Mifepristone polymorph with enhanced solubility, dissolution and oral bioavailability.

Mifepristone is one of potent anti-progesterone agents, which binds to progesterone receptors and glucocorticoid receptors. Until now, there are a lot of research focusing on enhancing the solubility and oral bioavailability of Mifepristone. However, poor solubility and oral bioavailability has some undesirable consequences. In this work, Mifepristone in form D was discovered for the first time and characterized by PXRD, TGA, DSC, FT-IR, SEM and SS NMR. Form D was a metastable crystal type which manifested favorable stability under ambient conditions. Form D had better dissolution characteristic compared with commercial Mifepristone in 0.5% SDS solution. In addition, Mifepristone in form D exhibited a 1.43-fold higher peak plasma concentration (Cmax) and 1.46-fold higher area under the curve (AUC) in rats. The work in this paper is a complement to the present understanding of drug polymorphism on the in vitro and in vivo behavior, and establishes the ground work for future development of Mifepristone in form D as a promising drug for the market.

The effect of GnRH antagonist cetrorelix on Wnt signaling members in pubertal and adult mouse ovaries.

Wide application of gonadotropin-releasing hormone (GnRH) agonists and antagonists for clinical purposes determines their effects on ovarian signaling pathways. Our study aimed to determine the localization, expression levels of Wnt signaling members in the pubertal and adult mouse ovary and the impact of GnRH antagonist cetrorelix on these signaling members. 0.5 mg/kg of cetrorelix was injected to 3-and 6-week-old mice for 2 weeks. At the end of injection, ovaries from 5 (5Ce)- to 8-week (8Ce)-old mice were embedded in paraffin for immunohistochemistry and homogenized for western blot to compare with control (5C-8C) and sham groups (5S-8S). WNT2 and WNT4 showed higher expression in thecal and stromal cells in adult mouse ovaries and only WNT4 expression was affected by cetrorelix. FZD1 was localized mainly in oocytes of pubertal ovaries and granulosa cells and oocytes of adult ovaries. FZD1 was reduced by cetrorelix in pubertal ovaries. FZD4 was abundantly localized in thecal and stromal cells of all groups and protein level was not affected by cetrorelix. LRP-6 was expressed mainly in oocytes and stromal cells of pubertal, oocytes of adult ovaries and its expression was reduced by cetrorelix in adult ovaries. CTNNB1 intensity in granulosa cells was the lowest in pubertal and the highest in adult ovaries and its expression was decreased by cetrorelix in adult ovaries. Cetrorelix affected the expression of specific members of the Wnt signaling depending on the developmental stage of mice, pointing out its possible interaction with gonadotropins during pubertal and adult stages.

Discovery of a Potent and Selective Steroidal Glucocorticoid Receptor Antagonist (ORIC-101).

The glucocorticoid receptor (GR) has been linked to therapy resistance across a wide range of cancer types. Preclinical data suggest that antagonists of this nuclear receptor may enhance the activity of anticancer therapy. The first-generation GR antagonist mifepristone is currently undergoing clinical evaluation in various oncology settings. Structure-based modification of mifepristone led to the discovery of ORIC-101 (28), a highly potent steroidal GR antagonist with reduced androgen receptor (AR) agonistic activity amenable for dosing in androgen receptor positive tumors and with improved CYP2C8 and CYP2C9 inhibition profile to minimize drug-drug interaction potential. Unlike mifepristone, 28 could be codosed with chemotherapeutic agents readily metabolized by CYP2C8 such as paclitaxel. Furthermore, 28 demonstrated in vivo antitumor activity by enhancing response to chemotherapy in the GR+ OVCAR5 ovarian cancer xenograft model. Clinical evaluation of safety and therapeutic potential of 28 is underway.

Editor's Highlight: Structure-Based Investigation on the Binding and Activation of Typical Pesticides With Thyroid Receptor.

A broad range of pesticides have been reported to interfere with the normal function of the thyroid endocrine system. However, the precise mechanism(s) of action has not yet been thoroughly elucidated. In this study, 21 pesticides were assessed for their binding interactions and the potential to disrupt thyroid homeostasis. In the GH3 luciferase reporter gene assays, 5 of the pesticides tested had agonistic effects in the order of procymidone > imidacloprid > mancozeb > fluroxypyr > atrazine. 11 pesticides inhibited luciferase activity of T3 to varying degrees, demonstrating their antagonistic activity. And there are 4 pesticides showed mixed effects when treated with different concentrations. Surface plasmon resonance (SPR) biosensor technique was used to directly measure the binding interactions of these pesticides to the human thyroid hormone receptor (hTR). 13 pesticides were observed to bind directly with TR, with a KD ranging from 4.80E-08 M to 9.44E-07 M. The association and disassociation of the hTR/pesticide complex revealed 2 distinctive binding modes between the agonists and antagonists. At the same time, a different binding mode was displayed by the pesticides showed mix agonist and antagonist activity. In addition, the molecular docking simulation analyses indicated that the interaction energy calculated by CDOCKER for the agonists and antagonists correlated well with the KD values measured by the surface plasmon resonance assay. These results help to explain the differences of the TR activities of these tested pesticides.

SPA70 is a potent antagonist of human pregnane X receptor.

Many drugs bind to and activate human pregnane X receptor (hPXR) to upregulate drug-metabolizing enzymes, resulting in decreased drug efficacy and increased resistance. This suggests that hPXR antagonists have therapeutic value. Here we report that SPA70 is a potent and selective hPXR antagonist. SPA70 inhibits hPXR in human hepatocytes and humanized mouse models and enhances the chemosensitivity of cancer cells, consistent with the role of hPXR in drug resistance. Unexpectedly, SJB7, a close analog of SPA70, is an hPXR agonist. X-ray crystallography reveals that SJB7 resides in the ligand-binding domain (LBD) of hPXR, interacting with the AF-2 helix to stabilize the LBD for coactivator binding. Differential hydrogen/deuterium exchange analysis demonstrates that SPA70 and SJB7 interact with the hPXR LBD. Docking studies suggest that the lack of the para-methoxy group in SPA70 compromises its interaction with the AF-2, thus explaining its antagonism. SPA70 is an hPXR antagonist and promising therapeutic tool.The xenobiotic-activated human pregnane X receptor (hPXR) regulates drug metabolism. Here the authors develop hPXR modulators, which are of potential therapeutic interest and functionally and structurally characterize the antagonist SPA70 and the structurally related agonist SJB7.

Uterus-targeted liposomes for preterm labor management: studies in pregnant mice.

Preterm labor caused by uterine contractions is a major contributor to neonatal morbidity and mortality. Treatment intended to reduce uterine contractions include tocolytic agents, such as indomethacin. Unfortunately, clinically used tocolytics are frequently inefficient and cross the placenta causing fetal side effects. Here we show for the first time in obstetrics the use of a targeted nanoparticle directed to the pregnant uterus and loaded with a tocolytic for reducing its placental passage and sustaining its efficacy. Nanoliposomes encapsulating indomethacin and decorated with clinically used oxytocin receptor antagonist were designed and evaluated in-vitro, ex-vivo and in-vivo. The proposed approach resulted in targeting uterine cells in-vitro, inhibiting uterine contractions ex-vivo, while doubling uterine drug concentration, decreasing fetal levels, and maintaining the preterm birth rate in vivo in a pregnant mouse model. This promising approach opens new horizons for drug development in obstetrics that could greatly impact preterm birth, which currently has no successful treatments.

Amylin Amyloid Inhibition by Flavonoid Baicalein: Key Roles of Its Vicinal Dihydroxyl Groups of the Catechol Moiety.

Amyloid formation of the 37-residue amylin is involved in the pathogenesis of type 2 diabetes and, potentially, diabetes-induced neurological deficits. Numerous flavonoids exhibit inhibitory effects against amylin amyloidosis, but the mechanisms of inhibition remain unclear. Screening a library of natural compounds uncovered a potent lead compound, the flavone baicalein. Baicalein inhibits amylin amyloid formation and reduces amylin-induced cytotoxicity. Analogue analyses demonstrated, for the first time, key roles of the vicinal hydroxyl groups on the A-ring. We provided mass spectrometric evidence that incubating baicalein and amylin leads to their conjugation, consistent with a Schiff base mechanism.

Gonadotropin-releasing hormone receptor-targeted paclitaxel-degarelix conjugate: synthesis and in vitro evaluation.

To increase the selectivity of chemotherapeutic agents, receptor-mediated tumor-targeting approaches have been developed. Here, degarelix [Ac-D-Nal-D-Cpa-D-Pal-Ser-Aph(L-Hor)-D-Aph(Cbm)-Leu-ILys-Pro-D-Ala-NH2], a gonadotropin-releasing hormone antagonist, was employed as a targeting moiety for paclitaxel (PTX). Five PTX-degarelix conjugates were synthesized, in which PTX was attached via disulfide bond to the different position in the degarelix sequence. All of the PTX-degarelix conjugates exhibited a half-life greater than 10 h determined in human serum. A fluorometric imaging plate reader assay showed that the conjugates LK-MY-9 and LK-MY-10 had an antagonism efficacy similar to that of degarelix. The in vitro cytostatic effects of the conjugates were determined by a (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay, and the 50% inhibitory concentration value of the conjugates on 3T3 mouse embryonic fibroblast cells were one order of magnitude higher than the 50% inhibitory concentration values of the conjugates on MCF-7 human breast cancer cells and HT-29 human colon cancer cells. Receptor saturation tests further demonstrated that pre-incubation of the cells with degarelix reduced the efficacy of LK-MY-10 in a concentration-dependent manner. In conclusion, degarelix is a valid and stable moiety that has great potential for targeting chemotherapy drugs.

Utilization of human nuclear receptors as an early counter screen for off-target activity: a case study with a compendium of 615 known drugs.

Off-target effects of drugs on nuclear hormone receptors (NHRs) may result in adverse effects in multiple organs/physiological processes. Reliable assessments of the NHR activities for drug candidates are therefore crucial for drug development. However, the highly permissive structures of NHRs for vastly different ligands make it challenging to predict interactions by examining the chemical structures of the ligands. Here, we report a detailed investigation on the agonistic and antagonistic activities of 615 known drugs or drug candidates against a panel of 6 NHRs: androgen, progesterone, estrogen α/ß, and thyroid hormone α/ß receptors. Our study revealed that 4.7 and 12.4% compounds have agonistic and antagonistic activities, respectively, against this panel of NHRs. Nonetheless, potent, unintended NHR hits are relatively rare among the known drugs, indicating that such interactions are perhaps not tolerated during drug development. However, we uncovered examples of compounds that unintentionally agonize or antagonize NHRs. In addition, a number of compounds showed multi-NHR activities, suggesting that the cross-talk between multiple NHRs co-operate to elicit in vivo effects. These data highlight the merits of counter screening drug candidate against NHRs during drug discovery/development.

Antihormonal potential of selected D-homo and D-seco estratriene derivatives.

Since many estrogen derivatives exhibit anti-hormone or enzyme inhibition potential, a large number of steroidal derivatives have been synthesised from appropriate precursors, in order to obtain potential therapeutics for the treatment of hormone-dependent cancers. In molecular docking studies, based on X-ray crystallographic analysis, selected D-homo and D-seco estratriene derivatives were predicted to bind strongly to estrogen receptor α (ERα), aromatase and 17,20 lyase, suggesting they could be good starting compounds for antihormonal studies. Test results in vivo suggest that these compounds do not possess estrogenic activity, while some of them showed weak anti-estrogenic properties. In vitro anti-aromatase and anti-lyase assays showed partial inhibition of these two enzymes, while some compounds activated aromatase. Aromatase activators are capable of promoting estrogen synthesis for treatment of pathological conditions caused by estrogen depletion, e.g. osteopenia or osteoporosis.

Oil-based formulation as a sustained-released injection for a novel synthetic peptide.

In this study, sustained-release of GnRH antagonist peptide LXT-101 was realized through oil formulation, and their releasing characteristics in vitro and in vivo were investigated. In this formulation, the static interaction between cationic charged peptide LXT-101 and the negative charged phospholipid led to the formation of the phospholipid-peptide complex, by which LXT-101 was completely dissolved in oils. This formulation was prepared by mixing an aqueous solution of LXT-101 and empty SUV (small unilamellar liposomes) containing EPC (phosphatidylcholine) and DPPG (1, 2-dipalmitog-sn-glycero-3- phosphoglycerol) at an appropriate ratio, the mixture was subsequently lyophilized, and the resultant was dissolved in the oil to form a clear oily solution containing solubilized peptide LXT-101. With atomic force microscopy combined with Langmuir-Blodgett technology, the morphology of the particles in the oily solution were examined to be oval-shaped and the mean particle size was 150 nm in diameter. In pure water at 37°C, about 70~90 % of LXT-101 was released slowly from the oily formulation over 7 days. An effective sustained suppression of testosterone in beagle dogs could be achieved over a period of seven days with this LXT-101 oily formulation, by i.m. at a dose of 0.2 mg/kg (2 mg/ml). This formulation dramatically improved the bioactivity of LXT-101 compared to its aqueous solution. It was also found that when the concentration of peptide LXT-101 was up to or over 10 mg/ml in aqueous solution, there was no significant difference between the oily formulation and aqueous solution. This fact meant that LXT-101 itself could conduct sustained release in vivo by self-assembly of nanofibers.

Synthesis and biological evaluation of 11' imidazolyl antiprogestins and mesoprogestins.

Antiprogestins with a 4' para imidazolylphenyl moiety were synthesized and their biochemical interactions with the progesterone and glucocorticoid receptor were investigated. Depending on the substitution pattern at the 17 position partial progesterone receptor (PR)-agonistic derivatives like compounds EC339 and EC336 or pure antagonists like compound EC317 were obtained. EC317 was investigated in vivo and found to be significantly more potent than RU 486 in cycling and pregnant guinea pigs. For testing the biological action progesterone receptor modulators (PRM), guinea pigs appears as a specific model when compare to pregnant human uterus. This model correlates to human conditions such as softening and widening of the cervix, the elevation of the uterine responsiveness to prostaglandins and oxytocin, and finally to induction of labor. The use of non-pregnant guinea pigs permitted the simultaneous assessment of PR-agonistic and PR-antagonistic properties and their physiological interactions with uterine and vaginal environment. These can histologically be presumed from the presence of estrogen or progesterone dominance in the genital tract tissues. The ovarian histology indicated the effects on ovulation. Corpora lutea in guinea pigs further reflects inhibitory effects of the progesterone-dependent uterine prostaglandin secretion. PRMs are initially synthesized as analogues of RU 486. They represent a heterogeneous group of compounds with different ratios of PR-agonistic and-antagonistic properties. PR-agonistic properties may be essential for uterine anti-proliferative effects. In various clinical studies these were also attributed to RU 486 or Ulipristal [1,2]. Adjusted PR-agonistic PRMs (EC312, EC313) [3] may be more effective in achieving a mitotically resting endometrium and superior uterine tumor inhibition. For the use in termination of pregnancy, progesterone-inhibitory effects are essentially needed. Even minor PR-agonistic properties compromise the therapeutic goals. Pure PR-antagonists, as EC317, clearly exceeded the gold standard RU 486 with respect to labor inducing effects. Mechanistically it is surprising that both types of compound may be potent inhibitors of ovulation.

Self-assembled nanostructures of long-acting GnRH analogs modified at position 7.

It is well known that GnRH analogs can self-assemble into amyloid fibrils and that the duration of action of GnRH analogs depends on the ability of the amyloid to slowly release active peptides. The aim of this study was to investigate the influence of the amino acid residues at position 7 of GnRH analogues on peptide self-assembly. It was found that the dominant shape of the nanostructure can be changed when the structures of the residues at position 7 differ significantly from that of leucine in Degarelix. When the backbone length was extended (peptide 9), or the side chain of the residue at position 7 was replaced by an aromatic ring (peptide 6), or the rotation of the amide bond was restricted (peptide 8), the nanostructure changed from fibrils to vesicles. The results also indicate that the increasing hydrophilicity had little influence on the nanostructure morphology. In addition, a suitable release rate was found to play a more important role for the duration of the peptide action by maintaining the equilibrium between the drug concentration and the persistent release time, while the nanostructure shape was found to exert little influence on the duration of the peptide action.

Stress-induced glucocorticoid signaling remodels neurovascular coupling through impairment of cerebrovascular inwardly rectifying K+ channel function.

Studies of stress effects on the brain have traditionally focused on neurons, without considering the cerebral microcirculation. Here we report that stress impairs neurovascular coupling (NVC), the process that matches neuronal activity with increased local blood flow. A stressed phenotype was induced in male rats by administering a 7-d heterotypical stress paradigm. NVC was modeled by measuring parenchymal arteriole (PA) vasodilation in response to neuronal stimulation in amygdala brain slices. After stress, vasodilation of PAs to neuronal stimulation was greatly reduced, and dilation of isolated PAs to external K(+) was diminished, suggesting a defect in smooth muscle inwardly rectifying K(+) (KIR) channel function. Consistent with these observations, stress caused a reduction in PA KIR2.1 mRNA and smooth muscle KIR current density, and blocking KIR channels significantly inhibited NVC in control, but not in stressed, slices. Delivery of corticosterone for 7 d (without stressors) or RU486 (before stressors) mimicked and abrogated NVC impairment by stress, respectively. We conclude that stress causes a glucocorticoid-mediated decrease in functional KIR channels in amygdala PA myocytes. This renders arterioles less responsive to K(+) released from astrocytic endfeet during NVC, leading to impairment of this process. Because the fidelity of NVC is essential for neuronal health, the impairment characterized here may contribute to the pathophysiology of brain disorders with a stress component.

Molecular docking revealed potential disruptors of glucocorticoid receptor-dependent reporter gene expression.

Glucocorticoids are an essential part of the endocrine system that is responsible for a variety of functions such as regulation of immune activity, appropriate brain function, and fetal development. Disturbance of glucocorticoid signaling can lead to various cardiovascular, inflammatory, and autoimmune diseases, so the identification of chemicals that can modulate activity of the glucocorticoid receptor (GR) is crucial. In this study, molecular docking was utilized to find new agonists and antagonists of the GR. The best hits were further tested on the in vitro model of MDA-kb2 cells expressing luciferase activity in a GR-dependent manner. Nine new potential modulators of the receptor, belonging to six structurally diverse classes, were identified. Six of them, tetramethrin and cypermethrin, diethyl hexyl phthalate and diphenyl isophthalate, naphthol AS-OL and dicumyl peroxide, induced luciferase activity; while the other three, bisphenol P, bisphenol M, and Antioxidant 425, suppressed luciferase activity. Of the nine potential GR modulators, only bisphenol M displayed appreciable binding affinity for the receptor.

Novel oxytocin receptor agonists and antagonists: a patent review (2002 - 2013).

INTRODUCTION: Oxytocin is a nine amino acid cyclic peptide hormone with a high degree of uterotonic activity. The oxytocin receptor (Oxtr) is most strongly expressed in the uterus and mammary gland, but can also be found in regions of the brain, showing a neurotransmitter-like activity. The Oxtr knockout mice have no obvious defects in fertility or sexual behavior, but display aberrant social behavior. All of these suggest that Oxtr may become an important therapeutic target for the treatment of a wide range of conditions. AREAS COVERED: This article will highlight the significant progress that has been made in the discovery and development of Oxtr agonists and antagonists in the patent literature between January 2002 and May 2013. EXPERT OPINION: In the past decade, cumulative evidence supports the idea that activation of the Oxtr can have a positive effect upon human cognition and social behavior. The authors suggest that new agonists and antagonists may play an important role in the treatment of disorders such as anxiety, autism or schizophrenia. It may even be that older Oxtr agonists and antagonists, which were used to overcome labor-related dysfunction, may also have a significant impact on human social behavior.

Oral delivery of oil-based formulation for a novel synthetic cationic peptide of GnRH (gonadotropin-releasing hormone) antagonist for prostate cancer treatment.

LXT-101, a cationic peptide is a novel antagonist of gonadotropin-releasing hormone (GnRH) for prostate cancer treatment. However, effective delivery of peptide drugs into the body by the oral route remains a major challenge due to their origin properties with high molecular weights, strong polarity and low stability in the gastrointestinal (GI) tract. In this study, we have developed a novel oral delivery of oil-based formulation in which therapeutic peptide LXT-101 are solubilized in oils and with this solution as oil phase, an optimum formulation of self-microemulsifying drug delivery system (SMEDDS) was developed. The peptide stability with the SMEDDS formulation in artificial gastric and intestinal fluid was tested in vitro. On the other hand, the testosterone level and plasma concentration of LXT-101 in rats after oral administration of the SMEDDS formulation were investigated in vivo. The data in vitro indicated that LXT-101 in the SMEDDS formulation was stable over 8 h in artificial gastric and intestinal fluid. LXT-101 can be absorbed in vivo and suppression of testosterone maintained in castration level within 12 h can be achieved effectively after SMEDDS formulation administered orally at a dose of 3.5 mg/kg. The approach can provide a potential way for delivery peptides by oral.

Design and validation of a homogeneous time-resolved fluorescence-based leptin receptor binding assay.

The pleiotropic cytokine hormone leptin, by activating its receptor OB-R, plays a major role in many biological processes, including energy homeostasis, immune function, and cell survival and proliferation. Abnormal leptin action is associated with obesity, autoimmune diseases, and cancer. The pharmacological characterization of OB-R and the development of synthetic OB-R ligands are still in their infancy because currently available binding assays are not compatible with ligand saturation binding experiments and high-throughput screening (HTS) approaches. We have developed here a novel homogeneous time-resolved fluorescence-based binding assay that overcomes these limitations. In this assay, fluorescently labeled leptin or leptin antagonist binds to the SNAP-tagged OB-R covalently labeled with terbium cryptate (Tb). Successful binding is monitored by measuring the energy transfer between the Tb energy donor and the fluorescently labeled leptin energy acceptor. Ligand binding saturation experiments revealed high-affinity dissociation constants in the subnanomolar range with an excellent signal-to-noise ratio. The assay performed in a 384-well format shows high specificity and reproducibility, making it perfectly compatible with HTS applications to identify new OB-R agonists or antagonists. In addition, fluorescently labeled leptin and SNAP-tagged OB-R will be valuable tools for monitoring leptin and OB-R trafficking in cells and tissues.

Update in TSH receptor agonists and antagonists.

The physiological role of the TSH receptor (TSHR) as a major regulator of thyroid function is well understood, but TSHRs are also expressed in multiple normal extrathyroidal tissues, and the physiological roles of TSHRs in these tissues are unclear. Moreover, TSHRs play a major role in several pathological conditions including hyperthyroidism, hypothyroidism, and thyroid tumors. Small molecule, "drug-like" TSHR agonists, neutral antagonists, and inverse agonists may be useful as probes of TSHR function in extrathyroidal tissues and as leads to develop drugs for several diseases of the thyroid. In this Update, we review the most recent findings regarding the development and use of these small molecule TSHR ligands.