Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/20—Hypnotics; Sedatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/24—Antidepressants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/26—Psychostimulants, e.g. nicotine, cocaine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• EP Published Patent Application No. 0126311 discloses substituted benzopyran compounds having blood pressure lowering activity, including 6-acetyl- trans-4-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-l-benzopyran-3-ol. Also EP-A-0 376524, EP-A-0205 292, EP-A-0250077, EP-A-0093 535, EP-A-0 150 202, EP-A-0076075 and WO/89/05808 (Beecham Group pic) describe certain benzopyran derivatives which possess anti-hypertensive activity.
• EP-A-0430 621 and EP-A-0385 584 (Beecham Group pic) describe the resolution of certain intermediates useful in the preparation of the compounds described in the above mentioned patent applications.
• EP-A-0 194 885 (E. Lilly) describes certain amino substituted benzopyran derivatives possessing anti-convulsant activity.
• EP-A-0509-762 (E.R. Squibb) describes certain indole and dihydroquinoline substituted derivatives which as disclosed as possessing inter alia anti-hypertensive activity.
• PCT/GB92/01045 (SmithKline Beecham pic; unpublished at the priority date), which describes certain fluorobenzoylamido benzopyrans, pyranopyridines and tetrahydronaphthylenes in which the 3 and 4 position substituents are trans to each other. These compounds are described as possessing inter alia anxiolytic and anti- convulsant activity.
• the present invention provides a method of treatment and/or prophylaxis of anxiety, mania, depression, the effects associated with withdrawal from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines; disorders treatable and/or preventable with anti-convulsive agents, such as epilepsy; and in the treatment or prevention of cerebral ischaemia, disorders resulting from sub- arrachnoid haemorrhage, Parkinson's Disease, migraine and/or psychosis, comprising administering to the sufferer in need thereof an effective or prophylactic amount of a compound of formula (I) or pharmaceutically acceptable salt thereof:
• P is a ring system selected from the following: a)
• R-P is hydrogen or C1.5 alkyl
• M a is R5;
• J is carbon and M is nitrogen and J a and M a are hydrogen;
• R] and R2 is hydrogen and the other is selected from the class of hydrogen, C3_g cycloalkyl, C1.5 alkyl optionally interrupted by oxygen or substituted by hydroxy, C ⁇ _ - alkoxy or substituted aminocarbonyl, C ⁇ . alkylcarbonyl, C ⁇ . alkoxycarbonyl, C ⁇ . alkylcarbonyloxy, C ⁇ .
• alkoxysulphonyl aryl, heteroaryl, arylcarbonyl, heteroaiylcarbonyl, phosphono, arylcarbonyloxy, heteroarylcarbonyloxy, arylsulphinyl, heteroarylsulphinyl, arylsulphonyl, heteroarylsulphonyl in which any aromatic moiety is optionally substituted, C ⁇ .(, alkylcarbonylamino, C ⁇ .
• alkoxycarbonylamino Cj.g alkyl-thiocarbonyl, C g alkoxy-thiocarbonyl, Cj.g alkyl-thiocarbonyloxy, 1-mercapto C2-7 alkyl, fo ⁇ nyl, or aminosulphinyl, aminosulphonyl or aminocarbonyl, any amino moiety being optionally substituted by one or two Cj.g alkyl groups, or C ⁇ .
• R5 is Cj_6 alkylcarbonyloxy, benzoyloxy, ONO2, benzyloxy, phenyloxy or C ⁇ . alkoxy and Rg and R9 are hydrogen or R5 is hydroxy and R*5 is hydrogen or C ⁇ .2 alkyl and R9 is hydrogen;
• R and R ⁇ are independently selected from hydrogen, alkyl, haloalkyl, cycloalkyl, aralkyl, cyano, nitro, COR1 , CONHR13, CONR1 R14 or halo where R13 and R14 are independently selected from hydrogen, alkyl, haloalkyl, aryl, aralkyl, cycloalkyl or (cycloalkyl)-alkyl;
• RjO is hydrogen, alkyl, haloalkyl, cycloalkyl, O-R15, cyano, nitro, CF3, halo, S-alkyl, COR ⁇ , COOR15, NR15CO alkyl or OCO alkyl where R15 is hydrogen, alkyl, haloalkyl, aryl, aralkyl, cycloalkyl or (cycloalkyl)-alkyl; m is O or 1; and the R7 group is cis or trans to the R5 group.
• All C . alkyl or C 1.4 alkyl or alkyl containing groups in formula (I) are preferably selected from methyl, ethyl, n - and iso -propyl, n -, iso -, sec - and tert-butyl.
• Suitable C3_g cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
• Suitable halo substituents include fluoro, chloro and bromo.
• Aryl whenever mentioned herein includes but is not limited to phenyl and naphthyl.
• Heteroaryl whenever mentioned herein includes a 5- or 6- membered monocyclic or 9- or 10- membered bicyclic of which 5- or 6- membered monocyclic heteroaryl is preferred.
• 5- or 6-membered monocyclic or 9- or 10-membered bicyclic heteroaryl preferably contains one, two or three heteroatoms which are selected from the class of oxygen, nitrogen and sulphur and which, in the case of there being more than one heteroatom, are the same or different.
• Examples of 5- or 6-membered monocyclic heteroaryl containing one, two or three heteroatoms which are selected from the class of oxygen, nitrogen and sulphur include furyl, thienyl, pyrryl, oxazolyl, thiazolyl, imidazolyl and thiadiazolyl, and pyridyl, pyridazyl, pyrimidyl, pyrazolyl and triazolyl.
• Preferred examples of such groups include furanyl, thienyl, pyrryl and pyridyl, in particular 2- and 3-furyl, 2- and
• 3-pyrryl, 2- and 3-thienyl, and 2-, 3- and 4-pyridyl examples of 9- or 10-membered bicyclic heteroaryl containing one, two or three heteroatoms which are selected from the class of oxygen, nitrogen and sulphur include benzofuranyl, benzothienyl, indolyl and indazolyl, quinolyl and isoquinolyl, and quinazolyl.
• Preferred examples of such groups include 2- and 3-benzofuryl, 2- and 3-benzothienyl, and 2- and 3-indolyl, and 2- and 3-quinolyl.
• Preferred variables are as those mentioned in the above mentioned patents which are incorporated herein by reference. Especially preferred variables are those mentioned in EP-0509762 which are incorporated herein by reference.
• the compounds of formula (I) may have chiral carbon atoms at positions around the saturated portion of ring system P especially at the atoms attached to R5 and the moiety R7 and therefore may exist as enantiomers.
• the present invention extends to each enantiomer and to mixtures thereof including racemates.
• the compound of formula (I) or a pharmaceutically acceptable salt thereof also includes solvates of such compounds, such as for example the hydrate.
• the administration to the mammal may be by way of oral or parenteral administration.
• a unit dose will normally contain 1 to 1000 mg, suitably 1 to 500 mg, for example an amount in the range of from 2 to 400 mg such as 2, 5, 10, 20, 30, 40, 50, 100, 200, 300 and 400 mg of the active compound.
• Unit doses will normally be administered once or more than once per day, for example 1, 2, 3, 4, 5 or 6 times a day, more usually 1 to 4 times a day, such that the total daily dose is normally in the range, for a 70 kg adult of 1 to 1000 mg, for example 1 to 500 mg, that is in the range of approximately 0.01 to 15 mg kg day, more usually 0.1 to 6 mg/kg/day, for example 1 to 6 mg/kg/day.
• the compound of formula (I) is administered in the form of a unit-dose composition, such as a unit dose oral, rectal, topical or parenteral (especially intravenous) composition.
• compositions are prepared by admixture and are suitably adapted for oral or parenteral administration, and as such may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, reconstitutable powders, injectable and infusable solutions or suspensions or suppositories.
• Orally administrable compositions are preferred, in particular shaped oral compositions, since they are more convenient for general use.
• Tablets and capsules for oral administration are usually presented in a unit dose, and contain conventional excipients such as binding agents, fillers, diluents, tabletting agents, lubricants, disintegrants, colourants, flavourings, and wetting agents.
• the tablets may be coated according to well known methods in the art.
• Suitable fillers for use include cellulose, mannitol, lactose and other similar agents.
• Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycollate.
• Suitable lubricants include, for example, magnesium stearate.
• Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulphate.
• Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
• Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.
• Oral formulations also include conventional sustained release formulations, such as tablets or granules having an enteric coating.
• fluid unit dose forms are prepared containing the compound and a sterile vehicle.
• the compound depending on the vehicle and the concentration, can be either suspended or dissolved.
• Parenteral solutions are normally prepared by dissolving the compound in a vehicle and filter sterilising before filling into a suitable vial or ampoule and sealing.
• adjuvants such as a local anaesthetic, preservatives and buffering agents are also dissolved in the vehicle.
• the composition can be frozen after filling into the vial and the water removed under vacuum.
• Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilised by exposure to ethylene oxide before suspending in the sterile vehicle.
• a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound of the invention.
• the compositions will usually be accompanied by written or printed directions for use in the medical treatment concerned.
• the present invention further provides a pharmaceutical composition for use in the treatment and/or prophylaxis of anxiety, mania, depression, disorders associated with a subarachnoid haemorrhage, neural shock, the effects associated with withdrawal from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines; disorders treatable or preventable with anti-convulsive agents, such as epilepsy; Parkinson's disease, psychosis, migraine and/or cerebral ischaemia, which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• the invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prophylaxis of anxiety, mania, depression and/or disorders associated with a subarachnoid haemorrhage, neural shock the effects associated with withdrawal from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines; disorders treatable or preventable with anti-convulsive agents, such as epilepsy; Parkinson's disease, psychosis, migraine and/or cerebral ischaemia.
• compositions may be prepared in the manner as hereinbefore described.
• the invention also extends to novel compounds of formula (I) and pharmaceutically acceptable salts thereof.
• compounds of formula (I) which have trans isomerisation between the groups attached to the atom attached to the group R and the group attached to the atom adjacent, on the right-hand side of the diagram as shown herein may be prepared by procedures generally described or analogous to those described in EP- 0126311, EP-0376524, EP-205292, EP-0250077, EP-0093535,
• compounds of formula (I) which have cjs isomeration between the groups attached to the atom attached to the group R7 and the group attached to the atom adjacent on the right-hand side of the diagram as shown herein may be prepared according to the procedures generally described on or analogous to those described in EP-A-0139992.
• Cis compound of formula (I) may also be prepared according to the procedures described by G. Burrell et al, Tet. Letters, 21, 3649-3652 (1996) or by the procedures described by U. Quast and E. Villhauer, Eur. J. Pharmacol, Molecular Pharmacology Section 245, 165-171 (1993).
• racemates for formula (I) may be resolved or enantiomerically purified compounds of formula (I) may be prepared using procedures conventional in the art and in particular using the procedures outlined in EP-0430631 and EP-0355584. Where appropriate it should also be appreciated that it is preferred that the compounds of formula (I) may be prepared in the required enantiomeric form by forming a chirally pure epoxide using catalysts and conditions generally outlined in WO91X14694 or WO 93X17026 and thereafter converted to the required compound of formula (I) using procedures outlined herein. Compounds of formula (I) may be prepared from readily available starting materials using the procedures outlined or analogous to those described in the above- mentioned patents.
• the invention also provides a pharmaceutical composition
• a pharmaceutical composition comprising novel compounds of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• the invention also provides the use of novel compounds of formula (I), or a pharmaceutically acceptable salt thereof as a therapeutic agent, in particular in the treatment and/or prophylaxis of anxiety, mania, depression, the effects associated with withdrawal from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines; disorders treatable and/or preventable with anti-convulsive agents, such as epilepsy; cerebral ischaemia disorders resulting from subarachnoid haemorrhage, Parkinson's disease, migraine and/or psychosis.
• Suitable examples of compounds of formula (I) include examples 1 to 4 in EP-A-0509762 which are incorporated herein by reference.
• mice Male Sprague - Dawley rats (Charles River, U.K., 250 - 300g) are singly housed for 3 days prior to testing. On the test day, the animals are then randomly assigned to groups of 8 - 16 and dosed orally at a dose volume of 1 ml/kg with various doses of compound (1 - 300 mg/kg) or vehicle. At min post dose the rats are placed with a weight - and treatment - matched pair male (encountered for the first time) in the social interaction box under high - light, unfamiliar conditions. The box is made of white perspex 54 x 37 x 26 cm with a transparent perspex front side. The floor is divided into 24 equal squares and is brightly lit (115 lux). Time spent (sees) in active social interaction (sniffing, grooming, following, mounting, climbing over or under, boxing, biting) is scored "blind" by remote monitoring as is the number of squares crossed (as an index of locomotion).
• the maximal electroshock seizure (MES) threshold test in rodents is particularly sensitive for detecting potential anticonvulsant properties 1 .
• anticonvulsant agents elevate the threshold to electrically - induced seizures whilst proconvulsants lower the seizure threshold.
• mice Male, Charles River, U.K. CD - 1 strain, 25 - 30g are randomly assigned to groups of 10 - 20 and dosed orally or intraperitoneally at a dose volume of 10 ml/kg with various doses of compound (0.3 - 300 mg/kg) or vehicle. Mice are then subjected at 30 or 60 min post dose to a single electroshock (0.1 sec, 50Hz, sine wave form) administered via corneal electrodes.
• the mean current and standard error required to induce a tonic seizure in 50% (CC50) of the mice in a particular treatment group is determined by the 'up and down' method of Dixon and Mood (1948) 2 . Stastical comparisons between vehicle - and drug - treated groups are made using the method of Litchfield and Wilcoxon (1949) 3 .
• the CC50 In control animals the CC50 is usually 14 - 18 mA. Hence the first animal in the control group is subjected to a current of 16 mA. If a tonic seizure does not ensue, the current is increased for a subsequent mouse. If a tonic convulsion does occur, then the current is decreased, and so on until all the animals in the group have been tested. The percentage increase or decrease in CC50 for each group compared to the control is calculated. Studies are carried out using a Hugo Sachs Electronik Constant Current Shock
• Drugs are suspended in 1% methyl cellulose.
• the X-maze test of anxiety examines the exploratory response of naive rats in an environment which offers both anxiogenic (open arms) and relatively non-anxiogenic (closed arms) areas. A selective increase in exploration of the open arms following drug pretreatment is therefore postulated to indicate anxiolytic effects.
• the X-maze was raised 70cm above the floor and consisted of two enclosed arms 45cm (long) x 15cm (wide) x 10cm (high) and two open arms 45 x 10 x 1cm arranged such that the two arms of each type were opposite each other. Both arm types were marked into two equal sections. Rats were placed onto the centre of the X-maze and observed for a period of 10 minutes during which time the following parameters were recorded: 1) the number of entries onto, and the time spent on, (a) open arms, (b) closed arms, (c) end of open arms and (d) end of closed arms. 2) the number of sections crossed. The fear-drive evoked in the open arms exceeds that in the enclosed arms and rats typically show a clear preference for the enclosed arms.
• Anxiolytic drugs increase the number of entries made onto, and the time spent on, the outer half of the open arms, and also the percentage of entries made onto, and the time spent on, the whole of the open arms. These four measures of anxiety, and also the total number of sections traversed, were calculated for each animal. Drugs are administered intraperitoneally or orally to groups of 6 to 12 rats 30 to 60 mins before testing. Statistical comparisons between vehicle- and drug-treated groups were made using a Mann- Whitney 'U' test (two tailed).
• End-tidal CO2 (et CO2) is monitored continuously and arterial blood gas analysis was performed periodically to assure stable and adequate ventilation throughout each experiment.
• Polyethylene cannulae are placed in the left external jugular vein and the right femoral artery and vein for drug administration, monitoring arterial blood pressure, and blood sampling, respectively.
• Transfemoral catheterization of the left vertebral artery is then performed via the left femoral artery using a 5 french Lehman dacron catheter ( Bard, Tewksbury MA). Anaesthesia is supplemented as needed with pentobarbital (5 mg/kg, i.v.) prior to the experimental period.
• the infusion of vehicle (10% polyethylene glycol 200) for 30 minutes has no effect on the acute vasospasm.
• the effect of a 30 minute infusion of test compounds on the reversal of acute vasospasm is observed in the basilar and anterior spinal arteries.
• the effects of the compounds of this invention are also examined in the chronic canine model of delayed cerebral vasospasm (two haemorrhage model of cerebral vasospasm).
• a control vertebral angiogram is obtained and autologous blood is administered intracisternally on day 1 (as above).
• On day 3 the intracisternal administration of blood is repeated and the severe delayed vasospasm is quantitated angiographically on day 7 in all animals.
• Transient forebrain ischemia is produced by bilateral carotid artery ligation under 2.5% isoflourane in 100% O2 anesthesia, the animals being placed onto a heating pad to maintain body temperature at 37° C.
• the common carotid arteries are exposed and aneurism clips are placed on both arteries for a certain period of time indicated in the figure legends.
• PBN dissolved in saline was administered intraperitoneally as a bolus 30 min before occlusion (pretreatments) or immediately after and again at 6 h of reperfusion, followed by the same dose b.i.d. for 2 days (post- treatment).
• animals are 5 sacrificed at 7 days postischemia and perfused with buffered formalin.
• Brains were removed, stored in formalin for 3 days, embedded in paraffin, cut at 7- ⁇ m-thick coronal sections (1.5-1.9 mm posterior to bregma ⁇ ) and stained with thionin. The number of intact neurons over a 750- ⁇ m length of the CA1 layer on both hippocampal sides of 3 10 sections is counted for each animal.
• the femoral artery is cannulated with polyethylene tubing (PE60; Clay Adams. Parsippany, NJ) extending just into the descending aorta.
• the tubing is lead subdermally from the artery and
• MCAO0 or sham surgery is carried out in the SHR, SD rats under sodium pentobarbital (65 mg/kg, i.p. and supplemented as needed) anesthesia. All animals are allowed free access to food and water prior to and after surgery. Body temperature is maintained at 37°C using a heating pad throughout the surgical procedure. Surgery is conducted similar to that described previously (2.4). The right dorsal surface to the head and shaved and prepped with providone-iodine, and the rat 5 placed in a stereotaxic device (David Kopf Instruments, Tujunga, CA) with the surgery (right) side of the head superior. A 1-2 cm incision was made between the orbit and the external auditory canal. The temporal muscle is dissected from the skull and retracted without damaging the zygomatic bond or mandibular nerve. Under an
• a 2-3 mm craniotomy is made just rostral to the zygomatic-squamosal skull suture.
• the dura is opened over the artery using the modified tip of a 30-gauge needle.
• electrocoagulation Force 2 Electrosurgical Generator, Valley Lab Inc., Boulder, CO
• rats are euthanized with an overdose of sodium pentobarbital.
• brains are removed and six coronal forebrain slices (2 mm thick) are made from the level of the olfactory bulbs to the cortical-cerebellar
• TTC triphenyltetrazolium chloride
• Infarct size which was expressed as the percent infarcted tissue in reference to the contralateral (normal) hemisphere and is calculated as:
• the swelling and infarct size are expressed in reference to the contralateral hemisphere (i.e., ipsilateral ischemic damage is normalized to the normal contralateral hemisphere).

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• 1992
  • 1992-12-11 GB GB929225859A patent/GB9225859D0/en active Pending
• 1993
  • 1993-12-08 JP JP6513937A patent/JPH08504433A/ja active Pending
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