Bile Acid Molecules Research Articles

Hydrophobicity and haemolytic potential of oxo derivatives of cholic, deoxycholic and chenodeoxycholic acids

The objective of this work was to study the effect of structure of bile acids on their membranolytic potential and extent of overlapping of the information about the membranolytic potential of bile acids and their physico-chemical parameters, namely: retention index RM0 (as a measure of bile acid hydrophobicity, reversed-phase thin-layer chromatography (RPTLC)), lecithin solubilisation (measure of the interaction of bile acids with phospholipids) and critical micellar concentration (CMC).It was found that bile acid concentrations at 100% lysis of erythrocyte membranes is described best by their CMC values, whereas at 50% lysis the parameter used is lecithin solubilisation. This indicates that different mixed micelles are formed in the membrane lysis at lower and higher concentrations of bile acids. Replacement of the hydroxyl (OH) group in the bile acid molecule with an oxo group yields derivatives with lowered hydrophobicity, power of lecithin solubilisation, tendency for self-aggregation as well as the membranolytic activity.

Cholesterol solubilization by oxo derivatives of selected bile acids and their membranotoxicity

This study is concerned with the effect of the structure of bile acids on the solubilization of cholesterol (cholesterol solubilizing capacity (CChm) and the equilibrium micellary solubilization of cholesterol (xChm)). It was found that the replacement of the hydroxy group in the bile acid molecule with an oxo group results in a decrease of the solubilization power of cholesterol. The examined bile acids form two linear groups at the plane of critical micellar concentration (CMC) and solubilization power of cholesterol (CChmorxChm). The group I is formed by bile acids with lower CMCs and higher cholesterol solubilites (deoxycholic (1), chenodeoxycholic (2), hyodeoxycholic (3), cholic (8), 12-oxolithocholic (4), and 7-oxolithocholic (5) acids). On the other hand, the group II is formed by bile acids with higher CMCs and lower cholesterol solubilites (7-oxodeoxycholic (9), 12-oxochenodeoxycholic (10), 12α-hydroxy-3,7-dioxocholanic (11), 7,12-dioxolithocholic (12), 3,7-dioxocholanic (6), and 3,12-dioxocholanic (7) acids). The common conformational characteristics of the bile acid molecules was determined (orientation of OH or oxo grups considered to the mean plane of the steroid skeleton). They form joint groups in the plane of CMC – (CChmorxChm), and the linear function (CChmorxChm) =f(CMC). The osmotic resistance of erythrocytes determines the membranotoxicity of bile acids. 12-Oxolithocholic acid represents the best compromise with regard to the solubilization of cholesterol and membranotoxicity.

NMR Studies Reveal the Role of Biomembranes in Modulating Ligand Binding and Release by Intracellular Bile Acid Binding Proteins

Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted.

Structures of Brucinium Cholate: Bile Acid and Strychnine Derivatives Meet in the Crystals

Single crystals concomitantly containing brucine 1a and a bile acid derivative (cholic acid 2H or 3-epicholic acid 3H) were successfully constructed. In these crystals, cholate 2− constructs inherent robust hydrogen bonded 21 helical assemblies and brucinium 1aH+ achieves 21 helical assemblies through weak interactions. These assemblies excellently synchronize helical pitches to one another. 3-Epicholate 3−, on the other hand, is unable to form a robust hydrogen-bonded motif because of inversion of the hydroxyl group at the 3 position. The present systems are the first example of steroidal bile acid molecules that cocrystallize with equal-sized alkaloidal molecules even when a salt bridge is formed.

Formation of hydrogen-bonded complexes between bile acids and lidocaine in the lidocaine transfer from an aqueous phase to chloroform

Bile acids are amphiphilic molecules, which, in addition to their physiological role, have also acquired increasingly more important pharmacological applications. It has been shown that these compounds have a promoting effect on the transport of many drugs through the cell membrane. Pharmacodynamic studies showed that they exerted a significant effect on the analgesic action of lidocaine. This study is concerned with the determination of the constants of hydrogen-bonded complexes formed between the investigated bile acids and lidocaine. It was found that a prerequisite for forming such a complex is the existence of at least two OH groups or one OH group and one keto group in the bile acid molecule at an appropriate mutual distance. If a keto group is involved in lidocaine binding, the resulting complex has a larger equilibrium constant. A model – multiple linear regression equation – was constructed, relating the molecular descriptors to the equilibrium constant of hydrogen-bonded complex. It was also shown how the complex formed between lidocaine and bile acid influences the rate constant of the decrease of lidocaine concentration in the aqueous phase during its transfer to the chloroform solution of a bile acid. It was found that the complex formed between lidocaine and bile acids plays an important role in the appearance of the depot effect of lidocaine.

Structural and Dynamic Determinants of Ligand Binding in the Ternary Complex of Chicken Liver Bile Acid Binding Protein with Two Bile Salts Revealed by NMR,

Bile acids are physiological detergents facilitating absorption, transport, and distribution of lipid-soluble vitamins and dietary fats;they also play a role as signaling molecules that activate nuclear receptors and regulate cholesterol metabolism. Bile acid circulation is mediated by bile acid binding proteins (BABPs), and a detailed structural study of the complex of BABPs with bile salts has become a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. The solution structure here reported describes, at variance with previously determined singly ligated structures, a BABP in a ternary complex with two bile acid molecules, obtained by employing a variety of NMR experiments. Exchange processes between the two bound chenodeoxycholate molecules as well as the more superficial ligand and the free pool have been detected through ROESY and diffusion experiments. Significant backbone flexibility has been observed in regions located at the protein open end, facilitating bile salts exchange. A detailed description of the protonation states and tautomeric forms of histidines strongly supports the view that histidine protonation modulates backbone flexibility and regulates ligand binding. This structure opens the way to targeted site-directed mutagenesis and interaction studies to investigate both binding and nuclear localization mechanisms.

Aqueous Accessibility of Transmembrane Domain 6 of the Human Apical Na+‐dependent Bile Acid Transporter is Modulated by Substrate Binding

Along the terminal ileum, the human apical Na+-dependent bile acid transporter (hASBT) couples internalization of two sodium ions to one bile acid molecule. Our previous study (Hussainzada et al. (2006) Mol. Pharmacol. 70:) defined transmembrane segment (TM) 7 in forming part of the putative substrate translocation pore using cysteine mutagenesis and thiol modification (SCAM). In the present study, SCAM analysis was extended to TM6, since it lies adjacent to TM7 according to a previously developed model (Zhang et al., (2004) Biochemistry 43:) and may therefore line another face of the translocation pore. Most cysteine substitutions in TM6 were well tolerated and all mutants were expressed at the plasma membrane. Two mutants, I229C and G237C were functionally inactive, suggesting a role for these residues during the transport cycle. Seven residues were sensitive to chemical labeling by various MTS reagents (Val235, Ser239, Leu240, Phe242, Arg246, Ala248 and Tyr253), of which four (Val235, Ser239, Phe242, Arg246) had altered MTS accessibility upon binding of Na+ and/or bile acid substrates. Most TM6 residues were significantly Na+-sensitive, suggesting involvement of this TM in Na+ interactions. In conclusion, TM6 may play a critical role in hASBT transport function by either directly interacting with Na+ ions or by transducing protein conformational changes occurring after Na+ binding. Supported by NIH RO1 DK61425.

Functional Modulation of Nuclear Steroid Receptors by Tauroursodeoxycholic Acid Reduces Amyloid β-Peptide-Induced Apoptosis

Tauroursodeoxycholic acid (TUDCA) prevents amyloid beta-peptide (Abeta)-induced neuronal apoptosis, by modulating both classical mitochondrial pathways and specific upstream targets. In addition, activation of nuclear steroid receptors (NSRs), such as the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) differentially regulates apoptosis in the brain. In this study we investigated whether TUDCA, a cholesterol-derived endogenous molecule, requires NSRs for inhibiting Abeta-induced apoptosis in primary neurons. Our results confirmed that TUDCA significantly reduced Abeta-induced apoptosis; in addition, the fluorescently labeled bile acid molecule was detected diffusely in both cytoplasm and nucleus of rat cortical neurons. Interestingly, experiments using small interfering RNAs (siRNAs) revealed that, in contrast to GR siRNA, MR siRNA abolished the antiapoptotic effect of TUDCA. Abeta incubation reduced MR nuclear translocation while increasing nuclear GR levels. Notably, pretreatment with TUDCA markedly altered Abeta-induced changes in NSRs, including MR dissociation from its cytosolic chaperone, heat shock protein 90, and subsequent translocation to the nucleus. Furthermore, when a carboxy terminus-deleted form of MR was used, nuclear trafficking of both MR and the bile acid was abrogated, suggesting that they translocate to the nucleus as a steroid-receptor complex. Transfection experiments with wild-type or mutant MR confirmed that this interaction was required for TUDCA protection against Abeta-induced apoptosis. Finally, in cotransfection experiments with NSR response element reporter and overexpression constructs, pretreatment with TUDCA significantly modulated Abeta-induced changes in MR and GR transactivation. In conclusion, these results provide novel insights into the specific cellular mechanism of TUDCA antiapoptotic function against Abeta-induced apoptosis and suggest targets for potential therapeutic intervention.

Hepatobiliary elimination of bile acid-modified oligodeoxynucleotides in Wistar and TR− rats: evidence for mrp2 as carrier for oligodeoxynucleotides

As therapeutic antisense tools, oligonucleotides (ODNs) must enter cells to bind to their target structures. ODNs distribute in nearly each tissue with relatively high concentrations in kidney and liver from where excretion into urine and bile occurs. To investigate mechanisms involved in hepatic ODN transport, normal mixed backbone phosphodiester/phosphorothioate ODNs (n-ODN) and two different bile acid-conjugated mixed backbone ODNs (1BA-ODN and 2BA-ODN) were applied to two different rat strains, normal Wistar rats and Wistar TR− rats. In normal Wistar rats, concentration-dependent hepatobiliary elimination of the ODNs was observed with a remarkable increase of excretion of the cholic acid BA-ODN conjugates. In contrast to normal Wistar rats, n-ODN excretion into bile by TR− rats, a mutant Wistar rat strain lacking a functional multidrug resistance-associated protein 2 (mrp2) at the canalicular membrane, was strongly diminished, whereas these rats excreted an ODN conjugated with two cholic acid molecules (2BA-ODN) into bile. Concomitant application of substrates transported by mrp2 such as bromosulfophthalein (BSP) or the synthetic chlorogenic acid derivative S 3025 significantly reduced the biliary appearance of normal ODN and 2BA-ODN in Wistar rats and also in TR− rats. To inhibit the expression of cRNA derived from the Na+-dependent taurocholate cotransporting polypeptide (Ntcp), antisense ODNs were constructed which fully retained the antisense properties when coupled with two bile acid molecules. The results indicate that ODNs are secreted via the mrp2 into bile. In the absence of mrp2, further excretory transport systems with affinity for bile acids seem to be relevant for their excretion. The results further indicate that bile acid tagged ODNs are useful tools for liver specific antisense therapy.

Conserved Residues in the Putative Catalytic Triad of Human Bile Acid Coenzyme A:Amino Acid N-Acyltransferase

Human bile acid-CoA:amino acid N-acyltransferase (hBAT), an enzyme catalyzing the conjugation of bile acids with the amino acids glycine or taurine has significant sequence homology with dienelactone hydrolases and other alpha/beta hydrolases. These enzymes have a conserved catalytic triad that maps onto the mammalian BATs at residues Cys-235, Asp-328, and His-362 of the human sequence, albeit that the hydrolases contain a serine instead of a cysteine. In the present study, the function of the putative catalytic triad of hBAT was examined by chemical modification with the cysteine alkylating reagent N-ethylmaleimide (NEM) and by site-directed mutagenesis of the triad residues followed by enzymology studies of mutant and wild-type hBATs. Treatment with NEM caused inactivation of wild-type hBAT. However, preincubation of wild-type hBAT with the substrate cholyl-CoA before NEM treatment prevented loss of N-acyltransferase activity. Substitution of His-362 or Asp-328 with alanine results in inactivation of hBAT. Although substitution of Cys-235 with serine generated an hBAT mutant with lower N-acyltransferase activity, it substantially increased the bile acid-CoA thioesterase activity compared with wild type. In summary, data from this study support the existence of an essential catalytic triad within hBAT consisting of Cys-235, His-362, and Asp-328 with Cys-235 serving as the probable nucleophile and thus the site of covalent attachment of the bile acid molecule.

Natural Bile Acids and Synthetic Analogues Modulate Large Conductance Ca2+-activated K+ (BKCa) Channel Activity in Smooth Muscle Cells

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid–induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BKCa channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid–induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BKCa channel activity is not due to Ca2+-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid–induced increase in BKCa channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid–induced increases in BKCa channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BKCa channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BKCa channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BKCa channel activity may explain their relaxing effect on smooth muscle.

Natural bile acids and synthetic analogues modulate large conductance Ca2+-activated K+ (BKCa) channel activity in smooth muscle cells.

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid–induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BKCa channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid–induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BKCa channel activity is not due to Ca2+-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid–induced increase in BKCa channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid–induced increases in BKCa channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BKCa channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BKCa channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BKCa channel activity may explain their relaxing effect on smooth muscle.

Comparative cytotoxic and cytoprotective effects of taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) in HepG2 cell line

This study was performed to compare the effects of two hydrophilic bile acids, taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA), on HepG2 cells. Cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations (50–800 μmol/l) of either bile acid, while their cytoprotective effect was tested in comparison with deoxycholic acid (DCA) (350 μmol/l and 750 μmol/l)-induced cytotoxicity. Culture media, harvested at the end of each incubation period, were analyzed to evaluate aspartate transaminase (AST), alanine transaminase and γ-glutamyltranspeptidase release. In addition, the hemolytic effect of THDCA and TUDCA on human red blood cells was also determined. At 24 h of incubation neither THDCA nor TUDCA was cytotoxic at concentrations up to 200 and 400 μmol/l. At 800 μmol/l both THDCA and TUDCA induced a slight increase in AST release. At this concentration and with time of exposure prolonged up to 72 h, THDCA and TUDCA induced a progressive increase of AST release significantly ( P<0.05) higher than that of controls being AST values for THDCA (2.97±0.88 time control value (tcv) at 48 h and 4.50±1.13 tcv at 72 h) significantly greater than those of TUDCA (1.50±0.20 tcv at 48 h and 1.80±0.43 tcv at 72 h) ( P<0.01). In cytoprotection experiments, the addition of 50 μmol/l THDCA decreased only slightly (−5%) AST release induced by 350 μmol/l DCA, while the addition of 50 μmol/l TUDCA was significantly effective (−23%; P<0.05). Higher doses of THDCA or TUDCA did not reduce toxicity induced by 350 μmol/l DCA, but were much less toxic than an equimolar dose of DCA alone. At the concentration used in this experimental model neither THDCA nor TUDCA was hemolytic; however at a very high concentration (6 mmol/l) both bile acids induced 5–8% hemolysis. We conclude that bile acid molecules with a similar degree of hydrophilicity may show different cytotoxic and cytoprotective properties.

Solution structure of ileal lipid binding protein in complex with glycocholate.

Ileal lipid binding protein (ILBP) is a cytosolic lipid-binding protein that binds both bile acids and fatty acids. We have determined the solution structure of porcine ILBP in complex with glycocholate by homonuclear and heteronuclear two-dimensional NMR spectroscopy. The conformation of the protein-ligand complex was determined by restrained energy minimization and simulated annealing calculations after docking the glycocholate ligand into the protein structure. The overall tertiary structure of ILBP is highly analogous to the three-dimensional structures of several other intracellular lipid binding proteins (LBPs). Like the apo-structure, the bile-acid complex of ILBP is composed of 10 anti-parallel beta-strands that form a water-filled clam-shell structure, and two short alpha-helices. Chemical shift data indicated that the bile acid ligand is bound inside the protein cavity. Furthermore, 13C-edited heteronuclear single-quantum correlation-NOESY experiments showed NOE contacts between several aromatic residues located in the proposed bile acid portal region and the 13C-labeled ligand. A single bile acid molecule is bound inside the protein, with the steroid moiety penetrating deep into the water-accessible internal cavity, such that ring A is located right above the plane of the Trp49 indole ring. The carboxylate tail of the ligand is protruding from the proposed bile acid portal into the surrounding aqueous solution. The body of the steroid moiety is oriented with the nonpolar face in contact with the mostly hydrophobic residues of beta-strands C, D and E, while the polar face shows contacts with the side-chains of Tyr97, His99, Glu110 and Arg121 in beta-strands H, I and J. Thus, the conformational arrangement of the ligand complex suggests that the binding affinity of ILBP for bile acid molecules is based mainly on strong hydrophobic interactions inside the protein cavity. Furthermore, this binding mode explains how ILBP can transport unconjugated and conjugated bile acids.

Substrate specificity of the ileal and the hepatic Na+/bile acid cotransporters of the rabbit. II. A reliable 3D QSAR pharmacophore model for the ileal Na+/bile acid cotransporter

To design a reliable 3D QSAR model of the intestinal Na+/bile acid cotransporter, we have used a training set of 17 inhibitors of the rabbit ileal Na+/bile acid cotransporter. The IC50 values of the training set of compounds covered a range of four orders of magnitude for inhibition of [3H]cholyltaurine uptake by CHO cells expressing the rabbit ileal Na+/bile acid cotransporter allowing the generation of a pharmacophore using the CATALYST algorithm. After thorough conformational analysis of each molecule, CATALYST generated a pharmacophore model characterized by five chemical features: one hydrogen bond donor, one hydrogen bond acceptor, and three hydrophobic features. The 3D pharmacophore was enantiospecific and correctly estimated the activities of the members of the training set. The predicted interactions of natural bile acids with the pharmacophore model of the ileal Na+/bile acid cotransporter explain exactly the experimentally found structure–activity relationships for the interaction of bile acids with the ileal Na+/bile acid cotransporter (Kramer et al. 1999. J. Lipid. Res. 40: 1604–1617).▪ The natural bile acid analogues cholyltaurine, chenodeoxycholyltaurine, or deoxycholyltaurine were able to map four of the five features of the pharmacophore model: a) the five-membered ring D and the methyl group at position 18 map one hydrophobic site and the 21-methyl group of the side chain maps a second hydrophobic site; b) one of the α-oriented hydroxyl groups at position 7 or 12 fits the hydrogen bond donor feature; c) the negatively charged side chain acts as hydrogen bond acceptor; and d) the hydroxy group at position 3 does not specifically map any of the five binding features of the pharmacophore model. The 3-hydroxy group of natural bile acids is not essential for interactions with ileal or hepatic Na+/bile acid cotransporters. A modification of the 3-position of a natural bile acid molecule is therefore the preferred position for drug targeting strategies using bile acid transport pathways.—Baringhaus, K-H., H. Matter, S. Stengelin, and W. Kramer. Substrate specificity of the ileal and the hepatic Na+/bile acid cotransporters of the rabbit. II. A reliable 3D QSAR pharmacophore model for the ileal Na+/bile acid cotransporter.

Bile acid secretion during synchronized rat liver regeneration

One major difficulty in interpreting the changes occurring during liver regeneration is the co-existence of non-activated cells and proliferating hepatocytes at all stages of differentiation. The aim of this study was to investigate bile acid (BA) secretion into bile during normal (NLR) and synchronized (SLR) liver regeneration in rats. Regeneration was synchronized by reversible inhibition of ribonucleotide reductase by 10 h treatment with hydroxyurea (HU) shortly after two-third partial hepatectomy. Total BA output as measured by GC–MS increased immediately after partial hepatectomy. This was followed by a further transient enhancement during the next day in the NLR. HU treatment did not significantly modify total BA output, but after releasing synchronized regeneration a marked reduction was observed. This was followed by a recovery to reach values close to those of NLR on day 7 of the regenerative process in SLR. Amidated BA output as measured by HPLC analysis revealed an early enhancement in the proportion of non-conjugated BAs in bile in NLR. However, the profile of conjugated BAs, which was not affected by HU treatment, matched that of total BAs during the first stage of SLR. By contrast, the increase in BA output observed on day 3 of the regenerative process in this group was accounted for by an enhancement in non-conjugated BA secretion. On day 7 of the regenerative process, the proportion of conjugated BA in bile was restored to approximately 100% in this group. Most BA molecules were conjugated with taurine rather than with glycine in all experimental groups, during both NLR and SLR. GC–MS determinations indicated that the magnitude of the cholic acid predominance in all bile samples was significantly modified during liver regeneration. This was increased early after partial hepatectomy and declined toward control values after few (2–3) days. Enhancement in the cholic acid predominance was due to a reduction in the proportion of all other major BAs, above all ursocholic acid and ω-muricholic acid. By contrast, minor BAs in normal control rat bile such as allo-cholic acid were increased during both NLR and SLR, and remained at detectable levels up to day 7. Changes in the proportion of secreted BA species were similar in SLR and NLR except that the early reduction in the proportion of BAs other than cholic acid was more pronounced in SLR and the quantitative importance of the diversity in BA species was recovered earlier in SLR than in NLR. In summary, these results indicate that profound changes in BA secretion during rat liver regeneration do occur. Most of them are probably related to the existence of retro-differentiation/re-differentiation processes which are speeded up by hydroxyurea-induced synchronization of the wave of hepatocyte proliferation.

The adsorption behavior of four bile salt species on cholesterol crystals in water—in connection with cholesterol solubilization

In order to identify the respective roles of bile salts (which are different depending on the species) in the solubilization of cholesterol (Ch) or in the dissolution of Ch-type gallstones, the adsorption behavior of bile salts on a Ch crystal surface in aqueous media was examined using four kinds of sodium salts: the deoxycholate (NaDC), the chenodeoxy-cholate (NaCDC), the ursodeoxycholate (NaUDC), and the cholate (NaC), mainly at 37°C.All the salts studied exhibit a two-step adsorption isotherm (assigned to type IV according to Brunauer's classification), but regarding the adsorption amount, these four bile salts are divided into two groups; the group comprising NaDC and NaCDC has a higher ability to adsorb at the Ch/water interface than the other group which comprises NaUDC and NaC. From the BET plot and/or the Langmuir plot, the maximum amounts of monolayer adsorption, the constants of adsorption equilibrium and the mean surface areas occupied by a bile salt molecule were determined. The amount of monolayer adsorption decreases in the order NaDC, NaCDC, NaUDC and NaC. These data were discussed in relation to the CMC, the hydrophobic indexes of the α and β sides of the bile acid molecule, and the solubilizing power (the molar ratio of solubilized cholesterol to bile salt). The species dependence of Ch solubilization was interpreted in terms of hydrophobic room space in a micelle constructed with aggregated bile salts.

Bile acid derived HMG-CoA reductase inhibitors

The target organ for HMG-CoA reductase inhibitors to decrease cholesterol biosynthesis in hypercholosterolemic patients is the liver. Since bile acids undergo an enterohepatic circulation showing a strict organotropism for the liver and the small intestine, the structural elements of an inhibitor for HMG-CoA reductase were combined with those for specific molecular recognition of a bile acid molecule for selective uptake by hepatocytes. Either, the HMG-CoA reductase inhibitors HR 780 and mevinolin were covalently attached to 3 ξ-( ω-aminoalkoxy)-7 α, 12 α-dihydroxy-5 β-cholan-24-oic acids to obtaine bile acid prodrugs, of the side chain of bile acids at C-17 was replaced by 3,5-dihydroxy-heptanoic acid — a structural element essential for inhibition of HMG-CoA reductase — to obtain hybrid bile acid: HMG-CoA reductase inhibitors. The prodrugs could, as expected, not inhibit rat liver HMG-CoA reductase to a significant extent, whereas the hybrid inhibitors showed a stereospecific inhibition of HMG-CoA reductase from rat liver microsomes with an IC 50-value of 0.7 μM for the most potent compound S 2467 and 6 μM for its diastereomere S 2468. Uptake measurements with isolated rat hepatocytes and ileal brush-border membrane vesicles from rabbit small intestine revealed a specific interaction of both classes of bile acid-derived HMG-CoA reductase inhibitors with the hepatocyte and ileocyte bile acid uptake systems. Photoaffinity labeling studies using 3-azi- or 7-azi-derivatives of taurocholate with freshly isolated rat hepatocytes or rabbit ileal brush-border membrane vesicles revealed a specific interaction of bile acid derived HMG-CoA reductase inhibitors with the respective putatative bile acid transporters in the liver and the ileum demonstrating the bile acid character of these derivatives, both for the prodrugs and the hybrids. Cholesterol biosynthesis in Hep G2 cells was inhibited by the bile acid prodrugs with IC 50-values in the range of 68 nM to 600 nM compared to 13 nM for HR 78 and 130 nM for mevinolin. Among the hybrid inhibitors, S 2467 was the most active compound with an IC 50-value of 16 μM compared to 55 μM for its diastereomere S 2468. Preliminary in vivo experiments showed an inhibition of hepatic cholesterol biosynthesis after oral dosage only with prodrugs such as S 3554, whereas the hybrid molecules were inactive after oral application. Measurement of inhibition of cholesterol biosynthesis by incorporation of [ 14C]octanoate or [ 3H]H 2O into the digitonin-precipitable sterol fraction in the liver and extrahepatic organs demonstrated a significant increase of liver selectivity of HMG-CoA reductase inhibitors such as HR 780 by coupling to bile acids. Taken together, these studies demonstrate that the liver selectivity of an HMG-CoA reductase inhibitor can be increased by combining with bile acid structural elements and making use of specific bile acid transport pathways of the liver.

Intestinal absorption of peptides by coupling to bile acids.

Poor intestinal absorption of peptides greatly limits their use as drugs for the treatment of chronic diseases. Since bile acids are efficiently absorbed by an active, Na(+)-dependent transport system in the ileum of mammals, model peptides of different chain length were attached to the 3-position of modified 3 beta-(omega-amino-alkoxy)-7 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid. These peptide-bile acid conjugates inhibited Na(+)-dependent [3H]taurocholate uptake into brush-border membrane vesicles isolated from rabbit ileum in a concentration-dependent manner. Furthermore, photoaffinity labeling of the bile acid-binding proteins of M(r) 93,000 and 14,000, identified as the protein components of the ileal Na(+)-dependent bile acid transport system in rabbit ileum (Kramer, W., Girbig, F., Gutjahr, U., Kowalewski, S., Jouvenal, K., Müller, G., Tripier, D., and Wess, G. (1993) J. Biol. Chem. 268, 18035-18046) by the photoreactive taurocholate analogue, (3,3-azo-7 alpha, 12 alpha-dihydroxy-5 beta [7 beta, -12 beta-3H]cholan-24-oyl)-2-aminoethanesulfonic acid, was inhibited by the peptide-bile acid conjugates. In contrast, the parent peptides and amino acids neither had a significant effect on [3H]taurocholate uptake by ileal brush-border membrane vesicles nor on photoaffinity labeling of the ileal bile acid-binding membrane proteins. The inhibitory effect of peptide-bile acid conjugates on [3H]taurocholate transport and photoaffinity labeling of the bile acid-binding proteins in rabbit ileal vesicles decreased with increasing chain length of the attached peptide radical. By in vivo ileum perfusion in anesthetized rats an intestinal absorption of the bile acid conjugate S3744 of the fluorescent oxaprolylpeptide 4-nitrobenzo-2-oxa-1,3-diazol-beta-Ala-Phe-5-Opr-Gly (S1037) and secretion of the intact compound into bile could be demonstrated, whereas the parent peptide S1037 or its t-butylester S4404 were not absorbed. The intestinal absorption of S3744 showed a similar temperature dependence as [3H]taurocholate absorption and was inhibited by the presence of taurocholate indicating a carrier-mediated uptake of S3744 via the ileal bile acid transporter. In conclusion, these results indicate that oligopeptides can be made enterally absorable by coupling to modified bile acid molecules making use of the specific intestinal absorption pathway for bile acids. This finding may be of great importance for the design and development of orally active peptide drugs.

Drug targeting to the liver with bile acids: THE “trojan horse” resurrected?

Bile acids are selectively taken up from portal blood into the liver by specific transport systems in the hepatocyte plasma membrane. Therefore, studies were performed to evaluate the potential of bile acids as shuttles to deliver drugs specifically to the liver. The alkylating cytostatic drug chlorambucii and the fluorescent prolyl-4-hydroxylase inhibitor 4-nitrobenzo-2-oxa-1,3-diazol-β-Ala-Phe-5-oxaproline-Gly were covalently linked via an amide bond to 7α, 12α,-dihydroxy-3β-(ω-aminoalkoxy)-5-β-cholan-24-oic acid. The chlorambucil-bile acid conjugates S 2521, S 2539, S 2567, and S 2576 inhibited Na+-dependent [3H]taurocholate uptake in a concentration-dependent manner both into isolated rat hepatocytes and rabbit ileal brush border membrane vesicles, whereas the parent drug chlorambucil showed no significant inhibitory effect. The chlorambucil-bile acid conjugates were able to prevent photoaffinity labeling of bile acid binding proteins in rat hepatocytes by the photolabile [3H]7,7-azo derivative of taurocholic acid indicating their bile acid character. The chlorambucil-bile acid conjugate S 2577 was able to alkylate proteins demonstrating the drug character conserved in the hybridmolecules. Liver perfusion experiments revealed a secretion profile of the chlorambucil-bile acid conjugate S 2576 into bile very similar to taurocholate compared to chlorambucil which is predominantly excreted by the kidney. 4-Nitrobenzo-2-oxa-1,3-diazol-β-Ala-Phe-5-oxaproline-Gly-t-butylester (S 4404), a fluorescent peptide inhibitor of prolyl-4-hydroxylase, was not transported in intact form from portal blood into bile in contrast to its bile acid conjugate S 3744; about 25% of the peptide-bile acid conjugate S 3744 was secreted in intact form into bile within 40 min compared with less than 4% of the parent oxaprolylpeptide S 4404. In conclusion, these studies reveal that modified bile acid molecules can be used as “Trojan horses” to deliver a drug molecule specifically into the liver and the biliary system. This offers important pharmacological options for the development of liver-specific drugs. This study was designed to determine whether the conjugation product of L-T3 with cholic acid would result in a liver-targeted compound (CGH 509A) with hypocholesterolemic (HC) activity significantly dissociable from cardiovascular (CV) and thyroxine-suppressing (TS) effects normally observed with thyroid hormone. Evaluation of HC activity in lipemic rats showed that CGH 509A was 6 times less potent than L-T3 with ED25 values estimated at 150 and 25 nmol/kg, respectively. CV function measured as changes in atrial rate, atrial tension and heart weight was determined in euthyroid rats. CGH 509A was at least 64 times less cardio-stimulant than L-T3, with minimum effective doses estimated at 2350 and 37 nmol/kg, respectively. TS activity was assessed in euthyroid rats as the potency of any compound to reduce plasma T4 levels. CGH 509A was 50 times less potent than L-T3 with ED50 values estimated at 900 and 18 nmol/kg, respectively. From these results, it is clear that, while L-T3 was equally potent on HC, CV and TS activities, the HC potency of CGH 509A was at least 15 and 6 times greater than its CV and TS potencies, respectively.