The Increased Dissolution and Oral Absorption of Itraconazole by Nanocrystals with an Endogenous Small-Molecule Surfactant as a Stabilizer.

The aim of this study was to develop cholic-acid-stabilized itraconazole nanosuspensions (ITZ-Nanos) with the objective of enhancing drug dissolution and oral absorption. A laboratory-scale microprecipitation-high-pressure homogenization method was employed for the preparation of the ITZ-Nanos, while dynamic light scattering, transmission electron microscope analysis, X-ray diffraction, differential scanning calorimetry, and high-performance liquid chromatography analysis were utilized to evaluate their physicochemical properties. The absorption and bioavailability of the ITZ-Nanos were assessed using Caco-2 cells and rats, with Sporanox® pellets as a comparison. Prior to lyophilization, the particle size of the ITZ-Nanos measured approximately 225.7 nm. Both X-ray diffraction and differential scanning calorimetry confirmed that the ITZ remained crystalline within the nanocrystals. Compared to the pellets, the ITZ-Nanos exhibited significantly higher levels of supersaturation dissolution and demonstrated enhanced drug uptake by the Caco-2 cells. The AUC(0-t) value for the ITZ-Nanos in rats was 1.33-fold higher than that observed for the pellets. These findings suggest that cholic acid holds promise as a stabilizer for ITZ nanocrystals, as well as potentially other nanocrystals.

Self-Association of the Anion of 7-Oxodeoxycholic Acid (Bile Salt): How Secondary Micelles Are Formed.

Bile acid anions are steroidal biosurfactants that form primary micelles due to the hydrophobic effect. At higher concentrations of some bile acid anions, secondary micelles are formed; hydrogen bonds connect primary micelles. Monoketo derivatives of cholic acid, which have reduced membrane toxicity, are important for biopharmaceutical examinations. The main goal is to explain why the processes of formation of primary and secondary micelles are separated from each other, i.e., why secondary micelles do not form parallel to primary micelles. The association of the anion of 7-oxodeoxycholic acid (a monoketo derivative of cholic acid) is observed through the dependence of the spin-lattice relaxation time on total surfactant concentration T1 = f(CT). On the function T1 = f(CT), two sharp jumps of the spin-lattice relaxation time are obtained, i.e., two critical micellar concentrations (CMC). The aggregation number of the micelle at 50 mM total concentration of 7-oxodeoxycholic acid anions in the aqueous solution is 4.2 ± 0.3, while at the total concentration of 100 mM the aggregation number is 9.0 ± 0.9. The aggregation number of the micelle changes abruptly in the concentration interval of 80-90 mM (the aggregation number determined using fluorescence measurements). By applying Le Chatelier's principle, the new mechanism of formation of secondary micelles is given, and the decoupling of the process of formation of primary and secondary micelles at lower concentrations of monomers (around the first critical micellar concentration) and the coupling of the same processes at higher equilibrium concentrations of monomers (around the second critical micellar concentration) is explained. Stereochemically and thermodynamically, a direct mutual association of primary micelles is less likely, but monomeric units are more likely to be attached to primary micelles, i.e., 7-oxodeoxycholic acid anions.

Noncovalent Bile Acid Oligomers as Facial Amphiphilic Antimicrobials.

New antimicrobial agents are needed to address the ever-growing risk of bacterial resistance, particularly for methicillin- and vancomycin-resistant Staphylococcus aureus (S. aureus). Here, we report a class of bile acid oligomers as facial amphiphilic antimicrobials, which are noncovalently fabricated by cholic acid (CA) and deoxycholic acid (DCA) with polyamines (e.g., diamines, diethylenetriamine, spermidine, and spermine). The antibacterial activities of these bile acid oligomers (CA/polyamines and DCA/polyamines) against S. aureus become stronger with increasing the amine group numbers of polyamines without obviously enhanced cytotoxicity and skin irritation. DCA/spermine, entirely composed of natural products, exhibits the best antibacterial activity but the lowest cytotoxicity and the weakest skin irritation. All CA/polyamines and DCA/polyamines form well-ordered ribbon-like aggregates, collecting numerous facial amphiphilic structures to significantly enhance the interactions with bacterial membranes. In particular, the biogenic polyamines with more than two amine groups provide extra positively charged sites, hence facilitating the binding of bile acid oligomers to the negatively charged outer membrane of the bacteria via electrostatic interaction. This in turn promotes more oligomeric bile acid units that can be inserted into the membrane through hydrophobic interaction between bile acids and lipid domains. The noncovalently constructed and separable amphiphilic antimicrobials can avoid the long-term coexistence of microorganisms and antibacterial molecules in different acting modes. Therefore, the noncovalent bile acid oligomers, especially those with higher oligomerization degrees, can be a potential approach to effectively enhance antibacterial activity, improve environmental friendliness, and reduce bacterial drug resistance.

Molecular Umbrella as a Nanocarrier for Antifungals.

A molecular umbrella composed of two O-sulfated cholic acid residues was applied for the construction of conjugates with cispentacin, containing a "trimethyl lock" (TML) or o-dithiobenzylcarbamoyl moiety as a cleavable linker. Three out of five conjugates demonstrated antifungal in vitro activity against C. albicans and C. glabrata but not against C. krusei, with MIC90 values in the 0.22-0.99 mM range and were not hemolytic. Antifungal activity of the most active conjugate 24c, containing the TML-pimelate linker, was comparable to that of intact cispentacin. A structural analogue of 24c, containing the Nap-NH2 fluorescent probe, was accumulated in Candida cells, and TML-containing conjugates were cleaved in cell-free extract of C. albicans cells. These results suggest that a molecular umbrella can be successfully applied as a nanocarrier for the construction of cleavable antifungal conjugates.

Access to New Cytotoxic Triterpene and Steroidal Acid-TEMPO Conjugates by Ugi Multicomponent-Reactions.

Multicomponent reactions, especially the Ugi-four component reaction (U-4CR), provide powerful protocols to efficiently access compounds having potent biological and pharmacological effects. Thus, a diverse library of betulinic acid (BA), fusidic acid (FA), cholic acid (CA) conjugates with TEMPO (nitroxide) have been prepared using this approach, which also makes them applicable in electron paramagnetic resonance (EPR) spectroscopy. Moreover, convertible amide modified spin-labelled fusidic acid derivatives were selected for post-Ugi modification utilizing a wide range of reaction conditions which kept the paramagnetic center intact. The nitroxide labelled betulinic acid analogue 6 possesses cytotoxic effects towards two investigated cell lines: prostate cancer PC3 (IC50 7.4 ± 0.7 µM) and colon cancer HT29 (IC50 9.0 ± 0.4 µM). Notably, spin-labelled fusidic acid derivative 8 acts strongly against these two cancer cell lines (PC3: IC50 6.0 ± 1.1 µM; HT29: IC50 7.4 ± 0.6 µM). Additionally, another fusidic acid analogue 9 was also found to be active towards HT29 with IC50 7.0 ± 0.3 µM (CV). Studies on the mode of action revealed that compound 8 increased the level of caspase-3 significantly which clearly indicates induction of apoptosis by activation of the caspase pathway. Furthermore, the exclusive mitochondria targeting of compound 18 was successfully achieved, since mitochondria are the major source of ROS generation.

Validation of Recombinant Chicken Liver Bile Acid Binding Protein as a Tool for Cholic Acid Hosting.

Bile acids (BAs) are hydroxylated steroids derived from cholesterol that act at the intestinal level to facilitate the absorption of several nutrients and also play a role as signaling molecules. In the liver of various vertebrates, the trafficking of BAs is mediated by bile acid-binding proteins (L-BABPs). The ability to host hydrophobic or amphipathic molecules makes BABPs suitable for the distribution of a variety of physiological and exogenous substances. Thus, BABPs have been proposed as drug carriers, and more recently, they have also been employed to develop innovative nanotechnology and biotechnology systems. Here, we report an efficient protocol for the production, purification, and crystallization of chicken liver BABP (cL-BABP). By means of target expression as His6-tag cL-BABP, we obtained a large amount of pure and homogeneous proteins through a simple purification procedure relying on affinity chromatography. The recombinant cL-BABP showed a raised propensity to crystallize, allowing us to obtain its structure at high resolution and, in turn, assess the structural conservation of the recombinant cL-BABP with respect to the liver-extracted protein. The results support the use of recombinant cL-BABP for the development of drug carriers, nanotechnologies, and innovative synthetic photoswitch systems.

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter.

The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

Bioinspired lipid-polysaccharide modified hybrid nanoparticles as a brain-targeted highly loaded carrier for a hydrophilic drug.

Lipid-polysaccharide modified biohybrid nanoparticles (NPs) are eminent drug carriers for brain targeting, owing to their ability to prolong the circulation time and penetrate the blood brain barrier (BBB). Biohybrid NPs particular interest arises from their potential to mimic biological components. Herein, we prepared bioinspired lipid polymeric NPs, either naked or surface modified by a synthesized biocompatible dextran-cholic acid (DxC). The nanoprecipitation method was tailored to allow the assembly of the multicomponent NPs in a single step. Modulating the solvent/antisolvent system provided lipid polymer hybrid NPs in the size of 111.6 ± 11.4 nm size. The NPs encapsulated up to 92 ± 1.2% of a hydrophilic anti-Alzheimer drug, rivastigmine (Riv). The brain uptake, biodistribution and pharmacokinetics studies, proved the efficient fast penetration of the bioinspired surface modified NPs to the brain of healthy albino rats. The modified nanocarrier caused a 5.4 fold increase in brain targeting efficiency compared to the drug solution. Furthermore, the presence of DxC increased Riv's brain residence time up to 40 h. The achieved results suggest that the fabricated biohybrid delivery system was able to circumvent the BBB and is expected to minimize Riv systemic side effects.

Structural and biochemical investigation of MARK4 inhibitory potential of cholic acid: Towards therapeutic implications in neurodegenerative diseases.

Microtubule affinity regulating kinase (MARK4) is considered as a potential drug target for diabetes, cancer, and neurodegenerative diseases. Since the role of MARK4 in the phosphorylation of tau protein and subsequently Alzheimer's disease has been established, therefore, we have investigated the binding affinity and MARK4 inhibitory potential of cholic acid (CHA) using both computational and spectroscopic methods. Molecular docking suggested a strong binding of CHA to the functionally important residues of MARK4. We further performed 500 ns molecular dynamics simulation which suggested the MARK4-CHA system was quite stable throughout the simulation trajectory. CHA potential binds to the MARK4 with a binding constant (K) of 107 M-1 at 288 K. Further, MARK4 activity was inhibited by CHA with an IC50 = 5.5 µM. Further insights into the thermodynamic parameters suggested that MARK4-CHA complex formation is driven by both electrostatic and van der Waals interactions. Overall study provides a rationale to use CHA in the drug development via MARK4 inhibition, towards possible therapeutic implications in neurodegenerative diseases.

Localized delivery of active targeting micelles from nanofibers patch for effective breast cancer therapy.

Nanofibers based transdermal drug delivery is a promising platform, and it effectively delivers the drug to tumor sites. The objective of the study was to fabricate stimuli-responsive polymeric nanofibers encapsulated with an active targeting micellar system for in situ drug delivery. Stimuli-responsive core-shell nanofibers release thedrug at target sites with minimum side effects to the other organs, decrease the drug administration concentration. Initially, we prepared CA conjugated PCPP polymeric micelles loaded with PTX. Then, core-shell nanofibers were prepared using PHM with coaxial electrospinning and distinct core-shell nanofibers formation confirm by SEM and TEM. Nanofibers showed a homogenous distribution of micelles inside the fiber mesh, diffusion, and erosion processes lead to a controlled release of PTX.In vitro drug release and swelling, revealed the pH based sustained release of the drug for 180 h from the nanofibers mat. Functional and stimuli-responsive nanofibers highly absorb H+ ions and repulsion of cations promoting maximum swelling to release more drugs in acidic pH. An increased transportation rate of 70% drug release through epidermis for 120 h. Nanofibers effectively internalize to the skin, and it confirmed by confocal microscopy. MCF-7 cells grown and spread over the nanofibers, which show the biocompatibility of nanofibers. Compared to PTX, drug-loaded nanofibers exhibited higher cytotoxicity for 8 days which was confirmed by the flow cytometry. These promising results confirm, the novel stimuli-responsive core-shell nanofibers actively target breast cancer cells and lead the way to safe cancer therapy.

Oral delivery of exenatide-loaded hybrid zein nanoparticles for stable blood glucose control and ß-cell repair of type 2 diabetes mice.

BACKGROUND: Exenatide is an insulinotropic peptide drug for type 2 diabetes treatment with low risk of hypoglycemia, and is administrated by subcutaneous injection. Oral administration is the most preferred route for lifelong treatment of diabetes, but oral delivery of peptide drug remains a significant challenge due to the absorption obstacles in gastrointestinal tract. We aimed to produce exenatide-loaded nanoparticles containing absorption enhancer, protectant and stabilizer using FDA approved inactive ingredients and easy to scale-up method, and to evaluate their long-term oral therapeutic effect in type 2 diabetes db/db mice. RESULTS: Two types of nanoparticles, named COM NPs and DIS NPs, were fabricated using anti-solvent precipitation method. In COM NPs, the exenatide was complexed with cholic acid and phosphatidylcholine to increase the exenatide loading efficiency. In both nanoparticles, zein acted as the cement and the other ingredients were embedded in zein nanoparticles by hydrophobic interaction. Casein acted as the stabilizer. The nanoparticles had excellent lyophilization, storage and re-dispersion stability. Hypromellose phthalate protected the loaded exenatide from degradation in simulated gastric fluid. Cholic acid promoted the intestinal absorption of the loaded exenatide via bile acid transporters. The exenatide loading efficiencies of COM NPs and DIS NPs were 79.7% and 53.6%, respectively. The exenatide oral pharmacological availability of COM NPs was 18.6% and DIS NPs was 13.1%. COM NPs controlled the blood glucose level of the db/db mice well and the HbA1c concentration significantly decreased to 6.8% during and after 7 weeks of once daily oral administration consecutively. Both DIS NPs and COM NPs oral groups substantially increased the insulin secretion by more than 60% and promoted the ß-cell proliferation by more than 120% after the 7-week administration. CONCLUSIONS: Both COM NPs and DIS NPs are promising systems for oral delivery of exenatide, and COM NPs are better in blood glucose level control than DIS NPs. Using prolamin to produce multifunctional nanoparticles for oral delivery of peptide drug by hydrophobic interaction is a simple and effective strategy.

Global chemical effects of the microbiome include new bile-acid conjugations.

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.

Efficient synthesis of cholic acid derivates through stereoselective C-H functionalization from hyodeoxycholic acid.

Five cholic acid derivatives (including allo-ω-muricholic acid and CDCA) were synthesized from hyodeoxycholic acid via selective oxidation of C3- or C6-hydroxyl groups by IBX and NBS oxidants and stereocontrolled conversion. The hydroxyl group could be introduced through hydrolyzing α-Br keto with K2CO3 aqueous solution or through oxidizing the double bond by monoperoxyphthalic acid. The reduction of C6-O6 carbonyl to methylene could undergo with PTSH, NaBH3CN and ZnCl2 only at 5ß configuration. A feasible synthetic route of CDCA from HDCA has been established to avoid the epimerization with the yield of 45% (8 steps). These strategies provided good yields, stereoselectivity and reproducibility for the preparation of cholic acid derivates and CDCA.

In Vitro Cellular Delivery of Peptide Nucleic Acid (PNA).

Cellular delivery methods are a prerequisite for cellular studies with PNA. This chapter describes PNA cellular delivery using cell-penetrating peptide (CPP)-PNA conjugates and transfection of PNA-ligand conjugates mediated by cationic lipids. Furthermore, two endosomolytic procedures employing chloroquine treatment or photochemical internalization (PCI) for significantly improving PNA delivery efficacy are described.

Cholic acid-based mixed micelles as siRNA delivery agents for gene therapy.

Gene therapy is a promising tool for the treatment of various cancers but is hindered by the physico-chemical properties of siRNA and needs a suitable vector for the delivery of siRNA to the target tissue. Bile acid-based block copolymers offers certain advantages for the loading and delivery of siRNA since they can efficiently complex siRNA and bile acids are biocompatible endogenous molecules. In this study, we demonstrate the use of lipids as co-surfactants for the preparation of mixed micelles to improve the siRNA delivery of cholic acid-based block copolymers. Poly(allyl glycidyl ether) (PAGE) and poly(ethylene glycol) (PEG) were polymerized on the surface of cholic acid to afford a star-shaped block copolymer with four arms (CA-PAGE-b-PEG)4. The allyl groups of PAGE were functionalized to bear primary or tertiary amines and folic acid was grafted onto the PEG chain end to increase cell uptake. (CA-PAGE-b-PEG)4 functionalized with either primary or tertiary amines show high siRNA complexation with close to 100% complexation at N/P ratio of 8. Uniform aggregates with diameters between 181 and 188 nm were obtained. DOPE, DSPE-PEG2k, and DSPE-PEG5k lipids were added as co-surfactants to help stabilize the nanoparticles in the cell culture media. Mixed micelles had high siRNA loading with close to 100% functionalization at N/P ratio of 16 and diameters ranging from 153 to 221 nm. The presence of lipids in the mixed micelles improved cell uptake with a concomitant siRNA transfection in HeLa and HeLa-GFP model cells, respectively.

Multifunctional nanoplatform based on star-shaped copolymer for liver cancer targeting therapy.

With high morbidity and death rates, liver cancer has become one of the most common cancers in the world. But, most chemotherapeutic anticancer drugs have high toxicity as well as low specificity. To improve the treatment modalities and enhance the therapeutic effect of liver cancer, a brand new liver-targeting nanoparticle (NP), Ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (5 F)-loaded cholic acid (CA)-functionalized star-shaped poly (lactic-co-glycolic acid) (PLGA)-polyethylene glycol (PEG)-lactobionic acid (LA) (5 F-loaded CA-PLGA-PEG-LA), was developed. The particle size, zeta potential, size distribution, surface morphology, drug loading content, drug encapsulation efficiency and drug release of 5 F-loaded NPs were characterized. Confocal microscopy and flow cytometry showed that the prepared NPs could be internalized by HepG2 cells. Furthermore, the cellular uptake efficiency of coumarin 6-loaded CA-PLGA-PEG-LA NPs was much better in compare with that of CA-PLGA-PEG and CA-PLGA NPs. Moreover, LA-conjugated NPs (CA-PLGA-PEG-LA NPs) enhanced fluorescence of HepG2 cells via ligand-mediated endocytosis. The antitumor effects of 5 F-loaded NPs were evaluated by the MTT assay in vitro and by a xenograft tumor model in vivo, demonstrating that targeted 5 F-loaded CA-PLGA-PEG-LA NPs were significantly superior to free 5 F and 5 F-loaded CA-PLGA-PEG NPs. All the results indicated the 5 F-loaded CA-PLGA-PEG-LA NPs can be employed as a novel potentially targeting drug delivery system for liver cancer therapy.

Antifouling activity of peracetylated cholic acid, a natural bile acid derivative.

The antifouling activity of peracetylated cholic acid (1), a bile acid derivative which was isolated in a previous work as a natural product from the Patagonian sponge Siphonochalina fortis, was evaluated in laboratory and field trials. Toxicity and settlement assays were performed with the mussel Mytilus edulis platensis, while the field trials were carried out by addition of the compound to experimental soluble-matrix paints, which were then tested in the sea. The results obtained in this work show that 1 has a good antifouling activity and low toxicity, and the paints aditivated with 0,6% Wt showed promissory performances in the field trials at the sea. These results confirm the previous hypothesis that the few acetylated and lipophilic bile acid derivatives isolated from marine invertebrates may act as natural antifoulants. Compound 1 is a natural, biodegradable product that can be easily prepared from cholic acid, which in turn can be isolated in industrial scale from cattle bile. All these facts make cholic acid a good scaffold for the preparation of derivatives, which can be natural product-like, effective and sustainable antifouling additives for marine paints and other applications.

Mimicking Enzymes: Asymmetric Induction inside a Carbamate-Based Steroidal Cleft.

Cholic acid has been elaborated into a carbamate-based tripodal architecture, which is able to promote an asymmetric organic transformation inside its chiral cavity. The nature of this steroidal catalyst has been disclosed by quantum-chemical calculations. It comprises the preorganization and confinement of the reagents within the cavity of the steroid to form a supramolecular complex held together by means of cooperative H-bond contacts. This operational mode resembles that of some enzymes.

Structural requirements of cholenamide derivatives as the LXR ligands.

A study of the structural requirements of cholic acid derivatives as liver X receptor (LXR) ligands was performed. A model of cholenamide derivative 1 complexed with LXR showed that the C24 carbonyl oxygen forms a hydrogen bond with His435 located close to Trp457. The N,N-dimethyl group is located in a hydrophobic pocket. Based on these data, we designed compounds with high affinity for LXRs. Cholenamide derivatives 1-11 were synthesized from 3ß-acetyl-Δ5-cholenic acid 20, and lactams 12-19 were synthesized from alcohol 25. Tertiary amides 3 and 4 showed higher activity in reporter assays, and compounds with hydrophobic residues exhibited the highest activity of all derivatives. The stereochemistry at C23 was found to be an important determinant of EC50 and gene transactivation, as each isomer exhibited different activity.

Conversion of chenodeoxycholic acid to cholic acid by human CYP8B1.

The human cytochrome P450 enzyme CYP8B1 is a crucial regulator of the balance of cholic acid (CA) and chenodeoxycholic acid (CDCA) in the liver. It was previously shown to catalyze the conversion of 7α-hydroxycholest-4-en-3-one, a CDCA precursor, to 7α,12α-dihydroxycholest-4-en-3-one, which is an intermediate of CA biosynthesis. In this study we demonstrate that CYP8B1 can also convert CDCA itself to CA. We also show that five derivatives of luciferin are metabolized by CYP8B1 and established a rapid and convenient inhibitor test system. In this way we were able to identify four new CYP8B1 inhibitors, which are aminobenzotriazole, exemestane, ketoconazole and letrozole.