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Neutrolide Neutrolide is a molecule designed to prevent or decrease the tissue damage caused by neutrophil infiltration after ischemia/reperfusion (I/R), which occurs as the result of stroke, coronary ischemia, trauma and other related conditions. Compounds called selectin blockers inhibit the effect of selectins and play an important role in this neutrophil-mediated injury. Furthermore, their simultaneous anti-inflammatory cytokine upregulation effect and Tumor Necrosis Factor inhibition ensures that several of inflammatory mediators are controlled during hypoxia. Drugs currently available for treatment have not resulted in optimal protective effects during clinical application. Preliminary studies in our laboratories already indicated that our recently discovered synthetic compounds , OC229, EC-234 and an improved OC229 (neutrolide) that incorporates the EC-234 nitroso group to the selectin blocker, provides protection against tissue damage in rats subjected to severe liver ischemia/reperfusion. Our results from the completed Phase II preclinical studies shows that neutrolide has a significantly protective effect when given at the time of reperfusion and at a dose of 25 mg/kg, for animals undergoing severe (90 min) liver ischemia. Functional and histological parameters remained near-normal in animals receiving neutrolide. Results of this innovative study could significantly improve the clinical outcome for patients at risk for suffering from ischemial/reperfusion injuries. Treatment with neutrolide will enhance the quality of life of millions of patients suffering from the large number of maladies associated with ischemia/reperfusion ischemic maladies, such as stroke, coronary infarction, peripheral vascular disease, and organ transplantation

Preliminary Data
Neutrolide provides protection against tissue damage in rats subjected to severe liver ischemia/reperfusion.
Our studies supported by SBIR Phase II award 1R43DK61910-03 evaluated Neutrolide for:

1. Drug Effectiveness.
2. Established best time of administration.
3. Conducted a comparative studies between selectin blockers drug effectiveness.
Supplement funding allowed us to conduct Non GLP basic toxicology studies to determine the feasibility of conducting GLP-toxicology and pharmacology studies for an IND authorization.

Best Dose of Administration

Best dose of administration for Neutrolide was 25 mg/kg. At the dose of 25 mg/kg a significant protection was noted in the group treated with Neutrolide. Histology showed to be in accordance with the best liver function tests, including ALT and AST serum levels that showed statistical differences with the control, the sham and the treated group when OC 229 was given at 25 mg/kg at the time of reperfusion (p-value = 0.0106, t = 2.92).

Neutrolide: Effects on Liver function, Histology after I/R and Modulation of Inflammatory molecular signal;ing pathways for NFkB and AP-1.

Mice subjected to 90 minutes of partial (70-80%) hepatic ischemia and three hours of reperfusion were divided into three groups (n=nine/group): Sham, Ischemic Control, and Treated group which received 25 mg/kg of the anti-selectin small molecule OC 229. These groups were studied when the treatment was given at the time of reperfusion (no pretreatment was given). The parameters measured at three hour of reperfusion included liver function tests (ALT and AST), liver histology and liver tissue electrophoretic mobility shift assay (EMSA) for NF?B and AP-1.
Results It was demonstrated that the multi-selectin inhibitor 229 offered significant protection for the ischemic liver when given at 25 mg/kg at the time of reperfusion. ALT and AST serum levels significantly decreased when the ischemic control and the group receiving OC 229 were compared (p=0.01). Treated animals demonstrated better histological findings as well. The EMSA showed dissociation of NF?B and AP-1 activity in the liver nuclear extracts after selectin inhibition treatment. A reduction on the activity of AP-1 and increment in NF-?B activation was seen.

We also measured HSP70 in the special knock-out mice developed by the University of Michigan. These animals lacked the gene of the fucosil transferase IV (FT4-null), fucosil transferase VII (FT7-null) and the combination of both deficiencies (FT4&7-null). The ligands for selectins are fucosilated molecules on the surface of the endothelium and leukocytes, so it makes sense that these animals could have a blocked selectin adhesion with potential protection from ischemic damage.
Using the same model of 90 minutes of partial (70-80%) hepatic ischemia and three hours of reperfusion were divided into five groups (n=6/group): Sham, Ischemic Control, and the Neutrolide treated with 25 mg/kg group that was studied at two times of administration: 15 minutes before reperfusion and at the time of reperfusion.

Effect of Neutrolide on tumor necrosis alpha (TNF- ) serum levels after I/R
Using the same model , we found a statistical significant inhibition in the serum expression of TNF-a in subjects treated with Neutrolide at the time of reperfusion

Neutrophil infiltration was studied through the measurement of myeloperoxidase (MPO) level in liver tissue homogenates by the method described by Bradley et al . An important increase was observed in MPO levels in the ischemic control group and a significant decreased of the activity of this enzyme in the Neutrolide treated animals when given at 25 mg/kg and at reperfusion only. The treated group not only had a decrease in the enzyme activity when compared with the I/R group, but also when compared to the sham group. The differences were statistically significant at alpha level of 0.05 .

Anti-Ischemic Resuscitation Fluid

Inflammation after ischemia and reperfusion (I/R) injury secondary to hemorrhagic shock is responsible for significant mortality and morbidity in the United States. Available intravenous fluids administered at the incident scene to treat hypovolemic shock do not completely prevent ischemia and reperfusion injury. Re-initiation of blood flow causes activation of several inflammatory mediators of the cytokine type, selectins and other molecules that rapidly mobilize the inflammatory response with subsequent global organ damage.
The goal of the proposed project is to prevent or reduce the deleterious effects of ischemic damage following resuscitation by optimizing the anti-inflammatory properties of the hypertonic saline (HS)(7.5%) resuscitation solution (recommended by "The Science of Fluid Resuscitation for Treating Combat Casualties and Civilian Injuries" Report ). The approach is based on the utilization of the anti-ischemic and anti-inflammatory anti-selectin compound Neutrolide developed by our laboratory, Anti Selectin blocker + nitroso pyrimidyl compund, which control adhesion molecules such as P-, L-, and E-selectins, 1, 2-, and 4 integrins, vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) . Our hypothesis is that this combination fluid will effectively regulate leukocyte recruitment and overcome the inflammatory and ischemic response by modulating undesirable molecular signaling pathways.

We propose to demonstrate, in a non-heparinized hybrid model of controlled-uncontrolled hemorrhagic shock resuscitation in the rat, an accepted model to study hypovolemic shock, that a small volume of 4 ml/kg of HS combined with anti-ischemic compound Neutrolide, will: 1) have a meaningful protective physiopathologic response to hemorrhage/hypovolemia by improving the end points of mean arterial pressure, arterial blood gases and liver function response, and 2) improve protection as determined by animal survival seen at 3-7 days after hemorrhagic shock. The study will also assess the response of the molecular pathways associated with hemorrhage and improved treatment.
A phase II study will further characterized the anti-inflammatory signaling mechanisms associated with the effect of the solution, and study tissue distribution, cytotoxicity, and evaluation of the physiological response to the optimal combination dose of the resuscitation fluid in a pig military-relevant model.

Neutrolide	Neutrolide is a molecule designed to prevent or decrease the tissue damage caused by neutrophil infiltration after ischemia/reperfusion (I/R), which occurs as the result of stroke, coronary ischemia, trauma and other related conditions. Compounds called selectin blockers inhibit the effect of selectins and play an important role in this neutrophil-mediated injury. Furthermore, their simultaneous anti-inflammatory cytokine upregulation effect and Tumor Necrosis Factor inhibition ensures that several of inflammatory mediators are controlled during hypoxia. Drugs currently available for treatment have not resulted in optimal protective effects during clinical application. Preliminary studies in our laboratories already indicated that our recently discovered synthetic compounds , OC229, EC-234 and an improved OC229 (neutrolide) that incorporates the EC-234 nitroso group to the selectin blocker, provides protection against tissue damage in rats subjected to severe liver ischemia/reperfusion. Our results from the completed Phase II preclinical studies shows that neutrolide has a significantly protective effect when given at the time of reperfusion and at a dose of 25 mg/kg, for animals undergoing severe (90 min) liver ischemia. Functional and histological parameters remained near-normal in animals receiving neutrolide. Results of this innovative study could significantly improve the clinical outcome for patients at risk for suffering from ischemial/reperfusion injuries. Treatment with neutrolide will enhance the quality of life of millions of patients suffering from the large number of maladies associated with ischemia/reperfusion ischemic maladies, such as stroke, coronary infarction, peripheral vascular disease, and organ transplantation
	Preliminary Data Neutrolide provides protection against tissue damage in rats subjected to severe liver ischemia/reperfusion. Our studies supported by SBIR Phase II award 1R43DK61910-03 evaluated Neutrolide for: 1. Drug Effectiveness. 2. Established best time of administration. 3. Conducted a comparative studies between selectin blockers drug effectiveness. Supplement funding allowed us to conduct Non GLP basic toxicology studies to determine the feasibility of conducting GLP-toxicology and pharmacology studies for an IND authorization. Best Dose of Administration Best dose of administration for Neutrolide was 25 mg/kg. At the dose of 25 mg/kg a significant protection was noted in the group treated with Neutrolide. Histology showed to be in accordance with the best liver function tests, including ALT and AST serum levels that showed statistical differences with the control, the sham and the treated group when OC 229 was given at 25 mg/kg at the time of reperfusion (p-value = 0.0106, t = 2.92). Neutrolide: Effects on Liver function, Histology after I/R and Modulation of Inflammatory molecular signal;ing pathways for NFkB and AP-1. Mice subjected to 90 minutes of partial (70-80%) hepatic ischemia and three hours of reperfusion were divided into three groups (n=nine/group): Sham, Ischemic Control, and Treated group which received 25 mg/kg of the anti-selectin small molecule OC 229. These groups were studied when the treatment was given at the time of reperfusion (no pretreatment was given). The parameters measured at three hour of reperfusion included liver function tests (ALT and AST), liver histology and liver tissue electrophoretic mobility shift assay (EMSA) for NF?B and AP-1. Results It was demonstrated that the multi-selectin inhibitor 229 offered significant protection for the ischemic liver when given at 25 mg/kg at the time of reperfusion. ALT and AST serum levels significantly decreased when the ischemic control and the group receiving OC 229 were compared (p=0.01). Treated animals demonstrated better histological findings as well. The EMSA showed dissociation of NF?B and AP-1 activity in the liver nuclear extracts after selectin inhibition treatment. A reduction on the activity of AP-1 and increment in NF-?B activation was seen. We also measured HSP70 in the special knock-out mice developed by the University of Michigan. These animals lacked the gene of the fucosil transferase IV (FT4-null), fucosil transferase VII (FT7-null) and the combination of both deficiencies (FT4&7-null). The ligands for selectins are fucosilated molecules on the surface of the endothelium and leukocytes, so it makes sense that these animals could have a blocked selectin adhesion with potential protection from ischemic damage. Using the same model of 90 minutes of partial (70-80%) hepatic ischemia and three hours of reperfusion were divided into five groups (n=6/group): Sham, Ischemic Control, and the Neutrolide treated with 25 mg/kg group that was studied at two times of administration: 15 minutes before reperfusion and at the time of reperfusion. Effect of Neutrolide on tumor necrosis alpha (TNF- ) serum levels after I/R Using the same model , we found a statistical significant inhibition in the serum expression of TNF-a in subjects treated with Neutrolide at the time of reperfusion Neutrophil infiltration was studied through the measurement of myeloperoxidase (MPO) level in liver tissue homogenates by the method described by Bradley et al . An important increase was observed in MPO levels in the ischemic control group and a significant decreased of the activity of this enzyme in the Neutrolide treated animals when given at 25 mg/kg and at reperfusion only. The treated group not only had a decrease in the enzyme activity when compared with the I/R group, but also when compared to the sham group. The differences were statistically significant at alpha level of 0.05 .
	Anti-Ischemic Resuscitation Fluid Inflammation after ischemia and reperfusion (I/R) injury secondary to hemorrhagic shock is responsible for significant mortality and morbidity in the United States. Available intravenous fluids administered at the incident scene to treat hypovolemic shock do not completely prevent ischemia and reperfusion injury. Re-initiation of blood flow causes activation of several inflammatory mediators of the cytokine type, selectins and other molecules that rapidly mobilize the inflammatory response with subsequent global organ damage. The goal of the proposed project is to prevent or reduce the deleterious effects of ischemic damage following resuscitation by optimizing the anti-inflammatory properties of the hypertonic saline (HS)(7.5%) resuscitation solution (recommended by "The Science of Fluid Resuscitation for Treating Combat Casualties and Civilian Injuries" Report ). The approach is based on the utilization of the anti-ischemic and anti-inflammatory anti-selectin compound Neutrolide developed by our laboratory, Anti Selectin blocker + nitroso pyrimidyl compund, which control adhesion molecules such as P-, L-, and E-selectins, 1, 2-, and 4 integrins, vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) . Our hypothesis is that this combination fluid will effectively regulate leukocyte recruitment and overcome the inflammatory and ischemic response by modulating undesirable molecular signaling pathways. We propose to demonstrate, in a non-heparinized hybrid model of controlled-uncontrolled hemorrhagic shock resuscitation in the rat, an accepted model to study hypovolemic shock, that a small volume of 4 ml/kg of HS combined with anti-ischemic compound Neutrolide, will: 1) have a meaningful protective physiopathologic response to hemorrhage/hypovolemia by improving the end points of mean arterial pressure, arterial blood gases and liver function response, and 2) improve protection as determined by animal survival seen at 3-7 days after hemorrhagic shock. The study will also assess the response of the molecular pathways associated with hemorrhage and improved treatment. A phase II study will further characterized the anti-inflammatory signaling mechanisms associated with the effect of the solution, and study tissue distribution, cytotoxicity, and evaluation of the physiological response to the optimal combination dose of the resuscitation fluid in a pig military-relevant model.