Doxorubicin coupled to lactosaminated albumin inhibits the growth of hepatocellular carcinomas induced in rats by diethylnitrosamine

Journal of Hepatology 43 (2005) 645–652 www.elsevier.com/locate/jhep

Doxorubicin coupled to lactosaminated albumin inhibits the growth of hepatocellular carcinomas induced in rats by diethylnitrosamine Luigi Fiume1,*, Luigi Bolondi2, Corrado Busi1, Pasquale Chieco3, Felix Kratz4, Marcella Lanza1, Alessandro Mattioli1, Giuseppina Di Stefano1 2

1 Department of Experimental Pathology, University of Bologna, via San Giacomo, 14 40126 Bologna, Italy Department of Internal Medicine and Gastroenterology, University of Bologna and St Orsola-Malpighi University Hospital, Bologna, Italy 3 Center for Applied Biomedical Research (CRBA), St Orsola-Malpighi University Hospital, Bologna, Italy 4 Tumor Biology Center, Freiburg, Germany

Background/Aims: The hepatocyte receptor for asialoglycoproteins internalizes galactosyl terminating macromolecules which can be used as hepatotropic drug carriers. Since this receptor is also expressed on the cells of well differentiated human hepatocellular carcinomas (HCCs), we studied whether conjugation of doxorubicin (DOXO) with lactosaminated human albumin (L-HSA) increases the drug efficacy on HCCs induced in rats by diethylnitrosamine (DENA). Methods: DENA was given in the drinking water for 8 weeks. One week after the last day of DENA administration, animals were randomly assigned to three groups. Each group was administered with either saline, free or coupled DOXO (1 mg/g). Rats received 4 weekly intravenous injections. One week after the last administration, rats were killed and HCC development was evaluated by counting the tumor nodules on the surface of hepatic lobes. Results: In rats treated with L-HSA coupled DOXO the number of neoplastic nodules was significantly lower (P!0.05) than that counted in animals injected with saline or with free DOXO. Coupled DOXO did not decrease body rat weight, which was markedly reduced by the free drug. Conclusions: Conjugation with L-HSA increased the antineoplastic efficacy and decreased the systemic toxicity of DOXO administered to rats with HCCs produced by DENA. q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Drug targeting; Asialoglycoprotein receptor; Hepatotropic conjugate of doxorubicin; Hepatocellular carcinoma targeted doxorubicin; Hepatocellular carcinoma chemotherapy

1. Introduction Hepatocellular carcinoma (HCC) is a major health problem worldwide, representing the third largest cause of cancer related death. Diagnosed at early stage, the tumor can be curable by resection, percutaneous ablation and liver transplantation [1]. To treat the vast majority of patients

Received 1 October 2004; received in revised form 16 November 2004; accepted 1 February 2005; available online 12 May 2005 * Corresponding author. Tel.: C39 512094721; fax: C39 512094746. E-mail address: [email protected] (L. Fiume).

who only receive a palliative therapy and to prevent recurrent HCC, an effective systemic chemotherapy is actively sought. The available anticancer drugs have a very limited efficacy on HCCs at the conventional doses, and dose escalation is hindered by unacceptable toxicity [2]. In the treatment of HCCs that maintain the receptor for asialoglycoproteins (ASGP-R), the chemotherapeutic index of the drugs which display the main side effects on extrahepatic tissues could be increased by coupling them to macromolecules that are taken up by this receptor [3–5]. ASGP-R is a glycoprotein present only on the surface of hepatocytes. It mediates uptake and lysosomal degradation

0168-8278/$30.00 q 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2005.02.045

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O

O N NH O

OH

N

2. Materials and methods 2.1. Synthesis and characterization of L-HSA-DOXO

O

CH2OH OH

The procedure of synthesis and the chemical characteristics of the conjugate L-HSA-DOXO are described in [19].

2.2. Animals OCH3 O

OH O

O

CH3 OH NH2 HCl Fig. 1. (6-Maleimidocaproyl)hydrazone of doxorubicin (DOXOEMCH).

of galactosyl terminating peptides [6,7], which can be used as vectors for selectively delivering the drugs to parenchymal liver cells [8,9]. In a study on the needle biopsies of 60 consecutive human HCCs, ASGP-R was histochemically detected on all the cells in 28 out of 35 (80%) well differentiated and in 5 out of 25 (20%) poorly differentiated forms of the tumor [10]. This result supported the attempts to develop a HCC chemotherapy through the ASGP-R. In line with this approach, we coupled doxorubicin (DOXO) to lactosaminated human albumin (L-HSA) using the (6-maleimidocaproyl)hydrazone derivative of the drug (DOXO-EMCH) (Fig. 1), first synthesized by Wilner et al. [11]. DOXO is a drug used in the chemoembolization of HCCs. Its derivative contains an acid sensitive hydrazone bond that is stable at the neutral pH of plasma, but allows DOXO to be intracellularly released from the carrier in the endosomal or lysosomal compartments [12]. L-HSA is a galactosyl terminating neoglycoprotein safely used as hepatotropic drug carrier in clinical studies [13–15]. The conjugate administered to mice accomplished a very efficient targeting of the drug in liver, where DOXO concentrations reached levels 7–20 times higher than those raised in extra-hepatic tissues [16]. In the experiments reported here we studied the anticancer efficacy of free and L-HSA coupled DOXO on the growth of HCCs induced in rats by diethylnitrosamine (DENA) [17]. These tumors internalize through the ASGPR amounts of the carrier L-HSA several times higher than those which entered in extra-hepatic tissues [18]. We have observed that in rats treated with coupled DOXO the number of HCC nodules was significantly lower compared to that counted in control animals administered with saline. In contrast, free DOXO, administered at the same dose as the coupled drug, did not exert any anticancer activity. Coupled DOXO did not decrease body weight of rats, which on the contrary was markedly reduced by the free drug.

Male Wistar rats (weighing 125–150 g) were used. They were obtained from Harlan Italy (Udine, Italy) and were maintained in an animal facility at the Department of Experimental Pathology, Bologna, receiving humane care, in accordance with European Legislation. The protocols of the experiments were approved by the Ethical Committee of the University of Bologna. Animals were fed a standard pellet diet ad lib.

2.3. Induction of hepatocellular carcinoma and evaluation of tumor growth Hepatocellular carcinomas (HCCs) were induced in thirty male Wistar rats (125–150 g), which received diethylnitrosamine (DENA) given in their drinking water (100 mg/l) for 8 weeks. One week after the last day of DENA administration, animals (weighing 270–320 g) were randomly assigned to three groups of ten animals. Each group was administered with either saline (NaCl 0.9%), free DOXO (1 mg/g) or coupled DOXO (1 mg/g), respectively. Compounds were injected weekly under isoflurane anesthesia in the dorsal vein of penis, in a volume of 10 ml/10 g. Four administrations were performed. No animal died during the experiment. One week after the last administration, rats were killed under isoflurane anesthesia. The livers were removed and lobes were separated. Tumor nodules R3 mm in diameter (measured with a digital caliper) were counted on the surface of each lobe and the difference in the nodule numbers in the three experimental groups was statistically evaluated (see legend to Fig. 4). A second evaluation of tumor growth was performed after the lobes were fixed in 10% formalin, which makes neoplastic nodules more evident. The upper and lower surfaces of each fixed lobe, together with a millimeter graded bar were photographed and tumor nodules R2 mm in diameter were blind counted on the digital enlargements of the photos. Lumps smaller than 2 mm were not counted since they could not be identified with certainty as nodules. In livers displaying a multinodular surface, identifiable nodules were counted to a maximum of 50, since the total number could not be reliably assessed. The evaluation of antitumor effect of the drugs was performed comparing the number of animals with more than 50 tumor masses in the different experimental groups (for statistical analysis, see legend to Fig. 6).

2.4. Histology and histochemistry Samples of all the lobes of the liver were fixed in 10% formalin, embedded in paraffin and routinely stained with hematoxylin and eosin (H&E). To verify whether preneoplastic and neoplastic foci are produced by DENA treatment, the expression of the placental form of glutathione S-transferase (GST-P), a marker of hepatocyte preneoplastic transformation in rats [20], was histochemically assessed [21].

3. Results 3.1. Chemical characteristics of L-HSA-DOXO conjugate The L-HSA preparation used for the synthesis of L-HSADOXO contained 24 galactosyl residues per protein molecule. The molar ratio DOXO/L-HSA in different conjugate preparations ranged from 5 to 7 (1 mg conjugate contained 36–50 mg DOXO). Three percent of the drug was not covalently linked to the protein. After lyophilization

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the conjugate easily dissolved in 0.9% NaCl to a concentration of 3 mg of coupled DOXO per ml, giving a completely clear solution.

Table 1 Areas under the curve (0–24 h) of DOXO concentrations in rat organs after intravenous administration of free or L-HSA coupled drug (1 mg/g)

3.2. Organ concentrations of DOXO in untreated and in DENA treated rats

Injected compound

Fig. 2 shows the concentrations of DOXO in organs of untreated rats, at different times after intravenous administration of 1 mg/g of free (frame A) or coupled drug (frame B). Also in the conjugate injected animals, the drug was determined as free DOXO (i.e. liberated from the carrier). The areas under curve from 0 to 24 h of the concentrations are shown in Table 1. The conjugate produced a very efficient liver targeting of DOXO. The drug concentrations in this organ were 7–20 times higher than those measured in extrahepatic tissues. On the contrary, in rats injected with free DOXO the area under curve of the drug levels in liver was A 50

Liver Heart

nmoles / g tissue

40

Intestine Kidney

30

Spleen

20

10

0 0

1 2

4

6

24

Time (h) B

Uncoupled DOXO L-HSA coupled DOXO

nmol!h/g tissue Liver

Heart

Intestine

Spleen

Kidney

42.46 400.70

65.30 19.63

55.79 29.71

109.60 58.19

95.25 26.15

Areas under the curve were calculated from the data of Fig. 2, using the software GraphPad Prism 3.02. L-HSA, lactosaminated human albumin; DOXO, doxorubicin.

slightly lower than those of the other organs. In conjugate treated rats the DOXO concentrations in liver were 9–10 times higher than those measured in the free drug administered animals. Conversely, the DOXO levels in extra-hepatic tissues were 2–3 times higher in the latter animals. The organ concentrations of DOXO in DENA treated animals measured at three time intervals after injection of free or coupled DOXO are reported in Table 2. In free drug injected animals the DOXO levels were similar to those measured at the same times in animals not treated with DENA. In conjugate injected rats, as observed in animals not treated with DENA, DOXO selectively accumulated in liver, with very low drug concentrations in extra-hepatic tissues. However, the liver concentration of DOXO 2 h after conjugate injection was significantly lower (P!0.05) than that measured in animals not treated with DENA. This result fits the finding that, 2 h after administration, the amounts of the carrier L-HSA taken up by the liver were smaller in DENA treated rats than in untreated animals [18] and can be explained by the extensive hepatic fibrosis caused by DENA (see Section 3.4), since the uptake of galactosyl terminating glycoproteins is retarded in fibrotic livers [22].

50

3.3. Effect of free and coupled DOXO on body weight and on tumor growth

40

30

20

10

0 0

2

4

6

8

24

Time (h) Fig. 2. DOXO concentrations in organs of rats after intravenous injection of 1 mg/g of the free drug (frame A) or 1 mg/g of L-HSA coupled DOXO (frame B). At different times after drug administration, animals were killed; organs were rapidly removed and tissue concentrations of DOXO were measured according to Bots et al. [35], with modifications [16]. Each entry is the mean value from determinations in three animals. CV (coefficient of variation) ranged from 7 to 30% of mean values.

The changes in body weight of DENA treated rats during administration of free or coupled DOXO are reported in Fig. 3. The conjugate practically did not affect the body weight, which, on the contrary, was significantly reduced by free DOXO. The anti-tumor effect of free and coupled DOXO was evaluated by counting the nodules on the liver lobes of rats of the three experimental groups (see Section 2.3). In Fig. 4, the number of tumor nodules R3 mm in diameter counted on non-fixed liver lobes are reported. In rats treated with free DOXO the number of nodules was not reduced compared to that of control animals injected with saline. On the contrary, in rats treated with L-HSA coupled DOXO the number of neoplastic nodules was significantly lower than that counted in animals injected with saline or with free DOXO. A second evaluation of the drug effect on the tumor growth was performed counting the nodules on the digital

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Table 2 Concentrations of DOXO in organs of DENA treated rats after intravenous administration of free or coupled DOXO (1 mg/g) Free DOXO

Liver Heart Intestine Kidney

Coupled DOXO

15 min

30 min

1h

2h

4h

6h

6.36G1.24 6.61G1.84 8.18G2.31 13.20G2.55

5.65G0.79 7.47G0.10 5.86G0.44 11.35G1.21

4.68G0.05 5.43G1.26 6.27G0.73 10.04G0.44

28.12G7.33 0.52G0.06 0.79G0.16 0.89G0.05

26.53G2.74 0.63G0.12 1.20G0.15 0.84G0.27

22.45G1.40 0.86G0.19 0.97G0.30 1.32G0.24

Data (nmol/g tissue) are mean valuesGSE from three animals. DOXO, doxorubicin; DENA, diethylnitrosamine. Eighteen rats received DENA in drinking water (100 mg/l) for 8 weeks. One week after the last day of DENA administration, they were injected in the dorsal vein of penis under isoflurane anesthesia with free or coupled DOXO and were killed at the indicated times after drug injection. Organs were rapidly removed and tissue concentrations of DOXO were measured according to Bots et al. [35], with modifications [16].

enlargements of the photos of formalin fixed liver lobes. Macroscopic views of fixed liver lobes are reported in Fig. 5. The anti-tumor efficacy of the drugs was assessed comparing the number of animals with more than 50 tumor masses in the different experimental groups (Fig. 6). In the group of rats treated with coupled DOXO only one animal had more than 50 tumor nodules, with a significant difference from the groups administered with saline (seven animals) or with the free drug (eight animals). In contrast, there was no difference between free DOXO treated rats and saline injected animals. 3.4. Histology and histochemistry

bile duct proliferation was particularly evident around micronodules. All morphological detected acidophilic foci and nodules were GST-P positive. However, GST-P immunohistochemical staining revealed several additional GST-P positive foci not detectable in HandE stained sections. A few foci composed of basophilic GST-P negative hepatocytes were also present in a few rat livers. In the animals killed after treatment with saline, free or coupled DOXO, all the nodules large enough to be macroscopically detectable, which were examined with light microscopy, were classified as hepatocellular carcinomas. In all the three experimental groups, most of them were of a solid poorly differentiated type, containing

At histological examination, the liver sections of the animals killed one week after the end of DENA administration, when the treatment with free or coupled DOXO was started, showed numerous foci of altered hepatocytes and several micronodular lesions. Foci and micronodules were mostly formed by large acidophilic hepatocytes showing various degrees of glycogen accumulation. Cellular and nuclear atypias suggestive of malignant progression were present only in the largest nodules. Increased fibrosis with

grams body weight

350

325

300

275

250

225 0

10

14

22

30

Days Fig. 3. Changes in rat body weight (mean valuesGSE) during the period of saline (C), free DOXO (:) and L-HSA coupled DOXO (&) administration. Differences were statistically evaluated by Bonferroni’s multiple comparison test after one-way ANOVA. Saline vs. coupled DOXO, PO0.05; Saline vs. free DOXO, P!0.01; coupled DOXO vs. free DOXO, P!0.05.

Fig. 4. Number of neoplastic nodules R3 mm in diameter counted on non-fixed liver of each rat. Since Bartlett’s test showed that in the three groups data have unequal variances, the statistical significance of intergroup differences was evaluated by the non-parametric Kruskal– Wallis test [36], followed by Dunn’s post test [37]. The number of nodules in coupled DOXO treated animals was significantly lower than those measured in saline and in free drug injected animals (P!0.05). There was no difference between free DOXO treated rats and control animals injected with saline.

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Fig. 5. Macroscopic views of formalin fixed liver lobes from one representative rat in the saline, free DOXO and coupled DOXO groups (upper, medium and lower set of photos, respectively).

enlarged hepatocytes, with a glossy, often basophilic cytoplasm and pleomorphic vesicular nuclei. A multinodular appearance, where small atypical nodules were sharply delineated by extensive fibrosis, was present in several liver sections from all the three experimental groups.

4. Discussion The expression of ASGP-R on the cells of the majority of the well differentiated forms of human HCCs [10] suggested the possibility of increasing DOXO value in the treatment of these cancers by coupling it to L-HSA, a ligand of the receptor [16]. In the experiments reported here we studied the anticancer activity of L-HSA-DOXO on HCCs induced in rats by DENA. In previous experiments we attempted to histochemically reveal the ASGP-R in these tumors. Unfortunately, in our hands the commercially available anti-ASGP-R antibody failed to detect the rat receptor. However, we obtained evidence of ASGP-R expression in HCCs induced by DENA by measuring the L-HSA uptake by the tumors [18]. Rat HCCs internalized more than 10 times higher amounts of L-HSA than extra-hepatic tissues.

A 20-times lower uptake of non-lactosaminated HSA indicated that penetration of L-HSA into HCCs occurred through the ASGP-R. In agreement with histochemical data on receptor expression in human HCCs [10], a correlation was found between the level of L-HSA uptake and the degree of tumor differentiation [18]. The finding of an enhanced penetration of L-HSA has a higher value than the histochemical detection of ASGP-R, since it demonstrates that: (1) the receptor is also functionally active and can internalize the ligand; (2) the capillarization occurring in HCCs [23] does not hinder the contact of L-HSA with the surface of the tumor cells. We have now observed that L-HSA coupled DOXO exerted an anticancer activity on rat HCCs whereas the free drug, administered at the same dose and with the same schedule, was completely ineffective. This finding indicated that L-HSA transported and released into HCC cells higher amounts of DOXO than those entered after administration of the free drug and that they were sufficient to produce the drug pharmacological activity. Moreover, coupled DOXO did not decrease the rat body weight, which on the contrary was markedly reduced by the treatment with the free drug. The lower DOXO concentrations in extra-hepatic tissues

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Fig. 6. Number of neoplastic nodules (R2 mm in diameter) counted on formalin fixed livers of each rat (&). The number of rats with more than 50 tumor nodules in the three experimental groups were compared (see Section 2.3). Statistical analysis was performed using Fisher’s exact test: L-HSA coupled DOXO vs. saline, PZ0.0198; vs. free DOXO, PZ0.0055.

after conjugate administration are most likely responsible for this result. L-HSA-DOXO appears to have advantages over two other vectors of DOXO, prepared to improve the systemic chemotherapy of HCCs. The first was addressed to liver tumors expressing the ASGP-R and was obtained by coupling the drug to galactosylated copolymers of N-(2hydroxypropyl)metacrylamide (pHPMA) by a peptide bond with a tetrapeptidyl spacer [4,24]. The copolymers forming the backbone of this conjugate are largely not biodegradable [25] and recent data [26] indicate that DOXO is scantily released from the tetrapeptidyl spacer of the pHPMA carriers in the cells. Therefore, the cytotoxicity of these complexes is probably due to a damage of the endosomal and lysosomal membranes caused by the drug in the coupled form [26,27] and, in the case of galactosylated pHPMA conjugate, is expected to be indiscriminately exerted on both normal and neoplastic hepatocytes. The second vector was prepared by encapsulating DOXO in polyethylene glycol coated liposomes (PLD). The rationale for the use of PLD in the treatment of solid tumors is based on their ability to extravasate through ‘leaky’ tumor vasculature, resulting in a preferential localization of DOXO in tumor tissue [28]. However, experiments in rats demonstrated that PLD remains entrapped in the sinusoidal space of liver and only small quantities of DOXO can gain access to hepatocytes [29]. In HCCs, where sinusoid capillarization with reduction of endothelial fenestrations and production of a capillary membrane occurs [23], a still lower contact

of DOXO is expected with the surface of neoplastic hepatocytes. Accordingly, in a recent phase II clinical study the response rate of patients with HCCs to PLD was not higher than that obtained with the free DOXO [30]. Two major issues should be addressed in future experiments to ascertain all the possible applications of L-HSA-DOXO in the treatment of human HCCs: (1) can the conjugate also be active on established tumors?; (2) will DOXO accumulation produced by the conjugate in nonneoplastic hepatocytes further impair the function of cirrhotic livers? As far as the first point is concerned, in the experiments reported here, histological examination of livers showed that only premalignant micronodules but not established tumors were formed at the time when L-HSADOXO treatment was started. Therefore, the effect caused by L-HSA-DOXO was to hinder HCC development rather than to reduce the tumor masses. We will now study the effect of L-HSA-DOXO on the size of established rat liver tumors by using micro-PET (positron emission tomography). In PET imaging of human HCCs, histopathological correlation suggests that well differentiated tumors are detected by 11C-acetate and the poorly differentiated types are detected by 18F-fluorodeoxyglucose [31]. If the same result can be obtained in rats, by using the two radio-tracers it might be possible to study separately the effect of L-HSADOXO on the size of well and poorly differentiated forms of HCCs. Some poorly differentiated rat HCCs internalize several times higher amounts of the L-HSA carrier than the extra-hepatic tissues [18], similarly to the well differentiated forms. Consequently, they might also respond to the anticancer activity of the conjugate. Likewise, 20% of the human poorly differentiated HCCs maintain the ASGP-R [10] and might therefore be a target for L-HSA-DOXO. Human HCCs expressing the receptor can be histochemically identified in needle biopsies or in resected tumor masses by the specific antibody to the human ASGP-R [10]. The second issue to be addressed is relevant since in the western countries the majority of human HCCs arise in cirrhotic livers [32]. We have shown that L-HSA-DOXO does not damage non-neoplastic hepatocytes, including regenerating cells in partially hepatectomized rats [33], but we have not studied the effect of the conjugate on cirrhotic livers. It is very difficult to reproduce in laboratory animals the mechanisms of the damage occurring in hepatocytes of human cirrhotic livers. As an approach to the problem, we will verify whether the conjugate worsens the damage and further impairs the function of cirrhotic livers in rats poisoned with carbon tetrachloride [34]. In conclusion, at present the available data suggest that L-HSA-DOXO might be usefully administered to patients without severely impaired liver function, as an adjuvant systemic chemotherapy to prevent recurrences after surgical removal or percutaneous ablation of the tumor. In this setting, the conjugate could make DOXO an effective drug, considering the enhanced anticancer activity of the coupled drug and the lower levels of DOXO raised in extra-hepatic

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tissues, which might permit increased numbers of drug administrations without producing cumulative cardiac toxic effects. The feasibility of clinical studies with L-HSADOXO is supported by the observation that L-HSA was found to be a safe and effective hepatotropic drug carrier in clinical studies. A L-HSA conjugate of the antiviral agent adenine arabinoside, administered for 28 consecutive days to patients with chronic hepatitis B infection, decreased viremia to the same extent as the free drug, without producing adverse reactions [14,15].

[12]

[13]

[14]

Acknowledgements [15]

This work was supported by research grants from MIUR (Italian Ministry of Education and University); University of Bologna and Fondazione Cassa di Risparmio in Bologna. Authors thank Prof G. Barbanti Brodano for critical review of the manuscript.

[16]

[17]

References [1] Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003;362:1907–1917. [2] Nowak AK, Chow PKH, Findlay M. Systemic therapy for advanced hepatocellular carcinoma: a review. Eur J Cancer 2004;40: 1474–1484. [3] Schneider Y, Abarca J, Aboud-Pirak E, Baurain R, Ceulemans F, Deprez-De Campeneere D. Drug targeting in human cancer chemotherapy. In: Gregoriadis G, Poste G, Senior J, Trouet A, editors. Receptor-mediated targeting of drugs. NATO ASI series A: life sciences, 82. New York: Plenum Press; 1984. p. 1–25. [4] O’Hare KB, Hume IC, Scarlett L, Chytry V, Kopeckova P, Kopecek J, et al. Effect of galactose on interaction of N-(2-hydroxypropyl) metacrylamide copolymers with hepatoma cells in culture: preliminary applications to an anticancer agents, daunomycin. Hepatology 1989;10:207–214. [5] Di Stefano G, Busi C, Mattioli A, Derenzini M, Trere` D, Fiume L. Inhibition of [3H]thymidine incorporation into DNA of rat regenerating liver by 2 0 ,2 0 -difluorodeoxycytidine coupled to lactosaminated poly-L-lysine. Biochem Pharmacol 1999;57:793–799. [6] Morell AG, Irvine RA, Sternlieb I, Scheinberg IH, Ashwell G. Physical and chemical studies on ceruloplasmin. V. Metabolic studies on sialic acid-free ceruloplasmin in vivo. J Biol Chem 1968;243: 155–159. [7] Ashwell G, Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem 1982;51:531–534. [8] Fiume L, Mattioli A, Balboni PG, Tognon M, Barbanti Brodano G, de Vries J, et al. Enhanced inhibition of virus DNA synthesis in hepatocytes by trifluorothymidine coupled to asialofetuin. FEBS Lett 1979;103:47–51. [9] Fiume L, Busi C, Mattioli A, Barbanti-Brodano G. Hepatocyte targeting of adenine-9-b-D-arabinofuranoside 5 0 -monophosphate (araAMP) coupled to lactosaminated albumin. FEBS Lett 1981;129: 261–264. [10] Trere` D, Fiume L, Badiali De Giorgi L, Di Stefano G, Migaldi M, et al. The asialoglycoprotein receptor in human hepatocellular carcinomas: its expression on proliferating cells. Br J Cancer 1999; 81:404–408. [11] Willner D, Trail PA, Hofstead SJ, King HD, Lasch SJ, Braslawsky GR, et al. (6-Maleimidocaproyl)hydrazone derivative of

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25] [26]

[27] [28]

[29]

[30]

651

doxorubicin—a new derivative for the preparation of immunoconjugates of doxorubicin. Bioconjug Chem 1993;4:521–527. Greenfield RS, Kaneko T, Daues A, Edson MA, Fitzgerald KA, Olech LJ, et al. Evaluation in vitro of adriamycin immunoconjugates synthesized using an acid-sensitive hydrazone linker. Cancer Res 1990;50:6600–6607. Fiume L, Bonino F, Mattioli A, Chiaberghe E, Torrani Cerenzia MR, Busi C, et al. Inhibition of hepatitis B virus replication by vidarabine monophosphate conjugated with lactosaminated serum albumin. Lancet 1988;2:13–15. Torrani Cerenzia MR, Fiume L, De Bernardi Venon W, Lavezzo B, Brunetto MR, Ponzetto A. Adenine arabinoside monophosphate coupled to lactosaminated albumin, administered for 4 weeks to patients with chronic type B hepatitis, decreased viremia without producing significant side effects. Hepatology 1996;23:657–661. Zarski JP, Barange K, Souvignet C, Bertini M, Marcellin P, Tran A, et al. Efficacy and safety of L-HSA-ara-AMP in chronic hepatitis B patients non responder to interferon therapy: a randomised clinical trial. J Hepatol 2001;34:487–488. Di Stefano G, Kratz F, Lanza M, Fiume L. Doxorubicin coupled to lactosaminated human albumin remains confined within mouse liver cells after the intracellular release from the carrier. Dig Liver Dis 2003;35:428–433. Rajewsky MF, Dauber W, Frankenberg H. Liver carcinogenesis by diethylnitrosamine in the rat. Science 1966;152:83–85. Di Stefano G, Fiume L, Bolondi L, Lanza M, Pariali M, Chieco P. Enhanced uptake of lactosaminated human albumin by rat hepatocarcinomas: implications for an improved chemotherapy of primary liver tumours. Liver Int; doi:10.1111/j.1478-3231.2005.1118x. Di Stefano G, Lanza M, Kratz F, Merina L, Fiume L. A novel method for coupling doxorubicin to lactosaminated human albumin by an acid sensitive hydrazone bond: synthesis, characterization and preliminary biological properties of the conjugate. Eur J Pharm Sci 2004;23: 393–397. Ito N, Tamano S, Shirai T. A medium-term rat liver bioassay for rapid in vivo detection of carcinogenic potential of chemicals. Cancer Sci 2003;94:3–8. Gramantieri L, Trere´ D, Chieco P, Lacchini M, Giovannini C, Piscaglia F, et al. In human hepatocellular carcinoma in cirrhosis proliferating cell nuclear antigen (PCNA) is involved in cell proliferation and cooperates with P21 in DNA repair. J Hepatol 2003;39:997–1003. Marshall JS, Williams S, Jones P, Hepner GW. Serum desialylated glycoproteins in patients with hepatobiliary dysfunction. J Lab Clin Med 1978;92:30–37. Kin M, Torimura T, Ueno T, Inuzuka S, Tanikawa K. Sinusoidal capillarization in small hepatocellular carcinoma. Pathol Int 1994;44: 771–778. Seymour LW, Ferry DR, Anderson D, Hesslewood S, Julyan PJ, Poyner R, et al. Hepatic drug targeting: phase I evaluation of polymerbound doxorubicin. J Clin Oncol 2002;20:1668–1676. Seymour LW. Soluble polymers for lectin-mediated drug targeting. Adv Drug Deliv 1994;14:89–111. Hovorka O, St’astny M, Etrych T, Subr V, Strohalm J, Ulbrich K, et al. Differences in the intracellular fate of free and polymer-bound doxorubicin. J Control Release 2002;80:101–117. Tritton TR, Yee G. The anticancer agent adriamycin can be actively cytotoxic without entering cells. Science 1982;217:248–250. Gabizon A, Martin F. Polyethylene glycol-coated (pegylated) liposomal doxorubicin. Rationale for use in solid tumours. Drugs 1997;54:15–21. Hilmer SN, Cogger VC, Muller M, Le Couteur DG. The hepatic pharmacokinetics of doxorubicin and liposomal doxorubicin. Drug Metab Dispos 2004;32:794–799. Hong RL, Tseng YL. A phase II and pharmacokinetic study of pegylated liposomal doxorubicin in patients with advanced

652

L. Fiume et al. / Journal of Hepatology 43 (2005) 645–652

hepatocellular carcinoma. Cancer Chemother Pharmacol 2003;51: 433–438. [31] Ho CL, Yu SCH, Yeung DWC. 11C-Acetate PET imaging in hepatocellular carcinoma and other liver masses. J Nucl Med 2003; 44:213–221. [32] Kern MA, Breuhahan K, Schirmacher P. Molecular pathogenesis of human hepatocellular carcinoma. Adv Cancer Res 2002;86:67–112. [33] Di Stefano G, Derenzini M, Kratz K, Lanza M, Fiume L. Livertargeted doxorubicin: effects on rat regenerating hepatocytes. Liver Int 2004;24:246–252.

[34] Perez Tamayo R. Is cirrhosis of the liver experimentally produced by CCl4 an adequate model of human cirrhosis? Hepatology 1983;3: 112–120. [35] Bots AM, Van Oort WJ, Noordoek J, Van Dijk A, Klein SW, Van Hoesel QG. Analysis of adriamycin and adriamycinol in micro volumes of rat plasma. J Chromatogr 1983;272:421–427. [36] Snedecor GW, Cochran WG. Statistical methods. Ames: Iowa State University Press; 1990. [37] Dunn OJ. Multiple contrasts using rank sums. Technometrics 1964;5: 241–252.