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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Groningen University Institute for Drug Exploration (GUIDE), Department of Pharmacokinetics and Drug Delivery, University of Groningen, Groningen, The Netherlands
Received December 5, 2005; accepted January 24, 2006.
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Abstract |
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-smooth muscle actin-stained area in liver sections from 8.5 ± 0.8 to 5.1 ± 0.9% (P < 0.01) and collagen-stained area from 13.1 ± 1.3 to 8.9 ± 1.5% (P < 0.05). DOX was coupled to M6PHSA, and the organ distribution of this construct (M6PHSA-DOX) was investigated. Twenty minutes after i.v. administration, 50 ± 6% of the dose was present in the livers, and colocalization of M6PHSA-DOX with HSC markers was observed. In addition, in vitro studies showed selective binding of M6PHSA-DOX to activated HSC. Moreover, M6PHSA-DOX strongly attenuated HSC proliferation in vitro, indicating that active drug is released after uptake of the conjugate. DOX inhibits liver fibrosis in BDL rats, and HSC-selective targeting of this drug is possible. This may offer perspectives for the application of antiproliferative drugs for antifibrotic purposes.
; Eng and Friedman, 2000; Friedman, 2000; Di Sario et al., 2002; Caligiuri et al., 2003; Kurikawa et al., 2003; Rombouts et al., 2003).
Since HSC proliferation is the hallmark of liver fibrosis, the question emerges what would happen if HSC proliferation is inhibited during the fibrotic process. Cytostatic drugs are among the most potent inhibitors of cell proliferation, and effects of these drugs on wound healing are well known (Bland et al., 1984). However, to our knowledge, no attempts to test cytostatic drugs in fibrotic animals have been made. Obviously, an important concern when using such drugs is the occurrence of serious side effects in nonhepatic tissues. Even within the liver, treatment with antiproliferative drugs may result in unwanted effects. For instance, inhibition of hepatocyte proliferation may be detrimental to the renewal of functional liver parenchyma, a crucial process in the fibrotic liver (Fausto, 2004).
In recent years, HSC-selective drug carriers have become available, which allow drugs to be delivered to HSC with increased specificity (Beljaars et al., 1999, 2000, 2003). Therefore, conjugating very potent inhibitors of cell proliferation to the HSC-selective drug carrier mannose-6-phosphate-modified human serum albumin (M6PHSA) has now become an option. M6PHSA has been shown to accumulate in HSC in vivo via binding to mannose-6-phosphate/insulin-like growth factor-II receptors that are up-regulated on the cell surface of activated HSC (de Bleser et al., 1995, 1996; Beljaars et al., 1999). Ligands bound to the receptor are subject to receptor-mediated endocytosis and are routed to the acidic lysosomal compartment, where degradation of the construct and subsequent release of the coupled drug may take place (Braulke and Mieskes, 1992; Dahms and Hancock, 2002). If cytostatic drugs can be targeted to the HSC by employing this strategy, this could result in a reduction of systemic side effects and may improve the applicability of potent antiproliferative agents for antifibrotic purposes in the future.
The present study first evaluates the antiproliferative effect of three cytostatic drugs in culture-activated rat HSC. Subsequently, the most promising drug from these experiments, doxorubicin (DOX), was tested in a rat experimental model for liver fibrosis to provide better rationale for its use as an antifibrotic drug. Finally, we use in vitro and in vivo techniques to investigate whether DOX can be selectively delivered to the HSC in a pharmacologically active form by coupling it to M6PHSA.
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Materials and Methods |
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). After isolation, HSC were cultured at 37°C in a 95% air, 5% CO2 atmosphere in Dulbecco's modified Eagle's medium with Glutamax-1 (Invitrogen, Carlsbad, CA), supplemented with 10% fetal calf serum (Cambrex Bio Science Walkersville, Inc., Walkersville, MD), 100 U/ml penicillin (Sigma, Gillingham, UK), and 100 µg/ml streptomycin (Sigma). The purity of the HSC culture was assessed after 9 days of culture in the described medium, during which HSC activation spontaneously occurs. In these cultures, always more than 95% of the cells were positive for the activated HSC marker -smooth muscle actin (-sma), as assessed by immunohistochemical staining and cell counting.
Effect of Drugs on HSC Proliferation in Vitro
After 9 days in culture, when HSC displayed an activated phenotype, cells were seeded in 24-well plates at a density of 30,000 cells per well. Cells were incubated with various concentrations of chlorambucil (Sigma), doxorubicin (Pfizer, Capelle aan den IJssel, The Netherlands), or cisplatin (Sigma) in the presence of 10% FCS, 50 ng/ml platelet-derived growth factor-BB and 10 µM 5-bromo-2'-deoxyuridine (BrdU; Sigma) for 24 h to allow detection of proliferating cells by immunohistochemistry. BrdU incorporation was subsequently quantified by cell counting as described previously (Greupink et al., 2005).
Antifibrotic Potential of Doxorubicin in Bile Duct-Ligated Rats
Experimental Animals. Male Wistar rats of 220 to 240 g were used. Animals had free access to tap water and standard lab chow (Harlan). All experiments were approved by the local committee for care and use of laboratory animals and were performed according to strict governmental and international guidelines for the use of experimental animals. Liver fibrosis was induced by ligation of the common bile duct as described previously (Beljaars et al., 1999).
Experimental Setup. Ten animals were subjected to bile duct ligation (BDL) under isoflurane/N2O/O2 anesthesia at day 0 of the protocol. On day 3, the animals were randomly divided into two groups and were treated with either PBS (n = 5) or DOX (n = 5) at a dose of 0.35 mg/kg/day. A total of seven i.v. injections were given, starting on day 3 of the protocol, and the animals were sacrificed 24 h after the last injection on day 10. Three additional rats were not subjected to bile duct ligation and served as control group.
Number of Activated HSC and the Extent of Liver Fibrosis. The number of activated HSC in the liver was assessed by staining for -sma (Sigma) on 4-µm cryostat sections, according to standard indirect immunohistochemical techniques. To study the extent of fibrosis, cryostat sections of livers were stained for collagen type III (Southern Biotechnology, Birmingham, UK). In addition, formalin-fixed paraffin-embedded tissue samples were stained with picrosirius red dye to stain all collagens (Sigma). Microphotographs of both stainings were taken at an original magnification of 4 x 10. The fibrotic area per liver section was quantified by morphometric analysis of the sections using the Image J software package (National Institutes of Health, Bethesda, MD). Secondary to the antifibrotic effects, adverse effects were also assessed. General toxicity was monitored by analysis of body weight (BW), whereas intrahepatic toxicity of DOX toward liver cells was examined by analysis of serum AST, ALT, AP, and 
-GT levels. Inflammatory cell influx in the livers was assessed by diaminobenzidine (DAB) staining as described previously, as well as by evaluation of hematoxylin/eosin-stained liver sections (Poelstra et al., 1990; Greupink et al., 2005). To evaluate the effect of DOX on the bile duct epithelial cell proliferation, staining with a monoclonal antibody raised against cytokeratin 7 (Santa Cruz Biotechnology, Santa Cruz, CA) was performed, according to standard indirect immunohistochemical techniques.
Synthesis and Characterization of M6PHSA-DOX. M6PHSA was synthesized as described by Beljaars et al. (1999). DOX was coupled to this drug carrier via cisaconitic acid, according to the method of Shen and Ryser (1981), covalently linking the drug and the carrier via an acid-sensitive spacer, which allows drug release within the acidic lysosomal compartment of cells (Shen and Ryser, 1981). M6PHSA-DOX was subsequently purified by dialysis against PBS and size-exclusion chromatography on a HiLoad 16/60 Superdex 200 column (Amersham Biosciences, Uppsala, Sweden). After further dialysis against water, the product was lyophilized and stored at –20°C until use.
Chemical Characterization M6PHSA-DOX. The total amount of coupled DOX was assessed by spectrophotometric analysis (Wirth et al., 1998; Griffiths et al., 2003). In brief, the conjugate was dissolved in PBS in a concentration of 1 mg/ml. The absorption at 480 nm was measured, and the total amount of DOX in the preparation was calculated from a calibration curve. The amount of free drug was subsequently assessed on an high-performance liquid chromatography system fitted with a pump (model 510; Waters, Milford, MA), a ThermoQuest 250 x 4.6-mm 5-µm Hypersil BDS C8 column (Thermo Electron Corporation, Waltham, MA), and a UV detector (model 441; Waters) at 254 nm. A solution of 6.7 g of trisodium citrate in 760 ml of water and 240 ml of acetonitrile (pH adjusted to 4 with formic acid) was used for elution at a flow of 1 ml/min. Monomeric protein content was analyzed by standard SDS-PAGE techniques, using a 10% polyacrylamide gel. The net-negative charge of M6PHSA-DOX and control proteins was assessed by anion-exchange chromatography on a Mono Q column as described previously (Beljaars et al., 1999).
In Vivo Distribution of DOX and M6PHSA-DOX
In Vivo Organ Distribution of 125I-Labeled M6PHSA-DOX. One week after BDL, animals were injected i.v. with a tracer dose of 125I-labeled M6PHSA-DOX under O2/N2O/isoflurane anesthesia. Twenty minutes after injection, blood samples were taken by heart puncture (5–10 ml), and the animals were subsequently sacrificed by bleeding after severing the aorta. Organs were harvested and processed as described previously. All of the urine present in the bladder was collected to measure the amount of radioactivity that was excreted into the urine. The organ-associated radioactivity was expressed per whole organ and was corrected for blood-derived radioactivity. The latter was calculated from the radioactivity present in each organ of BDL rats that were injected with 125I-labeled HSA, which is known to remain in the blood. The percentage of the dose of M6PHSA-DOX in blood was calculated from the amount of radioactivity in the 5- to 10-ml blood sample by extrapolation to the corresponding value for the total blood volume in rats (60 ml/kg) (Beljaars et al., 1999).
Localization of the Untargeted and Targeted Drug. To investigate the distribution of DOX, 20 min after i.v. injection of the uncoupled form (2 mg/kg), after injection of M6PHSA-DOX (30 mg/kg, which contains an amount of 2 mg/kg DOX), or after injection of PBS, the animals were sacrificed and 4-µm cryostat sections of liver, kidney, and heart were made. These sections were examined by fluorescence microscopy, without embedding the sections in a mounting medium first, because this may corrupt the original localization of DOX. Microphotographs were taken with an Olympus C5050 zoom digital camera and were converted to blue-scale images, so the localization of DOX could be better discerned.
Cellular Localization of M6PHSA-DOX. One week after BDL, animals were injected i.v. with 30 mg/kg M6PHSA-DOX. After 20 min, the animals were sacrificed and tissues were excised and frozen in isopentane at –80°C. Cryostat sections (4 µm) of liver, heart, and kidney were acetone-fixed and stained for the presence of the conjugate with an antibody directed against HSA (Cappel, Zoetermeer, The Netherlands). In addition, we investigated whether there was colocalization of the injected construct with HSC markers. To this end, double stainings were performed for HSA and HSC markers. As a marker for HSC, two monoclonal antibodies were combined: a mouse monoclonal IgG directed against desmin (Sigma) and mouse monoclonal IgG anti-glial fibrillary acidic protein (Neomarkers, Fremont, CA) (Geerts, 2001).
In Vitro Studies with M6PHSA-DOX. To investigate whether the in vivo binding to HSC was mediated by specific binding of M6PHSA-DOX to receptors on HSC, the protein backbone of the conjugate was labeled with 125I according to the Chloramine-T method. Culture-activated HSC were exposed to 100,000 cpm of 125I-labeled M6PHSA-DOX at 37°C, in the absence of a competitor for receptor binding or in the presence of either 1 mg/ml HSA or 1 mg/ml M6PHSA. After 2 h, the cell-associated radioactivity was measured on a -counter (Riastar; Packard Instruments, Palo Alto, CA). The effect of M6PHSA-DOX was investigated as described above for the uncoupled drugs. The cells were incubated with 10 µg/ml M6PHSA-DOX, an equivalent amount of control protein, or PBS.
Statistical Analysis. Results were expressed as the mean ± S.E.M. One-way analysis of variance was used to compare the means, and differences were considered statistically significant at P < 0.05. For statistical analysis, the SPSS software package was used (SPSS Inc., Chicago, IL).
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-smastained area from 8.5 ± 0.8% (PBS-treated BDL rats) to 5.1 ± 0.9% (P < 0.01). In normal livers, only 1.2 ± 0.2% of the total evaluated area of the liver was stained, mainly reflecting -sma-positive vascular smooth muscle cells in the vessel walls (Fig. 2, A–C and J). Treatment with DOX also reduced the amount of collagen, as reflected by a reduction in the collagen type III-stained area within liver sections to 8.9 ± 1.5%, compared with 13.1 ± 1.3% in PBS-treated BDL animals (P < 0.05). In livers of healthy control rats, only 4.9 ± 0.5% of the area was stained for collagen type III (Fig. 2, D–F and K). Sirius red staining confirmed the results of DOX on collagen deposition (Fig. 2, G–I).
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Besides the antifibrotic effect of DOX, treatment with this drug also resulted in significant body weight loss. Furthermore, DOX treatment elicited increased serum -GT levels but not AST and ALT levels, indicating that early hepatocyte or bile duct epithelial cell injury has occurred (Table 1). Yet, the absolute number of bile duct epithelial cells, as assessed by cytokeratin 7 staining, did not differ between the PBS- and DOX-treated groups (Table 1). Moreover, evaluation of DAB stainings revealed that the number of reactive oxygen species-producing cells was also increased in the livers of DOX-treated rats (Table 1). These DAB-positive cells consisted mainly of neutrophils, judged by the presence of polymorphic nuclei in these cells after evaluation of HE-stained liver sections and based on previous studies (Poelstra et al., 1990).
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Synthesis and Characterization of M6PHSA-DOX
To increase the HSC specificity of DOX, a conjugate of this drug and M6PHSA was synthesized (Fig. 3A). Spectrophotometric analysis of the construct revealed that, per milligram of construct, 66.7 µg of doxorubicin was present. Of the total amount of doxorubicin in the preparation, only 1% was present in uncoupled form, as assessed by high-performance liquid chromatography analysis. SDS-PAGE analysis revealed that no significant amounts of polymeric protein could be detected in the conjugate (Fig. 3B). Furthermore, the net-negative charge of the conjugate was increased compared with M6PHSA and HSA, which is in agreement with molecules covalently coupled to the positively charged amine groups of HSA (Fig. 3C). The retention times measured by anion-exchange chromatography were 18.9, 26.5, and 28.0 min for HSA, M6PHSA, and M6PHSA-DOX, respectively.
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In Vivo Distribution of DOX and M6PHSA-DOX
In Vivo Organ Distribution of 125I-Labeled M6PHSA-DOX. The organ distribution of M6PHSA-DOX in rats with liver fibrosis was assessed 20 min after i.v. injection of a radiolabeled tracer dose of the conjugate. In Fig. 4, it can be seen that already 50 ± 6% of the injected dose was distributed to the liver at that time. In heart and kidney, target organs for serious side effects of doxorubicin, only 0.2 ± 0.1 and 1.2 ± 0.5% of the dose accumulated, respectively. In the different parts of the intestine, less than 2% of the dose was found; in addition, in the bone marrow, no significant radioactivity could be demonstrated (data not shown).
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In Vitro Studies with M6PHSA-DOX. To test whether M6PHSA-DOX selectively binds to receptors on HSC, we performed binding studies on culture-activated HSC with 125I-labeled M6PHSA-DOX. In Fig. 6, it can be seen that binding to HSC was reduced by 75.2 ± 6.3% (P < 0.05) after coincubation with M6PHSA, which is an M6P/insulin-like growth factor-II receptor ligand. In contrast, the control protein HSA had no significant effect.
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; Di Sario et al., 2003; Rombouts et al., 2003; Greupink et al., 2005). The higher potency of DOX may be related to the fact that multiple intracellular mechanisms are implicated in its antiproliferative effect, in contrast to these other drugs (Gewirtz, 1999). In this article, we also show that treatment of BDL rats with doxorubicin reduces the number of activated HSC in the fibrotic rat liver and attenuates the fibrotic process. To our knowledge, this is the first study investigating the use of a cytostatic agent in an experimental model of liver fibrosis, and our data indicate that DOX, in principle, may form a new asset in the treatment of liver fibrosis for which, up until now, no pharmacotherapeutics are available. However, the observed side effects will most certainly impede its actual clinical application. In fact, toxicity is a typical problem that forms an obstacle in the application of many other promising antifibrotic drugs as well (Canbay et al., 2004; Bataller and Brenner, 2005; Hagens et al., 2005; Pinzani et al., 2005).
In this study, the toxicity of DOX was reflected by the loss in body weight, whereas the increase in serum -GT levels, but not AST, ALT, and AP levels, also suggested hepatocyte or bile duct epithelial cell injury. It is known that, during the early stages of hepatocellular injury, such an isolated -GT increase can precede AP, ALT, or AST elevation (Moss and Henderson, 1999). Very likely, the increased number of DAB-positive cells that we observed is the result of an immune response in reaction to this damage. It is known that drug-induced hepatic inflammation can lead to liver fibrosis, but usually this takes a prolonged period of time. In the present animal model, which is characterized by a very fast proliferation of fibrogenic cells, the antiproliferative effect of DOX on these cells apparently prevails, resulting in the observed antifibrotic effect. Therefore, it would be interesting to investigate the antifibrotic potential of DOX in animal models in which fibrosis progression is slower, for example, in the CCl4 model. In this BDL model, the antifibrogenic effect of DOX was most prominent in the parenchyma and less clear in the portal areas. Whether this is due to a reduced sensitivity of portal fibroblasts to DOX or due to lower drug concentrations in this highly fibrotic area remains to be established.
Although this DOX-induced hepatic inflammation does not result in a worsening of fibrosis, it should still be considered that an even more potent antifibrotic effect can be obtained when the influx of inflammatory cells is avoided. The same may be true for the observed weight loss seen in DOX-treated rats, because it has been shown that alcohol in combination with malnutrition causes more severe liver fibrosis in rats (Bosma et al., 1994). In this way, HSC-selective delivery of DOX may not only help to avoid general drug-related toxicity but also contribute to the antifibrogenic potential of DOX via the avoidance of profibrogenic side effects.
Analysis of circulating white blood cell counts revealed no significant differences between DOX-treated BDL rats and PBS-treated BDL rats (data not shown), suggesting that no relevant myelosuppressive effect was at hand at the present dose of DOX and duration of administration. This renders it unlikely that the antifibrotic effect of DOX we describe here is mediated via the known myelosuppressive effect of the drug.
Besides the proliferation of HSC, proliferation of ductular epithelial cells occurs in the liver after ligation of the common bile duct. Our data revealed that the ductular response of BDL rats was not significantly affected by treatment with DOX. A possible reason for this is the high expression of mdr1a drug efflux transporters in the apical membrane of the cholangiocytes (Gigliozzi et al., 2000). Because doxorubicin is a substrate for mdr1a, it is conceivable that the resistance of cholangiocytes to DOX is due to a high efflux of the drug from these cells.
To limit the effect of DOX to fibrogenic cells and to avoid the described extrahepatic and intrahepatic toxicity, we set out to investigate the targeted delivery of DOX to the HSC. From physicochemical analyses, we concluded that coupling of DOX to the HSC-selective drug carrier M6PHSA was successful. In vivo, the conjugate rapidly and selectively accumulated in the liver, whereas the untargeted drug also accumulated in organs that are prone to DOX toxicity. Within the liver, the uptake of M6PHSA-DOX was confined to the nonparenchymal cells and the observed colocalization of M6PHSA-DOX with HSC markers indicated that HSC were reached successfully. Next to the uptake in HSC, colocalization with Kupffer cell and endothelial cell markers also was observed. This very likely reflects scavenger receptor-mediated uptake of M6PHSA-DOX by these cells, because this is a highly negatively charged molecule. To attenuate uptake by these other nonparenchymal cells, the net charge of this construct should be modified. Nevertheless, we could clearly demonstrate that, although not 100% specific, injection with M6PHSA-DOX leads to a significant increase in stellate cell selectivity compared with the untargeted drug. It remains to be established whether the uptake of M6PHSA-DOX in Kupffer cells and liver endothelial cells results in toxicity or whether this contributes to the therapeutic effect of the conjugate. It is known from literature that attenuation of Kupffer cell functioning may attenuate fibrogenesis (Rivera et al., 2001).
The binding of M6PHSA-DOX to HSC was confirmed by studies performed with a 125I-labeled conjugate in cultures of activated HSC. The association of radiolabeled M6PHSA-DOX with the cells could be inhibited by an excess of M6PHSA but not by HSA, indicating that receptor-mediated uptake of the construct takes place. Moreover, the strong inhibition of cell proliferation by M6PHSA-DOX that we observed also indicates that active drug can be released from the conjugate after uptake by activated HSC.
Although these results show that the targeted delivery of a cytostatic drug to the HSC is possible, it is evident that chronic treatment of fibrotic rats is necessary to test the ultimate antifibrotic potential of this targeted strategy. However, before performing those studies, it will be necessary to better understand the pharmacokinetics of M6PHSA-DOX in fibrotic animals, as well as its intracellular kinetics within HSC. Explicitly, information on the mechanisms through which this new chemical entity is processed by activated HSC and the rate at which intracellular drug release and elimination occur will largely dictate the dosage regime of M6PHSA-DOX in chronically treated rats. Based on the expected differences in cellular handling between the targeted and untargeted drug, this may be very dissimilar for the two compounds. Yet, irrespective of the eventual dosing regimen required, it is likely that, by virtue of the improved organ and cell specificity and the resulting smaller volume of distribution, the total body dose of M6PHSA-DOX can be reduced compared with untargeted DOX, whereas still similar drug concentrations can be obtained within HSC.
In summary, we have shown that treatment of BDL rats with free DOX inhibits the progression of experimental liver fibrosis and strongly inhibits HSC proliferation in vitro. Subsequently, we succeeded in delivering this potent antiproliferative drug to HSC in vitro as well as in vivo. By doing so, accumulation of DOX in extrahepatic tissues as well as hepatocytes was avoided. We conclude that DOX exerts potent antifibrotic effects in bile duct-ligated rats. In conjunction with the here-described drug delivery strategy, this may offer perspectives for the use of potent but relatively toxic antiproliferative drugs in the pharmacological treatment of liver fibrosis.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: HSC, hepatic stellate cell(s); M6PHSA, mannose-6-phosphate-modified human serum albumin; DOX, doxorubicin; PBS, phosphate-buffered saline; BW, body weight; BDL, bile duct ligated/ligation; -sma, -smooth muscle actin; BrdU, 5-bromo-2'-deoxyuridine; ALT, alanine transaminase; AST, aspartate transaminase; AP, alkaline phosphatase; -GT, -glutamyltransferase; DAB, diaminobenzidine; PAGE, polyacrylamide gel electrophoresis; TNP-470, O-(chloroacetyl-carbamoyl) fumagillol.
Address correspondence to: Dr. R. Greupink, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. E-mail: a.h.greupink{at}rug.nl
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indicates P < 0.01 compared with normal rats; * indicates P < 0.05 compared with PBS-treated BDL rats; ** indicates P < 0.01 compared with PBS-treated BDL rats.
