Exploitation of bile acid transport systems in prodrug design

The enterohepatic circulation of bile acids is one of the most efficient recycling routes in the human body. It is a complex process involving numerous transport proteins, which serve to transport bile acids from the small intestine into portal circulation, from the portal circulation into the hepatocyte, from the hepatocyte into the bile, and from the gall bladder to the small intestine. The tremendous transport capacity and organ specificity of enterohepatic circulation combined with versatile derivatization possibilities, rigid steroidal backbone, enantiomeric purity, availability, and low cost have made bile acids attractive tools in designing pharmacological hybrid molecules and prodrugs with the view of improving intestinal absorption, increasing the metabolic stability of pharmaceuticals, specifically targeting drugs to organs involved in enterohepatic circulation, as well as sustaining therapeutically reasonable systemic concentrations of active agents. This article briefly describes bile acid transport proteins involved in enterohepatic circulation, summarizes the key factors affecting on the transport by these proteins, and reviews the use of bile acids and their derivatives in designing prodrugs capable of exploiting the bile acid transport system.     

Analysis of bile acids in human biological samples by microcolumn separation techniques: A review

Bile acids are a group of compounds essential for lipid digestion and absorption with a steroid skeleton and a carboxylate side chain usually conjugated to glycine or taurine. Bile acids are regulatory molecules for a number of metabolic processes and can be used as biomarkers of various disorders. Since the middle of the twentieth century, the detection of bile acids has evolved from simple qualitative analysis to accurate quantification in complicated mixtures. Advanced methods are required to characterize and quantify individual bile acids in these mixtures. This article overviews the literature from the last two decades (2000–2020) and focuses on bile acid analysis in various human biological samples. The methods for sample preparation, including the sample treatment of conventional (blood plasma, blood serum, and urine) and unconventional samples (bile, saliva, duodenal/gastric juice, feces, etc.) are shortly discussed. Eventually, the focus is on novel analytical approaches and methods for each particular biological sample, providing an overview of the microcolumn separation techniques, such as high-performance liquid chromatography, gas chromatography, and capillary electrophoresis, used in their analysis. This is followed by a discussion on selected clinical applications.     

Bile acids and their oxo derivatives: Potential inhibitors of carbonic anhydrase I and II,androgen receptor antagonists and CYP3A4 substrates

Some biological properties of bile acids and their oxo derivatives have not been sufficiently investigated, although the interest in bile acids as signaling molecules is rising. The aim of this work was to evaluate physico‐chemical parametar b (slope) that represents the lipophilicity of the examined molecules and to investigate interactions of bile acids with carbonic anhydrase I, II, androgen receptor and CYP450s. Thirteen candidates were investigated using normal‐phase thin‐layer chromatography in two solvent systems. Retention parameters were used in further quantitative structure–activity relationship analysis and docking studies to predict interactions and binding affinities of examined molecules with enzymes and receptors. Prediction of activity on androgen receptor showed that compounds 3α ‐hydroxy‐12‐oxo‐5β ‐cholanoic and 3α ‐hydroxy‐7‐oxo‐5β ‐cholanoic acid have stronger antiandrogen activity than natural bile acids. The inhibitory potential for carbonic anhydrase I and II was tested and it was concluded that molecules 3α ‐hydroxy‐12‐oxo‐5β ‐cholanoic, 3α ‐hydroxy‐7‐oxo‐5β ‐cholanoic, 3,7,12‐trioxo‐5β ‐cholanoic acid and hyodeoxycholic acid show the best results. Substrate behavior for CYP3A4 was confirmed for all investigated compounds. Oxo derivatives of bile acids show stronger interactions with enzymes and receptors as classical bile acids and lower membranolytic activity compared with them. These significant observations could be valuable in consideration of oxo derivatives as building blocks in medicinal chemistry.     

胆酸盐参与的自组装及微纳米材料制备

胆酸盐类物质可看作是一类阴离子型甾族生物表面活性剂,鉴于其特殊的两亲性骨架结构、独特的物理化学性质及其良好的生物相容性和环境友好性,其在溶液中能够参与超分子自组装形成有序聚集结构,且可以作为模板在微纳材料制备领域有着重要应用。本文结合我们课题组的研究工作,综述了近期国内外相关研究,详细介绍了生物小分子氨基酸对胆酸盐聚集行为的影响、胆酸盐参与形成的超分子凝胶及胆酸盐参与构筑的微纳米材料制备等方面的研究进展,以期对胆酸盐参与的自组装及微纳米材料制备领域的研究有更全面更深入的了解,为后续的应用研究提供坚实的基础。     

High-Performance Liquid Chromatographic Behavior of 2-,4- and 6-Hydroxylated Bile Acid Stereoisomers

Abstract

High-performance liquid chromatographic behavior has been investigated for 40 stereoisomeric 2-, 4- and 6-hydroxylated bile acids, most of which possess a vicinal glycol structure, as their 4-nitrophthalimidomethyl (NPM) ester derivatives on a reversed-phase column using methanol-water systems as a mobile phase. The bile acid NPM esters were further derivatized to the so-called “mixed” acetonide-NPM esters, whereby the stereochemical relationships (α, α-and β,β-cis, diaxial trans or diequatorial trans) of 2,3-, 3,4-and 6,7-diol groups in the molecules were reflected to changes in the capacity factors of bile acids.     

The use of tandem mass spectrometry for the differentiation of bile acid isomers and for the identification of bile acids in biological extracts

Tandem mass spectrometric (MS/MS) techniques hae been widely used for the differentiation of isomeric compounds, since their spectra may show differences sufficient to distinguish between them. There are several different ways by which the MS/MS data can be obtained depending on the energies of the ions and the collisions. In this paper MS/MS spectra have been obtained for a group of isomeric bile acids using: 1, low-energy ions and low-energy collisions in a triple quadrupole mass spectrometer by liquid chromatography/MS/MS; 2, high-energy ions and low-energy collisions in a hybrid mass spectrometer by fast-atom bombardment MS/MS. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) has also been used to identify the bile acids present in biological matrices such as bile extracts.     

新型饱和脂肪酸胆酸缀合物的合成及抗胆结石活性研究

胆结石是常见多发病, 但临床缺乏有效的治疗药物. 饱和脂肪酸与胆酸的缀合物能有效预防胆固醇结晶、溶解体内胆固醇结石. 以胆酸或熊去氧胆酸24位羧基为连接位点, 以氨基酸为连接子, 通过酰胺键将载体与具有溶石活性的饱和脂肪酸偶联, 设计合成了一系列新型脂肪酸胆酸缀合物, 其结构经元素分析, IR, 1H NMR和MS光谱分析确证. 通过测定化合物对模型胆汁溶液胆固醇结晶及模型小鼠胆结石的溶解活性, 研究了其体内外溶石活性.     

Investigation of side chain liquid crystal polymers bearing cholesterol and bile acid derivatives

Cholic acid (or 3a,7a,12a-trihydoxyl-5a-cholan-24-oic acid) and lithocholic acid (or 3a-hydroxyl-5a-cholanic-24-oic acid) are commonly occurring bile acids synthesized from cholesterol in the liver in mammals. They all possess a steroid skeleton containing four rings, three with six carbons and one with five carbons. The transformation of cholesterol to cholic acid results in two major structural changes that affect the steroid skeleton. The first is the hydrogenation of the double bond between C5 and C6 and the second is a conformational flip of ring A from the 5a-position to the 5a-position. In addition, one or more hydroxyl groups are added to the steroid skeleton. Outside of the ring system, C24 is converted from a saturated alkyl to a carboxylic acid group.Side chain polymers based on cholesterol moiety have been made as reported in the literature.Since bile acids and cholesterol are all in the family of steroid molecules, it is of interest to investigate whether bile acids may also act as mesogenic groups.Therefore, flexible spacer groups with 10 carbons are introduced between bile acid skeleton and the poymerizable double bonds. The monomers and polymers are compared with cholesterol and dihydrocholesterol monomers and polymers with the same spacers. Dihydrocholesterol is chosen to investigate the influence of the double bond in the formation of LC, given that both cholesterol and dihydrocholesterol have a planar structure but there is no double bond in the latter. These monomers and their corresponding polymers were characterized for their liquid crystalline (LC) properties by differential scanning calorimetry (DSC), polarizing optical microscopy (POM) and X-ray diffraction.It was found that only the compounds bearing the planar cholesterol moieties possess LC phases. It is concluded that the 5a-configuration between the first and second cycles on the steroid skeleton of bile acids does not favor proper alignment of the rigid part of the bile acid moieties. It is interesting to see the effect of a small variation in structure on the properties of the otherwise structurally-similar compounds and the materials made from them.     

Bile acids as stationary phase in liquid chromatography

Summary Bile acids chemically bonded onto aminopropylsilica have been employed as stationary phases in liquid chromatography. Bile acid aggregates were dynamically formed around molecules chemically anchored on the supports when the eluent contained bile salts. The bile salt aggregates achieved the separation of 1,1′-binaphthyl-2,2′-diyl-hydrogenphosphate enantiomers and dansyl amino acids.     

Self-assembly in aqueous bile salt solutions

The salts of bile acids (“bile salts”) self-assemble in aqueous solution, similar to classical amphiphiles. The micellization is not only driven by the hydrophobic effect, but also hydrogen binding. Moreover, instead of a small, hydrophilic head and a flexible, hydrophobic tail, bile salts are rigid, almost flat molecules with weakly separated hydrophobic and hydrophilic faces. This results in a complex self-assembly behaviour with very distinct aggregate properties. Some characteristics resemble the behaviour of classical amphiphiles, while others are very different and reminiscent of other classes of molecules, for example low-molecular weight gelators or chromonic materials. We review the peculiar properties of bile salt aggregates, concentrating on general trends rather than specific values and comparing them to classical amphiphiles.     

Improved method for the determination of the major neutral steroids and unconjugated bile acids in human faeces using capillary gas chromatography

An improved method has been developed for the determination of the major neutral steroids (cholesterol and 5 beta-cholestan-3 beta-ol) and unconjugated bile acids (deoxycholic acid and lithocholic acid) in human faeces, using capillary gas chromatography with flame ionization detection. The freeze-dried faecal sample was subjected to a two-stage Soxhlet extraction followed by an aqueous alkali-organic solvent partition step to separate neutral steroids from bile acids. The neutral steroids were analysed as their trimethylsilyl ether derivatives on an OV-1 capillary column. The bile acids were further purified on a Sep-Pak C18 cartridge and then fractionated on a Sep-Pak SIL cartridge. Unconjugated bile acids were analysed as their methyl ester-trimethylsilyl ether derivatives also on an OV-1 capillary column. Quantitation of neutral steroids and unconjugated bile acids was achieved by reference to appropriate internal standards, added to the faecal extract immediately after the Soxhlet extraction stage. The method is being used in a study of the effect of diet on the metabolic activity of human gut flora.     

Fluorescence high-performance liquid chromatographic determination of free and conjugated bile acids in serum and bile using 1-bromoacetylpyrene as a pre-labeling reagent

A fluorescence high-performance liquid chromatographic method is described for the determination of free and conjugated bile acids in serum and bile. Free and conjugated bile acids are extracted from serum or bile using a Sep-Pak C18 cartridge and then fractionated on a piperidinohydroxypropyl Sephadex LH-20 column. Free and glycine-conjugated bile acids are labeled with 1-bromoacetylpyrene in acetonitrile using dicyclohexyl-18-crown-6-ether as catalyst. Taurine-conjugated bile acids are hydrolyzed by cholylglycine hydrolase and then derivatized by the same reagent. Derivatized bile acids are separated stepwise on a reversed-phase column (Radial Pak A) using acetonitrile-methanol-water (A) (100 : 50 : 40) and (B) (100 : 50 : 20) as mobile phase. The eluate is monitored by a fluorophotometer at 370 nm (excitation) and 440 nm (emission). Linearities of fluorescence intensities (peak heights) with the amounts of free and conjugated bile acids were obtained between 50 pmol and 200 pmol for free bile acids and between 25 pmol and 100 pmol for glycine-conjugated bile acids, respectively. Recoveries from serum and bile samples are not less than 90%. This method is sensitive, reliable and useful for the simultaneous determination of free and conjugated bile acids in serum and bile.     

Differentiation of various traditional Chinese medicines derived from animal bile and gallstone: simultaneous determination of bile acids by liquid chromatography coupled with triple quadrupole mass spectrometry

Animal biles and gallstones are popularly used in traditional Chinese medicines, and bile acids are their major bioactive constituents. Some of these medicines, like cow-bezoar, are very expensive, and may be adulterated or even replaced by less expensive but similar species. Due to poor ultraviolet absorbance and structural similarity of bile acids, effective technology for species differentiation and quality control of bile-based Chinese medicines is still lacking. In this study, a rapid and reliable method was established for the simultaneous qualitative and quantitative analysis of 18 bile acids, including 6 free steroids (cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, hyodeoxycholic acid, and ursodeoxycholic acid) and their corresponding glycine conjugates and taurine conjugates, by using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This method was used to analyze six bile-based Chinese medicines: bear bile, cattle bile, pig bile, snake bile, cow-bezoar, and artificial cow-bezoar. Samples were separated on an Atlantis dC₁₈ column and were eluted with methanol–acetonitrile–water containing ammonium acetate. The mass spectrometer was monitored in the negative electrospray ionization mode. Total ion currents of the samples were compared for species differentiation, and the contents of bile acids were determined by monitoring specific ion pairs in a selected reaction monitoring program. All 18 bile acids showed good linearity (r² > 0.993) in a wide dynamic range of up to 2000-fold, using dehydrocholic acid as the internal standard. Different animal biles could be explicitly distinguished by their major characteristic bile acids: tauroursodeoxycholic acid and taurochenodeoxycholic acid for bear bile, glycocholic acid, cholic acid and taurocholic acid for cattle bile, glycohyodeoxycholic acid and glycochenodeoxycholic acid for pig bile, and taurocholic acid for snake bile. Furthermore, cattle bile, cow-bezoar, and artificial cow-bezoar could be differentiated by the existence of hyodeoxycholic acid and the ratio of cholic acid to deoxycholic acid. This study provided bile acid profiles of bile-based Chinese medicines for the first time, which could be used for their quality control.     

Chemoenzymatic route to macrocyclic hybrid peptide/polyketide-like molecules

Hybrid peptide-polyketides are a class of medically and biologically important natural products characterized by stereochemical and functional diversity. In their biosynthesis, hybrids are often macrocyclized to achieve rigid structures that populate bioactive conformations. We herein present a chemoenzymatic strategy to access the stereochemical and functional diversity found in macrocyclic hybrid natural products in a manner amenable to efficient library synthesis. Our method makes use of small building blocks in the form of Fmoc-protected epsilon-amino acids containing embedded polyketide functionality. The building block approach allows for combinatorial synthesis of linear molecules that can be activated as soluble thioesters or tethered to a solid-phase resin. We demonstrate that these linear molecules are substrates for macrocyclization by a tolerant catalyst, TycC TE, derived from a nonribosomal peptide synthetase. The method should allow for access to diverse structures with hybrid peptide-polyketide character that can be screened for improved or novel activities.     

Separation and quantitation of fatty acids, sterols and bile acids in feces by gas chromatography as the butyl ester-acetate derivatives

A system allowing the separation and quantitation of individual species of fecal fatty acids, sterols and bile acids in a single chromatographic step is described. The system is based on the butylation of carboxyl groups and acetylation of free hydroxyls of the compounds in fecal lipid extracts, followed by their resolution by temperature-programmed gas chromatography. As the butyl ester-acetate derivatives, fatty acids, sterols and bile acids elute separately and with no overlap on a variety of chromatographic columns, obviating the need for prior separation of each class by thin-layer or column chromatography. All common bile acids, a wide variety of sterols and keto-steroids, as well as saturated and unsaturated fatty acids may be routinely resolved. Quantitation is facilitated by the addition of the internal standards, heptadecanoic acid and nor-deoxycholic acid to each sample. With an automatic sample injector, the rapid assessment of a wide range of potential risk factors for colorectal cancer may be carried out in large numbers of samples.     

Novel Ligands Incorporating Phosphinoxide Groups as Particularly Efficient Sensitizers for Lanthanide Emission

Helical molecular assemblies of bile acids and their derivatives were systematically investigated. These molecules have a common asymmetric structure with three different directions, and form characteristic inclusion crystals with the following hierarchical structure. First, such molecules arrange along their helical axes to yield the helical tapes with a definite direction. Second, the helical tapes are combined in a parallel fashion by using their side-chains through hydrogen bonds to produce chiral sheets. Third, the sheets stack together through van der Waals forces in a parallel or antiparallel fashion. Fourth, there exist chiral spaces for including guests among their sheets. Such hierarchical structure enables us to explain a role of the side-chains with different hydrogen bonding groups and length.     

Effect of cocrystallization due to bile acid on molecular-ion sensitivity of biological phospholipids in Bi cluster time-of-flight secondary ion mass spectrometry measurements

Bi cluster time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a useful method for evaluating organic surfaces. However, its ability to detect large molecules is limited. One of the problems is that the sensitivities of macromolecules are lower than those of small molecules because larger molecules tend to exhibit lower ionization efficiencies and/or higher probabilities of fragmentation. Matrix-enhanced (ME)-SIMS is a sensitivity enhancement technique for intact molecular ions. The crystal structure of a mixed substance composed of an analyte and a matrix is known to affect the sensitivity of the analysis target. In this study, the effect of cocrystallization, which occurs due to the presence of bile acid, on the molecular-ion sensitivity was investigated using Bi cluster TOF-SIMS. Biological phospholipids and bile acids, which exhibit surfactant behaviors, were selected as the evaluated molecules and additives, respectively. The mass spectra indicated that the secondary-ion yields of phospholipids with bile acid were substantially greater than those of the pristine lipid. Specifically, samples with an analyte/bile acid ratio of 1:100 achieved approximately 60–100-fold sensitivity enhancement of [M + H]⁺ and [2M + H]⁺ molecular ions than the sensitivity achieved with the pristine samples. In the evaluation of molecular distribution, higher signal counts of intact ions were obtained from the cocrystallization area, although less-fragmented ions were emitted from these regions. Consequently, the results indicate that the cocrystallization due to the presence of bile acid provides an effective crystal structure for facilitating emission of larger molecules.     

Gas chromatographic determination of serum bile acids — Application of a novel extraction procedure

Summary The profile of serum bile acids is a result of their liver metabolism and enterohepatic circulation.In the present work size exclusion chromatography is used for extraction of serum bile acids to optimize the methodology for analyzing serum bile acids by high resolution gas chromatography.Compared to other extraction methods like adsorption-[1–3] or reversed phase chromatography [4,5], this novel technique yielded a satisfactory recovery (75–104%) with high reproducibility. Therefore a reliable determination of serum bile acids is possible.     

Synthesis of novel steroid-peptoid hybrid macrocycles by multiple multicomponent macrocyclizations including bifunctional building blocks (MiBs)

Two new groups of cholane-peptoid hybrid macrocycles were produced by implementing novel combinations of the MiB methodology. Steroid-based hybrid macrolactams including heterocycle and aryl moieties were obtained by utilizing cholanic dicarboxylic acids and diamines in a bidirectional double Ugi-Four-Component (Ugi-4CR) based macrocyclization protocol. Alternatively, N-substituted cyclocholamides were produced from a cholanic pseudo-amino acid by an Ugi-4CR-based cyclooligomerization approach. Both types of macrocycles are steroid-peptoid hybrid macrocycles containing exocyclic peptidic chains. These novel frameworks are a result of the use of bile acids bifunctionalized with carboxylic and amino functionalities as bifunctional building blocks of the Ugi-MiB approach.