver. Substantial co-localisation with the HSC markers was found. This should make it possible to deliver drugs to HSCs by entrapping hydrophilic drugs within the hydrophilic core of the liposome, without having to perform a conjugation reaction between the drug and the protein. In addition, hydrophobic drugs can be incorporated into the lipid bilayer of the liposome.

Cell-selective delivery of drugs can be achieved by coupling the chosen drug to the protein backbone through covalent conjugation to functional chemical groups in the side chains of amino acids within the protein. After the binding of a drug-targeting construct to its receptor, the conjugate may remain at the cell surface or, in case of binding to an internalising receptor, the drug carrier and the attached drug may be taken up by the cell through receptor-mediated endocytosis. By coupling drugs to the carrier proteins, biodegradable linkers, or linkers that cause slow release of the drug may be applied [28]. In this manner, optimal drug release may be obtained within the cell, whereas the chemical bond between the drug and the protein remains stable in the circulation. In the case of M6P-R [19], and collagen-VI receptors [16], the uptake occurs readily in the lysosomal compartment. This strategy is schematically depicted in Fig. 1.

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Fig. 1. Schematic representation of the employed targeting strategy. The process of receptor-mediated endocytosis leads to intracellular release of the targeted construct.

Haisma et al. showed that the adenoviral-mediated expression of a transgene in HSCs in vitro could be enhanced by coupling the PDGF-R-binding peptide to the virus [35]. Simultaneously, transfection of hepatocytes could be reduced. This may provide a new strategy to achieve adenoviral-mediated gene delivery to HSCs. Transcriptional retargeting (i.e., restricting gene expression to target cells by HSC-specific promoters) and transductional retargeting techniques (i.e., the HSC-specific targeting of the adenoviral vector) may be combined in the future to increase the HSC specificity of this gene delivery [36].

In conclusion, this review shows that fascinating progress has been made in the development of strategies that involve the cell-selective delivery of therapeutic agents to HSCs within the fibrotic liver. Eventually these efforts may lead to pharmacological strategies that effectively attenuate fibrogenesis, irrespective of the aetiology of the underlying disease and without displaying the adverse effects that are often associated with the use of potent antifibrotic compounds [37], [38].