Screening for Active Compounds Targeting Human Natural Killer Cell Activation Identifying Daphnetin as an Enhancer for IFN-γ Production and Direct Cytotoxicity.

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ+ NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments.

An overview of therapeutic potential of N-alkylated 1-deoxynojirimycin congeners.

Polyhydroxylated alkaloids display a wide range of biological activities, suggesting their use in the treatment of various diseases. Their most famous representative, 1-deoxynojirimycin (DNJ), is a natural product that shows α- and ß-glucosidase inhibition. This molecule has been since converted into two clinically approved drugs i.e., Zavesca® and Glyset®, targeting type I Gaucher's disease and type II diabetes mellitus, respectively. This review examines the therapeutic potential of important DNJ congeners reported in last decade and presents concise mechanism of glycosidase inhibition. A brief overview of substituents conjugation's impact on DNJ scaffold (including N-alkylated DNJ derivatives, mono-valent, di-valent and multivalent DNJ congeners, N-[5-(adamantan-1-yl-methoxy)-pentyl]-1-deoxynojirimycin (AMP-DNM) look alike DNJ based lipophilic derivatives, AMP-DNM based neoglycoconjugates, DNJ click derivatives with varying carboxylic acids and aromatic moieties, conjugates of DNJ and glucose, and N-bridged DNJ analogues) towards various enzymes such as α/ß glucosidase, porcine trehalase, as F508del-CFTR correctors, α-mannosidase, human placental ß-glucocerebrosidase, N370S ß-GCase, α-amylase and insect trehalase as potent and selective inhibitors have been discussed with potential bioactivities, which can provide inspiration for future studies.

1,3,5-Triaza-7-Phosphaadamantane (PTA) as a 31P NMR Probe for Organometallic Transition Metal Complexes in Solution.

Due to the rigid structure of 1,3,5-triaza-7-phosphaadamantane (PTA), its 31P chemical shift solely depends on non-covalent interactions in which the molecule is involved. The maximum range of change caused by the most common of these, hydrogen bonding, is only 6 ppm, because the active site is one of the PTA nitrogen atoms. In contrast, when the PTA phosphorus atom is coordinated to a metal, the range of change exceeds 100 ppm. This feature can be used to support or reject specific structural models of organometallic transition metal complexes in solution by comparing the experimental and Density Functional Theory (DFT) calculated values of this 31P chemical shift. This approach has been tested on a variety of the metals of groups 8-12 and molecular structures. General recommendations for appropriate basis sets are reported.

A comparative study of acarbose, vildagliptin and saxagliptin intended for better efficacy and safety on type 2 diabetes mellitus treatment.

As a complicated metabolic disorder, type 2 diabetes mellitus (T2DM) is becoming a major health concern worldwide. Drugs including acarbose, saxagliptin and vildagliptin are applied, but their efficacy is still required to be compared. Therefore, the study aimed to evaluate the efficacy and safety of acarbose, saxagliptin and vildagliptin in the treatment of T2DM. Ninety patients diagnosed with T2DM were treated with acarbose, saxagliptin and vildagliptin, respectively (30 patients for each drug). All patients were examined at 0, 4 and 12 weeks after treatment with vital signs recorded. Fasting blood glucose and blood biochemical indices were analyzed. In addition, fecal samples were taken for microbial macrogenome sequencing and safety evaluation within 12 weeks after treatment. Blood glucose level decreased at 4 and 12 weeks after treatment, and the total cholesterol (TC) and high-density lipoprotein (HDL) levels at 12 weeks were different. Genus abundance of intestinal flora was altered at different time points. Acarbose increased Butyricimonas level first and then decreased it during drug treatment. Saxagliptin increased Megamonas and decreased Turicibacter genus level gradually. Pseudomonas, Klebsiella, Blautia, Faecalibacterium and Roseburia levels fluctuated after Vildagliptin treatment, which increased fasting C-peptide level greater than the other two drugs. Saxagliptin showed higher adverse reactions than acarbose and vildagliptin. Collectively, acarbose, vildagliptin, and saxagliptin can effectively reduce the HbA1c level and affect the intestinal flora distribution in T2DM patients, and the adverse reactions of acarbose and vildagliptin are less than saxagliptin, providing alternative strategies for the treatment of T2DM.

Effect of stevia aqueous extract on the antidiabetic activity of saxagliptin in diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Stevia rebaudiana Bertoni is a perennial herb that belongs to the Asteraceae family. It is a natural sweetener plant known as "Sweet Leaf", "Sweet Herbs" and "Honey Leaf", which is estimated to be 300 times more sweetening than sugar cane. Stevia has been used as a traditional treatment for diabetes in many countries for hundreds of years. Several animal studies referred to the antihyperglycemic activity of stevia. However, the combined use of stevia with saxagliptin has not been studied so far, so this study has been done. The aim of the present study was to evaluate the antihyperglycemic effect of stevia alone and in combination with saxagliptin. MATERIALS AND METHODS: Diabetes was induced in rats by i.p. injection of streptozotocin and nicotinamide. Animals were divided into five groups, each contains eight rats. Group I: included negative controland group II: included diabetic control that received saline. Group III: included diabetic rats that received 400 mg/kg/day stevia aqueous extract. Group IV: included diabetic rats that received saxagliptin 10 mg/kg/day. Group V: included diabetic rats that received stevia 400 mg/kg + saxagliptin 10 mg/kg. Food and water intake were measured daily while body weight was measured weekly. After 3 weeks animals were sacrificed and blood and tissue samples were collected. Fasting blood glucose (FBG), serum insulin, serum dipeptidylepeptidase-4 (DPP-4), TC, TGs, LDL, HDL, GSH and MDA were measured in treated and control rats by colorimetric and ELISA methods. RESULTS: Both stevia and saxagliptin significantly reduced food, water intake, body weight and FBG. Stevia with saxagliptin produced more significant decrease in FBG. While serum insulin increased significantly in stevia, saxagliptin treated groups and their combination. Serum DPP-4 decreased significantly in all treated groups, concerning lipid profile, stevia and saxagliptin notably lowered TC, TGs, and LDL and increased HDL. Both stevia and saxagliptin remarkably decreased MDA and increased GSH compared to diabetic rats. In addition, stevia significantly improved the antidiabetic effects of saxagliptin. CONCLUSION: Stevia has an antihyperglycemic effect and could enhance the antidiabetic activity of saxagliptin. DPP-4 attenuation, antihyperlipidemic and antioxidant activity as well as improvement of insulin sensitivity may be involved in the antidiabetic action of stevia.

Neurotensin receptor 1 agonist provides neuroprotection in pre-diabetic rats.

Exogenous treatment of a neurotensin receptor 1 (NTR1) agonist exerted the neuroprotection in an obese and Alzheimer's model. However, the effects of NTR1 modulation on peripheral/hippocampal impairment and cognitive deficit following sustained HFD consumption are poorly understood. Forty rats received a normal diet (ND) or HFD for 16 weeks. At week 13, the ND group received a vehicle (n = 8). Thirty-two HFD-fed group were randomized into four subgroups (n = 8/subgroup) with a vehicle, 1 mg/kg of NTR1 agonist, 1 mg/kg of NTR antagonist, and combined treatment (NTR1 agonist-NTR antagonist) for 2 weeks, s.c. injection. Then, the cognitive tests and peripheral/hippocampal parameters were determined. Our findings demonstrated that NTR1 activator reversed obesity and attenuated metabolic impairment in pre-diabetic rats. It also alleviated hippocampal pathologies and synaptic dysplasticity, leading to deceleration or prevention of cognitive impairment progression. Therefore, NTR1 activation would be a possible novel therapy to decelerate or prevent progression of neuropathology and cognitive impairment in the pre-diabetes.

Hyperacmosins E-G, three new homoadamantane-type polyprenylated acylphloroglucinols from Hypericum acmosepalum.

Three new homoadamantane-type polyprenylated acylphloroglucinols, hyperacmosins E-G (1-3), with seven known compounds were isolated from the air-dried aerial parts of Hypericum asmosepalum. Their structures were determined by NMR, HRESIMS and experimental electronic circular dichroism (ECD) spectra. The hepatoprotective activity of these compounds were evaluated. Compounds 4 and 8 exhibited hepatoprotective activity against paracetamol-induced HepG2 cell damage.

The extracellular calcium-sensing receptor promotes porcine egg activation via calcium/calmodulin-dependent protein kinase II.

Extracellular calcium is required for intracellular Ca2+ oscillations needed for egg activation, but the regulatory mechanism is still poorly understood. The present study was designed to demonstrate the function of calcium-sensing receptor (CASR), which could recognize extracellular calcium as first messenger, during porcine egg activation. CASR expression was markedly upregulated following egg activation. Functionally, the addition of CASR agonist NPS R-568 significantly enhanced pronuclear formation rate, while supplementation of CASR antagonist NPS2390 compromised egg activation. There was no change in NPS R-568 group compared with control group when the egg activation was performed without extracellular calcium addition. The addition of NPS2390 precluded the activation-dependent [Ca2+ ]i rise. When egg activation was conducted in intracellular Ca2+ chelator BAPTA-AM and NPS R-568 containing medium, CASR function was abolished. Meanwhile, CASR activation increased the level of the [Ca2+ ]i effector p-CAMKII, and the presence of KN-93, an inhibitor of CAMKII, significantly reduced the CASR-mediated increasement of pronuclear formation rate. Furthermore, the increase of CASR expression following activation was reversed by inhibiting CAMKII activity, supporting a positive feedback loop between CAMKII and CASR. Altogether, these findings provide a new pathway of egg activation about CASR, as the extracellular Ca2+ effector, promotes egg activation via its downstream effector and upstream regulator CAMKII.

Potential of the zebrafish model for the forensic toxicology screening of NPS: A comparative study of the effects of APINAC and methiopropamine on the behavior of zebrafish larvae and mice.

The increased diffusion of the so-called novel psychoactive substances (NPS) and their continuous change in structure andconceivably activity has led to the need of a rapid screening method to detect their biological effects as early as possible after their appearance in the market. This problem is very felt in forensic pathology and toxicology, so the preclinical study is fundamental in the approach to clinical and autopsy cases of difficult interpretation intoxication. Zebrafish is a high-throughput suitable model to rapidly hypothesize potential aversive or beneficial effects of novel molecules. In the present study, we measured and compared the behavioral responses to two novel neuroactive drugs, namely APINAC, a new cannabimimetic drug, and methiopropamine (MPA), a methamphetamine-like compound, on zebrafish larvae (ZL) and adult mice. By using an innovative statistical approach (general additive models), it was found that the spontaneous locomotor activity was impaired by the two drugs in both species: the disruption extent varied in a dose-dependent and time-dependent manner. Sensorimotor function was also altered: i) the visual object response was reduced in mice treated with APINAC, whereas it was not after exposure to MPA; ii) the visual placing responses were reduced after treatment with both NPS in mice. Furthermore, the visual motor response detected in ZL showed a reduction after treatment with APINAC during light-dark and dark-light transition. The same pattern was found in the MPA exposed groups only at the dark-light transition, while at the transition from light to dark, the individuals showed an increased response. In conclusion, the present study highlighted the impairment of spontaneous motor and sensorimotor behavior induced by MPA and APINAC administration in both species, thus confirming the usefulness of ZL as a model for a rapid behavioural-based drug screening.

Kisspeptin and RF9 prevent paroxetine-induced changes in some parameters of seminal vesicle fluid in the male rats.

The aim of the study was to examine possible impacts of paroxetine and agomelatine on the levels of some components that constitute the seminal vesicle fluid. As a second purpose, it was also aimed to examine how possible negative effects induced by paroxetine on seminal vesicle fluid components were affected by kisspeptin and RF9 (an RFamide-related peptide antagonist, RFRP). Forty-two male rats, aged 21 days, divided into six groups; control, sham, paroxetine, agomelatine, paroxetine + kisspeptin and paroxetine + RF9. Paroxetine (3.6 mg/kg) and agomelatine (10 mg/kg) were administrated by oral gavage. Kisspeptin (1 nmol) and RF9 (20 nmol) were administered intracerebroventricular (i.c.v). The experiments were ended on post-natal 120 days; fructose, vitamin E, sodium, potassium and magnesium levels were measured in seminal vesicle fluid. Fructose, vitamin E, magnesium and potassium levels were significantly decreased in seminal vesicle fluid from the rats treated with paroxetine but did not show significant differences following agomelatine administration. The co-administration of kisspeptin or RF9 with paroxetine prevented the paroxetine-induced negative effects on seminal vesicle fluid components. These results suggest that reduction in sperm fertilising ability caused by changes in seminal vesicle fluid can be seen in long-term antidepressant use. RF-9 and kisspeptin might have positive effects on long-term antidepressant use-induced infertility.

Impact of baseline characteristics on glycemic effects of add-on saxagliptin or acarbose to metformin therapy: Subgroup analysis of the SMART study in Chinese patients with type 2 diabetes mellitus.

AIMS/INTRODUCTION: This secondary analysis of the 24-week SMART study examined the efficacy of add-on saxagliptin or acarbose to metformin across different patient subgroups with type 2 diabetes mellitus, based on baseline characteristics. MATERIALS AND METHODS: Randomized patients (n = 481) were classified into subgroups based on their baseline age (<65, ≥65 years), body mass index (BMI; <24, 24-<28, ≥28 kg/m2 ), glycated hemoglobin (HbA1c; <8%, 8-<9%, 9-<10%, ≥10%) and renal function (creatinine clearance 50-<80, ≥80 mL/min). Treatment effects on primary outcome (HbA1c) and key secondary outcomes of fasting plasma glucose (FPG), 2-h postprandial glucose and homeostatic model assessment of ß-cell function were assessed across patient subgroups. RESULTS: For saxagliptin, reductions in HbA1c from baseline to week 24 were consistent across different subgroups regardless of baseline age, body mass index, HbA1c and renal function (range -0.66 to -1.16%). Saxagliptin was associated with consistent reductions in FPG (-0.60 to -1.33 mmol/L) and 2-h postprandial glucose (-0.48 to -1.95 mmol/L) across the majority of subgroups studied. The efficacy of acarbose on FPG attenuated progressively with increasing baseline HbA1c (+0.86 to -1.43 mmol/L); an increase from baseline FPG was observed in patients with HbA1c >9%. The effect of acarbose on postprandial glucose was also variable (+0.23 to -3.38 mmol/L). CONCLUSIONS: As add-on to metformin, both saxagliptin and acarbose reduced HbA1c regardless of baseline HbA1c, age, body mass index and renal function; however, only saxagliptin was effective at a stable glycemic control (FPG and PPG). The efficacy of acarbose on FPG and PPG was significantly attenuated in patients with higher baseline HbA1c (≥8%).

RF9: May it be a new therapeutic option for hypogonadotropic hypogonadism?

Hypogonadotropic hypogonadism (secondary hypogonadism), congenital or acquired, is a form of hypogonadism that is due to problems with either the hypothalamus or pituitary gland affecting gonadotropin levels. Pulsatile secretion of gonadotropin-releasing hormone (GnRH) by hypothalamus is a primer step to initiate the release of pituitary gonadotropins. Kisspeptin and gonadotropin-inhibitory hormone (GnIH) are accepted as two major players in the activation and inhibition of GnRH regarding the neuroendocrine functioning of the hypothalamic pituitary gonadal axis. Kisspeptin is known as the most potent activator of GnRH. Regarding the inhibition of GnRH, RF-amide-related peptide-3 (RFRP-3) is accepted as the mammalian orthologue of GnIH in avian species. RF9 (1-adamantane carbonyl-Arg-Phe-NH2) is an antagonist of RFRP-3/GnIH receptor (neuropeptide FF receptor 1 (NPFFR1; also termed as GPR147). In recent years, several studies have indicated that RF9 activates GnRH neurons and gonadotropins in a kisspeptin receptor (Kiss1r, formerly known as GPR54) dependent manner. These results suggest that RF9 may have a bimodal function as both an RFRP-3 antagonist and a kisspeptin agonist or it may be a kiss1r agonist rather than an RFRP-3/GnIH receptor antagonist. These interactions are possible because Kisspeptin and GnIH are members of the RF-amide family, and both possibilities are not far from explaining the potent gonadotropin stimulating effects of RF9. Therefore, we hypothesize that RF9 may be a new therapeutic option for the hypogonadotropic hypogonadism due to its potent GnRH stimulating effects. A constant or repeated administration of RF9 provides a sustained increase in plasma gonadotrophin levels. However, applications in the same way with GnRH analogues and kisspeptin may result in desensitization of the gonadotropic axis. The reasons reported above contribute to our hypothesis that RF9 may be a good option in the GnRH stimulating as a kisspeptin agonist. We suggest that further studies are needed to elucidate the potential effects of RF9 in the treatment of the hypogonadotropic hypogonadism.

Opioid receptors inhibit the spinal AMPA receptor Ca2+ permeability that mediates latent pain sensitization.

Acute inflammation induces sensitization of nociceptive neurons and triggers the accumulation of calcium permeable (CP) α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) in the dorsal horn of the spinal cord. This coincides with behavioral signs of acute inflammatory pain, but whether CP-AMPARs contribute to chronic pain remains unclear. To evaluate this question, we first constructed current-voltage (IV) curves of C-fiber stimulus-evoked, AMPAR-mediated EPSCs in lamina II to test for inward rectification, a key characteristic of CP-AMPARs. We found that the intraplantar injection of complete Freund's adjuvant (CFA) induced an inward rectification at 3 d that persisted to 21 d after injury. Furthermore, the CP- AMPAR antagonist IEM-1460 (50 µM) inhibited AMPAR-evoked Ca2+ transients 21d after injury but had no effect in uninflamed mice. We then used a model of long-lasting vulnerability for chronic pain that is determined by the balance between latent central sensitization (LCS) and mu opioid receptor constitutive activity (MORCA). When administered 21 d after the intraplantar injection of CFA, intrathecal administration of the MORCA inverse agonist naltrexone (NTX, 1 µg, i.t.) reinstated mechanical hypersensitivity, and superfusion of spinal cord slices with NTX (10 µM) increased the peak amplitude of AMPAR-evoked Ca2+ transients in lamina II neurons. The CP-AMPAR antagonist naspm (0-10 nmol, i.t.) inhibited these NTX-induced increases in mechanical hypersensitivity. NTX had no effect in uninflamed mice. Subsequent western blot analysis of the postsynaptic density membrane fraction from lumbar dorsal horn revealed that CFA increased GluA1 expression at 2 d and GluA4 expression at both 2 and 21 d post-injury, indicating that not just the GluA1 subunit, but also the GluA4 subunit, contributes to the expression of CP-AMPARs and synaptic strength during hyperalgesia. GluA2 expression increased at 21 d, an unexpected result that requires further study. We conclude that after tissue injury, dorsal horn AMPARs retain a Ca2+ permeability that underlies LCS. Because of their effectiveness in reducing naltrexone-induced reinstatement of hyperalgesia and potentiation of AMPAR-evoked Ca2+ signals, CP-AMPAR inhibitors are a promising class of agents for the treatment of chronic inflammatory pain.

Thermodynamic stability and structure in aqueous solution of the [Cu(PTA)4]+ complex (PTA = aminophosphine­1,3,5­triaza­7­phosphaadamantane).

The chemistry of copper(I) with water-soluble phosphines is an emergent area of study which has the objective of finding ligands that stabilize copper in its lower oxidation state. Cu(I) has been found relevant in the mechanism of copper transports into cells, and the accessibility of this oxidation state has implications in oxidative stress processes. For these reasons the possibility to deal with stable, water soluble copper(I) is an attractive approach for devising new biologically relevant metal-based drugs and chelating agents. Here we present the X-ray absorption spectroscopy (XAS) and UV-visible spectrophotometric study of the [Cu(PTA)4]BF4 complex (PTA = aminophosphine­1,3,5­triaza­7­phosphaadamantane). In particular, we have studied the stability of the [Cu(PTA)n]+ species (n = 2-4) in aqueous medium, and their speciation as a function of the total [Cu(PTA)4]BF4 concentration by means of competitive UV-visible spectrophotometric titrations using metallochromic indicators. Also, the structure in solution of the Cu(I)/PTA species and the nature of the first coordination sphere of the metal were studied by transformed XAS. Both techniques allowed to study samples with total [Cu(PTA)4]BF4 concentration down to 68-74 µM, possibly relevant for biological applications. Overall, our data suggest that the [Cu(PTA)n]+ species are stable in solution, among which [Cu(PTA)2]+ has a remarkable thermodynamic stability. The tendency of this last complex to form adducts with N-donor ligands is demonstrated by the spectrophotometric data. The biological relevance of PTA towards Cu(I), especially in terms of chemotreatments and chelation therapy, is discussed on the basis of the speciation model the Cu(I)/PTA system.

Citrus bioflavonoids dipeptidyl peptidase-4 inhibition compared with gliptin antidiabetic medications.

This study compared dipeptidyl peptidase-4 (DPP-4) inhibitory activity of citrus bioflavonoid nutraceuticals compared with three gliptins. Citrus bioflavonoid standards and three commercially available citrus bioflavonoid supplements (Thompson's Super Bioflavonoid Complex®(SB), Ethical Nutrients Bioflavonoids Plus Vitamin C®(EN), and Country Life Citrus Bioflavonoids and Rutin®(CB)) were considered in this study. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis was undertaken to identify and quantitate the citrus bioflavonoids present in each supplement. The DPP-4 inhibitory activity was determined by fluorometric assay. All of the tested individual citrus flavonoids demonstrated DPP-4 inhibitory activity, with IC50 values ranging from 485 µM (rutin) to 5700 µM (hesperitin and eriodictyol). Similarly, the flavonoid supplements had IC50 values of 16.9 mg/mL (EN), 3.44 mg/mL (SB) and 2.72 mg/mL (CB). These values compare with gliptin IC50 values of 0.684 µM (sitagliptin), 0.707 µM (saxagliptin) and 2.286 µM (vildagliptin). The supplement flavonoid content varied from 11.98% (CB) to 5.26% (EN) and 14.51% (SB) of tablet mass, corresponding to daily flavonoid doses of around 300, 150 and 400 mg, respectively, with CB and SB containing rutin at levels of 7.0% and 7.5% of tablet mass, respectively. While our data demonstrated that citrus bioflavonoid based supplements do possess DPP-4 inhibitory activity, they are several orders of magnitude less potent than gliptins. Further studies using higher concentrations of citrus bioflavonoids, as well as investigations into antioxidant properties which may add additional benefit are warranted.

Calcium-sensing receptor (CASR) is involved in porcine in vitro fertilisation and early embryo development.

It has been demonstrated that extracellular calcium is necessary in fertilisation and embryo development but the mechanism is still not well understood. The present study mainly focussed on the extracellular calcium effector called the calcium-sensing receptor (CASR) and examined its expression in porcine gametes and embryos and its function during fertilisation and early embryo development. By using reverse transcription polymerase chain reaction, CASR was found to be expressed in porcine oocytes, spermatozoa and embryos at different developmental stages. Functionally, medium supplementation with a CASR agonist or an antagonist during in vitro fertilisation (IVF) and in vitro culture (IVC) was tested. During fertilisation, the presence of a CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilisation rate. During embryo development, for the IVF embryos, agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group. Furthermore, parthenogenetically activated embryos showed similar results with lower cleavage and blastocyst formation rates in the antagonist group than in the other groups. It was concluded that CASR, as the effector of extracellular calcium, modulates porcine fertilisation and early embryo development.

Nonclinical and clinical pharmacology evidence for cardiovascular safety of saxagliptin.

BACKGROUND: In the Saxagliptin Assessment of Vascular Outcomes Recorded in Patients with Diabetes Mellitus (SAVOR) trial in patients with type 2 diabetes mellitus (T2D) at high risk of cardiovascular (CV) disease, saxagliptin did not increase the risk for major CV adverse events. However, there was an unexpected imbalance in events of hospitalization for heart failure (hHF), one of six components of the secondary CV composite endpoint, with a greater number of events observed with saxagliptin. Here, we examined findings from nonclinical safety and clinical pharmacology studies of saxagliptin with the aim of identifying any potential signals of myocardial injury. METHODS: In vitro and in vivo (rat, dog, monkey) safety pharmacology and toxicology studies evaluating the potential effects of saxagliptin and its major active metabolite, 5-hydroxy saxagliptin, on the CV system are reviewed. In addition, results from saxagliptin clinical studies are discussed: one randomized, 2-period, double-blind, placebo-controlled single-ascending-dose study (up to 100 mg); one randomized, double-blind, placebo-controlled, sequential, multiple-ascending-high-dose study (up to 400 mg/day for 14 days); and one randomized, double-blind, 4-period, 4-treatment, cross-over thorough QTc study (up to 40 mg/day for 4 days) in healthy volunteers; as well as one randomized, placebo-controlled, sequential multiple-ascending-dose study in patients with T2D (up to 50 mg/day for 14 days). RESULTS: Neither saxagliptin nor 5-hydroxy saxagliptin affected ligand binding to receptors and ion channels (e.g. potassium channels) or action potential duration in in vitro studies. In animal toxicology studies, no changes in the cardiac conduction system, blood pressure, heart rate, contractility, heart weight, or heart histopathology were observed. In healthy participants and patients with T2D, there were no findings suggestive of myocyte injury or fluid overload. Serum chemistry abnormalities indicative of cardiac injury, nonspecific muscle damage, or fluid homeostasis changes were infrequent and balanced across treatment groups. There were no QTc changes associated with saxagliptin. No treatment-emergent adverse events suggestive of heart failure or myocardial damage were reported. CONCLUSIONS: The saxagliptin nonclinical and clinical pharmacology programs did not identify evidence of myocardial injury and/or CV harm that may have predicted or may explain the unexpected imbalance in the rate of hHF observed in SAVOR.

Screening of "spice" herbal mixtures: From high-field to low-field proton NMR.

Forty one samples of herbal spices intended to be introduced into the European market and seized by the French customs were analysed with high-field 1H NMR. Nine synthetic cannabinoids (MAM-2201, JWH-073, JWH-210, JWH-122, JWH-081, JWH-250, UR-144, XLR-11 and AKB-48-5F) were detected and quantified. The ability of a compact benchtop low-field NMR spectrometer for a rapid screening of the content of herbal blends was then successfully explored for the first time. Even if low-field 1H NMR spectra are much less resolved than high-field spectra, we demonstrate that they provide valuable clues on the chemical structures of synthetic cannabinoids with the detection of some typical signals.

Pharmacological Evaluation of Novel Bioisosteres of an Adamantanyl Benzamide P2X7 Receptor Antagonist.

Adamantanyl benzamide 1 was identified as a potent P2X7R antagonist but failed to progress further due to poor metabolic stability. We describe the synthesis and SAR of a series of bioisosteres of benzamide 1 to explore improvements in the pharmacological properties of this lead. Initial efforts investigated a series of heteroaromatic bioisosteres, which demonstrated improved physicochemical properties but reduced P2X7R antagonism. Installation of bioisosteric fluorine on the adamantane bridgeheads was well tolerated and led to a series of bioisosteres with improved physicochemical properties and metabolic stability. Trifluorinated benzamide 34 demonstrated optimal physicochemical parameters, superior metabolic stability (ten times longer than lead benzamide 1), and an improved physicokinetic profile and proved effective in the presence of several known P2X7R polymorphisms.

Identification of Eight Synthetic Cannabinoids, Including 5F-AKB48 in Seized Herbal Products Using DART-TOF-MS and LC-QTOF-MS as Nontargeted Screening Methods.

Synthetic cannabinoids are sprayed onto plant material and smoked for their marijuana-like effects. Clandestine manufacturers modify synthetic cannabinoid structures by creating closely related analogs. Forensic laboratories are tasked with detection of these analog compounds, but targeted analytical methods are often thwarted by the structural modifications. Here, direct analysis in real time coupled to accurate mass time-of-flight mass spectrometry (DART-TOF-MS) in combination with liquid chromatography quadruple time-of-flight mass spectrometry (LC-QTOF-MS) are presented as a screening and nontargeted confirmation method, respectively. Methanol extracts of herbal material were run using both methods. Spectral data from four different herbal products were evaluated by comparing fragmentation pattern, accurate mass and retention time to available reference standards. JWH-018, JWH-019, AM2201, JWH-122, 5F-AKB48, AKB48-N-(4-pentenyl) analog, UR144, and XLR11 were identified in the products. Results demonstrate that DART-TOF-MS affords a useful approach for rapid screening of herbal products for the presence and identification of synthetic cannabinoids.