Significant Dose-dependent Decreases Research Articles

Zodasiran, an RNAi Therapeutic Targeting ANGPTL3, for Mixed Hyperlipidemia

BackgroundAngiopoietin-like 3 (ANGPTL3) inhibits lipoprotein and endothelial lipases and hepatic uptake of triglyceride-rich lipoprotein remnants. ANGPTL3 loss-of-function carriers have lower levels of triglycerides, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and non-HDL cholesterol and a lower risk of atherosclerotic cardiovascular disease than noncarriers. Zodasiran is an RNA interference (RNAi) therapy targeting expression of ANGPTL3 in the liver.MethodsWe conducted a double-blind, placebo-controlled, dose-ranging phase 2b trial to evaluate the safety and efficacy of zodasiran in adults with mixed hyperlipidemia (fasting triglyceride level of 150 to 499 mg per deciliter and either an LDL cholesterol level of ≥70 mg per deciliter or a non-HDL cholesterol level of ≥100 mg per deciliter). Eligible patients were randomly assigned in a 3:1 ratio to receive subcutaneous injections of zodasiran (50, 100, or 200 mg) or placebo on day 1 and week 12 and were followed through week 36. The primary end point was the percent change in the triglyceride level from baseline to week 24.ResultsA total of 204 patients underwent randomization. At week 24, substantial mean dose-dependent decreases from baseline in ANGPTL3 levels were observed with zodasiran (difference in change vs. placebo, −54 percentage points with 50 mg, −70 percentage points with 100 mg, and −74 percentage points with 200 mg), and significant dose-dependent decreases in triglyceride levels were observed (difference in change vs. placebo, −51 percentage points, −57 percentage points, and −63 percentage points, respectively) (P<0.001 for all comparisons). Other differences in change from baseline as compared with placebo included the following: for non-HDL cholesterol level, −29 percentage points with 50 mg, −29 percentage points with 100 mg, and −36 percentage points with 200 mg; for apolipoprotein B level, −19 percentage points, −15 percentage points, and −22 percentage points, respectively; and for LDL cholesterol level, −16 percentage points, −14 percentage points, and −20 percentage points, respectively. We observed a transient elevation in glycated hemoglobin levels in patients with preexisting diabetes who received the highest dose of zodasiran.ConclusionsIn patients with mixed hyperlipidemia, zodasiran was associated with significant decreases in triglyceride levels at 24 weeks. (Funded by Arrowhead Pharmaceuticals; ARCHES-2 ClinicalTrials.gov number, NCT04832971.)

Developmental toxicity of short-chain chlorinated paraffins on early-stage chicken embryos in a shell-less (ex-ovo) incubation system

Short-chain chlorinated paraffins (SCCPs) are listed as a category of globally controlled persistent organic pollutants (POPs) by the Stockholm Convention in 2017. However, SCCP toxicity, particularly their developmental toxicity in avian embryos, has not been well studied. In this study, we observed the early development of chicken embryos (Gallus gallus domesticus) by applying a shell-less (ex-ovo) incubation system developed in our previous studies. After exposing embryos at Hamburger Hamilton stage (HHS) 1 to SCCPs (control, 0.1% DMSO; SCCPs-L, 200 ng/g; SCCPs-M, 2000 ng/g; SCCPs-H, 20,000 ng/g), we observed the development of embryos from the 3rd to 9th incubation day. Exposure to SCCPs-M and -H induced a significant reduction in survival, with an LD50 of 3100 ng/g on the 9th incubation day. Significant dose-dependent decreases in body length were observed from days 4–9. We also found that SCCPs-H decreased the blood vessel length and branch number on the 4th incubation day. Additionally, SCCPs-H significantly reduced the heart rate on the 4th and 5th incubation days. These findings suggest that SCCPs may have potential of developmental and cardiovascular toxicity during the early stages of chicken embryos. Quantitative PCR of the mRNA of genes related to embryonic development showed that SLC16A10 (a triiodothyronine transporter) level decreased in the SCCPs-H group, showing a significant positive correlation with the body length of embryos. THRA level, a thyroid hormone receptor, was significantly decreased in the SCCPs-H group, whereas that of DIO3 level, a deiodinase was significantly increased. These results suggest that SCCPs exposure induces developmental delays via the thyroxine signaling pathway. Analysis of thyroid hormones (THs) in blood plasma also indicated a significant reduction in thyroxine (T4) levels in the SCCPs-H group on the 9th incubation day of embryos. In conclusion, SCCPs induce developmental toxicity by disrupting thyroid functions at the early-life stage of chicken embryos.

Insulin Inhibits Autophagy by Inhibiting the Binding of FoXO1 to the Promoter Region of GABARAPL1.

Hyperinsulinemia and insulin resistance in T2D have a potent suppressive effect on hepatic autophagy, however, the underlying mechanisms remain unclear. To explore the effect of insulin on hepatic autophagy and its possible signaling pathways, HL-7702 cells were treated with insulin with or without insulin signaling inhibitors. The interaction between insulin and the promoter region of GABARAPL1 was assessed through luciferase assay and EMSA. There were significant dose-dependent decreases in the number of intracellular autophagosomes and the protein levels of GABARAPL1 and beclin1 in insulin-treated HL-7702 cells. Insulin signaling inhibitors reversed the inhibitory effect of insulin on rapamycin-induced autophagy and autophagy-related gene upregulation. Insulin blocks the binding of FoxO1 to putative insulin response elements in GABARAPL1 gene promoter, leading to the repressed transcription of GABARAPL1 gene and the suppression of hepatic autophagy. Our study identified GABARAPL1 as a novel target of insulin in suppressing hepatic autophagy.

Dose-dependent changes in real-life affective well-being in healthy community-based individuals with mild to moderate childhood trauma exposure

BackgroundChildhood trauma exposures (CTEs) are frequent, well-established risk factor for the development of psychopathology. However, knowledge of the effects of CTEs in healthy individuals in a real life context, which is crucial for early detection and prevention of mental disorders, is incomplete. Here, we use ecological momentary assessment (EMA) to investigate CTE load-dependent changes in daily-life affective well-being and psychosocial risk profile in n = 351 healthy, clinically asymptomatic, adults from the community with mild to moderate CTE.FindingsEMA revealed significant CTE dose-dependent decreases in real-life affective valence (p = 0.007), energetic arousal (p = 0.032) and calmness (p = 0.044). Psychosocial questionnaires revealed a broad CTE-related psychosocial risk profile with dose-dependent increases in mental health risk-associated features (e.g., trait anxiety, maladaptive coping, loneliness, daily hassles; p values < 0.003) and a corresponding decrease in factors protective for mental health (e.g., life satisfaction, adaptive coping, optimism, social support; p values < 0.021). These results were not influenced by age, sex, socioeconomic status or education.ConclusionsHealthy community-based adults with mild to moderate CTE exhibit dose-dependent changes in well-being manifesting in decreases in affective valence, calmness and energy in real life settings, as well as a range of established psychosocial risk features associated with mental health risk. This indicates an approach to early detection, early intervention, and prevention of CTE-associated psychiatric disorders in this at-risk population, using ecological momentary interventions (EMI) in real life, which enhance established protective factors for mental health, such as green space exposure, or social support.

Abstract 4981: The potent quadruplex-binding compound QN-302 down-regulates the S100P gene in in vitro and in vivo models of pancreatic cancer: a potential therapeutic target and biomarker for PDAC

Abstract The compound QN-302, a tetra-substituted naphthalene diimide (ND) derivative has been designed to have high affinity for G-quadruplex (G4) nucleic acids. It has single-digit nM anti-proliferative activity in a panel of human pancreatic cancer cell lines (Ahmed et al., ACS Med Chem Lett, 2020, 11, 1634-1644) and anti-tumor activity in xenograft, orthotopic and genetic (KPC) models for pancreatic cancer (PDAC). Its mode of action involves the targeting of G4-forming sites in promoter regions of cancer-associated genes, where their prevalence is over-represented compared to other genes. Transcriptome analyses of QN-302 in MIA-PACA2 cells have confirmed genes involved in cancer-associated pathways and carry G4 motifs in their promoters as targets. We have found that expression of the S100P gene, a well-authenticated indicator of PDAC disease and progression, is highly down-regulated in QN-302 treated cells. The S100P gene contains at least five putative G4 motifs, including a classic G4 promoter site just upstream of the transcription start site. We have observed that transcriptome analyses on tumor material from human PDAC patients, and those from poorly differentiated as well as more highly differentiated tumors, have shown that the S100P gene is highly over-expressed (especially in poorly differentiated tumors). We now report on gene expression analysis of S100P using control and treated tumor tissues from a therapeutic study in PDAC MIA-PACA2 xenografts in mice, by means of RT-qPCR and Western blot analyses. The data shows statistically significant dose-dependent decreases in expression of S100P at both the mRNA (to ca 25% of control values) and protein levels (to ca 22% of control values), giving added confidence to the concept that the S100P gene is a therapeutic target in PDAC and may also be useful as a marker of QN-302 response in future clinical use. QN-302 is bio-available at therapeutic doses and is well tolerated at these levels in the animal models. It is being developed for clinical evaluation by Qualigen Therapeutics Inc and is currently at the pre-IND stage. Citation Format: Nicole Williams, Jenny Worthington, Ahmed Ahmed, Tariq Arshad, Stephen Neidle. The potent quadruplex-binding compound QN-302 down-regulates the S100P gene in in vitro and in vivo models of pancreatic cancer: a potential therapeutic target and biomarker for PDAC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4981.

Toxicity of true indigo (Indigofera tinctoria) extract on haematology and oxidative stress enzymes of Nile tilapia (Oreochromis niloticus) juveniles

This study was conducted to ascertain the toxicity effect of Indigofera tinctoria on haematology and oxidative stress biomarker on the liver, kidney, gills, and tissue of Nile tilapia (Oreochromis niloticus) juveniles. Matured leaves of Indigofera tinctoria were collected, dried, and pulverized. Graded levels of the Indigofera tinctoria leaf powder were weighed and dissolved in 250 ml of water. Fish specimens were exposed to sub-acute concentrations of Indigofera tinctoria for 21 days. Significant changes and dose-dependent decreases in red blood cells, packed cell volume, hemoglobin values, and progressive increase in white blood cells were observed in Indigofera tinctoria exposed fish. There was an elevation in the activities of oxidative, in the liver, kidney, gill and tissue of the fish. The present findings show that disposal of leaves, effluent from Indigofera tinctoria should be done with extreme caution as it could threaten the life and existence of the aquatic organism.

Nicotinic Acetylcholine Receptor Subunit Alpha-7 Mediates PD-L1 and CTLA-4 Expression in HepG2 Cells

Background: The liver is the largest solid organ in the body and has several unique immunological roles. The alpha7 subtype of nicotinic acetylcholine receptor (α7nAChR) is expressed in the liver and has different immunoregulatory effects. Programmed death ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are two major checkpoints that act as a negative regulator of the immune system. In this study, we examined the effects of activation (with nicotine) and inhibition (with specific siRNA) of α7nAChR on the PD-L1 and CTLA-4 genes expression. Methods: Human hepatocellular carcinoma (HepG2) cell line was used to investigate the effects of treatment with low dose (1 μM) and high dose (10 μM) of nicotine on the PD-L1 and CTLA-4 expression. In addition to this, the effects of treatment with 100 nM concentration of specific siRNA targeting α7nAChR (α7-siRNA) were examined using quantitative real-time PCR. Results: Compared to untreated cells, the HepG2 cells treated with nicotine exhibited significant dose-dependent decreases in PD-L1 expression. Furthermore, nicotine has a biphasic effect on the CTLA-4 expression and decreased its expression in 10 μM concentration. The qRT-PCR revealed that α7-siRNA significantly reduced the mRNA levels of α7nAChR (P &lt; 0.01). Also, compared to control cells, α7-siRNA treated cells exhibited significant increases in both PD-L1 and CTLA-4 expression. Conclusion: These experiments determined that nicotine treatment leads to decreases in PD-L1 and CTLA-4 expression. These effects are reversed by treatment with α7-siRNA, which indicates the involvement of α7nAChR related mechanisms in these processes.

Assessment of anxiolytic potential and acute toxicity study of Combretum micranthum G. Don. leaves (Combretaceae)

Background: Combretum micranthum G. Don. (Combretaceae) is an ethnomedicinally valuable, undomesticated and indigenous shrub of West Africa. However, its anxiolytic potential have not been reported despite its ethanolic extract being used ethnomedicinally in the management of anxiety disorders.Aim: To determine the acute toxicity effect and assess the behavioural effects and anxiolytic potential of C. micranthum G. Don. leaves in mice.Settings: This study is an experimental design to evaluate the ethnomedicinal claim of Combretum micranthum G. Don using animal models of anxiety.Methods: Fifty-six male and female mice, ranging in weight between 20 g and 30 g were randomly distributed into three main groups. The first group of mice (n = 6) was assigned for toxicity assessment (LD50) study using the guideline of Organization for Economic Cooperation and Development (OECD). The second group of mice for behavioural study (n = 25) was further divided into five sub-groups. Sub-groups I, II and III were orally administered 500 mg/kg, 1000 mg/kg, 2000 mg/kg of ethanolic extract of C. micranthum (CmEE), respectively, whilst IV and V were intraperitoneally administered 1 mg/kg diazepam and normal saline 0.5 mL, respectively. They were thereafter evaluated for novelty-induced behaviours: locomotion, rearing and grooming using Open Field Test (OFT). The third group of mice (n = 25) was treated similar to the pattern used in behavioural study and evaluated for anxiolytic activity of CmEE using elevated plus maze (EPM) model. Data were expressed as mean ± standard error of mean (S.E.M) and analysed using Student’s-t test, and one-way analysis of variance (ANOVA) followed by Student–Newman– Keuls (SNK) test with values of p 0.05 considered significant.Results: The percentage yield of ethanolic leaf extract of C. micranthum was 14.28% weight/weight (w/w). Combretum micranthum showed no toxicity when administered orally to mice (LD50 ≥ 2000 mg/kg). Groups administered 500, 1000 and 2000 mg/kg of CmEE exhibited decreased locomotion (p 0.05) when compared with saline group. There was significant decrease in rearing at 2000 mg/kg but increase in grooming in mice administered 2000 mg/kg of CmEE was recorded. The groups administered 500, 1000 and 2000 mg/kg of CmEE showed increased percentage time spent in the open arm in a dose-dependent pattern (33.3%, 41.6% and 55.4%, respectively) when compared with the saline group. There were significant dose-dependent decreases in the indices of open arm avoidance at 1000 (48.9) and 2000 mg/kg (41.4) of CmEE.Conclusion: Combretum micranthum is non-toxic and preliminary data indicated that it possesses anxiolytic potential. However, it is recommended that further assays using other specific models of anxiety to determine its probable mechanism(s) of action should be explored.

Effects of 2.1 GHz Electromagnetic Radiation on Locomotor Activity, Recognition Memory, and Anxiety-Related Behavior in Rats

The effects of short-term (2 h per day for 7 days) exposures to different doses of 2.1 GHz radiofrequency electromagnetic radiation (RF-EMR) on cognition and behavior of rats were examined. The animals were divided into three equal groups, sham-exposed group 1 and two RF groups irradiated with different doses, groups 2 and 3, respectively. To assess locomotor activity, recognition memory, and anxiety-related behavior, the open field (OF), novel object recognition (NOR), and marble burying (MB) tests were used, respectively. Overall results showed that both 45 and 65 V/m doses of 2.1 GHz RF-EMR did not lead to significant modifications of locomotor activity in the OF test. At the same time, higher doses (65 V/m) significantly (about two times) decreased the exploration time and number of touches for a novel object in the NOR test. In the MB test, both RF-EMR doses led to significant dose-dependent decreases in the number of buried marbles, which was indicative of lower levels of anxiety. Further studies are needed, regarding this particular type and level of radiation, for the development of methods to provide protection of the brain from harmful effects of RF-EMR exposure.

Effects of Topical Tebufenozide Application to Choristoneura fumiferana Pupae (Lepidoptera: Tortricidae).

Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae) is a defoliating pest in Canada and the northeastern United States. Given its important ecological and economic effects in affected regions, several direct management techniques have been developed, including the application of the insect growth regulator tebufenozide (Mimic™, RH-5992) to feeding larval stages. While the effectiveness of tebufenozide, in this capacity, is understood, management programs of other lepidopteran pests have demonstrated the effectiveness of tebufenozide application when utilized against other life stages. Here, we investigated the toxicity of topically-applied tebufenozide to C. fumiferana pupae to determine if such a strategy could be feasible. We observed significant dose-dependent decreases in the likelihood of adult emergence, increases in the likelihood of pupal death or adult deformity at eclosion, and significant decreases in mean adult longevity. Estimated LD 50 (lethal dose) values for adult male and female C. fumiferana treated as pupae ≤ 4 days after pupation were approximately 1–3 and 2–3.5% ACI (active commercial ingredient) respectively. Estimated L-SD (lethal-sublethal) 50 doses for adult male and female C. fumiferana treated as pupae ≤4 days after pupation were <1, and <2% ACI, respectively. Mating success was also significantly lower in mating pairs containing adults treated as pupae. Although, the amounts required to cause appreciable pupal mortality were much higher than those currently applied operationally in the C. fumiferana system, our study illustrates the potential of tebufenozide to utilized against additional developmental stages in other lepidopteran pests.

Alpha7 Nicotinic Acetylcholine Receptor Mediates Nicotine-induced Apoptosis and Cell Cycle Arrest of Hepatocellular Carcinoma HepG2 Cells.

Purpose: The cytotoxic properties upon treatment with nicotine have been reported in several studies, but the underlying mechanisms remain not fully defined. The alpha7 nicotinic acetylcholine receptor (α7nAChR) is one of the important nicotinic receptors, which nicotine partly by binding to this receptor exerts its effects. The current study aimed to investigates the influences of nicotine on cellular proliferative and apoptotic activities and tried to determine the involvement of α7nAChR in these functions. Methods: Human hepatocellular carcinoma (HepG2) cell line was used to determine the individual or combined effects of treatments with nicotine (10 μM) and specific siRNA (100 nM) targeting α7nAChR expression. The MTT assay, DAPI staining assay, and flow cytometry assay were applied to measure the cell viability, apoptosis and cell cycle progression of the cells, respectively. In addition, the changes in the mRNA level of the genes were assessed by qRT-PCR. Results: Compared to control groups, the cells treated with nicotine exhibited significant dosedependent decreases in cell viability (log IC50 = -5.12±0.15). Furthermore, nicotine induced apoptosis and cell cycle arrest especially at G2/M Phase. The qRT-PCR revealed that nicotine increased the mRNA levels of α7nAChR as well as caspase-3 and suppressed the expression of cyclin B1. Treatment with α7-siRNA abolished these effects of nicotine. Conclusion: These experiments determined that upregulation of α7nAChR by nicotine inhibits HepG2 cells proliferation and induces their apoptosis. These effects blocked by treatment with α7-siRNA, which indicates the involvement of α7nAChR pathways in these processes.

Calibrated Photo-Stimulated Luminescence and E-Sensing Analyses Discriminate Korean Citrus Fruits Treated with Electron Beam

Korean citrus fruits (Jinjihyang and Chunggyun) were treated with electron beam (e-beam) at phytosanitary irradiation doses of 0, 0.4, and 1 kGy, and their irradiation status was screened after storage for 0, 10, and 20 days. Because of the inherent limitations of photo-stimulated luminescence (PSL), the first PSL measurement (PSL1) was not sufficient to distinguish the irradiated samples from the non-irradiated controls. The calibrated PSL approach clearly distinguished the e-beam-irradiated samples from the control based on PSL ratio (PSL2/PSL1): > 10, non-irradiated; < 10, irradiated. In addition to PSL, electronic-sensing techniques (E-tongue and E-nose) clearly discriminated the irradiated Jinjihyang and Chunggyun fruits samples from their non-irradiated counterparts based on principal component analysis of taste attributes and volatile patterns. The quantitative analysis of volatile in irradiated citrus fruits showed that limonene was present at the highest concentration among all the volatiles from peels, and significant dose-dependent decreases were observed in both citrus varieties (p < 0.05). Limonene from the irradiated pulps showed a dose-dependent increase on day 0.

Effects of Varying Doses of Cocos Nucifera Water on Body Weight and Blood Biochemistry of Broiler Chicks

Effect of 2 varying doses of Cocos nucifera water (coconut water) was investigated on body weight and blood biochemistry of broiler chicks. The body weight, dressed weight and organ weights were determined by the use of standard weighing balance. The coconut water was aseptically collected from each coconut by piercing through the eye with sharp rod. The sterile coconut water was transferred into labeled sample bottle, corked and kept for the experiment. A total of 60 two weeks old anak 2000 broiler chicks were assigned into three treatments of 20 chicks each and replicated four times of 5 chicks per replicate in a completely randomized design. Treatment one (control) received 0.00mgcoconut water/kg body weight of birds. Treatment two and three received 200mg and 400mg of coconut water per kg bodyweight of birds respectively through oral intubation. The administration of experimental material was terminated after 28 days following an overnight fast. Blood was collected by venu–puncture via external jugular vein and discarded into two sample sets of bottles (one without anticoagulant and another with anticoagulant). The result obtained revealed a non-significant (P&gt;0.05) non-dose dependent changes in live weight, dressed weight of broiler chicks with a significant (P&lt;0.05) but non-dose dependent dressing percentage. The organ weight characteristics showed a non-significant (P&gt;0.05), non-dose dependent weight changes in all the organs weighed except the gizzard that produced a significant (P&lt;0.05), dose dependent reduction in weight as dosage of administration of coconut water increased. For haematological indicesthe result showed dose dependent significant (P&lt;0.05) decrease in mean corpuscular haemoglobin concentration and relative white blood cell count but a significant (P&lt;0.05) non-dose dependent decrease in haemoglobin concentration. Anon-significant, non-dose dependent changes were observed for red blood cells count and parked cell volume for doses of coconut water tested. For blood lipids, significant dose dependent decreases in total cholesterol and triacylglycerol were observed but a significant and non-dose dependent decrease in very low-density lipoprotein cholesterol. The serum low density lipoprotein (LDL) and high-density lipoprotein cholesterol revealed non-significant, non-dose dependent changes with the dose of coconut water administered. Alanine aminotransferase showed a significant increase but non-dose dependent activities for the two doses of administration under investigation whereas aspartate aminotransferase exhibited non-significant but dose dependent changes in activity. However, alkaline phosphatase revealed a dose dependent significant reduction in activity for the doses tested. Having no detrimental effects on body weight and organ weights with a significant (P&lt;0.05) reduction in blood lipids, one will commend the use of coconut water in broiler production not above 400mg/kg body weight of birds due to its negative response to white blood cell count, haemoglobin concentration (though not correlated at all to RBC) and alanine aminotransferase activity in this study.

Organic mononitrites of 1,2-propanediol act as an effective NO-releasing vasodilator in pulmonary hypertension and exhibit no cross-tolerance with nitroglycerin in anesthetized pigs.

PurposeClinically available intravenous (IV) nitric oxide (NO) donor drugs such as nitroglycerin (GTN) cause systemic hypotension and/or tolerance development. In a porcine model, novel NO donor compounds – the organic mononitrites of 1,2-propanediol (PDNO) – were compared to GTN with regard to pulmonary selectivity and tolerance development. The vasodilatory effects of inorganic nitrite were investigated.Materials and methodsIn anesthetized piglets, central hemodynamics were monitored. At normal pulmonary vascular resistance (PVR), IV infusions of PDNO (15–60 nmol kg−1 min−1), GTN (13–132 nmol kg−1 min−1), and inorganic nitrite (dosed as PDNO) were administered. At increased PVR (by U46619 IV), IV infusions of PDNO (60–240 nmol kg−1 min−1) and GTN (75–300 nmol kg−1 min−1) before and after a 5 h infusion of GTN (45 nmol kg−1 min−1) were given.ResultsAt normal PVR, PDNO (n=12) and GTN (n=7) caused significant dose-dependent decreases in mean systemic and pulmonary arterial pressures, whereas inorganic nitrite (n=13) had no significant effect. At increased PVR, PDNO (n=6) and GTN (n=6) significantly decreased mean systemic and pulmonary pressures and resistances, but only PDNO reduced the ratio between pulmonary and systemic vascular resistances significantly. After the 5 h GTN infusion, the hemodynamic response to GTN infusions (n=6) was significantly suppressed, whereas PDNO (n=6) produced similar hemodynamic effects to those observed before the GTN infusion.ConclusionPDNO is a vasodilator with selectivity for pulmonary circulation exhibiting no cross-tolerance to GTN, but GTN causes non selective vasodilatation with substantial tolerance development in the pulmonary and systemic circulations. Inorganic nitrite has no vasodilatory properties at relevant doses.

Safety and in vivo immune assessment of escalating doses of oral laquinimod in patients with RRMS

BackgroundLaquinimod is an oral immunomodulator in clinical development to treat relapsing-remitting multiple sclerosis (RRMS). Laquinimod is in clinical development for the treatment of multiple sclerosis and Huntington Disease (HD). The objective of this study is to assess the safety, tolerability, pharmacokinetics (PK) and cytoimmunologic effects following escalating doses of laquinimod in patients with RRMS.MethodsOne hundred twelve patients were randomly assigned to laquinimod/placebo in a series of separate dose-escalating cohorts starting from a daily oral dose of 0.9 mg/1.2 mg escalating to 2.7 mg, in 0.3 mg increments.ResultsTwenty-eight patients received placebo and 84 received laquinimod ranging from 0.9 to 2.7 mg. No deaths occurred. One serious adverse event (SAE) of perichondritis was reported, which was unrelated to laquinimod (0.9 mg). There was no increased incidence of adverse events (AEs) with escalating doses. Laquinimod-treated patients showed more abnormal laboratory levels in liver enzymes, P-amylase, C-reactive protein (CRP), and fibrinogen, but most shifts were clinically non-significant. The exposure of laquinimod was dose proportional and linear in the tested dose range. An immunological substudy showed significant dose-dependent decreases in 6-sulpho LacNAc + dendritic cell (slanDC) frequency following laquinimod compared to placebo.ConclusionLaquinimod doses up to 2.7 mg were safely administered to patients with RRMS. An in vivo effect of laquinimod on the innate immune system was demonstrated.Trial registrationEudraCT Number: 2009-011234-99. Registered 23 June 2009.

In vitro effects of dalteparin on thrombin generation in canine plasma.

The calibrated automated thrombogram (CAT) is a functional thrombin generation (TG) assay that may provide a new approach for monitoring anticoagulant therapy in dogs. The effects of dalteparin on TG variables in dogs are unknown. Objectives were to establish normal TG variable ranges in dogs and measure the in vitro TG variables in canine pooled platelet-poor plasma (PPP) spiked with different dalteparin concentrations. In the first experiment, plasma samples from 25 adult healthy Beagle dogs and 11 client-owned healthy dogs of multiple breeds was measured individually for obtaining normal TG values. In the second experiment, separate pools of the remaining PPP from 24 of the 25 previous adult Beagles and from 45 different client-owned dogs were spiked with dalteparin at 9 concentrations with increasing anti-factor Xa (anti-FXa) activity. Activated partial thromboplastin time, tissue factor-induced TG, and anti-FXa activity were measured for each concentration. Concentration-response relationships were determined with ADAPT v.5, using various nonlinear regression models for stimulatory or inhibitory effects. Thrombin generation ranges of client-owned dogs and Beagles were equivalent only for time-to-peak (P < .05). In vitro dalteparin resulted in a concentration-dependent decrease in endogenous thrombin potential (ETP) in pooled PPP. The estimated dalteparin concentration that produced half the maximal inhibition of baseline ETP (IC50 ) was 0.289 U/mL. Thrombin generation and anti-FXa activity were more sensitive than APTT to detect the effects of dalteparin. The CAT assay can measure the effects of dalteparin in canine plasma, resulting in significant dose-dependent decreases in ETP, prompting further in vivo investigation.

Controlled Release of Second Generation mTOR Inhibitors to Restrain Inflammation in Primary Immune Cells.

Autoimmune disease occurs when the immune system incorrectly targets the body's own tissue. Inflammatory CD4+ T cell phenotypes, such as TH1 and TH17, are key drivers of this attack. Recent studies demonstrate treatment with rapamycin-a key inhibitor of the mTOR pathway-can skew T cell development, moving T cell responses away from inflammatory phenotypes and toward regulatory T cells (TREGS). TREGS are important in inducing and maintaining tolerance to self-antigens, creating new potential to treat autoimmune diseases more effectively and specifically. Next generation analogs of rapamycin, such as everolimus and temsirolimus, confer increased potency with reduced toxicity, but are understudied in the context of autoimmunity. Further, these drugs are still broadly-acting and require frequent treatment due to short half-lives. Thus, there is strong interest in harnessing the unique properties of biomaterials-controlled drug release and targeting, for example, to improve autoimmune therapies. Using second generation mTOR inhibitors and rapamycin, we prepared sets of degradable polymer particles from poly(lactide-co-glycolide). We then used these materials to assess physicochemical properties and the ability to control autoimmune inflammation in a primary cell co-culture model. Treatment with particle formulations resulted in significant dose-dependent decreases in dendritic cell activation, T cell proliferation, inflammatory cytokines, and frequencies of inflammatory TH1 phenotypes. Considering the current limitations of rapamycin, and the potential of next-generation analogs, this work provides a screening platform for biomaterials and sets the stage for in vivo evaluation, where delivery kinetics, stability, and targeting could improve autoimmune therapies through biomaterial-enabled delivery.

Phase 2 Study of Relugolix vs Placebo in Heavy Menstrual Bleeding Associated With Uterine Fibroids [17H

INTRODUCTION: Relugolix is a potent nonpeptide gonadotropin-releasing hormone receptor antagonist. This dose-finding study explored the ability of relugolix to decrease the heavy menstrual bleeding (HMB) associated with uterine fibroids (UF). METHODS: This randomized, double-blind, placebo-controlled, phase 2 study was designed to evaluate relugolix 10 mg, 20 mg and 40 mg administered orally once daily for 12 weeks in Japanese women with UF and HMB defined as a Pictorial Blood Loss Assessment Chart (PBAC) score of at least 120. The primary endpoint was the proportion of women with a total PBAC score of less than 10 from Week 6 to 12. Secondary endpoints included the proportion of women with amenorrhea, and myoma and uterine volumes. RESULTS: Patients (N = 216) were randomized to placebo or one of three relugolix groups. The proportion of patients with PBAC score of less than 10 from Week 6 to 12 was 0% in placebo group, and 20.8%, 43.6% and 83.6% in relugolix 10 mg, 20 mg and 40 mg groups (P &lt; 0.001 vs placebo).74% of patients in relugolix 40 mg group achieved amenorrhea, and a dose-dependent decrease in myoma and uterine volumes were observed. The incidence of headache, metrorrhagia, menorrhagia, and hot flush were more than 10% higher in relugolix 20 mg and 40 mg groups than in placebo group; these AEs were mild or moderate in severity. CONCLUSION: Treatment with relugolix showed significant dose-dependent decreases in menstrual blood loss compared with placebo and was generally well-tolerated with AEs consistent with the drug’s mechanism of action.

Neuropathic pain-induced enhancement of spontaneous and pain-evoked neuronal activity in the periaqueductal gray that is attenuated by gabapentin.

Neuropathic pain is a debilitating pathological condition that is poorly understood. Recent evidence suggests that abnormal central processing occurs during the development of neuropathic pain induced by the cancer chemotherapeutic agent, paclitaxel. Yet, it is unclear what role neurons in supraspinal pain network sites, such as the periaqueductal gray, play in altered behavioral sensitivity seen during chronic pain conditions. To elucidate these mechanisms, we studied the spontaneous and thermally evoked firing patterns of ventrolateral periaqueductal gray (vlPAG) neurons in awake-behaving rats treated with paclitaxel to induce neuropathic pain. In the present study, vlPAG neurons in naive rats exhibited either excitatory, inhibitory, or neutral responses to noxious thermal stimuli, as previously observed. However, after development of behavioral hypersensitivity induced by the chemotherapeutic agent, paclitaxel, vlPAG neurons displayed increased neuronal activity and changes in thermal pain-evoked neuronal activity. This involved elevated levels of spontaneous firing and heightened responsiveness to nonnoxious stimuli (allodynia) as well as noxious thermal stimuli (hyperalgesia) as compared with controls. Furthermore, after paclitaxel treatment, only excitatory neuronal responses were observed for both nonnoxious and noxious thermal stimuli. Systemic administration of gabapentin, a nonopioid analgesic, induced significant dose-dependent decreases in the elevated spontaneous and thermally evoked vlPAG neuronal firing to both nonnoxious and noxious thermal stimuli in rats exhibiting neuropathic pain, but not in naive rats. Thus, these results show a strong correlation between behavioral hypersensitivity to thermal stimuli and increased firing of vlPAG neurons in allodynia and hyperalgesia that occur in this neuropathic pain model.

Evacetrapib alone or in combination with statins lowers lipoprotein(a) and total and small LDL particle concentrations in mildly hypercholesterolemic patients

Potent CETP inhibitors reduce plasma concentrations of atherogenic lipoprotein biomarkers of cardiovascular risk. To evaluate the effects of the cholesteryl ester transfer protein (CETP) inhibitor evacetrapib, as monotherapy or with statins, on atherogenic apolipoprotein B (apoB)-containing lipoproteins in mildly hypercholesterolemic patients. VLDL and LDL particle concentrations and sizes (using nuclear magnetic resonance spectroscopy) and lipoprotein(a) concentration (using nephelometry) were measured at baseline and week 12 in a placebo-controlled trial of 393 patients treated with evacetrapib as monotherapy (30mg/d, 100mg/d, or 500mg/d) or in combination with statins (100mg plus simvastatin 40mg/d, atorvastatin 20mg/d, or rosuvastatin 10mg/d; Clinicaltrials.gov Identifier: NCT01105975). Evacetrapib monotherapy resulted in significant placebo-adjusted dose-dependent decreases from baseline in Lp(a) (up to -40% with evacetrapib 500mg), total LDL particle (LDL-P) (up to -54%), and small LDL particle (sLDL) (up to -95%) concentrations. Compared to statin alone, coadministration of evacetrapib and statins also resulted in significant reduction from baseline in Lp(a) (-31%), LDL-P (-22%), and sLDL (-60%) concentrations. The percentage of patients with concentrations above optimal concentrations for LDL-P (>1000nmol/L) and sLDL (>600nmol/L) decreased from 88% and 55% at baseline, respectively, to 20% and 12% at week 12, for patients treated with evacetrapib plus statins. Evacetrapib, alone or with statins, significantly increased LDL-P size. Evacetrapib, as monotherapy or with statins, significantly reduces the concentrations of atherogenic apoB-containing lipoproteins, including Lp(a), LDL-P, and sLDL.