Histological and immunohistochemical study of the effect of ozone versus erythropoietin on induced skeletal muscle ischemia-reperfusion injury in adult male rats.

Ischemia reperfusion (IR) injury of skeletal muscles is a serious problem because of its local and systemic complications. Previous studies reported that ozone and erythropoietin could alleviate IR effect on several organs. The current research is established to evaluate the possible protective role of ozone versus erythropoietin following IR injury of the gastrocnemius muscle. Fifty rats were equally divided into five groups: I control, II ischemia reperfusion (IR), III post-reperfusion ozone treated, IV post-reperfusion erythropoietin-treated, and V recovering post-reperfusion without treatment groups. The right femoral arteries of all rats were clamped for three hours to induce ischemia then clamps were released to allow reperfusion for two hours. Rats of group II were scarified immediately after reperfusion period. Rats of group III were injected with ozone just after reperfusion for 14 days. Animals of group IV were injected with erythropoietin just after reperfusion for 14 days. Rats of group V rats were kept for 2 weeks following reperfusion without treatment. Blood samples were obtained to estimate lactate dehydrogenase (LDH) and creatine kinase (CK) enzymes. Gastrocnemius muscle was processed for measurement of tissue malondialdehyde (MDA), as well as examination by light and electron microscopes. iNOS and PCNA immunohistochemistry and statistical analysis were applied. The current results indicated that both ozone and erythropoietin could be used as protective agents reducing the muscular damage induced by IR injury.

Epidermal growth factor attenuates lingual papillae lesions in a rat model of sialoadenectomy.

Salivary epidermal growth factor (EGF) plays an important role in the maintenance of the oral and gastro-esophageal mucosa. Sialoadenectomy delays healing of oral wounds and affects lingual papillae. In this work, we aimed to determine the effect of EGF deficiency induced by sialoadenectomy and evaluate the effect of exogenous EGF administration on the lingual papillae and taste buds in rats. Thirty male adult Wistar albino rats were equally divided into 3 groups; sham-operated control group, sialoadenectomy group and group of sialoadenectomy + EGF. EGF was given 8 weeks after sialoadenectomy in a dose of 1 µg /ml/day in drinking water for 2 weeks. The anterior two-thirds of the tongue was dissected and cut longitudinally into two halves; one half for light microscope and the other for electron microscope examinations. Saliva and blood were collected to determine salivary and plasma EGF. Our results revealed that sialoadenectomy significantly reduced plasma and saliva levels of EGF which resulted in severe disruption of the architecture of lingual papillae. These changes were effectively improved by the exogenous EGF administration. In conclusion, EGF supplementation reversed the effects of sialoadenectomy and restored almost normal architecture of lingual papillae and taste buds.

Bone marrow-derived mesenchymal stem cells ameliorate liver injury in a rat model of sepsis by activating Nrf2 signaling.

Sepsis is a fatal condition that leads to serious systemic inflammation and multiple organ dysfunction syndromes. This study was designed to investigate the possible therapeutic effect of bone marrow-derived mesenchymal stem cells (BMSCs) on sepsis-induced liver injury. We also aimed to examine the role of Nrf2 activation in modulating the response to sepsis following BMSCs treatment. Twenty-four adult male albino rats were assigned to: control, lipopolysaccharide (LPS) and LPS-stem cell groups. Liver samples were processed for light and electron microscope examinations. Immunohistochemical localization of BAX, proliferating cell nuclear antigen and nuclear factor-erythroid 2-related factor 2 (Nrf2) was carried out. Liver homogenates were prepared for assessment of reduced glutathione, glutathione peroxidase, tumor necrosis factor-alpha and interleukin-6 and also real-time PCR analysis of Nrf2 expression. BMSCs treatment improved the histopathological changes of the liver, enhanced tissue regeneration and decreased apoptosis following sepsis. We reported highly significant enhancement in Nrf2 expressions at mRNA and protein levels in the LPS-stem cell group compared with the LPS group. The up regulation of Nrf2 was probably implicated in decreasing inflammatory cytokine levels and counteracting oxidative stress induced by sepsis. Thus, BMSCs therapies could be a viable approach to treat sepsis-induced liver damage by activating Nrf2 signaling.

Effect of Food on the Pharmacokinetics of Single- and Multiple-Dose Hydrocodone Extended Release in Healthy Subjects.

BACKGROUND AND OBJECTIVES: Food intake can alter the pharmacokinetics of certain medications, including changes in their oral bioavailability, which is of particular concern for extended-release (ER) opioids because of the high drug loads. Two randomized, open-label studies assessed the effect of food on the pharmacokinetics of single and multiple doses of hydrocodone ER formulated with CIMA® Abuse-Deterrence Technology. METHODS: Healthy subjects in fed and fasted states received single 90-mg doses of hydrocodone ER (Studies 1 and 2) or multiple doses of hydrocodone ER (45 mg twice daily on days 2-3, 60 mg twice daily on days 4-5, 90 mg twice daily on days 6-10, and 90 mg once in the morning on day 11) (Study 2). Naltrexone was administered to minimize opioid-related adverse events. Pharmacokinetic parameters included maximum hydrocodone plasma concentration (C max) and area under the concentration-versus-time curve from time 0 to infinity (AUC0-∞) in Study 1 (day 1) and for one dosing interval at steady state (AUCτ,ss) in Study 2 (day 11). Before conducting the multiple-dose study, single-dose data were fitted with a population pharmacokinetic methodology. RESULTS: In total, 40 subjects were randomized to Study 1 and 43 subjects were randomized to Study 2. While overall exposure (AUC0-∞) was relatively similar (least squares mean ratio [90% CI]: 1.11 [1.06-1.16]), results indicated that the single-dose C max was 40% higher under fed versus fasted conditions (least squares mean ratio [90% CI]: 1.40 [1.31-1.51]; Study 1). Modeling of single-dose data predicted that the effect of food would be much less at steady state [predicted fed:fasted C max at steady state (C max,ss) and AUCτ,ss ratios of 1.18 and 1.09, respectively]. The multiple-dose study results validated these predicted ratios and indicated that the steady-state 90% CIs were within 0.80-1.25 for the fed:fasted C max,ss (1.14 [1.07-1.21]) and AUCτ,ss (1.11 [1.04-1.17]) parameters, indicating that clinically meaningful food effects at steady state are not expected. CONCLUSION: No evidence of an effect of food was found on the pharmacokinetics of hydrocodone ER after multiple days of twice-daily dosing.

Effect of orally administered zinc oxide nanoparticles on albino rat thymus and spleen.

This study aimed to evaluate the toxicological effects of oral intake of Zinc oxide nanoparticles (ZnO NPs) on the structure of thymus and spleen. Twenty-four young male Wistar albino rats were assigned into two groups: group I (control) and group II (ZnO NPs treated group).The thymus and spleen were analyzed biochemically, histopathologically and immunohistochemically. After ZnO NPs intake, hematologically, the total leucocytic count was significantly increased while the RBCs and platelets counts and Hb % were significantly decreased. Biochemically, a significant decrease in serum total antioxidant capacity and anti-inflammatory cytokines including interleukin 4 and 10 (IL-4 and IL-10) levels was noted. While a significant increase in splenic and thymic malondialdehyde (MDA) and DNA shearing, as well as the studied proinflammatory cytokines; IL-1ß, tumor necrotic factor (TNF-α) and interferon (INF-γ) levels was detected. Notably, we noted upregulation of the immunomodulatory [CD3, CD11b, heme oxygenase (HO-1)] and the inflammatory [toll-like receptor 4 and 6 (TLR4 and TLR6)] genes. Histopathologically, degenerative changes were detected in thymus and spleen of ZnO NPs treated group. While the immunohistochemical analysis of the ZnO NPs treated group revealed a decrease in the number of cells expressed positive reactions of anti-PCNA and an increase in the number of cells expressed positive reaction of anti-p53 in the thymus and spleen. In conclusion, ZnO NPs induced obvious immunotoxicity in the thymus and spleen, where oxidative/inflammatory pathway may be the potential mechanism underlying this immunotoxicity. © 2017 IUBMB Life, 69(7):528-539, 2017.

Pharmacokinetics and Safety of Single-Dose Inhaled Loxapine in Children and Adolescents.

This multisite open-label study sought to characterize the pharmacokinetics and safety of a single dose of inhaled loxapine in children and adolescents. Loxapine powder for oral inhalation was administered via a single-use handheld drug device to children and adolescents (aged 10-17 years) with any condition warranting chronic antipsychotic use. Patients were dosed according to body weight and cohort (<50 kg [n = 15], 2.5 or 5 mg; ≥50 kg [n = 15], 5 or 10 mg); the first 6 patients (cohort 1) enrolled in each weight group received the lower dose. Patients were enrolled in the higher-dose group (cohort 2) after an interim pharmacokinetic and safety analysis of data from cohort 1. Blood samples were collected for 48 hours after dosing to determine the pharmacokinetic profile of loxapine and its metabolites. Safety was assessed using adverse event (AE), laboratory value, physical/neurologic examination, vital sign, electrocardiogram, suicidality, and extrapyramidal symptom assessment. Thirty patients were enrolled and evaluable for pharmacokinetics. Loxapine plasma concentrations peaked by 2 to 5 minutes in most patients; systemic exposure increased with dose in both weight subgroups. Loxapine terminal elimination half-life was ∼13 to 17 hours. The most common AEs were sedation and dysgeusia. Sedation was severe in 1 patient in the <50-kg group (2.5-mg dose) and 1 patient in the ≥50-kg group (5-mg dose). No AEs indicative of bronchospasm or other serious AEs were reported. Inhaled loxapine was rapidly absorbed and generally well tolerated in pediatric patients; no new safety signals were observed.

An experimental study of a rat model of emphysema induced by cigarette smoke exposure and the effect of Survanta therapy.

The present study was performed to test the therapeutic effects of Survanta (an exogenous surfactant) on a Wistar rat model of emphysema. Thirty-five adult male Wistar rats were divided randomly into the following groups; control subgroups Ia&b (n=14); emphysematous model subgroups IIa,b&c (n=21) exposed to cigarette smoke (CS), received phosphate buffer solution (PBS) and Survanta respectively. The levels of serum myeloperoxidase (MPO), lung tissue lactate dehydrogenase (LDH), alkaline phosphatase (ALP) as well as antioxidants: catalase (CAT), superoxide dismutase (SOD) and oxidative stress: malondialdehyde (MDA) markers were measured. Immunohistochemical staining of the lung was applied with anti-P53, anti- tumor necrosis factor (TNFα) and anti-proliferating cell nuclear antigen (PCNA) to reveal the changes of the lung structure. The mean linear intercepts (MLI) of alveoli were measured to assess alveolar size. In emphysematous rats, the serum level of MPO and tissue LDH, ALP & MDA were significantly increased while; CAT and SOD were significantly decreased. Pictures analysis for all immunostains was clearly increased. In Survanta treated group, a significant improvement in all previously mentioned findings while; no improvement in alveolar diameter was detected. These results conclusively demonstrate that Survanta administration improves the inflammatory biochemical and histochemical parameters of the emphysematous lung.

Clinical Trial Simulations and Pharmacometric Analysis in Pediatrics: Application to Inhaled Loxapine in Children and Adolescents.

BACKGROUND AND OBJECTIVES: Loxapine for inhalation is a drug-device combination product approved in adults for the acute treatment of agitation associated with schizophrenia or bipolar I disorder. The primary objective of this study was to develop a clinical trial protocol to support a phase I pharmacokinetic study in children aged 10 years and older. In addition, this report details the results of the clinical study in relation to the predicted likelihood of achieving the target exposure associated with therapeutic effect in adults. METHODS: A nonlinear mixed-effects population pharmacokinetic model was developed using adult data and was adjusted for the targeted pediatric age groups by applying allometric scaling to account for body size effects. Based on this pediatric model, age-appropriate regimens to achieve loxapine exposures similar to the ones associated with therapeutic effect in the adult studies were identified via trial simulation. D-optimal design and power analysis were conducted to identify optimal pharmacokinetic sampling times and sample size, respectively. RESULTS: The developed clinical trial design formed the basis of a phase I study to assess the safety and pharmacokinetics of loxapine for inhalation in children aged 10 years and older (ClinicalTrials.gov ID: NCT02184767). CONCLUSION: The results of the study indicated that overall loxapine exposures were consistent with what had been predicted by the trial simulations. The presented approach illustrates how modeling and simulation can assist in the design of informative clinical trials to identify safe and effective doses and dose ranges in children and adolescents.

Therapeutic effect of mesenchymal stem cells on experimentally induced hypertensive cardiomyopathy in adult albino rats.

Hypertensive heart diseases affect millions of people worldwide. We aimed to investigate the hypertensive left ventricular histological changes and assess the effectiveness of bone marrow derived mesenchymal stem cells (MSCs) therapy in the treatment of hypertensive cardiomyopathy. Adult male albino rats were assigned into two groups: group I (control), group II (Experimental) subdivided into subgroup IIa (hypertensive) and subgroup IIb (stem cell therapy). Left ventricles (LVs) were processed for light and electron microscope. Mallory's trichrome and immunostaining for caspase-3 and desmin were carried out. Hypertension caused left ventricular histological and immunohistochemical changes that had been effectively improved by MSCs therapy.

Therapeutic effect of spermatogonial stem cell on testicular damage caused by lead in rats.

OBJECTIVE: To investigate the possible therapeutic effect of spermatogonial stem cells (SSCs) on lead-induced apoptosis and consequently infertility in adult male rats. MATERIALS AND METHODS: Sixty-six Sprague Dawley adult male rats were divided into three groups: control group, lead (Pb) acetate exposed group received (20mg Pb/kg) for 3weeks, and SSCs treated group. Each group included twenty-two rats. Serum testosterone level, 3 beta-hydroxysteroid dehydrogenase (3ß-HSD), 17 beta-hydroxysteroid dehydrogenase (17ß-HSD), proliferating cell nuclear antigen (PCNA) genes expression by RT-PCR, caspase 3 expression by immunohistochemistry and testicular histological findings were tested. RESULTS: Pb acetate exposed rats showed a significant decrease in the epididymal sperm count, motility, viability, serum testosterone level and testicular expression of 3ß-HSD, 17ß-HSD and PCNA compared to the control group, while treatment with SSCs attenuated Pb acetate induced decrease for these variables. Moreover, the increasing apoptosis of germinal cells as well as the high expression of caspase-3 induced by Pb acetate was reduced by SSCs treatment. CONCLUSION: SSCs exhibited therapeutic effect on reproductive system by inhibiting Pb-induced excessive cell apoptosis.

Neuroprotective effects of placenta-derived mesenchymal stromal cells in a rat model of experimental autoimmune encephalomyelitis.

BACKGROUND: Current therapies for multiple sclerosis (MS) are largely palliative, not curative. Mesenchymal stromal cells (MSCs) harbor regenerative and immunosuppressive functions, indicating a potential therapy for MS. A preparation of MSCs derived from full-term human placenta (PDMSCs) is a new approach in the treatment of patients with MS. OBJECTIVE: This study aimed to rule out the possible therapy by PDMSCs in experimental autoimmune encephalomyelitis (EAE), a rat model of MS. METHODS AND RESULTS: Thirty-five female Wistar rats were classified into the following groups: I, control; II, EAE untreated; III and IV, EAE treated with phosphate-buffered saline (PBS) at 9 and 16 days post-immunization (dpi), respectively; V and VI, EAE treated with PDMSCs at 9 and 16 dpi, respectively. Intravenous administration of PDMSCs at 9 or 16 dpi significantly ameliorated the disease course, decreasing brain inflammation and degenerating neurons. A reduction of axonal damage as well as an increase of oligodendrocyte precursors were recorded. Moreover, there was an engraftment of the PDMSCs into the brain tissue. Human brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin 3 (NTF3) were significantly expressed in brains of rats treated by PDMSCs. CONCLUSIONS: Human PDMSCs have demonstrated striking therapeutic effects when delivered at the onset or at the peak of the disease. PDMSCs have direct neurotrophic support after their engraftment within the lesion through expression of the neurotrophins.

Mesenchymal stromal cell injection protects against oxidative stress in Escherichia coli-induced acute lung injury in mice.

BACKGROUND AIMS: Stem cells may be a promising therapy for acute respiratory distress syndrome. Recent in vivo and in vitro studies suggested that the mesenchymal stromal cells (MSCs) have anti-oxidative stress properties. We hypothesized that intravenous injection of bone marrow-derived mesenchymal stem cells (MSCs) could attenuate Escherichia coli-induced acute lung injury (ALI) in mice by controlling the oxidative stress status. METHODS: Eighty mice were randomly divided into four groups: group 1 (control group) received 25 µL of saline as a vehicle; group 2 contained E coli-induced ALI mice; group 3 included mice that received MSCs before induction of ALI; group 4 included mice that received MSCs after induction of ALI. Lung samples were isolated and assayed for oxidative stress variables and histopathologic analysis. Total anti-oxidant capacity was measured in broncho-alveolar lavage. RESULTS: Pre- and post-injury MSC injection increased survival, reduced pulmonary edema and attenuated lung injuries in ALI mice. Histologically, MSCs exhibited a considerable degree of preservation of the pulmonary alveolar architecture. An increase of anti-oxidant enzyme activities and a decrease of myeloperoxidase activity and malondialdehyde levels in the MSC recipient groups versus the ALI group were found. Furthermore, the total anti-oxidant capacity and reduced glutathione levels were significantly increased in MSCs recipient groups versus the ALI group. Weak +ve inducible nitric oxide synthase immuno-expression in groups that received MSCs was detected. Pre-injury MSC injection showed better effects than did post-injury MSC injection. CONCLUSIONS: Systemic bone marrow-derived MSC injection was effective in modulating the oxidative stress status in E coli-induced acute lung injury in mice.

Differential effects of eugenol against hepatic inflammation and overall damage induced by ischemia/re-perfusion injury.

Liver injuries, liver tumor resection, and liver transplantation are known to be responsible for ischemia/reperfusion (I/R) injury that, in turn, gives rise to liver damage. This study was undertaken to investigate the possible protective effect of eugenol against the damage induced by I/R in rat livers as well as to explore possible mechanisms of action. Male rats were divided into four groups: sham-operated, I/R only, and two groups that received 10 or 100 mg eugenol/kg/day (Eug10 and Eug100, respectively) for 15 days by gavage and were then subjected to I/R, i.e. an ischemia induced for 45 min followed by re-perfusion for 6 h. The rats were euthanized and liver tissues and blood collected for examination. The results showed that I/R induced massive hepatic structural and functional damage. Eug10-treated rats had improvement in both liver function and structure, and inhibition of I/R-induced increases in serum myeloperoxidase (MPO), tumor necrosis factor (TNF)-α, as well as hepatic nuclear factor-κB (NF-κB) p65 and caspase-3 expression. Eug10 treatment also inhibited the degree of loss in reduced glutathione (GSH) and of rise in malondialdehyde (MDA) levels in liver tissues induced by I/R. In contrast, augmentation of liver damage induced by I/R was noted in Eug100-treated rats, with these hosts displaying significant increases in oxidant, inflammatory, and apoptotic markers relative to levels seen in I/R-only rats. The results of the present study provide the first evidence that a low dose of eugenol may protect the liver against I/R injury in part by decreasing levels of lipid peroxidation, down-regulating inflammatory mediators, and inhibiting apoptosis, and that a larger dose amplifies the liver injury via oxidant and inflammatory effects.

Hit to lead studies on (hetero)arylpyrimidines--agonists of the canonical Wnt-beta-catenin cellular messaging system.

A series of (hetero)arylpyrimidines agonists of the Wnt-beta-catenin cellular messaging system have been prepared. These compounds show activity in U2OS cells transfected with Wnt-3a, TCF-luciferase, Dkk-1 and tk-Renilla. Selected compounds show minimal GSK-3beta inhibition indicating that the Wnt-beta-catenin agonism activity most likely comes from interaction at Wnt-3a/Dkk-1. Two examples 1 and 25 show in vivo osteogenic activity in a mouse calvaria model. One example 1 is shown to activate non-phosphorylated beta-catenin formation in bone.

(1-(4-(Naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine: a wingless beta-catenin agonist that increases bone formation rate.

A high-throughput screening campaign to discover small molecule leads for the treatment of bone disorders concluded with the discovery of a compound with a 2-aminopyrimidine template that targeted the Wnt beta-catenin cellular messaging system. Hit-to-lead in vitro optimization for target activity and molecular properties led to the discovery of (1-(4-(naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine (5, WAY-262611). Compound 5 has excellent pharmacokinetic properties and showed a dose dependent increase in the trabecular bone formation rate in ovariectomized rats following oral administration.