Novel bile acid derivatives (BANBs) with cytostatic activity obtained by conjugation of their side chain with nitrogenated bases.

Drug targeting may contribute to overcoming resistance to chemotherapy and to reducing side effects. Here, by conjugating a nitrogenated base (NB) to the side chain of a bile acid (BA) moiety, we have synthesized and evaluated six novel compounds, designated BANB-1 to -6, with potential cytostatic activity and vectoriality toward enterohepatic tumors. These compounds were purified by liquid chromatography and their purity was checked by TLC and HPLC before being chemically characterized using IR, (1)H/(13)C NMR and FAB-MS. Using several cell lines - HepG2 (human hepatoblastoma), LS 174T and Caco-2 (human colon adenocarcinoma), Hepa 1-6 (mouse hepatoma), McA-RH7777 (rat hepatoma), CCRF S-180 II (mouse sarcoma) and CHO (Chinese hamster ovary) - their effect on cell viability was measured with the formazan test after drug exposure for 6h (cytotoxic effect) or 72h (cytostatic effect). A weak cytostatic effect of BANB-1, BANB-2 and BANB-3 was detected even in CHO cells stably transfected with rat bile acid transporters (Ntcp and Oatp1/1a1). In contrast, BANB-4, BANB-5 and BANB-6, similarly to cisplatin, showed strong cytostatic effects, together with mild non-specific toxicity. BANB-6 was effective even against Hepa 1-6/R cells, which were partly resistant to cisplatin. Treatment with BANB-6, but not cisplatin, was able to prolong the life span of Nude mice bearing tumors formed by Hepa 1-6/R cells orthotopically implanted in the liver. In conclusion, our results support the hypothesis that cytostatic bile acid derivatives such as BANB-6 may offer a useful pharmacological strategy for the treatment of tumors of the enterohepatic circuit.

Inhibition of the intestinal absorption of bile acids using cationic derivatives: mechanism and repercussions.

To pharmacologically interrupt bile acid enterohepatic circulation, two compounds named BAPA-3 and BAPA-6, with a steroid structure and 1 or 2 positive charges, were obtained by conjugation of N-(3-aminopropyl)-1,3-propanediamine with one or two moieties of glycocholic acid (GC). Both BAPA-3 and BAPA-6 inhibited Na+-dependent taurocholate (TC) uptake by Xenopus laevis oocytes expressing rat Asbt, with Ki values of 28 and 16 microM, respectively. BAPA-3 reduced Vmax without affecting Km. In contrast, BAPA-6 increased Km, with no effect on Vmax. Uptake of [14C]-GC by the last 10 cm of the rat ileum, perfused in situ over 60 min, was inhibited to a similar extent by unlabeled GC, BAPA-3 and BAPA-6. However, the intestinal absorption of these compounds was lower (BAPA-6) or much lower (BAPA-3) than that of GC. When administered orally to mice, both compounds (BAPA-3>BAPA-6) reduced the bile acid pool size, which was accompanied by up-regulation of hepatic Cyp7a1 and Hmgcr and intestinal Ostalpha/Ostbeta. A tendency towards a decreased expression of hepatic Ntcp and an enhanced expression of intestinal Asbt was also observed. Serum biochemical parameters were not affected by treatment with these compounds, except for a moderate increase in serum triglyceride concentrations. In sum, our results suggest that these compounds, in particular BAPA-3, are potentially useful tools for inhibiting the intestinal absorption of bile acids in a non-competitive manner.

Sensitivity of bile acid transport by organic anion-transporting polypeptides to intracellular pH.

We investigated the influence of intracellular pH (pHi) on [14C]-glycocholate (GC) uptake by human hepatoblastoma HepG2 cells that express sodium-independent (mainly OATP-A and OATP-8), but not sodium-dependent, GC transporters. Replacement of extracellular sodium by choline (Chol) stimulated GC uptake but did not affect GC efflux from loaded cells. Amiloride or NaCl replacement by tetraethylammonium chloride (TeACl) or sucrose also increased GC uptake. All stimulating circumstances decreased pHi. By contrast, adding to the medium ammonium or imidazole, which increased pHi, had no effect on GC uptake. In Chinese hamster ovary (CHO) cells expressing rat Oatp1, acidification of pHi had the opposite effect on GC uptake, that is, this was reduced. Changes in extracellular pH (pHo) between 7.40 and 7.00 had no effect on GC uptake at pHi 7.30 or 7.45 when pHopHi. Inhibition was not proportional to the pHo-pHi difference. Intracellular acidification decreased V(max), but had no effect on K(m). In sum, sodium-independent GC transport can be affected by intracellular acidification, possibly due both to modifications in the driving forces and to the particular response to protonation of carrier proteins involved in this process.

Relationship between asymptomatic hypercholanaemia of pregnancy and progesterone metabolism.

The aim of this study was to identify a subgroup of pregnant women with asymptomatic hypercholanaemia of pregnancy (AHP), in which the relationship between alterations in the level and pattern of serum bile acids (BAs) and of progesterone plus progesterone metabolites could be investigated in the absence of overt impairment of hepatobiliary function. Cholanaemia and serum concentrations of progesterone were assayed by an enzymic technique and by ELISA respectively, while BA molecular species and progesterone metabolites were measured by GC-MS, in the serum of 411 healthy pregnant women. Samples were collected after an overnight fast in the final week of each trimester of gestation. Two pregnant women were excluded because of the suspicion of intrahepatic cholestasis of pregnancy (ICP). Cholanaemia was found to increase progressively throughout pregnancy, but with normal mean values lower than 3.0 microM. Thus in our series AHP was defined arbitrarily as the presence of serum total BA concentrations 2-fold higher than this value, i.e. 6 microM, in the absence of hepatobiliary disease or symptoms of ICP. The prevalence of this condition was observed to increase with gestational age. Changes in the pattern of serum BAs in AHP were also found. These were reflected in a marked increase in the proportion of cholic acid together with a decrease in that of deoxycholic acid, while the proportions of chenodeoxycholic acid and lithocholic acid changed only moderately. When groups at the same gestational age were compared, serum progesterone levels were always significantly lower, while those of progesterone metabolites were higher, in women with AHP. Our results suggest that AHP is a relatively common condition in our geographical location, where ICP is rarely diagnosed. Changes in the serum BA pattern in hypercholanaemia resemble these described in ICP. The simultaneous finding of lower serum total progesterone levels along with an increase in its metabolites supports the hypothesis that a primary defect in progesterone metabolism may be involved in the aetiology of ICP.

Changes in the pool of bile acids in hepatocyte nuclei during rat liver regeneration.

BACKGROUND/AIMS: To investigate changes in nuclear bile acids (BAs) during rat liver regeneration. METHODS: Nuclei were isolated from control rat livers and after two-thirds partial hepatectomy (PH). BAs in bile, liver homogenate and nuclei were measured by gas chromatography-mass spectrometry. Nuclear translocation of radiolabeled BAs was determined using fresh isolated hepatocytes from control donors. RESULTS: Liver BA concentrations were transiently reduced after PH. Relative increases in: -MCA at 1 day, deoxycholic acid at 7 days and cholic acid (CA) at 3 and 14 days were found. Nuclear BAs accounted for <0.5% of liver BAs. Contamination with cytosolic BAs during nuclei isolation was <4%. Unconjugated- and conjugated-CA were able to reach the nucleus with similar efficiency. The pattern of nuclear BAs--CA (80%) and ursodeoxycholic acid (UDCA) (8.5%) being the most abundant--did not match that found in liver or bile. A transient decrease in CA/UDCA ratio, in absence of significant change in total BA content, was observed in nuclei after PH. "Flat" BA species were only detected in homogenate, but not in nuclei, at 1 day after PH. CONCLUSIONS: BA pool in nuclei of rat hepatocytes, whose composition is different to that of total liver BA pool, undergoes important changes during liver regeneration.