Novel cationic and neutral glycocholic acid and polyamine conjugates able to inhibit transporters involved in hepatic and intestinal bile acid uptake.

To obtain novel drugs able to inhibit transporters involved in bile acid uptake, three compounds were synthesized by conjugating N-(3-aminopropyl)-1,3-propanediamine (PA) with one (BAPA-3), two (BAPA-6), or three (BAPA-8) moieties of glycocholic acid (GC) through their carboxylic group. The expected net charge in aqueous solutions was 2+ (BAPA-3), 1+ (BAPA-6), and 0 (BAPA-8). They were purified by liquid chromatography and their purity checked by HPLC before being chemically characterized by elemental analysis, NMR, and FAB-MS. Using brush-border membranes isolated from rat ileum; their ability to inhibit [(14)C]-GC transport (BAPA-3>BAPA-6>BAPA-8) was suggested. This was further investigated 48h after injecting Xenopus laevis oocytes with the mRNA of rat sodium/taurocholate (TC)-cotransporting polypeptide (Ntcp), rat apical sodium-dependent bile salt transporter (Asbt), or the human isoforms OATP-C/1B1 and OATP8/1B3 of organic anion-transporting polypeptides, when maximal functional expression was detected. BAPA-8, BAPA-6, and BAPA-3 induced no inhibition of OATP8/1B3-mediated [(3)H]-TC uptake, but dramatically reduced [(3)H]-TC uptake by OATP-C/1B1. In the cases of Ntcp- and Asbt-mediated [(3)H]-TC uptake, these were sodium-dependent and were inhibited by BAPA-6>BAPA-8>BAPA-3 and BAPA-8>BAPA-6>BAPA-3, respectively. In conclusion, our results suggest that these compounds are potentially interesting research tools for the selective modulation of liver and intestinal uptake of bile acids and other cholephilic compounds. Moreover, they may be of pharmacological usefulness to prevent the acute toxicity of compounds reaching liver cells through specific transporters or to enhance both fecal elimination of bile acids and hence cholesterol consumption for the 'de novo' synthesis of bile acids.

Relationship between tumor cell load and sensitivity to the cytostatic effect of two novel platinum-bile acid complexes, Bamet-D3 and Bamet-UD2.

Based on the organotropic characteristics of bile acids towards the liver and the intestine, two novel compounds of the Bamet family, containing at least one bile acid moiety bound to platinum(II), have been synthesized and their cytostatic effect compared to their ability to become accumulated in tumor cells of hepato-intestinal origin. Bamet-UD2 [cis-diammine-bis-ursodeoxycholic platinum(II)] induced a marked inhibition of cell growth, which was more marked in human hepatoblastoma HepG2 and mouse hepatoma Hepa 1-6 cells than in rat hepatoma McA-RH7777 and human colon adenocarcinoma LS 174T cells. This effect was similar to that observed for cisplatin and stronger than that previously reported for other members of this family, such as Bamet-H2 and Bamet-R2. By contrast, Bamet-D3 [(N'N'' cis-dichloro N(3-3-amminepropylammine)propyl) glycocholamide platinum (II)] was only effective in reducing growth in human hepatoblastoma HepG2 cells. Because the in vitro DNA-reactivity was approximately 5-fold higher for Bamet-D3 than for Bamet-UD2, an additional cause for the difference in their cytostatic abilities was sought, investigating the relationship between cell load and the cytostatic effect of the drugs. Drug uptake by two cell lines, Hepa 1-6 and HepG2, with different sensitivities to these compounds was measured. The cellular uptake of Bamet-D3 and Bamet-UD2 was several-fold higher than that of cisplatin. No significant difference in the amount of both drugs taken up by these cell types was found. A study on sodium-dependency and substrate specificity indicated that Hepa 1-6 cells take up Bamet-D3 and Bamet-UD2 via similar mechanism(s), whereas these compounds do not seem to share the uptake pathways in HepG2 cells. Measurement of cell viability by formazan formation from tetrazolium salts and by neutral red uptake, after short-term (6 h) exposure to the desired drug, indicated that no acute toxic effect occurs in the presence of cisplatin or Bamet-D3 in either HepG2 or Hepa 1-6 cells. By contrast, in both cell lines Bamet-UD2 induced acute cell toxicity in a dose-dependent fashion. In sum, the results indicate that tumor cells efficiently take up these two novel compounds of the Bamet family. Although the exact uptake mechanism remains unknown, it seems to be dependent on the cell type. However, the cell load does not account for the differences in the anti-proliferative properties of the drugs. The strong and promising cytostatic activity of Bamet-UD2 is additionally related to its ability, absent in Bamet-D3, to acutely alter cellular functions other than proliferation.

Evidence for dual effects of DNA-reactive bile acid derivatives (Bamets) on hepatitis B virus life cycle in an in vitro replicative system.

A liver targeting strategy to direct antiviral drugs toward hepatitis B virus (HBV) was investigated. As model drugs we used cisplatin-bile acid derivatives (Bamets) to determine the production of virions by HBV-transfected hepatoblastoma cells (HepG2 2.2.15). Drug uptake was determined using flameless atomic absorption spectrometry to measure platinum cell contents. Cytotoxic effect was determined by formazan formation and neutral red uptake tests. The release of viral surface protein was evaluated by ELISA. The abundance of HBV-DNA in the medium was determined by quantitative real-time PCR and its structure by Southern blot analysis. The uptake of Bamets by HepG2 2.2.15 cells was higher than that of cisplatin. At concentrations lower than 10 microM, distinct Bamets have no toxic effect on host cells, whereas cisplatin dramatically reduced cell viability at concentrations higher than 1 microM. All the drugs tested inhibited the release of viral proteins to the medium, but induced a marked and progressive dose-dependent increase in the amount of viral DNA in the medium. This was mainly due to the release of short fragments of HBV-DNA in the case of cisplatin. On the contrary, Bamets induced an enhanced release of circular forms of HBV-DNA. These findings suggest the existence of a dual effect of Bamets on HBV life-cycle by enhancing the production of DNA replicative intermediates but reducing the secretion of complete virions. Altogether these characteristics recommend consideration of these compounds as a useful experimental tool in the investigation of novel liver targeted therapeutic agents based on bile acid derivatives for the treatment of HBV infections, or to carry out further studies on the HBV life cycle.