In vitro and in vivo antitumour effects of novel, orally active bile acid-conjugated platinum complexes on rat hepatoma

European Journal of Pharmacology 549 (2006) 27 – 34 www.elsevier.com/locate/ejphar

In vitro and in vivo antitumour effects of novel, orally active bile acid-conjugated platinum complexes on rat hepatoma Cecilia Barbara a,1 , Paola Orlandi a,1 , Guido Bocci a,⁎, Anna Fioravanti a , Antonello Di Paolo a , Gianfranco Natale b , Mario Del Tacca a , Romano Danesi a a

Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, Via Roma, 55 I-56126 Pisa, Italy b Department of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy Received 10 May 2006; received in revised form 26 July 2006; accepted 4 August 2006 Available online 16 August 2006

Abstract (NH3)2Pt(triacid) and (PPh3)2Pt(dehydrocholate)2 are novel bile acid-conjugated platinum complexes administered by oral route. The aims of the present study were to evaluate their in vitro cytotoxic activities on rat hepatoma cell line N1–S1, the in vivo antitumour effects in a syngeneic and orthotopic rat hepatoma model and the drug-related toxicities. Cisplatin, carboplatin and mitoxantrone were used as control drugs. In vitro experiments showed a concentration- and time-dependent antiproliferative activity of bile-conjugated platinum complexes. (NH3)2Pt(triacid) had similar effects on cell growth of cisplatin and carboplatin (e.g. at 48 h, IC50 0.7 ± 0.05 μM vs. 0.63 ± 0.28 μM and 1.1 ± 0.3 μM, respectively; mean ± S.D.). (NH3)2Pt (triacid) was able to inhibit tumour growth in a dose-dependent extent, reaching the maximum inhibitory effect at the 80 mg/kg dose (1.95 ± 0.5 g vs. 13.85 ± 3.9 g of control tumour weight). By contrast, despite the promising in vitro antiproliferative activity, (PPh3)2Pt(dehydrocholate)2 showed no significant in vivo antitumour effect. The toxicity profile of (NH3)2Pt(triacid) resulted favourable with minimal loss of weight and no gastrointestinal or neurological symptoms. Instead, (PPh3)2Pt(dehydrocholate)2 showed dose-dependent signs of severe weight loss and neurological alterations. In conclusion (NH3)2Pt(triacid) is a tolerable and active platinum derivative endowed by a preclinical antitumour activity by oral route. © 2006 Elsevier B.V. All rights reserved. Keywords: Hepatoma; Platinum complex; Bile acid; Cisplatin; Carboplatin; Mitoxantrone

1. Introduction Cisplatin (cis-diamminedichloroplatinum(II)) was introduced into clinical practice in the early 1970s and it is one of the most active antineoplastic agents currently used in medical oncology. Cisplatin has a broad spectrum of activity against epithelial cancers and has become the foundation of curative regimens in testicular and ovarian cancers, demonstrating significant activity also against lung, head and neck, oesophagus, bladder, cervix, and endometrial cancers (Ho et al., 2003). However, the clinical use of cisplatin is limited by important toxicities, including severe renal, neurological, gastrointestinal side effects, and intrinsic or acquired resistance (Momekov et al., 2005). Therefore attention has focused on new platinum compounds with improved ⁎ Corresponding author. Tel.: +39 050 830148; fax: +39 050 562020. E-mail address: [email protected] (G. Bocci). 1 These authors equally contributed to this work. 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.08.015

pharmacological and antitumour properties such as carboplatin and oxaliplatin (Haller, 2004; Muggia and Fojo, 2004). More than 30 platinum complexes have entered clinical trials in recent years to circumvent the side effects and the problem of tumour resistance to cisplatin (Galanski et al., 2005). New synthetic approaches (Zhang and Lippard, 2003) and a better knowledge of the molecular mechanism of platinum complexes (Wang and Lippard, 2005) provide the basis for a rational design of promising anticancer platinum coordination compounds. In clinical practice cisplatin, carboplatin and oxaliplatin are administered by intravenous infusion but platinum drugs that could be effectively administered orally are strongly desirable in order to increase their potential clinical uses in the outpatient setting (Ho et al., 2003). The synthesis of platinum analogues conjugated with carriers that increase drug uptake by the liver after oral administration may represent a therapeutic advantage for abdominal carcinomas, particularly those localized to the liver such as hepatocarcinomas

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(Macias et al., 1999; Marin et al., 1998a). Due to the high efficiency of hepatocytes to take up bile acids, these endogenous compounds or their analogues can be considered as possible carriers for delivering drugs to the liver (Criado et al., 1997b; Monte et al., 1999). Marin and colleagues have synthesized and characterized several members of a new family of compounds, named Bamets, by binding molecules containing a transition metal to bile acids (Criado et al., 1997a; Marin et al., 1998a). Bamet-R2 and Bamet-UD2 revealed a marked liver organotropism (Larena et al., 2001; Macias et al., 1999), a strong in vitro cytotoxic and proapoptotic effect (Monte et al., 2005, 1999) and an antitumour activity in orthotopically implanted hepatomas in mouse liver (Dominguez et al., 2001). (NH3)2Pt(triacid) and (PPh3)2Pt(dehydrocholate)2 are two novel platinum compounds obtained by conjugating dehydrocholic acid with two different platinum coordination complexes (Fig. 1). The (NH3)2Pt(triacid) is constituted by a molecule of bile acid obtained by synthesis (triacid) and conjugated with the amino platinum coordination complex Pt-(NH3)2; (PPh3)2Pt (dehydrocholate)2 is synthesized by the union of two molecules of dehydrocholic acid linked to the phosphinic platinum compound Pt-(PPh3)2. The aims of the present study were to evaluate i) the in vitro cytotoxic activities on rat hepatoma cell line N1–S1, ii) the in vivo antitumour effects in a syngeneic and orthotopic rat hepatoma model and iii) the drug-related toxicities of the two novel bile acid-conjugated platinum complexes administered by oral route.

Fig. 2. Graphic representations of (A) the oral administration of (NH3)2Pt (triacid) and (PPh3)2Pt(dehydrocholate)2 and (B) intravenous injections of cisplatin, carboplatin and mitoxantrone in a syngeneic and orthotopic rat hepatoma model. Arrow, administration of the drug dose.

(cisplatin, carboplatin and mitoxantrone) for in vivo per os and i.v. administration, respectively. All other reagents were from Sigma Chemicals Co. (Milan, Italy).

2. Materials and methods 2.2. Cell line and animals 2.1. Chemicals (NH3)2Pt(triacid) and (PPh3)2Pt(dehydrocholate)2 (Fig. 1) were generous gifts from Abiogen Pharma (Pisa, Italy). Drugs for comparison were carboplatin (Bristol-Myers Squibb S.r.l., Rome, Italy), cisplatin (Pharmacia Italia S.p.A., Milan, Italy), mitoxantrone (Wyeth Lederle S.p.A., Catania, Italy). Drugs were dissolved in sterile culture medium immediately before their in vitro use or in peanut oil vehicle [(NH3)2Pt(triacid) and (PPh3)2Pt(dehydrocholate)2] and in sterile saline solution

Sprague–Dawley male rat hepatoma N1–S1 cells were purchased from American Type Culture Collection (ATCC; Manassas, MA, USA). N1–S1 were routinely cultured in Medium 199 supplemented with 10% foetal bovine serum (FBS), 4 mM glutamine, 50 μg/ml streptomycin and 100 IU/ml penicillin G and kept in a humidified atmosphere of 5% CO2 at 37 °C. Cells were harvested with a solution of 0.25% trypsin–0.03% EDTA when they were in log phase of growth, and maintained at the abovedescribed culture conditions for all experiments. Sprague–Dawley male rats, weighing 225–250 g, were supplied by Harlan Nossan (Milan, Italy) and were allowed unrestricted access to food and tap water. Housing and all procedures involving animals were performed according to the protocol approved by the local Committee for the animal experimentation of the University of Pisa, in accordance with the European Community Council Directive 86-609, recognised by the Italian government, on animal welfare. Each experiment employed the minimum number of rats needed to obtain statistically meaningful results. 2.3. Cytotoxicity assay

Fig. 1. Chemical structure of the bile acid-conjugated platinum complexes, (NH3)2Pt(triacid) and (PPh3)2Pt(dehydrocholate)2.

In vitro chemosensitivity testing was performed on singlecell suspensions of N1–S1 cells (5 × 103 cells/well) plated in 24well sterile plastic plates and allowed to attach overnight. The treatment protocol was designed so that each drug concentration

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Fig. 3. In vitro effect of (NH3)2Pt(triacid) (A), (PPh3)2Pt(dehydrocholate)2 (B), cisplatin (C), carboplatin (D) and mitoxantrone (E) on rat hepatoma N1–S1 cell proliferation after 6, 12 and 48 h of drug incubation. Symbols and bars, mean values ± S.D., respectively; ⁎P b 0.05 vs. controls.

was represented by at least nine wells. Cells were treated with increasing concentrations of (NH3)2Pt(triacid), (PPh3)2Pt (dehydrocholate)2, cisplatin, carboplatin (each at 0.01– 100 μM) or mitoxantrone (0.0001–1 μM) for 6, 12 and 48 h and then media were replaced with drug-free medium and cells cultured for additional 96 h. At the end of the experiment, cells were harvested with trypsin/EDTA and counted with a haemocytometer. Cell viability was assessed by trypan blue dye exclusion. The survival of treated cells was expressed as a percentage of control (vehicle treated) cultures. The concentration of drugs that reduced cell survival by 50% (IC50) as compared to controls was calculated. Results are expressed as

the percentage of cell proliferation vs. controls and are the mean of three separate experiments ± S.D. 2.4. In vivo experiments Briefly, N1–S1 hepatoma cells were harvested and cell viability was assessed by the trypan blue exclusion test and 1 × 106 N1–S1 monodispersed cells were orthotopically injected in the right lobe of the liver of anaesthetized Sprague–Dawley male rats after carrying out a midline laparatomy. The animals were randomly divided into nine groups. After 24 h, the nine animals per group were gavaged with vehicle (peanut oil, control

Table 1 Calculated concentrations of (NH3)2Pt(triacid), (PPh3)2Pt(dehydrocholate)2, cisplatin, carboplatin and mitoxantrone that reduced cell proliferation by 50% (IC50) vs. controls at the indicated exposure times Duration of treatment

IC50 values of drugs (mean ± S.D., μM) (NH3)2Pt(triacid)

(PPh3)2Pt(dehydrocholate)2

Cisplatin

Carboplatin

Mitoxantrone

6h 12 h 48 h

10.9 ± 0.51 4.6 ± 0.62 0.7 ± 0.05

29.6 ± 4.18 9.8 ± 4.4 3.8 ± 2.96

7.6 ± 2.76 3.8 ± 2.02 0.63 ± 0.28

19 ± 3.28 6.5 ± 2.02 1.1 ± 0.3

0.04 ± 0.01 0.008 ± 0.002 0.001 ± 0.0005

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group), (NH3)2Pt(triacid) 10–20–40–60–80 mg/kg and (PPh3)2Pt (dehydrocholate)2 20–40–80 mg/kg every other day until day 11 (Fig. 2A). For comparison, the same experimental conditions were used in an additional experiment involving five groups of nine rats inoculated with the same number of N1–S1 cells; rats were treated by intravenous injections with vehicle alone (saline), mitoxantrone 1 mg/kg (Los et al., 1990), carboplatin 30 mg/kg (Kaneko et al., 1997) or cisplatin 3 mg/kg (Authier et al., 2003) every five days until day 16 (Fig. 2B). The experimental period ended 5 days after the last injection of drug and rats were sacrificed by an anaesthetic overdose. Body weight was recorded and tumours were explanted and weighted. Results are expressed as the mean of tumour weights of the nine animals ± S.D. and as the percentage of the mean control weight. Moreover, the T / C value has been calculated as follows (Suto et al., 2006): (mean tumour weight of the treated group / mean tumour weight of the control group)× 100. 2.5. Toxicity assessment 2.5.1. Physiological status of the animals The physiological status of control and treated rats was evaluated by weighing each animal immediately before drug injection and at the end of the experiment. A general physiological evaluation was performed including the presence or absence of diarrhoea, the detection by clinical examination of the bladder size scored with Ridet's grades (0, large-sized; 2, medium-sized; 4, small-sized bladder) (Ridet et al., 2000) (as a proof of neurotoxicity), the aggressive behaviour during rat handling and toxic deaths.

followed by the Student–Newman–Keuls test, using PRISM (Version 4.0, GraphPad, San Diego, USA). The level of significance was set at P b 0.05. 3. Results 3.1. Cytotoxic effects of (NH3)2Pt(triacid) and (PPh3)2Pt (dehydrocholate)2 on rat hepatoma cells In vitro experiments (Fig. 3 and Table 1) showed a concentration and time-dependent antiproliferative activity of bileconjugated platinum complexes. Indeed, Fig. 3A demonstrates that (NH3)2Pt(triacid) is already significantly cytotoxic after a 6 hexposure (51.3 ± 4.9% vs. 100% of controls, mean ± S.D.; P b 0.05) at the concentration of 10 μM as well as after 48 h at the lower concentration of 0.1 μM (80.1 ± 5.6% vs. 100% of controls; P b 0.05). (PPh3)2Pt(dehydrocholate)2 shows a similar cytotoxic profile (Fig. 3B) although with calculated IC50 values higher than (NH3)2Pt(triacid) at all the experimental time points (Table 1). Interestingly, (NH3)2Pt(triacid) has similar effects on N1–S1 cell growth of cisplatin and carboplatin (Fig. 3C and D)

2.5.2. Motor functions In order to properly evaluate the observed motor disorders of the hindlimbs of some treated rats, the motor function was assessed at the end of the experiment in the control and treated groups by a modified Tarlov score, as previously described (Ridet et al., 2000). Briefly, each hindlimb was rated according to the following criteria: 0 1 2 3 4 5

no movement of hindlimb (complete paralysis); movement in response to hindlimb pinch; minimal spontaneous movement, no weight-bearing, no walking; ability to support weight (i.e., raise abdomen above surface), no walking; ability to support weight and to walk with spasticity and lack of coordination; normal walking.

All animals were evaluated independently and blindly by two observers. The final score was expressed as the mean value of right and left hindlimb scores. 2.6. Statistical analysis Results are reported as means ± S.D. Statistical significance of differences was assessed by analysis of variance (ANOVA),

Fig. 4. In vivo effects of multiple oral escalating doses of (NH3)2Pt(triacid) (A), (PPh3)2Pt(dehydrocholate)2 (B) and i.v. 30 mg/kg carboplatin and 1 mg/kg mitoxantrone (C) in a syngeneic, orthotopic rat hepatoma model. Columns and bars, mean values ± S.D., respectively; ⁎P b 0.05 vs. controls.

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inhibit tumour growth in a dose-dependent extent, and its therapeutic effect was significant starting with the oral dose of 10 mg/kg as compared to controls (6.94 ± 2.2 g vs. 13.85 ± 3.9 g, respectively; P b 0.05; Fig. 4A) and reaching the maximum inhibitory effect at the dose of 80 mg/kg (1.95 ± 0.5 g; Fig. 4A). In contrast, in the group of animals receiving the treatment with (PPh3)2Pt(dehydrocholate)2, the reduction in tumour weight was not significant at the tested oral doses when compared to controls (e.g. 11.72 ± 2.6 g vs. 13.85 ± 3.9 g of controls at the 80 mg/kg dose; P N 0.05; Fig. 4B). The tumour weights of the carboplatin

Fig. 5. In vivo effects of (NH3)2Pt(triacid), (PPh3)2Pt(dehydrocholate)2 and i.v. 30 mg/kg carboplatin and 1 mg/kg mitoxantrone in a syngeneic, orthotopic rat hepatoma model expressed as T / C values. T / C value = (mean tumour weight of the treated group / mean tumour weight of the control group) × 100. Columns, T / C values × 100.

with similar IC50s (Table 1). In contrast, mitoxantrone shows a much greater cytotoxic activity in all the experimental settings (Fig. 3E; Table 1) with an initial significant antiproliferative effect at the concentration of 0.0001 μM administered for 48 h (85.0 ± 4.5% vs. 100% of controls; P b 0.05). 3.2. In vivo antitumour activity of (NH3)2Pt(triacid) and (PPh3)2Pt(dehydrocholate)2 on rat hepatoma Syngeneic N1–S1 cells injected orthotopically in Sprague– Dawley rat liver grew rapidly and tumours showed a progressive enlargement in their dimensions, with a mean tumour weight of 12.56 ± 2.80 g and 13.85 ± 3.90 g reached at the end of the experimental period in control animals treated with i.v. saline and p.o. peanut oil, respectively (Fig. 4). (NH3)2Pt(triacid) was able to Table 2 In vivo antitumour effects of different drugs expressed as the percentage of the tumour weight of vehicle alone-treated rats

Vehicle alone Carboplatin 30 mg/kg Mitoxantrone 1 mg/kg (NH3)2Pt(triacid) 10 mg/kg (NH3)2Pt(triacid) 20 mg/kg (NH3)2Pt(triacid) 40 mg/kg (NH3)2Pt(triacid) 60 mg/kg (NH3)2Pt(triacid) 80 mg/kg

Mean ± S.D.

Statistical comparison

100 4.94 ± 2.38 40.2 ± 9.55 50.1 ± 15.88 36.46 ± 14.51 34.37 ± 3.61 19.49 ± 5.77 14.08 ± 3.61

a,b,c,d,e f,g,h,i,l a,f b,g c,h d,i e,l

Carboplatin 30 mg/kg vs. (NH3)2Pt(triacid) 10 mg/kg, P b 0.05. Carboplatin 30 mg/kg vs. (NH3)2Pt(triacid) 20 mg/kg, P b 0.05. c Carboplatin 30 mg/kg vs. (NH3)2Pt(triacid) 40 mg/kg, P b 0.05. d Carboplatin 30 mg/kg vs. (NH3)2Pt(triacid) 60 mg/kg, P b 0.05. e Carboplatin 30 mg/kg vs. (NH3)2Pt(triacid) 80 mg/kg, P b 0.05. f Mitoxantrone 1 mg/kg vs. (NH3)2Pt(triacid) 10 mg/kg, P N 0.05. g Mitoxantrone 1 mg/kg vs. (NH3)2Pt(triacid) 20 mg/kg, P N 0.05. h Mitoxantrone 1 mg/kg vs. (NH3)2Pt(triacid) 40 mg/kg, P N 0.05. i Mitoxantrone 1 mg/kg vs. (NH3)2Pt(triacid) 60 mg/kg, P b 0.05. l Mitoxantrone 1 mg/kg vs. (NH3)2Pt(triacid) 80 mg/kg, P b 0.05. The statistical significance of the differences was performed by ANOVA, followed by the Student–Newman–Keuls test on the relative antitumour activity in vivo (vs. 100% of controls) of the (NH3)2Pt(triacid) doses in comparison with carboplatin and mitoxantrone ones. a

b

Fig. 6. Body weight of N1–S1 tumour-bearing rats treated with (A) p.o. (NH3)2Pt(triacid) (B) p.o. (PPh3)2Pt(dehydrocholate)2 and (C) i.v. cisplatin, carboplatin and mitoxantrone compared to controls. Symbols and bars, mean values ± S.D., respectively; arrows, administration of the drug dose; ⁎P b 0.05 vs. controls.

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Table 3 Toxic effects in control and treated rats Drug

Diarrhoea

Motor function (mean Tarlov score)

Aggressive behaviour

Detection of bladder (mean Ridet grade)

Number of toxic deaths

Control ( p.o.) Vehicle (peanut oil) p.o. (n = 9)

–

5

–

4

–

(NH3)2Pt(triacid) 10 mg/kg p.o. (n = 9) 20 mg/kg p.o. (n = 9) 40 mg/kg p.o. (n = 9) 60 mg/kg p.o. (n = 9) 80 mg/kg p.o. (n = 9)

– – – – –

5 5 5 5 5

– – – – –

4 4 4 4 4

– – – – –

(PPh3)2Pt(dehydrocholate)2 20 mg/kg p.o. (n = 9) 40 mg/kg p.o. (n = 9) 80 mg/kg p.o. (n = 9)

– – –

3 2 0

+ + +

2 2 0

– – 2

Control (i.v.) Vehicle (saline) i.v. (n = 9)

–

5

–

4

–

Mitoxantrone 1 mg/kg i.v. (n = 9)

–

5

+/−

4

–

Carboplatin 30 mg/kg i.v. (n = 9)

–

5

+/−

4

–

Cisplatin 3 mg/kg i.v. (n = 9)

–

0

+

0

8

n, number of animals for single dose and control group; –, absence of manifestation; +++, maximum manifestation; i.v., intravenous; p.o., per os; for Tarlov score and Ridet grade see Materials and methods section.

and mitoxantrone i.v. treated groups showed a significant decrease during the 16-day schedule if compared to the control ones (Fig. 4C). It is noteworthy that the carboplatin i.v. administration resulted in an almost complete regression of tumour mass (0.62 ± 0.3 g vs. 12.56 ± 2.8 g of controls, P b 0.05; Fig. 4C). These results are clearly compared in Fig. 5 that shows the data as T / C values. Moreover, a statistical analysis performed on the relative antitumour activity of (NH3)2Pt(triacid) in comparison with the other cytostatics (Table 2) demonstrated that carboplatin 30 mg/ kg has a greater effect in vivo than (NH3)2Pt(triacid) at all the tested doses, whereas mitoxantrone 1 mg/kg has superimposable effects or less activity than the bile acid-conjugated platinum complex (Table 2). Interestingly, the previously published i.v. dose of 3 mg/kg cisplatin showed, in our experimental setting, a severe toxicity profile with the loss of 8 / 9 animals during the experiment (see the next paragraph) thus making not possible a quantification of its antitumour activity.

significant degree (Fig. 6B) already after the second oral administration and clear signs of neurological toxicity, including complete paralysis or minimal spontaneous movement of the hindlimbs (Tarlov score 0–2) and medium/large size bladder (Ridet grade 0–2) (Table 3). Furthermore, animals showed a marked aggressive behaviour during the experimental period and 2 / 9 died after the last dose of 80 mg/kg. Fig. 6C shows the body weight curves of carboplatin, cisplatin and mitoxantrone treated rats. Carboplatin and mitoxantrone i.v. schedules determined a severe weight loss if compared to i.v. vehicle ones; however, no clinical signs of neurological disorders or toxic deaths were noticed (Table 3). In sharp contrast, cisplatin 3 mg/kg determined the toxic death of 8 / 9 animals after the second injection. Moreover, cisplatin treated rats showed a severe neurological impairment with a marked aggressiveness already after the initial dose (Table 3). 4. Discussion

3.3. Drug-related toxicity in treated rats The toxicity profile of (NH3)2Pt(triacid) resulted extremely favourable in terms of loss of weight (Fig. 6A) and of gastrointestinal and neurological symptoms. Indeed, no hindlimb paralysis was observed at all the tested doses and no neurologic bladder dysfunctions were recorded in the animals during the treatment period (Table 3). Moreover, no toxic deaths occurred even at the highest doses. By contrast, (PPh3)2Pt(dehydrocholate)2 induced dose-dependent signs of weight loss up to a severe and

The present study provides evidence, for the first time, of the in vitro and in vivo antitumour activity of the two novel bile acidconjugated platinum complexes (NH3)2Pt(triacid) and (PPh3)2Pt (dehydrocholate)2 on a syngeneic and orthotopic hepatoma model. In particular, (NH3)2Pt(triacid) demonstrated both a high cytotoxic activity on N1–S1 hepatoma cells and a significant in vivo antitumour effect without signs of toxic effects. Recently, research attention has been focused on platinum complexes as potential anticancer drugs because of the success

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of cisplatin and carboplatin. Biological carriers conjugated to cisplatin analogues have experimentally improved organotropism and specificity for tumour tissue, reducing side effects and drug resistance (Zhang and Lippard, 2003). Among new metal complexes with potential therapeutic effects, the orally active platinum compounds have attracted a great interest for their administration flexibility and antitumour activity comparable to those parenterally administered (Ho et al., 2003). Indeed satraplatin, an oral platinum (IV) complex, is currently undergoing phase III clinical trials in Europe for ovarian and hormone-refractory prostate cancer (Sternberg et al., 2005). Based on this background, we have tested in vitro and in vivo two novel bile acid-conjugated platinum complexes that have been orally administered in a rat hepatoma model. In vitro antiproliferative activity of bile acid-conjugated platinum complexes has been previously reported on human leukemic K562 cells (Bianchi et al., 2000) and on rat hepatoma McA-RH7777 cells or on mouse hepatoma Hepa 1–6 cells (Marin et al., 1998b). In particular, Bamet-R2 and Bamet-UD2 revealed a strong in vitro cytotoxic and proapoptotic effect (Monte et al., 2005, 1999). Both (NH3)2Pt(triacid) and (PPh3)2Pt(dehydrocholate)2 showed high cytotoxic effects on hepatoma cells similar to those determined by cisplatin and carboplatin. These data seem to confirm the activity of bile acidconjugated platinum compounds for the experimental treatment of liver carcinomas. Moreover, results from in vitro experiments have shown the capacity of liver-derived tumour cells to take up bile acids and their derivatives (Monte et al., 1999). Bile acids can be considered as carriers for drugs toward hepatocytes and liver in general because they are taken up by hepatocytes and undergo enterohepatic recirculation (Dominguez et al., 2001). Bamet compounds demonstrated a significant antitumour activity in orthotopically implanted hepatomas in mouse liver with a low toxicity profile (Dominguez et al., 2001). It has been demonstrated that the extent of the cisplatininduced cytostatic effect is closely associated with the presence of platinum in the tumour (Ishikawa et al., 1996). Therefore, the efficiency of the drug delivery system is a key factor in the usefulness of platinum-based chemotherapy. The enhanced uptake of Bamet compounds by liver tumour cells seems to be consistent with the existence in these cells of transport systems for cholephilic compounds (Dominguez et al., 2001). The significant in vivo effect of the bile acid-conjugated platinum complex (NH3)2Pt(triacid) compared also to mitoxantrone could be explained with the possible chemical splitting of the bile acid-conjugates. Indeed, the orally administered compound can be directly carried to the liver and split, determining high concentrations of platinum inside hepatomas with a nonreversible formation of adducts with DNA in proliferating cells. Similarly, oral (NH3)2Pt(triacid) dose-dependently inhibited liver tumour growth without a significant toxicity, particularly the neurological one, even at the highest dose. Interestingly, the therapeutic effects were similar to those obtained with carboplatin and mitoxantrone but with a lower weight loss. In contrast, despite the promising in vitro results, (PPh3)2Pt(dehydrocholate)2 showed no significant antitumour activity with a marked neurological and general toxicity. In previous animal studies, behavioural assess-

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ments have shown sensory (thermal hypoalgesia) and motor (coordination and motor force decrease) disorders after repeated injections of cisplatin in rats and mice, including a significant decrease in body weight (Authier et al., 2003). Platinated cholic acid derivatives showed to induce apoptosis in human testicular cancer cell line (Paschke et al., 2003) and, more recently, Monte et al. (2005) have demonstrated that Bamet compounds have a strong cytostatic activity due to the combination of a very low pronecrotic and a marked proapoptotic effect in vitro. The cytostatic effect due to the induction of necrosis may lead to the appearance in vivo of inflammation-associated side effects, whereas a proapoptotic effect of these compounds could account for a high activity combined to a very low toxicity to the liver, kidney and nervous system. These previously published data could help to elucidate our findings. Indeed, the bile acidconjugated platinum complex (NH3)2Pt(triacid) demonstrated a cytostatic activity in vitro probably due to its high proapoptotic effects on hepatoma cells; this event could explain its high antitumour activity with a very low toxicity profile in vivo. In conclusion, the significant antitumour activity of the oral bile acid-conjugated platinum complex (NH3)2Pt(triacid) together with its lack of toxicity may represent an orally active agent for future development in the treatment of human liver tumours. Acknowledgements This work was performed under an unrestricted research grant from Abiogen (Pisa, Italy). The authors thank Dr. Fabio Baschiera for the helpful experimental suggestions and Bruno Stacchini for his excellent technical assistance. References Authier, N., Gillet, J.P., Fialip, J., Eschalier, A., Coudore, F., 2003. An animal model of nociceptive peripheral neuropathy following repeated cisplatin injections. Exp. Neurol. 182, 12–20. Bianchi, N., Ongaro, F., Chiarabelli, C., Gualandi, L., Mischiati, C., Bergamini, P., Gambari, R., 2000. Induction of erythroid differentiation of human K562 cells by cisplatin analogs. Biochem. Pharmacol. 60, 31–40. Criado, J.J., Herrera, M.C., Palomero, M.F., Medarde, M., Rodriguez, E., Marin, J.J., 1997a. Synthesis and characterization of a new bile acid and platinum (II) complex with cytostatic activity. J. Lipid Res. 38, 1022–1032. Criado, J.J., Macias, R.I., Medarde, M., Monte, M.J., Serrano, M.A., Marin, J.J., 1997b. Synthesis and characterization of the new cytostatic complex cisdiammineplatinum(II)-chlorocholylglycinate. Bioconjug. Chem. 8, 453–458. Dominguez, M.F., Macias, R.I., Izco-Basurko, I., de La Fuente, A., Pascual, M.J., Criado, J.M., Monte, M.J., Yajeya, J., Marin, J.J., 2001. Low in vivo toxicity of a novel cisplatinursodeoxycholic derivative (Bamet-UD2) with enhanced cytostatic activity versus liver tumors. J. Pharmacol. Exp. Ther. 297, 1106–1112. Galanski, M., Jakupec, M.A., Keppler, B.K., 2005. Update of the preclinical situation of anticancer platinum complexes: novel design strategies and innovative analytical approaches. Curr. Med. Chem. 12, 2075–2094. Haller, D.G., 2004. Recent updates in the clinical use of platinum compounds for the treatment of gastrointestinal cancers. Semin. Oncol. 31, 10–16. Ho, Y.P., Au-Yeung, S.C., To, K.K., 2003. Platinum-based anticancer agents: innovative design strategies and biological perspectives. Med. Res. Rev. 23, 633–655. Ishikawa, H., Kikkawa, F., Tamakoshi, K., Matsuzawa, K., Kawai, M., Suganuma, N., Tomoda, Y., 1996. Distribution of platinum in human gynecologic tissues and lymph nodes after intravenous and intraarterial neoadjuvant chemotherapy. Anticancer Res. 16, 3849–3853.

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