Introduction

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The undesired side effects of cisplatin, cis-diamminedichloroplatinum(II), mainly, nephrotoxicity, myelotoxicity, and neuropathy (Lipp and Bokemeyer, 1999), often limit the usefulness of this powerful cytostatic drug against solid tumors (Loeher and Einhorn, 1984). This has encouraged the search for improved cytostatic derivatives with lower toxicity to extratumoral tissues. Although many different compounds have been synthesized, few of them are currently used in clinical practice (Bradner et al., 1980). The design of drugs targeting solely the tumor cell population is one of the main goals in the field of modern cancer chemotherapy. In this context, several strategies have been investigated; among them, the usefulness of organotropic molecules to target DNA-reactive platinum(II)-containing drugs to the desired organ where the tumor is located or to enhance drug elimination from the body (Macias et al., 1998, 1999). Bile acids are endogenous steroids synthesized by the liver. Owing to specific carrier proteins located in the plasma membrane of both liver and ileal cells (Meier, 1995), bile acids remain in the so-called enterohepatic circulation, which determines minor daily fecal loss and permits the maintenance of very low concentrations of these compounds in the systemic blood (Hofmann, 1994). The liver and intestinal organotropism of bile acids has prompted several investigators to propose the possible usefulness of this interesting characteristic for the use of bile acids or their analogs as shuttles for drugs toward tissues located in the enterohepatic circulation (Ho, 1987; Betebenner et al., 1991; Stephan et al., 1992; Kramer and Wess, 1996; Monte et al., 1999). In this sense, by binding molecules containing a transition metal to bile acids our group has synthesized and characterized several members of a new family of compounds named Bamets with cytostatic activity (Marin et al., 1998). Previous preclinical investigation of two of the most promising compounds of the Bamet family reported to date, Bamet-R2 (Criado et al., 1997) and Bamet-UD2 (Criado et al., 2000), revealed that these compounds maintain both the liver organotropism of bile acids (Macias et al., 1998; Larena et al., 2001) and the strong cytostatic effect of cisplatin (Marin et al., 1998; Martinez-Diez et al., 2000). Moreover, ursodeoxycholic acid (UDCA) increases hepatocyte levels of glutathione and thiol-containing proteins, which may account for hepatoprotective effect of UDCA against common mechanisms of liver damage such as oxidative injury (Mitsuyoshi et al., 1999; Trauner and Graziadei, 1999). Therefore, although the effect of UDCA on cisplatin-induced toxicity has not been established, the possibility that hepatoprotective properties of the leaving moiety in Bamet-UD2, i.e., UDCA, may endow the complex with additional beneficial properties cannot be ruled out. Results from in vitro experiments carried out by others (Kullak-Ublick et al., 1997) and us (Monte et al., 1999) have afforded evidence for the capacity of liver-derived tumor cells to take up bile acids and their derivatives. Because the prolonged tissue retention of platinum is relevant to long-term toxicity (Tothill et al., 1992), the aim of this work was to further evaluate at preclinical level Bamet-R2 and Bamet-UD2 effectiveness on orthotopically located liver tumors in vivo. Accumulation of these drugs in several different tissues, including the tumor was checked and nephrotoxicity, hepatotoxicity, myelotoxicity, and neurotoxicity was evaluated after repeated doses simulating chemotherapeutic treatment in tumor-free rats.

	Materials and Methods

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Chemicals. Cisplatin and Dulbecco's modified Eagle's medium were purchased from Sigma Chemical Co. (St. Louis, MO). Bamet-R2 and Bamet-UD2 were synthesized and chemically characterized as previously reported (Criado et al., 1997, 2000). All other reagents were from Merck (Darmstadt, Germany).

Animals and Cells. Male Wistar rats were obtained at 4 weeks of age from the Animal House at the University of Salamanca, Spain. Male Nude mice (Ico: Swiss nu/nu) were from Iffa Credo (Barcelona, Spain) and were maintained under pathogen-free conditions. Animals were fed on commercial rat or mouse pelleted food from Panlab (Madrid, Spain), as appropriate, and water ad libitum. Temperature (20°C) and the light/dark cycle (12:12 h) in the room were controlled. All animals were handled in accordance with recommendations of the University of Salamanca Animal Care Committee, which are based on the Guide for the Care and Use of Laboratory Animals (National Institute of Health Publication 80-23, revised 1985). The mouse hepatoma cell line Hepa 1-6 was obtained from the American Type Culture Collection (Rockville, MD) and was cultured in DMEM supplemented with 2 mM glutamine, 25 mM glucose, 26.2 mM NaHCO₃, 25 mM Hepes, 10% fetal calf serum, and antibiotics, in a CO₂, air (5:95%) atmosphere at 37°C.

In Vivo Antitumor Activity. Hepa 1-6 mouse hepatoma cells cultured as described above were harvested. Cell viability was assessed by the trypan blue exclusion test, and 10⁷ monodispersed cells were injected subcutaneously into the back of an athymic Nude mouse used as host. After 2 weeks, the subcutaneous tumor (2 cm in diameter) was surgically removed and cut into small cubic fragments of approximately 1 mm³. These were implanted in the liver of different animals following an adaptation of previously described methods (Yang et al., 1992). In brief, mice were anesthetized by i.p. injection of sterile pentobarbital solution (50 mg/kg of body weight, Nembutal; Abbot, Madrid, Spain). A ventral laparotomy was carried out and the left lateral lobe of the liver exposed. A small superficial incision in the liver was made with an 11 surgical blade at an angle of 30° to the liver surface. A piece of absorbable gelatin sponge was placed in the incision. After 1 to 2 min, the hemostatic sponge was removed and one of the 1-mm³ tumor fragments was inserted into the pocket. The hepatic lobe was returned to the peritoneal cavity and the abdominal wall was closed. The animals were randomly divided into five groups, including at least six mice per group that were treated by i.p. injection of 10 doses (on days 1, 4, 8, 12, 16, 20, 24, 28, 32, and 36 after liver implantation of tumor cells) of sterile Bamet-R2 (15 nmol/g of body weight), Bamet-UD2 (15 nmol/g of body weight), or cisplatin (5 or 15 nmol/g of body weight) dissolved in sterile 150 mM NaCl. Owing to its low solubility in this medium, Bamet-UD2 was administered as a suspension. A control group received only the vehicle. Intraperitoneal administration was chosen on the basis of previous investigations that revealed that following a single intravenous injection of Bamet-R2 (Macias et al., 1999) or Bamet-UD2 (Larena et al., 2001) liver uptake and excretion into bile with no major biotransformation was very efficient, although not so efficient as that for bile acids. This considerably reduces, although does not rule out, the existence of difference in the magnitude of the effect of similar doses of Bamets when administered i.p. instead of i.v. due to a first-pass effect. Animal survival was monitored daily and the mean survival time (MST) was calculated for each experimental group. The T/C value, the MST of the treated animals divided by that of the control animals, was determined. To investigate the evolution of the implanted liver tumors, six animals from each group were sacrificed on day 20, tumor size was measured with a sliding caliper, and tumor volume was calculated by the formula (length × width²)/2 (Carlsson et al., 1983). Tumor and liver tissue samples were collected from these animals to carry out measurements of platinum contents.

Evaluation of Toxicity in Rats. The therapeutic protocol used in the mice was simulated in male Wistar rats to evaluate several aspects of the potential toxicity of these compounds. Tumor-free rats received i.p. administration of 10 doses (once every 4 days) of 7.5 nmol/g of body weight cisplatin or Bamet-R2 dissolved in sterile saline or Bamet-UD2 as a suspension. Control animals received only the vehicle. Neurotoxicity was evaluated using an electrophysiological technique that allows detection of changes in nerve conduction velocity (De Koning et al., 1987). The method was a modification of that previously reported by Stanley (1981), and consisted in stimulating the sciatic nerve and recording electromyographic (EMG) responses from the plantar muscle. The first response, referred to as the M-wave, results from direct stimulation of motor axons, whereas the second, or H-wave, results from indirect stimulation of motoneurons mediated by sensory fibers. Nerve conduction velocity was determined in the animals before starting treatment (4 weeks of age) and at 7, 9, and 10 weeks of age. Recordings were carried out in the left hind limb of i.p.-anesthetized animals (pentobarbital, 50 mg/kg of body weight, Nembutal). The responses were evoked by percutaneous stimulation with a needle electrode (25-gauge) using a 1-ms square wave. EMG responses in the plantar muscle were recorded with a surface electrode. The system was grounded by a piece of braided wire wrapped around the paw proximal to the recording electrode. The signal was amplified, filtered, and recorded on a computerized oscilloscope system. The recording procedure was completed within 15 min and animals were allowed to recover from anesthesia in a warmed cabinet. To calculate nerve conduction velocity, the latency to the maximum amplitude point of the M-wave and the distance between the nerve stimulation site and the EMG recording electrode with the limb fully extended were measured. At the end of the experimental protocol, animals were anesthetized again, blood samples were collected from the cava vein, and tissue samples were extracted and weighed.

Analytical and Statistical Methods. Creatinine and urea levels were measured in serum and urine to investigate renal function integrity. Other parameters indicative of the general health state of the animals were measured in blood, serum, and urine by routine automated methods used in clinical chemistry (Coulter maxm; Izasa, Madrid, Spain, and Hitachi 747; Roche, Barcelona, Spain). After digesting the samples with nitric acid, platinum contents were measured by flameless atomic absorption spectrophotometry (Z-8100 Polarized Zeeman apparatus with a graphite furnace; Hitachi, Pacisa, Madrid, Spain). Results are expressed as means ± S.E. To calculate the statistical significance of differences among groups, the Bonferroni method of multiple range testing or the paired t test was used as appropriate. Statistical analyses were performed on a Macintosh PowerPC 6200/200 computer (Apple Computer, Inc., Cupertino, CA).

	Results

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Antitumor Effect on Nude Mice. Following implantation of mouse hepatoma Hepa 1-6 cells in the liver of Nude mice, 100% of the animals developed a single ovoid nodule of approximately 0.5 cm in diameter on day 14 (Fig. 1), which thereafter increased in size (Fig. 2). Features typically seen in some patients with liver tumors were also observed in this experimental model. In mice that received only vehicle (control group), tumor evolution was characterized by progressive local tumor growth, followed by regional invasion and spreading to the peritoneal cavity, intestine, kidney, and lung, and the development of bloody ascitis. The observations made in animals sacrificed 3 weeks after implantation are shown in Table 1. The cause of death, which occurred in the untreated group on approximately day 26 (Table 1), was attributable to multiple organ failure, mainly involving the liver because signs of jaundice and cachexia were observed on the days prior to death. This model was therefore considered as a suitable one to evaluate the antitumor activity against "in situ" liver tumors of Bamet-R2 and Bamet-UD2.

View larger version (131K): [in this window] [in a new window]   Fig. 1.   Single tumor in the liver of a Nude mouse in which a fragment of approximately 1 mm3 of Hepa 1-6 mouse hepatoma tumor was implanted 14 days previously.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Tumor size 20 days after orthotopic implantation of a fragment of approximately 1 mm3 of Hepa 1-6 mouse hepatoma tumor in Nude mice. The animals (six per group) were treated by repeated i.p. injection of either the vehicle (sterile 150 mM NaCl) in the control group or Bamet-R2, Bamet-UD2, or cisplatin at the indicated dose on days 1, 4, 8, 12, and 16. Results are expressed as means + S.E. *p < 0.05 compared with the control group by the Bonferroni method of multiple range testing.

View this table: [in this window] [in a new window]   TABLE 1 Tumor evolution and survival of tested mice Maximal survival, MST, and T/C values as well as body weight (total weight  tumor weight), and the presence of ascitis and metastasis in the indicated organs on day 20 following 1-mm3 Hepa 1-6 tumor implantation in the liver of nude mice, which were afterwards treated with 10 i.p. doses (once every 4 days) of saline (control), cisplatin, Bamet-R2, or Bamet-UD2 at the indicated doses. Values are expressed as means ± S.E.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Kaplan-Meier curves for the survival, as percentages of the initial number of animals per group, following orthotopic implantation of a fragment of approximately 1 mm3 of mouse Hepa 1-6 hepatoma tumor in Nude mice. The animals were treated i.p. once every 4 days with either 15 nmol/g of body weight of Bamet-R2 (n = 6), Bamet-UD2 (n = 8), or cisplatin (n = 11) or 5 nmol/g of body weight cisplatin (n = 5). The control group (n = 10) received only vehicle (sterile saline). Treatment was maintained for 5 weeks.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Normal liver tissue and tumor platinum contents 20 days after orthotopic implantation of a fragment of approximately 1 mm3 of Hepa 1-6 mouse hepatoma tumor in Nude mice. The animals (six per group) were treated by repeated i.p. injection of either Bamet-R2, Bamet-UD2, or cisplatin at the indicated dose on days 1, 4, 8, 12, and 16. Values are expressed as means ± S.E. Results in tumor tissue were compared with those obtained in normal liver tissue by the paired t test and with those obtained in the group treated with cisplatin at 15 nmol/g of body weight by the Bonferroni method of multiple range testing. N.S., p > 0.05; *p < 0.05.

Toxicity and Tissue Accumulation of Drugs in Rats. To study the toxicity of Bamet-R2 and Bamet-UD2 in comparison to cisplatin, rats with no implanted tumors were preferred as an experimental model for two reasons. On one hand, electrophysiological measurements and the collection of urine and serum samples were much easier. On the other hand, the absence of tumor prevented the appearance of artifacts in the parameters used to evaluate the health state of the animals. Nerve conduction velocity was calculated by measuring of the delay in the time elapsed between stimulation (S) and response (M) along a segment of known length in the rat sciatic nerve (Fig. 5A). Nerve conduction velocity increased in normal rats between 4 and 10 weeks of age (Fig. 5B). This normal development was not impaired by treatment of the animals with Bamet-R2 or Bamet-UD2 but was abolished by treatment with cisplatin (Fig. 5B). Clear signs of toxicity to the bone marrow, such as decreased numbers of leukocytes and platelets were observed in animals receiving cisplatin, but not Bamet-R2 or Bamet-UD2 (Table 2). Serum total bilirubin, alkaline phosphatase, or glutamic-oxalacetic transaminase-aspartate aminotransferase and glutamic-pyruvic transaminase-alanine aminotransferase transaminase levels were not significantly elevated in any group (Table 2). By contrast, signs of severe kidney damage were observed in rats treated with cisplatin (Table 2). Urea and creatinine concentrations were decreased in urine and increased in serum. The urinary excretion of Na⁺, K⁺, and Cl was also altered. Taking together, these results can presumably be accounted for by a significant reduction in kidney function, as revealed by the decreased creatinine clearance (Table 2). A more moderate degree of nephrotoxicity was also observed in animals receiving Bamet-R2. By contrast, no signs of kidney impairment were seen in rats treated with Bamet-UD2 (Table 2).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effect of cisplatin, Bamet-R2, and Bamet-UD2 on the age-related evolution of nerve conduction velocity in rats. A, typical recording of nerve conduction velocity measurements. B, time course of the development of nerve conduction velocity in rats following i.p. treatment with 10 doses (once every 4 days) of cisplatin, Bamet-R2 or Bamet-UD2 (7.5 nmol/g of body weight). Results are expressed as means ± S. E., p < 0.05, compared with initial nerve conduction velocity by the paired t test. *p < 0.05 compared with the control group by the Bonferroni method of multiple range testing. S, stimulus; M, direct response; H, indirect response.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Platinum content in several rat tissues following i.p. treatment with 10 doses (once every 4 days) of cisplatin, Bamet-R2, or Bamet-UD2 (7.5 nmol/g of body weight). Results are expressed as means ± S.E.; n = 5 for each group. *p < 0.05 compared with the group receiving cisplatin by the Bonferroni method of multiple range testing. The inset is a magnification of the results obtained in tissues with low platinum contents.

	Discussion

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Tumor xenografts in several animal models, such as the one used in the present work, (i.e., the immunodepressed nude mouse lacking thymus and hence T cells), have been used in many relevant investigations aimed at gaining insight into the clinical activity of new anticancer drugs because the implanted cells retain the characteristics of the parental ones. This accounts for the good correlation seen between the tumor response to antineoplastic drugs in this animal model and the situation in clinical practice (Unger, 1996). Our results support that intrahepatic implantation of mouse Hepa 1-6 hepatoma is a valuable experimental tool for the assessment of the inhibitory effect of drugs with liver vectoriality, such as Bamet-R2 and Bamet-UD2, on liver tumor growth.

In this model, the life span of tumor-bearing mice is usually measured as MST and compared in both treated (T) and nontreated control (C) groups. The results can therefore be expressed as T/C (%). According to the National Cancer Institute it is generally accepted that when a T/C value of 135% is obtained in preclinical experiments, similar to those included in the present study, positive indication of antitumor activity can be assigned to the tested drug. Based on this criterion, the results of the present work clearly point to the beneficial effect of two cytostatic agents that were obtained by binding cisplatin to different bile acid moieties. Both Bamet-R2 and Bamet-UD2 were effective in reducing liver tumor growth whereas low or no signs of toxic effects on the organs normally affected by treatment with cisplatin and its derivatives were seen. The present and previous results from our laboratory (Marin et al., 1998; Macias et al., 1999), suggest that the beneficial effects of these Bamets can be accounted for by a reduced drug accumulation in tissues other than those against which they are directed. These are the liver tumor, in which they are expected to exert their activity (Marin et al., 1998), and the normal parenchymal liver tissue, which is expected to efficiently eliminate the drug from the body, first by taking up the drug from plasma, then by secreting it into the bile, and finally by eliminating it into feces (Macias et al., 1999).

It has been demonstrated in several solid tumors that the extent of the cisplatin-induced cytostatic effect is closely associated with the presence of platinum in the tumor (Ishikawa et al., 1996). Thus, the efficiency of the drug delivery system is a key factor in the overall usefulness of the chemotherapy. The enhanced uptake of both Bamet-R2 and Bamet-UD2 by liver tumor cells compared with cisplatin is consistent with the existence in liver-derived tumor cells of transport systems for cholephilic compounds other than sodium-dependent specific bile acid transport systems of the NTCP family. These carriers, probably belonging to the organic anion-transporting polypeptide family (Buscher et al., 1988; Von Dippe and Levy, 1990; Marchegiano et al., 1992; Kullak-Ublick et al., 1996; Monte et al., 1999), are also able to take up bile acid derivatives. We believe that Bamets do not enter cells through NTCP. It has been shown that unchanged negatively charged side chain of bile acid moiety, which is not the case for Bamets, is important for bile acid derivatives to be transported by both NTCP and ileal bile acid transporter (Kramer et al., 1993). This is consistent with previously reported results by our group (Monte et al., 1999), which revealed that uptake of Bamet-R2 by hepatocytes was lower than that of glycocholic acid. By contrast, in tumor liver cells that were isolated during chemically induced liver carcinogenesis, similar uptake of Bamet-R2 and glycocholic acid was found. Moreover, this was also similar to Bamet-R2 uptake by normal hepatocytes. These results suggested that the efficiency of transport systems present in liver tumor cells, and therefore probably different from NTCP, responsible for Bamet uptake is lower than that of carriers accounting for bile acid uptake in normal hepatocytes but much higher than that of processes involved in cisplatin uptake. Although a similar degree of drug targeting to liver tumor was achieved with both Bamet-UD2 and Bamet-R2, the antitumor activity of the former was stronger than that of the latter. Indeed, the in vivo cytostatic capacity of Bamet-UD2 was similar to that of cisplatin. Higher DNA reactivity of cisplatin compared with Bamet-UD2 (Martinez-Diez et al., 2000) probably accounts for the fact that reduction in tumor size was similar for both drugs in spite of the lower amount of cisplatin than Bamet-UD2 found in tumor tissue. However, the overall beneficial effect of Bamet-UD2 was more marked than that of either Bamet-R2 or cisplatin. This was in part due to the absence of Bamet-UD2-induced toxicity on kidney, liver, bone marrow, or nerve. Several studies carried out on patients receiving cisplatin treatment have reported platinum accumulation in many different tissues. The highest levels were found in kidney and liver (Lange et al., 1973). As with its antitumor effect, cisplatin-induced side effects are closely associated with accumulation of the metal in the affected organ. For all assayed compounds the retention of platinum expressed as the total amount was higher in liver than in kidney. However, hepatotoxicity is a rare clinical problem in cisplatin treatment. This is probably due to the high detoxifying capability of this organ. No signs of toxic effect of either Bamet-UD2 or Bamet-R2 on the liver were found in the present work. By contrast, nephrotoxicity is a major problem in patients treated with cisplatin. Our results confirm the previously reported (McKeage et al., 1993) nephrotoxicity of cisplatin in rodents, although the exact mechanism by which cisplatin produces renal damage is still unknown. Although impairment of the S3 segments was first believed to be the cause of cisplatin-induced nephrotoxicity, changes in the function and structure of the entire nephron have been suggested to occur (Sheikh-Hamad et al., 1997). The functional data collected in the present work point to a mild degree of nephrotoxicity of Bamet-R2 and the absence of this side effect for Bamet-UD2. Blood analyses indicated that this was also the case of the sensitivity of bone marrow function to these compounds. Another important dose-dependent side effect of cisplatin is the development of peripheral neuropathy (Thompson et al., 1984). This is of axonal nature and mainly affects large myelinated fibers, which is responsible for predominant sensory alterations, although electrophysiological studies have also established the involvement of motor nerves (Chaudhry et al., 1994). A linear relationship has been observed between platinum levels and the cumulative doses of cisplatin, the highest platinum levels being found in patients with clinical and histopathological evidence of neurotoxicity (Gregg et al., 1992). Our results confirm both the accumulation and the neurotoxic effect of cisplatin in rodents, which contrasts with the reduced amount of platinum in nerves and the absence of neurotoxicity in animals treated with either Bamet-R2 or Bamet-UD2. Recent developments in supportive care for patients receiving cancer chemotherapy have focused on attempts to provide selective protection of normal tissues from the toxicity of the antineoplastic agent without impairing the cytostatic effectiveness of the therapy. In this regard, Bamet-UD2 meets both requirements. This prodrug results in both a free molecule of ursodeoxycholic acid and another one carrying the DNA-reactive group, i.e., ursodeoxycholate-diammine aquo platinum(II). The interesting possibility that the ability of the leaving ursodeoxycholic acid moiety from the Bamet-UD2 complex to increase liver protection against oxidative stress (Mitsuyoshi et al., 1999) may play a role in preventing the injury to normal cells exposed to the active strong anticancer form of ursodeoxycholic acid moiety bound to cisplatin cannot be ruled out. When possible, liver resection is considered to be the best therapy available for patients with hepatocellular carcinoma or hepatic metastasis from colorectal cancer. However, even in these cases, the high rate of recurrence recommends the use of adjuvant chemotherapy after resection (Kemeny et al., 1993; Bignami et al., 1995). The systemic toxicity of these types of treatment represents an additional risk factor for patient outcome. The strong antitumor activity of Bamet-UD2 together with its lack of toxicity may provide a valuable pharmacological tool for the design of future strategies aimed at treating unresectable liver tumors or for use in adjuvant treatments.