Male CD-1 Mice Research Articles

Dynamics of temperature and body weight in irradiated mice: the dose-effect and time-effect relationships

In an experiment on outbred male ICR CD-1 mice irradiated at a wide range of doses of gamma irradiation 60Co from 7.4 to 9.4 Gy with a 30-day survival rate from 100 to 5%, the dose-effect and time-effect relationships in relation to animals’ temperature and body mass index during acute radiation sickness were studied. The latent phase of acute radiation sickness is already related to a decrease in body temperature, which is determined using a remote infrared thermometer, in presence of body weight loss. A maximum dose-dependent fall in body temperature was observed in the critical phase of acute radiation sickness, when animals die in large numbers. In the recovery phase, body temperature returned to normal with subsequent body weight gain. Body temperature of irradiated mice is considered as an important criterion for their asthenia and can be used both in theoretical and applied studies.

Allolobophora caliginosa coelomic fluid and extract alleviate glucocorticoid-induced osteoporosis in mice by suppressing oxidative stress and regulating osteoblastic/osteoclastic-related markers

Allolobophora calignosa (Ac) is a folk medicine for millennia, as it possesses many biological activities. This study aimed to investigate the chemo-preventive activity of A.calignosa coelomic fluid (AcCF) and A.calignosa extract (AcE) on glucocorticoid-induced osteoporosis (GIOP) in mice. Characterization and in vitro biological activity of AcE and AcCF has been assessed. Male CD-1 mice were subcutaneously received dexamethasone (DEX) (1 mg/kg, 5 times/week) and concurrently intraperitoneally treated with either AcCF (20 mg/kg) or AcE (45 mg/kg) every other day for 28 days. Serum and bone homogenates were subjected for qPCR and biochemical analysis. AcE and AcCF treatment significantly increased bone mineral density (BMD), bone mineral content (BMC), calcium (Ca), phosphorus (P), and calcitonin levels, whereas activity of serum alkaline phosphatase (ALP), bone alkaline phosphatase (BALP), serum acidic phosphatase (ACP), bone acidic phosphatase (BACP) and parathyroid hormone (PTH) levels were significantly reduced compare with untreated GIOP mice. Treatment with AcE and AcCF modulates oxidative stress and downregulated Rank and Mmp9 expression, as well as increased glycosaminoglycan content in the organic bone matrix, resulting in osteoclastogenesis inhibition. Overall, AcCF and AcE show a chemo-preventive activity against GIOP by inhibiting oxidative stress and regulating expression and/or activity of osteoblast/osteoclast-related markers.

Depilatory double-disc mouse model for evaluation of vesicant dermal injury pharmacotherapy countermeasures.

Sulfur mustard (SM) is a chemical warfare vesicant that severely injures exposed eyes, lungs, and skin. Mechlorethamine hydrochloride (NM) is widely used as an SM surrogate. This study aimed to develop a depilatory double-disc (DDD) NM skin burn model for investigating vesicant pharmacotherapy countermeasures. Hair removal method (clipping only versus clipping followed by a depilatory), the effect of acetone in the vesicant administration vehicle, NM dose (0.5-20 μmol), vehicle volume (5-20 μl), and time course (0.5-21 days) were investigated using male and female CD-1 mice. Edema, an indicator of burn response, was assessed by biopsy skin weight. The ideal NM dose to induce partial-thickness burns was assessed by edema and histopathologic evaluation. The optimized DDD model was validated using an established reagent, NDH-4338, a cyclooxygenase, inducible nitric oxide synthase, and acetylcholinesterase inhibitor prodrug. Clipping/depilatory resulted in a 5-fold higher skin edematous response and was highly reproducible (18-fold lower %CV) compared to clipping alone. Acetone did not affect edema formation. Peak edema occurred 24-48 h after NM administration using optimized dosing methods and volume. Ideal partial-thickness burns were achieved with 5μmol of NM and responded to treatment with NDH-4338. No differences in burn edematous responses were observed between males and females. A highly reproducible and sensitive partial-thickness skin burn model was developed for assessing vesicant pharmacotherapy countermeasures. This model provides clinically relevant wound severity and eliminates the need for organic solvents that induce changes to the skin barrier function.

Binding preference at the μ-opioid receptor underlies distinct pharmacology of cyclopropyl versus valeryl analogs of fentanyl

Illicitly manufactured fentanyl is driving the current opioid crisis, and various fentanyl analogs are appearing in recreational drug markets worldwide. To assess the potential health risks posed by fentanyl analogs, it is necessary to understand structure-activity relationships for these compounds. Here we compared the pharmacology of two structurally related fentanyl analogs implicated in opioid overdose: cyclopropylfentanyl and valerylfentanyl. Cyclopropylfentanyl has a three-carbon ring attached to the carbonyl group on the fentanyl scaffold, whereas valerylfentanyl has a four-carbon chain at the same position. In vitro assays examining μ-opioid receptor (MOR) coupling to G proteins in CHO cells showed that cyclopropylfentanyl is a full agonist (EC50 = 8.6 nM, %Emax = 113%), with potency and efficacy similar to fentanyl (EC50 = 10.3 nM, %Emax = 113%). By contrast, valerylfentanyl is a partial agonist at MOR (EC50 = 179.8 nM, %Emax = 60%). Similar results were found in assays assessing MOR-mediated β-arrestin recruitment in HEK cells. In vivo studies in male CD-1 mice demonstrated that both fentanyl analogs induce naloxone-reversible antinociception and respiratory suppression, but cyclopropylfentanyl is 100-times more potent as an antinociceptive agent (ED50 = 0.04 mg/kg, s. c.) than valerylfentanyl (ED50 = 4.0 mg/kg, s. c.). Molecular simulation results revealed that the alkyl chain of valerylfentanyl cannot be well accommodated by the active state of MOR and may transition the receptor toward an inactive state, converting the fentanyl scaffold to a partial agonist. Taken together, our results suggest that cyclopropylfentanyl presents much greater risk of adverse effects when compared to valerylfentanyl. Moreover, the summed findings may provide clues to the design of therapeutic opioids with reduced adverse side effects.

Murine toxicology and pharmacokinetics of lead next generation galeterone analog, VNPP433-3β

VNPP433-3β (compound 2, (3β-(1H-imidazole-1-yl)-17-(1H-benzimidazole-1-yl)-androsta-5,16-diene), a multitarget anticancer agent has emerged as our lead next generation galeterone analogs (NGGA). Compound 2 is currently in development as potential new therapeutic for prostate and pancreatic cancers. The preliminary toxicity study reveals that the compound 2 was better tolerated by the normal male CD-1 mice than the male Nude mice. The maximum tolerated dose (MTD) in the Nude mice was estimated to be between 25 < 50 mg/kg. After oral dosing of compound 2 to male and female rats, the plasma concentration versus time curves were very consistent between animals and the AUClast increased with dose. Many plasmas concentration versus time curves profiles were nearly flat over 24 hr., suggesting extended absorption from the GI tract. Consequently, reliable values for half-life and AUCinf were not determined. Calculated oral bioavailability (using oral AUClast and excluding the outlier IV animal) ranged from 32 to 47 %. This should be considered a minimum value since the contribution to true AUC beyond 24 hr. is clearly not zero. Clearly, these toxicology and pharmacokinetics parameters pave the way for understanding the anticancer pharmacological actions and provide a meaningful basis for further preclinical development and eventual clinical development.

The Old and the New: Cardiovascular and Respiratory Alterations Induced by Acute JWH-018 Administration Compared to Δ9-THC-A Preclinical Study in Mice.

Several new psychoactive substances (NPS) are responsible for intoxication involving the cardiovascular and respiratory systems. Among NPS, synthetic cannabinoids (SCs) provoked side effects in humans characterized by tachycardia, arrhythmias, hypertension, breathing difficulty, apnoea, myocardial infarction, and cardiac arrest. Therefore, the present study investigated the cardio-respiratory (MouseOx Plus; EMKA electrocardiogram (ECG) and plethysmography TUNNEL systems) and vascular (BP-2000 systems) effects induced by 1-naphthalenyl (1-pentyl-1H-indol-3-yl)-methanone (JWH-018; 0.3-3-6 mg/kg) and Δ9-tetrahydrocannabinol (Δ9-THC; 0.3-3-6 mg/kg), administered in awake CD-1 male mice. The results showed that higher doses of JWH-018 (3-6 mg/kg) induced deep and long-lasting bradycardia, alternated with bradyarrhythmia, spaced out by sudden episodes of tachyarrhythmias (6 mg/kg), and characterized by ECG electrical parameters changes, sustained bradypnea, and systolic and transient diastolic hypertension. Otherwise, Δ9-THC provoked delayed bradycardia (minor intensity tachyarrhythmias episodes) and bradypnea, also causing a transient and mild hypertensive effect at the tested dose range. These effects were prevented by both treatment with selective CB1 (AM 251, 6 mg/kg) and CB2 (AM 630, 6 mg/kg) receptor antagonists and with the mixture of the antagonists AM 251 and AM 630, even if in a different manner. Cardio-respiratory and vascular symptoms could be induced by peripheral and central CB1 and CB2 receptors stimulation, which could lead to both sympathetic and parasympathetic systems activation. These findings may represent a starting point for necessary future studies aimed at exploring the proper antidotal therapy to be used in SCs-intoxicated patient management.

Can amphotericin B and itraconazole be co-delivered orally? Tailoring oral fixed-dose combination coated granules for systemic mycoses

The incidence and prevalence of invasive fungal infections have increased significantly over the last few years, leading to a global health problem due to the lack of effective treatments. Amphotericin B (AmB) and itraconazole (ITR) are two antifungal drugs with different mechanisms of action. In this work, AmB and ITR have been formulated within granules to elicit an enhanced pharmacological effect, while enhancing the oral bioavailability of AmB. A Quality by Design (QbD) approach was utilised to prepare fixed-dose combination (FDC) granules consisting of a core containing AmB with functional excipients, such as inulin, microcrystalline cellulose (MCC), chitosan, sodium deoxycholate (NaDC) and Soluplus® and polyvinyl pyrrolidone (PVP), coated with a polymeric layer containing ITR with Soluplus® or a combination of Poloxamer 188 and hydroxypropyl methyl cellulose-acetyl succinate (HPMCAS). A Taguchi design of experiments (DoE) with 7 factors and 2 levels was carried out to understand the key factors impacting on the physicochemical properties of the formulation followed by a Box-Behnken design with 3 factors in 3 levels chosen to optimise the formulation parameters. The core of the FDC granules was obtained by wet granulation and later coated using a fluidized bed. In vitro antifungal efficacy was demonstrated by measuring the inhibition halo against different species of Candida spp., including C. albicans (24.19–30.48 mm), C. parapsilosis (26.38–27.84 mm) and C. krusei (11.48–17.92 mm). AmB release was prolonged from 3 to 24 h when the AmB granules were coated. In vivo in CD-1 male mice studies showed that these granules were more selective towards liver, spleen and lung compared to kidney (up to 5-fold more selective in liver, with an accumulation of 8.07 µg AmB/g liver after twice-daily 5 days administration of granules coated with soluplus-ITR), resulting in an excellent oral administration option in the treatment of invasive mycosis. Nevertheless, some biochemical alterations were found, including a decrease in blood urea nitrogen (∼17 g/dl) and alanine aminotransferase (<30 U/l) and an increase in the levels of bilirubin (∼0.2 mg/dl) and alkaline phosphatase (<80 U/l), which could be indicative of a liver failure. Once-daily regimen for 10 days can be a promising therapy.

Açai Berry Administration Promotes Wound Healing through Wnt/β-Catenin Pathway

Recently, wound healing has received increased attention from both a scientific and clinical point of view. It is characterized by an organized series of processes: angiogenesis, cell migration and proliferation, extracellular matrix production, and remodeling. Many of these processes are controlled by the Wnt pathway, which activates them. The aim of the study was to evaluate the molecular mechanism of açai berry administration in a mouse model of wound healing. CD1 male mice were used in this research. Two full-thickness excisional wounds (5 mm) were performed with a sterile biopsy punch on the dorsum to create two circular, full-thickness skin wounds on either side of the median line on the dorsum. Açai berry was administered by oral administration (500 mg/kg dissolved in saline) for 6 days after induction of the wound. Our study demonstrated that açai berry can modulate the Wnt pathway, reducing the expression of Wnt3a, the cysteine-rich domain of frizzled (FZ)8, and the accumulation of cytosolic and nuclear β-catenin. Moreover, açai berry reduced the levels of TNF-α and IL-18, which are target genes strictly downstream of the Wnt/β-catenin pathway. It also showed important anti-inflammatory activities by reducing the activation of the NF-κB pathway. Furthermore, Wnt can modulate the activity of growth factors, such as TGF-β, and VEGF, which are the basis of the wound-healing process. In conclusion, we can confirm that açai berry can modulate the activity of the Wnt/β-catenin pathway, as it is involved in the inflammatory process and in the activity of the growth factor implicated in wound healing.

Vanadium inhalation effects on the corneal ciliary neurotrophic factor (CNTF): study in a murine model

Purpose Air pollution is a public health problem caused by predatory human activities and the indiscriminate burning of fossil fuels that liberate particulate matter (PM) into the atmosphere. Vanadium (V) adheres to them and reaches the bloodstream and different organs such as the eye when inhaled. Another way to reach the eye is by direct contact, and the cornea is the first layer exposed. Ciliary neurotrophic factor (CNTF) is secreted by the corneal nerves and some of its functions include self-renewal maintenance and wound healing by the activation of STAT3. Previous reports from our group indicate the activation of STAT3 after the inhalation of V, adhered to PM. Objective To analyse the effect of V inhalation in the expression of CNTF. Method: CD-1 male mice were exposed for 4 and 8 weeks to V inhalation. The eyes were removed, and the corneas were processed for immunohistochemistry for CNTF and analysed by densitometry. The same slides were used to evaluate histological modifications and to measure the corneas’ anterior epithelial and endothelial thickness. Results A decrease in CNTF expression in the anterior epithelium in the 8th week, as well as an increase in the endothelial and corneal thickness and disarray of all the layers of the anterior epithelium. Conclusion V inhalation disturbs the architecture of the cornea and modifies the presence of CNTF which might modify the renewal of the corneas after exposure to PM air pollution.

Evaluation of renal damage in a bleomycin-induced murine model of systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease of unknown etiology with a high mortality rate. Renal crisis has been reported as one of the predictors of early mortality in these patients. The present study was performed to evaluate bleomycin-induced SSc using an osmotic minipump as a possible model for the analysis of renal damage in SSc. Male CD1 mice were implanted with osmotic minipumps loaded with saline or bleomycin and sacrificed at 6 and 14 days. Histopathological analysis was performed through hematoxylin and eosin (H&E) and Masson's trichrome staining. The expression of endothelin 1 (ET-1), inducible nitric oxide synthase (iNOS), transforming growth factor β (TGF-β), and 8-hydroxy-2-deoxyguanosine (8-OHdG) was also evaluated by immunohistochemistry. The administration of bleomycin induced a decrease in the length of Bowman's space (3.6 μm, P<0.001); an increase in collagen deposition (14.6%, P<0.0001); and an increase in the expression of ET-1 (7.5%, P<0.0001), iNOS (10.8%, P<0.0001), 8-OHdG (161 nuclei, P<0.0001), and TGF-β (2.4% µm, P<0.0001) on Day 6. On Day 14, a decrease in the length of Bowman's space (2.6 μm, P<0.0001); increased collagen deposition (13.4%, P<0.0001); and increased expression of ET-1 (2.7%, P<0.001), iNOS (10.1%, P<0.0001), 8-OHdG (133 nuclei, P<0.001), and TGF-β (0.6%, P<0.0001) were also observed. Systemic administration of bleomycin via an osmotic minipump produces histopathological changes in the kidneys, similar to kidney damage in SSc. Therefore, this model would allow the study of molecular alterations associated with SSc-related renal damage.

Effect of wheat-grain moxibustion on Wnt/β-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition

To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) on Wnt/β-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition, and to explore the possible mechanism of wheat-grain moxibustion in treating bone marrow inhibition. Forty-five SPF male CD1(ICR) mice were randomly divided into a blank group, a model group and a wheat-grain moxibustion group, 15 mice in each group. The bone marrow inhibition model was established by intraperitoneal injection of 80 mg/kg of cyclophosphamide (CTX). The mice in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), 3 moxa cones per acupoint, 30 s per moxa cone, once a day, for 7 consecutive days. The white blood cell count (WBC) was measured before modeling, before intervention and 3, 5 d and 7 d into intervention. After intervention, the general situation of mice was observed; the number of nucleated cells in bone marrow was detected; the serum levels of interleukin-3 (IL-3), interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF) were measured by ELISA; the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc in bone marrow cells was measured by Western blot and real-time PCR method. Compared with the blank group, the mice in the model group showed sluggish reaction, unstable gait, decreased body weight, and the WBC, number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were decreased (P<0.01), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was decreased (P<0.01). Compared with the model group, the mice in the wheat-grain moxibustion group showed better general condition, and WBC, the number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were increased (P<0.01, P<0.05), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was increased (P<0.05). Wheat-grain moxibustion shows therapeutic effect on bone marrow inhibition, and its mechanism may be related to activating Wnt/β-catenin signaling pathway in bone marrow cells, improving bone medullary hematopoiesis microenvironment and promoting bone marrow cell proliferation.

Rosiglitazone Protects against Acetaminophen-Induced Acute Liver Injury by Inhibiting Multiple Endoplasmic Reticulum Stress Pathways.

Excessive acetaminophen (APAP) use can lead to acute liver injury (ALI) by inducing endoplasmic reticulum stress (ERS). We previously found that pretreatment with the peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand rosiglitazone (RSG) alleviated ALI in APAP-treated mice. To examine if RSG-mediated hepatoprotection is associated with ERS suppression. Forty-eight male CD-1 mice were randomly divided into control, RSG, APAP 4 h, APAP 24 h, RSG + APAP 4 h, and RSG + APAP 24 h groups. The RSG and RSG + APAP groups received RSG (20 mg/kg) by gavage 48, 24, and 1 h before intraperitoneal injection of 300 mg/kg APAP, while the APAP group received APAP alone and the control group received only normal saline. Animals were sacrificed immediately (RSG and control groups), 4 h (APAP 4 h and RSG + APAP 4 h), or 24 h (APAP 24 h and RSG + APAP 24 h) post-APAP injection. Liver tissues were collected for hematoxylin-eosin staining, TUNEL staining, and Western blotting for ERS-associated proteins. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were also measured. A second cohort received APAP or RSG + APAP as described and were monitored for survival over one week. At 4 and 24 h following APAP injection alone, serum ALT and AST levels were significantly elevated, and central lobular necrosis of the liver was observed. Necrosis area reached 21.7% at 4 h and 32.1% at 24 h post-APAP, while apoptotic fractions reached 25.6% and 32.4%. Further, 50% of mice in the survival analysis cohort died within one week post-APAP. At 4 h post-APAP, the ERS marker glucose-regulated protein-78 (GRP78) and ERS-associated proteins pJNK, GRP78, p-eIF2α, pPERK, and pIRE were all significantly upregulated. Pretreatment with RSG significantly reduced serum ALT and AST, liver necrosis area, apoptosis rate, and expression of ERS-associated proteins compared to APAP alone, while increasing survival to 80%. Rosiglitazone pretreatment can alleviate APAP-induced ALI by suppressing three branches of ERS signaling.

Ketamine preservative benzethonium chloride potentiates hippocampal synaptic transmission and binds neurotransmitter receptors and transporters

Benzethonium chloride (BZT) is an excipient used in numerous products including (R,S)-ketamine (ketamine) drug formulations for human and veterinary use. Emerging evidence indicates BZT is pharmacologically active. BZT may therefore contribute to some of the clinical or preclinical effects observed with ketamine. In the present study, we evaluated: (i) the affinity of BZT for neurotransmitter receptors and transporters, (ii) the effects of BZT on hippocampal synaptic transmission in vitro, and (iii) plasma and brain concentrations of BZT following its intraperitoneal administration to male CD1 mice. Radioligand binding assays determined the affinity of BZT for neurotransmitter targets. Effects of BZT on field excitatory postsynaptic potentials (fEPSPs) were established via electrophysiological recordings from slices collected from male C57BL/6J mice. The binding assays revealed that BZT binds to numerous receptors (e.g., σ2 Ki = 7 nM) and transporters (e.g., dopamine transporter Ki = 545 nM). Bath application of BZT potentiated hippocampal fEPSPs in mouse hippocampal slices with an EC50 of 2.03 nM. Following intraperitoneal administration, BZT was detected in the plasma, but not in the brain of mice. These data highlight that studies measuring peripheral endpoints or directly exposing systems, in vitro, intracerebroventricularly, or intracortically, to BZT-containing formulations should account for the direct effects of BZT. Our findings also suggest that earlier data attributing pharmacological effects to ketamine may be confounded by BZT and that additional investigation into the functional impact of BZT is warranted.This article is part of the Special Issue on ‘Ketamine and its Metabolites’.

High-Frequency Local Field Potential Oscillations May Modulate Aggressive Behaviors in Mice.

Aggressive behavior is one of congenital social behaviors in many species, which could be promoted by social neglect or isolation in the early stages of life. Many brain regions including the medial prefrontal cortex (mPFC), medial amygdala (MeA) and ventromedial hypothalamus (VMH) are demonstrated to relate to aggressive behavior; however, the dynamic patterns of neural activities during the occurrence of this behavior remain unclear. In this study, 21-day-old male CD-1 mice were reared in social isolation conditions and cohousing conditions for two weeks. Aggressive behaviors of each subject were estimated by the resident-intruder test. Simultaneously, the local field potentials of mPFC, MeA and VMH were recorded for exploring differences in the relative power spectra of different oscillations when aggressive behaviors occurred. The results showed that the following: (1) Compared with the cohousing mice, the socially isolated mice exhibited more aggression. (2) Regardless of "time condition" (pre-, during- and post- attack), the relative power spectra of beta band in the cohousing mice were significantly greater than those in the socially isolated mice, and inversely, the relative power spectra of gamma band in the cohousing mice were significantly smaller than those in the socially isolated mice. (3) The bilateral mPFC exhibited significantly smaller beta power spectra but greater gamma power spectra compared with other brain areas regardless of rearing patterns. (4) For the right VMH of the socially isolated mice, the relative power spectra of the gamma band during attacks were significantly greater than those before attack. These results suggest that aggressive behaviors in mice could be shaped by rearing patterns and that high-frequency oscillations (beta and gamma bands) may engage in mediating aggressive behaviors in mice.

Liposome Formulation of Carfilzomib with Improved Activity As a Monotherapy and in Combination with Vinblastine

Introduction: Carfilzomib (CFZ) is an epoxyketone-based irreversible proteasome inhibitor that is one of the leading treatments for multiple myeloma (MM). Cardiotoxicity is the primary limitation for the clinical use of CFZ. We developed a novel liposome nanoparticle formulation of CFZ, a compound with low water solubility (<5 µg/mL) and low pKa (5.14) (LS-CFZ) that dramatically reduces the toxicity, increases the plasma half-life, and improves the antitumor efficacy compared to the commercial formulation. In addition, CFZ was combined in the same liposome, with vinblastine (VBL) an autophagy inhibitor. The combination exhibited synergistic anti-tumor cell activity in vitro and in vivo using a colon cancer (C26) mouse tumor model. Methods: CFZ was screened for cytotoxicity in combination with various cell lines including MM and C26 with a focus on autophagy inhibitors. VBL was co-loaded into the same liposomes with CFZ, and also loaded into separate particles. Formulations with various lipids, trapping agents and drug to lipid ratios were screened for drug loading efficiency. The lead formulation was selected based on mouse plasma half-life after IV injection in male CD1 mice. The plasma concentration of CFZ, VBL and a fluorescent lipid (liposome) label (DiIC18(5)) were measured by HPLC. The dose ratio of the CFZ and VBL was by determined by the MTD in tumor-free mice and the optimum dose and ratio was used for the efficacy study. The lead formulations were evaluated for anti-tumor efficacy in the C26 colon cancer using a single treatment (day 1) when the tumor volume was 200 mm3 for all liposome formulations and two treatments (day 1 and day 2) (optimum for CFZ) for the unencapsulated CFZ and VBL. Results: Cytotoxicty studies in C26 cells indicated the optimum ratio of CFX/VBL was between 5:1 and 20:1. All liposomes used had a diameter of 110-130 nm. Ammonium sucrose octasulfate yielded the longest retention of CFZ and VBL in the liposome after IV injection in mice. The best lipid composition was sphingomyelin/Cholesterol/PEG-2000-PE (55/45/2.8, mol/mol/mol) with a particle size of 110-130 nm in diameter. Cryo TEM images of the LS-CFZ show a dense amorphous mass of drug precipitate in the center of the liposomes. The drug to lipid ratio chosen for use in the efficacy study was 101 g/mol lipid for CFZ and 103 g/mol lipid for VBL with a loading efficiency >90%. Using the optimized trapping agent and lipid composition the %ID of CFZ is nearly identical to that of the liposome carrier up to 6 h; an average of 98.4±5.4% of the drug is retained up to 6 h. The %ID of CFZ decreases below that of the liposome between 6 and 24 h and most (>98%) of the CFZ has been released from the liposomes by the 24 h point. The AUC for LS-CFZ (2 mg/kg) is 1584 fold higher than CFZ administered as SBCD-CFZ (sulfobutyl cyclodextrin formulated, Kyprolis®) (5 mg/kg). CFZ administered as SBCD-CFZ has a t1/2 of 0.2 h compared to 3.0 h for LS-CFZ. VBL had slightly better stability than CFZ with a plasma half-life of 4.9 h. LS-CFZ had single dose MTD of 30 mg/kg for L-CFZ, while SBCD-CFZ given QDx2 had an MTD 6 mg/kg. The dramatic decrease in toxicity is likely due to lower Cmax of the bioavailable CFZ and reduced cardiotoxicity. The increased tolerability of LS-CFZ allowed for a CFZ/VBL ratio of 20:1 (18/0.9 mg/kg, CFZ/VBL) for the liposome formulation; the unencapsulated CFZ and VBL were administered at a ratio of 5:1 (5/1 mg/kg, CFZ/VBL). The anti-tumor efficacy of LS-CFZ/VBL combination was far superior to all other treatments. LS-CFZ was nearly identical to LS-VBL, and both single agents had more activity than any unencapsulated drug. As a monotherapy, LS-CFZ was far more efficacious than SBCD-CFZ, which is commonly used for treatment of multiple myeloma patients. Conclusion: Liposome formulation of CFZ increased the tolerability by 5-fold compared to the commercial CFZ formulation. The described liposome technology enables significant improvement in anti-tumor efficacy for CFZ as a monotherapy or in combination against a C26 mouse model. The improved activity in solid tumors warrants investigation of LS-CFZ for MM treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 10389: Interleukin-1 Blockade With Anakinra Restores Cardiac Contractility Reserve and Exercise Capacity in a Mouse Model of Ischemic Heart Failure

Background: Acute myocardial infarction (AMI) is associated with an intense inflammatory response promoting adverse cardiac remodeling, impaired cardiac reserve, and reduced exercise capacity, hallmarks of heart failure. Interleukin-1β (IL-1β) is the prototypical inflammatory cytokine that is increased in AMI and is sufficient to impair cardiac reserve when administered exogenously in mice. Whether blocking IL-1β signaling with recombinant IL-1 receptor antagonist, anakinra, can restore cardiac contractile reserve and improve exercise capacity in AMI is unknown. Methods: We induced ischemic heart failure due to large non-reperfused anterior AMI in mice via surgical permanent ligation of the left anterior descending artery via lateral thoracotomy in 10-week-old male CD1 mice (N=30, 32-37 g in weight) followed for 10 weeks. Anakinra was given as intraperitoneal injection of 10 mg/kg, 0.2 ml, for three consecutive days. Cardiac contractile reserve was measured assessing changes in left ventricular fractional shortening with isoproterenol challenges (N=5). A subgroup of mice that had been previously trained to run on treadmill were used to measure interval changes in exercise capacity 10 weeks after AMI and with three consecutive days of anakinra treatment (N=6). Data are presented as mean and standard error of mean. T-test for paired group analysis was used to compare values. Results: Cardiac contractile reserve was impaired at -10±5% and restored with anakinra given for three days to +49±11% (p=0.008). Exercise capacity was also significantly reduced with AMI at 191±39 m and restored to 247±46 m after anakinra treatment (p=0.03). Conclusion: Ischemic heart failure due to large non-reperfused AMI leads to a severe reduction in cardiac contractility reserve and exercise capacity that are reversed by IL-1 blockade with anakinra.

The 5-HT1A receptor biased agonist, NLX-204, shows rapid-acting antidepressant-like properties and neurochemical changes in two mouse models of depression

Activation of cortical serotonin 5-HT1A receptors may be a promising strategy to achieve rapid-acting antidepressant (RAAD) activity. NLX-204 is a selective 5-HT1A receptor biased agonist that, in naïve mice, robustly decreases immobility in the forced swim test (FST), and preferentially phosphorylates extracellular signal-regulated kinase (ERK1/2), involved in antidepressant activity. Here, we evaluated the properties of NLX-204 in two mouse models of depression. Male CD-1 mice were subjected to unpredictable chronic mild stress (UCMS) for 4-weeks or to repeated corticosterone (CORT, 20 mg/kg s.c./day) for 3-weeks before receiving acute administration of NLX-204 (2 mg/kg, p.o.). Depressive-like behavior was assessed in the FST, anhedonia-like behavior in the sucrose preference test (SPT) and locomotor activity was also recorded. Phosphorylation of ERK1/2 (pERK1/2) and cAMP response binding element (pCREB) were measured ex vivo in hippocampus and prefrontal cortex (PFC). UCMS or CORT treatment increased immobility in the FST, elicited a sucrose preference deficit, and decreased pERK1/2 and pCREB levels in PFC and hippocampus. NLX-204 reduced depressive-like behavior in the FST in CORT and UCMS mice, and normalized sucrose preference in CORT mice, suggesting anti-anhedonic activity. NLX-204 increased pERK1/2 levels in PFC of UCMS mice. NLX-204 also increased pCREB levels in PFC of CORT mice. These data suggest that NLX-204 has RAAD-like properties not only in naïve mice, but also in mice in a “depressive-like” state, and that these involve changes in PFC and hippocampal pERK1/2 and pCREB levels. These data provide additional evidence that activation of 5-HT1A receptors by selective biased agonists, such as NLX-204, may constitute a promising RAAD strategy.

Montelukast reduces grey matter abnormalities and functional deficits in a mouse model of inflammation-induced encephalopathy of prematurity

Encephalopathy of prematurity (EoP) affects approximately 30% of infants born < 32 weeks gestation and is highly associated with inflammation in the foetus. Here we evaluated the efficacy of montelukast, a cysteinyl leukotriene receptor antagonist widely used to treat asthma in children, to ameliorate peripheral and central inflammation, and subsequent grey matter neuropathology and behaviour deficits in a mouse model of EoP. Male CD-1 mice were treated with intraperitoneal (i.p.) saline or interleukin-1beta (IL-1β, 40 μg/kg, 5 μL/g body weight) from postnatal day (P)1–5 ± concomitant montelukast (1–30 mg/kg). Saline or montelukast treatment was continued for a further 5 days post-injury. Assessment of systemic and central inflammation and short-term neuropathology was performed from 4 h following treatment through to P10. Behavioural testing, MRI and neuropathological assessments were made on a second cohort of animals from P36 to 54. Montelukast was found to attenuate both peripheral and central inflammation, reducing the expression of pro-inflammatory molecules (IL-1β, IL-6, TNF) in the brain. Inflammation induced a reduction in parvalbumin-positive interneuron density in the cortex, which was normalised with high-dose montelukast. The lowest effective dose, 3 mg/kg, was able to improve anxiety and spatial learning deficits in this model of inflammatory injury, and alterations in cortical mean diffusivity were not present in animals that received this dose of montelukast. Repurposed montelukast administered early after preterm birth may, therefore, improve grey matter development and outcome in EoP.

The acute effects of antimicrobials and lipopolysaccharide on the cellular mechanisms associated with neurodegeneration in pubertal male and female CD1 mice

Exposure to stressors during puberty can cause enduring effects on brain functioning and behaviours related to neurodegeneration. However, the mechanisms underlying these effects remain unclear. The gut microbiome is a complex and dynamic system that could serve as a possible mechanism through which early life stress may increase the predisposition to neurodegeneration. Therefore, the current study was designed to examine the acute effects of pubertal antimicrobial and lipopolysaccharide (LPS) treatments on the cellular mechanisms associated with neurodegenerative disorders in male and female mice. At five weeks of age, male and female CD-1 mice received 200 μL of broad-spectrum antimicrobials or water, through oral gavage, twice daily for seven days. Mice received an intraperitoneal (i.p.) injection of either saline or LPS at 6 weeks of age (i.e., pubertal period). Sickness behaviours were recorded and mice were euthanized 8 h post-injection. Following euthanasia, brains and blood samples were collected. The results indicated that puberal antimicrobial and LPS treatment induced sex-dependent changes in biomarkers related to sickness behaviour, peripheral inflammation, intestinal permeability, and neurodegeneration. The findings suggest that pubertal LPS and antimicrobial treatment may increase susceptibility to neurodegenerative diseases later in life, particularly in males.

The Effect of an Extract and Fractions of Date Pits on Some Plasma Constituents, Reproductive Hormones, and Testicular Histology in Male Mice.

Pits of dates (Phoenix dactylifera L.) have numerous nutritional benefits that could have wide-ranging applications. This study aimed to examine the effects of administering three extracts from powdered date pits on some basic physiological parameters, plasma constituents, reproductive hormones, and testicular histology in CD1 male mice. Three groups received doses of 100 mg/kg/day of lyophilized extract, a nonpolar fraction, and a polar fraction of date pits by oral gavage for 28 consecutive days. For the control, one group was administered a 1 mL/kg concentration of distilled water. The three different extracts significantly increased the plasma testosterone level but showed no significant effect on estradiol or luteinizing hormone levels, except for estradiol reduction in the polar extract group. The measured physiological or biochemical parameters or testicular histology also demonstrated no significant differences between the control mice and those mice treated with the three extracts, except for reductions in plasma urea in all extracts and in total protein in the nonpolar extract. Therefore, date pit extracts may potentially be used as a safe and effective dietary supplement. However, further investigation is needed.