UGT-dependent regioselective glucuronidation of ursodeoxycholic acid and obeticholic acid and selective transport of the consequent acyl glucuronides by OATP1B1 and 1B3.

Ursodeoxycholic acid (UDCA) is a major effective constituent of bear bile powder, which is widely used as function food in China and is documented in the Chinese pharmacopoeia as a traditional Chinese medicine. UDCA has been developed as the only accepted therapy by the US FDA for primary biliary cholangitis. Recently, the US FDA granted accelerated approval to obeticholic acid (OCA), a semisynthetic bile acid derivative from chenodeoxycholic acid, for primary biliary cholangitis. However, some perplexing toxicities of UDCA have been reported in the clinic. The present work aimed to investigate the difference between UDCA and OCA in regard to potential metabolic activation through acyl glucuronidation and hepatic accumulation of consequent acyl glucuronides. Our results demonstrated that the metabolic fates of UDCA and OCA were similar. Both UDCA and OCA were predominantly metabolically activated by conjugation to the acyl glucuronide in human liver microsomes. UGT1A3 played a predominant role in the carboxyl glucuronidation of both UDCA and OCA, while UGT2B7 played a major role in their hydroxyl glucuronidation. Further uptake studies revealed that OATP1B1- and 1B3-transfected cells could selectively uptake UDCA acyl glucuronide, but not UDCA, OCA, and OCA acyl glucuronide. In summary, the liver disposition of OCA is different from that of UDCA due to hepatic uptake, and liver accumulation of UDCA acyl glucuronide might be related to the perplexing toxicities of UDCA.

Role of vitamin C transporters and biliverdin reductase in the dual pro-oxidant and anti-oxidant effect of biliary compounds on the placental-fetal unit in cholestasis during pregnancy.

Maternal cholestasis causes oxidative damage to the placental-fetal unit that may challenge the outcome of pregnancy. This has been associated with the accumulation of biliary compounds able to induce oxidative stress. However, other cholephilic compounds such as ursodeoxycholic acid (UDCA) and bilirubin have direct anti-oxidant properties. In the present study we investigated whether these compounds exert a protective effect on cholestasis-induced oxidative stress in placenta as compared to maternal and fetal livers, and whether this is due in part to the activation of anti-oxidant mechanisms involving vitamin C uptake and biliverdin/bilirubin recycling. In human placenta (JAr) and liver (HepG2) cells, deoxycholic acid (DCA) similar rates of free radical generation. In JAr (not HepG2), the mitochondrial membrane potential and cell viability were impaired by low DCA concentrations; this was partly prevented by bilirubin and UDCA. In HepG2, taurocholic acid (TCA) and UDCA up-regulated biliverdin-IX alpha reductase (BVR alpha) and the vitamin C transporter SVCT2 (not SVCT1), whereas bilirubin up-regulated both SVCT1 and SVCT2. In JAr, TCA and UDCA up-regulated BVR alpha, SVCT1 and SVCT2, whereas bilirubin up-regulated only SVCT2. A differential response to these compounds of nuclear receptor expression (SXR, CAR, FXR and SHP) was found in both cell types. When cholestasis was induced in pregnant rats, BVR alpha, SVCT1 and SVCT2 expression in maternal and fetal livers was stimulated, and this was further enhanced by UDCA treatment. In placenta, only BVR alpha was up-regulated. In conclusion, bilirubin accumulation and UDCA administration may directly and indirectly protect the placental-fetal unit from maternal cholestasis-induced oxidative stress.

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.

Micronuclei induction, cell cycle delay and apoptosis as markers of cellular stress caused by ursodeoxycholic acid in human lymphocytes.

Ursodeoxycholic acid (UDCA) is a bile acid (BA) used for cholesterol gallstone dissolution. Since epidemiological evidence indicates that BAs can be involved in the etiology of colorectal cancer, we investigated the effects of UDCA and its physiologically produced taurine conjugate tauroursodeoxycholic acid (TUDCA) on human lymphocyte cultures in terms of genetic damage in the form of micronuclei (MN) production, cell cycle modifications and induction of apoptosis. With respect to controls, treatment with UDCA (from 10 microg/ml) caused a dose-related increase in MN, whereas TUDCA caused no significant increase (up to 1000 microg/ml). Fluorescence in situ hybridization (FISH) analysis using pancentromeric probes suggested that UDCA exerts aneugenic activity. Bromodeoxyuridine/Hoechst flow cytometry showed that both BA significantly inhibit cell cycle progression (UDCA at 100 microg/ml, and TUDCA, more markedly at 300-1000 microg/ml). Neither UDCA nor TUDCA affected induction of apoptosis, as evaluated by the Annexin-V-Fluos assay. We conclude that UDCA is potentially genotoxic. However, taking into account the characteristics of other physiological BA, our findings are in line with the concept that long-term UDCA treatment may be safely administered. The multi-assay approach reported here could be useful in the toxicological evaluation of newly developed BA analogs as candidates for pharmacological use.

Low in vivo toxicity of a novel cisplatin-ursodeoxycholic derivative (Bamet-UD2) with enhanced cytostatic activity versus liver tumors.

Cisplatin-bile acid derivatives belonging to the Bamet-family maintain both liver organotropism and cytostatic activity. "In vivo" toxicity and usefulness as chemotherapeutic agent versus liver tumors of a novel drug, Bamet-UD2 [cis-diamminechlorocholylglycinate platinum (II)], with enhanced "in vitro" cytostatic activity was investigated. Using orthotopically implanted mouse Hepa 1-6 hepatoma in the liver of Nude mice, the antitumor effect of Bamet-UD2 was compared with that of a previously characterized compound of this family, Bamet-R2 [cis-diamminebis-ursodeoxycholate platinum(II)], and cisplatin. Life span was significantly prolonged in mice treated with both Bamets (Bamet-UD2 > Bamet-R2), compared with animals receiving saline or cisplatin. All these drugs inhibit tumor growth (Bamet-UD2 = cisplatin > Bamet-R2). However, toxicity-related deaths only occurred under cisplatin treatment. Using rats maintained in metabolic cages, organ-specific toxicity and drug accumulation in tissues were investigated. The amount of both Bamets in the liver was severalfold higher than that of cisplatin. By contrast, a significantly higher amount of cisplatin in kidney and nerve was found. In lung, heart, muscle, brain, and bone marrow the amount of drug was small and also significantly lower in animals receiving Bamets. Signs of neurotoxicity (altered nerve conduction velocity), nephrotoxicity (increased serum urea and creatinine concentrations and decreased creatinine clearance), and bone marrow toxicity (decreased platelet and white blood counts) in animals treated with cisplatin but not with the Bamets were found. These results indicate that, owing to strong antitumor activity together with absence of side effects, Bamet-UD2 may be useful in the treatment of liver tumors.

Overcoming cisplatin resistance in vitro by a free and liposome-encapsulated bile acid derivative: BAMET-R2.

Low water solubility and development of resistance are important drawbacks in the use of cisplatin as a cytostatic agent. A novel bile acid-cisplatin complex, Bamet-R2 [cis-diamminechlorocholylglycinateplatinum (II)], with liver vectoriality, has been synthesized. Our aim was to investigate the usefulness of this compound to overcome cisplatin resistance and to determine whether its encapsulation into liposomes increases its water solubility, uptake by liver tumor cells and cytostatic activity. Highly efficient incorporation of Bamet-R2 into liposomes permitted an increase in the concentration of the drug compared with that in the initial free solution by more than 6 x 10(6)-fold, which is 10(3)-fold higher than the encapsulation obtained for cisplatin. A partially cisplatin-resistant (87-fold) monoclonal cell line (Hepa 1-6/10R) was obtained by 2 subcloning steps of a population of mouse hepatoma Hepa 1-6 cells grown in step-wise increasing cisplatin concentrations up to 10 microM. Decreased sensitivity to cisplatin was accompanied by a 3.2-fold lower drug accumulation compared to wild-type cells. Uptake was markedly increased by the binding of cisplatin to glycocholic acid in both Hepa 1-6 and Hepa 1-6/10R cells. This probably accounts for the partial overcoming (-82%) of resistance when used on Hepa 1-6/10R cells. Inclusion of Bamet-R2 into liposomes further increased the amount of the drug accumulated in both cell types and, hence, enhanced its cytostatic activity. Since both plain liposomes and Bamet-R2 have little toxicity, the formulation of this compound in liposomes may offer a substantial advantage over cisplatin in the treatment of tumors resistant to this anti-neoplastic agent.

Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture.

Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.

Metabolic fate and hepatocyte toxicity of reverse amide analogs of conjugated ursodeoxycholate in the rat.

Reverse amide analogs of conjugated bile acids were tested for their effects on the viability of cultured primary rat hepatocytes, for their transport and metabolism in the intact rat, and for their susceptibility to hydrolysis by intestinal bacteria. Succinylnorursodeoxycholanylamide (SNUDCN) and its parent C23 amine showed the same general lack of toxicity toward hepatocytes as the normal conjugates of ursodeoxycholic acid, at concentrations up to 500 microM. The 3alpha,7alpha,12alpha-trihydroxy analog and its parent amine were more toxic than the corresponding dihydroxy compounds, although their effects were similar to those observed for the normal conjugates of cholic acid. Following intraduodenal infusion, greater than 80% of administered SNUDCN appeared in the bile of bile fistula rats. Analysis of bile fractions indicated the presence of SNUDCN (81.5 mol% of original amount) and two metabolites, the taurine conjugate of SNUDCN (9.4 mol%) and SNUDCN containing an additional hydroxy group (9.1 mol%). Although SNUDCN underwent an efficient first pass enterohepatic circulation, it displayed a shorter biological half life than taurocholate (T1/2: 8.9 h vs 39.6 h, respectively). The reverse amide analogs were not hydrolyzed by any of a variety of intestinal bacteria known to hydrolyze normal conjugated bile acids. Despite the shorter half-life, the reverse amide analogs may be of potential use in the targeting of therapeutic bile acids to the colon.

Inhibitory effect of sodium ursodeoxycholate on basal and stimulated short-circuit current across the isolated toad skin.

The effect of sodium ursodeoxycholate (U) on short-circuit current (SCC), an index of basal and stimulated net ion transport across isolated skins of Bufo arenarum toads, was tested. U inhibited basal SCC when added to the epidermal side of the skins. The inhibitory effect was reversible after rinsing the preparation during 60 min. U also inhibited the natriferic response to oxytocin, db-cAMP and theophylline by 82%, 49% and 47%, respectively. Inhibition of SCC by exposure to U was reversed by the polyene antibiotic nystatin. In turn, SCC induced by nystatin in the amiloride-treated skin was insensitive to U and blocked by ouabain, a Na+, K(+)-ATPase inhibitor. These results strongly suggest that the effect of U is exerted at the apical membrane of sodium transporting cells, and rule out the existence of an additional site of inhibitory action of U.