Dose-related Manner Research Articles

PM2.5 induce neurotoxicity via iron overload and redox imbalance mediated-ferroptosis in HT22 cells

PM2.5 is an important risk factor for the development and progression of cognitive impairment-related diseases. Ferroptosis, a new form of cell death driven by iron overload and lipid peroxidation, is proposed to have significant implications. To verify the possible role of ferroptosis in PM2.5-induced neurotoxicity, we investigated the cytotoxicity, intracellular iron content, iron metabolism-related genes, oxidative stress indices and indicators involving in Nrf2 and ferroptosis signaling pathways. Neurotoxicity biomarkers as well as the ferroptotic cell morphological changes were determined by Western Blot and TEM analysis. Our results revealed that PM2.5 induced cytotoxicity, lipid peroxidation, as indicated by MDA content, and neurotoxicity via Aβ deposition in a dose-related manner. Decreased cell viability and excessive iron accumulation in HT-22 cells can be partially blocked by ferroptosis inhibitors. Interestingly, GPX activity, Nrf2, and its regulated ferroptotic-related proteins (i.e. GPX4 and HO-1) were significantly up-regulated by PM2.5. Moreover, gene expression of DMT1, TfR1, IRP2 and FPN1 involved in iron homeostasis and NCOA4-dependent ferritinophagy were activated after PM2.5 exposure. The results demonstrated that PM2.5 triggered ferritinophagy-dependent ferroptotic cell death due to iron overload and redox imbalance. Activation of Nrf2 signaling pathways may confer a protective mechanism for PM2.5-induced oxidative stress and ferroptosis.

Modulation by Withania somnifera of stress-induced anxiogenesis and airway inflammation in rats.

Stress is an aversive stimulus which disrupts the biological milieu of the organism and a variety of emotional and environmental stressors are known to influence allergic and immunological disorders like bronchial asthma but the pharmacological basis of such interactions is not clearly defined. Withania somnifera (ashwagandha) is a potent anti-stress agent used widely in Indian traditional medicine and the present experimental study evaluated the effects of W.somnifera extract (WSE) on chronic stress-induced neurobehavioral and immunological responses in an experimental model of allergic asthma in rats. Wistar rats (200-250 g) were immunized and challenged with ovalbumin (OVA) and exposed to restraint stress (RS) and WSE treatments for 15days. Following this, anxiety behavior was assessed by the elevated plus maze (EPM) test, and blood and BAL fluid samples were collected for measuring of inflammatory/immune markers by ELISA and biochemical assay. The data of the various treatment groups were analyzed by ANOVA and Tukey's test. Restraint stress (RS) induced anxiogenic behavior in the (EPM) test in OVA immunized rats, and this was attenuated by WSE (200 and 400 mg/kg), in a dose related manner. Examination of blood and BAL fluid in these RS exposed rats also resulted in elevations in IgE, TNF-α andIL-4 levels, which were also attenuated by WSE pretreatments. Further, WSE pretreatment neutralized the such RS induced changes in oxidative stress markers viz. elevated MDA and reduced GSH levels. The data pharmacologically validates role of stress in asthma and suggests that adaptogens like WSE could be a potential complementary agent for reducing anxiety as well as airway inflammation by a multi-targeted and holistic approach. The study also highlights the significance of integration of traditional and modern medical concepts in such chronic disorders.

Sexual Invigorating Potentials of a Combined Extract of Sabicea calycina and Carpolobia lutea on Male Wistar Rats in Crude Oil Challenged Environment

The effect of 50, 100 and 200 mg/kg of combined ethanol extract of S. calycina and C. lutea were investigated on mating behavioural parameters and sex/reproductive hormone boosting potentials in both non-bonny light crude oil (NBLCO) and bonny light crude oil (BLCO) environmentally challenged rats. Forty sexually experienced rats weighing 120-135g were arbitrarily allocated into two groups of twenty rats as NBLCO and BLCO respectively, each group were further subdivided into four groups of five rats and administered orally with 50 mL distilled water (control) and the extract of S. calycina and C. lutea at doses of 50, 100 and 200 mg/kg body weight. After 21 days of treatment protocols, the animals were observed for both precopulatory and copulatory activities, sex hormone was also evaluated. The copulatory activities monitored showed that mount latency, intromission latency and post ejaculatory intervals were significantly decreased by the 100 and 200 mg/kg of the extract in both the NBLCO and BLCO-challenged rats (P < 0.05). All doses significantly increased (P < 0.05) the mount frequency, intromission frequency, and ejaculatory frequency in the NBLCO groups while only the 100 and 200 mg/kg body weight was able. Copulatory efficiency was significantly increased (P < 0.05) by all doses in a dose-related manner in the NBLCO groups while only the 200 mg/kg dose level was able to significantly increase (P < 0.05) the copulatory efficiency in the BLCO groups. The hormonal assay showed that all doses produced a significant increase (P < 0.05) in testosterone, luteinizing hormone, follicle-stimulating hormone, and progesterone, with a concomitantly significant decrease (P < 0.05) in prolactin in the NBLCO groups, however, only the 100 and 200 mg/kg was able to able to achieve significant change (P < 0.05) in these hormonal levels in the BLCO groups. Findings from this study showed that the extract has aphrodisiac potentials and also the ability to restore hyposexual activity in rats challenged with crude oil. 

Dopaminergic and serotoninergic systems as preferential targets of the pyrethroid tefluthrin exposure in the rat brain

The monoaminergic systems dopamine (DA) and serotonin (5-HT) play important roles in neuromodulation, such as motor control, cognitive, affective, and neuroendocrine functions. In the present research study, we addressed the hypothesis that exposure to Type I pyrethroid tefluthrin may specifically target the dopaminergic and serotoninergic systems. Tefluthrin could modify brain monoamine neurotransmitters, DA and 5-HT levels as well as dopaminergic and serotoninergic signaling pathways. Adult male Wistar rats were treated with tefluthrin [2.2, 4.4 and 5.5 mg/kg bw, equivalent to 1/10, 1/5 and 1/4 of the acute oral rat lethal dose 50 (LD50) value] by oral gavage, six days. After last dose of tefluthrin, DA and 5-HT and metabolites levels were determined in brain regions (striatum, hippocampus, prefrontal cortex and hypothalamus). Tefluthrin induced a decrease of DA, 5-HT and metabolites contents, in a brain regional- and dose-related manner. The major decreases in DA and 5-HT contents were observed in prefrontal cortex tissue. Here, we studied that in vivo exposure to tefluthrin may alter DA and 5-HT neurotransmission in prefrontal cortex. Transcripts related to (i) dopaminergic [dopamine transporter 1 (Dat1), tyrosine hydroxylase (TH), dopamine receptors (Drd1, Drd2)], (ii) serotoninergic [serotonin transporter (SERT), tryptophan hydroxylase 2 (TPH2), serotonin receptors (5-HT1A, 5-HT2A)] and (iii) DA and 5-HT degradation [monoamine oxidases (MAOA, MAOB)] signaling pathways were investigated. Results showed that tefluthrin induced down-regulation of transcripts responsible for the synthesis and action of DA (TH, Drd1, Drd2) and 5-HT (SERT, TPH2). In contrast, tefluthrin treatment induced up-regulation of genes involved in DA transporter (Dat1), 5-HT receptors (5-HT1A, 5-HT2A) and monoamine oxidases (MAOA, MAOB). Given the integral roles of mitochondrial dysfunction and dopaminergic and serotoninergic alterations as hallmarks of neurodegenerative diseases, our data suggest that tefluthrin may be a candidate for pesticides contributing to neurodegenerative disorders pathogenesis by causing damage to the DA and 5-HT systems.

Amelioration by Withania somnifera of neurobehavioural and immunological markers in time dependent sensitization induced post traumatic stress disorder in rats.

Posttraumatic stress disorder (PTSD) is a complex neuropsychiatric pathophysiology with an unmet need for safe, effective, and sustainable therapeutic modalities. Thus, the present study evaluated the effects of Withaniasomnifera (WS, Ashwagandha) on an experimental model of PTSD in rats. Wistar rats (200-250 g) were used and time-dependent sensitization (TDS) was used as the experimental model of PTSD. Standardized WS root extract (100 and 300 mg/kg, p.o. for 15 days) was administered with TDS and their effects were observed on neurobehavioral (anxiety) and brain cytokines, corticosterone, and oxidative stress markers. Exposure to TDS resulted in anxiogenic behavior in the elevated plus maze (EPM) test, i.e., reductions in open arm entries and open arm time, as compared to the control group. Pretreatment with WS extract (100 and 300 mg/kg × 14 days) attenuated the TDS-induced anxiogenic activity in a dose-related manner, and these WS effects were comparable to those seen after the comparator drug fluoxetine (10 mg/kg). Assay of brain homogenates showed that TDS also resulted in elevations in brain interleukin-6 and reduction in corticosterone levels in both the hippocampus and prefrontal cortex (PFC), which were reversed after WS pretreatments. Further, WS pretreatment also reversed the TDS-induced changes in brain oxidative stress markers, namely elevated malondialdehyde and reduced glutathione levels in both the hippocampus and PFC. These results suggest that WS could have potential as a therapeutic agent for treating PTSD by attenuating anxiogenesis, neuroimmune axis activation, and oxidative stress.

Assessment of groundnut milk intake on nutritional status and some biochemical indices of normal rats

Groundnut milk also known as peanut milk, is an increasingly popular based beverage that serves as a potential alternative to cow’s milk. It is rich in essential nutrients such as proteins, healthy fats, vitamins, and minerals but limited research has explored its effect on nutritional status and biochemical indices. Therefore, this study assessed the effect of groundnut milk intake on nutritional status and some biochemical indices of Wistar rats. The Wistar rats (n=20), with body weight ≤50g were randomly divided into four groups of five rats each: Group 1 served as control. Groups 2, 3, and 4 were administered groundnut milk at daily doses of 247.2, 494.3, and 988.6 mg/kg respectively for eight weeks. Significant increase in abdominal circumference (P<0.005) between groups was observed. However, increase in the BMI and thoracic circumference between groups were insignificant (P>0.05). The levels of TC, TG and LDL-c were decreased significantly (P<0.005). However, HDL-c showed no significant difference. The decrease in atherogenic index, atherogenic coefficient and cardiac risk ratio indices were insignificant (P>0.05). The groundnut milk had no effect on serum total protein, adiponectin, and albumin while it increased serum leptin level significantly (P<0.005). The blood glucose levels of group 2, 3 and 4 were lowered dose-dependently (P<0.005) compared to the control. Groundnut milk showed no effect on liver and kidney-to-body weight ratio (P>0.05), but showed effect on heart-to-body weight ratio (P<0.005). The ratio of food and water intake of groups 2, 3, and 4 decreased significantly (P<0.005) compared to the control group. The results revealed that groundnut milk could play a positive role nutritionally and positively influence lipid profile, glucose, and leptin levels in a dose-related manner.

Ionomic analysis, polyphenols characterization, analgesic, antiinflammatory and antioxidant capacities of Cistus laurifolius leaves: in vitro, in vivo, and in silico investigations

This study aims to investigate the chemical and mineral composition, antioxidant, analgesic, and anti-inflammatory effects of the aqueous extract of Cistus laurifolius var. atlanticus Pit. (Cistaceae). Additionally, molecular docking interactions of various ligands with antioxidant protein target urate oxidase (1R4U) and anti-inflammatory protein target cyclooxygenase-2 (3LN1), revealing potential dual activities and highlighting specific residue interactions. The chemical characterization focused at first glance on the mineral composition which showed that C. laurifolius extract is a mineral-rich source of potassium (K), magnesium (Mg), manganese (Mn), sodium (Na), phosphorus (P), and zinc (Zn). We next performed, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, the latter showed various polyphenols in C. laurifolius extract including Gallic acid as the predominant polyphenol. Isoquercetin, Taxifolin and Astragalin were also among the major flavonoids detected. The antioxidant capacity of C. laurifolius leaves was tested using 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1- picrylhydrazyl (DPPH) and reducing power (RP) assays. In vitro analysis of the anti-inflammatory property of C. laurifolius leaves was conducted by the albumin denaturation test and the in vivo was assessed in the sequel by carrageenan-induced paw edema test. The analgesic activity was evaluated in vivo using tail flick, acetic acid-induced contortion, and plantar tests. The findings showed that the leave extract had a powerful antioxidant activity with an IC50 values of 2.92 ± 0.03 µg/mL (DPPH) and 2.59 ± 0.09 µg/mL (in RP test). The studied extract strongly abolished the induced inflammation (82%). Albumin denaturation test recorded an IC50 value of 210 µg/mL. Importantly, the oral administration of C. laurifolius extract considerably reduced the nociceptive effect of acetic acid in rats, showing a significant analgesic effect in a dose-related manner. Altogether, our results showed that C. laurifolius can be a promising source of phytochemicals for drug development potential.

A Phase 1 First-in-Human Monotherapy Study of F182112, a B-Cell Maturation Antigen (BCMA)-CD3 Bispecific Antibody, in Patients with Relapsed or Refractory Multiple Myeloma

Introduction: Outcomes of relapsed or refractory Multiple Myeloma（RRMM） are generally poor after receiving current arsenal of therapies. Several novel kinds of drugs targeting BCMA are being developed to overcome resistance in this heavily treated patient population. F182112 is a BCMA x CD3 bispecific antibody that redirects CD3 + T cells to mediate T-cell activation and subsequently caused lysis of BCMA-expressing myeloma cells. A phase 1 clinical trial, NTP-F182112-001, has been launched to evaluate the safety and efficacy of F182112 in patients with RRMM. Shan Dong New Time Pharmaceutical Co. Ltd provides the funding support for the research. Clinical trial information: NCT04984434. Methods: This is a first-in-human, open-label, multicenter, phase 1 study. It has enrolled patients with RRMM who aged ≥ 18 years and previously received at least 2 prior lines of therapy including at least a proteasome inhibitor and an immunomodulatory agent. F182112 was administered intravenously once a week. First four dose cohorts of F182112 (0.01, 0.1, 0.3 and 1μg/kg) were planned for accelerated titration phase and the following four dose cohorts (3, 10, 20, and 30 μg/kg) were planned for i3+3 dose escalation phase . Primary objectives were to access safety and tolerability by monitoring adverse events (AEs) per CTCAE v5.0, with exception of CRS grading per ASTCT 2018. The target rate of dose-limiting toxicity (DLT) was 25% (equivalence interval± 5%). In addition, soluble BCMA, cytokines and immunophenotyping were analysed in peripheral blood. The pharmacokinetics and immunogenicity of F182112 were also explored. Results : As of July 24, 2023, 22 patients have been treated with F182112 (0.01-30 μg/kg) at eight escalating dose levels. Median age was 64.5 years (range 52-74). All of patients were followed up at least five weeks and the median follow-up was 3 months (range 1-18). Fifteen (68.1%) patients received ≥4 prior lines of therapy and 15 (68.1%) patients were refractory to last lines of therapy. Treatment-related AEs (TRAEs) were reported in 18 (82%) pts, with grade ≥3 and serious AEs occurring in 14 (64%) and 4 (18.2%) pts, respectively. The most common TRAEs(≥20%) were CRS (72.7%), lymphopenia (68.2%), neutropenia (54.6%), leukopenia (50%) and anemia (31.8%). All CRS was grade 1 or 2 and the median duration time was 2 days (1-5). One dose-limiting toxicities (ALT elevation, grade 3) occurred at 10 μg/kg dose level. No patients required F182112 dose reduction due to AEs. As of this date cut-off, 20 of the 22 patients were evaluated for response（Figure 1）. The overall response rate (ORR) was 45% (9/20), 30% (6/20) patients had stable disease (SD) and 25% (5/20) patients experienced disease progression (PD). At dose cohorts of 10ug/kg and 20ug/kg （n=9），ORR was 77.8%（7/9）with very good partial remission or better (≥VGPR) rate of 44% (4/9) and stringent complete remission (sCR) rate of 33% (3/9). At the dose cohort of 30ug/kg (n=4), ORR was 25% (1/4), one patient of this dose cohort with extramedullary mass larger than 5cm in diameter at baseline experienced disease progression due to the mass enlargement, though his serum M protein decreased by 60.27%. F182112 exhibited a favorable PK profile in its target patient population of RR MM, with F182112 exposures increasing in a dose-related manner. Conclusions: F182112 in patients with RRMM is well tolerated with preliminary antitumor activity and a favorable PK profile in patients，supporting further evaluation.

The neutral CB1 antagonist AM6527 reduces ethanol seeking, binge-like consumption, reinforcing, and withdrawal effects in male and female mice.

Alcohol use disorder (AUD) is a debilitating physiological and psychiatric disorder which affects individuals globally. The current pharmacological interventions to treat AUD are limited, and hence there is an urgent need for a novel pharmacological therapy which can be effective and safe across the population. We aimed to investigate a novel neutral cannabinoid receptor-1 (CB1R) antagonist, AM6527, in several preclinical models of ethanol consumption using male and female C57BL6/J mice. Independent groups of male and female mice were subjected to repeated cycles of drinking in the dark (DID), or intermittent access to alcohol (IAA) procedures. Twenty minutes prior to ethanol access in each procedure, animals were treated with intraperitoneal injections of either 1, 3, and 10 mg/kg of AM6527 or its respective vehicle. Acamprosate (100, 200, 300, and 400 mg/kg) or its respective vehicle was used as a positive control. Separate groups of male mice were subjected to a chain schedule of ethanol reinforcement to gain access to ethanol wherein completion of a fixed interval (FI; 5 min) schedule (link 1: "Seeking") was reinforced with continuous access to ethanol (fixed ratio; FR1) for up to 1.8 g/kg (link 2: "consumption"). All the animals were treated with 1, 3, and 10 mg/kg of AM6527 or its respective vehicle 20 mins prior to the start of the FI chain of the procedure. Separately, AM6527 was also evaluated in male and female mice undergoing acute ethanol withdrawal following 8 weeks of intermittent or continuous access to 20% ethanol drinking. In both DID and IAA procedures, AM6527 reduced ethanol consumption in a dose-related manner in both male and female mice. AM6527 produced no tolerance in the DID procedure; mice treated with 3 mg/kg of AM6527 for 3 weeks continuously drank significantly smaller amounts of ethanol as compared to vehicle-treated mice over a period of three DID cycles. Moreover, in the IAA procedure, AM6527 caused an increase in water intake over the 24-h period. Acamprosate transiently reduced ethanol intake in male mice in both the DID and the IAA procedures but failed to produce any significant effect in female mice. AM6527 also produced a decrease in the FI responding ("ethanol seeking") in animals trained to self-administer ethanol. Lastly, AM6527 mitigated neurological withdrawal signs, i.e., handling induced convulsions(HIC) in mice undergoing acute ethanol withdrawal. Current findings support previous studies with CB1R neutral antagonist in reducing voluntary ethanol intake and seeking behavior. Based on results shown in this work, AM6527 can be developed as a first in class CB1R neutral antagonist to treat AUD in both males and females.

L-methionine protects against nephrotoxicity induced by methotrexate through modulation of redox status and inflammation

ABSTRACTObjective: Methotrexate (MTX) is a drug used in the treatment of cancer and autoimmune disorders; however, its clinical use is limited because of serious side effects including renal toxicity. This study aimed to investigate the protective effect of Lmethionine (L-Met) on MTX toxicity in the kidneys of rats.Methods: Thirty male rats were divided equally into five groups: control (saline), Met400 (400 mg/kg L-Met), MTX (20 mg/kg MTX), MTX-Met300 (300 mg/kg L-Met and 20 mg/kg MTX), and MTX-Met400 (400 mg/kg L-Met and 20 mg/kg MTX). Rats were euthanized one day after the last dose administration (day 16) and serum and renal tissue samples were collected. Renal function and injury indices, oxidative stress/antioxidant indices and proinflammatory cytokines were evaluated.Results: The results showed that L-Met could effectively counteract the nephrotoxic effects of MTX, in a dose-related manner, by improving most of the tested parameters. Furthermore, the higher dose of L-Met was able to restore several parameters to normal levels. In addition, investigation of MTX-induced hematological changes revealed a corrective potential of L-Met.Conclusion: L-Met can be an effective adjuvant therapy to modulate renal toxicity associated with MTX because of its antioxidant and antiinflammatory effects.

Machine Learning Technology for EEG-Forecast of the Blood-Brain Barrier Leakage and the Activation of the Brain's Drainage System during Isoflurane Anesthesia.

Anesthesia enables the painless performance of complex surgical procedures. However, the effects of anesthesia on the brain may not be limited only by its duration. Also, anesthetic agents may cause long-lasting changes in the brain. There is growing evidence that anesthesia can disrupt the integrity of the blood-brain barrier (BBB), leading to neuroinflammation and neurotoxicity. However, there are no widely used methods for real-time BBB monitoring during surgery. The development of technologies for an express diagnosis of the opening of the BBB (OBBB) is a challenge for reducing post-surgical/anesthesia consequences. In this study on male rats, we demonstrate a successful application of machine learning technology, such as artificial neural networks (ANNs), to recognize the OBBB induced by isoflurane, which is widely used in surgery. The ANNs were trained on our previously presented data obtained on the sound-induced OBBB with an 85% testing accuracy. Using an optical and nonlinear analysis of the OBBB, we found that 1% isoflurane does not induce any changes in the BBB, while 4% isoflurane caused significant BBB leakage in all tested rats. Both 1% and 4% isoflurane stimulate the brain's drainage system (BDS) in a dose-related manner. We show that ANNs can recognize the OBBB induced by 4% isoflurane in 57% of rats and BDS activation induced by 1% isoflurane in 81% of rats. These results open new perspectives for the development of clinically significant bedside technologies for EEG-monitoring of OBBB and BDS.

C-TYPE LECTIN-2D RECEPTOR CONTRIBUTES TO HISTONE-INDUCED VASCULAR BARRIER DYSFUNCTION DURING BURN INJURY.

Severe burns are associated with massive tissue destruction and cell death where nucleus histones and other damage-associated molecular patterns are released into the circulation and contribute to the pathogenesis of multiple-organ dysfunction. Currently, there is limited information regarding the pathophysiology of extracellular histones after burns, and the mechanisms underlying histone-induced vascular injury are not fully understood. In this study, by comparing the blood samples from healthy donors and burn patients, we confirmed that burn injury promoted the release of extracellular histones into the circulation, evidenced by increased plasma levels of histones correlating with injury severity. The direct effects of extracellular histones on human endothelial monolayers were examined, and the results showed that histones caused cell-cell adherens junction discontinuity and barrier dysfunction in a dose-related manner. Like burn patients, mice subjected to a scald burn covering 25% total body surface area also displayed significantly increased plasma histones. Intravital microscopic analysis of mouse mesenteric microcirculation indicated that treatment with a histone antibody greatly attenuated burn-induced plasma leakage in postcapillary venules, supporting the pathogenic role of extracellular histones in the development of microvascular barrier dysfunction during burns. At the molecular level, intrigued by the recent discovery of C-type lectin domain family 2 member D (Clec2d) as a novel receptor of histones, we tested its potential involvement in the histone interaction with endothelial cells. Indeed, we identified abundant expression of Clec2d in vascular endothelial cells. Further proximity ligation assay demonstrated a close association between extracellular histones and endothelial expressing Clec2d. Functionally, in vivo administration of an anti-Clec2d antibody attenuated burn-induced plasma leakage across mesenteric microvessels. Consistently, Clec2d knockdown in endothelial cells partially inhibited histone-induced endothelial barrier dysfunction. Together, our data suggest that burn injury-induced increases in circulating histones contribute to microvascular leakage and endothelial barrier dysfunction via a mechanism involving the endothelial Clec2d receptor.

THU046 Repeated Treatment With AZP-3813, A Bicyclic, 16-Amino Acid Peptide Antagonist Of The Human Growth Hormone Receptor, Induces Enhanced Suppression Of IGF1 In Beagle Dogs

Abstract Disclosure: G. Ravel: None. C. Chalmey: None. C. Berardet: None. D. Duracher: None. H. Kurasaki: None. T. Tomiyama: None. P. Reid: None. M.D. Culler: None. Medical treatment of acromegaly is based on either suppressing pituitary growth hormone (GH) secretion or inhibiting GH action by preventing interaction with its receptor in order to suppress the elevated levels of insulin-like growth factor 1 (IGF1). AZP-3813 is a 16-amino acid, bicyclic peptide antagonist of the GH receptor (GHR) derived from peptide sequences discovered using a unique, cell-free in vitro transcription-translation system screened against the human GHR, and optimized by rational design to increase binding affinity, solubility and half-life. The KD of AZP-3813 for the human GHR is 1.9nM. In previous studies, AZP-3813 was demonstrated to suppress IGF1 secretion in juvenile rats in a dose-related manner, and to maintain IGF1 suppression when given daily for extended periods. Recently, we demonstrated that AZP-3813 is also very effective in suppressing IGF1 in normal Beagle dogs. Dogs receiving a single 0.1, 1 and 10 mg/kg subcutaneous injection of AZP-3813 responded with a dose-related 2.2 + 7.1, 21.4 + 2.7 and 27.8 + 1.5% decrease in IGF1 after 24 hours, respectively, and the decrease in IGF1 attained with a single 10 mg/kg dose was maintained for over 72 hours. The relatively small further increase in IGF1 suppression with 10 versus 1 mg/kg suggests that 10 mg/kg AZP-3813 may induce near maximal GHR antagonism. To examine the effect of repeated and higher dose treatment of dogs with AZP-3813, young adult, non-fasted, Beagle dogs were injected subcutaneously at rotated dorsal sites with AZP-3813 at doses of 10, 30 or 60 mg/kg (n=2, one male and one female) daily for seven days. Blood samples, collected prior to dosing to establish baseline IGF1 levels and 24 hour after the final, seventh AZP-3813 injection, were assayed for total IGF1 content by radioimmunoassay. Baseline IGF1 levels were lower in the female dogs (136.7 + 36.1 ng/ml) than in the male dogs (197.0 + 11.3 ng/ml); however, the percent IGF1 suppression resulting from AZP-3813 treatment was similar for both sexes. AZP-3813, injected for seven days at 10, 30 and 60 mg/kg, induced highly significant 69.0 + 4.0, 70.5 + 9.5 and 75.0 + 1.0% decreases in IGF1, respectively. The similar magnitude of IGF1 suppression attained with the three doses of AZP-3813 implies that maximal GH antagonism is essentially achieved with the 10 mg/kg dose. The substantial increase in IGF1 suppression achieved by repeated 10 mg/kg injection of AZP-3813, compared to the previously observed suppression after a single 10 mg/kg injection, likely represents compound accumulation (under investigation) as well as a progressive effect of maximal GH antagonism on IGF1 production. These results demonstrate that the potent GHR antagonist activity of AZP-3813 translates to a highly effective suppression of IGF1 in normal dogs and further support the development of AZP-3813 as a potential therapy for acromegaly. Presentation: Thursday, June 15, 2023

Ventilatory Effects of Fentanyl, Heroin, and d-Methamphetamine, Alone and in Mixtures in Male Rats Breathing Normal Air .

The number of drug overdoses and deaths has increased significantly over the past decade and co-use of opioids and stimulants is associated with greater likelihood of overdose and decreased likelihood of accessing treatment, compared with use of opioids alone. Potential adverse effects of opioid/stimulant mixtures, particularly methamphetamine, are not well characterized. Two structurally different drugs with agonist properties at µ-opioid receptors (MOR), fentanyl and heroin, and d-methamphetamine, alone and in mixtures, were assessed for their effects on ventilation in rats breathing normal air. Whole-body phethysmography chambers were equipped with a tower and swivel allowing infusions to indwelling intravenous catheters. After a 45-minute habituation period, saline, fentanyl, heroin, or d-methamphetamine, alone and in mixtures, was administered. Five minutes later, the opioid receptor antagonist naloxone or vehicle was injected. Fentanyl (0.0032-0.1 mg/kg) and heroin (0.32-3.2 mg/kg) decreased ventilation [frequency (f) and tidal volume (VT)] in a dose-related manner whereas d-methamphetamine (0.1-3.2 mg/kg) increased f to >400% of control and decreased VT to <60% of control, overall increasing minute volume (product of f and VT) to >240% of control. When combined, d-methamphetamine (0.1-3.2 mg/kg) attenuated the ventilatory depressant effects of fentanyl (0.1 mg/kg) and heroin (3.2 mg/kg). d-Methamphetamine did not alter the potency of naloxone to reverse the ventilatory depressant effects of fentanyl or heroin. These studies demonstrate that d-methamphetamine can attenuate the ventilatory depressant effects of moderate doses of opioid receptor agonists while not altering the potency of naloxone to reverse opioid hypoventilation. SIGNIFICANCE STATEMENT: Co-use of opioids and stimulants is associated with greater likelihood of overdose and decreased likelihood of accessing treatment, compared with use of opioids alone. Potential adverse effects of opioid/stimulant mixtures are not well characterized. This study reports that 1) d-methamphetamine attenuates the ventilatory depressant effects of moderate doses of two structurally different opioid receptor agonists, fentanyl and heroin, and 2) d-methamphetamine does not alter potency or effectiveness of naloxone to reverse the ventilatory depressant effects of these opioid receptor agonists.

Effect of herbicides on cellular growth, microbiological and physiological activities of an identified rhizosphere isolate Rhodococcus erythropolis

ABSTRACT Considering the excessive pesticide pollution in the environment, atrazine (ATZ), butachlor (BCR) and quizalofop-p-ethyl (QUIZ) were selected to assess their toxic effect on Rhodococcus erythropolis PSB-6 (NCBI Accession No. MG028649). MIC values of ATZ, BCR and QUIZ to R. erythropolis were determined to be 100, 200 and 150 μM, respectively. Biomarker enzymatic assays including LPO, LDH and oxidative stress (CAT) induced by herbicides represented significant (p ≤ 0.005) toxicity towards strain PSB-6. Herbicide-induced morphological changes viz. aberrant margins; cellular cracking and distortion/damage in R. erythropolis cells were apparent under SEM observation. Furthermore, herbicide-treated and DAPI (4’,6-diamidino-2-phenylindole)-stained cells showed concentration-dependent reduction in cellular permeability as revealed under CLSM. Furthermore, herbicides displayed toxicity towards bioactive molecules of PSB-6 in a dose-related manner. Among them, ATZ imparted maximum negative effect, where it reduced the bacterial production of IAA, ACC deaminase and 2, 3-DHBA by 68% (p ≤ 0.001), 75% (p ≤ 0.001), and 83% (p ≤ 0.005), respectively, over control. Additionally, following herbicide exposure, bacterial counts (log10 CFU mL−1) were reduced. Higher concentrations of ATZ and BCR completely reduced the growth patten of strain PSB-6. The current investigation provides an insight into a mechanistic approach of chemical herbicide-induced hazard toward a beneficial soil isolate. Careful monitoring is therefore necessary before agricultural application of pesticides.

Disgusted snails, oxytocin, and the avoidance of infection threat

Disgust is considered to be a fundamental affective state associated with triggering the behavioral avoidance of infection and parasite/pathogen threat. In humans, and other vertebrates, disgust affects how individuals interact with, and respond to, parasites, pathogens and potentially infected conspecifics and their sensory cues. Here we show that the land snail, Cepaea nemoralis, displays a similar “disgust-like” state eliciting behavioral avoidance responses to the mucus associated cues of infected and potentially infected snails. Brief exposure to the mucus of snails treated with the Gram-negative bacterial endotoxin, lipopolysaccharide (LPS), elicited dose-related behavioral avoidance, including acute antinociceptive responses, similar to those expressed by mammals. In addition, exposure to the mucus cues of LPS treated snails led to a subsequent avoidance of unfamiliar individuals, paralleling the recognition of and avoidance responses exhibited by vertebrates exposed to potential pathogen risk. Further, the avoidance of, and antinociceptive responses to, the mucus of LPS treated snails were attenuated in a dose-related manner by the oxytocin (OT) receptor antagonist, L-368,899. This supports the involvement of OT and OT receptor homologs in the expression of infection avoidance, and consistent with the roles of OT in the modulation of responses to salient social and infection threats by rodents and other vertebrates. These findings with land snails are indicative of evolutionarily conserved disgust-like states associated with OT/OT receptor homolog modulated behavioral avoidance responses to infection and pathogen threat.

The antidepressant drugs vortioxetine and duloxetine differentially and sex-dependently affect animal well-being, cognitive performance, cardiac redox status and histology in a model of osteoarthritis

Osteoarthritis represents a leading cause of disability with limited treatment options. Furthermore, it is frequently accompanied by cardiovascular and cognitive disorders, which can be exacerbated by osteoarthritis or drugs used for its treatment. Here, we examined the behavioral and cardiac effects of the novel antidepressant vortioxetine in an osteoarthritis model, and compared them to duloxetine (an established osteoarthritis treatment). Osteoarthritis was induced in male and female rats with an intraarticular sodium-monoiodoacetate injection. Antidepressants were orally administered for 28 days following induction. During this period the acetone, burrowing and novel-object-recognition tests (NORT) were used to assess the effects of antidepressants on pain hypersensitivity (cold allodynia), animal well-being and cognitive performance, respectively. Following behavioral experiments, heart muscles were collected for assessment of redox status/histology. Antidepressant treatment dose-dependently reduced cold allodynia in rats with osteoarthritis. Duloxetine (but not vortioxetine) depressed burrowing behavior in osteoarthritic rats in a dose-related manner. Osteoarthritis induction reduced cognitive performance in NORT, which was dose-dependently alleviated by vortioxetine (duloxetine improved performance only in female rats). Furthermore, duloxetine (but not vortioxetine) increased oxidative stress parameters in the heart muscles of female (but not male) rats and induced histological changes in cardiomyocytes indicative of oxidative damage. Vortioxetine displayed comparable efficacy to duloxetine in reducing pain hypersensitivity. Furthermore, vortioxetine (unlike duloxetine) dose-dependently improved cognitive performance and had no adverse effect on burrowing behavior (animal surrogate of well-being) and cardiac redox status/histology. Our results indicate that vortioxetine could be a potential osteoarthritis treatment (with better characteristics compared to duloxetine).

Phytochemical Screening, Analgesic and Anti-Inflammatory Activity of the Ethanol Extract of the Cnidoscolus Phyllacanthus Leaves

Objective: The present study aimed to investigate the analgesic and anti-inflammatory activity of the Cnidoscolus phyllacanthus (family Euphorbiaceae) ethanolic leaves extract in the laboratory using in vivo methods to justify its traditional use in the above-mentioned pathological conditions. Methods: Phytochemical screening was done to find the presence of various secondary metabolites of the plant. In vivo, analgesic activity was performed employing the acetic acid-induced writhing test and tail immersion test on Swiss albino mice at doses of 50, 100 and 200 mg/kg body weight. An anti-inflammatory activity test was done on Wistar rats at three doses (50, 100 and 200 mg/kg body weight) using a carrageenan-induced paw oedema test. Data were analyzed by one-way analysis of variance (ANOVA), and Dunnett's t-test was used as the significance test. P value was considered as the minimum level of significance.  Results: Phytochemical screening revealed the presence of coumarin, flavonoids, monoterpenes, diterpenes and naphthoquinones in the extract. In the acetic acid-induced writhing test, the per cent inhibition of writhing response by the extract was 12.82%, 25.64% and 76.67% at 50, 100 and 200 mg/kg doses, respectively (p &lt; 0.01) and showed an increase in pain threshold in tail immersion method. The extract significantly (p &lt; 0.05 and p &lt; 0.01) inhibited carrageenan-induced inflammatory response in rats in a dose-related manner. Per cent, inhibition in paw oedema was 16.39 %, 29.50 % and 52.45 % at 50, 100 and 200 mg/kg doses, respectively.  Conclusion: The results obtained from the tests indicate that the plant might have one or more secondary metabolite(s) having central and peripheral analgesic and anti-inflammatory activity.

Toxic effects of sodium dodecyl sulfate on planarian Dugesia japonica.

Sodium dodecyl sulfate (SDS) is an anionic surfactant, which is widely used in various fields in human life. However, SDS discharged into the water environment has a certain impact on aquatic organisms. In this study, planarian Dugesia japonica (D. japonica) was used to identify the toxic effects of SDS. A series of SDS solutions with different concentrations were used to treat planarians for the acute toxicity test , and the results showed that the semi-lethal concentration (LC50) of SDS to D. japonica at 24 h, 48 h, 72 h, and 96 h were 4.29 mg/L, 3.76 mg/L, 3.45 mg/L, and 3.20 mg/L respectively. After the planarians were exposed to 0.5 mg/L and 1.0 mg/L SDS solutions for 1, 3, and 5 days, the activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) content were measured to detect the oxidative stress and lipid peroxidation in planarians. Random amplified polymorphic DNA (RAPD) analysis was performed to detect the genotoxicity caused by SDS to planarians. The results showed that the activities of SOD, CAT, and MDA content increased after the treatment, indicating that SDS induced oxidative stress in planarians. RAPD analysis showed that the genomic template stability (GTS) values of planarians treated by 0.5 mg/L and 1.0 mg/L SDS for 1, 3, and 5 days were 67.86%, 64.29%, 58.93%, and 64.29%, 60.71%, 48.21%, respectively. GTS values decreased with the increasing of SDS concentration and exposure time, indicating that SDS had genotoxicity to planarians in a time and dose-related manner. Fluorescent quantitative PCR (qPCR) was used to investigate the effects of SDS on gene expression of planarians. After the planarians were exposed to 1.0 mg/L SDS solution for 1, 3, and 5 days, the expression of caspase3 was upregulated, and that of piwiA, piwiB, PCNA, cyclinB, and RAD51 were downregulated. These results suggested that SDS might induce apoptosis, affect cell proliferation, differentiation, and DNA repair ability of planarian cells and cause toxic effects on planarian D. japonica.

Safety, Tolerability, and Pharmacokinetics of Single and Multiple Ascending Intravenous Infusions of PF-07304814 (Lufotrelvir) in Participants Hospitalized With COVID-19.

An urgent need remains for antiviral therapies to treat patients hospitalized with COVID-19. PF-07304814-the prodrug (lufotrelvir) and its active moiety (PF-00835231)-is a potent inhibitor of the SARS-CoV-2 3CL protease. Eligible participants were 18 to 79 years old and hospitalized with confirmed COVID-19. This first-in-human phase 1b study was designed with 2 groups: single ascending dose (SAD) and multiple ascending dose (MAD). Participants could receive local standard-of-care therapy. In SAD, participants were randomized to receive a 24-hour infusion of lufotrelvir/placebo. In MAD, participants were randomized to receive a 120-hour infusion of lufotrelvir/placebo. The primary endpoint was to assess the safety and tolerability of lufotrelvir. The secondary endpoint was to evaluate the pharmacokinetics of lufotrelvir and PF-00835231. In SAD, participants were randomized to receive 250 mg lufotrelvir (n = 2), 500 mg lufotrelvir (n = 2), or placebo (n = 4) by continuous 24-hour infusion. In MAD, participants were randomized to receive 250 mg lufotrelvir (n = 7), 500 mg lufotrelvir (n = 6), or placebo (n = 4) by continuous 120-hour infusion. No adverse events or serious adverse events were considered related to lufotrelvir. At doses of 250 and 500 mg, concentrations for the prodrug lufotrelvir and active moiety PF-00835231 increased in a dose-related manner. Unbound concentrations of the lufotrelvir active metabolite reached steady state approximately 2- and 4-fold that of in vitro EC90 following 250- and 500-mg doses, respectively. These safety and pharmacokinetic findings support the continued evaluation of lufotrelvir in clinical studies. Clinical Trials Registration. ClinicalTrials.gov NCT04535167.