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NEUROPHARMACOLOGY
Cerebral Vascular Disease Research Center, Department of Neurology, University of Miami School of Medicine, Miami, Florida (J.J.L., A.V., L.B., W.Z., R.B., L.K., M.D.G.); and Department of Chemistry, Florida International University, Miami, Florida (D.A.B.)
Received January 10, 2005; accepted February 10, 2005.
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Abstract |
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7 h. STAZN tissue levels at 2 to 3 h were, on average, 2.5% of blood levels in forebrain, 56% in myocardium, and 41% in kidney. STAZN was concentrated in liver with initial concentrations averaging 5.2-fold above blood levels and a subsequent linear decline of 40% between 24 and 72 h. These results establish that STAZN confers enduring ischemic neuroprotection, has a long circulating half-life, and penetrates well into brain and other organs—characteristics favoring its potential therapeutic utility.
), myocardium (Lefer and Granger, 2000), kidney (Nath and Norby, 2000), and bowel (Schoenberg and Beger, 1993). As such, antioxidant strategies represent an appealing approach for achieving organ protection.
Nitrone-based antioxidants have recently emerged as promising therapeutic agents for pathological conditions involving free radical-driven oxidative stress (Hensley et al., 1997). Nitrone spin-trap agents are important chain-breaking antioxidants in that they capture reactive paramagnetic species to form stable nitroxides. One class of nitrones—the 
-phenyl nitrones, represented by -phenyl-N-tert-butyl nitrone (PBN) (Novelli et al., 1986) and its 2,4-bis sodium sulfonate derivative, disodium 4-[(tert-butylimino)methyl] benzene-1,3-disulfonate N-oxide (NXY-059) (Kuroda et al., 1999)—have been widely tested as antioxidant therapeutics.
The azulenyl nitrones are a novel chemical class of chain-breaking antioxidants, first synthesized by Becker (1996, 1998), which possess several important properties that are thought to favor their potential efficacy as therapeutic neuroprotectants (Klivenyi et al., 1998). First, azulenyl nitrone antioxidants have low oxidation potentials, lying within the physiological range (Buettner, 1993; Becker et al., 1998); this facilitates electron transfer from these electron-rich nitrones to oxidizing radicals, resulting in the more efficient formation of nonreactive nitroxide spin adducts (Klivenyi et al., 1998). Second, azulenyl nitrones tend to be highly lipophilic, a property favoring increased blood-brain barrier permeability. In previous experimental studies, the first-generation azulenyl nitrone, AZN, exhibited significant neuroprotective efficacy in models of transient forebrain ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity, and axotomy-induced retinal injury (discussed in Becker, 1999; Ginsberg et al., 2003).
Stilbazulenyl nitrone (STAZN) is a second-generation azulenyl nitrone with high lipophilicity and antioxidant properties superior to those of AZN (Becker et al., 2002; Mojumdar et al., 2004). We have shown that STAZN reduces brain damage in a rat model of fluid percussion traumatic brain injury (Belayev et al., 2002). We next explored the protective efficacy of STAZN in focal ischemic stroke using a rat model of 2-h middle cerebral artery occlusion that gives rise to a large cortical plus subcortical infarct and substantial neurobehavioral deficit (Belayev et al., 1996). This study, comprising three separate series, demonstrated marked infarct volume reduction and neurobehavioral improvement at 3-day survival (Ginsberg et al., 2003). In over one-half of STAZN-treated animals, cortical infarction was virtually abolished (i.e., >99% reduction) and in 91% of cases, it was reduced by 60% or more compared with vehicle-treated controls. This degree of protection equaled or exceeded that observed with other highly effective strategies such as hypothermia (Huh et al., 2000), high-dose human albumin (Belayev et al., 2001), and anti-inflammatory strategies (Yrjanheikki et al., 1999) and is superior to the protection obtained by such strategies as N-methyl-D-aspartate antagonism and calcium channel blockade. The objective of the present study was 2-fold: 1) to test the hypothesis that the acute STAZN-induced neuroprotection in focal ischemia endures in animals allowed to survive for several weeks after focal ischemic stroke, and 2) to characterize the pharmacokinetics and tissue levels of STAZN.
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), and its purity was confirmed via reference to archived spectroscopic data for this compound (Becker et al., 2002). No impurities were detected.
Animal Preparation. Male Sprague-Dawley rats weighing 280 to 320 g were studied following an overnight withdrawal of food. All studies were approved by the University of Miami's Animal Use Committee. Animals were tested neurologically before anesthesia to confirm normal neurological function (described below). Anesthesia was induced with 3% halothane, 70% nitrous oxide, and a balance of oxygen, and following orotracheal intubation (2.1 mm o.d. x 45 mm B&D Insyte catheter tubing; Becton Dickinson Infusion Therapy Systems Inc., Sandy, UT), animals were ventilated with a rodent respirator (Stoelting Co., Wood Dale, IL) on a mixture of 70% nitrous oxide, 1.0 to 1.5% halothane, and a balance of oxygen passed through a humidifier containing 1 ml of Mucomyst-10 (acetylcysteine) in water. Animals were immobilized with pancuronium bromide (0.75 mg/kg i.v. and 0.35 mg/kg i.v. every half-hour), and atropine, 0.15 mg/kg i.p., was given to diminish secretions. Femoral arteries and veins were cannulated with PE-50 polyethylene tubing. Arterial blood pressure was continuously monitored (model RS3400 polygraph; Gould Instrument Systems Inc., Cleveland, OH). Arterial blood gases (pO2, pCO2, pH) (model ABL 330; Radiometer America, Inc., Westlake, OH) were monitored periodically and kept in the normal range by ventilatory adjustments. Plasma glucose (model 2300 Stat; YSI Inc., Yellow Springs, OH) and hematocrit were also measured. Rectal temperature was monitored by a thermistor and maintained at 36.5 ± 0.5°C by a heating pad beneath the animal (CMA/150 Temperature Controller; CMA/Microdialysis AB, Stockholm, Sweden). We have shown that brain temperature is a critical variable determining extent of ischemic brain injury (Busto and Ginsberg, 1998). Thus, cranial temperature was also monitored by a thermocouple probe (Omega Engineering, Stamford, CT) implanted in the temporalis muscle and was regulated at 36.2–36.7°C by a warming lamp placed near the head. Full details are presented in previous publications (Belayev et al., 1996, 2001).
Middle Cerebral Artery Occlusion. The proximal middle cerebral artery (MCA) was reversibly occluded for 2 h by the widely used method of intraluminal suture insertion as modified by our group. In this modification, the suture is coated with poly-L-lysine solution prior to use (Belayev et al., 1996); this leads to a larger and more consistent cortical and subcortical infarct that closely resembles the lesion resulting from proximal MCA occlusion in stroke patients.
Under an operating microscope, the right common carotid artery was carefully exposed; the occipital branches of the external carotid artery were coagulated, and the internal carotid artery was isolated. A 4-cm length of 3-0 monofilament, poly-L-lysine-coated nylon suture was inserted through the proximal external carotid artery into the internal carotid artery and was advanced a distance of 20 to 22 mm (according to the animal's weight) to occlude the MCA (Belayev et al., 1996). Animals were extubated and awakened at 50 to 60 min for neurological testing (as described below) to confirm a high-grade neurological deficit (total score = >10). In the present series, this was always observed. Animals were then reanesthetized (using a facemask) and the MCA suture withdrawn after 2 h of MCA occlusion. Incisions were closed, and rats were returned to their cages. Rectal temperature was measured repeatedly throughout the entire 30-day survival period (probe insertion depth, 5–6 cm).
Drug Treatment. STAZN or vehicle was administered at four time points after the onset of MCA occlusion: at 2 (i.e., at onset of recirculation), 4, 24, and 48 h. At each time point, the STAZN group (n = 16) received 0.6 mg/kg STAZN dissolved in dimethyl sulfoxide (DMSO, 0.3 mg/ml) intraperitoneally. This dosing regimen was chosen to resemble that previously shown by us to confer acute neuroprotection (Ginsberg et al., 2003). The vehicle-treated group (n = 11) received a comparable volume of DMSO i.p. at each time point. Randomization to treatment group was performed by an investigator who was not involved in the conduct of the animal studies.
Neurobehavioral Evaluation. A standardized neurobehavioral test battery was conducted repeatedly to document the initial deficit and to follow the subsequent neurological course over the 30-day survival period (Belayev et al., 1996). Except for the initial examination, all testing was performed by an observer blinded to the treatment group allocation. The test battery consisted of two components. 1) A postural reflex test was used to assess upper-body posture; this test is sensitive to both cortical and striatal lesions. Rats were suspended by the tail 1 m above the floor. Intact rats (score = 0) extended both forelimbs toward the floor. Abnormal responses included flexion of one or both forelimbs (score = 1), or inability to resist equally a lateral force applied behind the shoulders in either direction while placed on a sheet of plastic-coated paper (score = 2). 2) The second test assessed forelimb placing responses to visual, tactile, and proprioceptive stimuli, reflecting sensorimotor integration. To test visual placing, the animal was slowly lowered from each side toward a tabletop and forelimb extension assessed. Tactile placing was judged by lightly contacting the dorsal and then the lateral surface of the rat's forepaw to a table edge while obscuring its view of the table. Proprioceptive placing involved pressing the rat's paw against the table edge to stimulate the limb muscles. For each of these tests, a score of 0 was given for normal, immediate placing, a score of 1 if the placing was delayed or incomplete, and a score of 2 for absent placing. Total neurological score ranged from a normal score of 0 to a maximal possible score of 12. Neurobehavioral testing was conducted during MCAo (at 60 min), 3.45 h after treatment and 1, 2, 3, 10, 20, and 30 days after the ischemic insult.
Histopathology. Following a 30-day survival, animals were deeply anesthetized with halothane and perfused transcardially (via a polyethylene catheter ligated in the root of the aorta, with right atrial tip excised) at a pressure of 100 to 120 mm Hg with isotonic saline for 3 to 5 min followed by 40% formaldehyde, glacial acetic acid, and absolute methanol (1:1:8 by volume) for 20 min. Skulls were placed in a refrigerator for 24 h; the brain was then removed and immersed in formaldehyde/glacial acetic acid/methanol for an additional 24 h at 4°C. Brains were then paraffin-embedded. Coronal sections (10-µm thick) were obtained at nine standard intervals and were stained by hematoxylin and eosin (H&E) and examined by brightfield microscopy.
Morphometry and Image Analysis. Microscopic slides were also imaged by a high-resolution CCD camera interfaced to an MCID image analysis system (Imaging Research, Inc., St. Catherines, ON, Canada). Image analysis was conducted by an operator blinded to the treatment group assignment. As the chronic histopathology of untreated focal ischemic infarction involves extensive tissue loss with residual cavitation, cystic alterations, and ex vacuo ventricular dilatation, image analysis consisted of outlining and measuring the areas of noninfarcted ipsilateral and contralateral hemispheres. Volumes were then computed across coronal levels by numerical integration using Simpson's method (Carnevale, 1986). Quantitative histopathological infarct frequency maps were computer-generated for each treatment group using image analysis methods previously described (Zhao et al., 1996), and these image data sets were compared by pixel-based statistics to identify regions exhibiting neuroprotection.
STAZN Pharmacokinetics and Tissue Levels. The technical challenges of reliable detection and quantification of STAZN in rat blood and tissues subsequent to administration of STAZN in DMSO required methods that maximized the concentration of STAZN present in the samples analyzed. In preliminary studies, STAZN concentrations were compared in samples of whole blood, plasma, and red blood cells, to which STAZN in DMSO (0.3 mg/ml) had been added. Plasma samples, on average, contained 75% the STAZN concentration of whole blood. In subsequent assays, whole blood was used.
Reliable quantification of STAZN in rats receiving the drug in DMSO necessitated the use of large blood volumes. Sprague-Dawley rats, anesthetized and monitored as described above, received 0.6 mg/kg STAZN (0.3 mg/ml in DMSO) by intraperitoneal injection. In separate animals, the maximal blood volume (15 ml) was removed either 2, 3, 24, or 48 h after administration. Rats were then perfused with saline via the aortic root for 5 min, and the brain, heart, liver, and kidneys were removed for tissue analysis.
Whole blood samples were extracted with 3 volumes of ethyl acetate, which was evaporated under a stream of nitrogen. Samples were redissolved in 50 µl of methanol and analyzed by high-performance liquid chromatography (HPLC, Varian 9010 solvent delivery system; Varian 9050 variable wavelength UV-visible detector). The stationary phase was a Supelco Discovery C-18 reverse-phase column. Samples were eluted with a mobile phase of pure methanol. Chromatograms were made by observing the absorbance at 420 nm for 20 min. A standard curve made by plotting the area under the curve to known STAZN concentrations yielded a linear relationship over a concentration range of 0.5 µM to 0.1 mM. To ensure the correct identification of the retention time and peak area of STAZN for each sample, 0.15 µl of each sample was spiked with 0.15 µl of a standard solution of STAZN (0.01 mM), and the resulting 30-µl spiked solution was analyzed after every sample. Using the retention time of the spiked sample, the concentration of STAZN in each sample was calculated by using the area under the curve of the peak.
Tissues were frozen at –80°C. Subsequently, each organ was placed in liquid nitrogen and ground with a mortar and pestle. The organs were extracted with 3 volumes of ethyl acetate using glass-to-glass homogenizers for thorough mixing. The ethyl acetate was evaporated under a stream of nitrogen, and the samples were redissolved in 50 µl of methanol. Owing to the large amount of lipophilic material extracted from the liver, it was necessary to redissolve liver samples in 1 ml of methanol. Tissue samples were analyzed in a manner identical to that described above for the blood samples.
Measured concentrations of STAZN in the blood and tissues were corrected for STAZN extraction efficiency. This was determined by spiking blood and tissue samples with 5 µl of a solution of STAZN in DMSO (0.3 mg/ml) and analyzing as described above.
The large blood volume needed for the quantification of STAZN after i.p. administration in DMSO required one rat for each time point sampled and therefore precluded multiple sampling of the time course of STAZN concentration in a single rat. In addition, the solubility of STAZN in DMSO is limited to 0.3 mg/ml, larger volumes of DMSO could not be given i.p., and DMSO is unsuitable for intravenous drug delivery. For all these reasons, a vehicle was sought that would be suitable for intravenous administration of higher concentrations of STAZN and that would allow for detection of STAZN in the brain and in smaller volumes of blood. STAZN is moderately soluble in ethanol and can be dissolved in water only with the aid of a solubilizer. To produce whole blood levels of STAZN sufficient for HPLC detection, we dissolved STAZN at a concentration of 1.2 mg/ml in a vehicle consisting of 30% Solutol HS 15, 30% absolute ethanol, and 40% saline.
Further studies were conducted in another group of 19 normal rats anesthetized and monitored as described above. Rats received a continuous i.v. infusion of 5 mg/kg STAZN in the Solutol/ethanol/saline vehicle over a 1-h period. To minimize blood loss, two separate subgroups were studied. In one group (n = 9), blood samples (0.5 ml) were drawn at closely spaced time points (four to six time points per animal) spanning the first 3 h after onset of infusion. After the final sample, animals were perfused with saline delivered via the aortic root for 5 min, and the brain, heart, liver, and kidneys were removed for tissue analysis. In the second group (n = 7), 0.5 ml of blood samples were taken at 1, 2, 4, 6, 12, 24, 48, and 72 h after onset of STAZN infusion.
The samples were extracted with ethyl acetate and analyzed as described above. Extraction efficiency was determined by spiking tissue samples with 0.11 µg of STAZN (18.6 µmol solution of STAZN in 30% ethanol, 30% Solutol, and 40% saline) and analyzing by HPLC. Repeated analysis of single samples showed low intrasample variation (±1%).
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Mortality
Three rats in the STAZN group died prematurely (on days 1, 3, and 7, respectively), as did three animals of the vehicle group (on days 2, 3, and 4, respectively). These animals were eliminated from the neurobehavioral and histopathological analyses.
Neurobehavior
When tested prior to MCAo, all animals were neurologically normal (score = 0). At 60 min of MCAo, all rats exhibited a consistent, marked neurobehavioral deficit (score = 11 of a possible 12; Fig. 1). Within only 1 h 45 min of receiving the initial dose, STAZN-treated rats showed a significant neurological improvement compared with vehicle-treated animals. This improvement persisted throughout the 30-day survival period (Fig. 1).
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15% relative increment in the proportion of noninfarcted tissue. The topography of tissue preservation is shown by image frequency mapping in Fig. 4.
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Figure 5 presents low power photomicrographs of centrally located H&E-stained coronal sections from DMSO vehicle- and STAZN-treated rats, rank-ordered by increasing fraction of normal ipsilateral hemisphere volume relative to contralateral volume. Seven (of total n = 11) vehicle-treated rats showed moderate to large zones of cystic necrosis involving neocortex and extending to subcortical structures. In contrast, only two (of total n = 16) STAZN-treated animals showed comparable changes, and these were less extensive (Fig. 5).
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Microscopic Observations. In vehicle-treated brains, microscopic examination confirmed the cystic necrosis of cortex and subcortex described above and revealed noncontiguous, variably sized foci of thalamic necrosis in 5 of 11 brains. In STAZN-treated brains, as noted above, frank cystic cortical necrosis was present in only 2 of 16 animals. In another eight brains, neocortex was structurally intact but exhibited subtle to prominent hypercellularity attributable to increases in microglial nuclei; however, no frankly necrotic or obvious apoptotic neurons were apparent. The ventral thalamus contained a focus of necrosis or rarefaction in three animals and a small glial nodule in a fourth rat.
Pharmacokinetics
Intraperitoneal STAZN in DMSO. The mean concentration of STAZN in whole blood after i.p. administration of 0.6 mg/kg STAZN in DMSO (0.3 mg/ml) increased from 0.022 µg/ml 2 h after injection to 0.060 µg/ml 3 h after injection. This slow absorption from the intraperitoneal space is consistent with earlier results (Ginsberg et al., 2003). The subsequent decline was well described by a monoexponential function with computed half-life of 13.9 h (Fig. 6, inset). At 72 h, STAZN could not be detected by this method. These results were used to predict the time course of STAZN blood levels in the four-dose i.p. paradigm used in the in vivo studies. This curve is also shown in Fig. 6; mean predicted STAZN blood level over 0 to 72 h in the four-dose paradigm was 0.062 µg/ml.
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; Fujioka et al., 2003), and the neuroprotective efficacy shown with short survival periods may not necessarily endure with longer survival (Dietrich et al., 1993). It is possible, but as yet untested, that extending STAZN therapy beyond 48 h might confer additional protection.
The focal ischemia model employed here gives rise to a substantial, consistent cortical plus subcortical infarct that closely resembles, in extent and severity, the large hemispheric infarcts resulting from proximal MCA and internal carotid artery occlusions in patients. These characteristics have led to its widespread use in studies of ischemic neuroprotection (e.g., Huh et al., 2000; Belayev et al., 2001). Essential to the consistency of the model is the close monitoring and control of physiological variables, including brain temperature. Brain temperature control is particularly important, as it is an essential determinant of the extent of ischemic brain injury (Busto and Ginsberg, 1998).
STAZN is highly lipophilic and, hence, soluble in DMSO but virtually insoluble in water. STAZN dissolved in DMSO (0.3 mg/ml) is well tolerated when administered intraperitoneally to rats (Belayev et al., 2002; Ginsberg et al., 2003), and our previous study established that the DMSO vehicle itself does not confer neuroprotection (Ginsberg et al., 2003). In our pilot studies, four i.p. STAZN doses given over 3 days offered greater neuroprotection than a single dose; this was the rationale for the dosing used in the present experiments.
We have developed a useful method for the quantification of STAZN using HPLC. However, the concentration of STAZN in the blood after i.p. administration of 0.6 mg/kg STAZN in DMSO was so low that it was necessary to sample very large blood volumes. A greater volume of DMSO could not be safely given, and the concentration of STAZN in DMSO could not be increased. To increase the dose and allow for more frequent sampling with smaller blood volumes, a new drug delivery vehicle was devised using 30% Solutol HS 15, 30% ethanol, and 40% saline (v/v/v). Solutol HS 15 (consisting of polyoxyethylene esters of 12-hydroxystearic acid) is a nonionic surfactant with low in vivo toxicity, whose desirable clinical properties include its ability to reverse multidrug resistance of human carcinoma cells to antineoplastic agents (Coon et al., 1991). It has been used clinically to solubilize propanidid, vitamin K1, and diclofenac (Strickley, 2004).
Pharmacokinetic analysis in rats administered STAZN i.p. in DMSO revealed circulatory absorption over several hours followed by monoexponential decay with a circulating half-life of 14 h (Fig. 6); these results predicted a time-averaged whole blood STAZN concentration of 0.06 µg/ml over 72 h with the four-dose paradigm used in our in vivo studies. When STAZN was administered by i.v. infusion in Solutol/ethanol/saline, rapid initial clearance was followed by a slow decline with half-life of 7 h. These results, taken together, support a prolonged circulatory half-life of STAZN, a property desirable from the standpoint of neuroprotection in that infrequent dosing is sufficient to achieve therapeutic blood levels.
An additional advantage of STAZN is its penetrability across the intact blood-brain barrier. In the present study, forebrain STAZN levels averaged 2.5% of plasma levels at 3 h after i.v. infusion (Fig. 8). This result would predict that STAZN is capable of antagonizing reactive oxygen species, not only within the intravascular compartment, but also within brain parenchyma. STAZN levels were even more substantial in heart and kidney, amounting to 56 and 41% of plasma levels, respectively (Fig. 8). These pharmacokinetic data suggest that STAZN might have potential utility in combating oxidative stress in the settings of myocardial and/or renal injury (Lefer and Granger, 2000; Nath and Norby, 2000).
The present results show that STAZN is initially concentrated in the liver and that this is followed by its slow removal (Fig. 9)—results consistent with hepatic metabolism as a clearance pathway for STAZN. In this regard, it is of interest that the nitrone spin-trap PBN is hydroxylated in the liver, and although glucuronide and sulfonate conjugates are passed into the bile, these conjugates are cleaved by bacterial glucuronidase and sulfatase enzymes in the gut. The hydroxylated PBN is then absorbed and returned to the circulation (Reinke et al., 2000). Aromatic hydroxylation does not abrogate the radical scavenging properties of nitrones but may prolong their therapeutic effect.
In contrast to STAZN and other lipophilic nitrones, hydrophilic nitrones such as NXY-059 and S-PBN are quickly eliminated unchanged in the urine and have short half-lives dependent on renal function (Marklund et al., 2001; Strid et al., 2002). Furthermore, these hydrophilic nitrones normally penetrate the blood-brain barrier poorly (Kuroda et al., 1999), although there is in vitro evidence that they may do so under hypoxic and ischemic conditions (Dehouck et al., 2002).
When compared with other lipophilic nitrones (Liu et al., 1999), STAZN shows a similar tissue distribution, including the ability to penetrate the blood-brain barrier (Chen et al., 1990; Cheng et al., 1993). However, the circulatory half-life observed for STAZN is much longer than the 2 h half-life for PBN in rats (Trudeau-Lame et al., 2003). Less than 0.5% of a dose of PBN can be found unchanged in the urine, but rather, most of this drug is metabolized in the liver (Trudeau-Lame et al., 2003; Lame et al., 2004). The metabolic fate of STAZN is presently unknown and therefore warrants further study.
There is considerable current interest in developing nitrone spin traps as antioxidant therapeutics for a variety of disease states. The index nitrone, PBN, has shown protective efficacy in experimental models circulatory shock, reperfusion-induced myocardial damage, global and focal brain ischemia, age-related cognitive impairments, neuronal glutamate toxicity, and traumatic brain injury (reviewed in Ginsberg et al., 2003). Its sodium-sulfonated derivative, NXY-059 (AstraZeneca Pharmaceuticals LP, Wilmington, DE) is currently in clinical trials for acute ischemic stroke (Lees et al., 2003), despite the fact that it is less lipophilic than PBN, possesses only slight blood-brain barrier permeability (Kuroda et al., 1999), and requires massive, sustained dosing. In our previous study (Ginsberg et al., 2003) and in the present study, substantial STAZN neuroprotection was achieved with STAZN doses 300- to 600-fold lower than required for NXY-059 (Kuroda et al., 1999; Sydserff et al., 2002). In comparative in vitro antioxidant assays, STAZN was two to three orders of magnitude more potent than PBN and 300-fold more potent than NXY-059 (Becker et al., 2002). A further key advantage of STAZN is its low oxidation potential as measured by cyclic voltammetry, lying within the oxidation-potential range of biologically relevant antioxidants such as vitamin E and more than a full volt below that of PBN (and, by extrapolation, NXY-059) (Becker et al., 2002). In addition, pharmacokinetic properties such as clearance, half-life, and bioavailability are likely to be important in contributing to the neuroprotective efficacy of STAZN and other nitrones (Lame et al., 2004).
In summary, the present results establish that STAZN confers enduring brain protection in focal ischemic stroke, exhibits favorable pharmacokinetics with prolonged circulatory half-life, and penetrates well into the brain and other organs. These findings further support its therapeutic potential for ischemic disorders.
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ABBREVIATIONS: PBN, -phenyl-N-tert-butyl nitrone; NXY-059, disodium 4-[(tert-butylimino)methyl] benzene-1,3-disulfonate N-oxide; AZN, azulenyl nitrone; STAZN, stilbazulenyl nitrone; MCA, middle cerebral artery; DMSO, dimethyl sulfoxide; MCAo, middle cerebral artery occlusion; HPLC, high-performance liquid chromatography.
Address correspondence to: Dr. Myron D. Ginsberg, Department of Neurology (D4-5), University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101. E-mail: mdginsberg{at}stroke.med.miami.edu
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