Patent Application: US-30417505-A

Abstract:
an antipsychotic and glyt1 inhibitor combination is disclosed . the antipsychotic and glyt1 inhibitor combination may be employed in the prevention and treatment of symptoms of schizophrenia . pharmaceutical compositions and treatments comprising an antipsychotic and a glyt1 inhibitor for treating symptoms of schizophrenia are thus also disclosed .

Description:
it has now been found that combining an antipsychotic with a glyt1 inhibitor in the treatment of symptoms of schizophrenia results in an unexpected increase in extracellular glycine . the present invention is directed to an antipsychotic / glyt1 inhibitor combination . the antipsychotic and the glyt1 inhibitor of the combination may each be administered separately or may be together in a single pharmaceutical composition . the antipsychotic / glyt1 inhibitor combination may be used in the treatment of disorders such as schizophrenia , dementia , depression , alzheimer &# 39 ; s , adhd , substance abuse and anxiety . a number of compounds , including compound no . 1 , compound no . 2 , compound no . 3 , bind to and inhibit glycine uptake through glyt1 . glyt1 inhibitors that may be used in accordance with the invention therefore include : compound no . 1 , which is disclosed in u . s . pat . nos . 6 , 426 , 364 ; 6 , 525 , 085 ; and 6 , 579 , 987 . compound no . 2 , which is disclosed in u . s . pat . nos . 6 , 426 , 364 ; 6 , 525 , 085 ; and 6 , 579 , 987 . additional glyt1 inhibitors that may be used in accordance with the invention are disclosed in u . s . pat . nos . 6 , 426 , 364 ; 6 , 525 , 085 ; and 6 , 579 , 987 , the entire contents of which are hereby incorporated by reference . antipsychotics may be used in accordance with the invention include atypical and typical antipsychotics . atypical antipsychotics include , but are not limited to : olanzapine , 2 - methyl - 4 -( 4 - methyl - 1 - piperazinyl )- 1 oh - thieno [ 2 , 3 - b ][ 1 , 5 ] benzodiazepine , is a known compound and is described in u . s . pat . no . 5 , 229 , 382 as being useful for the treatment of schizophrenia , schizophreniform disorder , acute mania , mild anxiety states , and psychosis . u . s . pat . no . 5 , 229 , 382 is herein incorporated by reference in its entirety ; clozapine , 8 - chloro - 11 -( 4 - methyl - 1 - piperazinyl )- 5h - dibenzo [ b , e ][ 1 , 4 ] diazepine , is described in u . s . pat . no . 3 , 539 , 573 , which is herein incorporated by reference in its entirety . clinical efficacy in the treatment of schizophrenia is described ( hanes , et al ., psychopharmacol . bull ., 24 , 62 ( 1988 )); risperidone , 3 -[ 2 -[ 4 -( 6 - fluoro - 1 , 2 - benzisoxazol - 3 - yl ) piperidino ] ethyl ]- 2 - methyl - 6 , 7 , 8 , 9 - tetrahydro - 4h - pyrido -[ 1 , 2 - a ] pyrimidin - 4 - one , and its use in the treatment of psychotic diseases are described in u . s . pat . no . 4 , 804 , 663 , which is herein incorporated by reference in its entirety ; sertindole , 1 -[ 2 -[ 4 -[ 5 - chloro - 1 -( 4 - fluorophenyl )- 1h - indol - 3 - yl ]- 1 - piperidinyl ] ethyl ] imidazolidin - 2 - one , is described in u . s . pat . no . 4 , 710 , 500 . its use in the treatment of schizophrenia is described in u . s . pat . nos . 5 , 112 , 838 and 5 , 238 , 945 . u . s . pat . nos . 4 , 710 , 500 ; 5 , 112 , 838 ; and 5 , 238 , 945 are herein incorporated by reference in their entirety ; quetiapine , 5 -[ 2 -( 4 - dibenzo [ b , f ][ 1 , 4 ] thiazepin - 11 - yl - 1 - piperazinyl ) ethoxy ] ethanol , and its activity in assays which demonstrate utility in the treatment of schizophrenia are described in u . s . pat . no . 4 , 879 , 288 , which is herein incorporated by reference in its entirety . quetiapine is typically administered as its ( e )- 2 - butenedioate ( 2 : 1 ) salt ; and ziprasidone , 5 -[ 2 -[ 4 -( 1 , 2 - benzoisothiazol - 3 - yl )- 1 - piperazinyl ] ethyl ]- 6 - chloro - 1 , 3 - dihydro - 2h - indol - 2 - one , is typically administered as the hydrochloride monohydrate . the compound is described in u . s . pat . nos . 4 , 831 , 031 and 5 , 312 , 925 . its activity in assays which demonstrate utility in the treatment of schizophrenia are described in u . s . pat . no . 4 , 831 , 031 . u . s . pat . nos . 4 , 831 , 031 and 5 , 312 , 925 are herein incorporated by reference in their entirety . typical antipsychotics are conventional antipsychotics , including but not limited to , phenothiazine , butryophenones , thioxantheses , dibenzoxazepines , dihydroindolones , and diphenylbutylpiperidines . also included are pharmaceutically acceptable salts thereof , pharmaceutically acceptable esters thereof , and enantiomeric forms of the atypical or typical antipsychotics . dopamine transmission microdialysis studies were conducted to determine if compound no . 1 affected dopamine transmission in the brain . drugs inhibiting dopamine transmission are to date the most effective medications against schizophrenia . likewise , it is known that most drugs effective in schizophrenia antagonize d2 dopamine autoreceptors and thereby elevate extracellular dopamine ( ferre et al ., 1995 ). to determine if compound no . 1 was synergistic with this action , compound no . 1 was combined with the antipsychotic risperidone and effects on both glycine and dopamine were quantified in the striatum . a cumulative dose - response curve for compound no . 1 revealed the expected dose - dependent increase in extracellular glycine levels in the striatum . while the lowest dose ( 0 . 63 mg / kg ) was without effect , the highest dose of compound no . 1 ( 10 mg / kg ) caused a 2 . 5 - fold increase in glycine . although without affect on glycine , the lowest dose of compound no . 1 produced a significant reduction in extracellular dopamine , and following administration of 2 . 5 mg / kg the levels of dopamine were normalized and remained unaltered even by the highest dose of compound no . 1 . risperidone produced a dose - dependent elevation in both extracellular dopamine and glycine . while the effect on dopamine was expected due to blockade of d2 autoreceptors , the marked rise in glycine was unexpected . similar to dopamine , the elevation in glycine occurred at a threshold dose of 0 . 16 mg / kg risperidone . indeed risperidone was equally effective at producing a rise in extracellular glycine as compound no . 1 , as indicated by a 2 . 5 - fold increase in glycine after 2 . 5 mg / kg risperidone . the effects of each drug alone were additive when the drugs were given together . that is to say , the combination of compound no . 1 and risperidone produced a greater increase in glycine than either drug alone , reaching a 6 - fold increase following a combination of 1 mg / kg risperidone and 10 mg / kg compound no . 1 . also , commensurate with the biphasic reduction in dopamine by compound no . 1 , it was found that the combination of low dose compound no . 1 ( 0 . 63 mg / kg ) tended to inhibit the increase produced by 1 mg / kg risperidone , while the higher dose of compound no . 1 did not alter risperidone - induced elevations in dopamine . compounds ( as a na salt ) were stored , dissolved , and administered according to detailed instructions accompanying each compound . compounds were dissolved in a solvent consisting of 10 % bcd ( beta cyclodextrin ) and administered subcutaneously . male sprague dawley rats weighing 275 - 325 g at the start of the experiment were individually housed in a temperature - controlled colony room with a 12 - h light / dark cycle . food and water was available ad libitum throughout the experiment . the housing conditions and care of the animals were in accordance with the “ guide for the care and use of laboratory animals ” ( institute of laboratory animal resources on life sciences , national research council , 1996 ). guide cannula were stored in 95 % etoh prior to surgery , whereas surgical tools underwent heat sterilization ( 250 0 c ) immediately before each surgery . rats were anesthetized using a ketamine hydrochloride ( 100 mg / kg , ip ) xylazine ( 12 mg / kg ) mixture . after adequate anesthesia had been determined ( using toe and tail pinch procedures ), rats were placed into a stereotaxic instrument . the skull region was wiped with a 2 % betadine solution and a rostrocaudal incision was made to expose the surface of the skull . bilateral guide cannula ( 20 gauge ; plastics one ) were chronically implanted over the medial striatum ( a / p : + 0 . 5 , m / l : ± 2 . 5 , d / v : − 2 . 0 ; paxinos & amp ; watson , 1998 ) and secured using four skull screws and cranioplastic cement . the cannula need to be implanted at an angle to obtain the minimum inter - cannula distance needed for our probe leash used during microdialysis sampling . following surgery , body temperature was maintained using a heating pad and the rats were monitored until fully conscious . rats were then individually housed and assessed daily by monitoring general activity , body weight , and feces . rats were monitored for signs of an infection and cefazolin ( 100 mg / kg ; intramuscular ) was available as needed . notation was made of any animal administered antibiotic . rats were given at least five days to recover prior to microdialysis sampling . approximately 18 hr prior to sampling , a microdialysis probe ( 24 gauge ; 2 - 3 mm exposed membrane ; 13000 mwco ) encased in a spring leash and attached to a liquid swivel connected to a balancing arm was inserted into the guide cannula of an awake rat . the probe was secured in place by screwing a threaded portion of the probe leash onto the guide cannula . the rat was then placed into a behavioral chamber ( omnitech , columbus ohio ) equipped with a fan and house light ( 10w ), and food and water was available ad libitum . on the day of the experiment , dialysis buffer consisting of 5 mm glucose , 140 mm nacl , 1 . 4 mm cacl 2 , 1 . 2 mm mgcl 2 , and 0 . 15 % phosphate buffer saline , ph 7 . 4 , was perfused through the probe ( 2 . 0 μl / min ) at least two hr prior to sample collection . twenty - min dialysis samples were then collected for two hr to determine basal glycine levels . rats were then injected ( intraperitoneal ) with vehicle or one dose of the test compound , and 30 - min samples were collected for up to 10 hr . samples were split for separate chromatographic evaluation of glycine and dopamine , and frozen (− 80 0 c ) until analyzed . rats , with the dialysis probes in place , were given an overdose of pentobarbital , and the brains fixed by intracardiac infusion of pbs - formalin . coronal brain sections were 100 μm thick and stained with cresyl violet to verify probe placements . probes were left in place as the animal was perfused in order to check for the presence of blood in the probe tract . the concentrations of glycine and dopamine in dialysate samples was determined using a waters alliance 2690 hplc system with fluorometeric detection or an esa coulometric electrochemical detection hplc system , respectively . dialyis samples were split between the two systems enabling both dopamine and glycine to be measured in each sample . a waters spherisorb ods2 column ( 5 μm , 4 . 6 × 250 mm ) was used to separate the amino acids . glycine was detected using a waters 474 fluorescence detector with an excitation wavelength of 320 nm and an emission wavelength of 400 nm . the mobile phase consisted of 80 % h 2 o , 20 % acetonitrile , 0 . 1 m na 2 hpo 4 , and 0 . 1 mm ethylenediamine - tetraacetic acid ( ph to 5 . 8 with phosphoric acid ; 0 . 2 μm filter ) with a flow rate of 0 . 75 ml / min . samples were placed into the refrigerated autosampler ( 4 0 c ) and precolumn derivatization of the amino acids with o - phthaldehyde was performed using the waters alliance system . a total of 15 μl ( 5 μl sample plus 15 μl opa ) was injected onto the column . all samples collected 2 hr before and after treatment were analyzed . for the samples collected during post - treatment hrs 2 - 24 , one 20 - min sample / hr was analyzed ; the other two samples were retained for analysis of compound within the sample . glycine peak heights were compared to an external standard curve for quantification . a new standard curve was generated each day . for dopamine analysis , samples were placed in an esa ( chelmsford , mass .) model 540 autosampler connected to an hplc system with electrochemical detection . separation was achieved by pumping the samples through a 15 cm c 18 reversed phase column ( esa , inc .) and then samples were reduced / oxidized using coulometric detection . three electrodes were used : a guard cell (+ 400 mv ), a reduction analytical electrode (− 150 mv ) and an oxidation analytical electrode (+ 250 mv ). peaks were recorded and the area under the curve measured by a computer running esa chromatography data system . these values were normalized by comparison to an internal standard curve for isoproteronol and quantified by comparison to an external standard curve . peak heights were compared to an external standard curve for quantification . the data was normalized to percent change from baseline ( mean of 3 30 - min samples prior to treatment ). in addition , raw data was furnished and differences were reported along with the normalized data . all data was evaluated using a one - way anova with repeated measures over time using the statview program on a g4 macintosh . experiment # 1 . extracellular levels of dopamine and glycine in the striatum of the rat ( n = 5 ) were assessed following administration of risperidone at three ( 3 ) doses ( ascending ; 0 . 16 , 0 . 63 and 2 . 5 mg / kg ). dialysis samples were obtained every 30 min , each dose being assessed for 2 hours ( 8 hours total ). fig1 a - 1d show the effect of risperidone on glycine and dopamine . the data are shown both as amount of analyte per sample , as well as normalized to the percent change from the average of the baseline values ( i . e ., samples obtained before the first drug injection ). risperdone produced the expected elevation in extracellular dopamine , with the lowest dose eliciting a threshold elevation of approximately 50 %, and the two higher doses producing a 3 - 4 fold increase in dopamine . surprisingly , a similar elevation in extracellular glycine was observed following risperidone . although the lowest dose was without effect , the two higher doses of risperidone elicited a dose - dependent elevation in glycine up to a maximum 2 . 5 - fold increase . experiment # 2 . extracellular levels of dopamine and glycine in the striatum of the rat ( n = 5 ) were assessed following administration of compound no . 1 at three ( 3 ) doses ( ascending ; 0 . 63 , 2 . 5 and 10 mg / kg ). dialysis samples were obtained every 30 min , each dose being assessed for 2 hours ( 8 hours total ). fig2 a - 2d show the results of this experiment . as expected , compound no . 1 elicited a dose - dependent elevation in extracellular glycine . a threshold effect was seen after 0 . 63 mg / kg , and 10 mg / kg produced a 2 . 5 - fold elevation in glycine . the effect of compound no . 1 on extracellular dopamine was biphasic with respect to dose ( n = 7 ). the lowest dose of compound no . 1 produced a nearly 50 % reduction in extracellular dopamine . the levels of dopamine returned to normal following injection an injection of 2 . 5 mg / kg and remained unaltered by the highest dose of compound no . 1 . experiment # 3 . the data generated from experiments # 1 and # 2 was assessed to determine the best combination of doses of compound no . 1 and risperidone in order to determine synergism or antagonism between the two compounds . two dosing regimens were identified . in order to evaluate the effect of the low dose of compound no . 1 on dopamine ( see fig2 a - 2d ), a combination of 0 . 63 mg / kg compound no . 1 and 1 . 0 mg / kg risperidone was administered in a single bolus injection . in order to examine for a potential synergism between compound no . 1 and risperidone in elevating extracellular glycine ( see fig1 a - 1d and 2 a - 2 d ), 10 mg / kg compound no . 1 and 1 . 0 mg / kg risperidone was administered in a single bolus injection . effects on glycine : fig3 a - 3d illustrates the effect of both drug combinations on extracellular glycine in the striatum . the lower dose of compound no . 1 ( 0 . 63 mg / kg ) and the dose of risperidone examined each produce a modest rise in glycine when given alone ( see fig1 a - 1d and 2 a - 2 d , respectively ). when the two doses of drug were co - administered ( n = 5 ) there was an approximate doubling of extracellular glycine that was consistent with the effect of risperidone alone . however , combining risperidone with the higher dose of compound no . 1 ( 10 mg / kg ) caused a clear additive effect ( n = 6 ). thus , while each drug alone produced a 2 - 3 fold elevation in glycine , combined there was a 6 - fold increase in extracellular glycine . effects on dopamine : fig3 a - 3d illustrate the effect of both drug combinations on extracellular dopamine in the striatum . the upper panels illustrate the effect of combining the lower dose of compound no . 1 ( 0 . 63 mg / kg ) with risperidone ( n = 6 ). this dose of compound no . 1 reduced extracellular dopamine , and it can be seen that , although a significant increase was measured , compound no . 1 partly antagonized the increase in dopamine expected following 1 . 0 mg / kg risperidone . thus , the expected 300 % increase in dopamine following this dose of risperidone ( see fig1 c - 1d ) was reduced to 150 % when given in combination with compound no . 1 ( 0 . 63 mg / kg ). in contrast , the higher dose of compound no . 1 ( 10 mg / kg ) alone was without effect on dopamine ( fig2 c - 2d ), and when co - administered did not alter the capacity of risperidone to elevate extracellular dopamine ( n = 6 ). these data reaffirm the capacity of the glyt1 antagonist compound no . 1 to produce a dose - dependent elevation in extracellular glycine , and demonstrate that at lower doses , this drug reduces extracellular dopamine . the present data also affirm the findings of others that risperidone elevates extracellular dopamine , and makes the surprising and important observation that risperidone produces a dose - dependent elevation in extracellular glycine . moreover , in combination , the two drugs appear additive in their effects on glycine , indicating separate mechanisms of action . compound no . 1 and dopamine . it was surprising that compound no . 1 reduced extracellular dopamine . the fact that this was observed only at lower doses may indicate a separate mechanism of action than glyt1 blockade . regardless of the mechanism , this effect is synergistic with known therapeutic actions of antipsychotic medications . thus , reducing dopamine transmission in the striatum may be indicative of a mechanism for reducing dopamine receptor tone that is distinct from the classic d2 receptor blockade associated with most antipsychotic drugs . while this effect in the striatum ( especially ventral striatum ) is thought to be an important therapeutic action of antipsychotic drugs , reducing dopamine transmission in the prefrontal cortex would be expected to exacerbate the cognitive impairment associated with schizophrenia . however , there are known instances where pharmacological and environmental challenges differentially affect prefrontal cortical and striatal dopamine transmission , notably in relation to nmda receptor blockade ( cabib and puglisi - allegga , 1996 ; moghaddam and adams , 1998 ), and the effects of compound no . 1 on extracellular dopamine in striatum may not predict effects in the prefrontal cortex . this would be especially true if the effects on dopamine were indirect since the synaptic organization of the prefrontal cortex differs markedly from the striatum . the slight antagonism by low dose compound no . 1 ( 0 . 63 mg / kg ) of the effect of risperidone to elevate dopamine in the striatum is potentially important , especially if the effect of compound no . 1 is distinct in the cortex and striatum . thus , antagonism of risperidone in the striatum , but not in the cortex could have therapeutically beneficial impact , given that risperidone actions in the striatum are thought to mediate untoward motor side - effects . the elevation in glycine by risperidone was unexpected . the mechanism remains unclear . given that elimination of glycine from the extracellular space is primarily by glycine uptake , antagonism of the transporter is one option . while it is unlikely that risperidone binds directly to glyt ( goff and coyle , 2001 ), it is possible that blockade of dopamine ( or serotonin ) receptors may regulate glyt . regardless of the mechanism by which risperidone elevates glycine , there is a clear additive effect between compound no . 1 and risperidone with regard to elevating extracellular glycine . inasmuch as schizophrenia may in part result from reduced nmda conductance , the additive effect on extracellular glycine may provide therapeutic benefit by indirectly potentiating nmda conductances . thus if in fact elevating glycine is of therapeutic benefit , combining compound no . 1 with risperidone may permit the use of lower doses of risperidone . this study identified two novel actions of compound no . 1 and risperidone . low doses of compound no . 1 reduced dopamine levels and risperidone produced a dose - dependent elevation in glycine . while the cellular mechanisms mediating these actions remain unclear , they result in potentially , important interactions between the two drugs . thus , compound no . 1 ( 0 . 63 mg / kg ) slightly antagonized the capacity of risperidone to elevate dopamine , while the capacity of both drugs to elevate glycine was additive . inasmuch as dopamine and glutamate are involved in the etiology or symptomatology of schizophrenia the interactions of compound no . 1 with the known antipsychotic risperidone is therapeutically relevant . although only particular embodiments of the invention are specifically described above , it will be appreciated that modifications and variations of the invention are possible without departing from the spirit and intended scope of the invention . adams et al . 1998 . corticolimbic dopamine neurotransmission is temporarily dissociated from the cognitive and locomotor effects of phencyclidine . j . neuroscience , 18 ( 14 ): 5545 - 5554 . aragon et al . 2003 . structure , function and regulation of glycine neurotransmitters . european journal of pharmacology , 479 : 249 - 262 . bergeron et al . 1998 . modulation of n - methyl - d - aspartate receptor function by glycine transport . proc . natl . acad . sci . u . s . a ., 95 ( 26 ): 15730 - 15735 . cabib s , publisi - allegra s . 1996 . stress , depression and the mesolimbic dopamine system . psychopharmacology , 128 : 331 - 342 . danysz et al . 1998 . glycine and n - methyl - d - aspartate receptors : physiological significance and possible therapeutic applications . d &# 39 ; 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