Metabolic Profile of Obeticholic Acid and Endogenous Bile Acids in Rats with Decompensated Liver Cirrhosis.

Obeticholic acid (OCA) is a semisynthetic bile acid (BA) analog and potent farnesoid X receptor agonist approved to treat cholestasis. We evaluated the biodistribution and metabolism of OCA administered to carbon tetrachloride-induced cirrhotic rats. This was to ascertain if plasma and hepatic concentrations of OCA are potentially more harmful than those of endogenous BAs. After administration of OCA (30 mg/kg), we used liquid chromatography-mass spectrometry to measure OCA, its metabolites, and BAs at different timepoints in various organs and fluids. Plasma and hepatic concentrations of OCA and BAs were higher in cirrhotic rats than in controls. OCA and endogenous BAs had similar metabolic pathways in cirrhotic rats, although OCA hepatic and intestinal clearance were lower than in controls. BAs' qualitative and quantitative compositions were not modified by a single administration of OCA. In all the matrices studied, OCA concentrations were significantly lower than those of endogenous BAs, potentially much more cytotoxic.

Pathophysiology and pharmacotherapy of cholelithiasis.

Several factors are involved in the development of gallstone formation: formation of supersaturated bile; nucleation; formation, retention and adhesion of cholesterol crystals and eventually stone growth. The dynamics of the gallbladder may play a key role in the overall process. The pathophysiologic theory of cholesterol gallstone formation and the knowledge of their physico-chemical properties support the modern concept of gallstone therapy. Chenodeoxycholic and ursodeoxycholic have been widely used as cholesterol gallstone dissolving agents and evaluated in terms of efficacy and safety.

Effects of a salt of cholestyramine and 2-[4-(p-chlorobenzoyl)phenoxy]2-methyl propionic acid (alpha-1081) on biliary lipid secretion in rats.

1 Hypolipidaemic agents may increase biliary cholesterol in man, inducing a supersaturated bile. 2 To evaluate this possible side-effect, we have studied bile lipid secretion over a period of 8 h with intact enterohepatic circulation and 4 h with complete interruption in rats treated for two months with a salt of cholestyramine and 2-[4-(p-chlorobenzoyl)-phenoxy]2-methyl propionic acid (alpha-1081, 1.150 g/kg body wt., daily), cholestyramine (1.125 g/kg body wt. daily), procetofenic acid (25 mg/kg body wt. daily) and saline respectively (six rats for each group). 3 Cholesterol saturation index significantly (P less than 0.005) increased (from 0.21 +/- 0.01 to 0.39 +/- 0.09, mean +/- s.d.), in rats fed with procetofenic acid but it did not in alpha-1081- and cholestyramine-treated animals. 4 Procetofenic acid and, to a lesser extent, cholestyramine increased the bile flow. Procetofenic acid increased cholesterol secretion from 0.45 +/- 0.17 to 0.94 +/- 0.19 mumol kg-1 body wt. h-1 (mean +/- s.d.). 5 Cholestyramine increased both serum cholesterol and bile acid secretion from 0.45 +/- 0.17 to 0.68 +/- 0.10 and 25.8 +/- 9.48 to 39.96 +/- 6.68 mumol kg-1 body wt. h-1 respectively; alpha-1081, on the contrary, had no effect on bile lipid secretion. 6 These data suggest that alpha-1081 may be used as a new hypolipidaemic drug without any risk of increasing cholesterol in bile.

Relationship between structure and intestinal absorption of bile acids with a steroid or side-chain modification.

UNLABELLED: A structure-activity relationship for bile acid (BA) intestinal absorption is known to exist. To better understand the BA structural requirements for optimal BA intestinal absorption, rabbit ileal perfusion studies were performed. Unconjugated BA: Ursodeoxycholic (UDCA) and ursocholic acid (UCA) with methyl (6MUDCA and 6MUCA) or fluoro group (6FUDCA and 6FUCA) in the 6 position and UCA with a methyl group in 23 position (23MUCA) were compared with unconjugated UDCA, UCA, deoxycholic (DCA), chenodeoxycholic (CDCA), hyocholic (HCA), and hyodeoxycholic (HDCA) acid. BA lipophilicity was evaluated by their octanol-water partition coefficient. Conjugated BA: Taurine-conjugated UDCA and UCA with a methyl group in the 23 position (T23MUDCA and T23MUCA) were compared with the corresponding taurine-conjugated natural analogs. An analog of glycine-conjugated UDCA with the C24 amide bond replaced by a -CO-CH2- in the 24 position (24PUDCA) was studied and results were compared with the natural form (GUDCA). Unconjugated BA absorption was dose dependent (i.e., passive) and followed their lipophilicity: DCA > 6MUDCA > CDCA > HDCA > UDCA > HCA > 6FUDCA > 6MUCA > 6FUCA > UCA. Conjugated BA absorption was active, and Vmax was in the following order: TCA > TUDCA > TUCA > T23MUCA > T23MUDCA > 24PUDCA > GUDCA. 24PUDCA transport was also active and higher than GUDCA. CONCLUSION: Passive transport is dependent on BA lipophilicity. Conjugated BAs are actively transported, and the presence of a 23-C methyl group does not improve transport when compared with the natural analogs. The substitution of the C24 amide bond with a -CO-CH2-still affords interaction of the BA with the intestinal transport carrier.

A radioimmunoassay for lithocholic acid conjugates in human serum and liver tissue.

A sensitive and specific radioimmunoassay for glycine and taurine conjugates of lithocholic acid (CLCA) has been developed. 3H-glycolithocholic acid (S.A. = 17Ci/mmol) was used as tracer. Separation of free from antibody-bound bile acid was carried out using ammonium sulphate (saturated solution). The antiserum showed high specificity for both glyco and tauro conjugated lithocholate (100% cross reaction) and lithocholic acid (25% cross reaction). The sensitivity of the assay (1 pmole/tube), was adequate for measuring CLCA in peripheral blood and hepatic tissue in man.

Competition in liver transport between chenodeoxycholic acid and ursodeoxycholic acid as a mechanism for ursodeoxycholic acid and its amidates' protection of liver damage induced by chenodeoxycholic acid.

BACKGROUND: Ursodeoxycholic acid has been widely used as a therapeutic agent in cholesterol gallstones and liver disease patients, but its mechanism of action is still under investigation. AIMS: The protective effect of ursodeoxycholic acid, both free, taurine and glycine conjugated, against hepatotoxic bile acids such as chenodeoxycholic acid and its taurine amidate was studied in bile fistula rats and compared with the cholic and taurocholic acid effect. METHODS: Tauroursodeoxycholic acid, glycine ursodeoxycholic acid, ursodeoxycholic acid, taurocholic acid and cholic acid were infused iv over 1 hour (8 micromol/min/kg) together with an equimolar dose of either taurochenodeoxycholic acid or chenodeoxycholc acid. Bile flow, total and individual bile acid and biliary lactate dehydrogenase and alkaline phosphatase enzymes were measured. RESULTS: Taurochenodeoxycholic acid and chenodeoxycholc acid caused cholestasis and liver damage associated with a decreased bile flow, total and individual bile acids secretion accompanied by a biliary leakage of lactate dehydrogenase and alkaline phosphatase enzymes. Tauroursodeoxycholic acid, glycine ursodeoxycholic acid, ursodeoxycholic acid and taurocholic acid, on the contrary, were choleretic, inducing an opposite effect on biliary parameters. Simultaneous infusion of taurochenodeoxycholic acid and the protective bile acid resulted in a functional and morphological improvement of the above parameters in the following order: glycine ursodeoxycholic acid > tauroursodeoxycholic acid > ursodeoxycholic acid followed by taurocholic acid; cholic acid was ineffective. CONCLUSIONS: The results show the protective effect of glycine ursodeoxycholic acid, ursodeoxycholic acid and tauroursodeoxycholic acid. This may be due to a facilitated transport of the toxic bile acid into bile; conjugation with taurine is less effective than glycine. Finally, the better protective effect of ursodeoxycholic acid and its amidates with respect to cholic acid and its taurine conjugated form seems to be related to their different lipophilicity and micellar forming capacity.

[Therapy of cholesterol lithiasis with chenodeoxycholic acid (Chenoxyl)]. / La terapia della litiasi colesterolica con acido chenodesossicolico (Chenossil)

Chenodesoxycholic acid (500-750 mg/day Chenoxyl) was employed over a period of 9 months in the treatment of 40 patients with radiotransparent calculi in a functioning gallbladder and 8 with choledochal lithiasis. Radiological examination and complete hepatological exploration were carried out before and after the treatment in all cases. Complete dissolution was obtained in 6 and 2 cases respectively, with reduction of size in 13 and 2, and no change in 21 and 4. No significant variations were notes in the blood lipid picture or in liver function. There were no cases of diarrhoea.

[Changes in bile and blood lipid levels following chenodeoxycholic acid therapy]. / Modificazioni dell'assetto lipidico biliare ed ematico dopo terapia con acido chenodesossicolico.

Original and known methods and ordinary routine tests were used to evaluate changes in bile salts, phospholipids and cholesterol and in serum lipids induced in 50 patients during treatment with chenodeoxycholic acid (2-12 mg/Kg/day) for about 15 months. No significant differences in serum lipids, though a clear fall in triglycerides was noted in those who received 7-12 mg/Kg/day as opposed to 2-7 mg/Kg/day. There was a marked fall in the molar percentage of biliary cholesterol, followed by a significant reduction in the saturation index of Metzger. Once again, greater reduction was noted in patients receiving the higher dose range. A correlation between the dose used and the percentage of cholesterol in the bile was thus evident. After treatment, chenodeoxycholic acid was the main bile acid and there was a significant fall in cholic and deoxycholic acid. The desired drug effect can be obtained with low doses (7-12 mg/Kg/day) and a biliary chenodeoxycholic value well below 90%.

[Results of chenic acid therapy in patients with cholesterol calculi]. / Risultati della terapia con acido chenico nei pazienti portatori di calcoli di colesterolo.

58 patients suffering from cholesterol gallstones were treated with chenodesoxycholic acid at a dose of 2-15 mg/Kg/die for about 15 months so as to determine its effettiveness and establish a minimum effective dose. 8 of these patients presented stones in the common bile duct. The results showed: a complete dissolution of the stones in 16 of the 50 patients considered to be suffering from cholecystic lithiasis; partial dissolution in 15; no change in the remaining 19. Of the 8 patients with bile duct stones, 3 presented complete dissolution, 2 partial, and 3 no change. An improvement in digestive function was observed in most cases. The liver function tests carried out in each patient before, during and after treatment with chenodesoxycholic acid were completely normal.

[Correlation of malabsorption of bile acids, bile lipid composition and calculi]. / Correlazione tra il malassorbimento degli acidi biliari, la composizione lipidica biliare e i calcoli.

The relation between malabsorption of bile acids, the bile lipid composition, and biliary stones was examined in 8 patients subjected to ileal resection (particularly for Crohn's disease), 6 with ileal bypass for morbid obesity, and 10 healthy controls. The 1-14C-cholylglycine breath test was employed to detect of the absorption and deconjugation of bile acids. Bile lipid composition was expressed according with Metzger's saturation index. Healthy subjects gave normal findings in all respects, whereas ileal resection was accompanied by malabsorption, increased deconjugation, and faecal loss of 14C. These changes, particularly malabsorption, were more evident after ileal bypass. Preoperative saturation values rose to more than 1 in all cases, especially after resection. Liver disease (steatosis and cirrhosis) 6 months after bypass, together with cholesterol lithiasis in 2/6 patients.

[Medical treatment of cholesterol cholelithiasis using chemodeoxycholic acid in man]. / Il trattamento medico della calcolosi biliare colesterolica nell'uomo con acido chenodesossicolico.

The results of a tiral using chenodeoxycholic acid in 400 patients with cholesterol gallstones are reported. The "qualifying points" of such treatment are compared with the literature data in clinical and laboratory terms. 54% of 300 in clinical and laboratory terms. 54% of 300 patients who received less than 12 mg/kg/day achieved dissolution in a mean time of 11 months, while 64% of the remainder (12-15 mg/kg/day) did so in an average of 8 months. Microcalculi proved most sensitive to treatment (65% of positive results). Lithiasis over 5 years standing and over-weight (10% over the ideal figure) were factors that imposed more protracted treatment. Careful selection of candidates was proved important by the results of quarterly liver and intestine performance examinations. These were more extensive and more clearly aimed than those proposed by other workers. They showed that the acid is neither hepato nor enterotoxic. Indeed, no serious side-effects were noted.

A new gastric-emptying mouse model based on in vivo non-invasive bioluminescence imaging.

BACKGROUND: Different techniques were used to assess gastric emptying (GE) in small animals; most of them require sophisticated equipment, animal sacrifice and are expensive. In the present investigation a simple, non-invasive method based on bioluminescence imaging (BLI) is reported to study GE, using light-emitting Escherichia coli cells as a marker of the gastric content. METHODS: A new thermostable red-emitting luciferase was chosen as reporter gene to transform E. coli cells. Bioluminescent (BL) bacteria were administered to fasting mice, after a solid meal, and in response to different doses of metoclopramide (MET) and hyoscine butylbromide (HY). Bioluminescence imaging allowed to evaluate the real time 2D spatial and temporal distribution of bacteria along the gastrointestinal tract in animals and to calculate GE rate in basal conditions and following pharmacological stimulation. KEY RESULTS: The administered BL bacteria were easily imaged and localized in the stomach and subsequently followed in the duodenum and upper intestine allowing to accurately calculate GE. Gastric emptying after the test meal was significantly slower (T(1/2) 16 ± 3 min) than that obtained in fasting conditions (T(1/2) 2 ± 1 min); administration of HY (1 mg kg(-1) b.w.) significantly (P < 0.05) increased T(1/2) that was delayed up to 25 ± 4 min; MET (1 mg kg(-1) b.w.) significantly (P < 0.05) accelerated T(1/2), that was achieved within 8 ± 2 min. CONCLUSION & INFERENCES: The reported model is simple, inexpensive, reliable, sensitive and accurate; it can detect both acceleration and slowdown of GE. The model is useful in the investigation of new drug-induced alterations of gastric motility allowing to reduce the number of experimental animals.

Generation of a novel antibody probe to the apical sodium-dependent bile acid transporter that inhibits ileal bile acid absorption.

Intestinal bile acid absorption is mediated by a sodium-dependent transporter located in the brush border apical membrane of ileocytes. The transmembrane topology and the role of individual amino acid residues in the bile acid transport process have been investigated by means of various experimental approaches, leading to multiple hypotheses. We raised a monoclonal antibody against a segment of the transporter comprising vicinal cysteine residues, in order to evaluate its functional role. A 14 amino acid peptide, corresponding to amino acids 104-117 of the transporter, was synthesized, and a monoclonal anti-peptide antibody was raised. In vitro uptake-inhibition studies in the presence of the monoclonal anti-peptide antibody were performed using ileal brush border membrane vesicles. Rabbit ileum was perfused in vivo with 5 mM taurocholic acid in the presence of the monoclonal antibody, and bile acid absorption inhibition was evaluated. The anti-peptide monoclonal antibody significantly reduced the in vitro uptake and in vivo absorption of taurocholic acid. The present data demonstrate the functional relevance of the 104-117 peptide segment and report the generation of a novel antibody against the apical sodium-dependent bile acid transporter (ASBT) that may be used as a therapeutic agent in hypercholesterolemia and in cholestatic pruritus.

Systemic antibiotic therapy on multiple intracerebral abscesses of unknown origin.

A 68-year old patient presented with increasing right-sided weakness, dysarthria, pyrexia and a deteriorating general condition. CT and MRI showed about 20 round hyperdense lesions with peripheral enhancement of contrast material up to 1.5 cm in diameter. Antibiotic triple-therapy using ceftriaxone, gentamycin and metronidazole for 39 days and followed by antibiotic double-therapy using ceftriaxone and metronidazole for a further 22 days resulted in a radiologically proven reduction of the abscesses to very small remnants which disappeared completely during the follow-up period of two years. Simultaneously the general condition of the patient improved significantly. Repeated attempts at isolation of bacteria, fungi, protozoa and parasites from a subdural empyema failed. The histological examination of an abscess, which was entirely removed on occipital craniotomy, showed a structure resembling actinomycosis.

Bile acid structure and intestinal absorption in the animal model.

A close structure-activity relationship exists between the transport of bile acids (BA) in the liver and intestine; hepatic and intestinal BA transport can be evaluated and compared by using perfused liver and perfused intestine in the rabbit. The passive intestinal absorption is limited to the unconjugated BA, which occurs throughout the small bowel and colon, and is conditioned by the apical membrane lipid composition. A higher diffusion component is found in the terminal ileum compared to the jejunum, and seems to be related to the higher cholesterol-to-phospholipid ratio of the ileal brush border membranes. The active transport system is well characterized and the brush border membrane receptor, cytosolic BA binding proteins and basolateral anion exchange protein have been identified. Recently, the ileal BA transporter has been cloned from the hamster and human ileum and the main cytosolic BA binding protein was cloned from the rat ileum. In the liver, the active transport predominates on the passive diffusion both for conjugated and unconjugated BA. The maximal transport capacity in the liver is tenfold higher than in the intestine, while the Km values are of the same order of magnitude, i.e. in the millimolar range. Neither system operates at its maximum transport rate with prevalent concentrations of BA in portal blood or luminal content.

New insights in the physiology and molecular basis of the intestinal bile acid absorption.

Intestinal bile acid absorption is a fundamental step in the enterohepatic circulation and metabolism of these endogenous compounds. The physiology of the active, sodium coupled transport system for bile acids in the terminal ileum has been extensively studied and characterized. Structure-activity studies have elucidated the requirements for the ileal transport system, and studies with photolabile bile acid derivatives identified the putative ileal bile acid transport proteins. Characterization of the functional sites of the transport system elucidated some of the possible mechanisms which allow the interaction of bile acids and sodium ions with the ileal transporter. Considerable progress has been made during recent years, after the ileal apical and cytosolic bile acid transport proteins have been cloned and characterized. The role of point mutations in bile acid malabsorption has been studied, and the knowledge of the amino acid sequence of the transport proteins will be of help in the investigation of the transport mechanisms.