Safety and toxicology assessment of sodium nitrite administered by intramuscular injection.

Intramuscular (IM) injection of nitrite (1-10 mg/kg) confers survival benefit and protects against lung injury after exposure to chlorine gas in preclinical models. Herein, we evaluated safety/toxicity parameters after single, and repeated (once daily for 7 days) IM injection of nitrite in male and female Sprague Dawley rats and Beagle dogs. The repeat dose studies were performed in compliance with the Federal Drug Administration's (FDA) Good Laboratory Practices Code of Federal Regulations (21 CFR Part 58). Parameters evaluated consisted of survival, clinical observations, body weights, clinical pathology, plasma drug levels, methemoglobin and macroscopic and microscopic pathology. In rats and dogs, single doses of ≥100 mg/kg and 60 mg/kg resulted in death and moribundity, while repeated administration of ≤30 or ≤ 10 mg/kg/day, respectively, was well tolerated. Therefore, the maximum tolerated dose following repeated administration in rats and dogs were determined to be 30 mg/kg/day and 10 mg/kg/day, respectively. Effects at doses below the maximum tolerated dose (MTD) were limited to emesis (in dogs only) and methemoglobinemia (in both species) with clinical signs (e.g. blue discoloration of lips) being dose-dependent, transient and reversible. These signs were not considered adverse, therefore the No Observed Adverse Effect Level (NOAEL) for both rats and dogs was 10 mg/kg/day in males (highest dose tested for dogs), and 3 mg/kg/day in females. Toxicokinetic assessment of plasma nitrite showed no difference between male and females, with Cmax occurring between 5 mins and 0.5 h (rats) or 0.25 h (dogs). In summary, IM nitrite was well tolerated in rats and dogs at doses previously shown to confer protection against chlorine gas toxicity.

Assessment of ICAM-1 N-glycoforms in mouse and human models of endothelial dysfunction.

Endothelial dysfunction is a critical event in vascular inflammation characterized, in part, by elevated surface expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is heavily N-glycosylated, and like other surface proteins, it is largely presumed that fully processed, complex N-glycoforms are dominant. However, our recent studies suggest that hypoglycosylated or high mannose (HM)-ICAM-1 N-glycoforms are also expressed on the cell surface during endothelial dysfunction, and have higher affinity for monocyte adhesion and regulate outside-in endothelial signaling by different mechanisms. Whether different ICAM-1 N-glycoforms are expressed in vivo during disease is unknown. In this study, using the proximity ligation assay, we assessed the relative formation of high mannose, hybrid and complex α-2,6-sialyated N-glycoforms of ICAM-1 in human and mouse models of atherosclerosis, as well as in arteriovenous fistulas (AVF) of patients on hemodialysis. Our data demonstrates that ICAM-1 harboring HM or hybrid epitopes as well as ICAM-1 bearing α-2,6-sialylated epitopes are present in human and mouse atherosclerotic lesions. Further, HM-ICAM-1 positively associated with increased macrophage burden in lesions as assessed by CD68 staining, whereas α-2,6-sialylated ICAM-1 did not. Finally, both HM and α-2,6-sialylated ICAM-1 N-glycoforms were present in hemodialysis patients who had AVF maturation failure compared to successful AVF maturation. Collectively, these data provide evidence that HM- ICAM-1 N-glycoforms are present in vivo, and at levels similar to complex α-2,6-sialylated ICAM-1 underscoring the need to better understand their roles in modulating vascular inflammation.

A randomized clinical trial testing the anti-inflammatory effects of preemptive inhaled nitric oxide in human liver transplantation.

Decreases in endothelial nitric oxide synthase derived nitric oxide (NO) production during liver transplantation promotes injury. We hypothesized that preemptive inhaled NO (iNO) would improve allograft function (primary) and reduce complications post-transplantation (secondary). Patients at two university centers (Center A and B) were randomized to receive placebo (n = 20/center) or iNO (80 ppm, n = 20/center) during the operative phase of liver transplantation. Data were analyzed at set intervals for up to 9-months post-transplantation and compared between groups. Patient characteristics and outcomes were examined with the Mann-Whitney U test, Student t-test, logistic regression, repeated measures ANOVA, and Cox proportional hazards models. Combined and site stratified analyses were performed. MELD scores were significantly higher at Center B (22.5 vs. 19.5, p<0.0001), surgical times were greater at Center B (7.7 vs. 4.5 hrs, p<0.001) and warm ischemia times were greater at Center B (95.4 vs. 69.7 min, p<0.0001). No adverse metabolic or hematologic effects from iNO occurred. iNO enhanced allograft function indexed by liver function tests (Center B, p<0.05; and p<0.03 for ALT with center data combined) and reduced complications at 9-months (Center A and B, p = 0.0062, OR = 0.15, 95% CI (0.04, 0.59)). ICU (p = 0.47) and hospital length of stay (p = 0.49) were not decreased. iNO increased concentrations of nitrate (p<0.001), nitrite (p<0.001) and nitrosylhemoglobin (p<0.001), with nitrite being postulated as a protective mechanism. Mean costs of iNO were $1,020 per transplant. iNO was safe and improved allograft function at one center and trended toward improving allograft function at the other. ClinicalTrials.gov with registry number 00582010 and the following URL:http://clinicaltrials.gov/show/NCT00582010.

Ethosomes and ultradeformable liposomes for transdermal delivery of clotrimazole: A comparative assessment.

THE OBJECTIVE OF WORK WAS TO FORMULATE, EVALUATE AND COMPARE THE TRANSDERMAL POTENTIAL OF NOVEL VESICULAR NANOCARRIERS: ethosomes and ultradeformable liposomes, containing clotrimazole (CLT), an anti-fungal bioactive. The ethosomal formulation (ET4) and ultradeformable liposomal (UL) formulation (TT3) showed highest entrapment 68.73 ± 1.4% and 55.51 ± 1.7%, optimal nanometric size range 132 ± 9.5 nm and 121 ± 9.7 nm, and smallest polydispersity index 0.027 ± 0.011 and 0.067 ± 0.009, respectively. The formulation ET4 provided enhanced transdermal flux 56.25 ± 5.49 µg/cm(2)/h and decreased the lag time of 0.9 h in comparison to TT3 formulation (50.16 ± 3.84 µg/cm(2)/h; 1.0 h). Skin interaction and FT-IR studies revealed greater penetration enhancing effect of ET4 than TT3 formulation. ET4 formulation also had the highest zone of inhibition (34.6 ± 0.57 mm), in contrast to TT3 formulation (29.6 ± 0.57 mm) and marketed cream formulation (19.0 ± 1.00 mm) against candidal species. Results suggested ethosomes to be the most proficient carrier system for dermal and transdermal delivery of clotrimazole.