Cancer Therapy Vol 3, 57-64, 2005

 

Targeting of cytostatic bile acid derivatives toward tumours of the enterohepatic circuit

Review Article

 

Jose J.G. Marin*, Marta R. Romero, Marta Vallejo, Maria J. Monte

Laboratory of Experimental Hepatology and Drug Targeting, University of Salamanca, Salamanca, Spain

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*Correspondence: Dr. Jose J.G. Marin, Department of Physiology and Pharmacology, Campus Miguel de Unamuno, E.D. S-09, 37007-Salamanca, Spain; Phone: 34 923 294674; Fax: 34 923 294669; E-mail: jjgmarin@usal.es

Key words: Cancer, Chemotherapy, Drug targeting, Intestine, Liver

Abbreviations: bile salt export pump, (BSEP); cis-diammine-bisursodeoxycholate-platinum(II), (Bamet-UD2); cis-diamminechloro-cholylglycinate-platinum(II), (Bamet-R2); ileal bile acid transporter, (IBAT); Na+-taurocholate cotransporting polypeptide (NTCP); organic anion-transporting polypeptides, (OATP)

 

Received: 8 February 2005; Accepted: 11 February 2005; electronically published: February 2005

 

Summary

Two important limitations to the chemotherapy available for the treatment of tumours of the enterohepatic circuit are low patient response to cytostatic agents, together with the appearance of undesirable side effects. One interesting strategy to overcome these drawbacks consists of using drugs with enhanced tumour cell selectivity. In this sense, based upon the existence in the plasma membrane of hepatocytes, cholangiocytes and cells of the intestinal mucosa of proteins able to carry out efficient uptake of bile acids and their derivatives, several targeted agents have been synthesized by coupling cytostatic moieties to these natural acid steroids. The best results in preclinical screenings have been obtained for cisplatin derivatives such as cis-diamminechloro-cholylglycinate-platinum(II) (Bamet-R2) and, in particular, cis-diammine-bisursodeoxycholate-platinum(II) (Bamet-UD2). These compounds are efficiently incorporated into hepatic and intestinal tumour cells and they are also taken up and efficiently excreted into bile by normal hepatocytes. The potential clinical usefulness of Bamets as anti-tumour agents is based on their strong ability to form adducts with DNA, which results in pro-apoptotic and cytostatic activity and in vivo anti-tumour effects, together with enhanced liver and intestinal organotropism, which probably accounts for minor non-specific toxicity and a marked overall increase in the life span of nude mice bearing liver implants of wild-type or cisplatin-resistant liver or colon tumours. This beneficial effect is enhanced by the encapsulation of Bamets into liposomes. In conclusion, novel cytostatic agents with enhanced targeting towards enterohepatic tumours are now available for further investigations in the preclinical stage and, eventually, in clinical trials.

 

I. Introduction

A large proportion of diagnosed cancers are found in tissues of the enterohepatic circuit, which includes the liver, the biliary tree, the gallbladder and the intestine, and the incidence of some of them, such as hepatocellular carcinoma, is rapidly increasing (Cahill et al, 2004). In addition, their prognosis is usually very poor and some of them are quite difficult to treat (Jarnagin et al, 2003; O'Connell et al, 2003; Si et al, 2003). Current therapy for these types of tumour includes surgery, when feasible, and this is often combined with radiotherapy and/or chemotherapy (Cahill et al, 2004). However, the latter alternative very often faces two major drawbacks that seriously limit its clinical usefulness. These are the appearance of noxious side effects and the development of resistance to the cytostatic drug (Canon et al, 1990). Thus, in order to treat tumours of the enterohepatic circuit, more efficient anticancer drugs are needed. One way to increase the beneficial characteristics of novel drugs is to enhance their cell selectivity.

The objective of drug targeting, especially for cancer chemotherapy, is to enhance the effectiveness of the drug by concentrating it at the target site and minimizing its effects at non-target sites. This is one of the main aims of modern research in the field of gut and liver cancer chemotherapy. To achieve this goal, many different strategies have been devised and several targeting systems, usually based on the biological properties of several molecules coupled to cytostatic agents, have been proposed.

Examples of research into intestinal drug targeting include studies on the usefulness of cisplatin-encapsulated transferrin-polyethylene glycol liposomes to treat the peritoneal dissemination of gastric cancer (Iinuma et al, 2002). Moreover, several targeted drug delivery systems for the delivery of anticancer drugs to the colon have been reviewed recently (Minko, 2004). One of these possibilities is the targeting/drug delivery of celecoxib using guar gum as a carrier (Krishnaiah et al, 2002). Regarding liver targeting, owing to the existence in parenchymal hepatic cells of specific plasma membrane receptors for asialoglycoproteins there exists the posibility of coupling antitumour drugs to moieties containing branched galactose residues (Yu et al, 2000; Ohya et al, 2001; Wu et al, 2002). Other interesting alternatives include albumin-conjugates (Stehle et al, 1997), combinations with biotinyl saccharides (Ouchi et al, 2004), the gelatin powder (Gelfoam) used in regional chemotherapy (Pohlen et al, 2001) and nanoparticles (Yamada et al, 2003). Intracellular targets have been also explored, and include the use of anti-sense oligonucleotides and ribozymes (Huesker et al, 2002). Furthermore, plasma membrane proteins expressed in both hepatic and intestinal cells and involved in the efficient uptake of the so-called cholephilic organic anions, such as bile acids (Meier et al, 2002), have been investigated by our group, and others, for use as molecular targets for directing anticancer drugs towards tumours of the enterohepatic circuit (Marin et al, 2001).

 

II. Enterohepatic organotropism of bile acid derivatives

Bile acids are synthesized from cholesterol in hepatocytes. Proteins of the superfamily of ATP binding cassette proteins or ABC proteins, such as the bile salt export pump (BSEP, gene symbol ABCB11) are involved in bile acid secretion into bile (Meier et al, 2002). Most bile acid molecules secreted into bile are re-absorbed at the intestine, mainly in the ileum, by very efficient uptake mechanisms, including the ileal bile acid transporter (IBAT, gene symbol SLC10A2). Thus, a minor daily faecal loss (approximately 5% of the total bile acid pool) occurs, which is replaced by de novo synthesis. The sodium-dependent transporter NTCP (gene symbol SLC10A1), and several members of the organic anion-transporting polypeptides (OATP) family, in particular OATP-C or according to the new nomenclature (Hagenbuch et al, 2004) OATP1B1 (gene symbol SLCO1B1), and OATP8 or OATP1B3 (gene symbol SLCO1B3) are responsible for the efficient uptake of bile acids from sinusoidal blood (Meier et al, 2002). As a result, only a small proportion of the bile acid pool escapes from the enterohepatic circuit to the systemic circulation, where bile acid concentrations are usually lower than 10 mM.

The marked enterohepatic organotropism of these compounds has prompted several investigators to propose the possible usefulness of bile acids or their analogues as shuttles for drugs towards tissues located in the enterohepatic circuit (Ho, 1987; Betebenner et al, 1991; Stephan et al, 1992; Kramer et al, 1996). The most evident advantage of this strategy in cancer chemotherapy is that selective drug uptake will be enhanced if the resulting molecule is recognized as a substrate by plasma membrane carrier proteins expressed in tumours of the enterohepatic circuit. Therefore, a key aspect in designing these new drugs is to know at exactly which point of the bile acid molecule the active agent should be bound.

In principle, the possibilities for conjugating a drug to a bile acid include the hydroxyl groups, in particular the one located at the 3a-position, and the carboxyl group on the side chain (Figure 1).

 

 

Figure 1. Schematic representation of a typical bile acid (cholic acid) showing potential positions for coupling to pharmacologically active moieties. (A) Chemical structure (3a, 7a, 12a-trihydroxy-5b-cholanoic acid). (B) Spatial representation of the amphipathic characteristics endowing bile acid derivatives the ability to be efficiently encapsulated into liposomes.

Some bile acid transporters, such as NTCP in hepatocytes and IBAT in cells of the intestinal epithelium and cholangiocytes, which carry out efficient co-transport of bile acids with sodium (Figure 2A) seem to interact with the side chain of the bile acid molecule (Kramer et al, 2001), and therefore this part of the molecule must not be used to bind the active agent if it is desired that the resulting drug be taken up by these transporters. In contrast, bile acid derivatives obtained by coupling an active agent to the bile acid side chain are efficiently taken up by several members of the human OATP family when expressed in Xenopus laevis oocytes (Briz et al, 2002).

 

 

Figure 2. Schematic representation of the plasma membrane carriers involved in normal uptake/secretion of bile acids (BA) by normal hepatocytes (A) and targeting of cytostatic bile acid derivatives to liver tumour cells (B). NTCP, Na+-taurocholate-cotransporting polypeptide; OATP, organic anion-transporting polypeptide; A-, exchanged anion; BSEP, bile salt export pump; MRP2, multidrug resistance-associated protein-2.

 

 

The whole of the strategy described above is based on the hypothesis that proteins able to take up bile acids are expressed in enterohepatic tumours. But is this in fact true? Although markedly reduced, the expression of carrier proteins and the ability to take up cholephilic organic anions that are typically substrates of OATPs and NTCP are still present in several, but not all, tumour cell lines of enterohepatic origin (Kroker et al, 1978; Von Dippe et al, 1990; Marchegiano et al, 1992; Kullak-Ublick et al, 1996). Moreover the specific expression of several of these transporters has been found in human hepatocellular carcinomas (Kullak-Ublick et al, 1997). Using real-time quantitative RT-PCR we have also recently detected the expression of these transporters in biopsies collected from intestinal adenomas and carcinomas (unpublished results). Moreover, the ability of liver tumour cells to take up bile acids has been confirmed in laboratory animals. Thus, cells isolated from the liver of rats undergoing a protocol of hepatocarcinogenesis induction were able to take up bile acid derivatives in a selective and sodium-independent way (Monte et al, 1999). This was consistent with the major role of members of the OATP family in this process.

Although the efficiency of tumour cells to take up bile acids could be expected to be lower than that of normal epithelial cells, it should be noted that tumour cells are not polarized, and hence drug uptake might not be accompanied by efflux to a similar extent (Figure 2B). Indeed, when overall drug accumulation was analyzed after administering bile acid derivatives to nude mice that had previously been ortothopically implanted with murine liver tumour cells, the amount of drug in the tumours was higher than that found in healthy tissue of the same liver (Briz et al, 2003). Moreover, as expected, there is a relationship between tumour cell load and sensitivity to the cytostatic effect of these compounds (Larena et al, 2002).

Although originally the main reasons for synthesizing cytostatic bile acid derivatives were to use them against tumours located in tissues of the enterohepatic circuit (Marin et al, 2001) or to enhance their water miscibility (Maeda et al, 1990), an additional interest coming from their organotropic characteristics is to extend their use to regional treatment of tumours located outside to the enterohepatic circuit. In these cases, the pharmacological advantage of these compounds would be due to the ability of the liver to efficiently take up and eliminate into bile the drug that, escaping from the tumour, might reach the general circulation during regional therapy (Macias et al, 1998; Larena et al, 2001).

 

III. Coupling bile acids to transition metals to generate cytostatic agents

Although the possibilities of targeting cytostatic agents to the enterohepatic circuit are very broad, and in this respect the coupling of bile acids to organic antitumour drugs, such as chlorambucil (Kramer et al, 1992) as well as other organic moieties (Eunok et al, 2001) has been achieved and investigated, we shall focus the present review on the family of compounds for which this targeting strategy has been most deeply explored so far, i.e., Bamets. This name is an acronym for BA (bile acids) and MET (metal), since the characteristic shared by all members of this family of compounds is that they contain a bile acid-like moiety and an atom of a DNA-reactive transition metal.

The reason for choosing this tandem is the small size of the resulting molecule, which would increase the probability of maintaining both the substrate properties as regards bile acid transporters and the reactivity versus DNA, and hence the antiproliferative effect of these metals, in particular platinum(II) such as in cisplatin - cis-diamminedichloro platinum(II) - (Muggia, 1991). Bamets containing transition metal atoms other than platinum, such as gold (Carrasco et al, 2001) are less efficient cytostatic agents than those containing Pt(II) in the reactive moiety.

Regarding the organic moiety of the molecule, two types of variable have been assayed. These are the bile acid moiety and the nature of the linker placed between this and the transition metal atom alone or as part of cisplatin-derivatives (Marin et al, 2001). More recently, other groups have expanded the list of variations in the Bamet family by synthesizing several carboplatin-bile acid derivatives (Paschke et al, 2003). Two of the best studied and most promising compounds of the Bamet family are cis-diamminechloro-cholylglycinate platinum(II) (Bamet-R2) (Criado et al, 1997) and cis-diammine-bisursodeoxycholate platinum(II) (Bamet-UD2) (Criado et al, 2000).

 

IV. Mechanism of the cytostatic effect

The mechanism of DNA-Bamet interactions is expected to be similar to that of the moiety endowing them with cytostatic ability; namely, cisplatin in the case of Bamet-R2 and Bamet-UD2. Cisplatin forms DNA adducts mainly at the guanine-N7 position (Horacek et al, 1971; Pinto et al, 1985). By different approaches, indirect evidence for the existence of Bamet-induced changes in DNA structure have also been found (Marin et al, 1998a; Martinez-Diez et al, 2000). Thus, in vitro experiments have shown that Bamets are able to change the eletrophoretic mobility of supercoiled covalently-closed and open forms of the circular plasmid pUC18 from Escherichia coli. Moreover, incubation of double-strand DNA with Bamets resulted in an increase in the DNA melting temperature, suggesting the existence of stronger interactions between both strands. This would presumably be due to the formation of inter-strand Bamet-DNA adducts. Scatchard analysis of the results obtained from ethidium bromide displacement assays reveal a marked reduction in the number of DNA sites for ethidium bromide intercalation after in vitro incubation of DNA with Bamets.

The Bamet-DNA interaction probably plays an important role in the ability of these compounds to induce the inhibition of DNA synthesis by proliferating cells, as has been determined by measurement of the incorporation of radiolabeled thymidine into DNA using rat hepatocytes in primary culture and several tumour cell lines (Marin et al, 1998a; Martinez-Diez et al, 2000). The sensitivity to the cytostatic effect of Bamets is not similar for all compounds and all cell lines assayed. However, in general, in the case of Bamet-R2 and Bamet-UD2 this ability is comparable to that of cisplatin.

In addition to blocking DNA replication, the cytostatic effect of Bamets is believed to be due to both a mild pro-necrotic effect and strong pro-apoptotic activity. This is a relevant issue, since cytostatic activity due to the induction of necrosis may lead to the appearance in vivo of noxious inflammation-associated side effects. Apoptosis has been suggested to be an important mechanism in cell killing by cisplatin (Ormerod et al, 1994).

In studies on enterohepatic tumour cells, the release of lactate dehydrogenase to the culture medium and caspase 3 activity in treated cells have been measured to evaluate pro-necrotic versus pro-apoptotic effects, respectively. Apoptosis was confirmed by DNA fragmentation as studied by DNA-ladder formation, TUNEL, single-cell electrophoresis or ÒcometÓ assays, Hoechst-33258 staining, and flow cytometry. Bamet-UD2, Bamet-R2, and cisplatin induce a similarly low degree of necrosis. However, pro-apoptotic ability in tumour cells is particularly high for Bamet-UD2. When administered orally, this compound is also able to induce an increase in the rate of apoptosis observed in normal intestinal mucosa of mouse small intestine, although this effect is mild enough as to not affect the structure and function of this organ (Marin et al, 2004).

 

V. Efficient fecal elimination and low systemic toxicity

As mentioned above, two important drawbacks, also limit the clinical usefulness of chemotherapy based on cisplatin-related cytostatic drugs (Muggia, 1991). These are the development of resistance by tumour cells (Canon et al, 1990), and the appearance of undesirable side effects, which mainly include nephrotoxicity, but also myelotoxicity, and neurotoxicity (Von Hoff et al, 1979; Zhang et al, 1994). By coupling cisplatin to bile acids these drawbacks can be markedly reduced.

One of the most interesting properties of certain members of the Bamet family is their low overall toxicity. However, this characteristic is not shared by all Bamets. For instance, Bamet-D3, which proved to be highly effective as a cytostatic agent was found to induce renal toxicity in rats (Larena et al, 2001). The side effects induced by Bamet-R2 are significantly lower that those induced by cisplatin in rats (Dominguez et al, 2001). Moreover, and more importantly, despite the marked anti-tumour activity of Bamet-UD2, no toxicity to the liver, kidney, bone marrow or nervous system has been detected for this drug in in vivo studies (Dominguez et al, 2001). The reason for the low toxicity of Bamets, in particular Bamet-UD2, as compared to cisplatin is most probably related to the organotropic properties of these compounds, which are assumed to be due to the presence of the bile acid moiety in the molecule. In addition, in the case of Bamet-UD2 the bile acid moiety is ursodeoxycholic acid, a bile acid with well-known cytoprotective properties (Botla et al, 1995; Saksena et al, 1997) that cooperate to reduce the noxious effects of the cisplatin moiety on normal cells exposed to the drug.

When after multiple drug administration to rats for 40 days, the platinum contents in different tissues were measured by flameless atomic absorption spectrometry, the amounts of platinum found in kidney, nerve, brain, bone marrow, lung, heart and muscle were significantly lower in rats treated with Bamet-R2 and Bamet-UD2 than in those receiving cisplatin. By contrast, platinum liver contents at short-term (3 h after administration of a single dose) were higher in Bamet-treated rats than in animals receiving cisplatin. Nevertheless, the level of platinum in liver tissue was more efficiently reduced later on (14 days after administration of a single dose) in animals receiving Bamets.

This particular time course in the liver handling of these drugs is probably due to the following three features: i) The ability of hepatocytes to take up Bamets (Macias et al, 1998); ii) Bamets are efficiently secreted into bile, with no major biotransformation (Macias et al, 1998; Larena et al, 2001); iii) Although Bamets are absorbed in the intestine, such absorption is significantly lower than the biliary excretion rate (Marin et al, 1996). Thus, Bamets differ from the parent drug cisplatin in their cholephilic characteristics, which are responsible for an important shift in the predominance of faecal versus renal pathways of elimination from the body (Marin et al, 1998b; Macias et al, 1998; Palomero et al, 1998; Macias et al, 1999; Larena et al, 2001).

 

VI. Circumvention of resistance to chemotherapy

Another interesting aspect of cytostatic bile acid derivatives is their potential ability to circumvent resistance of tumour cells to chemotherapy. To evaluate this possibility, monoclonal cell lines of enterohepatic origin with several-fold enhanced resistance to cisplatin have been obtained. In all cases, resistance was characterized as mainly being due to an up-regulation of the export pumps of the ABC superfamily of proteins; namely multidrug resistance-associated protein-2 (MRP2), which is believed to play a key role in cisplatin resistance (Cui et al, 1999; Leonard et al, 2003).

As free drugs, Bamet-R2 and Bamet-UD2 are not able to overcome cisplatin resistance in cells of non-enterohepatic origin, such as resistant COR-L23/R non-small-cell lung cancer cells. In contrast, both drugs were equally effective as cytostatic agents in wild-type and cisplatin-resistant cells derived from intestinal and liver tumours. This can probably be explained in terms of the ability of these cells to strongly reduce the intracellular content of cisplatin, whereas they are not able to prevent the efficient uptake of Bamet-R2 and Bamet-UD2 (Briz et al, 2000).

In addition to their interaction with carrier proteins located in the plasma membrane of normal and tumour cells, which is responsible for the specific drug targeting of cytostatic bile acid derivatives, they have another physicochemical characteristic of great interest in the fight to overcome resistance to chemotherapy: as they are amphypathic compounds, that is, they have a hydrophilic region while another part of the molecule is hydrophobic, they can be efficiently encapsulated into liposomes. Liposomes can be used both to encapsulate hydrophilic compounds in the aqueous core and to load lipophilic compounds in the hydrophobic layers, while amphipathic molecules can be loaded in both regions of the liposomes. This is particularly interesting in the case of multilamellar liposomes, in which the encapsulation of Bamets is several fold more efficient than that obtained for cisplatin (Briz et al, 2003). When cisplatin-resistant tumour cells were treated with Bamet-R2 or Bamet-UD2 encapsulated in different types of liposomes the cytostatic effect was enhanced to values similar or even higher than those found for cisplatin in wild-type cells (Briz et al, 2003).

 

VII. In vivo antitumoral activity

Although cisplatin-related drugs are among the most effective cytostatic agents against certain types of tumours (Boulikas et al, 2003), so far regimens including these drugs have had only moderate success in the treatment of hepatic and intestinal tumours (Lee et al, 2003; Mancini et al, 2003; O'Dwyer et al, 2003). Enhanced targeting of cisplatin using Bamets may change this situation, and such expectations are indeed supported by preclinical investigation in laboratory animals.

Subcutaneous implantation of human colon carcinoma cells or mouse hepatoma cells into the backs of nude mice resulted in the formation of single tumours. When these animals were treated by intratumour injection of saline, cisplatin, Bamet-R2 or Bamet-UD2, cisplatin, as well as the Bamets assayed, significantly reduced tumour growth. The order of antitumour activity at all doses used was cisplatin>Bamet-UD2>Bamet-R2. However, at the most effective anti-tumour dose, cisplatin caused 33% mortality, versus no deaths recorded in any of the groups of animals during treatment with Bamets (Dominguez et al, 2001). Similar results have been obtained in a model closer to the clinical situation. After subcutaneous implantation and growth in the back of a donor nude mouse wild-type or resistant enterohepatic tumours were harvested and re-implanted in the livers of several nude mice. These animals were then treated by intraperitoneal injection of saline, cisplatin or Bamet-UD2. Both cisplatin and Bamet-UD2 were able to inhibit tumour growth. However, although some mice treated with cisplatin survived longer than those receiving only saline, no significant differences in mean survival times were found between these two groups. By contrast, treatment with Bamet-UD2 significantly prolonged the survival of mice bearing a tumour in their livers. This difference in antitumour effectiveness was particularly evident when cisplatin-resistant tumours were implanted. The life span of animals treated with Bamet-UD2 was markedly increased when the mice were treated with Bamet-UD2 encapsulated in anionic liposomes, and even more so when this drug was loaded into cationic liposomes (Briz et al, 2003).

 

Acknowledgements

This study was supported in part by the Ministerio de Ciencia y Tecnologia, Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica (Grant BFI2003-03208) and the Junta de Castilla y Leon (Grant SA013/04), Spain.

The group is member of the Spanish Network for Cooperative Research on Hepatitis. Instituto de Salud Carlos III, Spain (Grant G03/015).

Secretarial help by M.I. Hernandez, technical help by E. Flores and English revision of the manuscript by N. Skinner are gratefully acknowledged.

 

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