3α,7-Dihydroxy-14(13→12)abeo-5ß,12α(H),13ß(H)-cholan-24-oic Acids Display Neuroprotective Properties in Common Forms of Parkinson's Disease.

Parkinson's Disease is the most common neurodegenerative movement disorder globally, with prevalence increasing. There is an urgent need for new therapeutics which are disease-modifying rather than symptomatic. Mitochondrial dysfunction is a well-documented mechanism in both sporadic and familial Parkinson's Disease. Furthermore, ursodeoxycholic acid (UDCA) has been identified as a bile acid which leads to increased mitochondrial function in multiple in vitro and in vivo models of Parkinson's Disease. Here, we describe the synthesis of novel C-nor-D-homo bile acid derivatives and the 12-hydroxy-methylated derivative of lagocholic acid (7) and their biological evaluation in fibroblasts from patients with either sporadic or LRRK2 mutant Parkinson's Disease. These compounds boost mitochondrial function to a similar level or above that of UDCA in many assays; notable, however, is their ability to boost mitochondrial function to a higher level and at lower concentrations than UDCA specifically in the fibroblasts from LRRK2 patients. Our study indicates that novel bile acid chemistry could lead to the development of more efficacious bile acids which increase mitochondrial function and ultimately cellular health at lower concentrations proving attractive potential novel therapeutics for Parkinson's Disease.

Studies on the Importance of the 7α-, and 12α- hydroxyl groups of N-Aryl-3α,7α,12α-trihydroxy-5ß-cholan-24-amides on their Antigermination Activity Against a Hypervirulent Strain of Clostridioides (Clostridium) difficile.

Chenodeoxycholic acid (CDCA) is a natural germination inhibitor for C. difficile spores. In our previous study (J. Med. Chem., 2018, 61, 6759-6778), we identified N-phenyl-3α,7α,12α-trihydroxy-5ß-cholan-24-amide as an inhibitor of C. difficile strain R20291 with an IC50 of 1.8 µM. Studies of bile salts on spore germination have shown that chenodeoxycholate, ursodeoxycholate and lithocholate are more potent inhibitors of germination compared to cholate. Given this, we created amide analogs of chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids using amines identified from our previous studies. We found that chenodeoxy- and deoxycholate derivatives were active with potencies equivalent to those for cholanamides. This indicates that only 2 out of the 3 hydroxyl groups are needed for activity and that the alpha stereochemistry at position 7 is required for inhibition of spore germination.

Synthesis and Biological Activity of New Brassinosteroid Analogs of Type 24-Nor-5ß-Cholane and 23-Benzoate Function in the Side Chain.

Brassinosteroids are polyhydroxysteroids that are involved in different plants' biological functions, such as growth, development and resistance to biotic and external stresses. Because of its low abundance in plants, much effort has been dedicated to the synthesis and characterization of brassinosteroids analogs. Herein, we report the synthesis of brassinosteroid 24-nor-5ß-cholane type analogs with 23-benzoate function and 22,23-benzoate groups. The synthesis was accomplished with high reaction yields in a four-step synthesis route and using hyodeoxycholic acid as starting material. All synthesized analogs were tested using the rice lamina inclination test to assess their growth-promoting activity and compare it with those obtained for brassinolide, which was used as a positive control. The results indicate that the diasteroisomeric mixture of monobenzoylated derivatives exhibit the highest activity at the lowest tested concentrations (1 × 10-8 and 1 × 10-7 M), being even more active than brassinolide. Therefore, a simple synthetic procedure with high reaction yields that use a very accessible starting material provides brassinosteroid synthetic analogs with promising effects on plant growth. This exploratory study suggests that brassinosteroid analogs with similar chemical structures could be a good alternative to natural brassinosteroids.

Glabrescione B delivery by self-assembling micelles efficiently inhibits tumor growth in preclinical models of Hedgehog-dependent medulloblastoma.

Aberrant activation of the Hedgehog (Hh) pathway leads to the development of several tumors, including medulloblastoma (MB), the most common pediatric brain malignancy. Hh inhibitors acting on GLI1, the final effector of Hh signaling, offer a valuable opportunity to overcome the pitfalls of the existing therapies to treat Hh-driven cancers. In this study, the toxicity, delivery, biodistribution, and anticancer efficacy of Glabrescione B (GlaB), a selective GLI1 inhibitor, were investigated in preclinical models of Hh-dependent MB. To overcome its poor water solubility, GlaB was formulated with a self-assembling amphiphilic polymer forming micelles, called mPEG5kDa-cholane. mPEG5kDa-cholane/GlaB showed high drug loading and stability, low cytotoxicity, and long permanence in the bloodstream. We found that mPEG5kDa-cholane efficiently enhanced the solubility of GlaB, thus avoiding the use of organic solvents. mPEG5kDa-cholane/GlaB possesses favorable pharmacokinetics and negligible toxicity. Remarkably, GlaB encapsulated in mPEG5kDa-cholane micelles was delivered through the blood-brain barrier and drastically inhibited tumor growth in both allograft and orthotopic models of Hh-dependent MB. Our findings reveal that mPEG5kDa-cholane/GlaB is a good candidate for the treatment of Hh-driven tumors and provide relevant implications for the translation of GlaB into clinical practice.

Physical PEGylation to Prevent Insulin Fibrillation.

Insulin is one of the most marketed therapeutic proteins worldwide. However, its formulation suffers from fibrillation, which affects the long-term storage limiting the development of novel devices for sustained delivery including portable infusion devices. We have investigated the effect of physical PEGylation on structural and colloidal stability of insulin by using 2 PEGylating agents terminating with polycyclic hydrophobic moieties, cholane and cholesterol: mPEG5kDa-cholane and mPEG5kDa-cholesterol, respectively. Microcalorimetric analyses showed that mPEG5kDa-cholane and mPEG5kDa-cholesterol efficiently bind insulin with binding constants (Ka) of 3.98 104 and 1.14 105 M-1, respectively. At room temperature, the 2 PEGylating agents yielded comparable structural stabilization of α-helix conformation and decreased dimerization of insulin. However, melting studies showed that mPEG5kDa-cholesterol has superior stabilizing effect of the protein conformation than mPEG5kDa-cholane. Furthermore, the fibrillation study showed that at a 1:1 and 1:5 insulin/polymer molar ratios, mPEG5kDa-cholesterol delays insulin fibrillation 40% and 26% more efficiently, respectively, as compared to mPEG5kDa-cholane which was confirmed by transmission electron microscopy imaging. Insulin was released from the mPEG5kDa-cholane and mPEG5kDa-cholesterol assemblies with comparable kinetic profiles. The physical PEGylation has a beneficial effect on the stabilization and shielding of the insulin structure into the monomeric form, which is not prone to fibrillation and aggregation.

In Vitro and in Vivo Behavior of Liposomes Decorated with PEGs with Different Chemical Features.

The colloidal stability, in vitro toxicity, cell association, and in vivo pharmacokinetic behavior of liposomes decorated with monomethoxy-poly(ethylene glycol)-lipids (mPEG-lipids) with different chemical features were comparatively investigated. Structural differences of the mPEG-lipids used in the study included: (a) surface-anchoring moiety [1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), cholesterol (Chol), and cholane (Chln)]; (b) mPEG molecular weight (2 kDa mPEG45 and 5 kDa mPEG114); and (c) mPEG shape (linear and branched PEG). In vitro results demonstrated that branched (mPEG114)2-DSPE confers the highest stealth properties to liposomes (∼31-fold lower cell association than naked liposomes) with respect to all PEGylating agents tested. However, the pharmacokinetic studies showed that the use of cholesterol as anchoring group yields PEGylated liposomes with longer permeance in the circulation and higher systemic bioavailability among the tested formulations. Liposomes decorated with mPEG114-Chol had 3.2- and ∼2.1-fold higher area under curve (AUC) than naked liposomes and branched (mPEG114)2-DSPE-coated liposomes, respectively, which reflects the high stability of this coating agent. By comparing the PEGylating agents with same size, namely, linear 5 kDa PEG derivatives, linear mPEG114-DSPE yielded coated liposomes with the best in vitro stealth performance. Nevertheless, the in vivo AUC of liposomes decorated with linear mPEG114-DSPE was lower than that obtained with liposomes decorated with linear mPEG114-Chol. Computational molecular dynamics modeling provided additional insights that complement the experimental results.

Synthesis and Structural Determination of New Brassinosteroid 24-Nor-5α-Cholane Type Analogs.

Natural brassinosteroids possess a 22R, 23R configuration that appears essential for biological activity. It is, therefore, interesting to elucidate if the activity of brassinosteroids with a short side chain depends on the C22 configuration. Herein, we describe the synthesis of new brassinosteroids analogs with 24-norcholane type of side chain and R configuration at C22. The initial reaction is the dihydroxylation of a terminal olefin that leads to S/R epimers. Three different methods were tested in order to evaluate the obtained S/R ratio and the reaction yields. The results indicate that Upjohn dihydroxylation is the most selective reaction giving a 1.0:0.24 S/R ratio, whereas a Sharpless reaction leads to a mixture of 1.0:0.90 S/R with 95% yield. Using the latter mixture and following a previous reported method, benzoylated derivatives and both S and R brassinosteroids analogs were synthesized. All synthesized compounds were completely characterized by NMR spectroscopy, and HRMS of new compounds are also given. In conclusion, a synthetic route for preparation of new analogs of brassinosteroids of 24-norcholane type and R configuration at C22 were described. It is expected that this will help to elucidate if a configuration at C22 is a structural requirement for hormonal growth activity in plants.

Transcriptome Analysis of Dual FXR and GPBAR1 Agonism in Rodent Model of NASH Reveals Modulation of Lipid Droplets Formation.

Non-alcoholic steatohepatitis (NASH) is a progressive, chronic, liver disease whose prevalence is growing worldwide. Despite several agents being under development for treating NASH, there are no drugs currently approved. The Farnesoid-x-receptor (FXR) and the G-protein coupled bile acid receptor 1 (GPBAR1), two bile acid activated receptors, have been investigated for their potential in treating NASH. Here we report that BAR502, a steroidal dual ligand for FXR/GPBAR1, attenuates development of clinical and liver histopathology features of NASH in mice fed a high fat diet (HFD) and fructose (F). By RNAseq analysis of liver transcriptome we found that BAR502 restores FXR signaling in the liver of mice feed HFD-F, and negatively regulates a cluster of genes including Srebf1 (Srepb1c) and its target genes-fatty acid synthase (Fasn) and Cell death-inducing DFF45-like effector (CIDE) genes, Cidea and Cidec-involved in lipid droplets formation and triglycerides storage in hepatocytes. Additionally, BAR502 increased the intestinal expression of Fgf15 and Glp1 and energy expenditure by white adipose tissues. Finally, exposure to BAR502 reshaped the intestinal microbiota by increasing the amount of Bacteroidaceae. In conclusion, we have shown that dual FXR/GPBAR1 agonism might have utility in treatment of NASH.

Introduction of Nonacidic Side Chains on 6-Ethylcholane Scaffolds in the Identification of Potent Bile Acid Receptor Agonists with Improved Pharmacokinetic Properties.

As a cellular bile acid sensor, farnesoid X receptor (FXR) and the membrane G-coupled receptor (GPBAR1) participate in maintaining bile acid, lipid, and glucose homeostasis. To date, several selective and dual agonists have been developed as promising pharmacological approach to metabolic disorders, with most of them possessing an acidic conjugable function that might compromise their pharmacokinetic distribution. Here, guided by docking calculations, nonacidic 6-ethyl cholane derivatives have been prepared. In vitro pharmacological characterization resulted in the identification of bile acid receptor modulators with improved pharmacokinetic properties.

Control of Peptide Aggregation and Fibrillation by Physical PEGylation.

Peptide therapeutics have the potential to self-associate, leading to aggregation and fibrillation. Noncovalent PEGylation offers a strategy to improve their physical stability; an understanding of the behavior of the resulting polymer/peptide complexes is, however, required. In this study, we have performed a set of experiments with additional mechanistic insight provided by in silico simulations to characterize the molecular organization of these complexes. We used palmitoylated vasoactive intestinal peptide (VIP-palm) stabilized by methoxy-poly(ethylene glycol)5kDa-cholane (PEG-cholane) as our model system. Homogeneous supramolecular assemblies were found only when complexes of PEG-cholane/VIP-palm exceeded a molar ratio of 2:1; at and above this ratio, the simulations showed minimal exposure of VIP-palm to the solvent. Supramolecular assemblies formed, composed of, on average, 9-11 PEG-cholane/VIP-palm complexes with 2:1 stoichiometry. Our in silico results showed the structural content of the helical conformation in VIP-palm increases when it is complexed with the PEG-cholane molecule; this behavior becomes yet more pronounced when these complexes assemble into larger supramolecular assemblies. Our experimental results support this: the extent to which VIP-palm loses helical structure as a result of thermal denaturation was inversely related to the PEG-cholane:VIP-palm molar ratio. The addition of divalent buffer species and increasing the ionic strength of the solution both accelerate the formation of VIP-palm fibrils, which was partially and fully suppressed by 2 and >4 mol equivalents of PEG-cholane, respectively. We conclude that the relative freedom of the VIP-palm backbone to adopt nonhelical conformations is a key step in the aggregation pathway.

Disruption of TFGß-SMAD3 pathway by the nuclear receptor SHP mediates the antifibrotic activities of BAR704, a novel highly selective FXR ligand.

Liver fibrosis, a major health concern worldwide, results from abnormal collagen deposition by activated hepatic stellate cells (HSCs) in an injured liver. The farnesoid-x-receptor (FXR) is a bile acid sensor that counteracts HSCs transdifferentiation. While targeting FXR holds promise, 6-ethyl-CDCA known as obeticholic acid, the first in class of FXR ligands, causes side effects, partially because the lack of selectivity toward GPBAR1, a putative itching receptor. Here, we describe the 3-deoxy-6-ethyl derivative of CDCA, BAR704, as a highly selective steroidal FXR agonist. METHODS: Liver Fibrosis was induced in mice by carbon tetrachloride (CCl4). MAIN RESULTS: In transactivation assay BAR704 activated FXR with and EC50 of 967 nM while exerted no agonistic activity on other receptors including GPBAR1. In naïve mice, BAR704 modulated the expression of FXR target genes in the liver of wild type mice but not in FXR-/- mice. In cirrhotic mice, administration of BAR704, 15 mg/kg for 9 weeks, spared the liver biosynthetic activity (bilirubin and albumin plasma levels), reduced liver fibrosis score (Sirius red staining), expression of pro-fibrogenetic (Colα1α, TGFß and αSMA) and inflammatory genes (IL-1ß, TNFα) and portal pressure. From mechanistic stand point, we have found that exposure of LX2 cells, a human HSCs line, to BAR704 increased the transcription of the short heterodimer partner (SHP) and induced the binding of this nuclear receptor to SMAD3, thus abrogating the binding of phosho-SMAD3 to the TGFß promoter. CONCLUSIONS AND APPLICATIONS: BAR704 is a selective FXR agonist that reduces liver fibrosis by interfering with the TGFß-SMAD3 pathway in HSCs. Selective FXR agonists may represent an attractive strategy for the treatment of liver fibrosis.

Synthesis and activity evaluation of a series of cholanamides as modulators of the liver X receptors.

The Liver X receptors (LXRs) are members of the nuclear receptor family, that play fundamental roles in cholesterol transport, lipid metabolism and modulation of inflammatory responses. In recent years, the synthetic steroid N,N-dimethyl-3ß-hydroxycholenamide (DMHCA) arised as a promising LXR ligand. This compound was able to dissociate certain beneficial LXRs effects from those undesirable ones involved in triglyceride metabolism. Here, we synthetized a series of DMHCA analogues with different modifications in the steroidal nucleus involving the A/B ring fusion, that generate changes in the overall conformation of the steroid. The LXRα and LXRß activity of these analogues was evaluated by using a luciferase reporter assay in BHK21 cells. Compounds were tested in both the agonist and antagonist modes. Results indicated that the agonist/antagonist profile is dependent on the steroid configuration at the A/B ring junction. Notably, in contrast to DMHCA, the amide derived from lithocholic acid (2) with an A/B cis configuration and its 6,19-epoxy analogue 4 behaved as LXRα selective agonists, while the 2,19-epoxy analogues with an A/B trans configuration were antagonists of both isoforms. The binding mode of the analogues to both LXR isoforms was assessed by using 50 ns molecular dynamics (MD) simulations. Results revealed conformational differences between LXRα- and LXRß-ligand complexes, mainly in the hydrogen bonding network that involves the C-3 hydroxyl. Overall, these results indicate that the synthetized DMHCA analogues could be interesting candidates for a therapeutic modulation of the LXRs.

BAR502, a dual FXR and GPBAR1 agonist, promotes browning of white adipose tissue and reverses liver steatosis and fibrosis.

Non-alcoholic steatohepatitis (NASH) is a highly prevalent chronic liver disease. Here, we have investigated whether BAR502, a non-bile acid, steroidal dual ligand for FXR and GPBAR1, reverses steato-hepatitis in mice fed a high fat diet (HFD) and fructose. After 9 week, mice on HFD gained ≈30% of b.w (P < 0.01 versus naïve) and were insulin resistant. These overweighting and insulin resistant mice were randomized to receive HFD or HFD in combination with BAR502. After 18 weeks, HFD mice developed NASH like features with severe steato-hepatitis and fibrosis, increased hepatic content of triacylglycerol and cholesterol and expression of SREPB1c, FAS, ApoC2, PPARα and γ, α-SMA, α1 collagen and MCP1 mRNAs. Treatment with BAR502 caused a ≈10% reduction of b.w., increased insulin sensitivity and circulating levels of HDL, while reduced steatosis, inflammatory and fibrosis scores and liver expression of SREPB1c, FAS, PPARγ, CD36 and CYP7A1 mRNA. BAR502 increased the expression of SHP and ABCG5 in the liver and SHP, FGF15 and GLP1 in intestine. BAR502 promoted the browning of epWAT and reduced liver fibrosis induced by CCl4. In summary, BAR502, a dual FXR and GPBAR1 agonist, protects against liver damage caused by HFD by promoting the browning of adipose tissue.

A novel combined strategy for the physical PEGylation of polypeptides.

Poly(ethylene glycol) (PEG) may be covalently conjugated to peptide drugs to overcome their rapid clearance but in doing so their potency can be lost. Here, a non-covalent approach was used to conjugate PEG bearing a terminal cholanic moiety (mPEG5kDa-cholane) to a 28 amino acid peptide, vasoactive intestinal peptide (VIP). Palmitoylation of the peptide was essential to facilitate physical interaction via a single binding site involving two mPEG5kDa-cholane molecules with an affinity constant of ~3·10(4)M(-1); these calorimetry data corroborating Scatchard analysis of dissolution data. The peptide/polymer complex (below 10-12nm diameter) provided for up to 5000-fold greater solubility of the peptide at pH7.4 (4µg/mL) and markedly increased peptide solution stability at 25°C over 30days. Mannitol enabled the complex to be lyophilized to yield a freeze-dried formulation which was efficiently reconstituted albeit with an ~10% decrease in solubility. The predominantly α-helical conformation of the peptide alone at pH5-6.5 was lost at pH7.4 but fully recovered with 2 molar equivalents of mPEG5kDa-cholane. After lyophilization and reconstitution an ~10% loss of α-helical conformation was observed, which may reflect the equivalent decrease in solubility. Pharmacokinetic studies following subcutaneous administration of the peptide (0.1mg/Kg) alone and with 2 molar equivalents of polymer showed that mPEG5kDa-cholane dramatically increased peptide concentration in the systemic circulation. This is the first demonstration of non-covalent PEGylation of acylated peptides, an important biologic class, which improves in vitro and in vivo properties, and thereby may prove an alternative to covalent PEGylation strategies.

Impaired Itching Perception in Murine Models of Cholestasis Is Supported by Dysregulation of GPBAR1 Signaling.

BACKGROUND & AIMS: In cholestatic syndromes, body accumulation of bile acids is thought to cause itching. However, the mechanisms supporting this effect remain elusive. Recently, GPBAR1 (TGR5) a G-protein coupled receptor has been shown to mediate itching caused by intradermal administration of DCA and LCA. 6α-ethyl-3α, 7α-dihydroxy-24-nor-5ß-cholan-23-ol (BAR502) is a non-bile acid dual ligand for FXR and GPBAR1. METHODS: Cholestasis was induced in wild type and GPBAR1-/- mice by administration of α-naphthyl-isothiocyanate (ANIT) or 17α-ethynylestradiol. RESULTS: In naïve mice skin application of DCA, TLCA, 6-ECDCA, oleanolic and betulinic acid induces a GPBAR1 dependent pruritogenic response that could be desensitized by re-challenging the mice with the same GPBAR1 agonist. In wild type and GPBAR1-/- mice cholestasis induced by ANIT fails to induce spontaneous itching and abrogates scratching behavior caused by intradermal administration of DCA. In this model, co-treatment with BAR502 increases survival, attenuates serum alkaline phosphatase levels and robustly modulates the liver expression of canonical FXR target genes including OSTα, BSEP, SHP and MDR1, without inducing pruritus. Betulinic acid, a selective GPBAR1 ligand, failed to rescue wild type and GPBAR1-/- mice from ANIT cholestasis but did not induced itching. In the 17α-ethynylestradiol model BAR502 attenuates cholestasis and reshapes bile acid pool without inducing itching. CONCLUSIONS: The itching response to intradermal injection of GPBAR1 agonists desensitizes rapidly and is deactivated in models of cholestasis, explain the lack of correlation between bile acids levels and itching severity in cholestatic syndromes. In models of non-obstructive cholestasis, BAR502 attenuates liver injury without causing itching.

Exploitation of cholane scaffold for the discovery of potent and selective farnesoid X receptor (FXR) and G-protein coupled bile acid receptor 1 (GP-BAR1) ligands.

Nuclear and G-protein coupled receptors are considered major targets for drug discovery. FXR and GP-BAR1, two bile acid-activated receptors, have gained increasing consideration as druggable receptors. Because endogenous bile acids often target both receptor families, the development of selective ligands has been proven difficult, exposing patients to side effects linked to an unwanted activation of one of the two receptors. In the present study, we describe a novel library of semisynthetic bile acid derivatives obtained by modifications on the cholane scaffold. The pharmacological characterization of this library led to the discovery of 7α-hydroxy-5ß-cholan-24-sulfate (7), 6ß-ethyl-3α,7ß-dihydroxy-5ß-cholan-24-ol (EUDCOH, 26), and 6α-ethyl-3α, 7α-dihydroxy-24-nor-5ß-cholan-23-ol (NorECDCOH, 30) as novel ligands for FXR and GP-BAR1 that might hold utility in the treatment of FXR and GP-BAR1 mediated disorders.

A novel soluble supramolecular system for sustained rh-GH delivery.

Methoxy-poly(ethylene glycol)s bearing a terminal cholanic moiety (mPEG(5kDa)-cholane, mPEG(10kDa)-cholane and mPEG(20kDa)-cholane) were physically combined with recombinant human growth hormone (rh-GH) to obtain supramolecular assemblies for sustained hormone delivery. The association constants (Ka) calculated by Scatchard analysis of size exclusion chromatography (SEC) data were in the order of 10(5)M(-1). The complete rh-GH association with mPEG(5kDa)-cholane, mPEG(10kDa)-cholane and mPEG(20kDa)-cholane was achieved with 7.5 ± 1.1, 3.9 ± 0.4 and 2.6 ± 0.4 w/w% rh-GH/mPEG-cholane, respectively. Isothermal titration calorimetry (ITC) yielded association constants similar to that calculated by SEC and showed that rh-GH has 21-25 binding sites for mPEG-cholane, regardless the polymer molecular weight. Dialysis studies showed that the mPEG-cholane association strongly delays the protein release; 80-90% of the associated rh-GH was released in 200 h. However, during the first 8h the protein formulations obtained with mPEG(10kDa)-cholane and mPEG(20kDa)-cholane showed a burst release of 8 and 28%, respectively. Circular dichroism (CD) analyses showed that the mPEG(5kDa)-cholane association does not alter the secondary structure of the protein. Furthermore, mPEG(5kDa)-cholane was found to enhance both the enzymatic and physical stability of rh-GH. In vivo pharmacokinetic and pharmacodynamic studies were performed by subcutaneous administration of rh-GH and rh-GH/mPEG(5kDa)-cholane to normal and hypophysectomised rats. The study showed that mPEG(5kDa)-cholane decreases the maximal concentration in the blood but prolongs the body exposure of the protein, which resulted in 55% bioavailability increase. Finally, rh-GH formulated with mPEG(5kDa)-cholane yielded prolonged weight increase of hypophysectomised rats as compared to rh-GH in buffer or formulated with mPEG(5kDa)-OH. After the second administration the weight of the animals treated with rh-GH formulated with mPEG(5kDa)-cholane was about 2 times higher than that obtained with equal dose of non-formulated rh-GH.

Bazedoxifene-scaffold-based mimetics of solomonsterols A and B as novel pregnane X receptor antagonists.

Pregnane X receptor (PXR), a member of the NR1I nuclear receptor family, acts as a xenobiotic sensor and a paramount transcriptional regulator of drug-metabolizing enzymes and transporters. The overexpression of PXR in various cancer cells indicates the importance of PXR as a drug target for countering multidrug resistance in anticancer treatments. We describe the discovery of novel bazedoxifene-scaffold-based PXR antagonists inspired by the marine sulfated steroids solomonsterol A and B as natural leads. A luciferase reporter assay on a PXR-transfected HepG2 cell line identified compounds 19-24 as promising PXR antagonists. Further structure-activity relationship studies of the most active PXR antagonist from the series (compound 20, IC50 = 11 µM) revealed the importance of hydroxyl groups as hydrogen-bond donors for PXR antagonistic activity. PXR antagonists 20 and 24 (IC50 = 14 µM), in addition to the downregulation of PXR expression, exhibited inhibition of PXR-induced CYP3A4 expression, which illustrates their potential to suppress PXR-regulated phase-I drug metabolism.

Design, synthesis, and biological evaluation of potent dual agonists of nuclear and membrane bile acid receptors.

Bile acids exert genomic and nongenomic effects by interacting with membrane G-protein-coupled receptors, including the bile acid receptor GP-BAR1, and nuclear receptors, such as the farnesoid X receptor (FXR). These receptors regulate overlapping metabolic functions; thus, GP-BAR1/FXR dual agonists, by enhancing the biological response, represent an innovative strategy for the treatment of enteroendocrine disorders. Here, we report the design, total synthesis, and in vitro/in vivo pharmacological evaluation of a new generation of dual bile acid receptor agonists, with the most potent compound, 19, showing promising pharmacological profiles. We show that compound 19 activates GP-BAR1, FXR, and FXR regulated genes in the liver, increases the intracellular concentration of cAMP, and stimulates the release of the potent insulinotropic hormone GLP-1, resulting in a promising drug candidate for the treatment of metabolic disorders. We also elucidate the binding mode of the most potent dual agonists in the two receptors through a series of computations providing the molecular basis for dual GP-BAR1/FXR agonism.

Solomonsterol A, a marine pregnane-X-receptor agonist, attenuates inflammation and immune dysfunction in a mouse model of arthritis.

In the present study we provide evidence that solomonsterol A, a selective pregnane X receptor (PXR) agonist isolated from the marine sponge Theonella swinhoei, exerts anti-inflammatory activity and attenuates systemic inflammation and immune dysfunction in a mouse model of rheumatoid arthritis. Solomonsterol A was effective in protecting against the development of arthritis induced by injecting transgenic mice harboring a humanized PXR, with anti-collagen antibodies (CAIA) with beneficial effects on joint histopathology and local inflammatory response reducing the expression of inflammatory markers (TNFα, IFNγ and IL-17 and chemokines MIP1α and RANTES) in draining lymph nodes. Solomonsterol A rescued mice from systemic inflammation were assessed by measuring arthritis score, CRP and cytokines in the blood. In summary, the present study provides a molecular basis for the regulation of systemic local and systemic immunity by PXR agonists.