Bile Acid Structure Research Articles

Novel NTCP ligand dimeric bile acid conjugated with ASO reduce hepatitis B virus surface antigen in vivo

Hepatitis B virus (HBV) specifically infects hepatocytes and causes severe liver diseases. However, functional cure is rarely attainable by current treatment modalities. Anti-sense oligonucleotide (ASO), which targets pregenomic RNAs to reduce hepatitis B virus (HBV) antigen production and viral replication, has been studied as a novel treatment strategy for HBV cure and can be conjugated with N-acetylgalactosamine (GalNAc), thereby enhancing hepatocyte uptake via the asialoglycoprotein receptor (ASGPR). In comparison to GalNAc-ASO conjugation, clinical research indicates that unconjugated ASO is more effective in reducing hepatitis B virus surface antigen level. Recent studies have revealed that human sodium taurocholate co-transporting polypeptide (NTCP) is a functional cellular receptor for hepatitis B virus (HBV), and the bivalent bile acid structure may interact with multiple binding sites on NTCP, yielding much stronger interaction and substantially improved binding affinity. In this study, we synthesized NTCP ligand-antisense oligonucleotide (ASO) conjugation and evaluated the potential of antiviral therapy specifically reduction of HBV antigenemia in mice in vivo.

Lactobacillus plantarum CCFM1180 attenuates obesity induced by estrogen deficiency by activating estrogen receptor alpha in abdominal adipose tissue and regulating gut microbiota-derived metabolitesestrogen

Lipid metabolism disorders commonly occur during menopause. Estrogen deficiency has been shown to lead to excessive energy intake and abnormal lipid metabolism in ovariectomized rats, resulting in obesity. Probiotics exhibit anti-obesity properties, and their underlying mechanism has been widely reported. In this study, we demonstrated the metabolic benefits of Lactobacillus plantarum CCFM1180 in suppressing appetite, controlling body weight, correcting obesity-induced abnormalities, enhancing liver lipid metabolism, and protecting liver function in estrogen-deficient rats. The mechanisms associated with the anti-obesity and anti-dyslipidemia effects of CCFM1180 on estrogen-deficient rats were clarified. The results showed that CCFM1180 dramatically reduced food intake by activating the expression of estrogen receptor alpha (ERα) and increasing the level of leptin in abdominal adipose tissue. These changes, combined with the increased butyrate concentration and recovered bile acid structure, helped enhance lipid metabolism. Additionally, CCFM1180 treatment was found to be safer than exogenous estrogen supplementation. Thus, L. plantarum CCFM1180 could be considered a new therapeutic strategy for preventing and alleviating menopausal lipid abnormalities.

Chemical exploration of TGR5 functional hot-spots: Synthesis and structure-activity relationships of C7- and C23-Substituted cholic acid derivatives

The activation of TGR5 bestows on bile acids the ability to modulate nongenomic signaling pathways, which are responsible of physiological actions including immunosuppressive and anti-inflammatory properties as well as the regulation of glucose metabolism and energy homeostasis. TGR5 agonists have therefore emerged in drug discovery and preclinical appraisals as promising compounds for the treatment of liver diseases and metabolic syndrome. In this study, we have been devising site-selected chemical modifications of the bile acid scaffold to provide novel chemical tools able to modulate the functions of TGR5 in different tissues. Biological results of the tested collection of semisynthetic cholic acid derivatives were used to extend the structure-activity relationships of TGR5 agonists and to clarify the molecular basis and functional role of TGR5 hot-spots in the receptor activation and selectivity. Some unexpected properties deriving from the molecular structure of bile acids have been unveiled as relevant to the receptor activation and may hence be used to design novel, selective and potent TGR5 agonists.

The 16α-hydroxylated Bile Acid, Pythocholic Acid Decreases Food Intake and Increases Oleoylethanolamide in Male Mice.

Modulation of bile acid (BA) structure is a potential strategy for obesity and metabolic disease treatment. BAs act not only as signaling molecules involved in energy expenditure and glucose homeostasis, but also as regulators of food intake. The structure of BAs, particularly the position of the hydroxyl groups of BAs, impacts food intake partly by intestinal effects: (1) modulating the activity of N-acyl phosphatidylethanolamine phospholipase D, which produces the anorexigenic bioactive lipid oleoylethanolamide (OEA) or (2) regulating lipid absorption and the gastric emptying-satiation pathway. We hypothesized that 16α-hydroxylated BAs uniquely regulate food intake because of the long intermeal intervals in snake species in which these BAs are abundant. However, the effects of 16α-hydroxylated BAs in mammals are completely unknown because they are not naturally found in mammals. To test the effect of 16α-hydroxylated BAs on food intake, we isolated the 16α-hydroxylated BA pythocholic acid from ball pythons (Python regius). Pythocholic acid or deoxycholic acid (DCA) was given by oral gavage in mice. DCA is known to increase N-acyl phosphatidylethanolamine phospholipase D activity better than other mammalian BAs. We evaluated food intake, OEA levels, and gastric emptying in mice. We successfully isolated pythocholic acid from ball pythons for experimental use. Pythocholic acid treatment significantly decreased food intake in comparison to DCA treatment, and this was associated with increased jejunal OEA, but resulted in no change in gastric emptying or lipid absorption. The exogenous BA pythocholic acid is a novel regulator of food intake and the satiety signal for OEA in the mouse intestine.

Bile Acids: Physiological Activity and Perspectives of Using in Clinical and Laboratory Diagnostics.

Bile acids play a significant role in the digestion of nutrients. In addition, bile acids perform a signaling function through their blood-circulating fraction. They regulate the activity of nuclear and membrane receptors, located in many tissues. The gut microbiota is an important factor influencing the effects of bile acids via enzymatic modification. Depending on the rate of healthy and pathogenic microbiota, a number of bile acids may support lipid and glucose homeostasis as well as shift to more toxic compounds participating in many pathological conditions. Thus, bile acids can be possible biomarkers of human pathology. However, the chemical structure of bile acids is similar and their analysis requires sensitive and specific methods of analysis. In this review, we provide information on the chemical structure and the biosynthesis of bile acids, their regulation, and their physiological role. In addition, the review describes the involvement of bile acids in various diseases of the digestive system, the approaches and challenges in the analysis of bile acids, and the prospects of their use in omics technologies.

Bile acid detergency: permeability, inflammation, and effects of sulfation.

Bile acids are amphipathic, detergent molecules. The detergent effects of di-α-hydroxy-bile acids are relevant to several colonic diseases. The aims were to review the concentrations of bile acids reaching the human colon in health and disease, the molecular structure of bile acids that determine detergent functions and the relationship to human diseases (neuroendocrine tumors, microscopic colitis, active celiac disease, and ulcerative colitis, Crohn's disease and ileal resection), the relationship to bacterial uptake into the mucosa, mucin depletion, and epithelial damage, the role of bile acids in mucosal inflammation and microscopic colitis, and the role of sulfation of bile salts in detoxification or prevention of the detergent effects of bile acids. The concentrations of bile acids reaching the human colon range from 2 to 10 mM; di-α-hydroxy bile acids are the only bile acids with detergent effects that include mucin depletion, mucosal damage, bacterial uptake, and microscopic inflammation that may be manifest in diseases associated with no overt inflammation of the mucosa, such as bile acid diarrhea, ileal diseases such as neuroendocrine tumors, ileal resection, and nonalcoholic steatohepatitis. Sulfation inactivates colonic secretion due to primary bile acids, but it may render secondary bile acids proinflammatory in the colon. Other evidence in preclinical models of inflammatory bowel disease (IBD) suggests reduced sulfation causes barrier dysfunction, inflammation, or carcinogenesis. These advances emphasize relevance and opportunities afforded by greater understanding of the chemistry and metabolism of bile acids, which stands to be further enhanced by research into the metabolic interactions of microbiota with bile acids.

Analysis of fecal bile acids and metabolites by high resolution mass spectrometry in farm animals and correlation with microbiota

There is a growing interest in the named “acidic sterolbiome” and in the genetic potential of the gut microbiome (GM) to modify bile acid (BA) structure. Indeed, the qualitative composition of BAs in feces correlates with the bowel microorganisms and their collective genetic material. GM is responsible for the production of BA metabolites, such as secondary and oxo-BAs. The specific BA profiles, as microbiome-host co-metabolic products, could be useful to investigate the GM-host interaction in animals under physiological conditions, as well as in specific diseases. In this context, we developed and validated an ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry method for the simultaneous analysis of up to 21 oxo-BAs and their 9 metabolic precursors. Chromatographic separation was achieved in 7 min with adequate analytical performance in terms of selectivity, sensitivity (LOQ from 0.05 to 0.1 µg/mL), accuracy (bias% < 5%), precision (CV% < 5%) and matrix effect (ME% < 10%). A fast solvent extraction protocol has been fine-tuned, achieving recoveries > 90%. In parallel, the gut microbiota assessment in farming animals was evaluated by 16S rRNA next-generation sequencing, and the correlation with the BA composition was performed by multivariate analysis, allowing to reconstruct species-specific associations between the BA profile and specific GM components.

Melancholé: The Dark Side of Bile Acids and Its Cellular Consequences.

The first bile acid, cholic acid, was discovered in 1848 from ox gallbladder, and the bile acid structure solved by Otto Wieland was recognized with the 1927 Nobel Prize in Chemistry.1 Primary bile acids are synthesized in the liver from cholesterol and conjugated to taurine or glycine for biliary secretion. Bile acid synthesis is catalyzed by approximately 17 enzymes via the classic or alternative pathways. The classic bile acid synthesis pathway is initiated by cholesterol 7α-hydroxylase (CYP7A1), followed by sterol 12α-hydroxylase (CYP8B1).

The Gut Microbiota-Bile Acids-TGR5 Axis Mediates Eucommia ulmoides Leaf Extract Alleviation of Injury to Colonic Epithelium Integrity.

Eucommia ulmoides leaves (EL) are rich in phenolic acids and flavonoids, showing enhancing intestinal health effects. The intestinal microbiota-bile acid axis plays important roles in the occurrence and recovery of inflammatory bowel disease (IBD). However, whether EL extract (ELE) has regulatory effects on the intestinal microbiota, bile acid metabolism, and IBD is still unclear. To fill this gap, 2% dextran sulfate sodium (DSS)-induced mild IBD in a C57BL/6J mouse model that was treated with 200 or 400 mg/kg (intake dose/body weight) ELE was used. Oral ELE supplementation alleviated DSS-induced shortening of colon and colonic epithelial injury. Compared with the DSS group, ELE supplementation significantly decreased Toll-like receptor 4 (TLR4) and interlukin-6 (IL-6) and increased occludin and claudin-1 mRNA expression level in the colon (p < 0.05). Combined 16S rRNA gene sequencing and targeted metabolomic analyses demonstrated that ELE significantly improved the diversity and richness of the intestinal microbiota, decreased the abundance of Bacteroidaceae, and increased Akkermansiaceae and Ruminococcaceae abundance (p < 0.05) compared with DSS-induced IBD mice. Moreover, ELE significantly increased the serum contents of deoxycholic acid (DCA) and tauroursodeoxycholic acid (TUDCA), which were highly positively correlated with Akkermansia and unidentified_Ruminococccaceae relative to the DSS group. We then found that ELE increased Takeda G-protein coupled receptor 5 (TGR5), claudin-1, and occludin mRNA expression levels in the colon. In the Caco-2 cell model, we confirmed that activation of TGR5 improved the reduction in transepithelial electoral resistance (TEER) and decreased the permeability of FITC-dextran on monolayer cells induced by LPS (p < 0.05). siRNA interference assays showed that the decrease in TGR5 expression led to the decrease in TEER, an increase in FITC-dextran permeability, and a decrease in claudin-1 protein expression in Caco-2 cells. In summary, ELE alleviated IBD by influencing the intestinal microbiota structure and composition of bile acids, which in turn activated the colonic TGR5 gene expression in the colon and promoted the expression of tight junction proteins. These findings provide new insight for using ELE as a functional food with adjuvant therapeutic effects in IBD.

The Physiological Importance of Bile Acid Structure and Composition on Glucose Homeostasis

Studies have identified several effects of bile acids (BAs) in glucose homeostasis, energy expenditure, and body weight control, through receptor-dependent and independent mechanisms. BAs are produced from cholesterol and characterized by their structures, which result from enzymes in the liver and the gut microbiota. The aim of this review is to characterize the effects of BA structure and composition on diabetes. The hydroxyl groups of BAs interact with binding pockets of receptors and enzymes that affect glucose homeostasis. Human and animal studies show that BA composition is associated with insulin resistance and food intake regulation. The hydroxylation of BAs and BA composition contributes to glucose regulation. Modulation of BA composition has the potential to improve glucose metabolism.

Diet-induced remission in chronic enteropathy is associated with altered microbial community structure and synthesis of secondary bile acids

BackgroundThe microbiome has been implicated in the initiation and persistence of inflammatory bowel disease. Despite the fact that diet is one of the most potent modulators of microbiome composition and function and that dietary intervention is the first-line therapy for treating pediatric Crohn’s disease, the relationships between diet-induced remission, enteropathy, and microbiome are poorly understood. Here, we leverage a naturally-occurring canine model of chronic inflammatory enteropathy that exhibits robust remission following nutritional therapy, to perform a longitudinal study that integrates clinical monitoring, 16S rRNA gene amplicon sequencing, metagenomic sequencing, metabolomic profiling, and whole genome sequencing to investigate the relationship between therapeutic diet, microbiome, and disease.ResultsWe show that remission induced by a hydrolyzed protein diet is accompanied by alterations in microbial community structure marked by decreased abundance of pathobionts (e.g., Escherichia coli and Clostridium perfringens), reduced severity of dysbiosis, and increased levels of the secondary bile acids, lithocholic and deoxycholic acid. Physiologic levels of these bile acids inhibited the growth of E. coli and C. perfringens isolates, in vitro. Metagenomic analysis and whole genome sequencing identified the bile acid producer Clostridium hiranonis as elevated after dietary therapy and a likely source of secondary bile acids during remission. When C. hiranonis was administered to mice, levels of deoxycholic acid were preserved and pathology associated with DSS colitis was ameliorated. Finally, a closely related bile acid producer, Clostridium scindens, was associated with diet-induced remission in human pediatric Crohn’s disease.ConclusionsThese data highlight that remission induced by a hydrolyzed protein diet is associated with improved microbiota structure, an expansion of bile acid-producing clostridia, and increased levels of secondary bile acids. Our observations from clinical studies of exclusive enteral nutrition in human Crohn’s disease, along with our in vitro inhibition assays and in vivo studies in mice, suggest that this may be a conserved response to diet therapy with the potential to ameliorate disease. These findings provide insight into diet-induced remission of gastrointestinal disease and could help guide the rational design of more effective therapeutic diets.

1965-P: Glucagon Receptor Agonism Stimulates Body Weight Loss in Antibiotic Treatment in Obese Mice

Glucagon, an essential regulator of glucose and lipid metabolism, also promotes weight loss in diet-induced obese (DIO) mice. Glucagon’s role as a primary counterregulatory hormone to insulin action has long received scientific attention, yet its broader therapeutic potential is an evolving research interest. We reported that chronic glucagon receptor (GCGR) activation increased energy expenditure and plasma bile acids (BA) in an farnesoid X receptor (FXR) dependent manner. Glucagon stimulates the conversion of cholesterol to BAs, with an increase in cholic and chenodeoxycholate species, critical regulators of FXR activity. Gut microbiota are known to alter the chemical structure of bile acids to produce secondary and tertiary BA. Thus, in these studies we tested the hypothesis that GCGR agonism regulates the gut microbiome to alter circulating BAs and stimulate FXR. GCGR agonism stimulated a profound change in the gut microbiome of DIO mice. These changes were largely associated with an increase in Actinobacteria and a decrease in Deferribacteria phylums. Assessment of functional microbiome composition via PICRUSt uncovered significant impacts on 130 out of 160 total KEGG pathways, including those involved in BA synthesis. To investigate the role of the gut microbiome on GCGR-stimulated weight loss we treated DIO mice with an antibiotic (AB) cocktail (ampicillin, vancomycin, metronidazole, neomycin, and amphotericin B) or vehicle. Mice were then treated with the GCGR agonist, IUB288, or vehicle for 14d to stimulate weight-loss. Surprisingly, AB-treated mice lost a similar amount of body-weight when treated with IUB288, suggesting that the gut microbiome may be dispensable for the weight loss effect. However, post hoc analysis of the cecal microbiome identified only a partial ablation of microbiota in AB-treated mice, with unexpected increases in Proteobacteria and Tenericutes. Thus, it is possible that GCGR-agonism acts via one of the resistant microbes to regulate weight loss. Disclosure T. Kim: None. J.P. Antipenko: None. S. Nason: None. N. Presedo: None. W.J. Van Der Pol: None. B. Finan: Employee; Self; Novo Nordisk A/S. R. DiMarchi: Employee; Self; Novo Nordisk Inc. C.D. Morrow: None. K.M. Habegger: Consultant; Self; Glyscend, Inc., Novo Nordisk Inc. Research Support; Self; Glyscend, Inc. Stock/Shareholder; Self; Glyscend, Inc. Funding National Institutes of Health

Evaluating Hepatobiliary Transport with 18F-Labeled Bile Acids: The Effect of Radiolabel Position and Bile Acid Structure on Radiosynthesis and In Vitro and In Vivo Performance.

Introduction An in vivo determination of bile acid hepatobiliary transport efficiency can be of use in liver disease and preclinical drug development. Given the increased interest in bile acid Positron Emission Tomography- (PET-) imaging, a further understanding of the impact of 18-fluorine substitution on bile acid handling in vitro and in vivo can be of significance. Methods A number of bile acid analogues were conceived for nucleophilic substitution with [18F]fluoride: cholic acid analogues of which the 3-, 7-, or 12-OH function is substituted with a fluorine atom (3α-[18F]FCA; 7β-[18F]FCA; 12β-[18F]FCA); a glycocholic and chenodeoxycholic acid analogue, substituted on the 3-position (3β-[18F]FGCA and 3β-[18F]FCDCA, resp.). Uptake by the bile acid transporters NTCP and OATP1B1 was evaluated with competition assays in transfected CHO and HEK cell lines and efflux by BSEP in membrane vesicles. PET-scans with the tracers were performed in wild-type mice (n = 3 per group): hepatobiliary transport was monitored and compared to a reference tracer, namely, 3β-[18F]FCA. Results Compounds 3α-[18F]FCA, 3β-[18F]FGCA, and 3β-[18F]FCDCA were synthesized in moderate radiochemical yields (4–10% n.d.c.) and high radiochemical purity (>99%); 7β-[18F]FCA and 12β-[18F]FCA could not be synthesized and included further in this study. In vitro evaluation showed that 3α-FCA, 3β-FGCA, and 3β-FCDCA all had a low micromolar Ki-value for NTCP, OATP1B1, and BSEP. In vivo, 3α-[18F]FCA, 3β-[18F]FGCA, and 3β-[18F]FCDCA displayed hepatobiliary transport with varying efficiency. A slight yet significant difference in uptake and efflux rate was noticed between the 3α-[18F]FCA and 3β-[18F]FCA epimers. Conjugation of 3β-[18F]FCA with glycine had no significant effect in vivo. Compound 3β-[18F]FCDCA showed a significantly slower hepatic uptake and efflux towards gallbladder and intestines. Conclusion A set of 18F labeled bile acids was synthesized that are substrates of the bile acid transporters in vitro and in vivo and can serve as PET-biomarkers for hepatobiliary transport of bile acids.

Therapeutic targeting of bile acids.

The first objectives of this article are to review the structure, chemistry, and physiology of bile acids and the types of bile acid malabsorption observed in clinical practice. The second major theme addresses the classical or known properties of bile acids, such as the role of bile acid sequestration in the treatment of hyperlipidemia; the use of ursodeoxycholic acid in therapeutics, from traditional oriental medicine to being, until recently, the drug of choice in cholestatic liver diseases; and the potential for normalizing diverse bowel dysfunctions in irritable bowel syndrome, either by sequestering intraluminal bile acids for diarrhea or by delivering more bile acids to the colon to relieve constipation. The final objective addresses novel concepts and therapeutic opportunities such as the interaction of bile acids and the microbiome to control colonic infections, as in Clostridium difficile-associated colitis, and bile acid targeting of the farnesoid X receptor and G protein-coupled bile acid receptor 1 with consequent effects on energy expenditure, fat metabolism, and glycemic control.

Influence of the structure of bile acids on their partition coefficient in dibutyl ether and chloroform

Bile acids are well known natural surfactants able to modify the per?meability of biological membranes. The logarithm of partition coefficient between, tradi?tionally used, n-octanol and water is a measure of lipophilicity as a predictor of solute membrane partitioning. The aim of this work was to determine partition coefficients of bile acids in a mixture of water and chloroform and dibutyl ether at different pH values and with addition of different concentrations of sodium ions, and to examine the influence of the structure of bile acid nucleus on measured partition coefficients. Partition coefficients of three bile acid salts were determined using shake-flask method and the concentration of bile acids was determined after twelve hours of shaking at the room temperature in aqueous and organic layer using reversed phase HPLC with DAD detector on 210 nm. For all three analysed bile acid salts values of logP are lower in dibutyl ether than in chloroform. At certain pH values, curves representing the dependence of partition coeffi?cient on pH value intersect, and these are the pH values for which partition coefficients are the same for both solvents. Increasing the solution ionic strength, this intersection is shifted toward lower pH values. It is found that, for both organic solvents, after the addition of hy?droxyl group in the steroid nucleus (i.e. if the bile acid is less hydrophobic) the value of logP falls, especially if more hydroxyl groups are present. With chloroform as a solvent, system quickly comes to excess with electrolyte ions than with dibutyl ether.

Metabolic Effects of Bile Acids in the Gut in Health and Disease

In the last decade, it became clear that bile acids, in addition to their role in intestinal absorption of lipids and fat-soluble vitamins, are major regulators of metabolism. They activate signal transduction pathways through binding to the specific bile acid receptors TGR5 and FXR. Indirectly, bile acids influence metabolism via modification of the gut microbiota ecosystem. The relation between bile acid metabolism and gut microbiota composition is very complex whereas gut microbiota modulates bile acid structure, creating a complex bile acid pool consisting of a mixture of differentially structured species, bile acids alter gut microbiota by disturbing bacterial membrane integrity. In addition, to the effects on glucose and energy homeostasis, recent literature ascribed a role for bile acid signaling in control of inflammation and regulation of the nervous system. In this review, we discuss a selection of recent published studies describing the effects of intestinal bile acid signaling on health and disease.

Hydrophobicity and Self-association of Bile Acids with a Special Emphasis on Oxo Derivatives of 5-&amp;#946; Cholanic Acid

The article is an extensive review of the influence of the structure of bile acids on their hydrophobicity as expressed via the retention parameters of reverse phase chromatography and via the 1-octanol/water partition coefficients. The emphasis of the discussion is on the relationship beween the structure of the steroid skeleton of bile acids and their self-association. Finally, the lipohilicity of bile acids and its role in their interaction with biological systems is also discussed.

A contribution to the study of hydrophobicity (lipophilicity) of bile acids with an emphasis on oxo derivatives of 5β-cholanoic acid

Due to their promotory action on the transport of some drugs through various membranes (lipophilic barriers), oxo derivatives of bile acids have recently been increasingly used in biopharmacy. These compounds exhibit also a lower membranolytic (toxic) activity than their hydroxy analogues. Because of that it is of special importance to find out the descriptors that would adequately describe the structure of bile acids and their biological activity and be used to model the quantitative structure-activity relationship. In view of this, the present work is concerned with the application of the chromatographic parameter RM0 obtained by normal-phase thin-layer chromatography in the solvent system toluene-butanol and silica gel as stationary phase to describe the lipophilicity of bile acids. Also, the work introduces a new molecular descriptor (ND) that reflects both 2D and 3D topological characteristics of the molecule. Between the retention constant, RM0 and the descriptor ND there is a good correlation, and both RM0, and ND describe sufficiently well the structural (conformational) changes that arise in the process of oxidation of the OH group of the steroid skeleton to an oxo group. On the other hand, the in silico descriptors of lipophilicity, logP (atomic-based prediction) and ClogP (fragment-based prediction) predict the hydrophobicity of bile acid oxo derivatives with a certain error.

Simulating Fasted Human Intestinal Fluids: Understanding the Roles of Lecithin and Bile Acids

The purpose of this work is to evaluate the roles of lecithin and bile salts in a new generation of fasted simulated small intestinal fluid (FaSSIF-II), thus enhancing the closer mimic of simulated fluids to the real human intestinal fluids (HIF) in drug discovery and drug product development. To assess the effects of lecithin in FaSSIF-II, solubility studies were conducted at 37 °C using four media including first generation simulated intestinal fluid (FaSSIF-I), FaSSIF-II, phosphate pH 6.5 buffer, and HIF. A total of 24 model compounds representing a wide range of biopharmaceutic properties were included. The drug solubility values measured in the FaSSIF-II were compared with those in FaSSIF-I, pH 6.5 buffer and HIF. To assess the effects of bile acids, solubility was measured for 4 compounds in the FaSSIF-I containing five different bile acids of various concentrations. The lecithin concentration in the FaSSIF-II is lowered from 0.75 mM to 0.2 mM. The results suggested that the FaSSIF-II is a better medium to reflect HIF, compared with pH 6.5 phosphate buffer and FaSSIF-I. Solubility of neutral compounds including atovaquone, carbamazepine, cyclosporine, danazol, diethylstilbestrol, felodipine, griseofulvin and probucol in FaSSIF-II showed improvement in predicting the in vivo solubility. The relative standard deviation (SD) of solubility measurement in FaSSIF-II is comparable with FaSSIF-I. For the acidic and basic tested compounds, the FaSSIF-II performs similarly to the FaSSIF-I. Experimental results showed that the level of bile salts typically is less than 5 mM under fasted state. Among the five studied bile acids, the conjugation (glycine or taurine) has no impact on the drug solubilization, while there may be a minimal effect of the degree of hydroxylation of the steroid ring system on solubilization. The lecithin concentration of 0.2 mM in FaSSIF-II has been demonstrated to closely represent HIF, for both neutral and ionizable compounds. In the composition of simulated intestinal fluids, the structure of bile acids has minimal effect, providing the flexibility of choosing one bile salt to represent complex in vivo bile acids.

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.