Cholephilic characteristics of a new cytostatic complex of cisplatin with glycocholate (Bamet-R2)

Abstract

The aim of this work was to investigate both the existence of enterohepatic circulation of cisplatin-cholylglycinate complex, Bamet-R2, and the relevance of biliary versus urinary excretion of this compound. Two experimental models were used: (i) intraluminal perfusion of `in situ' ileum in anaesthetized rats bearing a biliary catheter that permitted bile sample collection and (ii) conscious rats in which a permanent intraarterial catheter had been implanted to carry out sequential blood sampling after intravenous (i.v.) or intragastric (i.g.) drug administration. Total platinum in serum, bile, ileum, liver, urine and feces was measured by flameless atomic absorption spectroscopy. Serum concentration versus time curves obtained after i.v. administration of 1 μmol Bamet-R2 or cisplatin revealed that the area under the curve was significantly higher for Bamet-R2 than for cisplatin (+48%). Non-ultrafiltrable platinum accounted for 54.8 and 48.4% of serum platinum 168 h after cisplatin and Bamet-R2 i.v. administration, respectively. When the animals received i.g. 1 μmol cisplatin or Bamet-R2, serum concentrations of total platinum were markedly higher (three-fold) after Bamet-R2 than after cisplatin administration. The area under the curve was, also in this case, significantly higher for Bamet-R2 than for cisplatin (+28%). This was in part due to the enhanced intestinal absorption of Bamet-R2, as confirmed in experiments on perfused rat ileum, where a markedly higher amount of the drug was found in ileum tissue and bile after perfusion with media containing Bamet-R2 as compared with experiments where cisplatin instead of Bamet-R2 was added to perfusion media. Moreover, after i.v. administration to conscious rats, excretion of Bamet-R2 by the kidney was three-fold lower than that of cisplatin, while elimination of the former compound into feces was four-fold higher than that of the latter. In summary, these results indicate that in addition to the previously reported cytostatic activity of Bamet-R2, this complex has interesting cholephilic characteristics typical of bile acids, such as low urinary excretion together with enhanced intestinal absorption and biliary secretion, probably endowed by the cholylglycyl moiety included in the Bamet-R2 molecule.

Introduction

Since the use of bile acid carrier mechanisms to enhance the hepatic and small intestinal absorption of drugs was first proposed [1], several attempts in this direction have been made 2, 3, 4, among them the synthesis of Bamet-R2 [5]. The existence of specific bile acid carrier proteins in the plasma membranes of hepatocytes and ileocytes [6], which are not present in most cell types, makes these cells ideal targets for bile acid-labeled drugs. Bamet-R2 can be considered one of the many second-generation platinum-containing compounds that have been developed in an effort to obtain new cytostatic drugs having fewer toxic side effects than cisplatin but with comparable chemotherapeutic efficacy. What makes Bamet-R2 peculiar is the use of endogenous compounds with marked liver organotropism – i.e., bile acids – to direct a cisplatin-derived drug toward the hepatobiliary system. These derivatives are expected to be more efficiently taken up by the liver than their parent drug [7]. The strategy also endows certain of these derivatives with enhanced hepatobiliary excretion. Owing to the previously reported cytostatic activity [5]and liver organotropism [8]of Bamet-R2, one could speculate that this compound might be potentially useful in the chemotherapy of liver tumors. However, it has been reported that bile acids cannot be envisaged as useful shuttles for cytostatic drugs toward liver-derived tumors because of the absence in liver tumor cell lines of efficient bile acid uptake, which is a phenotypical characteristic of differentiated hepatocytes [9]. This seems to be true, but only in part. Thus, it has been reported that Na⁺-dependent bile acid uptake is to a certain extent lost in rat hepatoma cells 10, 11, 12, 13. However, some degree of Na⁺-independent uptake is still present in liver tumor cells 13, 14, thus accounting for an efficient bile acid uptake, although lower than that of hepatocytes 13, 15.

Because only anionic bile acids are efficiently taken up by the Na⁺-dependent bile acid transport system located at the sinusoidal membrane of the hepatocyte, Bamet-R2, which is a neutral compound, is not expected to be a good substrate for this carrier. Nevertheless, efficient Bamet-R2 liver uptake has been reported [8]and the possibility of this occurring via a Na⁺-independent bile acid transport system has been suggested. This means that the absence of Na⁺-dependent bile acid transport systems in liver tumor cells will probably not affect the ability of these cells to take up Bamet-R2 to any great extent.

A second possibility to be considered is the use of Bamet-R2 in regional chemotherapy. Using isolated perfused rat livers, Bamet-R2 has been shown to be rapidly taken up from the perfusate and secreted into bile. Thus, efficient biliary elimination is also expected to occur after Bamet-R2 leaves the tumoral area during intraarterial administration.

The existence of carrier systems for bile acids in the intestine (mainly in the ileum) account for an efficient re-uptake of molecules that have reached the gut with bile [6]. Reabsorbed bile acids return to the liver via the portal vein. Thus, only a minor fraction of the bile acid pool is lost every day in the feces, while the rest remains sequestered within the so-called enterohepatic circulation. Whether Bamet-R2 shares this behavior with natural bile acids is an interesting question involving two important pharmacological issues: the re-exposure to Bamet-R2 of tumors located in the enterohepatic circuit and the ability of the body to eliminate this drug. The aim of this work was therefore to investigate the existence of enterohepatic circulation of Bamet-R2 and the relevance of biliary versus urinary excretion of this complex by using two experimental models: (i) intraluminal perfusion of `in situ' ileum in anaesthetized rats bearing a biliary fistula that permitted bile sample collection and (ii) conscious rats to which a permanent intraarterial catheter had been implanted to carry out sequential blood sampling after drug administration. At the end of these experiments, serum ultrafiltrable platinum was determined. However, in the rest of the study total platinum was used because this is related to both toxicity and anti-tumor efficacy 16, 17, 18, 19. Moreover, the use of ultrafiltrable platinum, although preferred by some investigators [20], has been questioned by others [21]due to the ill-defined mixture of species included in this term and to the unreproducible values found by different laboratories for this parameter, since different techniques to prepare and filter the samples have been used.