in the following paragraphs , the present invention will be described in detail by way of example with reference to the figures . throughout this description , the preferred embodiment and examples shown should not be considered as limiting the scope of the present invention . the whole - cell patch clamp technique was used in mouse cortical neuronal cultures in order to screen for nmdar antagonists in danshen , the root of salvia milthiorriza bunge . danshen water extracts contained high potency , non - competitive nmdar antagonists with a readily reversible mode of action . the nmdar antagonists were identified as tanshinones . tanshinones are plant - derived diterpenoid quinones . compounds of the generic structures shown in fig5 and 6 are also expected to work in the present inventory and can be tested using various assays described herein or known in the art . chemical definitions of terms used in fig5 and 6 are defined below . as used herein , the term “ alkyl ” denotes branched or unbranched hydrocarbon chains containing between one and six , preferably one and four , carbon atoms , such as , e . g ., methyl , ethyl , n - propyl , iso - propyl , n - butyl , sec - butyl , iso - butyl , tert - butyl , and 2 - methylpentyl . these groups may be optionally substituted with one or more functional groups which are attached commonly to such chains , such as , e . g ., hydroxyl , bromo , fluoro , chloro , iodo , mercapto or thio , cyano , alkylthio , heterocycle , aryl , heteroaryl , carboxyl , alkoxycarbonyl , alkyl , alkenyl , nitro , amino , alkoxyl , amido , and optionally substituted isothioureido , amidino , guanidino , and the like to form alkyl groups such as trifluoromethyl , 3 - hydroxyhexyl , 2 - carboxypropyl , 2 - fluoroethyl , carboxymethyl , 4 - cyanobutyl , 2 - guanidinoethyl , 3 - n , n ′- dimethylisothiouroniumpropyl , and the like . the term “ alkenyl ” denotes an alkyl group as defined above having at least one double bond , e . g ., allyl , 3 - hydroxy - 2 - buten - 1 - yl , 1 - methyl - 2 - propen - 1 - yl and the like . the term “ alkynyl ” denotes an alkyl group as defined above having at least one triple bond . the term “ aryl ” denotes a chain of carbon atoms an which form an least one aromatic ring having preferably between about 6 - 14 carbon atoms , such as , e . g ., phenyl , naphthyl , indenyl , and the like , and which may be substituted with one or more functional groups which are attached commonly to such chains , such as , e . g ., hydroxyl , bromo , fluoro , chloro , iodo , mercapto or thio , cyano , cyanoamido , alkylthio , heterocycle , aryl , heteroaryl , carboxyl , alkoxycarbonyl , alkyl , alkenyl , nitro , amino , alkoxyl , amido , and the like to form aryl groups such as biphenyl , iodobiphenyl , methoxybiphenyl , anthryl , bromophenyl , iodophenyl , chlorophenyl , hydroxyphenyl , methoxyphenyl , formylphenyl , acetylphenyl , trifluoromethylthiophenyl , trifluoromethoxyphenyl , alkylthiophenyl , trialkylammoniumphenyl , amidophenyl , thiazolylphenyl , oxazolylphenyl , imidazolylphenyl , imidazolylmethylphenyl , cyanophenyl , pyridylphenyl , pyrrolylphenyl , pyrazolylphenyl , triazolylphenyl , tetrazolylphenyl and the like . the term “ amino ” denotes the group nrr ′, where r and r ′ may independently be alkyl , aryl or acyl as defined above , or hydrogen . the term “ carbonyl ” denotes compounds with a carbon to oxygen double bond ( c ═ o ), including aldehydes and ketones . the term “ halide ” denotes a binary combination of a halogen with another element , such as potassium iodide ki , or an organic compound in which halogen atoms replace one or more hydrogen atoms , for example ch 3 cl . description cite ischemic damage koroshetz w . j ., moskowitz m . a . emerging treatments for stroke in humans . tips 1996 ; 17 : 227 - 233 . animal models hunter a . j ., mackay k . b ., rogers d . c . to what extent have functional studies of ischemia in animals been useful in the assessment of potential neuroprotective agents . tips 1999 ; 19 : 59 - 66 . neurodegeneration lipton s . a ., rosenberg p . a . excitatory amino acids as a final common pathway for neurologic disorders . n . engl . j . med . 1994 mar 3 ; 330 ( 9 ): 613 - 22 . glaucoma and aids lipton s . a . retinal ganglion cells , glaucoma and related dementia neuroprotection . prog . brain res . 2001 ; 131 : 712 - 8 . kaul m ., garden g . a ., lipton s . a . pathways to neuronal injury and apoptosis in hiv - associated dementia . nature 2001 apr 19 ; 410 . lipton s . a . neuronal injury associated with hiv - 1 : approaches to treatment . annu . rev . pharmacol . todicol . general nicotera p ., lipton s . a . excitotoxins in neuronal apoptosis and necrosis . j . cereb . blood flow metab . 1999 jun ; 19 ( 6 ): 583 - 91 . jonas s ., ayigari v ., viera d ., waterman p . neuroprotection against cerebral ischemia . a review of animal studies and correlation with human trial results . ann . n . y . acad . sci . 1999 ; 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( eds ) experimental models of epilepsy : a manual for the laboratory worker . raven press , new york ( 1972 ) pp . 407 - 432 . kindled rat seizure girgis m . kindling as a model for limbic epilepsy . neurosci . ( 1981 ) 6 : 1695 - 1706 . pinel j . p . j ., rovner l . i . experimental epileptogenesis : kindling - induced epilepsy in rats . exper . neurol . ( 1978 ) 58 : 190 - 202 . genetic animal löscher , w . genetic animal models of epilepsy as a unique models of epilepsy resource for the evaluation of anticonvulsant drugs . a review . meth . fing . exptl . clin . pharmacol . ( 1984 ) 6 : 531 - 547 . oguro k ., ito m ., tsuda h ., mutoh k ., shiraishi h ., shirasaka y ., mikawa h . associated of nmda receptor sites and seizures e1 mice . epilepsy res . ( 1991 ) 9 : 255 - 230 . seyfried t . n . audiogenic seizures in mice . fed . proc . ( 1979 ) 38 : 2399 - 2404 . dba / 2 mouse carling r . w ., leeson p . d ., moore k . w ., smith j . d ., moyes anticonvulsant c . r ., mawer i . m ., thomas s ., chan t ., baker r ., foster a . c . 3 - nitro - 3 , 4 - dihydro - 2 ( 1h )- quinolones . excitatory amino acid antagonists acting at glycine - site nmda and ( rs )- alpha - amino - 3 - hydroxy - 5methyl - 4 - isoxazolepropionic acid receptors . j . med . chem . ( 1993 ) 36 ( 22 ): 3397 - 408 . neuropathic pain inoue t ., mashimo t ., shibata m ., shibuta s ., yoshiya i . radip development of nitric oxide - induced hyperalgesia depends on an alternate to the cgmp - mediated pathway in the rat neuropathic pain model . brain res . ( 1998 ) 792 ( 2 ): 263 - 70 . stevens c . w . an amphibian model for pain research . lab animal ( 1995 ) 24 : 32 - 36 . stevens c . w . alternatives to the use of mammals for pain research . life sciences ( 1992 ) 50 : 901 - 912 . kavaliers m . k ., ossenkopp k . p ., sanberg p . r . ( eds ), animal models of nociception and pain ( 1997 ) r . g . landes co . : austin . the particular compound that affects the disorder of interest can be administered to a patient either by themselves , or in pharmaceutical compositions where it is mixed with suitable carriers or excipient ( s ). in treating a patient exhibiting a disorder of interest , a therapeutically effective amount of a agent or agents such as these is administered . a therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient . toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals , e . g ., for determining the ld 50 ( the dose lethal to 50 % of the population ) and the ed 50 ( the dose therapeutically effective in 50 % of the population ). the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio ld 50 / ed 50 . compounds which exhibit large therapeutic indices are preferred . the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human . the dosage of such compounds lies preferably within a range of circulating concentrations that include the ed 50 with little or no toxicity . the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized . for any compound used in the method of the invention , the therapeutically effective dose can be estimated initially from cell culture assays . for example , a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the ic 50 as determined in cell culture ( i . e ., the concentration of the test compound which achieves a half - maximal disruption of the protein complex , or a half - maximal inhibition of the cellular level and / or activity of a complex component ). such information can be used to more accurately determine useful doses in humans . levels in plasma may be measured , for example , by hplc . the exact formulation , route of administration and dosage can be chosen by the individual physician in view of the patient &# 39 ; s condition . ( see e . g . fingl et al ., in the pharmacological basis of therapeutics , 1975 , ch . 1 p . 1 ). it should be noted that the attending physician would know how to and when to terminate , interrupt , or adjust administration due to toxicity , or to organ dysfunctions . conversely , the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate ( precluding toxicity ). the magnitude of an administrated dose in the management of the oncogenic disorder of interest will vary with the severity of the condition to be treated and to the route of administration . the severity of the condition may , for example , be evaluated , in part , by standard prognostic evaluation methods . further , the dose and perhaps dose frequency , will also vary according to the age , body weight , and response of the individual patient . a program comparable to that discussed above may be used in veterinary medicine . depending on the specific conditions being treated , such agents may be formulated and administered systemically or locally . techniques for formulation and administration may be found in remington &# 39 ; s pharmaceutical sciences , 18th ed ., mack publishing co ., easton , pa . ( 1990 ). suitable routes may include oral , rectal , transdermal , vaginal , transmucosal , or intestinal administration ; parenteral delivery , including intramuscular , subcutaneous , intramedullary injections , as well as intrathecal , direct intraventricular , intravenous , intraperitoneal , intranasal , or intraocular injections , just to name a few . for injection , the agents of the invention may be formulated in aqueous solutions , preferably in physiologically compatible buffers such as hanks &# 39 ; s solution , ringer &# 39 ; s solution , or physiological saline buffer . for such transmucosal administration , penetrants appropriate to the barrier to be permeated are used in the formulation . such penetrants are generally known in the art . use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention . with proper choice of carrier and suitable manufacturing practice , the compositions of the present invention , in particular , those formulated as solutions , may be administered parenterally , such as by intravenous injection . the compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration . such carriers enable the compounds of the invention to be formulated as tablets , pills , capsules , liquids , gels , syrups , slurries , suspensions and the like , for oral ingestion by a patient to be treated . agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art . for example , such agents may be encapsulated into liposomes , then administered as described above . liposomes are spherical lipid bilayers with aqueous interiors . all molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior . the liposomal contents are both protected from the external microenvironment and , because liposomes fuse with cell membranes , are efficiently delivered into the cell cytoplasm . additionally , due to their hydrophobicity , small organic molecules may be directly administered intracellularly . pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose . determination of the effective amounts is well within the capability of those skilled in the art , especially in light of the detailed disclosure provided herein . in addition to the active ingredients , these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically . the preparations formulated for oral administration may be in the form of tablets , dragees , capsules , or solutions . the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known , e . g ., by means of conventional mixing , dissolving , granulating , dragee - making , levitating , emulsifying , encapsulating , entrapping or lyophilizing processes . pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water - soluble form . additionally , suspensions of the active compounds may be prepared as appropriate oily injection suspensions . suitable lipophilic solvents or vehicles include fatty oils such as sesame oil , or synthetic fatty acid esters , such as ethyl oleate or triglycerides , or liposomes . aqueous injection suspensions may contain substances which increase the viscosity of the suspension , such as sodium carboxymethyl cellulose , sorbitol , or dextran . optionally , the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions . pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient , optionally grinding a resulting mixture , and processing the mixture of granules , after adding suitable auxiliaries , if desired , to obtain tablets or dragee cores . suitable excipients are , in particular , fillers such as sugars , including lactose , sucrose , mannitol , or sorbitol ; cellulose preparations such as , for example , maize starch , wheat starch , rice starch , potato starch , gelatin , gum tragacanth , methyl cellulose , hydroxypropylmethyl - cellulose , sodium carboxymethylcellulose , and / or polyvinylpyrrolidone ( pvp ). if desired , disintegrating agents may be added , such as the cross - linked polyvinyl pyrrolidone , agar , or alginic acid or a salt thereof such as sodium alginate . dragee cores are provided with suitable coatings . for this purpose , concentrated sugar solutions may be used , which may optionally contain gum arabic , talc , polyvinyl pyrrolidone , carbopol gel , polyethylene glycol , and / or titanium dioxide , lacquer solutions , and suitable organic solvents or solvent mixtures . dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses . pharmaceutical preparations which can be used orally include push - fit capsules made of gelatin , as well as soft , sealed capsules made of gelatin and a plasticizer , such as glycerol or sorbitol . the push - fit capsules can contain the active ingredients in admixture with filler such as lactose , binders such as starches , and / or lubricants such as talc or magnesium stearate and , optionally , stabilizers . in soft capsules , the active compounds may be dissolved or suspended in suitable liquids , such as fatty oils , liquid paraffin , or liquid polyethylene glycols . in addition , stabilizers may be added . dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the kinase modulating effects , or minimal effective concentration ( mec ). the mec will vary for each compound but can be estimated from in vitro data ; e . g ., the concentration necessary to achieve a 50 - 90 % inhibition of the kinase using the assays described herein . dosages necessary to achieve the mec will depend on individual characteristics and route of administration . however , hplc assays or bioassays can be used to determine plasma concentrations . dosage intervals can also be determined using the mec value . compounds should be administered using a regimen which maintains plasma levels above the mec for 10 - 90 % of the time , preferably between 30 - 90 % and most preferably between 50 - 90 %. 578902vl several preferred embodiments in accordance with the invention are described using the following examples for illustration : nmda - evoked currents in cultured mouse cortical neurons were blocked effectively in the presence of 1 % ( v / v ) aqueous danshen extract ( fig1 a ). the major components in aqueous extracts of the salvia miltiorrhiza roots are water - soluble compounds such as tanshinol , rosmarinic acid , and salvianolic acid ( table 1 ; fig1 b ). however , none of these major chemical components of the aqueous extracts showed nmdar antagonist activity even at millimolar concentration . aqueous danshen extracts contain only very small amounts of tanshinones . in order to investigate whether tanshinones were the effective components in the water extracts , a series of purified tanshinones were tested in the patch - clamp assay . cryptotanshinone , miltirone , tanshinone i , tanshinone iia , and tanshinone iib produced a strong , but readily reversible inhibition of the nmda - induced currents similar to the effect of the aqueous extracts ( fig1 c , d ). the present example examined whether tanshinones inhibited currents of ampa and gaba a receptors evoked by ampa or gaba , respectively . currents activated by ampa ( 10 μm ) in mouse cultured cortical neurons showed either no , or only a small , inhibition by tanshinones ( 6 ± 3 % at 100 nm tanshinone iia ). similarly , currents activated by gaba ( 20 μm ) were blocked by 10 ± 3 % ( n = 3 ). the concentration dependence of the steady - state block of nmda - induced currents was determined in order to obtain the dose / response curve of the nmdar antagonist action of the tanshinones ( fig2 ). the data were fit according to a logistic equation and the concentration at which 50 % of the nmda - evoked response was blocked ( ic 50 ) was calculated from the equation . the ic 50 values ( 95 % confidence interval ) were 1 . 1 nm ( 0 . 5 - 1 . 6 nm ) for tanshinone iib , 1 . 7 nm ( 1 - 2 . 4 nm ) for cryptotanshinone , 2 . 1 nm ( 1 . 5 - 2 . 6 nm ) for miltirone , 3 . 4 nm ( 2 . 6 - 4 . 2 nm ) for tanshinone ii a and 3 . 7 nm ( 2 . 7 - 4 . 7 nm ) for tanshinone i . the electrophysiological determined ic50 values of the tanshinones are up to two orders of magnitude smaller than those of other nmdar antagonists . concentration - response curves were obtained in the presence of a saturating dose of tanshinone and increasing amounts of nmda in cultured mouse cortical neurons ( fig3 a ). the data indicated that the degree of inhibition of the nmda - evoked current by a fixed dose of tanshinone was independent of the dose of co - applied agonist . similarly , nmdar inhibition by tanshinones was not affected by increasing concentrations of co - agonist glycine . thus , tanshinones act as non - competitive blockers of nmdars . consistent with the lack of competition with nmda and glycine , no whole - cell currents were observed when danshen water extracts were either co - applied with nmda ( 200 μm ) in the absence of glycine or with glycine in the absence of nmda . these data indicate that it is unlikely that tanshinones act as partial agonists at the glutamate or glycine recognition sites . the voltage - dependence of the inhibitory effects of the tanshinones was assessed by constructing current / voltage ( i / v ) plots in the presence or absence of the drugs ( fig3 b ). the reversal potential of nmda - induced currents was close to 0 mv . similar to the effect of the water extract , the inhibition of the nmda - induced whole - cell currents by cryptotanshinone , miltirone , and tanshinones ii a , ii b , and i was voltage - independent . tanshinones had no discernible effect on nmda - evoked currents when they were added to the patch - pipette internal solution . the lack of voltage - dependence of inhibition makes it unlikely that the tanshinones act as channel blockers . this example also compared the blocking efficacy of the tanshinones in transiently transfected human embryonic kidney ( hek ) 293 cells expressing recombinant nmdars containing either the nr1 / nr2b or nr1 / nr2d subunit combinations . addition of danshen water extract ( 1 % v / v ) or purified tanshinones ( 20 nm ) reversibly inhibited approximately 80 - 90 % of the nmda - induced current in the absence of tcm in recombinant nmdars composed of the nr1 and nr2b subunits . in contrast , only little blocking activity was observed in nmdars composed of the nr1 / nr2d subunits upon co - application of nmda and glycine in the absence of mg 2 + with either cryptotanshinone ( 100 nm ; 10 ± 1 %, n = 3 ), miltirone ( 100 nm ; 11 ± 2 %, n = 3 ), or tanshinone i ( 100 nm ; 4 ± 1 %, n = 3 ). even concentrations that were two orders of magnitude higher than the ic 50 of these compounds in nr2b containing receptors did not significantly block nr2d containing receptors . however , tanshinones ii a ( 100 nm ) and ii b ( 100 nm ) blocked 61 ± 11 % and 52 ± 11 % of the nmda - induced current in nr2d containing nmdars ( fig3 c ). the inhibition produced by tanshinone iia in recombinant receptors containing the nr2d subunit was voltage - dependent with greatly reduced efficacy at positive potentials ( fig3 c ). this is in contrast to the voltage - independent inhibitory effect of the drug in recombinant nmdars containing the nr2b subunit suggesting a subunit specific molecular blocking mechanism of the drug in the two types of nmdars . one skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned , as well as those inherent therein . the molecular complexes and the methods , procedures , treatments , molecules , specific compounds described herein are presently representative of preferred embodiments and are exemplary and are not intended as limitations on the scope of the invention . changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention . it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention . all patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains . all patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference . the invention illustratively described herein suitably may be practiced in the absence of any element or elements , limitation or limitations which is not specifically disclosed herein . thus , for example , in each instance herein any of the terms “ comprising ”, “ consisting essentially of ” and “ consisting of ” may be replaced with either of the other two terms . the terms and expressions which have been employed are used as terms of description and not of limitation , and there is no intention that in the use of such terms and expressions indicates the exclusion of equivalents of the features shown and described or portions thereof . it is recognized that various modifications are possible within the scope of the invention . thus , it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features , modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art , and that such modifications and variations are considered to be within the scope of this invention . one skilled in the art will appreciate that the present invention can be practiced by other than the preferred embodiments which are presented in this description for purposes of illustration and not of limitation , and the present invention is limited only by the claims that follow . it is noted that equivalents for the particular embodiments discussed in this description are also within the scope of the present invention .
the present invention is based in part on the surprising discovery that tanshinones from salvia miltiorrhiza act as allosteric high - potency n - methyl - d - aspartate receptor antagonists . pharmacological blockade of excessive activation of n - methyl - d - aspartate receptors greatly reduces ischemic injury of neurons in cell culture and animal models . tanshinones thus represent a novel class of compounds with nmda receptor blocking activities with potential for the development of safe neuroprotective drugs for therapy of stroke and other neurodegenerative and neuropsychiatric disorders .