Each year, 750,000 strokes occur in the United States. Approximately 80% of the strokes are ischemic, and 20% are hemorrhagic. Hemorrhagic stroke commonly occurs after rupture of aneurysms or head trauma. There is an initial period of cerebral ischemia lasting several hours due to a rise in cerebral spinal fluid (CSF) pressure and acute cerebral vasospasm. One-half of the patients that survive later develop delayed vasospasm. The mortality in the 1st month hovers around 50%. The majority of deaths (>60%) occur during the first 2 days and is associated with ischemic brain injury (Broderick et al., 1994). The factors that contribute to the acute fall in cerebral blood flow following subarachnoid hemorrhage (SAH) remain uncertain.

Ischemic strokes are caused by blockage of cerebral arteries. In the ischemic neuronal tissue, there is depletion of ATP, cellular acidification (Sarvary et al., 1994), and a rise in intracellular Na⁺ and Ca²⁺ concentrations (Kristian et al., 1998). With prolonged ischemia (0.5–2 h), intracellular organelles rupture, there is a release of proteolytic enzymes, and neurons die. There is also release of vasoconstrictors and neurotransmitters by ischemic neurons (Werling et al., 1993; Tseng et al., 1999; Guyot et al., 2001). The release of the neurotransmitters contributes to brain injury by increasing metabolic demand, whereas the vasoconstrictors oppose the recruitment of collateral flow. The size of the infarct can be minimized if blood flow is restored within 2 h after the onset of the stroke or if the metabolic demand of the tissue is reduced.

Current treatments for hemorrhagic stroke focuses on the repair of aneurysms and reductions intracranial pressure. Osmotic agents and steroids have also been tried to reduce cerebral edema and pressure. However, these treatments have proven to be largely ineffective.

Recent studies have indicated that 20-HETE may contribute to the development of vasospasm following SAH. In this regard, 20-HETE is a potent constrictor of cerebral arteries (Gebremedhin et al., 1998, 2000), and the levels of 20-HETE increase in CSF following SAH (Kehl et al., 2002; Cambj-Sapunar et al., 2003). Inhibition of the synthesis of 20-HETE, with 17-octadecynoic acid (17-ODYA) or HET0016, or blockade of its vasoconstrictor actions with WIT0002 prevents the acute fall in cerebral flow following SAH (Kehl et al., 2002; Yu et al., 2004). There is also evidence that blockade of the synthesis of 20-HETE may also reverse delayed vasospasm in dogs (Hacein-Bey et al., 2004a).

The only approved treatment for ischemic stroke is the administration of tissue plasminogen activator to dissolve clots and restore cerebral blood flow (The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995). There are no approved agents to increase collateral flow following ischemic stroke. Since the level of arachidonic acid (AA) in the CSF was high during ischemic stroke (Pilitsis et al., 2003), it is possible that the production of 20-HETE in cerebral arteries increases and that this might contribute to brain injury by opposing the recruitment of collateral flow. 20-HETE also activates several signal transduction pathways (Muthalif et al., 1998; Sun et al., 1999; Randriambozvonjy et al., 2003) involved in apoptosis and cell death. Thus, blockade of the formation of 20-HETE may reduce infarct size following ischemic stroke similar to its ability to restore cerebral blood flow following SAH. However, little progress has been in developing 20-HETE inhibitors for the treatment of stroke. The current available compounds, dibromododecinoic acid (DDMS), 17-ODYA, 1-aminobenzotriazol (ABT), or sodium 10-undecynyl sulfate (10-SUYS), are not very potent, and/or selective inhibitors for synthesis of 20-HETE (Su et al., 1998; Wang et al., 1998; Miyata et al., 2001; Xu et al., 2002) and 20-HETE antagonists are ineffective because they do not cross the blood brain barrier. HET0016 is the most selective inhibitor of the synthesis of 20-HETE (Miyata et al., 2001). However, this compound is not very soluble and has a limited half-life (Nakamura et al., 2003). Thus, new and more specific inhibitors are needed to evaluate the role of 20-HETE in various models of stroke. The present study characterize the effects of a new derivative of hydroxyformamidine, TS-011 (Fig. 1), to inhibit the formation of 20-HETE, prevent the fall in CBF following SAH and in reducing infarct size in a rat model of ischemic stroke.

Materials and Methods

General. Experiments were performed on 5-week-old spontaneously hypertensive rats (SHRs) and 8-week-old IGS rats purchased from Charles River Laboratories, Inc. (Wilmington, MA), 9- to 11-week-old male Sprague-Dawley rats from Harlan (Indianapolis, IN) and 9- to 11-week-old male SLC Wistar rats (Nippon SLC, Shizuoka, Japan). The rats were housed in approved animal care facilities either at the Medical College of Wisconsin or Taisho Pharmaceutical Co., Ltd. (Saitama, Japan) and had free access to food and water throughout the study. All protocols were reviewed by the Animal Care Committee of Taisho Pharmaceutical Co., Ltd. or the Medical College of Wisconsin (Milwaukee), and they conformed to the recommendations for the care of laboratory animals established by the National Institutes of Health and the Japanese Experimental Animal Research Association (1987).

Characterization of TS-011 as a Selective Inhibitor of the Formation of 20-HETE by CYP4A and 4F Enzymes. The effects of TS-011 on the formation of EETs and 20-HETE by human and rat renal microsomes were studied since they are rich mixed source of the P450 isoforms (CYP2C, CYP2J, CYP2E, CYP4A, CYP4F, and CYP4B) known to metabolize AA. The structure of TS-011 is presented in Fig. 1 and was synthesized in the Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd.. Human renal microsomes were purchased from the Human Cell Culture Center (Laurel, MD). Microsomes were also prepared from the kidneys of six male SHRs as follows. The rats were anesthetized with pentobarbital (50 mg/kg i.p.), the kidneys were removed, and the renal cortex was homogenized in a 20 mM HEPES buffer, pH 7.4, containing 1 mM EDTA, 100 μM p-(amidinophenyl)-methanesulfonyl fluoride, and 250 mM sucrose. The homogenate was centrifuged at 600g for 10 min and 16,000g for 30 min. The supernatant was collected and centrifuged at 200,000g for 30 min. The resulting pellet was suspended in 50 mM 3-(N-morpholino)propanesulfonic acid buffer.

Microsomes (200 μg/ml protein) were preincubated with various concentrations of TS-011 [10^-12 to 10^-4 M for 10 min at 37°C in 100–200 μl of a 50 mM 3-(N-morpholino)propanesulfonic acid buffer, pH 7.4] containing 5 mM MgCl₂ and 1 mM EDTA. [³H]Arachidonic acid (2 μCi/ml, 0.01 μM) and NADPH (1 mM) were added, and the reactions were incubated for 20 min for EET or 1 h for 20-HETE for the rat microsomes and for 6 h for the human microsomes. The reactions were stopped by the addition of formic acid, pH 3.5. Acetonitrile was added to adjust the final concentration to 50%, and the samples were separated by HPLC on a 150- × 4.6-mm Bio-sil C18HL90–5S column (Absorbosphere C18; Alltech Associates, Deer-field, IL) at a flow rate of 1.0 ml/min using a linear elution gradient ranging from acetonitrile:water:acetic acid (50:50:0.1 v/v) to acetonitrile:acetic acid (100:0.1) over 30 min. The metabolites formed were monitored using a radioactive flow detector (Ramona Star; Raytest, Pittsburgh, PA). The identity of each metabolite was confirmed by comigration with known standards.

The ability of TS-011 to inhibit the formation of 20-HETE by the major P450 isoforms known to produce 20-HETE in man were also examined. Microsomes prepared from baculovirus-infected insect cells that express human recombinant CYP4A11, 4F2, 4F3A, and 4F3B enzymes (BD Gentest, Woburn, MA) and [¹⁴C]arachidonic acid (5 μCi/ml, 10 μM) were preincubated for 5 min at 37°C with various concentrations of TS-011 (10^-9 to 10^-4 M) or vehicle in 1 ml of 100 mM potassium phosphate buffer, pH 7.4, containing 10 mM MgCl₂. After the preincubation period, 1 mM NADPH was added, and the reactions were incubated for 20 min at 37°C. The reaction was terminated by acidification with formic acid, the metabolites were extracted with ethyl acetate, and the metabolites were separated and monitored by HPLC as described above.

Effects of TS-011 on CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 Activities. The selectivity of TS-011 as an inhibitor of P450 isoforms involved in the formation of 20-HETE was further tested by studying the ability of this compound to inhibit the activity of the major human hepatic P450 enzymes involved in drug metabolism. In these experiments, 0.5 to 3 pmol microsomes prepared from baculovirus infected insect cells expressing recombinant human CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 enzymes (BD Gentest) were incubated for 15 to 45 min with an appropriate fluorescent substrate, 0.08 or 1.3 mM NADPH, and TS-011 (10^-8 to 10^-4 M) or vehicle. The substrates used were 0.5 pmol (enzyme) and 5 μM 3-cyano-7-ethoxycoumarin for CYP1A2, 75 μM 7-methoxy-4-trifluoromethyl-coumarin for CYP2C9, 1.5 μM 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7methoxy-4-methylcoumarin for CYP2D6, 25 μM 3-cyano-7-ethoxycoumari for CYP2C19, and 40 μM 7-benzyloxyquinoline for CYP3A4. The formation of the fluorescent metabolites were monitored using the following excitation and emission wavelengths: CYP1A2, 405 and 460 nm; CYP2C9, 405 and 535 nm; CYP2C19, 405 and 460 nm; CYP2D6, 390 and 460 nm; and CYP3A4, 405 and 535 nm, respectively. The experiments were repeated six times for each enzyme, and IC₅₀ values for TS-011 on each enzyme were determined according to the method of Crespi et al. (1997).

Characterization of the Effects of TS-011 on Various Receptors and Enzymes. These binding and enzyme assays were performed as a contract study by CEREP (Celle l'Evescault, France). A list of the assays employed is provided in Table 1. All experiments were performed at least in duplicate.

Effects of Pretreatment of Rats with TS-011 on the Acute Fall in Cerebral Blood Flow after SAH: Prevention Protocol. These experiments were performed on 9- to 12-week-old male Sprague-Dawley rats. The rats were anesthetized with ketamine (20 mg/kg i.m.) and thiobutabarbital (Inactin; 50 mg/kg i.p.) and surgically prepared for induction of SAH and measurement of CBF through an intact cranial window as previously described (Kehl et al., 2002; Cambj-Sapunar et al., 2003). The head of the rat was placed in a stereotaxic apparatus and the atlantooccipital membrane was exposed by separating the muscle layers at the base of the skull. A cannula was placed in the cisterna magna for injection of blood, and a pulled PE-10 cannula was placed under the dura for measurement of intracranial pressure (ICP). CBF was measured using laser-Doppler flowmetry after thinning the bone on the parietal cortex. A cannula was placed in the trachea, and the rats were artificially ventilated with a mixture of 30% O₂ in N₂. End-tidal P_CO2 was maintained at 35 mm Hg. Cannulas were also placed in the femoral artery and vein for infusion of drugs and measurement of mean arterial blood pressure (MAP). Body temperature was maintained at 37°C, and the rats received an i.v. infusion of 0.9% NaCl solution containing 1% bovine serum albumin at a rate of 3 ml/h to replace fluid losses.

After surgery and a 30-min equilibration period, baseline CBF and ICP were measured. The rats then received an i.v. injection of vehicle (n = 17) (11% sulfobutylether-β-cyclodextrin) or TS-011 at doses of 0.001 (n = 5), 0.01 (n = 6), or 0.1 (n = 12) mg/kg, and CBF and ICP were measured for an additional 30 min. After the control value of CBF was established, 0.3 ml of unheparinized arterial blood was infused into the cisterna magna at a rate of 30 μl/min over a 10-min period. Control animals received an equal volume of aCSF. CBF and ICP were recorded at 10, 20, 30, 60, 90, and 120 min after induction of SAH. At the end of most experiments, CSF (∼100 μl) was withdrawn from the cannula placed in the cisterna magna, transferred to glass vials, and stored at -80°C. 20-HETE concentration in these samples was measured with a fluorescent HPLC assay as previously described (Maier et al., 2000).

Effects of TS-011 on the Fall in CBF following SAH: Reversal Protocol. Experiments were performed to determine whether TS-011 could not only prevent, but also reverse, the fall in CBF following SAH. CBF was measured with laser-Doppler flowmetry for a control period and for 30 min after induction of SAH. The rats then received an i.v. injection of TS-011 (0.1 mg/kg, n = 7) or an equal volume of vehicle (n = 7), and CBF was followed for an additional 120 min.

Effects of TS-011 on Infarct Size following Transient Occlusion of the MCA of Rats. Male Wistar rats were anesthetized with 1% halothane. A catheter was placed in the femoral artery for blood sampling and measurement of MAP. Transient middle cerebral artery occlusion (MCAO) was produced by the intraluminal suture method described in detail elsewhere (Nagasawa and Kogure, 1989). The junction of the right common carotid artery, external cerebral artery, and internal cerebral artery were exposed. An 18-mm length of nylon suture (4-0; Nitcho Kogyo Co., Ltd., Tokyo, Japan) coated with silicon (Xantopren VL plus; Heraeus Kulzer Dental Products Division, South Bend, IN) was introduced into the internal cerebral artery and advanced 13 mm to the origin of the right MCA. The neck wounds were closed, anesthesia was withdrawn, and successful occlusion of the MCAO was tested by the appearance of left side hemiparesis. Rats that did not demonstrate hemiparesis were excluded from analysis. After 60 min of occlusion of the MCAO, the rats were reanesthetized with halothane, and the sutures blocking the MCA were withdrawn, allowing reperfusion. TS-011, at doses of 0.001, 0.01, 0.1, or 1.0 mg/kg i.v., or vehicle (10% hydroxypropyl-β cyclodextrin) was administered to approximately 20 rats per group just prior to reperfusion of the MCA. Thirty minutes after reperfusion of the MCA, the rats were allowed to awake from anesthesia. Twenty-four hours later, the rats were anesthetized with diethyl ether, decapitated, and the brains were removed and cut into seven 2-mm-thick coronal sections (from +4 mm to -8 mm bregma). The sections were immersed in a 2% (w/v) 2,3,5-triphenyltetrazolium chloride (TTC) solution at 37°C for 30 min and subsequently fixed with 10% (v/v) buffered formalin solution. Cortical and subcortical infarct areas were measured using NIH Image Analysis software.

Effect of Carotid Artery Infusion of 20-HETE on Infarct Volume in Rats. Experiments were performed to determine whether an increase in the levels of 20-HETE in the cerebral circulation could produce a cerebral infarct similar to that seen following transient occlusion of the MCA. The experiments were performed according to the method by Shirakura et al. (1992). Rats were anesthetized with 1% halothane. The right carotid artery was exposed via a midline incision, and a nonoccluding catheter was introduced. 20-HETE (8 or 12 mg/kg) dissolved in 0.1 ml of 0.1 M Na₂CO₃ solution, or an equal volume of vehicle was infused over a 15-s period.

The dose of 20-HETE was decided from the results of preliminary studies to obtain the reproducible infarct volume and neurologic deficits. The catheter was then removed, the neck closed, and the animal was allowed to recover from surgery. Twelve hours later, circling behavior, beam walking, and forelimb extension tests were performed to access the degree of motor dysfunction. Higher neurologic deficit score denotes more severely impaired function. The rats were then anesthetized with diethyl ether, and the brains were collected and stained with TTC to access infarct volume.

Effects of TS-011 on Infarct Size following Collagenase-Induced Intracerebral Hemorrhage. We also examined the effects of TS-011 on infarct size in a collagenase-induced model of intracerebral hemorrhage (Del Bigio et al., 1996). Briefly, 8-week-old Sprague-Dawley rats purchased from Charles River Laboratories, Inc. were anesthetized with thiopental (30 mg/kg i.p.), and the head was placed in a stereotaxic apparatus. The small hole was drilled in the skull (3 mm left, 0.2 mm posterior to bregma), and a 30-gauge needle was inserted 6 mm below the surface of the skull into the caudate nucleus. A solution of collagenase (0.7 μl; 200 units/ml in saline) was infused into the brain over a 5-min period. The needle was withdrawn, the hole in the skull was sealed with bone wax, and the skin incision was closed. TS-011, at doses of 0.001, 0.01, and 0.1 mg/kg i.v., or vehicle (10% hydroxypropyl-β cyclodextrin) was administered immediately after collagenase injection. Twenty-four hours later, circling behavior, beam walking, and forelimb extension tests were performed to access the degree of motor dysfunction according to the method of Peeling et al. (1998). Higher neurologic deficit score denotes more severely impaired function. The brain was removed to access infarct volume as described above.

Statistical Analysis. Mean values ± S.E. are presented. Significance of differences in mean values within and between groups was examined by a two-way analysis of variance for repeated measures followed using a Dunnett's test for planned comparisons. P < 0.05 was considered to be significant.

Results

Characterization of TS-011 as a Selective Inhibitor of the Formation of 20-HETE by CYP4A and CYP4F Enzymes. Microsomes prepared from human kidney only produced 20-HETE when incubated with AA. TS-011 inhibited the formation of 20-HETE by human renal microsomes with an IC₅₀ (95% confidence interval) of 8.42 nM (4.28–16.58 nM; Fig. 2a). Microsomes prepared from the kidneys of SHR produced both 20-HETE and 11,12-EET. TS-011 selectively inhibited the formation of 20-HETE formation by rat renal microsomes with an IC₅₀ of 9.19 nM (3.93–21.50 nM; Fig. 2b). It had no effect on the formation of EETs even at a concentration of 100 μM (Fig. 2b).

The effects of TS-011 on the P450 isoforms (CYP4F2, 4F3A, 4F3B, and 4A11) reported to produce 20-HETE in man are presented in Fig. 2, c–f. Microsomes prepared from baculovirus infected insect cells that express these isoforms only produced 20-HETE when incubated with AA. TS-011 inhibited the production of 20-HETE by these isoforms in a concentration-dependent manner, and IC₅₀ values (95% confidence interval) averaged 30.4 nM (28.0–33.0 nM) for CYP4F2, 42.6 nM (38.2–47.5 nM) for CYP4F3A, 43.0 nM (33.8–54.6 nM) for 4F3B, and 188 nM (116–303 nM) for 4FA11 (Fig. 2, c–f).

Effects of TS-011 on CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 Activities. TS-011 had little effect on the activities of these human hepatic P450 isoforms involved in drug metabolism. The IC₅₀ for CYP1A2-catalyzed substrate oxidation was 60.8 μM. The IC₅₀ values for CYP2C9-, CYP2C19-, CYP2D6-, and CYP3A4-catalyzed substrate oxidation reactions were all >100 μM (Table 2).

Characterization of the Effects of TS-011 on Other Receptors and Enzymes. The results of these experiments are summarized in Table 1. TS-011 was screened for binding to a broad range of receptors and for inhibitory effects on activity of many enzymes. TS-011 exhibited little or no affinity for any of the receptors tested. It also had no significant effect on the activity of any of the enzymes tested at a concentration of 10^-6 M.

Effect of TS-011 on CBF and 20-HETE Levels in CSF after SAH. The effects of pretreatment of the rats with various doses (0.001–0.1 mg/kg) of TS-011 on the changes in CBF following SAH in rats is presented in Fig. 3a. Injection of aCSF into the cisterna magna had no effect of CBF. In contrast, CBF fell by 60% in vehicle-treated rats during the injection of blood into CSF (Fig. 3a). This corresponded with a rise in ICP from about 10 to 60 mm Hg (Fig. 3b), whereas MAP was unaltered. Within 10 min after the infusion of blood into the CSF, ICP returned to 30 mm Hg and CBF rose to 70% of control. It remained at this level for the 2-h course of the experiment. TS-011 had no effect on baseline CBF, and it did not affect the initial fall in CBF or rise in ICP seen after induction of SAH. However, pretreatment of rats with TS-011 at doses of 0.01 and 0.1 mg/kg prevented the sustained fall in CBF following SAH. In these animals, CBF rapidly returned to values not significantly different from control after induction of SAH. CBF also recovered in the rats pretreated with the lowest dose (0.001 mg/kg) of TS-011, but it took longer to fully recover.

A comparison of the effects of TS-011 (0.1 mg/kg) on CBF when given 30 min before or 30 min after induction of SAH is presented in Fig. 4. TS-011 returned CBF completely to control within 2 h when given therapeutically, 30 min after induction of SAH when regional cerebral blood flow (rCBF) was already reduced.

The effects of TS-011 on the changes in 20-HETE levels in CSF seen following induction of SAH are presented in Fig. 5. 20-HETE levels in CSF were relatively low and on average, <100 ng/ml in sham-operated control animals in which we did not prepare a thinned cranial window for measurement of CBF. The baseline levels of 20-HETE in CSF were higher in rats prepared with a thinned cranial window. 20-HETE levels increased markedly 2 h after induction of SAH. In contrast, the levels of 20-HETE remained unaltered in rats that received an injection of aCSF in the CSF. Administration of TS-011 given either 30 min before or 30 min after induction of SAH had a similar effect to reduce the levels of 20-HETE in CSF following SAH. Thus, the results from these two groups were pooled and presented together in Fig. 5.

Effects of TS-011 on Infarct Size following Transient Occlusion of the MCA in Rats. The results of these experiments are summarized in Fig. 6. Total, cortical, and subcortical infarct volumes in the vehicle-treated rats 24 h after transient occlusion of the MCA averaged 194 ± 19, 136 ± 15, and 57.1 ± 4.0 mm³, respectively (n = 22). Total and cortical infarct volumes were significantly reduced in rats given 0.01 and 0.1 mg/kg TS-011. Subcortical infarct volume was also significantly reduced in rats given 1 mg/kg TS-011.

Effect of Intra-Arterial Infusion of 20-HETE on Infarct Volume in Rats. The results of the experiments to determine whether an increase in the levels of 20-HETE in the cerebral circulation could produce an ischemic infarct that resembles that seen following transient occlusion of the MCA is presented in Fig. 7. No infarct or neurological deficits were observed in control rats that received an infusion of vehicle in the carotid artery. In contrast, large cortical infarcts and severe neurologic deficits were observed in the rats that received an infusion of 20-HETE into the carotid artery (Table 3).

Effects of TS-011 on Infarct Size following Collagenase-Induced Intracerebral Hemorrhage. The results of these experiments are presented in Table 4. The area of cerebral infarct in the control rats averaged 42 ± 5 mm², and these rats exhibited severe motor neurological deficits, including circling behavior, the inability to walk across a beam, and muscle weakness in the front forepaws. TS-011 dose dependently reduced infarct size up to 30% and reduced the degree of motor deficit.

Discussion

The present study characterized the effects of TS-011 on the formation of 20-HETE by human and rat renal microsomes and recombinant CYP4A and 4F enzymes as well as its potential effects to prevent cerebral vasospasm following SAH and to reduce infarct size following transient occlusion of the MCA. The results indicate that TS-011 is the most potent and selective inhibitor of the formation of 20-HETE that has yet to be identified. The IC₅₀ values for inhibition of the formation of 20-HETE by rat and human renal microsomes averaged 9.19 and 8.42 nM, respectively (Fig. 2, a and b). This is an order of magnitude lower concentration than the reported IC₅₀ for HET0016 (Miyata et al., 2001). TS-011 is a 100 to 1000 times more potent inhibitor of the formation of 20-HETE than DDMS, ABT, 17-ODYA, or 10-SUYS (Wang et al., 1998; Su et al., 1998; Miyata et al., 2001; Xu et al., 2002). TS-011 exhibited a 1000-fold selectivity for inhibition of the formation of 20-HETE versus other P450 enzymes that metabolize AA. It had no effect on the formation of EETs by rat renal microsomes even when used at a concentration of 100 μM. We demonstrated that TS-011 is a potent and specific inhibitor of all of the CYP4F and CYP4A isoforms that are known to produce of 20-HETE in man (Lasker et al., 2000) and that it had no effect on the activity of the human hepatic P450 enzymes involved in drug metabolism at concentrations up to 60 μM (Table 2). TS-011 was also screened for binding to known classes of receptors and for inhibitory effects on a broad range of enzymes. TS-011 did not bind to any receptor type strongly, and it had no significant effect on any enzyme activity at concentrations of up to 10^-6 M. We also confirmed that 1 μM TS-011 had no effect on 20-HETE-induced vasoconstriction in isolated canine basilar artery with endothelium (supplemental Data). This result indicates that TS-011 has no direct inhibitory effect on 20-HETE-induced response. Thus, it seems that TS-011 is a potent and selective inhibitor of CYP4F and CYP4A enzymes that catalyze the formation of 20-HETE in a variety of tissues in both man and rats.

Recent studies have indicated that the levels of 20-HETE increase in the CSF of rats (Kehl et al., 2002; Cambj-Sapunar et al., 2003), dogs (Hacein-Bey et al., 2004a), and man (Hacein-Bey et al., 2004b) following SAH and inhibition of the synthesis of 20-HETE, with 17-ODYA or HET0016 (Kehl et al., 2002; Cambj-Sapunar et al., 2003), or blockade of its vasoconstrictor actions with WIT0002 (Yu et al., 2004) can prevent the acute fall in CBF. There is also evidence that blockade of the synthesis of 20-HETE can even reverse delayed vasospasm in dogs subjected to the dual hemorrhage model (Hacein-Bey et al., 2004a). These studies suggest that blockade of the synthesis and/or actions of 20-HETE may be useful in limiting cerebral ischemia and injury to the brain following SAH, head injury, and other conditions associated with intracranial bleeding. Nevertheless, the currently available 20-HETE inhibitors have limited potential as therapeutics agents for the treatment of stroke and vasospasm. For example, DDMS, 17-ODYA, ABT, and 10-SUYS are not very potent or selective inhibitors of the synthesis of 20-HETE (Wang et al., 1998; Su et al., 1998; Miyata et al., 2001; Xu et al., 2002). They inhibit both the synthesis of 20-HETE and EETs. DDMS, 17-ODYA, and WIT0002 also avidly bind to plasma proteins and have minimal bioavailability when delivered systemically. Finally, HET0016 has a limited half-life and is poorly soluble (Nakamura et al., 2003). Thus, the present study evaluated the therapeutic potential of TS-011 in preventing acute cerebral vasospasm following SAH. Pretreatment of rats with TS-011 significantly lowered the levels of 20-HETE in the CSF after SAH and prevented the sustained fall in CBF. TS-011 also reversed acute vasospasm and returned rCBF to control when given therapeutically, 30 min after the induction of SAH. These studies suggest that TS-011 is effective at opposing acute vasospasm and minimizing cerebral ischemia and neurologic deficits following SAH. The half-life of TS-011 in rats was 10 min when TS-011 was administered i.v., and the main metabolite of TS-011 had no inhibitory effect on 20-HETE synthesis in human renal microsomes.

The present study also examined the potential of TS-011 on minimizing infarct size following transient occlusion of the MCA since previous studies have indicated that there is release of fatty acids including AA following ischemic stroke (Pilitsis et al., 2003). The results indicate that administration of 0.01 or 0.1 mg/kg of TS-011 significantly reduced infarct volume in rats following transient occlusion of the MCA by 35%. In this regard, blockade of 20-HETE formation with TS-011 is as or more effective than N-methyl-d-aspartate (Liu et al., 1996a), endothelin (Patel et al., 1996), leukotriene (Aspey et al., 1997), platelet-activating factor (Liu et al., 1996b), and Ca channel blockers (Shino et al., 1991) in reducing infarct size in this model. Effect of TS-011 on minimizing infarct volume was stronger in cortex than subcortex area. Thus, it is possible that the production of 20-HETE in cerebral arteries in cortical penumbra region might increase following ischemic stroke and inhibition of 20-HETE formation by TS-011 might rescue the cortical damage. However, future investigation is necessary to clarify the exact mechanism by which TS-011 improves the infarct volume and possible combination therapy with tissue plasminogen activator that is the only approved drug for ischemic stroke. In further studies, we found that injection of 20-HETE into the carotid artery of rats produced a massive cerebral infarct that resembled that seen following transient occlusion of the MCA. These studies suggest that the release of AA and elevations in 20-HETE levels in the brain compromise CBF and contribute to the cerebral injury following ischemia/reperfusion of the brain. AA is known to induce endothelial damage and edema in the brain (Shirakura et al., 1992). However, future study is necessary to clarify the precise mechanism by which 20-HETE caused infarct volume and neurologic deficits.

Finally, the potential beneficial effects of TS-011 in reducing infarct volume in collagenase-induced model of ICH were examined. Administration of TS-011 at a dose of 0.1 mg/kg significant reduced infarct size and the degree of neurological deficit in rats following this model of ICH. The neurological deficits in this model is thought to be due the destruction of brain tissue secondary to cerebral edema and reduced blood flow in the affected tissue (Jenkins et al., 1989; Del Bigio et al., 1996). Although the mechanism of the beneficial effect of TS-011 remains to be determined, one likely possibility is that TS-011 might improve the rCBF following ICH by preventing acute vasospasm similar to its effects following SAH.

In summary, the present results indicate that TS-011 is the most potent and selective inhibitor of the synthesis of 20-HETE that has been developed to date. TS-011 prevents acute cerebral vasospasm following SAH and provides the first evidence that blockade of the synthesis of 20-HETE can reduce infarct size in rat models of ischemic stroke and ICH.