Lazaroid U-74389G Research Articles

Effect of lazaroid U-74389G and Sildenafil, alone or in combination, on pulmonary function during ischamia-reperfusion injury of myocardium: An exploratory study

Background: This experimental study was designed to evaluate the effects of the pharmaceutical substances U-74389G (Lazaroid) and Sildenafil, administered separately or in combination, during anaesthesia in relation to pulmonary function, in a myocardial ischaemia-reperfusion procedure. Methods: We sought to determine the effect of sildenafil and lazaroid U-74389G maleate, alone or in combination, on systemic and pulmonary tissue inflammation and oxidative stress in a pig model of myocardial ischaemia-reperfusion injury. A total of 56 pigs were divided into seven groups according to the use of sildenafil, lazaroid, or their combination, and the time of administration (prior to the induction of myocardial ischaemia or after reperfusion). Results: Both sildenafil and lazaroid U-74389G maleate, administered either 10 minutes before the induction of myocardial ischaemia or immediately after the start of reperfusion, resulted in a significant effect on several markers of systemic inflammation (p<0.05). In addition, a significant reduction in lung tissue expression of tumour necrosis factor-alpha (TNF-a) (p<0.05) was observed with sildenafil, lazaroid U-74389G maleate, and their combination, when administered either before the induction of myocardial ischaemia or after the induction of reperfusion. However, no effect on tissue oxidative stress was demonstrated. Conclusion: Sildenafil, lazaroid U-74389G maleate, and their combination led to a significant amelioration of both systemic and pulmonary tissue-specific inflammatory responses. Notably, they did not exert any significant effect against tissue oxidative stress in this model. Human mechanistic studies are required to confirm these observations.

Abstract 13235: Ischemic Preconditioning and Lazaroid (u-74389g) Administration Similarly Attenuate Ischemia-Reperfusion-Induced Spinal Cord Inflammatory and Apoptotic Responses by Targeting the S100-Receptor for Advanced Glycation End Product Pathway

Introduction: Spinal cord injury and subsequent paraplegia represents a devastating complication of thoracoabdominal aortic surgery. Pro-inflammatory cytokine expression and spinal cord neuronal apoptosis have been implicated in inflammation and neurodegeneration respectively after ischemia. Hypothesis: We investigated the neuroprotective effects of the steroid antioxidant Lazaroid U-74389G versus conventional ischemic preconditioning (IPC) in a pig model of spinal cord ischemia-reperfusion injury by examining the expression of pro-inflammatory and pro-apoptotic S100 members and the receptor for advanced glycation end products (RAGE). Methods: Spinal cord ischemia was induced by thoracoabdominal aortic occlusion (TAO). A total of 27 pigs were randomized in four groups. Group I (n=5) sham operation, group II (n=7) TAO for 45min and received placebo, group III (n=8) received 3 doses of Lazaroid (3 mg/kg) 60min and 30min before and at 30min during TAO (duration 45min), and group IV (n=6) TAO for 20min followed by additional TAO for 45min and 80 minutes later. Neurologic evaluation was performed according to Tarlov score (0-4, 4=normal). The lumbar spinal cords were harvested at 7th postoperative day for the expression of inflammatory and apoptotic markers by real-time RT-PCR. Results: Group III and IV had better neurologic outcome postoperatively compared to group II where all animals developed paraplegia (Tarlov scores: 3.5 versus 3.6 versus 0.33, respectively, P<0.001). Group II showed marked pro-inflammatory responses as quantified by 2-4-fold increases in the mRNA of pro-inflammatory S100A6, S100A8 and high-mobility group box 1, and RAGE, compared to group I. Additionally, group II, exhibited apoptosis as quantified by a 40-60% decrease in the mRNA of anti-apoptotic BCL2 and BIRC2, and 2-3-fold increases in pro-apoptotic BAX mRNA and caspase 3 activity. Compared to group II, inflammatory and apoptotic responses were highly attenuated albeit to a similar degree in groups III and IV. Conclusions: In a large animal model very close to the clinical setting, we demonstrated that in addition to IPC, Lazaroid represents an alternative yet effective pharmacologic intervention in reducing spinal cord ischemia-reperfusion injury following TAO.

Lazaroid U-74389G Decreases Testis Tissue Injury Induced by Testicular Torsion Detorsion: An Experimental Study

Lazaroid U-74389G Decreases Testis Tissue Injury Induced by Testicular Torsion Detorsion: An Experimental Study

The Rat Uterus after U-74389G Process

Aim: This study co-evaluated the 4 quoted histologic variables after the lazaroid U-74389G (L) drug administration. The calculation was based on the results of 2 preliminary studies, each one evaluating two respective histologic variables of Endometrial Edema (EE) and Uterus Inflammation (UI) or Endometrial Karyorrhexis (EK) and Uterus Congestion (UC); in an induced ischemia reperfusion animal experiment. Materials and methods: The 2 main experimental endpoints at which the EE, UI and EK, UC scores were evaluated was the 60th reperfusion min (for the groups A and C) and the 120th reperfusion min (for the groups B and D). Specially, the groups A and B were processed without drugs, whereas the groups C and D after L administration. Results: The first preliminary study showed that L has a non-significant recessing potency for EE and UI histologic parameters at the “without lesions” grade 0.2636364±0.14594051 (p-values=0.0698). The second preliminary study showed that L has a non-significant recessing potency for EK and UC histologic parameters at the “without lesions” grade 0.1253529 ± 0.08529668 (p-values=0.1373) since they were co-evaluated together. These 2 studies were co-evaluated since they came from the same experimental setting. This study co-evaluated the combined diagnostic values of the four variables together. Conclusion: L administration and reperfusion time together non-significantly accentuated the 4 histologic variables within the “without lesions alterations” score 0.0758471 [-0.1464624 - +0.2981566] (p-value=0.4940).

Lazaroid U-74389G in liver ischemia-reperfusion injury: A swine model.

Reactive oxygen species have a key role in liver ischemia-reperfusion (I/R) injury. In the present study, the effect of the anti-oxidant compound lazaroid U-74389G in preventing liver I/R injury was investigated in a swine model. Ischemia was produced by portal vein occlusion. Two sets of experiments were performed, each with two groups (n=7 per group). In the first group, the potential protective effect of an intracaval injection of U-74389G after a 30-min ischemia, followed by a 60-min reperfusion period was assessed (biopsies at 0, 15, 30 and 90 min experimental time). In the second set, the effect of intracaval U-74389G injection after 30 min of ischemia, followed by a longer reperfusion period of 120 min was determined (biopsies at 0, 15, 30 and 150 min experimental time). Liver malondialdehyde, hepatocyte vacuolation-degeneration, venous congestion, inflammatory cell infiltration, sinus congestion-dilation and Chiu score of intestinal damage were determined at up to 150 min of reperfusion. In the second set of experiments, the Chiu score of intestinal damage was improved by the administration of U-74389G (3.17±0.40 vs. 4.33±0.21; P=0.030). However, in the two sets of experiments, the liver inflammatory reaction was more pronounced in the U-74389G groups (P=0.017 for the first set, P=0.021 for the second set). No significant effect of U-74389G on any other parameters was detected. In conclusion, intestinal damage due to portal venous congestion and reflow appears to be mitigated by the lazaroid U-74389G; however, intracaval administration of U-74389G does not appear to exert any protective effects against liver I/R-induced inflammation.

Lazaroid (U-74389G) ameliorates lung injury due to lipid peroxidation and nitric oxide synthase-dependent reactive oxygen species generation caused by remote systematic ischemia-reperfusion following thoracoabdominal aortic occlusion

IntroductionLung ischemia-reperfusion injury after thoracoabdominal aortic occlusion represents a major complication, which increases morbidity and mortality. In the present study we hypothesized that lazaroid U-74389G intravenous administration protects from lung ischemia-reperfusion injury through lipid peroxidation inhibition. Materials and methodsA total of 24 pigs were randomized in three groups. Group I (n = 8) underwent sham operation, group II (n = 8) underwent thoracoabdominal aortic occlusion for 45min and received placebo and group III (n = 8) received 3 doses of lazaroid (3 mg/kg) 60 and 30min before thoracoabdominal aortic occlusion and at 30min during thoracoabdominal aortic occlusion (duration 45min). Aortic occlusion was performed with aortic balloon-catheters under fluoroscopic guidance. All animals were sacrificed at the 7 t h postoperative day and lung specimens were harvested for molecular analysis. ResultsmRNA levels of leukotrienes LB4 (LTB4R2), LC4 (LTC4S) and nitric oxide synthase (NOS) isoforms including iNOS, nNOS and eNOS were determined with real-time RT-qPCR. Nitric oxide can either induce (iNOS) or inhibit (nNOS and eNOS) lipid peroxidation based on its specific isoform origin. Group III showed significantly reduced mRNA levels of LTB4R2 (−63.7%), LTC4S (−35.9%) and iNOS (−60.2%) when compared with group II (P < 0.05, for all). The mRNA levels of nNOS was significantly increased (+37.4%), while eNOS was slightly increased (+2.1%) in group III when compared with group II (P < 0.05 and P = 0.467 respectively). ConclusionLazaroid U-74389G may represent an effective pharmacologic intervention in reducing lung ischemia-reperfusion injury following thoracoabdominal aortic occlusion.

Experimental Hemorrhagic Shock Protocol in Swine Models: The Effects of 21-Aminosteroid on the Small Intestine

BackgroundThe protective potential of lazaroids has been reported in previous studies on ischemia/reperfusion and induced hemorrhagic shock protocols. ObjectivesThe present study is the first experimental protocol on the effects of the antioxidant factor U-74389G on the small intestine of swine models in a hemorrhagic shock protocol and resuscitation with 3 different types of fluids. MethodsThe study included 49 Landrace breed swine that were divided into groups of 7 each. Hemorrhage was provoked 45 minutes after starting the surgical protocol (0 minutes), followed by resuscitation starting 30 minutes after haemorrhage ceased by using 3 different fluids. Three groups (Group A, resuscitation using blood; Group B, resuscitation with Ringer’s lactate solution; and Group C, resuscitation with hypertonic saline solution) underwent resuscitation with fluid alone, and another 3 groups (named A', B,' and C') were administered lazaroid U-74389G in addition to fluid. Control Group S underwent all the surgical procedures without hemorrhagic shock. Vital signs, complete blood count, and biochemical markers were analyzed, and tissue samples of the small intestine were collected from all animals. Further, malondialdehyde, tumor necrosis factor-α, and levels of inflammation in the tissue sample were measured. ResultsIn Group S-A-A' and Group S-C-C', the analysis did not show statistically significant differences in the percentage changes of histopathology, malondialdehyde, and tumor necrosis factor-α through time. In Group S-B-B', the malondialdehyde levels in the small intestine were reduced in both the B and B' groups, without lazaroid (Group B) (P = 0.038) and lazaroid (Group B') (P = 0.011), compared with Group S (control), but the group without lazaroid (Group B) had greater reduction in malondialdehyde levels than the group treated with lazaroid (Group B'). With regard to the biochemistry results, 24% reduction was observed for alkaline phosphatase (P = 0.022) in Group A' treated with lazaroid compared with that in the untreated group. Lastly, for the complete blood count parameters, a 14% reduction in white blood cells was observed in Group B', which was treated with lazaroid in all phases (P = 0.015) (absolute value = 6.23) compared with Group B (absolute value = 13.74). ConclusionsDespite few initial findings of this study suggesting that administration of lazaroid U-74389G may have some potential in attenuation of the effects of hemorrhagic shock in the small intestine of swine models, no differences remained after correction for multiple comparisons was made. Therefore, further research is required to investigate this result thoroughly.

Lazaroid U-74389G for cardioplegia-related ischemia–reperfusion injury: an experimental study

BackgroundThe adverse effects of myocardial ischemia and reperfusion during cardiopulmonary bypass (CPB) have been thoroughly described. Lazaroid U-74389G, a 21 aminosteroid, has been shown to attenuate ischemia and reperfusion injury and improve recovery in a variety of experimental models. MethodsSixteen male swine were randomly divided in two groups. All animals underwent 45 min of ischemic cardioplegic arrest, with U-74389G addition to the standard cardioplegic solution, whereas controls underwent the same procedure without U-74389G. Creatine kinase-MB isoenzyme (CK-MB) and cardiac troponin T levels were measured immediately before CPB (time point 0), during the ischemic period (time point 1) and 30 (time point 2), 60 (time point 3), and 120 (time point 4) min after reperfusion. Myocardial biopsies were obtained at time points 0 and 4. ResultsCK-MB levels (in U/L) at time points 0-4 were 205 (186-235) versus 219 (196-269; P = 0.72), 215 (167-248) versus 253 (193-339; P = 0.23), 234 (198-255) versus 338 (249-441; P = 0.02), 244 (217-272) versus 354 (269-496; P = 0.01), and 285 (230-321) versus 439 (432-530; P < 0.01) in lazaroid-treated animals versus controls, respectively. Cardiac troponin T levels (in ng/L) at time points 0-4 were 58 (26-287) versus 237 (26-395; P = 0.72), 129 (61-405) versus 265 (145-525; P = 0.23), 261 (123-467) versus 474 (427-1604; P = 0.04), 417 (204-750) versus 841 (584-1818; P = 0.11), and 643 (353-1259) versus 1600 (1378-2313; P < 0.01), respectively. Necrosis grades at time point 4 were 0.0 (0.0-1.0) versus 1.5 (1.0-2.0; P < 0.01) in lazaroid-treated animals versus controls, respectively. ConclusionsThe present study, in addition to reconfirming the well-described adverse effects of CPB, demonstrates the efficacy of the newer generation lazaroid U-74389G in alleviating these effects.

Lazaroid U-74389G inhibits the osteoblastic differentiation of IL-1β-indcued aortic valve interstitial cells through glucocorticoid receptor and inhibition of NF-κB pathway

BackgroundAortic valve calcification is characterized as the active process of aortic valve interstitial cells (AVICs), and considered as an inflammatory disease. As an antioxidant, the anti-inflammatory activity of Lazaroid has been exhibited in various models. We hypothesized that Lazaroid U-74389G would inhibit the osteoblastic differentiation of AVICs induced by IL-1β. MethodsNormal tricuspid aortic valve leaflets were collected from patients with acute aortic dissection (Type A) undergoing the Bentall procedure. AVICs were isolated and stimulated with IL-1β in presence or absence of U-74389G in culture. Cell lysates were analyzed for osteogenic markers and nuclear factor-κB using real-time PCR and Immunoblotting. Culture media was analyzed for IL-6 and IL-8 with enzyme-linked immunosorbent assay. Alizarin Red Staining was adopted to demonstrate the calcium deposition. ResultsThe expression of alkaline phosphatase and bone morphogenetic protein, accompanied by the production of IL-6 and IL-8, was up-regulated in response to IL-1β and was inhibited by the addition of U-74389G. The NF-κB pathway was activated by IL-1β and involved in the suppression of U-74389G on osteoblastic differentiation in AVICs. The negative effects of U-74389G on ostengenic gene expression and mineralization of AVICs were blocked by glucocorticoid receptor antagonist mifepristone and the NF-κB inhibitor Bay 11-7082. ConclusionsU-74389G inhibits the pro-osteogenic response to IL-1β stimulation in AVICs. The osteoblastic differentiation and mineralization of AVICs were inhabited by U-74389G though the modulation of NF-κB activation, and this pathway could be potential therapeutic targets for medical treatment of calcified aortic valve disease.

Effectiveness of sildenafil and U-74389G in a rat model of colitis

Crohn’s disease is still incurable. Compounds with anti-inflammatory and/or antioxidative effects are tested in various preclinical models of the disease. Our aim was to investigate the effects of sildenafil and lazaroid U-74389G on an experimental rat model with trinitrobenzenesulfonic acide-induced colitis. Trinitrobenzenesulfonic acid was instilled into the colon of all male Wistar rats except for those belonging to the first group. For some days, the animals in group 3 received daily sildenafil orally; the rats in group 4 received daily U-74389G intravenously, and the rats in group 5 were co-administered daily sildenafil orally and intravenous U-74389G. The rats in groups 1 and 2 were not given any treatment. During the study, the weight of the rats was recorded as a marker of their clinical condition. The colon damage was evaluated using the macroscopic colonic mucosa damage index (CMDI), microscopic (Geboes score), and biochemical methods (tissue tumour necrosis factor [TNF]-a and malondialdehyde [MDA]). Sildenafil reduced TNF-a tissue levels and increased the body weight. U-74389G reduced TNF-a, the macroscopic index of mucosal damage score (CMDI) and increased the body weight. The combined treatment with sildenafil and U-74389G reduced tissue levels of both TNF-a and MDA, lowered CMDI and microscopic Geboes score, and increased the body weight. U-74389G demonstrated a significant anti-inflammatory activity related to its ability to reduce colonic TNF-a, CMDI score, and improve weight change. We confirmed that sildenafil has an anti-inflammatory capacity in terms of reducing colonic TNF-a and improving body weight. Finally, the combined treatment showed superior effects by reducing colonic TNF-a, colonic MDA, the CMDI score, Geboes score, and by improving weight.

Lazaroid U-74389G Administration in Pancreatic Ischemia-Reperfusion Injury: A Swine Model Encompassing Ischemic Preconditioning.

The potential of lazaroid U-74389G in attenuating injury after ischemia and reperfusion has been reported in various organs. The present study focuses specifically on the pancreas and aims to examine any effects of U-74389G in a swine model of pancreatic ischemia and reperfusion, encompassing ischemic preconditioning. Twelve pigs, weighing 28-35 kg, were randomized into two experimental groups. Group A (control group, n=6): Two periods of ischemic preconditioning (5 min each) separated by a 5-min rest interval; then ischemia time 30 min and reperfusion for 120 min. Group B (n=6): the same as above, with U-74389G intravenous injection in the inferior vena cava immediately prior to the initiation of reperfusion. Blood sampling and pancreatic biopsies were conducted at 0, 30, 60, 90 and 120 min after reperfusion. Repeated-measures ANOVA was undertaken to evaluate differences between the two study groups. No statistically significant differences were noted concerning the histopathological parameters in the control and therapy groups (P=0.563 for edema, P=0.241 for hemorrhage, P=0.256 for leukocyte infiltration, P=0.231 for acinar necrosis and P=0.438 for vacuolization). In accordance with the above, serum metabolic data (glucose, creatinine, urea, total and direct bilirubin, total calcium, amylase, lipase, SGOT/AST, SGPT/ALT, ALP, GGT, LDH, CRP, insulin) were not significantly different between the two groups; similarly, tumor necrosis factor-α values (P=0.705) and tissue malondialdehyde levels (P=0.628) did not differ between the two groups. This swine model of pancreatic ischemia and reperfusion, encompassing preconditioning, indicates that U-74389G lazaroid does not seem to exert protective effects from pancreatic damage.

Effects of lazaroid U-74389G on intestinal ischemia and reperfusion injury in porcine experimental model

Background: The readmission of molecular oxygen into an ischemic tissue promotes the oxidation of resuscitated tissue with certain pathophysiologic mechanisms. Materials and methods: Twenty four pigs (male or female) were randomized in this study. The animals were allocated to four groups with an equal number (n = 6) in each group: 1) control group-ischemia for 30 min and reperfusion for 60 min. 2) control group-ischemia for 30 min and reperfusion for 120 min. 3) ischemia for 30 min and immediate iv injection of lazaroid U-74389G and reperfusion for 60 min. 4) ischemia for 30 min and immediate iv injection of lazaroid U-74389G and reperfusion for 120 min. Results: We investigated further the role of an antioxidant molecule such as U-74389G and we concluded that there is statistically significant relation in MDA (malondialdeyde), TNF –α (tumor necrosis factor-α) measurement in tissue, while the histological score in the groups that the lazaroid was administered was improved. Conclusions: In many emergency clinical situations, such as reperfusion of the intestine, the role of U-74389G can be protective.

Effectiveness of sildenafil and U-74389G in a rat model of colitis

BackgroundCrohn disease is still incurable. Compounds with anti-inflammatory and/or antioxidative effects are tested in various preclinical models of the disease. Our aim was to investigate the effects of sildenafil and lazaroid U-74389G in an experimental rat model of trinitrobenzenesulfonic acid–induced colitis. Materials and methodsTrinitrobenzenesulfonic acid was instilled into the colon of all male Wistar rats except for the rats belonging to the first group. For 6 days, the animals in group 3 were administered daily sildenafil orally, the rats in group 4 were administered daily U-74389G intravenously, and the rats in group 5 were coadministered daily sildenafil orally and intravenous U-74389G. The rats in groups 1 and 2 were not administered any treatment. During the study, the weights were recorded as a marker of clinical condition. The colon damage was evaluated using macroscopic colon mucosal damage index (CMDI), microscopic (Geboes score), and biochemical methods (tissue tumor necrosis factor [TNF]-α and malondialdehyde [MDA]). ResultsSildenafil reduced TNF-α tissue levels and increased body weight. U-74389G reduced TNF-α, the macroscopic index of mucosal damage score (CMDI) and increased body weight. The combined treatment with sildenafil and U-74389G reduced tissue levels of both TNF-α and MDA, lowered CMDI and microscopic Geboes score, and increased body weight. ConclusionsU-74389G demonstrated a significant anti-inflammatory activity related to its ability to reduce colonic TNF-α, CMDI score, and improve weight change. We confirmed that sildenafil has anti-inflammatory capacity by reducing colonic TNF-α and by improving body weight. Finally, the combined treatment showed superior effects by reducing colonic TNF-α, colonic MDA, CMDI score, Geboes score, and by improving weight.

Effects of lazaroid U-74389G liposomes in a glioblastoma mouse model.

2098 Background: Lazaroid U-74389G (LAZ) is a 21-aminosteroid that has radioprotective effects against radiation-induced lipid peroxidation. Also in vitro antiproliferative effects have been reported against glioblastoma cell lines. The aim of this study was to use a liposomal formulation to evaluate LAZ radioprotective and antiproliferative effects in a glioblastoma mouse model. Methods: LAZ PEGylated liposomes (Lipo G) were developed at the University of Houston, College of Pharmacy. Glioblastoma cell line U87-expressing firefly luciferase reporter gene (100,000 cells in 2 μL) was injected intracranially in each male SCID hairless outbred mouse. There were 4 treatment groups (n=8-9, each): brain model (M) without treatment (control), radiation 2Gy weekly (M+R), Lipo G at 5 mg/kg dose IP twice per week (M+L) and radiation with Lipo G (M+R+L). Treatment lasted three weeks. Tumor size was monitored using non-invasive bioluminescence imaging (BLI), in each mouse. Mice were sacrificed after 3 weeks. Brain was harvested for immunohistochemistry examinations. Lipid peroxidation of brain tissues was quantified by measuring malondialdehyde (MDA) as a surrogate biomarker. Survival was evaluated using Kaplan Meier analysis at P= 0.05. Results: The relative BLI intensity was 4002.03±1737.67, 2034±737.72, 1387.36±684.53 and 2498.89±2521.32 % for M, M+R, M+L and M+R+L, respectively. The tumor size of the M+L group was reduced by 65% compared to control . For the radiation treated groups (M+R and M+R+L), there was no significant difference in tumor size compared to control group. MDA brain concentration in M+L and M+R+L groups was significantly less than in M+R group (8.27±0.78 and 10.37±3.30 µM/gm vs. 23.09± 3.79 µM/gm). The survival mean was 22.67, 25.33, 25.22 and 27.13 days for M, M+R, M+R+L and M+L groups, respectively. Mean survival of LAZ treated groups (M+L and M+R+L) was significantly longer than that of the control group Conclusions: LAZ liposomal formulations administered at 5 mg/kg dose reduced tumor growth by 65%. LAZ also protected brain tissue from radiation-induced lipid peroxidation by reducing MDA concentration by 50%. These provocative data warrant further investigation of LAZ as a radiation protectant and chemotherapeutic agent.

Activation of acetylcholinesterase after U-74389G administration in a porcine model of intracerebral hemorrhage

Spontaneous intracerebral hemorrhage (ICH) accounts for 10-15% of all strokes. Despite high incidence, morbidity and mortality, the precise pathophysiology of spontaneous ICH is not fully understood, while there is little data concerning the mechanisms that follow the primary insult of the hematoma formation. The cholinergic system, apart from its colossal importance as a neurotransmission system, seems to also play an important role in brain injury recovery. It has been recently suggested that the brain possesses a cholinergic anti-inflammatory pathway that counteracts the inflammatory responses after ICH, thereby limiting damage to the brain itself. We, herein, report the findings of our study concerning the role of acetylcholinesterase (AChE; a crucial membrane-bound enzyme involved in cholinergic neurotransmission) in a porcine model of spontaneous ICH, with a focus on the first 4 and 24 h following the lesion's induction, in combination with a study of the effectiveness of the lazaroid antioxidant U-74389G administration. Our study demonstrates the activation of AChE activity following U-74389G administration. The lazaroid U-74389G seems to be an established neuroprotectant and this is the first report of its supporting role in the enhancement of cholinergic response to the induction of ICH.

Abstract 5460: Dual efficacy of Lazaroid U-74389G liposomes in glioblastoma mouse model

Abstract Purpose: Lazaroid U-74389G (LAZ) is a 21-aminosteroid that has radioprotective effect against radiation-induced lipid peroxidation. In-vitro antiproliferative effect was reported against Glioblastoma primary cell lines. The aim of this study was to use a liposomal formulation in a proof-of-concept efficacy study to evaluate LAZ radioprotective and antiproliferative effects in Glioblastoma mouse model. Methods: LAZ PEGylated liposomes (Lipo G) were developed in our laboratory. Lipo G size was 80-90 nm with Zeta potential of -22mv. Glioblastoma cell line U87-Lexpressing firefly luciferase reporter gene (100,000 cells in 2 μL) was injected intracranially in each male SCID hairless outbred mouse. The mice were randomized among 4 treatment groups (n=8-9, each): brain model (M) without treatment (control), radiation of 2Gy once a week (M+R), Lipo G at 5 mg/kg dose IP twice a week (M+L) and radiation with Lipo G (M+R+L). The treatment lasted three weeks. The tumor size was monitored using non-invasive bioluminescence imaging (BLI), in each mouse, expressed as the relative BLI photon intensity on the second and third week to that of the first measurement. The mice were sacrificed at the end of the third week and the whole brain was harvested for immunohistochemistry examinations. Lipid peroxidation of brain tissues was quantified by measuring malondialdehyde (MDA) as a surrogate biomarker. The survival was evaluated using Kaplan Meier analysis at P = 0.05. Results: The relative BLI intensity was 4002.03±1737.67, 2034±737.72, 1387.36±684.53 and 2498.89±2521.32 % for M, M+R, M+L and M+R+L, respectively. The tumor size of the M+L group was reduced by 65% compared to the control group. For the radiation treated groups (M+R and M+R+L), there was no significant difference in the tumor size suppression compared to the control group. MDA brain concentration in M+L and M+R+L groups was significantly less than that in M+R group (8.27±0.78 and 10.37±3.30 µM/gm vs. 23.09± 3.79 µM/gm). The survival mean was 22.67, 25.33, 25.22 and 27.13 days for M, M+R, M+R+L and M+L groups, respectively. The mean survival duration of LAZ treated groups (M+L and M+R+L) was significantly longer than that of the control group (27.13 and 25.22 day vs. 22.67 day, respectively). Conclusion: LAZ liposomal formulations administered at 5 mg/kg dose delivered a therapeutic concentration of LAZ to the brain for the efficacy of suppressing Glioblastoma tumor growth (65% reduction). The liposomal LAZ also protected the brain tissue from radiation-induced lipid peroxidation by reducing MDA concentration (lipid peroxidation surrogate) to 50%. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5460. doi:10.1158/1538-7445.AM2011-5460

The effects of lazaroid U-74389G in a rat sepsis model

To examine the protective effects of a lazaroid, 21-aminosteroid U-74389G, in a rat septic shock model. Male Sprague-Dawley rats (n = 60) aged 6-8 months. Groups were exposed to 500 cGy radiation followed by E. coli inoculation, and either placebo or lazaroid injection (10 mg/kg intraperitoneal) 5 days after irradiation. Hemodynamic measurements, arterial blood gases, serum lactate, total antioxidative capacity, and cytokine levels were measured at specific time intervals. Treatment with the lazaroid U-74389G maintained cardiac output and mean aortic pressure. Lazaroid treatment also prevented the increase in serum lactate seen in placebo-treated rats. Cytokine serum levels in lazaroid-treated rats were not significantly different from those in placebo-treated rats at any time point. Lazaroid treatment of E. coli-inoculated septic animals lessens the hemodynamic deterioration seen in sepsis.

Abstract 2652: Formulation and pharmacokinetics of Lazaroid U-74389G (LAZ) liposomes in rats

Abstract Purpose Lazaroid U74389G (LAZ) is a 21-aminosteroid that has a radioprotective and antioxidant effects. It prevents the damage in the tissues after radiation by a selective action on vascular endothelium cells in normal tissues. The aim of this study is to develop liposomal formulations of LAZ with favorable pharmacokinetic (PK) profiles for potential therapeutic merits in an animal model. Methods Conventional (LipoB) and pegylated (LipoG) liposomes were prepared with various phosphatidylcholine: cholesterol ratios and lipid: LAZ ratio of 10:1. The liposomes were characterized with the size, charge and release profile. The PK study was performed in Sprague-Dawley rats. The rats were randomized into 3 groups (n=4 each) prior to the dosing of the formulations (LAZ solution, LipoB and LipoG) at a dose of 1 mg/kg by injection through the cannula at jugular veins. At pre-determined time points, blood samples were collected from the tail vein. The LAZ concentrations in plasma were quantified by a validated UPLC/MS/MS assay using Daidazine as the internal standard, with a lower limit of quantification (LLOQ) of 4 ng/ml for LAZ. The PK profiles were constructed by plotting the plasma concentration versus time and the PK parameters were derived using compartmental models of WinNonlin Ver 3.3 software. Results The liposomal preparations with the least amount of cholesterol had the lowest Encapsulation Efficiency (EE=26%). However, an increase of cholesterol ratio up to 20% increased the EE up to 58%. The size of the liposomes was in the range of 90-118 nm. In in-vivo study, the plasma profile of the solution fits 1-compartment model while the liposomal formulations fit 2-compartment model. The solution group yielded a higher Cmax and faster clearance (4.27 μg/ml and 546 ml/hr) than those of LipoB and LipoG (0.97 and 2.03 μg/ml and 380 and 216 ml/hr, respectively). The t1/2's of LAZ from LipoB and LipoG were 5 folds of that from the solution (0.64 and 0.52 hr vs 0.10hr). The solution showed a significantly smaller AUC compared to those of LipoB and LipoG (0.56 μg.hr/ml vs. 0.79 and 1.42 μg.hr/ml, respectively). The volume of distribution was small for the solution compared to LipoB and LipoG (73 ml vs. 1173 and 589 ml, respectively). The half-lives of distribution and elimination, t1/2 α and t1/2 β, were comparable between LipoB and LipoG (0.22 vs. 0.16 hr and 3.17 vs. 2.55 hr, respectively). Conclusion The liposomal formulations modified the pharmacokinetics of LAZ solution. The liposomal formulations resulted in a slower clearance and wider distribution of LAZ in-vivo compared to the solution. LAZ systemic exposures in rats after administration of LAZ liposomes were 1.4 −2.5 folds higher than that of solution. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2652.

Protective effect of U-74389G on paraquat induced pneumotoxicity in rats

Paraquat is a very toxic herbicide and a dangerous pollutant of the environment. It forms reactive oxygen species and increases the lipid peroxidation in the pulmonary cells. Our aim in this study was to estimate the protective effects of the lazaroid U-74389G possessing antilipidperoxidation activity and membrane-stabilizing effect. The experiment was carried out with 96 male Wistar rats. Paraquat dichloride was administered orally at 80 mg/kg. The lazaroid U-74389G was injected intraperitoneally twice – 2 h before receiving the paraquat with 10 mg/kg and four hours later with 5 mg/kg. Isolated application of paraquat increased enzyme activities of lactate dehydrogenase (LDH) and acid phosphatase (AcP) and the total protein content in bronchoalveolar lavage fluid (BALF). In the same experimental group the number of polymorphonuclear cells (PMNs) in BALF is elevated significantly on days 1 and 3. The combined treatment with paraquat and U-74389G did not increase the total protein content and the number of PMNs and it elevated the enzyme activities of LDH and AcP significantly less than the alone application of paraquat. It is concluded that the lazaroid U-74389G reduces the pneumotoxic effects of paraquat, estimated by sensitive cytologic and biochemical markers in BALF. The protective effect of U-74389G is well-expressed until day 3 after the treatment.

A lazaroid mitigates postresuscitation myocardial dysfunction

Lazaroids, a series of 21-aminosteroids, reduce free radical mediated injury after ischemia and reperfusion. We hypothesized that the lazaroid U-74389G would minimize postresuscitation myocardial dysfunction and thereby improve neurologically meaningful survival in a rodent model after resuscitation from 8 mins of ventricular fibrillation. Randomized, controlled laboratory study. University-affiliated research institute. Sprague-Dawley rats. Ventricular fibrillation was electrically induced in ten anesthetized Sprague-Dawley rats. The lazaroid agent U-74389G in a dose of 1 mg.kg-1 or its vehicle serving as a placebo was injected into the right atrium after 7 mins of untreated ventricular fibrillation. One minute after injection of the compound, precordial compression was begun together with mechanical ventilation and continued for 6 mins before attempted electrical defibrillation. All animals were successfully resuscitated. Postresuscitation cardiac index, left ventricular end-diastolic pressure, the rate of left ventricular pressure increase measured at a left ventricular pressure of 40 mm Hg, and the maximum rate of left ventricular pressure decline were significantly less impaired in lazaroid-treated animals. This contrasted with control animals, which had significantly greater myocardial impairment, greater neurologic deficit, and lesser duration of survival. The lazaroid compound U-74389G, administered during cardiac arrest, mitigated postresuscitation myocardial dysfunction and improved survival.