Search

While nerve cells rapidly die in stroke, and patients do initiate a neurogenic response to replace these cells, there has been no attempt to develop pleiotropic cytoprotective compounds to further promote neurogenesis in stroke patients, and there are no neurogenic or cytoprotective drugs approved by the FDA. In this study, we focused on the parent compound J-147, a curcumin derivative that is a 5-LOX inhibitor, and has potent neuroprotective and neurogenic activities. J-147 rescues HT22 cells from iodoacetic acid challenge (EC 50 50 nM), and oxytosis (EC 50 115 nM). J-147 is neuroprotective against glutamate excitotoxcity in E18 cortical neuron culture (EC 50 27 nM), and prevents cell death in an embryonic cortical cell trophic factor withdrawal assay (EC 50 25 nM). J-147 can rescue a clone of the hippocampal nerve cell line HT22 expressing TrkB, from serum starvation under conditions where the cells can be protected by BDNF (EC 50 50 nM). In mice, J-147 is neurogenic and can stimulate cell division in germinal regions of the brain (increase BrdU + DCX + labeled cells), enhances endogenous neurotrophic factors and energy metabolism. With this basis, we tested J-147 in 2 translational stroke models. First, using a rodent intraluminal suture model, in combination with a quantal analysis method to determine the effect of increasing ischemia duration (5-90 minutes) on behavioral deficits, IV J-147 treatment (10 mg/kg) administered 5 minutes following the end of occlusion (5-90 minutes), significantly (P=0.045) reduced stroke-induced behavioral deficits and increased the P 50 value to 36.37 ± 1.72 (n=19) minutes from P 50 = 20.1 ± 4.07 minutes (n=19) for the control group. J-147 also reduced infarct volume following IV administration (p50 value (behavior) to 1.88 ± 0.10 mg (n=20) compared to P 50 = 1.07 ± 0.07 (n=19) for control(p1/2 5.89-8.56 hours). These data support the development of J-147 and neurogenic analogs as a novel class of drug to treat stroke.

Introduction: DS-1040 is an inhibitor of the activated thrombin-activatable fibrinolysis inhibitor (TAFIa) being developed for the treatment of acute ischemic stroke (AIS). We evaluated the effect of DS-1040 infusiontreatment on rabbit TAFIa activity; plasma concentrations and we also determined if DS-1040 treatment affected behavioral outcome following embolization using the standard rabbit small clot embolic stroke model (RSCEM). Safety was assessed by measuring the effect of DS-1040 on intracerebral hemorrhage (ICH) and mortality. Methods: The inhibitory effect of DS-1040 on TAFIa was evaluated using hippuryl-Arg, a substrate of carboxypeptidase. The study was conducted randomized and blinded per RIGOR guidelines. Male New Zealand white rabbits (2-2.5kg) were embolized by injecting a suspension of small blood clots into the brain via an indwelling carotid catheter. Saline (control), tPA (3.3 mg/kg, i.v., 10% bolus/90% infusion for 0.5 h), high and low doses of DS-1040 (approx. 940 mg/per rabbit and 470 mg/per rabbit, respectively) were given 1 h following embolization as an i.v. bolus followed by a s.c. infusion for 47 h. Behavioral analysis was conducted 48 h following embolization, resulting in the calculation of an effective stroke dose (P50) or clot amount (mg) that produces neurological deficits in 50% of the rabbits. ICH was also assessed in all treatment groups. Results: DS-1040 inhibited rabbit TAFIa with an IC50 value of 11.7 ng/mL. Using the dosing regimen described above, the mean plasma concentration of DS-1040 at 48 h was 1838 ng/mL (high dose) and 591 ng/mL (low dose). Rabbits treated with high and low doses of DS-1040 had P50 values of 1.61 ± 0.23 mg (p Conclusions: DS-1040 significantly improved behavioral function in small clot embolized rabbits, suggesting that DS-1040 should be further pursued as a treatment for stroke victims.

Introduction: After vascular occlusion, there are two major zones of injury: the infarct core that rapidly dies, and the surrounding penumbra, which is hypoxic and at risk for infarction. Restoring blood flow by vascular recanalization (ie. endovascular +/- IV tPA) to prevent the penumbra from infarcting has become the new standard of care for acute ischemic stroke patients presenting with salvageable tissue. However, not all patients benefit from recanalization. Regardless of successful or partial reperfusion, secondary hypoxic events within the rescued penumbra can cause selective neuronal loss (SNL) that may account for suboptimal clinical recovery. OMX is a breakthrough oxygen delivery protein tuned to release oxygen specifically in hypoxic tissue and reduce hypoxia to prevent SNL within the reperfused penumbra. Methods: To mimic the clinical situation whereby occlusion is followed by gradual reperfusion after spontaneous or therapeutic thrombolysis, we used the endothelin-1 induced middle cerebral artery occlusion (MCAO) model. OMX was administered intravenously up to 2h post-MCAO. Short and long-term histological and behavioral outcomes were used to assess hypoxia, apoptosis, gliosis, SNL, infarct volume and sensorimotor functions. Results: Despite reperfusion, hypoxic tissue persists and progressively infarcts in vehicle-treated rats. However, OMX-treated rats showed no infarct expansion over 7d, indicating that OMX prevents infarction of the majority of hypoxic tissue. OMX reduces hypoxia and caspase 3/9 activity in a dose-dependent manner and significantly prevents SNL and gliosis. When administered post-MCAO, OMX significantly improves both sensory and motor functions (∼30-80% improvement, * p Conclusions: OMX is a promising therapeutic candidate that can be administered in combination with reperfusion therapies to stabilize the at-risk hypoxic tissue and ameliorate long-term clinical outcomes in stroke patients.

Background and Purpose: Transcranial laser therapy (TLT) and tissue plasminogen activator (tPA) improve behavior when administered following clot-induced strokes. tPA increases cerebral reperfusion and blood flow and TLT also enhances cerebral blood flow and activates mitochondrial function. We hypothesize that a combination therapy may be more effective than individual therapies and that efficacy may be mediated by enhancing mitochondrial function. Therefore, the goal of the present study is to evaluate the effect of combined TLT and tPA therapy on behavioral outcome and cellular ATP content using the rabbit small clot embolic stroke model (RSCEM). Methods: The study was conducted randomized and blinded. Male New Zealand white rabbits were embolized by injecting a suspension of small blood clots into brain via a carotid catheter. Using the RSCEM, we studied the effects of continuous wave TLT (7.5 mW/cm2, 2 minutes) alone or in combination with standardized intravenous (IV) tPA (3.3mg/kg) applied 1 hour post-embolization on 3 endpoints: 1) behavioral function measured 2 days [effective stroke dose (P50 in mg) producing neurological deficits in 50% of embolized rabbits], 2) intracerebral hemorrhage (ICH) rate, and 3) cortical adenosine-5'-triphosphate (ATP) content was measured 6 hours following embolization. Results: TLT and tPA significantly (p Conclusion: TLT and tPA both effectively and safely improve behavioral outcome in embolized rabbits and attenuate stroke-induced ATP deficits; TLT-tPA did not offer additional clinical benefit, but the TLT-tPA combination produced a synergistic effect on ATP levels. The study suggests that tPA-induced reperfusion in combination with TLT neuroprotection therapy may optimally protect viable cells in the cortex measured using ATP levels as a marker.

Introduction: We studied the pharmacokinetics and pharmacology of intravenously administered CNB-001, a pleiotropic anti-inflammatory molecule with neurotrophic properties in two rabbit neuro-ischemia models. Hypothesis: In vitro, we have found that CNB-001 can significantly reduce cell death mediated by glutamate, iodoacetic acid, ATP depletion, oxidative stress (glutathione depletion) and oxygen-glucose depletion. We tested the hypothesis that CNB-001 would attenuate ischemia-induced behavioral deficits in vivo. Methods: Abiding by RIGOR guidelines, the study was randomized and the neuro-examiners were naive (blinded) to treatment. CNB-001 (10mg/kg) was administered IV 45 minutes prior to balloon of occlusion of the infrarenal aorta, which reliably produces spinal cord ischemia (SCI) depending upon the occlusion time. Using TARLOV scores and quantal analysis, we quantified the ischemia duration (P50 in minutes) producing SCI in 50% of study rabbits. Second, using a rabbit embolic stroke model (RSCEM), CNB-001(1-25mg/kg) was applied IV 1 hour post-ischemia and behavioral function measured. We calculated the effective stroke clot dose (P50 in mg) producing neurological deficits in 50% of embolized rabbits. Control groups and positive-control drugs were also studied in parallel in both rabbit ischemia models. All behavioral endpoints were measured 2days after ischemia. Results: Pharmacokinetic analysis of CNB-001 in normal male rabbits indicated that the t1/2 was 3.37 ± 0.52 hours and clearance rate was 44.2 ml/min/kg in rabbits. In the SCI model, CNB-001 (10mg/kg) significantly increased P50 values to 19.38 ± 2.03 min (n=16) from 13.58 ± 0.90min (n=14; p Conclusions: CNB-001 can be provided to patients prior to cardiaovascular surgery to reduce the probability of SCI. Moreover, CNB-001 can reduce behavioral deficits associated with brain ischemia when given after an embolic stroke.

On June 20 and 21, 2012, a National Institute of Neurological Disorders and Stroke-sponsored workshop on “Optimizing the Predictive Value of Preclinical Research” was held in Washington, DC.1 The primary objective of the workshop was to review deficiencies in preclinical and translational research and provide recommendations or guidelines to improve the rigor of preclinical research. The following is based on open discussions held at the meeting and panel recommendations for the application of basic principles of experimental design and reporting transparency that are currently standard of practice for stroke clinical trials. This article, which includes author interpretations of discussions, emphasizes the need for good laboratory practices, transparent scientific reporting, and the use of translational research models with clinically relevant end points to develop new strategies to treat stroke. There is a critical medical need for new therapeutic strategies to treat acute ischemic stroke and hemorrhagic stroke to reduce mortality and improve the quality of life for stroke victims. Current US statistics indicate that stroke is the fourth leading cause of death and the leading cause of adult disability in the United States, with upward of 795 000 victims annually who suffer a new or recurrent stroke, despite improved diet, and control of diabetes mellitus, hypertension, and hypercholesterolemia. Currently, the only Food and Drug Administration-approved treatment for stroke is the thrombolytic, tissue plasminogen activator (tPA) if administered within 3 hours of a stroke. Recently, the therapeutic window for tPA has been increased and shown to be beneficial if patients are treated within 4.5 hours of an ischemic stroke.2–4 However, the Food and Drug Administration (FDA) has not yet approved the extension of the time window >3 hours. Although thrombolysis is now widely accepted as a standard of care for acute ischemic stroke, only a minority (6%) of …

Background and Purpose— Because photon energy delivered using a low-energy infrared laser may be useful to treat stroke, we determined whether transcranial laser therapy would improve behavioral deficits in a rabbit small clot embolic stroke model (RSCEM). Methods— In this study, the behavioral and physiological effects of laser treatment were measured. The RSCEM was used to assess whether low-energy laser treatment (7.5 or 25 mW/cm 2 ) altered clinical rating scores (behavior) when given to rabbits beginning 1 to 24 hours postembolization. Behavioral analysis was conducted from 24 hours to 21 days after embolization, allowing for the determination of the effective stroke dose (P 50 ) or clot amount (mg) that produces neurological deficits in 50% of the rabbits. Using the RSCEM, a treatment is considered beneficial if it significantly increases the P 50 compared with the control group. Results— In the present study, the P 50 value for controls were 0.97±0.19 mg to 1.10±0.17 mg; this was increased by 100% to 195% (P 50 =2.02±0.46 to 2.98±0.65 mg) if laser treatment was initiated up to 6 hours, but not 24 hours, postembolization (P 50 =1.23±0.15 mg). Laser treatment also produced a durable effect that was measurable 21 days after embolization. Laser treatment (25 mW/cm 2 ) did not affect the physiological variables that were measured. Conclusions— This study shows that laser treatment improved behavioral performance if initiated within 6 hours of an embolic stroke and the effect of laser treatment is durable. Therefore, transcranial laser treatment may be useful to treat human stroke patients and should be further developed.

Background and Purpose— It has been proposed that antioxidants and spin-trap agents may be neuroprotective after acute ischemia stroke. Although the antioxidant ebselen is currently in clinical trials, little is known about the effectiveness of ebselen, which has glutathione peroxidase–like and anti-inflammatory properties in embolic stroke models. Therefore, we determined the effects of ebselen when administered alone or with the thrombolytic tissue plasminogen activator (tPA), the only Food and Drug Administration–approved pharmacological agent for the treatment of stroke. Methods— Male New Zealand White rabbits were embolized by injection of a suspension of small blood clots into the middle cerebral artery via a catheter. Five minutes after embolization, ebselen (10 to 50 mg/kg) was infused intravenously. Control rabbits received infusions of the vehicle required to solubilize ebselen. In additional rabbits, ebselen (20 mg/kg) was administered 60 minutes after embolization, either alone or in combination with tPA (0.9 or 3.3 mg/kg tPA). Behavioral analysis was conducted 24 hours after embolization, allowing determination of the effective stroke dose (P 50 ) or clot amount (mg) that produces neurological deficits in 50% of the rabbits. Results— A drug is considered neuroprotective if it significantly increases the P 50 compared with the vehicle-treated control group. The P 50 of controls 24 hours after embolization was 1.35±0.30 mg. Rabbits treated 5 minutes after embolization with 10, 20, or 50 mg/kg ebselen had P 50 values of 2.12±0.56, 2.82±0.75 ( P 50 =1.69±0.32 mg). When tPA (3.3 mg/kg) was infused 60 minutes after embolization and ebselen (20 mg/kg) was injected at either 5 (P 50 =2.98±0.18 mg) or 60 (P 50 =3.60±0.79 mg) minutes, there was no additional neuroprotective effect compared with tPA alone (P 50 =3.38±0.55 mg). However, if ebselen (20 mg/kg) was administered concomitantly with low-dose tPA (0.9 mg/kg) 60 minutes after embolization, the P 50 was 3.52±0.73 mg ( P 50 =1.69±0.32 and 1.54±0.36 mg, respectively). Conclusions— This study indicates that ebselen may be neuroprotective when administered shortly after an embolic stroke, but the time- and dose-response analyses suggest that it has a narrow therapeutic window. Nevertheless, ebselen may be beneficial if administered concomitantly with a thrombolytic because it significantly enhanced the neuroprotective activity of low-dose tPA.

Background and Purpose — It has been proposed that the novel spin trap agent disodium-[(tert-butylimino)methyl]benzene-1,3-disulfonate N -oxide (NXY-059) may be useful in the treatment of ischemic stroke. However, there is little information concerning the neuroprotective properties of NXY-059 when administered after an embolic stroke. Moreover, there is no information available concerning the combination of NXY-059 with the only Food and Drug Administration–approved pharmacological agent for the treatment of acute stroke, the thrombolytic tissue plasminogen activator (tPA). Thus, we determined the effects of NXY-059G, a generic form of NXY-059, on behavioral outcome after an embolic stroke when administered alone or in combination with tPA. Methods — Male New Zealand White rabbits were embolized by injecting a suspension of small blood clots into cerebral circulation via a carotid catheter. NXY-059G (100 mg/kg) was infused intravenously 5 minutes or 3 hours after embolization, whereas control rabbits received infusions of the saline vehicle. In tPA studies, the thrombolytic was administered intravenously starting 60 minutes or 3 hours after embolization (3.3 mg/kg). In combination studies, NXY-059G was given 5 minutes after embolization, followed by the administration of tPA beginning either 60 minutes or 3 hours after embolization. Behavioral analysis was conducted 24 hours after embolization. Results — In the vehicle control group, the ES 50 value (calculated as the amount of microclots [milligrams] that produce neurological dysfunction [impairment] in 50% of the rabbits within a specific treatment group) measured 24 hours after embolism was 1.04±0.31 mg, and this was increased by 153% to 2.54±0.72 mg if NXY-059G was administered beginning 5 minutes after embolization. However, if NXY-059G was administered beginning 3 hours after embolization, the ES 50 was 2.01±0.40 mg. The rabbits treated with tPA alone had an ES 50 of 2.64±0.66 or 0.63±0.35 mg if tPA administration started 60 minutes or 3 hours after embolization, respectively. If tPA was administered after NXY-059G (started at 5 minutes), the ES 50 values were 3.15±0.50 or 2.66±0.82 if tPA administration started 60 minutes or 3 hours after embolization, respectively. Conclusions — This study suggests that NXY-059G is neuroprotective and can increase behavioral ratings if administered early after an embolic stroke. In addition, the study shows that NXY-059G can be used in combination with tPA without negative side effects. The drug combination can improve behavioral function and increase ES 50 values. However, during the short time course of the behavioral analysis, the combination was not statistically better than either drug alone.

Future stroke therapy will require pleiotropic drugs with multi-mechanisms of action. We studied a safe pyrazole compound, CNB-001, which has multiple potent neuroprotective and neurotrophic activities. Previously, we found that CNB-001 improves clinical function following small clot embolic stroke in rabbits, increase cortical brain-derived neurotrophic factor (BDNF) content and activates BDNF signaling pathways. Abiding by RIGOR guidelines (blinding and randomization), we studied CNB-001 in rat stroke model [filament middle cerebral artery occlusion (MCAo)] where Sprague-Dawley (SD) rats were subjected to 3 hours MCAo. After a delay of 1 hour during reperfusion, CNB-001 (10 mg/kg) or vehicle (n=12 per group) was infused through the jugular vein over 5 mins as a single dose. One week after stroke onset, these animals were tested using a cylinder test, a measure of limb-use-asymmetry, and further cylinders tests were done at week 2 and 4 following stroke, and then followed by Barnes Maze analysis, to quantitate spatial learning and memory. CNB-001-treated rats showed significant improvement (p50 value (minutes), which represents the occlusion time required to induce behavioral deficits in 50% of the population. IV CNB-001 treatment administered 5 minutes following the end of occlusion, significantly (p50 value to P 50 = 46.71±7.58 minutes (n=21) compared with the control group (P 50 = 24.9±6.07 minutes, n=20). Moreover, CNB-001 significantly reduced infarct volume by 60-95%(p

Background and Purpose —Tissue plasminogen activator (tPA) is an effective treatment for stroke, but its utility is limited by fear of cerebral hemorrhage. Tenecteplase (TNK), a genetically modified form of wild-type tPA, exhibits a longer biological half-life and greater fibrin specificity, features that could lead to fewer cerebral hemorrhages than wild-type tPA in stroke patients. Methods —We injected radiolabeled blood clots into the cerebral circulation of New Zealand White rabbits. One hour later, we administered tPA (n=57), 0.6 mg/kg TNK (n=43), 1.5 mg/kg TNK (n=27), or vehicle control (n=37). A blinded observer examined the brains for macroscopic hemorrhage using a semiquantitative score. We estimated thrombolysis by assessing the amount of radiolabel remaining in the cerebral vessels postmortem. Results —Both wild-type tPA and TNK caused thrombolysis in most subjects. Hemorrhage was detected in 26% (6/23) of the control group, 66% (27/41) of the wild-type tPA group, 55% (16/29) in the 0.6-mg/kg TNK group, and 53% (9/17) in the 1.5-mg/kg TNK group ( P 2 test). The tPA group was statistically significantly different from the control group, but the TNK and tPA groups did not differ from each other. Neither TNK nor tPA affected the size of the hemorrhages. Conclusions —TNK shows comparable rates of recanalization compared with wild-type tPA in a model of embolic stroke. While tPA increases hemorrhage rate, the hemorrhage associated with TNK treatment is not statistically different compared with controls or the tPA group. These findings suggest that TNK shows promise as an alternative thrombolytic treatment for stroke, but we could not demonstrate improved safety compared with wild-type tPA.

Background and Purpose —It has been proposed that spin trap agents such as N -t-butyl-phenylnitrone (PBN) may be useful as neuroprotective agents in the treatment of ischemia and stroke. However, to date, there is little information concerning the effectiveness of spin trap agents when administered in combination with the only Food and Drug Administration–approved pharmacological agent for the treatment of stroke, the thrombolytic tissue plasminogen activator (tPA). Thus, we determined the effects of PBN when administered before tPA on hemorrhage and infarct rate and volume. We also compared the effects of PBN with those of 2,2,6,6-tetramethylpiperidine- N -oxyl (TEMPO), another spin trap agent that has a different chemical structure and trapping profile, on the incidence of infarcts and hemorrhage. Methods —One hundred sixty-five male New Zealand White rabbits were embolized by injecting a blood clot into the middle cerebral artery via a catheter. Five minutes after embolization, PBN or TEMPO (100 mg/kg) was infused intravenously. Control rabbits received saline, the vehicle required to solubilize the spin traps. In tPA studies, rabbits were given intravenous tPA starting 60 minutes after embolization. Postmortem analysis included assessment of hemorrhage, infarct size and location, and clot lysis. Results —In the control group, the hemorrhage rate after a thromboembolic stroke was 24%. The amount of hemorrhage was significantly increased to 77% if the thrombolytic tPA was administered. The rabbits treated with PBN in the absence of tPA had a 91% incidence of hemorrhage compared with 33% for the TEMPO-treated group. In the combination drug–treated groups, the PBN/tPA group had a 44% incidence of hemorrhage, and the TEMPO/tPA group had a 42% incidence of hemorrhage. tPA, PBN/tPA, and TEMPO/tPA were similarly effective at lysing clots (49%, 44%, and 33%, respectively) compared with the 5% rate of lysis in the control group. There was no significant effect of drug combinations on the rate or volume of infarcts. Conclusions —This study suggests that certain spin trap agents may have deleterious effects when administered after an embolic stroke. However, spin trap agents such as PBN or TEMPO, when administered in combination with tPA, may improve the safety of tPA by reducing the incidence of tPA-induced hemorrhage. Overall, the therapeutic benefit of spin trap agents for the treatment of ischemic stroke requires additional scrutiny before they can be considered “safe” therapeutics.

Background and Purpose —Dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) may function as neurotrophic or neuroprotective factors to protect central nervous system (CNS) neurons against a variety of insults, including excitotoxicity. The present study evaluated the pharmacological effects of DHEAS in a reversible spinal cord ischemia model. Methods —DHEAS was administered (50 mg/kg IV) 5 or 30 minutes after the start of occlusion to groups of rabbits exposed to ischemia induced by temporary (15 to 60 minutes) occlusion of the infrarenal aorta. The group P 50 represents the duration of ischemia (in minutes) associated with 50% probability of resultant permanent paraplegia. Results —The P 50 of the vehicle-treated control group, when behavioral analysis was assessed 18 hours after aortal occlusion, was 28.8±2.0 minutes. Neuroprotection was demonstrated if a drug significantly prolonged the P 50 compared with the vehicle-treated control group. Treatment with DHEAS at 5 minutes significantly ( P 50 of the group to 36.8±3.9 minutes. In addition, the DHEAS effect appeared durable, because a significant difference between the control and DHEAS-treated groups was still measurable at the 4-day time point. At 4 days, the P 50 of the control group was 26.1±2.2 minutes, whereas the P 50 for the DHEAS-treated group was 38.6±5.9 minutes. DHEAS was not neuroprotective if administered 30 minutes after occlusion. In addition, the GABA A antagonist bicuculline abolished the neuroprotective effect of DHEAS. Conclusions —The present study suggests that neurosteroids may have substantial therapeutic benefit for the treatment of ischemic stroke.

Multiple plant (fruits, vegetables) and spice (turmeric)-derived natural products, which have pleiotropic activities (anti-inflammatory, antioxidant, anti-apoptotic, neurogenic) are also neuroprotective in vitro against numerous chemical insults and promote behavioral recovery in vivo following embolic strokes. We have focused on 2 types of natural products: 1) curcumin-based compounds such as CNB-001 and 2) flavone-based compounds such as fisetin and baicalein. We have synthesized a new library of flavonoid molecules to improve their pharmacological characteristics and have selected candidates using a battery of vitro screening assays with stringent criteria: 1) HT22 hippocampal cell survival (in vitro ischemia model using 20µM iodoacetic acid); 2) primary mouse cortical neurons cell survival (in vitro excitotoxicity assay using 10µM glutamate); 3) HT22 hippocampal cell survival assay (oxytosis using 5 mM glutamate to induce non-NMDA mediated cell death due to the absence of NMDA receptors on HT22 cells). EC50 ≤ 100 nM and survival ≥80% in the IAA assay, ≥35% survival in the cortical neuron assay and EC 50 ≤ 300 nM and ≥50% survival in the oxytosis assay. All compounds were screened in triplicate using concentrations up to 10 µM. We chemically modified fisetin to improve its potency, increase blood brain barrier penetration (CLogP, an indicator of blood brain barrier penetration) and reduce tPSA (topographical polar surface area). We have developed a structure activity relationship (SAR) indicating that the absence of a hydroxyl group on the A ring of fisetin enhances its potency. Two compounds PM-001 and PM-004) are 6 fold more potent than fisetin. Altering a hydroxyl group at the 3-position of the B ring has no impact on activity. The addition of multiple hydrophobic groups to various positions on the A ring, such as the addition of methyl groups at the 6 and/or 7 position improved potency ∼10-100 fold over fisetin (e.g. compound PM-010) while the addition of a more hydrophobic naphthalene group (PM-002) and (PM-003) improved the potency ∼40 fold over fisetin and increased the ClogP and reduced the tPSA. SAR studies on the C ring suggested that hydrogen bond accepting groups such as a methoxy at the 4' position and a hydrogen bond donating group such as a hydroxyl at the 3' position further enhanced potency. For example compound CMS-064, CMS-069 and CMS-092 are some of the most potent compounds, with EC 50 s below 40 nM. These compounds also have CLogPs and tPSAs consistent with enhanced brain penetration. In conclusion, we have used a rational medicinal chemistry approach in combination with in vitro screening analysis to identify new flavonoid molecules to optimally promote neuroprotection. The molecules which will be discussed in detail are being further characterized and de-risked to treat stroke.

Background and Purpose— It has been proposed that the novel thrombolytic microplasmin may be useful in the treatment of ischemic stroke. In the present study the effects and safety profile of microplasmin were evaluated in 2 rabbit embolic stroke models that have been used successfully to develop tissue plasminogen activator (tPA) as the only Food and Drug Administration-approved treatment for stroke. The rabbit small clot embolic stroke model (RSCEM) and rabbit large clot embolic stroke model (RLCEM) were used to determine the potential neuroprotective properties and safety profile of microplasmin, respectively, after an embolic stroke. Methods— Rabbits were embolized by injecting small blood clots (RSCEM) or large blood clots (RLCEM) into the cerebral circulation. For the RSCEM, 126 rabbits were included, with behavioral analysis conducted 24 hours later, allowing for determination of the effective stroke dose (ES 50 ) or clot amount (milligrams) that produces severe neurological deficits in 50% of rabbits. For RLCEM safety study analysis, 47 rabbits were included, with postmortem analyses consisting of assessment of hemorrhage and infarct rate and size. In test animals microplasmin was infused intravenously 60 minutes after embolization, whereas control rabbits were given infusions of the saline/Plasma-Lyte vehicle with all assessments performed in a blinded fashion. Results— In the RSCEM, a drug is considered neuroprotective if it significantly increases the ES 50 compared with the vehicle-treated control group. The ES 50 of the vehicle-treated control group 24 hours after embolization was 1.36±0.42 mg (n=38). Microplasmin, infused starting 60 minutes after embolization, increased the ES 50 to 2.32±0.57 (n=21), 1.89±0.48 (n=21), 2.81±0.55 (n=22), and 1.89±0.28 mg (n=24) for the 1-, 2-, 4-, and 8-mg/kg doses, respectively. There was a statistically significant behavioral improvement in the 4-mg/kg dose arm ( P =0.040). The microplasmin dose of microplasmin that was statistically significant (4 mg/kg) was subsequently determined to be safe in the RLCEM because it did not increase the incidence of hemorrhages (56%) compared with vehicle-treated rabbits (63%), nor did it significantly alter hemorrhage volume, infarct rate, or infarct volume. Conclusions— The present study shows that microplasmin improves behavioral rating scores in the RSCEM when administered 60 minutes after embolization, at a dose that does not increase hemorrhages in the RLCEM. This is in contrast to tPA, which significantly enhances the hemorrhage rate in the RLCEM.

Background and Purpose — It has been proposed that the novel spin trap agent disodium-[(tert-butylimino)methyl]benzene-1,3-disulfonate N -oxide (NXY-059) may be useful in the treatment of ischemia and stroke. To date, there is little information concerning the safety of NXY-059 when administered in combination with the only Food and Drug Administration–approved pharmacological agent for the treatment of stroke, the thrombolytic tissue plasminogen activator (tPA). Thus, we determined the effects of NXY-059G, a generic form of NXY-059, on hemorrhage and infarct rate and volume when administered alone or in combination with tPA. In addition, we determined whether NXY-059G affected 2 physiological variables, blood glucose levels and body temperature. Methods — Male New Zealand White rabbits were embolized by injecting a large blood clot into the middle cerebral artery via a catheter. Five minutes after embolization, NXY-059G (100 mg/kg) was infused intravenously; control rabbits received infusions of saline, the vehicle required to solubilize NXY-059G. In tPA studies, the thrombolytic was administered intravenously starting 60 minutes after embolization (20% bolus injection/80% infusion over 30 minutes). Body temperature and blood glucose levels were measured throughout the study. Postmortem analysis included assessment of hemorrhage and infarct rate, size, and location. Results — In the vehicle control group, the hemorrhage rate after a thromboembolic stroke was 52% (n=23), and this was increased by 67% if tPA was administered (n=15). The rabbits treated with NXY-059G in the absence of tPA had a 79% incidence of hemorrhage (n=19), an increase of 52% over the control group. In the combination drug–treated groups, the NXY-059G/tPA group had a 47% incidence of hemorrhage (n=15). There was a decrease of hemorrhage volume in the NXY-059G+tPA group compared with the other 3 groups included in the study. There was no significant effect of NXY-059G either alone or in combination with tPA on infarct rate or volume. NXY-059G did not significantly alter the physiological variables that were measured. Conclusions — This study suggests that NXY-059G may affect the integrity of the cerebral vasculature when administered immediately after an embolic stroke, as evidenced by an increase in hemorrhage rate. However, when NXY-059G is administered in combination with tPA, it may improve the safety of tPA by reducing the incidence of tPA-induced hemorrhage. The mechanism(s) involved in the NXY-059G–induced increase in hemorrhage rate and reduction of tPA-induced hemorrhage rate remains to be elucidated.

It has been proposed that the novel spin trap agent disodium-[(tert-butylimino)methyl]benzene-1,3-disulfonate N-oxide (NXY-059) may be useful in the treatment of ischemic stroke. However, there is little information concerning the neuroprotective properties of NXY-059 when administered after an embolic stroke. Moreover, there is no information available concerning the combination of NXY-059 with the only Food and Drug Administration-approved pharmacological agent for the treatment of acute stroke, the thrombolytic tissue plasminogen activator (tPA). Thus, we determined the effects of NXY-059G, a generic form of NXY-059, on behavioral outcome after an embolic stroke when administered alone or in combination with tPA.Male New Zealand White rabbits were embolized by injecting a suspension of small blood clots into cerebral circulation via a carotid catheter. NXY-059G (100 mg/kg) was infused intravenously 5 minutes or 3 hours after embolization, whereas control rabbits received infusions of the saline vehicle. In tPA studies, the thrombolytic was administered intravenously starting 60 minutes or 3 hours after embolization (3.3 mg/kg). In combination studies, NXY-059G was given 5 minutes after embolization, followed by the administration of tPA beginning either 60 minutes or 3 hours after embolization. Behavioral analysis was conducted 24 hours after embolization.In the vehicle control group, the ES50 value (calculated as the amount of microclots [milligrams] that produce neurological dysfunction [impairment] in 50% of the rabbits within a specific treatment group) measured 24 hours after embolism was 1.04+/-0.31 mg, and this was increased by 153% to 2.54+/-0.72 mg if NXY-059G was administered beginning 5 minutes after embolization. However, if NXY-059G was administered beginning 3 hours after embolization, the ES50 was 2.01+/-0.40 mg. The rabbits treated with tPA alone had an ES50 of 2.64+/-0.66 or 0.63+/-0.35 mg if tPA administration started 60 minutes or 3 hours after embolization, respectively. If tPA was administered after NXY-059G (started at 5 minutes), the ES50 values were 3.15+/-0.50 or 2.66+/-0.82 if tPA administration started 60 minutes or 3 hours after embolization, respectively.This study suggests that NXY-059G is neuroprotective and can increase behavioral ratings if administered early after an embolic stroke. In addition, the study shows that NXY-059G can be used in combination with tPA without negative side effects. The drug combination can improve behavioral function and increase ES50 values. However, during the short time course of the behavioral analysis, the combination was not statistically better than either drug alone.

Background and Purpose — Platelet activation and deposition in brain microvessels appear to be key events in the pathogenesis of ischemia-induced neuronal degeneration and behavioral deficits. It has been hypothesized that activated platelets in combination with polymorphonuclear leukocytes and fibrin may play a role in vessel reocclusion leading to the “no-reflow” phenomenon after administration of the thrombolytic tissue plasminogen activator (tPA). We studied the effects of the novel glycoprotein IIb/IIIa platelet receptor antagonist SM-20302 when administered in combination with tPA on infarct and hemorrhage rate and volume to determine whether activated platelets are involved in either infarct or hemorrhage generation after a thromboembolic stroke. Methods — One hundred thirty-two male New Zealand White rabbits were included in the present study. Rabbits were embolized by injecting a blood clot into the middle cerebral artery via a catheter. Five or 65 minutes after embolization, SM-20302 (5 mg/kg) was infused intravenously. In drug combination studies, tPA was infused intravenously for 30 minutes starting 60 minutes after embolization, and SM-20302 was administered 5 or 65 minutes after embolization. Postmortem analysis included assessment of hemorrhage, infarct size and location, and clot lysis. Results — In the vehicle control group, the hemorrhage rate after a thromboembolic stroke was 33%. There was a significant increase (109%) in the hemorrhage rate in the group of rabbits treated with the thrombolytic tPA. SM-20302 by itself did not significantly alter the embolism-induced hemorrhage rate when administered either 5 or 65 minutes after embolism. The SM-20302 groups had a 42% and 33% incidence of hemorrhage in the 5- and 65-minute groups, respectively. In groups treated with a combination of drugs, the SM-20302/tPA group had a 31% and 71% incidence of hemorrhage when SM-20302 was administered 5 and 65 minutes after embolization, respectively. SM-20302 in combination with tPA also significantly increased infarct rate, but not hemorrhage or infarct volume. Conclusions — This study suggests that treatment of thromboembolic stroke with the combination of a platelet inhibitor and tPA may have a beneficial outcome on the basis of the following: First, acute administration of SM-20302 did not significantly increase hemorrhage rate. Second, SM-20302 in combination with tPA significantly reduced tPA-induced intracerebral hemorrhage. Third, there appears to be a specific window of opportunity when a platelet inhibitor must be administered to produce a beneficial effect. Overall, on the basis of our results, we hypothesize that the increased rate of intracerebral hemorrhage observed after tPA administration may be partly due to increased reocclusion of cerebral vessels following lysis of the emboli and that reocclusion can be controlled by administration of a platelet inhibitor.